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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.

This study was aimed at exploring biological properties of PoP for their potential use as an ingredient of choice in nutraceuticals and ethnic drug formulations. Pomegranate peel extracts (PoPx), derived from hydro-alcoholic extracts were evaluated for antioxidant, antimicrobial and potential antiulcer properties. Higher levels of total phenolics (427.19 mgGAE/g of extracts) recovered from hydro-alcoholic extracts exhibited significant (P

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

Pomegranates have health-promoting benefits because of their polyphenol constituents. Previous studies have demonstrated the antimicrobial activity of aqueous and organic extracts of pomegranate components and by-products. We sought to determine the antimicrobial activity against 40 foodborne pathogens representing eight bacterial species using juice itself. In addition, we sought to determine the synergistic antimicrobial activity between pomegranate juice and other plant products displaying antimicrobial activity. The antimicrobial activity of pomegranate juice was dependent on the test organism, which varied to highly susceptible (four Gram-positive species) to unaffected (Salmonella and Escherichia coli O157:H7). Two Gram-negative species, which were inhibited were Helicobacter pylori and Vibrio parahemolyticus. No synergistic antimicrobial activity was seen between pomegranate and either barberry, oregano, or cranberry. The antimicrobial activity of pomegranate juice is dependent on the test organism and extraction method. The sensitivity of H. pylori suggests that pomegranate juice may be an alternative or supplemental treatment for gastric ulcers caused by this organism.

Apple juice from four cultivars were fermented for 0, 24, 48 and 72 h using Lactobacillus acidophilus and their effects related to management of hyperglycemia, hypertension, inhibition of Helicobacter pylori and proliferation of probiotic Bifidobacterium longum were evaluated using in vitro models. All the assays were carried out at fermented acidic pH and by adjusting the pH to 6.0–7.0. Total soluble phenolics, 2,2-diphenyl-1-picrylhydrazyl-linked free radical-scavenging activity and α-amylase inhibition decreased over 72 h. Overall, α-glucosidase inhibition and hypertension-relevant angiotensin-converting enzyme inhibition decreased after 72 h for pH-adjusted samples, whereas contrasting results were obtained for fermented acidic pH samples. Only at 48- and 72-h fermented acidic pH samples had H. pylori inhibitory activity. Further investigations into mechanisms using proline revealed that H. pylori inhibition was not through inhibition of proline oxidation via proline dehydrogenase. No inhibition was observed when samples that had H. pylori inhibition were further tested for their effect on probiotic Bifidobacterium longum growth. PRATICAL APPLICATIONS In this study, for the first time, fermentation of apple juice using lactic acid bacteria was shown to have relevance in managing hyperglycemia and hypertension using in vitro models. This approach was also shown to have potential to inhibit stomach ulcer relevant bacterium Helicobacter pylori. Further, we also established that fermented extracts will not inhibit the beneficial intestinal lactic acid bacteria. This research provides biochemical rationale for further in vivo studies based on the in vitro enzyme assay models used in this study. Further, this study can provide biochemical rationale for the development of fermented food design strategies that can have potential to reduce hyperglycemia linked to type 2 diabetes and related chronic diseases. This research also provides new approaches to develop low-cost, natural antimicrobials with no side effects, which can be used as whole food dietary bioactive to manage and/or prevent infectious and diet-linked chronic diseases.

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.

Staphylococcus aureus, a Gram-positive organism, is responsible for numerous infections ranging in severity from skin and soft tissue infections to endocarditis and septic arthritis. Further, Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In this study, several single-seed origin clonal lines of herbs from Lamiaceae family were screened for antimicrobial applications against S. aureus. These clonal lines have consistent total phenolic content, phenolic profile and antioxidant activity as opposed to heterogenous phenolic profiles of typical herbal extracts. The clonal extracts of specific herb species were evaluated for anti- S. aureus activity. Water extracts of clonal lemon balm had the highest anti- S. aureus activity followed by clonal extracts of rosemary RoLA, sage, oregano, rosemary Ro-6, rosemary RoK-2 and chocolate mint. The anti- S. aureus activity of the water extracts correlated with gallic acid and caffeic acid content, which...

Infection by Helicobacter pylori is associated with gastric and duodenal ulcers. Conventional treatments to eradicate it have side-effects such as diarrhea and dizziness. The excessive use of antibiotics could also lead to antibiotic-resistant bacteria. The use of plant phenolic phytochemicals can be an alternative because of their health benefits due to both antioxidant activity and antimicrobial activity. The pea (Pisum sativum), the world's second most important pulse crop, produces phenolic phytochemicals with antimicrobial potential. Because the synthesis of phenolic compounds increases with stress, we investigated the anti-H. pylori effectiveness of extracts from pea sprouts, germinated in the dark condition following treatment with either distilled water or acetyl salicylic acid. The peas were germinated for 8 days and the sprouted samples were measured for total soluble phenolic content, antioxidant and guaiacol peroxidase activity. Subsequently, the sprout extracts were tested for anti-H. pylori activity using the agar diffusion method and the effective dose was determined based on phenolic content. The results showed that both acetyl salicylic acid-treated and untreated pea sprouts at days 5 and 8 had anti-H. pylori activity. The minimum volume for inhibition was 50 μL of extracts. The inhibitory effects were dose dependent. From this study, the potential to use natural phenolic phytochemicals from pea sprouts to control H. pylori was found to be promising. This provides a strategy and foundation to design legume phenolics as functional ingredients against H. pylori.

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 is one of the most severe food-borne bacterial infections causing Listeriosis. As L. monocytogenes can survive harsh adverse conditions – such as low pH, high NaCl, and refrigeration temperatures – as well as resist current antimicrobial measures such as the use of disinfectants and antibiotics, there is a need for alternative anti-Listeria strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via dark-germination sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus or Lentinus edodes were investigated for in vitro antibacterial activity against L. monocytogenes. L. monocytogenes growth was inhibited most effectively by R. oligosporus bioprocessed soybean extracts, which showed anti-Listeria activity at total phenolic concentrations as low as 10 µg 100 µL−1. In both sprouted soybean extract and L. edodes-bioproc...

Plants have been used medicinally throughout history. Before the beginning of the 19th century, many herbs were considered conventional medicines and were included in medical curricula and formularies. In the United States two important factors fostered a schism between the use of mainstream drugs and herbal therapies: (1) the development of a pharmaceutical industry and (2) the formation of the Food and Drug Administration which created regulatory changes. It is estimated that nearly 80% of the world's population use plants as drugs and one in three Americans use some type of herbal remedy. Consumer interest in “natural” therapies is on the rise in the U.S. and elsewhere and corporate researchers are searching for new drugs among ancient remedies and new classes of compounds to combat infectious diseases worldwide. Infectious diseases continue to be the foremost cause of death worldwide. The Center for Disease Control and Prevention (CDC) reported a 58 percent rise in deaths from infectious dise...

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.

Helicobacter pylori infection has been implicated as a major cause of gastric inflammation, peptic ulcer disease, and gastric cancer. While antibiotics have been the mainstay of current therapies for gastrointestinal disease linked to H. pylori infection, negative side-effects and antibiotic resistance issues have strengthened the need for alternative therapeutic strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus were investigated for in vitro antibacterial activity against H. pylori. Helicobacter pylori growth inhibition by soybean extracts was increased most effectively by 2 d sprouting or 2 d R. oligosporus bioprocessing. Anti-H. pylori activity was not associated with antioxidant activity, but was linked to extracts when activity of the phenolic-polymerizing enzy...

Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45°C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin ( cpe ) and alpha toxin ( cpa ) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa . None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe + strains in retail foods and the genetic diversity among nonoutbreak strains.

Food safety continues to be a major concern for the food industry in recent years. One of the industry's top priorities has been to find alternative ways to preserve their newly developed foods while satisfying the increasing consumer demand to produce safe, all-natural products. In order to achieve this “clean label”, much research has been devoted to the use of effective plant-based antimicrobials, such as those from herbs and spices, to replace chemical preservatives. However, due to the cross-pollination character of herbs and spices, there is a lot of genetic heterogeneity among different batches of the same plant species. This poses a problem for the routine use of plants, and their extracts, as a barrier towards microbial growth. In order to combat this, a unique tissue-culture-based selection strategy was used to isolate an elite phenolic phytochemical-producing clonal line of oregano (Origanum vulgare). Ethanol extracts of this elite clonal line of oregano were then used to study its inhibitory a...

Clostridium perfringens toxins are responsible for a variety of human and veterinary diseases. C. perfringens isolates are commonly classified into one of five types, A to E, based on their ability to produce four so-called lethal toxins, alpha, beta, epsilon, and iota. In addition to the enterotoxigenic nature of certain isolates, the two defining characteristics of C. perfringens’s role in foodborne illness are its potential growth rate and its ability to produce heat-resistant spores. Spore formation by C. perfringens is an important characteristic because (i) spores are able to survive cooking procedures while eliminating competitors, (ii) it is an essential classification tool, and (iii) enterotoxin formation is associated with sporulation. This chapter talks about in situ methods for cpe-positive C. perfringens, detection of enterotoxin, molecular methods, and location of the enterotoxin gene in C. perfringens isolates. The application of molecular techniques has had a tremendous impact on the diagnosis and investigation of C. perfringens food poisoning and nonfood-borne outbreaks. Reference laboratories are now able to determine rapidly and easily if isolates have the potential to cause diarrheal disease and through the use of discriminatory molecular typing methods provide evidence that cpe-positive strains from humans and foods have a common origin.

Several edible Hymenomycete mushroom species have been screened as possible inhibitors of human T4 leukemic cancer cells, HeLa cervical cancer cells, Plasmodium falciparum (a pyrimethamine resistant malarial parasite), and six pathogenic microorganisms. Grifola frondosa, Ganoderma lucidum, Lentinula edodes, and Laetiporus sulphureus demonstrated activity against human T4 leukemic cancer cells. Grifola frondosa, Ganoderma lucidum, Lentinula edodes, and Lentinula aurantiacum demonstrated activity against HeLa cervical cancer cells. Pofyporus umbellatus, Russula xerampelina, Ganoderma lucidum, Trametes versicolor, Lentinula aurantiacum, Laetiporus sulphureus, Boletus variipes. Boletus queletii, Grifola frondosa, and Lentinula edodes demonstrated activity against Plasmodium falciparum. No activity was observed against the pathogenic microorganisms.

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

Clostridium perfringens produced eight extracellular and two intracellular amylolytic activities when examined by zymograms following polyacrylamide gel electrophoresis under native conditions. The major intracellular amylase was isolated from vegetative cells of C. perfringens. It possessed an estimated molecular mass of 112 kDa. Sulfhydryl and phenol functional groups were essential to its activity. The amylase was endo-acting on starch and also hydrolyzed pullulan. Polyclonal antisera against a purified extracellular amylase did not cross-react with intracellular amylase and the two amylases were biochemically different. The distribution of extracellular amylolytic activities of sporulating cells was different from that of vegetative cells, whereas the distribution of intracellular amylolytic activities remained identical. A significant increase of a particular amylase (A8) occurred in the extracellular fluid during sporulation compared with that during vegetative growth. Regulation of the excretion of amylase(s) may be sporulation and enterotoxingenicity related.Key words: Clostridium perfringens, amylase, sporulation.

Clostridium perfringens is a leading cause of foodborne illness, though cases are widely under-reported because of the mild nature of the gastrointestinal illness, which consists of diarrhea and abdominal cramps. These appear 8–24 hours following ingestion of large numbers of vegetative cells in temperature-abused protein foods, typically meat and poultry. Cells sporulate in the small intestine, producing an enterotoxin. The ability of C. perfringens to grow at elevated temperatures, produce spores, and reproduce rapidly contributes to its role as an important cause of foodborne illness.

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.

An alpha-amylase (EC 3.2.1.1) secreted by Clostridium perfringens NCTC 8679 type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion-exchange and gel permeation chromatography. The enzyme exhibited maximal activity at pH 6.5 and 30 degrees C without the presence of calcium. The pI of the enzyme was 4.75. The estimated molecular weight of the purified enzyme was 76 kDa. The purified enzyme was inactivated between 35 and 40 degrees C, which increased to between 45 and 50 degrees C in the presence of calcium (5 mM). The purified enzyme produced a mixture of oligosaccharides as major end products of starch hydrolysis, indicating alpha-amylase activity.

We previously demonstrated the widespread presence of enterotoxigenic Bacillus cereus in marine foods. In view of the widespread consumption of raw fish, we sought to determine the ability of this organism to grow on the surface of wild Alaskan salmon at abusive temperatures (12, 16, and 20°C), using an isolate able to produce elevated levels of hemolysin BL enterotoxin and nonhemolytic enterotoxin. An incubation temperature of 37°C for colony formation was found to be selective for B. cereus grown on salmon held for up to 24 h at each temperature. A fivefold increase in log CFU per gram was observed after 26 and 22 h at 16 and 20°C, respectively, while a >4-log CFU/g increase occurred on salmon held at 12°C for 48 h. Generation times of 169.7, 53.5, and 45.6 min were observed at 12, 16, and 20°C. Nonhemolytic enterotoxin was detected when levels of B. cereus were in excess of 10(8) CFU/g. Nisin, at concentrations of 1 and 15 m g/g of salmon, reduced levels of B. cereus 2.5- and 25-fold, respectively. Our results indicate that fresh salmon can serve as an excellent substrate for enterotoxigenic B. cereus and that this organism can reach levels associated with foodborne illness following moderate temperature abuse.

The effect of glucose and other sugars on sporulation and extracellular amylase production byClostridium perfringens NCTC 8679 type A in a defined medium was studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity, while disaccharides such as lactose, maltose, and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10mm except for fructose (25mm). Levels of heat-resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in this strain ofC. perfringens.

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.

Tea polyphenolics such as catechins are known to have the potential to inhibit many bacterial pathogens. Helicobacter pylori has been identified as an etiologic agent in the development of gastric ulcer, peptic ulcer, gastritis, and many other stomach-related diseases. In this study, we investigated the effect of 9 tea extracts--3 different brands representing 4 different processed types (white, green, oolong, and black)--on the inhibition of H. pylori. Extraction times of 2 and 5 minutes were compared. Most 5-minute extracts showed H. pylori inhibition, whereas 2-minute extracts only of Choice darjeeling black and Tazo white showed inhibition. No recovery was observed after the addition of 0.5 and 5 mM proline, indicating that tea polyphenols do not inhibit H. pylori by inhibition of proline oxidation via proline dehydrogenase. Extracts that showed inhibition were further evaluated for their effect on beneficial lactic acid bacteria. None of the samples showed inhibition, suggesting that tea might be able to inhibit H. pylori without affecting the beneficial lactic acid bacteria. High-performance liquid chromatography indicated the presence of gallic acid, quercetin, caffeine, and tea catechins (including catechin, epicatechin, and epigallocatechin) in all the tea samples. Our study indicates that tea can be potentially used as a low-cost dietary support to combat H. pylori-linked gastric diseases without affecting the beneficial intestinal bacteria.

Spore coat proteins of several strains ofClostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent−) strain of this organism. The results indicated that spore coat proteins from many strains ofC. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulationspecific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393–410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.

All 47 food-borne isolates of Bacillus cereus sensu stricto , as well as 10 of 12 food-borne, enterotoxigenic isolates of Bacillus thuringiensis , possessed appendages. Spores were moderately to highly hydrophobic, and each had a net negative charge. These characteristics indicate that spores of food-associated B. thuringiensis and not only B. cereus sensu stricto have high potential to adhere to inert surfaces.

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.

Two extracellular proteases, E-A and E-B, produced by sporulating cells ofClostridium perfringens NCTC 8798, were isolated by ammonium sulfate fractionation followed by DEAE-Sephacel and Sephacryl S-300 chromatography. E-A was further purified to homogeneity following separation on casein-agarose. E-A and E-B possessed native molecular weights of 330 kDa and 96 kDa respectively. SDS-PAGE of E-A indicated that it was composed of one major 120-kDa subunit. Both E-A and E-B hydrolyzed N-succinyl-l-phenylalanine-p-nitroanilide and were inhibited by chymotrypsin but not antipain or leupeptin, indicating that they were chymotrypsin-like enzymes. Calcium but not dithiothreitol was effective in minimizing inactivation at 50°C. Comparative analysis of E-A and I-A-1, the principal intracellular protease, indicated that they were very similar but not identical.

Five intracellular proteases from sporulating cells of Clostridium perfringens type A were identified and three could be separated by DEAE–Sephacel. Two, I-A and I-B, had caseinolytic activity and one, I-C, was only active on N-benzoyl-DL-arginine-p-nitroanilide. I-A and I-B could each be further separated by Sephacryl S-300 into I-A-1 and I-A-2 and I-B-1 and I-B-2, respectively. I-A-1, a chymotrypsin-like enzyme, was the major intracellular protease, constituting 74% of the intracellular caseinolytic activity. In addition to cytoplasmic proteases both trypsin and chymotrypsin-like enzyme activity was associated with the membrane fraction. I-A-1 had a molecular weight of 330 000, with subunits of 120 000 and 138 000. I-A-1 cleaved a 1200 molecular weight peptide from C. perfringens enterotoxin. Early sporulating cell extracts of C. perfringens contained three presumptive enterotoxin precursors, which disappeared following treatment with I-A. Such cells also contained at least 10 spore coat related proteins, only one (51 500 molecular weight) of which was sensitive to I-A-1. The results indicate a possible role for the major intracellular protease in the processing of C. perfringens enterotoxin and a less important role, if any, in spore coat formation. Key words: Clostridium perfringens, protease, spores, sporulation.

For the period 1990 through 2003, seafood was the most commonly identified food linked to foodborne outbreaks in the United States. Fish as a commodity has rarely been examined for the presence of Bacillus cereus in particular. For the present study, 347 fresh and processed retail seafood samples were examined for the presence of Clostridium botulinum, Clostridium perfringens, and B. cereus. The presence of C. botulinum was not confirmed in any of the isolates, but C. perfringens was confirmed in 17 samples. One of the C. perfringens isolates possessed the enterotoxin gene, as determined by PCR. In contrast, 62 confirmed B. cereus isolates were obtained from separate samples at levels ranging from 3.6 to > 1,100 CFU/g. Thirty (48%) of 62 isolates produced both the hemolysin BL (HBL) and nonhemolytic (NHE) enterotoxins, and 58 (94%) and 31 (50%) produced NHE or HBL toxins, respectively. The presence of at least one of the three genes of the NHE complex was detected in 99% of the isolates; 69% of the isolates possessed all three genes. In contrast, 71% of the isolates possessed at least one of the three genes of the HBL complex, and 37% possessed all three HBL gene components. Fifty of the 62 B. cereus isolates were from imported seafood, and 19 (38%) of these samples were at levels > 100 CFU/g. Twelve of the 14 highest enterotoxin assay results were from isolates from imported food. Only one B. cereus isolate possessed the cereulide synthetase gene, ces; this isolate also possessed the genes for the three-component HBL and NHE complexes. A majority of enterotoxin-producing isolates were resistant to 2 of 10 antibiotics tested, ceftriaxone and clindamycin. Our results demonstrate the potential of seafood as a vehicle for foodborne illness caused by B. cereus, in particular the enterotoxin-producing genotype.

A peptidoglycan hydrolase (PGH), which causes the germination and lysis of the coat-stripped spores of Clostridium perfringens, was extracted from perfringens vegetative cells. The enzyme was partially purified and showed an MW of 125, 000 and a pI of 7.6. It was sensitive to a variety of monovalent and divalent cations and was inhibited by sulfhydryl-active agents. The sites of PGH action, the cell wall and the spore-cortex, were determined by the release of free amino groups from cell wall fragments and the release of soluble fluorescent products from fluorescamine-labelled cortical fragments of the C. perfringens spores. The enzyme appears distinct from the extracellular initiation protein produced by C. perfringens. PGH may be an autolysin with corticolytic activity.

Ulcer-associated dyspepsia is caused by infection withHelicobacter pylori. H. pyloriis linked to a majority of peptic ulcers. Antibiotic treatment does not always inhibit or killH. pyloriwith potential for antibiotic resistance. The objective of this study was to determine the potential for using phenolic phytochemical extracts to inhibitH. pyloriin a laboratory medium. Our approach involved the development of a specific phenolic profile with optimization of different ratios of extract mixtures from oregano and cranberry. Subsequently, antimicrobial activity and antimicrobial-linked urease inhibition ability were evaluated. The results indicated that the antimicrobial activity was greater in extract mixtures than in individual extracts of each species. The results also indicate that the synergistic contribution of oregano and cranberry phenolics may be more important for inhibition than any species-specific phenolic concentration. Further, based on plate assay, the likely mode of action may be through urease inhibition and disruption of energy production by inhibition of proline dehydrogenase at the plasma membrane.

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.