Probiotics and Antimicrobial Proteins最新文献

Bacterial foodborne contamination poses a dual challenge of chemical preservative risks and antibiotic resistance, drives the need for green production of natural antimicrobial alternatives. The reported cationic antimicrobial peptide (AMP) Spgillcin_177-189 derived from the Scylla paramamosain, has strong antimicrobial activity against Staphylococcus aureus and clinical isolation strains. To meet industry demand in future, large-scale production of Spgillcin_177- 189 is essential. In the study, Pichia pastoris expression system was established for production of the recombinant Spgillcin_177- 189 (rSpgillcin_177-189). Then, multicopy strategy was selectively designed by employing the Golden Gate assembly technology to efficiently construct multi-copy plasmids, which significantly enhanced the expression level of Spgillcin_177- 189. A yield of 126.1 mg/L was harvested with 2.75-fold higher that of the single-copy strain. In addition, the recombinant Spgillcin_{177 - 189} exhibited potent antibacterial activity against multiple foodborne pathogens within a MIC range of 5.25-84 µg/mL. It also showed effective bactericidal activity and anti-biofilm activity against Staphylococcus aureus and Vibrio parahaemolyticus. rSpgillcin_{177 - 189} exhibited good thermostability, with no obvious cytotoxicity and hemolytic activity. rSpgillcin_{177 - 189} may interact with microbial surface components via hydrogen bonding, which were vital for peptide activity in combating bacteria. The rSpgillcin_{177 - 189} specifically targeting the cell membrane, disrupted bacterial membrane integrity and leading to cell death. This study provided a very feasible genetic engineering strategy for large-scale production of rSpgillcin_{177 - 189}, which will be applied at a lower cost in agricultural and food industries in future.

This study investigated the oxidative stress alleviating effects of vegetable juice (VJ) fermented with Lactiplantibacillus plantarum WB3813 and L. plantarum WB3814 using a H₂O₂-induced A549 cell model. Both WB3813 and WB3814 strains exhibited stable fermentation characteristics in VJ, supporting their suitability as starter cultures for developing plant-based probiotic products. Fermentation enhanced antioxidant activity, with WB3814-VJ showing the highest total polyphenol content (TPC) (204 mg GAE/mL), while WB3813-VJ exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) (38%) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities (68%). Treatment with fermented VJ with WB3813 and WB3814 showed greater cytoprotective effects than LGG in H₂O₂-induced A549 cells and significantly reduced reactive oxygen species (ROS) production (89.74%-91.67%). Additionally, the expressions of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were downregulated, while that of antioxidant-related genes, including Nrf2, HO-1, and NQO1, were upregulated. These anti-inflammatory and antioxidant effects were closely associated with the modulation of the NF-κB and Keap1/Nrf2/HO-1 signaling pathways. These findings suggest that VJ fermented with L. plantarum WB3813 and WB3814 can mitigate oxidative stress through its anti-inflammatory and antioxidant activities, supporting its potential as a functional food for respiratory health.

A synbiotic composed of pomegranate peels extract, Saccharomyces (S.) cerevisiae, Lactococcus (L.) lactis, Lactobacillus (Lact) paracasei, and Lact. plantarum was fabricated in free (FPLYS) and (NPLYS) nano-encapsulated forms. The encapsulation efficiency, physichochemical properties, and antioxidant and antimicrobial activities of FPLYS and NPLYS were tested. For in vitro rumen fermentation study, a basal diet was supplemented with FPLYS-H (10⁹ CFU probiotics/g diet), NPLYS-H (10⁹ CFU probiotics/g diet), NPLYS-L (10⁷ CFU probiotics/g diet), 40 mg/kg diet monensin (MONS), or not supplemented (CON). The encapsulation efficiency of gum arabic-gelatin complex for prebiotic and probiotics was 75.26% and 91.80%, respectively. The size and zeta potential of gum arabic-gelatin were 27.72 nm and 11 mV, respectively. NPLYS showed two-time higher antioxidant activity than FPLYS. NPLYS showed antimicrobial activity against Escherichia (E.) coli and Staphylococcus (Staph.) aureus similar to gentamicin, while FPLYS showed lower activity (p < 0.05). NPLYS and FPLYS showed antimicrobial activity against Pseudomonas (Ps.) aeruginosa and antifungal activity against Aspergillus (A.) niger and A. flavus, but lower than getntamicin and fluconazol, respectively (p < 0.05). NPLYS-L significantly increased total gas production compared to CON, MONS, and NPLYS-H. Compared to CON, both MONS and FPLYS-H significantly reduced methane production, followed by NPLYS-H and NPLYS-L. Among treatments, MONS significantly increased propionate, while reducing butyrate and acetate/propionate ration. All treatments increased cellulose digestibility and protozoal count (p < 0.05). NPLYS-L significantly decreased rumen ammonia concentration compared to CON, however other treatments recorded intermediate values. The synbiotic developed in this study can be either used as an antimicrobial agent and/or a rumen fermentation modifier, replacing synthetic antibiotics used for these purposes. Nanoencapsulation process improved biological activities of the synbiotic.

Chronic stress is a critical risk factor for depression, and prolonged exposure to stressors increases the risk of metabolic dysregulation. Gut microbiota (GM) intervention is a promising therapeutic target for stress-related emotional and behavioral disorders. In the present study, we apply compound lactic acid bacteria (CLAB) (Lacticaseibacillus rhamnosus GG and Enterococcus faecium) administration therapy to investigate the effect of gut microbiota (GM) on chronic restraint stress (CRS)-depression and its related glucose-lipid metabolism disturbance. We established a non-human primate model of CRS depression to mimic the depressive state in humans and applied CLAB for gastric gavage treatment. CRS macaques exhibit marked depressive-like behavior and disrupted GM structure, decreased levels of C1q/tumor necrosis factor-related protein 9 (CTRP9) and increased levels of CTRP5, and induced insulin resistance (IR) and lipid metabolism disorders. Treatment with CLAB normalized GM structure in CRS macaques, increased the abundance of Lactobacillus murinus and Lactobacillus reuteri, and reduced depressive-like behavior in CRS macaques, with a rebound in CTRP9 and HDL-C levels, reduced CTRP5 and TG levels and improved IR. In addition, CLAB may serve as a promising non-pharmacological intervention that alleviates depression-related metabolic dysregulation by modulating the gut microbiota and improving lipid and glucose metabolism.

This study evaluated the viability and functional properties of postbiotics derived from Bifidobacterium animalis subsp. lactis BB-12 following thermal and ultrasound processing. Sixteen processing conditions were tested, including heat treatments (65-95 °C for 5-90 min) and ultrasound amplitudes (98-320 μm for 15-60 min). Postbiotics were assessed for antimicrobial activity, probiotic-stimulating effects on Lacticaseibacillus casei 431 and Bifidobacterium spp. cultures, and their capacity to enhance short-chain fatty acid (SCFA) production. Complete inactivation was achieved under all heat treatment conditions, whereas only 11 ultrasound conditions resulted in total loss of culturability, indicating greater microbial resistance at lower intensities or shorter exposures. Discrepancies between flow cytometry and colony counting in ultrasound-treated samples suggest the presence of viable but non-culturable cells (VBNC), highlighting the limitations of culture-based viability assessments, especially when assessing VBNC states. Postbiotics from heat-treatment showed significantly stronger probiotic-enhancing effects, with growth increases up to 2.9-fold for L. casei and 37.7-fold for Bifidobacterium spp. compared to controls, alongside greater antimicrobial activity, especially against Enterococcus faecalis. Both processing methods significantly increased SCFA levels (p < 0.05). In conclusion, it can be said that heat treatment was more effective than ultrasound in producing biologically active postbiotics from B. lactis BB-12. The enhanced functional properties observed in heat-inactivated preparations underscore thermal processing as a robust method for postbiotic production. These findings highlight the potential of heat-derived postbiotics as active bioingredients for microbiota-targeted functional foods and nutraceuticals. Further in vivo validation and standardization efforts are needed to fully demonstrate their therapeutic potential and support regulatory approval.

The search for effective probiotic alternatives to antibiotics in aquaculture has increased in recent years. This study evaluated the dietary supplementation of Saccharomyces cerevisiae and Pseudomonas aeruginosa on growth, feed utilization, intestinal morphology, and health of red tilapia (Oreochromis sp.) over a 60-day feeding trial. Fish were fed diets containing three graded levels of S. cerevisiae (1, 2, and 4 g kg⁻¹ feed) or P. aeruginosa (1, 2, and 3 mL kg⁻¹ feed), and compared with a control group. Both probiotics enhanced performance and physiological responses compared with the control. The highest inclusion levels (4 g kg⁻¹ for S. cerevisiae and 3 mL kg⁻¹ for P. aeruginosa) yielded superior growth rate, feed efficiency, and intestinal villus development, accompanied by improved muscle protein content and stable hematological profiles. No adverse effects were detected in water quality or fish health. These findings highlight the potential of S. cerevisiae and a non-pathogenic P. aeruginosa strain as promising probiotics for red tilapia culture, demonstrating complementary effects on nutrient utilization and physiological condition.

The progressive implementation of worldwide restrictions on antibiotic use has necessitated the development of sustainable alternatives for controlling bacterial diarrhea in piglets. This study investigated the effects of a cell-free supernatant (CFS) from Limosilactobacillus reuteri SBC5-3 on growth performance, intestinal inflammation, and microbiota in weaned piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC K88). Forty-eight piglets were randomly assigned to four groups: a control group (CON, basal diet + physiological saline), an ETEC-challenged group (ETEC K88, basal diet + ETEC K88), a prevention group (CFS + ETEC K88, basal diet with CFS supplementation + ETEC K88), and a CFS group (CFS, basal diet with CFS supplementation + physiological saline). The CFS was administered orally at 5 mL/kg body weight for 7 days prior to challenge. Results showed that CFS pretreatment significantly reduced diarrhea rate and fecal scores, improved average daily gain, and alleviated ETEC K88-induced villus damage and lymphocyte infiltration in the jejunum and ileum. Moreover, CFS downregulated the expression of intestinal mucosal pro-inflammatory cytokines (IL1B, IL6, TNFA) and suppressed the activation of the NF-κB pathway by reducing phosphorylation of TAK1, IκBα, and p65. Intestinal microbiota analysis revealed that CFS increased the abundance of Lactobacillus and reduced Escherichia-Shigella. These findings demonstrate that L. reuteri SBC5-3 CFS effectively mitigates ETEC K88-induced diarrhea and intestinal inflammation by inhibiting NF-κB signaling and modulating intestinal microbiota, highlighting its potential as an antibiotic alternative in piglet production.

The global rise in resistance to chemical fungicides and their strict regulation by the EU, has created an urgent need for alternative antifungal strategies in agriculture. Plant defensins represent promising alternatives owing to their broad-spectrum antifungal activity, structural stability, and low toxicity to mammalian cells and plants. In this study, we identified and characterized a novel antifungal defensin, K4CBP6, from Solanum lycopersicum L., along with its γ-core peptide derivatives, K4CBP6γ1 and K4CBP6γ2, as potential biofungicide agents. Protein database mining revealed a widespread distribution of K4CBP6 homologs within the Solanaceae family. Recombinant K4CBP6 (rK4CBP6) was successfully produced using a Komagataella phaffii-based expression system, while K4CBP6γ1 and K4CBP6γ2 were chemically synthesized. Structural analyses via electrospray ionization mass spectrometry and electronic circular dichroism spectroscopy confirmed a cysteine-stabilized α-helix β-strand folded structure for rK4CBP6. In vitro susceptibility assays demonstrated that both rK4CBP6 and K4CBP6γ2 exhibited antifungal activity against major tomato pathogens, Botrytis cinerea, Cladosporium herbarum, and Fusarium oxysporum with minimum inhibitory concentrations ranging from 12.5 to 25 µg ml^-1. Furthermore, neither rK4CBP6 nor K4CBP6γ2 exhibited cytotoxic effects on mammalian cell lines or adverse effects in animal and plant model systems even at concentrations of 200 and 400 µg ml^-1. Proof-of-concept experiments on tomato plants and fruits confirmed their protective efficacy against B. cinerea and C. herbarum. These findings highlight the potential of rK4CBP6 and K4CBP6γ2 as sustainable biofungicide candidates for plant disease management, owing to their antifungal efficacy both in vitro and in planta, along with their lack of cytotoxic effects.

The Pacific white shrimp (Litopenaeus vannamei) aquaculture faces emerging threats from novel pathogens and escalating antibiotic resistance. This study successfully isolated and identified the pathogenic bacterium Acinetobacter ursingii strain 31C2 from diseased L. vannamei using an integrated approach combining microbiological, biochemical, and molecular techniques. The pathogenicity of this strain was confirmed in L. vannamei and marine medaka (Oryzias melastigma) infection models, exhibiting a strong dose-dependent mortality, with median lethal doses (LD₅₀) of 2.83 × 10⁴ CFU/g shrimp and 2.58 × 10⁶ CFU/fish, respectively. Infection caused severe hepatopancreatic necrosis (tubular deformation and epithelial vacuolation) and intestinal villi destruction. Antimicrobial susceptibility testing revealed that the 31C2 strain was resistant to tetracycline and azithromycin. To identify effective agents targeting this strain, the antimicrobial peptide Scymicrosin_7-26 (derived from Scylla paramamosain) was evaluated. The peptide had potent antibacterial activity against A. ursingii 31C2 in vitro (MIC: 3-6 µM). In vivo application significantly enhanced survival of L. vannamei and O. melastigma infected with 31C2 by 30% and 20%, respectively. Treatment drastically reduced bacterial loads in the hepatopancreas and intestine, restored tissue integrity, and modulated the immune response by suppressing the hyperactivation of the Toll and IMD pathways and their downstream transcription factors, dorsal and relish, while upregulating penaeidin3 and propo expression. This study identified A. ursingii as an emerging shrimp pathogen and validated Scymicrosin_7-26 as a promising antibiotic-free therapeutic for disease control in aquaculture.

Probiotics are live microorganisms that provide health benefits to the host by improving digestion, enhancing nutrient absorption, and modulating the immune system. Among them, lactic acid bacteria are known for producing vitamins and short-chain fatty acids, both essential for intestinal health. In this in silico study, we performed high-fidelity (PacBio HiFi) whole-genome sequencing and comprehensive comparative genomic analysis of five Lactobacillales strains (Enterococcus lactis, Enterococcus mundtii, Ligilactobacillus agilis, Limosilactobacillus reuteri, Limosilactobacillus vaginalis) isolated from the intestinal microbiota of chickens and pigs. The assembled genomes ranged from 1.8 to 2.8 Mb, with more than 98% completeness and less than 1.31% contamination. Taxonomic classification, presence of antimicrobial resistance genes, bacteriocin biosynthetic potential, carbohydrate-active enzyme repertoires and vitamin biosynthesis pathways, and capacity to degrade plant polysaccharides were investigated. Functional characterization identified 65 families of carbohydrate-active enzymes, with E. mundtii presenting the greatest diversity (43 families) and absolute number (100 terms) of enzymes. Metabolic reconstruction suggested functional specialization among strains, with xylooligosaccharide degradation exclusive to E. mundtii and pectin utilization limited to E. lactis. Genes related to the biosynthesis of B-complex vitamins, including riboflavin, folate, and menaquinone, showed heterogeneous and complementary distribution among strains. These findings suggest the potential for metabolic complementarity and cross-feeding, where metabolites produced by one strain serve as precursors for biosynthetic pathways in others. Collectively, these genome-resolved insights offer a data-driven framework for designing multi-strain probiotics aimed at improving intestinal health and feed efficiency in poultry and swine.