Studies On The Bacteriocin From Bacillus Subtilis R21-4

In the research field of food preservation, an intense interest for scientists is focused on the isolation of antimicrobial substances from microbial metabolites. Natural biopreservatives derived from traditional fermented foods are generally recognized as safe. In this paper, one strain screened for its safety andbroad antimicrobial spectrum and its antimicrobial substance--bacteriocin are studied in detail. Themain results are as follows:1. One strain of Bacillus sp. is screened from fermented foods and some samples of beneficial microorganisms. The metabolites produced by the strain have high antimicrobial activity and broad antimicrobial spectrum. The strain and its metabolites are identified. The results show that the culture has a broad antimicrobial spectrum against some gram-negative and gram-positive bacteria, and moulds, especially against E.coli, Bacillus cereus, and Staphylococcus aureus. The isolated Bacillus R21-4 is identified as Bacillus subtilis through morphological, physiological and biochemical experiments. The metabolites of R21-4 with antimicrobial activity are identified as a kind of protein.2. The production of bacteriocin is greatly influenced by the composition of medium and culture condition. A number of experiments are done to evaluate the culture conditions on the antimicrobial activity, The results shows the optimum culture conditions are as follows: glucose 10g/L , peptone 20g/L, KH2PO41.5 g/L, MgSO4.7H2O1.5 g/L, NaCl 1.0 g/L, initial pH 7.0, temperature 37℃, seed age 6h, inoculation amount 1 %, the aeration 1 /5, incubating time 21 h.3. Isolation and purification of the bacteriocin are studied. The results show that 30% ammonium sulfate can precipitate most of the bacteriocin from the culture broth. Two protein peaks can be obtained by Bio-Gel P-6 chromatography and both peaks have antimicrobial activity. So the bacteriocin is consisted of two proteins with different MW. Only one protein with larger MW can be detected through Tricine-SDS-PAGE, and its MW is about 8,570Da. Study on the isolated bacteriocin's property shows that the bacteriocin has good stability against temperature and pH. The antimicrobial activity is not influenced disposed for 10 minutes under 80℃, and only 13% loss of activity is suffered under the temperature of 100℃ for 10min. The antimicrobial activity changes little from pH 3 to pH10. Some activity is lost disposed by organic solvent. The bacteriocin is sensitive to the proteinases. The titers to the indicator bacteria Bacillus cereus, E.coli, Staphylococcus aureus are 32, 8 and 8 respectively.4. Antimicrobial effect of the bacteriocin produced by R21-4 is studied in the food systems. The results show that the conspicuous antimicrobial effect in the preservation of fresh milk. The samples were stored under 37℃ and the bacteria concentration in the sample with bacteriocin was 3-5 order less than the sample without bacteriocin...