LIPASE PRODUCTION OF MICROORGANISM ISOLATED FROM FERMENTED UGBA

ABSTRACT

The use of starter cultures have generally been recognized as one major way of ensuring product consistency and to a reasonable extent eliminate the problem of food-borne pathogens. The aim of this study was to assess the lipase production of microorganisms isolated from (Pentaclethra macrophylla) fermented ugba. One (1) gram of the sample was thoroughly mashed with laboratory pestle and mortar and mixed with 9 ml of normal saline water as diluents in a McCartney bottle and the content was thoroughly shaken. 1 ml of solution from preceding concentration to 9 ml of diluents, was added using sterile syringe. After series of biochemical characterization the following genera of microorganisms were observed E.coli, Staphylococcus aureus, Proteus sp, Bacillus sp and Klebsiella sp. The occurrence of microorganism’s species in Ugba samples. The result shows that Bacillus subtilis 100%, Proteus 100% had the highest occurrence followed by E.coli 66.7%, S.aureus 66.7% while Klebsiella sp 33.3% had the lowest occurrence. The result of Lipase activity of the Bacillus isolates. Isolated from the rancid palmoil samples. The results show that all the isolates produced extra cellular Lipase enzyme but to varying extracts. The ability of these isolates from Ugba imply great potentials on waste oils in view of the potential application of these enzymes. Lipase are used in industry to hydrolyze fats and to produce many desirable esters. Certain environmental factors such as pH, temperature, and incubation time and aeration rate were observed to play a major role during enzyme production and metabolic activities.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgments                                                                                                                  iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii

CHAPTER ONE

1.0 INTRODUCTION                                                                                                          1

1.1 Aim and Objectives                                                                                                          2

1.9.1 Objectives                                                                                                                      3

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Lipase Production                                                                                                             4

2.2.1 General Description of Lipases                                                                                         6

2.2.2 Lipase as Biocatalysts                                                                                                      6

2.2.3 Bacterial Lipases                                                                                                              7

2.2.3.1 Fermentation Conditions                                                                                               9

2.2.3.2 Purification of Bacterial Lipases                                                                                    9

2.2.4 Properties of Lipases                                                                                                        10

2.2.5 Yeast Lipases                                                                                                                 12

2.2.6 Application and Uses of Lipases                                                                                   14

2.7 Bacillus Lipases                                                                                                                15

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Sample Collection                                                                                                            16

3.2 Media Used                                                                                                                       16

            3.3 Sterilization                                                                                                                      16

3.4 Microbiological Analysis                                                                                                 16

3.5 Identification of Bacteria Isolates                                                                                    17

3.6 Characterization of Isolates                                                                                              17

3.6.1 Gram Staining                                                                                                               17

3.6.2 Motility Test                                                                                                                  18

3.7 Biochemical and Cultural Characteristics                                                                                    18

3.7.1 Catalase Test                                                                                                                  18

3.7.2 Coagulase Test                                                                                                              18

3.7.3 Citrate Test                                                                                                                    19

3.7.4 Indole Test                                                                                                                     19

3.7.5 Triple Sugar Iron Test                                                                                                   19

3.7.6 Oxidase Test                                                                                                                  20

3.7.7 Urease Test                                                                                                                    20

3.8 Screening of Isolates for Lipase Production                                                                           20

CHAPTER FOUR

4.0 RESULTS                                                                                                                        22

CHAPTER FIVE

5.0 DICUSSION, CONCLUSION AND RECOMMEDNATION

5.1 Discussion                                                                                                                         27

5.2 Conclusion                                                                                                                        30

5.3 Recommendation                                                                                                             30

Reference                                                                                                                                31

LIST OF TABLES

Table                     Title                                                                Page

1                                                   Morphology and Biochemical Identification of isolates                    23     

2                                                   Occurrence of Isolates in Ugba                                                24     

3                                                   Test for Iipase Activity of Microorganisms Isolates from Ugba             25

4                                                   Influence of Incubation time on Iipase Activity of Microorganisms Isolated from Ugba                                                         26

CHAPTER ONE

1.0 INTRODUCTION

 Ugba is a product of alkaline fermentation of oil bean (Pentaclethra macrophylla) seeds which are utilized by the Ibos and other ethnic groups in southern Nigeria as a delicacy and food flavoring. It constitutes an important nutritional contribution mainly as a source of protein and   plays an economical, social and cultural role among the Ibos in the eastern part of Nigeria.  Published investigations on the biochemical changes during Ugba fermentation have shown that proteolysis is the main activity leading to a pronounced increase of free amino acids (FAA) such as lysine (Odunfa and Oyeyiola, 2015; Njoku and Okemadu, 2009).

Microbiology of the fermentation of African oil bean seeds have identified Bacillus species as the main micro-organisms responsible for its fermentation. The predominant species is Bacillus subtilis, but other species like B. pumilus, B. megaterium, B. lichenformis have also been found (Ogueke and Aririatu, 2004). Ogueke and Aririatu (2004) noted that the Bacillus species and Proteus species are proteolytic and dominate during the fermentation process and therefore are responsible for the observed increased free amino acids recorded during production of the product. Though proteolysis have generally been observed to be the main activity during the production of Ugba and the Bacillus species identified with this proteolytic activity, no detailed screening of the various fermenting organisms of Ugba for protease enzyme production have been reported in literature.  Most fermented foods in Nigeria are produced at household level and hygiene is a major concern. Their fermentation process is usually by spontaneous culture method where the inoculum is by chance. This has almost always led to the problem of inconsistent product quality and other attendant problems. The problem of occurrence and growth of pathogens in most of these fermented food products cannot be ruled out as the general hygienic conditions of the processors, the equipment used, water and other raw materials cannot be said to be free of potential pathogens.

The use of starter cultures have generally been recognized as one major way of ensuring product consistency and to a reasonable extent eliminate the problem of food-borne pathogens (Eman, 2009). Unfortunately, however, no lactic acid bacteria (LAB) starter cultures are commercially available yet for small scale processing of traditional African foods. The potential of starter cultures for fermentation on a household scale for most of our traditionally fermented foods has not yet been fully explored. A starter culture is applied to improve a fermentation process, be it a lactic, alcoholic or the other types of fermentation. The old tradition of using a portion of a fermented product to start a new batch resembles the principle of starter cultures in an empirical sense. However, most commercial starter cultures originated from those food substrates to which they are applied today.  The oil bean seed is mainly composed of proteins 42, lipids 43 and carbohydrates 15% (Odunfa and Oyeyiola, 2015; Njoku and Okemadu, 2009; Ogueke and Aririatu, 2004).  Selection of any organism(s) as starter culture(s) for the production of Ugba will therefore depend on the ability of such organism(s) to degrade these major components of the oil bean seed. Degradation of carbohydrate, lipid and protein requires that the organism must possess the capacity to produce amylase, lipase and protease enzymes which are required for carbohydrate, lipid and protein metabolism respectively.

1.1 AIM AND OBJECTIVES

The aim of this study is lipase production of microorganism’s isolated from (Pentaclethra macrophylla) fermented ugba.

1.2 Objectives

1.     To isolate and identify microorganisms from (Pentaclethra macrophylla) fermented ugba.

2.     To determine lipase producing microorganisms isolated from (Pentaclethra macrophylla) fermented ugba.

3.     To determine the biochemical changes of microorganisms isolated from (Pentaclethra macrophylla) fermented ugba.

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