Screening Of Se-enriched Lactobacillus And Optimization Of Fermentation Conditions

Selenium is an essential micronutrient for humans involved in functioning at immune response,cancer prevention,anti-oxidation and other significantly physiological functions.It is often supplied by natural food including grains,fruit and vegetables at levels too low for human dietary intakes.As inorganic selenium exists certain toxicity,microorganism is giving cause for concern notably in foreign scholars as an efficient and safe selenium supplementation carrier.The selected strain Lactobacillus plantarum Dfa-301 was mutagenized and optimized to enrich Se and finally a kind of Se-enriching lactobacillus tablet was made by its functional reseach.(1)A total of 4 species lactic acid bacteria were screened for their abilities to tolerate and enrich Se.Out of all the tested cultures,Lactobacillus plantarum was found to have the greatest ability to uptake Se.And then the selected strain was mutagenized respectively with UV irradiation,DES treatment and their combination.The optimal compound mutagenesis conditions were as follows:20 W UV lamp was irradiated for 60s 3 times and 20%DES ethanol was mutagenized for 20 min 4 rounds.The mutant strain with high biomass and selenium enrichment was identified as Dfa-301-30,and the content of selenium was 473.275 μg/g,which was 20.51%higher than that before mutagenesis.(2)The culture medium were optimized to understand the effect of different C source,N source and inorganic ions for selenium enrichment.The optimum fermentation medium(the modified MRS)were as follows:glucose 20.0 g/L;beef powder 24.0g/L;the additions of 20.00 g/L Mg2+、K+、NH4+、Ca2+ were 0.04mL、0.60 mL、0.40 mL、0.30 mL/20 mL(the culture medium).And single factor test including Na2SeO3 concentration,culture time,selenium adding time,initial pH,and inoculation volume were optimized with Box-behnken design.The optimum culture conditions were as follows:Na2SeO3 concentration 5.0 μg/mL,initial pH 5.5,selenium adding time 7.6 h,inoculation volume 3.0%,culture time 36.0 h.The enrichment of selenium was 839.635 μg/g after cultivated in optimized medium,which was 1.77 times that of the yield before.Then a fed-batch fermentation protocol was researched for selenium enrichment,which exhibited a potential for industrial application.The final dry cell biomass reached 3.8495 g/L and a Se uptake level of 940.329 μg/g was achieved in a 1 L fermentor after 92 h cultivation.(3)A kind of Se-enriching Lactobacillus plantarum tablet was prepared by screening crucial steps such as preparation of lyophilized powder and excipients,optimization process and tablet quality standards.The tablet formula was as follows:selenium-enriched lyophilized powder 45%(w/w),microcrystalline cellulose 49.6%(w/w),croscarmellose sodium 1:1 Inside and outside addition 5%(w/w),Magnesium Stearate 0.4%(w/w),5%PVP(alcohol solution)appropriate amout.The results showed that the prepared functional tablets was stable at a temperature of 40 ℃±2℃ and a relative humidity of 75%±5%for 6 months and has 4.87×107-5.23×107 cfu/g viable cells and 30.13 μg/tablet of the average selenium content in the three batches of samples.The traits were stable and the disintegration time and selenium content were in accordance with the requirements.(4)This study reports a green approach to biosynthesize SeNPs by Lactobacillus plantarum Dfa-301-30.SeNPs were characterized by different kinds of analysis method showing that the formation of SeNPs was monitored by UV-visible spectra with maximum absorption bands at 218 and 268 nm.TEM and SEM images reveal the spherical shape of biosynthesized SeNPs and a size range of 192.28±26.34 nm.The nocrystalline nature of SeNPs is confirmed by the XRD.Moreover,from the FTIR spectrum,the presence of highly stable biopolymer on the surface of SeNPs suggests a possible role as capping agent.An XPS spectrum of the Se 3d region(Se 3d5/2 and Se 3d3/2)suggests a strong interaction between Cell and SeNPs.