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BACKGROUND: Cholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor. METHODS: We synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied. RESULTS: Nano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001). CONCLUSION: Our study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.

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Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC's swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC-MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.

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Background and Objectives: Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and the third most deadly cancer in the world. According to recent experimental reports, probiotics and their derivatives protect CRC patients from treatment-related side effects. Therefore, the present study aimed to investigate the cytotoxic impact of the cell-free supernatant (CFS) of Lentilactobacillus buchneri on the HT-29 cancer cell line. Materials and Methods: In the current study, we used the L. buchneri CFS, which was well isolated and identified in our previous investigation from traditional yogurt in the Arak region of Iran. The apoptosis induction in HT-29 cancer cells was assessed by cell cytotoxicity, flow cytometry, and qRT-PCR. Results: L. buchneri CFS inhibited the proliferation of HT-29 cancer cells in a time- and dose-dependent manner. The apoptotic effect of CFS was further supported by the flow cytometry data, which showed that the maximum incidence of apoptosis was observed in HT-29 cancer cells treated with the IC50 concentration of CFS after 72 hours. CFS of L. buchneri also exerted the up-regulating effect on the expression of pro-apoptotic genes including BAX, CASP9, and CASP3. L. buchneri CFS at an IC50 dose induced cell cycle arrest in the G0/G1 phase in HT-29 cells. Conclusion: This study indicates that L. buchneri CFS can prevent colorectal cancer (CRC) development in patients by inducing cancer cell apoptosis. This finding suggests that the CFS of L. buchneri could be used as a therapeutic agent for the control of CRC.

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Helicobacter pylori is a prominent gastrointestinal pathogen associated with various gastrointestinal illnesses. It presents substantial health risks due to its antibiotic resistance. Therefore, it is crucial to identify alternative treatments for H. pylori infections. Limosilactobacillus spp exhibit probiotic properties with beneficial effects in humans; however, the mechanisms by which it counteracts H. pylori infection are unknown. This study aimed to evaluate the potential of Limosilactobacillus fermentum T0701 lyophilized cell-free supernatants (LCFS) against H. pylori. The LCFS has varying antimicrobial activities, with inhibition zones of up to 10.67 mm. The minimum inhibitory concentration and minimum bacterial concentration of LCFS are 6.25-25.00 mg/mL and 6.25 mg/mL to > 50.00 mg/mL, respectively, indicating its capability to inhibit H. pylori. There is morphological damage observed in H. pylori treated with LCFS. Additionally, H. pylori adhesion to AGS cells (human gastric adenocarcinoma epithelial cells) reduces by 74.23%, highlighting the LCFS role in preventing bacterial colonization. Moreover, LCFS exhibits no cytotoxicity or morphological changes in AGS cells, and with no detected virulence or antimicrobial resistance genes, further supporting its safety profile. L. fermentum T0701 LCFS shows promise as a safe and effective non-toxic agent against H. pylori, with the potential to prevent gastric colonization.

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The aim of this study was to evaluate the effect of curcumin combined with Lactobacillus rhamnosus GG cell-free supernatant (LGG CFS) on the proliferation and induction of apoptosis in SCC-9 oral squamous cell carcinoma (OSCC) cells. Curcumin (40 µg/ml) and 25% v/v LGG CFS (108 CFU/ml), both alone and in a combination regimen, significantly decreased the viability of SCC-9 cells and normal human gingival fibroblast (HGF) cells. Interestingly, the combination of low doses of curcumin (5 µg/ml) and 25% v/v LGG CFS (106 CFU/ml) had no effect on the HGF cells but significantly inhibited the viability of SCC-9 cells (p < 0.05). Flow cytometric analysis revealed that SCC-9 cells treated with the combination of low-dose curcumin and low-dose LGG CFS had a higher apoptotic rate than the cells in the control group and the single treatment groups (p < 0.05). The combined treatment also significantly increased the Bax/Bcl2 mRNA and protein expression in SCC-9 cells (p < 0.05) but not in HGF cells, indicating the underlying mechanism of the combination regimen. There was no significant difference in caspase-3 protein expression or the Bcl-xL/Bak and Mcl-1/Bak ratios between the treatment and control groups in both cell lines. These findings suggested that the coadministration of curcumin and LGG could exhibit anticancer effects in SCC-9 cells without causing toxicity to normal fibroblast cells.

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Breast cancer has emerged as the most widespread and dangerous type of malignancy among women worldwide. Postbiotics have recently emerged as a promising novel adjunct in breast cancer therapy, due to their immunomodulatory effects and the potential to mitigate the adverse effects of conventional treatments. This study aims to investigate the therapeutic effects of postbiotics derived from Lactobacillus brevis (CSF2) and Lactobacillus casei (CFS5), specifically examining their ability to inhibit cell proliferation and induce apoptosis in MCF-7 breast cancer cells. In the current study, the anticancer activity of the cell-free supernatant of L. brevis and L. casei was investigated against MCF-7 cells using MTT assay, flow cytometry, and qRT-PCR technique. Both bacteria showed a high potential for the induction of cell death in MCF-7 cells. However, CFS2 cytotoxicity was significantly higher than CFS5. Flow cytometry results showed significant induction of early apoptosis in cells treated with both CFS2 and CFS5 within 48 h. The induction was notably higher in cells treated with CFS2 compared to CFS5. Overall, CFS2 therapy resulted in a greater increase in BAX and CASP9 gene expression, as well as an elevated BAX/BCL2 ratio within 48 h. These findings indicate that the CFS2 treatment showed a higher level of apoptotic activity than the CFS5 treatment. High biocompatibility was demonstrated following treatment with CFS2 and CFS5. These CFSs may serve as adjunctive medications for suppressing the proliferation of cancer cells. The results of the current study highlight the potential of postbiotics in cancer treatment and suggest that supernatants may serve as effective agents for suppressing cancer cell growth and viability.

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Introduction: Pear Valsa canker, caused by Valsa pyri (V. pyri), poses a major threat to pear production. We aimed to assess the effectiveness of the cell-free supernatant (CFS) produced by Trichoderma virens (T. virens) to control the development of pear Valsa canker and reveal the inhibitory mechanism against the pathogenic fungi. Results: Using morphological characteristics and phylogenetic analysis, the pathogen G1H was identified as V. pyri, and the biocontrol fungus WJ561 was identified as Trichoderma virens. CFS derived from WJ561 exhibited strong inhibition of mycelial growth and was capable of reducing the pathogenicity of V. pyri on pear leaves and twigs. Scanning electron microscopy (SEM) observations revealed deformations and shrinkages in the fungal hyphae treated with CFS. The CFS also destroyed the hyphal membranes leading to the leakage of cellular contents and an increase in the malondialdehyde (MDA) content. Additionally, CFS significantly inhibited the activities of catalase (CAT) and superoxide dismutase (SOD), and downregulated the expression of antioxidant defense-related genes in V. pyri, causing the accumulation of reactive oxygen species (ROS). Artesunate, identified as the main component in CFS by liquid chromatograph-mass spectrometry (LC-MS), exhibited antifungal activity against V. pyri. Conclusion: Our findings demonstrate the promising potential of T. virens and its CFS in controlling pear Valsa canker. The primary inhibitory mechanism of CFS involves multiple processes, including membrane damage and negatively affecting enzymatic detoxification pathways, consequently leading to hyphal oxidative damage of V. pyri. This study lays a theoretical foundation for the utilization of T. virens to control V. pyri in practical production.

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The intestinal epithelial barrier can prevent the invasion of pathogenic microorganisms and food antigens to maintain a consistent intestinal homeostasis. However, an imbalance in this barrier can result in various diseases, such as inflammatory bowel disease, malnutrition, and metabolic disease. Thus, the aim of this study was to select probiotic strains with epithelial barrier-enhancing ability in cell-based model and further investigate them for their improving effects on colitis mouse and weaned piglet models. The results showed that selected specific cell-free fermentation supernatants (CFSs) from Ligilactobacillus salivarius P1, Lactobacillus gasseri P12, and Limosilactobacillus reuteri G7 promoted intestinal epithelial cell growth and proliferation, strengthening the intestinal barrier in an intestinal epithelial cell line Caco-2 model. Further, the administration of CFSs of L. salivarius P1, L. gasseri P12, and L. reuteri G7 were found to ameliorate DSS-induced colitis in mice. Additionally, spray-dried powders of CFS from the three strains were examined in a weaned piglet model, only CFS powder of L. reuteri G7 could ameliorate the feed/gain ratio and serum levels of D-lactate and endotoxin. In conclusion, a new potential probiotic strain, L. reuteri G7, was selected and showed ameliorating effects in both colitis mouse and weaned piglet models.

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The beneficial effects of probiotics, postbiotics, and paraprobiotics have already been registered in managing ischemic stroke-generated neuroinflammation and gut dysbiosis. Herein, we examined the impact of cell-free supernatant (CFS) obtained from probiotics (Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01) in a rat transient middle cerebral artery occlusion (MCAO) model of focal cerebral injury. Pre-MCAO supplementation of probiotics (2 × 109 CFU/mL) for 21 days or CFS (1 mL/rat) for 7 days protect the MCAO-induced somatosensory and motor impairments recorded at 24 h and 72 h after reperfusion in foot-fault, rotarod, adhesive removal, and vibrissae-evoked forelimb placing tests. We also noted the reduced infarct area and neuronal degradation in the right hemisphere of probiotics- and CFS-recipient MCAO-operated animals. Moreover, MCAO-induced altered concentrations of glial-fibrillary acidic protein, NeuN, zonula occludens-1 (ZO-1), TLR4, IL-1ß, IL-6, and TNF-α, as well as matrix metalloproteinase-9 (MMP9) were reversed in the treatment groups. Probiotics and CFS treatment ameliorated the elevated levels of IL-6, IL-1ß, and MMP9 in the blood plasma of rats. The disrupted microbial phyla, Firmicutes-to-Bacteroides ratio, villi/crypt ratio, and decreased mucin-producing goblet cells, ZO-1, and occludin in the colon of MCAO-operated rats were recovered following probiotics and CFS treatment. NMR characterization of CFS and rat blood plasma revealed the presence of several important bacterial metabolites. These findings suggest that the CFS obtained from Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01 has the propensity to improve MCAO-generated neurological dysfunctions in rats by dampening neuroinflammation and modulating the gut-brain axis modulators.

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This study delves into the production and antimicrobial characteristics of cell-free supernatants from Pediococcus acidilactici (CFSs-Pa). Antimicrobial activity was initially observed in CFS-Pa harvested after 12 h of incubation and increased up to the late stationary phase at 48 h. The increase in antimicrobial activity did not align with total protein content, pointing to other factors linked to the accumulation of organic acids, particularly lactic acid. The SDS-PAGE analysis also indicated that the expected proteinaceous compound (pediocin) was not observed in CFS-Pa. Further investigations suggested that the antimicrobial properties of CFS-Pa were exclusively due to organic acids. The MIC values confirmed potent antimicrobial activity, particularly at a 10% dilution of CFS-Pa in MRS broth. The time-kill assays demonstrated bactericidal activity against EHEC, Listeria monocytogenes, and Staphylococcus aureus by 12 h, 18 h, and 24 h using a 10% dilution of CFS-Pa. Additionally, CFS-Pa exhibited dose-dependent antioxidant activity, requiring a 70% (v/v) concentration to inhibit DPPH scavenging activity by 50%. All the experimental results suggested potential applications of CFS-Pa in food preservation. An attempt to incorporate CFS-Pa into bacterial cellulose (BC) for edible food packaging demonstrated promising antimicrobial results, particularly against L. monocytogenes and S. aureus, with room for optimization.

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Attention toward the preventive effects of postbiotics on metabolic diseases has increased because of greater stability and safety over probiotics. However, studies regarding the bioactive effects of postbiotics, especially from probiotic Bacillus strains, are relatively limited. The anti-obesity effects of the cell-free culture supernatant of Bacillus velezensis KMU01 (CFS-B.vele) were evaluated using high-fat-diet (HFD)-induced mice. HFD-induced mice (n = 8 per group) received equal volumes of (1) CFS-B.vele (114 mg/kg) in PBS, (2) Xenical in PBS, or (3) PBS alone by oral gavage daily for 13 weeks. The results demonstrated that CFS-B.vele changed the gut microbiota and showed anti-obesity effects in HFD-induced obese mice. The elevated Firmicutes/Bacteroidota ratio induced by HFD was decreased in the CFS-B.vele group compared to the other groups (p < 0.05). The CFS-B.vele intervention led to the enrichment of SCFA-producers, such as Roseburia and Eubacterium, in the cecum, suggesting their potential involvement in the amelioration of obesity. Due to these changes, the various obesity-related biomarkers (body weight, fat in tissue, white adipose tissue weight and size, serum LDL-cholesterol level, hepatic lipid accumulation, and adipogenesis/lipogenesis-related gene/protein expression) were improved. Our findings suggest that CFS-B.vele has potential as a novel anti-obesity agent through modulation of the gut microbiota.

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BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

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The growing popularity of probiotics has led to the generation of substantial by-products. Among these, cell-free supernatant is recognized for containing beneficial postbiotics. Here, we upcycled Lactobacillus casei-free supernatant (LFS) into cheese analogues using inulin (INU), locust bean gum (LBG), and kappa-carrageenen (kCG). In this system, LBG/kCG established the primary structure, while interstitial spaces were progressively filled by INU. Despite the absence of milk proteins and fats, the cheese analogue with 35% w/w INU, 0.2% w/w LBG, and 0.8% kCG exhibited a texture and appearance resembling commercial processed cheese, as determined by texture profile analysis and dynamic small amplitude oscillatory rheometry technique. This can be attributed to the effective fat-replacing activity of INU regarding texture and rheology. Furthermore, the potassium-dominated salt composition of LFS proved advantageous for the LBG/kCG-derived structure-forming. These findings hold significant promise for upcycling probiotics wastewater into low-fat vegan cheese analogues, enriched with both prebiotics and postbiotics.

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Free-living amoebae of the genus Acanthamoeba are infected by various bacteria in nature, and thus bacteria can protect themselves from adverse environmental conditions. Contrary to this ameba-bacteria relationship whether Acanthamoeba has antibacterial effects on bacteria is the different aspect of the relationship between these microorganisms. In this study, we investigate various Acanthamoeba strains have antibacterial effects on various Staphylococcus strains. Three environmental Acanthamoeba strains, isolated from various aquatic environments in Turkey, and Acanthamoeba castellanii ATCC 50373 standard strains were used in the study. The antistaphylococcal effect of cell-free supernatant (CFS) obtained from these amoebae against 12 different Staphylococcus bacteria was investigated by colony counting method. In addition, the pathogenicity of the tested Acanthamoeba strains was determined using osmotolerance and thermotolerance tests. CFSs obtained from Acanthamoeba were found to have varying degrees of antistaphylococcal effects on various Staphylococcus strains (0%-100%). It was determined that the CFS of the standard Acanthamoeba strain showed 100% inhibitory effect against one clinical methicillin-resistant Staphylococcus aureus strain (M2). Also, CFS of Ugöl strain showed 99.97% inhibitory effect against one clinical methicillin-sensitive Staphylococcus epidermidis strain (L3). It was determined that all Acanthamoeba isolates had no pathogenic potential. According to the results, it has been observed that Acanthamoeba produces antibacterial substance(s) against Staphylococcus bacteria and that the ameba-bacteria relationship may also result in the detriment of the bacteria. Furthermore, the current study indicates that new and natural antimicrobial agents from Acanthamoeba can be used as an alternative to infections caused by Staphylococcus.

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Alternaria alternata, a notorious phytopathogenic fungus, has been documented to infect several plant species, leading to the loss of agricultural commodities and resulting in significant economic losses. Lactic acid bacteria (LAB) hold immense promise as biocontrol candidates. However, the potential of LABs derived from fruits remains largely unexplored. In this study, several LABs were isolated from tropical fruit and assessed for their probiotic and antifungal properties. A total of fifty-five LABs were successfully isolated from seven distinct fruits. Among these, seven isolates showed inhibition to growth of A. alternata. Two strains, isolated from fruits: Ficus benghalensis, and Tinospora cordifolia exhibited promising antifungal properties against A. alternata. Molecular identification confirmed their identities as Lactiplantibacillus plantarum MYSVB1 and MYSVA7, respectively. Both strains showed adaptability to a wide temperature range (10-45°C), and salt concentrations (up to 7%), with optimal growth around 37 °C and high survival rates under simulated gastrointestinal conditions. Among these two strains, Lpb. plantarum MYSVB1 demonstrated significant inhibition (p < 0.01) of the growth of A. alternata. The inhibitory effects of cell-free supernatant (CFS) were strong, with 5% crude CFS sufficient to reduce fungal growth by >70% and complete inhibition by 10% CFS. Moreover, the CFS was inhibitory for both mycelial growth and conidial germination. CFS retained its activity even after long cold storage. The chromatographic analysis identified organic acids in CFS, with succinic acid as the predominant constituent, with lactic acid, and malic acid in descending order. LAB strains isolated from tropical fruits showed promising probiotic and antifungal properties, making them potential candidates for various applications in food and agriculture.

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Helicobacter pylori infection is the major risk factor associated with the development of gastric cancer. Currently, administration of standard antibiotic therapy combined with probiotics and postbiotics has gained significant attention in the management of H. pylori infection. In this work, the immunomodulatory effects of Lactobacillus crispatus-derived extracellular vesicles (EVs) and cell-free supernatant (CFS) were investigated on H. pylori-induced inflammatory response in human gastric adenocarcinoma (AGS) cells. L. crispatus-derived EVs were isolated by ultracentrifugation and physically characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, the protein content of L. crispatus-derived EVs was also evaluated by SDS-PAGE. Cell viability of AGS cells exposed to varying concentrations of EVs and CFS was assessed by MTT assay. The mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR. ELISA was used for the measurement of IL-8 production in AGS cells. In addition, EVs (50 µg/mL) and CFS modulated the H. pylori-induced inflammation by downregulating the mRNA expression of IL-1ß, IL-6, IL-8, and TNF-α, and upregulating the expression of IL-10, and TGF-ß genes in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with L. crispatus-derived EVs and CFS. In conclusion, our observation suggests for the first time that EVs released by L. crispatus strain RIGLD-1 and its CFS could be recommended as potential therapeutic agents against H. pylori-triggered inflammation.

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To confirm the antagonistic activity characterization of the strain Pseudoalteromonas SW-1 (P. SW-1), its cell-free supernatant (CFS) was studied against a clam pathogenic strain of Vibrio Alginolyticu MP-1 (V.MP-1). The CFS of P. SW-1 exhibited evident antagonistic activities against the pathogens, and the absorbance value (600 nm) of V. MP-1 remained at a lower level at 24 h when compared with the control. The results showed that the inhibitory activities of strain P. SW-1 CFS showed differences after treatment with heat, acid and alkali, and proteinase K. The CFS of P. SW-1 inhibitory activities decreased after treatment with heat, but the inhibitory activities of strain P. SW-1 CFS were still effective after treatment with proteinase K for 24 h. The acid and alkali treatments could increase the inhibitory activities of strain P. SW-1 CFS. Therefore, the ammonium sulfate precipitation test also indicated that P. SW-1 could produce some active protein compounds to antagonize pathogenic V. MP-1.

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This study investigated the impact of initial culture media pH on the antibacterial properties and metabolic profile of cell-free supernatants (CFSs) from Lactobacillus acidophilus BIOTECH 1900 (LAB1900). The CFSs harvested from LAB1900 grown in de Man, Rogosa, Sharpe broth with initial pH of 5.5 (CFS5.5) and 6.6 (CFS6.6) were tested. The two CFSs elicited varying degrees of activity against three gram-negative bacteria. In the agar-well diffusion against Pseudomonas aeruginosa, CFS5.5 and CFS6.6 recorded 14.36 ± 1.34 and 13.06 ± 1.29 mm inhibition, respectively. Interestingly, against Klebsiella pneumoniae, CFS5.5 showed 14.36 ± 1.56 mm inhibition which was significantly higher than the 12.22 ± 1.31 mm inhibition of CFS6.6 (p = 0.0464). While against Acinetobacter baumannii, significantly higher inhibition of 10.66 ± 0.51 mm was observed in CFS6.6 compared to the 7.58 ± 1.93 mm inhibition of CFS5.5 (p = 0.0087). Nonetheless, both CFSs were bactericidal, with a minimum inhibitory and bactericidal concentration range of 3.90625-7.8125 mg/mL. The varied antibacterial activities may be attributed to the metabolite compositions of CFSs. A total of 152 metabolites driving the separation between CFSs were noted, with the majority upregulated in CFS5.5. Furthermore, 15 were putatively identified belonging to acylcarnities, vitamins, gibberellins, glycerophospholipids, and peptides. In summary, initial culture media pH affects the production of microbial metabolites with antibacterial properties.

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BACKGROUND AND OBJECTIVE: Ensuring colon homeostasis is of significant influence on colon cancer and delicate balance is maintained by a healthy human gut microbiota. Probiotics can modulate the diversity of the gut microbiome and prevent colon cancer. Metabolites/byproducts generated by microbial metabolism significantly impact the healthy colonic environment. Hesperidin is a polyphenolic plant compound well known for its anticancer properties. However, low bioavailability of hesperidin after digestion impedes its effectiveness. CYP2W1 is a newly discovered oncofetal gene with an unknown function. CYP2W1 gene expression peaks during embryonic development and is suddenly silenced immediately after birth. Only in the case of some types of cancer, particularly colorectal and hepatocellular carcinomas, this gene is reactivated and its expression is correlated with the severity of the disease. This study aimed to investigate the effects of hesperidin-treated Lacticaseibacillus rhamnosus GG (LGG) cell-free supernatants on CaCo2 colon cancer cell viability and CYP2W1 gene expression. METHODS: Alamar Blue cell viability assay was used to investigate the cytotoxic effect of cell-free supernatant of LGG grown in the presence of hesperidin on CaCo2 cells. To observe the effect of cell-free supernatants of LGG on the expression of CYP2W1 gene, qRT-PCR was performed. RESULTS: Five times diluted hesperidin treated cell-free supernatant (CFS) concentration considerably reduced CaCo2 colon cancer cell viability. Furthermore, CYP2W1 gene expression was similarly reduced following CFS treatments and nearly silenced under probiotic bacteria CFS treatment. CONCLUSION: The CYP2W1 gene expression was strongly reduced by cell-free supernatants derived from LGG culture, with or without hesperidin. This suggests that the suppression may be due to bacterial byproducts rather than hesperidin. Therefore, the CYP2W1 gene in the case of deregulation of these metabolites may cause CYP2W1-related colon cancer cell proliferation.

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Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) infections are well-known hospital-borne infections and are a major contributing factor to global health concerns of antimicrobial resistance due to the formation of biofilms. Probiotics are known to assist in the healing of wounds through immunomodulation and also possess anti-pathogen properties via competitive inhibition. The probiotic bacterium, Lactiplantibacillus plantarum MTCC 2621 and its cell-free supernatant (Lp2621) have previously been reported to have antibacterial, excellent antioxidant, and wound healing activity in in vitro conditions and wounds contaminated with S. aureus in mice. Methods: In the current study, we evaluated its anti-MRSA, biofilm inhibition and eradication efficacy, immunomodulatory activity in THP-1 cells, and wound healing potential in wounds contaminated with MRSA infection in mice. Results: In agar well diffusion assay, Lp2621 showed anti-MRSA activity and revealed dose-dependent inhibition and eradication of biofilm by crystal violet assay as well as by Confocal Scanning Laser Microscopy (CLSM) analysis. Further, Lp2621 showed immunomodulatory activity at varied concentrations as measured by IL-6 and IL-10 gene expression in THP-1 cells. Similar findings were observed in serum samples of mice after treatment of excision wound contaminated with MRSA infection by Lp2621 gel, as evident by expression of IL-6 (pro-inflammatory) and IL-10 (anti-inflammatory) cytokines. Conclusions: Overall, our results show that Lp2621 has potent anti-MRSA and antioxidant properties and can prevent and eliminate biofilm formation. It also showed promise when applied to mice with MRSA-infected wounds.