ISOLATION AND SCREENING OF LACTIC ACID BACTERIA FROM FERMENTED MILK PRODUCT (FURA DE NUNU) FOR BACTERIOCIN PRODUCTION

                                                              ABSTRACT

Lactic acid bacteria (LAB) commonly used as starter cultures in dairy foods are known to produce antimicrobial substances such as bacteriocins, having great potential as food preservatives. The present study was aimed at isolating and screening LAB for bacteriocin production from fermented milk product (fura de nunu). Among the thirteen (13) isolates gotten, six (6) were chosen through screening for LAB and for bacteriocin production potentials. The bacteriocins produced by isolates NU06, NU07 and NU08 demonstrated good antimicrobial activity against the four indicator organisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyrogenes) while isolates NU01, NU02 and NU09 had moderate and minimal inhibitory effect against the indicator strains as well. This shows that bacteriocin producing LAB are beneficial in fermented milk products (fura de nunu) for shelf life extension and increase in their organoleptic properties.

TABLE OF CONTENTS

Title page                                                                                                                    i

Certification                                                                                                                ii

Dedication                                                                                                                   iii

Acknowledgements                                                                                                    iv

Table of contents                                                                                                        v

List of tables                                                                                                               viii

Abstract                                                                                                                      ix

CHAPTER ONE

1.0 Introduction                                                                                                          1

1.1 Aim                                                                                                                       4

1.2 Objectives                                                                                                             4

CHAPTER TWO

2.0 Literature Review                                                                                                 5

2.1 Preparation of fura de nunu                                                                                  5

2.1.1 Ingredients                                                                                                         6

2.1.2 Procedures                                                                                                         6

2.1.3 Nutritional Benefits of Fura de nunu                                                                7

2.1.4 Health Hazards Associated with Fura de nunu                                                 8

2.2 Lactic Acid Bacteria (LAB)                                                                                 9

2.2.1 General description of LAB                                                                              9

2.2.2 Classification of Lactic Acid Bacteria                                                               11

2.2.3 Metabolic Activities of Lactic Acid Bacteria                                                    13

2.3 Properties of Lactic Acid Bacteria                                                                       16

2.3.1 Preservative Property of LAB                                                                                       17

2.3.2 Acid Production                                                                                               17

2.3.3 Exopolysaccharide Formation                                                                           18

2.4 Lactic acid bacteria and Bacteriocin                                                          19

2.4.1 Classification of LAB Bacteriocins                                                           20

2.4.2 Mode of action of Bacteriocins                                                                         24

2.4.3 Applications of Bacterions                                                                                25

2.4.4 Advantages of LAB Bacteriocins                                                                     28

2.4.5 Disadvantages of LAB Bacteriocins                                                                 29

CHAPTER THREE

3.0 Materials and Methods                                                                                         31

3.1 Sample Collection                                                                                                 31

3.2 Determination of pH and Titratable Acidity                                                        31

3.3 Enumeration and Isolation of microorganisms                                                     33

3.4 Phenotypic Characterization                                                                                 33

3.5 Biochemical Characterization                                                                               33

3.6 Characterization of LAB                                                                                      35

3.7 Screening for Bacteriocin production                                                                   36

3.8 Antimicrobial Activity of Bacteriocin                                                                  36

CHAPTER FOUR

4.0 Results                                                                                                                  38

CHAPTER FIVE

5.1 Discussion                                                                                                             44

5.2 Conclusion                                                                                                            45

5.3 Recommendation                                                                                                  46

 References

                                                       LIST OF TABLES

Table                                      Title                                                                                         Page

1.     Most important bacteriocins produced by lactic acid bacteria (LAB).                            23

2.     Enumeration of microorganisms                                                                                      39

3.      Morphological and biochemical characterization of LAB from fura de nunu                40

4.     Screening of LAB isolates at different growth parameters                                              41

5.     Carbohydrate fermentation test by lactic acid bacteria                                                    42

6.    Inhibition produced by LAB bacteriocins against indicator strains                                  43

                                                      CHAPTER ONE

1.0              Introduction

Fermented foods are generally defined as those foods or beverages produced with the help of controlled microbial growth and enzymatic transformation of food components (Marco et al., 2017). Fermentation of food is one of the oldest method of food processing and preservation that is entrenched in traditional cultures and village life (Campbell-Platt, 1994). It has evolved a method of preserving foods during scarcity, imparting appropriate aroma and flavors to foods, decreasing products toxicity and generating product diversity. History suggests that fermented foods were consumed 7,000 years ago in Babylon. Fermented foods contribute immensely to human diet in many countries around the world. The process of fermentation is an inexpensive technology which preserves food, improves its safety and nutritional values and enhances its sensory properties (Murty and Kumar, 1995; Steinkraus, 1996).

Fermented milk products, also known as cultured dairy foods or cultured milk products, are dairy foods that have been fermented with lactic acid bacteria such as (Lactobacillus, Leuconostoc and Lactoccocus) which may be incorporated into the milk or naturally present in the milk. For even distribution, milk fat throughout the structure milk and milk products may or may not be mixed vigorously (homogenized) and must be pasteurized (Santosa et al., 2006). The fermentation process increases the shelf life of the product, while enhancing the taste and improving the digestibility of milk products. Fermented milk products are also main source of whey proteins like lactalbumin, lactoperoxidase, lactoglobulin, immunoglobulins and lactoferin. These proteins have exhibited a number of biological effects having various effects on the functions of digestion and anti-carcinogenic activities (Mclntosh et al., 1998).

Fura de nunu (fermented cereal mix) is a highly nutritious beverage which is a two in one product consisting of a cereal “fura” made from millet and “nono” a fermented milk product similar to yoghurt. The fura is mixed with nunu in a bowl for customers. Depending on the consistency, this milk product can be used as food, refreshing drink and a weaning food for infants (Belewu, 1999).

Typically, fresh cow milk is collected in the morning in calabashes, sieved and left to ferment for a minimum of 24 hours or a maximum of 48 hours depending on the season (Frank, 2001). During the hot season which is usually from March to June, high ambient temperatures promote acidification of the milk within 12 to 24 hours yielding the desired product while in cold season (November to January) where temperature of (15 -17)˚C are recorded, fermentation takes up to 48 hours. The fermented milk is then churned using a wooden ladle. Fat accumulates as a result of the churning, the fat is removed, execess whey is drained off to obtain a product with a thick consistency which is the “nunu” which is consumed with “fura”.

Lactic acid bacteria (LAB) are group of gram-positive, cocci or rods, which produce lactic acid as the major end product of the fermentation of carbohydrate (Axelson, 1998). LAB are used as natural or selected starters in food fermentation in which they perform acidification due to production of lactic acid flavor. Bacteria from the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus are the main species of LAB involved. Although several more have been identified but they play a minor role in lactic fermentations (Axelson, 1998). LAB are often inhibitory to other microorganisms and this is the basis of their ability to affect the keeping quality and safety of many food products. The principal factors, which contribute to this inhibition, are low pH, organic acids bacteriocins, hydrogen peroxide, ethanol nutrient depletion and low redox potential.

Bacteriocins are naturally occurring peptides produced by Gram-positive bacteria, that have antibiotic properties they are usually small such as 24 amino acids (Chatterjee et al., 2005). Many bacteriocin are active against food-borne pathogens especially against   Listeria monocytogenes. Several types of bacteriocins from food – associated lactic acid bacteria have been identified and characterized, of which the important ones are nisin, diploccocins, acidophilins, bulgarican, helveticins, lacticins and plantaricins. Maximal bacteriocin production could be obtained by supplementing a culture medium with growth limiting factors such as sugars vitamins and nitrogen sources or by regulating pH of the growth medium or by choosing the best adapted culture medium. At present, nisin produced by Lactococcus lactis the only bacteriocin commercially and marketed (Balasubramayam and varadaraj, 1998). It has been reported that nisin is more effective against Gram-positive bacteria, particularly the spore formers, (Delves- Broughton 1990). Other bacteriocins of Lactobacillus has been reported to be effective against closely related species of mesophilic Lactobacillus and therefore considered as potential natural food preservatives (Daeschel, 1993., De Vugst and Vandamme, 1994).

1.1 Aim

The aim of this research is to determine the effect of crude extracts (bacteriocin) from lactic acid bacteria isolated from fura de nunu against selected test organisms.

1.2 Objectives

1. To isolate lactic acid bacteria from fura de nunu.

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