This study aimed to investigate the effects of Bacillus amyloliquefaciens LFB112 on the growth performance, carcass traits, immune response, and serum biochemical parameters of broiler chickens. A total of 396 1 day old, mixed-sex commercial Ross 308 broilers with similar body weights were allotted into six treatment groups. The assigned groups were the CON group (basal diet with no supplement), AB (antibiotics) group (basal diet + 150 mg of aureomycin/kg), C+M group (basal diet + 5 × 10⁸ CFU/kg B. amyloliquefaciens LFB112 powder with vegetative cells + metabolites), C group (basal diet + 5 × 10⁸ CFU/kg B. amyloliquefaciens LFB112 vegetative cell powder with removed metabolites), M group (basal diet + 5 × 10⁸ CFU/kg B. amyloliquefaciens LFB112 metabolite powder with removed vegetative cells), and CICC group (basal diet + 5 × 10⁸ CFU/kg Bacillus subtilis CICC 20179). Results indicated that chickens in the C+M, C, and M groups had higher body weight (BW) and average daily gain (ADG) (p < 0.05) and lower feed conversion ratio (FCR) (p = 0.02) compared to the CON group. The C+M group showed the lowest abdominal fat rate compared to those in the CON, AB, and CICC groups (p < 0.05). Compared to the CON group, serum IgA and IgG levels in the C+M, C, and M groups significantly increased while declining in the AB group (p < 0.05). B. amyloliquefaciens LFB112 supplementation significantly reduced the serum triglyceride, cholesterol, urea, and creatinine levels, while increasing the serum glucose and total protein (p < 0.05). In conclusion, B. amyloliquefaciens LFB112 significantly improved the growth performance, carcass traits, immunity, and blood chemical indices of broiler chickens and may be used as an efficient broiler feed supplement.

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

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.

Solid-state microbial fermentation (SSMF) has been established as an effective bioprocessing strategy to augment the nutritional value of plant-derived feed substrates while reducing anti-nutritional factors (ANFs). However, there have been limited studies on the effects of microbial solid-state fermentation on the nutritional value and potential functional components in cottonseed hulls. This study investigated the nutritional enhancement of cottonseed hulls through anaerobic solid-state fermentation mediated by Candida utilis CU-3, while exploring the functional potential of the fermented feed by analyzing fungal community dynamics and metabolite profiling. The laboratory-preserved free gossypol-degrading strain Candida utilis CU-3 was inoculated into unsterilized, crushed, and screened cottonseed hulls for solid-state fermentation at room temperature for 10 days. The results demonstrated that, compared to the control group, the experimental group achieved a 61.90% increase in free gossypol degradation rate, a 27.78% improvement in crude protein content, and a 5.07% reduction in crude fiber, while crude fat showed no significant difference. During the fermentation process, microbial diversity decreased, and Candida utilis CU-3 became the dominant species. Untargeted metabolomics data revealed that cottonseed hulls inoculated with Candida utilis CU-3 produced functional bioactive compounds during fermentation, including chrysin, myricetin (anti-inflammatory, antibacterial, and antioxidant activities), and ginsenoside Rh2 (anticancer, antibacterial, and neuroprotective properties). This study demonstrates that inoculating Candida utilis CU-3 into cottonseed hulls enhances their health-promoting potential through the biosynthesis of diverse functional metabolites, providing a theoretical foundation for improving the nutritional profile of cottonseed hull-fermented feed.

[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.