Structural Properties and Anti-Inflammatory Activity of GLP-P, a Kefir-Derived Neutral Glycopeptide

Bioactive glycopeptides, commonly present in natural foods, exhibit notable immunomodulatory and neuroprotective effects. However, naturally occurring low-molecular-weight glycopeptides have rarely been reported, and their structural and functional properties remain insufficiently explored. In this study, a low-molecular-weight glycopeptide complex (GLP-P) was isolated from kefir. The structure of GLP-P was characterized via molecular weight (Mw) determination, monosaccharide and amino acid composition analysis, Fourier transform infrared spectroscopy, methylation analysis, and nuclear magnetic resonance spectroscopy. GLP-P had a molecular weight of 1192 Da and mainly consisted of four monosaccharides (glucose 64.7%, galactose 34.4%, and others 2.0%) and eight amino acids (asparagine 30.89 ± 0.01 μg/mg, threonine 8.71 ± 0.04 μg/mg, serine 9.5 ±0.08 μg/mg and others.). The primary chain structure of GLP-P contained β-D-Galp-(1→4)-β-D-Glcp-(1→4)-α/β-D-Glcp linkages, including both α- and β-D-Glcp isomers. Notably, the HMBC spectrum of GLP-P exhibits putative O-glycosylation characteristics. Functionally, GLP-P significantly reduced IL-6 and TNF-α expression while enhancing IL-10 in LPS-stimulated RAW264.7 macrophages. These effects were associated with inhibition of the TLR4/NF-κB pathway. These findings suggest that low-molecular-weight GLP-P has shown potential efficacy in the treatment of inflammation in vitro. These results provide a theoretical basis for kefir glycopeptide development of functional foods and as an adjuvant therapeutic agent for inflammation-related disorders in the future.