Oxidative damage to proteolytic enzymes: inactivation of papain and ficin by ferrylmyoglobin

 Ferrylmyoglobin [MbFe(IV)=O], formed by activation of metmyoglobin by hydrogen peroxide, inactivates the cysteine proteinases papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) more efficiently than hydrogen peroxide, but less efficiently than hydroxyl radicals as generated by peroxynitrite or the Fenton reaction. Metmyoglobin and oxymyoglobin could not inactivate papain and ficin. Oxidation of papain and ficin by ferrylmyoglobin occurs in enzyme/haem-protein complexes with binding constants of approximately 10⁵ l · mol^–1; inactivation of proteolysis by papain plateaus at neutral pH to about ¹/₃ whereas the inactivation under acidic conditions was larger. Received: 11 July 1997 / Revised version: 16 September 1997     

INACTIVATION OF PAPAIN PROTEOLYTIC ACTIVITY IN THE PRESENCE OF ASCORBIC ACID AND CU++ IONS

The simultaneous presence of ascorbic acid, Cu⁺⁺ ions and oxygen causes reversible inactivation of proteolytic activity of papain. Low concentrations of ascorbic acid and Cu⁺⁺ ions have no effect on their own. Ascorbic acid at concentrations higher than 2 × 10⁻⁴ m inactivates papain irreversibly in the presence of Cu⁺⁺ ions. Papain inactivation is not accompanied by the destruction of papain molecules. Irreversible papain inactivation does not occur in solutions free of oxygen and the intermediates of ascorbic acid oxidation, i.e. dehydroascorbic acid and hydrogen peroxide, do not cause papain inactivation. The irreversible inactivation of papain is most probably due to free radicals formed during ascorbic acid oxidation. Owing to the possible occurrence of both ascorbic acid and Cu⁺⁺ ions in beer, partial irreversible inactivation of papain can be expected even at very low oxygen concentrations.     

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     

INACTIVATION OF FICIN PROTEOLYTIC ACTIVITY IN THE PRESENCE OF ASCORBIC ACID AND Cu2+-IONS

The simultaneous presence of ascorbic acid, Cu²⁺ions and oxygen causes irreversible ficin inactivation. The degree of inactivation is dependent on the concentration of inhibitors. Relatively higher decrease of ficin proteolytic activity was found with high molecular substrate, hide powder azure, than with a low molecular one, N-benzoyl-L-arginine-p-nitroanilide. Gel chromatography on Sephadex G-50 showed that ficin inactivation is not due to the destruction of its molecule resulting in the decrease of its molecular weight. Inactivation of ficin proteolytic activity was associated with a partial drop in ficin immunoreactivity.     

Antioxidant peptides isolated from sea cucumber Stichopus Japonicus

The sea cucumber Stichopus japonicus protein was hydrolyzed by pepsin, trypsin, papain, acid protease and neutral protease, respectively, to get five kinds of peptide fractions: pepsin peptides (PP), trypsin peptides (TP), acid protease peptides (AP), neutral protease peptides (NP) and papain peptide (PAP). Antioxidative activities of all peptide fractions were evaluated by hydroxyl radical– (·OH) and Superoxide anion (O₂ ^{· −} )–scavenging activity. Trypsin peptide (TP) exhibited the highest antioxidative activity compared to other peptide fractions. In considering scavenging effects on hydroxyl radicals (·OH) and Superoxide anions (O₂ ^{· −} ), TP was employed for isolation, purification and identification of antioxidant peptide. To purify and characterize antioxidative peptide, two steps gel filtration, one-step ion-exchange column chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) were used. The purified antioxidative peptide TP2b-1 was a novel peptide and was sequenced as GPEPTGPTGAPQWLR, in which the low molecular weight and some amino acid constituents played important role in the radical-scavenging effects according reports. The IC₅₀ values of TP2b-1 were 138.9 μM on ·OH and 353.9 μM on O₂ ^{· −} .     

The inactivation of Bacillus subtilis spores at low concentrations of hydrogen peroxide vapour

Spores of the bacterium Bacillus subtilis were deposited onto the surface of membranes by a process of filtration and exposed to concentrations of hydrogen peroxide vapour between 10 and 90 mg/m³ (ppm) for times ranging from 1.5 to 48 h. The inactivation data obtained in this way was modelled using the Weibull, Series-Event and Baranyi inactivation models. The Weibull model provided the best fit, and its use was extended to previously published literature obtained at higher hydrogen peroxide concentrations to produce a correlation yielding D (decimal reduction value) values over a range from 10 to almost 4000 ppm.     

Kinetics of green asparagus ascorbic acid heated in a high-temperature thermoresistometer

 Thermal degradation of green asparagus ascorbic acid in high-temperature short-time conditions was studied by heating in a five-channel computer-controlled thermoresistometer. Ascorbic acid was heated to between 110  °C and 140  °C and the degradation kinetics were analyzed assuming that two different inactivation mechanisms were occurring, one aerobic and the other anaerobic. The two reactions followed first-order kinetics, with E _a=12.3(2.0) kcal/mol and k _125  °C=47.0(3.0)×10^–3 s^–1 for the aerobic oxidation, and E _a=6.1(1.4) kcal/mol and k _125  °C=4.1(0.2)×10^–3 s^–1 for the anaerobic degradation. Received: 30 January 1998 / Revised version: 11 June 1998     

UV inactivation of milk-related microorganisms with a novel electrodeless lamp apparatus

A novel UV apparatus based on Dean vortex technology is designed for inactivating bacteria in milk. In this apparatus, the milk flows through a helical quartz tube coiling around an electrodeless UV lamp (EUL) with a radio frequency of 2.65 MHz. Flow rate, inner diameter of quartz tube, different UV sources, and different types of bacteria have been found as the key factors for the valuable effects on bacterial inactivation. The EUL apparatus worked more efficiently in the UV inactivation of the predetermined populations of milk-related bacteria than the conventional low-pressure high-intensity mercury lamp. When the UV dose of 21.3 mJ/cm² was applied, the numbers of all the bacteria were reduced by more than 6 log₁₀ with a flow rate of 28.8 L/h and a tube’s inner diameter of 1.5 mm. Dean vortices were formed in the milk flow during the UV processing and played an important role in the UV inactivation of the bacteria. Another inactivation test with the apparatus applying the UV dose of 21.3 mJ/cm² was also done with raw cow’s milk containing indigenous microorganisms, including Salmonella and Shigella spp., Listeria monocytogenes, Staphylococcus spp., Enterobacteriaceae, lactic acid bacteria, pseudomonads, and the total aerobic bacteria were reduced by approximately 3–4 log₁₀. In short, the EUL apparatus requires smaller energy, occupies less space, and has simpler operating procedures than thermal pasteurization. Thus, the novel method provides a viable alternative to thermal pasteurization of milk for improving the microbial safety of milk and extending its shelf life.     

Inhibition of enzymes and Pseudomonas tolaasii growth on Agaricus bisporus following treatment with surface dielectric barrier discharge plasma

In this study the applicability of cold plasma, produced by surface dielectric barrier discharge (SDBD), to inactivate Pseudomonas tolaasii (P.tolaasii), polyphenol oxidase (PPO) and peroxidase (POD) enzymes, as well as its impact on quality parameters such as, color, texture, pH and weight loss were evaluated. The study evidently shows that treating with combination of 30% hydrogen peroxide vapor)with flow rate of 0.47 mL min⁻¹) and argon (H₂O₂ + Ar) for 180 s is capable of reducing the activity of PPO (0.17 U min⁻¹ g⁻¹ FW) and POD (0.21 U min⁻¹ g⁻¹ FW) and increasing the SOD enzyme (16.29 U g⁻¹ FW) in a 21-day storage period compared to control samples. This is while the quality characteristics of button mushroom are preserved during storage after such treatment.Industrial relevanceThis study provides information of A. bisporus storage during 21-day period after SDBD plasma treatment, which is rarely. Greater inactivation of P. tolaasii after 180 s treatment with combination of hydrogen peroxide vapor and air (H₂O₂ + air) was shown compared to control samples, but this treatment caused also slightly degradation of button mushroom color. A better reduction of PPO and POD enzyme activity as well as further increase of SOD enzyme activity was observed following treatment with 180 s of H₂O₂ + Ar gas in a 21-day storage period. This research work contributes to the understanding SDBD plasma induced effects on the shelf-life of button mushroom and could be a basis for a possible industrial implementation.     

Molecular characteristics of tara galactomannans: Effect of degradation with hydrogen peroxide

Tara gum (M_w = 1519 × 10³ g/mol) was degraded by hydrogen peroxide in the presence/absence of ascorbic acid, and properties of the products were investigated using gel permeation chromatography, Fourier transform infrared spectroscopy, atomic force microscope, and rheometer methods. Results showed that molecular weight of tara gum decreased with increasing concentration of hydrogen peroxide. The addition of ascorbic acid could sharply increase the degradation rate and degree of tara gum by hydrogen peroxide, which is also confirmed by atomic force microscope. The degradation products kept the structure characteristics of galactomannan. However, they performed different rheological properties. At 1% (w/v) of concentration, DP1 (941 × 10³ g/mol) and DP2 (335 × 10³ g/mol) prepared by H₂O₂ exhibited non-Newtonian behavior, while DP3 (35.1 × 10³ g/mol) and DP4 (14.2 × 10³ g/mol) obtained by hydrogen peroxide plus ascorbic acid displayed Newtonian property. These results may promote the application of tara gum in diverse fields.     

Ranking the efficacies of selected red wine phenolic anti-oxidants using reversed-phase HPLC

Several studies have assessed total anti-oxidant activity of wine or individual components in isolation using chemical-based assays. In this study, a quantitative approach was developed to assess the relative anti-oxidant efficacies of selected red wine phenolics via peak reduction, using reversed-phase HPLC. Both intact red wine and phenolic standard solutions were challenged with five oxidant model systems as follows: (1) hydrogen peroxide (H₂O₂); redox-active metal ions (2) Fe³⁺ and (3) Cu²⁺; and the Fenton reagents (4) H₂O₂ + Fe²⁺; and (5) H₂O₂ + Cu⁺. Treatment with oxidants (1–3) resulted in loss of 47–60% of phenolic standards, which increased to 66–89% for treatment with the Fenton systems, with quercetin exhibiting the optimal anti-oxidant activity. For intact red wine, treatment with oxidants (1–3) led to all phenolic compounds being oxidised (27–77% loss), with caffeic acid and quercetin as the most effective anti-oxidants. For both Fenton systems (4–5), activities in red wine were considerably enhanced for caffeic acid and quercetin, which exhibited the highest anti-oxidant efficacies with 100% peak reduction, while p-coumaric acid and gallic acid were less effective anti-oxidants with peak reductions of 60–68%. The ranking, facilitated by this new quantitative approach, allows comparison of the individual efficacies of the anti-oxidants in a complex matrix.     

Flavonols of lotus (<Emphasis Type="Italic">Nelumbo nucifera,</Emphasis> Gaertn.) seed epicarp and their antioxidant potential

Large amounts of fresh seed epicarp of Nelumbo nucifera Gaertn. (FSENN) are discarded in China without any utilization. The aim of this study was to identify the flavonols found in a fraction of an extract of FSENN, and to measure their levels and investigate antioxidant properties. Glycosylated flavonols in fraction 2 (Fr2) from the extract of FSENN and their aglycones were identified by HPLC-ESI–MS² (negative mode), and six glycosylated and one aglycone flavonols in Fr2 were found. We also quantified flavonol aglycones (myricetin, quercetin, kaempferol and isorhamnetin) using HPLC method. The result showed that the quercetin content (10.2 mg/g of dry fraction) was higher than that of other aglycones. Antioxidant properties of Fr2 were evaluated in vitro by a number of methods including 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, the β-carotene bleaching method, and hydroxyl radical and hydrogen peroxide scavenging ability using the chemiluminescence method. This antioxidant potential in terms of IC₅₀ values was 5.48, 40 ± 0.14 and 0.62 ± 0.05 μg (dry Fr2)/mL on DPPH radicals, hydroxyl radicals and hydrogen peroxide, respectively. The Fr2 also exhibited antioxidant property in the β-carotene bleaching assay. In total, it possesses high levels of flavonol compounds with high antioxidant potential, and it is beneficial for the treatment or prevention of a variety of diseases and has nutraceutical potential.     

Postharvest BTH treatment induced disease resistance and enhanced reactive oxygen species metabolism in muskmelon (Cucumis melo L.) fruit

Benzo (1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a novel synthetic elicitor capable of inducing disease resistance in many plants. Fruits were dipped in BTH at 100?mg/L for 10?min, and then stored at room temperature (22?±?2?°C, relative humidity 55–60?%). The results showed that BTH significantly (P?<?0.05) reduced the lesion area of fruits inoculated with Trichothecium roseum and the natural disease incidence of fruits during storage at room temperature. The treatment effectively maintained cell membrane integrity and decreased the production of lipid peroxidation and malondialdehyde. BTH treatment markedly enhanced the accumulation of hydrogen peroxide, (H₂O₂) while decreased the rate of superoxide anion (O ₂ ^·? ) production. Furthermore, BTH observably increased the activities of NADPH oxidase (NOX, EC1.6.3.1), superoxide dismutase (SOD, EC1.15.1.1), peroxidase (POD, EC1.11.1.7), ascorbate peroxidase (APX, EC1.11.1.11), and glutathione reductase (GR, EC1.6.4.2), while inhibited the catalase (CAT, EC1.11.1.6) activity. These results indicated that BTH treatment increased the disease resistance of muskmelon fruits by enhancing their antioxidant system activity after harvest, and suggested that postharvest treatment with BTH could be promising in reducing decay and reducing or/and substituting chemical fungicides to control postharvest diseases in fruits.     

Optimisation of hydrolysis of purple sea urchin (Strongylocentrotus nudus) gonad by response surface methodology and evaluation of in vitro antioxidant activity of the hydrolysate

BACKGROUND: Hydrolysates prepared from sea urchin (Strongylocentrotus nudus) gonad by enzymatic treatment showed strong 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging activity and reducing power. RESULTS: Hydrolysis of S. nudus gonad by the commercial protease papain was optimised for maximum degree of hydrolysis (DH) and trichloroacetic acid‐soluble peptide index (TCA‐SPI) using response surface methodology. Results showed that the optimal conditions were the following: temperature of 48.83 °C, pH of 6.92, enzyme‐to‐substrate ratio of 3143 U g^?1, and substrate concentration of 83.5 g L^?1. Under these conditions, a DH of 27.96 ± 0.54% and a TCA‐SPI of 57.32 ± 0.63% were obtained. The hydrolysate prepared in the optimal conditions was fractionated by an ultra‐filtration system and the resultant fraction below 10 kDa was found to effectively scavenge hydroxyl radical (EC₅₀ = 13.29 ± 0.33 mg mL^?1) and hydrogen peroxide (EC₅₀ = 16.40 ± 0.37 mg mL^?1), inhibit lipid peroxidation (EC₅₀ = 11.05 ± 0.62 mg mL^?1), chelate Fe²⁺ (EC₅₀ = 7.26 ± 0.44 mg mL^?1), and protect mice macrophages against death induced by tert‐butyl hydroperoxide. CONCLUSION: Hydrolysates prepared from S. nudus gonad have the potential to be applied as natural antioxidant agents. Copyright © 2012 Society of Chemical Industry     

Inactivation ofEscherichia coliO157:H7 by combinations of GRAS chemicals and temperature

Reported outbreaks of foodborne illness involvingEscherichia coliO157:H7 have increased in the United States during the last decade, with a variety of food products being implicated as vehicles of infection. Studies were carried out to determine the efficacy of combinations of various GRAS chemicals and moderate temperatures to killE. coliO157:H7. A five-strain mixture ofE. coliO157:H7 of approximately 10⁸cfu ml⁻¹was inoculated into 0·1% peptone solutions containing 1·0 or 1·5% lactic acid plus 0·1% hydrogen peroxide, 0·1% sodium benzoate or 0·005% glycerol monolaurate. The solutions were incubated at 8°C for 0, 15 and 30 min; at 22°C for 0, 10 and 20 min; or at 40°C for 0, 10 and 15 min; populations ofE. coliO157:H7 were determined at each sampling time. At 40°C, the pathogen was inactivated to undetectable levels within 10 min of incubation in the presence of 1·0 or 1·5% lactic acid plus hydrogen peroxide, and within 15 min of incubation in the presence of 1·5% lactic acid plus sodium benzoate or glycerol monolaurate. At 22°C, complete inactivation ofE. coliO157:H7 was observed after 20 min of exposure to 1·5% lactic acid plus 0·1% hydrogen peroxide, whereas a reduction of 5 log₁₀cfu ml⁻¹was observed with a treatment of 1·5% lactic acid plus glycerol monolaurate. None of the treatments resulted in total inactivation of the pathogen at 8°C. The aforementioned treatments could potentially be used to inactivate or reduceE. coliO157:H7 populations on raw produce.     

The first ESR observation of radical species generated under pulsed electric fields processing

Electron spin resonance (ESR) spectroscopy was used to investigate the generation of free radicals under pulsed electric field (PEF) processing. Spin trapping studies, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), showed the generation of hydrogen radicals in phosphate buffer and oleic acid emulsion. The concentration of hydrogen peroxide in phosphate buffer after PEF treatment was determined by spectrofluorometric method. Electric field strength has a significant effect on the concentration of H₂O₂ (p < 0.05). In PEF-treated samples with electric field strength of 30 kV/cm and 35 kV/cm, trace concentrations of H₂O₂ (0.177 × 10⁻⁶ and 2.035 × 10⁻⁶ mol/L, respectively) were detected.     

Oxidative behaviour of Blanquilla pears treated with 1‐methylcyclopropene during cold storage

The increasing use of 1‐methylcyclopropene (1‐MCP) to extend the commercial life of fruit constitutes an attractive way of improving packing house competitiveness. This compound prevents the effects of ethylene in a wide range of fruit and vegetables. However, despite the extensive literature relating to this action on ethylene, little is known about its other physiological effects. In this work, pears (Pyrus malus L cv Blanquilla) were treated with 100 ppb 1‐MCP immediately after harvest and stored in air for 5 months. Differences in oxidative stress and in antioxidant potential between controls and 1‐MCP‐treated fruits were established, determining the changes in the levels of hydrogen peroxide, ascorbate content and ionic leakage during storage. Activities of the H₂O₂‐generating enzyme superoxide dismutase (EC 1.15.11) and the H₂O₂‐scavenging enzymes catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11) and unspecific peroxidase (EC 1.11.1.7) were also determined. 1‐MCP‐treated fruits exhibited lower levels of hydrogen peroxide, ascorbate and lower ionic leakage during storage. In accordance with this result, 1‐MCP‐treated fruits also exhibited higher enzymatic antioxidant potential. These results challenge the hypothesis that the beneficial effects of 1‐MCP on ripening were not exclusively due to its action on ethylene but also to an increase in antioxidant potential in pear. Copyright © 2004 Society of Chemical Industry     

Preservation of hermaphrodite and female papaya fruits (Carica papaya L?., Cv Sunrise, Solo group) by freezing: physical, physico-chemical and sensorial aspects

 The physical, physico-chemical and sensorial changes that occur during the freezing and frozen storage of hermaphrodite and female papaya slices (Carica papaya L., cv Sunrise, Solo group) were studied. Samples were evaluated by panellists throughout a 1-year period of storage at –24°C; the panellists described the fruit in terms of physico-chemical measurements of texture (firnmess and total pectins content), colour [L, a _L , b _L , peroxidase (POD) and polyphenoloxidase (PPO) activities] and flavour/taste [soluble solids content, pH, tritatable acidity and quality index (^oBrix/acidity)], and ranked them in order of preference. Desserts prepared from frozen slices of hermaphrodite and female papaya fruits mixed with orange, lemon and sugar were also evaluated. The freezing process itself did not result in significant changes in the more commonly assessed quality parameters of texture, colour and flavour/taste. However, during storage at –24°C, changes in sensorial characteristics occurred that became more evident as storage continued (≤ 9 months). Nevertheless, these quality changes can be masked by using these papaya slices as an ingredient in fruit products, with frozen female papaya slices being especially suitable for this purpose. Received: 1 August 1997 / Revised version: 14 November 1997     

Listeria innocua Multi-target Inactivation by Thermo-sonication and Vanillin

Hurdle technology combining an emerging preservation technique such as low-frequency ultrasound is an alternative for processing juices that are susceptible to suffer a loss of quality due to traditional heat treatments. Predictive microbiology allows evaluation of the effectiveness of preservation techniques and its combinations in order to enhance both food quality and safety. Listeria innocua inactivation by thermo-sonication along with vanillin was investigated. Fermi model (R ² _adj= 0.970 ± 0.02) and surface response methodology (p < 0.05) were utilized in order to evaluate the survival of L. innocua to a multi-target treatment and to predict the interactions of studied techniques, high-intensity/low-frequency ultrasound (20 kHz/400 W) at selected wave amplitudes (60, 75, or 90 μm), temperature (40, 50, or 60 °C), and vanillin (200, 350, or 500 mg/kg). A combination of ultrasound, vanillin, and temperature enhanced L. innocua inactivation as described by Fermi parameters a and t _c, which decreased as the studied effects increased. A multi-target inactivation effect was observed for a temperature range of 45–55 °C.