Studies On The Mutation Breeding And Zymotechnique Of One Local Strain Sr-1 For High Bacteriocin-Producing L. Acidophilus

One local strain of Lactobacillus acidophilus (L. acidophilus) with broadspectrum antimicrobial activity was seperated and screened out from intestine of human and animals in the present studies. The antimicrobial substance (namely bacteriocin) secreted by the bacterium was studied in the aspects of biological character, extraction, mutagenesis and fermentation. Results lay foundation for developing and applying of food biological antisepsis and medicament for animal intestinal bacterial diseases.1. One local strain of Lactobacillus with antimicrobial activity seperated from feces of human and animals was confirmed as L. acidophilus by the tests on its morphological, physiological and biochemical character. Results of the biological character of the secreted antimicrobial substance showed that the antimicrobial substance secreted by the separated stain Sr-1 is protein. The antimicrobial substance was confirmed as bacteriocin for its broadspectrum antimicrobial property to both gram-positive and gram-negative bacteria such as E. coli, S.pullorum, S. aureus and A. sobria.2. Bacteriocin was precipitated by ammonium sulfate from fermented broth and further separated by Bio-Gel?P-6 chromatography, then the antimicrobial activity of all the collected fractions was tested by gel diffusion test, results showed that bacteriocin secreted by L. acidophilus Sr-1 contain two types of protein since two of them has antimicrobial property. Only the one with higher molecular weight can be showed on Tricine-SDS-PAGE gel, and its molecular weight is 8100Da or so. The purified bacteriocin has fine stability against heat alkale when pH is above 5.0. The antimicrobial activity stay still after being heated at 90℃for 30min, lost only 11.1% after being heated at 121℃for 20min, and lost to a certain extent after being digested by proteinase. The purified bacteriocin has perfect and broadspectrum antimicrobial effect.3. The segregated Lactobacillus acidophilus (Sr-1) with the capability of producing the bacteriocin as original strain was developed into a mutant stock (SUN-211) with higher production of bacteriocin by selected strictly, after repeated induction and mutation with the combination of UV administer radiation and NTG,the detection of SUN-211 metabolic product demonstrated bacteriocin valency induced by the broth increased 47.12%, compared with that absent of induction and mutation. The gained character was with excellent genetic stability, which was indicted after the strain passages.4. The composition of medium and condition of cultivate took great influence on the growth of Lactobacillus acidophilus Sr-1 and the product of bacteriocin were tested. The studies on optimization of media composition and cultivate condition for the fermentation of Lactobacillus acidophilus Sr-1 indicated the fermentation medium based on MRS could increase 53.6% and 35.3% of the growth of bacteria and the production of bacteriocin, respectively. The major composition of this fermentation medium included: soy peptone2.5%, yeast powder 0.4%, glucose 2% and vitamin 0.3%; the optical incipient pH of medium was 6.5; the optical temperature for induction of bacteriocin was 32℃; the best fermentation stage was 24h; and the giant molecule protein played the important role on the induction of bacteriocin.5. The examination of effect on bacteriocin crude extract secreted by Lactobacillus acidophilus Sr-1 to food antisepticise and cure of animal intestinal tract diseases demonstrated that bacteriocin induced the conspicuous antiseptic effect on the conservation of sterilized fresh milk: the number of bacteria in groups with bacteriocin differed in 4 to 5 order of magnitude from that in control groups under 37℃after 24, 48h, respectively, indicating bacteriocin good at antisepticising. Bacteriocin also showed the excellent prevention and cure effect on gastrointestinal tract diseases of economic animal and pig or other animals. Comparing with antibiotic, bacteriocin shorted 1-2 days of convalescence, showed the better growth after bacteriocin treatment.