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 223.5.1 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

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, 2003). 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 (Umeh et al., 2007). The tuber consist 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 Mings, 2006). The roots contains considerable quantities of antinutrients 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 who 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 this method, 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) (Achi and Akomas, 2006), and characteristic flavour developed through a pH decrease and organic acid production (Ampe et al., 1994). 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, 2006).

1.2 Aim 

The aim of this project work is to evaluate the enzymatic potentials of microorganisms from cassava retting.  

The objectives are:

i. To isolate microbes associated with cassava retting

ii. To evaluate the enzymatic potentials of microorganisms from cassava retting   

iii. To determine the antimicrobial susceptibility of the isolates associated with cassava retting. 

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