INVESTIGATION OF PROBIOTIC POTENTIAL OF LACTIC ACID BACTERIA AND ITS RESPONSE TO COLD STRESS

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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