Proteomic insights into stress responses and adhesion markers of co-encapsulated Lactobacillus reuteri DPC16 with Cyclocarya paliurus leaf extracts during in vitro gastrointestinal tract simulation (GITS)

Abstract

This study explores the mucin-adhesive properties and the proteomic changes in L. reuteri DPC16 co-encapsulated with Cyclocarya paliurus (CP) leaf extracts by hempseed protein isolate. The mucin adhesion ability of L. reuteri DPC16 was assessed and visualized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Novel microcapsules containing L. reuteri DPC16 and CP leaf extracts were subjected to sequential simulated gastrointestinal conditions, including gastric, duodenal, and ileal phases, with corresponding pH adjustments and digestive enzymes. Surface proteins from L. reuteri DPC16 were extracted at each stage, separated by SDS-PAGE, and analyzed using LC-ESI-MS/MS to identify and quantify differentially expressed proteins. Over 200 surface proteins were identified, with significant upregulation of stress-responsive proteins such as heat shock proteins and enzymes involved in cell wall synthesis. These, along with proteins associated with metabolic pathways, contributed to the stability and activity of the probiotic under gastrointestinal conditions. CP leaf extracts further enhanced oxidative stress resistance and modulated protein expression to boost probiotic efficacy. Notably, increased expression of mucosal adhesion-related proteins, including mucus-binding and moonlighting-binding proteins, suggested an enhanced ability of L. reuteri DPC16 to colonize the gut. Proteomic analysis provided comprehensive insights into the molecular dynamics of L. reuteri DPC16, uncovering its adaptive mechanisms and functional potential in diverse environments. These findings highlight the value of protein markers in validating the health-promoting properties of L. reuteri DPC16, supporting its application in functional foods and therapeutic supplements.