Kathleen Ann Glass

The objectives of this study were to validate experimentally derived D- and z-values for Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) in a pilot plant-scale meat system in 3 commercial products: deli-style uncured roast beef, uncured turkey breast, and cured ham, and to evaluate the efficacy of United States Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) guidelines (e.g., Appendix A for cooked beef, roast beef, and experimentally derived Time-Temperature Tables for Cooking Ready-to-Eat Poultry Products). Each product-pathogen combination was inoculated with 8.0 log colony-forming units (CFU)/g of the designated pathogen cocktail, stuffed into 10.61cm diameter casings, and cooked using either a step-up steam (roast beef, turkey breast) or wet-bulb/ dry-bulb (ham) thermal process. Validation experiments confirmed that heating to 71.1°C was sufficient to inactivate >6.5 log of all 3 pathogens in each of the products tested. Shiga toxin-producing Escherichia coli and Salmonella also had >7.0-log reductions in roast beef when heated to 62.8°C and held for 5 min. Less than 4.0 log CFU/g of Salmonella or STEC were inactivated in roast beef heated to 54.4°C and held for 2 h. A series of follow-up experiments confirmed that ≥ 4 h were needed to achieve a 5.0-log reduction of Salmonella in roast beef held at 54.4°C. The D54.4°C of 23.3 min for Salmonella in samples of roast beef with a slow come-up time was significantly greater (P < 0.05) than the D54.4°C of 10.3 min in control samples that were not subjected to a slow heating process, suggesting that extended come-up times during cooking can enhance thermotolerance of Salmonella. These results support the continued use of USDA, FSIS Appendix A guidelines for controlling Salmonella, L. monocytogenes, and STEC in deli-style roast beef, turkey breast, ham, and similar product types using cook processes with endpoint internal temperatures ≥ 62.8°C.

The United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) Appendix B guidelines identify cooling options to prevent growth of Clostridium perfringens in uncured meat and poultry products where Phase 1 cooling (between 49 and 27°C) is limited to 1 h and continued chilling to 4°C. This study screened several commercially available vinegar-based clean-label ingredients to inhibit C. perfringens in a model poultry product during extension of Phase 1 cooling. Eight uncured turkey products (75% moisture, 1.5% salt, pH 6.6 and 6.2) were formulated with no antimicrobial (control) or 1% dry vinegar (DV), dry vinegar-cultured sugar (DV-CS), or dry vinegar-fruit-spice-extract (DV-FSE). The batter was inoculated with 2.5-log CFU/g C. perfringens spores (3-strain mixture), vacuum- packaged (25 g/pouch), cooked to 70°C, and cooled according to the following schedule: Phase 1 (48.9 – 26.7°C) in 2, 3, 4, or 5 h; Phase 2 (26.7 – 12.8°C) in 5 h; and Phase 3 (12.8 – 4.4°C) in 5 h (total cooling 12, 13, 14, or 15 h). Triplicate samples were enumerated after cooking and at the end of each phase; each experiment was replicated twice. As expected, C. perfringens grew rapidly in control samples (> 1.5-log and > 6-log for the 2- and 5-h extended Phase 1 cooling, respectively). In contrast, treatments containing 1% DV or DV-FSE inhibited 1-log growth at pH 6.6 and 6.2 when Phase 1 cooling was extended to 2 and 3 h, respectively. Turkey containing 1% DV-CS at pH 6.6 and 6.2 inhibited C. perfringens to ≤ 1-log when Phase 1 was extended 4 and 5 h, respectively. This study confirms that DV-based clean-label antimicrobials can be used by manufacturers to extend Phase 1 cooling of uncured meat and poultry products beyond the recommendations in the 2021 USDA-FSIS Appendix B Option 2 Stabilization Guidelines. Further research is needed to compare the efficacy of similar ingredients among various suppliers.

Certain cheeses can be legally produced in the United States using raw milk, but they must be aged for at least 60 d to reduce pathogen risks. However, some varieties, even when aged for 60 d, have been shown to support growth of Listeria monocytogenes or survival of Shiga toxin-producing Escherichia coli (STEC). Thermization, as a subpasteurization heat treatment, has been proposed as a control to reduce the risk of pathogens in raw cheese milk while retaining some quality attributes in the cheese. However, the temperature and time combinations needed to enhance safety have not been well characterized. The objective of this research was to determine and validate decimal reduction values (D-values) for L. monocytogenes and STEC at thermization temperatures 65.6, 62.8, and 60.0°C; a D-value at 57.2°C was also determined for L. monocytogenes only. Nonhomogenized, pasteurized whole-milk samples (1 mL) were inoculated with 8-log cfu/mL L. monocytogenes or STEC (5- or 7-strain mixtures, respectively), vacuum-sealed in moisture-impermeable pouches, and heated via water bath submersion. Duplicate samples were removed at appropriate intervals and immediately cooled in an ice bath. Surviving bacteria were enumerated on modified Oxford or sorbitol MacConkey overlaid with tryptic soy agar to aid in the recovery of heat-injured cells. Duplicate trials were conducted, and survival data were used to calculate thermal inactivation rates. D_65.6°C-, D_62.8°C-, and D_60.0°C-values of 17.1 and 7.2, 33.8 and 16.9, and 146.6 and 60.0 s were found for L. monocytogenes and STEC, respectively, and a D_57.2°C-value of 909.1 s was determined for L. monocytogenes. Triplicate validation trials were conducted for each test temperature using 100 mL of milk inoculated with 3 to 4 log cfu/mL of each pathogen cocktail, A 3-log reduction of each pathogen was achieved faster in larger volumes than what was predicted by D-values (D-values were fail-safe). Data were additionally compared with published results from 21 scientific studies investigating L. monocytogenes and STEC in whole milk heated to thermization temperatures (55.0-71.7°C). These data can be used to give producers of artisanal raw-milk cheese flexibility in designing thermal processes to reduce L. monocytogenes and STEC populations to levels that are not infectious to consumers.

Acidification and nutrient depletion by dairy starter cultures is often sufficient to prevent outgrowth of pathogens during post-processing of cultured dairy products. In the case of cottage cheese, however, the addition of cream dressing to the curd and subsequent cooling procedures can create environments that may be hospitable for the growth of Listeria monocytogenes. We report on a non-bacterio-cinogenic Lacticaseibacillus rhamnosus strain that severely limits the growth potential of L. monocytogenes in creamed cottage cheese. The main mechanism underlying Listeria spp. inhibition was found to be caused by depletion of manganese (Mn), thus through competitive exclusion of a trace element essential for the growth of many microorganisms. Growth of Streptococcus thermophilus and Lactococcus lactis that constitute the starter culture, on the other hand, were not influenced by reduced Mn levels. Addition of L. rhamnosus with Mn-based bioprotective properties during cottage cheese production therefore offers a solution to inhibit undesired bacteria in a bacteriocin-independent fashion.

Biopreservatives are clean-label ingredients used to control pathogenic and spoilage microorganisms in ready-to-eat foods including cheese. The efficacies of six commercial biopreservatives in controlling L. monocytogenes growth at 4°C were tested in a high-moisture model cheese (pH 6.00, 56% moisture, 1.25% salt) made of cream, micellar casein, water, salt, lactose, lactic acid, and a single protective culture (PC-1, PC-2, or PC-3, 10 6 CFU/g target) or bacterial fermentate (CM-1 or CM-2 [cultured milk] or CSV-1 [cultured sugar-vinegar blend], 0.5% or 1.0% level). Cheeses were inoculated with 3-log CFU/g L. monocytogenes (5-strain cocktail), after which 25-g samples were vacuum-sealed and stored at 4°C for 8 weeks. L. monocytogenes populations from triplicate samples were enumerated weekly on Modified Oxford agar in duplicate trials. L. monocytogenes growth (≥1-log increase) was observed in approximately 1 week in control cheese and those formulated with 10 6 CFU/g PC-1 or PC-2. Growth was delayed to 2.5 weeks in model cheeses formulated with 10 6 CFU/g PC-3 or 0.5% CM-2 and to 3 weeks with 0.5% CM-1 or CSV-1. Growth was further delayed to 6.5-7.5 weeks in model cheeses formulated with 1.0% CM-1 or CM-2, while formulation with 1.0% CSV-1 inhibited L. monocytogenes growth for 8 weeks. In a second set of experiments, the combined effect of pH and 0.5% CSV-1 on L. monocytogenes inhibition was investigated. Incorporation of 0.5% CSV-1 delayed L. monocytogenes growth to 3, 6, and >10 weeks in cheeses of pH 6.00, 5.75, and 5.50, respectively, versus growth observed in 1, 1, and 3.5 weeks in control cheeses. These data suggest that certain fermentates have greater antilisterial activity than protective cultures in directly acidified cheeses with direct biopreservative incorporation and refrigerated storage. Further research is needed to optimize conditions to prevent listerial growth utilizing protective cultures in fresh, soft cheeses.

This study investigated the effectiveness of cooking processes that incorporated hydrated surface lethality (HSL) steps for ensuring the reduction of Salmonella on the surfaces of small-dimension meat and poultry products cooked using short-duration, high-temperature impingement oven processes. Whole-muscle chicken tenders (3% fat), beef patties (10% and 30% fat), pork patties (10% and 30% fat), and chicken patties (10% and 20% fat) were surface inoculated with a 5-strain mixture of Salmonella to yield 8 log colony-forming units/g, then cooked in a two-zone impingement oven using either dry heat or steam-humidified HSL processes. The HSL steps used steam injection to control the wet-bulb temperature at either 71.1°C or 82.2°C. Dry-heat cooking processes using a dry-bulb temperature of 204.4°C and no steam-injected HSL steps failed to consistently achieve a 6.5 log reduction of Salmonella on chicken tenders and the low-fat patty products (≤10% fat). In contrast, processes incorporating an HSL step using an 82.2°C wet-bulb temperature in one or both zones resulted in ≥6.5 log reductions of Salmonella for all products. Sufficient reductions were achieved regardless of whether this 82.2°C wet-bulb HSL step was incorporated before or after a dry-cook step. Processes that incorporated an HSL step using a 71.1°C wet-bulb temperature in both zones also resulted in reductions ≥6.5 log for all products. Processes using a 71.1°C wet-bulb HSL step in only one zone delivered ≥ 6.5 log reduction for all of the patty products. However, the one-zone 71.1°C HSL step achieved ≥6.5 log reduction in chicken tenders only if used in the first zone of the two-zone oven. When the 71.1°C HSL step was used in the second zone for chicken tenders after using dry heat in the first zone, the target reduction of 6.5 log was not achieved. This research successfully validated approaches to ensure ≥6.5 log reduction of Salmonella on product surfaces.

High-moisture have been shown to support Listeria monocytogenes growth during refrigerated storage. Prior studies suggest that organic acids vary in their antilisterial activity and that cheeses of lower pH delay growth longer than those of higher pH; however, no standard pH value for Listeria control in cheese exists. The objective of this research was to create a predictive model to include the effects of acid type, pH, and moisture on the growth of L. monocytogenes in a model cheese system. Cream, micellar casein, water, lactose, salt, and acid (citric, lactic, acetic, or propionic) were combined in 32 formulations targeting 4 pH values (5.25, 5.50, 5.75, and 6.00) and 2 moisture levels (50 and 56%). Each was inoculated with 3-log CFU/g L. monocytogenes (5-strain mixture) after which 25-g samples were vacuum-sealed and stored 8 weeks at 4ºC. Triplicate samples were enumerated on Modified Oxford agar weekly in duplicate trials. Model cheeses formulated with acetic and propionic acids inhibited growth (i.e. lactic > citric acids in antilisterial activity within the model cheese system developed and can be used in formulating safe high-moisture cheeses.