Litopenaeus vannamei protein (LVP) is a high-quality protein. However, its functional properties do not fully meet the needs of food processing. In this study, LVP-xylose conjugates were prepared by conventional wet heat method (GLVP) and ball-milling-assisted wet heat method (GBLVP), respectively. The changes in structure and functional properties of the glycosylated LVP were explored. The findings revealed that ball-milling pretreatment increased the grafting degree to 35.21%. GBLVP had a sparser surface structure and lower particle size than GLVP. FTIR spectra showed that xylose was grafted onto LVP successfully and GBLVP had the lowest α-helix content. Compared with GLVP, GBLVP had a decrease in intrinsic fluorescence intensity and surface hydrophobicity, and an increase in UV absorption intensity. Moreover, GBLVP had higher foaming capacity, solubility and water-holding capacity, and lower allergenicity than GLVP. However, ball-milling pretreatment had a negative impact on the vitro digestibility and oil-holding capacity of GBLVP. In conclusion, ball-milling-assisted treatment of glycosylation could effectively improve the functional properties of LVP, benefiting the broader application of LVP in the food industry.

In this study, we investigated the structural features of the polysaccharide obtained from Craterellus cornucopioides (CCP2) by high-performance liquid chromatography, Fourier transform infrared spectroscopy and ion chromatography. The results showed that CCP2 was a catenarian pyranose that principally comprised of mannose, galactose, glucose, and xylose in the ratio of 1.86: 1.57: 1.00: 1.14, with a molecular weight of 8.28 × 10⁴ Da. Moreover, the immunoregulation effect of CCP2 was evaluated both in vitro and in vivo. It displayed a remarkable immunological activity and activation in RAW264.7 cells by enhancing the phagocytosis of macrophages in a dose-dependent manner without showing cytotoxicity at the concentrations of 10–200 μg/mL in vitro. Additionally, Histopathological analysis indicated the protective function of CCP2 against immunosuppression induced by cyclophosphamide (Cy). Meanwhile, the intake of CCP2 had better immunoregulatory activity for immunosuppression BALB/c mice model. After prevention by CCP2, the spleen and thymus weight indexes of BALB/c mice model were significantly increased. The RT-qPCR and Western Blot results provided comprehensive evidence that the CCP2 could activate macrophages by enhancing the production of cytokines (IL-2, IL-6, and IL-8) and upregulating the protein expression of cell membrane receptor TLR4 and its downstream protein kinase (TRAF6, TRIF, and NF-κB p65) production of immunosuppressive mice through TLR4-NFκB p65 pathway. The results demonstrated that CCP2 could be a potential prebiotic and might provide meaningful information for further research on the immune mechanism.