Influence of pressure release rate and protein concentration on the formation of pressure-induced casein structures

The formation of pressure-induced casein structures (600 MPa for 30 min at 30 degrees C) was investigated for different pressure release rates (20 to 600 MPa min-1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min-1, while lower release rates resulted in less firm gels (200 MPa min-1) or liquid structures (20 MPa min-1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min-1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment.     

Effects of added whey protein aggregates on textural and microstructural properties of acidified milk model systems

Non-fat milk model systems containing 5% total protein were investigated with addition of micro- or nanoparticulated whey protein at two levels of casein (2.5% and 3.5%, w/w). The systems were subjected to homogenisation (20 MPa), heat treatment (90 °C for 5 min) and chemical (glucono-delta-lactone) acidification to pH 4.6 and characterised in terms of denaturation degree of whey protein, particle size, textural properties, rheology and microstructure. The model systems with nanoparticulated whey protein exhibited significant larger particle size after heating and provided acid gels with higher firmness and viscosity, faster gelation and lower syneresis and a denser microstructure. In contrast, microparticulated whey protein appeared to only weakly interact with other proteins present and resulted in a protein network with low connectivity in the resulting gels. Increasing the casein/whey protein ratio did not decrease the gel strength in the acidified milk model systems with added whey protein aggregates.     

Effect of pH at pressure treatment on the acid gelation of skim milk

Skim milk at pH between 6.4 and 7.3 was pressure treated at 200–600 MPa for 30 min and then slowly acidified with glucono-δ-lactone to form acid gels. Milks at low pH produced acid gels with low elastic moduli (final G′) and yield stresses and those at higher pH produced acid gels with higher final G′ and yield stresses. Pressure treatment disrupted the casein micelles at all pH and transferred high levels of casein to the serum phase. Denaturation of α-lactalbumin occurred at a pressure of 600 MPa only, and the level of denaturation increased with increasing pH. Denaturation of β-lactoglobulin (β-LG) occurred at all pressures, with the level of denaturation increasing with the magnitude of the pressure treatment and with pH. The denaturation of the whey proteins and the disruption of the casein micelles could not entirely account for the changes in the rheological properties of the acid gels, as denaturation of up to 50% of the whey proteins produced acid gels with very low final G′ and yield stresses. It is proposed that the pH and the magnitude of the pressure treatment affect the interactions of the denatured β-LG with the casein proteins in the pressure-treated milks, and that this affects the ability of the denatured β-LG to participate in the acid gel structures.Industrial relevanceThe control and manipulation of the firmness of acid skim milk gels is important in many dairy food applications such as yogurts and some types of cheeses. This study has demonstrated that acid gel firmness can be substantially manipulated when the milk is pH adjusted and pressure treated before acidification, and that these effects are different to those obtained through heating. The commercial uptake of high pressure processing in the dairy industry is dependent on this technology producing unique functional properties in milk when compared with traditional processing. The results of this study indicates that high pressure processing of milk may offer unique functional properties in acid gel applications which could be used for the development of new or improved dairy products.     

Effects of pH and ionic strength on the rheology and microstructure of a pressure-induced whey protein gel

The effects of pH and ionic strength (I) on the properties of a pressure-induced gel from a whey protein isolate (0.2 g mL^?1) were studied using a series of buffers with different pH and ionic strength (I). The rheological properties and water-holding capacity (WHC) of the gel changed in a complex pattern with pH and I but could be successfully modelled using an artificial neural network (ANN) model. The degree of cross-linking of S–S bonds was higher at pH 8.0 than at pH 5.0; consequently, a rigid gel was formed at alkaline pH values. The geometric dimensions of the microstructure decreased with increasing pH and decreasing ionic strength, and the WHC mainly depended on the geometric properties of the microstructure. These results indicate that pH and ionic strength influenced the gel properties by controlling the cross-linking reaction and phase separation that occur during gelation. They also confirmed the good potential application of ANN in studies of gelation.     

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.     

Influence of the Maillard reaction on the properties of cold-set whey protein and maltodextrin binary gels

The Maillard conjugation of proteins and reducing saccharides is used to modify the technological functionality of whey proteins. In this study, whey protein isolate (WPI) was conjugated with maltodextrin (at 1:1 ratio and two total solid contents of 100 and 200 mg mL⁻¹) through the Maillard reaction and used to form cold-set gels. The glycation reaction increased the strength of hydrogen bonding of whey proteins and preferentially modified α-lactalbumin, in comparison with β-lactoglobulin. It also increased the reducing power of binary protein-saccharide solution and allowed formation of self-standing cold-set WPI gel at a low protein content (i.e., ≈50 mg mL⁻¹). Microscopic imaging showed micro-phase separated maltodextrin domains, interrupting the protein network, in gels made of protein-maltodextrin physical mixtures, whereas Maillard conjugation resulted in more homogenous microstructures at both total solid contents. The Maillard reaction increased gel firmness and water-holding capacity and caused a reduction in the extent of gel swelling.     

The effect of calcium chloride addition on the microstructure and composition of Cheddar cheese

The addition of calcium is widely accepted as a tool in cheese-making but the effect on the microstructure of cheese during and following manufacture is not known. In this study, cheeses made with milk containing 200–600 mg L⁻¹ of additional CaCl₂ had significantly lower fat loss into the whey collected after cooking; however, the final fat composition or yield of cheese did not change. The microstructure of the gel with 300 or 600 mg L⁻¹ CaCl₂ addition was less porous and the cooked curd consisted of a denser protein network that may retain more fat during the early stages of manufacture. In contrast, the cheddared curd and cheese contained more micro-pores than cheeses with lower or no calcium addition. Such micro-pores could possibly be the channels by which fat escaped during pressing. This study shows that calcium addition altered the microstructure and pattern of fat loss during Cheddar manufacture.     

Heat and/or high-pressure treatment of skim milk: changes to the casein micelle size, whey proteins and the acid gelation properties of the milk

Skim milk was subjected to heat, pressure or combined processes. In general, higher levels of whey protein denaturation were observed for milk subjected to combined processes than those heat- or pressure-treated only. Heat treatment caused small changes to the casein micelle size. Pressure treatment decreased the casein micelle size; however, the effect was less marked when heat and pressure treatments were combined. Acidification of the skim milks produced gels with a range of firmness, yield stresses and yield strains depending on the treatments applied. These changes in acid gel properties were not related only to whey protein denaturation levels in the milks.     

Effect of high pressure - low temperature processing on composition and colloidal stability of casein micelles and whey proteins

The aim of this study was to identify the impact of high pressure treatments at sub-zero temperatures (high pressure - low temperature; HPLT) on milk proteins. Whey protein solutions, micellar casein dispersions and two mixtures (micellar caseins:whey proteins, 80:20 and 20:80, w/w) were pressure treated (100–600 MPa) at pH 7.0 or 5.8 at −15 °C, −35 °C and ambient temperature. Solubility data showed that whey proteins could only be affected by HPLT treatments at pH 7.0 if caseins were present, while effects could be induced at pH 5.8 without the presence of caseins. The caseins formed on the one hand large aggregates (flocs) and on the other hand the solubility was increased by the creation of smaller micelles. The formation of flocs could only be observed for HPLT treated samples, which indicates the formation of different protein interactions in milk protein based samples compared with common HP treatments.     

Rennet coagulation properties of UHT‐treated phosphocasein dispersions as a function of casein and NaCl concentrations

The renneting properties of whey protein‐free, UHT‐heated (140 °C/10 s) casein dispersions were investigated as a function of casein and NaCl concentration. It was found that the rennet coagulation time and gel firmness can be optimised when the whey protein‐free casein concentration is increased, while the added NaCl concentration is kept low. The strongest gel firmness occurs at 0.05 and 0.08 m NaCl addition and at a micellar casein concentration between 6.0 and 6.6 g/100 mL. Weak rennet gels were formed at 3.0–3.6 g/100 mL casein at all NaCl concentrations tested.     

Gelation of casein-whey mixtures: effects of heating whey proteins alone or in the presence of casein micelles.

The aim of the present work was to investigate the role of whey protein denaturation on the acid induced gelation of casein. This was studied by determining the effect of whey protein denaturation both in the presence and absence of casein micelles. The study showed that milk gelation kinetics and gel properties are greatly influenced by the heat treatment sequence. When the whey proteins are denatured separately and subsequently added to casein micelles, acid-induced gelation occurs more rapidly and leads to gels with a more particulated microstructure than gels made from co-heated systems. The gels resulting from heat-treatment of a mixture of pre-denatured whey protein with casein micelles are heterogeneous in nature due to particulates formed from casein micelles which are complexed with denatured whey proteins and also from separate whey protein aggregates. Whey proteins thus offer an opportunity not only to control casein gelation but also to control the level of syneresis, which can occur.     

Smoothing temperature and ratio of casein to whey protein: Two tools to improve nonfat stirred yogurt properties

Consumers are not always ready to compromise on the loss of texture and increased syneresis that nonfat stirred yogurts display compared with yogurts that contain fat. In this study, we investigated milk protein composition and smoothing temperature as a means to control nonfat yogurt microstructure, textural properties, and syneresis. Yogurts were prepared with different ratios of casein to whey protein (R1.5, R2.8, and R3.9). Yogurts were pumped through a smoothing pilot system comprising a plate heat exchanger set at 15, 20, or 25°C and then stored at 4°C until analysis (d 1, 9, and 23). Yogurt particle size and firmness were measured. Yogurt syneresis and water mobility were determined, respectively, by centrifugation and time domain low-frequency proton nuclear magnetic resonance (¹H-LF-NMR). Increasing the smoothing temperature increased gel firmness and microgel (dense protein aggregates) sizes independently of the whey protein content. Also, yogurt microgel sizes changed with storage time, but the evolution pattern depended on protein ratio. Yogurt R1.5 showed the largest particles, and their sizes increased with storage, whereas R2.8 and R3.9 had smaller microgels, and R3.9 did not show any increase in microgel size during storage. Micrographs showed a heterogeneous gel with the empty area occupied by serum for R1.5, whereas R2.8 and R3.9 showed fewer serum zones and a more disrupted gel embedding microgels. Induced syneresis reduced with greater whey protein content and time of storage. This is in agreement with ¹H-LF-NMR showing less bulk water mobility with increasing whey protein content during storage. However, ¹H-LF-RMN revealed higher values of spontaneous serum separation during storage for R1.5 and R3.9 yogurts, whereas these were lower and stable for R2.8 yogurt. Microgels play an important structural role in yogurt textural attributes, and their characteristics are modulated by whey protein content and smoothing temperature. Optimization of these parameters may help improve nonfat stirred dairy gel.     

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R² = 0.95).     

Effect of high hydrostatic pressure and whey proteins on the disruption of casein micelle isolates

High hydrostatic pressure disruption of casein micelle isolates was studied by analytical ultracentrifugation and transmission electron microscopy. Casein micelles were isolated from skim milk and subjected to combinations of thermal treatment (85 degrees C, 20 min) and high hydrostatic pressure (up to 676 MPa) with and without whey protein added. High hydrostatic pressure promoted extensive disruption of the casein micelles in the 250 to 310 MPa pressure range. At pressures greater than 310 MPa no further disruption was observed. The addition of whey protein to casein micelle isolates protected the micelles from high hydrostatic pressure induced disruption only when the mix was thermally processed before pressure treatment. The more whey protein was added (up to 5 g/l) the more the protection against high hydrostatic pressure induced micelle disruption was observed in thermally treated samples subjected to 310 MPa.     

Stability of oil–water primary emulsions stabilised with varying levels of casein and whey proteins affected by high-intensity ultrasound

This study evaluates physical and chemical stability of ultrasound-assisted grape seed oil primary emulsions stabilised by varying compositions of caseins to whey proteins (80:20, 60:40, 50:50 and 40:60) at different sono-operating conditions (81.9 and 117.0 J mL⁻¹). Physical and chemical stabilities were influenced by both sonication energy densities and milk protein compositions. Emulsions prepared at 81.9 J mL⁻¹ energy density with ≥40% whey protein fraction (60:40, 50:50, 40:60 and WPI) showed greater physical stability than the emulsions sonicated at 117.0 J mL⁻¹ which exhibited physical instability due to the depletion flocculation mechanism at the critical casein concentration (≥40%). The emulsion oxidative stability was found to be affected by sonication conditions as 117.0 J mL⁻¹ induced the oxidation reactions once the whey concentration exceeds 40%. Therefore, ultrasound prepared emulsions with casein to whey ratios of 60:40, 50:50, 40:60 and WPI at 81.9 J mL⁻¹ energy density was found to be stable for 10 days at 4 °C.     

Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates

Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding α_S1- and α_S2-casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels.     

Structural properties of stirred yoghurt as influenced by whey proteins

The effect of whey protein addition on structural properties of stirred yoghurt systems at different protein and fat content was studied using laser diffraction spectroscopy, rheology and confocal laser scanning microscopy (CLSM). The composition of heated milk systems affected micro- and macroscopic properties of yoghurt gels. Particle size increased as a function of increasing whey protein content and decreased as a function of increased fat level. Firmness (elastic modulus) and apparent viscosity of manufactured yoghurt samples increased as a function of increased interparticle interactions, mainly caused by self-aggregation of whey proteins or aggregated whey protein-coated fat globules, respectively. The resistance towards shear-induced disruption of yoghurt gels increased with an increasing proportion of casein protein in the protein mixture, whereas products with high whey protein level revealed lower resistance behaviour towards shear-forces. CLSM images illustrated that the presence of large whey protein aggregates and lower number of fat globules lead to the formation of an interrupted and coarse gel microstructure characterised by large interstitial spaces. The higher the casein fraction and/or the fat level, the less interspaced voids in the network were observed. However, it is evident that the addition of whey proteins reinforces firmness properties of low-fat yoghurts comparable to characteristics of full-fat yoghurt.     

酪蛋白与乳清蛋白比例对酸奶凝胶性质的影响

研究了乳中酪蛋白和乳清蛋白比例对凝固型酸奶流变学特性和微观结构的影响,结果表明,固定蛋白质质量分数、降低酪蛋白和乳清蛋白的比例,可以明显提高酸奶凝胶的质量.乳中蛋白质质量分数一致时,酸奶凝胶的硬度、黏度、持水力随着酪蛋白和乳清蛋白比例的减小而增大,凝胶网络结构变得更规则、致密,孔隙更小.在低蛋白质质量分数下,降低乳中酪...     

Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high-pressure treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PN-L, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti-beta-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti-cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to beta-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with beta-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.     

Gelation of casein-whey protein mixtures

Heated milk consists of a mixture of whey protein-coated casein micelles and soluble whey protein aggregates. The acid-induced gelation properties of heated milk are consistently different from those of unheated milk—i.e., a shift in gelation pH, stronger gels, and a different microstructure of the gels. In this study we investigated the role of the different fractions of denatured whey proteins on the acid-induced gelation, the gel hardness, and the microstructure. Both whey protein fractions contribute to the observed shift in gelation pH, although by a different mechanism. Obtaining gels with high gel hardness occurs most effectively when all denatured whey proteins are present as whey protein aggregates. It was observed that disulfide bridge exchange reactions during the acid-induced gelation at ambient temperature play an important role for both whey protein fractions. Additionally, disulfide interactions seem to occur between the aggregates and the casein micelles during the gel state. In this study, we show the development of a new approach for confocal scanning laser microscopy measurements—i.e., separate staining of the proteins in milk. By using this method, we were able to determine that, although whey protein aggregates are not linked to the casein micelles, they nevertheless gel at the same moment. This work adds to a better understanding of the role of denatured whey proteins during acid-induced gelation and could improve the effective use of whey proteins.