Denaturation Of Β-lactoglobulin Research Articles

Accessing the thermal and electric effects in β-lactoglobulin denaturation and interaction with phenolic compounds

Protein-phenolic interactions have been on the spotlight in food research since they can change the structural, functional and nutritional properties of both compounds. Electric field processing technologies, such as ohmic heating (OHM), can contribute to improving food safety, quality and to tailor functionality of foods and ingredients. In this study, the potential of OHM processing in modifying protein-phenolic interactions was accessed. β-lactoglobulin (β-Lg) solutions were processed at 70 °C and 90 °C by conventional heating and OHM (26 V cm−1 at 50 Hz and 20 kHz), followed by their association with different phenolic compounds – i.e., quercetin (Qu), malvidin-3-glucoside (M3G), trans-resveratrol (TR), p-coumaric acid (p-C) and tyrosol (Ty). The protein solutions and protein-phenolic complexes were characterized by UV-spectroscopy, fluorescence spectroscopy, circular dichroism, and antioxidant capacity. Protein-phenolic interactions were confirmed by changes in the UV-absorbance spectra, reduction of the surface hydrophobicity and changes in the secondary structure of the protein. The formation of stable complexes was established through fluorescence quenching analysis and the binding affinities were found to be in the order TR > Qu > p-C > M3G > Ty. Furthermore, the binding parameters and changes of the protein's structural features were impacted by the processing conditions, including those associated with OHM. This study demonstrates for the first time that OHM processing and the control of its variables can potentially be used to tune β-Lg-PhCs interactions.

Structural and gelling properties of whey proteins influenced by various acids: Experimental and computational approaches

Five different acids, i.e., hydrochloric acid (HCl), acetic acid, lactic acid, γ-aminobutyric acid (GABA) and taurine were applied to investigated their effects on heat-induced gelation properties of whey protein isolate (WPI) at pH = 5.5. All WPI gels were found to have self-similar structures with the fractal dimension, df = 2.40. Those denatured WPI formed strong physical gels that were susceptible to shear and strain. Of all the acids, HCl, GABA and taurine significantly strengthened WPI gels. Moreover, denser packing of protein aggregates was observed from the gels with GABA and taurine. Besides experimental approaches, molecular dynamics (MD) simulations were also applied to investigate the effects of various acids on denaturation of β-lactoglobulin (β-LG). GABA and taurine were found to protect β-LG from denaturation and delayed the molecular heat-induced unfolding. Moreover, presence of HCl, GABA and taurine conserved the rigidity of unfolded β-LG and localized the residue –SH121, which was believed to result in promoted reaction-limited clustering aggregations (RLCA) of β-LG molecules and strengthened WPI gels. Further studies suggested that there was no strong binding or aggregation between cations (Na+) or anions/zwitterions and β-LG molecule. Therefore, the effects from various acids (anions/zwitterions) on β-LG and WPI were supposed to result from long-distanced electrostatic interactions and/or their effects on the protein hydration shells.

Protein Stability─Analysis of Heat and Cold Denaturation without and with Unfolding Models.

Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The extraction of thermodynamic properties from these measurements requires the application of models. Differential scanning calorimetry (DSC) is less common, but is unique as it measures directly a thermodynamic property, that is, the heat capacity Cp(T). The analysis of Cp(T) is usually performed with the chemical equilibrium two-state model. This is not necessary and leads to incorrect thermodynamic consequences. Here we demonstrate a straightforward model-independent evaluation of heat capacity experiments in terms of protein unfolding enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T)). This now allows the comparison of the experimental thermodynamic data with the predictions of different models. We critically examined the standard chemical equilibrium two-state model, which predicts a positive free energy for the native protein, and diverges distinctly from the experimental temperature profiles. We propose two new models which are equally applicable to spectroscopy and calorimetry. The ΘU(T)-weighted chemical equilibrium model and the statistical-mechanical two-state model provide excellent fits of the experimental data. They predict sigmoidal temperature profiles for enthalpy and entropy, and a trapezoidal temperature profile for the free energy. This is illustrated with experimental examples for heat and cold denaturation of lysozyme and β-lactoglobulin. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defined thermodynamic context and are amenable to molecular dynamics calculations.

Effect of calcium on the reversible and irreversible thermal denaturation pathway of β-lactoglobulin

Thermal denaturation of β-lactoglobulin (BLG), boosted in the presence of calcium ions, is responsible for the dairy fouling issue in milk processing. However, the reported kinetic model concerning BLG denaturation was generally based on a simplified one-step reaction model where the unfolding of the protein was considered as a kinetically driven reaction. In such a model, the presence of break-slope phenomenon in the Arrhenius plot was justified by the fact that unfolding step constitutes a rate-limited step compared to aggregation at low temperatures. Nevertheless, there is accumulated experimental evidence implying that the unfolding step is much faster than aggregation regardless of the temperature. Therefore, in this paper, it is proposed that the thermal unfolding of BLG is thermodynamically controlled such that a chemical equilibrium between native and unfolded BLG species can be instantaneously established, followed by an irreversible aggregation of unfolded BLG. This novel approach allows to mathematically interprets the break-slope behavior in the Arrhenius plot. The developed model was applied to investigate the effect of Ca 2+ on the unfolding equilibrium and subsequent aggregation process of BLG in whey protein solutions. Ca 2+ was confirmed to have a protective role on the unfolding equilibrium of BLG by increasing the enthalpy Δ H u and decreasing the equilibrium constant K c , while it facilitated the aggregation of BLG by showing a reduction of the corresponding enthalpy and entropy which cannot be seen as reported using one-step reaction model. This approach allows us to be closer to reality when mimicking the heat-induced appearance/disappearance of different BLG species, including native, unfolded and aggregated ones. Accurate population balance of these foulant species during thermal processing could lead to new perspectives on fouling prediction. • Thermal unfolding of β-lactoglobulin can be considered to occur instantaneously. • Unfolding of β-lactoglobulin is thermodynamically driven. • New model mathematically interprets the break-slope phenomenon in Arrhenius plot. • Ionic calcium has a protective role on the unfolding of β-lactoglobulin. • Calcium favors β-lactoglobulin aggregation by showing decreased enthalpy.

Effect of pressure treatment of recombined whole milk on fat globule membrane composition and acid gelation functionality

Recombined whole milk was prepared by pressure treating skim milk (200 to 600 MPa/30 min) then homogenizing with milkfat (HPHO) or by homogenizing milkfat with skim milk then pressure treating the recombined whole milk (HOHP). β-Lactoglobulin denaturation increased at higher pressures. Low levels of α-lactalbumin were denatured at 600 MPa only. Denaturation was similar in the HPHO and HOHP milk. The HPHO milk had statistically similar levels of total protein, higher levels of whey protein and κ-casein and lower levels of αs-casein adsorbed to the fat globules compared with the HOHP milk. The HPHO milk had a higher proportion of β-casein directly at the interface at all pressures and a higher proportion of κ-casein and a lower proportion of denatured whey proteins at pressure up to 400 MPa than the HOHP milk. Acid gels prepared from the HOHP milk had higher final G′ and yield stresses than those from the HPHO milk. These differences are discussed in relation to the compositions of the proteins adsorbed to the fat globules and how these interact during acidification.

Effect of calcium on the thermal denaturation of whey proteins and subsequent fouling in a benchtop fouling device: An experimental and numerical approach

The key role of calcium in whey protein fouling behavior is well known in plate heat exchangers (PHEs), as it affects both the thermal denaturation of proteins and deposition reaction on the surface. However, the complex flow pattern and the closed configuration of PHEs make it hard to investigate this phenomenon in situ. In this paper, a microchannel benchtop fouling device was designed, making it possible to achieve a similar temperature profile to that performed in PHEs but in a laminar regime. A 3D simulation was developed to predict the thermal denaturation of β-lactoglobulin (BLG) as well as its deposition in the microchannel using computational fluid dynamics (CFD). The thermodynamics and heat transfer of the numerical model was validated by experimentally measuring the bulk fluid temperature profile in the microchannel using fluorescence microscopy, where Pyrromethene 556 was used as a temperature indicator. Results revealed a quasi-linear relationship between the pre-exponential factor of deposition reaction and the calcium concentration, implying that the fouling was built in such a pattern that only one calcium ion per one BLG molecule is involved. The imaging of the fouling deposit in situ showed spherical structures of deposits at low calcium levels, while denser and more rod-like ones were found at higher calcium concentrations. The fouling behavior was found to follow a crystallization-like pattern with preference upon the previously fouled layer instead of clean stainless steel surface. These findings confirm the essential role of ionic calcium on the formation of fouling deposits by anchoring the denatured BLG protein upon the surface as well as strengthening the protein-protein interactions for fouling build-up.

Interactions of casein and carrageenan with whey during pasteurization and their effects on protein deposition

Despite significant research to date to understand dairy fouling, most efforts have focused on milk ingredients, especially whey protein. In contrast, little is known about the effects of other milk proteins like casein and non-milk ingredients such as carrageenan gum. Therefore, the fouling mechanisms of complex dairy products are still elusive. In this study, different model dairy solutions were prepared with whey protein isolate (WPI; 2 wt%), casein (0.5 wt%) and κ-carrageenan (0.03 wt%), and their fouling behaviors during high-temperature-short-time pasteurization (~73 ºC) were characterized under well-controlled flow. The addition of casein into WPI solution significantly increased its fouling tendency because casein reduced the thermal stability of whey, but the deposits formed were more porous with apparent fractures. As evidenced by fluorescence and Raman spectroscopy analyses on the changes in protein structures and increased exposure of tryptophan residues, the presence of casein was confirmed to facilitate the denaturation of β-lactoglobulin. Adding κ-carrageenan increased the fouling of WPI solution with formation of more compact deposit. This work sheds light on the interactions of milk proteins with polysaccharides under thermal processing conditions and is expected to provide the dairy industry with knowledge of designing dairy formulation with reduced fouling tendency.

Quark-Type Cheese: Effect of Fat Content, Homogenization, and Heat Treatment of Cheese Milk.

The effect of homogenization and fat reduction in combination with variable heating conditions of cow milk on the characteristics of Quark-type cheese were investigated. The mean composition of full-fat cheeses was 71.96% moisture, 13.95% fat, and 10.31% protein, and that of its reduced-fat counterparts was 73.08%, 10.39%, and 12.84%, respectively. The increase of heat treatment intensity increased moisture retention and improved the mean cheese protein-to-fat ratio from 0.92 to 1. Homogenization increased the moisture and protein retention in cheese, but the effect was less intense for milk treated at 90 °C for 5 min. The extended denaturation of whey proteins resulted in harder, springier, and less cohesive cheese (p < 0.05). Treatment of milk at 90 °C for 5 min resulted in higher residual lactose and citric acid and lower water-soluble nitrogen contents of cheese (p < 0.05); the latter was also true for homogenization (p < 0.05). Storage did not affect the composition and texture but decreased galactose and increased citric acid and soluble nitrogen fractions (p < 0.05). In conclusion, heat treatment conditions of milk that induced a considerable denaturation of β-lactoglobulin and left a considerable amount of native α-lactalbumin was adequate for the manufacture of a “clean-label” Quark-type cheese, whereas homogenization was more effective for full-fat cheese.

Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.

Comparing different methods for estimating kinetic parameters of whey protein heat-induced denaturation in infant milk formulas

Abstract Modeling the heat-induced denaturation of milk proteins is a relevant issue because of heating processes in the manufacturing of several dairy products. In this study, four different parameter estimation methods were evaluated to estimate the kinetic parameters of the heat-induced protein denaturation of β-lactoglobulin (β-LG) and lactoferrin (LF) in infant milk formulas (IMF). The methods were: a two-step method, nonlinear least-squares (NLS), one-step linearized, and weighted least-squares (WLS). The WLS was the best alternative, avoiding biases observed when applying other methods besides producing consistently low residuals. Using the second proposed weight function, the WLS produced sum of squared errors and mean absolute percentage errors with average values of respectively 0.18 and 12.3% versus 0.27 and 13.3% considering all methods. However, the WLS requires good initial guesses for parameters, and previous knowledge of sampling and residuals variance. Those can be provided by previously performing the two-step and NLS methods, respectively.

New experimental set-up for testing microwave technology to continuously heat fouling-sensitive food products like milk concentrates

Microwave heating is an emerging alternative direct heating method to face deposit and particle formation upon heating fouling-sensitive food products. This study successfully demonstrated the implementation of a new set-up for continuous microwave heating at pilot scale (f = 2450 MHz, P = 0.6–6 kW). Reconstituted skim milk concentrate (DM = 27, 31.5 and 36 g/100 g) was heated (110–125 °C, 5 s holding time) through microwave technology and with a tubular heat exchanger in order to examine heat-related product changes and compare both technologies.The results showed significantly reduced whey protein denaturation (35–40% less β-lactoglobulin denaturation) and deposit formation (90% less fouling) of microwave heated reconstituted skim milk concentrate compared to tubular heating, whereas colour changes were insignificant for all experiments. Both methods resulted in comparable particle sizes at equal flow profiles, however, turbulent flow conditions were necessary in order to prevent elevated particle formation. Industrial relevanceMicrowave heating is a promising alternative heating method for fouling-sensitive dairy products. This study demonstrates that through short heat-uptake times gentle product handling is ensured, resulting in retained native product properties. Reduced fouling leads to extended running times, implying less and eventually shorter cleaning intervals. Moreover, compared to other direct heating methods like steam injection, no additional water/steam is added to the product. With its space-saving design and easy handling, continuous microwave heating is a rising technology with high industrial relevance.

Effect of casein/whey ratio on the thermal denaturation of whey proteins and subsequent fouling in a plate heat exchanger

Dairy fouling is a ubiquitous problem in food processing, however, the fouling mechanism is not fully understood and investigations arose mainly from experiments with model systems that contained only whey proteins, typically reconstituted from whey protein isolate powder (WPI). The effect of casein on fouling has been rarely considered despite it is the major component of milk proteins. To fill this gap, whey protein-based model fluids containing different casein concentrations and fixed content of added calcium were prepared, leading to various Casein/WPI mass ratios. The effect of Casein/WPI on β-lactoglobulin (BLG) denaturation at molecular level and subsequent fouling behavior in the pilot-scale plate heat exchanger during pasteurization treatment was investigated. It was shown that Casein/WPI significantly affects the fouling behavior: at low Casein/WPI, fouling mass dropped dramatically until a minimum value was reached located at Casein/WPI of 0.2. While at higher Casein/WPI, fouling mass increased with elevated Casein/WPI. Element mapping of the fouling layer also reveals that different structures and fouling mechanisms occur depending on Casein/WPI ratio. Finally, it was established that contrary to WPI solutions, BLG thermal denaturation is poorly correlated to decrease/extent of fouling for casein protein-based solutions showing that the presence of casein deeply modifies mineral and protein interactions and fouling build-up.

Comparative study on quality of whole milk processed by high hydrostatic pressure or thermal pasteurization treatment

Quality of whole milk pasteurized by high hydrostatic pressure processing (HPP: 600 MPa for 5 min) or HPP combined with microfiltration (MF + HPP) were compared with two conventional heat treatments, low temperature long time (LTLT: 63 °C for 30 min) and high temperature short time (HTST: 72 °C for 15 s). Inactivation of microorganisms, changes in volatiles, protein denaturation, gastric digestion, and sensory quality were evaluated after 8 days of storage at 5 °C. Total bacterial and E.coli counts were reduced to approximately the same level for both high pressure and heat treated milks. The denaturation of β-lactoglobulin was significantly higher for high-pressurized milks, while no significant differences were found in gastric in-vitro protein digestion compared with heat treated milks. The results of volatile compounds revealed that HTST and HPP-treated milks exhibited very similar aroma profiles, while LTLT-milks were characterized by higher levels of ketones. After 8 days storage, the sensory analysis showed that LTLT milk was the one that differed most from the other three milks with highest intensities of all the sensory attributes, while no tangible differences were found between HTST and any HPP and MF + HPP milk, except for Boiled Taste that was slightly but significantly lower for MF + HPP.

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating

Shearing is an embedded step during thermal processing of milk that can add additional impact on native proteins. Behaviour of native proteins in bovine skim milk was studied at different temperatures (70–100 °C) for 5 min with or without applying a constant shear rate at 1000 s−1. Shear stress generated generally decreased with increase in temperature due to decrease in milk viscosity which displayed a Newtonian fluid behaviour. β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La) denatured rapidly even at low temperature (70 °C) under the shearing and about 25% more of them denatured at 85 °C than those of the non-sheared counterparts. Shearing predominantly induced formation of thiol/disulphide aggregates mainly consisting of β-Lg and α-La, as well other minor whey proteins and caseins. These results highlighted the necessity of assessing impact of shearing on milk proteins when optimising thermal processing conditions.

Kinetics of denaturation and aggregation of highly concentrated β-Lactoglobulin under defined thermomechanical treatment

The influence of thermomechanical treatment (temperature 60 °C – 100 °C and shear rate 0.05 s−1 – 50 s−1) on the denaturation and aggregation of β-Lactoglobulin at high concentrations (50 % – 70 %) was investigated. While denaturation and aggregation reaction rate decreased at higher concentrations, a combination of heat and mechanical treatment resulted in an increase in reaction rates and consequently, in higher degrees of non-disulfide covalent bond aggregation. The kinetic parameters were determined by non-linear regression. Denaturation (combined effect of unfolding and aggregation) and covalent bond aggregation were found to follow 2.15 and 1.7 order kinetics, respectively. At the investigated conditions, the activation energy calculated for the denaturation and covalent bond aggregation reaction ranged from 25 to 283 kJ mol−1. Although the activation energy for the denaturation and aggregation reactions decreases with increasing shear rate, it was found that the mechanical treatment mainly affected the reactions at high temperatures, probably due to the increased molecular motion.

Influence of moderate electric fields in β-lactoglobulin thermal unfolding and interactions

In this study, the effects of moderate electric fields during thermal denaturation of β-lactoglobulin were examined through an in situ circular dichroism approach, complemented by intrinsic extrinsic fluorescence analysis. Results have shown that the effects of electric fields in protein unfolding were linearly dependent on the applied electric field intensity (V/cm) and increased by the use of low electric frequencies – i.e. 50 to 200 Hz. These electric effects caused significant changes on β-lactoglobulin melting temperature, unfolded conformation and subsequent intermolecular interactions, revealed by the increase of surface hydrophobicity (ANS affinity) and higher conservation of retinol binding. The obtained data provides a clear evidence that moderate electric fields contribute to distinct folding/unfolding of β-lactoglobulin, resulting in structural modifications. These findings are relevant for (bio)-technological applications involving electric fields processing, bringing new insights for the development of innovative strategies to control protein function and tune production of functional protein systems.

Kinetics of heat-induced denaturation of proteins in model infant milk formulas as a function of whey protein composition

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow’s milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, β-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when β-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of β-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing β-lactoglobulin.

Investigation on the influence of high protein concentrations on the thermal reaction behaviour of β-lactoglobulin by experimental and numerical analyses

In this study, treatments at various temperature–time profiles were performed for β-lactoglobulin samples at different concentrations (50–70%) using a special rheometer as processing device. Rheological measurements, offline protein chemical analyses, and molecular dynamics analyses were performed to investigate the influence of high protein concentrations and treatment temperature on the denaturation and aggregation behaviour of β-lactoglobulin. Under these conditions, the degree of denaturation and aggregation decreased with increasing protein concentration. This corresponded to a strongly decreased diffusion and increased stability of exposed surface protein regions at high concentrations. Irreversible denaturation was observed for temperatures above 60 °C. Increasing thermal treatment intensity resulted in an increase of aggregation. Depending on the thermal treatment conditions, different protein–protein interactions were measured. By increasing the treatment temperature, the resulting aggregates were increasingly stabilised by covalent bonds. In addition to disulphide bonds, non-disulphide covalent cross-links were formed at temperatures above 100 °C.

A comparison of pilot-scale supersonic direct steam injection to conventional steam infusion and tubular heating systems for the heat treatment of protein-enriched skim milk-based beverages

Abstract Direct supersonic steam injection, direct steam infusion, and indirect tubular heating were each applied to protein-enriched skim milk-based beverages with 4, 6 and 8% (w/w) total protein, and the effect of final heat temperature on the physical properties of these beverages was investigated. Supersonic steam injection resulted in significantly lower levels of denaturation of β-lactoglobulin (34.5%), compared to both infusion (76.3%) and tubular (97.1%) heating technologies. Viscosity, particle size and accelerated physical stability of formulations did not differ significantly between the heating technologies, while noticeable colour differences due to heat treatment (mainly attributed to increasing b* value) were observed, particularly for tubular heating. Overall, the extent of protein denaturation in high-protein dairy products was significantly influenced by the particular heating technology applied. The application of supersonic steam injection technology, with rapid heating and high shear characteristics, may enable differenciated product characteristics for ready-to-drink ambient-delivery high-protein dairy beverages. Industrial relevance The design and application of novel direct supersonic steam injection technology was comprehensively studied and found to provide significant benefits over direct steam infusion and indirect tubular heating technologies for skim milk-based protein beverages. This type of injection heating system resulted in heat-treated formulations with lower levels of denatured whey proteins, compared to tubular and infusion heating, offering an alternative opportunity to the industry in terms of producing shelf-stable dairy protein beverages.