Supplementary Table S4 (The lefse_results of N-vs-T in cohort 1.txt)

Supplementary Table S10 (The RNA sequencing data of MKN1_PBS-vs-MKN1_Fn(L))

Listeria monocytogenes, Klebsiella pneumoniae, and Bacillus cereus are common foodborne pathogens widely distributed across various food products. However, studies on the contamination risk posed by these three pathogens in prepared meat products remain limited. This study aimed to investigate the prevalence of these pathogens in prepared meat products collected from Guangzhou, China, and to analyze their genetic diversity, virulence gene profiles, and antibiotic resistance. A total of 43 L. monocytogenes, 87 K. pneumoniae, and 21 B. cereus strains were isolated from 55 prepared meat product samples, with L. monocytogenes exhibiting the highest contamination rate. Multilocus sequence typing (MLST) analysis of 151 isolates identified 67 distinct sequence types (STs), including 23 novel STs, indicating high genetic diversity. Multiple virulence-associated genes were detected in isolates from all three genera. Antimicrobial susceptibility testing revealed that four isolates of L. monocytogenes exhibited resistance to all 12 antibiotics tested. K. pneumoniae isolates showed high resistance to ampicillin, and 23.8% of B. cereus isolates displayed resistance to four antibiotic classes. Overall, the high contamination rates, along with the genetic diversity and multidrug resistance observed among the isolates, indicate potential food safety risks, underscoring the need for enhanced microbiological surveillance of prepared meat products.

Misidentification of poisonous mushrooms has become a growing global food safety threat, with rising incidence of intoxication and limited detection solutions. Current methods lack accuracy, speed, and field applicability, constrained by fungal diversity, toxin complexity, and incomplete knowledge of toxin biosynthetic pathways. This review addresses these gaps by systematically synthesizing the physiological and genetic basis of poisonous mushrooms—including classification, toxin mechanisms, and key toxin-related genes—while critically evaluating current molecular targets and outlining multi-omics-driven strategies for the discovery of novel candidates. Building on this foundation, it critically evaluates recent advances in nucleic acid-based detection strategies, with emphasis on molecular-level solutions that overcome existing bottlenecks. Key findings highlight four synergistic technological pillars converging toward next-generation molecular detection paradigms: (i) multi-omics for target discovery (ii) programmable nucleases (e.g., CRISPR-Cas, Argonaute); for high-specificity detection; (iii) integrated microfluidics for point-of-care testing; and (iv) artificial intelligence for target optimization and signal interpretation. The integration of these pillars represents the application potential of emerging technologies in poisonous mushroom molecular detection. This roadmap charts future poisonous mushrooms detection research and tool development toward intelligent, field-deployable detection platforms, ultimately strengthening preventive food safety systems and mitigating public health risks.

Supplementary Table S6 (The lefse_results of Ln-vs-Lp in cohort 1.txt)

Supplementary Table S8 (Pathological association of F. nucleatum enrichment score with GC patients)

Supplementary Table S7 (The progression-free survival time of GC patients in Cohort 1)

Vibrio parahaemolyticus is a prevalent foodborne pathogen in aquatic products, for which rapid detection is crucial to ensure food safety. While magnetic separation enables efficient target enrichment, its detection phase remains dependent on exogenous labels, leading to cumbersome procedures and susceptibility to matrix interference. To address these limitations, this study developed an exogenous label-free thermomagnetic responsive aptasensing strategy termed ELTRAS, which leverages a charge-regulated thermomagnetic dual-responsive (FSPMS@BSCR-Apt) nanoprobe for efficient capture and rapid visual detection of V. parahaemolyticus. The FSPMS@BSCR-Apt probe was constructed via a layer-by-layer assembly strategy, featuring a superparamagnetic Fe3O4 core sequentially coated with a SiO2 interlayer and a thermo-responsive PNIPAm-co-MAA shell, followed by directional modification of a high negative charge-density aptamer via the streptavidin-biotin system, thereby integrating magnetic separation, temperature-regulated aggregation, and specific recognition. In the absence of the target bacteria, the probe remains dispersed due to strong electrostatic repulsion; upon bacterial presence, target binding induces charge shielding and thermally triggered aggregation, causing a visible solution transition from turbid to clear, which allows quantitative detection by monitoring the absorbance change at 420 nm. The assay could be completed within 22 min after sample introduction. This strategy exhibited a wide linear range from 2.2 & times; 101 to 2.2 & times; 108 CFU/mL (R2 = 0.99278), with a detection limit as low as 25.63 CFU/mL, and demonstrated high specificity against non-target bacteria along with excellent repeatability. Spike-and-recovery experiments in complex food matrices such as shrimp, crab, and seawater confirmed its strong anti-interference capability and accuracy. This exogenous label-free, rapid, and user-friendly aptasensing platform provides a promising tool for on-site food monitoring with potential extensibility to other foodborne pathogens.

Bioactive peptides (BPs) and probiotics have attracted increasing attention in food and nutrition research for their roles in microbial metabolism and functional food development, with lactic acid bacteria (LAB) representing widely used probiotic microorganisms possessing well-characterized metabolic and peptide transport systems within the gut microbiota. This review summarizes current knowledge on food-derived BPs and their interactions with probiotic LAB, with a particular focus on peptide transport and utilization mechanisms, including oligopeptide permease (Opp) and di-/tripeptide permease (Dpp) systems. Sources and production methods of BPs are reviewed, along with experimental evidence describing peptide-supported microbial growth and metabolic responses. Relevant analytical approaches used for peptide characterization and functional assessment are also discussed. Most available evidence derives from controlled in vitro studies and primarily reflects microbial physiological responses rather than direct host-level effects. This review provides a mechanistic perspective on peptide-probiotic interactions in LAB and outlines research directions related to nitrogen utilization and microbial functional performance.

Cereulide, a heat-stable toxin produced by Bacillus cereus, is recognized as a major foodborne risk. However, the metabolic features associated with cereulide production and their biological relevance remain insufficiently characterized. This study aimed to prioritize candidate metabolites associated with cereulide-related phenotypes and to explore their potential pathway-level relationships. An integrated analytical pipeline combining RF-CNN-assisted feature prioritization, pathway enrichment, time-series profiling, and temporal association analysis was developed to support both classification performance and biological interpretability. Using fivefold cross-validation, nine candidate metabolites were prioritized. Based on pathway enrichment and temporal analyses across three growth stages, histidinol, L-leucine, and biotin were highlighted as candidate metabolites associated with cereulide-related phenotypes. A putative working network describing their potential relationships was then proposed. In exogenous supplementation experiments, L-leucine and biotin were associated with reduced cereulide production, whereas histidine showed the opposite trend, providing phenotypic support for the biological relevance of the related pathways under the tested conditions. Overall, this study establishes a metabolomics-guided strategy for prioritizing candidate metabolites and provides biologically interpretable and testable hypotheses for exploring the potential metabolic basis of B. cereus-associated phenotypes.

Cellular agriculture (CA), an emerging and sustainable agricultural paradigm, offers a promising solution to global challenges in food security and environmental sustainability. However, the large-scale manufacturing of CA is hindered by its dependence on serum-based culture media, which are costly and compositionally variable and raise biosafety and animal welfare concerns. Addressing this bottleneck requires the development of affordable, reliable, and ethically compliant serum-free media (SFM). This perspective elucidates the functional roles of serum components, summarizes recent advances in serum substitutes, and highlights microorganism-derived substitutes as particularly promising because of their low cost, compositional stability, and scalability. Furthermore, we outline the evolution of SFM formulation from empirical and design-of-experiment-based optimization to multiomics-driven formulation and artificial intelligence (AI)-assisted design. Overall, this review provides a focused framework for understanding current challenges, design strategies, and future directions of SFM development for CA.

Supplementary Table S9 (The clinical information of GC patients in Cohort 2)

For more than a century, model microorganisms have underpinned major biological explorations and facilitated pivotal advances in molecular genetics, biochemistry and biotechnology. These easy-to-manipulate microbes continue to drive technological progress in life sciences, ranging from the decoding of genetic code using Escherichia coli to the application of CRISPR-Cas9 genome editing in various microbial hosts. This article systematically reviews the latest research progress of model microorganisms, and highlights their transformation from basic research objects into programmable chassis for synthetic biology and industrial bioproduction. Benefiting from large-scale microbial big data resources, intelligent data mining technologies and iterative synthetic biology advances, researchers are now tapping into previously unexplored microbial biodiversity to enable customized strain design—developing novel chassis strains tailored for specific applications ranging from chronic disease intervention to targeted biomanufacturing. Combined with CRISPR editing, synthetic genome assembly and AI-assisted optimization, microbial models have evolved into programmable and personalized engineering platforms. Specialized chassis represented by Pseudomonas putida for environmental remediation and Streptomyces for antibiotic discovery further diversify the functional scenarios of modern microbial biotechnology. This review systematically summarizes recent advances in model microorganism research, focusing on the evolving functional positioning of microbial chassis from basic research tools to industrial engineering platforms. It also elaborates on prevailing bottlenecks including industrial scale-up difficulties, imperfect regulatory systems and biosafety risks. Such advanced engineering platforms offer promising solutions to key challenges in human health protection, sustainable agriculture and green biomanufacturing, fueling the sustainable innovation of modern life sciences and biotechnology.

Despite increasing evidence supporting the promising anti-diabetic potential of natural polysaccharides, studies on structurally well-defined polysaccharides and their systemic mechanisms of action in type 2 diabetes mellitus (T2DM) remain limited. Here, we characterize GFP-Z, a bioactive α-glucan (1760.0 kDa) isolated from Grifola frondosa, which features an α-1,4-linked backbone with α-1,6, α-1,3 and α-1,2 branches. Pharmacological evaluation in db/db mice showed that GFP-Z administration significantly alleviated hyperglycemia and diabetic symptoms, with glucose-lowering effects comparable to metformin under the tested conditions, without causing apparent hepatorenal toxicity. Integrated multi-omics analyses, biochemical assays, and a preliminary pharmacological attenuation experiment further suggest that GFP-Z may exert its effects, at least in part, through immunomodulation-associated metabolic regulation, as reflected by reduced hepatic M1-type macrophage infiltration, altered circulating cytokines and hepatic immunometabolites, activation of the hepatic JAK/STAT-PI3K/AKT signaling axis, and attenuation of GFP-Z-associated immune and glucose-lowering responses by tofacitinib. Taken together, our findings suggest that GFP-Z may represent a promising bioactive polysaccharide capable of improving metabolic disorders in T2DM, potentially through systemic immunomodulation-associated metabolic regulation.

The recovery and quantification of viable probiotic cells from food samples are essential for verifying product efficacy and quality. The traditional culture methods are time-consuming and laborious. In this study, 998 genomic sequences of LAB were analyzed and four novel species-specific molecular markers were mined for Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, and Lactobacillus acidophilus. The specificity of the genetic markers was evaluated using 15 LAB species. A new Propidium Monoazide-qPCR (PMA-qPCR) assay was developed to quantify four viable LAB species. The optimal PMA conditions were 40 mu mol/L with 30 min of light exposure, 20 mu mol/L with 20 min, 30 mu mol/L with 20 min, and 30 mu mol/L with 25 min for L. reuteri, L. rhamnosus, L. paracasei, and L. acidophilus, respectively. Four quantitative standard curves were established, demonstrating a robust correlation (R-2 > 0.99) with viable cell counts over an extensive concentration range (10(3)-10(8) CFU/mL). The detection limits were 8.45 x 10(3) CFU/mL, 4.30 x 10(2) CFU/mL, 7.40 x 10(3) CFU/mL, and 1.32 x 10(2) CFU/mL of pure culture for L. reuteri, L. rhamnosus, L. paracasei, and L. acidophilus, respectively. In 59 actual probiotic products, there was no significant difference between the results of PMA-qPCR and the traditional culture method (P > 0.05). The assay can accurately quantify viable bacteria in probiotic products within approximately 3 h and holds great promise for quality-control applications.

BACKGROUND:Helicobacter pylori is a globally prevalent gastric pathogen, and chronic infection accounts for most gastric cancer (GC) cases worldwide. Major oncogenic determinants, including CagA, VacA, and the type IV secretion system, show marked geographic heterogeneity, yet the evolutionary forces shaping this uneven distribution remain unclear. Prophages can mediate horizontal gene transfer and modulate bacterial fitness and virulence, but their contribution to H. pylori carcinogenicity has not been systematically evaluated. METHODS:We characterized prophage diversity, population structure, and virulence potential using 2379 H. pylori host genomes and 139 complete prophage genomes. Prophage population structure and intergenomic relatedness were inferred, and the prophage pangenome and protein-sharing network were reconstructed. Homology-based association analyses were performed to test enrichment of prophage orthologous groups (POGs) with major oncogenic virulence factors (CagA and/or VacA) across the 2379 host genomes. RESULTS:Prophages segregated into geographically structured populations. The EastAsia and EastAsia2 prophage groups were tightly coupled to high-risk hspEAsia hosts and exhibited the largest and most diverse accessory repertoires. Virulence-associated genes were strongly population-specific and were detected only in the EastAsia/EastAsia2 prophage populations. Moreover, carriage of POGs homologs from 1961P, HPy1R, and phiHP33 showed significant positive associations with CagA and/or VacA across the 2379 genomes, whereas no enrichment was observed for KHP30 or KHP40. CONCLUSIONS:H. pylori prophages are not passive genomic remnants but population-structured reservoirs whose gene repertoires track high-risk virulence backgrounds and may contribute to the bacterium's carcinogenic potential.

Cell culture is a fundamental technique in food biomanufacturing. However, the inherent limitations of traditional serum-containing culture systems hinder the scalable application of cell culture in the food industry, thus driving the development of serum-free cell culture. This review summarizes the current status and technical challenges of serum-free media (SFM) for cells relevant to food biomanufacturing. In this context, we systematically compile recent advances in the use of microbial resources as potential supplements for SFM, given that their functional ingredients are capable of meeting cellular nutritional requirements, alleviating cellular stress damage and maintaining cell functionality. Furthermore, we propose development strategies for microbial resource-based SFM, including the construction of candidate component pools, identification of cellular requirements and limiting factors, and the formulation and optimization of components based on a "resource-requirement matching" framework. This review provides methodological support for the rational design of SFM and contributes to advancing food biomanufacturing toward more cost-effective, controllable, and scalable production.