Recombinant Lactobacillus Acidophilus Expressing Oxidase Oxa And Its Characteristics

Zearalenone(ZEN)is an estrogen-like toxin produced by Fusarium,which can cause reproductive toxicity,genetic toxicity,immunotoxicity and other toxicity to human body and poultry.The growth,harvest,processing,transportation and storage of crops all could be polluted by ZEN that may pose a threat to human health worldwide.In view of this situation,this experiment were studied according to the biological detoxification method of the detoxification and degradation of ZEN.L.acidophilus is one of the probiotics widely studied in the field of Lactobacillus.Because of its strong adhesion ability in the gut,acid tolerance and bile salt tolerance,it could effectively regulating the environment of gastrointestinal flora,improving the immunity of the body and inhibiting pathogens.Until now,Lactobacillus acidophilus has been developed and applied in the field of dairy,beverage,feed and other food,as well as contributed to the medical research and application.Based on this,we chose L.acidophilus to obtain the ability of degrading Zen by gene cloning.In this paper,we designed primers P1 and P2 amplified Oxa gene isolated from Acinetobacter SM04 by using p PIC9K-Oxa as a template and inserted it into Lactobacillus shuttle vector p MG36 e,then transferred the recombinant vector into E.coli JM109 by chemical method to construct the recombinant plasmid p MG36 eOxa,and then cloned the recombinant plasmid into Lactobacillus acidophilus ATCC4356.The recombinant protein was obtained by the culture of the recombinant bacteria and the expression and degradation activity of the recombinant protein Oxa were verified by SDS-PAGE and ZEN-degradation experiments.The results showed that the recombinant Lactobacillus acidophilus could effectively express ZENdegrading enzyme Oxa,and the degradation rate of the recombinant protein Oxa was42.95% after reacting with ZEN at 37°C.In this experiment,the probiotic characteristics of recombinant Lactobacillus acidophilus were compared with Lactobacillus acidophilus further,the conclusions are as follows:(1)The logarithmic survival rate of recombinant Lactobacillus acidophilus ATCC4356-p MG36e-Oxa in MRS medium containing 0.3% bile salt for 6hours was 90.35%,slightly lower than 94.28% of Lactobacillus acidophilus under the same conditions.(2)The logarithmic survival rate of recombinant Lactobacillus acidophilus ATCC4356-p MG36e-Oxa was 89.99% after cultured in artificial gastric juice at p H 2.0 for 3 hours,and the logarithmic survival rate was 95.79% of Lactobacillus acidophilus under the same conditions.(3)We studied the bacteriostatic effect of Lactobacillus acidophilus and recombinant Lactobacillus acidophilus on different concentrations of Escherichia coli,Salmonella and Staphylococcus aureus,and the result showed that the recombinant Lactobacillus acidophilus has the capacity to inhibit the three pathogenic bacteria to varying degrees,even showed stronger bacteriostasis than Lactobacillus acidophilus.(4)The recombinant Lactobacillus acidophilus and Lactobacillus acidophilus labeled with FITC were cultured with Caco-2 cells to determine the adhesion ability by detecting the fluorescence intensity,the results showed that the fluorescence intensity of Caco-2 cells was 14207,32619,75465 respectively after FITC labeling with different concentrations of recombinant Lactobacillus acidophilus and was 13571,28776,69756 respectively after FITC labeling with different concentrations of Lactobacillus acidophilus,which were all much higher than 356 of the blank control(P < 0.05).In conclusion,the probiotic properties of recombinant Lactobacillus acidophilus fluctuated slightly,but it still retained good bile salt tolerance,acid tolerance and adhesion ability.