Survival of Inoculated Campylobacter jejuni and Escherichia coli O157:H7 on Kale During Refrigerated Storage.

Campylobacter and pathogenic Escherichia coliillnesses have been attributed to the consumption of fresh produce. The leafy green, kale, is increasingly consumed raw. In comparison to other leafy greens, kale has a longer shelf-life. Due to the extended shelf-life of kale, it is warranted to examine the survival of pathogenic Campylobacter jejuni and E. coli O157:H7 inoculated on the surface of kale stored in a controlled environment at 4 ± 1.4°C, and average humidity of 95 ± 1.9% over a 23-day period. At predetermined time points (days 0, 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21), inoculated kale was destructively sampled and the surviving bacteria determined by serial dilution and plating onto Tryptic soy agar, Charcoal cefoperozone deoxycholate agar, and Eosin methylene blue for total aerobic bacteria, C. jejuni, and E. coli O157:H7, respectively. Enrichment and PCR were used for detection when pathogens were not detected using serial dilution and plating. Aerobic heterotrophic bacteria increased over the 23-day period, in contrast, significant declines in the inoculated pathogens were observed. Inoculated E. coli O157:H7 survived longer on kale (up to 19 d); in comparison, C. jejuni was undetectable by day 13 using enrichment and PCR or plating. In conclusion, C. jejuni and E. coli O157:H7 declined on fresh kale over time when held at refrigerated temperatures but were still detected during the majority of the time when the kale would likely still be considered edible by consumers.

Prebiotic Inulin Supplementation and Peripheral Insulin Sensitivity in adults at Elevated Risk for Type 2 Diabetes: A Pilot Randomized Controlled Trial.

Prediabetes affects 84.1 million adults, and many will progress to type 2 diabetes (T2D). The objective of this proof-of-concept trial was to determine the efficacy of inulin supplementation to improve glucose metabolism and reduce T2D risk. Adults (n = 24; BMI: 31.3 ± 2.9 kg/m2; age: 54.4 ± 8.3 years) at risk for T2D were enrolled in this controlled feeding trial and consumed either inulin (10 g/day) or placebo (maltodextrin, 10 g/day) for six weeks. Assessments included peripheral insulin sensitivity, fasting glucose, and insulin, HOMA-IR, in vivo skeletal muscle substrate preference, Bifidobacteria copy number, intestinal permeability, and endotoxin concentrations. Participant retention was 92%. There were no baseline group differences except for fasting insulin (p = 0.003). The magnitude of reduction in fasting insulin concentrations with inulin (p = 0.003, inulin = Δ-2.9, placebo = Δ2.3) was attenuated after adjustment for baseline concentrations (p = 0.04). After adjusting for baseline values, reduction in HOMA-IR with inulin (inulin = Δ-0.40, placebo=Δ0.27; p = 0.004) remained significant. Bifidobacteria 16s increased (p = 0.04; inulin = Δ3.1e9, placebo = Δ-8.9e8) with inulin supplementation. Despite increases in gut Bifidobacteria, inulin supplementation did not improve peripheral insulin sensitivity. These findings question the need for larger investigations of inulin and insulin sensitivity in this population.

Manure-Based Amendments Influence Surface-Associated Bacteria and Markers of Antibiotic Resistance on Radishes Grown in Soils with Different Textures.

A controlled greenhouse study was performed to determine the effect of manure or compost amendments, derived during or in the absence of antibiotic treatment of beef and dairy cattle, on radish taproot-associated microbiota and indicators of antibiotic resistance when grown in different soil textures. Bacterial beta diversity, determined by 16S rRNA gene amplicon sequencing, bifurcated according to soil texture (P < 0.001, R = 0.501). There was a striking cross-effect in which raw manure from antibiotic-treated and antibiotic-free beef and dairy cattle added to loamy sand (LS) elevated relative (16S rRNA gene-normalized) (by 0.9 to 1.9 log10) and absolute (per-radish) (by 1.1 to 3.0 log10) abundances of intI1 (an integrase gene and indicator of mobile multiantibiotic resistance) on radishes at harvest compared to chemical fertilizer-only control conditions (P < 0.001). Radishes tended to carry fewer copies of intI1 and sul1 when grown in silty clay loam than LS. Composting reduced relative abundance of intI1 on LS-grown radishes (0.6 to 2.4 log10 decrease versus corresponding raw manure; P < 0.001). Effects of antibiotic use were rarely discernible. Heterotrophic plate count bacteria capable of growth on media containing tetracycline, vancomycin, sulfamethazine, or erythromycin tended to increase on radishes grown in turned composted antibiotic-treated dairy or beef control (no antibiotics) manures relative to the corresponding raw manure in LS (0.8- to 2.3-log10 increase; P < 0.001), suggesting that composting sometimes enriches cultivable bacteria with phenotypic resistance. This study demonstrates that combined effects of soil texture and manure-based amendments influence the microbiota of radish surfaces and markers of antibiotic resistance, illuminating future research directions for reducing agricultural sources of antibiotic resistance.IMPORTANCE In working toward a comprehensive strategy to combat the spread of antibiotic resistance, potential farm-to-fork routes of dissemination are gaining attention. The effects of preharvest factors on the microbiota and corresponding antibiotic resistance indicators on the surfaces of produce commonly eaten raw is of special interest. Here, we conducted a controlled greenhouse study, using radishes as a root vegetable grown in direct contact with soil, and compared the effects of manure-based soil amendments, antibiotic use in the cattle from which the manure was sourced, composting of the manure, and soil texture, with chemical fertilizer only as a control. We noted significant effects of amendment type and soil texture on the composition of the microbiota and genes used as indicators of antibiotic resistance on radish surfaces. The findings take a step toward identifying agricultural practices that aid in reducing carriage of antibiotic resistance and corresponding risks to consumers.

Use of dietary phytochemicals for inhibition of trimethylamine N-oxide formation.

Trimethylamine-N-oxide (TMAO) has been reported as a risk factor for atherosclerosis development, as well as for other cardiovascular disease (CVD) pathologies. The objective of this review is to provide a useful summary on the use of phytochemicals as TMAO-reducing agents. This review discusses the main mechanisms by which TMAO promotes CVD, including the modulation of lipid and bile acid metabolism, and the promotion of endothelial dysfunction and oxidative stress. Current knowledge on the available strategies to reduce TMAO formation are discussed, highlighting the effect and potential of phytochemicals. Overall, phytochemicals (i.e., phenolic compounds or glucosinolates) reduce TMAO formation by modulating gut microbiota composition and/or function, inhibiting host's capacity to metabolize TMA to TMAO, or a combination of both. Perspectives for design of future studies involving phytochemicals as TMAO-reducing agents are discussed. Overall, the information provided by this review outlines the current state of the art of the role of phytochemicals as TMAO reducing agents, providing valuable insight to further advance in this field of study.

Inactivation of Salmonella enterica and Enterococcus faecium NRRL B2354 on cumin seeds using gaseous ethylene oxide.

The objectives of this study were to investigate the effects of processing parameters (relative humidity (RH), temperature, and exposure time) on the ethylene oxide (EtO) microbial inactivation of Salmonella spp. and to evaluate Enterococcus faecium NRRL B2354 as a suitable surrogate for Salmonella inactivation on cumin seeds. Five grams of cumin seeds inoculated with either Salmonella or E. faecium were treated with EtO at different temperatures (46, 53, and 60 °C) and RH (30, 40, and 50%) levels for different exposure time to investigate the effects of process parameters on the microbial inactivation. The Weibull model fit the survival data of both bacteria with a shape parameter p < 1, which showed a tailing effect with concave shape indicating that the sensitive cells were inactivated first, and the sturdy ones survived at low RH treatment conditions. In general, the log reductions of both bacteria on cumin seeds increased with the increasing RH and temperature for EtO treatment. RH is a critical factor for successful EtO inactivation treatment. RH must be higher than 40% to implement a successful and efficient EtO decontamination of cumin seeds. E. faecium consistently showed lower log reductions than those of Salmonella under all EtO treatment conditions investigated in this study, demonstrating that E. faecium is a suitable surrogate for Salmonella. Twenty minutes of EtO treatment at 50% RH achieved ~5 log reductions of both bacteria at all three temperatures. A response surface model was developed to predict the log reductions of both bacteria under different treatment conditions and the contour plots representing log reductions were created. Inactivation is positively correlated to temperature and RH. Therefore, a higher temperature is required to achieve the desired log reduction at lower RH and vice versa. The developed response surface model is a valuable tool for the spice industry in identifying the possible combinations of EtO process parameters (temperature, RH, and exposure time) required to achieve a desired microbial reduction of Salmonella for ensuring microbial food safety of spices.

Thermal Resistance of Single Strains of Shiga Toxin-Producing Escherichia coli O121:H19 and O157:H7 Based on Culture Preparation Method and Osmolyte-Reduced Water Activity.

ABSTRACT: Bacterial exposure to stress, such as reduced water activity (aw), can increase thermal resistance. Pathogen thermal resistance studies on low-aw foods use a variety of methods to inoculate food, as well as strategies to reduce aw, which can influence observations. This study investigated effects of culture preparation method and osmolyte-induced aw on thermal resistance of two Shiga toxin-producing Escherichia coli (STEC) strains (O121:H19 and O157:H7) challenged with isothermal conditions, determining D- and z-values for each isolate (56, 59, and 62°C). Tryptic soy broth (TSB) and agar (lawn cultures) were compared. D-values of broth cultures were significantly and consistently larger than those of lawn cultures, and O121 was significantly more resistant than O157, but only at 56°C (P < 0.05). To compare potential effects of aw on STEC thermal resistance, cells were suspended in osmolyte solutions with varying aw: high (TSB, aw 0.99), intermediate (61% glycerol or 26% NaCl, aw 0.75), and low (82% glycerol, aw 0.5). In most instances, STEC strains in high-aw broth exhibited greater heat resistance compared to reduced-aw solutions, with the exception of the glycerol intermediate-aw solution (aw 0.75). Magnitudes varied with strain and temperature. The z-values of lawn cultures were significantly lower than those of broth cultures (P < 0.05), but there were few differences between high-aw and reduced-aw samples. There were no significant differences of z-values based on strain type. These results highlight that thermal resistance can be affected by culture preparation and that osmolyte-induced changes to aw influence thermal inactivation of STEC by varying magnitudes. These results emphasize the challenges of extrapolating results from laboratory inactivation kinetic experiments to determine the inactivation of low-aw foods, especially those considered dry in nature.

Thermal inactivation of Salmonella, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and a surrogate (Pediococcus acidilactici) on raisins, apricot halves, and macadamia nuts using vacuum-steam pasteurization.

Salmonella, Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes have been isolated from low water activity foods (LWAF), where they may survive for extended periods. The ready-to-eat nature of many LWAF, such as dried fruits and nuts, warrants effective post-harvest thermal treatment for the reduction of pathogens such as low-temperature, saturated steam, also known as vacuum-assisted steam pasteurization. The objective of this study was to determine reductions of Salmonella, STEC, L. monocytogenes, and a possible surrogate (Pediococcus acidilactici) on dried apricot halves, whole macadamia nuts, and raisins after treatment with vacuum-assisted steam at three temperatures (62 °C, 72 °C, or 82 °C) and multiple time intervals. Bacterial inactivation was variable between commodities, with higher temperatures and longer times necessary to achieve comparable reductions of pathogens on apricot halves and macadamia nuts compared to raisins. Reductions of the tested pathogens were comparable; therefore, one species was not more resistant than the others. Pathogens were reduced by 5-log CFU/g on apricot halves after 20 min at 72 °C and after 5 min at 82 °C. Longer treatment times were necessary to achieve reductions of each pathogen on macadamia nuts. Pathogens were reduced by nearly 5 log CFU/g on macadamia nuts after 38 min at 72 °C (4.6-6.5 log CFU/g) and after 12 min at 82 °C (4.9-5.7 log CFU/g). Reductions of pathogens on raisins were achieved at lower temperatures than necessary for the other foods. A 5-log reduction for each of the pathogens (CFU/g) on raisins occurred after 20 min at 62 °C and after 5 min at 72 °C. Overall, the reductions of the pathogens exceeded those of P. acidilactici on both the dried fruits and macadamia nuts. Statistically significant differences, indicating greater confidence as a conservative surrogate, were observed at lower treatment temperatures. Inactivation kinetics were modeled for each pathogen on each food type and temperature. Bacterial survival was best described by the Weibull model for raisins and macadamia nuts, while the Gompertz model best described reductions on apricot halves according to Akaike information criterion (AIC) and root-mean-square error (RMSE) evaluations. Water activity and moisture content were increased due to the treatments, which could be addressed through implementation of drying steps. Thermal inactivation kinetic models and 5-log reduction parameters can help food processors design and evaluate similar vacuum-assisted steam interventions to comply with FSMA regulations and preventive control plans. However, results or model predictions should not be extrapolated to assume the safety of other types of foods.

Compositional variability of food wastes and its effects on acetone-butanol-ethanol fermentation.

Converting food waste into butanol via acetone, butanol, and ethanol (ABE) fermentation provides the potential to recover energy and value-added chemicals from food waste. However, the high variability of food waste compositions has hindered the consistency and predictability of butanol production, impeding the development of a robust industrial fermentation process. This study characterized the compositional variation of collected food wastes and determined correlations between food waste compositional attributes and butanol yields for a better prediction of food waste fermentation with Clostridium. The total sugar, starch, fiber, crude protein, fat and ash contents (on dry basis) in the food waste samples were in a range of 0.5-53.5%, 0-25.2%, 0.6-26.9%, 5.5-21.5%, 0.1-37.9%, and 1.4-13.7%, respectively. The high variability of food waste composition resulted in a wide range (3.5-11.5 g/L) of butanol concentrations with an average of 8.2 g/L. Pearson's correlation analysis revealed that the butanol concentrations were strongly and positively correlated with equivalent glucose and starch contents in food waste, strongly and negatively correlated with fiber content, and weakly correlated with total sugar, protein, fat, and ash contents. The regression models constructed based on equivalent glucose and fiber contents reasonably predicted the butanol concentration, with the R2 of 0.80. Our study investigated the variability of food waste composition and, for the first time, unveiled relationships between food waste compositional attributes and fermentation yields, contributing to a greater understanding of food waste fermentation, which, in turn, assists in developing new strategies for increased consistency and predictability of food waste fermentation.

Effects of Dairy Manure-Based Amendments and Soil Texture on Lettuce- and Radish-Associated Microbiota and Resistomes.

Dairy cattle are routinely treated with antibiotics, and the resulting manure or composted manure is commonly used as a soil amendment for crop production, raising questions regarding the potential for antibiotic resistance to propagate from "farm to fork." The objective of this study was to compare the microbiota and "resistomes" (i.e., carriage of antibiotic resistance genes [ARGs]) associated with lettuce leaf and radish taproot surfaces grown in different soils amended with dairy manure, compost, or chemical fertilizer only (control). Manure was collected from antibiotic-free dairy cattle (DC) or antibiotic-treated dairy cattle (DA), with a portion composted for parallel comparison. Amendments were applied to loamy sand or silty clay loam, and lettuce and radishes were cultivated to maturity in a greenhouse. Metagenomes were profiled via shotgun Illumina sequencing. Radishes carried a distinct ARG composition compared to that of lettuce, with greater relative abundance of total ARGs. Taxonomic species richness was also greater for radishes by 1.5-fold. The resistomes of lettuce grown with DC compost were distinct from those grown with DA compost, DC manure, or fertilizer only. Further, compost applied to loamy sand resulted in twofold-greater relative abundance of total ARGs on lettuce than when applied to silty clay loam. The resistomes of radishes grown with biological amendments were distinct from the corresponding fertilizer controls, but effects of composting or antibiotic use were not measureable. Cultivation in loamy sand resulted in higher species richness for both lettuce and radishes than when grown in silty clay loam by 2.2-fold and 1.2-fold, respectively, when amended with compost.IMPORTANCE A controlled, integrated, and replicated greenhouse study, along with comprehensive metagenomic analysis, revealed that multiple preharvest factors, including antibiotic use during manure collection, composting, biological soil amendment, and soil type, influence vegetable-borne resistomes. Here, radishes, a root vegetable, carried a greater load of ARGs and species richness than lettuce, a leafy vegetable. However, the lettuce resistome was more noticeably influenced by upstream antibiotic use and composting. Network analysis indicated that cooccurring ARGs and mobile genetic elements were almost exclusively associated with conditions receiving raw manure amendments, suggesting that composting could alleviate the mobility of manure-derived resistance traits. Effects of preharvest factors on associated microbiota and resistomes of vegetables eaten raw are worthy of further examination in terms of potential influence on human microbiomes and spread of antibiotic resistance. This research takes a step toward identifying on-farm management practices that can help mitigate the spread of agricultural sources of antibiotic resistance.

Gamma Irradiation Influences the Survival and Regrowth of Antibiotic-Resistant Bacteria and Antibiotic-Resistance Genes on Romaine Lettuce.

Contamination of romaine lettuce with human pathogens, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) occurs during production. Post-harvest interventions are emplaced to mitigate pathogens, but could also mitigate ARB and ARGs on vegetables. The objective of this research was to determine changes to lettuce phyllosphere microbiota, inoculated ARB, and the resistome (profile of ARGs) following washing with a sanitizer, gamma irradiation, and cold storage. To simulate potential sources of pre-harvest contamination, romaine lettuce leaves were inoculated with compost slurry containing antibiotic-resistant strains of pathogenic (Escherichia coli O157:H7) and representative of spoilage bacteria (Pseudomonas aeruginosa). Various combinations of washing with sodium hypochlorite (50 ppm free chlorine), packaging under modified atmosphere (98% nitrogen), irradiating (1.0 kGy) and storing at 4°C for 1 day versus 14 days were compared. Effects of post-harvest treatments on the resistome were profiled by shotgun metagenomic sequencing. Bacterial 16S rRNA gene amplicon sequencing was performed to determine changes to the phyllosphere microbiota. Survival and regrowth of inoculated ARB were evaluated by enumeration on selective media. Washing lettuce in water containing sanitizer was associated with reduced abundance of ARG classes that confer resistance to glycopeptides, ß-lactams, phenicols, and sulfonamides (Wilcoxon, p < 0.05). Washing followed by irradiation resulted in a different resistome chiefly due to reductions in multidrug, triclosan, polymyxin, ß-lactam, and quinolone ARG classes (Wilcoxon, p < 0.05). Irradiation followed by storage at 4°C for 14 days led to distinct changes to the ß-diversity of the host bacteria of ARGs compared to 1 day after treatment (ANOSIM, R = 0.331; p = 0.003). Storage of washed and irradiated lettuce at 4°C for 14 days increased the relative abundance of Pseudomonadaceae and Carnobacteriaceae (Wilcoxon, p < 0.05), two groups whose presence correlated with detection of 10 ARG classes on the lettuce phyllosphere (p < 0.05). Irradiation resulted in a significant reduction (∼3.5 log CFU/g) of inoculated strains of E. coli O157:H7 and P. aeruginosa (ANOVA, p < 0.05). Results indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4°C are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and relative abundance of various ARG classes.

Effect of post-harvest interventions on surficial carrot bacterial community dynamics, pathogen survival, and antibiotic resistance.

Strategies to mitigate antibiotic-resistant bacteria (ARB), including human pathogens, on raw vegetables are needed to reduce incidence of resistant infections. The objective of this research was to determine the effectiveness of standard post-harvest interventions, sanitizer washing and cold storage, to reduce ARB, including antibiotic resistant strains of the human pathogen E. coli O15:H7 and a common spoilage bacterium Pseudomonas, on raw carrots. To provide a background inoculum representing potential pre-harvest carryover of ARB, carrots were dip-inoculated in dairy cow manure compost slurry and further inoculated with known ARB. Inoculated carrots were washed with one of three treatments: sodium hypochlorite (50 ppm free chlorine), peroxyacetic acid (40 ppm peroxyacetic acid; 11.2% hydrogen peroxide), tap water (no sanitizer), or no washing (control). Washed carrots were air dried, packaged and stored at 10 °C for 7d or 2 °C for up to 60 d. Enumeration was performed using total heterotrophic plate counts (HPC), HPCs on antibiotic-containing media ("ARBs"), E. coli O157:H7, and Pseudomonas sp. immediately after washing (0 d) and after 7 d of storage. In addition to the cultured bacteria, changes to the surficial carrot microbiota were profiled by sequencing bacterial 16S rRNA gene amplicons to determine the effect of sanitizer wash, storage temperature, and time of storage (0, 1, 7, 14 and 60 d). Storage temperature, addition of a sanitizer during wash, and duration of storage significantly affected the bacterial microbiota (Wilcoxon, p < 0.05). Inclusion of either sanitizer in the wash water significantly reduced the log CFU/g of E. coli O157:H7 and Pseudomonas sp., as well as HPCs enumerated on cefotaxime- (10 µg/ml), sulfamethoxazole- (100 µg/ml), or tetracycline (3 µg/ml) supplemented media compared to the unwashed control (ANOVA, p < 0.05). However, no significant reductions to bacteria resistant to vancomycin or clindamycin occurred after washing and storage. Members of the Proteobactetria, Firmicutes, Actinobacteria, Planctomycetes, and Acidobacteria comprised the bacterial carrot microbiota. The diversity of the carrot microbiota was significantly affected by the temperature of storage and by extended storage (60 d), when spoilage began to occur. There were no significant differences to the relative abundance of bacterial groups associated with the type of sanitizer used for washing. Results of this study indicate that inclusion of a sanitizer in wash water, followed by storage at 2 °C, might be an effective strategy to prevent re-growth of pathogenic E. coli O157:H7 and reduce levels of bacteria resistant to certain antibiotics on carrots.

Inactivation of Salmonella and Surrogate Bacteria on Cashews and Macadamia Nuts Exposed to Commercial Propylene Oxide Processing Conditions.

Propylene oxide (PPO), a chemical fumigant, has been validated to reduce Salmonella on bulk almonds but has not been evaluated for other tree nuts. There is a need to identify nonpathogenic surrogate microorganisms whose inactivation is comparable to that of Salmonella to assure effective PPO processing parameters in different packaging configurations without introducing Salmonella into the pasteurization facility. The objective of this research was to compare the reduction of Salmonella and three potential surrogate bacterial strains, Enterococcus faecium ATCC 8459, Pediococcus acidilactici ATCC 8042, or Staphylococcus carnosus ATCC 51365, on cashews and macadamia nuts processed by using PPO. Whole cashews and macadamia nuts were coinoculated with a five-strain cocktail of Salmonella and one surrogate, dried to the original water activity of 0.44 to 0.51 before being packaged in woven polypropylene bags (2.3 kg), and shipped overnight in Styrofoam containers under ambient conditions to a commercial facility for PPO treatment. Salmonella and surrogates were recovered by vigorous shaking in phosphate buffer (1:1, m/v), serial diluted, and plated onto tryptic soy agar with an overlay of xylose lysine Tergitol 4 for Salmonella or mannitol salt agar or bile esculin azide agar for each surrogate. The mean log reductions of Salmonella and each surrogate ( n = 18), within a sample and among all trials (three independent), were compared by using a matched pairs t test. Reduction in log CFU per gram of Salmonella was significantly greater than that of E. faecium on both macadamia nuts (7.3 ± 0.19 versus 6.4 ± 0.31) and cashews (5.4 ± 0.15 versus 5.1 ± 0.25) and significantly greater than P. acidilactici on both nuts (7.8 ± 0.22 versus 6.3 ± 0.33 on macadamia nuts and 4.9 ± 0.22 versus 4.1 ± 0.25 on cashews). Reduction of S. carnosus exceeded that of Salmonella. E. faecium and P. acidilactici may be considered as surrogates for Salmonella on whole macadamia nuts and cashews processed by using PPO.

Alterations to metabolically active bacteria in the mucosa of the small intestine predict anti-obesity and anti-diabetic activities of grape seed extract in mice.

Epidemiological and clinical studies suggest that grapes and grape-derived products may reduce the risk for chronic disease. Grape seed extract specifically has been gaining interest due to its reported ability to prevent weight gain, moderate hyperglycemia, and reduce inflammation. The purpose of this study was to examine the long-term effects of two doses of grape seed extract (10 and 100 mg kg-1 body wt per d in mice) on markers of metabolic syndrome in the context of a moderately high-fat diet. After 12 weeks, the lower dose of grape seed extract was more effective at inhibiting fat gain and improving glucose tolerance and insulin sensitivity. Neither the high fat diet nor grape seed extract altered skeletal muscle substrate metabolism. Most interestingly, when examining the profile of metabolically active microbiota in the mucosa of the small intestine, cecum, and colonic tissue, grape seed extract seemed to have the most dramatic effect on small intestinal tissue, where the population of Firmicutes was lower compared to control groups. This effect was not observed in the cecal or colonic tissues, suggesting that the main alterations to gut microbiota due to flavan-3-ol supplementation occur in the small intestine, which has not been reported previously. These findings suggest that grape seed extract can prevent early changes in glucose tolerance and alter small intestinal gut microbiota, prior to the onset of skeletal muscle metabolic derangements, when grape seed extract is consumed at a low dose in the context of a moderately high fat diet.

Changes in the gut microbial communities following addition of walnuts to the diet.

Walnuts are rich in omega-3 fatty acids, phytochemicals and antioxidants making them unique compared to other foods. Consuming walnuts has been associated with health benefits including a reduced risk of heart disease and cancer. Dysbiosis of the gut microbiome has been linked to several chronic diseases. One potential mechanism by which walnuts may exert their health benefit is through modifying the gut microbiome. This study identified the changes in the gut microbial communities that occur following the inclusion of walnuts in the diet. Male Fischer 344 rats (n=20) were randomly assigned to one of two diets for as long as 10 weeks: (1) walnut (W), and (2) replacement (R) in which the fat, fiber, and protein in walnuts were matched with corn oil, protein casein, and a cellulose fiber source. Intestinal samples were collected from the descending colon, the DNA isolated, and the V3-V4 hypervariable region of 16S rRNA gene deep sequenced on an Illumina MiSeq for characterization of the gut microbiota. Body weight and food intake did not differ significantly between the two diet groups. The diet groups had distinct microbial communities with animals consuming walnuts displaying significantly greater species diversity. Walnuts increased the abundance of Firmicutes and reduced the abundance of Bacteriodetes. Walnuts enriched the microbiota for probiotic-type bacteria including Lactobacillus, Ruminococcaceae, and Roseburia while significantly reducing Bacteroides and Anaerotruncus. The class Alphaproteobacteria was also reduced. Walnut consumption altered the gut microbial community suggesting a new mechanism by which walnuts may confer their beneficial health effects.

Processes to Preserve Spice and Herb Quality and Sensory Integrity During Pathogen Inactivation.

Selected processing methods, demonstrated to be effective at reducing Salmonella, were assessed to determine if spice and herb quality was affected. Black peppercorn, cumin seed, oregano, and onion powder were irradiated to a target dose of 8 kGy. Two additional processes were examined for whole black peppercorns and cumin seeds: ethylene oxide (EtO) fumigation and vacuum assisted-steam (82.22 °C, 7.5 psia). Treated and untreated spices/herbs were compared (visual, odor) using sensory similarity testing protocols (α = 0.20; ß = 0.05; proportion of discriminators: 20%) to determine if processing altered sensory quality. Analytical assessment of quality (color, water activity, and volatile chemistry) was completed. Irradiation did not alter visual or odor sensory quality of black peppercorn, cumin seed, or oregano but created differences in onion powder, which was lighter (higher L* ) and more red (higher a* ) in color, and resulted in nearly complete loss of measured volatile compounds. EtO processing did not create detectable odor or appearance differences in black peppercorn; however visual and odor sensory quality differences, supported by changes in color (higher b* ; lower L* ) and increased concentrations of most volatiles, were detected for cumin seeds. Steam processing of black peppercorn resulted in perceptible odor differences, supported by increased concentration of monoterpene volatiles and loss of all sesquiterpenes; only visual differences were noted for cumin seed. An important step in process validation is the verification that no effect is detectable from a sensory perspective.

The effect of prebiotic supplementation with inulin on cardiometabolic health: Rationale, design, and methods of a controlled feeding efficacy trial in adults at risk of type 2 diabetes.

Prediabetes is associated with low-grade chronic inflammation that increases the risk for developing type 2 diabetes (T2D) and cardiovascular disease (CVD). An elevated lipopolysaccharide concentration, associated with dysbiosis of the intestinal microbiota, has been implicated in the development of both T2D and CVD. Selective modulation of the intestinal microbiota with prebiotics reduces intestinal permeability and endotoxin concentrations, inflammation, and metabolic dysfunction in rodents. The effect of prebiotic supplementation on cardio-metabolic function in humans at risk for T2D is not known. The primary aim of this trial is to determine the influence of prebiotic supplementation with inulin on insulin sensitivity and skeletal muscle metabolic flexibility in adults at risk for T2D. We hypothesize that prebiotic supplementation with inulin will improve insulin sensitivity and skeletal muscle metabolic flexibility. We will randomize 48 adults (40-75 yrs) with prediabetes or a score ≥ 5 on the American Diabetes Association (ADA) risk screener to 6 weeks of prebiotic supplementation with inulin (10 g/day) or placebo. Subjects will be provided with all food for the duration of the study, to avoid potential confounding through differences in dietary intake between individuals. Intestinal permeability, serum endotoxin concentrations, insulin sensitivity, skeletal muscle metabolic flexibility, endothelial function, arterial stiffness, and fecal bacterial composition will be measured at baseline and following treatment. The identification of prebiotic supplementation with inulin as an efficacious strategy for reducing cardio-metabolic risk in individuals at risk of T2D could impact clinical practice by informing dietary recommendations and increasing acceptance of prebiotics by the scientific and medical community.

Inoculation Preparation Affects Survival of Salmonella enterica on Whole Black Peppercorns and Cumin Seeds Stored at Low Water Activity.

Salmonellosis has been increasingly associated with contaminated spices. Identifying inoculation and stabilization methods for Salmonella on whole spices is important for development of validated inactivation processes. The objective of this study was to examine the effects of inoculation preparation on the recoverability of Salmonella enterica from dried whole peppercorns and cumin seeds. Whole black peppercorns and cumin seeds were inoculated with S. enterica using one dry transfer method and various wet inoculation methods: immersion of spice seeds in tryptic soy broth (TSB) plus Salmonella for 24 h (likely leading to inclusion of Salmonella in native microbiota biofilms formed around the seeds), application of cells grown in TSB, and/or application of cells scraped from tryptic soy agar (TSA). Postinoculation seeds were dried to a water activity of 0.3 within 24 h and held for 28 days. Seeds were sampled after drying (time 0) and periodically during the 28 days of storage. Salmonella cells were enumerated by serial dilution and plated onto xylose lysine Tergitol (XLT4) agar and TSA. Recovery of Salmonella was high after 28 days of storage but was dependent on inoculation method, with 4.05 to 6.22 and 3.75 to 8.38 log CFU/g recovered from peppercorns and cumin seeds, respectively, on XLT4 agar. The changes in surviving Salmonella (log CFU per gram) from initial inoculation levels after 28 days were significantly smaller for the biofilm inclusion method (+0.142pepper, +0.186cumin) than for the other inoculation methods (-0.425pepper, -2.029cumin for cells grown on TSA; -0.641pepper, -0.718cumin for dry transfer; -1.998pepper for cells grown in TSB). In most cases, trends for reductions of total aerobic bacteria were similar to those of Salmonella. The inoculation method influenced the recoverability of Salmonella from whole peppercorns and cumin seeds after drying. The most stable inoculum strategies were dry transfer, 24-h incubation of Salmonella and spices in TSB (i.e., potential inclusion of Salmonella within native microbiota biofilms), and inoculation of Salmonella cells grown on TSA subsequent to drying. However, with the dry transfer method it was difficult to obtain the large amount of inoculum needed for inactivation studies.

Fibre digestibility, abundance of faecal bacteria and plasma acetate concentrations in overweight adult mares.

The purpose of the present study was to compare digestibility of grass hay, faecal and plasma volatile fatty acid (VFA) concentrations, and faecal bacterial abundance in overweight and moderate-condition mares. Five overweight adult mixed-breed mares and five adult mixed-breed mares in moderate condition were housed individually and limit-fed orchard grass (Dactylis glomerata) hay at 20 g/kg body weight (as fed) daily for 14 d. Forage DM and fibre digestibility were determined using AOAC methods; digestible energy was measured using bomb calorimetry; plasma and faecal VFA concentrations were determined by use of GC and MS; faecal Firmicutes, Bacteroidetes, Fibrobacter succinogenes, Ruminococcus flavefaciens and total bacteria abundance was determined by quantitative real-time PCR using previously designed phylum-specific 16S ribosomal RNA gene primers. No differences in hay digestibility, faecal VFA concentrations or faecal bacterial abundance were detected between overweight and moderate-condition mares. Mean plasma acetate concentrations were higher (P = 0·03) in overweight (1·55 (range 1·43-1·65) mmol/l) v. moderate-condition (1·39 (range 1·22-1·47) mmol/l) mares. We conclude that the higher plasma acetate in overweight mares should be further investigated as a potential link between gut microbes and obesity in horses.

Association of Campylobacter spp. levels between chicken grow-out environmental samples and processed carcasses.

Campylobacter spp. have been isolated from live poultry, production environments, processing facilities, and raw poultry products. Environmental sampling in a poultry grow-out house, combined with carcass rinse sampling from the same flock, may provide a relative relationship between pre- and postharvest Campylobacter contamination. Air samples, fecal/litter samples, and feed/drink line samples were collected from 4 commercial chicken grow-out houses in western Virginia between September 2011 and January 2012. Birds from each sampled house were the first flock slaughtered the following day and were then sampled by postchill carcass rinses. Campylobacter, from postenrichment samples, was detected in 27% (32/120) of house environmental samples and 37.5% (45/120) of carcass rinse samples. All environmental sample types from each house included at least one positive sample except the house 2 air samples. The sponge sample method was found to have a significantly higher (P < 0.05) proportion of Campylobacter-positive samples (45%) than the fecal/litter samples (20%) and air samples (15%) when sample types of all the houses were compared. The proportion positive for the fecal/litter samples postenrichment, for each flock, had the highest correlation (0.85) to the proportion of positive carcass rinse samples for each flock. Environmental samples from house 1 and associated carcass rinses accounted for the largest number of Campylobacter positives (29/60). The fewest number of Campylobacter positives, based on both house environmental (4/30) and carcass rinse samples (8/30), was detected from flock B. The results of this study suggest that environmental sampling in a poultry grow-out house, combined with carcass rinse sampling from the same flock, have the potential to provide an indication of Campylobacter contamination and transmission. Campylobacter qualitative levels from house and processing plant samples may enable the scheduled processing of flocks with lower pathogen incidence or concentrations, as a way to reduce postslaughter pathogen transmission.

Diversity of the spinach (Spinacia oleracea) spermosphere and phyllosphere bacterial communities.

The bacterial diversity of seeds, transmission of bacteria from seed to phyllosphere, and fate of seed-transmitted bacteria on mature plants are poorly characterized. Understanding the dynamics of microbial communities is important for finding bio-control or mitigation strategies for human and plant pathogens. Bacterial populations colonizing spermosphere and phyllosphere of spinach (Spinacia oleracea) seedlings and plants were characterized using pyrosequencing of 16S rRNA gene amplicons. Spinach seed microbiota was composed of three bacterial phyla: Proteobacteria, Firmicutes and Actinobacteria, belonging to > 250 different operational taxonomic units (OTUs). Seed and cotyledon bacterial communities were similar in richness and diversity. Richness of 3-4 leaf-stage of development plants increased markedly to > 850 OTUs classified within 11 phyla. Although some bacterial OTUs were detected on seeds, cotyledons and plants, the breadth of new sequences indicates the importance of multiple sources outside the seed in shaping phyllosphere community. Most classified sequences were from previously undescribed taxa, highlighting the benefits of pyrosequencing in describing seed diversity and phyllosphere bacterial communities. Bacterial community richness increased from 250 different OTUs for spinach seeds and cotyledons, to 800 OTUs for seedlings. To our knowledge this is the first comprehensive characterization of the spinach microbiome, complementing previous culture-based and clone library studies.