PROTEASE ACTIVITES OF LACTIC ACID BACTERIA ISOLATED FROM NUNU

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