| Summary: | Lyophilization is one of the most used methods for bacterial preservation. In this process, the cryoprotectant not only largely decreases cellular damage but also plays an important part in the conservation of viability during freeze-drying. This study investigated using cryoprotectant and a mixture of the cryoprotectant to maintain probiotic activity. Seven probiotic strains were considered: (<i>Limosilactobacillus reuteri</i> KUKPS6103; <i>Lacticaseibacillus rhamnosus</i> KUKPS6007; <i>Lacticaseibacillus paracasei</i> KUKPS6201; <i>Lactobacillus acidophilus</i> KUKPS6107; <i>Ligilactobacillus salivarius</i> KUKPS6202; <i>Bacillus coagulans</i> KPSTF02; <i>Saccharomyces cerevisiae</i> subsp. <i>boulardii</i> KUKPS6005) for the production of a multi-strain probiotic and the complex medium for the lyophilized synbiotic production. Cholesterol removal, antioxidant activity, biofilm formation and gamma aminobutyric acid (GABA) production of the probiotic strains were analyzed. The most biofilm formation occurred in <i>L. reuteri</i> KUKPS6103 and the least in <i>B. coagulans</i> KPSTF02. The multi-strain probiotic had the highest cholesterol removal. All the probiotic strains had GABA production that matched the standard of γ-aminobutyric acid. The lyophilized synbiotic product containing complex medium as a cryoprotectant and wall material retained a high viability of 7.53 × 10<sup>8</sup> CFU/g (8.89 log CFU/g) after 8 weeks of storage. We found that the survival rate of the multi-strain probiotic after freeze-drying was 15.37% in the presence of complex medium that was used as high performing wall material. Our findings provided a new type of wall material that is safer and more effective and, can be extensively applied in relevant food applications.
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