الفهرس | Only 14 pages are availabe for public view |
Abstract . Probiotics have shown promising results in prophylaxis and safe biotherapy. The search for a biologically active strain able to produce bioactive compounds of applied importance to meet the growing needs was the main target. To attain this target, buffalo colostrum samples was used as an isolation source of LAB. Out of the 53 LAB isolates, only 16 produced exopolysaccharides; of which, one isolate attained the highest productivity (3.8 g l-1) and was also able to produce L-asparaginase (ASNase). Such isolate was molecularly identified as Weissella paramesenteroides MN2C2 and deposited in the GenBank under number MK530206. The optimum conditions for maximal productivity of bioactive compounds were 24 h incubation at 35 °C and pH 6.5 for EPSs; 48 h at 37 °C, pH 7 and 10% inoculum concentration for ASNase. EPSs molecules were partially purified (PP) and physicochemical characterized; UV, FTIR spectra showed absorption bands related to many functional groups. 1HNMR polysaccharide spectrum consists mainly of three regions: ring proton of several sugar residues of the polysaccharides, anomeric proton region and the alkyl proton region. HPLC elucidated EPSs structure as consisting of 80% fructose, 9.3% glucose and 5.6% sucrose. SEM showed a three-dimensional structure of irregular highly compacted lumps with different sizes and a smooth surface. Energy dispersive X-Ray and mapping analyses revealed that PP-EPSs composed of carbon, nitrogen, oxygen, phosphorous and sulfur in high ratios of its weight reached 42.31, 10.11, 42.68, 4.13 and 0.78 %, respectively. As indicated by TEM and DLS analyses, the nanoparticle (NP) sizes of EPSs prepared from crude or PP using ultrasonication ranged from 37.3 to 105 nm for the crude CEPSs-NPs, and from 45.7 to 204 nm for the PPEPSs- NPs, PP-ASNase has a specialized activity of 72 units mg-1 protein and a molecular weight of ≈ 36 kDa using SDS-PAGE technique. PP-EPSs proved its superiority as anticancer with highly selectivity indices against liver HepG-2, colon (Caco-2) and breast MCF-7 cancer cells. The CEPSs-NPs had significant activity against breast cancer MCF-7; and the toxicity was increased in the nanoparticles prepared from partially purified polysaccharides. Both CEPS-NPs and PPEPSs-NPs also showed stronger DPPH antioxidant activity than CEPSs, PP-EPSs and PP-ASNase, respectively. Both PP-EPSs and CEPSs reduced Coxsackievirus (CVB3) yield by > 99%. The probiotic W. paramesenteroides MN2C2 were characterized by its survivability for 3 and 6 hours at pH ranging from 1.5 to 9 and 0.3% of bile salts; in the presence of pancreatic juice, under simulated stomach and intestinal conditions; besides its ability to adhere to intestinal walls. It also showed antimicrobial activity against B. subtilis and P. aeruginosa, with inhibition zones of 25 and 19 mm, respectively. The strain is considered as safe because of the absence of analytical activity in human blood and the high sensitivity to antibiotics tetracycline, fusidic acid, ampicillin and resistance to vancomycin and kanamycin. Also, W. paramesenteroides MN2C2 was able to ferment milk when mixed with probiotic commercial starter strains to produce a promising milk product that is suitable, practical, effective, preventative and with safe therapeutic properties |