KEANEKARAGAMAN BAKTERI ASAM LAKTAT PENGHASIL ANTIMIKROBIA SELAMA PROSES FERMENTASI BAKASANG

Helen Joan Lawalata,SPi.,MSi., Drs. Langkah Sembiring, M.Sc.,Ph.D.

2013 | Disertasi | S3 Biologi

Escherichia coli Penelitian mengenai keanekaragaman bakteri asam laktat penghasil antimikrobia selama proses fermentasi bakasang secara tradisional telah dilakukan. Bakteri penghasil asam laktat telah diisolasi dengan menggunakan media MRS yang ditambahkan dengan CaCO3. Sebanyak 200 isolat bakteri penghasil asam yang ditandai dengan terbentuknya zona jernih telah berhasil diisolasi dari sampel bakasang dan berdasarkan hasil uji konfirmasi sebagai bakteri asam laktat diperoleh 125 isolat bakteri asam laktat yang diseleksi berdasarkan kemampuan penghambatan oleh kultur sel dan supernatan isolat BAL terhadap pertumbuhan bakteri ATCC 35218, Staphylococcus aureus ATCC 25923 dan Pseudomonas fluorescens FNCC 0070 (bakteri patogenik, bakteri pembusuk, dan bakteri penghasil bioamin) dengan menggunakan metode difusi sumuran (Well difusion). Keseratus dua puluh lima isolat BAL memiliki kemampuan antagonistik terhadap pertumbuhan bakteri uji. Pada seleksi terhadap isolat BAL yang memiliki kemampuan antagonistik tertinggi terhadap pertumbuhan bakteri uji diperoleh 4 isolat BAL penghasil antimikrobia yang memiliki diameter penghambatan terbesar (15 mm) yaitu isolat BksC24, isolat BksJ21, isolat BksJ43, dan isolat BksK25. Karakterisasi dan identifikasi telah dilakukan terhadap keempat isolat terpilih dengan menggunakan pendekatan sistematik polifasik yaitu dengan menerapkan analisis sistematik numerik-fenetik, sistematik kimiawi (profil protein total sel dengan SDS-PAGE) dan sistematik molekular-fenetik (ARDRA) serta sistematik molekular filogenetik berdasarkan analisis sequence gen 16S rRNA. Hasil karakterisasi dan identifikasi menunjukkan bahwa isolat uji BksC24, isolat BksJ21, isolat BksJ43, dan isolat BksK25 diidentifikasi sebagai anggota spesies Pediococcus acidilactici karena sangat mirip dan berkerabat dekat dengan species Pediococcus acidilactici DSM 20284 T . Namun demikian, berdasarkan jumlah nukleotida yang berbeda diantara isolat uji dengan 17 strain acuan (anggota genus Pediococcus) menunjukkan bahwa isolat BksJ21 dan isolat BksK25 diduga kuat merupakan species baru (novel species) dalam genus Pediococcus sedangkan isolat BksC24 dan isolat BksJ43 diduga kuat merupakan strain baru (novel strain) anggota spesies P. acidilactici DSM 20284T. Hasil penelitian menunjukan bahwa keanekaragaman bakteri asam laktat penghasil antimikrobia yang diisolasi selama proses fermentasi bakasang dapat diungkapkan dengan menggunakan analisis sistematik polifasik. Dengan demikian, penelitian ini berhasil memperoleh isolat bakteri asam laktat penghasil senyawa antimikrobia yang diduga sebagai spesies baru dan strain baru yang berpotensi untuk dimanfaatkan sebagai kultur starter yang dapat digunakan sebagai bahan pengawet alami bahan makanan yang membuat makanan fermentasi tradisional menjadi makanan kesehatan (fungsional)

Escherichia coli The study of lactic acid bacteria diversity which produce antimicrobial agent during fermentation process of bakasang traditionally made in Manado, North Sulawesi has been conducted. Acid-producing bacteria were isolated using MRS media supplemented with CaCO3. As many as 200 isolates of acidproducing bacteria were isolated from samples bakasang and based on test results as a confirmation of lactic acid bacteria isolates obtained 125 will be selected based on the inhibition against growth of ATCC 35218, Staphylococcus aureus ATCC 25923 dan Pseudomonas fluorescens FNCC 0070 (pathogenic bacteria, spoilage bacteria and bioamine producing bacteria) using well diffusion methods. Hundred twenty-five isolates BAL has the ability antagonistic to the growth of test bacteria. Selection of the BAL isolates that have the highest antagonistic ability against growth of bacteria tests showed that four isolate BAL-producing antimicrobial were obtained which having the largest diameter of inhibition (15 mm), namely BksC24 isolates, BksJ21 isolates, BksJ43 isolates, and BksK25 isolates. Characterization and identification performed on the four strains selected were done by systematic polyphasic approach, namely numerical-phenetic systematics, chemosystematics (whole cell protein profiles by SDS-PAGE) and molecular systematics (fingerprinting ARDRA and molecular phylogenetic based on analysis 16S rRNA gene sequence. The results showed that the characterization and identification showed that the isolate BksC24, isolate BksJ21, isolate BksJ43 and isolate BksK25 was very similar and most closely related to the species of Pediococcus acidilactici DSM 20284T. However, results of 16S rRNA gene sequence analysis of the selected isolate on the basis of nucleotide differences along with 17 reference strains (members of the Pediococcus genera) showed that isolate BksJ21 and BksK25 were very likely to be a new species belonged to Pediococcus genus and isolate BksC24 and isolate BksJ43 were very likely to be a new strain member of P. acidilactici species. The results showed that identity, diversity and novelty status of the four isolate of lactic acid bacteria producing antimicrobial isolated from bakasang could be unravelled by approach of polyphasic systematic. Therefore, this study was succesfull to obtain the isolates lactic acid bacteria producing antimicrobial which very likely to be new species and new strains that potential to be used as starter cultures which could be used as natural preservative ingredients that make food of traditional fermented into healthy foods.

Kata Kunci : BAL, antimikrobia, bakasang, gen 16S rRNA, ARDRA