Enterococcus Faecium NRRL B-2354 Research Articles

Utilizing superheated steam to inactivate Enterococcus faecium NRRL B-2354 on various material surfaces used in food and produce industries

Traditional sanitation in dry food and produce manufacturing plants is challenging as residual moisture can harbor microbial contamination. Superheated steam (SHS) is produced when water is heated to temperatures (125–400 °C) beyond the boiling point. SHS does not lead to condensation on surfaces. There are limited studies available on the sanitation efficacy of SHS on different surfaces. This study was conducted to understand the sanitation efficacy of SHS to inactivate Enterococcus faecium NRRL B-2354 on different surfaces (stainless steel, concrete, plywood, leather, polytetrafluoroethylene (PTFE), silicon rubber, cotton, and cardboard). The surfaces were either spot inoculated or coated with a representative food matrix (baby formula) and treated with SHS at 150 °C for a total process time (come-up time + treatment time) of 3 min. Spot-inoculated surfaces showed a higher microbial inactivation (range: 9.2 ± 0.9 to 10.7 ± 0.7 log reduction/coupon) whereas surfaces with inoculum coated with baby formula had lower microbial inactivation (range: 7.8 ± 0.4 to 9.6 ± 0.4 log reduction/coupon). The effects of surface characteristics like thermal inertia, surface roughness, and hydrophobicity of microbial inactivation were studied. Prolonged usage of SHS (150 °C for 5 min once a week over 8 weeks) did not alter surface characteristics, particularly roughness of stainless steel, silicone rubber and plywood.

Inactivation of Salmonella, Enterococcus faecium and natural microbiota on dry food matrices with microwave-driven plasma-processed air

This study explores the efficacy of plasma-processed air (PPA) as a non-thermal treatment for decontamination of dry food surfaces, such as wheat grains, black peppercorns, and onion flakes. We compared the effect of various inoculum types of Enterococcus faecium, revealing a higher resistance of the powder form compared with liquid and slurry inocula on black peppercorns. Furthermore, Enterococcus faecium NRRL B-2354 appeared to be a suitable control indicator for Salmonella. The PPA efficacy varied across matrices, being higher on medium agar (7.0 ± 0.5 log10), followed by wheat grains (2.1 ± 0.2 log10), black peppercorns (1.5 ± 0.1 log10), and no reduction on onion flakes. Variation was also found across microorganisms, as the natural microbiota was reduced on wheat grains (1.6 ± 0.6 log10 for mesophilic count and 0.8 ± 0.3 log10 for spores), while on black peppercorns this was only achieved after upscaling (1.3 ± 0.2 log10 for mesophilic and 1.5 ± 0.1 log10 for spores). The findings of this study give guidance to further optimize the application of PPA treatment by changing the technical parameter settings of the plasma setup to increase the microbial efficacy and/or combining the PPA treatment with other methods to enhance microbial control.

Effect of temperature and relative humidity on ethylene oxide inactivation of Salmonella and Enterococcus faecium NRRL B-2354 on chia seeds and its influence on the quality of seeds

Ethylene oxide (EtO) sterilization is commonly used by the food industry to inactivate pathogens in spices, but its efficacy on edible seeds, a high-risk food commodity, remains unexplored. This study aimed to evaluate the antimicrobial efficacy of EtO gas on chia seeds and assess the impact of processing parameters on microbial reduction, byproduct formation, and seed quality. Further, Enterococcus faecium NRRL B-2354 was evaluated for its suitability as a surrogate for Salmonella. Chia seeds were inoculated with a cocktail of five strains of Salmonella enterica or E. faecium. The inoculated sample was treated with EtO (735.3 mg/L) at different combinations of relative humidity (30, 40, and 50%) and temperature (46, 53, and 60 °C) for exposure times ranging from 2 to 120 min. The results showed that EtO treatment effectively reduced the bacterial populations on chia seeds, with the inactivation trend following a non-linear pattern that was described well by the Weibull model. The treatment time required for a 5-log reduction of Salmonella was less than 10 min at 50% RH and 53–60 °C. Exposure of chia seeds to EtO for 2 min at 50% RH gave 3.15, 3.59, and 3.87 log reductions at 46, 53, and 60 °C, respectively. E. faecium was found to be a reliable surrogate for Salmonella during EtO treatment. The modified Bigelow model developed based on Weibull model parameters could be used to predict Salmonella inactivation (RMSE = 0.73 min) and E. faecium inactivation (RMSE = 0.87 min) on chia seeds during EtO treatment. Fatty acid composition, protein content, color, and germination ability of chia seeds were not impacted significantly (P > 0.05) by EtO fumigation. These findings suggest that EtO gas can effectively inactivate Salmonella on chia seeds while preserving their viability and quality.

Heat resistance of Lactobacillus brevis, Pediococcus acidilactici and Enterococcus faecium in buffer (pH 4), alcoholic and alcohol-free beer

Why was the work done: To measure the heat resistance of three vegetative bacteria in buffer (pH 4), alcoholic and alcohol-free beer. To verify that Pediococcus acidilactici ATCC 8042 and Lactobacillus brevis BSO 566 are as heat resistant as previously reported and to establish if Enterococcus faecium NRRL B-2354 could have application in validation studies for the pasteurisation of beer. How was the work done: : The heat resistance of L. brevis, P. acidilactici and E. faecium in buffer, alcoholic and alcohol-free beer was determined using two approaches - capillary test tubes and flask method. What are the main findings: E. faecium was the most heat resistant microorganism in all three liquids. D values were significantly greater, and z-values were similar or significantly greater than the corresponding values of L. brevis and P. acidilactici. Why is the work important: E. faecium is used in the food industry as a pathogenic surrogate for the validation of thermal and non-thermal processes. The work reported here suggests that E. faecium can also be used for the validation of pasteurisation of beer. Its high z-value suggests that at higher pasteurisation temperatures (>65°C) it may be more resistant than yeast ascospores and could therefore be used as an indicator for flash pasteurisation.

Reduction Foodborne Pathogens and Surrogate Microorganism on Citrus Fruits after Lab- and Pilot-scale Finishing Wax Application

After finishing waxes are applied, citrus fruits are typically dried at 32–60°C for 2–3 min before final packing. The survival of Listeria monocytogenes, Salmonella, and Enterococcus faecium NRRL B-2354 was evaluated under laboratory conditions on lemons after applying one of four finishing waxes (F4, F6, F8, and F15) followed by an ambient hold or heated (50 or 60°C) drying step. The reduction of inoculated microorganisms during drying was significantly influenced by wax type and temperature, with greater reductions at higher temperatures. Greater reductions after waxing and drying at 60°C were observed with L. monocytogenes (2.84–4.44 log) than with Salmonella (1.65–3.67 log), and with Salmonella than with E. faecium (0.99–2.93 log). The survival of Salmonella inoculated at 5.8–5.9 log/fruit on lemons and oranges after applying wax F6 and drying at 60°C was evaluated during storage at 4 and 22°C. The reductions of Salmonella after waxing and drying were 1.7 log; additional reductions during storage at 4 or 22°C were 1.40–1.43 or 0.18–0.29 log, respectively, on waxed lemons, and 0.56–1.02 or 0.54–0.57 log, respectively, on waxed oranges. Under pilot-scale packinghouse conditions with wax F4, mean and minimum reductions of E. faecium ranged from 2.15 to 2.89 and 1.64 to 2.12 log, respectively. However, E. faecium was recovered by whole-fruit enrichment (limit of detection: 0.60 log CFU/lemon) but not by plating (LOD: 1.3 log CFU/lemon) from uninoculated lemons run with or after the inoculated lemons. The findings should provide useful information to establish and implement packinghouse food safety plans.

Clean-in-place (CIP) validation of oil-based cleaning and sanitization for Salmonella-contaminated tubing: Evaluation of Enterococcus faecium as a surrogate

The safety of low-moisture foods is of continual concern due to the survival and cross-tolerance of certain bacterial pathogens under dry conditions. This study screened for Salmonella surrogates using a controlled bacterial desiccation system for testing against formulated antimicrobial oils. The effect of inoculum preparation on the heat resistance of Salmonella in peanut butter was also addressed. Thereafter, a tubing system was utilized to validate a dry cleaning and sanitation procedure at the temperature range of peanut butter processing. Tubes were contaminated by re-circulating peanut butter inoculated with a test organism to 108−9 CFU/g. The contaminated system was flushed with fresh canola oil under laminar flow for cleaning, followed by holding with acidified oil or water-in-oil (W/O) emulsion to sanitize. A linear relation (R2, 0.89) was observed between the oil-flush (cleaning) duration and surface cell removal, and the prevailing surface cleanliness dictated the efficacy of sanitization. System decontamination was achieved at 60 °C through a two-step oil flush (3.6 min) and acidified W/O emulsion treatment (30 min). The test organisms, S. Enteritidis phage type 30 and Enterococcus faecium NRRL B-2354, were reduced by this procedure to levels undetectable by enrichment of tubes (n = 9). The E. faecium showed less susceptibility than S. Enteritidis under all the conditions tested, suggesting that it may be used as a Salmonella surrogate to validate such a process in the food-processing environment. Adaptation of the proposed oil-based system would allow a shifted cleaning and sanitation paradigm precluding the necessity of traditional wet cleaning and application of flammable alcohol-based sanitizers.

Transfer of Enterococcus faecium and Salmonella enterica during simulated postharvest handling of yellow onions (Allium cepa)

Bacterial transfer during postharvest handling of fresh produce provides a mechanism for spreading pathogens, but risk factors in dry environments are poorly understood. The aim of the study was to investigate factors influencing bacterial transfer between yellow onions (Allium cepa) and polyurethane (PU) or stainless steel (SS) under dry conditions. Rifampin-resistant Enterococcus faecium NRRL B-2354 or a five-strain cocktail of Salmonella was inoculated onto onion skin or PU surfaces at high or moderate levels using peptone, onion extract, or soil water as inoculum carriers. Transfer from inoculated to uninoculated surfaces was conducted using a texture analyzer to control force, time, and number of contacts. Transfer rates (ratio of recipient surface to donor surface populations) of E. faecium (4–5%) were significantly higher than those of Salmonella (0.5–0.6%) at the high (7 log CFU/cm2) but not moderate (5 log CFU/cm2) inoculum levels. Significantly higher populations of E. faecium transferred from onion to PU than from PU to onion. The transfer rates of E. faecium were impacted by inoculum carrier (61% [onion extract], 1.6% [peptone], and 0.31% [soil]) but not by inoculation level or recipient surface (PU versus SS). Bacterial transfer during dry onion handling is significantly dependent on bacterial species, inoculation levels, inoculum carrier, and transfer direction.

Enhanced heat tolerance of freeze-dried Enterococcus faecium NRRL B-2354 as valid Salmonella surrogate in low-moisture foods

In microbial studies of low-moisture foods (LMFs, water activity less than 0.85), freeze-dried bacteria benefit us to inoculate LMFs without introducing extra water or altering food physiochemical properties. However, the freeze-drying process would bring unavoidable damage to bacterial cells and results in less-resistant inoculum that are unlikely to be qualified in microbial studies. Herein, we enhanced bacterial heat tolerance by subjecting the cells to mild heat (42-50 °C) to counteract the reduced heat tolerance and survivability of freeze-dried bacteria. Enterococcus faecium NRRL B-2354 (E. faecium), a Salmonella surrogate in LMFs, was used as the target microorganism because it was widely accepted in microbial validation of thermal pasteurizing LMFs. Three types of LMFs (peanut powder, protein powder, and onion powder) were used as LMFs models to validate the freeze-dried E. faecium in comparison with Salmonella enterica Enteritidis PT 30 (S. Enteritidis) prepared by the traditional aqueous method. The heat tolerance (D65℃ value) of E. faecium increased at all treatments and peaked (+31.48 ± 0.13%) at temperature-time combinations of 45 °C-60 min and 50 °C-5 min. Survivability of freeze-dried inoculum and its heat tolerance retained well within 50 d storage. The freeze-dried E. faecium was prepared in this study brought equal or higher heat tolerance (D85℃ or D75℃) than S. Enteritidis in tested LMFs models. For instance, the D85℃ of freeze-dried E. faecium (heat-treated at 50 °C for 5 min) and S. Enteritidis in whole egg powder are 35.56 ± 1.52 min and 28.41 ± 0.41 min, respectively. The freeze-dried E. faecium with enhanced heat tolerance appears to be a suitable Salmonella surrogate for dry-inoculating LMFs. Our protocol also enables industry-scale production of freeze-dried inoculum by broth-cultivation method combined with mild-heat treatment.

Thermal resistance of shiga toxin-producing Escherichia coli, Salmonella and Enterococcus faecium in peanut butter, oatmeal raisin, and chocolate chip cookies

Thermal-death-time characteristics, D and z-values of shiga toxin-producing Escherichia coli (STEC), Salmonella, and Enterococcus faecium NRRL B-2354 were investigated in peanut butter, oatmeal, and chocolate chip cookie formulations at three moisture levels. E. faecium was shown to be more resistant than Salmonella, followed by E. coli at all water activity levels. E. faecium was at least 1.5 times more heat resistant compared to Salmonella under the same moisture and temperature conditions for all cookie formulations. The E. faecium to Salmonella ratio was larger at higher moisture levels. E. faecium was found to be suitable as a surrogate for Salmonella in validating soft cookie baking. Among the three formulations, Salmonella was the most heat resistant in peanut butter cookies. At the lowest moisture and water activity level (8% moisture, aw 0.421) of peanut butter cookies the D-values for E. coli were 14.01, 3.56, 1.73 min at 65, 70, 75 °C, while the D-values for Salmonella were 4.97, 2.59, 1.23 min, and for E. faecium were 15.50, 5.02, 2.02 min at 75, 80, and 85 °C, respectively. The z-values for E. coli, Salmonella, and E. faecium, were 11.02, 16.45 and 11.31 °C, respectively.

Effect of Lactose and Milk Protein on Thermal Resistance of Enterococcus faecium NRRL B-2354 and Salmonella in Dairy Powders

Microbial challenge studies using nonpathogenic surrogates provide a practical means for validating thermally based pathogen controls for low-moisture foods. Because the relative thermal resistance, or kill ratio, of Enterococcus faecium NRRL B-2354 (a nonpathogenic surrogate) to Salmonella is greatly influenced by food composition, this study assessed relative thermal resistance of a five-strain Salmonella cocktail and E. faecium in skim milk powder (SMP), lactose-free skim milk powder (LSMP), 90% milk protein isolate (MPI), and lactose powder (LP). The impact of sugar composition (lactose versus glucose-galactose) on resuscitation of bacterial survivors, by using SMP and LSMP, was also determined. Dairy powders were inoculated with agar-grown cultures, mixed, preequilibrated at 0.25 water activity (aw), ground to achieve homogeneity, reequilibrated, and subjected to isothermal treatment. After enumeration on nonselective differential media, log-linear and Bigelow models were fit to the survivor data via one-step global regression. The aw changes and glass transition temperature were assessed at elevated temperatures by using uninoculated, equilibrated powder samples. Estimated D90°C-values were approximately two times higher for E. faecium (P < 0.05) than for Salmonella in SMP, LP, and MPI, but statistically similar (P > 0.05) in LSMP. Addition of sugars to recovery media did not influence survivor resuscitation from heat-treated SMP and LSMP, confirming that microbial inactivation was impacted primarily by the thermal treatment, not the recovery step. Thermally induced changes in aw were seen only for LP and MPI, with the glass transition temperature observed only for SMP and MPI. In conclusion, rather than always requiring greater lethality of E. faecium than Salmonella, these findings suggest that sufficient pathogen controls for low-moisture foods can also be validated by thoroughly documenting the appropriate kill ratios of E. faecium to Salmonella.

Media impacts recovery of Salmonella enterica and Enterococcus faecium NRRL B2354 from whole black peppercorns, basil leaves, and chia seeds treated with antimicrobial gasses

Salmonella enterica contamination of low water activity foods (LWAFs) has resulted in recalls of spices, herbs, and seeds and outbreaks of salmonellosis. To improve the safety of these ready-to-eat products, new treatment methods, including fumigation with chlorine dioxide (ClO2) or hydrogen peroxide (H2O2) gas are being explored, and effectiveness determined. To prevent overestimation of treatment effectiveness, it is vital that recovery methods should accurately quantify all viable cells, even those injured. This study evaluated different media and supplements for the recovery of multiple strains of S. enterica and Enterococcus faecium NRRL B2354, from ClO2 or H2O2 treated black peppercorns, dried basil leaves, and chia seeds. Also, this study aimed to compare the log reduction of these two microorganisms to evaluate E. faecium NRRL B2354, as a surrogate for S. enterica. On average, recovery of S. enterica was improved by 1 log CFU from ClO2 and H2O2 treated LWAFs when a non-selective but differential media containing tryptic soy agar with yeast extract, ammonium iron citrate and sodium thiosulfate (MTSAYE) was used, when compared to plating on XLD (p &amp;lt; 0.05). Furthermore, addition of sodium pyruvate, ferrous sulfate, or 3’3’-thiodiproionate supplements to MTSAYE did not show increased recovery of either S. enterica or E. faecium NRRL B2354 (p &amp;gt; 0.05). On each treatment and LWAF combination tested, there was no significant difference between the log reduction of S. enterica and E. faecium NRRL B2354, indicating its suitability as a surrogate under the test conditions.

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

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.

Survivability of Salmonella and Enterococcus faecium in chili, cinnamon and black pepper powders during storage and isothermal treatments

Outbreaks and recalls associated with foods containing spices suggest a need for risk assessment of Salmonella in spices. In this study, the survivability of Salmonella Enteritidis PT 30, Salmonella cocktail (S. Enteritidis PT 30, S. Tennessee K4643 and S. Agona 447967), and Enterococcus faecium NRRL B-2354 in chili, cinnamon and black pepper at water activities (aw) 0.3 and 0.5 were evaluated during one-year storage at 21 °C. The thermal resistance of Salmonella cocktail in spices was also evaluated at 70 °C before and after storage. At aw 0.5, 4-month storage caused 5 log reduction of Salmonella cocktail in chili, while 8 months led to the same level of reduction in cinnamon. But only 3 log reduction were observed in black pepper over one year. Storage at aw 0.3 caused less reduction in Salmonella cocktail during the same storage periods. Less than 2 log reduction of E. faecium were observed over the one year storage at both aw levels, except for in chili stored at 0.5 aw. The D70°C-values for Salmonella cocktail in chili, cinnamon and black pepper of aw 0.3 before storage were 15.4, 20.8 and 36.6 min, respectively. 21–50% drops in the D70°C-value were obtained after two-month of storage, mostly in chili and least in black pepper. The high D70°C-value in black pepper persisted over one-year storage. Based on these results, chili powder showed the highest antimicrobial effect, followed by cinnamon and black pepper powders during storage and isothermal treatments.

The effect of dry headspace on the thermal resistance of bacteria in peanut oil and peanut butter in thermal treatments

The objective of this study was to investigate the effect of a dry headspace on the thermal resistance of bacteria in lipid oil and peanut butter. Our recent study found Enterococcus faecium to be extremely resistant to heat in a thin oil layer when exposing to a dry environment. Thus, we hypothesized that peanut oil or peanut butter that is in contact with dry air during a thermal process may cause surface drying and, therefore, protect bacteria from heat inactivation. In this research, we treated peanut oil (aw = 0.31 at 80 °C) and peanut butter (aw = 0.38 at 80 °C) inoculated with Salmonella Enteritidis PT30 (S. Enteritidis) or Enterococcus faecium NRRL B-2354 (E. faecium) at 80 °C using two types of hermetically sealed sample holders, one with negligible headspace and the other with a large headspace. The results indicated that S. Enteritidis and E. faecium in peanut oil and peanut butter became significantly (P < 0.05) more resistant to heat when treated in sample holders with large headspace compared to in fully filled sample holders with negligible headspace. When the sample holder head space was switched from “Negligible” to “Large”, the thermal death time (D80-value) of E. faecium increased 16.7 times in oil and 4.3 times in peanut butter; the D80-value of S. Enteritidis increased 4.6 times in peanut oil and 2.6 times in peanut butter. When plotted vs. the material water activity measured at the treatment temperature (80 °C), the D80-value of both bacteria agreed well with reported data obtained from other low-moisture foods, indicating that the local water activity of the sample was the dominating factor and surface drying was the cause to the enhanced thermal resistance. This study illustrates the importance of selecting appropriate test cells in isothermal studies on thermal resistence of bacteria in high fat low moisture foods. It also suggests that food industry should be cautious about surface drying in the pasteurization of low-moisture products.

Predictive Microbial Modeling of Enterococcus faecium NRRL B-2354 Inactivation during Baking of a Multicomponent Low-Moisture Food

The use of baking ovens as a microbial kill step should be validated based on results of thermal inactivation models. Although traditional isothermal models may not be appropriate for these dynamic processes, they are being used by the food industry. Previous research indicates that the impact of additional process conditions, such as humidity, should be considered when validating thermal processes for the control of microbial hazards in low-moisture foods. In this study, the predictive performance of traditional and modified thermal inactivation kinetic models accounting for process humidity were assessed for predicting inactivation of Enterococcus faecium NRRL B-2354 in a multi-ingredient composite food during baking. Ingredients (milk powder, protein powder, peanut butter, and whole wheat flour) were individually inoculated to achieve ∼6 log CFU/g, equilibrated to a water activity of 0.25, and then mixed to form a cookie dough. An isothermal inactivation study was conducted for the dough to obtain traditional D- and z-values (n = 63). In a separate experiment, cookies were baked under four dynamic heating conditions: 135°C, high humidity; 135°C, low humidity; 150°C, high humidity; and 150°C, low humidity. Process humidity measurements; time-temperature profiles for the product core, surface, and bulk air; and microbial survivor ratios were collected for the four conditions at six residence times (n = 144). The traditional isothermal model had a high root mean square error (RMSE) of 856.51 log CFU/g, significantly overpredicting bacterial inactivation during the process. The modified model accounting for the dynamic time-temperature profile and process humidity data was a better predictor with an RMSE of 0.55 log CFU/g. These results indicate the importance of accounting for additional process parameters in baking inactivation models and that model performance can be improved by utilizing model parameters obtained directly from industrial-scale experimental data.

Persistence of Salmonella enterica and Enterococcus faecium NRRL B-2354 on Baby Spinach Subjected to Temperature Abuse after Exposure to Sub-Lethal Stresses.

The exposure of foodborne pathogens such as Salmonella enterica to a sub-lethal stress may protect bacterial cells against distinct stresses during the production of leafy greens, which can constitute potential health hazards to consumers. In this study, we evaluated how the prior exposure of S. enterica to sub-lethal food processing-related stresses influenced its subsequent persistence on baby spinach under cold (4 °C for 7 days) and temperature abuse (37 °C for 2 h + 4 °C for 7 days) conditions. We also compared the survival characteristics of pre-stressed S. enterica and Enterococcus faecium NRRL B-2354 as its surrogate on baby spinach. A cocktail of three S. enterica serovars, as well as S. Typhimurium ATCC 14028 wild type and its ΔrpoS mutant, and E. faecium NRRL B-2354, was first exposed to sub-lethal desiccation, oxidation, heat shock, and acid stresses. Afterward, baby spinach was inoculated with unstressed or pre-stressed cells at 7.0 log CFU/sample unit, followed by 7-day storage under cold and temperature abuse conditions. The unstressed S. enterica (fresh cells in sterile 0.85% saline) decreased rapidly within the first day and thereafter persisted around 5.5 log CFU/sample unit under both conditions. The desiccation-stressed S. enterica showed the highest bacterial counts (p < 0.05) compared to other conditions. The unstressed S. enterica survived better (p < 0.05) than the oxidation- and acid-stressed S. enterica, while there were no significant differences (p > 0.05) between the unstressed and heat-shocked S. enterica. Unlike the wild type, temperature abuse did not lead to the enhanced survival of the ΔrpoS mutant after exposure to desiccation stress, indicating that the rpoS gene could play a critical role in the persistence of desiccation-stressed S. enterica subjected to temperature abuse. E. faecium NRRL B-2354 was more persistent (p < 0.05) than the pre-stressed S. enterica under both conditions, suggesting its use as a suitable surrogate for pre-stressed S. enterica by providing a sufficient safety margin. Our results demonstrate the merit of considering the prior exposure of foodborne pathogens to sub-lethal stresses when validating the storage conditions for leafy greens.

Thermal Inactivation Kinetics of Salmonella and Enterococcus faecium NRRL-B2354 on Whole Chia Seeds (Salvia hispanica L.)

Intervention technologies for inactivating Salmonella on whole chia seeds are currently limited. Determination of the thermal inactivation kinetics of Salmonella on chia seeds and selection of an appropriate nonpathogenic surrogate will provide a foundation for selecting and optimizing thermal pasteurization processes for chia seeds. In this study, chia seed samples from three separate production lots were inoculated with a five-strain Salmonella cocktail or Enterococcus faecium NRRL-B2354 and equilibrated to a water activity of 0.53 at room temperature (25°C). After equilibration for at least 3 days, the inoculated seeds were subjected to isothermal treatments at 80, 85, or 90°C. Samples were removed at six time points, and surviving bacteria were enumerated. Whole chia seeds were diluted in a filter bag at 1:30 because bacterial recovery with this method was similar to that obtained from ground seeds. Survivor data were fitted to consolidated models: one primary model (log linear or Weibull) and one secondary model (Bigelow). E. faecium had higher thermal resistance than did Salmonella, suggesting that E. faecium may be a suitable conservative nonpathogenic surrogate for Salmonella. The Weibull model was a better fit for the survivor data than was the log-linear model for both bacteria based on the lower root mean square error and corrected Akaike's information criterion values. Lipid oxidation measurements and fatty acid concentrations were significantly different from those of the control samples, but the overall magnitude of the differences was relatively small. The thermal inactivation kinetics of Salmonella and E. faecium on chia seeds may be used as a basis for developing thermal pasteurization processes for chia seeds.

Gaseous chlorine dioxide technology for improving microbial safety of spices

In this study, black peppercorns and cumin seeds were inoculated with a five-serotype cocktail of Salmonella or its surrogate Enterococcus faecium NRRL B-2354. The inoculated samples (ca. 107 CFU/g) were treated by ClO2 gas at different gas concentrations (5, 10, and 15 mg/L) and relative humidities (60, 70, and 80%) at room temperature (25 °C) at five gas exposure times (60, 120, 180, 240, 300 min). More than 5-log reduction of Salmonella was achieved in both spices with 300 min of ClO2 gas treatment at 15 mg/L and 80% RH. The corresponding values for E. faecium were 4.36 and 4.17 log CFU/g in black peppercorns and cumin seeds, respectively, which were significant lower than Salmonella (p < 0.05). Gas concentration, RH, and exposure time showed significantly positive linear associations with Salmonella inactivation. E. faecium appears a suitable surrogate for Salmonella and can be used for future process validations using comparable conditions.

Thermal inactivation of Salmonella, Listeria monocytogenes and Enterococcus faecium NRRL B-2354 in desiccated shredded coconut

Desiccated shredded coconut (DSC) products have been implicated in multiple Salmonella outbreaks and Listeria monocytogenes recalls. The objective of this study was to evaluate the thermal resistance of Salmonella and L.monocytogenes in DSC as impacted by water activity (aw), and the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for these foodborne pathogens during thermal processing of DSC. The inactivation kinetics of each strain in DSC fitted the log-linear model well; their thermal tolerances were inversely related to aw. The D80°C-, D85°C-, and D90°C-values of Salmonella were 38.7, 15.5, and 6.0 min at aw 0.45 compared to 53.2, 28.0, and 12.5 min at aw 0.25 under respective temperatures. For aw 0.25 and 0.45 DSC, D-values of E. faecium ranged from 49.6 to 6.5 min and 85.5 to 24.2 min at 80–90 °C, which were 1.4–1.9 and 1.1–1.3 times of those of Salmonella at the tested temperatures, indicating E. faecium is an appropriate surrogate of Salmonella during thermal processing of DSC. Compared to Salmonella, L. monocytogenes exhibited less thermal resistance in DSC. This study provides useful information for the food industry to develop thermal inactivation strategies to control Salmonella and L.monocytogenes during the post-drying process of DSC.

Radiofrequency pasteurization against Salmonella and Listeria monocytogenes in cocoa powder

In this study, thermal processes were developed for cocoa powder against Salmonella and Listeria monocytogenes in a pilot Radiofrequency (RF) unit and validated using their respective surrogates Enterococcus faecium NRRL B-2354 and Listeria innocua. For 1.0 kg cocoa powder samples, RF heating to 90 °C provided 1.74–1.84 log reduction of E. faecium. A 48 min holding post-RF heating provided an additional 3.65 and 2.24 log10 CFU/g reduction of E. faecium for insulated and non-insulated samples, respectively. Compared to E. faecium, L. innocua was less resistant to RF heating in aw 0.45 cocoa powder; RF heating to 75 °C coupled with 24 min insulated holding resulted in an additional 5.64 log10 CFU/g reduction. These data suggested that E. faecium was a conservative surrogate strain for controlling Salmonella and L. monocytogenes during RF processing of cocoa powder. To achieve maximal microbial reduction and improve energy efficiency, an insulated holding following RF heating is highly recommended. According to the predictive line obtained from the Bigelow model, a 7.6 min RF heating plus 48 min insulated holding attained a 5-log reduction of E. faecium without a negative impact on the color of cocoa powder.