Carbon Source Substitution During Submerged Fermentation Alters Cordyceps militaris Extracellular Vesicle Metabolite Profiles and Anti-Photoaging Activity

Abstract Background: Cordyceps militaris extracellular vesicle-enriched preparations, operationally designated CMEVs in this study, are being explored as food-derived nanomaterials, but the effects of source material, fermentation stage, and carbon source on their characteristics and biological activity remain unclear. We compared CMEVs obtained from fruiting bodies and submerged liquid fermentation, selected a collection time, and examined the effects of four carbon sources on CMEV characteristics and protection against UVB-induced injury.Results: Compared with fruiting body-derived CMEVs, fermentation-derived CMEVs yielded more BCA-detectable protein per gram of source dry mass, had a more negative zeta potential, and showed a narrower particle-size distribution. In UVB-damaged HaCaT cells, fermentation-derived CMEVs resulted in higher cell viability, lower MMP1 and IL6 expression, and higher COL1A2 expression. The two source groups had similar particle-to-protein ratios. Although the BCA-measured protein concentration was highest on fermentation day 5, CMEVs collected on day 4 had a higher particle-to-protein ratio and a more uniform particle-size distribution. Day 4 was therefore selected for the subsequent experiments. Tre-CMEVs and Glc-CMEVs had similar protein concentrations, particle-to-protein ratios, and zeta potentials, while Tre-CMEVs showed a relatively narrow particle-size distribution. In UVB-damaged HaCaT cells, Tre-CMEVs reduced ROS and MDA, decreased IL6 and MMP1 expression, and increased COL1A2 expression. Tre-CMEVs also reduced wrinkle formation, epidermal thickening, lipid peroxidation, inflammation, and collagen loss in UVB-exposed mice. Untargeted metabolomics distinguished Tre-CMEVs from Glc-CMEVs and identified differential features related to amino acid and energy metabolism, including signals putatively annotated as arginine, glutamate, and tricarboxylic acid-cycle intermediates.Conclusions: Day 4 was selected as the collection point based on particle characteristics and cellular activity. Tre-CMEVs reduced oxidative stress, inflammatory responses, and matrix damage in HaCaT cells and alleviated UVB-induced skin injury in mice. These findings support further evaluation of Tre-CMEVs as a UVB-protective preparation.