UNIVERSITY OF BUEA

FACULTY OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

A Research Project Submitted to the Faculty of Science, University of Buea, in Partial

Fulfilment of the Requirements of the Award of a Bachelor of Science (B.Sc.) Degree

in Biochemistry.

By

NGOMONWING CLOVIS AFESE

(SCO7A157)

SUPERVISED BY

Michael A. G. Boyo M.Sc. (Leeds)

Senior Clinical Biochemist

JULY, 2010 .

SWEET POTATO’S beta-AMYLASE HYDROLYSIS OF

CASSAVA STARCH FROM THREE CASSAVA VARIETIES IN

THE SOUTH WEST REGION - CAMEROON

UNIVERSITY OF BUEA

FACULTY OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY AND MICROBIOLOGY

A Research Project Submitted to the Faculty of Science, University of Buea, in Partial

Fulfilment of the Requirements of the Award of a Bachelor of Science (B.Sc.) Degree

in Biochemistry.

By

NGOMONWING CLOVIS AFESE

(SCO7A157)

SUPERVISED BY

Michael A. G. Boyo M.Sc. (Leeds)

Senior Clinical Biochemist JULY, 2010.

SWEET POTATO’S beta-AMYLASE HYDROLYSIS OF

CASSAVA STARCH FROM THREE CASSAVA VARIETIES IN

THE SOUTH WEST REGION - CAMEROON

ii

DEDICATION

I whole heartedly dedicate this work to my parents, Mr. Ngomonwing David Afese and Mrs.

Ngomonwing Elizabeth Nchang.

iii

UNIVERSITY OF BUEA

FACULTY OF SCIENCE

CERTIFICATION

This is to certify that the research project entitled “Sweet Potato’s beta-Amylase Hydrolysis of

Cassava Starch from Three Cassava Varieties in the South West Region - Cameroon’’

by

Ngomonwing Clovis Afese (SCO7A157)

was carried out in the Department of Biochemistry and Microbiology under the supervision of Mr.

Michael A. G. Boyo

Supervisor................................................. Date................................

Mr. Michael A. G. Boyo

Head of Department.................................. Date................................

Dr. Fidelis Cho-Ngwa

iv

ACKNOWLEDGEMENTS

I am very grateful to my supervisor, Mr. Michael A. G. Boyo, for his advice and thorough evaluation

of this research work. You have inspired me positively and made me learn more about research.

Thank you once again.

Thanks to the lab coordinators of the University of Buea, for allowing me use their lab equipment. I

am grateful to Mr. Nchouanyo Martin, an agronomic engineer at IRAD – Ekona, for providing me the

improved cassava varieties.

All of my gratitude goes to my parents, Mr. / Mrs. Ngomonwing D. Afese, for raising and taking care

of me. I acknowledge their perseverance and endurance over the years. I also thank them for

providing all the finance for this work. I love you so much.

I am thankful to Mr. /Mrs. Nkembe John C., especially for welcoming me and giving me a place to

stay in your home during my three years of schooling in Buea. Thanks to Mr. / Mrs. Ngomonwing

Ignatius. A., for helping me financially to complete this work.

Thanks to my course mates: Ngome Macdonald, Ngam Hyginus and Ngwana Theodore, for sharing

their ideas with me and for their cooperation. I won’t forget to thank my cousin, Chinepoh Clinton,

for helping me during the preparation of cassava starch.

To Pechele Stephani, for editing this work, I say you are wonderful. To Mrs. N. Margarate Ngwing,

Mr. / Mrs. Pekwang Philip., N. Doreen, N. Akewoh, Mbako P., N. Seraphine, Lem A., I say thanks

for your moral support and encouragement.

Above all, I give all the glory to God Almighty for making this research work successful.

v

ABSTRACT

Three improved cassava varieties (8061, 8034 and 8017) were selected. They were obtained from the

Institute of Research and Agricultural Development (IRAD) – Ekona and processed for starch. The

starch (used as substrate) was then hydrolyzed using β-amylase (extracted from sweet potato) by the

Caraway-Somogyi iodine/potassium iodide (IKI) method (1959), over a period of 50min at 10min

intervals. The rate of enzymatic hydrolysis gave the following result: -96.62mg/l/min, -87.03mg/l/min

and -24.19mg/l/min for the 8061, 8034 and 8017 cassava varieties respectively. This result showed

that the 8061 variety hydrolysis easily, while the 8017 variety is resistant to hydrolysis. The 8024

variety is intermediate. Apart from these three cassava varieties, there exist several other varieties in

the South West Region whose rate of enzymatic hydrolysis can also be determined.

vi

TABLE OF CONTENTS

Title Page

DEDICATION.............................................................................................................................ii

CERTIFICATION.....................................................................................................................iii

ACKNOWLEDGEMENTS.......................................................................................................iv

ABSTRACT.................................................................................................................................v

TABLE OF CONTENTS...........................................................................................................vi

LIST OF FIGURES.................................................................................................................viii

LIST OF TABLES..............................................................................…...................................ix

CHAPTER1: INTRODUCTION AND LITERATURE REVIEW

1.1 INTROUDUCTON.................................................................................................................1

1.2 LITERATURE REVIEW......................................................................................................2

1.2.1 Biology of Cassava.............................................................................…...............................2

1.2.2 Cassava: Food and Industry...................................................................................................4

1.2.3 Starch Biochemistry...............................................................................................................5

1.2.4 Cassava Starch.......................................................................................................................5

1.2.5 Starch Hydrolysis...................................................................................................................6

1.2.6 Resistant Starch......................................................................................................................7

1.2.7 Amylases..................................................................................................................................8

1.2.8 beta- Amylase...........................................................................................................................8

1.3 Hypothesis................................................................................................................................9

1.4 Statement of Problem................................................................................................................9

1.5 Significance/Rationale of Study................................................................................................9

vii

1.6 Study Objectives..........................................................................................................................9

CHAPTER TWO: MATERIALS AND METHODS

2.1 MATERIALS............................................................................................................................10

2.2 METHODS...............................................................................................................................11

2.2.1 Cassava Varieties ...................................................................................................................11

2.2.2 Preparation of Cassava Starch Flour ......................................................................................12

2.2.3 Preparation of Reagents..........................................................................................................13

2.2.4 Extraction of Crude β-amylase from Sweet Potato.................................................................14

2.2.5 Preparation of Pure Starch Standard Stock..............................................................................14

2.2.6 Measurement of Absorbance of Pure Starch.............................................................................15

2.2.7 Preparation of 1.00g/l Stock of Cassava Starch Varieties.......................................................15

2.2.8 Measurement of Initial Absorbance of Cassava Varieties.......................................................15

2.2.9 Rate of Enzymatic Hydrolysis................................................................................................16

CHAPTER 3: RESULTS.......................................................................................................17

CHAPTER4: DISCUSSION, CONCLUSION AND RECOMMENDATION

4.1 Discussion.................................................................................................................................22

4.1.1 Activity of Crude β-amylase Extract......................................................................................22

4.1.2 Measurement of Absorbance and Lugol’s iodine test.......................................................…...22

4.1.3 Rate of Hydrolysis...................................................................................................................23

4.2 Conclusion.................................................................................................................................23

4.3 Recommendations....................................................................................................................23

REFERENCES...............................................................................................................................24

viii

LIST OF FIGURES

Title Page

Figure1: Procedure for Cassava Starch Extraction..............……….............................................13

Figure2: A Plot of Starch Concentration against Time for the 8061 Cassava Variety..................19

Figure3: A Plot of Starch Concentration against Time for the 8034 Cassava Variety………......20

Figure4: A Plot of Starch Concentration against Time for the 8017 Cassava Variety...................21

ix

LIST OF TABLES

Title Page

Table1: Enzymatic Hydrolysis of Crude amylase Extract for the 8061 Variety.....................17

Table2: Enzymatic Hydrolysis of Crude amylase Extract for the 8034 Variety.....................18

Table3: Enzymatic Hydrolysis of Crude amylase Extract for the 8017 Variety.....................18

1

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Cassava (Manihot esculenta Crantz) is one of the main staple food crops for sub-Saharan African

people and it has been estimated that it provides food for over 500million people in the world

(Abu et al., 2006; Cock, 1982). It is perennial tropical root crop, cultivated for its starchy

tuberous roots (FAO, 2006). Cassava is basically a carbohydrate (starch) food, with the edible

part of a fresh cassava root containing 75-80% moisture, 32-35% carbohydrate, 2-3% protein,

0.70-2.50% ash and 0.1% fat, fibre (Ihekoronye and Ngoddy, 1985; Oluwole et al., 2004). As a

source of starch, cassava is highly competitive. The roots contain more starch by dry weight,

than almost any other food crop and its starch is easy to extract using simple local technologies

(FAO, 2006).

According to the report by Kendi (2002), there are five varieties of cassava grown in the South

West Region: two local (local red and local white) and three improved (8061, 8034 and 8017)

varieties. Local farmers have different uses for these varieties and this has lead to the planting of

more than one variety of cassava in a farmer’s farm. Kendi further mentioned that the improved

varieties out-yield the local ones and respond better to fertilizer application. The average tons per

hectare for the local varieties range from 8-15, while that of the improved varieties go as high as

26tons per hectare (Basong, 1989)

About 10% of starch comes from cassava roots and over the past years, the global demand for

cassava starch has increased (FAO, 2006). Despite the fact that cassava serves primarily as a

2

food crop, it is used as a raw material for most starchy industries such as brewing and food

industries (Balagopalan, 2002; Cock, 1982). Sin-Yie and Chun-Ling (1983) showed that the

alcohol yield for cassava starch is more than those of other starchy tubers such as potato.

However, the concept of resistant starch has evoked new interest on starch and resistant starch is

now considered to give functional properties. This is applicable in many brewing and food

industries (Englyst et al., 1982; Visser et al., 1997). Using β-amylase extracted from sweet potato

tubers, different varieties of cassava starches will hydrolyze or digest at different rates. In this

study, the rate of hydrolysis of three improved cassava varieties (8061, 8034 and 8017) in the

South West Region of Cameroon would be compared.

1.2 LITERATURE REVIEW

1.2.1 Biology of Cassava

Cassava (Manihot esculenta Crantz) belongs to the dicotyledonous family Euphorbiaceae (Alves,

2002). It is a shrub, characterized by sympodial branching and grows between 1- 4m in height.

The main stem of a cassava plant is mostly divided into 2-4 branches, that may also have

branches on their own and these branchings are followed shortly by flower. However, in flower

buds are aborted before maturity, giving the impression that cassava stem branching don’t

produce flowers (Alves, 2002).

Cassava plants undergo C3 photosynthesis (Edwards et al., 1990). It has a relatively high rate of

photosynthesis of 43μmolCO2/m2/s and is photosynthetically efficient at temperatures as high as

450C (Angelov et al., 1993; Edwards et al., 1990, Hunt et al., 1977). According to El-Sharkawy

and Cock (1990), the optimal temperature for photosynthesis for field grown cassava is 350C but

3

the range for optimal photosynthesis is 250C-45

0C. Thus cassava is well adapted to the tropical

environment.

Cassava organs contain cyanogenic glycosides: linamarin (95%) and lotaustralin (lesser

amounts). Cassava cultivars with 100-500mg CN equivalents Kg -1

are classified as “bitter”

cassava, while those with less than 100mg CN equivalents Kg -1

are classified as “sweet”

cassava (Wheatley et al, 1993). McMahon et al (1995) further showed that the amount of

cyanogenic glycoside is dependent on the plant’s age, cultivar, cultural practice and

environmental conditions. The hydrolysis of linamarin leads to the release of the poisonous

compound hydrogen cyanide (HCN) (Cooke and Coursey, 1981).

The main carbohydrates storage is the root. In the first 75 days after the planting of cassava,

carbohydrates accumulation is mainly in the leaves rather than in the stems and roots.

Afterwards, carbohydrate is mainly allocated to the storage roots reaching 60% of the total dry

matter after 4 months (Howeler and Cadavid, 1983). The fully developed cassava storage root is

made up of three sections: the periderm (bark), cortex (peel) and the parenchyma. The periderm

comprises about 3% of the total mass of the root and it is a thin layer that can be easily removed

from the exterior of the root. The parenchyma, which is the edible part, consists mainly of starch

and makes up about 85% of the total root mass (Wheatley and Chuzel, 1993). The shape and size

of cassava roots is dependent on the cultivar (variety) and environmental factors.

Post-harvest deterioration is a major constraint for the marketing of fresh cassava roots. The

roots are highly perishable and deteriorate within 24-74 hours after harvest (FAO, 2006). This

deterioration is manifested biochemically by the production of phenolic compounds whose

polymerization leads to the discolouration of the roots (Wheatley and Chuzel, 1993). Wheatley

4

and Chuzel, 1993 further concluded that this process of deterioration is an oxygen dependent

process and it can be inhibited by placing the roots in an anaerobic environment. Tissue

dehydration at the site of mechanical damage on the roots promotes the rapid onset of

deterioration (Ghosh et al., 1988). Secondary deterioration is caused by microbial infection of

damaged tissues (Wheatley and Chuzel, 1993).

1.2.2 Cassava: Food and Industry

Cassava serves as the main staple food for over 500million people in the world since it is a

perennial root crop (Abu et al, 2006; Balagopalan, 2002). It also contributes significantly to the

livelihood of these people. Cassava is used as food in various ways across the world. In West

Africa, cassava roots are fermented in a pot for 4-7 days, boiled and pounded into “Fufu” and

eaten with vegetable soup (Lancaster, 1982). In Cameroon, Nigeria, Ghana, Garri is one of the

most important cassava food. Cassava roots are peeled, grated and dewatered in a sack and

allowed to ferment for 2-4 days. This is followed by sieving, then fried in a shallow iron pan and

stirred continuously until it becomes dry and crisp. Palm oil is added sometimes during frying

(Balagopalan, 2002). Cassava Macaroni, is prepared by blending cassava flour, groundnut and

wheat semolina in the ratio 60:12:15. This food is used to feed children because of its high

protein content (Balogapalan, 2002). Other cassava foods include Farina, eaten in South America

and West Indies. Cassava is also important in animal feeds. Various parts of the plant such as the

leave stem and roots are used to feed animals. The high energy value of cassava makes it a good

source of carbohydrate in animals diets (Omole, 1977).

Cassava is an important commodity in industries, mainly because of its starch which is used in

the production of various items. Cassava starch which is used in the production of adhesives on

5

stamps, stiffening agents in textiles, coating on pills and papers, binder in concrete, bottle-

labelling adhesives. It is also used as raw material for making of ethanol, sugar-syrups and

bakery products (Balagopalan, 2002; FAO, 2006).

1.2.3 Starch Biochemistry

Starch is one of the most abundant substances in nature. They are synthesized naturally in a

variety of grains and root crops such as cassava, corn, potato, wheat, rice (FAO, 2006).

Furthermore, starch is the most important form of carbon reserve in plants with respect to the

amount produced, its commercial importance and its distribution among different plant species

(Martin and Smith, 1995).

Starch comprises of two types of glucan polymers: amylase and amylopectin (Martin and Smith,

1995), because of the existence of two types of linkages: the α-1, 4 and the α-1, 6 glycosidic

linkages. Amylose is made up of linear chains of α-1, 4 linkages of glucose subunits which are

typically about 500-2000 residues long. It makes up about 30% of total starch. On the other

hand, amylopectin is highly branched and accounts for about 70% of total starch in plants. This

is due to the presence of α-1, 6 glycosidic linkages. The degree of branching in amylopectin is

approximately 25 glucose units in the unbranched segment (Martin and Smith, 1995; Okita,

1992; Smith et al., 1995; Stryer, 5th

edition).

1.2.4 Cassava Starch

Cassava starch is stored in the amyloplast of thickened roots. The starch content of a mature

cassava root ranges from 74-85% of the dry weight (Munyikwa et al, 1997). An 18 months study

of the growth of cassava starch shows that growth continues up to six months. No further growth

is observed after six months (Moorthy and Ramanujan, 1986).

6

“Cassava makes a really excellent starch” says Danila Mejia, an agricultural engineer with

FAO’s Agricultural Support System Division. “Compared to starches derived from most other

plants, it has greater clarity and viscosity, and it’s very stable in acidic food products. It has

excellent properties for use in non-food products such as pharmaceuticals and thermoplastics.”

(FAO, 2006).

Cassava starch comprises 14-24% amylose (Kawabata et al., 1984; Ketiku and Oyenuga, 1972).

On the bases of x-ray diffraction patterns, starch is classified into three types: A, B and C.

Cassava starch comprises mainly the A-type that is characteristic of cereal starch (Guilbot and

Mercier, 1985). This A-type pattern is typically characterised by closely packed helices,

compared to the more open B-type arrangement.

Cassava starch consists of 0.08-1.54% crude fat, 0.03-0.6% crude protein and 0.75-4%

phosphorus (Munyikwa et al., 1997; Soni et al., 1985). About 10% of starch is gotten from

cassava (FAO, 2006).

Cassava has many advantages for starch production: high level of purity; a neutral taste;

excellent thickening characteristics; desirable textural characteristics and it is a relatively cheap

source of raw material, containing a high concentration of starch that can be greater than or equal

to the properties offered by other starches like maize, rice, wheat, sweet potato (Cassavabiz).

1.2.5 Starch Hydrolysis

Starch determination methods are broadly grouped into acid hydrolysis or enzymatic hydrolysis

(Anon, 1987). Starch can be hydrolyzed by HCl completely (Poonam and Dahel, 1995).

However, low glucose yield, the formation of large amounts of salt and the need to use corrosive

resistant equipment are some of the disadvantages of using acid hydrolysis.

7

On the other hand, enzymatic hydrolysis of starch has the following advantages: specificity of

the enzyme allows the production of sugars with well-defined chemical and physical properties,

and the milder enzymatic reaction conditions result in few site reactions and less browning (Haki

and Rakshit, 2003a: Poonam and Dahel, 1995)

1.2.6 Resistant Starch

The term “resistant starch” was first mentioned by Englyst et al. (1982). The concept of resistant

starch has evoked new interest on starch and resistant starch is now considered to give functional

properties and this finds application in many different brewing and food industries.

Berry (1986) classified starch according to their behaviour when incubated with enzymes

without prior exposure to dispersing agent, as follows:

i) Rapidly digestible starch (RDS): It is found in high amount in starch food cooked by moist

heat, such as bread, potatoes. It is measured chemically as the starch which is easily converted to

the constituent glucose molecules in 20 minutes of enzymatic digestion.

ii) Slowly digestible starch (SDS): It is measured chemically as starch converted to glucose after

a further 100 minute of enzymatic digestion.

iii) Resistant starch (RS): It is starch not hydrolyzed after 120 minutes of incubation with an

enzyme (Englyst et al., 1992). Some factors influencing the formation of resistant starch include

granules structure (Holm et al., 1987) amylose:amylopectin ratio (Berry, 1986), linearization of

amylopectin.

8

Resistant starch has received much attention for its potential functional properties. Being a

functional fibre, its fine particles and bland taste has made possible the formulation of a number

of food and brewing product with better consumer acceptability (Sajilata et al., 2006).

1.2.7 Amylases

Enzymes that break down or convert starches into sugars are called amylases (Schegel, 2003).

They are known as the most important of carbohydrate degrading enzyme produced by various

bacteria species (Syn and Chen, 1997), yeast (Moreira et al., 2001), fungi (Tani et al., 1986) and

plants (Mahmona, 1993). Frangui (2001) further reported that amylases of plant origin have the

highest productivity. Amylases are classified into three types: α-amylase, β-amylase and γ-

amylase. Foods that contain much starch, but little sugar such as rice, sweet potato, germinating

corn, taste slightly sweet as they are chewed because amylase turns some of their starch into

sugar.

1.2.8 beta-Amylase

β-amylase (EC 3.2.1.2), has alternate names: 1,4-α-D-glucan maltohydrolase, glycogenase and

saccharogen amylase. It is a form of amylase which catalyses the hydrolysis (from the non-

reducing end) of the second α-1, 4-glycosidic bond, cleaving off two glucose units (maltose) at a

time.

With the understanding of the nature of β-amylase and its hydrolytic potential, the use of this

enzyme has been extended to various fields such as brewing industries (Haq et al., 2002). This

enzyme is extensively used in starch liquefaction and alcohol production (Dhanya and Swetha,

2009).

9

1.3 Hypothesis

It is hypothesized that starches are classified into three types (RDS, SDS and RS), according to

their behaviour when incubated with enzymes, without prior exposure to dispersing agents

(Berry, 1986).

1.4 Statement of Problem

Different cassava varieties show different resistances to hydrolysis by β-amylase (Englyst et al.,

1992).

1.5 Significance / Rationale of Study

The use of cassava starch in brewing industries, for the production of alcohol, food industries

and in biofuel has gained much importance in recent years (P. Prema, 1986). In addition, the

consumption of alcohol , especially in developing countries, has increased drastically in recent

decades (S. Mesaki, 1995).This study would enable local producers of alcohol to know which

cassava varieties are most suitable for use to produce alcohol and bakery products, with the least

time and lowest cost of production.

1.6 Study Objectives

Main Objective: The main objective is to hydrolyze cassava starch from three cassava

varieties.

Specific Objectives: The specific objectives are:

i. Identification of three cassava varieties in the South West Region – Cameroon.

ii. Extraction of cassava starch flour.

iii. Extraction of crude amylase from sweet potato.

10

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

A. Reagents:

5.00g/l pure starch standard (BDH laboratory supplies; GPRTM

, prod. 302644k)

1.00g/l starch solution (for the 3 cassava varieties)

10% HCl – used as stopping reagent to stop the hydrolysis reaction.

1:10 dilution ratio of Lugol’s iodine solution – used as working iodine stock, to

test for the presence of starch.

Tap water

B. Equipment:

Digital weighing balance (Scout ProSPU402) – used for weighing.

Spectrophotometer (JENWAY 6300Spectrophotometer) – used for measuring

absorbance.

Centrifuge (Model 0408-2)

Oven (HotBox Oven with fan, Size 3; Gallenkamp) – used to dry starch.

Beakers (200ml, 1000ml)

11

Test tubes (20ml)

Centrifuge tubes (15ml)

Droppers (2ml, 3ml)

Volumetric flask (250ml)

Pipette (10ml)

Cuvette (3ml)

Plastic dishes

Tin containers (1200g Ovaltine, two 2500g Peak milk tins) – used during the

drying process.

Bunsen burner – used to boil water.

Spatula

2.2 METHODS

2.2.1 Cassava Varieties:

Three improved cassava varieties: 8061, 8034 and 8017 (about 2.5kg per variety), were gotten

from the Institute of Research and Agricultural Development (IRAD) - Ekona. They were freshly

harvested and processed for cassava starch production the same day.

12

2.2.2 Preparation of Cassava Starch Flour

Cassava starch flour was prepared according to the steps adopted by Ikegwu et al. (2009). The

process is summarized in Figure 1. The roots of the three cassava varieties were labelled

accordingly and peeled with a knife. Next, they were washed with tap water and chipped into

small pieces of irregular shapes and ground using a cassava milling machine. After grinding, the

pulp (mash) was put inside a salt bag and sieved by pouring water inside the bag, while shaking

with the hand. The filtrate was then left to settle for 12 hours in a bucket. After 12 hours, the

supernatant was decanted and discarded and the sediment put in a tin container. This was

followed by drying in an oven for 2 days at 80°C. After they were dry, they were ground using a

corn mill machine, to form cassava starch flour. The flour of the cassava varieties were then put

into a plastic paper, labelled accordingly and kept for the experiment.

13

Fresh cassava roots

Peeling

Washing

Rasping (Milling)

Filtering (Sieving) in a bag

Settling (Sedimentation) (12hours)

Decantation

Drying (800C)

Milling

Cassava starch flour

Figure 1. Procedure for cassava starch extraction.

2.2.3 Preparation of Reagents

10% HCl was used as the stopping reagent. A 250ml stock of it was prepared by adding 25ml of

concentrated HCl into 200ml of tap water. Tap water was again added to reach the 250ml mark.

It was then mixed.

14

A 1:10 dilution ratio of lugol’s solution (composed of 5.0g I2 and 10.0g KI, dissolved in 100.0ml

of distilled water) was prepared and used as working I2 solution. This is to test for the presence

of starch. The 1:10 dilution ratio was prepared by measuring 1ml of lugol’s solution and diluting

in 9ml of tap water.

2.2.4 Extraction of Crude β-amylase from Sweet Potato

The sweet potato (Ipomoea batatas) variety used was the improved variety TIB1 (Tropical

Ipomoea Batatas number 1). This is the “yellow” type. The extraction procedure is based roughly

on Methods in Enzymology (Abelson and Simon, 1992). Three small tubers of sweet potato were

bought from the Muea market, peeled and washed. They were then chipped into small pieces and

ground using a milling machine. 200.0g of the pulp (ground) was weighed in a 200ml beaker

using a digital weighing balance and 100.00g of tap water (weighed in a 200ml beaker) added.

The mixture was then mixed using a spatula and put into six 15ml centrifuge tubes. It was then

spun at 3000rpm for 20mins. After 20mins of spinning, the supernatant was collected using a

3ml dropper and put into 2 test tubes. This supernatant is suspected to contain the crude β-

amylase.

2.2.5 Preparation of Pure Starch Standard Stock

5.00g of pure starch standard was weighed using a digital weighing balance and put inside a

200ml beaker. 50.0ml of tap water was then added, by measuring 5 times using a 10.0ml pipette.

The mixture was then stirred with a spatula for 1min and poured into a 1000ml beaker containing

900ml of warm water. The resulting 950ml solution was then transferred into a 1000ml

measuring cylinder, then tap water added to the 1000ml mark. It was then poured into a plastic

15

dish, well stirred and labelled. This is the stock of pure starch standard with a concentration of

5.00g/l.

2.2.6 Measurement of the Absorbance of Pure Starch

The absorbance was measured against a blank of air at 620nm. An empty dry cuvette was

inserted into the spectrophotometer and zeroed. 2.0ml of the 5.00g/l pure starch stock prepared

was pipette into a test tube and 1ml of lugol’s iodine solution added to it. This solution was then

shaken and poured into a cuvette. The absorbance was measured using a spectrophotometer at

620 nm wavelength.

2.2.7 Preparation of 1.00g/l Stock of Cassava Starch Varieties

This process is known as gelatinization or liquefaction. For the 8061 variety, 1.00g of it was

weighed using a digital weighing balance, put into a 200ml beaker and 50.0ml of tap water added

and stirred for 1min. It was then transferred into a 1000ml beaker containing 900ml boiling

water. The resulting 950ml solution was then poured into a 1000ml cylinder and tap water added

to reach the 1000ml mark. The 1000ml solution was then poured into a plastic dish, stirred and

labelled. The same was done for the other varieties and labelled accordingly.

2.2.8 Measurement of the Initial Absorbance of Cassava Varieties

The initial absorbencies of the three cassava varieties were determined at 620nm without the

enzymatic reaction. This was taken as absorbance at time 0min. 2.0ml of the 1.00g/l stock of the

8061 cassava variety was pipetted, after stirring the stock. Next, 1ml of lugol’s iodine solution

was added and shaken. The mixture was then poured into a cuvette and the absorbance measured

using a spectrophotometer. The same procedure was done for the 8034 and 8017 varieties.

16

2.2.9 Rate of Enzymatic Hydrolysis

The β-amylase activity of the crude extract and the rate of enzymatic hydrolysis were determined

using the Caraway-Somogyi iodine/potassium (IKI) method (1959), with some modifications

such as no buffer was used. The experiment was performed at 250C. According to this method,

the first step is known as gelatinization which involves preparation of the cassava varieties stocks

in 2.2.7.

For the 8061 cassava variety, 5.0ml of 1.00g/l stock was pipette and put into 5 different test

tubes. 2.0ml of the crude enzyme extract was then added to each of the 5 test tubes and the times

noted. The incubation with crude enzyme was allowed to proceed for 50min at 10min intervals

from the first to the fifth test tube. After every 10mins, the hydrolysis reaction was stopped by

adding 2ml of 10% HCl into the test tube. 2.0ml of the resulting 9ml was then put into another

test tube and 1ml of lugol’s iodine solution added. The mixture was then poured into a cuvette

and the absorbance measured at 620nm. The same procedure was done for the 8034 and 8017

stock.

17

CHAPTER THREE

RESULTS

The results obtained for the hydrolytic ability of the crude β-amylase extract from sweet potato,

monitored over a 50mins period at 10mins intervals is presented in Table 1, Table 2 and Table 3,

for the 8061, 8034 and 8017 varieties respectively.

Table 1. Enzymatic hydrolysis of crude β-amylase for 8061 variety.

Time (min) Absorbance Concentration (mg/l)

0 1.325 8461

10 0.874 5581

20 0.715 4566

30 0.630 4023

40 0.560 3576

50 0.472 3014

18

Table 2. Enzymatic hydrolysis of crude β-amylase for 8034 variety.

Time (min) Absorbance Concentration (mg/l)

0 1.292 8250

10 0.832 5313

20 0.617 3940

30 0.572 3653

40 0.543 3467

50 0.520 3321

Table 3. Enzymatic hydrolysis of crude β-amylase for 8017 variety.

Time (min) Absorbance Concentration (mg/l)

0 0.609 3889

10 0.532 3397

20 0.504 3218

30 0.476 3040

40 0.446 2848

50 0.401 2561

19

Figure 2. A plot of starch concentration against time for the 8061 cassava variety.

20

Figure 3. A plot of strach concentration against time for the 8034 cassava variety.

21

Figure 4. A plot of starch concentration against time for the 8017 cassava variety.

NB: Series1 shows the time course of starch hydrolysis by crude extract from sweet potato.

22

CHAPTER FOUR

DISCUSSION, CONCLUSION AND RECOMMENDATION

4.1 DISCUSSION

4.1.1 Activity of Crude β-amylase Extract

The results showed that the concentration of the three cassava varieties decreases with time from

8461 - 3014mg/l, 8250 - 3321mg/l and 3889 - 2561 mg/l for the 8061, 8034 and 8017 varieties

respectively. This implied that sweet potato contains a considerable amount of β-amylase which

degrades starch.

4.1.2 Measurement of Absorbance and Lugol’s iodine test

The measurement of starch absorbance at 620nm showed that with time, the absorbencies

decreased as hydrolysis proceeded. An iodine test involves the reaction between large chains of

starch and iodine to form a blue-black colour. The darker and more intense the blue-black colour

is, the longer the chains and/or the higher the concentration of starch (Noonnan, 1996). In

addition, Noonan (1996) reported that an iodine test is used to check for the completion of starch

conversion. It is not required to perform such a test but it is seen as a good practice as it will

indicate the brewer, for example, of problems during mashing such as improper temperature.

23

4.1.3 Rate of Hydrolysis

From the graphs in Figure 2, Figure 3 and Figure 4, the rate of hydrolysis for the cassava

varieties used was gotten to be -96.62mg/l/min, -87.03mg/l/min and -24.19mg/l/min for the

8061, 8034 and 8017 varieties respectively. The negative sign signifies that the hydrolysis

reaction decreased with time. This means more sugars were formed as the reaction time went on.

This was indicated by the formation of a faint blue-black colour.

4.2 CONCLUSION

Sweet potato contains a good quantity of the enzyme β-amylase which can be used by local

alcohol producers to degrade starch to sugars (maltose). Furthermore, the best variety of cassava

which can be exploited by local alcohol producers and local food factories, to produce alcohol or

bakery products is the 8061 variety. The cuttings of this variety or cultivar can be purchased from

IRAD and cultivated.

4.3 RECOMMENDATIONS

I. This work was carried out without the use of a buffer. So in order to get quality

results, the Caraway-Somogyi iodine/potassium iodide (IKI) method (1959) of starch

hydrolysis should be followed and optimum working conditions respected.

II. According to Mr. Nchouanyo Martin, an agronomic engineer at IRAD – Ekona, there

exist over 80 varieties of cassava in the South West Region. These other varieties can

be studied to determine and compare their rate of hydrolysis.

24

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