Postbiotics, as a novel class of functional components, have garnered considerable scholarly and industrial interest due to their distinctive advantages in food processing applications and their positive impact on human health. Although postbiotics have demonstrated potential in alleviating constipation, their specific mechanism of action and bioactive components remain unclear. This study aimed to investigate the ameliorative effects and potential mechanisms of postbiotics derived from Bifidobacterium animalis subsp. lactis BL-99 (BL-99) on FC using both in vivo and in vitro models. The findings revealed that both BL-99 and its postbiotics significantly mitigated FC symptoms, as evidenced by enhanced intestinal motility, and elevated fecal water content. Additionally, treatment with BL-99 postbiotics was associated with an increase in the thickness of the intestinal muscular layer and a reduction in apoptosis of intestinal smooth muscle cells (SMCs). Mechanistically, BL-99 postbiotics were found to enhance the contractile response and promote the proliferation of intestinal SMCs. Furthermore, untargeted metabolomics analysis identified two key bioactive peptides, Glu-Val and Glu-Leu, as the active components in BL-99 responsible for regulating SMC function. Collectively, these findings highlight the potential of BL-99 postbiotics as a promising functional food ingredient for alleviating FC, providing a novel and effective strategy for the developing dietary interventions targeting this condition.

Background/Objectives: Functional constipation (FC) commonly affects middle-aged and older adults, but current pharmacological treatments have limitations. Postbiotics may offer safety advantages, but clinical evidence is limited. This randomized controlled trial evaluated the efficacy and safety of a heat-inactivated two-strain Lacticaseibacillus paracasei fermented milk in adults with FC. Methods: One hundred adults aged 45–75 years with Rome IV-defined FC received the fermented milk or placebo for 4 weeks. The primary outcome was the change in weekly spontaneous bowel movement (SBM) frequency from baseline to week 4. Secondary outcomes included complete spontaneous bowel movement (CSBM) frequency, whole-gut transit time (WGTT), constipation symptom scores, quality of life, serum biomarkers, and adverse events. Primary analysis was per-protocol (n = 96); intention-to-treat analysis was applied to primary and key secondary outcomes. Results: Dropout was 4% (n = 4, 2 per group), and adherence was >80% in both groups. The intervention showed no significant benefit over placebo for the primary outcome or for most secondary clinical outcomes. Although both groups improved within-group, no significant between-group differences were observed at week 4 for changes in SBM (MD = −0.14, 95% CI: −0.85, 0.57; p = 0.683), CSBM (MD = 0.27, 95% CI: −0.61, 1.15; p = 0.543), or WGTT (MD = −1.55 h, 95% CI: −7.65, 4.55; p = 0.614). Symptom and quality-of-life scores also did not differ between groups. Exploratory biomarker analyses showed significantly greater increases in serum VIP and ACh in the intervention group (VIP: MD = 105.23 ng/L, p < 0.001; ACh: MD = 42.95 ng/L, p = 0.035). No adverse events were reported. Conclusions: Four weeks of this postbiotic was safe but did not significantly improve bowel function or symptoms in the overall FC population. The increases in serum VIP and ACh suggest engagement of neurotransmitter-related pathways; however, these exploratory findings do not imply causation or clinical efficacy and warrant confirmation in longer-duration trials (Clinical Trial Registry: ChiCTR2500111771).

Staphylococcus aureus (S. aureus) is a common foodborne pathogen that can cause the severe contamination of dairy products. Therefore, there is an urgent need for rapid detection methods. In this study, a paper-based biosensor integrating a dual-recognition strategy using aptamers and antibodies was developed for the sensitive, rapid, and on-site detection of S. aureus in complex milk matrices. The biosensor combines a milk matrix-adapted aptamer with polyclonal antibodies (pAbs) and utilizes colloidal gold nanoparticles (AuNPs) as visual signal reporters. Using a SELEX process tailored to the milk matrix, the high-affinity aptamer SA2-1 was selected to specifically bind S. aureus, while pAbs enabled multi-epitope capture on the paper substrate. The aptamer–AuNP conjugates generated visual signals, achieving a detection limit of 10² CFU/mL within 15 min without an instrument. This dual-recognition strategy synergistically enhances both sensitivity and specificity, offering a cost-effective solution for dairy safety monitoring.

Background: Functional constipation is a common gastrointestinal disorder that impairs quality of life. Although probiotics have shown benefits in constipation, whether live and heat-killed forms of the same strain act through similar mechanisms remains unclear. This study compared the effects of live and heat-killed Lacticaseibacillus paracasei K56 in experimental constipation. Methods: Loperamide-treated mice received live or heat-killed L. paracasei K56. Gastrointestinal motility, colonic histology, neurotransmitters, aquaporin expression, gut microbiota, short-chain fatty acids, and metabolomic profiles were evaluated. Results: Both preparations relieved constipation, as reflected by shorter defecation latency, increased fecal output and moisture, and faster intestinal transit, with live K56 producing greater improvement. Colonic injury was alleviated, accompanied by reduced Aqp2 and Aqp3 expression and normalization of 5-hydroxytryptamine, substance P, and vasoactive intestinal peptide. The treatments increased Lactobacillus and Lachnospiraceae, promoted butyrate production, and altered tryptophan metabolism. Heat-killed K56 was mainly associated with changes in phenylalanine/tyrosine- and methionine-related metabolism, whereas live K56 showed more pronounced alterations in arginine/polyamine, tryptophan, and bile acid metabolism. Conclusions: Live and heat-killed L. paracasei K56 improved constipation through shared and distinct mechanisms involving gut microbiota, microbial metabolism, enteric neurotransmission, and intestinal water homeostasis. Live K56 produced stronger effects, whereas heat-killed K56 retained substantial biological activity.

The decay of smooth muscle contractility is a major trigger of aging-related intestinal hypomotility, which imposes a substantial healthcare burden. Despite this, effective targeted therapy for this condition remains unavailable. Here, we identified the probiotic Bifidobacterium animalis subsp. lactis BL-99 as a microbiota modulator that restores gut motility through the bile acid (BA)-mediated rescue of smooth muscle contractile function. In a randomized, double-blind clinical trial (ChiCTR2300077583, with the primary endpoint of whole-gut transit time [WGTT], and secondary endpoints including bowel movement frequency, symptom severity, and quality-of-life measures) and naturally aged mouse models, BL-99 intervention significantly reduced the WGTT and concurrently remodeled the gut microbiota. This suppressed bile salt hydrolase-expressing bacteria, which preserved conjugated BA pools, including taurochenodeoxycholic acid (TCDCA). Mechanistic investigations revealed that BL-99 was associated with enhanced expression of the serum response factor (SRF)–myocardin (Myocd) transcriptional axis in colonic smooth muscle cells (SMCs), restoring contractile apparatus components (e.g., α-SMA, SM-MHC). Crucially, TCDCA administration recapitulated BL-99 regulation of intestinal SMC contractile protein expression by inhibiting the PI3K/AKT pathway. Our findings established a microbiota-BA-SMC signaling axis that ameliorates age-related intestinal hypomotility, positioning both BL-99 and TCDCA as precision therapeutic candidates targeting SMC-related dysmotility.