ENZYMATIC ACTIVITIES OF SPECIES OF MICROORGANISMS ASSOCIATED WITH CASSAVA RETTING

ABSTRACT

The enzymatic potentials of microorganisms isolated from retting cassava were evaluated for the purpose of cellulase and amylase activities. Cassava tubers of 12 months old were collected from 3 different sources: Ahieke, Ndoru, Umudike, Awawom and Umuariga in Abia State. They were hand peeled, cut into cylinders, washed, submerged into water and allowed to ret. After retting a dilution of the retted tubers was inoculated into different media plates: De-Man Rogosa Sharp agar, Sabouraud Dextrose agar, Nutrient agar, Mannitol salt agar and MacConkey agar. Thirty- Five (35) isolates were identified in the retting cassava samples which were bacteria 25(71.43%) and fungi 10 (28.57%). The bacterial isolates identified include Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Lactobacillus spp and Proteus spp while fungi isolated were Aspergillus niger, Rhodotorula spp and Saccharomyces cerevisiae. The total viable counts of the isolates increased as the retting hours increased. Lactobacillus spp and Saccharomyces cerevisiae dominated the later stage of the fermentation. Bacillus subtilis has the highest percentage occurrence 8(22.9%) while Rhodotorula spp has the lowest percentage occurrence 2(5.7%). The production of amylase enzyme was recorded with all the isolates with the exception of Staphylococcus aureus. Production of cellulase and pectinase enzymes was selective among the isolates. Results from this study shows that enzymes like amylase, pectinase and cellulase from microorganisms played an important role in retting of cassava tubers

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

Lists of Tables                                                                                                                        viii

List of Figures                                                                                                                         ix

Abstract                                                                                                                                   x

CHAPTER ONE

1.0  Introduction                                                                                                                     1

1.1 Aim and Objectives                                                                                                          2

1.2 Objectives                                                                                                                         2

CHAPTER TWO

2.0 Literature Review                                                                                                                         3

2.1 Fermentation                                                                                                                     3

2.2 Cassava Fermentation                                                                                                       4

2.2.1 Submerged Fermentation                                                                                              5

2.3 Physical and Biochemical Changes during Cassava Fermentation                                5

2.3.1 Physical Changes                                                                                                           5

2.3.1.1 Dry Matter                                                                                                                  5

2.3.1.2 Softening of the Roots                                                                                                6

2.3.2 Biochemical Changes                                                                                                    6

2.3.2.1 pH                                                                                                                               6

2.3.2.2 Titrable Acidity                                                                                                          7

 2.3.2.3 Organic Acid                                                                                                             7

2.3.2.4 Vitamins and Minerals                                                                                               7

 2.3.2.5 Fiber                                                                                                                           7

2.3.2.6 Sugar and Starch Content                                                                                           8

2.4 Microbiology of Cassava Fermentation                                                                           8

2.5 Cassava Retting                                                                                                                10

2.6 Microbial Community of Cassava Retting                                                                       11

2.7 Enzymes Involved In Detoxification and Root Softening During Cassava Retting          13

2.7.1  Amylase                                                                                                                        13

2.7.2 β-Amylases                                                                                                                    14

2.7.3 Hemicellulases                                                                                                               14

 2.7.4 Pectinases                                                                                                                     16

CHAPTER THREE

3.0 Materials and Methods                                                                                                     17

3.1  Sample Collection                                                                                                           17

3.2 Media Used                                                                                                                       17

3.3 Sterilization                                                                                                                      17

3.4 Sample Processing and Retting Procedure                                                                       17

3.6 Microbial Enumeration and Analysis                                                                               18

3.7 Identification and Characterization of Isolates                                                                 18

3.7.1 Gram staining                                                                                                                           19

3.7.2 Motility Test                                                                                                                  19

3.8 Biochemical and Cultural Characteristics                                                                        20

3.8.1 Catalase test                                                                                                                   20

3.8.2 Coagulase Test                                                                                                               20

3.8.3      Citrate Test                                                                                                                 20

3.8.4 Indole Test                                                                                                                     21

3.8.5 Methyl Red Test                                                                                                            21

3.8.6 Oxidase Test                                                                                                                  21

3.8.7 Urease Test                                                                                                                    22

3.8.8 Voges-proskaeur Test                                                                                                    22

3.8.9 Hydrogen Sulphide Test                                                                                                23

3.8.10 Sugar Fermentation Test                                                                                             23

3.9 Identification of Fungal Isolates                                                                                       24

3.11 Determination of Activities of Microbial Enzymes during Cassava Retting                        24

3.11.1 Screening for Enzyme Production                                                                               24

3.11.2 Production of cellulase enzyme                                                                                   25

3.11.3 Screening of isolates for the pectinase activity                                                           25

CHAPTER FOUR

4.0 Results                                                                                                                              26

CHAPTER FIVE

5.0  Discussion, Conclusion And Recommendation                                                              36

5.2 Conclusion                                                                                                                        38

5.3 Recommendation                                                                                                              38

LIST OF TABLES

Table                        Title                                                                Page

1.         Identification and characterize of bacterial Isolate from retting cassava samples          27

2.         Identification and characterization of fungal isolates                                                 29

3.         Total viable count                                                                                                       31

4.         Percentages Occurrence of bacteria Isolates from the Retting Cassava Samples        33

5.         Enzymatic activities of isolates from cassava retting (mm)                                       35

 CHAPTER ONE

1.0    INTRODUCTION

Cassava (Manihot esculenta crantz) is a potential shrub with an edible starchy root, which grows in the tropical and sub-tropical areas of the world (Burell, 2013). It is one of the staple foods consumed in Africa and other parts of the world. It was estimated that the crop provides about 40% of all the calories consumed in Africa and ranks second only to cereal grains as the chief source of energy in Nigerian diet (Roger , 2014). The tuber consists of 64-87% starch depending on the stage of the growth or maturity of the tuber but very limited quantities of protein, fats, vitamins, and minerals (Alloys and Ming, 2016). The roots contain considerable quantities of anti-nutrients factors, cyanogenic glucoside. The cyanogenic potential of cassava is by far the single factor that adversely constraints the use of cassava as food and feed for animals. This is as a result of the toxic effect of cyanide on humans and animals that rely on cassava as food. Cassava has bitter and sweet varieties. The presence of cyanogenic compounds which predominates in bitter varieties and processes to reduce them were recently reviewed by (Montagnac et al., 2009).

Different processing techniques are used to reduce cassava toxicity and selected antinutrients such as, boiling, drying, steaming, baking, frying, soaking, fermentation, steam distillation, etc. Fermentation is the common method of cassava processing and through it, cassava can be processed into different food products such as Fufu, garri, Lafun, chikwangue, etc. Cassava retting (fermentation) is a technique involving long soaking of cassava roots in water to affect the breakdown of tissues. Retting is one of the simplest and lactic acid fermentation process for the processing of cassava tubers into various African staple foods. It simply involves steeping of cassava roots in water until they soften. However, this takes about three to four days under optimal condition. In other conditions retting may take considerable longer for example, tubers older than 24months or during the colder seasons of the year. During the consequent fermentation, roots are softened by the activities of microorganisms producing various enzyme, the endogenous cyanogenic glycosides (linamarin and lotaustralin) are subsequently hydrolyzed to glucose and cyanohydrins, which easily break down to ketone and hydrogen cyanide (HCN)  and characteristic flavour developed through a pH decrease and organic acid production (Ampe  and Brauman, 2014). The fermentation process (retting) is characterized by the activities of certain microorganisms which produces enzyme such as pectinases, amylase, etc resulting in the breakdown of cassava tissues. The presence of unspecified microorganisms complicates the control of the fermentation process and lead to the production of objectionable odours (Achi and Akomas, 2016)

1.1 AIM AND OBJECTIVES

The aim of this study is to determine microorganisms associated with cassava retting and their enzymatic activities.

1.2 OBJECTIVES

1.  To isolate microorganisms associated with cassava retting.
2. To identify and characterize microorganisms associated with cassava retting.
3. To determine the enzymatic potentials of microorganisms associated with cassava retting.

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