Effects of Steam Explosion and Microbial–Enzyme Fermentation on Free Gossypol Content, Fermentation Quality, and Bacterial Community Structure of Cottonseed Hulls

The use of cottonseed hulls as a feed resource is constrained by their high fiber content and residual free gossypol. This study evaluated the effects of steam explosion combined with fermentation using a microbial–enzyme preparation on the nutrient composition, fermentation quality, bacterial communities, and in vitro digestion and fermentation characteristics of cottonseed hulls. Four treatments were evaluated using physicochemical analyses, 16S rRNA gene sequencing, and in vitro incubation: an untreated control (CK), steam explosion (BP), fermentation with a microbial–enzyme preparation (FJ), and steam explosion followed by fermentation with the same preparation (BF). All processing treatments significantly reduced free gossypol content (p < 0.05), with BF showing the lowest concentration (70.42 mg/kg), representing a 77.89% reduction relative to CK. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents of BF were 573.38 and 446.23 g/kg dry matter (DM), respectively, both significantly lower than those of CK (p < 0.05) but not significantly different from those of BP. Crude protein content did not differ significantly among treatments. BF had the highest lactic acid content (27.94 g/kg DM) and the lowest endpoint pH (4.12). Bacterial community analysis showed that Firmicutes was the dominant phylum in all three fermented groups. At the end of fermentation, Lentilactobacillus was the dominant genus in the BF group, with a relative abundance of 97.55%. No associations between bacterial taxa and fermentation parameters remained significant after correction for multiple testing. KEGG functional prediction indicated differences in potential functional composition among treatments. In vitro DM digestibility in BF reached 43.99% at 48 h; however, NDF digestibility did not differ significantly among treatments at any incubation time. Cumulative gas production and total volatile fatty acid concentration in BF at 48 h were 154.28 mL/g DM and 64.58 mmol/L, respectively, neither of which differed significantly from FJ. In conclusion, steam explosion combined with microbial–enzyme fermentation reduced free gossypol content, promoted lactic acid accumulation, and altered bacterial community composition, providing experimental evidence to support further development of cottonseed hulls as a feed resource.