Effect of microwave pretreatment on the kinetics of ascorbic acid degradation and peroxidase inactivation in different parts of green asparagus (Asparagus officinalis L.) during water blanching

In this study, the effects of two blanching conditions on ascorbic acid (AA) and peroxidase (POD) in different segments of asparagus (bud, upper, middle, and butt) were investigated. The blanching treatments were: blanching in water at 70, 80 and 90 °C (WB); microwave heating (900 W, 30 s) followed by water blanching (MW + WB). AA degradation and POD inactivation in all segments of asparagus for both treatments are well described by first-order models. The degradation rate of AA and POD is gradually increased from butt to bud segment of asparagus. In addition, MW pre-treatment could increase the E_a of AA degradation and decrease the E_a of POD inactivation during water blanching of asparagus. Therefore, it is recommended that the different segments of asparagus should be subjected to different blanching times, and MW pre-treatment could be applied for alleviating AA degradation and accelerating POD inactivation during blanching, cooking and pasteurisation in water.     

Peroxidase and polyphenol oxidase thermal inactivation by microwaves in green coconut water simulated solutions

Enzymes from coconut water such as peroxidase (POD) and polyphenol oxidase (PPO) when in contact with oxygen begin reactions causing nutritional and color losses. Solutions simulating the chemical constituents of coconut water were submitted to a batch process in a microwave oven. PPO and POD inactivation data could be characterized by: PPO/water D_93 °C=16.5 s (z=35.5 °C); PPO/sugars D_91 °C=18 s (z=33°C); POD/water D_91.5 °C=44 s (z=24 °C) and POD/sugars D_92 °C=20.5 s (z=19.5 °C). The contact between salts and enzymes promoted a drastic reduction of the initial activity. After the incidence of microwave energy at temperatures above 90 °C, enzymes activity was not detected. These results can indicate an adequate choice of temperature conditions to inactivate coconut water enzymes. The knowledge of how green coconut water constituents influence POD and PPO activity will supply useful information about microwave processing of coconut water.     

Thermal inactivation of peroxidase during blanching of butternut squash

The kinetics of thermal inactivation of peroxidase in butternut squash during water blanching was studied. Thin slices of squash were immersed in water at 60-90 °C for 0-60 min. For treatments below 70 °C, a biphasic, first-order kinetics model was proposed for peroxidase inactivation that would correspond to heat-labile and heat-resistant fractions of the enzyme. Parameters of the first-order model for the heat-labile and heat-resistant fractions were k_L,ref=24.8733 min⁻¹, E_aL=14023 J/mol and k_R,ref=0.0729 min⁻¹, E_aR=15794 J/mol, respectively. For treatments above 70 °C, a monophasic first-order kinetics model was proposed. Parameters were k_ref=8.6425 min⁻¹, E_a=149900 J/mol. Decreases in ascorbic acid contents due to blanching were analyzed. Blanching processes at high temperature and short time resulted in higher ascorbic acid retention. A linear relationship between ascorbic acid retention and processing temperature was found.     

Steam blanching effect on polyphenoloxidase,peroxidase and colour of mango (Mangifera indica L.) slices

The heat stability of peroxidase (POD) and polyphenoloxidase (PPO) was investigated in mango (Mangifera indica L.) slices, and the relative colour was studied after different steam blanching times. There was complete inactivation after 5 min for POD and 7 min for PPO. Steam blanching of 3 min gave residual activity of 2.85% and 8.33% for PPO and POD, respectively, and when compared with samples blanched for 5 min had no effect on colour over 20 days of storage. Correlation was found between activities of PPO, POD and colour change over 20 days. After 7 min steam blanching the browning index was stable but less than at 3 and 5 min because non-enzymic browning had occurred. This research suggests that yellowness (b) and lightness (L) values contribute positively to the browning index (BI), compared to redness (a).     

Effects of gamma irradiation on bio-chemical and physico-chemical parameters of fresh-cut red beet (Beta vulgaris L. var. conditiva) root

Red beet (Beta vulgaris L. var. conditiva) root is a popular item present in ready to eat salads and minimally processed foods. In this research, the effect of low doses of gamma radiation (1 and 2 kGy) on peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities, as well as on the levels of compounds related to the response to the oxidative stress of plant metabolism, the changes in colour and the mechanical behaviour of fresh-cut red beet root were analyzed, with the purpose of understanding the influence of the processing on tissue characteristics. Cell wall modifications were also studied through sequential extractions of polysaccharides from the alcohol-insoluble residues (AIR) obtained from each tissue. Irradiation seemed to contribute to higher cell–cell adhesion through increasing of calcium-cross linking at the middle lamellae regions, in addition to an increment of cross-links of polymers into the cell wall. Chemical modifications produced in the cell walls as a response to higher levels of H₂O₂ and subsequent POX mediated effects, were visualized structurally as a more elastic behaviour of irradiated tissues and rigidification of cell walls of treated roots, though puncture test did not reveal significant differences. Microscopy showed a continuum of thick cell walls in beet root tissue, which suffered slight modifications after irradiation, coherent with the bio-chemical results obtained.     

Inactivation kinetics and secondary structural change of PEF-treated POD and PPO

Effects of pulsed electric fields (PEF) on the activity of peroxidase (POD) and polyphenol oxidase (PPO) in buffered solution were studied while the corresponding changes to their secondary structures was demonstrated by far-UV Circular dichroism (CD). The relative residual activity of POD and PPO decreased with the increase in electric field strength and treatment time, and PPO was more susceptible than POD to PEF treatment. The greatest reduction of the activity was achieved for POD at 25 kV/cm for 1740 μs and PPO at 25 kV/cm for 744 μs with reductions of 32.2% and 76.2%, respectively. The inactivation kinetic parameters D-value and Z_E value were calculated. The D-values of PPO were smaller than those POD at higher electric field strength, and Z_E values of POD and PPO were 36.9 and 16.2 kV/cm, respectively. The secondary structures of the two enzymes were changed following treatment by PEF. The intensity of negative peaks in the CD spectra decreased, and the CD spectra of PPO changed more significantly than that of POD; the reduction of the relative α-helix fractions for POD at 25 kV/cm for 124 μs was 22.63% while it was 50.72% for PPO at 25 kV/cm for 52 μs. The inactivation of PEF-treated POD and PPO was in close agreement with their secondary structure changes.     

Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape (Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes (Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K_m and V_max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD_400 nm/min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E_a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.     

Polyphenol oxidase from bayberry (Myrica rubra Sieb. et Zucc.) and its role in anthocyanin degradation

Polyphenol oxidase (PPO) was extracted from bayberry (Myrica rubra Sieb. et Zucc. cv. Biqi), and its partial biochemical characteristics were studied. Stable and highly active PPO extracts were obtained using insoluble polyvinylpolypyrrolidone (PVPP) in sodium phosphate, pH 7.0, buffer. The highest PPO activity was observed in the ripe fruits. Optimum pH and temperature for bayberry PPO activity were pH 6.0 and T = 30 °C with 0.1 M catechol as substrate. PPO showed activity using the substrates of catechol, gallic acid and protocatechuic acid, but no activity with the substrates (+)-catechin, p-coumaric acid or caffeic acid. K_m and V_max values were 313 mM and 3.26ΔOD/min/g FW, respectively, with catechol as the substrate. Bayberry PPO did not act directly on cyanidin 3-glucoside but the rate of anthocyanin degradation was stimulated by the addition of gallic acid.     

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.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

Thermal inactivation of Salmonella spp. during conching

Although chocolate is a microbiologically stable product it has been described as a vehicle for Salmonella spp. Because of the low water activity (a_w) and the high fat content of chocolate Salmonella spp. shows an increased heat resistance, even during the thermal process of chocolate making. The aim of this study was to evaluate the thermal inactivation of Salmonella spp. during conching in various masses of chocolate and cocoa butter at different temperatures (50-90 °C). The effect of thermal treatment on Salmonella spp. was determined with the MPN (Most-Probable-Number) method. Results of thermal treatment showed approximate D-values for cocoa butter from D_50°C = 245 min to D_60°C = 306 min, for cocoa liquor from D_50°C = 999 min to D_90°C = 26 min and for dark chocolate of D_50°C = 1574 min. z-values were found to be z = 20 °C in cocoa liquor and z = 14 °C in dark chocolate. This study demonstrates that the conching process alone does not ensure the inactivation of Salmonella spp. in different chocolate masses and that an additional decontamination step at the beginning of the process as well as an HACCP concept is necessary during chocolate production to guarantee the absence of Salmonella spp. in chocolates and related products.     

Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris)

Characterisation and tissue distribution of polyphenol oxidase (PPO) was studied in deepwater pink shrimp (Parapenaeus longirostris) post mortem. PPO activity was the highest in the carapace, followed by that in the abdomen exoskeleton, cephalotorax, pleopods and telson. No PPO activity was found in the abdomen muscle and in the pereopods and maxillipeds using the enzymatic assay. Storage of whole shrimps and of the different organs showed that melanosis (blackening) required the presence of the cephalotorax to be initiated, indicating that its development depends on other factors in addition to the PPO levels. Further characterisation was carried out in extracts partly purified using 40–70% ammonium sulfate fractionation. The enzyme had the highest activity at pH 4.5 and was most stable at pH 4.5 and 9.0. No clear maximum was observed in the 15–60 °C range but the higher stability was achieved at 30–35 °C. Apparent kinetic constants in the partly purified PPO from carapace were K_M = 1.85 mM and V_max = 38.5 U/mg of protein, pointing to a high affinity and reactivity of the enzyme when assayed with DOPA. Electrophoretic mobility was studied in native PAGE and non-reducing SDS–PAGE followed by staining with DOPA. Approximate MW of 500 kDa and 200 kDa were observed, respectively. These two forms could correspond to aggregates of minor PPO subunits that could not be resolved in these electrophoretic systems. The peptide mass fingerprinting obtained by MALDI-TOF analysis showed some peptides whose homology with hemocyanins and different PPO subunit precursors has already been demonstrated in the same species.     

Dual mode diffusion and sorption of sodium chloride in pork meats under cooking conditions

This study aims to obtain insight into mechanisms of NaCl diffusion in pork meats under cooking conditions: the loins at 5 (raw), 63 (pre-cooked) and 98 °C (pre-cooked), the mince at 98 °C (pre-cooked), and the filet at 98 °C (pre-cooked). It has been generally presumed that NaCl in any of pork meats diffuses with a constant Fick's diffusion coefficient, D, through liquid water channel imbibed in them. However in the present study, we experimentally obtained skewed bell shape variations of D in all of the above meats with respective maxima at certain low NaCl concentrations. These variations were interpreted in terms of a dual mode sorption and diffusion theory, which had been successfully applied to NaCl diffusion behaviors in Japanese radish and solidified egg white. This interpretation gives a thermodynamic diffusion coefficient, D_T(p) for the partition species of NaCl and another one, D_T(L) for the Langmuir type sorption species, both in the water swollen substrates in the meats. It was found that D_T(p) values are sizably smaller than corresponding D_T(L) values. This difference was ascribed to the lower water content in the p region than that in the L region. With the two D_Ts and additional equilibrium parameters, the theory explained the remarkable decrease of D value with C at 21 °C found by Guiheneuf et al. and nearly constant D values in the higher C range at 5 °C reported by other researchers. Experimentally obtained sorption isotherms of NaCl, which were slightly convex upward in the low C range, were satisfactorily reproduced with the parameters and the fractions of water swollen substrates in the whole meats.     

Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless)

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from a table grape (Crimson Seedless) using Triton X-114 and characterized using spectrophotometric methods. Both PPO and POD were activated by acid shock. However, in the presence of the anionic detergent sodium dodecil sulphate (SDS), PPO was activated whereas POD was inactivated. The enzymes were kinetically characterized and both followed Michaelis–Menten kinetics, although with different values of their kinetic parameters. The V_m/K_m ratio showed that Crimson Seedless grape PPO presents a similar affinity for 4-tert-butyl-catechol (TBC) whether activated by acid shock (0.018 min⁻¹) or SDS (0.023 min⁻¹). With regards to POD, the K_m and V_m values for 2,2′-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) were 0.79 mM and 1.20 μM/min, respectively. In the case of H₂O₂, the K_m and V_m value were 0.4 mM and 0.93 μM/min, respectively. PPO and POD showed similar thermostability, losing >90% of relative activity after only 5 min of incubation at 78 °C and 75 °C, respectively. In addition, PPO´s activation energy was similar to that obtained for POD (295.5 kJ/mol and 271.9 kJ/mol, respectively).     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Inactivation kinetics of Bacillus spores in batch- and continuous-heating systems

Kinetic parameters for the thermal inactivation of Geobacillus stearothermophilus ATCC 7953 and Bacillus flexus 1316 spores were determined for temperatures ranging from 100 to 130 °C and 100 to 125 °C, respectively. Ringer's solution (pH = 7.1) was used as the heating medium. A batch-heating system, in which the samples are kept in small tubes that are heated with steam, and a continuous-heating system, which is based upon a heat exchanger and enables high temperature short time heating, were used. Experiments were conducted in both systems and the heat resistances of the two species were determined. Additionally, a comparison of the two heating systems was carried out. The reaction rate constant at the reference temperature, k_ref, the activation energy, E_a, the D-value and the z-value were calculated for the two species. The D-values at 121 °C for G. stearothermophilus and for B. flexus in the batch-heating system were found to be 42 and 4.2 s, respectively. The z-values were calculated as 13 K for G. stearothermophilus and 16 K for B. flexus in the batch-heating system. The results from both systems differed significantly, wherein the continuous-heating system had been more lethal than the batch-heating system.     

Theoretical analysis on pulsed microwave heating of pork meat supported on ceramic plate

Theoretical analysis has been carried out to study the role of ceramic plates (alumina and SiC) and pulsed microwave heating of pork meat (Pork Luncheon Roll (PLR) and White Pudding (WP)) samples. Spatial hot spots occur either at the center of the sample or at the outer face or at the face attached with alumina plate and application of pulsing minimizes formation of hot spots within meat samples. Pulsing of microwave is characterized by set point for temperature difference (ΔT_S) and on–off constraints for temperature (T′). It is found that alumina plate with higher ΔT_S and lower T′ may be recommended for thick meat samples (both WP and PLR) whereas for thin meat samples, lower ΔT_S with alumina plate/without plate may be preferred. It is also observed that SiC plate may be selectively used with ΔT_S = 20 K for both the pork meats. The distributed microwave incidence is found to be effective due to lesser degree of thermal runaway in absence of pulsing for both meat samples.     

Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D_MS value (decimal reduction time value) of C. sakazakii in standard conditions (35 °C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D_MS = 0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D_MS = 0.61 min), Listeria monocytogenes (D_MS = 0.86 min), and Enterococcus faecium (D_MS = 1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37 °C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D_MS values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D_MS values was found, the relationship between static pressure and D_MS values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D_MS values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.     

Thermal inactivation kinetics of Rabdosia serra (Maxim.) Hara leaf peroxidase and polyphenol oxidase and comparative evaluation of drying methods on leaf phenolic profile and bioactivities

Inactivation kinetics of peroxidase and polyphenol oxidase in fresh Rabdosia serra leaf were determined by hot water and steam blanching. Activation energy (52.30 kJ mol⁻¹) of polyphenol oxidase inactivation was higher than that (20.15 kJ mol⁻¹) of peroxidase. Water blanching at 90 °C or steam blanching at 100 °C for 90 s was recommended as the preliminary treatment for the retention of phenolics. Moreover, comparative evaluation of drying methods on the phenolics profiles and bioactivities of R. serra leaf were conducted. The results indicated that only intact leaf after freeze drying retained the initial quality. The sun- and air-dried leaves possessed identical phenolic profiles. The homogenised leaf (after freeze-drying) possessed a lower level of phenolics due to enzymatic degradation. Good antioxidant activities were detected for the sun- and air-dried leaves. There was insignificant difference in anti-tyrosinase and anti-α-glucosidase activities among sun-, air-, and freeze-dried leaves.