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

Dedication

Acknowledgement iii

Table of Contents iv

List of Tables vii

List of Figures viii

Abstract ix

CHAPTER ONE 1

1.0. 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

3.0. 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 37oC, 45oC and 50oC 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

Table Title Page

1 Colonial and biochemical identification of Lactic acid bacteria species 28

2 Proteolytic activities of isolates 29

LIST OF FIGURES

Figure Title Page

1 Effect of temperature on the proteolytic activities of L. lactis and L. plantarum 30

2 Effect of pH on the proteolytic activities of L. lactis and L. plantarum 31

3 Effect of salinity on the proteolytic activities of L. lactis and L. plantaru