Mechanism analysis of calcium-induced low freeze-drying survival of probiotic encapsulated in alginate

Calcium alginate is widely used for probiotics encapsulation. In our previous report, low freeze-drying survival of probiotics encapsulated in calcium alginate was presented due to the damage by calcium with increased concentration during freeze-drying. The damage makes freeze-dried probiotic-loaded calcium alginate difficult to use in commercial foods. Therefore, it is urgent to analyze the mechanism of calcium-induced low survival of probiotics. In the present work, this mechanism was analyzed using Lactobacillus rhamnosus GG (LGG) as a research model. The results showed that calcium led to the emergence of 491 differential genes (300 upregulated genes), 309 (175 up-regulated metabolites, positive ion) and 129 differential metabolites (33 upregulated metabolites, negative ion). For one of the mechanisms, GO and KEGG analysis of differential genes indicated that calcium increased cardiolipin by downregulating glpO and upregulating LGG-RS07795 and cls genes in the glycerophospholipid metabolism pathway, increasing the fluidity of cell membrane, thereby causing the catabolism of cell membrane components. Additionally, the disturbances of differential genes/metabolites co-enrichment pathways by calcium were other mechanisms. Specifically, the correlation analysis of differential genes/metabolites that had upstream/downstream relationships in co-enrichment KEGG pathways showed consistently strong correlations, among which hisC gene regulated histidine metabolism pathway and phenylalanine, tyrosine and tryptophan biosynthesis pathway, ndk gene regulated pyrimidine metabolism pathway and purine metabolism pathway, and 3-phosphoserine, phenylalanine, and tyrosine were involved in multiple pathways including other differential genes/metabolites co-enrichment KEGG pathways. Based on the above mechanism analysis, the addition of small molecule sugar/sugar alcohol and down-regulated differential metabolites (ergothioneine and cytidine) in the co-enrichment pathway were effective strategies to improve the freeze-drying survival of LGG encapsulated in calcium alginate. Overall, these findings are expected to promote the application of dried calcium-based probiotics foods in the food industry.