Evaluation of Salmonella thermal inactivation model validity for slow cooking of whole-muscle meat roasts in a pilot-scale oven.

Sublethal heating can increase subsequent thermal resistance of bacteria, which may compromise the validity of thermal process validations for slow-roasted meats. Therefore, this research evaluated the accuracy of a traditional log-linear inactivation model, developed via prior laboratory-scale isothermal tests, and a novel path-dependent model accounting for sublethal injury, applied to pilot-scale slow cooking of whole-muscle roasts. Irradiated turkey breasts, beef rounds, and pork loins were inoculated with an eight-serovar Salmonella cocktail via vacuum tumble marination in a salt-phosphate marinade. The resulting initial Salmonella population in the geometric center (core) was 7.0, 6.3, and 6.3 log CFU/g for turkey, beef, and pork, respectively. Seven different cooking schedules representing industry practices were evaluated in a pilot-scale, moist-air convection oven. Core temperatures recorded during cooking were used to calculate lethality real-time via the log-linear model. The path-dependent model reduced the bias (mean residual) and root mean square error by 4.24 and 4.60 log CFU/g respectively, in turkey; however, the new model did not reduce the prediction error in beef or pork. Overall, results demonstrated that slow-cooked roasts, processed to a computed lethality at or near that required by the regulatory performance standards, as calculated with a state-dependent model, may be underprocessed.

Evaluating the predictive ability of a path-dependent thermal inactivation model for salmonella subjected to prior sublethal heating in ground turkey, beef, and pork.

Pathogen thermal inactivation models currently available to and used by industry consider only the present state of the product when predicting inactivation rates. However, bacteria subjected to sublethal thermal injury can develop partial protection against lethal temperatures. The objective of this study was to extend the capabilities of a previously published path-dependent Salmonella inactivation model by accounting for longer sublethal heating periods and different substrates and to test this new model against independent data. Ground samples of irradiated (> 10 kGy) turkey breast, beef round, and pork loin were inoculated with an eight-serovar Salmonella cocktail and subjected to 53 nonisothermal treatments (in triplicate) that combined a linear heating rate (1, 2, 3, 4, or 7 K/min), a variable length sublethal holding period (at 40, 45, or 50°C), a lethal holding temperature (55, 58, 61, or 64°C), and a nominal target kill (3- or 5-log reductions) (n = 159 for each meat species). When validated against nonisothermal data from similar treatments, traditional state-dependent model predictions resulted in root mean squared errors (RMSEs) of 2.9, 2.2, and 4.6 log CFU/g for turkey, beef, and pork, respectively. RMSEs for the new path-dependent model were 0.90, 0.81, and 0.82 log CFU/g for the same species, respectively, with reductions in error of 63 to 82 % relative to the state-dependent model. This new path-dependent model can significantly reduce error from the state-dependent model and could become a useful tool for assuring product safety, particularly relative to slow heating processes.

A mathematical risk model for Escherichia coli O157:H7 cross-contamination of lettuce during processing.

A stochastic simulation modelling approach was taken to determine the extent of Escherichia coli O157:H7 contamination in fresh-cut bagged lettuce leaving the processing plant. A probabilistic model was constructed in Excel to account for E. coli O157:H7 cross contamination when contaminated lettuce enters the processing line. Simulation of the model was performed using @Risk Palisade© Software, providing an estimate of concentration and prevalence in the final bags of product. Three different scenarios, named S1, S2, and S3, were considered to represent the initial concentration on the contaminated batch entering the processing line which corresponded to 0.01, 1 and 100 cfu/g, respectively. The model was satisfactorily validated based on Standard Error of Prediction (SEP), which ranged from 0.00-35%. ANOVA analysis performed on simulated data revealed that the initial concentration in the contaminated batch (i.e., S1, S2, and S3) did not influence significantly (p=0.4) the E. coli O157:H7 levels in bags derived from cross contamination. In addition, significantly different (p<0.001) prevalence was observed at the different levels simulated (S1; S2 and S3). At the lowest contamination level (0.01 cfu/g), bags were cross-contaminated sporadically, resulting in very low E. coli O157:H7 populations (mean: ≤2 cfu/bag) and prevalence levels (<1%). In contrast, higher average prevalence levels were obtained for S2 and S3 corresponding to 3.05 and 13.39%, respectively. Furthermore, the impact of different interventions on E. coli O157:H7 cross-contamination (e.g., pathogen testing, chlorination, irradiation, and cleaning and disinfection procedures) was evaluated. Model showed that the pathogen was able to survive and be present in the final bags in all simulated interventions scenarios although irradiation (0.5 KGy) was a more effective decontamination step in reducing prevalence than chlorination or pathogen testing under the same simulated conditions.

Single directional migration of Salmonella into marinated whole muscle turkey breast.

Irradiated, whole muscle turkey breasts were cut into blocks measuring 10 by 10 by 6 cm and exposed on one side to a marinade inoculated to contain a cocktail of eight Salmonella serovars at 10(8) CFU/ml. After exposure for 5, 10, or 20 min with or without vacuum (101.3 kPa), cylindrical cores perpendicular to the exposed surface were removed from the blocks with a hand-coring device and subdivided into 1-cm segments. Each segment was macerated, serially diluted in sterile peptone water, and plated to quantify Salmonella. Bacterial migration was greater under vacuum, compared with nonvacuum marination, at 20 min (P < 0.05). When all time levels were pooled within the vacuum and nonvacuum treatments, vacuum processing during marination increased bacterial migration into turkey breast (P < 0.05). This study provides evidence that if bacteria are present on the surface of the muscle, they could migrate into the intact muscle with or without the aid of vacuum.

R-phycoerythrin as a time-temperature integrator to verify the thermal processing adequacy of beef patties.

The objective of this study was to relate R-phycoerythrin (PE) fluorescence decay to the inactivation of Salmonella in beef patties cooked using adequate and inadequate thermal processes as defined by the U.S. Department of Agriculture (USDA) safe harbor requirements and lethality standards. Ground beef containing 4.8 or 19.1% fat was inoculated with an eight-strain cocktail of Salmonella and formed into 113-g patties. Capillary tubes containing PE in borate buffer at pH 9.0 were attached to a thermocouple and inserted horizontally into the patties. Patties (n = 43) were cooked on a grill maintained at 177 degrees C for 6 to 13 min and reached internal temperatures ranging from 57 to 77 degrees C. Patties were analyzed for Salmonella survivors and for fluorescence decay of PE. The thermal lethality of each process was calculated at a reference temperature of 65 degrees C. Twenty-four of the 43 high-fat patties met the USDA safe harbor regulations, with thermal lethalities of >66 s, whereas only 20 of these patties met the proposed 5-log10 lethality standard. Three of the 20 low-fat patties that met USDA regulations did not meet the proposed lethality standard. A normalized PE fluorescence value of about 0.3 (confidence interval = 99%) indicated that patties had been processed sufficiently to reduce Salmonella by 5 log10 cycles. PE has the potential for use as a marker to verify processing adequacy in food-processing plants and in other settings in which the use of the target pathogen is inappropriate.

Incidence and seasonal variation of Listeria species in bulk tank goat's milk.

Four hundred and fifty raw goat's milk samples obtained from the bulk tanks of 39 goat farms were analyzed for Listeria spp. over a 1-year period. Modified versions of the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) and Food and Drug Administration (FDA) protocols were used for recovery of Listeria. Overall, 35 (7.8%) samples yielded Listeria spp. with Listeria monocytogenes identified in 17 of the 35 (3.8%) Listeria-positive samples. Listeria innocua was detected in 26 (5.8%) samples. Eight milk samples contained both L. monocytogenes and L. innocua. Milk samples from 18 of the 39 (46.2%) farms were positive for Listeria at least once during this 1-year study. The modified USDA-FSIS method, which used Listeria repair broth rather than University of Vermont (UVM) broth for primary enrichment followed by a 4-h nonselective incubation period, yielded more Listeria-positive samples (77.1%) than the FDA method (51.4%). All L. monocytogenes isolates belonged to serotypes 1 (62.6%) or 4 (37.4%). Moreover, five different Listeria ribotypes were identified from 34 selected L. monocytogenes isolates, 2 of which were deemed to be of clinical importance. Listeria isolation rates were markedly higher during winter (14.3%) and spring (10.4%) as compared to autumn (5.3%) and summer (0.9%) with these trends similar to those previously reported for cow's milk.

Fate of Listeria monocytogenes, Salmonella typhimurium DT104, and Escherichia coli O157:H7 in Labneh as a pre- and postfermentation contaminant.

Commercially pasteurized milk (approximately 2% milkfat) was heated at 85 to 87 degrees C/30 min, inoculated to contain 2,000 to 6,000 CFU/ml of Listeria monocytogenes, Salmonella typhimurium DT104, or Escherichia coli O157:H7, cultured at 43 degrees C for 4 h with a 2.0% (wt/wt) commercial yogurt starter culture, stored 12 to 14 h at 6 degrees C, and centrifuged to obtain a Labneh-like product. Alternatively, traditional salted and unsalted Labneh was prepared using a 3.0% (wt/wt) starter culture inoculum, similarly inoculated after manufacture with the aforementioned pathogens, and stored at 6 degrees C and 20 degrees C. Throughout fermentation, Listeria populations remained unchanged, whereas numbers of Salmonella increased 0.33 to 0.47 logs during the first 2 h of fermentation and decreased thereafter. E. coli populations increased 0.46 to 1.19 logs during fermentation and remained that these levels during overnight cold storage. When unsalted and salted Labneh were inoculated after manufacture, Salmonella populations decreased >2 logs in all samples after 2 days, regardless of storage temperature, with the pathogen no longer detected in 4-day-old samples. Numbers of L. monocytogenes decreased from 2.48 to 3.70 to < 1.00 to 1.95 logs after 2 days with the pathogen persisting up to 15 days in one lot of salted/unsalted Labneh stored at 6 degrees C. E. coli O157:H7 populations decreased from 3.39 to 3.7 to < 1.00 to 2.08 logs during the first 2 days, with the pathogen no longer detected in any 4-day-old samples. Inactivation rates for all three pathogens in Labneh were unrelated to storage temperature or salt content. Unlike L. monocytogenes that persisted up to 15 days in Labneh, rapid inactivation of Salmonella typhimurium DT104 and E. coli O157:H7 suggests that these emerging foodborne pathogens are of less public health concern in traditional Labneh.

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.

Isolation and identification of cheese-smear bacteria inhibitory to Listeria spp.

A newly developed hydrophobic grid membrane method was used to rapidly screen 105 traditional French cheeses for surface smear microorganisms inhibitory to Listeria monocytogenes strain V7. Approximately 125,000 colonies comprising a wide variety of bacteria were examined of which less than 0.1% produced visible zones of inhibition. Isolates possessing antilisterial activity consisted of various strains of Enterococcus faecalis, Staphylococcus xylosus, Staphylococcus warneri and coryneform bacteria, including one orange coryneform resembling Brevibacterium linens. All strains of E. faecalis and the orange coryneform that inhibited L. monocytogenes V7 exhibited strong inhibition against a panel of 21 Listeria strains comprised of L. monocytogenes (14 strains), L. innocua (two strains), L. ivanovii (two strains), L. seeligeri (two strains) and L. welshimeri (one strain). The remaining cheese isolates showed moderate to weak inhibition towards the same set of Listeria strains. Inhibitory substances produced by all strains except the orange coryneform were sensitive to one or more of five proteolytic enzymes tested and were therefore classified as bacteriocin-like inhibitory agents.

"New" food-borne pathogens of public health significance.

Recent work by epidemiologists and microbiologists has uncovered several hitherto unrecognized food-borne bacterial pathogens of public health significance. One of these, Listeria monocytogenes, has attracted considerable attention because of two major cheese-related outbreaks of listeriosis that were characterized by cases of meningitis, abortion, and perinatal septicemia. Thus far, L. monocytogenes has been responsible for well over 300 reported cases of food-borne listeriosis, including about 100 deaths, and has cost the dairy industry alone more than 66 million dollars as a result of product recalls. The ability of L. monocytogenes to grow at refrigeration temperatures, coupled with appearance of the pathogen in raw and processed meats, as well as poultry, vegetables, and seafood, makes this bacterium a serious threat to susceptible consumers and to the entire food industry. Yersinia enterocolitica, another psychrotrophic food-borne pathogen of recent concern, was linked to several outbreaks of yersiniosis associated with consumption of both raw and pasteurized milk, as well as contaminated water. Food-borne infections involving Y. enterocolitica typically result in enterocolitis, which may be mistaken for acute appendicitis. Unfortunately, inadvertent removal of healthy appendixes from victims of food-borne yersiniosis is all too common. Although known for many years, Campylobacter jejuni has only recently been recognized as a food-borne pathogen and a leading cause of gastroenteritis in the United States. Notable outbreaks of campylobacteriosis linked to consumption of raw milk, cake icing, eggs, poultry, and beef have underscored the need for thorough cooking and proper handling of raw products.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavior of Listeria monocytogenes during manufacture and ripening of brick cheese.

Brick cheese was made by the washed-curd procedure from pasteurized whole milk inoculated to contain ca. 1 x 10(2) to 1 x 10(3) Listeria monocytogenes [strain Scott A, Ohio, V7, or California]/ml. Cheeses were ripened (15 degrees C/95% relative humidity) with a surface smear for 2, 3, or 4 wk to simulate production of mild, aged, or "Limburger-like" brick cheese, respectively, and then stored an additional 20 to 22 wk at 10 degrees C. Populations of strains Scott A, Ohio, V7, and California increased 1.89, 1.72, .83, and .86 orders of magnitude, respectively, following completion of brining ca. 32 h after the start of cheese making. All four L. monocytogenes strains leached from cheese into brine during 24 h and survived in brine at 10 degrees C at least 5 d after removal of cheese. Strains Scott A and Ohio grew rapidly during the initial 2 wk of smear development and attained maximum populations of ca. 6.6 and 6.2, 7.0 and 6.9, and 5.6 and 5.1 log10/g in 4-wk-old slice (pH 6.0 to 6.5), surface (pH 6.5 to 6.9), and interior (pH 5.6 to 6.2) samples of cheese, respectively. Numbers of strains Scott A and Ohio generally decreased 1- to 7-fold during 20 to 22 wk at 10 degrees C. Strains V7 and California failed to grow appreciably in any cheese during or after smear development, despite pH of 6.8 to 7.4 in fully ripened cheese; the strains were never isolated from 2- and 3-wk-old cheese and with direct plating were detected sporadically at levels generally less than or equal to 4.0 log10/g in cheese aged greater than or equal to 4 wk. Cold enrichment of slice, surface, and interior samples of cheeses aged greater than or equal to 4 wk generally yielded positive results for L. monocytogenes; strains V7 and California were detected in all cheeses after 20 to 22 wk at 10 degrees C. At 10 ppm, methyl sulfide, dimethyl disulfide, or methyl trisulfide (compounds commonly produced during ripening of brick and Limburger cheese) failed to inhibit appreciably growth of L. monocytogenes.

Methods for improved recovery of Listeria monocytogenes from cheese.

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.

Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camemberti.

Wheys from making Camembert cheese were either uncultured or cultured with Penicillium camemberti, adjusted to pH 5.0, 5.2, 5.4, 5.6, 6.2, and 6.8, and filter sterilized. Whey samples were inoculated to contain 100 to 500 Listeria monocytogenes (strains Scott A, V7, CA, or OH) cfu/mL and incubated at 6 degrees C. Counts of L. monocytogenes were obtained by surface plating appropriate dilutions on Tryptose Agar. Listeria monocytogenes failed to grow at or below pH 5.4; except for strains Scott A and OH which grew in cultured whey at pH 5.4 and attained populations of 7.8 x 10(3) and 5.4 x 10(4) cfu/mL, respectively, after 35 d of storage. In uncultured whey at pH 5.6, 6.2, and 6.8, populations of L. monocytogenes increased from 7.20 to 7.81, 7.51 to 8.23, and 7.48 to 8.08 log10 cfu/mL, respectively, after 35 d of storage at 6 degrees C. In cultured whey at pH 5.6, 6.2, and 6.8, numbers of L. monocytogenes increased from 7.53 to 8.13, 7.82 to 8.55, and 7.95 to 8.80 log10 cfu/mL, respectively, after 35 d of storage. Generation times for L. monocytogenes at 6 degrees C in uncultured whey at pH 5.6, 6.2, and 6.8 ranged between 25.3 and 31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the Listeria strain. In contrast, generation times were significantly (p less than 0.05) shorter in cultured whey and ranged between 16.6 and 27.4 h, 10.3 and 16.6 h, and 17.4 and 16.3 h at pH values of 5.6, 6.2, and 6.8, respectively.

Survival of Listeria monocytogenes in Cold-Pack Cheese Food During Refrigerated Storage.

Duplicate lots of cold-pack cheese food were manufactured, according to nine different formulations, inoculated to contain ca. 5 × 102 Listeria monocytogenes (strains Scott A, V7, California or Ohio) colony forming units (CFU)/g and stored at 4°C. L. monocytogenes in cheese food was enumerated by surface-plating appropriate dilutions made in tryptose broth containing 2% (w/v) sodium citrate (TBC) on McBride Listeria Agar (MLA). Initial TBC dilutions were stored at 3°C and surface-plated on MLA after 2, 4, 6 and 8 weeks if the organism was not quantitated by direct plating of the original samples. Selected Listeria colonies were confirmed biochemically. Populations of L. monocytogenes in cheese food manufactured without preservatives or acidifying agents generally decreased less than 10-fold after 182 d of storage. However, numbers of L. monocytogenes steadily decreased in cheese food containing 0.30% sorbic acid or 0.30% sodium propionate and which was acidified to pH 5.0 to 5.1 with lactic and/or acetic acid. In cheese food preserved with 0.30% sorbic acid, L. monocytogenes survived an average of 130 d in non-acidified cheese food and 112, 93 or 74 d in cheese food acidified with lactic acid, lactic plus acetic acid, or acetic acid, respectively. When 0.30% sodium propionate was substituted for sorbic acid, L. monocytogenes survived an average of 142 d in non-acidified cheese food and 118, 103 or 98 d in cheese food acidified with lactic, acetic, or lactic plus acetic acid, respectively.