Optimizing postbiotic production through solid-state fermentation with Bacillus amyloliquefaciens J and Lactiplantibacillus plantarum SN4 enhances antibacterial, antioxidant, and anti-inflammatory activities.

Background: Postbiotics are an emerging research interest in recent years and are fairly advanced compared to prebiotics and probiotics. The composition and function of postbiotics are closely related to fermentation conditions. Methods: In this study, we developed a solid-state fermentation preparation method for postbiotics with antimicrobial, antioxidant, and anti-inflammatory activities. The antibacterial activity was improved 3.62 times compared to initial fermentation conditions by using optimization techniques such as single factor experiments, Plackett-Burman design (PBD), steepest ascent method (SAM), and central composite design (CCD) methods. The optimized conditions were carried out with an initial water content of 50% for 8 days at 37°C and fermentation strains of Bacillus amyloliquefaciens J and Lactiplantibacillus plantarum SN4 at a ratio of 1:1 with a total inoculum size of 8%. The optimized SSF medium content ratios of peptide powder, wheat bran, corn flour, and soybean meal were 4, 37.4, 30, and 28.6%, respectively. Results: Under these optimized conditions, postbiotics with a concentration of 25 mg/mL showed significant broad-spectrum antibacterial capabilities against Escherichia coli, Salmonella, and Staphylococcus aureus and strong antioxidant activity against ABTS, DPPH, and OH radicals. Moreover, the optimized postbiotics exhibited good anti-inflammatory ability for reducing nitric oxide (NO) secretion in RAW 264.7 macrophage cells in response to LPS-induced inflammation. Furthermore, the postbiotics significantly improved intestinal epithelial wound healing capabilities after mechanical injury, such as cell scratches in IPEC-J2 cells (p < 0.05). Conclusion: In brief, we developed postbiotics through optimized solid-state fermentation with potential benefits for gut health. Therefore, our findings suggested that the novel postbiotics could be used as potential functional food products for improving body health.

Optimization of Exopolysaccharide Produced by Lactobacillus plantarum R301 and Its Antioxidant and Anti-Inflammatory Activities.

In this study, the yield of exopolysaccharide (EPS) from Lactobacillus plantarum R301 was optimized using a single-factor experiment and response surface methodology (RSM). After optimization, the EPS yield was increased with a fold-change of 0.85. The significant factors affecting EPS production, as determined through a Plackett-Burman design and Central Composite Design (CCD), were MgSO4 concentration, initial pH, and inoculation size. The maximum yield was 97.85 mg/mL under the condition of 0.01% MgSO4, an initial pH 7.4, and 6.4% of the inoculation size. In addition, the EPS exhibited strong antioxidant activity, as demonstrated by its ability to scavenge DPPH, ABTS, and hydroxyl radicals. The scavenging rate was up to 100% at concentrations of 4 mg/mL, 1 mg/mL, and 2 mg/mL, respectively. Moreover, the EPS also exhibited reducing power, which was about 30% that of ascorbic acid when both tended to be stable with the increased concentration. These results suggest that L. plantarum R301 EPS possesses different antioxidant mechanisms and warrants further investigation. In addition to its antioxidant activity, the EPS also demonstrated good anti-inflammatory activity by inhibiting the inflammation induced by lipopolysaccharide (LPS) in RAW 264.7 cells, which could decrease nitric oxide (NO) production and expression of the proinflammatory cytokine Il-6. These findings suggest that L. plantarum R301 EPS could be used as a potential multifunctional food additive in the food industry.

Soybean Oil Regulates the Fatty Acid Synthesis II System of Bacillus amyloliquefaciens LFB112 by Activating Acetyl-CoA Levels.

[Background] Bacillus LFB112 is a strain of Bacillus amyloliquefaciens screened in our laboratory. Previous studies found that it has a strong ability for fatty acid metabolism and can improve the lipid metabolism of broilers when used as feed additives. [Methods] This study aimed to confirm the fatty acid metabolism of Bacillus LFB112. Sterilized soybean oil (SSO) was added to the Beef Peptone Yeast (BPY) medium, and its effect on fatty acid content in the supernatant and bacteria, as well as expression levels of genes related to fatty acid metabolism, were studied. The control group was the original culture medium without oil. [Results] Acetic acid produced by the SSO group of Bacillus LFB112 decreased, but the content of unsaturated fatty acids increased. The 1.6% SSO group significantly increased the contents of pyruvate and acetyl-CoA in the pellets. Furthermore, the mRNA levels of enzymes involved in the type II fatty acid synthesis pathway of FabD, FabH, FabG, FabZ, FabI, and FabF were up-regulated. [Conclusions] Soybean oil increased the content of acetyl-CoA in Bacillus LFB112, activated its type II fatty acid synthesis pathway, and improved the fatty acid metabolism level of Bacillus LFB112. These intriguing results pave the way for further investigations into the intricate interplay between Bacillus LFB112 and fatty acid metabolism, with potential applications in animal nutrition and feed additive development.

Effects of Clostridium butyricum as an Antibiotic Alternative on Growth Performance, Intestinal Morphology, Serum Biochemical Response, and Immunity of Broilers.

The current study aimed to investigate the effects of Clostridium butyiricum on growth performance, intestinal morphology, serum biochemical response, and immunity in broiler chickens. A total of 330 commercial one-day-old, mixed-sex Ross 308 broilers were randomly divided into five treatment groups with six replicates per group. The broilers were fed the basal diet (CON), the basal diet with 150 mg/kg of aureomycin (AM), the basal diet with C. butyricum at 2 × 108 CFU/kg (CBL), the basal diet with C. butyricum at 4 × 108 CFU/kg (CBM), and the basal diet with C. butyricum at 8 × 108 CFU/kg (CBH). Results showed that the final body weight (BW) (p < 0.01; p < 0.05), ADG from day 22 to 39 (p < 0.05), and ADG from day 1 to 39 (p < 0.01; p < 0.05) were improved in a linear and quadratic response with the inclusion of C. butyricum. There were no differences in feed conversion rate (FCR) among all groups (p > 0.05). Supplementation with C. butyricum quadratically reduced the crypt depth at day 21 (p < 0.01), linearly improved the villus height in the jejunum at day 39 (p < 0.001), and linearly and quadratically increased the villus height to crypt depth (V/C) ratio in the jejunum at day 21 (p < 0.01) and day 39 (p < 0.01; p < 0.001). Dietary C. butyricum affected the thymus index at day 21 and day 39 (linear, p < 0.01), and the bursa of Fabricius index at day 39 (quadratic, p < 0.05). Compared to the AM group, the serum urea contents were decreased (p < 0.05) but the IgG contents were increased in the CBL and CBH groups at day 21 (p < 0.01); in addition, serum albumin (ALB) concentrations in all the C. butyricum-supplemented groups (p < 0.01) and IgG concentrations in the CBM group were augmented at day 39 (p < 0.05). In conclusion, dietary C. butyricum could enhance growth performance by improving jejunal morphology and stimulating immunity organ development in broilers, and could be an alternative to antibiotics in poultry feeds.

Biofilm and Small Colony Variants-An Update on Staphylococcus aureus Strategies toward Drug Resistance.

Recently, the drawbacks arising from the overuse of antibiotics have drawn growing public attention. Among them, drug-resistance (DR) and even multidrug-resistance (MDR) pose significant challenges in clinical practice. As a representative of a DR or MDR pathogen, Staphylococcus aureus can cause diversity of infections related to different organs, and can survive or adapt to the diverse hostile environments by switching into other phenotypes, including biofilm and small colony variants (SCVs), with altered physiologic or metabolic characteristics. In this review, we briefly describe the development of the DR/MDR as well as the classical mechanisms (accumulation of the resistant genes). Moreover, we use multidimensional scaling analysis to evaluate the MDR relevant hotspots in the recent published reports. Furthermore, we mainly focus on the possible non-classical resistance mechanisms triggered by the two important alternative phenotypes of the S. aureus, biofilm and SCVs, which are fundamentally caused by the different global regulation of the S. aureus population, such as the main quorum-sensing (QS) and agr system and its coordinated regulated factors, such as the SarA family proteins and the alternative sigma factor σB (SigB). Both the biofilm and the SCVs are able to escape from the host immune response, and resist the therapeutic effects of antibiotics through the physical or the biological barriers, and become less sensitive to some antibiotics by the dormant state with the limited metabolisms.

Novel Hybrid Peptide Cathelicidin 2 (1-13)-Thymopentin (TP5) and Its Derived Peptides with Effective Antibacterial, Antibiofilm, and Anti-Adhesion Activities.

The increasing numbers of infections caused by multidrug-resistant (MDR) pathogens highlight the urgent need for new alternatives to conventional antibiotics. Antimicrobial peptides have the potential to be promising alternatives to antibiotics because of their effective bactericidal activity and highly selective toxicity. The present study was conducted to investigate the antibacterial, antibiofilm, and anti-adhesion activities of different CTP peptides (CTP: the original hybrid peptide cathelicidin 2 (1-13)-thymopentin (TP5); CTP-NH2: C-terminal amidated derivative of cathelicidin 2 (1-13)-TP5; CTPQ: glutamine added at the C-terminus of cathelicidin 2 (1-13)-TP5) by determining the minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), propidium iodide uptake, and analysis by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy). The results showed that CTPs had broad-spectrum antibacterial activity against different gram-positive and gram-negative bacteria, with MICs against the tested strains varying from 2 to 64 µg/mL. CTPs at the MBC (2 × MIC 64 µg/mL) showed strong bactericidal effects on a standard methicillin-resistant Staphylococcus aureus strain ATCC 43300 after co-incubation for 6 h through disruption of the bacterial membrane. In addition, CTPs at 2 × MIC also displayed effective inhibition activity of several S. aureus strains with a 40-90% decrease in biofilm formation by killing the bacteria embedded in the biofilms. CTPs had low cytotoxicity on the intestinal porcine epithelial cell line (IPEC-J2) and could significantly decrease the rate of adhesion of S. aureus ATCC 43300 on IPEC-J2 cells. The current study proved that CTPs have effective antibacterial, antibiofilm, and anti-adhesion activities. Overall, this study contributes to our understanding of the possible antibacterial and antibiofilm mechanisms of CTPs, which might be an effective anti-MDR drug candidate.

Targeting the TLR2 Receptor With a Novel Thymopentin-Derived Peptide Modulates Immune Responses.

The innate and adaptive immune systems act in concert to protect us from infectious agents and other harmful substances. As a state of temporary or permanent immune dysfunction, immunosuppression can make an organism more susceptible to infection, organ injury, and cancer due to damage to the immune system. It takes a long time to develop new immunomodulatory agents to prevent and treat immunosuppressive diseases, with slow progress. Toll-like receptor 2 (TLR2) agonists have been reported as potential immunomodulatory candidates due to their effective activation of immune responses. It has been demonstrated that thymopentin (TP5) could modulate immunity by binding to the TLR2 receptor. However, the fairly short half-life of TP5 greatly reduces its pharmacological potential for immunosuppression therapy. Although peptide cathelicidin 2 (CATH2) has a long half-life, it shows poor immunomodulatory activity and severe cytotoxicity, which seriously hampers its clinical development. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In this study, to overcome all these challenges faced by the parental peptides, six hybrid peptides (CaTP, CbTP, CcTP, TPCa, TPCb, and TPCc) were designed by combining the full-length TP5 with different active fragments of CATH2. CbTP, the most potent TLR2 agonist among the six hybrid peptides, was effectively screened through in silico analysis and in vitro experiments. The CbTP peptide exhibited lower cytotoxicity than either CATH2 or TP5. Furthermore, the immunomodulatory effects of CbTP were confirmed in a CTX-immunosuppressed mouse model, which showed that CbTP has increased immunopotentiating activity and physiological stability compared to the parental peptides. CbTP successfully inhibited immunosuppression and weight loss, increased immune organ indices, and improved CD4+/CD8+ T lymphocyte subsets. In addition, CbTP significantly increased the production of the cytokine TNF-α and IL-6, and the immunoglobulins IgA, IgM, and IgG. The immunoenhancing effects of CbTP were attributed to its TLR2-binding activity, promoting the formation of the TLR2 cluster, the activation of the TLR2 receptor, and thus activation of the downstream MyD88-NF-кB signaling pathway.

Design and Immunological Evaluation of a Hybrid Peptide as a Potent TLR2 Agonist by Structure-Based Virtual Screening.

Immunity is a versatile defensive response that is involved in protecting against disease by identifying and destroying self and non-self harmful substances. As a state of temporary or permanent immune dysfunction, immunosuppression can make an organism more susceptible to infection, organ injury, and cancer due to damage to the immune system. It has taken a long time to develop new immunomodulatory agents to prevent and treat immunosuppressive diseases. In recent years, Toll-like receptor 2 (TLR2) agonists have been reported to have profound effects on the immune system, and they are regarded as potent immunomodulatory candidates. TP5 and LL-37, the potent immunomodulatory agents, have been reported to produce a robust innate immune response by binding to TLR2. However, their development has been weakened by several concerns, such as potential cytotoxicity, weak physiological stability and poor immunomodulatory activity. To overcome these challenges, hybridization has been proposed. Therefore, six hybrid peptides (LTPa, LTPb, LTPc, TPLa, TPLb, and TPLc) were designed by combining the full-length TP5 with a characteristic fragment of LL-37 that included LL-37 (13-36), LL-37 (17-29), and LL-37 (13-31). LTPa, the most potent TLR2 agonist, was simply and effectively screened by molecular docking and in vitro experiments. Furthermore, the immunomodulatory effects of LTPa were confirmed by a CTX-immunosuppressed murine model, which demonstrated that LTPa successfully inhibit immunosuppression, increased immune organ indices, enhanced DC maturation, regulated T lymphocyte subsets, and increased cytokine and Ig contents. Our study also revealed that the immunomodulatory effects of LTPa are associated with binding to TLR2, forming TLR2 clusters, and activating the NF-κB signaling pathway.

Association of polymorphisms in Pit-1 gene with growth and feed efficiency in meat-type chickens.

OBJECTIVE: The pituitary specific transcription factor-1 (Pit-1) gene is responsible for pituitary development and growth hormone expression and is regarded as a pivotal candidate gene for growth and production in chickens. Therefore, the aim of this study was to investigate the association of Pit-1 polymorphisms with growth and feed efficiency traits in yellow meat-type chickens. METHODS: In the present study, five single nucleotide polymorphisms (SNPs) of Pit-1 were selected and genotyped by high-throughput matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in 724 meat-type chickens. RESULTS: Association analysis showed that rs13687126 of Pit-1 was strongly associated with body weight gain (BWG) and feed intake (FI) (p<0.05), and that rs13687128 was significantly correlated with body weight at 70 days of age (BW70), BWG and feed conversion ratio (FCR) (p<0.05). SNP rs13905622 was strongly related to BW70 and FCR (p<0.05). Furthermore, birds with the GG genotype of rs13687126 had larger BWG and FI than those with the AG genotype (p<0.05). Individuals with the TT genotype of rs13687128 were significantly higher BW70 and BWG than those of the CT and CC genotype, while FCR was just the opposite (p<0.05). For rs13905622, the AA chickens showed strongly larger BW70 and lower FCR compared with the AT and TT chickens (p<0.05). Additionally, an ACA haplotype based on rs13687126, rs13687128, and rs13905622 had significant effects on BW70 and FCR (p<0.05). CONCLUSION: Our studies thus provide crucial evidence for the relationship between polymorphisms of Pit-1 and growth and feed efficiency traits which may be useful for meat-type chicken breeding programs.