Lactobacillus paracasei ET-22 and derived postbiotics reduce halitosis and modulate oral microbiome dysregulation - a randomized, double-blind placebo-controlled clinical trial.

Oral microbial dysbiosis is the primary etiologic factor for halitosis and may be the critical preventive target for halitosis. This study included randomized controlled trials (RCTs) assessing the effects of Lactobacillus paracasei ET-22 live and heat-killed bacteria on halitosis and the related oral microbiome. 68 halitosis subjects were divided into placebo, ET-22 live (ET-22.L) and ET-22 heat-killed (ET-22.HK) groups. Subjects took different lozenges three times a day for 4 weeks and underwent saliva collection and assessment of breath volatile sulfur compound (VSC) levels at the beginning and end of the intervention. Salivary volatile organic compounds were measured using HS-SPME-GC/MS, and the microbiome profile was determined by 16S rRNA gene amplicon sequencing. A positive decrease in breath volatile sulfur compound (VSC) levels was observed in the means of both ET-22.L and ET-22.HK groups after 4 weeks of intervention, being more marked in the ET-22.L group (p = 0.0148). Moreover, ET-22.L and ET-22.HK intervention remarkably changed the composition of total salivary volatile organic compounds (VOCs) and aroma-active VOCs. Key undesirable VOCs, such as indole, pyridine, nonanoic acid, benzothiazole, and valeric acid, were significantly reduced. Meanwhile, ET-22.L or ET-22.HK also altered the taxonomic composition of the salivary microbiome. The halitosis pathogens Rothia and Streptococcus were significantly reduced in the ET-22.HK group and the pathogenic Solobacterium and Peptostreptococcus were significantly inhibited in the ET-22.L group. Collectively, our study suggests that both ET-22.L and ET-22.HK can significantly inhibit the production of undesirable odor compounds in subjects with halitosis, which may be related to the changes of the oral microbiome.

Postbiotics Derived from L. paracasei ET-22 Inhibit the Formation of S. mutans Biofilms and Bioactive Substances: An Analysis.

Globally, dental caries is one of the most common non-communicable diseases for patients of all ages; Streptococcus mutans (S. mutans) is its principal pathogen. Lactobacillus paracasei (L. paracasei) shows excellent anti-pathogens and immune-regulation functions in the host. The aim of this study is to evaluate the effects of L. paracasei ET-22 on the formation of S. mutans biofilms. The living bacteria, heat-killed bacteria, and secretions of L. paracasei ET-22 were prepared using the same number of bacteria. In vitro, they were added into artificial-saliva medium, and used to coculture with the S. mutans. Results showed that the living bacteria and secretions of L. paracasei ET-22 inhibited biofilm-growth, the synthesis of water-soluble polysaccharide and water-insoluble polysaccharide, and virulence-gene-expression levels related to the formation of S. mutans biofilms. Surprisingly, the heat-killed L. paracasei ET-22, which is a postbiotic, also showed a similar regulation function. Non-targeted metabonomics technology was used to identify multiple potential active-substances in the postbiotics of L. paracasei ET-22 that inhibit the formation of S. mutans biofilms, including phenyllactic acid, zidovudine monophosphate, and citrulline. In conclusion, live bacteria and its postbiotics of L. paracasei ET-22 all have inhibitory effects on the formation of S. mutans biofilm. The postbiotics of L. paracasei ET-22 may be a promising biological anticariogenic-agent.

Bifidobacterium animalis subsp. lactis BL-99 ameliorates colitis-related lung injury in mice by modulating short-chain fatty acid production and inflammatory monocytes/macrophages.

Pulmonary inflammation as one of the extraintestinal manifestations of ulcerative colitis (UC) has attracted extensive attention, and its pathogenesis is closely related to gut dysbiosis. Bifidobacterium animalis subsp. lactis BL-99 (BL-99) can alleviate osteoporosis caused by UC, but less research has been done on other extraintestinal manifestations (EIM) caused by UC. This study aimed to explore the role and potential mechanisms of BL-99 on DSS-induced pulmonary complications in colitis mice. The results showed that BL-99 decreased weight loss, disease activity index score, colonic pathology score, and the production of pro-inflammatory cytokines (e.g., TNF-α, IL-1ß, and IL-6) in colitis mice. BL-99 also alleviated DSS-induced lung pathological damage by suppressing the infiltration of pro-inflammatory cytokines, inflammatory monocytes, and macrophages. Furthermore, 16S rRNA gene sequencing showed lower abundances of several potentially pathogenic bacteria (e.g., Burkholderia, Shigella, and Clostridium perfringens) and enrichment in specific beneficial bacteria (e.g., Adlercreutzia and Bifidobacterium animalis) in colitis mice with BL-99 treatment. Targeted metabolomics suggested that BL-99 intervention promoted the production of intestinal acetate and butyrate. Finally, we observed that the pulmonary expression of primary acetate and butyrate receptors, including FFAR2, FFAR3, and, GPR109a, was up-regulated in BL-99-treated mice, which negatively correlated with inflammatory monocytes and macrophages. Altogether, these results suggest that BL-99 might be utilized as a probiotic intervention to prevent the incidence of colitis-related lung injury owing to its ability to shape the intestinal microbiota and suppress inflammation.

Effects of Coix seed extract, Lactobacillus paracasei K56, and their combination on the glycolipid metabolism in obese mice.

Coix seed extract (CSE) and probiotics have been reported to regulate glycolipid metabolism through different modes of action. We tested the effects of CSE, Lactobacillus paracasei K56, and their combination to determine whether they have synergistic effects on glycolipid metabolism of obese mice. We fed male C57BL/6J mice with high-fat diet for 8 weeks to establish an obesity model. The obesity mice were selected and divided into five groups: the model control group and four intervention groups. After 10 weeks of continuous gavage intervention, the mice in the intervention groups exhibited lower body weight (lower about 2.31-4.41 g, vs. HFD 42.25 g, p < 0.01), and epididymal (lower about 0.58-0.92 g, vs. HFD 2.50 g, p < 0.01) and perirenal fat content (lower about 0.24-0.42 g, vs. HFD 0.88 g, p < 0.05); decreased fasting blood glucose, total cholesterol, triglycerides, and VLDL; and increased HLDL, respiratory exchange ratio, energy expenditure, and amount of exercise performed. K56 + CSE-combined intervention groups were more effective in lowering blood glucose, IL-1ß, and TNF-α levels than the CSE and K56 alone interventions. The content of fatty acid synthase and SREBP-1c protein in liver tissue was lower. The combination has synergistic effects on weight control, fat reduction, and blood glucose regulation by improving the chronic inflammatory state and reducing the content of lipid synthesis-related enzymes of obese mice, which can hinder chronic disease progression. PRACTICAL APPLICATION: Coix seed extract can be used in obese people to regulate abnormal glucose and lipid metabolism and delay the development of chronic diseases.

Lacticaseibacillus paracasei K56 Attenuates High-Fat Diet-Induced Obesity by Modulating the Gut Microbiota in Mice.

This study investigated the effects of Lacticaseibacillus paracasei K56 (L. paracasei K56) on body weight, body composition, and glycolipid metabolism in mice with high-fat diet-induced obesity and explored the underlying mechanisms. Male C57BL/6J mice were fed a high-fat diet for 8 weeks to induce obesity; then, the obese mice were gavaged with or without L. paracasei K56 for 10 weeks. The body weight, body composition, fat mass, blood lipid, blood glucose, and hormones of the mice were evaluated. Moreover, the fatty acid synthesis (FAS) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions in the liver were detected via Western blotting. 16S rRNA gene sequencing was adopted to determine the gut microbiota alterations. The high-fat diet successfully induced obesity, as indicated by the abnormal increase in body weight, visceral fat, fat mass, blood lipids, fasting blood glucose, and insulin-resistance. Moreover, the FAS expression in the liver was significantly increased, whereas the PPAR-γ expression was significantly decreased. The relative abundance of Proteobacteria, Actinobacteria and Patescibacteria was also significantly increased, and that of Verrucomicrobia was significantly decreased. However, these indicators of mice supplemented with L. paracasei K56 were significantly opposite to those of obese mice. The Ruminococcuaceae_UCG-013, Akkermansia, Prevotellaceae_UCG-001, Muribaculum, and Lachnospiraceae_NK4A136 groups were significantly negatively correlated with body weight, blood lipids, and blood glucose-related indicators, whereas Coriobacteriaceae_UCG-002, Enterorhabdus, Raoultibacter, Acinetobacter, Romboutsia, Leuconostoc, and Erysipelatoclostridium were significantly positively correlated with these indicators. L. paracasei K56 might be a promising probiotic strain that could effectively slow down the body weight gain, reduce fat accumulation, alleviate insulin-resistance, and restore pancreatic ß-cell function in obese mice by regulating the gut microbiota.

Effects of Coix Seed Extract, Bifidobacterium BPL1, and Their Combination on the Glycolipid Metabolism in Obese Mice.

Coix seed extract (CSE) and probiotics have been reported to regulate glycolipid metabolism via different modes of action. We tested the effects of CSE, Bifidobacterium BPL1, and their combination to determine their effects on glycolipid metabolism in obese mice. Male C57BL/6J mice were fed a high-fat diet for 8 weeks to establish an obesity model. Obese mice were selected and divided into four groups: the model control group and three intervention groups. After 10 weeks of continuous gavage intervention, the mice in the intervention groups exhibited lower body weight (lower about 2.31 g, vs. HFD mice 42.23 g) and epididymal (lower about 0.37 g, vs. HFD mice 2.5 g) and perirenal fat content (lower about 0.47 g, vs. HFD mice 0.884 g); decreased fasting blood glucose, total cholesterol, triglycerides, and VLDL; and increased HLDL, respiratory exchange ratio, energy expenditure, and amount of exercise performed. CSE, BPL1 and their combination can effectively control the weight gain in obese mice, reduce fat content, and regulate blood lipids and abnormal blood sugar. These results may be related to reduce the chronic inflammatory states, improve energy metabolism, exercise, relieve insulin sensitivity, and reduce lipid synthesis via the intervention of CSE, BPL1 and their combination. Compared with the single use of CSE alone, the combination of CSE + BPL1 can better exert the regulation function of intestinal flora, and change in the abundance of bacteria that could improve the level of inflammatory factors, such as increasing Bifidobacterium, reducing Lactococcus. Compared with the use of BPL1 alone, the combination of CSE and BPL1 can better regulate pancreatic islet and improve blood sugar. CSE may act directly on body tissues to exert anti-inflammatory effects. BPL1 and CSE + BPL1 may improve the structure and function of the intestinal flora, and reduce tissue inflammation.

In vitro Study of Bifidobacterium lactis BL-99 With Fructooligosaccharide Synbiotics Effected on the Intestinal Microbiota.

Probiotics and prebiotics relieve constipation by altering the composition of the intestinal microbiota. However, their synergistic mechanism of action remains unclear. Herein, an in vitro fermentation model was constructed to examine the synergistic effects of Bifidobacterium lactis BL-99 and fructooligosaccharide (FOS) on the regulation of intestinal microbiota from a population with constipation. The utilization of FOS was promoted by BL-99, and the increase rate being 22.33%. Relative to the BL-99 and the FOS groups, the BL-99_FOS group showed a highly significant increase in acetic acid content (P < 0.01) and a marked decrease in CO2 and H2S contents (P < 0.01) in the fermentation broth. In addition, the BL-99_FOS combination significantly changed the structure of the intestinal microbiota, enhanced the relative abundances of beneficial bacteria that relieved constipation, including Bifidobacterium, Fecalibacterium, Lactobacillus, Subdoligranulum, and Blautia, and decreased those of the harmful bacteria, including Bilophila and Escherichia-Shigella. These findings suggested that BL-99 and FOS synergistically regulated the composition and structure of the intestinal microbiota from the population with constipation and increased acetic acid and decreased CO2 and H2S levels, thereby providing a theoretical basis for the application of synbiotics.

Bifidobacterium infantis strain YLGB-1496 possesses excellent antioxidant and skin barrier-enhancing efficacy in vitro.

Atopic dermatitis (AD) is a recurring allergic skin disease that has a high incidence. Orally applied Bifidobacteria ameliorate signs of irritated skin and enhance the skin barrier. The present study investigated the safety and efficacy of a topically used cell-free culture supernatant (CFS) from a Bifidobacterium infantis strain using in vitro evaluation methods. The results showed that CFS had strong free radical scavenging activity on DPPH, ABTS, ·OH and O2 -radicals. CFS treatment fundamentally reduced the intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) contents and improved the activities of antioxidant enzymes (CAT, SOD and GSH-Px) in H2 O2 -treated HaCaT cells. Notably, the upregulation of skin physical barrier gene (FLG, LOR, IVL, AQP3 and TGM1) expression and skin antimicrobial peptide gene (CAMP, hBD-2 and hBD-3) expression by CFS might contribute to skin barrier resistance. CFS was non-irritating to the skin and eyes. CFS from the Bifidobacterium infantis strain had strong antioxidant properties on the skin and strengthened skin barrier function, and it was safe for topical use.

Bifidobacterium lactis BL-99 protects mice with osteoporosis caused by colitis via gut inflammation and gut microbiota regulation.

Patients diagnosed with inflammatory bowel disease or related conditions also frequently suffer from osteoporosis as a consequence of changes in the intestinal microenvironment and consequent dysbiosis. We hypothesized that anti-inflammatory probiotic treatment would be sufficient to alleviate intestinal inflammation and thereby prevent the development of osteoporosis. To that end, the ability of Bifidobacterium lactis BL-99 administration to protect against bone loss in an experimental model of dextran sodium sulfate-induced ulcerative colitis (UC) was analyzed, and the underlying molecular mechanisms were interrogated in detail. The results of these analyses revealed that BL-99 administration suppressed colitis-associated weight loss (P < 0.05), disease activity index scores, and the production of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17) (P < 0.05). Colon tissue pathological sections similarly revealed BL-99-mediated reductions in tissue injury severity. Micro-computed tomography (Micro-CT) analyses further exhibited significant improvements in percent bone volume (BV/TV) as well as trabecular number and thickness in BL-99-treated animals (P < 0.05). Such probiotic supplementation also resulted in pronounced changes in the composition of the gut microbiota. Moreover, BL-99 intervention markedly increased the expression of intestinal barrier-related proteins (Claudin-1, MUC2, ZO-1, and Occludin). Together, these results suggest that BL-99 can be utilized as a beneficial probiotic preparation to prevent the incidence of osteoporosis in UC patients owing to its ability to shape the intestinal microflora and to suppress inflammatory cytokine production.

Maternal diet associated with infants' intestinal microbiota mediated by predominant long-chain fatty acid in breast milk.

Introduction: Long-chain fatty acids in breast milk are affected by the mother's diet and play an important role in the growth, development, and immune construction of infants. This study aims to explore the correlation between maternal diet, breast milk fatty acids (FAs), and the infant intestinal flora. Methods: We enrolled 56 paired mothers and their infants; both breast milk samples and infants' fecal samples were collected to determine the long-chain FA content of breast milk by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS), and metagenomic technology was applied to determine the microbial composition of infant feces. The maternal diet was also investigated using a 24-h dietary recall. Results: The results indicated that the fat contribution rates of edible oils in the maternal diet are significantly positively correlated with the contents of certain long-chain fatty acids (C16:0, C18:1, C16:1, and C22:4) in breast milk, which mainly regulate the abundance of Lacticaseibacillus rhamnosus, Lacticaseibacillus fermentum, and Lacticaseibacillus paracasei in the infant gut. Through KEGG pathway analysis, our data revealed that the long-chain FAs in different groups of breast milk were significantly correlated with the pathways of biotin metabolism, glycerolipid metabolism, and starch and sucrose metabolism. Discussion: The results of this study suggest a pathway in which the diets of lactating mothers may affect the composition of the infant intestinal microbiota by influencing breast milk FAs and then further regulating infant health.

Safety Evaluation of Bifidobacterium lactis BL-99 and Lacticaseibacillus paracasei K56 and ET-22 in vitro and in vivo.

Probiotics have been reported to play a major role in maintaining the balance of microbiota in host. Consumption of food with probiotics has increased with consumer concerns regarding healthy diets and wellness. Correspondingly, safety evaluation of probiotics for human consumption has become increasingly important in food industry. Herein, we aimed to test the safety of Bifidobacterium lactis BL-99 and Lacticaseibacillus paracasei K56 and ET-22 strains in vitro and in vivo. In results, these strains were found to be negative for mucin degradation and platelet aggregation test. Additionally, the three strains were susceptible to eight antibiotics. In accordance with bacterial reversion mutation (Ames) assay, the tested strains had no genetic mutagenicity. Finally, it was confirmed that there were no dose-dependent mortality and toxicity throughout multidose oral toxicity tests in rats. Our findings demonstrated that B. lactis BL-99 and L. paracasei K56 and ET-22 can achieve the generally recognized as safe (GRAS) status as probiotics in the future.

Alteration of behavior and monoamine levels attributable to Lactobacillus plantarum PS128 in germ-free mice.

Probiotics, defined as live bacteria or bacterial products, confer a significant health benefit to the host, including amelioration of anxiety-like behavior and psychiatric illnesses. Here we administered Lactobacillus plantarum PS128 (PS128) to a germ-free (GF) mouse model to investigate the impact of the gut-brain axis on emotional behaviors. First, we demonstrated that chronic administration of live PS128 showed no adverse effects on physical health. Then, we found that administration of live PS128 significantly increased the total distance traveled in the open field test and decreased the time spent in the closed arm in the elevated plus maze test, whereas the administration of PS128 had no significant effects in the depression-like behaviors of GF mice. Also, chronic live PS128 ingestion significantly increased the levels of both serotonin and dopamine in the striatum, but not in the prefrontal cortex or hippocampus. These results suggest that the chronic administration of PS128 is safe and could induce changes in emotional behaviors. The behavioral changes are correlated with the increase in the monoamine neurotransmitters in the striatum. These findings suggest that daily intake of the L. plantarum strain PS128 could improve anxiety-like behaviors and may be helpful in ameliorating neuropsychiatric disorders.

Psychotropic effects of Lactobacillus plantarum PS128 in early life-stressed and naïve adult mice.

Ingestion of specific probiotics, namely "psychobiotics", produces psychotropic effects on behavior and affects the hypothalamic-pituitary-adrenal axis and neurochemicals in the brain. We examined the psychotropic effects of a potential psychobiotic bacterium, Lactobacillus plantarum strain PS128 (PS128), on mice subjected to early life stress (ELS) and on naïve adult mice. Behavioral tests revealed that chronic ingestion of PS128 increased the locomotor activities in both ELS and naïve adult mice in the open field test. In the elevated plus maze, PS128 significantly reduced the anxiety-like behaviors in naïve adult mice but not in the ELS mice; whereas the depression-like behaviors were reduced in ELS mice but not in naïve mice in forced swimming test and sucrose preference test. PS128 administration also reduced ELS-induced elevation of serum corticosterone under both basal and stressed states but had no effect on naïve mice. In addition, PS128 reduced inflammatory cytokine levels and increased anti-inflammatory cytokine level in the serum of ELS mice. Furthermore, the dopamine level in the prefrontal cortex (PFC) was significantly increased in PS128 treated ELS and naïve adult mice whereas serotonin (5-HT) level was increased only in the naïve adult mice. These results suggest that chronic ingestion of PS128 could ameliorate anxiety- and depression-like behaviors and modulate neurochemicals related to affective disorders. Thus PS128 shows psychotropic properties and has great potential for improving stress-related symptoms.

Genome architecture of Lactobacillus plantarum PS128, a probiotic strain with potential immunomodulatory activity.

BACKGROUND: Clinical and preclinical observations indicate that Lactobacillus plantarum has anti-inflammatory activity and may regulate the immune responses of its hosts when ingested. Recently, modification of teichoic acids (TAs) produced by L. plantarum was reported as a key to regulating the systemic immune response in mice. However, data linking TA-related genetic determinants and the immunomodulatory effect are limited. To provide genomic information for elucidating the underlying mechanism of immunomodulation by L. plantarum, we sequenced the genome of L. plantarum strain PS128. RESULTS: The PS128 genome contains 11 contigs (3,325,806 bp; 44.42% GC content) after hybrid assembly of sequences derived with Illumina MiSeq and PacBio RSII systems. The most abundant functions of the protein-coding genes are carbohydrate, amino acid, and protein metabolism. The 16S rDNA sequences of PS128 are closest to the sequences of L. plantarum WCFS1 and B21; these three strains form a distinct clade based on 16S rDNA sequences. PS128 shares core genes encoding the metabolism, transport, and modification of TAs with other sequenced L. plantarum strains. Compared with the TA-related genes of other completely sequenced L. plantarum strains, the PS128 contains more lipoteichoic acid exporter genes. CONCLUSIONS: We determined the draft genome sequence of PS128 and compared its TA-related genes with those of other L. plantarum strains. Shared genomic features with respect to TA-related subsystems may be important clues to the mechanism by which L. plantarum regulates its host immune responses, but unique TA-related genetic determinants should be further investigated to elucidate strain-specific immunomodulatory effects.

Calophyllolide content in Calophyllum inophyllum at different stages of maturity and its osteogenic activity.

Calophyllum inophyllum is a coastal plant rich in natural substances. Its ingredients have been used for the development of an anti-human immunodeficiency virus (HIV) drug. In this study, we collected C. inophyllum fruit, and the ethanol extract of the fruit was chromatographically separated using silica gel and Sephadex LH-20 columns to obtain the major compound, calophyllolide. The fruits were harvested from September to December in 2011; a quantitative analysis of the calophyllolide content was conducted using HPLC to explore the differences between the different parts of the fruit during the growing season. The results showed that in fruits of C. inophyllum, calophyllolide exists only in the nuts, and dried nuts contain approximately 2 mg·g-1 of calophyllolide. The calophyllolide levels in the nuts decreased during maturity. In addition, calophyllolide dose-dependently enhanced alkaline phosphatase (ALP) activity in murine osteoblastic MC3T3-E1 cells, without significant cytotoxicity. The expression of osteoblastic genes, ALP and osteocalcin (OCN), were increased by calophyllolide. Calophyllolide induced osteoblasts differentiation also evidenced by increasing mineralization and ALP staining.

Effect of Lactobacillus plantarum Strain K21 on High-Fat Diet-Fed Obese Mice.

Recent studies have demonstrated beneficial effects of specific probiotics on alleviating obesity-related disorders. Here we aimed to identify probiotics with potential antiobesity activity among 88 lactic acid bacterial strains via in vitro screening assays, and a Lactobacillus plantarum strain K21 was found to harbor abilities required for hydrolyzing bile salt, reducing cholesterol, and inhibiting the accumulation of lipid in 3T3-L1 preadipocytes. Furthermore, effects of K21 on diet-induced obese (DIO) mice were examined. Male C57Bl/6J mice received a normal diet, high-fat diet (HFD), or HFD with K21 administration (10(9) CFU in 0.2 mL PBS/day) for eight weeks. Supplementation of K21, but not placebo, appeared to alleviate body weight gain and epididymal fat mass accumulation, reduce plasma leptin levels, decrease cholesterol and triglyceride levels, and mitigate liver damage in DIO mice. Moreover, the hepatic expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) related to adipogenesis was significantly downregulated in DIO mice by K21 intervention. We also found that K21 supplementation strengthens intestinal permeability and modulates the amount of Lactobacillus spp., Bifidobacterium spp., and Clostridium perfringens in the cecal contents of DIO mice. In conclusion, our results suggest that dietary intake of K21 protects against the onset of HFD-induced obesity through multiple mechanisms of action.