| The antibacterial substances produced by Bacillus subtilis WL17 had a broad antibacterial spectrum, which were insensitive to temperature and pH. The main components of antibacterial substances were peptides with small molecule weight. The antibacterial substances, as a natural preservative, had a grand development potential in food industry. In this paper, fermentation optimization, isolation and purification, antibacterial mechanism and preservation application of antibacterial substances produced by Bacillus subtilis WL17 were studied.Fermentation optimization of Bacillus subtilis WL17 was studied. First, Plackett-Burman design was undertaken to evaluate the effects of the six factors in liquid fermentation, and the significant factors affecting the inhibitory zone diameter of produced antibacterial substance were determined as follows:Initial pH, Broth volume and C/N. Then, a response surface methodology was used to optimize the above critical internal factors, the optimal conditions of the variables were as:Initial pH 6.91, Broth volume 47.59ml/250ml and C/N 2.22. Under this condition, the inhibitory zone diameter of produced antibacterial substance was increased from 16.8mm to 23.14mm.Antibacterial activity and stability of antibacterial substances were studied. Antibacterial substances showed significant antagonistic activities against many food pathogens, such as Salmonella typhimurium, S.aureus and so on. The antimicrobial peptides were very stable against high temperature pH, trypsin and pepsin. But under the treatments of high temperature and strong acid or strong base, the antimicrobial activity decreased significantly. In addition, the antimicrobial peptides were stable against pulsed light treatment.The study on antibacterial mechanism of antibacterial substances showed that the antibacterial substance of Bacillus subtilis WL17 had remarkable inhibition effect to the growth of E. coli. The MIC values of antibacterial substance to E. coli was 0.614 mg/mL. Adding antibacterial substance in lag phase and lag phase could have better effect than that in the stable phase during the growths of E. coli. The experiments of FDA and Fluo 3 AM suggested that antibacterial substances could increase the cell membrane permeabi- lity, SEM and TEM showed that the cell surface of E.coli. was coarse and cell membrane was ruptured.Antimicrobial substances were isolated and purified by Sephadex LH-20 and preparative liquid chromatography. Four fractions were isolated from antimicrobial crude extract by Sephadex LH-20, and the fourth fraction showed strong antibacterial activity. Three antimicrobial components were obtained from the fourth fraction by preparative liquid chromatography. Mass spectrum analysis showed that this antimicrobial components contained three kinds of fengycin homologues and two kinds of surfactin homologues,furthermore, some new antimicrobial compounds were also found in this components.The effects of adding different concentrations of antimicrobial substances obtained from bacillus subtilis on the bacterial count, TBA value, TVB-N value, hardness, springiness, cohesiveness of fish-ball during storage process were studied. And the synergetic preservation effects on the fish-ball between antimicrobial substance and pulsed light treatment were also researched. Results showed that antimicrobial substances at a concentration of 2mg/g caused 1.23 log cfu/ml reduction of the microbial count, whereas the TBA value and TVB-N value were both at the low-level with no significance texture change of fish-ball. There were synergistic effects between antimicrobial substance and pulsed light treatment, which showed remarkable preservation on the fish-ball after treated with pulsed light as well as adding 2mg/g antimicrobial substance. |
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