| The bacteriostatic substance produced by Bacillus spores has been used as a natural bacteriostatic substance in many aspects due to its obviously inhibitory effect on a variety of foodborne bacteria.In this study,Bacillus ZYCHH-01 and E.coli were used as experimental strain and indicator strain respectively.And spot-on lawn method was used to assess size of the inhibition zone indicating its activity.In addition,fermentation medium and conditions of the experimental strains were optimized.Biochemistry characteristics of bacteriostatic substances were observed and bacteriostatic substances were separated and purified so as to explore their application potential.In order to improve the activity of bacteriostatic substance produced by Bacillus ZYCHH-01,we measured the concentration of carbon source and nitrogen source in varying fermentation medium.We found that optimal carbon source and nitrogen source were from glucose and peptone respectively,and that additional soybean meal powder is capable of improving the activity of bacteriostatic substance.In our study,the best fermentation medium was shown in parentheses: beef extract 3 g/L,peptone 10 g/L,sodium chloride 5g/L,glucose 5 g/L,soybean meal 4 g/L.The fermentation conditions of Bacillus ZYCHH-01 were optimized as well.By single factor test,we simulated the potential condition shown in parentheses: the p H of the culture medium was 8.0,the inoculum was 3%,the shaking speed was 190 r/min,the fermentation temperature was 37 ?,the seed age was 24 h,and the fermentation time was 27 h.We further used the response surface method to study the influence of p H,fermentation temperature,fermentation time on the concentration of Bacillus ZYCHH-01 bacteria and the activity of antibacterial substances and found the optimal fermentation conditions shown in parentheses: 7.78 of medium p H,3% of inoculum,190 r/min of shaker speed,fermentation temperature at 36 ?,seed age for 24 h,fermentation time for 27.5 h.based on multiple parallel experiments could reach 21.02 mm,which was consistent with the predicted value of 21.20 mm.The results of biochemical experiments indicated that the minimum inhibitory concentration of the supernatant was 1:64,and that bacteriostatic substances were sensitive to proteases instead of alpha amylase or cellulose enzyme.It was also shown that bacteriostatic substances were storage resistant and stable to organic solvents and some surfactants.Furthermore,bacteriostatic substances had a desired bacteriostatic activity within the broad p H range from 5.0 to 9.0 and hold thermal stability within p H 6.0-8.0.It still had an intriguingly antibacterial activity against foodborne pathogens even when exposed to a high temperature environment of 100 ? for 30 minutes.Ammonium sulfate precipitation method and ethanol precipitation method were used to extract the antibacterial substances.and the protein content was determined using Bradford method was used to assess protein content.The experimental results showed that both two extraction methods were capable of extracting antibacterial substances,but the ammonium sulfate precipitation approach was significantly better than the ethanol precipitation method.The effects of different ammonium sulfate saturations on the activity of bacteriostatic substances in the salting out process were investigated,and it was suggested that the activity of bacteriostatic substances extracted with 80% ammonium sulfate was the highest.The antimicrobial substances were desalted by ultrafiltration centrifuge tube and separated and purified by DEAE-Sepharose FF ion exchange chromatography.Then the antibacterial activity of the four absorption peaks was measured.The results showed that peak 1 had the most obvious antibacterial activity. |
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