UNIVERSITI PUTRA MALAYSIA PHYSICO-CHEMICAL AND MICROBIOLOGICAL CHANGES DURING FERMENTATION AND STORAGE OF NIPA SAP (Nypa fruticans Wurmb) NUR AIMI BINTI RADI IPPH 2013 1
PHYSICO-CHEMICAL AND MICROBIOLOGICAL CHANGES DURING FERMENTATION AND STORAGE OF NIPA SAP (Nypa fruticans Wurmb) By NUR AIMI BINTI RADI Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science. June 2013
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science PHYSICO-CHEMICAL AND MICROBIOLOGICAL CHANGES DURING FERMENTATION AND STORAGE OF NIPA SAP (Nypa fruticans Wurmb) Chairman Faculty : Fatimah Abu Bakar, PhD By NUR AIMI BINTI RADI June 2013 : Halal Products Research Institute The purpose of this study is to evaluate the changes in the physico-chemical and microbiological profiles of nipa sap during natural fermentation. Natural fermentation of nipa sap involves the breakdown of carbohydrate materials under anaerobic condition with the activity of microorganisms and enzymes present. The process of fermentation is responsible for various changes of properties of nipa sap. The development of ethanol in the sap has always been the main concern related to the halal requirement and standard quality of the sap. Therefore, it is important to determine the basic properties of the sap that can be a reference to the producer. The sap from nipa palm (Nypa fruticans) was collected from local collectors. Samples were fermented at 30 0 C for 63 days. Physical, chemical, and microbiological analyses were carried out at seven-day intervals, starting from day 0 (fresh tapping) until day 63. Physical (colour and transmittance value) and chemical analyses (ash, moisture, crude protein content, ph, total titratable acidity, total soluble solid, ethanol, sugar and organic i
acid contents) were carried out. The ethanol content was analysed using Headspace Gas Chromatography Mass Spectrometer (GC-MS), while the sugar and organic acid contents were analysed using High Performance Liquid Chromatography (HPLC) with Refractive Index and UV detectors, respectively. Results showed that the physical and chemical properties of fresh nipa sap were significantly different (P<0.05) compared to the fermented sap. The ph value decreased from the initial ph of 7.25 in fresh nipa sap to 3.16 in the fermented sap. The decrease in ph value correlated with the increase in organic acids content, which consisted mainly of lactic, acetic, and succinic acids. Total acidity was recorded to be 1.18% (v/v) in fresh nipa sap, and 4.59% (v/v) in the 63 day fermented sap. Succinic acid became the main contributor to the acidic condition in fermented nipa sap, with 1.83% v/v during the 63 days of fermentation, followed by lactic acid (1.67% v/v), acetic acid (0.98% v/v), tartaric acid (0.10% v/v), and pyruvic acid (<0.01% v/v). The initial concentration of ethanol in fresh nipa sap was 0.11% (v/v). Drastic increase in the ethanol content for the sample was recorded during the first seven days of fermentation (6.66% v/v), before beginning to drop slightly from day 21 (6.43% v/v) of fermentation until day 63 (5.72% v/v). The changes in other volatile compounds were also observed during fermentation, including higher alcohols, acetoin, diacetyl, and esters. Total sugar in fresh nipa sap was 16.73% (w/v), with sucrose as the main sugar present (13.33% w/v), followed by fructose (1.40% w/v), glucose (1.27% w/v), and maltose (0.73% w/v). The sugar concentration declined over the fermentation process, giving a different sugar composition in each interval day. Fructose (0.80% w/v) became the main sugar produced on the 63 rd day of fermentation, followed by sucrose (0.60% w/v), glucose ii
(0.34% w/v), and maltose (0.33% w/v). Identification of microbial species was done using the Analytical Profiling Identification system, API. The five species of yeast were identified, namely Saccharomyces cerevisiae, Cryptococcus humicola, Candida guilliermondii, Kloeckera spp., and Stephanoascus ciferii. Five species of lactic acid bacteria identified were Lactobacillus plantarum, Pediococcus spp., Streptococcus thermophilus, and Lactobacillus brevis. Acetic acid bacteria were isolated at the later stage of fermentation and identified as Acetobacter and Gluconobacter. It is concluded that the microbial species were responsible for giving the different characteristics of nipa sap during the fermentation process. It also appeared that activities brought about by microorganisms in the early stages helped the activities in the successive stages. Three stages of fermentation were revealed, including alcoholic, lactic, and acetic acid fermentation. Each stage showed specific properties and importance in order to ensure the complete cycle of the fermentation process. Further studies with a large number of samples and from various sources should be carried out in order to increase the precision of the study. iii
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains PERUBAHAN FIZIKO-KIMIA DAN MICROBIOLOGI DALAM FERMENTASI DAN PENYIMPANAN NIRA NIPAH (Nypa fruticans Wurmb) By Pengerusi Fakulti : Fatimah Abu Bakar, PhD NUR AIMI BINTI RADI Jun 2013 : Institut Penyelidikan Produk Halal Tujuan kajian ini adalah untuk menilai perubahan fiziko-kimia dan profil mikrobiologi nira nipah semasa berlangsungnya fermentasi semulajadi. Proses fermentasi semulajadi nira nipah melibatkan penguraian bahan-bahan karbohidrat dalam keadaan anaerobik dengan kehadiran dan aktiviti pelbagai mikroorganisma dan enzim. Fermentasi yang berlaku menyebabkan perubahan pelbagai sifat air nira nipah. Penghasilan ethanol dalam nira sentiasa menjadi perhatian utama yang berkaitan dengan keperluan halal dan kualiti standard nira. Oleh itu, adalah penting untuk menentukan sifat-sifat asas nira yang boleh dijadikan sebagai rujukan kepada pengeluar. Nira dari pokok nipah (Nypa fruticans) telah dikumpulkan daripada pembekal tempatan. Sampel telah difermentasi pada 30ºC selama 63 hari. Analisis fizikal, kimia dan mikrobiologi telah dijalankan pada selang setiap tujuh hari, bermula dari hari 0 (sejurus selepas pengumpulan nira segar) sehingga 63 hari. Analisis fizikal (warna dan nilai transmitans) dan analisis kimia (kandungan abu, kelembapan, dan kandungan protein iv
mentah, ph, jumlah keasidan tertitrat, jumlah pepejal larut, kandungan etanol, kandungan gula, dan kandungan asid organik) telah dijalankan. Kandungan etanol dianalisis menggunakan Headspace Gas Chromatography Mass Spectrometer (GC- MS), manakala kandungan gula dan asid organik dianalisis menggunakan High Performance Liquid Chromatography (HPLC) dengan masing-masing menggunakan pengesan Indeks Biasan dan UV. Hasil analisis menunjukkan sifat fizikal dan kimia nira nipah segar adalah berbeza secara signifikan (P <0.05) dengan nira fermentasi. Nilai ph menurun dari ph awal 7.25 nira segar kepada 3.16 nira fermentasi. Penurunan dalam nilai ph boleh dikaitkan dengan peningkatan kandungan asid organik, yang utamanya terdiri daripada asid laktik, asetik, dan succinic. Keasidan jumlah adalah 1.18% (v/v) bagi nira segar, manakala 4.59% (v/v) bagi nira terfermentasi hingga hari ke-63. Asid suksinik merupakan penyumbang utama kepada keadaan asid nira nipah terfermentasi (1.83% v/v) selama 63 hari, diikuti oleh asid laktik (01.67% v/v), asid asetik (0.98% v/v), asid tartarik (0.10% v/v), dan asid piruvik (<0.01% (v/v). Kepekatan awal kandungan etanol nira nipah segar adalah 0.11% (v/v). Peningkatan drastik kandungan etanol untuk sampel dicatatkan dalam tempoh tujuh hari pertama fermentasi (6.66% v/v), sebelum mula jatuh sedikit selepas 21 hari fermentasi sehingga 63 hari (5.72% v/v). Perubahan sebatian lain yang tidak menentu turut diperhatikan semasa penapaian, termasuk alkohol yang lebih tinggi, acetoin, diacetyl, dan ester. Jumlah gula nira nipah segar dianalisis adalah sebanyak 16.73%( w/v), dengan sukrosa sebagai gula utama (13.33% w/v), diikuti oleh fruktosa (1.40% w/v), glukosa (1.27% w/v), dan maltosa (0.73% w/v). Kepekatan gula penurun terhasil akibat fermentasi, memberikan komposisi gula yang berbeza pada setiap v
peringkat penilaian. Fruktosa (0.80% w/v) menjadi gula utama wujud pada hari ke-63 penapaian, diikuti oleh sukrosa (0.60% w/v), glukosa (0.34% w/v), dan maltosa (0.33% w/v). Pengenalpastian spesies mikrob telah dilakukan menggunakan system Analytical Profiling Identification, API. Lima spesies yis telah dikenalpasti, iaitu Saccharomyces cerevisiae, Cryptococcus humicola, Candida guilliermondii, Kloeckera sp, dan Stephanoascus ciferii. Lima spesis bakteria asid laktik dikenalpasti iaitu Lactobacillus Plantarum, Pediococcus spp., Streptococcus thermophilus, dan Lactobacillus brevis. Bakteria asid asetik telah diperincikan pada peringkat akhir fermentasi dan dikenalpasti sebagai Acetobacter dan Gluconobacter. Kesimpulannya, bahawa spesis mikrob bertanggungjawab menyebabkan perubahan ciriciri nira nipah semasa fermentasi. Aktiviti mikroorganisma pada peringkat awal membantu aktiviti-aktiviti mikroorganisma di peringkat seterusnya. Tiga peringkat fermentasi telah dinamakan, iaitu fermentasi asid alkohol, laktik, dan asetik. Setiap peringkat memberikan ciri-ciri tertentu dan penting untuk menghasil kitaran lengkap proses fermentasi. Kajian lanjut dengan sebilangan besar sampel dan dari pelbagai sumber perlu dijalankan dalam usaha untuk meningkatkan ketepatan kajian. vi
ACKNOWLEDGEMENTS I would like to express my sincere appreciation and deepest gratitude to Professor Dr. Fatimah Abu Bakar and Mr. Dzulkifly Mat Hashim, for their valuable advice, endless guidance, and encouragement to complete this study. My acknowledgment also goes to all the laboratory staff of Halal Products Research Institute and the Faculty of Food Science and Technology, Universiti Putra Malaysia for their technical support. My acknowledgement also goes to my husband, Muhammad Bin Mohd Sabri, my father, Radi Bin Yahaya, my mother, Ariyati Binti Ahamad, my brothers, Mohammad Badri and Ahmad Fakhruddin, and my sisters, Nur Zawani, Nur Hanisah, and Nur Afiqah, for their moral support and love. This study was fully financed by the Ministry of Science, Technology and Innovation (MOSTI), under the project tittle: Modelling of the Intoxication Effect of Selected Fermented Alcohol Containing Foods. vii
I certify that an Examination Committee has met on date of viva to conduct the final examination of name of student on his degree thesis entitled "title of thesis" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Janna Ong Abdullah, PhD Associate Professor Dr. Faculty of Biotechnology and Bio molecular Sciences Universiti Putra Malaysia (Chairman) Shuhaimi Mustafa, PhD Professor Halal Products Research Institute Universiti Putra Malaysia (Internal Examiner) Abdul Karim Sabo Mohamed, PhD Associate Professor Dr. Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Mohd Khan Ayob, PhD Associate Professor Dr. Universiti Kebangsaan Malaysia (External Examiner) Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date : viii
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Fatimah Abu Bakar, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Dzulkifly Mat Hashim Lecturer Halal Products Research Institute Universiti Putra Malaysia (Member) BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date : ix
DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at other institution. NUR AIMI BINTI RADI Date : x
TABLE OF CONTENTS ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS Page i iv vii viii x xv xvii xx CHAPTER 1 INTRODUCTION 1.1 Background of the Study 1 1.2 Justification of the Study 3 1.3 Research Hypothesis 4 1.4 Research Objectives 4 1.5 Significance of the Study 5 2 LITERATURE REVIEW 2.1 Background of Nypa fruticans 6 2.2 Palm Sap 7 2.3 Tapping Process of Palm Sap 8 2.4 Fermentation of Palm Sap 12 2.5 Metabolism and Biotransformation of Nipa Sap 15 2.5.1 Yeasts 16 2.5.2 Lactic Acid Bacteria 24 2.5.3 Acetic Acid Bacteria 29 2.6 Alcohol in Islam 33 2.7 Alcoholic Fermentation 33 2.8 Organic Acid Production 35 3 PHYSICO-CHEMICAL ANALYSIS DURING FERMENTATION OF NIPA SAP 3.1 Introduction 37 3.2 Objectives 38 3.3 Materials and Methods 38 3.3.1 Sample Collection 38 3.3.2 Fermentation of Nipa Sap 39 3.3.3 Physical Properties Measurement 40 3.3.4 Chemical Properties Measurement 40 xi
3.3.5 Statistical Analysis 44 3.4 Results and Discussion 45 3.4.1 Physical Properties Measurement 45 3.4.2 Chemical Properties of Nipa Sap 48 3.5 Summary 54 4 DETERMINATION OF VOLATILE COMPOUNDS DURING FERMENTATION OF NIPA SAP 4.1 Introduction 55 4.2 Objectives 56 4.3 Materials and Method 56 4.3.1 Reagents and Materials 56 4.3.2 Headspace Gas Chromatography-Mass Spectrometry Analysis 57 4.3.3 Method Validation 58 4.3.4 Statistical Analysis 60 4.4 Results and Discussion 60 4.4.1 Volatile Compounds from GC-MS Analysis 60 4.4.2 Method Validation 67 4.5 Summary 68 5 DETERMINATION OF SUGARS DURING FERMENTATION OF NIPA SAP 5.1 Introduction 69 5.2 Objective 70 5.3 Materials and Methods 70 5.3.1 Reagents and Materials 70 5.3.2 Sample Preparation 71 5.3.3 Instrument 71 5.3.4 HPLC Conditions 72 5.3.5 Method Validation 72 5.3.6 Statistical Analysis 73 5.4 Results and Discussion 73 5.4.1 Sugar Identification 73 5.4.2 Sugar Content in Fresh and Fermented Nipa Sap 73 5.4.3 Total Sugar and Reducing Sugar Content in Fresh and Fermented Nipa Sap 77 5.4.4 Method Validation 78 5.5 Summary 79 xii
6 DETERMINATION OF ORGANIC ACIDS DURING FERMENTATION OF NIPA SAP 6.1 Introduction 80 6.2 Objective 80 6.3 Materials and Method 81 6.3.1 Reagents and Materials 81 6.3.2 Sample Preparation 81 6.3.3 Instrument 81 6.3.4 HLC Conditions 82 6.3.5 Method Validation 82 6.3.6 Statistical Analysis 83 6.4 Results and Discussion 83 6.4.1 Organic Acids Content in Fresh and Fermented Nipa Sap 84 6.4.2 Method Validation 88 6.5 Summary 89 7 TENTATIVE ISOLATION AND IDENTIFICATION OF MICRO-FLORA DURING FERMENTATION OF NIPA SAP 7.1 Introduction 90 7.2 Objective 90 7.3 Materials and Methods 91 7.3.1 Enumeration of Yeasts and Bacteria 91 7.3.2 Characterization of Isolates 92 7.3.3 Morphology and Physiological Properties of Yeasts 92 7.3.4 Biochemical Characterization and Identification of Yeasts 92 7.3.5 Morphology and Physiological Properties of Lactic Acid Bacteria 93 7.3.6 Biochemical Characterization of Lactic Acid Bacteria 94 7.3.7 Identification of Lactic Acid Bacteria 95 7.3.8 Morphology and Physiological Properties of Acetic Acid Bacteria 96 7.3.9 Statistical Analysis 96 7.4 Results and Discussion 97 7.4.1 Observation on Vertically Upright Bottle 97 7.4.2 Enumeration of Yeasts, Lactic Acid Bacteria, and Acetic Acid Bacteria 97 7.4.3 Tentative Identification of Yeasts 101 7.4.4 Morphological, Physiological and Biochemical Properties of the Identified Yeasts 104 xiii
7.4.5 Tentative Identification of Lactic Acid Bacteria 110 7.4.6 Morphological, Physiological and Biochemical Properties of the Identified 112 Lactic Acid Bacteria 7.4.7 Tentative Identification of Acetic Acid Bacteria 117 7.4.8 Morphology and Physiology Characteristics of Acetic Acid Bacteria 121 7.5 Summary 122 8 GENERAL DISCUSSION 8.1 Fresh and Fermented Nipa Sap Profiling 123 8.2 Relationship among Chemical and Microbiological 124 Characteristics of Fermented Nipa Sap 9 CONCLUSION AND RECOMMENDATION 9.1 Conclusion 129 9.2 Recommendation 131 REFERENCES 133 APPENDICES 150 BIODATA OF STUDENT 168 PUBLICATION 169 xiv