Isolasi dan Identifikasi Molekuler Bakteri Asam Laktat dari Larva Black Soldier Fly (Hermetia illucens) pada Media yang Berbeda

Abstract

Usage of Antibiotic Growth Promoters (AGP) in livestock is banned due to the negative impact on the livestock productivity and consumers. Probiotic is an alternative for AGP providing microorganisms that give a benefit for health of the host. This study aims to isolate and molecularly identify the Lactic acid bacteria (LAB) from digestive tract of Black Soldier Fly (BSF) larvae in broiler manure and palm kernel meal as substrates, as probiotic candidates, using 16S ribosomal RNA (rRNA) gene with Polymerase Chain Reaction (PCR) technique. The active carbohydrate enzyme and types of secondary metabolites are predicted. The LAB isolates were morphologically identified including color and diameter of colony. Probiotic potency is observed by using antimicrobial test against pathogenic bacteria (Escherichia coli and Salmonella typhimurium), and hemolysis activity. Three selected isolates were molecularly identified followed by construction of phylogenic tree. Identification of predicted carbohydrate enzymes and secondary metabolite types was analyzed from the closer relationship that has a complete genome. The pH value and result of antimicrobial test against pathogenic bacteria were analyzed by T-test using SPSS 25.0. Five isolates from BSF larvae reared on chicken manure substrate and three isolates from BSF larvae reared on palm kernel meal substrate were identified as gram-positive bacteria. Those had the pH value around 4.34 – 4.6 and 4.1 – 4.4, respectively. Isolates of LAB from BSF larvae reared on chicken manure and palm kernel meal substrates had inhibition zones against pathogenic bacteria (E. coli and S. typhimurium) which were not significantly different. The inhibition zone of LAB isolates from BSF larvae reared on chicken manure and palm kernel meal substrates against E. coli are categorized as strong and medium, respectively. Category of inhibition zone of all LAB isolates from BSF larvae reared on different substrates against S. typhimurium are medium. All isolates showed no hemolysis activity. By using 16S rRNA gene sequencing 3 isolates (A3, A4, and B1) is Enterococcus faecalis with their percentage of similarities, are 99.90%, 99.79%, and 100%. Isolate A4 is predicted to have active enzyme groups including Glycoside Hydrolase, while isolate B1 have grouped active enzymes including Glycoside Hydrolase, GlycosylTransferase, and Carbohydrate Esterase. The species have secondary metabolite types including Lanthipeptide class II and Cyclic lactone autoinducer. In conclusion, three isolates of which the isolation result of digestive tract from The isolate A3, A4, and B1 are potentially used as probiotics.

Penggunaaan Antibiotic Growth Promoters (AGP) di bidang peternakan dilarang dikarenakan adanya dampak negatif yang menyebabkan penurunan produktivitas ternak dan berpengaruh pada konsumen yang mengonsumsinya, sehingga diperlukan adanya alternatif sebagai pengganti AGP. Probiotik merupakan salah satu alternatif AGP dengan pemberian mikroorganisme hidup yang dapat menguntungkan bagi kesehatan inang. Penelitian ini bertujuan mengisolasi dan mengidentifikasi Bakteri Asam Laktat (BAL) dari saluran pencernaan larva Black Soldier Fly (BSF) yang dipelihara dari media feses ayam broiler dan bungkil sawit sebagai kandidat probiotik, serta mengidentifikasi molekuler isolat BAL menggunakan primer gen 16S ribosomal RNA (rRNA) dengan teknik Polymerase Chain Reaction (PCR). Penelitian ini juga bertujuan mengidentifikasi prediksi enzim aktif karbohidrat dan senyawa metabolit sekuender melalui pendekatan kekerabatan berdasarkan urutan nukleotida. Isolat BAL diidentifikasi secara morfologi yaitu warna dan diameter koloni. Potensi probiotik diamati dari uji antimikrobial BAL terhadap bakteri patogen yaitu Eschericia coli dan Salmonella typhimurium, serta uji aktivitas hemolisis BAL. Setelah itu, isolat terpilih diidentifikasi molekuler dan mendapatkan hasil urutan gen dan diidentifikasi homologi sekuens di Genbank, hasil identifikasi digambarkan menjadi pohon filogenetik. Identifikasi molekuler dilanjutkan dengan identifikasi prediksi enzim karbohidrat melalui DataBase for automated Carbohydrate-active enzyme Annotation (dbCAN) dan senyawa metabolit sekunder melalui Antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) dari hasil kekerabatan yang memiliki complete genome. Analisis data dilakukan pada parameter pH dan aktivitas antimikroba BAL terhadap bakteri patogen dengan uji T menggunakan aplikasi SPSS versi 25.0, untuk menganalisis perbedaan nyata isolat BAL yang berasal dari larva BSF media feses ayam dengan media bungkil sawit. Hasil penelitian didapat isolat BAL terpilih asal larva BSF media feses ayam broiler dan bungkil sawit masing-masing sebanyak 5 dan 3 isolat. Isolat BAL asal larva BSF media feses ayam memiliki pH medium berkisar 4,34 – 4,6 dan isolat BAL asal larva BSF media bungkil sawit berkisar 4,1 – 4,4. Isolat BAL asal larva BSF media feses ayam maupun bungkil sawit memiliki zona hambat terhadap Eschericia coli dan Salmonella typhimurium yang tidak berbeda nyata. Kategori zona hambat isolat BAL asal larva BSF media feses ayam dan bungkil sawit terhadap E coli yaitu kuat dan sedang. Kategori hambatan isolat BAL asal larva BSF media feses ayam maupun bungkil sawit terhadap S. typhimurium yaitu sedang. Isolat BAL asal larva BSF dengan media yang berbeda tidak memiliki aktivitas hemolisis. Ketiga isolat yang berpotensi sebagai kandidat probiotik (A3, A4, dan B1) teridentifikasi kemiripannya dengan spesies Enterococcus faecalis dengan persentase kemiripan masing-masing 99,90%, 99,79%, dan 100%. Isolat A4 teridentifikasi secara molekuler memiliki kelompok enzim karbohidrat Glycoside Hydrolase dan isolat B1 teridentifikasi memiliki kelompok enzim karbohidrat Glycoside Hydrolase, GlycosylTransferase, dan Carbohydrate Esterase. Isolat A4 asal larva BSF media feses ayam teridentifikasi memiliki senyawa metabolit sekunder Lanthipeptide class II dan isolat B1 asal larva BSF media bungkil sawit teridentifikasi memiliki senyawa metabolit sekunder Cyclic lactone autoinducer.