Screening Of Agro-Wastes As Growth Substrate, Quantification And Antimicrobial Activity Of Bioactive Metabolites Of Lactobacillus Species

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Date
2015-06
Authors
ALI HASSAN, HASSAN PYAR
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Abstract
The objective of this study was to screen and identify a suitable agro-waste in the development of a selective growth medium for Lactobacillus. The bioactive metabolites produced by Lactobacillus were quantified and the antimicrobial activity determined. Prior to the commencement of study, the identity of twenty probiotic bacteria provided by School of Industrial Technology, USM, was confirmed as Lactobacillus using morphological and biochemical studies. From the twenty bacteria cultures, nine that exhibited viable count of above 9.0 log10 CFU/mL were selected for acid and bile tolerance tests. Five bacteria culture that exhibited viable counts of more than 9.0 log10 CFU/mL in acidic medium of pH 1.5 and in a medium with 0.5%w/v bile salt at pH less than 4.5, were identified using SEM, API 50 CHL and 16S rDNA methods. The identity of the lactobacilli was confirmed to be L. brevis, L. plantarum and L. casei. The nutritional values of six easily available, economical, and abundant agro-wastes, namely honeydew skin, guava skin, mango skin, cabbage leave, pineapple skin and papaya skin, were determined through proximate analysis and screened as growth substrate. All the three lactobacilli grew in the six agrowastes. The extracellular fluid (cell-free fluid) demonstrated a significantly higher antimicrobial activity than intracellular fluid (lysate fluid) for the three Lactobacillus species cultured in all the six agro-waste substrates. The viable count of lactobacilli was the highest when cultured in pineapple skin. The influence of substrate nutrients, glucose, YSBP (combination of yeast extract, skim milk, beaf extract and pepton at ratio 1:1:1:1), dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, Tween 80, magnesium sulphate and manganese sulphate on the growth of L. brevis, L. plantarum and L. casei was evaluated. The optimum growth was achieved with 1.0%w/v glucose, 1.0%w/v YSBP, 0.1%w/v K2HPO4, 0.1%w/v sodium acetate, 0.2%w/v triammonium citrate, 0.075%w/v Tween 80, 0.20%w/v MgSO4 and 0.005%w/v MnSO4. A new and selective medium was developed using pineapple skin as platform enriched with the essential nutrients. The viable count of L. brevis, L. plantarum and L. casei cultured in the newly developed more economical selective pineapple skin medium was comparable with Man-Rogosa–Sharpe (MRS) medium. The bioactive metabolites of extracellular fluid of L. brevis, L. plantarum and L. casei, which included lactic acid, lipid, diacetyl, exopolysaccharide, hydrogen peroxide and protein, were quantified. It was found that the content of lactic acid was the highest followed by protein. The bioactive protein in the extracellular fluid of Lactobacillus casei was selected for identification using LC-MS/MS Orbitrap. Ubiquitin and casein were found to be the major proteins in the extracellular fluid of Lactobacillus casei. The antimicrobial activity of extracellular fluid, lactic acid and bioactive protein against S. typhimurium, P. aeruginosa, E. coli, K. pneumonia, S. aureus and C. albicans was determined by comparing the zone of inhibition. The bioactive protein (bacteriocin) showed comparatively higher antimicrobial activity than lactic acid. The IC50, MIC and MBC of bacteriocin were closely comparable to erythromycin for S. typhimurium, P. aeruginosa, K. pneumonia and S. aureus. The bioactive protein (bacteriocin) was more effective than erythromycin against E. coli, but less effective than clotrimazole against C. albicans. In conclusion, a selective and economical pineapple skin medium for lactobacilli was successfully developed. The use of agro-waste as growth medium could reduce waste accumulation and environmental pollution. Furthermore, the bioactive protein (bacteriocin) could be potential source for the discovery of novel antimicrobial agents.
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Screening Of Agro-Wastes As Growth Substrate, Quantification And Antimicrobial Activity , Of Bioactive Metabolites Of Lactobacillus Species
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