Purification, Characteristics And Molecular Structure Of Bifidocin A,A New Bacteriocin Produced By Bifidobacterium

Bateriocin is non-toxic, no residue and safety as a kind of natural preservative. Some researches have found that many lactobacillus strains can produce bacteriocin. But there are a few studies about bifidobacterium which are predominant bacteria in human intestinal microflora. This paper aimed at obtaining an excellent bifidobacterium strain inhibiting other bacteria from breast-fed babies faeces sample which is of our own intellectual property right. Optimum conditions of producing bacteriocin, a rapid and efficient procedure of purification and characteristics and molecular structure have been studied.Results are as follows.(1) This paper obtained a good bacteriocin-producing bifidobacterium strain which was identified as Bifidobacterium animalis named as BB04. The antimicrobial effect was demonstrated to be induced from bacteriocin after excluding interference. The bacteriocin was named as bifidocin A.(2) The optimum conditions of producing bacteriocin obtained by single-factor experiment and response surface method were: optimum incubation time of 40.5h,optimum incubation temperature of 35 ℃, optimum incubation pH of 6.75. After optimization procedure titer of bacteriocin reached 787.3 AU/mL which is 1.3-fold than before.(3) A four-step purification method was achieved: ammonium sulfate precipitation, SP Sepharose Fast Flow cation-exchange chromatography, gel filter chromatography of Sephadex G-10 and reverse phase high-performance liquid chromatography(RP-HPLC)according to cationic character and hydrophobic property of bifidocin A. The specific activity after the last step was 5535 AU/mg, increasing by 115-fold.(4) The results of research about characteristics of bifidocin A by Bifidobacterium animalis BB04 showed that the bacteriocin could remain 80% activity after 30 min at50-80 ℃ and 50% activity after being heated at 121 ℃ for 20 min. The effect stayed unchanged between pH3.0 and pH8.0 and remained over 60%. The bacteriocin was sensitive to proteinase K, pepsin and acid proteinase, but inactive partially by trypsin and neutral proteinase. Bifidocin A had a broad inhibitory spectrum of G+bacteria, G-bacteria and fungus, such as Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus aereu, Bacillus and Saccharomyces. What’s more, the mode of action was bactericidal action. MIC and MBC were 640AU/mL and 2560AU/mL, respectively. The absorption ofbacteriocin onto/from indicator bacteria cells was influenced by pH and concentration of salt ions, while not by temperature and time.(5) The molecular mass of bacteriocin bifidocin A was test of 1198.7Da. What is more,the front ten amino acids of N-terminal sequence of bifidocin A was Val--Phe--His--Pro--Gly--Thr--Gly--Pro--Pro--Gly(VFHPGTGPPG). The result of second structure of bifidocin A indicated over 58% random coil, 27% beta and 12% turn structure which illustrated the tertiary structure of bifidocin A was a new structure different from protein structure known before.