Abstract: To overcome the limitations of traditional chemical cryoprotectants, such as toxic residues and inadequate cryoprotective effect, this study developed a green composite cryoprotectant consisting of natural, non-toxic tannic acid (TA) and trehalose (Tre). Various analysis techniques such as rheological analysis were employed to investigate the effects of different TA/Tre molar ratios on the physicochemical properties and storage stability of the composite cryoprotectant, and its cryoprotective effect on Lactiplantibacillus plantarum XJ-25 (LPXJ-25), isolated from wine. The plate counting method was used to determine the cell viability of LPXJ-25 after cryoprotectant treatment. The results showed that TA and Tre formed a stable supramolecular network structure via hydrogen bonding, endowing the composite cryoprotectant with typical non-Newtonian fluid characteristics. Notably, its glass transition temperature reached −63.321 19 ℃, which was comparable to that of glycerol, a conventional cryoprotectant. The spot assay results confirmed that the optimal molar ratio of TA to Tre was 1:1. After treatment with this composite cryoprotectant and cryopreservation at −80 ℃ for 30 days, the viability of LPXJ-25 reached 76.67%, which was significantly higher than that (34%) observed when using glycerol (P < 0.001). The green composite cryoprotectant eliminates the safety risks associated with traditional chemical cryoprotectants, thereby providing a novel approach for the green cryopreservation of L. plantarum and other food-related probiotics.

Key words: tannic acid; trehalose; Lactiplantibacillus plantarum; cryoprotectant; cell viability