Effect of postbiotics derived from kefir lactic acid bacteria-mediated bioconversion of citrus pomace extract and whey on high-fat diet-induced obesity and gut dysbiosis.

The objective of this study was to develop a highly bioactive postbiotic for weight management by bioconversion of whey (WHE) and polyphenol-rich citrus pomace extract (CPX) using kefir lactic acid bacteria (LAB). WHE and CPX bioconverted by kefir LAB (CPB) were fed to C57BL/6J mice on high-fat diets for five weeks and compared with oral administrations of saline (CON), WHE, CPX, and kefir LAB. Hesperetin, a potential therapeutic agent for obesity, was increased in the CPB after bioconversion from an inactive precursor. Compared with the CON group, the CPB group showed significantly reduced body weight gain, adipose tissue weight/body weight ratio, hypertriglyceridemia, and adipocyte diameter along with increased gene expression related to energy expenditure in adipose tissue (p < 0.05). Interestingly, the abundance of gut microbiota related to butyrate production was significantly altered in the CPB group compared with the CON group. There was a significant correlation between obesogenic biomarkers and the abundance of butyrate-producing and obesogenic gut microbiota. In conclusion, kefir LAB-derived bioconversion of WHE and CPX may be effective in combating obesity and obesity-related diseases.

Effect of folic acid supplementation on proliferation and apoptosis in bovine mammary epithelial (MAC-T) cells.

Folic acid (FA) is known to be an important micronutrient in humans; however, information regarding the effect of FA supplementation on bovine mammary epithelial (BME) cells is insufficient. FA supplementation is reported to increase milk production in dairy cows, but the underlying molecular mechanisms are unknown. This study examined the effects of FA supplementation on the proliferation and apoptosis of a BME cell line (MAC-T). MAC-T cells were treated with various concentrations (deficient in FA (DF) < 0.01 ng/mL; low-level FA (LF) 3.1 ng/mL; normal FA (NF) 15.4 ng/mL; and high-level FA (HF) 30.8 ng/mL) based on serum folate (10-20 ng/mL) in milking cows. HF treatment significantly increased the proliferation of MAC-T cells. Cellular apoptosis was observed mainly in the DF group. The number of apoptotic cells in DF media was significantly higher than that in NF media. The bcl-2/bax mRNA expression ratio was significantly increased in the HF group compared to that in the DF group. FA supplementation significantly increased the ratio of Bcl-2/Bax protein levels in MAC-T cells. FA supplementation increases proliferation and decreases apoptosis in these cells. This study might provide information regarding the molecular mechanism through which FA supplementation is associated with increased milk yield.

Evaluation of Ceftazidime as an Antibiotic Supplement in Mannitol-Yolk-Polymyxin B Agar Used for Enumeration of Bacillus cereus in Ready-to-Eat Vegetables.

ABSTRACT: The Bacillus cereus group of bacteria, which causes foodborne diseases, can be detected by culture on selective media. However, the presence of competing flora is the most common factor preventing the accurate enumeration of B. cereus on selective agars. In this study, we improved the selectivity of mannitol-yolk-polymyxin B agar (MYPA) and its modified version containing trimethoprim (mMYPA) developed in our previous study by supplementation with ceftazidime (16 µg/mL). Ceftazidime-supplemented MYPA (C-MYPA16) and mMYPA (C-mMYPA16) were evaluated for bacteria recovery and selectivity with three types of ready-to-eat vegetables. Four B. cereus and one Bacillus thuringiensis strains were mixed and artificially inoculated into vegetable salad, radish sprouts, and sprout mix and then recovered on MYPA, mMYPA, C-MYPA16, and C-mMYPA16. In all tested vegetables, mMYPA, C-MYPA16, and C-mMYPA16 culture resulted in similar recovery of B. cereus and B. thuringiensis (P > 0.05), whereas radish sprout and sprout mix colonies grown on MYPA were undistinguishable. C-mMYPA16 was the most selective medium because it eliminated most of the competing flora, especially that in sprouts, without negatively affecting the recovery of B. cereus and B. thuringiensis. Our results indicate that supplementation of mMYPA with ceftazidime may improve the selectivity of this medium for B. cereus and B. thuringiensis in food testing.

Synergistic Effects of Heat-Killed Kefir Paraprobiotics and Flavonoid-Rich Prebiotics on Western Diet-Induced Obesity.

The synergistic anti-obesity effect of paraprobiotic heat-killed lactic acid bacteria (HLAB) and prebiotics has not been studied. To determine the anti-obesity properties of prebiotic polyphenol-rich wine grape seed flour (GSF) and paraprobiotic HLAB, C57BL/6J mice were administered a high-fat and high-fructose diet (HFFrD) with 5% microcrystalline cellulose (CON), HFFrD supplemented with 2.5% GSF, HFFrD with orally administered HLAB, or HFFrD with a combination of GSF and orally administered HLAB (GSF+HLAB) for 8 weeks. Compared with the CON group, the GSF and HLAB groups both showed significant reductions in HFFrD-induced body weight gain and adipose tissue weights (p < 0.05). Interestingly, combined supplementation with GSF and HLAB revealed statistically significant synergistic effects on body weight gain, visceral adiposity, and plasma triacylglycerol concentrations (p < 0.05). The synergistic action was significantly related to a decreased adipocyte gene expression in fatty acid synthesis and inflammation metabolism. In conclusion, the combination of prebiotic GSF and paraprobiotic kefir HLAB is potentially useful, as natural food ingredients, in the prevention of obesity and obesity-related diseases, especially for immunocompromised individuals.

Antiobesity Effect of Prebiotic Polyphenol-Rich Grape Seed Flour Supplemented with Probiotic Kefir-Derived Lactic Acid Bacteria.

The interaction between prebiotics and probiotics may exert synergistic health benefits. This study investigated the combined effects of polyphenol-rich wine grape seed flour (GSF), a prebiotic, and lactic acid bacteria (LAB) derived from kefir, a probiotic, on obesity-related metabolic disease in high-fat diet (HFD) induced obese (DIO) mice. DIO mice were fed with HFD with 6% microcrystalline cellulose (CON) or HFD supplemented with GSF (5% or 10% GSF), HFD with LAB orally administrated (LAB), or HFD with a combination of GSF and LAB orally administrated (GSF+LAB) for 9 weeks. The vehicle, saline, was also orally administered to the CON and GSF groups. In comparison to CON, all GSF and LAB groups showed a reduction ( P < 0.05) in HF-induced weight gain, liver and adipose tissue weights, plasma lipid concentrations, insulin resistance, and glucose intolerance. The combination of 10% GSF and LAB showed synergistic effects ( P < 0.05) on body weight gain, plasma insulin and total cholesterol concentrations, and cecum propionate contents. Plasma zonulin and cecum propionate concentrations and intestinal FXR gene expression were ( P < 0.05) correlated with body weight gain. A pathway analysis of microarray data of adipose tissue showed that the combination of GSF and LAB affected genes involved in metabolic and immunological diseases, including inflammasome complex assembly ( P < 0.05). In conclusion, a combination of GSF and LAB inhibited HF-induced obesity and inflammation via alterations in intestinal permeability and adipocyte gene expression.

Presence of Stenotrophomonas maltophilia exhibiting high genetic similarity to clinical isolates in final effluents of pig farm wastewater treatment plants.

Although the prevalence of community-acquired Stenotrophomonas maltophilia infections is sharply increasing, the sources and likely transmission routes of this bacterium are poorly understood. We studied the significance of the presence of S. maltophilia in final effluents and receiving rivers of pig farm wastewater treatment plants (WWTPs). The loads and antibiotic resistance profiles of S. maltophilia in final effluents were assessed. Antibiotic resistance determinants and biofilm formation genes were detected by PCR, and genetic similarity to clinical isolates was investigated using multilocus sequence typing (MLST). S. maltophilia was recovered from final effluents at two of three farms and one corresponding receiving river. Tests of resistance to antibiotics recommended for S. maltophilia infection revealed that for each agent, at least one isolate was classified as resistant or intermediate, with the exception of minocycline. Furthermore, multidrug resistant S. maltophilia susceptible to antibiotics of only two categories was isolated and found to carry the sul2 gene, conferring trimethoprim/sulfamethoxazole resistance. All isolates carried spgM, encoding a major factor in biofilm formation. MLST revealed that isolates of the same sequence type (ST; ST189) were present in both effluent and receiving river samples, and phylogenetic analysis showed that all of the STs identified in this study clustered with clinical isolates. Moreover, one isolate (ST192) recovered in this investigation demonstrated 99.61% sequence identity with a clinical isolate (ST98) associated with a fatal infection in South Korea. Thus, the pathogenicity of the isolates reported here is likely similar to that of those from clinical environments, and WWTPs may play a role as a source of S. maltophilia from which this bacterium spreads to human communities. To the best of our knowledge, this represents the first report of S. maltophilia in pig farm WWTPs. Our results indicate that nationwide epidemiological investigations are needed to examine the possible link between WWTP-derived S. maltophilia and hospital- and community-acquired infections.

Chardonnay Grape Seed Flour Ameliorates Hepatic Steatosis and Insulin Resistance via Altered Hepatic Gene Expression for Oxidative Stress, Inflammation, and Lipid and Ceramide Synthesis in Diet-Induced Obese Mice.

To identify differentially expressed hepatic genes contributing to the improvement of high-fat (HF) diet-induced hepatic steatosis and insulin resistance following supplementation of partially defatted flavonoid-rich Chardonnay grape seed flour (ChrSd), diet-induced obese (DIO) mice were fed HF diets containing either ChrSd or microcrystalline cellulose (MCC, control) for 5 weeks. The 2-h insulin area under the curve was significantly lowered by ChrSd, indicating that ChrSd improved insulin sensitivity. ChrSd intake also significantly reduced body weight gain, liver and adipose tissue weight, hepatic lipid content, and plasma low-density lipoprotein (LDL)-cholesterol, despite a significant increase in food intake. Exon microarray analysis of hepatic gene expression revealed down-regulation of genes related to triglyceride and ceramide synthesis, immune response, oxidative stress, and inflammation and upregulation of genes related to fatty acid oxidation, cholesterol, and bile acid synthesis. In conclusion, the effects of ChrSd supplementation in a HF diet on weight gain, insulin resistance, and progression of hepatic steatosis in DIO mice were associated with modulation of hepatic genes related to oxidative stress, inflammation, ceramide synthesis, and lipid and cholesterol metabolism.

Evaluation of Tazobactam-Supplemented, Modified Charcoal-Cefoperazone-Deoxycholate Agar for Qualitative Detection of Campylobacter from Chicken Carcass Rinse.

Overgrowth of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) is the most common confounding factor for the isolation of Campylobacter from poultry samples. mCCDA modified by supplementation with tazobactam, an ESBL inhibitor, was evaluated for Campylobacter isolation from chicken carcass rinse with regard to isolation rate and selectivity. In total, 120 whole chicken carcasses purchased from retail stores were rinsed with buffered peptone water enriched with 2× blood-free Bolton broth at 42°C for 48 h and then inoculated onto mCCDA with and without tazobactam supplementation (mCCDA or T-mCCDA) at 42°C for 48 h under microaerobic conditions. Suspect colonies were subcultured and confirmed by colony PCR. Plates with tazobactam exhibited a higher Campylobacter isolation rate (56.7% vs. 30.8%, p < 0.05) and selectivity (0.8 vs. 83.3% plates contaminated with non-Campylobacter, p < 0.05) than mCCDA. Thus, tazobactam-supplemented mCCDA would be a useful option for qualitative detection of Campylobacter in chicken carcass rinse.

Modification of Karmali agar by supplementation with potassium clavulanate for the isolation of Campylobacter from chicken carcass rinses.

The detection ability and selectivity of Karmali agar was improved by supplementation of an extended-spectrum ß-lactamase inhibitor, potassium clavulanate. The optimum concentration of potassium clavulanate (0.5 µg/ml) in Karmali agar was determined by inoculation of 50 Campylobacter and 30 extended-spectrum ß-lactamase-producing E. coli strains onto normal and modified Karmali agar containing various concentrations of the agent. Eighty retail carcasses were rinsed with 400 ml of buffered peptone water. The rinse samples were enriched in 2 × blood-free Bolton enrichment broth at 42°C for 48 h and then were streaked onto normal and modified Karmali agar containing 0.5 µg/ml potassium clavulanate. The suspicious colonies were subcultured on Columbia blood agar and confirmed by colony PCR. In chicken carcass samples, the modified Karmali agar showed a significantly greater isolation rate than normal Karmali agar (42.5 versus 21.3%; P < 0.05). Furthermore, the selectivity of the modified Karmali agar was also significantly higher (P < 0.05) than that of the normal Karmali agar, as seen by comparison of the number of contaminated agar plates (83.8 versus 97.5%) and the growth index (1.67 versus 2.91) of the non-Campylobacter colonies.

Evaluation of potassium-clavulanate-supplemented modified charcoal-cefoperazone-deoxycholate agar for enumeration of Campylobacter in chicken carcass rinse.

Potassium-clavulanate-supplemented modified charcoal-cefoperazone-deoxycholate agar (C-mCCDA) that was described in our previous study was compared with original mCCDA for the enumeration of Campylobacter in pure culture and chicken carcass rinse. The quantitative detection of viable Campylobacter cells from a pure culture, plated on C-mCCDA, is statistically similar (P > 0.05) to mCCDA. In total, 120 chickens were rinsed using 400 mL buffered peptone water. The rinses were inoculated onto C-mCCDA and mCCDA followed by incubation at 42 °C for 48 h. There was no statistical difference between C-mCCDA (45 of 120 plates; mean count, 145.5 CFU/mL) and normal mCCDA (46 of 120 plates; mean count, 160.8 CFU/mL) in the isolation rate and recovery of Campylobacter (P > 0.05) from chicken carcass rinse. The Pearson correlation coefficient value for the number of Campylobacter cells recovered in the 2 media was 0.942. However, the selectivity was much better on C-mCCDA than on mCCDA plates (P < 0.05). Significantly fewer C-mCCDA plates (33 out of 120 plates; mean count, 1.9 CFU/mL) were contaminated with non-Campylobacter cells than the normal mCCDA plates (67 out of 120 plates; mean count, 27.1 CFU/mL). The C-mCCDA may provide improved results for enumeration of Campylobacter in chicken meat alternative to mCCDA with its increased selectivity the modified agar possess.

Improvement of modified charcoal-cefoperazone-deoxycholate agar by addition of potassium clavulanate for detecting Campylobacter spp. in chicken carcass rinse.

The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in raw poultry is one of the most common factors that interfere with the isolation of Campylobacter by cefoperazone-based selective agar. The performance of modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by addition of an ESBL inhibitor, potassium clavulanate (0.5 mg/L). The ability of the supplemented medium (C-mCCDA) to detect Campylobacter species from chicken carcass rinse was compared with that of normal mCCDA. The isolation rate using C-mCCDA was significantly (p<0.05) higher compared with that using mCCDA (C-mCCDA, 67 out of 120; mCCDA, 38 out of 120). Furthermore, the selectivity of the C-mCCDA as assessed by comparing the number of contaminated plates (C-mCCDA, 44 out of 120; mCCDA, 110 out of 120) and growth index (C-mCCDA, 1.76; mCCDA, 2.79) of competing flora was also better (p<0.05) than that of mCCDA.

Improvement of modified charcoal-cefoperazone-deoxycholate agar by supplementation with a high concentration of polymyxin B for detection of Campylobacter jejuni and C. coli in chicken carcass rinses.

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate Campylobacter jejuni and C. coli from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (P < 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.