Optimization Of Fermentation Process For Zearalenone Degrading Enzyme And Analysis Of Degradation Product

Zearalenone(ZEN)is a mycotoxin,a secondary metabolite of Fusarium that is widespread in nature and has an estrogenic effect and was obtained early from moldy corn.The mycotoxins show a strong reproductive toxicity to humans and animals,especially pigs.Therefore,various methods for degrading zearalenone have been explored.In this article,ZENL09,a strain capable of degrading ZEN,was identified,the fermentation conditions for ZENL09-producing zearalenone degrading enzymes were optimized,the products of enzymatic degradation of zearalenone were analyzed,and the cytotoxicity of degradation products was discussed.The details are as follows:(1)The study found that the fermentation supernatant of strain ZENL09 had a better degradation effect on zearalenone,and the degradation rate was 53.59%.By observing the morphology and physicochemical characteristics of the strain,the 16 S rRNA sequence was determined,and the BLAST comparison on NCBI was performed to initially identify strain ZENL09 as Bacillus subtilis.(2)The fermentation process conditions of the zearalenone degrading enzyme produced by the strain were optimized,and the optimum fermentation conditions were: fermentation period 24 h,inoculation amount 3%,shaker Speed 200r/min,liquid volume 25mL/250 mL,fermentation temperature 33°C,initial pH of fermentation medium 8.0;optimum fermentation medium: sucrose 20(g/L),peptone 26.67(g/L),yeast Powder 13.33(g/L).Under the optimal fermentation conditions,the ZEN degradation rate of the ZENL09 fermentation supernatant reached 88%.(3)The fermentation supernatant of ZENL09 was treated with proteinase K and a boiling water bath and the degradation rate of ZEN was found to be significantly reduced,demonstrating that the ZENL09 fermentation supernatant is an enzyme for the degradation of ZEN.The centrifuged supernatant was centrifuged and concentrated with 3KD and 10 KD ultrafiltration tubes.The degradation rates of the entrapped fractions obtained were comparable,and the degradation rate was not significantly different.This indicated that the enzyme capable of degrading ZEN was trapped after centrifugation by a 10 KD ultrafiltration tube.Partial and degradative enzymes have a molecular weight greater than 10,000 Da.The SDS-PAGE test of the retained fractions revealed that there were two distinct bands in the electrophoretogram.It was speculated that the molecular weight of the ZEN-degrading enzyme may be 25.0 KDa or 66.2 KDa.(4)The effect of different flow relative HPLC analysis on the product of degradation of ZEN by the fermentation supernatant of ZENL09 was studied.Finally,the optimum mobile phase was determined as follows: acetonitrile/water = 35/65(v/v).Under the mobile phase conditions,the product peak at a retention time of 9.5 min.The product was analyzed with a high-resolution liquid chromatography and mass spectrometer.The relative molecular weight of the degradation product was determined to be 256.The degradation product was then analyzed by tandem mass spectrometry and the possible structure of the degradation product was preliminarily presumed.(5)The toxicity of degradation products on HepG2 cells was studied.The toxicity of degradation products on HepG2 cells was reduced compared with that of ZEN.