| A large number of side effects,such as the increase of antibiotic resistant strains in bacteria,the imbalance of normal intestinal flora in livestock and poultry,the decline of animal immunity and so on,can be caused by the abuse of antibiotics in livestock production industries.The probiotics as effective substitutes for antibiotics,owing to the advantages of no pollution,no resistance and no drug residue,have been widely used in animal husbandry and aquaculture industry.So developing efficient probiotics is urgent.Enterococcus faecium is part of human and animal intestinal flora,with strong regulation function of intestinal flora balance,which is directly feed variety of microorganism and has been clearly permitted by the Ministry of Agriculture.1 The preliminary screening of fermentative supernatant fluid against indicated bacteriaThe 76 Enterococcus faecium strains were confirmed by PCR amplifying with the species-specific fragment ddlE faecium.The tests of fermentative supernatant fluid against indicated bacteria showed that the 76 strains had different antimicrobial activities,and two of them displayed obvious antimicrobial activities against the indicated bacteria Escherichia coli K88 and Salmonella choleraesuis,compared with the control strain TC3(p<0.05),which were named as XC2 and WH10.2 The safety evaluation of Enterococcus faecium XC2 and WH10(1)The results of PCR amplifying the virulence genes cylA,gelE,esp,as,ace,efaAfm showed that only efaAfm was existed in XC2 and WH10 strains.(2)The results of antibiotic sensitivity suggested that XC2 and WH10 strains were highly sensitivity to most antibiotics including vancomycin.(3)The results of acute oral toxicity test showed that there were no obvious abnormalities such as behavior,activity,fur luster,no illness and death in the experimental group mice,which were gavaged with XC2 and WH10 strains respectively,and the morphological structure of viscera and tissue were normal,compared with the control group.Therefore,Enterococcus faecium XC2 and WH10 were not pathogenic bacteria.3 The functional characteristics assessment of Enterococcus faecium XC2 and WH10(1)The determination of growth characteristics showed that the lag phases of XC2 and WH10 were short and quickly entered the logarithmic growth period,experienced the stationary phase at 8h,which were suitable for industrial production.(2)The tolerance tests demonstrated that XC2 and WH10 strains could tolerate the artificial gastric fluid of pH2.5,the artificial intestinal fluid of pH6.8,the bile salt concentration of 0.3%and the temperature of 70?.(3)The adhesion tests showed that the adhesion rate of strain XC2(7.62±0.16%)was higher than that of strain WH 10(3.54±0.47%)and which showed the most significant difference(p<0.01).(4)The adherence inhibition tests discovered that the competitive inhibition percent,exclusive inhibition percent and displacement of K88 adhesion to IPEC-J2 cells by the XC2 strain was 91.8%,73.9%and 3.0%respectively;at the same time the competitive inhibition percent,exclusive inhibition percent and displacement of K88 adhesion to IPEC-J2 cells by the WH10 strain was 82.0%,82.1%and 22.4%;both them could effectively inhibit the adhesion of pathogenic Escherichia coli K88.All the above results indicated that the screening of Enterococcus faecium XC2 and WH10 had good antibacterial effect,high safety,strong proliferative capacity and excellent tolerance in this study,which provided a solid material foundation for developing the independent intellectual property rights of efficient microecological preparation. |
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