PRODUCTION OF AMYLASE BY MICROCOCCUS SPECIES ISOLATED FROM FERMENTED UGBA

ABSTRACT

In this study, Micrococcus species have been implicated as microbial population of fermented African oil bean seed ‘ugba’. The organism was catalase positive and could not utilize citrate and was coagulase and methyl red negative. Micrococcus isolates were screened for amylase enzyme production using the activity zone technique with iodine solution. Micrococcus sp was able to produce enzyme amylase and therefore could utilize the carbohydrate content of ugba. The quantity of amylase produced were relatively very small (0.824 IU/ml, 1.412 IU/ml, 1.427 IU/ml) which points that African oil bean seed contains relatively small amounts of carbohydrate. However, the enzyme producing ability of Micrococcus confirms that it plays a role in ugba fermentation and could contribute to its taste and flavor. Proper handling of ugba or a modern attempt in production may impend the participation of Micrococcus species in ugba spoilage and enzyme activity.

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

1.0 Introduction                                                                                                          1

1.1 Aims and Objectives                                                                                             3

Chapter two

2.0 Literature Review                                                                                                 4

2.1 Fermented Foods                                                                                                  4

2.2 Pentaclethramacrophylla                                                                                      5

2.3 Uses and Application of Pentaclethramacrophylla                                              6

2.4 Methods of Production                                                                                         8

2.4.1 Traditional Method                                                                                            8

2.4.2 Laboratory Method                                                                                            9

2.5 Microorganisms Involved in Ugba Fermentation                                                 9

2.6 Micrococcus Spp                                                                                                   11

2.7 Description and Significance of Micrococcus                                                      12

2.8 Genome Structure of Micrococcus                                                                       12

2.9 Cell Structure and Metabolism of Micrococcus                                                   13

2.10 Ecology of Micrococcus                                                                                     15

2.11 Pathology of Micrococcus                                                                                  15

2.12 Pathogenesis of Micrococcus                                                                              16

2.13 Industrial Uses of Micrococcus                                                                          16

2.14 Amylase                                                                                                              17

2.15 Industrial Applications and Uses of Amylase                                                    17

2.16 Microorganisms Involved in Amylase Production                                             18

Chapter Three

3.0 Materials and Methods                                                                                         19

3.1 Study Area                                                                                                            19

3.2 Sample Collection                                                                                                 19

3.3 Production of Ugba                                                                                              19

3.4 Preparation and Sterilization of Media                                                                 20

3.5 Isolation of Micrococcus spp from Ugba                                                             20

3.6 Identification of Isolate                                                                                        20

3.7 Gram Staining                                                                                                       21

3.8 Biochemical Tests                                                                                                 21

3.9 Screening of Micrococcus spp for Amylase Production                                       23

3.10 Assay of Amylase Enzyme.                                                                                24

Chapter Four

4.0 Results                                                                                                                  25

4.1 Identification of The Isolate                                                                                 27

4.2 Growth and Amylase Production                                                                         27

Chapter Five

5.1 Discussion                                                                                                             28

5.2 Conclusion                                                                                                            29

     References

LIST OF TABLES

Table                                                  Title                                                                Page

  1        Medicinal uses of Pentaclethramacrophylla                                                   7

2.1       Characteristics of the Micrococcus spisolate.                                                25

2.2       Amylase activities of Micrococcus spisolated from ugba.                              26

LIST OF FIGURES

Figure                                                 Title                                                                Page

1                                  African Oil Bean Seed                                                            6

2                                  Processes Involved in Traditional Ugba Production              8

3                                  Tetrad arrangement of Micrococcus                                       14

4                                  Pigmented colonies of Micrococcus.                                      14

CHAPTER ONE

1.0  INTRODUCTION

Fermented foods whether from plants or animal origin, are an intricate part of the diet of people in all parts of the world. Fermented products remain of interest since they do not require refrigeration during distribution and storage. The traditional condiments have not attained commercial status due to the very short life, objectionable packaging materials, stickiness and the characteristic putrid odor (Oyedeji and Ijigbade, 2016).

Nigerian indigenous fermented foods constitute a group of foods that are produced in homes, villages and small-scale cottage industries. The fermented foods are derived from substrates like roots, legumes, cereals, oil seeds, nuts, meat, fish, milk, palm tree, sap, etc. (Iwuoha and Eke1996).These various plant seeds that are fermented and used as food in some rural parts of Nigeria, are: ‘iru’ from African locust bean (Parkiabiglobosa), ‘ogiri’ from castor bean (Ricunuscommunis), ‘okpei’ from mesquite seed (ProsopsisAfricana) and ‘ugba’ from African oil bean (Pentaclethramacrophylla) (Enujiugha, 2009).

Ugba(orUkpaka as it is called in Igbo language) popularly called African salad, is a ready-to-eat food which is produced by the fermentation of African oil bean seeds (Pentaclethramacrophylla). African oil bean seed is from a woody plant predominant in the rain forest areas of West and Central Africa belonging to the family Leguminosaea, sub-family Mimosoidae (Anyanwu et al., 2016).As an important nutritional item, Ugba is very rich in protein. The production of ugba is usually produced as a family business that has become an art that is handed over from one generation to another (Olasupoet al., 2016). It is an essential food item for various traditional ceremonies where it is mixed with slices of boiled stock fish (Ugba and Okpoloko). The natural fermentation of the seed which at present is still done at the household level, renders the production nutritious, palatable and non-toxic (Oyediji and Ijigbade, 2016).

Microbial population of Ugba is introduced through the air, water, utensils, banana leaves or the handler. No starter culture is used for the traditional method. Microorganisms involved are predominantlyBacillus, Micrococcus and Lactobacillus.  Other organisms isolated from Ugba includes Pseudomonas, Staphylococcus,            Enterobacters, Lueconostoc, CorynebactriumandAlkalegenes(Nwaguet al., 2011).These organisms utilize some of the nutrients inherent in the seed while their metabolic activities detoxify the seed, soften it and also produce flavor and aroma compounds which impact the characteristic tastes and flavor (Nwagu and Amadi, 2013).

Micrococcus spdo not play active role during microbial fermentation of Ugba (Nwaguet al., 2011). Bacillus and Micrococcus spp (now Kocuriarosea) were especially found to persist during the spoilage of Ugba, highly alkaline and unfavourable conditions notwithstanding. And was able to utilize soluble starch and Bambara flour for their growth and metabolic activities. The secretion of amylase (A primary metabolite) was possibly induced in the presence of starch sources which had to be hydrolyzed to simple sugar prior to their assimilation into the cell (Nwagu and Amadi, 2013).

Carbohydrates are the major nutrient content in Ugba and the ability to produce hydrolytic enzymes signifies that Micrococcus spp can break down these complex polymers to simpler units in order to assess nutrients for their growth and metabolism (Nwagu and Amadi,2013).

Amylase is a hydrolyzing enzyme in function which causes hydrolyses of molecules. In biotechnology, amylases are the most important enzymes used. The main use of amylase includes hydrolysis of starch to yield glucose syrup, amylase rich flour and in the formation of dextrin during backing in food industries (Vermaet al., 2011). Amylase from microbial sources can be produced in large quantities to meet the high industrial and market demands, and the diversity of microbes as the source for bio-based products is gaining ground every day (Adeoyoet al., 2016).The interest in microbial amylase production is increasing because of its economical bulk potentials in a wide range of industrial applications and additives in food processing.

1.1 AIMS AND OBJECTIVES

        i.            To isolate Micrococcus spfrom fermented ugba.

      ii.            To assess the growth and amylase production of Micrococcus spfrom fermented Ugba.

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