| Bacterial peptidoglycan hydrolases (autolysins) degrade the peptidoglycan of cell walls, causing cell autolysis. Autolysis of lactococci used as starter cultures in the manufacture of fermented milk results in the leakage of lipases, proteases and peptidases and other intracellular components, which play an important role in flavor development during ripening. Hence, autolysis properties of lactic acid bacteria are crucial for their applications as dairy starters.To establish a rapid and sensitive method to detect autolysis rate of lactic acid bacteria, A Flow Cytometric Method (FCM) can be employed. Cell suspensions were dyed 30 min by PI-PBS (20 mmol/L) in 4℃dark environment. A 630 nm long pass filter was used to collect the red fluorescence (FL3 488 nm), and 1×105 cells were collected each sample. Data were analyzed by CellQuest online analysis system. Test time depended on different concentrations of cell suspensions, were about 40 s to 100 s. FCM enumerations were accurate down to a concentration of 104 cells/mL. Correlation test results reported that there were no significant difference between FCM test and other normal methods for the determination of autolysis rate of lactic acid bacteria. FCM by use of fluorescent probes propidium iodide (PI) is a good analytical method that allows the rapid and sensitive measurement of viability of lactic acid bacteria. This FCM method provides tools to assess the viability of different lactic acid bacteria in fermentation starters and probiotic products.In order to obtain different autolysis rate lactic acid bacteria, Streptococcus salivarius ssp., Lactobacillus delbrueckii ssp. Bulgaricus, Lactobacillus lactis, Lactobacillus casei, and Lactococcus lactis ssp. Lactis were mutated by 50 keV N+ ions implantation. The results indicate that the survival rate curve took a saddle shape and there was a high positive mutation rate by the dose of 4×2.6×1013 ion/cm2. The survival rate was 25%～33% under this implantation condition. Strains which have different autolyisis rates were obtained among the mutated strains whose mutation rate in the range of -51.96%～+151.24%. Fermentation property of these strains were stable after 7 generation transfer inoculation. It was indicated that the ion implantation technique is a feasible method in breeding lactic acid bacteria starter strains.Autolysis rate of different strains were determined under various conditions of incubated temperature and pH values, as these factors were closely related to cell viability and flavor development in the products. Autolysis rate increased when temperature and pH values were rasing in a certain range. Over higher or lower temperature and pH value would inhibit autolysis of strains. But there were some strains indicate high autolysis in extreme low pH. Mg2+ could affect autolysis rate of lactic acid bacteria. Strains showed higher autolysis when they were suspended in 0.1 mol/L magnesian phosphate buffer. Lipase and protease activities were closely related to the sort of strains. Time of reaching the highest activities for lipase and protease depended on the autolytic degree of strains. All Lactococcus lactis ssp. Lactis strains showed higher lipase activities, and protease activities in Lactobacillus delbrueckii ssp. Bulgaricus, were mostly higher than other strains. These properties should be considered when commercial starters are selected.The occurrence of the acmA gene, encoding the major peptidoglycan hydrolase of lactic acid bacteria, has been determined in 14 Lactococcus lactis ssp. Lactis and Lactococcus lactis ssp. cremoris strains. On the basis of PCR amplification of the gene acmA, 12 strains exhibited a single 1,100 bp fragment highly correlated with N-acetylmuramidase of Lactococcus lactis. There was discrepancy between the high autolysis level strains and low auloysis level strains. Using fluorescent quantitative real-time PCR testified that autolysis rate was closely related with the transcript level of N-acetylmuramidase.Coagulation time decreased significantly by using high autolysis rate lactic acid bacteria fermented yogurt, were positivly related to acid producing ability of lactic acid bacteria starters. There was no raletive correlation between autolysis rate of strains and water holdup, viscosity and flavor compounds of yogurt. However, yogurt acidities were significant infuluenced by autolysis rate of lactic acid bacteria staters, especially in the simulated shelf life of 10℃~ 15℃storage conditions. High autolysis rate starters could decrease acidification level of yogurt.A dratic orthogonal rotary composite design is employed to study the effects of characters of yogurt fermented by high autolysis strains in mixed starters. According to regression analysis to strains GS1-A8, LD3-A3 and S15 -A3 coding value (X1, X2, X3) as independent variables, the shelf life of yogurt acidity changing degree (Δ0T) Y1 and sensory evaluation scores Y2 as the dependent variable, a regression equation were established. The optimum combinated scheme of three strains of programs was selected according the model: GS1 91.46%, LD3-A3 2.44%, S15-A3 6.10%. Yogurt fermented by the selected optimum starter has favorable sensory properties, good flavor and texture and lower acidification level.
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