Sojae Semen Praeparatum Fermented by <i>Rhizopus chinensis</i> and <i>Bacillus</i> sp. DU‐106 Alleviates Loperamide‐Induced Constipation in Rats via Regulating Calcium Signaling Pathways and the Gut Microbiota

ABSTRACT To investigate the effects of different processing methods of sojae semen praeparatum (SSP) on constipation, we prepared SSP through fermentation using Rhizopus chinensis and Bacillus sp. DU‐106 as a starter culture, and examined its laxative effects and mechanisms in loperamide‐induced constipated Sprague‐Dawley (SD) rats. The results showed that SSP effectively alleviated loperamide‐induced constipation compared to the model control group. Specifically, SSP intake increased fecal water content, decreased fecal hardness, shortened small intestinal transit time, improved intestinal propulsion, and restored colonic epithelial integrity. It elevated serum levels of gastrointestinal hormones (substance P, motilin, gastrin, and serotonin) and regulated colonic NOS and ATPase activity. RT‐qPCR and Western blot (WB) results showed that after SSP intervention, the mRNA levels of ADRB2, IP3R, PKC, PLCβ, and SERCA, as well as the protein expression levels of ADRB2, IP3R, PKC, PLCβ, p‐PKC, and SERCA, were all significantly down‐regulated. 16S rRNA sequencing revealed that SSP restored gut microbiota composition, increasing beneficial bacteria like Bacteroidota, Lactobacillus, Weissella , and Blautia , while reducing Actinobacteriota and Staphylococcus . SSP also elevated total short‐chain fatty acids (SCFAs) and increased the abundance of NK4A214 group, and Blautia , thereby promoting gut motility and SCFA production, while reducing diarrhea‐associated harmful Staphylococcus . These results demonstrate that dual‐fermented SSP may alleviate constipation in rats by modulating calcium signaling pathways and the gut microbiota. This study provides a novel direction for the value‐added processing and innovative application of traditional SSP, while also establishing a theoretical foundation for developing SSP‐derived functional products.