Food concepts for specific target groups: The role of ... · essentieller Aminosäuren bei gleichem kalorischen Wert Isolierung von Lactoferrin und anderen minoren Proteinen als Functional

Food and Bioprocess Engineering

1

„Food concepts for specific target groups:

The role of dairy components and processing technologies”

Prof. Dr.-Ing. U. Kulozik

Technical University of Munich

Freising-Weihenstephan


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3

Original citation from: “此物胜肉远矣” 健体强身的乳制品

该书指出：“牛乳最宜老人，平补血脉、益心

、长肌肉，令人身体康强、润泽、面目光悦，

志不衰。”

书中强调指出:“故为人子者当须供之，以为常

食，或为乳饼…，恒使恣意充足为度。此物胜

肉远矣！

元代邹宏著《寿亲养老新书》中有关于牛奶的食疗方

—“食治老人益气牛乳方”。


4

During the Yuan-Dynasty (1206-1368) Zou Hong published a medical text book (which is

still being referred to in modern China), entitled

“The new book about the theory, how to nurture your parents, for them to live a long life”

In this book a diet is proposed: The recipe to strengthen the body´s energy flow by

means of bovine milk. It says:

“Cow milk is particularly good for the elderly. It can strengthen the blood circulation, it

is good for the heart and build-up of muscles. Milk makes mentally and physically

strong and yields a brilliant appearance.“

„Therefore, one should prepare dishes for the parents using milk products, either a

liquid milk or bakery products containing milk in sufficient amounts. Dairy products

are much better then meat.“

“Food with a value far above that of meat”

- Dairy products that can strengthen the body

Milk is a preventively therapeutic substance as part of the Traditional Chinese Medicine (TCM)


5

Target areas of the food and pharma industries in public health

Well-Being/

Appearance/

Performance

Age

‚Adding life to years‘

Vision for foods for the developed world (EU strategy)

Products with a positive effect on health and quality of life in the last quarter of life.


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Prozess-

gestaltung/

- bedingungen

Moleküleigen-

schaften

biotischer

Matrices

Prozess-Struktur-

Funktion,

Reaktionskinetik

Struktur-

Eigenschafts-

Funktion

Humansensorische

Funktion

Wissenschaftl.

Verständnis

Vorhersagbares

Verhalten

Arbeitsmethodik Life Science-Verfahrenstechnik

Verknüpfung von Substrat, Verfahrenstechnik und Analytik

Lebensmittel-

verfahrenstechnik

Physiologische

Funktion

Molekulare Struktur;

Interaktionspotenziale

Funktionalitätsursachen

Prozesstechnische

Stoffumwandlung

Biologischer

Effekt

Ernährungs-

wissenschaft


7

A non-dairy example: Healthy eggs concepts across age groups (1990ies)


8

Target groups for personalized nutrition

• Pregnant women

• Pre-term born infants

• Infants, Small children

• School children

• Sports people

• Genetically predisposed people (e.g.

Phenylketonurie, PKU; Allergenicity)

• Reconvalescent patients

• Normal people (reducing the risk of

developing certain diseases)

• Elderly people (Cognitive performance,

Sarcopenia, Dysphagia)

• …


9

Methods for enrichment or isolation of single milk or whey fractions

Classical coagulation/aggregation reactions

- enzymatic

- thermal

- isoelectric

Chromatography

Ultrahigh pressure

Centrifugation

Membrane technologies

- Mikrofiltration (0,1 – 2 µm)

- Ultrafiltration (5 – 300 kDa)

- Nanofiltration (200 – 5.000 Da)

Combination methods/

Hybrid technologies

DF-Medium: UF-Permeate –lactose/salt

MF-Retentate – micellar casein UF-Retentate – native whey

Micellar casein Molke

Spiral woundmodul

(10 kDa)

Ig- rich whey

Skim milk/ Colostrum

Pore size(0,1 µm)

Ideal Whey

CFU Igs


10

WPI

UF/Diafiltration

0,5-2,0% Protein

Heat treatment

105°C/5 s; pH 7,5

MF

0,1 µm

Retentate

aggregated b-Lg Permeate

natives a-La

m in5 7 .5 10 12 . 5 1 5 1 7. 5 20 22 . 5

0

100

200

300

400

500

native

heated

a-La b-Lg B

b-Lg A

pH adjustment

Method and process to obtain isolated a-La and microparticulated ß-Lg

Potential medical applications of a-Lactalbumin

• Induction of apoptose of tumor cells

(„HAMLET/BAMLET“)

• Treatment of skin papilloma

• Increased serotonin release and induction of

anxiolytic effects; Good mood under stress

• Ca-/Zn-/Fe-binding properties


11

Whey protein

solution

UF-Diafiltration

20 % ß-Lg, Citrat

Heat treatment

56 °C/40 min; pH 3,6

Diafiltration

(UF/Water)

Microfiltration

0,1 µm

Retentate

denat. a-La Permeate

natives ß-Lg

native

heated

RO of UF-

Permeate

Method and process to obtain isolated ß-Lg

and microparticulated a-La


12

Effect of HAMLET/BAMLET on human skin papillomas

Gustafsson et al (2005). J. Nutrition 135, 1299-1303

Delgado et al. (2015): FEBS Open Bio 5, 397-404

Hoque et al. (2015) Biochimica Biophysica Acta 1850, 1729–39

HAMLET/BAMLET“: Human/Bovine Alpha-Lactalbumin Made LEthal to Tumor cells

Mechanism: Protein acts as emulsifier to expose oleic acid to the cell membrane, where it disrupts

or disturbs the cell wall structure of the tumor cell.


13

2500 g/10 rpm/15 L·h-1

b-Lactoglobulin: Process for removal from whey

for the manufacture of humanized infant food products

Selective thermal aggregation Centrifugal separation Chromatographic purification/

isolation

85 °C/ 15 s/180 L·h-1 pH 7/50 °C/10 CV·min-1

1 2 3

Yield (α -La) = 89%

Removal (β-Lg) = 95%

Yield (α -La) = 83%

Removal (β-Lg) = 98%

Reason: b-Lg does not exist in human milk. It is considered the main allergen in milk.


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Examples of some natural bioactive substances in milk

and their reported functions

Casein macropeptide

Lactoferrin

Immunoglobulins

Growth factors

Insulin

Cytokines

Milk fat globule

membrane proteins

b-Casomorphins

Somatostatin

Prolactin

Delta-sleep peptides

Some bioactive peptides/proteins

native to milk and colostrum

The big question: Are the native

bioactive peptides in milk enough

to benefit the consumer?

Functions

• Mineral binding

• Hypertensive

• Immuno peptides

• Antimicrobial

• Anti-inflammatory

Intestinal

or Plasma-

Enzymes

Microbial

enzymes

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Release of caseinomacropeptide during renneting

Dalgleish et al. 2004

• 50 % of CMP unglycosylated

• 50 % of CMP glycosylated (GMP)

• Very heat stable in comparison

to whey proteins

• High amount of essential amino acids

• High amount of branched-chain amino acids

• Absence of all aromatic amino acids (Phe, Tyr, Trp)

CMP properties

GalNAc

NeuNAc

Gal

Amino acid

chain


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Physiological and biological properties of CMP

Phenylketonuria (PKU)

• Metabolic disease: Phe can not be metabolised

• CMP contains no Phe tolerable natural protein source

Enrichment of CMP in

dietetic products

Hyperthreoninemia

• High content of Threonin Amino acid imbalance

in infant milk formulas

Absence of CMP in

infant milk formulas

Gut microbiota

• CMP acts as prebiotic substance promoting Bifidobacteria

• Defence against pathogens

Other Properties

• CMP acts as anticariogenic substance

• Anti-inflammatory properties

• Positively supporting treatments of certain liver diseases

Recio et al. (2009): Glycosylated dairy components- Their roles in nature … and

functionality in dairy products. In. Dairy-based ingredients, CRC Press


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Fractionation of CMP/GMP by IEX-Chromatography

Time [min]

0,0 0,5 1,0 1,5 2,0

Ab

so

rptio

n a

t 2

18

nm

[m

Au

]

0

500

1000

1500

2000

2500

Co

nd

uctivity [

mS

/cm

]

0

20

40

60

80

100

Conductivity Aglyco-CMP Glyco-CMP


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Separation principle of Cross-Flow Electro membrane filtration (CFEMF)

Variables:

• Position of the electrodes

• Voltage

• pH

• Temperature

• Crossflow/pressure

conditions

18

Diss. Holder, Univ. of Hohenheim, 2014


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Enzymatic pre-treatment and fractionation of CMP from whey proteins

Met - Ala - Ile - Pro - Pro - Lys - Lys - Asn - Gln - Asp - Lys - Thr - Glu - Ile - Pro -

Thr - Ile - Asn - Thr - Ile - Ala - Ser - Gly - Glu - Pro - Thr - Ser - Thr - Pro - Thr -

Ile - Glu - Ala - Val - Glu - Ser - Thr - Val - Ala - Thr - Leu - Glu - Ala - Ser - Pro -

Glu - Val - Ile - Glu - Ser - Pro - Pro - Glu - Ile - Asn - Thr - Val - Gln - Val - Thr -

Ser - Thr - Ala - Val - OH

Nominal MW 7 kDa

(too close to other whey proteins

to be separated by size)

No aromatic amino acids

High level of the amino acid threonin

Contains Glutamin and Lysin Crosslinking

by enzyme Transglutaminase possible


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Crosslinking of CMP by the enzyme Transglutaminase

CMP A

CMP B

untreated

10 min

120 min

Incubation time [min]

Rem

ain

ing C

MP

[%

]

Glycosylated CMP

0

20

40

60

80

100

0 20 40 60 800

20

40

60

80

100

0 20 40 60 80

WPC

Cpr = 6 %

TG: 3 U/g protein

= 40 °C

At this point removal of crosslinked CMP-

aggregates by microfiltration or centrifugation

CMP-free rennet whey

Assessment of CMP aggregates as

functional ingredient


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Origin, structure and composition of the Milk Fat Globule Membrane (MFGM)

(Lopez et al., 2008)

Monolayer

Bilayer

Components MFGM-

DM

Proteins 70%

Phospholipids 25%

(Walstra et al., 2006)


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SD

S-P

AG

E in

ten

sit

y (

MF

GM

pro

tein

s)

[%]

0

20

40

60

80

100

XO/XDH

BTN

PAS 6/7 PAS 6/7: 3.17

PAS 6/7: 40

MFGM isolates compared to native BM

(<1% skim milk proteins)

Cream washing Coagulation method

XO/X

DH

XO/X

DH

PAS 6/7

PAS 6/7

BTN

BTN

Increase of MFGM protein yield by optimizing the isolation method:

Evaluation of washing method and coagulation method

Significantly higher protein yield using the coagulation method by avoiding

losses of the outer double layer.

(≙ Diafiltration of BM whey)

MF

GM

pro

tein

yie

ld


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Immunoglobulins in colostrum and milk

Reported medical treatments

• ETEC (14 strains enterotox. E. coli)

• HIV-1 gp140

• Clostridium difficile

• Influenza virus

with success rates between 85-95%


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Fractionation of immunoglobulins from milk

• After 6 diafiltration steps IgG is below 10% on the MF-retentate side

Combination of MF and UF operated in diafiltration mode opens the opportunity to

produce Ig-rich WPC at industrial scale




Spiral woundmodul

(10 kDa)

Ig- rich whey


Pore size(0,1 µm)

Ideal Whey

CFU Igs


26

Heat sensitivity of immunoglobulins: Iso-effect lines for IgG and IgA in milk


27

Immunoglobulins from vaccinated cows: Basic Idea

Immunizing the cow to

obtain high titers against

hospital germs

Use for the treatment

of human health

issues resulting from

MRS

Process for IgG

isolation

Need for alternative methods for the treatment of antibiotic resistant, pathogenic organisms

Mixed mode chromatography




Spiral woundmodul

(10 kDa)

Ig- rich whey


Pore size(0,1 µm)

Ideal Whey

CFU Igs

Membrane fractionation (MF/DF)


28

Target areas of the food and pharma industries in public health

Well-Being/

Appearance

Level

Age

‚Adding life to years‘

Vision for foods for the developed world (EU strategy)

Products with a positive effect on health and quality of life in

the last quarter of life.


29

Chair for Food and Bioprocess Engineering

&

Technology Section of the ZIEL Munich

Garching

Freising-Weihenstephan

River Isar

Munich airport


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Conclusions

• Milk is a healthy juice and a good base for new product concepts also outside of

conventional dairy product segments

• Basic insights into nutritional and physiological effects are there and appear to be

reliable.

• Screening methods for more indepth assessment of physiological effects are

established

• Controlled studies, preferably human clinical studies, should be conducted

following the rules and principles of „Good Clinical Study Practice“

• Nutritional supplements and product concepts for special target groups are

extendable market segments, even outside the food sector.

• So, what is missing? Prospective, double blind, controlled in vivo studies?

• Who wants to go this way? Positioning in the market place in/out of food

segment?


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Categories from conventional foods to functional foods

and prescription drugs

(Calvo et. al. 2016, Development of novel functional ingredients: …

Trends in Food Sci.&Technol. 54, 197-203

Frei verkäufliche Rezeptpflichtige

Arzneimittel


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Zielprodukte für Fraktionen von Molkenproteinen

Abreicherung von β-Lactoglobulin für

Babynahrung:

An Humanmilch angenäherte Protein-

zusammensetzung mit erhöhtem Anteil

essentieller Aminosäuren bei gleichem

kalorischen Wert

Isolierung von Lactoferrin und anderen minoren Proteinen als Functional

Food oder Food for Special (Medical) Purposes


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Milk protein 100 %

Milk fat globule

membrane

Casein

80 %

Whey proteins

(soluble at pH 4.6)

a - Casein

15 - 19 g/l

b - Casein

9 - 11 g/l

k - Casein

2 - 4 g/l

a - Lactalbumin

0,6 - 1,7 g/l

b - Lactoglobulin

2 - 4 g/l

Proteose Pepton 3 ( Lactophorin )

HFPP ( 300 mg/l) Hydrophobic fraction

of proteose peptones

Immuno

globuline

Proteose Pepton 5 b - CN - 5P

Proteose Pepton 8f b - CN - 4P

Proteose Pepton 8s b - CN - 1P

Caseinomakropeptid

CMP

para - k - Casein ~ 2 g/l

Serumalbumin

0,4 g/l

Gesamtanteil:

1,2 g/l

a S1

- Casein 12 - 15 g/l

a S2

- Casein 3 - 4 g/l

Osteopontin 3 - 10 mg/l

b 2 - Microglobulin

( Lactollin ) 9,5 mg/l

Vitamin bindende Proteine

Angiogenine

Angiogenin 1

4 - 8 mg/l

Angiogenin 2

Kininogen

Lactogenin

Glycolactin

Metall bindende Proteine

Minore Glykoproteine

Lactoperoxidase LP

30 mg/l

Xanthin Oxidase XO

M - 1

Orcosomucoid

( a 1 - Säure GP) ~ 20 mg/l

Betacellulin restl. Mucine

Transferrin Lactoferrin Ceruloplasmin ( Ferroxidase )

Prosaposin 6 mg/l

Folat bP 6 - 10 mg/l

Vitamin D bP 16 mg/l

Vitamin B 12

bP ( Transcobalamin )

0,1 - 0,2 mg/l

Riboflavin bP

Niedermolekulares Kin

Bradykinin

Hochmolekulares Kin

Lactoferricin B

Immunoglobulin G1 (IgG1)

300 - 600 mg/l

Immunoglobulin G2 (IgG2)

50 mg/l

Immunoglobulin M ( IgM )

90 mg/l

Immunoglobulin A ( IgA )

10 mg/l

Sekretkomponente (SC) 20 - 100 mg/l

Immunoglobulin

E ( IgE )

Immunoglobulin D ( IgD )

Lysozym

0,3 mg/l

Wachstumsfaktoren

Hormone

Phosphopeptide

b - Casomorphine

Glutathion Peroxidase GPX

Superoxid Dismutase SOD

Catalase

Oxidoreduktasen

Hydrolasen

(Alkalische Phosphatase )

Transferasen

Lyasen

Isomerasen

Ligasen

Enzyme

Angiotensin - 1 - Converting

Enzyme Inhibitor - Peptide

(ACEI)

b - CM - 7

b - CM - 9

b - CM - 13

b - Neocasomorphin - 6

b - CM - 21

b - CN - 4P

a S1

- CN - 5P

Lactoferroxine

Albutensin A

Serorphin

Laktokinine

Casoxin A,B,C

Casoplatelin

Casocidin - I

Casokinine

PAS - 1

PAS - 3

PAS - 4

Butyrophilin

BRCA - 1

MUC - 1

CD - 36

Isradicin

Casecidin

Casecidin

b - Lactorphin a - Lactorphin

a S1

- Casein Exorphin

© by Markus Kreuß & Nadja Siegert 2006

= Source/Group

= Protein

= Peptid

= Glykoprotein

Casopiastrin

Morphiceptin

Insulin

NGF

TGF - a

TGF - b

EGF ~ 2 ng/ml

Pareathyroid -

Hormon

Motilin Somatostatin

Gastrin -

releasing P.

Gonadotropin -

releasing P

IGF - II 75 ng/ml

IGF - I

30 ng/ml


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References

• Tolkach, A, Kulozik, U: Fractionation of whey proteins and caseinomacropeptide by means of enzymatic crosslinking and membrane separation

techniques. J Food Eng, 67 (1-2) 13-20, 2005

• Tolkach, A, Kulozik, U: Optimization of thermal pre-treatment conditions for the separation of native a-lactalbumin from whey protein concentrates by

means of selective denaturation of b-lactoglobulin. J Food Sci, 70 (9) 557-566, 2005

• Thomä-Worringer, C, Sorensen, J, Lopez-Fandino, R: Health effects and technological features of caseinomacropeptide. Int Dairy J, 16 (11) 1324-33,

2006

• Tolkach, A, Kulozik, U: Reaction kinetic pathway of reversible and irreversible thermal denaturation of b-Lactoglobulin. J Dairy Sci Technol, 87, 301-

315, 2007

• Kreuß, M, Krause, I, Kulozik, U: Separation of a glycosylated and non-glycosylated fraction of caseinomacropeptide using different anion-exchange

stationary phases. J Chromatography A, 12 (1-2) 126-132, 2008

• Kreuß, M, Kulozik, U: Separation of glycosylated caseinomacropeptide at pilot scale using membrane adsorption in direct-capture mode. J

Chromatography A, 12 (19) 50, 8771-77, 2009

• Cheison, SC, Leeb, E, Sierra Toro, J, Kulozik, U: Influence of hydrolysis temperature and pH on the selective trypsinolysis of whey proteins and

potential recovery of native b-lactalbumin. Int Dairy J, 21 (3) 166-171, 2011

• Leeb, E, Kulozik, U, Cheison, SC: Thermal pre-treatment of β-Lactoglobulin as a tool to steer enzymatic hydrolysis and control the release of

peptides. Procedia Food Sci, Vol. 1, 1540–46, 2011

• Voswinkel, L, Kulozik, U: Fractionation of whey proteins by means of membrane adsorption chromatography. Procedia Food Sci, 1, 900-907, 2011

• Betz, M, Steiner, B, Schantz, V, Oidtmann, J, Mäder, C, Richling, E, Kulozik, U: Antioxidant capacity of bilberry extract microencapsulated in whey

protein hydrogels. Food Res Int, 47, 51–57, 2012

• Heidebach, T, Först, P, Kulozik, U: Microencapsulation of probiotic cells for food applications. Crit Rev Food Sci Nutr, 52 (4) 291-311, 2012

• Toro-Sierra, J, Tolkach, A, Kulozik, U: Fractionation of α-Lactalbumin and β-Lactoglobulin from whey protein isolate using selective thermal

aggregation: An optimized membrane separation procedure and resolubilization techniques at pilot plant scale, Food Bioprocess Technol, 6 (4) 1032-

43, 2013

• Voswinkel, L, Kulozik, U: Fractionation of all major and minor whey proteins with radial flow membrane adsorption chromatography at lab and pilot

scale, Int Dairy J, 39, 209-214, 2014

• Leeb, E, Holder, A, Letzel, T, Cheison, SC, Kulozik, U, Hinrichs, J: Fractionation of dairy based functional peptides using ion-exchange membrane

adsorption chromatography and cross-flow electro membrane filtration, Int Dairy J, 38, 116-123, 2014

• Holzmüller, W, Gmach, O, Griebel, A, Kulozik. U: Casein precipitation by acid and rennet coagulation of buttermilk: Impact on the isolation of milk fat

globule membrane proteins. Int Dairy J, 63, 115-123, 2016

• Holzmüller, W, Kulozik, U: Isolation of milk fat globule (MFGM) material by coagulation and diafiltration of buttermilk. Int Dairy J, 63, 88-91, 2016

• Würth, R, Foerst, P, Kulozik, U: Development and evaluation of a spray drying microencapsulation process for water-insoluble milk protein capsules.

Int Dairy J 61, 99-106

• Voswinkel, L, Etzel, M, Kulozik, U: Adsorption of β-lactoglobulin in anion exchange membrane chromatography versus the contacting mode and

temperature. LWT Food Sci Technol, 79, 78-83, 2017

• Würth, R, Wiesner, S, Foerst, P, Kulozik, U: Impact of the CaCl2 content in the rehydration media on the capsule formation out of spray dried capsule

precursors for the immobilization of probiotic bacteria. Int Dairy J, 68, 75-79. 2017

Documents

Food concepts for specific target groups: The role of ... · essentieller Aminosäuren bei gleichem kalorischen Wert Isolierung von Lactoferrin und anderen minoren Proteinen als Functional