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Proceeding of International Conference on Biodiversity for Sustainable Industries Proceeding of International Conference on Biodiversity 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 COCOA BEAN QUALITY Yusya’ Abubakar*, Heru P. Widayat, Martunis, Murna Muzaifa, Rizka Try Gustina Department of Agricultural Product Technology, Faculty of Agriculture, Syiah Kuala University, 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 sustainability. 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 pulp (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, microscope, and microscopic digital camera, micro pipet, vortex, and glassware. 80 | Proceeding ICBSI: 79-83 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 Abubakar et al | 81 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 82 | Proceeding ICBSI: 79-83 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
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