Screening And Mechanism Analysis Of Probiotics For The Inhibition Of Fusarium And Degradation Of Two Mycotoxins

Fusarium graminearum is the most common Fusarium strain for grain contamination, which can produce a group of toxic secondary metabolites, including deoxynivalenol, zearalenone and Toxin-2. Many side effects such as vomiting, estrogenic, immunotoxic, mutagenisity, cytotoxic and genotoxic can be caused by consumption of cereals contaminated with these mycotoxins.Food and feed spoiling moulds cause great economic losses worldwide. A serial of physical and chemical treatments can be applied to prevent or minimize the spoilage of food and contaminations from mycotoxins. However, the efficiency of conversional treatments is low, and a risk of introducing harmful breakdown chemicals is concerned.In the present research, firstly, we used Fusarium graminearum as the contamination indicator, and probiotic strains of Lactobacillus. salivarius ssp. Salivarius, L. fermentum and L. crispatus were screened out for their inhibitory ability on the indicator growth. We also tested the inhibitory effect of L. fermentum and L. crispatus on the spore germination and mycelial growth, which have not been reported so far. Results showed that the spore germination and mycelial growth can be inhibited by L. fermentum and L. crispatus, and the inhibitory capacity was decreased with the prolonging of time. Principal components analysis found that the bacteriocins, proteins and organic acids from the supernatant of culture suspension are the main inhibitor.Secondly, the contamination status of deoxynivalenol and zearalenone in maize and wheat was investigated in Henan and Anhui province. Deoxynivalenol and zearalenone were found in 72.3%and 73.4%of random selected samples respectively in these provinces. Deoxynivalenol was detected 379.2μg per kilogram, which was lower than the national standard, and Zearalenone was 178.1μg per kilogram, which was much higher than the limitation of the national standard.Thirdly, Bacillus subtilis ZZ and B. licheniformis DY were screened out for their degradation ability of deoxynivalenol from 59 Lactobacillus and Bacillus strains by using competitive indirect ELISA. Mechanism analysis showed that the heat sensitive protein substances produced by Bacillus rather than cell itself played the major role. B. subtilis ZZ and B. licheniformis DY can degrade 98% and 30.7% Zearalenone respectively in broth after incubated for 12 h under the optimum condition (37℃,180 rpm).Fourthly, B. cereus was screened out with the ability of degrading zearalenone from 186 bacteria strains including Lactobacillus, Bacillus, Bifidobacterium and rumen microorganisms isolates by competitive indirect ELISA. Meanwhile, three other strains isolated from samples of maize, wheat and soil (collected from Henan and Anhui province) were screened out for their degradation ability in selective medium. They were identified as Brachybacterium rhamnosum, Ralstonia metallidurans, Enterococcus faecalis, B. cereus, Micrococcus luteus and Burkholdeia cepacia. Then the growth condition of B. cereus and M. luteus were optimized since they showed better ability of zearalenone degradation. The B. cereus can completely degrade the zearalenone in liquid BA (pH=6.5) after incubated for 96 h under the optimum condition (37℃, 180 rpm), the protein substances produced by B. cereus were anticipated to be the major degrader. M. luteus can degrade 99% zearalenone in liquid LB (pH=7.0) with 0.05M MnCl2 after incubated for 120 h under the optimum condition (37℃,180 rpm).Finally, a 795 bp DNA fragment (ZEN-jjm) from Gliocladium roseum coding lactonohydrolase was obtained. Sequencing results showed a 9-base difference in the amplified gene compared with reported gene zhd101, resulting in 3 amino acid difference in lactonohydrolase. The degradation capability of expressed protein in the supernatant of disrupted cells was verified by HPLC, and a similar biological function was observed in the expressed protein.