Heat-killed Lacticaseibacillus casei OS1423 retains immunoprotective functionality across ingredient and adlay tea formulations.

The development of functional microbial ingredients that retain probiotic-associated benefits without reliance on cellular viability is of growing interest in functional food design. In this study, heat-killed preparations of Lacticaseibacillus casei OS1423 and L. paracasei OS1213 were evaluated to determine whether they could retain immunoprotective activity after heat inactivation while offering advantages for food application. Using a cyclophosphamide (CP)-induced immunosuppressed mouse model, both live and heat-killed forms were assessed for their ability to modulate selected immune parameters. The heat-killed preparations partially restored selected cytokine responses, splenocyte proliferative activity, and systemic immunoglobulin production, indicating that strain-associated immunomodulatory activity was retained in selected readouts despite the loss of viability. Supplementation was also associated with gut microbial reorganization, particularly through partial recovery of bacterial groups commonly linked to intestinal homeostasis and SCFA-producing microbial guilds, although functional predictions were interpreted as inferred and exploratory. To evaluate food application feasibility, heat-killed L. casei OS1423 was incorporated into an adlay (Coix lacryma-jobi L.) tea matrix. The fortified beverage retained selected immunomodulatory activities of HK_OS1423, particularly mucosal cytokine recovery, splenocyte proliferation, and serum IgA restoration, supporting its applicability in grain-based functional beverage matrices. Collectively, these findings support the potential of heat-killed probiotics as functional microbial ingredients and highlight their practical relevance for grain-based beverages designed to support immune health.