ABSTRACT
This study investigates the probiotic
potential of lactic acid bacteria (LAB) and their response to cold stress. The
aim is to isolate LAB strains, assess their survival under cold conditions, and
evaluate their probiotic capabilities. LAB are essential microorganisms found
in fermented foods and human gastrointestinal tracts, with known health
benefits due to their antimicrobial activity and role in promoting gut health.
Samples of Ogiri and Ugba, traditional Nigerian fermented foods, were collected
from local vendors in Umuahia metropolis for LAB isolation. The study involved
a series of microbiological techniques, including serial dilution, culture,
sub-culturing, and biochemical identification. The isolates were characterized
through cultural, microscopic, biochemical, and sugar utilization tests. The
study also examined LAB’s survival in cold temperatures, which is crucial for
maintaining their functionality in industrial food production and
pharmaceutical applications.
LAB have evolved adaptive mechanisms
to survive under stress, including cold conditions, which is significant for
their viability during storage and processing. The study revealed that LAB
strains from the fermented foods exhibited resilience to cold stress, which is
crucial for enhancing their use in probiotics and functional foods. The
potential of LAB to improve human health, particularly by contributing to the
balance of gut microflora and reducing pathogenic bacteria, was also
highlighted. The research concluded that understanding LAB’s response to cold
stress could improve their application in food processing and storage, as well
as in therapeutic products. Recommendations were made to encourage the consumption
of fermented products in low-income communities due to their health benefits.
Furthermore, freezing LAB to preserve their probiotic properties for longer
periods has significant potential for food and pharmaceutical industries.
TABLE
OF CONTENTS
CHAPTER ONE
1.0 Introduction
1.1. Background
of Study
1.2. Aim
of Study
1.3.
Objective
CHAPTER TWO
LITERATURE REVIEW
2.1.
Background of Study
2.1. Classification
and Uses of Lactic Acid Bacteria
2.1.1. Lactobacillus
2.1.2. Bifidobacteria
and Propionibacteria
2.2. Lactic
Acid Bacteria And Stress: Basic Concepts
2.3. Cold
Sensors
2.4. Cold-Stress
Proteins
2.5. Freezing
And Cryoprotection: An Industrial Issue
2.6. Role
Of Probiotic Lab In Fermented Food
2.7. Stress
Resistance Of Probiotic Lab
2.8. Application
Of Lactic Acid Bacteria In Health And Disease
CHAPTER
THREE
MATERIALS AND METHODS
3.1. Sample
Collection
3.2. Materials
and Apparatus
3.2.1. Sterilization
of Materials
3.2.2.
Normal
Saline Preparation
3.2.3. Media
Preparation for Isolation of Lactic Acid Bacteria from the Ogiri and ugba
Samples
3.3.
Isolation
of Lactic Acid Bacteria
3.3.1.
Sub-Culturing
3.4. Characterization
and Identification of Lactic Acid Bacterial Isolates
3.4.1.
Gram
Staining Techniques
3.4.2.
Motility
Test
3.5.
Biochemical
Test
3.5.1.
Catalase
Test
3.5.2.
Methyl
Red Test
3.5.4.
Indole
Test
3.5.5.
Citrate
Test
3.5.6.
Oxidase
Test.
3.6. Determination
of Lactic Acid Bacteria Response to Different Cold Stress
3.7.
Probiotic
Properties Analysis
3.7.1.
Determination
of Sugar Fermentation
3.5.2.
Assay
for NaCl Tolerance
3.5.3.
Acid and Bile Salt Tolerance
3.5.4.
Antimicrobial
Activity
CHAPTER
FOUR
RESULTS
AND DISCUSSIONS
4.1
Results
4.1.1 Identification of the Isolated Test Organisms
(Lab Species)
4.1.4
Antimicrobial
Activity of the Isolated Test Species (Lab Species)
4.2
Discussion
CHAPTER
FIVE
CONCLUSION
AND RECOMMENDATION
5.1 Conclusion
5.2
Recommendation
References
LIST
OF TABLES
Table
4.1 Shows the identification of the
isolated test organisms
Table 4.2. Total viable counts of the LAB species after 2 hours storage at
different temperature.
Table 4.3 Shows the OD (in nanometer, nm) of the lab
species broth cultures
Table
4.4.2 Antimicrobial activity for Leuconostoc mesenteriodes
Table 4.1.5 Acid and bile tolerance of Lactobacillius
bulgaricus (mean ± standard deviation, n=3 )
CHAPTER
ONE
INTRODUCTION
1.1 BACKGROUND
OF STUDY
Lactic
acid bacteria (LAB) constitute a heterogeneous group of bacteria which are
found in diverse environments from the human and animal body to plants. These
bacteria have been used for long to produce various fermented foods from
products derived from animals (milk, meat, fish, etc.) or plants (vegetables,
wine, olives, etc.). The industrialization of food bio-transformations
increased the economic importance of LAB. Although LAB are a low cost
ingredient of the food transformation processes, they play a crucial role in
the development of the organoleptique and hygienic quality of fermented
products. Therefore, the reliability of starter cultures in terms of quality
and functional properties (important for the development of aroma and texture),
but also terms of growth performance and robustness has become essential for
successful fermentations. Therefore LAB strains were selected for resistance
against bacteriophages, for fast growth and acidification, for proteolytic
properties, for bacteriocin resistance, etc. However, in addition these strains
must also resist the adverse conditions encountered in industrial processes,
for example during starter handling and storage (freeze-drying, freezing or
spray-drying).The development of new applications such as live vaccines and
probiotic foods reinforces the need for robust LAB since they may have to
survive in the digestive tract, resist the intestinal flora, eventually
colonize the digestive or uro-genital mucosa and express specific functions in
conditions unfavorable to growth (for example, during stationary phase or
storage). Except probiotic strains for which high tolerance to acid and bile
was used as a selection criteria. LAB have seldomly been selected for stress
resistance.
However,
bacteria are not only submitted to potentially stressful environmental changes
in industrial processes, but also in nature where the ability to quickly
respond to stress is essential for survival (Stortz et al., 2000) It is now well established that LAB, like other
bacteria, evolved stress-sensing systems and defenses against stress which
allow them to withstand harsh conditions and sudden environmental changes.
Stress defenses are good examples of such integrated regulation systems. Bacterial
stress responses rely on the coordinated expression of genes which alter
different cellular process (cell division, DNA metabolism, housekeeping, membrane
composition, transport, etc.) and act in concert to improve the bacteria stress
tolerance (stortz et al., 2000) The
integration of these stress responses is accomplished by networks of regulators
which allow the cell to react to various and complex environmental shifts. The
current knowledge on the environmental stress responses in LAB varies between
species and depending on the type of stress. The best studied are acid, heat
and cold stress, although for the latter most of the studies focused on a
specific family of proteins instead of the whole response. It is interesting to
outline how the changes of food characteristics during the fermentation process
can be described as dynamic fluctuations of the food environment itself and, at
the same time, stress source for the microorganisms involved, such as LAB. In
fact, whenever autochthonous bacteria are adapted and competitive in their
respective environment, the environment can be described as stressful for LAB.
The fermentation parameters, including temperature, water activity (Aw),
oxygen, pH, as well as the concentration of starter cultures, affect the
regulatory mechanism and the response mechanisms of LAB, as well as their
effects on the final products properties.
Members
of LAB have a long traditional history as starter cultures in food and beverage
fermentations from ancient times. They contribute to the rapid acidification of
food products and also improve the flavor, texture, and nutritional composition
of fermented foods (Ross et al.,
2002). It was at the beginning of the twentieth century when Elie Metchnikoff
first proposed the scientific rationale that specific bacteria were thought to
be beneficial to health (Stanton et al.,
2003). Foods containing probiotics belong to the functional food category, and
such foods provide specific health benefits over and above their nutritional
value (Stanton et al.,
2005).Functional foods include those containing bioactive ingredients such as
probiotics; bioactive peptides such as bacteriocins, which are small
antimicrobial peptides; bioactive fatty acids such as conjugated linoleic acid
(CLA); and organic acids (Stanton et al.,
2001).
1.3.
AIM OF STUDY:
The
aim of the study is to investigate the probiotic potential of lactic acid
bacterial and its response of to cold stress
OBJECTIVE:
·
To isolate and identify
strains of lactic acid bacterial from known sample.
·
To determine the effect
of cold temperature on the survival of lactic acid bacterial.
·
To determine its
probiotic potential.
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