Abstract
Lactic acid bacteria are important organisms that have been used as starter cultures for the fermentation of food and food products. This study was aimed at evaluating the potentials of Lactic acid bacteria (LAB) isolated from ogi for development of starter culture and amylase production. Isolation of the LAB isolates was done on de Mann Rogosa Sharpe (MRS) agar using standard microbiological techniques. The isolates were evaluated for use as starter cultures by assaying for acidification, production of lactic acid and hydrogen peroxide. Amylase activity was determined by the spectrophotometric method employing starch as substrate and 3, 5 dinitrosalicyclic acid (DNS) as coupling reagent. The identification of Lactic acid bacteria isolates obtained from samples of ogi indicated that Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus plantarum were the main organisms involved in the fermentation of ogi. From the results obtained, it was evident that the quantity of lactic acid produced increased following a decline in pH as fermentation time increased. It was observed that L. plantarum produced the highest quantity of lactic acid (3.85 gL-1) at 48 hrs compared to all other LAB species used in this work with a strain of L. fermentum having the lowest yield 2.65 gL-1) after 48 hrs of incubation. The highest (1.24 g/l) concentration of hydrogen peroxide was produced by L. fermentum at 24 hrs of incubation. Increasing incubation resulted in a reduction in the quantity of hydrogen peroxide produced. Hydrogen peroxide produced by the LAB strains ranged between 0.46 to 1.24 g/l. The results of this study showed that amylase production increased with increase in incubation time linearly till the 96th hours. Maximum amylase (3.47U/g) production was obtained at 96hrs. The study revealed that the strains isolated had an optimum pH of between 3.0 to 3.5 with a maximum enzyme activity of 4.28 U/g. Maximum enzyme production was obtained with maize bran as substrate recording 2.48 U/g after 96 hrs of incubation. The results of this study showed that L. plantarum and L. fermentum can be used as potential starter cultures in the production of ogi and cultivation of these isolates using a cheap substrate (maize bran) will result in increased amylase synthesis for several applications.
TABLE
OF CONTENTS
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgment v
Table of Contents vi
List of Tables xi
List of Figures xii
Abstract xiii
CHAPTER 1: INTRODUCTION
1
1.1 Statement
of Problems 3
1.2 Justification
4
1. 3 Aim and Objective 4
1.3.1 Specific Objectives 4
CHAPTER 2: LITERATURE
REVIEW 5
2.1 Historical
Perspective of Fermentation 5
2.2 Fermentation Techniques 7
2.3 Fermented
Foods 8
2.4 Lactic
Acid Bacteria (LAB) 10
2.5 Starter Cultures 12
2.5.1 Functions of starter cultures 13
2.5.2 Factors to Consider in selecting Lactic acid bacteria starter
cultures for cereal fermentation 14
2.5.3 Starter cultures In African cereal fermentation
17
2.6 Cereal-Based Functional Foods 17
2.6.1 Ogi 19
2.6.2 Uji 21
2.6.3 Mawe 22
2.7 Antimicrobial
Compounds Produced By Lactic Acid Bacteria 23
2.7.1 Organic acids and low pH 24
2.7.2 Hydrogen peroxide 25
2.7.3 Diacetyl
26
2.7.4 Bacteriocins 27
2.8 The
Role of Fermentation In Food Safety 28
2.8.1 Safety of Lactic acid bacteria 30
2.9 Health Benefits of Fermented Foods and
Beverages 31
2.9.1 Bioactive compounds 31
2.10 Amylases
31
2.10.1 Microorganisms associated with α-amylase production
33
2.10.2 Bacteria
as a source material for α-amylase production 33
2.10.3 The Production Process of α-amylase 34
2.10.4 Nitrogen sources used for the production of
α-amylase 35
2.10.5 Carbon sources used for the production of
α-amylase 35
2.10.6 Metal ions 35
2.11 Amylolytic Lactic Acid Bacteria (ALAB) 35
2.11.1 Amylase action on starch 37
2.11.2 Effect of Temperature and pH on α-amylase
Activity 37
2.11.3 Industrial Uses of α-amylase
38
2.11.3.1 Glucose and fructose
industry 39
2.11.3.2 Bakery and anti-salting
industries 39
2.11.3.3 Detergent industry 39
2.11.3.4 Alcohol industry 39
2.11.3.5 Textile desizing 40
2.11.3.6 Paper industry 40
2.11.3.7 Feed industry 40
2.11.4 Application
of ALAB in Starch-Based Food Processing 41
CHAPTER 3: MATERIALS AND METHODS 42
3.1 Sample
Collection 42
3.2 Isolation
of Lactic Acid Bacteria 42
3.2.1 Pure Cultures of Isolated Lactic Acid
Bacteria 43
3.3 Identification of Isolated
Lactic Acid Bacteria 43
3.3.1 Gram Staining 43
3.3.2 Spore Staining 43
3.4 Biochemical Tests 44
3.4.1 Sugar fermentation
test 44
3.4.2 Catalase Test 45
3.4.3 Indole Test 45
3.4.5 Citrate Utilization Test 45
3.5 Screening of Isolated LAB for Amylase
Enzyme Production Potential 45
3.6. Molecular Identification 46
3.6.1. DNA
extraction (Boiling method) 46
3.6.2. DNA
quantification 46
3.6.3. 16S rRNA
Amplification 46
3.6.4.
Sequencing 47
3.6.5.
Phylogenetic Analysis 47
3.7 Optimization of
Process Parameters for α-Amylase Production 48
3.7.1 Effect of incubation period 48
3.7.2 Effect of selected carbon sources 48
3.7.3 Effect of pH 48
3.8 Extraction of Crude α-Amylase from
Isolated Organisms 49
3.9. Measurement of Amylase Activity 49
3.10 Development of
Starter Culture by Lactic Acid Bacteria (LAB). 50
3.10.1 Determination of
lactic acid production by isolated LAB 50
3.10.2 Determination of
hydrogen peroxide production by isolated bacteria 50
3.11 Use of Isolated Lactic Acid Bacteria as
Starter Culture for Ogi Production 50
3.11.1 Preparation of Innocula 50
3.12 Physicochemical
Analysis 51
CHAPTER 4: RESULTS
AND DISCUSSION
4.1 Results
52
4.1.1 Phenotypic Characterization of Lactic Acid
Bacteria Isolates 52
4.1.2 Acidification Activity of Lactic Acid
Bacteria 52
4.1.3 Optimized culture conditions for lactic acid
production 52
4.1.4 The Amount of Hydrogen Peroxide Produced by
Lactic Acid Bacteria Isolates 53
4.1.5 Preliminary
Screening of α-amylase Produced by the Isolates 53
4.1.6 Enzyme Optimization Studies 53
4.1.7 Physio-Chemical
Properties of Fermenting Ogi 54
4.2 Discussion 66
CHAPTER 5:
CONCLUSION AND RECOMMENDATION 73
REFERENCES
74
APPENDIX:
Sequences Result February 2020 Sequencing result in
FASTA format and corresponding ID after BLAST ANALYSIS on NCBI website 96
LIST OF TABLES
Table Title Page
4.1:
Colonial and Biochemical Characteristics of the Lactic acid bacteria Isolates55
4.2:
Preliminary Screening of α-amylase Produced by the Isolates. 56
4.3:
Acidification Activity of the Isolates at Different Incubation Time 57
4.4: Amount of Lactic Acid Production by the Isolates 58
4.5:
Amount of Hydrogen peroxide (H2O2) Production by the
Isolates 59
4.6: pH and Titratable Acidity Values of Ogi fermented with Starter Cultures 60
LIST OF FIGURES
Figure Title Page
4.1: Optimization of pH for α-amylase Production
by the Isolates 61
4.2: Effect of Incubation Time on
α-amylase Production by the isolates 62
4.3:
Effect of Sucrose on α-amylase Production by the Isolates 63
4.4:
Effect of Maize Bran substrate on α-amylase production by the isolates 64
4.5:
Effect of Glucose on α-amylase Production by the Isolates 65
CHAPTER 1
1.0
INTRODUCTION
Fermentation of food typically involves
the application of microorganisms (either from the environment, that is, spontaneous
process or inoculation in a controlled environment) that produces certain
enzymes which changes the chemical attributes of the food from its original
form or state (Lovet and Numomoipre, 2017). Many African foods are fermented
before consumption (Nwachukwu and Ijeoma, 2010). Fermented foods are consumed throughout the
world and offer many opportunities
for diversification as
a result of
their global popularity (Kimaryo et al., 2000). The demand for
many fermented cereal-based
products is increasing worldwide,
as they suit
social and cultural
culinary traditions in
many parts of the
world (Kimaryo et al., 2000).
Lactic acid bacteria (LAB) have been
associated from time immemorial with fermentation of foods and their
preservation. Today they are clearly the most important group of industrial
microorganisms (Narvhus and Gadaga,
2003). The production of fermented foods presently is based on the
use of LAB as starter cultures, in order to initiate and provide controlled and
predictable fermentation (Herich and Levkut, 2002). A starter culture is a
microbial preparation of large numbers of cells of at least one microorganism
to be added to a raw material to produce a fermented food by accelerating and
steering its fermentation process. The starter cultures can contribute to the
microbial safety or offer one or more organoleptic, nutritional, or health
advantages (Patrigani et al., 2006).
Examples are starter cultures that produce antimicrobial substances, sugar
polymers, sweeteners, aromatic compounds, vitamins or useful enzymes or that
have probiotic properties. Also a few
exhibit amylolytic activity and are qualified as amylolytic lactic acid
bacteria (ALAB) which are able to decompose starchy materials through the
amylases production during the fermentation processes (Asoodeh et al.,
2010).
The group of Lactic acid bacteria (LAB)
occupies a central role in these processes and has a long and safe history of
application and consumption in the production of fermented foods and beverages
(Caplice and Fitzgerald, 1999). They cause rapid acidification of the raw
production of organic acids mainly lactic acids. Also, their production of
acetic acid, ethanol, aroma compounds, bacteriocins, exopolysaccharides and
several enzymes is of importance (Lore et al., 2005). In this way, they
enhance shelf life and microbial safety, improve texture and contribute to the
pleasant sensory profile of the end product (Nasib et al., 2006). They
are able to inhibit many microorganisms including spoilage and pathogenic organisms.
They are generally recognized as safe and are increasingly being recognized for
their health and nutritional benefits hence some strains are used as
probiotics.
Among the many African traditionally
fermented foods, “ogi” (acid
fermented cereal gruel) is a locally prepared food from fermented maize,
sorghum or millet in Nigeria. It is a staple food and serves as weaning foods
for infants, as well as, dietary food for adults in West Africa (Abioye and
Aka, 2015). The traditional preparation of ogi
involves soaking of maize grains, fermentation, wet milling, sieving and
boiling. Ogi is often marketed as a
wet cake wrapped in leaves or transparent polythene bags. It can be diluted and
boiled into a pap, or cooked and turned into a stiff gel called “agidi” prior
to consumption (FAO, 1996). The fermentation
process is often
carried out on
small or household
scales and are characterized by
the use of
simple, non-sterile equipment,
random or natural inoculums, unregulated
conditions, sensory fluctuations,
poor durability and unattractive packaging of the
processed products (Olanrewaju
et al., 2009). In general, a
wide spectrum of microorganisms is involved during the fermentation process but
a few types usually determine the quality of the end product (Patrigani et al., 2006; Mathara et al., 2008).
1.1
STATEMENT OF PROBLEMS
A study conducted by Olayiwola et al. (2012) showed that ogi was the most consumed maize based
dish (73.5%) in four agro-ecological
zones of Nigeria. The fermentation process of ogi production and other fermented products is still being carried
out by crude method (Achi, 2005). The fermentation is usually achieved by the
use of indigenous microflora or the addition of fermented materials from
previous production (Oyewole and Isah, 2012). Thus, spontaneous (crude)
fermentation processes are difficult to control and are not predictable in
terms of length of fermentation and quality of products and are exposed or
liable to contamination by pathogens.
With increasing urbanisation, there is a
gradual shift from a traditional based society to a modern society in several
African countries. As a consequence, some successful attempts have been made
towards upgrading traditional processing technology, including the use of starter culture and implementation of
quality assurance measures. The development of starter culture will optimise
the process of ogi fermentation and provide a means for controlling the
quality of the product.
1.2
JUSTIFICATION
The necessity to reduce fermentation time
as well as to guarantee product consistency and quality of fermented products
justifies the decision for the evaluation of lactic acid bacteria isolated from
ogi for development of starter
culture and enzyme production potential. Hence, there is a need to apply modern
biotechnological techniques such as
use of starter cultures in improving traditional ogi processing technology for commercialization.
1.3 AIM AND OBJECTIVE
The aim of this study is
to evaluate the potentials of Lactic acid bacteria isolated from ogi for development of starter culture
and enzyme production potential. To achieve this aim, certain specific
objectives were targeted.
1.3.1 SPECIFIC OBJECTIVES
1.
To isolate and identify Lactic acid bacteria
from traditional fermented Ogi.
2.
To determine the potentials of isolated lactic
acid bacteria for the production of starter culture.
3.
To use the
isolated Lactic acid bacteria strains in the fermentation of maize to produce ogi.
4.
To determine the enzyme production potential of
isolated Lactic acid bacteria from ogi.
5.
To study different growth conditions of the isolates for optimal enzyme (amylase) production.
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