UNIVERSITI PUTRA MALAYSIA

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1 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

2 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

3 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 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 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

4 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

5 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 Pengerusi: Suraini Abd Aziz, PhD Oleh LISA ONG GAIK AI DECEMBER 2006 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 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 10 6 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

6 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

7 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

8 I certify that an Examination Committee has met on 26 December 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 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

9 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

10 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 ix

11 TABLE OF CONTENTS Page ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ii iv vi viii ix xiii xv xix CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 2.1 Palm Kernel Cake (PKC) General Characteristic and Nutritional Values of Palm Kernel Cake Cell Wall Composition of Palm Kernel Cake Treatment of Palm Kernel Cake as Effective Feed Utilisation of PKC in Livestock 2.2 Solid Substrate Fermentation (SSF) Microorganism Substrate Biomass Measurement Environmental Factors Application of SSF 3 GENERAL MATERIALS AND METHODS 3.1 Materials Palm Kernel Cake (PKC) Microorganism Source of Chemicals and Reagents 3.2 Methods General Plan of the Experimental Work Inoculum Preparation Solid Substrate Fermentation (SSF) Submerged Fermentation (SmF) Enzymatic Hydrolysis Mannanase Assay Soluble Protein Assay Reducing Sugar Assay x

12 4 SCREENING AND PROFILING OF ENZYMES PRODUCED DURING SOLID SUBSTRATE FERMENTATION 4.1 Introduction 4.2 Materials and Methods Pre-germinated Aspergillus niger Solid Substrate Fermentation (SSF) Enzyme Extraction Enzymes Assays Sample Analyses 4.3 Results and Discussion Enzyme Screening and Profiling The effect of Inoculum Type and Extraction Condition 4.4 Conclusion 5 OPTIMISATION OF MANNANASE PRODUCTION IN SOLID SUBSTRATE AND SUBMERGED FERMENTATION 5.1 Introduction 5.2 Materials and Methods Solid Substrate Fermentation for Mannanase Production Submerged Fermentation for Mannanase Production Sample Analyses 5.3 Results and Discussion Optimisation of Mannanase Production Parameters in Solid Substrate Fermentation Optimisation of Mannanase Production Parameters in Submerged Fermentation 5.4 Conclusion 6 PRODUCTION AND CHARACTERISATION OF MANNANASE USING OPTIMISED CONDITION IN SUBMERGED AND SOLID SUBSTRATE FERMENTATION 6.1 Introduction 6.2 Materials and Methods Solid Substrate Fermentation for Mannanase Production in Optimised Condition Submerged Fermentation for Mannanase Production in Optimised Condition Sample Analyses Characterisation of Mannanase 6.3 Results and Discussion Enzyme Production in Submerged Fermentation xi

13 6.3.2 Enzyme Production in Solid Substrate Fermentation Enzyme Decay Experiment Enzyme Characteristics 6.4 Conclusion 7 ENZYMATIC HYDROLYSIS OF PALM KERNEL CAKE USING CRUDE ENZYME 7.1 Introduction 7.2 Materials and Methods Production of Mannanase in Submerged Fermentation and Solid Substrate Fermentation Optimisation of the Parameters for the Hydrolysis Reducing Sugar Assay 7.3 Results and Discussion The Effect of Substrate Concentration on Hydrolysis The Effect of Enzyme Concentration on Hydrolysis The Effect of Initial ph on Hydrolysis The Effect of Operating Temperature Enzymatic Hydrolysis of Palm Kernel Cake 7.4 Conclusion GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK 146 REFERENCES APPENDICES BIODATA OF THE AUTHOR LIST OF PUBLICATIONS xii

14 LIST OF TABLES Table Page 2.1 Proximate analysis (%) of palm kernel cake Amino acid content of PKC Characteristics of PKC surveyed in Physicochemical properties of endo-mannanases from some different microbial sources 2.5 Effect of mannan degrading enzyme on broiler s performance Nutritive value of fermented PKC Comparison of ration composition using fermented PKC and typical diet Recommended levels of PKC in livestock feeds Comparison between submerged and solid substrate fermentation Main groups of microorganisms involved in SSF processes Main application of SSF processes in various economical sectors Composition of raw palm kernel cake Cellulases and hemicellulases produced by A. niger through SSF 4.2 The production of mannanase using different types of inoculum and extraction temperature 5.1 The mannanase production by A. niger FTCC 5003 from various inoculum concentrations in solids substrate fermentation 5.2 The mannanase production by A. niger FTCC 5003 from various initial moisture content in solid substrate fermentation 5.3 The mannanase production by A. niger FTCC 5003 from various temperature in solid substrate fermentation xiii

15 5.4 The mannanase production by A. niger FTCC 5003 from various percentages of nitrogen in urea in solid substrate fermentation 5.5 The mannanase production by A. niger FTCC 5003 from various inoculum concentrations in submerged fermentation 5.6 The mannanase production by A. niger FTCC 5003 from various agitation speeds in submerged fermentation 5.7 The mannanase production by A. niger FTCC 5003 from various temperatures in submerged fermentation 5.8 The mannanase production by A. niger FTCC 5003 from various percentage of palm kernel cake in submerged fermentation 5.9 Optimum condition obtained for solid state and submerged fermentation 6.1 Half-life (t½) for crude mannanase that produced obtained from SSF and SmF 7.1 Reducing sugar production and productivity obtained from various PKC concentrations through the hydrolysis 7.2 Reducing sugar production and productivity obtained from various enzyme concentrations through the hydrolysis of PKC 7.3 Reducing sugar production and productivity obtained from various initial phs through the hydrolysis of PKC 7.4 Reducing sugar production and productivity obtained from various operating temperatures through the hydrolysis of PKC 7.5 Reducing sugar production and productivity obtained from optimised condition through the hydrolysis of PKC xiv

16 LIST OF FIGURES Figure Page 2.1 Mechanical extraction of palm kernel oil Raw palm kernel cake The preliminary study of SSF The optimisation process for mannanase production The enzymatic hydrolysis of PKC Influence of the extraction condition on mannanase activity Growth morphology of spore suspension of A. niger as inoculum in solid substrate fermentation after 48 hours of fermentation 4.3 Growth morphology of per-germinated A. niger as inoculum in solid substrate fermentation after 48 hours of fermentation 5.1 Mannanase productions by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration 5.2 Soluble protein concentration by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration 5.3 Reducing sugar concentration by A. niger FTCC 5003 in solid substrate fermentation at different inoculum concentration 5.4 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different initial moisture content 5.5 Soluble protein concentration by A. niger FTCC 5003 in solid substrate fermentation at different initial moisture content 5.6 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different incubation temperature 5.7 Mannanase production by A. niger FTCC 5003 in solid substrate fermentation at different percentage of nitrogen source from urea 5.8 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different inoculum concentration xv

17 5.9 Soluble protein concentration produced by A. niger FTCC 5003 in submerged fermentation at different inoculum concentration 5.10 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different agitation speed 5.11 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different agitation speed 5.12 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different agitation speed 5.13 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different temperature 5.14 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different temperature 5.15 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different temperature 5.16 Mannanase production by A. niger FTCC 5003 in submerged fermentation at different % of PKC 5.17 Soluble protein concentration by A. niger FTCC 5003 in submerged fermentation at different % of PKC 5.18 Reducing sugar concentration by A. niger FTCC 5003 in submerged fermentation at different % of PKC 6.1 Mannanase productions by A. niger in submerged fermentation 6.2 Mannanase productions by A. niger in solid substrate fermentation 6.3 Heat inactivation of mannanase activity of four different temperature Effect of ph on mannanase activity Effect of temperature on mannanase activity 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

18 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 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 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 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 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 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 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 Hydrolysis of PKC using optimum condition 143 xvii

19 LIST OF ABBREVIATIONS A w BSA Water activity Bovine serum albumin CMCase Carboxymethyl Cellulase DNS DP EU FPase FTCC MARDI PKC PUF SmF SSF Dinitrosalicylic acid solution Degree of polymerisation European Union Filter paper assay Food Technology Culture Collection Malaysia Agricultural Research and Development Institute Palm kernel cake Polyurethane foam Submerged fermentation Solid substrate fermentation xviii

20 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 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.

21 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

22 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

23 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. iv. partially characterise the crude enzymes produced, optimise the parameters for the hydrolysis of PKC using the crude enzymes preparation obtained from both techniques of fermentation. 4

24 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

25 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 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