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Nowadays, fortified vegetable oils with vitamin D3 are widely available in different countries and are consumed daily. The reduction rate of added vitamin D3 in fortified canola oil during heating process, the changes in oxidative status, and the thermal kinetic degradation of vitamin D3 in the fortified oil were investigated. For this purpose, canola oil was fortified at two levels of vitamin D3 with 5.625 µg/mL (low concentration or LC) and 13.585 µg/mL (a high concentration or HC). Samples were heated isothermally at 100, 150, and 180 °C for 30 min. The vitamin D3 concentration was determined by the high-performance liquid chromatographic method. The retention of vitamin D3 in samples treated at 100 °C for 30 min showed no significant reduction. Samples treated at 150 and 180 °C depending on the initial concentration showed the retention of 67.5% to 72.97% and 33.16% to 40.35% of vitamin D3 , respectively. An inverse relationship was found between the increment of lipid oxidation products (peroxide and anisidine values) and the retention of vitamin D3 . Kinetic parameters such as rate constant, activation energy, decimal reduction time, and quotient indicator were also calculated. An Arrhenius relationship was used for the assessment of temperature dependence of vitamin D3 degradation. Activation energies for vitamin D3 in LC and HC between 100 and 180 °C were found to be 44.01 and 38.77 kJ/mol, respectively. PRACTICAL APPLICATION: The oil can be fortified with vitamin D3 at low cost and offers a good bioavailability. A high-temperature cooking method may not be appropriate for the fortified products containing high lipid content.

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q10 values of inulinase dropped below one. At 70 and 80 °C, t1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.

Individual metakaolin-based alkali activated binder (AAB) was utilized to optimize binary and ternary ones having f e l d s p a r / m e t a k a o l i n and s l a g / f e l d s p a r + m e t a k a o l i n ratios of 0.3 and 0.4, respectively. These three AABs had the ability to directly solidify 10.0 (FMK0-10R), 8.0 (FMK3-8R) and 12.0% (S4FMK3-12R) of the nuclear grade KY-2 beads, respectively, recording compressive strength values greater than twice the waste acceptance criteria. Leaching of 134Cs, 60Co and 152+154Eu, whether singularly or multiply loaded, was assessed. The multi-radionuclidic systems recorded greater leached fractions in the order of: 152+154Eu>134Cs>60Co. Among the studied systems, S4FMK3-12R formulations recorded the lowest diffusion coefficient values (D). Gamma-irradiation made a desired influence on all studied leaching systems with inverse relationships with the applied irradiation doses. Irradiating the optimized ternary AAB with 3.0 KGy (S4FMK3-12R-ɣ3) yielded the lowest D value (6.65 × 10−13 cm2/s), when single component-60Co was diffused. The leachability indexes of all irradiated AABs were not only greatly exceeded the value of 6 but also sometimes be twice such value. XRD, FT-IR and SEM examinations of S4FMK3, S4FMK3-12R and S4FMK3-12R-ɣ3 reflected their multi-layered semicrystalline natures and to what extent these AABs and the solidified beads had good and poor radiation stabilities, respectively, with a proposed three-step mechanism of such instability.

Personalized medicine asks if a new treatment will help a particular patient, rather than if it improves the average response in a population. Without a causal model to distinguish these questions, interpretational mistakes arise. These mistakes are seen in an article by Demidenko [2016] that recommends the “D-value,” which is the probability that a randomly chosen person from the new-treatment group has a higher value for the outcome than a randomly chosen person from the control-treatment group. The abstract states “The D-value has a clear interpretation as the proportion of patients who get worse after the treatment” with similar assertions appearing later. We show these statements are incorrect because they require assumptions about the potential outcomes which are neither testable in randomized experiments nor plausible in general. The D-value will not equal the proportion of patients who get worse after treatment if (as expected) those outcomes are correlated. Independence of potential outcomes is unrealistic and eliminates any personalized treatment effects; with dependence, the D-value can even imply treatment is better than control even though most patients are harmed by the treatment. Thus, D-values are misleading for personalized medicine. To prevent misunderstandings, we advise incorporating causal models into basic statistics education.

Improper food processing is one of the major causes of foodborne illness. Accurate prediction of the thermal destruction rate of foodborne pathogens is therefore vital to ensure proper processing and food safety. When bacteria are subjected to pH and thermal stresses during growth, sublethal stresses can occur that may lead to differences in their subsequent tolerance to thermal treatment. As a preliminary study to test this concept, the current study evaluated the effect of prior pH and thermal stresses on thermal tolerance of Salmonella and Staphylococcus using a tryptic soy broth supplemented with yeast extract. Bacteria incubated at three pH values (6.0, 7.4, and 9.0) and four temperatures (15, 25, 35, and 45°C) for 24 hr were subjected to thermal treatments at 55, 60, and 65°C. At the end of each treatment time, bacterial suspensions were surface‐plated on standard method agar for quantification of bacterial survival and further calculation of the thermal death decimal reduction time (D‐value) and thermal destruction temperature (z‐value). The effect of pH stress alone during the incubation on the thermal tolerance of both bacteria was generally insignificant. An increasing pattern of D‐value was observed with the increment of thermal stress (incubation temperature). The bacteria incubated at 35°C required the highest z‐value to reduce the 90% in D‐values. Staphylococcus mostly displayed higher tolerance to thermal treatment than Salmonella. Although further research is needed to validate the current findings on food matrices, findings in this study clearly affirm that adaptation of bacteria to certain stresses may reduce the effectiveness of preservation procedures applied during later stage of food processing and storage.

The traditional log-linear inactivation kinetics model considers microbial inactivation as a process that follows first-order kinetics. A basic concept of log reduction is decimal reduction time (D-value), which means time/dose required to kill 90% of the relevant microorganisms. D-value based on the first-order survival kinetics model is insufficient for reliable estimations of bacterial survivors following inactivation treatment. This is because the model does not consider the inactivation curvature and variability in bacterial inactivation. However, although the D-value has some limitations, it is widely used for risk assessment and sterilization time estimation. In this study, stochastic inactivation models are used in place of the conventional D-value to describe the probability of a population containing survivors. As representative bacterial inactivation normally follows a log-linear or log-Weibull model, we calculate the time required for a specific decrease in the number of cells and the number of survival cells as a probability distribution using the stochastic inactivation of individual cells in a population. We compare the probability of a population containing survivors calculated via the D-value, an inactivation kinetics model, and the stochastic formula. The stochastic calculation can be approximately estimated via a kinetic curvature model with less than 5% difference below the probability of a population containing survivors 0.1. This stochastic formula indicates that the D-value model would over- or under-estimate the probability of a population containing survivors when applied to inactivation kinetics with curvature. The results presented in this study show that stochastic analysis using mathematical models that account for variability in the individual cell inactivation time and initial cell number would lead to a realistic and probabilistic estimation of bacterial inactivation.

A prototype of microwave pasteurizer has been proposed as an alternative for holder pasteurization (HP) routinely used in Human Milk Bank (HMB), ensuring microbiological safety of human milk (HM). It was shown that the time of heat generation was about 15–16 min shorter by applying the microwave than in HP. Total inactivation of heat-sensitive bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis, suspended in milk, occurred in the temperature 62–72 °C in HP. In the case of heat-resistant enterococci the level of inactivation depended on the conditions of the process and the properties of the strains. The application of microwave heating allows to obtain lower D-value than those achieved during HP. The using of microwave heating at 62.5 or 66 °C for 5 or 3 min, respectively, allows to inactivation of HM microbiota. Appropriate microbiological quality of milk is critical for the effectiveness of the pasteurization process. Industrial relevance Looking for new methods of donor human milk (HM) preservation is dictated by the necessity of providing microbiological safety and, at the same time, maintain its high nutritional and biological value. The holder pasteurization used in the Human Milk Banks (HMB) (heating at 62 °C for 30 min) leads to inactivation of all vegetative forms of microorganisms. Unfortunately, this method causes significant reduction of health benefitting properties of HM. The paper demonstrates the possibility of using the new microwave pasteurizer for preservation of HM, allowing for quick heating of milk to the appropriate temperature and maintaining it in these conditions for a required time. It was shown that the decimal reduction times (D) for strains inoculated to UHT or human milk are several times shorter by using microwave heating than in the commercial pasteurization method. The total inactivation of HM microbiota is obtained after heating at 62.5 and 66 °C for 5 and 3 min, respectively.

Human noroviruses (HNoVs) cause significant gastrointestinal disease outbreaks worldwide. Tulane virus (TV) is a cultivable HNoV surrogate widely used to determine control measures against HNoVs. The objective of this study was to determine the heat inactivation kinetics (D- and z-values) of TV in cell-culture media and on spiked homogenized spinach using the first-order and Weibull models. TV in cell-culture media at approximately 7 log PFU/mL (PFU-plaque forming unit) in 2-mL glass vials was heated at 52, 54, and 56 °C for up to 10 min in a circulating water bath. Survivors were enumerated using confluent host LLC-MK2 cells in six-well plates by plaque assay. Data from three replicate treatments assayed in duplicate were analyzed statistically. D-values by the first-order model for TV in cell-culture media at 52, 54, and 56 °C were 4.59 ± 0.05, 2.91 ± 0.05, and 1.74 ± 0.07 min, respectively, with a z-value of 9.09 ± 0.01 °C (R2 = 0.997). The Weibull model showed td = 1 values of 2.53 ± 0.08, 1.99 ± 0.10, and 0.57 ± 0.64 min, respectively, at the same temperatures. The D-values for TV in spinach were 7.94 ± 0.21, 4.09 ± 0.04, and 1.43 ± 0.02 min and the z-value was 10.74 ± 0.01 °C (R2 = 0.98) by the first-order model and 4.89 ± 0.02, 3.21 ± 0.45, and 0.25 ± 0.38 min for the Weibull model at 50, 54, and 58 °C, respectively. In comparison to previously reported results for the cultivable HNoV surrogate, murine norovirus -1, TV in cell-culture media and spiked on spinach homogenates showed lower D- and z-values. TV may not be an ideal HNoV surrogate for heat inactivation studies in cell-culture media or homogenized spinach in vacuum bags.

Fermented fish, including rusip and bekasam, contains lactic acid bacteria, some of which have probiotic properties. Considering the beneficial effect, the count of viable cells in probiotic carrier foods must be at least 106 CFU/g throughout the product shelf-life. The processing and storage condition significantly affect the viability of probiotics in carrier foods. This research was conducted to determine the heat resistance of probiotic candidate Enterococcus faecalis R22B isolated from rusip in three different media: bekasam, rusip, and physiological saline solution (NaCl 0.85%). E. faecalis R22B was heated in each medium at a temperature of 70 °C for 0, 2, 4, 8, and 12 min. The media characteristics showed differences within the parameters included pH values of 4.41-6.99; aw 0.67-0.92; moisture content 72.56-98.09%; ash content 0.69-12.32%; protein content 0-14.7%; fat content 0-0.84%; and carbohydrate content from 0-4.93%. The results showed that the difference in the heating medium had no significant effect (p>0.05) on the heat resistance of the bacteria, as indicated by the D70 value. The required average heating time at 70 oC to decrease E. faecalis R22B cells by 1log10 (D70 value) in bekasam, rusip, and physiological saline media were 3.17, 3.29, and 3.55 min, respectively. Despite having a lower DT-value, the reduction of bacteria cells in three media heated at 70 oC showed no difference (p>0.05); therefore, bekasam and rusip are suitable for probiotic carrier food.

Aichi virus (AiV) that results in gastroenteritis worldwide, is spread through contaminated shellfish and water. The resistance/tolerance of AiV to common inactivation processes along with the absence of commercially available vaccines makes it necessary to study its thermal inactivation kinetics. This research evaluated the heat inactivation of AiV in cell-culture media using 2-ml sterile glass vials by the linear and Weibull models. Heat treatments of AiV titers of 7 log plaque forming units (PFU)/ml were conducted thrice in a water-bath at 50, 54, and 58 °C for up to 90 min. Plaque assays for each dilution in duplicate were used to determine infectious virus titers. Linear model D-values for AiV at 50 ± 1 °C (± = standard error) (come-up time = 68 s), 54 ± 0.7 °C (130 s), and 58 ± 0.6°C (251 s) were 43.3 ± 4.23 (R2 = 0.40, RMSE = 0.56), 5.69 ± 0.28 (R2 = 0.80, RMSE = 0.43), and 1.20 ± 0.63 min (R2 = 0.69, RMSE = 0.39), respectively, and the linear model z-value was 5.14 ± 0.39°C (R2 = 0.99, RMSE = 0.08). For the same temperatures, the Weibull model td = 1 values were 20.98 ± 8.8 (R2 = 0.62, RMSE = 0.46, α (scale parameter) = 2.30, β (shape parameter) = 0.38), 3.84 ± 0.69 (R2 = 0.85, RMSE = 0.38, α = 1.08, β = 0.66), and 0.87 ± 0.10 min (R2 = 0.80, RMSE = 0.32, α = 0.22, β = 0.61), respectively and the z-value (using Td = 1 ) was 5.79 ± 0.22 °C (R2 = 1.0, RMSE = 0.03). A better fit was obtained with the Weibull model for log reductions versus time with higher R2 and lower RMSE values. Application of AiV inactivation parameters can help reduce the risk of AiV outbreaks.

Animal intestines are the source of edible sausage casings, which are traded worldwide and may come from areas where notifiable infectious animal diseases are prevalent. To estimate the risks of virus contamination, knowledge about the quantity of virus and decimal reduction values of the standard preservation method by salting is of great importance. A literature search, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was performed in search engine CAB Abstracts to determine the viral load of 14 relevant animal viruses in natural casings or intestines. Only a very limited number of scientific publications per virus were found and viral loads in the intestines varied from high for ASFV (five publications), BVDV (3), CSFV (6), PPRV (3), RPV (2) and TGEV (3) to moderate for PEDV (2) and SVDV (3), low for HEV (2) and FMDV (5), very low for VESV (1) and negative for PrV (2) and VSV (1). PRRSV was found in intestines, however, viral titers were not published. Three viruses (BVDV, CSFV and PPRV) with high viral loads were selected to search for their inactivation kinetics. For casings, no inactivation data were found, however, thermal inactivation data of these viruses were available, but differed in quantity, quality and matrices. In conclusion, important data gaps still exist when it comes to the quantitative inactivation of viruses in sausage casings or livestock intestines.

Salt is among the most harmful agents that negatively influences crop yield. Alfalfa is an important perennial forage crop that exhibits wide cultivar variations in salt tolerance. Developing salt-tolerant alfalfa plants is a promising way to utilize salinized land. A comprehensive method was developed to achieve reliable and effective evaluation of alfalfa salt resistance. This included principal components, membership functions and cluster and stepwise regression analyses. These were used to analyse the salt tolerance coefficients of 14 traits and to evaluate 20 diverse alfalfa cultivars at the seedling stage. The various morphological root parameters of six alfalfa cultivars with contrasting salt tolerance were also tested by a scanning apparatus. According to the comprehensive evaluation value (D value), one highly salt-tolerant, two salt-tolerant, four moderately salt-tolerant and 13 salt-sensitive alfalfa cultivars were screened. A mathematical equation for the evaluation of alfalfa salt tolerance was established: D' = -0.126 + 0.667SFW + 0.377SDW + 1.089K+ /Na+ + 0.172SFW/RFW (R2 = 0.988; average forecast accuracy of 96.95%), where four indices were closely related to the salt tolerance: shoot fresh weight, ratio of shoot fresh weight to root fresh weight, shoot dry weight and ratio of K+ to Na+ in the shoot. We also found that SSA correlated strongly with SFW, SDW, K+ /Na+ , D values, while SRV correlated obviously with SFW, SFW/RFW and D values after 150 mm NaCl treatment. In conclusion, the SFW, K+ /Na+ , SDW, SFW/RFW, SSA and SRV could be used as indicators of salt tolerance in alfalfa seedlings grown under 150 mm NaCl treatment.

Efficient methods to achieve the safe decontamination of agricultural products are needed. Here, we investigated the decontamination of citrus fruits to test the antifungal potential of a novel non-thermal gas plasma apparatus, termed a roller conveyer plasma instrument. This instrument generates an atmospheric pressure dielectric barrier discharge (APDBP) plasma on a set of rollers. Penicillium venetum was spotted onto the surface of the fruit or pericarps, as well as an aluminium plate to act as a control, before performing the plasma treatment. The results showed that viable cell number of P. venetum decreased with a decimal reduction time (D value or estimated treatment time required to reduce viable cell number by 90%) of 0.967 min on the aluminium plate, 2.90 min and 1.88 min on the pericarps of ‘Kiyomi’ (Citrus unshiu × C. sinensis) and ‘Kawano-natsudaidai’ (C. natsudaidai) respectively, and 2.42 min on the surface of ‘Unshu-mikan’ (C. unshiu). These findings confirmed a fungicidal effect of the plasma not only on an abiotic surface (aluminium plate) but also on a biotic surface (citrus fruit). Further development of the instrument by combining sorting systems with the plasma device promises an efficient means of disinfecting citrus fruits during food processing.

Adequate surrogate identification is critical for validating in-plant thermal process controls for Salmonella inactivation in different food matrices. This study compared the thermal inactivation parameters (D- and z-values) and evaluated the heat resistance of Enterococcus faecium (8459) as a surrogate for a 5-serovar Salmonella cocktail in cornmeal. The cornmeal was spray inoculated with the respective bacteria to achieve ~9 log CFU/g population and set to the desired moisture contents (16, 22, and 28% w.b.). The inoculated cornmeal was then heat-treated at pre-determined temperatures (60, 64, and 68°C) in sealed aluminum thermal-death-time disks in hot water baths for pre-determined time intervals. Injury-recovery media [brain heart infusion (BHI) agar overlaid with xylose lysine deoxycholate (XLD) agar for Salmonella or BHI agar overlaid m-enterococcus agar for E. faecium] were used for microbial enumeration to account for thermally injured bacterial cells. The D-values of Salmonella in cornmeal at 16, 22, and 28% moisture content were 37.5, 8.4, and 2.4 min at 60°C, 19.9, 3.5, and 1.1 min at 64°C, and 10.1, 1.4, and 0.5 min at 68°C, respectively. Whereas the D-values of E. faecium in cornmeal at 16, 22, and 28% moisture content were 140.4, 18.9, and 3.3 min at 60°C, 78.4, 7.1, and 1.6 min at 64°C, and 37.3, 2.8, and 0.8 min at 68°C, respectively. The z-values of E. faecium and Salmonella in cornmeal at 16, 22, and 28% moisture content were 13.9, 9.7, and 12.5°C, and 14.0, 10.4, and 11.7°C, respectively. These results indicated similar or higher thermal resistance (D-values) and equivalent thermal sensitivity (z-values) of E. faecium compared to Salmonella at different moisture contents and respective temperatures (P ≤ 0.05). Therefore, E. faecium could be used as a surrogate for Salmonella during thermal process validation of cornmeal processing.

A predictive model was made for the logarithm of the thermal decimal reduction time (logD) of Salmonella enterica (D = time to 90% reduction by inactivation). The model was fitted with multiple linear regression from 521 logD-values reported in literature for laboratory media and foods highly varying in water activity and pH. The single regression model with temperature as the only variable had a high residual standard error (RSE) of 0.883 logD and no predictive value (fraction of variance explained (R2) < 0.001). Adding water activity, sugar content and pH as predictors resulted in a model with a lower RSE of 0.458 logD and an adjusted R2 of 0.73. The model was validated by comparing 985 predicted with observed logD for S. enterica from other publications. The model was subsequently validated with 1498 published logD-values for inactivation of vegetative cells of nine other pathogenic bacteria genera (mainly Listeria monocytogenes, Escherichia coli, Clostridium perfringens, Cronobacter spp., Staphylococcus aureus, Yersinia enterocolitica) in or on a variety of laboratory media, meat, fish, dairy, nuts, fruits and vegetables. Regression analyses for validation with the 985 logD of S. enterica and 2483 logD of all genera show deviations from the expected slope of 1 (both 0.81) and the expected intercept of 0 (0.04 and 0.19 logD respectively). However, only 0.7% and 2% respectively of the new logD (expected: 0.5%) were observed above the 99% prediction interval of the original S. enterica model based on 521 logD. The findings suggest that i) the variability of thermal resistance of strains within species is larger than between genera and species; ii) one generic predictive model, also accounting for variability, suffices for designing the thermal inactivation of a variety of vegetative pathogenic bacteria in many food types.

In the present study, Enterobacter aerogenes KCTC2190 was isolated from soil around a cattle shed area, which was capable of producing intracellular β-galactosidase. Partially purified β-galactosidase was immobilized by entrapment method in agar-agar gel matrix. Agar-agar entrapped beads were prepared by dropping the enzyme-agar solution to ice-cooled toluene-chloroform ((3:1 (v/v)). 45.88±0.11% activity of partially purified β-galactosidase was retained after immobilization (bead shape). Maximum immobilization yield was observed in the presence of 2.5% agar-agar concentration. After immobilization, optimum temperature required for the enzyme-substrate reaction was shifted from 50 to 60 °C and the optimum reaction time was shifted from 15 to 25 min. The optimum pH for both free and immobilized β-galactosidase was pH 7. Free enzyme showed lower activation energy in comparison with the immobilized one. For free as well as immobilized β-galactosidase thermal deactivation, rate constant (kd) increased with increasing temperature while the values of decimal reduction time (D-values) and half-lives (t1/2) decreased. Immobilization process increased the t1/2 and D-values of β-galactosidase while it decreased the kd. Thermostability of immobilized β-galactosidase was higher as they showed higher enthalpy (ΔΗ0) and Gibb’s free energy (ΔG0)value than those of the free β-galactosidase. The negative entropy (ΔS0) of free and immobilized β-galactosidase established that both were in a more ordered state within the temperature range (50 to 70 °C) studied. Immobilized β-galactosidase was able to retain 51.65±1.61% of its initial activity after 7 batches of enzyme-substrate reaction. Immobilized β-galactosidase showed 78.09±3.69% of its initial activity even after 40 days of storage at 4 °C.

International audience; The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0–5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4–0.2 or 1.1–0.8 s on plastic, 0.9–0.7 or 1.4–0.8 s on steel and 1.6–1.7 or 3.2–4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.

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. HIGHLIGHTS

The presence of Listeria monocytogenes in Mozzarella di Bufala Campana Protected Designation of Origin cheeses may depend on curd stretching conditions and post contaminations before packaging. To avoid cross-contamination, thermal treatment of water, brines and covering liquid may become necessary. The present study aimed to improve knowledge about L. monocytogenes thermal resistance focusing on the influence of some cheese making operations, namely curd stretching and heat treatment of fluids in contact with cheese after molding, in order to improve the safety of the cheese, optimize efficacy and sustainability of the processes. Moreover, the role that cheese curd stretching plays in L. monocytogenes inactivation was discussed. The 12 tested strains showed a very heterogeneous heat resistance that ranged from 7 to less than 1 Log10 Cfu/mL reduction after 8 min at 60°C. D-values (decimal reduction times) and z-values (thermal resistance constant) calculated for the most heat resistant strain among 60 and 70°C were highly affected by the matrix and, in particular, heat resistance noticeably increased in drained cheese curd. As cheese curd stretching is not an isothermal process, to simulate the overall lethal effect of an industrial process a secondary model was built. The lethal effect of the process was estimated around 4 Log10 reductions. The data provided may be useful for fresh pasta filata cheese producers in determining appropriate processing durations and temperatures for producing safe cheeses.

Thermal stability (D-value and pasteurization) and gastric acid resistance of spore forming and nonspore forming probiotic strains were evaluated in this study. Bacillus coagulans MTCC 5856 spores showed highest thermal resistance (D-value 35.71 at 90 °C) when compared with other Bacillus strains and Lactobacillus species. B. coagulans strains exhibited significantly higher resistance to simulated gastric juice (pH 1.3, 1.5, and 2.0) compared to Lactobacillus strains. It also showed high resistance to cooking conditions of chapati (whole wheat flour-based flatbread) (88.94% viability) and wheat noodles (and 94.56% viability), suggesting remarkable thermal resistance during food processing. Furthermore, B. coagulans MTCC 5856 retained 73% viability after microwave cooking conditions (300 s, at 260 °C) and 98.52% in milk and juice at pasteurization temperature (420 min, at 72 °C). Thus, B. coagulans MTCC 5856 clearly demonstrated excellent resistance to gastric acid and high temperature (90 °C), thereby suggesting its extended application in functional foods (milk, fruit juices, chapati, and wheat noodles) wherein high temperature processing is involved.

To ensure safe reuse, biosolids are stabilized to reduce pathogens, odor, and volatile solids. Stabilization by lime addition have high material costs, high transportation costs, and loss of ammonia. Decreasing required lime additions would improve the sustainability of biosolids reuse. This study examined combining lime and heat treatment to reduce lime additions needed for required fecal coliform destruction. In contrast to the current best management practice requiring a pH of 12, fecal coliform reduction for final concentrations

Decontamination practices, which often involve thermal treatments, are routinely performed in beef packing plants and have generally improved the safety of meat in North America. We investigated whether Escherichia coli in the beef production chain is becoming more heat resistant due to those treatments. Cattle isolates (n = 750) included seven serogroups (O157, O103, O111, O121, O145, O26, and O45) which were collected between 2002 and 2017. Beef plant isolates (n = 700) from carcasses, fabrication equipment, and beef products were included. Heat resistance was determined in Luria-Bertani broth at 60°C and by PCR screening for the locus of heat resistance (LHR). The decimal reduction for E. coli at 60°C (D(60ºC) values) ranged from 0 to 7.54 min, with 97.2% of the values being 2 min was not significantly different (P > 0.05) among cattle and meat plant isolates. E. coli from equipment before sanitation (median, 1.03 min) was more heat resistant than that after sanitation (median, 0.9 min). No significant difference in D(60ºC) values was observed among E. coli isolates from different years, from carcasses before and after antimicrobial interventions, or from before and during carcass chilling. Of all isolates, 1.97% harbored LHR, and the LHR-positive isolates had greater median D(60ºC) values than the LHR-negative isolates (3.25 versus 0.96 min). No increase in heat resistance in E. coli was observed along the beef production chain or with time. IMPORTANCE The implementation of multiple hurdles in the beef production chain has resulted in substantial improvement in the microbial safety of beef in Canada. In this study, we characterized a large number of Escherichia coli isolates (n = 1,450) from various sources/stages of beef processing to determine whether the commonly used antimicrobial interventions would give rise to heat-resistant E. coli on meat, which in turn may require alternatives to the current control of pathogens and/or modifications to the current cooking recommendations for meat. The findings show that the degree and rate of heat resistance in E. coli did not increase along the production chain or with time. This furthers our understanding of man-made ecological niches that are required for the development of heat resistance in E. coli.

Aluminum based reflective nanolens arrays were developed via a series of aluminum electropolishing and anodization steps with subsequent selective dissolution of anodic aluminum oxide (AAO). The diameter of nanolenses (d) on arrays can be controlled by altering electrolytes and voltages used for aluminum anodization. The d values of arrays produced by anodization in 0.3 M oxalic acid at 40, 60, and 80 V, and in 1.0 M phosphoric acid at 100, 110, and 120 V were 71.94, 121.90, and 161.53 nm, and 220.16, 252.06, and 274.78 nm, respectively. The effectiveness of UV (254 nm) inactivation of Escherichia coli O157:H7 and Listeria monocytogenes at concentrations of 5–6 log CFU/mL in water and in a 10% (w/v) sucrose solution was improved using a nanolens array having a d value of 252.06 nm. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10068-020-00765-z) contains supplementary material, which is available to authorized users.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p

The inactivation and damage of histamine-forming bacteria (HFB), Enterobacter aerogenes and Staphylococcus capitis, in a 0.1 M potassium phosphate buffer (pH 6.8) and marlin meat slurry by high hydrostatic pressure (HHP) treatments were studied using viability measurement and scanning electron microscopy (SEM). HHP treatments showed first order destruction kinetics to E. aerogenes and S. capitis during the pressure holding period. HFB in marlin meat slurry had higher D values and were more resistant to HHP treatments than in phosphate buffer. In phosphate buffer, E. aerogenes had higher D values than S. capitis at &gt, 380 MPa of pressure, whereas the reverse trend was noticed at lower pressures (&lt, 380 MPa). In marlin meat slurry, S. capitis had a higher D value than E. aerogenes at the same treatment pressure, indicating that S. capitis was more resistant to HHP treatment. To our knowledge, this is the first report to demonstrate that HHP can be used to inactivate HFB, E. aerogenes, and S. capitis, by causing disruption to bacterial cell membrane and cell wall as demonstrated by SEM micrographs.

In this study, 12 upland cotton seedlings were used as the material, and four treatments were designed (15% PEG for 6 h, 250 mM NaCl for 3 h, 15% PEG for 6 h after 250 mM NaCl pretreatment, and blank control). Various physiological indicators, including the malondialdehyde (MDA) and proline (Pro) contents and superoxide dismutase (SOD) and peroxidase (POD) activities, and the relative electrolyte leakage (REL), were measured during exposure to the aforementioned stresses, and three stress-related transcription factors (GhHsfA, GhbZIP, and GhNAC) were used to assess the differences in the drought resistance of cotton during exposure to PEG stress and NaCl/PEG combined stress. The analyses of the physiological and biochemical indicators revealed that the cotton seedlings exposed to NaCl/PEG combined stress exhibited the highest relative changes in the SOD and POD enzyme activities, while the relative changes in the MDA content and REL were relatively small. The cluster analysis showed that the treatments could be ranked as follows based on degree of damage exhibited by the exposed cotton seedlings: PEG > NaCl > NaCl/PEG. The exposure of cotton to NaCl/PEG combined stress resulted in a lower degree of damage than that obtained after exposure to PEG alone, which indicated that an appropriate amount of NaCl could partially relieve the adverse effects of drought on cotton seedlings. In addition, the relative expression levels of GhHsfA, GhbZIP, and GhNAC were significantly correlated with multiple physiological and biochemical indicators under different stresses, and the principal component analysis identified these transcription factors as important indicators. Based on these findings, these three transcription factors can be used as molecular indicators for the identification of drought resistance. A comprehensive D value cluster analysis ranked the 12 cotton varieties based on their drought resistance, and the most drought-resistant variety was ND359-5. This study provides new methods and materials for research on drought resistance in cotton.

Background and objective Atmospheric pressure plasma jet (APPJ) gained great interest due to its effectiveness as selective non-lethal technique with low operational costs. In this study, argon APPJ system was designed and the generated cold plasma was applied in disinfection of microbial cells. Materials and methods Argon APPJ was generated by blowing argon through capillary metallic tube inserted in alumina and powered by 8-25 kHz sinusoidal voltage waveform. The plasma applied in inactivation of microbes by direct exposure of cell suspension, approximately 10 mm below jet nozzle, for different intervals. Interference of organics in exposure medium, on lethal activity of plasma was investigated. Results APPJ jet induced high levels of reactive oxygen (ROS) and nitrogen species (RNS). Jet length increased with applied voltage and flow rate in laminar mode, but decreased with flow rate in turbulent mode. Percent reduction in living cell count was 98.3 and 94.1%, for E. coli and S. aureus suspended in water after 30s of exposure, respectively, with 2.7- and 2-folds increase in plasma lethal activity, as compared with LB broth medium. D-values (Decimal Reduction Time) were increased from 34-333, 37-476 and 139-385 s for E. coli, S. aureus and C. albicans in water and complex liquid organic media, respectively. Conclusion Designed argon APPJ system can be used in disinfection of different microbes. Plasma antimicrobial activity drastically decreased in presence of organic matter. The generated plasma can be promising approach for treatment of diseases, especially caused by antibiotic-resistant pathogens.

Understanding the thermal inactivation of biopreservatives in food matrixes is essential for their proper use in the food industry, enabling reduction of heating time and optimization of heating temperature. With this aim in mind, the effect of heat treatment on the activity and thermostability of commercial nisin from Lactococcus lactis was investigated. Residual activity tests performed for 90 min in the 70–86 °C temperature range at pH 3.0 demonstrated that bacteriocin thermoinactivation followed first-order kinetics. Low values of the inactivation rate constant (0.0089–0.0892 min−1) and high values of the decimal reduction time (25.8 ≤ D-value ≤ 258.4 min) indicated excellent nisin thermostability in the lower end of the investigated temperature range, but the thermal resistance constant (z-value) was only 15.4 °C, which pointed out poor thermoresistance at the higher end. Thermodynamic parameters estimated for nisin irreversible thermal inactivation were energy, enthalpy and Gibbs free energy of activation of 155.1, 152.2–152.3 and 83.5–86.6 kJ mol−1, respectively, and activation entropy of 188.6–192.5 J mol−1 K−1. If on the one hand these results taken together confirm the thermostability of nisin as a biopreservative in use in the food industry, on the other hand suggest the importance of looking for new more thermostable antimicrobials to use in operations at higher temperatures.

This paper aims to compare the physical properties of panels made of lignocellulosic raw materials and cement. Density (D), water absorption (WA), and thickness swelling (TS) after 2 and 24 h of immersion in water were analyzed. These lignocellulosic materials were used to produce panels: sugarcane bagasse, eucalyptus, banana pseudostem, coconut shell, and coffee husk. Three panels of each material were produced and each sample was arranged with the following dimensions: 150 × 150 × 15 mm. Each sample was submerged in water for 2 and 24 h, and the WA and TS capacity was measured in both conditions. Coconut and banana pseudostem panels presented both the lowest D values and the highest values of WA after 2 h and 24 h of immersion. The coffee panel showed the maximum D value. About TS the eucalyptus panels presented the highest value. Sugarcane panels showed the lowest TS at 2 h according to the recommended standards related to TS for 2 h. TS resistance tends to decrease with the increase in D, so panels produced with smaller and thicker particles tend to be slenderer and to present higher resistance to perpendicular traction.

In many food products, the population of microorganisms present after initial manufacturing stages is too high. As such, an intervention treatment is required to reduce the microbial load of these products. Thermal treatments are still by far the most common methods for microbial inactivation. The appropriate use of these technologies is aided by using mathematical models that describe the effect of temperature and other conditions on microbial responses. These kinetic models are subcategorised as primary models that describe the evolution of the population with time and secondary models that describe the effect of the environmental conditions on the parameters of the primary models. This chapter offers a comprehensive discussion of primary and secondary models for thermal microbial inactivation.

The objective of the study was the evaluation of the efficacy of plasma activated water (PAW) for Escherichia coli inoculated on soybean seed surface. PAW was prepared by high-power atmospheric pressure air plasma jet. The inactivation kinetics, explained by Bigelow log-linear and Weibull models, showed the significant antibacterial potential of PAW in comparison to the control (sterile distilled water). PAW treatment for 25 min let to log10 CFU/g reduction of E. coli from 6.8 to 3.6 and its antibacterial effect persisted further 44 h (2.7 log10 CFU/g). Decimal reduction time (D-value) for tested bacteria was 7 min. Furthermore, the influence on PAW on the polyphenolics content and antioxidant activity was evaluated. To identify the PAW chemistry, physical properties, including pH, redox potential, and conductivity were estimated. The levels of H2O2, nitrate (NO3−) and nitrite anions (NO2−) as long-lived species were measured and compared to SDW without plasma activation.

Milk inoculated with Clostridium botulinum spores (PA9508B) (107 CFU/mL) was subjected to high-pressure high-temperature treatment (HPHT) (700–900 MPa; 90–110 °C for 0–24 min) using an insulated sample chamber, as well as in a water bath (90–100 °C) (conventional) and surviving spores were enumerated. Destruction kinetics were well described by a first order kinetic model. Conventional heating D values ranged from 156 to 12 min with a z value of 7.8 °C. During HPHT, higher pressures and temperatures resulted in accelerated spore destruction rates. At 900 MPa, the associated D values were 14.5, 1.8 and 0.35 min at 90, 100 and 110 °C, respectively, much lower than the conventional D values. The pressure dependent ZT values were 11.2, 12.3, 12.4 °C at 700, 800, 900 MPa respectively, increasing with pressure. The temperature dependent ZP values were 470, 630, and 800 MPa at 90, 100, and 110 °C, respectively. By comparing ZT with ZP values, it was clear the spores were relatively more sensitive at higher temperatures than at higher pressures in this temperature-pressure range. D value trends beyond 120 °C indicated thermal inactivation was the principal mode of spore destruction and high pressure can protect spores under these conditions.

Salmonella Enteritidis is a major foodborne pathogen exposed to various environmental and preservation stresses in the food chain. Because adaptive responses of viable bacterial cells in the presence of sublethal stress can induce cross-protection against different stresses, we investigated the heat resistance of Salmonella Enteritidis at 60 °C under prolonged exposure to acid-salt combined stress and subsequent refrigeration. Salmonella Enteritidis was grown in tryptic soy broth at four pH values (4.5, 5.4, 6.4, and 7.3) and four NaCl concentrations (0%, 1%, 2%, and 3%) at 37 °C for 24 h and then incubated at 4 °C for 0, 1, 4, or 7 days. For 0 and 1 day-refrigerated cultures, previous adaptation to single stresses (acid or salt stress) increased the heat resistance of Salmonella Enteritidis, resulting in increased D-values, whereas the combination of acid and salt stress reduced heat tolerance; acid stress played a more critical role in mediating this effect than salt concentration. To elucidate the related mechanisms, the expression levels of heat shock sigma factors (rpoH) and heat shock genes (dnaK and groEL) were analyzed and found to be associated with the heat resistance of Salmonella Enteritidis. The refrigeration period was negatively correlated (P 0.01) with the D-value (r = -0.505) and with the transcript levels of rpoH (r = -0.654), dnaK (r = -0.652), and groEL (r = -0.645). Our findings demonstrated that acid-salt combined preservation techniques and subsequent refrigeration may prevent S. Enteritidis survival in heat-pasteurized food products caused by cross-protection of acid or salt adapted cells.

In-shell pecans are susceptible to microbial contamination. This study was performed to investigate feasibility of using hot water treatment as a kill-step for food-borne pathogens during pecan shelling. In-shell pecans were subjected to hot water at 70, 80 or 90 °C for 1, 2, 3, 4 or 5 min. The time-temperature treatments to achieve a 5-log reduction of Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and non-pathogenic Enterococcus faecium were determined. Thermal death values were determined for each tested condition. L. monocytogenes was most susceptible to heat treatment and were reduced by 4.6 ± 0.35 log CFU/g at 70 °C for 5 min, while 3–5 min at 80 and 90 °C treatments was required to achieve a similar reduction level for S. enterica, E. coli O157:H7, and E. faecium. S. enterica were most resistant and required 4 min treatment time to achieve a 5-log reduction at 80 and 90 °C. The D-values ranged from 1.15 to 1.72, 0.83 to 1.19, and 0.41–0.92 min at 70, 80 and 90 °C, respectively. E. faecium had the highest D-value (1.72 min at 70 °C), indicating a potential surrogate for process validation for pecan industries. Utilizing proper hot water treatment during pecan shelling could reduce food safety risk.

Thermal processing is usually used by the fruit juice industry for enzyme and microbial inactivation, thus limiting organoleptic changes that affect shelf life and quality of products. Mainly the inactivation of enzymes is necessary to avoid quality changes related to cloud loss, using mild temperature that allows minor alterations in product’s sensory as well as in nutritive traits. Pineapple juice, a product obtained by diluting the concentrate juice and pasteurizing afterword shipped to Europe, has a high residual pectin methylesterase (PME) activity. In this view, the present work provides a detailed kinetic study of the effect of pasteurization on inactivation of PME in pineapple juice. The fresh juice was treated with temperatures ranging from 70 to 95 °C, for various times, until the complete denaturation of PME occurred. Results of thermal stabilization treatments showed that enzyme inactivation behaviour follows a first-order kinetic process, in the studied temperature range. Moreover, the reaction rate constants for denaturation of PME were determined; in particular, activation energy (Ea), decimal reduction time (Dref), and z value (z), were 78.2 ± 4.5 kJ/mol, 16 s (at reference temperature of 106 °C) and 36 ± 3 °C, respectively. Based on the thermal resistance data here reported to inactivate PME, a treatment of 106 °C with a mean holding time of 1 min should be employed in order to preserve cloudiness of pasteurized pineapple juice.

Decimal reduction time ( D-value) was calculated for six non-O157 Shiga toxin-producing Escherichia coli (STEC) in a laboratory medium and ground beef. For the laboratory medium, an overnight culture of each strain of STEC was divided into 10-mL sample bags and heated in a water bath for a specific time on the basis of the temperatures. Survival curves were generated by plotting the surviving bacterial population against time, and a linear-log primary model was used to estimate the D-values from survival curves. The z-values (the temperature raised to reduce the D-value by one-tenth) were calculated by plotting the log D-values against temperature. Similarly, for ground beef, six fat contents, 5, 10, 15, 20, 25, and 30% of ground beef were formulated for this study. Inoculated meat was divided into 5-g pouches and submerged in a water bath set at specific temperatures (55, 60, 65, 68, and 71.1°C). The average D-value for these strains in a laboratory medium was 17.96 min at 55°C, which reduced significantly ( P0.05) to 1.58 min at 60°C, and then further reduced ( P0.05) to 0.46 min at 65°C. In ground beef, a negative correlation ( P0.05) between fat content of ground beef and D-values was observed at 55°C. However, at temperatures greater than 60°C, there was no impact ( P0.05) of fat content of ground beef on the thermal resistance of non-O157 STECs. Irrespective of the fat content of ground beef, the D-values ranged from 15.93 to 11.69, 1.15 to 1.12, and 0.14 to 0.09 min and 0.05 at 55, 60, 65, and 68°C, respectively. The data generated from this study can be helpful for the meat industry to develop predictive models for thermal inactivation of non-O157 STECs in ground beef with varying fat content.

Heat treatments can cause degradation in tomatoes of lycopene which has important antioxidant effects. No information about decimal reduction time (D100) of lycopene is available. D-value is the time required at a given temperature to reduce 90% of the molecule. This study for the first time determine the kinetic of lycopene thermal degradation. The content was measured at regular intervals of pasteurization using canned tomato paste to determinate D value. Microbiological analysis was carried out to verify product stability after packaging. Yeasts, molds and lactic acid bacteria were determined. The pasteurization time allowed to observe a loss of the red color. Lycopene content, after an increase at 8 min, decreased at 32 min of pasteurization. D100 value was calculated at 75 min; a diminution of 90% in lycopene content in the concentrated tomato paste was observed. Microbiological analysis confirmed the stability of products after 8 min of pasteurization.

Heat treatments are widely used in food processing often with the aim of reducing or eliminating spoilage microorganisms and pathogens in food products. The efficacy of applying heat to control microorganisms is challenged by the natural diversity of microorganisms with respect to their heat robustness. This review gives an overview of the variations in heat resistances of various species and strains, describes modeling approaches to quantify heat robustness, and addresses the relevance and impact of the natural diversity of microorganisms when assessing heat inactivation. This comparison of heat resistances of microorganisms facilitates the evaluation of which (groups of) organisms might be troublesome in a production process in which heat treatment is critical to reducing the microbial contaminants, and also allows fine-tuning of the process parameters. Various sources of microbiological variability are discussed and compared for a range of species, including spore-forming and non-spore-forming pathogens and spoilage organisms. This benchmarking of variability factors gives crucial information about the most important factors that should be included in risk assessments to realistically predict heat inactivation of bacteria and spores as part of the measures for controlling shelf life and safety of food products.

Cloudy apple juice has been treated by high pressure carbon dioxide (HPCD) as non-thermal technology to inactive polyphenoloxidase and pectinmethylesterase in batch mode. Stirring speed (from 200 to 600 rpm) induced an increase in the enzyme inactivation rate while a triple cycle of pressurization/depressurization led to the same enzyme inactivation efficiency. Enzyme inactivation kinetics were determined at different temperatures (from 35 to 45 °C) and pressures (from 10 to 20 MPa). Data were described by the first order kinetic model and the Weibull model. For the first order kinetic model, decimal reduction time for HPCD treatment was found to be smaller than for mild heating, in the same temperature range. The same tendency was observed for the first decimal reduction time in the Weibull model. HPCD treatment resulted in a homogenization effect reflected in the shifting of the particle size distribution towards smaller diameters after treatment. HPCD treatment did not result in a change of water and oxalate soluble pectin content, total phenolic compounds and hidroxymethylfurfural content., Spanish Government (MINECO) and the European Regional Development Fund for financial support of project CTQ2015-64396-R and AEI’s contract.

Liquid cultures are commonly used to inoculate low-moisture foods. However, liquid-inoculation may generate varied results due to differences in preparation methods, and the introduction of liquid into a low-moisture matrix may cause stickiness and clumping. The objectives of this study were to develop a dry-inoculation method using freeze-dried Enterococcus faecium NBRL-2354, and to evaluate the stability of freeze-dried inoculum over time (35 days at −20 °C) in terms of survival and heat resistance. Two protectants, freeze-drying buffer and skim milk, mixed separately with liquid E. faecium were freeze-dried and their preservative effects were compared. Heat resistance of freeze-dried inoculum was determined by decimal reduction time at 80 °C (D80) in wheat flour (water activity 0.45 at room temperature). Freeze-drying buffer and skim milk formed slimy layers around bacterial cells and yielded freeze-dried inoculum with high survivability. D80-values of freeze-dried E. faecium protected by freeze-drying buffer or skim milk were 6.67 ± 0.22 min and 5.92 ± 0.39 min, respectively. Freeze-dried inoculum was stable with a high population concentration (>10.0 log CFU/g), while skim milk-protected inoculum showed stable heat resistance throughout storage. In summary, freeze-dried E. faecium protected by skim milk was the optimum dried inoculum and can be used as a substitute for liquid-inoculum in low-moisture foods.