Development of a risk-ranking framework to evaluate potential high-threat microorganisms, toxins, and chemicals in food.

Through a cooperative agreement with the U.S. Food and Drug Administration, the Institute of Food Technologists developed a risk-ranking framework prototype to enable comparison of microbiological and chemical hazards in foods and to assist policy makers, risk managers, risk analysts, and others in determining the relative public health impact of specific hazard-food combinations. The prototype is a bottom-up system based on assumptions that incorporate expert opinion/insight with a number of exposure and hazard-related risk criteria variables, which are propagated forward with food intake data to produce risk-ranking determinations. The prototype produces a semi-quantitative comparative assessment of food safety hazards and the impacts of hazard control measures. For a specific hazard-food combination the prototype can produce a single metric: a final risk value expressed as annual pseudo-disability adjusted life years (pDALY). The pDALY is a harmonization of the very different dose-response relationships observed for chemicals and microbes. The prototype was developed on 2 platforms, a web-based user interface and an Analytica(R) model (Lumina Decision Systems, Los Gatos, Calif., U.S.A.). Comprising visual basic language, the web-based platform facilitates data input and allows use concurrently from multiple locations. The Analytica model facilitates visualization of the logic flow, interrelationship of input and output variables, and calculations/algorithms comprising the prototype. A variety of sortable risk-ranking reports and summary information can be generated for hazard-food pairs, showing hazard and dose-response assumptions and data, per capita consumption by population group, and annual p-DALY.

Standardization of a method to determine the efficacy of sanitizers in inactivating human pathogenic microorganisms on raw fruits and vegetables.

The efficacy of sanitizers in killing human pathogenic microorganisms on a wide range of whole and fresh-cut fruits and vegetables has been studied extensively. Numerous challenge studies to determine the effects of storage conditions on survival and growth of pathogens on raw produce have also been reported. Results of these studies are often difficult to assess because of the lack of sufficient reporting of methods or, comparatively, because of variations in procedures for preparing and applying inocula to produce, conditions for treatment and storage, and procedures for enumerating pathogens. There is a need for a standard method to accurately determine the presence and populations of pathogenic microorganisms on produce. The adoption of standard, well-characterized reference strains would benefit a comparative assessment of a basic method among laboratories. A single protocol will not be suitable for all fruits and vegetables. Modifications of a basic method will be necessary to achieve maximum recovery of pathogens on various types of produce subjected to different sanitizer or storage treatments. This article discusses parameters that must be considered in the course of developing a basic standard method against which these modifications could be made.

Sublethal sanitizer stress and adaptive response of Escherichia coli O157:H7.

The effect of sublethal exposure to peroxyacetic acid (PAA) sanitizer on adaptation to peroxidative stress and development of thermal cross-resistance was investigated in Escherichia coli O157:H7. Acute sublethal PAA sanitizer exposure was used to represent a contact scenario. Cultures were grown in Trypticase soy-yeast extract broth. Acute treatment cultures were pretreated with 0.1% PAA, then all cultures were challenged at either 80 mM H202 or 54 degrees C. Acute and peroxide control cultures showed substantially increased peroxidative tolerance (D80mM > 2 h) versus negative control cultures not exposed to sanitizer (D80mM = 0.19+/-0.03 h). The inactivation rate of the acetic acid control (D80mM = 0.21+/-0.05 h) was similar to the negative control rate. Acute (D54 degrees C = 0.55+/-0.07 h) cultures did not exhibit increased thermal resistance versus the control (D54 degrees C = 0.54+/-0.07 h). Thermal injury was determined as difference in D54 degrees C value (deltaD54 degrees c) obtained on pyruvate and deoxycholate media. Thermal-induced injury was not observed in either control (deltaD54 degrees C = 0.04 h) or acute (deltaD54 degrees C = 0.05 h) cultures.

The role of probiotic cultures in the prevention of colon cancer.

Risk factors for colon cancer include both hereditary and environmental factors. Dietary patterns represent controllable risk factors for the development of colon cancer. Much attention has focused on decreasing colon cancer risk through increasing intake of dietary fiber; recently, this has included interest in the consumption of prebiotics and probiotics. Because factors involved in the initiation and promotion of colon cancer might be separated in time from actual tumor development, it is difficult to choose "outcomes" or "end points" that are definitive indicators of efficacy of probiotics or prebiotics. Studies that have explored the cause-effect relationship directly have used animal models. In this review, we have confined our discussion to animal studies from the last 10 years that have examined most directly the relationship between prebiotic and probiotic consumption and colon cancer development. To present the consensus of these studies first, it appears that probiotics with or without prebiotics have an inhibitory effect on the development of aberrant crypts (precancerous lesions) and tumors in animal models. The effect is not completely consistent and is small in some studies, but this may represent a dose or time effect.

Consumption of exogenous bifidobacteria does not alter fecal bifidobacteria and breath hydrogen excretion in humans.

The hypothesis that consumption of bifidobacteria by humans would increase colonic bifidobacteria and decrease breath hydrogen excretion was examined. A commercially available strain of bifidobacteria was tracked through the gastrointestinal tract. We determined that a 12-d feeding period of 10(10) cells of exogenous bifidobacteria daily was adequate to achieve a stable number of exogenous bifidobacteria in the colon. A 12-d washout period was chosen because the exogenous bifidobacteria could no longer be detected at that time. A double-blind crossover study used both male and female subjects. The order of treatment with skim milk alone or skim milk + bifidobacteria was randomized. Breath hydrogen excretion (micromol/L) and fecal counts of total bifidobacteria [log colony forming units (CFU)/g feces] were not significantly different between males and females and were not affected by consumption of exogenous bifidobacteria. Calculations based on the numbers of exogenous bifidobacteria consumed and the fecal numbers of exogenous bifidobacteria excreted suggested that numbers of the exogenous strain increased within the gastrointestinal tract. These data suggest that it is difficult to permanently alter total colonic bifidobacteria and affect physiologic function (net hydrogen in the colon as reflected by breath hydrogen) by feeding bifidobacteria, although the percentage of the total bifidobacteria represented by the exogenous strain can be affected.

Differentiation of ingested and endogenous bifidobacteria by DNA fingerprinting demonstrates the survival of an unmodified strain in the gastrointestinal tract of humans.

Consumption of bifidobacteria as a dietary adjunct has received considerable attention for its possible role in the maintenance of gastrointestinal health. However, speculation exists about these presumed health benefits because of an inability to assess the fate and mechanism of action of ingested bifidobacteria. Thus, our objective was to examine the fate of ingested bifidobacteria through the gastrointestinal tract. Variations in the highly conserved 16S ribosomal DNA (rDNA) of bifidobacteria from six male subjects (18 to 35 y old) were assessed by restriction fragment length polymorphism (RFLP) analysis. During the 16-d study, 10(10) colony-forming units (CFU) of a commercially available bifidobacteria were delivered to subjects in fluid milk for each of 8 d. During the remaining 8 d, subjects consumed milk without bifidobacteria. Feces were collected at 4-d intervals and plated on selective media. For each subject, 10-15 colonies were randomly selected and used as template for PCR-amplification of 16S rDNA. 16S rDNA was restriction digested and resolved by electrophoresis. The 16S rDNA-RFLP of the ingested bifidobacteria was unique compared with bifidobacteria found in subjects prior to the feeding study. When subjects consumed bifidobacteria, a 16S rDNA-RFLP identical to that of the ingested bifidobacteria was observed in feces. The concentration of the ingested bifidobacteria in feces increased to 67.2 +/- 8.5% (mean +/- SEM) of total bifidobacteria. After feeding stopped, the ingested bifidobacteria diminished and became undetectable. Using this molecular approach to monitor ingested bifidobacteria, we demonstrate the kinetics of passage of this organism through the gastrointestinal tract of healthy humans.

Probiotics, cecal microflora, and aberrant crypts in the rat colon.

Our hypothesis was that administration of bifidobacteria, Lactobacillus acidophilus or both to rats will minimize the numbers of aberrant crypts in the distal colon that develop in response to the carcinogen 1,2-dimethylhydrazine (DMH). A series of experiments was designed to test this hypothesis where the treatments used were as follows: skim milk controls (Skim-Basal), skim milk + bifidobacteria (Bifido-Basal), skim milk + fructooligosaccharide (Skim-FOS), and skim milk + bifidobacteria + fructooligosaccharide (Bifido-FOS). In two experiments, bifido-bacteria + FOS administration significantly decreased the number of aberrant crypts that developed, but there was no clear relationship of aberrant crypts to numbers of bifidobacteria or Clostridium perfringens. In the third experiment, the Bifido-FOS treatment led to significantly fewer aberrant crypts and aberrant crypt foci than the Bifido-Basal treatment. The Skim-FOS group had significantly more cecal bifidobacteria than the Skim-Basal group and significantly fewer C. perfringens than the Skim-Basal and Bifido-Basal. In a fourth experiment, L. acidophilus was added as an additional treatment. The number of aberrant crypts was not significantly different among the groups. However, the number of C. perfringens was significantly decreased by the addition of bifidobacteria, L. acidophilus or the combination of the two, whereas the numbers of bifidobacteria and L. acidophilus were not affected by treatment. A significant correlation (R2 = 0.84, P < 0.01) was noted between the body weight of rats at DMH administration and the magnitude of the difference in aberrant crypts between the Skim-Basal rats and the Bifido-FOS rats. The results suggest that there is variability in the effects of bifidobacteria and L. acidophilus administration on both aberrant crypt formation and C. perfringens.

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils.

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low aw of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105 degrees C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.

Resistance of Neosartorya fischeri to wet and dry heat.

Dry heat resistance parameters for Neosartorya fischeri ascospores were obtained at 90 degrees C and 95 degrees C under 30%, 40%, 50%, 60%, and 75% relative humidity (RH) conditions. The dry heat treated spores were exposed to saturated water vapor (for 20-24 h at 4 degrees C) prior to recovery in buffer held at two temperatures (0 degrees C and 80 degrees C). Approximately the same level of recovery was obtained at the two buffer temperatures except at the shortest heating times for the heat treatment carried at 30% and 40% RH, where the number of survivors was significantly higher for spores placed in the buffer held at 80 degrees C. The effect of this high temperature was attributed to heat activation of the ascospores that remained dormant during the dry heat treatment conditions mentioned above. The wet heat resistance of N. fischeri ascospores was also determined at temperatures ranging from 82.5 degrees C to 95 degrees C. The results indicate that as the RH decreased, the heat resistance of the ascospores increased. There were about four orders of magnitude difference in the heat resistance between wet heat (100% RH) and the lowest dry heat treatment condition (30% RH).

Factors affecting recovery of Neosartorya fischeri ascospores after exposure to dry heat.

Recovery of Neosartorya fischeri ascospores subjected to a dry heat treatment (DHT) at 95 degrees C, 50% relative humidity (RH) for 60 minutes increased exponentially as the initial temperature of the recovery buffer increased. Different diluents were evaluated and the same recovery pattern was obtained when water or dilute buffers were used to recover the DHT spores. However, when glycerol was added to the buffer, the number of spores recovered in solutions held in ice water increased with increasing glycerol concentration. When the DHT spores were exposed to an atmosphere saturated with water vapor (100% RH) before being placed in the buffer, the recovery was independent of the initial temperature of the buffer. This occurred even if the spores were subsequently dried before being introduced into the buffer. It is hypothesized that the temperature-dependent recovery was due to injury of the DHT spores during the sudden rehydration in dilute solutions at low temperatures.

Evaluation of a Visual DNA Probe for Enterotoxigenic E. coli Detection in Foods and Wastewater by Colony Hybridization.

A visual DNA probe for the detection of enterotoxigenic E. coli (LT EEC) by colony hybridization was evaluated to determine its efficacy as a more restrictive, routine indicator method for foods and wastewater. The E. coli heat labile enterotoxin (LT) gene was used as the DNA probe to detect LT EEC. In control experiments the reliability of the probe was demonstrated with food spiked with LT EEC. Raw oysters and wastewater examined for naturally occurring LT EEC showed significant levels of probe positive isolates. Despite some problems, for example background noise associated with food type, the DNA probe proved satisfactory as a method for indicating the presence of enterotoxigenic Enterobacteriaceae .

Effect of thermal treatments in oils on bacterial spore survival.

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.

Tailing of survivor curves of clostridial spores heated in edible oils.

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7.2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.

Association of [32P] with Clostridium botulinum 52A Vegetative Cells Following Growth in a Medium Containing Sodium Dihydrogen [32P]-Pyrophosphate.

The association of [32P] with Clostridium botulinum 52A vegetative cells following growth in a medium containing either sodium dihydrogen [32P]-pyrophosphate ([32P]-SAPP) or sodium dihydrogen [32P]-orthophosphate ([32P]-orthophosphate) was studied. Absorbency measurements at 630 nm were used in addition to [32P] recovery in determining [32P] association with cellular growth and metabolism. Radiolabeling experiments showed [32P]-orthophosphate was associated with vegetative cells during logarithmic growth, yet was released once stationary phase was attained or upon lysis. [3P]-SAPP was also associated with cells during growth, but was not released once stationary phase was attained. Results suggested [32P]-SAPP continued to bind cells or other metabolic materials following attainment of the stationary phase of cells. Fractionation of 24 and 48 h-old cultures grown in the presence of [32P]-SAPP showed a higher percentage of [32P] associated with the RNA fraction (3.91 and 2.48%, respectively) compared to the DNA fraction (0.09 and 0.07%, respectively).

Histamine Levels in Commercially Processed Fish in Morocco.

Histamine levels were determined in 248 samples of fish commercially processed in Morocco. Concentrations ranging from <0.01 to 694 mg/100 g of fish (mg%) were observed. The mean value was 12.33 mg% (sardines, 9.75; mackerel, 13.74; tuna 9.86) and the standard deviation was 55.28 mg% (sardines, 43.21; mackerel, 71.99; tuna, 25.05). The bulk of the samples (85.5%) had low histamine levels (<10 mg%); 26 samples (10.5%) had levels within the range 10-50 mg% and should be classified as not from fresh fish or of low quality; 10 samples (4%) had toxicologically significant levels, above 50 mg%. Tuna fish was more susceptible to histamine development than were sardines or mackerel; 7% of tuna fish samples contained levels above 50 mg% as compared to 3.7% and 3.2% for sardines and mackerel, respectively. The percentage of samples containing levels above 50 mg% was somewhat higher for fish processed in the central region (7.1%) than the southern (4.3%) or northern (1.3%) regions; however, statistically the regional differences were not significantly different. Histamine development in sardines demonstrated first-order kinetics. Reaction rates ranged from 0.00200 to 0.000421 mn-1. Refrigeration controlled histamine development. Fish held at 8°C showed a shelf life 12 h longer than fish held at 17°C. A combination of salting and refrigeration was more effective. Fish held at 8°C and salted at a level of 5 or 8% showed a shelf life 35 h longer than fish held at 17°C with no salt.

Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate.

Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy. Reduction in pH from 7 to 5.5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B. At pH 5.5, 225 mg/l of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6.5, 225 mg/l of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A. Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5.5, but the addition of sorbate (225 mg/l undissociated sorbic acid) completely inhibited outgrowth. Sorbate inhibited germination of Cl. botulinum and Bacillus cereus spores triggered to germinate by amino acids. Inhibition occurred after germinant binding, as measured by commitment to germinate.

Inhibition of germinant binding by bacterial spores in acidic environments.

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.

Inhibition of Clostridium botulinum 52A toxicity and protease activity by sodium acid pyrophosphate in media systems.

The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP.

Influence of a Minimal Change in pH on Germination of Clostridium botulinum 52A in Media Containing Sodium Acid Pyrophosphate and Potassium Sorbate.

The influence of 0.4% sodium acid pyrophosphate (SAPP) or 0.26% potassium sorbate (PS) on Clostridium botulinum 52A growth and toxicity from spores was studied at two pH levels 5.55 and 5.85. Absorbancy measurements at 630 nm were used in combination with microscopic evaluations and toxin analysis to compare effects of additives on normal cell development. Treatment cultures containing 0.4% SAPP and 0.26% PS at a higher pH of 5.85 showed no increase in absorbancy and no sign of toxicity, but elongated vegetative cells (≥9 µm) were observed using phase contrast microscopy rather than scanning electron microscopy. The SAPP-PS treatment culture at a lower pH of 5.55 displayed no signs of growth spectrophometrically or microscopically, as well as no toxicity. These data suggest that a SAPP-PS combination in a laboratory medium at pH 5.85 does not halt germination and outgrowth, yet may prevent cell division; whereas, the same treatment at pH 5.55 inhibits normal spore germination.