ENZYMATIC POTENTIALS OF MICROORGANISMS FROM CASSAVA RETTING

ABSTRACT

The enzymatic potentials of microorganisms isolated from retting cassava were evaluated for the purpose of pectinase, cellulase and amylase activities. Cassava tubers of 12months old were collected from 3 different sources: 5 from Ahieke, 5 from Ndoru and 5 from Umuariga, they were handpeeled, cut into cylinders, washed, submerged into water and allowed to ret. After retting a dilution of the retted tubers were inoculated into different media plates: De-Man Rogosa Sharp agar, Sabouraud Dextrose agar, Nutrient agar, Mannitol salt agar and MacConkey agar and incubated at 300C for 3-5days. A total of 35 isolates was identified in the retting cassava samples which are bacteria 25(71.43%) and fungi 10(28.57%). The bacteria isolates identified include Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Lactobacillus spp and Proteus spp. While the 2 fungal isolates and 1yeast was identified which include Aspergillus niger, Rhodotorula spp and Saccharomyces cerevisiae respectively. The total viable count of the isolates in heterotrophic plate count, fungal plate count and lactic acid bacteria plate count were increasing as the retting hours increases from 1.94x106cfu/ml to 2.52x106cfu/ml, 1.823x106cfu/ml to 2.808x106cfu/ml and 1.712x106cfu/ml to 2.897x106cfu/ml respectively while coliform plate count and Staphylococcus plate count were decreasing from 2.014x106cfu/ml to 7.47x105cfu/ml and 2.131x106cfu/ml to 7.76x105cfu/ml respectively. The distribution of the isolates shows that Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Proteus spp dominated the beginning of the fermentation period while Lactobacillus spp, Saccharomyces cerevisiae and Aspergillus niger dominated the later stage of 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   that produced none of the enzymes. Some of the isolates were able to produce cellulase enzyme while some produce pectinase enzyme. Proteus spp neither produced cellulase nor pectinase. Antimicrobial susceptibility pattern of the bacteria isolates shows that Rifampicin and Gentamycin have a high level of sensitivity to all the isolates with 4(16.0%) and 5(20.0%) while chloramphenicol, Norfloxacin and amoxil have a high resistance to the isolates with 3(33.0%), 2(22.0%) and 2(22.0%) respectively. 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

List of tables ﾿ viii

List of figures ﾿ ix

Abstract ﾿ x

CHAPTER ONE

1.0 ﾿ Introduction ﾿ 1

1.1 ﾿ Aim and Objectives ﾿ 2

CHAPTER TWO

2.0 ﾿ Literature Review ﾿ 4

2.1 ﾿ Nutritional and Anti-Nutritional Properties ﾿ 4

2.2 ﾿ Cyanogenic Glycosides ﾿ 4

2.3 ﾿ Toxicity of Cyanogens ﾿ 9

2.4 ﾿ Diseases Associated with Cassava Toxicity ﾿ 10

2.4.1 ﾿ Tropical Ataxic Neuropathy (TAN) ﾿ 10

2.4.2 ﾿ Konzo ﾿ 11

2.4.3 ﾿ Hyperthyroidism ﾿ 11

2.4.4 ﾿ Spastic Paraparesis ﾿ 12

2.4.5 ﾿ Other Diseases ﾿ 12

2.5 ﾿ Cassava  Processing ﾿ 12

2.6 ﾿ Soaking (Retting)                                  ﾿ 13

2.7 ﾿ Fermentation ﾿ 13

2.7.1 ﾿ Solid State Fermentation (SSF) ﾿ 14

2.7.2 ﾿ Submerged Fermentation (SMF) ﾿ 14

2.8 ﾿ Enzymes Involved in Cassava Retting ﾿ 16

2.8.1 ﾿ Amylase ﾿ 17

2.8.2 ﾿ Pectinases ﾿ 19

2.8.3 ﾿ Microbial Linamarase (β-glucosidase) ﾿ 20

CHAPTER THREE

3.0 ﾿ Materials and Methods ﾿ 21

3.1 ﾿ Sample Collection ﾿ 21

3.2 ﾿ Retting Procedure ﾿ 21

3.3 ﾿ Sample Preparation for Microbial Enumeration ﾿ 21

3.4 ﾿ Microbial Population Studies ﾿ 22

3.4.1 ﾿ Media used                                                ﾿ 22

3.5 ﾿ Identification of Isolates ﾿ 22

 3.5.1 ﾿ Bacteria ﾿ 22

3.5.2 ﾿ Fungal identification ﾿ 26

3.6 ﾿ Determination of Activities of Microbial Enzymes During Cassava Retting ﾿ 26

3.6.1 ﾿ Production of Amylase enzyme ﾿ 27

3.6.2 ﾿ Production of Cellulases enzyme ﾿ 27

3.6.3 ﾿ Screening of Isolate for pectinase Activity ﾿ 27

3.7 ﾿ Antibiotic Susceptibility Tests ﾿ 28

CHAPTER FOUR

4.0 ﾿ Results ﾿ 29

CHAPTER FIVE

5.0 ﾿ Discussion, Conclusion and Recommendations ﾿ 40

5.1 ﾿ Discussion ﾿ 40

5.2 ﾿ Conclusion ﾿ 42

5.3 ﾿ Recommendations ﾿ 43

References ﾿

LIST OF TABLES

Table ﾿ Title ﾿          Page 

4.1.    ﾿ Identification and characterize of bacterial Isolate from retting cassava samples ﾿   31

4.2.    ﾿ Identification and characterize of Fungal Isolates ﾿   32

4.3.  ﾿ Total viable microbial count of retting cassava samples ﾿   33

4.4. ﾿ Distribution of Isolates from the retting cassava samples ﾿   36

4.5. ﾿ Percentages occurrence of isolates from the retting cassava samples ﾿   37

4.6. ﾿ Enzymatic Activities of Isolates ﾿   38

4.7. ﾿ Antimicrobial susceptibility of bacterial isolates from retting cassava sample ﾿   39

LIST OF FIGURES

Figure ﾿ Title ﾿        Page 

2.1: ﾿ Chemical Structures of Some Cyanogenic Glycosides ﾿ 6

2.2: ﾿ Location of Cyanogenic glycoside and Linamarase in the plant cell ﾿ 8