Profiling of Effective Microorganisms (EM) and Shelf Life Assesment of EM Fortified Fish Feed

Nanthini, Usha (2019) Profiling of Effective Microorganisms (EM) and Shelf Life Assesment of EM Fortified Fish Feed. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Effective Microorganisms (EM) is a microbial blend consists nearly 80 different microorganisms mainly from five groups of microorganisms being lactic acid bacteria, photosynthetic bacteria, yeast, fermenting fungi and actinomycetes. The EM has been commonly used in agriculture, environmental and waste water treatment sectors. The benefits of EM in aquaculture including improving feed conversion rate, water quality and microbial diversity have sparked local interest. Exploring microbial content in the EM including lactic acid bacteria and the feasibility of them being included in fish feed have been objectives of my study. The EM product provided by private industry was subjected to microbial isolation using nutrient agar (NA) and de Man, Rogosa and Sharpe (MRS) agar. Bacterial isolates were then grouped and characterized macroscopically, microscopically, and biochemically. The targeted bacteria were incorporated into fish feed with oven drying and freeze drying of bacterial inoculants compared and evaluated for shelf-life. A total of 25 isolates have been isolated from NA and 20 isolates from MRS respectively. Morphological groupings have streamlined the isolates into 9 (3 isolates from MRS, 6 isolates from NA). Microscopic characterizations and biochemical tests have revealed that all isolates are Gram-positive, non-spore forming bacteria with mixtures of rod and cocci shaped cells. Three bacteria isolates from MRS agar namely AK1, RF2 and SE3 were inoculated into fish feed containing 50 pellets. Bacterial isolates that were inoculated onto fish feed has yielded average of 107 CFU/ml four weeks after inoculation as compared to 0.5 MacFarland standard of the initial inoculant. Freeze drying recorded to have overall higher CFU counts as compared to oven drying. AK1 isolate was having the highest counts close to 108 CFU/ml and is significantly higher than SE3 isolate. Bacterial isolates subjected to oven drying method have recorded consistent CFU counts averaging 107 CFU/ml and significant difference was not observed among all three isolates. All these have indicated that both oven dried and freeze dried methods were not affecting the CFU counts of all three isolates. All three isolates have consistent CFU counts indicating consistent shelf-life 4 weeks after inoculation. Three isolates from MRS agar have been subjected to identification. The DNA extraction using Phenol Chloroform Isoamylalcohol (PCI) method has yielded purity range of 1.7 to 2.11 and DNA concentration range between 260 ng to 500 ng/μl. PCR amplification revealed the band for isolate SE3 is observed at around 1500 bp while no band was observed for isolate AK1 and RF2. Due to less concentration of the PCR purified product of SE3, the isolate could not be sequenced succesfully. Keywords : Effective microorganisms, lactic acid bacteria, Lactobacillus spp, fish feed, drying.