Whey Protein Denaturation Research Articles

Rheological Properties of Kefir as Affected by Heat Treatment and Whey Protein Addition

Abstract The effects of heat treatment and whey protein addition on the fermentation time and the rheological properties of kefir determined using a pneumatic tube viscometer of novel design, were investigated. Kefir samples were prepared with and without the addition of whey protein concentrates (WPC) at varying concentrations, while heat treatment of the milk was made prior or after WPC addition (resulted to native or denatured whey proteins, respectively). Increasing WPC concentration resulted to increased lactic acid concentration and reduced fermentation time. The flow curves of the samples suggested that kefir behaves as a pseudoplastic fluid. The apparent viscosity of kefir samples was increased with increasing WPC concentration. However, the “state” of added whey proteins greatly affected the rheological properties of kefir. Particularly, denatured whey proteins resulted to higher consistency index values and thus apparent viscosity and lower flow behavior index values, when compared with native whey proteins. Practical Applications Kefir is a fermented beverage, which is reported of being beneficial to health. The large number of different microorganisms found in it and the variety of bioactive compounds produced during fermentation distinguishes it from other probiotic products. The study of the rheological properties of kefir as well as the factors that have an effect on them is of great scientific and commercial interest, because they can affect the quality of the final product and thus its acceptance by the consumers, the promotion of its standardization, and allow the correct selection of equipments like pumps and filling machines in the industry. The addition of whey protein concentrates (WPC) can enhance rheological properties of kefir and thus improve the quality of the final product. Furthermore, the “state” of the added whey proteins greatly affects the impact degree on the rheological behavior of kefir.

Effect of pre- and post-heat treatments on the physicochemical, microstructural and rheological properties of milk protein concentrate-stabilised oil-in-water emulsions

The effect of heat treatment on the physical stability of milk protein concentrate (MPC) stabilised emulsions was investigated; 3% (w/w) MPC dispersions were preheated at 90 °C for 5 min at neutral pH prior to emulsification. Heat-treated (120 °C, 10 min) emulsions stabilised by preheated MPC had slightly fewer droplet–droplet interactions than that stabilised by unheated MPC because the whey proteins were pre-denatured (∼90% denaturation of the total whey proteins), which led to a reduction in subsequent heat-induced droplet–droplet and droplet–protein interactions. Emulsions stabilised by calcium-depleted MPC were also investigated. The presence of some non-micellar casein fractions gave better emulsification and may have conferred a protective stabilising effect on whey protein aggregation, in both the dispersed phase and the continuous phase during the secondary heat treatment. It was concluded that calcium manipulation and thermal modification of MPC can be utilised to control the functionality in oil-in-water emulsions.

Physical properties of pizza Mozzarella cheese manufactured under different cheese-making conditions

The effect of manufacturing factors on the shreddability and meltability of pizza Mozzarella cheese was studied. Four experimental cheeses were produced with 2 concentrations of denatured whey protein added to milk (0 or 0.25%) and 2 renneting pH values (6.4 or 6.5). The cheeses were aged 8, 22, or 36d before testing. Shreddability was assessed by the presence of fines, size of the shreds, and adhesion to the blade after shredding at 4, 13, or 22°C. A semi-empirical method was developed to measure the matting behavior of shreds by simulating industrial bulk packaging. Rheological measurements were performed on cheeses with and without a premelting treatment to assess melt and postmelt cheese physical properties. Lowering the pH of milk at renneting and aging the cheeses generally decreased the fines production during shredding. Adding whey protein to the cheeses also altered the fines production, but the effect varied depending on the renneting and aging conditions. The shred size distribution, adhesion to the blade, and matting behavior of the cheeses were adversely affected by increased temperature at shredding. The melting profiles obtained by rheological measurements showed that better meltability can be achieved by lowering the pH of milk at renneting or aging the cheese. The premelted cheeses were found to be softer at low temperatures (<40°C) and harder at high temperatures (>50°C) compared with the cheeses that had not undergone the premelting treatment. Understanding and controlling milk standardization, curd acidification, and cheese aging are essential for the production of Mozzarella cheese with desirable shreddability and meltability.

The effect of pre-denatured whey proteins on the textural and micro-structural properties of model processed cheese spreads

Whey proteins (WP) were heated in a scraped-surface heat exchanger to produce products with different levels of denaturation. Model processed cheese spreads containing the WP were prepared. Rheological measurements showed that higher levels of denaturation in the WP produced softer (lower elastic modulus) cheeses. Temperature scans indicated that the cheeses prepared with high levels of native whey proteins did not melt (the elastic modulus was higher than the viscous modulus at all temperatures), whereas those prepared with high levels of denatured whey proteins melted on increasing the temperature. Microstructural examination of selected cheeses indicated that, when denatured whey proteins were used in processed cheeses, the whey proteins were incorporated as large aggregates dispersed within the cheese matrix. These aggregates did not contribute to, and may disrupt the structure of the cheese. When native whey proteins were used, they were incorporated into the cheese matrix, producing a denser network structure.

Encapsulation of Lactobacillus rhamnosus GG in microparticles: Influence of casein to whey protein ratio on bacterial survival during digestion

Abstract Encapsulation of Lactobacillus rhamnosus GG in various microparticles made of only milk proteins (casein, native whey and/or denatured whey proteins) was done. The microparticles obtained were rather similar in shape (mostly round) and size (around 60 μm) whatever the formulation but the obtained gel presented different elasticity (varying between 61 and 96 Pa). An original equipment involving a granulo-morphometer coupled to a thermostated reactor was developed and validated to visualize in situ the microparticles during digestion. Although the initial particles were similar, their disintegration in simulated gastric media was totally different and characterized by two stages. An initial decrease in particle size more or less quick depending on the protein composition was followed by a stable phase characterized by the particle size and shape retention. At the end of gastric digestion, a significant amount of intact particles was still noticeable for each formulation. Nevertheless, the formulation containing a mix of casein and denatured whey presented the best bacterial survival (99%) and encapsulation rate (97%) in comparison with formulations containing either only casein or casein and native whey or casein in mixture with native and denatured whey proteins. Industrial relevance This paper is part of a global project entitled “Structured dairy matrices to enhance probiotic efficiency”. The entire project will provide milk structured matrices allowing the stabilization and the vectorization of Lactobacillus rhamnosus GG (LGG). This project will consist of four main axes: milk constituent's interactions with LGG, stabilization process implementation, and structural and functional characterization of the matrices obtained. The scientific objective is to propose models connecting process parameters, matrix structure (from an atomic, molecular to a macro scale) and their functionality. This implies the in-depth study of interactions between milk components and probiotic strain. For this purpose, the use of genetically modified strain of LGG will allow the identification of biomolecules interacting with milk matrices. The industrial aims are to optimize and control the processes to suit the needs of industrial criterions: encapsulation rate, gastric resistance, intestinal release, storage in the final food….

Effect of Transglutaminase on the Mechanical and Barrier Properties of Whey Protein/Pectin Films Prepared at Complexation pH

The behavior of pectin and thermally denatured whey proteins at both different protein/polysaccharide ratios and different pH values was investigated. Our findings suggest the formation at pH 5.1 (complexation pH) of transglutaminase-catalyzed cross-links among soluble ionic whey protein/pectin complexes, which could be responsible for the observed increase of both tensile strength (2-fold) and elongation to break (10-fold) of films obtained in the presence of enzyme. Conversely, a significant reduction of elasticity, probably due to the formation of covalent bonds among single whey protein molecules, was observed when the films were prepared in the presence of the enzyme at pH 6.0. In addition, the presence of the enzyme at complexation pH significantly reduced film permeability. Atomic force and scanning electron microscopy revealed significant changes in the microstructure of the films prepared in the presence of TGase as well as in the morphology of their surface.

Denaturation of whey proteins as a function of heat, pH and protein concentration

This study examined the influence of pH and protein concentration on whey protein (WP) denaturation during heating at 140 °C. The denaturation of β-Lg followed first-order reaction kinetics and depended on protein concentration, being minimum at 10%. However, the lowest rate of denaturation was observed at acidic pH, suggesting increased stability at pH 4, which was confirmed by significantly increased solubility and low turbidity, as well as the electrophoretic patterns of the major WP. The results of differential scanning calorimeter tests revealed the concentration-dependence of WP denaturation. Such information may be useful for defining conditions (pH, protein concentration, temperature) required to produce microparticulated whey proteins with improved functional characteristics.

Effect of phospholipid molecular structure on its interaction with whey proteins in aqueous solution

Because most dairy processes require heat treatment to ensure microbiological safety during storage, dairy industry has invested a lot of effort to acquire knowledge related to all the changes that may take place during heating. More precisely, measures have to be taken to avoid undesired changes that can badly affect the quality of the final product. Whey protein denaturation is one of the major changes that take place upon sterilization. The purpose of the present study was to investigate and compare the degree and type of interaction that occurs between whey protein isolate and different kinds of dialkyl phospholipids, as a function of their molecular structure, upon heating at different temperatures. Circular Dichroism (CD) experiments were performed to assess the extent of secondary structural changes of β-lactoglobulin upon incubation in the presence of both anionic and zwitterionic phospholipids. The far-UV spectra of the former revealed a significant change in ellipticities in the region between 208 and 222 nm, which represents the α-helical content of peptides and proteins upon incubation in the presence of anionic phospholipids at temperatures above their phase transition temperature. On the other hand, these changes were not evident upon incubation with phospholipids in the gel state or when applying zwitterionic phospholipids. Nuclear Magnetic Resonance (NMR) has been used to investigate the whey protein–surfactant interaction into more detail. The chemical shift of the surfactant methyl groups toward a more hydrophobic region in the NMR spectra revealed a hydrophobic whey protein–phospholipid interaction. This finding was further supported by Saturation Transfer Difference-NMR (STD-NMR) measurements. The NMR experiments also revealed that the lack of protein structural reorganization upon incubation in the presence of zwitterionic (lyso)-phospholipids could not be ascribed to a lack of interaction, but was caused by the fact that the interaction did not affect the protein's structure.Overall, this work indicated that phospholipids can modify the secondary structure of whey proteins (i.e. when using anionic phospholipids in the liquid crystalline state) due to hydrophobic interactions. In addition, the results prove that the heat stability of whey protein containing products may be optimized by appropriate selection of the phospholipid composition.

Optical backscatter method for determining thermal denaturation of β-lactoglobulin and other whey proteins in milk

The heat denaturation of whey proteins affects the functional properties of milk. Correlations of β-lactoglobulin (β-LG) denaturation to gelation time, gel firmness, and gel moisture content have been widely documented. Currently, no technique is available for quantifying β-LG denaturation in milk without altering its native composition or requiring a laborious procedure. The goal of this study was to establish if an optical backscatter response of whey protein denaturation during milk heat treatment could be determined that would be the basis for an inline optical measurement technology. The experimental design consisted of 1 factor (time at 80°C) and 6 levels (0, 3, 5, 7, 12, and 25min). Physicochemical analysis performed indicated that β-LG denaturation followed a first-order response during thermal treatment. The light backscatter response, measured as a ratio of two 25-nm wave bands (832.5nm/382.5nm), significantly correlated to β-LG denaturation and had a 14% increase for milk with 75% β-LG denaturation. The strength of the optical response at the proposed wave bands and their correlation to denaturation suggests that light backscatter could potentially be used to measure β-LG and other whey protein denaturation inline.

High-pressure treatment of milk in industrial and pilot-scale equipments: effect of the treatment conditions on the protein distribution in different milk fractions

The effects of two different high-pressure (HP) equipments, operating at industrial- and pilot scales, and of the HP-release rate on the contents of non-sedimentable proteins and denatured whey proteins were investigated after treatments of skim milk—from 250 to 650 MPa. Non-sedimentable caseins and denatured whey proteins significantly increased with the pressure level. The industrial-scale equipment produced lower micellar disintegration than the pilot-scale equipment with similar degrees of whey protein denaturation. Ultracentrifugation supernatants obtained from skim milk at 100,000×g and 20 °C for 1 h were also HP-treated for comparative purposes, showing that, in skim milk, the presence of casein promoted the denaturation of whey proteins, although the extent of whey protein denaturation did not influence the release of casein to the soluble phase. Furthermore, most denatured whey proteins remained soluble after treatment in both equipments. In the pilot-scale equipment, the pressure-release rate influenced casein solubilization and whey protein denaturation.

Colloidal properties of concentrated heated milk

The colloidal properties of casein micelles in heated milk as a function of volume fraction are largely unknown. Heat-treatment of milk causes denaturation and unfolding of whey proteins, which then react with caseins to form a complex mixture of soluble whey protein aggregates and whey protein coated casein micelles. The distribution of the complexes between the soluble and the micellar phase depends on the pH of milk before heating. This work focused on the contribution of the heat induced complexes to the colloidal properties of concentrated casein micelles. Concentration was performed using osmotic stressing. Although there were differences in the apparent diameter after heating milk at pH 6.4, 6.8 or 7.0, redilution of the concentrated milk showed no irreversible aggregation of the casein micelles with concentration. Above 70 g L−1 protein, there was a decrease in the mean square displacement slope measured by light scattering, suggesting that above this concentration, the casein micelles were no longer free diffusing. The viscosity of the concentrated milk could be predicted using a rheological model for hard spheres assuming constant voluminosity.

The effect of various heat treatments on the antioxidant capacity of milk before and after simulated gastrointestinal digestion

Various reactions take place between compounds of milk during heat treatment including denaturation and aggregation of whey proteins, and formation of new complexes. In this study, the antioxidant capacities of raw, pasteurised and sterilised milk were determined both before and after an enzymatic in vitro digestion process, using a radical cation decolorisation assay. The mean values of raw, pasteurised and sterilised milk before simulated digestion were 4.02, 4.47 and 4.18 mM trolox/g, respectively, and these did not differ significantly. The antioxidant capacity of all milk types increased significantly at the end of the simulated gastrointestinal digestion, being 11.13, 12.33, 11.88 mM trolox/g for raw, pasteurised and sterilised milk, respectively.

In vitro interactions between probiotic bacteria and milk proteins probed by atomic force microscopy

Interactions between microbial cells and milk proteins are important for cell location into dairy matrices. In this study, interactions between two probiotic strains, Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, and milk proteins (micellar casein, native and denatured whey proteins) were studied. The bacterial surface characterization was realized with X-ray photoelectron spectroscopy (XPS) to evaluate surface composition (in terms of proteins, polysaccharides and lipid-like compounds) and electrophoretic mobility that provide information on surface charge of both bacteria and proteins along the 3–7 pH range. In addition, atomic force microscopy (AFM) enabled the identification of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. These specific events appeared to be more important for the GG strain than for the GR-1 strain, showing that matrix interaction is strain-specific. Furthermore, our study highlighted that in addition to the nature of the strains, many other factors influence the bacterial interaction with dairy matrix including the nature of the proteins and the pH of the media.

A comparative study on conventional and microwave-assisted extraction for microencapsulation of Garcinia fruit extract

(−)-Hydroxycitric acid (HCA) is the principal acid present in the fruit rinds of certain species of Garcinia and is reported to have various health benefits. However, HCA is highly hygroscopic in nature and becomes lactonised during evaporation and drying. To reduce the lactonisation of HCA, Garcinia cowa fruit extract was obtained through two different extraction techniques, autoclave and microwave-assisted extractions, and was then microencapsulated using whey protein isolate (WPI) and denatured whey protein isolate (DWPI) with a 1:1 wall-to-core ratio using a spray drying technique. The microwave-assisted extracts encapsulated with WPI and DWPI had higher free HCA and net HCA recovery than the autoclaved extract encapsulated with similar wall materials. Furthermore, the microwave-assisted extracts and the associated encapsulated samples had higher antioxidant activity than their counterparts. The encapsulation of the microwave-assisted extracts with both wall materials had little variations in their free (55.04 and 54.58% for WPI and DWPI, respectively) and net HCA recovery (84 and 82%) and antioxidant activity (13.3 and 13.6%), which signified a smaller influence of the wall materials. These results indicated that microwave-assisted extraction had a higher extraction efficiency, encapsulation efficiency and antioxidant activity with both wall materials.

Influence of heat and pH on structure and conformation of whey proteins

The aim of this study was to understand the fundamental interactions responsible for aggregation of whey proteins (WPs) at pH 6 and 3 during heating at 140 °C for 30 s in the presence of different acidulants. The conformational changes in the various heat-treated WP dispersions were studied using chemical bond blockers and analysed using differential scanning calorimeter thermograms, polyacrylamide gel electrophoresis and turbidity measurements. Overall, the results indicated that WPs were denatured mainly by disruption of hydrophobic interactions, and that the extent of WP denaturation at pH 3 was affected by the type of acidulant used. The type of acidulant affected the extent of formation of additional high or medium molecular weight aggregates during heating at pH 3, while the types of interactions involved in the formation of such aggregates were affected by the pH at heating.

Effect of the high-pressure-release phase on the protein composition of the soluble milk fraction

This paper describes a study on the influence of the high-pressure-release rate on the protein distribution between the soluble and colloidal fractions of milk. Skim milk, without or with sulfhydryl-blocking agents, was pressure treated at 250 and 350MPa for 5 to 15min at 25°C, applying different pressure-release rates (pressure-release times between 0.07 to 10min). The liberation of caseins to the soluble phase and the denaturation of whey proteins were assessed. A significantly higher increase in the content of soluble casein took place during the pressure-release phase as compared with a pressure-holding phase. Denaturation of β-lactoglobulin mainly took place by -SH-S-S exchange reactions during the holding phase. The present results, thus, show a negligible influence of whey proteins on the increase in the content of nonsedimentable casein in pressure-treated milk and provide evidence for the importance of the pressure-release rate in this process, so that the slower the pressure release rate, the higher the level of soluble casein.

Effect of whey protein – alginate wall systems on survival of microencapsulated Lactobacillus plantarum in simulated gastrointestinal conditions

Whey protein isolate is a suitable carrier system for probiotics in addition to sustained and targeted release in gastrointestinal tract. In this study two different wall materials namely, whey protein isolate with sodium alginate and denatured whey protein isolate with sodium alginate were employed for microencapsulation of Lactobacillus plantarum (mtcc 5422) using spray drying and freeze drying techniques. Microencapsulated powders final moisture content, cell survival (before and after drying), particle morphology and stability in simulated acidic and bile conditions were investigated. It was observed that the moisture content of the spray dried powder was lower than freeze dried powder and spray drying method produced almost 9–12% less cell survival compared to freeze drying method. In simulated acidic and bile conditions denatured whey protein encapsulated cells showed better stability than undenatured whey protein. This study indicated that combination of denatured whey protein isolate and sodium alginate wall matrix was able to deliver probiotics with improved survival rate and suitable for controlled core release applications.

Heat induced casein–whey protein interactions at natural pH of milk: A comparison between caprine and bovine milk

This paper is a study on the distribution of the denatured whey proteins and κ-casein in soluble and micelle-bound complexes in heat treated caprine and bovine milk (90°C, 10min) at natural pH (6.71). Proteins were fractionated using fractionation technique based on renneting and were analysed by three electrophoretic techniques: native PAGE, SDS-PAGE under reducing and non-reducing conditions. Lower than 3% of the total β-LGs remained stable after heat treatment of both milk species, but bovine α-LA was more heat stable than its counterpart in caprine milk (29.6% against 3.82%). Denatured caprine whey proteins (>95%) were part of micelle-bound complexes whereas soluble complexes were not observed. Conversely, about 30% of denatured bovine whey proteins were involved in soluble complexes. About 24.2% of total κ-CN was included into complexes formed in heat-treated bovine milk whereas in heat-treated caprine milk this percentage is about three times higher. Caprine micelle-bound complexes, apart from whey proteins and κ-casein included also β-casein and αs2-casein, which were not found in their bovine counterparts. This knowledge could be very useful in understanding the differences in technological–functional properties of caprine and bovine milk and to enable better control of dairy processes.

Effect of heat treatment of rennet skim milk induced coagulation on the rheological properties and molecular structure determined by synchronous fluorescence spectroscopy and turbiscan

Heat treatment applied to milk induces denaturation of whey proteins, leading to a complex mixture of whey protein and whey protein coated casein micelles. The present paper investigates the effects of heat treatment (60 and 80°C during 20min) and rennet-induced coagulation temperature (30 and 40°C) determined by rheology, synchronous fluorescence spectroscopy (SFS) and turbiscan measurements. The gelation times determined by rheology and SFS increased with the increase of heat treatment applied to milk. The rise in temperature induced a decrease in the maximum curd firming rate and an increase in the viscosity of the investigated milk samples. The principal component analysis (PCA) applied, separately, to the SF and turbiscan spectra showed a clear discrimination between: (i) raw milks and heated milks; and (ii) milks renneted at 30°C from those renneted at 40°C. The results showed the ability of SFS as a rapid and non-destructive technique for the: (i) monitoring network structure and molecular interaction during the coagulation process; and (ii) determination of gelation time of rennet-induced coagulation of studied milk samples.

Acid-induced gelation behavior of casein/whey protein solutions assessed by oscillatory rheology.

Gelation process of acid-induced casein gels was studied using response surface method (RSM). Ratio of casein to whey proteins, incubation and heating temperatures were independent variables. Final storage modulus (G') measured 200min after the addition of glucono-δ-lactone and the gelation time i.e. the time at which G' of gels became greater than 1Pa were the parameters studied. Incubation temperature strongly affected both parameters. The higher the incubation temperature, the lower was the G' and the shorter the gelation time. Increased heating temperature however, increased the G' but again shortened the gelation time. Increase in G' was attributed to the formation of disulphide cross-linkages between denatured whey proteins and casein chains; whilst the latter was legitimized by considering the higher isoelectric pH of whey proteins. Maximum response (G' = 268.93Pa) was obtained at 2.7% w/w, 25°C and 90°C for casein content, incubation and heating temperatures, respectively.