ISSN: 2347-467X, Vol. 10, No. (3) 2022, Pg.

918-927

Current Research in Nutrition and Food Science

www.foodandnutritionjournal.org

Performance of Mixed-Microbial Culture from Civet Fecal

Suspensions on Physicochemical Composition of Wet
Fermented Arabica Coffee
DARWIN*, AMI MULIAWATI and RAMAYANTY BULAN

Department of Agricultural Engineering, Syiah Kuala University, Banda Aceh Indonesia.

Abstract
This study aimed to evaluate the effects of mixed microbial culture
from civet fecal suspension used as the inoculum for the fermentation
of Arabica coffee. The type of Arabica coffee used for the research was Article History
the unpeeled coffee or the Arabica coffee cherries. Different proportion Received: 08 October
of inoculum introduced was thoroughly evaluated to assess the appropriate 2022
concentration of inoculum (0-40% inoculums represented in treatment 0-4 Accepted: 09 December
or T0 to T4) that would be applied to the fermentation of Arabica coffee 2022
cherries. Results revealed that treatment 4 (T4) containing 40% of the
Keywords
inoculum could degrade the sugar of the coffee beans faster than that of the Arabica Coffee;
other treatments in which within 24 hours of the incubation approximately Civet Fecal Suspension;
84% of the sugar was converted. T4 also reached the lowest caffeine content Coffee Cherries;
Decaffeination;
(1.8%) of the fermented coffee beans among other that of other treatments In-Vitro-Fermentation.
while the control had higher caffeine content (2.2%).This was substantially
significantas the Arabica coffee cherries fermented with mixed microbial
civet fecal suspensions can remarkably reduce the sugar and caffeine
content of the Arabica coffee beans.

Introduction seeds, method of processing, mixture, and roasting

Currently, coffee has been one of the most notable processes. 2,3 One of the essential processing
commercially traded products in the world market methods incoffee production is the fermentation
since it has become the most popular beverage process. The key purpose of the fermentation
world wide. 1 The various tastes of the coffee process of coffee production is to remove the pulp
beverage are typically influenced by some factors and mucilage layer, reduce water content, and also
including a plant variety or cultivar, the areas to improve the quality of the coffee beans.4
of cultivation, cultivation methods, quality of the plant

CONTACT Darwin [email protected] Department of Agricultural Engineering Syiah Kuala University, Banda
Aceh Indonesia.

© 2022 The Author(s). Published by Enviro Research Publishers.

This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Doi: http://dx.doi.org/10.12944/CRNFSJ.10.3.9
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 919

Coffee cherry is unprocessed coffee that still has pulp experiment was the unpeeled coffee or the Arabica
and mucilage surrounding its beans. The coffee pulp coffee cherries. Different proportion of inoculum
typically contained crude carbohydrates, proteins, introduced was investigated to evaluate the suitable
free amino acids, fatty acids, and minerals5,6 while concentration of inoculum that would be applied
mucilage contained polysaccharides such as pectin, to the fermentation of Arabica coffee cherries.
cellulose, and starch.7 During the fermentation
of coffee microbial consortia involved would convert Materials and Methods
pulp and mucilage to produce metabolites that would Coffee Bean Collection
affect the flavors and aroma of the coffee.8 Some The type of coffee used in this study was coffee
studies revealed that the process of fermentation cherries of Arabica coffee (Coffea arabica) beans.
in coffee would enhance the variety of the compounds They were obtained from the coffee plantation
of coffee aroma and flavors. 9,10 The activities situated in the Jeget Ayu Village, Jagong Jeget
of microorganisms and the extent of fermentation Subdistrict, Central Aceh Regency, Aceh Province,
would stipulate the free sugar concentration and Indonesia. After collection, the coffee cherries were
free amino acids, which proceed to cover the bean immediately sorted as well as cleaned to remove
and then assist to generate Maillard compounds and some undesired materials, such as leaves, stalks,
volatile during the process of roasting.11 sand, and gravel that could hinder the processes
of fermentation. To evaluate the effectiveness
The current practice in coffee fermentation conducted of fermentation processes inoculated by the
amongst coffee producers is natural fermentation. digestive civet microbes, the different proportion
This process highly depends on the indigenous of inoculum was introduced to the coffee beans
microbiota related to the coffee farm microbiome.12 including 5, 10, 20, and 40% of inoculums.
This method would generate some variations and The control was set up in which the coffee bean was
thereby require further study to evaluate its effect on naturally fermented without adding any inoculum
the quality of coffee.13 Currently, the starter cultures of civet fecal suspension.
introduced to the fermentation of coffee have
been claimed to succeed in conventional natural Inoculum Preparation
processes. This method is significant since it would The coffee beans were fermented by using civet
make the fermentation of coffee more consistent, feces suspension, which was selectively enriched
predictable, and restrained.14 The limitation of the from the mixed microbial cultures of civet feces.
method is that the fermentation process is conducted The civet feces covering coffee beans were obtained
in open chambers, which are susceptible to the and/or collected from the coffee plantation in the
contamination of the natural microbiota.12 Jagong Jeget Subdistrict, the District of Central
Aceh. Before using the civet feces they were warmed
Many studies have been carried out on the at 37 ± 0.5°C to reactivate the microorganisms.
fermentation of coffee beans using specific
microorganisms, such as yeast and lactic acid For the selective enrichment processes, 100
bacteria.15,16 The use of pure culture to perform g of civet feces were mixed with culture media
fermentation of coffee would tend to generate consisting of 100 ml of DI water, 1 g of NaHCO3
specific metabolites (i.e. ethanoland lactic acid). (Merck, Germany), 1 g of Bacto Peptone (Merck,
This would be different if the fermentation was carried Germany), and 1 g of Glucose Anhydrous (Merck,
out using mixed microbial culture, which could Germany) as a substrate. The cultivation process
generate various fermentation end-products. 7,17 was carried out at a fed-batch mode reactor at 37 ±
This would significantly affect the physicochemical 0.5°C.The reactor was daily topped up with 100 ml
properties of the coffee bean and potentially enrich of culture media containing substrate until it reached
its flavors and aroma. The purpose of the current the volume of 1 liter. The physical characteristics
research is to study the effects of mixed-microbial of civet fecal suspension used as inoculum for the
culture from civet fecal suspensions as the mixed fermentation of Arabica coffee beans were presented
culture starters used for the fermentation of Arabica in Table 1.
coffee. The type of Arabica coffee used for the
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 920

Table 1: Physicochemical properties of civet of civet feces, samples were analyzed using UV-VIS
fecal suspension used as inoculum Spectrophotometer Lab Equipment 325-1000nm
4nm 722N Ultraviolet (Yuchengtech, China).
Parameters Unit Civet fecal The wavelength applied for the optical density (OD)
suspension analysis of microbial growth was 600 nm. DI water
was used as a blank in a spectrophotometer prior
pH - 7.01 to the start of the OD analysis.
Electrical Conductivity µS/cm 21.8
Total Dissolved Solids mg/L 10.9 Analysis of Solid, Moisture, and Ash Contents
Optical Density OD600 0.3 Analysis of solid and moisture contents was
conducted by using a laboratory oven (Thermolyne
Experimental Design and Procedures Drying Oven, USA) at 105ºC for about 24 hours.
The inoculum used for the in vitro fermentation To determine the ash content, samples obtained
of coffee beans was taken from the selective from the solid analysis were subsequently put
enrichment processes. The proportion of inoculums in the laboratory furnace (Thermolyne Muffle
and the coffee beans were labeled as treatments furnace, USA) under 500ºC for about 30 minutes.
from 1 to 4. Treatment 1 (T1) consisted of 10 ml All procedures of the analysis were performed
of inoculums and 190 g of coffee beans. Treatment according to the Standard Methods.19,22,23
2 (T2) contained 20 ml of inoculums and 180 g
of coffee beans. Treatment 3 (T3) consisted of 40 ml Acid Content Determination
of inoculums and 160 g of coffee beans. Treatment Titratable acidity (TTA) was analyzed to assess
4 (T4) contained 80 ml of inoculums and 120 g the total amount of acids generated during the
of coffee beans. Treatment 0 was a control containing fermentation of coffee beans. The titratable acidity
200 g of coffee beans without adding inoculums. was measured by using a Lab Standard pH meter
To assure the in vitro fermentation performing (Milwaukee, USA). The titrant filled in the burette
effectively, the operational temperature was set up tube was 100 mM of NaOH (Merck, Germany).
close to the body temperature of the civet, which was Before titration was started, some drops of 100 mM
about 37 ± 0.5°C.18 The fermentation of the coffee phenolphthalein (Merck, Germany) were added
beans was carried out in a series of batch reactors to the analyte. To ensure reproducible data,
with the working volume of 200 ml. To ensure the all measurements were repeated.
fermentation process of coffee beans performing
completely, the process was carried out for 48 hours. Soluble Carbohydrate Determination
The soluble carbohydrate content of the coffee
Analytical Methods beans was measured by using the Glucometer, Bio
pH, Total Dissolved Solids (TDS), and Electrical Sensor AGM-2100 (Gluco Dr.auto, South Korea)
Conductivity (EC) Determination with an assay method of electrochemical method
To evaluate the effectiveness of in-vitro fermentation (Gold electrode). To determine glucose concentration,
of the coffee beans, each sample was regularly taken the glucometer was in fitting to the test strips
and analyzed including samples before and after (Gluco Dr Biosensor Test Strips, South Korea).
fermentation. To monitor fermentation parameters The determination of sugar concentration was
including pH, and total dissolved solids (TDS) performed at room temperature. To prevent an
of the culture during the cultivation and fermentation overlooked assessment of sugar concentration, each
processes, samples were measured by using sample was diluted ten times. All procedures applied
a portable pH meter Model: MW 102 (Milwaukee, for the determination of sugar content infermented
USA). Prior to the start of the analysis, pH meter products were conducted according to the study by
was calibrated with some buffer solutions including Darwin.24
4, 7, and 10.19,20,21
Caffeine Determination
Microbial Growth Determination The determination of caffeine inthe coffee beans was
To assess the microbial growth during the conducted by using a Laboratory Ultra violet-Visible
cultivation processes of the mixed microbial cultures Spectrophotometer(Yuchengtech, China).150 ml
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 921

of hot DI water was used to dissolve 1 g of the 5% (α = 0.05) level of significance to assess the
milled coffee. The mixture was filtered using 150 influence of inoculums concentration towards
mm filter paper (Cat No 1001 150, Whatman, China) the physicochemical characteristics of fermented
and placed into the flask. The coffee solution was Arabica coffee beans.
mixed with 1.5 g of CaCO3(Merck, Germany), and
subsequently extracted 4 times by using 25 mL Results and Discussion
of chloroform (Merck, Germany). The bottom layer The results of the current study showed that pH in
of the mixture was separated and heated to remove the cultivated culture gradually decreased from 7.5
the chloroform residue. For the caffeine analysis, to 6.2 at the early stage of the cultivation process
t h e e x t r a c t w a s d i l u t e d w i t h D I w a t e r. (from day 1 to day 3). The growth of microbes
The measurement of caffeine was conducted represented in the OD value started to increase
at the wavelength of 275 nm.25,26 onday 2 of the process (Figure1). The OD value
of the culture increased from 0.076 to 0.299 and
Statistical Analysis reached its maximum value of 0.3 onday 9 of the
Experimental data collected during fermentation cultivation process. Besides, at the end of the
processes were statistically analysis with single process pH of the culture was around 6.5 suggesting
factorial of analysis of variance (ANOVA). The data that the mixed microbial culture used for the
sample taken was conducted in triplicate. Besides, in-vitro coffee fermentation was ready for the acid
the data analyzed using ANOVA were tested with stage processes.27,28

Fig. 1: Microbial growth as measured by absorbance (OD600) and pH of the culture during the
cultivation process of civet fecal suspension(P<0.05)

The physicochemical properties of Arabica coffee potential substrates that could be used for the
cherries utilized in in-vitro fermentation of civet are microbes as their source of energy and minerals to
depicted in Table 2. Results revealed that the pH perform the fermentation processes.23,30 The sugar
of the coffee cherries was quite low at around 5.30 content of the coffee cherries was about 17 mM
indicating that the fermentation process would be which would also be used as a source of carbon
performed under acidic conditions.29 As depicted forthe microbes and may be converted to alcohols,
in Table 2, some parameters of the coffee cherries organic acids, and other metabolites that could
including total solid (52%) ash content (8%) and enhance the quality of the fermented coffee beans.
caffeine (2.20%) indicated that they contained
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 922

Table 2: Physicochemical properties of Arabica suspension as inoculum (T0) had the lowest pH
coffee cherries before fermentation at the beginning of the fermentation period amongst
other samples of coffee cherries fermented using
Parameters Unit Arabica Coffee civet fecal suspension as inoculum. Further, the pH
Bean of the control or T0 dropped sharply from 5.2 to 3.9
within 4 hours of incubation. This is highly different
Moisture content % 48.40 ± 0.02 from the sample of T4 in which its pH did not drop
Ash content % 7.95 ± 0.01 until 8 hours of incubation. This suggested that the
Total Solid % 51.60 ± 0.03 use of civet fecal suspension as inoculum in the
Glucose mmol/L 17.00 ± 0.02 fermentation of Arabica coffee cherries may enhance
Alkalinity mmol/L 3.13 ± 0.01 the buffer capacity of the fermentation culture.
Caffeine % 2.20 ± 0.02 At the end of the fermentation period (48 h), the
pH 5.30 ± 0.03 pH of all tested samples was too acidic, which was
approximately between 4.4 and 4.1. Some studies
(P<0.05) reported that lactic acid-producing bacteria would
dominate the fermentation process when the pH
The results showed that all fermented coffee cherries of the culture was below 4.5.29,31 Hence, at this pH
had low pH between 4.0 and 5.2 indicating that level lactic acid would be the dominant fermentation
the fermentation process occurred at acid stage end-product generated from the fermentation of
condition (Fig. 2). The sample of coffee cherries coffee cherries using inoculum of fecal suspension.
naturally fermented without using civet fecal

Fig. 2: pH of the coffee beans during the fermentation process (P<0.05)

The results showed that within 4 hours of incubation to the coffee cherries may enhance the formation
the titratable acidity of all treatments applied of acids during their fermentation processes.
to the Arabica coffee cherries increased from This occurred because the pulps of Arabica coffee
approximately 2.0 to 2.5% (Figure 3). Treatment cherries were quite rich in carbohydrates and/or
4 (T4) generated the highest titratable acidity polysaccharides, and thereby could be used as
amongst the other treatments in which within 48 h substrates and/or source of energy for the growth
of incubation it had reached 3% of titratable acidity of microbes.32 Further, during the fermentation, the
while others only reached between 2.2 and 2.3% substrate would be converted into fermentation
of titratable acidity. This suggested that high end-products that may significantly enrich the taste
proportion of fecal suspension as inoculum added of the fermented coffee beans.6,17
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 923

Fig. 3: Titratable acidity of the Arabica coffee cherries during the fermentation process (P<0.05)

The results showed that the addition of civet that period the sugar content was lower (0 mM)
fecal suspension as inoculum to the fermentation than that of the coffee beans (0.3 mM) that were
of Arabica coffee cherries effectively reduced their in-vivo digested by civet (Table 3). This may be
glucose content. As depicted in Fig. 4, within 4 hours indicated that above 24 hours of incubation the
of incubation the sugar concentration of the coffee glucose concentration of the fermented coffee beans
beans decreased significantly from approximately was completely degraded, and suggested that the
17 mM to around 12 mM. The study also revealed use of civet fecal suspension as inoculum would
that the more inoculum concentration was added the accelerate the sugar conversion.33 This is because
faster glucose was converted. In this study, treatment during the fermentation sugar content presented
4 was able to degrade the sugar of the coffee beans in the pulp and mucilage was reduced and consumed
faster than that of the other treatments in which by microorganisms while metabolites (i.e. volatile
within 24 hours of the incubation approximately and non-volatile organic acids and alcohols) were
84% of the sugar was converted. Besides,within formed, thereby would enhance the quality of the
32 hoursof incubation T4 had completely degraded coffee including its flavor and aroma.34
sugar in the fermented coffee beans, and within

Fig. 4: Glucose concentration of the Arabica coffee beans during the fermentation process (P<0.05)

The results of the present study showed that the to the fermentation of Arabica coffee cherries the
civet fecal suspension used as inoculum effectively lower concentration of caffeine inthe fermented
reduced the caffeine content of the fermented coffee beans was obtained. In this study, treatment
Arabica coffee beans. As shown in Fig. 5, the 4 containing 40% of the inoculum had the lowest
higher concentration of the inoculum was added caffeine content (1.8%) of the fermented coffee
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 924

beans among the other treatments. This is quite culture of civet fecal suspension successfully
different from the control naturally fermenting Arabica decreased their caffeine content. This result
coffee cherries that were not using fecal civet reaffirmed the previous study,which revealed that
suspension as inoculum had higher caffeine content the use of civet fecal suspension as inoculum
(2.2%). The caffeine content of the tested samples had effectively reduced the caffeine content of the
was still higher than that of the original luwak fermentable coffee beans.17 They also reported that
coffee beans digested in-vivo by civet, which were the use of civet fecal suspension as inoculum could
about 1.34% (Table 3). Overall, the fermentation degrade almost 90% of caffeine of the Robusta
of Arabica coffee cherries using mixed-microbial green coffee beans.

Fig. 5: Caffeine of the coffee beans fermented with different proportions of inoculum (P<0.05)

Table 3: Physicochemical properties of coffee content of the fermented coffee beans. The results
bean fermented in-vivo by civet (luwak coffee) of the current study also revealed that the higher
the proportion of the inoculum introduced to the
Parameters Unit Luwak Coffee fermentation of Arabica coffee cherries the lower
Bean the concentration of the total solid of the fermented
coffee beans generated. This is because the high
Total Solid % 48.20 concentration of inoculum may be represented as
Glucose mmol/L 0.28 a significant amount of microbes involved in the
Caffeine % 1.34 fermentation processes.35 They would enhance the
pH - 4.70 fermentation rates and increase substrate utilization
attributed to the decrease ofthe solid content
(P<0.05) of the coffee pulp. The substrate was subsequently
converted into valuable fermentation end-products
The results showed that the moisture content that could improve the quality of the fermented coffee
of the fermented coffee beans increased when the beans.36 In this study, the total solid of the fermented
concentration of inoculum added was increased coffee beans using civet fecal suspension as
(Fig. 6). The moisture content of the coffee inoculum were quite lower (26-37 %) in comparison
beans fermented with civet fecal suspension was to the original luwak coffee beans in-vivo fermented
higher (63-74%) than that of the control (60%). by civet (48%). This suggested that the use of civet
This occurred since the inoculum used was in the fecal suspension as inoculum would effectively
form of liquid. Hence, an increasing proportion of the degrade the solid content of the coffee pulp.
inoculum would consequently increase the moisture
DARWIN et al., Curr. Res. Nutr Food Sci Jour., Vol. 10(3) 918-927 (2022) 925

Fig. 6: Moisture content and total solid concentration of the coffee beans fermented with
different proportions of inoculum (P<0.05)

Conclusion Acknowledgments
The quality of Arabica coffee beans had been The authors acknowledge the Directorate of
improved through the fermentation processes Research and Community Service, the Ministry
using civet fecal suspensions. The fermentation of Education, Culture, Research and Technology,
of Arabica coffee cherries using mixed microbial culture Indonesia.
of civet fecal suspension successfully reduced
their glucose content. Within above 24 hours of the Funding
fermentation process, the addition of 40% inoculum This research is funded by the scheme of the
to the Arabica coffee cherries was able to convert the National Competitive Basic Research PDKN
sugar content of the coffee beans. The use of mixed (grant number: 39/UN11.2.1/PT.01.03/DRPM/2022).
microbial culture of civet fecal suspension for the
fermentation of Arabica coffee cherries significantly Conflict of Interests
reduced their caffeine content from 2.20% to 1.84%. The authors stated that they have no conflict
This is substantially significant since the fermentation of interest to publish the article.
of Arabica coffee cherries using civet fecal suspension
could enhance the quality of the coffee beans
by reducing caffeine and sugar content.

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