Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation

A dimeric serine protease Neriifolin S of molecular mass 94 kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (K_m of 0.195 mM) and physiological efficiency (K_cat/K_m of 144.5 mM s). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45 °C. Its proteolytic activity is strongly stimulated in the presence of Ca⁺² ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent T_m, was found to be 65 °C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54 kJ mol⁻¹, 24.89 kJ mol⁻¹, −82.34 kJ mol⁻¹ and 337.20 J mol⁻¹ K⁻¹.     

Degradation kinetics of anthocyanins in acerola pulp: Comparison between ohmic and conventional heat treatment

Degradation kinetics of monomeric anthocyanins in acerola pulp during thermal treatment by ohmic and conventional heating was evaluated at different temperatures (75–90 °C). Anthocyanin degradation fitted a first-order reaction model and the rate constants ranged from 5.9 to 19.7 × 10⁻³ min⁻¹. There were no significant differences between the rate constants of the ohmic and the conventional heating processes at all evaluated temperatures. D-Values ranged from 116.7 to 374.5 for ohmic heating and from 134.9 to 390.4 for conventional heating. Values of the free energy of inactivation were within the range of 100.19 and 101.35 kJ mol⁻¹. The enthalpy of activation presented values between 71.79 and 71.94 kJ mol⁻¹ and the entropy of activation ranged from −80.15 to −82.63 J mol⁻¹ K⁻¹. Both heating technologies showed activation energy of 74.8 kJ mol⁻¹ and close values for all thermodynamic parameters, indicating similar mechanisms of degradation.     

Thermal inactivation of lactoperoxidase in goat,sheep and bovine milk – A comparative kinetic and thermodynamic study

Using isothermal heating, inactivation of lactoperoxidase (LPO) in goat, sheep and cow milk was studied in the temperature range of 70–77 °C. Kinetic and thermodynamics studies were carried out at different time–temperature combination in order to evaluate the suitability of LPO as marker for the heat-treatment of milk and dairy products from different species. The thermal inactivation of LPO followed the first-order kinetics. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius and thermal death time models. The corresponding z-values were 3.38 ± 0.013, 4.11 ± 0.24 and 3.58 ± 0.004 °C in goat, sheep and cow milk, respectively. Activation energy values varied between milk species with 678.96 ± 21.43 kJ mol⁻¹ in goat milk, 560.87 ± 28.18 kJ mol⁻¹ in sheep milk and 641.56 ± 13.12 kJ mol⁻¹ in cow milk, respectively.     

Purification and characterization of olive (Olea europaea L.) peroxidase – Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18–20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 °C, and apparent K_m values of 41.0 and 0.53 mM, for phenol and H₂O₂, respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 °C, 411.2 and −13.6 kJ mol⁻¹ K⁻¹. The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol⁻¹, corresponding to a calculated temperature coefficient (Q₁₀) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase.     

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.     

Kinetics of the formation of radicals in meat during high pressure processing

The kinetics of the formation of radicals in meat by high pressure processing (HPP) has been described for the first time. A threshold for the radicals to form at 400 MPa at 25 °C and at 500 MPa at 5 °C has been found. Above this threshold, an increased formation of radicals was observed with increasing pressure (400–800 MPa), temperature (5–40 °C) and time (0–60 min). The volume of activation (ΔV^#) was found to have the value −17 ml mol⁻¹. The energy of activation (E_a) was calculated to be 25–29 kJ mol⁻¹ within the pressure range (500–800 MPa) indicating high independence on the temperature at high pressures whereas the reaction was strongly dependent at atmospheric pressure (E_a = 181 kJ mol⁻¹). According to the effect of the processing conditions on the reaction rate, three groups of increasing order of radical formation were established: (1) 55 °C at 0.1 MPa, (2) 500 and 600 MPa at 25 °C and 65 °C at 0.1 MPa, and (3) 700 MPa at 25 °C and 75 °C at 0.1 MPa. The implication of the formation of radicals as initiators of lipid oxidation under HPP is discussed.     

Properties of polyphenol oxidase from Anamur banana (Musa cavendishii)

Polyphenol oxidase (PPO) was extracted from Anamur banana, grown in Turkey, and its characteristics were studied. The optimum temperature for banana PPO activity was found to be 30 °C. The pH-activity optimum was 7.0. From the thermal inactivation studies, in the range 60–75 °C, the half-life values of the enzyme ranged from 7.3 to 85.6 min. The activation energy (E_a) and Z values were calculated to be 155 kJ mol⁻¹ and 14.2 °C, respectively. K_m and V_max values were 8.5 mM and 0.754 OD₄₁₀ min⁻¹, respectively. Of the inhibitors tested, ascorbic acid and sodium metabisulphite were the most effective.     

Effects of light,temperature and water activity on the kinetics of lipoxidation in almond-based products

Lipoxidation in almond-derived products was investigated using the chemiluminescence (CL) and thiobarbituric acid-reactive substances (TBARS) methods to detect the first and later reaction products, respectively. The effects of light during storage at 5 °C, 22 °C and 40 °C were studied, as well as the effects of combined heat/water activity treatments in the 60–120 °C and 0.38–0.72 range. During storage, light was found to enhance the CL and TBARS values, and specific responses were observed in almond paste and the final Calisson product. During the heating of almond paste, as the initial water activity (a_w) increased, the CL rate constants increased during heating to 60 °C and 80 °C, but interestingly, these values decreased during further heating to 120 °C, whereas the maximum TBARS rate constants occurred at a_w 0.57 at all the heating temperatures tested. The activation energies, based on the CL and TBARS values, decreased specifically when the a_w increased from 0.38 to 0.72, giving overall values ranging from110 kJ mol⁻¹ to 60 kJ mol⁻¹. Likewise, in the same water activity range, the temperature-dependent rate constant enhancing factor (Q₁₀) decreased from 3.3 to 1.6.     

Quantification and characterisation of polyphenol oxidase from vanilla bean

Polyphenol oxidase (PPO) of Vanilla planifolia Andrews beans was extracted and purified through ammonium sulphate precipitation, dialysis, and gel filtration chromatography. PPO activity was measured by improved UV technique using 4-methylcatechol and catechol as substrates increasing substantial sensitivity of previous procedure. The optimum pH and temperature for PPO activity were found to be 3.0 and 3.4 and 37 °C, respectively. K_m and V_max values were found to be 10.6 mM/L and 13.9 OD₃₀₀ min⁻¹ for 4-methylcatechol and 85 mM/L and 107.2 OD₃₀₀ min⁻¹ for catechol. In an inhibition test, the most potent inhibitor was found to be 4-hexylresorcinol followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and z values were calculated as 92.10 kJ mol⁻¹ and 21 °C, respectively.     

Kinetics of formation of acrylamide and Schiff base intermediates from asparagine and glucose

From the concentration of glucose and asparagine as reactants and of acrylamide as product each determined by LC–MS during reaction in an acetonitrile/water (68:32) model system at pH 7.6 (0.04 M phosphate buffer) and from the relative concentration of the Schiff base intermediate, the decarboxylated Schiff base intermediate, the Amadori product and aminopropionamide determined in the same reaction mixtures at 120 °C, 140 °C, 160 °C and 180 °C for up to 16 min, the energy of activation for formation of the Schiff base intermediate was found to have the value 50 ± 2 kJ mol⁻¹, while the apparent activation energy for formation of acrylamide was 64.4 ± 0.6 kJ mol⁻¹, for formation of the decarboxylated Schiff base intermediate 92 ± 2 kJ mol⁻¹, and for formation of the Amadori compound 59 ± 4 kJ mol⁻¹, respectively. At high temperature conditions, formation of the Schiff base is accordingly rate determining, while at lower temperatures, decarboxylation becomes rate determining. Aminopropionamide was only detected at reaction times at which acrylamide formation already is significant in favor of, a reaction path including direct formation of acrylamide from the decarboxylated Schiff base, rather than including dissociation of ammonia from aminopropionamide.     

Morphology and release profile of microcapsules encapsulating peppermint oil by complex coacervation

The gelatin/gum Arabic microcapsules encapsulating peppermint oil were prepared by complex coacervation using transglutaminase as hardening agent. The release of microcapsules was investigated in different dispersing media. The release of microcapsules separately followed first order release kinetics model in hot water and zero order release kinetics model in high-temperature oven. The release of microcapsules in hot water exhibited initial rapid release phase and following slow release phase, where effect of core/wall ratio on the release rate was different due to the increase of loading and particle size with the core/wall ratio. The release kinetics rate constant, K, was analyzed by an Arrhenius plot and found to fit well with a linear relation between in ln(K) and 1/T. The activation energy was evaluated to be 32.6 kJ mol⁻¹. The microcapsules were only release about 7% of peppermint oil during the storage of 40 days in cold water, exhibiting excellent storage stability.     

Application of molecular modelling and spectroscopic approaches for investigating binding of vanillin to human serum albumin

In the present study, the interaction of vanillin and human serum albumin (HSA) has been characterised by molecular modelling, fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic methods. The results of molecular modelling suggested that vanillin was located within the binding pocket of subdomain IIA of HSA mainly by hydrophobic forces. The quenching of HSA fluorescence takes place with a binding constant (K) of 8.8, 7.7, 5.7, 4.2 × 10⁴ M⁻¹ at four different temperatures (288, 298, 308, 318 K), respectively. Meanwhile, the number of binding site (n ≈ 1) was also obtained from fluorescence titration data. The enthalpy change ΔH⁰ and the entropy change ΔS⁰ were calculated to be −20 kJ mol⁻¹ and 5.8 J mol⁻¹ K⁻¹ according to the Van’t Hoff equation. Furthermore, the alterations of protein secondary structure in the presence of vanillin were explored by FT-IR and CD spectra.     

Effects of temperature,solid content and pH on the stability of black carrot anthocyanins

Anthocyanin stability of black carrots was studied at various solid contents (11, 30, 45 and 64° Brix) and pHs (4.3 and 6.0) during both heating, at 70–90 °C, and storage at 4–37 °C. Monomeric anthocyanin degradation fitted a first-order reaction model. Degradation of monomeric anthocyanins increased with increasing solid content during heating, while it decreased during storage. For example, at pH 4.3, half-life periods for anthocyanins at 30, 45 and 64° Brix were, respectively, 8.4, 6.9 and 5.2 h during heating at 80 °C and 18.7, 30.8 and 35.9 weeks during storage at 20 °C. At 30–64° Brix, increasing pH from 4.3 to 6.0 enhanced the degradation of anthocyanins during heating. The effect of pH on thermal stability of anthocyanins was also studied at six different pHs (2.5–7.0) in citrate-phosphate buffer solutions and significant decrease in anthocyanin stability was observed at pHs above 5.0. Higher activation energies (E_a) were obtained during heating than during storage with increasing solid contents. At 30–64° Brix, E_a values ranged from 68.8 to 95.1 kJ mol⁻¹ during heating and from 62.1 to 86.2 kJ mol⁻¹ during storage. Q₁₀ values at 20–37 °C were as high as 3.1 at 45° Brix and 3.6 at 64° Brix.     

Kinetic and thermodynamic study of thermal inactivation of the antimicrobial peptide P34 in milk

The knowledge on thermal inactivation of biopreservatives in a food matrix is essential to allow their proper utilisation in food industry, enabling the reduction of heating times and optimisation of heating temperatures. In this work, thermal inactivation of the antimicrobial peptide P34 in skimmed and fat milk was kinetically investigated within the temperature range of 90–120 °C. The inactivation kinetic follows a first-order reaction with k-values between 0.071 and 0.007 min⁻¹ in skimmed milk, and 0.1346 and 0.0119 min⁻¹ in fat milk. At high temperatures, peptide P34 was less resistant in fat milk, with a significant decrease in residual activity as compared with skimmed milk. At temperatures below 110 °C, the fat globules seem to have protective effect to the peptide P34. Results suggest that peptide P34 is heat stable in milk with activation energy of 90 kJ mol⁻¹ in skimmed milk and 136 kJ mol⁻¹ in fat milk.     

Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices

Naringinase, induced from Aspergillus niger CECT 2088 cultures, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) (PVA) hydrogel, cryostructured in liquid nitrogen, to obtain biocatalytically active beads. The effects of matrix concentration, enzyme load and pH on immobilization efficiency were studied. Between 95% and 108% of the added naringinase was actively entrapped in PVA cryogel, depending on the conditions of immobilization used. The optimal conditions were: 8% (w/v) PVA at pH 7 and 1.6–3.7 U ml⁻¹ of enzyme load. The pH/activity profiles revealed no change in terms of shape or optimum pH (4.5) upon immobilization of naringinase. However, the optimum temperature was shifted from 60 °C to 70 °C and the activation energy of reaction, E_a, was decreased from 8.09 kJ mol⁻¹ to 6.36 kJ mol⁻¹ by immobilization. The entrapped naringinase could be reused through six cycles (runs of 24 h at 20 °C), retaining 36% efficacy for the hydrolysis of naringin in simulated juice.     

Weibull distribution for modeling air drying of coroba slices

Application of Weibull distribution model was investigated for describing the moisture content of coroba slices during air drying. One set of experiments was performed following a full factorial design at three levels for air temperature (71, 82 and 93 °C) and velocity (0.82, 1.00 and 1.18 m/s). The set was designed to assess the adequacy of the Weibull model to describe water losses. The high regression coefficients (R² > 0.99) and low reduced chi-square indicated the acceptability of Weibull model for predicting moisture content. Values of scale parameter ranged from 41.77 to 71.52 (min) and values of shape parameter ranged from 1.06 to 1.21. Temperature sensitivity of scale parameter increased with increasing air velocity from 0.82 m/s (E_a = 210.45 J/mol) to 1.00 m/s (E_a = 214.93 J/mol) and then decreased with increasing velocity to 1.18 m/s (E_a = 139.03 J/mol). The normalized Weibull model was investigated for determining the effective diffusion coefficient (D_e). The D_e values ranged approximately from 2.51 × 10⁻¹² m²/s to 4.27 × 10⁻¹² m²/s.     

Effect of pH on chlorophyll degradation and colour loss in blanched green peas

Effect of pH on the chlorophyll degradation and visual green colour loss in blanched green peas were studied at 70, 80, 90 and 100 °C in buffered solutions of pH 5.5, 6.5 and 7.5. The degradation of chlorophylls a and b followed a first-order reaction and the temperature-dependence of these reactions was modelled by the Arrhenius equation. The activation energies ranged from 4.80 ± 0.91 to 14.0 ± 0.71 kcal mol⁻¹ for chlorophyll a and from 6.84 ± 0.29 to 11.0 ± 1.06 for chlorophyll b with varying pH values. The visual green colour degradation, as represented by the change of the −a (greenness), the ratio −a/b and hue (h) values measured by tristimulus colorimeter, also followed a first-order reaction. Activation energies for −a values ranged from 8.13 ± 0.71 to 12.0 ± 1.07 kcal mol⁻¹, and for −a/b values ranged from 8.77 ± 1.34 to 12.0 ± 1.07 kcal mol⁻¹ with varying pH values at 70, 80, 90 and 100 °C.     

An approximate shelf life prediction of elaborated lager beer in terms of degradation of its iso-α-acids

A 3-l laboratory-scale beer production protocol was designed and developed to produce beer. Concentrations of cis-/trans-iso-α-acids were quantified by HPLC. The trans-/cis-ratio varied from 0.62 to 0.49 after accelerated aging from 4 °C to 50 °C. The degradation of trans-iso-α-acids in elaborated pale lager beers followed first order reaction and this degradation conforms to the Arrhenius equation. The activation energy (E_a) and frequency factor (A) for trans-iso-α-acids in elaborated pale lager beer ranged from 69.2 kJ mol⁻¹ and 1.88 × 10¹⁰ days⁻¹ for trans-isocohumulone to 74.4 kJ mol⁻¹ and 1.3642 × 10¹¹ days⁻¹ for trans-isoadhumulone. The higher value of E_a demonstrated greater temperature sensitivity of trans-iso-α-acids during accelerated storage. The average half life of trans-iso-α-acids in elaborated pale lager beer was found to decrease from 471 days to 12 days when temperature increased from 4 °C to 40 °C. Using the activation energy of trans-α-acid degradation and the temperature profile of the accelerated aging, a mathematical model was employed to predict the loss of iso-α-acids, when the initial concentration of iso-α-acids in the product is known. The results obtained in the investigation can be of great importance to the industry in predicting the alteration of beer bitterness during warm periods and in tropical countries where summer temperature can reach 40 °C.     

Fitting Fick’s model to analyze water diffusion into chickpeas during soaking with ultrasound treatment

Fick’s model together with Arrhenius relationship were successfully used to evaluate water absorption of chickpea during soaking at a temperature range of 20-97 °C with 25 kHz 100 W, 40 kHz 100 W and 25 kHz 300 W ultrasound treatments. Use of ultrasound, increase in ultrasound power and soaking temperature significantly (P < 0.05) increased the water diffusion coefficient (D_eff) of chickpea during soaking. Average gelatinization temperature of chickpea was found as 61.47 °C. Activation energy (E_a) values of chickpea for below and above gelatinization temperature were found to be 28.69 and 9.34 kJ mol⁻¹, respectively. Ultrasound treatments significantly decreased the soaking time of chickpea.     

Water absorption and starch gelatinization in whole rice grain during soaking

Hydration of rough rice grain in hot water as a function of time was studied at temperature range 25-90 °C. A simple model which considers simultaneous unsteady-state water diffusion and first-order irreversible water-starch reaction phenomenon, was used to evaluate the kinetics parameters from experimental curves. The values of the diffusion coefficients and reaction rate constants were between 1.40×10⁻¹¹ and 9.36×10⁻¹¹ m² s⁻¹ and 2.29×10⁻¹⁰ and 3.72×10⁻⁵ s⁻¹, respectively. Both parameters followed a Arrhenius-type equation with distinct activation energies below and above a break temperature of 60 °C. It was 25.4 and 289.3 kJ mol⁻¹ for the activation energies of diffusion and reaction, respectively, below 60 °C. Above this temperature the respective values of the activation energies of diffusion and reaction were 30.0 and 16.6 kJ mol⁻¹. This break temperature was in agreement with the gelatinization temperature determined experimentally.