ABSTRACT
Fruits and vegetable juices are proving to be promising carriers for probiotics. The development of these plant-based probiotic beverages is owed to the issues of lactose intolerance and cholesterol content associated with fermented dairy foods. Thus, tigernut was exploited for the development of a functional beverage. The starter cultures employed for this development of functional tigernut milk beverage were Lactobacillus fermentum CS19 and Lactococcus lactis isolated from “Ogi” and Yoghurt respectively. These starter cultures were characterized. They were subjected to Gram Staining and biochemical tests. Their probiotic potentials were determined. They were screened for their acid tolerance, salt tolerance, milk fermentation capacity and antimicrobial activity. Then molecular characterization of the starter cultures was done by DNA extraction and quantification, 16S rRNA amplification, sequencing and phylogenetic analysis. 1.5 x 108 CFU/ml of each identified isolate were encapsulated in gelatin matrix and used for fermentation of the pasteurized milk extracted from the tigernut. There was a control sample which was left uninoculated. The fermentation of the tigernut milk was carried out at 30oC for 72 hours. The physicochemical and proximate compositions of the tigernut milk samples were determined before and after fermentation. After 72hours of fermentation, the viability counts of the starter cultures were determined and the fermented tigernut milk samples were stored at different temperatures, 4, 28 and 40oC for 4 weeks. Samples were taken weekly to determine the changes in the physicochemical and proximate compositions of the fermented tigernut milk samples. Effects of storage temperature and storage time of the fermented tigernut milk beverage on viability of the starter cultures: Lactobacillus fermentum CS19 and Lactococcus lactis were evaluated. The sensory properties of the functional tigernut milk product was also evaluated. There was decrease in pH and increase in TTA of the fermented tigernut milk samples with increase in storage time. There was increase in the moisture and protein contents of the fermented samples while decrease in fat and ash contents were observed. However, no fibre was detected after fermentation. Lactobacillus fermentum CS19 had more viability count with value of 1.7x 1010 CFU/ml than Lactococcus lactis which had 1.9 X 109 CFU/ml viable cells. There was significant reduction in the lactic acid bacteria counts as storage period increased. At the end of the 4weeks, there was reduction by 1.0 X 105 CFU/ml in all counts between the first and fourth weeks, with exception of the samples stored at 40oC as they had approximately 1.0 x 106CFU/ml reduction. At storage temperature of 4oC, the samples fermented by Lactobacillus fermentum CS19 and Lactococcus lactis and had viability counts of 1.3 X 1010 and 1.3 X 1010 in the first week of storage and reduced to 1.3 X 105 and 1.7 X 105 respectively, in the fourth week. While at storage temperature of 40oC, the samples fermented by Lactobacillus fermentum CS19 and Lactococcus lactis and had viability counts of 1.7 X 109 and 1.3 X 109 CFU/ml in the first week of storage and reduced to 7.0 X 103 and 6.7 X 103 CFU/ml. The tigernut milk sample fermented by Lactobacillus fermentum CS19 had the best sensory acceptability. From this study, despite the encapsulation of the starter cultures, the minimum biovalue of 1.0 X 106 CFU/ml required for beverages to be considered functional was not met at the end of the fourth week of storage. More studies to achieve this are therefore recommended.
TABLE OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables ix
List
of Figures x
Abstract xi
CHAPTER
1: INTRODUCTION 1
1.1 Statement of Problem 3
1.2 General
Objective 3
1.3 Specific Objectives 3
1.4 Significance
of Study 4
CHAPTER 2: LITERATURE
REVIEW 5
2.1 Tigernuts 5
2.2 Lactic
Acid Bacteria as Starter Cultures 6
2.3 Functional
Foods of Plant Origin 7
2.4 Tigernut Milk as a Functional Beverage 9
CHAPTER 3: MATERIALS AND
METHODS 15
3.1
Sample Collection 15
3.2
Source of Starter
Cultures 15
3.3 Microbiological Analysis 15
3.4 Characterization of Isolates 16
3.5 Screening for Potential Starters 16
3.5.1
Acid tolerance test 16
3.5.2
Salt tolerance test 16
3.5.3
Milk fermentation test 17
3.5.4
Antimicrobial activity test 17
3.6 Molecular Characterization of Isolates 18
3.6.1 DNA extraction (boiling method) 18
3.6.2 DNA quantification 18
3.6.3 16S
rRNA amplification 19
3.6.4 Sequencing 19
3.6.5 Phylogenetic analysis 19
3.7
Preparation of Starter
Cultures 20
3.8
Preparation and
Fermentation of Tigernut Milk 20
3.9
Physicochemical and Proximate Analysis of
Tigernut Milk Samples
Before and After
Fermentation of Tigernut Milk 22
3.10
Effect of Storage Temperature and Storage Time of Fermented
Tigernut
Milk on Viability of Lactobacillus
fermentum CS19 and
Lactococcus
Lactis 22
3.11
Sensory
Evaluation of Fermented Tigernut Milk Samples 23
3.12 Statistical
Analysis 23
CHAPTER 4: RESULTS AND
DISCUSSION 24
4.1 Results 24
4.2 Discussion 55
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 61
5.1 Conclusion 61
5.2 Recommendations 61
References
LIST
OF TABLES
4.1: Phenotypic
characterization of lactic acid bacteria isolates 25
4.2: Acid
tolerance of the isolates 27
4.3: Salt
tolerance of the isolates 29
4.4: Milk
fermentation capacity 31
4.5: Antimicrobial
activities of the isolates against selected pathogens 33
4.6: Hydrogen
ion concentration (pH) and total titratable acidity
(TTA) of tigernut
milk samples after 72 hours of fermentation 38
4.7: pH
of fermented tigernut milk samples stored at different
temperatures for 4 weeks 40
4.8: TTA
of fermented tigernut milk samples stored at different
temperatures for 4 weeks 41
4.9: Proximate
composition of tigernut milk samples after 72 hours of
fermentation 44
4.10: Moisture
content of fermented tigernut milk samples stored at
different temperatures for 4 weeks 45
4.11: Protein
content of fermented tigernut milk samples stored at
different temperatures for 4 weeks 46
4.12: Fat
content of fermented tigernut milk samples stored at
different temperatures for 4 weeks 47
4.13: Ash
content of the tigernut milk samples for storage period of 4
weeks 48
4.14: Lactobacillus fermentum CS19 and Lactococcus Lactis viability
count
in the fermented milk samples after 72 hours fermentation 50
4.15: Viability count (CFU/ml) of the Lactobacillus fermentum CS19 and
Lactococcus Lactis in the
fermented milk
samples during the 4-week storage
period 52
4.16:
Sensory evaluation of the fermented
tigernut milk samples 54
LIST
OF FIGURES
2.1: The three varieties of tigernut
6
3.1: Flow
chart for preparation of tigernut milk 21
4.1: Agarose
gel electrophoresis of the 16S rRNA gene of some selected
bacterial isolates. 35
4.2: Phylogenetic tree showing the evolutionary
distance between the bacterial isolates 36
CHAPTER 1
INTRODUCTION
The
development of functional foods and beverages is continuously emerging, with
fruits and vegetable juices proving to be promising carriers for probiotics
(Dimitrovski et al., 2015). The
fermentation of foods using probiotic microorganisms has gained popularity
because of their special effect on human health, and thus has been applied in
various products in the food industry (Kechagia et al., 2013).
Most
functional foods available today are milk-based but consumers' preference now
tend towards botanical products which are either free from, or have minimal
cholesterol content; lactose intolerance and cholesterol content are two major
drawbacks linked to fermented dairy products (Vasudha and Mishra, 2013).
Fruits
and vegetables are considered health foods and ideal substrates for the growth
of probiotic microorganisms. In contrast to dairy products, they lack
allergens, lactose and cholesterol that adversely affect certain groups of the
population (Aspri et al., 2020).
Fermentation
of various raw materials (including fruits and vegetables) using probiotic
microorganisms is ancestral. This fermentation is explored worldwide and
recognized as the most suitable way to increase daily consumption of fresh-like
fruits and vegetables (Rosello-Soto et al.,
2018).
Tigernut
(Cyperus esculentus), is a weed plant
of the tropical and mediterranean regions, highly appreciated for its health
benefits and nutritive value (Sanchez-Zapata et al., 2012). It is an underutilized crop belonging to the family
Cyperaceae, known in Nigeria by the Hausas as aya, Yorubas as ofio and
by the Igbos as aki awusa, where the
yellow and brown varieties are preferred to the black variety, but the yellow
variety due to its big size, attractive colour and freshness is most preferred.
Tigernuts have highly nutritious starch content, dietary fibre and
carbohydrate, and are rich in sucrose, fat, protein and important mineral
elements such as potassium, sodium, calcium, magnesium, zinc and traces of
copper (Adejuyitan, 2011). Tigernut can be exploited for the development of
functional beverages. Tigernut milk beverages are highly valued; they have been
reported to prevent heart diseases, colon cancer and thrombosis, and activate
blood circulation (Said et al.,
2017).
The
lactic acid bacteria group of probiotics are a component of various fermented
(functional) foods. They have properties which are highly of importance. They
have: ability to produce antimicrobials, antitumoural activity, and ability to
inhibit pathogens; they are able to prevent colon cancer, stimulate the immune
system, stabilize gut microflora and alleviate lactose intolerance (Chang et al., 2015). Lactic acid bacteria
consist of a group of organisms belonging to the genera, Leuconostoc, Lactococcus, Pediococcus,
Oenococcus, Streptococcus, Enterococcus (Wedajo, 2015). They are widely
applied in the food fermentation industry because of their generally recognized
as safe (GRAS) status. When applied in food fermentations, they enhance food
safety and impart positively on the sensory attributes of the foods (Widyastuti
et al., 2014).
The
lactic acid fermentation has been used for extended periods of time for the
preservation of dairy, vegetable and meat products, and thus utilized today in
industrial fermentation (Malo and Urquhart, 2016). They have been found to play
major roles in food fermentation processes, with their major metabolic action
being the acidification of the food and production of many beneficial compounds
such as organic acids, polyols, exopolysaccharides and antimicrobials, thus
have many applications in the food industry (Bintsis, 2018).
1.1
STATEMENT OF PROBLEM
There
are drawbacks associated with the consumption of dairy-based fermented foods.
For this reason, consumers are looking towards consumption of plant-based
fermented foods which have minimal or no cholesterol and also do not have
lactose which would affect intolerant people. There also difficulties in
obtaining animal proteins in developing countries. Animal proteins are scarce
and expensive and alternatives are therefore sought after.
1.2 GENERAL OBJECTIVE
To determine the
suitability of tigernut milk as a substrate for production of probiotic
beverage by Lactic Acid Bacteria.
1.3 SPECIFIC OBJECTIVES
i.
Isolation and
characterization of Lactic Acid Bacteria from different sources for use as
starter cultures.
ii.
Screening of isolated
Lactic Acid Bacteria for functional properties of probiotics.
iii.
To determine the changes
in physicochemical and proximate composition of fermented tigernut milk sample.
iv.
To evaluate the effects
of storage temperature and storage time of fermented tigernut milk beverage on
viability of lactic acid bacteria and shelf life of the fermented products.
v.
To evaluate the sensory properties
of the functional tigernut milk product.
1.4
SIGNIFICANCE
OF STUDY
Tigernuts
milk beverage is a drink acceptable to everyone without allerginicity
associated with its consumption. The problem of its commercialization centers
on the extension of its shelf life and ensuring its safety. With the right
treatment and incorporation of probiotics (the lactic acid bacteria), it can be
converted into a commercial functional drink, a healthy alternative to the
conventional dairy products, for especially the lactose-intolerant and
vegetarians.
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