Interlaboratory Evaluation of Enterococcus faecium NRRL B-2354 as a Salmonella Surrogate for Validating Thermal Treatment of Multiple Low-Moisture Foods.

ABSTRACT: This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P < 0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P < 0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P > 0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision.

Evaluation of Hot-Air Drying To Inactivate Salmonella and Enterococcus faecium on Apple Pieces.

ABSTRACT: Hot-air drying processes are used to provide specific quality attributes to products, such as dehydrated apple pieces. To comply with the U.S. Food and Drug Administration Food Safety Modernization Act, there is a need to understand microbial lethality during these processes. The objective of this study was to determine the level of inactivation provided by hot-air drying on a Salmonella cocktail inoculated onto apple cubes and to evaluate the performance of Enterococcus faecium as a surrogate. A cocktail of Salmonella serovars (Agona, Tennessee, Montevideo, Mbandaka, and Reading) and E. faecium were individually inoculated onto cored, peeled Gala apple cubes at 9.2 ± 0.3 and 8.8 ± 0.1 log CFU per sample, respectively. Apple cubes were dried at 104 or 135°C in ∼1.5-kg batches using a hot-air dryer with a vertically directed heat source and without mixing. Three subsamples, consisting of four inoculated cubes, were enumerated at each time point (n ≥ 5) from multiple product bed depths. Water activity decreased throughout the duration of the study, with samples drying faster at 135 than 104°C. Samples at the bottom bed depth, closer to the heat source, dried faster than those at the higher bed depth, regardless of temperature. Significant microbial inactivation was not seen immediately. It took >10 min at the bottom bed depth or >40 min of drying at the top bed depth, regardless of temperature (P < 0.05). By the end of drying, average Salmonella inactivation of greater than 5 log CFU per sample was achieved. At temperature conditions evaluated, E. faecium inactivation was slower than Salmonella, indicating that it would likely serve as a good surrogate for in-plant validation studies. Case hardening did not inhibit microbial inactivation in the conditions tested. Hot-air drying under the conditions evaluated may provide a preventive control in the production of dehydrated products, such as apples.

Effect of High Pressures in Combination with Temperature on the Inactivation of Spores of Nonproteolytic Clostridium botulinum Types B and F.

The impact of high pressure processing on the inactivation of spores of nonproteolytic Clostridium botulinum is important in extended shelf life chilled low-acid foods. The three most resistant C. botulinum strains (Ham-B, Kap 9-B, and 610-F) were selected for comparison of their thermal and pressure-assisted thermal resistance after screening 17 nonproteolytic C. botulinum strains (8 type B, 7 type E, and 2 type F). Spores of strains Ham-B, Kap 9-B, and 610-F were prepared using a biphasic media method, diluted in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.00) to 105 to 106 CFU/mL, placed into a modified sterile transfer pipette, heat sealed, and subjected to a combination of high pressures (600 to 750 MPa) and high temperatures (80 to 91°C) using laboratory and pilot-scale pressure test systems. Diluted spores from the same crops were placed in nuclear magnetic resonance tubes, which were heat sealed, and subjected to 80 to 91°C in a Fluke 7321 high precision bath with Duratheram S oil as the heat transfer fluid. After incubation for 3 months, survivors in both studies were determined by the five-tube most-probable-number method using Trypticase-peptone-glucose-yeast extract broth. The highest (>5.0) log reductions in spore counts for Ham-B, Kap 9-B, and 610-F occurred at the highest temperature and pressure combination tested (91°C and 750 MPa). Thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 87°C, decreasing to <1.0 min at 87°C for these strains. Pressure-assisted thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 91°C with any pressure combination and decreased to <1.0 min as the pressure increased from 600 to 750 MPa at 91°C. Based on the pressure-assisted thermal D-values, pressure exerted a more protective effect on spores of Ham-B, Kap 9-B, and 610-F when processed at 83 to 91°C combined with pressures of 600 to 700 MPa when compared with thermal treatment only. No protective effect was observed when the spores of Ham-B, Kap9-B, and 610-F were treated at lower temperatures (80 to 83°C) in combination with 750 MPa. However, at higher temperatures (87 to 91°C) in combination with 750 MPa, a protective effect was seen for Ham-B, Kap9-B, and 610-F spores based on the calculated pressure-assisted thermal D-values.

Effect of sporulation temperature on the resistance of Clostridium botulinum type A spores to thermal and high pressure processing.

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinum type A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase-peptone-glucose-yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41°C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P < 0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P < 0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinum spores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinum spores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

Cleaning and sanitation of Salmonella-contaminated peanut butter processing equipment.

Microbial contamination of peanut butter by Salmonella poses a significant health risk as Salmonella may remain viable throughout the product shelf life. Effective cleaning and sanitation of processing lines are essential for preventing cross-contamination. The objective of this study was to evaluate the efficacy of a cleaning and sanitation procedure involving hot oil and 60% isopropanol, ± quaternary ammonium compounds, to decontaminate pilot-scale processing equipment harboring Salmonella. Peanut butter inoculated with a cocktail of four Salmonella serovars (∼ 7 log CFU/g) was used to contaminate the equipment (∼ 75 L). The system was then emptied of peanut butter and treated with hot oil (90 °C) for 2 h followed by sanitizer for 1 h. Microbial analysis of food-contact surfaces (7 locations), peanut butter, and oil were conducted. Oil contained ∼ 3.2 log CFU/mL on both trypticase soy agar with yeast extract (TSAYE) and xylose lysine deoxycholate (XLD), indicating hot oil alone was not sufficient to inactivate Salmonella. Environmental sampling found 0.25-1.12 log CFU/cm(2) remaining on processing equipment. After the isopropanol sanitation (± quaternary ammonium compounds), no Salmonella was detected in environmental samples on XLD (<0.16 log CFU/cm(2)). These data suggest that a two-step hot oil clean and isopropanol sanitization treatment may eliminate pathogenic Salmonella from contaminated equipment.

Growth and survival of Salmonella in ground black pepper (Piper nigrum).

A four serovar cocktail of Salmonella was inoculated into ground black pepper (Piper nigrum) at different water activity (aw) levels at a starting level of 4-5 log cfu/g and incubated at 25 and at 35 °C. At 35 °C and aw of 0.9886 ± 0.0006, the generation time in ground black pepper was 31 ± 3 min with a lag time of 4 ± 1 h. Growth at 25 °C had a longer lag, but generation time was not statistically different from growth at 35 °C. The aw threshold for growth was determined to be 0.9793 ± 0.0027 at 35 °C. To determine survival during storage conditions, ground black pepper was inoculated at approximately 8 log cfu/g and stored at 25 and 35 °C at high (97% RH) and ambient (≤40% RH) humidity. At high relative humidity, aw increased to approximately 0.8-0.9 after approximately 20 days at both temperatures and no Salmonella was detected after 100 and 45 days at 25 and 35 °C, respectively. Under ambient humidity, populations showed an initial decrease of 3-4 log cfu/g, then remained stable for over 8 months at 25 and 35 °C. Results of this study indicate Salmonella can readily grow at permissive aw in ground black pepper and may persist for an extended period of time under typical storage conditions.

Effect of growth on the thermal resistance and survival of Salmonella Tennessee and Oranienburg in peanut butter, measured by a new thin-layer thermal death time device.

In published data the thermal destruction of Salmonella species in peanut butter deviates from pseudo-first-order kinetics. The reasons for such deviation are unknown. This study examined both the method used to measure the thermal destruction rate and the method of growth of the microorganisms to explain variations in destruction kinetics. Growth on a solid matrix results in a different physiological state that may provide greater resistance to adverse environments. In this study, Salmonella Tennessee and Oranienburg were grown for 24 h at 37°C under aerobic conditions in broth and agar media to represent planktonic and sessile cell growth, respectively. Peanut butter was held at 25°C and tested for Salmonella levels immediately after inoculation and at various time intervals up to 2 weeks. Thermal resistance was measured at 85°C by use of a newly developed thin-layer metal sample holder. Although thermal heat transfer through the metal device resulted in longer tau values than those obtained with plastic bags (32.5 ± 0.9 versus 12.4 ± 1.9 s), the bags have a relative variability of about 15 % compared with about 3 % in the plates, allowing improved uniformity of sample treatment. The two serovars tested in the thin-layer device showed similar overall thermal resistance levels in peanut butter regardless of growth in sessile or planktonic states. However, thermal destruction curves from sessile cultures exhibited greater linearity than those obtained from planktonic cells (P = 0.0198 and 0.0047 for Salmonella Oranienburg and Salmonella Tennessee, respectively). In addition, both Salmonella serovars showed significantly higher survival in peanut butter at 25°C when originally grown on solid media (P = 0.001) with a <1-log loss over 2 weeks as opposed to a 1- to 2-log loss when grown in liquid culture. Consequently, the use of cells grown on solid media may more accurately assess the survival of Salmonella at different temperatures in a low-water-activity environment such as peanut butter.

Identification of a Mycoplasma ovis-like organism in a herd of farmed white-tailed deer (Odocoileus virginianus) in rural Indiana.

Mycoplasma ovis is a hemoplasma parasite of sheep, goats, and reindeer; however, natural hemoplasma infection in white-tailed deer has not previously been reported. Subsequent to finding many coccoid, bacillary, and ring-shaped organisms, consistent with hemotropic mycoplasmas, on RBCs from a 72-day-old female white-tailed fawn, we sought to (1) identify the putative hemoplasma observed in blood from the fawn, (2) evaluate others in the herd for hemoplasma infection, and (3) identify clinicopathologic characteristics of hemoplasma-infected white-tailed deer. EDTA-anticoagulated whole blood was collected from the fawn and 8 apparently healthy does in the same herd. CBCs were performed on 7 nonclotted samples from the fawn and 6 does. DNA was extracted from all samples, followed by PCR amplification of bacterial (16S rDNA) and protozoal (18S rDNA) genes. The nearly complete 16S rDNA product from the fawn's sample was directly sequenced and compared with known sequences in the GenBank database. Samples from the fawn and 7 of 8 does were PCR-positive using hemoplasma-specific and M ovis-specific protocols. The fawn was PCR-negative for Anaplasma spp., Babesia spp., and Theileria spp. The 16S rDNA sequence from the fawn (GenBank accession number, FJ824847) was most closely related to M ovis (AF338268), having 98.5% sequence identity. The fawn had a mild nonregenerative anemia, a neutrophilic left-shift with toxic change, aspiration bronchopneumonia, and gastrointestinal disease. Hematologic values, including blood film evaluation, in infected does were unremarkable. The M ovis-like organism may have acted as either an opportunistic or primary pathogen in the fawn. The high occurrence of subclinical infections in the does suggests that white-tailed deer may act as wildlife reservoirs for M ovis.