Whey Protein Denaturation Research Articles

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels.

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.

High pressure microfluidization treatment of heat denatured whey proteins for improved functionality

Solutions (5% protein) of a whey protein concentrate (WPC) in fresh acid whey or in water, as well as the fresh whey alone, were adjusted to pH 5.8, 4.8 or 3.8, heat treated at 90 °C for 10 min and further exposed to high pressure (150 MPa) microfluidization treatment. The volumes of sediment after centrifugation were recorded as a measure of the degree of insolubility of the proteins. Microfluidization disrupted the heat-induced aggregates into non-sedimenting whey protein polymers so that in some cases, especially at pH 3.8, the products studied were almost completely resistant to sedimentation after the microfluidization treatments. Heat denatured/microfluidized whey proteins reaggregated upon subsequent heating, with the pH having a major impact on the amount of sediment produced. Microfluidization of aqueous WPC solutions heat-treated before spray- or freeze-drying substantially increased the solubility of the powders upon reconstitution. Heat-induced viscoelastic gels were produced from freeze-dried microfluidized samples processed at pH 3.8 and reconstituted to solutions containing 12% (w/w) protein.

Bound water measurements for aqueous protein solutions and food gels

The role of aqueous media in the stabilization of globular proteins and formation of gels was studied by absorption millimeter spectroscopy. This method allowed to measure bound water, the fraction of water which had decreased rotational mobility owing to the presence of solute. Hydration data for globular proteins were compared with data obtained previously for low-weight molecules and groups. It was found that rotational mobility of water molecules in the hydration shells of various kinds of solutes (groups) decreased in the following order: water structure breaking compounds>polar groups>unfolded proteins>globular proteins>non-polar groups. Time courses of the storage modulus were determined for the chemical acidification by glucono-δ-lactone (GDL) of milk samples prepared from skimmed milk powder (SMP). Gelation of unheated milk was a monotonous process that started at pH 4.9. Heat-treated milk from SMP (16 and 14 g per 100 ml) acidified by GDL (3 g per 100 ml) at 43 °C gave non-monotonous kinetics of gelation with two phases corresponding to the mechanisms induced by denatured whey proteins at pH>5 and by casein–casein interactions at pH 4.8–4.9. For heat-treated milk, measurement of bound water gave two stages of decrease in water mobility. Additional hydration of SMP during acidification gave 0.15–0.2 g and 0.8 g bound H 2O per gram of SMP for unheated and heat-treated milk, respectively.

Formation of soluble and micelle-bound protein aggregates in heated milk.

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.

Firmness of pressure-induced casein and whey protein gels modulated by holding time and rate of pressure release

The main protein fractions of milk, casein and whey protein, may react differently to processing due to their amino acid sequence and conformation and their ability to build up intermolecular covalent and non-covalent bonds. Gels made from solutions of micellar casein and whey protein isolate were induced by applying ultra-high pressure (600 MPa/30 °C). The pressure built up was held constant at 200 MPa min −1, while the holding time of 0.15 and 30 min was varied as well as the pressure release rate (20, 200, 600 MPa min −1). The firmness of the pressure-induced whey protein gel was essentially influenced by the degree of whey protein denaturation and determined by the amount of disulfide bonds stabilizing the gel microstructure. In contrast, the firmness of pressure-induced casein-based gels was mainly influenced by pressure release rate. A slow pressure release produced a rough and weak gel. A high release rate, however, caused a homogeneous microstructure to be formed that created a high level of firmness.

Impaired Rennetability of Heated Milk; Study of Enzymatic Hydrolysis and Gelation Kinetics

Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of κ-casein. During cheese production rennet cleaves κ-casein into casein macropeptide and para-κ-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of κ-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.

Heat induced gelation of acid milk: balance between weak and covalent bonds.

Gelation of acidified milk at pH[ges ]5, after heat treatments is a well known phenomenon, due to the precipitation of whey proteins, and especially β-lactoglobulin onto κ-casein (Sawyer, 1969). High heat treatments cause denaturation of whey proteins which associate with κ-casein through disulphide interchange reactions (Hill, 1989). Since their charge is reduced, the denatured proteins associated with casein micelles become susceptible to aggregation when milk is then acidified, which promotes enhanced protein–protein interactions (Lucey et al. 1997). The gelation phenomenon involves disulphide bonds (Hashizume & Sato, 1988; Goddard, 1996) which are responsible for the gel firmness (Goddard, 1996). However, other interactions between proteins can occur, such as hydrogen and hydrophobic bonds, especially at the initial stage of interactions (Haque et al. 1987; Haque & Kinsella, 1988; Jang & Swaisgood, 1990). It is therefore relevant to investigate a possible contribution of weak linkages to the gel structure and firmness.

Quantification of heat-induced casein–whey protein interactions in milk and its relation to gelation kinetics

This paper describes a quantitative study on the distribution of denatured whey proteins over whey protein aggregates and whey proteins associated with the casein micelles in heated milk. This was done by combination of enzymatic fractionation and capillary electrophoresis. More severe heat treatment at the natural pH of milk caused more denaturation, but the ratio of denatured whey proteins associated with the casein micelle and present in aggregates remained constant. The distribution is related to changed acid-induced gelation properties and textural properties of milk-derived products. We clearly demonstrated that the observed shift in gelation pH of heated milk is linearly correlated with the distribution of denatured whey proteins. Quantitative description will allow better control and tuning of the final gel properties of milk-derived products.

Low-Fat Set Yogurt Made from Milk Subjected to Combinations of High Hydrostatic Pressure and Thermal Processing

The combined use of high hydrostatic pressure (300 to 676MPa, 5min) and thermal treatment (85°C, 30min) in milk for the manufacture of low-fat yogurt was studied. The objective was to reduce syneresis and improve the rheological properties of yogurt, reducing the need for thickeners and stabilizers. The use of high hydrostatic pressure alone, or after thermal treatment, reduced the lightness and increased the viscosity of skim milk. However, milk recovered its initial lightness and viscosity when thermal treatment was applied after high hydrostatic pressure. The MALDI-TOF spectra of skim milk presented monomers of whey proteins after a treatment of 676MPa for 5min. Yogurts made from skim milk subjected to 400 to 500MPa and thermal treatment showed increased yield stress, resistance to normal penetration, and elastic modulus, while having reduced syneresis when compared to yogurts from thermally treated or raw milks. The combined use of thermal treatment and high hydrostatic pressure assures extensive whey protein denaturation and casein micelle disruption, respectively. Although reaggregation of casein submicelles occurs during fermentation, the net effect of the combined HHP and thermal treatment is the improvement of yogurt yield stress and reduction of syneresis.

Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk.

Skim milk was adjusted to pH values between 6.5 and 6.7 and heated (80, 90, and 100 degrees C) for up to 60 min. Changes in casein micelle size, level of whey protein denaturation, and level of whey protein association with the micelles were monitored for each milk sample. Changes in casein micelle size were markedly affected by the pH at heating. At low pH (6.5-6.55), the casein micelle size increased markedly during the early stages of heating, and the size plateaued on prolonged heating. The maximum increase in size was approximately 30-35 nm. In contrast, at high pH (6.7), much smaller changes in size were observed on heating and the maximum increase in size was only approximately 10 nm. An intermediate behavior was observed at pH values between these two extremes. The rate of denaturation of the major whey proteins, alpha-lactalbumin and beta-lactoglobulin, was essentially unaffected by the pH at heating for the small pH changes involved in this study, and the changes in casein micelle size were poorly related to the level of whey protein denaturation. In contrast, the level of denatured whey proteins associating with the micelles was markedly dependent on the pH at heating, with high levels of association at pH 6.5-6.55 and low levels of association at pH 6.7. Changes in casein micelle size were related to the levels of denatured whey proteins that were associated with the casein micelles, although there was a small deviation from linearity at low levels of association (<15%). Further studies on reconstituted and fresh milk samples at smaller pH steps confirmed that the association of whey proteins with the casein micelles was markedly affected by the pH at heating. These results indicate that the changes in casein micelle size induced by the heat treatment of skim milk were a consequence of the whey proteins associating with the casein micelles and that the level of association was markedly influenced by small pH changes of the milk. It was not possible to determine whether the association itself influenced the casein micelle size or whether parallel reactions involving micellar aggregation caused the increase in micelle size as whey protein association progressed.

Transfer of Protein from Milk to Cheese

This report concerns measurement of paracasein in milk and transfer of protein from milk to cheese. In the main experiment, two vats of Cheddar cheese were made from each of 11 lots of milk from one large herd over a period of 7 mo.Exclusion of solutes from moisture in paracasein micelles in milk and cheese was central to estimation of paracasein and to the transfer of protein from milk to cheese and whey. Solute-exclusion by paracasein and its changes during cheesemaking could be visualized by considering paracasein micelles to be a very fine sponge. The sponge excludes solutes, especially the large solutes like whey proteins. The sponge shrinks during cheesemaking and expels solute-free liquid, thereby slightly diluting the whey surrounding the micelles inside the curd.Paracasein N in milk was calculated as the difference between total milk N and rennet whey N, the latter adjusted to its level in milk. Adjustment used appropriate solute-exclusion factors (h) of the protein fractions of whey and 1.08 for paracasein and associated salts. They were combined to give a factor Fpc, which adjusted the level of rennet whey N to its level in milk: 1.001×(1−1.01×FM/100−Fpc×pc/100), where FM = fat in milk, pc = estimated paracasein, and 1.001 = dilution of milk by chymosin and CaCl2. The mean Fpc was 3.03. Differences in values were small among different procedures for calculating paracasein, but they are considered to be important because they represent biases, which, in turn, are important in analyses commercially.We conclude that solute exclusion by moisture in paracasein must have decreased during cheesemaking because the ratio of moisture to paracasein in the final cheese was 1.5, much less than the h of 2.6 for serum proteins by paracasein. Release of solute-excluding moisture from paracasein during cooking was likely the reason for lower total N in cheese whey than in the rennet whey in the paracasein analysis.Paracasein, estimated to be in cheese, curd fines, salted whey, and whey during cheddaring, was 98.21, 0.20, 0.25 and 0.19%, respectively, of the paracasein in milk for a total of 98.85% (SD of 22 vats = 0.46); the location of the missing paracasein is not known. On the other hand, recovery of milk N in cheese and wheys was 99.92% (SD = 0.37%). Nitrogen in paracasein and its hydrolysis products in cheese was estimated to be 98.51% of total cheese N.Proteose-peptone from milk appeared not to be included with the paracasein in appreciable amounts. Some was apparently included with denatured serum proteins during Rowland fractionation of whey, perhaps as a coprecipitate. Measured paracasein would include fat globule membrane proteins in milk containing fat, and denatured whey proteins in heated milks. It was concluded that the method of measurement and the associated calculations are integral parts of the definition and quantification of paracasein in milk.

Association of denatured whey proteins with casein micelles in heated reconstituted skim milk and its effect on casein micelle size

When skim milk at pH 6.55 was heated (75 to 100 degrees C for up to 60 min), the casein micelle size, as monitored by photon correlation spectroscopy, was found to increase during the initial stages of heating and tended to plateau on prolonged heating. At any particular temperature, the casein micelle size increased with longer holding times, and, at any particular holding time, the casein micelle size increased with increasing temperature. The maximum increase in casein micelle size was about 30-35 nm. The changes in casein micelle size were poorly correlated with the level of whey protein denaturation. However, the changes in casein micelle size were highly correlated with the levels of denatured whey proteins that were associated with the casein micelles. The rate of association of the denatured whey proteins with the casein micelles was considerably slower than the rate of denaturation of the whey proteins. Removal of the whey proteins from the skim milk resulted in only small changes in casein micelle size during heating. Re-addition of beta-lactoglobulin to the whey-protein-depleted milk caused the casein micelle size to increase markedly on heat treatment. The changes in casein micelle size induced by the heat treatment of skim milk may be a consequence of the whey proteins associating with the casein micelles. However, these associated whey proteins would need to occlude a large amount of serum to account for the particle size changes. Separate experiments showed that the viscosity changes of heated milk and the estimated volume fraction changes were consistent with the particle size changes observed. Further studies are needed to determine whether the changes in size are due to the specific association of whey proteins with the micelles or whether a low level of aggregation of the casein micelles accompanies this association behaviour. Preliminary studies indicated lower levels of denatured whey proteins associated with the casein micelles and smaller changes in casein micelle size occurred as the pH of the milk was increased from pH 6.5 to pH 6.7.

Properties of casein micelles reformed from heated mixtures of milk and ethanol

Addition of an aqueous solution of ethanol (>30% ethanol) to skim milk, followed by heating to >60 °C, resulted in the mixture becoming translucent; objectively, its Hunter L-value was reduced. The degree of translucence increased with increasing ethanol concentration and temperature; the effect was not fully reversible on cooling, as cooled mixtures had higher turbidity values than unheated mixtures. Following removal of ethanol by vacuum rotary evaporation and readjustment to the original milk solids concentration, the milk contained large protein aggregates (referred to as reformed micelles), the properties of which were studied. Milk containing reformed micelles had a type B heat coagulation time–pH profile and was less stable to ethanol than control milk. The mechanism responsible for these changes in the properties of milk appeared to be independent of calcium or other milk salts, but was probably linked to ethanol-induced denaturation of whey proteins.

Texture of acid milk gels: formation of disulfide cross-links during acidification

Denaturation of whey proteins during pasteurization of milk results in the formation of whey protein aggregates and whey protein-coated casein micelles. After cooling a substantial number of thiol groups remains exposed. Formation of larger disulfide-linked protein structures during acidification at ambient temperature was demonstrated by analytical methods. The time-dependent formation of these structures attributed significantly to the mechanical properties of acid milk gels, resulting in gels with an increased storage modulus and hardness. Addition of the thiol-blocking agent N-ethylmaleimide prevented the formation of disulfide-linked structures. The mechanical properties are shown to be the result of the contribution of denatured whey proteins to the protein network as such and the additional formation of disulfide bonds. Surprisingly, these sulfhydryl group-disulfide bond interchange reactions take place at ambient temperature and under acidic conditions. Therefore, the disulfide cross-linking is highly relevant for textural properties of acid-milk products, like yoghurt.

Casein–whey protein interactions in heated milk: the influence of pH

Heat-treatment of milk causes denaturation of whey proteins, leading to a complex mixture of whey protein aggregates and whey protein coated casein micelles. In this paper we studied the effect of pH-adjustment of milk (6.9–6.35) prior to heat-treatment on the distribution of denatured whey proteins in aggregates and coating of casein micelles. Proteins were fractionated using an alternative fractionation technique based on renneting. Acid- and rennet-induced gelation of these milks were used to obtain more information on the characteristics of the milk. Acid-induced gelation appeared to be mainly influenced by the presence of whey protein aggregates. Rennet-induced gelation was determined by the whey protein coating of the casein micelles. Both the quantity of whey proteins present on the surface of the casein micelles and the homogeneity of the coating were determining the renneting properties. These results extend the current knowledge on pH dependent casein–whey protein interactions. In order to present a clear picture of the changes occuring during heat treatment of milk at various pH, the results are summarized in a model. In this model we propose that heating at a pH>6.6 lead to a partial coverage of the casein micelles and the formation of separate whey protein aggregates. Heating at a pH<6.6 lead to an attachment of all whey proteins to the casein micelles. At pH 6.55 the coverage is rather homogeneous but lowering the pH further lead to an inhomogeneus coverage of the casein micelles. Surprisingly small changes of the pH at which the milk was heated had considerable effects on the gelation behaviour both in renneting and in acid gelation.

Effect of pre-heating on the foaming properties of whey protein isolate using a membrane foaming apparatus

Abstract The effect of the thermal denaturation of whey proteins on the formation, stability and structure of their respective foams was examined. A membrane foaming apparatus, which is a very gentle foaming method, was used to produce the foams. It was shown that the denaturation of the β -lactoglobulin, the main component in whey protein isolate, strongly improves the foam stability. At a denaturation degree >70%, it is possible to reduce drainage to a large extent. The image analysis demonstrated that higher levels of denaturation of the proteins, and thus higher viscosities of the protein solution, produced coarser foam textures with larger bubbles. The incorporation of the bubbles was more difficult when the viscosity of the continuous phase was high.

Preliminary observations on the effects of milk fortification and heating on microstructure and physical properties of stirred yogurt

The aim of this work was to study how milk fortification and heating affect yogurt microstructure (micellar characteristics, protein network) and physical properties (viscosity, water-holding capacity (WHC), and graininess). Milk was fortified with skim milk powder (control), whey protein concentrate (WPC), caseinate, or a mixture of caseinate and whey protein. Two heat treatments were applied, giving average whey protein denaturation levels of 58% and 77%. For caseinate-enriched yogurts, the heating effect was negligible. When milk was enriched with WPC, heating led to a high level of cross-linking within the gel network. Heating increased yogurt viscosity and WHC, but also graininess. When milk was fortified with a blend of WPC and caseinate giving a whey protein-to-casein ratio of 0.20, the yogurt viscosity was greatly improved, while graininess was kept low. The results show a relationship between micelle solvation and yogurt microstructure, as well as micelle size in milk base and yogurt graininess.

Thermal modifications of structure and co-denaturation of alpha-lactalbumin and beta-lactoglobulin induce changes of solubility and susceptibility to proteases.

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.

Whey protein aggregation under shear conditions – effects of pH‐value and removal of calcium

The effects of pH‐value and a reduction in calcium content on the kinetics of whey protein denaturation and the aggregation behaviour, under shear in a scraped surface heat exchanger, were examined. The denaturation rate of β‐lactoglobulin at 80 °C is considerably retarded as the pH‐value decreases from pH 6.7 to 4.5. Aggregates which are produced under shear between pH 4 and 5.5 reveal a small particle size (&lt;5 μm) irrespective of the lactose content and the heating temperature. This is attributed to the low reactivity of the thiol groups and the small net charge of the proteins in this pH‐range. At a reduced calcium concentration the heat‐ and shear‐treatment resulted in a gritty structure with large rubber‐like particles. These are not to be taken as primary whey protein aggregates but as fragments of a fine‐stranded gel.

Gelation of Mixed Gels Containing κ‐Carrageenan and Skim Milk Components

ABSTRACT: The gelation characteristics of mixed gels containing κ‐carrageenan and skim milk or milk fractions (skim milk permeate or retentate) obtained by ultrafiltration were examined. Increasing the skim milk solids content of mixtures containing carrageenan increased setting temperatures and gel strength. The milk proteins contributed to gel strength but did not influence the setting temperature of mixtures. The binding of denatured whey proteins to casein micelles affected gel network formation of milk‐carrageenan mixtures containing 10% milk solids. Network formation in mixed gels containing carrageenan and milk or milk fractions was initiated by the carrageenan component and dictated primarily by the ionic content of the mixtures.