Lysosomes function as major intracellular calcium reservoirs, with the lysosomal cation channel transient receptor potential mucolipin 1 (TRPML1) serving as a oxygen species (ROS)-sensitive Ca2+ release pathway under oxidative stress. Nevertheless, the involvement of lysosomal Ca2+ release-mediated mitochondrial apoptosis in modulating lamb meat quality during postmortem aging has yet to be elucidated. In this study, the specific TRPML1 inhibitor Mucolipin-specific synthetic inhibitor 3 (ML-SI3) or normal saline (control) was injected into the longissimus dorsi muscle of lamb, and relevant indicators were measured at 0, 6, 12, 24, 72, and 120 h postmortem. The results showed that increased ROS levels altered lysosomal membrane permeability (LMP), upregulated TRPML1. Following lysosomal permeabilization, Ca2+ were transferred to mitochondria through membrane contact sites, significantly increasing the mitochondrial Ca2+ content (P < 0.05) and triggering apoptosis. This was evidenced by increased opening of the mitochondrial permeability transition pore (MPTP) and increased release of cytochrome c (Cyt-c), along with the upregulation of caspase-9 and caspase-3 (P < 0.05). Consequently, during postmortem aging, the myofibrillar fragmentation index (MFI) increased while shear force decreased (P < 0.05), indicating improved meat tenderness. However, cooking loss also increased (P < 0.05), suggesting reduced water-holding capacity (WHC). Compared with the control, ML-SI3 treatment inhibited TRPML1 expression, reduced lysosomal Ca2+ release, alleviated mitochondrial calcium overload, and decreased Cyt-c release, resulting in significant suppression of caspase-3/9 activities and apoptosis (P < 0.05). Meanwhile, cooking loss was significantly reduced (P < 0.05), indicating that ML-SI3 delayed tenderization and improved WHC.
Bile acids (BAs) are cholesterol-derived steroids present in animal-derived foods. Whether structurally related BAs differ in host-directed mechanisms affecting SARS-CoV-2 entry remains unclear. Here, six BAs: chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA), and tauroursodeoxycholic acid (TUDCA) were compared using Vero E6 cytopathic-effect assays, bio-layer interferometry (BLI), farnesoid X receptor (FXR) coactivator recruitment, RT-qPCR, and western blotting. CDCA, UDCA, and GUDCA reduced SARS-CoV-2 Omicron XBB.1.16-induced cytopathic effects under post-infection and pre-post-infection regimens with limited cytotoxicity. All six BAs bound ACE2 and reduced spike-ACE2 binding in a concentration-dependent manner; molecular docking and molecular dynamics simulations provided a plausible structural rationale. Reciprocal binding assays against the Omicron BA.4/BA.5 spike RBD showed that under the conditions tested, all six BAs exhibited higher apparent affinity for ACE2 than for RBD. CDCA, GCDCA, and TCDCA acted as FXR agonists and increased ACE2 expression, whereas UDCA, GUDCA, and TUDCA showed antagonist-like behaviour under CDCA stimulation, reducing ACE2 mRNA and protein levels. Together, these findings are consistent with a model in which each BA’s in vitro anti-entry activity reflects the combined influence of direct ACE2 engagement and FXR-associated regulation of ACE2 abundance; this model remains to be tested experimentally. UDCA and GUDCA combined the more favourable profiles, showing both direct ACE2 engagement and an FXR antagonist-like reduction in ACE2 expression. These findings provide an in vitro basis for evaluating selected BAs as candidate host-directed bioactive components, whose diet-derived exposure and efficacy would remain to be validated in vivo.
Solid-state fermentation using Jiuqu starters is a promising approach for modifying wheat bran fiber, but the relationships among fiber conversion, dough functionality, and microbial communities remain unclear. Here, 17 Jiuqu starters were evaluated for dietary fiber composition and whole-wheat dough rheology. Six representative starters with distinct soluble dietary fiber (SDF) changes and insoluble dietary fiber (IDF)–component conversion patterns were further analyzed for fiber structure, microbial composition, predicted functional potential, physicochemical associations, and bacterial–fungal co-occurrence patterns. Twelve starters increased SDF by 1.06–5.69 g/100 g DM, and SDF content was positively correlated with dough G′ and G″ at 1 Hz (P < 0.001). Structural analyses showed starter-dependent disruption of bran microstructure without alteration of the cellulose I crystalline type, while 103–104 Da fractions predominated in SDF (61.55%–67.93%). Fiber-conversion outcomes were associated with recurrent microbial signatures and genus-level co-occurrence patterns, while microbial α-diversity alone did not explain the observed phenotypic differences. Among the representative starters, those exhibiting high SDF accumulation showed higher network connectivity than those with negative SDF increments. Starters with intermediate SDF accumulation displayed heterogeneous microbial composition and network organization rather than a uniform intermediate pattern. These findings link distinct fiber-conversion phenotypes to structural modification, dough rheology, and community-level microbial organization, providing a basis for identifying Jiuqu starters with targeted fiber-modifying potential.
Intestinal calcium absorption is central to mineral homeostasis, yet strain-level evidence linking probiotic-derived short-chain fatty acids (SCFAs) to epithelial calcium transport remains limited. Here, Lactiplantibacillus plantarum GXS77, a candidate strain with probiotic-associated in vitro traits, was characterized by whole-genome analysis, gastrointestinal tolerance, Caco-2 adhesion, antimicrobial activity, Cell Counting Kit-8 (CCK-8) viability testing, SCFA quantification, Fluo-4 AM calcium imaging, RNA sequencing, and Western blotting. GXS77 showed 83.18% survival in simulated gastric fluid, 60.26% survival in simulated intestinal fluid, and 28.6% adhesion to differentiated Caco-2 monolayers. Its cell-free supernatant inhibited Listeria monocytogenes (>5 mm), Salmonella spp. (2–5 mm), and Staphylococcus aureus (0–2 mm), while live-bacteria exposure did not reduce the CCK-8 signal relative to control. After 48 h fermentation, GXS77 produced 10.95 g/L acetate and 0.14 g/L butyrate. Both GXS77 cell-free supernatant and a matched SCFA mixture enhanced intracellular calcium responses versus the pH-matched control, indicating an effect beyond acidification alone. RNA-seq identified 108 genes meeting |log2FC| > 1 and Benjamini-Hochberg-adjusted p < 0.05, with enrichment of xenobiotic, steroid hormone, retinol, and related metabolic pathways. Western blotting confirmed higher Calbindin-D9k, TRPV6, and Claudin-2 levels, supporting activation of both transcellular and paracellular calcium transport routes. These findings provide an in vitro molecular framework linking GXS77-derived metabolites to epithelial calcium transport.
Antibiotic-associated diarrhea (AAD) is one of the most common adverse effects during antibiotic therapy, with gut microbiota dysbiosis serving as its core underlying cause, while probiotics can restore gut microbial balance. This study investigated the regulatory effects of Lactiplantibacillus plantarum GOLDGUT-LP1024 (LP1024) and two multi-strain probiotics (WLFX-1, WLFX-2) on alleviating cefotaxime-induced diarrhea in AAD mice. The severity of diarrhea was assessed by diarrhea status scores (DSSs) in mice. Serum levels of inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). The protein and gene expressions related to inflammation and intestinal barrier were examined by western blotting (WB) and reverse transcription-quantitative real-time PCR (RT-qPCR), respectively. Short-chain fatty acid (SCFAs) content and gut microbiota profile in feces were determined by gas chromatography (GC) and metagenomic sequencing. Three probiotic groups reduced cefotaxime-induced DSSs. The two multi-strain probiotics restored serum interleukin-4 (IL-4) levels and increased colon Il10 mRNA expression, while decreasing the p-p38/p38 ratio and reducing colon Il6 and Il1b mRNA expression. Three probiotic groups repaired the intestinal barrier and enhanced the expression of zonula occludens-1 (ZO-1), Occludin, and mucin 2 (MUC2) in the colon. WLFX-1 restored acetic acid’s content, and WLFX-2 restored acetic acid and propionic acid levels. Three probiotic groups effectively ameliorated the disturbed gut microbiota. Probiotics alleviate diarrhea by enhancing intestinal mucosal barrier function, reducing inflammation, and restoring gut microbial balance. Two multi-strain probiotics were more effective than LP1024. These results provide evidence and theoretical support for using probiotics in treating antibiotic-associated side effects.
