ISOLAT ION AND CHARACTERISATION OF PROTEOLYTIC LACTIC ACID BACTERIA FROM INDIGENOUS FERMENTED FOODS

ABSTRACT

This study was aimed at the isolation and identification of proteolytic lactic acid bacteria from indigenous fermented foods. 10 grams of each of the samples (Ugba, Kunu, Yoghurt and Ogi) were aseptically added into 90 ml of sterile 0.9% NaCl. From this study, a total of 5 species were isolated and they are Lactobacillus plantarum, Enterococcus spp., Lactobacillus brevis, Lactobacillus lactis and Streptoccus spp were all isolated. All of isolated bacterial strains showed the proteolytic activity on skim milk agar. Among them, Enterococcus spp has the highest proteolytic activity of 23.0mm as indicated by the widest clear zone while Lactotococcus lactis has the least proteolytic activity of 17.0mm. The study indicated that the different combinations of LAB strains can positively influence the flavor development of indigenous fermented foods. Several properties of LAB i.e. acid production, proteolysis, production of diacetyl and autolysis can be explored for their use as starters or adjuncts.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    2

CHAPTER TWO

            Literature Review                                                                                                       3

2.1       Lactic Acid Bacteria (LAB)                                                                                       3

2.2       Proteinases                                                                                                                  4

2.3       Peptide Uptake Systems                                                                                             6

2.4       Regulation of the Proteolytic Systems                                                                       7

2.5       Technological Aspects of Proteolysis in Diary Products                                           9

2.6       Traditional Fermented Foods                                                                                     10

2.6.1    Ogi                                                                                                                              11

2.6.2    Tempe kedele                                                                                                             11

2.6.3    Natto, tu-si, tao-si, tnua-nao                                                                                       12

2.6.4    Kenkey                                                                                                                        12

CHAPTER THREE

3.0       Materials and Methods                                                                                               13

3.1       Collection of Samples                                                                                                13

3.2       Sterilization of Materials                                                                                            13

3.3       Isolation of Lactic Acid Bacteria                                                                               13

3.4       Identification of Lactic Acid Bacteria                                                                                    14

3.4.1    Gram Staining                                                                                                            14

3.4.2    Biochemical Test                                                                                                        14

3.4.2.1 Catalase Test                                                                                                               14

3.4.2.2 Citrate Test                                                                                                                 15

3.4.2.3 Methyl Red (MR)                                                                                                       15

3.4.2.3 Voges Proskauer (VP)                                                                                                            15

3.4.2.3 Indole Test                                                                                                                  15

3.4.2.4 Sugar Fermentation Test                                                                                            15

3.5       pH Determination                                                                                                       16

3.6       Measurement of Growth                                                                                             16

3.7       Determination of Lactic Acid                                                                                     16

CHAPTER FOUR

4.0       Results                                                                                                                        18

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation                                                          23

5.1       Conclusion                                                                                                                  24

5.2       Recommendation                                                                                                       24

References                                                                                                                  25

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Proteases constitute one of the most important groups of enzymes both industrially academically. Proteases are the enzymes that hydrolyse proteins by addition of water across peptide bonds and catalyse peptide synthesis in organic solvents with low water (Ishtiaq et al., 2010). Proteases are essential constituents of all forms of on earth, including bacteria, fungi, actinomycetes, plant and animals. Proteases are classified according to their structure or the properties of the activity site such as serine, metallo, carboxyl, acidic, neutral, and alkaline (Nihan and Elif, 2011). They are generally used in detergents (Ishtiaq et al., 2010) food industries meat processing, cheese making, silver recovery from photographic film (Hamid mukhtar and Ikram, 2018) production of digestive and certain medical treatments of inflammation and virulent wounds.

Lactic Acid Bacteria (LAB) are Gram positive, fastidious, acid tolerant, generally non-sporulating, catalase negative, devoid of cytochrome, and non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics that produce lactic acid as a major or sole product of fermentative metabolism. Lactic acid bacteria (LAB) have been used for the fermentation of food and feed products since ancient days and today their major applications are still in the food and feed industry as starter cultures.

They also have medical pharmaceutical applications (Beg et al., 2003). Lactic acid bacteria are widely distribution in the nature. The genera, Lactobacillus, Streptococcous, Lactococcus, Leuconostol, Bifidobacterium, Carnobacterium, Enterococcus and Sporolactobacillus are known as lactic acid bacteria. They can be divided into species, subspecies, variants and strains. Each geneus and species has different characteristics but they are generally Gram positive, non-sporing, cocci or rod, non-motile bacteria that produce lactic acid as a major end-product during fermentation of carbohydrates (Myat, 2008). Lactic acid bacteria ferment various carbohydrate mainly lactate and acetate into lactic acid and acetic acid. LAB that only produce lactic acid an end product are called homofermentative, those that also produce acetic acid, ethanol and carbondioxide are termed heterofermentative (Hnin et al., 2015).

1.1       AIM AND OBJECTIVES

The aim of this study is to isolate, screen and identify proteolytic lactic acid bacteria from indigenous fermented foods. The objectives are:

1.         To isolate and identify lactic acid bacteria from fermented food product (yogurt and fermented milk).

2.         To determine the percentage occurrence of the isolates.

3.         To determine the proteolytic activities of selected fermented food

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