CHEMICAL AND MICROBIOLOGICAL CHARACTERISTICS OF …

Cocoa Beans from Pidie District, Aceh Province, Indonesia

113 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University

CHEMICAL AND MICROBIOLOGICAL CHARACTERISTICS OF

COCOA BEANS FROM PIDIE DISTRICT, ACEH PROVINCE,

INDONESIA

Martina Fitri Wahyuni1*, Dewi Yunita1, Yusriana1, Yuliani Aisyah1, Rabya A Lahmer2,

Diirisa Mugampoza3

1Department of Agricultural Product Technology, Faculty of Agriculture, Syiah Kuala University,

Jl. Tgk. Hasan Krueng Kalee No.3, Darussalam, Banda Aceh 23111, Indonesia 2Department of Food Science and Technology, Faculty of Agriculture, University of Tripoli,

Tripoli, Libya 3Department of Food Technology, Faculty of Science, Kyambogo University, Kampala, Uganda

*Corresponding e-mail: [email protected]

ABSTRACT

This research aimed to examine the chemical and microbiological characteristics of cocoa beans which

are used as a raw material in a small-scale chocolate factory in Pidie Jaya District, Aceh Province, Indonesia. The

information would be used to improve the overall quality of the cacao beans produced in the country. 8 triplicate

samples were collected from a local farmer in Padang Tiji Sub-District, Pidie District, Aceh Province, Indonesia

starting from Day 0 to Day 5 fermentation, after drying, and after roasting. The samples were then analysed for

water content, pH, lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeast, using standard methods. The

results showed that the water content slightly decrease during fermentation from 66.60 % to 56.40 % and continued

to decline drastically to only 1 % after roasting. Also, the pH declined from 4.91 at Day 0 to 3.93 at Day 3 of

fermentation suggesting the growth of LAB and AAB. Then, the pH increased again at Days 4 (4.15) and Days 5

(4.41) this increase occurs because the existing citric acid is used up during fermentation. Before fermentation, the

total cell count of yeast was dominant. The highest LAB and AAB growth occurred on the second day of

fermentation. These results indicate that the microflora of raw cacao beans and at the end of fermentation is

dominated by yeast. It is suspected that the increase in yeast growth at the end of this fermentation is because

certain types of yeast dominate.

Keywords: cacao beans, Aceh cacao, LAB, AAB, yeast.

INTRODUCTION

Cocoa ( Theobroma cacao L.) is a plantation crop that is widely planted in Indonesia

and it is one of the leading export commodities in the country besides coffee. Cocoa plays an

important role in employment, provides income to farmers and it is also the major country's

foreign exchange source (insert reference). Aceh is one of the many provinces in Indonesia that

has developed its agro-industry sector, marked by the existence of a chocolate factory in Pidie

Jaya District. Cacao producers need to pay attention to the demands of the current cocoa market,

which requires good quality of cocoa beans with a distinctive chocolate flavor and taste (Rubiyo

and Siswanto, 2012). According to the Ministry of Industry in Indonesia (2007), the processing

of cocoa fruit determines the quality of the final product because during the process, a

distinctive taste of cocoa is formed. Fermentation is the most vital processing stage to guarantee



the end product with a good chocolate flavor. Ardhana and Fleet (2003) states that the growth

of yeast, lactic acid bacteria and acetic acid bacteria in the cocoa pulp plays an important role

in biochemical changes during cocoa fermentation so that the color, texture, aroma, taste, and

physical appearance are changed to the desired levels. In addition, the fermentation process will

reduce the bitter and acid tastes of the raw cocoa beans (Susanto and Saneto, 1994).

Physical, chemical and microbiological characteristics are important factors that

determine the quality of cocoa beans. Physical characteristics such as color, acidity and size of

seed are used to classify cocoa bean quality based on physical appearance, while the level of

acidity and water content in seeds are used to describe their chemical characteristics (Krähmer

et al., 2015). The acidity of the cocoa bean seeds produced by microorganisms during

fermentation affects their taste. The water content affects the efficacy of further processing and

also influences their shelf life. Therefore, this study aimed to examine the chemical and

microbiological characteristics of the cocoa beans that are used as raw materials in the chocolate

factory Pidie Jaya District, Aceh Province, Indonesia. This information would be used to

improve the overall quality of Aceh cocoa beans in Indonesia.

MATERIALS AND METHODS

Bulk cocoa lindak were obtained from a local farmer in the Padang Tiji Sub-district,

Pidie District, Aceh Province, Indonesia. The cocoa beans from this area are normally supplied

to a chocolate factory in Pidie Jaya Sub-district. The media used were Potato Dextro Agar

(PDA; Merck) for enumeration yeast, de Man Ragosa Sharpe (MRS; Merck) for lactic acid

bacteria and Yeast Glucose Calcium Carbonate (YGC; Merck & Himedia) for acetic acid

bacteria.

Sampling

Samples for microbial and chemical analyses were collected from days 0 to 5 of

fermentation, after drying, and after roasting. Fermentation was carried out for 5 days with

stirring every 24 h, dried traditionally for 5 days and roasted at 120oC for 30 min. Cocoa beans

(300 g) were taken at 4 different points in the fermentation vat. The sample was put in a sterile

bottle and transported to the laboratory in an ice box to be analyzed within 8 h.

Water Content

Water content was analysed using the method of Apriyantono et al. (1989), the cocoa

sample (5 g) was placed in an empty cup which was dried in an oven (EYELA WF100) at 105oC



for 15 min, and cooled in a desiccator and weighed. The cup containing the sample was put in

the oven at 105oC for 6 h. The cup was then cooled in a desiccator and weighed to the constant

weight (± 0.1 g). Water content was calculated with a wet base formula.

pH

Measurement of pH was carried out based on the method of Apriyantono et al. (1989),

using a digital pH meter (LAMOTTE). Cocoa beans (1 g) were mixed into 10 ml of distilled

water. Samples were measured with a pH meter which had been calibrated with buffer solutions

of pH 7.

Total Cell Counts

Total cell counts were enumerated based on procedure described by Collins et al.

(1995). The sample (1 g) was crushed using mortal and homogenized in 9 ml sterile peptone

water (Oxoid) to obtain a dilution factor of 10-1. Further dilutions up to 10-10 were prepared for

each sample. Each dilution was spread in duplicate on specific media. The inoculated media

was then incubated at 37°C for 48 hand the total colony growth was counted by a colony counter

(Suntex).

RESULTS AND DISCUSSION

Chemical Characteristics of Cocoa Beans

Water content

The water content of unfermented cocoa beans decreased slightly from 66.60 % at day

0 to 56.40 % at the end of fermentation (5 days) and continued to decrease drastically after

roasting to only 1.0 % (Figure 1). The decrease in water content during fermentation could be

due to increased microbial and enzyme activity so that an increase in temperature could cause

the pulp to become dilute and detached (Nasution et al. 1985). In addition, components in seeds

such as water, polyphenols and proteins are degraded in the form of simpler organic

compounds. The destruction of the pulp from the seeds causes the pores of the seed to open and

this facilitates the removal of free water so as to facilitate/allow the drying process (Nasution

et al. 1985). According to Beckett (2009), acetic acid bacteria play a role in converting alcohol

to acetic acid. The conversion is due to strong exothermic reaction which contributes to the

increase in temperature. In some cases, fermentation temperature can reach 50°C or higher. The

increase in temperature also results in acid and heat diffusion into the seeds which results in the

death of seeds which then begins the process of forming colors, aromas, and flavors (Beckett,



2009; Schwan & Wheals. 2004). These enzymatically driven biochemical processes involve

sugars, amino acids, and peptide peptides as substrates (Schwan & Wheals, 2004).

Figure 1. changes in water content of cocoa beans during the fermentation and roasting.

P: After drying. S: After roasting.

The water content of dried cocoa beans did not meet the standards set by Indonesian

Standard Board (2008) which requires that cocoa beans should not exceed the maximum

standard of 7.5% water content. According to Rahmadi (2008), the water content of cocoa beans

is an important factor in maintaining the quality of cocoa beans during storage. In this study,

the moisture content of cocoa beans after roasting reached 1% and that is low water content for

roasted cocoa beans. roasted cocoa beans that have been cooked are characterized by the aroma

of chocolate, the seed coat is taken back and crushed, the color of the seed skin becomes pale

and the seed moisture content reaches 2 - 3.5% (Lada et al., 2016). However, the results of the

research by Ramlah (2016) showed that the roasting temperature of 120oC, also employed in

this study, produces good aroma and flavor in chocolate, compared to chocolate roasted at 40oC.

According to Klahors and Suanne (2005), when roasting is done using high temperatures there

is flavonoid oxidation which helps in flavor formation, and causes a distinctive chocolate flavor

that gets sharper.

Drying and roasting are one of the processes that determine the quality of cocoa

produced. Roasting aims to develop flavor, aroma, color, ease the release of the skin of the seed,

reduce the moisture content and loosen the skin so that it can easily be separated from the seed

coat (Agricultural Research Agency, 2013). Drying of the cocoa beans leads to oxidation of

66.6061.50

59.90 60.40 57.3056.40

13.50

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0 1 2 3 4 5 P S

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)

Fermentation time (Days)



polyphenols which is catalyzed by polyphenol oxidase, resulting in new components and loss

of membrane integrity, causing the formation of brown color (Puziah, 2005). This helps reduce

bitterness and astringency and also enhances the development of brown color from well

fermented cocoa beans. The biochemical oxidation of acetic acid from seeds continues during

drying which also contributes to (Afoakwa, 2010).

pH/ Acidity

The pH of cocoa beans decreased from 4.91 at day 0 to 3.93 on the 3rd day of

fermentation and at the end of fermentation (day 5) pH increased to 4.41. After drying and

roasting, the pH of the cocoa beans became 5.24 (Figure 2).

Figure 2. Changes in pH of cocoa beans during the fermentation and roasting.

P: After drying. S: After roasting.

The pH decrease at the beginning of fermentation indicates microbial activity.

According to Nasution et al. (1975), decrease in pH occurs because of the activity of lactic acid

bacteria that convert sugars into lactic acid. Also, acetic acid bacteria convert alcohol (ethanol)

to acetic acid. Decrease in pH is characterized by an increase in acid levels due to increase in

the population of acetic acid bacteria. As the anaerobic phase in the cocoa fermentation begins

to stop, microbial succession by acetic acid bacteria continues which turns ethanol into acetic

acid (Lopez & Dimick 1995). Acetic acid diffuses into the layer of cocoa beans, so that the

acidification process occurs. Diffused acids into cell walls will cause the death of cocoa bean

cells (Biehl et al. 1985). Acetic acid is one of the potential flavors for the quality of cocoa beans.

Drysdale and Fleet (1988) reported that acetic acid bacteria can play a role in the synthesis of

4.914.58

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4.154.41

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pH

Val

ue

Fermentation Time (Days )



compounds associated with the aroma and flavor of cocoa such as the aldehydes, ketones and

other volatile compounds.

The pH increase at the end of fermentation is due to decrease in acid content due to

decreased activity of acetic acid bacteria and lactic acid bacteria (Apriyanto et al., 2016). In

addition, once the pulp substrates on the seeds used by microorganisms are exhausted, the pH

subsequently rises (Karinawantika, 2015). According to Lagunes et al. (2007), cocoa pulp has

a low initial pH (3.0-3.5) because it contains citric acid. As citric acid is used up during

fermentation, the pH increases until it reaches pH 5-6 after 6 days of fermentation.

In this study, the pH of cocoa beans at the end of fermentation was low pH implying

that the beans had high acidity. Overall, the pH values obtained in this study were lower than

the results of Senanayake et al. (1996), which showed that the pH of cocoa beans at the end of

fermentation ranges from 4.8 to 4.9 at 5 days. Our results were also different from what has

been reported by Muzaifa et al. (2017), where the pH of cocoa beans on the 5th day of

fermentation is 5.84. According to Afoakwa et al. (2011), the level of acidity affects the

characteristics of flavor and aroma of chocolate products produced. Cocoa beans with a pH of

5.0–5.5 produce a higher aroma potential compared to pH 4.0–4.5 which gives a lower aroma.

Microbial Characteristics

Microbes such as yeast, lactic acid bacteria, acetic acid bacteria and molds are involved

in traditional and spontaneous fermentation of cocoa (Leal et al., 2008). These microorganisms

develop sequentially according to the availability of nutrients in the mass of cocoa during

fermentation. The initial flora is dominated by yeast but the final microbiota of fermented cocoa

beans comprises of LAB and AAB. The type and population of these microorganisms are

strongly related to the sources of existing and developing microorganisms at the local level

(Muzaifa et al., 2017).

The results of microbial growth observed during cocoa bean fermentation showed that

yeast increased from 7.18 at day 0 to 8.51 log cfu/g at day 2 of fermentation . LAB and AAB

increased from 4.14 log cfu/g and 3.70 log cfu/g at day 0 to 4.60 log cfu/g and 5.32 log cfu/g

on the 2nd day of fermentation. On the 3rd day, microbial counts began to decline until the end

of fermentation (Figure 3). However, at the end of fermentation, the number of yeast increased

slightly (8.30 Log CFU/g) than LAB (7.61 Log CFU/g) and AAB (7.81 Log CFU/g) (Figure

3).



Figure 3. changes in Total Cell Counts of cocoa beans during the fermentation and roasting.

P: After drying. S: After roasting. LAB: Lactic Acid Bacteria. AAB: Acetic Acid Bacteria.

At the beginning of fermentation, the yeast dominated the microflora of cocoa beans

until the second day of fermentation, this was different from the results of Muzaifa et al. (2017),

who reported that the yeast growth pattern immediately decreases after 24 hours. As for the

highest LAB and AAB, growth occurred on the second day of fermentation. On the third day

of yeast growth, lactic acid bacteria and acetic acid bacteria began to decline until the fifth day

of fermentation. This decrease could have been caused by competition between these microbial

populations. According to Nurhidayat (1984) and Thompson et al. (2001), changes in the

atmosphere of fermentation are due to yeast activity in breaking the sugars contained in the

pulp into alcohol. In this condition, alcohol is disinfectant for yeast so it could have become a

limiting factor for the growth of yeast itself. Yeast growth also produced mainly acetic acid,

sufficient aeration and acidity of seeds due to the diffusion of acetic acid into seeds. Aeration

encourages the growth of acetic acid bacteria which suppresses the growth of yeast.

The pattern of yeast growth at the end of fermentation experienced a slight increase.

This is different from what was previously reported by Muzaifa et al. (2017), where the pattern

of yeast growth until the end of fermentation decreased. It is suspected that the increase of

yeast growth at the end of fermentation in this study is because certain types of yeast dominate.

Ardhana and Fleet (2003) stated that Saccharomyces cerevisiae and Candida tropicalis are

dominant yeasts during cocoa fermentation because of their high survival rate. After drying and

roasting, there were no LAB and AAB detected because the cocoa beans went through a high

temperature process that killed the microbes.

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Tota

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t (L

og

CFU

/g)

Fermentation Time (Days )

PDA (Yeast) MRS (LAB) YGC (AAB)



CONCLUSION

The results showed that the water content decreases during fermentation until after

roasting. The moisture content of dry cocoa beans was higher than 7.5%, the standard set by

BSN (National Standardization Agency). Similarly, the water content of roasted cocoa beans

was lower than the optimal moisture content of cocoa beans for the next stage of processing.

Cocoa beans at the end of fermentation entered the low pH category which would give a lower

aroma of cocoa beans. At the end of fermentation, yeast growth was slightly higher than LAB

and AAB, presumably because certain types of yeast predominate. As for the highest LAB and

AAB, growth occurred on the 2nd day of fermentation. Judging from the chemical and

microbiological characteristics, it is necessary to analyze the flavour compounds of the cocoa

beans. Therefore, identification of flavour compounds of the cocoa beans will be investigated

in further research.

ACKNOWLEDGMENT

This work was supported by Syiah Kuala University under the project “Hibah Dana Awal

Laboratorium” 2018.

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