Liaocheng University

Dietary oils significantly influence the modification of milk lipid profiles and volatile compounds (VOCs). In this study, donkeys were fed varying ratios of soybean oil to linseed oil, and the milk were analyzed for lipids and VOCs. A total of 1,167 lipid species were detected in donkey milk, belongs to 20 sub-classes. Sixty-four different lipids were screened and were most relevant to glycerophospholipid metabolism and linoleic acid metabolism. The level of C18:3 increased with higher linseed oil supplementation. In addition, 406 VOCs were identified in donkey milk, categorized into 15 sub-classes, mainly 80 hydrocarbons and 56 heterocyclic compounds. Eighty-nine differential VOCs were identified, mainly heterocyclic compounds, hydrocarbons, and alcohols. These VOCs were characterized by fruity, oily, grassy, and sweet aromas. A 2:3 dietary ratio of soybean oil to linseed oil significantly enhances the lipid quality and modulates the profile of VOCs in donkey milk. This study provides comprehensive insights on the impact of soybean and linseed oils blends on the lipid and VOC profiles, improving donkey milk's nutritional quality.

The interaction mechanism between chlorogenic acid (CGA) and/or selamectin (SEI) and immunoglobulin G (IgG) was studied by multispectral techniques and molecular docking, and the effect of the complex formation on the protein's functional properties was then evaluated. The results showed that CGA or SEI binds to the different site on IgG chain through different binding forces, which induces different influence on the protein's secondary structure. The interaction of IgG with CGA is higher than that of IgG with SEI due to the multiple hydroxyl groups, the flexible and compact structure of CGA. When two drugs coexist, the pre-added drug can all decrease the interaction intensity between the subsequently added drug and IgG. The results of the biological activity experiments showed that CGA exhibits excellent antioxidant and antidiabetic activities, while SEI displays excellent antibacterial and antidiabetic performance. These biological activities were affected in varying degrees by the drug combination and the related complex formation with IgG. In addition, the antiviral potential of CGA and SEI was preliminary predicted by molecular docking, and their strong binding to SARS-CoV-2 main protease (M^pro) may indicate the good antiviral activity of the drug combination. It is expected to provide theoretical support for developing more efficient antibacterial and antiviral drugs by the results of this research, and for rational drug co-administration in treating specific diseases.

Milk-derived extracellular vesicles (MEVs) modulate metabolic and immune processes. Donkey milk, given its compositional similarity to human milk and documented bioactivities-including antioxidant, anti-inflammatory, and anti-cancer properties-represents a promising source for therapeutically relevant EVs. However, standardized protocols for efficient donkey milk EVs isolation remain limited. This study compared two isolation methods for donkey milk EVs: conventional ultracentrifugation (UC) and the ultrafast isolation system EXODUS (DUS). We systematically evaluated MEV morphology, particle size distribution, and marker protein expression profiles. Subsequently, we performed comprehensive proteomic characterization using 4D label-free quantitative proteomics. Both isolation methods yielded MEVs with characteristic spherical or elliptical membrane structures. UC-isolated MEVs showed significantly higher particle concentrations compared to DUS-isolated vesicles; however, UC extraction introduced polymer precipitates and protein particle contaminants. Proteomic analysis identified 1220 proteins across both isolation methods. Gene Ontology annotation revealed distinct protein enrichment patterns: DUS-isolated MEVs showed enhanced representation of proteins associated with extracellular regions, extracellular matrix, and extracellular exosomes, while UC-isolated MEVs were enriched in proteins related to GTP binding, GTPase activity, and early endosome functions. These findings suggest that DUS extraction preserves greater MEV structural integrity. KEGG pathway enrichment analysis demonstrated that differentially expressed proteins from each extraction method exhibit distinct biological functionalities. These findings reveal that DUS extraction preserves superior structural integrity and functional capacity, while UC provides higher yield at the cost of increased contamination. This systematic framework enables researchers to align extraction strategy with specific applications, advancing both fundamental investigation of donkey milk bioactivities and industrial-scale production of therapeutically relevant EVs.