Development and functional assessment of a broad-spectrum cryoprotectant for preserving next-generation probiotics

While next-generation probiotics (NGPs) are increasingly recognized for their health benefits, their incorporation into food products is severely hindered by challenges in preservation. Freeze-drying (lyophilization) is the standard method for preserving probiotics, yet oxygen exposure and freeze-drying stresses substantially reduce bacterial survival rates, particularly for anaerobic probiotics. In this study, we developed a broad-spectrum lyophilization medium (LM) for both conventional and next-generation probiotics. The formulation, consisting of skim milk, cryoprotective sugars and antioxidants, was systematically optimized, and evaluated for its cryoprotective efficacy and underlying mechanisms across a panel of conventional (Lactobacillus and Bifidobacterium strains) and emerging (Akkermansia muciniphila, Bacteroides uniformis, Gordonibacter urolithinfaciens-G. uro) probiotics. Results show that, first, the optimal LM significantly increased the survival rates of all strains, particularly oxygen-sensitive strains, with nearly 20% increase in G. uro. All freeze-dried bacteria showed no significant viability loss after six months of storage at 4 °C. Next, scanning electron microscopy and flow cytometry analyses confirmed retained cell membrane integrity during freeze-drying, and the significantly attenuated oxidative stress in anaerobes with ascorbic acid supplementation. Functional assessments demonstrated preservation of key bacterial metabolic activities, including efficiency in short-chain fatty acid production and polyphenol catabolism, comparable to fresh cultures. Lastly, mouse study confirmed that freeze-dried G. uro remains metabolically active in producing urolithins at comparable levels to fresh cultures. To our knowledge, this work presents the first broad-spectrum LM that effectively preserves the viability and metabolic activity of strict anaerobes, providing a scalable solution for the development of NGP-integrated functional foods and health products.