PRODUCTION AND EVALUATION OF BREAD USING WHOLE WHEAT FLOUR

AND BLACK TURTLE BEANS FLOUR

BY

ADEGBOLA, ADEDOYIN SIMILOLUWA

MATRIC NO: 184067

A PROJECT PROPOSAL
SUBMITTED TO THE

DEPARTMENT OF FOOD SCIENCE AND ENGINEERING,

FACULTY OF ENGINEERING AND TECHNOLOGY,

LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY,

OGBOMOSO, OYO STATE

OCTOBER, 2023
 DEDICATION

This project is dedicated to Almighty God, the creator of all creations. Also to my daring and

loving parent Mr. and Mrs Adegbola, for their efforts towards my educational career in terms of

finance and parental advice.

ii
 ACKNOWLEDGEMENT

My appreciation goes to my project Supervisor, Dr. M.A Ojo, who guided me through the process

and techniques behind writing a project thesis. Thank you for your support sir.

iii
 CERTIFICATION

This is to certify that the proposal will be carried out by ADEGBOLA ADEDOYIN
SIMILOLUWA (MATRICULATION NUMBER: 184067), in the Department of Food Science
and Engineering, Faculty of Engineering and Technology, Ladoke Akintola University of
Technology, Ogbomoso.

Dr. M.A Ojo Date

Supervisor

Dr. J.A Adejuyitan Date

Head of Department

iv
 TABLE OF CONTENT

DEDICATION ii
ACKNOWLEDGEMENT iii
CERTIFICATION iv
TABLE OF CONTENT v
SUMMARY vii
CHAPTER ONE 8
INTRODCUTION 8
1.1 Background of the study 8
1.2 Justification of Study 9
1.3 Problem Statement 9
1.4 Aim and Objectives 10
1.4.1 Aim 10
1.4.2 Objectives 10
1.5 Scope of the Study 10
CHAPTER TWO 11
LITERATURE REVIEW 11
2.1 Bread Wheat 11
2.1.1 Origin of Bread Wheat 11
2.1.2 Whole Flour 11
2.1.3 Consumer Acceptance 13
2.1.4 Cultivation and Production of Wheat 13
2.1.5 Characteristics of Wheat Bread 14
2.1.6 Importance of Wheat 16
2.1.7 Nutritional Composition of Bread Wheat 17
2.2 Black beans 18
2.2.1 Origin and Distribution of Black Beans 18
2.2.2 Cultivation of Black Beans 19
2.2.3 Nutritional Quality of Black Beans 21
2.2.4 Physicochemical Properties of Black Beans 24
2.2.5 Consumer Acceptability 25
2.2.6 Phytochemicals and Antioxidants Present in Black Beans 26
CHAPTER THREE 28
MATERIALS AND METHODS 28
3.1 Materials 28
3.2 Preparation of Sample 28
3.2.1 Preparation of black beans flour (Phaseolus vulgaris L.) 28
3.2.2 Preparation of black beans blended Bread 28
3.3 Proximate Analysis 33
3.3.1 Determination of moisture content 33
3.3.2 Ash Content 34
3.3.3 Determination of lipid content 34
3.3.4 Protein determination 35
3.3.5 Crude fiber 36
3.3.6 Carbohydrate determination 36
3.4 Physical Examination 37
3.4.1 Loaf Weight 37
3.4.2 Loaf Volume 37
3.4.3 Specific Volume 37
3.4.4 Color Analysis 37
3.4.5 Textural property 38
3.5 Determination of total bacterial count (TBC) 38
3.5.2 Enumeration of yeast and mold count 38
3.5.3 Determination of coliform bacteria 38
3.6 Sensory Analysis 39
3.7 Statistical Analysis 39
REFERENCES 40

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 SUMMARY

Bread, with ancient roots dating back to the Neolithic era, is a staple across many cultures,

with varying levels of consumption around the world. Despite the staple nature of bread, there

exists a significant health challenge, particularly in Nigeria: Protein-Energy Malnutrition (PEM).

This study aims to combat PEM by enriching bread with black beans, a leguminous crop known

for its protein and nutritional benefits. The goal is not just to offer carbohydrate-rich bread, as with

traditional wheat bread, but to enhance taste, extend shelf life, and significantly boost the bread's

nutritional value, thus providing a fortified dietary solution to a pervasive problem.

Freshly harvested black beans will be processed into flour using the Alibas et al. (2022)

method, and blended with whole wheat flour and other baking ingredients. Post-production, the

bread will undergo various chemical analyses such as moisture content, ash content, lipid content,

protein determination, crude fiber, and carbohydrate determination. Additionally, bread quality

evaluation metrics such as loaf weight, volume, specific volume, color analysis, and textural

properties will be measured. Lastly, the bread will be assessed for microbial content, and sensory

analysis will be conducted using a panel of students from Ladoke Akintola University of

Technology, Nigeria. All data gathered will be statistically analyzed using the SPSS software with

one-way ANOVA to understand the significance of the findings.

This study presents a strategic and innovative approach to addressing Protein-Energy

Malnutrition in Nigeria and potentially other regions facing similar nutritional challenges. By

integrating nutrient-rich black beans into a widely consumed staple like bread, the research offers

a viable and sustainable dietary solution to a profound health issue.

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 CHAPTER ONE

INTRODCUTION

1.1 Background of the study

Bread is a baked food dating back to 2300 BC, this era is other-wisely referred to as the

Neolithic era. This era was known as the rise of the first Agricultural revolution. The highest

consumption of bread per capital is reported in Turkey (104 kg) and Bulgaria (95 kg), while the

lowest one is in Great Britain (approximately 32 kg). European residents consume on average 59

kg of bread per year and this level was stable in recent years (Meenu et al., 2023).

Furthermore, legume incorporation in bread might address some of the challenges faced in

promoting legume consumption. While legumes are nutritionally valuable, their consumption in

many parts of the world is limited due to factors like long cooking times and concerns about

digestibility. By integrating legumes like black beans into commonly consumed products like

bread, it could provide a seamless way to increase legume intake in the daily diet (Rachwa-Rosiak

et al., 2015).

Black beans (Phaseolus vulgaris) are a variety of the common bean family, alongside other

popular types like kidney beans, pinto beans, and navy beans. Native to the Americas, black beans

have been a staple in the diets of various cultures, particularly in Latin America, for thousands of

years. Renowned not only for their rich, earthy flavor and velvety texture but also for their myriad

of health benefits, black beans have garnered attention and appreciation worldwide (Fabbri et al.,

2016). The use of whole or Whole flour in combination with black beans in bread production not

only offers an avenue to explore the nutritional benefits but also challenges in terms of taste,

texture, and consumer acceptability.

8
1.2 Justification of Study

The aim of this study is to develop a bread that isn't just carbohydrate-heavy like the typical

wheat flour bread but also to enhance its taste, shelf-life, and overall nutritional content by

incorporating black beans. By blending black beans with regular bread ingredients, not only can

we potentially prolong the bread's shelf life due to the natural antioxidants found in black beans,

but we can also offer a richer flavor profile and deliver essential nutrients such as protein, minerals,

and vitamins in every slice. Over the years, the food industry has recognized the importance of

nutritionally balanced bread and has seen numerous innovations in this regard (Jideani and Vogt,

2015). However, standard wheat flour bread remains dominant in the market. Through this study,

we intend to fortify traditional wheat bread with black bean powder, offering a more nutritionally

rounded option for consumers.

1.3 Problem Statement

Protein-Energy Malnutrition (PEM) remains a pervasive health challenge in Nigeria,

leading to distressingly high mortality rates, particularly in tertiary health facilities (Ubesie and

Ibeziakor, 2012). Beyond Nigeria, the broader African continent faces a deeply concerning rate of

child mortality for those under five, primarily attributed to PEM (Beshir, 2019). A potential avenue

to combat this nutritional deficiency is through the inclusion of alternative nutrient-rich sources in

staple foods. Black beans, known for their rich protein content and a range of essential

micronutrients, stand out as a promising solution. With their widespread availability and ease of

integration into diverse diets, black beans have the potential to be a consistent and reliable nutrient

source. The challenge then, is to adeptly incorporate black beans into bread, offering a fortified

food option that can contribute to addressing the profound issue of protein-energy malnutrition.

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1.4 Aim and Objectives

1.4.1 Aim

The aim of this study is to produce bread from blends of whole wheat flour and black beans

flour.

1.4.2 Objectives

The specific objectives are;

i. to produce bread from a blend of whole flour and black beans.

ii. to determine the physical properties of black beans fortified bread.

iii. to determine the consumer acceptability of produced blends of black turtle beans flour

and whole wheat flour.

iv. to determine the physical properties of bread produced from blends of black turtle flour

and whole wheat flour

1.5 Scope of the Study

This research work will focus on the fortification of wheat bread with black turtle beans

flour. The research will produce samples of bread in different percentage of blended black turtle

bean flour and wheat flour. Evaluations such as proximate analysis, microbial analysis and sensory

analysis will be carried out on the samples.

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 CHAPTER TWO

LITERATURE REVIEW

2.1 Bread Wheat

Bread is prepared by baking in an oven at a controlled temperature as determined by the

ingredient of the bread. Bread is a staple food prepared, usually by baking, from flour and water

dough. The major ingredient for bread making is the wheat flour which becomes the most desirable

because of its high gluten content. This makes bread a mainly carbohydrate food as wheat, which

is the major ingredient of bread, is a cereal. Hence, major research is being focused on how to

supplement the nutrient of bread.

2.1.1 Origin of Bread Wheat

The first cultivation of wheat occurred about 10 000 years ago, as part of the ‘Neolithic

Revolution’, which saw a transition from hunting and gathering of food to settled. These earliest

cultivated forms were diploid (genome AA) (einkorn) and tetraploid (genome AABB) (emmer)

wheats and their genetic relationships indicate that they originated from the south-eastern part of

Turkey. Cultivation spread to the Near East by about 9000 years ago when hexaploid bread wheat

made its first appearance (Kimber and Sears, 2015).

2.1.2 Whole Flour

Whole flour originates from grinding the entire grains — inclusive of the bran, endosperm,

and germ. It holds a pivotal place in nutrition, culinary arts, and health studies (Smith et al., 2018).

Over the years, an increasing body of literature has examined the attributes, advantages, and

challenges of whole flour.

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 Whole flour's nutritional content distinguishes it from other types of flour. Beyond

containing the endosperm, whole flour integrates the vast nutrient spectrum present in the bran and

germ. Its enriched profile includes dietary fiber, essential fatty acids, a suite of vitamins (especially

B vitamins), and crucial minerals such as magnesium, zinc, and iron (Jones et al., 2015).

Furthermore, bioactive compounds like antioxidants and phytochemicals, which offer numerous

health benefits, are also present in commendable amounts (Gómez et al., 2020).

A series of studies have emphasized the profound health benefits linked to whole flour

consumption. Products crafted from whole flour have been consistently associated with a

diminished risk of several chronic ailments. For instance, research led by Zong et al. (2016)

highlighted that heightened whole grain intake was correlated with decreased risks of

cardiovascular diseases, select cancers, and overall mortality. Similarly, the ample fiber content

inherent in whole flour facilitates digestion, satiety promotion, and the maintenance of stable blood

sugar levels — attributes that are especially salient for those with diabetes or its precursors

(McKeown et al., 2017).

From a culinary perspective, while whole flour presents clear nutritional advantages, it also

introduces certain challenges in baking. The bran and germ's inclusion can disrupt gluten

development, often resulting in baked goods that are denser than those made with wheat flours

(Brown et al., 2019). Consequently, many culinary experts and food scientists have been driven to

experiment with diverse methodologies and additives to refine the texture and leavening of whole

flour-based products (Patel and Sharma, 2020).

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2.1.3 Consumer Acceptance

There's a growing shift among health-conscious consumers towards whole grains and

alternative flours. This trend is driven by increasing awareness of the health implications of Whole

flour and the benefits of a diet rich in whole grains (Reynolds et al., 2019).

Whole flour, with its undeniable presence in various food products worldwide, offers certain

advantages like extended shelf life and a fine texture suited for many culinary applications.

However, its compromised nutritional profile and associated health concerns have garnered

scrutiny in recent years. With the current trajectory favoring nutritionally-rich and health-

promoting foods, the role and consumption patterns of Whole flour are likely to evolve, influenced

by both scientific findings and shifting consumer preferences (Martinez et al., 2020).

2.1.4 Cultivation and Production of Wheat

Shewry (2019) submitted that the earliest cultivated forms of wheat were essentially

landraces selected by farmers from wild populations, presumably because of their superior yield

and other characteristics, an early and clearly non-scientific form of plant breeding! However,

domestication was also associated with the selection of genetic traits that separated them from their

wild relatives. The spread of wheat from its site of origin across the world has been elegantly

described by Kimber and Sears (2013) thus; The main route into Europe was via Anatolia to Greece

(8000 BP) and then both northwards through the Balkans to the Danube (7000 BP) and across to

Italy, France and Spain (7000 BP), finally reaching the UK and Scandanavia by about 5000 BP.

Similarly, wheat spread via Iran into central Asia reaching China by about 3000 BP and to Africa,

initially via Egypt. It was introduced by the Spaniards to Mexico in 1529 and to Australia in 1788.

Shewry (2019) discovered that about 95 % of the wheat grown worldwide is hexaploid bread

wheat, with most of the remaining 5 % being tetraploid durum wheat. The latter is more adapted

13
to the dry Mediterranean climate than bread wheat and is often called pasta wheat to reflect its

major end-use.

However, it may also be used to bake bread and is used to make regional foods such as

couscous and bulgar in North Africa. Small amounts of other wheat species (einkorn, emmer, spelt)

are still grown in some regions including Spain, Turkey, the Balkans, and the Indian subcontinent.

In Italy, these hulled wheats are together called faro (Szabo´ and Hammer, 2016) while spelt

continues to be grown in Europe, particularly in Alpine areas (Lodhi et al. 2020). The recent

interest in spelt and other ancient wheats (including kamut, a tetraploid wheat of uncertain

taxonomy, related to durum wheat) as healthy alternatives to bread wheat (Abdel-Aal et al., 2018)

may also lead to wider growth for high value niche markets in the future

2.1.5 Characteristics of Wheat Bread

Marijia et al., (2012) evaluated the quality characteristics of Wheat, they discovered that

energy value of variety Altana varies in the range of 75.4 – 148.0 cm², according to the region of

cultivation. Energy of wheat EMS varies in range of 79.5 – 125.3 cm² and energy of wheat Pobeda

varies in the range of 70.3 – 98.7 cm². The obtained results showed that the best rheological

behavior and highest quality was determined in the Altana wheat variety harvested in the Novaci

region. It was characterized with high extensograph energy of 148 cm², 1000 AE maximum

viscosity and mixolab index 3-58-889 Dmitry et al., (2021) analyzed the relationship between the

characteristics of bread wheat grains, storage time and germination, their work showed that

genotype significantly affects the variability of all seed traits, and the year of harvesting affects

about 80 % of them (including all the traits of shape and size).

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 No significant trend was detected for the grain shape/size traits, while 90 % of the color

traits demonstrated such a trend. The most significant negative correlations were found between

the harvesting year and the traits of grain redness: the greater the storage time, the more intensive

is red color component for the grains. At the same time, it was shown that grains of longer storage

time (earlier harvesting year) have lighter coat.

Analysis of linear correlations between germination of wheat seeds of different genotypes

and harvesting years and their seed traits revealed a negative linear relationship between the red

component of coat color and germination: the redder the grain s, the lower their germination rate.

he results obtained demonstrate manifestations of metabolic changes in the coat of grains

associated with storage time and their relationship with a decrease of seed viability (Dmitry et al.,

2021).

Mustapha et al., (2016) analyzed the quality characteristics of bread wheat based on genetic

and agronomic diversity, they observed a significant association between morphological and bread

wheat quality traits while the correlation was weak with the genetic data. Molecular variance

between old (released before the year 2000) and new cultivars accounted for 1 % of the total

variation and the variance was 3 % between national and foreign cultivars. Their results showed

that the number of alleles was lower in national and new cultivars compared to foreign and old

cultivars. Therefore, breeding sources do not appear to improve the genetic base of wheat cultivars

in Turkey. Introducing new variation sources may be needed to broaden the narrowed gene pool

of bread wheat (Mustapha et al.,2016).

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2.1.6 Importance of Wheat

Akbari et al., (2016) submitted that a Bread wheat is one of the most important crop species,

responsible for the emergence and development of agriculture and has fed, and continues to feed,

a large part of the world’s population across many centuries. Wheat has been improved by man

over the last 8000 to 10,000 years ago when the species first arose. Initially it happened in an

unconscious way, then intentionally, but empirically, and then, for more than a century, based on

scientific knowledge (Charmet, 2011). Wheat breeding, as for many other crops, has been evolving

fast, both in terms of basic science, methods and tools. The literature on wheat breeding is vast,

including countless scientific papers, reviews and even dense book collections already published.

Therefore, all relevant aspects and examples cannot be covered in a single text.

On the contrary, we do encourage readers to go through this bibliographic ever growing

wealth for a deeper understanding on any given topic. Thus, the objective of this review is to

provide a brief and valuable synthesis on some selected aspects related to the history, but

especially, current advances in wheat breeding, devoted especially to students and researchers with

little or even no knowledge on the theme. Through this review, the reader can have a quick and

general overview on the discussed topics and, when necessary, get a direction to start searching

for further literature, as we have tried to cite the most important and recent papers on each topic.

Therefore, in the next sections we show the origin of this species and how it became so important

with a brief history of wheat cultivation and breeding. Priorities and particularities of wheat

breeding are presented. Special consideration is given to new approaches and tools that are

currently under development, and the ones that lately reappeared. Finally, the promising future

and perspectives are discussed (Venske et al., 2019).

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2.1.7 Nutritional Composition of Bread Wheat

Khan et al., analyzed the nutritional composition of wheat. They discovered that wheat

grains of different varieties contain a net protein level of 9.15 %-10.27 %, 2.15 %-2.55 % total

fats, 1.72 %- 1.85 % dietary fibers, 77.65x10(-6)-84.25x10(-6) of potassium and 7.70x10(-6)-

35.90x10(-6) of sodium ions concentration, 0.24x10(-6)-0.84x10(-6) of phosphorus, 1.44 %-2.10

% ash, 31.108- 43.602 g of thousand grain mass (TGM) and 8.38 %-9.67 % moisture contents.

This study is significant in providing an opportunity to explore the available wheat varieties and

to further improve their nutritional excellence and also essential for setting nutritional regulations

for domestic and export purposes.

Pawan K. (2011) analyzed the nutritional and medicinal composition of Wheat and found

out that Wheat has several medicinal virtues; starch and gluten in wheat provide heat and energy;

the inner bran coats, phosphates and other mineral salts; the outer bran, the much-needed roughage

the indigestible portion that helps easy movement of bowels; the germ, vitamins B and E; and

protein of wheat helps build and repair muscular tissue. The wheat germ, which is removed in the

process of refining, is also rich in essential vitamin E, the lack of which can lead to heart disease.

The loss of vitamin and minerals in the Whole wheat flour has led to widespread prevalence of

constipation and other digestive disturbances and nutritional disorders. The whole wheat, which

includes bran and wheat germ, therefore, provides protection against diseases such as constipation,

ischemic, heart disease, disease of the colon called diverticulum, appendicitis, obesity and diabetes

(Pawan, 2011).

To enhance the quality as well as the quantity of proteins/starches, and the content of

vitamins, essential amino acids, minerals and other healthy components of wheat, it is essential to

understand the molecular and genetic control of various aspects of plant growth and development.

17
Shewry et al., (2015) evaluated the nutritional composition of wheat, their work proves that in

addition to being a major source of starch and energy, wheat also provides substantial amounts of

a number of components which are essential or beneficial for health, notably protein, vitamins

(notably B vitamins), dietary fiber, and phytochemicals. Of these, wheat is a particularly important

source of dietary fiber, with bread alone providing 20 % of the daily intake in the UK, and well‐

established relationships between the consumption of cereal dietary fiber and reduced risk of

cardio‐vascular disease, type 2 diabetes, and forms of cancer (notably colorectal cancer). Wheat

shows high variability in the contents and compositions of beneficial components, with some

(including dietary fiber) showing high heritability. Hence, plant breeders should be able to select

for enhanced health benefits in addition to increased crop yield.

2.2 Black beans

2.2.1 Origin and Distribution of Black Beans

Black beans (Phaseolus vulgaris L.) are among the diverse and globally significant

legumes. Renowned for their dense nutrient profile and culinary versatility, they have become

integral in numerous cultural diets. Understanding their origins and distribution patterns provides

insights into their evolutionary journey and global importance.

The domestication of black beans can be traced back to Mesoamerica, particularly areas of

modern-day Mexico and Central America (Rodríguez and Menezes, 2019). Early agrarian societies

began cultivating these beans approximately 7,000 years ago. Evidence from archaeological sites,

especially in regions of the Tehuacán Valley in Mexico, indicates the presence of black bean

cultivation in ancient agrarian practices (Serrano-Serrano et al., 2020).

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 The spread of black beans from their center of origin in Mesoamerica to other parts of the

world was facilitated by both natural and human-mediated processes. As populations migrated or

traded, black beans were introduced to new regions, leading to genetic diversification. Recent

genetic studies have highlighted specific markers indicating multiple domestication events and

subsequent hybridization, contributing to the bean's wide genetic diversity (Martínez-Castillo et

al., 2021).

From its Mesoamerican origins, black bean cultivation spread to South America and

eventually to North America. Spanish and Portuguese explorers played a pivotal role in

introducing black beans to Europe and subsequently to Africa and Asia in the 15th and 16th

centuries (Oliveira et al., 2018). Today, black beans are grown globally, with significant

production centers in Brazil, India, China, and several African countries. The bean's adaptability

to diverse climatic conditions, from the temperate zones of North China to the tropical regions of

Brazil, underscores its resilience and versatility (Zhao and Chung, 2019).

As black beans spread across continents, they integrated into local cuisines and cultures. In

Latin America, for instance, black beans are a staple, often paired with rice. Similarly, in various

parts of Africa and Asia, they've been incorporated into traditional dishes, highlighting their global

culinary relevance (González-Salazar et al., 2020).

2.2.2 Cultivation of Black Beans

The history of black bean cultivation is rooted deeply in Mesoamerican agricultural

practices. Ancient civilizations, such as the Aztecs and Mayans, cultivated these beans as one of

their primary protein sources, often alongside maize and squash in the classic "Three Sisters"

method of intercropping (Gonzalez et al., 2017).

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 Black beans thrive in well-draining soils, with a pH level ranging from 6.0 to 7.5. While

they're somewhat adaptable, they prefer warmer climates, requiring a growing season with

temperatures between 60 °F and 70 °F. Adequate sunlight is crucial for optimal growth, as is a

moderate amount of rainfall, particularly during the flowering and pod-filling stages (Mendes et

al., 2018).

Direct seeding is the most common method for sowing black beans, with a recommended

depth of 1-2 inches. Crop rotation is vital to prevent soil-borne diseases and maintain soil fertility.

Integrating black beans into a rotation with cereals like maize can reduce the risk of pest

infestations and improve overall yield (Silva et al., 2019).

Black beans face threats from various pests and diseases. Common pests include aphids, bean

weevils, and whiteflies. Diseases like bean rust, anthracnose, and bacterial blight can also impact

yields. Integrated pest management (IPM), involving the combined use of biological, cultural,

physical, and chemical measures, has been recommended to manage these challenges sustainably

(Ramirez-Villegas et al., 2020). Black beans are typically ready for harvest 90-150 days after

sowing, depending on the variety and growing conditions. Once the pods are fully mature and have

a dry appearance, they're harvested. Post-harvest, the beans are threshed, cleaned, and dried to a

moisture content of about 10 % to ensure safe storage and prevent fungal growth (Diaz et al.,

2021).

With global climate change posing challenges for agriculture, the need for resilient and

sustainable crops intensifies. Black beans, given their adaptability, could play a significant role in

ensuring food security. Research focusing on drought-resistant and high-yield varieties, coupled

with sustainable cultivation practices, is the way forward (Torres et al., 2022).

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2.2.3 Nutritional Quality of Black Beans

Black beans are predominantly recognized for their high protein content. On average,

cooked black beans contain around 21 % protein, which is a considerable amount for a plant-based

source (Martinez et al., 2015). Additionally, they are rich in complex carbohydrates, specifically

starches and fibers, which contribute to satiety and digestive health. Notably, black beans have a

low fat content, with the fats present being primarily unsaturated (Arboleda et al., 2018). A

significant portion of black beans' carbohydrate content comes from dietary fiber, both soluble and

insoluble. The soluble fiber, in particular, has been associated with improved cholesterol levels,

while the insoluble fiber aids in digestion and promotes gut health (Fabbri et al., 2016).

These beans are rich in bioactive compounds, including antioxidants like anthocyanins, which

contribute to their dark color. These compounds help counteract oxidative stress in the body,

reducing the risk of chronic diseases (Santos et al., 2019). Additionally, the presence of

phytonutrients, such as phytosterols, has been associated with potential health benefits, including

cholesterol-lowering properties (Meenu et al., 2023). While black beans are nutritionally dense,

they also contain anti-nutritional factors like lectins, phytates, and tannins. These compounds can

interfere with the absorption of certain nutrients.

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 Table 2.1: Nutritional Information in 100g of Black Beans

Calories Carbohydrates Protein Fibre Calcium Iron Magnesium Phosphorus Potassium Zinc Copper

6.5 2.9 0.783

331 Cal 60 g 21 g 21.8g 111mg 188 mg 471 mg 1416 mg
mg mg mg

Source: TACO (2005)

22
Plate 2.1: Black beans.
Source: Nkenmeni et al. (2019)

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 However, traditional preparation methods, like soaking and cooking, significantly reduce

these anti-nutritional factors, mitigating their effects (Meenu et al., 2023).

The nutritional composition of black beans offers various health benefits. Regular

consumption has been linked to reduced risks of chronic diseases, such as heart disease and type

2 diabetes. Their fiber and antioxidant content contribute to heart health, while their low glycemic

index makes them suitable for blood sugar regulation (Díaz-Batalla et al., 2018).

2.2.4 Physicochemical Properties of Black Beans

Black beans (Phaseolus vulgaris L.) are an integral part of many diets globally. Beyond

their nutritional attributes, understanding their physicochemical properties is pivotal for diverse

applications in food science, storage, and processing. This review sheds light on the key

physicochemical characteristics of black beans, drawing from a plethora of scientific studies.

Black beans exhibit a characteristic kidney shape with a smooth texture and a deep black

hue. The size of the beans varies among cultivars but generally falls within a range of 5-7 mm in

length (Cordova et al., 2017). Understanding the morphology and size of black beans is crucial for

sorting, grading, and processing applications. Like many legumes, black beans have the inherent

ability to absorb water, which is significant during soaking processes. The water absorption index

indicates how much water the beans can imbibe, directly impacting their swelling capacity. Factors

such as seed coat integrity, internal structure, and cultivar differences influence this property

(Munoz et al., 2018).

Black beans generally possess a neutral to slightly alkaline pH. The pH, coupled with the

bean's inherent acidity, impacts its preservation, microbial stability, and taste when incorporated

24
into dishes (Torres and Frias, 2019). The physicochemical properties of black beans underscore

their versatility and applicability in diverse culinary and food processing scenarios. From their

water absorption capacity, which influences soaking and cooking times, to their functional

properties, determining their role in food formulations, black beans' physicochemical attributes are

as essential as their nutritional ones. As the global demand for plant-based foods continues to

surge, a comprehensive understanding of these properties will remain pivotal for maximizing the

potential of black beans in the food industry.

2.2.5 Consumer Acceptability

The texture and taste of black beans play a significant role in their gastronomic appeal.

Being soft and mildly flavored, they are often regarded as a versatile ingredient in various dishes,

from soups to salads (Santos et al., 2017). Their ability to absorb flavors makes them a favorite in

savory dishes, while their rich, dark hue enhances the visual appeal of meals. Black beans have a

longstanding history in Latin American cuisines, from Brazil's feijoada to Mexico's frijoles negros

(Diaz et al., 2018). The cultural significance of black beans in these regions enhances their

acceptability. Moreover, as global culinary boundaries blur, the integration of black beans into

other cuisines, such as in Asian salads or European stews, indicates a growing cultural

acceptability (Martinez et al., 2019).

The affordability of black beans, especially compared to animal-based protein sources,

contributes to their wide acceptance, especially in developing countries (Torres and Lopez, 2021).

Their shelf stability and ease of storage also play into their economic acceptability, as they can be

stored for extended periods without significant nutrient loss.

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 While there are multiple facets endorsing the acceptance of black beans, challenges persist.

The presence of anti-nutrients, like phytates, and the potential for causing flatulence in some

individuals can deter consistent consumption (Alvarez et al., 2022). Proper preparation methods

can mitigate these challenges to an extent.

2.2.6 Phytochemicals and Antioxidants Present in Black Beans

Black beans, scientifically classified as Phaseolus vulgaris L. have transcended their

traditional role as a protein source to be recognized for their dense array of phytochemicals and

antioxidants. Their rich flavonoid content, especially anthocyanins responsible for their distinctive

dark hue, has been pinpointed as a primary contributor to their antioxidant properties (Vargas et

al., 2017). Alongside flavonoids, black beans also harbor phytosterols, which structurally resemble

cholesterol and have been associated with reducing cholesterol levels in the bloodstream (Reyes-

Moreno et al., 2018). Another notable group of phytochemicals in black beans are saponins.

Beyond their antioxidant capabilities, saponins have shown promise in boosting immunity and

may also possess anti-carcinogenic effects (Martinez et al., 2019).

The free radical scavenging ability of black beans is especially noteworthy, largely

attributed to their phytochemical composition, notably the anthocyanins (Gomez et al., 2020).

Moreover, specific compounds in these beans have demonstrated metal chelating capacities,

binding excess metal ions like iron and copper, instrumental in curbing oxidative stress within the

body (Silva et al., 2017). Another intrinsic trait of black beans is their electron-donating capacity,

crucial for neutralizing harmful free radicals. This reducing power is primarily credited to the

bean's rich ensemble of polyphenols and flavonoids (Oliveira et al., 2018).

26
 The health ramifications of these bioactive compounds in black beans span various domains.

For instance, the prevention of LDL cholesterol oxidation, facilitated by their antioxidant

properties, plays a pivotal role in mitigating cardiovascular ailments (Cruz et al., 2019).

Additionally, several of the bean's phytochemicals, anthocyanins in particular, exhibit potent anti-

inflammatory actions, potentially reducing the onslaught of chronic ailments (Lopez et al., 2020).

Preliminary studies even suggest a role for black beans in cancer prevention, with some

phytochemicals inhibiting the proliferation of certain cancer cells, although this area warrants

deeper investigation (Perez et al., 2021).

27
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials

Freshly harvested black beans and whole wheat flour will be obtained from Eke market,

Enugu, Nigeria. The wheat flour will be obtained from Sabo market, Ogbomoso, Nigeria. Other

ingredients used for baking which includes yeast, butter, sugar will also be bought from new Wazo

market, Ogbomoso. All the reagents will be of analytical grade

3.2 Preparation of Sample

3.2.1 Preparation of black beans flour (Phaseolus vulgaris L.)

The method of Yilmaz & Alibas, (2022) for flour production will be employed in the

production of the black beans flour. Carefully selected and freshly harvested black beans plant will

be cleaned of all post-harvest dirts and all foreign objects will be removed. Afterwards, the black

beans will be dried using the cabinet dryer at the Food Processing Lab. Afterwards, the dried black

beans will be grounded into fine particles using a model as shown in Figure 3.1,

3.2.2 Preparation of black beans blended Bread

Following the method of Sanful & Darko (2010), samples of black beans blended bread – the

proportion of which will be 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30 (wheat flour and black

turtle beans flour respectively) as shown in Table 3.1. The samples shall be prepared by mixing

black beans flour with wheat flour using a model. The mixing will be done to ensure a

homogeneous mixture of the samples. The sample formulations will be baked using the straight

dough method (Kinton and Ceserani, 2008). All ingredients will be mixed in a mixer for

28
approximately 5 min. The dough will be left in bowls to prove covered with damp clean muslin

cloth for approximately 55 min at room temperature (29 °C), the dough will then be knocked back

to and molded into a loaf, placed in a loaf tin and further proved in a proving cabinet for 90 min at

30 ’C, 85 % relative humidity and baked at 250 °C for 30 min (Giami et al., 2004). It will thereafter

be removed and placed on a tray to cool as shown in Figure 3.2.

29
 Black Beans

Cleaning

Drying
(40-70’C; 30-15h)

Grinding

Sieving (particle size)

Black Beans Flour

Fig 3.1: Flowchart for the production of black bean flour

Source: Sánchez-Toledano et al., (2023)

30
 Table 3.1: Formulation ratio of whole wheat flour and black beans flour
Sample A (%) B (%) C(%) D (%) E (%) F (%) G (%)

Whole 100 95 90 85 80 75 70

Wheat

flour

Black 0 5 10 15 20 25 30

Beans

Source: USDA (2019)

31
 Weighing of ingredients

Mixing

Kneading

Proving

Baking
(180 ’c – 200 ’c, 40
mins)

Cooling

Black Beans fortified

wheat bread

Fig 3.2: Flow chart for the production of black beans fortified bread

Source: Mohammed et al., (2023)

32
3.3 Proximate Analysis

The following analysis will be carried out to determine the proximate composition of bread

blended with black beans;

3.3.1 Determination of moisture content

This method is based on moisture evaporation. Here, the aluminum dishes will be washed

dried in oven and in desiccators for cooling. The weight of each dish will be taken 5.0 g of ground

samples will be weighed into a sterile aluminum dish, weight of the dish and weight of un-dried

sample (in duplicate) will be taken. This will be transferred into an oven set at 80 °C for 2 hours

and at 100 °C for 3 hours respectively. This will be removed and cooled in desiccators. Then the

weight will be measured using a measuring scale balance. It will be transferred back into the oven

for another one hour and then reweighed. The process will continue until a constant weight is

obtained. The difference in weight between the initial weight and the constant weight gained

represents the moisture content.

Calculation: The loss in weight multiplied by 100 over the original weight is percentage moisture

content.

%moisture=

loss in weight
x 100___________________________________________(1)
weight of sample taken

W2-W3
%moisture= x 100___________________________________________________________(2)
W2-W1

Where W1= initial weight of empty crucible, W2= weight of crucible + food before drying, W3 =

final weight of crucible + food after drying.

33
% Total solid (Dry matter) (%) = 100- moisture (%)

(AOAC 2019)

3.3.2 Ash Content

Twenty grams (20 g) of each of the samples will be weighed into a clean dried and cooled

platinum crucible. It will be put into a furnace set at 550 °C and incinerated for 3 hours. It will

then be brought out and allowed to cool in desiccators and weighed again.

Calculation: Percentage weight is calculated as weight of ash multiplied by 100 over original

weight of the samples used.

Weight of ash
%Ash Content = x 100_______________________________(3)
weight of original sample used

Weight of ash
%Ash Content = x 100_______________________________(4)
weight of original sample used

Where W1 = weight of empty crucible

W2 = weight of crucible + food before drying or ashing

W3 = weight of crucible + ash

(AOAC 2019)

3.3.3 Determination of lipid content

The method employed was the Soxhlet extraction technique described by AOAC (922.06).

15 g of the samples were weighed and carefully placed inside a fat free thimble. This was covered

with cotton wool to avoid the loss of sample. Loaded thimble was put in the Soxhlet extractor,

about 200 ml of petroleum ether were poured into a weighed fat free Soxhlet flask and the flask

34
was attached to the extractor. The flask was placed on a heating mantle so the petroleum ether in

the flask refluxed. Cooling was achieved by a running tap connected to the extractor for at least

6hrs after which the solvent was completely siphoned into the flask. Rotary vacuum evaporator

was used to evaporate the solvent leaving behind the extracted lipids in the Soxhlet. The flask was

removed from the evaporator and dried to a constant weight in the oven at 60 °C. The flask was

then cooled in a desiccator and weighed. Each determination was done in triplicate. The amount

of fat extracted was calculated by difference.

Ether Extract (100 g of dry matter) =

Weight of extracted lipids

x 100_______________________(5)
Weight of dry sample

3.3.4 Protein determination

Total protein will be determined by the Kjeldahl method as modified by Williams (1964).

The analysis of a compound of its protein content by Kjeldahl method is based upon the

determination of the amount of reduced nitrogen present. About 20 g of the samples will be

weighed into a filter paper and put into a Kjeldahl flask, 10 tablets of Na2SO4 were added with 1

g of CuSO4 respectively. Twenty milliliters (20 ml) of conc. H2SO4 will be added and then digested

in a fume cupboard until the solution becomes colorless. It was cooled overnight and transferred

into a 500 ml flat bottom flask with 200 ml of water. This will then be cooled with the aid of packs

of ice block. About 60 to 70 ml of 40 % of NaOH will be poured into the conical flask which will

be used as the receiver with 50 ml of 4 % boric acid using 3 days of screened methyl red indicator.

The ammonia gas will then be distilled into the receiver until the whole gas evaporates. Titration

will be done in the receiver with 0.01M HCl until the solution becomes colorless.

Calculation: The percentage protein is calculated as follows:

35
Vs – Vb x 0. 01401x N acid (6.25) x 100 Original wt. of sample used ---------------------- (6)

Where Vs = Vol (ml) of acid required to titrate sample, Vb = Vol (ml) of acid required to titrate

blank, N acid = normality of acid (AOAC 2019).

3.3.5 Crude fiber

The bulk of roughages in food is referred to as fiber and is estimated as crude fiber. Twenty

grams (20 g) of the different samples will be defatted with diethyl ether for 8 h and boiled under

reflux for exactly 30 min with 200 ml of 1.25 % H2SO4. It will be then filtered through cheese

cloth on a flutter funnel. This will be later washed with boiling water to completely remove the

acid. The residue will be then boiled in a round bottomed flask with 200 ml of 1.25 % sodium

hydroxide (NaOH) for another 30 min and filtered through previously weighed couch crucible.

The crucible will be then dried with samples in an oven at 100 °C, left to cool in a desiccator and

later weighed. This will be later incinerated in a muffle furnace at 600 °C for 2 to 3 hours and

later allowed to cool in a desiccator and weighed. (AOAC 2019)

Calculation = Weight of fiber = (C2-C3) y

C2-C3
% fibre = x 100 ________________________________(7)
weight of original sample

3.3.6 Carbohydrate determination

Carbohydrate content will be determined by ‘difference’ following the AOAC method. That

is, the percentage composition of other proximate analysis will be deducted from 100 to get the

percentage composition for carbohydrate.

Available carbohydrate (%) = 100- (protein (%) + Moisture (%) + Ash (%) + Fibre (%) + Fat(%)

36
3.4 Physical Examination

3.4.1 Loaf Weight

The loaf weight will be determined by weighing the bread loaves after baking using the

laboratory scale and recorded in grams (g).

3.4.2 Loaf Volume

The loaf volume will be determined by using Rape seed displacement method described by

AACC (2000). This will be done by loading millet grains into an empty box with calibrated mark

until it reached the marked level and unloaded back. The bread sample will be put into the box and

the measured millet will be loaded back again. The remaining millet grains left outside the box

will be measured using measuring cylinder and recorded in cm3.

3.4.3 Specific Volume

The specific volume (volume to mass ratio) (cm3 /g) will be calculated. (Ibidapo et al.,2015).

Specific volume cm3/g= Loaf volume/ Loaf weight

3.4.4 Color Analysis

Crust colour analysis of the bread samples will be done by using Hunter Lab colorimeter

(model SM-3001476 micro sensors, New York). The instrument will be calibrated with user

supplied black plate calibration standard used for zero setting and white calibration plates for white

calibration settings. Sample measurements will be taken at three different points and readings will

be displayed as L*, a* and b* colour parameters according to CIELAB system of colour

measurement. The value of a* ranged from -100 (redness) to +100 (greenness), b* values ranged

37
from -100 (blueness) to +100 (yellowness) while L* value which indicates lightness, ranged from

0 (black) to 100 (white) (Bolarinwa et al., 2020).

3.4.5 Textural property

Texture analyzer (TA HD Plus, stable micro-Systems, Godalming, Surrey, UK) will be used

to measure the hardness, cohesiveness, chewiness and springiness of the bread samples according

to AACC (2004) procedure.

3.5 Determination of total bacterial count (TBC)

Nutrient agar media will be used to determine the total bacterial count. Nutrient agar plates

will be dried and labeled for appropriate dilutions to be used for dilutions and spread plate method.

Plates will be inoculated and incubated at 37 % for 18-24 hours. Total number of bacteria cfu/g of

sample will be calculated and recorded for interpretation of the result (Khanom et al., 2016).

3.5.2 Enumeration of yeast and mold count

Diluted samples will be inoculated into sabouraud dextrose agar (SDA) medium

supplemented with chloramphenicol (40 mg/l) by spread plate method. The plates will be

incubated at 25 ℃ for 48-72 hours. Visible colonies will be counted and calculated as the total

yeast and mold and recorded as cfu/g (Khanom et al., 2016).

3.5.3 Determination of coliform bacteria

Membrane faecal coliform (mFC) media will be used to detect the coliform bacteria by

using spread plate method. Plates will be inoculated and incubated at 37 ℃ for 24 hours. All blue

colored colonies will be enumerated to calculate total coliform as cfu/g (Khanom et al., 2016).

38
3.6 Sensory Analysis

The sensory analysis will be carried out by twenty untrained sensory panelists of undergraduate

students of Ladoke Akintola University of Technology, Nigeria. Each panelist will be served with

all the bread samples including the control and enriched wheat fiber bread. Water will be provided

for rinsing between the samples. The organoleptic attributes to be assessed include colour, taste,

aroma, and overall acceptance of the bread using the 9-point hedonic scale with 1 dislike extremely

and 9 like extremely.

3.7 Statistical Analysis

All experiments including organoleptic analyses will be replicated. Statistical analysis will be

performed using SPSS (Statistical Package for Social Sciences). One-way ANOVA (Analysis of

variance at the level of significance p≤ 0.05) will be performed using Duncan multiple range tests

to ascertain the significance of the means.

39
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