EXTRACTION DES PROTÉINES DE CANOLA PAR DES SOLUTIONS AQUEUSES ÉLECTRO-ACTIVÉES,
OPTIMISATION DES CONDITIONS D’EXTRACTION ET ÉTUDE DE LEURS
PROPRIÉTÉS TECHNO-FONCTIONNELLES
Thèse
Alina Gerzhova
Doctorat en sciences et technologie des aliments
Philosophiae Doctor (Ph. D.)
Québec, Canada
© Alina Gerzhova, 2016
EXTRACTION DES PROTÉINES DE CANOLA PAR DES SOLUTIONS AQUEUSES ÉLECTRO-ACTIVÉES,
OPTIMISATION DES CONDITIONS D’EXTRACTION ET ÉTUDE DE LEURS
PROPRIÉTÉS TECHNO-FONCTIONNELLES
Thèse
Alina Gerzhova
Sous la direction de :
Mohammed Aider, directeur de recherche Martin Mondor, codirecteur de recherche
iii
Résumé
Le tourteau de canola représente une importante source de protéines de haute valeur
nutritionnelle et pourrait faire l’objet de multiples utilisations dans l’alimentation humaine.
Toutefois, l’extraction conventionnelle de ces protéines, généralement réalisée en milieu
fortement alcalin, altère leurs qualités et fonctionnalités. De plus, l'engouement pour les
technologies vertes, justifié par la pollution générée par l’industrie agroalimentaire et la
consommation accrue de l’énergie par celle-ci, incite les industriels à innover en utilisant des
méthodes d'extraction alternatives respectueuses des principes du développement durable.
Dans ce contexte, l’électro-activation, une technologie novatrice et prometteuse pour
l’industrie alimentaire, a fait l’objet d’une étude détaillée sur son utilisation pour produire
des solutions d’extraction ayant un pouvoir extractif élevé. Elle consiste à appliquer un
champ électrique pour activer des solutions aqueuses, légèrement salines, pour les utiliser
dans la réalisation de différentes réactions chimiques, y compris l’extraction de protéines.
Ce projet vise à démontrer la preuve du concept de l’efficacité de l’électro-activation
à être utilisée comme technologie d’extraction sans utilisation d’acides et de bases et à étudier
son potentiel pour l’extraction des protéines à partir de tourteau de canola.
Le premier objectif consistait à étudier l'effet du pH, de la concentration du tourteau
de canola et de sel sur le taux d'extraction des protéines par la méthode d’extraction
conventionnelle. Il en est ressorti que seul le pH a un effet significatif sur le taux d’extraction.
Cette étude a servi de témoin pour la comparaison avec l’extraction par la technologie de
l’électro-activation en solution.
Le deuxième objectif visait à étudier les propriétés alcalines des solutions électro-
activées (SEA) en fonction du type de configuration du réacteur d’électro-activation,
l'intensité du courant électrique, la concentration du sel ajouté et le temps d’électro-activation
(de traitement). Les résultats obtenus ont montré que toutes les solutions électro-activées
après 10 minutes ont toutes été caractérisées par un pH très alcalin (pH > 10), mais leur
alcalinité titrable a varié de manière significative en fonction du temps d’électro-activation
et de l'intensité du courant appliqué (les facteurs les plus importants), ainsi que de la
concentration du sel et du type de configuration. Cette étude a montré qu'en modifiant les
paramètres d’électro-activation, il était possible de moduler les propriétés alcalines des
iv
solutions aqueuses électro-activées pour une éventuelle utilisation comme conditions
optimales pour l'extraction des protéines à partir de tourteau de canola.
Le troisième objectif consistait à évaluer et comprendre l’effet de l’électro-activation
sur le taux d'extraction des protéines du tourteau de canola et leurs propriétés physico-
chimiques. Dans cette optique, les solutions électro-activées (SEA) optimisées et une solution
de NaOH (témoin) ont été utilisées. Les résultats obtenus ont montré que le taux d'extraction
était plus élevé avec l’utilisation des SEA. De plus, les résultats obtenus par l’analyse SDS-
PAGE indiquaient que les protéines extraites par les SEA ont été nettement moins dénaturées
comparées à celles extraites avec du NaOH. L’analyse par FTIR a révélé la présence de pics
plus étroits, témoignant de la structure native secondaires des protéines extraites par électro-
activation.
Le quatrième objectif visait l’étude des propriétés fonctionnelles des protéines de
canola extraites par électro-activation et leur comparaison avec les propriétés de protéines
extraites par la méthode conventionnelle avec du NaOH. Les résultats obtenus ont montré
des améliorations dans les propriétés de surface telles que l'émulsification et la taille des
particules pour les protéines extraites par des SEA. En outre, les protéines extraites par
électro-activation étaient plus efficaces pour abaisser la tension de surface liée à leur taux de
flexibilité et taux d'adsorption plus élevé.
Le cinquième objectif a fait l’objet d’une étude dans laquelle les protéines extraites
par électro-activation sous forme de concentrés et d'isolats ont été ajoutées à une formulation
de biscuits sans gluten afin d'étudier leur comportement dans une réelle matrice alimentaire.
La formulation de biscuits sans gluten est à base d’une combinaison de farine de sarrasin
biologique cultivé au Québec et de farine de riz. Les résultats obtenus ont permis de
démontrer la pertinence d’ajouter des protéines de canola dans ce genre de produit. En effet,
par rapport à une farine sans gluten seule, l'ajout des protéines de canola a nettement amélioré
la texture des biscuits, la valeur nutritionnelle et l’acceptabilité générale du produit, ce qui a
était soutenu par une évaluation sensorielle.
Finalement et compte tenu des résultats obtenus à chacune des étapes ce projet de
doctorat, il est possible de conclure que les solutions aqueuses électro-activées (SEA) sont
des agents d'extraction puissants qui offrent des perspectives prometteuses pour l'extraction
de protéines de canola et pourraient être appliquées à d'autres tourteaux oléagineux. En plus,
v
leur utilisation comme substitut d’agents chimiques d’extraction en fait la preuve de
l’adéquation totale avec les concepts du développement durable.
vi
Abstract
Canola meal is an alternative source of valuable proteins which can be valorized for
human consumption. However, conventional extraction which implies the use of chemical
alkali and acids has a serious impact on their quality and functionality. In addition, today’s
trend towards “eco-friendly” technologies urges scientists to look for alternative methods of
protein extraction.
A novel technology of electro-activation (EA) has been reported to produce solutions
with high extraction potential by subjecting dilute salt solutions to the energy of external
electric field. Therefore the aim of the work was to study the efficiency of electro-activated
solutions for protein extraction from canola meal as a “green” emerging approach.
The first objective was to find the optimal conditions for higher extractability from
canola meal by studying the effects of pH, meal to solvent ratio, and salt concentration. It
was found that pH of the extracting medium had the most significant effect on protein
extractability. This study served as a reference template for further comparison with
extraction by electro-activated solutions (EAS).
Second objective aimed to study the properties of electro-activated solutions
depending on the type of configuration, current intensity, salt concentration, and the time of
treatment. All the solutions after 10 min treatment were characterized by highly alkaline pH
(>10) gradually attaining pH ~12, yet their titratable alkalinity differed significantly
depending on the time and the current intensity as the most important factors as well as on
the salt concentration and cell configuration. However, most importantly, it was shown that
by changing the parameters of electro-activation, optimal conditions for proteins extraction
can be modulated.
Third objective focused on protein extraction from canola meal. Electro-activated
solutions optimized in the second objective were compared to chemical base in terms of
protein extractability, and their effect on the physico-chemical properties and secondary
structure. The results showed comparably higher extractability rates for electro-activated
solutions as well as less damaged proteins as was reflected in the FTIR spectra and SDS-
PAGE analyses.
The fourth objective was to study the proteins` functionality, which showed certain
improvements in surface related properties such as emulsion activity index and droplet size
vii
for proteins extracted by electro-activated solutions. Also they were more efficient in
lowering the surface tension which pointed at their higher flexibility and adsorption rate.
Finally, for the fifth objective the extracted proteins in the form of concentrates and
isolates were added to gluten-free biscuit formulation in order to investigate their behavior
in a real food matrix. In comparison with gluten free flour blend consisting of the mixture of
rice and buckwheat flours, the addition of canola proteins markedly improved the texture of
biscuits, their nutritional value, and general acceptance which was supported by sensorial
test. Considering the results obtained at each of the stages it is possible to conclude that
electro-activated solutions are powerful extracting agents providing promising perspectives
for the extraction of proteins from canola and other oilseeds. Moreover their utilization will
allow to eliminate the use of chemical bases for the extraction step which well corresponds
to the aims stated by the concept of sustainable development.
viii
Table of contents
Résumé .............................................................................................................................................. iii Abstract ............................................................................................................................................. vi Table of contents ............................................................................................................................. viii List of tables ..................................................................................................................................... xii List of figures .................................................................................................................................. xiv
List of abbreviations ....................................................................................................................... xvii Acknowledgments ........................................................................................................................... xix
Préface ............................................................................................................................................... xx
Introduction ......................................................................................................................................... 1
1. CHAPTER 1: Literature review .................................................................................................. 4
Proteins ............................................................................................................................... 4
1.1.1. General information .................................................................................................... 4
1.1.2. Role of proteins in nutrition and their nutritive value ................................................ 5
1.1.3. Functional properties .................................................................................................. 5
1.1.4. Sources of proteins .................................................................................................... 18
Canola ................................................................................................................................ 20
1.2.1. Historical remarks ..................................................................................................... 20
1.2.2. Production, economical interest ............................................................................... 23
1.2.3. Meal processing ........................................................................................................ 24
1.2.4. Composition, nutritive and biological value of canola meal ..................................... 25
1.2.5. Proteins of canola ...................................................................................................... 31
1.2.6. Methods of protein extraction .................................................................................. 33
1.2.7. Effect of processing conditions on the quality of proteins ....................................... 36
1.2.8. Possible application ................................................................................................... 39
Electro-activation as an emerging technology .................................................................. 41
1.3.1. History and development .......................................................................................... 41
1.3.2. Principles ................................................................................................................... 42
1.3.3. Phenomenon of EAS .................................................................................................. 47
1.3.4. Reactors design ......................................................................................................... 49
1.3.5. Application of EAS ..................................................................................................... 53
2. CHAPTER 2: Problematic, research hypothesis and objectives ............................................... 58
2.1. Problematic ....................................................................................................................... 58
Research hypothesis.......................................................................................................... 58
Main objective ................................................................................................................... 58
Specific objectives ............................................................................................................. 59
3. CHAPTER 3: Total dry matter and protein extraction from canola meal as affected by pH and salt addition and the use of Zeta-potential/turbidity to optimize the extraction conditions .............. 60
Contextual transition ......................................................................................................... 60
ix
Résumé .............................................................................................................................. 61
Abstract ............................................................................................................................. 62
Introduction ...................................................................................................................... 63
Materials and methods ..................................................................................................... 65
3.6.1. Materials ................................................................................................................... 65
3.6.2. Proximate analysis ..................................................................................................... 65
3.6.3. Extraction .................................................................................................................. 66
3.6.4. Protein precipitation ................................................................................................. 66
3.6.5. Total dry matter and protein extractability .............................................................. 66
3.6.6. Turbidity and Zeta Potential (ζ) measurements ........................................................ 67
3.6.7. SDS-Gel electrophoresis ............................................................................................ 67
3.6.8. Experimental design and statistical analysis ............................................................. 68
Results and discussion ....................................................................................................... 69
3.7.1. Extractability .............................................................................................................. 69
3.7.2. Protein and ash composition of extracts .................................................................. 77
3.7.3. Precipitability based on Zeta Potential and Turbidity ............................................... 78
3.7.4. Gel Electrophoresis ................................................................................................... 81
Conclusion ......................................................................................................................... 83
Acknowledgments ............................................................................................................. 84
4. CHAPTER 4: Monitoring of pH and alkalinity changes in the electro-activated aqueous solutions generated in the cationic compartment. The effect of salt concentration, current intensity, time and cell configuration ................................................................................................................ 85
Contextual transition ......................................................................................................... 85
Résumé .............................................................................................................................. 86
Abstract ............................................................................................................................. 87
Introduction ...................................................................................................................... 88
Materials and methods ..................................................................................................... 91
4.5.1. Chemicals .................................................................................................................. 91
4.5.2. Ion-exchange membranes ......................................................................................... 91
4.5.3. Reactor design. Configurations of electro-activation cells ....................................... 91
4.5.4. Protocol of electro-activation ................................................................................... 92
4.5.5. Analysis methods ...................................................................................................... 92
4.5.6. Statistical analysis ...................................................................................................... 92
Results and discussion ....................................................................................................... 93
4.6.1. Configuration 1 .......................................................................................................... 93
4.6.2. Configuration 2 .......................................................................................................... 97
4.6.3. Configurations 3 and 4 ............................................................................................ 101
4.6.4. The effect of type of configuration ......................................................................... 102
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Conclusion ....................................................................................................................... 106
Acknowledgments ........................................................................................................... 107
5. CHAPTER 5: A comparative study between the electro-activation technique and conventional extraction method on the extractability, composition and physicochemical properties of canola protein concentrates and isolates ..................................................................................................... 108
Contextual transition ....................................................................................................... 108
Résumé ............................................................................................................................ 109
Abstract ........................................................................................................................... 110
Introduction .................................................................................................................... 111
Materials and methods ................................................................................................... 113
5.5.1. Chemicals ................................................................................................................ 113
5.5.2. Ion-exchange membranes ....................................................................................... 113
5.5.3. Configurations of the electro-activation reactor .................................................... 114
5.5.4. Experimental ........................................................................................................... 114
5.5.5. Chemical analysis .................................................................................................... 115
5.5.6. Total dry matter and protein extractability ............................................................ 116
5.5.7. SDS PAGE ................................................................................................................. 116
5.5.8. FTIR .......................................................................................................................... 117
5.5.9. Statistical analysis .................................................................................................... 117
Results and discussion ..................................................................................................... 117
5.6.1. Changes in pH .......................................................................................................... 118
5.6.2. Extractability ............................................................................................................ 122
5.6.3. Composition ............................................................................................................ 125
5.6.4. SDS PAGE ................................................................................................................. 128
5.6.5. FTIR .......................................................................................................................... 132
Conclusion ....................................................................................................................... 137
Acknowledgments ........................................................................................................... 138
6. CHAPTER 6: Study of the functional properties of canola protein concentrates and isolates extracted by electro-activated solutions .......................................................................................... 139
Contextual transition ....................................................................................................... 139
Résumé ............................................................................................................................ 140
Abstract ........................................................................................................................... 141
Introduction .................................................................................................................... 142
Materials and Methods ................................................................................................... 144
6.5.1. Raw materials and extraction methods .................................................................. 144
6.5.2. Nitrogen solubility index ......................................................................................... 144
6.5.3. Water absorption capacity (WAC) ........................................................................... 145
6.5.4. Fat absorption capacity (FAC) ................................................................................. 145
6.5.5. Surface characteristics ............................................................................................ 145
xi
6.5.6. Surface active properties ........................................................................................ 147
6.5.7. Statistical analysis .................................................................................................... 148
Results and discussion ..................................................................................................... 149
6.6.1. Nitrogen solubility index ......................................................................................... 149
6.6.2. Water absorption capacity ...................................................................................... 150
6.6.3. Fat absorption capacity ........................................................................................... 152
6.6.4. Surface characteristics ............................................................................................ 153
6.6.5. Surface active properties ........................................................................................ 157
Conclusion ....................................................................................................................... 165
Acknowledgments ........................................................................................................... 166
7. CHAPTER 7: Incorporation of canola proteins extracted by electro-activated solutions in the gluten free biscuit formulation of rice-buckwheat flour blend: Assessment of quality characteristics and textural properties of the product. ............................................................................................ 167
Contextual transition ....................................................................................................... 167
Résumé ............................................................................................................................ 168
Abstract ........................................................................................................................... 169
Introduction .................................................................................................................... 170
Materials and methods ................................................................................................... 172
7.5.1. Materials ................................................................................................................. 172
7.5.2. Proximate analysis ................................................................................................... 173
7.5.3. Cookie preparation .................................................................................................. 173
7.5.4. Analytical tests ........................................................................................................ 174
7.5.5. Statistical analysis .................................................................................................... 175
Results and discussion ..................................................................................................... 175
7.6.1. Proximate analysis ................................................................................................... 175
7.6.2. Dimensions and cookie spread ratio ....................................................................... 176
7.6.3. Dough characteristics .............................................................................................. 179
7.6.4. Cookie characteristics ............................................................................................. 181
7.6.5. Surface colour ......................................................................................................... 183
7.6.6. Moisture content and water activity ...................................................................... 185
7.6.7. Microstructure analysis ........................................................................................... 187
7.6.8. Preliminary sensory evaluation ............................................................................... 189
Conclusion ....................................................................................................................... 191
Acknowledgments ........................................................................................................... 193
8. CHAPTER 8: Conclusion and perspectives ............................................................................ 194
General conclusion .......................................................................................................... 194
Perspectives .................................................................................................................... 196
References ....................................................................................................................................... 198
xii
List of tables
Table 1.1: Indispensable amino acid requirements (FAO/WHO/UNU, 2007). ..................... 4
Table 1.2: Various protein functionalities and their application in food. Adapted from Fennema (1996). ..................................................................................................................... 6
Table 1.3: Worldwide production of major oilseeds and their meals in MMT and their protein contents. Adapted from Ramachandran et al. (2007); USDA (2013). ..................... 20
Table 1.4: Proximate composition of canola meal. Adapted from Newkirk et al. (2003). .. 25
Table 1.5: Amino acid composition of canola. Adapted from Newkirk et al. (2003), Downey (1990). .................................................................................................................... 26
Table 1.6 : Carbohydrate content of canola meal. Adapted from Newkirk (2011). ............. 27
Table 1.7: Mineral composition of canola and soybean meal. Adapted from Bell et al. (1999); Downey (1990); Newkirk (2011). ........................................................................... 28
Table 1.8: Extraction methods and conditions used for canola and rapeseed. ..................... 37
Table 3.1: Proximate chemical composition of canola oil cake on a dry weight basis. ....... 69
Table 4.1: Variables and their coded values. ........................................................................ 97
Table 4.2: Comparison of pH and alkalinity between two configurations for different current intensities within 0.01 M NaCl concentration. ....................................................... 103
Table 4.3: Comparison of pH and alkalinity between two configurations for different current intensities within 0.1 M NaCl concentration. ......................................................... 104
Table 4.4 : Comparison of pH and alkalinity between two configurations for different current intensities within 1 M NaCl concentration. ............................................................ 105
Table 5.1: Proximate chemical composition of canola oil cake on a dry weight basis. ..... 118
Table 5.2: Comparison of pH and alkalinity between two configurations for I = 0.3 A and within different NaCl concentrations. ................................................................................ 119
Table 5.3: Comparative characteristics of protein concentrates. ........................................ 125
Table 5.4: Comparative characteristics of protein isolates. ................................................ 127
Table 6.1: Absorption capacities of electro-activated protein isolate (EAPI), conventional protein isolate (CPI), electro-activated protein concentrate (CPC), and conventional protein concentrate (CPC)............................................................................................................... 151
Table 6.2 : Surface pressure at air-protein solution interface. ............................................ 154
Table 6.3 : Interfacial tension between oil and protein solution. ....................................... 155
Table 6.4: Foaming capacity of protein isolates and concentrates. .................................... 159
Table 7.1: Control biscuit formulation. .............................................................................. 173
Table 7.2: Proximate analysis of flours and proteins. ........................................................ 176
Table 7.3: Physical parameters of cookies made from flour blend of rice and dark (toasted) buckwheat and rice and green (non-toasted) buckwheat. ................................................... 177
Table 7.4: Changes in surface colour of biscuits prepared from the blend of rice and dark buckwheat with the addition of canola proteins. ................................................................ 183
xiii
Table 7.5: Changes in surface colour of biscuits prepared from the blend of rice and green buckwheat with the addition of canola proteins. ................................................................ 184
xiv
List of figures
Figure 1.1: Major destabilization processes in emulsions. Adapted from Tadros (2013). ... 12
Figure 1.2 : Triangle of U showing the relationship of major Brassica species. Adapted from Ahuja et al. (2010). ...................................................................................................... 21
Figure 1.3: Decrease in glucosinolates and erucic acid levels in rapeseed/canola. Adapted from Daun et al. (2011). ....................................................................................................... 22
Figure 1.4: Number of varieties of canola in Canada. Adapted from Daun et al. (2011). ... 22
Figure 1.5: Processing of canola by prepress solvent extraction. Adapted from Daun (2004). .............................................................................................................................................. 24
Figure 1.6: Dissociation of 12S globulin under external conditions. Adapted from Mieth et al. (1983) ............................................................................................................................... 32
Figure 1.7: Basic electrolytic cell. Adapted from Zeng and Zhang (2010). ......................... 44
Figure 1.8 : Electrolytic cell showing the passage of electrons and some products of electrode reactions. Adapted from Chaplin (2000). ............................................................. 46
Figure 1.9: Flow type electrochemical modules. Adapted from Leonov et al. (1999). ........ 50
Figure 1.10: A schematic representation of a three-call electro-activation reactor. Adapted from (Liato et al., 2015a). ..................................................................................................... 51
Figure 1.11: Example of a CEM with fixed SO3 groups. Adapted from (Bazinet et al., 1998a). .................................................................................................................................. 53
Figure 1.12 : Electro-activation unit for protein extraction form sunflower oil cake, where 1-tank with oilcake; 2 - cathodic chamber; 3 - membrane; 4 – anodic chamber; 5 – tank with anolyte; 6 – peristaltic pump; 7 – anode; 8 – cathode; 9 – filter; 10 – oilcake; 11 – extracting solution. Adapted from Plutakhin et al. (2005). .................................................. 56
Figure 3.1: Influence of the solution pH on the total dry matter extractability from canola meal. ..................................................................................................................................... 70
Figure 3.2: Influence of the solution pH on the protein extractability from canola meal. ... 70
Figure 3.3: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 10. .......................................................................................................................... 73
Figure 3.4: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 11. .......................................................................................................................... 75
Figure 3.5: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 12. .......................................................................................................................... 77
Figure 3.6: Protein and ash content of extracts obtained with 10% canola meal concentration. ....................................................................................................................... 78
Figure 3.7: The influence of the solution pH on the Zeta potential of the canola protein. ... 79
Figure 3.8: The influence of the solution pH on the turbidity of canola proteins solution. . 80
Figure 3.9: SDS-PAGE of canola proteins A) without NaCl addition and B) with an addition of 1 M NaCl. ........................................................................................................... 82
Figure 4.1: Configuration 1 of the electro-activation reactor used for the generation of the electrolysed water. ................................................................................................................ 93
xv
Figure 4.2: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 1. ................................................. 94
Figure 4.3 : The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M). ........... 96
Figure 4.4: Configuration 2 of the electro-activation reactor used for the generation of the electrolysed water. ................................................................................................................ 97
Figure 4.5: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 2. ................................................. 99
Figure 4.6: The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M). ......... 101
Figure 4.7: Configuration 3 (a) and 4 (b) of the electro-activation reactor used for the generation of the electrolysed water. .................................................................................. 102
Figure 5.1 : Configurations of the reactor used for EA corresponding to: a) Configuration 1 (C1); b) Configuration 2 (C2). ............................................................................................ 114
Figure 5.2: Changes in pH during EA for I = 0.3 A: a) C1; b) C2. .................................... 119
Figure 5.3: Changes in pH during extraction by EAS: a) C1, 0.01 M NaCl; b) C1, 0.1 M NaCl; c) C1, 1 M NaCl; d) C2, 0.01 M NaCl; e) C2, 0.1 M NaCl; f) C2, 1 M NaCl. ....... 120
Figure 5.4: Total dry matter extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2. ................................................ 123
Figure 5.5: Protein extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2. ................................................................. 124
Figure 5.6 : Molecular profiles as analyzed by SDS PAGE: a) EAPC extracted with 0.01 M electro-activated NaCl solution; b) EAPC extracted with 1 M electro-activated NaCl solution; c) CPC extracted with 0.01M and 1 M NaCl solutions. ...................................... 129
Figure 5.7 : Molecular profiles as analyzed by SDS PAGE: a) EAPI extracted with 0.01 M electro-activated NaCl solution; b) EAPI extracted with 1 M electro-activated NaCl solution; c) CPI extracted with 0.01 M NaCl solution; d) CPI extracted with 1 M NaCl solution. .............................................................................................................................. 132
Figure 5.8: Deconvulved FTIR spectra: a) Protein concentrates extracted with 0.01 M NaCl; b) Protein concentrates extracted with 1 M NaCl. ................................................... 133
Figure 5.9 : Deconvulved FTIR spectra: a) Protein isolates extracted with 0.01 M NaCl; b) Protein isolates extracted with 1 M NaCl. .......................................................................... 137
Figure 6.1: The solubility behavior of canola proteins as a function of pH, where EAPI is electro-activated protein isolate, CPI is conventional protein isolate, EAPC is electro-activated protein concentrate and CPC is conventional protein concentrate. ..................... 150
Figure 6.2: Surface hydrophobicity of protein isolates and concentrates as a function of pH. ............................................................................................................................................ 157
Figure 6.3: Foam stability as a function of time: A) FS of proteins isolates, B) FS of protein concentrates. ....................................................................................................................... 160
xvi
Figure 6.4: Emulsifying properties of canola protein isolates and concentrates: A) Emulsion activity index; B) Droplet size. ........................................................................................... 162
Figure 6.5: Emulsion stability index of the proteins. ......................................................... 163
Figure 6.6: Creaming stability of canola protein-stabilized emulsions. ............................. 165
Figure 7.1: Cookies prepared with rice and dark buckwheat flours (1), rice and green buckwheat flours (2) with the incorporation of electro-activated canola protein concentrate and isolate: (A) Control; (B) 3% EAPC; (C) 6% EAPC; (D) 9% EAPC; (E) 3% EAPI; (F) 6% EAPI; (G) 9% EAPI. .................................................................................................... 178
Figure 7.2: Dough characteristics: (A) dough prepared with grey buckwheat; (B) dough prepared with green buckwheat. ......................................................................................... 180
Figure 7.3 : Cookie characteristics: (A) cookies prepared with grey buckwheat; (B) cookies prepared with green buckwheat. ......................................................................................... 182
Figure 7.4 : Moisture of baked biscuits. ............................................................................. 186
Figure 7.5: Water activity (aw) levels for baked biscuits. ................................................... 186
Figure 7.6: Scanning electron microscopy at 1000x magnification of: (A) rice flour; (B) grey buckwheat flour; (C) green buckwheat flour; (D) EAPC; (E) EAPI at 300x magnification; and (F) EAPI at 1000x magnification. ....................................................... 187
Figure 7.7 : Scanning electron microscopy of the cross section of selected biscuits at 1000x magnification: (A) 3% EAPC; (B) 6% EAPC; (C) 9% EAPC; (D) 3% EAPI; (E) 6% EAPI; (F) 9% EAPI; (G) control. .................................................................................................. 189
Figure 7.8 : Preliminary sensory characteristics of canola protein enriched cookies......... 191
xvii
List of abbreviations
A Alkalinity Å Angstrom a* CIE color space co-ordinate: degree of greenness/redness ACE Angiotensin-i converting enzyme ADF Acid detergent fiber AEM Anion-exchange membrane ANC Acid neutralization capacity ANOVA Analysis of the variance ANS Anilino-1-naphthalenesulfonic acid AOAC Association of analytical communities b* CIE color space co-ordinate: degree of blueness/yellowness C1 Configuration 1 C2 Configuration 2 CEM Cation-exchange membrane CM Canola meal CMC Carboxymethyl cellulose CPC Conventional protein concentrate CPC_10 Conventional protein concentrate extracted at pH 10 CPC_12 Conventional protein concentrate extracted at pH 12 CPI Conventional protein isolates CS Creaming stability DIR Direct alkaline extraction EA Electro-activation EAI Emulsion activity index EAPC Electro activated protein concentrate EAPC_C1_60 Electro-activated protein concentrate, treated in the configuration 1 for 60 min EAPC_C2_60 Electro-activated protein concentrate, treated in the configuration 2 for 60 min EAPC_C1_10 Electro-activated protein concentrate, treated in the configuration 1 for 10 min EAPC_C2_10 Electro-activated protein concentrate, treated in the configuration 2 for 10 min EAPI Electro activated protein isolate EAS Electro-activated solutions ESI Emulsion stability index FAC Fat absorption capacity FC Foaming capacity FID Flame ionization detector FS Foaming stability FTIR Fourier transform infrared spectroscopy
xviii
GC Gas chromatography GF Gluten free HE Height of the emulsion HS The height of the serum layer I Electric current IEM Ion exchange membrane kDa Kilo Dalton L* CIE color space co-ordinate: degree of lightness LAL Lysinoalanine LDL Low-density lipoprotein LDV Laser Doppler velocimetry MMT Million metric tones MW Molecular weight NDF Neutral detergent fiber NSI Nitrogen solubility index NSPS Nonstarch polysaccharides NTU Nephelometric turbidity units PDCCAS Protein digestibility corrected amino acid score PER Protein efficiency ratio pI Isoelectric point PMM Protein micellar mass RFI Relative fluorescence intensity SD Standard deviation SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Scanning electron microscopy SHMP Sodium hexametaposphate TPA Texture profile analysis TRIS Tris(hydroxymethyl)aminomethane UV Ultraviolet WAC Water absorption capacity
xix
Acknowledgments
It has been incredible 3 years of experience, meetings, challenges and achievements. And it
would not be possible without people to whom I want to express my sincere gratitude.
Hereby, I want to thank my professor Mohammed Aider for the opportunity he offered me
as well as for his faith in me and encouragements especially in the beginning of my thesis. I
also want to thank for his guidance, support and the ability to turn me back to the topic
without deviating from the subject and dissipating my attention on things less relevant. I also
want to thank my co-director Martin Mondor for reading and correcting my articles as well
as for his precise recommendations and pieces of advice. I kindly appreciate the participation
of Dr. Marzouk Benali and would like to acknowledge the members of the committee Dr.
Charles Lavigne and Dr. Wassef Ben Ounis for reading and evaluating my thesis.
I cannot forget the precious contribution of Diane Gagnon, who helped a lot thorough all
these years of my thesis spent in the lab. Thank you for your kindness, sympathy and
responsiveness, not to mention the candies in your office. I want to thank Pascal Lavoie and
Pierre Côté for the technical support in the lab, and Ahmed Gomaa for his help with
understanding FTIR.
I would like to express my sincere gratitude to the friends I met here from all over the world.
Truly they are real specialists passionate about what they do. So many times when I needed
a piece of advice they were there for me. Thank you also for sharing our best moments here.
Valerie Carnovale, Shyam Suwal, Sergei Mikhaylin, Attara Hell, Alex Kastyuchik, Stanislav
Stefanovski, Mayank Pathak, Mathieu Persico, Nassim Naderi, and many others. I have never
seen such a bright multinational company. You become our family here.
I am who I am thanks to my dear mom and my sister. There are no words capable of
describing my gratitude and my feelings to what you mean to me. While writing this part I
tended to write “we” or “us” because all of this would be impossible without my dearest
husband, my best friend and advisor Cheslav Liato. Thank you for your love, support, and
inspiration. Next to you I forget that I am so far from my homeland.
Finally, I thank you, my dear reader for appreciating my work.
xx
Préface
Cette thèse de doctorat est composée de huit chapitres et les résultats sont présentés
sous forme d’articles scientifiques soumis ou publiés dans des revues internationales avec
comité de lecture.
Le premier chapitre est une revue de littérature détaillée dans laquelle trois éléments
principaux ont été traités : le tourteau de canola avec le potentiel qu’il présente en tant que
source de protéines de haute valeur nutritionnelle et technologique, la technologie d’électro-
activation comme alternative à l’élimination d’agents chimiques utilisés pour la valorisation
du tourteau de canola et finalement le potentiel offert par les protéines de canola pour la
production de produits sans gluten.
Le deuxième chapitre est consacré à énoncer l’hypothèse de recherche qui découlait
du premier chapitre, de l’objectif général du présent projet de doctorat et des objectifs
spécifiques qui serviront à atteindre l’objectif principal et à confirmer l’hypothèse de
recherche.
Le troisième chapitre est un article scientifique qui a été rédigé suite à la réalisation
du premier objectif spécifique et qui consistait à étudier l'effet du pH, de la concentration du
tourteau de canola et de sel sur le taux d'extraction des protéines par la méthode d’extraction
conventionnelle. Cette étude a servi de témoin pour la comparaison avec l’extraction par la
technologie de l’électro-activation en solution. Cet article est publié dans Food Chemistry.
Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of total
dry matter and protein extraction from canola meal as affected by the pH, salt addition and
use of Zeta-potential/turbidimetry analysis to optimize the extraction conditions. Food
Chemistry, 15, 243-252.
Le quatrième chapitre est un article scientifique consacré à la réalisation du
deuxième objectif spécifique qui visait à étudier les propriétés alcalines des solutions électro-
activées (SEA) en fonction du type de configuration du réacteur d’électro-activation,
l'intensité du courant électrique, la concentration du sel ajouté et le temps d’électro-activation
(de traitement). Cet article est soumis à Engineering in Food and Agriculture.
Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of a
self-generation process of alkaline aqueous solutions by using the electro-activation
technology. Food Bioscience, Soumis.
xxi
Le cinquième chapitre est un article scientifique qui a traité du troisième objectif
spécifique de cette thèse de doctorat et consistait à évaluer et comprendre l’effet de l’électro-
activation sur le taux d'extraction des protéines du tourteau de canola et leurs propriétés
physico-chimiques. Cet article est publié dans Food Bioscience.
Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Study of the
functional properties of canola protein concentrates and isolates extracted by electro-
activated solutions as non-invasive extraction method. Food Bioscience 12, 128-138.
Le sixième chapitre est un article scientifique qui a traité du quatrième objectif
spécifique de ce projet. Il visait l’étude des propriétés fonctionnelles des protéines de canola
extraites par électro-activation et leur comparaison avec les propriétés de protéines extraites
par la méthode conventionnelle avec du NaOH. Cet article est publié dans Food Bioscience.
Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). A
comparative study between the electro-activation technique and conventional extraction
method on the extractability, composition and physicochemical properties of canola protein
concentrates and isolates. Food Bioscience 11, 56-71.
Le septième chapitre est également réalisé sous forme d’un article scientifique qui a
traité du cinquième objectif spécifique de cette thèse de doctorat. Il a fait l’objet d’une étude
dans laquelle les protéines extraites par électro-activation sous forme de concentrés et
d'isolats ont été ajoutées à une formulation de biscuits sans gluten afin d'étudier leur
comportement dans une réelle matrice alimentaire. Cet article est publié dans International
Journal of Food Science and Technology.
Alina Gerzhova, Martin Mondor, Marzouk Benali, Mohammed Aider. (2015). Incorporation
of canola proteins extracted by electro-activated solutions in gluten free biscuit formulation
of rice-buckwheat flour blend: Assessment of quality characteristics and textural properties
of the product. International Journal of Food Science and Technology, Accepted.
Le huitième chapitre est une conclusion générale et des recommandations
spécifiques pour une meilleure valorisation des résultats de cette thèse de doctorat. En effet
et compte tenu des résultats obtenus à chacune des étapes de ce projet de doctorat, il est
possible de conclure que l’hypothèse de recherche énoncée au début de ce projet a été vérifiée
et validée par la réalisation de cinq objectifs spécifiques.
xxii
Madame Alina Gerzhova, candidate au doctorat en Sciences et technologie des
aliments, est l'auteure principale des cinq articles scientifiques présentés dans cette thèse de
doctorat. Elle a été responsable de la conception, la planification et de la réalisation des
expériences ainsi que de la rédaction et de la correction des articles. Dr Mohammed Aider,
directeur de recherche de la candidate et responsable du projet, a activement participé à toutes
les étapes de réalisations de ce projet de doctorat. Dr Martin Mondor, codirecteur de
recherche de la candidate, a activement pris part à toutes les étapes de réalisation de cette
thèse de doctorat. Dr Marzouk Benali, membre actif et participant à ce projet, a activement
participé à la réalisation de toutes les étapes du projet.
1
Introduction
Nutrition has always played an important role in human`s life with its main aim being to
provide with all the essential ingredients required for healthy existence. Unfortunately, the
problem of malnutrition and hunger is still a serious worldwide concern. According to The
United Nations Food and Agriculture Organization (FAO) about 795 million out of 7.3
billion people in the world were suffering from chronic undernourishment in 2012-2014
(FAO, 2014). It is an oppressive statistic, in spite of the significant decrease in the number
of undernourished people in comparison with the period 1990–1992, and it is aggravated by
the constant increase of the world population. According to United Nations Statistical Office,
the world population has grown from 2.5 billion in 1950 to 7.2 billion people in the mid-2013
and is predicted to increase by almost one billion people within the next twelve years reaching
10.9 billion by 2100. This however is not accompanied with the corresponding increase in
food sources, proteins in particular. Malnutrition implies first of all the protein energetic
deficiency which is a lack of proteins and calories in the diet. Proteins are indispensable for
the normal growth and development and their inadequate intake may lead to failure to thrive
in children and serious health disorders in adults.
That is why there is an increased interest worldwide towards non-conventional protein
sources the most cheap and abundant represented by plant proteins. In spite of that fact the
utilization of plant proteins in human consumption is rather limited. Most of them are used
as forage for animals to obtain conventional sources of proteins such as meat, eggs, and milk
which is substantially inefficient. Due to the animal metabolism 6 kg of plant proteins are
needed in order to produce 1 kg of animal proteins, thus 85 % of possible protein and energy
sources are wasted and turned into polluting emissions coming from animals` metabolism
(Arntfield, 2004; Wu et al., 2014). Furthermore, a hundred times more water is needed to
produce 1 kg of animal proteins in comparison with plant proteins
(http://www.canolacouncil.org, 2015a). The land used to produce animal feed could be
reduced tenfold if it was aimed for human consumption only. Regarding this and an increase
in population a more efficient utilization of plant proteins is needed. Indeed, the situation will
become critical when the protein production from animal sources reaches its maximum
capacity (Agriculture and Agri-Food Canada, 2014). This urges the food industry to search
and develop alternative sources.
2
Oilseeds such as soybean, sunflower, canola and especially the press cake left after oil
extraction are interesting protein sources which can be valorized (Rodrigues et al., 2012).
Among them canola is the leading crop in Canada and the second largest oilseed crop in the
world, left behind by soybean only (Dessureault, 2012; http://www.canolacouncil.org,
2015b). The seed of canola consists of 40% oil approximately and 17-26% protein, whereas
canola meal (a by-product of canola oil extraction) has up to 50% protein on a dry basis
(Aider and Barbana, 2011). Until relatively recently canola (rapeseed) press-cake has been
regarded and utilized mostly in animal nutrition due to the presence of anti-nutritive factors;
however the emergence of low glucosinolates and low erucic acid cultivar opened new
possibilities for its utilization as a food grade protein supplement. Its balanced amino-acid
composition and adequate digestibility can compete with such a widely consumed plant
based protein source as soybean (Tan et al., 2011a). The most promising utilization of canola
proteins is in the form of isolated protein powder, characterized by increased shelf life, higher
microbial stability, lower weight and improved portability (Arntfield, 2004).
The idea of utilization of canola proteins in food industry is supported by their interesting
functional properties including foaming, gelling, water holding, and emulsification properties
(Aachary and Thiyam, 2011; Aluko and McIntosh, 2001; Khattab and Arntfield, 2009). As
an ingredient it can potentially replace whey proteins, casein, and egg yolk in such widely
consumed products as mayonnaise (Morris, 1992), emulsion-type meat products (Cumby et
al., 2008; Mansour et al., 1996; Thompson et al., 1982; Yoshie-Stark et al., 2006), bread
(Kodagoda et al., 1973a; Shahidi, 1990), and pasta (Alireza Sadeghi and Bhagya, 2008).
However, the successful utilization of canola proteins is challenged by the choice of
extracting technology.
Conventional extraction with the use of sodium hydroxide and subsequent precipitation with
hydrochloric acid have a detrimental effect on quality and functionality of proteins
(Rodrigues et al., 2012). In addition it creates a large amount of effluents which need to be
further processed. The use of chemical acids and bases cause environmental, safety and
health hazards and stimulate researchers to develop alternative “green” methods (Shi et al.,
2012). One of the promising techniques for protein extraction implies the energy of electric
current in the form of electro-activated aqueous solutions. The conversion of the electric
energy into chemical creates a necessary conditions such as pH and alkalinity which can be
3
utilized for proteins extraction (Aider et al., 2012b). No chemical bases are therefore required
which make the EA an environmentally friendly technology.
The aim of the project was to investigate the use of electro-activated solutions for protein
extraction from canola meal as well as its effect on structural and functional characteristics.
This will expand the current knowledge on the utilization of EAS and possibly become a
novel extracting method allowing to valorize canola oil cake for its application in the food
sector.
4
1. CHAPTER 1: Literature review
Proteins
1.1.1. General information
The word protein is defined as “any of a group of complex organic compounds, consisting
essentially of combinations of amino acids in peptide linkages that contain carbon, hydrogen,
oxygen, nitrogen, and usually, sulfur” (Morris, 1992). All of them regardless of their origin
are formed of twenty amino-acids. It is their order and conformation that provide such a great
variety resulting in more than 1010 different species of naturally occurred proteins (Walstra,
2002).
Some amino-acids were found to be more important than others. In the early studies in rats
performed by Rose (1947) the lack of certain amino acids resulted in poor growth, loss in
weight and eventually death, whereas other did not have that impact. It was found that these
amino acids could not be synthesized by organism and were called essential. Requirements
for protein consumption are basically the need in essential amino-acids which must be
satisfied. Daily requirements for adults are presented in the Table 1.1.
Table 1.1: Indispensable amino acid requirements (FAO/WHO/UNU, 2007).
Amino acid Indispensable amino-acid requirements mg/kg per day mg/g protein
Histidine 10 15 Isoleucine 20 30 Leucine 39 59 Lysine 30 45 Methionine+cysteine 15 22 Methionine 10 16 Cysteine 4 6 Phenylalanine+Tyrosine 25 38 Threonine 15 23 Trpytophan 4 6 Valine 26 39
Total indispensable amino acids 184 277
5
1.1.2. Role of proteins in nutrition and their nutritive value
Among other foodstuffs supplying energy to a human body proteins were characterized as
“unquestionably the most important of all known substances in the organic kingdom” (Lewis,
1948). Derived from a Greek word “protein” means “first” or “primary” due to its important
role in sustaining life since it contains nitrogen, essential for the structure of the cell, a basic
component of protoplasm (Li-Chan, 2004). It is difficult to overestimate the role of proteins
in human nutrition. Dietary proteins as a source of amino-acids are used by the human body
to synthesize its proper proteins. Apart from being the builders of new tissues and cells they
play specific role in the maintenance of body functions, in controlling and regulating body
processes (metabolism, osmotic regulation), catalyzing reactions, contracting muscles,
transporting and storing nutrients, protecting against oxidative stress, infection, and bleeding
etc. (Fennema, 1996; Wu et al., 2014).
The lack of proteins in a diet may lead to serious health problems in adults. In children, who
are more susceptible to protein malnutrition, protein deficiency causes impaired growth and
results in decreased immune function and increased susceptibility to infectious disease (Wu
et al., 2014).
1.1.3. Functional properties
Apart from nutritional aspect proteins are widely used in food technology performing specific
functions. Food preferences are normally based on the sensory characteristics such as texture
and mouthfeel, flavour, colour, and appearance. Sensory characteristics of food are the result
of multiple interactions between various functional ingredients and proteins have a great
influence on the sensorial perception. For a protein to be useful as an ingredient in certain
type of foods e.g. cakes, biscuits, sauces, etc. it must possess multiple functionalities or
functional properties. Kinsella and Melachouris (1976b) describes functional properties as
“physicochemical property which affects the processing and behavior of protein in food
systems”. Functional properties of proteins used in various types of foods are shown in Table
1.2.
Functional properties greatly vary depending on the type of protein, methods of extraction
and precipitation, drying, concentration, or modification (enzymatic, alkaline, or acid
hydrolysis, chemical), as well as environmental conditions, notably temperature, pH, and
6
ionic strength. In addition food proteins are normally composed of several proteins each
having their proper functionalities (solubility, isoelectric point, susceptibility to denaturation,
etc.) and thus a specific protein preparation will not always reflect the properties of the total
protein but rather of one of the components (Kinsella and Melachouris, 1976b).
Table 1.2: Various protein functionalities and their application in food. Adapted from Fennema (1996). Functionality Mechanism Food
Solubility Hydrophilicity Beverages
Viscosity Water binding, hydrodynamic size
and shape
Soups, gravies, and salad
dressings, deserts
Water binding Hydrogen bonding, ionic hydration Meat sausages, cakes, and
breads
Gelation Water entrapment and
immobilization, network formation
Meats, gels, cakes,
bakeries, cheese
Cohesion
adhesion
Hydrophobic, ionic, and hydrogen
bonding
Meats, sausages, pasta,
baked goods
Elasticity Hydrogen bonding, disulfide
crosslinking
Meats, bakery
Emulsification Adsorption and film forming at
interfaces
Sausages, bologna, soup,
cakes, dressings
Foaming Interfacial adsorption and film
formation
Whipped toppings, ice
cream, cakes, desserts
Fat and flavor
binding
Hydrophobic bonding, entrapment Bakery products,
doughnuts
Physical and chemical properties are involved in the formation of protein functionality
including size; shape; amino acid composition and sequence; net charge and distribution of
charges; hydrophobicity/hydrophilicity; secondary, tertiary and quaternary structures;
molecular flexibility / rigidity; and finally ability to interact with other components. A
challenging task is the prediction of functional properties through the analyses of molecular
properties. Until now it has not been completely succeeded despite all the available
information mostly because the behavior in model system differs from the one in real foods.
7
Nevertheless, considerable progress has been achieved towards the understanding of the
structure-function relationships. It is not an easy task to correlate the physical and chemical
properties with a specific functionality. However, in general terms functional properties can
be regarded as a set of three molecular aspects depending on the interactions (Fennema, 1996;
Gauthier, 2012):
• Hydration properties – based on protein-water interactions: solubility,
dispersibility, wettability, swelling, thickening, water absorption and water-holding
capacity
• Surface related properties – based on protein-interface interaction: emulsification,
foaming, flavor binding, pigment binding
• Hydrodynamic/rheological properties – based on protein-protein interactions:
elasticity, viscosity, cohesiveness, chewiness, adhesion, stickiness, gelation, dough
formation, texturization.
1.1.3.1. Hydration properties
The majority of foods contain water and thus interactions of macromolecules such as proteins
with water are of great importance. Most of the proteins functional properties e.g. solubility,
water absorption and water holding ability, viscosity, emulsification, foaming, gelation
depend on the water-protein interactions.
1.1.3.1.1. Water absorption and water holding capacity
The ability to absorb or bind and hold water is very important in several food preparations
especially in low and intermediate moisture foods such as baked and comminuted meat
products. In the absence of sufficient water they do not dissolve but rather imbibe water. This
characteristic plays a major role in the texture and consistence and confers body, thickening
and viscosity to such products. As an example the ability to bind and retain meat juices in
beef patties and frankfurter type meat emulsions enhances mouthfeel and flavor, in bakery
products it prolongs the shelf life (Kinsella, 1979a). When substituting one protein for
another in formulations the information on the water absorption and holding is crucial since
without any adjustments there is a risk to obtain a product with non-acceptable organoleptic
properties, either too dry or too moist.
8
Methods of analysis
The capacity to absorb or bind water is measured by subjecting the dried protein powder to
water vapor of a known relative humidity when it reaches its equilibrium. It is governed by
the amount of polar and non-polar groups exposed at the surface and related to the amino-
acid composition of the protein as well as its conformation. The ability to retain water against
gravitational flow known as water holding capacity (sometimes also referred in the literature
as water absorption capacity) is however even more important. It is analyzed by subjecting
the protein to the excess of water and centrifuging the protein dispersion (Fennema, 1996).
Factors affecting water absorption
It is dependent on the environmental factors such as pH, ionic strength, type of salts, and
temperature. By changing the pH of the medium the net charge of the protein molecule
changes too which impacts protein-water interactions. At isoelectric point (pI) they are
minimal increasing above and below pI. Water hydration properties reach their maximum at
pH 9-10 in most proteins (Fennema, 1996). An increase in temperature normally decreases
the water bonding capacity due to the decrease in hydrogen bonding. However a denatured
protein might have higher water absorption capacity due to the protein unfolding and
liberating previously buried groups unlike protein solubility (Cheftel et al., 1985). Low salt
concentrations (<0.2M) enhance protein hydration, however at higher concentrations it
usually decreases due to the increased water-salt interactions.
1.1.3.1.2. Solubility
Proteins solubility is one of the most important parameters as it influences other properties
such as foaming, emulsifying, gelling and thickening. Good solubility gives wider
possibilities of the potential applications of proteins and is one of the most practical indexes
of the extent of denaturation (Kinsella and Melachouris, 1976b). Good protein solubility
initially is required in some functional properties such as emulsification or foam formation
in order to allow fast migration to the oil/water or air/water interfaces. On the contrary for
some properties such as water absorption high solubility is not beneficial. Thus, it has been
shown that water absorption capacity can be improved by preliminary denaturation (Cheftel
et al., 1985).
9
Methods of analysis
Most of the time nitrogen solubility index (NSI) is used as a practical and quick test. Proteins
are complex structures with great variety of forms and conformations. They possess both
hydrophobic and hydrophilic areas, which determine their behavior in solution. Therefore,
the solubility of a protein depends on the properties of the groups that are situated at the
surface level (Rodrigues et al., 2012). Hydrophobic patches are involved in protein-protein
interactions which are accompanied by decreased solubility, whereas hydrophilic ones are
responsible for protein-water interactions and therefore improve solubility. According to the
solubility in different solvents, proteins are classified into: albumins that are soluble in water;
globulins, soluble in diluted salt solutions; glutelins, soluble only in acid or alkaline solutions;
and prolamins that are soluble in 70% ethanol. Two latter groups are highly hydrophobic
(Fennema, 1996).
Factors affecting the solubility
Solubility depends on many factors such as pH of the solutions, ionic strength, temperature
or the presence of organic solvents.
pH: The pH of the medium is one of the factors having the highest impact on solubility. It is
closely related to the isoelectric point of each protein where they carry a net charge equal to
zero. Below the isoelectric point (pI) proteins are charged positively whereas above the pI
the net charge is negative. Under such conditions solubility is provided by electrostatic
repulsions and by the hydration of the charged residues. For most proteins minimum
solubility is observed at isoelectric point where they show low interaction with water so that
the solubility curve gains the U-shaped form when plotted against pH. The solubility
measurements in a wide pH range give a good indication of a potential or limitation for a
protein as a functional ingredient (Kinsella and Melachouris, 1976b).
Temperature: When proteins are heated to the temperatures higher than 50°C solubility
decreases as links implied in protein stabilization are disrupted and protein unfold which
liberates the hydrophobic residues buried inside the molecule in its native state. Unfolding
alters the protein-solvents interactions making protein-protein interactions more favorable
and resulting in protein aggregation (Cheftel et al., 1985).
10
Ionic strength: Ionic strength of the solution greatly impacts the protein solubility. At low
salt concentrations the solubility is increased due to the interactions between ions of salt and
protein charges thus decreasing electrostatic interactions between charged groups of a protein
molecule. This is known as “salting-in” effect. On the contrary, high salt concentrations
decrease the solubility due to the competition for the water molecules between salt and
proteins and lead to protein precipitation known as “salting-out” (Arakawa and Timasheff,
1985).
1.1.3.2. Surface related properties
Many food products are composed of two immiscible layers, a hydrophilic and a hydrophobic
one (e.g. water and oil or water and air) and can be classified as emulsion-type products or
foam-type products. These food systems are rather unstable because of a great surface tension
between two phases and need a surface active agent capable of bringing two phases together.
Proteins are amphiphilic substances by nature, which means they contain both hydrophilic
and hydrophobic patches and therefore are capable of lowering the surface tension between
two immiscible phases performing emulsion or foaming properties. In spite of the fact that
all proteins are ampiphilic by their nature their surface activity varies greatly depending on
the ratio of hydrophobic and hydrophilic patches. In addition protein conformation which
includes the flexibility of the polypeptide chain, adaptability to the environmental changes,
disposition of the hydrophobic and hydrophilic residues is also of great importance
(Fennema, 1996). There are three main features that determine a good surfactant: (1) ability
to rapidly migrate to the interface; (2) ability to rapidly unfold and adsorb at the interfaces;
(3) ability to form a strong and cohesive film that can withstand the thermal or mechanic
stress (Dickinson, 1998). The speed of migration depends on the size of a molecule, small
and compact proteins will migrate faster. The distribution of hydrophilic and hydrophobic
amino acids on a protein surface influences the rapidity of protein adsorption at the interfaces.
Hydrophobic proteins migrate to the interface and adsorb more readily (Nakai, 1983). During
adsorption protein molecule unfold and orient its hydrophobic parts towards air or oil phase
and hydrophilic towards water. Structural rigidity or flexibility of a folded protein thus has a
great impact. Compact globular proteins stabilized by covalent bonds will take time to unfold
whereas proteins with random coil type of structural organization will unfold and adsorb
11
much more readily. It may also be affected by extrinsic factors, such as the ionic strength,
pH and temperature. Upon unfolding new liberated protein areas also might affect the
functionality at the interfaces (Damodaran, 1989). Viscoelastic film that is formed after
influences the stability of the system. It is favoured by some degree of surface denaturation,
which amplifies protein-protein interaction and enhances cohesive forces between the
proteins in the film (Kinsella, 1981).
Interfacial properties can be evaluated by measuring the interfacial tension. Surface
hydrophobicity was also successfully employed in predicting the proteins functionality at the
interfaces (Damodaran, 1989).
Although the principles of foam and emulsion formation are similar, the requirements for a
surfactant differ and thus the protein that performs well at air-water interfaces may not act
the same at oil-water interfaces.
1.1.3.2.1. Emulsion properties
Emulsions are two-phase systems consisting of water and oil. Their ratio determines the type
of emulsion to be formed either oil in water or water in oil. When the dispersed phase is oil
and the continuous phase is water the oil in water emulsion is formed and vice versa.
Examples of oil in water emulsion would be mayonnaise or vinaigrette whereas butter
represents a water in oil emulsion (Gauthier, 2012). To form an emulsion an adequate amount
of energy is needed to be applied which is generally achieved by using mixers, homogenizers,
or other appliance in order to disperse one phase in another. For two immiscible phases such
state is thermodynamically unfavorable due to the high free energy of the interface between
the two phases. As all the systems tend to equilibrium reached when the contact area between
two immiscible phases is minimal, various destabilization processes arise leading to phase
separation. Various forces are involved in phase separation and may take place
simultaneously making it more difficult to distinguish them.
Destabilization processes in emulsions
The most common destabilization processes are shown in the Figure 1.1. Creaming and
sedimentations are caused by external forces usually gravitational or centrifugal. Due to the
differences in the density, droplets with lesser density will move to the top, or if their density
is higher will move to the bottom. Flocculation results from droplet aggregation due to van
12
der Waals forces when there is no sufficient repulsion to keep them apart. Although being
immiscible two liquids normally have mutual solubility up to a certain degree. In emulsion
with polydisperse distribution smaller droplets disappear with time as they diffuse to the bulk
and become deposited on the larger droplets. This phenomenon is known as Ostwald
ripening. Coalescence takes place when droplets approach and fusion together in order to
reduce the surface area and is related to the strength of the film formed around the droplets.
When it is not strong enough it becomes thin and finally disrupts leading to the formation of
larger droplets. Finally phase inversion may take place with time or if conditions were
changed transforming oil in water emulsion into water in oil emulsion and vice versa (Tadros,
2013).
Figure 1.1: Major destabilization processes in emulsions. Adapted from Tadros (2013).
Emulsifiers can slow down the phase separation. According to Kinsella (1979a) the
stabilization of emulsified droplets is achieved by formation of a charged layer around the
oil droplets creating mutual repulsions and/or by the formation of film around the droplets
by solutes such as proteins.
Methods of analysis
There is no standardized test for analyzing the emulsification properties (McWatters and
Cherry, 1981). Three different methods such as emulsifying capacity, emulsion stability,
emulsifying activity, and droplet size distribution are most commonly used for the analysis
of the emulsifying properties of proteins (Kinsella and Melachouris, 1976b).
13
Emulsifying capacity is determined as the amount of oil that can be emulsified by a protein.
It is measured by conductivity or viscosity measurements under constant addition of oil until
the phase inversion occurs. The collapse of emulsion is accompanied by a sudden drop in
viscosity or conductivity, or an increase in resistance (Kinsella and Melachouris, 1976b).
Emulsion activity is measured as the available contact area between two phases by estimating
the turbidity of a diluted emulsion (Karaca et al., 2011a). This method was proposed by
Pearce and Kinsella (1978) based on the Mie theory of light scattering, which states that the
interfacial area of an emulsion is twice its turbidity (Cameron et al., 1991).
Emulsion stability can be evaluated by several methods. Most of them subject the emulsion
to various external perturbations such as heating, centrifugation, etc. and note the amount of
oil released after centrifugation (Kinsella and Melachouris, 1976b). Another approach is to
measure the changes in droplet size (Tan et al., 2014) or turbidity (Zhu et al., 2010) with the
time. The degree of creaming has been monitored by using the creaming index (Karaca et al.,
2011b). Emulsions are stored in the glass cylinder for a certain period of time until the
creamed layer became visible on the top of the emulsion. Creaming index was measured as
the relation of total height of the emulsion (HE) and the height of the serum layer (HS). New
generation of instruments such as turbiscan can directly provide with the information about
the degree of creaming, sedimentation, flocculation or coalescence (Álvarez Cerimedo et al.,
2010).
Droplet size is an important characteristic of an emulsion and the most important factor to
predict the coalescence. Smaller droplets are less prone to coalescence because of the smaller
film area between them which lowers the probability of its rupture. Smaller droplets also
decrease the rate of creaming (Fennema, 1996).
Factors affecting emulsion properties
Emulsifying properties can be influenced by several factors including intrinsic (pH, ionic
strength, presence of other substances, type of protein, etc.) and extrinsic factors (type of
appliance used to prepare the emulsion, the energy input, rate of shear, etc.) (Fennema, 1996).
Protein solubility and hydrophobicity: Protein solubility facilitates emulsion formation,
however high solubility (100%) is not required as normally high soluble proteins as well as
low-soluble proteins do not perform well as emulsifiers. Hence 25-80% solubility range is
suitable in most cases, however the minimum solubility required for good emulsification
14
varies depending on the protein (Fennema, 1996). Emulsifying properties are also correlated
with the surface hydrophobicity which can be used to predict emulsifying properties of some
proteins (Damodaran, 1989). Partial denaturation may improve emulsification as it facilitates
protein unfolding due to increased molecular flexibility and surface hydrophobicity
(Fennema, 1996).
pH and ionic strength: Proteins are normally low-soluble at their isoelectric point which
hampers the emulsion formation. Most of proteins have a compact structure at their pI which
also impedes the unfolding and adsorption, yet it can stabilize the film already created and
enhance emulsion stability (Cheftel et al., 1985; Fennema, 1996). On the other hand, the
interfacial films formed by proteins are relatively thin and electrically charged and therefore
are particularly sensitive to pH and ionic strength of the medium. At pH proximate to
isoelectric point and when the ionic strength exceeds a particular level they tend to flocculate
as there is not enough electrostatic repulsion between the droplets to overcome the various
attractive interactions (McClements, 2004). Experimental data on different proteins are rather
contradicting showing that one proteins have the optimal emulsifying properties at their pI
whereas other performs better at pH far from isoelectric point (Cheftel et al., 1985).
Protein concentration: Protein concentration affects differently the emulsion properties.
Emulsion capacity and efficiency in general will decrease above a critical concentration
whereas emulsion stability will increase (Kinsella, 1979a; Kinsella and Melachouris, 1976b).
Type of appliance: As mentioned before small droplets give more stable emulsions. The size
of droplets is influenced by the energy input and the type of appliance used to prepare an
emulsion. High pressure homogenizers are the most effective in producing fine droplets and
thus resulting in a more stable emulsion. It is followed by sonication (using ultrasound) and
mechanical stirrers such as Ultraturrax (Walstra, 1993).
1.1.3.2.2. Foaming properties
Foams are also two-phase systems composed of gas bubbles (air) dispersed in liquid (water)
and requiring a surface active agent such as protein. Many food products are foams by their
nature e.g. cakes, bread, meringue, whipped cream, mousses, marshmallow, ice cream, etc.
Their smooth texture and mouthfeel are due to these dispersed micro bubbles (Fennema,
1996). In order to create foam it is necessary to provide the energy to the system same as for
15
creating emulsions. It can be done by whipping, bubbling, or shaking a protein solution.
Foams differ from emulsion by their much bigger contact area as the dispersed phase (gas)
occupies a much bigger volume than in emulsion. Due to that fact the majority of foams are
instable (Cheftel et al., 1985).
Destabilization processes in foams
There are three major destabilization processes notably draining, gas diffusion, and rupture
of liquid lamella. Draining occurs due to the gravitational effect as the liquid is heavier than
the gas it tends to drain towards the bottom with the time. Difference in pressure might also
cause draining. If the pressure in gas bubbles is high they tend to press on one another making
the liquid drain. Evaporation that takes place during storage might also be the cause of
draining. When the foam surface becomes dry, the films around the bubbles lose their
elasticity, which makes them weaker and favors their approach. Gas diffusion from small to
large bubbles takes place due to the dissolution of gas in the liquid phase. Finally, protective
films tend to become thin and fragile resulting in the collapse of the foam (Cheftel et al.,
1985; Gauthier, 2012).
Similar to the emulsion formation when air is purged through the liquid the air bubbles tend
to coalesce to minimize surface exposure. This, however may be prevented by the presence
of an appropriate surfactant capable of forming an interfacial film barrier (Kinsella, 1981).
Formation of foams is similar to emulsion formation. First proteins diffuse to the air-water
interface to reduce the interfacial tension, after that they unfold and orient their polar parts
towards water and non-polar towards air and finally they form a continuous film which
protects air bubbles and stabilizes the foam (Kinsella and Melachouris, 1976b).
Analysis of foaming properties
Foaming properties are analyzed by several methods. Foaming capacity, foam expansion or
overrun is the ability to form foams and refers to the maximum volume increase of protein
dispersion, achieved by the incorporation of air by whipping, stirring, or aeration. Foaming
power measures the increase in volume, when the gas is purged through the protein solution.
Basically all of them are based on the measurement of the initial volume of a protein solution
and the final volume of the foam followed by stirring or gas introduction. Foam stability is
16
the ability of a formed foam to retain its volume which is measured as a volume decrease
with the time (Kinsella and Melachouris, 1976b).
Factors affecting foaming properties
A number of factors influence foaming properties such as type of protein and its composition,
its solubility, hydrophobicity, concentration, method of preparation, external factors such as
pH, temperature, the presence of salts, sugars, lipids, and finally, the method of measurement
(Kinsella and Melachouris, 1976b).
Internal factors or molecular properties
The stability of foams is governed by three principal factors: low interfacial tension, high
viscosity of the liquid phase and strong, cohesive, and elastic films (Cheftel et al., 1985).
Protein-based foams first of all depend on the intrinsic molecular properties of the protein
such as conformation, flexibility, molecular size, shape, amino acid sequence, surface
polarity and hydrophobicity, charge, etc. (Kinsella and Melachouris, 1976b). However, foam
formation and foam stabilization require somewhat different properties, hence some of the
properties desired for foam formation, do not provide the stability whereas molecular
characteristics which impart foam stability may slow down the foam formation. Thus,
foaming capacity mostly depends on the rate of adsorption, hydrophobicity and flexibility of
the molecule, whereas the stability is influenced by the rheological properties of the film
formed around the bubble (Fennema, 1996). To perform well in foams surfactant should be
soluble in the liquid phase and capable of rapid migration and orientation to form an
interfacial film around newly developed gas bubbles. Same as in emulsions flexible proteins
are better foaming agents. On the other hand they are poorly structured (low in secondary
and tertiary structures) which results in coarse rapidly-formed and less stable foams
(Kinsella, 1981). Foam stability on the other hand requires strong and viscoelastic film,
impermeable for air which can be provided by globular proteins with high molecular weight
and more rigid structure partially resistant to unfolding (Gauthier, 2012). Globular proteins
due to their rigid structure will unfold and spread much slower so that it will take more time
for them to build foam however it will be more stable. Surface viscosity is an important factor
influencing the stability. High surface viscosity and high film yield result in strong and
viscoelastic foams that are resistant to stress as they can expand and compress if necessary.
17
On the other hand high viscosity is not desirable during the initial foam formation as it slows
down the process of migration and adsorption (Kinsella, 1981).
External factors
pH: Foamability and foam stability is high at the isoelectric point for proteins which are
soluble at their pI due to the lack of repulsive forces which favors the formation of protein-
protein interactions and of a viscous film at the interface. In addition at pI more proteins are
adsorbed at the interface because of the lack of repulsions between the interface and the
adsorbing molecules. For proteins that are poorly soluble at their pI only those that are soluble
will participate in foam formation. Insoluble fraction, however, can also be adsorbed and
contribute to foam stabilization. Under such conditions the volume will be low but the
stability will be high. At pH other than isoelectric point the foaming capacity of proteins is
good but the stability is not (Fennema, 1996).
Salts: For some proteins foamability and foam stability increases with an increase in salt
concentration due to the neutralization of charges by ions of salt. However, it depends on the
type of the salt present and the solubility characteristics of the protein in the presence of that
salt. For some proteins it can have the opposite effect. When the salting-in effect is implied
it decreases the surface denaturation (unfolding) and as a result foaming properties. Sodium
chloride also can reduce surface viscosity and rigidity of protein films (Kinsella, 1981).
Sugars: The addition of sugars in general improves the foam stability as it increases the bulk
phase viscosity which decreases the drainage. On the other hand it decreases the foaming
ability.
Lipids: The presence of even traces of lipids (0.1%) significantly impairs the foaming
properties. Polar and surface active lipids adsorb themselves at the interfaces obstructing the
protein adsorption. The lack of viscoelastic properties for such foams result in rapid collapse
during whipping (Cheftel et al., 1985; Fennema, 1996).
Protein concentration: An increase in protein concentration results in the formation of stiff
foams made of small bubbles and high viscosity. Higher viscosity promotes the formation of
firm films at the interfaces which improves foam stability (Cheftel et al., 1985).
Temperature: Limited heat treatment without thermal coagulation provokes partial
unfolding and facilitates foam formation. The stability of such foams however is reduced due
to decreased viscosity. It also depends on the type of a protein and its structural flexibility.
18
The greater the rigidity of native protein, the greater the improvement caused by heating
(Kinsella, 1981).
The method of foaming: In order to form a foam the time and intensity of whipping should
be chosen in such a way that they allow sufficient unfolding and adsorption of proteins. Too
fast agitation may result in protein denaturation, aggregation and precipitation due to
increased shear rate and thus decrease foaming properties (Fennema, 1996).
1.1.3.3. Other properties
1.1.3.3.1. Oil binding capacity
The ability of protein to bind fat is a very important characteristic in certain foods such as
meat replacers, cakes, cookies as it is associated with the flavor retention and improved
mouthfeel (Kinsella and Melachouris, 1976b). It is based on protein-lipid interactions and is
measured similarly to water absorption capacity by the addition of liquid oil to the protein
powder, mixing and holding for a specific amount of time, centrifuging and determining the
amount of absorbed oil. Such method accounts for the amount of oil physically entrapped by
the protein. It is influenced by the type of protein, its solubility, hydrophobicity, and protein
concentration. Proteins having higher solubility exhibited lower fat absorption. Increase in
protein concentration increased the oil binding by proteins as observed by Hutton and
Campbell (1981).
1.1.4. Sources of proteins
Conventional sources of proteins are still represented by animal sources such as meat, egg,
and fish. However, the interest towards alternative protein sources has grown dramatically
over past years. The world’s situation regarding malnutrition in developing countries, high
cost of proteins from animal sources, health concerns such as intolerances to animal proteins
or conscious refuse from consuming animal proteins has led to a substantial search for
proteins from alternative sources which could fully replace those from animal ones.
Alternative sources have been thoroughly studied in recent years with proteins derived from
plants being the most cheap and abundant.
19
1.1.4.1. Cereals
Wheat, rice, barley, oats, sorgo, corn are the principal cereal crops. Protein content of cereals
is rather low in both quantity and quality. The highest amount of proteins is found in wheat
(12-14% on a dry basis), followed by millet and sorghum (10.5%), maize (9%) and rice
having the lowest amount of proteins (5-7%) (USDA, 2012). Beside that cereals are deficient
in some essential amino-acids, particularly in lysine. Almost all of the cereal proteins have it
in insufficient quantity. Apart from lysine other amino acids such as tryptophan, threonine,
or methionine appear to be limiting depending on the type of cereal. Another limiting factor
of cereal as a major source of proteins is their high content of carbohydrates which leads to
low protein-to-calories ratio (Bressani and Elias, 1968).
1.1.4.2. Legume seeds
Legumes represent another group of protein-rich material. The most commonly cultivated
legumes are pea, chick-pea, broad bean, lentils and the common bean. Their protein content
may reach up to 30% on a dry basis and their overall protein quality is accepted to be higher
than those of cereal proteins. Contrary to the cereal proteins legumes are a good source of
lysine, however, they lack sulfur-containing amino-acids and tryptophan (Bressani and Elias,
1968). On the other hand the digestibility of legume proteins is lower due to the presence of
poorly digestive carbohydrates which cause certain inconveniences such as flatulence. These
problems are related to the fact than the human digestive system lacks the enzymes
responsible for the hydrolysis of these sugars which leads to their anaerobic fermentation in
the large intestine with gas release (Rodrigues et al., 2012).
1.1.4.3. Oilseeds
Oilseeds are plants used in the industry for oil production due to their high oil content.
Soybean, rapeseed, flaxseed, cotton, peanut and sunflower are the most widely produced
oilseeds. Apart from high oil content they are characterized by the elevated percentage of
proteins and balanced content of carbohydrates. Protein content of the oil cake left after oil
extraction depending on the seed ranges between 35% and 60% on a dry basis (Moure et al.,
2006). Nutritive value of oilseeds proteins is generally higher in comparison with legume
and cereal proteins. Soybean is limited in sulfur-containing amino-acids but at the same time
is considered to be a good source of lysine. Peanut, sesame and cottonseed are limited in
20
lysine although they have high content of methionine. Soybean is the most studied and the
most widely spread oilseed culture used in both human and animal diet. However, the
proteins of canola are also being extensively studied and their quality is not inferior to the
soybean ones (Bressani and Elias, 1968). Rapeseed or canola is widely used in oil production.
Worldwide production of major oilseeds as well as the protein content of their meals is shown
in the Table 1.3.
Table 1.3: Worldwide production of major oilseeds and their meals in MMT and their protein contents. Adapted from Ramachandran et al. (2007); USDA (2013).
Soybean Canola Cottonseed Sunflower
Oilseed production, MMT 281.66 66.49 44.39 41.76
Oil cake production, MMT 188.06 36.92 15.66 16.30
Oil cake protein content, % 47.5 39.9 40.3 34.1
Canola
1.2.1. Historical remarks
Canola and rapeseed are the part of the most widespread family of plants cultivated by human
– the Brassicaceae or Cruciferae. Cabbage, cauliflower, turnip, Brussel sprouts, and radish
all belong to this family. Although being the part of one family they are greatly differed by
seed size, seed color, and chemical composition. Rapeseed is tolerant to the low temperatures
and thus can be grown in regions which are not suitable for soybean and sunflower.
One of the earliest cultivated species of rapeseed is suggested to date back as much as 3000
years ago in India, later being introduced to China and Japan (Shahidi, 1990). The wide
spread of cultivars is believed to be due to the birds migration who were attracted by small
size seeds rich nutrients. Unlike other oilseeds rapeseed comes from several species. Figure
1.2 shows the three basic ones: B.nigra, B.oleracea, and B.rapa which by natural
hybridization centuries ago gave rise to the other species, B. carinata, B. juncea, and B. napus
(Daun et al., 2011).
21
Figure 1.2 : Triangle of U showing the relationship of major Brassica species. Adapted from Ahuja et al. (2010).
In Canada rapeseed appeared just before the World War II and by the end of the war
significant amounts were produced in Western Canada. It quickly adapted to the Canadian
prairies and soon became the major oilseed crop, producing seeds not only for domestic use
but also for export. In 1970 about 70 to 90% of the edible oils were produced in Canada by
harvesting around 1 million tons of rapeseed. Yet there was a concern about the composition
of fatty acids and erucic acid in particular. Some works from Europe demonstrated that the
high intake of erucic acid could lead to heart lesions. Apart from erucic acid first cultivars
were high in glucosinolates which prevented their use as high protein supplement for animal
rations. After various breeding programs and due to the introduction of gas chromatography
which allowed to separate fatty acids and to study the genetics behind the fatty acid
composition of rapeseed the new cultivar low in erucic acid and glucosinolates was
introduced in 1974 by Dr. Baldur Stefansson at the University of Manitoba. The development
of low glucosinolates and erucic acid rapeseed crops is shown in the Figure 1.3.
22
F
Figure 1.3: Decrease in glucosinolates and erucic acid levels in rapeseed/canola. Adapted from Daun et al. (2011).
Figure 1.4: Number of varieties of canola in Canada. Adapted from Daun et al. (2011).
The further transformation from low erucic rapeseed to canola as it is known today took
several decades of constant improving and releasing of new varieties. The major tasks apart
from reducing anitutritive factors were to improve yield of crops by producing herbicide
resisting cultivars. For 30 years the amount of varieties has increased by around a hundred
times since 1980 (Figure 1.4). Today canola oil is recognized as GRAS (generally
recognized as safe) and as one of the healthiest oils due to its lowest composition of saturated
fatty acids (Daun et al., 2011). Therefore canola is the world's only "Made in Canada" crop
23
and its greatest agricultural success story. Name canola comes from the Canadian Canola
Association and is a contraction of ‘Canadian oil, low acid’ (Rodrigues et al., 2012).
To be named canola, an oilseed plant must meet this internationally regulated standard "Seeds
of the genus Brassica (Brassica napus, Brassica rapa or Brassica juncea) from which the oil
shall contain less than 2% erucic acid in its fatty acid profile and the solid component shall
contain less than 30 micromoles of any one or any mixture of 3-butenyl glucosinolate, 4-
pentenyl glucosinolate, 2-hydroxy-3 butenyl glucosinolate, and 2-hydroxy- 4-pentenyl
glucosinolate per gram of air-dry, oil-free solid" (http://www.canolacouncil.org, 2015b). The
term rapeseed does not have any regulatory basis and in general is used in relation to oilseed
rape cultivars with 45% or more erucic acid in oil and seed meals that are either high or low
in glucosinolates and mainly used for non-edible purposes such as lubricants and hydraulic
fluids (Raymer, 2002). However, this designation is not used worldwide and there are
rapeseed cultivars that have low erucic acid and glucosinolates (Arntfield Susan, 2011).
1.2.2. Production, economical interest
Today canola is Canada`s most valuable crop, bringing $19.3 billion to the Canadian
economy each year, providing more than 249 000 Canadian jobs and $12.5 billion in wages.
From 14 crushing and refining plants about 10 million tons of canola seed can be processed
giving about 3 million tons of canola oil and 4 million tons of canola meal annually. Around
90% of totally produced canola is exported as seeds, oil or meal to 55 markets around the
world including United States (65% of oil exports, 90% meal export), China, Japan, and
Mexico (http://www.canolacouncil.org, 2015a). Worldwide canola/rapeseed is the second-
largest oilseed grown crop after soybeans (Arntfield Susan, 2011; Daun et al., 2011).
The use of canola is not restricted to the oil production; the press cake left after oil extraction
is a good source of protein for poultry, fish and cattle and potentially a source for edible
protein for human. It also can be used as a food ingredient due to its functionalities as
texturized protein, emulsion or foaming agent, as a basis for edible protein films, etc. It also
found application in the biodiesel production. The possible technical application includes
fiber production for textile, asphalt emulsions, packaging films, detergents, etc. (Thiyam-
Hollaender et al., 2013; Wanasundara, 2011).
24
1.2.3. Meal processing
The processing of seeds which eventually results in canola meal or oil cake – a basis for the
protein extraction starts with flaking (to facilitate the oil removal) and cooking in order to
inactivate the enzyme myrosinase which catalyzes the hydrolysis of glucosinolates. It is
performed in stack cookers with temperatures reaching as high as 100-120 °C (Thiyam-
Hollaender et al., 2013). After that the flakes are transported to the screw press, also known
as an expeller allowing to extract about 70% of the oil. The remaining oil is extracted with
the help of solvents, normally hexane, using counter-current extraction process. Thereby
obtained “white flakes” contain less than 1% of oil (Daun, 2004). After oil extraction the
meal is desolvenized and toasted, where the residual solids are removed by steam heating the
meal at 130 °C, followed by drying and cooling. The general scheme of canola oil extraction
is shown in the Figure 1.5.
Figure 1.5: Processing of canola by prepress solvent extraction. Adapted from Daun (2004).
25
1.2.4. Composition, nutritive and biological value of canola meal
1.2.4.1. Nutritive and biological value of canola meal
Canola meal or oil cake (presscake) is what is left after oil extraction and constitutes 60 % of
the seed. In Canada a blend of Brassica napus, B. rapa, and B. juncea are used for canola oil
production by prepress solvent extraction. The nutrient composition of canola meal is
affected by environmental conditions during growth and harvest conditions as well as
processing of the seed and meal (Newkirk, 2011).
Table 1.4: Proximate composition of canola meal. Adapted from Newkirk et al. (2003).
Component Average
Crude protein (Nx6.25: %) 36 Oil (%) 3.5 Linoleic acid (%) 0.6 Ash 6.1 Crude fiber (%) 12 Tannins (%) 1.5 Sinapine (%) 1.0 Phytic acid (%) 3.3 Glucosinolates (µmol/g) 7.2
Proteins
Protein is the most abundant fraction in canola meal, depending on variety it can reach up to
40% on a dry basis. The quality of a protein can be characterized by the protein efficiency
ratio (PER) which is a gain in body mass related to protein intake. According to Friedman
(1996) the PER below 1.5 describes a protein of low or poor quality; between 1.5 and 2.0, an
intermediate quality; and above 2.0, good to high-quality. For canola proteins the PER has
been estimated as 2.64, which is higher than PER of soybean meal equal to 2.19 (Tan et al.,
2011a). Canola has been reported to be a source of well-balanced amino acids (Table 1.5),
which makes it attractive for the utilization as feed but also as a protein source for human
diet. Similarly to other oilseeds canola is limited in lysine, however rich in cystine and
methionine (http://www.canolacouncil.org, 2015b). It is also rich in sulfur-containing amino
acids such as glutathione and homocysteine, which are effective regulators in antioxidant
defense processes (Fleddermann et al., 2013). Another way to characterize the quality of
26
proteins is by analyzing the digestibility of amino acids by PDCCAS (Protein Digestibility
Corrected Amino Acid Score). The investigations showed that canola meal meets the
requirements for essential and non-essential amino acids in adults and 10-12 years old
children according to the PDCCAS. In the work of Klockeman et al. (1997) the PDCCAS of
canola amino acids were >1.00 for 10-12-year-olds and adults. Another work showed the
PDCCAS for rapeseed proteins equal to 0.83-0.93 which is comparable to animal sources
such as egg (1.00), milk (1.00), and fish (1.00) (Fleddermann et al., 2013). Nitrogen
digestibility of canola (rapeseed) proteins is equal to 93.3% - 97.3% which is comparable to
animal proteins notably egg (98%), casein (95%), and collagen (95%) and higher than plant
proteins of wheat (91%) and beans (78%) (Fleddermann et al., 2013).
Table 1.5: Amino acid composition of canola. Adapted from Newkirk et al. (2003), Downey (1990).
Amino-acid Canola/rapeseed* Canola/rapeseed** Soybean** Alanine 1.57 4.36 4.2 Arginine 2.08 5.78 7.2 Aspartate+asparagine 2.61 7.25 11.7 Cystine 0.86 2.39 1.6 Glutamate+glutamine 6.53 18.14 18.7 Glycine 1.77 4.92 4.2 Histidine 1.12 3.11 2.6 Isoleucine 1.56 4.33 4.5 Leucine 2.54 7.06 7.8 Lysine 2.00 5.56 6.4 Methionine 0.74 2.06 1.3 Methionine+cystine 1.60 4.44 ND Phenylalanine 1.38 3.83 5.0 Proline 2.15 5.97 5.1 Serine 1.44 4.00 5.1 Threonine 1.58 4.39 4.0 Tryptophan 0.48 1.33 1.3 Tyrosin 1.16 3.22 3.2
*Amounts shown are in grams per hundred grams of canola meal with 36% of protein ** Amounts shown are in grams per hundred grams of protein
In addition, a first cross over study in humans showed that the bioavailability of canola
proteins was similar to soybean proteins and that they can be used as effectively in human
nutrition (Fleddermann et al., 2013).
27
Carbohydrates
Carbohydrates compose a relatively small part of canola seed. The carbohydrate`s content is
rather complicated and is mostly represented by pectins (50%), cellulose (24.1%), and
pentosans (25.9%) (Table 1.6) (Bell, 1984). Total dietary fiber was estimated to be around
27.3-30.1% (Slominski et al., 1994). Comparatively to soybean canola meal has higher
amount of fiber from the hull which unlike soybean is not eliminated and represents about
30% of meal weight. It is, however, poorly digestible and its removal would significantly
enhance the digestibility and metabolizing energy (Bell, 1993). Cellulose constitutes the
major part of hull, followed by hemicellulose and lignin. Canola has been reported to contain
8-10% sucrose, 2-3% oligosaccharides, 20-22% nonstarch polysaccharides (NSP), and 5-8%
lignin and polyphenols. Starch is presented only in trace levels. Among low molecular weight
carbohydrates soluble sugars constitute about 48% and include D-glucose, D-fructose, D-
galactose, sucrose melibiose, raffinose, manninotriose and stachyose as reserve
carbohydrates, most of them however will be washed out during processing due to their
solubility. These compounds are known as the principal cause of flatulence. (Bell, 1984).
Table 1.6 : Carbohydrate content of canola meal. Adapted from Newkirk (2011).
Component Average Starch (%) 5.1 Sugars (%) 6.7
Sucrose (%) 6.2 Fructose + glucose (%) 0.5
Cellulose (%) 4.5 Oligosaccharides (%) 2.2 NSPs (%) 15.7 Soluble NSPs (%) 1.4 Insoluble NSPs (%) 14.4 Crude fiber (%) 11.7 ADF (%) 16.8 Acid detergent lignin (%) 5.1 NDF (%) 20.7 Total dietary fiber (%) 32.3
NSPS, nonstarch polysaccharides
ADF, acid detergent fiber
NDF, neutral detergent fiber
28
Vitamins and minerals
Mineral content of canola meal greatly depends on the location and the soil where it was
grown reflecting the uptake of soil minerals. Overall canola meal is a good source of essential
minerals and vitamins such as choline, biotin, folic acid, niacin, riboflavin, and thiamine
(Table 1.7) (Newkirk, 2011).
Table 1.7: Mineral composition of canola and soybean meal. Adapted from Bell et al. (1999); Downey (1990); Newkirk (2011). Mineral Average
Canola meal Average Soybean meal
Calcium (%) 0.64 0.3
Phosphorus (%) 1.12 0.63
Magnesium (%) 0.56 0.27
Copper (mg/kg) 6.2 23
Zinc (mg/kg) 68.2 43
Iron (mg/kg) 188 119
Manganese (mg/kg) 55 29
Biotin (mg/kg) 0.96 0.3
Choline (mg/kg) 6500 2614
Folic acid (mg/kg) 2.3 0.7
Niacin (mg/kg) 156 28
Pantothenic acid (mg/kg) 9.3 16.3
Pyridoxine (mg/kg) 7 6
Riboflavin (mg/kg) 5.7 2.9
Thiamine (mg/kg) 5.1 5.6
Vitamin E (mg/kg) 13 3
1.2.4.2. Bioactive properties
Canola proteins are also one of the promising sources of bioactive compounds. Thus, the
isolates itself and their hydrolysates were analyzed for Angiotensin-I Converting Enzyme
(ACE) inhibitory activity, antioxidant properties, bile acid-binding capacity, and anti-
thrombotic activity (Aider and Barbana, 2011).
1.2.4.2.1. ACE inhibitory activity
ACE is known to participate in the blood pressure regulation by converting the inactive
peptide Angiotensin I into Angiotensin II which is the peptide associated with the
29
development of various cardiovascular diseases. The inhibition of its formation was
successfully used for the treatment of hypertension and related organ damages. However, the
use of synthetic ACE-inhibitory drugs has been reported to have the side effects (Aachary
and Thiyam, 2011). The natural inhibitory action of peptides derived from canola
hydrolysates was first studied by Marczak et al. (2003), and later by Yoshie-Stark et al.
(2006) and Wu and Muir (2008). Both studies showed strong inhibitory potential, however
further studies in vivo were required.
1.2.4.2.2. Antioxidant capacity
It is known that the oxidation processes in the body give rise to the production of free radicals
which can damage cell structure and be precursors of serious disorders such as oxidative
stress-originated diseases (e.g., cardiovascular and neurodegenerative diseases), and cancer
(Apak et al., 2013). The results of Xue et al. (2009) and Cumby et al. (2008) proved that
rapeseed protein hydrolysates could be utilized as a source of bioactive peptides with
antioxidant properties. The antioxidant capacity was reported to be dependent upon peptide
concentration and composition. Also other minor substances such as phenolics which
possibly were co-extracted also could have contributed to the free radicals scavenging
activity of canola proteins.
1.2.4.2.3. Bile acid binding capacity
It has been demonstrated that the intake of dietary plant protein has hypocholesterolemic
effect; this is to say it reduces the LDL (low-density lipoprotein) cholesterol in the blood
vessels, responsible for various heart diseases and atherosclerosis (Aachary and Thiyam,
2011). The hypocholesterolemic effects of plant proteins might be attributed to its bile acid
binding capacity. As the body uses cholesterol to produce bile-acids, their binding promotes
further cholesterol decomposition to produce more bile acids to replace those that have been
lost (Pandolf and Clydesdale, 1992). The study of Yoshie-Stark et al. (2006) and Yoshie-
Stark et al. (2008) showed that canola proteins were able to bind the bile acids. They also
noted that precipitated protein isolate had higher bile acid binding capacity in comparison
with ultrafiltered which could be due to the higher concentration of fiber.
In addition, antidiabetic (Mariotti et al., 2008a), antithrombotic (Zhang et al., 2008),
anticancer (Xue et al., 2010), and antiviral (Yust et al., 2004) properties have been noted for
canola/rapeseed proteins.
30
1.2.4.3. Antinutritive factors
1.2.4.3.1. Glucosinolates
Canola is distinguished by low content of glucosinolates (10-12 µmol/g and lower) in
comparison with rapeseed cultivars (120–150 µmol/g) which is one of the major
improvements achieved by breeders (Bell, 1993). Total glucosinolate content of Canadian
canola meal was reduced to about 7.2 µmol/g (Daun et al., 2011; Newkirk et al., 2003). There
are also cultivars with the content in glucosinolates as low as 1.66 and 0.53 µmol/g (Bell et
al., 1991). Glucosinolates on their own are non-toxic, the problem arises during their
hydrolysis into nitriles, isothicyonates, and thiocyanates catalyzed by the enzyme myrosinase
(Bell, 1993). High glucosinolates rapeseed cultivars lead to reduced feed intake, enlarged
thyroid, reduced plasma thyroid hormone levels, and may as well affect organ liver and
kidney (Aachary and Thiyam, 2011; Bell, 1984). The glucosonolate`s content of today`s
canola meal was claimed to be of little consequence (Newkirk, 2011). A two-solvent oil
extraction method was developed which allowed to reduce the amount of glucosinolates in
meals to trace levels (Naczk et al., 1985). On the other hand both intact glucosinolates and
their metabolites have the ability to give various types of biological effects (Bernhoft, 2010).
Reduced risk of cancer due to their consumption has been reported (Song and Thornalley,
2007; Verhoeven et al., 1996).
1.2.4.3.2. Phytic acid
In canola as well as in many other plants and oilseeds phosphorous is stored mainly in the
form of phytic acid which is considered an antinutritive factor reducing the bioavailability of
proteins as well as the absorption of minerals, especially Zn. It is known to react with basic
proteins by the formation of insoluble protein-phytate complexes and to inhibit the action of
the digestive enzymes, such as pepsin, trypsin and α-amylase (Rodrigues et al., 2012).
However, the inhibition of α-amylase decreased the glycemic index of the food which is
beneficial for diabetics as it may aid them to control blood glucose (Harland and Morris,
1995). Also it binds di-and trivalent metals, such as calcium, magnesium, zinc, and iron, to
form poorly soluble compounds that are not readily absorbed from the intestine (Liener,
1994). Out of 1.22% of total P in canola meal 0.53 % is phytate-bound. In comparison with
soybean, canola however, provides twice as much non-phytate phosphorus (Bell, 1993). On
31
the other hand, phytic acid was reported to have a positive effect and was claimed to act as
an antioxidant and an anticancer agent (Harland and Morris, 1995; Lott et al., 2000).
1.2.4.3.3. Phenolic compounds
Phenolic compounds include simple phenols, phenolic acids, coumarins, flavonoids,
stilbenes, hydrolysable and condensed tannins, lignans, and lignins (Naczk and Shahidi,
2004). Phenolic acids and condensed tannins are the predominant types of phenolics in
canola/rapeseed. The effect of tannins on palatability and protein digestibility in canola meal
has been reported to have less negative effects as compared to other plants (Daun et al., 2011).
Phenolic acids are present in the form of free, esterified and insoluble-bound acids and are
derivatives of benzoic and cinnamic acids (Naczk et al., 1998b). They have traditionally been
associated with a bitter flavor, dark colour and astringency of canola meal. In addition they
can form complexes with proteins and essential amino acids making them less bioavailable.
Sinapine is the most abundant phenolic acid ester in canola meal and is considered to be a
major contributor to the unpleasant flavor (Naczk et al., 1998a). However, it has been stated
that at the levels found in canola meal it did not affect feed intake or growth rate (Daun et
al., 2011). Also during the conventional processing of canola meal to obtain protein
concentrates and isolates, the content of phenolics was reduced by 60-83% (Naczk and
Shahidi, 2004).
1.2.5. Proteins of canola
1.2.5.1. Structure
Rapeseed/canola is known to have a complicated protein composition with wide distribution
of isoelectric points and molecular weights (Gillberg and Törnell, 1976). Quinn and Jones
(1976) reported around 30 different protein species in rapeseed meal with isoelectric points
situated mostly in neutral region. By solubility in various solvents canola protein consists of
approximately 70% of salt soluble globulins, up to 20% of alcohol soluble prolamins, and
10% to 15% water-soluble albumins (Tan et al., 2011a). They are also classified by the
sedimentation coefficient in Svedberg units (S) which shows the speed of sedimentation of a
macromolecule in a centrifugal field. Canola proteins are mainly composed of high-
molecular weight and low-molecular weight units determining their nutritive and functional
32
properties. According to the corresponding sedimentation coefficient canola consists of two
main classes of storage proteins: 12S cruciferin, which is a salt-soluble globulin and a 2S
napin, which is a water-soluble albumin (Karaca et al., 2011a). They account for 60% and
20% respectively of the total protein in a seed (Wu and Muir, 2008). They differ not only by
their molecular mass but also by the types of molecular interactions involved in structure
stabilization which determines their functionalities.
1.2.5.1.1. Globulin 12S cruciferin
Cruciferin is an oligomeric protein with high molecular mass (300 - 310 kDa) and several
subunits. The quarternary structure of a protein obtained by x-ray scaterring represents a
trigonal antiprism consisting of 6 ordered subunits composed of two polypeptide chains (α =
30 kDa and β = 20 kDa) stabilized by intramolecular disulfide bridges and noncovalent
interactions. It has been stated that the quarternary structure and subunit was similar to other
11/12-S seed proteins revealing certain degree of homology between these proteins (Prakash
and Narasinga Rao, 1984; Prakash and Rao, 1986; Schwenke, 1994). Similar to other oilseed
proteins of this group it can dissociate under the influence of external factors. The scheme of
dissociation is shown in the Figure 1.6.
Figure 1.6: Dissociation of 12S globulin under external conditions. Adapted from Mieth et al. (1983)
The dissociation to the 7S fraction which is presented as a half 12S fraction indicates the
oligomerisation of canola globulin. The 2-3S units may also split under the effect of
detergents such as sodium dodecylsulphate and reducing agents such as mercaptoethanol to
smaller units of 18000 and 31000 g/mol molar masses (Mieth et al., 1983). Cruciferin is
classified as a “neutral protein” with the isoelectric point equal to 7.25 ± 0.10 (Schwenke et
al., 1981). However, upon dissociation numerous subunits were found to have isoelectric
points in a wide range of pH from 4.75 to 9.15 (MacKenzie, 1975). The type of a cultivar
may also have an impact on the ratio of acidic to basic amino-acids influencing the isoelectric
point as well as the secondary structure. Secondary structure was mostly characterized by
33
circular dichroism. According to the findings it is low in α-helix (11%) but is relatively high
in β-pleated structure (50%) (Tan et al., 2011a).
1.2.5.1.2. Albumin 2S napin
A small molecular weight protein fraction named napin is a basic protein with the isoelectric
point situated in the pH range of 9-10 and molar mass between 12 and 17.7 kDa. It consists
of two polypeptide chains of 4.5 kDa and 10 kDa linked by inter- and intra-molecular
disulphide bonds which ensures the maintenance of the conformation at high and low pH
values as well as at different electrolyte concentrations (Mieth et al., 1983). For napin α-
helical structure is predominant (40% to 46%) while β-sheet conformation accounts for only
12%. The high degree of amidation of the amino acids is responsible for the strongly basic
character of the low-molecular rapeseed protein (Schwenke, 1994). It is a hydrophilic protein
by nature which was shown by measurements through fluorescent probes (Jyothi et al., 2007).
The disulfide links are the major stabilizing bonds (Wanasundara, 2011).
1.2.6. Methods of protein extraction
1.2.6.1. Protein solubilization
Method of extraction influences first of all the composition and the structure profile of the
obtained product which is in close relationship with functional properties. Protein extraction
from canola meal same as from other plants involves two consecutive processes such as
solubilization in the aqueous medium followed by separation of solubilized proteins from the
residual meal. Solubilization is conventionally performed in alkaline medium adjusted with
sodium hydroxide to pH 9-12. The time of extraction varies from 30 min to 2 h in most cases
with constant stirring or shaking. This method allowed obtaining up to 80% of proteins from
laboratory prepared meal (low temperatures treatment) (Gillberg and Törnell, 1976; Tzeng
et al., 1990b), whereas industrially obtained meals normally gave lower rates due to the fact
that an important part of proteins was denatured during oil extraction and desolvenization.
After that the proteins are centrifuged in order to separate the supernatant with solubilized
proteins from the meal residues and either oven dried or lyophilized. The speed and the time
of centrifugation also vary. Such extraction has been named direct alkaline extraction (DIR).
Although being rather effective in terms of protein yield, this method has its drawbacks.
Harsh alkaline medium have negative impact on the quality of extracted proteins leading to
34
their denaturation, reduced digestibility, and damage to some amino acids (lysine and
cysteine). It was reported that proteins prepared by this procedure have poor solubility thus
being ill-suited for using as food ingredients (Tan et al., 2011b). Moreover, it leads to salt
formation during the pH adjustment which is not desirable (Sari et al., 2013). Salts are also
extensively used in order to increase the protein extractability due to salting-in effect. In most
cases NaCl is the salt of choice, using different molarities from 0.2 to 2M. They gave lower
total extractabilities and needed an additional step such as dialysis to remove salts. The use
of polyphosphates notably sodium hexametaposphate (SHMP) as an effective complexing
agents in protein isolation has been reported (Tzeng et al., 1988b). However the protein
extractability was largely dependent on the type of meal preparation and was reduced for
industrially prepared meal (Thiyam-Hollaender et al., 2013). Another method is known as
Osborne scheme based on the consecutive extraction of proteins in water, salt solution,
ethanol, and alkaline solution. Thus, albumins, globulins, prolamins and glutenins are
extracted one after another (Osborne, 1912). This method is particularly useful in case where
specific protein fractions are needed. On the other hand, it is not economical to scale up at
the industrial level due to long processing time, high water consumption and the need for
dialysis to separate salts used for globulin extraction. Extraction by the use of enzymes has
also been utilized as an alternative to alkaline extraction as it is held in milder conditions and
thus has less negative impact on the environment (Sari et al., 2013).
1.2.6.2. Protein separation
1.2.6.2.1. Isoelectric precipitation
As the aqueous extract together with proteins contain other substances, it is subsequently
subjected to other treatments in order to remove the impurities. After extraction and
centrifugation the supernatant is subsequently treated with acid (most often HCl) in order to
precipitate the solubilized proteins at their isoelectric point or subjected to ultrafiltration and
diafiltration. The majority of works report 2 isoelectric points, one situated in pH acid region
(pH 3-5) and the other in neutral region (pH 6-8) (Ghodsvali et al., 2005; Pedroche et al.,
2004; Quinn and Jones, 1976; Tan et al., 2011b). However, commercial canola meals might
have only one point of minimum solubility situated in acidic region (between pH 4-5) as a
result of heat treatment during meal processing (Naczk et al., 1985). Isoelectric precipitation
35
is widely used in the industry as it allows to obtain 96-99% of protein in the precipitate, is a
rapid and effective technique that might be easily adapted to a large scale, does not require
sophisticated equipment and is relatively inexpensive (Zaman et al., 1999). However, the use
of acids brings about the formation of salts which further should be separated. The following
washing step results in the formation of substantial amounts of chemical effluents. Also it
may result in protein denaturation impairing its further utilization as a food ingredient and
decreasing its nutritive value. After centrifugation and washing to remove salts the
precipitates are dried or lyophilized prior to utilization.
1.2.6.2.2. Ultrafiltration and diafiltration
Ultrafiltration can be used instead of isoelectric precipitation. It serves to separate protein
molecules from low molecular substances which were co-extracted and is performed under
pressure ranging from 1-15 bar using membranes with cut-off values of 2 - 300 kDa (Lewis,
1996). Diafiltration is an extension of ultrafiltration performed by the addition of water and
further filtration so as to wash out the low-molecular weight substances which might still be
found after ultrafiltration. It allows to obtain proteins of higher solubility and better
functional properties. In addition, it allows the reducing of nutritive factors which have
smaller molecular weight comparing to proteins (Rodrigues et al., 2012).
Despite the aforementioned advantages it found few applications in the industry first of all
due to the economics of the process, cost of membranes and their maintenance (Brennan et
al., 2006). Secondly, as opposed to the separation of dairy proteins where ultrafiltration is
extensively used, plant proteins require preliminary extraction as the starting meal is a solid
material and proteins need to be pulled out first. In general the extraction is performed at high
pH values where vegetable proteins have their maximum solubility and where their
hydrolysis might occur. The ultrafiltration in such conditions may therefore lead to the loss
of nitrogen (Lewis, 1996). In addition, most of the proteins in the extract are close to their
solubility limits so the following concentration might enable them to precipitate leading to
membranes fouling. This phenomenon appearing on the surface of the membrane results in
the flux decline with the time as feed components accumulate in the membrane pores as well
as on the membrane surface. In addition it significantly reduces the working life of the
membrane. Membrane fouling is a very important factor limiting the use of membranes in
the separation of food proteins. In some cases, a flux decline may be so important that it
36
makes membrane processes unpractical for protein isolation. Problem of membrane fouling
has been reported by Sayed Razavi et al. (1996), Bazinet (2005), Alibhai et al. (2006). A
substantial review on membrane fouling was presented by Fane and Fell (1987) and Marshall
et al. (1993).
1.2.7. Effect of processing conditions on the quality of proteins
The quality of a product which implies nutritive value of canola meal and extracted proteins
as well as functional properties strongly depends on the processing conditions starting from
milling, oil extraction, desolvenization and protein isolation. Each step can bring about
changes in quality some of which are irreversible. These changes are accompanied by the
structural transformations due to the changes in conformation and interactions with other
seed compounds and may lead to protein denaturation (Mieth et al., 1983).
1.2.7.1. Effect of meal processing
Being an efficient process for oil extraction it is detrimental for the quality of the meal due
to high temperatures and the use of solvents. High temperatures have by far the most drastic
effect on the quality of the meal. While processing the meal is subjected several times to the
action of high temperatures. During milling the temperature can increase due to the shear
stress, causing protein denaturation. Two methods of canola meal preparation, a conventional
screw-pressed and a cold-milled techniques were compared by Manamperi et al. (2012).
Results showed that milling temperature have significant effect on protein quality, yield and
functionality. Meal desolvenization is the most critical step for the protein quality where the
product is exposed to temperatures over 100°C for a long term (1 to 2h) inevitably leading to
their damage (Thiyam-Hollaender et al., 2013). The results on protein extractability of
industrially toasted meal showed that it had a significantly higher amount of non-extractable
proteins in the residues in comparison with the other meal samples not subjected to heat,
irrespective of protein extraction method (Tan et al., 2011b). In the work of Samadi et al.
(2013) the seeds were heat treated and the changes in secondary structure were monitored
showing important changes in the content of α-helix and β-sheet indicating protein
denaturation. A significant decrease in the availability and the digestibility of amino-acids
due to heating process has also been reported. A 7-11% reduction in lysine content has been
found in toasted meal in comparison with non-toasted presumably due to the Maillard
37
reactions which occur during toasting which is the final step of desolvenisation (Newkirk et
al., 2003).
Table 1.8: Extraction methods and conditions used for canola and rapeseed.
Source and type of defatting
Extraction solution
pH Time Meal to solvent ratio
Maximum Protein,%
Precipitation technique
Reference
Laboratory defatted rapeseed
H2O adjusted with NaOH, CaCl2 (aq)
1-12 30 min 1:20 >80 HCl, pH 2.5-6 (Gillberg and Törnell, 1976)
Laboratory defatted Canola
5% NaOH 9.5-12 30 min 1:18 69.5 HCl, pH=3.5, 7.5, ultrafiltration
(Ghodsvali et al., 2005)
Laboratory defatted Ethiopian mustard
H2O adjusted with NaOH
10-12 60 min, twice
10% 79.2 0.5 N HCl pH=3.5;5 CaCl2
(Pedroche et al., 2004)
Canola laboratory defatted
0.1M NaOH - 20 min 1:10 No data 0.1M HCl, pH=4
(Aluko and McIntosh, 2001)
Commercial/ laboratory defatted canola
H2O adjusted with NaOH
10.5-12.5 30 min-2h
1:18 >80 HCl, pH=3.5, Ultrafiltration, diafiltration combined
(Tzeng et al., 1990b)
Commercial/ laboratory defatted rapeseed
0-3% SHMP* or NaOH (aq)
1-12 30 min 1:6-1:21 >80 HCl, pH=3.5, CaCl2, ultrafiltration, diafiltration
(Tzeng et al., 1988b; Tzeng et al., 1988a)
Soybean, Rapeseed microalgue
Enzyme assisted Depending on enzyme
3h-24h 1:25 80, 15-30
- (Sari et al., 2013)
Commercial/ laboratory defatted canola meal
Osborne scheme/ 0.1NaOH
- 1h each solvent
1:10 No data vacuum-filtered, dialyzed, 1M HCl, pH 4.0
(Tan et al., 2011b)
Laboratory defatted canola
Osborne scheme - 4h, 4h, 1h
1:5 No data vacuum-filtered, dialyzed
(Manamperi et al., 2012)
laboratory defatted rapeseed
10%NaCl, 0.01M sodium pyrophosphate
pH 7 90 min 3 times
1.3:35, 1:50
67 dialysis (Bhatty et al., 1968)
Commercial canola meal
0-1M NaCl or CaCl2,
0.1 to 0.4% NaOH
2-12 60 min 5% 92.9 Acetic acid, pH 3.5
(Klockeman et al., 1997)
Commercial canola meal
0.01-0.1M NaC1/NaH2PO4
5.5-6.5 60 min No data No data ultrafiltration (Ismond and Welsh, 1992)
*SHMP – sodium hexametaphosphate
38
Important changes have also been observed in the studied functional properties of canola
proteins as shown by Khattab and Arntfield (2009). Boiled and roasted meal had significantly
lower nitrogen solubility, emulsion and foaming capacities comparing to raw meal. Reduced
nitrogen solubility of protein isolates obtained from toasted meal as compared to non-toasted
meal was reported by Naczk et al. (1985).
The alternative technologies include aqueous extraction and the use of enzymes which
significantly increases the price of processing and is hardly competitive with the conventional
technology in economic sense (Embong and Jelen, 1977; Thiyam-Hollaender et al., 2013).
For the production of canola meal and oil the major point is the economic side of the question
that is why although alternative techniques to conventional processing exist the finished
product is not compatible in terms of price. Recently the use of fluidized-bed desolvenizing
system introduced by Dr. Weigel Anlagenbau GmbH has been utilized for gentle solvent
removal under reduced temperatures (Adem et al., 2014; Thiyam-Hollaender et al., 2013).
Meals were significantly lighter in colour and had higher protein dispersibility index.
Implementation of a new technology will open new perspectives for the industrial production
of high quality meal.
1.2.7.2. Effect of protein production
The meal subjected to high temperatures on the stage of oil extraction has already partially
damaged proteins and thus a high quality product cannot be obtained even if mild conditions
are to be used for protein extraction.
The harsh alkaline treatment extensively used for protein isolation in order to maximize the
yield could additionally damage the amino-acids such as a cystine, lysine, arginine and
possibly serine. During extraction step the formation of amino acid derivatives such as
lysinoalanine (LAL) which drastically decreases the nutritive value as well as digestibility
has been noted (De Groot and Slump, 1969). Also it has been reported to result in amino
acids racemization making them unavailable for a human body (Maga, 1984). The effect of
pH, temperature and time of treatment with regard to amino acid degradability and LAL
formation was analyzed by De Groot and Slump (1969) in various samples and by Deng et
al. (1990) in rapeseed meal and isolates particularly. The combined effects of pH,
temperature and long treatment resulted in the most significant changes in amino-acids.
However, the samples which were not subjected to temperatures and prolonged treatment,
39
contained only low levels of LAL. Less than 500 µg/g of LAL which is similar to that found
in commercially produced casein and soybean protein isolates was detected in rapeseed
proteins extracted even at pH 12. Klockeman et al. (1997) also did not detect any LAL in
canola protein isolates extracted with 0.4% NaOH. Therefore, alkali extracted protein isolates
were claimed to not represent a significant health hazard due to the LAL content (Deng et
al., 1990). On the other hand extraction at high pH is accompanied by the oxidation of
phenolic compounds which gives bitter taste and dark colour to the isolates. The dark colour
is also a concern when it comes to food utilization. Many researches were seeking the ways
to avoid the darkening or to decolorize the product. Hydrogen peroxide and sodium sulphite
were mostly used as decolorizing agents (El-Kadiri et al., 2013; Keshavarz et al., 1977; Tzeng
et al., 1990b).
Functional and physico-chemical properties were largely affected by the type and pH of
extracting medium (Mwasaru et al., 1999a; Mwasaru et al., 1999b). Higher pH of the
extracting medium allows to extract more protein but of reduced quality. The reduced
solubility and precipitability are the indication of protein alteration. Pedroche et al. (2004)
compared the functional properties of meal to those extracted at pH 10 and pH 12. In general,
the functional properties except water binding and relative foam stability impaired with the
increase in pH of extraction explained by the protein denaturation. Only isolates obtained at
pH 10 had functional properties comparable to those of the defatted meal.
1.2.8. Possible application
Proteins as already mentioned have been used as food ingredients due to their functional
properties which is their ability to confer specific characteristic to the food, either sensorial
(texture and mouthfeel) or technological (foaming, emulsifying, gelling, etc.). The area of
possible protein application is based on their functional properties but is not limited to them.
The knowledge of functional properties of isolated protein is not a guaranteed knowledge of
its behavior in food systems where numerous factors might apply. Thus, an interaction with
other components as well as processing conditions have a great influence and are able to
either enhance or impair the performance of a protein in a food matrix (Arntfield, 2004).
Hence the behavior in model systems might differ from their behavior in real products
(Luyten et al., 2004).
40
The addition of protein isolates to food formulations has two aims, first – increase the
nutritive value, and second – improve the texture. There are few works devoted to the
feasibility of canola proteins utilization in various food matrixes. One of them is comminuted
meat products, where the gelling capacity of canola proteins was utilized. The addition of
protein isolates improved cooking yield, reduced the shrinkage and gave more tender meat
patties (Thompson et al., 1982). Several authors showed improved water holding capacity
and improved cooking yield in meat products (Cumby et al., 2008; Mansour et al., 1996) as
well as improved taste and aroma (Yoshie-Stark et al., 2006).
Another possible application is baked goods or extruded products. The studies on the
inclusion of canola proteins in baked goods are scarce and mostly refer to the improvement
of nutritive value. Incorporation of canola proteins in bread has been attempted up to 18-20%
of flour with further increase resulting in denser bread and decreased sensorial properties
(Kodagoda et al., 1973a; Shahidi, 1990). Bread systems might be challenging as the
distinctive texture, loaf volume as well as sensorial characteristics of bread are attributed to
the gluten of wheat. However, in other baked systems such as cookies which do not rely on
gluten visco-elastic properties the incorporation of other proteins may add to the textural
properties, especially in those systems which do not contain wheat (gluten free products).
Such products mostly based on starchy flours have low nutritional value as well as poor
texture. The utilization of proteins thus can perform two roles. So far the possibility of
improving texture in baked gluten-free products by canola proteins has not been regarded.
A recent trend of functional food, such as protein enriched products show that in future they
will play an important role in our lives. More and more enriched products are coming to the
market. With the continuous supply of food grade oil there will be a constant supply of
protein from oilseeds. The range of products where oilseed proteins can be incorporated is
wide, however their utilization in human nutrition should be corroborated by the relevant
studies to receive the commercial recognition as in case of soybeans (Arntfield, 2004).
Canola meal is a highly potential source of edible proteins and a potential ingredient
possessing interesting functional properties which can be implemented in the food
production. However its performance is greatly perturbed during processing such as
conventional extraction. Alternative technologies have been proposed, however all of them
are not competitive with conventional extraction in terms of price. That is one of the reasons
41
why the production of protein from canola has not become commercially available for
purchase. Hence, a method that will combine the effectiveness and the cost of a conventional
technique and at the same time will give the product of a higher quality might solve this
problem
Electro-activation as an emerging technology
In recent years there has been a significant increase in the development of new extraction and
separation techniques with the emphasis on using “green technologies”. The majority of
conventional technologies imply the utilization of organic and inorganic chemicals such as
solvents, acids, bases, etc. in order to extract the targeted components from plant materials
some of which drastically affects the quality of a product. Strict regulations on the
employment of such techniques due to the environmental, safety and health concerns and an
increased awareness of consumers about the use of chemical agents urge the food industry to
search for alternative methods. It is amplified by the demand for a higher quality product that
traditional processing cannot satisfy. “Green” methods on the contrary are environmentally
friendly and cause less air pollution and industrial waste (Shi et al., 2012).
Also an increase in scientific interest in using the conventional sources of energy such as
electric power for non-conventional purposes has been noted (Aider et al., 2012b). Thus, an
application of a direct electric current in separation processes could be an alternative method
to the existing conventional technologies. The electric energy transformed into the chemical
energy allows to decrease the use of chemical acids and alkali providing environmentally
friendly “green” technology for improved food processing application.
1.3.1. History and development
The use of electric current in technology has been known since 1802 when a Russian scientist
Petrov observed that the liberation of gases near the electrodes was accompanied by the
acidification of water near the anode and alkalization near the cathode. Thirty years later
Faraday stated the world-known electrolysis laws. This gave a rise to a new branch of science
known as electrochemistry (Tomilov, 2002). Electro-activation (EA) as a technology itself
accounts for more than a 100 years, when its unique physico-chemical properties were
observed by Russian scientists while working with drilling mud (Prilutskii and Bakhir, 1999).
42
In 1972 a Russian engineer Bakhir noted that electro-activated solutions (EAS) prepared
from low concentrated salt solutions had properties different from chemically alkalized or
acidified solutions. However, he also observed that these properties were not constant and
tended to decrease with the time finally becoming equal to their chemical counterparts. Due
to these observations such solutions were called electro-activated (Tomilov, 2002). Since that
time EAS were utilized in many fields mostly as a powerful disinfecting agent (Lelianov et
al., 1991; Prilutskii et al., 1996; Sato et al., 1989; Sergunina, 1968). However, the lack of
theoretical and experimental studies hindered the development of EA as a technology and
prevented its broader application (Gnatko et al., 2011). In recent years the number of studies
devoted to Electro-activation technology increased dramatically showing its high potential
for the utilization in food and other industries (Aït-Aissa and Aïder, 2015; Aït Aissa and
Aïder, 2013b; Huang et al., 2008; Kastyuchik, 2015; Koffi et al., 2014; Liato, 2015; Liato et
al., 2015c; Thorn et al., 2012).
1.3.2. Principles
Electro-chemical activation occurs as a result of physico-chemical processes during which
the water and the dissolved particles find themselves in the nonequilibrium thermodynamic
state characterized by abnormal physico-chemical properties (Plutakhin et al., 2013). It takes
place in the near-electrode layer after subjecting water or brine solutions to the action of
external electric field (Aider et al., 2012b; Plutakhin et al., 2013; Tomilov, 2002). Water
molecules activated by the electric field fall into a metastable state resulting in higher activity
and modified physico-chemical properties such as pH, oxido-reduction potential, electro
conductivity, etc. With the time, however, the solutions are prone to relaxation, finally
coming into equilibrium (Aider et al., 2012b). The non-equilibrium processes are the
quintessence of electro-activation. When in most electro-chemical processes the equilibrium
state is sought, for EA the parameters are selected in such a way allowing to move as far as
possible from the equilibrium conditions of the flow of electro-chemical reactions (Tomilov,
2002). As a technology EA is based on the well-known phenomenon of electrolysis (Aider
et al., 2012b; Plutakhin et al., 2013; Tomilov, 2002).
43
1.3.2.1. Water electrolysis
Water electrolysis is its decomposition with the liberation of hydrogen and oxygen gases as
a result of the passage of electric current (Chaplin, 2000). The decomposition of water is
performed in the electrolytic cell, a basic example of which is shown in the Figure 1.7
consisting of two electrodes immersed in the aqueous solution known as electrolyte. The
electrodes are connected to the positive and negative charges of electric source and are termed
anode and cathode respectively. When water or a brine solution is subjected to the direct
electric current via two immersed electrodes, positively and negatively charged ions start to
migrate through the solutions towards oppositely charged electrodes to transfer the charge
(Bazinet, 2005). At the electrode`s surface a number of reactions occur leading to the
liberation of hydrogen and oxygen gases. Electric energy is transformed into the chemical
energy which gives rise to the reactions that cannot be performed under normal conditions
(Plutakhin et al., 2013). This also leads to the formation of new substances and to the
structural and compositional changes in water structure (Petrushanko and Lobyshev, 2001).
1.3.2.2. Charge transfer
The set of reactions which takes place as a result of applied current is linked to the transport
of electric charge (Bard and Faulkner, 2000). For electrolysis the electric charge is carried
out by two different species - electrons in the electrode and migrating ions in the electrolyte.
Due to the presence of two phases (electrode-electrolyte) and the fact that the charge is
transported by different carriers the change of charge carrier takes place at the interface. On
one side charge carriers of one type transfer the charge to the carriers of the other type. This
is possible due to the chemical reactions which imply both charge carriers, known as electro
chemical or electrode reactions. On the anode, as a result of chemical reaction electrons are
generated and pass from the solution to the electrode. On the cathode electrons are transferred
from the electric circuit to the electrolyte. The withdrawal of electrons on the anode is
equivalent to the oxidation of an element whereas the addition of electrons equals to the
reduction process (Bagotsky, 2006).
44
Figure 1.7: Basic electrolytic cell. Adapted from Zeng and Zhang (2010).
1.3.2.3. Electrode reactions
Due to the presence of different cationic and anionic species as well as molecules a number
of different processes can take place simultaneously at the electrodes. The type of reactions
depends on the electrode material, ionic species in the solution, and other factors. Depending
on the material some electrodes can participate in the reactions (Bagotsky, 2006), however it
is not the subject of the current study as for electro-activation non-consumable (inert)
electrodes are utilised. They serve primarily as a stock and a sink of electrons and permit the
electron transfer without participating themselves in the reactions (Brett and Brett, 1993).
The principal reactions which take place during water electrolysis are water oxidation on the
anode with O2 liberation (Eq. 1.1; 1.2) and water reduction on the cathode with H2 release
(Eq. 1.3; 1.4). It is also accompanied by pH changes near anode and cathode. As a result an
acidic solution is generated in the near anode layer and an alkaline solution is produced in
the near cathode layer. In order to obtain two solutions with different properties such as acidic
45
anolyte and alkaline catholyte the electrolytic cell can be separated with the porous
diaphragm or an ion-exchange membrane (Aider et al., 2012b).
Anode oxidation:
2Н2О (l) − 4ē→ 4Н+ (aq) + О2 (g) ↑ (Eq. 1.1)
4OH–(aq) → O2↑ (g) + 2H2O (l) + 4ē (Eq. 1.2)
Cathode reduction:
2Н2О (l) + 2ē→ Н2↑ (g) + 2ОН −(aq) (Eq. 1.3)
2H+ (aq) + 2ē → H2↑ (g) (Eq. 1.4)
where (l), (g) and (aq) are liquid, gas or aqueous solution.
Provided that only hydrogen and oxygen molecules as well as charged species participate in
electrode reactions twice as much hydrogen is liberated compared to oxygen gas (Aider et
al., 2012b). The near electrode layer is the area of high intensity where the voltage can reach
up to hundred thousand volts, yet it is rather thin. This layer is also known as double diffusive
layer (Tomilov, 2002).
As the water itself poorly conducts the electric current, to facilitate the charge transfer and
to decrease the system resistance salts are usually added (Chaplin, 2000). Most often salts
like NaCl, KCl, Na2SO4, and NaHCO3 are used. In the solution they dissociate and exist as
ionic species e.g. Na+, K+, Cl-,SO42-, HCO3
-, etc., carrying positive and negative charges
(Bagotsky, 2006). When different anionic and cationic species as well as not dissociated
molecules are present in the solution several different reactions might occur at the same time
due to the competition between ions which move towards the electrodes (Anshitz et al.,
2008). This yields new solutes and compounds which can further react between each other
(Figure 1.8). The electrode reactions are numerous and rather complex. Some of the
reactions products are unstable and quickly undergo further transformations (Chaplin, 2000).
46
Figure 1.8 : Electrolytic cell showing the passage of electrons and some products of electrode reactions. Adapted from Chaplin (2000).
1.3.2.3.1. Cathode reactions
The sequence of the redox reactions in the cathodic compartment depends on the activity of
metals. All metals can be divided in three groups: (1) Active metals (from Li to Al), (2)
medium active (Al to H), and (3) low active (metals after H). This classification is known as
the reactivity series and was made according to the magnitudes of the electrode potentials. In
this series metals are placed from the most active (having the lowest electrode potential) to
the least active ones (having the highest electrode potential). With an increase of the standard
electrode potentials the reducing capacity of metals decreases. Thus all the metals placed
before hydrogen (first and second groups) have more negative potential and can expel the
hydrogen from the diluted mineral acids (Vapirov et al., 2000). Metals of the first group
hence are not prone to reduction. On the cathode instead the hydrogen is reduced according
to Eq. 1.3. Cations of the second group are reduced together with the hydrogen ions (Eq. 1.3
and Eq.1.5). Finally, the cations of low active metals reduce themselves on the cathode (Eq.
1.5).
Men+ + nē → Me0 (Eq. 1.5)
If cations of different ions are present in the solution, then first metal to be reduced will be
the one with the highest potential followed by the metals with lower potentials (Anshitz et
al., 2008). When NaCl salt are used cathode reactions also yield sodium hydroxides as well
47
as a number of highly active reducers and metastable aquacomplexes of hydroxyl anion (ОН-
), hydroperoxide anion (НО2-), molecular oxygen ion-radical (O2
-), oxygen anion (O2-),
peroxide anion (О22-) and metastable aquacomplexes of hydrogen superoxide (НO2), free
hydrogen radical (Н•), free peroxide radical (НO2•) (Belovolova et al., 2006; Gnatko et al.,
2011). Catholyte thus saturated with the reducers gains the enhanced adsorbing capacity
(Leonov et al., 1999).
1.3.2.3.2. Anode reactions
At the anode oxidation of the acid residuals takes place. Those that do not contain oxygen
oxidize themselves e.g. Eq. 1.6. Those that contain oxygen are not oxidized at the anode,
instead the oxygen from the water is liberated (Eq. 1.1).
2Cl− - 2ē → Cl2↑ (Eq. 1.6)
Simultaneously a number of active oxidizing compounds are generated presented by a
mixture of peroxide compounds such as НO•– (hydroxyl radical); НО2− (peroxide anion); О2
(singlet molecular oxygen); О2− (superoxide anion); O3 (ozone); O• (atomic oxygen) and
chlorine-oxygen compounds notably HClO (hypochlorous acid); ClO− (hypochlorite ion);
ClO• (hypochlorite radical); and ClO2 (chlorine dioxide). Some of them can further react
with each other neutralizing themselves, e.g. reaction of hypochlorous acid with hydrogen
peroxide (Eq. 1.7) or reaction of hydrogen peroxide with ozone (Eq.1.8) (Bakhir, 2012b;
Gnatko et al., 2011).
HClO + H2O2 →O2↑+ H2O + HCl (Eq. 1.7)
H2O2+ O3→ 2O2↑+ H2O (Eq. 1.8)
1.3.3. Phenomenon of EAS
It is difficult to highlight a single reason of the electro-activation phenomenon. Instead there
is a number of factors explaining the high reactivity of EAS. As was already mentioned it
can be done only when the system is in thermodynamically unstable state which is achieved
by the use of external electric field. The electric field supplies the energy required for the
electro-chemical reactions to launch. This energy is the highest near the electrodes surface,
where they contact with the electrolyte (Plutakhin et al., 2013). As a result of treatment
48
anolyte and catholyte acquire such properties that cannot be modulated in the solutions not
subjected to electro-chemical effect (Sprinchan et al., 2011b).
The activated state of the solutions is not perpetual. With the time the anomalous properties
vanish and the thermodynamic equilibrium is established with the pH and oxido-reduction
potential inherent to typical solutions known as relaxation (Sprinchan et al., 2011b). The
phenomenon of relaxation demonstrates the presence of metastable particles and the fact of
the existence of activated state. Thus EAS exist only during the period of relaxation
(Prilutskii and Bakhir, 1999).
One of the suggested explanations is the formation of various products during water
electrolysis. Some of them are rather stable and can exist in the medium for a certain amount
of time, other immediately enter the subsequent reactions (Gnatko et al., 2011). Several
groups of reactive substances can be discerned. First group represents stable reaction
products such as bases and acids. They explain the alkaline properties of the catholyte and
acid properties of the anolyte. Second group is highly active metastable compounds such as
molecular ions НО2-, О2
-, ОН- as well as free radicals. And third refers to the long-lived quasi-
stable structures formed in the near electrode surface area which could be presented as free
structural complexes, hydrated ion shells, molecules, or radicals (Sprinchan et al., 2011b).
The factors of the second group enhance the redox properties of the catholyte such as an
electron donor. Finally the third group of factors is responsible for the catalytic activity of
the catholyte. Specifically the products of the second and third groups cause the modification
of the activation energy barriers between the components of the system. Their generation is
possibly only under the condition of electro-chemical treatment (Sprinchan et al., 2011b).
Next possible explanation is connected to the water state and the modification of its structural
and energetic characteristics (Gnatko et al., 2011; Petrushanko and Lobyshev, 2001). When
EA is performed under the condition of the low mineral concentration the water electrolysis
at the electrodes surface is performed under high voltages which cause the structural
modification of water molecules. As a result of hydrogen bonds rupture the water structure
gains electron donor and electron acceptor properties near cathode and anode. Such water
easily forms aqua complexes (hydrated ionic complexes) which enhance the reaction and
catalytic activity of EAS. In the solutions with high mineral salt concentrations the voltage
is low and water structure is less prone to changes, thus the properties of such solutions
49
mainly depend on the products of electrolysis reaction. (Prilutskii and Bakhir, 1999). The
resonance effect of water clusters resulting from co-vibrating dipoles of water molecules as
well as charged species is also one of the factors used to explain the non-contact EA. Such
dipole systems are unstable and rapidly collapse in static, whereas in dynamic state provided
by electric field it becomes possible (Aider et al., 2012b; Shironosov, 2000). Finally the
formation and the presence of gaseous microbubbles also favors the anomalous properties of
solutions in activated state (Gnatko et al., 2011).
The evidence of the existence of activated state was shown by Pastukhov and Morozov
(2000) who studied the EAS by means of Raman spectroscopy. According to the study the
vibrational spectra of electrochemically treated solutions and their chemical analogues were
different in the spectral region between 700 and 2700 cm-1. A broad peak was obtained for
EAS in this region which was not present for their chemical counterparts. The intensity of
the peak also decreased with the time indicating the relaxation. The spectra of EAS were
similar to those of concentrated alkali and acids in spite of the fact that they had low
mineralization. This led to the assumption that EAS might have properties similar to those of
concentrated acids and bases (Aider et al., 2012b; Plutakhin et al., 2013).
1.3.4. Reactors design
As already mentioned the process of electro-activation takes place in the near electrode layer,
where the resistance can reach up to several thousand volts per cm. This layer is
comparatively thin (around 0.1 µm), thus a design and a construction of a reactor with optimal
characteristics was not an easy task (Tomilov, 2002).
1.3.4.1. Flow-type reactor
First attempts to design a reactor for the production of electro-activated solutions were made
in 1974 – 1975. Twenty years later the reactor has been released. It was a flow type reactor
made of several parallel placed flow electrochemical modules (Figure 1.9) with external
tubular and internal rod-like electrodes. A tubular ceramic ultrafiltration diaphragm was
placed between them. Such configuration allows to treat the volumes of water equally
saturating them with metastable active particles and provides unipolar water treatment when
it passes through anodic and cathodic chambers (Leonov et al., 1999). The volumetric flow
rates usually range between 0.5–1.9 l/min (Aider et al., 2012b). As a result two solutions are
50
obtained notably anolyte and catholyte with physico-chemical properties determined by the
current intensity, mineral composition and the volume of treated solution. Since that time a
number of technologically improved reactors appeared not only for technological purposes
but also for domestic use producing electro-activated solutions with disinfecting, sterilizing,
stabilizing, conservation, extracting, and healing properties (Bakhir, 2012a).
Figure 1.9: Flow type electrochemical modules. Adapted from Leonov et al. (1999).
Many other types of reactors have been used worldwide. In 1999, only in Japan over 30
different types of reactors were functioning. More on different types of reactors can be found
in Aider et al. (2012b); Tanaka et al. (1999).
1.3.4.2. Stationary type of reactors
Together with flow type reactors the stationary types are also used. In this case the solutions
are treated in batches of 1-15 l and the time of EA varies between 3 and 115 min. The voltages
of 9–120 V are used for electro-activation with the current intensity being set from 0.7 to 20
A (Aider et al., 2012b).
Outlet C
Outlet A
Anode
Diaphragm
Outlet A
Outlet C
Inlet A Inlet C
Direction ofwater flows
Anode
Outlet A
Diaphragm
Outlet C
Cathode
Cathode
51
For large scale production flow- type reactors are more practical, whereas in the lab mostly
a stationary type of cell is used for electro-activation (Aider et al., 2012b). A three-
compartment reactor consisting of the anodic, cathodic and central chambers was used in the
works of Aït Aissa and Aïder (2013b), Koffi et al. (2014), and Liato et al. (2015b) (Figure
1.10). Anodic and cathodic compartments separated by the ion-exchange membranes allow
to produce solutions with oxidizing (acid anolyte) and reducing (alkaline catholyte)
properties. By changing the parameters such as type of configuration, current intensity, time
of treatment, types of salts and their concentrations the solutions with desired properties can
be obtained. Central compartment has also been utilised for the analysis of a non-contact
EAS (electro-activated solution) (Liato et al., 2015b).
Figure 1.10: A schematic representation of a three-call electro-activation reactor. Adapted from (Liato et al., 2015a).
1.3.4.3. Requirements for reactors and its functional parts
In order to be efficient reactors must meet the following requirements. First, a large active
electrode area is needed per unit volume. Second is a uniform distribution of the electric
potential on the electrode surface. Finally, rather high current density, good mass transport
towards the electrodes, and low power consumption are required (Gnatko et al., 2011).
Certain requirements exist for the electrodes and membranes as they are the functional parts
of the reactor.
52
1.3.4.3.1. Electrodes
A very important characteristic is the chemical stability of the electrodes (Aider et al., 2012b).
They must not participate in the chemical reactions themselves being chemically inert as well
as insoluble in the products of electrolysis Thus typical materials used for electrodes are
platinum or titanium coated with porous layers of a metal oxide catalyst (e.g. RuO2, TiO2,
SnO2, IrO2) due to their high stability, selectivity, electro-chemical reactivity, corrosion
resistance and operating life (Thorn et al., 2012; Trasatti, 1987; Trasatti, 2000). Chemically
inert graphite electrodes also have been utilised, however they have some disadvantages such
as their porosity and friability. Gas bubbles can accumulate in the pores thus reducing the
active working surface of electrodes (Plutakhin et al., 2013).
1.3.4.3.2. Membranes
Membranes are used in order to separate the reaction products and increase the effectiveness
of the process. There are several types of membranes used in electro-activation, however the
most utilised are ion-exchange membranes such as cation exchange membranes (CEM) and
anion exchange membranes (AEM) as they allow to separate the ionic species according to
their charge. Both of them are monopolar, this is to say that they allow only one type of ions
to pass. CEM is permeable for cations and rejects anions, for AEM it is the opposite. They
are normally made of a macromolecular material known as skeleton with fixed ionisable
groups which can be ion-exchange resins (Bazinet et al., 1998a). The skeleton can be made
completely from the ion-exchange material, in this case membranes are called homogenous
or the filler can be added in order to increase the mechanical strength, such membranes are
called heterogeneous (Shaposhnik, 1999). Typical groups fixed on the CEM are sulfonic
(SO3-), carboxylic (COO-), arsenic (AsO3
2-), or phosphoric (PO32-) groups and alkyl
ammonium groups (NR3+, NHR2
+, NH2R+) for AEM (Bazinet, 2005). When the membrane
is placed in the solution it is neutralized by the ions of the opposite charge known as counter
ions or compensator ions. These ions are used to carry the electric current in the membrane.
For CEM counter ions are positive, whereas for the AEM they are negative (Figure 1.11)
(Bazinet et al., 2010). Ions move inside the membrane through its pores and canals and
include the processes of sorption on the surface of the membrane, transport, and desorption
(Yaroslavtsev and Nikonenko, 2009). The selectivity of membranes depends on the pore
sizes which can vary from 10 to 100 Å. Higher pore size is less selective and allow more ions
53
with the same charge to pass than do membranes with small pores. Typically pore size of 10
- 20 Å are used (Bazinet et al., 1998a).
Figure 1.11: Example of a CEM with fixed SO3 groups. Adapted from (Bazinet et al., 1998a).
1.3.5. Application of EAS
Unique properties of EAS open doors to their wide application in many fields including water
preparation, chemical industry, agriculture, livestock breeding, medical and food industries
(Golokhvast et al., 2011). As it was aforementioned anolyte and catholyte possess largely
differing properties determined by their composition. The presence of strong oxidizers in the
anolyte makes it a powerful cleaning and disinfecting agent having highly pronounced
biocide properties. The catholyte on the other hand rich in reducers gains considerable
absorption and catalytic activity as well as detergent capacity (Prilutskii and Bakhir, 1999).
Hence, their utilisation is dictated by the stated goals.
1.3.5.1. Cleaning and disinfection
The utilisation of EAS as disinfectant has several advantages. They were shown to be more
environmentally friendly than traditional cleaners. Furthermore they are not harmful for
human health e.g. not corrosive to skin, mucous membrane, or organic material as compared
to chemical acids and bases (Huang et al., 2008). No changes in body weight nor any
abnormal effects were found in mice after digesting electro-activated solutions (Morita et al.,
2011).
Antimicrobial properties of the anolyte also known as strongly acidic electrolyzed water,
electrolyzed strong acid aqueous solution, superoxidized water, electrolyzed oxidizing water,
etc. have been utilized by many authors as an alternative to chlorine and hydrogen peroxide
54
(Gnatko et al., 2011; Huang et al., 2008). It showed the high antimicrobial effect against
numerous pathogens, some of them are: Pseudomonas aeruginosa, Staphylococcus aureus,
S. epidermidis, E. coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Bacillus
cereus, Listeria monocytogenes, Mycobacterium tuberculosis, Campylobacter jejuni, etc.
(Drees et al., 2003; Kim et al., 2000; Sato et al., 1989; Vorobjeva et al., 2004). It also showed
the inhibitory effect on fungal species such as: Alternaria spp., Bortrytis spp., Cladosporium
spp., Fusarium spp., Aspergillus spp., and yeasts: Saccharomyces cerevisiae (Guillou and El
Murr, 2002), Kloeckera and Candida spp (Lustrato et al., 2006), and many other. Various
mechanisms were proposed, however the main is the high oxidation potential of electro-
activated water which leads to damage of cell membranes. There are also indications of
antiviral capacity against blood borne pathogenic viruses such as hepatitis B virus (HBV),
hepatitis C virus (HCV), and human immunodeficiency virus (HIV) (Kitano et al., 2003;
Sakurai et al., 2003; Selkon et al., 1999) by destruction of viral surface proteins, nucleic acids
and RNA (Huang et al., 2008). Thus, electro oxidized water have been used for disinfection
of processing equipment (Abe, 1999; Ayebah and Hung, 2005; Walker et al., 2005), washing
freshly cut fruits and vegetables (Al-Haq et al., 2001; Gil et al., 2015; Izumi, 1999; Park et
al., 2001), disinfecting poultry, meat, and seafood (Ozer and Demirci, 2006; Park et al.,
2002; Yang et al., 1999). A substantial review on the application of electro oxidized water as
a disinfectant is presented by Huang et al. (2008). Electro-activated solutions have also been
used in combination with other treatments such as sterilisation of canned vegetables in order
to decrease the sterilisation temperatures (Genois, 2014; Liato et al., 2015c).
1.3.5.2. Other applications
Other than disinfection EA was used for the stabilisation of the maple sap soft drink by its
acidification directly in the anodic compartment of a 3-cell reactor (Koffi et al., 2014). EAS
were used in dough instead of tap water for making bread and macaroni (Prilutskii and
Bakhir, 1999). Their activity on yeast activation for bread baking resulted in lower
fermentation time and higher bread volume (Nabok and Plutakhin, 2009). Other examples
include the catalysis of starch hydrolysis (Leonov et al., 1999), the germination of barley
(Plutakhin et al., 2014), thermal coagulation of beetroot pectin (Shazzo et al., 2005), acid
mine drainage neutralization (Kastyuchik, 2015), etc.
55
The utilisation of catholyte is based on its catalytic and reducing properties. Catalytic
properties were utilised for synthesis of lactulose from lactose in a three cell stationary
reactor (Aider and Gimenez-Vidal, 2012; Aït Aissa and Aïder, 2013b). In the other study the
possibilities of simultaneous separation of protein concentrate and lactulose synthesis were
analyzed by EA of whey in a flow type reactor (Sprinchan et al., 2011b).
As an extracting agent EAS were used for hop extraction (Khrapenkov et al., 2004), pectin
production from apple and beet cake, extraction of propolis and ginseng (Prilutskii and
Bakhir, 1999).
1.3.5.3. Protein production
Extraction of proteins by the use of electro-activated solutions is another perspective
application which can be used for the valorisation of proteins from vegetable sources and by-
products or for improving existing technologies on the protein preparation from conventional
sources. For protein extraction and precipitation necessary alkaline and acid conditions are
created in the cathodic and anodic chambers.
Electro acidification of soy protein extract using the technology of electro dialysis was
performed by Bazinet et al. (1998b); Bazinet et al. (1997). Protein extract was treated in the
electrochemical cell separated from electrodes by the ion exchange membranes to precipitate
the proteins. The obtained isolates were higher in protein and lower in ash content, and
showed equal or improved functional properties in comparison with commercial samples. At
the same time the quality of isolates were improved (less denatured) regarding the solubility
profiles. The method was improved later in the work of Mondor et al. (2004) by adding the
ultrafiltration membrane which helped to obtain more pure isolates but also increased the
cost of extraction. Although the mentioned technology used for acidification is
fundamentally different from electro-activation as in this case protein precipitation was based
only on the pH of the medium and not on the metastable state of the solution, it gives the
indication of the possibility of performing the EA technique for protein extraction.
Electro-coagulation of whey performed in electrolyser with gradual increase and decrease in
pH in order to separate whey proteins was realized and patented by Bologa et al. (1996). The
thermal coagulation of proteins from Lucerne (alfalfa) leaf juice using EA technology was
performed by Plutakhin et al. (2004). Later, protein isolates were produced from sunflower
oilcake in a flow type reactor by the same author - Plutakhin et al. (2005). The schematic
56
representation of extracting unit is shown in the Figure 1.12. The sunflower meal was added
with water (1:10) in a tank (1) equipped with capronic filter. Tank (1) was connected to the
peristaltic pump (6) which pumped the solution through cathodic chamber until required pH
was reached. A 5-time passage was needed to reach pH 11. In the anodic chamber at the same
time the NaCl solution with the concentration of 50 mg/l was circulated. After extraction the
protein solution was pumped through the anolyte chamber until the pH reached 4
(corresponding to the isoelectric point). In order to prevent the thermal coagulation of
proteins the parameter of current intensity and the speed of circulation were chosen in such
a way that the temperature did not exceed 40 °C. As a result 34% of protein was extracted
using electro-activation compared to 39% obtained using alkaline extraction with NaOH. The
losses of protein were summoned by the local overheating due to the contact with the surface
of electrodes which could be solved by modification of the unit construction (increasing the
surface of electrodes) and by increasing the solvent to meal ratio (Plutakhin et al., 2005).
Figure 1.12 : Electro-activation unit for protein extraction form sunflower oil cake, where 1-tank with oilcake; 2 - cathodic chamber; 3 - membrane; 4 – anodic chamber; 5 – tank with anolyte; 6 – peristaltic pump; 7 – anode; 8 – cathode; 9 – filter; 10 – oilcake; 11 – extracting solution. Adapted from Plutakhin et al. (2005).
Other works on protein extraction include a series of patents differing by technical
characteristics and conditions of extraction (Koschevoi et al., 1997; Koshchaev et al., 2005;
Petenko et al., 2003; Plutakhin et al., 2006a, b). Regarding all the aforementioned information
the advantages of using EAS over traditional approach become evident. First of all, they
allow to reduce the use of chemical bases and acids and potentially produce protein products
of higher quality. The conditions, required for protein extraction and precipitation are
generated using the electric field by the passage of electric current through the salt solution.
57
Secondly, the amount of preparative steps is also reduced. In comparison with conventional
extraction which includes preparation of NaOH and acid separately, EA needs only the
preparation of salt solution. Third, the conditions of extraction can be monitored by
controlling the parameters of electro-activation. However, there is a lack of studies on the
relationship between the parameters of the EA process and properties of electro-activated
solutions (Aider et al., 2012b). Also the effects of EAS on the quality of obtained proteins
such as structural characteristics and functional properties have not been studied.
58
2. CHAPTER 2: Problematic, research hypothesis and objectives
2.1. Problematic
The literature review showed increasing demand for alternative protein sources to satisfy the
needs of continuously growing world`s population. Proteins from renewable plant sources
are considered as the most promising. These sources are being properly studied in order to
show that they are suitable for human consumption and the idea of their valorization is close
to its realization. New protein sources require appropriate extraction techniques based on the
knowledge of the physico-chemical properties, spatial arrangement, process requirements, as
well as techno-functional properties of these proteins. While conventional processing
significantly alters the quality and nutritional value of the extracted proteins, new processing
techniques are needed. In addition, regarding the environmental situation ``green`` methods
(eco-friendly) are welcomed and encouraged worldwide. Human start taking after the
nature`s best phenomena, which leads us to eco-friendly techniques, one of which is electro-
activated solutions. With their help the utilization of chemical acids and alkali can be
significantly reduced or even completely eliminated which will as well be positively reflected
on the quality of extracted proteins. Such proteins can be further incorporated into different
food matrices conferring required properties. But before implementing any novel
methodology or technology, a proper research should be conducted to confirm that such
technique is beneficial compared to conventional processing.
Research hypothesis
The use of electro-activated aqueous solutions, as alkali replacement, will allow to extract
proteins from canola press-cake with their further utilisation as a product ingredient.
Main objective
The main objective of this doctoral thesis is to study the electro-activated solutions in terms
of their generation, protein extraction capacity and their impact on proteins physico-chemical
and techno-functional properties.
59
Specific objectives
In order to verify the stated hypothesis and to achieve the main objective, the following
specific objectives were highlighted:
1. To study and to find the optimal conditions for protein extraction from canola meal
by performing conventional alkaline technique as a comparative basis for the electro-
activation method.
2. To investigate the process of generating electro-activated aqueous solutions
depending on the influence of different operating conditions in order to obtain
solutions with alkaline properties as a replacement of chemical alkali.
3. To perform the extraction of canola proteins by using electro-activated aqueous
solutions
4. To study and analyse the impact of electro-activation on the physico-chemical and
functional properties of canola proteins.
5. To incorporate the proteins extracted by the electro-activation technology in a gluten
free food matrix (biscuits) so as to verify their behaviour in real food systems.
60
3. CHAPTER 3: Total dry matter and protein extraction from canola
meal as affected by pH and salt addition and the use of Zeta-
potential/turbidity to optimize the extraction conditions
Contextual transition
Nowadays there is a need in alternative protein sources with high nutritive value and adequate
functional properties. One of the possible sources is canola, a leading oilseed crop in Canada.
Although there are works devoted to the extraction of canola proteins the amount of cultivars,
different pre-treatments techniques and tests make it difficult to choose the optimal extracting
conditions and give sometimes contradictory information. In our case canola meal as a by-
product of oil production was directly obtained from the industry and was used for all the
analysis. The aim was to screen the number of factors affecting the extractability and to see
which protein profiles are extracted. It was also of interest to verify whether conditions
optimal for protein extraction also give the highest dry matter extractability.
This chapter is presented as an article entitled: “Total dry matter and protein extraction from
canola meal as affected by pH and salt addition and the use of Zeta-potential/turbidity to
optimize the extraction conditions”.
The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:
scientific supervision, article correction and revision), Marzouk Benali (Scientific
collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder
(Thesis director: scientific supervision, article correction and revision).
61
Résumé
La matière sèche et les protéines ont été extraites à partir de tourteau de canola sous
différentes conditions. Nous avons observé que le pH du milieu d'extraction, la concentration
du tourteau de canola et la concentration du sel ont des effets différents sur l’extractibilité de
la matière sèche totale et des protéines, avec le pH ayant l’effet le plus significatif. Le plus
fort taux d'extractibilité de la matière sèche (42.8 ± 1.18%) a été obtenu avec 5% de tourteau
de canola à pH 12 sans addition de sel, tandis que l'extractibilité maximale des protéines
(58.12 ± 1.47%) a été obtenue avec 15% de tourteau sous les mêmes conditions. La plus
faible quantité de matière sèche extraite (26.63 ± 0.67%) a été obtenue avec 5% de tourteau
de canola à pH 10 sans addition de sel, alors que la plus faible quantité des protéines a été
obtenue avec 10% de tourteau à pH 10 et 0.01 M NaCl. Les analyses de turbidité et du
potentiel zeta ont démontré que le rendement maximal de protéine est atteint à pH 4 – 5;
intervalle de pH correspondant au point isoélectrique de la majeure partie des protéines de
canola. Les analyses réalisées par SDS-PAGE ont démontré que l'addition du sel augmente
la solubilité des protéines de canola, particulièrement la fraction de la cruciférine, alors que
le sel pourrait également réduire l'extractibilité de la napine.
62
Abstract
Total dry matter and proteins were extracted from canola meal (CM) under different
conditions. It was observed that pH of the extraction medium, CM concentration, and salt
concentration influenced the extractability of total dry matter and proteins, with the pH of the
extracting medium having the most significant effect. The maximum extractability of total
dry matter (42.8 ± 1.18%) was obtained with 5% CM at pH 12 without salt addition whereas
the maximum total protein extractability (58.12 ± 1.47%) was obtained with 15% CM under
the same conditions. The lowest amount of dry matter (26.63 ± 0.67%) was obtained with
5% CM at pH 10 without salt addition and the lowest protein amount was observed with 10%
CM at pH 10 in 0.01 M NaCl. Turbidity and ζ-potential measurements indicated that the
optimum pH for obtaining the maximum yield of protein was within the range of pH 4 and
pH 5. In our investigation, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) showed that although, addition of salt increased the solubility of canola proteins
specifically the cruciferin fraction, it reduced the extractability of napin.
63
Introduction
Canola is Canada's one of the most important and valuable crop. Canada is the largest
canola producer and exporter in the world. It is not only a principal source of vegetable oil
but also the second most important oilseed crop after soybean (Pan et al., 2011). It is used for
producing edible oils with a high content of monounsaturated fatty acids and low levels of
saturated fatty acids. Canola meal is a by-product of oil extraction with a high protein content
and dietary fiber. Depending on the type of meal it can contain up to 50% protein (dry basis)
and 20% carbohydrate (Aider and Barbana, 2011; Yoshie-Stark et al., 2008). Canola protein
content and amino-acid composition are similar to soybean and it can be used not only as
forage for livestock, but in the food industry owing to its interesting functional properties
(Aider and Barbana, 2011; Aluko and McIntosh, 2001; Fleddermann et al., 2013). Canola
protein has also been attributed with bioactive properties (Aider and Barbana, 2011; Khattab
et al., 2010; Wu and Muir, 2008; Yoshie-Stark et al., 2006; Yoshie-Stark et al., 2008).
However, the complex composition of canola proteins (Gillberg and Törnell, 1976;
Kodagoda et al., 1973b; Tzeng et al., 1990b; Wu and Muir, 2008) makes the standardization
of extraction conditions a challenging task. Here it is important to note that a high alkaline
medium (pH values ≥10) significantly increases the protein extractability. In addition, it has
been also stated that the extraction of phytic acid (an anti-nutritional factor) remains low at
high alkaline conditions (Ghodsvali et al., 2005; Tzeng et al., 1990b; Tzeng et al., 1988a).
The effect of extraction conditions on the composition, amino-acids and functional
properties of the extracted protein isolates have been previously investigated (Aluko and
McIntosh, 2001; Khattab and Arntfield, 2009; Pedroche et al., 2004; Tzeng et al., 1988b;
Tzeng et al., 1988a; Xu and Diosady, 1994; Yoshie-Stark et al., 2006). There also have been
attempts to reduce the amount of anti-nutritional factors (glucosinolates, polyphenols and
phytic acid) present in the meal as they might negatively impact the organoleptic and
nutritional properties of the final product (Diosday et al., 1984; El-Kadiri et al., 2013;
Ghodsvali et al., 2005). The influence of the solvent used for defatting the meal on the protein
yield has also been investigated (Tzeng et al., 1990b). The polypeptide profile and the
composition of canola proteins has been studied by Aluko and McIntosh (2001); Klockeman
et al. (1997); Mimouni et al. (1990); Wu and Muir (2008). The two most abundant protein
fractions present in canola meal are the 12S globulin known as cruciferin and 2S albumin
64
known as napin. Since canola proteins mainly consist of water soluble albumins and salt
soluble globulins, the most common extraction technique employed are alkaline extraction
using aqueous NaOH solution and extraction with salt solutions such as CaCl2, sodium
hexametaphosphate (SHMP) and NaCl (Tan et al., 2011a; Tzeng et al., 1988b; Tzeng et al.,
1988a). A method known as protein micellar mass (PMM) has also been reportedly used,
where the meal is dissolved in a buffer containing 0.1 M NaCl/0.1 M NaH2PO4 (Ismond and
Welsh, 1992). An extraction method based on solubility of canola proteins in different
solvents (water, salt solution, acid or alkali and aqueous alcohol) known as Osborne scheme
has also been performed (Manamperi et al., 2012; Tan et al., 2011b). Nevertheless, despite
the numerous methods developed for the extraction of canola protein the conventional
extraction method, commonly known as direct alkaline extraction (DIR) that uses alkaline
medium, remains the most effective and utilised method. In addition, it is also a reference
method when newly developed techniques and processes are to be compared for their
efficiently and efficacy (He et al., 2013).
Alkaline extraction is traditionally followed by isoelectric precipitation. Canola
proteins have two isoelectric points, one in the range of pH 3-5 and the other between pH 6-
8 (Ghodsvali et al., 2005; Pedroche et al., 2004; Quinn and Jones, 1976; Tan et al., 2011b).
Isoelectric point is related to the composition of the side chains of proteins, and because the
type of protein can vary based on the type of canola meal investigated, contradictory
information regarding isoelectric points has been found in the literature. Ultrafiltration and
diafiltration can also be used following alkaline extraction for separating the extracted
proteins from the solvent (Tzeng et al., 1990b). Most of the studies done concerning
isoelectric point (pI) determination, use either isoelectric focusing or titration of the
supernatant with HCl with subsequent determination of nitrogen in precipitate in relation to
the total nitrogen extracted and plotting the values vs pH (Ghodsvali et al., 2005; Pedroche
et al., 2004; Tan et al., 2011b).
The quality of literature available highlights the importance of this topic; however,
most studies mainly focus on the characterization of the obtained protein isolates, and not on
the extractability of proteins and dry matter. Furthermore, apart from optimizing the
parameters for protein extraction, extraction of carbohydrates is also of particular interest as
it may have beneficial impact on the functional properties (Shao et al., 2013). This study
65
therefore, has been done to optimize parameters for protein extractability and total
extractability. Additionally it also compares the conditions optimal for protein and total
extractability. Specifically, the first objective was to determine the optimal conditions (pH,
ionic strength and solvent to meal ratio) for canola extraction and ensure maximum
extractability, to also understand the influence of these parameters on the total and protein
extractability, using the conventional technique. The second objective was to analyze and
understand the dynamics of precipitation of canola proteins at their isoelectric point by
studying the charge on the surface of particles (zeta potential) in suspension, in combination
with turbidity. This would provide useful information concerning the optimal conditions for
protein precipitation. The chosen method is not only efficient but also it is faster compared
to other conventional methods reported in the literature.
Materials and methods
3.6.1. Materials
Defatted canola meal was kindly provided by Bunge ETGO, Becancour, Québec,
Canada. Sodium hydroxide (NaOH) was purchased from VWR International LLC (West
Chester, PA, USA). Concentrated hydrochloric acid (HCl) was purchased from Fisher
Scientific (Mississauga, ON, Canada) and sodium chloride (NaCl) was purchased from
Caledon Laboratories LTD (Georgetown, ON, Canada).
3.6.2. Proximate analysis
Proximate analysis of the industrial canola meal was performed according to AOAC
International (2012). Moisture and total dry matter were determined by drying the weighed
sample in a Fisher Isotemp Vacuum Oven (Fisher Scientific, Montreal, QC, Canada)
according to method 925.09. Ash content was determined according to method 923.03 by
combusting a weighed portion of 5 g in the muffle oven until constant weight and the ash
content was expressed as the ratio between the weight sample after combustion and its dry
weight before combustion. Crude protein was determined following the Dumas method
992.23 with LECO Truspec FP-428 (Leco Corp., St. Joseph, MI, USA) using a conversion
factor of 6.25. Residual oil in the defatted meal was determined by Soxhlet extraction
66
according to method 925.85 using petroleum ether as extraction solvent. Carbohydrates were
determined by difference (Karaca et al., 2011a).
3.6.3. Extraction
Industrially defatted canola meal was weighed and dispersed in distilled water to give
final concentrations of 5%, 10% and 15% (w/w). For the experiments aiming to determine
the influence of the ionic strength on the extraction, canola meal was dissolved in NaCl
solution of the following concentrations: 0.01; 0.1 and 1 M. For the other values, the
extractability has been interpolated assuming the linear behavior. Extractions were carried
out at room temperature, for 1 h, under constant agitation using a magnetic stirrer. The pH of
the solutions was adjusted by the addition of 0.1–2 M NaOH solution dropwise so as to reach
the pH values of 10, 11 and 12 and maintained constant by the addition of aqueous NaOH as
required.
The slurry was subsequently centrifuged at 10 000 g during 30 min at room temperature so
as to separate insoluble material using Sorvall centrifuge RC-5C (Thermo Fisher Scientific,
Waltham, Massachusetts, USA). The supernatant was collected and the residues were freeze-
dried. All the extractions were performed in triplicate and the mean values were determined.
3.6.4. Protein precipitation
For the determination of turbidity and zeta potential the extract was prepared using
the following concentrations: 5% (w/w) of canola meal, dispersed in water with subsequent
pH adjustment with NaOH until a value of 12 is reached. These conditions were chosen as
those giving high percent of extractable nitrogen and total dry matter. Total time of extraction
was 1 hour. The slurry was centrifuged as described before. The supernatant was collected
and the isoelectric precipitation was performed by continuous addition of HCl with different
molarities (1 M-0.01 M) depending on the pH (higher concentrations were needed while
operating with the extremums) so as to reach the pH values from 12 to 2 with decrement of
1 pH unit and subsequently left for 2 h to allow aggregation.
3.6.5. Total dry matter and protein extractability
In order to determine the total extractability the aliquote of the supernatant in each
case was weighed and dried in the oven until constant weight. The extractability was
67
calculated as a ratio of dry matter in supernatant over total dry matter content. In order to
exclude the minerals added during the extraction the ash was determined in each case and
subtracted from the total extractable matter. Nitrogen content of the supernatant was
measured using Dumas method with LECO Truspec FP-428 (Leco Corp.). The aliquote of
the supernatant was weighed and oven dried overnight at 60°C before being analyzed. The
extractability was expressed as a function of nitrogen content in supernatant in relation to
nitrogen content in canola meal on a dry basis.
3.6.6. Turbidity and Zeta Potential (ζ) measurements
Turbidity was measured in nephelometric turbidity units (NTU) using The Hach
Model 2100AN Laboratory Turbidimeter (HACH LANGE GmbH Willstätterstr. 11, D-
40549 Düsseldorf, Germany). Samples adjusted to pH from 12 to 8 were used without
dilution whereas sample adjusted to pH values from 7 to 2 were analyzed using the dilution
factor of 2. All measurements were taken in triplicate and average values were recorded.
The zeta potential was measured as described by Narong and James (2006) using Zetasizer
2000 (Malvern Instruments Ltd., UK). This instrument measures the electrophoretic mobility
and the zeta potential on the particles in the solution using Laser Doppler velocimetry (LDV).
Samples adjusted to values from pH 12 to pH 2 were diluted to a concentration of
approximately 0.01% (w/w) using distilled water prior to the analysis, left for stabilization
for 2 h and subsequently analyzed. The signal recorded by the equipment was converted to
zeta potential according to Henry`s equation:
= ( ) (Eq. 3.1)
Where - Zeta potential, - electrophoretic mobility, - viscosity of the solution, -
dielectric constant, ( ) – Henry`s function that measures the ratio between the radius of
the particle and the thickness of the electric double layer. For the aqueous media and
moderate electrolyte concentration ( ) is 1.5, used in the Smoluchowski approximation.
The experiments were done at room temperature (23°C).
3.6.7. SDS-Gel electrophoresis
To assess the impact of tested conditions on the polypeptide profile of extracted
proteins the Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was
68
performed following the method of Aluko and McIntosh (2001). Based on the results of
proteins yield at various conditions 4 samples were chosen for the test such as canola meal
and canola proteins extracted at pH 10, 11 and 12 with and without the addition of 1 M NaCl.
Specifically, 1% protein solutions were prepared and left overnight, followed by dilution with
deionized water so that the amount of proteins loaded onto the gel was approximately 20 µg.
Subsequently, diluted samples were dissolved in Tris-HCl buffer solution containing SDS
for non-reduced conditions. For reduced conditions samples were dissolved in Tris-HCl
buffer with addition of 5% (v/v) β-mercaptoethanol. Afterwards, the samples were boiled for
5 min, vortexed and 10 µl of each sample were loaded onto 4–20% gradient gels (#456-1093,
Bio-Rad, Canada). Precision plus Protein Kaleidoscope Standards of 10-250 kDa (#161-
0375, BioRad, Canada) were used as molecular weight standards. After staining and
destaining the gels were scanned using Chemi Doc XRS 170-807 (Bio-Rad, Hercules, CA,
United States of America).
3.6.8. Experimental design and statistical analysis
A full factorial experimental design was used to study the effect of different
experimental conditions on total extractability of the dry matter from canola meal obtained
after oil extraction from canola seed harvested in the Becancour region, Quebec, Canada.
The same conditions were studied regarding their effect on differential protein extractability.
The obtained data were used to optimize the experimental conditions. Zeta potential and
turbidity measurements were used to obtain the most favorable conditions for high protein
recovery from the total dry matter extracted from the meal. Each treatment was carried out
in a triplicate and mean values ± SD were calculated and used for the comparisons between
the different treatments at 95% confidence level with a protected LSD test. Detailed analysis
of the variance (ANOVA) was performed by using SAS software (Version 9.1, SAS
Institute, Cary, NC, USA). A decomposition of the ANOVA was also used to validate the
significance of each individual independent variable. Minitab software (Version 16, Minitab
Inc, State College, PA, USA) was used for the optimization of the extraction conditions.
69
Results and discussion
Proximate chemical composition of canola meal is presented in the Table 3.1. The results
are in accordance with those reported in the literature before (Pedroche et al., 2004; Tan et
al., 2011b).
Table 3.1: Proximate chemical composition of canola oil cake on a dry weight basis.
Moisture (%)
Protein (N*6.25,%)
Ash (%)
Fat (%)
Carbohydrate (%, by difference)
Total phosphorus (%)
10.51 ± 0.01 43.36 ± 0.71 7.19 ± 0.01 3.58 ± 0.07 35.36 2.21 ± 0.48
3.7.1. Extractability
In order to determine the influence of different parameters the total and protein
extractability were measured as a function of pH, canola meal concentration (solvent to meal
ratio) and salt concentration (ionic strength). In the next sections the impact of each of these
parameters on the extractability will be discussed.
3.7.1.1. Effect of pH
In the first experiment the effect of pH on the total dry matter and protein
extractability was studied. It has been previously shown that pH has a major influence on the
extractability of canola proteins (Ghodsvali et al., 2005; Klockeman et al., 1997; Nioi et al.,
2012). More alkaline medium allows more protein to be solubilized and this effect is more
important at low CM concentration. Similar tendency can be noticed regarding total dry
matter extractability. However, the gain in total extractability with an increase of the
extraction pH is less important than the one observed for protein extractability as shown in
Figures 3.1 and 3.2.
70
* CM – canola meal
Figure 3.1: Influence of the solution pH on the total dry matter extractability from canola meal.
* CM – canola meal
Figure 3.2: Influence of the solution pH on the protein extractability from canola meal.
0
10
20
30
40
50
60
10 11 12
Ext
ract
abili
ty,%
pH
CM 5%
CM 10%
CM 15%
0
10
20
30
40
50
60
10 11 12
Ext
ract
abili
ty, %
pH
CM 5%
CM 10%
CM 15%
71
There is a significant increase in protein extractability, by as much as 2.5 times, when the pH
is increased from 10 to 12 and by as much as 1.7 times for total extractability. Earlier studies
however reported higher results (Gillberg and Törnell, 1976; Quinn and Jones, 1976; Tzeng
et al., 1988a). In such conditions they managed to extract more than 85% of the proteins. In
our case this range of pH allowed to extract from 22 to 58% of canola proteins and from 26
to 43% of total dry solids which is supported by more recent works (Ghodsvali et al., 2005;
Pedroche et al., 2004).
On the other hand, extraction at pH > 10 has a negative effect on proteins
functionality. Some authors reported poorer functional properties of the proteins obtained at
harsh alkaline conditions such as pH 12 as a result of protein denaturation (Jyothirmayi et
al., 2006; Pedroche et al., 2004). These authors reported that only protein isolates obtained
at pH 10 had functional properties comparable to those of the defatted meal. In this context
the extraction at pH=10 would result in proteins with better functionality but would at the
same time result in the lowest yield.
3.7.1.2. Effect of canola meal concentration
Extractable protein increased from 22.4% to 28.5% and the total dry matter increased
from 26.6% to 29% as the concentration of CM increased from 5% to 15% for pH 10. For
pH 11 and 12 the corresponding increases were 29.5% to 39.8%, 30% to 33.1%, 53.9% to
58.2 and 42.8% to 42%, respectively (Figures 3.1 and 3.2). Most authors used 5%
concentration of CM or 1:18 solvent to meal ratio (Ghodsvali et al., 2005; Klockeman et al.,
1997; Tzeng et al., 1990b; Tzeng et al., 1988a) or 10% (Aluko and McIntosh, 2001; Pedroche
et al., 2004). As it might be noticed CM concentration influences the total extractability
mainly at lower pH values (pH 10) whereas at pH 11 this effect is reduced and at pH 12 there
is even a slight decrease in total extractability. Protein extractability increases with an
increase in CM concentration at all tested pH values. As the reduced use of solvents is more
desirable the 15% concentration seems more attractive. On the other hand extraction at such
a high concentration is complicated by its high viscosity. So a concentration of 10% would
be optimal.
3.7.1.3. Effect of salt concentration
It is generally recognized that salt in low concentrations enhances the protein
solubility due to “salting-in” effect which is explained by the interactions between ions in the
72
solution and the protein charges. These interactions weaken the protein-protein interactions
and increase the protein solubility. At higher salt concentration, on the contrary, the salting-
out effect takes place resulting in lower solubility due to the binding of the water molecules
by the ions of salt.
There is limited information about the effect of salt on the extractability of canola
proteins. Most of the studies analyzed the influence of ionic strength on their precipitability.
Thus Tzeng et al. (1990b) reported high protein yield due to “salting-in” effect that allowed
them to obtain 80% yield. In their study the 0.15 M CaCl2 was added during the precipitation
step in order to obtain the soluble protein. Ghodsvali et al. (2005) used the addition of 15%
CaCl2 also during the precipitation step. In the study of Aluko and McIntosh (2001) 0.02 M
and 0.15 M CaCl2 was added after the pH adjustment to 6. A lower protein yield in calcium
precipitated proteins in comparison with acid precipitated proteins has been reported. Thus,
the 6-16% reduction of protein yield was observed with salt-coagulated proteins compared
to those of acid-coagulated proteins (Soetrisno and Holmes, 1992). The authors explained
that fact due to higher pH used for salt-coagulated proteins (pH 6 compared to pH 4 for acid-
coagulated proteins).
Regarding the extractability of the proteins from other than canola sources, it was
reported that the addition of salt (0.1-0.5 M) broadened the pH range of the minimum
solubility and shifted it to the lower values (Eromosele et al., 2008; Jyothirmayi et al., 2006).
The salt addition has significantly increased the protein extractability at neutral pH region
and even at the range of minimum solubility close to the isoelectric point as shown in the
work of Oomah et al. (1994) for flaxseed meal. In the study of Lazos (1992) the protein
extractability from pumpkin seeds was significantly increased (from 4.5-8.7% to 12-55% )
at their minimum solubility with an increase in sodium chloride concentration. However, the
decrease in protein solubility was noticed in the range of pH 8-11. The work of Jyothirmayi
et al. (2006) showed higher nitrogen extractability at isoelectric point when sodium chloride
was added (60% compared to 26.8%). On the other hand the authors note that at pH 12 the
amount of nitrogen extracted was insensitive to the ionic strength. This might be explained
by the denaturation of globulin proteins at harsh alkaline medium, by various conformations
of protein molecules at different pH values, and by their interactions with other compounds
present in the meal (phytates, sugars, fibres). However, an increase in solubility usually is
73
associated with a decrease in the recovery yield of the precipitate (Gillberg and Törnell,
1976). The authors reported that only 25% of the dissolved proteins were recovered. As it
can be seen in Figures 3.3 to 3.5, our results revealed that the addition of salt influenced in
a different manner the total and protein extractability. In order to exclude the influence of
pH, the data will be compared within constant pH values at pH 10, 11 and 12, respectively.
3.7.1.3.1. Extraction at pH 10
Figure 3.3: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 10.
a
b
74
Figure 3.3 shows the extrapolated results of the influence of NaCl concentration at
pH 10. Globally, the addition of salt had a positive effect on the extractability at all CM
concentrations. For protein extractability 5% CM concentration and 1 M NaCl will be the
most favorable conditions. In comparison with salt-free extraction the 0.01 M concentration
of NaCl didn`t influence the extractability at CM concentration of 5% (22.38 to 22.39% for
protein extractability and 26.63 to 26.90% for total extractability) and even a minor decrease
was observed in our case for 10% and 15% CM concentration (25.62 to 22.12; 28.03 to 27.36
and 28.53 to 25.74; 29.08 to 27.94 for protein and total extractability and CM concentration
of 10% and 15% respectively.), (data not shown). The 0.1 M concentration had some positive
effect (32.51 ± 0.31 % of total dry matter and 24.89 ± 0.91 % of proteins with 5% CM
concentration) whereas with 1 M NaCl at pH 10 and the concentration of CM 5% there was
a double increase in protein extractability and almost double increase in total extractability
22.39 ± 2.52 % to 40.87 ± 3.48 % and 26.63 ± 0.77 % to 39.88 ± 0.48 % respectively).
Therefore, the maximum yield was observed at the maximum concentration of salt e.g. 1 M
and at the concentration of canola meal of 5%. The optimal conditions at pH 10 will be 0.5
– 1 M NaCl and 5 – 6% CM concentration.
3.7.1.3.2. Extraction at pH 11
The extrapolated results for pH 11 are shown in Figure 3.4. As it can be seen the
conditions the most favorable for total extractability are not optimal for protein solubility.
Thus, the optimal conditions for total extractability are similar to those ones obtained at pH
10 e.g. 0.7 – 1 M NaCl concentration and 5 – 7% CM concentration where the increase in
protein extractability is not significant. For proteins the optimum is in the range of 8 – 15%
of CM concentration and NaCl 0.3 – 0.9 M. However, in comparison with the extraction at
pH 10 the increase observed with the addition of salt was lower and a 10% increase was
revealed in this case with the maximum yield for total dry matter at 5% CM concentration
and 1 M NaCl (from 29.98 ± 1.22% to 39.37 ± 3.13 %). Concerning the extractability of
proteins a 12% increase was observed in the whole range of CM concentration whereas
within one concentration the increase was only 4%. Maximal values were obtained at 0.1 M
NaCl and 15% CM concentration (40.57 ± 0.35 %), although with no salt added and under
75
the same conditions the results were (39.79 ± 3.54%) showing no statistically significant
difference (data not shown).
Figure 3.4: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 11.
a
b
76
3.7.1.3.3. Extraction at pH 12
The effect of salt addition at pH 12 is shown in Figure 3.5. The pattern observed for
both extractabilities is also different. For the total extractability the tendency was similar to
those observed at pH 10 and 11 with the optimum in the range of maximal salt concentration
(0.6 – 1 M) and minimal CM concentration (5%). As for the protein extractability the
decrease is observed with an increase in salt concentration being minimal within the range of
0.4 – 1 M NaCl concentration and 5 – 10% CM concentration. In this case the maximal yield
is obtained with no salt added and at 15% CM concentration (58.12 ± 1.18%) (data not
shown). This value decreases to 42.99 ± 0.24% with an increase in salt concentration for 15%
CM concentration and from 53.91 ± 5.18% to 34.91 ± 0.93% and 54% to 39% for 5% and
10% CM concentration respectively (data not shown). In spite of the decrease the percentage
of extracted matter in general exceeds the levels extracted at lower pH values. However this
effect is due to the influence of pH rather than the influence of salt addition.
Our results demonstrated that the salt addition had a positive impact on extractability
only at pH 10 whereas at pH 11 this effect was minor and at pH 12 a negative effect was
observed. This is consistent with the data in the literature (Oomah et al., 1994). However, in
the work of Klockeman et al. (1997) an increase in protein extractability with an increase in
salt concentration at pH 11 has been noted. The NaCl concentration of 1 M does have an
effect on the extractability at lower pH values whereas lower concentrations such as 0.1 and
0.01 M have less pronounced influence. In spite of the positive effect of NaCl addition the
maximum protein extractability was observed in salt-free extraction at pH 12. Therefore, salt
extraction would be useful when a highly alkaline medium is not desired as in case when
functional properties need to be preserved.
77
Figure 3.5: The effect of salt concentration on total dry matter (a) and protein extractability (b) at pH 12.
3.7.2. Protein and ash composition of extracts
Protein content of extracts obtained at pH 10 without NaCl ranged between 27-32% with ash
being ~12% on a dry basis. When 1 M NaCl was used the percentage of protein was lower
(8-16% on a dry basis) due to the high content of salt added during extraction (60-70% of
a
b
78
ash) (Figure 3.6). At pH 11 and 12 the protein content was 32-37% and 39-45% respectively,
whereas ash composed 13-15% and 14-19% of the total dry solids. For 1M NaCl
concentration extract obtained at pH 11 had 8-19% protein and 55-75% ash and extract
obtained at pH 12 had 8-21% of protein and 56-66% of ash on a dry basis. The utilization of
1 M NaCl for protein extraction requires further desalting in order to purify the extracted
fractions.
Figure 3.6: Protein and ash content of extracts obtained with 10% canola meal concentration.
3.7.3. Precipitability based on Zeta Potential and Turbidity
The extract obtained during the first step represents a colloid system. This means that
solubilized particles are dispersed in the solution. Various repulsive and attractive forces are
involved and interactions between particles influence the stability of the system. The
character of these interactions dictated by the surface charge of the particles predetermines
the behavior of the system. The stability of such a system depends first of all on the pH of
the medium. It is a well-known fact that proteins are able to change their surface charge
depending on the pH of the medium. As the proteins have acid and basic groups their charge
depends on the pH and ionic strength of the medium (Benítez et al., 2007).
In order to obtain a desired protein yield the understanding of the precipitation
behavior and electro-kinetic characteristics is needed. One of the readily measurable electro-
kinetic characteristics is zeta potential which is an expression of the charge found on the
0
10
20
30
40
50
60
70
80
90
pH
10
pH
11
pH
12
pH
10
pH
11
pH
12
pH
10
pH
11
pH
12
pH
10
pH
11
pH
12
no NaCl 0.01 M NaCl 0.1 M NaCl 1 M NaCl
Pro
tein
an
d a
sh c
on
ten
t o
n a
dry
ba
sis,
%
Ash
Proteine
79
surface of the suspended particle. When the net charge of the particles is the same, the
repulsive forces are predominant which prevents their aggregation so that they remain in
suspension. On the other hand, when the net charge becomes neutral, the repulsive forces
become less important which results in the aggregation and precipitation of suspended
particles. It is known as isoelectric point precipitation.
Our study was not aimed to determine the effect of ionic strength on the zeta potential
so the measurements were taken under the same extraction conditions (5% CM concentration
and pH 12). The effect of pH on the zeta potential is shown in Figure 3.7. For canola meal
as it might be seen, the proteins carry net negative charge in the alkaline medium with
maximum value of net charge at pH 10. Further decrease in pH values causes a decrease in
net charge leading to the isoelectric point. However, at more alkaline medium there is an
increase in zeta potential which is probably due to the nature of proteins. As a matter of fact
one of canola`s main proteins is napin which is characterized by strong alkalinity due to a
high level of amidation of its amino acids (Aider and Barbana, 2011).
Figure 3.7: The influence of the solution pH on the Zeta potential of the canola protein.
The minimum net charge is observed at pH values around 4.3 which indicates that the
isoelectric point is in the range of pH 4-5. This is in agreement with previous works where
the highest precipitability has been reported at pH values between 4.5 and 5.5 (Ghodsvali et
al., 2005; Tan et al., 2011b). On the other hand, Quinn and Jones (1976) reported minimum
solubility for canola proteins at pH values 3.7-4.0 and 7.7-8.0 and Pedroche et al. (2004)
-70
-60
-50
-40
-30
-20
-10
0
10
20
30
2 3 4 5 6 7 8 9 10 11 12
Ze
ta p
ote
nti
al,
mV
pH
80
indicated two isoelectric points at pH 3.5 and 5. This might be due to different types of meal
used for the experiments with different profiles composition.
Another way of evaluating the maximum precipitability is by measuring the turbidity
of the suspension. Turbidity shows the amount of colloidal material present in the suspension.
The increase in turbidity is a reflection of an increase in flocculation rate. Therefore, the
maximum turbidity is supposed to be in the range of the minimal solubility of the proteins.
The turbidity has been measured over a range of pH values from 12 to 2. As it can be seen in
Figure 3.8 the maximum turbidity is in the range of pH 4-6 with its peak at pH 5. Initially
with no pH adjustment at pH 12 the turbidity of the slurry is close to zero. In the beginning
there are almost no changes in turbidity over the range of pH 12 to pH 9. The alkaline medium
corresponds to maximum solubility of the proteins; the repulsive forces are strong enough
which prevents the aggregation of the particles. When the pH becomes lower than 9 a rapid
increase in turbidity is observed which continues until reaching a pH of 5. Further decrease
in pH results in decrease of turbidity.
Figure 3.8: The influence of the solution pH on the turbidity of canola proteins solution.
0
2000
4000
6000
8000
10000
12000
2 3 4 5 6 7 8 9 10 11 12
Tu
rbid
ity
, N
TU
pH
81
The correlation coefficient between the zeta potential and the turbidity is -0.8 with
p=0.003 indicating strong linear relationship which can be noticed from Figure 3.7 and
Figure 3.8. The maximum turbidity is found at pH values between 4 and 5, similarly the
minimum net charge is also found at pH values between 4 and 6. This means that close to the
isoelectric point there is no energy barrier preventing flocculation so the turbidity of
suspension at this point is the highest. Slightly higher values of the turbidity might correspond
to complex protein composition of canola proteins. The range of pH 4 – 6 is the zone where
the proteins are the least stable. Even minor fluctuations in pH result in changes on the surface
of the particle.
3.7.4. Gel Electrophoresis
The results of SDS PAGE are presented in Figure 3.9A and 3.9B. Reducing and non-
reducing conditions were followed by 2 tests with and without NaCl addition. The aim was
to verify whether or not the salt addition influences the protein profiles extracted under pH
10, 11 and 12 and to which extent. As it can be seen, pH 10 and 11 give more prominent
protein profiles in comparison with pH 12 which is due to the protein denaturation in highly
alkaline medium leading to poor protein solubility.
It is known that canola`s main protein fractions are napin and cruciferin which differ
by their molecular weights, sedimentation coefficients, and functionalities. Napin is a 2S
albumin, whereas cruciferin is a 12S globulin. They also differ by their isoelectric points
which complicate their production (Manamperi et al., 2012).
The influence of salt addition can be regarded at pH 10 and 11 showing more
pronounced bands in Figure 3.9A for the molecular weights of 45-50 kDa and 27-30 kDa.
The former band disappeared under reducing conditions indicating the presence of disulfide
bonds whereas the latter became more intense due to formation of new polypeptides implying
other than disulfide linking. Fraction of 18 kDa becomes more distinct under reducing
conditions unlike the study of Aluko and McIntosh (2001) in which this fraction disappeared.
These bands can be assigned to cruciferin fraction which according to the literature is
presented by following molecular weight profiles: 50 kDa, 29.5 kDa and a minor band of 44
kDa (Wu and Muir, 2008).
82
Figure 3.9: SDS-PAGE of canola proteins A) without NaCl addition and B) with an addition of 1 M NaCl.
83
Napin is a low molecular weight basic protein that consists of two polypeptide chains
linked together by disulfide bonds. It usually constitutes from 15 to 45% of total canola
proteins with the molecular weight ranging from 12.5-14.5 kDa. Napin’s fraction of 15 kDa
and a minor band of 27.5 kDa (a dimer of napin) being water soluble is clearer in the Figure
3.9A. Under reducing conditions the bands fade due to the cleavage of disulfide links which
are main stabilizing bonds for napin. New bands of 9 and 13 kDa appeared under reducing
conditions due to the formation of new polypeptides. They are more intense in the first case
where no salt was added, suggesting that these bands are the part of water soluble proteins
linked by disulfide bonds. This is in accordance to the works of Nioi et al. (2012) and Wu
and Muir (2008) where napin and its dimer appeared with their molecular weights of 14 kDa
and 27.5 kDa from purified fraction and dissociated into small polypeptide chains under
reducing conditions highlighting the presence of disulfide links.
On the whole, salt addition contributed to higher solubility of canola proteins
specifically cruciferin fraction although it reduced napin extraction. Therefore, the extraction
conditions should be chosen with regards to the aims of the work as it will have a direct
impact on the functional properties of obtained product.
Conclusion
The impact of extraction conditions on total and protein extractability was studied.
Among all the parameters tested, the changes in pH had the strongest influence on both
extractabilities. Maximum extractability was obtained with pH 12. Extraction with NaCl
significantly increased the extractability only at pH 10. Taking into account the information
that functional properties might be affected in case of highly alkaline medium, the
combination of low pH (i.e. pH 10) with addition of NaCl would be a better alternative to
obtain a high protein yield. However, this could also imply to remove the added salt prior to
use of the ingredient for food formulation. The study of zeta potential and turbidity revealed
a strong correlation between both parameters and might be further used in the determination
of isoelectric points when different extraction procedures are tested since both methods are
fast in comparison with titration and protein dosage on the supernatant or precipitate. The
SDS-PAGE analysis showed that salt addition contributes to higher solubility of canola
proteins specifically cruciferin fraction although it reduces napin extraction. Therefore, the
84
extraction conditions should be chosen with regards to the aims of the work as it will have a
direct impact on the functional properties of obtained product.
Acknowledgments
This work was financially supported by the innovation in food support program that
was funded by contracts through the "Growing Forward" Program that occurred between the
Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of
Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".
85
4. CHAPTER 4: Monitoring of pH and alkalinity changes in the electro-
activated aqueous solutions generated in the cationic compartment.
The effect of salt concentration, current intensity, time and cell
configuration
Contextual transition
The previous section showed that pH and salt concentration have the major impact on protein
extractability from industrially defatted canola meal. In the following chapter such conditions
were modulated by electro-activated solutions generated in the cathodic compartment of a 2-
and a 3-cell reactor. The study of factors such as type of configuration, time of treatment,
current intensity, and salt concentration on the pH and alkalinity of solutions is presented
hereafter.
This chapter is submitted as an article entitled: “Monitoring of pH and alkalinity changes in
the electro-activated aqueous solutions generated in the cationic compartment. The effect of
salt concentration, current intensity, time and cell configuration”.
The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:
scientific supervision, article correction and revision), Marzouk Benali (Scientific
collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder
(Thesis director: scientific supervision, article correction and revision)
86
Résumé
Les propriétés des solutions aqueuses électro-activées à l’interface cathode/solution
(catholyte), notamment le pH et l'alcalinité, ont été étudiées. Un plan factoriel complet a été
appliqué pour examiner les effets de l'intensité du courant, de la durée du traitement et de la
concentration du sel sur le pH et l'alcalinité de solutions électro-activées traitées selon 4 types
de configuration du réacteur d’électro-activation. Les configurations de réacteur à deux et
trois compartiments, séparés par une membrane échangeuse d'anions (AEM) ou une
membrane échangeuse de cations (CEM) ou par les deux, ont été testés. L'intensité du courant
électrique et le temps du traitement avaient les effets les plus significatives pour toutes les
configurations testées. D'autre part, la concentration du sel a un effet significatif seulement
lorsque les configurations 2 et 4 ont été appliquées en raison de la disposition des membranes
dans le réacteur. Les valeurs maximales de pH et d'alcalinité ont été obtenues avec I = 0.2 A,
t = 60 min à toutes les concentrations de NaCl pour les configurations 1 et 3. Les mêmes
valeurs pour les configurations 2 et 4 ont été obtenues uniquement avec la concentration
maximale de NaCl de 1 M. Malgré le pH fortement alcalin (pH ~ 12), l’alcalinité de la
solution était comparable à celle du NaOH dilué (~ 0.03 M).
87
Abstract
The properties of catholyte (pH and alkalinity) were investigated in the current study. A full-
factorial design was applied in order to examine the effects of current intensity, time of
treatment and salt concentration on the pH and alkalinity of electro-activated solutions treated
in 4 types of configurations. Two and three-cell reactor configurations were tested with
compartments separated by either anion exchange membrane (AEM) or cation exchange
membrane (CEM) or by both of them. Current intensity and time were found to be highly
significant for all tested configurations. On the other hand, the salt concentration had a
significant effect only when configurations 2 and 4 were applied due to the membrane
disposition in the reactor. Maximal values of pH and alkalinity (25.46 ± 0.31mmol/l) were
obtained with I = 0.2A, t = 60 min with all NaCl concentrations for the configurations 1 and
3. Whereas the same values for the configurations 2 and 4 were achieved only with maximal
NaCl concentration of 1M. Despite highly alkaline medium (pH~12) the strength of the
solution was weaker than chemical alkali solutions and was comparable to dilute NaOH
(~0.03M).
88
Introduction
Not long ago water was regarded as a biochemically passive substance. Its major role
was to act as a solvent where the transformations of active substances took place. Only the
interactions of organic and inorganic substances with water were studied while water
structure and changes of its properties were out of interest. However, about 50 years ago a
theory of a structured water was developed and the possibility of changing its properties
without any chemicals was recognized (Prilutskii and Bakhir, 1999). During the analysis, the
water subjected to magnetism, sound, illumination, electric field, heating or freezing showed
new properties. These properties had an impact on the kinetics of chemical reactions taking
place in the water, on its biological, solubilizing, and cleaning activity. Such water treated by
one of the listed methods was called “activated”, which comprises the sum of effects or new
properties appeared during technological treatments and without elemental chemical changes
(Aider et al., 2012b; Prilutskii and Bakhir, 1999).
Thus, the water treated under the influence of external electric field was called the
“electro-activated” (EA) water. The presence of an electric field supplies an external energy
making possible non-conventional chemical reactions due to the high reactivity of the
solutions (Aider et al., 2012b). Electro activated water found numerous utilization in
environmental and food industry (Huang et al., 2008) and currently is widely used mostly as
a disinfectant. Its bactericidal effect on many pathogenic bacteria was reported in the
literature (Kiura et al., 2002; Rahman et al., 2010). In addition, improved extractive and
catalytic function were also stated (Aider et al., 2012b; Aït Aissa and Aïder, 2013a).
Electroactivation as a technology of producing activated water with specific
properties is based on a well-known process of electrolysis with its main principles studied
in the early 19th century (Aider et al., 2012b). The simplest scheme comprises two electrodes
anode and cathode immersed in the aqueus solutions and a current (power) supply. A
potential difference between two electrodes enables the reaction to occur (Bazinet, 2005).
The changes in water activity are closely related to the oxido-reduction electrode reactions.
The main reactions which take place in the electrolyzing cell are water oxidation on the anode
with O2 liberation (Eq. 4.1), water reduction on the cathode with H2 release (Eq. 4.2),
formation of the gaseous chlorine on the anode (Eq. 4.3), formation of very active oxidants
in the anodic compartment such as Cl2 O, ClO2, ClO−, HClO, Cl•, O2•, O3, HO2, OH• and
89
formation of overactive reducing agents such as ОН−, Н3−О2
−, Н2, НО2•, НО2
−, О2− in the
cathodic compartment (Tomilov, 2002).
2Н2О − 4е→ 4Н+ + О2 ↑ (Eq. 4.1)
2Н2О + 2е→ Н2↑ + 2ОН− (Eq. 4.2)
2Cl− − 2е→ Сl2↑ (Eq. 4.3)
Thus, under the application of external electric field the two types of solutions are
generated. An electrolyzed basic solution is produced on the cathode side possessing strong
reducing potential and electrolyzed acid solution posessing strong oxidating potential is
generated on the anode side (Hsu, 2005).
The main drawback of such a scheme is the fact that the products of reactions mix. In
order to separate them different types of membranes are used such as porous non-permeable
diaphragme or permselective anion or cation exchange membranes or both (Mani, 1991). The
membranes properties and their selectivity are well-explained in the literarure and can be
found elsewhere (Baker, 2012).
External electric field creates unique prerequisites for the reactions which under usual
conditions cannot be performed. As an example the simple reaction of NaCl hydrolysis under
normal condition will flow in the opposite direction but the application of external energy
allowed chlorine and NaOH production, which is currently used in the industry. A two-cell
reactor and a CEM are used for such a reaction. As a result chlorine ions are discharged with
gas release on the anode and Na+ ions migrate to the adjacent compartment where H+ ions
form gaseous H2 while Na+ ions together with OH- form alkali (Shaposhnik, 1999).
The differences between electroactivation and electrodialysis are not evident at first
glance. However, when water splitting by electricity is a physico-chemical transformation of
the medium composition leading to the appearance of H+ and OH- ions, acids, hydroxides,
peroxides, radicals and hypochlorites, the electroactivation assumes the acquisition of new
properties which cannot be explained by usual chemical reactions. Thus, the anolite and
catholite gain such properties namely pH and oxido-reduction potential which cannot be
modulated in usual chemical solutions (Leonov et al., 1999; Tomilov, 2002). In general,
while the electric field is applied the composition of the water near anode and cathode
changes and it is accompanied with appropriate reactions. The maximum intensity is reached
in the near electrode layer called double diffusive layer where actually the process of
90
activation takes place. As a result after excitation by an external electric field the water falls
into a metastable state characterized by abnormal physico-chemical characteristics due to the
electrons activity in the water (Aider et al., 2012b; Tomilov, 2002).
Properties of electro-activated solution (EAS) have been extensively studied
(Belovolova et al., 2006; Petrushanko and Lobyshev, 2001). The vibrational spectra of EAS
were studied by means of Raman Spectroscopy (Pastukhov and Morozov, 2000). Their
results showed a significant difference in Raman spectra between 700 and 2700 cm −1 of the
electro-activated water taken in the near-anode (anolyte) and near-cathode (catholyte)
regions and chemically acidified and alkalinized water. The results of Raman spectroscopy
showed the presence of H+ or OH− ions in the anolyte and catholyte respectively which are
known to be responsible for acidic and alkaline properties. Furthermore, concentrated acid
and alkaline solutions showed the bands similar to the Raman Spectra bands of anolyte and
catolyte. As a result it was assumed that properties of electroactivated solutions may be
similar to those of concentrated acids and alkali (Aider et al., 2012b). Leonov et al. (1999)
also stated that electrochemically activated solutions have properties similar to those of
conventional acids and alkalis. In order to verify the feasibility of such a suggestion the
properties of catholyte generated under chosen conditions were analyzed in the current study.
Knowing the main parameters that influence the process of electroactivation and
using the specific configuration the desired degree of EA can be achieved. However, the
studies on the correlation between constructional characteristics of the electrolyzers and
technological parameters of the process on one side, and functional properties of
electroactivated solutions on another side, are scarcely reported in scientific literature. The
empirical adjustment of parameters is used in most sources (Aider et al., 2012b).
The aim of this study was to investigate the parameters that influence the pH and the
strength of the generated solution namely alkalinity in the cathodic compartment by studying
the effect of cell configuration, salt concentration, current intensity and the time of treatment
and to compare the obtained solutions with conventional base such as NaOH.
91
Materials and methods
4.5.1. Chemicals
Sodium chloride (NaCl) was purchased from Caledon Laboratories LTD
(Georgetown, ON, Canada). Sodium hydroxide (NaOH) was purchased from VWR
International LLC, West Chester, PA, USA. Concentrated hydrochloric acid (HCl) and
sodium sulphate (Na2SO4) was purchased from Scientific Fisher, Canada, CAS.
4.5.2. Ion-exchange membranes
Anion (AM-40) and cation (CM-40) exchange membranes were purchased from the
Publicly Traded Company Schekina-Azot (Shchekina, Russian Federation). Prior to
utilisation both membranes were washed with 96%-ethanol to remove the wax and prepared
according to manufacturer’s instructions before their use in the electro-activation reactor.
4.5.3. Reactor design. Configurations of electro-activation cells
Electro-activation reactor was made of transparent plexiglass columns with the
dimensions of L50xW50xH120 mm. Three- and two-compartment electro-activation
reactors were used resulting in 4 different configurations. For the first configuration
consisting of three chambers and shown in the Figure 4.1 AEM separated the anode and
central parts, whereas CEM was placed on the cathodic side, separating it from central
section. Second configuration had the same three compartments but this time the membranes
were swapped places (Figure 4.4). Third and fourth configurations had two compartments
separated by CEM and AEM, respectively (Figure 4.7).
The RuO2–IrO2–TiO2 electrodes 120x34x1 mm with working active area of 40 cm2 were
placed at the extremities of the cells and were connected to the positive side of a direct electric
current power supply for the anodic compartment and to the negative side for the cathodic
one. The values of the applied voltage and the intensity of the electric current were measured
using a current power supply (CSI12001X, Circuit Specialists, Inc, USA).
92
4.5.4. Protocol of electro-activation
This study primarily focused on analyzing the catholyte and its properties. A full
factorial design was used to test the effect of cell configuration, salt concentration, current
intensity and time on two parameters, namely, pH and alkalinity of the solutions. The EA
was carried out at three constant electric current intensities of 0.05, 0.1 and 0.2 A providing
the galvanostatic state with total time of treatment of 10, 30 and 60 min.
To ensure the current flow salt solutions were circulated in all compartments. The
cathodic compartment was filled with NaCl solution of following concentrations 0.01, 0.1
and 1 M. Anodic and central (where applicable) compartments were always filled with
Na2SO4 solution of constant concentration corresponding to 0.25 M to avoid the liberation
of toxic chlorine (Cl2) on the anode.
4.5.5. Analysis methods
During each treatment, samples of 5 ml were taken every 2 min within the first 10
min and then every 10 min. The pH of the samples was measured using a pH meter (Model
SR 601 C SympHony, VWR Scientific Products, USA) after which the samples were
returned back to the cell to minimize the impact of sampling.
To determine the alkalinity simple titration method was used (Clesceri, 2005) with some
modifications. The electroactivated solution was titrated with 0.1M HCl until neutral pH was
reached. The volume of acid used to neutralize the alkali was noted and used for the
calculation of alkalinity:
= ( )∗ ∗ , / , (Eq. 4.4)
Where is alkalinity, ( ) is molar concentration of acid used for titration, is volume
of tested solution taken for analysis, is volume of HCl used to neutralize the alkali.
4.5.6. Statistical analysis
The analysis of the variance (ANOVA) at 95% confidence level was performed using
MiniTab software to test the significance (p ≤ 0.05) of each independent input variable on
pH and alkalinity of the catholyte.
93
Results and discussion
4.6.1. Configuration 1
Configuration 1 is shown in the Figure 4.1 together with main reactions which take
place during the electro-activation. This configuration is characterised by two main
processes: desalination in the central compartment and concentration in the compartments
found at the extremeties. The cathodic compartment is separated from the central one by
CEM. This configuration allows the transfer of cations from the central compartment and at
the same time prevents anions from leaving the cathodic compartment. Water electrolysis
takes place on the cathode with H2 release so over some period of time the OH- ions are
accumulated leading to a pH increase.
Figure 4.1: Configuration 1 of the electro-activation reactor used for the generation of the electrolysed water.
4.6.1.1. pH evolution
It is known that pH is a measure of the hydrogen ions expressed as a negative logarithm of
H+ concentration. In other words, it is a measure of the acidity or basicity of an aqueous
solution. Initial pH values varied from 5.6-5.9 depending on the distilled water used in the
laboratory. When the direct current was applied, the pH of the solution increased drastically
within the first few minutes and after 10 minutes of electroactivation it reached a plateau.
94
Figure 4.2: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 1.
a b c
d e f
95
The effect of salt concentration and current intensity on the pH of the cathodic solution, over
time, is shown in Figure 4.2 All figures have the same tendency. One can notice from
Figures 4.2a - 4.2c that for all three concentrations the higher the current intensity, the higher
the pH obtained after 60 minutes of operation. In addition, an alkaline pH (e.g. pH=10) for
I=0.2 A is reached within one minute of EA, within 2.5 min for I=0.1 A and within 5 min for
I=0.05 A. This is to say that with higher current intensity a pH increase was performed faster
in comparison with lower current intensities. As it can be seen from Figures 4.2d-4.2f there
is no significant difference between the curves indicating that the salt concentration does not
affect the pH.
4.6.1.2. Alkalinity
Alkalinity is a measure of the capacity to neutralize acids, also called acid
neutralization capacity (ANC). The major substances that will affect the water alkalinity are
hydroxide, carbonate, and bicarbonate. Alkalinity is often confused with the pH, assuming
solutions which are alkaline have pH higher than 7. However, to have high alkalinity a
solution should not always have high pH values. A solution containing carbonate alkalinity
will have pH around 8.3. In the current study, only hydroxide alkalinity is tested since the
other aforementioned components are not present in the solutions. Since the hydroxide ion
OH- is a strong base, high alkalinity will result in high pH (>10). The total alkalinity of a
solution is its ability to bind protons which also means an ability to resist changes in pH by
neutralizing acids. In other words the alkalinity measurements can be used for evaluating the
buffering capacity of the solutions (Warfvinge, 2011). Hence, it provides with useful
information concerning the strength of the EAS making possible their comparison with
conventional bases.
When analyzing the alkalinity of obtained solutions significant changes were found.
The Figure 4.3 shows the effect of tested parameters on the alkalinity of the catholyte. The
graph is depicted in coded values which are explained in Table 4.1. At the beginning the
titrated solution is low-sensitive to the changes in the concentrations of H+ ions (i.e. to the
addition of acid) and its pH is changing slowly. But close to the equivalent point even
marginal amounts of acid result in the drastic and quick changes in pH. Only the time and
the current intensity had a significant effect on the alkalinity (p<0.05). The effect of time and
concentration is supported by the first Faradey’s law which states that the mass of the
96
substance liberated on the electrode is directly proportional to the amount of electricity
flowing through the electrolyte and to the time (Damaskin et al., 2008). Regardless of the
NaCl concentration maximum alkalinity (26.84 ±1.24 mmol/l) was attained at maximum
current intensity (I=0.2 A) and within maximum time (t=60 min). Other values obtained after
60 min of treatment were 13.28 ± 0.48 and 6.65 ± 0.24 for current intensities of 0.1A and
0.05A (Figure 3c). The NaCl concentration did not have a significant influence on the
alkalinity of the solution (Figures 3a, 3b).
Figure 4.3 : The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M).
97
Table 4.1: Variables and their coded values.
Variable Coded values -1 0 1
NaCl concentration, M 0.01 0.1 1 Current intencity, A 0.05 0.1 0.2 Time, min 10 30 60
With an increase in current intensity the water electrolysis on the cathode becomes
more intensive as well as the formation of other active reducers, therefore more H+ and OH-
ions are generated. The former in turn are reduced on the electrode surface in the form of gas
and the latter are accumulated leading to a pH increase. Similarly more OH – ions are
accumulated with the time flow. Since the CEM separates two compartments hydroxyl ions
which tend to reach anode cannot escape the compartment being rejected by the membrane
according to Donnan exclusion and thus enhancing the alkaline strength of the solution. The
Na+ ions easily permeate through the membrane from the central compartment and together
with hydroxyls form strong base NaOH.
4.6.2. Configuration 2
Figure 4.4: Configuration 2 of the electro-activation reactor used for the generation of the electrolysed water.
Second configuration is shown in the Figure 4.4. It is similar to the previous one but
the membranes are swapped places. This modification leads to changes in the behavior of the
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charged species. Contrary to the configuration 1, the middle compartment is a zone of
concentration whereas the two extremities compartments are zones of depletion. Anions now
can permeate through AEM while cations from the middle compartment are retained.
4.6.2.1. pH evolution
The evolution of the pH in the cathodic compartment for the configuration 2 is shown
in Figure 4.5. Similar to the previous configuration the pH increases as a function of time.
However, regarding the current intensity not all the curves follow the same tendency. The
difference is noticed at the lowest salt concentration. Thus, in low NaCl concentration the
current intensity has an impact only for 10 and 30 min of treatment while after it the curves
become almost identical (superimposed). For higher concentrations the curves are well-
separated and higher current intensities result in higher pH values as it was observed for the
configuration 1. Similarly, at constant low current intensity there is no difference between
solutions pH for different concentrations, however one can notice that for I=0.1A and 0.2A,
pH values are slightly lower at low NaCl concentration (0.01 M).
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Figure 4.5: The effect of current intensity and salt concentration over time on the pH monitoring of electro-activated solutions, configuration 2.
a b c
d e f
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4.6.2.2. Alkalinity
The alkalinity evolution is shown in Figure 4.6. Difference in configuration revealed
new parameter affecting the alkalinity of the solutions. While time and current intensity have
the same effect as in previous configuration (Figure 4.6c) a marked deviation is observed
concerning the effect of NaCl concentration (Figures 4.6a, 4.6b). The alkalinity changes
drastically within the range of NaCl concentration considered in this study. Thus, with
minimal quantity of salt present (0.01M) for 0.05A the alkalinity is 5.12 ± 0.25, increasing
to 6.90 ± 0.37 for 0.1M NaCl and 7.45 ± 0.78 for 1M NaCl. With 0.1A current intensity these
values are 8.47±0.04, 13.14 ± 0.06 and 14.25 ± 0.35. And finally for 0.2A the values of 11.55
± 0.07, 24.15 ± 0.49 and 28.15 ± 0.64 were obtained respectively for 0.01, 0.1 and 1M
concentrations of NaCl (Figure 4.6a). The increase is more important within the range of
salt concentrations of 0.01M and 0.1M, while between 0.1M and 1M it is less pronounced.
The effect of the type of configuration is more important with minimal salt
concentration and maximal current intensity in this compartment. Due to different
membranes disposition, the cathodic compartment becomes a zone of depletion. When
0.01M concentration is used there is a lack of current carriers which can be noticed by higher
voltage (data not shown). In addition, chlorine and hydroxyl ions are leaving the cathodic
compartment under the effect of external electric field. In order to compensate the lack of
ions in the compartment water dissociation on the membrane takes place so as to provide the
solution with new ions (Tanaka, 2010). Therefore newly generated H+ and OH- ions become
current carriers. In this case, the higher the water dissociation the lesser the alkalinity is.
Water dissociation leads to H+ and OH- ions generation, the latter by turn leave the
compartment and the former neutralize OH- ions formed during the water electrolysis. With
the time more ions are accumulated in the compartment and the voltage starts slowly to
decrease, less water is dissociated and the alkalinity increases.
When higher NaCl concentrations are used in the cathodic compartment the higher alkalinity
is observed as there are enough current carriers. Therefore, the concentration is as important
as time and current intensity for this configuration.
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Figure 4.6: The effects of salt concentration, current intensity and time on the alkalinity: a) time hold constant at maximum value (60 min); b) current intensity hold constant at maximum value (0.2 A); c) concentration hold constant at maximum value (1 M).
4.6.3. Configurations 3 and 4
Third configuration is a two-compartment reactor composed of an anodic and a
cathodic chambers separated by a CEM (Figure 4.7a). Due to a closer electrodes disposition
than for the first two configurations, the voltage is lower (data not shown). There is less
distance to cover while transporting the electric charges between the electrodes. For the
cathodic compartment, this configuration is similar to the one of configuration 1. Basically,
the same reactions take place as it was presented for the first configuration. The only
difference is the absence of the middle compartment. However, contrary to the first
configuration there is a transfer of H+ ions from the anodic compartment together with Na+
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ions to the cathodic compartment. It is less favorable as hydrogen ions neutralize OH- thus
reducing the alkalinity. Overall, in terms of pH and alkalinity the results observed for that
configuration were similar to the one discussed for the first configuration (data not shown).
No decrease neither in pH nor alkalinity was observed despite the additional H+ ions
migration for this configuration presumably due to the range of chosen conditions. However,
it is possible that the difference will occur if the time of treatment and the current intensity
are increased.
Figure 4.7: Configuration 3 (a) and 4 (b) of the electro-activation reactor used for the generation of the electrolysed water.
The fourth configuration (Figure 4.7b) is also a two-compartment cell with an AEM
separating both electrode compartments. The study of pH and alkalinity showed the same
tendency as with configuration 2. However, the absence of the middle compartment resulted
in additional release of chlorine (Cl2) on the anode due to the migration of Cl- from the
cathodic compartment. Apart from that fact and a lesser distance between the electrodes no
difference was noticed. Similar to the second configuration all three parameters were
significant for the changes in alkalinity (not shown).
4.6.4. The effect of type of configuration
Due to the aforementioned information among 4 tested configurations only the first
two will be discussed. The major difference between configuration 1 and configuration 2 is
the impact of salt concentration on the alkalinity which is not significant in the first case but
have a pronounced impact in the second case. Table 4.2 shows the increase in alkalinity and
pH with an increase in time for the two configurations. As it can be noticed the pH changes
progressively with an increase in time and current intensity. Regarding pH no important
a b
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difference between the two configurations has been observed. Concerning the alkalinity the
situation is quite different.
Table 4.2: Comparison of pH and alkalinity between two configurations for different current intensities within 0.01 M NaCl concentration.
C (NaCl) = 0.01 M, I = 0.050 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 10.56 0.02 0.92 0.11 10.75 0.07 1.00 0.06
30 11.02 0.13 3.20 0.06 11.00 0.02 2.73 0.10
60 11.34 0.05 6.90 0.14 11.42 0.06 5.12 0.25
C (NaCl) = 0.01 M, I= 0.1 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10,00 10.88 0.03 2.34 0.05 10.91 0.01 2.15 0.13
30,00 11.24 0.06 6.50 0.57 11.23 0.02 4.75 0.21
60,00 11.54 0.02 12.85 0.78 11.39 0.04 8.47 0.04
C (NaCl) = 0.01 M, I=0.2 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 11.05 0.01 4.85 0.78 11.20 0.06 3.50 0.28
30 11.60 0.02 14.07 0.04 11.33 0.02 7.30 0.28
60 11.81 0.07 25.46 0.31 11.49 0.04 11.55 0.07
The data presented in the table show that for the first configuration within 0.01M
concentration of NaCl the maximum alkalinity of 25.46 ± 0.31 is reached within 60 minutes.
It increases gradually with an increase in time and current intensity. For the Configuration 2
an increase is also observed but it is not that abrupt. For the same time and current intensity
it was only 11.55 ± 0.07, more than half as much compared to the Configuration 1. Therefore
for the minimal NaCl concentration the alkalinity in configuration 1 reached its maximum
but in configuration 2 did not.
With an increase in the NaCl concentration the alkalinity of the first configuration was not
statistically different from the one observed at 0.01 M concentration, however the second
configuration showed significant difference and by its values the alkalinity was closer to the
first configuration (Table 4.3). Finally, at maximum concentration of 1M in the cell using
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the second type of configuration the maximum alkalinity was reached, whereas no significant
changes were observed in the cell with the first type of configuration (Table 4.4).
Table 4.3: Comparison of pH and alkalinity between two configurations for different current intensities within 0.1 M NaCl concentration.
C(NaCl) = 0.1M, I=0.05 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 10,68 0,01 0,93 0,04 10,83 0,04 1,27 0,01
30 11,22 0,00 3,28 0,03 11,29 0,05 3,25 0,07
60 11,56 0,04 6,62 0,17 11,56 0,04 6,90 0,37
C (NaCl) = 0.1 M, I= 0.1 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10,00 11,03 0,08 2,03 0,10 11,08 0,01 2,16 0,20
30,00 11,48 0,01 7,08 0,03 11,55 0,02 6,77 0,24
60,00 11,76 0,01 13,80 0,08 11,81 0,01 13,14 0,06
C (NaCl) = 0.1 M, I=0.2 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 11,38 0,01 4,35 0,21 11,39 0,01 4,45 0,07
30 11,81 0,02 14,77 0,10 11,82 0,01 13,05 0,21
60 12,03 0,02 27,90 0,14 12,03 0,01 24,15 0,49
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Table 4.4 : Comparison of pH and alkalinity between two configurations for different current intensities within 1 M NaCl concentration.
C (NaCl) = 1 M, I = 0.050 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 10,62 0,03 0,96 0,06 10,66 0,08 1,07 0,04
30 11,12 0,01 3,03 0,04 11,11 0,08 3,35 0,13
60 11,40 0,01 6,42 0,40 11,49 0,04 7,45 0,78
C (NaCl) = 1 M, I= 0.1 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10,00 10,92 0,04 2,02 0,03 11,01 0,02 2,31 0,07
30,00 11,36 0,00 6,77 0,04 27,99 23,37 7,20 0,00
60,00 11,74 0,03 13,20 0,14 11,72 0,01 14,25 0,35
C (NaCl) = 1 M, I=0.2 A
Configuration 1 Configuration 2
τ (min) pH Sd ± Alkalinity Sd ± pH Sd ± Alkalinity Sd ±
10 11,24 0,01 4,39 0,01 11,28 0,03 4,77 0,18
30 11,74 0,02 13,32 0,11 11,80 0,02 14,40 0,03
60 12,00 0,01 27,15 0,35 11,96 0,00 28,15 0,64
Therefore, it seems that the second configuration is less effective in comparison with
the configuration 1 mostly due to the fact that targeted OH- ions leave the compartment
through the AEM. Configuration 1 prevents hydroxyl ions from leaving the compartment and
in addition allows the transfer of Na+ ions from the central compartment forming NaOH.
Solutions generated with the first configuration are independent of the salt concentration and
thus do not require high content of salt. For the second configuration the similar alkalinity
can be reached when 1M NaCl concentration is used.
The study of alkalinity of EAS carries precious information about the strength of the
obtained solutions. While no important variation was observed in terms of pH for the
solutions generated under different conditions significant differences were noted in terms of
alkalinity. This can be explained by activation energy of solutions and their metastable non-
equilibrium state which is the heart of electro-activation. Alkaline catholyte having the excess
of negative charges does not contain counter-ions and locally is electronegative even though
the system in total is electrically neutral (Belovolova et al., 2006). The activation energy of
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EAS comes from the effect of an electric field on dipole moments of water resulting in the
appearance of resonance microclasters (G, 2006; Plutakhin et al., 2013). This high resonance
energy of co-vibrating dipoles and charged ions in the near-electrode spaces explains the
abnormal properties of EAS. When compared to conventional base such as NaOH maximal
values of alkalinity obtained under tested conditions can give a solution equivalent to 0.03M
concentration of NaOH. If this concentration is to be increased, higher values of current
intensity as well as longer treatments are needed. On the other hand the product of EA is not
a concentrated solution but an activated one in a metastable state which significantly
increases their reactivity (Plutakhin et al., 2013). Such solutions need further investigations
in terms of their reactivity. Their behavior in various physico-chemical and biological
reactions is the subject of further analysis.
Conclusion
The effect of current intensity, time and salt concentration on the pH and alkalinity of aqueous
solutions obtained in the reactor of 4 different configurations has been investigated. Among
the four tested configurations a pairwise similarity was observed. Thus, configurations 1 and
3 were similar as were configurations 2 and 4. The time of treatment was the most important
parameter for the changes in pH of all EA solutions. High alkaline pH was achieved during
the first 10 minutes, yet further treatment did not result in marked distinction. Regarding the
current intensity, the salt concentration and the type of configuration the differences were
minor. The situation was quite different for the alkalinity. Although the pH of solutions in all
cases indicated highly alkaline medium, the alkalinity studies showed a significant difference
for different time and current intensity (for all 4 configurations) and salt concentration (for
configurations 2 and 4). Alkalinity was higher under higher current intensity and longer time
of treatment. For configurations 2 and 4 the alkalinity increased with an increase in salt
concentration which was explained by the membrane disposition. In general, the
configurations 1 and 3 seem more effective under the tested conditions as they keep the
required ions in the cell whereas other two configurations allow their transfer to the adjacent
compartment.
Despite highly alkaline pH which indicated the presence of OH- groups the generated
solutions according to maximally achieved alkalinity corresponded to a 0.03M NaOH
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solution. Under the tested conditions the solutions comparable to strong NaOH were not
obtained. A substantial increase in current intensity and time of treatment is therefore
required in order to increase the alkalinity.
Particular interest is shown by the correlation between the pH and alkalinity. Solutions
obtained in configurations 1 and 2 did not show an important difference in terms of pH,
whereas in terms of alkalinity a substantial increase was observed. Further analyses on the
utilization of EAS are needed. Alkaline properties of the catholyte can be used in
technological processes where conventional bases are normally used. Potentially the
technology of EA can become a better alternative to the utilization of conventional bases in
many technological processes as it allows to refuse from chemical reagents and therefore is
a “green” and environmentally friendly technology.
Acknowledgments
This work was financially supported by the innovation in food support program that
was funded by contracts through the "Growing Forward" Program that occurred between the
Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of
Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".
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5. CHAPTER 5: A comparative study between the electro-activation
technique and conventional extraction method on the extractability,
composition and physicochemical properties of canola protein
concentrates and isolates
Contextual transition
Based on the results obtained in previous two chapters the following section aimed to study
the electro-activated solutions for their capacity to extract proteins from canola meal and to
compare them to the traditional technique which uses the NaOH solutions. Also the effect of
such treatment was assisted by studying the physico-chemical properties of extracted
proteins.
This chapter is presented as an article entitled: “A comparative study between the electro-
activation technique and conventional extraction method on the extractability, composition
and physicochemical properties of canola protein concentrates and isolates”.
The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:
scientific supervision, article correction and revision), Marzouk Benali (Scientific
collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder
(Thesis director: scientific supervision, article correction and revision)
This article was published in the “Food Bioscience” (2015), Volume 11, 1, Pages 56-71
The results were presented at The World Congress of Food Science & Technology (IUFoST)
2014 in Montreal as a moderated poster, at The International Congress on Engineering and
Food (ICEF) 2015, and IFT conference 2015.
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Résumé
La technologie d’électro-activation en solution a été utilisée avec succès pour l'extraction des
protéines à partir de tourteau de canola. Une solution alcaline a été générée dans le
compartiment cathodique du réacteur d’électro-activation sous l'influence d’un champ
électrique continu. Cette solution aux propriétés alcalines a été utilisée comme substitut du
NaOH vu ces propriétés d'extraction améliorées par rapport aux solutions chimiquement
alcalinisées. L'étude vise à vérifier l'efficacité de la solution électro-activée pour l'extraction
des protéines à partir de tourteau du canola en analysant son effet sur le taux d'extraction, la
composition et la structure secondaire des protéines extraites. Les paramètres testés
comprenaient la concentration de NaCl (0.01-1 M), la durée d'électro-activation (10-60 min)
et l'intensité du courant électrique (0.2 ; 0.3 A). L'électro-activation a été réalisée dans un
réacteur d’électro-activation à trois compartiments, séparés par des membranes échangeuses
d'ions, après quoi les solutions obtenues ont été utilisées pour l'extraction pendant une heure.
Le taux d’extractibilité maximale des protéines était de 34.32 ± 1.21% et a été obtenue avec
une solution électro-activée générée avec un courant électrique de 0.3 A ; et ce, quel que soit
le temps d'activation. L'extraction conventionnelle dans les mêmes conditions (pH 7-10) a
donné un taux d’extraction de 31.18 ± 1.89% de protéines. Les profils électrophorétiques des
concentrés des protéines et les isolats électro-activés analysés par SDS-PAGE ont été plus
clairs et visibles par rapport à ceux obtenus par la méthode d’extraction conventionnelle avec
du NaOH. L’étude réalisée par FTIR a révélé des différences importantes dans les structures
secondaires des protéines dépendamment des conditions de traitements utilisés ; notamment
le pH et la concentration du sel. Aussi, les analyses par FTIR ont clairement montré que les
protéines obtenues avec des solutions électro-activées étaient nettement moins dénaturées
que celles obtenues par la méthode conventionnelle d’extraction avec du NaOH.
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Abstract
A novel technology of electro-activation was used for protein extraction from canola
meal. An alkaline solution was generated in the cathodic compartment under the influence of
electric field. It has been reported to have improved extractive properties when compared to
chemically alkalized solutions. The study aims to verify the efficiency of electro-activated
solutions for protein extraction from canola oil cake by analyzing the effect of extraction
method on the extractability rates, composition, and secondary structure of extracted
proteins.
The tested parameters included NaCl concentration (0.01-1M), duration of electro-
activation (10-60 min), and current intensity (0.2, 0.3 A). The electro-activation was
performed in a three-compartment cell separated by ion exchange membranes, after which
the obtained solutions were used for 1-hour extraction. Maximal protein extractability was
34.32 ± 1.21% obtained with the electro-activated solution generated under 0.3 A irrespective
of the activation time. The conventional extraction under the same conditions (pH 7-10)
yielded 31.18 ± 1.89% of proteins. Electrophoretic profiles of electro-activated protein
concentrates and isolates analyzed by SDS-PAGE were clearly more distinguishable
compared to those obtained by conventional method. FTIR study revealed considerable
difference in proteins’ secondary structures between different treatment conditions (pH and
salt concentration) as well as between conventional and electro-activated samples, showing
less denatured spectra for the latter.
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Introduction
The interest towards new protein sources has grown dramatically in the past few years.
Increasing malnutrition in developing countries, high cost of proteins from animal sources,
health concerns such as intolerance to animal proteins and a conscious choice of many to
refrain consuming animal proteins has led to a substantial search for alternative sources of
proteins which could replace conventional ones. Alternative sources have been thoroughly
studied in recent years with proteins derived from plants, bacteria and yeasts being the most
promising ones. Among them oilseeds are interesting options as the protein rich oil cake left
after oil extraction is a byproduct which can be valorized.
Canola has become an important agricultural crop in Canada and around the world. It
was developed in Canada primarily as a source of edible oil. Canola is also used for the
production of biodiesel and its byproduct, the protein rich canola oilcake also finds use as
forage for livestock. Other possible applications of canola meal proteins include adhesives,
plastics, and biocomposites (Gillberg and Törnell, 1976). The protein content of an oilcake
left after oil extraction accounts for 20-50% on a dry weight basis, similar to soybean which
is extensively used in food industry (Tan et al., 2011a). However, at present its utilization is
limited to the production of animal feed. Numerous studies done on canola proteins’
physicochemical, functional and bioactive properties indicate potential for it to be used in the
food industry (Aider and Barbana, 2011; Ghodsvali et al., 2005; Khattab and Arntfield, 2009;
Moure et al., 2006; Rodrigues et al., 2012; Wanasundara, 2011; Yoshie-Stark et al., 2006;
Yoshie-Stark et al., 2008). Currently the most common extraction technique is a direct
alkaline extraction method which comprises protein solubilization at a highly alkaline pH
≥10 with subsequent precipitation either at its isoelectric point or by the use of membrane
technologies. Processing in a highly alkaline medium allows to extract up to 60% (Ghodsvali
et al., 2005) or even up to 80% of total proteins (Gillberg and Törnell, 1976; Pedroche et al.,
2004) depending on the canola variety but at the same time causes undesirable modifications
such as protein denaturation, reduction of digestibility and loss of essential amino acids
(Rodrigues et al., 2012; Sari et al., 2013). Furthermore, the presence of antinutritive factors
is another limiting factor. However, the amount of antinutritive factors can be significantly
reduced by the use of ultrafiltration and diafiltration (Ali et al., 2011; Xu and Diosady, 2002).
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In order to increase the protein yield and improve the qualitative characteristics of the
protein extracted from different vegetable sources, the effect of various conditions and
reagents such as salt (Eromosele et al., 2008; Karaca et al., 2011a), temperature (Gillberg and
Törnell, 1976), time of extraction, and meal to solvent ratio (Nioi et al., 2012) has been
studied. Alternative methods to direct alkaline extraction have also been investigated such
as protein micellar mass (Ismond and Welsh, 1992), the use of enzymes (Sari et al., 2013),
and Osborne scheme (Manamperi et al., 2012; Tan et al., 2011a, b).
One of the most promising methods for the protein extraction is the use of direct electric
current. Water subjected to an electric field known as electro activated solution (EAS) was
claimed to possess improved extracting, cleaning and disinfecting properties (Aider et al.,
2012b). The energy supplied by electricity transforms water to a metastable state,
characterized by abnormal physicochemical properties (Leonov et al., 1999; Plutakhin et al.,
2013). Electro-activation (EA) may be explained by the phenomenon of water electrolysis
and oxido-reduction reactions which take place on the electrodes under the influence of
electric field. This leads to drastic changes in physicochemical properties in the near
electrode layer, resulting in the formation of an acid solution in the anodic side and an
alkaline solution in the cathodic side. The products of oxido-reduction reactions responsible
for physicochemical activity of the solutions have been identified: 1) stable products of
electro-chemical reactions responsible for pH changes; 2) non-stable over reactive substances
such as ОН−, Н3−О2
−, Н2, НО2•, НО2
−, О2− ; 3) other products formed near the electrode
surface in the form of free structural complexes or hydrated shells of ions, molecules, radicals
and atoms (Sprinchan et al., 2011a). The simultaneous formation of aqua complexes which
can also increase the reactivity of the medium has also been documented (Leonov et al.,
1999). The abnormal properties of EAS were studied by analyzing the Raman spectra and
fluorescence and comparing them with that of chemically alkalized water (Belovolova et al.,
2006; Pastukhov and Morozov, 2000). The authors concluded that the metastable state of the
near-electrode solutions was the cause of EAS activity (Aider et al., 2012b). It was also
reported that the physicochemical properties of EAS could be manipulated by using various
configurations of the reactor, membranes, and the time of treatment (Liato et al., 2015b). The
EAS as mentioned before in addition to their acid and alkaline properties contain other
substances which increase their reactivity (Prilutskii and Bakhir, 1999; Tomilov, 2002).
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These substances are very unstable, yet they might contribute to the extraction processes. In
the food industry electro-activation has been used for protein extraction from sunflower seed;
protein solution was pumped through the cathodic chamber to extract and subsequently
through the anodic chamber to precipitate the extracted proteins (Plutakhin et al., 2005).
Comparing the yield, the authors found that chemical extraction at (10.2%), was not
significantly higher than electro-chemical extraction (8.9%) showing the sufficiently high
efficiency of extraction of sunflower proteins by means of EA.
The aim of the current study was to use the catholyte from cathodic compartment for
protein extraction from canola oilcake and to compare them with conventional extraction in
terms of protein and total dry matter extractability, subunit composition and secondary
structure of extracted proteins, amount of total phosphorus (as an estimate of phytic acid
content), and the amount of free amino-acids. In order to find the optimal conditions the
effects of the type of configuration, salt concentration, time of treatment, and current intensity
were studied. Conventional extraction was put through similar conditions in order to compare
the efficiency of new technique.
Materials and methods
5.5.1. Chemicals
Defatted canola meal was kindly provided by Bunge ETGO, Becancour, Québec,
Canada. Sodium hydroxide (NaOH) was purchased from VWR International LLC (West
Chester, PA, USA). Concentrated hydrochloric acid (HCl) and sodium sulphate (Na2SO4)
were purchased from Fisher Scientific (Mississauga, ON, Canada) and sodium chloride
(NaCl) was purchased from Caledon Laboratories LTD (Georgetown, ON, Canada).
5.5.2. Ion-exchange membranes
Anion exchange membrane (AEM) AM-40 and cation exchange membrane (CEM)
CM-40 were purchased from the Publicly Traded Company Schekina-Azot (Shchekina,
Russian Federation). Prior to utilisation both membranes were washed with 96%-ethanol to
remove the wax and prepared according to manufacturer’s instructions by soaking them in
the salt solutions before their use in the electro-activation reactor.
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5.5.3. Configurations of the electro-activation reactor
Electro-activation reactor was made of transparent Plexiglas columns with the
dimensions of 50x50x120 mm (LxWxH). Two types of configurations of the three-
compartmental reactor were used (Figure 5.1). For the first configuration referred as C1
further in the text, the AEM separated the anode and central parts whereas CEM was placed
on the cathodic side separating it from central section. The second configuration (C2) had the
same three compartments but this time the membranes were swapped places. The RuO2–
IrO2–TiO2 electrodes 120x34x1 mm with working active area of 40 cm2 were placed at the
extremities of the cells and were connected to the positive side of a direct electric current
power supply for the anodic compartment and to the negative side for the cathodic one. The
values of the applied voltage and the intensity of the electric current were measured using a
current power supply (CSI12001X, Circuit Specialists, Inc. Mesa, AZ, USA).
Figure 5.1 : Configurations of the reactor used for EA corresponding to: a) Configuration 1 (C1); b) Configuration 2 (C2).
5.5.4. Experimental
5.5.4.1. Protocol of EA
The EA was carried out at two constant electric current intensities of 0.2 and 0.3 A with
total time of treatment of 10, 30, and 60 min. To ensure the current flow salt solutions were
circulated in all three compartments. The cathodic compartment was filled with NaCl
solution of the following concentrations 0.01, 0.1, and 1M. Anodic and central compartments
were always filled with Na2SO4 solution of 0.25M concentration.
a b
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5.5.4.2. Extraction by EA
Industrially defatted canola meal was weighed and dispersed in EAS, 10% (w/w).
Extractions were carried out at room temperature for 1 hr, under constant agitation using a
magnetic stirrer. After that the slurry was centrifuged at 10,000 x g during 30 min at room
temperature so as to separate insoluble material using Eppendorf centrifuge 5804R
(Eppendorf AG, Hamburg, Germany). The supernatant was collected as electro activated
protein concentrate, further referred as EAPC. All the extractions were performed in triplicate
and the mean values were determined. To obtain electro activated protein isolate, further
referred as EAPI the pH of the supernatant was adjusted to pH 4.3. The precipitate was
collected by centrifugation (10,000 x g, 10 min with the help of Eppendorf centrifuge 5804R)
and washed three times with distilled water. The pellets were freeze-dried.
5.5.4.3. Conventional extraction
Industrially defatted canola meal was dispersed in aqueous NaCl solutions of the
following concentrations 0.01M, 0.1M, and 1M. The pH of the slurries was adjusted by
adding 2M NaOH solution dropwise so as to reach pH 7-10 and the extraction was held 1 hr
with agitation. After centrifugation the supernatant was collected as conventional protein
concentrate (CPC). The conventional protein isolates (CPI) were obtained by isoelectric
precipitation as was aforedescribed for EAPI.
5.5.5. Chemical analysis
Proximate analysis of the industrial canola meal was performed according to AOAC
International (2012). Moisture and total dry matter were determined by drying the weighed
sample in a Fisher Isotemp Vacuum Oven (Fisher Scientific, Montreal, QC, Canada)
according to method 925.09. Ash content was determined according to method 923.03 by
combusting a weighed portion of 5 g in the muffle oven until constant weight and the ash
content was expressed as the weight ratio between the sample after and before combustion.
Crude protein was determined following the Dumas method with LECO Truspec FP-428
(Leco Corp., St. Joseph, MI, USA) according to the method 992.23 using a conversion factor
of 6.25. Residual oil in the defatted meal was determined by Soxhlet extraction according to
method 925.85 using petroleum ether as extraction solvent. Carbohydrates were determined
by difference (Karaca et al., 2011a). Phytic acid being the principal storage form of
116
phosphorus in canola was estimated from the ash residues (Stack, 1996) as the total
phosphorus content and was analyzed according to Plaami and Kumpulainen (1991). The
amount of free amino-acids was analyzed by EZ-Faast (Phenomenex, Torrance, USA) using
GC-FID. All analyses were conducted in triplicate and mean values were calculated
5.5.6. Total dry matter and protein extractability
In order to determine the total dry matter extractability the aliquot of the supernatant in
each case was weighed and dried in the oven until constant weight. The extractability was
calculated as a ratio of dry matter in supernatant excluding the salt added during the
extraction over total dry matter content of the canola meal. Nitrogen content of the
supernatant was measured using Dumas method with LECO Truspec FP-428. The protein
extractability was expressed as a function of nitrogen content in the supernatant in relation
to nitrogen content in canola meal on a dry weight basis.
5.5.7. SDS PAGE
To assess the impact of tested conditions on the polypeptide profiles of extracted
proteins the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was
performed following the method of Aluko and McIntosh (2001). Initially protein
concentrates were dialyzed at 4 °C using Spectra/Por 3 dialysis tubing with 3.5 kDa cutoff
against water refreshing several times. After that, concentrates were freeze-dried and 1%
protein solutions (w/w) were prepared and left overnight, followed by dilution with deionized
water so that the amount of proteins loaded onto the gel was approximately 20 µg. Similarly
1% solutions were prepared from protein isolates. Subsequently, diluted samples were
dissolved in Tris-HCl buffer solution containing SDS for non-reduced conditions. For
reduced conditions samples were dissolved in Tris-HCl buffer with the addition of 5% (v/v)
β-mercaptoethanol. Afterwards, the samples were boiled for 5 min, vortexed and 10 µl of
each sample were loaded onto 4–20% gradient gels (#456-1093, Bio-Rad, Canada). Precision
plus Protein Kaleidoscope Standards of 10-250 kDa (#161-0375, BioRad, Canada) were used
as molecular weight standards and the electrophoresis was run at 15 mA until the tracking
dye reached the bottom of the gel. After staining in 2.5% Coomassie Brilliant Blue R250 in
water–ethanol–acetic acid (4:5:1, v/v/v) and destaining with water–methanol–acetic acid
117
(10:4:1, v/v/v) the gels were scanned, using Chemi Doc XRS 170-807 (Bio-Rad Laboratories,
Inc, Hercules, CA, United States of America).
5.5.8. FTIR
FTIR was performed as described by Ellepola et al. (2005). The infrared spectra were
recorded at room temperature (22°C) using a Nicolet Magna IR 560 spectrometer (Thermo
Scientific, Madison, WI, USA) with continuous nitrogen supply. Canola protein dispersions
(5% w/w) were dissolved in 0.01 mol/l deuterated phosphate buffer (pD 7.4). All protein
dispersions were prepared in D2O instead of H2O since D2O has greater transparency in the
infrared region, 1600–1700 cm -1. To ensure complete H/D exchange, samples were prepared
the day before and left overnight at 4°C prior to infrared measurements. Samples were placed
between two CaF2 windows separated by 25 mm polyethylene terephthalate film spacer for
FTIR measurement. A total of 256 scans were averaged at 4 cm -1 resolution. To study the
amide I` region of the protein, Fourier self-deconvolutions were performed using the software
OMNIC 9.2.98. Band narrowing was achieved with a full width at half maximum of 22 cm−1
and with a resolution enhancement factor of 2.2 cm−1.
5.5.9. Statistical analysis
All tests were performed in triplicate, and the means ± standard deviations were used.
Data collected were subjected to analysis of variance at 5% confidence level. When
significant difference between treatments was found by ANOVA analysis, Tukey's multiple
range test was carried out for multiple comparison of the means using Minitab Software.
Results and discussion
Proximate chemical composition of canola oil cake is shown in the Table 5.1. On the
whole these values are well correlated with those reported in the literature before. It had
43.36% of proteins which is in accordance with those reported by Pedroche et al. (2004) and
Tan et al. (2011b). When harvested, canola seeds contain 19.0% of protein and 54.2% of fat
(Yoshie-Stark et al., 2008). After defatting procedure (made industrially) in our case the oil
cake still had fat traces in the amount of 3.58%. Ash and moisture were also in the range of
values reported for canola oil cake.
118
Phytic acid is the major storage form of phosphorus in canola. Depending on the variety
it accounts for 3.0–6.0 g/100 g (Bell, 1993; Gilani et al., 2012; Tan et al., 2011a). In spite of
its known adverse effect such as binding minerals and proteins leading to mineral depletion
and deficiency it was also reported to have a beneficial effect. Phytic acid is an effective
chelator of iron and it prevents the formation of free radicals, thus working as an antioxidant.
In addition it seems to bind heavy metals such as cadmium and lead helping to prevent their
accumulation in the body (Lott et al., 2000).
Table 5.1: Proximate chemical composition of canola oil cake on a dry weight basis.
Moisture
(%)
Protein
(N*6.25,%)
Ash
(%)
Fat
(%)
Carbohydrate
(%, by difference) Total phosphorus (%)
10.51 ± 0.01 43.36 ± 0.71 7.19 ± 0.01 3.58 ± 0.07 35.36 2.21 ± 0.48
5.6.1. Changes in pH
5.6.1.1. pH increase during EA
During the EA pH in the cathodic chamber increases drastically within first 10 min
regardless of the reactor`s configuration (Figure 5.2) due to the oxido-reduction reactions on
the electrodes. After 10 min further increase is also observed, however it is not that abrupt.
In previous study the effect of current intensity, time of treatment, salt concentration and type
of configuration on the pH and the strength of the catholyte (alkalinity) was studied
(Gerzhova et al.). It was shown that time of treatment and current intensity had the most
important impact on the alkalinity of the catholyte for C1. For C2, in addition to the
aforementioned factors, salt concentration had a significant impact. The pH increase for the
C1 was mostly dependent on the time of treatment and to a lesser extent on the current
intensity. The effect of salt concentration was also present for the C2. Properties of chosen
solutions for I = 0.3 A are shown in the Table 5.2. Properties of the solutions obtained under
lower current intensities were shown previously (Gerzhova et al.). The most important
differences between two different configurations are observed at minimal NaCl concentration
of 0.01M.
119
time of EA, min0 10 20 30 40 50 60 70
pH
5
6
7
8
9
10
11
12
13
0.01M NaCl
0.1M NaCl
1M NaCl
time of EA, min0 10 20 30 40 50 60 70
pH
5
6
7
8
9
10
11
12
13
0.01M NaCl
0.1M NaCl
1M NaCl
Figure 5.2: Changes in pH during EA for I = 0.3 A: a) C1; b) C2.
Table 5.2: Comparison of pH and alkalinity between two configurations for I = 0.3 A and within different NaCl concentrations.
Configuration 1 Configuration 2
C(NaCl) τ(min) pH ± Sd Alkalinity ± Sd pH ± Sd Alkalinity ± Sd
0.01
10 11.32 ± 0.01 8.5 ± 0.99 11.19 ± 0.01 3.95 ± 0.35
30 11.78 ± 0.06 21.04 ± 0.08 11.45 ± 0.04 9.54 ± 0.03
60 12.09 ± 0.01 44.5 ± 0.99 11.65 ± 0.02 14.27 ± 0.04
0.1
10 11.38 ± 0.01 7.07 ± 0.04 11.57 ± 0.01 6.29 ± 0.21
30 11.81 ± 0.02 21.64 ± 0.03 11.95 ± 0.01 17.90 ± 0.28
60 12.03 ± 0.02 43.30 ± 0.57 12.13 ± 0.01 32.35 ± 0.35
1
10 11.44 ± 0.02 7.03 ± 0.01 11.62 ± 0.04 6.90 ± 0.06
30 11.94 ± 0.04 21.05 ± 0.07 12.09 ± 0.01 20.40 ± 0.28
60 12.25 ± 0.01 43.21 ± 0.28 12.33 ± 0.01 43.04 ± 0.06
5.6.1.2. pH decrease during extraction
Solutions chosen for the extraction all had alkaline pH. During the extraction a pH
decrease was observed which can be explained by the buffer capacity of the canola oilcake.
The pH decrease is shown in the Figure 5.3.
a b
120
Figure 5.3: Changes in pH during extraction by EAS: a) C1, 0.01 M NaCl; b) C1, 0.1 M NaCl; c) C1, 1 M NaCl; d) C2, 0.01 M NaCl; e) C2, 0.1 M NaCl; f) C2, 1 M NaCl.
121
Solutions obtained under I = 0.2 A had the same tendency as those obtained under I =
0.3 A but were significantly weaker, so not to overload the paper only the results obtained
for I=0.3 A were shown. No statistically significant difference was observed for the C1
between different salt concentrations, whereas for the C2 a considerable difference was noted
between 0.01M NaCl and the other two concentrations. Regarding 0.1 and 1M NaCl
concentrations for the C2 only the solution treated 60 min was significantly different from
the other ones.
Time of treatment had the major impact on the changes in pH during extraction. A 10-
min solution was the weakest, which was in agreement with alkalinity measurements (Table
5.2). Its pH decreased dramatically down to pH 6 once it was mixed with canola oilcake and
was kept within this value during the extraction. Such behaviour was observed for all treated
solutions regardless of their NaCl concentration or the type of configuration. For the 30-min
solution there was a difference between configurations. This solution was twice as strong as
the 10-min according to its alkalinity. The pH of the 30-min solution dropped down not that
abruptly and was maintained around pH 8 for C1. For C2 there was a significant difference
between all three concentrations. Finally, the solutions treated 60 min were the strongest for
the C1 which was also well correlated with their alkalinity. In C2 the strength of the solutions
increased with an increase in salt concentration. In this case it seems that the salt
concentration was the most important parameter as even after 60 min of treatment the pH of
the solution made with 0.01M NaCl decreased faster and to a lesser value in comparison with
solutions of higher salt content (Figures 5.3d, 5.3e, 5.3f). Thus, the strength of 60-min treated
solution in C2 was lower than the strength of solutions treated 30 min in the same
configuration but with higher salt content. Comparing two configurations it is possible to
conclude that for the C1 the higher the time of treatment and the current intensity the lesser
the pH decrease is, which means the stronger the solution is. For C2 only the solution with
1M NaCl solution could maintain the pH within the scope of C1. The ability to maintain the
pH is related to the strength of the solution, to its alkalinity or buffering capacity in other
words. Although the concentration of OH- is sufficient for producing the same value of pH
as in chemical analogues it is insufficient to maintain it within the constant value, its buffering
capacity is lower in comparison with strong alkali such as NaOH. In order to increase the
pH of extraction so as to simulate the conditions similar to the conventional extraction the
122
higher current intensity and the longer time of treatment are required. The high pH can
possibly be maintained when there is a constant generation of OH- ions. This can be reached
when performing the extraction directly in the cell. However, there is a risk of membrane
fouling or precipitating on the electrodes.
5.6.2. Extractability
According to the previous study the pH has the major effect on the extractability which
is in accordance with other publications (Ghodsvali et al., 2005; Klockeman et al., 1997; Nioi
et al., 2012). Canola proteins are characterized as very complicated due to the diversity of
their molecular weights and isoelectric points. Thus, Quinn and Jones (1976) reported over
30 protein species with two major proteins - cruciferin being a neutral protein of a high
molecular weight (300-310 kDa) and an isoelectric point around pH 7 and napin, a small
molecular weight protein (12.5-14.5 kDa) characterized by strong alkalinity with an
isoelectric point around pH 10-11 (Aider and Barbana, 2011; Karaca et al., 2011a). Minimal
solubility was reported to be around pH 4.3 and the majority of plant proteins have their
isoelectric points within slightly acid pH region (pH 4-5). With an increase in pH of
extracting medium the amount of extracted proteins increases dramatically especially in the
pH range of 10-12 due to the increase in proteins ionization.
5.6.2.1. Total dry matter extractability
Total dry matter extractability is shown in Figure 5.4. Both configurations: C1 and C2
(Figures 5.4b, 5.4c) did not show an important difference in comparison with conventional
extraction (Figure 5.4a). Extractability increases with an increase in NaCl concentration,
however the increase is not considerable. Between different times of EA the difference was
also not important which means that even with the solution obtained after 10 min of EA good
results can be obtained. The effect of current intensity was also present. Minimal amounts of
extracted dry matter under I=0.2 A was 19.67 ± 1.83% obtained in C1 with 10-min solution
of 0.01M NaCl which increased to 29.52 ± 1.30% for 60-min solution of 1M NaCl. Second
configuration showed slightly lower results, however the difference was not significant (not
shown). Higher current intensity (I=0.3 A) showed the same tendency and the results were
24.15 ± 0.51% to 32.03 ± 4.97% for C1 and 23.69 ± 0.18% to 30.54 ± 1.35% for C2 (Figures
5.4b, 5.4c).
123
Figure 5.4: Total dry matter extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2.
5.6.2.2. Protein extractability
The tested parameters impacted the protein extractability comparatively more than the
total dry matter with the current intensity being the most significant factor (p < 0.0001).
Maximum extractability under I = 0.2 A was 19.93 ± 1.39% obtained in C1, 1M NaCl
concentration and with the solution treated 60 min which was significantly lower in
comparison with conventional extraction. Other parameters with 0.2 A current intensity gave
even lower values (not shown). With I = 0.3 A results were comparable to those obtained
with conventional extraction (Figure 5.5). Overall, the tendency of the changes in
extractability as a function of other parameters (time of EA, salt concentration, type of
configuration) was the same for both tested current intensities, that is why only the higher
one (I = 0.3 A) will be discussed. The effect of time of EA was lower than expected.
According to the alkalinity studies the strength of the 10 min and 30 min solutions increased
by 3 times and for the 30 min and 60 min solutions - by 2 times. However, the difference in
the amount of proteins extracted by each of these solutions was not significant. Considering
the pH of the extraction medium as a key factor these results become logic. Apparently, the
protein extractability has low sensitivity to pH changes within the region of pH 7-10 and the
increase of the extractability is observed at pH values higher than 10. Thus, the pH of the
solution, electro-activated during 10 min was maintained around 7 during the extraction and
the values obtained in the first configuration are perfectly correlated with extraction held at
pH 7 adjusted by the addition of aqueous NaOH. For C1 the extractability was 26.29 ± 0.25%
for 0.01M NaCl, 31.18 ± 1.20% for 0.1M NaCl and 32.75 ± 2.59% for 1M NaCl.
Conventional extraction held at pH 7 gave 24.84 ± 0.93%, 28.41 ± 1.88% and 31.18 ± 1.89%
for the same NaCl concentrations. A 30 min solution maintained pH around 8 and allowed
pH
7 8 9 10
Ext
racta
bili
ty,
%
0
10
20
30
40
500.01 M
0.1 M
1 M
a time, min
10 30 60
Ext
rac
tab
ility
, %
0
10
20
30
40
50 0.01 M
0.1 M
1 M
b time, min
10 30 60
Ext
rac
tab
ility
, %
0
10
20
30
40
50 0.01 M
0.1 M
1 M
c
124
to extract 27.72 ± 0.92%, 30.82 ± 0.61% and 32.07 ± 2.42% whereas the results for the
conventional extraction held at pH 8 were 23.28 ± 0.90%, 25.30 ± 0.52% and 27.78 ± 0.27%
which is slightly lower. Finally, the 60-min solution with pH around 9-10 gave 33.82 ±
0.59%, 36.10 ± 1.24% and 38.06 ± 0.13% which was substantially higher in comparison with
conventional extraction at pH 10 (23.67 ± 0.19%, 27.98 ± 0.47% and 29.97 ± 1.69%).
Figure 5.5: Protein extractability for I = 0.3 A: a) conventional extraction; b) extraction with EAS in C1; c) extraction with EAS in C2.
An increase in protein extractability with an increase in salt concentration is due to the
”salting-in” effect for conventional extraction and for the solutions obtained in the C1. As
for C2, more pronounced increase is observed (Figure 5.5c). The positioning of the AEM
and CEM also significantly affected the protein extractability by the EAS. In C2, alkalinity
of the solution increased with an increase in salt concentration as described in previous work
(Gerzhova et al.). Briefly, an anion exchange membrane which was placed in the cathodic
chamber, separating it from the middle section allowed the migration of hydroxyl ions,
responsible for the alkalinity into the neighboring compartment. Cathodic chamber was the
zone of depletion (desalting) that led to water dissociation on the membrane in order to supply
the current carriers. A generation of H+ ions in the cathodic compartment limited the increase
in alkalinity by neutralizing OH- ions. This was especially pronounced when 0.01M NaCl
concentrations was used. With an increase in salt concentration the effect of water
dissociation decreased which is supported by an increased alkalinity and the higher amount
of extracted proteins (Figures 5.5). When 1M NaCl concentration was used the effect of ion
migration through the membrane was minor for such high salt concentration. This explains
the difference in the amount of extracted proteins between the two configurations.
pH
7 8 9 10
Ext
racta
bili
ty,
%
0
10
20
30
40 0.01 M
0.1 M
1 M
a time, min
10 30 60
Ext
rac
tab
ility
, %
0
10
20
30
40
0.01 M
0.1 M
1 M
b time, min
10 30 60
Ext
rac
tab
ility
, %
0
10
20
30
400.01 M
0.1 M
1 M
c
125
5.6.3. Composition
5.6.3.1. Composition of protein concentrates
The composition of protein concentrated is shown in Table 5.3. The proteins extracted
through conventional method (conventional protein concentrates) were designated with the
code CPC followed by the pH of extraction (e.g. CPC_10 corresponds to conventional protein
concentrate extracted at pH 10). The protein extracted with EAS (electro-activated protein
concentrates) were designated EAPC, followed by the type of configuration, and the time of
treatment (e.g. EAPC_C1_10 means conventional protein concentrate, obtained by extraction
with EAS, treated in the configuration 1 for 10 min).
Table 5.3: Comparative characteristics of protein concentrates.
Protein content, % Ash, % Total phosphorus, %
Free amino acids (total), %
C(NaCl), M 0.01 1 0.01 1 0.01 1 0.01 1
CPC_10 35.11 ±
1.29b
14.22 ±
0.62b
11.68 ±
1.68b
65.98 ±
1.10a
0.41 ±
0.02b
0.03 ±
0.02d
3.57 ±
0.32a
0.59 ±
0.23a
CPC_12 49.52 ±
1.17a
17.12 ±
0.81a
16.15 ±
0.52a
63.92 ±
0.84a
0.35 ±
0.05b
0.06 ±
0.01c
2.46 ±
0.42a
0.49 ±
0.16a
EAPC_1_10 29.88 ±
0.17c
12.75 ±
0.77b
12.40 ±
1.37b
69.01 ±
0.57a
0.74 ±
0.26b
0.15 ±
0.02a
4.51 ±
0.97a
0.76 ±
0.06a
EAPC_1_60 34.04 ±
2.53b
13.74 ±
1.41b
11.31 ±
1.75b
66.58 ±
3.45a
0.67 ±
0.33b
0.04 ±
0.02c,d
3.54 ±
0.4a
0.62 ±
0.05a
EAPC_2_10 27.95 ±
1.60c
12.78 ±
0.78b
11.36 ±
0.27b
63.69 ±
4.90a
1.33 ±
0.24a
0.09 ±
0.00b
4.90 ±
1.03a
0.77 ±
0.1a
EAPC_2_60 29.16 ±
0.76c
14.37 ±
0.40b
9.87 ±
0.42b
66.97 ±
1.05a
0.80 ±
0.19b
0.03 ±
0.00d
4.20 ±
0.55a
0.67 ±
0.16a
* Results represent the average of three determinations ± SD, values in the same column with different letters
are significantly different (p< 0.05)
The highest protein concentration was obtained by conventional extraction at pH 12.
First configuration after 60 min treatment yielded the amount of proteins statistically similar
to conventional one at pH 10. All the other treatments were not statistically different. All
protein concentrates extracted with 1M NaCl by either conventional method or by electro-
activation had significantly lower amounts of proteins on a dry weight basis. This can be
126
explained by the presence of the considerable amount of salt and thus an increased mineral
content. Only concentrates extracted by conventional extraction at pH 12 had statistically
higher values, whereas no difference was observed for EA concentrates and conventional
ones at pH 10. Ash content of the extracts treated with the addition of 1M NaCl ranged
between 63.69 ± 4.90% and 69.01 ± 0.57% in comparison with 9.87 ± 0.42% and 16.15 ±
0.52% for 0.01M NaCl. Although the amount of total phosphorus in the sample treated with
0.01M NaCl was statistically equal apart from the EAPC_C2_10, a slight decrease with an
increase in pH can be noticed. According to Gillberg and Törnell (1976) and Ghodsvali et al.
(2005) the extraction of phytic acid and nitrogen takes place concomitantly and nitrogen
extractability is influenced by the phytic acid. Ghodsvali et al. (2005) stated that maximum
interactions were at pH lower than pI, when anionic groups of phytic acid bind to cationic
groups of proteins as proteins are charged positively and the phytic acid is charged
negatively. In neutral region it reacts with metal ions and proteins to form a soluble ternary
phytic acid–cation–protein complex. At alkaline pH, both the protein and the phytic acid are
negatively charged and the protein–phytate interaction takes place only in the presence of
multivalent cations and through a salt linkage or an alkaline-earth ion bridge (Champagne et
al., 1985). At high pH (> 11) the interaction between protein and phytic acid decreases and
in the presence of sufficient amount of Ca or Mg the ternary complex will precipitate
(Champagne et al., 1985). In addition, it has been reported that at high pH, the insoluble
phytate exists in a fine colloidal suspension which can be removed by centrifugation (Tzeng
et al., 1990a). Lower amounts of total phosphorus (phytic acid) in extracts treated with 1M
NaCl solution can be explained by the presence of considerable amounts of salt and decreased
protein content. Working in high salt concentration also weakens the electrostatic interactions
of phytates with the proteins (Schwenke, 1994) which explains the lower amounts of phytic
acid in concentrates extracted with 1M NaCl solution in comparison with 0.01M. The
amount of free amino acids can be useful and practical indices for evaluating the protein
quality. No statistically significant difference was noticed within the same salt concentration.
However, smaller values were obtained in the solutions with 1M NaCl compared to 0.01M
which can be related to the total amounts of proteins as the latter had considerably higher
protein contents.
127
5.6.3.2. Composition of protein isolates
The protein, mineral and total phosphorus contents of protein isolates are shown in
Table 5.4. The conventional isolates were named similarly to concentrates: CPI followed by
the pH of extraction. Electro activated protein isolates were designated EAPI, followed by
the type of configuration and the time of treatment.
Table 5.4: Comparative characteristics of protein isolates.
Protein content, % Ash, %
Total phosphorus, %
C(NaCl), M 0.01 1 0.01 1 0.01 1
CPI_10 89.67 ±
0.90a
97.95 ±
1.60a
1.43 ±
0.06b
1.54 ±
0.09b
1.17 ±
0.38a
0.85 ±
0.11b
CPI_12 86.16 ±
0.54b
89.99 ±
1.88c
1.96 ±
0.04a
2.32 ±
0.47a
1.68 ±
0.41a
0.97 ±
0.22b
EAPI_1_10 90.87 ±
1.00a
94.51 ±
0.49a,b
0.84 ±
0.08c
1.19 ±
0.15b
1.58 ±
0.1a
1.72 ±
0.01a
EAPI_1_60 82.43 ±
0.47c
94.16 ±
0.46a,b
1.13 ±
0.4b,c
1.28 ±
0.02c
1.13 ±
0.23a
0.82 ±
0.1b
EAPI_2_10 91.62 ±
0.32a
90.08 ±
0.43b,c
0.30 ±
0.05d
1.11 ±
0.25b
1.26 ±
0.04a
1.04 ±
0.07a,b
EAPI_2_60 85.27 ±
0.04b
91.67 ±
0.37b,c
2.28 ±
0.17a
1.18 ±
0.01b
1.42 ±
0.29a
0.62 ±
0.03b
* Results represent the average of three determinations ± SD, values in the same column with different letters
are significantly different (p< 0.05)
The protein content of all isolates was rather high and the ash content was reduced
significantly in comparison with concentrates which means a higher purity of final product.
Interestingly, the higher protein content was observed in samples with milder treatment
conditions. Thus CPI_10, EAPI_C1_10 and EAPI_C2_10 had higher protein content
compared to other isolates. Regarding samples treated with 1M NaCl CPI_10, EAPI_C1_10
and EAPI_C1_60 had the highest protein content. In addition samples of 1M NaCl
concentration had significantly higher protein amount in comparison with those of 0.01 M
NaCl. High amounts of total phosphorus (phytic acid) can be explained by the higher purity
of the sample compared to protein extracts which contained other interfering substances such
as salts and carbohydrates. In addition higher salt concentrations help to reduce the
128
electrostatic interactions of phytates with proteins resulting in lesser amounts of phytic acid
found in the isolate extracted with 1M NaCl (Schwenke, 1994).
5.6.4. SDS PAGE
5.6.4.1. SDS PAGE of protein concentrates
Figure 5.6 shows polypeptide compositions of canola protein concentrates with their
major components analyzed by SDS-PAGE in the absence (non-reducing) and presence
(reducing) of β-mercaptoethanol.
Major bands in non-reducing conditions were identified between 37 and 50 kDa (as 45
kDa), two between 26 and 37 kDa (27 kDa and 32 kDa), and two between 15 and 20 kDa (as
16 kDa and 18 kDa). This was consistent with Tan et al. (2011b) who observed 5 main
fraction with the following molecular weights: 16, 18, 26, 30, 45, and 53 kDa. However, the
last subunit of 53 kDa was not present in our proteins. After comparing all three graphs it is
noteworthy that in spite of different extraction techniques the proteins have more or less
similar composition, apart from the CPC_12 that is distinguished by weaker marked bands
or their absence. However, it had a band of higher molecular weight around 250 kDa which
was not present in other fractions. Similar phenomenon was noticed by Tan et al. (2011b)
who used different solvents in order to extract separately albumin, globulin, glutelin and
prolamin fractions. The authors managed to extract higher molecular weight subunits in
glutelin fraction which were not observed in albumin and globulin fractions. Therefore, it is
possible that under higher pH, subunits of higher molecular weight could be extracted (Quinn
and Jones, 1976). The smeared pattern of CPC_12 is an indication of reduced protein
solubility as a result of protein denaturation due to harsh alkaline conditions. The similarity
in obtained profiles is due to the complexity of canola protein composition and the
simultaneous co-extraction of different protein profiles. It is known that canola proteins have
complicated composition. Its two major components 12S globulin (cruciferin, 300 kDa) and
2S albumin (napin, 14 kDa) considerably differ by their molecular weight, isoelectric point
and functionalities (Manamperi et al., 2012; Wu and Muir, 2008). Cruciferin being salt-
soluble however tend to be co-extracted together with albumin during the conventional
extraction as well as napin was found in extracts obtained with 1M NaCl concentration. That
is why minor difference was noticed between profiles extracted with 0.01M NaCl and 1M
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NaCl. Thereby extracted fractions, in fact, are a mixture of two main proteins found in canola
such as cruciferin and napin.
Figure 5.6 : Molecular profiles as analyzed by SDS PAGE: a) EAPC extracted with 0.01 M electro-activated NaCl solution; b) EAPC extracted with 1 M electro-activated NaCl solution; c) CPC extracted with 0.01M and 1 M NaCl solutions.
There was a noticeable difference in SDS-PAGE profiles between the non-reduced and
reduced state. Under the reducing conditions two of the most distinctive profiles of 16 and
45 kDa disappeared and smaller fractions of 10, 12, and 22 appeared indicating that the
former fractions were linked by disulfide bonds and the latter are the result of other than S-S
interactions. Other bands of 18, 27 and 32 kDa were not affected by the presence of β-
mercaptoethanol and became more pronounced under reducing conditions which means that
other than S-S bonds are predominant. In reducing conditions new bands are more marked in
a b
c
130
0.01M NaCl concentrations. This was consistent with the work of Tan et al. (2011b) who
stated that globulin bands had less intensity compared to the polypeptide profiles of albumins
but on the whole polypeptide profiles of globulin and albumin fractions were very similar.
A comparison with conventional extracting technique revealed the difference in terms of 27,
32 and 44 kDa fractions which are similar in all EAPC and conventional ones extracted at
pH 10 (CPC_0.01_10 and CPC_1_10) but disappears at pH 12. Napin was reported to appear
on the gel in 14 kDa and 27.5 kDa (a dimer of napin) fractions according to Wu and Muir
(2008) who studied the purified samples of both major proteins from canola. In our case it
explains why the bands at 27 and 32 kDa are more pronounced with a lesser amount of salt.
The 14 kD fraction was not observed in our study however the band observed at 16 kDa is
presumably napin that upon the contact with β-mercaptoethanol give rise to two new bands
of 10 kDa and 6 kDa. It is also present in conventional concentrate extracted at pH 12 which
is explained by napin`s rigid structure low prone to structural changes at different pH
(Krzyzaniak et al., 1998). The band at 44 kDa is present in all EAPC and in CPC_10; however
it disappears in the extract obtained under higher pH (pH 12). This phenomenon is linked to
the protein structure as 12 S globulin is an oligomeric protein which dissociates into smaller
subunits under the influence of pH or ionic strength (Schwenke, 1994).
5.6.4.2. SDS PAGE of protein isolates
The non-reduced (without β-mercaptoethanol) and reduced (with β-mercaptoethanol)
SDS-PAGE patterns for protein isolates and canola meal displayed as CM are presented in
Figure 5.7. Similar to protein concentrates the protein isolates profiles look comparable,
however new bands of higher molecular weight revealed on the gel. These bands of 75 kDa,
100 and 110 kDa were not present in the concentrates and could result from protein
aggregation during the precipitation with HCl. These aggregates are linked together by
disulfide bonds which can be confirmed by their absence in reduced condition. In addition a
partial hydrolysis of high molecular weight proteins may take place with an increase in
extracting pH. Also the 12S subunits were reported to dissociate in the presence of 2-8M urea
solutions or at pH levels below 3.5 (Goding et al., 1970). As it can be noted in the Figure
5.7c and Figure 5.7d the band corresponding to 100 kDa disappeared at pH 12 and is lighter
at pH 11 in comparison with conventional extraction held at pH 10 and to those performed
with EAS. This was also observed by Jarpa-Parra et al. (2014) when lentil protein was
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subjected to different pH during extraction. In their case an alkaline pH created a progressive
dissociation of the 11S protein to lower molecular weight subunits (7S and 2S).
An evident difference between protein profiles of CPI and EAPI can be noticed which
increases with an increase in pH suggesting proteins denaturation (Figures 5.7a, b and 5.7c,
d). In the work of Mwasaru et al. (1999a) subunit composition of proteins isolates from
cowpea and pigeon pea was not affected by the extraction technique. In the study the authors
used micellar isolation and isoelectric protein methods and concluded that regardless of the
extraction technique and pH conditions extracted proteins exhibited similar electrical
mobility. In the other work where the influence of pH of extraction on the molecular profiles
was tested samples extracted at pH 8, 9, and 10 showed similar SDS-PAGE patterns. The
authors suggested that the protein compositions of the three extracts were similar and mainly
composed of globulin proteins (Jarpa-Parra et al., 2014). Consequently, it can be concluded
that protein composition is not influenced by the extraction technique in the range of pH 7-
10, however further pH increase provokes proteins denaturation which can be observed by
fading away of the bands in Figures 5.7c and 5.7d.
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*MW standard: molecular weight standard
**CM: canola meal
Figure 5.7 : Molecular profiles as analyzed by SDS PAGE: a) EAPI extracted with 0.01 M electro-activated NaCl solution; b) EAPI extracted with 1 M electro-activated NaCl solution; c) CPI extracted with 0.01 M NaCl solution; d) CPI extracted with 1 M NaCl solution.
5.6.5. FTIR
Although total protein concentration is an important parameter the understanding of
protein secondary structure is crucial for the understanding of its nutritive quality,
functionalities, availability and digestive behavior which determines its further utilization.
Even if the protein concentration is the same, the quality may differ due to the difference in
the secondary structures. For the proteins to maintain its biological and functional values it
should be folded in the same way as in the native state. The changes in environment influence
protein conformation and changes in pH and ionic strength are strong factors affecting it.
a b
c d
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5.6.5.1. Secondary structure of protein concentrates
Figure 5.8 shows the infrared spectrum of canola protein concentrates in the amide I`
region (1700-1600 cm-1).
Figure 5.8: Deconvulved FTIR spectra: a) Protein concentrates extracted with 0.01 M NaCl; b) Protein concentrates extracted with 1 M NaCl.
It depends on the secondary structure of the backbone and is hardly affected by the
nature of the side chain, thereby being the most commonly used region in monitoring the
changes in secondary structure. It is composed mainly of C=O stretching vibrations and is
extremely sensitive to changes in hydrogen bonding so that even subtle changes in the
secondary structure can be observed (Barth, 2007; Kong and Yu, 2007). Bands of β-pleated
sheets arise in easily distinctive peaks between approximately 1620 and 1640 cm-1 and in
some cases even below 1620 cm-1, showing more than one component and one weaker band
between 1670 and 1680 cm-1. Amide I` bands disposed between approximately 1650 and
1658 cm-1 are generally considered to be a characteristic of α-helical structures. The peaks
around 1616 ± 3 and 1689 ± 4 cm-1 are normally due to the formation of an intermolecular
hydrogen-bonded antiparallel β-sheet structure. Finally, the peak around 1645 cm-1 points at
a b
134
the presence of a disordered structure known as random coil (Dong et al., 1995; Surewicz et
al., 1993; Susi and Byler, 1988; Tang and Ma, 2009).
Five main bands were observed in the spectra after deconvultion, which, according to
the literature are β-structures predominantly (Figure 5.8). The main protein fractions in
canola proteins are cruciferin and napin as shown previously in the SDS PAGE profiles. The
secondary structure of cruciferin is performed mainly by β-sheet (50%) and low content of
α-helix (10%) conformations and was found similar to other 11S globulins (Schwenke et al.,
1983). Napin, on the other hand is characterized by a high content of α-helix structure (40-
46%) and a low content of β-sheet conformation (12%) (Schwenke, 1994). The quality of
protein can be judged by their peak intensities, a protein in the native state would have clear
peaks revealing α- and β-forms. A decrease in peak intensity, band shifting, an appearance
of new bands associated with aggregation or an increase in random coil structure would
indicate protein unfolding. When the protein is completely unfolded it loses its secondary
structure and together with it its value and functionalities. An evident decrease in peak
intensities can be observed in the Figure 5.8a from the top to the bottom as reflected by the
protein unfolding. The most pronounced secondary structure is carried by two top proteins
EAPC_C1_0.01_10 and EAPC_C2_0.01_10, their bands are identical, and therefore
secondary structures are comparable. The most prominent peak is the one at 1634 cm-1
attributed to β-sheet structure, followed by 1651 cm-1 associated with α-helix structure. The
presence of two peaks at 1618 cm-1 and 1688 cm-1 was claimed to be due to the formation of
an intermolecular hydrogen-bonded antiparallel β-sheet structure (Chehín et al., 1999; Dong
et al., 1995). Thus, protein aggregation judged by an increase in non-native intermolecular
β-sheet structures is the beginning of the loss of native protein which is a common reaction
to the application of thermal, physical or chemical stresses (Chehín et al., 1999; Chi et al.,
2003). The growth of aggregation bands is accompanied by the rise of another broad band
at 1645 cm-l, which can be assigned to unordered structure or random coil (Dong et al., 1995).
This was noticed for CPC_0.01_10 (Figure 5.8a), which still has some secondary structure;
however the peaks are significantly diminished and the appearance of a broad platform at
1645 cm-1 is observed. Finally, at pH 12 (CPC_0.01_12) more loss in the secondary structure
was observed, the protein was denatured with a broad spectrum within Amide I`, distributed
between 1600 and 1700 cm-1. Structural differences in Figure 5.8a are induced by the
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different characteristics of the solvent which in one case is EAS and in the other case saline
solutions adjusted to desired pH. Clearly, the properties of a solvent play an important role.
The pH of a solution determines the charge on the protein molecule. Proteins are normally
stable against aggregation over narrow pH ranges and may aggregate rapidly in solutions
with pH outside these ranges. Thus, in the work of Jarpa-Parra et al. (2014) the pH change in
the range of 7-9 did not have an impact on the secondary structure of lentil proteins.
However, at extreme values protein unfolded exposing its buried sites and changing the
electrostatic interactions (Chehín et al., 1999). As the pH changes the number of charged
groups increases which by turn increases the charge repulsions. This is sufficient to overcome
the attractive forces (mostly hydrophobic and dispersive) resulting in protein destabilization
and partial unfolding (Chi et al., 2003). First it was thought that aggregation arose from
completely unfolded protein, however further research showed that aggregates were formed
from partly unfolded proteins (Chi et al., 2003). The protein`s native conformation is flexible
and does not exist as a single structure but as an ensemble of conformations and the
aggregation may take place even at physiological conditions (Chi et al., 2003). Aggregation
band and the presence of random coil both characterize protein unfolding, however, when
the presence of aggregates is an intermediate state and protein molecule can refold again as
it still has secondary structure, the random coil means the loss of secondary structure and its
properties. Therefore the unfolded state is normally devoid of any native structure and exists
as a structureless random coil (Salahuddin, 1984; Tanford, 1968). Figure 5.8b shows the
spectroscopic characteristics of protein concentrates in the presence of 1M NaCl. The bands
of the spectra are similar to those shown in Figure 5.8a, however all bands are shifted to the
higher values which indicates the weakening of H-bonding (Zhao et al., 2008). This was also
observed by Ma et al. (2001) who studied the effect of different salts on the conformation of
oat globulins. In addition, an appearance of a new band in EAPC_C1_1_10 which was not
present in Figure 5.8a was noted. This band centered at 1644 cm-1 as previously discussed
was assigned to a random coil conformation and the presence of this band indicates a higher
level of disorder in protein`s secondary structure. Similar to Figure 5.8a this band grows
from the top protein to the bottom one. The decrease in 1672, 1654, and 1635 cm-1 and an
increase in 1690, 1644 and 1618 cm-1 peak intensities suggest transition from ordered
conformation to random coils and aggregated strands. Salts are known to perturb protein
136
conformation by affecting both electrostatic and hydrophobic interactions via a modification
of water structure (Ma et al., 2001). Over 100 years ago Hofmeister discovered that adding
salts to egg white protein could alter its solubility (Kunz et al., 2004; Schwartz et al., 2010).
The degree of the solubility is governed by the type of ions, their concentration and their
ability to increase or decrease the hydrophobic interaction, resulting in “salting-in” or
“salting-out” effects. This phenomenon became known as ̀ `Hofmeister series`` or ̀ `lyotropic
series``. Salts with the ability of the ions to reduce the hydrophobic interaction may enhance
the unfolding tendency of proteins were called chaotropes or structure breakers and salts
which can increase the hydrophobic interactions were called ``kosmotropes``, or ``structure
makers`` (Schwartz et al., 2010). However such classification was argued (Zangi, 2009).
NaCl depending on its concentration can cause both ``salting-in`` and ``salting-out effects``,
at low concentrations working as a chaotropic salt but with an increase in molar concentration
it acts as a kosmotrope and stabilizes protein conformation. Thus, it could precipitate proteins
only in the presence of 3.5N and higher salt concentration (Kunz et al., 2004). In the current
work the salt concentration was chosen in order to increase the extractability of proteins
which was reflected on the protein spectra.
5.6.5.2. Secondary structure of protein isolates
The spectra of protein isolates were quite different in shape and peak intensities,
indicating that proteins were more stable in concentrates. Broader and flatter surface suggests
that protein`s native state was more affected in protein isolates in comparison with protein
concentrates. As opposed to concentrates all the isolates were subjected to isoelectric
precipitation which could explain the difference in their secondary structure. The net charge
on the protein due to the titration of all the ionizing groups led to intramolecular repulsion,
sufficient to overcome the attractive forces resulting in at least partial unfolding of the
protein. This is supported by the presence of the peak close to 1645 cm-1 corresponding to a
random coil which was not present in the concentrates (Figure 5.9a). The Figure 5.9b was
characterized by the higher level of disordered structure and the band shifting towards the
higher frequencies, similar to what was observed in the concentrates and explained by the
presence of NaCl. On the whole, the tendency was similar in protein isolates and
concentrates. With an increase in the severity of treatment the aggregation bands and those
related to random coil increased in intensities, while those related to the native structure
137
decreased. In spite of acidic precipitation step which had certain negative effect on both
conventional and EA isolates, the latter managed to retain more of the native structure.
..
Figure 5.9 : Deconvulved FTIR spectra: a) Protein isolates extracted with 0.01 M NaCl; b) Protein isolates extracted with 1 M NaCl.
Conclusion
EAS proved to be an effective and a better medium for protein extraction from canola
meal. In contrast to conventional techniques which use chemicals, EA is a “green
technology” as it utilizes electric current to generate solutions possessing desired properties.
Both quantitative and qualitative analyses were performed comparing the extraction
with EAS with the conventional method. Proteins extracted by EAS were of higher quality
as seen in the SDS PAGE and FTIR results, also, the protein extractability was higher when
compared to conventional extraction for same process parameters (pH and time). It was noted
that the protein yield could be increased by increasing the current intensity, time of treatment
and salt concentration. Another option is to perform the extraction inside the EA cell, which
a b
138
would ensure a constant generation of OH-. However, it would influence the quality of
extracted proteins, resulting in the conformational changes and possibly higher denaturation
rates. In addition, there is a risk of membrane fouling or precipitation on the electrodes.
The structure of proteins dictates its quality and functional properties which determines their
further industrial utilization and commercial significance. Therefore, next objective should
be to analyze and compare the functional properties of proteins obtained by EAS and
conventional extraction.
Acknowledgments
This work was financially supported by the innovation in food support program that
was funded by contracts through the "Growing Forward" Program that occurred between the
Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of
Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".
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6. CHAPTER 6: Study of the functional properties of canola protein
concentrates and isolates extracted by electro-activated solutions
Contextual transition
Following the study of physico-chemical properties in the previous chapter which gave us
some indications of improved quality of proteins extracted by electro-activated solutions in
comparison with conventional method, the effect of electro-activated solutions on the
functional properties was monitored in the current chapter.
This chapter is presented as an article entitled: “Study of the functional properties of canola
protein concentrates and isolates extracted by electro-activated solutions”.
The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:
scientific supervision, article correction and revision), Marzouk Benali (Scientific
collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder
(Thesis director: scientific supervision, article correction and revision)
This article was accepted for the publication in the “Food Bioscience” (2015), Volume 12, 1, Pages 128-138
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Résumé
Cette étude a comparé les propriétés fonctionnelles des isolats et des concentrés de protéines
de canola produits par l’utilisation de solutions électro-activées par rapport à des protéines
extraites par la méthode alcaline conventionnelle. Pour les isolats, les résultats n’ont montré
aucune différence significative en termes de solubilité des protéines, le caractère hydrophobe
de la surface, l’absorption de l’eau et la capacité d'absorption du gras. Toutefois, pour les
concentrés de protéines, une plus grande capacité d'absorption du gras a été observée pour
les protéines extraites par des solutions électro-activées. En outre, certaines propriétés tensio-
actives ont également été améliorées, notamment l'indice d'activité d’émulsification qui était
plus élevé pour les protéines extraites par électro-activation. Également, la taille des
particules était plus petite pour les protéines extraites par la méthode d’électro-activation.
Dans tous les cas, les propriétés fonctionnelles ont été fortement dépendantes de pH du milieu
et le comportement des isolats et des concentrés était tout à fait différent dans la gamme de
pH proche du point isoélectrique, ainsi que dans les régions neutres et alcalines. Pour
l'ensemble des fonctionnalités, en général les résultats ont montré que les concentrés sont
plus efficaces à pH 4, tandis que les isolats donnent des meilleurs résultats à pH 7 et pH 9.
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Abstract
This study compared the functional properties of canola protein isolates and
concentrates produced by electro-activated solutions and conventional alkaline extraction. In
the case of the isolates, the results showed no significant difference in terms of protein
solubility, surface hydrophobicity, water absorption and fat absorption capacity. However,
for the protein concentrates, higher fat absorption capacity was observed for the proteins
extracted by electro-activated solutions. Moreover, some surface active properties were also
enhanced, notably higher emulsion activity index and smaller droplet size were observed for
the proteins extracted by the electro-activation method. In all cases, the functional properties
were strongly dependent on the pH of the medium and the behavior of the isolates and
concentrates was quite different in the pH range close to isoelectric point, in neutral and
alkaline regions. For all the functionalities, generally the results showed that the concentrates
were more effective at pH 4, whereas isolates performed better at pH 7 and pH 9.
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Introduction
Proteins in the form of isolates or concentrates are important ingredients in many food
products, where they perform specific functions (Kinsella and Melachouris, 1976a). The
quality of a food protein is established by its nutritional and functional properties (Schwenke,
2001). Nutritional properties include nutritive value, which is the amount of essential amino
acids, their bioavailability, or in other words “properties affecting the body after passage of
food into the alimentary canal” (Akiva, 1981). Functional properties could be defined as
“physical and chemical properties which affect the behavior of proteins in food systems
during processing, storage, preparation and consumption” (Kinsella and Melachouris, 1976a)
or “properties influencing foods prior to entering the body” (Akiva, 1981). This term has
been widely used in close relationship to the industrial utilization of proteins in various food
components. The range of functional properties is very wide and shows whether the protein
can be incorporated in a food matrix so as to impart some specific property; e.g. organoleptic
(colour, flavor, mouthfeel), textural (viscosity, adhesion), rheological (gelation, dough
formation, elasticity), surface active (emulsification, foaming), etc. (Kinsella and
Melachouris, 1976a). These properties may be more important than nutritional properties
when the protein is applied not as a main component but as an ingredient in a complex food
system (Schwenke, 2001). Nutritive value is of little importance if the protein or the product
in which it is incorporated is not acceptable for eating due to inappropriate texture, mouthfeel,
appearance, or flavor (Kinsella and Melachouris, 1976a). Functional properties of a protein
are governed by the qualitative/quantitative content and the sequence of amino acids,
building long polypeptide chains of different conformations. Molecular size, shape, and net
surface charge are no less important. Yet not only the intrinsic protein properties but also
their interactions with other food ingredients will determine their further utilization. The
majority of food systems are multicomponent and their functionality is the result of numerous
interactions between the different components. Thus, emulsion includes the interplay
between proteins, water, and lipids, whereas foams are formed from water, protein and air.
In addition, the environmental conditions such as temperature, pH and ionic strength play a
significant role (Kinsella, 1981).
Among new and developing sources for food applications, proteins from oilseeds
deserve particular consideration. Although canola is mostly regarded as a source of healthy
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oil, it has also been reported to be rich in balanced proteins, which could be found in the oil
cake left after oil extraction (Khattab and Arntfield, 2009; Ohlson and Anjou, 1979). Many
studies were devoted to various types of protein extraction and characterization and the
results were quite inspiring (Ghodsvali et al., 2005; Tzeng et al., 1988a). Its nutritive value
is similar to soybean proteins which are extensively used in food processing (Bell, 1993;
Delisle et al., 1984). Amino acid composition is balanced and highly suitable for use in
products for 10-12-year-olds and adults (Klockeman et al., 1997; Pedroche et al., 2004; Tan
et al., 2011a). Apart from having high biological value, canola proteins were found to possess
highly interesting functional properties which imparts their further utilization in the industry
so as to potentially substitute animal proteins (Aluko and McIntosh, 2001; Ghodsvali et al.,
2005).
The type of protein isolation technique directly influences its quality and composition
as well as functional properties by acting directly on their conformation and on the types of
proteins being extracted. A conventional direct alkaline extraction (DIR) which comprises
extraction at highly alkaline medium (pH >10) using diluted NaOH solutions or deionized
water with pH adjustment with precipitation at the isoelectric point has been reported to have
adverse effects on proteins. Harsh conditions used during extraction launch a set of
undesirable reactions such as protein denaturation, dissociation and amino acid racemization
(Moure et al., 2006; Pedroche et al., 2004). In addition, the use of chemicals generates large
amounts of pollutants which need further waste management and which negatively affect the
environment. Thus, a milder extraction method is required, which could solubilize proteins
without damaging their native conformation, maintain their activity and at the same time
would give high percentage of extracted proteins. In our previous work (Gerzhova et al.,
2015a), a novel technology that uses Electro-activated solutions (EAS) was used for protein
and total dry matter extraction from canola meal. This method uses an electric field to
produce alkaline solutions on the basis of water electrolysis and which have been claimed to
possess good extractive properties. To assess the quality of proteins extracted by EAS and
DIR, the analysis of secondary structure and physicochemical properties were performed and
revealed certain differences. Changes in protein secondary structure may cause changes in
the hydrophobicity or hydrophilicity, and structural stability of protein which are in close
144
connection with the functional properties such as water holding capacity, surface
hydrophobicity and emulsifying activity.
Considering that data on the functional properties would be useful in terms of
verifying the possibility of their incorporation in food matrix (Zhu et al., 2010). The aim of
this study was therefore to evaluate and compare the functional properties of protein isolates
and concentrates extracted by conventional technique and by means of alkaline electro-
activated solutions generated in an electro-activation reactor.
Materials and Methods
6.5.1. Raw materials and extraction methods
Protein isolates and concentrates were produced from defatted canola meal (kindly
provided by Bunge ETGO, Becancour, Québec, Canada). NaCl, HCl, NaOH were purchased
from Laboratoires MAT Inc (Montreal, Canada). Two extraction methods were used: the
conventional alkaline extraction at pH 10 in 0.01 M NaCl and the extraction by EAS
(reactor`s configuration I, 0.01 M NaCl solution in the cathodic compartment, current
intensity 0.3 A, electro-activation time 60 min) as described in our previous study (Gerzhova
et al., 2015a). For both methods, the extraction was conducted during 60 min. The protein
concentrates were obtained by freeze drying of the supernatant straight after extraction and
centrifugation at 10,000 x g, while the protein isolates were obtained by precipitation of the
proteins from the supernatant with 1 M HCl followed by freeze drying. Protein isolates and
concentrates extracted by EAS will be referred to as ``electro-activated protein isolate``
(EAPI) and ``electro-activated protein concentrate`` (EAPC) accordingly, and those
extracted by the conventional alkaline extraction as conventional protein isolate (CPI) and
conventional protein concentrate (CPC).
6.5.2. Nitrogen solubility index
Protein solubility was analyzed according to the method of Jarpa-Parra et al. (2014).
Concentrates and isolates were dissolved in distilled water to obtain a 0.5% protein
concentration and the pH of the suspension was adjusted to 2–10 by using 0.1 M HCl or
NaOH solutions. Afterwards they were stirred for 1 h and centrifuged during 10 min at 10,000
x g at 23°C with the help of Eppendorf centrifuge 5804R (Eppendorf AG, Hamburg,
145
Germany). After that, the supernatant was carefully decanted and the protein content was
analyzed by the bicinchoninic acid (BCA) protein assay (Fisher Scientific, Waltham, MA,
USA) with bovine serum albumin (BSA) as the protein standard. All the analyses were
performed in triplicate. Protein solubility was calculated as nitrogen solubility index (NSI)
as follows:
(%) = ∗ ( )
( )∗ (%) ∗ 100 (Eq. 6.1)
6.5.3. Water absorption capacity (WAC)
Water absorption capacity of the canola protein concentrates and isolates was
measured according to Ghodsvali et al. (2005). Sample of 1 g of each material was carefully
mixed with 10 ml deionized water in 15 ml centrifuge tubes with the help of a glass rode
during 30 s. This was done every 10 min, and after 30 min the tubes were centrifuged at 2,000
x g in the Eppendorf centrifuge 5804R for 15 min at ambient temperature (23°C). The
supernatant was decanted and the tube was inverted and drained for 15 min before being
weighed. The WAC was expressed as the amount of water absorbed per gram of sample.
6.5.4. Fat absorption capacity (FAC)
Fat absorption capacity was measured as described by Pedroche et al. (2004). Sample
of 0.5 g of each material was mixed with 6 ml of canola oil during 30 s with a glass rode
every 5 min and after 30 min, the tube was centrifuged in the Eppendorf centrifuge 5804R at
1,600 x g for 25 min at 23°C. Afterwards, the free oil was decanted and the FAC was
expressed as the amount of absorbed oil per gram of sample by weight difference.
6.5.5. Surface characteristics
Dispersions of 0.2% (w/w) protein content of isolates and concentrates were prepared
by solubilizing them in deionized water at room temperature (23°C) for 1 h at pH 4, 7, 9 and
left overnight.
6.5.5.1. Surface tension
Surface tension at the air-water interfaces was obtained by surface tension
measurements according to the Wilhelmy Plate method using KRÜSS 2570 Processor
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Tensiometer (KRÜSS, Hamburg, Germany) at 20°C. The platinum plate and the sample
vessel were thoroughly cleaned at the end of each analysis and flamed with the help of a
Bunsen burner.
6.5.5.2. Interfacial tension
Interfacial tension between solutions and canola oil was determined by the Du Noüy
ring method using KRÜSS 2570 Processor Tensiometer (KRÜSS, Hamburg, Germany) at
20°C. A protein solution was first added to the vessel after which canola oil as a lighter phase
was carefully added and the measurements were taken every 2 min with a total of 10
measurements after layering of the oil. The interfacial tension between water (without
protein) and canola oil was also measured.
6.5.5.3. Surface hydrophobicity
Average surface hydrophobicity was determined using the fluorescent probe, 8-
anilino-1-naphthalenesulfonic acid (ANS) according to the method of Kato and Nakai (1980)
with some modifications as described by Karaca et al. (2011a). Protein dispersions of 0.01%
were solubilized in appropriate buffers at pH 4 (sodium phosphate citrate buffer), pH 7
(phosphate buffer), pH 9 (borate buffer) and diluted with the same buffer to obtain 0.002%,
0.004%, 0.006%, 0.008%, and 0.01 % (w/w) pure protein concentrations. All the solutions
were prepared in duplicate. After that, 10 µl of ANS (8.0 mM in 0.1 M phosphate buffer, pH
7) was added to one tube at each concentration containing a 2 ml sample and vortexed for 10
s, while 10 µl of a phosphate buffer was added to the other tube as a control which was also
vortexed. After keeping each sample in the dark for 15 min, the relative fluorescence intensity
(RFI) of each sample and control was measured with a Cary Eclipse Fluorescence
Spectrophotometer (Varian inc., Palo Alto, CA, USA) at excitation and emission
wavelengths (λex, λem) of 390 and 470 nm, respectively. The excitation and emission slit
widths were 5 nm. The net RFI at each protein concentration was determined by substracting
the corresponding blank and plotted versus protein concentration. Initial slope (S0) of the line
was calculated by linear regression analysis and used as an index of protein surface
hydrophobicity (H0).
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6.5.6. Surface active properties
6.5.6.1. Foaming properties
Foaming properties were analyzed according to the method of Zhu et al. (2010).
Canola protein concentrates and isolates samples (0.2 g) were dispersed in 20 ml of deionized
H2O and the pH was adjusted to 4, 7, 9, by 0.01-1 N HCl or NaOH followed by 1 h stirring
with a magnetic stirrer at ambient temperature (23°C). The solutions were homogenized with
the help of Ultra Turrax T-25 (VWR International, RADNOR, PA, USA) at 22000 rpm
during 2 min. Foaming capacity (FC) was expressed as the volume difference before and
after whipping:
(%) = (Eq. 6.2)
Where V0 is the initial volume and Vt is a volume measure at different times after whipping.
Foaming stability (FS) was measured after 2, 5, 10, 20, 30, 40, 50, and 60 min and expressed
as:
(%) = (Eq. 6.3)
Where FC0 is a foaming capacity at 0 min that is to say right after whipping.
6.5.6.2. Emulsifying properties
Emulsifying properties were determined as Emulsion activity index (EAI) and
Emulsion stability index (ESI) by the turbidimetric technique of Pearce and Kinsella (1978).
First, aqueous protein solutions were prepared by dissolving protein samples to get 1% total
concentration in appropriate buffers as for the surface hydriophobicity analysis with pH 4, 7,
9. The suspension was then stirred overnight. Each preparation also contained 0.05% sodium
azide to prevent microbial growth. An oil-in-water emulsion was obtained by emulsifying 30
ml of protein solution with 10 ml of canola oil using UltraTurrax T-25 equipped with a S25
N-18G dispersing tool at 7500 rpm for 1 min, followed by stirring at 14500 rpm for another
minute. A 10µl sample was taken from the bottom immediately after the emulsion was
prepared and after 10 min of static storage and diluted with 0.1% SDS to give absorbance
between 0.1 and 0.6 at 500 nm. The absorbance was measured by HP 8453 UV visible
spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) with SDS solution as a
148
blank using plastic cuvettes (1 cm path length). The EAI and ESI were then calculated as
follows:
= ∗( )∗ ∗ (Eq. 6.4)
= . (Eq. 6.5)
= (Eq. 6.6)
Where T is turbidity, DF - dilution factor, φ -volume of oil fraction, C- weight of protein per
unit volume of the aqueous phase (g/mL), 10 000 - the correction factor for square meters, l-
path length of the cuvette (1 cm), A0 is absorbance measured at 500 nm at 0 min, ∆A is
absorbance difference between 0 min and 10 min, (Fennema, 1996).
6.5.6.3. Creaming stability
Creaming stability was measured as described by Karaca et al. (2011a). The same
emulsion as for the EAI analysis was prepared, poured in glass tubes, kept at room
temperature, and was visually observed after 24 h. The height of the creaming layer was
measured and the creaming stability (CS) was expressed as follows:
(%) = ∗ 100 (Eq. 6.7)
Where Ht is the height of the turbid layer and He is the total height of the emulsion.
6.5.6.4. Droplet size
Average droplet size of emulsions was determined using the method described by Tan
et al. (2014). Emulsions were prepared as for the EAI analysis at three different pH values
(4, 7, and 9). The mean droplet size of the studied emulsions was analyzed by mean of
Mastersizer Nano 3000 (Malvern Instruments Ltd., Malvern, U.K) equipped with Hydro EV
dispersion unit. Refractive index and absorption were set at 1.467 and 0.001 respectively.
The obscuration in all measurements was kept at around 14%.
6.5.7. Statistical analysis
All the experiments were performed at least in duplicate and the data presented are
means ± standard deviation. Analysis of variance (ANOVA) at 95% confidence level was
performed with the Tukey`s test by using Minitab statistical program (Minitab Inc., State
College, PA, USA).
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Results and discussion
6.6.1. Nitrogen solubility index
The solubility of canola protein isolates and concentrates is shown in Figure 6.1. For
all the samples, the lowest solubility with average values of 8.25 ± 0.90% for EAPI, 8.09 ±
0.85% for CPI, 22.08 ± 0.04% for EAPC and 20.88 ± 1.04% for CPC, respectively was
observed at the pH range between 4 and 5 which corresponds to pH range of isoelectric point
for canola proteins (Gerzhova et al., 2016; Tan et al., 2011a). For proteins, a U-shaped
relationship with the lowest point situated close to the isoelectric point has been noted
(Cheftel et al., 1985). Consistent to what was reported by other authors, the solubility
increases in both senses towards acid and alkaline pH and reaching its maximum at pH 10
with mean values of 56.73 ± 3.08%, 56.82 ± 3.57% for EAPI and CPI and 66.71 ± 4.15%
and 63.78 ± 3.37% for EAPC and CPC, respectively. These results are in good agreement
with the data reported previously in other works on canola or related to canola proteins (Aider
et al., 2012a; Mao and Hua, 2012; Pedroche et al., 2004; Zhu et al., 2010). The solubility of
concentrates was significantly higher within the whole tested range of pH, indicating more
hydrophilic character of the extracted proteins or less damaged structure. Isolates were
additionally subjected to the influence of acid in order to precipitate them, which could cause
the further unfolding of the molecules with the liberation of hydrophobic patches. As shown
in our previous study, the isolates and concentrates were quite different in their secondary
structures with the isolates being more unfolded as indicated by the presence of some peaks
which were not observed in the concentrates (Gerzhova et al., 2015a) . In the literature, it has
been stated that solubility can serve as an index of the extent of protein denaturation (Kinsella
and Melachouris, 1976a; Zhu et al., 2010). Moreover, proteins having low solubility over a
broad pH range are an indication of severe denaturation which have been shown to markedly
affect the functional properties of such proteins (Mwasaru et al., 1999a). Also part of soluble
proteins could have been lost during the precipitation step. In the present study, there was no
significant difference (p > 0.05) between the proteins extracted by electro-activated solutions
and conventional alkaline extraction. This can be explained by the fact that the pH of
extraction process in both methods was 10. Thus, it is possible to conclude that EAS did not
influence the solubility profiles within the selected range. However, it is important to mention
150
that protein isolate extracted by the electro-activation method had higher solubility in the pH
range from 5 to 9. This particularity makes it possible to enhance the use of canola proteins
in some applications where the solubility is a key point. This is justified by the fact that
among all the functionalities, solubility is probably one of the most important one, as it will
impact in almost all the other functional properties. High degree of solubility of the
ingredients predetermines its successful industrial utilization whereas poor solubility
significantly decreases the range of potential applications.
pH
0 2 4 6 8 10 12
NS
I, %
0
10
20
30
40
50
60
70
80
EAPI
CPI
EAPC
CPC
Figure 6.1: The solubility behavior of canola proteins as a function of pH, where EAPI is electro-activated protein isolate, CPI is conventional protein isolate, EAPC is electro-activated protein concentrate and CPC is conventional protein concentrate.
6.6.2. Water absorption capacity
The results on the water absorption capacity of the canola protein concentrates and
isolates produced by both methods (electro-activation vs conventional) are summarized in
the Table 6.1 and are in accordance with the other results already published on such or related
to canola proteins (Dev and Mukherjee, 1986; Pedroche et al., 2004; Thompson et al., 1982).
Statistical analysis of the obtained data did not show any significant difference (p > 0.05)
between the WAC of canola proteins extracted by EAS and DIR.
151
Table 6.1: Absorption capacities of electro-activated protein isolate (EAPI), conventional protein isolate (CPI), electro-activated protein concentrate (CPC), and conventional protein concentrate (CPC).
WAC FAC
EAPI 1.25 ± 0.05a 0.97 ± 0.01a
CPI 1.30 ± 0.13a 1.11 ± 0.04a
EAPC ND* 3.46 ± 0.16c
CPC ND* 3.02 ± 0.02b
*ND – not determined
** Results represent the average of three determinations ± SD, values in the same column with different
letters are significantly different (p< 0.05)
***WAC - Water absorption capacity
****FAC - Fat absorption capacity
Although some authors reported good WAC for canola or related to canola proteins
(Lin et al., 1974; Sosulski et al., 1976), high water absorption is not always a good indication,
since a compound with very high WAC may bind too much of water and as a result dehydrate
other components in the system (Xu and Diosady, 1994). No data on WAC of concentrates
has been obtained for both concentrates in the present study due to their high water solubility.
This was also previously reported in the works of Xu and Diosady (1994) and Manamperi et
al. (2012). In the case of Xu and Diosady (1994), highly soluble protein isolate with low
WAC was obtained by membrane processing from the water soluble protein fraction. It is the
insoluble proteins mainly that contribute to higher WAC as they tend to swell and imbibe
water, a phenomenon which was also observed by Zhu et al. (2010). If conventional
extraction is used, the WAC increases with an increase in pH of extraction. Some authors
reported inverse relationship with protein solubility (Mwasaru et al., 1999a). On the other
hand, the review of Hutton and Campbell (1981) on WAC showed that most of the studies
agree that there is no direct correlation between the ability to absorb water and the solubility
of proteins. Kinsella and Melachouris (1976a) studied the influence of pH on water
absorption capacity and stated that the pH had little effect on this parameter, which indicates
an absence of a direct correlation between water binding and protein solubility.
For practical considerations, solubility measurements do not provide with the
information on whether a protein will bind more or less water or contribute to the texture
profile of a product (Hermansson, 1979). Thus, WAC is used in such case. For example,
152
WAC is extremely important in a variety of meat products because it contributes to the
binding and retaining of meat juices, enhances mouthfeel and flavor of meat products. In
bakery products and cheeses, the WAC helps to prolong the product shelf life (Kinsella,
1979b). In other studies, protein has been reported to be primarily responsible for water
absorption. The ability of proteins to absorb water is related to their surface hydrophilicity as
the protein-water interactions starts at polar amino-acid sites on the protein molecule; i.e.
carbonyl, hydroxyl, amino, carboxyl, and sulfhydryl groups. However, the environment
which affects the protein conformation (secondary structure) is also very important. The
conformational transformation during unfolding may liberate the groups buried inside the
protein molecule thereby increasing or decreasing its hydrophilicity, depending on the nature
of those groups (Hutton and Campbell, 1981). Size, shape of the protein molecule as well as
lipids, carbohydrates and tannins associated with proteins also have an impact (Mao and Hua,
2012). It is noteworthy that the presence of carbohydrates may add to higher WAC due to
the presence of hydrophilic parts such as polar or charged side chains (Zhu et al., 2010).
6.6.3. Fat absorption capacity
Fat absorption capacity of the studied canola protein concentrates and isolates is
presented in the Table 6.1. Statistical analysis of the obtained results showed that
significantly higher values (p < 0.001) were obtained for canola protein concentrates
produced by both direct conventional alkaline and electro-activation methods than for the
isolates. At the same time, the results showed that there was no significant difference (p >
0.05) between FAC values of the isolates produced by the two methods. However, for the
concentrates, significantly (p < 0.001) higher values were obtained for protein produced by
the use of the electro-activation based extraction method.
Data obtained in this study on the FAC values of the canola protein concentrates and
isolates match up with the values obtained by Pedroche et al. (2004), Yoshie-Stark et al.
(2006), and Xu and Diosady (1994). The observed differences between the two extraction
methods in regards to the fat absorption capacity of the extracted materials can be explained
by the fact that FAC is regarded as the physical entrapment of oil and is highly affected by
protein content and protein conformation (Kinsella, 1979b). In this regard, in our previous
study we showed that the canola proteins extracted by EAS have significant conformational
153
differences in comparison with those extracted by the conventional method (Gerzhova et al.,
2015a) . The results on the capacity of canola proteins to bind fat materials are very important
for food applications. Indeed, protein-lipid interactions are involved in many other functional
properties. For example, lipoprotein complexes are functional components of egg yolk,
meats, milk, coffee whiteners, dough, and cake batters. This interaction enhances the
organoleptic acceptability of foods in which proteins are incorporated and impart better
flavor retention, texture and mouthfeel. However, in some types of foods such as doughnuts
or pancakes, high FAC may not be desired and the addition of soy flour is sometimes used
to prevent the excessive fat absorption during cooking (Hutton and Campbell, 1981).
6.6.4. Surface characteristics
Surface characteristics such as surface tension or interfacial tension can be used in
the evaluation of the surface activity of proteins. The force that acts at the interface between
two liquids or a solid and a liquid is called the interfacial tension or surface tension when it
acts between a gas and a liquid. Protein molecules being of amphiphilic nature and having
both hydrophobic and hydrophilic residues possess the ability to lower the tension between
two immiscible substances by migrating towards the interface and aligning there. As a result,
hydrophobic and hydrophilic residues enter aqueous and non-aqueous phases by unfolding
and stretching at the interface (Cheftel et al., 1985). The parameters such as the shape, size
of the molecule as well as its speed at which it can reach the interface are crucial. Main
surface characteristics of the studied proteins for both extraction methods are discussed
below.
6.6.4.1. Surface tension
The behavior of canola protein concentrates and isolates at the air-protein solution
interface is shown in the Table 6.2. Statistical analysis of the data showed that pH had minor
effect (p > 0.05) on the changes in the surface pressure. Nevertheless, proteins solubilized at
pH 7 and pH 9 had slightly lower values, i.e. better surface active properties in comparison
with pH 4, which can be explained by lower protein concentration as a result of lower
solubility at pH 4. Within the same pH, the results were as follows: no significant difference
(p> 0.05) between samples at pH4. At pH 7, EAPC and EAPI gave significantly lower values,
154
which means their surface characteristic were better compared to conventional ones. Finally,
at pH 9 only EAPI was significantly different.
Table 6.2 : Surface pressure at air-protein solution interface.
Surface Tension, mN/m
Sample pH 4 pH 7 pH 9
EAPI 46.97 ± 1.69a* 41.45 ± 0.56a** 42.57 ± 0.05a**
CPI 47.77 ± 1.23a* 45.43 ± 0.54bc* 45.50 ± 1.59b*
EAPC 49.37 ± 1.31a* 44.10 ± 0.2ab** 45.59 ± 0.46b**
CPC 48.67 ± 0.31a* 47.22 ± 0.01c* 47.95 ± 1.21b*
*Results represent the average of three determinations ± SD, values in the same column with different
letters are significantly different (p< 0.05)
6.6.4.2. Interfacial tension
A pH factor had significant effect (p < 0.001) on the interfacial tension at the oil-
protein solution interface as shown in Table 6.3. In comparison with pure water which gave
an interfacial tension value of 21.23 ± 0.12 mN/m, all of the produced canola protein samples
significantly reduced the interfacial tension. Proteins solubilized at pH 4 managed to lower
the tension by 50% and samples at pH 7 and pH 9 by 75%. It is important to mention that no
statistically significant difference (p > 0.05) between proteins extracted by EAS and by DIR
was found. It should also be noted that protein concentrates had slightly lower interfacial
tension values in comparison with isolates. Lower surface activity at pH 4 can be attributed
to the lower protein solubility, as aforementioned (Figure 6.1). High protein solubility is
necessary for rapid migration and adsorption at the oil-water interface (Karaca et al., 2011a).
In addition, poor solubility resulted in lower protein concentration which also contributed to
observed lower results. The percentage of proteins adsorbed at the interfaces was measured
by Liang and Tang (2013) and the results they reported showed that the least amount of
proteins was adsorbed at pH close to the pI, corresponding to the lowest solubility, a fact
which explained the higher interfacial tension and poorer functional properties.
155
Table 6.3 : Interfacial tension between oil and protein solution.
Interfacial tension, mN/m
Sample pH 4 pH 7 pH9
EAPI 10.65 ± 0.27abc* 5.52 ± 0.31a** 4.55 ± 0.50a**
CPI 11.82 ± 0.81ac* 5.95 ± 0.41a** 4.65 ± 0.43a***
EAPC 10.28 ± 0.39b* 5.24 ± 0.42a** 5.42 ± 0.19a**
CPC 11.84 ± 0.80c* 5.29 ± 0.36a** 5.83 ± 0.36a**
* Means followed by the same letter in the columns are not significantly different (P > 0.05). Values given are
means of duplicate determinations.
6.6.4.3. Surface hydrophobicity
Results on the surface hydrophobicity are shown in the Figure 6.2 and are in good
agreement with those of Wu and Muir (2008) who reported hydrophobic value of 950 for
canola protein isolate at pH 7. The analysis of the obtained data in this study showed no
significant difference (p > 0.05) between the EAPI and CPI, presumably due to the acidic
precipitation step in the protein isolation process which compensated the effects of extraction
treatment. However, a higher level of surface hydrophobicity was observed for CPC in
comparison with EAPC at pH 4. At pH 4, the S0 of the canola protein concentrates was
almost twice as much in comparison with the isolates. This result shows that at this pH, the
isolates and concentrates are in different conformations. As was shown in the previous study
(Gerzhova et al., 2015a), the FTIR spectra revealed considerable differences in the secondary
structures of isolates and concentrates, a factor which could significantly influence their
hydrophobicity. However, at pH 7 and pH 9, their surface hydrophobicity was smaller than
those of the isolates showing more hydrophilic character. Generally, the effect of the pH was
highly significant for all the samples (p < 0.001). A progressive decrease of the hydrophobic
character was observed with an increase in pH. The maximum hydrophobicity was obtained
at pH 4. This has been reported to be due to the different conformation of canola protein at
acidic conditions (Folawiyo and Apenten, 1996). An increase in hydrophobicity with a
decrease in pH has also been reported by Das and Kinsella (1989) and by Paulson and Tung
(1987) who observed a steep decrease in hydrophobicity from pH 3.5 to pH 6.5 and a gradual
decrease with further pH increase. In addition, hydrophobicity and solubility are closely
related. As aforementioned, solubility corresponds to the number of exposed hydrophilic
156
groups, whereas the hydrophobicity comes from the number of exposed hydrophobic groups.
The lowest solubility was reported to be around pH 4-5 for all the samples, therefore the
hydrophobicity was expected to be the highest at this pH interval. Although highly soluble
proteins usually perform better in most applications, for some functions a certain degree of
hydrophobicity is required.
Structure-function relationship approach has been used by chemists for years in order
to better understand and be able to predict certain functional properties by regarding protein
structure. Hydrophobicity is the one that is closely correlated with many functional
properties. However, the surface hydrophobicity rather than the average one should be
considered. In fact, the nonpolar amino acids which are buried inside the protein
molecule are unable to participate in any interactions and the surface hydrophobicity is
not correlated with the average one (Keshavarz and Nakai, 1979). The surface
hydrophobicity can give a good indication of protein solubility as well as surface properties
such as foaming and emulsifying. Kato and Nakai (1980) by measuring the surface
hydrophobicity managed to explain the surface properties of proteins by using analytical
protocols. Moreover, hydrophobicity has been reported to be negatively correlated with the
interfacial tension (Keshavarz and Nakai, 1979). The authors stated that the hydrophobic
proteins should be able to interact easily with oil and water molecules simultaneously.
However, the results presented in the current work show that this statement should be treated
with caution. In our case, the most hydrophobic proteins at pH 4 did not show the best surface
active properties. Lower protein solubility, and as a result lower protein concentration in the
solution, was insufficient to decrease the interfacial tension. Therefore, it is not the
hydrophobicity but a reasonable balance between the hydrophilic and lipophilic parts that
determines the good surface active properties and higher hydrophobicity will not necessarily
result in better functionalities (Nakai, 1983).
157
pH 4 pH 7 pH 9
Hydro
phobic
ity
0
1000
2000
3000
4000
5000
6000EAPI
CPI
EAPC
CPC
aa
b
b
a abb b a
ab b
* Bars with different letters are significantly different for a given pH.
Figure 6.2: Surface hydrophobicity of protein isolates and concentrates as a function of pH.
6.6.5. Surface active properties
Surface activity is the ability to lower the interfacial tension. The greater it is the faster
the molecule can migrate and absorb between the layers so as to decrease the tension. The
mobility of molecule is of great importance because the surface tension is affected by surface
concentration and increases with an increase of the latter (Kinsella and Melachouris, 1976a).
Most important from technological point of view surface properties are presented below.
6.6.5.1. Foaming properties
The results on foaming capacity (FC) of the concentrates and isolates produced by
both extraction methods are shown in the Table 6.4 and those of the foam stability (FS) are
shown in the Figure 6.3. For isolates, maximum foaming capacity was obtained with proteins
solubilized at pH 9. Slightly lower values were obtained at pH 7 and much lower values
obtained at pH 4. For the concentrates, the situation was the opposite with maximum values
obtained at pH 4, followed by pH 7 and, finally pH 9. As aforementioned, a lot of factors
influence the foaming capacity and the solubility known to be highly dependent on the pH of
the medium is one of them. The curves of pH versus the FC as well as the pH versus the
solubility were observed to be parallel (Mao and Hua, 2012). An increase in net charge on
the surface of the protein molecule weakens the hydrophobic interactions and favors the foam
158
formation by increasing the flexibility of the molecule and allowing it to spread at the
interface. Another important factor is the hydrophobicity. Townsend and Nakai (1983) who
studied the relationship between the hydrophobicity, solubility, and the foaming capacity
claimed that the optimum foaming capacity would be associated with those proteins whose
dispersibility is more than 40% and hydrophobicity more than 700 which is associated with
a good balance of hydrophilic and hydrophobic groups necessary for effective stabilization
of the air bubbles. Hydrophobicity was found maximum at pH 4 for both isolates and
concentrates where the solubility reached its minimum values (less than 10% for isolates and
around 20% for the concentrates). The results on FC are consistent with the aforementioned
statement for the isolates, whose foamability was the lowest at pH 4. However, the
concentrates showed quite a different tendency. In spite of the low solubility, the concentrates
showed the highest FC at this pH value. This can be related to the fact that their solubility at
pH was higher in comparison with the isolates, showing that 20% solubility in combination
with high hydrophobicity was sufficient to provide good foaming capacity. Similar
information was reported by Townsend and Nakai (1983) who obtained good results for
proteins with solubility around 20% and hydrophobicity above 500, a fact which led to the
conclusion that insoluble proteins might also contribute to the stabilization of air bubbles in
foams in which they can serve as a physical barrier to their coalescence. A decrease in the
foaming capacity at pH 7 and pH 9 for the concentrates is due to their decreased
hydrophobicity. In spite of their high solubility in this pH range, their hydrophobicity is lower
than 500, which suggests that hydrophobicity is a more important parameter for the FC than
solubility. This is in a good agreement with the statement that irrespective of the degree of
solubility, proteins with low hydrophobicity show poor foaming capacity (Townsend and
Nakai, 1983). The extraction method had significant effect (p < 0.001) on the foaming
properties of the canola proteins. The obtained results showed higher values in terms of FC
and FS for isolates extracted with the electro-activation based method in comparison with
those separated by the conventional alkaline extraction method for pH 4. Canola proteins and
napin in particular have been reported to possess good foaming properties (Aluko and
McIntosh, 2001; Nitecka et al., 1986; Nitecka and Schwenke, 1986). However, it is important
to consider that many other factors could have influenced resulting in higher values starting
from the type of meal used, extraction conditions and the method of analysis. Food foams
159
represent a medium where two immiscible substances such as gas (air) and liquid are mixed
with the help of a surface active agent (protein). They are usually measured as the volume
expansion upon the incorporation of air. To perform well in foams a protein should be soluble
in the liquid phase and be capable of rapid migration to the interface to form a film around
nascent gas bubbles (Kinsella and Melachouris, 1976a). The rate of migration of the
proteins is important, however the inherent surface activity and the ability of the protein
to unfold and reorient at the interface is crucial for foam formation. This is closely related
to the intrinsic molecular properties such as molecular size, shape, flexibility,
hydrophobicity, charge, etc. Thus, protein with higher surface hydrophobicity adsorbs more
readily at the interfaces. Flexible and loose molecules can unfold faster, whereas globular
proteins with rigid structure will take more time to adsorb and lower the interfacial tension
(Kinsella and Melachouris, 1976a).
Table 6.4: Foaming capacity of protein isolates and concentrates.
Foaming capacity, %
Sample pH 4 pH 7 pH 9
EAPI 28.92 ± 2.35a 60.46 ± 2.41a 73.64 ± 3.19a
CPI 19.62 ± 0.33b 57.83 ± 1.52a 66.91 ± 5.8a
EAPC 77.38 ± 5.23c 36.14 ± 3.99b 24.73 ± 3.53b
CPC 78.49 ± 8.63c 33.02 ± 2.76b 31.76 ± 3.33b
* Means followed by the same letter in the columns are not significantly different (P > 0.05). Values given are
means of duplicate determinations
The results on FS are shown in the Figure 6.3. The volume of the foam was reduced
gradually within 60 min of observation, showing that foams produced by canola proteins
were not stable. Overall stability of isolates was higher compared to the concentrates with
the exception of those solubilized at pH 4. FS also was higher at higher pH values. The results
are consistent with those obtained by Aluko and McIntosh (2001) who measured the FS after
30 min storage at room temperature for foams at pH 7 prepared from acid precipitated
isolates. Foaming stability is the ability to withstand gravitational and mechanical stress and
is critical if proteins are intended to be used as foaming agents. Not always properties needed
for a good foaming capacity are desirable for a good foaming stability. In general proteins
that possess good foaming properties exhibit poor foaming stability and vice versa. It is
160
noteworthy that foamability and stability are governed by two different sets of molecular
properties. While the foaming capacity depends on the rate of adsorption, hydrophobicity
and flexibility of the molecule, the stability mostly relies on the rheological properties of the
film formed around the bubble (Fennema, 1996). High surface viscosity and high film yield
are required for foam stabilization as they reflect strong cohesion between film forming
molecules. However, it is not desirable in the initial foam formation step (Kinsella, 1981).
time, min
0 10 20 30 40 50 60 70
FS
, %
0
20
40
60
80
100
120EAPI (pH4)
CPI (pH4)
EAPI (pH7)
CPI (pH7)
EAPI (pH9)
CPI (pH9)
time, min
0 10 20 30 40 50 60 70
FS
, %
0
20
40
60
80
100
120EAPC (pH4)
CPC (pH4)
EAPC (pH7)
CPC (pH7)
EAPC (pH9)
CPC (pH9)
Figure 6.3: Foam stability as a function of time: A) FS of proteins isolates, B) FS of protein concentrates.
6.6.5.2. Emulsifying Properties
6.6.5.2.1. Emulsion activity index and droplet size
Results on EAI are shown in the Figure 6.4A. For isolates, the EAI increased with an
increase in pH to attain its maximum at pH 9 with mean values of 22.10 ± 1.74 m²/g of
emulsifier and 19.01 ± 0.20 m²/g for EAPI and CPI, respectively, showing the highest contact
area between the two phases. Simultaneously, the data on the droplet size (Figure 6.4B) show
that the finest droplets were also obtained at the highest pH with average values of 22.37 ±
0.40 µm and 26.15 ± 2.32 µm for EAPI and CPI, respectively. The interfacial tension was
also the smallest at pH 9 showing better surface active properties of proteins at that pH (Table
6.3). The largest droplet size was obtained at pH close to the isoelectric point. Low solubility
at isoelectric point and the lack of repulsive forces results in low protein concentration which
is insufficient to cover the surface of all the droplets to act as a bridge between the oil droplets
leading to their flocculation (Wang et al., 2010). The formation of salt bridges at the
isoelectric point has been reported by Tornberg et al. (1997). The biggest droplet size around
A B
161
isoelectric point has also been reported by Tan et al. (2014) and Liang and Tang (2013) and
decreased when the pH deviated from it. Concentrates showed a different behaviour with an
increase in the emulsifying properties towards the lower pH value. The same tendency was
noticed when foaming properties were analyzed and explained by higher hydrophobicity at
pH 4 and higher solubility in comparison with isolates. Proteins soluble at their isoelectric
points should perform well because protein adsorption and viscoelasticity at an oil-water
interface is maximum near or at isoelectric pH (Kinsella and Melachouris, 1976a). Proteins
with a certain degree of solubility at isoelectric point have the highest EAI, whereas low-
soluble proteins show poor EAI (Fennema, 1996). On the other hand the emulsifying
properties of proteins tends to decrease when protein concentration is increased above a
critical concentration (Kinsella and Melachouris, 1976a). When the concentration of soy
proteins was relatively high an increased density made them less flexible to adsorb and unfold
at the limited oil droplet surface quick enough (Wang et al., 2010). Lower EAI of
concentrates at pH 7 and 9 could be explained due to the aforementioned phenomenon. In
general canola proteins have poor emulsifying properties and collapse easily (Aluko and
McIntosh, 2001). Low EAI could be due to high molecular weight of canola proteins and the
presence of disulfide bonds which reduce their flexibility and ability to unfold and adsorb at
the interfaces. Separated canola proteins` albumin and globulin fractions showed
comparatively better EAI (Tan et al., 2014). Low emulsion activity could also be due to
denaturation of the extracted proteins and a loss of functionality. No significant difference
was observed between protein isolates and concentrates extracted with EAS in comparison
with the proteins extracted by conventional method except for pH 9 where EAPI performed
significantly better than CPI which could be an indication of less damaged structure. This is
consistent with the previous study where the FTIR and SDS PAGE revealed that more native
structure was retained by proteins extracted by EAS (Gerzhova et al., 2015a). Emulsions of
fat and water are systems frequently encountered in food processing which, similar to foams,
are thermodynamically unstable and require a surfactant capable of stabilizing it.
Stabilization of emulsified droplets is performed by formation of a charged layer or a film
around the fat globules which provoke mutual repulsion. The kinetics of film formation is
greatly affected by protein composition and conformation; flexible molecules would unfold
easily, showing high surface activity. This lowers the interfacial tension and prevents droplet
162
coalescence (Kinsella, 1979b). Among the diversity of methods EAI estimates the contact
area between the two phases. According to Mie theory of light scattering, the turbidity of a
dilute suspension of spherical particles is related to its interfacial area which was used by
Pearce and Kinsella (1978) as the basis of the emulsifying activity index (Cameron et al.,
1991).
pH 4 pH 7 pH 9
EA
I
0
5
10
15
20
25EAPI
CPI
EAPC
CPC
aa
bb a
a
b b
a
b
c c
pH 4 pH 7 pH 9
Dro
ple
t siz
e,
µm
0
10
20
30
40
50
60
70EAPI
CPI
EAPC
CPC
a
b
cc
a aa
a
a
bab
bc
* Bars with different letters are significantly different (P < 0.05) for a given pH.
Figure 6.4: Emulsifying properties of canola protein isolates and concentrates: A) Emulsion activity index; B) Droplet size.
Data on the droplet size of emulsions reported in the literature on canola proteins
widely varies which can be explained mostly by varying emulsifying conditions. Droplet size
distribution is strongly dependent on the instrument used, intensity, and time and finer
droplets can be obtained when using vacuum homogenizers or sonication (Tornberg et al.,
1997). The variety of methods of analysis and the absence of a standardized technique make
it difficult to compare the results obtained in other studies. Same turbidimetric technique was
used in the works of Tan et al. (2014), Aluko and McIntosh (2001), and Karaca et al. (2011a).
Although higher results on EAI have been reported by Tan et al. (2014), the discrepancy in
the formula by which the EAI was calculated was found. The method of measuring the EAI
by turbidimetric technique was first introduced by Pearce and Kinsella (1978) and in the
original article the equation uses φ in the denominator instead of (1-φ). However, φ means
the volume fraction of the oil while (1-φ) represents the mass of protein in the unit volume
of emulsion and should be used instead (Fennema, 1996). The validity of such correction was
shown by Cameron et al. (1991). When recalculated the results obtained on protein isolates
at pH 4, 7, and 9 were similar to those reported by Tan et al. (2014).
A B
163
pH 4 pH 7 pH 9
ES
I
0
5
10
15
20EAPI
CPI
EAPC
CPC
ac
bcbc
c a
b
aa a
a a a
* Bars with different letters are significantly different (P < 0.05) for a given pH.
Figure 6.5: Emulsion stability index of the proteins.
6.6.5.2.2. Emulsion stability index
Data presented in the Figure 6.5 show that all the emulsions prepared with canola
proteins possessed low stability and underwent rapid coalescence within few minutes after
they were formed. It is an indication of the weak interfacial film formed around the oil
droplets. The thicker the film the lower is the coalescence rate. In addition, the coalescence
stability of droplets is closely related to the visco-elasticity and integrity of the interfacial
films (Liang and Tang, 2013). Low stability of emulsions can also be attributed to the rather
high droplet size. Smaller droplets are less susceptible to the coalescence due to smaller film
area between them and as a result a lower risk of its rupture (Fennema, 1996). For all the
emulsions, the average oil droplet size was greater than 10 µm, which was consistent with
the information reported by Wu and Muir (2008) (Figure 6.4B). The dependence of
coalescence stability on the droplet size has been also shown by Das and Kinsella (1989) who
found that the larger the droplet size, the higher the coalescence rate.
Generally, emulsions are thermodynamically unstable systems due to the high free
energy of the interface between the two phases. The equilibrium is reached when the contact
area between two immiscible substances is minimal. Having high surface area, the droplets
tend to reduce it and hence are subjected to various destabilization processes such as
creaming, flocculation, coalescence, and oiling off (Cameron et al., 1991). As the emulsion
activity can be estimated from the contact area between two phases, the stability of an
164
emulsion should be related to the constancy of the interfacial area. Stable emulsions would
possess the constant turbidity, providing that the interfacial area does not change with time
(Pearce and Kinsella, 1978). Changes in the average droplet size influence the turbidity
causing its decrease and hence can be interpreted as the coalescence. Coalescence is
accompanied by an increase in viscosity, increase in droplet size and decrease in surface area.
Among different destabilization processes that can occur in the emulsion, the changes in the
interfacial area point to the presence of coalescence and do not provide with the information
about creaming or flocculation as creaming itself does not cause a reduction in the interfacial
area of the whole emulsion and flocculation is reversed when the emulsion is diluted with
SDS solution (Pearce and Kinsella, 1978).
6.6.5.3. Creaming stability
Data on creaming stability is presented in Figure 6.6. No significant difference (p >
0.05) was found between the samples treated at different pH or different extraction
techniques, except for the CPI and CPC solubilized at pH 4 and pH 9. The visually observed
separation started almost right after the emulsification and two layers were clearly
distinguishable within one hour after the emulsion was prepared. Such rapid creaming could
be explained by the emulsifying efficiency of the device used to form the emulsion (Ultra
Turrax) which have been reported to give the most rapid creaming of the emulsion formed
when compared with other emulsifiers, notably valve homogenizers and sonication technique
(Tornberg et al., 1997). The rate of creaming depends on the average droplet size, with finer
droplets being more stable against creaming (Das and Kinsella, 1989; Fennema, 1996).
Creaming is one of the destabilization mechanisms characterized by the gravitational phase
separation; i.e. rising of the droplets that have lower density to the top which can be visually
observed. As a result, a turbid layer is formed at the top of emulsion with a transparent layer
at the bottom.
165
pH 4 pH 7 pH 9
Cre
am
ing s
tabili
ty
0
10
20
30
40
50EAPI
CPI
EAPC
CPC a
a
a
a
a
a a a
aa a a
* Bars with different letters are significantly different (P < 0.05) for a given pH.
Figure 6.6: Creaming stability of canola protein-stabilized emulsions.
Conclusion
The current study showed the effect of the isolation technique on the functional
properties of canola proteins. Two methods were compared; namely the isolation by electro-
activated solutions and conventional alkaline extraction and their effect on functional
properties such as total proteins solubility, water and fat absorption capacity, as well as their
performance at the interfaces has been studied. The results did not show significant difference
between the two methods in terms of protein solubility, surface hydrophobicity, WAC and
FAC, except for the concentrates separated by EAS that showed significantly higher fat
absorption capacity. In addition, proteins separated by EAS performed better at the interfaces,
EAPI and EAPC at pH 7 and 9 showed lower surface tension at air-protein solution interface
compared to CPI and CPC which points to their higher flexibility and higher adsorption rate.
Nevertheless their foaming properties were statistically equal. As for the emulsifying
properties, there was a significant difference between the EAPI and CPI at pH 9, where
isolate extracted by electro-activated solution showed higher emulsion activity index than the
conventional isolate. Also, isolates produced by electro-activated solutions created emulsions
with significantly smaller droplet size at pH 4 and pH 9 when compared to the conventional
process. However, the stability of the emulsions prepared from the electro-activated isolates
at pH 4 and pH 7 was slightly lower compared to the conventional ones at the same pH
166
values. Considerably different behavior between the protein isolates and concentrates at
different pH was noticed in spite of the fact that their surface activity as well as
hydrophobicity showed the same tendency. In general, protein concentrates performed better
at the isoelectric point in foams and emulsions, where isolates showed much poorer
properties. One of the reasons could be the loss of soluble proteins in the isolates at the
isoelectric point during acid precipitation procedure. On the other hand, isolates were
superior in terms of emulsifying activity index and foaming capacity at other pH which can
be explained by the hydrophobicity-hydrophilicity rate of proteins.
Acknowledgments
This work was financially supported by the innovation in food support program that
was funded by contracts through the "Growing Forward" Program that occurred between the
Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of
Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".
167
7. CHAPTER 7: Incorporation of canola proteins extracted by electro-
activated solutions in the gluten free biscuit formulation of rice-
buckwheat flour blend: Assessment of quality characteristics and
textural properties of the product.
Contextual transition
In previous chapters the behaviour of extracted proteins was shown following the model
systems. However, the behaviour in model systems might differ from those encountered in
real foods. That is why in the current study the extracted proteins were incorporated into the
matrix of biscuits as one of the possible applications of canola proteins.
This chapter is presented as an article entitled: “Incorporation of canola proteins extracted by
electro-activated solutions in gluten free biscuit formulation of rice-buckwheat flour blend:
Assessment of quality characteristics and textural properties of the product”.
The authors are: Alina Gerzhova (Ph. D. candidate: planning and realization of the
experiments, results analysis and manuscript writing), Martin Mondor (Thesis co-director:
scientific supervision, article correction and revision), Marzouk Benali (Scientific
collaborator of the project: correction and revision of the manuscript) and Mohammed Aïder
(Thesis director: scientific supervision, article correction and revision)
This article was accepted for the publication in the “International Journal of Food Science
and Technology” on 23.11.2015.
168
Résumé
Les protéines de canola extraites par des solutions électro-activées ont été incorporées dans
une formulation de biscuits sans gluten faits à base d’un mélange des farines de riz et de
sarrasin biologique. Les propriétés physiques et texturales ont été influencées de manière
significative par l’ajout des protéines de canola. Une augmentation du diamètre et d'épaisseur
des biscuits et une diminution des taux de propagation ont été notées pour tous les
échantillons supplémentés en comparaison avec le témoin fait de la même formulation mais
sans ajout de protéines de canola. La dureté des biscuits a diminué avec l'ajout de protéines.
Les modifications de dureté du biscuit (résistance à la rupture) ont été corrélées avec les
variations de dureté de la pâte. Les biscuits enrichis en protéines avaient une texture plus
légère et plus aérée comme observée par la microscopie électronique à balayage et avaient
moins d'amidon gélatinisé. Cette observation ainsi que l’épaisseur améliorée des biscuits sont
une indication de l'amélioration de la capacité de rétention de gaz et d'une structure plus
stable de la matrice. En outre, les biscuits ont été caractérisés par rapport à leur teneur en
humidité et l'activité de l'eau qui était plus faible. Enfin, le test sensoriel a montré des
améliorations significatives dans la texture et la sensation en bouche du produit expérimental.
Le goût, l'arôme et l'acceptabilité globale étaient également plus élevés par rapport au produit
témoin. En général, les résultats ont montré que les protéines de canola peuvent être utilisées
dans la formulation de biscuits sans gluten afin d'améliorer leurs caractéristiques texturales,
nutritionnelles et sensorielles.
169
Abstract
Canola proteins extracted by electro-activated solutions were incorporated into the gluten
free biscuits made from the blend of rice and buckwheat flours. The physical and textural
properties were significantly influenced by the addition of proteins. An increase in diameter
and thickness and a decrease in spread ratio were noted for all of the supplemented samples
in comparison with the control one. The hardness of biscuits decreased with the addition of
proteins as compared to the control. The changes in biscuit hardness or fracture strength were
in line with the changes in dough hardness. Protein enriched biscuits had lighter, more aerated
texture which was observed by scanning electron microscopy and had lesser amounts of
gelatinized starch. This and an increased thickness are an indication of improved gas holding
capacity and a more stabilized structure. Furthermore, they were characterized by lower
moisture and lower water activity. Finally, the sensorial test showed significant
improvements regarding textural characteristic and mouthfeel, the taste, aroma and overall
acceptability were also higher as compared to the control. Overall, the results showed that
canola proteins can be utilized in biscuit formulation in order to improve their textural,
nutritional, and sensorial characteristics as well as control the spread in cookies.
170
Introduction
A growing interest towards functional foods has encouraged researches to discover
new ingredients and new ways for their incorporation into the daily diet. In many aspects,
food now is regarded not only from nourishing point of view but also as a preventive or even
treating way of different diseases. Thus, gluten free (GF) products can be regarded as
functional food for those people who are gluten intolerant or suffer from celiac disease. As it
is known celiac disease is a lifelong disorder that arises in genetically predisposed people as
a reaction to gliadin fraction of wheat when consuming products containing gluten such as
bread, pasta or cookies. The list of cereals causing reaction also includes rye, barley and
possibly oats as they share a common taxonomy (Schober et al., 2003). The only known
treatment today is the lifelong adherence to a gluten free diet which created a separate market
niche. Although celiac disease according to different sources strikes around 1% of the
population (many cases are believed to be left undiagnosed), the demand for gluten free
products has increased drastically in recent years. Apart from celiacs (1%) and gluten
intolerants (6%), 22% of Canadians avoid eating gluten containing products due to various
reasons. In Canada, an annual growth rate of gluten free consumption of more than 26% has
been recorded from 2008 to 2012 and is expected to grow at least by 10% each year through
to 2018 (Agriculture and Agri-Food Canada, 2014). In spite of the growing popularity of GF
products and the choice offered in supermarkets, the quality of such products leaves much to
be desired. Mostly based on starchy flours like rice or corn/maize, they contain carbohydrates
but are poor in proteins and other essential nutrients such as vitamins and minerals as well as
fibers (Gallagher, 2008; Hager et al., 2012). In addition, the texture is also poor. The absence
of gluten, a structure making protein, results in a sandy mouthfeel and crumbly texture as
well as the lack of volume which poses a serious challenge for bread making industry
(Arendt, 2009). Biscuits on the contrary do not need developed gluten network and therefore
can serve a suitable matrix for incorporation of functional ingredients such as proteins to
gluten free food matrices. Moreover, they have relatively long shelf-life, good eating quality
and are also widely consumed (Yamsaengsung et al., 2012).
The research towards GF products aims to improve their textural and organoleptic
properties to give people who follow the GF diet better choices. In order to balance the
nutrients and increase the nutritive value, the addition of protein has been regarded by
171
different researchers. Biscuits were supplemented with milk proteins (Conforti and Lupano,
2004; Gallagher et al., 2005), safflower protein isolate (Ordorica-Falomir and Paredes-
López, 1991), soy protein isolate (Rababah et al., 2006) as well as other flours with high
protein content (Gambuś et al., 2009; Tyagi et al., 2007; Yamsaengsung et al., 2012). The
use of plant proteins is attractive due to several reasons and the cost is one of the most
important ones. Also, the search for alternative protein sources reveals new cultures which
can diversify the human diet. Among them, canola can rank with such a widely consumed
plant protein as soybean. A study in human showed that canola proteins can be considered to
be as efficient as soy proteins for a postprandial amino acid response (Fleddermann et al.,
2013). Being rich in highly digestible proteins as well as amino acids, it also possesses certain
important functional properties (Aider and Barbana, 2011; Tan et al., 2011a). In previous
works, canola proteins were isolated with the help of an emerging technology named electro-
activation and their physico-chemical, structural, and functional properties were investigated
(Gerzhova et al., 2015a, b). The results showed less damaged secondary structure and the
improvement in certain functional properties in comparison with proteins extracted with the
conventional alkaline extraction method (Aider et al., 2012b; Gerzhova et al.).
The use of composite flours in the production of GF cookies has been regarded.
Among them, rice and buckwheat were chosen as the base for the incorporation of canola
proteins. Numerous benefits of buckwheat flours have been noted such as lowering
cholesterol, blood lipid, blood sugar and reducing the occurrence of hyperlipidemia, obesity,
and diabetes (Cai et al., 2004). In addition it is characterized by high content of valuable
protein with a biological value equivalent to 93% of defatted milk and 81.5% of egg protein
as well as high content of minerals, organic acids, vitamins, resistant starch, total and soluble
dietary fibers, and polyphenols (Gambuś et al., 2009). Buckwheat flour has a strong flavor
due to the presence of phenolic compounds which can enhance the organoleptic properties
and improve the flavor (Sakač et al., 2015). The evaluation of buckwheat supplemented
cookies (30; 40; 50% of wheat flour) showed improved organoleptic properties in
comparison with control wheat cookie (Filipčev et al., 2011b). Rice, on the other hand, is the
common flour used in many GF formulations due to its bland taste, white colour, and highly
digestible carbohydrates (Rosell and Marco, 2008). The mixture of rice and buckwheat flours
has already been tested for the production of cookies in terms of sensorial properties (Sakač
172
et al., 2015; Torbica et al., 2012). A blend of rice and buckwheat flours (90/10; 80/20; 70/30)
showed good results giving significantly higher scores for almost all the tested sensorial
attributes when compared to wheat based samples (Torbica et al., 2012). However, some
works have claimed the decrease in sensorial characteristics of biscuits getting the lowest
score for taste, colour and texture characterizing them as crumbly and having a strong
aftertaste (Baljeet et al., 2010; Kaur et al., 2015; Yamsaengsung et al., 2012). Therefore, it
can be concluded that buckwheat does have a specific aroma which influence the
organoleptic properties. Sensorial tests conducted by different researches showed that
customer acceptance regarding buckwheat supplemented or buckwheat based cookies vary
greatly. However, its health benefits and the absence of gluten cannot be argued which
justifies the choice of buckwheat flour for its use in gluten free formulations.
Considering the aforementioned information, the main objective of this work was to
study the behavior of canola protein concentrates and isolates in the matrix of biscuit dough
and cooked biscuits by supplementing the rice-buckwheat flour blend with different
concentrations of canola proteins. The inclusion of canola proteins into the biscuit matrix
therein has two goals; notably to increase the nutritive value and to improve the textural
properties of the end product.
Materials and methods
7.5.1. Materials
Rice flour was purchased from Erawan Marketing (Erawan Marketing Co., LTD,
Bangkok, Thailand). Organic dark grey buckwheat flour (T60) and green buckwheat flour
(T40) were purchased from Aliments Trigone (Aliments Trigone Inc., QC, Canada). Electro-
activation extracted canola protein isolate (EAPI) and concentrate (EAPC) were produced as
described in (Gerzhova et al., 2015a) by using the following parameters: reactor`s
configuration # 1, concentration of NaCl in the cathodic compartment 0.01 M, time of
electro-activation 60 min, and current intensity of 0.3 A. Margarine, sugar, honey, sodium
bicarbonate and salt were purchased from the local market.
173
7.5.2. Proximate analysis
Moisture (925.09), ash (923.03), fat (925.85), and protein content (992.23) were
determined according to the methods described in the 19th edition of AOAC International
(2012). Carbohydrates were determined by difference.
7.5.3. Cookie preparation
The control cookie was prepared according to Torbica et al. (2012) by mixing the rice
and the buckwheat flours at 80/20 ratio with slight modifications (Table 7.1). Carboxymethyl
cellulose (CMC) was not added to the biscuit dough in order to exclude its effect and to test
the effect of canola proteins on the texture. The incorporation of canola proteins was done by
substituting the part of rice flour by 3, 6 and 9%. First, dry ingredients were mixed together
and then combined with liquids. Honey was dissolved in water before adding it to the dry
mixture. The amount of water was decreased for samples containing 6 and 9% of EAPI or
EAPC to obtain the dough with good handling properties. Cookie dough was prepared in a
Kitchen Aid Professional mixer 550 Plus (St. Joseph, MI, USA) at speed # 2 for 10 min using
a K-beater. After that, the dough was sheeted to the thickness of 7 mm using a rolling pin
and two gauge stripes after which the cookies were cut to pieces of 60 mm in diameter, baked
for 12 min at 180 °C in a Whitefficiency Rational Self Cooking Center (Rational AG, Iglinger
Straße 62, Landsberg am Lech, Germany) and left to cool down for 2 h before sealing them
in zip lock bags for storage and further analyses which were performed 24 h after preparation.
Table 7.1: Control biscuit formulation.
Ingredient Amount, g % of the total Buckwheat flour 60 10.00 Rice flour 240 39.99 Sugar 75 12.50 Margarine 100 16.66 Salt 2.1 0.35 Sodium bicarbonate 3 0.50 Honey 15 2.50 Water 105 17.50 Total 600.1 100.00
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7.5.4. Analytical tests
7.5.4.1. Texture profile analysis (TPA)
Dough test was performed with the help of TPA using TA-XT2 Texture Analyser
(Texture Technologies Corp., NY, USA) according to Wehrle et al. (1999). A 25 mm cylinder
aluminium probe and a 5 kg load cell were run at a test speed of 1 mm/s with 5 s recovery
between two strokes and a compression distance of 3 mm. TPA curves were analyzed for
hardness, springiness and cohesiveness.
7.5.4.2. Biscuit fracture strength
Biscuit fracture strength was determined using a TA-XT2 Texture Analyser (Texture
Technologies Corp., NY, USA) equipped with a 3-point Bending Rig and a 5 kg load cell. A
biscuit was placed on two base beams with 40 mm distance between them. The analyzer was
set at a return to start cycle, with a pre-test speed of 1.0 mm/s, test speed of 3.0 mm/s, and
post-test speed of 10.0 mm/s. The maximum force required to break the cookie and the travel
distance until the maximum force was reached were recorded using 9 biscuits.
7.5.4.3. Moisture content and Water activity (aw)
Moisture content in biscuits was analyzed according to the oven dry method (Sathe,
1999). Water activity was analyzed with the help of Aqualab 3TE water activity meter
(Decagon Devices, Inc., Pullman, WA, USA). A set of 3 biscuits taken from different parts
of the oven (front, medium, back) were ground in a Magic Bullet Single Shot blender prior
to the analysis in three replicates.
7.5.4.4. Colour
Top and bottom surface colour of biscuits were measured by using Minolta CR-300
Chroma Meter (Sensing Inc., Osaka, Japan) in triplicates 24 h after baking. The instrument
was calibrated using a standard light white reference tile. The results were expressed in terms
of CIE* values: L* (lightness), a* (redness to greenness - positive to negative values,
respectively), and b* (yellowness to blueness - positive to negative values, respectively)
values.
7.5.4.5. Diameter, thickness and spread ratio
The average of 6 biscuits was taken when measured the weight, diameter and
thickness of cookies. After cooling, the diameter of biscuits was measured using Vernier
caliper, after which they were rotated 90° and re-measured. The thickness of the biscuits was
175
measured by stacking them on top of each other and dividing by 6. An average of two
measurements was taken after re-stacking and re-measuring of the biscuits. Spread factor was
calculated by dividing the average value of diameter by the average value of thickness of
biscuits.
7.5.4.6. Scanning electron microscopy (SEM)
Microstructures of the flours and biscuits were analyzed with the help of Jeol, JSM-
6360LV Scanning Electron Microscope (Tokyo, Japan). Flour samples and biscuits were
prepared as described in Torbica et al. (2012) by mounting them on aluminum specimen stubs
with the help of double‐sided adhesive tape and were sputter-coated with gold. The analysis
of samples was performed at an accelerating voltage of 15 kV under high vacuum.
7.5.4.7. Preliminary sensory evaluation
Preliminary sensory evaluation of the cookies was conducted by a group of 8
volunteers from students of different nationalities both male and female who were habitual
cookie consumers. Cookie samples were served in a random order and evaluated according
to the acceptability of their texture, taste, aroma and overall appreciation using a 9-point
hedonic scale with 9 being “like extremely”, 8 – “like very much”, 7 – “like moderately”, 6
– “like slightly”, 5 – “neither like nor dislike”, 4 – “dislike slightly”, 3 – “dislike moderately”,
2 – “dislike very much”, and 1 – “dislike extremely”. Water was provided between each
sample.
7.5.5. Statistical analysis
Analysis of variance (ANOVA) was used in order to test the effect of protein addition
for the significance of differences. When it was detected, the Multiple Comparisons test
(Holm-Sidak method) (p<0.05) versus Control Group was performed using Sigma Plot
software (Systat Software, Inc., San Jose, CA, USA).
Results and discussion
7.6.1. Proximate analysis
Proximate analysis of the flours and proteins on a dry basis is shown in Table 7.2.
Rice flour had significantly lower amounts of protein, ash, and fat in comparison with
buckwheat flours. Dark (toasted) buckwheat was characterized by higher amounts of protein,
ash and fat as compared to green (non-toasted) buckwheat. The data on the composition of
176
rice and buckwheat flours is consistent with what has been reported before (Altındağ et al.,
2015; Kaur et al., 2015).
Table 7.2: Proximate analysis of flours and proteins.
Sample Moisture,
(%)
Protein,
(N*6.25,%)
Ash,
(%)
Fat,
(%)
Carbohydrate,
(by difference, %)
Rice flour 10.75 ± 0.13 8.53 ± 0.21 0.37 ± 0.01 0.27 ± 0.02 80.08 ± 0.37
Buckwheat flour
(dark) 10.54 ± 0.07 11.32 ± 0.19 1.71 ± 0.00 2.15 ± 0.06 74.28 ± 0.32
Buckwheat flour
(green) 11.66 ± 0.33 9.56 ± 0.02 1.48 ± 0.01 1.81 ± 0.04 75.49 ± 0.4
EAPC 4.82 ± 0.05 34.04 ± 2.53 11.31 ± 1.75 2.60 ± 0.01 47.23 ± 4.34
EAPI 1.83 ± 0.04 82.43 ± 0.47 1.13 ± 0.4 8.02 ± 0.02 6.59 ± 0.93
7.6.2. Dimensions and cookie spread ratio
Physical parameters of cookies such as diameter, thickness and spread ratio are
summarized in Table 7.3. The diameter and the thickness of cookies increased with the
addition of either canola protein isolate or concentrate at all concentrations. The spread ratio
on the contrary was the highest for the control cookies in both green and grey buckwheat and
decreased with an addition of canola proteins. Significant difference (p < 0.001) was noted
for diameter, thickness and spread ratio for the samples containing 6% EAPC and 9% EAPC
versus control cookie. For green buckwheat cookies, the diameter and the thickness were
lower in comparison with the dark buckwheat cookies whereas the spread ratio was higher
for all of the samples.
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Table 7.3: Physical parameters of cookies made from flour blend of rice and dark (toasted) buckwheat and rice and green (non-toasted) buckwheat.
Grey buckwheat Green buckwheat
Sample Diameter, mm
Thickness, mm
Spread ratio Diameter,
mm
Thickness,
mm
Spread
ratio
Control 6.10± 0.12d 0.88± 0.06c 6.98± 0.57a 5.95± 0.05c 0.83± 0.05e 7.20± 0.37a
3% EAPC 6.23± 0.08c 1.00± 0.07b 6.27± 0.39c 6.12± 0.06ab 0.92± 0.07d 6.67± 0.43c
6% EAPC 6.33± 0.07ab 1.19± 0.11a 5.37± 0.45e 6.26± 0.05a 1.13± 0.05b 5.56± 0.20e
9% EAPC 6.40± 0.06a 1.21± 0.06a 5.29± 0.20f 6.27± 0.05a 1.23± 0.05a 5.12± 0.16f
3% EAPI 6.20± 0.04c 0.96± 0.01bc 6.46± 0.05b 6.16± 0.03ab 0.87± 0.16e 7.18± 1.29a
6% EAPI 6.23± 0.1c 1.02± 0.02b 6.12± 0.22cd 6.18± 0.02ab 1.00± 0.01c 6.20± 0.08d
9% EAPI 6.27± 0.12ab 1.05± 0.04b 6.00± 0.09d 6.25± 0.01a 1.03± 0.04c 6.08± 0.23de
During the baking process, a set of transformations occur such as volume increase of
the product due to the gas production from leavening agents and water vaporization which
results in weight decrease (Chevallier et al., 2000a). Spread factor is a relation of biscuit
diameter to its thickness. It is considered to be a characteristic of the quality of the biscuit
and should be controlled. Depending on the type of cookies, high or low spread is desirable.
High spread ratio is expected in short dough biscuits which are high in both fat and sugar
content, whereas in semi-sweet formulations with lower amounts of sugar and fat, biscuits
may not flow or even shrink (Manley, 2011). As reported by Filipčev et al. (2011a), the well-
developed crumb is easier to achieve at low spread ratio. Control cookies were flat and soft
on the surface whereas those enriched with EAPC at 6% and 9% levels formed a nice crust
and resembled homemade cookies. The addition of EAPC at levels of 6% and 9% also
promoted the development of fine cracks on the surface (Figure 7.1) related to the
recrystallization of sucrose on the cookie surface during baking (Pareyt et al., 2008). On the
other hand, they were less friable and better maintained their integrity during storage. The
reduction of spread factor with an addition of canola proteins can be related to the
significantly increased thickness of biscuits. This increase was more pronounced with the
addition of protein concentrate. An increase in cookie thickness with the incorporation of
canola proteins indicates their ability to entrap and hold the CO2 leading to the expansion of
the cookies and thus acting as a structural element. Increase in thickness and reduction of
spread factor could also be related to the oil absorption capacity of canola proteins. As
reported by Sudha et al. (2007), the spread of the biscuits reduced significantly when fat
178
amount was lowered whereas a significant increase in the thickness was observed. In our
case, higher values for oil absorption capacity for canola protein concentrate were obtained
in comparison with protein isolate (3.02 ± 0.02 versus 0.97 ± 0.01) (Gerzhova et al., 2015b).
Figure 7.1: Cookies prepared with rice and dark buckwheat flours (1), rice and green buckwheat flours (2) with the incorporation of electro-activated canola protein concentrate and isolate: (A) Control; (B) 3% EAPC; (C) 6% EAPC; (D) 9% EAPC; (E) 3% EAPI; (F) 6% EAPI; (G) 9% EAPI.
The increase in cookies thickness and the reduction of the spread factor with
incorporation of safflower proteins has been reported by Ordorica-Falomir and Paredes-
López (1991). The addition of soy protein isolate also decreased the spread ratio of biscuits
(Rababah et al., 2006). In the other work the diameter of the biscuits increased with the
incorporation of sodium caseinate but decreased with the addition of whey proteins, whereas
both proteins decreased the thickness of the cookies (Gallagher et al., 2005). This information
shows that the behavior of the cookie during baking greatly depends on the type and
properties of its ingredients. Different proteins and different flours may have different impact
179
on the cookies dimensions. For more details, the effect of flour properties on cookie quality
has been elucidated in the work of Mancebo et al. (2015).
7.6.3. Dough characteristics
With the addition of canola proteins the handling characteristics of the dough
changed. As shown in the Figure 7.2, hardness of the dough significantly decreased with an
addition of 3% of canola protein concentrate and isolate comparatively to the control.
However, an increase in hardness was observed when the levels of protein concentrate was
increased. This could be related to the amount of water in the formulation as water in the
dough acts as a plasticizer (Faridi and Faubion, 1990). Water content was kept constant for
control cookie, 3% EAPI and 3% EAPC, however, the addition of 6% EAPC and 9% EAPC
as well as 6% EAPI and 9% EAPI required less water to obtain the dough of good
consistence. If the amounts of water were not adjusted the dough would become too liquid
and impossible to handle. The amount of water needed for the dough is related to the water
absorption capacity of its components. In bread making, rice flour based doughs require
very high hydration in comparison with wheat flour doughs to achieve an appropriate
consistency (Rosell and Marco, 2008). Rice flour is rich in starch, responsible for higher
water absorption as around 46% of flour’s total absorption is associated with the starch
(Hazelton et al., 2004; Mancebo et al., 2015). Protein concentrate on the contrary is highly
soluble and poorly binds water as was observed in our previous study (Gerzhova et al.,
2015a). The addition of canola protein isolate decreased the dough hardness, however further
increase in protein isolate concentration did not have any significant impact. This observation
is in good agreement with the literature, since a decrease in dough hardness with an increase
in protein concentration was noted by Gallagher et al. (2005) who studied the addition of
dairy protein powders. They remarked that dough became softer and more workable and at
the same time more mechanically resistant. Another study on the addition of some protein
isolates to the gluten free dough showed that the addition of soybean protein isolate caused a
decrease in the hardness, whereas the addition of pea protein increased this parameter (Marco
and Rosell, 2008). The role of gluten in the cookie system has also been studied and some
contradictory results have been reported. Even though the gluten network is not developed
in biscuits, its levels influence the dough hardness. Thus, in the work of Pareyt et al. (2008)
higher gluten levels decreased the dough hardness whereas in the work of Fustier et al. (2008)
180
the opposite has been found which could be due to different formulations used and different
concentration of the components.
Figure 7.2: Dough characteristics: (A) dough prepared with grey buckwheat; (B) dough prepared with green buckwheat.
The springiness increased with the addition of 3% of canola proteins for both isolates
and concentrates, and decreased with an increase in the level of proteins added. Springiness
refers to the elastic properties of the dough and its ability to spring back, recover after
deformation. Usually the elastic properties are referred to the presence of gluten and the
development of strong gluten network is undesirable in cookies formulation. On the other
hand, a certain degree of springiness should be present as it makes the dough easier to handle.
Since our formulation is gluten free, it can be concluded that canola proteins confer certain
00,10,20,30,40,50,60,70,80,91
0
500
1000
1500
2000
2500
3000
Hardness Springiness CohesivenessA
00,10,20,30,40,50,60,70,80,91
0
500
1000
1500
2000
2500
3000
Hardness Springiness CohesivenessB
181
elasticity to the dough and works as a structural agent. Similar observation was made by
Schober et al. (2003) on the addition of the soybean flour rich in proteins to the gluten free
dough and biscuits. In our case the addition of canola proteins made dough softer and
springier, easier to handle. The decrease in springiness with further increase in protein
concentration same as for hardness could be explained by the lesser amount of water used to
prepare the dough.
Cohesiveness is a parameter that could explain the structural integrity of the product.
Dough experiences a number of stresses before being baked. Thus, dough having adequate
cohesiveness will be more tolerant of handling during sheeting and cutting. No significant
difference was found between the compared samples. Cohesiveness varied from 0.22 to 0.28
for both types of buckwheat flours. Cohesiveness values of 0.2 – 0.3 have been reported for
biscuits supplemented with whey protein concentrate, which however increased to 0.3 – 0.35
with the addition of 10 and 15% of sodium caseinate (Gallagher et al., 2005).
7.6.4. Cookie characteristics
The hardness of the cookies, expressed in terms of force required to break it, is
presented in Figure 7.3. The hardness of the cookies decreased with an addition of canola
protein concentrate at 3% level but increased with an addition of 6% and 9% EAPC for both
grey and green buckwheat. The addition of EAPI at all levels significantly (p < 0.001)
decreased the hardness of biscuits. Similar tendency was noted for the dough hardness, which
is in accordance with Sudha et al. (2007) who found a high positive correlation between the
dough and the cookie hardness. Biscuits prepared from the flours higher in protein have been
reported to have harder structure as a result of strong adherence between proteins and starch
(Altındağ et al., 2015). It has been previously reported that biscuits supplemented with 10
and 15% of sodium caseinate were significantly harder than the control, whereas the addition
of whey proteins resulted in lower hardness similar to the control (Gallagher et al., 2005).
The authors explained this observation by the difference in water bonding capacities of tested
proteins. In the works of Conforti and Lupano (2004) and (Tyagi et al., 2007) the addition of
proteins also resulted in decreased hardness. Another positive correlation has been revealed
between biscuit thickness and the peak force required to break the biscuits (snap test)
(Gallagher et al., 2003). The results obtained in the current work match with the
182
aforementioned statement as the hardness of cookies increased with an increase in
incorporation level of EAPC same as the thickness of cookies increased.
Figure 7.3 : Cookie characteristics: (A) cookies prepared with grey buckwheat; (B) cookies prepared with green buckwheat.
As shown in Figure 7.3 the distance was significantly higher (p < 0.001) for all the
samples containing canola proteins in comparison with the control and it increased with an
increase in protein level. The distance represents the bending characteristics of the cookie. A
biscuit which is more flexible will bend before it breaks resulting in a longer distance. EAPC
had more pronounced bending characteristics (flexibility) than EAPI. The snap characteristic
of biscuits has been stated to be largely influenced by their dimensions. Thus, an increase in
0
2
4
6
8
10
12
14
0200400600800
10001200140016001800
Dis
tanc
e, m
m
For
ce, g
Force DistanceA
0
2
4
6
8
10
12
14
0200400600800
10001200140016001800
Dis
tanc
e, m
m
For
ce, g
Force DistanceB
183
distance was observed with an increase in the thickness of the biscuit and this observation
was in good agreement with the literature (Wehrle et al., 1999).
7.6.5. Surface colour
Table 7.4: Changes in surface colour of biscuits prepared from the blend of rice and dark buckwheat with the addition of canola proteins.
Parameter L* a* b*
Surface upper bottom upper bottom upper bottom
Control 58.66 ± 0.32a 44.06 ±
0.33a
4.37 ± 0.24a 8.04 ±
0.29d
23.31 ± 0.94c 18.28 ± 0.94c
3% EAPC 58.09 ± 0.60a 46.59 ±
3.66a
3.10 ± 0.37c 8.74 ±
0.14c
26.46 ± 0.49ab 21.02 ± 1.76a
6% EAPC 58.63 ± 0.83a 44.49 ±
1.32a
2.53 ± 1.93d 9.05 ±
0.28b
27.97 ± 0.53a 21.27 ± 2.70a
9% EAPC 57.27 ± 0.25a 42.46 ±
0.47b
4.65 ± 0.21a 9.33 ±
0.33a
27.46 ± 0.51a 19.14 ±
0.98ab
3% EAPI 58.87 ± 2.82a 45.47 ±
0.36a
3.73 ± 0.58b 8.59 ±
0.00c
22.71 ± 1.28c 19.14 ±
0.03ab
6% EAPI 57.89 ± 1.58a 45.56 ±
0.10a
3.81 ± 0.23b 8.47 ±
0.30c
22.07 ± 1.50c 19.29 ±
0.27ab
9% EAPI 58.08 ± 0.14a 45.54 ±
0.55a
3.13 ± 0.29c 9.09 ±
0.02b
22.17 ± 0.37c 19.35 ±
0.07ab
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Table 7.5: Changes in surface colour of biscuits prepared from the blend of rice and green buckwheat with the addition of canola proteins.
Parameter L* a* b*
Surface upper bottom upper bottom upper bottom
control 59.31 ± 0.73a 45.49 ± 0.22a 2.88 ± 0.18b 7.98 ± 0.11d 32.75 ± 0.71ab 20.50 ± 1.41a
3% EAPC 56.98 ± 1.00b 41.38 ± 3.57c 2.50 ± 0.39b 9.21 ±
0.39ab
34.16 ± 1.05a 21.01 ± 2.26a
6% EAPC 58.04 ± 2.14ab 41.95 ±
2.66bc
2.51 ± 0.32b 9.46 ± 0.13a 32.16 ± 4.36ab 20.79 ± 2.77a
9% EAPC 57.17 ± 2.33b 38.86 ± 1.11d 3.49 ± 0.09a 9.53 ± 0.06a 31.23 ± 2.47ab 18.01 ±
1.37ab
3% EAPI 59.61 ± 0.14a 44.06 ±
2.96ab
2.26 ± 0.19c 8.80 ±
0.19bc
29.53 ± 3.78b 21.75 ± 2.44a
6% EAPI 58.82 ± 0.04a 44.19 ±
1.19ab
2.31 ± 0.13c 8.90 ± 0.11b 29.04 ± 1.09b 21.05 ± 1.01a
9% EAPI 57.93 ± 1.37ab 44.21 ± 3.55a 3.37 ± 0.15a 9.04 ± 0.86b 27.80 ± 0.58c 20.79 ± 2.62a
Colour properties of the cookies prepared with blends of rice and buckwheat flours
with a supplementation with canola proteins are summarized in Tables 7.4, 7.5. For the top
surface of cookies prepared with grey buckwheat flour, no significant difference for L* and
a* parameters was found. However b* values indicating the yellowness showed significant
difference for cookies added with EAPC at all levels versus control cookie (p < 0.001). The
yellowness of the cookies increased with an addition of canola protein concentrate whereas
isolate did not bring any changes. This could be related to the colour of the added proteins
and the high contents of soluble sugars in the canola protein concentrate. These sugars may
enhance the intensity of the Maillard reactions during the baking process. Moreover, canola
protein concentrate is yellowish whereas the isolate is closer to neutral white colour when
hydrated which can be due to the presence of other compounds including polyphenols in the
concentrate. For the bottom surface, significant difference (p < 0.001) was found within a*
(redness) values for all cookies apart from those supplemented with 3% EAPI and 6% EAPI,
whereas lightness and yellowness of all the samples were statistically similar. The redness of
the bottom surface of the biscuits containing canola protein concentrate was comparatively
higher than the control or those containing canola protein isolate. In comparison with the top
surface, the bottom surface had higher a*, b*, and L* values in all the samples. Similar
185
observation has been reported by Torbica et al. (2012) for a*. For cookies prepared from
green buckwheat no significant difference (p > 0.05) was found for the top surface. For the
bottom surface, the a* parameter was significantly different (p < 0.001) for cookies prepared
with 6% EAPC and 9% EAPC, respectively.
Colour development during baking comes not only from the colour of ingredients
used in formulation but also from the non-enzymatic browning known as Maillard reactions
which take place between reducing sugars and amino groups of specific amino acids. Thus,
higher protein content of enriched biscuits can contribute to the darker colour (Gallagher et
al., 2003). Other reactions which impart the development of specific colour during baking of
the final product are sugar caramelization and starch dextrinization (Chevallier et al., 2000a).
The lesser amounts of water in biscuits prepared with canola protein concentrate could also
enhance the caramelization of sugars giving the darker surface with reddish-brown hues
(Mancebo et al., 2015). The amount of available water (free water) and its distribution are
also important factors for Maillard browning and a strong positive correlation has been
established between L* values and water activity levels (Gallagher et al., 2003). The effect
of protein addition on the crust colour could have been more pronounced if the formulation
did not contain honey which is high in fructose which is a reducing sugar participating in
Maillard reactions.
7.6.6. Moisture content and water activity
Moisture content and water activity showed similar tendency (Figures 7.4 and 7.5)
decreasing with the addition of protein concentrate, and not showing significant difference
with the addition of canola protein isolate. Biscuits containing 9% EAPC had the lowest
moisture content (5.75 ± 0.26% and 6.04 ± 0.49% for grey and green buckwheat flour,
respectively) as well as the lowest water activity (0.48 ± 0.01% and 0.48 ± 0.04%) which is
a measure of food dryness and susceptibility of a product to spoilage (Filipčev et al., 2011b).
As mentioned before, canola protein concentrate is highly soluble and do not actively bind
water which is why the initial amount of water was reduced in the formulation for those
samples. This explains the reduction in moisture content with an increase in EAPC
concentration. Other works on the contrary reported increased moisture content with the
incorporation of protein products or other flours explained by their higher water retention
capacity (Torbica et al., 2012; Tyagi et al., 2007). Lower water activity in biscuits
186
supplemented with protein is due to the less available water in the system which has also
been observed by Gallagher et al. (2005) on the addition of whey protein concentrate and
sodium caseinate. The high positive correlation (0.928) between moisture and water activity
has been reported by Schober et al. (2003) which led to the conclusion that it is the amount
of water in the baked biscuit which had the strongest effect on the available water.
Figure 7.4 : Moisture of baked biscuits.
Figure 7.5: Water activity (aw) levels for baked biscuits.
0
2
4
6
8
10
Grey Buckwheat Green buckwheat
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
Grey buckwheat Green buckwheat
187
7.6.7. Microstructure analysis
Microstructure of rice and buckwheat flours as well as canola proteins is shown in
Figure 7.6. The most abundant and visible ingredient in rice and buckwheat flours is starch.
Rice flour is composed of fine polygonal-shape starch granules which are less than 5 µm and
grouped in agglomerates. Both grey and green buckwheat flours are represented by round
shape starch granules densely packed in clusters. This is in a good agreement with Mariotti
et al. (2008b), Torbica et al. (2012), and (Hager et al., 2012). The images obtained for green
and grey buckwheat flours are very similar showing no difference on the microstructural
level. EAPC is composed of uneven fragments of different shapes and sizes whereas EAPI
is represented by rather large particles of irregular shape.
Figure 7.6: Scanning electron microscopy at 1000x magnification of: (A) rice flour; (B) grey buckwheat flour; (C) green buckwheat flour; (D) EAPC; (E) EAPI at 300x magnification; and (F) EAPI at 1000x magnification.
The micrographs of baked biscuits enriched with EAPC are presented in Figures
7.7A-C, those enriched with EAPI in Figures 7.7D-F and control sample is shown in Figure
7.7E. As the microstructures of green and dark buckwheat were identical only the biscuits
made with green buckwheat are presented here. The matrix of a baked cookie has been
characterized as a composite of fat, sugar, and proteins in which intact or partially damaged
starch granules are embedded. The sugar that melts during baking and become glassy after
cooling provides the structure cohesiveness and link fat, proteins and starch granules
188
(Chevallier et al., 2000a). The starch granules when heated in water undergo the swelling and
the disruption of molecules while losing its crystallinity which is known as gelatinization.
The further heating leads to the formation of a continuous phase of solubilized molecules and
a discontinuous phase of swollen, amorphous starch granules (Arendt et al., 2008). It has
been stated that in biscuit system starch granules are mostly left intact due to low moisture
content and the presence of sugars which prevents starch gelatinization (Chevallier et al.,
2000a; Chevallier et al., 2000b). In Figures 7.7A, D, and G more starch granules are
gelatinized in comparison with other micrographs which can be due to the higher water
content of these samples. In the Figures 7.7A, B, C, and F more intact granules can be
discerned. Also the amount of gelatinized starch is not the same in the center and on the top
surface. Chevallier et al. (2000a) compared the center of biscuits and the top surface and
found that in the biscuit center some starch granules were swollen and some granules have
partially lost their birefringence whereas the top surface due to water evaporation from the
surface during baking showed more intact starch granules. Filipčev et al. (2011a) compared
the microstructures of the dough and the cooked biscuits and found them very similar.
Torbica et al. (2012) also reported intact starch granules in cookies based on rice/buckwheat
formulation. The presence of carboxymethyl cellulose (CMC) due to its hydration capacity
could be the reason of lower water content in the biscuit system preventing starch
gelatinization.
189
Figure 7.7 : Scanning electron microscopy of the cross section of selected biscuits at 1000x magnification: (A) 3% EAPC; (B) 6% EAPC; (C) 9% EAPC; (D) 3% EAPI; (E) 6% EAPI; (F) 9% EAPI; (G) control.
The surface of the cross sections of biscuits supplemented with canola proteins looks
more uneven including some cavities and presumably revealing some structure capable of
holding air cells during baking resulting in thicker biscuits. The control sample as well as
those containing 3% EAPI and 3% EAPC has less pronounced cavities. Regarding the
volume increase during baking (Table 7.5), the control sample was the densest followed by
a 3% EAPI and 3% EAPC supplemented cookies which showed the minimal volume
increase.
7.6.8. Preliminary sensory evaluation
Even though the health aspect and nutritive value is of great importance the sensory
characteristics remain the most important ones in terms of customer acceptance and thus
cannot be neglected. The results of the sensorial test are presented in the Figure 7.8. Only
190
the cookies made with the green buckwheat were taken for the test as such having milder
taste and more appealing yellowish colour (based on the preliminary test). In general, all of
the samples were rated between 6 and 7 for their appearance with no statistical difference
between them (p > 0.05). Aroma and taste are sometimes difficult to distinguish for most
people and as was shown by the analysis the scores for these two characteristics were very
similar. As judged by the panelists the taste and aroma of supplemented cookies were
increased with the addition of protein isolate and 9% EAPI got the highest appreciation.
Protein concentrate was admitted to add a specific flavor and aftertaste which was not equally
judged by different panelists. However, the taste and the aroma are not critical and can be
easily corrected by the addition of spices. The most crucial difference was noted in terms of
texture which also had a great impact on the overall appreciation. The control cookie had the
lowest score (2.50 ± 0.76) and was characterized as hard and dry. All the samples got
significantly higher marks compared to control (p < 0.001). The highest scores for texture
was assigned to the cookie with 9% EAPC (8.13 ± 0.64), characterized as crunchy and
slightly moist and making a specific noise when bitten. These features made it pleasant to
bite and resulted in the highest acceptance among the panelists. Other samples, notably 3%
EAPC and 6% EAPC got 5.00 ± 1.60 and 7.25 ± 1.83, respectively. Cookies supplemented
with protein isolates were similar to EAPC supplemented cookies and were rated accordingly
(5.88 ± 1.25, 6.38 ± 0.92, and 7.75 ± 1.04, for 3% EAPI, 6% EAPI, and 9% EAPI
respectively). Regarding overall acceptability the sample with 9% EAPI scored the maximum
among all the others (7.00 ± 0.76), whereas the control cookie got the minimum (4.38 ± 1.06).
The results of the test showed improved sensorial characteristics of gluten free enriched
cookies. Not only the protein addition resulted in a healthy gluten-free snack but was also
positively evaluated by judges in terms of organoleptic properties.
191
Figure 7.8 : Preliminary sensory characteristics of canola protein enriched cookies.
Conclusion
Canola protein concentrates and isolates were successfully incorporated into the
gluten free biscuits made from a blend of rice and buckwheat flours at the ratio of 80/20 with
3, 6 and 9% protein as substituting levels of the rice flour. The addition of canola proteins
needed the recipe adjustment for water content. Less water was needed for cookies enriched
with either protein isolates or concentrates in order to achieve the dough which can be
192
handled. Analyses showed significant difference for quality parameters of dough and
biscuits. The thickness and the diameter of biscuits enriched with 6% EAPC and 9% EAPC
were significantly higher in comparison with the control which resulted in lower spread ratio.
The addition of canola proteins produced significantly thicker biscuits with more aerated
texture which is an indication of improved gas holding capacity of the dough. This was also
observed in the micrographs of protein supplemented cookies. The addition of canola
proteins significantly changed the textural properties of dough and biscuits making them less
hard but springier. The amount of water in the initial formulation had a great impact on
textural parameters as well as on the moisture and water activity. Higher amount of water led
to the softer and less springy dough with higher moisture content and water activity. As the
addition of proteins required less water, the hardness increased with further increase in
protein concentration. The results on the colour changes showed a significant increase in the
product yellowness with the addition of EAPC. Other than that, the addition of canola
proteins did not bring significant changes for the lightness or redness/greenness of the end
product. The changes in biscuit hardness were in line with the changes in dough hardness.
Indeed, hardness of the cookies, expressed in terms of force required to break it decreased
with addition of canola protein concentrate at 3% level but increased with an addition of 6%
and 9% EAPC for both grey and green buckwheat. The addition of EAPI at all levels
significantly decreased the hardness of biscuits making them softener and more pleasant for
tasting. From sensorial point of view, addition of canola proteins to cookies made from a
blend of rice and buckwheat flours makes it possible to produce gluten free product with
good acceptability. Indeed, sensorial test showed improved overall acceptability as well as
taste and aroma of protein supplemented cookies; however the most significant changes were
noted in terms of texture. Gluten free rice and buckwheat cookies in the absence of a structure
making agents were hard, dry, and brittle whereas canola proteins imparted crunchiness,
slight moistness and as a result a pleasant mouthfeel.
Finally, the current work showed that canola proteins can be added to the gluten free
biscuit formulation in order to improve its nutritive value and texture, and control the spread.
However, the optimization of the formulation is needed followed by the sensorial analysis in
order to produce biscuits with acceptable organoleptic quality. Further work should be aimed
193
for the optimization of the formulation which can significantly improve the organoleptic and
handling properties
Acknowledgments
This work was financially supported by the innovation in food support program that
was funded by contracts through the "Growing Forward" Program that occurred between the
Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (Ministry of
Agriculture, Fisheries and Food of Quebec) and "Agriculture and Agri-Food Canada".
194
8. CHAPTER 8: Conclusion and perspectives
General conclusion
Current work focused on two main objectives, one of which was to investigate the properties
of electro-activated solutions regarding protein extraction from an industrial by-product of
oil production which is canola meal. The number of benefits of canola protein has been
reported among which are balanced amino-acid content and functional properties which can
be exploited in the production of many conventional foods. As for EAS their disinfecting and
catalytic functions are being extensively studied and there are indications of their extractive
ability as well.
Although canola proteins have been previously studied the amount of cultivars and the
various techniques used for the extraction of proteins made it difficult to choose the best
suitable extraction parameters. Furthermore the data on the simultaneous extraction of dry
matter has not been reported. Which is why the first objective was directed at the selection
of optimal parameters for maximal protein extraction and the verification whether such
parameters will result in the highest dry matter extraction or not. Meal to solvent ratio, pH
and a salt concentration were tested in connection with it. The most significant parameter
was pH of the extracting solution which was in accordance with published results. Salt
concentration did not have a significant impact on protein extractability in spite of the fact
that the main fraction is represented by globulins. SDS PAGE showed numerous fractions
with molecular weights varying from 7 to 100 kD revealing complicated protein composition.
A new approach was chosen for the determination of isoelectric point which combined the
turbidity and zeta-potential measurements. As was shown maximum turbidity was correlated
with minimal ζ-potential corresponding to zero net charge on the surface of protein molecule.
Second objective was aimed to modulate the parameters optimal for protein extraction from
canola meal with regards to results obtained in the first objective. Although electro-activated
solutions are becoming more and more popular due to their unique properties literature
review failed to provide with information required for choosing such parameters.
Considering that fact 4 types of configurations, three salt concentrations, three different times
of treatment and three current intensities were analyzed for modulating the extraction
conditions. Their effects on the pH and alkalinity propagation have been studied. The results
195
showed a significant difference between these two parameters. Alkalinity was shown to be
the most important parameter describing the force of electro-activated solutions. It was
largely dependent on the time of treatment and on the current intensity. The ionic strength of
the solution was significant only for two configurations showing the importance of the ion-
exchange membrane disposition in electro-activation reactor. More specifically the choice of
the membrane can improve or impair the efficiency of alkalinity generation as was shown
with configuration 2 where the withdrawal of anions from cathodic compartment enabled
water dissociation on the membranes surface neutralizing the alkalinity. Also for two and
three compartment cells similar results were obtained, which allowed us to reduce the number
of configuration for the next objective.
In the third objective the catholyte generated in the three-cell reactor has been investigated
for its extraction capacity and its effect on protein composition, secondary structure and
functional properties. Same analyses were performed on the proteins obtained following the
conventional technique. In general the effect of extracting solutions was comparable,
however certain improvements of the new technique were observed. Thus, the SDS PAGE
test showed clearer and more distinguishable bands which signified that proteins were less
denatured. Furthermore the FTIR study also proved that statement by providing narrower
peaks associated with native structure for protein extracted with EAS. As in case of
conventional technique where the quality of proteins following the FTIR peaks and SDS
PAGE bands was reduced with an increase in pH of extraction, the quality of proteins
extracted with EAS decreased with an increase in time of treatment.
In terms of functionalities studied in the fourth objective, proteins extracted by EAS showed
improved surface active properties suggesting their utilization in related food systems. Also
protein isolates and concentrates showed different behaviour depending on the pH at which
they were studied. Concentrates were more soluble within all the tested pH range and
performed better at their isoelectric point. Isolates were comparatively less soluble due to the
additional acid treatment for protein precipitation and to the loss of soluble proteins during
precipitations step.
Last objective was to verify the behaviour of proteins in real foods systems where gluten free
biscuits were chosen as an example of application. Baked goods are mostly prepared from
wheat flour, which is not suitable for people sensitive to gluten. Growing demand for gluten-
196
free products has been attributed to the increasing awareness of customers, improved
diagnostics as well as progressive marketing. The biggest problem in gluten free products is
the lack of structure, associated with lower sensorial characteristics. Also most of them are
poor in nutriments. The addition of canola proteins exhibited two roles, increased the
nutritive value and enhanced the structure. As a result of protein addition the textural and
sensorial characteristics were significantly improved.
Overall the results answered the stated hypothesis and showed the potency of EAS as an
extracting agent. The study revealed some benefits of using EAS as alternatives to
conventional extracting method, however it poses some new questions and leaves room for
further investigations.
Perspectives
Although this PhD work endeavoured to cover as much as possible, some of the observations
were restricted by the limits of chosen parameters. The addition of more points and more
factors might supply with the important information which would substantially improve the
quality parameters. For further perspectives there are several directions. First is adjusting the
parameters of EA and testing their effect on same physico-chemical and functional
properties. For example, the extraction of proteins using EAS treated for lesser amounts of
time e.g. 10-45 min might extract the proteins of higher quality, although reduce the
extractability rate. Other extracting parameters might also allow to extract different fraction
of proteins. Since canola consists of a mixture of proteins with largely varying properties, the
possibility of their fractionation while extracting seems highly appealing.
Furthermore, the protein precipitation can be performed by using the EAS treated in anodic
compartment respectively. This will allow to use efficiently both solutions anolyte and
catholyte at the same time. Another possibility is to perform the protein extraction directly in
the cathodic compartment while being connected to the electric power. For this the constantly
generated products of electrodes reactions will immediately react with meal which should
influence the extraction process.
It will be of interest to try EAS on other oilseed proteins such as soybean, sunflower or
leguminous such as pea.
197
The gelling properties have not been investigated within the scope of the current research
project, however gelling systems are frequently encountered in food systems, and their study
will broaden the spectrum of possible industrial application.
The possibility of protein incorporation in other food systems (pasta, bread, simulated meat
products) should also be studied and possibly compared to the protein that already found a
wide application, such as soybean.
The bioavailability of proteins themselves and in food products should also be checked
primarily in order to see their degradation in digestive tract, which can be performed by
performing simulated digestion. Finally, clinical tests on tolerance and allergenicity will be
needed to provide the data on safety of such preparation so that it could reach the commercial
scale.
198
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