Lactiplantibacillus plantarum ZDY2013 Inhibits the Development of Non-Alcoholic Fatty Liver Disease by Regulating the Intestinal Microbiota and Modulating the PI3K/Akt Pathway.

Non-alcoholic fatty liver disease (NAFLD) is a common chronic hepatic condition whose impact on human health is increasingly significant. The imbalance of the gut microbiome, linked to insulin resistance, heightened intestinal permeability, and pro-inflammatory reactions, may be the linchpin in the development of NAFLD. In our research, the impact of Lactiplantibacillus plantarum ZDY2013 administration for 12 weeks on gut microbiota dysbiosis induced by a high-fat, high-fructose, high-cholesterol (FHHC) diet in male C57BL/6n mice was investigated. Research results presented that the intervention of L. plantarum ZDY2013 in mice fed with the FHHC diet could restore their liver function and regulate oxidative stress. Compared to mice in the model group, the intervention of L. plantarum ZDY2013 significantly regulated the gut microbiota, inhibited the LPS/NF-κB pathway, and led to a lower level of colonic inflammation in the mice administered with L. plantarum ZDY2013. It also improved insulin resistance to regulate the PI3K/Akt pathway and lipid metabolism, thereby resulting in reduced fat accumulation in the liver. The above results suggest that the intervention of L. plantarum ZDY2013 can hinder the progression of diet-induced NAFLD by reducing inflammation to regulate the PI3K/Akt pathway and regulating gut microbiota disturbance.

The flavor substances changes in Fuliang green tea during storage monitoring by GC-MS and GC-IMS.

To study the effect of storage (for 0, 3, 6, and 12 months) on the flavor of green tea (GT), we monitored the volatile organic compounds (VOCs) in GT through gas chromatography (GC) combined with ion mobility spectrometry and headspace solid-phase micro extraction, GC-MS (mass spectrometry). Then, relative odor activity value (ROAV) was applied to analyze the aroma contribution of the VOCs. During storage, the polyphenol and caffeine contents gradually decreased from 22.38 % to 18.51 % and from 4.37 % to 3.74 %, respectively, and the total soluble sugar first increased and then decreased (from 4.89 % to 7.16 % and then 5.02 %). Although the total free amino acid contents showed a fluctuating trend, the content of cysteamine increased gradually. The contents of VOCs with positive contribution to GT aroma, including linalool, geraniol, nonanal, and 6-methyl-5-hepten-2-one, decreased. They also contributed less in the ROAV after storage. The ROAVs of nonanal, linalool, and geraniol decreased from 3.37 to 0.79, from 100 to 38.21, and from 2.98 to 1.8, respectively, after 12 months of storage. Principal component analysis can be used to identify the samples with different storage durations based on these data. Given the increase in amount of cysteamine and decrease in that of linalool oxide, oxidation may be not the only factor responsible for tea quality in storage.

Targeting the gut microbiota to investigate the mechanism of Lactiplantibacillus plantarum 1201 in negating colitis aggravated by a high-salt diet.

High-salt diet (HSD) affects the composition and function of the intestinal microbiota and cause health problems. This study confirmed that HSD aggravates dextran sulphate sodium (DSS)-induced colitis by changing the relative abundance of the gut microbiota, activating the NF-κB pathway, and up-regulating the mRNA levels of inflammatory factors. We explored the effect of L. plantarum 1201 in negating DSS-induced ulcerative colitis, which is aggravated by HSD for the first time. Results show that L. plantarum 1201 rebuilt the balance of intestinal flora by decreasing the ratio of Firmicutes/Bacteroidetes and increasing the relative abundance of Bifidobacterium, Lactobacillus and butyric-producing bacteria. Moreover, L. plantarum 1201 inhibited the up-regulation of inflammatory cytokines (e.g., IL-1ß, TNF-α, IL-6, IL-22, and IFN-γ) mRNA levels, increased colonic tight junction protein (ZO-1, ocludin, and claudin-3) expression, and increased serum levels of beneficial metabolites, including alpha-tocopherol (α-T) and D-mannose. By reconstructing an animal model of colitis, we further discovered that α-T and D-mannose inhibited the NF-κB pathway, improved tissue injury, and decreased the expression of pro-inflammatory cytokines (e.g., IL-1ß, TNF-α, and IL-6). This study proves for the first time that L. plantarum 1201 attenuates high-salt-aggravated colitis by increasing the serum concentrations of endogenic D-mannose in mice serum and inhibiting the consumption of α-T through intestinal flora. Therefore, regulating the gut microbiota is a potential treatment for high-salt-aggravated colitis.

Effect of Bifidobacterium animalis subsp. lactis SF on enhancing the tumor suppression of irinotecan by regulating the intestinal flora.

The gut microbiota plays a role in tumor therapy by participating in immune regulation. Here, we demonstrated through 8-day probiotic supplementation experiments and fecal microbiota transplantation experiments that Bifidobacterium animalis subsp. lactis SF enhanced the antitumor effect of irinotecan and prevented the occurrence of intestinal damage by modulating the gut microbiota and reducing the relative abundance of pro-inflammatory microbiota. Therefore, the intestinal inflammation was inhibited, the TGF-ß leakage was reduced, and the PI3K/AKT pathway activation was inhibited. Thus, the tumor apoptotic autophagy was finally promoted. Simultaneously, the reduction of TGF-ß relieved the immunosuppression caused by CPT-11, promoted the differentiation of CD4+ and CD8+ T cells in tumor tissue, and consequently inhibited tumor growth and invasion. This study disclosed the mechanism of B. lactis SF assisting CPT-11 in antitumor activity and suggested that B. lactis SF plays a new role in anticancer effects as a nutritional intervention.

Antagonistics of Lactobacillus plantarum ZDY2013 against Helicobacter pylori SS1 and its infection in vitro in human gastric epithelial AGS cells.

In this study, the anti-Helicobacterpylori activity of Lactobacillusplantarum ZDY2013 was investigated and Lactobacillusrhamnosus GG was used as a positive control. The anti-H. pylori mechanism in vitro was also examined. Results revealed that either the viable cells or supernatant of L. plantarum ZDY2013 could suppress the growth or urease activity of H. pylori. The inhibitory effects of L. plantarum ZDY2013 were relatively higher than those of L. rhamnosus GG (P < 0.05), and such effects might be a result of their lactic acid production (e.g., 51.105 ± 0.097 mmol/L for L. plantarum ZDY2013 and 33.113 ± 0.063 mmol/L for L. rhamnosus GG). The anti-adhesion capacity of L. plantarum ZDY2013 against H. pylori was also stronger than that of L. rhamnosus GG in terms of inhibition, competition, and displacement. Among these inhibitory strategies, competition exhibited the best performance, with an inhibition ratio of 92.65%. Upon inhibition and anti-adhesion, the cells and supernatant of L. plantarum ZDY2013 significantly strengthened the expression of the anti-inflammatory cytokine IL-10, but attenuated the expression of the pro-inflammatory cytokine TNF-α in AGS cells induced by H. pylori SS1. Remarkably, the supernatant of ZDY2013 achieved a relatively higher anti-inflammatory effect than that exerted by its cells. With excellent lactic acid yield and antagonistic and anti-inflammatory effects against H. pylori SS1 infection, L. plantarum ZDY2013 shows potential to be used as a probiotics candidate.

Evaluation of probiotic properties of Lactobacillus plantarum WLPL04 isolated from human breast milk.

Lactobacillus plantarum WLPL04, a specific strain isolated from human breast milk, was investigated for its survival capacity (acid and bile salt tolerance, survival in simulated gastrointestinal tract, inhibition of pathogens, antibiotic susceptibility, yield of exopolysaccharides) and probiotic properties (antiadhesion of pathogens, protection from harmful effect of sodium dodecyl sulfate, and antiinflammatory stress on Caco-2 cells). The results showed that Lb. plantarum WLPL04 had broad-spectrum activity against gram-positive strains (Listeria monocytogenes CMCC54007, Bacillus cereus ATCC14579, and Staphylococcus aureus CMCC26003) and gram-negative strains (Pseudomonas aeruginosa MCC10104, Shigella sonnei ATCC25931, Enterobacter sakazakii ATCC29544, Salmonella typhimurium ATCC13311, and Escherichia coli O157:H7). Antibiotic susceptibility tests showed that Lb. plantarum WLPL04 was susceptible to 8 of 14 antibiotics (e.g., erythromycin and nitrofurantoin) and resistant to 6 of 14 antibiotics (e.g., kanamycin and bacitracin). Lactobacillus plantarum WLPL04 was able to survive at pH 2.5 for 3h and at 0.45% bile salt for 12h, suggesting that it can survive well in the gastrointestinal tract. In addition, the exopolysaccharide yield of Lb. plantarum WLPL04 reached 426.73 ± 65.56 mg/L at 24h. With strategies of competition, inhibition, and displacement, Lb. plantarum WLPL04 reduced the adhesion of E. coli O157:H7 (35.51%), Sal. typhimurium ATCC 13311 (8.10%), and Staph. aureus CMCC 26003 (40.30%) on Caco-2 cells by competition, and subsequently by 59.80, 62.50, and 42.60%, respectively, for the 3 pathogens through inhibition, and by 75.23, 39.97, and 52.88%, respectively, through displacement. Lactobacillus plantarum WLPL04 attenuated the acute stress induced by sodium dodecyl sulfate on Caco-2 cells and significantly inhibited the expression of inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor-α) on Caco-2 cells but increased IL-10 expression in vitro compared with the Salmonella-treated group. In summary, Lb. plantarum WLPL04 from breast milk could be considered as a probiotic candidate for dairy products to promote human health.

Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress.

Acid tolerance responses (ATR) in Lactobacillus plantarum ZDY2013 were investigated at physiological and molecular levels. A comparison of composition of cell membrane fatty acids (CMFA) between acid-challenged and unchallenged cells showed that acid adaptation evoked a significantly higher percentage of saturated fatty acids and cyclopropane fatty acids in acid-challenged than in unchallenged cells. In addition, reverse transcription-quantitative PCR analysis in acid-adapted cells at different pH values (ranging from 3.0 to 4.0) indicated that several genes were differently regulated, including those related to proton pumps, amino acid metabolism, sugar metabolism, and class I and class III stress response pathways. Expression of genes involved in fatty acid synthesis and production of alkali was significantly upregulated. Upon exposure to pH 4.5 for 2 h, a higher survival rate (higher viable cell count) of Lactobacillus plantarum ZDY2013 was achieved following an additional challenge to 40 mM hydrogen peroxide for 60 min, but no difference in survival rate of cells was found with further challenge to heat, ethanol, or salt. Therefore, we concluded that the physiological and metabolic changes of acid-treated cells of Lactobacillus plantarum ZDY2013 help the cells resist damage caused by acid, and further initiated global response signals to bring the whole cell into a state of defense to other stress factors, especially hydrogen peroxide.

Changes in gastric microbiota induced by Helicobacter pylori infection and preventive effects of Lactobacillus plantarum ZDY 2013 against such infection.

Helicobacter pylori is a gram-negative pathogen linked to gastric ulcers and stomach cancer. Gastric microbiota might play an essential role in the pathogenesis of these stomach diseases. In this study, we investigated the preventive effect of a probiotic candidate Lactobacillus plantarum ZDY 2013 as a protective agent against the gastric mucosal inflammation and alteration of gastric microbiota induced by H. pylori infection in a mouse model. Prior to infection, mice were pretreated with or without 400 µL of L. plantarum ZDY 2013 at a concentration of 10(9) cfu/mL per mouse. At 6 wk postinfection, gastric mucosal immune response and alteration in gastric microbiota mice were examined by quantitative real-time PCR and high-throughput 16S rRNA gene amplicon sequencing, respectively. The results showed that L. plantarum ZDY 2013 pretreatment prevented increase in inflammatory cytokines (e.g., IL-1ß and IFN-γ) and inflammatory cell infiltration in gastric lamina propria induced by H. pylori infection. Weighted UniFrac principal coordinate analysis showed that L. plantarum ZDY 2013 pretreatment prevented the alteration in gastric microbiota post-H. pylori infection. Linear discriminant analysis coupled with effect size identified 22 bacterial taxa (e.g., Pasteurellaceae, Erysipelotrichaceae, Halomonadaceae, Helicobacteraceae, and Spirochaetaceae) that overgrew in the gastric microbiota of H. pylori-infected mice, and most of them belonged to the Proteobacteria phylum. Lactobacillus plantarum ZDY 2013 pretreatment prevented this alteration; only 6 taxa (e.g., Lachnospiraceae, Ruminococcaceae, and Clostridiaceae), mainly from the taxa of Firmicutes and Bacteroidetes, were dominant in the gastric microbiota of the L. plantarum ZDY 2013 pretreated mice. Administration of L. plantarum ZDY 2013 for 3 wk led to increase in several bacterial taxa (e.g., Rikenella, Staphylococcus, Bifidobacterium), although a nonsignificant alteration was found in the gastric microbiota. Overall, this study demonstrated that L. plantarum ZDY 2013 pretreatment played an important role in preventing gastric mucosal inflammation and gastric microbiota alteration induced by H. pylori infection, and the selective modulation in gastric microbiota posed by this intervention suggested that targeting gastric microbiota through oral administration of probiotics might be an alternative strategy to prevent H. pylori infection.

Antibacterial activity and mechanism of action of ε-poly-L-lysine.

ε-Poly-L-lysine (ε-PL)(2) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 µg/mL (90% mortality for 5 µg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS)(3) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR)(4) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response)(5) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.

Identification of an outer membrane protein of Salmonella enterica serovar Typhimurium as a potential vaccine candidate for Salmonellosis in mice.

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine.

Mitochondrial mutation impairs cytoplasmic male sterility rice in response to H2O2 stress.

Cytoplasmic male sterility (CMS) is a phenomenon widely observed in various plant species characterized with disrupted anther development caused by mitochondrial mutation. CMS is becoming a model system for the investigations of nucleus-cytoplasmic interaction. To reveal the possible effects of CMS genes on plant growth in adverse environment, plant development and biochemical characters of mitochondria from Honglian (HL)-CMS line Yuetai A and maintainer Yuetai B treated with H(2)O(2) were analyzed. Results showed that 40-60mM H(2)O(2) significantly inhibits rice seedling development and growth. When treated with H(2)O(2), ATP content and mitochondrial membrane potential in Yuetai A decreased significantly faster than those of Yuetai B. These biochemical changes were accompanied by the severe nuclear DNA fragmentation and the release of mitochondrial cytochrome c in the leaf cells of Yuetai A. In addition, the antioxidative enzyme activities and mitochondrial electron transfer chain complexes were significantly down-regulated. Disturbance of the biochemical indexes indicate that HL-CMS line is more susceptible to H(2)O(2) stress than the maintainer line, the deleterious effects caused by the CMS-related ORFH79 peptide compromises the adaptability of HL-CMS line to the adverse environment.

A Honglian CMS line of rice displays aberrant F0 of F0F1-ATPase.

Honglian (HL) is one of the three major types of cytoplasmic male sterility (CMS) of rice (Oryza sativa L) that has been commercially used in hybrid production. In previous studies, the CMS in HL is shown to be associated with a chimeric gene orfH79 that is cotranscribed with an extra atp6 in mitochondria. This study demonstrated that the intact F0F1-ATPase in HL CMS line was specifically reduced in both of its protein quantity and enzyme activity, whereas its F1 sector was not affected. It implies that the F0 sector presents a labile linkage with F1. In the presence of fertility restorer gene, F0F1-ATPase can be recovered. Furthermore, orfH79 transcripts were preferentially polyadenylated, and consequently degraded rapidly in florets of the restored hybrid plants, indicating that the atp6-orfH79 is involved in the sterile phenotype. With inhibition of cytochrome pathway of electron transfer chain, the biomass of the sterile plants grown in dark was significantly lower than that of the fertile lines. However, the respiration measurements showed an increase in the electron transferring capacity in the sterile plants, suggesting that the reduction of biomass in sterile line was caused by the disruption of F0F1-ATPase.

Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice.

One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.