Thermal Inactivation Kinetics Research Articles

Kinetic study on peroxidase inactivation and anthocyanin degradation of black cherry tomatoes (Solanum lycopersicum cv. OG) during blanching

Summary Introduction Blanching is a necessary treatment stage in processing of tomato products to inactivate enzymes. However, it may cause the degradation of nutrients. Objective In this study, the kinetics of thermal peroxidase inactivation and anthocyanin degradation in black cherry tomatoes (cv. OG) were determined to predict the quality changes during the blanching. Methods Tomatoes were blanched at five levels of temperature (75–95oC) for five time periods (30–150 s). Results It was found that as the blanching temperature increased and the blanching time is prolonged, more peroxidase was inactivated and the greater number of anthocyanins was lost. The thermal peroxidase inactivation and anthocyanin degradation showed an apparent first-order reaction with the activation energy of 129.96 kJ/mol and 65.99 kJ/mol, respectively. Peroxidase and anthocyanin in black cherry tomatoes were found to be heat-sensitive. Conclusion These kinetic parameters were necessary to select and design appropriate blanching conditions for black cherry tomatoes on larger scale processing.

Strain Variability in Growth and Thermal Inactivation Characteristics of Listeria monocytogenes Strains after Acid Adaptation

Given the importance of strain variability to predictive microbiology and risk assessment, this study aimed to quantify the magnitude of strain variability in growth and thermal inactivation kinetics behaviors after acid adaptation. Thirty-three Listeria monocytogenes strains were exposed to acid-adapted tryptic soy broth supplemented with yeast extract (TSBYE; pH 5.5) and non-acid-adapted TSBYE (pH 7.0) for 20 h. Next, the growth parameters of these adapted and nonadapted strains that grew in nonbuffered TSBYE at 25°C were estimated. The tested strains were inactivated at 60°C in nonbuffered broth to obtain the heat resistance parameters. The results revealed that strain variability was present in the growth and thermal inactivation characteristics. The maximum specific growth rate ranged from 0.21 to 0.44 h-1 and from 0.20 to 0.45 h-1 after acid and nonacid adaptation, respectively. The lag times were from 0.69 to 2.56 h and from 0.24 to 3.36 h for acid-adapted and non-acid-adapted cells, respectively. The apparent D-values at 60°C of the pathogen ranged between 0.56 and 3.93 min and between 0.52 and 3.63 min for the presence and absence of acid adaptation condition, respectively. Acid adaptation significantly (P < 0.05) increased the magnitude of strain variability in the thermal inactivation characteristics of L. monocytogenes, with the coefficient of variation increasing to 0.17, whereas acid adaptation did not significantly (P ≥ 0.05) influence the variabilities in the growth parameters of the tested strains. Furthermore, the subsequent growth behaviors of all strains did not exhibit significant (P > 0.05) changes after exposure to acidic broth. However, the thermal resistance of most (25 of 33) of the tested strains increased (P < 0.05) after growing in acid-adapted broth. The relevant data generated in the present study can be used to describe the strain variability in predictive microbiology and to deeply understand the behavioral responses of different strains to acid adaptation.

Thermal inactivation kinetics for Acidovorax citrulli on watermelon seeds as influenced by seed component, temperature, and water activity

Knowledge of the thermal inactivation kinetics of Acidovorax citrulli is essential to design an effective heat treatment protocol for its control on watermelon seeds. The coat and embryo of watermelon seeds were selected as two components to compare the heat resistance of A. citrulli. The embryo was chosen to investigate the influence of different water activity levels using a heating block system under a fixed heating rate of 5 °C min−1, which aimed to simulate radio frequency heating. By combining the thermal death kinetics of A. citrulli with the germination rate curves of the seeds, practical treatment protocols were proposed. The results indicated that the heat resistance during A. citrulli inoculation for seed embryo increased with decreasing water activity. The inactivation data for A. citrulli inoculated on the embryo fitted the Weibull model (R2 = 0.965–0.999, RMSE = 0.023–0.304) better than a first-order kinetic model (R2 = 0.927–0.988, RMSE = 0.164–0.467). The Mafart's modified Bigelow model and the cumulative lethal time model showed that under given conditions thermal death kinetics could be applied to reliably predict population reductions of A. citrulli. Thermal treatment protocols were proposed under different heating uniformity index values. These findings could be used to develop an effective treatment protocol based on the germination rate of watermelon seeds and microbial inactivation level.

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.

Influence of drying conditions, food composition, and water activity on the thermal resistance of Salmonella enterica

Salmonella contamination of low-water activity (aw) foods poses a serious concern worldwide. The present study was conducted to assess the effects of drying conditions, food composition, and water activity on the desiccation tolerance and thermal resistance of S. Enteritidis FUA1946, S. Senftenberg ATCC43845 and S. Typhimurium ATCC13311 in pet food, binder formulation, and skim milk powder. The samples were wet inoculated with the individual Salmonella strains and were equilibrated to aw 0.33 and 0.75, followed by an isothermal treatment at 70 °C. The thermal inactivation data was fitted to the Weibull model. Irrespective of the aw, food composition and physical structure of the selected foods, strain S. Enteritidis FUA1946 displayed the highest desiccation and thermal resistance, followed by S. Senftenberg ATCC43845 and S. Typhimurium ATCC13311. The food matrix and strain type significantly (p < 0.05) influenced the thermal resistance of microorganisms in foods along with aw change during thermal treatments. To further study the effect of food composition, an additional set of experiments using dry inoculation of the resistant Salmonella strain in the low-aw foods was designed. Significant (p < 0.05) matrix-dependent interaction on Salmonella reduction was observed. The water adsorption isotherms of selected low-aw foods were measured at 20 and 70 °C to relate the thermal inactivation kinetics with the change in the aw. The characterization of thermal resistance of the Salmonella serovars in low-aw products with different compositions and aw in this study may be used for the validation of thermal challenge studies.

Bacteria and Mold Spore Heat Resistance in Guava Juice and Its Control by pH and Sodium Benzoate.

Heat-resistant bacteria and molds can survive the pasteurization conditions used in high-acid fruit juices. The objective of this study was to evaluate the log reductions and thermal inactivation kinetics of spores of Bacillus subtilis bacteria and ascospores of Talaromyces flavus and Eupenicillium javanicum molds under influence of pH and sodium benzoate preservative. The spores were suspended in guava juice, processed at 90-100°C for B. subtilis and at 80-90°C for T. flavus and E. javanicum, and decimal reduction (D) values were estimated from the log survivor curves. Next, the effects of pH change (3.5-4.5) and 0.015% sodium benzoate addition on the D values of spores were investigated. Lower D values were obtained at higher temperatures (D100°C value of 2.32 min vs. D90°C value of 15.33 min for B. subtilis, D90°C value of 2.96 min vs. D80°C value of 59.52 min for T. flavus, and D90°C value of 1.58 min vs. D80°C value of 21.32 min for E. javanicum). The D values decreased further (to 1.8 min at 100°C for B. subtilis, to 2.33 min at 90°C for T. flavus, and to 1.49 min at 90°C for E. javanicum) when the pH of guava juice was decreased from 4.1 to 3.5. Inclusion of sodium benzoate in pH 3.5 juice enhanced the thermal inactivation of spores (D100°C value decreased to 1.4 min for B. subtilis, to 1.98 min for T. flavus, and to 1.34 min for E. javanicum). To conclude, the combination of low pH and sodium benzoate provided the best method for spore inactivation, which could enhance food safety and extend food's shelf life.

Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol−1, and from 179 to 359 kcal mol−1 K−1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M−1 and ΔGo value was −8.3 kcal mol−1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases.

Thermal Inactivation Kinetics of Kudzu (Pueraria lobata) Polyphenol Oxidase and the Influence of Food Constituents.

The thermal inactivation kinetics of kudzu (Pueraria lobata) polyphenol oxidase (PPO) were investigated in model and food systems. PPO in kudzu tissue (tPPO) showed a higher thermostability than that of PPO in crude extract (cPPO) and purification fractions (pPPO). The PPO inactivation rate constant (k) increased with an increase in temperature, and tPPO showed the lowest k value, followed by that of cPPO and pPPO at the same temperature, indicating that PPO in the food system was more resistant to thermal treatment. Food constituents (pectin, starch, sucrose, and bovine serum albumin) in the food system decreased the activity of PPO but increased the thermostability of PPO, among which pectin exhibited the strongest protective effect against thermal inactivation, and the influence of sucrose was much slighter than that of other macromolecules. Fluorescence emission spectra indicated that pPPO exhibited stronger interactions with pectin than sucrose, and pPPO with pectin showed a more stable conformation under thermal treatment.

Integrated calculation of pasteurization time: A case study for thermal inactivation kinetics of a mixed fruit beverage

AbstractThe impact of batch thermal treatment (50–95°C/0–10 min) on various quality attributes of a mixed fruit beverage (pomegranate–amla–muskmelon juice = 57:5:38) was evaluated. The responses include microbial count (aerobic mesophiles [AM], yeast and mold [YM], psychrotrophs [PSY], and lactic acid bacteria [LAB]); enzyme activity (polyphenol oxidase [PPO] and peroxidase [POD]); and bioactive (ascorbic acid [AA], total phenolics [TPC], antioxidants [AOC], anthocyanins [TAC], and flavonoids [TFC]). Weibull model indicated a tailing behavior (β = 0.8) for LAB, while others showed the shouldering behavior (β &gt; 1). YM was the most resistant microbial group. Enzyme inactivation and bioactive degradation were discussed with thenth‐order model. POD was more resistant than PPO. The corresponding activation energy (Ea) values for PPO and POD were 61.8 and 65.1 kJ/mol, respectively. Among bioactive compounds, AA was most sensitive toward thermal stress. At 95°C, the 90% inactivation of enzyme activity (t90value) was achieved after the total process time (come up + holding + cooling time) of 1.5 + 4.1 + 0.3 min. The corresponding losses (%) for AA, TPC, and AOC, att90condition, were 39, 27, and 34, respectively.Practical applicationsThe study attempts to introduce the expression for calculating the time required for 5‐log cycle reduction in resistant microbes (t5Dvalue) and 90% reduction in enzyme activity (t90value) of the sample. The kinetic model based on the cumulative approach of come‐up time (tCUT), holding time (tHOLD), and cooling time (tCOOL) will undoubtedly help in designing the thermal pasteurization process of this mixed fruit juice and thereby pave the pathway for similar counterparts.

On optimum dynamic temperature profiles for thermal inactivation kinetics determination.

Determination of inactivation kinetics, associated with thermal processing of foods and obtained from dynamic temperature experiments, requires carefully designed experiments, the primary element being the selection of the appropriate temperature profile along with a carefully planned sampling schedule. In the present work, a number of different dynamic temperature profiles were investigated in terms of their ability to generate accurate kinetic parameters with low confidence intervals (CIs). Although alternative models have been also tested, our work was concentrated on thermal inactivation kinetics that could be described by the classical D-z values. A pair of D and z values was assumed, and for each temperature profile tested, concentration data at different processing times were generated through the appropriate models. Next, an error (up to ±2.5% or ±5%) was introduced on these theoretical values to generate pseudo-experimental data, and the back-calculation of the assumed kinetic parameters by non-linear regression was performed. The accuracy and the 95% CIs of the estimated kinetic parameters were evaluated; joint confidence regions were also constructed to investigate parameters correlation. The effect of temperature profile pattern, level of error, number of experimental points, and reference temperature was assessed. A stepwise increasing and a single triangle-pattern temperature profile were the best profiles among those tested. As a general observation, based on different kinetic models investigated, temperature profiles and sampling intervals that result in concentration versus time diagrams having shapes as suggested by the primary model used when isothermally applied are not considered appropriate for parameter estimation.

Thermal inactivation kinetics of seven genera of vegetative bacterial pathogens common to the food chain are similar after adjusting for effects of water activity, sugar content and pH

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.

Aichi virus inactivation by heat in 2‐ml glass vials

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.

Inactivation and Subsequent Growth Kinetics of Listeria monocytogenes After Various Mild Bactericidal Treatments.

This study was carried out to investigate the effects of mild heat, lactic acid, benzalkonium chloride and nisin treatments on the inactivation, sublethal injury, and subsequent growth of Listeria monocytogenes. Results showed that the Bigelow model successfully described the thermal inactivation kinetics, while the Log-linear model with tail consistently offered the most accurate fit to LA, BC, and nisin inactivation curves of cells. Differential plating indicated that percentage of sublethal injury for nisin treated cells was significantly higher than that for the other three treatments. Compared to non-treated cells, significant extension of lag time was observed for all treated cells. The longer exposures to heat treatment contributed to the extended lag time of the survivors. While for LA, BC and nisin treated cells, the longest lag time was not observed at the most severe treatment conditions. The correlation analysis of sublethal injury percentage on the duration of lag time revealed that only heat treatment showed the significant correlation. Overall, the lag time analysis could evaluate a wide range of bacterial injury. Lag time of treated cells was significantly influenced by stress treatments and temperatures of recovery, however, there were not any significant changes in the maximum specific growth rate between treated and non-treated cells under isothermal recovery conditions. The information generated from this study is valuable for utilizing intervention strategies in the elimination or growth inhibition of L. monocytogenes.

Partial purification, characterization and kinetics of thermal inactivation of pectin methylesterase and polygalacturonase enzymes from Indian lemon (Citrus limon (L.)

The extraction, purification, and characterization of PME and PG were studied for lemon fruit. Enzymes were partially purified by anion-exchange chromatography using UNOsphere™ Q anion exchanger for lemon PME and DEAE Cellulose anion exchanger for lemon PG. A single band with a fold purity of 20.95 and 15.75 for PME and PG respectively was observed on SDS-PAGE. Molecular weights of partially purified fractions of PME and PG as exhibited by SDS-PAGE were 48 kDa and 95 kDa respectively. Optimum pH and temperature of partially purified lemon PME and PG were pH 9, pH 5 and 45 °C. The crude PME and PG extracts exhibited D-values of 120.57–63.79 and 185.72–29.18 min; Z-values of 15.11 and 16.50 °C; and activation energies (Ea) of 58.23 and 57.18 kJ/mol, respectively. The enzyme stability was not significantly influenced by the fruit matrix as reflected by Z and Ea values. D and Z-values of both the enzymes and microorganisms should be considered while designing thermal treatments to obtain a product with better shelf life.

A Comparison of Three Methods for Determining Thermal Inactivation Kinetics: A Case Study on Salmonella enterica in Whole Milk Powder

Different methods for determining the thermal inactivation kinetics of microorganisms can result in discrepancies in thermal resistance values. In this study, thermal resistance of Salmonella in whole milk powder was determined with three methods: thermal death time (TDT) disk in water bath, pouches in water bath, and the TDT Sandwich system. Samples from three production lots of whole milk powder were inoculated with a five-strain Salmonella cocktail and equilibrated to a water activity of 0.20. The samples were then subjected to three isothermal treatments at 75, 80, or 85°C. Samples were removed at six time points and cultures were enumerated for survivors. The inactivation data were fitted to two consolidated models: two primary models (log linear and Weibull) and one secondary model (Bigelow). Normality testing indicated that all the model parameters were normally distributed. None of the model parameters for both consolidated models were significantly different (α = 0.05). The amount of inactivation during the come-up time was also not significantly different among the methods (α = 0.05). However, the TDT Sandwich resulted in less inactivation during the come-up time and overall less variation in model parameters. The survivor data from all three methods were combined and fitted to both consolidated models. The Weibull had a lower root mean square error and a better fit, according to the corrected Akaike's information criterion. The three thermal treatment methods produced results that were not significantly different; thus, the methods are interchangeable, at least for Salmonella in whole milk powder. Comparisons with more methods, other microorganisms, and larger varieties of food products using the same framework presented in this study could provide guidance for standardizing thermal inactivation kinetics studies for microorganisms in foods.

Thermal Inactivation of Myrosinase from White Mustard Seeds

Myrosinases (thioglucosidases) catalyze the hydrolysis of a class of compounds called glucosinolates, of which the aglycones show various biological functions. It is often necessary to minimize the loss of myrosinase activity during thermal processing of cruciferous vegetables. Myrosinase was isolated from a popular spice, white mustard (Sinapis alba), and its thermal inactivation kinetics was investigated. The enzyme was extracted from white mustard seeds and purified by a sequential processes of ammonium sulfate fractionation, Concanavalin A-Sepharose column chromatography, and gel permeation chromatography. At least three isozymes were revealed by Concanavalin A-Sepharose column chromatography. The purity of the major myrosinase was examined by native polyacrylamide gel electrophoresis and on-gel activity staining with methyl red. The molecular weight of the major enzyme was estimated to be 171 kDa. When the consecutive step model was used for the thermal inactivation of the major myrosinase, its inactivation energy was 44.388 kJ/mol for the early stage of destruction and 32.019 kJ/mol for the late stage of destruction. When the distinct two enzymes model was used, the inactivation energy was 77.772 kJ/mol for the labile enzyme and 95.145 kJ/mol for the stable enzyme. The thermal inactivation energies lie within energy range causing nutrient destruction on heating.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella in ground black pepper at different water activities

Thermal inactivation kinetics of Salmonella in low moisture foods are necessary for developing proper thermal processing parameters for pasteurization. The effect of water activity on thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 in ground black pepper has not been studied previously. Identification of a suitable surrogate assists in conducting in-plant process validations. Ground black pepper was inoculated with a 5-serotype Salmonella cocktail or E. faecium NRRL B-2354, equilibrated to water activities of 0.25, 0.45 or 0.65 in a humidity-controlled chamber, and isothermally treated at different temperatures. The survivor data were used for fitting the log-linear models to obtain the D and z-values of Salmonella and E. faecium in ground black pepper. Modified Bigelow models were developed to evaluate the effects of temperature and water activity on the thermal inactivation kinetics of Salmonella and E. faecium. Water activity and temperature showed significant negative effects on the thermal resistance of Salmonella and E. faecium in ground black pepper. For example, significantly higher D values of Salmonella were observed at water activity of 0.45 (D70°C = 20.5 min and D75°C = 7.8 min) compared to water activity of 0.65 (D70°C = 3.9 min and D75°C = 2.0 min). D-values of E. faecium were significantly higher than those of Salmonella at all three water activities, indicating that E. faecium is a suitable surrogate for Salmonella in thermal processing validation.

Challenges of dry hazelnut shell surface for radio frequency pasteurization of inshell hazelnuts

Thermal inactivation kinetics (D and z values) of Salmonella and Enterococcus faecium in fresh hazelnut shell and kernel were determined. Aluminum pouches containing either 1.5 g hazelnut shell powder or 2 g of ground kernel inoculated with either bacterium at their original water activities (aw-shell = 0.91 ± 0.01, aw-kernel = 0.93 ± 0.01) were isothermally treated in a thermal death time (TDT) sandwich at 55, 60, 65, 70 and 75 °C for determining D values. A single layer inshell hazelnuts with one inoculated nut located at the center (cold spot) of a tray was subjected to hot air assisted radio frequency (HARF) heating in a 6-kW 27.12-MHz RF system with hot air temperature at ~75 °C for 10, 15 and 20 min. The log-linear model fits survival data of both Salmonella and E. faecium in hazelnut shell and kernel well. The D-values of both bacteria in hazelnut kernel were higher than those in hazelnut shell. Higher D-values of E. faecium demonstrated that it is a suitable surrogate for Salmonella in both hazelnut shell and kernel. There was a large variation between calculated (based on F-value) and experimental log reductions of Salmonella. Only 1.0 ± 0.1 log reduction of Salmonella was achieved in inshell hazelnut after 20 min of HARF heating. Because the RF pasteurization is a simultaneous drying process, the F-value methodology could not give an accurate and reliable lethality prediction due to the increased thermal resistance of Salmonella caused by moisture loss. Around one log increase in Salmonella inactivation was achieved after spraying 0.1% buffered peptone water (BPW) on inshell hazelnuts for the same RF treatment condition. While the water activity of the ground shell was 0.91 ± 0.01, the water activity of top surface layer of the shell where bacteria were inoculated for the RF treatment was 0.17 ± 0.01 before RF treatment. This very dry shell surface was the major factor for the deviation of calculated F-value from the observed microbial reduction in this RF microbial study. The dry shell surface creates a challenge for thermal inactivation of pathogens on inshell hazelnuts and other nuts with a similar shell structure. An efficient pasteurization protocol for inshell hazelnuts needs to be developed and optimized by combining surface disinfectants/salt solution and RF process conditions to enhance microbial food safety and quality of hazelnuts. This study provides valuable information and direction to the hazelnut industry for improving safety and quality of hazelnuts.

Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential.

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (kcat/Km) and the Michaelis–Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

Kinetic stability of the water-forming NADH oxidase from Giardia lamblia: implications for biotechnological processes

NAD(P)H oxidases (NOXs) catalyze the oxidation of NAD(P)H by reducing molecular O2 to H2O2 or H2O. Water-forming NOXs have been highlighted as promising biocatalyst systems for regeneration of NAD+ or NADP+ in industrial applications. The NADH oxidase of Giardia lamblia (GlNOX) uses naturally both nicotinamide cofactors producing only water as byproduct; therefore, it can be considered a multipurpose and innocuous regeneration system for redox reactions using either NAD+ or NADP+. From a biotechnological perspective, kinetic stability (and not thermodynamic stability) must be the most important aspect to consider for enzymes involved in industrial applications; therefore, understanding the kinetic stability of a technologically interesting enzyme is an indispensable step toward its application. In this work, we analyze the thermal inactivation kinetics of GlNOX and the influence of diverse additives on it. A combination of 1,4-Dithiothreitol as reducing agent and the disaccharide trehalose protects remarkably GlNOX from inactivation. Structural studies indicate that inactivation is not related to denaturation, whereas overoxidation of the active site cysteine seems to be the main factor related to loss of the enzyme activity. It is concluded that understanding the factors that influence the protein inactivation of GlNOX allowed to improve its kinetic stability, making it biotechnologically valuable.