| Bacillus subtilis, a kind of non-pathogenic bacteria widespread in nature, can produce antimicrobial substances in the process of growth and metabolism, mainly including organic acids, lipopeptide and antibacterial proteins. These antimicrobial substances have strong suppression and sterilization ability to spoilage and pathogenic bacteria in food, and are susceptible to be degraded by protease in people and animals, that are not absorbed by the body and do not have side effects on human body, and have broad application in the aspect of food preservation. Currently, the study of biological preservatives of microbial source is very mature and they are already produced in industry and they have very wide range of applications. But so far, the studies of producing antimicrobial substances by using food-grade material are rarely. So antimicrobial substances produced by using food-grade material are safe, and have non-toxic effects, and can be used as food additives added directly to food, and can reduce the cost of production.This research studied on the optimization of food-grade medium and fermentation conditions for production of antibacterial substances, by Bacillus subtilis PNG27 fermentation broth pretreatment, antibacterial proteins purification and physicochemical properties, aimed at provideing evidence for developing a new biological preservatives. The main results are as follows.1. Optimizational research of Food-grade medium and fermentation conditions for production of antibacterial substances by Bacillus Subtilis PNG27. Single-factor test was used to screening out the most suitable carbon-nitrogen sources, growth factors and fermentation conditions. Four key factors are screened out by Plackett-Burman experimental design, which were skimmed milk powder, tomato juice, fermentation time and fermentation temperature. Then the Box-Behnken design and response surface analysis were combined to optimize the key factors. The optimal medium composition and fermentation conditions were 15.0g/L of wheat flour,20.0 g/L of skimmed milk powder, 200mL/L of soybean flour enzymatic solution,40 mL/L of tomato juice, 10.0g/L of sodium chloride.The optimal fermentation conditions were 50 mL of liquid volume,32.14℃ of incubation temperature and 60 h of incubation time. Under this condition, Pseudomonas fluorescens inhibition zone diameter of 23.24 mm; Bacillus pumilus inhibition zone diameter of 17.70 mm. The result of verification experiment showed that Pseudomonas fluorescens inhibition zone diameter 23.08 mm, that the inhibitory activities are increased by 1.47 times; Bacillus pumilus inhibition zone diameter 17.22 mm, that the antibacterial activities are increased by 1.42 times, indicating that the model in the range of factor levels is valid.2. Optimizational research of a flocculation method was used to fermentation broth by Bacillus Subtilis PNG27 Three key factors affecting flocculation were screened out by single factor experiments, which are flocculant dosage, pH, addition of diatomaceous earth, and then the key factors were optimized by orthogonal test. According to the orthogonal experiment, the best combination of flocculation condition were as below: 3.0 mL/L of flocculant dosage, pH 9, 10g/L of diatomite dosage,120 min of coagulation time,33℃of flocculation reaction temperature. The recoveries of antibacterial substances were 91.42% and 89.28%, and the residual number of colonies in fermentation broth was 0 cfu/mL.3. Studies on the purifation and characteristics of antibacterial protein produced by Bacillus subtilis PNG27. A protein with molecular weight of about 40 KDa, isolated from cell cultures of strain Bacillus subtilis PNG27. The purification procedure consisted of 20～60% ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and Sephadex G-75 column chromatography. The inhibitory spectrum of the antimicrobial protein from Bacillus subtilis PNG27 on bacteria were determined The results indicated that the antibacterial protein had a wide antibacterial activity, in which it exhibited significant antibacterial activity on Gram-positive bacteria and Gram-negative bacteria. Research result of physicochemical properties of antibacterial proteins showed that the activity of antibacterial protein decreased while the pH incrased in the range of pH2-10. Results of thermal stability study showed that the antibacterial activity of antibacterial protein were lost after treatment at 80℃ for 1.0 h, indicating that the antibacterial protein are more sensitive to temperature; Results also indicated that the antibacterial protein have resistance to trypsin and proteinase K,but more sensitive to alkaline and pepsin protease... |
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