antimicrobial activity of goat milk casein generated
TRANSCRIPT
ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN GENERATED BY LACTIC ACID BACTERIA USED AGAINST
Cronobacter sakazakii
IBRAHIM MUSTAFA ELSHAAFI
UNIVERSITI SAINS ISLAM MALAYSIA
ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN GENERATED BY LACTIC ACID BACTERIA USED AGAINST
Cronobacter sakazakii
Ibrahim Mustafa Elshaafi (Matric. No. 3090103)
Thesis submitted in fulfillment for the degree of master of science
Faculty of Science and Technology UNIVERSITI SAINS ISLAM MALAYSIA
NILAI
January 2015
i
APPROVAL
The thesis entitled “ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN GENERATED BY LACTIC ACID BACTERIA USED AGAINST Cronobacter sakazakii”submitted by Mr. Ibrahim Mustafa Elshaafi (Matric. No. 3090103) for the Degree of Master in Science was duly approved by the following academic authorities:
ZAITON HASSAN, Ph.D, Faculty of Science and Technology, Universiti Sains Islam Malaysia Date: January 2015
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AUTHOR’S DECLARATION
بسم اهللا الرحمن الرحيم
I hereby declare that the work in this thesis entitled “ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN GENERATED BY LACTIC ACID BACTERIA USED AGAINST Cronobacter sakazakii” and submitted for the Degree of Master in Science and Technologyis my own except for quotations and summaries which have been duly acknowledged.
Date: Signature: Name: IBRAHIM MUSTAFA ELSHAAFI Matric No: 3090103 Address: Faculty of Science & Technology Universiti Sains Islam Malaysia (USIM) 71 800 Nilai, Negeri Sembilan, Malaysia
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BIODATA OF AUTHOR
Ibrahim Mustafa Elshaafi (3090103) was born on the 8th February 1987 in Misurata, Libya (828398). He completed his primary and secondary education in Misurata, a city located in the north west of Libya. He is holder of the degree in Microbiology, after a 3-year program at the Faculty of Sciences of the University of Misurata (7th October), Misurata, from 2005 to 2008. He is at present master student majoring in food microbiology.
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ACKNOWLEDGEMENTS
بسم اهللا الرحمن الرحيم
Grateful to Allah SWT and His Messenger Muhammad SAW. I would like first and foremost to extend my deepest gratitude to Assoc. Prof Dr. Zaiton Hassan who accepted to supervise this thesis work and for the provision of all needed supports despite her various other duties. Dear Assoc. Prof. Dr. Zaiton Hassan, I have learned from you more than I can humanly acknowledge here. I extend my heartfelt thanks to you for your patience, guidance and inspirational skills. I express my sincere gratitude to Dr. Hanina Mohd Noor for her commendable advices and guidance in her capacity of co-supervisor. Your advices and teachings have been instrumental in the implementation of my study program. My special acknowledgement and appreciation to Dr. Norakkiah Abdullah Sani from UKM who co-supervise this thesis and share her invaluable experience in Microbiology particularly in Cronobacter sakazakii. My deepest appreciations go to my colleagues Sharifah, Shahidah. Emdakim, Sa’adu Lawal, Dahimi, Belal, Honoré and Aween for their help and friendship during my studies at USIM. Special regards are extended to my beloved elder brothers Ahmed, Mohammad and Abdullah, I thank my father, Mr. Mustafa Ali Elshaafi, and mother, Mrs Turkeyah Elttomi, for all the sacrifices consented for my education.
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ABSTRACT
ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN GENERATED BY LACTIC ACID BACTERIA USED AGAINST
Cronobacter sakazakii
Cronobacter sakazakii is a foodborne pathogen that has been implicated to be the main cause of neonate’s infections from reconstituted powdered infant formula and powdered milk. Attention is being focused on production of physiologically active compounds derived from milk proteins with antimicrobial property that are effective against the foodborne pathogens are increasing. This study was carried out to investigate the capabilities of 30 lactic acid bacteria (LAB) isolates obtained from different sources to hydrolyse goat’s milk casein (GMC) and produce peptides with antimicrobial activity that can inhibit the growth C. sakazakii and other pathogens. Eight isolates showed good proteolytic activity with clear zones ranging from 7 to 12 mm on skim milk-MRS agar. Initial screening of GMC fermented with these LAB isolates showed that three of them produced hydrolysates that showed antimicrobial activity against the target pathogens C. sakazakii ATCC 25944, C. sakazakii E4 and C. sakazakii O531G and other pathogens. The three LAB were identified as Lactobacillus casei CF2, Enterococcus faecium F.F.B, and Lactobacillus plantarum S by bio-chemical tests, API CHL50, and 16s DNA sequencing. The inhibitory activity of goat milk casein hydrolysate (GMCH) against multi antibiotic resistant pathogens were significantly (P<0.05) affected by LAB isolates, fermentation time, and concentration of GMCH. Killing of target pathogen was observed within 2 h at 0.7 % GMC (v/v). Amino acid profiles of the GMCH varied in the concentration and types of amino acids depending on the LAB used to hydrolyse the GMC. The main amino acid present in the three LAB-GMCH was lysine. The lysine to arginine ratio of GMCH using L. casei-CF2 was 100:0, E. faecium-F.F.B was 91.3: 4.54, L. plantarum-S was 87: 1.58. The ratio of hydrophilic to hydrophobic amino acid of the GMCH also varies with the organism used to generate the peptides. The amino acid profile of the GMCH consisted of hydrophilic amino acid: 69.69% for L. casei-CF2-GMCH, 86.95% for E. faecium-F.F.B-GMCH and 79.68% for L. plantarum-S-GMCH. The results of this study showed potential antimicrobial activity against C. sakazakii by peptides generated from goat milk casein by LAB.
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ABSTRAK
Cronobacter sakazakii adalah pathogen bawaan makanan yang telah dibabitkan sebagai punca utama penyebab infeksi bayi daripada serbuk isian semula formula bayi dan susu tepung. Perhatian keatas penghasilan kompoun aktif fisiologi daripada protein susu yang mempunyai ciri antimikrob berkesan ke atas patogen bawaan makanan adalha meningkat. Kajian ini telah dijalankan untuk menyiasat kebolehan 30 bakteria asid laktik (LAB) yang dipencilkan daripada pelbagai sumber untuk menghidrolisis casein susu kambing (GMC) dan menghasilkan peptida yang mempunyai aktiviti antimikrob dan mampu merencat pertumbuhan C. sakazaki dan patogen lain. Lapan pencilan telah menunjukkan aktiviti proteolitik yang baik dengan zon cerah antara 7 ke 12 mm pada agar MRS-susu rendah lemak. Saringan awal ke atas GMC yang telah ditapai dengan pencilan LAB menunjukkan tiga daripadanya menghasilkan hidrolisat yang menunjukkan aktiviti antimikrob ke atas patogen sasaran C. sakazakii ATCC 25944, C. sakazakii E4 and C. sakazakii O531G dan patogen lain. Ketiga tiga LAB telah dikenalpasti sebagai Lactobacillus casei CF2, Enterococcus faecium F.F.B, dan Lactobacillus plantarum S menggunakan ujian bio-kimia, API CHL50, susunan 16s rDNA. Aktiviti perencatan kasein susu kambing hidrolisat (GMCH) keatas patogen pelbagai rintangan antibiotik adalah signifikan (p<0.05) dipengaruhi oleh isolate LAB, masa penapaian dan kepekatan GMCH. Pembunuhan patogen sasaran telah diperhatikan dalam jangkmasa 2 jam pada 0.7 % GMC (v/v). Profil asid amino GMCH berbeza pada kadar kepekatan dan jenis asid amino bergantung kepada LAB yang digunakan untuk menghidrolisis GMC. Asid amino utama yang hadir pada ketiga-tiga LAB-GMCH adalah lisin. Nisbah lisin kepada arginin GMCH menggunakan L. casei-CF2 adalah 100:0, E. faecium-F.F.B adalah 91.3: 4.54, L. plantarum-S adalah 87: 1.58. Nisbah asid amino hidrofilik kepada hidrofobik GMCH juga berbeza pelbagai dengan organisma yang digunakan untuk menghasilkan peptida. Profil asid amino GMCH terdiri daripada asid amino hidrofilik : 69.69% untuk L. casei-CF2-GMCH, 86.95% untuk E. faecium-F.F.B-GMCH dan 79.68% untuk L. plantarum-S-GMCH. Keputusan kajian ini menunjukkan kebolehan aktiviti antimikrob terhadap C. sakazakii melalui peptide yang terhasil daripada casein susu kambing oleh LAB.
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الملخص
االهتمام . السبب الرئيسي لعدوي حديثي الوالدة من حليب األطفال المجفف Cronobacter sakazakiiتعتبر بكتريا اجريت . بدأ يتزايد علي المرآبات النشطة وظيفيا المستمدة من بروتينات الحليب مع خواصها المضادة للميكروبات
ازين حليب الماعز بكتيريا حمض الالآتيك عزلت من مصادر مختلفة لتقوم بتحليل آ 30هذه الدراسة للتحقق من قدرة - 7ثمانية عزالت أظهرت نشاط تحليلي مع مناطق واضحة تتراوح مابين . إلنتاج مكونات لها نشاط مضاد للبكتيريا
الفرز االولي لكازين حليب الماعز المخمر بواسطة بكتيريا حمض الالآتيك . MRSملم علي اقار الحليب و ال 12 C. sakazakiiونات لها القدرة علي تثبيط نمو بكتيريا المستهدفة عزالت لها القدرة علي انتاج مك 3اظهر أن
ATCC 25944, C. sakazakii E4 and C. sakazakii O531G. تم تعريف . وبكتيريات ممرضة أخري Lactobacillus casei CF2, Enterococcus faecium F.F.B, andالبكتيريا حمض الالآتيك علي انها Lactobacillus plantarum S نظام بواسطة اختبارات آيميائية ،API CHL 50 وبواسطة تسلسل الحامض
النشاط المثبط لكازين حليب الماعز ضد مسببات االمراض المقاومة للمضادات الحيوية آان مميز . DNAالنووي قتل . اعز، وقت التخمير وترآيز مهضوم آازين حليب الممتأثرا بحسب نوع عزلة البكتيريا (p<0.05)احصائيا عند
ملف االحماض االمينية لثالث مهضومات آازين ). ح/ح( 0.7الممرض المستهدف لوحظ خالل ساعتين عند ترآيز علي آل االحوال ترآيز ونوع االحماض . حليب الماعز اختبر وأظهر انماط مختلفة لهضم الكازين الي ببتيدات
الاليسين لوحظ بأنه الحامض . المستخدمة للهضماالمينية المنتجة تختلف علي حسب نوع بكتريا حمض الالآتيك ، L. casei-CF2 100:0نسبة الاليسين الي االرجينين للعزلة . االميني المتوفر بشكل اآبر في المهضومات الثالثة
E. faecium- FFB 91.3:4.54 ،L. plantarum-S 87:1.58 نسبة الهيدروفيلك الي الهيدروفلك تختلف . ارجنين %69.69مهضوم آازين حليب الماعز يحتوي علي احماض امينية هيدروفيلكية بنسبة . المستخدمة علي البكتيريا
. L. plantarum-Sلبكتيريا %79.68و E. faecium-FFBلبكتيريا %86.95و L. casei-CF2لبكتريا
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CONTENTS
CONTENTS Page APPROVAL i AUTHOR DECLARATION ii BIODATA OF AUTHOR iii ACKNOWLEDGEMENTS iv ABSTRACT v ABSTRAK vi vii الملخصCONTENT vii LIST OF TABLES xii LIST OF FIGURES xiv LIST OF APPENDICES xvi LIST OF ABBREVIATION AND SYMBOLS xvii CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITTERATURE REVIEW 4
2.1 Cronobacter and Its Significance for Safety 4
2.1.1 Taxonomy of Enterobacter sakazakii 4 2.1.2 Health hazards of Cronobacter in Infants 6 2.1.3 Characteristics of Cronobacter Species 8 2.1.4 Susceptibility of Cronobacter spp. to Antibiotics 10 2.1.5 Characteristics of Cronobacter Infections 10 2.1.6 Presence of Cronobacter in Infant Formula Milk 11 2.1.7 Inactivation and Inhibition of Cronobacter in Infant Formula Milk 13
2.2 Antimicrobial Compounds Produced by Lactic Acid Bacteria 14 2.3 Occurrence of Bioactive Peptides in Various Milk and Milk Products 18
2.3.1 Health Aspects 182.3.2 Antimicrobial Agents 20
2.4 Production and Functionality of Bioactive Peptides 21
2.4.1 Enzymatic Hydrolysis 22 2.4.2 Microbial Fermentation 23
2.5 Milk as a Source of Antimicrobial Peptides 24 2.6 Lactose 25
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CHAPTER 3: ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF LACTIC ACID BACTERIA FROM DIFFERENT SOURCES
26
3.1 INTRODUCTION 26 3.2 MATERIALS AND METHODS 29
3.2.1 Isolation of LAB from Different Sources 29 3.2.2 Proteolytic ActivityDetection 30 3.2.3 Acid and Bile Tolerance of LAB Isolates 30
3.2.3.1 Bile Tolerance 30 3.2.3.2 Tolerance to Different Acidic pH Values 31
3.2.4 Sodium Chloride Tolerance 31 3.2.5 Phenotypic Identification of LAB Using API 50 CHL Assay 32 3.2.6 Genotypic Characterization 32
3.2.6.1 DNA Extraction 323.2.6.2 PCR Amplification of LAB 34 3.2.6.3 PCR Product Clean Up 35 3.2.6.4 Gel Extraction 36 3.2.6.5 PCR Product Sequencing 37
3.3 RESULTS 37
3.3.1 Proteolytic Activity of LAB Isolated from Different Sources 37 3.3.2 Bile salt, Acid and Sodium Chloride Tolerance of LAB Isolates 39 3.3.3 Phenotypic Identification of LAB Using API 50 CHL Assay 43 3.3.4 Molecular Characterization for LAB Isolates 45
3.3.5 PCR of Lactic Acid Bacteria 46
3.4 DISCUSSION 47 3.5 CONCLUSION 49
CHAPTER 4: ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN HYDROLYSATE AGAINST MULTI ANTIBIOTIC RESISTANT Cronobacter sakazakii AND SELECTED PATHOGENIC BACTERIA
50
4.1 INTRODUCTION 50 4.2 MATERIALS AND METHODS 53
4.2.1 LAB Cultures 53 4.2.2 Goat and Bovine Milk Casein Preparation 53 4.2.3 Generation of Protein Hydrolysate 54 4.2.4 Protease Activity Assay (PA) 54 4.2.5 Antibiotic Resistant Test of Target Bacteria 56
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4.2.6 Antibacterial Activity of GMC and BMC Hydrolysate 56
4.3 RESULTS 57 4.3.1 Protease Activity Assay (PA) 57 4.3.2 Multiple Antibiotic Resistant (MAR) Patterns of Tested Pathogens 57
4.3.3 Antimicrobial Activity of GMC and BMC Hydrolysates against Selected Pathogens
61
4.4 DISCUSSION 62 4.5 CONCLUSION 64
CHAPTER 5: ANTIMICROBIAL ACTIVITY OF GOAT MILK CASEIN HYDROLYSATE DERIVED FROM FERMENTATION OF GOAT MILK CASEIN WITH LACTIC ACID BACTERIA
65
5.1 INTRODUCTION 65 5.2 MATERIALS AND METHODS 67
5.2.1 Microorganisms and Culture Conditions 67 5.2.2 Lactose Content 67 5.2.3 Detection of Proteolytic Activity by O-Phthalaldehyde (OPA) Spectrophotometric Assay
68
5.2.4 Protein Content of GMCH 69 5.2.5 Determination of pH of GMC Hydrolysates 70 5.2.6 Antimicrobial Kinetics of AMP 70 5.2.7 Amino Acid Composition 71 5.2.8 Statistical Analyses 72
5.3 RESULTS 72
5.3.1 Effect of Lactose Concentration and Fermentation Time on Proteolytic Activity (Pa) and pH of GMCH
72
5.3.2 Time-Kill Assay of GMCH 77 5.3.3 Amino Acid Composition of GMCH 84
5.4 DISCUSSION 86 5.5 CONCLUSION 88
CHAPTER 6: CONCLUSION AND RECOMMENDATIONS 89
6.1 CONCLUSION 89 6.2 RECOMMENDATIONS 90
REFERENCES 91
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APPENDICES 134 LIST OF PUBLICATONS 147
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LIST OF TABLES
Page
TABLE 1: Characteristic of LAB Isolated from Different Sources 38
TABLE 2: Growth of Selected LAB in MRS Broth with 0.3% of Bile Salt Incubated at 37oC Measured at OD560 nm
40
TABLE 3: Survival of pH-Stressed LAB-CF2 in MRS Broth Incubated at 37oc for 24h Measured at OD560 nm
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TABLE 4: Survival of pH-Stressed LAB-F.F.B in MRS Broth Incubated at 37oC for 24 h Measured at OD560 nm
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TABLE 5: Survival of pH-Stressed LAB-S in MRS Broth Incubated at 37oC for 24 h Measured at OD560 nm
42
TABLE 6: Survival of Selected LAB in MRS Broth with Different NaCl Concentrations Incubated at 37oC
42
TABLE 7: Carbohydrates Fermentation by Lactic Acid Bacteria Isolates Using API 50 CHL Identification System
43
TABLE 8: Quantification of Purified DNA Extracts 45
TABLE 9: Protease Activity of Selected LAB on Hammersten Casein Incubated at 45oC for 30 min
57
TABLE 10: Antibacterial Activity of Selected Antibiotics Against Pathogenic Bacteria Measured by Diameter of Inhibition Zone around the Discs
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TABLE 11: Antibacterial Activity of Selected Antibiotics Against Target C. sakazakii spp. Measured by Diameter of Inhibition Zone around the Discs
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TABLE 12: Antimicrobial Activity of GMCH Against Selected Pathogens Incubated at 30oC for 24 h
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TABLE 13: CFU Reduction of C. sakazakii ATCC 25944, C. sakazakii E4, andC.sakazakii O531G using different LAB-GMCH Concentrations Evaluated at Different Time Intervals
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TABLE 14: Initial Number of Target Pathogens at Different LAB-GMCH Concentrations
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TABLE 15: Amino Acid Composition of GMCH Fermented by Selected LAB at 37oC for 48 h Incubation
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LIST OF FIGURES
Page
FIGURE 1: Growth of LAB on MRS Agar with CaCO3 Showing Clear Zones around the Colonies
37
FIGURE 2: LAB Isolates as Seen Under the Microscope Showing the Shape and Gram Reaction
38
FIGURE 3: Clear Zones around LAB Colonies Indicating Proteolytic Activity on MRS Skim Milk Agar
39
FIGURE 4: DNA Bands of LABs on the 1.0% Agarose Gel 46
FIGURE 5: The Effect of Lactose Concentration on PA and pH of Fermentation after 24 h at 37oC with L. casei-CF2
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FIGURE 6: The Effect of Lactose Concentration on PA and pH of Fermentation after 48 h at 37oC with L. casei-CF2
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FIGURE 7: The Effect of Lactose Concentration on PA and pH of Fermentation after 24 h at 37oC with E. faecium-FFB
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FIGURE 8: The Effect of lactose Concentration on PA and pH of Fermentation after 48 h at 37oC with E. faecium-FFB
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FIGURE 9: The Effect of Lactose Concentration on PA and the pH of Fermentation after 24 h at 37oC with L. plantarum-S
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FIGURE 10: The Effect of Lactose Concentration on PA and the pH of Fermentation after 48 h at 37oC with L. plantarum-S
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FIGURE 11: Time-kill Curve of GMCH-L. casei CF2 on Survival of (A) C. sakazakii ATCC 25944, (B) C. sakazakii O531G and (C) C. sakazakii E4 after 24 h Fermentation at 30oC
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FIGURE 12: Time-kill Curve of GMCH-L. casei CF2 on Survival of (A) C. sakazakii ATCC 25944, (B) C. sakazakii O531G and (C) C. sakazakii E4 after 48 h Fermentation at 30oC
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FIGURE 13: Time-kill Curve of GMCH-E. faecium F.F.B on Survival of (A) C. sakazakii ATCC 25944, (B) C. sakazakii O531G and (C) C. sakazakii E4 after 48 h Fermentation at 30oC
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FIGURE 14: Time-kill Curve of GMCH-L. plantarum S on Survival of (A) C. sakazakii ATCC 25944, (B) C. sakazakii O531G and (C) C. sakazakii E4 after 48 h Fermentation at 30oC
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LIST OF APPENDICES
Page
APPENDIX A Partial sequence of LAB isolates DNA (A): L. casei CF2, (B): E. faecium F.F.B and (C): L. plantarum S.
137
APPENDIX B Figure 1: Amino acid composition profile of GMCH fermented by LAB at 37oC (A): standard amino acids, (B): Control GMC, (C): L. casei CF2 and (D): E. faecium F.F.B, E: L. plantarum S.
140
APPENDIX C ANOVA Output for the Effect of LAB, Fermentation Time and Lactose Concentration on Proteolytic Activity
143
APPENDIX D Least mean Square Comparison for pH and Antimicrobial Activity of GMCH
144
APPENDIX E ANOVA Output for the Effect of GMCH, GMCH Concentration and Time on the Inhibitory Activity Against Cronobacter spp.
145
APPENDIX F Least Mean Square Comparison for pH and Antimicrobial Activity of GMCH
146
APPENDIX G ANOVA Output for the Effect of GMCH, GMCHC and Kill-Time on the Inhibitory Activity Against Cronobacter spp.
147
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LIST OF ABBREVIATIONS AND SYMBOLS
°C degree celsius
µl microliter
µm micrometer
ACE angiotensin-I converting enzyme
AMPs antimicrobial peptides
BMC bovine’s milk caseinate
bp base pair
CaCO3 Calcium carbonate
CCl3COOH trichloroacetic acid
CEP cell envelop-associated proteinase
CFU colony-forming unit
CH3COONa sodium acetate
CH3COONa.3H2O sodium acetate trihydrate
cm centimeter
CPPs calcium-binding phosphopeptides
dH2O distilled water
ddH2O double-distilled water
DNA deoxyribonucleic acid
FeSO4.7H2O iron (II) sulfate heptahydrate
FT fermentation time
g gram
GABA �- aminobutyric acid
GI gastrointestinal
GMC goat’s milk casein
GMCH goat’s milk casein hydrolysate
GMCHs goat’s milk casein hydrolysates
GMP glycomacropeptide
v
h hour
H2O water
HCl hydrochloric acid
IFM Infant formula milk
LAB lactic acid bacteria
LC lactose concentration
M mole
mg milligram
MgSO4.7H2O magnesium sulfate
min minute
ml milliliter
mm millimeter
mM millimole
MnSO4.H2O manganese (II) sulfate monohydrate
MRS Man, Rogosa and Sharpes
Na2CO3 sodium carbonate
NaCl sodium chloride
NaOH sodium hydroxide
ng nanogram
nm nano meter
OPA O-phthalaldehyde
PA Protease activity assay
PBS phosphate-buffer saline
PCR polymerase chain reaction
pmole picomole
RC regenerated cellulose
RNA ribonucleic acid
sec second
TCA trichloroacetic acid
u unit
UV ultraviolet
vi
v/v volume/volume
w/v weight/volume
α-la α-lactalbumin
β-lg β-lactoglobulin
μg microgram
93
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Fermented Goat’s milk. USIM 1st Postgraduate Colloquium (2010). Univirsiti Sains Islam Malaysia. Oral
2) Elshaafi I.M., Zaiton H, Hanina M.N, Norrakiah A.S., and Asma S.A. Milk-Derived
Bioactive compouds Generated by Lactic Acid Bacteria used against Cronobacter sakazakii. Interational Postgraduate Conference on Biotechnology (IPCB 2011) Institute of Marine Biotechnology, UMT and Soka UN, (INTERNATIONAL). Oral. pp. 25.
3) Elshaafi I.M., Zaiton H, Hanina M.N, Norrakiah A.S. Antimicrobial Activity of
Fermented Goat’s Milk. Pertandingan Inovasi Pelajar (SIC’ 11). 2011. Univirsiti Sains Islam Malaysia. Oral
4) Elshaafi I.M., Zaiton H, Hanina M.N, Norrakiah A.S. Antibacterial Activity of Goat’s
Milk Casein Hydrolysate Generated by Lactic Acid Bacteria Used against Cronobacter sakazakii strains. International Conference on Beneficial Microbes 2014 (ICOBM 2014) Penang, Malaysia. Oral
5) Hassan Z., Elshaafi I.M, Mohd Noor H, Sani N.A. Antibacterial Activity of Goat Milk
Casein Fermented with selected Lactic Acid Bacteria against Selected Pathogens. 2nd International Congress on food Technology; November 05-07, 2014 Kusadas / Turkey Association of Food Technology/Turkey & Ankara University/Turkey
POSTERS
1) Elshaafi I.M., Zaiton H, Hanina M.N. Antimicrobial Activity of LAB-Fermented
Goat’s milk. Student’s Innovation Competiotion (SIC2011). Universiti Sains Islam Malaysia.
2) Elshaafi I.M., Zaiton H, Hanina M.N, Norrakiah A.S. Antimicrobial Activity of LAB-
Fermented Goat’s milk Casein. International Conference on Postgraduate Esucation (ICPE-5 2012). Universiti Teknologi Malaysia (UTM).