Synthesis of resistant starch in three Rwandese cassava varieties and its applicability in

yoghurt processing

By:

Herve Mwizerwa

A56/81986/2015

A proposal submitted as a partial fulfillment for the award of Masters of Science

Degree in Food Science and Technology of the University of Nairobi

Department of Food Science, Nutrition and Technology

2016

1
PROJECT NAME:EFFECTS OF SOAKING AND BLANCHING THE
CYANIDE CONTENT IN PROCESSING OF CASSAVA CRISPS

PRESENTED BY:FREDRICK MAINA THIGA

INDEX NUMBER: 2071011438

INSTITUTION : THIKA TECHNICAL TRAINING INSTITUTE

COURSE CODE : 2403

EXAMINER : KNEC

COURSE : DIPLOMA IN FOOD SCIENCE AND PROCESSING

TECHNOLOGY

SERIES : NOVEMBER 2019

SUPERVISOR : MADAM JOAN

This project is submitted to the Kenya National Examination Council in
partial fulfillment for the award in Diploma in food science technology.
i
ii
 Abstract

Starch is the most plentiful granular polysaccharide in plants where it is found in the chloroplast of

leaves and the amyloplast. Based on digestibility starch is classified into three types: rapidly

digestible starch, slowly digestible starch and resistant starch. Resistant starch (RS) is defined as

the sum of starch and products of starch breakdown which are not absorbed in the small intestine

of humans. In bowel, RS serve as a substrate for microbial fermentation that produce carbon

dioxide, methane and short chain fatty acids known to prevent colon diseases. Cassava is a staple

food crop in Rwanda where it is mainly utilized as flour for paste (Ugali) or consumed raw, boiled,

fried or roasted and its leaves are consumed as vegetables. Differences in amounts of RS present in

popular cassava varieties in Rwanda and effects of applying different heat treatments on cassava

starch is not yet known. On the other hand, popular products such as yoghurt rely heavily on

imported relatively expensive corn starch, pectin and gelatin for their production in Rwandan

context. Application of cassava starch with good component of beneficial healthy RS in their

processing as a thickening agent is yet to be tested. There is also a wide gap in cassava value

addition in Rwanda where its industrialization is solely limited to manufacturing of packaged flour.

The current proposed project seeks to determine the levels of RS in popular cassava varieties in

Rwanda and to demonstrate opportunity in adding value to cassava starch by synthesizing RS and

testing its applicability in processing acceptable yoghurt. This study will provide scientific

information on which cassava variety and treatment can produce large amount of RS and will

demonstrate the opportunity for cassava product diversification as well as providing yoghurt rich

in probiotics and prebiotics (RS) as a preventive measure against colon cancer.

Key words: Resistant starch, Cassava, yogurt

iii
 Abbreviations and Acronyms

AACC: American Association of Cereal Chemists

AMG: Amyloglucosidase

ANOVA: Analysis of Variance

AOAC: Association of Official Analytical Chemists

EFSA: The European Food Safety Authority

FAO: Food and Agriculture Organization

GOPOD: Glucose Oxidase–Peroxidase Reagent

HMT: Heat Moisture Treatment

ISO: International Organization for Standardization

M.C: Moisture Content

MINAGRI: Ministry of Agriculture and Animal Resources

MT: Metric Ton

NISR: National Institute of Statistics of Rwanda

RAB: Rwanda Agriculture Board

RDB: Rwanda Development Board

Rpm: Rotation per Minute

RS: Resistant Starch

SCFA: Short Chain Fatty Acids

USAID: United States Agency for International Development

WAI: Water Absorption Index

WSI: Water Solubility Index

iv
 Terminology

Cassava (Munihor esculenta) is a woody plant in the Euphorbiaceae family. It is widely

cultivated as an annual crop in tropical and subtropical regions.

Starch is a natural polymer occurring in plant organisms as an indirect product of photosynthesis

synthesized from glucose, which is formed from carbon dioxide and water and is the major

component of most of plant-originated foodstuffs.

Resistant starch is the sum of starch and products of starch degradation which have not been

absorbed in the small intestine of healthy humans. They are transported to the large intestine where

they are fermented by microorganisms to yield SCFAs, carbon dioxide and methane.

Starch Gelatinization is a process of breaking down the intermolecular bonds of starch molecules

in the presence of water and heat which irreversibly damages the starch granule in water.

Starch retrogradation is a process in which disaggregated amylose and amylopectin chains in

gelatinized starch reassociate to form more ordered structures as the cooked starch cools.

Hydrothermal modification of starch refers to physical modification of starch without

gelatinization or damage of the starch granules in terms of size, shape or birefringence through a

controlled application of heat and moisture.

v
Starch annealing refers to alterations within the crystallites of the granules which consist of

incubation of granular starch in excess water for a period of time at a temperature below the

gelatinization temperature.

Heat-moisture treatment refers to hydrothermal treatment which consists of treatment of starch

granules at restricted levels of moisture (<35%) during a given time (15minutes–16hours) and

0
temperature (84–120 C) above the glass transition temperature but below the gelatinization

temperature.

vi
 Table of content

ABSTRACT ..................................................................................................................................... III

ABBREVIATIONS AND ACRONYMS ........................................................................................ IV

TERMINOLOGY ............................................................................................................................. V

TABLE OF CONTENT .................................................................................................................. VII

CHAPTER ONE: INTRODUCTION ................................................................................................ 1

1.1. Background information ........................................................................................................ 1

1.2. Statement of the problem ....................................................................................................... 3

1.3. Justification of the study ........................................................................................................ 4

1.4. Objectives .............................................................................................................................. 6

1.4.1. Main objective ................................................................................................................... 6
1.4.2. Specific objectives ............................................................................................................. 6

CHAPTER TWO: LITERATURE REVIEW .................................................................................... 7

2.1. Cassava .................................................................................................................................. 7

2.2. Starch ..................................................................................................................................... 7

2.2.1. Rapidly digestible starch (RDS) ........................................................................................ 8
2.2.2. Slowly digestible starch (SDS) .......................................................................................... 8
2.2.3. Resistant starch .................................................................................................................. 8

2.3. Types of resistant starch......................................................................................................... 8

2.3.1. Resistant starch I ................................................................................................................ 8
2.3.2. Resistant starch II ............................................................................................................... 8
2.3.3. Resistant starch III ............................................................................................................. 9
2.3.4. Resistant starch IV ............................................................................................................. 9

2.4. Starch content of Cassava ...................................................................................................... 9

2.5. Resistant starch in cassava ................................................................................................... 10

2.6. Importance of resistant starch .............................................................................................. 10

2.6.1. Prebiotic effect of resistant starch .................................................................................... 10
2.6.2. Prevention of colon cancer............................................................................................... 11
2.6.3. Control of glycemic response .......................................................................................... 11
2.6.4. Enhancement of satiety .................................................................................................... 11
2.6.5. Production of Short Chain Fatty Acids ............................................................................ 11
vii
 2.6.6. Effects on nutrients absorption ........................................................................................ 11

2.7. Effect of processing on resistant starch ............................................................................... 12

2.7.1. Heat moisture treatment of starch .................................................................................... 12
2.7.2. Annealing of starch .......................................................................................................... 12

2.8. Applications of resistant starch in food industry ................................................................. 13

2.9. Yoghurt manufacturing ........................................................................................................ 13

2.9.1. Application of thickeners in yoghurt ............................................................................... 13

CHAPTER THREE: MATERIALS AND METHODS .................................................................. 15

3.1. Determination of the levels of RS in three Rwandese cassava varieties ............................. 15

3.1.1. Raw material acquisition.................................................................................................. 15
3.1.2. Design .............................................................................................................................. 15
3.1.3. Methodology .................................................................................................................... 15
i. Starch Extraction .............................................................................................................. 15
ii. Resistant starch determination ......................................................................................... 15
3.1.4. Data analysis .................................................................................................................... 16

3.2. Evaluation of the effect of hydrothermal modification on the RS content in starch extracted
from Rwandese cassava varieties..................................................................................................... 16
3.2.1. Design .............................................................................................................................. 16
3.2.2. Methodology .................................................................................................................... 16
i. Heat-moisture treatment (HMT) ...................................................................................... 16
ii. Annealing ......................................................................................................................... 17
iii. Starch profiling ................................................................................................................ 17
a. Moisture content and dry matter content determination .............................................. 17
b. Total starch determination ........................................................................................... 17
c. Water absorption index (WAI) and Water Solubility Index (WSI) ............................. 17
d. Pasting profile of starch ............................................................................................... 17
e. Texture analysis ........................................................................................................... 18
3.2.3. Data Analysis ................................................................................................................... 18

3.3. Effect of addition of RS on the quality of yoghurt .............................................................. 18

3.3.1. Design .............................................................................................................................. 18
3.3.2. Methodology .................................................................................................................... 19
i. Yogurt manufacturing ...................................................................................................... 19
ii. Analysis of yoghurt quality .............................................................................................. 20
a. Texture analysis ........................................................................................................... 20
b. Viscosity analysis......................................................................................................... 20
c. Syneresis ...................................................................................................................... 20
d. Acidity and pH ............................................................................................................. 20
e. Total solids ................................................................................................................... 20
f. Levels of RS ................................................................................................................. 20
iii. Sensory evaluation ........................................................................................................... 20
3.3.3. Statistical analysis ............................................................................................................ 21
viii
4.0 WORK PLAN.............................................................................................................................22

5.0 PROPOSED BUDGET.............................................................................................................. 23

6.0 REFERENCES...........................................................................................................................24

ix
 CHAPTER ONE: INTRODUCTION

1.1. Background information

Starch is the most plentiful granular polysaccharide in plants where it is found in the chloroplast of leaves

and the amyloplast (Ellis et al., 1998). It is the main source of energy for many people and in the diet it is

contributed mainly by cereal grains, root crops and tubers (Vegh, 2009).

Based on digestibility starch is classified into three types: rapidly digestible starch, slowly digestible

starch and resistant starch (Pereira and Magali, 2014). Resistant starch (RS) is defined as the sum of starch

and products of starch breakdown which are not absorbed in the small intestine of healthy humans

(Englyst et al., 1992). From there, it is conveyed to the large intestine and it is fermented by

microorganisms into short chain fatty acids (SCFAs), methane and carbon dioxide. Starch’s resistance to

digestion is contributed by different reasons which lead to description of four groups. These are: resistant

starch I (RSI) that is unreachable to digestion by being surrounded in a indigestible medium; resistant

starch II (RSII) which is un-gelatinized starch that is in a granular form and resistant to enzyme digestion;

resistant starch III (RSIII) that is retrograded starch formed through cooling of gelatinized starch and

resistant starch IV (RSIV) which is modified starch due to chemical cross-bonding with reagents, ethers

and esters (Sajilata et al., 2006; Nugent, 2005).

In bowel, resistant starch serve as a substrate for microbial fermentation that produce carbon dioxide,

methane and short chain fatty acids like acetic, propionic and butyric acid that are known to prevent colon

diseases (Noor-Aziah et al., 2011, Nugent, 2005). As a result, resistant starch has got good impact on

diabetes, cardiovascular diseases, colonic health and obesity (Lunn and Buttriss, 2007; Nugent, 2005;

Sajilata et al., 2006; Morales-Medina et al., 2014). Researches in nutrition have demonstrated that RS

1
increases lipid and cholesterol breakdown, cut down the threat of ulcerative colitis, colorectal cancer,

constipation, Type II diabetes and fixes toxins, bile acids, and carcinogens (Haralampu, 2000).

The level of RS in different crops and food products have been studied by Moongngarm (2013) in

Thailand; Pereira and Leonel (2014) in Brazil; Ogbo and Okafor (2015) in Nigeria and these levels in

cassava were found to range from 0.19% to 9.69% of total starch.

RS has also been perceived in different food like breads, breakfast cereals, biscuits, maize, mashed

potatoes and legumes (Pereira and Magali, 2014). The main obstacle to the food industry is the production

of foods with sufficient RS resulting in an important enhancement of consumer’s health (Chung et al.,

2011), and cassava could be one of the important sources that can be incorporated in consumer friendly

foods like yoghurt.

Cassava is a staple food crop in Rwanda with 3,161,470 MT of annual production on 195,910 ha of land in

2014 with an average annual increase of 7.28% since 2010 (FAOSTAT, 2014). Cassava is mainly utilized

in Rwanda as flour for paste (Ugali) or consumed raw, boiled, fried or roasted and its leaves are consumed

as vegetables (Adekunle, 2007; Umuhozariho et al., 2011).The current industrial utilization of cassava in

Rwanda is solely for packaged flour and starch production is planned for the future. This indicates a gap in

value addition to cassava which may not only promote industrial development in Rwanda but also increase

incomes of both farmers and processors. Post-harvest losses for cassava are estimated at more than 30% in

Rwanda (USAID, 2010), therefore, diversification of cassava products can be a solution to post-harvest

losses that may arise from the increased production of this commodity.

The dairy sector in Rwanda has experienced an extreme revolution as a result of many programmes which

have led to an increased milk production from 372,619 tons in 2006 to 706,030 tons in 2014 (NISR,

2015). The National Dairy Strategy envisages an increase of milk production of 13% per year and milk

2
exports is to reach 20 million US dollars per year by 2017(RDB, 2012; MINAGRI, 2013). Value-added

products like cheese and shelf-stable yoghurt, which is currently an emerging milk product, offer attractive

market opportunities though impeded by import of packaging material and some ingredients (MINAGRI,

2013). Hence, sourcing ingredients within the country would lower the cost of production of value-added

milk products. The current proposal is therefore designed to monitor the variations of resistant starch III

content in popular Rwandese cassava varieties and assess its applicability in yoghurt processing.

1.2. Statement of the problem

Levels of RS vary with plant sources and technological treatment preceding their use (Sajilata et al.,

2006).Differences in amounts of RS present in popular cassava varieties in Rwanda and effects of

applying different heat treatments on cassava starch is not yet known. On the other hand, popular products

such as yoghurt rely heavily on imported relatively expensive corn starch, pectin and gelatin for their

production in Rwandan context. Application of cassava starch with good component of beneficial healthy

RS in their processing as a thickening agent is yet to be tested. The rates of colorectal cancer are

dramatically increasing in sub-Saharan Africa (Graham et al., 2012) and many epidemiological studies

confirm the importance of probiotics against cancer (Kumar, 2010). Consequently, there is an increased

demand for low cost prebiotics and probiotics especially among the middle class in Africa, Rwanda

included (Reid et al., 2014, Monachese et al., 2011). Postharvest loss of cassava in Rwanda is estimated at

more than 30% (USAID, 2010) and is due to availability of few appropriate post-harvest preservation

facilities, perishability of the product and limited manufacture of cassava based products. There is also a

wide gap in cassava value addition in Rwanda where its industrialization is solely limited to

manufacturing of packaged flour. The current proposed project seeks to determine the levels of RS in

popular cassava varieties in Rwanda and to demonstrate opportunity in adding value to cassava starch by

synthesizing RS and testing its applicability in processing acceptable yoghurt.

3
 1.3. Justification of the study

Cassava is a staple food crop in Rwanda ranking second after plantains in terms of production quantity

with 3,161,470 MT and third in terms of monetary value with USD. 307,969,520 in 2013 behind plantains

and potatoes (FAOSTAT, 2013). With an annual growth of 7.29% from 2010, cassava was put among the

priority food crops in Rwanda together with maize, wheat, rice, Irish potato, soybean and bean

(MINAGRI, 2012). In Rwanda, cassava is mainly utilized as flour for paste (Ugali) for household

consumption or consumed raw, boiled, fried or roasted and its leaves are consumed as vegetables

(Adekunle, 2007; Umuhozariho et al., 2011). Several studies indicated that manufacturing of cassava

value added products is lucrative and has potential for food security achievement, improved revenue and

decreased post-harvest losses (Saediman et al., 2015; Ukpongson et al., 2011; Ezeh et al., 2011). Cassava

is highly perishable and production of resistant starch, a highly stable product, provides an opportunity to

preserve the produce and add value. Furthermore, diversification of cassava products can be a solution to

the increased production of this commodity and empowering the investment in industrial processing of

cassava products hence enhancing the livelihood of population that rely on cassava farming as their

income.

The dairy sector in Rwanda has experienced an extreme revolution as result of many programmes (RDB,

2012). Introduction of new cattle breeds, insemination program, active diseases eradication program and

the “One Cow per Poor Family” program has increased ownership of dairy cows among Rwandans. This

has led to an increased milk production from 372,619 tons in 2006 to 706,030 tons in 2014 (NISR, 2015)

and the national dairy strategy anticipates an increase of 13% every year (MINAGRI, 2013) hence an

obvious need for milk products diversification. Yoghurt is currently one of the most promising products

that can be produced locally and cost-effectively if all raw materials are from inside the country.

Overreliance on imported corn starch, gelatin or pectin as thickening agent in yoghurt manufacturing can

be reduced by demonstrating possibility of using starch from local cassava in production of healthy starch.
4
Thickening agents are added to yoghurt to provide an acceptable texture, improve its viscosity and

mouthfeel and reduce syneresis (Gonçalvez, 2005). Starch is the mostly used thickener, for it is not

expensive, and if used at below 5%, it does not noticeably affect the taste (Saha and Battacharya, 2010). A

similar effect may be achieved by adding cassava starch. The nutritional value of the added starch can be

enhanced by using it in the form of RS.

Resistant starch is a pre-biotic that when mixed with pro-biotic yoghurt culture will result into symbiotic

and healthy product which has the advantage of providing health enhancing bacteria and the promoting the

growth of naturally occurring microflora thus preventing the occurrence of some gut diseases (Collins and

Gibson, 1999). Those products are expensive and rarely found in developing countries (Monachese et al.,

2011) like Rwanda; therefore the yoghurt enriched with resistant starch III may serve as an alternative

source of prebiotics and probiotics as well as a cost-effective way to cut down the occurrence of such

diseases.

Nutrition contributes to the incidences of colorectal cancer in world (Graham et al., 2012). Though the

prevalence of colorectal cancer in Africa is very little compared to developed countries, there is a trend

toward westernized lifestyle which increases the risk of Africans developing colorectal cancer (Graham et

al., 2012). The treatment and management of this cancer imposes a large financial burden on both health-

care systems and the families of diseased patients. The availability of yoghurt rich in probiotics and

prebiotics (RS) may serve as a preventive measure against colon cancer as well as a relief for the

government and family expenses on colon cancer and other gut diseases treatment.

Currently, cassava is used mainly for flour. Its utilization for starch and ethanol production is still low.

There is an opportunity for development of other innovative high valued products such as resistant starch

as a way of product diversification.

5
Consumers and processors are currently not aware of how local processing conditions lead to development

of RS and its health importance. This study will provide scientific information on which cassava variety

and treatment can produce large amount of RS for both processors and consumers.

1.4. Objectives

1.4.1. Main objective

The overall objective is to synthesize RS from three Rwandese cassava varieties and evaluate its

applicability in yoghurt processing.

1.4.2. Specific objectives

1. To determine the levels of RS in three Rwandese cassava varieties namely NASE14, THE

I92/0057 and GARUKUNSUBIRE.

2. To evaluate the effect of hydrothermal treatment on the RS content in cassava starch extracted

from Rwandese cassava varieties.

3. To develop yoghurt with cassava starch high in RS and test its quality (Texture and viscosity) and

sensory acceptability.

1.5. Hypotheses

1. There is no significant difference in RS content among the three Rwandese cassava varieties

2. Heat-moisture treatment and annealing treatment do not increase the RS content in cassava starch.

3. RS synthesized from cassava starch does not alter the texture, viscosity and sensory properties of

yoghurt.

6
 CHAPTER TWO: LITERATURE REVIEW

2.1. Cassava

Cassava (Manihot esculenta) is the major basis of energy for millions of human beings in third world

st
(Fakir et al. 2012). In 21 century, as a result of enlargement of international trade of cassava based food

and its robust evolution in Africa, cassava has gained an significant position in global agriculture where in

2012 it has reached to more than 280 million tons of harvest, a 60 % increase since 2000 (FAO, 2013).

Cassava root is reach in carbohydrate ranging between 32% and 35% on a fresh weight basis, and

from 80% to 90% on a dry matter basis (Zvinavashe et al., 2011). Cassava contains very little fat

(0.1%) and protein (2-3%) and it is relatively rich in vitamin C and calcium and it has considerable

amounts of B-vitamins group (Ooye et al., 2014).

Variety of cassava is an important factor in production of diversified food products due to the fact that

many parameters like amylase, starch, cyanide, whiteness and sweetness differ from variety to variety

(Jisha et al., 2010; Zhang et al., 2010). In Rwanda, cassava serves as a revenue generating and food

security produce (Mushimiyimana et al., 2011).

2.2. Starch

Starch is a natural macromolecule occurring in plant organisms and is the main make up of most plant-

originated foods and numerous industrial raw materials (Leszczyñski, 2004). It is an indirect product of

photosynthesis as it is synthesized from glucose, which is formed from carbon dioxide and water

(Leszczyñski, 2004). Starch is made up of chains of α-D-glucose linked by the activity of enzymes by 1-4

carbon atoms and 1 - 6 in some cases. The linear chain, known as amylose, contains exclusively α -1, 4

bonds. The branched polymer, amylopectin, contains branches of glucose molecules linked at carbons 1

and 6 (Pierna et al., 2005).

7
Based on digestibility, starch is grouped into three types: rapidly digestible starch, slowly digestible starch

and resistant starch (Pereira and Magali, 2014).

2.2.1. Rapidly digestible starch (RDS)

RDS is the amorphous starch which is chemically determined as the starch converted to the integral

glucose units in 20 minutes of breakdown by enzyme (Sajilata et al., 2006).

2.2.2. Slowly digestible starch (SDS)

SDS is the amorphous starch which is physical inaccessible and due to different reasons is digested

slowly. It is measured as starch transformed to glucose units after 100 minutes of enzymatic treatment

(Sajilata et al., 2006).

2.2.3. Resistant starch

Resistant starch was introduced by Englyst et al. (1982) to designate minor portions of starch that was

unaffected by α-amylase and pullulanase hydrolysis in vitro. Currently, resistant starch is referred as the

sum of starch and products of starch degradation which have not been absorbed in the small intestine of

healthy humans (Englyst et al., 1992).

2.3. Types of resistant starch

2.3.1. Resistant starch I

Resistant starch I (RS I) is the grain starch which is protected by thick cell wall or protein matrix that

prevent diffusion of water into the starch and this, inhibit gelatinization and swelling because of absence

of moisture which makes it unsusceptible to enzymatic hydrolysis (Birt et al., 2013).

2.3.2. Resistant starch II

RSII is the natural starch that is shielded from digestion by the granules arrangement which make it is

distinctive since it maintains its form and resistance during food preparation (Nugent, 2005). RS II has got

8
 o
a gelatinization temperature exceeding 100 c and when cooked below this temperature, it remains

unchanged and resistant hydrolysis (Birt et al., 2013).

2.3.3. Resistant starch III

RSIII is normally made during starch retrogradation (Nugent, 2005). This form has a gelatinization

o
temperature as higher as 170 C and is not affected by cooking (Birt et al., 2013). Amylase enzyme is not

able to hydrolyse RSIII due to the double helices structure formed when starchy foods are cooled and

amylose and amylopectin lose the water-binding capacity which makes the starch molecules to not fit into

the binding site of the enzyme (Birt et al., 2013).

2.3.4. Resistant starch IV

RSIV is a chemically modified starch by etherisation, esterification or cross-bonding with compounds in

order to decline its digestibility (Nugent, 2005).

2.4. Starch content of Cassava

Starch content in mature cassava is around 30 % (Tonukari, 2004). However, this is dependent of cassava

variety (Jisha et al., 2010). The study by Fakir et al. (2012) on starch content of seven cassava varieties

indicated a significant difference in starch content of these varieties where it varied between 15.04% and

24.97% on fresh weight basis. In another study by Richardson (2013) on six varieties of cassava root from

six different countries, reported their starch content to vary from 26.285to 31.9%.

In the food industry, cassava starch is mainly used for the following purposes: custard, thickener, filler,

pharmacological products, binder and stabilizer (Yongfeng, 2013; Ozemoya et al., 2007, Oladunmoye et

al., 2014, Da et al., 2008, Alarcón and Dufour 2012).

Cassava starch is preferred to corn starch due to the nonexistence of unwanted cereal flavor therefore it

finds application in food industry for its unique flavor and its physicochemical performance when heated 9
in liquid medium since it produces better clarity and viscosity as well a low retro-gradation tendency and

gelatinization temperature in comparison to other starches (Demiate and Kotovic, 2011). However it has

got shortcomings including instability to acidic conditions and cooking (Takizawa et al., 2004).

2.5. Resistant starch in cassava

The level of RS in different crops and food products have been studied by Moongngarm (2013) in

Thailand; Pereira and Leonel (2014) in Brazil; Ogbo and Okafor (2015) in Nigeria and these levels in

cassava were found to range from 0.19% to 9.69g/100g of total starch.

Ogbo and Okafor (2015) found that RS in six improved cassava varieties namely, TMS30555, TMS

30572, TMS 693, TMS98/0505, TMS 4(2)1425 and TME varies from 5.7 to 7.07 g/100mg while

Moongngarm (2012) found 9.69% as RS content in cassava root. Pereira and Leonel (2014) found a

significant difference in RS content of 25 different cassava flours varying from 0.19% to 2.2% on dry

weight basis.

Resistant starch type III is estimated to 2.4% in cassava starch and this value can be raised up to 41.3% by

enzymatic treatment followed by Annealing (Lertwanawatana et al., 2015).

2.6. Importance of resistant starch

2.6.1. Prebiotic effect of resistant starch

A food ingredient is qualified as prebiotic when it can escape digestion and intestinal absorption attaining

the large intestine where it changes the native microflora make-up and activity which results in evident

health enhancement properties (Gibson et al., 2004).

RS has been demonstrated to regulate the bowel bacterial content through elevating bacteria that produce

amylolytic and short chain fatty acids (Kovatcheva-Datchary et al., 2009), hence it is recognised as a

prebiotic (Li, 2010).

10
 2.6.2. Prevention of colon cancer

Nutrition researches reported a connection between food, colon bacteria and colon cancer especially in

animal where feeds with high resistant starch have shown evidences of colon cancer prevention (Ridlon

and Hylemon, 2006).

In South Africa, a research on people who eat good amount RSIII containing maize and few dietary fibre

has demonstrated that they exhibited lower frequencies of colorectal cancer in comparison to others who

consume higher amount of dietary fibre, but lower resistant starch content (Ahmed, 2000).

2.6.3. Control of glycemic response

Researches have demonstrated the ability of RS to cut down the insulin response and it was confirmed by

the European Food Safety Authority (EFSA, 2011). The recent studies by Johnston et al. (2010) and Maki

et al. (2012) indicated an enhancement in insulin sensitivity up to 72% after 30g of RSII was

supplemented to the foods of pre-diabetics.

2.6.4. Enhancement of satiety

Though this mechanism is not fully explained yet, many studies reported the importance of RS to

improve satiety (Willis et al., 2009, Anderson et al., 2010). It was reported to reduce the total food

consumption by 15% when the food with 66% RSII HAMS was ingested (Anderson et al., 2010).

2.6.5. Production of Short Chain Fatty Acids

Resistant starch is thus initially broken down by bacterial amylases to produce glucose which is further
broken down into organic acids especially short chain fatty acids (SCFA) like butyrate and lactic acid as

well as CO2, H2 and CH4 (Eliasson, 2004).

2.6.6. Effects on nutrients absorption

It has been demonstrated that the replacing some digestible starch by RS reduces blood glucose (Eliasson,

2004), and it is suggested that RS has the ability to increase calcium and magnesium uptake by improving

11
their solubility in the intestine through acidic condition created by fermentation of RS (Younes et al.,

1993).

2.7. Effect of processing on resistant starch

It has been proven that there is possibility to raise RS up to 20% by varying a number of input process

variables like water content, pH, temperature and time, heating and cooling rounds, freezing and drying

(Englyst et al., 1987). Cooking, autoclaving, parboiling, baking, microwave irradiation, extrusion cooking

and storage were testified to rise up the RS content at different extents (Hódsági et al., 2012; Faraj et al.,

2004; Yadav et al, 2010). Milling, fermentation and germination are thought to reduce the formation of

resistant starch (Devi et al., 2009; Nigudkar, 2014; Abd-Elmoneim et al., 2004; Buddrick et al., 2015).

2.7.1. Heat moisture treatment of starch

There are evidences that native starches can treated by different heat moisture conditions to increase their

RS content as in the attempt to prepare the low calorie cassava pearl by Vijayakumari et al. (2014), heat

moisture treatment of cassava starch for 48 hours has raised the RS content from 1.9% to 27.1% . Heat

moisture treatment has also been applied to jackfruit seeds where their natural starch had 32% RS and it

0
was increased up to 52% by heating these seeds at 80 C for 16 hours and the moisture content was 25%

(Kittipongpatana and Kittipongpatana, 2015). Sankhon et al. (2014) reported that heat moisture treatment

of African locust bean starch increased its RS content from 33.38% to 50.14%.

2.7.2. Annealing of starch

Vijayakumari et al. (2014) reported that annealing of cassava starch for 72 hours increased its RS content

up to 28.6% while Lertwanawatana et al. (2015) reported an increase from 2.4% to 41.3% of Resistant

starch type III in cassava starch treated by pullulanase, a debranching enzyme and high pressure

annealing.

Annealing was also reported to increase the RS type III in amylomaize, barley, pea and lentil starch from

9% to 19% (Vasantahn and Bhatty, 1998).

12
 2.8. Applications of resistant starch in food industry

A high consumption of ultra-processed plant products may cause a decrease in dietary fibre intake which

makes the production and application of resistant starch in food to become inevitable (Leszczyñski, 2004).

In contrast to the conventional dietary fibers which can change the general food organoleptic qualities, RS

is natural, flavorless, white, small size particle, high gelatinization temperature, good extrusion qualities

and low water binding capacity (Sajilata et al., 2006). This permits the production of food products with

appreciable texture, appearance and mouth feel and can serve in designing of new functional foods or

probiotics encapsulation (Homayouni et al., 2013).

RS has been included in many foods like cheese, ice cream, yoghurt, milk, bread, corn flakes, cakes,

muffins, pasta and batter (Homayouni et al., 2013).

2.9. Yoghurt manufacturing

Yogurt is mostly liked for being highly nutritive good sensory properties (Sfakianakis and Tzia, 2014).

During its manufacturing, some changes occur which lead to the development flavor and texture of yogurt

and it involves three main stages, homogenization, pasteurization and fermentation (Lee and Lucey, 2010),

2014).

2.9.1. Application of thickeners in yoghurt

Thickeners are applied in yogurt in order to improve the texture of yogurt, which is an important factor to

determine yogurt quality (Gonçalvez et al., 2005). Starch, gelatin and pectin are the most used thickeners

in yogurt manufacturing with starch being more preferred due to a number of factor including being

cheap, simplicity of processing and it does not affect the sensory properties (Saha and Battacharya, 2010).

These thickeners interact with the casein present in yogurt to make a strong structure which permits the

development of rheological behavior (Gámbaro, 2002).

The study by Gonçalvez et al. (2005) reported that gelatin and starch applied in different proportions,

positively affect the viscosity, creaminess and ropiness of yogurt and reduced significantly the occurrence

13
of syneresis. However it was reported that high concentration of pectin may affect the perception of aroma

in yogurt (Routray and Mishra, 2011). Pancar et al. (2015) indicated a correlation between viscosity of

yogurt and the amount of thickeners used.

Resistant starch has been used to enrich yogurt by Aryana et al. (2015) and they have reported that this

enrichment with RSII produced an acceptable yogurt and RSII was not affected by yogurt heat treatment.

2.10. Identified gaps

 Researches on cassava in Rwanda have emphasized on productivity and resistance to diseases

leaving out the postharvest utilization of cassava.

 The improvement of cassava resistant starch by hydrothermal treatment has not been tested in

Africa despite it producing half of the world production.

 The cross link of two value chains; cassava and dairy products, has not been tested.

14
 CHAPTER THREE: MATERIALS AND METHODS

3.1. Determination of the levels of RS in three Rwandese cassava varieties

3.1.1. Raw material acquisition

Cassava roots samples from three different cassava varieties (NASE14, THE I92/0057 and

GARUKUNSUBIRE) of 10 months in the field will be collected from Rwanda Agriculture Board field

research stations of Rubona and Rilima. The samples will be brought to laboratory for analysis which will

be consisted of first extracting starch followed by isolating and measurement of RS.

3.1.2. Design

Three replicates of analysis will be conducted for each cassava variety and the mean value will be

recorded.

3.1.3. Methodology

i. Starch Extraction

Wet method will be applied for starch extraction as per Benesi et al. (2004). After washing, peeling and

chopping, cassava roots will be pulverized for 5 minutes in a blender. The mash will be put in 1:10 ratio of

water volume, agitated for 5 minutes followed by filtering with a cotton cloth. The sediment will be

allowed to settle for 2 hours and the top fluid will be discarded. The process will be repeated until clear

water is observed. Sediment starch will be dried for 24 hours and kept.

ii. Resistant starch determination

RS will be determined using AOAC Method 2002.02 (2003). Briefly described, non-resistant starch will

be hydrolyzed to glucose by the collective activity of pancreatic α-amylase (4mL) and amyloglucosidase

(AMG) (3U/mL) for 16 hours at 37°C. This reaction will be ended by adding ethanol (4mL) and RS will

be recuperated as pellet by centrifugation. RS in the pellet will be dissolved in 2M KOH (2mL) by

strongly stirring in an ice–water bath. This solution will be buffered with acetate buffer and the starch will

15
 be hydrolyzed to glucose with AMG. The aliquot (0.1 mL) will be put in duplicate into glass test tubes (16

× 100 mm), and then treated with 3.0 mL of glucose oxidase–peroxidase reagent (GOPOD) and the tube

contents will be shaken on a Vortex mixer and incubated at 50°C for 20 minutes. The spectrophotometer

will be set to 0 with the blank solution and the absorbance of every solution will be measured at 510nm

against the blank solution. The average of duplicate absorbance values will be recorded.

3.1.4. Data analysis

The sample will be analysed in triplicate and the mean value will be considered. The mean values of RS in

three cassava varieties will be compared by means of T-test at 5% significance level

3.2. Evaluation of the effect of hydrothermal modification on the RS content in starch

extracted from Rwandese cassava varieties

3.2.1. Design

In order to evaluate its effect on RS levels, three varieties of cassava will be treated with two methods of

hydrothermal modification; annealing and Heat-Moisture treatment (HMT) respectively. Three HMT

(18%, 24% and 30% moisture content) and two annealing treatments (45°C and 55°C) will be applied to

starch from each of the three cassava varieties. This will be treated in 3x5 factorial design of 3 cassava

varieties and 5 hydrothermal treatments.

3.2.2. Methodology

i. Heat-moisture treatment (HMT)

HMT will be applied as per the method by Franco et al. (1995). The 45°C and 55°C moisture contents of

the samples will be increased up to 18% and 24% respectively by the addition of the suitable quantity of

distilled water and mixed; the samples will be hermetically closed in glass jars and heated in an oven at

100°C for 16 hours. Starch samples will be cooled, air dried and subjected to RS measurement.

16
 ii. Annealing

The method of Knutson (1990) will be applied for starch annealing with slight modification. Starch (100g)

will be heated in excess water at 45°C and 55°C for 24 hours. Samples will be centrifuged to eliminate

excess water and air-dried and thereafter subjected to RS determination.

iii. Starch profiling

a. Moisture content and dry matter content determination

To determine the moisture content and dry matter content, the ISO 1660 (1996) method will be applied

which consists of drying the sample at 130 (± 3) °C for a period of 90 minutes.

b. Total starch determination

The total starch content will be determined as per AOAC (1996) Official Method 996.11Starch (Total) in

Cereal Products Amyloglucosidase – α-Amylase Method.

c. Water absorption index (WAI) and Water Solubility Index (WSI)

(WAI) and (WSI) will be determined as per Anderson (1982) with minor adaptation. Starch (3.0 g) will be

mixed with 30 ml distilled water and heated at 60°C for 1 hour in a water bath, then centrifuged at 3000× g

for 15 minutes and the supernatant will be recuperated in a pre-weighed dry aluminum bowls. The

remaining deposits will be drained off by leaving tubes containing samples to stand inverted for 10

minutes. The supernatant will be dried in an air oven at 105°C for 12 hours for WSI measurement.

d. Pasting profile of starch

Pasting properties of the flours will be measured with a Brabender Viscograph using AACC Approved

Method 61.02. Flour (40 g) and water (420 ml) will be weighed and mixed to make slurry.

17
The slurry will be heated from 30°C and temperature increased at a constant rate of 1.5°C per minute until

93°C where it will be held for 15 minutes, after the temperature will be decreased at a constant rate of

1.5°C per minutes until 30°C and then held at this temperature for 15 minutes. The resistance to stirring as

a viscosity curve will be recorded. The pasting temperature, peak viscosity, hot paste viscosity and cold

paste viscosity will be determined from the pasting curve. The breakdown viscosity which is the peak

viscosity minus trough viscosity, setback which is equivalent to cold paste viscosity minus trough

viscosity and consistency which equals to cold paste viscosity minus peak viscosity will be also

calculated.

e. Texture analysis

Texture Analyser will be used to objectively evaluate the texture of starch according to the method by

Kaszab et al. (2002).

3.2.3. Data Analysis

Three varieties will be treated with 4different hydrothermal modification which will make a 3x4 factorial

experiment. Each experiment will be done in triplicate which will make and be analysed in a factorial

design of 36 treatments and the analysis of variance (ANOVA) will be performed at P ≤ 0.05 using

GenStat application.

3.3. Effect of addition of RS on the quality of yoghurt

3.3.1. Design

Yogurt will be processed as per figure 1 below. Cassava starch will be applied in three different

proportions, 0.1%, 0.5% and 1% respectively. Corn starch will be applied as a control at 0.6%.

The processed yogurt will be analysed for its quality and sensory parameters. Sensory evaluation data will

be analysed in a complete randomised design.

18
 3.3.2. Methodology

i. Yogurt manufacturing

The yoghurt will be processed as per Gonçalvez (2005). Commercial skim UHT milk will be standardized

at 11.1 % (w /w) total solids using 8 % white commercial sugar and dried skimmed milk. Thickeners will

be added in the following proportions: 0, 0.1%, 0.5% and 1% of cassava starch. All the components will

be mixed and heated at 90ºC for 15 minutes. The solution will be cooled at 42 ºC and inoculated with a

commercial starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) then incubated at

42°C awaiting the pH to reach 4.5. The yogurt will be cooled to 5°C and kept at the same temperature for

5 days before testing. Sample without thickener will be used as control.

The RS contents in yoghurts (Treated with cassava starch high in RS and ordinary yoghurt) will be

measured as per AOAC (2003): Official Method Number: 2002.02.

Preliminary treatment of milk (standardization)

Homogenization

Starch
Heat treatment (950C for 15 Min)

Cooling (430C)
Starter culture

Inoculation with starter culture

Stirred yogurt

Packaging (250C)

Cooling and storage (50C)

Sensory Evaluation

Figure 1: Generalized scheme for Yoghurt manufacturing

19
 ii. Analysis of yoghurt quality

a. Texture analysis

Texture Analyser will be used to objectively evaluate the texture of modified yoghurt as per Kaszab et al.

(2002).

b. Viscosity analysis

Viscosity will be measured by Viscometer as per Djurdjević et al. (2002) which consist of comparing the

apparent viscosity of the yogurt to the apparent viscosity of the reference liquid (Water).

c. Syneresis

The method by Gonçalvez et al. (2005) will be used. Yogurt will be centrifuged and the supernatant will

be removed and measured as percentage syneresis.

d. Acidity and pH

Titratable acidity of Fermenting yogurt will be determined according to AOAC method 942.15 (2000).

Yogurt (10 ml) will be titrated with 0.1 N NaOH using phenolphthalein as an indicator. The titratable

acidity will be recorded as percent lactic acid. pH will recorded using a pH meter.

e. Total solids

Total solids in yogurt will be determined as per ISO 13580: 2005 method.

f. Levels of RS

Levels of RS in yogurt treated with modified starch will be determined as per AOAC Method 2002.02

(2003).

iii. Sensory evaluation

A panel of 12 people will be tasked to evaluate the quality characteristics (taste, colour, flavour, mouthfeel

and overall acceptability) of all yoghurt samples treated with different thickeners using a 7-point hedonic

scale where 7= like very much, 6= like moderately, 5= like slightly, 4= dislike slightly, 3= dislike

moderately, 2= dislike very much and 1= dislike extremely.

20
 3.3.3. Statistical analysis

Sensory evaluation data will be analysed in a complete randomised design when evaluating the overall

acceptability of the yogurt treated with cassava starch containing high RS content. Yoghurt quality

parameters will be measured in triplicate and the mean value will be considered and the ANOVA will be

performed at 5% significance level.

21
 4.0 Work plan

2016 2017
SN Activities Sub/activities Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr
Proposal write up
1
Proposal presentation
2
Purchasing of
3 materials
Starch
Pre-testing
4 extraction
5 Sample collection
Starch
extraction
Proximate
analysis
Laboratory analysis Application
6
of RS in
yogurt
Sensory
evaluation
Data analysis
7
Report writing
8
Application for
9 publication
Dissertation
10 presentation

22
 5.0 Proposed Budget

SN Items Quantity Unit Price (USD) Total price (USD)

Reagents
1 Megazyme Resistant starch assay kit 1 400 400
2 Megazyme starch assay kit 1 250 250
3 D-Glucose standard solution 100 ml 80 80
4 Ethanol 500 ml 100 100
5 Potassium Hydroxide 1 Kg 50 50
6 Sodium acetate buffer 1L 40 40
7 Sodium maleate buffer 50g 110 110
Sub Total1 1030
Processing Materials
8 Skim UHT milk 100L 0.5 50
9 Blender/chopper 1 50 50
10 Cassava Samples 150 0.5 75
11 Weighing scale 1 20 20
12 Filter cloth 5m 14 70
13 Pectin 500g 30 30
14 Gelatine 500g 20 20
15 Corn starch 1 kg 20 20
16 Yogurt starter culture 1 Pack 60 60
Sub Total2 395
Write up requirements
17 Internet (GB) 50GB 2 100
18 Airtime 50 0.5 25
19 Printing 400 0.1 40
20 Binding 4 5 20
Sub Total3 185
Others
21 Transport 3 100 300
22 Laboratory fees 20 days 5 100
23 Accommodation at field work 9 10 90
24 Panelist allowances 20 5 100
25 Research assistant (BSc) 20 days 5 100
26 Ethical clearance 1 50 50
27 Research permit 1 50 50
28 Data processing 1 50 50
Sub Total4 840
Sub Total 2450
29 Contingency (5%) 122.5
Total 2572.5

23
 6.0 References

1. AACC International. Approved Methods of Analysis, 11th Ed. Method 61-02.01. Determination of

the Pasting Properties of Rice with the Rapid Visco Analyser. Approved September 06, 2011

AACC International, St. Paul, MN, U.S.A. http://dx.doi.org/10.1094/AACCIntMethod-61-02.01.

2. Abd-Elmoneim O.E., Schiffler B, Bernhard R. (2004). Effect of fermentation on the starch

digestibility, resistant starch and some physicochemical properties of sorghum flour.

Nahrung/Food. 48(2):91–4.

3. Adekunle J. (2007). Culture and Customs of Rwanda. Greenwood Publishing Group. London, UK.

4. Ahmed R. (2000). Fermentation of dietary starch in humans. American Journal of

Gastroenterology. 95(4):1017-1020.

5. Alarcón, F. and Dufour D. (2012). Sour Cassava Starch in Colombia. In “Cassava in the third

millennium: modern production, processing, use, and marketing systems” (Ed. by Ospina B and

Ceballos H. ). International Center for Tropical Agriculture (CIAT), Cali, Colombia.

6. Anderson R.A. (1982). Water absorption and solubility and amylograph characteristics of roll-

cooked small grain products. Cereal Chemistry. 59: 265-271.

7. Anderson R.L., Ternes S.B., Strand K.A. and Rowling M.J. (2010). Vitamin D homeostasis is

compromised due to increased urinary excretion of the 25-hydroxycholecalciferol-vitamin D-

binding protein complex in the Zucker diabetic fatty rat. American Journal of Physiology

Endocrinology and Metabolism. 299: 959–967.

8. Anon, (2000). AACC Board holds midyear meeting. Cereal Foods World 45: 325.

9. Aryana K., Greenway F., Dhurandhar N., Tulley R., Finley J., Keenan M., Martin R., Pelkman C.,

Olson D. and Zhen J. (2015). A resistant-starch enriched yogurt: fermentability, sensory

characteristics and a pilot study in children. F1000Research, 4(139):1-12.

24
10. Behall K.M. and Hallfrisch, J. (2002). Plasma glucose and insulin reduction after consumption of

breads varying in amylose content. European Journal of Clinical Nutrition, 56:913-920.

11. Benesi I.R.M., Labuschagne M.T., Dixon A.G.O. and Mahungu N.M. (2004). Stability of native

starch quality parameters, starch extraction and root dry matter of cassava genotypes in different

environments. Journalof the Science of Food and Agriculture, 84:1381-1388.

12. Berry C. S. (1986). Resistant starch: Formation and measurement of starch that survives

exhaustive digestion and amylolytic enzymes during the determination of dietary fiber. Journal of

Cereal Science, 4: 301-314.

13. Birt D.F., Boylston T., Hendrich S., Jay-Lin J., Hollis J., Li L., McClelland J., Moore S., Phillips

G.J., Rowling M., Schalinske K., Paul Scott M. and Whitley E.M. (2013). Resistant Starch:

Promise for Improving Human Health. American Society for Nutrition. Advances in Nutrition 4:

587–601, doi:10.3945/an.113.004325.

14. Brown I.L. (2004). Applications and uses of resistant starch. Journal of AOAC International

87:727–732.

15. Buddrick O., Oliver A.H.J., Hughes J.G., Kong I. and Small D.M. (2015).The effect of

fermentation and addition of vegetable oil on resistant starch formation in whole grain breads.

Food Chemistry, 180:181-185.

16. Chung H.J., Donner E. and Liu Q. (2011). Resistant starch in foods. In: Comprehensive

biotechnology: agriculture and related biotechnologies (Ed. by M. Moo-Young), pp.527-534.

http://dx.doi.org/10.1016.

17. Collins M.D. and Gibson G.R. (1999). Probiotics, Prebiotics and Synbiotics: Approaches For

Modulating The Microbial Ecology Of The Gut. American Journal of Clinical Nutrition.

69(5):1052-1057.

18. Da G., Dufoura D., Marouzéa C., Le Thanh M., Maréchalc P.A. (2008).Cassava Starch Processing

at Small Scale in North Vietnam. Starch/Stärke, 60:358–372. doi:10.1002/star.200800202. 25

19. Demiate I.M. and Kotovicz V. (2011). Cassava starch in the Brazilian food industry. Ciência e

Tecnologia de Alimentos.Campinas, 31(2): 388-397.

20. Devi A.F., Fibrianto K., Torley P.J. and Bhandari B. (2009). Physical properties of cryomilled rice

starch. Journal of Cereal Science, 49:278–284.

21. Djurdjević J.D., Maćej O. and Jovanović S. (2002). Viscosity of Set-Style Yogurt As Influenced By

Heat Treatment of Milk and Added Demineralized Whey Powder. Journal of agricultural sciences,

47(1):45-56.

22. Eliasson A.C. (2004), Starch in food Structure, function and applications. CRC press, Woodhead

Publishing Limited, Cambridge, England.

23. Ellis R.P., Cochrane M.P., Dale M.F.B., Duffus C.M., Lynn A., Morrison I.M., Prentice R.D.M.,

Swanston J.S. and Tiller S.A. (1998). Starch production and industrial use. Journal of the Science

of Food and Agriculture, 77: 289–311.

24. Englyst H.N., Kingman S.M. and Cummings J.H. (1992). Classification and measurement of

nutritionally important starch fractions. European Journal of Clinical Nutrition 46(2):33-50.

25. Englyst H.N., Trowell H., Southgate D.A.T. and Cummings J.H. (1987). Dietary fiber and resistant

starch. American Journal of Clinical Nutrition, 46:873–874.

26. Ezeh C.I., Obioma N.Q. and Onuabuobi J.C. (2011). Consumer Behaviour and Preferences to

Cassava Value Added Products in Ikwuano Local Government Area (L.G.A) of Abia State,

Nigeria. International Journal of Social Science and Humanity, 1(4): 274-277.

27. Fakir M. S. A., Jannat M., Mostafa M.G. and Seal H. (2012). Starch and flour extraction and

nutrient composition of tuber in seven cassava accessions. Journal of the Bangladesh Agricultural

University,10 (2): 217–222.

28. FAO (2000). The World Cassava Economy: Facts, Trends and Outlooks. Food and Agriculture

Organization, Rome, Italy.

26
29. FAO (2013). Save and Grow Cassava: A guide to sustainable production intensification. Food and

Agriculture Organization, Rome, Italy.

30. FAOSTAT. (2014). Food and Agricultural Commodities Production; Available online:

http://faostat3.fao.org/browse/rankings/commodities_by_country/E

31. Faraj A., Vasanthan T., and Hoover R. (2004). The effect of extrusion cooking on resistant starch

formation in waxy and regular barley flours. Food Research International, 37:517 – 525.

32. Franco M.L., Preto S.J.R., Ciacco F.C. and Tavares D.Q. (1995). Effect of the Heat Moisture

Treatment on the Enzymatic Susceptibility of Corn Starch Granules. Starch/Starke, 47: 233-228.

33. Gámbaro A.,Varela P., Ares G. (2003). Effect of Thickeners on the Texture of stirred Yogurt.

Alimentos e Nutrição Araraquara, 16(3): 207-211.

34. Gibson G.R., Probert H.M., Loo J.V., Rastall R.A. and Roberfroid M.B. (2004). Dietary

modulation of the human colonic microbiota: updating the concept of prebioticis. Nutrition

Research Reviews, 17:259-275.

35. Gonçalvez D., Pérez C., Reolon G., Segura N., Lema P., Gámbaro A., Ares G., Varela P. (2005).

Effect of Thickeners on the Texture of Stirred Yogurt. Alimentos e Nutrição Araraquara, 16 (3):

207-211.

36. Graham, Adeloye D., Grant L., Theodoratou E., Campbell H. (2012). Estimating the incidence of

colorectal cancer in sub-Saharan Africa: A systematic analysis. Journal of Global Health,

2(2):020404. doi: 10.7189/jogh.02.020204.

37. Haralampu S.G. (2000). Resistant starch - A review of the physical properties and biological

impact of RS3. Carbohydrate Polymers, 41:285–292.

38. Hódsági M., Jámbor É., Juhász E. and Gergely S. (2012). Effects of microwave heating on native

and resistant starches. Acta Alimentaria, 41:233–247.

27
39. Homayouni A., Amini A., Khodavirdiv A. Keshtiban, Mohammad A., Mortazavian, Esazadeh

K.and Pourmoradian S. (2013). Resistant starch in food industry: A changing outlook for consumer

and producer. Starch/Stärke, 65:1 –13.

40. ISO 13580: 2005 Yogurt - Determination of total solids content, International Organisation for

Standardisation, Geneva, Switzerland.

41. ISO 1660:1996 Starch−Determination of moisture content − Oven drying methods, International

Organisation for Standardisation, Geneva, Switzerland.

42. Jisha S., Padmaja G. and Sajeev M. S. (2010). Nutritional and textural studies on dietary fiber-

enriched muffins and biscuits from cassava-based composite flours. Journal of Food Quality, 33:

79–99.

43. Johnston K.L., Thomas E. L., Bell J. D., Frost G. S. and Robertson M. D. (2010). Resistant starch

improves insulin sensitivity in metabolic syndrome. Diabetic Medicine, 27(4):391-397.

44. Kaszab T., Csima G., Lambert-Merete A. Fekete A. (2002). Food Texture Profile Analysis by

Compression Test. Corvinus University of Budapest, Faculty of Food Science, Department of

Physics and Control. Budapest, Hungary.

45. Kittipongpatana O.S. and Kittipongpatana N. (2015). Resistant Starch Contents of Native and

Heat-Moisture Treated Jackfruit Seed Starch. Scientific World Journal, 2015 (519854) doi:

10.1155/2015/519854

46. Knutson C.A. (1990). Annealing of Maize Starches at Elevated Temperatures. Cereal Chemistry,

67(4):376-384.

47. Kovatcheva-Datchary P., Egert M., Maathuis A., Rajilic-Stojanovic M., de Graaf A.A., Smidt H.,

de Vos W.M. and Venema K. (2009). Linking phylogenetic identities of bacteria to starch

fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing.

Environmental Microbiology, 11:914-926.

28
48. Kumar M., Kumar A., Nagpal R., Mohania D., Behare P., Verma V., Kumar P., Poddar D.,

Aggarwal P.K., Henry C.J., Jain S. and Yadav H. (2010). Cancer-preventing attributes of

probiotics: an update. International Journal of Food Science and Nutrition. 61(5):473-96. doi:

10.3109/09637480903455971.

49. Kumari M., Urooj A., Prasad N.N. (2007). Effect of storage on resistant starch and amylose

content of cereal–pulse based ready-to-eat commercial products. Food Chemistry, 102(4):1425–

1430.

50. Lee1 W. J. and Lucey J. A. (2010) Formation and Physical Properties of Yogurt. Asian-Australian

Journal of Animal Sciences, 23(9) : 1127 – 1136.

51. Lertwanawatana P., Frazier R, A. and Niranjan K. (2015).High pressure intensification of cassava

resistant starch (RS3) yields. Food chemistry, 181: 85-93. doi:10.1016/j.foodchem.2015.02.005

52. Leszczyñski W. (2004). Resistant Starch – Classification, Structure, Production. Polish Journal of

Food and Nutrition Sciences, 13(54):37–50.

53. Li L. (2010). Assessing prebiotic effects of resistant starch on modulating gut microbiota with an

in vivo animal model and an in vitro semi-continuous fermentation model. Graduate Theses and

Dissertations. Iowa State University. Paper 11655.

54. Lunn J. and Buttriss J.L. (2007). Carbohydrates and dietary fibre Nutrition Bulletin. 32:21-64.

55. Maki K.,Pelkman C.L., Finocchiaro E. T., Kelley K.M., Lawless A. L., Schild A. L., and Rains

T.M. ( 2012). Resistant Starch from High-Amylose Maize Increases Insulin Sensitivity in

Overweight and Obese Men. Journal of Nutrition, 142: 717–723. doi: 10.3945/jn.111.152975.

56. MINAGRI (Ministry of Agriculture and Animal Resources). (2013). National Dairy Strategy.

http://www.minagri.gov.rw/index.php?id=678

57. Monachese M., Cunningham-Rundles S., Diaz M.A., Guerrant R., Hummelen R., Kemperman R.,

Kerac M., Kort R., Merenstein D., Panigrahi P., Ramakrishna B., Safdar N., Shane A., Trois L.

29
 andReid G. (2011). Probiotics and prebiotics to combat enteric infections and HIV in the

developing world: a consensus report. Gut Microbes, 2: 198-207.

58. Moongngarm A. (2013). Chemical compositions and resistant starch content in starchy foods.

American journal of Agricultural and Biological Sciences, 8 (2):107–113.

59. Morales-Medina R., Muñio M.D.M., Guadix E.M., and Guadix A. (2014). Produciton of resistant

starch by enzymatic debranching in legume flours. Carbohydrate Polymers, 101:1176-1183.

http://dx.doi.org/10.1016/j.carbpol.2013.10.027.

60. Mushimiyimana I., Hakizimana E., Gashaka G., Sallah P.Y.K., Kalisa S., Gatunzi F., Asiimwe T.,

Kahia J., Gahakwa D. (2011) Micro-propagation of a disease resistant cassava variety in Rwanda.

Rwanda Journal, Agricultural Sciences. 24(E 2011):49–57.

61. Nigudkar M.R. (2014). Estimation of Resistant Starch Content of Selected Routinely Consumed

Indian Food Preparations. Current Research in Nutrition and Food Science. 2(2):73-83.

62. Nilsson A.C., Ostman E.M., Holst J.J. and Bjorck I.M.E. (2008). Including indigestible

carbohydrates in the evening meal of healthy subjects improves glucose tolerance, lowers

inflammatory markers, and increases satiety after a subsequent standardized breakfast. Journal of

Nutrition, 138:732-739.

63. NISR (National Institute of Statistics of Rwanda). (2015). Statistical Yearbook 2015.

http://statistics.gov.rw/publication/statistical-yearbook-2015

64. Noor-Aziah A.A., Ho L.H., Komathi C.A. and Bhat R. (2011). Evaluation of resistant starch in

crackers incorporated with unpeeled and peeled pumpkin flour. American Journal of Food

Technology, 6:1054-1060.

65. Nugent A.P. (2005). Health properties of resistant starch. Nutrition Bulletin, 30: 27-54.

66. Official Methods of Analysis of AOAC International (2000): AOAC International, Gaithersburg,

MD, USA, AOAC Official Method 942.15-2000.

30
67. Official Methods of Analysis of AOAC International (2003): AOAC International, Gaithersburg,

MD, USA. Official Method 2002.02-2005.

68. Official Methods of Analysis of AOAC International (2003): AOAC International, Gaithersburg,

MD, USA, AOAC Official Method 996.11-2005.

69. Ogbo F.C. and Okafor E.N. (2015). The resistant starch content of some cassava based Nigerian

foods. Nigerian Food Journal, 33: 29–34.

70. Oladunmoye O.O., Ogugua C.A., Bussie Maziya-D., Ochuko L.E.and Elemo G.N. (2014).

Chemical and functional properties of cassava starch, durum wheat semolina flour, and their

blends. Food Science and Nutrition, 2(2): 132– 138. doi:10.1002/fsn3.83

71. Ooye D.A, Oso G.K, Olalumade B.B. (2014). Effects of Different Processing Methods on the

Proximate and Cyanogenic Composition of Flour from Different Cassava Varieties. Research and

Reviews: Journal of Agriculture and Allied Sciences, 3(3): 1-6.

72. Pancar E. D., Andiç S. and Boran G. (2016). Comparative Effects of Fish and Cow Gelatins and

Locust Bean Gum on Chemical, Textural, and Sensory Properties of Yogurt. Journal of Aquatic

Food Product Technology, 0(00):1-11. http://dx.doi.org/10.1080/10498850.2014.944293.

73. Pereira B. L.B. and Magali L. (2014). Resistant starch in cassava products. Food Science and

Technology Campinas, Doi: http://dx.doi.org/10.1590/fst.2014.0039.

74. Pereira, K. D. (2007). Resistant starch, the latest generation of energy control and healthy

digestion. (Amido Resistente, a Última Geraçãono Controle de Energia e Digestão Saudável).

Ciênciae Tecnologia deAlimentos, 27: 88 –92.

75. Pierna J.A.F., Volery P., Besson R., Baeten V. and Dardenne P. (2005). Classification of Modified

Starches by Fourier Transform Infrared Spectroscopy Using Support Vector Machines. Journal of

Agricultural and Food Chemistry, 53(17): 6581-6585. DOI: 10.1021/jf0501544.

76. RDB, (2012). Ideal condition for milk production: Investment Opportunity in the Dairy Sector.

www.rdb.rw/fileadmin/user_upload/Documents/Agriculture/Dairy.pdf Visited on
th
18 /05/2016. 31
77. Reid G., Nduti N., Sybesma W., Kort R., . Kollmann T.R, Adam R., Boga H., Brown E.M.,

Einerhand A., El-Nezami H., Gloor G.B., Kavere I.I, Lindahl J., Manges A., Mamo W., Martin R,

McMillan A., Obiero J., Ochieng P.A., Onyango A., Rulisa S., Salminen E., Salminen S., Sije A.,

Swann J.R., Treuren W., Waweru D., and Kemp S.J. (2014). Harnessing microbiome and probiotic

research in sub-Saharan Africa: recommendations from an African workshop. Microbiome, 2(12):

1-13. doi: 10.1186/2049-2618-2-12.

78. Richardson K.V.A. (2013). Quality Characteristics, Root Yield And Nutrient Composition Of Six

Cassava (Manihot Esculenta Crantz) Varieties. Gladstone Road Agricultural Centre Crops

Research Report No. 18. Nassau, Bahamas.

79. Ridlon J.M. and Hylemon P.B. (2006). A Potential Role for Resistant Starch Fermentation in

Modulating Colonic Bacterial Metabolism and Colon Cancer Risk. Cancer Biology and Therapy,

5(3): 273-274.

80. Routray W. and Mishra H.N. (2011). Scientific and Technical Aspects of Yogurt Aroma and Taste:

A Review. Comprehensive Reviews in Food Science and Food Safety, 10. (2011). Doi

10.1111/j.1541-4337.2011.00151.x

81. Saediman H., Amini A., Basiru R. and Nafiu L.O. (2015). Profitability and Value Addition in

Cassava Processing in Buton District of Southeast Sulawesi Province, Indonesia. Journal of

Sustainable Development, 8(1): 226-234.

82. Saha D. and Bhattacharya S. (2010). Hydrocolloids as thickening and gelling agents in food: a

critical review. Journal of Food Science and Technology, 47(6):587–597. doi: 10.1007/s13197-

010-0162-6.

83. Sajilata M.G., Singhal R.S. and Kulkarni P.R., (2006). Resistant starch - A review. Comprehensive

Review in Food Science and Food Safety, 5:1-17.

32
84. Sankhon A., Amadou I., Yao W., Wang H., Qian H. and Mlyuka E. (2014). Effect of Different

Heat-moisture Treatments on the Physicochemical Properties of African Locust Bean (Parkia

biglobosa) Starches. Journal of Agricultural Science and Technology, 16: 331-342.

85. Schulz A.G.M., Van Amelsvoort J.M.M. and Beynen A.C. (1993). Dietary native resistant starch

but not retrograded resistant starch raises magnesium and calcium absorption in rats. Journal of

Nutrition, 123:1724-1731.

86. Sfakianakis P. and Tzia C. (2014). Conventional and Innovative Processing of Milk for Yogurt

Manufacture; Development of Texture and Flavor: A Review. Foods, 3:176-193.

doi:10.3390/foods3010176.

87. Takizawa F.F. Graziela O.S.; konkel F.E.; Demiate I.M. (2004).Characterization of tropical

starches modified with potassium permanganate and lactic acid. Brazilian Archives of Biology and

Technology, 47(6):921-931.http://dx.doi.org/10.1590/S1516-89132004000600012

88. Tonukari N.J. (2004). Cassava and the future of starch. Electronic Journal of Biotechnology,

7(1):58.

89. Ukpongson M.A., Chikaire J., Anaeto F.C., Nwakwasi R.N., Aja O.O and Ike C.L. (2011). Effects

of Cassava Processing and Value Added Products on Sustainable Poverty Alleviation in Ikwuano

Area of Abia State, Nigeria. New York Science Journal, 4(10): 73-77.

90. Umuhozariho M.G., Shayo N.B., Msuya J.M., Sallah P.Y.K. (2011). Utilization of Cassava Leaves

as a Vegetable in Rwanda. African Journal Online, Rwanda Journal, 24 (E2011).

91. USAID (2010). Baseline Survey Report, Rwanda Dairy Competitiveness Program. United States

Agency for International Development, Washington DC, USA. Available on

http://pdf.usaid.gov/pdf_docs/PA00K5CS.pdf.

92. Vasanthan T. and Bhatty R.S. (1998). Enhancement of Resistant Starch (RS3) in Amylomaize,

Barley, Field Pea and Lentil Starches. Starch, 50(7): 286–291.

33
93. Vijayakumari A., Alummoottil N.J., Gourikutty P., Jaffer T. S. and Kadirvelu J. (2014). Preparation

and Characterization of a Low-calorie Cassava Pearl (Sago) from Physically Modified Cassava

Starch. Trends in Carbohydrate Research, 6(3):33-40.

94. Willis H.J., Eldridge A.L., Beiselgel J., Thomas W. and Slavin J.L. (2009).Greater satiety response

with resistant starch and corn bran in human subjects. Nutrition Research, 29:100–105.

95. Yadav B. S., Sharma A. and Yadav R.B. (2010). Effect of storage on resistant starch content and in

vitro starch digestibility of some pressure-cooked cereals and legumes commonly used in India.

International Journal of Food Science and Technology, 45(12): 2449–2455.

96. Yongfeng A. (2013). Structures, properties, and digestibility of resistant starch. Phd Dissertation,

Iowa State University. Ames, Iowa, USA.

97. Younes H., Levrat M.A, Demigne Â.C., Re Âme Â.C. (1993). Relationship between fermentations

and calcium in the cecum of rats fed digestible or resistant starch. Annals of Nutrition and

Metabolism, 37: 311-319.

98. Zhang P., Wang W.Q., Zhang G.L., Kaminek M., Dobrev P., Xu J. and Gruissem W. (2010).

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought

Stress Resistance and Alters Cytokinin Metabolism in Cassava. Journal of Integrative Plant

Biology, 52(7): 653–669.

99. Zvinavashe E., Elbersen H. W., Slingerland M., Kolijn S. and Sanders J.P.M. (2011). Cassava for

food and energy: exploring potential benefits of processing of cassava into cassava flour and

bioenergy at farmstead and community levels in rural Mozambique. Biofuels, Bioproducts and

Biorefining. 5 (2): 151–164.

34