Identification of safe putative probiotics from various food products.

The objective of this study was to isolate, identify, and assess the safety and functionality in vitro of putative probiotic bacterial strains. Isolation procedures were based on standard methods using elective and selective media. The isolates were identified by comparative 16S rRNA sequencing analysis while their safety was determined according to the safety tests recommended by the FAO/WHO such as antibiotic resistance, hemolysin, and biogenic amine production. Most of the isolates did not pass the in vitro safety tests; therefore, only Lactiplantibacillus plantarum (from ant intestine and cheese), Lacticaseibacillus paracasei (from goat milk and kimchi), Enterococcus faecium (from chili doenjang and vegetables with kimchi ingredients), Limosilactobacillus fermentum (from saliva), and Companilactobacillus alimentarius (from kimchi) were identified and selected for further studies. The isolates were further differentiated by rep-PCR and identified to the strain level by genotypic (16S rRNA) and phenotypic (Gen III) approaches. Subsequently, the strain tolerance to acid and bile was evaluated resulting in good viability after simulated gastrointestinal tract passage. Adhesion to mucin in vitro and the presence of mub, mapA, and ef-tu genes confirmed the adhesive potential of the strains and the results of features associated with adhesion such as hydrophobicity and zeta potential extended the insights. This study reflects the importance of fermented and non-fermented food products as a promising source of lactic acid bacteria with potential probiotic properties. Additionally, it aims to highlight the challenges associated with the selection of safe strains, which often fail in the in vitro tests, thus hindering the possibilities of "uncovering" novel and safe probiotic strains.

Lactobacillus johnsonii BFE6154 Ameliorates Diet-Induced Hypercholesterolemia.

The functional characteristics of Lactobacillus johnsonii BFE6154, first isolated from Maasai traditional fermented milk, were previously identified in vitro, but its cholesterol-lowering properties have not been verified yet. In this study, we investigated the effect of L. johnsonii BFE6154 on cholesterol regulation and the mode of action. Stimulation of Caco-2 intestinal epithelial cells with L. johnsonii BFE6154 downregulated the gene expression of Niemann-Pick C1-like 1 (NPC1L1) through the activation of liver X receptor (LXR). Also, stimulation of HepG2 cells with the metabolites produced by L. johnsonii BFE6154 revealed an increase in the gene expression of low-density lipoprotein receptor (LDLR). Oral administration of L. johnsonii BFE6154 in mice receiving a high-fat and high-cholesterol diet (HFHCD), reduced total cholesterol and low-density lipoprotein-cholesterol (LDL) and increased high-density lipoprotein-cholesterol (HDL) in the blood, compared to the control. Diet-induced hypercholesterolemic mice receiving L. johnsonii BFE6154 showed a suppression of cholesterol absorption under the control of NPC1L1 in the intestine. Furthermore, L. johnsonii BFE6154 consumption ameliorated the hepatic cholesterol level and LDLR expression, which was reduced by HFHCD. These molecular modulations led to the increase of cholesterol excretion and the decrease of cholesterol levels in the feces and liver, respectively. Taken together, these results suggest that L. johnsonii BFE6154 may protect against diet-induced hypercholesterolemia through the regulation of cholesterol metabolism in the intestine and liver.

Two putative probiotic strains improve diet-induced hypercholesterolemia through modulating intestinal cholesterol uptake and hepatic cholesterol efflux.

AIMS: Two putative probiotic strains, Lacticaseibacillus (Lc.) rhamnosus BFE5264 and Lactiplantibacillus (Lp.) plantarum NR74, have been shown to suppress cholesterol uptake and promote cholesterol efflux in Caco-2 cells. However, an in vivo beneficial effect of these strains on plasma cholesterol levels has not been verified yet; neither have the underlying mechanisms of regulating cholesterol metabolism clarified thus far. This study has focused on these two aspects. METHODS AND RESULTS: A murine model has been used, and the animals receiving a high-fat/high-cholesterol diet showed elevated plasma cholesterol levels. However, supplementation of Lc. rhamnosus BFE5264 and Lp. plantarum NR74 resulted in the down regulation of Niemann-Pick C1-like 1 (NPC1L1) in the intestine in addition to counteracting the diet-induced suppression of low-density lipoprotein receptor expression in the liver. ATP Binding Cassette Subfamily A Member 1 (ABCA1) was only significantly increased upon administration of Lc. rhamnosus BFE5264. CONCLUSIONS: The present findings demonstrate that supplementation with Lc. rhamnosus BFE5264 and Lp. plantarum NR74 may improve diet-induced hypercholesterolemia by suppression of cholesterol absorption in the small intestine and by supporting the regulation of cholesterol metabolism in the liver. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to understanding the beneficial effects of probiotics on host cholesterol metabolism and underlying mechanisms related to hypercholesterolemia.

Pilot Study on the Forehead Skin Microbiome and Short Chain Fatty Acids Depending on the SC Functional Index in Korean Cohorts.

Dry skin is one of the indicators of a compromised skin barrier. An intact skin barrier is not only important to reserve the hydration within the epidermal tissue but also to protect our skin from environmental stressors and inhibit pathogen invasion; damage to the skin barrier may lead to inflammatory skin diseases. Some microbial metabolites such as short chain fatty acids may inhibit or destroy harmful bacteria and regulate the host immune system. The impact of the skin microbiome and short chain fatty acids on skin barrier function was studied in two groups of 75 participants each. The cohort was equally divided in dry and moist skin types, based on stratum corneum (SC) functionality index (SCFI), reflecting the ratio of transepidermal water loss (TEWL). A dry group represents a low SCFI and a moist group a high SCFI. Compared with the dry skin group, propionate and Cutibacterium levels (previously known as Propionibacterium acnes) were significantly higher (p < 0.001) in the moist group. Levels of Cutibacterium were negatively correlated with those of Staphylococcus (p < 0.0001) in both dry and moist groups. The moist group also had a significantly higher propionate concentration (p < 0.001). This study showed that the microbial community and short chain fatty acid concentration may be considered as significant determinants of the SCFI of the skin.

Assessment of the safety and anti-inflammatory effects of three Bacillus strains in the respiratory tract.

Chronic respiratory diseases are part of accumulating health problems partly due to worldwide increase in air pollution. By their antimicrobial and immunomodulatory properties, some probiotics constitute promising alternatives for the prevention and treatment of chronic respiratory diseases. We have isolated Bacillus strains from Korean fermented foods and selected three potentially probiotic strains (two Bacillus subtilis and one Bacillus amyloliquefaciens) based on safety, antimicrobial efficacy, activity against airborne pathogens and their immunomodulatory properties in vivo. Safety evaluation included in silico analysis for confirming absence of virulence genes. Safety for the respiratory tract was confirmed by an in vivo pathogenicity test using a murine model. Antimicrobial activity was displayed against several airborne pathogens. Potential antimicrobial metabolites such as 2,3-butanediol and propylene glycol were identified as possible antagonistic agents. Immunomodulatory properties in vitro were confirmed by upregulation of IL-10 expression in a macrophage cell line. Intranasal instillation and inhalation in an ovalbumin (OVA)-induced lung inflammation murine model reduced T helper type 2 (Th2) cytokines at transcriptional and protein levels in the lungs. The safety and potentially beneficial role of these Bacillus strains could be demonstrated for the respiratory tract of a murine model.

Safety Evaluation and In vivo Strain-Specific Functionality of Bacillus Strains Isolated from Korean Traditional Fermented Foods.

Unveiling and understanding differences in physiological features below the species level may serve as an essential fast-screening tool for selecting strains that can promote a specific probiotic effect. To study the intra-species diversity of Bacillus, a genus with a wide range of enzyme activities and specificity, 190 Bacillus strains were isolated from traditional Korean fermented food products. Altogether, in the preliminary safety screening, 8 of these strains were found negative for lecithinase and hemolysis activity and were selected for further investigations. On the basis of different levels of enzyme functionalities (high or low proteolytic, amylolytic, and lipolytic (PAL) activities), two Bacillus subtilis strains were selected for an in vivo study. Each of the two strains was separately administered at a level of 1 × 108 CFU per day to C57BL/6 mice that were fed 60% high-fat diet ad libitum for 8 weeks, while Xenical, an anti-obesity drug, was used as a positive control in the experimental setup. B. subtilis M34 and B. subtilis GS40a with low and high amylolytic activities, respectively, induced significantly different and contrasting physiological effects. The production of short-chain fatty acids appeared to be closely associated with a shift in the gut microbiota.

Modulation of the Gut Microbiome and Obesity Biomarkers by Lactobacillus Plantarum KC28 in a Diet-Induced Obesity Murine Model.

Lactobacillus plantarum KC28 showed a beneficial (anti-obesity) effect in a diet-induced obese (DIO) C57BL/6 murine model receiving an intermediate high-fat diet (IF). This diet was selected for probiotic studies by prior comparisons of different combinations of basic (carbohydrate, protein and fat) components for optimized induction of dietary obesity in a murine model. Prior selection of Lact. plantarum strain KC28 was based on different physiological tests for safety and functionality including cell line adhesion and anti-adipogenic activity. The strain was administered at 5.0 × 109 CFU/mouse/day to the DIO mice (control mice received a normal diet). The anti-obesity effect of KC28 and the well-known probiotic strains Lact. rhamnosus GG (LGG) and Lact. plantarum 299v was assessed over 12 weeks. Xenical served as anti-obesity control. The high-fat diet groups receiving strains KC28 and LGG and the control Xenical group showed significant weight loss and notable changes in some obesity-related biomarkers in the liver (significant up-regulation of PGC1-α and CPT1-α only by KC28; p < 0.05) and mesenteric adipose tissue (significant down-regulation of ACOX-1, PPAR-γ, and FAS; KC28 p < 0.001 for PPAR-γ and FAS), compared with the IF control. Favourable changes in the studied biomarkers suggest a similar beneficial influence of Lact. plantarum KC28 on the alleviation of obesity comparable with that of the two well-studied probiotic strains, LGG and 299v. This probably resulted from a modulation in the cecal microbiota of the IF group by either probiotic strain, yet in a different manner, showing a highly significant increase in the families Desulfovibrionaceae and Lactobacillaceae only in the group receiving Lact. plantarum KC28.

Do Your Kids Get What You Paid for? Evaluation of Commercially Available Probiotic Products Intended for Children in the Republic of the Philippines and the Republic of Korea.

A wide range of probiotic products is available on the market and can be easily purchased over the counter and unlike pharmaceutical drugs, their commercial distribution is not strictly regulated. In this study, ten probiotic preparations commercially available for children's consumption in the Republic of the Philippines (PH) and the Republic of Korea (SK) have been investigated. The analyses included determination of viable counts and taxonomic identification of the bacterial species present in each formulation. The status of each product was assessed by comparing the results with information and claims provided on the label. In addition to their molecular identification, safety assessment of the isolated strains was conducted by testing for hemolysis, biogenic amine production and antibiotic resistance. One out of the ten products contained lower viable numbers of recovered microorganisms than claimed on the label. Enterococcus strains, although not mentioned on the label, were isolated from four products. Some of these isolates produced biogenic amines and were resistant to one or several antibiotics. Metagenomic analyses of two products revealed that one product did not contain most of the microorganisms declared in its specification. The study demonstrated that some commercial probiotic products for children did not match their label claims. Infants and young children belong to the most vulnerable members of society, and food supplements including probiotics destined for this consumer group require careful checking and strict regulation before commercial distribution.

Amelioration of Alcohol Induced Gastric Ulcers Through the Administration of Lactobacillus plantarum APSulloc 331261 Isolated From Green Tea.

Gastric inflammation is an indication of gastric ulcers and possible other underlying gastric malignancies. Epidemiological studies have revealed that several Asian countries, including South Korea, suffer from a high incidence of gastric diseases derived from high levels of stress, alcoholic consumption, pyloric infection and usage of non-steroidal anti-inflammatory drugs (NSAIDs). Clinical treatments of gastric ulcers are generally limited to proton pump inhibitors that neutralize the stomach acid, and the application of antibiotics for Helicobacter pylori eradication, both of which are known to have a negative effect on the gut microbiota. The potential of probiotics for alleviating gastrointestinal diseases such as intestinal bowel syndrome and intestinal bowel disease receives increasing scientific interest. Probiotics may support the amelioration of disease-related symptoms through modulation of the gut microbiota without causing dysbiosis. In this study the potential of Lactobacillus plantarum APSulloc 331261 (GTB1TM), isolated from green tea, was investigated for alleviating gastric inflammation in an alcohol induced gastric ulcer murine model (positive control). Treatment with the test strain significantly influenced the expression of pro-inflammatory and anti-inflammatory biomarkers, interleukin 6 (IL6) and interleukin 10 (IL10), of which the former was down- and the latter up-regulated when the alcohol induced mice were treated with the test strain. This positive effect was also indicated by less severe gastric morphological changes and the histological score of the gastric tissues. A significant increase in the abundance of Akkermansia within the GTB1TM treated group compared to the positive control group also correlated with a decrease in the ratio of acetate over propionate. The increased levels of propionate in the GTB1TM group appear to result from the impact of the test strain on the microbial population and the resulting metabolic activities. Moreover, there was a significant increase in beta-diversity in the group that received GTB1TM over that of the alcohol induced control group.

Alteration of Microbiome Profile by D-Allulose in Amelioration of High-Fat-Diet-Induced Obesity in Mice.

Recently, there has been a global shift in diet towards an increased intake of energy-dense foods that are high in sugars. D-allulose has received attention as a sugar substitute and has been reported as one of the anti-obesity food components; however, its correlation with the intestinal microbial community is not yet completely understood. Thirty-six C57BL/6J mice were divided in to four dietary groups and fed a normal diet (ND), a high-fat diet (HFD, 20% fat, 1% cholesterol, w/w), and a HFD with 5% erythritol (ERY) and D-allulose (ALL) supplement for 16 weeks. A pair-feeding approach was used so that all groups receiving the high-fat diet would have the same calorie intake. As a result, body weight and body fat mass in the ALL group were significantly decreased toward the level of the normal group with a simultaneous decrease in plasma leptin and resistin. Fecal short-chain fatty acid (SCFA) production analysis revealed that ALL induced elevated total SCFA production compared to the other groups. Also, ALL supplement induced the change in the microbial community that could be responsible for improving the obesity based on 16S rRNA gene sequence analysis, and ALL significantly increased the energy expenditure in Day(6a.m to 6pm). Taken together, our findings suggest that 5% dietary ALL led to an improvement in HFD-induced obesity by altering the microbiome community.

Safety Evaluation and Whole-Genome Annotation of Lactobacillus plantarum Strains from Different Sources with Special Focus on Isolates from Green Tea.

Lactobacillus plantarum shows high intraspecies diversity species, and has one of the largest genome sizes among the lactobacilli. It is adapted to diverse environments and provides a promising potential for various applications. The aim of the study was to investigate the safety and probiotic properties of 18 L. plantarum strains isolated from fermented food products, green tea, and insects. For preliminary safety evaluation the L. plantarum strains were tested for their ability to produce hemolysin and biogenic amines and for their antibiotic resistance. Based on preliminary safety screening, four strains isolated from green tea showed antibiotic resistance below the cut-off MIC values suggested by EFSA, and were selected out of the 18 strains for more detailed studies. Initial selection of strains with putative probiotic potential was determined by their capacity to survive in the human GIT using an in vitro simulation model, and for their adhesion to human Caco-2/TC-7 cell line. Under simulated GIT conditions, all four L. plantarum strains isolated from green tea showed higher survival rates than the control (L. plantarum subsp. plantarum ATCC 14917). All studied strains were genetically identified by 16S rRNA gene sequencing and confirmed to be L. plantarum. In addition, whole-genome sequence analysis of L. plantarum strains APsulloc 331261 and APsulloc 331263 from green tea was performed, and the outcome was compared with the genome of L. plantarum strain WCFS1. The genome was also annotated, and genes related to virulence factors were searched for. The results suggest that L. plantarum strains APsulloc 331261 and APsulloc 331263 can be considered as potential beneficial strains for human and animal applications.

Amelioration of obesity-related biomarkers by Lactobacillus sakei CJLS03 in a high-fat diet-induced obese murine model.

Recent progresses in clinical diagnostic analyses have demonstrated the decisive influence of host gut microbiota on the status of metabolic disorders. Short chain fatty acids (SCFAs) produced by gut microbiota, in particular, are considered as a key biomarker, both of communication between gut microbiota and the host, and of impact on host metabolic homeostasis. Microbiota modulation and concomitant anti-obesity effects of probiotics have been reported by different researchers. However, the underlying modulatory functions of probiotics on gut microbiota towards host metabolic homeostasis are still not fully understood. In this study, the impact of Lactobacillus sakei CJLS03 (isolated from Korean kimchi) on obesity-related biomarkers was investigated using a diet-induced obese mouse model. Body weight increase, SCFAs, the gut microbiota and various obesity-associated biomarkers were significantly and beneficially influenced by L. sakei CJLS03 administration compared to the control groups. Analytical data on faecal samples support the role of the colonic microbial population in SCFA production. The composition of the latter may be influenced by modulation of the distal gastro-intestinal microbiota by putative probiotics such as L. sakei CJLS03.

Differences in Anxiety Levels of Various Murine Models in Relation to the Gut Microbiota Composition.

Psychobiotics are probiotic strains that confer mental health benefits to the host through the modulation of the gut microbial population. Mounting evidence shows that the gut microbiota play an important role in communication within the gut⁻brain axis. However, the relationship between the host genetics and the gut microbiota and their influence on anxiety are still not fully understood. Hence, in our research, we attempted to draw a connection between host genetics, microbiota composition, and anxiety by performing an elevated plus maze (EPM) test on four genetically different mice. Four different breeds of 5-week-old mice were used in this experiment: Balb/c, Orient C57BL/6N, Taconic C57BL/6N, and Taconic C57BL/6J. After 1 week of adaptation, their initial anxiety level was monitored using the EPM test via an EthoVision XT, a standardized software used for behavorial testing. Significant differences in the initial anxiety level and microbial composition were detected. Subsequently, the microbiota of each group was modulated by the administration of either a probiotic, fecal microbiota transplantation, or antibiotics. Changes were observed in host anxiety levels in correlation to the shift of the gut microbiota. Our results suggest that the microbiota, host genetics, and psychological symptoms are strongly related, yet the deeper mechanistic links need further exploration.

Correction: Cholesterol-lowering effect of Lactobacillus rhamnosus BFE5264 and its influence on the gut microbiome and propionate level in a murine model.

[This corrects the article DOI: 10.1371/journal.pone.0203150.].

Cholesterol-lowering effect of Lactobacillus rhamnosus BFE5264 and its influence on the gut microbiome and propionate level in a murine model.

Thanks to recent scientific progress a relationship between the intestinal microbiota and metabolic diseases could be established. A deeper understanding of underlying mechanisms has opened ways towards new approaches for alleviating conditions associated with metabolic diseases. Dysbiosis appears to be a major underlying factor associated with metabolic syndrome and related adverse health conditions. A major focus has therefore shifted to controlling of the gut microbiota through administration of functional lactic acid bacteria (LAB). The scope for health promotion and/or support by probiotics such as LAB has thereby been widened beyond the improving of intestinal health, also to include anti-obesity, anti-diabetic and cholesterol-lowering effects. In this study we investigated the cholesterol-lowering and microbiota modulatory potential of a LAB strain, Lactobacillus rhamnosus BFE5264, isolated from Maasai fermented milk. A mouse model receiving a high-cholesterol diet served as model for evaluating its functionality. The administration of L. rhamnosus BFE5264 resulted in a significant reduction of the serum cholesterol level that was accompanied by changes in intestinal microbiota and the production of short chain fatty acid (SCFA) in comparison to the control group. This strain also beneficially influenced the regulation of cholesterol metabolism in the liver in a pattern similar to that resulting from statin treatment, a drug inhibiting cholesterol biosynthesis in the liver.

Modulation of Active Gut Microbiota by Lactobacillus rhamnosus GG in a Diet Induced Obesity Murine Model.

Gut microbiota play a key role in the development of metabolic disorders. Defining and correlating structural shifts in gut microbial assemblages with conditions related to metabolic syndrome have, however, been proven difficult. Results from 16S genomic DNA and 16S ribosomal RNA analyses of fecal samples may differ widely, leading to controversial information on the whole microbial community and metabolically active microbiota. Using a C57BL/6J murine model, we compared data from 16S genomic DNA and ribosomal RNA of the fecal microbiota. The study included three groups of experimental animals comprising two groups with high fat diet induced obesity (DIO) while a third group (control) received a low fat diet. One of the DIO groups was treated with the probiotic Lactobacillus rhamnosus GG (LGG). Compared to the data obtained by DNA analysis, a significantly higher abundance of OTUs was accounted for by RNA analysis. Moreover, rRNA based analysis showed a modulation of the active gut microbial population in the DIO group receiving LGG, thus reflecting a change in the induced obesity status of the host. As one of the most widely studied probiotics the functionality of LGG has been linked to the alleviation of metabolic syndrome, and, in some cases, to an impact on the microbiome. Yet, it appears that no study has reported thus far on modulation of the active microbiota by LGG treatment. It is postulated that the resulting impact on calorie consumption affects weight gain concomitantly with modulation of the functional structure of the gut microbial population. Using the 16S rRNA based approach therefore decisively increased the precision of gut microbiota metagenome analysis.

Protective effects of Bacillus probiotics against high-fat diet-induced metabolic disorders in mice.

Recently, modulation of gut microbiota by probiotics treatment has been emerged as a promising strategy for treatment of metabolic disorders. Apart from lactic acid bacteria, Bacillus species (Bacillus spp.) have also been paid attention as potential probiotics, but nevertheless, the molecular mechanisms for their protective effect against metabolic dysfunction remain to be elucidated. In this study, we demonstrate that a probiotic mixture composed of 5 different Bacillus spp. protects mice from high-fat diet (HFD)-induced obesity, insulin resistance and non-alcoholic fatty liver disease (NAFLD). Probiotic Bacillus treatment substantially attenuated body weight gain and enhanced glucose tolerance by sensitizing insulin action in skeletal muscle and epididymal adipose tissue (EAT) of HFD-fed mice. Bacillus-treated HFD-fed mice also exhibited significantly suppressed chronic inflammation in the liver, EAT and skeletal muscle, which was observed to be associated with reduced HFD-induced intestinal permeability and enhanced adiponectin production. Additionally, Bacillus treatment significantly reversed HFD-induced hepatic steatosis. In Bacillus-treated mice, hepatic expression of lipid oxidative genes was significantly increased, and lipid accumulation in subcutaneous and mesenteric adipose tissues were significantly decreased, commensurate with down-regulated expression of genes involved in lipid uptake and lipogenesis. Although, in Bacillus-treated mice, significant alterations in gut microbiota composition was not observed, the enhanced expression of tight junction-associated proteins showed a possibility of improving gut barrier function by Bacillus treatment. Our findings provide possible explanations how Bacillus probiotics protect diet-induced obese mice against metabolic disorders, identifying the treatment of probiotic Bacillus as a potential therapeutic approach.

Long-term fermented soybean paste improves metabolic parameters associated with non-alcoholic fatty liver disease and insulin resistance in high-fat diet-induced obese mice.

Recently, Korean traditional fermented soybean paste, called Doenjang, has attracted attention for its protective effect against diet-related chronic diseases such as obesity and type 2 diabetes. Long-term fermented soybean pastes (LFSPs) are made by fermentation with naturally-occurring microorganisms for several months, whereas short-term fermented soybean pastes (SFSPs) are produced by shorter-time fermentation inoculated with a starter culture. Here, we demonstrate that administration of LFSP, but not SFSP, protects high-fat diet (HFD)-fed obese mice against non-alcohol fatty liver disease (NAFLD) and insulin resistance. LFSP suppressed body weight gain in parallel with reduction in fat accumulation in mesenteric adipose tissue (MAT) and the liver via modulation of MAT lipolysis and hepatic lipid uptake. LFSP-treated mice also had improved glucose tolerance and increased adiponectin levels concomitantly with enhanced AMPK activation in skeletal muscle and suppressed expression of pro-inflammatory cytokines in skeletal muscle and the liver. LFSP also attenuated HFD-induced gut permeability and lowered serum lipopolysaccharide level, providing an evidence for its probiotic effects, which was supported by the observation that treatment of a probiotic mixture of LFSP-originated Bacillus strains protected mice against HFD-induced adiposity and glucose intolerance. Our findings suggest that the intake of LFSP, but not SFSP, offers protection against NAFLD and insulin resistance, which is an effect of long-term fermentation resulting in elevated contents of active ingredients (especially flavonoids) and higher diversity and richness of Bacillus probiotic strains compared to SFSP.