Isolasi, purifikasi, identifikasi, dan optimasi medium fermentasi antibiotik yang dihasilkan oleh aktinomisetes laut

Abstract

Isolation and purification of active compounds produced by marine actinomycetes has been carried out. Marine sediment samples were obtained from 3 different places in Banten West Coast, Cirebon North Coast, and Yogyakarta South Coasts. A total of 40 actinomycetes isolates were obtained 4 isolates were active against Escherichia coli ATCC 25922, 5 isolates were active against Staphylococcus aureus ATCC25923, 4 isolates were active against Bacillus subtilis ATCC 66923, 4 isolates were active against Pseudomonas aeroginosa ATCC27853, 4 isolates were active against Candida albican BIOMCC00122, and 4 isolates were active against Aspergillus niger BIOMCC00134. A11 isolate showed the most active to Gram-positive and Gram-negative bacteria. Species identification using 16S rRNA gene sequencing showed that A11 isolate is Streptomyces sp. Elucidation of its molecular formula and structure using LC-MS, 1H NMR, 13C NMR, and 13C DEPT NMR showed the antibiotic was cyclo(tyrosyl-prolyl), molecule formula was C14H16N2O3 which has a melting point of 140 °C. Minimum Inhibitory Concentration (MIC) of the antibiotic was determined against 4 bacterial test strains, namely Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 66923, which were inhibited at 27, 69, 80, and 74 μg mL-1, respectively. Fermentation profile of Streptomyces sp. A11 showed a lag phase which occurred until 8 hours, a log phase from 9 until 48 hours and a stationary phase from 48 until 144 hours. The growth phase showed maximum specific growth rate (μ) of 0.04 hourand the rate of substrate conversion into biomass (Y) of 0.6 max-1 x/sgram biomass per gram substrate. The optimum temperature and pH of cyclo(tyrosyl-prolyl) fermentation were 30 °C and 6.5-7.5, respectively. Optimum composition of fermentation medium was determined with three independent variables: dextrin as a carbon source, peptone as nitrogen source, and a mixture of mineral salts using Response Surface Methodology. The results showed that the three variables significantly affected the activity of cyclo(tyrosyl-prolyl). Peptone gave the strongest effect compared to dextrin and mineral salts. Interaction was found between dextrin and peptone. On the contrary, no interaction was observed between peptone and mineral salts, and between dextrin and mineral salts. Using a mathematical model, the most optimum composition of the medium were found to be dextrin (32.55 g L), peptone (11.22 g L), and -1-1mineral salt (8.65 mL), in which 51.54 g L cyclo(tyrosyl-prolyl) was produced. -1Verification of the model in laboratory showed the cyclo(tyrosyl-prolyl) activity to be 50.04 mg L. Thus, the difference between the result of the experiment and -1the expected response value was 2.9%.

Kebutuhan antibiotik, anti fungal, maupun anti kanker baru masih sangat diperlukan, terutama yang efektif melawan bakteri resisten, virus, protozoa, fungi atau kanker. Untuk mendapatkan antibiotik baru, para peneliti telah banyak melakukan berbagai cara seperti eksplorasi senyawa aktif dari mikroba, tumbuhan, maupun sintesis secara kimia. Salah satu mikroba yang banyak diteliti untuk diambil senyawa aktifnya adalah aktinomisetes. Indonesia merupakan negara kepulauan yang memiliki bentangan laut yang luas, kurang lebih 3,1 juta km2 atau hampir 2 kali lipat dibandingkan luas daratannya. Karakteristik laut yang bermacam-macam mengindikasikan biodiversitas hayati yang besar, khususnya biodiversitas mikroba laut. Namun demikian potensi ini belum banyak dimanfaatkan. Penelitian ini bertujuan untuk mendapatkan senyawa aktif yang berpotensi sebagai antibiotik dan memproduksinya dalam skala laboratorium yang dihasilkan oleh aktinomisetes laut melalui isolasi, penapisan, pemurnian, identifikasi dan optimasi medium fermentasi.