43 Accepted: 12/12/2023, Revised: 30/01/2024, Published: 31/01/2024
MICROBIAL SUCCESSION DURING THE FERMENTATION OF SEMERU ARABICA COFFEE IN WINE COFFEE PRODUCTION
Feronika Heppy Sriherfyna, Neysa Nurjananah Khairanny, Mochamad Nurcholis, Jaya Mahar Maligan
Departemen Ilmu Pangan dan Bioteknologi – Fakultas Teknologi Pertanian Universitas Brawijaya
Jl. Veteran Malang 65145 East Java Indonesia
*Penulis Korespondensi, Email : [email protected]
ABSTRACT
The current trend in coffee product diversification is wine coffee, which has a wine-like flavor and aroma. Reviews of wine coffee are still limited, especially those related to microbial succession during the fermentation process. The objective of this research was analyzing the microbial population and the effect of microbial succession during fermentation process of Arabica Semeru coffee. The analysis was conducted by identifying microbes microscopically and macroscopically. The results of microbial identification during the 30-day fermentation showed populations of yeast, lactic acid bacteria, and acetic acid bacteria. Along with the length of fermentation time, the temperature decreased and the pH fluctuated and stabilized after 16 days of fermentation. There was an increase in sugar and protein content, from 0.61% to 2.87% and 2.13% to 2.23%, respectively. The ethanol content reached 0.047% at the end of fermentation. The microbial exploration was required for further investigation in understanding its role during fermentation process.
Keywords: Arabica Semeru, Bacteria, Fermentation, Succession; Wine coffee; Yeast
INTRODUCTION
Currently, there are many kinds of coffee derivative products due to the increasing trend of people consuming coffee. The normal coffee processing produced a coffee flavor and aroma, whereas the utilization of fermentation method produce coffee which has another flavor and aroma. Therefore, this research was important to carried out. Coffee wine is one of the coffee diversification products obtained from the fermentation process and has a taste and aroma that resemble wine (Widayat et al., 2021). One of the coffee varieties that has the potential to be processed into coffee wine is the Arabica coffee variety obtained from the slopes of Mount Semeru. Arabica Semeru coffee produces compounds with taste notes of green apple, caramel, and brown sugar (Maligan et al., 2020). Wine coffee fermentation lasts for 30–60 days under facultative anaerobic conditions and involves indigenous microbes such as yeasts, lactic acid bacteria, and acetic acid bacteria (Sulaiman and Hasni, 2022). Microbes utilize the mucilage layer, rich in pectin and sugar, as a substrate. The high activity of microbes in producing metabolites has an impact on the concentration of reducing sugar, protein, ethanol, pH, and temperature during fermentation (Dinata et al., 2021).
During wine coffee fermentation, the substrate will be degraded by yeast and produce ethanol, carbon dioxide, organic acids, and flavor-forming precursor compounds. Lactic acid bacteria will grow and utilize the remaining substrate degraded by yeast to produce lactic acid, acetic acid, and other acidic compounds. Acetic acid bacteria predominantly grow through the oxidation of ethanol produced by yeast and partly from lactic fermentation (Sarbu
& Csutak, 2019). It is known that in Gayo Arabica wine coffee fermentation, yeast is the
44 dominant microbe at the beginning of fermentation and will initiate the growth of lactic acid bacteria and acetic acid bacteria (Sulaiman & Hasni, 2022). Reviews related to microbial growth patterns in wine coffee from Arabica Semeru coffee varieties are still limited. Recently, the investigation of growth profiles in wine coffee was conducted from Gayo Arabica, Bourbon Arabika, Brazil Arabika, and Robusta. Therefore, this study aims to determine the types and populations of microbes involved during the fermentation process of Arabica Semeru coffee and to determine the effect of microbial succession on environmental conditions and compounds produced during the fermentation process.
METHODOLOGY
Materials
The main ingredients used for this research include fresh Arabica Semeru coffee cherries obtained from Alir Coffee, Singosari, Malang. Microbial growth media used included YPGA (Yeast Peptone Glucose Agar), Chloramphenicol, MRSA (deMann Rogosa Sharpe Agar), Nystatin, and GYC (Yeast Glucose Ethanol Acetic Bacteria). Chemicals used included 70% alcohol, spirtus, distilled water, n-butanol, kjehdahl tablets, concentrated H2SO4, PP indicator, methyl red indicator, boric acid, 30% NaOH, 0.1 N HCl, Nelson reagent, and arsenomolibdat reagent.
Tools
The tools used for this research include an autoclave (GEA), a spectrophotometer (Shimadzu), a kjeldahl deconstruction device, a kjeldahl distillation device, a fume hood, gas chromatography, laminar air flow, a stomacher (Seward), an incubator (Binder), a colony counter (Stuart), a microscope (Olympus), a thermometer, a pH meter (Hanna), a magnetic stirrer (5 cm), a hot plate stirrer (Caliesys), an analytical balance (Scout Pro), an electric stove (Maspion), a micropipette, an ose needle, a spreader, a Petri dish, a test tube, a vortex (Thermo Scie), and Whatmann filter paper.
Research Design
This research is an experimental study by conducting a fermentation process on Arabica Semeru coffee cherries for 30 days of fermentation and analyzing the fermentation days 0; 2; 4; 6; 8; 10; 12; 14; 16; 20; and 30 for observations in the form of total microbial analysis, microscopic identification, pH, and temperature (Modified from Sulaiman & Hasni, 2022). Observations of sugar, protein, and ethanol content were made before and after wine coffee fermentation took place.
Research Stages
Arabica Semeru coffee cherries were obtained from Alir Coffee, Singosari, Malang.
A total of 5 kg of coffee cherries were then sorted to separate superior quality coffee cherries and low quality coffee cherries as well as from leaves and other impurities. The superior quality coffee was used for this research. Coffee cherries were fermented in plastic bags.
Observations including number of colonies, colony identification, pH and temperature were carried out on fermentation days 0; 2; 4; 6; 8; 10; 12; 14; 16; 20 and 30. Meanwhile, reducing sugar content, protein content and ethanol content were analyzed on day 0 and day 30 of fermentation.
Method
The parameters observed were total yeast, total lactic acid bacteria (LAB), total acetic acid bacteria (AAB), pH, temperature (Sulaiman & Hasni, 2022), microscopic and macroscopic identification (Sari, 2020), reducing sugar content (Fauzi et al., 2013), protein content (Chang & Zhang, 2017), and ethanol content (Hermanto et al., 2020).
45 RESULT AND DISCUSSION
1. Microbial Growth During Fermentation
The results of microbial number testing during the fementation process of coffee wine from Semeru Arabica coffee cherries show in Figure 1. The fermentation process was conducted for 30 days.
Figure 1. Changes in microbial population during the wine coffee fermentation process.
Based on Figure 1, changes in the microbial population occurred at each observation interval. At the beginning of fermentation, yeast becomes the dominant microbe and plays a role in breaking down the main sugar and other polysaccharides into alcohol and carbon dioxide. The carbon dioxide produced during the breakdown of sugar will bind with water to form organic acid compounds in the coffee wine. Therefore, lactic acid bacteria and acetic acid bacteria will begin to dominate during the fermentation of coffee wine. In this case, lactic acid bacteria will ferment sugar into lactic acid, acetic acid, and other compounds that cause the pH to decrease and fluctuate, as well as the synthesis of pectinolytic enzymes that act as precursors for the formation of flavors and aromas in coffee. Acetic acid bacteria will oxidize alcohol and lactic acid into acetic acid, which also plays a role in the formation of flavor precursors in coffee (Sarbu & Csutak, 2019).
The presence of lactic acid bacteria with high growth rates compared to other microbes is related to the low oxygen conditions during the fermentation process, which cause high amylolytic activity that will degrade starch into glucose. Meanwhile, yeast and enterobacteria were found in low numbers and tended to decrease until the end of fermentation (Siregar et al., 2020).
2. Macroscopic and Microscopic Identification of Microbes
The results of microscopic identification of microbial isolates in coffee wine at 1000x magnification present in Figure 2. The identification include microbial shapes and colors of acetic acid bacteria, yeast, and lactic acid bacteria.
Based on Figure 2, the LAB isolates that grow on the fermentation of Arabica Semeru wine coffee have similar shapes and colors of colonies with lactic acid bacteria from the genus Streptococcus, Lactococcus, or Leuconostoc (Azhara et al., 2022; Sulmiyati et al., 2018). The AAB isolates that grow in the fermentation of Arabica Semeru wine coffee, the shape and color of the colonies are similar to those of acetic acid bacteria from the genera Enterobacteriaceae and Acinetobacter (Martati, 2007). Meanwhile, yeast isolates that grow on fermented Semeru Arabica wine coffee have similar shapes and colors of colonies with yeast from the genus Torulaspora delbruckii and Saccharomyces (Kurtzman et al., 2011). These microorganisms was predicted has a contribution in wine flavor and aroma production.
01 23 45 67 89 1011
0 2 4 6 8 10 12 14 16 20 30
Microbial Number ( Log CFU/ml)
Fermentation Time (Days)
AAB LAB Yeast
46 Figure 2. Microscopic Observation Results of wine coffee microbial isolates 1000x
magnification Description:
LAB-C: Coccus-shaped lactic acid bacteria isolate; LAB-B: Bacil-shaped lactic acid bacteria isolate; AAB-C: Coccus-shaped acetic acid bacteria isolate; K-C: Coccus-shaped yeast isolate
2. Changes in pH and Temperature During Fermentation
The result of pH and temperature changing present in Figure 3. The fermentation process was conducted for 30 days.
Figure 3. The changes in pH and temperature during fermentation
4,50 4,60 4,70 4,80 4,90
0,00 10,00 20,00 30,00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
pH
Temperature (°C)
Fermentation Time (days) Temperature pH
Acetic acid bacteria t4 Yeast t2
Coccus Coccus
(AAB-C) (K-C)
Lactic acid bacteria t0 Lactic acid bacteria t0
Coccus Bacili
(LAB-C) (LAB-B)
47 During fermentation, the temperature decreases and the pH tends to fluctuate until the end of fermentation. An increase in temperature and a decrease in pH during fermentation indicate the breakdown of sugar from coffee grounds into alcohol by yeast, and the growth of lactic acid bacteria will increase, which produces organic acid products (Sulaiman & Hasni, 2022). Organic acid compounds produced in coffee will affect the quality as well as the aroma and distinctive taste produced from coffee. One of the dominant acid compounds in coffee is chlorogenic acid, which reaches 6-7% in coffee beans and 2.6% in coffee fruit pulp before fermentation. The content of chlorogenic acid in coffee fruit will change when fermented, with an average content of 5-7.5% (Mulyara, 2021). In the fermentation process of Semeru Arabica wine coffee, the fermentation environment temperature is less than 30°C, so microbial growth during fermentation is less than optimal.
Chamir, as the dominant microbe that grows at the beginning of fermentation, has a growth temperature range of 25–30 °C (Zaini, 2016).
4. Reducing Sugar Content
The test results of reducing sugar, protein, and ethanol content during fermentation present in Table 1. The analysis was conducted at the beginning and end of fermentation time.
Table 1. Results of Biochemical Analysis of Wine Coffee from Semeru Arabica Coffee Cherries
No Analysis Length of fermentation
0 day (%)
30 days (%)
1 Reduced Sugar Content 0.61 2.87
2 Protein Content 2.13 2.23
3 Ethanol Content 0.04 0.05
When compared to the microbial growth at the beginning of fermentation of Arabica Semeru wine coffee, namely yeast, the microbial population at the beginning of fermentation was higher than the microbial population at the end of fermentation. This high microbial population is related to the consumption of sugar in mucilage, which is utilized by microbes to grow (Elhalis et al., 2021). However, in this study, the total reducing sugar at the beginning of fermentation was lower than at the end of fermentation. Based on Table 1, there was an increase in reducing sugar content during fermentation. The high level of reducing sugar can occur due to enzymatic reactions during the fermentation process. Polysaccharides are broken down by the activity of polygalacturonase and pectinase enzymes produced during the yeast fermentation process (Ribeiro et al., 2017).
5. Protein Content
Based on Table 1, there is an increase in protein content, which indicates high microbial activity in breaking down proteins. Proteins will be broken down by proteolytic bacteria, which produce protease enzymes, into essential amino acids. This protein component will affect the flavor of the final coffee wine product (Zaini, 2016). The content of protein in coffee fruit produces different protein content depending on the maturity level of the fruit. The protein content in coffee fruit harvested before it is fully ripe will produce lower protein content (Sitorus, 2019). Proteins formed during the fruit ripening process will increase enzymes that stimulate protein synthesis and increase nutrient reserves such as carbohydrates, protein, and fat (Ifmalinda et al., 2014).
6. Ethanol Content
Based on Table 1, there was an increase in ethanol content during fermentation. In wine coffee processing, coffee fruit is fermented without stripping the pulp with seeds so that the substrate contained in the mucilage will be greater and utilized by microbes (Saripah et al., 2021). Yeast is a heterofermentative microbe that will form alcohol by
48 breaking down simple sugars into form alcohol through the breakdown of simple sugars into pyruvic acid and will undergo decarboxylation into acetaldehyde. The yeast that grows on wine coffee is thought to be able to degrade complex carbohydrates into simple compounds that are utilized for growth and alcohol production (Zaini, 2016). Optimization of fermentation process required to enhance the ethanol content and the quality of wine coffee.
CONCLUSION
During the fermentation of Semeru Arabica wine coffee, three types of microbes were found, namely yeast that initiated the growth of lactic acid bacteria and acetic acid bacteria.
Changes in temperature and pH occurred, respectively, in the range of 19.25°C–25.92°C and 4.54–4.75. The increase in protein content during fermentation was 2.13%–2.23%. The reducing sugar content was 0.61%–2.87%, and the ethanol content was 0.044%–0.047%.
The exploration of yeast, lactic acid bacteria, and acetic acid bacteria was required for further investigation in understanding its role during fermentation process.
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