SKRINING DAN IDENTIFIKASI MOLEKULER BAKTERI ASAM LAKTAT PENGHASIL BAKTERIOSIN PADA BERBAGAI PRODUK FERMENTASI IKAN

HERU PRAMONO, Dr. Ir. Murwantoko, M.Si.

2013 | Tesis | S2 Bioteknologi

Tujuan penelitian ini adalah melakukan skrining dan identifikasi bakteri asam laktat pada produk fermentasi ikan tradisional Indonesia. Dua ratus bakteri asam laktat (BAL) yang telah diisolasi asal 26 sampel petis, terasi dan peda di skrining keberadaan bakteri penghasil bakteriosinnya dengan metode disc diffusion, agar spot test, agar well diffusion assay, dan critical dilution method terhadap bakteri indicator Pediococcus acidilactici LB 42. Tujuh puluh isolate menunjukkan aktivitas penghambatan ketika diuji dengan metode disc diffusion assay. Diantara 70 isolat, 15 isolat menunjukkan aktivitas penghambatan pada uji spot test dan 10 isolat diantara 15 isolat menunjukkan aktivitas penghambatan pada uji agar well diffusion assay. Aktivitas antimikrobia ke 10 isolat telah diuji dengan critical dilution method dan empat isolate menunjukkan aktivitas tertinggi (1.800 AU/ml), yaitu PDM20B12, PD5AM7 dan PD5M3 asal peda dan THL09 asal terasi. Seluruh bakteriosin yang dihasilkan oleh isolat tersebut menghambat Pseudomonas fluorescens namun Eschericia coli dan Staphylococcus aureus hanya terhambat lemah (< 2 mm). Seluruh bakteriosin tersebut stabil pada suhu tinggi (100 o C for 20 min), rentang pH luas (3-11) dan perlakuan tween 20, tween 80, dan kloroform. Seluruh bakteriosin tersebut kehilangan aktivitas ketika diuji menggunakan proteinase K, namun hanya bakteriosin PDM20B12 yang kehilangan aktivitas ketika diuji dengan pepsin, papain kasar, dan tripsin. Berdasarkan karakteristik yang diamati, bakteriosin yang diproduksi oleh isolat PDM20B12, PD5AM7, PD5M3, dan THL09 termasuk bakteriosin kelas II, bakteriosin berukuran kecil dan stabil pada suhu tinggi. Identifikasi menggunakan metode Bergey’s manual identification menunjukkan seluruh isolat penghasil bakteriosin tersebut termasuk dalam genus Pediococcus. Identifikasi molekuler dengan sekuensing 16S rDNA mengkonfirmasi bahwa isolat BAL tersebut merupakan Pediococcus acidilactici.

The objectives of this research were to screen and identify lactic acid bacteria from Indonesian traditional fermented fish. Two hundred lactic acid bacteria (LAB) isolated from 26 samples of petis, terasi and peda were screened for the presence of bacteriocin-producer strain using disc diffusion method, agar spot test, agar well diffusion assay, and critical dilution assay against Pediococcus acidilactici LB 42 as an indicator strain. Seventy isolates showed inhibitory activity when tested using disc diffusion assay. Among 70 isolates, 15 isolates showed inhibitory activity used agar spot test and 10 isolates from those 15 isolates showed inhibitory activity on agar well diffusion assay. The antibacteria activity from those 10 isolates were tested with critical dilution method and 4 isolates showed highest activity (1.800 AU/ml), i.e. PDM20B12, PD5AM7 and PD5M3 that were isolated from peda and THL09 isolated from terasi. All of bacteriocin produced by those four isolates inhibited Pseudomonas fluorescens but Eschericia coli and Staphylococcus aureus inhibited weak (< 2 mm). All of those four bacteriocins were stable at high temperature (100 o C for 20 min), wide range of pH (3-11) and treatment of tween 20, tween 80 and chloroform. All of bacteriocins lost their activity when tested using proteinase K, but only bacteriocin produced by isolate PDM20B12 that lost of activity in treatment of pepsin, crude papain, and trypsin. Based on observed characteristics, bacteriocins produced by isolates PDM20B12, PD5AM7, PD5M3, and THL09 belonged to class II bacteriocin, a low molecular weight and heat stable bacteriocin. Identification using Bergey’s manual identification method was resulted that all of isolates were genus Pediococcus. Molecular identification confirmed that LAB isolates were Pediococcus acidilactici.

Kata Kunci : Bakteri asam laktat, Fermentasi ikan, Identifikasi molekuler, Bakteriosin, Pseudomonas fluorescence, Pediococcus acidilactici