Isolation Of A Novel Lactic Acid Bacteria For Feed And Preliminary Study On Its Antibacterial Material

During transport and storage, the feed can not avoid contamination by pathogenic bacteria, including Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, and so on. These microorganisms may gain entry to animals and develop enteric infection disease, even pose a threat to their health. To solve this problem, antibiotic is widly used in the feed industry. However, with the use of antibiotic, antibiotic tolerance of pothagenic bacteria is more and more serious. At the same time, the antibiotic residues in the feed also attracted much attention. Therefore, the use of antibiotics is limited in some developed countries. Exploiting green feeding additives for feed become urgent. It was of great practical value for developing green feeding additives for feed since the lactic acid bacteria（LAB） strains could inhibit the growth of pathogenic bacteria, resisit to acid, bile salt and feed processing and determine its antibacterial material.Four LAB strains were capable to inhibit the growth of feedstuff, by excluding the influence of organic acid and H2O2. Among these four LAB strains, the strain R16 was found to be resistant to acid, bile salt and high temperature and thus was identified as target bacteria. It was further identified as Pediococcus acidilactici on the base of physiological and biochemical characteristic and 16 SrDNA gene sequences similarities. The strain R16 was named Pediococcus acidilactici R16.Antibiotic substance was purified from fermented liquid of Pediococcus acidilactici R16 by ultrafiltration, followed by anion-exchange chromatography, gel filtration chromatography and reversed phase liquid chromatography. To indentify this antibiotic substance, it was analyzed by liquid chromatography-mass spectrometry. The results showed that the antibiotic substrance is a peptide. Molecular weight and isoelectric point of this peptide is 705.63 Da and 3.58, with the sequence of ENGEEE, respectively. This peptide was named lactein R16.The optimal pH for antibiotic activity of lactein R16 was pH 3.0⁵.0. The activity of lactein R16 reduced at pH 6.0⁸.0. Lactein R16 could retain when treated at 80℃ or below for 30 min, but lost approximately 21.95%, 49.47% and 59.98% when treated at 90, 100, 121℃ for 30, 30,15 min, respectively. Its antibiotic activity was not affected when treated with protase. Lactein R16 showed high antibactra activity toword gram-positive bacterium, with minimal inhibitory concentration to Escherichia coli, Listeria monocytogenes, and Salmonella enteritidis of 6.40 mg?mL^-1, 6.40 mg?mL^-1, 3.20 mg?mL^-1, respectively. Howerve, it showed low inhibiting activity to gram-negative bacterium. When Pediococcus acidilactici R16 and lactein R16 was used in the solid state fermentation process of soybeam meal that contaminated by E.coli, the E.coli count could reduced by 17.20% and 33.44%, respectively. Lactein R16 has positive effect on the proliferation of Saccharomyces cerevisiae and Lactobacillus plantarum. Lactein R16 showed certain capacity of scavenging H2O2, hydroxyl free radical, DPPH? free radicals and superoxide anion. Clearance ration was 25.65%, 70.13%, 79.42%, 10.11%, respectively.