Studies On Biological Characteristics And Protoplasts Formation Of Lactobacillus Bulgaricus

This paper studied the biological characteristics and protoplast formation of Lactobacillus (L. bulgaricus), which established the good study foundation about the culture and protoplast fusion of the strains.Series I The study on the biological characteristics of L.bulgaricus1. The observation of L.bulgaricus colony form and cell shape. According obversation, L.bulgaricus is gram-positive bacteria, non-spore-forming, slim rod. MRS broth is homogeneous cloudy. The colony form changes with the gas condition in which L.bulgaricus cultured.2. In the carbohydrate fermentation test, L.bulgaricus can ferments glucose, milk sugar, D-fructose, D-mannose, sucrose, it can' t fermented cellobiose, mannose, arabinose, galactose, raffinose, maltose.3. L.bulgaricus was cultured in various gas environment, through observing the colony morphology and cell shape, studying the gas effects. In oxygen environment, the colony morphology is translucence,small; the cell shape is slim, curve, long rod. In CO₂ enviroment, the colony morphology is translucence, small; the cell shape is slim curve, long rod. In no-oxygen environment, the colony morphology is wax white, humid, large, the cell shape is thick, straight and short rod. The result showed the optimum growth gas environment is no-oxygen.4. L.bulgaricus was cultured at various pH, through observing the growth of L.bulgaricus, studying the pH effect. The result showed: L.bulgaricus can grow best at pH6 , growing better at pH4, growing worse at pH 4,while it can't survive at pH 2 or pH8. so, the optimum pH is 6.5. L.bulgaricus was cultured at various temperature, through observing the growth of L.bulgaricus, studying the temperature effect. The result showed: L.bulgaricus can grow best at 37℃and 42℃, growing worse at 55℃, while it can't survive at 20℃or 60℃. so, the optimum growth temperature is 37℃and 42℃.6. The preliminary study of growth kinetic of L.bulgaricus has been done through determined OD₆₀₀ and pH in different cultivation time. The curves showed: the lag phase is 0-2h; log phase is 2-16h; the stationary phase and decline phase were afer 16h. During the course of culturing, the pH declines with the culture time goes, when falling below4.2, the pH don't change.7. The effects of various storage methods on the viability and stability of L.bulgaricus were studied. L.bulgaricus was stored with MRS broth, skim milk and glycerin freezing methods. Examining its viability and stability every other time through carbohydrate fermentation acid test. The result showed: the viability time is more long and the stability is more high of storing with skim milk and glycerin freezing.Series II The preliminary study on the protoplast preparation of L. bulgaricus1. The comparing of two judging methods about the rate of L. bulgaricus protoplast formation. Calculating the rate of protoplast formation in same hour with the methods of plate dilution and hematocyte count. The result indicated that the production frequency above 10% with plate dilution to with hematocyte count. Because the process of preparation and operation was much trouble, the hematocyte count was adopted.2. The selecting of the optimum concentration of mutanolysin. Formation protoplast with different concentration of mutanolysin. After 30min, Calculating the rate of protoplast formation immedicately. The result showed: the rate of proplast was 31.5% 56.1% 100% respectively, while the concentration of mutanolysin was 2ug/ml, 4ug/ml, 6ug/ml respectively. At las, the 4ug/ml of concentration was used.3. The selecting of the optimum time. Calculating the rate of protoplast in different time while the concentration of mutanolysin was 4ug/ml. The rate increases with the time going, Afer 75min, the number don't increase. Becase the time and regeneration rate were inverse ratio, the 45-60min was the optimum time.4. The effect of glycine on production rate of protoplast. Adding glycine (1mg/ml) to the MRS broth during the course of cultivation of L. bulgaricus, without glycine as the control. Comparing the rate of protoplast formation of adding glycine and the control at the same condition. The results was that the formation rate of adding glycine higher than without , Which indicated glycine can promote obviously the rate of protoplast formation of L. bulgaricus.