Enhanced preservation of viability and species stratification in Lacticaseibacillus group using levan-fortified skim milk as a cryoprotectant during freeze-drying

Ensuring the viability and accurate stratification of Lacticaseibacillus group (LCG) species after freeze-drying is essential for their effective use as probiotics. This study investigates the use of reconstituted skim milk (RSM) as a cryoprotectant base, supplemented with fructans such as inulin and diverse forms of levan from Halomonas smyrnensis, to maintain the viability of Lacticaseibacillus casei ATCC 334, Lacticaseibacillus paracasei ATCC 25302, and Lacticaseibacillus rhamnosus ATCC 53103. Cellular viability was enhanced with levan-based cryoprotectants, motivating the use of levan-based hydrogels (gHLs) for freeze-drying LCG species. Throughout freeze-drying, the species-specific molecular masses (m/z) were preserved irrespective of the cryoprotectant used, with markers 3445 and 6664 m/z identified as potential species-specific molecular mass indicators for Lc. paracasei and Lc. rhamnosus, respectively. This study is the first to utilize levan in various forms as a cryoprotective agent alongside RSM, highlighting its promise as an effective cryoprotectant for LCG and potentially other probiotics.