ABSTRACT
The B vitamins are essential in the human diet. Many food-grade bacteria such as lactic acid bacteria produce excess of B vitamins such as folate (vitamin B9) and cobalamin (vitamin B12). This unique property of bacteria offers the possibility to fortify raw food materials such as ugba with B vitamins by natural means. This study was aimed at determining the folate and cobalamin levels in cultures of lactic acid bacteria from Pentaclethra macrophylla seeds. Four LAB species were isolated from Pentaclethra macrophylla of which were Lactobacillus fermentum, Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides.based on morphological, biochemical and physiological characteristics. The levels of folate and cobalamin in the cultures of LAB from Pentaclethra macrophylla were analyzed using High Performance Liquid Chromatography (HPLC) method. This showed the concentration of both B vitamins in LAB fermented Pentaclethra macrophylla to be 0.1125 mg/ml (folate) and 0.1154 mg/ml (cobalamin) and that of unfermented Pentaclethra macrophylla to be 0.3112 mg/ml (folate) and 0.0624 mg/ml. From this study, it was seen that the isolated LAB used in fermentation of Pentaclethra macrophylla were able to increase the folate and cobalamin levels by 72.26% and 45.93% respectively. Therefore, it has been shown that increased folate and cobalamin levels in fermented Pentaclethra macrophylla (ugba) are possible through judicious selection of the microbial species and cultivation conditions.
TABLE OF CONTENTS
Title
page i
Certification ii
Dedication iii
Acknowledgment iv
Table
of contents v
List
of tables viii
List
of figures ix
Abstract x
CHAPTER ONE
INTRODUCTION 1
1.1 Aim and Objectives of the Study 2
1.1.1 Aim 2
1.1.2 Objectives 2
CHAPTER TWO
LITERATURE REVIEW 3
2.1 Fermentation 3
2.2 Ugba 4
2.2.1 Nature
of the Plant and the Seeds 5
2.2.2 Chemical
Composition of Seeds 5
2.3 Preparation
of Ugba 7
2.4 Microorganisms Involved in the
Fermentation of African Oil Bean Seed 9
2.5 Nutritional Value of Ugba 10
2.6 Lactic
Acid Bacteria 11
2.6.1 Characteristics
of Lab 12
2.6.2 Use of Lactic Acid Bacteria in Food 13
2.7 B-Vitamins
14
2.8 Folate or Folic Acid 15
2.8.1 Production of Folate by Lactobacillus 16
2.9 Vitamin B12 17
CHAPTER THREE
MATERIALS AND METHOD 19
3.1 Study Area 19
3.2 Collection of Samples 19
3.3 Culture Media Preparation and Sterilization 19
3.4 Preparation of Homogenate Ugba Samples 20
3.5 Inoculation of Samples 20
3.6 Colony Count of Isolates 20
3.7 Characterization and Identification of
Isolates 21
3.8 Identification of Lactic Acid Bacteria 21
3.8.1 Biochemical
Characterization 21
3.8.1.1 Gram Staining 21
3.8.1.2 Catalase Test 22
3.8.1.3 Spore
Stain Test 22
3.8.1.4 Citrate Test 23
3.8.1.5 Carbohydrate
Fermentation Test 24
3.8.1.6 Haemolytic Activity 24
3.9 Physiological
Characterization of Lactic Acid Bacteria 24
3.9.1 Growth
at Different Temperatures 24
3.9.2 Salt
Tolerance Test 24
3.9.3 Growth
at Different pH 25
3.10 Laboratory Fermentation of Ugba Using Lab Isolates for Vitamin
Production 25
3.11 Evaluation
of the Folate and Cobalamin Levels of the Isolated LAB 25
CHAPTER FOUR
RESULTS 27
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION 36
5.1 Discussion 36
5.2 Conclusion and Recommendation 39
References 40
Appendix I 46
Appendix II 47
Appendix III 48
Appendix IV 49
LIST
OF TABLES
|
TABLE
|
TITLE
|
PAGE
|
|
4.1
|
Morphological, Biochemical and Physiological
Characteristics of Lactic Acid Bacteria Isolated from Pentaclethra macrophylla
|
30
|
|
4.2
|
Carbohydrate Fermentation Profile of Lactic Acid Bacteria
Isolated from Pentaclethra macrophylla
|
31
|
|
4.3
|
Folate
and Cobalamin levels in Unfermented and Fermented Ugba Seed Fermented by
Cultures of Lactic Acid Bacteria
|
36
|
LIST
OF FIGURES
|
FIGURE
|
TITLE
|
PAGE
|
|
4.1
|
HPLC
Analysis of Vitamin B9 Standard
|
32
|
|
4.2
|
HPLC
Analysis of Vitamin B9 from Ugba Fermented by LAB Isolates
|
33
|
|
4.3
|
HPLC
Analysis of Vitamin B12 Standard
|
34
|
|
4.4
|
HPLC
Analysis of Vitamin B12 from Ugba Fermented by LAB Isolates
|
35
|
CHAPTER ONE
INTRODUCTION
In
dairy and food industries, lactic acid bacteria (LAB) and related organisms
have been used as starter culture and they play very imperative role in
fermentation. They may improve nutritional, organoleptic, technological and
shelf-life characteristics in diverse fermented foods and beverages (Shah and
Prajapati, 2013; Capozzi et al.,
2012). LAB have capability to synthesize water-soluble vitamins specifically
those included in the B-group (folic acid, riboflavin, cobalamin and biotin),
since they are produced in minute quantity not much information is available
regarding their amount of production.
There is renewed interest in folates and
cobalamin due to recent findings that long-term
supplementation with folic acid and vitamin B12 in elderly
subjects resulted in effects on DNA methylation of several genes, among which
genes implicated in developmental processes. Folate supplementation has
been associated with lowered risk of neural tube defects during pregnancy (Collins,
1994). Folates may also play a significant role in the reduction of cancer,
since they function as cofactors in reactions involving 1-carbon transfer
during purine and pyrimidine biosynthesis (Ames, 1999; Sarma and Duttagupta,
1995). Vitamin B12 (cyanocobalamin) is
necessary for hematopoiesis, neural metabolism, DNA and RNA production, and
carbohydrate, fat and protein metabolism. B12
improves iron functions in the metabolic cycle and assists folic
acid in choline synthesis. B12 metabolism is interconnected with
that of folic acid. Boushey et al. (1996) and Morrison et
al. (1995) also reported that an increased consumption of folic acid
substantially lowers the plasma homocysteine, an important factor for the
emergence of coronary heart disease. Deficiencies of folate and vitamin B12
may be a public health problem affecting millions of people according to a recent World
Health Organization review for example; a low folate and vitamin B12
status increases the risks of anaemia and birth defects, and these risks are
reduced by supplying these vitamins. Microbial vitamin production is a
convenient strategy to achieve natural enrichment of vitamins in fermented
foods, notably from vegetable sources. Such an LAB as L. plantarum is known for folate production in a high concentration,
while L. reuteri is known for vitamin
B12 production in high concentrations.
1.1 Aim and Objectives of the
Study
1.1.1 Aim
This study is aimed at determining the folate
and cobalamin levels in cultures of lactic acid bacteria from Pentaclethra macrophylla seeds.
1.1.2 Objectives
1.
To isolate and identify lactic acid bacteria from Pentaclethra macrophylla seeds
2.
To carry out laboratory fermentation of Pentaclethra macrophylla with the LAB isolates
3.
To determine the levels of folate and cobalamin in the cultures of the
LAB isolated from Pentaclethra
macrophylla
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