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UNIVERSITI PUTRA MALAYSIA
ANAHITA KHORAMNIA
FBSB 2012 15
MICROBIAL LIPASE PRODUCTION OPTIMIZATION, CHARACTERIZATION AND APPLICATION IN
COCONUT OIL MODIFICATION
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MICROBIAL LIPASE PRODUCTION OPTIMIZATION, CHARACTERIZATION AND APPLICATION IN
COCONUT OIL MODIFICATION
By
ANAHITA KHORAMNIA
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
July 2012
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Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of requirement for the degree of Doctor of Philosophy
MICROBIAL LIPASE PRODUCTION OPTIMIZATION, CHARACTERIZATION AND APPLICATION
IN COCONUT OIL MODIFICATION
By
ANAHITA KHORAMNIA
July 2012
Chair: Professor Lai Oi Ming, PhD
Faculty: Biotechnology and Biomolecular Sciences
Lipases are the enzymes that catalyze the hydrolysis of fats and oils and can be found
widely in nature. These lipases, especially when from microbial sources are preferred
and extensively used especially in biotechnological applications and commercial
industries.
Nowadays, the use of antibiotics have been thought to contribute to the emergence of
antibiotic-resistant microorganisms and a new market for natural food additives with
antimicrobial properties has been opened. One of the most popular elements with
strong anti microbial activities are medium-chain fatty acid glycerides and medium-
chain free fatty acids (MCFA) particularly lauric acid.
Coconut oil is considered as a good source of MCFA as it consisted of about 50%
lauric acid in triglyceride form. Medium chain triglycerides (MCTGs) could be
hydrolyzed to medium chain glyceride derivatives and MCFAs including lauric acid
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using lipases. Therefore, in this study bacterial and fungal lipases were produced,
optimized and characterized. Certain possible applications in coconut oil
modification were also investigated. Different fermentation systems were also
developed to get the highest lipolytic activity and functionality in coconut oil
modification.
Among different lipase producing bacterial isolates, three Gram-positive cocci
(Staphylococcus xylosus, S. sciuri, S. aureus) and one Gram-negative short rod
(Acinetobacter sp.) were selected based on their highest lipase production activity
and better lipase characteristics. Bacterial isolates were found to be new strains
according to the BIOLOG and DSMZ identification. All strains were able to produce
lipase in submerged fermentation (SmF) but only Acinetobacter sp. showed the same
capability on coconut solid state fermentation (SSF).
The use of fungi for the production of commercially important products has
increased rapidly over the past half century. Two lipolytic filamentous fungal strains,
Geotrichum candidum ATCC 34614 and G. candidum local isolate were investigated
in the case of lipase production in both SmF and SSF. Lipase production for G.
candidum ATCC 34614 was 15 times higher than the bacterial isolates while, for the
local strain it was in the same range with bacterial isolates. Both fungal strains
revealed great potential on coconut SSF.
Modeling studies on culture parameters optimization for lipase production of these
microorganisms were performed using response surface methodology (RSM) and
artificial neural networks (ANNs). Based on the obtained optimum conditions,
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lipases were produced in SmF for characterization studies. The results demonstrated
good characteristics for all lipases as they were found to be thermostable, acid-base
tolerant and solvent-detergent stable. Lipases were then used to perform hydrolysis
reaction on coconut oil.
A coconut solid-state fermentation system was developed in order to apply the
produced lipase directly on substrate without any downstream processing. Both
fungal strains SSF lipases showed high functionality on coconut oil MCTG
conversion into MCDG, MCMG and MCFA. The oil conversion percentages reached
to 78% and 76% after optimization for G. candidum ATCC 34614 and G. candidum
local strain, respectively. The local strain showed higher lipase functionality under
extreme conditions of moisture and oil content compared to the former strain that
revealed no activity in those conditions. Microscopic studies demonstrated that the
local strain can grow faster and better on coconut solid culture with penetration
capability compared to G. candidum ATCC 34614. Coconut oil was successfully
modified by G. candidum lipases under solid state fermentation of coconut. The
bacterial strain (Acinetobacter sp.) did not reveal any activity in coconut oil
modification due to its short shelf life on coconut solid culture.
Modified coconut oils obtained from direct fermentation of fungal lipase during SSF
process were characterized for its antimicrobial activity and thermal characteristics.
The optimized modified coconut oils extracted from G. candidum ATCC 34614 and
G. candidum local strain revealed 95 and 90 % antimicrobial activity against S.
aureus and 90 and 85% against E. coli, respectively. The contributions of high level
lauric acid together with medium chain mono- and di- glycerides in modified
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coconut oils were the key factors for antimicrobial activity. The solid and submerged
fermented coconuts also showed bactericidal effects. Differential scanning
calorimetry of modified coconut oils showed lower melting points compared to the
normal coconut oil.
G. candidum ATCC 34614 cultures analysis using HS-SPME/GC-MS also showed
that 46 and 37 aromatic compounds were produced during the SmF and SSF,
respectively. The produced aromatic compounds were mainly esters with fruity and
flora notes in the modified coconut samples indicating successful hydrolysis of the
lipases.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
LIPASE MIKROB PENGHASILAN PENGOPTIMUMAN, PENCIRIAN DAN DALAM
PENGUBAHSUAN MINYAK KELAPA APLIKASI
Oleh
ANAHITA KHORAMNIA
Julai 2012
Pengerusi: Profesor Lai Oi Ming, PhD
Fakulti: Bioteknologi Dan Sains Biomolekul
Lipase adalah enzim yang memangkinkan tindak balas terutamanya hidrolisis lemak
dan minyak. Lipase boleh didapati secara meluas di alam sekitar. Walau
bagaimanapun, lipase dari samber mikrob lebih digemari dan digunakan secara
meluas terutamanya dalam industri biotecknologi dan industri komersil.
Pada masa kini, pengunaan antibiotik telah menyebabkan kemunculan
mikroorganisma yang rintans-antibiotic dan ini membuka pasaran baru kepada
makanan aditif yang mempunyai ciri-ciri anti-mikrob. Salah satu elemen yang paling
popular yang mempunyai cirri-ciri anti-mikrob yang kuat ialah gliserida acid lemak
rantai sederhana dan acid lemak rantai sederhana (MCFA) terutamanya asik lauric.
Minyak kelapa dianggap sebagai sumber MCFA yang terdiri daripada kira-kira 50%
acid laurik dalam bentuk trigliserida. A trigliserida rantai sederhan boleh dihidrolisis
kepada gliserida terbitan rantai sederhana dan MCFA dengan menggunakan enzim
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lipase. Dalam kajian ini, lipase dari bacteria dan kulat telah dihasil, dioptimum dan
dicirikan untuk diaplikasi dalam pengubahsuaian minyak kelapa. Sistem fermentasi
yang berbeza telah dibangunkan untuk mendapat amaun lipase yang paling tinggi
dan sejurusnya digunakan untuk pengubasuaian minyak kelapa.
Antara bakteria yang didapati boleh menghasilkan lipase ialah: 3 Gram positif cocci
(Staphylococcus xylosus, S. sciuri, S. aureus) dan 1 Gram negative rod pendek
(Acenitobacter sp.). Bakteria-bakteria ini dipilih berdasarkan kemampuan mereka
menghasilkan kandungan lipase yang tinggi dan mempunyai ciri-ciri yang lebih baik.
Isolat bakteria ini merupakan strain baru berdasarkan kepada sistem BIOLOG dan
DSMZ. Semua strain mampu menghasilkan lipase dalam fermentasi tenggelam
manakala hanya Aceinitobacter sp. nenunjukkan keupayaan yang sama dalam sistem
penapaian pepejal kelapa.
Pengunaan fungi dalam pengeluaran komersil produk adalah penting dan telah
meningkat dengan pesat sejak setengah abad yang lalu. Dua strain lipolitic kulat
Geotrichum candidum ATCC 34614 dan G. candidum tempatan disiasat dalam
kebolehan untuk menghasilkan lipase di kedua-dua sistem SmF dan SSF. G.
candidum ATCC 36414 dapat menghasilkan lebih 15 kali ganda kandungan lipase
berbanding dengan isolat bakteria manakala strain tempatan adalah dalam
lingkungan yang setara dengan isolat bakteria. Kedua-dua strain fungi ini
mempunyai potensi untuk digunakan dalam sistem fermentasi pepejal.
Kajian pemodelan untuk menyiasat parameter yang optimum bagi pengeluaran lipase
daripada microorganisma telah dijalankan dengan menggunakan kaedah gerak balas
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permukaan (RSM) dan rangkaian neural tiruan (ANN). Lipase telah dihasilkan
dalam sistem fermentasi pepejal untuk tujuan pencirian, berdasarkan keadaan
optimum yang diperolehi. Keputusan menujukkan lipase ini mempunyai semua ciri-
ciri yang baik seperti tahan panas, toleran kepada asid,bes dan pelarut-detergen.
Dengan ini, lipase ini telah digunakan untuk menjalankan tindak balas hidrolisis ke
atas minyak kelapa.
Sistem fermentasi pepejal telah dibangunkan untuk menghasilkan lipase secara
langsung di atas substrat tanpa sebarang pemprosesan hiliran. Kedua-dua strain kulat
yang menghasilkan lipase dalam penapaian pepejal menunjukkan fungsi yang tinggi
dalam penukaran minyak kepada MCTG, MCDG, MCMG dan MCFA. Penukaran
minyak sampai 78% dan 76% didapati selepas pengoptimuman bagi ATCC 36414
dan G. candidum strain tempatan. Strain tempatan menunjukkan fungsi lipase yang
lebih tinggi dalam keadaan kelembapan dan minyak kandungan yang tinggi
berbanding dengan strain G. candidum tempatan yang tidak menunjukkan sebarang
aktiviti lipase. Kajian mikroskopik menunjukkan bahawa strain tempatan tumbuh
lebih cepat and baik berbanding dengan strain ATCC 36414. Minyak kelapa berjaya
ditukar dengan menggunakan lipase G. candidum dalam sistem fermentasi pepejal.
Bakteria strain (Acinetobacter sp.) lipase tidak menunjukkan sebarang aktiviti dalam
pengubahsuaian minyak kelapa disebabkan oleh hayat yang singkat apabila
diaplikasikan dalam sistem fermentasi pepejal.
Minyak kelapa yang diubahsuai daripada kulat dalam kaedah fermentasi pepejal
telah dicirikan dan menunjukkan ciri anti-mikrob dan toleransi terhadap suhu.
Miinyak kelapa yang diubahsuai dengan menggunakan G. candidum ATCC 34614
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dan G. candidum tempatan mennujukkan 95 dan 90% aktiviti anti-mikrob masing –
masing manakala S.aureus dan E.coli mempunyai aktiviti anti-mikrob sebanyak 90%
and 85% masing-masing. kandunsan asid laurik yang tinggi bersama-sama dengan
rantai serderhana mono- dan di-gliserida merupakan faktor yang menyebabkan
aktiviti anti-mikrob ini. Fermentasi pepejal dan fermentasi tenggelam juga
menunjukkan aktiviti anti-mikrob. Analisa DSC menunjukkan bahawa minyak
kelapa yang diubahsuai mempunyai tahap lebur yang sedikit berbanding dengan
minyak kelapa biasa dan berpotensi untuk dijadikan bahan pengemulsi.
G. candidum ATCC 36414 di analsia dengan mengunakan HS-SPME dan GC-MS
dan menunjukkan bahawa 46 dan 37 bahan aroma wujud bagi kedua-dua sistem
fermentasi pepejal and penapaian tenggelam. Bahan aroma ini tediri daripada ester
dengan nota buah-buahan dan bunga-bungaan dalam sampel minyak kelapa yang
diabahsuci, menunjukan lipase berjaya menghidrolisis sampel tersebut.
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ACKNOWLEDGEMENTS
In the Name of God, the Most Merciful and Most Beneficent
All praises do to God, Lord of the universe. Only by his grace and mercy this thesis
would have been impossible if not for the assistance and direct involvement of so
many kindhearted individuals.
This work has been performed at Bioprocess and instrumentation laboratories,
Department of Bioprocess, Faculty of Biotechnology and Biomolecular sciences and
Institute of Bioscience in the Universiti Putra Malaysia (UPM), Malaysia.
Hereby, I would like to address my special thanks to:
My Supervisor, Professor Dr. Lai Oi Ming who accepted me as her graduate student
and guiding me through my study with her enthusiasm, encouragement and deep
knowledge in biotechnology. I would also like to acknowledge her generous
guidance, kindness, thoughtfulness and helpful and valuable support shown to me
throughout my study path. I will never forget her moral support, attention and
cooperation during my pregnancy. Without her, it may not have been possible to
enjoy my study and motherhood at the same time. Further, I would like to extend my
gratitude to my co-supervisor; Professor Dr. Tan Chin Ping for his professional
guidance and helpfulness throughout my research. I would also like to acknowledge
Professor Dr. Norhani Abdullah and Associate Professor Dr. Rosfarizan Mohammad
for providing me the required facilities.
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I cannot forget to give my heartiest thanks to my research group in Institute of
Bioscience (Boon Kee, Charles, Yee Ying and Teckim) and my fellow friends in
Biotech 3 (Perabhu) for their help, support, sharing their knowledge and working
experience and also various things about life. I specially appreciate my best friend
Zahra as I owe her so much for her friendship, trust, collaboration and endless
support through these years.
Special thanks are also due to all the staff of Biotech 3 and MPP (Puan Alluyah, Ms.
Renuga and Mr. Rosli) for their kind assistance in all the matters.
I am grateful especially to Boon kee Beh, Alex Kok whye and Sami Saadi who
helped me to better interpretation of my results in fermentation products.
I am indebted to my beloved parents, my brother Amir and my sister Arezoo for their
tolerance, sacrifices and patience as they were unable to see me at all during this PhD
career. Special thanks to my beloved mother for her invaluable support. I would also
like to acknowledge my dear mother-in-law for her kindness and thoughtfulness.
Last but not the least, I wish especially to acknowledge my beloved husband, Afshin
and my genius son, Artin for their unbelievable love, support, patience and
understanding for every situation I am in. My dearest Afshin, thank you for your
scientific and moral supports.
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I certify that an Examination Committee has met on 02.07.2012 to conduct the final examination of Anahita Khoramnia on his Doctor of Philosophy thesis entitled “Microbial lipases production: optimization characterization and application in coconut oil modification” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Doctor of Philosophy.
Members of the Thesis Examination Committee were as follows: Rosfarizan binti Mohamad, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Thomas Choong Shean Yaw, PhD Associate Professor Faculty of Engineering Universiti Putra Malaysia (Internal Examiner) Ling Tau Chuan, PhD Professor Faculty of Science Universiti Malaya (Internal Examiner) Shieh Chwen-Jen, PhD Professor Biotechnology Center National Chung Hsing University Taiwan (External Examiner)
SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date: 23.09.2012
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Lai Oi Ming, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Tan Chin Ping, PhD Professor Faculty of Food Sciences Universiti Putra Malaysia (Member) Lo Seong Koon, PhD Lecturer MARDI Universiti Putra Malaysia (Member)
BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or other institutions.
ANAHITA KHORAMNIA
Date: 02 July 2012
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TABLE OF CONTENTS
Page ABSTRACT ii ABSTRAK vi ACKNOWLEDGEMENTS x APPROVAL xii DECLARATION xiv LIST OF TABLES xx LIST OF FIGURES xxii LIST OF ABBREVIATIONS xxv
CHAPTER 1 INTRODUCTION
1.1 Background 1.2 Objectives
1
2 LITERATURE REVIEW 4 2.1 Lipases 4 2.1.1 Lipase function 6 2.1.2 Lipases reactions 8 2.1.3 Sources of lipases 10 2.1.3.1 Microbial lipases 11 2.2 Isolation and screening of lipase producing microorganisms 13 2.3 Geotrichum candidum lipases 14 2.4 Microbial lipases Characteristics 16 2.4.1 pH optima and stability 16 2.4.2 Temperature optima and stability 17 2.4.3 Effect of calcium ion 18 2.4.4 Effect of organic solvents 18 2.4.5 Specificity of lipase 19 2.5 Lipase production 22 2.5.1 Factors affecting microbial lipase production 22 2.5.1.1 Effect of chemical factors on lipase production 22 2.5.1.2 Effect of physical factors on lipase production 24 2.5.2 Lipase production in different fermentation systems 25 2.5.2.1 Submerged fermentation (SmF) 26 2.5.2.2 Solid state fermentation (SSF) 27 2.6 Strategies for improving fermentation process (Optimization
techniques) 30
2.6.1 Response surface methodology (RSM) 30 2.6.1.1 Application of RSM in fermentation studies 32 2.6.2 Artificial Neural Network (ANN) 32 2.6.2.2 Application of ANN in fermentation 37
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2.7 Applications of lipases 37 2.8 Role of fermentation in food 40 2.8.1 SSF and fermented food 42 2.9 Role of Geotrichum candidum in food 43 2.10 Coconut as a potent fermented food 46
3 BACTERIAL LIPASE PRODUCTION IN SUBMERGED FERMENTATION
49
3.1 Introduction 49 3.2 Materials and Methods 51 3.2.1 Isolation and screening of lipase producing bacteria 51 3.2.2 Preparation of tributyrin agar plates 52 3.2.3 Preparation of Rhodamin B agar plates 52 3.2.4 Gram characterization 53 3.2.5 Bacterial strains 53 3.2.6 Composition of lipase production medium 54 3.2.7 Lipase activity assay 54 3.2.8 Experimental design 54 3.2.9 Response surface methodology analysis 55 3.2.10 Artificial neural network analysis 55 3.2.11 Validation of the optimized condition 56 3.2.12 Effect of pH and temperature on Staphylococci lipases activity
and stability 57
3.3 Results and Discussion 3.3.1 Isolation, screening and identification of lipase producing bacteria
58 58
3.3.2 Design and modelling 59 3.3.3 Response surface methodology analysis of lipase production 61 3.3.4 Artificial neural network analysis and modelling 64 3.3.5 Comparison of RSM and ANN predicted values 65 3.3.6 Main effects and interactions between parameters 67 3.3.6.1 Staphylococcus xylosus 67 3.3.6.2 Staphylococcus sciuri 70 3.3.6.3 Staphylococcus aureus 72 3.3.7 Importance of parameters on lipase production 72 3.3.8 Characterization of Staphylococci lipases 75 3.3.8.1 Temperature and pH 75 3.3.8.2 Effects of pH on lipase activity and stability 75 3.3.8.3 Effects of temperature on lipase activity and stability 79 3.3.8.4 Effects of calcium on lipase activity 82 3.3.8.5 Specificity of lipase enzymes toward natural oils 84 3.4 Conclusion 85 88
4 BACTERIAL LIPASE PRODUCTION IN SOLID STATE FERMENTATION
89
4.1 Introduction 89 4.2 Materials and Methods 91 4.2.1 Bacterial strain 91
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4.2.2 Lipase production in SmF 92 4.2.3 Substrate preparation and lipase production in SSF 93 4.2.4 Lipase activity assay 94 4.2.5 Experimental design 95 4.2.6 Artificial neural network analysis 95 4.2.7 Validation of the optimized condition 96 4.2.8 Characterization of Acinetobacter sp. lipase 97 4.2.8.1 Effect of pH and temperature on the lipase activity and
stability 97
4.2.8.2 Effect of organic solvents on lipase activity 98 4.2.8.3 Effects of surfactants on lipase activity 98 4.2.8.4 Effect of calcium on the lipase activity 98 4.3 Results and Discussion 99 4.3.1 Production of lipase in SmF and SSF systems 99 4.3.2 Artificial neural network analysis and modeling 100 4.3.3 Optimum conditions and verification study 104 4.3.4 Main effects and interactions between parameters in SmF and
SSF lipase production 105
4.3.5 Comparison of lipase production by Acinetobacter sp. in different fermentation systems
109
4.3.6 Partial characterization of Acinetobacter sp. lipase 111 4.3.6.1 Effects of pH and temperature on the lipase activity and
stability 111
4.3.6.2 Effects of organic solvents on lipase activity 113 4.3.6.3 Effects of surfactants on lipase activity 115 4.3.6.4 Effect of calcium on the lipase activity 116 4.4 Conclusion 117
5 OPTIMIZATION OF FUNGAL LIPASE PRODUCTION IN SUBMEGED FERMENTATION
119
5.1 Introduction 119 5.2 Materials and methods 121 5.2.1 Fungal strains 121 5.2.2 Media composition 121 5.2.3 Stock culture 122 5.2.4 Inoculum development and lipase production 122 5.2.5 Lipase assay 122 5.2.6 Crude enzyme preparation 123 5.2.7 Characterization study of fungal lipases 123 5.2.7.1 Effect of temperature on lipase activity 123 5.2.7.2 Effect of pH on lipase activity 123 5.2.7.3 Effect of calcium chloride concentration on lipase activity 124 5.2.7.4 Specificity of lipase towards vegetable oils 124 5.2.7.5 Effect of solvents on lipase activity 124 5.2.7.6 Regioselectivity 125 5.2.8 Application of G. candidum submerged fermentation in coconut
oil modification 126
5.3 Results and Discussions 126
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5.3.1 Morphology of filamentous G. candidum strains in submerged cultivation
126
5.3.2 Effect of media composition on lipase production 127 5.3.3 Effect of incubation time on lipase production 128 5.3.4 Effect of agitation on lipase production 129 5.3.5 Effect of temperature on lipase production 129 5.3.6 Effect of oil content on lipase production 131 5.3.7 Effect of starter culture elimination on lipase production 132 5.3.8 Effect of inoculum size on lipase production 133 5.3.9 Effect of pH on lipase production 133 5.3.10 Glucose contribution in the production medium 133 5.3.11 Sodium chloride contribution in the spore suspension 135 5.3.12 Optimization of G. candidum ATCC 34614 lipase production
using RSM and ANN 135
5.3.12.1 Design and modeling 135 5.3.12.2 Response surface methodology analysis of lipase
production 136
5.3.12.3 Artificial neural network analysis and modeling 140 5.3 12.4 Verification of estimated data 140 5.3.12.5 Importance analysis 141 5.3.12.6 Main effects and interactions between parameters 143 5.3.13 Optimization of lipase production of local G. candidum 145 5.3.13.1 Lipase production analysis 145 5.3.13.2 Response Surface Methodology model 146 5.3.13.3 Artificial Neural Network model 147 5.3.13.4 Importance of effective parameters on lipase production
148
5.3.13.5 Main effects and interactions between parameters 149 5.3.14 Comparison of optimum conditions 150 5.3.15 Characterization of G. candidum ATCC 34614 and local lipases 151 5.3.15.1 Specificity of lipase towards vegetable oils 155 5.3.15.2 Effect of different solvents on lipase stability 158 5.3.15.3 Effect of calcium chloride concentration 160 5.3.15.4 Application of G. candidum strains lipases 162 5.4 Conclusion 165
6 MICROBIAL MODIFICATION OF COCONUT OIL USING LIPASES OF G. CANDIDUM STRAINS IN SOLID STATE FERMENTATION SYSTEM
169
6.1 Introduction 169 6.2 Materials and Methods 171 6.2.1 Fungal strains 171 6.2.2 Inoculum preparation 171 6.2.3 Solid state fermentation for lipase production 171 6.2.4 Lipase activity in SSF 172 6.3.5 Experimental design and statistical analysis 172 6.2.6 SSF culture condition for lypolitic partial hydrolysis 173 6.2.7 Artificial Neural Network 173
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6.2.8 Verification of the ANN model 173 6.2.9 Oil extraction from SSF 174 6.2.10 Acylglycerol composition analysis using HPLC 174 6.2.11 Electron microscopy 176 6.3 Results and discussion 177 6.3.1 Direct modification of coconut oil via solid-state fermentation 177 6.3.2 Extracellular lipase production in SSF by G. candidum strains 177 6.3.3 Lipase preservation study in SSF 179 6.3.4 Optimization of G. candidum strains coconut oil modification in
SSF 181
6.3.4.1 Effect of variables and ranges 181 6.3.4.2 Modeling and optimization of coconut oil conversion by G.
candidum strains lipases in SSF 183
6.3.4.3 Three dimensional plots 188 6.3.4.4 Comparison of G. candidum strains function in SSF 191 6.3.4.5 Feasibility of direct modification of coconut oil process 198 6.3.5 Coconut ultrastructure study by SEM 200 6.3.6 SEM study and comparison of G. candidum strains growths on
coconut SSF 200
6.3.7 Microscopic level explanation for different fungal functionality in SSF
207
6.3.8 Improvement of G. candidum ATCC 34614 function 209 6.3.9 Contribution of glycerol in SSF 209 6.4 Conclusion 211
7 CHARACTERIZATION OF FERMENTED COCONUT AND ITS MODIFIED OIL
214
7.1 Introduction 214 7.2 Materials and Methods 216 7.2.1Materials 216 7.2.2 Acylglycerol composition analysis 216 7.2.3 Antimicrobial activity studies 216 7.2.4 Preparation of bacterial inoculums 216 7.2.5 Determination of anti-bacterial activity 217 7.2.6 Headspace solid-phase micro-extraction (HS-SPME) 217 7.2.7 Gas chromatography-mass spectrometry (GC–MS) analysis 218 7.2.8 Diferential scanning calorimetry (DSC) analysis 219 7.3 Results and Discussion 220 7.3.1 Antimicrobial effects of modified coconut oils 220 7.3.1.1 Mechanism of antimicrobial action 225 7.3.1.2 The product’s side effects 225 7.3.2 Identification of aroma compounds in fermented products 226 7.3.2.1 Comparison of aroma profiles of G. candidum cultures in
SmF and SSF 232
7.3.3 Thermal characteristic of the MCOs using DSC 237 7.4 Conclusion
244
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8 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH
246
8.1 Summary 246 8.2 Conclusions 248 8.3 Recommendations for future research 250
9 REFERENCES 251 APPENDICES 271 BIODATA OF STUDENT 282 LIST OF PUBLICATIONS 283