Synthesis of acetylated raffinose and its mechanism of action in regulating gut microbiota to alleviate cyclophosphamide-induced immunosuppression in mice.

Raffinose (Raf) exhibits antioxidant, hepatoprotective, and immune-enhancing activities; however, its inferior bioactivity and poor prebiotic performance constrain its industrial applications in food, pharmaceuticals and related fields. To enhance the bioactivity of Raf and simultaneously increase the high-value utilization of agricultural waste streams-specifically cottonseed processing waste liquid-this study employed Raf isolated and purified from cottonseed processing waste liquid to prepare acetylated raffinose (Ac-Raf) via acetylation modification. The maximum degree of substitution (DS) of Ac-Raf was 0.670, and structural characterization revealed that acetyl groups were preferentially attached to the C-1 position of glucose/galactose units. Subsequently, the mechanism by which Ac-Raf ameliorates cyclophosphamide (CTX)-induced immunosuppression was investigated. The results demonstrated that Ac-Raf reshaped the gut microbiota balance, increased short-chain fatty acid (SCFA) levels, modulated immune factor expression, and repaired intestinal barrier function, exhibiting significantly superior efficacy compared to unmodified Raf. Furthermore, correlation analysis of immune factors revealed the key mediating role of the "microbiota-SCFAs-immunity" axis. Collectively, acetylation modification markedly enhances the prebiotic activity of Raf, providing a theoretical support for the development of targeted immunomodulatory products derived from cottonseed processing waste liquid, and also offers technical ideas and methodological references for improving the biological activity of oligosaccharides through modification strategies.