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Abstract Background We aimed to explore the association and potential causality between polyunsaturated fatty acids concentrations and the risk of periodontal disease. Materials and methods Data were collected from the 2011–2014 National Health and Nutrition Examination Survey (NHANES). Weighted logistic regression analysis and restricted cubic spline (RCS) analysis were used to analyse the associations of the concentrations of omega-3 and omega-6 fatty acids and the omega-6/omega-3 fatty acids ratio with the risk of periodontitis. E-value and propensity score matching (PSM) analyses were used for sensitivity analyses. In addition, two-sample Mendelian randomisation (MR) analyses were performed to assess the potential causal impact of the concentrations of those fatty acids on periodontitis risk. Results A total of 2462 participants from the NHANES were included. Logistic regression analysis revealed that high omega-3 fatty acids levels were negatively associated with the risk of developing periodontitis (P 0.05). Conclusion Although omega-3 fatty acids and the omega-6/omega-3 fatty acids ratio were associated with the risk of periodontitis in cross-sectional studies, the MR results did not support a causal relationship between them. Therefore, there is no indication that an increase in the omega-3 fatty acids concentration or a decrease in the omega-6/omega-3 fatty acids ratio may be beneficial for preventing periodontitis.

Abstract Background Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood. Methods We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice. Results Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice. Conclusion Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

The addition of glycerin, vitamin C, and niacinamide to pig diets increased the redness of longissimus dorsi; however, it remains unclear how these supplements affect gut microbiota and metabolites. A total of 84 piglets (20.35 ± 2.14 kg) were randomly allotted to groups A (control), B (glycerin-supplemented), C (vitamin C and niacinamide-supplemented), and D (glycerin, vitamin C and niacinamide-supplemented) during a feeding experiment. Metagenomic and metabolomic technologies were used to analyze the fecal compositions of bile acids, metabolites, and microbiota. The results showed that compared to pigs in group A, pigs in group D had lower virulence factor expressions of lipopolysaccharide (p < 0.05), fatty acid resistance system (p < 0.05), and capsule (p < 0.01); higher fecal levels of ferric ion (p < 0.05), allolithocholic acid (p < 0.01), deoxycholic acid (p < 0.05), tauroursodeoxycholic acid dihydrate (p < 0.01), glycodeoxycholic acid (p < 0.05), L-proline (p < 0.01) and calcitriol (p < 0.01); and higher (p < 0.05) abundances of iron-acquiring microbiota (Methanobrevibacter, Clostridium, Clostridiaceae, Clostridium_sp_CAG_1000, Faecalibacterium_sp_CAG_74_58_120, Eubacteriales_Family_XIII_Incertae_Sedis, Alistipes_sp_CAG_435, Alistipes_sp_CAG_514 and Methanobrevibacter_sp_YE315). Supplementation with glycerin, vitamin C, and niacinamide to pigs significantly promoted the growth of iron-acquiring microbiota in feces, reduced the expression of some virulence factor genes of fecal pathogens, and increased the fecal levels of ferric ion, L-proline, and some secondary bile acids. The administration of glycerol, vitamin C, and niacinamide to pigs may serve as an effective measure for muscle redness improvement by altering the compositions of fecal microbiota and metabolites.

ObjectiveAs a pseudokinase, Tribbles Pseudokinase 3 (TRIB3) is implicated in a wide array of biological processes, including cell signal transduction, metabolic regulation, stress responses, and immune regulation. While its significant role in the immune regulation of certain cancers is well-established, the specific functions and impact of TRIB3 in head and neck squamous cell carcinoma (HNSC) remain unclear.MethodsThe data of RNA-sequence was acquired from the TCGA database to analyze the expression patterns of TRIB3 and elucidate its prognostic value in HNSC patients. Furthermore, the correlation between TRIB3 and tumor mutation burden, clinical data, immune checkpoint genes, and immune cell infiltration was explored. Moreover, the TRIB3 location in tumor tissues and subcellular structures was identified via Tisch in the HPA database, and the potential protein interaction molecules for TRIB3 were elucidated in the STRING database. The potential TRIB3 gene function was assessed using gene set enrichment analysis (GSEA), whereas the TRIB3 expression levels in clinical HNSC samples were verified by RT-qPCR and immunohistochemistry. the role of TRIB3 in enhancing the malignant behavior of HNSC cells was validated in vitro through a series of methods including RT-qPCR, CCK8 assay, wound healing assay, and transwell assay.ResultsIt was revealed that TRIB3 was significantly overexpressed in the nucleus and cytoplasm of HNSC. Furthermore, this overexpression markedly enhanced the migration ability of tumor cells. As an independent prognostic factor, TRIB3 was associated with advanced tumor T stage and was significantly involved with tumor mutation burden and immune cell infiltration in HNSC. Moreover, it was observed that TRIB3 was not a predicted factor for PD1/PDL1 and ATL4 inhibitor treatment; however, it was substantially correlated with various immune evasion-related genes in HNSC.ConclusionTRIB3 could serve as a potential prognostic marker for HNSC and might be a key gene mediating HNSC immune evasion.

Herein, the NiFe2O4/CeO2 composite photocatalysts were successfully synthesized via a facile one-pot hydrothermal method. We found that the one-pot synthesized composite photocatalysts exhibited better photocatalytic degradation performance than sole photocatalyst or conventional hand ground composite photocatalyst under visible light irradiation, and the degradation efficiency of methylene blue for the one-pot photocatalyst with 10% CeO2 reached 82% in 10 h. The enhanced photocatalytic degradation performance can be explained by the improved adsorption and charge transfer of the reaction site induced by the CeO2 phase. This study can guide the development of alternative photocatalyst for photocatalytic degradation of wastewater.

Background: This retrospective cohort study aimed to evaluate the effect of lifestyle factors (e.g., smoking, drinking, physical exercise, and sleep duration) on the long-term survival of gastric cancer (GC) patients after radical resection. Materials and Methods: GC patients after radical resection were enrolled from January 2016 to December 2017. Their baseline clinical data, lifestyle factors, and prognosis were collected. The primary endpoint was all-cause death. The relationship between the variables and survival was examined using the Cox proportional hazards model. Results: A total of 309 patients were enrolled and 296 patients were followed up for a median of 54.0 months, with 130 confirmed deaths. Older age (>60 years) (hazard ratio [HR]: 1.307, 95% confidence interval [CI]: 1.056–2.161, P = 0.006), advanced tumor, node, and metastasis stage (P < 0.05), poorly pathological differentiation (HR: 1.765, 95% CI: 1.080–2.884, P = 0.023), history of smoking (P < 0.001), never physical exercise (HR: 2.057, 95% CI: 1.170–3.617, P = 0.012), long sleep duration (≥8 h) (HR: 4.160, 95% CI: 1.501–11.533, P = 0.006), and short sleep duration (

The objective of this study was to determine the influence of supplementing the diet of growing–finishing pigs with glycerin and/or a mixture of vitamin C and niacinamide on carcass traits and pork quality. Eighty-four weaned piglets with an initial average body weight of 20.35 ± 2.14 kg were assigned, at random, to four groups for a 103-day feeding experiment: control; glycerin-supplemented group; vitamin C and niacinamide-supplemented group; and glycerin, vitamin C and niacinamide-supplemented group. At the end of the experiment, three pigs/group were randomly selected and slaughtered, and samples were collected for analysis. The results indicated that supplementing crossbred pigs with glycerin, vitamin C and niacinamide simultaneously increased the redness (a*) value (p < 0.05), glycerol content (p < 0.01) and myristoleic acid content (p < 0.01) in the longissimus dorsi and tended to increase the level of flavor amino acids, linoleic acid, linolenic acid and erucic acid, as well as the percentage and density of type I myofibers in the longissimus dorsi and the semimembranosus muscle. Glycerin had an influence (p < 0.01) on the erucic acid content in the longissimus dorsi and the semimembranosus muscle, and vitamin C and niacinamide had an interaction effect (p < 0.05) on the redness (a*) value of the longissimus dorsi. Glycerin, vitamin C and niacinamide supplementation in the diet of crossbred pigs improved the color, flavor and nutritional value of pork, which contributed to an increased intent to purchase this product.

Background and purposeThere was little evidence to study the relationship between hypocalcemia and mortality among critically ill patients with intracerebral hemorrhage (ICH) aged ≥16 years. This study aimed to determine the potential association between hypocalcemia and in-hospital and ICU mortality in patients with ICH in the United States.MethodsWe analyzed 1,954 patients with ICH from the e-Intensive Care Unit Collaborative Research Database and divided them into hypocalcemia and non-hypocalcemia groups. Hypocalcemia was defined as albumin-adjusted total calcium below 8.4 mg/dl. The primary and secondary outcomes were hospital and ICU mortality, respectively. We performed multivariable regression and subgroup analyses to evaluate the association of hypocalcemia with hospital and ICU mortality. Cumulative survival rate analysis was performed using Kaplan–Meier curves with log-rank statistics.ResultsWe enrolled 1,954 patients with ICH who had been hospitalized in ICU for >24 h and were older than 16 years (average age, 61.8 years; men, 56.7%). We noted that 373 (19%) hospital mortality occurred, including 235 (12%) ICU mortality. In this sample, 195 patients had hypocalcemia. Multivariable logistic regression analyses showed that hypocalcemia was associated with a 67% increased risk of in-hospital and a 72% increased risk of ICU mortality. This association was consistent across subgroup analyses.ConclusionsHypocalcemia was associated with a high risk of hospital and ICU mortality among critically ill patients with ICH. Future prospective, randomized, controlled studies are needed to confirm our results.

Neonatal Hoxa1−/− piglets were characterized by dyspnea owing to the Hoxa1 mutation, and maternal administration with ATRA alleviated the dyspnea of neonatal Hoxa1−/− piglets. The purpose of this experiment was to explore how maternal ATRA administration rescued the abnormal fetal lungs of Hoxa1−/− piglets. Samples of the lungs were collected from neonatal Hoxa1−/− and non-Hoxa1−/− piglets delivered by sows in the control group, and from neonatal Hoxa1−/− piglets born by sows administered with ATRA at 4 mg/kg body weight on dpc 12, 13, or 14, respectively. These were used for the analysis of ELISA, histological morphology, immunofluorescence staining, immunohistochemistry staining, and quantitative real-time PCR. The results indicate that the Hoxa1 mutation had adverse impacts on the development of the alveoli and pulmonary microvessels of Hoxa1−/− piglets. Maternal administration with ATRA at 4 mg/kg body weight on dpc 14 rescued the abnormal lung development of Hoxa1−/− piglets by increasing the IFN-γ concentration (p < 0.05), airspace area (p < 0.01) and pulmonary microvessel density (p < 0.01); increasing the expression of VEGFD (p < 0.01), PDGFD (p < 0.01), KDR (p < 0.01), ID1 (p < 0.01), and NEDD4 (p < 0.01); and decreasing the septal wall thickness (p < 0.01) and the expression of SFTPC (p < 0.01) and FOXO3 (p < 0.01). Maternal administration with ATRA plays a vital role in rescuing the abnormal development of lung of Hoxa1−/− fetal piglets.

ObjectivesWe aimed to investigate the association between post-procedure cerebral blood flow velocity (CBFV) and severity of brain edema in patients with acute ischemic stroke (AIS) who received early endovascular therapy (EVT).MethodsWe retrospectively included patients with AIS who received EVT within 24 h of onset between February 2016 and November 2021. Post-procedure CBFV of the middle cerebral artery was measured in the affected and the contralateral hemispheres using transcranial Doppler ultrasound. The severity of brain edema was measured using the three-level cerebral edema grading from the Safe Implementation of Thrombolysis in Stroke-Monitoring Study, with grades 2–3 indicating severe brain edema. The Association between CBFV parameters and severity of brain edema was analyzed.ResultsA total of 101 patients (mean age 64.2 years, 65.3% male) were included, of whom 56.3% (57/101) suffered brain edema [grade 1, 23 (22.8%); grade 2, 10 (9.9%); and grade 3, 24 (23.8%)]. Compared to patients with non-severe brain edema, patients with severe brain edema had lower affected/contralateral ratios of systolic CBFV (median 1 vs. 1.2, P = 0.020) and mean CBFV (median 0.9 vs. 1.3, P = 0.029). Multivariate logistic regression showed that severe brain edema was independently associated with affected/contralateral ratios of systolic CBFV [odds ratio (OR) = 0.289, 95% confidence interval (CI): 0.069–0.861, P = 0.028] and mean CBFV (OR = 0.278, 95% CI: 0.084–0.914, P = 0.035) after adjusting for potential confounders.ConclusionPost-procedure affected/contralateral ratio of CBFV may be a promising predictor of brain edema severity in patients with AIS who received early EVT.

Abstract Objective Many clinical studies evaluating the relationship between metabolic syndrome and esophageal cancer yielded uncertain results. The purpose of this study is to systematically assess the relationship between metabolic syndrome and esophageal cancer. Methods We searched clinical studies on metabolic syndrome and esophageal cancer risk in PubMed, Embase, and the Cochrane Library. Meta-analysis was conducted by RevMan 5.3 softwares. Results A total of four cohort studies and two case–control studies met eligibility criteria and were included in the meta-analysis. Meta-analysis using a fixed-effect model indicated that MetS was related with a higher risk of EC (OR: 1.16, 95% CI 1.08–1.25). Subgroup analyses grouped by pathological types showed that MetS was related with a higher risk of EAC (OR: 1.19, 95% CI 1.10–1.28). Subgroup analyses grouped by metabolic conditions showed hyperglycemia (OR: 1.12, 95% CI 1.03–1.21),hypertension (OR: 1.23, 95% CI 1.04–1.46), obesity (OR: 1.40, 95% CI 1.22–1.60, P

Flavonoids played critical roles in stabilizing microbial homoeostasis when animals suffered exoteric stresses. However, whether flavonoids attenuated heat stress of dairy cows is still not clear. Therefore, in the present article, flavonoids extracted from honeycomb were supplemented to investigate the production, digestibility, and rumen microbial metabolism responses of cows under heat stress conditions. A total of 600 multiparous dairy herds were randomly allotted into the control treatment (CON), the heat stress (HS) treatment, and the honeycomb flavonoids supplement under heat stress conditions (HF) treatment for a 30-day-long trial. Each treatment contains 4 replicates, with 50 cows in each replicate. Production performances including dry matter intake (DMI), milk production, and milk quality were measured on the basis of replicate. Furthermore, two cows of each replicate were selected for the measurement of the nutrient digestibility, the ruminal fermentable parameters including ruminal pH, volatile fatty acids, and ammonia-N, and the rumen microbial communities and metabolism. Results showed that HF effectively increased DMI, milk yield, milk fat, and ruminal acetate content (p < 0.05) compared with HS. Likewise, digestibility of NDF was promoted after HF supplement compared with HS. Furthermore, relative abundances of rumen microbial diversities especially Succiniclasticum, Pseudobutyrivibrio, Acetitomaculum, Streptococcus, and Succinivibrio, which mainly participated in energy metabolism, significantly improved after HF supplement. Metabolomic investigation showed that HF supplement significantly upregulated relative content of lipometabolic-related metabolites such as phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine, while it downregulated biogenic amines. In summary, HF supplement helps proliferate microbial abundances, which further promoted fiber digestibility and energy provision, and ultimately enhances the production performances of dairy cows under heat stress conditions.

This experiment was conducted to compare the antibacterial ability and to identify the antibacterial components of different fermented compound Chinese medicine feed additives in order to develop one fermented compound Chinese medicine feed additive product that can effectively alleviate metritis, vaginitis, and mastitis of sows. The Oxford cup method and double dilution method were used to compare the antibacterial ability of three fermented compound Chinese medicine feed additives (A, B, and C). UHPLC-QE-MS-based untargeted metabolomics was used to identify the antibacterial components of fermented compound Chinese medicine feed additives. Results showed that among fermented compound Chinese medicine feed additives A, B, and C, additive A had the strongest ability to inhibit the growth of Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, and Streptococcus agalactiae. The MIC and MBC of additive A were the lowest for Staphylococcus aureus compared to that for the other three pathogens. The concentrations of 23 Chinese medicine ingredients (ellagic acid, guanine, camphor, L-valine, sinapine, dipropylphthalate, 3-hydroxy-5-isopropylidene-3,8-dimethyl-2,3,3a,4,5,8a-hexahydro-6(1H)-azulenone, 7-dihydroxy-2-(4-hydroxyphenyl)-8-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-6-(3,4,5-trihydroxyoxan-2-yl)chromen-4-one, acetylcholine, farrerol, pyrogallol, ethyl gallate, demethylwedelolactone, methyl gallate, kaempferide, gallic acid, eriodictyol, threonic acid, inositol, 3′,4′,7-trihydroxyflavanone, taxifolin, asiatic acid, and isorhamnetin) in additive A were significantly (p < 0.05 or p < 0.01) higher than those in additive B, respectively. It is concluded that the mixture composed of 23 active components in fermented compound Chinese medicine feed additive A plays an important role in inhibiting the growth of Staphylococcus aureus, Salmonella cholerae suis, Escherichia coli, and Streptococcus agalactiae.

Background: Geese are conventionally considered to be herbivorous, which could also be raised with concentrate feeding diets without green grass because of the similar gastrointestinal tract with other poultry. However, the geese gut microbiota profiles and their interactions with epithelial cells are still of limited study. Flavonoids were well-documented to shape gut microbiota and promote epithelial barrier functions individually or cooperatively with other metabolites. Therefore, in the present study, honeycomb flavonoids (HF) were supplemented to investigate the effects on growth performances, intestinal development, and gut microbiome of geese.Material and Methods: A total of 400 1-day-old male lion-head geese with similar birth weight (82.6 ± 1.4 g) were randomly divided into five treatments: the control treatment (CON) and the HF supplementation treatments, HF was supplemented arithmetically to increase from 0.25 to 1%. Growth performance, carcass performances, and intestines' development parameters were measured to determine the optimum supplement. Junction proteins including ZO-1 and ZO-2 and cecal microbiota were investigated to demonstrate the regulatory effects of HF on both microbiota and intestinal epithelium.Results: Results showed that 0.5% of HF supplement had superior growth performance, carcass performance, and the total parameters of gastrointestinal development to other treatments. Further research showed that tight junction proteins including ZO-1 and ZO-2 significantly up-regulated, while Firmicutes and some probiotics including Clostridiales, Streptococcus, Lachnoclostridium, and Bifidobacterium, remarkably proliferated after HF supplement. In conclusion, HF supplement in concentrate-diet feeding geese effectively increased the growth performances by regulating the gut microbiota to increase the probiotic abundance to promote the nutrient digestibility and fortify the epithelial development and barrier functions to facilitate the nutrient absorption and utilization.

Different untargeted metabolomics approaches were used to identify the differential metabolites between milk samples collected from two groups. Sows were supplemented with fermented compound Chinese medicine feed additive at levels of 0 g/d/sow (control group, n = 10) and 50 g/d/sow (experimental group, n = 10), respectively, from d 104 of gestation to d 25 of lactation, samples of colostrum and mature milk were collected. Data indicated that supplementing fermented compound Chinese medicine feed additive to sows significantly increased the concentrations of quercetin, pinocembrin, chlorogenic acid, methyl succinic acid, L-tryptophan, adenosine, guanine, arteannuin, ferulic acid, echimidine N-oxide, pogostone and kynurenine in the colostrum and inosine, guanosine, benzene-1,2,4-triol, hypoxanthine, adenine, trehalose 6-phosphate in mature milk, respectively. Seven pathways (flavone and flavanol biosynthesis, galactose metabolism, phenylpropanoid biosynthesis, stilbenoid and gingerol biosynthesis, flavonoid biosynthesis, ABC transporters and purine metabolism) in colostrum and two pathways (sucrose metabolism and retrograde endocannabinoid signaling) in mature milk were significantly enriched in the experimental group compared to control group, respectively. The supplementation of fermented compound Chinese medicine feed additive to sows increased the level of antibacterial and anti-inflammatory ingredients in milk and the findings of this study hint that supplementation with fermented compound Chinese medicine feed additive in sows is beneficial for the improvement of milk quality.

Administration of all-trans retinoic acid (ATRA) to pregnant sows improves developmental defects of Hoxa1–/– fetal pigs, and this study aimed to explore the influence of maternal ATRA administration during pregnancy on gut microbiota of neonatal piglets. Samples of jejunal and ileal meconium of neonatal piglets before suckling were collected including 5 Hoxa1–/– and 20 non-Hoxa1–/– (Hoxa1+/+ and Hoxa1+/−) neonatal piglets from the control group and 5 Hoxa1–/– and 7 non-Hoxa1–/– neonatal piglets from the experimental group. Results indicated that Hoxa1 mutation shaped the bacterial composition of the jejunum and ileum of neonatal piglets and Hoxa1–/– neonatal piglets had significantly higher diversity and species richness, higher relative abundance of phylum Bacteroidetes, lower relative abundances of phylum Firmicutes and genus Lactobacillus, and lower ratio of Firmicutes to Bacteroidetes than non-Hoxa1–/– neonatal piglets. After maternal ATRA administration, Hoxa1–/– neonatal piglets had significantly higher diversity and species richness, higher relative abundances of two bacterial phyla (Bacteroidetes and Proteobacteria), and lower relative abundances of phylum Firmicutes and genus Lactobacillus in the jejunum than non-Hoxa1–/– neonatal piglets. Hoxa1–/– neonatal piglets delivered by sows with maternal ATRA administration had lower diversity and species richness and higher relative abundance of phylum Firmicutes in the jejunum than Hoxa1–/– neonatal piglets born by sows with no maternal ATRA administration. Non-Hoxa1–/– neonatal piglets delivered by sows with maternal ATRA administration had higher diversity and species richness and significantly lower relative abundances of phyla Firmicutes and Actinobacteria and genus Lactobacillus in the ileum than non-Hoxa1–/– neonatal piglets born by sows with no maternal ATRA administration. Hoxa1 mutation decreased the expression of bacterial genes involved in ABC transporters, purine metabolism, and aminoacyl-tRNA biosynthesis and increased the expression of bacterial genes involved in two-component system, starch and sucrose metabolism, and arginine and proline metabolism. Maternal ATRA administration decreased the expression of bacterial genes involved in arginine and proline metabolism, peptidoglycan biosynthesis, and fatty acid biosynthesis. Hoxa1 mutation resulted in bacterial dysbiosis of the small intestine of Hoaxa1–/– neonatal piglets, and maternal ATRA administration restored the bacterial dysbiosis of Hoxa1–/– neonatal piglets and altered the bacterial composition of the small intestine of non-Hoxa1–/– neonatal piglets.

Growth and health responses of pigs fed fermented liquid diet are not always consistent and causes for this issue are still not very clear. Metabolites produced at different fermentation time points should be one of the most important contributors. However, currently no literatures about differential metabolites of fermented liquid diet are reported. The aim of this experiment was to explore the difference of metabolites in a fermented liquid diet between different fermentation time intervals. A total of eighteen samples that collected from Bacillus subtilis fermented liquid diet on days 7, 21 and 35 respectively were used for the identification of metabolites by gas chromatography time of flight mass spectrometry (GC-TOF-MS). Fifteen differential metabolites including melibiose, sortitol, ribose, cellobiose, maltotriose, sorbose, isomaltose, maltose, fructose, d-glycerol-1-phosphate, 4-aminobutyric acid, beta-alanine, tyrosine, pyruvic acid and pantothenic acid were identified between 7-d samples and 21-d samples. The relative level of melibiose, ribose, maltotriose, d-glycerol-1-phosphate, tyrosine and pyruvic acid in samples collected on day 21 was significantly higher than that in samples collected on day 7 (P

A L9(34) orthogonal experiment was conducted to evaluate the influence of 9 mixtures which consisted of Cu2+, Zn2+, Fe2+ and I− ions at different ion concentrations on fungal diversity and toxigenic fungal growth in a Bacillus subtilis-fermented liquid feed (FLF) using pyrosequencing. The maximal Chao estimator and Shannon index were achieved in the FLF with a mixture of Cu2+ (200 mg/kg), Zn2+ (160 mg/kg), Fe2+ (150 mg/kg) and I− (2.4 mg/kg). The minimal relative abundance of Aspergillus was achieved when a mixture of Cu2+ (200 mg/kg), Zn2+, Fe2+ and I− was added to the FLF. Compared with Zn2+, Fe2+ and I−, Cu2+ was the most important ion in inhibiting Aspergillus growth. Adding Zn2+ (160 mg/kg), Cu2+, Fe2+ and I− to the FLF minimized the relative abundance of Fusarium. It was Zn2+ instead of Cu2+ played a critical role in suppressing the growth of Fusarium. The proper use of the mixture of Cu2+, Zn2+, Fe2+ and I− in FLF contributes to inhibit the growth of mycotoxin-producing fungi during storage. The new findings of this study help farmers properly use the mixture of Cu2+, Zn2+, Fe2+ and I− to inhibit the growth of mycotoxin-producing fungi in the production of high quality FLF and alleviate mycotoxins damages to animals and humans.