Efficacies of Potential Probiotic Candidates Isolated from Traditional Fermented Korean Foods in Stimulating Immunoglobulin A Secretion.

The aim of this study was to evaluate efficacies of selected lactic acid bacteria (LAB) in inducing immunoglobulin A (IgA) secretion. Twenty-five different LAB isolated from traditional fermented Korean foods were characterized for their probiotic properties and screened to identify those that could stimulate lamina propria cells (LPCs) from Peyer's patch to secret IgA in vitro. Among them, four strains (Lactiplantibacillus plantarum CJW55-10, Lactiplantibacillus pentosus CJW18-6, L. pentosus CJW56-11, and Pediococcus acidilactici CJN2696) were found to be strong IgA inducers. The number of IgA positive B cells and soluble IgA level were increased when LPCs were co-cultured with these LAB. Expression levels of toll-like receptor (TLR) such as TLR2 and TLR4 and secretion of interleuckin-6 were augmented in LPCs treated with these LAB. Further, we determined whether oral intake of these LAB enhanced IgA production in vivo. After one-week of daily oral administration, these LAB feed mice increased mucosal IgA and serum IgA. In conclusion, selected strains of LAB could induce systemic IgA secretion by activating lamina propria B cells in Peyer's patch and oral intake of selected strains of LAB can enhance systemic immunity by inducing mucosal IgA secretion.

Monitoring Cellular Immune Responses after Consumption of Selected Probiotics in Immunocompromised Mice.

Probiotics are currently considered as one of tools to modulate immune responses under specific clinical conditions. The purpose of this study was to evaluate whether oral administration of three different probiotics (Lactiplantibacillus plantarum CJLP243, CJW55-10, and CJLP475) could evoke a cell-mediated immunity in immunodeficient mice. Before conducting in vivo experiments, we examined the in vitro potency of these probiotics for macrophage activation. After co-culture with these probiotics, bone marrow derived macrophages (BMDMs) produced significant amounts of proinflammatory cytokines including interleukin-6 (IL-6), IL-12, and tumor necrosis factor-α (TNF-α). Levels of inducible nitric oxide synthase (inos) and co-stimulatory molecules (CD80 and CD86) were also upregulated in BMDMs after treatment with some of these probiotics. To establish an immunocompromised animal model, we intraperitoneally injected mice with cyclophosphamide on day 0 and again on day 2. Starting day 3, we orally administered probiotics every day for the last 15 d. After sacrificing experimental mice on day 18, splenocytes were isolated and co-cultured with these probiotics for 3 d to measure levels of several cytokines and immune cell proliferation. Results clearly indicated that the consumption of all three probiotic strains promoted secretion of interferon-γ (IFN-γ), IL-1ß, IL-6, IL-12, and TNF-α. NK cell cytotoxicity and proliferation of immune cells were also increased. Taken together, our data strongly suggest that consumption of some probiotics might induce cell-mediated immune responses in immunocompromised mice.

Effects of Lactobacillus plantarum CJLP55 on Clinical Improvement, Skin Condition and Urine Bacterial Extracellular Vesicles in Patients with Acne Vulgaris: A Randomized, Double-Blind, Placebo-Controlled Study.

Lactobacillus plantarum CJLP55 has anti-pathogenic bacterial and anti-inflammatory activities in vitro. We investigated the dietary effect of CJLP55 supplement in patients with acne vulgaris, a prevalent inflammatory skin condition. Subjects ingested CJLP55 or placebo (n = 14 per group) supplements for 12 weeks in this double-blind, placebo-controlled randomized study. Acne lesion count and grade, skin sebum, hydration, pH and surface lipids were assessed. Metagenomic DNA analysis was performed on urine extracellular vesicles (EV), which indirectly reflect systemic bacterial flora. Compared to the placebo supplement, CJLP55 supplement improved acne lesion count and grade, decreased sebum triglycerides (TG), and increased hydration and ceramide 2, the major ceramide species that maintains the epidermal lipid barrier for hydration. In addition, CJLP55 supplement decreased the prevalence of Proteobacteria and increased Firmicutes, which were correlated with decreased TG, the major skin surface lipid of sebum origin. CJLP55 supplement further decreased the Bacteroidetes:Firmicutes ratio, a relevant marker of bacterial dysbiosis. No differences in skin pH, other skin surface lipids or urine bacterial EV phylum were noted between CJLP55 and placebo supplements. Dietary Lactobacillus plantarum CJLP55 was beneficial to clinical state, skin sebum, and hydration and urine bacterial EV phylum flora in patients with acne vulgaris.

Sub-clinical detection of gut microbial biomarkers of obesity and type 2 diabetes.

BACKGROUND: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression. METHODS: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background. RESULTS: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities. CONCLUSIONS: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions.

Effect of temperature and relative humidity on the survival of foodborne viruses during food storage.

Millions of people suffer from foodborne diseases throughout the world every year, and the importance of food safety has grown worldwide in recent years. The aim of this study was to investigate the survival of hepatitis A virus (HAV) and viral surrogates of human norovirus (HuNoV) (bacteriophage MS2 and murine norovirus [MNV]) in food over time. HAV, MNV, and MS2 were inoculated onto either the digestive gland of oysters or the surface of fresh peppers, and their survival on these food matrices was measured under various temperature (4°C, 15°C, 25°C, and 40°C) and relative humidity (RH) (50% and 70%) conditions. Inoculated viruses were recovered from food samples and quantified by a plaque assay at predetermined time points over 2 weeks (0, 1, 3, 7, 10, and 14 days). Virus survival was influenced primarily by temperature. On peppers at 40°C and at 50% RH, >4- and 6-log reductions of MNV and HAV, respectively, occurred within 1 day. All three viruses survived better on oysters. In addition, HAV survived better at 70% RH than at 50% RH. The survival data for HAV, MS2, and MNV were fit to three different mathematical models (linear, Weibull, and biphasic models). Among them, the biphasic model was optimum in terms of goodness of fit. The results of this study suggest that major foodborne viruses such as HAV and HuNoV can survive over prolonged periods of time with a limited reduction in numbers. Because a persistence of foodborne virus on contaminated foods was observed, precautionary preventive measures should be performed.

Inhibitory effects of the κ-casein macropeptide isolated from milk protein on the biofilm formation and virulence of Listeria monocytogenes.

We demonstrate the inhibitory effects of κ-casein macropeptide (CMP) on the biofilm formation and virulence of Listeria monocytogenes Scott A. The inhibition of biofilm formation by CMP was initially investigated by using the protocol applied for the 96-well microtiter plate assay. Low concentrations of CMP (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) that were tested resulted in a profound inhibitory effect on biofilm formation at a concentration of 0.4 mg/mL. CMP also significantly repressed the transcription of inlA (encoding internalin A) that was responsible for the initial adhesion and invasion event, and prolonged the survival of Caenorhabditis elegans infected by L. monocytogenes. Two-dimensional gel electrophoresis showed that newly identified proteins in the presence of CMP were involved in the stress response and metabolic processes that have important roles in developing listerial biofilms. Our results suggest that CMP from milk protein would be capable of eliminating biofilm formation and virulence by L. monocytogenes in the food industry.

Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments.

We employed Caenorhabditis elegans as a model to study the effectiveness of sanitizers in killing pathogenic Escherichia coli strains ingested by free-living nematodes. Adult worms that had fed on six pathogenic E. coli strains (highly persistent in the nematode intestine) were treated with three chemical solutions. In planktonic cells, none of the H2O2 and acetic acid treatments influenced the survival of the pathogenic E. coli strains, whereas sodium hypochlorite critically decreased the viability of the strains. Importantly, the survival of the E. coli strains was dramatically increased by persistence in the C. elegans gut under 0.1% sodium hypochlorite, and several strains could survive at a concentration of 0.5%. In addition, all pathogenic E. coli strains in the C. elegans gut survived on the lettuce for 5 days even though they were washed with 0.1% sodium hypochlorite. Taken together, our results indicate that pathogenic E. coli ingested by C. elegans may be protected against washing treatment with commercial sanitizers on raw food materials.

Bacillus licheniformis isolated from Korean traditional food sources enhances the resistance of Caenorhabditis elegans to infection by Staphylococcus aureus.

We investigated whether Bacillus spp., newly isolated from Korean traditional food resources, influence the resistance of hosts to foodborne pathogens, by using Caenorhabditis elegans as a surrogate host model. Initially, we selected 20 Bacillus spp. that possess antimicrobial activity against various foodborne pathogens, including Staphylococcus aureus. Among the selected strains, six strains of Bacillus spp. used in preconditioning significantly prolonged the survival of nematodes exposed to S. aureus. Based on 16S rRNA gene sequencing, all six strains were identified as B. licheniformis. Our findings suggest that preconditioning with B. licheniformis may modulate the host defense response against S. aureus.

Susceptibility of Listeria monocytogenes biofilms and planktonic cultures to hydrogen peroxide in food processing environments.

Recent studies have indicated that Listeria monocytogenes formed biofilms on the surface of food processing equipment, and may survive sanitization treatments. The purpose of this study was to compare the susceptibility of L. monocytogenes grown in either a biofilm or planktonic culture when exposed to hydrogen peroxide (H(2)O(2)). Twelve strains of biofilm-forming L. monocytogenes and their planktonic counterparts were treated with various concentrations of H(2)O(2) (1, 6, and 10%), and the cell survival was then determined at 10-min exposure intervals. When grown as a biofilm, L. monocytogenes was significantly more resistant to H(2)O(2) than under planktonic culture conditions. Planktonic L. monocytogenes strains exhibited significantly different susceptibility to 1% H(2)O(2). Equally interestingly, biofilms of the 12 L. monocytogenes strains also inhibited different survival rates after being treated with 6 and 10% H(2)O(2). However, most of the biofilms recovered to a population of 2-9 log CFU/glass fiber filter (GFF) after a 24-h re-growth period. These results indicate that there was no significant correlation between the H(2)O(2) resistance of biofilm- and planktonic-cultured cells, and suggest that different mechanisms for the resistance to sanitation or disinfection underly the persistence of certain strains in food-processing environments.

Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity.

In the current study, the probiotic potential of approximately 350 strains of lactic acid bacteria isolated from Korean infant feces and Kimchi was investigated. Common probiotic properties of the bacterial strains, such as acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells (HT-29 cells), were examined. Some strains were found to have immune modulatory and antimicrobial properties. Antagonistic activity against a panel of pathogenic bacteria was found to be strain dependent. To evaluate the immune modulatory activity of the strains, lymphocyte interferon (IFN)-γ secretion was determined in conjunction with cell proliferation. Some strains of Lactobacillus gasseri, L. fermentum and L. plantarum exhibited increased IFN-γ levels and lymphocyte proliferation. To evaluate the effects of these immune modulating lactobacilli on host life span, Caenorhabditis elegans was used as an in vivo model. Nematodes that were supplied heat-killed lactobacilli as a food source exhibited obvious differences in life span compared with those fed Escherichia coli OP50. The mean life span (determined as mean percent survival) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 13.89% and 13.69% greater, respectively, than that of control nematodes after 21 days (P=0.036 and 0.043, respectively). In addition, some of safety profiles, including hemolytic type, gelatin hydration and degradation of urea, were found to be positive. These newly identified lactobacilli hold promise for use as probiotic agents, feed additives and/or in food applications.

Genetic and proteomic analysis of factors affecting serum cholesterol reduction by Lactobacillus acidophilus A4.

This article identifies novel factors involved in cholesterol reduction by probiotic bacteria, which were identified using genetic and proteomic approaches. Approximately 600 Lactobacillus acidophilus A4 mutants were created by random mutagenesis. The cholesterol-reducing ability of each mutant was determined and verified using two different methods: the o-phthalaldehyde assay and gas chromatographic analysis (GC). Among screened mutants, strain BA9 showed a dramatically diminished ability to reduce cholesterol, as demonstrated by a 7.7% reduction rate, while the parent strain had a more than 50% reduction rate. The transposon insertion site was mapped using inverse PCR (I-PCR), and it was determined using bioinformatic methods that the deleted region contained the Streptococcus thermophilus catabolite control protein A gene (ccpA). In addition, we have shown using two-dimensional gel electrophoresis (2-DE) that several proteins, including a transcription regulator, FMN-binding protein, major facilitator superfamily permease, glycogen phosphorylase, the YknV protein, and fructose/tagatose bisphosphate aldolase, were strongly regulated by the ccpA gene. In addition, in vivo experiments investigating ccpA function were conducted with rats. Rats fed wild-type L. acidophilus A4 showed a greater than 20% reduction in total serum cholesterol, but rats fed BA9 mutant L. acidophilus showed only an approximately 10% reduction in cholesterol. These results provide important insights into the mechanism by which these lactic acid bacteria reduce cholesterol.