1

UGWU, FRANCA NWAKAEGO

MICROBIAL AND BIOCHEMICAL CHARACTERISATION OF

CASSAVA RETTING PROCESS FOR FUFU PRODUCTION

BIOLOGICAL SCIENCES

MICROBIOLOGY

MADUFOR, CYNTHIAC.

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

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TITLE PAGE

MICROBIAL AND BIOCHEMICAL

CHARACTERISATION OF CASSAVA

RETTING PROCESS FOR FUFU

PRODUCTION

BY

UGWU, FRANCA NWAKAEGO

PG/M.Sc/05/39479

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

SEPTEMBER, 2012

3

CERTIFICATION

Ugwu, Franca Nwakaego, Reg. No. PG/M.Sc/05/39479, a postgraduate student

in the Department of Microbiology, has satisfactorily completed the

requirements for the Degree of Master of Science [M.Sc] in Microbiology

(Food). The work embodied in this dissertation is original and has not been

submitted in part or in full for any other diploma or degree of this or any other

University.

……………………………. ……………………………

Prof. (Mrs.) I.M. Ezeonu Prof. J.O. Ugwuanyi

Head Supervisor

Department of Microbiology Department of Microbiology

University of Nigeria, Nsukka University of Nigeria, Nsukka

……………………………

Prof. A.N. Moneke

Supervisor

Department of Microbiology

University of Nigeria, Nsukka

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DEDICATION

This research project is dedicated to Almighty God for

His extravagant Grace, to my beloved husband,

Mr. Ugwuoke Chima and my little angel Ugwuoke Amarachukwu.

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ACKNOWLEDGEMENT

One tree cannot make a forest. A lot of people have in one way or the

other contributed immensely to my academic attainment and deserves special

recognition.

My immense thanks to my supervisors: Prof. J.O. Ugwuanyi and Prof.

A.N. Moneke, who through their constructive criticisms and thoughtful advice

shaped my ambition into a more concrete one. I am also grateful to Dr. E.A. Eze

for his noble advice and friendship in the course of this research. I would not

forget the hand that fed me during the drought season; therefore my heartfelt

thanks and appreciation go to Dr. F.S. Ire, who rendered his unreserved

technical assistance. I am also grateful to Mr. C.I. Nnamchi for his concern.

My special gratitude goes to my beloved parents, Mr. and Mrs. Gabriel

Ugwu (Ikuku 230) for their wonderful parental advice, support and

encouragement. I am also indebted to my brothers and sisters: Bar. Queen

(Esq), Engr. Ugochukwu, Engr. Maxwell, Pharmacist Ernest, Ogochukwu and

master Kaosisochukwu for their unconditional love.

Thanks to my invaluable friends and colleagues; Doris Onyeka,

Rosemary Nwonah, Nkechinyere Ejike, Ikechukwu Eze (Bishop), and Pat.

Onyeacholam, for their understanding and encouragement.

Finally, I have every cause to feel particularly grateful to my lovely

husband and friend Chima Ugwuoke for lending me his shoulder to lean on.

Conclusively, to God be the glory. His grace is sufficient for me. I

worship you, Lord.

Franca Nwakaego Ugwu

September, 2012

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TABLE OF CONTENT

CONTENT PAGE

COVER PAGE

TITLE PAGE - - - - - - - - - i

CERTIFICATION - - - - - - - - ii

DEDICATION - - - - - - - - - iii

ACKNOWLEDGEMENT - - - - - - - iv

TABLE OF CONTENT - - - - - - - - v

LIST OF TABLES - - - - - - - - x

LIST OF FIGURES - - - - - - - - xii

ABSTRACT - - - - - - - - - xiii

CHAPTER ONE

1.0 Introduction - - - - - - - 1

1.1 Literature Review - - - - - - - 4

1.1.1 Overview on Cassava (Manihot Esculenta Crantz) - - 4

1.1.1.1 Botany and Cultivation - - - - - - 5

1.1.1.2 Pest and Diseases - - - - - - - 6

1.1.1.3 Economic Importance - - - - - - - 7

1.1.1.4 Composition - - - - - - - - 9

1.1.2 Toxic Compounds in Cassava - - - - - 10

7

1.1.2.1 Classification of Cassava Based on its Cyanogenic Potentials - 14

1.1.2.2 Measurement of Cyanogenic Compounds - - - 15

1.1.2.3 Toxic Effects and Diseases Associated with Cyanide Exposure- 16

1.1.2.4 Diseases Related to Cassava Toxicity - - - - - 18

1.1.3 Processing into Different Products - - - - - 21

1.1.3.1 Cassava Leaf Consumption - - - - - - 23

1.1.4 Cassava Processing Techniques - - - - - - 24

1.1.5 Fermentation - - - - - - - - 27

1.1.5.1 Physical and Biochemical Changes during Cassava Fermentation 30

1.1.5.2 Retting - - - - - - - - - 33

1.1.5.3 Microbial Community of Cassava Retting - - - 34

1.1.5.4 Enzymes Involved In Detoxification and Root Softening during

Cassava Retting. - - - - - - - 36

1.1.5.4.1 Amylase - - - - - - - - - 37

1.1.5.4.2 Hemicellulases: - - - - - - - - 39

1.1.5.4.3 Pectinases - - - - - - - - 40

1.1.5.5 Fermentation Modulation - - - - - - 41

CHAPTER TWO

2.0 Materials and Method - - - - - - 43

2.1 Plant Material - - - - - - - - 43

2.2 Retting Procedure - - - - - - - 43

2.3 Sample Preparation - - - - - - - 43

8

2.4 Microbiological Population Studies - - - - 44

2.4.1 Media Used - - - - - - - - 44

2.4.2 Sub-Culturing - - - - - - - - 44

2.5 Identification - - - - - - - - 45

2.5.1 Bacteria - - - - - - - - - 45

2.5.1.1 Catalase Test - - - - - - - - 46

2.5.1.2 Indole Tests - - - - - - - - 46

2.5.1.3 Citrate Utilization Test: - - - - - - 46

2.5.1.4. Potassium Cyanide Test - - - - - - 47

2.5.1.5 Carbohydrate Fermentation Test - - - - - 47

2.5.2 Motility test - - - - - - - - - 48

2.6 Fungal Identification - - - - - - - 48

2.6 Determination of Time Profile of Enzyme Activities in

Cassava Retting- - - - - - - 49

2.6.1 Amylase Activity Assay - - - - - - 49

2.6.2 Pectinase Activity - - - - - - - 49

2.6.2.1 Pectin Lyase Activity Assay - - - - - - 50

2.6.2.2 Polygalacturonase Activity Assay - - - - - 50

2.6.2.3 Pectinesterase Activity Assay - - - - - 51

2.6.3 Cellulase Activity - - - - - - - 51

2.7 Physiochemical Parameters - - - - - - 51

2.7.1 pH - - - - - - - - - - 51

9

2.7.2 Titrable Acidity - - - - - - - - 52

2.7.3 Cyanide Content - - - - - - - 52

2.8 Fermentation Modulation - - - - - - 52

2.8.1 Effect of Temperature - - - - - - - 53

2.8.2 Effect of Cations - - - - - - - 53

2.8.3 Effect of Added Chemical Agents - - - - - 54

2.9 Strength of Cassava Cuttings - - - - - - 54

CHAPTER THREE

3.0 Results - - - - - - - - - 56

3.1 Microbial Counts - - - - - - - 56

3.2 Evolution of Microbial Flora during Cassava Retting - - 73

3.3 Percentage Isolates on Respective Media - - - - 75

3.4 Determination of Time Profile of Enzymatic Activities in Cassava

Retting - - - - - - - - - 82

3.4.1 Effect of Enzymatic Activities on Cassava Retting Time - - 82

3.4.2 Effect of Pectinases Activities - - - - - - 82

3.5 Effect of pH , Titrable Acidity (%) And Cyanide Content on Cassava

Retting - - - - - - - - - 85

3.6 Effect of Cassava Cuttings on Retting Time - - - 89

3.7 Fermentation Modulation - - - - - - 91

3.7.1 Effect of Temperature on Cassava Retting - - - 91

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3.7.2 Effect of Cations on Cassava Retting - - - - 91

3.7.3 Effect of Added Chemical Agents on Renting Time - - 95

3.7.3.1 Effect of Temperature(s) on Cassava Retting Time in the Presence

of Chemical agents - - - - - - - - 95

3.7.3.2 Effect of Added Chemicals on Daily pH during Cassava Retting 95

CHAPTER FOUR

4.0 Discussions and Conclusions- - - - - 98

4.1 Discussion - - - - - - 98

4.2 Conclusion - - - - - - - 106

References - - - - - - - - - 112

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LIST OF TABLES

Table 1: Microbial Count (C.F.U/Ml) on Potato Dextrose agar (PDA)- 57

Table 2: Microbial Count (C.F.U/Ml) On De Mann, Rogosa and

Sharpe agar (MRS) - - - - - - - 58

Table 3: Microbial Count (C.F.U/Ml) On Violet Red Bile Glucose agar

(VRBG)- - - - - - - - - 59

Table 4: Microbial Count (C.F.U/Ml) On Plate Count agar (PCA) - 60

Table 5: Morphological, Physiological, Biochemical and Sugar Fermentation

Characteristics of Isolates on Plate Count agar (PCA) - 61

Table 6: Morphological, Physiological, biochemical and fermentation

characteristics of Isolates on de Mann, Rogossa and Sharpe

(MRS) - - - - - - - - 64

Table 7: Morphological, Physiological, biochemical and sugar fermentation

characteristics of isolates on VRBG agar - - - - 68

Table 8: Identification of Fungal Isolates - - - - - 72

Table 9: Percentage occurrence of isolates on Plate Count agar (PCA) 75

Table 10: Percentage occurrence of Isolates on Violet Red Bile

Glucose agar (VRBGA) - - - - - - 76

Table 11: Percentage occurrence of Isolates on de Mann, Rogosa &

Sharpe agar (MRs agar) - - - - - - 77

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Table 12: Percentage occurrence of Isolates on Potato Dextrose agar (PDA)78

Table 13: Percentage occurrence of Bacterial Isolates - - - 79

Table 14: Percentage occurrence of Fungal Isolates - - - 80

Table 15: Microbial Succession at the different Fermentation Periods- 81

Table 16: Size of Cassava cuts on Retting time- - - - 90

Table 17: Effect of Temperature on Cassava retting time - - 92

Table 18: Effect of Added Cations on Cassava Retting Time - - 93

Table: 19 Cationic Contents of Cassava Tubers Before and After

Soaking - - - - - - - - 94

Table 20: Effect of Temperature(s) on Cassava retting time in

the presence of Chemical agents- - - - - 96

Table 21: Effect of Added Chemicals on the daily pH of samples

during Cassava Retting - - - - - - 97

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LIST OF FIGURES

Fig 1 Profile of Enzymatic Activities during Retting - - - 83

Fig. 2: Profile of Pectinase Activities - - - - - - 84

Fig. 3: Evolution of pH during Cassava Retting - - - - 86

Fig. 4: Evolution of Titrable Acidity during Cassava Retting - - 87

Fig. 5: Cyanide Content during cassava retting - - - - - 88

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ABSTRACT

A total of 27 isolates tentatively identified in the retting of cassava were bacteria

18 (66.7%) and fungi 9 (33.33%). Bacterial isolates identified comprised of

Shigella sonnei (4.79%), Escherichia coli (9.59%), Enterobacter spp (19.18%),

Klebsiella pneumoniae (11.64%), Lactobacillus spp (30.82%), Streptococus spp

(7.53%), Bacillus spp (9.59%), and Leuconostoc mesenteroides (6.85%). Fungal

isolates comprised of Aspergillus spp (47.69%), Fusarium spp (7.69%), Mucor

spp (6.15%), Penicillium spp (12.31%), Candida spp (20.00%), and Rhizopus

spp (6.15%). Microbial succession was determined by the sensitivities of

microorganisms to the very acidic conditions that developed during the retting

process. The enzymatic activities studied showed that there was decrease in

cellulolytic activities, decrease in amylolytic activities after 48h and increase in

pectinase activities. Significant activities of deploymerizing enzymes; pectin

lyase (PL) and polygalacturonase (PG) was detected after 24h of retting, while

there was increase in pectinesterase (PE) actitivites. The trend in pH values was

opposite to the titrable acidity. pH decreased from 6.52 to 4.52, while the

titrable acidity increased from 0.010% to 0.054%. The cyanide content of the

cassava roots decreased progressively during the retting process from 1.4 x 103

to 3.1 x 102

mg/kg. This therefore indicates detoxification. The smaller the cut

size, the shorter the retting time. Optimum retting temperature of 350C -40

0C

was observed. Addition of cations (Ca2+

and Mg2+

), and chemical agents

15

(kerosene and nail) effectively reduced the retting time. Results from this study

suggest that microorganisms, pH and most importantly temperature are the

major factors affecting cassava retting.

CHAPTER ONE

4.0 INTRODUCTION

Cassava, Manihot esculenta Crantz, is an important root crop in Africa, Asia,

South America and India (Padmaja, 1995). Cassava is a staple food for at least

500 million people in the tropics (Bradbury, 2006) that provides carbohydrates,

or energy. It counts as a higher producer of carbohydrates per hectare than the

main cereal crops and can be grown at a considerably lower cost (Taiwo, 2006).

The percentage of dry mater (starch content) in the harvested root is an

important criterion of quality both for human consumption and for processing

uses. 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 & Mings, 2006).

Additionally, the roots contain considerable quantities of antinutrient factor

cyanide. Cyanides occur in cassava in the form of two cyanogenic glucosides,

linamarin and a small amount of methyllinamarin – lotaustralin – located

inside the plant cells together with a specific hydrolytic enzyme, linamarase,

located in the cell wall (Bradbury, 2006). However, under normal conditions,

16

they are separated from the substrate. Any process that ruptures the cell walls

will bring the enzymes into contact with the glycosides and will thus release

free cyanide and reduce the glycosides‟ content of the final product. Based on

the amount of cyanides in the cassava roots, cassava has been classified into

“bitter” – and “sweet” – cassava. Thus the sweet varieties can be eaten boiled

while the bitter varieties have to be processed before it can be consumed

(Tewe,1992).

The various fermentation processes have been broadly categorized into

submerged fermentation process, which involves the soaking of the roots under

water as in fufu production (retting), and the solid fermentation process, which

does not involve soaking as in the gari production (Oyewole, 2001).

Traditionally, cassava roots are processed in a number of ways that vary from

region to region. The processing methods involve several steps including

peeling, soaking, grinding, steeping in water or in the air to allow fermentation

to occur, drying, milling, roasting, steaming, pounding, and mixing in cold or

hot water (Taiwo, 2006). A very popular processing method in Nigeria is

soaking of the roots under water for 3-4 days to effect softening (retting). The

different techniques of processing cassava roots have one common goal: the

reduction of cyanogenic compounds in order to obtain a safe food. All the

traditional processes for processing cassava permit the enzyme linamarase to

interact with cyanogenic compound to release HCN (hydrocyanic acid). The

17

HCN then dissolves in water or escapes into the air. However, it is often

impossible to remove all the cyanogenic compounds through processing.

Cassava toxicity in humans is a well-documented problem. Cassava tubers vary

widely in their content of cyanogenic glycosides, although the normal range of

cyanogenic glycoside content is from 15 to 400 mg HCN/kg fresh weight.

Cyanide doses up to 50 to 100mg/kg fresh peeled tuber are reported to be

moderately poisonous to adults whereas over 100mg HCN/kg fresh peeled tuber

is dangerously poisonous (Alloys and Ming, 2006). Several diseases are

associated with the consumption of inadequately processed cassava roots, such

as hypergoitre, tropical ataxic neuropathy, epidemic spastic paraparensis (Tewe,

1992) and konzo (Bradbury, 2006). Sublethal doses of cyanogenic compounds

are usually detoxicated in the body by conversion to thiocyanate, a sulphur-

containing compound with goitrogenic properties if in excess, which is excreted

in the urine (Tewe, 1992). A chronic overload of thiocyanate in conjuction with

low iodine intake, however, results in goiter and, in extreme cases, in cretinism

in children (Oke, 1994).

Retting as a very popular cassava processing method, is a spontaneous lactic

fermentation of cassava roots, a key step in the preparation of foo-foo (cassava

mash) in Nigeria. This process has been described and optimized in terms of

product quality and retting speed. During the process, cyanogenic compounds

are degraded, flavor compounds are elaborated and the roots softened (Ampe &

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Brauman, 1994). The role of microorganisms such as lactic acid bacteria, yeasts,

molds, and aerobic mesophiles has been extensively studied (Brauman et al.,

1996; Coulin et al., 2006). The combined action of several enzymes (pectinases,

amylases, linamarase, cellulases, and xylanase) during retting has been

extensively studied too (Ampe & Brauman, 1994).

This research work seeks to: (a) evaluate the microbial and biochemical

characterization of cassava retting process for fufu production, (b) determine the

time profile of the enzyme activities, (c) determine the changes in the

physicochemical parameters and (iv) to modulate the retting process.

1.1 LITERATURE REVIEW

1.1.1 Overview on Cassava (Manihot esculenta Crantz)

Cassava is a major crop in the tropics and over half a billion of the world‟s

population depends on it. It has been estimated by Food and Agricultural

Organization that 70 million tons of cassava was grown in Africa and it

continues to be staple food of energy for millions of people (Aryee et al., 2006).

The origin of cassava has been considered to be in Venezuela but a scientific

research has discovered that cassava originated from the southern border of the

Amazon River basin in Brazil. Cassava was introduced to Africa during the 16th

century by the Portuguese settlers (Pandley et al., 2000). On a worldwide basis

cassava counts as the ninth important source of energy in human diet; in

19

developing countries especially in the tropic it is ranked as the fourth supplier of

dietary energy after rice, sugar, and maize (Bokanga, 2001).

1.1.1.1 Botany and Cultivation

Cassava is a perennial shrub of the family Euphorbiaceae, and is mainly grown

for its starchy roots and raw material for industrial uses. Cassava is a higher

producer of carbohydrates per hectare than the main cereal crops and can be

grown at a considerably lower cost. Cassava is well adapted to poor soils with

marginal nutritional status and pH from 4 to 9 (Tewe, 1992). Cassava also

grows under sub- optimal conditions: it is tolerant of soil infertility, drought

stress and most pest and diseases (Bokanga, 2001) and can be stored

underground for several months after maturation (Taiwo, 2006). The mature

cassava roots may range in length from 15 to 100 cm and weigh 0.5 to 2.5 kg. In

favored areas, the root can be harvested as early as six to seven months after

planting, but most of the local varieties attain maximum yield after 18 months.

Improved varieties reach their maximum starch content after 12 – 15 months

(Hillocks, 2002). Among major food producing plants, cassava is one of the

most drought resistant. The crops can be grown in areas with an annual rainfall

as low as 500mm. At the onset of a dry period, the plant reduces its leaf area by

20

shedding some of its older leaves and by ceasing growth. The tolerance of

drought of up to 6 months mostly depends on the efficient use of water

(Onwueme and Charles, 1994).

Cassava can be grown on soils with low fertility to give reasonable yields but

fertilizers are often needed to reach the maximum production potential.

However, due to high cost and lack of availability, application of chemical

fertilizers is limited. Only about 3% of cassava fields in Africa are fertilized

compared to 2% of banana/plantain, 11% of rice, 15% of maize and 20% of

yam (Nweke, 1994).

Once the roots have been harvested, they start deteriorating within 2 to 3 days

and rapidly become of little value for consumption or industrial applications.

Two types of deterioration have been known to occur. The first is primary

deterioration, which consists of physiological changes characterized by an

internal root discoloration called vascular streaking or vascular discoloration

(Bokanga, 2001). Mechanical damages are unavoidable during harvesting and

handling operations. Wounds and bruises are the triggers of primary

deterioration and also constitute points of entry for microorganisms leading to

the second stage of cassava root spoilage, known as “secondary deterioration”.

This is induced by microorganisms that cause rotting, may be fungi or bacteria

especially Bacillus species (Bokanga, 2001).

1.1.1.2 PEST AND DISEASES

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Pests attack cassava mainly during dry season when cassava is one of the few

feed available. The cassava mealybug (Phenacoccus manihot) was introduced

into Africa in the early nineteen seventies (1970s) from South America. It

spread rapidly throughout Africa. This pest can induce severe defoliation

(Nwanze et al., 1979), causing substantial yield losses.

The cassava green mite (Mononychellus tanajoa) was also introduced from

South America into Africa in the 1970ies. It can cause up to 60% of chlorophyll

depletion and a reduction of leaf area of about 50% (Ayanru and Sharma, 1984).

The African cassava mosaic virus destroyed 80% of Uganda‟s crop within six

years (Nweke et al., 2004). Thresh and Otim-Nape, (1994) reported that about

28 to 40% of the production was lost in 1990 due to African cassava mosaic

virus. According to Wydra and Verdier (2002) control of this virus is possible

using an integrated weeding approach and planting of mixed varieties.

Cassava bacterial blight (Xanthomonas campestris pv. manihotis) is a major

constraint to cassava cultivation, and crops loss can reach 50 to 75% when

highly susceptible varieties are grown. To reduce cassava bacterial blight,

weeding, mixing varieties, crop rotation and intercropping with maize are

recommended (Wydra and Verdier, 2002). Additionally, several resistant

cassava varieties are available, which are also resistant to the cassava mosaic

disease (Onwueme and Charles, 1994).

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1.1.1.3 Economic Importance

For more than 500 million people in Africa, Asia and South America, cassava

provides income, employment and food security. The world production has

steadily increased in the last 35 years, and the worldwide cassava production

doubled (Plucknett et al., 2001). In 2002, about 186 million tons of cassava was

produced. More than half of that amount was produced in Africa (55%), the rest

in Asia (28%) and South America (17%) (FAO, 2005).

In Africa, the contribution of cassava to total energy intake is greater than for

maize or sorghum (Hillocks, 2002). Table 1 summarizes the per capita supply of

cassava for selected countries in Africa. In the Democratic Republic of Congo,

54% of the total energy intake comes from cassava; in Mozambique it

corresponds to about 36% and in Angola to 32%. The average energy supply

from cassava in Africa is about 8.5%.

Table 1: Cassava per capita supply 2002

Per capita supply

Kg/y kJ/d Total kJ/d % of total kJ/d

Africa 77.9 867 10153 8.5

Congo, D.R. 286.5 3580 6695 53.5

Mozambique 240 3010 8704 36.4

Angola 242 2763 8721 31.7

23

Ghana 212.9 2659 11166 23.8

Cote d‟Ivoire 93.4 1176 11011 10.7

(FAO, 2005)

1.1.1.4 COMPOSITION

The mature cassava plant consists of 6% leaves, 44% stem and 50% storage

roots (Tewe, 1992). Cassava roots mainly contain carbohydrates of which 80%

is starch (Bokanga, 2001) depending on the stage of growth or maturity of the

tuber. Cassava starch contains 17% amylose content compared to other starchy

carriers. Amylose and amylopectin constitute about 99% or more of dry

cassava starch. Root bulking usually begins between 45 and 60 days after

planting and the starch content reaches a maximum after eight to twelve

months. Thereafter lignification leads to an increase in fibre formation and a

decrease in starch content. Roots also contain small amounts of maltose,

fructose, glucose and sucrose (Tewe, 1992).

In addition to carbohydrates, other nutrients such as lipids, proteins, minerals

and vitamins, and water exist in varying concentration in cassava roots. Cassava

proteins are deficient in many essential amino acids, especially the sulphur

amino acids (methionine and cysteine), lysine, and tryptophan (Alloys & Ming,

2006). The low levels of sulphur amino acids were further exacerbated by losses

resulting from the involvement of cystine or cysteine in the detoxification

24

process of cyanogenic glycoside present in the cassava by which the cyanide is

converted to thiocyanate (Vetter, 2000).

A 50% to 87% protein loss has been reported in the preparation of food stuff

from cassava roots in Cameroon, while vitamin C, niacin and thiamine were

almost entirely lost (Bokanga, 2001). Cassava root is rich in calcium, ascorbic

acid, thiamine, riboflavin, and niacin (Alloys & Ming, 2006). Riboflavin has

been found in higher quantities in some fermented cassava products than in

fresh cassava roots, and it has been suggested that this vitamin may be

synthesized during fermentation (Bokanga, 2001).

1.1.2 TOXIC COMPOUNDS IN CASSAVA

There are at least 2650 species of plants that produce cyanoglycosides and their

corresponding hydrolytic enzymes (beta-glycosidase), which are brought

together when the cell structure of the plant is disrupted by predator, with

subsequent breakdown to a sugar and cyanohydrin, that rapidly decomposes to

hydrogen cyanide and an aldehyde or a ketone (Haque & Bradbury, 2002;

Curtis et al., 2002). The generation and release of cyanide is termed

cyanogenesis. The presence of cyanogenic glycoside in crop plant such as

cassava can present health problems for people that subsist on these plants. One

proposed function of cyanogenesis is to protect the plant against herbivory

(Mkong et al., 1990).

25

Cassava produces two cyanogenic glycosides, linamarin (2-

hydroxyisobutyronitrile-β-D-glucopyranoside) and lotaustralin (2-dydroxy-2-

methylbutyronitrile-β-D-glucopyranoside) (Petruccioli et al., 1999), in about 10

to 1 ratio. The amino acids valine and isoleucine are the precursors used in the

synthesis of linamarin and lotaustralin respectively. The metabolic pathway for

converting valine to linamarin has been elucidated (Bokanga, 2001).

All cassava tissues, with the exception of seeds, contain the cyanogenic

glycosides linamarin (> 90% total cyanogens) and lotaustralin (> 10% total

cyanogens). Leaves have the highest cyanogenic glycoside level of 5.0g

linamarin/kg fresh weight, whereas roots have approximately 20-fold lower

linamarin levels (White et al., 1998). The cyanogenic glycosides are stored in

the cytoplasmic vacuole while the enzyme capable of degrading them is located

in the cell wall outside the cytoplasm. Therefore, in intact cells the breakdown

of cyanogenic glycosides would not occur. When cassava tissues are bruised

and the cellular structures are disrupted, linamarin is degraded (Bokanga, 2001).

Linamarin is a rather stable compound, which is not changed by boiling the

cassava. Rupture of the cassava vacuole releases linamarin, which is hydrolysed

by linamarase, a cell wall-associated β-glycosidase. The breakdown of

linamarin leads to the formation of acetone cyanohydrins and glucose (White et

al., 1998). At pH above 5 or temperature > 350C (White et al., 1998), the

acetone cyanohydrin will spontaneously breakdown into acetone and hydrogen

cyanide (HCN).This breakdown also may be catalysed by the enzyme

26

hydroxynitrile lyase (HNL), which also is present in cassava. Once HCN is

produced, it will dissipate in the air since its boiling temperature is 25.70C

(Bokanga, 2001; Alloys & Ming, 2006).

In spite of the relative instability of cyanohydrins, it coexists with intact

glycoside and HCN in differently processed cassava products. It is therefore

clear that the cyanide in cassava products exists in three forms: (i) the

glycosides (Linamarin and lotaustralin), (ii) the cyanohydrins and, (iii) the free

hydrocyanic acid (HCN) (Tewe, 1992). Two factors are required for the

production of HCN: HCN precursors and catabolic enzymes in the cyanogenic

plant (Cock, 1985).

Reaction 1

Linamarin Glucose

Reaction 2

+

Cyanohydrin Acetone

Fig 1: Catabolism of linamarin to produce cyanohydrins and hydrocyanic acid

(HCN) (adapated from McMahon et al., 1995).

CH2OH

OH OH

O OH

OH

CN

C CH3

CH3 HO

+ H2 +

Cyanohydrin

CH2OH

CN

OH OH

O O

C

CH3

OH

CH3

CN

C CH3

CH3

HO HCN

+

pH > 4 H3C C

CH3

Hydrocyanic acid

27

The term “free cyanide” is used by some authors to refer to hydrocyanic acid

and by others to the sum of hydrocyanic acid and cyanohydrins. Some authors

use the term “bound cyanide” to refer to cyanogenic glycosides, while others

may use it to refer to hydrocyanic acid bound to albumin and other blood

proteins as part of in vivo cyanide detoxification process. The stability of the

cyanohydrin has been studied and found that at 300C and pH 6 it had a half-life

of about 30 minutes, and that alkaline pH favoured its dissociation, while acid

pH favoured its stability (Bokanga, 2001).

Hydrocyanic acid (HCN) is a volatile compound. It evaporates rapidly in air at

temperatures over 280C and dissolves readily in water. It may easily be lost

during transport, storage and analysis of specimens. The normal range of

cyanogen content of cassava tubers falls between 15 and 400mg HCN/kg fresh

weight. The concentration varies greatly between varieties and also with

environmental and cultural conditions. The concentration of the cyanogenic

glycosides increases from the center of tuber outwards, generally, the cyanide

contents is substantially higher in the cassava peel (Tewe, 1992). The low

expression of hydroxynitrile lyase in roots is believed to be an important factor

in a disease – Konzo. Low activity of hydroxynitrile lyase may also contribute

to the accumulation of high amounts of cyanohydrin in certain cassava flours

(White et al., 1998).

The activity of linamarase varies significantly between leaves, cortex and

parenchyma roots. The activity in root parenchyma is about 5 to 50 times lower

28

than in the leaves and root cortex (Nambisan & Sundaresan, 1994). The highest

linamarase activity has been observed at temperatures between 40 and 450C and

at pH 5.5 to 6.0; though Ampe and Brauman (1994) reported that linamarase

activity was still very high at pH 4.0. Linamarase is inactivated at temperatures

above 700C (Nambisan, 1994).

1.1.2.1 Classification of Cassava based on its Cyanogenic Potentials

Cyanogenic compounds in cassava are found in all tissues of the plant,

with the exception of seeds. The highest quantities are encountered in the leaves

and the cortex (peel) of the tuber (Balagopian, 2002; Bradbury and Egan, 1992).

In addition, the amount of cyanogenic glycosides not only depends on the

variety but also on climatic and cultivation conditions (Cook, 1985). The

amount of cyanogenic glycosides varies with part of the plant, its age, variety,

and environmental conditions such as soil, moisture, and temperature. Based on

the amount of cyanogenic glycoside, a tentative classification has been made as

follows: << Innocuous >> (less than 50 mg HCN/kg fresh peeled tuber),

<<Moderately poisonous>> (50 – 100mg HCN/kg fresh peeled tuber), or

<<Dangerously poisonous>> (over 100 mg HCN/kg fresh peeled tuber)

(Alloys & Ming, 2006).

Additionally, cassava is also grouped into “sweet”, mainly used as thirst

quenchers and snacks, and “bitter” for processing to flour and flour products;

with low and high levels of cyanogens, respectively (Petruccioli et al., 1999).

29

Varieties with cyanide of less than 100 mg/kg fresh weight (fwt) are considered

to be sweet varieties; those with a higher content than 100mg/kg are bitter

varieties. This also refers to the taste of raw roots, which is used by the farmers

to distinguish between sweet and bitter varieties. In older literature, cassava

(Manihot esculenta Crantz) was divided into a bitter species (Manihot

palamata) and a sweet species (Manihot aipi) (Rawel & Kroll, 2003), despite

the fact that there is no morphological difference (Mkumbira et al., 2003). A

study carried out on the cassava peel has shown that the peel of „bitter‟ cassava

variety contains on average 650 ppm and the pulp to contain 310 ppm total

cyanide while the corresponding values for „sweet‟ variety were 200 ppm and

38 ppm, respectively. The above classification is conveniently based on the

cyanide content; with the sweet variety having most cyanide in the cortex and

skin and little or no cyanide in the pulp, whereas the bitter varieties, more or

less, have an even distribution of cyanide throughout the tuber. For this reason

the former can be eaten boiled while the latter has to be processed before it can

be consumed (Tewe, 1992).

1.1.2.2 MEASUREMENT OF CYANOGENIC COMPOUNDS

Different methods are available for qualitative, semi-qualitative and quantitative

determinations of cyanogenic compounds. Several methods have been

developed for direct quantification of cyanogenic glycosides (Vetter, 2000).

30

However, the majority of methods used are indirect approach following three

separate steps:

Extraction of cyanogens from plant material,

Hydrolysis of cyanogens to cyanohydrins and subsequently to HCN;

Determination of HCN.

The first step, extraction of cyanogens, is normally carried out in dilute acid

(Bradbury et al., 1994) to stop the degradation of cyanogenic compounds. The

second step can be achieved either by autolysis, which relies on the endogenous

linamarase, by enzymatic hydrolysis by adding exogenous linamarase or by acid

hydrolysis (Bradbury et al., 1991). Various methods have been developed for

the third step, such as the picrate paper test or a related field colour tests, liquid

phase quantitative methods, an enzyme-based amperometric sensor, a β-

glycosidase electrode, membrane introduction mass spectrometry (MIMS) to

quantify the carbonyl compounds that accompany HCN liberations, and a

quantitative procedure based on the UV-vis spectrum of the sodium picrate-

cyanide complex (Curtis et al., 2002).

In all the three step methods, the most commonly used method is the picrate

paper test. While these methods are fairly easy to use, they often require long

sample incubation periods and the picrate paper test is particularly prone to

interferences giving rise to false positives (Curtis et al., 2002).

31

1.1.2.3 TOXIC EFFECTS AND DISEASES ASSOCIATED WITH

CYANIDE EXPOSURE.

Cassava toxicity in humans is a well-documented problem. However, opinions

on its importance vary greatly. On one hand, cyanide exposure can cause

diseases in human that may occasionally be serious or fatal. Reported toxic

effects of cassava are relatively rare in comparison with its wide use as a staple

(Bokanga, 2001). Cyanide poisoning could occur, if a population uses bitter

varieties for the first time, without sufficient knowledge on processing, or if

short-cuts in processing are introduced due to increased market demand or food

shortages. Increased cyanide levels in the roots due to drought may also lead to

increasing cyanide exposure (Rosling et al., 1994). High and continuous

consumption of cassava have been associated with various diseases and

nutritional disorders. They include: tropical ataxic neutropathy (White et al.,

1998; Bokanga, 2001), goiter and cretinism – hyperthyroidism (Bokanga, 2001;

Tewe, 2003), spastic paraparesis (Bokanga, 2001) or konzo (Bradbury, 2006;

White et al., 1998; Bokanga, 2001).

Cyanide poisoning from high cyanogenic cassava is typically associated with

insufficient consumption of cysteine and methionine in diet (White et al., 1998).

The human body has developed a certain tolerance towards cyanide exposure.

Cyanide is detoxified in the body by conversion to thiocyanate, a sulphur-

containing compound with goitrogenic properties (Tewe, 1992). Upon

consumption, cyanohydrins can readily decompose into cyanide, but cyanogenic

32

glycosides are partly excreted unchanged in the urine (Bokanga, 2001).

Rhodanase (thiosulphate cyanide sulphur transferase) catalyzes the conversion

of cyanide to thiocyanate. This enzyme is present in most tissues in human

(White et al., 1998) with the highest concentration being in the liver and kidney

(Bokanga, 2001). To a lesser extent, mercaptopyruvate cyanide sulphur

transferase, which is present in the red blood cells also catalyses the conversion

of cyanide to thiocyanate. The essential substrates for conversion of cyanide to

thiocyanate are thiosulphate and 3-mercaptopyruvate, and derived mainly from

cystine, cysteine and methionine, the sulphur-containing amino acids. Vitamin

B12 in the form of hydroxycobalamine probably influences the conversion of

cyanide to thiocyanate. Thiocyanate is widely distributed throughout body

fluids including saliva, in which it can readily be detected (Tewe, 1992).

Thoicyanate has a known goitrogenic effect: it interferes with the ability of the

body to use a limited supply of dietary iodine. However, a high thiocyanate load

does not show a goitrogenic effect if the dietary iodine intake is adequate

(Bokanga, 2001).

In normal health, a dynamic equilibrium between cyanide and thiocyanate is

maintained. A low protein diet, particularly one which is deficient in sulphur-

containing amino acids may decrease the detoxification capacity and thus make

a person more vulnerable to the toxic effect of cyanide (Tewe, 1992).

1.1.2.4 DISEASES RELATED TO CASSAVA TOXICITY

33

A) Tropical Ataxic Neuropathy (TAN):

TAN is similar to konzo. TAN has been attributed to cyanide intake from

insufficiently processed cassava roots. It is a paralytic disease with a slow onset,

which is observed in the elderly people who follow a monotonous cassava diet.

The clinical picture is dominated by damage to one of the sensory tracts in the

spinal cord resulting in an uncoordinated gait called ataxia. Symptoms include

balance disturbance due to degradation of the spinal cord, deafness and loss of

vision (Hewlett, 1994).

B) Konzo:

There is increasing evidence to link prolonged consumption of insufficiently

processed cassava with a newly described disease named Konzo (Bokanga,

2001). The disease has only been reported in poor, rural areas in Africa. Konzo

is an irreversible paralysis of the legs in women of childbearing age and

children, which occurs in Mozambique, United Republic of Tanzania,

Democratic Republic of Congo, Central African Republic and Cameroon

(Bradbury, 2006). But this has not yet been reported in Western Africa, since

they may have less access to animal protein and sufficiently large diversity of

food (Boivin, 1997).

Three prerequisites for the occurrence of konzo are: a farming system

dominated by bitter cassava, insufficient cassava processing that leaves high

residual level of cyanogens in cassava foods, and a protein deficient diet

(Bokanga, 2001). Reduced processing times are believed to be a major factor

34

leading to high cyanide loads from cassava flour. Flours consumed in konzo-

affected villages contained mean cyanide content of 32 mg/kg in rural Zaire and

26 to 186 mg/kg in northern Mozambique (Tylleskar et al., 1992).

C) Hyperthyroidism:

Thiocyanate has the same molecular size as iodine and interferes with iodine

uptake by the thyroid gland. Under conditions of high ingestion of insufficiently

processed cassava, there may be a chronic cyanide overload leading to a high

level of serum thiocyanate of 1 to 3mg/100ml, compared to a normal level of

about 0.2mg/100ml (Tewe, 1992).Endemic goiter occurs only when the iodine

intake is below about 100ml per day. A thiocyanate/iodine, SCN/I, ratio, of

lower than two, portends a risk of endemic cretinism, a condition characterized

by severe mental retardation and severe neurologic abnormalities. It has been

found that thiocyanate can cross the placental barrier and affect the foetus in

pregnant women and very little thiocyanate in breask milk indicating that the

mammalian gland does not concentrate thiocyanate (Tewe, 1992).

D) Spastic Paraparesis:

This disease affects mainly women and children. It damages the nerve tract in

the spinal cord that transmits signals for movement, thus causing a spastic

paralysis of both legs. Outbreak has been reported in Zaire, Mozambique and

Tanzania. Symptoms include nausea, vertigo and confusion. Sufferers also show

a high serum thiocyanate level and a urinary thiocyanate excretion of about ten

times than that of non-cassava-eaters (Tewe, 1992).

35

E) Other Diseases:

Exposure to cyanide may also be linked to other diseases such as protein

malnutrition or diabetes. High cyanide intake may contribute to protein

malnutrition, since essential amino acids are required for detoxification of

cyanohydrin to thiocyanate. The low protein content of cassava further

contributes to low protein intake (Rosling et al., 1994).

1.1.3 PROCESSING INTO DIFFERENT PRODUCTS

Two factors limit the direct utilization of fresh cassava. First, the high moisture

content leads to rapid deterioration in a few days and poor shelf life. Second,

some cassava varieties contain high amounts of cyanide and cannot be

consumed directly. Processing not only helps to improve shelf life and

decreases the amount of cyanide but also reduces the bulk of the roots, thus

reducing transportation cost.

Cassava forms a substrate for a wide variety of fermented foods in Africa, Asia,

and Latin America (Alloys & Ming, 2006). The fermented foods are gari, fufu,

chickwangue, lafun, agbelima, and farinha. The first three will be briefly

discussed.

A) Gari:

In West Africa, gari is by far the most popular form of cassava consumed by

about 200 million people daily (Okafor et al., 1998). The cassava tuber is

36

harvested, peeled, washed, grated, and packed into coarsely knit bags. A

weight is put on the bag to express some of the juice, and then it is left to

undergo natural fermentation for 2 - 4 days. The grated cassava, after sieving to

remove any coarse lump and impurities, is heated by means of constant turning

over a heated steel pan. On garifying, the grated cassava is dried to about 10%

moisture content and the starch is probably partially gelatinized. At this stage, a

little palm oil may be added to give it colour. Final dry granular product is gari

(Aloys & Ming, 2006; Taiwo, 2006; Bokanga, 2001).Gari is consumed in a

variety of ways. The granular product can be added to soup or stew, or like in

Nigeria, a product call eba can be prepared by mixing gari with hot water to

obtain a thick paste (Bokanga, 2001).

Cassava fermentation to gari is associated with a community of microorganisms

including yeasts and bacteria. Microorganisms revealed include

Corynebacterium sp. for acid production, Geotrichum candidum for flavor

production, Leuconostoc sp., Alcaligenes, Lactobacillus plantarum, Candida,

where Leuconostoc was the most frequently occurring organism (Alloys &

Ming, 2006).

B) Fufu:

The term „fufu‟ covers very different products depending on the region. In

Nigeria, fufu is the name of a food made from cassava roots soaked for 3 to 5

days (ret), mashed and directly cooked into dough. In Central Africa, fufu is

obtained by mixing cassava flour with hot water. The flour is made either by

37

sun drying whole roots or chips and then milling or by soaking roots in water

for three to five days, where fermentation occurs then milling and drying

(Bokanga, 2001).

Lactobacillus bacteria have been revealed to be involved in fufu production

with Lactobacillus and Leuconostoc species dominating the spectrum (Oyewole

& Odunfa, 1990; Brauman et al., 1996). The succession among the Lactic acid

bacteria (LAB) isolates revealed the dominance of Lactobacillus plantarum. It

also was revealed that Saccharomyces cerevisiae; Lactobacillus brevis;

Streptococcus faecalis and E. coli appeared to be important in the fermentation

of fufu (Alloys & Ming, 2006).

C) Chickwangue:

Chickwangue is the most popular fermented cassava product in Central Africa,

particularly in the Congo and in Cameroon where it is called miondo and

Bobolo. To prepare chickwangue, soaking or steeping in water for 3 days

ferments cassava roots, after which it is mashed and steamed. The steamed

mash is kneaded into smooth dough, which is wrapped in leaves and steamed.

After steaming, the wrapped cassava is allowed to cool. Its shelf life is about 3

to 7 days at room temperature if the wrapping is not open. Otherwise it will dry

up and become unfit for eating (Bokanga, 2001; Alloys and Ming, 2006).

1.1.3.1 CASSAVA LEAF CONSUMPTION

In many tropical countries, cassava leaves constitute a highly prized vegetable.

Young tender leaves are usually selected, pounded and boiled for 15 to 30

38

minutes and then various ingredients are added to taste. There have been no

reports of toxicity associated with the consumption of cassava leaves, even

though the concentration of cyanogenic glycosides in it is 5 to 10 times greater

than that of the root parenchyma (Bokanga, 2001).

Cassava roots are well recognized as deficient in protein, the leaves contain 7 to

10% protein. Much of these proteins are made up of the enzyme linamarase, its

activity was found to be about 200 times greater in the leaves than it was in the

roots. Cassava leaves have a food potential as a source of protein in animal feed

(Bokanga, 2001).

1.1.4 CASSAVA PROCESSING TECHNIQUES

Cassava is a highly perishable, starchy root crop, which starts to deteriorate

within two to three days after harvest if not processed (Aryee et al., 2006).

Traditionally, cassava roots are processed by a number of methods that vary

widely from region to region (Alloys & Ming, 2006). Processing is under taken

primarily to detoxify the cassava product to improve its palatability and to

convert it into a storable form (Aryee et al., 2006; Alloys & Ming, 2006; Tewe,

1992; Bokanga, 2001). It is widely claimed that there is inter-and-intra-

communal variability in cassava processing techniques due to a clear lack of

standardization in the production of foods with variable organoleptic properties

and different levels of residual cyanohydrins and hydrogen cyanide, HCN

(Agbor-Egbe & Mbome, 2006). These methods consist of different combination

39

of peeling, chopping, grating, soaking, drying, boiling and fermentation (Tewe,

1992; Alloys and Ming, 2006).

A) Peeling

The first step in processing of cassava roots is often to remove the peel, which

results in great reduction of the cyanogenic potential of the raw material

because the peel represents about 15% of the weight of the root and its

cyanogens content is usually 5-10 times greater than that of the root

parenchyma (Alloys & Ming, 2006; Bokanga, 2001). Peeling is usually done by

hand using knife; the process is slow and labour-intensive, averaging 25kg per

man-hour, but it gives the best results as against mechanical peeler (Bokanga,

2001). Peeling, therefore, can be an effective way to reduce the cyanide content

by at least 50% in cassava tubers. However, it should be noted that while the

peel contains high glycoside content relative to the pulp, the glycosidase level is

higher in the pulp (Tewe, 1992).

B) Grating and Chopping

This process takes place after peeling and is sometimes applied to whole tubers.

Grating of the whole tuber ensures the even distribution of the cyanide in the

product, the concentration depends on the time during which the glycoside and

the glycosidase interact in an aqueous medium. Grating also, obviously,

provides a greater surface area for fermentation to take place (Tewe, 1992). At

the home level, cassava roots are chopped manually using a knife. This process

is slow and produces large irregular chips that take 3 to 7 days to dry and impart

40

a sour and musty taste, actually preferred by some consumers, to the food made

from the dry chips (Bokanga, 2001).

C) Soaking

Soaking of cassava roots normally precedes cooking or fermentation (Tewe,

1992). Soaking in water has been reported to leach out the soluble cyanogenic

glycosides from cassava. It has been found that soaking cassava roots in water

for one day decreases the cyanide from 108.2 ppm to 59.5 ppm, while soaking

for five days has reduced it further to 2.9 ppm (Alloys & Ming, 2006). A

variation of the soaking technique is known as retting. The process involves

prolonged soaking of cassava roots in water to affect the breakdown of tissue

and extraction of the starchy mass (Tewe, 1992).

D) Drying (sun- and oven-drying)

Cassava roots contain about 61% water, coupled with the solubility of its

cyanogenic glycoside component (Tewe, 1992). Drying process or technique

eliminates cyanide from the cassava tuber and also improves the shelf life of

processed or dried cassava tubers. Dehydration is achieved either through sun-or

oven- drying. Sun drying has been reported to be more effective than the oven

drying as the former results in a greater loss of total cyanide compared to

laboratory oven drying at 600C for 48hours. Oven drying apparently affects the

stability of linamarase, which decomposes at 720C. Second, sun drying tends to

produce greater loss of bound cyanide due to slower drying rate. It also allows a

41

longer contact time between the glycosidase and glycoside in the aqueous

medium (Alloys & Ming, 2006; Tewe, 1992).

Sun drying facilitates the continuation of the fermentation process. More

importantly, it is cost effective, but slow and often encourages the growth of

mould and other microorganisms including Aspergillus flavus (pathogenic), A.

fumigatus; A. niger; A. japonicus and Penicillium rubrum. Exposure to this

microbial growth leads to Aflatoxicosis and/or mycotoxic infection (Tewe,

1992; Alloys and Ming, 2006).The effects on cyanide removal of different

drying temperature have been studied by Nambisan and Sundaresan (1985).

E) Boiling:

Boiling of cassava pieces to remove cyanide was studied by Nambisan and

Sundarsan (1985). Samples of different size (A: 6x3x3cm, B: 3x2x1cm, C:

1x0.5x0.5cm) were boiled in water for 30 min. Reduction was highest for the

smallest size C, 69-75% of the initial level, followed by size B and size A,

where reductions were 50% and 255% respectively. Most of the cyanide

removed was recovered in the water.

With soaking, the free cyanide of cassava chips is rapidly lost in boiling water.

About 90% of the free cyanide is removed within 15 minutes of boiling fresh

cassava chips, compared to a 55% reduction in bound cyanide after 25 minutes.

Cooking destroys the enzymes, linamarase, at about 720C thus leaving a

considerable portion of the glycoside intact (Tewe, 1992). The percentage

42

reduction of cyanide in the cassava chips depends on the boiling time, volume

of water, and the tuber piece size (Alloys & Ming, 2006).

1.1.5 FERMENTATION

Fermentation is one of the oldest applied biotechnologies, having been used in

food processing and preservation as well as beverages production for over 6,000

years (Oboh, 2006). A recent survey of the modes of utilization of cassava in

Africa has revealed that nearly three out of four cassava-based foods

encountered in the survey were fermented products (Bokanga, 2001).

Fermentation improves the nutritional quality of foods by improving the

nutrient density and increasing the bioavailability of nutrients. This is achieved

by the production of phytases, degradation of antinutritional factors,

predigestion of certain foods components, synthesis of promoters for

absorption, and enhancement of the uptake of nutrients by the mucosa (Mensah

& Tomkins, 2003). Fermentation is a microbial degradative process whereby

the firm tissue of cassava is broken down into a soft, easily, disintegratable

lump (Alloys & Ming, 2006). Three types of fermentation have been generally

distinguished. (1) A submerged fermentation, in which cassava roots, whole

or in large pieces, are steeped in water for a period of 3 to 5 days; (2) A mash

fermentation, in which a mash is obtained by grating or rasping fresh cassava

roots, and the mash is left to ferment in a container for several days; and (3) A

low-moisture fermentation whereby peeled cassava roots are heaped together

43

and fungal growth is allowed to develop at the surface of the roots (Bokanga,

2001).

Cassava roots are fermented in several parts of Africa for a number of reasons.

One of the most important reasons is to obtain a desired sour product, e.g. gari,

agbelima, fufu. A second reason for fermentation of cassava is to remove

considerable amount of cyanide from the high cyanide cassava varieties through

processes such as retting or submerged fermentation and heap fermentation of

roots. A third reason for fermenting of cassava roots in some parts of Africa is

to modify the texture of the product (Obilie et al., 2003). Cassava fermentation

inevitably takes place in two stages, the first is retting or disintegration of the

cell membranes facilitated by microorganisms and the second is microbial

fermentation. Microbial fermentation has been practiced traditionally from

many years in all parts of the world to improve palatability and textural quality

and to upgrade nutritive value by enriching with protein and reducing toxic

factors (Alloys & Ming, 2006). Dry fermentation is also practiced in parts of

Africa during the drought season and in the south Pacific as a means of reducing

cyanide. Cassava roots are heap covered until molds grow and roots soften.

Mold growth has been reported to be contributing to cyanogens loss due to the

enzymes-induced cellular disruption releasing β-glycosidase with linamarase

activity (Alloys & Ming, 2006).

The microorganism associated with cassava fermentation are mostly

Lactic acid bacteria (Lactobacillus plantarum, Streptococcus faecium and

44

Leuconostoc mesenteroides) and spore-forming bacteria such as Bacillus sp.

Lactic acid bacteria are mainly responsible for the rapid acidification that

characterizes cassava fermentation. The Bacillus sp. seems to be responsible for

inducing the retting of cassava root tissues during the submerged fermentation

of whole roots. Other microorganisms, such as Corynebacterium sp.,

enterobacteriacease, yeast and moulds have been reported, but they are usually

present in low numbers and their role is not clearly understood (Bokanga,

2001).

Additionally, root fermentation has been attributed to lactic acid bacteria and it

is essential for the development of the sensory attributes and the preservation of

cassava foods. It has long been assumed that cassava fermentation is one of the

principle mechanisms of cyanide removal, supported by the reported ability of

certain organisms to hydrolyze linamarin (Agbor-Ebge & Mbome, 2006)).

Despite the fact tha presence of natural flora has the ability to hydrolyze

linamarin; it has been shown that this is not a prerequisite for linamarin

elimination. It has been demonstrated that during natural fermentation of

cassava roots soaked in water, microbial growth was essential for efficient

cyanogens elimination (Agbor-Ebge & Mbome, 2006).

1.1.5.1 PHYSICAL AND BIOCHEMICAL CHANGES DURING

CASSAVA FERMENTATION.

PHYSICAL CHANGES

45

A) Dry Matter:

In a number of cases, dry matter had decreased. The decrease can be due partly

to the reduction in sugar and starch and the leaking of soluble constituents. In

addition, the softening of the tubers may lead to increased absorption of water

contributing to the reduction in dry weight (Alloys & Ming, 2006).

B) Softening of the Roots:

During the soaking of roots for the production of gari, or lafun, the texture of

the roots undergoes noticeable change and the roots are rendered soft. It has

been observed that the softening of tubers sets in during the second day and

continues until the fourth day. Bacillus subtilis has been shown to have the

highest softening capability among the different microbial cultures tried for fufu

production (Alloys & Ming, 2006). Softening of tissues during fermentation can

be attributed to enhanced action of pectinolytic and cellulotic enzymes produced

by microorganisms (Alloys & Ming, 2006). Root softening during soaking has

been ascribed to an increase in the activity of cell-wall degrading enzymes. It

has been reported that the rate of acidification increased with decreasing cassava

roots sizes (<15mm) during the first 48h of fermentation for the production of

Nigerian fufu, which also affected product organoleptic qualities. The reduction

in cyanogens levels has been found to strongly correlate to the degree of root

size reduction, which is also directly related to the rate of softening and

acidification (Agbor-Ebge & Mbome, 2006).

BIOCHEMICAL CHANGES

46

i) pH:

Microorganisms in the fermenting medium convert sugar and starch in the tuber

into organic acids, which decreases the pH. However, the level to which the pH

falls and the period taken for the pH reduction varies not only with the type of

fermentation but also with the conditions used for fermenatatoin (Alloys &

Ming, 2006). Brauman et al (1996) found out that lactic acid bacteria produced

high amounts of lactic acid leading to rapid drop in pH to around 4.5 as

previously found during the preparation of fufu or lafun, a similar product.

ii) Titrable Acidity:

Titrable acidity undergoes changes during fermentation. The acidity has been

expressed as lactic acid or total acidity percent. While the pH decreases, the

titrable acidity increases (Alloys & Ming, 2006). The level of acidification has

been reported to increase with increasing period of fermentation (Oyewole &

Ogundele, 2001).

iii) Organic Acid:

The microorganisms present in the fermentation medium convert starch and

sugars in the tuber to organic acids, which impart the characteristic odor and

taste to different fermented products. Lactic acid is one of the common aids

reported in almost all types of cassava fermentation (Alloys & Ming, 2006).

iv) Vitamins and Minerals:

Vitamins and minerals decrease during cassava fermentation. These are due to

leaching out into the steep water or to microbial utilization. Riboflavin has been

47

reported to remain unchanged during gari fermentation. Also there is reduction

in the protein content due to the leaching loss in the steep liquor or microbial

utilization (Padmaja et al., 1994).

v) Fiber:

Fiber content in the fermenting tuber rise with the time of fermentation

(Oyewole & Ogundele, 2001) and the effect is uniform. The increase in fibre

content is due to the action of pectinolytic and cellulolytic enzymes produced by

the fermenting microorganisms, which break down the cell membranes (Alloys

& Ming, 2006).

vi) Sugar and Starch Content:

Reducing sugars increase on the first day of gari fermentation and subsequently

decreased during the next days of fermentation. The increase during the first day

has been attributed to the breakdown of the starch by starch-splitting enzymes,

with the sugar produced being further utilized by the organisms (Alloys &

Ming, 2006). A reduction in starch content has been attributed to the conversion

to sugars during fermentation. When there is depletion of sugars, the organism

starts breakdown of the starch for sugar availability (Alloys & Ming, 2006).

1.1.5.2 RETTING

Retting is one of the simplest methods for the processing of cassava (Mannihot

esculenta Crantz) tuber into various African staple foods (Ogbo, 2006). Retting,

a spontaneous lactic fermentation of cassava roots is the key step in the

48

preparation of foo-foo (cassava flour) and chickwangue (cassava bread, the

main cassava-based foods of Central Africa (Ampe & Brauman, 1995).

Retting of cassava entails steeping roots in water for 3 to 4 days (Brauman et

al., 1996) under optimal conditions. On other conditions, retting may take

considerably longer, for example, with tubers older than 24 months or during

the colder seasons of the year. In addition, up to 20% of tubers steeped under

these conditions may fail to soften (Ogbo, 2006). During the consequent

fermentation, roots are softened, 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 & Akomas, 2006), and characteristic flavours developed (Brauman et al.,

1996) through a pH decrease and organic acid production (Ampe et al., 1994).

The fermentation process is initiated as a result of chance inoculation by

microorganisms from the environment.The presence of unspecified

microorganisms complicates the control of the fermentation process and lead to

the production of objectionable odours (Achi & Akomas, 2006). Softening is

indispensable for further processing of the roots but the essential mechanisms

involved are not fully understood (Ampe & Brauman, 1994). Increase in the

fibre conent of „fufu‟ has been reported as fermentation time increased. The

increase in the digestibility of the fermented product may be connected with the

increasing retting that aids „fufu‟ particle extraction from the cassava. The

dispensability is associated with decreasing particle size of the food material

49

(Oyewole & Ogundele, 2001). It has been reported that the most influential

factor affecting retting time is temperature, with optimum at 300C (Alloys &

Ming, 2006).

1.1.5.3 Microbial Community of Cassava Retting

Westby and Choo (1994) studied the role of microorganisms in the fermentation

of soaked roots. They immersed roots in either untreated water or in water

containing an antibiotic to prevent microbial growth. The cyanide reduction

exceeded 90% after 3 days in the untreated water, where fermentation took

place, whereas a reduction of less than 50% was found in the absence of

microbial growth. The reduction strongly correlated with root softening and

release of linamarin into the surrounding water. In view of this finding, growth

of microorganisms appears to be important for the loss of cell structure of

cassava and subsequently leaching of cyanogens, although other mechanisms,

such as β-glucosidase activity of microorganisms, may also intervene.

Other authors attributed the decrease in cyanogenic compounds to β-glucosidase

activity of microorganisms in spontaneously fermented products (Kimaryo et

al., 2000; Okafor and Ejiofor, 1990; Ikediobi and Onyike, 1982). However, the

experiments do not show whether the decrease was due to β-glycosidase activity

or to root softening, which also leads to a more intimate contact between

cyanogenic glycosides and endogenous linamarase (Westby and Choo, 1994;

Ampe and Brauman, 1994).

50

Cassava fermentation has been shown to be a complex microbial process in

which a small amount of lactic acid bacteria (LAB) namely Lactococcus latics,

Leucoconostoc mesenteroides and Lactobacillus plantarum, rapidly replace the

epiphytic microflora and governed the retting of the cassava roots. Factors

favouring their dominance are: presence of oxygen at the onset of retting, high

amounts of acids (e.g. lactic acid) leading to a rapid drop in pH; resistance to

free cyanide at high concentration which inhibits other aerobic organisms and

production of bacteriotoxins (Brauman et al., 1996). Clostridium sp. has also

been studied. In cassava retting this organism has contributed to the flavor of

cassava fermented products. Clostridium sp. produces typical fermentation

products like butyrate and to a lesser extent, propionate. They might also play

an important role in the destruction of plant cell walls, as strains with

pectinolytic activities have been isolated. Butyrate, propionate and ethanol seem

to be characteristic of retting, since these products have not been reported in

other cassava fermentation, such as that used for gari production (Brauman et

al., 1996). It has also been reported that the yeast flora which population

increased with increase in period of fermentation contributes significantly to the

odour of fermented cassava (Oyewole & Ogundele, 2001). Yeast could play an

important role in the case of prolonged storage (Brauman et al., 1996).

51

1.1.5.4 ENZYMES INVOLVED IN DETOXIFICATION AND ROOT

SOFTENING DURING CASSAVA RETTING.

1.1.5.4.1 AMYLASE

The amylases family of enzymes is a diverse group of starch degrading

enzymes. There are 3 types of amylases:

α-Amylase (Endo- α-1,4-glucan-4-glucanohydrolase)

β-Amylase (α-1,4-glucan-malto-hydrolase)

Amyloglucosidase (glucoamylase) or (1,4- α-D-glucan glucohydrolases).

The first and second types of amylase will be discussed here.

α-Amylases

These are endoglucanases widely distributed in animals, plants, bacteria and

fungi. They possesses many isoforms based on immunological properties,

proteolytic finger prints, isoelectric point (PI) and sensitivity to Calcium (Ca2+

)

and pH (Marchylo & Mac-Gregory, 1983; Deikman & Jones, 1986). These are

the principal enzymes involved in carbohydrate hydrolyses (Sanwo &

Demazon, 1992). α-Amylases are multiproteins which belong to the family of

(β/α)8 – barrel or Tim barrel enzymes (Svensson & SФgard, 1992). The average

molar mass is about 50,000 Daltons. They are stable within a pH range of 4.5 –

8.0; with an optimum activity between pH 4.8 and 6.5. The temperature range is

between 350C and 90

0C. Many α-amylases are calcium metallo-enzymes

containing at least one calcium atom per molecule of enzymes essential for their

conformational stabilization. Fungal α-amylases usually have lower pH options

52

compared to bacterial α-amylases. α-Amylases are endo splitting enzymes that

catalyze the hydrolysis of the internal α-1, 4-glucosidic linkages of starch,

glycogen, amylose, amylopectin and various related polysaccharides (Hara &

Miwa, 1990), with the liberation of glucose and oligosaccharides. The

oligosaccharides may partially be degraded to a mixture of maltose, glucose,

maltotriose and α-dextrin. The enzyme does not attack α-1, 6-linkages but can

bypass it. The action of the enzyme on starch causes the rapid reduction in

viscosity and iodine staining power of starch with the slow release of reducing

sugars (Sun & Henson, 1991).

β-Amylases

β-Saccharifying amylases are widely distributed in plants (Higashihara &

Okada, 1974) and have recently been found to be widespread in the microbial

world (Priest, 1992). Microbial β-amylases have a molar mass range between

35,000 and 50,000 Daltons (Fogarty and Kelly, 1979a; 1979b), with an

exception of the enzymes from several Bacillus species which have

considerably higher molar mass (Shinke et al., 1975). A pH optimum of 5-7 has

been reported for most microbial β-amylases and a temperature optimum of 5-7

has been reported for most microbial β-amylases and a temperature optimum of

45-600C (Obi & Odibo, 1984). They are generally inhibited by sulphydryl

reagents such as p-chloromercuribenzoate (Takashi, 1975), N-ethylemelimide

(Thoma, 1974) and by Schardinger dextrins (Thoma & Roshland, 1960).

53

β-Amylases are exosplitting enzymes which attack alternate α-1,4 glucoside

bonds in amylose, amylopectin and glycogen starting from the non-reducing

end of the chain with the release of maltose in β-configuration (Sun & Henson,

1991; Kwan et al., 1994). These enzymes do not cleave the α-1, 6-glucosidic

bond that form the branching points in amylopectin, thereby leaving residue α-

limit dextrins and slowly decreases the turbidity and viscosity of the starch.

They cannot bypass the α-1, 6-glucosidic branch point. The smallest molecule

readily attacked is maltotetraose (Gacesa & Hubble, 1987).

1.1.5.4.2 HEMICELLULASES

The primary plant cell wall consists of cellulose, hemicelluloses, pectin and

protein. Celluloses and hemicelluloses are the main load – bearing

polysaccharides in the cell wall, maintaining the cell shape and turgor pressure.

Cell elongation relies on the selective enzymatic modification of the load-

bearing hemicelluloses molecules. Pectin is not load-bearing, but may control

the mobility and access of enzymes to load-bearing hemicelluloses molecules;

thereby modulating cell elongation. Pectin contains negatively charged

galacturonic acid residues, which contribute to the cell wall cation exchange

capacity (Wehr et al., 2004).

Hemicellulases are group of enzymes that are defined and classified according

to their substrate hemicelluloses. The hemicelluloses are polymers of xylose,

galactose, mannose, arabinose, other sugars and their uronic acids. These are

54

usually classified according to the sugar residue present. Examples: D-xylan, D-

galactan, D-mannan, L-arabinan. However, they do not occur as homoglucans

but rather as heteroglycans containing different types of sugar residues often as

short appendages linked to the main backbone chain. Examples of these include

L-arabino-D-xylan (wheat flour), L-arabino-D-glucurono-D-xylan (grass), D-

glucurono-D-xylan (wood) etc (Ghose & Bisaria, 1987).

The hemicellulases defined as glycan hydrolases (EC 3.2.1) attack the backbone

chain of the hemicelluloses but are not responsible for cleavage of side-branch

sugar appendages (mono- or oligosaccharide). The hemicellulases best

characterized are 1, 4-beta-D-xylanases and, in particular, those derived from

fungal sources presumably because their substitutes, xylan, constitute the largest

proportion of hemicelluloses in pasture plant. The term “xylanases” refers to

those enzymes, which are capable of hydrolyzing the 1-4-beta-D-xylopyranosyl

linkages of the 1,4-beta-D-xylans, namely, arabinoxylan,

arabinoglucuronoxylan, arabino-4-O-methyl-D-glucoronoxylan and

glucuronoxylan. D-xylanases of this type have been assigned the enzyme

commission numbers 3.2.1.8 (1, 4-beta-D-xylan xylanohydrolase, endo-

xylanase) and 3.2.1.37 (1, 4-beta-D-xylan xylohydrolase, exo-xylanase) (Ghose

& Bisaria, 1987).

1.1.5.4.3 PECTINASES

Pectic substances are ubiquitous in the plant kingdom and their efficient

utilization could enhance the economic competitiveness of bioconversion

55

processes. The enzymes that hydrolyse pectin substances are broadly termed a

pectinases (Kapoor & Kuliad, 2002). Pectinases constitute a complex enzymatic

system that includes polygalacturonase (PG; EC 3.2.1.15), pectinesterase (PE;

EC 4.2.2.10). PE and PG must act together to degrade completely the pectin

molecule, and they liberate methanol as a byproduct of PE action. PL can

depolymerise pectin by the beta – elimination mechanism without the

complementary action of the two other enzymes (Taragano & Pilosof, 1999).

The presence of high pectinase activities together with the absence of cellulase

and xylanase indicating that the former are responsible for the softening of

cassava has been reported (Brauman et al., 1996). Pectinesterases (PE) and

polymethylgalaturonate lyases (PGL) have only been found in cassava

inoculated with Corynebacterium sp. However, no pectinoltic activities and

especially no PE were found in fresh roots. Surprising, some researchers found

no pectinolytic activity in retting inoculated with Bacillus sp., although

softening occurred. Moreover the presence of extracellular pectin methyl

esterase during retting has been shown (Ampe & Brauman, 1994).

Cassava softening can be characterized by the dissociation of cellulose fibres

from the pectin cement because of the action of enzymes, such as hydrolases

and lyases, on the pectin glycosidic linkages. The fact that softening begins on

the second day of retting indicates that the bacteria responsible for this

phenomenon are acid-tolerant anaerobes (Brauman et al., 1996). Various

bacteria are able to produce pectinases. Some LAB, which are important in

56

retting, such as Lactobacillus plantarum and Leuconostoc mesenteroides,

produces PG or PGL (Ampe & Brauman, 1994).

1.1.5.5 FERMENTATION MODULATION

In Nigeria and other parts of West Africa, local processors have responded to

this challenge of addition of various chemical substances to water for steeping

of the cassava roots. Chemical substances commonly employed include

kerosene, trona and common wire nails. The use of these substances has their

origins in local belief, kerosene as a solvent, while trona and nails as tenderizers

during cooking, but has not been shown scientifically to achieve intended

purpose (Ogbo, 2006).

57

CHAPTER TWO

5.0 MATERIALS AND METHODS

2.1 Plant Material

Cassava tubers were collected from a farm in Nru, Nsukka Local Government

Area in Enugu State. The cassava plants were 12 – 15 months old.

2.2 Retting Procedure

The cassava tubers were collected from the farm and taken to the laboratory

immediately where they were cleaned, hand peeled and cut into cylinders of

2.5cm height and washed. They were completely submerged in tap water, and

left at ambient temperature (30 ± 20C) until retting (softening) occurred. Retting

was observed by hand feel and floating of the cassava cuts on the steep water.

2.3 Sample Preparation for Microbial Enumeration

Samples of the cassava were collected for microbial enumeration at 6 hourly

intervals until retting was completed. Ten grams of the sample were

homogenized using laboratory mortar (sterilized by cleaning with 70% ethanol

and flaming of the surface) under aseptic conditions. Ten (10) fold serial

dilution containing 90 ml of 0.1% peptone water and 10g cassava homogenized

sample was prepared and spread-plated for the enumeration of viable counts

from dilution factors of 10-2

, 10-4

, 10-6

, 10-8

, and 10-10

.

58

2.4 Microbiological Population Studies

2.4.1 Media Used

The following media were used for the isolation of microorganisms from the

retting tubers:

de Man, Rogosa and Sharp agar (MRS) – for enumeration of Lactic

acid bacteria.

Potato dextrose agar (PDA) – for enumeration of yeast and molds.

Violet red bile glucose agar (VRBG) – for enumeration of

Enterobacteriaceae.

Plate count agar (PCA) – for enumeration of aerobic mesophiles.

0.1ml of the diluted sample from the respective dilution factor was inoculated

onto the respective media plates and incubated. MRS plates were incubated

microaerobically in candle jar at 300Cfor 3-5 days; PDA plates were incubated

aerobically at room temperature (25±20C) for 2-3 days; VRBG plates were

incubated for 2 days at 350C and PCA plates were incubated at 35

0C for 3 days.

Colonies were counted from each plate of the respective media after 2-3 days of

incubation.

2.4.2 SUB-CULTURING:

Isolates from respective media were sub-cultured until pure cultures were

obtained and stored as slants on their respective media.

59

2.5 IDENTIFICATION OF ISOLATES

2.5.1 Bacteria

Identification was implemented on the basis of Gram staining and biochemical

characteristics of the isolates.

A) Microscopic Examination of Cell morphology

i) Gram Staining:

Gram staining was done as described by Chessbrough (2000). Gram staining

reagents include: crystals violet stain, Lugol‟s iodine, acetone-alcohol

decolourizer, and neutral red (1 g/1 [0.1% w/v] or safaranin. A smear of each

culture (isolates) was prepared on a clean grease-free glass slide, spreads evenly

covering an area of about 15-20mm diameter on a slide. The smears were heat

fixed by passing three times through the flame of a Bunsen burner and allowed

to cool before staining. The fixed smears were covered with crystals violet stain

for 30-60 seconds and rapidly washed off with clean water. Then, the smears

were covered with Lugol‟s iodine for 30-60 seconds and the iodine was washed

off with clean water. The smears were decolourized rapidly with acetone-

alcohol for few seconds and was washed immediately with clean water. The

smears where covered with neutral red stain or safaranin for 2 min and the stain

was washed of with clean water. The back of the slide was wiped clean, and

placed in a draining rack for the smears to air-dry.The smears were then

examined under a light microscope using oil immersion objective (X100).

60

ii) Biochemical Tests

The following tests were carried out on the isolates:

a) Catalase test

b) Indole production

c) Simmon‟s Citrate test (citrate as carborn source)

d) Potassium cyanide test

e) Sugar fermentation

iii) Motility test

2.5.1.1 Catalase Test:

A few drops of hydrogen peroxide (3% w/v) was placed on a clean grease-free

glass slides. The isolates were then emulsified on the slide. Positive test was

indicated by effervescence.

2.5.1.2 Indole Tests:

One mililitre of Kovac‟s reagent was added to the 24hrs culture of the SIM

medium for motility. The tubes were gently shaken and pink colour for

positivity in a ring around the interface between the medium and the alcohol

reagent, which rises to the surface, was observed.

2.5.1.3 Citrate Utilization Test:

Simmons citrate agar was used to determine the utilization of citrate as a sole

carbon source. 24.2g of Simmons citrate agar which consist of: magnesium

sulphate, 0.2g; monoammonium phosphate, 1.0g; Dipotassium phosphate, 1.0g;

sodium citrate, 2.0g; sodium chloride, 5.0g; bromothymol blue, 0.08g; and Agar

61

(Bi-tec), 15.0g; was added to IL of reagent-grade distilled water and autoclaved

at 15 p.s.i in tubes. The isolates were streaked on the medium and incubated at

370C for 24hr-48hr. Citrate-utilizing isolates were observed by changing of the

medium colour from green to Prussian blue.

2.5.1.4 Potassium Cyanide Test:

This was determined in potassium cyanide (KCN) broth, which consists of:

peptone, 0.6g; NaCl, 1.0g; potassium dihydrogen phosphate, 0.05g; disodium

hydrogen phosphate (Na2HPO4.2H2O), 1.13g in 200ml-distilled water. 0.5g w/v

of potassium cyanide was added to the medium after autoclaving and allowed to

cool. The isolates were inoculated and incubated at 370C for 48hrs. Turbidity

was observed indicating a positive reaction.

2.5.1.5 Carbohydrate Fermentation Test:

This test was carried out to determine the ability of the isolates to metabolize

sugars with the production of acid and / or gas. The following sugars were used

for the test: glucose, lactose, mannitol, sucrose, xylose, maltose, arabinose,

sorbitol, galactose and melibiose.

Phenol Red Peptone water indicator was used, which consists of: peptone,

10.0g; sodium chloride, 5.0g; and phenol red indicator, 0.025g. 15g phenol red

peptone water indicator was dissolved in 1L of distilled water. It was mixed

well and distributed into tubes and bottles containing inverted Durham‟s tubes.

Each bottles and tubes contained 5ml aliquots of 1g of each of the sugars in

100ml of the phenol red peptone water indicator. The bottles and tubes were

62

then sterilized by autoclaving, cooled and then inoculated with pure isolates

using sterile loops, before being incubated at 300C for 48hrs to 12 days. Change

in color from yellow to pink observed indicates acid production, while gas

production was observed by the downward displacement of liquids in the

Durham‟s tubes.

iii) Motility Test:

The test was carried out using Sulphur Indole Motility (SIM) medium which

comprises of: tryptone, 20g: peptone, 6.1g: ferrous ammonium sulpahte, 0.2g;

sodium thiosuphate, 0.2g; Agar No. 1, 3.5g. The media was prepared as butt

tubes with a dept of 5cm and autoclaved. The isolates were inoculated by

stabbing a straight wire loop carrying the inoculum once vertically into the

centre of the agar butt to a depth of approximately 2cm. The tubes were

incubated overnight at 370C. Motility was evident as haze of growth extends

into the agar from the stab line.

2.5.2 FUNGAL IDENTIFICATION

Lactophenol cotton blue mount was used for the microscopic preparation of

fungal isolates. A drop of lactophenol cotton blue was placed on a clean glass

slide. Using a sterile wire loop, a small portion of the colony was cut from the

culture and placed to the drop of lactophenol cotton blue. The preparation was

covered with a cover slip and pressed gently. It was gently heated to remove air

bubbles and to spread the fungus evenly throughout the preparation. It was then

examined under the microscope using x10 and x40 objectives.

63

2.6 DETERMINATION OF TIME PROFILE OF ENZYME

ACTIVITIES IN CASSAVA RETTING

The following enzymes were assayed at 12 hourly intervals

1. Amylase

2. Pectinase

3. Cellulase

2.6.1 Amylase Activity Assay

The amylase activity was assayed as described by Okolo et al., (1995). The

amylase activity was determined by incubating 0.5ml of retting juice with 0.5ml

of 1% (w/v) of soluble starch in 0.1ml citrate – phosphate buffer pH 6.0 and

0.1ml toluene. After incubation for 10mins at 400C, the reaction was stopped by

the addition of 1ml of 3, 5 dinitrosalicyclic acid reagents (DNS). The reaction

mixture was then heated for 10mins in boiling water and cooled. After the

addition of 5ml of deionized water to the mixture, the absorbance of the

coloured solution was measured spectrophotometrically at wavelength of

540nm.

One unit of amylase activity is defined as the amount of enzyme that produced

reducing sugars equivalent to 1µmol of glucose from retting juice per min at

400C.

2.6.2 Pectinase Activity

Pectinase activity was assayed as described by Ampe and Brauman (1994) by

titrating 1ml of the retting juice in 1% pectin, 0.1M NaCl and ImM NaN3. Then

64

brought to pH 7 by adding 0.1M NaOH at 300C. The following activities were

assayed:

Pectin lyase activity

Pectinesterase activity

Polygalacturonase activity

2.6.2.1 Pectin Lyase Activity Assay

Pectin lyase activity was assayed as described by Delgado et al., (1992). The

reaction mixture consisted of 1.5ml of 0.5% of pectin in 40mM acetate buffer

pH 4.5. After incubation for 15min at 370C, the reaction was mixed with 50µl of

the appropriate extract dilution (retting juice). The reaction was stopped by

adding 0.15ml of 1N HCl (for the blank, the HCl was added first). One unit of

pectin lyase activity was defined as the amount of enzyme needed to cause a

difference of optical density OD235 of 1, in the condition of the assay.

2.6.2.2 Polygalacturonase Activity Assay

Polygalacturonase activity was assayed as described by Taragano and Pilosof

(1999). The reaction mixture consisted of 0.5% of pectin in 40mM acetate

buffer, pH 4.5. After incubation for 15min at 370C, the reaction was mixed with

25µl of the retting juice. The absorbance of the solution was measured

spectrophotometrically at wavelength of 510nm. One unit of polygalacturonase

activity was defined as the amount of enzyme needed to release 1µl of reducing

sugar in the condition of the assay.

65

2.6.2.3 Pectinesterase Activity Assay

Pectinesterase (pectin pectlhydrolase) activity was assayed as described by

Ampe and Brauman (1994). The activity was assayed by titrating 1ml retting

juice in 1% of pectin, 0.1M NaCl and 1mm NaN3, brought to pH 7 with 0.01M

NaOH at 300C. One unit was defined as the amount neutralizing 1µmol of COO

-

/min.

2.6.3 Cellulase Activity

Cellulose activity was assayed as described by Ampe and Brauman (1994). The

activity of cellulose was assayed by incubating 100ml of retting juice with

100ml of 100mg/ml of microcrystalline cellulose and 50ml of sodium phosphate

buffer at pH 6.0, and then quantified spectrophotometrically at 400nm.

2.7 PHYSIOCOCHEMICAL PARAMETERS

The following physiocochemical parameters were also determined at the same

time interval.

- pH

- Titrable acidity

- Cyanide content

2.7.1 pH

A 10ml sample of the retting juice was collected at 12 hourly intervals for

estimation of pH using pH meter.

66

2.7.2 Titrable Acidity

This is the percentage lactic acid. 0.1N NaOH was titrated against 10ml

supernatant of homogenized cassava cut, using phenolphthalein indicator at 12

hourly intervals. The volume was divided by 100.

2.7.3 Cyanide Content

The cyanide content was determined as described by Onwuka (2005). Five

grams of the cassava tuber were soaked in 50ml of distilled water in six

containers separately. The first was allowed for 24hr, after which it was filtered.

One mililitre of the filtrate was put into a test tube in which 4ml of picrate (for

cyanide) was added. Blank sample was also prepared by adding 1ml of distilled

water to 4ml of picrate in a test tube. The two test tubes were allowed to stand

for 90mins after which 1:10 dilution was made out of the two tubes and read

colorimetrically at wavelength of 470nm. The subsequent five containers were

analyzed as same above at 12hr intervals for 3 days. A standard curve for

cyanide was established from which units of activity was calculated.

2.8 FERMENTATION MODULATION

This was done to determine the effect of the following on cassava retting:

Effect of temperature on cassava retting

Effect of cations (Ca2+

and Mg2+

ribbon ) on cassava retting

Effect of Added chemical agents (kerosene, and concrete nail [3

inches])

67

2.8.1 Effect of Temperature

Cassava roots of age between 12 to 15 months were used. Roots were peeled,

cut into cylinders of 2.5cm height and washed. Retting was performed by

completely submerging 1.5g of the cassava pieces in tap water and incubated at

different temperatures (300C, 35

0C, 40

0C, and 45

0C) until retted. Retting was

observed by floating of the cassava cuts in water and by hand feeling.

2.8.2 Effect of Cations

Cassava roots were peeled, cut into cylinders and washed. 1.5kg of the cassava

pieces were submerged in tap water in three different labeled containers. The

cations were added to the water immediately after soaking. The cations include

calcium carbonate CaCO3 (2 grams), magnessium ribbon (9cm) and control (no

cation). The cassava was allowed to ret at room temperature of 25±270C.

ii) Cation levels of cassava before and after soaking

This was carried out as described by Oyewole and Asagbra (2003). Cassava

roots was peeled, cut and washed. 1.5kg of the cassava pieces were soaked in

tap water in three different labeled containers for 48hrs (primary fermentation),

after which the steep liquor was decanted. Fresh tap water was added to the

containers in which the cations were added immediately to the respective

containers and allowed to steep for 48hrs (secondary fermentation). One piece

of cassava cut from the respective containers was analysed for natural cations

immediately it was soaked by ash method for Ca2+

and Mg2+

. After retting, one

68

piece of cassava cut from each cation-contained container was also analysed for

their respective cation level by the same method mentioned above.

a) Ash method

This was carried out as described by Onwuka (2005). The cassava cuts were

oven dried and milled. 1.0g of the oven-dried milled cassava was weighed out

and put into in a crucible. The milled samples were ashed in a murfled furnace

at 6000C for 4hr. When the residue turned black in colour, small amount of

water was added to dissolve salts. The moistened residue was dried in an oven

and the ashing process was repeated. It was then cooled in desiccators and

reweighed.

2.8.3 Effect of Added Chemical Agents

Cassava roots were peeled, cut into cylinders and washed. 1.5kg of the cassava

cuts was soaked in different containers for kerosene, 6 inches nail and control

(no chemical agent). The agents were added immediately to the cassava steep at

the concentration of 0.1ml; 0.3ml; 0.5ml kerosene and concrete nail (3 inches).

It was allowed to ret at different temperatures using the incubator. The pH of

each experiment was also determined daily.

2.9 STRENGTH OF CASSAVA CUTTINGS

The cassava roots were cleaned, hand peeled and cut into cylinders of 2.5cm,

5.0cm, 7.5cm, 10.0cm and 12.5cm height. Then, the pieces were washed and

completed submerged in tap water in respective containers, which was left at

69

ambient temperature (30±20C) until retting was observed. Retting was observed

by hand feel and floating of the cassava pieces on the tap water.

70

CHAPTER THREE

3.0 RESULTS

3.1 MICROBIAL COUNTS

The results in Table 1 revealed that the total fungal count increased from

3.9×108 c.f.u/ml to 6.2×10

8 c.f.u/ml after 72h of retting. However, the lactic acid

bacterial counts shown in Table 2 revealed that there was no growth from 0h –

6h. There was gradual decrease from 5×107

c.f.u/ml to 4.6×108 c.f.u/ml after 24h

but afterward increased to 9×107

c.f.u/ml after 24h and later decreased from

7.6×108 c.f.u/ml to 3.9×10

8 c.f.u/ml after 72h. The counts of Enterobacteriaceae

as shown in Table 3 also increased from 5×107 c.f.u/ml to 9.2×10

8 c.f.u/ml after

12h and later decreased from 7.7×108 c.f.u/ml to 4.8×10

8 c.f.u/ml after 72h. The

counts of aerobic mesophiles decreased from 7.2×108

c.f.u/ml to 1.5×108

c.f.u/ml after 72h of retting, as shown in Table 4.

71

EVOLUTION OF MICROBIAL FLORA DURING CASSAVA RETTING

TABLE 1: MICROBIAL COUNT (C.F.U/ML) ON POTATO DEXTROSE AGAR (PDA)

Dilution Cell concentration (cfu/ml) at time (h):

0 6 12 18 24 30 36 42 48 54 60 66 72

10-2

9×104 6.2×10

4 5.8×10

4 4.7×10

4 3.8×10

4 4.0×10

4 4.2×10

4 5.6×10

4 6.6×10

4 7.6×10

4 8.6×10

4 8.8×10

4 1.2×10

5

10-4

8.3×106 3.2×10

6 5.0×10

6 4.1×10

6 2.8×10

6 2.9×10

6 3.3×10

6 4.8×10

6 5.1×10

6 5.7×10

6 6.1×10

6 7.6×10

6 1.0×10

6

10-6

3.9×108 1.8×10

8 1.6×10

8 3.8×10

8 1.9×10

8 2.1×10

8 1.7×10

8 1.8×10

8 5.1×10

8 3.0×10

8 2.1×10

8 4.2×10

8 6.2×10

8

10-8

3.7×1010

2.6×1010

1.1×1010

2.5×1010

1.1×1010

6×109 1.4×10

10 1.5×10

10 4.0×10

10 1.3×10

10 1.8×10

10 3.1×10

10 4.8×10

10

10-10

2.2×1012

1.8×1012

6×1011

1.5×1012

6×1011

1×1012

1.7×1012

1.4×1012

2.9×1012

1.1×1012

1.5×1012

3.3×1012

3.9×1012

72

TABLE 2: MICROBIAL COUNT (C.F.U/ML) ON de MANN, ROGOSA AND SHARPE AGAR (MRs)

Dilution Cell concentration (cfu/ml) at time (h):

0 6 12 18 24 30 36 42 48 54 60 66 72

10-2

NG NG 4.4×104 1.1×105 9.2×104 1.8×105 1.1×105 1.2×105 1.56×105 2.1×105 1.7×105 1.6×105 1.1×105

10-4

NG NG 7.0×105 1.2×107 5.2×106 3.2×106 2.5×106 7.4×106 7.5×106 7.5×106 1.6×106 1.3×106 8.8×107

10-6

NG NG 5.0×107 4.8×108 4.6×108 1.4×108 2.7×108 9.0×107 7.6×108 2.5×108 1.3×108 4.8×108 3.9×108

10-8

NG NG 2.0×109 3.3×1010 4.1×1010 1.4×1010 6.4×1010 5.0×109 3.7×1010 1.7×1010 2.2×1010 1.7×1010 5.1×1010

10-10

NG NG 5.0×1011 1.9×1012 1.6×1012 3×1011 2.9×1012 7.1×1012 5.0×1012 2.6×1012 2.8×1012 1.3×1012 2.6×1012

Key: Ng – No growth.

73

TABLE 3: MICROBIAL COUNT (C.F.U/ML) ON VIOLET RED BILE GLUCOSE AGAR (VRBG)

Dilution Cell concentration (cfu/ml) at time (h):

0 6 12 18 24 30 36 42 48 54 60 66 72

10-2

2.2×104 5.0×104 6.5×104 6.9×104 8.5×104 9.0×104 9.7×104 1.1×105 1.01×105 1.1×105 1.2×105 1.3×105 1.5×105

10-4

2.4×106 4.0×106 4.8×106 6.1×106 7.0×106 5.7×106 9.1×106 8.8×106 7.9×106 8.0×106 9.1×106 9.5×106 1.0×107

10-6

5.0×107 3.2×108 9.2×107 5.9×108 7.7×108 2.3×108 7.3×108 2.8×108 5.8×108 7.1×108 5.0×108 6.6×108 4.8×108

10-8

5.0×109 8.0×109 1.7×1010 1.5×1010 4.9×1010 1.3×1010 4.1×1010 5.4×1010 4.8×1010 6.8×1010 6.5×1010 4.2×1010 3.9×1010

10-10

1.0×1011 5.0×1011 6.2×1012 9.0×1011 2.1×1012 5.0×1011 3.7×1012 4.3×1012 2.7×1012 5.4×1012 4.1×1012 2.5×1012 3.8×1012

74

TABLE 4: MICROBIAL COUNT (C.F.U/ML) ON PLATE COUNT AGAR (PCA)

Dilution Cell concentration (cfu/ml) at time (h):

0 6 12 18 24 30 36 42 48 54 60 66 72

10-2

1.2×105 8.9×104 8.0×104 7.1×104 6.5×104 5.9×104 4.2×104 4.0×104 3.9×104 2.8×104 2.2×104 2.0×104 2.5×104

10-4

8.6×106 7.2×106 6.5×106 6.2×106 5.4×106 4.9×105 4.1×106 3.7×106 2.8×106 2.2×106 1.6×106 1.9×106 2.1×106

10-6

7.2×108 6.0×108 5.4×107 5.1×108 4.0×108 4.5×108 3.1×108 3.1×108 1.8×108 1.8×108 1.4×108 1.0×108 1.5×108

10-8

6.9×1010 4.2×1010 3.4×1010 4.8×1010 3.1×1010 4.0×1010 2.8×1010 3.1×1010 1.7×1010 1.2×1010 1.3×1010 6.0×109 7.0×109

10-10

5.1×1012 2.2×1012 2.3×1012 3.6×1012 3.0×1012 3.7×1012 2.6×1012 3.0×1012 1.5×1012 9.0×1011 8.0×1011 6.0×1011 6.0×1011

75

Table 5: Morphological, Physiological, Biochemical and Sugar Fermentation Characteristics of Isolates on Plate Count Agar (PCA)

Test P0 – 4 P0– 10 P6 – 2 P6 – 4 P6 – 6 P6 – 8 P12 – 4 P12 – 10 P18 – 2 P18 – 6 P18 – 8 P18 – 10 P24 – 4 P24 – 6 P24 – 8

Grain reaction - + - + + - - - + - + + - + +

Microscopic Appearance SR C SR C C SR SR SR C SR C C C C C

Cellular Arrangement Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs

Mortility + - + - - + + + - + - - - - -

Indole + - + - - + + + - + - - - - -

Catalase + - + - - + + + - + - - - - -

Gas from D-Glucose + - + - - + + + - + - - - - -

Carbohydrate Fermentation

Acid from:

D – Glucose - + - + + - - - + - + + + + +

Lactose + + + + + + + + + + + + + + +

Mannitol + - + - - + + + - + + - - + -

Sucrose W - W - + W W W - W + - + + +

Xylose W + W + - W W W + W - + - - -

Maltose + + + + + + + + + + + + + + +

Arabinose + - + - - + + + - + - - - - -

D – sorbitol + D + d - + + + d + + d - + -

Probable Organism E. co

li

L. la

ctis

E. co

li

L. la

ctis

Strep

toco

cus S

p.

E.co

li

E.co

li

E.co

li

L.la

ctis

E.co

li

Strep

toco

cus S

p.

L.la

ctis

Strep

toco

cus S

p.

Strep

toco

cus S

p.

Strep

toco

cus fa

ecalis

76

Table 5: Cont’d

Test P30 – 6 P30 – 10 P36 – 2 P36 – 6 P36 – 8 P42 – 4 P42 – 6 P42 –10 P48 – 6 P48 – 8 P48 – 10 P54 – 2 P54 – 6 P54 – 8 P54 – 10

Grain reaction - + + + + - + + + + + + + + +

Microscopic Appearance SR C R C CR SR C R C R C R C R R

Cellular Arrangement Pairs Pairs Singly Pairs Singly Pairs Pairs Singly Pairs Singly Pairs Singly Pairs Singly Singly

Motility + - + - + + - + - + - + -\

+ +

Indole + - - - - + - - - - - - - - -

Catalase + - + - + + - + - + - + - + +

Gas from D-Glucose + - - - + + - + - + - + - - +

Carbohydrate Fermentation

Acid from:

D – Glucose - + + + + - + + + + + + + + +

Lactose + + - + - + + - + - + - + - -

Mannitol + - + + + + - + - + - + + + +

Sucrose W - - + - W + - - - - - + - -

Xylose W + + - + W - + + + + + - + +

Maltose + + - + - + + - + - + - + - -

Arabinose + - + - + + - + - + - + - + +

D – sorbitol + D - + - + - - - - d - + - -

Probable Organism

E. co

li

L. la

ctis

B. p

um

ula

n

S. fa

ecalis

B. m

acera

ns

E.co

li

Strep

toco

ccus

. sp

B. m

acera

ns

Strep

toco

ccus

. sp

B. m

acea

ns

L. la

ctis

B. m

acera

ns

Strep

toco

cus

Sp

.

B. m

acera

ns

B. m

acera

ns

77

Table 5: Cont’d

Test P60 – 2 P60 – 4 P60 – 8 P66 – 4 P66 -6 P66 – 10 P72 – 4 P72 –8 P72 –10

Grain reaction + + + + + + + + +

Microscopic Appearance C R R R R C R R R

Cellular Arrangement Pairs Pairs Singly Pairs Singly Pairs Pairs Singly Pairs

Motility - + + + + - + + +

Indole - - - - - - - - -

Catalase - + + + + - - - -

Gas from D-Glucose - - + + - - + - -

Carbohydrate fermentation

Acid from:

D – Glucose + + + + + + + + +

Lactose + - - - - + - - -

Mannitol + + + + + + - + +

Sucrose + - - - - + - - -

Xylose - + + + + - + + +

Maltose + - - - - + - - -

Arabinose - + + + + - + + +

D – sorbitol + - - - - + - - -

Probable Organism Strep

toco

ccus

faeca

lis

B .su

btilis

B. m

acera

ns

S. m

acera

ns

B. su

btilis

S. fa

ecalis

B. m

acera

ns

B. su

btilis

B. su

btilis

key: P – Plate count agar plate, SR – short rod, C- cocci, W- weak, D- not detected, P0 – at 0hr, P-2

– dilution factor, - - negative, + -

positive

78

Table 6: Morphological, Physiological, Biochemical and Sugar Fermentation Characteristics of Isolates on De Man, Rogoss and Sharpe (MRS).

Test M12 – 2 M12 – 6 M12 – 8 M12 – 10 M18– 2 M18– 4 M18– 6 M18– 8 M18– 10 M24– 2 M24– 4 M24– 6 M24– 8 M24– 10 M30– 2

Gram reaction + + + + + + + + + + + + + + +

Microscopic Appearance C SR SR SR C SR C C C SR C SR C C SR

Cellular Arrangement Pairs Singly Singly Pairs Pairs Singly Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs

Motility - - - - - - - - - - - - - - -

Spore - - - - - - - - - - - - - - -

Cetalase - - - - - - - - - + - + - - -

Hydrolysis of Arginine - + + + - + - - - + - + - - +

Growth in 6.5% NaCI - + + - - + - - - + - - - - +

Growth in 10% NaCl - + + - - + - - - + - - - - -

Gas from D – Glucose - - - + - - - - - - - + - - +

Carbohydrate fermentation

Acid from:

D – Glucose + + + + + + + + + + + + + + +

Lactose + - - + + - + + + + + + + + W

Mannitol - - - - - - - - - + - - - - W

Sucrose - + + - - + - - - d - - - - d

Xylose + - - d + - + + + + + d - - d

Maltose + d d + + d + + + + + + + + +

Arabinose - - - d - - - - - + - d - - +

Sorbital d - - - d - d d d d d - - - -

Galactose + W W + + + + + - + + + + W

Melibiose d - - + d - d d d + d + - - +

Probable organism La

ctoco

ccus

lactis

La

ctob

acillu

s

delh

mectkii

L. d

elbru

eckii

La

ctob

acillu

s

fermen

tum

L. la

ctis

L. d

elbru

eckii

L. la

ctis

L. la

ctis

L. la

ctis

La

ctob

acillu

s Sp

L. la

ctis

L. ferm

entu

m

L. la

ctis

L. la

ctis

La

ctob

acillu

s

brevis

79

Table 6: Cont’d

Test M30 – 4 M30 – 6 M30 – 8 M30 – 10 M36– 2 M36– 4 M36– 6 M36– 8 M36– 10 M42– 2 M42– 4 M42– 6 M42– 8 M42– 10 M48– 2

Gram reaction + + + + + + + + + + + + + +

Microscopic Appearance SR SR C CB C C SR B CB SR C SR SR B C

Cellular Arrangement Pairs Singly Pairs Pairs Pairs Singly Pairs Chains Pairs Pairs Chains Pairs Pairs Chains Chains

Motility - - - - - - - - - - - - - - -

Spore - - - - - - - - - - - - - - -

Catalase - - - - - - - - - - - - - - -

Hydrolysis of Arginine + + - + - - + - - + - + + + -

Growth in 6.5% OaNI - + d + d D - + + - D + + + d

Growth in 10% NaCl - + - - - - - + W - - + - + -

Gas from D – Glucose + - - - - - + - - + - - + - -

Carbohydrate fermentation

Acid from:

D – Glucose + + + + + + + + + + + + + + +

Lactose + - W d W W W + + + W + W - W

Mannitol - - d - d D W + + - D + W - d

Sucrose - + + d + + d + d - + d d + +

Xylose d - d W d D d W + d D + d - d

Maltose + D + + + + + + + + + + + d +

Arabinose d - + - + + + + + d + + + - +

Sorbitol - - - d - - - + d - - - - - -

Galactose + W + - + + d + + + + - W W +

Melibiose + - d - d D + + + + d + + - d

Probable organism L. ferm

entu

m

L. d

elbru

eckii

leuco

no

stoc

mesen

teriod

es

La

ctic. Sp

L. m

esenterio

des

L. m

esenterio

des

L. b

revis

L. p

lan

taru

m

La

ctic Sp

L. ferm

entu

m

L. m

esenterio

des

La

ctic Sp

L. b

revis

L. d

elbru

eckii

L. m

esenterio

des

80

Table 6: Cont’d

Test M48 – 4 M48 – 8 M48 – 10 M54 – 4 M54– 6 M60– 10 M60– 2 M60– 4 M60– 6 M60– 8 M60– 10 M66– 2 M66– 4 M66– 6 M66– 8

Gram reaction + + + + + + + + + + + + + +

Microscopic Appearance CB C C C B B SR SR B B B C SR B B

Cellular Arrangement Pairs Pairs Pairs Chains Chains Chains Pairs pairs Chains Chains Chains Chains Pairs Chains Chains

Motility - - - - - - - - - - - - - - -

Spore - - - - - - - - - - - - - - -

Cetalase - - - - - - - - - - - - - - -

Hydrolysis of Arginine + - - - - - + + - - - - + - -

Growth in 6.5% OaNI + D d d + + - + + + + d + + +

Growth in 10% NaCl - - - - + + - + + + + - + + +

Gas from D – Glucose - - - - - - + - - - - - - - -

Carbohydrate fermentation

Acid from:

D – Glucose + + + + + + + + + + + + + + +

Lactose d W W W + + + + + + + W + + +

Mannitol - D d d + + - + + + + d + + +

Sucrose d + + + + + - d + + + + d + +

Xylose W D d d W W W + W W W d + W W

Maltose + + + + + + + + + + + + + + +

Arabinose - + + + + + W + + + + + + + +

Sorbital - - - - + + - - + + + - - + +

Galactose - + + + + + + - + + + + - + +

Melibose - W W W + + + + + + + d + + +

Probable organism La

ctic sp

L. m

esenterio

de

L. m

esenterio

de

L. m

esenterio

de

L. p

lan

taru

m

L. p

lan

taru

m

L. ferm

entu

m

La

ctic sp

L. p

lan

taru

m

L. p

lan

taru

m

L. p

lan

taru

m

L. m

esenterio

des

La

ctic spp

L. p

lan

tatru

m

L. p

lan

taru

m

81

Table 6: Cont’d

Test M66 – 10 M72 – 2 M72 – 4 M72 – 6 M72– 10

Gram reaction + + + + +

Microscopic Appearance CB B C B B

Cellular Arrangement Singly Chains Pairs Chains Chains

Motility - - - - -

Spore - - - - -

Cetalase - - - - -

Hydrolysis of Arginine + - - - -

Growth in 6.5% OaNI + + d + +

Growth in 10% NaCl - + - + +

Gas from D – Glucose - - - - -

Carbohydrate fermentation

Acid from:

D – Glucose + + + + +

Lactose D + W + +

Mannitol - + d + +

Sucrose D + + + +

Xylose W W d W W

Maltose + + + + +

Arabinose - + + + +

Sorbital - + - + +

Galactose - + + + +

Melibose - + d + +

Probable organism La

ctic sp

La

ctic sp

L. m

esenterio

de

L. p

lan

taru

m

L. p

lan

taru

m

M – MRS agar plate, R – Rod, C – cocci/rod, C – cocci/bacilli, B – bacilli, M12 – at 12hr, M-2

– dilution factor

82

Table 7: Morphological, Physiological, biochemical and sugar frmentation characteristics of isolates on VRBGA medium

Test V0 – 2 V0 – 4 V0 – 6 V0 – 8 V0– 10 V6– 2 V6– 4 V6– 6 V6– 8 V6– 10 V12– 2 V12– 4 V12– 6 V12– 8 V12– 10

Gram reaction - - - - - - - - - - - - - - -

Microscopic Appearance R SR SR R SR R SR R SR R R R R R SR

Cellular Arrangement Singly Pairs Pairs Singly Pairs Pairs Pairs Singly Pairs Pairs Singly Pairs Pairs Pairs Chains

Motility - + + - + + + - + + - + + + -

Indole Production - + + - + - + - + - - - - - -

Growth in KCN medium - - - - - + - - - + - + + + +

Gitrate as C source - - - - - + - - - + - + + + +

Catalase + + + + + - + + + - + + + - -

H2S Production - - - - - - + - - - - - - - -

Gas from D – Glucose - + + - + + + - + + - + + + +

Carbohydrate fermentation

Acid from:

D – Glucose - - - - - + - - - + - + + + -

Lactose W + + W + + + W + + W + + + +

D – mannitol + + + + + + + + + + + + + + +

Sucrose - W W - W + W - W + + + + + +

Xylose - W W - W d W - W d - d + d +

Maltose + + + + + d + + + d + d + d +

Arabinose + + + + + + + + + + + + + + +

Sorbital - + - + - - + - + - - - + - +

Probable organism Sh

igella

son

nei

Esch

erichia

coli

E. co

li

S. so

nn

ei

E. co

li

En

terob

acter

saka

zaki

E. co

li

S. so

nn

ei

E.co

li

E. sa

kaza

ki

S. so

nn

ei

E. sa

kaza

ki

En

terob

acter

cloa

cae

E. sa

kaza

ki

Kleb

siella

ph

enu

mon

iae

Key : V – Violet red bile glucose agar plate, V0 – at 0hr, V-2

– dilution factor, w – weak, d – not detected

83

Table 7: Cont’d

Test V18 – 2 V18 – 4 V18 – 6 V18 – 8 V18– 10 V24– 2 V24– 4 V24– 6 V24– 8 V24– 10 V30– 2 V30– 4 V30– 10 V36– 4 V36– 6

Gram reaction - - - - - - - - - - - - - - -

Microscopic Appearance SR SR R R SR R R R SR SR R R R R R

Cellular Arrangement Pairs Chains Singly Pairs Pairs Singly Singly Pairs Chains Chains Pairs Pairs Pairs Pairs Pairs

Motility + - - + - - - + - - + + + + +

Indole Production + - - - - - - - - - - - - - -

Growth in KCN medium - + - + + - - + + + + + + + +

Citrate as C source - + - + + - - + + + + + + + +

Catalase + - + + - + + + - - + + + - -

H2S Production - - - - - - - - - - - - - - -

Gas from D – Glucose + + - + + - - + + + + + + + +

Carbohydrate fermentation

Acid from:

D – Glucose - - - - - - - - - - - - - - -

Lactose + + W + + W W + + + + + + + +

D – Mannitol + + + + + + + + + + + + + + +

Sucrose W + - + + - - + + + + + + + +

Xylose W + - + + - - + + + + + + d d

Maltose + + + + + + + + + + + + + d d

Arabinose + + + + + + + + + + + + + + +

Sorbitol + + - + + - - + + + + + + - -

Probable organism E. co

li

K. p

neu

mo

nia

e

S. so

nn

ei

En

terob

acter S

p.

K. p

neu

mo

nia

e

S. so

nn

ei

S. so

nn

ei

En

terob

acter S

p.

K. p

neu

mo

nia

K. p

neu

mo

nia

E. clo

aca

e

E. cla

oca

e

E. sa

kazza

ki

E.sa

kazza

ki

E. sa

kazza

ki

84

Table 7: Cont’d

Test V36 – 8 V42 – 2 V42 – 4 V42 – 6 V42– 8 V42– 10 V48– 4 V48– 6 V48– 8 V48– 10 V54– 4 V54– 6 V54– 8 V60– 2 V60– 4 V60– 6

Gram reaction - - - - - - - - - - - - - - - -

Microscopic Appearance R R R SR SR R SR SR R SR R R R SR R R

Cellular Arrangement Pairs Pairs Pairs Chains Chains Pairs Pairs Pairs Pairs Pairs Pairs Pairs Pairs Chains Pairs Pairs

Motility + + + - - + - - + - + + + - + +

Indole Production - - - - - - - - - - - - - - - -

Growth in KCN medium + + + + + + + + + + + + + + + +

Citrate as C source + + + + + + + + + + + + + + + +

Catalase - + + - - + - - - - - + + - + +

H2S Production - - - - - - - - - - - - - - - -

Gas from D – Glucose + + + + + + + + + + + + + + + +

Carbohydrate fermentation

Acid from:

D – Glucose + + + - - - - - - - - + + - - -

Lactose + + + + + + + + + + + + + + + +

D – mannitol + + + + + + + + + + + + + + + +

Sucrose + + + + + + + + + + + + + + + +

Xylose d + + + + + + + D + d + + + + +

Maltose d + + + + + + + D + d + + + + +

Arabinose + + + + + + + + + + + + + + + +

Sorbitol - + + + + + + + - + - + + + + +

Probable organism E sa

kaza

kii

E. sa

kaza

ki

E. clo

aca

e

K. p

neu

mo

nae

K. p

neu

mo

nia

e

En

terob

acter S

p.

k. pn

eum

on

iae

K. p

neu

mo

nia

e

E. sa

kaza

ki

K. p

neu

mo

nia

e

E. sa

kaza

ki

En

terob

acter S

p.

E. C

loca

cae

K. p

neu

mo

nia

e

En

terob

acter S

p.

En

terob

acter S

p.

85

Table 7: Cont’d

Test V60 – 8 V60 – 10 V66 – 2 V66 – 4 V66– 6 V66– 8 V66– 10 V72– 2 V72– 4 V72– 6 V72– 8 V72– 10

Gram reaction - - - - - - - - - - - -

Microscopic Appearance SR SR R R SR SR R SR SR R R R

Cellular Arrangement Pairs Pairs Pairs Pairs Chains Pairs Pairs Chains Chains Pairs Pairs Pairs

Motility - - + + - - + - - + + +

Indole Production - - - - - - - - - - - -

Growth in KCN medium + + + + + + + + + + + +

Citrate as C source + + + + + + + + + + + +

Catalase - - + + - - - - - + + +

H2S Production - - - - - - - - - - - -

Gas from D – Glucose + + + + + + + + + + + +

Carbohydrate fermentation

Acid from:

D – Glucose - - + + - - + - - + + +

Lactose + + + + + + + + + + + +

D – mannitol + + + + + + + + + + + +

Sucrose + + + + + + + + + + + +

Xylose + + + + + + d + + + + +

Maltose + + + + + + d + + + + +

Arabinose + + + + + + + + + + + +

Sorbitol + + + + + + - + + + + +

Probable organism K. p

neu

mo

nia

e

K. p

neu

mo

nia

e

E. clo

cae

E. clo

aca

e

K. p

neu

mo

nia

e

K. p

neu

mo

niea

E. sa

kaza

ki

K. p

neu

mo

nia

e

K. p

neu

mo

niea

E. clo

aca

e

E. clo

aca

e

E. clo

aca

e

Key: V – Violet red bile glucose agar plate, V0 – at 0hr, V-2

– dilution factor

86

Table 8: Identification of Fungal Isolates

Dilut

ion

facto

r

0hr 6hr 12hr 18hr 24hr 30hr 36hr 42hr 48hr 54hr 60hr 66hr 72hr

10-2

Asperg

illus

niger

Penicil

lium sp

Asperg

illus

fumiga

tes

Fusar

ium

sp

Penicil

lium sp

Fusar

ium

sp

Penicil

lium sp

A.

fumigat

es

A.

fumig

ates

A.

fumig

atus

C.

tropic

alis

A.

niger

C.

tropic

alis

10-4

Fusari

um sp

A.

niger

A.

niger

Muco

r sp

A.

fumiga

tes

Muco

r sp

Fusari

um sp

A.

fumigat

es

A.

fumig

ates

Candi

da

tropic

alis

A.

niger

A.

fumiga

tes

A.

fumig

ates

10-6

Asperg

illus

flavus

Penicil

lum sp

A.

flavus

Candi

dia

krusie

Penicil

lium sp

A.

fumig

ates

A.

niger

Rhizop

us sp

C.

krusie

Rhizo

pus sp

C.

tropic

alis

Penicil

lium sp

C.

tropic

alis

10-8

A.

flavus

A.

niger

A.

niger

Muco

r sp

A.

fumiga

tes

A.

fumig

ates

A.

niger

PenicilI

lium sp

A.

niger

C.

krusie

A.

niger

A.

niger

Rhizo

pus sp

10-10

Mucor

sp

A.

flavus

A.

flavus

A.

fumig

atus

C.

krusie

A.

niger

Fusari

um sp

Penicill

ium sp

Rhizo

pus sp

C.

tropic

alis

C.

tropic

alis

C.

krusie

C.

tropic

alis

87

3.3 EVOLUTION OF MICROBIAL FLORA DURING CASSAVA

RETTING

Cassava retting has been observed as succession of microbial population

and this succession is dependent on the sensitivities of micro-organisms to

acidic conditions that developed during the process. Table 9 summarizes the

percentages occurence of isolates on Plate Count Agar (PCA). Bacillus

macerans and E.coli had the highest frequency 23.085% and 20.51%,

respectively of the total isolates obtained. The percentage occurence of isolates

on Violet Red Bile Glucose Agar (VRBG) are summarized in Table 10.

Klebsiella pneumoniae showed the highest frequency of occurrence (29.31%),

E.coli and Enterobacter sp. showed the lowest, 10.34% and 10.34%,

respectively. Table 11, summarizes the percentage occurence of isolates

obtained on de Mann, Rogosa and Sharpe agar (MRS). Leuconostoc

mesenteriodes showed the highest frequency of occurrence (20.4%),

Lactobacillus brevis having lowest frequency (06.12%). The percentages

occurence of isolates on Potato Dextrose Agar (PDA) are summarized in Table

12. Aspergillus niger and Aspergillus fumigatus had the highest frequency of

occurrence 20.00% and 20.00%, respectively. Table 13, summarizes the

percentage bacterial isolates characterized. Lactic acid bacteria (Lactobacillus

sp.) had 21.23% out of eighteen (18) bacterial isolates obtained. These results

suggest their acid resistance capacity. The percentage of fungal isolates

88

characterized is summarized in Table 14. Aspergillus species had highest

frequency of occurrence (47.69%). These results suggest their abundance in

nature. The microbial successions at the different retting periods are

summarized in Table 15. These reveal that microbial successions are dependent

on time and pH of the retting medium.

89

3.2 PERCENTAGE OCCURRENCE OF ISOLATES ON

RESPECTIVE MEDIA

Table 9: Percentage occurence of Isolates on Plate Count agar (PCA)

Organism (s) Freq. of occurence Frequency (%)

Escherlichia coli 08 20.51

Lactococcus lactis 06 15.38

Streptococcus sp. 07 17.95

Streptococcus faecalis 04 10.26

Bacillus pumulan 01 02.56

Bacillus macerans 09 23.08

Bacillus subtilis 04 10.26

Total 39

90

Table 10: Percentage Occurences of Isolates on Violet Red Bile Glucose

agar (VRBG)

Organism (s) Freq. of occurence Frequency (%)

Shigella sonnei 07 12.07

Escherlichia coli 06 10.34

Enterobacter sakazaki 10 17.24

Klebsiella pneumonia 17 29.31

Enterobacter sp. 06 10.34

Entrobacter cloacae 12 20.69

Total 58

91

Table 11: Percentage Occurrence of Isolates on de Mann, Rogosa & Sharpe

agar (MRS)

Organism (s) Freq. of occurence Frequency (%)

Lactococcus lactis 08 16.33

Lactobacillus delbruecki 05 10.20

Lactobacillus fermentum 05 10.20

Lactobacillus sp. 09 18.37

Lactobacillus brevis 03 06.12

Leuconostoc mesenteriodes 10 20.41

Lactobacillus plantarum 09 18.37

Total 49

92

Table 12: Percentage Occurrence of Isolates on Potato Dextrose agar

(PDA)

Organism (s) Freq. of occurence Frequency (%)

Aspergillus niger 13 20.00

Aspergillus flavus 05 07.69

Aspergillus fumigatus 13 20.00

Fusarium sp. 05 07.69

Mucor sp. 04 06.15

Penicillum sp. 08 12.31

Candida krusie 05 07.69

Candida tropicalis 08 12.31

Rhizopus sp. 04 06.15

Total 65

93

Table 13: Percentage Occurrence of Bacterial Isolates

Organism (s) Freq. of Occurence Frequency (%)

Shigella sonnei 07 04.79

Echerlichia coli 14 09.59

Enterobacter sp. 28 19.18

Klebsiella pneumonia 17 11.64

Lactococcus lactis 14 09.59

Streptococcus sp. 11 07.53

Bacillus sp. 14 09.59

Lactobacillus sp. 31 21.23

Leuconostoc mesenteriodes 10 06.85

Total 146

94

Table 14: Percentage occurence of Fungal Isolates

Organism (s) Freq. of occurence Frequency (%)

Aspergillus sp. 31 47.69

Fusarium sp. 05 07.69

Mucor sp. 04 06.15

Penicillum sp. 08 12.31

Candida sp. 13 20.00

Rhizopus sp. 04 06.15

Total 65

95

Table 15: Microbial Succession at the different Fermentation Periods

TIME INTERVALS (HR)

Isolate (s) 0 6 12 18 24 30 36 42 48 54 60 66 72

Escherlichia coli 4 4 2 2 1 1

Lactococcus lactis 1 1 1 6 3 1 1

Shigella sonnie 2 1 1 1 2

Aspergillus niger 1 2 2 1 2 1 2 2

Fusarium sp. 1 1 1 2

Aspergillus flavus 2 1 2

Mucor sp. 1 2 1

Streptococcus sp. 1 1 2 1 1 1

Enterobacter sakazaki 2 2 1 3 1 1 1 1

Penicillum sp. 2 2 1 2

Lactobacillus delbrueckii 2 1 1 1

Lactobacillus fermentum 1 1 1 1 1

Enterobacter cloacae 1 2 1 1 1 3

Klebsiella pneumonia 1 2 2 2 3 3 2 2

Aspergillus fumigatus 1 1 2 2 2 2 1 1 1

Enterobacter sp. 1 1 1 1 2

Candida krusie 1 1 1 1 1

Streptococcus faecalis 1 1 1 1

Lactobacillus sp. 1 1 1 1 1 1 2 1

Lactobacillus brevis 1 1 1

Leuconostoc mesenteriodes 1 2 1 3 1 1 1

Bacillus pumulans 1

Bacillus macerans 1 1 1 3 1 1 1

Lactobacillus plantarum 1 1 3 2 2

Rhizopus sp. 1 1 1 1

Candida tropicalis 2 3 3

Bacillus subtilis 1 1 2

96

3.4 DETERMINATION OF TIME PROFILE OF ENZYMATIC

ACTIVITIES IN CASSAVA RETTING

3.4.1 Effect of Enzymatic Activities on Cassava Retting Time

The amylase, pectinase and cellulase activities are depicted in Fig. 1. The

amylase activities were maximal at 48h while at 12h the activities were

minimal. Pectinase activities were maximal at 72hr but cellulase activities were

maximal at 12hr. Analysis of variance data confirmed the dependence of the

enzyme activities during cassava retting on retting time.

3.4.2 Effect of pectinases Activities

Pectinases activities (Pectin lyase (PL), Polygalacturonase (PG) and

Pectinesterase (PE)) are illustrated in fig. 2. Pectin lyase (PL) and

polygalacturonase (PG) exhibited optimum activities at 24h and no significant

activity at 0 – 12h. Pectinesterase activity (PE) was maximal at 72h. These

results suggest PL and PG being dependent on microbial activities and PE is

dependent on low pH. Analysis of variance data confirmed the dependence of

pectinases activity during cassava retting on retting time.

97

Fig. 1: Profile of Enzymatic Activities during Retting

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 50 60 70 80

EZY

ME

AC

TIV

ITIE

S U

MO

L^1

Time (HR)

Amylase

Pectinase

Cellulase

98

PECTINASES ACTIVITIES

Fig. 2 Profile of Pectinase Activities

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50 60 70 80

EZY

MA

TIC

AC

TIV

ITIE

S U

MO

L^1

TIME (HR)

PL

PG

PE

99

3.5 EFFECT OF pH , TITRABLE ACIDITY (%) AND CYANIDE

CONTENT ON CASSAVA RETTING.

The physiocochemical parameters such as pH, titrable acidity and cyanide

content of the retting cassava were determined. As shown in Fig 3, a fast

decrease of the pH was observed in the root from 6.5 to 4.5 after 72h. Titrable

acidity increased strongly from 0.010% at 12hr to 0.054% at 72hr as shown in

Fig 4. These results are an indication that cassava retting is made in acidic

medium. The cyanide content in cassava root decreased progressively during

retting, from 1.46×103 to 3.1×10

2. This result is shown in fig 5. The cyanide

content varied very little during the first 36h of retting, but decreased drastically

from 9×102 to 3.1×10

2 after 48h of retting. This result suggests that the retting

process was a detoxification process.

100

Fig. 3: Evolution of pH during Cassava Retting

0

1

2

3

4

5

6

7

0 12 24 36 48 60 72

pH

Time (Hr)

101

Fig. 4: Evolution of Titrable Acidity during Cassava Retting

0

0.01

0.02

0.03

0.04

0.05

0.06

0 12 24 36 48 60 72

Titr

ab

le a

cid

ity

(%)

Time (Hr)

102

Fig. 5: Cyanide Activities during Cassava Retting

0

1

2

3

4

5

6

7

8

9

10

0 12 24 36 48 60

CN

(m

g/kg

-1)

Time (Hr)

103

3.6 EFFECT OF SIZE OF CASSAVA CUTTINGS ON RETTING

TIME

The effect of size of cassava cuttings on retting was determined in an

attempt to know or determine if the size of the cuts affects retting time. The

result is shown in Table 16. It can be observed that sizes 2.5cm and 5.0cm cuts

retted in 3 days, but that of 12.5cm took longer than 3 days to ret. This is an

indication that size of cassava cuttings affects retting time.

104

Table 16: Size of Cassava cuts on Retting time

Size of cassava cylinder (cm) Retted (days)

2.5cm 3 days

5.0cm 3 days

7.5cm 4 days

10.0cm 4 days

12.5cm 5 days

105

3.7 FERMENTATION MODULATION

3.7.1 Effect of Temperature on Cassava Retting

Varying temperatures were used to determine its effect on cassava retting.

These results were reflected in Table 17. The optimum temperature was

between 350C - 45

0C. At 45

0C, retting occurred in less than 2 days. These

results reveal that cassava retting depended highly on temperature.

3.7.2 Effect of Cations on Cassava Retting

As summarized in Table 18, the two (2) cations such as Ca2+

and Mg2+

had effect on the cassava retting. The three cations efficiently affected retting

time. The cationic content of the cassava cuts were determined before soaking

and after soaking. These are shown in Table 19.This could suggest that during

the retting process the micro-organisms involved utilized Ca2+

and Mg2+

.

106

Table 17: Effect of Temperature on Cassava retting time

Temperature (0C) Retted

(days)

30 3 days

35 2 days

40 2 days

45 <2days

107

Table 18: Effect of Added Cations on Cassava Retting Time

Cation (s) Retted (days)

Control

Ca2+

3 days

2 days

Mg2+ 2 days

108

Table: 19 Cationic Contents of Cassava Tubers Before and After Soaking

Mg2+

Ca2+

Before soaking 0.0060%(6mg/100g) 0.0060% (6mg/100g)

After soaking 0.00097%(0.97mg/100g) 0.00012%(1.2mg/100g)

109

3.7.3 EFFECT OF ADDED CHEMICAL AGENTS ON RETTING TIME

3.7.3.1 Effect of Temperature(s) on Cassava retting time in the

presence of Chemical agents.

As summarized in Table 20, two (2) chemical agents namely kerosene

and 1g concrete nail (3 inches) were used to determine their effect on retting

time at different temperatures. At temperature 350C - 40

0C there was stable

reduction in retting time but at 450C, addition of 0.5ml kerosene and 1g 3 inches

nail reduced retting time to less than 2 days. These results suggest that addition

of chemical agents during cassava retting efficiently reduced the retting time but

this is highly depended on the temperature of the retting medium and the size of

cassava to be retted.

3.7.3.2 Effect of Added Chemicals on the Daily pH during Cassava

Retting

The pH of the retting medium with the addition of chemical agents was

determined. These results are shown in Table 21. The pH of the medium

containing 0.1ml/L kerosene, 0.3ml/L and 0.5ml/L kerosene decreased

drastically at the day 3, but that of nail gradually decreased from 6.0 – 4.7.

These results reveal that the quantity of the chemical agents to be added during

cassava retting depended on the size or quantity of cassava to be retted.

110

Table 20: Effect of Temperature(s) on Cassava retting time in the presence

of Chemical agents.

Retting time at different temperature(s)

Agent(s) 25±270C 30

0C 35

0C 40

0C 45

0C

0.1ml kerosene 3days 3days 2days 2days 2days

0.3ml kerosene 2 days 2days 2days 2days 2days

0.5ml kerosene 2 days 2days 2days 2days <2days

3 inches nail 2 days 2days 2days 2days <2days

Control 3 days 3 days 2 days 2 days 2 days

111

Table 21: Effect of Added Chemicals on the daily pH of samples during

Cassava Retting

Agents Day 0 Day 1 Day 2

Day3

0.1ml kerosene 6.2 4.8 4.6 4.4

0.3ml kerosene 6.2 4.6 4.4 4.3

0.5ml kersoene 6.2 4.7 4.5 4.4

Nail 6.0 5.5 5.3 4.7

Control 6.0 4.9 4.7 4.4

112

CHAPTER FOUR

4.0 DISCUSSIONS AND CONCLUSIONS

4.1 DISCUSSION

During the process of retting of cassava, the total viable count of aerobic

mesophiles were high within 12hr of soaking and decreased after 24hr of

soaking. The decrease in count could be due to increase in acidity of the

medium. The proliferation of E. coli especially in the early and intermediate

stages of retting is a characteristic of mixed acid fermentation. This result shows

that E. coli and Streptococcus spp did not survive at a certain point in the retting

state due to acidic condition that developed during the retting process and

perhaps also due to increase in cyanide content in the retting liquor. Presence of

Streptococcus faecalis at the latter stage could be attributed to the water used for

soaking of the cassava roots. Bacillus spp were detected at the later stage of the

retting process. The increasing acidity of the medium could have resulted in the

decreasing growth of these species. According to Oyewole (1992), Bacillus

subtilis and Klebsiella spp contributed significantly to the rotting of cassava

roots. Kobawila et al. (2005) reported that Bacillus subtilis produces amylase

enzymes that are necessary for the breakdown of starch to sugar, which are

needed for the growth of other fermenting microorganism, including lactis.

Certain strains of Bacillus, notably Bacillus pumilus, have the capacity to use

cyanohydric acid for their nutrition. Thus, this can contribute to the reduction of

the cyanide content in the fermentation medium (Kobawila et al., 2005). The

113

studies by Obilie et al. (2003) showed the involvement of Bacillus species in the

textural modification of cassava tissue during fermentation. Essers et al. (1995)

also reported Bacillus spp. as one of the isolates from heap fermenting cassava

roots which was able to cause a softening of cassava tissues.

Enterobacteriaceae were detected during the fermentation. Entrobacteria

mainly Entrobacter sakazaki, Enterobacter cloacae and Klebsiella pneumoniae

were present in high numbers in the retting liquor. The increasing acidity during

the fermentation resulted in their decreasing growth or number at the latter stage

of retting. This might be due to the high number of competing microorganisms

especially lactic acid bacteria.

Out of seven (7) lactic acid bacteria (LAB) isolates, cocci showed the

highest occurence and were mainly Leuconostoc mesenteriodes which

represented 20.41% of the total lactic acid bacteria. There was enhanced

occurence of homofermenting Lactococcus lactis in the early stage of retting

representing 16.33% of the LAB. This agrees with Brauman et al. (1996) that

the rapid growth of Lactococcus lactis in the early stage of retting could be due

to its high resistance to cyanide together with linamarase activity. Lactobacillus

delbrueckii, an obligate homofermentative lactobacillus was also isolated. This

species with Lactobacillus salivarious have been identified in other fermented

plant materials and are important for the acidification process (Coulin et al.,

2006). Lactobacillus fermentum and Lactobacillus brevis were the main species

of the obligate heterofermentative group isolated. Amoa-Awua et al. (1996)

114

have reported the occurrence of L. brevis, L. mesenteriodes and Streptococcus

spp in cassava fermentation. Lactobacillus plantarum, facultative

heterofermentative LAB, was prominent at the end of the fermentation process.

This is consistent with Ben Omar et al. (2000) who reported that L. plantarum

strains are well-known to develop in vegetable fermentations to dominate in the

later phases of fermentation and terminate many of the spontaneous lactic

fermentation as in silage and vegetable fermentation. This agrees well with the

results on the microbial succession in this study. L. plantarum has been known

to be more acid resistant than Leuconostoc spp or many other Lactobacillus spp,

which is one of the reasons why strains of L. plantarum often predominate in

the late stages of vegetable fermentation (Figueroa et al., 1995). This present

study is in agreement with the three-step microbial succession trend observed

by Brauman et al. (1996) where the epiphytic homofermenting microflora was

rapidly supplemented by Lactococcus lactis and then by the heterofermenting

Leuconostoc mesenteriodes, which governed the process and finally

Lactobacillus plantarum became the dominant flora in the last hours.

Aspergillus, Fusarium, Mucor, Penicillum and Rhizopus species were

isolated from the retting cassava. Aspergillus species had the highest frequency

(47.69%) of the total fungal count. This may be probably due to the abundance

of the organism in nature (Fagbemi and Ijah, 2005). Mucor was also isolated

which represents 6.15% of the fungal counts. According to Petrucioli et al.

(1999), the β-glucosidase from Mucor circinelloides showed a broad substrate

115

spectrum, thus indicating great potential as a detoxifying agent for many foods

and feed cyanogenic plants. Yeasts of the genera Candida were also isolated

towards the end of the fermentation. Candida species consisted about 20% of

the total fungal count. Candida tropicalis was the predominate species,

representing 12.3% of the total identified yeast isolates, followed by Candida

krusei. This agrees with Oyewole (2001), who reported that Candida tropicalis,

Candida krusei and Saccharomyces cerevisiae were common in tropical

fermented foods and contribute considerably to development of the final

flavours of products. Oyewole (1990) in his earlier work demonstrated that one

of the yeast strains from cassava fermentations, C. krusei, had a significant

influence on the typical odour of „fufu‟. According to Oyewole (1990), the

occurrence of wild yeasts at high numbers is more alarming than high numbers

of Enterobacteriaceae during cassava processing, due to their more immediate

effect regarding sensory characteristics. The appearance of yeasts only at the

end of the retting process suggests that they could not have played a significant

role in the fermentation (Brauman et al., 1996). Yeasts play a major role in

odour development and, where high yeast biomass is encouraged protein-

enriched products are not (Oyewole, 1992).

The progressive increase in the frequency of LAB and fungi observed

during the retting process (later stage of fermentation) may probably be due to

increased acidity of the medium, which favoured the growth of the

microorganisms. Traditional sour cassava starch fermentation has been reported

116

to be succession of microbial population (Figueroa et al., 1995). Ampe et al.

(2001) suggested that this succession is determined by the sensitivities of

microorganisms to the very acidic conditions that develop during the process.

In this study, the pH of the retting liquor (steep water) was acidic after

24h of fermentation. This explains the high count of lactic acid bacteria and

fungi in the later stage of the fermentation, probably due to increased acidity of

the medium, which favored the growth of the microorganisms. The decrease in

pH recorded throughout the fermentation period may be associated with the

production of some organic acids by the associated microorganism during

fermentation.

There was significant increase in amylase activity at 48h. Fermentation of

cassava tubers is accompanied by a gradual decrease in pH, increase in amylase

activity in the steep liquor, and increased microbial load and lactic acid

concentration (Olukayode & Olusola, 1987).

Cellulase activity decreased drastically to 0.006mg/ml which could be

accepted as insignificant. This agrees with Ampe and Brauman (1994) findings,

that no cellulase or xylanase was observed in their cassava fermentation.

From the pectinase results, pectolytic enzymes of microbial origin are

closely indispensable for the softening to be completed. The depolymerizing

enzymes (PG and PL) were not detected at the 12h but significant activities

were detected after 24h of fermentation. This agrees with Brauman et al. (1995)

that PG and PL are dependent on microbial activity while PE is dependent on

117

low pH. There was significant increase in PE activity. The significant activity of

pectin esterases (PE) in the retting juice suggests its involvement in softening

(Ampe and Brauman, 1994). Softening of tissues during fermentation can be

attributed to enhanced action of pectinolytic and cellulolytic enzymes produced

by microorganisms (Ampe et al., 1995; Agbor-Egbe et al., 1995).

Data from the pH determination showed that there was a significant

decrease in pH of the retting liquor from 6.52 to 4.52 after 72h.This agrees with

Kobawila et al. (2005) that cassava root retting is made in acid medium. This

corresponds with McMahon et al. (1995) report that cyanohydrin breakdown

non-enzymatically to HCN (hydrocyanic acid) and acetone at pH above 4 or

temperature above 350C. Ogunsua (1980) reported that microorganisms in the

fermenting medium convert sugars and starch in the tubers into organic acids,

which decreased the pH.

The titrable acidity (TTA) increased during the fermentation process.

This agrees with the fact that cassava root fermentation is made in acid medium.

Many authors have observed that the trend in pH was opposite to the titrable

acidity; while the pH decreased, the titrable acidity increased (Moorthy and

George, 1998; Ogunsua, 1980; Blanshard et al., 1994 and Alloys and Mings,

2006). The result obtained in this study is consistent with these observations.

Cyanide content in the cassava roots decreased progressively during

fermentation process from 1.46 ×103 to 3.1×10

2mg/kg. The fermentation is thus

a detoxification process. The cyanide content varied very little during the first

118

36h of fermentation, but decreased significantly from 9.4×102 to 3.1×10

2mg/kg

-

1 after 72h of fermentation. The cyanide content of the fermenting cassava

decreased probably due to the presence of cyanide-degrading activities of the

fermenting microorganisms. Microorganisms can grow in cyanide-containing

substrate due to their anaerobic metabolism, their alternative metabolism

regarding the respiratory chain and their capacity to detoxify cyanide by

splitting the CN-radical into carbon and nitrogen (Cereda and Mattos, 1996).

Esssier et al. (1995) have reported that the genus Bacillus possesses linamarase

enzymes by which they are capable of reducing the initial hydrogen cyanide

concentration from 2.2 to 0.7mg/kg within 72hr, thus helping in the

detoxification process. Fusarium, Mucor, Escherichia and Bacillus have been

reported by many authors to possess specific enzyme systems and pathways to

degrade cyanide. The reduction in cyanogen levels has been found to be

strongly correlated to the degree of root size reduction, which is also directly

related to the rate of softening and acidification (Agbor-Egbe and Mbome,

2006).

Strength of cassava cutting was further studied. Cassava cubes/cuts of

size 2.cm and 5.0cm retted efficiently at 3 days while cube size of 12.5cm was

longer (5 days). This indicates that the smaller the cut, the quicker the retting

time. This agrees with Okafor et al. (1984) report that small pieces of cassava

allow retting to be completed more quickly than when whole roots are used.

119

The results of the effect of temperature on cassava retting showed that the

optimum temperature was between 350C - 40

0C. This is an indication that

temperature had a very strong effect on the retting time. At 450C, the retting

time was less than 2 days which is not good for detoxification to be efficient.

These results agree with the report of Ampe et al. (1994) that the temperature of

400C was found to be the upper limit for retting to occur and thus the profile

closely matches that of the mesophilic bacterial growth. Ogbo (2006) in his

work reported that most influential factor affecting retting time is temperature.

A temperature of 300C has been found to be on its own a factor strong enough

to abolish observations of any contributions towards retting speed from other

factors. Analysis of retting time data at 250C to 30

0C showed that addition

of 0.3ml kerosene/L, 0.5ml/L and 6 inches nail (1g) significantly shortened

retting time of cassava which, corresponded to retting time at 350C – 40

0C. At

350C – 40

0C however, all samples showed the same retting time during the test.

A 0.3ml/L and 0.5ml/L kerosene at 25 – 300C shortened the retting time when

compared to 0.1ml/L kerosene at the same temperature. The results of this study

disagree with the report of Ogbo (2006), that kerosene was not efficacious in

shortening retting time of cassava.

Nail, or in chemical terms the element, iron, placed in retting water

underwent several chemical reactions and brought about a remarkable influence

on retting. Nail being in contact with the cassava water made it to rust which at

120

the end changed the steep water colour to black. This could be explained thus,

brown hydrated iron (III) oxide produced initially as rust probably reduced by

products of microbial activity in the retting liquor to iron (II) oxide (Ogbo,

2006). Iron (11) oxide is more readily utilized by almost all microorganism but

ferric iron (Fe3+

) and its derivatives are extremely insoluble thus making the

iron uptake difficult (Prescott et al., 2005).

4.2 CONCLUSION

Conclusively, this study has shown that soaking of cassava roots in water

is the essential feature for the efficient processing and the most effective

technique to maximize cyanide reduction. Adequate succession of the micro

flora during the retting period, especially the predominance of lactic acid

bacteria during the latter stage gives the product a better shelf life due to the

acidic nature of the final product. It is also evident that, the degree of root size

reduction, the pH values of the steep liquor, adequate temperature and the

enzymatic activities influenced the break down of cyanohydrins which led to the

softening (retting) of the roots. Addition of chemical agents to aid in faster

retting time is essential but on the contrary the side effects has to be checked.

121

Appendix A

ANOVA for Amylase, Pectinase and cellulose

Sources of variation Sum of squares df Mean square F Sig.

Amylase activity Between groups 1.451 5 .290 348154.960 .000

Within groups .000 6 .000

Total 1.451 11

Pectinase activity Between groups 2.918 5 .584 5835.200 .000

Within groups .001 6 .000

Cellulose activity Between Groups 2.902 5 .580 1393040.840 .000

Within groups .000 6 .000

Total 2.902 11

Homogenous Subsets

Amylase activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 12hr 2 .0080

24hr 2 .1240

36hr 2 .1440

72hr 2 .7010

60hr 2 .7375

48hr 2 .8815

Sig 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogenous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000

122

Pectinase activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 12hr 2 02000

24hr 2 .2500

36hr 2 .5000

48hr 2 1.0100

60hr 2 1.1000

72hr 2 1.5600

Sig 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogenous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000

Cellulose activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 72hr 2 .0060

60hr 2 .1290

48hr 2 .1760

36hr 2 8590

24hr 2 .9195

12hr 2 1.3300

Sig 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000

123

One Way Analysis of Variance for PL, PG, and PE activities

Sources of variation Sum of squares df Mean square F Sig.

PG activity Between groups 39975.429 6 6662.571 18655.200 .000

Within groups 2.500 7 .357

Total 39977.929 13

PL activity Between groups 50134.429 6 8355.738 23396.067 .000

Within groups 2.500 7 .357

Total 50136.929 13

PE activity Between Groups 3.913 6 .652 9861.222 .000

Within groups .000 7 .000

Total 3.914 13

Homogeneous Subsets

PL activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 0hr 2 .0000

12hr 2 .0000

72hr 2 51.0000

60hr 2 55.0000

48hr 2 89.0000

36hr 2 120.5000

24hr 2 178.5000

Sig 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000.

124

PG activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 0hr 2 .0000

12hr 2 .0000

60hr 2 50.0000

72hr 2 50.0000

48hr 2 92.0000

PL activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6

Duncan a 0hr 2 .0000

12hr 2 .0000

72hr 2 51.0000

60hr 2 55.0000

48hr 2 89.0000

36hr 2 120.5000

24hr 2 178.5000

Sig 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

36hr 2 135.0000

24hr 2 139.0000

Sig. 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000.

125

PE activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6 7

Duncan a 0hr 2 .1500

12hr 2 .2200

24hr 2 .49000

36hr 2 1.0125

48hr 2 1.1075

60hr 2 1.4300

72hr 2 1.565

Sig 1.000 1.000 1.000 1.000 1.000 1.000 1.000

Mean for groups in homogeneous subsets are displayed.

PE activity

Subset for alpha = 0.05

Time of fermentation N 1 2 3 4 5 6 7

Duncan a 0hr 2 .1500

12hr 2 .2200

24hr 2 .49000

36hr 2 1.0125

48hr 2 1.1075

60hr 2 1.4300

72hr 2 1.565

Sig 1.000 1.000 1.000 1.000 1.000 1.000 1.000

Means for groups in homogeneous subsets are displayed.

a. Uses Harmonic Mean Sample Size = 2.000

126

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