Modulation of the intestinal microbiota of dogs by kefir as a functional dairy product.

Kefir is a traditional dairy product with multiple probiotic characteristics derived from its associated microorganisms, including more than 50 species of lactic acid bacteria and yeast. For centuries, many people have produced kefir for human consumption; its consumption and potential role as a probiotic supplement in companion animals have never been tested. The present study explored the potential application of kefir as a probiotic supplement for dogs. Kefir was orally administered to healthy adult dogs (n = 6) for 2 wk. On d 0 and 14 (before and after kefir consumption, respectively), gut microbiota was analyzed comprehensively using quantitative PCR and 16S rDNA amplicon-based community analysis using fresh fecal samples. The 16S rDNA amplicon-based community analysis showed that the relative abundance of the phylum Fusobacteria was significantly decreased after kefir consumption. Furthermore, the relative abundance of the families Prevotellaceae, Selenomonadaceae, and Sutterellaceae increased significantly, whereas that of the families Clostridiaceae, Fusobacteriaceae, and Ruminococcaceae decreased significantly. The quantitative PCR assay showed that kefir consumption significantly increased the population of lactic acid bacteria and the lactic acid bacteria:Enterobacteriaceae ratio and significantly decreased the Firmicutes:Bacteroidetes ratio. In summary, 2-wk kefir administration successfully modified the gut microbiota without causing any clinically evident adverse effects. Therefore, kefir could be further developed as a novel probiotic food supplement for dogs to improve the quality of life of dogs.

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.

Contamination Level of Hygiene Indicator and Prevalence of Foodborne Pathogens in Retail Beef in Parallel with Market Factor.

In this study, the contamination levels of hygienic indicators and foodborne pathogens in retail meat products were investigated in relation to the various market factors including processing temperature, processing area, and market type. Ground beef samples (n=80) were purchased from 40 meat markets and investigated for microbiological quality. Beefs processed below 20℃ had significantly lower numbers of total coliforms (TC) than these processed over 20℃ (2.01 vs. 2.79 log CFU/g; p<0.05). Interestingly, separation of processing area did not affect the contamination levels. Remarkably, the contamination levels of hygienic indicator differ among market types, indicating that not only processing condition but distribution structure that is directly related with storage period could affect the final microbiological loads of the meat products. In addition, the prevalences of Listeria monocytogenes (a psychrotroph), Enterococcus faecium, and Enterococcus faecalis were 7.5% (6/80), 10.0% (8/80), and 20.0% (16/80), respectively, which is irrelevant to market factors except meat products from wholesale markets where no L. monocytogenes were found among 30 samples. The results of this study indicate that the contamination level of hygiene indicator and foodborne pathogens in retail beef is more related with processing temperature and storage period than other environmental factors.

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.

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.

Improvement of Polymyxin-Egg Yolk-Mannitol-Bromothymol Blue Agar for the Enumeration and Isolation of Bacillus cereus in Various Foods.

A modified polymyxin-egg yolk-mannitol-bromothymol blue agar (mPEMBA) was developed by supplementing polymyxin-egg yolk-mannitol-bromothymol blue agar (PEMBA) with trimethoprim to improve the selectivity for and recoverability of Bacillus cereus from naturally and artificially contaminated food samples. The number of B. cereus in mPEMBA was significantly higher than in PEMBA, indicating better recoverability (P < 0.05) in red pepper powder (PEMBA 0.80 ± 0.22 log CFU/g versus mPEMBA 1.95 ± 0.17 log CFU/g) and soybean paste (PEMBA 2.19 ± 0.18 log CFU/g versus mPEMBA 3.09 ± 0.13 log CFU/g). In addition, mPEMBA provided better visual differentiation of B. cereus colonies than PEMBA, which is attributable to the reduced number of competing microflora. We conclude that the addition of trimethoprim to PEMBA could generate a synergistic effect to improve selectivity for B. cereus .

Modulation of gut microbiota and increase in fecal water content in mice induced by administration of Lactobacillus kefiranofaciens DN1.

Lactobacillus kefiranofaciens is the key probiotic bacterium in kefir. In this study, we investigated the effects of oral consumption of L. kefiranofaciens on the fecal quality and intestinal microbiota of mice. Four-week-old Balb/c mice were divided into two groups (n = 8 each) and administered 0.2 mL of saline (control group) or saline containing 2 × 108 cfu L. kefiranofaciens DN1 (LKF_DN1 group) for two weeks. At the end of the experiment, their fecal samples were collected and the fecal quality and microbiota were assessed. The LKF_DN1 group exhibited higher total fecal weight and fecal weight per stool sample than the control group (p < 0.05). Interestingly, the fecal water content was significantly higher in the fecal samples of the LKF_DN1 group than in those of the control group (p < 0.05). The numbers of total bacteria, Firmicutes, Bacteroidetes, Lactobacillus, and Prevotella were significantly higher in the LKF_DN1 group than in the control group (p < 0.05). In contrast, the number of opportunistic pathogens, including Proteobacteria and Enterobacteriaceae, and the percentage of genus Clostridium among the total bacteria were significantly reduced in the LKF_DN1 group (p < 0.05). Our data suggest that regular L. kefiranofaciens DN1 administration could alleviate constipation and improve gut microbiota.

A Single-Step Enrichment Medium for Nonchromogenic Isolation of Healthy and Cold-Injured Salmonella spp. from Fresh Vegetables.

Culture-based detection of nontyphoidal Salmonella spp. in foods requires at least four working days; therefore, new detection methods that shorten the test time are needed. In this study, we developed a novel single-step Salmonella enrichment broth, SSE-1, and compared its detection capability with that of commercial single-step ONE broth-Salmonella (OBS) medium and a conventional two-step enrichment method using buffered peptone water and Rappaport-Vassiliadis soy broth (BPW-RVS). Minimally processed lettuce samples were artificially inoculated with low levels of healthy and cold-injured Salmonella Enteritidis (100 or 101 colony-forming unit/25 g), incubated in OBS, BPW-RVS, and SSE-1 broths, and streaked on xylose lysine deoxycholate (XLD) agar. Salmonella recoverability was significantly higher in BPW-RVS (79.2%) and SSE-1 (83.3%) compared to OBS (39.3%) (p < 0.05). Our data suggest that the SSE-1 single-step enrichment broth could completely replace two-step enrichment with reduced enrichment time from 48 to 24 h, performing better than commercial single-step enrichment medium in the conventional nonchromogenic Salmonella detection, thus saving time, labor, and cost.

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.

Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 µg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.

Antimicrobial Activity of Kefir against Various Food Pathogens and Spoilage Bacteria.

Kefir is a unique fermented dairy product produced by a mixture of lactic acid bacteria, acetic acid bacteria, and yeast. Here, we compared the antimicrobial spectra of four types of kefirs (A, L, M, and S) fermented for 24, 36, 48, or 72 h against eight food-borne pathogens. Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa, and Cronobacter sakazakii were used as test strains, and antibacterial activity was investigated by the spot on lawn method. The spectra, potencies, and onsets of activity varied according to the type of kefir and the fermentation time. The broadest and strongest antimicrobial spectrum was obtained after at least 36-48 h of fermentation for all kefirs, although the traditional fermentation method of kefir is for 18-24 h at 25℃. For kefir A, B. cereus, E. coli, S. Enteritidis, P. aeruginosa, and C. sakazakii were inhibited, while B. cereus, S. aureus, E. coli, S. Enteritidis, P. aeruginosa, and C. sakazakii were inhibited to different extents by kefirs L, M, and S. Remarkably, S. aureus, S. Enteritidis, and C. sakazakii were only inhibited by kefirs L, M, and S, and L. monocytogenes by kefir M after fermentation for specific times, suggesting that the antimicrobial activity is attributable not only to a low pH but also to antimicrobial substances secreted during the fermentation.

Growth Inhibition of Cronobacter sakazakii in Experimentally Contaminated Powdered Infant Formula by Kefir Supernatant.

Kefir is a type of fermented milk containing lactic and acetic acid bacteria and yeast. In this study, we evaluated the antimicrobial activity of kefir supernatant against Cronobacter sakazakii in powdered infant formula (PIF). In a spot-on-lawn test, the growth of 20 C. sakazakii strains, including 10 clinical and 10 food isolates, was completely inhibited in the presence of kefir supernatant. Significant differences in the diameters of inhibition zones were observed upon treatment with kefir compared with the results for Lactobacillus kefiri and Candida kefyr culture supernatants or solutions of lactic and acetic acid and ethyl alcohol in the agar well diffusion test (P < 0.05). The addition of 100 µl of kefir supernatant to 1 ml of nutrient broth completely inhibited the growth of C. sakazakii, as evaluated by spectrophotometry. The antimicrobial activity of kefir supernatant in experimentally contaminated PIF was also tested; we found no viable C. sakazakii cells remaining in PIF rehydrated with 30% kefir supernatant solution for 1 h, demonstrating that the antimicrobial activity of kefir supernatant against C. sakazakii could be applied in real food samples.