ABSTRACT
Tomato is one of the most popular consumed vegetable crops worldwide mainly as fresh or processed products. The antimicrobial potentials of lactic acid bacteria metabolites against tomato paste spoilage microorganisms were evaluated. Cow milk and Tomato samples were purchased. The tomato samples were processed into tomato paste. Standard microbiological methods were used in isolation, microbial count and identification of organisms. Cow milk samples were used for the isolation of lactic acid bacteria. Antimicrobial effects of the isolated lactic acid bacteria metabolites were carried out on the spoilage isolates from tomato paste samples using the agar well diffusion method, while shelf-life study was done on the tomato paste samples using the lactic acid bacteria metabolites as biopreservative agents for a period of 14 days. The results showed that total viable count ranged from 2.81 x 107Cfu/ml - 1.3 x 106Cfu/ml, the coliform count from 6.5 x 104Cfu/ml – 1.1 x 104Cfu/ml, while the fungal count was from 3.0 x 104Cfu/ml – 1.5 x 104Cfu/ml. The bacterial isolates from the tomato samples were Bacillus sp and Pseudomonas sp, while the fungal isolates were Rhizopus sp, Saccharomyces sp, Fusarium sp, Apergillus sp and Mucor sp. Lactic acid bacteria isolates from the cow milk samples were Lactobacillus fermentum M6, Lactobacillus fermentum LF-12 and Lactobacillus fermentum M8.The strains of Lactobacillus fermentum were able to grow at different temperature, pH and NaCl concentration and also produced antimicrobial compounds with Lactobacillus fermentum LF-12 recording the highest lactic acid (0.98 g/l), diacetyl (1.77 g/l) and hydrogen perioxide (1.60 g/l). The metabolites from strains of Lactobacillus fermentum were all potent against the spoilage microorganisms with zones of inhibition ranging from 2 mm to 12 mm. There were significant differences (p<0.005) in the activities of the metabolites of the strains of Lactobacillus fermentum. The result of the shelf-life study revealed a great reduction in bacterial load and growth over a period of 14 days of storage. This shows that strains of Lactobacillus fermentum can be used as bioperservative agents in preserving tomato paste as an alternative to chemical preservatives.
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/Justification 2
1.2 Aim 2
1.3 General
Objective 2
1.4 Specific
Objectives 2
CHAPTER 2:
LITERATURE REVIEW 4
2.1 Spoilage Microorganisms of Tomatoes 5
2.2 Lactic Acid Bacteria 9
2.2.1 Taxonomy of
lactic acid bacteria (LAB) 10
2.2.2 Role of lactic acid bacteria in fermented
food 11
2.2.3 Potential probiotic benefits 16
2.2.4 The mechanism of action of
lactic acid bacteria 16
2.2.5 Selection criteria of lactic acid bacteria 17
2.2.6
Antimicrobial activity of lactic acid
bacteria species 18
2.2.7 Antimicrobial compounds produced by lactic
acid bacteria 19
2.2.7.1 Diacetyl
20
2.2.7.2 Hydrogen peroxide 21
2.2.7.3 Reuterin 22
2.2.7.4 Bacteriocin 22
CHAPTER 3: MATERIALS AND METHODS 25
3.1 Sample Collection 25
3.2 Preparation of Media and Diluents 25
3.3 Preparation of Tomato Paste Samples and
Isolation of Spoilage
Organisms 25
3.4 Determination of Microbial Population 26
3.5 Characterization and Identification of
Microbial Isolates 26
3.5.1 Gram
staining test 26
3.5.2 Spore
staining 26
3.5.3 Motility
test 27
3.5.4 Biochemical
characterization of bacteria isolation 27
3.6 Identification of Fungal Isolates 27
3.7 Isolation and Characterization of
Lactic Acid Bacteria (LAB) from
Cow
Milk Samples 28
3.7.1
Cultural isolation and
characterization of lactic acid bacteria 28
3.7.2
Molecular identification
of the lactic and bacteria 29
3.7.3
Determination of
the effect of NaCl concentration on the growth of
lactic acid bacteria isolates
29
3.7.4
Determination of
the effect of temperature on the growth of lactic acid
bacterial isolates 29
3.7.5
Molecular
identification of the lactic acid bacteria 29
3.8 Test Microorganisms Isolates from Tomato
Samples 31
3.9 Preparation of Cell-Free
Filtrate of Lactic Acid Bacteria 31
3.10 Determination of Lactic Acid Bacteria
Metabolites 32
3.11 Antimicrobial Activity of
Lactic Acid Bacteria Metabolites Against
Spoilage Organisms 33
3.12 Shelf
Life/Biopreservative Study of Tomato Paste 34
3.13 Statistical Analysis 34
CHAPTER
4: RESULT AND DISCUSSION 35
4.1 Result 35
4.2 Discussion
51
CHAPTER
5: CONCLUSION AND RECOMMENDATION 55
5.1 Conclusion 55
5.2 Recommendation 55
References
Appendices
LIST OF TABLES
4.1 The
cultural and biochemical characteristics of the bacterial
isolates from the tomato samples 36
4.2 The total microbial loads of isolates from
the tomato samples in
colony-forming units per milliliter (cfu/ml) 38
4.3 The colonial and microscopic morphology of
the fungal isolates
from the tomato samples 40
4.4 The
cultural and biochemical characteristics of lactic acid 42
bacteria
isolates from cow milk samples
4.5 Antimicrobial
metabolites produced by strains of lactobacillus
fermentum 46
4.6
The antimicrobial sensitivity
pattern of the metabolites of lactic acid
bacteria
isolated from cow milk against some selected tomato spoilage organisms (mm) 48
4.7 The
shelf life study of the tomato paste samples inoculated with 50 the metabolites of lactic acid bacteria on the bacterial
population
LIST OF FIGURES
2.1 Tomato
plants and parts 4
2.2 Differentiation
of species according to the recent taxanomy 10
4.1 The
result of molecular analysis of the lactic acid bacteria 44
from the diary
product (cow milk) sample
CHAPTER 1
INTRODUCTION
Tomato (Solanum lycopersicum)
is one of the most popular and extensively consumed vegetable crops worldwide.
The health benefits of tomatoes are attributed to the abundant antioxidant
components present in tomatoes (Willcox et
al., 2003), such as lycopene and provitamin A, ascorbic acid, vitamin E and
other flavonoids. Tomatoes are consumed mainly as fresh or processed products. Tomatoes
are processed for wide ranges of products such as canned and sun-dried
tomatoes, ketchups, pastes, purees, salads, sauces, soups, and juice products,
supplying significant sources of vitamins and minerals to the consumers (Tsen et al., 2008).
Lycopene (a carotenoid), an
essential component of tomato with potent antioxidant activity, has been
reported and accumulating
evidence has shown an inverse correlation between the consumption of tomato
products rich in lycopene and the risk of several types of cancer and
cardiovascular disease (Talvas et al.,
2010). Studies have shown that lycopene level in plasma increased only after
the consumption of red tomato paste and purified lycopene (Talvas et al., 2010). It has also been documented
that the absorption of lycopene is greater from the processed tomatoes than
from fresh tomatoes, since processing breaks down the tomato cell matrix and
makes lycopene more available. Fermentation used with a purpose of the
extension of shelf life and enhanced safety of foods by the use of natural or
controlled microbiota and/or antimicrobial compounds is an approach to the solution
of food preservation that has gained increased attention in recent years. Many lactic
acid bacteria (LAB) strains are able to produce protein compounds, such as antimicrobial
peptides (bacteriocins), and organic acids (lactic and acetic), hydrogen
peroxide with efficient antimicrobial effect (Cleveland et al., 2001; DeVuyst and Leroy, 2007).
1.1 STATEMENT OF PROBLEM/JUSTIFICATION
Tomato is one of the highly nutritious food ingredients used in
the preparation of food all over the world (Ogunniyi and Oladejo, 2011;
Ogunbanwo et al., 2014). Its
utilization as an
ingredient in vegetable
salads, other dishes and
its processing into
different products like puree,
ketchups and juice
is well documented (Ogunbanwo et al., 2014).
Nutritionally, it contains a
large amount of
water, niacin, calcium
and vitamins especially A, C, and
E
which are important
in the metabolic activities
of man and
protects the body
against diseases (Bernard et al.,
1999).
The high water content
in tomatoes makes
it very susceptible to
spoilage bacteria and
fungi during storage, harvesting
and transportation (Spadaro
and Gullino, 2004). Because of
this use of lactic acid bacteria could help to increase the shelf life as well
as reduce the pathogens of the fermented tomato paste during processing and
storage.
1.2 AIM
This work is designed to evaluate the antimicrobial potentials of
lactic acid bacteria metabolites against
tomato paste spoilage microorganisms.
1.3 GENERAL
OBJECTIVE
To
evaluate the antimicrobial potentials of metabolites of lactic acid bacteria isolated
from diary product (cow milk) on tomato paste.
1.4 SPECIFIC OBJECTIVES
1.
To isolate
and identify the Lactic acid bacteria isolates from the dairy product (cow
milk) using phenotypic and molecular characteristics
2.
To isolate
and characterize spoilage microorganisms of tomato paste samples.
3.
To assay the
antimicrobial activities of metabolites of the lactic acid bacteria isolated
from cow milk on the spoilage organisms of tomato paste.
4.
To check the
bacterial load of tomato paste samples during the period of shelf-life study.
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