Identifikasi dan karakterisasi isolat Ligilactobacillus ruminis BR2-12 dari feses bayi sebagai probiotik

Lorentz, Rachel Theoni (2023) Identifikasi dan karakterisasi isolat Ligilactobacillus ruminis BR2-12 dari feses bayi sebagai probiotik. Bachelor thesis, Universitas Pelita Harapan.

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Abstract

Berbagai Lactobacillus spp. banyak dimanfaati sebagai probiotik. Salah satu sumber isolasi Lactobacillus spp. adalah feses bayi. Penelitian mengenai Lactobacillus spp. pada feses bayi di Indonesia dengan evaluasi potensinya sebagai probiotik masih minim. Oleh karena itu, penelitian ini bertujuan untuk mengidentifikasi dan mengkarakterisasi isolat BR2-12 yang sebelumnya dari feses dan akan dianalisis baik genus dan spesies yang mungkin kandidat probiotik. Metode penelitian ini mencakup, yaitu remajaan dan purifikasi BR2-12; identifikasi secara morfologi koloni dan morfologi sel dengan pewarnaan Gram dan endospore; kemudian, identifikasi molekuler sekuens 16S-rRNA dengan tahapan, ekstraksi DNA, amplifikasi PCR; kurva pertumbuhan BR2-12 dilakukan pada media MRS selama 48 jam dengan enumerasi (CFU); setelah itu, dilakukan uji biokimia, aktivitas katalase (H2O2), dan kemampuan fermentasi terhadap berbagai gula; dan yang terakhir adalah analisis karakterisasi dasar probiotik dilakukan terhadap fenol (0,2% dan 0,5% selama 24 jam), NaCl (1,5%, 2,5% dan 3,5% selama 24 jam), gastric juice pada pH 2 (30, 60 dan 120 menit) dan garam empedu (0,3% dan 0,5% selama 0, 1, 2, dan 3 jam). Hasil percobaan menunjukkan morfologi koloni seperti berukuran kecil berwarna putih, tekstur smooth, elevasi convex, bentuk bulat dan margin entire, lalu morfologi sel bersifat Gram-positif, endospora negative dan memiliki morfologi basil memanjang; analisis sekuens 16S-rRNA BR2-12 menunjukkan isolat BR2-12 identik dengan Ligilactobacillus ruminis; hasil kurva pertumbuhan yang dilakukan pada media MRS menunjukkan fase eskponensial pada jam ke 24; uji aktivitas katalase menunjukkan hasil negatif dan uji fermentasi gula terhadap sukrosa, raffinose dan selobiosa menunjukkan positif dan sesuai dengan kemampuan fermentasi Lgb. ruminis; analisis karakterisasi dasar probiotik, terhadap ketahanan fenol pada konsentrasi 0,2% dan 0,5% memiliki survival rate masing-masing 93% dan 13% selama 24 jam, ketahanan terhadap NaCl pada konsentrasi 1,5%, 2,5% dan 3,5% memiliki survival rate masing-masing 89%, 81% dan 50% selama 24 jam, pada uji larutan gastric juice pH 2 pada 0, 30, 60 dan 120 menit memiliki nilai viabilitas (CFU, 10-6) masing-masing adalah 3,06, 3,75, 4,72 dan 4,11, pada uji garam empedu konsentrasi 0,3% dan 0,5% pada jam ke 0, 1, 2, dan 3 jam diperoleh nilai viabilitas masing-masing 4,30, 3,83, 3,45 dan 3,11, dan 4,75, 4,40, 3,97 dan 3,53. Dari hasil percobaan menunjukkan Ligilactobacillus ruminis BR2-12 selanjutnya memiliki potensi dikembangan sebagai probiotik./Lactobacillus spp. are widely utilized as probiotics. One of the sources of isolating Lactobacillus spp. is infant feces. Research on Lactobacillus spp. in infant feces in Indonesia limited. Therefore, this study aims to identify and characterize the isolate BR2-12 previously obtained from infant feces and assess potential as a probiotic. The research methods included culturing and purifying BR2-12; morphological identification of colonies and cells using Gram staining and endospore staining; molecular identification through 16S-rRNA sequencing, which involved DNA extraction and PCR amplification; the growth curve of BR2-12 was determined on MRS media for 48 hours with the colony-forming units (CFU) method; then, biochemical tests were performed, including catalase activity (H2O2) and fermentation; lastly, a basic probiotic characterization analysis was conducted for phenol (0.2% and 0.5% for 24 hours), NaCl (1.5%, 2.5%, and 3.5% for 24 hours), gastric juice at pH 2 (30, 60, and 120 minutes), and bile salt (0.3% and 0.5% for 0, 1, 2, and 3 hours). The results showed that the colony morphology was small, white, with smooth texture, convex elevation, round shape, and entire margin. The cell morphology was Gram-positive, non-endospore-forming, and had elongated bacillus morphology. The 16S rRNA sequence analysis revealed that the BR2-12 isolate was identical to Ligilactobacillus ruminis. The growth curve on MRS media indicated an exponential phase at 24 hours. The catalase activity test yielded negative results, and the fermentation test on sucrose, raffinose, and cellobiose showed positive results, consistent with Lgb. ruminis fermentation ability. The basic probiotic characterization analysis showed that the survival rate of BR2-12 was 93% and 13% for phenol concentrations of 0.2% and 0.5%, respectively, after 24 hours. The survival rates for NaCl concentrations of 1.5%, 2.5%, and 3.5% were 89%, 81%, and 5%, respectively, after 24 hours. In the gastric juice solution at pH 2, the viabilities (x 10 6 CFU/mL) were 3.06, 3.75, 4.72, and 4.11 at 0, 30, 60, and 120 minutes, respectively. In the bile salt test at concentrations of 0.3% and 0.5%, the viabilities (x 10 6 CFU/mL) at 0, 1, 2, and 3 hours were 4.30, 3.83, 3.45, and 3.11, respectively, and 4.75, 4.40, 3.97, and 3.53, respectively. From the experimental results, Ligilactobacillus ruminis BR2-12 has the potential for further development as a probiotic.

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