KEMAMPUAN ISOLAT GABUNGAN ASAL SPONS DAN MANGROVE DALAM MENGHASILKAN SENYAWA ANTIVIBRIO

IFHAN DWINHOVEN, Dr. Ir. Triyanto, M.Si; Noer Kasanah, Apt., M.Si, Ph.D.

2019 | Tesis | MAGISTER BIOTEKNOLOGI

Vibriosis merupakan masalah utama dalam akuakultur yang menyebabkan kematian ikan dalam jumlah yang besar dan waktu yang cepat. Pengobatan vibriosis masih terbatas pada antibiotik yang sama pada manusia dan hewan. Untuk itu diperlukan senyawa antibakteri baru yang lebih spesifik dan selektif dengan eksplorasi sumber daya laut. Penelitian ini bertujuan untuk mendapatkan isolat bakteri gabungan asal spons dan sedimen mangrove yang bersifat antivibrio serta mengetahui profil senyawa bioaktif antivibrio tersebut. Isolat bakteri gabungan didapatkan dari sedimen mangrove berasal dari Situbondo, Cilacap, Rembang, dan Palembang dan ekstrak spons berasal dari perairan Manado. Semua isolat dikultur pada media NB. Supernatan hasil fermentasi metabolit sekunder dari isolat bakteri gabungan pada berbagai media dilakukan uji bioassay dengan 96 well plate terhadap Vibrio alginolyticus, Vibrio harveyi dan Vibrio parahaemolyticus. Deteksi senyawa bioaktif dengan metode kromatografi lapis tipis (KLT). Produk fermentasi metabolit sekunder dari isolat bakteri gabungan terpilih diekstraksi menggunakan etil asetat dan dilakukan uji Minimum Inhibitor Concentration (MIC), bioautografi dan analisis H-NMR. Hasil skrining antivibrio diperoleh semua sampel memiliki aktivitas terhadap bakteri uji dan hasil pada KLT menunjukkan bahwa isolat bakteri gabungan bakteri CIL-10-2016-01/02 dan TPMD-13-01/02 memiliki kandungan senyawa peptida dengan perubahan warna lembayung setelah pewarnaaan menggunakan ninhidrin. Nilai MIC pada isolat bakteri gabungan bakteri CIL-10-2016-01/02 dapat menghambat pertumbuhan V. parahaemolyticus pada konsentrasi 2,5 mikroliter sedangkan isolat bakteri gabungan bakteri TPMD-13-01/02 pada konsentrasi 10 mikroliter. Hasil bioautografi menunjukkan bahwa isolat bakteri gabungan bakteri CIL-10-2016-01/02 aktif melawan bakteri V. harveyi dan V. parahaemolyticus pada nilai Rf 0.16 sedangkan isolat bakteri gabungan bakteri TPMD-13-01/02 pada nilai Rf 0.57, yang merupakan senyawa peptida. Hasil analisis spektra H-NMR mengindikasikan terdapat senyawa peptida pada isolat bakteri gabungan bakteri CIL-10-2016-01/02 dan TPMD-13-01/02. Sehingga didapatkan bahwa isolat bakteri gabungan bakteri CIL-10-2016-01/02 dan TPMD-13-01/02 dapat menghasilkan senyawa peptida yang bersifat antivibrio.

Vibriosis is a major concern in aquaculture causing rapid large amounts of fish death and economic loose. Treatment of Vibriosis is limited to the use same antibiotics for human and animal. Thus, the new antibacterial compounds which are more specific and selective for Vibrio sp. are needed through the exploration of marine resources to discover the bioactive compounds. The aim of this study were to obtain bacterial combination isolates from sponges and mangrove sediments possessing bioactivity against Vibrio sp., and to determine the chemical profiles of antivibrio compounds. The mangrove sediment originated from the coastal of Situbondo, Cilacap, Rembang, and Palembang, while the sponges were collected from Manado. All of the isolates were cultured in NB medium. The secondary metabolites fermentation was carried out in various media, then the supernatants was tested for microtiter bioassay against V. alginolyticus, V. harveyi and V. parahaemolyticus in 96-well plate. Detection of bioactive compounds were done by Thin Layer Chromatography (TLC) with indicators of UV 254 nm and UV 366 nm, and TLC staining using ninhydrin, p-anisaldehyde, and H2SO4 reagents. The selected combination bacteria isolates were being extracted with ethyl acetate and further examined for the Minimum Inhibitor Concentration (MIC), bioautography assay and H-NMR analysis. The screening results showed that all the samples possessed bioactivity against Vibrio sp.. TLC results showed that the CIL-10-2016-01/02 and TPMD-13-01/02 were predicted to contain peptide compounds with pink discoloration on the TLC spot after dyeing with ninhydrin. Based on the MIC assay CIL-10-2016-01/02 was abled to inhibit the growth of V. parahaemolyticus at a concentration of 2.5 microliter, while the TPMD-13-01/02 was at a concentration of 10 microliter. The results of bioautography assay showed that CIL-10-2016-01/02 and TPMD-13-01/02 were active against V. harveyi and V. parahaemolyticus at Rf 0.16 and at Rf 0.57, respectively, which indicated that the compound is a peptide molecule. H-NMR spectra indicated that there were peptide compounds in the combination bacterial isolates CIL-10-2016-01/02 and TPMD-13-01/02. Thus , the results showed that the combination bacterial isolates of CIL-10-2016-01/02 and TPMD-13-01/02 was capable to produce antivibrio compound.

Kata Kunci : Antivibrio, Peptida, Senyawa Bioaktif, Vibrio