ABSTRACT
In the present study, spontaneous and induced fermentation techniques were used in fermenting different fruit juices (Pineapple fruit juice, Watermelon fruit juice and Pineapple+Watermelon fruit juices) to determine their microbial and physicochemical changes. A higher bacterial count (8.6±0.58a-9.0±2.19c) log(cfu)ml-1 than yeast count (7.6±3.18a-7.9±1.76e) log(cfu)ml-1 were observed in the spontaneous fermentation. In the induced fermentation a higher yeast count (7.7±2.33a-8.3±5.29c) log(cfu)ml-1 than bacterial count(8.5±1.53a-8.9±6.25d) log(cfu)ml-1 was observed. A higher Lactic acid bacterial count (7.0±1.53a -8.9±2.73e) log(cfu)ml-1was recorded during the spontaneous fermentation. pH and sugar content (0Brix) decreased while TTA and alcohol content increased both in spontaneous and induced fermentation of the different fruit juices. Nine bacterial species, namely Staphylococcus spp, Bacillus spp, Enterobacter cloacae, Providencia vermicola, Cronobacter sakazakii, Ignatschineria indica, Comamonas testosteroni, Lactobacillus plantarurum and Lactobacillus fermentum and two yeast species, namely Rodotorular spp. and Saccharomyces spp were identified. Also, three yeast species were isolated from Palm wine which is a readily available raw material for the induced fermentation. This study gives an insight into the microbial and physicochemical changes during the fermentation processes of different beverages.
TABLE OF CONTENTS
Title Page i
Declaration
ii
Dedication
iii
Certification
iv
Acknowledgements
v
Table of Contents
vi
List of Tables
x
List of Figures
xi
Abstract
xii
CHAPTER 1: INTRODUCTION 1
1.1 Fruits
and Fermentation in Food Industries 1
1.2 Justification for the Research 3
1.3 Main Objectives 3
1.3.1 Specific objectives 4
CHAPTER 2: LITERATURE REVIEW 5
2.1 Fruits and Fruit Juices 5
2.2 Pineapple 6
2.2.1 Origin and distribution of pineapple 6
2.2.2 Morphology of pineapple 7
2.2.3 Climatic requirement of pineapple 7
2.2.4 Variety of pineapple cultivated 8
2.3 Watermelon
9
2.3.1 Origin and distribution of
watermelon 9
2.3.2 Climatic requirement of watermelon 10
2.3.3 Microbiological assessment of watermelon
fruit / juices 11
2.4 Palm Wine
12
2.4.1 Origin
12
2.5 Fermentation
12
2.5.1 Definition of fermentation 12
2.5.2 History of fermentation 14
2.5.3
Fermentation of fruit juices 15
2.6 Factors Affecting Fermentation 17
2.6.1 Effect of pH on fermentation and wine
quality 17
2.6.2 Effect of sugar content on
fermentation 18
2.6.3 Effect of acid on fermentation and wine
quality 18
2.6.4 Relationship between sugar content and
alcohol production 19
CHAPTER 3: MATERIALS AND METHODS 21
3.1
Collection of Samples 21
3.2
Sterilization of Materials and Equipment 21
3.3
Media Preparation
21
3.4
Preparation of Juice 21
3.5
Microbiological Analysis
22
3.5.1
Identification of isolates 23
3.5.1.1
Gram staining 23
3.5.1.2 Sugar fermentation test 23
3.5.1.3 Catalase test 24
3.5.1.4 Oxidase test 24
3.5.1.5 Coagulase test 24
3.5.1.6 Methyl red test 24
3.5.1.7 Motility test 25
3.6 Physicochemical analysis 25
3.6.1 pH detection 25
3.6.2 Titratable acidity 25
3.6.3 Sugar content 25
3.6.4 Alcohol content 25
3.7 Molecular
Characterization
26
3.8
Starter Culture for Induced Fermentation 27
3.8.1
Source and collection of yeast isolates 27
3.8.2
Isolation of yeast from palm wine 27
3.8.3
Microscopy examination 28
3.8.4
Biochemical tests 28
3.8.4.1 Sugar fermentation test 28
3.8.4.2 Carbon
assimilation test 29
3.8.4 Multiplication of starter 29
3.8.4.3 Nitrogen assimilation 29
3.8.4.4 Urea hydrolysis 29
3.8.5 Multiplication
of starter 30
3.8.6
Preparation of juice for induced fermentation 30
3.8.7
Preparation of the yeast starter cultures 30
3.8.8 Induced fermentation
31
3.8.9 Microbiological analysis
31
3.8.10
Identification of isolates 32
3.9
Statistical Analysis
32
CHAPTER 4: RESULTS
AND DISCUSSION 33
4.1
Results
33
4.1.1
Microbiological results of the spontaneous fermentation of fruit juices 33
4.1.2
Physicochemical results of the spontaneous fermentation of fruit juices 38
4.1.3 Identification of isolates during the
spontaneous fermentation of fruit
juice 43
4.1.4 Identification of isolates from palm wine
for induced fermentation 50
4.1.5 Microbiological results of the induced
fermentation of fruit juices 52
4.1.6 Physicochemical results of the induced
fermentation of fruit juices 57
4.1.7 Identification of
isolates of fruit juices during induced fermentation 62
4.2
Discussion
64
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 67
5.1
Conclusion
67
5.2
Recommendations
67
References
68
Appendix 76
LIST OF TABLES
4.1: Total
viable count (TVC) (Mean±S.E) of fermented fruit juice
(spontaneous fermentation) samples for
various time interval 35
4.2: Total lactic acid bacterial count (TLC)
(Mean± S.E) of fermented
fruit juice (spontaneous fermentation)
samples for various time interval 36
4.3: Total yeast count (TYC) (Mean± S.E) of fermented fruit juice
(spontaneous fermentation) samples for
various time interval 37
4.4: Gram
reaction, cell morphology and biochemical characteristics of
bacterial isolates 44
4.5: Morphology, microscopy and biochemical tests
of some fungal species 45
4.6: Molecular identification of isolates
46
4.7: Characteristics and identification of yeasts 51
4.8: Total viable count (TVC) (Mean ±S.E) of
fruit juice
(induced fermentation) samples for various
time interval 54
4.9: Total lactic acid bacterial
count (TLC) (Mean ±S.E) of fruit juice
(induced fermentation)
samples for various time interval 55
4.10: Total yeast count (TYC) (Mean± S.E) of fruit juice (induced fermentation)
samples for various time interval 56
4.11: Gram reaction, cell morphology and biochemical
characteristics of
bacterial isolates
(induced fermentation) 63
4.12: Morphology, microscopy and biochemical tests
of some fungal species 65
LIST OF FIGURES
4.1: pH
changes of fermenting Fruit juices (spontaneous fermentation) 39
4.2: TTA
changes of some fermenting fruit juices (spontaneous
fermentation) 40
4.3: 0Brix
changes of fermenting fruit juices (spontaneous fermentation) 41
4.4:
Alcohol content changes of some fermented fruit juices
(spontaneous fermentation) 42
4.5: pH
changes of some fermenting fruit juices (induced fermentation) 58
4.6: TTA
changes of some fermenting fruit juices (induced fermentation) 59
4.7: 0Brix changes of some
fermenting fruit juices (induced fermentation) 60
4.8: Alcohol content changes of some
fruit juices (induced fermentation) 61
CHAPTER 1
INTRODUCTION
1.1 FRUITS
AND FERMENTATION IN FOOD INDUSTRIES
Different
flavours of wine which are distinctive come from different types of raw
materials during alcoholic and malolactic fermentation (Cole and Noble, 1995). In the world, various raw
materials are used to produce alcoholic beverages traditionally. Alcoholic beverages
consumed in all parts of the world vary in accordance to location and
ingredients (Allan, 1983). The use of
fermentation techniques is the oldest and most economical methods of producing
and preserving foods (Steinkraus et al.,
1983; Campbell-platt, 1994; Mugochi et al., 1999).
Fermentation has many advantages such as inhibition of growth of spoilage and
pathogenic microorganism by producing organic acids and other compounds for
improving organoleptic properties, producing different flavour compounds and
often making the nutritional value of the original foods better (Kingamko et al., 1994; Lorri and Svanberg, 1994
Mugochi et al., 1999).
Over the
years there has been controversy over the relative advantages of spontaneous
fermentations and induced fermentations. Research has shown that spontaneous
fermentation gave a more complex aromatic quality (Benda,1982), a subsequent
study concluded that there is significant preference for wine produced with
selected yeast (Smihitte et al., 1984; Shuts and Gather, 1993).
Pineapple
(Ananas comosus) belongs to the
Bromeliaceae family and is grown in different parts of Nigeria. In homes, pineapples
can be sliced and consumed fresh, made into fruit juice or concentrates for
future use. Fruit crop like pineapple, has a lot of nutritional, medicinal,
industrial and economic importance (Sarah et
al., 1997). According to Dull (1971),
pineapple which serves as food for human contains the following: moisture
(81.2-86.2%), total solids having mainly sucrose, glucose and sucrose (13-19%),
fibre (2-3%) and lipids and nitrogenous compounds (0.15). Pineapple is also
rich in vitamin C. The fruit richly contains calcium (Ca), having proteolytic
activity because of enzyme bromelin. Dull (1971) stated that when pineapple is
consumed, it helps to detoxify the human body thereby preventing blood
clotting. It also helps to prevent problems with the kidney, heart and stomach.
Large quantities of ripped pineapples are usually wasted as a result of poor
handling and inadequate storage facilities. To avoid wastage and also to make
it available all year round, fermentation of pineapple juice for wine
production is required.
Watermelon
(Citrullus lanatus) belonging to the
Cucurbitaceae family is a tropical fruit which grows in almost all parts of
Africa including Nigeria and South-East Asia (Koocheki et al., 2007). It is a
good source of vitamins and often used as appetizer or snack, depending on how
it is prepared (Enukainure et al., 2010). Watermelon is a good source of
phytochemical and lycopene which is a red carotenoid pigment acting as
antioxidant during normal metabolism as well as for protection against cancer (Perkins-Veazie
and Collins, 2004). Large quantities of watermelon are however wasted as a
result of poor storage facilities and conditions. In Nigeria, watermelon is blended,
fermented and drank as juice (Enukainure et al., 2010; Onyeleke and Olaniyan,
2007).
Palm
wine, known as a refreshing alcoholic beverage is mainly taken in the Southern
part of Nigeria, Asia and Southern part of America (Elijah et al., 2010). It is
obtained from the sap of the palm trees like oil palm (Elasis guiniensis) and
Raphia Palm ( Raphia hookeris and Raphia vinifera) (Okafor,
2007). Palm wine comes in variety of flavours ranging from sweet (unfermented)
and sour (fermented) and vinegary. It is produced by succession of
microorganisms, which include Gram- negative bacteria, lactic acid bacteria,
yeasts as well as acetic acid bacteria. Yeasts isolated from palm wine have
been confirmed as coming from genera Saccharomyces,
Pichia, Schizosaccharomyces, Kloekera,
Endomycopsis, Saccharomycoides and Candida
which get into the wine from a variety of sources including air, tapping
utensils, previous brew and the tree. Hence, palm wine serves as source of
single cell protein and vitamins (Fleet, 2003; Okafor 2007; Duarte et al.,
2010; Adedayo and Ajiboye, 2011; Ogbonna et al., 2013). Twenty indigenous
strains of Saccharomyces cerevisiae
which have been tagged genetically different from the strains used to make wine
from grapes have undertaken the major fermentation and this due to the fact
that they are able to continue fermentation process at high ethanol level of
18% making them ideal for producing ethanol (Ezeogu and Emeruwa 1993).
1.2 JUSTIFICATION
FOR THE RESEARCH
Fermentation
in food industry is important as this fermented food constitutes a major part
of the diet of African homes. In Nigeria, there are large farms of Pineapples
and watermelons respectively. The
production of fruit juices and alcoholic beverages in particular from these
fruits are important as they help to reduce post-harvest loss, provide us with
essential nutrients and eventually provide employment opportunities. hence, spontaneous fermentation and induced
fermentation using yeast isolated from palm wine was carried out.
1.3 MAIN
OBJECTIVES
To
determine the microbial and physicochemical changes involved in the spontaneous
fermentation and induced fermentation of Pineapple fruit juice, Watermelon
fruit juice and Pineapple+Watermelon (mixed) fruit juice.
1.3.1 Specific
objectives
i.
To
extract fruit juices from pineapple fruits and watermelon fruits.
ii.
To
determine microbial load as well as to isolate and identify bacteria and yeasts
during spontaneous fermentation of Pineapple fruit juice, Watermelon fruit juice
and mixed fruit juices.
iii.
To
isolate and identify yeast from palm wine which will in turn be used for the
induced fermentation.
iv.
To
determine physicochemical properties of Pineapple fruit juice, Watermelon fruit
juice and mixed fruit juices during Spontaneous fermentation.
v.
To
determine the microbial load of the fruit juice as well to isolate and identify
bacteria and yeast during the induced fermentation of Pineapple fruit juice,
Watermelon fruit juice and mixed fruit juices.
vi.
To determine
physicochemical properties of Pineapple fruit juice, Watermelon fruit juice and
mixed fruit juice during induced fermentation.
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