Proceeding of International Conference on for Sustainable Industries

VOL.1/JUNE/2017 ISBN:978-602-61830-0-2

presented by:

P R O C E E D I N G ISBN: 978-602-61830-0-2 ICBSI June 2017

ISOLATION AND IDENTIFICATION OF YEAST INVOLVED IN COCOA FERMENTATION TO BE USED AS A STARTER TO IMPROVE QUALITY

Yusya’ Abubakar*, Heru P. Widayat, Martunis, Murna Muzaifa, Rizka Try Gustina Department of Agricultural Product , Faculty of , Syiah Kuala , Jl. Tgk. H. Krueng Kalee 3, Banda Aceh, Aceh. Indonesia *Email: [email protected]

ABSTRACT - Indonesia has been known as the third largest cocoa (Theobroma cacao L.) producer in the world. However, most of Indonesian cocoa is considered as low in quality due to unfermented bean. Bean fermentation has been introduced to smallholder farmers in Aceh, but the quality of bean produced is not consistent. Among factors determining a succesfull fermentation is involvment of major microorganisms, including yeast which is grown naturally around cocoa beans in the first 24 hours of fermentation. Therefore, this study is aimed to isolate and identify the type of yeasts involved in the cocoa fermentation process. Then, the yeast is being used as starter in cocoa fermentation to improve the bean quality. Improving cocoa quality is very important to improve farmer competitiveness, which in turn will enhance cocoa production . Samples of microorganisms taken from the best cocoa mass fermentation were isolated to obtain pure yeast culture. The culture was identified conventionally by observing (testing) its morphological and biochemical properties. The results showed that the six isolates predominantly grown fall into four different yeasts genus. Yet, only three generas has been identified succesfully which are Candida sp., Saccharomyces sp. and Kluyveromyces sp.

Keywords : cocoa, fermentation, isolation, identification, yeast

INTRODUCTION

Cocoa (Theobroma cacao L.) is one of the plantation products mostly produced by smallholder farmer in Aceh. In general, cocoa tree is suitable to the land and growing environment in Aceh. However, after harvest most farmers dried their cocoa bean directly without fermentation process, so that the bean quality is considered low. Fermentation has a very importan role in developing flavor and aroma that influencing cocoa bean quality. Fermentation process in cocoa mass usually occured spontaneosly triggered by microorganisms that present in the surrounding of the fermentation box/container. During 5-7 days of fermenttion, yeast is grows in the first 24 hours of fermentation (Leerían dan Patterson, 1983; Schwan dkk., 1998; Ardhana dan Fleet, 2003). Then lactic acid bacteria start to grow producing lactic acid which favoring environment for the growth of acetic acid bacteri that converting alcohol into acetic acid. Types of yeast usually found in cocoa fermentation are Saccharomyces sp., Pichia fermentans, Torulopsis sp., Kloeckera spiculata and Hansenula anomal I. These yeast involved in in a fermentation that producing alcohol, carbon disoxide, and heat while at the same time they degraded cocoa (Ardhana, 2003; Nielsen, 2006). Unfortunately, information about type of microorganisms growing during cocoa bean fermentation in Aceh is very limited, so that modification to improve the fermentation process is very difficult. Therefore, it is necessary to isolate and identify types of miscroorganisms (including their physiological and morphological characteristics) playing important roles in the fermentation process. With proper handling, the pure strains isolated could be used as a starter in the fermentation process. Introduction of starter is one of techniques that could be explored to improve the fermentation process, which in turn will improve quality of cocoa bean in Aceh.

MATERIALS AND METHODS

This research was conducted in Industrial Microbiology Laboratory, and Plant Desease Laboratory at Faculty of Agriculture, and Microbiology and Histology Laboratory at Faculty of Veterinar, Syiah Kuala University, in 2015. Cocoa bean used in this research was obtained from Pidie Jaya District, Aceh Province, Indonesia, and fermented in a laboratory in Faculty of Agriculture, Syiah Kuala Univerity as outlined by Abubakar (2015). Chemicals used for analysis are aquades, peptone, MEA (malt extract agar), Alcohol 70% dan 96%, glucose, lactose, maltose, manitol, metilen blue, and safranin. Instrument and equipment used were wood fermentation box, refrigerator, autoclave, incubator, laminar flow cabinet, oven, qaubec colony counter, , and microscopic digital camera, micro pipet, vortex, and glassware.

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Cocoa bean fermentation and drying Bean fermentation was conducted in a Laboratory at Faculty of Agriulture, Syiah Kuala Univerity. Fermentation was held in wooden box, for 5 days. About 40 kg wet bean was poured into each box (40 cm x 40 cm x 40 cm) and then wrapped with banana leaves. The cocoa mass were agitated every 24 hours for 5 days. After fermentation was completed, the bean was dried using direct sunlight till the water content fall below 7%. The dried fermented bean was kept in jute-sack. Cocoa mass samples were taken everyday from four representative points in the fermentation box.

Yeast isolation and identification Analytical procedure was adopted from Ardhana and Fleet (2003) and Nielsen et al. (2006) with modification. Samples as much as 20 g from cocoa mass was diluted into 180 ml 0.1% peptone, homogenized, and vortexed for 3 minutes. About 1 ml sample was serially diluted into 0.1% pepton until 10-7. Then, 0.1 ml aliquot was plated on MEA (malt extract agar) and incubated at 30 0C for 48 hours. Then the plate was observed, and a single colony having the most resambles properties to yeast was chosen to be purified using streak method (Yarrow, 1998). The choosen colony was taken and streaked on a new plate and then incubated for 2x24 hours at 300C. Once pure isolate was obtained, its morphological and biochemical properties was observed. Morphological observation included color, texture, and sel shape of the colony. Biochemical and physiological test included sugar fermentation test, and growth test at 300C, 370C, 400C dan 420C. The results were then compared to the one obtained by Ebabhi (2008), Ghosh (2011), Jumiati (2013), and Mpofu (2008), to draw conclusion about their genus.

RESULTS AND DISCUSSIONS

R1 R5

Figure 1. Shape and cell morphology of R1 and R5. Observation was conducted with Gram coloring and viewed under microscope magnified 1000x

R2 R3

Figure 2. Shape and cell morphology of R2 and R3. Observation was conducted with Gram coloring and viewed under microscope magnified 1000x

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R6

Figure 3. Shape and cell morphology of R6. Observation was conducted with Gram coloring and viewed under microscope magnified 1000x

R4

Figure 4. Shape and cell morphology of R4. Observation was conducted with Gram coloring and viewed under microscope magnified 1000x

Table 1. Morphological shape of yeast identified Isolate Colony Surface Degree of Shining Colony edge Surface Shape Color Cell shape

R1 Fine Shiny/Glossy Flat Bumpy Cream Long cylinder R2 Fine Shiny/Glossy Irregular Flat White, cream Long oval R3 Curly Did not shiny Wavy Flat White, cream Long cylinder R4 Curly Did not shiny Wavy Flat Cream Long with cell wall formation R5 Like string Did not shiny Irregular Flat Milk White Long cylinder R6 Like grain Did not shiny Irregular Bumpy Cream Round

Sugar Fermentation Test

Table 2. Ability of isolates to ferment sugar Isolate Glucose Sucrose Maltose Lactose Manitol

R1 + + + +/- + R2 + - + - - R3 - - + - - R4 - - + - - R5 - - + - - R6 + + + + +/ *(+) = grow ; (-)= did not grow

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Table 3. Isolates’ ability to grow at several temperatures Isolate 30oC 37oC 40oC 42oC

R1 + + - - R2 + - + - R3 + + + - R4 + - - - R5 + + + + R6 + + + +

Six isolates having different shape have been found. The six isolates were chosen because they growed dominantly during isolation process. The isolates cell morphology were observed, and then they were tested on growth resistency at certain temperatures, and ability to ferment several type of sugars. Results from these tests were compared to several related literatures to strengthen the yeast genus identified. Morphological observation of the isolates coud be seen on Table 1.

Sugar fermentation test Sugar fermentation test showed that two isolates were able to ferment glucose, sucrose, maltose, laktose, and mannitol. Indicators used to observe the fermentation process was change in medium color from purple to yellow. The test results were presented in Table 2.

Test of ability to grow in certain temperatures In this test, yeast isolate were exposed to several incubaton temperatures i.e. 300C, 370C, 400C and 420C. The wide range of temperatures were chosen because several yeasts were able to grow at high temperatures, while several others only able to grow at its optimum temperature.

Yeast identification Genus of Candida sp. Based on observation and identification using morphology, physiology and biochemical properties, genus of yeast isolate R1 dan R5 were Candida sp. (Figure 1). These results also very similar to previous works conducted by severeal researchers (Ghosh, 2011; Mpofu, 2008; Ebabhi, 2013; Jumiati, 2013). The researcher mentioned that Candida sp. has cell with variable shape, ranging from round, round and long, oval, long oval, cylindris, and elongated cylindris. It is very rare that the cell shape as apiculate, ogival, bottle, and tringular. Candida genus also has fine colony surface, shiny, flat colony edge, bumpy surface, and colony color from white until cream. This yeast also able to ferment glucose and maltose perfectly, and grow very well at 370C until 420C. Ardhana and Fleet (2004) reported that Candida sp. present in cocoa fermentation in Indonesia.

Genus of Saccharomyces sp. Observation and identification using morphology, fisiology and biochemical properties, indicated strongly that yeast isolate R2 and R3 were come from genus Saccharomyces sp. (Figure 2). This results also resemble closely with previous research conducted by Jumiyati (2012) and Ebabhi (2013). Saccharomyces genus has fine colony surface, shiny, and with pale cream color. They were able to ferment glucose (but not lactose) and grow well at 400C.

Genus of Kluyveromyces sp. Based on observation and identification using morphology, fisiology and biochemical properties, it could be concluded that yeast isolate R6 was come from genus Kluyveromyces sp. (Figure 3). This results were based on previous research conducted by Ebabhi (2013), especially on cell shape, colony morphology and fermentation ability and growing ability at certain temperatures. Kluyveromyces genus has several cell shapes including round, egg round, cylindris and ellips. It has a grainy and bumpy surface, colony did not shiny, irregular edge, and with cream color. It was able to ferment glucose, sucrose, lactose, mannitol, and maltose, and could grow well at 300C, 370C, 400C dan 420C. Thompson (2001) and Nielsen et al. (2005) reported that Kluyveromyces genus present in cocoa fermentation.

Unidentified yeast genus Based on observation of colony morphology, cell, and test on fermentation ability and growing ability in certain temperatures, isolate R4 (Figure 4) could not yet be groupep into one of the genus, because comparison of parameters did not match to the one reported by Ghosh (2011), Mpofu (2008), Ebabhi (2013), and Jumiati (2013). Isolate R4 had curly colony surface, did not shiny, wavy colony edge, flat elevation, and cream in color. Its cell had long shape and as if had splitted wall. It was only able to ferment maltose, and grew at 30oC.

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The four yeast isolates that had been identified from the cocoa fermentation process in Aceh, will be stored properly and refreshed regularly to maintain they viability. The isolates later will be propogated and then used as a starter in controled cocoa bean fermentation, to improve dried cocoa bean quality.

CONCLUSION

During fermentation of cocoa bean in Aceh, six yeast isolates having different morphologies had been isolated. They fell into four genus, i.e. Candida sp, Kluyveromyces sp., and Saccharomyces sp., while the fourth one could not be identified yet using the morphological comparison methods used in this research.

ACKNOWLEDGMENTS

The author would like to acknowledge Syiah Kuala University for providing research facilities, Directorate General of Higher Education, Ministry of Education and Cultural, Indonesia, as source of funding of university priority research scheme.

REFERENCES

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