SCREENING FOR PROTEOLYTIC ACTIVITIES OF LACTIC ACID BACTERIA ISOLATED FROM RIPENED FRUITS

ABSTRACT

Different samples of fruits were analyzed for the presence of Lactic acid bacteria species with proteolytic activity. 13 isolates were recovered and identified as Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc mesenteroides and Wiesella cibaria based on morphological, physiological and biochemical analysis. The proteolytic activities of these isolates were investigated using modified agar supplemented with 1% gelatin and was quantified by measurement of the diameter of zone of clearance (mm). When tested on the media, isolates identified as Lactococcus lactis and Lactobacillus plantarum were found to exhibit the highest proteolytic activity. Furthermore, the two potential protease producers that showed higher proteolytic activities were subjected to growth at different optimized conditions of temperature, pH and salinity. Lactococcus lactis showed its maximum proteolytic activities at 37oC, pH of 7 and 2% NaCl. Lactobacillus plantarum also followed the same trend and exhibited maximum proteolytic activities at 37oC, pH 7 and 2% NaCl. Reduced proteolytic activities were observed at 28oC, 45oC, pH of 4 and 10 for both isolates. However there was total absence of proteolytic activity at 7% NaCl concentration.

TABLE OF CONTENTS

Certification i

Dedication ii

Acknowledgement iii

Table of Contents iv

List of Tables vii

List of Figures viii

Abstract ix

CHAPTER ONE 1

INTRODUCTION 1

1.1. AIMS AND OBJECTIVES 3

CHAPTER TWO 4

2.0. LITERATURE REVIEW 4

2.1. LACTIC ACID BACTERIA 4

2.2. OCCURRENCE OF LACTIC ACID BACTERIA 5

2.3. GENERAL CHARACTERISTICS OF LACTIC ACID BACTERIA 5

2.4. CLASSIFICATION OF LACTIC ACID BACTERIA 6

2.4.1. Homofermentative Lactic Acid Bacteria 6

2.4.2. Heterofermentative Lactic Acid Bacteria 7

2.5. PROTEOLYTIC ENZYMES 7

2.6. OCCURRENCE OF PROTEOLYTIC ENZYMES 8

2.6.1. Plants 8

2.6.2. Animals 8

2.6.3. Microorganisms 9

2.7. PURIFICATION OF PROTEOLYTIC ENZYMES 12

2.8. PROTEOLYTIC SYSTEMS OF LACTIC ACID BACTERIA 12

2.9. APPLICATIONS OF PROTEOLYTIC ENZYMES 13

2.9.1. Applications in Food Industry 13

2.9.2. Role in Pathological Processes 14

2.9.3. Nucleic Acid Isolation 14

2.9.4. Applications in Detergent industries 15

2.9.5. Recovery of Silver from used Films 15

2.10. FUTURE SCOPE 16

CHAPTER THREE 18

MATERIALS AND METHODS 18

3.1. SAMPLE COLLECTION 18

3.2. MEDIA PREPARATION 20

3.2.1. De Man Ragosa Sharpe (MRS) Agar 20

3.2.2. Nutrient Agar 20

3.2.3. De Man Ragosa Sharpe (MRS) Agar Supplemented with 1% (1g) Gelatin 20

3.3. STERILIZATION AND PREPARATION OF FRUIT SAMPLES 20

3.4. ISOLATION OF LACTIC ACID BACTERIA 21

3.5. IDENTIFICATION OF LACTIC ACID BACTERIA ISOLATES 21

3.5.1 Macroscopy 21

3.5.2. Microscopy 21

3.5.2.1. Gram Staining 21

3.5.2.2. Motility Test 22

3.5.3. Biochemical Tests 22

3.5.3.1. Catalase Test 22

3.5.3.2. Carbohydrate Fermentation Test. 22

3.5.4. Growth at 5% and 7% NaCl 23

3.5.5. Growth at the Temperature of 37^oC, 45^oC and 50^oC 23

3.6. PRELIMINARY ASSAY FOR PROTEOLYTIC ACTIVITY 23

3.7. QUANTIFICATION OF PROTEOLYTIC ACTIVITY AT OPTIMIZED

CONDITIONS 24

3.7.1. Effect of Temperature 24

3.7.2. Effect of pH 24

3.7.3. Effect of Salinity 24

3.8. DATA ANALYSIS 25

CHAPTER FOUR 26

4.0. RESULTS 26

4.1. ISOLATION OF BACTERIA 26

CHAPTER FIVE 33

5.0. DISCUSSION AND CONCLUSION 33

5.1. DISCUSSION 33

5.2. CONCLUSION 35

REFRENCES 36

LIST OF TABLES

LIST OF FIGURES

CHAPTER ONE

1.0.  INTRODUCTION

Microorganisms excrete a wide variety of proteolytic enzymes which are molecules of relatively small size and are compact, spherical structures that catalyze the peptide bond cleavage into proteins; they hydrolyze peptide bonds and therefore lead to the disassembly of proteins (Polgar, 1989). Some bacteria and fungi, particularly pathogenic, food spoilage and soil microorganisms use proteins as their source of carbon and energy. They secrete enzymes called proteases that hydrate proteins to amino acids, which are transported into the cell and catabolized (Joanne et al., 2011).

Fresh fruits are part of the normal human diet and are consumed in large quantities in most civilizations. Traditionally, fruits have been regarded as microbiologically safer than other unprocessed foods such as meat, milk, poultry and sea food. These products are rich in carbohydrate and poor in proteins with pH level from 7.0 to slightly acidic and provide a suitable niche to several bacterial, yeast and mould (Wiessinger et al., 2000; Trias et al., 2008).

Many fruits posses a natural defense mechanism. Fruits contain organic acids in quantities adequate enough to contribute a pH level of 4.6 to lower. The pH and the type of acid itself are the major influence that selects for the predominant flora present in fruits. Acid tolerant microbes, like fungi and lactic acid bacteria are demonstrated as a part of autochthonous micro flora of some fruits owing to their low ph and organic acids (Brackett, 1988; Sajur et al., 2006).

Lactic acid bacteria in fruits produce secrete proteolytic enzymes as part of their strategy for penetrating into plant host cell wall. The activities of these enzymes results in  metabolic changes that occur in fruits such as ripening, change of colour and change in texture.

The proteolytic system of lactic acid bacteria has received special interest because of the role of Lactic acid bacteria in the food and dairy industry. Since Lactic acid bacteria are unable to synthesize amino acids required for their metabolism, they rely on an active system of proteolytic enzymes which enables them to utilize the nitrogen sources present in their environments (Pritchard and Coolbear, 1993). Besides enabling them to grow in environments such as milk, the proteolytic system of Lactic cid bacteria also confers organoleptic improvements in fermented foods for which they act as starters (Savijoki et al., 2006). Proteases of other Lactic acid bacteria members such as the Lactococci, Lactobacilli and the Streptococci have been well-studied while those of the Pediococci are less reported in the literature. For example, strains of Pediococcus acidilactici isolated from sausages have been reported to possess protease and peptide-hydrolyzing enzymes (Benito et al., 2007).

The suitability of an enzyme for industrial processes depends on its unique characteristics such as optimum temperature, pH, mode of action, etc. (Zhang et al., 2011). Therefore, characterization and purification of an enzyme is highly important in appreciating its potential uses. A better understanding of the protease produced by lactic acid bacteria would allow for further improvements in its use as starter culture in meats and other fermentations and also enable us to identify other potential industrial applications of the enzyme.

The activities of proteolytic enzymes are easily detected by screening for their actions on gelatin. Gelatin is a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bones, and connective tissues of animals. Generally, gelatin dissolves readily at high temperature, and sets to gel on cooling. In the presence of proteolytic enzymes, gelatin cannot gel because of the protease enzymatic breakdown of the gelatin.

1.1. AIMS AND OBJECTIVES

ü To isolate, identify and characterize the species of lactic acid bacteria from ripened fruits.

ü To screen the isolates for proteolytic activities.

ü To determine the optimal conditions for the proteolysis by Lactic acid bacteria.

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