TRI RATNA SULISTIYAN
SEKOLAH PASCASARJANA INSTITUT PERTANIAN BOGOR
BOGOR 2014
menghasilkan senyawa antimikrob dan bersifat toksik terhadap embrio ikan zebra berpotensi sebagai penghasil kandidat obat.
Saran
Kajian tentang keragaman bakteri endofit yang tidak dapat dikulturkan (unculturable) dari tanaman kunyit putih perlu dilakukan untuk melengkapi informasi tentang keragaman bakteri endofit dari tanaman kunyit putih. Langkah lebih lanjut perlu dilakukan untuk mengetahui lebih dalam peran bakteri endofit tanaman kunyit putih dalam menghasilkan senyawa aktif. Hal ini dapat dilakukan dengan membandingkan aktivitas senyawa aktif yang diperoleh dari bakteri endofit tanaman kunyit putih terpilih, tanaman kunyit putih bebas endofit yang diperoleh dari kultur jaringan tanaman dan tanaman kunyit putih asli yang diperoleh dari alam. Selain itu perlu dilakukan uji lanjut tentang senyawa yang diduga antikanker dari empat bakteri endofit terpilih dari tanaman kunyi putih.
6 DAFTAR PUSTAKA
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Lampiran 1 Komposisi media
Media Nutrient Agar (NA) dalam setiap liter media Yeast extract 3.0 g
Pepton 5.0 g
Agar 18.0 g
Media Water-Yeast Extract Agar (WYE) dalam setiap liter media Yeast extract 0.3 g
K2HPO4 0.5 g
Lampiran 2 Skema uji toksisitas akut ekstrak terhadap embrio ikan zebra
Embio ikan zebra
Telur terfertilisasi
Pre-conditioning
Penambahan ekstrak Seleksi telur
Lampiran 3 Karakteristik koloni bakteri endofit asal tanaman kunyit putih Kode
isolate Uji
KOH Bentuk Tepian Elevasi Permukaan Warna
DB.S.1 + Wrinkled Undulate Umbonate Dull/opaque Kuning terang DB.S.2 + Circular Entire Convex Shiny/opaque Kuning DB.S.3 - Irregular Entire Raised Shiny/translucent Putih DB.S.4 - Circular Entire Convex Shiny/opaque Kuning DB.P.1 - Irregular Undulate Convex Shiny/opaque Putih
DB.P.2 - Irregular Undulate Convex Shiny/opaque Orange terang BB.P.1 + filamentous Serated Flat Dull/translucent Putih BB.P.3 - Circular Entire Convex Shiny/opaque Putih
BB.P.4 - Irregular Erose Flat Shiny/translucent Putih kekuningan BB.S.1 + Irregular Entire Raised Shiny/opaque Kuning
BB.S.3 + Circular Undulate Convex Shiny/opaque Kuning BB.S.5 + Irregular Entire Convex Shiny/opaque Kuning terang BB.S.6 + Irregular Entire Convex Shiny/opaque Orange BB.S.7 + Circular Entire Pulvinate Shiny/opaque Kuning tua BB.S.8 + Circular Entire Flat Shiny/translucent Putih transparent RB.P.1 + Irregular Undulate Flat Dull/translucent Putih
RB.P.2 - Irregular Undulate Raised Shiny/opaque Putih
RB.P.3 - Circular Entire Flat Shiny/translucent Putih transparent RB.P.4 + Wrinkled Undulate Umbonate Dull/opaque Kuning terang RB.S.2 - Irreegular Entire Umbonate Shiny/opaque Kuning RB.S.3 - Irregular Undulate Raised Shiny/opaque Putih
RB.S.4 + Irregular undulate Convex Shiny/opaque Kuning terang RB.S.5 - Circular Entire Raised Shiny/opaque Kecoklatan DL.P.1 + Circular Entire Pulvinate Shiny/opaque Orange DL.P.2 - Circular Entire Convex Shiny/opaque Kecoklatan DL.P.3 - Circular Entire Flat Shiny/translucent Putih transparent DL.P.4 - Irregular Undulate Convex Shiny/translucent Putih transparent DL.P.5 + Circular Undulate Raised Shiny/opaque Putih keruh DL.P.6 + Circular Erose Raised Shiny/opaque Putih keruh DL.S.1 - Puntiform Entire Pulvinate Shiny/opaque Merah
RL.P.1 - Circular Entire Convex Shiny/opaque Putih kecoklatan RL.P.4 - Puntiform Entire Pulvinate Shiny/opaque Kuning keorange RL.S.1 + Circular Undulate Raised Shiny/opaque Putih keruh RL.S.2 - Circular Entire Convex Shiny/opaque Kekuningan RL.S.3 + Puntiform Entire Convex Shiny/opaque Merah muda BL.P.1 - Circular Entire Raised Shony/opaque Kecoklatan BL.P.2 + Circular Erose Raised Shiny/opaque Putih keruh BL.S.1 - Irregular Erose Umbonate Shiny/translucent Putih terang BL.S.2 + Irregular Entire Convex Shiny/opaque Orange
DI.P.1 - Irregular Undulate Convex Shiny/translucent Putih transparent DI.P.2 + Circular Undulate Convex Shiny/dull Kecoklatan DI.P.3 - Irregular Entire Convex Shiny/dull Putih susu DI.P.4 - Circular Undulate Umbonate Shiny/dull Putih susu DI.S.1 - Circular Undulate Raised Shiny/translucent Bening DI.S.2 + Puntiform Entire Pulvinate Dull/opaque Kuning DI.S.3 + Circular Entire Umbonate Shiny/dull Orange DI.S.4 + Circular Entire Convex Shiny/dull Kuning DI.S.5 - Circular Undulate Raised Shiny/dull Kekuningan RI.P.1 - Circular Entire Pulvinate Shiny/opaque Kehijauan RI.P.2 - Circular Entire Raised Shiny/translucent Kekuningan RI.P.3 - Circular Entire Convex Shiny/opaque Kuning RI.P.4 + Circular Entire Raised Shiny/opaque Kuning keruh RI.P.5 + Irregular Lobat Flat Dull/opaque Putih RI.P.6 - Circular Undulate Convex Shiny/opaque Kekuningan
Lampiran 3 Karakteristik koloni bakteri endofit asal tanaman kunyit putih (Lanjutan)
Kode isolate
Uji
KOH Bentuk Tepian Elevasi Permukaan Warna
RI.P.7 - Circular Entire Raised Shony/opaque Kecoklatan RI.P.8 - Circular Lobat Convex Shiny/opaque Kekuningan RI.S.2 - Irregular Undulate Convex Shiny/opaque Putih keruh RI.S.3 + Circular Entire Pulvinate Shiny/opaque Putih RI.S.4
- Irregular Undulate Flat Shiny/translucent Bening tak berwarna RI.S.5 - Circular Entire Raised Shiny/opaque Putih
RI.S.6 - Irregular Undulate Raised Shiny/translucent Putih kekuningan RI.S.7 - Circular Undulate Convex Shiny/opaque Kuning terang RI.S.8 - Circular Entire Pulvinate Shiny/opaque Putih kecoklatan BI.P.1 + Puntiform Entire Pulvinate Dull/opaque Kuning
BI.P.2 - Circular Entire Convex Shiny/opaque Kuning keputihan BI.P.3 - Circular Entire Raised Shiny/opaque Putih terang BI.P.4 + Circular Entire Pulvinate Shiny/opaque Putih keruh BI.P.6 + Circular Entire Raised Shiny/opaque Kuning keruh BI.P.7 + Irregular Erose Raised Shiny/opaque Putih susu BI.S.1 - Circular Undulate Raised Shiny/opaque Kuning terang BI.S.2 - Irregular Undulate Pulvinate Shiny/opaque Putih
BI.S.3 - Circular Entire Raised Shiny/opaque Putih terang BI.S.4 - Irregular Undulate Pulvinate Shiny/opaque Putih
Lampiran 4 Hasil uji toksisitas embrio ikan zebra ekstrak bakteri C. freundii (BI.S.3)
Konsentrasi (ppm)
Fase ekstrak etil asetat Fase air
Rerata % kematian Rerata % kematian
0 0.0 0.0 0.7 13.4 50 2.0 40.0 1.0 20.0 100 3.3 66.6 2.3 46.6 150 4.3 86.6 2.7 53.4 200 4.7 93.4 3.3 66.6 250 5.0 100.0 5.0 100.0 Contoh perhitungan LC 50: y = 87.74x - 108,2 50 = 87.74x - 108,2 50 + 108.2 = 87.74x x = 158.2/87.74 x = 1.803 LC50 = 101.803 LC50 = 63.5 ppm
Fase ekstrak etil asetat bakteri C. freundii (BI.S.3)
Lampiran 5 Hasil uji toksisitas embrio ikan zebra ekstrak bakteri B. subtilis (RB.P.1)
Konsentrasi (ppm)
Fase ekstrak etil asetat Fase air
Rerata % kematian Rerata % kematian
0 0.0 0.0 0.7 13.4 50 3.3 66.6 1.7 33.4 100 3.7 73.4 2.0 40.0 150 4.0 80.0 2.0 40.0 200 4.3 86.6 2.3 46.6 250 5.0 100.0 2.7 53.4 Contoh perhitungan LC 50: y = 43.39x - 10.45 50 = 43.39x - 10.45 50 + 10.45 = 43.39x x = 60.45/43.39 x = 1.393 LC50 = 101.393 LC50 = 24.7 ppm
Fase ekstrak etil asetat bakteri B. subtilis (RB.P.1)
Lampiran 6 Hasil uji toksisitas embrio ikan zebra ekstrak bakteri P. otitidis (BL.S.1)
Konsentrasi (ppm)
Fase ekstrak etil asetat Fase air
Rerata % kematian Rerata % kematian
0 0.0 0.0 0.7 13.4 50 3.7 73.4 0.7 13.4 100 4.0 80.0 1.3 26.6 150 4.3 86.6 2.0 40.0 200 4.7 93.4 3.3 66.6 250 5.0 100.0 5.0 100.0 Contoh perhitungan LC 50: y = 37.11x + 8.197 50 = 37.11x + 8.197 50 - 8.197 = 37.11x x = 41.803/37.11 x = 1.126 LC50 = 101.126 LC50 = 13.4 ppm
Fase ekstrak etil asetat bakteri P. otitidis (BL.S.1)
Lampiran 7 Hasil uji toksisitas embrio ikan zebra ekstrak bakteri B. cenocepacia (RI.P.1)
Konsentrasi (ppm)
Fase ekstrak etil asetat Fase air
Rerata % kematian Rerata % kematian
0 0.0 0.0 0.7 13.4 50 3.7 73.4 0.0 0.0 100 4.3 86.6 2.0 40.0 150 5.0 100.0 2.7 53.4 200 5.0 100.0 4.0 80.0 250 5.0 100.0 5.0 100.0 Contoh perhitungan LC 50: y = 41.34x + 4.554 50 = 41.34x + 4.554 50 - 4.554 = 41.34x x = 45.446/41.34 x = 1.099 LC50 = 101.099 LC50 = 12.6 ppm
Fase ekstrak etil asetat bakteri B. cenocepacia (RI.P.1)
TRI RATNA SULISTIYANI. Keragaman Bakteri Endofit Tanaman Kunyit Putih (Curcuma zedoaria) dan Toksisitasnya terhadap Embrio Ikan Zebra. Dibimbing oleh YULIN LESTARI dan PUSPITA LISDIYANTI.
Kunyit putih merupakan tanaman herbal yang secara tradisional banyak digunakan untuk pengobatan penyakit yang berhubungan dengan kanker. Keragaman dan potensi bakteri endofit dalam tanaman kunyit putih perlu dikaji. Penelitian ini bertujuan untuk mengkaji keragaman bakteri endofit yang berasosiasi dengan tanaman kunyit putih (Curcuma zedoaria) dan toksisitasnya terhadap embrio ikan zebra.
Tanaman kunyit putih diambil dari tiga lokasi yang berbeda di Bogor, Jawa Barat, Indonesia. Isolasi bakteri endofit dilakukan menggunakan empat media yang berbeda (Nutrient Agar (NA), NA yang mengandung ekstrak kunyit putih (NAT), Water Yeast Extract Agar (WYEA), WYEA yang mengandung ekstrak kunyit putih), dan dua metode isolasi yaitu metode spread plate dan plant piece. Populasi bakteri endofit dihitung dengan menumbuhkan bakteri pada 4 jenis media yang berbeda dan identifikasi isolat terseleksi menggunakan analisis molekuler berdasarkan 16S rDNA. Uji aktivitas antimikrob pada bakteri terseleksi dilakukan terhadap enam mikrob uji yaitu Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, dan Sacharomyces cerevisiae. Empat bakteri endofit penghasil senyawa antimikrob selanjutnya dilihat potensinya dalam menghasilkan senyawa toksik melalui uji toksisitas terhadap embrio ikan zebra.
Sebanyak 207 isolat bakteri endofit telah berhasil diisolasi dari bagian akar, batang dan daun tanaman kunyit putih, dan 73 isolat yang berbeda diperoleh berdasarkan perbedaan karakteristik morfologi. Tiga puluh dua persen isolat diperoleh dari kebun Bojong Gede, 22% isolat dari kebun percobaan Pusat Penelitian Biologi LIPI Cibinong dan 46% isolat berasal dari kebun koleksi tanaman obat Pusat Penelitian Biofarmaka IPB Dramaga. Bakteri endofit didominasi oleh bakteri dari rimpang yaitu sebanyak 38%, dari batang sebanyak 32%, dan dari daun sebanyak 30%. Berdasarkan hasil analisis sekuen 16S rDNA, bakteri yang diperoleh termasuk dalam kluster Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, dan Actinobacteria, dengan 23 genus yang berbeda yaitu Stenothropomonas, Pseudomonas, Enterobacter, Providencia, Klebsiella, Dickeya, Pantoea, Bacillus, Acinetobacter, Citobacter, Mycobacterium, Cellulomonas, Microbacterium, Methylobacterium, Penylobacterium, Roseomonas, Agrobacterium, Bosea, Xanthobacter, Rhizobium, Burkholderia, Ralstonia, and Alcaligenes. Bakteri dari kluster Gammaproteobacteria mendominasi semua sampel kunyit putih dan bakteri genus Pseudomonas, Klebsiella, Bacillus, Enterobacter, Microbacterium ditemukan dari semua bagian tanaman sampel.
Berdasarkan hasil uji antimikrob, enam belas isolat dari 73 bakteri endofit