A comparative study of polyphenoloxidases from taro (Colocasia esculenta) and potato (Solanum tuberosum var. Romano)

Taro (C. esculenta) is a staple food in many tropical regions. A comparative study of crude polyphenoloxidases from taro (tPPO) and potatoes (pPPO) was carried out to provide information useful for guiding food processing operations. Crude PPO was prepared by cold acetone precipitation using ascorbic acid as antioxidant. The PPO content of taro acetone powder was 770±17 units (mg protein)⁻¹ as compared with 3848±180 units (mg protein)⁻¹ in potato acetone powder. The pH-activity optimum was pH 4.6 for tPPO and pH 6.8 for pPPO. Both enzymes retained >80% activity after incubation at pH 4.5–8 but there was rapid activity loss at pH < 4. The temperature-activity optimum (T_opt) was 30°C for tPPO and 25°C for pPPO with 75 and 27% of their respective maximum activity retained at 60°C. Both tPPO and pPPO were irreversibly inactivated by 10 min heating at 70°C. The activation enthalpy (ΔH^#) and activation entropy (ΔS^#) for tPPO heat-inactivation were 87.4 (±0.1) kJ mol⁻¹ and −56.2 (±4) J mol⁻¹ K⁻¹, respectively. For pPPO, ΔH^# was 59.1 (±0.1) kJ mol⁻¹ whilst ΔS^# was −141 (±4) J mol⁻¹ K⁻¹. The apparent substrate specificity was established from values V_max/K_m as: 4-methylcatechol>chlorogenic acid>dl-dopa>catechol>pyrogallol> dopamine>>caffeic acid for tPPO. There was no detectable activity towards caffeic acid. The substrate specificity for pPPO was: 4-methylcatechol>caffeic acid>pyrogallol>catechol>chlorogenic acid >dl-dopa>dopamine. According to the order of inhibitor effectiveness (sodium metabisulphite>ascorbic acid>NaCl≈ (EDTA), there was a significant lag-phase before increases occurred in the absorbance at 420 nm. Preincubation of PPO with inhibitors increased the extent of inhibition, indicating a direct effect on the structure of the enzyme.     

REVISED CONFORMATIONAL STABILITY PARAMETERS FOR BETA-LACTOGLOBULIN ISOTHERMAL DENATURATION BY UREA

Isothermal unfolding studies for beta‐lactoglobulin (β‐Lg) usually apply a denaturation model suitable for a single submit protein. A method is described for correcting such results in retrospect. This leads to accurate estimates for the Gibbsfree energy of stabilization of native β‐Lg aimer. The denaturation of β‐Lg aimer is described by a dissociation coupled unfolding (DCU) process; Dimer → monomer → unfolded state. The Gibbs free energy change for DCU (ΔG°_DCU) was 80 kJ mol^?1 at pH 7 and 97 kJ mol^?1 for β‐Lg at pH 2.6. With the addition of a range of salts (pH 6.85) then ΔG°_DCU increased to 105 kJ mol^?1. The literature is replete with denaturation studies employing β‐Lg as the principal protein. In many instances, it would be prudent to reevaluate stability data for β‐Lg in line with techniques outlined here.     

The initial stage of high‐pressure induced β‐lactoglobulin aggregation: the long‐run simulation

Research objective of this study was to clarify how the initial stage of high‐pressure induced aggregation of β‐lactoglobulin takes place. For this purpose a special simulation method was developed. Distinctive features of this approach are: the lowest resolution model of protein particles, the local interaction potential and the abandonment of the continuous simulation of particle trajectories. Relatively short molecular dynamics trajectories are used only in order to find the average time step between the collisions. Results of particle collisions that occur with some probability, are separately (‘statically’) modelled using a random variable. This allows the analysis of the process which takes place within 10²–10³ s real‐time, with an existing probability of successful collision of 10^?10–10^?11. Modelling results confirm that at the initial stage of aggregation of 0.5–2% solutions with a neutral pH‐value only dimers as well as trimers arise due to SH/S–S interaction. In addition it was shown that aggregation follows the general principles of the reaction‐limited aggregation of colloids. The proposed approach could further be used in research projects examining the aggregation of β‐lactoglobulin or similar systems.     

Spectroscopic and molecular docking studies of the interactions of sunset yellow and allura red with human serum albumin

Binding interactions of human serum albumin (HSA) with sunset yellow (SY) and allura red (AR), two food colorants, were investigated at the molecular level through fluorescence and UV absorption as well as molecular docking. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the two dyes and HSA. When compared with AR (293 K: Ksv = (4.21 ± 0.36) × 10⁴ L·mol⁻¹; K_b = (0.30 ± 0.23) × 10⁶ L·mol⁻¹), SY (293 K: Ksv = (6.80 ± 0.10) × 10⁴ L·mol⁻¹; K_b = (3.11 ± 2.01) × 10⁶ L·mol⁻¹) had stronger quenching ability and higher affinity for HSA due to less steric hindrance. It can be deduced that the energy transfer from HSA to the two dyes occurred with high probability based on the Förster resonance energy transfer theory (r < 7 nm, 0.5 R₀ < r < 2.0 R₀). The spectral analysis suggested that the formation of the dye-HSA complex resulted in the change in microenvironment around Tyr and Trp residues and in the secondary structure of the protein. According to molecular docking simulation, the two structural analogs almost bound to the same site of HSA, near Sudlow's Site I, but significant difference existed in the number and location of hydrogen bond (H-bond) formed between the dyes and HSA. From the molecular docking along with the thermodynamic parameters (AR: ΔH^o = −(58.79 ± 15.24) kJ·mol⁻¹, ΔS^o = −(115.1 ± 31.10) J·mol⁻¹·K⁻¹; SY: ΔH^o = −(52.24 ± 3.15) kJ·mol⁻¹, ΔS^o = −(50.07 ± 11.14) J·mol⁻¹·K⁻¹), it could be inferred that H-bond and van der Waals forces were the major binding forces involved in formation of the dye-HSA complexes.     

Effects of chloride,thiocyanate and sulphate salts on β‐lactoglobulin–pectin associative complexes

Protein–polysaccharide complexes are used to improve protein stability and encapsulate high‐value ingredients, yet the influence of different salts on their formation has not been investigated. Using light scattering and turbidimetry, effects of chloride, sulphate and thiocyanate salts on β‐lactoglobulin and pectin complexes (protein/pectin ratio = 2:1 and 4:1) were determined in relation to effects of pH and ionic strength. Effects of anions on complex formation were significant at 25 mmol kg^?1 added ionic strength. Cation effects were not significant. At 100 mmol kg^?1 ionic strength, pH of complex formation increased with sulphate salts (pH 5.1) relative to chloride and thiocyanate salts (pH 4.9), while pH of coacervation increased with sulphate salts (pH 4.7) and decreased with thiocyanate salts (pH 4.4) relative to chloride salts (pH 4.6). Pure β‐lactoglobulin stability was otherwise reduced with thiocyanate salts below pH 5, implying a significant effect of pectin interactions.     

Kinetic and thermodynamic investigation of mancozeb degradation in tomato homogenate during thermal processing

BACKGROUND: The kinetic and thermodynamic parameters of mancozeb degradation in tomato homogenates under the conditions prevailing in the manufacture of tomato products (at 60–100 °C for 0–60 min) were investigated. A gas chromatography–mass spectrometry method was used to analyse residual mancozeb in tomato homogenate. Ethylenethiourea (ETU), the main toxic degradation product of mancozeb, was measured by high‐performance liquid chromatography (HPLC)–with photodiode array detector (PDA). RESULTS: The degradation of mancozeb and the formation of ETU in tomato homogenates were adequately described as first‐order kinetics. Dependence of the rate constant followed the Arrhenius relationship. Apparent activation energies, temperature coefficients, half time and time to reduce to 90% of the initial value of mancozeb were calculated as kinetic parameters. The thermodynamic parameters of mancozeb were also described as Δg^d = ? 2.440 and 7.074 kJ mol^?1; Δh^d = ? 32.555 and ? 42.767 kJ mol^?1; Δs^d = ? 0.090 and ? 0.150 kJ mol^?1 K^?1; K_e = 0.414 and 9.797 L g^?1 for 333 and 373 K respectively. CONCLUSION: Current findings may shed light on the reduction of mancozeb residue and its toxic degradation product during thermal processing of tomatoes and may also be valuable in awareness and prevention of potential risks from dietary exposure. Copyright © 2011 Society of Chemical Industry     

Aqueous solubility of calcium citrate and interconversion between the tetrahydrate and the hexahydrate as a balance between endothermic dissolution and exothermic complex formation

Aqueous solubility of calcium citrate tetrahydrate was found to decrease with increasing temperature, while solubility of hexahydrate increased with a transition temperature at 51.6 °C. Excess citrate increased calcium citrate solubility but decreased the calcium ion activity of the saturated solution with an initial solubility overshooting to form supersaturated solutions indicating binding of calcium to citrate with an association constant of 3.6 ± 0.1 × 10⁴, ΔHº = −5.07 ± 0.04 kJ mol⁻¹, ΔSº = 70.3 ± 0.3 J mol⁻¹ K⁻¹ at 25 °C. Dissolution of the tetrahydrate and hexahydrate was found to have ΔHº = 27 ± 9 kJ mol⁻¹, ΔSº = −218 ± 30 J mol⁻¹ K⁻¹ and ΔHº = 57 ± 7 kJ mol⁻¹, ΔSº = −126 ± 24 J mol⁻¹ K⁻¹, respectively, as determined from the temperature dependence of solubility corrected for complex formation. The exothermic complex formation results in inverse solubility only for the tetrahydrate with its moderate endothermic dissolution, which also precipitates at ambient temperature rather than the less soluble hexahydrate.     

Lysyl oxidase from jumbo squid (Dosidicus gigas) muscle: purification and partial characterization

Lysyl oxidase (LOX; E.C.1.4.3.13) was purified from jumbo squid muscle (Dosidicus gigas) with 1900‐fold and yield 1.9%, and characterized for the first time. The purification procedure consisted of fractionation with urea and a combination of size‐exclusion and anion‐exchange chromatography. The enzyme had a molecular weight of 32 kDa, as estimated by SDS‐PAGE. Using a specific LOX substrate (1,5‐diaminopentane), its optimum activity was determined at pH 8.2 and 65 °C. Activation energy (E_a) of the enzyme was 69.94 kJ K^?1 mol^?1. The enzyme was strongly inhibited by β‐aminopropionitrile fumarate (BAPN), a specific LOX inhibitor. Moreover, purified LOX was able to work at different temperatures (20–90 °C) at pH 8.2. Although further research is needed, the results from this work suggest that based on LOX activity, this enzyme may be of practical use in preventing textural changes in jumbo squid during storage or processing.     

Kinetic behaviour and thermal inactivation of pectinlyase used in food processing

Kinetic properties and thermal inactivation of pectinlyase (PL) were assayed in commercial pectinase preparations (Rapidase C80, Pectinase CCM, Pectinex 3XL and Grindamyl 3PA) by using apple pectin as substrate. The PL activity of Rapidase C80 showed substrate inhibition, while the other enzyme preparations followed typical Michaelis–Menten kinetics. The optimum pH and temperature values for PL activity lay within the range of 5.5–6.5 and 35–40 °C, respectively. PL was heat‐inactivated with simple first order kinetics. With respect to this, thermodynamic activation parameters (ΔH^#, ΔS^# and ΔG^#) using a non‐linear Arrhenius plot were calculated. The Pectinase CCM and Pectinex 3XL PL showed a half‐life (t_1/2) at 50 °C of 2.0 min and 11.8 min, respectively.     

Beta-lactoglobulin denaturation by dissociation-coupled unfolding

Many food proteins have multiple subunits. Beta-lactoglobulin (β-lg) dimer was assessed for its resistance to subunit dissociation and unfolding in 0–8 M urea. Equilibrium denaturation profiles were monitored by ultraviolet difference spectrophotometry. The results were analysed by a new dissociation coupled unfolding (DCU) model. The Gibbs free energy change for denaturing β-lg (ΔG_DCU⁰) was 60 (±2.3) kJ mol⁻¹ at pH 7 and 72 (±1.6) kJ mol⁻¹ at pH 2.6. By comparison, the dissociation free energy for β-lg dimer (ΔG_D) is 26.0 kJ mol⁻¹ at pH 7 and 22.6 kJ mol⁻¹ at pH 2.6. Hence, dimerization accounts for 43% (pH 7) and 32% (pH 2.6) of the stability of native β-lg dimer. Such results indicate a 3-state denaturation process in urea with native β-lg monomer acting as a stable intermediate during DCU. The issues raised by a study of β-lg may be relevant to other multisubunit proteins in food systems.     

Production,partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger

In order to achieve a higher added value of two galactomannan‐containing wastes, copra paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of producing β‐mannanase by SSF. A 2³ factorial design was used to select the best interaction among the two fungi, the two substrates and two fermentation times. The treatment ‘A niger–copra–2.5 days’ produced a significantly higher (p < 0.05) β‐mannanase activity, having five different isoforms of the enzyme, one of which was partially purified to a specific activity of 764 U mg⁻¹ (U = nmol of mannose released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose ratio (14:1) than SCW (6:1), and low oil content, which led to higher β‐mannanase production from SSF. A β‐mannanase from SSF of copra produced by A oryzae was highly purified using acetone precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of 110 kDa, a pI between 3.5 and 4.5 and a specific activity of 1760 U mg⁻¹; purification achieved was 90.7 times. The temperature and pH for optimal activity were 40 °C and 6.0 respectively. The optimal temperature was lower and the optimal pH higher than others previously reported (produced by submerged fermentation), which could be important for viscosity reduction of concentrated coffee extract in instant coffee manufacture. Copra is an interesting alternative for β‐mannanase production, since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan content and may be used for monogastric animal feed. © 2000 Society of Chemical Industry     

Urea and Heat Unfolding of Cold-Adapted Atlantic Cod(Gadusmorhua)Trypsin and Bovine Trypsin

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypins from Atlantic cod (cod PMS-trypsin) and cattle (bovine PMS-trypsin) were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. For urea unfolding at 25°C, the free energy change at zero concentration of urea (ΔG(H₂O)) for cod PMS-trypsin was 11(±4·4) kJ mol⁻¹ compared with 18(±1·14) kJ mol⁻¹ for bovine PMS-trypsin, while the mid-point concentration for urea unfolding curve ([urea]_1/2) was 3·0(±0·57) M and 4·1(±0·16) M, respectively. From studies of enzyme heat unfolding, the mid point temperature of the thermal unfolding curve ( T _m ) was 46(±1·4)°C for cod PMS-trypsin compared with 57(±2)°C for bovine PMS-trypsin. The standard free energy change (Δ G° ) for reversible thermal unfolding of cod PMS-trypsin was 9(±1) kJ mol⁻¹ compared with 19(±1) kJ mol⁻¹ for bovine PMS-trypsin. Values for the enthalpy (Δ H _m ), entropy (Δ S _m ) and heat capacity (Δ C _p ) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a significantly lower conformational stability than bovine PMS-trypsin.     

Moisture sorption isotherms and thermodynamic properties of apple Fuji and garlic

The moisture equilibrium isotherms of garlic and apple were determined at 50, 60 and 70 °C using the gravimetric static method. The experimental data were analysed using GAB, BET, Henderson–Thompson and Oswin equations. The isosteric heat and the differential entropy of desorption were determined by applying Clausius–Clapeyron and Gibbs–Helmholtz equations, respectively. The GAB equation showed the best fitting to the experimental data (R² > 99% and E% < 10%). The monolayer moisture content values for apple were higher than those for garlic at the studied temperatures; the values varied from 0.050 to 0.056 and from 0.107 to 0.168 for garlic and apple, respectively. The isosteric heat and the differential entropy of desorption were estimated in function of the moisture content. The values of these thermodynamic properties were higher for apple (in range 48–100 kJ mol^?1 and 14–150 J mol^?1 K^?1) than for garlic (in range 43–68 kJ mol^?1 and 0–66 J mol^?1 K^?1). The water surface area values decreased with increasing temperature. The Kelvin and the Halsey equations were used to calculate the pore size distribution.     

Characterization of polyphenoloxidase from 2 peach (Prunus persica L.) varieties grown in Argentina

Polyphenoloxidase was extracted from September peach (_SEPPO) and Summerset peach (_SUPPO) and its physicochemical characteristics were analyzed. The optimum pH was 6.5 for _SEPPO and 5.5 for _SUPPO. The optimum temperature was 35°C for _SEPPO and 39.4°C for _SUPPO. Activation energy (Ea) from thermal activation was 41.5 kJ/mol for _SEPPO and 37.5 kJ/mol for _SUPPO. Heating at 60°C by 5 min, _SUPPO was denatured whereas _SEPPO retained 2.6% of activity. Activation enthalpy (ΔH^#) and activation entropy (ΔS^#) for _SEPPO heat-inactivation were 69.9 J/mol and −83.5 kJ/mol·K for _SUPPO, ΔH^# was 91.8 J/mol while ΔS^# was −21.0 kJ/mol·K. Substrate specificity (V_max/K_M) was 4-methylcatechol>catechol>pyrogallol for _SEPPO and 4-mehtylcatechol>pyrogallol>catechol for _SUPPO. For both enzymes, the order of inhibition effectiveness using reductor agents was metabisulphite>ascorbic acid. Benzaldehyde, 4-hydroxybenzaldehyde, and dl-dopa were competitive inhibitors, and their K_I values were 38.86, 8.43, and 2.08 mM, respectively.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Purification and some properties of a novel raw starch‐digesting amylase from Aspergillus carbonarius

A medium was developed to obtain the maximum yield of raw starch‐digesting amylase from Aspergillus carbonarius (Bainier) Thom IMI 366159 in submerged culture with raw starch as the sole carbon source. The amylase was purified to apparent homogeneity by sucrose concentration and ion exchange chromatography on S‐ and Q‐Sepharose (fast flow) columns. SDS‐PAGE revealed two migrating protein bands corresponding to relative molecular masses of 31.6 and 32 KDa. The enzyme was optimally active at pH 6.0–7.0 and 40 °C, was uninfluenced across a relatively broad pH range of 3.0–9.0 and retained over 85% activity between 30 and 80 °C after 20 min incubation. The enzyme was strongly activated by Co²⁺ and only slightly by Fe²⁺, while Ca²⁺, Hg²⁺, EDTA and N‐bromosuccinamide elicited significant repression of the enzyme activity. The enzyme hydrolysed amylopectin (K_m 0.194 mg ml ⁻¹), glycogen (K_m 0.215 mg ml ⁻¹), pullulan (K_m 0.238 mg ml ⁻¹), amylose (K_m 0.256 mg ml ⁻¹) and raw potato starch (K_m 0.260 mg ml ⁻¹), forming predominantly maltose and relatively smaller amounts of glucose. © 2000 Society of Chemical Industry     

Phytosterol content in seven oat cultivars grown at three locations in Sweden

The total and individual sterol content in 21 oat samples (seven cultivars grown at three different locations in Sweden) were analysed by gas chromatography after acid hydrolysis. The total sterol content in these oat cultivars varied between 350–491 µg g⁻¹ of dry weight of kernel. The most abundant phytosterol was β‐sitosterol (237–321 µg g⁻¹) followed by campesterol (32–46 µg g⁻¹), Δ⁵‐avenasterol (15–47 µg g⁻¹) and stigmasterol (11–21 µg g⁻¹). There was a statistically significant difference in total sterol content between cultivars (p < 0.05) but no effect was found for cultivation location. Furthermore when contents of Δ⁵‐avenasterol in hexane‐extracted oat oil and acid‐hydrolysed oat samples were compared, it was noticed that the content of Δ⁵‐avenasterol was lowered due to acid hydrolysis. © 1999 Society of Chemical Industry     

The effect of pH and calcium ion on rheological behaviour of β‐lactoglobulin‐basil seed gum mixed gels

Effect of pH (4.5–7.5) and Ca²⁺ (0.01–0.5 m ) on gelation of single and mixed systems of 10% β‐lactoglobulin (BLG) and 1% basil seed gum (BSG) was investigated. The gelling point of BLG and BSG gels was strongly pH‐dependent, and stiffer gels formed at higher pH. The BLG gels were formed upon heating to 90 °C and reinforced on cooling to 20 °C; however, the gelation of BSG occurred at temperatures below 70 °C. By increasing Ca²⁺ concentration, storage modulus of BLG and BSG gels were increased, although pH had a greater effect than Ca²⁺. In contrast, mixed systems showed two distinct types of behaviour: BLG gel formation and BSG network, suggesting that phase‐separated gels were formed. In addition, higher strength was obtained for BLG‐BSG mixture at higher Ca²⁺ concentration.     

Oxidatively induced chemical changes and interactions of mixed myosin, β‐lactoglobulin and soy 7S globulin

The effects of oxidation on the chemical characteristics of myosin, β‐lactoglobulin and soy 7S globulin were investigated in a free radical‐generating system (FeCl₃/H₂O₂/ascorbate). Oxidised myosin exhibited a marked increase (>10‐fold) in carbonyls, a small increase in amines and conversion of some free sulphhydryls to disulphide bonds. Oxidised β‐lactoglobulin and 7S globulin also showed a major increase in the carbonyl content (five‐ and threefold respectively), but other chemical changes were relatively minor. In the oxidised myosin/β‐lactoglobulin composites, some cross‐linked aggregates were formed. Aggregation was also evidenced in the myosin/7S globulin composites exposed to the oxidising agents. The results indicated that oxidation enhanced interactions of the non‐muscle proteins with myosin apparently through the modification of amino acid side chains. These physicochemical changes may influence the functionality of processed muscle foods. © 2000 Society of Chemical Industry     

Determination of some biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir)

Polyphenol oxidase (PPO) was extracted from Emir grapes grown in Turkey and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. The optimum pH and temperature for grape PPO were found to be 4.2 and 25 °C respectively using catechol as substrate. K_m and V_max values were found to be 25.1 ± 2.72 mmol L⁻¹ and 0.925 ± 0.04 OD₄₁₀ min⁻¹ respectively. Of the inhibitors tested, the most potent was sodium metabisulfite, followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and Z values were calculated as 251.4 kJ mol⁻¹ (r² = 0.996) and 8.92 °C (r² = 0.993) respectively. Copyright © 2006 Society of Chemical Industry