Improved Viability of Limosilactobacillus reuteri under Freeze-Drying, Simulated Gastrointestinal, Thermal, and Storage Conditions through Optimized Cryoprotectant Formulations

Viability loss of probiotics during the freeze-drying process can significantly reduce their functional performance in drug and food products. In the present study, the simplex lattice design was utilized to optimize cryoprotective formulations to enhance the survival of Limosilactobacillus reuteri on freeze-drying, under simulated gastrointestinal, thermal, and storage conditions. Maltodextrin, mannitol, and saccharose were selected as cryoprotectants. The results revealed that the optimal microencapsulation efficiency (97.25%), freeze-drying yields (97.74%), and viability under in vitro gastric and intestinal conditions (85.84%) were achieved with a formulation consisting of 66.61% maltodextrin, 15.16% mannitol, and 18.23% saccharose. The absence of cryoprotective agents during freeze-drying led to a 3.88% reduction in L. reuteri viability. The highest stability was observed in the optimized composite coated with O-carboxymethyl chitosan, where only 1.06 log CFU/g of bacterial loss occurred after 42 days of storage at 4°C. The presence of the maltodextrin–mannitol– saccharose composite formed a protective layer that improved the microcapsule structure, enhanced the resistance of probiotics to thermal stress, and significantly improved their stability during storage. Overall, the results indicate that the optimized composite formulation can be an effective strategy for improving the viability and stability of probiotics under various adverse conditions.