View
2
Download
0
Category
Preview:
Citation preview
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
© COPYRIG
HT UPM
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
© COPYRIG
HT UPM
COPYRIGHT
All material contained within the thesis, including without limitation text, logos,
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
© COPYRIG
HT UPM
i
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
© COPYRIG
HT UPM
ii
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
© COPYRIG
HT UPM
iii
(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.
© COPYRIG
HT UPM
iv
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
© COPYRIG
HT UPM
v
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
© COPYRIG
HT UPM
vi
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.
© COPYRIG
HT UPM
vii
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”.
© COPYRIG
HT UPM
viii
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 :
© COPYRIG
HT UPM
ix
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 :
© COPYRIG
HT UPM
x
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 :
© COPYRIG
HT UPM
xi
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
© COPYRIG
HT UPM
xii
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
© COPYRIG
HT UPM
xiii
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
© COPYRIG
HT UPM
xiv
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
Recommended