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