| Lactic acid bacteria is one of the most common probiotic which is widely distributed in nature. This kind of bacteria can be used in food production, health care and feed industry. Lactic acid bacteria can produce lots of bioactive substances, such as bacteriocins, organic acids, kinds of enzymes, and many of these active substances are able to inhibit bacterial. With the social progress and the improvement of living standards, people pay more and more attention on food safety. Chemical additives and antibiotics are usually used in food production, and they seriously affect human’s health. Therefore, looking for safe, stable and efficient antibacterial substance is the main research direction of food additives now. As production of probiotic, the antibacterial substances produced by lactic acid bacteria are generally recognized as safe. Using this kind of substances as food additives will greatly improve China’s food security.Laboratory preserved lactic acid bacteria fmb-9which isolated from kimchi was able to inhibit bacterial. The aim of this research was to screen, activate and also identify lactic acid bacteria fmb-9, analyze the physical and chemical properties of the antibacterial substances produced by this strain, initially study on the separation and purification methods of the antibacterial substances, optimize the medium of production antibacterial substances. This research could provide a theoretical foundation for further development and utilization. The results were described as followed:1. To activate and rejuvenate lactic acid bacteria fmb-9which was in preservation and to identify the activated strain. Colony morphology observation showed typical characteristics of lactic acid bacteria; result of Gram staining experiment was positive; physical and chemical properties research proved this strain was initially identified belonging to Lactobacillus. In the16SrDNA sepuence analysis, the sequence fragment of1437bp16Sribosomal DNA was amplified from total DNA of strain fmb-9by PCR, then the sequence analysis was down by the application of BLAST on the websites of NCBI and the phylogenetic tree of this strain was drawn and analyzed based on the results. The results showed that fmb-9shared99%16SrDNA homology with lots of strains and lactic acid bacteria fmb-9was a species of Lactobacillus rhamnosus.2. The inhibition experiments showed that the antibacterial substance produced by fmb-9had a broad antibacterial spectrum to bacterial, such as Bacillus pumilus. Staphylococcus aureus, Micrococcus luteus, Escherichia coli and many other pathogens, but had no inhibitory effect on fungi. Growth curve results showed that lactic acid bacteria fmb-9growed into logarithmic phrase in4h and growed into stable phrase in16h; the pH of medium continued to decline in fermentation, eventually stabilized at pH3.8; strain began producing antibacterial substances in2h and the amout of antibacterial substances researched the highest value. Exclude protease inhibition experiments and sensitivity experiments showed that antibacterial ability of the fermentation broth was unrelated to organic acid and catalase, and the broth was sensitive to trypsin, a-chymotrypsin, pepsin, and proteinase K, suggesting that this antibacterial substance might have the characteristics of protein. Stability experiments showed that the antibacterial substance produced by fmb-9was stable with treatment of temperature, pH and UV, and it could show antibacterial activity in the range of pH2.0-5.0. This substance would lose its activity when pH was higher than5.0.3. Purification experiments showed that antibacterial substance produced by fmb-9could be purified by acid precipitation, extraction and column chromatography. This substance could not be settled in pH2.0, was soluble in methanol, but hardly in n-hexane and ethyl acetate, and dextran gel column chromatography could separate antibacterial substances in effect. Preliminary separation process:First, adjust the fermentation broth to pH2.0for acid precipitation, then extract broth with n-hexane and ethyl acetate, reconstitute the concentrated raffinate with methanol solution, separate the solution by column chromatography on Sephadex in last. The maximum UV absorption peak of the antibacterial substance was at222nm. It is revealed that this substance had a large polarity by HPLC analysis.4. Tomato medium No.1was chosen to be the initial medium. The experiments of medium components screening showed that the most appropriate fermentation materials were tomato juice, potato juice and corn juice, the optimum carbon source and nitrogen source were glucose and peptone. Glucose and peptone were chosen as key factors in single factor experiments and key factor screening experiments. According to the steepest ascent experiment, central composite experimental and response surface analysis, the optimum amount of each component in the medium were:glucose50.74g/L, soy peptone47.80g/L, tomato juice10ml/L, potato juice20ml/L, corn juice10ml/L, Tween801.0ml/L, K2HPO40.5g/L. In this situation, the diameter of antibiotic circle reached13.55mm which was increased by15.80%over the original fermentation condition. |
PDF Full Text Request