CHAPTER ONE

1 INTRODUCTION

Cocoa bean is the dried and fully fermented fatty seed of the cocoa tree, from which

chocolate is made. (The word "cocoa" is derivative of "cacao".) "Cocoa" can often also

refer to the drink commonly known as hot chocolate. In any cocoa processing company,

cocoa seeds are used to manufacture cocoa powder used in the production of all food

with chocolate flavour. Cocoa butter is used in the cosmetics and pharmaceutical

preparations. Cocoa jelly is made on a small scale form cocoa, and sweet is sometimes

allowed, fermenting to produce cocoa wine. Cocoa, however, fulfils two primary

functions in foods, as a colorant and as a flavour ingredient.

Cocoa shells are the waste by-product of the roasting of cocoa beans and have no value

in chocolate manufacturing. In the season 2008/09 cocoa harvest in Ghana was 703,000

tonnes (GBC News, 2009), meaning about 84,360 - 105,450 tonnes of cocoa bean shells

were produced as waste. The West African Mills Company Limited (WAMCO) utilizes

about 110 tonnes of cocoa beans per day with about 14% being the shells. Therefore,

any process in which cocoa bean shells can be utilized can be of supreme importance.

1.1 Main objective

The main objective of the project is to develop a process for the utilization of waste

cocoa bean shells.

1.2 Specific objectives

The specific objectives of this project are as follows:

i. to do literature review,

ii. to work on the methodology,

iii. to do cost estimation for the project,

iv. to extract pectin from the cocoa bean shells,

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v. to test the quality of the product, and

vi. to draw conclusions on the work done.

2

CHAPTER TWO

2 LITERATURE REVIEW

2.1 The cocoa tree

Cocoa is essentially the botanical name and refers to the tree, the pods and the

unfermented beans from the pods. Cocoa belongs to the family Sterculiaceae and the

common cocoa is classified as Theobroma cacao. Cocoa (Theobroma cacao) is a native

of the Amazon region of South America. The majority of cocoa is produced in the

tropical areas of the African continent. There are over 20 species in the genus but the

cocoa tree Theobroma cacao is the only one cultivated widely. 

Being of very delicate and sensitive in nature, the cocoa tree needs protection from wind

and requires a fair amount of shade, especially in its first four years of growth. With

pruning and careful cultivation, the trees of most strains will begin bearing fruit in the

fifth year.

A perennial that yields several harvests annually, it was first cultivated in South

America, introduced into Europe during the 16th century and today grown mainly in

western Africa. The average cocoa tree attains a height of about 6 m, has shiny leaves as

long as 30 cm and small pink flowers on the trunk and older branches. Commonly

called cocoa beans, the seeds are surrounded by a yellow or reddish-brown pod up to 30

cm long. Cocoa beans are either purple or off-white and resemble almond.

(http://dacnet.nic.in/cashewcocoa/ctech.htm).

2.1.1 Varieties

The cocoa industry has traditionally recognized three main production types of the tree.

These trees are based mainly on the properties of the beans they produce. Cocoa

consists of three main varieties namely, Criollo, Forastero and Trinitario.

3

(a) Criollo has large, white or very pale purple beans which are more or less round in

cross section. The fermentation time is short and the roasted beans give a very high

quality product lacking in astringency. The trees lack vigour and tend to be susceptible

to diseases. They are probably not truly wild, but the product of ancient cultivation by

the Mayas. Two geographical populations can be recognized, Central America criollos

and South American criollos. The South American population was introduced into

north-eastern South American by Capuchin monks (Soria, 1970). Criollos are now

rarely cultivated because of their lack of vigour. Consequently there has been

considerable genetic erosion from this population and they are a priority group for

collection into gene banks.

(b) Forastero is a large group of wild, semi-wild and cultivated populations found

throughout the Amazon basin. The beans are flattened with violet pigmentation in the

cotyledons. The trees are usually vigorous; the pods have a hard pericarp and are not

conspicuously warty. The beans require up to a week for adequate fermentation and

produce a roasted product that may be astringent. The bulk of the world’s cocoa

production is from this population type.

(c) The trinitario population arose from initial crosses between criollo and forstero

populations. Trinitarios are not found in the wild. They display a wide variability

ranging from criollo to forastero characteristics. They retain some of the productive

vigour of the forastero types and combine this with the good flavour characteristics of

the criollos. The industry refers to them as “fine cocoa” (Smartt and Simmonds, 1995).

2.2 Cultivation of cocoa

The cultivation of cocoa from the planting stage to the harvesting stage is affected by

certain climatic conditions such as rainfall, temperature and humidity of the

surroundings.

4

Average rainfall of 1250-3000 mm per annum and preferably between 1500-2000 mm

with a dry season of not more than 3 months with less than 100 mm rain per month for

the plant is ideal, but the quantity is less important than distribution. Rainfall can be

supplemented with irrigation during dry months.

Temperature varying between 30-32oC mean maximum and 18-21oC mean minimum

but around 25oC is considered to be favourable. It can’t be grown commercially in areas

where the minimum temperature falls below 10oC and annual average temperature is

less than 21oC.

Humidity is also uniformly high in cocoa-growing areas, often 100% at night, falling to

70-80% by day, sometimes low during the dry season. The most marked effect of

humidity is on leaf area. Plants growing at low humidity (50-60%) having larger leaves

and greater leaf area than plants growing at medium (70-80%) and high (90-95%)

humidity under the latter conditions leaves are small and tend to be curled and withered

at the tip. Another effect of humidity concerns the spread of fungal diseases and the

difficulties of drying and storage. (http://dacnet.nic.in/cashewcocoa/ctech.htm).

Soil

Cocoa is grown on a wide range of soil types and the standards for soil suitable for

cocoa vary considerably. Cocoa trees are more sensitive to moisture stress than other

tropical crops. In addition cocoa trees are sensitive to water logging. While they can

withstand flooding, they will not tolerate stagnant, water logged conditions. The depth

of the soil should be at least 1.5 m. The best soil for cocoa is forest soil rich in humus.

The soil should be such as allowing easy penetration of roots capable of retaining

moisture during summer and allowing circulation of air and moisture. Clay loams and

sandy loams are suitable. Shallow soils should be avoided. Cocoa is grown on soils with

a wide range of pH from 6-7.5 where major nutrients and trace elements will be

5

available. Cocoa does not come up in coastal sandy soils where coconut flourishes.

(http://dacnet.nic.in/cashewcocoa/ctech.htm).

Harvesting and curing

The development of the pod takes 5-6 months from fertilizing the flower to full

ripening. Harvesting involves removing the ripe pods from the trees and opening them

to extract the wet beans. As they ripen, the pods change colours, green pods becoming

orange, yellow and red pods turning orange. Each pod will have 25-45 beans embedded

in white pulp (mucilage). Generally cocoa gives two main crops in a year during

September – January and April-June, though off-season crops may be seen almost all

through the year especially under irrigated condition. Only ripe pods have to be

harvested without damaging the flower cushions by cutting the stalk with the help of

knife. The harvesting is to be done at regular intervals of 10-15 days. The damaged,

unripe and infested pods have to be separated out to ensure better quality of beans after

processing. The harvested pods should be kept for a minimum period of two days before

opening for fermentation. However, pod should not be kept beyond four days.  Curing is

the process by which cocoa beans are prepared for the market, which requires beans of

good flavour and of high quality. The curing process involves fermentation followed by

drying. Fermentation involves keeping the mass of cocoa beans well insulated so that

heat is retained, while at the same time air is allowed to pass through the mass. The

process lasts up to 7 days and followed immediately by drying. Cocoa bean mass under

the process of fermentation has to be overturned regularly to maintain the uniform

specified temperature all over the mass. (http://dacnet.nic.in/cashewcocoa/ctech.htm).

Pest and diseases

Organized research into cocoa as a group began in about 1930. Emphasis has always

been on the control of pest and diseases since each producing area has severe limitations

imposed upon it by specific pathogens. In South and Central America, the two main

6

diseases are witches’-broom disease (Crinipellis perniciosa) and monila pod

(Moniliopthora roreri). West Africa has a viral disease called swollen shoot disease and

is severely troubled by capsids. (Smartt and Simmods, 1995).

In cocoa, three species of Phytophthora cause diseases commonly referred to as black

pod rot, and account for approximately 450,000 MT of losses worldwide. Phytophthora

species are found in all three growing regions, but the most aggressive and fast moving

is phytophthora megakarya which is found in West Africa (Duffey, 2009).

Phytophthora palmivora is pan- tropical and causes both pod rot and bark canker. Vast

research effort has been devoted to control of these problems through plant breeding

and through chemical control. (Smartt and Simmods, 1995).

2.3 The cocoa bean

The cocoa bean is comprised of an inner nib portion covered by an outer shell, also

referred to as the hull. On a dry basis, the shell of the bean comprises about 12-15% of

the weight of the bean, while the nib and residual moisture amounts to approximately

85-88%. Typical analytical data ranges for chemical compositions of cocoa nib are

shown in Table 1.

Table 1: Chemical composition of cocoa nib

Chemical composition Amount, %

Fat 48-57

Theobromine 0.8-1.3

Caffeine 0.1-0.7

Total nitrogen 2.2-2.5

Ash 2.6-4.2

Moisture 2.3-3.2

(http://www.freepatentonline.com/6312753.html)

7

Various processes are traditionally employed to extract cocoa butter and cocoa solids

from cocoa beans. Typical methods of processing cocoa beans include the steps of

cleaning the bean, roasting, winnowing, grinding the nib, liquor pressing to produce

cocoa butter and cocoa cake, also referred to as partially defatted cocoa solids,

optionally alkalizing and milling the cake.

(http://www.freepatentonline.com/6312753.html)

2.4 Cocoa producing countries

Nearly 70% of the world’s cocoa is grown in West Africa. The world’s largest cocoa

producer is Ivory Coast, Ghana being the second largest producer. Table 2 shows the

share of production of the cocoa growing countries in the world. Ivory Coast cocoa is an

essential part of many chocolate products whereas cocoa from Ghana is more prevalent

in Europe and prized for its consistent quality. Almost 90% of the world’s cocoa comes

from small holdings owned by an individual or family and are typically 5 ha or 12 acres

in size. An estimated 14 million people are employed in the cocoa industry worldwide.

In Ghana alone, 3.2 million people work with cocoa, second only to the Ivory Coast.

The cocoa crop covers about 28% of the cropped land in Ghana. The average cocoa

farm covers 1.2 ha but there are few large plantations. Farmers normally burn small

parts of secondary forest to open up new cocoa land. They intercrop with maize, yams,

plantains and cassava. The advantage of the intercropping is to provide shade for the

young cocoa plant. After 5 years farmers grow the crop as a monoculture because the

cocoa plants have developed a closed canopy. Without fertilizing, yields decrease after

20 years, but up to 50 years production is still possible. Potentially the yield could range

between 1 -1.5 t /ha but the average yield is about 300 kg/ ha.

(http://www.cocoainitiative.org/cocoa-producing-countries.html)

8

Table 2: Share of cocoa producing countries in the total cocoa bean production in

2005/2006

(http://www.cocoainitiative.org/cocoa-producing-countries.html)

2.5 The production process

A cocoa processing plant transforms cocoa beans into three main components: cocoa

liquor, cocoa butter and cocoa powder. These components can be used to make different

products. Cocoa liquor is used with other ingredients to produce chocolate. Chocolate is

used as a product on its own or combined with other ingredients to form confectionery

products. Cocoa butter is used in the manufacture of chocolate. It is also widely used in

cosmetic products such as moisturizing creams and soaps and in the pharmaceutical

industry in the making of suppositories and oral medications in capsule form. Cocoa

powder is used as an ingredient in foodstuff. For example, it is used in chocolate

flavoured drinks, chocolate flavoured desserts, chocolate spreads and sauces, cakes and

biscuits. (http://www.eurococoa.com/cocoa/story/trade.htm).

9

COUNTRY SHARE OF PRODUCTION, %

Cote d’Ivoire 38

Ghana 21

Indonesia 13

Cameroon 5

Nigeria 5

Brazil 4

Ecuador 3

Malaysia 1

Other 10

From bean to masse

The processing of raw cocoa beans into cocoa masse contains a number of stages.

Before arrival at the factory the raw cocoa beans have been fermented and dried, with

the first quality control measures already taken place at the port. On arrival at the

processing factory the beans are subjected to another thorough inspection, thereafter

cleaned and mixed into the desired blend for roasting to take place. Roasting of the

beans is done within a temperature range of 140-145oC for about 70 min. The roasted

beans are transported into a silo for cooling. The cooled beans are fed into a winnower

where the beans are mechanically thrown against an impact surface and broken into

coarse pieces called nibs. The nibs are then conveyed directly to the sieving and

aspiration section. Through vibration of the winnower, dust, other light object and shells

are trapped, which are sent to the shell silo by an elevator. The nibs are heat-treated to

eliminate possible bacteria and subsequently roasted and ground into a liquid cocoa

masse. The nibs are alkalized before, during or after the roasting process. This

determines the colour and taste of the cocoa masse, which act as an intermediate or semi

finished product supplied to the chocolate industry and also a basis for the production of

cocoa powder and cocoa butter. (http://www.eurococoa.com/cocoa/story/trade.htm).

From masse to butter and cake

Fat is pressed out of the cocoa masse under high pressure within the range of 40-50

MPa. The butter is subsequently filtered, to remove the last remnants of solid cocoa

ingredients. The manufacturer supplies the cocoa butter in liquid form in tankers, or in

solidified form in cardboard boxes. If the client so wishes, cocoa butter can be made

odourless by means of steam and vacuum extraction. What remains after the removal of

the cocoa butter through pressing are cocoa cakes, disks with a thickness of

approximately 5 cm. These cakes are broken up and ground into a fine cocoa powder.

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Each manufacturer supplies its own type of powder, with its own distinctive colour,

aroma, pH-value and fat content. (http://www.eurococoa.com/cocoa/story/trade.htm).

From masse to chocolate

Chocolate is made from cocoa masse with sugar, cocoa butter and, optionally, milk

added. The resulting mixture is rolled and ‘conched’. Conching is a treatment whereby

chocolate is kept in continuous movement to allow the cocoa masse to thicken and to

develop into a homogenous substance. This process also allows volatile acids to escape,

whereby the aroma is improved. Depending on the desired taste, other ingredients may

be added. Finally, the hot chocolate masse must be allowed to cool slowly. This process,

called 'tempering', is important for the right crystallization of the cocoa butter. After

tempering, the chocolate can be poured into any desired form and hardened. During the

hardening process the volume of the chocolate is reduced, allowing the chocolate to

come out of the mould automatically.

(http://www.eurococoa.com/cocoa/story/trade.htm)

2.6 Cocoa bean shell

The cocoa bean shell comprises of about 12 - 15% of the weight of the bean of which

the nibs are being processed. These shells come off the bean during the roasting process

and are separated from the beans by strong air action after being cooled. The chemical

composition of the cocoa bean shell is shown in Table 3. Some of the products that can

be obtained from waste cocoa bean shells include fertilizer, animal feed, antioxidant,

activated carbon, dye, glue, theobromine and pectin.

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Table 3: Chemical composition of cocoa bean shell

Component Amount, %

Cellulose 41

Pectin 13

Protein 12

Fats 12

Minerals 8

Water 6

Sugar 5

Theobromine 1

Polyphenol 1

Starch 1

Source: Mahro, 2007

2.6.1 Fertilizer

To grow and develop normally, plants require many different substances. About 60

elements are present in the compounds of which plants are made up. They must all be

contained in the soil and the atmosphere surrounding the plants.

Fertilizers are substances which are introduced into the soil to increase the yield of

agricultural crops. With each harvest, part of the nutrients is withdrawn from the soil,

thereby gradually exhausting it and this result in reduced crop yield. To obtain stable

harvest these losses must be offset, for example by a fertilizer. In addition to increasing

the amount of agricultural produce, fertilizers also improve their quality. Often the

starch, sugar, protein and oil content of plants are raised and also effects on the strength

of the plants to diseases, drought, low temperatures and other conditions could have

been improved.

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Carbon, hydrogen, oxygen, nitrogen, potassium, phosphorus, magnesium, sulphur,

calcium and iron are the most important elements for plant growing. These ten elements

account for over 99% of the plant weight. Normally nitrogen, phosphorus and

potassium are the main elements because the lack of these nutrients is the limiting factor

in most soils. To make rational use of fertilizers, one must know the composition of the

soil and the nutrient requirements of the individual agricultural crops. This makes it

possible to introduce fertilizers into the soil in the amounts essential for normal

development of the plant. Fertilizers are divided into two groups as organic fertilizer

(manure, peat, industrial waste, green manure etc) and inorganic fertilizer (specially

manufactured product). Compound fertilizers are fertilizers that contain more than one

mineral. They have the biggest proportion followed by ammonium sulphate and muriate

of potash.

Normally compound fertilizer contains nitrogen, phosphorus and potassium. Therefore

products are sold given the percentage of the N-P-K concentration. These elements are

not added themselves but as NH3, P2O5 and K2O.

Nowadays fertilizers are used in all sectors of agricultural production, especially the oil-

palm, tobacco, cotton and rice sector consuming major parts of it. In 2001 Ghana

imported 80,800 tonnes of fertilizers and compound fertilizer formed nearly 50 % of it

(FAO, 2005).

In Ghanaian soils the level of organic carbon, nitrogen and available phosphorus are

generally very low. Potassium is the most abundant. The Cocoa Research Institute of

Ghana found out that the ideal fertilizer for cocoa plantations should contain 18%

phosphorus, 22% potassium, calcium, sulphur and magnesium. The cocoa bean shell is

already used as a fertilizer, but as an organic one added with other organic materials.

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Normally it is sold with the composition 3-1-3.5 plus 1% magnesium and different trace

elements. (Onlinegartencenter, 2008)

2.6.2 Animal feed

Animal feed given to livestock contains essential nutrients such as protein, carbohydrate

and minerals, which are good for an effective and productive growth of the animals. It

has been found out that waste cocoa bean shells contain such nutrients which can be

used as animal feed. According to the Cocoa Research Institute of Nigeria (CRIN),

waste cocoa bean shells have been evaluated for use in feeding laying hens and broiler

starters in replacement of maize in the feed of the layers. (http://web.catie.ac.cr/disco-

cacao/ingles/13session.pdf)

2.6.3 Antioxidant

Antioxidants, which inhibit oxidation processes, are used in the food industry, cosmetic

industry, in medicine production and plastic production. They stop reaction processes of

several molecules. Mostly antioxidants react with free radicals so that chain reactions

with other substances are blocked and the product is protected. Antioxidants are part of

a long list of natural foods like garlic, red wine, broccoli, rosemary, etc. They appear

also in breast milk of mothers and protect the child against infections. The most

common antioxidant is vitamin C which is available in all citrus fruits. Substances

which are oxidized more easily than others are called antioxidants too. Ascorbic acid

(vitamin C) is a good example for such a reducing substance. Besides, there is another

group which supports the antioxidant effect by forming complexes with ions of metals.

Tetra-natrium-ethylen-di-amin-tetra-acetate is one of these substances.

Parts of the cocoa bean shell are polyphenols and epicatechin which are common

antioxidants. A normal extraction process could be used to obtain the substances. Water,

methanol or ethanol would be needed for such a process. The amount of antioxidants in

14

the sold product is normally less than 0.5% of it. Nevertheless, the worldwide

production in 2007 has been more than 880,000 tonnes and 37 billion US$ (Ceresana,

2008).

2.6.4 Activated carbon

Generally, activated carbon is used for the removal of undesirable odours, colours,

tastes of gases, vapours or liquids. In water treatment it also removes bacteria, chlorine

or ozone. A big advantage of activated carbon is the fact that it can be reactivated

thermally. Therefore the carbon is heated or reactivated with water vapour. Activated

carbon has a highly porous structure where the pores are linked similarly as in a sponge.

The interior surface ranges between 300-2000 m2/g coals. The density is within 200 -

600 kg/m3. The distribution of the size of the pores influences the properties of

adsorption.

Activated carbon is a product of plant-based, animal-based, mineral-based or

petrochemical substances. Feed material can be for example nutshell, glance coal, bones

etc. The raw material can be made either with dehydration materials like zinc-chloride

or phosphoric acid and temperatures between 500 - 900˚C or with dry distillation. The

raw material is then activated with water vapour, CO2 or even air and temperature

between 700 - 1000˚C. During this process some parts of the coal are converted in CO

so that new pores are created and the surface increase. Depending on the use, more

process steps can be added.

The possible usage of activated carbon is spread over the whole sector of chemical

engineering for example as component in medicine, water and waste water treatment

and in different industries as a substance for adsorption (Berlitz, 1999).

15

2.6.5 Dye

Chemical compounds, which are able to change the colour of other materials, are called

dyes. They are used to dye fabrics, paper, plastics etc. Dyes are found in nature but can

also be produced artificially. Some natural dyes are produced by animals such as purple

from the purple snake and other dyes as indigo or carotene are produced by plants.

To be a dye the molecule needs a specific structure. Molecules with σ-bonds absorb

electromagnetic energy in the field of X-ray to UV-radiation. Molecules with electron in

π-bonds are already stimulated by the energy of electromagnetic waves (photons).

These interactions are the origin of colours. Substances are used as dyes if the physical

and chemical properties are sufficient and constant (Fabian, 1980).Cocoa bean shell

contains natural dye. Experiments showed that wool which is dyed by cocoa bean

extracts is colourfast, non-fading and resistant of sweats and can therefore be one of the

possible utilization of the waste cocoa bean shell. The colour of the cocoa bean shell

dye is brown (Mahro, 2007).

2.6.6 Glue

By definition glue is a non metallic material which is able to connect two things by

cohesion or adhesion (EN 923). The majority of glues used are organic compounds but

also synthetic adhesives are also known. Glues are divided into groups through the

mechanism of adhesion or their chemical structure. Organic compounds can be for

example proteins. It is called bio-adhesion if a protein is used as glue. The cocoa bean

shell has high amount of proteins (12%) and can therefore be used as an adhesive. It is

confirmed that extracted proteins have good glue properties if compared with other

water soluble paper glues (Mahro, 2007).

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2.2.7 Theobromine

Theobromine or 3,7-dimethylxanthin (C7H8N4O2) is a white solid substance which is

hardly soluble in water. It is an alkaloid from the group of methylxanthins where

caffeine (1,3,7-trimethylxanthin) is the most known one.

Theobromine is a weak acid (pKs= 9.9) and normally found fixed to hydrochloric acid

or a tannin substance. The process of roasting or fermentation releases the alkaloid. It

has a similar effect on the human body as caffeine since its chemical structure is close to

it. The effects are vasodilation, stimulation to the heart and relaxation of unlined

muscles. Theobromine is also emotionally elevating and general stimulation reactions

are described. The presence of theobromine is provable with the Murexid-reaction.

(Berlitz, 1999).

2.6.8 Pectin

Pectins are plant based polysaccharides, which are essentially α-1,4-glycosidic linked

D-galacturonic acid units. The pectins, more exactly, are the methylated esters of

galacturonic acid and depending on the degree of esterification they are divided in two

major groups. High methoxyl pectin has an esterification ratio higher than 50%

(typically 60 – 75%) and low methoxyl pectin has an esterification ratio lower than 50%

(typically 20 – 40%).They occur in higher plants, in all stronger parts of it like the stem,

the leaves, etc. The functions of the pectins are the regulation of water level and

strengthening in plants. They make up about one third of the cell wall usually in its dry

state. The pectin composition differs from plant to plant and its stage in life. Especially

citrus fruits contain high amount of pectin. Citrus peel contains 20 – 30% on dry matter

basis, apple pomace 10 – 15% and cocoa been shell approximately 13% (May, 1990).

High methoxyl pectin requires a minimum amount of soluble solids and a pH within a

narrow range, around 3.0, in order to form gels and their gels are thermally reversible.

17

In general, high methoxyl pectin is hot water soluble and often contains a dispersion

agent such as dextrose to prevent lumping. Low methoxyl pectin produces gels

independent of sugar content. It is also not as sensitive to pH as the high methoxyl

pectin is. Low methoxyl pectin requires the presence of a controlled amount of calcium

or other divalent cations for gelation. During the extraction, pectin is modified

chemically therefore the pectin occurring in the plant is named protopectin. As different

raw materials are used for the extraction and also the method of extraction verifies,

different types of pectin are produced.

Pectin extraction is based on the method proposed by De Giorgi, Tomasicchio, and

Andreotti (1985). The basic extraction includes hot water as the solvent, the separation

of the pectin with alcohol, filtration, cleaning and standardization. Depending on the

raw material and the use, other steps may be included.

Non-traditional pectin sources have been investigated and cocoa shells was investigated

as one of the potential source of it (Kroyer, 1995; Schieber, et al., 2001).

The chemical structure of pectin is based on α-D-galacturonic acid (at least 65 % of the

mass), which is primarily linked α-1-4-glycosidic but can also be linked ß-1-4-

glycosidic.

This linear structure, called backbone, is interrupted by α-L-rhamnose. Moreover the

rhamnose molecules often have oligomer side chains of different sugarmolecules as

shown in the basic structure of pectin molecule in Figure 1.

Figure 1: Basic structure of a pectin molecule

18

Galacturonic acid

Rhamnose

In Figure 1 two galacturonic acid molecules are followed by one rhamnose molecule

linked with a 1,2-bond. The appearance of rhamnose is not regular but if it appears, it is

in large numbers and very concentrated. Therefore these parts of the molecule are called

hairy regions. The linear regions without rhamnose are called smooth regions. The

hydroxyl groups of the galacturonic acid are partly acetyled and the carboxyl groups are

esterified with methanol. The ratio of esterification fluctuates from plant to plant but it

has a high importance on the plant properties. Therefore the ratio of esterification is

used for the classification of pectin.

The suitability of pectins for different purposes is determined by certain characteristics

of the pectin such as the galacturonic acid content, methoxyl content, degree of

esterification and acetyl content (Ranganna, 1986). The higher galacturonic acid and

lower ash content are the two criteria governing the pectin’s purity (Hwang et al.,

1992). The spreading quality and gel grade of pectin is dependent on their methoxyl

content. A higher degree of methylation increases the capacity to form gels, whereas a

higher degree of acetylation inhibits gelling (Whistler and BeMiller, 1997). Gel grade is

the weight of sugar with which one part by weight of pectin will under suitable

conditions, form a satisfactory jelly. This is the most important character that

determines the value of pectin in international market. (McCready, R.M et al, 1955)

Generally, pectin is used in the pharmaceutical and food industry as a thickening agent,

a gelling agent and a colloidal stabiliser. The worldwide production is about 40,000

tonnes per year.

(http://www.journal.su.ac.th/index.php/jf60135a012suij/article/viewFile/48/48)

19

CHAPTER THREE

3 METHODOLOGY

The main raw material for the extraction of pectin for this project is cocoa bean shells

from WAMCO in Takoradi. The block diagram summarizing the processes is shown in

Figure 2.

3.1 Raw material preparation

The cocoa bean shells from WAMCO was taken through a series of cleaning in order to

remove all foreign material such as dust, twines, sticks and their likes. The cleaning of

the shells was done to enhance efficiency in the extraction and also the quality of the

resulting pectin. The shells were put on a 1 mm sieve to separate the clean shell from

dust. This was done for several times. The shells were then washed with some distilled

water on the sieve to remove the remaining dust stacked on the shells. These shells were

then dried in an oven at 95oC for 15 min.

(http://www.worldcocoafoundation.org/scientific-research/documents/Mollea2007.pdf)

An amount of the cleaned dry shells were taken and ground using a cutting mill in order

to increase the surface area of the shells for effective extraction process. Grinding also

ensures maximum amount of the pectin to be extracted from the shell. 500 g of the

smoothly ground cocoa bean shells was weighed with an electric balance and

transferred into a 10 L stainless cooking pot. Sodium bisulphite solution was prepared

by adding 5 g of solid NaHSO3 to a small amount of distilled water in a 5 L beaker and

stirred using a stirrer till the solid dissolved. The solution was then topped to obtain 5 L

NaHSO3 1000 ppm solution in the beaker. 1 L of the prepared solution was then added

to the weighed ground shells in the stainless pot and allowed to stand for about 30 min

to enhance cleaning and isolation of the pectin.

20

(http://global-agricultureblogspot.com/2009/02/technology-utilization-of-skin-

pectin.html)

Cocoa bean shell

Raw extract

Precipitate

Filtered cake

Pectin

21

1000 ppm NaHSO3

CleaningDistilled water

Drying

Grinding

Extraction

pH 1.5 ;70 – 80 °C 1 M HCl

Fitration Residue

Precipitation95% Ethanol

FiltrateFiltration

95% Ethanol Washing

Drying

40 - 60°C

Figure 2: Block diagram for the extraction of pectin

3.2 Extraction process

1.5 L of distilled water was then added to the mixture in the stainless pot and stirred

until a homogenous mixture was formed. 1 M HCl was added gradually to the

homogenous mixture until a pH of 1.5 was attained checking with a pH meter. The

mixture was then heated to a temperature range 70-80oC and stirred continuously for 60-

90 min. This condition was used because it corresponds to the maximum quantity of

pectin that is extractable. (Berardini, Knodler, Schieber, & Carle, 2005)

After extraction, the slurry was passed through a Buchner funnel under vacuum with a

90 mm Whatman No.1 qualitative filter paper to separate the residue from the raw

extract containing the pectin. The raw extract was then transferred into another stainless

pot and boiled at a temperature of 95-97oC while stirred intensively with a stirrer until

the volume was half its initial volume to make it more concentrated. The concentrated

filtrate was then cooled to room temperature.

(http://global-agricultureblogspot.com/2009/02/technology-utilization-of-skin-

pectin.html)

3.3 Precipitation process

Pectin, which is the main extract of the process, was in solution therefore the need to

precipitate it out. An acid-alcohol solution was prepared by mixing 95% ethanol with 2

ml of concentrated HCl. 1.5 L of the acid-alcohol was added to 1 L of the concentrated

filtrate in that proportion into a 5 L beaker. The mixture in the beaker was covered with

a clean cloth and allowed to stay for 10-14 h for sedimentation of pectin to take place.

The sediment pectin was then filtered through a fourfold polyester cloth. The residue,

which is the pectin acid, was separated from the cloth and transferred into a 600 ml

beaker. For every 1 L pectin acid taken, 1-2 L of 95% ethanol was added to wash it to

enhance the purity of the pectin. The wet pectin was then dried at a temperature of 40-

22

60oC with a dryer to reduce its moisture content. The coagulated pectin can also be

solubilised with 0.05 M NaOH for further test to be carried.

(http://global-agricultureblogspot.com/2009/02/technology-utilization-of-skin-

pectin.html)

3.4 Testing of pectin

Option 1

The presence of pectin can be proved with Zwikker-reagent solution. The Zwikker-

reagent solution is prepared by mixing 40 ml of a 10% CuSO4 solution with 50 ml of

water and 10 ml of pyridine. The produced Zwikker solution is then added dropwise to

the test solution. If pectin is present, a blue precipitate can be observed after a while.

(Hoffmann, 2004).

Option 2

This detection reaction can prove pectin concentration up to 0.01%. 14 g of hydroxyl-

amin-hydrochloride is dissolved in 100 ml of water. 1-2 ml of this hydroxyl-amin-

hydrochloride solution is added to the test solution. After adding 1 ml of 14% NaOH

solution the tube is stirred. Two minutes later 1 ml of 25% HCl and a few drops of 10%

FeCl3 solution are added. If pectin is present a red coagulate is visible. Depending on

the ratio of esterification the colour is less or more intensive (Hoffmann, 2004).

3.5 Quality test

Galacturonic acid content

5 g of the pectin is weighed with an analytical balance, and transferred to a 250 ml

beaker. A mixture of 5 ml of 2.7 N HCl and 100 ml of 60% ethanol is added to the

sample in the beaker and stirred for 10 min. It is then transferred to a fritted-glass filter

tube (30-60 ml capacity). It is then washed with six 15 ml portions of the same

23

HCl/ethanol mixture prepared earlier, followed by 60% ethanol until the filtrate is free

of chlorines. It is finally washed with 20 ml of ethanol. It is then dried in an oven at

105oC for 2.5 h, which after is cooled and weighed. Exactly one-tenth of the total net

weight of the now ash-free dried sample is weighed and transferred to a 250 ml conical

flask. The sample is then moistened with 2 ml of ethanol. 100 ml of freshly boiled and

cooled distilled water is added. It is stoppered and swirled occasionally until a complete

solution is formed. 5 drops of the indicator phenolphthalein TS is added to the solution

and titrated with 0.1 N NaOH until a sharp pink colour is observed. The volume

obtained is recorded as the initial titre (V1).

Exactly 20 ml of 0.5 N NaOH is added to the solution obtained after the first titration. It

is stoppered, shaken vigorously and allowed to stand for 15 min. Exactly 20 ml 0.5 N

HCl is added and shaken until the pink colour disappears. It is then titrated with 0.1 N

NaOH to a faint pink colour that persists after vigorous shaking. The volume obtained is

recorded as the saponification titre (V2).

Exactly one-tenth of the total net weight of the dried sample is transferred to a 50 ml

beaker, and moistened with 2 ml of ethanol. The pectin is then dissolved in 25 ml of

0.125 M NaOH. The solution is allowed to stand for 1 hr, with agitation at room

temperature. The saponified pectin solution is transferred to a 50 ml volumetric flask,

and diluted to the 50 ml mark with distilled water. 25 ml of the diluted pectin solution is

transferred to a distillation apparatus. 20 ml of Clark’s solution, which consists of 100 g

of magnesium sulphate heptahydrate (MgSO4.7H2O) and 0.8 ml of H2SO4 and distilled

water to a total of 180 ml. The distillation apparatus consists of a steam generator

connected to a round-bottom flask to which a condenser is attached. Both the steam

generator and the round-bottom flask are equipped with heating mantles. The distillation

is started by heating the round-bottom flask containing the sample. The first 15 ml of

distillate is collected separately in a measuring cylinder. Then the steam supply is

24

started and continued distilling until 150 ml of distillate has been collected in a 200 ml

beaker. The distillates are combined and titrated with 0.05 N NaOH to a pH of 8.5, and

the volume required recorded in mm as ‘S’. A blank determination is performed using

20 ml distilled water. The required volume is recorded as B; the acetate ester titre (S –

B) is recorded as V4.

Weight of galacturonic acid in milligrams is calculated by the formula,

Weight of galacturonic acid = 19.41 (V1 + V2 - V4)

The weight of galacturonic acid obtained in this way is the content of it in one-tenth of

the weight of the washed and dried sample. To calculate the percentage galacturonic

acid on a moisture and ash-free basis, multiply the number of milligram obtained by

1000/x in which x is the weight, in milligram of the washed and dried sample.

(http//www.nap.edu/html/fcc/pectins.pdf)

Degree of methylation and acetylation

Duplicate of approximately 30 mg samples of pectin is weighed (noting actual weight)

and each placed into a centrifuge tube fitted with gas-tight cap or lid. A control tube

containing no pectin is set up and carried through the procedure. 1 ml

isopropanol/NaOH solution, 4oC (50% (v/v) isopropanol/0.4 M NaOH, 4oC) is added to

each tube. It is capped and mixed gently. The mixture is allowed to stand for 2 h at

room temperature. It is then centrifuged 10 min at 2000 × g, at room temperature. The

supernatant is immediately removed and placed in a small vial with a septum and then

sealed immediately.

15 µl clear supernatant is removed preferably through the septum (or alternatively an

HPLC (High-performance liquid chromatography)), and injected into an HPLC system.

A 5 mM H2SO4 solvent system set at a flow rate of 0.6 ml/min and a temperature of

25

30oC is used. A refractive index detector at 40oC is set. 15 µl aliquots of methanol and

acetic acid standard (0 - 5 mg of each) is used and injected into the HPLC system to

prepare a standard curve.

Area from HPLC trace (response area) is plotted on the y-axis versus concentration of

methanol or acetic acid (mg/ml) on the x-axis. Methanol and acetic acid peaks are

identified in sample chromatogram by comparison with retention time for appropriate

standard. The amount of methanol and acetic acid in the sample is calculated from

sample response area using standard curve. The degree of methylation is given by

Degree of methylation=millimoles methanolmillimoles uronic

× 100

The degree of acetylation is also given by,

Degree of acetylation =millimoles acetic acidmillimoles uronic

× 100

(Voragen et al., 1986)

(http/www.nshtvn.org/ebook/molbio/Current Protocols/CPFAC/fae0304.pdf)

26

CHAPTER FOUR

4 RESULTS AND DISCUSSIONS

4.1 Influence of the extraction pH on pectin yield

The extraction of pectin from cocoa bean shells was done at different pH conditions.

Pectin which was leached from 500 g of ground cocoa bean shells was done under pH

of 1.5, 2.0 and 2.5 within a constant time period. Figure 3 shows the obtained results

expressed as yield by shells dry weight. The shell residue after the leaching process was

not easy to separate because they were soft by that time. Also, the viscosity of the

filtrate increased with pectin concentration and molecular weight.

Extraction pH of 1.5

A first set of leaching which was performed under pH of 1.5 yielded an amount of 107.3

g of dried pectin powder. The volume of 1 M HCl added to the homogeneous mixture in

the stainless pot to attain pH of 1.5 for the leaching process is shown in Table 5 in the

appendix and its graph shown in Figure 4. The amount of filtrate containing the pectin

after the separation of residue was about 2.5 L of which 3.75 L of acid alcohol was

added to precipitate the pectin out. The pectin product obtained had a sweet fruity-like

smell which could be attributed to the methyl and acetyl esterification of some of the

carboxyl groups in the galacturonic acid component of the pectin molecular structure.

The dried pectin obtained also had a light greyish colour.

Extraction pH of 2.0

However, at the pH of 2.0, the amount of filtrate obtained was about 600 ml of which

900 ml of the acid alcohol was added to precipitate out the pectin. The weight of the

dried pectin at this condition was 50.8 g and also had a fruity-like smell. The colour of

this dried pectin was light brown.

27

Extraction pH of 2.5

Also at the pH of 2.5, the dried pectin powder obtained under this condition was 47.2 g

and also had a fruity-smell for the same reason at pH of 1.5.Under this condition, 540

ml of the acid alcohol was used to precipitate out pectin from 360 ml of the filtrate

obtained after separation of the shell residue. The colour of the dried pectin powder

observed was also light brownish and also it was very easy to attain the pH condition

from the table drawn for the volume of 1 M HCl needed for pH of 1.5.

1.4 1.6 1.8 2 2.2 2.4 2.6

0

20

40

60

80

100

120

Figure 3: Pectin extracted (expressed as yield) from cocoa bean shells

28

pH

Yield, g

1 1.5 2 2.5 3 3.5 4 4.5 5

0

500

1000

1500

2000

2500

3000

3500

Figure 4: Volume of 1 M HCl expressed as pH

4.2 Test results

A preliminary test done on the pectin sample gave a blue precipitate, which indicates

the presence of pectin.

Galacturonic acid content

Test done on the dried pectin sample obtained at pH of 1.5, 2.0 and 2.5 gave a

percentage galacturonic acid content of 34.16, 31.44 and 30.28% respectively.

4.3 Conclusion

In conclusion, the project experiment was successful and that the extraction of pectin

from waste cocoa bean shells is feasible and economically beneficial. It can therefore be

considered as a possible raw material for pectin production. The viscosity of the

resulting pectin filtrate solution as well as the resulting pectin yield obtained increased

with decreasing extraction pH.

29

Volume, ml

pH

RECOMMENDATION

A longer time of about 3 h during the extraction process is suggested, which could have

an effect on the quantity and purity of the pectin.

A quality test to determine the degree of methylation and acetylation of the pectin was

not performed due to lack of HPLC with a refractive index detector. Hence the

acquisition of the instrument is suggested.

However, further quality test such as the total insoluble, residual solvent, nitrogen

content, ash content, loss on drying of the pectin among other test need to be conducted

to account for the purity and quality of the pectin is also suggested.

A process for the optimization of the extraction of pectin from waste cocoa bean shells

is also suggested.

30

COST ESTIMATION

ItemAmount per

unit

Unit price,

GH¢

Amount

needed

Gross Price,

GH¢

Conc. HCl 2.5 L 45 1 L 18

95% ethanol 2.5 L 45 5 L 90

NaOH 1 kg 80 1 kg 80

CuSO4 500 g 60 4 g 0.48

FeCl3 500 g 75 10 g 1.5

NaHSO3 1 kg 80 5 g 0.4

Pyridine 500 ml 120 10 ml 2.4

Phenolphthalein TS

indicator100 g 45 20 g 9.0

MgSO4 1 kg 80 100 g 8.0

Conc. H2SO4 2.5 L 52 100 ml 2.08

Isopropanol 2.5 L 150 50 ml 3.0

Methanol 2.5 L 45 100 ml 1.8

Acetic acid 2.5 L 75 100 ml 3.0

Stainless pot 10 L 30 2 60

White polyester cloth 1 yard 3 2 yards 6

Whatman No 1 filter

paper

1 box

(100 pieces)25 50 pieces 12.5

Total 298.16

REFERENCES

31

Berlitz, H., Grosch, W. (1999). Lehrbuch der Lebensmittelchemie (Study book for

food chemistry). Springer Verlag, Berlin

Ceresana Research (2008). Marktstudie Antioxidantien (Market study antioxodants).

Konstanz, Deutschland. Available from:

http://www.ceresana.com/upload/Marktstudien/brochueren/Ceresana_Resear

ch_-_Broschuere_Marktstudie_Antioxidantien_UC-705D.pdf / [Accessed

2009 October 2]

Duffey, T. (2009), Managing Pest and Diseases. Available from:

http://blog.worldcocoafoundation.org/2009.03- [Accessed 2009 November

14]

EN 923. Adhesives – Terms and Definitions

Fabian, J and Hartmann, H (1980), Light Absorption of Organic Colorants,

Theoretical Treatment and Empirical Rules, Springer Verlag, Berlin

FAO, 2005 (Food and agriculture organization of the United Nations), Fertilizer use

by crop in Ghana. Rome. Available from:

http://www.fao.org/docrep/008/a0013e/a0013e00.htm [Accessed 2009

October 14]

GBC News. http://www.modernghana.com/news/242453/1/ghana-cocoa-

production-up-703000-tonnes.html [Accessed 2009 October 8]

Hoffmann H, Mauch W., Untze W., (2004). Zucker und Zuckerware (Sugar and

sugar products), Behr Verlag, Hamburg.

http://dacnet.nic.in/cashewcocoa/ctech.htm [Accessed 2009 October 9]

http://global-agricultureblogspot.com/2009/02/technology-utilization-of-skin-

pectin.html [Accessed 2009 October 9]

http://www.cocoainitiative.org/cocoa-producing-countries.html [Accessed 2009

October 12]

32

http://www.eurococoa.com/cocoa/story/trade.htm [Accessed 2009 October 9]

http://www.freepatentonline.com/6312753.html [Accessed 2009 October 20]

http://www.worldcocoafoundation.org/scientific-research/documents/

Mollea2007.pdf [Accessed 2009 October 29]

http: //web.catie.ac.cr/discocacao/ingles/13session.pdf [Accessed 2009 October 29]

Mahro B., Timm M, Henrichs R (2007). Möglichkeiten der Nutzung von biogenen

Reststoffen der Lebensmittelindustrie als Biomasse-Ressource (Possible

usage of waste products of the food industry as biomas resource). Institut für

Umwelt- und Biotechnik, Universität Bremen

May, C.D (1990), Industrial pectins: Sources, production and applications,

Carbohydrate Polymers, 12, p: 79-99. Journal of Food Engineering, Volume

44

Mc Cready R.M., Reeve R.M. (1955). Journal of Agricultural and Food Chemistry.

Available from: http://pubs.acs.org. [Accessed 2009 November 13]

Onlinegartencenter (2008). Available from:

http://www.onlinegartencenter.net/product_info.php?products_id=58

[Accessed 2009 October 16]

Smartt, J and Simmonds, N.W (1995), Evolution of crops 2nd edition, p: 472-474.

Longman Singapore Publisher (Pte) Ltd, Singapore; John Wiley & Sons,

Weinheim.

Sriamornsak P.; Chemistry of Pectin and its Pharmaceutical Uses: A Review.

Silpakorn University, Thailand. Available from:

http://www.journal.su.ac.th/index.php/suij/article/viewFile/48/48 [Accessed

2009 Obtober 29]

Wikipedia Pectin (2008). Available from:

<commons.wikimedia.org/wiki/File:Pektin3.svg> [Accessed 2009 November 02]

33

APPENDIX

Table 4: Molar masses

34

Chemical formula Molar mass, g/mol

NaHSO3 104

HCl 36.5

NaOH 40

CuSO4 159

C5H5N 79.1

H2SO4 98

MgSO4.7H2O 246

Source: Carey, 2003

Table 5: Volume of 1M HCl used in attaining pH of 1.5

Volume,

ml

300 600 900 120

0

1500 1800 2100 2400 2700 3000 3300

pH 4.37 3.2

5

2.26 1.85 1.77 1.66 1.60 1.57 1.55 1.52 1.49

Table 5 shows the volume of 1 M HCl added to the homogeneous mixture of ground

shells and water in attaining the extraction pH condition of 1.5

CALCULATIONS

Preparation of 60% ethanol from 96% ethanol

Using the dilution factor, C1V1=C2V2

V2 ¿C1V1/C2

Where C1= concentration of the less concentrated ethanol, %

C2= concentration of concentrated ethanol used = 96%

V1= final volume of less concentrated ethanol = 1000 ml

V2= volume of concentrated ethanol required, ml

35

V 2=0.60 ×1000

0.96=625 ml

Therefore 625 ml of 96% ethanol was measured into 1000 ml volumetric flask and top

up with distilled water to the 1000 ml mark.

To prepare 95% ethanol from 96% ethanol

V 2=0.95 ×1000

0.96=990 ml

Also 990 ml of 96% ethanol was measured into 1000 ml volumetric flask and top up

with distilled water to the 1000 ml mark.

Preparation of lower concentrated HCL from 11.5 M HCL

From the equation

V 1=C2V 2

C1

For 1M HCL in 1000 ml

C1= 1M C2= 11.5 M V1 = 1000 ml

V 1=1× 1000

11.5=87.34 ml

For 2.7 M HCL in 500 ml

C1= 2.7 M C2= 11.5 M V1 = 500 ml

V 1=2.7 ×500

11.5=118 ml

Preparation of lower concentrated NaOH solution using NaOH pellet

36

m=C ×V × M

For 0.1 M NaOH solution mass of pellet required in 500 ml

C = 0.1 M M = 40 g/mol V = 500 ml

m=0.1 × 40 ×0.5=2g

For 0.5 M NaOH solution mass of pellet required in 500 ml

C = 0.5 M M = 40 g/mol V = 500 ml

m=0.5 × 40 ×0.5=10 g

For 0.05 M NaOH solution mass of pellet required in 500 ml

C = 0.05 M M = 40 g/mol V = 500 ml

m=0.05 × 40 ×0.5=1g

For 0.125 M NaOH solution mass of pellet required in 500 ml

C = 0.125 M M = 40 g/mol V = 500 ml

m=0.125 × 40 ×0.5=2.5 g

Galacturonic acid content calculations

Weight of galacturonic acid content, W=19.41(V1+V2-V4)

galacturonic acid ,%=W ×1000

x

Where x= 5 g = 5000 mg

Sample at pH of 1.5

37

V1 = 1.5 ml V2 = 8.5 ml V4 = S – B = 1.3 - 0.1 = 1.2 ml

W = 19.41(1.5+8.5-1.2) = 170.808 mg

Therefore

galacturonic acid ,%=170.808×10005000

=34.16 %

Sample at pH of 2.0

V1 = 1.2 ml V2 = 8.3 ml V4 = S – B = 1.5 - 0.1 = 1.4 ml

W = 19.41(1.2+8.3 - 1.4) = 157.221 mg

Therefore

galacturonic acid ,%=157.221×10005000

=31.44 %

Sample at pH of 2.5

V1 = 1.2 ml V2 = 8.2 ml V4 = S – B = 1.7 - 0.1 = 1.6 ml

W = 19.41(1.5+8.5-1.2) = 151.398 mg

Therefore

galacturonic acid ,%=151.398×10005000

=30.28 %

38