Analysis of the thermally induced structural changes of bovine lactoferrin.

Bovine lactoferrin (LF) is subjected to thermal processing during isolation for commercial use and while preparing milk products intended for infant nutrition. The present study is focused on the heat-induced structural changes of LF in buffer solution. Fluorescence spectroscopy, molecular modeling, and enzymatic hydrolysis studies were combined to extensively characterize LF thermal behavior. The temperature-induced changes induced on LF conformation were analyzed through intrinsic and ANS fluorescence parameters (intensity, maximum position, and parameter A value), the phase diagram method, and quenching experiments using acrylamide and iodide. A higher exposure of hydrophobic residues was highlighted through the molecular modeling approach, with a decrease in α-helix content from 23.5% to 21.2% when increasing the temperature from 25 °C to 80 °C. The experimental results demonstrate a more flexible conformation of the protein at higher temperature, thus facilitating the enzymatic hydrolysis by thermolysin.

Headspace fingerprinting as an untargeted approach to compare novel and traditional processing technologies: a case-study on orange juice pasteurisation.

As a rule, previous studies have generally addressed the comparison of novel and traditional processing technologies by a targeted approach, in the sense that only the impact on specific quality attributes is investigated. By contrast, this work focused on an untargeted strategy, in order to take into account unexpected and unintended effects of (novel) processing, and to possibly uncover unknown compounds resulting from alternative processing. The potential of headspace GC-MS fingerprinting was explored as a tool to compare the impact of thermal, high pressure (HP) and pulsed electric field (PEF) processing for mild pasteurisation of orange juice. This study demonstrated that when processing conditions are selected based on equivalent microbial safety, the impact of heat, HP and PEF pasteurisation on the volatile profile of orange juice can be considered comparable. During refrigerated storage, however, indirect impact differences were revealed, which were attributed to differences in degree of enzyme inactivation.

High-pressure treatment reduces the immunoreactivity of the major allergens in apple and celeriac.

SCOPE: The impact of thermal and high pressure (HP) processing on the immunoreactivity of the allergens Mal d 1, Mal d 3 and Api g 1 has been investigated in apple and celeriac tissue, respectively. METHODS AND RESULTS: The extracted proteins were assessed using SDS-PAGE and Western blot. The results showed that Mal d 1 was subject to loss of immunoreactivity as soon as the apple tissue was disrupted although it was remarkably resistant to both thermal and HP processing. This is in contrast to the Mal d 1 structural homolog from celeriac, Api g 1, that was susceptible to thermal processing. The other major allergen in apple, Mal d 3, was found to be resistant to chemical modification and thermal processing in apple, which is in contrast to behavior in solution. However, the combination of pressure and temperature significantly reduced its immunoreactivity. Pectin was found to protect Mal d 3 from thermal denaturation in solution and is a possible candidate for the protective effect of the fruit. CONCLUSION: The conclusion to be drawn from these results is that the combination of HP and thermal processing is an effective method to reduce the allergenicity of both apple and celeriac.

The effect of high pressure-high temperature processing conditions on acrylamide formation and other Maillard reaction compounds.

The effect of high pressure-high temperature (HPHT) processing on the formation of acrylamide and other Maillard-type reaction compounds was investigated in order to elucidate the impact of HPHT conditions on the different stages of the Maillard reaction. This study was performed in equimolar asparagine-glucose model systems that were treated at various HP/HT conditions (100-115 °C, 400-700 MPa, 0-60 min), and, for comparison, the model system was also heat-treated at ambient pressure. On the treated samples, the concentration of acrylamide, reactants, hydroxymethylfurfural, organic acids, and melanoidins was determined and the pH prior to and after treatment was measured. Based on the measured responses, the retarding effect of high pressure on the overall Maillard reaction was demonstrated; no or little differences were observed between 400 and 700 MPa. The study was conducted in two types of buffer, i.e. phosphate and MES buffer. In case of acrylamide, aspartic acid and browning, a higher concentration was generated in the MES buffer system, but these differences with the phosphate buffer system could be ascribed to pH changes resulting from the application of combined high pressure and high temperature. Based on the results, acrylamide formation is not expected to pose a major hazard to HPHT-treated products.

High pressure, thermal and pulsed electric-field-induced structural changes in selected food allergens.

SCOPE: The effects of high-pressure/temperature treatment and pulsed electric field treatment on native peanut Ara h 2, 6 and apple Mal d 3 and Mal d 1b prepared by heterologous expression were examined. METHODS AND RESULTS: Changes in secondary structure and aggregation state of the treated proteins were characterized by circular dichroism spectroscopy and gel-filtration chromatography. Pulsed electric field treatment did not induce any significant changes in the structure of any of the allergens. High-pressure/temperature at 20 °C did not change the structure of the Ara h 2, 6 or Mal d 3 and resulted in only minor changes in structure of Mal d 1b. Ara h 2, 6 was stable to HPP at 80 °C, whereas changes in circular dichroism spectra were observed for both apple allergens. However, these changes were attributable to aggregation and adiabatic heating during HPP. An ELISA assay of temperature treated Mal d 3 showed the antibody reactivity correlated well with the loss of structure. CONCLUSION: In conclusion, novel-processing techniques had little effect on purified allergen structure. Further studies will demonstrate if these stability properties are retained in foodmatrices.

Investigating the potential of Bacillus subtilis alpha-amylase as a pressure-temperature-time indicator for high hydrostatic pressure pasteurization processes.

The potential of Bacillus subtilis alpha-amylase (BSA) as a pressure-temperature-time indicator (pTTI) for high pressure pasteurization processing (400-600 MPa; T(i) 10-40 degrees C; 1-15 min) was investigated. A stepwise approach was followed for the development of an enzyme-based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA-MES 0.05 M pH 5.0) was kinetically calibrated under isobaric-isothermal conditions. Time dependent changes in activity could be modeled best by a first-order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T-dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two-sectional planes of a vertical, single vessel system.

Investigation of the influence of different moisture levels on acrylamide formation/elimination reactions using multiresponse analysis.

The influence of water activity on the formation and elimination reactions of acrylamide was examined by means of multiresponse modeling on two different levels of complexity: basic equimolar asparagine-glucose systems and equimolar potato-based asparagine-glucose systems. To this end, model systems were first equilibrated to initial water activities in the range of 0.88-0.99 (corresponding roughly to the moisture gradient observed in French fries) and then heated at temperatures between 120 and 200 degrees C during different reaction times. For each sample, the concentration of acrylamide, glucose, asparagine, and aspartic acid was measured, as well as the extent of browning. A mechanistic model was proposed to model the five measured responses simultaneously. For both types of model systems, the model prediction was quite adequate, with the exception of the extent of browning, especially in the case of the potato-based model system. Moreover, the corresponding estimated kinetic parameters for acrylamide formation and elimination did not change significantly (based on a 95% confidence level) within the range of water activities tested, nor between the systems in the absence or presence of the potato matrix. The only remarkable difference was observed for the activation energy of acrylamide elimination, which was lower in the presence of the potato matrix, although not always significant. In general, these results confirm the generic nature of the model proposed and show that the influence of different moisture levels on acrylamide formation and elimination is minimal and that the addition of a potato matrix has little or no influence on the kinetic model and corresponding kinetic parameters.

Kinetics of acrylamide formation/elimination reactions as affected by water activity.

The influence of water activity on the kinetics of acrylamide formation and elimination reaction was investigated using low-moisture equimolar asparagine-glucose model systems, which were heated at temperatures between 120 and 200 degrees C for variable heating times. To determine the water content corresponding to the water activities tested, a sorption moisture isotherm was constructed experimentally. The acrylamide concentrations measured at different water activities could be modeled on the basis of a reaction scheme including not only acrylamide formation and elimination reactions but also an alternative Maillard reaction between both reactants. The corresponding rate constants and activation energies were estimated using nonlinear regression analysis. Whereas the rate constant for acrylamide formation varied only slightly with the initial water activity of the model system, the elimination rate constant showed a clear minimum around a water activity of 0.82. The opposite trend, namely, a maximum at a water activity of 0.82, was found for the Maillard reaction rate constant as a function of water activity, which confirms data from literature. The activation energies for the different reactions changed in a comparable way as the corresponding rate constant with water activity.

Effect of moisture content during dry-heating on selected physicochemical and functional properties of dried egg white.

This article addresses the effect of moisture content (0.8-9.9%) during dry-heating (80 degrees C) on selected physicochemical (solubility, turbidity, residual denaturation enthalpy, aggregation, surface hydrophobicity, and sulfhydryl content) and functional (foaming ability, foam density, and stability) properties of freeze-dried egg white (FDEW). Moisture content during dry-heating proved to be a parameter determining the functionality of the resulting egg white powder. The degree of conformational changes induced in the egg white proteins by dry-heating was strongly dependent on the amount of water present. Preferentially, dry-heating at 80 degrees C should be performed on egg white powder with a moisture content below 6.8%, as the loss of protein solubility above this value is extensive. In addition to insoluble aggregates, soluble, strongly stabilized aggregates were also formed, especially at higher moisture contents. The decrease in denaturation enthalpy, increase in surface hydrophobicity, and exposure of SH groups previously hidden in the protein core and their subsequent oxidation were more pronounced at prolonged dry-heating times and at higher moisture contents. These conformational changes resulted in improved foaming ability and foams with lower density. No effect of dry-heating on the foam stability was observed.

Impact of pH on the kinetics of acrylamide formation/elimination reactions in model systems.

The effect of pH on acrylamide formation and elimination kinetics was studied in an equimolar (0.1 M) asparagine-glucose model system in phosphate or citrate buffer, heated at temperatures between 120 and 200 degrees C. To describe the experimental data, a simplified kinetic model was proposed and kinetic parameters were estimated by combined nonlinear regression and numerical integration on the data obtained under nonisothermal conditions. The model was subsequently validated in a more realistic potato-based matrix with varying pH. By increasing acidity, the reaction rate constants at T(ref) (160 degrees C) for both acrylamide formation and elimination can significantly be reduced, whereas the temperature dependence of both reaction rate constants increases. The introduction of a lyophilized potato matrix (20%) did not affect the acrylamide formation reaction rate constant at reference temperature (160 degrees C) as compared to the asparagine-glucose model system; the elimination rate constant at T(ref), on the contrary, was almost doubled.

Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment.

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.

Kinetic study on the changes in the susceptibility of egg white proteins to enzymatic hydrolysis induced by heat and high hydrostatic pressure pretreatment.

A kinetic study was conducted on the effect of heat pretreatment in the temperature range of 50-85 degrees C at atmospheric pressure and of high hydrostatic pressure pretreatment (100-700 MPa) at four temperatures (10, 25, 40, and 60 degrees C) on the susceptibility of egg white solutions (10% v/v, pH 7.6) to subsequent enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Both heat pretreatment at atmospheric pressure and high-pressure pretreatment resulted in an increase in degree of hydrolysis (DH) after 10 min of enzymatic reaction (DH10) of egg white solutions, as measured using the pH-stat method, which could be described by a fractional conversion model (based on an apparent first-order reaction kinetic model). The temperature dependence of the corresponding rate constants could be described by the Arrhenius equation. At elevated pressure, a negative apparent activation energy was obtained, implying an antagonistic effect of pressure and temperature. The pressure dependence of the rate constants could be described by the Eyring equation, and negative activation volumes were observed, which demonstrates the positive effect of pressure on the susceptibility of egg white solutions to subsequent enzymatic hydrolysis.

Trypsin inhibition activity of heat-denatured ovomucoid: a kinetic study.

A kinetic study was conducted on the effect of heating in the temperature range of 75-110 degrees C on the trypsin inhibition activity of ovomucoid. Heat treatment of isolated ovomucoid resulted in a time-dependent decrease in trypsin inhibition activity that could accurately be described by a first-order kinetic model. The magnitude and the temperature dependence of the rate constants was affected by the pH during heat treatment. The heat stability of ovomucoid was the lowest at pH 7.6. Heat treatments intended to decrease the trypsin inhibition activity should therefore be carried out as soon as possible after laying, because the ovomucoid was inactivated faster at the pH of fresh egg white (pH 7.6). The presence of the other egg white constituents decreased the heat stability of ovomucoid compared to that of the model system of ovomucoid in buffer, presumably by the formation of ovomucoid-lysozyme complexes in the former.

Heat-induced changes in the susceptibility of egg white proteins to enzymatic hydrolysis: a kinetic study.

A kinetic study was conducted on the effect of heating in the temperature range of 50-92 degrees C, on the susceptibility of ovalbumin and albumen solutions to enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Heat treatment resulted in an increase in degree of hydrolysis after 10 min of enzymatic reaction of both ovalbumin and albumen, as measured using the pH-stat method. The time-dependent change in the susceptibility to enzymatic hydrolysis after heat treatment was described by a fractional conversion model (based on an apparent first-order reaction kinetic model). Different end levels of degree of hydrolysis were obtained after heating for a long time at different temperatures, which suggests that the final degree of unfolding of the protein is temperature dependent.