ISOLASI, KARAKTERISASI DAN UJI POTENSI BAKTERIOSIN BAKTERI ASAM LAKTAT DARI UDANG

Romadhon, Prof. Dr. Ir. Sebastian Margino,

2013 | Tesis | S2 Bioteknologi

Penggunaan pengawet sintesis untuk menghambat kerusakan makanan berbahaya bagi kesehatan. Oleh sebab itu perlu dicari pengawet alami dan aman jika dikonsumsi. Bakteriosin salah satu pengawet alami sebagai agen biopreservatif potensial digunakan sebagai antibakteri. Penelitian ini bertujuan untuk mengisolasi dan menyeleksi bakteri asam laktat, mengoptimasi produksi dan purifikasi bakteriosin, mengidentifikasi isolat terpilih, dan karakterisasi bakteriosin. INTISARI Isolasi Bakteri asam laktat dilakukan pada usus udang berasal dari laut. Seleksi dilakukan secara kualitatif dan kuantitatif. Isolat terpilih dioptimasi produk bakteriosinnya meliputi variasi konsentrasi inokulum, sumber C dan N, pH medium, agitasi dan kinetika pertumbuhan. Purifikasi bakteriosin dengan adsorbsi desorbsi dan dilanjutkan fraksinasi pada kolom kromatografi ion exchange dengan matriks DEAE-cellulose, Identifikasi isolat terpilih berdasarkan karakteristik morfologi, biokimiawi, fisiologis, dan molekuler (gen 16S rRNA). Karakterisasi bakteriosin hasil purifikasi meliputi berat molekul, pH, suhu, enzim proteolitik, bakteri patogen dan pembusuk. Hasil isolasi memperoleh 209 isolat BAL. Dari 209 isolat, 54 isolat diantaranya menghasilkan bakteriosin. Seleksi secara kualitatif dan kuantitatif didapatkan 8 isolat unggulan. Satu diantaranya isolat SFE-7 (33) yang terbaik dengan aktivitas bakteriosin 72.415 Au/ml. Optimalisasi produksi bakteriosin dengan inokulum 5 % (v/v), sumber C glukosa 20 g/l, sumber N yeast extract 20 g/l, pH 5, suhu 37 o C, kecepatan agitasi 100 rpm, dan waktu produksi 15 jam. Hasil kinetika pertumbuhan µ max 0,065/h, (Yb/x) 0,4 [ x10 6 AU (g CDM) -1 ], (Yx/s) 62,33 [ g CDM (g gula) -1 ] , (Yb/s) 50 [ x 10 6 AU (g gula) -1 ]. Ekstraksi bakteriosin dengan metode adsorbs-desorbsi menghasilkan aktivitas spesifik yaitu 312,773 Au/ml dengan tingkat kemurnian 56,45 kali. Hasil Fraksinasi kolom DEAE-cellulose memperoleh aktivitas bakteriosin tertinggi pada fraksi ke-36 yaitu 96,072 Au/ml, sedangkan aktivitas spesifik bakteriosin sebesar 337,500 Au/ml dengan tingkat kemurnian 60,91 kali. Identifikasi isolat unggulan dinyatakan sebagai Pedioccocus accidilactici. Berat molekul bakteriosin yaitu 2.76 kDa. Bakteriosin aktif pada pH 2- 10; suhu 60-121oC; enzim pepsin, tripsin 0.1 mg/ml, papain (crude) 0.1 dan 1 mg/ml. Bakteriosin dapat menghambat bakteri patogen E coli, Staphylococcus aureus.

The use of synthetic preservative was aimed to inhibit food deterioration Therefore, it is necessary to look for natural and safe preservative. Bacteriocin is one of natural preservatives as a potential biopreservative agent. Asa an antibakteria. This research was aimed to isolate and select lactic acid bacteria (LAB), bacteriocin production optimization and bacteriocin purification characterization, and identifying selected isolate. Isolation of LAB was carry out from sea shrimp’s intestine. The selection was done based on qualitatively and quantitatively bacteriocin production. Selected isolate was optimized. Purification of bacteriocin with adsorption- desorption method then be continued purification step using chromatography column of ion exchange with DEAE-cellulose matrix.Characterization of purified bacteriocin included molecular weight, pH, temperature, proteolytic enzyme, pathogenic bacteria. The identification of selected isolate was based on characteristics of morphology, biochemical, physiological, and molecular (gene 16S rRNA). The result of isolation was 209 BAL isolates. Was fifty four among of them could produce bacteriocin. Based on the selections qualitatively and quantitatively, results found 8 selected isolates, one of them was SFE-7 (33) isolate which had bacteriocin activity of 72.415 Au/ml. Optimization of bacteriocin production with inoculums of 5% (v/v), source C of glucose of 20 g/l, yeast extract of source N of 20 g/l, pH of 5, temperature of 37˚C, agitation velocity of 100 rpm, and time of 15 hours. , resulted in the maximum µ growth kinetics of 0.065/h, (Yb/x) 0.4 [x10 6 AU (g CDM) -1 ], (Yx/s) 62.33 [g CDM (g sugar) -1 ], (Yb/s) 50 [x 10 6 AU (g sugar) -1 ]. Extraction of bacteriocin with adsorption-desorption method resulted specific activity was 312.773 Au/ml with the level of purity 56.45 times. The result of fractionation column DEAE-cellulose was obtained the highest bacteriocin activity at the 36 th fraction that was 96.072 Au/ml, while bacteriocin specific activity was by 337.500 Au/ml with the purity level of 60.91 times., bacteriocin has molecular weight of 2.76 kDa. Characterization of purified bacteriocin showed it’s action on pH of 2-10; temperature of 60-121˚ C; pepsin enzyme, trypsine of 0.1 mg/ml, papain (crude) of 0.1 and 1 mg/ml. Bacteriocin can inhibit the pathogenic bacteria, such as Escherici coli and Staphylococcus aureus. Identification of isolate SFE-7 (33) was identified as Pedioccocus accidilactici

Kata Kunci : udang, Pedioccocus Accidilactici, Purifikasi, bakteriosin