| Lactic acid bacteria and their metabolites which could inhibit the growth and reproduction of some pathogenic bacteria or spoilage bacteria have great potential for development and application as antibacterial agents.So far,there are only several bacteriostatic agents derived from lactic acid bacteria are allowed to be used in the food industry,such as Nisin,Natamycin,and Kojic acid.Lactic acid bacteria with antibacterial ability were selected from the isolated strains which were preserved by the key laboratory of dairy biotechnology and safety control of Jiangsu province.And the antibacterial properties of the supernatant and the surface of the bacteria were analyzed.The results were as followed:(1)Screening,classification and identification of lactic acid bacteria with antibacterial ability.Using Bacillus subtilis,Staphylococcus aureus,Bacillus cereus,Salmonella,Escherichia coli and Pseudomonas as the indicator bacteria,65 lactic acid bacteria with antibacterial ability were selected from 102 strains and the ratio was 63.72%.Then,6 strains with high inhibitory effect on Bacillus subtilis and Salmonella were reselected by regulating the pH to 5.0.Meanwhile,strain YT showed the highest antibacterial ability to the indicator strains.By morphology observation and 16S rDNA identification.strain YT was identified to be Lactobacillus rhamnosus and named as Lactobacillus rhamnosus YT.(2)Analysis of the antibacterial properties of Lactobacillus rhamnosus YT culture supernatant.The effect of pH,temperature and enzyme treatment on the antibacterial ability of the culture supernatant was measured.The antibacterial ability of the culture supernatant was proved to be narrow pH tolerance and valid as the pH lower than 5.0.Moreover,the antibacterial ability of the culture supernatant was stable at 3 0??100?,and the antibacterial ability against to Bacillus subtilis and Salmonella were still retained 95.44%and 95.99%after the treatment of 100? for 15 min.Furthermore,the antibacterial ability of the culture supernatant was showed low sensitivity to the treatment of pepsin,papain and proteinase K and obvious sensitity to catalase.Finally,the supernatant was extracted by ethyl acetate and the obtained the substance with strong antibacterial ability was obtained.Using Sephadex-G100,the 30th?36th tubes with antibacterial ability were seperated and then lactic acid(44.07 mg/mL)was detected by HPLC.Combing with the reverse complementation test,it was suggested that the antibacterial ability of the culture supernatant was caused by lactic aicd as well as other low-molecular antibacterial substances.(3)The effect of carbon and nitrogen sources and culture conditions on the antibacterial ability of Lactobacillus rhamnosus YT cells.The antibacterial activity of bacteria fermented with maltose as carbon source is higher than that of glucose,lactose and rhamnose.The number of viable bacteria and the antibacterial ability of the bacteria of Lactobacillus rhamnosus YT fermented with tryptone and soybean peptone as nitrogen sources were higher than that of fish meal peptone.When the and the culture temperature is,the of the strain cells showed the highest antibacterial ability when the cells were cultivated at 27? with initial pH 7.0.Furthermore,the surface of Lactobacillus rhamnosus YT bacteria was covered with a thick transparent material by staining observation.Then,phenol,LiCl and ultrasonic treatments were used to extract active substances from the cell surface.And substances extracted by ultrasound was proved to be strong antibacterial ability.The ultrasonic extract and the indicator bacteria were co-incubated,the lag periods of Bacillus subtilis and Salmonella were delayed by 4 h and 2 h,respectively.Simultaneously,the growth rate and biomass of the indicator bacteria were significantly lower than that of the control group.(4)Analysis of the physical and chemical properties of the ultrasonic extract of Lactobacillus rhamnosus YT bacteria.Ultrasonic extract was verified to be sensitive to high temperature owing to its antibacterial ability to indicator bacteria decreased by 20.57%-30.18%with the treatment at 80??100? for 15 min.The antibacterial ability of the ultrasonic extract is stable under pH 3.0?9.0 and the antibacterial ability at pH 7.0?9.0 buffer was slightly higher than that of pH 3.0-5.0.The antibacterial ability of the ultrasonic extract was demonstrated unsensitive to the treament of pepsin,trypsin,papain,?-amylase and ?-amylase.The ultrasonic extract was able to significantly reduce the surface tension of the liquid(72.771 mN/m?51.011 mN/m)and its critical micelle concentration(CMC)value was 6.88 mg/mL.Moreover,the ultrasonic extract was showed emulsifying activity to n-hexane,isooctane,xylene,rapeseed oil and olive oil.In addition,the ultrasonic extract was performed strong emulsification stability to p-xylene.After static placing for 120 h and 24 h,the emulsification activity was only reduced by 5.77%.It was indicated that the antibacterial substance extracted from Lactobacillus rhamnosus YT cell surface of bacteria was a kind of bio surfactant. |
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