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UNIVERSITI PUTRA MALAYSIA
PRODUCTION OF PROCESSED CHEESE USING LIPASE-CATALYZED TRANSESTERIFIED PALM KERNEL OLEIN
AND ANHYDROUS MILK FAT MIXTURES
MARIAM MOHAMED ISMAIL
FSMB 1999 2
PRODUCTION OF PROCESSED CHEESE USING LIPASE-CATALYZED TRANSESTERIFIED PALM KERNEL OLEIN AND ANHYDROUS MILK
FAT MIXTURES
MARIAM MOHAMED ISMAIL
MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA
1999
PRODUCTION OF PROCESSED CHEESE USING LIPASE-CATALYZED TRANSESTERIFIED PALM KERNEL OLEIN AND ANHYDROUS MILK
FAT MIXTURES
By
MARIAM MOHAMED ISMAIL
Thesis submitted in Fulfillment of the Requirements for the Degree of Master of Science in the Faculty of
Food Science and Biotechnology U niversiti Putra Malaysia
July 1999
To those who are proud of my success
my life partner,
Fareg
and my beloved daughter,
Eman
ACKNOWLEDGEMENT
All praise due to Allah, the Most Gracious and Merciful, for giving me the
strength, health and determination to complete my study.
Then, I would like to express my deepest gratitude, honor and sincere
appreciation to my supervisor Professor Dr. Hasanah Mohd Ghazali for her invaluable
guidance, encouragement, constructive suggestions throughout the study period, and for
giving me total freedom to explore my research. Her enthusiasm and patience have left a
feeling of indebtedness that can not be fully expressed. I would like to thank her for
sponsoring my study.
My heartfelt appreciation and gratitude go to the members of my supervisory
committee, Dr. Mohd. Yazid Abd. Manap, Associate Professor Dr. Salmah Yusof, Dr.
Kamariah Long and Mr. Dzulkifly Mat Hashim for their generous guidance, valuable
comments during the study and for reviewing my manuscript and making valuable
comments from time to time.
My special appreciation is also extended to Dr. Lai Oai Meng for her warm
friendship and helpful comments while she was a graduate student at the department of
Biotechnology, Faculty of Food Science and Biotechnology. I wish to express my
sincere appreciation to my colleagues, namely, Maryam, Hanan, Tri Haryati, Gabby,
Tan Chin Ping, Shuhaimi and Khalid for their friendship and support.
111
My deep appreciation is accord to the Dean, staff members, technicians and my
colleagues, Faculty of Food Science and Biotechnology for their support and
cooperation. Special thanks to Mr. Mohd Amran bin Suratman, Food Processing Lab.
for his assistance in the formulation of plastic casein. Mrs. Asmarani Abdullah at the
Deputy Dean's Office for her assistance.
I also wish to extend my sincere gratitude and thanks to the staff of the Palm Oil
Research Institute Malaysia (PORIM) for allowing me to formulate the experimental
processed cheese at their labs. The assistance and support by Dr. Chong Chiew, Mrs.
Karimah Ahmad and their co-workers at the Physics and Processing Labs are deeply
appreciated.
Most of all I am grateful to my husband, Fareg Mohamed Aldebrez and my
daughter Eman for their encouragement, endless patience, and sacrifice all the way
throughout this work. Without the benefit of their loving support and dedication, I
would not have had the wonderful opportunities that I have experienced.
Last but not least, heartfelt appreciation goes to the Government of Malaysia and
Universiti Putra Malaysia for providing the financial support for this study through the
IRP A program (No. : 03-02-04-0025) awarded to Prof Dr. Hasanah Mohd Ghazali.
IV
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS... . . . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..... 111 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vu LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX LIST OF PLATES . . . ... . .. ... ... . .. ... . .. . . . . . . . .. ... ... .. . . . . ... ... . . . . . . ... .. ... Xl LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xu ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVll
CHAPTER
I
II
ill
INTRODUCTION ....................................................... . 1
LITERATURE RE�W .............................................. . 6
Fats and Oils . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. 6 Structure and Composition of Fats and Oils . .. . . . . . . ... . . . . . . .. 6 Types of Fats and Oils . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Reactions of Fats and Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Lipases . . . ........ ... ... ... ...... ... ... ... ... ... ... ... ... ... ... ... ... ... 17 Definition of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 17 Lipase Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Reactions of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 23 Applications ofLipases . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 29
Cheese . . . . . . . . . ... . . . .. . ... ... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 32 History and Background . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 32 Definition of Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Types of Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Techniques and Procedures Used in Cheese Production . .. ... 34 Factors Affecting The Physical Properties of Cheese . . . . . . .. . . . 35 Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Sensory Evaluation and Texture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Sensory Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 40 Texture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
MATERIALS AND METHODS 44
Enzymatic Transesterification of Palm Kernel Olein and Anhydrous Milk Fat . .. ... . . . . . . . . . . . . . . . . . . . . . . . 44
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
v
IV
v
Formulation of Processed Cheese Using Transesterified Fat . . . . . . . . . . . 5 1 Preparation of Starter Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 Preparation of Plastic Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 Formulation of Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Chemical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Analysis of Plastic Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Analysis of Produced Processed Cheese Blends . . . . . . . . . . . 67
Sensory Evaluation and Texture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Sensory Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Texture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
RESULTS AND DISCUSSION 71
Enzymatic Transesterification of Palm Kernel Olein and Anhydrous Milk Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Triglyceride Compositions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Degree of Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Slip Melting Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Solid Fat Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , 8 1 Crystal Forms of Trans esterified PKO and AMP . . . . . . . . . . . 84 Thermal Behaviour . . . . . . . . . . .. .. ... . . . . . . . . . . .. . .. . . . . . .... . . . . . , . 89
Production of Processed Cheese Using Transesterified Fat . . . . . . . . . . . 99 Plastic Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Sensory Evaluation and Texture Analysis of Produced Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Sensory Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Texture Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Relating Sensory and Non-Sensory Data . . . . . . . . . . . . . . . . . . . , 1 1 0
CONCLUSION AND RECOMMENDATIONS 1 12
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 12 Recommendations for Further Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 5
BmLOGRAPHY 1 1 6
APPENDIX
APPENDIX A 137
APPENDIXB 141
APPENDIX C 144
VITA 147
VI
Table
1
2
3
4
5
6
7
8
9
1 0
1 1
12
1 3
1 4
1 5
1 6
LIST O F TABLES
Chemical Characteristics of Palm Kernel Olein
Physical Properties of Palm Kernel Olein
Fatty Acids Composition of Anhydrous Milk Fat . . . . . . . . . . . . . . . . . . . . . .
Physical and Chemical Properties of Anhydrous Milk Fat
Amount of Ingredients in the Final Product . . . . . . . . . . . . . . . . . . . . . . . . . . .
X-Ray Diffiaction Analysis of Pure PKO and AMF
X-Ray Diffiaction Analysis of the 3 : 7 PKO:AMF
X-Ray Diffiaction Analysis of the 1 : 1 PKO:AMF
X-Ray Diffiaction Analysis of the 7 :3 PKO:AMF
Last Melting Peak (LMP) and Slip Melting Point (SMP) of Pure PKO and AMF Fats . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . . . . . . . . . . . .
Last Melting Peak (LMP) and Slip Melting Point (SMP) of the 3 :7 PKO:AMF Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . .
Last Melting Peak (LMP) and Slip Melting Point (SMP) of the 1: 1 PKO:AMF Reaction Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . .
Last Melting Peak (LMP) and Slip Melting Point (SMP) of the 7:3 PKO:AMF Reaction Mixture . . . . . . , . . . . . . . . . . . . . . , . . . . . .
Chemical Analyses of Commercial Cheddar Cheese and Prepared Plastic Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemical Analysis of Processed Cheese Blends
Texture Analysis of Processed Cheese Blends
V11
Page
10
1 1
14
15
60
86
86
87
87
96
97
97
98
1 00
104
1 10
1 7 Sensory Data Vs Texture Analyzer Data of Processed Cheese Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 8 Degree of Hydrolysis of the Palm Kernel Olein and Anhydrous Milk Fat and their Reaction Mixtures before and after
1 9
20
2 1
22
23
24
Transesterification
Slip Melting Point of Palm Kernel Olein and Anhydrous Milk Fat and their Reaction Mixtures before and after Transesterification
Solid Fat Content of Pure PKO and AMF
Solid Fat Content of the 3 : 7 PKO:AMF Reaction Mixture
Solid Fat Content of the 1 : 1 PKO:AMF Reaction Mixture
Solid Fat Content of the 7 :3 PKO:AMF Reaction Mixture
Amount of Ingredients in the Final Product . . . . . . . . . . . . . . . . . . . . . . . . . . .
viii
1 1 1
137
1 38
1 38
1 39
1 39
140
143
Figure
1
2
3
4
5
6
7
8
9
1 0
1 1
12
1 3
1 4
1 5
1 6
LIST OF FIGURES
The Lipase Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Positional Specificity of Microbial Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Flow Chart of the Preparation Process of Plastic Casein
Determination of Total Protein Using Micro Kjeldahl Method . . . . . . .
Triglyceride Profile of Palm Kernel Olein
Triglyceride Profile of Anhydrous Milk Fat
Triglyceride Profile of the 3 : 7 PKO:AMF Reaction Mixture before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Triglyceride Profile of the 1 : 1 PKO:AMF Reaction Mixture before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Triglyceride Profile of the 7:3 PKO:AMF Reaction Mixture before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Free Fatty Acid Content ofPKO, AMF and their Reaction Mixtures before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Slip Melting Point ofPKO&AMF and their Reaction Mixtures . . . . . .
Solid Fat Content of Pure PKO and AMF Fats
Solid Fat Content of the 3 : 7 PKO:AMF Reaction Mixture
Solid Fat Content of the 1 : 1 PKO:AMF Reaction Mixture
Solid Fat Content of the 7:3 PKO:AMF Reaction Mixture
Heating Thermogram ofPKO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
lX
Page
1 8
2 1
5 8
65
72
72
73
74
75
78
80
82
82
83
83
90
1 7
1 8
1 9
20
2 1
22
23
24
25
26
27
28
29
Heating Thermogram of AMF
Heating Thermogram of the 3 : 7 PKO:AMF before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heating Thermogram of the 1 : 1 PKO:AMF before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heating Thermogram of the 7 :3 PKO:AMF before and after Transesterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Titrable Acidity of Plastic Casein during Preparation Process . . . . . . . . .
Multiple Comparison Test for Processed Cheese Sample
Texture Profile of Internal Control Processed Cheese
Texture Profile of Non-Transesterified Fat Processed Cheese . . . . . . . . .
Texture Profile of Transesterified Fat Processed Cheese
Texture Profile of Commercial Processed Cheese
Sensory Data Vs Texture Analyzer Measurement Relationship . . . . . . .
Panelists to Numerical Scores Conversion Scale
Schematic Diagram of Texture Profile of the Cheese Sample
x
9 1
93
94
95
101
1 07
1 08
108
109
109
1 1 1
146
146
Plate
1
2
3
4
5
6
7
8
9
1 0
1 1
12
1 3
14
1 5
1 6
1 7
LIST OF PLATES
Prepared Starter Sub-Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ingredients of Plastic Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pasteurization of Reconstituted Skimmed Milk Powder
Addition of Sub-Culture
Addition of Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . ' "
Cutting the Curd with Cheese Knife . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cheddaring of the Curd . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Molding the Curd with a Muslin Cloth before Pressing
Pressing the Plastic Casein using Manual Screw Press
Major Ingredients of Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stephan Direct Steam Injector Stainless Steel Cooker . . . . . . . . .
Paired Comparison Test of Processed Cheese Samples
Multiple Comparison Test of Processed Cheese
Stable Micro System (SMS) Texture Analyzer
Internal Control Prepared Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transesterified Fat Processed Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Non-Transesterified Fat Processed Cheese
Xl
Page
53
54
54
55
55
56
56
57
57
6 1
61
68
69
70
1 04
105
1 05
LIST OF ABBREVIATIONS
Symbol Meaning
AMF Anhydrous Milk Fat
AOCS American Oil Chemist Society
BS British Standard
C 12 :0 Lauric Acid
C 14:0 Myristic Acid
C 1 6:0 Palmitic Acid
C I 6 : I Palmitoleic Acid
C 1 8 :0 Stearic Acid
C I 8 : I Oleic Acid
C 1 8 :2 Linoleic Acid
C 1 8 : 3 Linolenic Acid
C22 : I Erucic Acid
Ca2+ Calcium
CBS Cocoa Butter Substitute
DG Diglycerides
DSC Differential Scanning Calometry
FA Fatty Acids
FFA Free Fatty Acids
xu
HMG
HPLC
IDF
IV
LMG
LMP
LOO
LOP
MG
MMG
Na+
N a3C6Hs07
NBD
NMR
NTF
000
pH
PKO
POP
PORIM
POS
PPP
High Melting Glycerides
High Performance Liquid Chromatography
International Dairy Federation
Iodine Value
Low Melting Glycerides
Last Melting Peak
l -linoleoyl-dioleoyl glycerol
1 -linoleoyl-2-oleoyl-palmitoyl glycerol
Monoglycerides
Medium Melting Glycerides
Sodium
Sodium Citrate
Neutralized Bleached Deodorized
Nuclear Magnetic Resonance
Non Transesterified Fat
Triolein
Hydrogen Ion
Palm Kernel Olein
1 ,3-dipalmitoyl -2-oleoyl glycerol
Palm Oil Research Institute of Malaysia
I -palmitoyl-2-oleoyl-stearoyl glycerol
Tripalmitin
Xlll
PPS 1,2-dipalmitoyl-stearoyl glycerol
r Correlation Coefficient
RBD Refined Bleached Deodorized
rpm Revelation Per Minute
SFC Solid Fat Content
SMP Slip Melting Point
SMS Stable Micro System
Sp Species
TA Titrable Acidity
TF Transesterified PKO and AMF Fat
TG Triglyceride
TLC Thin Layer Chromatography
v/v Volume Per Volume
w/w Weight Per Weight
XRD X-Ray Diffraction
XIV
Abstract of thesis presented to the Senate ofUniversiti Putra Malaysia in fulfillment of the requirements for the degree of Master of Science.
PRODUCTION OF PROCESSED CHEESE USING LIPASE-CATALYZED TRANSESTERIFIED PALM KERNEL OLEIN AND ANHYDROUS MILK
FAT MIXTURES
By
MARIAM MOHAMED ISMAIL
July 1999
Chairman: Professor Hasanah Mohd. Ghazali, Ph.D.
Faculty: Food Science and Biotechnology
In recent years, lipase-catalyzed transesterification has received considerable
attention from the fats and oils industry due to its potential to modify the physical and
chemical properties of edible fats and oils. In this study, enzymatic transesterification
of three different ratios of palm kernel olein (PKO) and anhydrous milk fat (AMF)
was carried out using immobilized lipase from Rhizomucor miehei (Lipozyme 1M 60)
to modify their chemical and physical properties, such that they can be used in the
production of processed cheese.
After transesterification for 8 hours, there were either increases or decreases
in the concentrations of several existing triglycerides (TG) of the reaction mixtures
and, in some cases, the formation of new peaks. The results also showed that
increasing the reaction time, increased the % free fatty acid (FF A) between 1 to 2%.
xv
The slip melting points (SMP) of the fat mixtures, which were determined after
removal of free fatty acids, declined after the transesterification process. The solid fat
content (SFC) decreased with respect to the control samples. At 40°C, the PKO:AMF
(7:3) transesterified fat mixture was completely melted, making it a potential
candidate in the formulation of processed cheese. Thermal profiles showed that the
original peaks of the transesterified mixtures have either shifted or completely
changed and some new peaks were also formed. It was also found that the
transesterified fat mixtures were in the �'
form, compared to a predominantly � form
before transesterification.
After the preparation of experimental processed cheese, several chemical and
physical analyses of this final product were carried out. The results showed that the
experimental processed cheese had a satisfactory aroma and texture compared to the
commercial sample. It also recorded a high total solid content and a low amount of
moisture, with a good workability and overall general behaviour at cool, ambient, and
elevated temperatures.
Sensory evaluation and texture analysis studies were also conducted on the
final product. The experimental processed cheese samples achieved an acceptable
response from the panelists, where there was no significant difference between the
samples in terms of colour and texture. The texture profiles indicated that the
experimental processed cheese samples exhibited close texture profiles compared to
their counterpart commercial sample.
XVI
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains.
PENGHASILAN KEJU TERPROSES MENGGUNAKAN CAMPURAN OLEIN ISIRONG KELAPA SA WIT DAN LEMAK SUSU TANPA AIR YANG
DITRANSESTERIFKASI MELALUI PEMANGKINAN BERLIPASE
Oleh
MARIAM MOHAMED ISMAIL
Julai 1999
Pengerusi : Profesor Hasanah Mohd. Ghazali, Ph.D.
Faculti : Sains Makanan dan Bioteknologi
Pada masa kini, transesterifikasi yang dimangkin oleh lipase telah menerima
perhatian dari industri minyak dan lemak disebabkan potensinya untuk mengubah
ciri-ciri fizikal dan kimia minyak dan lemak. Dalam kajian ini. transesterifikasi
secara ber enzim di antara campuran minyak sawit isirong olein (PKO) dan lemak
susu anhidrus (AMF) pada tiga nisbah yang berbeza telah dijalankan menggunakan
lipase tersekat-gerak dari Rhizomucor miehei (Lipozyme 1M 60) untuk mengubah
ciri-ciri kimia dan fizikal dalam campuran untuk membolehkan campuran minyak
untuk menghasilkan keju terproses.
XVll
Selepas transesterifikasi selama 8 hours, terdapat pengurangan atau
penambahan kepekatan beberapa trigliserida yang menang wujud dalam campuran
yang bertindakbalas, dan pembentukan puncak-puncak baru dalam kes-kes tertentu.
Keputusan juga menunjukkan bahawa peningkatan masa tindakbalas telah
menyebabkan penambahan % asid lemak bebas di antara 1 hingga 2%. Titik lebur
(SMP) campuran-campuran lemak yang ditentukan setelah dikeluarkan asid-asid
lemak bebas didapati menurun selepas proses transesterifikasi. Kandungan pepejal
lemak (SFC) telah berkurangan berbanding dengan sampel-sampel kawalan. Pada
suhu 40°C, campuran lemak PKO:AMF (7:3) tertransesterifikasi telah lebur
sepenuhnya. 1ni menjadikan campuran tersebut berpotensi dalam formulasi keju
terproses. Gambarajah terma menunjukkan bahawa puncak-puncak asal dalam
campuran-campuran tertransesterifikasi telah samada beralih tempat atau berubah
sepenuhnya dan beberapa puncak baru telah terbentuk. Selain itu, keputusan juga
menunjukkan bahawa campuran-campuran lemak tertransesterifikasi menghasilkan
bentuk kristal jenis f3', dimana bentuk f3 adalah dominan sebelum transesterifikasi.
Selepas penyediaan keju terproses, beberapa analisis kimia dan fizikal produk
akhir telah dilakukan. Keputusan yang diperolehi menunjukkan behawa keju terproses
yang disediakan mempunyai aroma yang memuaskan dan tekstur yang setanding
dengan sampel komersial. Produk juga merekodkan kandungan pepejal yang tinggi
dan kuantiti lembapan yang rendah dengan kebolehan keIja yang baik dan sifat umum
keseluruhan pada suhu-suhu sejuk, ambien dan tinggi.
XVI11
Kajian penilaian deria dan analisis tekstur juga dijalankan pada produk akhir.
Sarnpel-sarnpel keju terproses yang diediakan telah mencapai respon yang boleh
diterima daripada para ahli panel dari segi warna dan tekstur sifat sarnpel-sampel ini
tidak menunjukkan perbezaan yang bererti. Sarnpel-sampel keju terproses
menunjukkan sifat tekstur yang hampir sarna dengan sarnpel komersial.
XIX
CHAPTER I
INTRODUCTION
Fats and oils are complex mixtures of simple and mixed triglycerides that
function as energy reserves. Triglycerides differ according to the types and composition
of fatty acids and their distribution along the glycerol backbone. These characteristics
play an important role in the physical, rheological, and nutritional properties of fats and
oils.
In recent years, the use of lipases as catalysts has received considerable attention
because of its potential ability to modify the physical and chemical properties of edible
fats and oils (Kyotani et al. 1 988; Yamane et al. 1989; Miller et aI . , 1 99 1 ; Choo and
Rhee, 1 993 ; Foglia et aI . , 1 993 ; Ghazali et aI . , 1 995b; Lai et aI., 1 998). The main
technical application of lipases is to modify the fatty acid compositions of triglycerides
by interesterification.
Lipases are a group of enzymes that preferentially catalyze the hydrolysis and
synthesis of triglycerides. Immobilized lipases show many advantages over traditional
inorganic catalysts (Eigtved et at . , 1 986; Hansen et at . , 1 986; Miller et aI . , 1 988). They
1
2
have large catalytic activity under mild operating conditions. They show large selectivity
to the desired product with no significant side reactions, leading to products of high
purity. They are easily recovered from the reaction mixture and can be reused. There is
no contamination of the final product, saving time and cost in the purification stage
(Coteron et aI. , 1998).
Transesterification with lipase provides a useful way to improve the properties of
triglyceride. Through enzymatic transesterification, it is possible to incorporate a desired
acyl group onto specific positions of the glycerol, whereas chemical transesterification
does not possess this regiospecificity due to the random nature of the reaction
(Sreenivasa, 1978; Norris, 1982; Gunstone and Norris, 1983). Thus, lipase-catalyzed
transesterification can provide regio- or sterospecific structured lipids for nutritional,
medical, and food applications (Akoh, 1995 ; Lee et aI. , 1997; 1998).
Transesterification reaction is most often confirmed by closely monitoring
changes in the product's physico-chemical properties (Goh et aI. , 1993; Kerry, et aI. ,
1993). Physical properties such as melting characteristics, crystallization, and thermal
behaviour are commonly used as specifications for fats and oils. The melting
characteristics including melting point and solid fat content of fat are among the most
important predictors of its functionality used in industry today, and many fat ingredients
are selected for applications based on these characteristics (Kaylegian et aI. , 1993). The
SFC profile over a given temperature range is an important characteristic in fat
functionality that can be used to predict how the fat will behave at different stages of
manufacturing (rolling and baking of doughs). Another important indicator of fat
3
functionality is its crystallization behaviour, which dictates the crystal form present and
the respective stabilities of crystals. Fats, in particular their component triglyceride
molecules, can crystallize in more than one polymorphic forms which differ from each
other in terms of melting point, heat of fusion and crystallization, and density. The a
form is a form that crystallises from the melt, has a strong single short spacing at
0.4 1 5nm and exhibits an enantiotropic transition on further cooling Hagemann ( 1 988).
According to Hagemann ( 1988), D'Souza et ai. ( 1990) and Timms ( 1 991 ), the �' form is
a form exhibiting two strong short spacing bands at 0 .42 and 0.38nm, and the � form
exhibits three strong short spacings bands at 0.46, 0.39 and 0.37nm. Thermal analyses of
fats and oils are also indicators of their physical properties. Differential scanning
calorimetry (DSC) is the most convenient method for determining the thermal behaviour
of fats and oils (Hagemann, 1 988; Che Man et aI. , 1 995; Haryati et aI. , 1996). It can
provide much information on the behaviour of fats and oils.
Cheese is a commercial product, which is widely consumed and almost the one
food of good keeping ability, high in protein with fat, calcium phosphorus, riboflavin,
and other vitamins, in a concentrated form (Sukumar, 1 980). Cheeses of various kinds
always have been important sources of nutrients wherever milk-producing animals could
be raised. It has been an art handed down from generation to generation and was known
as a staple food with bread since 6000BC (Scott, 1 986). Natural cheese is made from the
curd of the milk of cows and other animals, the curd being obtained by the coagulation
of milk casein with an enzyme (usually rennin), an acid (usually lactic acid), and with or
4
without further treatment of the curd by heat, pressure, salt, and ripening with selected
microorganisms (Sukumar, 1 980).
Progress in the development of cheese production, combined with a considerable
increase in the quality of the finished product, made its appearance at the end of the 19th
century (Kosikowski, 1 982). However, there are some constraints (from a nutritional
viewpoint) on the use of cheese depending on the interpretation of the word 'cheese'
(Meyer, 1 973). Therefore, some fresh curds still hold lactose in the free whey and this
may cause allergic reactions in susceptible persons, while riper cheese with no free
lactose is safe. Extremely ripe cheese, where the protein is well broken down to
peptones and amino acids, can be absorbed very quickly and so create difficulty in some
persons, who should, therefore, avoid overripe cheese. Although these difficulties when
consuming cheese do exist for a minority of the population, the individual has usually a
wide assortment of varieties from which to choose so that some of the difficulties can be
avoided (Potter, 1 978) . However, the cheese may reach a condition that makes it unfit
for consumption. This is due to uncontrollable protein decomposition, where all types of
cheese, including the hard cheeses, are unsuitable for long storage, in the sense of
forming an "iron ration". In addition, the harder cheeses are also dependent on
temperature, for instance, it is a well-known fact that hard and semi-hard cheese in
tropical countries become unfavorably altered in flavour and texture (Meyer, 1 973).
To overcome the shortcoming of the natural cheese, the processed cheese
becomes a solution. The invention of processed cheese in 1 9 1 1 has been a very
important step forward in the dairy field (Kosikowski, 1 982). Processed cheese is the