EVALUATION OF CAFFEINE CONTENT IN COCOA BEANS FROM IKOM

LOCAL GOVERNMENT AREA OF CROSS RIVER STATE

A PROJECT REPORT SUBMITTED TO

THE DEPARTMENT OF PLANT SCIENCE AND BIOTECHNOLOGY IN

FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF B.SC. (HONS)
DEGREE,

FACULTY OF BIOLOGICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA.

BY

MATTHEW, IFEANYI VICTOR

REG. NO. 2018/247173

SUPERVISOR: PROF. N. E. ABU

OCTOBER, 2023.

1
 TITLE PAGE

EVALUATION OF CAFFEINE CONTENT IN COCOA BEANS FROM IKOM LOCAL

GOVERNMENT AREA OF CROSS RIVER STATE.

i
 CERTIFICATION

This is to certify that MATTHEW, IFEANYI VICTOR with registration number

2018/247173, an undergraduate student of the Department of Plant Science and

Biotechonology, Faculty of Biological Sciences, University of Nigeria, Nsukka, has

satisfactorily completed the requirements for the research work for the Bachelor of Science

(B.Sc.) degree in Plant Science and Biotechnology. The work embodied in this report is

original and has not been submitted in part or full for any other diploma or degree of this or

any other University.

---------------------------------------------- -----------------------------------------------

PROF. N. E. ABU PROF. C. C. ONYEKE

(Project Supervisor) (Head of Department)

DATE ______________________ DATE ______________________

------------------------------------------------------------------------

EXTERNAL EXAMINER

DATE __________________

ii
 DEDICATION

This Project work is dedicated to God almighty.

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 ACKNOWLEDGEMENT

First and foremost, I want to acknowledge and thank God for his infinite mercies, grace and

goodness for giving me the abilities to undertake this work. My heartfelt appreciation and

profound gratitude goes to my supervisor Prof. N. E. Abu who guided me through this

writing, Mr. Udoh, Obiora, for his advice, encouragement and hard work all through the

period of this work. I would like to appreciate the Head of my department, Prof. C. C.

Onyeke and all the lecturers of this department for their efforts in providing a conducive

studying environment for me to carry out my research. I will not fail to acknowledge the

unending support of my lovely and ever supportive parents, Mr and Mrs Matthew Onuh for

their immense contribution in prayers, financial aids, advices and my siblings for all the

prayers and encouragement throughout the period of this work. I would also like to thank in a

special way my colleagues for their advice and contributions to help make this work a

success; God bless you all.

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 TABLE OF CONTENT

Title page i

Certification page ii

Dedication iii

Acknowledgements iv

Table of Content v

List of Tables viii

List of Figures ix

List of Plates x

Abstract xi

CHAPTER ONE: INTRODUCTION

1.1 Background of study 1

CHAPTER TWO: LITERATURE REVIEW

2.1 Taxonomy 4

2.2 Botanical description 4

2.3 Origin of Cocoa 7

2.4 Harvesting 7

2.5 Cocoa Pre-processing 8

2.6 Types of Cocoa 8

2.7 Uses of cocoa 10

2.8 Disease of Cocoa 11

2.9 Production and Consumption 11

2.10 Constraints and Breeding 12

v
2.11 Bioactive compound on Cocoa beans 12

2.11.1 Caffeine 14

CHAPTER THREE: MATERIALS AND METHODS

3.1 Sample collection 16

3.2 Sample preparation 16

3.3 Preservation of Specimen 16

3.4 Chemicals and reagents 17

3.5 Instruments and Apparatus 17

3.6 Calibration solutions preparation 17

3.7 Extraction of caffeine 17

3.8 Statistical analysis 19

CHAPTER FOUR: RESULTS

4.1 Presentation of Results 20

CHAPTER FIVE: DISCUSSION AND CONCLUSION

5.1 Discussion 22

5.2 Conclusion 23

REFERENCES 24

vi
 LIST OF TABLE

Table 1: Caffeine analysis of cocoa (Theobroma cacao) beans from communities in Ikom

Local Government Area of Cross River State.

21

vii
viii
 LIST OF FIGURE

Figure 1: Chemical structure of caffeine 14

ix
 LIST OF PLATE

Plate 1: Picture of Cocoa pod 6

Plate 2: An open cocoa pod showing the cocoa seed 6

Plate 3: Boiling of the sample in a water bath (DK-420) 18

Plate 4: Weighing of Sodium carbonate 18

Plate 5: Taking of absorbance reading using a UV/Vis spectrometer 19

x
 ABSTRACT
Cocoa beans, the primary raw material for chocolate production, have gained significant
attention for their rich flavor and potential health benefits. Among the many compounds
present in cocoa beans, caffeine is a key component known for its stimulant properties and
contribution to the overall sensory profile of chocolate products. This research was aimed to
conduct a comprehensive analysis of caffeine levels in cocoa beans sourced from Ikom, a
renowned cocoa-producing area in Nigeria. The result of the analysis revealed significant
variation among the cocoa beans sample from different locations in Ikom. The caffeine
content analysis showed that samples from Lasmotor and Cocoa research institute showed the
lowest caffeine content (3.00 ± 0.00) when compared to samples from Ajijinkpor (4.00 ±
0.000), Ikom (4.00 ± 1.414), Nde (4.50 ± 0.707) and Ekparanbong (5.00 ± 0.000). The
evaluation of caffeine content in cocoa beans from the Ikom L.G.A has provided valuable
insights into the factors influencing caffeine concentrations and their potential impact on the
chocolate industry. The significant variability in caffeine content emphasizes the need for
nuanced approach to cocoa bean selection and management. The findings underscore the
importance of sustainable farming practices that consider factors affecting caffeine levels.

xi
 CHAPTER ONE

Introduction

1.1 Background of study

The demand for cocoa increases every year, which is the reason, for decades the cocoa

industry has been affected by its sustainability. About 10 tons of wet waste and cocoa

byproducts are disposed of for every ton of dry beans, making waste management extremely

complex and expensive (Mariatti et al., 2021). Consumers are more concerned about the

possible health benefits of their diet and this fact has increased the demand for foods that

contain biologically active compounds (Teplova et al., 2018). The new challenge for the food

industry is to enrich its products for human consumption with compounds that have

functional properties such as antioxidant compounds. Specifically, polyphenol compounds

are of great interest due to their possible positive effects on human health, such as anticancer,

antioxidant, and anti-inflammatory activity (Manuela et al., 2020). Consequently, food

science research is focused on finding new and sustainable sources of antioxidants,

optimizing extraction and purification methods as well as developing innovative health-

promoting functional foods (Soares and Oliveira, 2020). It has been projected that, by 2050,

the population of the planet will be around 9.8 billion people; it is necessary to use resources

properly, recycle and reuse by-products. This population increase necessitates the search for

new food sources (Ribeiro et al., 2022).

Cocoa originates from beans of the cocoa tree (Theobroma cacao L.) and is the main

ingredient in chocolate manufacturing. The popularity, value and quality of cocoa products

are much related to unique and complex flavours contributed by various volatile and non-

volatile compounds (Rahmat et al., 2019). These volatile compounds comprised of nitrogen

and oxygen heterocyclic compounds, aldehydes and ketones, esters, alcohols, hydrocarbons,

nitriles and sulphides, pyrazines, ethers, furans, thiazoles, pyrones, acids, phenols, imines,

1
amines, oxazoles, and pyrroles (Afoakwa et al., 2008). Cocoa is the leading agricultural

export crop of the country and Nigeria is currently the world’s fourth largest producer of

cocoa, after Cote d’Ivoire, Indonesia and Ghana and the third largest exporter, after Cote

d’Ivoire and Ghana (Verter and Becrarova, 2018). Cocoa is therefore a major commodity

crop cultivated in Nigeria and is a major raw material used in the production of cocoa powder

(for beverage drink), various chocolate based products, biscuits and confectioneries.

Processed cocoa bean is also used to make sweets, sweetening products, cocoa butter (used in

making chocolate), perfume, and in pharmaceuticals (Adejeobi et al., 2011). Locally, cocoa

bean is used in cooking soup that has resemblance of okra and in treating various abdominal

problems or ailments (Opeke, 2005). Proceeds from export also provide substantial revenue

for the governments’ capital development projects (Aikpokpodion, 2010). The production of

cocoa in Nigeria has witnessed a downward trend since the early 1970s due to numerous

factors like ageing trees, ageing farmers, wrong application of recommended agronomic

techniques by farmers, effects of pests and diseases and deficiencies in macro and micro

nutrients in the soils (Adejeobi et al., 2011).

Cocoa contains approximately 380 known chemicals and 10 psychoactive compounds and is

a natural psychoactive substance that belongs to a group of chemical compounds called

purine alkaloids (Faudone et al., 2021). It is an organic chemical compound whose sum

formula is C8H10N4O2 (Patil, 2012). It is found in various plants such as coffee, tea, cocoa,

guarana, and yerba mate. It is a common ingredient in many beverages, including coffee, tea,

energy drinks, and sodas (Fiani et al., 2021). Caffeine acts as a stimulant, increasing alertness

and arousal levels, improving attention span, and reducing fatigue and drowsiness. Therefore,

it is commonly used as a stimulant to enhance mental and physical performance, which

attracts people who want to improve their performance in sports or training (Mclellan et al,.

2016). The concentration of caffeine in cocoa beans can vary depending on several factors,

2
including bean variety, geographic origin, and processing methods. On average, cocoa beans

contain about 0.1% to 0.7 % caffeine by weight (Franco et al., 2013). However, the actual

caffeine content in cocoa products can be influenced by various factors, such as processing

techniques and formulation.

3
 CHAPTER TWO

Literature review

2.1 Taxonomy

Domain: Eukaryota

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Phylum: Spermatophyta

Subphylum: Angiospermae

Class: Magnoliopsida

Subclass: Dilleniidae

Order: Malvales

Family: Sterculiaceae

Genus: Theobroma

Species: T. cacao

Source: Rajana, 2018.

2.2 Botanical description:

The chocolate tree Cocoa Theobroma cacao L. is cultivated as an economic crop in 58

countries and on more than 17 million acres (6.9 million ha) worldwide, with 72 % of the

production in West and Central Africa (Clay, 2004). About six million people depend on its

farming for their livelihoods (Baah and Garforth, 2008). Theobroma cacao L. is an important

tropical rainforest tree, previously classified in the Sterculiaceae and presently recognized as

4
a member of the family Malvaceae (Bayer and Kubitzki, 2003) that originated in tropical

South America (Bartley, 2005; Wood and Lass, 2001). The tree is commonly known as cacao

tree, while the term cocoa is reserved for the products made from the dried and fermented

seeds. Theobroma cacao encompasses many morphologically variable populations with a

marked potential for inter and intra-mating (Bartley, 2005).

The cocoa tree typically reaches a height of 4 to 8 meters and 4 to 6 meters of diameter of the

crown, although it can grow taller under favourable conditions. It has a straight trunk with a

greyish-brown bark, which becomes rough and furrowed with age (Bartley, 2005). The tree

branches horizontally, forming a broad and rounded crown. The leaves of the cocoa tree are

simple, alternate, and oblong in shape. They have a leathery texture and measure about 10 to

30 centimetres in length. The leaves are dark green on the upper surface and paler green on

the underside. They have prominent venation and entire margins (Wood and Lass, 2001).

The cocoa tree produces small, fragrant flowers that emerge directly from the trunk and

branches. The flowers are borne in clusters and have a unique appearance, with five small

sepals and five petals that are creamy white to pale yellow in colour. The flowers have

numerous stamens and a single pistil at the centre. The fruit of a cocoa tree is a large,

ellipsoid or ovoid-shaped structure called a pod (Plate 1). The pods are typically 15 to 30

centimetres long, although their size can vary depending on the variety. They have a thick,

rough, and woody rind that matures from green to yellow, red, or purple, depending on the

cultivar (Lele, 2013).

5
Plate 1: Cocoa pod

The main pollinating agent of cocoa are a small group of insects belonging to the

Ceratopogonidea family, genus Forcipomyia. Inside the cocoa pod, there are numerous seeds

embedded in a sweet, juicy pulp. These seeds are commonly referred to as cocoa beans or

cocoa seed (Plate 2) (Wood and Lass, 2001). The beans are flattened and have a somewhat

oval or rectangular shape. Usually the fruit when immature is green, and yellow when ripe.

They are covered by a thin, brownish seed coat, which is easily removed during processing.

Each pod contains around 20 to 50 cocoa beans (Lele, 2013).

Plate 2: An open cocoa pod showing the cocoa seed.

6
2.3 Origin of Cocoa

The native range of cocoa is designated as south-western Amazon, including Peru, Ecuador,

Colombia and Bolivia (Motamayor et al., 2002). Its accepted centre of origin and diversity is

near the headwaters of the Amazon River Basin near Iquitos, Peru at the confluence of the

Caquetá, Napo and Putumayo rivers (Sereno et al., 2006). The greatest known variation in

morphological and physiological characters of cocoa occurs in this region (Motamayor et al.,

2002). The eastern Cordillera of the Andes is the boundary between the early plantings of

Criollo (native) and Forastero (exotic) cocoa. Amerindian settlers from the Amazon region

are believed to have migrated from South to Central America, taking pale-coloured, plump

cocoa beans (least astringent) with them (Cheesman, 1944).

Cocoa cultivation was extended southwards from Central America by the Spaniards, who

promoted the consumption of cocoa in the sixteenth century (Badrie et al., 2015). From mid-

seventeenth century and onwards, cocoa was introduced into Peninsular Malaysia, Indonesia,

Ceylon (now Sri Lanka), and the Philippines from South America (Brazil and Venezuela) and

Central America and Trinidad (Badrie et al., 2015). It is documented as reaching Nigeria in

1874, Cameroon in 1876 and Ghana in 1878, as Amelonado cocoa from Brazil (Opoku et al.,

2007).

2.4 Harvesting

Theobroma cacao normally begins to bear berries after 3 years and the yield reaches a

maximum after 8-9 years. Trees simultaneously bear flowers, developing berries and mature

fruits. The pods develop on the trunk and branches. After about 5-6 months’ development the

pods ripen and turn yellow or orange. Harvesting is carried out at varying frequencies (1-4

weeks). The pods are then opened either on the same day or after a few days to allow a

sufficient quantity to accumulate for the fermentation stage. Beans are removed and separated

7
from the placenta. At this stage, they are covered in a sweet mucilaginous pulp (Poonam,

1999).

2.5 Cocoa Pre-Processing

After harvesting, the fruits are washed and opened manually, and the placenta separated from

the seeds. This step allows a more adequate fermentation, generating seeds with better

sensory characteristics. After removing the pulp, the seeds have about 65% moisture (Cruz,

2002). To avoid undesirable chemical reactions and the development of spoilage micro-

organisms, the interval between opening the fruit and fermentation should not exceed 24

hours (Efraim et al., 2011). Fermentation lasts between 2 and 8 days, due to the type of cocoa

and the environmental characteristics (Afoakwa, 2016). During this process, the seeds are

stirred frequently to optimize air penetration and improve product homogeneity (Owusu et

al., 2013).

Finally, the cocoa beans go into drying and storage. There are two techniques for this step:

natural or artificial. The natural technique is quite simple, requiring only the space available

to perform it. The artificial one uses dryers in a warehouse (Cruz, 2002). One of the most

important parameters to control is the water removal rate. If high, the inside of the cocoa

beans will remain moist, promoting the proliferation of fungi/micro-organisms and reducing

the quality of the product. If slow, the product will lose weight, increasing the fragility of

cocoa beans (Lopes, 2000). The drying stage is intended not only to eliminate the water in

excess (final moisture close to 7%) (Lopes et al., 2003), but also to reduce the acidity of the

cocoa beans, following the biochemical changes initiated in the fermentation, which will give

the characteristic flavour and aroma of chocolate (Lopes et al., 2003). This whole process

takes place near the fruit harvest site.

8
2.6 Types of Cocoa

Three major classes of cocoa are traditionally recognized in the cocoa trade: Forastero (Upper

and Lower Amazon, Amelonado and Guianese), Criollo, and hybrids of Criollo and Forastero

referred to as Trinitario (Lachenaud and Oliver 2005). This classification is based on

morphological traits, particularly those of the fruit (pod) and seed (bean).

Criollo cocoa is currently grown throughout Central America, in countries such as Mexico

and in South America. Authentic or pure Criollo trees are rare globally because Criollo

varieties are typically susceptible to diseases and display low vigor and criollo hybrids are

slightly more common (Bekele and Phillips, 2019). Motamayor et al. (2002) and Motilal

et al. (2010) reported on the low genetic diversity and heterozygosity found among the

Criollo germplasm they studied.

Forastero cocoa is the most widely cultivated and abundant type of cocoa. Forastero beans

tend to have higher butterfat contents than Criollo cocoa (Khan et al., 2008). This increases

their value in the cocoa trade because cocoa butterfat attracts an attractive price. Forastero

cocoa trees currently yield 95% of traded cocoa, referred to as bulk cocoa in the market

(Bekele and Phillips, 2019). Forastero cocoa beans have a purplish color and a strong, robust

flavor profile (Ayodele et al., 2019). Iwaro et al. (2003) observed that a relatively larger

proportion of individuals from this group are resistant to diseases, such as black pod disease,

compared to other groups.

Trinitario cocoa is a cross between Criollo and Forastero (Johnson et al., 2009).

Hybridization that produced Trinitario cocoa is believed to have first occurred in Trinidad

hence the designation (Motamayor et al., 2003). The Trinitario group currently comprises a

wide range of hybrids between Criollo and Amazon Forastero cocoa found in the Caribbean

region, South and Central America, Papua New Guinea and wherever there has been a

mixture of Criollo and Amazon Forastero cacao (Bekele et al. 2009). This is thus a very

9
heterogeneous group, displaying extreme phenotypic and genetic diversity. Trinitario and

Criollo cocoa produce Fine or Flavor cocoa.

Refractario cocoa is another recognized group of cocoa, and has a distinctive genetic

background when compared to Nacional cocoa from Ecuador, Amazon Forasteros from Peru

and Trinitarios (Zhang et al. 2008). Research findings of Motamayor et al. (2003)

revolutionized the classification of cacao in that they revealed ten genetic clusters of cacao.

These genetic groups are Amelonado, Contamana, Criollo, Curaray, Guiana, Iquitos,

Marañon, Nacional, Nanay and Purús.

2.7 Uses of Cocoa

The most well-known and commercially significant use of the cocoa tree is the production of

chocolate and cocoa-based products. The cocoa beans are processed to produce cocoa

powder, cocoa butter, and chocolate liquor, which are utilized in confectionery, baking, and

dessert production (Ramos et al., 2021). Additionally, cocoa beans can be consumed directly

or used to make cocoa beverages. Cocoa butter, a fat extracted from cocoa beans, is widely

used in the cosmetic industry for its moisturising and emollient properties. It is a common

ingredient in various skincare products, such as lotions, creams, lip balms, and soaps, due to

its ability to hydrate and nourish the skin (Afoakwa et al., 2014). The cocoa tree also plays a

vital role in agroforestry systems and environmental sustainability. It can serve as a shade

tree for other crops, providing shade and protection against excessive sunlight. The

cultivation of cocoa in agroforestry systems promotes biodiversity, soil conservation, and

carbon sequestration, making it environmentally beneficial (Ruf et al., 2020). It provides

employment and income opportunities for millions of farmers, contributing to rural

development and poverty reduction (Vaast et al., 2018). The global cocoa industry has a

substantial impact on trade, generating revenue for both producing and consuming countries.

10
Apart from economic importance, intake of cocoa products has positive for health (Wollgast

and Anklam, 2000). Polyphenolic compounds of cocoa beans exhibit anti-carcinogenic

(Ioannone et al., 2015), non-inflammatory and skin-protective (anti-aging) properties, which

is associated with their anti-oxidative potential (Martorell et al., 2011).

2.8 Disease of cocoa

The most dangerous diseases are frosty pod and witches’ broom, that are caused by related

hemibiotrophic basidiomycetes, Moniliophthora roreri and Moniliophthora perniciosa,

respectively (Aime and Phillips-Mora, 2005). Frosty pod is twice as damaging as black pod

and more difficult to control than witches’ broom (Evans, 2002). Witches’ broom disease is

one of the most important diseases of cocoa (Theobroma cacao L.) in Latin America and the

Caribbean islands, causing large scale losses (De Arruda et al., 2003). M. perniciosa

basidiospores can infect any meristematic tissues of cocoa and are the only known infective

propagules for this pathogen (Griffith et al., 2003). The disease shows two distinct stages: a

biotrophic and a necrotrophic/saprotrophic phase. In the biotrophic phase the fungus exists as

an intercellular monokaryotic mycelium, that causes hypertrophy and hyperplasia of the

tissues, loss of apical dominance, and proliferation of axillary shoots, which results in the

formation of abnormal stems called a green broom. In the second stage, the fungus changes to

the saprotrophic phase, with the spread of intracellular dikaryotic mycelium, which causes

necrosis and death of infected tissues distal to the original infection site, thus forming the dry

broom (Scarpari et al., 2005).

2.9 Production and consumption

Cocoa production occurs primarily in tropical regions, with the largest producers being Côte

d'Ivoire, Ghana, and Indonesia. The 2020/2021 cocoa year saw global cocoa production reach

11
approximately 4.7 million metric tons, with Côte d'Ivoire alone accounting for around 43% of

the total production. Other major cocoa-producing countries included Ghana, Indonesia,

Nigeria, Cameroon, and Ecuador (ICCO, 2021). According to International Cocoa

Organisation (2021), cocoa consumption is driven by the demand for chocolate and cocoa-

based products worldwide. In the 2020/2021 cocoa year, global cocoa bean grindings, which

serve as an indicator of cocoa consumption, amounted to about 4.2 million metric tons. The

main cocoa grinding countries included the Netherlands, Côte d'Ivoire, Germany, the United

States, and Malaysia (Rahmat et al., 2019).

2.10 Constraints to Breeding

Cacao breeding has been constrained to date by the long breeding cycle of the crop. The

production of sufficient fruits in progeny to be evaluated may not be realized until a plant is

about 2–5 years old. A single selection cycle frequently takes more than a decade (Monteiro

et al., 2009), and two or more cycles of selection are often required before a new variety can

be released to farmers with an expectation of good performance (Phillips-Mora et al., 2013).

The relationship between genetics and the environment to which cacao varieties must be

adapted in the future must be understood when designing breeding programs. The projected

impact of climate change must be taken into consideration as well as the management

practices and specific growing environments in which new varieties will be distributed

(Bekele and Phillip, 2019).

2.11 Bioactive compounds on cocoa beans

The bioactive effect of cocoa beans and their processing products results mainly from the

presence of polyphenols in their composition. Although the beans also contain other

12
ingredients, such as methylxanthine (mainly caffeine and theobromine) (Bogumila et al.,

2019).

Polyphenols, being secondary metabolites, are one of the most plentiful and widespread

groups of naturally occurring substances in the plant kingdom. They are synthesized

biogenetically in two major primary pathways of synthesis: shikimate pathway and acetate

pathway (Sirerol et al., 2016). Cocoa beans are rich in polyphenols (ca. 15% of beans dry

matter), as are wine, vegetables or tea (Krähmer et al., 2015). Three groups of polyphenols

may be identified in cocoa beans: proanthocyanins (ca. 58%), catechins or favan-3-ols (ca.

37%) and anthocyanins (ca. 4%) (Wollgast and Anklam, 2000). Moreover, greater

antioxidant and pro-health effect is attributed to polyphenols, and their qualitative and

quantitative composition changes significantly under the influence of high temperature, pH,

or access to oxygen. (Bogumila et al., 2019).

The major and predominating catechin of cocoa beans is epicatechin which represents ca.

35% of total Catechin is a component of cocoa tannins which are responsible for colour

changes and for astringent after-taste of beans and products made of them. Bitterness of

epicatechins is undesirable; hence, producers try to reduce the amount of this favonoid in the

finished product (Pedan et al., 2016). The make-up of polyphenols very strongly depends on:

origin, genotype, beans processing and ripeness degree (Kothe et al., 2013).

Methylxanthines, like theobromine (C7H8N4O2, 3,7-dimethylxanthine or 3,7-dihydro-3,7-

dimethyl-1H-purine-2,6-dione) and caffeine (C8H10N4O2, 1,3,7-trimethylpurine-2,6-dione),

represent another group of bioactive compounds of cocoa beans (Franco et al., 2013). The

content of methylxanthines and the theobromine, caffeine ratio varies depending on cocoa

genotype. (Afoakwa et al., 2008).

13
2.11.1 Caffeine

Caffeine (1,3,7-trimethylxanthine) is a heterocyclic organic compound with a purine base

called xanthine, consisted of a pyrimidine ring (Zulak et al., 2006). Caffeine is known as an

alkaloid because it is a secondary plant metabolite derived from purine nucleotides, with a

heterocyclic nitrogen atom (Zulak et al., 2006). It is an organic chemical compound whose

sum formula is C8H10N4O2 (Patil, 2012).

Figure 1: Chemical structure of caffeine.

Source : DePaula and Farah, 2019.

It is abundantly found in cocoa (Theobroma cacao), coffee, Camellia sinesis (tea) guaraná

(Pauliniacupana) seeds, and in kola (Cola sp.) nuts (Chou and Benowitz, 1994). It is a

common ingredient in many beverages, including coffee, tea, energy drinks, and sodas (Fiani

et al., 2021).

Caffeine acts as a stimulant, increasing alertness and arousal levels, improving attention span,

and reducing fatigue and drowsiness. Therefore, it is commonly used as a stimulant to

enhance mental and physical performance, which attracts people who want to improve their

performance in sports or training (Mclellan et al., 2016). Thus, it is often added to dietary

supplements for its ability to increase metabolism and aid in weight loss. It can also help to

reduce appetite increasing the body’s energy expenditure due to increased heat production.

This can help with fat-burning and weight loss (DePaula and Farah, 2019). However, it is

14
essential to remember that excessive caffeine consumption can lead to unwanted side effects,

such as insomnia, irritability, anxiety, and even heart problems (Bessada et al., 2019).

15
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Sample Collection

The samples used for the laboratory work which are Theobroma cacao seed were collected as

pods. Fresh Theobroma cacao pods were harvested and obtained from different communities

located at Ikom Local Government Area of Cross River state, Nigeria. They were properly

identified and authenticated by Mr. Felix Nwafor, a plant taxanomist of the Department of

Plant Science and Biotechnology, University of Nigeria, Nsukka.

3.2 Sample preparation

The cocoa pods were cut open using a knife and the seeds collected and dried in a

commercial dehydrator (dehydrator ST-02, China) at 500C. The seeds were grouped into two;

for planting and laboratory analysis. The seeds for laboratory analysis were further ground

using a blender (KENWOOD 6000 BT, China) and stored in an air-tight amber bottle until

use (Vuletic et al., 2021).

3.3 Preservation of specimen

The seeds were dried with a dehydrator to reduce the moisture content in it. The dried

specimen was then ground into a powdery form and stored in an air-tight amber bottle. A

label was glued to the body of the bags indicating the type and name of the specimen (Vuletic

et al., 2021).

16
3.4 Chemicals and reagents

The chemicals used in this study include Sodium carbonate (Na2CO3), Chloroform (CHCl3)

and distilled water. All reagent used in this study were of analytical grade and all solutions

were prepared by using distilled water (Vuletic et al., 2021).

3.5 Instruments and apparatus

The instruments used in this study include: test-tube, spatula, water bath (DK-420), UV/Vis

spectrometer, weighing balance, filter paper, aluminium foil, blender (KENWOOD 6000 BT,

China), dehydrator (dehydrator ST-02, China), separation funnel (Vuletic et al., 2021).

3.6 Calibration solutions preparation

Caffeine stock solution (100 ppm) was prepared by dissolving 0.01 g of recrystallized

caffeine in 100 ml of chloroform in the volumetric flask. From the caffeine stock solution,

following dilutions were prepared: 1 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm and 25 ppm.

Their absorbance was measured at the wavelength of 274 nm in quartz cuvettes three times

for each dilution (Vuletic et al., 2021).

3.7 Extraction of caffeine

Exactly 1 gram of each cocoa bean sample was weighed and put in a test tube. 10 ml of

distilled water was added to the sample and was put in a water bath (DK-420). The content

was heated and boiled for 10 minutes in the water bath (DK-420) (Plate 3). The content was

put in a centrifuge for 10 minutes. A total of 1 gram of Sodium Carbonate was weighed (Plate

4) and added to each sample for precipitating tannins and put in a centrifuge tube for 10

minutes (Vuletic et al., 2021).

17
Plate 3: Boiling of the sample in a water bath (DK-420).

Plate 4: Weighing of sodium carbonate

The samples were filtered and filtrate was collected and stored in a vacutainer. From the

given sample, caffeine was extracted by adding 5 ml of chloroform in the separation funnel.

Caffeine was extracted by stirring in the separation funnel for a few minutes. The lower

caffeine-containing layer was separated and analysed for caffeine content with UV/Vis

spectrometer. The Absorbance reading was measured at 274 nm and the reading were taken

(Plate 5). Three samples of each specimen were analysed for caffeine content (Vuletic et al.,

2021).

18
Plate 5: Taking of the absorbance reading using a UV/Vis spectrometer.

3.8 Statistical Analysis

In this course of analysis, one-way analysis of variance (ANOVA) was used to statistically

analyse the data used obtained in the study. The level of statistical significance was set at p<-

0.05 (95% confidence interval). The software used is SPSS version 23.

19
 CHAPTER FOUR

4.1 Presentation of Results

This chapter shows the results of the caffeine analysis done during this study. The result of

the caffeine analysis of cocoa (Theobroma cacao) beans obtained from different communities

in Ikom local government area of Cross river state are presented (Table 1). The results of

some samples got from same community showed significantly no difference while others

from same community showed significantly high difference between the samples.

The samples obtained from Ajijinkpor showed no significant difference within the

community with a mean value of 4.00 ± 0.000 at p≤0.05. Likewise, samples obtained from

Lasmotor showed no difference with a mean value of 3.00 ± 0.000. The samples obtained

from Ekparabong showed a significant difference at p≤0.05 within the community with a

highest mean value as 5.00 ± 0.000. The samples obtained from Ikom also showed variability

within the community. The mean values obtained from Ikom ranged from 3.00 ± 0.000 to

4.00 ± 1.414. Significant difference was also observed among samples from Nde community

with a mean range of 3.00 ± 0.000 to 4.50 ± 0.707. Four samples from the Cocoa research

institute showed no significant difference with a mean value of 3.00 ± 0.000 while in two

samples from within the same research institute significant variation was observed. The mean

ranges from 4.00 ± 0.000 to 4.50 ± 0.707.

However, when compared with the samples from the different communities in Ikom L.G.A,

samples from Lasmotor showed the lowest caffeine content (3.00 ± 0.000) when compared to

samples obtained from Ajijinkpor (4.00 ± 0.000), Ikom (4.00 ± 1.414), Nde (4.50 ± 0.707)

and Ekparabong (5.00 ± 0.000).

20
Table 1: Caffeine analysis of cocoa (Theobroma cacao) beans from communities in Ikom

Local Government Area of Cross River State

Community Samples Caffeine values

Ajijinkpor AJI-IKO-TL 4.00 ± 0.000ab

AJI-IKO-SC 4.00 ± 0.000ab

Ekparabong EK-IKO-SC 3.00 ± 0.000b

EK-IKO-TL 5.00 ± 0.000a

Ikom IKO-IKO-TL 3.00 ± 0.000b

IKO-IKO-SC 4.00 ± 1.414ab

Lasmotor LAS-IKO-TL 3.00 ± 0.000b

LAS-IKO-SC 3.00 ± 0.000b

Nde NDE-IKO-SC 3.00 ± 0.000b

NDE-IKO-TL 4.50 ± 0.707a

Cocoa Research Institute CRIN-ABIL 3.00 ± 0.000b

CRIN-TRI 3.00 ± 0.000b

CRIN-SG 3.00 ± 0.000b

CRIN-EXT 3.00 ± 0.000b

CRIN-F3HY 4.00 ± 0.000ab

CRIN-SGa 4.50 ± 0.707a

Values expressed as mean +- standard deviation. Means with same letters as superscripts are

not significantly different at p <- 0.05.

21
 CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1 Discussion

In this research, the result obtained showed that samples obtained from Ajijinkpor, Lasmotor

communities and Cocoa research institute showed no significant difference in the caffeine

content, while samples from Ekparabong, Ikom and Nde communities of the same local

government high significant variation was observed. However, significant difference in the

caffeine content was observed when the samples from the different communities in Ikom

local government area was compared with each other. The reason for the variations observed

can be attributed to the genetic influence on caffeine content in cocoa beans. Post-harvest

processing methods, such as fermentation and drying could be another factor for the variation

in this study. This observation aligns with Martin (2017), who stated that certain cocoa

varieties are naturally predisposed to higher caffeine concentrations, while others have lower

levels due to genetic influence in cocoa beans. Considering the post-harvest processing

method, Nielsen et al. (2018), demonstrated that variations in post-harvest processing

techniques can alter the original physical and chemical properties of the seed which leads to

changes in caffeine levels. Proper fermentation and drying practices can potentially mitigate

caffeine fluctuations in cocoa beans.

Furthermore, considering the influence of caffeine on sensory attributes, particularly

bitterness, the consumer's preference for certain chocolate flavours may also impact their

daily caffeine intake. Some individuals may consume more chocolate to achieve a

satisfaction, inadvertently increasing their caffeine consumption. Research has shown that

daily caffeine consumption can vary widely based on dietary habits, cultural practices, and

personal preferences (Mitchell et al., 2014). The caffeine level observed from Ikom L.G.A

are well below the recommended daily consumption for caffeine intake. The daily limit of

22
caffeine consumption is 300 - 400 mg per day and varies among individuals (Siva et al.,

2020). It is vital for both consumers and producers to be aware of the caffeine content in

cocoa beans and their derived products, emphasizing the importance of transparency and

informed choices in daily dietary practices.

5.2 Conclusion:

In conclusion, the evaluation of caffeine content in cocoa beans from the Ikom L.G.A has

provided valuable insights into the factors influencing caffeine concentrations and their

potential impact on the chocolate industry. The significant variability in caffeine content

emphasizes the need for nuanced approach to cocoa bean selection and management. The

findings underscore the importance of sustainable farming practices that consider factors

affecting caffeine levels. Farmers in the Ikom region can optimize their cultivation and post-

harvest processing methods to achieve desired caffeine levels, thereby contributing to the

overall quality of cocoa-based products. The observed variability in caffeine content

emphasizes the need for consumers to be conscious of potential caffeine levels in chocolate

products.

23
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