Inactivation of Shiga toxin-producing Escherichia coli in lean ground beef by gamma irradiation.

In this study the radiation resistance of 40 Shiga Toxin-Producing Escherichia coli (STEC) isolates which contained various combinations of the shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eae), and hemolysin (ehx) genes were determined. The STEC were suspended in lean ground beef and irradiated at 4 °C. D10 values, the radiation dose needed to reduce 1 log (90%) of a microorganism, ranged from 0.16 to 0.48 kGy, with a mean of 0.31 kGy for the 40 isolates. Isolates associated with illness outbreaks had a mean D10 of 0.27 kGy, while non-outbreak isolates had a mean D10 of 0.36 kGy (p < 0.05). The presence or absence of stx1, stx2, or both stx1 and 2 had no affect on D10 (p > 0.05). The presence (0.30 kGy) or absence (0.35 kGy) of ehx had no affect on D10 (p > 0.05). However, the mean D10 of isolates lacking eae (0.37 kGy) were significantly higher than those containing eae (0.27 kGy) (p < 0.05). There was no difference in D10 for isolates lacking eae regardless of whether or not they were associated with a foodborne illness outbreak (p > 0.05). It may be possible to use some of the STEC isolates which lacked eae, ehx, or both (D10 > 0.30) as avirulent surrogates in food irradiation research. The data presented in this study provides risk assessors data for metagenomic analysis as well as food and radiation processors with valuable information to control of STEC in meat.

Survival after cryogenic freezing of Campylobacter species in ground Turkey patties treated with polyphosphates.

The use of polyphosphate-based marinades in the processing of poultry has been previously shown to increase the survival of Campylobacter species present in the exudates derived from these products. This study investigates the effects that some of the same polyphosphates have on the survival of Campylobacter species within a ground turkey product subjected to cryogenic freezing. Ground turkey patties with two different polyphosphate formulations added in two different concentrations were artificially contaminated with known concentrations of Campylobacter jejuni or Campylobacter coli. The patties were cryogenically frozen at -80°F (-62.2°C) with liquid nitrogen vapor and held at -20°C for 7 or 33 days, after which the number of Campylobacter surviving in the patties was determined. On average the cryogenic freezing resulted in a 2.5-log decrease in the survival of C. jejuni cells and a 2.9-log decrease in C. coli cells present in the turkey patties. Additionally, the presence of polyphosphates in the turkey patties had no effect on Campylobacter survival up to the maximum allowed concentration (0.5%) for polyphosphates in poultry marinades. Finally, it was determined that the added polyphosphates had little effect on the pH of the ground turkey meat; an effect which previously had been implicated in the enhancement of Campylobacter survival due to the presence of polyphosphates.

Effect of anolyte on background microflora, Salmonella, and Listeria monocytogenes on catfish fillets.

Near-neutral electrolyzed water (anolyte), having a pH of 6.0 to 6.5 ± 0.02, oxidation reduction potential of greater than 700 mV, and a residual chlorine level of 10 to 200 ppm, was reported to have a potential use to decontaminate food surfaces. An electrolyzing cell was developed that is capable of producing neutral electrolyzed water containing a chlorine level of greater than 700 ppm in the form of hypochlorous acid (anolyte). Anolyte with a chlorine level of 300 ppm was used to determine its effect on Salmonella and Listeria monocytogenes cells after a 3-min contact. Transmission electron micrograph results showed disruption of the outer cellular membrane for both bacteria. The anolyte (300 ppm) was used as a washing solution to decontaminate catfish fillets inoculated with either Salmonella or L. monocytogenes. After a 3-min contact time with the anolyte, there was a 1-log reduction for Salmonella, and after 8 days of refrigerated storage (4°C), this bacterial reduction was maintained. There was no reduction of L. monocytogenes on the catfish fillet surfaces. The anolyte was an effective wash solution for Salmonella reduction on the catfish fillet surfaces.

Thermal inactivation of Escherichia coli O157:H7 and Salmonella on catfish and tilapia.

Thermal inactivation kinetics of individual cocktails of Escherichia coli O157:H7, or of Salmonella meat isolates or seafood isolates were determined in catfish and tilapia. Determinations were done at 55, 60 and 65 °C using a circulating-water bath and calculated using linear regression analysis. Salmonella seafood and meat isolates D-10 values on the finfish were the same and ranged from 425 to 450, 27.1 to 51.4, 2.04-3.8 s (z = 4.3 °C) at 55, 60 and 65 °C, respectively. The E. coli O157:H7 D-10 values ranged from 422 to 564, 45.2 to 55.5 and 3.3-4.2 s (z = 4.3 °C) at 55, 60 and 65° C, respectively. The only statistical difference (P ≤ 0.05) was found when comparing the D-10 values for E. coli O157:H7 at 55 °C on catfish and tilapia. The other D-10 values for the Salmonella at all temperatures and E. coli O157:H7 at 60 and 65 °C on the catfish or tilapia showed no statistical difference. D-10 values for the catfish and tilapia were significantly lower than the reported values in other food systems, but the z-values were within the literature reported range. These D-10 values can be used to determine cooking parameters of finfish.

Radiation inactivation of foodborne pathogens on frozen seafood products.

Foodborne illness due to consumption of contaminated seafood is, unfortunately, a regular occurrence in the United States. Ionizing (gamma) radiation can effectively inactivate microorganisms and extend the shelf life of seafood. In this study, the ability of gamma irradiation to inactivate foodborne pathogens surface inoculated onto frozen seafood (scallops, lobster meat, blue crab, swordfish, octopus, and squid) was investigated. The radiation D(10)-values (the radiation dose needed to inactivate 1 log unit of a microorganism) for Listeria monocytogenes, Staphylococcus aureus, and Salmonella inoculated onto seafood samples that were then frozen and irradiated in the frozen state (-20°C) were 0.43 to 0.66, 0.48 to 0.71, and 0.47 to 0.70 kGy, respectively. In contrast, the radiation D(10)-value for the same pathogens suspended on frozen pork were 1.26, 0.98, and 1.18 kGy for L. monocytogenes, S. aureus, and Salmonella, respectively. The radiation dose needed to inactivate these foodborne pathogens on frozen seafood is significantly lower than that for frozen meat or frozen vegetables.

Use of 1% peroxyacetic acid sanitizer in an air-mixing wash basin to remove bacterial pathogens from seeds.

To achieve the production of pathogen-free sprouts, there must be appropriate mixing of liquid sanitizer with the seeds to assure contact. Commercial treatments by irradiation or ozone gas of Salmonella spp. artificially inoculated seeds were compared, and these resulted in a 1 log reduction after all treatments. Use of peroxyacetic acid (1%) sanitizer on Salmonella spp. or Escherichia coli O157:H7 inoculated alfalfa seeds consistently resulted in a greater than 1 log reduction. In addition, during these studies debris was noted after the seeds were removed. Based on this observation, an air-mixing wash basin was developed for commercial use. Validation was done by commercial growers using 1% peroxyacetic acid sanitizer to wash seeds in the air-mixing basin, followed by sprouting the seeds. No positive or false-positive pathogen results were reported after the required testing of the sprout water (run-off during sprouting). Use of 1% peroxyacetic acid sanitizer in the air-mixing wash basin does provide the sprout grower an effective means of sanitizing sprout seeds.

Radiation D10-values on thawed and frozen catfish and tilapia for finfish isolates of Listeria monocytogenes.

With the popularity of catfish and tilapia in the healthy diet, the consumption and harvesting of farm-raised finfish have increased. Since 1987 the pathogenic bacterium Listeria monocytogenes has been isolated from seafood, particularly farm-raised catfish in the United States. Seafood isolates of L. monocytogenes are now available. In order to maintain the raw finfish product, nonthermal interventions to remove bacterial pathogens need to be evaluated using these isolates. A nonthermal intervention process, irradiation, was used to determine the destruct values of the L. monocytogenes seafood isolates along with a nonpathogenic Listeria strain and an L. monocytogenes strain previously studied. The irradiation destruct values were obtained for each individual isolate inoculated on raw and frozen catfish or tilapia irradiated at 4 or -10 degrees C. The Dradiation values obtained for L. monocytogenes inoculated on raw or frozen catfish did not differ (P > 0.05) from the values obtained for strains inoculated on the raw or frozen tilapia. The Dradiation-values ranged from 0.48 to 0.85 kGy, with an average of 0.62 +/- 0.09 kGy, which is typical for Listeria. The data obtained have identified a multi-isolate cocktail that can be used for future radiation inactivation studies for L. monocytogenes inoculated on finfish.

Effect of gamma or beta radiation on Salmonella DT 104 in ground pork.

Mixtures of six Salmonella Typhimurium DT 104 strains were inoculated into three ground pork products to determine the effect of fat content on the radiation resistance of Salmonella DT 104. The ground pork products were 90% lean, 50:50 fat:lean, and 100% fat. Inoculated products were irradiated using a gamma radiation source in a self-contained 137Cesium irradiator or a 10 MeV accelerator producing electrons (e-beam). The radiation D10-values (dose required for a 90% inactivation of viable CFU) for Salmonella DT 104 inoculated into 90% lean ground pork, 50:50 fat/lean ground pork, and 100% pork fat and subjected to beta radiation were 0.42 kGy, 0.43 kGy, and 0.43 kGy, respectively. The corresponding radiation D10-values for Salmonella DT 104 subject to gamma radiation were 0.56, 0.62, and 0.62 kGy, respectively. There was no statistical significant difference (P = 0.3) in radiation D10-values for Salmonella in the three products subject to either radiation treatment. Therefore, fat content had no effect. There was a significant difference (P = 0.001) between the radiation D10-values obtained with the two radiation sources. The radiation D10-values were within the reported range for irradiation destruction of Salmonella contaminated raw meat products.

Effect of alfalfa seed washing on the organic carbon concentration in chlorinated and ozonated water.

The bioassays assimilable organic carbon (AOC) and coliform growth response are better indexes than biological oxygen demand to determine water quality and water's ability to support the growth of bacteria. Ozonated (5 mg/liter) and chlorinated tap water were used to wash alfalfa seeds for 30 min. After washing in the ozonated tap water, the AOC concentration increased 25-fold, whereas the dissolved ozone decreased to undetectable levels. The AOC levels for the chlorinated water after washing the seeds also increased. These increases are due to ozone's strong oxidizing ability to break down refractory, large-molecular-weight compounds, forming smaller ones, which are readily used as nutrient sources for microorganisms. This same phenomenon was observed when using ozone in the treatment of drinking water. The AOC value increased from 1,176 to 1,758 micrograms C-eq/liter after the reconditioned wastewater was ozonated. When the ozonated wastewater was inoculated with Salmonella serotypes, the cells survived and increased generation times were observed. The increased nutrients would now become more readily available to any pathogenic microorganisms located on alfalfa seed surface as seen with the increase in the inoculated levels of Salmonella in the ozonated wastewater. If the washing process using ozonated water is not followed by the recommended hypochlorite treatment or continually purged with ozone, pathogen growth is still possible.

Simplified qualitative method for canavanine in seeds and sprouts.

The major stored nitrogen compound in alfalfa seeds is canavanine. To identify this nonprotein amino acid from seed extract and sprout water, a qualitative micro-thin-layer chromatography method was developed. Successful separation and identification was achieved using microsilica plates, a 70:30 ethyl alcohol-water solvent system, and 1% ammonium disodium pentacyanoammineferrate II for color development. This quick method was used to identify canavanine (sensitivity 50 microg) from irradiated and nonirradiated alfalfa and clover seed extracts and alfalfa sprout water. Broccoli and radish seed extracts were negative for canavanine. This simple method is useful to track the release and decrease of canavanine in the sprout water.

Irradiation D-values for Escherichia coli O157:H7 and Salmonella sp. on inoculated broccoli seeds and effects of irradiation on broccoli sprout keeping quality and seed viability.

Like alfalfa sprouts, broccoli sprouts can be a vehicle for bacterial pathogens, which can cause illness when they are consumed. The gamma irradiation process was used to reduce numbers of bacterial pathogens on broccoli sprouts and seeds, and the effect of this process on the seeds was studied. The irradiation destruct values for Salmonella sp. and for strains of Escherichia coli O157:H7 inoculated on broccoli seeds were determined. Results obtained in this study indicate that a dose of 2 kGy reduced total background counts for broccoli sprouts from 10(6) to 10(7) CFU/g to 10(4) to 10(5) CFU/g and increased the shelf life of the sprouts by 10 days. Yield ratio (wt/wt), germination percentage, sprout length, and thickness were measured to determine the effects of various irradiation doses on the broccoli seeds. Results show a decreased germination percentage at a dose level of 4 kGy, whereas the yield ratio (wt/wt), sprout length, and thickness decreased at the 2-kGy dose level. The radiation doses required to inactivate Salmonella sp. and strains of E. coli O157:H7 were higher than previously reported values. D-values, dose required for a 1-log reduction, for the nonvegetable and vegetable Salmonella sp. isolates were 0.74 and 1.10 kGy, respectively. The values for the nonvegetable and vegetable isolated strains of Escherichia coli O157:H7 were 1.43 and 1.11 kGy, respectively. With the irradiation process, a dose of up to 2 kGy can extend the shelf life of broccoli sprouts. A dose of > 2 kGy would have an adverse effect on the broccoli seed and decrease the yield of broccoli sprouts.

Warm water treatment in combination with modified atmosphere packaging reduces undesirable effects of irradiation on the quality of fresh-cut iceberg lettuce.

Fresh-cut iceberg lettuce dipped in either 5 or 47 degrees C water for 2 min was packaged in modified atmosphere film bags and then exposed to 0, 0.5, 1, or 2 kGy gamma-radiation. Dipping cut lettuce in 47 degrees C water for 2 min prior to irradiation reduced antioxidant and phenolic accumulations induced by irradiation. Irradiation at 2 kGy increased cellular leakage and sogginess of cut lettuce dipped in both temperatures. Samples irradiated at 0.5 and 1 kGy had similar firmness and vitamin C and antioxidant contents as the controls after 14 and 21 days of storage except 1 kGy samples dipped at 47 degrees C had lower antioxidant contents than controls at 14 days of storage. Lettuce dipped at 47 degrees C and irradiated at 0.5 and 1 kGy had better overall visual quality and less tissue browning than corresponding irradiated samples dipped at 5 degrees C. These results suggest lettuce treated with warm water and irradiated at 0.5 or 1 kGy had the best sensory quality without significant loss in texture, vitamin C, or total antioxidants.

Inactivation of Escherichia coli O157:H7 and Salmonella by gamma irradiation of alfalfa seed intended for production of food sprouts.

Inonizing irradiation was determined to be a suitable method for the inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the production of food sprouts. The radiation D (dose resulting in a 90% reduction of viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on alfalfa seeds were higher than the D-values for their inactivation on meat or poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for vegetable-associated isolates were not significantly different. The D-values for nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55 +/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the relatively high D-values were not due to the low moisture content or the low water activity of the seed. The D-values for Salmonella on alfalfa seeds from two different sources did not differ significantly, even though there were significant differences in seed size and water activity. The increased moisture content of the seed after artificial inoculation did not significantly alter the D-value for the inactivation of Salmonella. The results of this study demonstrate that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing radiation, which will permit satisfactory commercial yields of sprouts from alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.

Growth of Salmonella spp. and Vibrio cholerae in Reconditioned Wastewater †.

Many food-processing plants are looking to increase the use of reconditioned water beyond the currently approved uses for initial cleaning (vegetables) and scalding water (meat and poultry). The preliminary survey showed that the reconditioned water from a local meat plant could support bacterial growth. The growth potential of Salmonella spp. and Vibrio cholerae (starting level of 3 to 4 log CFU/ml) in the reconditioned wastewater from this plant (with and without added thiosulfate) was studied at temperatures from 5 to 42°C. Bioassays for the assimilable organic carbon and coliform growth response suggest that this reconditioned water contained sufficient nutrients to support bacterial growth. Both pathogens grew in the unchlorinated reconditioned and chlorinated reconditioned water containing 10 mg of sodium thiosulfate per ml to neutralize the residual chlorine. Cell counts declined rapidly in chlorinated water without thiosulfate. The results of this study emphasized the importance of maintaining residual chlorine levels (0.2 mg/liter) in both reconditioned and potable waters to prevent pathogen growth.

Growth of Escherichia coli O157:H7 at Fluctuating Incubation Temperatures.

Temperature abuse of foods is often transitory and little information is available describing the response of the foodborne pathogen, Escherichia coli O157:H7, to nonisothermal and/or fluctuating temperature storage. Growth responses were determined for a mixture of three E. coli O157:H7 strains in brain heart infusion (BHI) broth as a function of temperature (static and fluctuating), initial pH (5, 6, and 7), and NaCl content (0.5, 1, 2, and 3%). Five 6-h "square-wave" fluctuating temperature regimes were used: 4 to 12, 4 to 19, 4 to 28, 8 to 19, and 12 to 28°C and compared with growth at 8, 10, 12, 19, and 28°C. The growth curves obtained from fitting the Gompertz equation for the fluctuating temperatures were compared to those obtained for the static temperatures. Increased NaCl concentration decreased growth temperature both for the fluctuating temperature growth curves and the static growth data. The cells grew or remained viable for up to 21 days under all conditions and fluctuating temperatures. Growth kinetics at fluctuating temperatures more closely approximated the higher temperature than the midpoint temperature of each cyclic range. The results indicate that transitory abuse could lead to more rapid growth than expected of E. coli O157:H7 in foods and that given sufficient time E. coli O157:H7 can grow at as low as 8°C.

Detectability of Erythromycin in Eight Milk Products Using the AOAC Bacillus stearothermophilus Disc Assay.

A comparison was made of the ability of the AOAC Bacillus stearothermophilus disc method using Antibiotic Medium 4(A4) and Penicillin in Milk (PM) assay agar to detect erythromycin in eight milk products. PM assay agar outperformed A4 agar by 470%, producing an average detectable level of 0.26 ppm (range 0.16-0.37 ppm), and is the medium of choice for the detection of erythromycin by this method.

Characteristics of Selected Strains of Bacillus cereus.

Identification techniques for Bacillus cereus are unsettled even though there is an increased awareness of the organism's potential public health implications. Biochemical and morphological characteristics of 17 strains of B. cereus , including 10 isolated from confirmed foodborne outbreaks, were studied by routine methods. Of the characteristics attributed to B. cereus , two strains were negative for the Voges-Proskauer reaction and nitrate reduction; three did not utilize salicin; and five exhibited rhizoidal growth on nutrient agar. Heat resistance of the strains was determined using the serum bottle technique. In demineralized water D- values at 100°C ranged from 0.6 to 27 min, with z-values from 7.4 to 14.5°C. Mean growth constant (k/h) determined turbidimetrically in nutrient broth at 15, 21, 25, 35, 40, 45 and 50°C was 0.15, 0.39, 0.89, 1.54, 1.99, 2.54 and 2.08, respectively. No single feature typified pathogenic strains. At 7 or 11°C, sixteen strains produced hemolysin on blood agar plates, whereas at 45°C, only two strains were hemolytic. Phospholipase activity measured on egg yolk agar plates was evident for three strains at 7°C, for all strains at 35°C, and for only two strains at 45°C.

Applicability of the Bacillus stearothermophilus Disc Assay for Penicillin Residues in Casein/Caseinates.

Six buffer systems were examined as hydrating solutions for assaying antibiotic residues in bovine casein or caseinates by the qualitative Bacillus stearothermophilus disc assay method. Formic acid and 1% potassium phosphate buffer systems were suitable in that they did not react adversely with the B. stearothermophilus spores or cause degradation of penicillin. With the formic acid buffer, a 20% casein slurry and a 10% caseinate slurry were sufficiently fluid to allow capillary saturation of a 12.7-mm paper disc. Casein and caseinate rehydrated with 1% potassium phosphate buffer were too viscous to permit saturation of a disc. The detectable level in a 20% casein slurry and a 10% caseinate slurry was ≥0.004 IU penicillin G/ml. Casein and caseinate prepared from potassium or procaine penicillin G-contaminated skim milk contained no detectable level of antibiotics as determined by the B. stearothermophilus disc assay method.

Detectability Levels of Four Beta-Lactam Antibiotics in Eight Milk Products Using the AOAC Bacillus stearothermophilus Disc Assay.

The 50% dectability level (ED50) of the Bacillus stearothermophilus disc assay in raw, pasteurized whole, protein-fortified lowfat, lowfat, and skim milks, half-and-half, heavy cream and goat's milk was determined for penicillin G, ampicillin, cloxacillin and cephapirin. Results demonstrate a lower level of detectability with PM agar than with A4 agar for ampicillin, cloxacillin and penicillin. Ranges of detection using PM agar at 64°C were 0.0025 to 0.0042 IU/ml (0.0016 to 0.0026 µg/ml) for penicillin G, 0.0021 to 0.0042 µg/ml for ampicillin, 0.0030 to 0.0059 µg/ml for cephapirin and 0.0167 to 0.0334 µg/ml for cloxacillin. Liquid penicillinase is recommended when performing the confirmation test for beta-lactam identification.