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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

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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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COPYRIGHT

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icons, photographs and all other artwork, is copyright material of Universiti Putra

Malaysia unless otherwise stated. Use may be made of any material contained within

the thesis for non-commercial purposes from the copyright holder. Commercial use

of material may only be made with the express, prior, written permission of

Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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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)

By

NUR AIMI BINTI RADI

June 2013

Chairman : Fatimah Abu Bakar, PhD

Faculty : 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 300C 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

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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 63rd

day of fermentation, followed by sucrose (0.60% w/v), glucose

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(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.

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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

NUR AIMI BINTI RADI

Jun 2013

Pengerusi : Fatimah Abu Bakar, PhD

Fakulti : 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

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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

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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 ciri-

ciri 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.

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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”.

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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


(Internal Examiner)

Abdul Karim Sabo Mohamed, PhD


Faculty of Food Science and Technology


(Internal Examiner)

Mohd Khan Ayob, PhD


Universiti Kebangsaan Malaysia

(External Examiner)

___________________________________

Professor and Deputy Dean

School of Graduate Studies


Date :

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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


(Chairman)

Dzulkifly Mat Hashim

Lecturer

Halal Products Research Institute


(Member)

___________________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date :

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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 :

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TABLE OF CONTENTS

Page

ABSTRACT

i

ABSTRAK

iv

ACKNOWLEDGEMENTS

vii

APPROVAL

viii

DECLARATION

x

LIST OF TABLES

xv

LIST OF FIGURES

xvii

LIST OF ABBREVIATIONS

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

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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



4.4.1 Volatile Compounds from GC-MS Analysis 60


4.5 Summary 68

5 DETERMINATION OF SUGARS DURING


5.1 Introduction 69

5.2 Objective 70



5.3.2 Sample Preparation 71

5.3.3 Instrument 71

5.3.4 HPLC Conditions 72




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


5.5 Summary 79

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6 DETERMINATION OF ORGANIC ACIDS DURING


6.1 Introduction 80

6.2 Objective 80

6.3 Materials and Method 81


6.3.2 Sample Preparation 81

6.3.3 Instrument 81

6.3.4 HLC Conditions 82




6.4.1 Organic Acids Content in Fresh and

Fermented Nipa Sap 84


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


7.3.1 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


Lactic Acid Bacteria 93

7.3.6 Biochemical Characterization of Lactic Acid

Bacteria 94

7.3.7 Identification of Lactic Acid Bacteria 95


Acetic Acid Bacteria 96



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

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7.4.5 Tentative Identification of Lactic Acid

Bacteria 110

7.4.6 Morphological, Physiological and

Biochemical Properties of the Identified

Lactic Acid Bacteria

112

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

Characteristics of Fermented Nipa Sap 124

9 CONCLUSION AND RECOMMENDATION

9.1 Conclusion 129

9.2 Recommendation 131

REFERENCES

133

APPENDICES

150

BIODATA OF STUDENT

168

PUBLICATION

169

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