Molecular Identification of Lactic Acid Bacteria Isolated from Gastrointestinal Tract of Oreochromis Spp. (Red Tilapia) and Viability Upon Storage

Jasmi, Mas Rohayu (2020) Molecular Identification of Lactic Acid Bacteria Isolated from Gastrointestinal Tract of Oreochromis Spp. (Red Tilapia) and Viability Upon Storage. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

According to the Department of Fisheries Malaysia, red tilapia is the number two most produced fish after the catfish species in the year 2017 contributing for about 49.37 % to the Malaysia’s production value. There are variety of choices for commercial probiotics available in the market for the supplementation to the aquaculture diet, however, it has been reported that the best choice of probiotic dietary supplementation for fish is bacteria which is originated and isolated from the host species themselves. This is because there are greater opportunity for the native bacteria sources to compete against pathogens residing in the gastrointestinal tract (GIT). Thus, the objective of this project was to identify three potential probiotic lactic acid bacteria (LAB) strains isolated from the gastrointestinal tract of Oreochromis spp. (red tilapia) based on 16S rRNA gene sequencing and to analyze the storage effect on probiotic coated feeds. Genomic DNA of the three isolates were extracted using Geneaid Presto Mini gDNA Bacteria kit. The concentrations of the extracted DNA were 36 ng/μL, 27.5 ng/μL and 62.1 ng/μL for isolates LAB1, LAB2 and LAB3 respectively. The purity of the DNA extracted from the A260/A280 ratio readings were 1.97, 1.84 and 2.04 for LAB1, LAB2 and LAB3 respectively. The 16S rRNA gene sequence coding region were amplified by polymerase chain reaction (PCR) using the universal primers; 27F for the forward primer and 1492R for the reverse primer. An approximately 1400bp fragments of the amplicon size were obtained for the three isolates. The amplified products were then subjected to Sanger Sequencing. The isolates were identified by comparing the sequences with known sequences in GenBank using nucleotide BLAST (Basic Local Alignment Search Tools) program. The bacterial isolates showed 98.95%, 98.59% and 96.60% sequence similarity with Leuconostoc pseudomesenteroides strain 3832 (LAB1), Leuconostoc lactis strain KCCM202369 (LAB2) and Leuconostoc mesenteroides strain MU3 (LAB3) respectively. Next, a rapid and cost-effective study prior for further exploration on the supplementation of isolated probiotics to fish feed was evaluated i.e., assessing the viability of the respective LAB upon top coating during storage. The coating of feed pellets with respective probiotics (LAB 1, LAB2 and LAB3) revealed no major decrease in viability of the supplemented probiotics over 21 days of storage at 4°C, with viable cells of about 108 CFU/ml. In conclusion, identification of the characterized probiotic strains and the optimization of preparation and storage (condition and duration) elucidated from the current study would warrant further application assessment of the probiotic-supplemented red tilapia fish feed.