Dong-Hyeon Kim

Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: Targeted and untargeted community analysis with correlation of biomarkers

Kefir is a probiotic beverage containing over 50 species of lactic acid bacteria and yeast. In this study, the anti-obesity and anti-non-alcoholic fatty liver disease (NAFLD) effects of kefir were comprehensively addressed along with targeted and untargeted community analysis of the fecal microbiota in a high-fat diet (HFD)-induced obese mouse model. HFD-fed C57BL/6 mice were orally administrated either kefir or milk (control) once a day for 12 weeks, and body and organ weight, fecal microbiota and mycobiota, histopathology, blood cholesterol and cytokines and gene expressions were analyzed. Compared to the control, mice in the kefir group exhibited a significantly lower body weight (34.18 g vs. 40.24 g; p=0.00004) and histopathological liver lesion score (1.13 vs. 3.25; p=0.002). Remarkably, the kefir-fed mice also harbored more Lactobacillus/Lactococcus (7.01 vs. 6.32 log CFU/g), total yeast (6.07 vs. 5.01 log CFU/g) and Candida (5.56 vs. 3.88 log CFU/g). Kefir administration also up-regulated genes related to fatty acid oxidation, PPARα and AOX, in both the liver and adipose tissue (PPARα, 2.95- and 2.15-fold; AOX, 1.89- and 1.9-fold, respectively). The plasma concentration of IL-6, a proinflammatory marker, was significantly reduced following kefir consumption (50.39 pg/ml vs. 111.78 pg/ml; p=0.03). Strikingly, the populations of Lactobacillus/Lactococcus, total yeast and Candida were strongly correlated with PPARα gene expression in adipose and hepatic tissue (r=0.599, 0.580 and 0.562, respectively). These data suggest that kefir consumption modulates gut microbiota and mycobiota in HFD-fed mice, which prevents obesity and NAFLD via promoting fatty acid oxidation.

Dual function of Lactobacillus kefiri DH5 in preventing high-fat-diet-induced obesity: direct reduction of cholesterol and upregulation of PPAR-α in adipose tissue

SCOPE Kefir consumption inhibits the development of obesity and non-alcoholic fatty liver disease (NALFD) in mice fed 60% high-fat diet (HFD). To identify the key contributor of this effect, we isolated lactic acid bacteria (LAB) from kefir and examined their anti-obesity properties from in vitro screening and in vivo validation. METHODS AND RESULTS Thirteen kefir LAB isolates were subjected to survivability test using artificial gastrointestinal environment and cholesterol-reducing assay. Lactobacillus kefiri DH5 showed 100% survivability in gastrointestinal environments and reduced 51.6% of cholesterol; thus, this strain was selected for in vivo experiment. Compared to the HFD-saline group, the HFD-DH5 group showed significantly lower body weight (34.68 versus 31.10 g; p < 0.001), epididymal adipose tissue weight (1.39 versus 1.05 g; p < 0.001), blood triglyceride (38.2 versus 31.0 mg/dL; p < 0.01) and LDL-cholesterol levels (19.4 versus 15.7 mg/dL; p < 0.01). In addition, L. kefiri DH5 administration significantly modulated gut microbiota of HFD-fed mice. The hepatic steatosis was significantly milder (Lesion score, 2.1 versus 1.2; p < 0.001) and adipocyte diameter was significantly smaller (65.1 versus 42.2 μm; p < 0.001) in the HFD-DH5 group. L. kefiri DH5 upregulated PPAR-α, FABP4, and CPT1 expression in the epididymal adipose tissues (2.29-, 1.77-, and 2.05-fold change, respectively), suggesting a reduction in adiposity by stimulating fatty acid oxidation. CONCLUSION L. kefiri DH5 exerts anti-obesity effects by direct reduction of cholesterol in the lumen and upregulation of PPAR-α gene in adipose tissues.

Modulation of intestinal microbiota in mice by kefir administration

Kefir is a multi-species probiotic consisting of lactic and acetic acid bacteria and yeast. The modulatory effects of kefir on gut microfloral profiles of mice were investigated using group-specific quantitative real-time polymerase chain reaction. At the end of 3 week oral kefir administration period, the numbers of Firmicutes, Proteobacteria, and Enterobacteriaceae were significantly (p<0.05) decreased, and the numbers of Bacteroidetes, Lactobacillus, and Lactococcus, and total yeast were significantly (p<0.05) increased in the kefir group, compared with the control group. Kefir consumption significantly (p<0.05) suppressed proliferation of the opportunistic pathogen Enterobacteriaceae, compared with saline administration, with an increased proportion of Lactobacillus and Lactococcus to total bacteria, suggesting lactobacilli-Enterobacteriaceae antagonism. Kefir consumption significantly (p<0.05) reduced the Firmicutes/Bacteroidetes (F/B) ratio, compared with controls, suggesting oral administration of kefir could be beneficial in the treatment of obesity.

Antimicrobial and anti-biofilm activities of Lactobacillus kefiranofaciens DD2 against oral pathogens

ABSTRACT Background: Streptococcus mutans and Streptococcus sobrinus are major causative bacterial pathogens of dental caries. Objective: We investigated the applicability of three Lactobacillus strains (L. kefiranofaciens DD2, DD5, and DD6) isolated from kefir and three commercial Lactobacillus strains (L. plantarum ATCC 10012, L. johnsonii JCM 1022, and L. rhamnosus ATCC 7469) as potential oral probiotics with respect to their survivability in an experimental oral environment, antimicrobial activity, and anti-biofilm formation activity against S. mutans and S. sobrinus. Results: Strains DD2, ATCC 10012, ATCC 7469, and JCM 1022 had the best oral survivability, including aerotolerance and enzymatic resistance, and inhibited the growth and biofilm formation of S. mutans and S. sobrinus. In particular, DD2 suppressed all three classes of biofilm formation-associated genes: those associated with carbohydrate metabolism and those encoding regulatory biofilm and adhesion proteins. Conclusions: These results indicate that the novel kefir isolate L. kefiranofaciens DD2 effectively and directly inhibits S. mutans and S. sobrinus.

Heat resistance of Salmonella Enteritidis under prolonged exposure to acid-salt combined stress and subsequent refrigeration

Salmonella Enteritidis is a major foodborne pathogen exposed to various environmental and preservation stresses in the food chain. Because adaptive responses of viable bacterial cells in the presence of sublethal stress can induce cross-protection against different stresses, we investigated the heat resistance of Salmonella Enteritidis at 60 °C under prolonged exposure to acid-salt combined stress and subsequent refrigeration. Salmonella Enteritidis was grown in tryptic soy broth at four pH values (4.5, 5.4, 6.4, and 7.3) and four NaCl concentrations (0%, 1%, 2%, and 3%) at 37 °C for 24 h and then incubated at 4 °C for 0, 1, 4, or 7 days. For 0 and 1 day-refrigerated cultures, previous adaptation to single stresses (acid or salt stress) increased the heat resistance of Salmonella Enteritidis, resulting in increased D-values, whereas the combination of acid and salt stress reduced heat tolerance; acid stress played a more critical role in mediating this effect than salt concentration. To elucidate the related mechanisms, the expression levels of heat shock sigma factors (rpoH) and heat shock genes (dnaK and groEL) were analyzed and found to be associated with the heat resistance of Salmonella Enteritidis. The refrigeration period was negatively correlated (P < 0.01) with the D-value (r = -0.505) and with the transcript levels of rpoH (r = -0.654), dnaK (r = -0.652), and groEL (r = -0.645). Our findings demonstrated that acid-salt combined preservation techniques and subsequent refrigeration may prevent S. Enteritidis survival in heat-pasteurized food products caused by cross-protection of acid or salt adapted cells.

Modern perspectives on the health benefits of kefir in next generation sequencing era: Improvement of the host gut microbiota

ABSTRACT Kefir is a natural complex fermented milk product containing more than 50 species of probiotic bacteria and yeast, and has been demonstrated to have multiple properties conferring health benefits, including antiobesity, anti-hepatic steatosis, antioxidative, antiallergenic, antitumor, anti-inflammatory, cholesterol-lowering, constipation-alleviating, and antimicrobial properties. To better understand the underlying mechanisms of these benefits, we here review research on the effect of kefir (and kefir microorganisms) consumption to modulate the host gut microbiota. Owing to its excellent gastrointestinal resistance and colonization ability and wide ranges of microbial interaction, kefir has shown significant and wide-spectrum modulatory effects on the host gut microbiota. In particular, as a bacteria- and yeast-containing food, kefir can modulate both the gut microbiota and mycobiota. Since the association of this modulation with health benefit has only been addressed in a small number of recent studies thus far, further studies are needed to determine the precise mechanisms of the beneficial effects of kefir in relation to the modulation of the gut microbiota and mycobiota. Gaining this insight will surely help to take full advantage of this unique probiotic food.

Prevalence and toxin type of Clostridium perfringens in beef from four different types of meat markets in Seoul, Korea

Beef is the primary source of foodborne poisoning caused by Clostridium perfringens. We investigated the prevalence of C. perfringens in retail beef from four different types of meat markets in Seoul using a standard culture method and real-time PCR assay. From June to September 2015, 82 beef samples were collected from 6 department stores (n=12), 14 butcher shops (n=28), 16 traditional markets (n=32), and 5 supermarkets (n=10). The culture method and real-time PCR assay revealed that 4 (4.88%) and 10 (12.20%) samples were positive for C. perfringens, respectively. The beef purchased from the department store showed the highest prevalence (16.67%), followed by the traditional market (3.12%), butcher shop (3.57%), and supermarket (0%) (p>0.05). All isolates were type A and negative for the enterotoxin gene. In conclusion, the real-time PCR assay used in this study could be useful for rapid detection and screening of C. perfringens in beef.

Evaluation of cephamycins as supplements to selective agar for detecting Campylobacter spp. in chicken carcass rinses

Although cefoperazone is the most commonly used antibiotic in Campylobacter-selective media, the distribution of cefoperazone-resistant bacteria such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is increasing. Here we evaluated the potential of cephamycins for use as supplements to improve modified charcoal-cefoperazone-deoxycholate agar (mCCDA) by replacing cefoperazone with the same concentrations (32 mg/L) of cefotetan (modified charcoal-cefotetan-deoxycholate agar, mCCtDA) and cefoxitin (modified charcoal-cefoxitin-deoxycholate agar, mCCxDA). In chicken carcass rinse samples, the number of mCCDA plates detecting for Campylobacter (18/70, 26%) was significantly lower than that of mCCtDA (42/70, 60%) or mCCxDA plates (40/70, 57%). The number of mCCDA plates (70/70, 100%) that were contaminated with non-Campylobacter species was significantly higher than that of mCCtDA (20/70, 29%) or mCCxDA plates (21/70, 30%). The most common competing species identified using mCCDA was ESBL-producing E. coli, while Pseudomonas species frequently appeared on mCCtDA and mCCxDA.

Improvement of Enterobacteriaceae enrichment broth by supplementation with sodium citrate for detection of Cronobacter sakazakii using real-time PCR

Enterobacteriaceae enrichment (EE) broth, a selective enrichment medium for Cronobacter sakazakii detection, has been shown to contain polymerase chain reaction (PCR) inhibitors. Therefore, a modified enrichment broth was developed, wherein possible PCR inhibitors, brilliant green and bile salts, were substituted with sodium citrate. Investigations were conducted to determine whether realtime PCR detection of C. sakazakii in powdered infant formula and dried vegetables with this modified medium improved compared with conventional culture methods. The detection rate of the modified EE broth was significantly higher (p<0.05) than that of the conventional EE broth for both types of food. For dried pumpkin samples, real-time PCR after culturing in modified EE broth yielded significantly higher detection rates and selectivity than those achieved using conventional culture in Druggan-Forsythe-Iversen agar. These results show that real-time PCR after enrichment in the modified EE broth may be an effective screening method for detection of C. sakazakii.