Isolasi dan karakterisasi Yeast Amilolitik dari Ragi Tape

NURHARTADI, Edhi, Dr.Ir. Endang S. Rahayu, MS

2005 | Tesis | S2 Ilmu dan Teknologi Pangan

Ragi tape merupakan agensia yang digunakan untuk memfermentasi bahan pangan berpati seperti ketela pohon dan beras ketan. Di dalam ragi tape terdapat banyak mikrobia amilolitik dan fermentatif. Tujuan penelitian ini adalah untuk mengisolasi dan mengidentifikasi yeast amilolitik dari ragi tape sebagai sumber enzim amilolitik dari mikrobia. Isolasi dilakukan dengan metode spread plate menggunakan media PGYS (pepton-glukosa-yeast ekstrak-soluble starch), ditambah chloromycetin. Inkubasi dilakukan pada 30(simbol)C, selama 2 hari. Koloni dengan zona hidrolisis dimurnikan dengan metode goresan pada media yang sama. Kemudian dilanjutkan dengan identifikasi dan karakterisasi berdasarkan pengamatan morfologi sel dan pengujian sifat fisiologisnya sampai tingkat spesies. Pengamatan morfologi meliputi bentuk dan ukuran sel, reproduksi vegetatif, pembentukan pseudomiselium dan true miselium dan pembentukan spora. Pengamatan fisiologis meliputi uji fermentasi dan pembentukan asam, pertumbuhan pada media tanpa vitamin, media 50% glukosa, media 10% NaCl-5% glukosa, uji urease, uji pertumbuhan pada suhu tertentu, dan uji asimilasi dari berbagai sumber karbon dan nitrogen. Isolasi yeast amilolitik yang dilakukan terhadap empat macam merk ragi tape yaitu NKL, MK, 66 dan tanpa merk diperoleh 15 isolat terdiri dari 9 isolat dari ragi NKL, 2 isolat dari ragi 66 dan 4 isolat dari tanpa merk. Hasil identifikasi diperoleh dugaan Debaryomyces vanriji (4 isolat); Saccharomycopsis fibuligera (3 isolat); Pichia burtonii (8 isolat). Dari hasil diketahui bahwa S. fibuligera dan P. burtonii terdapat di semua merk ragi tape. Seleksi isolat yeast amilolitik yang potensial dilakukan berdasarkan rasio amilolitik yaitu rasio diameter zona hidrolisis dengan diameter koloni. Diperoleh hasil isolat E-3 (Saccharomycopsis fibuligera) mempunyai rasio amilolitik tertinggi. Pengaruh variasi kadar pati terlarut terhadap pertumbuhan sel isolat E-3 diperoleh hasil yaitu dengan kadar pati terlarut 1,5% sudah cukup untuk memberikan jumlah sel yang banyak. Hubungan antara kadar biomassa, kadar pati terlarut dan kadar gula reduksi dengan lama fermentasi diperoleh hasil bahwa kadar biomassa dan kadar gula reduksi akan meningkat, sedangkan kadar pati terlarut akan menurun. Enzim amilolitik yang dihasilkan selama fermentasi diketahui sangat besar aktivitasnya di hari pertama fermentasi, selanjutnya mengalami penurunan di hari kedua dan ketiga fermentasi. Dengan kadar pati terlarut 2% diperoleh aktivitas enzim glukoamilase tertinggi yaitu 44,13 mg glukosa/L/menit/1 ml lar.enzim dan aktivitas enzim (simbol)-amilase tertinggi yaitu 261,09 mg maltosa/L/jam/1 ml lar. enzim.

Ragi tape was an agent that used to ferment starchy foods like tapioca and glutinous rice. Inside ragi tape there were many amylolytic and fermentative microbial. The research aim was to isolate and characterize amylolytic yeast from ragi tape as other origin of amylolytic enzyme from microbial. Isolation was done with spread plate method with PGYS (peptone-glucoseyeast extract-soluble starch) medium, added with chloromycetin, incubated at 30􀁱C for 2 days. Colony with hydrolytic zone then purified with streak method on same medium. Identification based on cell morphology and physiology test observation. Observation on cell morphology included size and shape of cell, vegetative reproduction, pseudomycelium and true mycelium form, spores form. Physiological observation include fermentation and acid form, growth on vitamin-free medium, growth on 50% glucose medium, growth on 10% NaCl-5% glucose medium, urease test, growth on different temperatures, and assimilation many carbon and nitrogen source. Isolation of amylolytic yeast was done on four ragi tape: NKL, MK, 66 and without merk gave result 15 isolates. 9 isolates from ragi NKL, 2 isolates from ragi 66, and 4 isolates from no merk. From identification give suspected result: Debaryomyces vanriji (4 isolates); Saccharomycopsis fibuligera (3 isolates); Pichia burtonii (8 isolates). This result showed that S. fibuligera and P. burtonii were on all merk of ragi tape. Selection of potential isolate amylolytic yeast based on amylolytic ratio that was ratio of size of hydrolytic zone and size of colony. From ratio amylolytic give result isolate E-3 has the highest amylolytic ratio among the others. Observation on isolate E-3 growth curve with variation soluble starch concentration give result that with 1,5% soluble starch was enough to get high cells. Relation between cell, soluble starch and reduction sugar concentration with time fermentation give result that cells and reduction sugar concentration will increase, while soluble starch concentration will decrease. Amylolyitic enzyme that produce on fermentation on 1st day was high, then will decrease on 2nd and 3rd day of fermentation. Glucoamylase activity was highest at 44.13 mg glucose/L/min/1 ml enzyme solution with soluble starch concentration on 2%. This result show that glucoamylase activity was high with high of soluble starch concentration. 9Symbol)-amylase activity was highest at 261.09 mg maltose/L/min/1 ml enzyme solution with 2% soluble starch. This result show too that (symbol)-amylase activity will high with high of soluble starch concentration.

Kata Kunci : Fermentasi Pangan,Ragi Tape,Yeast Amilolitik, isolation, amylolytic yeast, soluble starch concentration, enzyme activity