Whey Protein Denaturation Research Articles

Ultrasonic process affecting interactions between sodium caseinate and whey proteins

Ultrasound has been widely explored for several applications, such as emulsification or structural modification of food materials such as proteins. In this work, the effect of ultrasound on the control of whey proteins (WPI) aggregation was evaluated in the presence of sodium caseinate (NaCas). Solutions of NaCas, WPI and both (1:1) were treated with ultrasound under different power and time conditions and were initially evaluated in terms of particle size distribution, charge density, pH and polyacrylamide gel electrophoresis. Three pairs of conditions were adopted to provide the same energy density - A1 (450 W / 300 s, 6750 MJ/m3), A2 (150 W / 900 s, 6750 MJ/m3), A3 (600 W / 300 s, 900 MJ/m3), A4 (202.5 W / 900 s, 9112.5 MJ/m3), A5 (742.5 W / 300 s, 11137.5 MJ/m3) and A6 (247.5 W / 900 s, 11137.5 MJ/m3). Best conditions of transmitted energy - A1, A3 and A5 - were studied for surface hydrophobicity, circular dichroism and infrared spectroscopy. The decrease of surface hydrophobicity of NaCas:WPI mixtures pointed to a protective effect of NaCas against WPI denaturation, confirmed by the presence of more ordered structures by FTIR analysis that were not observed in the absence of NaCas. Finally, the effect of these structural changes on the gelation capacity of the ultrasound-treated proteins was assessed. Ultrasound was able to reduce the stress at rupture from 1988.59 Pa (control) to 1655.31 Pa (A3) and 1871.24 Pa (A5), and more markedly increase the Young modulus from 113.69 kPa (control) to 243.30 kPa (A3) and 392.44 kPa (A5). This study identified that higher power values with shorter times were able to provide greater protein changes that affected gelation properties, showing that the modulation of ultrasound conditions can produce ingredients with different techno-functional properties.

Determination and evaluation of whey protein content in matured cheese via liquid chromatography

To date, there is no valid analytical method available to determine the total whey protein (TWP) content in cheese. Therefore, the aim of this study was to develop HPLC methods for whey protein determination in mature cheese. For that purpose, foil-ripened whey protein-enriched model cheese and traditional Edam-type cheese were produced and analyzed. Suitable protein extraction methods and subsequent analytical methods to determine the acid-soluble whey protein (ASWP) and TWP content in cheese were developed. To characterize the influence of proteolysis on native and denatured whey proteins, the ASWP and TWP contents were determined throughout the ripening process. Both chromatographic methods showed that the individual whey proteins (α-lactalbumin and β-lactoglobulin) were not degraded during ripening. However, the analyzed ASWP content increased by up to 25% throughout ripening. Compared to traditional Edam-type cheese, the β-lactoglobulin content in whey protein-enriched cheese (containing 30% high-heat milk) increased by a factor of 3.5. To evaluate the chromatographic results, two different calculation models were used to estimate a reference value for the TWP content in the manufactured cheese. Further studies are required to optimize the quantification of TWP content in hard, semihard, soft, and cream cheeses.

Correlation between free fatty acids content and textural properties of Gouda cheese supplemented with denatured whey protein paste

In this study, the denatured whey protein paste (DWPP) was used to improve the texture characterizations of Gouda cheese. Five treatments of cheese were manufactured by adding 0, 1, 2, 3 and 4% of DWPP to cheese curd. Fortification of Gouda cheese with DWPP increased values of moisture, salt in moisture, water-soluble nitrogen/total nitrogen and non-protein nitrogen/total nitrogen whereas decreased values of density and free oil. The cheese contained DWPP was lighter and more yellowish compared to the control. The cheese samples contained 1 and 2% DWPP exhibited a significant increase in hardness, cohesiveness, springiness, gumminess and chewiness values while, the cheese samples that contained 3 and 4% DWPP exhibited a significant decrease. Adding DWPP to cheese lowered saturated fatty acids and raised unsaturated fatty acid (USFA) values which partially caused a lowering in cheese hardness at high levels of DWPP because of the low melting points of USFA. Based on these results, supplementation of Gouda cheese with 1 or 2% DWPP improved the texture properties.

Spray-Dried Infant Formula Emulsion Stability as Affected by Pre-Heat Treatment and Total Solids Content of the Feed.

Pre-spray-drying processing may affect stability after reconstitution of emulsion-based powders, such as infant formulas. This study aimed to evaluate the effects of pasteurization temperature and total solids (TS) of the feed on the stability of the emulsions obtained from the reconstituted powders. Four infant formula powders (50%-75 °C, 50%-100 °C, 60%-75 °C, and 60%-100 °C) were produced at pilot scale, from emulsions with 50 or 60% TS pasteurized at 75 or 100 °C for 18 s. Both the emulsion feeds and the emulsions from the reconstituted powders (12.5% TS) were analyzed. The results showed that feeds with 60% TS were flocculated, as indicated by the large particle size and viscosity and the pseudoplastic behavior. Light microscopy revealed that, during spray drying, the flocs were disrupted in 60%-100 °C, while the 60%-75 °C emulsion remained flocculated, reducing its stability post-reconstitution. Although all four emulsions were mainly stabilized by caseins, the presence of β-lactoglobulin was also detected at the oil-water interface, in native state in the formulas preheated at 75 °C and aggregated in the formulas preheated at 100 °C. In conclusion, both the degree of whey protein denaturation (resulting from pasteurization) and the TS of the concentrates during infant formula production affected the emulsion stability of the reconstituted powders.

Structural changes induced by pasteurisation and/or high-pressure treatment of skim caprine milk

The effects of pasteurisation, high-pressure (HP), and a combination of pasteurisation and high-pressure (PHP) on the physicochemical properties and protein structure of caprine skim milk was investigated. Samples treated by PHP generally had a higher pH, whey protein denaturation, surface hydrophobicity, and intrinsic fluorescence than those treated only with heat or pressure. In contrast, the size of skim milk casein micelles decreased significantly with an increase in pressure level and time; however, the effect was less marked when heat and pressure treatments were combined. For the PHP and HP samples, as the level and time of pressure increased, the α-helix and β-turn content reduced, whereas β-sheet and random coil were induced. Thus, PHP treatment could be used as a good alternative technology in the dairy industry to promote the functional properties of skim caprine milk.

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.

Sedimentation in UHT high-protein dairy beverages: Influence of sequential preheating coupled with homogenisation or supplementation with carbohydrates

Consumer demand for UHT high-protein dairy beverages is growing all around the world. To overcome the potential low stability of these products during processing and storage remains a great challenge for dairy industries. In this context, the potential of preheating treatment coupled with homogenisation or the addition of sucrose or corn soluble fibre on reducing the sedimentation level of UHT dairy beverages containing 10% w/w of protein and equivalent proportion of caseins to whey proteins were evaluated. The samples supplemented with sucrose or corn soluble fibre displayed a reduced overall particle size distribution, but equivalent amount of sediment compared with that in the control sample. The applied pretreatment promoted intense whey protein denaturation and reduced the overall particle size distribution and the amount of material sedimented compared with the control sample. The applied pretreatment has potential to improve the stability of UHT high-protein dairy beverages.

Heating affects protein digestion of skimmed goat milk under simulated infant conditions

The objective of this research was to analyse the effects of heating on digestion of skimmed goat milk proteins. Most previous goat milk digestion studies evaluated the digestion only based on the supernatant. In this study, digestion of skimmed goat milk was studied in both supernatant and gastric clot. The results indicated that, compared to mild temperature heated samples (≤75 °C), samples heated at ≥80 °C showed more extensive gastric clot formation with a higher protein digestion rate, but also resulted in a larger amount of undigested whey proteins due to its severe aggregation. For the peptidome, β-casein was the major source of bioactive peptides. The samples heated at 65 °C showed higher bioactive peptide abundances, whereas at temperatures higher than 75 °C, it was reduced due to cleavage into smaller peptides. Overall, this study showed that different heating temperatures induced different whey protein denaturation degrees, which affected their digestion in skimmed goat milk..

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.

Unraveling molecular interactions in whey protein-kefiran composite films to understand their physicochemical and mechanical properties

To enhance health promoting properties of edible whey protein films, the inclusion of kefiran to develop composite films was studied. Physicochemical and mechanical properties of films prepared with different wpi:kefiran ratios, different percentage of plasticizer and subjected or not to a heat treatment were analyzed. The inter- and intra-molecular interactions produced by the incorporation of kefiran into whey-protein films were studied to understand the effect exerted by kefiran on the protein network that may affect the properties of the films. Differential-scanning-calorimetry and Fourier-transform–infrared-spectroscopy studies revealed that kefiran does not affect proteins denaturation, but rather modifies protein-protein interactions in the films, exhibiting a change in wpi secondary structure and a drop in films' melting temperature. Composite wpi:kefiran films had higher opacity, lower water content and lower water activity compared to single wpi films. Incorporation of kefiran into the films decreases tensile strength and elastic modulus and produces an increment in the elongation at break value. It was confirmed that the addition of a whey-protein denaturation step produces an improvement in the protein network that is crucial for the mechanical behavior of the films. Understanding the molecular interactions within composite films greatly aids in obtaining novel formulations with tailor-made physicochemical properties.

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).

Solubilization of concentrated protein dispersion: Effect of hydrogen peroxide (H2O2) and sodium hexametaphosphate (SHMP)

In the present study, the functionality of H2O2 and sodium hexametaphosphate (SHMP) on solubilization of whey protein hydrolysate (WPH) and isolate (WPI), resistant to sterilization temperature at various concentrations, was investigated. The physical state of the treated WPH and WPI dispersions at the presence of various concentrations of H2O2 and SHMP was related to their colloidal structures and thermal stability. Using optimum concentration of H2O2, both dispersions stabilized against heat treatment likely because free SH groups blocked by H2O2. The solubilization range by SHMP was comparably low (up to 6 and 15% w/w for WPI and WPH, respectively). Moreover, the desirable stability was reached when H2O2 and SHMP were simultaneously used. The pH adjustment (7.2), prior to sterilization, also improved the stability range. This research highlights the potential of these substances to restrain the denaturation of whey proteins. Further investigations are still required to elucidate the accurate mechanism of solubilization.

Deciphering the impact of whey protein powder storage on protein state and powder stability

Whey protein concentrate powders can be strongly affected by storage conditions inducing both chemical and structural protein modifications. In the present work, a combination of different storage conditions were evaluated, leading to the evaluation of 6 different batches that were chosen in order to evaluate the impact of the following parameters: storage temperature, storage duration and storage heat load (°C.days). In each set the relative humidity was maintained at 70%. To estimate the impact of the three parameters, whey protein lactosylation, denaturation and aggregation were followed. It was shown that the highest temperatures (60 vs 55.5 °C and 41 vs 36 °C) led to greater protein modification, whereas the duration (17 vs 14 days and 33 vs 30 days) and the total heat load (low vs high C°.days) did not affect protein modification. Lactose content is known to be an important parameter for powder browning. Here, we show that if lactose content is crucial for the level of initial process induced browning of the powder, the additional browning that occurs during storage is mostly independent of lactose content. In addition, during storage, detected lactosylated proteins were found to be inversely proportional to powder browning indicating the occurrence of advanced Maillard reaction steps ( e.g. dicarbonyl and AGEs formation and production of melanoidins). Protein aggregates contained both β-lactoglobulin and α-lactalbumin and were mostly formed by non-disulphide covalent bonds. Interestingly, despite protein structure being strongly impacted, good powder rehydration properties were obtained for the studied conditions. • Whey powder functionality is not impacted by storage. • Maillard-induced protein modification is repeatable between powder productions. • Initial protein lactosylation content drives powder aging. • Browning during storage is not related to lactose content.

Developing an optical backscatter method for determining casein micelle particle size in heated milk

A plethora of different factors, such as heat treatment, pH, soluble calcium and phosphate concentrations, colloidal calcium phosphate, ionic strength, redox potential, etc., affect functionally of critical milk components such as casein micelles, fat globules and whey proteins. These physicochemical changes induce fat- or protein-protein interactions that would be associated to changes in particle size that might be revealed using light backscatter measurements. We hypothesized that inline, simple, low-cost light backscatter measurements might have the potential to provide functionally related information, representing an interesting opportunity for process control. Casein micelle particle size and near infrared light backscatter spectra were measured in milks heat treated at 80 and 90 °C and pH 6.3, 6.7 and 7.1 in order to obtain prediction models for estimating changes in casein micelle particle size during milk heat treatment. Light intensity was measured over a spectral range of 200–1100 nm using a simple optical backscatter sensor and was implemented into models for particle size predictions as a function of heat treatment temperature and pH. Models which included an exponential factor containing a ratio of two specific wavebands were found to improve R2 when compared to single wavelength models. The best model exhibited an R2 of 0.993 and SEP of 2.36 nm. The developed prediction models show promise for in-line monitoring of whey protein denaturation and casein micelle particle size.

Thermal Denaturation of Milk Whey Proteins: A Comprehensive Review on Rapid Quantification Methods Being Studied, Developed and Implemented

Heat treatment of milk signifies a certain degree of protein denaturation, which modifies the functional properties of dairy products. Traditional methods for detecting and quantifying the denaturation of whey proteins are slow, complex and require sample preparation and qualified staff. The world’s current trend is to develop rapid, real-time analytical methods that do not destroy the sample and can be applied on/in-line during processing. This review presents the rapid methods that are being studied, developed and/or applied to determine and quantify the thermal denaturation of whey proteins, including spectroscopic, electrochemical and miniaturized methods. The selected methods save a significant amount of time and money compared to the traditional ones. In addition, the review emphasizes the methods being applied directly to milk and/or that have potential for on/in/at-line application. There are interesting options to quantify thermal denaturation of whey proteins such as biosensors, nanosensors and microchips, which have fast responses and could be automated. In addition, electrochemical sensors are simple to use and portable, while spectroscopy alternatives are suitable for on/in/at-line process.

Tryptophan front-face fluorescence and functional properties of whey: A preliminary study

This study investigated the potential of front-face fluorescence spectroscopy to predict the functional properties of whey. Whey has a commercial interest due to its excellent nutritional value and versatile functional properties. However, its attributes depend dramatically on heat treatment, which may alter its suitability for different food applications. Tryptophan front-face fluorescence of whey and its functional properties, i.e., foaming, gel-forming, and emulsifying properties, were evaluated after milk heat treatment (at 80 °C with seven holding times) to detect correlations between tryptophan fluorescence and functional parameters and generate predictive models. Whey samples were obtained by isoelectric precipitation of caseins from reconstituted skim milk powder enriched with whey protein isolate. As expected, heat treatments induced an increase in whey protein denaturation, as well as a decrease in total whey protein concentration and tryptophan fluorescence intensity. Gel-forming and emulsifying properties of whey significantly correlated with the maximum intensity of tryptophan (P < 0.001). Concerning foaming properties, only the foam stability index revealed a weak correlation with tryptophan maximum intensity (P < 0.05). Consequently, models presented strong significances (P < 0.001). Specifically, using only tryptophan fluorescence allowed successful prediction of emulsifying properties but was not enough for foaming and gel forming properties.

Droplet drying and whey protein denaturation in pulsed gas flow - A modeling study

Pulse combustion drying is a novel advanced drying technique that uses pulsating gas flow generated by intermittent combustion. The technique has been used for fine powder production from various materials, including heat sensitive materials such as foods and pharmaceuticals. In this study, denaturation of whey protein isolate (WPI) during drying in pulsated gas flow is investigated numerically. A mathematical model has been developed by coupling a single droplet drying model with a kinetic model of whey protein denaturation, both validated experimentally. To understand the influence of operating conditions on process dynamics and product properties, variations of initial droplet size, initial solid content, gas temperature, pulsation frequency, and gas velocity components are simulated. It is found that small droplets undergo drying and denaturation faster, and stay longer in the drying chamber. The influence of initial solid content is complex and depends on other variables. Gas pulsation has been shown to enhance drying with small reduction in final product activity. It also reduces the discrepancies in product quality and energy consumption in case of different initial droplet size distributions. The influence of frequency and velocity amplitude weakens at higher magnitudes of the same. Average flow velocity has a negligible effect on the properties of dried product, but it determines the droplet residence time. For WPI it is better to dry slowly at moderate temperatures than to dry faster at high temperatures, to prevent too much denaturation. It is also shown that employing pulsation is a better strategy than increasing temperature to enhance the drying process while preserving product quality. • Whey protein denaturation during droplet drying in pulsating flow is modelled. • Denaturation is faster at higher temperature, stronger pulsation, and smaller droplet size. • Small droplets stay longer in drying chamber, getting more dried and denatured. • Pulsation reduces quality differences due to different initial droplet size distributions. • Product quality is retained better by pulsating flow than by increasing temperature.

Effects of heating temperatures and pH of skim milk fortified with milk protein concentrate on the texture and microstructure of high-protein yoghurts

Texture and microstructure of high-protein yoghurts manufactured from milk protein concentrate (MPC) fortified skim milk heated at different temperatures (75, 80, 85, 95 °C for 10 min) and pH (6.5, 6.7, 6.9, 7.1) were studied. Whey protein (WP) denaturation increased from 16% to 89% with increasing heating temperatures, while WP binding to casein micelles decreased from 95% to 21% with increasing heating pH. Gelation pH during fermentation increased with increasing heating temperatures and pH. Gel network became denser when heating temperatures and pH increased. Firmness and coagulum particle size of yoghurts increased with increasing heating temperatures and pH. Water-holding capacity reached above 87%, and increased slightly with increasing heating temperatures and pH, followed by slight decreases during storage. Reducing heating temperatures and pH of milk base offers potential strategies to obtain a desirably smooth texture without excessive syneresis for high-protein yoghurts manufactured by concentration before fermentation.

Effect of skim milk addition and heat treatment on characteristics of cow milk Ricotta cheese manufactured from Cheddar cheese whey

The effect of varying levels of Cheddar cheese whey to skim milk (70:30, 80:20, 90:10) ratio and heat treatments (95, 90 and 85 °C holding for 10, 20 and 30 min each) on the properties of cow milk Ricotta cheese were investigated. The composition, yield, solids recoveries, colour, spreadability and sensory properties were significantly influenced. The composition and solids recoveries increased with increasing skim milk quantity and severity of heat treatment, but higher skim milk had adverse effect on colour, spreadability and sensorial acceptability. The lower proportion of skim milk (90:10) and higher heating temperature (95 °C) resulted in Ricotta with high moisture, fat, protein recovery, colour and spreadability and sensory acceptability, as also corroborated by principal component analysis (PCA). PCA extracted five principal components which explained 92.02% variance and grouped cheeses according to the level of cheese milk and heating temperature which are having similar properties. It is possible to produce whiter, uniform, easily spreadable, shiny, soft, creamy, granular, adhesive, and highly acceptable Ricotta from mixing 90 part of Cheddar whey and 10 parts skim milk in cheese milk and heat-treating at 95 °C/10 min. In this formulation, whey proteins denaturation and association with casein micelle were validated by RP-HPLC and SDS-PAGE.

The Effect of Heat Treatment on Cow's Milk Protein Profiles.

Milk is a food of high nutritional value processed by heat treatment. Heat treatment of milk is a technological process designed to inhibit the growth of microorganisms and extend the shelf life of products. The heating process directly affects the molecular structure of whey proteins by the process of denaturation. It leads to the formation of a whey protein–casein polymer complex. Based on these facts, milk heat-treatment conditions should be controlled during milk processing. This work focuses on describing the whey protein denaturation process and formation of the complex of whey protein with casein. The effect of heat treatment on individual milk protein fractions alpha-casein (α-cas), beta-casein (β-cas), kappa-casein (κ-cas), beta-lactoglobulin (β-lg) and alpha-lactalbumin (α-la) was studied by SDS-PAGE. Formation of the whey protein–casein polymer complex increased significantly (p < 0.05) on increasing the temperature and duration of the heat treatment.