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The role of spices as vehicles of foodborne illness prompted an examination of bacterial spores in these products. Here, we report on the levels and characteristics of spores of Clostridium perfringens associated with 247 U.S. retail spices. Forty-three confirmed isolates from 17% of samples were obtained, present at levels ranging from 3.6 to 2,400/g. Twenty-seven (63%) of C. perfringens isolates were positive for the enterotoxin gene ( cpe). Seven random spice isolates produced enterotoxin at levels of between 4 and 16 ng/mL, compared with three outbreak (control) strains that each produced enterotoxin levels of >1,024 ng/mL. D95°C levels (1.0 to 3.3 min) of spores of four randomly selected spice isolates suggests a plasmid-localized cpe, while one had D95°C (>45 min) consistent with chromosomally located cpe. Five of the 43 isolates possessed the epsilon toxin gene ( etx, as well as cpe). Foods could easily become contaminated with spores of cpe-positive C. perfringens by the addition of spices. Because of its spore-forming ability, its rapid generation times at elevated temperatures, improper heating, cooling, and holding conditions could lead to elevated levels of C. perfringens in foods, a requirement for its implication in foodborne outbreaks.

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D 95°C values ranging from 0.64 to 3.53 min while two emetic strains had D 95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10 7 and 3.8 × 10 7 CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10 4 CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10 8 /g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log 10 less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.

Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts ( 107 CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were

Bacillus cereus is a gram-positive, spore-forming, facultative anaerobe that is responsible for two types of gastrointestinal diseases: emesis and diarrhea. A significant difference in the D(95 degrees C)-values of spores of the emetic and the diarrheal types was initially determined. A mixture of B. cereus spores of the diarrheal type was inoculated into cooked rice. At inoculation levels of 2.5 x 10(2) spores per g of rice, cell numbers of 6.64 log were detected after 22 h at 20 degrees C and 6.81 log after 34 h at 17 degrees C, whereas at 12 degrees C the counts did not go above 4.0 log even after 48 h. When added to raw rice before cooking at inoculum levels of 10(3)/g, the number of viable spores decreased by 2 log, and a

Bacillus cereus is a gram-positive, endospore forming pathogenic bacterium that is ubiquitous in the environment and is frequently associated with emetic and diarrheal types of foodborne illness. In this study, 178 samples of raw rice from retail food stores were analyzed for the presence of B. cereus spores. Spores of Bacillus species were found in 94 (52.8%) of the rice samples with an average concentration of 32.6 CFU/g (3.6-460 CFU/g for B. cereus and 3.6-23 CFU/g for Bacillus thuringiensis). Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. Bacillus mycoides (240 CFU/g) was the predominant isolate in one rice sample. Using PCR the isolates were checked for the presence of the cereulide synthetase gene (ces), the hblA and hblD genes of the hemolysin BL (HBL) complex and the nheA and nheB genes of the nonhemolytic (NHE) enterotoxin complex. The ces gene was not identified in any of the isolates. By contrast 47 (56.6%) B. cereus isolates possessed the hblA and hblD genes and 74 (89.1%) isolates possessed the nheA and nheB genes. As determined by commercial assay kits, forty four (53.0%) of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 78 (93.9%) were positive for either one or the other. Protein toxin crystals were detected visually in the 11 B. thuringiensis isolates. PCR analysis revealed 10 (90.9%) of those 11 isolates carried the cry gene. All the B. thuringiensis isolates were positive for NHE and HBL enterotoxins. Our results suggest that foodborne illness in the U.S. due to B. cereus with rice as the vehicle would be most likely associated with the diarrheal-type syndrome.

Clostridium perfringens is a foodborne disease agent that produces a sporulation-specific enterotoxin. To produce enterotoxin for experimental purposes or spores for challenge or physiological studies, the use of a convenient sporulation medium is required. The most commonly used is Duncan-Strong medium. Few isolates sporulate at high levels in this medium. We investigated the effectiveness of peptones from a variety of sources on the sporulation of this organism compared with the peptone in the original formulation, proteose peptone (control). Seven strains were used to screen 32 peptones, with starch or raffinose as the carbohydrate source. In most cases, raffinose was more effective than starch in stimulating sporulation, confirming our previous study. Two promising peptones, potato peptone, and Proteose Peptone no. 3, were selected and tested against 49 additional enterotoxin-positive and -negative strains, with raffinose as the carbohydrate. For 49 strains, 5 sporulated best (>10%) in the control peptone, 6 sporulated best in Peptone no. 3, and 23 sporulated best in the potato peptone. Of the 23 strains, 16 sporulated at levels 25% more than the control peptone. The increase in sporulation rates was reflected in the enterotoxin and heat-resistant spore levels. The methylxanthines caffeine and theobromine were effective in increasing the sporulation of less than half of 19 enterotoxin-positive strains. Our results suggest that the replacement of proteose peptone with potato peptone be considered if difficulty in obtaining spores of specific strains of C. perfringens is encountered.

Several food additives are used to extend the shelf life of food, and delay or inhibit the growth of pathogenic microorganisms. Extracts of plants, particularly herbs and spices, are rich in essential oils and phenolic secondary metabolites and several possess antimicrobial activity. Some of these plant extracts are used in the aroma and flavor industry, and are classified as “generally regarded as safe” (GRAS). Oregano and cranberry extract mix provide a GRAS-type phytochemical source with the potential to develop a natural and effective antimicrobial strategy against Vibrio parahaemolyticus . Different ratios of extract mixtures were optimized from oregano and cranberry, and evaluated for antimicrobial activity in laboratory medium and seafood products. The results indicated that the antimicrobial activity was enhanced in response to extract mixtures than individual extracts of each species. The efficacy was further improved by combination with lactic acid, which is also considered GRAS. These results show the potential of plant extracts to be antimicrobial and, when combined with lactic acid, they can be used as a more effective multiple-barrier food preservation system. Such a synergistic ingredient approach can have wide implications for improvement of food safety. Industrial relevance Phenolic phytochemicals are excellent sources of antioxidants and antimicrobials in the diet, which contribute positively to the management of oxidation-linked and infectious diseases. Traditionally, individual phenolics have been targeted as antimicrobials with low efficacy. In this manuscript, we have shown how mixtures of phenolic phytochemicals from cranberry and oregano, in synergistic combination with lactic acid, can inhibit V. parahaemolyticus in seafood systems. Such a strategy can be used for enhancing food safety in food industry.

Ulcer-associated dyspepsia is caused by an infection of Helicobacter pylori . H. pylori is a food-related pathogen that infects the stomach and weakens the stomach lining and is linked to a majority of peptic ulcers. Antibiotic treatment does not always inhibit or kill H. pylori as it has side effects with potential for antibiotic resistance. Previous research has indicated that phytochemical-enriched wines have therapeutic benefits. The objective of this study was to determine the potential of phenolic phytochemical-enriched wine and vodka to inhibit H. pylori in laboratory medium. This offers a novel approach to couple antioxidant-enriched benefits of alcoholic beverages with synergistic antimicrobial effectiveness and can be considered as ‘generally regarded as safe’ (GRAS). This approach involved the development of phenolic phytochemical-enriched alcoholic beverages through release from dry botanicals enclosed in tea bags. Phenolic phytochemical-enriched alcoholic beverages were then assayed for total phenolics, antioxidant activity, and phenolic profile by HPLC. Various phenolic-enriched concentrations were then used for antioxidant assay and corresponding antimicrobial activity against H. pylori . Results indicate that total phenolics increased from 80 μg/ml in control to an average of 186 μg/ml in phenolic-enriched white wine. Corresponding antioxidant activity increased from 38% 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical inhibition in control to an average of 83% in phenolic-enriched white wines. Total phenolics increased from 5.7 μg/ml in control to an average of 289 μg/ml in phenolic-enriched vodka. The antioxidant activity increased from 2% DPPH inhibition in control to 69% inhibition in phenolic-enriched vodka. Phenolic-enriched wine enhanced the inhibitory activity against H. pylori but there was no concentration-dependent correlation. Raspberry, cinnamon and peppermint-enriched wines had the highest antimicrobial activity. In the case of phytochemicals-enriched vodka, raspberry was most inhibitory. Results indicate that the synergistic contribution of phenolics and antioxidant activity may be more important for inhibition than any specific phenolic concentration. This research has implications for diet-based management of H. pylori .

Listeria monocytogenes, Vibrio parahaemolyticus and Escherichia coli 0157:H7 are important food-borne pathogens. Control of these pathogens using synthetic antimicrobials such as currently approved antibiotics is challenging due to potential development of resistance. A profile of antimicrobials compared to a single compound could be potentially more effective. Cranberry pomace is a byproduct of the cranberry processing industry. Solid-state bioprocessing of cranberry pomace using the food grade fungus Rhizopus oligosporus improves phenolic and antioxidant profiles. We hypothesize that these phytochemicals mobilized during pomace bioprocessing could improve the antimicrobial functionality. The objective of this research was to use the food grade fungus R. oligosporus to release phenolic aglycones and relate its antioxidant functionality and diphenyl mobilization to antimicrobial activity against L. monocytogenes, V. parahaemolyticus and E. coli 0157:H7. Bioprocessing of pomace was done for 20 days with R. oligosporus. Total phenolics in water extracts were assayed using a Folin-Ciocalteu method. The antioxidant potential was measured using a β-carotene oxidation system (APF) and 1,1-diphenyl-2-picrylhydrazyl-radical (DPPH) system. Changes in phenolic profiles were analyzed using HPLC. Antimicrobial activities of the extracts during growth were tested using standard disk assays. Soluble phenolics, antioxidant activity and ellagic acid concentrations were enriched by bioprocessing and antimicrobial activity of the extracts against L. monocytogenes and V. parahaemolyticus correlated with highest soluble phenolics and APF in the same extracts. For E. coli 0157:H7 inhibition correlated with the extracts corresponding to highest DPPH and ellagic acid concentration. The bioprocessing-based antimicrobial activity depended on different phenolic functional properties of the extracts. Sensitivity towards soluble phenolics reflected in DPPH activity suggested inhibition by the disruption of the membrane by hyperacidification. Whereas, sensitivity to APF and ellagic acid suggests potential antimicrobial activity by membrane-transport disruption. The variation in sensitivity of pathogens has implications for designing new food grade antimicrobials. This bioprocessing strategy can be an innovative approach to produce broad spectrum antimicrobials against important food-borne pathogens.

Cranberry pomace is a byproduct of the cranberry processing industry. Solid-state bioprocessing of cranberry pomace using food grade fungus Lentinus edodes improved phenolic profile and antioxidant activity. We investigated whether these phenolic phytochemicals mobilized during bio-processing of the pomace would improve the antimicrobial functionality. Listeria monocytogenes , Vibrio parahaemolyticus and Escherichia coli-O157 : H7 are important food borne pathogens. Control of these pathogens using synthetic antimicrobials such as currently approved antibiotics have limitations due to potential development of resistance. We believe a profile of antimicrobials compared to a single compound could be potentially more effective. The objective of this research was to use food grade fungus Lentinus edodes to release phenolic aglycones and relate its antioxidant functionality and ellagic acid mobilization to antimicrobial activity against Listeria monocytogenes , Vibrio parahaemolyticus and Escherichia coli-O157: H7 . Bioprocessing of pomace was done for 20 days with Lentinus edodes . Total phenolics in water extracts were assayed using Folin-Ciocalteu method. The antioxidant potential was measured using a β-carotene oxidation system (APF) and 1,1-diphenyl-2-picrylhydrazyl-radical (DPPH) system. Changes in phenolic profiles were analyzed using HPLC. Antimicrobial activities of the extracts during growth were tested using standard disk assays. Results showed that the antimicrobial activities of the extracts were enriched by bioprocessing. The bioprocessing-based antimicrobial activity depended on different phenolic functional properties of the extracts. Inhibition of L. monocytogenes by the extracts correlated well with the increase in soluble phenolics, antioxidant activity and enrichment of ellagic acid during solid-state bioprocessing. Inhibition of V. parahaemolyticus and E. coli O157 : H7 by the extracts correlated with highest ellagic acid and/or APF. Sensitivity towards soluble phenolics and the antioxidant activity measured by DPPH radical inhibition assay suggests inhibition by the disruption of the membrane by localized hyper-acidification and potentially disruption of H + -ATPase. Whereas, APF and ellagic acid dependent antimicrobial activity suggests potential inhibition by membrane-transport and/or electron transport disruption. The variation in sensitivity of pathogens has implications for designing new food-grade antimicrobials. This solid-state bioprocessing strategy is an innovative approach to produce broad-spectrum antimicrobials against important food borne pathogens.

Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe. C. perfringens counts varied from

The growth of two enterotoxin-positive (ent + ) and two enterotoxin-negative (ent-) strains of Clostridium perfringens were examined in two laboratory media, fluid thioglycollate and fluid thioglycollate with beef, and in autoclaved ground beef stored at 37, 41, 43, 46, and 48°C. There was no association between enterotoxigenicity and optimum growth temperature, which varied depending on strain and medium. Experimentally obtained values for generation times at 43°C were as low as 8.7 and 6.3 min in thioglycollate both for ent + and ent- strains respectively. In autoclaved ground beef the shortest generation times, 7.1 min and 9.2 min, were obtained at 41 and 46°C for two ent + strains. Forent- strains in autoclaved ground beef a generation time of 6.6 min was observed at 43°C. Temperature optima obtained experimentally were the same as those obtained by the Gompertz model. The rapid growth of the ent + strains between 41 and 46°C underlines previous recommendations for proper holding and cooling of protein foods such as meat and poultry known to support the growth of this organism.

Clostridium perfringens strain FD-1041 was grown in Duncan-Strong medium containing raffinose for 5 h at 43°C. Enterotoxin in the cell extract was purified 5.5-fold by a modified ammonium sulfate-gel filtration method using Sephacryl S-200. The enterotoxin could be purified to homogeneity with 50% recovery in less than 48 h.

The absence in the supernatant fluid of either extracellular amylase in cultures grown at 46°C or propionic acid has been proposed for the presumptive identification of enterotoxin-positive strains of Clostridium perfringens . However we found that propionic acid was formed by several enterotoxin-positive, as well as enterotoxin-negative, strains of this organism. In addition 4 of 13 ent+ stains produced extracellular amylase during growth at 46°C. These results indicate that neither trait can be used as a reliable indicator of the enterotoxigenicity of isolates of this organism.

Four selective and eight non-selective plating media were evaluated for their ability to enumerate six strains of heat-activated and heat-injured spores of Clostridium perfringens . Trypticase-sulfite-neomycin (TSN) agar and sulfite-polymyxin-sulfadiazine (SPS) agar gave higher counts of heat-activated spores than non-selective media. In the case of heat-injured spores, wide variation in recovery was obtained depending on strain and medium. Higher counts of heat-injured spores were obtained by incubating plates at 37°C than at 45°C, although, except for one strain, no significant difference between the two temperatures was observed using heat-activated spores.

A fluorescamine assay for the detection of a spore-lytic enzyme from Clostridium perfringens is described. The substrate is prepared by treatment of cortical fragments with fluorescamine which reacts with amino terminal groups in the peptidoglycan which are not cross-linked, presumably diaminopimelic acid. Treatment of the labelled substrate with lytic enzymes results in the release of soluble fluorescent products which can be easily measured in a basic fluorometer. The assay is very sensitive, inexpensive and reproducible. As little as 1 μg of lysozyme can be detected by this assay.

An initiation protein which promoted genermination of dithiothreitol-sodium dodcyl suflate-trated spores of clostridium perfringens was purified from 7-h culture supernatant fluid of cell of Cl. perfringenes . A 3100-fold purification was obtained after cellulose-phosphate and Sephadex G-100 chromatography. The isolated product had an apparent molecular weight of 100 000, an isoelectric point of 7.9, was reversibly inhibited by HgCl 2 and lysed isolated cortical fragments from Cl. perfringens spores.

A number of media have been proposed for the enumeration and confirmation of Clostridium perfringens in food and water. Most of these employ sulfite and iron together with selective antibiotics. This report discusses these various media and conditions for their use.

Protein samples were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and stained with colloidal gold. Three artifact bands appeared demonstrating apparent molecular weights of 58,000, 63,000, and 65,000. The appearance of these bands was due to oxidized dithiothreitol used to denature the proteins prior to electrophoresis. The appearance of the artifact bands could be avoided by the addition of excess iodoacetamide to the denatured protein sample and by limiting staining time to 1.5 h at 13 V/Cm.

Effectiveness of a betalains/sorbate curing system versus a conventional nitrite system for color development and control of microbial growth in chicken frankfurters was tested. Normal heat processing involved in the manufacture of frankfurters was successful in reducing cell levels of indigenous aerobic mesophiles, and spore levels of inoculated Clostridium perfringens and Clostridium sporogenes by 1 to 2 log cycles. There were no significant differences in numbers of C. perfringens spores for either treatment after 9 d of storage at 4 or 20°C. The two curing systems tested were also equally effective in controlling growth of C. sporogenes when tested at 20°C over a 9-d storage period. The betalains/sorbate system proved to be more effective in inhibiting growth of aerobic mesophiles over the 9-d storage period at both 4 and 20°C. It is concluded that in chicken frankfurters under the conditions of this experiment, a betalains/sorbate curing system provided the same level of microbiological protection as a nitrite curing system.