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 ﾿

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