Co-enzymatic hydrolysis of camel milk casein releases active peptides through gut microbiota remodeling and immune restoration

Camel milk casein is a potential high-quality source of bioactive peptides; however, its immunomodulatory potential and mechanisms of action remain unclear. This study employed a multi-enzyme synergistic hydrolysis strategy aimed at efficiently releasing immunologically active peptides from camel milk casein. Firstly, a composite evaluation index (CE) based on degree of hydrolysis (DH) and RAW 264.7 macrophage proliferation rate was established. Combining this with response surface methodology (RSM) to optimise the process, papain and α-chymotrypsin (2:1) were identified as the optimal enzyme system, with the optimal conditions being pH 6.5, 50 °C, 2.2 h of enzymatic hydrolysis (predicted CE value 88.22, validated value 85.32 ± 0.96). In vitro experiments demonstrated that the enzymatic hydrolysate (M) significantly promoted macrophage proliferation, inhibited excessive NO release, and bidirectionally regulated the balance between pro-inflammatory (TNF-α, IL-6) and anti-inflammatory (IL-10) factors. In a cyclophosphamide-induced immunosuppressed mouse model, intervention with M effectively reversed weight loss and atrophy of immune organs, inhibited splenic lymphocyte apoptosis, and restored serum immunoglobulin (IgG, IgA, IgM) levels. Notably, 16S rRNA sequencing revealed that M exerts its immunoprotective effects by reshaping the gut microbiota, increasing the abundance of Lactobacillus and Bifidobacterium genera. In summary, this study not only established an efficient process for the proteolytic hydrolysis of camel milk but also elucidated the potential mechanism by which it improves immune function via the ‘gut microbiota–immune axis’, thereby providing a theoretical basis for the high-value development of camel milk resources.