Proteolytic specificity of cathepsin D on bovine F-actin

Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na acetate buffer, pH 5.5, at 37°C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by cathepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Peptides (2% trichloroacetic acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C(18) column using an acetonitrile/water gradient and identified from their N-terminal sequence and mass. Cathepsin D cleavage sites were identified at Cys(12)-Asp(13), Gly(22)-Phe(23), Arg(30)-Ala(31), Thr(79)-Asn(80), Ile(87)-Trp(88), Thr(91)-Phe(92), Phe(92)-Tyr(93), Arg(97)-Val(98), His(103)-Pro(104), Leu(107)-Thr(108), Thr(108)-Glu(109), Lys(120)-Met(121), Leu(144)-Tyr(145), Ile(153)-Val(154), Leu(155)-Asp(156), Ile(167)-Tyr(168), Leu(180)-Asp(181), Met(192)-Lys(193), Leu(195)-Thr(196), Arg(208)-Glu(209), Arg(212)-Asp(213), Leu(223)-Asp(224), Lys(240)-Ser(241), Thr(262)-Leu(263), Trp(342)-Ile(343), Arg(349)-Ser(350), Trp(358)-Ile(359), and Lys(375)-Cys(376). In general, cathepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degraded extensively by cathepsin D with peptides released from numerous locations in the protein molecule.     

荠菜过氧化物酶热失活动力学的研究

目的 研究热烫处理引起荠菜过氧化酶(POD)失活的速率常数和动力学规律。方法 对荠菜在80～100℃的热水处理一定时间条件,对荠菜过氧化酶的残余活性进行测定,并用一级动力学模型对过氧化物酶失活动力学进行拟合,分析失活速率常数。结果 热烫处理钝化新鲜荠菜POD活性效果明显,在80～100℃处理范围内,随着处理温度的上升,荠菜POD 失活速率常数k 值从0.01702增加到0.14260,反应活化能为114 kJ/mol。一级动力学模型拟合决定系数R²＞0.99。结论 荠菜POD 的热失活动力学符合一级动力学模型。     

Thermal inactivation kinetics of alkaline phosphatase in equine milk

Alkaline phosphatase (ALP) is widely used as an indicator of proper pasteurization in bovine milk. Due to interest in the use of equine ALP as a time/temperature integrator (TTI) for evaluation of the efficacy of thermal processing of equine milk, its inactivation kinetics were evaluated in whole and skimmed equine milk. Experimentally determined decimal reduction times showed that equine ALP is more readily inactivated in equine milk than its bovine counterpart. Thus, considering the required 6 D reduction of pathogens and the rather low enzyme level present in equine milk, equine ALP will not be suitable as indicator for correct pasteurization of equine milk under the conditions currently used in the reference method for the determination of ALP in milk-based products.     

α-淀粉酶在棉织物上的热失活动力学及稳定性

研究3种热稳定剂以及α-淀粉酶质量浓度、汽蒸温度、汽蒸时间和酶液pH值对中温α-淀粉酶在棉织物上热稳定性的影响.探讨中温α-淀粉酶在棉织物上的热失活的动力学.结果表明,棉织物的结构本身对中温α-淀粉酶就具有很好的热稳定效果,醋酸钙、乳酸钠和低分子质量壳聚糖(LWCS)都能进一步提高α-淀粉酶在棉织物上的热稳定性,其中以LWCS的效果最好;棉织物α-淀粉酶退浆优化工艺为:α-淀粉酶0.1 g/L,LWCS0.5 mmol/L,酶液pH值6.100℃汽蒸10 min.按此工艺对棉织物进行处理后,退浆率达到97.2%.     

Thermal inactivation kinetics of Bacillus stearothermophilus spores using a linear temperature program.

A systematic study of the inactivation kinetics of Bacillus stearothermophilus spores was carried out in nonisothermic heating conditions using a linear temperature increase program and analyzing the experimental data by means of a one-step nonlinear regression. The D and z values estimated are close to those obtained in isothermic conditions and estimated by using a two-step model, first D values are calculated, and then in the second step a z value is deduced (D(121 degrees C) = 3.08 and 4.38 min, respectively, and z = 7 and 7.9 degrees C, respectively). No convergence problems were observed when using the one-step nonlinear regression proposed. The results indicated that the methodology applied in this study can be used to obtain kinetic data for bacterial spores, which could mean a significant reduction in the amount of experimental work employed to generate these data.     

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd

The use of raw milk in the processing of buffalo Mozzarella cheese is permitted, but the heat treatment used for stretching the curd must ensure that the final product does not contain pathogens such as Shiga toxin-producing Escherichia coli (STEC) that may be present on buffalo dairy farms. This study carried out challenge tests at temperatures between 68°C and 80°C for 2 to 10 min to simulate curd temperatures during the stretching phase. Curd samples were inoculated with 2 STEC strains (serotypes O157 and O26), and their inactivation rates were assessed in the different challenge tests. The curd samples were digested with papain to ensure a homogeneous dispersion of bacteria. The STEC cells were counted after inoculation (range 7.1–8.7 log cfu/g) and after heat treatments using the most probable number (MPN) technique. A plot of log MPN/g versus time was created for each separate experiment. The log linear model with tail was used to provide a reasonable fit to observed data. Maximum inactivation rate (k_max, min⁻¹), residual population (log MPN/g), decimal reduction time (min), and time for a 4D (4-log₁₀) reduction (min) were estimated at each temperature tested. A 4D reduction of the O26 STEC strain was achieved when curd was heated at 68°C for 2.6 to 6.3 min or at 80°C for 2.1 to 2.3 min. Greater resistance was observed for the O157 strain at 68°C because k_max was 1.48 min⁻¹. The model estimates can support cheesemakers in defining appropriate process criteria needed to control possible STEC contamination in raw milk intended for the production of Mozzarella.     

Proteolysis of bovine F-actin by cathepsin B

The proteolytic specificity of cathepsin B on bovine F-actin was investigated. Actin (0.5 mg/ml) was incubated with cathepsin B (1.65 U/ml) for 6 h at 37°C and samples were taken periodically for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). During incubation, actin was hydrolysed with the simultaneous appearance of three peptides detectable by SDS–PAGE with molecular masses of 35, 33, and 29 kDa. These peptides were electroblotted from SDS–PAGE gels onto polyvinylidene difluoride membranes and their N-terminal sequence determined by Edman degradation. Principal cleavage sites of cathepsin B activity on actin were identified at Met₄₉–Gly₅₀, Thr₆₈–Leu₆₉ and Leu₁₀₇–Thr₁₀₈. Reverse-phase high performance liquid chromatography (RP-HPLC) was performed on 2% trichloroacetic acid-soluble fractions of the 6 h hydrolysate. Thirteen peptides separated by RP-HPLC were collected and identified from their N-terminal sequence and, in some cases, from their mass (as determined by mass spectrometry). Cleavage sites were identified at: Gly₂₂–Phe₂₃, Ala₂₄–Gly₂₅, Arg₃₀–Ala₃₁, Lys₇₀–Tyr₇₁, His₇₅–Gly₇₆, Gly₇₆–Ile₇₇, Thr₇₉–Asn₈₀, Lys₈₆–Ile₈₇, Phe₉₂–Tyr₉₃, Arg₉₇–Val₉₈, Thr₁₀₅–Leu₁₀₆, Thr₂₅₁–Ile₂₅₂, Ala₃₂₁–Leu₃₂₂, Leu₃₂₂–Ala₃₂₃, Ile₃₂₉–Lys₃₃₀, Lys₃₃₀–Ile₃₃₁, and Glu₃₆₃–Tyr₃₆₄. The results of this study showed that actin was degraded extensively by cathepsin B with the majority of the peptides released arising from the N- and C-termini of the protein.     

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

This 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 (D_83.6) 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.     

Thermal inactivation kinetics of Salmonella spp. within intact eggs heated using humidity-controlled air.

The heat resistance of six strains of Salmonella (including Enteritidis, Heidelberg, and Typhimurium) in liquid whole egg and shell eggs was determined. Decimal reduction times (D-values) of each of the six strains were determined in liquid whole egg heated at 56.7 degrees C within glass capillary tubes immersed in a water bath. D-values ranged from 3.05 to 4.09 min, and significant differences were observed between the strains tested (alpha = 0.05). In addition, approximately 7 log10 CFU/g of a six-strain cocktail was inoculated into the geometric center of raw shell eggs and the eggs heated at 57.2 degrees C using convection currents of humidity-controlled air. D-values of the pooled salmonellae ranged from 5.49 to 6.12 min within the center of intact shell eggs. A heating period of 70 min or more resulted in no surviving salmonellae being detected (i.e., an 8.7-log reduction per egg).     

Thermal and high-pressure inactivation kinetics of carrot pectinmethylesterase: from model system to real foods

Pectinmethylesterase (PME) was extracted from carrots (Daucus carrota) and subsequently purified by affinity chromatography on a CNBr-Sheparose-PME inhibitor (PMEI) column. Detailed kinetic studies on the inactivation of purified carrot PME by thermal and high-pressure processing were performed. Next to the model systems, the thermal and high-pressure inactivation of PME in real products (carrot juice and carrot pieces) was investigated. The inactivation was performed under isothermal and isothermal–isobaric conditions. Under all conditions investigated, a first-order kinetic model could describe the inactivation data. It was found that carrot PME is much more thermostable and pressure-stable in carrot pieces than in carrot juice or purified form.     

Thermal inactivation kinetics of peroxidase and lipoxygenase from fresh pinto beans (Phaseolus vulgaris)

 Thermal inactivation kinetics of crude peroxidase (POX) and lipoxygenase (LOX) in fresh pinto beans were studied over the temperature range of 55–90°C. The inactivation of both enzymes followed first-order kinetics. The biphasic inactivation curves for POX indicate the existence of several isoenzymes of varying heat stability. In the temperature range of 55–70°C, the activation energies (E _a) of POX were 46.5 kcal·mol^–1 for the heat-labile portion and 37.6 kcal·mol^–1 for the heat-stable portion. On the other hand, the LOX enzyme had an E _a value of 42.26 kcal·mol^–1 at 55–75°C and 49.1 kcal·mol^–1 at 55–90°C. Received: 28 July 1997 / Revised version: 16 October 1997     

Thermal inactivation kinetics of peroxidase and lipoxygenase from fresh pinto beans (Phaseolus vulgaris)

 Thermal inactivation kinetics of crude peroxidase (POX) and lipoxygenase (LOX) in fresh pinto beans were studied over the temperature range of 55–90°C. The inactivation of both enzymes followed first-order kinetics. The biphasic inactivation curves for POX indicate the existence of several isoenzymes of varying heat stability. In the temperature range of 55–70°C, the activation energies (E _a) of POX were 46.5 kcal·mol^–1 for the heat-labile portion and 37.6 kcal·mol^–1 for the heat-stable portion. On the other hand, the LOX enzyme had an E _a value of 42.26 kcal·mol^–1 at 55–75°C and 49.1 kcal·mol^–1 at 55–90°C. Received: 28 July 1997 / Revised version: 16 October 1997     

Hypochlorite inactivation kinetics of lactococcal bacteriophages

Ten heat-resistant lactococcal phages in M17 broth were inactivated by exposure to a range of different hypochlorite concentrations (2000-5000 mg l⁻¹). Deviations from first-order kinetics as sigmoidal shapes were observed in the survival curves of all bacteriophages. Therefore, an empirical model with four parameters was used to describe hypochlorite inactivation. The model provided good fit for all phages; however, it was observed that there was a high correlation between the parameters of this model. That is why, the number of parameters was reduced from four to two with a slight loss of goodness-of-fit and this reduced model produced fits comparable to the original model. Alternative D and z values were also proposed for the model. By comparing the times necessary to reduce the number of phages six- or seven-logs, the most hypochlorite-resistant phages were found as: φpld67 37, φpll47 21 and φpld66 36. Demonstration of this model may also be used for other bacteriophages inactivated by other biocides.     

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics,inactivation and potential application

High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.     

Pressure inactivation kinetics of Yersinia enterocolitica ATCC 35669

The survival curves of Yersinia enterocolitica ATCC 35669 inactivated by high hydrostatic pressure were obtained at four pressure levels (300, 350, 400, and 450 MPa) in sodium phosphate buffer (0.1 M, pH 7.0) and four pressure levels (350, 400, 450, 500 MPa) in UHT whole milk. Tailing was observed in all the survival curves. A linear model and three nonlinear models were fitted to these data and the performances of these models were compared. The linear regression model for survival curves at four pressure levels had regression coefficients (R2) values of 0.785-0.962 and mean square error (MSE) of 0.265-0.893. A residual plot strongly suggested that a linear regression function was not appropriate as there was strong curvature in the plotted data. The nonlinear regression model using the log-logistic had R2 values of 0.946-0.982 and MSE values of 0.110-0.320. The Weibull model had R2 values of 0.944-0.975 and MSE values of 0.153-0.349. These results indicated that both were better models to describe the pressure inactivation kinetics of Y. enterocolitica in milk and buffer. Among the three nonlinear models studied, the modified Gompertz model produced the poorest fit to data. The number of parameters of the log-logistic model was reduced from four to two so that the model was greatly simplified. The reduced log-logistic model still produced a fit comparable to the full model. Since pressure had no significant effect on the shape factors of the Weibull model at the pressure levels of 300-400 MPa for buffer and 400-500 MPa for milk, models were developed to predict survival curves of Y. enterocolitica at pressures different from the experimental pressures.     

Pressure inactivation kinetics of phage lambda cI 857

Inactivation curves of phage lambda cI 857 inactivated by high hydrostatic pressure were obtained at three pressure levels (300, 350, and 400 MPa) in buffered media and ultrahigh-temperature 2% reduced fat milk. Pressurization of phage lambda in buffered media at 300 MPa for 300 min, 350 MPa for 36 min, and 400 MPa for 8 min reduced the titer of phage lambda by 7.5, 6.7, and 7.7 log, respectively. Pressurization of phage lambda in milk at 300 MPa for 400 min, 350 MPa for 80 min, and 400 MPa for 20 min reduced the titer of phage lambda by 5.4, 6.4, and 7.1 log, respectively. Tailing was observed in all inactivation curves, indicating that the linear model was not adequate for describing these curves. Among the three nonlinear models studied, the Weibull and log-logistic models consistently produced best fits to all inactivation curves, and the modified Gompertz model the poorest. Because there were no significant differences in the values of shape factor (n) for suspension medium buffer, we reduced the number of parameters in the Weibull model from two to one by setting n at the mean value. The simplified Weibull model produced a fit comparable to the full model. Additionally, the simplified Weibull model allowed predictions to be made at pressures different from the experimental pressures. Menstruum was found to significantly affect the pressure resistance of phage lambda. Comparison of pressure inactivation of hepatitis A virus and phage lambda indicated that phage lambda is more sensitive to pressure than hepatitis A virus in Dulbecco's modified Eagle medium with 10% fetal bovine sera.     

Thermal inactivation of polyphenoloxidase in pineapple puree

Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 °C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy (E_a) as well as Decimal reduction time (D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (40-90 °C) of the PPO followed first-order kinetics.     

Thermal inactivation of Campylobacter jejuni in broth

New Zealand has a high rate of reported campylobacteriosis compared with other developed countries. One possible reason is that local strains have greater heat tolerance and thus are better able to survive undercooking; this hypothesis is supported by the remarkably high D-values reported for Campylobacter jejuni in The Netherlands. The objective of this study was to investigate the thermal inactivation of isolates from New Zealand in broth, using strains that are commonly found in human cases and food samples in New Zealand. Typed Campylobacter strains were heated to a predetermined temperature using a submerged-coil heating apparatus. The first-order kinetic model has been used extensively in the calculation of the thermal inactivation parameters, D and z; however, nonlinear survival curves have been reported, and a number of models have been proposed to describe the patterns observed. Therefore, this study compared the conventional first-order model with eight nonlinear models for survival curves. Kinetic parameters were estimated using both one- and two-step regression techniques. In general, nonlinear models fit the individual inactivation data sets better than the log-linear model. However, the log-linear and the (nonlinear) Weibull models were the only models that could be successfully fitted to all data sets. For seven relevant New Zealand C. jejuni strains, at temperatures from 51.5 to 60°C, D- and z-values were obtained, ranging from 1.5 to 228 s and 4 to 5.2°C, respectively. These values are in broad agreement with published international data and do not indicate that the studied New Zealand C. jejuni strains are more heat resistant than other strains, in contrast with some reports from The Netherlands.