This study describes the optimization of the formulation of cryoprotectants for Bifidobacterium lactis Z-1 using response surface methodology (RSM). Using the Plackett-Burman design, trehalose, sucrose, L-cysteine hydrochloride concentration were identified as factors with a significant influence on the viable count of lyophilized Bifidobacterium lactis Z-1. Subsequently, the optimal response ranges of the three factors were determined using the steepest ascent method. The levels of these factors were further optimized using RSM with Box-Benhnken design. As a result, a regression model was fitted into the experimental data. The optimal levels of trehalose, sucrose and L-cysteine hydrochloride were determined as 200.51, 46.16 and 2.31 g/L, respectively, while the other ingredients were the same as those in the traditional recipe (skimmed milk powder 100 g/L, whey protein powder 20 g/L, glycerol 4 g/L, sodium isoascorbate 10 g/L and sodium glutamate 8 g/L).

A total of 30 food-grade Lactobacillus strains isolated from naturally fermented products from Tibetan, Xinjiang and Qinghai regions including Lactobacillus rhamnosus LGG (as a control) were evaluated for acid and bile salt tolerance, hydrophobicity, surface charge and auto-aggregation ability. Out of these, 16 strains were selected and further evaluated for tolerance to artificial gastric and intestinal fluids. The results showed that the survival rates of 4 of the 16 strains were greater than 14.44% after 60 min of incubation in artificial gastric fluid and were 3.91%–100.00% after 30 min. For all 16 selected strains, the survival rate was 31.73%–112.83% after 5 h, and Z-1 had the strongest tolerance to artificial gastric fluid of these strains with a 93.07% survival rate after 60 min. The survival rate of Z-1 was 106.5% after 5 h of incubation in artificial intestinal fluid, hydrophobicity 97.52%, surface charge 97.35% and 24.67% in the presence of chloroform and ethyl acetate, respectively, and auto-aggregation ability 11.26%.