Abstract:[Background] Synergetic metabolism of multi-microbial strains is one of the essential feature of fermentation process for Chinese strong aroma Baijiu. [Objective] To study the relationship between functional microbial organisms in the fermentation process of multi-grain strong aroma Baijiu, which could provided theoretical references for optimization of fermentation process. [Methods] The typical yeasts and Lactobacillus acetotolerans in the fermentation process of multi-grain strong aroma Baijiu were isolated by culture-dependent method, and the relationship between these yeasts and L. acetotolerans strains and variation of major volatile metabolites were also discussed. [Results] Three typical yeast strains (Kazachstania humilis Z1, Pichia kudriavzevii Z2, Candida ethanolica Z3) and one Lactobacillus acetotolerans strain (L. acetotolerans W) were isolated. The number of L. acetotolerans W in the co-culture medium of K. humilis Z1 and L. acetotolerans W (Z1&W) was significantly less than that in the single culture medium of L. acetotolerans W. All of three yeast strains could inhibit the lactic acid production ability of L. acetotolerans W, and no lactic acid was produced in co-culture medium of Z1&W. The volatile profile of single Z1 was similar with that in Z1&W. Obvious difference in volatile profile was monitored between single Z2 and Z2&W, in which, concentrations of ethyl acetate and ethyl lactate in Z2&W were significantly increased. [Conclusion] Under the condition of co-culture system, ethanol-producing yeast had inhibition effect on lactic acid metabolism of L. acetotolerans, and L. acetotolerans had certain influence on ethanol metabolism of yeast. It was of great significance for the quality control of multi-grain strong aroma Baijiu and its relationship with the microflora.