Abstract: 【Objective】In order to obtain optimization formulation of pure cultures in Tibetan kefir. 【Method】The influence of the different mixture of five strains in pure cultures in Tibetan kefir on flavor components in fermented milk were studied using Mixture Design. Regression model on microorganism composition and main metabolites was established.【Results】The results suggested that the predictable production of lactic acid reached the maximum, 8.16g/L,the most predictable production of diacetyl was 77.23mg/L, the most predictable production of ethanol was 4259mg/L,and the production and CO2 was 2.12g/L. Based on these, response values satisfied all expectation were optimized, and the most excellent combination was Lactococcus lactis 27%, Leuconostoc mesenteroides 37%, Lactobacillus kefiri 11%, Lactobacillus casei 10% and Kluyveromyces marxianus 15%.【Conclusion】With the aid of analysis software (Design-Expert6.0.5), formulation of pure cultures in Tibetan kefir could be optimized for several responses and the best formulation could be gotten.

Key words: Mixture Design, Tibetan kefir, pure cultures, optimization