UNIVERSITI PUTRA MALAYSIA

PRODUCTION OF MANNAN-DEGRADING ENZYMES BY ASPERGILLUS NIGER IN SOLID STATE AND SUBMERGED

FERMENTATION FOR HYDROLYSIS OF PALM KERNEL CAKE

LISA ONG GAIK AI

FBSB 2006 39

PRODUCTION OF MANNAN-DEGRADING ENZYMES BY ASPERGILLUS NIGER IN SOLID STATE AND SUBMERGED FERMENTATION FOR

HYDROLYSIS OF PALM KERNEL CAKE

By

LISA ONG GAIK AI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of

Philosophy

DECEMBER 2006

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

PRODUCTION OF MANNAN-DEGRADING ENZYMES BY ASPERGILLUS NIGER IN SOLID STATE AND SUBMERGED FERMENTATION FOR

HYDROLYSIS OF PALM KERNEL CAKE

By

LISA ONG GAIK AI

DECEMBER 2006

Chairman: Suraini Abd Aziz, PhD Faculty: Biotechnology and Biomolecular Sciences The production of mannan degrading enzymes under solid substrate

fermentation and liquid fermentation by Aspergillus niger FTCC 5003 were

carried using palm kernel cake (PKC) as the sole carbon source. The crude

enzymes produced were then used in the enzymatic hydrolysis of PKC. In

the preliminary study, it was observed that mannanase was the highest

enzyme activity produced. Other enzymes produced were, amylase,

xylanase, polygalacturonase and cellulases. Types of inoculum and

extraction temperature play an important role in the production of mannanase

under solid substrate fermentation (SSF). The optimum parameters for the

mannanase production in submerged fermentation (SmF) under shake flask

condition were 1×104 spores/mL, with 2% (w/v) of PKC incubated at 35ºC,

and agitated at 200 rpm. While the optimum parameters for mannanase

production in SSF were 1×106 spores/mL, with 50% (v/w) of initial moisture

content, 2% of nitrogen in urea and incubated at 30ºC. Mannanase

produced under optimum condition was 104 U/mL (SmF) and 1705 U/g PKC

(SSF). Mannanase obtained from SSF was more heat stable as compared to

ii

those obtained from SmF. Both types of mannanase had the same optimum

pH, which was pH 7 and both had two different optimum temperatures, which

were 30°C and 50°C for SmF; and 35°C and 55°C for SSF. For the

enzymatic hydrolysis of PKC, the optimum condition for the hydrolysis was

10% of PKC, 100 U/mL enzyme concentration, at 45°C and pH 6.5 when

mannanase obtained from SmF was used. On the other hand, when

mannanase from SSF was used the optimum condition was obtained at 45°C,

pH 7.5 with 10% PKC and 50 U/mL enzyme concentration.

iii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENGHASILAN ENZIM PENGHURAIAN MANAN MELALUI FERMENTASI KEADAAN PEPEJAL DAN CECAIR OLEH ASPERGILLUS NIGER UNTUK

MENGHIDROLISIS HAMPAS ISIRONG KELAPA SAWIT

Oleh

LISA ONG GAIK AI

DECEMBER 2006

Pengerusi: Suraini Abd Aziz, PhD Fakulti: Bioteknologi dan Sains Biomolekul Penghasilan enzim penghuraian manan oleh Aspergillus niger melalui

fermentasi substrate pepejal (SSF) and fermentasi cecair (SmF) dengan

menggunakan hampas isisarong kelapa sawit sebagai sumber karbon utama.

Enzim kasar yang dihasilkan digunakan untuk menghidrolisiskan PKC.

Dalam permulaan penyelidikan ini, didapati mananase adalah enzim yang

tertinggi dihasilkan. Enzim lain seperti amilase, xylanase, poligalaktoranase

and celulase juga dihasiklan. Jenis inokulum dan suhu pnegekstrakan

memainkan peranan penting dalam penghasilan mananase melalui SSF.

Parameter optimum untuk penghasilan mananase melalui SmF dalam

keadaan kelalang kon dengan menggunakan spora kepekatan 1 × 104

spore/mL dengan 2% (i/b) PKC, pengeraman pada suhu 35°C dan

digoncang pada 200 rpm. Manakala parameter optimum untuk penghasilan

mananase melalui SSF adalah dengan menggunakan spora kepekatan 1 x

106 spora/mL, 50% (i/b) kandungan kelembapan permulaan, 2% sumber

nitrogen daripada urea dan pengeraman pada 30°C. Pada keadaan

optimum, aktiviti mananase yang diperolehi adalah 104 U/mL (LF) dan 1705

iv

U/g PKC (SSF). Mananase yang dihasilkan melalui SSF adalah lebih tahan

kepada suhu bebanding dengan proses SmF. Selain itu, didapati bahawa

kedua-dua mananase mempunyai pH optimum pada pH 7 dan kedua-dua

enzim mempunyai dua suhu optimum, iaitu pada 30°C and 50°C untuk SmF;

dan pada 35°C serta 55°C untuk SSF. PKC seterusnya digunakan untuk

proses hidrolisis berenzim menggunakan enzim mannanase kasar yang

dihasilkan melalui SmF dan SSF. Keadaan optimum untuk proses hidrolisis

berenzim dengan menggunakan manannase yang dihasilkan melaluli proses

SmF adalah 10% PKC, 100 U/mL enzim, pada suhu 45°C dan pH 6.5.

Manakala keadaan optimum proses hidrolisis berenzim dengan

menggunakan manannase yang diperolehi melaluli SSF adalah pada 45°C

dan pH 7.5 dengan 10% PKC dan 50 U/mL enzim.

v

ACKNOWLEDGEMENTS

First and foremost I would like to thank GOD for guiding me all these years.

It is by His grace that I was able to complete this thesis.

I would like to express my sincere appreciation to my advisor, Associate

Professor Dr. Suraini Abd Aziz for her constant guidance and supervision

during my research and studies. I am obliged to her for her encouragement

and support not only in my research and study, but also in my career.

I would also like to extend my appreciation to Professor Dr. Mohd Ali Hassan

and Professor Dr. Mohamed Ismail Abdul Karim who have been guiding me

in this work and were always available for constructive comments and

invaluable assistance while serving as committee members.

I would like to thank my family members for giving me the encouragement

and support in my study. I appreciate the support given to me by Puan

Noraini Samat, Dr. Mohd Jaafar, Mr. Abdul Rahman Abdul Razak and those

from the Bioprocess Laboratory in MARDI and I would like to thank my fellow

laboratory mates and friends for helping me in so many little ways.

Last but not least, I would like to thank the Ministry of Science, Technology

and Innovation for providing the NDB top-down grant.

vi

I certify that an Examination Committee has met on 26 December 2006- to conduct the final examination of Lisa Ong Gaik Ai on her Doctor of Philosophy thesis entitle “Production of Mannan-degrading Enzymes by Aspergillus niger in Solid State and Submerged Fermentation for Hydrolysis of Palm Kernel Cake” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the Candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Mohd Arif Syed, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Norhani Abdullah, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Arbakariya Ariff, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Ashok Pandey, PhD Professor Biotechnology Division Regional Research Laboratory, CSIR Trivandrum, India (External Examiner)

HASANAH MOHD. GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date : 17 MAY 2007

vii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: Suraini Abd-Aziz, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Mohd Ali Hassan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Mohamed Ismail Abdul Karim, PhD Professor Faculty of Engineering International Islamic University Malaysia (Member)

________________________ AINI IDERIS, PhD

Professor/Dean School of Graduate Studies Universiti Putra Malaysia

Date: 14 JUNE 2007

viii

DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

__________________ LISA ONG GAIK AI

Date: 23 APRIL 2007

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TABLE OF CONTENTS

Page

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

iiivvi

viiiix

xiiixvxix

CHAPTER

1 INTRODUCTION

1

2 LITERATURE REVIEW 2.1 Palm Kernel Cake (PKC)

2.1.1 General Characteristic and Nutritional Values of Palm Kernel Cake

2.1.2 Cell Wall Composition of Palm Kernel Cake

2.1.3 Treatment of Palm Kernel Cake as Effective Feed

2.1.4 Utilisation of PKC in Livestock 2.2 Solid Substrate Fermentation (SSF)

2.2.1 Microorganism 2.2.2 Substrate 2.2.3 Biomass Measurement 2.2.4 Environmental Factors 2.2.5 Application of SSF

55

6

9

1321242732333743

3 GENERAL MATERIALS AND METHODS 3.1 Materials

3.1.1 Palm Kernel Cake (PKC) 3.1.2 Microorganism 3.1.3 Source of Chemicals and Reagents

3.2 Methods 3.2.1 General Plan of the Experimental Work 3.2.2 Inoculum Preparation 3.2.3 Solid Substrate Fermentation (SSF) 3.2.4 Submerged Fermentation (SmF) 3.2.5 Enzymatic Hydrolysis 3.2.6 Mannanase Assay 3.2.7 Soluble Protein Assay 3.2.8 Reducing Sugar Assay

4545454646464649495051515253

x

4 SCREENING AND PROFILING OF ENZYMES PRODUCED DURING SOLID SUBSTRATE FERMENTATION 4.1 Introduction 4.2 Materials and Methods

4.2.1 Pre-germinated Aspergillus niger 4.2.2 Solid Substrate Fermentation (SSF) 4.2.3 Enzyme Extraction 4.2.4 Enzymes Assays 4.2.5 Sample Analyses

4.3 Results and Discussion 4.3.1 Enzyme Screening and Profiling 4.3.2 The effect of Inoculum Type and

Extraction Condition 4.4 Conclusion

54545555555556616161

6269

5 OPTIMISATION OF MANNANASE PRODUCTION IN SOLID SUBSTRATE AND SUBMERGED FERMENTATION 5.1 Introduction 5.2 Materials and Methods

5.2.1 Solid Substrate Fermentation for Mannanase Production

5.2.2 Submerged Fermentation for Mannanase Production

5.2.3 Sample Analyses 5.3 Results and Discussion

5.3.1 Optimisation of Mannanase Production Parameters in Solid Substrate Fermentation

5.3.2 Optimisation of Mannanase Production Parameters in Submerged Fermentation

5.4 Conclusion

707072

72

757777

77

92111

6 PRODUCTION AND CHARACTERISATION OF MANNANASE USING OPTIMISED CONDITION IN SUBMERGED AND SOLID SUBSTRATE FERMENTATION 6.1 Introduction 6.2 Materials and Methods

6.2.1 Solid Substrate Fermentation for Mannanase Production in Optimised Condition

6.2.2 Submerged Fermentation for Mannanase Production in Optimised Condition

6.2.3 Sample Analyses 6.2.4 Characterisation of Mannanase

6.3 Results and Discussion 6.3.1 Enzyme Production in Submerged

Fermentation

112112113

113

114114114115

115

xi

6.3.2 Enzyme Production in Solid Substrate Fermentation

6.3.3 Enzyme Decay Experiment 6.3.4 Enzyme Characteristics

6.4 Conclusion

118121124128

7 ENZYMATIC HYDROLYSIS OF PALM KERNEL CAKE USING CRUDE ENZYME 7.1 Introduction 7.2 Materials and Methods

7.2.1 Production of Mannanase in Submerged Fermentation and Solid Substrate Fermentation

7.2.2 Optimisation of the Parameters for the Hydrolysis

7.2.3 Reducing Sugar Assay 7.3 Results and Discussion

7.3.1 The Effect of Substrate Concentration on Hydrolysis

7.3.2 The Effect of Enzyme Concentration on Hydrolysis

7.3.3 The Effect of Initial pH on Hydrolysis 7.3.4 The Effect of Operating Temperature 7.3.5 Enzymatic Hydrolysis of Palm Kernel

Cake 7.4 Conclusion

129129130

130

130132132

132

134138140

142145

8 GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK

146

REFERENCES APPENDICES BIODATA OF THE AUTHOR LIST OF PUBLICATIONS

150

178

190

191

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LIST OF TABLES

Table

Page

2.1 Proximate analysis (%) of palm kernel cake

7

2.2 Amino acid content of PKC

8

2.3 Characteristics of PKC surveyed in 1989

8

2.4 Physicochemical properties of endo-mannanases from some different microbial sources

16

2.5 Effect of mannan degrading enzyme on broiler’s performance

19

2.6 Nutritive value of fermented PKC

20

2.7 Comparison of ration composition using fermented PKC and typical diet

21

2.8 Recommended levels of PKC in livestock feeds

24

2.9 Comparison between submerged and solid substrate fermentation

26

2.10 Main groups of microorganisms involved in SSF processes

29

2.11 Main application of SSF processes in various economical sectors

44

3.1 Composition of raw palm kernel cake

45

4.1 Cellulases and hemicellulases produced by A. niger through SSF

62

4.2 The production of mannanase using different types of inoculum and extraction temperature

66

5.1 The mannanase production by A. niger FTCC 5003 from various inoculum concentrations in solids substrate fermentation

79

5.2 The mannanase production by A. niger FTCC 5003 from various initial moisture content in solid substrate fermentation

84

5.3 The mannanase production by A. niger FTCC 5003 from various temperature in solid substrate fermentation

88

xiii

5.4 The mannanase production by A. niger FTCC 5003 from various percentages of nitrogen in urea in solid substrate fermentation

91

5.5 The mannanase production by A. niger FTCC 5003 from various inoculum concentrations in submerged fermentation

94

5.6 The mannanase production by A. niger FTCC 5003 from various agitation speeds in submerged fermentation

99

5.7 The mannanase production by A. niger FTCC 5003 from various temperatures in submerged fermentation

103

5.8 The mannanase production by A. niger FTCC 5003 from various percentage of palm kernel cake in submerged fermentation

107

5.9 Optimum condition obtained for solid state and submerged fermentation

111

6.1 Half-life (t½) for crude mannanase that produced obtained from SSF and SmF

122

7.1 Reducing sugar production and productivity obtained from various PKC concentrations through the hydrolysis

134

7.2 Reducing sugar production and productivity obtained from various enzyme concentrations through the hydrolysis of PKC

136

7.3 Reducing sugar production and productivity obtained from various initial pHs through the hydrolysis of PKC

140

7.4 Reducing sugar production and productivity obtained from various operating temperatures through the hydrolysis of PKC

142

7.5 Reducing sugar production and productivity obtained from optimised condition through the hydrolysis of PKC

143

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LIST OF FIGURES

Figure

Page

2.1 Mechanical extraction of palm kernel oil

6

3.1 Raw palm kernel cake

45

3.2 The preliminary study of SSF

47

3.3 The optimisation process for mannanase production

48

3.4 The enzymatic hydrolysis of PKC

49

4.1 Influence of the extraction condition on mannanase activity

63

4.2 Growth morphology of spore suspension of A. niger as inoculum in solid substrate fermentation after 48 hours of fermentation

67

4.3 Growth morphology of per-germinated A. niger as inoculum in solid substrate fermentation after 48 hours of fermentation

67

5.1 Mannanase productions by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration

78

5.2 Soluble protein concentration by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration

80

5.3 Reducing sugar concentration by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration

82

5.4 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different initial moisture content

85

5.5 Soluble protein concentration by A. niger FTCC 5003 in solid substrate fermentation at different initial moisture content

85

5.6 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different incubation temperature

88

5.7 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different percentage of nitrogen source from urea

91

5.8 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different inoculum concentration

94

xv

5.9 Soluble protein concentration produced by A. niger FTCC 5003 in submerged fermentation at different inoculum concentration

95

5.10 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different agitation speed

98

5.11 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different agitation speed

99

5.12 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different agitation speed

100

5.13 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different temperature

104

5.14 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different temperature

104

5.15 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different temperature

105

5.16 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different % of PKC

108

5.17 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different % of PKC

109

5.18 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different % of PKC

110

6.1 Mannanase productions by A. niger in submerged fermentation

116

6.2 Mannanase productions by A. niger in solid substrate fermentation

119

6.3 Heat inactivation of mannanase activity of four different temperature

123

6.4 Effect of pH on mannanase activity

125

6.5 Effect of temperature on mannanase activity

125

7.1 The effect of different PKC concentrations on the hydrolysis using crude mannanase obtained from submerged fermentation using standard condition of 75 U/ml enzyme concentration, pH 7 and at 40°C

133

xvi

7.2 The effect of different PKC concentrations on the hydrolysis using crude mannanase obtained from solid substrate fermentation using standard condition of 75 U/ml enzyme concentration, pH 7 and at 40°C

133

7.3 The effect of different enzyme concentrations on the hydrolysis of palm kernel cake using crude mannanase obtained from submerged fermentation using standard condition of 6% PKC concentration, pH 7 and at 40°C

135

7.4 The effect of different substrate concentration on the hydrolysis of palm kernel cake using crude mannanase obtained from solid substrate fermentation using standard condition of 6% PKC concentration, pH 7 and at 40°C

136

7.5 The effect of different initial pHs on the hydrolysis of palm kernel cake using crude mannanase obtained from submerged fermentation using standard condition of 75 U/ml enzyme concentration, 6% PKC concentration and at 40°C

139

7.6 The effect of different initial pHs on the hydrolysis of palm kernel cake using crude mannanase obtained from solid substrate fermentation using standard condition of 75 U/ml enzyme concentration, 6% PKC concentration and at 40°C

139

7.7 The effect of different operating temperatures on the hydrolysis of palm kernel cake using crude mannanase obtained from submerged fermentation using standard condition of 75 U/ml enzyme concentration, 6% PKC concentration and pH 7

141

7.8 The effect of different operating temperatures on the hydrolysis of palm kernel cake using crude mannanase obtained from solid substrate fermentation using standard condition of 75 U/ml enzyme concentration, 6% PKC concentration and pH 7

142

7.9 Hydrolysis of PKC using optimum condition

143

xvii

xviii

LIST OF ABBREVIATIONS

Aw Water activity

BSA Bovine serum albumin

CMCase Carboxymethyl Cellulase

DNS Dinitrosalicylic acid solution

DP Degree of polymerisation

EU European Union

FPase Filter paper assay

FTCC Food Technology Culture Collection

MARDI Malaysia Agricultural Research and Development Institute

PKC Palm kernel cake

PUF Polyurethane foam

SmF Submerged fermentation

SSF Solid substrate fermentation

CHAPTER 1

INTRODUCTION Malaysia is the world’s largest producer of palm oil. According to MPOB

(2005), oil palm alone covered an area of 3.80 million hectares in 2003. Most

of the hectare belongs to the private sector which account for about 59% of

total hectare while another 31% are government sponsored schemes. The

smallholder constitutes only 10%. The by product from the palm oil industry

especially in the extraction of palm kernel oil is the palm kernel cake (PKC).

Most of the PKC are exported or being used locally as supplement in animal

feed, which is normally used for ruminant. From the countries that import

PKC, European Union (EU) countries absorb more than 85% of Malaysian

PKC annually and the Netherlands is the biggest importer of Malaysian PKC

among the EU countries. Asian countries which import Malaysian PKC are

South Korea and Japan. In 2000, approximately 1.1 million tonnes or 90% of

Malaysian PKC exports to the EU were taken by the Netherlands (Aspar,

2001).

The by-product of oil palm that is palm kernel cake is fibrous in nature.

Degradation of fibre into simpler carbohydrate improves the nutritive value of

the by-products for poultry. Natural degradation of fibre in the open and in

the rumen involves many different microbes (Tenkanen et al., 1997; Mohd-

Jaafar et al., 2001). Numerous sources of microbial cocktails are available

commercially for speeding up the degradation process. Under proper

fermentation condition, the degradation period may be reduced further with

possible increase in efficiency.

PKC is obtained as a by-product after solvent or mechanical extraction of the

oil from the palm kernel. In Malaysia, mechanical extraction by screw press

is the most widely used. The solvent extraction process is generally not used

currently due to its higher cost (Aspar, 2001). The 5-8 % fat content (Alimon,

2004) makes PKC as an energy feed. PKC also contains approximately 16%

fibre. Fibre is considered an essential nutrient for dairy cattle, since cattle fed

on insufficient fibre often develop metabolic or digestive problems (Miller and

O’Dell, 1969). PKC is a valuable feed meal and a useful source of protein

and energy for livestock and it is commonly used in animal feed for ruminants

(Hutagulung, 1981; Awaludin, 2001), swine and poultry (Siew and Noraini,

1992).

Fishmeal and maize (corn), which constitute the major portion of poultry

rations, are rather expensive and are the main reason for the high cost of

diets. Attempts to cut cost have revolved around finding cheap and available

substitutes, generally of agro-industrial origin. Brewers dried grains, wheat

bran, oil palm slurry, and poultry manure are among the by-products that

have been tried and have shown great promise (Osei and Amo, 1987). PKC

has largely been neglected as feed ingredient for poultry. Nevertheless

Nwokolo et al. (1976) found PKC to be a good source of protein for poultry.

McDonald et al. (1982) suggested that PKC in the diets of poultry should be

limited to 20%.

PKC has become a draw back for monogastric livestock especially poultry

because the cell wall of PKC is composed of mainly linear and high

crystalline mannan and a small quantity of galactomannan (Mohd-Jaafar and

2

Jarvis, 1992). Utilisation of filamentous fungi in solid substrate fermentation

(SSF) for fibre digestibility improvement has been widely applied (Raimbault,

1998). Aspergillus and Rhizopus sp. are among the most important

microorganisms used in SSF process due to their physiological,

enzymological and biochemical properties and also due to the wide

application in food and feed production (Noraini et al., 2001). Besides that,

Aspergillus has been reported by Ong et al. (2004), to produce abundant

mannanase in the presence of PKC as sole carbon source in SSF.

Enzymatic degradation of PKC now becomes an important and popular

method in increasing PKC digestibility. The application of enzymes in

hydrolysing mannan and galactomannan in poultry feed has been reported

by Noraini et al. (2000).

A large number of industrial processes in the areas of industrial,

environmental and food biotechnology utilise enzymes at some stage or the

other. Current developments in biotechnology are yielding new applications

for enzymes. SSF holds tremendous potential for the enzymes production. It

can be of special interest in those processes where the crude fermented

product may be used directly as the enzyme source (Pandey et al., 1999). In

addition to the conventional applications in food and fermentation industries,

microbial enzymes have attained significant role in biotransformation

involving organic solvent media, mainly for bioactive compounds (Pandey,

1992a; Pandey et al., 1999). This research was therefore concerned with the

cultivation of selected fungus on PKC under submerged (SmF) and solid

substrate (SSF) fermentation conditions. It was expected that the fungus will

utilise the PKC by producing enzymes to degrade the polysaccharide content.

3

Hence, the objectives of this study were to:

i. screen for hydrolytic enzymes produced by Aspergillus

niger under solid substrate fermentation (SSF) using

palm kernel cake (PKC) as substrate,

ii. optimise the production of mannan-degrading enzyme

through submerged and solid substrate fermentation by

A. niger using PKC as substrate,

iii. partially characterise the crude enzymes produced,

iv. optimise the parameters for the hydrolysis of PKC using

the crude enzymes preparation obtained from both

techniques of fermentation.

4

CHAPTER 2

LITERATURE REVIEW 2.1 Palm Kernel Cake (PKC) Palm kernel cake (PKC), is a by-product from kernel oil extraction, which

constitutes about 50% of the original palm kernel. It can be obtained by

either solvent or mechanical extraction. The cake produced by solvent

extraction is dry, gritty and unpalatable to animals. However, it is less

subjected to rancidity due to lower oil content and it can be stored for longer

time than that processed by screw press (Yeong et al., 1981). The left over

residue is usually dark brown in colour and has a pleasant odour (Babjee et

al., 1985).

PKC or sometimes referred to as palm kernel expeller (PKE) is obtained as a

by product from the extraction of palm kernel oil via the mechanical process.

The production of PKC involves the grinding of palm kernels followed by

screw pressing with or without an intermediary flaking and cooking stages

(Tang, 2001). Figure 2.1 showed the simplified flow chart on the mechanical

extraction of palm kernel oil.

5

Cleaning

Grinding

Flaking

Steam conditioning

Screw pressing

Coarse screen filter

Palm kernel cake Filter press Storage Palm kernel oil

Figure 2.1 Mechanical extraction of palm kernel oil

2.1.1 General Characteristic and Nutritional Values of Palm

Kernel Cake PKC is valuable in supplying protein and energy as the proximate analyses of

PKC are shown in Table 2.1. It is being exported to Europe for feeding dairy

cattle. PKC contains about 16 - 18% crude protein, which is higher than that

in rice bran. The nutritive values are found to be close to rice bran, wheat

bran and coconut oil meal (Yeong et al., 1981; Mat, 1983). Nevertheless, the

protein content of PKC is considered sufficient to meet the requirements of

most ruminants. To some extent, the protein level in PKC may also meet the

requirement of certain classes of poultry, such as breeder and layer hens,

provided that lysine and methionine are supplemented (Alimon, 2004).

6