Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log10 BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log10 BA spores/mL of milk and the 1.4- μm membrane removed 4.50 ± 0.35 log10 BA spores/ mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log10 spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log10 BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization

Evaluation of post-fermentation heating times and temperatures for controlling Shiga toxin-producing Escherichia coli cells in a non-dried, pepperoni-type sausage

Coarse ground meat was mixed with non-meat ingredients and starter culture (Pediococcus acidilactici) and then inoculated with an 8-strain cocktail of Shiga toxinproducing Escherichia coli (ca. 7.0 log CFU/g). Batter was fine ground, stuffed into fibrous casings, and fermented at 35.6°C and ca. 85% RH to a final target pH of ca. pH 4.6 or ca. pH 5.0. After fermentation, the pepperoni- like sausage were heated to target internal temperatures of 37.8°, 43.3°, 48.9°, and 54.4°C and held for 0.5 to 12.5 h. Regardless of the heating temperature, the endpoint pH in products fermented to a target pH of pH 4.6 and pH 5.0 was pH 4.56±0.13 (range of pH 4.20 to pH 4.86) and pH 4.96±0.12 (range of pH 4.70 to pH 5.21), respectively. Fermentation alone delivered ca. a 0.3- to 1.2-log CFU/g reduction in pathogen numbers. Fermentation to ca. pH 4.6 or ca. pH 5.0 followed by post-fermentation heating to 37.8° to 54.4°C and holding for 0.5 to 12.5 h generated total reductions of ca. 2.0 to 6.7 log CFU/g

Thermal Inactivation of Salmonella spp. Within Refrigerated or Frozen Turkey Burgers Following Pan Frying

Turkey burgers (ca. 1.25 or 2.5 cm thick) were inoculated (ca. 6.5 log CFU/g) with a Salmonella spp. cocktail, stored at 4 °C (18 h) or –20 °C (30 d), and then cooked in 15 or 30 mL of canola oil. Regardless of oil volume, cooking refrigerated 1.25 cm thick burgers to 57.2, 65.6, 73.9, or 82.2 °C delivered reductions of ca. 4.8 to > 6.0 log CFU/g compared to ca. 3.0 to >5.0 log CFU/g for frozen burgers. Cooking refrigerated 2.5 cm thick burgers to 57.2 to 82.2 °C delivered reductions of ca. 2.8 to > 6.1 log CFU/g compared to ca. 2.4 to >5.1 log CFU/g for frozen burgers. Average internal temperatures for refrigerated or frozen burgers cooked to 57.2, 65.6, 73.9, or 82.2 °C ranged from 38.3 to 96.2, 48.0 to 99.4, 55.2 to 98.5, or 59.4 to 98.3 °C, respectively. Thus, pan frying refrigerated or frozen Turkey burgers to >73.9 °C delivered a >5.0-log reduction of Salmonella

Viability of Listeria monocytogenes and Salmonella spp. on Slices of a German-Style Bologna Containing Blends of Organic Acid Salts During Storage at 4 or 12°C

Viability of cells of Listeria monocytogenes or Salmonella spp. was quantified on slices of a German-style bologna manufactured by a local butcher to contain no added antimicrobials or to include 0.9% or 1.3% of a blend of potassium acetate and sodium diacetate (K-Ace) or 2.5% of a blend of potassium lactate and sodium diacetate (K-Lac) as ingredients. After slicing (ca. 7.1 cm L by 6.7 cm W, ca. 0.5 cm thick, ca. 22.4 g each), a single slice of bologna was placed into a nylon–polyethylene bag and surface inoculated with 250 µL per side of a five-strain mixture of either cells of L. monocytogenes or Salmonella spp. to achieve an initial level of ca. 3.5–4.0 log CFU/slice. The packages were vacuum-sealed and then stored at 4 or 12°C for 90 and 30 days, respectively. Without antimicrobials added to the formulation, L. monocytogenes numbers increased by ca. 5.4 and 6.0 log CFU/slice at both 4 and 12°C during the entire 90- and 30-day storage period, respectively. Likewise, levels of Salmonella also increased by ca. 6.0 log CFU/slice at 12°C in the absence of added antimicrobials; however, levels of this pathogen decreased by ca. 1.7 log CFU/slice after 90 days at 4°C. With the inclusion of 0.9% or 1.3% K-Ace or 2.5% K-Lac in the bologna formulation, levels of L. monocytogenes decreased by ca. ≤0.7 log CFU/slice after 90 days at 4°C, whereas levels of Salmonella decreased by ca. 1.6–2.3 log CFU/slice. After 30 days at 12°C, levels of L. monocytogenes increased by ca. ≤3.4 log CFU/slice on product containing 0.9% K-Ace or 2.5% K-Lac but remained relatively unchanged on slices formulated with 1.3% K-Ace. For Salmonella, in the presence of 0.9% or 1.3% K-Ace or 2.5% K-Lac, pathogen levels decreased by ca. ≤0.7 log CFU/slice at 12°C after 30 days. Our data validate that the inclusion of K-Ace (0.9% or 1.3%) or K-Lac (2.5%) as ingredients is effective for controlling L. monocytogenes and Salmonella on slices of bologna during refrigerated storage

Inactivation of Listeria monocytogenes and Salmonella spp. During Cooking of Country Ham and Fate of L. monocytogenes and Staphylococcus aureus During Storage of Country Ham Slices

Thermal inactivation studies were undertaken on Listeria monocytogenes and Salmonella spp. inoculated on the surface of country ham. Hams (average = ca. 3.4 ± 0.5 kg each; average = ca. ≥18% shrinkage) were used as provided by the processor (i.e., “salted hams”), desalted in tap water (i.e., “desalted hams”), or dried for an additional period (i.e., “extra-dried hams”). Hams were surface inoculated (ca. 9.5 log CFU/ham) with a multistrain cocktail of L. monocytogenes or Salmonella spp. and cooked within a bag in a circulating water bath to an internal temperature of 130°F (54.4°C) instantaneous, 145°F (62.8°C) and held for 4 min, 153°F (67.2°C) and held for 34 s, or 160°F (71.1°C) instantaneous. Regardless of ham type, all four time and temperature combinations tested herein delivered a ≥6.7-log reduction of cells of L. monocytogenes or Salmonella spp. Differences in product pH, moisture content, or aw did not have an appreciable impact on the thermal inactivation of L. monocytogenes or Salmonella spp. on country ham. In addition, shelf-life studies were undertaken using slices of “salted” country ham that were surface inoculated (ca. 5.5 log CFU/slice) with a multistrain cocktail of L. monocytogenes or Staphylococcus aureus and then stored at 20°C. Levels of S. aureus increased by ca. ≤1.4 log CFU/slice during storage for 90 days, whereas levels of L. monocytogenes remained relatively unchanged (≤0.2 log CFU/slice increase). Our data validated that cooking parameters elaborated in the U.S. Department of Agriculture’s Food Safety and Inspection Service Cooking Guideline for Meat and Poultry Products (Revised Appendix A) are sufficient to deliver significant reductions (ca. ≥6.8 log CFU/ham) in levels of L. monocytogenes and Salmonella spp. on country ham. In addition, in the event of postprocessing contamination, country ham may support the outgrowth of S. aureus or survival of L. monocytogenes during storage at 20°C for 90 days

Genetic diversity and profiles of genes associated with virulence and stress resistance among isolates from the 2010-2013 interagency Listeria monocytogenes market basket survey.

Whole genome sequencing (WGS) was performed on 201 Listeria monocytogenes isolates recovered from 102 of 27,389 refrigerated ready-to-eat (RTE) food samples purchased at retail in U.S. FoodNet sites as part of the 2010-2013 interagency L. monocytogenes Market Basket Survey (Lm MBS). Core genome multi-locus sequence typing (cgMLST) and in-silico analyses were conducted, and these data were analyzed with metadata for isolates from five food groups: produce, seafood, dairy, meat, and combination foods. Six of 201 isolates, from 3 samples, were subsequently confirmed as L. welshimeri. Three samples contained one isolate per sample; mmong the 96 samples that contained two isolates per sample, 3 samples each contained two different strains and 93 samples each contained duplicate isolates. After 93 duplicate isolates were removed, the remaining 102 isolates were delineated into 29 clonal complexes (CCs) or singletons based on their sequence type. The five most prevalent CCs were CC155, CC1, CC5, CC87, and CC321. The Shannon's diversity index for clones per food group ranged from 1.49 for dairy to 2.32 for produce isolates, which were not significantly different in pairwise comparisons. The most common molecular serogroup as determined by in-silico analysis was IIa (45.6%), followed by IIb (27.2%), IVb (20.4%), and IIc (4.9%). The proportions of isolates within lineages I, II, and III were 48.0%, 50.0% and 2.0%, respectively. Full-length inlA was present in 89.3% of isolates. Listeria pathogenicity island 3 (LIPI-3) and LIPI-4 were found in 51% and 30.6% of lineage I isolates, respectively. Stress survival islet 1 (SSI-1) was present in 34.7% of lineage I isolates, 80.4% of lineage II isolates and the 2 lineage III isolates; SSI-2 was present only in the CC121 isolate. Plasmids were found in 48% of isolates, including 24.5% of lineage I isolates and 72.5% of lineage II isolates. Among the plasmid-carrying isolates, 100% contained at least one cadmium resistance cassette and 89.8% contained bcrABC, involved in quaternary ammonium compound tolerance. Multiple clusters of isolates from different food samples were identified by cgMLST which, along with available metadata, could aid in the investigation of possible cross-contamination and persistence events