ABSTRACT

The protease activity of lactic acid bacteria from fresh cow milk (Nunu) was evaluated. Three species of Lactic acid bacteria were isolated from the Nunu. The identities of the isolates were confirmed through Gram staining and other biochemical tests. Several parameters such as pH, incubation temperature and incubation time were checked to determine the optimal condition for protease enzyme production and activity. The three LAB isolates had varying levels of prevalence and were identified to be Lactobacillus acidophilus with 66.7% occurrence, Lactobacillus casei also with 66.7% and Lactobacillus bulgaricus 100%. Preliminary tests for protease activity was conducted using  agar well diffusion method in 1% casein infused medium, Lactobacillus bulgaricus had the highest activity with a mean halo zone size of 7.1 mm while Lactobacillus casei had the least 5.3 mm, the halo zone size of protease activity of Lactobacillus acidophilus was an average of 5.6 mm. The optimum growth and enzyme production was recorded at pH 6.0 with protease halo zone size in the range of 5.33 mm (L. casei) to 6.33 mm (L. bulgaricus) while L. acidophilus had a mean halo zone size of 5.67 mm. The optimal incubation temperature for protease enzyme production and activity was observed to be 40oC in which Lactobacillus bulgaricus had the highest level of enzyme activity with halo zone size of 7.00± 0.50 (mm) while the other isolates each recorded 5.33 mm halo zone size. The optimal incubation time was recorded at 48 hours for all three LAB isolates, L. bulgaricus had the highest level of protease activity with a halo zone size of 7.00mm the other isolates had the same level of activity of 6.17 mm halo zone size. The result obtained in the work indicated that the widest clear zones were produced at pH 6 after 48 hrs of incubation period at 40°C for all 3 LAB isolates indicating the optimal conditions for protease enzyme production. 

TABLE OF CONTENT

Title Page i

Certification ii

Dedication iii

Acknowledgement iv

Table of contents v

Lists of Tables vii

Lists of Figures ix

Abstract x

CHAPTER ONE

1.0 Introduction 1

1.1 Aim and objectives 3

CHAPTER TWO

2.0 Literature Review 4

2.1 Sources of Proteolytic Enzymes 5

2.1.1 Plant Proteases 5

2.1.2 Animal Proteases 6

2.1.3 Microbial Proteases 6

2.1.3.1 Bacteria 7

2.1.3.2 Fungi 7

2.1.3.3 Viruses 8

2.2 Physiological Functions of Proteases 8

2.3 Industrial Applications 9

2.3.1 Detergent Industry 9

2.3.2 Leather Industry 10

2.3.3 Food Industry 10

2.3.4 Pharmaceutical Industry 10

2.3.5 Waste Treatment 11

CHAPTER THREE

3.0 Materials and Methods 12

3.1 Collection of Samples 12

3.2 Isolation of Lactic Acid Bacteria 13

3.3 Screening for Protease Enzyme 14

3.4 Characterization of Lactic Acid Bacteria Isolates 14

3.4.1 Colony Features 14

3.4.2 Microscopic Features 14

3.4.3 Biochemical Tests 14

3.4.3.1Catalase Test 14

3.4.3.2 Coagulase Test 15

3.4.3.3 Oxidase Test 15

3.4.3.4 Citrate Test 15

3.4.3.5 Carbohydrate Utilization 16

3.5 Identification of Lactic Acid Bacteria Isolates 16

3.6 Determination of Protease Activity 16

3.7 Optimization of Parameters for Protease Enzyme Production and Activity 17

3.7.1 Effect of pH on Enzyme Activity 17

3.7.2 Effect of Temperature on Enzyme Activity 17

3.7.3 Effect of Incubation Time on Enzyme Activity 17

CHAPTER FOUR

4.0 Results 18

CHAPTER FIVE

5.0 Discussion, Conclusion and Recommendation 28

5.1 Discussion 29

5.2 Conclusion 30

5.3 Recommendation 30

References 31

LIST OF TABLES

Table           Title Page

1: Biochemical characteristics of LAB isolates. 21

LIST OF FIGURES

Figure Title        Page

1:  Sketch for serial dilution of Nunu sample  12

2: Occurrence of lab isolates from Nunu 22

3: Proteolytic activity of LAB isolates of Nunu 23

4: Effect of pH on protease activity of LAB isolates 24

5: Effect of incubation temperature on LAB isolates 25

6: Effect of incubation time on LAB isolates 26