Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk / Purificação parcial da enzima isomerase do ácido linoleico da bactéria Lactobacillus paracasei isolada do leite

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 µg.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Clinical, microbiological and immunological short, medium and long-term effects of different strains of probiotics as an adjunct to non-surgical periodontal therapy in patients with periodontitis. Systematic review with meta-analysis.

Introduction/objectives: Probiotics have been proposed as adjuncts to non-surgical periodontal therapy (NSPT), however, the effect of their use remains unclear. The aim of this systematic review and meta-analysis was to analyze the evidence regarding the use of probiotics as an adjunct to NSPT in patients with periodontitis at a clinical, microbiological and immunological level. Data/sources: A comprehensive search to identify clinical studies investigating the use of probiotics as an adjunct to NSPT in patients treated for periodontitis was performed. The data were grouped according to probiotic strain, frequency, form and duration of the probiotic intake. Study selection: A total of 25 articles were included, all articles analysed clinical parameters, 10 included also microbiological findings and only 4 had immunological findings. The difference in probing depth (PD) between the test and the control group was statistically significant in favour of the test group when the probiotics were in the form of lozenges, administered twice a day and when the strain was L. reuteri. In terms of Clinical Attachment Level (CAL) gain the difference was statistically significant in the short and in the medium term but not in the long term. Due to the heterogeneity of the data, it was not possible to compare trough a meta analysis the immunological and the microbiological findings that were therefore analysed only descriptively. Conclusions: The use of probiotics as an adjunct to NSPT in patients with periodontitis appears to provide additional clinical benefits that depend on the duration, the frequency, the form and the strain of probiotic used. Clinical significance: This review not only shows data on the efficacy of probiotics in non-surgical periodontal therapy, but provides important information on their effects over time and which forms of probiotic administration might be most clinically useful.

Lactobacillus fermentum MCC2760 abrogate high-fat induced perturbations in the enterohepatic circulation of bile acids in rats.

AIM: This study in hyperlipidemic rats elucidated the effect of Lactobacillus fermentum MCC2760 on intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic BA transporters. MAIN METHODS: Diets rich in saturated fatty acids [coconut oil (CO)] and omega-6 fatty acids [sunflower oil (SFO)] at 25 g fat/100 g diet were fed to rats with or without MCC2760 (109 cells/kg body weight). After 60 days of feeding, intestinal BA uptake and expression of Asbt, Osta/b mRNA and protein, and hepatic expression of Ntcp, Bsep, Cyp7a1, Fxr, Shp, Lrh-1, and Hnf4a mRNA were measured. Hepatic expression of HMG-CoA reductase protein and its activity and total BAs in serum, liver, and feces were assessed. KEY FINDINGS: Hyperlipidaemic groups (HF-CO and HF-SFO) had: 1) increased intestinal BA uptake, Asbt and Osta/b mRNA expression, and ASBT staining 2) increased BA in serum, 3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, and NTCP staining 4) increased activity of HMG-CoA reductase, 5) increased hepatic expression of Fxr and Shp mRNA, 6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and 7) decreased BA in Feces when compared to their respective controls (N-CO and N-SFO) and experimental groups (HF-CO + LF and HF-SFO + LF). Immunostaining revealed increased intestinal Asbt and hepatic Ntcp protein expression in the HF-CO and HF-SFO groups compared to control and experimental groups. SIGNIFICANCE: Incorporating probiotics like MCC2760 abrogated hyperlipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in rats. Probiotic MCC2760 can be used to modulate lipid metabolism in high-fat-induced hyperlipidemic conditions.

Integrative ATAC-seq and RNA-seq analyses of IPEC-J2 cells reveals porcine transcription and chromatin accessibility changes associated with Escherichia coli F18ac inhibited by Lactobacillus reuteri.

Escherichia coli is the main cause of postweaning diarrhea in pigs, leading to economic loss. As a probiotic, Lactobacillus reuteri has been used to inhibit E. coli in clinical applications; however, its integrative interactions with hosts remain unclear, especially in pigs. Here, we found that L. reuteri effectively inhibited E. coli F18ac adhering to porcine IPEC-J2 cells, and explored the genome-wide transcription and chromatin accessibility landscapes of IPEC-J2 cells by RNA-seq and ATAC-seq. The results showed that some key signal transduction pathways, such as PI3K-AKT and MAPK signaling pathways, were enriched in the differentially expressed genes (DEGs) between E. coli F18ac treatment with and without L. reuteri groups. However, we found less overlap between RNA-seq and ATAC-seq datasets; we speculated that this might be caused by histones modification through ChIP-qPCR detection. Furthermore, we identified the regulation of the actin cytoskeleton pathway and a number of candidate genes (ARHGEF12, EGFR, and DIAPH3) that might be associated with the inhibition of E. coli F18ac adherence to IPEC-J2 cells by L. reuteri. In conclusion, we provide a valuable dataset that can be used to seek potential porcine molecular markers of E. coli F18ac pathogenesis and L. reuteri antibacterial activity, and to guide the antibacterial application of L. reuteri.

Lactobacillus gasseri LG-G12 Restores Gut Microbiota and Intestinal Health in Obesity Mice on Ceftriaxone Therapy.

Gut microbiota imbalance is associated with the occurrence of metabolic diseases such as obesity. Thus, its modulation is a promising strategy to restore gut microbiota and improve intestinal health in the obese. This paper examines the role of probiotics, antimicrobials, and diet in modulating gut microbiota and improving intestinal health. Accordingly, obesity was induced in C57BL/6J mice, after which they were redistributed and fed with an obesogenic diet (intervention A) or standard AIN-93 diet (intervention B). Concomitantly, all the groups underwent a treatment phase with Lactobacillus gasseri LG-G12, ceftriaxone, or ceftriaxone followed by L. gasseri LG-G12. At the end of the experimental period, the following analysis was conducted: metataxonomic analysis, functional profiling of gut microbiota, intestinal permeability, and caecal concentration of short-chain fatty acids. High-fat diet impaired bacterial diversity/richness, which was counteracted in association with L. gasseri LG-G12 and the AIN-93 diet. Additionally, SCFA-producing bacteria were negatively correlated with high intestinal permeability parameters, which was further confirmed via functional profile prediction of the gut microbiota. A novel perspective on anti-obesity probiotics is presented by these findings based on the improvement of intestinal health irrespective of undergoing antimicrobial therapy or not.

Change in the Gut Microbiome and Immunity by Lacticaseibacillus rhamnosus Probio-M9.

With the exploding growth of the global market for probiotics and the rapid awakening of public awareness to manage health by probiotic intervention, there is still an active debate about whether the consumption of probiotics is beneficial for nonpatients, which is due to the lack of systematic analysis based on time series multiomics data sets. In this study, we recruited 100 adults from a college in China and performed a random case-control study by using a probiotic (Lacticaseibacillus rhamnosus Probio-M9) as an intervention for 6 weeks, aiming to achieve a comprehensive evaluation and understanding of the beneficial effect of Probio-M9 consumption. By testing advanced blood immunity indicators, sequencing the gut microbiome, and profiling the gut metabolome at baseline and the end of the study, we found that although the probiotic intervention has a limited impact on the human immunity and the gut microbiome and metabolome, the associations between the immunity indicators and multiomics data were strengthened, and further analysis of the gut microbiome's genetic variations revealed inhibited generation of single nucleotide variants (SNVs) by probiotic consumption. Taken together, our findings indicated an underestimated influence of the probiotic, not on altering the microbial composition but on strengthening the association between human immunity and commensal microbes and stabilizing the genetic variations of the gut microbiome. IMPORTANCE Although the global market for probiotics is growing explosively, there is still an active debate about whether the consumption of probiotics is beneficial for nonpatients. In this study, we recruited 100 adults from a college in China and performed 6 weeks of intervention for half of the volunteers. By analyzing the time series multiomics data in this study, we found that the probiotic intervention (i) has a limited effect on human immunity or the global structure of the gut microbiome and metabolome, (ii) can largely influence the correlation of the development between multiomics data and immunity, which was not able to be discovered by conventional differential abundance analysis, and (iii) can inhibit the generation of SNVs in the gut microbiome instead of promoting it.

Exopolysaccharides of Lactobacillus rhamnosus GG ameliorate Salmonella typhimurium-induced intestinal inflammation via the TLR4/NF-κB/MAPK pathway.

BACKGROUND: Salmonella typhimurium (S.T), as an important foodborne bacterial pathogen, can cause diarrhea and gastroenteritis in humans and animals. Numerous studies have confirmed that exopolysaccharides (EPSs) have various biological functions, but the mechanism through which EPSs improve the immunity of animals against the invasion of pathogenic bacteria is unclear. Here, we explored the protective effect of EPSs of Lactobacillus rhamnosus GG (LGG) on the S.T-infected intestine. METHODS: Mice received adequate food and drinking water for one week before the start of the experiment. After 7 d of prefeeding, 2×108 CFU/mL S.T solution and an equivalent volume of saline (control group) were given orally for 1 d. On the fourth day, the mice were treated with 0.5 mg/mL EPSs, 1.0 mg/mL EPSs, 2.0 mg/mL EPSs, or 2.0 mg/mL penicillin for 7 d. Finally, the body and relative organ weight, histological staining, and the levels of antioxidant enzyme activity and inflammatory cytokines were determined. RESULTS: The S.T-infected mice exhibited symptoms of decreased appetite, somnolence, diarrhea and flagging spirit. Treatment with EPSs and penicillin improved the weight loss of the mice, and the high dose of EPSs showed the best therapeutic effect. EPSs significantly ameliorated S.T-induced ileal injury in mice. High-dose EPSs were more effective than penicillin for alleviating ileal oxidative damage induced by S.T. The mRNA levels of inflammatory cytokines in the ileum of mice showed that the regulatory effects of EPSs on inflammatory cytokines were better than those of penicillin. EPSs could inhibit the expression and activation of key proteins of the TLR4/NF-κB/MAPK pathway and thereby suppress the level of S.T-induced ileal inflammation. CONCLUSIONS: EPSs attenuate S.T-induced immune responses by inhibiting the expression of key proteins in the TLR4/NF-κB/MAPK signaling pathway. Moreover, EPSs could promote bacterial aggregation into clusters, which may be a potential strategy for reducing the bacterial invasion of intestinal epithelial cells.

Antibiofilm activity and NMR-based metabolomic characterization of cell-free supernatant of Limosilactobacillus reuteri DSM 17938.

The microbial biofilm has been defined as a "key virulence factor" for a multitude of microorganisms associated with chronic infections. Its multifactorial nature and variability, as well as an increase in antimicrobial resistance, suggest the need to identify new compounds as alternatives to the commonly used antimicrobials. The aim of this study was to assess the antibiofilm activity of cell-free supernatant (CFS) and its sub-fractions (SurE 10 K with a molecular weight <10 kDa and SurE with a molecular weight <30 kDa), produced by Limosilactobacillus reuteri DSM 17938, vs. biofilm-producing bacterial species. The minimum inhibitory biofilm concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) were determined via three different methods and an NMR metabolomic analysis of CFS and SurE 10K was performed to identify and quantify several compounds. Finally, the storage stability of these postbiotics was evaluated by a colorimetric assay by analyzing changes in the CIEL*a*b parameters. The CFS showed a promising antibiofilm activity against the biofilm developed by clinically relevant microorganisms. The NMR of CFS and SurE 10K identifies and quantifies several compounds, mainly organic acids and amino acids, with lactate being the most abundant metabolite in all the analyzed samples. The CFS and SurE 10 K were characterized by a similar qualitative profile, with the exception of formate and glycine detected only in the CFS. Finally, the CIEL*a*b parameters assess the better conditions to analyze and use these matrices for the correct preservation of bioactive compounds.

Metformin alleviates liver fibrosis in mice by enriching Lactobacillus sp. MF-1 in the gut microbiota.

BACKGROUND: Liver fibrosis is associated with gut dysbiosis. Metformin administration has emerged as a promising method for the treatment of organ fibrosis. We aimed to investigate whether metformin ameliorates liver fibrosis by enhancing the gut microbiota in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and the underlying mechanism. MATERIALS AND METHODS: A liver fibrosis mouse model was established, and the therapeutic effects of metformin were observed. We administered antibiotic treatment and performed fecal microbiota transplantation (FMT), and 16S rRNA-based microbiome analysis to evaluate the effects of the gut microbiome on metformin-treated liver fibrosis. We isolated the bacterial strain preferably enriched by metformin and assessed its antifibrotic effects. RESULTS: Metformin treatment repaired the gut integrity of the CCl4-treated mice. It reduced the number of bacteria in colon tissues and reduced the portal vein lipopolysaccharide (LPS) levels. The FMT performed on the metformin-treated CCl4 mice alleviated their liver fibrosis and reduced their portal vein LPS levels. The markedly changed gut microbiota was screened out from the feces and named Lactobacillus sp. MF-1 (L. sp. MF-1). In the CCl4-treated mice, daily gavage of L. sp. MF-1 maintained gut integrity, inhibited bacterial translocation, and reduced liver fibrosis. Mechanistically, metformin or L. sp. MF-1 inhibited the apoptosis of intestinal epithelial cells and restored CD3+ intestinal intraepithelial lymphocytes in the ileum and CD4+Foxp3+ lamina propria lymphocytes in the colon. CONCLUSIONS: Metformin and its enriched L. sp. MF-1 can reinforce the intestinal barrier to alleviate liver fibrosis by restoring immune function.

Exopolysaccharides from vaginal lactobacilli modulate microbial biofilms.

BACKGROUND: Exopolysaccharides (EPS) secreted by beneficial lactobacilli exert a plethora of positive activities, but little is known about their effects on biofilms of opportunistic vaginal pathogens and especially on biofilms of lactobacilli themselves. Here, the EPS produced by six vaginal lactobacilli, belonging to Lactobacillus crispatus (BC1, BC4, BC5) and Lactobacillus gasseri (BC9, BC12, BC14) species were isolated from cultural supernatants and lyophilized. RESULTS: Lactobacillus EPS were chemically characterized in terms of monosaccharide composition by liquid chromatography (LC) analysis coupled to UV and mass spectrometry (MS) detection. Moreover, the ability of EPS (0.1, 0.5, 1 mg/mL) to stimulate the biofilm formation of lactobacilli and to inhibit the formation of pathogens' biofilms was evaluated by crystal violet (CV) staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Isolated EPS (yields 133-426 mg/L) were heteropolysaccharides mainly composed of D-mannose (40-52%) and D-glucose (11-30%). For the first time we demonstrated that Lactobacillus EPS were able to stimulate in a dose-dependent manner (p < 0.05) the formation of biofilms of ten strains belonging to L. crispatus, L. gasseri and Limosilactobacillus vaginalis species, in terms of cell viability (84-282% increase at 1 mg/mL) and especially biofilm biomass (40-195% increase at 1 mg/mL), quantified with MTT assay and CV staining, respectively. EPS released from L. crispatus and L. gasseri were found to better stimulate the biofilms of the same producer species rather than that of other species, including producing strains themselves and other strains. Conversely, the biofilm formation of bacterial (Escherichia coli, Staphylococcus spp., Enterococcus spp. and Streptococcus agalactiae) and fungal (Candida spp.) pathogens was inhibited. The anti-biofilm activity was dose-dependent and was more marked for L. gasseri-derived EPS (inhibition up to 86%, 70%, and 58% at 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL, respectively), whilst L. crispatus-derived EPS resulted overall less efficient (inhibition up to 58% at 1 mg/mL and 40% at 0.5 mg/mL) (p < 0.05). CONCLUSIONS: Lactobacilli-derived EPS favour the biofilm formation of lactobacilli preventing, at the same time, that of opportunistic pathogens. These results support the possible employment of EPS as postbiotics in medicine as a therapeutic/preventive strategy to counteract vaginal infections.

The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees.

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin (vg) and superoxide-dismutase-1 (sod-1) genes and mortality of bees infected with N. ceranae. Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased (p < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

Effects of Probiotic Lactobacilli plantarum in Treatment of Experimentally Induced Periodontal Disease in Rabbits.

The aim of this study was to evaluate the therapeutic effects of probiotic Lactobacillus plantarum in experimentally induced periodontal disease in rabbits. The incisor teeth of 24 rabbits were scaled under general anesthesia. Two weeks later, silk ligatures were placed at the gingival margin of the incisor teeth to induce periodontal disease. After confirming the presence of periodontal disease by periodontal probing four weeks later, incisor mucogingival flaps were created and gingival pocket lavage and debridement was performed. The rabbits were randomly divided into four groups. Group 1: Control; Group 2: Microencapsulated form of the probiotic; Group 3: Planktonic form of the probiotic; and Group 4: Biofilm form of the probiotic. The rabbits were euthanized eight weeks later, and gingival connective tissue and epithelium were resected for histopathological and histomorphometric evaluation. The results showed that the rate of epithelial regeneration was lower and bone regeneration was significantly higher in the treatment groups compared to the Control group. The highest level of bone regeneration was in Group 2 (Microencapsulated probiotic). There was no significant difference in bone regeneration observed between the biofilm and planktonic probiotic groups. This study showed that applying the probiotic Lactobacillus plantarum in microencapsulated form improved bone regeneration in experimentally induced periodontal disease in rabbits.

Antimicrobial and immunoregulatory effects of Lactobacillus delbrueckii 45E against genitourinary pathogens.

BACKGROUND: Lactobacilli are essential microbiota that maintain a healthy, balanced vaginal environment. Vaginitis is a common infection in women during their reproductive years. Many factors are associated with vaginitis; one of them is the imbalance of microbiota in the vaginal environment. This study aimed to evaluate the antimicrobial properties of Lactobacillus delbrueckii 45E (Ld45E) against several species of bacteria, namely, Group B Streptococcus (GBS), Escherichia coli, Klebsiella spp., and Candida parapsilosis, as well as to determine the concentration of interleukin-17 (IL-17) in the presence of Ld45E. METHODS: The probiotic characteristics of Ld45E were evaluated by examining its morphology, pH tolerance, adhesive ability onto HeLa cells, hemolytic activity, antibiotic susceptibility, and autoaggregation ability. Then, the antimicrobial activity of Ld45E was determined using Ld45E culture, cell-free supernatant, and crude bacteriocin solution. Co-aggregation and competition ability assays against various pathogens were conducted. The immunoregulatory effects of Ld45E were analyzed by measuring the proinflammatory cytokine IL-17. A p-value less than 0.05 was considered statistical significance. RESULTS: Ld45E is 3-5 mm in diameter and round with a flat-shaped colony. pH 4 and 4.5 were the most favorable range for Ld45E growth within 12 h of incubation. Ld45E showed a strong adhesion ability onto HeLa cells (86%) and negative hemolytic activities. Ld45E was also sensitive to ceftriaxone, cefuroxime, ciprofloxacin, and doxycycline. We found that it had a good autoaggregation ability of 80%. Regarding antagonistic properties, Ld45E culture showed strong antimicrobial activity against GBS, E. coli, and Klebsiella spp. but only a moderate effect on C. parapsilosis. Cell-free supernatant of Ld45E exerted the most potent inhibitory effects at 40 °C against all genital pathogens, whereas bacteriocin showed a robust inhibition at 37 °C and 40 °C. The highest co-aggregation affinity was observed with GBS (81%) and E. coli (40%). Competition ability against the adhesion of GBS (80%), E. coli (76%), Klebsiella (72%), and C. parapsilosis (58%) was found. Ld45E was able to reduce the induction of the proinflammatory protein IL-17. CONCLUSIONS: Ld45E possessed antimicrobial and immunoregulatory properties, with better cell-on-cell activity than supernatant activity. Thus, Ld45E is a potential probiotic candidate for adjunct therapy to address vaginal infections.

Clinical effects of combined Lactobacillus paracasei and kestose on canine atopic dermatitis.

Probiotics and prebiotics are viable bacteria with beneficial effects on the host and components that selectively act on the beneficial commensal bacteria, respectively. The combined use of probiotics and prebiotics is termed synbiotics. Probiotic intake improves dysbiosis in the intestinal microbiota and can positively affect canine atopic dermatitis (CAD). However, clinical studies on improvements in CAD using synbiotics remain limited. In this study, 15 dogs with CAD who received prednisolone, a synthetic glucocorticoid (GC) used in the treatment of CAD, for more than 90 days were continuously treated with Lactobacillus paracasei M-1 from fermented food as a probiotic, and trisaccharide kestose as a prebiotic, for 90 days to determine their synbiotic effects on CAD. The CAD symptoms were evaluated using the canine atopic dermatitis lesion index (CADLI) and pruritus visual analog scores (PVAS) at 30, 60 and 90 days after synbiotic administration. The total prednisolone use for 90 days pre- and post-administration was also evaluated. Synbiotic administration significantly reduced the CADLI (pre: median, 28.0 [22.0-32.0]; 30 days: median, 20.0 [20.0-28.0]; 60 days: median, 20.0 [10.0-21.0]; 90 days: median, 12.0 [10.0-19.0]) and PVAS (pre: median, 6.0 [5.0-7.0]; 30 days: median, 3.0 [3.0-3.5]; 60 days: median, 3.0 [3.0-3.5]; 90 days: median, 2.0 [2.0-3.5]) scores, and reduced the total prednisone use over 90 days (pre: 112.0 [25-450] mg; post: 80.0 [18.-300.0] mg; p⟨0.001) in the 15 dogs. Thus, the synbiotic activity of L. paracasei M-1 and trisaccharide kestose can improve CAD.

Effects of citric acid and heterofermentative inoculants on anaerobic co-fermentation of Chinese cabbage waste and wheat bran.

The effects of Lactobacillus buchneri, Lactobacillus hilgardii and citric acid on organic acid production, substrate consumption, protein degradation and microbial community were investigated in this study. The results indicated that combined inoculants induced a significant increase in levels of lactic acid (43 g/kg dry matter), acetic acid (14 g/kg dry matter), butyric acid (5 g/kg dry matter), total organic acid (60 g/kg dry matter) and ammonia nitrogen (20 g/kg total nitrogen). Furthermore, citric acid addition into the combined inoculants caused a significant increase in levels of acetic acid (12 g/kg dry matter), water-soluble carbohydrate (12 g/kg dry matter) and a reduction in ammonia nitrogen formation (22 g/kg total nitrogen). Microbiologically, combining inoculants and citric acid enriched Lactobacillus buchneri and Lactobacillus hilgardii and upregulated the functional pathways related to acid production and resistance. Collectively, combining citric acid and heterofermentative inoculants was beneficial to recycle Chinese cabbage waste in producing organic acids.

Herbal formula BaWeiBaiDuSan alleviates polymicrobial sepsis-induced liver injury via increasing the gut microbiota Lactobacillus johnsonii and regulating macrophage anti-inflammatory activity in mice.

Sepsis-induced liver injury (SILI) is an important cause of septicemia deaths. BaWeiBaiDuSan (BWBDS) was extracted from a formula of Panax ginseng C. A. Meyer, Lilium brownie F. E. Brown ex Miellez var. viridulum Baker, Polygonatum sibiricum Delar. ex Redoute, Lonicera japonica Thunb., Hippophae rhamnoides Linn., Amygdalus Communis Vas, Platycodon grandiflorus (Jacq.) A. DC., and Cortex Phelloderdri. Herein, we investigated whether the BWBDS treatment could reverse SILI by the mechanism of modulating gut microbiota. BWBDS protected mice against SILI, which was associated with promoting macrophage anti-inflammatory activity and enhancing intestinal integrity. BWBDS selectively promoted the growth of Lactobacillus johnsonii (L. johnsonii) in cecal ligation and puncture treated mice. Fecal microbiota transplantation treatment indicated that gut bacteria correlated with sepsis and was required for BWBDS anti-sepsis effects. Notably, L. johnsonii significantly reduced SILI by promoting macrophage anti-inflammatory activity, increasing interleukin-10+ M2 macrophage production and enhancing intestinal integrity. Furthermore, heat inactivation L. johnsonii (HI-L. johnsonii) treatment promoted macrophage anti-inflammatory activity and alleviated SILI. Our findings revealed BWBDS and gut microbiota L. johnsonii as novel prebiotic and probiotic that may be used to treat SILI. The potential underlying mechanism was at least in part, via L. johnsonii-dependent immune regulation and interleukin-10+ M2 macrophage production.

Lactobacillus plantarum HF02 alleviates lipid accumulation and intestinal microbiota dysbiosis in high-fat diet-induced obese mice.

BACKGROUND: Obesity is closely associated with lipid accumulation and intestinal microbiota dysbiosis. It has been proved that probiotics supplement contributes to alleviate obesity. The objective of this study was to investigate the mechanism by which Lactobacillus plantarum HF02 (LP-HF02) alleviated lipid accumulation and intestinal microbiota dysbiosis in high-fat diet-induced obese mice. RESULTS: Our results showed that LP-HF02 ameliorated body weight, dyslipidemia, liver lipid accumulation, and liver injury in obese mice. As expected, LP-HF02 inhibited pancreatic lipase activity in small intestinal contents and increased fecal triglyceride levels, thereby reducing dietary fat hydrolysis and absorption. Moreover, LP-HF02 ameliorated the intestinal microbiota composition, as evidenced by the enhanced ratio of Bacteroides to Firmicutes, the decreased abundance of pathogenic bacteria (including Bacteroides, Alistipes, Blautia, and Colidextribacter) and the increased abundance of beneficial bacteria (including Muribaculaceae, Akkermansia, Faecalibaculum, and Rikenellaceae_RC9_gut_group). LP-HF02 also increased fecal short-chain fatty acids (SCFAs) levels and colonic mucosal thickness, and subsequently decreased serum lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels in obese mice. Additionally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot results demonstrated that LP-HF02 ameliorated hepatic lipid accumulation via activating the adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway. CONCLUSION: Therefore, our results indicated that LP-HF02 could be considered as a probiotic preparation for preventing obesity. © 2023 Society of Chemical Industry.

Probiotic characterization and comparison of broiler-derived lactobacillus strains based on technique for order preference by similarity to ideal solution analysis.

A total of 10 lactobacillus strains were isolated from broiler chickens and their probiotic properties including tolerance to gastrointestinal fluids and heat treatment, antimicrobial activity, adhesion capacity to intestinal cells, surface hydrophobicity, autoaggregation, antioxidative activity, and immunomodulatory effects on chicken macrophages were evaluated. The Limosilactobacillus reuteri (LR) was the most frequently isolated species, followed by Lactobacillus johnsonii (LJ) and Ligilactobacillus salivarius (LS). All isolates showed good resistance to simulated gastrointestinal conditions and antimicrobial activity against 4 indicator strains including Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis LR 21 exhibited excellent performances on autoaggregation, hydrophobicity and adhesion capacity to Caco-2 intestinal cells. In the meantime, this strain also possessed considerable tolerance to heat treatment, which indicated great potential to be used in the feed industry. However, LJ 20 strain had the highest free radical scavenging activity compared with the other strains. Furthermore, qRT-PCR results revealed that all isolated strains significantly increased the transcriptional levels of proinflammatory genes and tended to induce the M1-type polarization on HD11 macrophages. Particularly, the technique for order preference by similarity to ideal solution (TOPSIS) was adopted in our study to compare and select the most promising probiotic candidate based on in vitro evaluation tests.

Heat-killed Lactobacillus murinus confers neuroprotection against dopamine neuronal loss by targeting NLRP3 inflammasome.

The intestinal flora has become very active in studies related to Parkinson's disease (PD) in recent years. The microbe-gut-brain axis is closely related to the maintenance of brain homeostasis as well as PD pathogenesis. Alterations in gut bacteria can contribute to neuroinflammation and dopamine (DA) neurodegeneration. Lactobacillus murinus, a gram-positive bacterium, is a commensal gut bacteria present in the mammalian gut and considered as a potential probiotic due to its beneficial effects, including anti-inflammatory and antibacterial actions. In this study, the effects of live L. murinus and heat-killed L. murinus on DA neuronal damage in rats and the underlying mechanisms were investigated. Data showed that heat-killed L. murinus ameliorated 6-hydroxydopamine-induced motor dysfunctions and loss of substantia nigra DA neurons, while no protection was shown in live L. murinus treatment. At the same time, heat-killed L. murinus reduced the activation of NLRP3 inflammasome in microglia and the secretion of pro-inflammatory factors, thus inhibiting the development of neuroinflammation. Furthermore, heat-killed L. murinus failed to display its original neuroprotective properties in NLRP3 inflammasome knockout mice. Together, heat-killed L. murinus conferred neuroprotection against DA neuronal loss via the inhibition of microglial NLRP3 inflammasome activation. These findings provide a promising potential for future applications of L. murinus, and also beneficial strategy for PD treatment.