Isolation and Identification of Microorganisms from Fermented Glutinous Rice Using Black Bamboo (Gigantochloa Atroviolacea) and Sweet Bamboo (Gigantochloa Atter) Gladys Delarosa Purnomo1), Lindayani2) and Laksmi Hartayanie2) 1) Students; Food Technology Department; Agricultural Technology Faculty; Soegijapranata Catholic University 2) Lecturer; Food Technology Department; Agricultural Technology Faculty; Soegijapranata Catholic University [email protected] ABSTRACT Fermented glutinous rice is a kind of traditional food from glutinous rice fermented with ragi. The typically containers used for fermentation glutinous rice are banana leaves, plastic bottles, etc. Some efforts to increase local knowledge or use natural resources from environment is very important, for the example is to use bamboo. Traditional Chinnese food was made using bamboo to get a spesific flavor, so in this research bamboo was used as a container of fermented glutinous rice. The purpose of this study was to identify the microorganisms found in fermented glutinous rice. There was a preliminary test (sensory evaluation) to determine the best ragi and packaging by 30 panelists. The results of sensory evaluation showed that local ragi tape NKL and black bamboo obtained from Ungaran were the best result. The methods of this study include chemical analysis (total sugars, total acids and pH) and microbiological analysis (isolation and identification of lactic acid bacteria, molds and khamir). Identification tests of lactic acid bacteria were done by characteristics morphology test (cell shape, gram staining and spore formation), catalase activity test, motility test, gas production test, growth capabilities test at various temperatures, pH and NaCl. Identification of lactic acid bacteria used API 20 STREP test kit. The results shown that isolate identified as Aerococcus viridans 3 and Aerococcus viridans 2. Mold identification obtained 5 isolates from Aspergillus sp. and 2 isolate from Mucor sp. Yeast identification using API 20 C AUX identified as Saccharomyces cerevisiae 1 and Candida utilis. Keyword: fermented glutinous rice, lactic acid bacteria, molds, yeast, ragi, bamboo INTRODUCTION The heterogeneous of ethnic and cultural diversity enriched Indonesia with various types of traditional foods. The Indonesian traditional fermented foods such as soy sauce, tempe, oncom, tape and saurkraut. One of fermented foods widely known is tape. Many types raw material that used to production tape in Indonesia, such as cassava, sweet potato, sorghum, banana, and glutinous rice (black and white). The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013 Page 1 Fermented Glutinous Rice (“Tape Ketan”) is a traditional food made from glutinous rice (Oryza sativa var glutinosa) and fermented with ragi. Cronk et al. (1977) explain that tape ketan is a fermented food, have sweet and sour taste and likely rice pasta contains little alcohol. According Lestario (1998) in Atmodjo (2006), good tape ketan have soft grain texture, sticky, sweet and slightly sour taste and contains little alcohol and not sting. In general, the process of making tape using anaerobic fermentation process. After rice blend with ragi, put into containers, then stored in sealed containers for 2-3 days at temperature 26-280C. Tape inculated with anaerobic fermentation have sweeter taste than aerobic fermentation, because microorganisms in tape ketan does not need many oxygen (Tarigan, 1988). Tape ketan usually fermented in plastic containers. In specific areas, tape ketan wrapped with banana leaves. Some efforts to increase local knowledge or use natural resources in environment is very important, for example is bamboo. Bamboo is still limited to used, such as for structural materials and cooking for chinese food. Traditional Chinnese food used bamboo to get spesific flavor. So in this research wants to used bamboo as packaging in fermented tape ketan. Fermentation process involves microorganisms to change components in foods from macro nutrient molecules into simple molecules. This foods become easy to digested and absorbed by intestine. Microorganisms involves in fermentation process are yeast, fungi and bacteria (Volk & wheeler, 1984). According Hesseltine et al. (1988), ragi tape have at least one species of yeast, one species mold (Mucor, Rhizopus, or Amylomyces) and one or two coccus bacteria in each sample. Gunadya & Between (1997) found, microorganisms in ragi tape such as Aspergillus, Saccharomyces, Candida, Hansenula and Acetobacter. According to Sujaya et al. (2001), Lactic Acid Bacteria that found in tape ketan are Weissella, Lactobacilli, Enterococci and Pediococci. Microorganisms in ragi tape can influenced microorganisms founded in tape ketan because ragi acts to start fermentation. There are many factors that influence fermentation process such as time, moisture, temperature, oxygen and substrates (Gaman and Sherrington, 1994) so microorganisms in fermented foods can be different from region to other region. The objectives in this research were to isolate and to identify microorganisms in tape ketan which fermented in black bamboo and sweet bamboo. MATERIALS AND METHODS The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013 Page 2 Making of Tape Ketan Glutinous rice Figure 1. Making Process of Tape Ketan Preliminary Test In preliminary test performed by sensory analysis with rating test hedonic to determine level of consumer preference tape ketan with variety local yeast. Testing by filling out questionnaires provided. The panelists must give score 1 (one) to 5 (five), which score one indicates the lowest value (strongly dislike) and score five indicates highest value (really like) (Resurrecion, 1998). Organoleptic parameters tested included flavor, aroma, texture and color. Panelists to be used is untrained panelists in UNIKA Soegijapranata students (30 people). There were 12 samples tested in preliminary test that is combination of packaging (black bamboo and sweet bamboo), ragi local brands (Na Kok Liong, Gedang and Sidojoyo), and concentrated ragi (0.2% and 0.3%). Preliminary tests to determine the best concentration ragi, ragi local brands and packaging to be used in main test. Main Test In main test, sample used is the best results sample from preliminary test, that is tape ketan saved in sweet bamboo, used NKL local ragi brand and the concentration is 0,2%. The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013 Page 3 Microbiological analyzes conducted in 2 times and physicochemical analyzes conducted in 3 times. Physicochemical testing conducted in day 3, 5, 7 and 9. Tape Ketan Figure 2. Main Research Flowchart Design The methods of measuring total acid is titration method (Simbolon, 2008). Measurement pH using pH meter. And for total sugars using phenol method (Apriyantono et al., 1989). Isolation molds, yeast and LAB using same methods that is isolate colony until form a single colony. Colony purification method using spread plate method. Dilution for purification colony molds up to 10-5. Medium for mold is PDA with addition 0,1% chloramfenicol. Incubation for mold during 5 days at temperature 280C (Chiang et al., 2006; Lay, 1994). For purification method in LAB performed by diluting until 10-8. Media used is MRS with addition 1% of CaCO3. Incubation of BAL during 48 hours at temperature 370C (Chiang et al., 2006; Rahayu & Margino, 1997). In purification colony of yeast used MEA agar and dilution until 10-5. Incubation yeasts during 2-4 days at temperature 280C. Identification of fungi was conducted by slides culture and incubated for 2-3 days. After incubation, molds observed using microscope and compairing morphological appearance with Introduction to Food Borne Fungi (Lay, 1994; Samson et al., 1984). Identification tests of LAB were done by morphology test (cell shape, gram staining and spore formation), catalase activity test, motility test, gas production test, and growth capabilities test at various temperatures (100C, 450C and 500C), pH (4.4 and 9.6) and NaCl (6.5% and 18%) (Lay, 1994; Rahayu & Margino, 1997; Sneath et al., 1984). Testing of species LAB do with API 20 Strep test kit (Pelinescu et al., 2009). Identification of yeast done by API 20 C AUX test kit (Kurtzman & Fell, 1998). RESULTS AND DISCUSSION Preliminary Test The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013 Page 4 From the results of preliminary test known that local brand ragi NKL with concentration 0.2% and sweet bamboo are the best results (Table 1, Table 2). Then, NKL ragi local brands with concentration ragi 0.2% and sweet bamboo used for main test (physicochemical and isolation identification of microorganisms). Table 1. The Sensory of Tape Ketan based on NKL, Gedang and Sidojoyo Concentration Merk ragi 0,20% 0,30% NKL 3,12 2,96 gedang 3,07 3,03 sidojoyo 3,03 3,01 Table 2. The Sensory of Tape Ketan based on Bamboo Species Bamboo species Average hitam 3,03 legi 3,1111 Note: shown sample have the best results Physicochemical Analyzes Chemical analyses consist of total sugar, pH and total acid on days 3, 5, 7 and 9. Based on the results in Graph 1, known that total sugar contained in tape ketan decreased from day 3 to day 9. In results of pH, the value is going decreased from day 3 to day 9 (Graph 2). This is caused by