Thermal Inactivation Kinetics Research Articles

Contribution to modeling the kinetics of phosphatase thermal inactivation

This article focuses on modeling the kinetics of thermal inactivation of acid phosphatases extracted from breadfruit (Artocarpus communis L). By examining the stability of these enzymes at different temperatures, the study highlights their importance in the plant kingdom for fruit preservation, and in the animal kingdom for medical diagnostics. The study demonstrated that the kinetics of thermal inactivation of acid phosphatases P1A, P1B and P2 are influenced by temperature and the substrate used. Analyses revealed that more complex kinetic models, such as two-step models, offer a better representation of inactivation for certain substrates, notably PPNa and pNPP at specific temperatures. This indicates the formation of partially active enzymes in some cases. Understanding these inactivation mechanisms is crucial for optimizing the use of phosphatases in plant applications, such as fruit preservation, and in medical diagnostic contexts. The results obtained provide a solid basis for future research aimed at improving the stability and efficacy of phosphatases in various fields.

Exploring the catalytic potential of watermelon urease: Purification, biochemical characterization, and heavy metal precipitation

Bioactive urease from watermelon (Citrullus lanatus) seeds was purified using acetone fractionation, anion-exchange, and size-exclusion chromatography, achieving a 121-fold increase and specific activity of 3216 U/mg. The enzyme appeared as a single band on native and SDS-PAGE, with a molecular mass of 480 ± 10 kDa and subunit mass of 80 ± 10 kDa, indicating six identical subunits. Atomic absorption spectroscopy revealed 1.46 nickel ions per subunit. Watermelon urease exhibited serological similarities with jack bean and pigeon pea ureases, an optimal pH of 7.3, an activation energy of 3 kcal/mol, Vmax of 3571 μmol/min/mg, and Km of 0.16 mM. The enzyme displayed biphasic thermal and pH inactivation kinetics, a strong preference for urea, and a half-life of 70 days with 1 mM DTT. This study highlights watermelon urease's role in bioremediation by facilitating the precipitation of heavy metals as stable carbonates, promoting environmental sustainability.

Immobilization of laccase on mesoporous metal organic frameworks for efficient cross-coupling of ethyl ferulate.

Laccases act as green catalysts for oxidative cross-coupling of phenolic antioxidnt compounds, but low stability and non-recyclability limit its application. To address that, metal-organic frameworks Cu-BTC and Cr-MOF were synthesized as supports to immobilize the efficient laccase from Cerrena sp. HYB07. The Brunauer-Emmett-Teller surface area of Cu-BTC and Cr-MOF were 1213.2 and 907.1m2/g, respectively. The two carriers respectively presented pore diameters of 1.2-10nm and 1.4-12nm as octahedron, indicating nano-scale mesoporosity. These Cu-BTC and Cr-MOF carriers could adsorb laccase with enzyme loading of 1933.2 and 1564.4U/g carrier, respectively. The stability and organic solvent tolerance of Cu-BTC-laccase and Cr-MOF-laccase were both obviously improved compared to free laccase. Thermal inactivation kinetics showed that both the two immobilized laccases displayed lower thermal inactivation rate constants. Importantly, the Cu-BTC-laccase and Cr-MOF-laccase both showed much higher activity for cross-coupling of ethyl ferulate than free laccase, which had 2.5-fold higher cross-coupling efficiency than that by free laccase. The ethyl ferulate coupling product was also analyzed by mass spectroscopy and the synthesis pathway of ethyl ferulate dimer was proposed. The cross coupling of ethyl ferulate required the formation of radical intermediates of ethyl ferulate generated by laccase mediated oxidation. This work paved the way for MOFs immobilized laccase for cross coupling of antioxidant phenols.

Thermal inactivation kinetics of Salmonella and Campylobacter in chicken livers

Salmonella and Campylobacter are major foodborne pathogens that can cause outbreaks associated with contaminated chicken liver. Proper cooking is necessary to avoid the risk of illness to consumers. This study tested the thermal inactivation of a four-strain Salmonella cocktail and a three-strain Campylobacter cocktail in chicken livers separately at temperatures ranging from 55.0–62.5°C. Inoculated livers were sealed in aluminum cells and immersed in a water bath. The decimal reduction time (D-values) of Salmonella in chicken livers were 9.01, 2.36, 0.82, and 0.23 min at 55.0, 57.5, 60.0, and 62.5°C, respectively. The D-values of Campylobacter ranged from 2.22 min at 55.0°C to 0.19 min at 60.0°C. Salmonella and Campylobacter had similar z-values in chicken livers of 4.8 and 4.6°C, respectively. Chicken livers can be heated to internal temperatures of 70.0–73.9°C for at least 1.6–0.2 s to achieve a 7-log reduction of Salmonella. Validation tests demonstrated that heating chicken livers to internal temperatures of 70.0–73.9°C for 2–0 s resulted in a reduction of Salmonella exceeding 7 logs. Collectively, these data show that Salmonella exhibits higher heat resistance than Campylobacter in chicken livers. Therefore, Salmonella could be considered as the target pathogen when designing thermal treatments or cooking instructions for liver products. These findings will aid in designing effective thermal processing for both industrial and home cooking to eliminate Salmonella and Campylobacter, ensuring consumer safety when consuming chicken liver products.

Bootstrapping for Estimating the Conservative Kill Ratio of the Surrogate to the Pathogen for Use in Thermal Process Validation at the Industrial Scale

A surrogate is commonly used for process validations. The industry often uses the target log cycle reduction for the test (LCRTest) microorganism (surrogate) to be equal to the desired log cycle reduction for the target (LCRTarget) microorganism (pathogen). When the surrogate is too conservative with far greater resistance than the pathogen, the food may be overprocessed with quality and cost consequences. In aseptic processing, the Institute for Thermal Processing Specialists recommends using relative resistance (DTarget)/(DTest) to calculate LCRTest (product of LCRTarget and relative resistance). This method uses the mean values of DTarget and DTest and does not consider the estimating variability. We defined kill ratio (KR) as the inverse of relative resistance.The industry uses an extremely conservative KR of 1 in the validation of food processes for low-moisture foods, which ensures an adequate reduction of LCRTest, but can result in quality degradation. This study suggests an approach based on bootstrap sampling to determine conservative KR, leading to practical recommendations considering experimental and biological variability in food matrices. Previously collected thermal inactivation kinetics data of Salmonella spp. (target organism) and Enterococcus faecium (test organism) in Non-Fat Dried Milk (NFDM) and Whole Milk Powder (WMP) at 85, 90, and 95°C were used to calculate the mean KR. Bootstrapping was performed on mean inactivation rates to get a distribution of 1000 bootstrap KR values for each of the treatments. Based on minimum temperatures used in the industrial process and acceptable level of risk (e.g., 1, 5, or 10% of samples that would not achieve LCRTest), a conservative KR value can be estimated. Consistently, KR increased with temperature and KR for WMP was higher than NFDM. Food industries may use this framework based on the minimum processing temperature and acceptable level of risk for process validations to minimize quality degradation.

The effect of water activity on thermal resistance of Salmonella in chocolate products with different fat contents

Several instances of Salmonella outbreaks have been documented in association with chocolates. Conching, an important chocolate manufacturing step, may be designed for control of Salmonella if proper product temperature is maintained for sufficient time. A better understanding of thermal inactivation kinetics of Salmonella in different chocolate products is, however, highly desirable in evaluating the effectiveness of an existing conching process for Salmonella reduction or assessing the need for modification of manufacturing operations. Our previous study on milk chocolate (41.8% fat content) noted that aw and temperature are two key factors influencing Salmonella inactivation in a thermal process. In this research we studied the effect of different fat levels on Salmonella inactivation in chocolates at 80 °C. White and dark chocolates (fat content: 53.0% and 74.3%) were used in this study. The samples were conditioned to three water activity (aw) levels, 0.23, 0.33, and 0.43, at room temperature. The changes of aw in these samples were quantified when heated to 70 °C and 80 °C to reflect the real relative humidity conditions during the process. The data indicated an increase in the aw in the chocolate products, correlating with elevated temperatures. The thermal inactivation tests showed that D-values (time to achieve one log reduction at a fixed temperature) of Salmonella were between 33.9 and 46.5 min when treated at 80 °C, the corresponding aw,80 °C were 0.29 and 0.28 in white and dark chocolate samples, respectively. Our data suggests that the higher fat content contributed to a less increase in aw, leading to a higher heat tolerance of bacteria in the chocolates. The research results may offer useful insights for the chocolate manufactures to utilize conching process for the pathogen control of chocolate products made with different recipes.

Thermal inactivation kinetics of Listeria monocytogenes in milk under isothermal and dynamic conditions

Thermal processing is a widely used method to ensure the microbiological safety of milk. Predictive microbiology plays a crucial role in quantifying microbial growth and decline, providing valuable guidance on the design and optimization of food processing operations. This study aimed to investigate the thermal inactivation kinetics of Listeria monocytogenes in milk under both isothermal and dynamic conditions. The thermal inactivation of L. monocytogenes was conducted under isothermal and non-isothermal conditions in sterilized and pasteurized milk, with and without background microbiota, respectively. Furthermore, a secondary model was developed between the shoulder effect and temperature, which was then integrated into the dynamic model. The results showed that L. monocytogenes grown in Tryptic Soy Yeast Extract Broth (TSBYE) prior to thermal inactivation exhibited higher heat resistance compared to cells grown in sterilized milk at isothermal temperatures of 60.0, 62.5, and 65℃. Moreover, the presence of background microbiota in milk significantly enhanced the heat resistance of L. monocytogenes, as evidenced by the increased D-values from 1.13 min to 2.34 min, from 0.46 min to 0.53 min, and from 0.25 min to 0.34 min at 60.0, 62.5, and 65 °C, respectively, regardless of whether the background microbiota was inactivated after co-growth or co-inactivated with L. monocytogenes. For non-isothermal inactivation, the one-step dynamic model based on the log-linear with shoulder model effectively described the microbial inactivation curve and exhibited satisfactory model performance. The model developed contributes to improved risk assessment, enabling dairy processors to optimize thermal treatment and ensure microbiological safety.

Thermal Inactivation of Escherichia Phage OSYSP and Host Strain Escherichia coli O157:H7 EDL933: A Comparative Kinetic Analysis

Lytic bacteriophages are promising biocontrol agents against pathogenic bacteria for food and therapeutic applications. Investigating the feasibility of combining phage and physical lethal agents, such as heat, as an effective hurdle combination could lead to beneficial applications. The current research was initiated to compare the thermal inactivation kinetics of a lytic phage (Escherichia phage OSYSP) and its host (Shiga toxin-producing Escherichia coli O157:H7 EDL933), considering they have different critical thermal targets in their structures. To provide a basis for comparison, thermal inactivation kinetics were determined on suspensions of these agents in buffered peptone water using a thermally controlled circulating water bath. Results showed that the bacteriophage virions have a remarkable heat resistance (p < 0.05) compared to their host cells. The D-values of the populations of phage (PFU/mL) and EDL933 strain (CFU/mL) were 166.7 and 7.3 min at 55°C, compared to 44.4 and 0.3 min at 60°C, respectively. Additionally, D-values were significantly (p < 0.05) more influenced by temperature changes in the case of E. coli O157:H7 EDL933 (z-value 3.7°C) compared to that for phage OSYSP (z-value 7.7°C). When the phage suspension was heat-treated in a thermal cycler instead of a water bath, no significant differences between the two treatment procedures (p > 0.05) in estimating virus D- and z-values were observed. Based on these findings, it may be feasible to combine phage OSYSP with mild heat during processing of food to selectively inactivate E. coli O157:H7 EDL933 and subsequently maintain product safety during storage by the surviving phage population; however, the feasibility of this application needs to be investigated. Additionally, the relatively heat-resistant phage OSYSP could qualify as a biological indicator to validate thermal treatments of minimally processed foods in which E. coli O157:H7 EDL933 is the pathogen-of-concern.

Comparative study on the impact of equally stressful environmental sporulation conditions on thermal inactivation kinetics of B. subtilis spores

Control of bacterial spores continues to be one of the main challenges for the food industry due to their wide dissemination and extremely high resistance to processing methods. Furthermore, the large variability in heat resistance in spores that contaminate foods makes it difficult to establish general processing conditions. Such heterogeneity not only derives from inherent differences among species and strains, but also from differences in sporulation environments that are generally ignored in spores encountered in foods. We evaluated heat inactivation kinetics and the thermodependency of resistance parameters in B. subtilis 168 spores sporulated at adverse temperatures, water activity (aw), and pH, applying an experimental approach that allowed us to quantitatively compare the impact of each condition. Reduction of incubation temperature from the optimal temperature dramatically reduced thermal resistance, and it was the most influential factor, especially at the highest treatment temperatures. These spores were also more sensitive to chemicals presumably acting in the inner membrane. Reducing sporulation aw increased heat resistance, although the magnitude of that effect depended on the solute and the treatment temperature. Thus, changes in sporulation environments varied 3D100°C values up to 10.4-fold and z values up to 1.7-fold, highlighting the relevance of taking such a source of variability into account when setting heat processing conditions. UV-C treatment and sodium hypochlorite efficiently inactivated all spore populations, including heat-resistant ones produced at low aw.

Characterization of a fungal α-galactosidase and its synergistic effect with β-mannanase for hydrolysis of galactomannan

An acid stable α-galactosidase was produced and purified from mannolytic fungal strain, Penicillium aculeatum APS1. Enzyme was produced using wheat bran and copra cake moistened with corn steep liquor by solid state fermentation. APS1αgal having molecular weight of 65.4 kDa was purified to electrophoretic homogeneity by three phase partitioning and gel permeation chromatography with high enzyme recovery. APS1αgal was found to be maximally active at 55 °C and pH 4.5, having high stability at acidic pH. Thermal stability and thermal inactivation kinetics of APS1αgal were also studied. APS1αgal was found to effectively hydrolyse oligosaccharides as well as polysaccharides having α-1,6 linked galactose. Abolishment of enzyme activity in N-brommosuccinimide revealed an important role of tryptophan residue in catalysis. APS1αgal had shown outstanding tolerance to NaCl and proteases. MALDI-TOF MS/MS analysis indicated that enzyme is probably a member of family GH27. Synergistic interaction between APS1αgal and β-mannanase for hydrolysis of galactomannan was very clear and maximum 2.0° of synergy was found under simultaneous mode of action. This study reports a new source of α-galactosidase with biochemical properties suitable for applications in food and feed industries.

Thermal inactivation kinetics of Paucilactobacillus wasatchensis SH05 in whole milk

Paucilactobacillus wasatchensis is a non-starter lactic acid bacterium that causes late-blowing defects in aged cheeses, such as Cheddar. As P. wasatchensis is a less studied spoilage bacterium, this work was conducted to understand its heat resistance. Four P. wasatchensis strains were initially compared for their heat resistance, and strain SH05 was selected for the thermal inactivation kinetics experiment using aluminum thermal-death-time (TDT) disks and hot water baths. Five TDT disks, each containing 5 mL of sterilized whole milk inoculated with P. wasatchensis, were submerged in a pre-heated water bath set at respective temperatures and sampled at pre-determined time intervals. The D-values of P. wasatchensis SH05 in whole milk were D50 = 1.9, D52 = 1.0, D54 = 0.6, and D56 = 0.3 min, with the z-value of 7.7 °C.

Kinetic study of thermal inactivation of enterococci and clostridial spores

In order to better understand the fate of clostridia and enterococci during the heat treatment of livestock effluents, a first approach consisted in modelling their persistence under specific conditions (culture media, pure strain). The objective of this study is to establish the thermal inactivation kinetics of strains belonging to two species of enterococci (E. faecalis and E. faecium), to the species C. difficile and to the species C. novyi (used as a non-toxic model of C. botulinum group III), by evaluating in particular the scales imposed by European regulations.

Thermal Inactivation of Salmonella enterica and Nonpathogenic Bacterial Surrogates in Wheat Flour by Baking in a Household Oven

Wheat flour has been implicated in recalls and outbreaks linked to Salmonella and pathogenic Escherichia coli. An instructional online video posted on a popular YouTube channel with over 20 million subscribers claimed that safe raw cookie dough could be made from flour baked in a household oven at 177°C (350°F) for 5 min, but no evidence in support of that claim was provided. This study was conducted to assess thermal inactivation of two Salmonella strains, as well as Enterobacter aerogenes and Pantoea dispersa in wheat flour during home oven baking. Wheat flour was inoculated with Salmonella Enteritidis PT 30, Salmonella Typhimurium PT 42, or their potential surrogates at high concentrations (4.8 to 6.1 log CFU/g) before baking in a consumer-style convection oven (toaster oven) at 149, 177, and 204°C (300, 350, or 400°F) for up to 7 min. Flour was heated in an aluminum tray, with a maximum depth of ∼2 cm. Heated wheat flour samples (5 g each) were enumerated in triplicate, and the microbial concentration was expressed in log CFU per gram. Thermal profiles of the geometric center of the wheat flour pile and air in the oven during the baking were recorded. Water activity of wheat flour samples was also measured before and after baking. The water activity of wheat flour decreased, as baking temperature and time increased. Water activity values ranged from 0.30 to 0.06 after 7 min, as oven temperature increased from 149 to 204°C. Thermal inactivation kinetics were linear until counts approached the limit of detection for all microorganisms. D-values for Salmonella and potential surrogate strains ranged from 1.86 to 2.13 min at 149°C air temperature, 1.66 to 1.92 min at 177°C air temperature, and 1.12 to 1.38 min at 204°C air temperature. Both Salmonella strains and surrogates showed similar inactivation patterns. Baking of wheat flour in household toaster ovens has potential as an inactivation treatment of pathogenic bacteria in consumer homes, despite its low water activity.

Experimentally Implementing the Linear Nonisothermal Equation for Simultaneously Obtaining D- and z-Values of Salmonella Senftenberg in Skim Milk with a Differential Scanning Calorimeter

For bacteria with log-linear thermal inactivation kinetics in food, D-values are obtained in multiple isothermal inactivation experiments at different temperatures, and the z-value is obtained from these D-values. In a previous work, the cumulative lethality integral was mathematically solved in closed form when temperature in the food increased linearly with time. The solution revealed that each nonisothermal experiment could yield both D- and z-values, eliminating the need for getting multiple D-values to get a z-value. The present study reports on the first experimental implementation of this method of obtaining D- and z-values for Salmonella Senftenberg suspended in skim milk for which a differential scanning calorimeter (DSC) provided the required constant heating rate. The resulting D- and z-values were compared with those obtained from an isothermal method with capillary tubes. No significant differences in z-values were found between the two methods. The D-values also agreed but only after correcting the nonisothermal value for temperature lag in the DSC caused by the large sample size required. A 5 K/min heating rate was used in this comparison. Other rates were also investigated: 1, 3, 7.5, and 10 K/min. Although D- and z-values should be independent of DSC heating rate, heating rates of 1 and 10 K/min yielded values that were significantly different from the others; therefore, these rates cannot be recommended for use in this nonisothermal method.

Thermal inactivation kinetics of Aeromonas hydrophila in soymilk of varying pH and sugar concentrations

AbstractTackling microbial risk in food requires a good knowledge of the behavior of an organism in specific foods. The effect of temperature (50, 52.5, 55, 57.5, 60, 62.5, and 65°C), pH (6.0, 6.5, and 7.0) and sugar concentration (0, 5.0 and 10% w/v) on survival of A. hydrophila in soymilk was investigated. Heat resistance studies were carried out using conventional screw‐capped tube procedure with heating at constant temperature. Survival plots were made from experimental data, and D‐ and z‐values obtained ranging between 18.15–0.57 min and 9.71–9.55°C respectively. Significant effects were observed in D‐ and z‐values with variations in pH and sugar concentrations of soymilk. This hurdle combination proved effective in preserving soymilk, aimed at optimizing nutrient retention, organoleptic quality and destruction of A. hydrophila. The data obtained would be useful in effective thermal processing, establishing appropriate pasteurization schedules with reference to A. hydrophila contamination in soymilk.Novelty impact statementAeromonas hydrophila has increasingly been recognized as an emerging food borne pathogen that poses threat to human health. Although inactivation studies of A. hydrophila in several foods have been reported, there is limited information on its inactivation in soymilk using multiple hurdles. In this study, thermal inactivation kinetics of Aeromonas hydrophila from a water source was carried out in soymilk of varying pH and sugar concentrations and was observed to exhibit apparently linear survival curves, and higher decimal reduction times (D‐value) and z‐values than reported for other studied liquid foods. The parameters obtained in this study will be useful in the optimization of thermal processing of soymilk in combination with other hurdles to ensure safety from this organism and similar gram‐negative bacteria.

Thermal Inactivation Kinetics and Radio Frequency Control of Aspergillus in Almond Kernels.

Mold infections in almonds are a safety issue during post-harvest, storage and consumption, leading to health problems for consumers and causing economic losses. The aim of this study was to isolate mold from infected almond kernels and identify it by whole genome sequence (WGS). Then, the more heat resistant mold was selected and the thermal inactivation kinetics of this mold influenced by temperature and water activity (aw) was developed. Hot air-assisted radio frequency (RF) heating was used to validate pasteurization efficacy based on the thermal inactivation kinetics of this target mold. The results showed that the two types of molds were Penicillium and Aspergillus identified by WGS. The selected Aspergillus had higher heat resistance than the Penicillium in the almond kernels. Inactivation data for the target Aspergillus fitted the Weibull model better than the first-order kinetic model. The population changes of the target Aspergillus under the given conditions could be predicted from Mafart’s modified Bigelow model. The RF treatment was effectively used for inactivating Aspergillus in almond kernels based on Mafart’s modified Bigelow model and the cumulative lethal time model.

Thermal inactivation kinetics of Salmonella enterica and Enterococcus faecium NRRL B-2354 as a function of temperature and water activity in fine ground black pepper

Fine ground black pepper generally consumed as a seasoning without any further processing has been associated with Salmonella enterica outbreaks. Thermal inactivation kinetics data is necessary to develop a pasteurization process for fine ground black pepper. This study investigates the influence of temperature and water activity on thermal inactivation kinetics of Salmonella in fine ground black pepper. It also assesses the suitability of Enterococcus faecium as a surrogate for Salmonella. Fine ground black pepper of varying water activities, aw (0.40, 0.55, 0.70) was subjected to isothermal treatments at different temperatures (65–80 °C) for five equidistant time points with intervals ranging from 18 s to 250 min. The survival data were used to fit two primary models (log-linear and Weibull) and two secondary models (response surface and Modified Bigelow). Results indicated that among the two primary models, the Weibull model explained the thermal inactivation kinetics better with lower RMSE (0.24 – 0.56 log CFU/g) and AICc values at all aw and temperatures. Water activity and treatment temperature significantly enhanced the thermal inactivation of Salmonella. E. faecium NRRL B-2354 was found to be a suitable surrogate for Salmonella in fine ground black pepper at all tested treatment conditions. The developed modified Bigelow model based on the Weibull model could be applied to predict the inactivation kinetics of Salmonella in black pepper and would benefit the spice industry in identifying process parameters for thermal pasteurization of fine ground black pepper.

Thermal inactivation kinetics parameters of browning enzymes in starfruit (Averrhoa carambola L.) juice

SummaryThis study aimed to evaluate the thermal inactivation kinetics of polyphenol oxidase (PPO) and peroxidase (POD) in starfruit juice. It followed the Malaysia Food Regulations 1985 and CODEX STAN 247‐2005. Glucose, fructose and sucrose were the main sugars in starfruit juice. The total soluble solids, pH, titratable acidity, and total phenolics content of the starfruit juice produced were 8.13 ± 0.25 °Brix, 3.80 ± 0.05, 0.43% ± 0.02% malic acid, and 93.67 ± 4.96 mg GAEL−1, respectively. Thermal inactivation kinetics of PPO and POD followed the first‐order kinetic model. The decimal reduction time at 83.6 °C (D83.6) of PPO and POD was 198.48 and 98.4 s, respectively, while the thermal resistance constant (z value) of PPO and POD was 12.8 and 5.4 °C, respectively. In conclusion, PPO might be a suitable signal for thermal processing on starfruit juice since it has higher heat resistance than POD.

Radio frequency inactivation of E. coli O157: H7 and Salmonella Typhimurium ATCC 14028 in black pepper (piper nigrum) kernels: Thermal inactivation kinetic study and quality evaluation

In recent years, E. coli O157: H7 and Salmonella Typhimurium outbreaks in black peppers have caused intestinal diseases among human being, which calls for emergence of pasteurization processes for black pepper. Radio frequency (RF) energy has the potential of pasteurizing low moisture spices within a short time. In this study, thermal inactivation kinetics studies of E. coli O157: H7 and Salmonella Typhimurium ATCC 14028 on black pepper kernels were conducted, respectively. Then, RF inactivation experiments were conducted on inoculated black pepper kernels to achieve the required lethality. Results showed the number of log reduction for E. coli O157: H7 was > 6 log CFU/g after 7.0 min when heating to 90 °C in RF. Meanwhile, Salmonella Typhimurium ATCC 14028 reached > 6 log CFU/g reduction after 8.0 min RF heating and reaching 100 °C. From the quality point of view, the residual amount of Caryophyllene, 3-Carene, d-Limonene, β-Pinene in black pepper after RF pasteurization was 0.0060, 0.0038, 0.0034, 0.0016 mg/g, respectively. Comparatively, after steam pasteurization, Caryophyllene, 3-Carene, d-Limonene, β-Pinene content in black pepper was 0.0043, 0.0023, 0.0020, 0.0009 mg/g, respectively. Compared with steam heating, flavor substances in black pepper were better retained after RF heating. Thus, RF heating could effectively inactivate E. coli O157: H7 and Salmonella Typhimurium ATCC 14028 without significant influence on the quality of black pepper kernels.

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S.

Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.