Category Archives: Enterococcus faecium

Resaerch- Evaluation of Various Lactic Acid Bacteria and Generic E. coli as Potential Non-pathogenic Surrogates for In-Plant Validation of Biltong Dried Beef Processing


Validation studies conducted within a food processing facility using surrogate organisms could better represent the manufacturing process than controlled laboratory studies with pathogenic bacteria on precision equipment in a BSL-2 lab. The objectives of this project were to examine potential surrogate bacteria during biltong processing, conduct biltong surrogate validation lethality studies, and measure critical factors and intrinsic parameters during processing. Beef pieces (1.9 cm × 5.1 cm × 7.6 cm) were inoculated with four-strain mixtures of Carnobacterium divergens/C. gallinarumPediococcus acidilactici/P. pentosaceous, and Biotype 1 E. coli ATCC BAA (-1427, -1428, -1429, and -1430), as well as a two-strain mixture of Latilactobacillus sakei and other commercially available individual bacterial cultures (P. acidilactici Saga200/Kerry Foods; Enterococcus faecium 201224-016/Vivolac Cultures). Inoculated beef was vacuum-tumbled in marinade and dried in a humidity-controlled oven for 8–10 days (24.9 °C; 55% relative humidity). Microbial enumeration of surviving surrogate bacteria and evaluation of intrinsic factors (water activity, pH, and salt concentration) were performed post inoculation, post marination, and after 2, 4, 6, 8, and 10 days of drying. Trials were performed in duplicate replication with triplicate samples per sampling time and analyzed by one-way RM-ANOVA. Trials conducted with E. faeciumPediococcus spp., and L. sakei never demonstrated more than 2 log reduction during the biltong process. However, Carnobacterium achieved a >5 log (5.85 log) reduction over a drying period of 8 days and aligned with the reductions observed in previous trials with pathogenic bacteria (Salmonella, E. coli O157:H7, L. monocytogenes, and S. aureus) in biltong validation studies. Studies comparing resuspended freeze-dried or frozen cells vs. freshly grown cells for beef inoculation showed no significant differences during biltong processing. Carnobacterium spp. would be an effective nonpathogenic in-plant surrogate to monitor microbial safety that mimics the response of pathogenic bacteria to validate biltong processing within a manufacturer’s own facility. View Full-Text

Research – Desiccation survival in Salmonella enterica, Escherichia coli and Enterococcus faecium related to initial cell concentration and cellular components

Journal of Food Protection

Salmonella enterica is well-known for its ability to survive and persist in low-moisture environments.  Previous studies have indicated a link between the initial cell concentration and the population of Salmonella that survive upon desiccation and subsequent storage; however, how the initial cell concentration affects survival is unknown.  This study examined the basis of this phenomena and whether it occurred in other microorganisms, specifically Shiga toxigenic Escherichia coli (STEC), and Enterococcus faecium . Salmonella, STEC, and E. faecium were grown as lawns on TSAYE and harvested using buffered peptone water (BPW). To determine recovery at different initial cell levels, cultures were diluted to 9, 7, and 5 log CFU/mL and applied to filters.  Filters were dried for 24 h, then stored for 28 d at 25°C/33% RH. During storage, cells were recovered from filters using BPW and cultivated on TSAYE.  Both Salmonella and E. coli , but not E. faecium , showed non-proportional recovery. Less viability remained with lower initial starting population after 24 h desiccation such that ≥10 log CFU/mL were recovered when 11 log CFU/mL was desiccated, but ❤ log CFU/mL were recovered when 5 log CFU/mL was desiccated. Once dried, persistence did not appear affected by initial cell concentration. When dead cells (heat-treated) were added to the diluent, recovery of Salmonella was proportional with respect to the initial cell concentration. To further examine the response on desiccation, Salmonella was diluted in BPW containing one of 11 different test cell components related to quorum sensing or known to affect desiccation resistance to assess recovery and persistence. Of the 11 additions only cell debris fractions, cell-free extract, and peptidoglycan improved recovery of Salmonella . Desiccation survival appears related to cell wall components, however, the exact mechanism affecting survival remains unknown.

Research- Predictive microbial modeling of E. faecium NRRL B-2354 inactivation during baking of a multi-component low-moisture food

Journal of Food Protection

Validating baking ovens as a microbial kill step, using thermal inactivation models, is desirable; however, traditional isothermal models may not be appropriate for these dynamic processes, yet they are being used by the food industry. Previous research indicates that the impact of additional process conditions, such as process 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 bacterial 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), individually inoculated to ~6 logCFU/g and equilibrated to a water activity of 0.25, were mixed to form a 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℃/high humidity, 135℃/low humidity, 150℃/high humidity, and 150℃/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 poor 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 yielded a better predictive performance with a RMSE of 0.55 log CFU/g. The results demonstrate 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.

Research – Thermal Inactivation Kinetics of Salmonella and Enterococcus faecium NRRL-B2354 on whole chia seeds (Salvia hispanica L.)

Journal of Food Protection

Intervention technologies for inactivating Salmonella in whole chia seeds are currently limited. The determination of the thermal inactivation kinetics of Salmonella o n chia seeds and selection of an appropriate nonpathogenic surrogate will provide a knowledge 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 0.53 aw at room temperature (25 °C). After equilibration for at least three days, the inoculated seeds were subjected to isothermal treatments at 80, 85, or 90 °C. Samples were taken out at six timepoints and enumerated for survivors. Initial dilution of whole chia seeds was performed in a filter bag at a 1:30 ratio after it was shown to have similar recovery to grinding the seeds. Survivor data were fitted to consolidated models consisting of a primary model (log-linear or Weibull) and one secondary model (Bigelow). E. faecium exhibited higher thermal resistance than Salmonella , suggesting its suitability as a conservative nonpathogenic surrogate. The Weibull model was a better fit for the survivor data than the log-linear model for both bacteria due to its lower root mean square error and corrected Akaike’s Information Criterion values. Measurements of lipid oxidation and fatty acid content indicated a few statistically different values compared to the control samples, but the overall difference in magnitudes were relatively small. The thermal inactivation kinetics of Salmonella and E. faecium o n chia seeds as presented in this study can serve as a basis for developing thermal pasteurization processes for chia seeds.

Research – Evaluation of Hot-air Drying to Inactivate Salmonella spp. and Enterococcus faecium on Apple Pieces

Journal of Food Protection

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 Salmonella cocktail ( S. Agona, S. Tennessee, S. Montevideo, S. Mbandaka and S. 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/sample, respectively . Apple cubes were dried at 104°C 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 4 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 at 135°C drying faster 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/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.

Research -Determination of Enterococcus faecium thermal reduction in normal and high oleic peanut products

Wiley Online

During processing, peanut butter can become contaminated with pathogenic bacteria (e.g., Salmonella ). The introduction of an additional heat treatment step after roasting can help inactivate these microorganisms. In this study, trials were conducted to determine Enterococcus faecium (Salmonella surrogate) reduction rates during the roasting of high oleic (HO) peanuts and heat‐treatment of normal oleic (NO) and HO peanut butters. HO peanuts were inoculated with E. faecium and roasted in a convection oven at 190°C. There was a 2 and 6 log CFU/g reduction at 300 and 480 s, respectively. ‐values for HO peanut butter at 110, 120, and 125°C were 438.9, 165.1, and 80.6 s, respectively. The ‐value was calculated to be 20.8°C. There was no significant difference in ‐values and ‐values between NO and HO peanut butter. In a pilot scale experiment, HO peanut butter was inoculated with E. faecium and agitated in a heated mixer for 21.5 min. E. faecium was reduced by 5.1 log CFU/g after 16.5 min with no apparent change in viscosity or texture. This study demonstrated that significant reductions in E. faecium can be achieved during roasting and through an additional heat‐treatment step.


Research – Thermal Resistance of Foodborne Pathogens and Enterococcus faecium NRRL B-2354 on Inoculated Pistachios



Process control validations require knowledge of the resistance of the pathogen(s) of concern to the target treatment and, in some cases, the relative resistance of surrogate organisms. Selected strains of Escherichia coli O157:H7 (five strains), Listeria monocytogenes (five strains), and Salmonella enterica (five strains) as well as Salmonella Enteritidis phage type (PT) 30 and nonpathogenic Enterococcus faecium NRRL B-2354 were inoculated separately (as individual strains) onto inshell pistachios. The thermal tolerance of each strain was compared via treatment of inoculated pistachios to hot oil (121°C) or hot water (80°C) for 1 min. Survivor curves in hot oil or hot water (0.5 to 6 min, n = 6 to 15) were determined for one or two of the most resistant strains of each pathogen, as well as E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30, and the Weibull model was fit to the data. A pilot-scale air-impingement oven was used to compare the thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30 on pistachios with or without a brining pretreatment and at either dry (no steam) or 30% humidity (v/v) oven conditions. No significant difference in the time to a 4-log reduction in hot oil or hot water was predicted for any of the strains evaluated, on the basis of the 95% confidence interval. In the pilot-scale oven, E. faecium NRRL B-2354 was more thermally resistant than Salmonella in a broad set of differing treatments, treatment times, and temperatures. Salmonella is a suitable target pathogen of concern in pistachios for thermal processes because no other pathogen tested was more thermally resistant under the conditions evaluated. E. faecium NRRL B-2354 was at least as thermally resistant as Salmonella under all conditions evaluated, making it a good potential surrogate for Salmonella on pistachios.

  • Salmonella Enteritidis PT 30 is a reasonable target strain for laboratory-based studies.
  • E. coli O157:H7 or L. monocytogenes were less thermally resistant than Salmonella.
  • E. faecium NRRL B-2354 was at least as thermally resistant or more so than Salmonella.
  • E. faecium NRRL B-2354 is a good potential thermal-treatment surrogate for pistachios.