PRODUCTION OF AMYLASE FROM FERMENTED UGBA USING MICROCOCCUS SPECIES

        TABLE OF CONTENTS

Title page                                                                                                                           i

Certification                                                                                                                    ii

Dedication                                                                                                                         iii

Acknowledgements        iv

Table of Contents         v

List of Tables                                                                                                                     viii

List of Figures         ix

Abstract         x

CHAPTER ONE

1.1 INTRODUCTION                                             1

1.2 Aims and Objectives                                                                                                     3

CHAPTER TWO

2.0 LITERATURE REVIEW         4

2.1 Amylase (History and Discovery)         4

2.1.1 Uses and Applications of Amylases                                                                          5

2.2 Microorganisms and Amylases         6

2.3 Production of Amylases         8

2.4 Fermented foods                                                                                                          10

2.5 Method of production of Ugba                                                                                   11

2.6 Microorganisms involved in Ugba Fermentation        12

2.7 Micrococcus species in Ugba        13

2.7.1 Industrial uses of Micrococcus         13

CHAPTER THREE

3.0 MATERIALS AND METHODS                                                                                   15

3.1 Materials and equipment                                                                                               15         

3.1.1 Source of Materials (Sample collection)           15

3.2 Samples/Media Preparation                       15

Isolation of Micrococcus                                                                                                16  

3.4 Characterization of isolate for identification of Micrococcus species                            18

3.5 Identification of isolates                                                                                                 19

3.5.1 Gram staining                                                                                                               19

3.5.2 Spore staining                                                                                                               19

3.6 Biochemical tests                                                                                                             19

3.7 Screening of isolate for enzyme production                                                                    21                  

3.8 Determination of Amylase activity                                                                                  22

3.8.1 Determination of temperature effect on enzyme production                                        23

3.8.2 Determination of pH effect on enzyme production                                                      23

CHAPTER FOUR

4.0 RESULTS                    24

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION             32

5.1 Discussion             32

5.2 Conclusion 33

5.3 Recommendation 34

REFERENCES                                                                                                                     35

APPENDIX                                                                                                                           39

LIST OF TABLES

Tables                                                        Titles                                                                                Pages

1              Morphological and cultural appearance of the isolate                               26                                       

2              Occurrence of Micrococcus specie in ugba                                                     28

3              Amylase activity                                                                                             29

4                       Amylase activity at different temperatures                                                    39

5                       Amylase activity at different temperatures                                                    40

LIST OF FIGURES

FIGURES                                       TITLES                                                                      PAGES

1              Serial dilution process                                                                                    17

2              Graphical presentation of amylase activities at different temperature           30

3              Graphical presentation of amylase activities at different pH                         31

ABSTRACT

Ugba is a fermented product gotten from the fermentation of African oil bean seeds (Pentaclethra macrophylla Benth). It is an important source of carbohydrate, protein and lipids. Microorganisms such as Micrococus roseus, Micrococus luteus, Leuconostoc mesenteroides, Staphylococcus epidermidis, Bacillus substilis, Bacillus megatarium, and Bacillus lichenifornis are predominant in its fermentation process where they secrete enzymes that break down the biomolecules of ugba. Micrococcus was aseptically isolated from ugba samples. 80% of the five different samples screened contain Micrococcus species. Iodine starch test colorimetric method was employed in the determination of amylase production by the isolate. Micrococcus was aseptically inoculated in a soluble starch solution and incubated for 48hours at different temperature and pH range, a test tube containing no Micrococcus was used as a control. After incubation, Micrococcus hydrolysed the starch by producing enzyme, the spectrophotometric absorbance of the blue black coloration was read; the reduction in starch activity as shown by the reduced absorbance relative to the control measured the enzyme activity signifying utilization and reduction in the starch incorporated in the media. Optimum production of amylase was seen at 60⁰C and pH value of 6. Temperature and pH had significant effect on the rate of enzyme “amylase” production by Micrococcus. The ability of the Micrococcus isolated in this study to break down starch shows its role in amylase production which breaks down the starch molecules in Ugba and supports the use of microorganisms as starter cultures in fermentation, breaking down of complex organic molecules and in enzyme production.

CHAPTER ONE

1.1 INTRODUCTION

Enzymes are the large biomolecules that are required for the numerous chemical inter-conversions that sustain life. They accelerate all the metabolic process in the body and carry out a specific task. They are biological substances or macromolecules that are produced by a living organism which acts as a catalyst in a chemical reaction. Almost all chemical reactions in chemical reactions in a biological cell need enzymes in order to occur at rates sufficient for life. They are highly selective catalyst which can greatly accelerate both the rate and specificity of metabolic reactions, which range from the digestion of food to the synthesis of DNA. Amylases are enzymes that break down starch or glycogen (Murthy et al., 2009).

Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells. Although amylase can be derived from several sources, including plants, animals and micro-organisms, once produced by microorganisms generally meet industrial demands (Thippeswamy et al, 2006). Currently, a large number of microbial amylases are available commercially, having almost completely replaced the chemical hydrolysis of starch in the starch-processing industry.

Amylases are classified based on how they break down starch molecules as thus α-amylase (alpha-amylase), ß-amylase (Beta-amylase), Amyloglucosidase (AMG); but microbial amylases contain a mixture of these amylases. In most cases the enzymatic process is inhibited by pH, temperature, media, high substrate and product concentration and also instability of the enzyme under repetitive or prolonged use Ramakrushna, et al., 1982.

Humans exploit microbial amylases for different purposes such as high fructose corn syrup preparation, Additives to detergents for removing stains, Saccharification of starch for alcohol production and brewing, they hydrolyse starch to a range of products such as glucose and maltose or to specific malto-oligosaccharide or mixed malto-oligosaccharides. They are employed in industries for different purposes; glucose and maltose-forming amylases in alcohol fermentation and sugar syrup formulation, and malto-oligosaccharide forming amylases in food processing (Ramakrushna et al., 1982).

Microbial amylases have successfully replaced the chemical hydrolysis of starch in starch-processing industries and are potentially useful in the pharmaceutical and fine chemical industries. Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger, Bacillus subtilis, B. cereus, B. polmyxa, B. amyloliquefaciens, B. coagulans, Lactobacillus, Escherichia, Proteus, B. lincheniformis, Bacillus steriothermophilu, Bacillus megaterium, Strepotmyces species., Pseudomonas species, Micrococcus species (Obeta, 1982). 

Traditionally, amylase is produced by submerged fermentation (SMF). In recent years, however, solid state fermentation (SSF) processes have been utilized more and more for the production of this enzyme.  It is known that commercial carbon sources such as glucose and starch are not advantageous for production of amylase since they are very expensive. Therefore, several investigators have described the utilization of cheap and easily available agricultural residues such as ugba, wheat straw, wheat bran, coffee waste, banana waste, potato peel and sugarcane bagasse as carbon source in the production of amylase (Pandey, et al., 2000).

Ugba is a fermented product gotten from fermentation of African oil bean seeds (Pentaclethra macrophylla benth). The oil bean seeds are largely composed of proteins, carbohydrates and oil. The ability to break down these major components of the seed is an important characteristic of organisms able to ferment the seed. Most organisms isolated from fermented ugba possessed these characteristics. Micrococcus and Bacillus species are known to break down the components of ugba. They secrete amylases to the outside of their cells to carry out extra-cellular digestion. When they have broken down the insoluble starch contained in ugba, the soluble end products such as (glucose or maltose) are absorbed into their cells (Frazier, 1976). Fermentation detoxifies the African oil bean seed with subsequent increase in nutrient availability and digestibility.

Reports by Mbajunwa, et al., (1998) indicates that micrococcus species do not play an active role during microbial fermentation of ugba. However, further work on spoilage association of ugba showed that the population of micrococcus species increased with increase in keeping time of ugba. This indicates the ability of micrococcus species to thrive in the alkalophilic environment while constituting as a spoilage organism to produce protease, amylase, or lipase able to utilize protein, carbohydrate or lipid content of ugba as source of nutrition (Njoku, et al., 1990).

AIMS AND OBJECTIVES

This work is aimed at the following

· Isolating Micrococcus from fermented ugba

· Producing amylase from fermented Ugba using Micrococcus specie.

· Optimizing the amylase production

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