Screening and Isolation of Indigenous Cellulolytic and

Proteolytic Yeasts from Fermented Dried Cocoa Beans

Dalia Sukmawati1,2, Shabrina Nida Al Husna3, Desty Saszieta1, Almira Marvella
Priskaningrum1, Axel Mareta Mutiani1, Ariza Budi Tunjung Sari4
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Hasyim Ashari Building, 9th floor,
Universitas Negeri Jakarta, Jakarta, Indonesia.
2
Universitas Negeri Jakarta Culture Collection (UNJCC), Faculty of Mathematics and Natural Sciences,
Universitas Negeri Jakarta, Rawamangun, Indonesia; Dalia-Sukmawati@unj.ac.id
3
Department of Microbiology, School of Life Science and Technology, Institut Teknologi Bandung, Bandung,
Indonesia
4
Coffee and Cocoa Research Center Indonesia, Jl. PB Sudirman No.90, Wetan Ktr., Jemberlor, Kec. Patrang,
Kabupaten Jember, Jawa Timur 68118, ariza.bts@gmail.com

*Corresponding author: Dalia-sukmawati@unj.ac.id

Abstract. Fermentation is a biochemical process that requires various microorganisms. The activity of
microorganisms in the cocoa bean fermentation process is important to the success of enzymatic molecule
breakdown. Yeast is a functional microorganism involved in cocoa bean fermentation, functioning as a glucose and
ethanol producer, amongst many other things. Yeast has many other abilities aside from its primary role in sugar
breakdown. One of these is its ability to produce cellulase and protease enzymes, which are both frequently used in
industry. The objective of this study was to discover yeast isolates that can degrade cellulose and proteases from the
fermentation of Sulawesi 1 and TSH 858 cocoa beans. The study was carried out in January 2022 at the Laboratory
of Microbiology, FMIPA UNJ. The method used is descriptive. Data were obtained by isolating up to 20 g of yeast
from each fermentation. The results showed that 231 yeast isolates, including 136 from Sulawesi 1 and 95 from TSH
858. Cellulolytic screening revealed 29 isolates from Sulawesi 1 and 27 isolates from TSH 858, whereas the
proteolytic screening discovered three isolates and no isolate from the TSH 858 that produced proteolytic enzymes.
Keywords: Cellulolytic, Proteolytic, Yeast, Cocoa Bean Fermentation

INTRODUCTION
Indonesia is known as one of the largest cocoa producers in the world with a total cocoa production of
around 200,000 tons in 2019-2020 (International Cocoa Organization, 2022). Seed is one component of cocoa
that is mainly used in the industry. Cocoa beans must be fermented during the processing process. Fermentation
is the process by which large molecules are broken down into simpler molecules with the assistance of
microorganisms. This fermentation intends to promote the content of vitamins, essential amino acids, protein,
flavor, aroma, and appearance in food (Sharma et al., 2020). Several microorganisms, such as lactic acid
bacteria (LAB), acetic acid bacteria (BAA), and yeast, are involved in the fermentation of cocoa beans (De
Vuyst & Weckx, 2016). Under anaerobic and acidic conditions (pH 4.0), yeasts that grow in fermented cocoa
beans degrade the pulp and produce ethanol from glucose (Camu et al., 2007).
Yeast has been shown to have a lot of potential, such as the potential to manufacture cellulase and protease
enzymes (Wilson, 2011; Baur et al., 2015; Anoop Kumar et al., 2019).
Cellulase enzymes are categorized as extracellular enzymes into three types: 1,4-β-endoglucanase, 1,4-β-
exoglucanase, and β-glucosidase (β-D-glucoside glucohydrolase or cellobiase) (Gupta et al., 2012). Cellulase is
often used in biotechnology and industry, such as in the pulp and paper industry, the textile industry, the
production of beverages such as wine, waste decomposers, and as a lignocellulosic degrader in bioethanol
production (Ali et al., 2012; Chakraborty et al. al., 2016; Singh et al., 2016; Ingle et al., 2017; An et al., 2021).
Protease enzymes, as well known as proteolytic enzymes, hydrolyze peptide bonds. Proteases are hydrolase
enzymes (EC 3) that belong into the subclass of peptide hydrolases or peptidases (EC 3.4) (Mótyán et al., 2013).
Proteases are also used in the food, bioremediation, and pharmaceutical industries (Mamo & Assefa, 2018).
The purpose of this study was to obtain and test the ability of yeasts isolated from fermented Sulawesi 1 and
TSH 858 cocoa beans to produce cellulase and protease enzymes.

RESEARCH METHODOLOGY
 Yeast Isolation
The yeast used in this study was obtained from the chocolate fruit of Sulawesi 1 and TSH 858 varieties. The
isolation process followed the Palm (2018) method with several modifications. The isolated part of the
chocolate fruit is the seeds that still have pulp. The chocolate fruit was cut under aseptic conditions and the pulp
was transferred to a sterile box (there are 2 boxes each containing 1 kg of pulp). Then the box is closed for the
fermentation stage.
In each box, 20 grams of pulp was taken and put into YMB with a fermentation period of 0, 24, 48, and 72
hours, then shaken for 60 minutes. Then, 10 -2 , 10-3, 10-4 and 10-5 graded dilutions were carried out. From the
dilutions of 10-4 and 10-5, 0.1 mL was taken and inoculated by the spread plate method on YMA. The purpose of
the multilevel dilution is to minimize or reduce the number of microbes suspended in the liquid. Then incubated
at 28 °C for 48 hours.

Yeast Colony Purification

Colony purification using the method of Murtiningsih et al. (2017), was carried out by taking colonies that
grew apart and showed different morphological characters by inoculating isolates on YMA media using the
quadrant streak method to obtain a single colony. Incubation was carried out at 28 °C for 48 hours. Single
colonies in petri dishes were then inoculated into the media so that YMA was tilted as stock using a loop ose.
Incubation was carried out at 28 °C for 48 hours.

Cellulase Activity Assay

Screening of cellulase-producing yeasts based on the method of Sari et al, (2017) with several modifications.
The yeast isolate to be tested was obtained from YMA agar stock, then streaked with 5 lines on a petri dish
containing Carboxyl Methyl Cellulose (CMC) media. The composition of CMC media per 100 mL contains
0,02 g MgSO4. 7H2O; 0,075 g KNO3; 1 gram of CMC; 0,05 g K2HPO4; 0,002 g FeSO4; 0,004 g CaCl2; 0,2 g
yeast extract; 1,8 g agar; 0,1 g glucose. Incubation was carried out at 28 °C for 48 hours. Cellulolytic activity
testing was carried out using the Congo Red method. Congo Red solution (0,1% w/v) was poured into the
culture and allowed to stand for 15 minutes. The solution was then discarded and rinsed with 0,2 M NaCl for 15
minutes three times. This washing aims to remove Congo Red which is not bound to polysaccharides. Next,
yeast was incubated at 28 °C for 48 hours to complete the formation of the clear zone, then observed the clear
zone formed. Yeast isolates that were able to decompose CMC were indicated by the formation of a clear zone
around the colony after being tested by the Congo Red method.

Protease Activity Assay

Screening of protease enzyme-producing yeasts based on the Chen et al. (2018) with several modifications.
Yeast isolates were streaked in 5 lines on a petri dish containing media for protease assay. The composition of
the media used in the protease enzyme activity test were skim milk (28 g/L), peptone (5 g/L), yeast extract (2,5
g/L), glucose (1 g/L), and agar (15 g/L). Subsequently, all isolates were incubated at 28 °C for 48 hours. The
isolates which had the greatest cellulase enzyme activity were seen from the clear zone.

RESULTS AND DISCUSSION

Yeast Isolation
TABLE 1. Obtaining yeast isolates isolated from chocolate fruit on YMA medium,
incubation at 28 °C for 48 hours
Fermentation Period (h) Yeast isolates
Variety
0 24 48 72 obtained
Sulawesi 1
30 32 40 34 136 (58,9%)
(SK)
TSH 858
16 26 41 12 95 (41,1%)
(TK)
Total 231 (100%)
 The isolation results obtained as many as 231 yeast isolates consisting of 136 yeast isolates from chocolate
variety Sulawesi 1 (58.9%) and 95 yeast isolates from chocolate variety TSH 858 (41.2%) (Table 1). Based on
table 1, it can be seen that the yeast has grown since the fermentation has not taken place (fermentation 0 hours).
Yeast is a pioneer in cocoa fermentation with species including Saccharomyces cerevisiae, Candida rugosa and
Kluyveromyces marxianus (Schwan et al., 2004).
Yeast undergoes an adaptation phase at the 0 hour incubation time, then the yeast enters the logarithmic
phase from 0 to 48 hours, at 48 hours yeast growth reaches the optimum point because at 48 hours the
fermentation temperature reaches a temperature of 30 °C which is included in the optimum temperature range
for yeast growth. The optimal temperature range for yeast growth according to the research of Hardianto et al.
(2018) that the optimum temperature for yeast growth is generally 28-30 °C. After the optimum growth of yeast
has decreased due to entering the death phase causing limiting factor for yeast growth, the alcohol which
produced by yeast will increase and become disinfectant for the yeast itself (Muzaifa et al., 2017).

Screening of Cellulase Producing Yeast Isolates

TABLE 2. Number of positive yeast isolates for cellulase and protease enzymes
Cocoa variety Potential yeasts
Screening assay
Sulawesi 1 (SK) TSH 858 (TK) obtained
Cellulolytic yeast 33 26 59
Proteolytic yeast 3 0 3
 FIGURE 1. Yeast isolates that were positive for cellulase activity were indicated by the
presence of a clear zone. : (A1) SK1-5(24).21; (A2) SK1-3(72).21; (A3) SK1-3(72).22; (A4) SK1-4.9;
(A5) SK1-4.10; (A6) SK1-5.11; (A7) SK1-5.12; (B1) TK2-5.1; (B2) TK2-5.2; (B3) TK2-5.3; (B4)
TK2-5.4; (B5) SK1-5.12; (B6) SK1(1)-5(72).2; (B7) SK1(1)-5(72).3; (B8) SK1(1)-5(72).4; (C1) SK1-3(72).19;
(C2) SK1-3(72).20; (C3) SK1-3(72).13; (C4) SK1-3(72).14; (C5) SK1-3(72).15; (C6) SK1-3(72).16; (C7)
SK1-3(72).17; (C8) SK1-3(72).18; (D1) TK1-3.13; (D2) TK1-3.16; (D3) TK1-3.15; (D4) TK1-3.14;
(D5) TK1-3.17; (D6) TK1-3.21; (D7) TK1-3.19; (D8) TK1-3.18

Based on the results of the screening that has been carried out, it was found that 29 isolates from chocolate
variety SK and 27 isolates from chocolate variety TK were declared as producing cellulase enzymes (Table 2),
which isolates were declared positive if there was a clear zone around the isolate. The formation of a clear zone
around the yeast colony indicates the activity of the cellulase enzyme that degrades the CMC medium (Subowo,
2015). Talanta et al. (2018) said that the size of the clear zone formed is an early indication of the presence of
the cellulase enzyme produced where the larger the clear zone formed, the greater the possibility of the cellulase
produced or having high enzyme activity. The formation of a clear zone shows that the cellulose in the medium
is hydrolyzed by the cellulase enzyme into a simpler compound, namely cellobiose which is then converted into
two glucose molecules (Perez et al., 2002). Yosmar et al. (2013) said that yeast can decompose cellulose
compounds and form a clear zone on CMC medium due to the process of breaking the 1,4 - Glycoside bond in
CMC medium by the activity of the cellulase enzyme produced by the yeast.
 Screening of Protease Producing Yeast Isolates

FIGURE 2. Protease positive isolates were characterized by the presence of a clear

zone
The proteolytic activity assay was carried out using skim milk media. The results of the qualitative test
shown in table 2, showed that 3 isolates from chocolate variety SK were proteolytic yeasts because they were
able to produce protease enzymes which were characterized by the formation of a clear zone around the yeast
colonies (Figure 2). The presence of a clear zone that appears indicates the breakdown of protein molecules
contained in skim milk in the media. Proteolytic yeasts are able to produce extracellular protease enzymes,
which are protein-breaking enzymes that are produced inside the cell and then released out of the cell
(Simamora et al., 2018). Skimmed milk contains casein which functions as an enzyme substrate. Hydrolysis of
casein was used to show the hydrolytic activity of the protease enzyme (Susanti, 2002).
Based on the results of the tests, it was discovered that isolates from Sulawesi 1: SK1(-5)(72)1, SK1(-5)
(72)2, and SK1(-5)(72)4, had the ability to produce cellulose and protease. These three isolates were isolated
after 72 hours of fermentation (Table 3.)

CONCLUSION
The results of this study indicate that yeast isolates from the Sulawesi 1 cocoa variety were known to be
more capable of producing cellulase and protease enzymes than yeast isolates from the TSH 858 cocoa variety.
Sulawesi 1 contained one of 135 yeast isolates known to be capable of producing 29 cellulase enzymes and
three isolates that have the potential to produce protease enzymes. Meanwhile, out of 95 yeast isolates found in
the TSH 858, 27 were known to be capable of producing cellulase enzymes and none were capable of producing
protease enzymes. Three yeast isolates with the ability to produce cellulase and protease enzymes were obtained
from the total isolates.

REFERENCE

1. International Cocoa Organization. (2022). Quarterly Bulletin of Cocoa Statistics, Vol. XLVII, No. 2, Cocoa
year 2021/22. https://www.icco.org/wp-content/uploads/Production_QBCS-XLVIII-No.-2.pdf
2. Sharma, R., Garg, P., Kumar, P., Bhatia, S. K., & Kulshrestha, S. (2020). Microbial fermentation and its
role in quality improvement of fermented foods. Fermentation, 6(4), 106.
3. De Vuyst, L., & Weckx, S. (2016). The cocoa bean fermentation process: from ecosystem analysis to starter
culture development. Journal of Applied Microbiology, 121(1), 5-17.
4. Camu, N., De Winter, T., Verbrugghe, K., Cleenwerck, I., Vandamme, P., Takrama, J. S., ... & De Vuyst,
L. (2007). Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved
in spontaneous heap fermentation of cocoa beans in Ghana. Applied and environmental microbiology,
73(6), 1809-1824.
5. Wilson, D. B. (2011). Microbial diversity of cellulose hydrolysis. Current opinion in microbiology, 14(3),
259-263. doi:10.1016/j.mib.2011.04.004.
6. Baur, C., Krewinkel, M., Kranz, B., von Neubeck, M., Wenning, M., Scherer, S., ... & Fischer, L. (2015).
Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk.
International Dairy Journal, 49, 23-29.
7. Anoop Kumar, V., Suresh Chandra Kurup, R., Snishamol, C., & Nagendra Prabhu, G. (2019). Role of
cellulases in food, feed, and beverage industries. Green bio-processes, 323-343.
8. Gupta, P., Samant, K., & Sahu, A. (2012). Isolation of cellulose-degrading bacteria and determination of
their cellulolytic potential. International journal of microbiology, 2012.
https://doi.org/10.1155/2012/578925.
9. Ali, H., Hashem, M., Shaker, N., Ramadan, M., El-Sadek, B., & Hady, M. A. (2012). Cellulase enzyme in
bio-finishing of cotton-based fabrics: effects of process parameters. Research Journal of Textile and
Apparel.
10. Chakraborty, S., Gupta, R., Jain, K. K., Gautam, S., & Kuhad, R. C. (2016). Cellulases: application in wine
and brewery industry. In New and Future Developments in Microbial Biotechnology and Bioengineering
(pp. 193-200). Elsevier.
11. Singh, S., Singh, V. K., Aamir, M., Dubey, M. K., Patel, J. S., Upadhyay, R. S., & Gupta, V. K. (2016).
Cellulase in pulp and paper industry. In New and future developments in microbial biotechnology and
bioengineering (pp. 152-162). Elsevier.
12. Ingle, A. P., Rathod, J., Pandit, R., da Silva, S. S., & Rai, M. (2017). Comparative evaluation of free and
immobilized cellulase for enzymatic hydrolysis of lignocellulosic biomass for sustainable bioethanol
production. Cellulose, 24(12), 5529-5540.
13. An, X., Chen, X., Wang, Y., Zhao, X., Xiao, X., Long, H., ... & Zhang, Q. (2021). Cellulolytic bacterium
characterization and genome functional analysis: An attempt to lay the foundation for waste management.
Bioresource Technology, 321, 124462. https://doi.org/10.1016/j.biortech.2020.124462.
14. Mótyán, J. A., Tóth, F., & Tőzsér, J. (2013). Research applications of proteolytic enzymes in molecular
biology. Biomolecules, 3(4), 923-942.
15. Mamo, J., & Assefa, F. (2018). The role of microbial aspartic protease enzyme in food and beverage
industries. Journal of Food Quality, 2018.
16. Palm, Y. O. T. (2018). Isolasi, Identifikasi Dan Produksi Etanol Khamir Indigenous Nira Siwalan
(Borassus flabellifer L.) Dari Tuban, Jawa Timur, Indonesia. Jurnal Biotropika| Vol, 6(1), 6.
17. Murtiyaningsih, H., & Hazmi, M. (2017). Isolasi dan uji aktivitas enzim selulase pada bakteri selulolitik
asal tanah sampah. Agritrop: Jurnal Ilmu-Ilmu Pertanian (Journal of Agricultural Science), 15(2).
18. Sari, S. L. A., Setyaningsih, R., & Wibowo, N. F. A. (2017). Isolation and Screening of Cellulolytic Fungi
from Salacca edulis Leaf Litter. Biodiversitas, 18(3), 1282–1288.
19. Chen, P. H., Chen, R. Y., & Chou, J. Y. (2018). Screening and evaluation of yeast antagonists for biological
control of Botrytis cinerea on strawberry fruits. Mycobiology, 46(1), 33-46.
20. Schwan, R. F., & Wheals, A. E. (2004). The microbiology of cocoa fermentation and its role in chocolate
quality. Critical reviews in food science and nutrition, 44(4), 205-221.
21. Hardianto, Muhibuddin, A., & Sektiono, A. (2018). Optimalisasi Fosfat untuk Meningkatkan Pertumbuhan
Kerapatan Populasi dan Kemampuan Antagonis Saccharomyces cerevisiae terhadap Fusarium sp.
SAINTEKBU, 10(2), 27-41. https://doi.org/10.32764/saintekbu.v10i2.206
22. Muzaifa, M., Abubakar, Y., & Haris, F. (2017). Profil Pertumbuhan Mikroorganisme pada Fermentasi Biji
Kakao Aceh. Jurnal Teknologi Dan Industri Pertanian Indonesia, 9(2), 50–54.
https://doi.org/10.17969/jtipi.v9i1.5975
23. Subowo, YB. (2010). Uji Aktivitas Enzim Selulase dan Ligninase dari beberapa Jamur dan Potensinya
sebagai Pendukung Pertumbuhan Tanaman Terong (Solarium melongena). Berita Biologi, 10(1), 1-6.
24. Talanta, VM., Mariana, Lambui, O., dan Suwastika, IN. (2018). Uji Aktivitas Selulase dari Jamur
Selulolitik Asal Tanah Danau Kalimpa’a Central Sulawesi. Journal of Science and Technology, 7(3), 323-
333. ISSN-e: 2541-1969.
25. Perez, J., Dorado, MJ., Rubia, T., Martinez, J. (2002). Biodegradation and Biological Treatments of
Cellulose, Hemicellulose, and Lignin : An Overview. Journal of International Microbiology. Vol. 5:53-63.
26. Yosmar, R., Suharti, N., Rasyid, R. (2013). Isolasi dan Uji Kuantitatif Hidrolisat Jamur Penghasil Enzim
Selulase dari Tanah Tumpukan Ampas Tebu. Jurnal Farmasi Andalas, 1(1), 3-12. ISSN: 2302-8254.
27. Simamora, C. J. K., & Sukmawati, S. (2018). Identification and Characterization of PrTK-2 Bacterial
Isolate Producing Extracellular Protease Enzyme from Rubber Seeds Tempeh. Bioscience, 2(1), 79-88.
28. Susanti, E. (2002). Petunjuk Praktikum Biokimia. Malang: Jurusan Kimia FMIPA UM.