THE EFFECT OF EXOPOLYSACCHARIDES OF LACTIC ACID BACTERIA ON SOME FOOD BORNE PATHOGENS

ABSTRACT

The inhibition of food borne pathogens by exopolysaccharides of lactic Acid Bacteria isolates from Ugba was investigated. Food borne pathogens were isolated from three food types, Rice, beans and soup including Staphylococcus aureus, Escherichia coli, species of Proteus, Pseudomonas, Shigella, Salmonella and Bacillus. The prevalence of these isolates varied with Staphylococcus species being in all foods (100%), Bacillus (56%), Shigella(44.49%), Pseudomonas (33.3%), E.coli (22.2%), Proteus and Salmonella (11.1%) each. Three Lactic Acid Bacteria, L. plantarum, L. casei  and L. fermemtum were isolated from Ugba samples and had Exopolysaccharides yields of 121.67mg/l, 116.67mg/l and 98.33mg/l respectively. The antibacterial activity tests showed limited but significant activity of the EPS in the range of 8.33mm to 15.0mm diameter of inhibitions. Lactobacillus casei EPS had the highest level of inhibition against the isolates ranging from 9.00mm(Bacillus) to 15.0mm (Proteus) while L. plantarum EPS had least activity in the range of 0.00(Bacillus) to 12.67mm (Proteus). Comparatively, the Gram positive isolates were less susceptible to EPS (0.00mm to 11.67mm) than the Gram negative isolates (9.33mm to 15.0mm).There were also variations in the levels of activities of the EPS extracts from the different levels of activities of the EPS extracts from the different LABs from fermented foods like Ugba has potentials as good sources of bioactive metabolites with high utility value especially as supplement if not alternatives to commercial antibiotics.

TABLE OF CONTENTS

Title page                                                                                                                                i          

Dedication                                                                                                                              ii

Acknowledgements                                                                                                                iii        

Table of Contents                                                                                                                   iv        

Abstract                                                                                                                                   v         

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim                                                                                                                             4

1.2       Objectives                                                                                                                   4

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Ugba

2.2       Lactic Acid Bacteria                                                                                                   7

2.3       Taxonomical Classification of Lactic Acid Bacteria                                                 7

2.4       Occurrence of Lactic Acid Bacteria in Nature                                                           8

2.5       Exopolysaccharide                                                                                                     8

2.6       Exopolysaccharide Producing Bacteria                                                                     9

2.7       Biosynthetic Pathways Leading To Exopolysaccharide Synthesis in Lab              11

2.8       Classification and Chemical Composition of Exopolysaccharide                                    13

2.8.1    Homo-exopolysaccharide                                                                                           14

2.8.2    Hetero-exopolysaccharide                                                                                          16

2.9       Exopolysaccharide Yields Produced By Lactic Acid Bacteria                                    17

2.10     Health Properties of Exopolysaccharide                                                                    18

2.11     Novel Applications of Exopolysaccharides from Lactic Acid Bacteria                      19

2.12     Foodborne Bacterial Pathogens                                                                                  20

2.12.1  Salmonella                                                                                                                  20

2.12.2  Staphylococcus aureus                                                                                               20

2.12.3  Clostridium botulinum                                                                                                21

2.12.4  Shigella                                                                                                                       21

CHAPTER THREE

3.0       Materials and Methods                                                                                               21

3.1       Source of Materials                                                                                                    21

3.2       Media and Sample Preparations                                                                                 21

3.2.1    Media Preparation                                                                                                      21

3.2.2    Sample Preparation                                                                                                    22

3.3       Isolation of Food Borne Pathogens                                                                            22

3.4       Characterization and Identification of Food Borne Pathogens.                                    23

3.4.1    Colony morphology.                                                                                                   23

3.4.2    Microscopic characteristics                                                                                        23

3.4.3    Biochemical Reaction Tests                                                                                       23

3.4.3.1 Catalase Test                                                                                                               24

3.4.3.2 Indole Test                                                                                                                  24

3.4.3.3 Coagulase Test                                                                                                           24

3.4.3.4 Oxidase Test                                                                                                               24

3.4.3.5 Motility, Indole, Urease (MIU)                                                                                  25

3.4.4    Carbohydrate utilization test                                                                                      25

3.5       Isolation of Lab from Ugba                                                                                        26

3.6       Extraction of Exopolysaccharide                                                                               26

CHAPTER FOUR

4.0       Results                                                                                                                        27

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendations                                                        35

5.1       Discussion                                                                                                                   35

5.2       Recommendation                                                                                                       37

5.3       Conclusion                                                                                  37

            References                                                                              38

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Lactic acid bacteria are widely exploited in medicine, and traditional dairy products, as well as in biotechnological and industrial fermentation processes as a well established starter culture (Gorska et al., 2010). Lactic acid bacteria have shown a significant importance in health complications with increasing number of health beneficial microflora in the intestinal tract, along with an ability to synthesize functional exopolysaccharides. In addition, an important role of lactic acid bacteria has been noticed in the food fermentation, since lactic acid bacteria-derived fermented foods display increased rate of hygienic safety, storage stability and attractive sensory properties. Traditional differentiation of lactic acid bacteria species can be accomplished through their identification and detection by employing various molecular methods as potential alternatives in order to assess their quality control measures in dairy products (Habibi et al., 2011). Due to the versatile potentiality of microbial exopolysaccharides to work as a texturizer, viscosifer, emulsifier and syneresis-lowering agent, as well as due to their pseudo-plastic rheological behavior and water binding capacity, they have shown demanding Industrial need especially in food industry. A wide range of different lactic acid bacteria produce different types of chemically-structured forms of exopolysaccharides. Since, there is no confirmed reports available on the harmful effects of lactic acid bacteria so far, they are classified as Generally Regarded as Safe (GRAS) microorganisms. The microbial exopolysaccharides play a vital role to conceal the bacterial surface facilitating an adhesive interaction at the surface of other bacteria. Moreover, they also work as a substance in the rhizosphere community for bacterial aggregation as well as environment protective agents (Kim et al., 2007). Since exopolysaccharides significantly contribute to the specific rheology and smooth textural properties of fermented and milk products, they have become major targets of ongoing research especially in food processing industry (Kim et al., 2013). Production of exopolysaccharidesis considered a unique feature of lactic acid bacteria in the formation of starters for fermented milk products. In addition, EPSs have shown number of health beneficial effects in human beings especially in the treatment of gastrointestinal, tumor and bowl diseases. Lactic acid bacteria-derived exopolysaccharides, although produced in a very less amount in fermented yogurt, play a crucial role in improved smooth and creamy texture of yogurt, one of the very important aspects of yogurt quality. These EPSs have also shown industrial effectiveness in the development of improved quality low-milk-solid yogurt, low-fat yogurt, and cream yogurt with various health beneficial properties. In addition, various health beneficial attributes of lactic acid bacteria-derived exopolysaccharides have been confirmed previously either as non-digestible food fractions or being natural candidates to treat cancer, ulcer and immune modulation along with their potent ability to reduce blood cholesterol levels (Lynch et al., 2014). Lactic acid bacteria and lactic acid bacteria-derived exopolysaccharides also have significant economic and therapeutic potential for the development of nutrient rich functional food products with prolonged human health beneficial effects. Interestingly, exopolysaccharides may also play an important role by interacting with human immune system serving as vital component of functional foods as well as provide healing effects in bowel disease by working as prebiotics. A few selected lactic acid bacteria display exopolysaccharide production in the form of glucans or fructans by utilizing sucrose as sole carbon source through the action of glycosyl-transferase enzymes (Orr et al., 2009). In addition, although, exopolysaccharides show potential ability to colonize dental surfaces by Streptococci, non-significant importance has been given on the relevance to the ecological niche of gastrointestinal biome lactic acid bacteria. Lactic acid bacteria-derived exopolysaccharides in composition might exist as a single type of sugar monomer (homo-polysaccharides) or in the combination of several types of monomers (hetero-polysaccharides). However, variations in sugar composition, chain length, degree of branching, or sugar linkages in the exopolysaccharides produced by different lactic acid bacteria have been observed as leading factors, which assist in the termination of the rheological and healthpromoting potential of lactic acid bacteria-derived exopolysaccharides (Rather et al., 2013). Based on the chemical composition of lactic acid bacteriaderived exopolysaccharides, they have been classified in two chemical classes, homo-exopolysaccharide and hetero-exopolysaccharide. Homoexopolysaccharides are the chemical structures of single type of monosaccharide, whereas, hetero-exopolysaccharides contain regular repeating units of 3-8 different carbohydrate moieties synthesized from intracellular sugar nucleotide precursors. However, biosynthesis process of both homoand hetero-exopolysaccharides differs from each other. Synthesis of homo-exopolysaccharide occurs through the enzymatic reactions of glucansucrase or levansucrase by using sucrose (Rather et al., 2014), whereas, hetero-exopolysaccharide synthesis completes in four major steps involving sugar transportation, sugar nucleotide synthesis, repeating unit synthesis, and polymerization of the repeating units (Gorska et al., 2010).

Generally, exopolysaccharides are produced either in a bioreactor or in situ through proper down-stream processing for their further practical applications as a functional food additives and in fermentation purposes. Since lactic acid bacteria are often used in the preparation of fermented mixed starter cultures for dairy fermented food products, application of exopolysaccharides as bio-ingredients in food industry depends on the recovery rate and economic yield production (Sanchez et al., 2006). Lactic acid bacteria are the predominant microbiota and play an important role in natural fermentation of meat and vegetables and are used as mixed starters in controlled fermentation process. In addition, mesophilic lactobacilli bacteria as a secondary microbiota have also shown significant role in the development of unique flavor and texture of cheese products (Habibi et al., 2011). Biotechnological advances have led to the discovery of lactic acid bacteria-derived biopolymers or exopolysaccharide molecules with confirmed evidences of industrial and medical usefulness to mankind. Enriched with biocompatibility, being GRAS and non-toxic ability of lactic acid bacteria-derived exopolysaccharides have made them a first-line choice in the treatment of various chronic diseases including tissue engineering, drug delivery system, and disease healing ability as compared to the plants and algal-based polysaccharides (Kim et al., 2007). Reports have confirmed that a few selected biopolymers can be degraded in vivo; they might be possible alternatives to synthetic biopolymers for using in tissue replacement and controlled drug release strategies (Lynch et al., 2014).

1.1       AIM

To determine the inhibitory effect of exopolysaccharides from Lactic acid bacteria from fermented Pentaclethra macrophylla (UGBA) on some food borne pathogens.

1.2       OBJECTIVES

1.     To isolate Lactic acid bacteria from fermented Ugba

2.     To isolate some pathogenic bacteria species from spoilt foods

3.     To extract exopolysaccharides from Lactic acid bacteria isolates

4.     To determine the antibacterial activity of the exopolysaccharides against the isolated food pathogens.

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