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
Cocoa Beans from Pidie District, Aceh Province, Indonesia
114 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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
Cocoa Beans from Pidie District, Aceh Province, Indonesia
115 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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,
Cocoa Beans from Pidie District, Aceh Province, Indonesia
116 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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
1.00
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
0 1 2 3 4 5 P S
Wat
er C
on
ten
t (%
)
Fermentation time (Days)
Cocoa Beans from Pidie District, Aceh Province, Indonesia
117 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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
4.243.93
4.154.41
5.24 5.24
0.00
1.00
2.00
3.00
4.00
5.00
6.00
0 1 2 3 4 5 P S
pH
Val
ue
Fermentation Time (Days )
Cocoa Beans from Pidie District, Aceh Province, Indonesia
118 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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).
Cocoa Beans from Pidie District, Aceh Province, Indonesia
119 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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.
7.1
8
8.4
8
8.5
1
7.3
3
7.0
9 8.3
0
0.0
0
0.0
0
4.1
4
4.6
0
8.8
2
7.3
0
7.5
3
7.6
1
0.0
0
0.0
0
3.7
0
5.3
2
8.5
9
7.5
5
8.0
1
7.8
1
0.0
0
0.0
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
0 1 2 3 4 5 P S
Tota
l Cel
l Co
un
t (L
og
CFU
/g)
Fermentation Time (Days )
PDA (Yeast) MRS (LAB) YGC (AAB)
Cocoa Beans from Pidie District, Aceh Province, Indonesia
120 Proceeding of The 8th AIC: Health and Life Sciences 2018 – Syiah Kuala University
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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