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

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

CHAPTER ONE

1.0 INTRODUCTION

Proteases constitute one of the most important groups of enzymes both industrially and academically (Hnin et al., 2015). Proteases are the enzymes that hydrolyze proteins by addition of water across peptide bonds and catalyze peptide synthesis in organic solvents with low water.

Proteases are essential constituents of all forms of life on earth, including bacteria, fungi, actinomycetes, plant and animals. Reports also show that nearly 67% of proteases are of microbial origin (Wang et al., 2013) and there is a growing demand for industrial enzymes of microbial origin due to its relative ease in production and wide applications in different industrial processes. Agarwal et al., (2004) reported that proteases of microbial origin possess considerable industrial potentials due to fair biochemical diversity and wide applications in industries including; food industries, medical formulations, detergents and processes like waste treatment recovery of silver and resolution of amino acid mixtures. While proteases are one of the three largest groups of enzymes applied industrially, they are reported to account for about 60% of world sales of enzymes (Rajkumar et al., 2011). Proteases were initially classified into Endopeptidases and Exopeptidases. However the availability of structural and mechanistic information on these enzymes facilitated new classification schemes (Carlos and Judith, 2008).

Lactic acid bacteria (LAB) are a group of Gram-positive, non-spore forming, cocci or rod shaped, catalase negative and fastidious organisms, considered as 'Generally Recognized as Safe' (GRAS) organisms, they are generally associated with habitats rich in nutrients, such as various food products (milk, meat, vegetables), but some are also members of the flora of the mouth, intestine and vagina of mammals (Khalid 2011).

Alessandro et al., (2010) observed a key feature in Lactic Acid Bacteria that is their ability to synthesize porphyrin groups as heme, this makes lactic acid bacteria devoid of a “true” catalase and cytochromes when grown in laboratory growth media, which lack hematin or related compounds. The taxonomy of Lactic Acid Bacteria has been based on the Gram reaction and the production of lactic acid from various fermentable carbohydrates (Delphine et al., 2011). The classification of lactic acid bacteria into different genera is largely based on morphology, mode of glucose fermentation, growth at different temperatures, and lactic acid produced (Khalid, 2011). LAB has been exploited over the years for the production of fermented foods because of their ability to produce desirable changes in taste, flavour and texture. Ayad et al., (2004) observed that LABs are the most important groups of microorganisms in fermented foods and noted the consistent presence of LABs in dairy products.

Nunu is a natural milk from animals mostly cattle. It is a normal secretion from the mammary gland of females which is secreted during gestation in the udder (i.e. breast) and used during lactation to feed their young. According to Adebolu, (2007) natural milk is sterile from the animal body but soon receives an influx of microorganisms once out of the body. This is attributed to its rich nutritional composition which makes it fertile for microbial habitation and proliferation. Milk contamination can come from varied sources including air, soil, the handles and psychrotrophic microorganisms as external contaminants. Majorie and Kathlean, (2006) observed that many different species of microorganisms are found in Nunu some of which are potential pathogens but lactic acid bacteria represents a substantial percentage of the microflora of raw milk (Adebolu, 2007). This makes Nunu, natural milk a good source of lactic acid bacteria for use in this project work. Nunu is natural milk gotten from cow that is utilized as food often in combination with processed cereals like maize, sorghum, millet as Fura de Nunu, a delicacy of most part of northern Nigeria (Umoh et al., 1988). Fura the fermented cereal balls which is mixed with Nunu in the preparation of Fura de Nunu is reported to house diverse microorganisms including LABs (Inyang and Zakari, 2008). The fermentation is carried out by Lactic acid bacteria includes members of the genera Lactobacillus, Pediococcus, Streptococcus, Leuconostoc, and Enterococcus. The most commonly isolated specie is Lactobacillus plantarum (Olasupo et al., 1997) Okunye et al., (2015) observed some medical benefits of Fura de Nunu asserting it as a complete food containing carbohydrate, protein, fats, vitamins and minerals as well as enzymes and probiotics.

1.1 AIM AND OBJECTIVES

The aim of this project work is to assess the protease activity of crude enzyme from LABs isolated from Nunu.

The objective of the study includes the under listed;

· To isolate, characterize and identify LAB from Nunu (raw cow milk) sample.

· To evaluate the protease activities of the LAB isolates

· To determine the optimal conditions suitable for maximum production of protease enzyme by the LABs in terms of pH, temperature and incubation time.

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