Pediococcus pentosaceus ZJUAF-4 relieves oxidative stress and restores the gut microbiota in diquat-induced intestinal injury.

Lactic acid bacteria (LAB) play a key role in promoting health and preventing diseases because of their beneficial effects, such as antimicrobial activities, modulating immune responses, maintaining the gut epithelial barrier and antioxidant capacity. However, the mechanisms with which LAB relieve oxidative stress and intestinal injury induced by diquat in vivo are poorly understood. In the present study, Pediococcus pentosaceus ZJUAF-4 (LAB, ZJUAF-4), a selected probiotics strain with strong antioxidant capacities, was appointed to evaluate the efficiency against oxidative stress in diquat-induced intestinal injury of mice. Alanine transaminase (ALT) and aspartate aminotransferase (AST) were analyzed to estimate the liver injury. The intestinal permeability was evaluated by 4 kDa fluorescein isothiocyanate (FITC)-dextran (FD4), D-lactate (DLA), and diamine oxidase (DAO) levels. Jejunum reactive oxygen species (ROS) production was examined by dihydroethidium (DHE) staining. Western blotting was used to detect the expression of nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and its downstream genes in jejunum. The gut microbiota was analyzed by high-throughput sequencing method based on the 16S rRNA genes. The results showed that ZJUAF-4 pretreatment was found to protect the intestinal barrier function and maintain intestinal redox homeostasis under diquat stimulation. Moreover, oral administration of ZJUAF-4 increased the expression of Nrf2 and its downstream genes. High-throughput sequencing analysis indicated that ZJUAF-4 contributed to restoring the gut microbiota influenced by diquat. Our results suggested that ZJUAF-4 protected the intestinal barrier from oxidative stress-induced damage by modulating the Nrf2 pathway and gut microbiota, indicating that ZJUAF-4 may have potential applications in preventing and treating oxidative stress-related intestinal diseases. KEY POINTS: • ZJUAF-4 exerted protective effects against diquat-induced intestinal injury. • Activation of Nrf2 and its downstream targets towards oxidative stress. • ZJUAF-4 administration restoring gut microbiota.

Effects of dietary metabolizable energy and crude protein levels on laying performance, egg quality and fecal microbiota of Taihe Silky Fowl during the peak laying period.

Objective: The experiment aimed to study the effect of dietary metabolizable energy (ME) and crude protein (CP) on laying performance, egg quality, serum routine biochemical and lipid metabolism indicators, the apparent digestibility of nutrients, and fecal microbiota of Taihe Silky Fowl (TSF) during the peak laying period. Methods: A total of 540 26-week-old TSF female fowls were randomly allocated to 9 groups with 5 replicates per group and 12 fowls per replicate. The fowls were fed with a 3 × 3 factorial arrangement of treatments diets (ME:10.88,11.30, or 11.72 MJ/kg; CP: 15, 16, or 17%). Results: With the increasing CP level, the egg weight (EW) (P=0.023), egg production (EP) (p=0.047), and egg mass (EM) (p=0.022) enhanced, while the feed conversion rate (FCR) (p=0.023) decreased. As the ME levels grew, the average daily feed intake (ADFI) (p<0.001) and FCR (p=0.045) decreased. With enhanced ME, the triglycerides (TG) (p=0.037), total cholesterol (TCHO) (p=0.041), and high-density lipoprotein cholesterol (HDL-C) (p=0.028) increased, whereas the low-density serum lipoprotein cholesterol (LDL-C) (p=0.039) decreased. The apparent digestibility of crude protein increased as the ME level increased (p= 0.029) and as the CP level decreased (p=0.027). At the same time, the apparent digestibility of gross energy increased as the ME level increased (p=0.018). Different levels of ME or CP changed the composition of fecal microbiota, 17% CP increased the abundance of Bifidobacterium. Conclusion: It is suggested that 10.88 MJ/kg dietary ME and 17% CP level are suitable for the nutritional requirements of TSF during the peak laying period.

An alternative ZnO with large specific surface area: Preparation, physicochemical characterization and effects on growth performance, diarrhea, zinc metabolism and gut barrier function of weaning piglets.

High-dose ZnO is widely used to prevent diarrhea and promote growth of weaning piglets, which has led to serious problems of animal toxicity, bacterial resistance and environmental pollution. In this study, a novel alternative ZnO (AZO) was prepared and its physicochemical properties were characterized. Animal experiments were further conducted to evaluate the effects of the ZnO forms, the dose of AZO and the combinations with AZO on the growth performance, diarrhea, zinc metabolism and gut barrier function of weaning piglets. The results showed that the AZO, compared with ordinary ZnO (OZO), nano ZnO (NZO) and porous ZnO (PZO), had the largest surface area and reduced the release of Zn2+ into the gastric fluid. AZO showed better antibacterial activity on Escherichia coli K88, Staphylococcus aureus and Salmonella enteritidis but lower cytotoxicity on porcine intestinal epithelial cells. Animal experiments suggested that low-dose AZO, NZO and PZO (300 mg/kg) improved growth performance and reduced diarrhea in weaning piglets as well as high-dose OZO (3000 mg/kg). Notably, low-dose AZO had the lowest diarrhea incidence. Additionally, low-dose AZO in combination with probiotics improved digestibility and digestive enzyme activities. Low-dose AZO in combination with probiotics also upregulated the expression of the intestinal zinc transporter proteins ZIP4 and DMT1, increased zinc bioavailability, reduced faecal zinc emissions, and avoided zinc overload in the liver and oxidative damage caused by high-dose ZnO. Moreover, low-dose AZO in combination with probiotics improved the gut barrier function of weaning piglets by promoting the expression of tight junction proteins, mucins and antimicrobial peptides and increasing gut microbiota diversity and beneficial Lactobacillus. This study proposed a novel strategy to replace high-dose ZnO and antibiotics with low-dose AZO and probiotics in weaning piglets, which effectively improved growth performance and prevented diarrhea while reducing animal toxicity, bacterial resistance, heavy metal residues and zinc emission pollution.

Integrated rumen microbiome and serum metabolome analysis responses to feed type that contribution to meat quality in lambs.

BACKGROUND: Lifestyle factors, such as diet, are known to be a driver on the meat quality, rumen microbiome and serum metabolites. Rumen microbiome metabolites may be important for host health, the correlation between rumen microbiome and production of rumen metabolites are reported, while the impact of rumen microbiome on the serum metabolome and fatty acid of meat are still unclear. This study was designed to explore the rumen microbiome, serum metabolome and fatty acid of meat in response to the grass diet and concentrate diet to lambs, and the relationship of which also investigated. METHODS: In the present study, 12 lambs were randomly divided into two groups: a grass diet (G) and a concentrate diet (C). Here, multiple physicochemical analyses combined with 16S rRNA gene sequences and metabolome analysis was performed to reveal the changes that in response to feed types. RESULTS: The concentrate diet could improve the growth performance of lambs compared to that fed with the grass diet. The microbiome composition was highly individual, compared to the concentrate group, the abundance of Rikenellaceae_RC9_gut_group, F082_unclassified, Muribaculaceae_unclassified, Ruminococcaceae_NK4A214_group, Bacteroidetes_unclassified, and Bacteroidales_UCG-001_unclassified were significantly (P < 0.05) lower in the grass group, while, the abundance of Succinivibrio, Succinivibrionaceae_UCG-002, Fibrobacter and Christensenellaceae_R-7_group were significantly (P < 0.05) higher in the grass group. Serum metabolomics analysis combined with enrichment analysis revealed that serum metabolites were influenced by feed type as well as the metabolic pathway, and significantly affected serum metabolites involved in amino acids, peptides, and analogues, bile acids, alcohols and derivatives, linoleic acids derivatives, fatty acids and conjugates. Most of the amino acids, peptides, and analogues metabolites were positively associated with the fatty acid contents. Among the bile acids, alcohols and derivatives metabolites, glycocholic was positively associated with all fatty acid contents, except C18:0, while 25-Hydroxycholesterol and lithocholic acid metabolites were negatively associated with most of the fatty acid contents. CONCLUSION: Correlation analysis of the association of microbiome with metabolite features, metabolite features with fatty acid provides us with comprehensive understanding of the composition and function of microbial communities. Associations between utilization or production were widely identified among affected microbiome, metabolites and fatty acid, and these findings will contribute to the direction of future research in lamb.

Bacillus amyloliquefaciens 40 regulates piglet performance, antioxidant capacity, immune status and gut microbiota.

Probiotics can improve animal growth performance and intestinal health. Bacillus species, Lactobacillus species, Bifidobacterium species, yeast etc. are the common types of probiotics. However, understanding the effects of probiotics on the immune status and gut microbiota of weaning piglets and how the probiotics exert their impact are still limited. This study aimed to investigate the effects of Bacillus amyloliquefaciens 40 (BA40) on the performance, immune status and gut microbiota of piglets. A total of 12 litters of newborn piglets were randomly divided into 3 groups. Piglets in control group were orally dosed with phosphate buffered saline; BA40 group and probiotics group were orally gavaged with resuspension BA40 and a probiotics product, respectively. The results showed that BA40 treatment significantly decreased (P < 0.05) the diarrhea incidence (from d 5 to 40), diamine oxidase, D-lactate, interleukin (IL)-1ß and interferon-γ concentrations compared with control group and probiotics group. Meanwhile BA40 dramatically increased the total antioxidant capacity, IL-10 and secretory immunoglobulin-A concentrations in contrast to control group. For the microbial composition, BA40 modulated the microbiota by improving the abundance of Bacteroides, Phascolarctobacterium (producing short-chain fatty acids) and Desulfovibrio and reducing the proliferation of pathogens (Streptococcus, Tyzzerella, Vellionella and paraeggerthella). Meanwhile, a metabolic function prediction explained that carbohydrate metabolism and amino acid metabolism enriched in BA40 group in contrast to control group and probiotics group. For correlation analysis, the results demonstrated that BA40-enriched Phascolarctobacterium and Desulfovibrio provide insights into strategies for elevating the health status and performance of weaned piglets. Altogether, BA40 exerted stronger ability in decreasing diarrhea incidence and improved antioxidant activity, gut barrier function and immune status of piglets than the other treatments. Our study provided the experimental and theoretical basis for the application of BA40 in pig production.

The Role of Probiotics in Alleviating Postweaning Diarrhea in Piglets From the Perspective of Intestinal Barriers.

Early weaning of piglets is an important strategy for improving the production efficiency of sows in modern intensive farming systems. However, due to multiple stressors such as physiological, environmental and social challenges, postweaning syndrome in piglets often occurs during early weaning period, and postweaning diarrhea (PWD) is a serious threat to piglet health, resulting in high mortality. Early weaning disrupts the intestinal barrier function of piglets, disturbs the homeostasis of gut microbiota, and destroys the intestinal chemical, mechanical and immunological barriers, which is one of the main causes of PWD in piglets. The traditional method of preventing PWD is to supplement piglet diet with antibiotics. However, the long-term overuse of antibiotics led to bacterial resistance, and antibiotics residues in animal products, threatening human health while causing dysbiosis of gut microbiota and superinfection of piglets. Antibiotic supplementation in livestock diets is prohibited in many countries and regions. Regarding this context, finding antibiotic alternatives to maintain piglet health at the critical weaning period becomes a real emergency. More and more studies showed that probiotics can prevent and treat PWD by regulating the intestinal barriers in recent years. Here, we review the research status of PWD-preventing and treating probiotics and discuss its potential mechanisms from the perspective of intestinal barriers (the intestinal microbial barrier, the intestinal chemical barrier, the intestinal mechanical barrier and the intestinal immunological barrier) in piglets.

Dynamics of defatted rice bran in physicochemical characteristics, microbiota and metabolic functions during two-stage co-fermentation.

Defatted rice bran (DFRB) is an inexpensive and easily available agricultural byproduct. Existence of anti-nutritional factors (ANFs), high fiber and low protein content, susceptible to oxidation and rancidity make DFRB currently underutilized. In this study, Bacillus subtilis with high enzyme activities, Saccharomyces cerevisiae with high single-cell proteins concentration and Lactiplantibacillus plantarum with excellent acid secreting capacity were screened to co-fermented DFRB with phytase, and multiple physicochemical analyses combined with high-throughput sequencing were applied to provide insights into the dynamics of the physicochemical characteristics and the complex microbiome during the two-stage co-fermentation of DFRB. The results showed that co-fermentation effectively improved the nutritional value by degrading ANFs (trypsin inhibitors and phytic acid), fiber (acid detergent fiber and neutral detergent fiber) and allergenic protein, and increasing the trichloroacetic acid soluble protein, amino acids and organic acid. In addition, co-fermentation prevented lipid oxidation by enhancing antioxidant activity and reducing the activity of lipase and lipoxygenase. High-throughput sequencing results suggested that co-fermentation optimized microbial community of DFRB by increasing desirable Lactobacillus, Pediococcus, Saccharomyces and Talaromyces and reducing undesirable bacteria (Enterobacter and Pseudomonas) and animal and plant-pathogenic fungi (Blumeria, Alternaria, Fusarium, etc.). Furthermore, high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) were adopted to predict microbial metabolic functions and metabolic pathways during whole DFRB co-fermentation.

Co-fermented yellow wine lees by Bacillus subtilis and Enterococcus faecium regulates growth performance and gut microbiota in finishing pigs.

Fermented yellow wine lees (FYWL) are widely used to increase feed utilization and improve pig performance. Based on the preparation of co-FYWL using Bacillus subtilis and Enterococcus faecalis, the purpose of this study was to investigate the effects of co-FYWL on growth performance, gut microbiota, meat quality, and immune status of finishing pigs. 75 pigs were randomized to 3 treatments (5 replicates/treatment), basal diet (Control), a basal diet supplemented with 4%FYWL, and a basal diet supplemented with 8%FYWL, for 50 days each. Results showed that the 8% FYWL group significantly reduced the F/G and increased the average daily weight gain of pigs compared to the control group. In addition, 8% FYWL improved the richness of Lactobacillus and B. subtilis in the gut, which correlated with growth performance, serum immune parameters, and meat quality. Furthermore, acetate and butyrate in the feces were improved in the FYWL group. Simultaneously, FYWL improved the volatile flavor substances of meat, increased the content of flavor amino acids, and played a positive role in the palatability of meat. In addition, FYWL increased serum IgA, IgM, IL-4 and IL-10 levels. Overall, the growth performance, the gut microbiota associated with fiber degradation, meat quality, and immune status were improved in the 8% FYWL group.

Co-fermented defatted rice bran alters gut microbiota and improves growth performance, antioxidant capacity, immune status and intestinal permeability of finishing pigs.

Based on preparation of co-fermented defatted rice bran (DFRB) using Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus plantarum and phytase, the present study aimed to evaluate the effects of co-fermented DFRB on growth performance, antioxidant capacity, immune status, gut microbiota and permeability in finishing pigs. Ninety finishing pigs (85.30 ± 0.97 kg) were randomly assigned to 3 treatments (3 replicates/treatment) with a basal diet (Ctrl), a basal diet supplemented with 10% unfermented DFRB (UFR), and a basal diet supplemented with 10% fermented DFRB (FR) for 30 d. Results revealed that the diet supplemented with FR notably (P < 0.05) improved the average daily gain (ADG), gain to feed ratio (G:F) and the digestibility of crude protein, amino acids and dietary fiber of finishing pigs compared with UFR. Additionally, FR supplementation significantly (P < 0.05) increased total antioxidant capacity, the activities of superoxide dismutase and catalase, and decreased the content of malonaldehyde in serum. Furthermore, FR remarkably (P < 0.05) increased serum levels of IgG, anti-inflammatory cytokines (IL-22 and IL-23) and reduced pro-inflammatory cytokines (TNF-α, IL-1ß and INF-γ). The decrease of serum diamine oxidase activity and serum D-lactate content in the FR group (P < 0.05) suggested an improvement in intestinal permeability. Supplementation of FR also elevated the content of acetate and butyrate in feces (P < 0.05). Moreover, FR enhanced gut microbial richness and the abundance of fiber-degrading bacteria such as Clostridium butyricum and Lactobacillus amylovorus. Correlation analyses indicated dietary fiber in FR was associated with improvements in immune status, intestinal permeability and the level of butyrate-producing microbe C. butyricum, which was also verified by the in vitro fermentation analysis. These findings provided an experimental and theoretical basis for the application of fermented DFRB in finishing pigs.

Bifidobacterium longum, Lactobacillus plantarum and Pediococcus acidilactici Reversed ETEC-Inducing Intestinal Inflammation in Mice.

Microecological preparation could relieve Enterotoxigenic Escherichia coli (ETEC) K88-induced diarrhea in piglets, but which bacteria play a key role and the mitigation mechanism have not been fully clarified. In this study, 36 male mice were randomly divided into six groups (CON, K88, BK (Bifidobacterium longum + K88), LK (Lactobacillus plantarum + K88), PK (Pediococcus acidilactici + K88), and MK (mixed strains + K88)) to explore the prevention mechanisms. Three probiotic strains and their mixtures (TPSM) significantly relieved the weight loss and restored the ratio of villus height to crypt depth in the jejunum. Except for Bifidobacterium longum, other strains significantly decreased interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α) in mice serum. The TPSM treatment significantly downregulated the mRNA expression of the inflammatory cytokines and the Toll-like receptor and downstream gene (TLR4, MyD88, NF-κB) in jejunum induced by ETEC. Furthermore, the TPSM could restore dysbiosis of the intestinal microbiota caused by ETEC. The intestinal microbiota analysis demonstrated that Bifidobacterium longum enriched the Bifidobacterium genus (p < 0.05), Lactobacillus plantarum enriched the Lactobacillus genus (p < 0.05), Pediococcus acidilactici enriched the Coriobacteriaceae_UCG-002 and Christensenellaceae_R-7_group genus (p < 0.05), mixed bacteria enriched the Akkermansia genus (p < 0.05), but ETEC enriched the Desulfovibrio genus (p < 0.05). Meanwhile, the starch and sucrose metabolism, galactose and fructose metabolism, mannose metabolism and ABC transporters were increased with probiotics pre-treatment (p < 0.05). To sum up, the microecological preparation alleviated ETEC-induced diarrhea by regulating the immune response, rebalancing intestinal microbiota and improving carbohydrate metabolism.

Effect of Porcine Clostridium perfringens on Intestinal Barrier, Immunity, and Quantitative Analysis of Intestinal Bacterial Communities in Mice.

Clostridium perfringens (C. perfringens) is one of the main pathogens which can cause a range of histotoxic and enteric diseases in humans or animals (pigs, or broilers). The Centers for Disease Control and Prevention (CDC) estimates these bacteria cause nearly 1 million illnesses in the United States every year. For animal husbandry, necrotizing enteritis caused by C. perfringens can cost the global livestock industry between $2 billion and $6 billion per year. C. perfringens-infected animals can be isolated for its identification and pathology. A suitable animal model is one of the essential conditions for studying the disease pathogenesis. In previous studies, mice have been used as subjects for a variety of Clostridium perfringens toxicity tests. Thus, this study was designed to build a mouse model infected porcine C. perfringens which was isolated from the C.perfringens-infected pigs. A total of 32 6-week-old male C57BL/6 mice were randomly divided into four groups. Control group was orally administrated with PBS (200 µL) on day 0. Low group, Medium group, and High group were gavaged with 200 ul of PBS resuspension containing 8.0 × 107 CFU, 4.0 × 108 CFU, and 2.0 × 109 CFU, respectively. We examined growth performance, immune status, intestinal barrier integrity, apoptosis-related genes expression, and copies of C. perfringens in mice. The results showed that the growth performance declined and intestinal structure was seriously damaged in High group. Meanwhile, pro-inflammatory factors (IL-1ß, TNF-α, and IL-6) were significantly increased (P < 0.05) in High group compared to other groups. The tight junctions and pro-apoptosis related genes' expression significantly decreased (P < 0.05) in High group, and high dose caused a disruption of intestinal villi integrity and tissue injury in the jejunum of mice. In addition, the enumerations of C. perfringens, Escherichia coli, and Lactobacillus explained why the gut of High group mice was seriously damaged, because the C. perfringens and Escherichia coli significantly enriched (P < 0.05), and Lactobacillus dramatically decreased (P < 0.05). Overall, our results provide an experimental and theoretical basis for understanding the pathogenesis and exploring the effects of porcine C. perfringens on mice.

Screening of Bacteria Inhibiting Clostridium perfringens and Assessment of Their Beneficial Effects In Vitro and In Vivo with Whole Genome Sequencing Analysis.

Various countries and organizations call for banning the use of antibiotic growth promoters (AGPs) as prophylaxis and for growth promotion in the livestock industry. Hence, seeking a substitute for antibiotics is strongly required by the livestock industry to maintain the productivity level and profits. Probiotics could represent one viable solution because of their beneficial effects on host health and maintaining the intestinal microbiota balance. In the present study, we aimed to isolate bacterial strains with probiotics properties from JinHua pig (a Chinese native pig breed) gastrointestinal tract that have antagonistic activity against to common disease-causing bacteria on farms. The four most potent strains were isolated (PP31, BA11, BA40, BV5) by the agar well diffusion method and further characterized by acid, bile salt, trypsin tolerance, whole genome sequencing (WGS), and suppressing Clostridium perfringens adhesion to IPEC-J2 cells. According to these results, BA40 had the highest number and variety of probiotic secondary metabolic secretion genes and capacity to exclude the attachment of Clostridium perfringens to IPEC-J2 cells as same as PB6. The animal experiment in vivo illustrated that BA40 and PB6 could reduce the phenomenon induced by Clostridium perfringens challenge of body weight loss, colon length decrease, pro-inflammatory cytokine increase, and Clostridium perfringens and Escherichia coli increase. The present study provides evidence that BA40 could represent a novel probiotic candidate as PB6, which exhibited some probiotic features and mitigated the burden of Clostridium perfringens associated gut disease.

Bacillus subtilis and Enterococcus faecium co-fermented feed regulates lactating sow's performance, immune status and gut microbiota.

Fermented feed (FF) is widely applied to improve swine performance. However, the understandings of the effects of FF on the immune status and gut microbiota of lactating sows and whether probiotics are the effective composition of FF are still limited. The present study aimed to investigate the performance, immune status and gut microbiota of lactating sows fed with a basal diet supplemented with Bacillus subtilis and Enterococcus faecium co-fermented feed (FF), with the probiotic combination (PRO) of B. subtilis and E. faecium and control diet (CON) as controls. Compared with the CON group, FF group remarkably improved the average daily feed intake of sows and the weight gain of piglets, while significantly decreased the backfat loss, constipation rate of sows and diarrhoea incidence of piglets. The yield and quality of milk of sows in FF group were improved. Besides, faecal acetate and butyrate were promoted in FF group. Additionally, FF increased the level of IgG, IgM and IL-10 and decreased the concentration of TNF-α in serum. Furthermore, FF reduced the abundance of Enterobacteriaceae and increased the level of Lactobacillus and Succiniclasticum, which were remarkably associated with growth performance and serum immune parameters. Accordingly, microbial metabolic functions including DNA repair and recombination proteins, glycolysis and gluconeogenesis, mismatch repair and d-alanine metabolism were significantly upregulated, while amino acid metabolism was downregulated in FF group. Overall, the beneficial effects of FF were superior to PRO treatment. Altogether, administration of FF during lactation improved the performance and immune status, and modulated gut microbiota of sows. Probiotics are not the only one effective compound of FF.

Protective Effects of Bacillus amyloliquefaciens 40 Against Clostridium perfringens Infection in Mice.

This study aimed to investigate the protective effects of Bacillus amyloliquefaciens (BA40) against Clostridium perfringens (C. perfringens) infection in mice. Bacillus subtilis PB6 was utilized as a positive control to compare the protective effects of BA40. In general, a total of 24 5-week-old male C57BL/6 mice were randomly divided into four groups, with six mice each. The BA40 and PB6 groups were orally dosed with resuspension bacteria (1 × 109 CFU/ml) once a day, from day 1 to 13, respectively. In the control and infected groups, the mice were orally pre-treated with phosphate-buffered saline (PBS) (200 µl/day). The mice in the infected groups, PB6 + infected group and BA40 + infected group, were orally challenged with C. perfringens type A (1 × 109 CFU/ml) on day 11, whereas the control group was orally dosed with PBS (200 µl/day). The results showed that the BA40 group ameliorated intestinal structure damage caused by the C. perfringens infection. Furthermore, the inflammatory responses detected in the infected groups which include the concentrations of IL-1ß, TNF-α, IL-6, and immunoglobulin G (IgG) in the serum and secretory immunoglobulin (SigA) in the colon, and nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity in the jejunum, were also alleviated (P < 0.05) by BA40 treatment. Similarly, cytokines were also detected by quantitative PCR (qPCR) in the messenger RNA (mRNA) levels, and the results were consistent with the enzyme-linked immunosorbent assay (ELISA) kits. Additionally, in the infected group, the mRNA expression of Bax and p53 was increasing and the Bcl-2 expression was decreasing, which was reversed by BA40 and PB6 treatment (P < 0.05). Moreover, the intestinal microbiota imbalance induced by the C. perfringens infection was restored by the BA40 pre-treatment, especially by improving the relative abundance of Verrucomicrobiota (P < 0.05) and decreasing the relative abundance of Bacteroidetes (P < 0.05) in the phyla level, and the infected group increased the relative abundance of some pathogens, such as Bacteroides and Staphylococcus (P < 0.05) in the genus level. The gut microbiota alterations in the BA40 group also influenced the metabolic pathways, and the results were also compared. The purine metabolism, 2-oxocarboxylic acid metabolism, and starch and sucrose metabolism were significantly changed (P < 0.05). In conclusion, our results demonstrated that BA40 can effectively protect mice from C. perfringens infection.

Variations of Soybean Meal and Corn Mixed Substrates in Physicochemical Characteristics and Microbiota During Two-Stage Solid-State Fermentation.

Corn germ meal (CGM) and corn gluten feed (CGF) are the two main corn byproducts (CBs) obtained from corn starch extraction. Due to their high fiber content, low protein content, and severe imbalance of amino acid, CBs are unable to be fully utilized by animals. In this study, the effect of microorganism, proteases, temperature, solid-liquid ratio, and time on nutritional properties of CB mixture feed (CMF) was investigated with the single-factor method and the response surface method to improve the nutritional quality and utilization of CBs. Fermentation with Pichia kudriavzevii, Lactobacillus plantarum, and neutral protease notably improved the nutritional properties of CMF under the fermentation conditions of 37°C, solid-liquid ratio (1.2:1 g/ml), and 72 h. After two-stage solid-stage fermentation, the crude protein (CP) and trichloroacetic acid-soluble protein (TCA-SP) in fermented CMF (FCMF) were increased (p < 0.05) by 14.28% and 25.53%, respectively. The in vitro digestibility of CP and total amino acids of FCMF were significantly improved to 78.53% and 74.94%, respectively. In addition, fermentation degraded fiber and provided more organic acids in the CMF. Multiple physicochemical analyses combined with high-throughput sequencing were performed to reveal the dynamic changes that occur during a two-stage solid-state fermentation process. Generally, Ascomycota became the predominant members of the community of the first-stage of fermentation, and after 36 h of anaerobic fermentation, Paenibacillus spp., Pantoea spp., and Lactobacillales were predominant. All of these processes increased the bacterial abundance and lactic acid content (p < 0.00). Our results suggest that two-stage solid-state fermentation with Pichia kudriavzevii, Lactobacillus plantarum, and protease can efficiently improve protein quality and nutrient utilization of CMF.

Dynamics of the Physicochemical Characteristics, Microbiota, and Metabolic Functions of Soybean Meal and Corn Mixed Substrates during Two-Stage Solid-State Fermentation.

Substantial annual economic loss in livestock production is caused by antinutritional factors in soybean meal and corn mixed substrates, which can be degraded by microbial fermentation. Although considerable efforts have been made to explain the effects of fermentation on soybean meal and corn-based feed, the dynamics of the physicochemical characteristics, microbiota, and metabolic functions of soybean meal and corn mixed substrates during solid-state fermentation remain unclear. Here, multiple physicochemical analyses combined with high-throughput sequencing were performed to reveal the dynamic changes that occur during a novel two-stage solid-state fermentation process. Generally, inoculated bacteria rapidly proliferated in the initial 12-h aerobic fermentation (P = 0.002). Notably, most nutritional changes occurred during 12 to 24 h compared to 0 to 12 h. Second-stage anaerobic fermentation increased the bacterial abundance and lactic acid content (P < 0.00). Bacillus spp., Enterococcus spp., and Pseudomonas spp. were predominantly involved in the maturation of the fermented mixed substrates (P < 0.05). Additionally, the available phosphorus exhibited the greatest interaction with the microbial community structure. Cellular processes and environmental information processing might be the main metabolic processes of the microbiota during this fermentation. An in vivo model further evaluated the growth-promoting effects of the fermented products. These results characterized the dynamic changes that occur during two-stage solid-state fermentation and provided potential references for additional interventions to further improve the effectiveness and efficiency of solid-state fermentation of feed.IMPORTANCE Solid-state fermentation (SSF) plays pivotal roles not only in human food but also farm animal diets. Soybean meal (SBM) and corn account for approximately 70% of the global feed consumption. However, the nutritional value of conventional SBM and corn mixed substrates (MS) is limited by antinutritional factors, causing substantial economic loss in livestock production. Although emerging studies have reported that SSF can improve the nutritional value of SBM-based substrates, the dynamic changes in the physicochemical features, microbiota, and metabolic functions of MS during SSF remain poorly understood, limiting further investigation. To provide insights into the dynamics of the physicochemical characteristics and the complex microbiome during the two-stage SSF of MS, multiple physicochemical analyses combined with high-throughput sequencing were applied here. These novel insights shed light on the complex changes that occur in the nutrition and microbiome during two-stage SSF of MS and are of great value for industrial feed-based practices and metabolomic research on SSF ecosystems.

Solid-state fermentation of distilled dried grain with solubles with probiotics for degrading lignocellulose and upgrading nutrient utilization.

Solid-state fermentation (SSF) was carried out in this study to improve the nutritional digestibility of two types of distilled dried grain with solubles (DDGS) by inoculating probiotic combinations. The fermented DDGS (FDDGS) contained more crude protein, small peptides and total amino acids than did unfermented DDGS. The concentrations of fiber indexes significantly declined after fermentation. The amounts of probiotics, enzymes and organic acids were significantly improved after fermentation. Microscopy revealed that SSF disrupted the surface structure and increased small fragments of DDGS substrate, thereby facilitating in vitro digestibility of FDDGS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography indicated the breakdown of macromolecular protein and lignocellulose, which contributed to the increase of small peptides and monosaccharides. These findings suggested the great potential of SSF to promote the nutritional quality and digestibility of the two DDGS and to expand their utilization.