Validation of a Simulated Commercial English Muffin Baking Process to control Salmonella Contamination.

A validation study was conducted to investigate the effect of the English muffin baking process to control Salmonella contamination and to study the thermal inactivation kinetic parameters (D- and z-values) of Salmonella in English muffin dough. The unbleached bread flour was inoculated with 3 serovar Salmonella cocktail (Salmonella serovars viz., Newport, Typhimurium, and Senftenberg), and dried back to its preinoculated water activity levels with 7.46 ± 0.12 log CFU/g of Salmonella concentration. The Salmonella inoculated flour was used to prepare English muffin batter and baked at 204.4°C (400°F) for 18 min and allowed to cool at ambient air for 15 min. The English muffins reached 99 ± 0°C (211.96 ± 0.37°F) as their maximum mean internal temperature during baking. The pH and aw of English muffin dough were 5.01 ± 0.01 and 0.947 ± 0.003, respectively. At the end of the 18-min baking period, the Salmonella inoculated English muffins recorded a more than 5 log CFU/g reduction on the injury-recovery media. The D-values of 3 serovar cocktails of Salmonella at 55, 58.5, and 62°C were 42.0 ± 5.68, 15.6 ± 0.73, and 3.0 ± 0.32 min, respectively; and the z-value was 6.2 ± 0.59°C. The water activity (aw) of the English muffin crumb (0.947 ± 0.003 to 0.9557 ± 0.001) remained statistically unchanged during baking, whereas the aw of the muffin crust decreased significantly (0.947 ± 0.003 to 0.918 ± 0.002) by the end of 18 min of baking. This study validates and documents the first scientific evidence that baking English muffins at 204.4°C (400°F) for 18 min acts as an effective kill step by controlling Salmonella population by >5 log CFU/g.

Validation of baking as a kill-step for controlling Shiga toxin-producing Escherichia coli during traditional crust pizza baking process.

A study was conducted to validate a simulated traditional crust pepperoni pizza baking process to control Shiga toxin-producing Escherichia coli (STEC) and to determine the heat resistance characteristics of STEC in pizza dough. Pizza dough and pepperoni slices were inoculated with 7 strains STEC cocktail and baked at 500°F (260°C) for 12 min using a conventional kitchen oven followed by 15 min of ambient air cooling. The mean internal temperature of the pizza reached 209.32 ± 1.94°F by the end of 12 min of baking and dropped to 137.90 ± 2.88°F after the 15 min ambient air cooling. The aw and pH of the traditional crust pizza did not alter significantly during the baking process. The STEC population decreased by >5 log CFU/g in traditional crust pizza after 12 min of baking. Where pepperoni slices were used as a source of STEC introduction, a reduction of >6.5 log CFU/g was observed. The D-values of STEC cocktail in pizza dough at 55, 58, 61°C were 49.5 ± 4.10, 15.3 ± 0.68, and 2.8 ± 0.31 min, respectively. The z-value of STEC was 4.8 ± 0.16°C. This study validated that a typical traditional crust pizza baking process with ~209°F internal temperature for at least 12 min will result in 5 log reductions in STEC population.

Survivability and thermal resistance of Salmonella and Escherichia coli O121 in wheat flour during extended storage of 360 days.

Foodborne pathogens like Salmonella and Escherichia coli O121 can endure the harsh low water activity (aw) environment of wheat flour for elongated periods of time and can proliferate when hydrated for baking or other purposes. This study determined the survivability and thermal tolerance (D- and z-values) of Salmonella and Escherichia coli O121 in wheat flour and muffin batter (prepared from inoculated flour on the days of analyses) during the storage period of 360 days. The Salmonella and E. coli O121 studies were conducted as two independent experiments. Both studies were designed as randomized complete block with three replications as blocks. All experimental data were analyzed using one-way ANOVA and Tukey's test in Minitab® software, and P ≤ 0.05 was considered significant. The wheat flour was spray inoculated individually with 7-isolate Salmonella or 3-isolate E. coli O121 cocktail and then dried back to the original aw levels. On each analysis day, inoculated wheat flour (~5 g) or muffin batter (~2.5 g) was placed inside the TDT disks, heat treated at set temperatures in hot water baths, and sampled at predetermined time intervals for determining the survival microbial population. The population of E. coli O121 and Salmonella cocktails in wheat flour at day 1 were 7.6 ± 0.18 and 7.8 ± 0.07 log CFU/g, respectively, which decreased to 2.0 ± 0.40 and 2.8 ± 0.59 log CFU/g on day 360, respectively. The D-values of Salmonella and E. coli O121 cocktails in inoculated flour and muffin batter prepared from inoculated flour (on the day of analysis) were determined on days 1, 30, 90, 180, 270, and 360 [given enough surviving bacterial population (~3 to 4 log CFU/g) was present in the flour]. The population of Salmonella and E. coli O121 in wheat flour decreased by 5.0 and 5.6 log CFU/g, respectively, during the storage period of 360 days. The D70°C, D75°C, and D80°C values of Salmonella in wheat flour remained similar during the storage period. Whereas, for E. coli O157:H7 in wheat flour, the D70°C value decreased from 20.3 ± 2.82 to 7.1 ± 2.82 min, and D75°C decreased from 10.2 ± 2.14 to 2.7 ± 0.27 min, during the storage period of 180 days. The z-values of Salmonella or E. coli O157:H7 remained similar during the storage period. The D- and z-values from this research can be employed for validation of thermal process to ensure safety of wheat flour.

Thermal inactivation of Salmonella during hard and soft cookies baking process.

This study validated a simulated commercial baking processes for hard and soft cookies to control Salmonella, and determined D- and z-values of 7-serotype Salmonella (Newport, Senftenberg, Tennessee, Typhimurium, and three isolates from dry pet food) cocktail in cookie doughs. Cookie doughs were prepared using flour mist-inoculated with the Salmonella cocktail. Hard and soft cookies were baked at 185 °C for 16 min and 165.6 °C for 22 min, respectively, followed by 30 min of ambient air cooling. D-values of the cocktail in cookie doughs were determined using thermal-death-time disks. Studies were designed as randomized complete blocks with three replications as blocks (α = 0.05). Salmonella populations decreased by > 5 log CFU/g in hard and soft cookies at 11.5 and 20.5 min of baking, respectively. Salmonella was not detected in hard cookies at the end of baking (as determined by enrichment), whereas in soft cookies, 0.6 log CFU/g Salmonella was present at the end of baking and cooling. Salmonella D-values in hard cookie dough at 60, 65 and 70 °C were 59.6, 28.1 and 11.9 min, respectively; while in soft cookie dough they were 62.3, 28.6 and 14.4 min, respectively. The Salmonella z-values in hard and soft cookie doughs were 14.5 and 15.8 °C, respectively.

Validation of brownie baking step for controlling Salmonella and Listeria monocytogenes.

Pathogens, such as Salmonella and Listeria monocytogenes, can survive under the dry environment of flour for extended periods of time and could multiply when flour is hydrated to prepare batter or dough. Therefore, inactivation of these pathogens during the cooking/baking step is vital to ensure the microbiological safety of bakery products such as brownies. The aim of this research was to validate a simulated commercial baking process as a kill-step for controlling Salmonella and L. monocytogenes in brownies and to determine thermal inactivation parameters of these pathogens in brownie batter. Independent studies were conducted in a completely randomized design for each pathogen. All-purpose flour was inoculated with a 5-serovar Salmonella and 3-strain L. monocytogenes cocktails. For baking validation, brownie batters were prepared from inoculated flour, and cooked in the oven set at 350°F (176.7°C) for 40 min followed by 15 min of ambient air cooling. For calculating D-values, brownie batter was transferred into thermal-death-time disks, sealed, and placed in hot-water baths. The samples were held for pre-determined time intervals in hot-water baths and immediately transferred to cold-water baths. Microbial populations were enumerated using injury-recovery media. At the end of baking, Salmonella and L. monocytogenes populations decreased by 6.3 and 5.9 log CFU/g, respectively. D-values of Salmonella and L. monocytogenes cocktails were 53.4 and 37.5 min at 64°C; 27.2 and 16.9 min at 68°C; 10.7 and 9.1 min at 72°C; and 4.6 and 7.3 min at 76°C; respectively. The z-values of Salmonella and L. monocytogenes cocktails were 11.1 and 16.4°C, respectively. This study can be used as a supporting document for the validation of similar brownie baking processes to control Salmonella and L. monocytogenes. The data from this study can also be employed for developing basic prediction models for the survival and thermal resistance of these pathogens during brownie baking step.

Comparison of survival and heat resistance of Escherichia coli O121 and Salmonella in muffins.

This study was conducted to validate a simulated commercial baking process for plain muffins against E. coli O121 (isolated from the recent illness outbreak associated with flour), and compare the thermal inactivation parameters (D- and z-values) of cocktails of four isolates of E. coli O121 and three serovars of Salmonella (Newport, Typhimurium, and Senftenberg) in muffin batter. Flour samples were spray inoculated with the E. coli O121 or Salmonella cocktails, dried back to the pre-inoculation weight to achieve ~7 log10 CFU/g, and used to prepare muffin batter. For the muffin baking validation study using E. coli O121, muffin batter was baked at 375 °F (190.6 °C) oven temperature for 21 min followed by 30 min of ambient cooling. The E. coli O121 population decreased by >7 log10 CFU/g in muffins by 17 min of baking, and was completely eradicated after 21 min of baking and ambient cooling. The D-values of E. coli O121 and Salmonella cocktails in muffin batter at 60, 65 and 70 °C were 42.0 and 38.4, 7.5 and 7.2, and 0.4 and 0.5 min, respectively; whereas the z-values of E. coli O121 and Salmonella were 5.0 and 5.2 °C, respectively.

Evaluation of thermal inactivation parameters of Salmonella in whole wheat multigrain bread.

This study was conducted to validate a simulated commercial whole wheat multigrain bread baking process at 375 °F (190.6 °C) oven temperature for 35 min to inactivate Salmonella, and to determine the thermal inactivation parameters of a 7-serovar Salmonella cocktail in whole wheat multigrain bread dough. A ≥5-log CFU/g reduction in Salmonella population was achieved by 15 min, and no viable Salmonella was detected after enrichment plating by 16 min. The aw of the bread crumb (0.96) after baking and 60 min of cooling was similar to that of pre-baked bread dough, whereas the aw of bread crust decreased to 0.81 at the end of baking and cooling. The D-values of the Salmonella cocktail in bread dough were 59.6, 20.0 and 9.7 min at 50, 52 and 55 °C, respectively; and the z-value was 6.5 °C.

Validation of a nut muffin baking process and thermal resistance characterization of a 7-serovar Salmonella inoculum in batter when introduced via flour or walnuts.

This study was conducted to validate a commercial nut muffin baking process and to compare the survival of a 7-serovar Salmonella cocktail when contaminated via inoculated flour or walnuts. Enriched wheat flour or walnut pieces were mist inoculated with the Salmonella cocktail and dried back to the pre-inoculation weight, resulting in a Salmonella population level of 6.9 and 8.4 log CFU/g, respectively. Nut muffin batters were prepared separately using inoculated flour or walnuts, followed by baking at 375 °F (190.6 °C) oven temperature for 21 min and post-bake ambient air-cooling (B + C). During baking, >5-log CFU/g reductions in the Salmonella population in nut muffins was achieved in 17 min, and Salmonella was not detected by direct plating (<0.2 log CFU/g detection limit) but was recovered by enrichment at the end of 21 min of baking and B + C. In a separate baking study using an extended baking time (24 min) at 375 °F, Salmonella was detected after 24 and 22 min using enrichment plating of nut muffins prepared from inoculated flour and walnuts, respectively. The D-values of the Salmonella cocktail in nut muffin batters prepared from inoculated flour were 24.0, 4.0 and 0.6 min at 60, 65 and 70 °C; whereas, corresponding D-values in batters prepared from inoculated walnuts were 22.0, 3.6 and 1.7 min. The z-values of the Salmonella cocktail in nut muffin batters were 6.1 and 9.0 °C for inoculated flour and walnuts, respectively. This simulated commercial nut muffin baking study utilizing an oven temperature of 190.6 °C for at least 17 min validates that the process will eliminate Salmonella populations by ≥5 log CFU/g if pre-baking contamination occurs via flour or walnut ingredients.

Validation of a Simulated Commercial Frying Process to Control Salmonella in Donuts.

This study validated a typical commercial donut frying process as an effective kill-step against a 7-serovar Salmonella cocktail (Newport, Typhimurium, Senftenberg, Tennessee, and three dry food isolates) when contamination was introduced through inoculated flour. The bread and pastry flour mix (3:1) was inoculated with the Salmonella cocktail, and subsequently dried back to original preinoculation moisture content, achieving a Salmonella population of 7.6 log CFU/g. Inoculated flour was used to prepare a typical commercial donut batter, which was fried using 375°F (190.6°C) oil temperature. No viable Salmonella was detected using an enrichment plating protocol in the donuts after 2 min of frying, resulting in >7-log reduction in Salmonella population. The internal donut temperature increased from ∼30°C to ∼119°C at the end of 2 min of frying. The water activities of the donut crumb and crust after 2 min of frying, followed by 30 min of ambient air cooling, were 0.944 and 0.852, respectively. The donut pH after ambient-air cooling was 5.51. The D- and z-values of the Salmonella cocktail in donut dough were determined using thermal-death-time disks and temperature-controlled water baths. The D-values of the cocktail were 8.6, 2.9, and 2.1 min at 55°C, 58°C, and 61°C, respectively, whereas the z-value was 10°C. This study validated that >7-log reduction could be achieved if donuts are fried for at least 2 min in the oil at 190.6°C, and calculated D- and z-values present the heat resistance of Salmonella in donut dough at the start of the frying processes. However, results from this study should not be extrapolated when donut composition and frying parameters are changed significantly.

Validation of the baking process as a kill-step for controlling Salmonella in muffins.

This research investigates the potential risk of Salmonella in muffins when contamination is introduced via flour, the main ingredient. Flour was inoculated with a 3-strain cocktail of Salmonella serovars (Newport, Typhimurium, and Senftenberg) and re-dried to achieve a target concentration of ~8logCFU/g. The inoculated flour was then used to prepare muffin batter following a standard commercial recipe. The survival of Salmonella during and after baking at 190.6°C for 21min was analyzed by plating samples on selective and injury-recovery media at regular intervals. The thermal inactivation parameters (D and z values) of the 3-strain Salmonella cocktail were determined. A ≥5logCFU/g reduction in Salmonella population was demonstrated by 17min of baking, and a 6.1logCFU/g reduction in Salmonella population by 21min of baking. The D-values of Salmonella serovar cocktail in muffin batter were 62.2±3.0, 40.1±0.9 and 16.5±1.7min at 55, 58 and 61°C, respectively; and the z-value was 10.4±0.6°C. The water activity (aw) of the muffin crumb (0.928) after baking and 30min of cooling was similar to that of pre-baked muffin batter, whereas the aw of the muffin crust decreased to (0.700). This study validates a typical commercial muffin baking process utilizing an oven temperature of 190.6°C for at least 17min as an effective kill-step in reducing a Salmonella serovar population by ≥5logCFU/g.

Validation of Baking To Control Salmonella Serovars in Hamburger Bun Manufacturing, and Evaluation of Enterococcus faecium ATCC 8459 and Saccharomyces cerevisiae as Nonpathogenic Surrogate Indicators.

This study was conducted to validate a simulated commercial baking process for hamburger buns to destroy Salmonella serovars and to determine the appropriateness of using nonpathogenic surrogates (Enterococcus faecium ATCC 8459 or Saccharomyces cerevisiae) for in-plant process validation studies. Wheat flour was inoculated (∼6 log CFU/g) with three Salmonella serovars (Typhimurium, Newport, or Senftenberg 775W) or with E. faecium. Dough was formed, proofed, and baked to mimic commercial manufacturing conditions. Buns were baked for up to 13 min in a conventional oven (218.3°C), with internal crumb temperature increasing to ∼100°C during the first 8 min of baking and remaining at this temperature until removal from the oven. Salmonella and E. faecium populations were undetectable by enrichment (>6-log CFU/g reductions) after 9.0 and 11.5 min of baking, respectively, and ≥5-log-cycle reductions were achieved by 6.0 and 7.75 min, respectively. D-values of Salmonella (three-serovar cocktail) and E. faecium 8459 in dough were 28.64 and 133.33, 7.61 and 55.67, and 3.14 and 14.72 min at 55, 58, and 61°C, respectively, whereas D-values of S. cerevisiae were 18.73, 5.67, and 1.03 min at 52, 55, and 58°C, respectivly. The z-values of Salmonella, E. faecium, and S. cerevisiae were 6.58, 6.25, and 4.74°C, respectively. A high level of thermal lethality was observed for baking of typical hamburger bun dough, resulting in rapid elimination of high levels of the three-strain Salmonella cocktail; however, the lethality and microbial destruction kinetics should not be extrapolated to other bakery products without further research. E. faecium demonstrated greater thermal resistance compared with Salmonella during bun baking and could serve as a conservative surrogate to validate thermal process lethality in commercial bun baking operations. Low thermal tolerance of S. cerevisiae relative to Salmonella serovars limits its usefulness as a surrogate for process validations.

Stored-product insects carry antibiotic-resistant and potentially virulent enterococci.

A total of 154 enterococcal isolates from 95 stored-product insects collected from a feed mill, a grain storage silo, and a retail store were isolated and identified to the species level using PCR. Enterococcus casseliflavus represented 51% of the total isolates, followed by Enterococcus gallinarum (24%), Enterococcus faecium (14%), Enterococcus faecalis (7%), and Enterococcus hirae (5%). Many isolates were resistant to tetracycline (48%), followed by streptomycin (21%), erythromycin (14%), kanamycin (13%), ciprofloxacin (12%), ampicillin (4%), and chloramphenicol (<1%). Enterococci carried genes coding for virulence factors, including the gelatinase gene gelE (26% of isolates), an enterococcal surface protein gene esp (1%), and the cytolysin gene cylA (2%). An aggregation substance (asa1) gene was detected in six out of 10 E. faecalis isolates and five of these were positive for the aggregation substance. Enterococci were positive for hemolytic (57% of isolates) and gelatinolytic (23%) activity. The filter-mating assay showed that the tetracycline resistance gene, tetM, was transferable among E. faecalis by conjugation. These data demonstrated that stored-product insects can serve as potential vectors in disseminating antibiotic-resistant and potentially virulent enterococci.

Survival of Enterococcus faecalis OG1RF:pCF10 in poultry and cattle feed: vector competence of the red flour beetle, Tribolium castaneum (Herbst).

Laboratory experiments were designed to determine the survival of Enterococcus faecalis OG1RF:pCF10 in poultry and cattle feed and its acquisition and transmission by adults of the red flour beetle, Tribolium castaneum (Herbst), to sterile feed. Adult T. castaneum beetles were introduced into poultry and cattle feed inoculated with E. faecalis OG1RF:pCF10 and incubated at 28 degrees C with 65% relative humidity for 7 days in a growth chamber. E. faecalis survived in both poultry and cattle feed during the 7-day test period. There was a logarithmic decrease in E. faecalis concentration in poultry and cattle feed and in and on the insects. E. faecalis persisted on the surface and within T. castaneum adults for 7 days when adults were released on E. faecalis-inoculated poultry feed and for only 5 days on E. faecalis-inoculated cattle feed. The concentration of E. faecalis decreased more slowly on poultry feed than on cattle feed, and this may explain why adult T. castaneum insects were more successful in acquiring and transferring E. faecalis from inoculated poultry feed to sterile poultry feed during the 7-day test period. However, T. castaneum adults reared on inoculated cattle feed were unable to contaminate sterile cattle feed on day 7. To our knowledge, this is the first report documenting T. castaneum to successfully acquire antibiotic-resistant enterococci from animal feed and transfer them to sterile feed. Management of T. castaneum through effective integrated pest management program is therefore important to prevent the spread of antibiotic-resistant and virulent enterococci in animal feed and feed manufacturing environments.