Isolation And Breeding Of Lactic Acid Bacteria From Tibet Kefir Milk

Lactic acid bacteria is the most direct and important factor in the production of fermented milk,which determines the nutritional and sensory indexes of fermented milk.Tibet kefir is a kind of traditional fermented milk in Linzhi area of Tibet,which is a little different from the famous Kefir,while their essence and structure are similar.Tibet kefir has a unique flavor and regional characteristics,so it is very feasible and practical to find a potential probiotic Lactic acid bacteria strain as a milk fermental starter.In this study,79 strains of lactic acid bacteria were isolated from Tibet kefir milk.The M14,M16 and G60 of three strains of lactic acid bacteria produce transparent rings in the casein plate,which mean that they produced protease;the morphological characteristics and physiological and biochemical characteristics and identification of three strains of Lactobacillus casei,Lactobacillus plantarum and Lactobacillus pentosus.The maximum concentration of three strains could reach 10¹⁰ cfu/mL;All of the three strains of lactic acid bacteria could be grown in pH 1.5 environment,and could tolerate 1.2% bile salts.The activity of protease of M16 was relatively stronger in three strains,and the protease activity was 17.163 ?g/?mL·min?at pH 7.2.The optimum conditions for the preparation and regeneration of M16 protoplasts were as follows: the lysozyme concentration was 0.5 g/mL,the treatment temperature was 35 ?,and the treatment time was 30 min.Under these conditions,the rate of protoplast preparation was low,55.6%,while the regeneration rate was relatively high,42.2%.The lethal dose of UV mutagenesis was investigated.The 90% lethal dose was 15 W,the distance from ultraviolet lamp was 30 cm,and the treatment time was 280 s.Adding lithium chloride would increase the positive mutation rate,so the addition amount was determined,0.5%.With M16 as the starting strain,the protoplast was induced by UV-LiCl combined mutagenesis.The colonies were screened from casein plate,which the colonies of transparent rings were larger than that of the strain M16.A total of 24 positive mutanted strains were identified and numbered M1601-M1624.Among them,M1624,M1611 and M1608 three strains had high OD600 value of their medium,means vigorous growth,compared with the starting strain M16 increased significantly;Among five strains of lactic acid bacteria,acid producing of M1619 and M1624 were significantly lower than start strain M16;acid tolerance and bile salt tolerance ability of five strains and start strain M16 showed no significant difference;five strains of lactic acid bacteria had the genetic stability protease activity,almost no decline in five generations.Protease activity of M1611 was highest in five strains of lactic acid bacteria,which was 21.463 ?g/?mL·min?in the fifth generation,increased by 25% relatived to start strain M16;while the capability of producing acid,low pH and bile salt tolerence of M1611 and start strain M16 showed no significant difference.