A proteomic approach to detect lactosylation and other chemical changes in stored milk protein concentrate

Milk proteins undergo chemical changes such as lactosylation, deamidation and protein cross-linking during processing and storage of milk products. A proteomic technique combining two-dimensional gel electrophoresis and mass spectrometry was used to investigate chemical modifications to proteins, in milk protein concentrate (MPC80), during storage. Lactosylation, deamidation and protein cross-linking were observed on 2-DE gels. They were storage temperature-, humidity- and time-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The masses of the spots varied by multiples of 324, indicating the attachment of lactose to lysine residues in the proteins. The trypsin-digested spots of α-lactalbumin were analysed by MALDI-TOF mass spectrometry, which indicated multiple lactosylation sites. The lactose adducts on gels were quantified by image analysis, allowing development of adducts over time to be monitored. The results show that proteomics can be used for the detection and quantification of chemical modifications to proteins in stored MPC80.     

Studies on the impact of glycation on the denaturation of whey proteins

It could be shown for technologically relevant whey protein powders that denaturation of β-lactoglobulin (β-Lg) is affected significantly by the extent of covalent modification of lysine residues by lactose. The amount of acid soluble β-Lg as measured via RP-HPLC with UV detection after heating for 10 min at 80 °C increased from 40% (4.6% lysine modification) to 82% (22.4% lysine modification). An increase in glycation leads to a slower denaturation-induced oligomerisation, as shown by SDS-PAGE. Concomitant with an increase in lysine modification, the denaturation temperature increased from 79.5 to 84 °C, as measured by differential scanning calorimetry (DSC). Covalent attachment of lactose to whey proteins during preparation or storage significantly improves the heat stability of whey proteins, which may be of particular importance for the technological use of whey proteins varying in the degree of lysine modification.     

Heat-Induced Aggregation Properties of Whey Proteins as Affected by Storage Conditions of Whey Protein Isolate Powders

The control of storage as any other manufacturing steps of dairy powders is essential to preserve protein functional properties. This study aimed to determine the effects of different storage conditions on both protein denaturation and protein lactosylation in whey protein isolate (WPI) powder, and also on heat-induced aggregation. Two different storage temperature conditions (20 and 40 °C) were studied over 15 months. Our results showed that protein lactosylation progressively increased in WPI powders over 15 months at 20 °C, but heat-induced aggregation properties did not significantly differ from non-aged WPI. On the other hand, powders presented a high level of denaturation and aggregation at 40 °C from the first 2 weeks of storage, involving first protein lactosylation and then aggregation in the dry state. This was correlated with an increasing Browning Index from 15 days of storage. These changes occurred with a decrease in aggregate size after heat treatment at 5.8?≤?pH?≤?6.6 and modification of heat-induced aggregate shapes.     

Spray-dried whey protein/lactose/soybean oil emulsions. 1. Surface composition and particle structure

Emulsions made of whey protein, lactose and soybean oil were spray-dried and the chemical surface composition of the dried powders estimated by electron spectroscopy for chemical analysis. In particular, the ability of whey protein to encapsulate fat was highlighted. Additionally, the structure of the spray-dried powder particles was studied by scanning electron microscopy. The powders were examined after storage in both dry and humid atmospheres (relative humidity 75%, 4 days). It was found that the ability of whey protein to encapsulate soybean oil is rather low compared with sodium caseinate, with a large part of the powder surface covered by fat after spray-drying. After storage in humid atmosphere there is a release of encapsulated oil onto the powder surface in most cases, and an increase in fat coverage. The release offat onto the powder surfaces causes the particle structure to change dramatically for powders containing a critical amount of lactose. Such powders agglomerate and lose structure completely. In comparison, powders containing no lactose storage under humid conditions also cause a release of fat onto the powder; however, in this case particle structure remains intact. Powders containing only a small amount of lactose, up to ~25% of emulsion dry weight, do not exhibit the release of fat onto the powder surfaces after storage under humid conditions and the structure of these powder particles does not change. The presence of lactose in whey protein-stabilized emulsions, however, does not increase fat encapsulation by whey protein, as reported earlier for sodium caseinate-stabilized emulsions that were spray-dried. During spray-drying of whey protein/lactose solutions there is a strong overrepresentation of surface-active whey protein on the powder surface. Whey protein coverage increases even further when the powders are stored under humid conditions, also making them lose structure.     

Protein lactosylation in UHT milk during storage measured by Liquid Chromatography–Mass Spectrometry and quantification of furosine

The initial stage of the Maillard reaction, protein lactosylation, occurs during heat treatment of milk and continues during subsequent storage. We compared the initial lactosylation as well as the rate of lactosylation of milk proteins during storage in UHT milk subjected to direct or indirect heat treatment using liquid chromatography (LC) coupled with electrospray injection mass spectrometry (ESI‐MS). Furosine content was used as an overall marker to allow for a quantitative correlation of lactosylation measured by LC‐ESI‐MS in the UHT milks. Protein lactosylation increased during the storage period of 6 months at 20 °C. Both the initial extent and the rate of lactosylation positively correlated with the number of lysine residues in the different proteins. An exponential or linear correlation with furosine concentration could be established for major and minor lactosylated proteins, respectively.     

Assessment of physical characteristics and dissolution behavior of protein based powders

Proteins are main constituents of a broad range of food products in powder form, from dairy to dough-based foods. Water is the main responsible of physical properties modifications in powders and many scientific studies have been focusing on characterizing the role of water content and water activity in carbohydrates powders. This work deals with the assessment of physical properties and dissolution behavior of milk proteins powders and their interactions with carbohydrates (lactose). In the first part, micellar caseins, native and denatured β-lactoglobulin and Na-caseinate powders were selected and equilibrated at different water activities. In the second part, binary mixtures of proteins with lactose were prepared in different proportions (25:75 and 75:25). Sorption isotherms were built at 25 °C and water adsorption kinetics was found to be faster in proteins than in carbohydrates. Thermal analysis (DSC) showed that micellar caseins and Na-caseinate exhibit a clear glass transition, while the measurement was not easy for BLG powders. In binary systems, proteins delayed lactose crystallization, up to a different extent depending on the kind of protein. Dissolution behavior was measured by conductimetry; the protein structure (native vs denatured) and its initial water activity played an important role, especially influencing powder's wettability. The presence of lactose strongly accelerated the dissolution process, in a more important way for BLG. Scientific findings could lead to improved powder engineering, in order to optimize dissolution behavior and storage stability of protein-based foods.     

Apparent chemical composition of nine commercial or semi-commercial whey protein concentrates, isolates and fractions

Summary Analytical results are given for whey powders prepared on a commercial or semi-commercial scale by three companies. Altogether, five preparations enriched in β-lactoglobulin, four whey protein isolates and a fraction enriched in α-lactalbumin were analyzed for protein composition, including %β-lactoglobulin, α-lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native β-lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true β-lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimated true β-lactoglobulin weight % of 70.9 ± 1.1, 62.0 ± 3.4 and 39.5 ± 4.9. Physico-chemical properties of the group of samples are reported elsewhere (Holt et al ., 1999).     

Spray-dried whey protein/lactose/soybean oil emulsions. 2. Redispersability, wettability and particle structure

In the present paper redispersion and wettability experiments of spray-dried whey protein-stabilized emulsions are presented. Emulsion droplet size after redispersion gives information about eventual coalescence between emulsion droplets in the powder matrix during drying or storage, resulting in an increase in emulsion droplet size after redispersion. Results from redispersion experiments are combined with previously presented knowledge about powder surface composition and particle structure to elucidate internal processes in the powder matrix and external processes on the powder surface during drying and storage of whey protein powder. The results show that with addition of lactose to whey protein-stabilized emulsions, emulsion droplet structure remains intact in the powder matrix during drying since the emulsion droplet size in the redispersed spray dried emulsion is unchanged. In the absence of lactose there is a growth in emulsion droplet size after redispersion of the spray-dried whey protein-stabilized emulsion, showing that a coalescense of emulsion droplets occurs during the drying or redispersion process. Storage of the whey protein-stabilized powders in a humid atmosphere (relative humidity 75%, 4 days) induces changes in some powders. When the powder contains a critical amount of lactose there is a remarkable increase in emulsion droplet size after redispersion of humid stored powders compared with the emulsion before drying and with the redispersed dry stored powder. In addition, there is a release of encapsulated fat after humid storage of lactose-containing powders detected by electron spectroscopy for chemical analysis. For powders which do not contain any lactose there is no increase in emulsion droplet size after storage in a humid atmosphere compared with the redispersed dry stored emulsion. Addition of only a small amount of lactose prevents coalescence of emulsion droplets and the subsequent increase in droplet size during drying. If the lactose content is kept rather low neither an effect on the droplet size after storage under humid conditions nor a release of fat onto powder surfaces is detected. Furthermore, wettability of the spray-dried whey protein-stabilized emulsions by water is presented. It is concluded that it is beneficial to wettability in water to have as high a coverage of lactose on the powder surface as possible. In addition, a review of particle structure for powders of various composition is presented.     

Improving the structure and rheology of high protein,low fat yoghurt with undenatured whey proteins

The objective of this study was to investigate the effects of whey protein denaturation and whey protein:casein-ratio on the structural, rheological and sensory properties of high protein (8% true protein), low fat (<0.5% fat) yoghurt. Yoghurt milk bases were made by adding undenatured whey proteins from native whey protein concentrate (NWPC) to casein concentrate in different whey protein:casein-ratios. The degree of whey protein denaturation was then controlled by the temperature treatment of the yoghurt milk bases. Addition of NWPC in low (whey protein:casein-ratio 25:75) or medium levels (whey protein:casein-ratio 35:65) in combination with heat treatment at 75 °C for 5 min gave yoghurts with significantly lower firmness, lower storage modulus (G′), and better sensory properties (less coarse and granular and more smooth), compared with corresponding yoghurts produced from yoghurt milk bases heat-treated at 95 °C for 5 min or with control yoghurts (no addition of NWPC).     

The prediction of moisture sorption isotherms for dairy powders

Moisture sorption isotherms were measured for whey protein isolate, high micellar casein and a milk protein concentrate powder. No temperature dependence was observed over the temperature range of 4–37 °C. At 50 °C the powders absorbed less moisture than observed at the lower temperatures. These isotherms were used to predict the isotherms for freeze-dried amorphous lactose/casein/whey protein powders. An isotherm for micellar casein was predicted using a simple additive isotherm model and was used along with isotherms for whey protein and amorphous lactose to predict moisture sorption isotherms for commercial dairy powders. Predicted isotherms compared well with measured isotherms indicating that this simple additive isotherm model is suitable for predicting moisture sorption isotherms of dairy powders. Delayed lactose crystallisation was observed in lactose/whey protein powders when compared to lactose/casein powders over the same water activity range.     

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 (<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.     

Microencapsulation properties of soy protein isolate: Influence of preheating and/or blending with lactose

Properties and storage stability of spray-dried emulsions stabilized by unheated and preheated (95 °C, 15 min) soy protein isolates, alone or in combination with lactose, were investigated. In general, the heat pretreatment greatly improved retention efficiency (RE), redispersion behavior, glass transition temperature (T_g) and thermal stability of the emulsion powders, but accelerated instability of the reconstituted emulsions. Additional blending with lactose further considerably improved the RE and dissolution behavior, but significantly decreased the stability of reconstituted emulsions and T_g. Storage at 75% relative humidity resulted in considerably increased droplet size of reconstituted emulsions, as well as decreased RE, wettability and T_g, especially in the powders containing lactose. Microscopic observations confirmed that the changes in properties and stability of the powders upon storage were closely related to rupture of particle structure, and/or particle agglomeration. These findings provide fundamental understanding for the development of microencapsulated products using soy proteins as the wall materials.     

Influence of particle size on the physicochemical properties and stickiness of dairy powders

The compositional and physicochemical properties of different whey permeate (WPP), demineralised whey (DWP) and skim milk powder (SMP) size fractions were investigated. Bulk composition of WPP and DWP was significantly (P < 0.05) influenced by powder particle size; smaller particles had higher protein and lower lactose contents. Microscopic observations showed that WPP and DWP contained both larger lactose crystals and smaller amorphous particles. Bulk composition of SMP did not vary with particle size. Surface composition of the smallest SMP fraction (<75 μm) showed significantly lower protein (−9%) and higher fat (+5%) coverage compared with non-fractionated powders. For all powders, smaller particles were more susceptible to sticking. Hygroscopicity of SMP was not affected by particle size; hygroscopicity of semi-crystalline powders was inversely related to particle size. This study provides insights into differences between size fractions of dairy powders, which can potentially impact the sticking/caking behaviour of fine particles during processing.     

Rheological properties and structure of inulin–whey protein gels

The rheological properties, structure and synergistic interactions of whey proteins (1–7%) and inulin (20% and 35%) were studied. Gelation of whey proteins was induced with Na⁺. Inulin was dissolved in preheated whey protein solutions (80 °C, 30 min). Inulin gel formation was strongly affected by whey proteins. The presence of whey proteins at a level allowing for protein gel network formation (7%) significantly increased the G′ and G″ values of the gels. Scanning electron micrographs showed a thick structure for the mixed gel. Whey proteins at low concentrations (1–4%) were not able to form a gel; further, these low concentrations partly or wholly impaired formation of a firm inulin gel. Although interactions between inulin and whey proteins may be concluded from hydrophobicity measurements, the use of an electrophoretic technique did not show any inulin–whey protein complexes.     

Comparison of micellar casein isolate and nonfat dry milk for use in the production of high-protein cultured milk products

High protein levels in yogurt, as well as the presence of denatured whey proteins in the milk, lead to the development of firm gels that can make it difficult to formulate a fluid beverage. We wanted to prepare high-protein yogurts and explore the effects of using micellar casein isolate (MCI), which was significantly depleted in whey protein by microfiltration. Little is known about the use of whey protein-depleted milk protein powders for high-protein yogurt products. Microfiltration also depletes soluble ions, in addition to whey proteins, and so alterations to the ionic strength of rehydrated MCI dispersions were also explored, to understand their effects on a high-protein yogurt gel system. Yogurts were prepared at 8% protein (wt/wt) from MCI or nonfat dry milk (NDM). The NDM was dispersed in water, and MCI powders were dispersed in water (with either low levels of added lactose to allow fermentation to achieve the target pH, or a high level to match the lactose content of the NDM sample) or in ultrafiltered (UF) milk permeate to align its ionic strength with that of the NDM dispersion. Dispersions were then heated at 85°C for 30 min while stirring, cooled to 40°C in an ice bath, and fermented with yogurt cultures to a final pH of 4.3. The stiffness of set-style yogurt gels, as determined by the storage modulus, was lowest in whey protein-depleted milk (i.e., MCI) prepared with a high ionic strength (UF permeate). Confocal laser scanning microscopy and permeability measurements revealed no large differences in the gel microstructure of MCI samples prepared in various dispersants. Stirred yogurt made from MCI that was prepared with low ionic strength showed slow rates of elastic bond reformation after stirring, as well as slower increases in cluster particle size throughout the ambient storage period. Both the presence of denatured whey proteins and the ionic strength of milk dispersions significantly affected the properties of set and stirred-style yogurt gels. Results from this study showed that the ionic strength of the heated milk dispersion before fermentation had a large influence on the gelation pH and strength of acid milk gels, but only when prepared at high (8%) protein levels. Results also showed that depleting milk of whey proteins before fermentation led to the development of weak yogurt gels, which were slow to rebody and may be better suited for preparing cultured milk beverages where low viscosities are desirable.     

Developments in whey protein and lactose permeate production processes and their relationship to specific product attributes

This paper reviews recent developments in whey processing and the production of whey protein concentrate and lactose permeate powders with particular reference to a new whey processing facility in the Netherlands. The processes have been designed to optimise product quality with the whey protein concentrate targeted for use in nutritional applications and the permeate powder for use in lactose replacement.     

Glycosylation and expanded utility of a modified whey protein ingredient via carbohydrate conjugation at low pH

Whey protein, at one time considered a by-product of the cheese-making process, is now commonly used in foods for its thickening and emulsifying properties. Currently, approximately 30% of these proteinaceous resources remain under-utilized. Previously, an acidified, thermally treated whey protein concentrate (mWPC) was developed to produce a cold-set thickening ingredient. Mass spectroscopy revealed an approximate 2.5-fold decrease in the lactosylation of β-lactoglobulin in mWPC starting materials compared with commercial whey protein concentrates, manufactured at a higher pH. Potentially, this should increase the number of reactive sites that remain available for carbohydrate attachment. With this study, the formation of glycoprotein complexes was demonstrated between the mWPC ingredient and lactose, naturally occurring in mWPC powders, or between mWPC protein components with dextran (35 to 45 and 100 to 200 kDa) materials at low pH. In fact, additional dry heating of mWPC powders showed a 3-fold increase in the amount of lactosylated β-lactoglobulin. Evidence of Maillard reactivity was suggested using colorimetry, o-phthaldialdehyde assays, and sodium dodecyl sulfate PAGE followed by glycoprotein staining. Resultant glycoprotein dispersions exhibited altered functionality, in which case steady shear and small amplitude oscillatory rheology parameters were shown to be dependent on the specific reducing sugar present. Furthermore, the emulsion stability of mWPC-dextran fractions was 2 to 3 times greater than either mWPC or commercial WPC dispersions based on creaming index values. The water-holding capacity of all test samples decreased with additional heating steps; however, mWPC-dextran powders still retained nearly 6 times their weight of water. Scanning electron microscopy revealed that mWPC-dextran conjugates formed a porous network that differed significantly from the dense network observed with mWPC samples. This porosity likely affected both the rheological and water-binding properties of mWPC-dextran complexes. Taken together, these results suggest that the functionality of mWPC ingredients can be enhanced by conjugation with carbohydrate materials at low pH, especially with regard to improving the emulsifying attributes.     

Stability of oil‐in‐water emulsions as influenced by thermal treatment of whey protein dispersions or emulsions

Properties of whey protein concentrate stabilised emulsions were modified by protein and emulsion heat treatment (60–90 °C). All liquid emulsions were flocculated and the particle sizes showed bimodal size distributions. The state and surface properties of proteins and coexisting protein/aggregates in the system strongly determined the stability of heat‐modified whey protein concentrate stabilised emulsions. The whey protein particles of 122–342 nm that formed on protein heating enhanced the stability of highly concentrated emulsions. These particles stabilised protein‐heated emulsions in the way that is typical for Pickering emulsions. The emulsions heated at 80 and 90 °C gelled due to the aggregation of the protein‐coated oil droplets.     

Hydration of Whey Powders as Determined by Different Methods

Four methods for evaluating water hydration of 15 whey derivative powders were compared, and results are discussed with respect to the chemical composition of the powders. Hydration capacities between 0.21 and 4.64 mL water/g of powder were obtained, depending on the method used. The filtration/centrifugation method gave the highest hydration capacity, whereas the paste-water retention method gave the lowest. The Baumann test and the paste-water retention method were well correlated with protein and lactose content of the powders, enabling differentiation between hydration capacities of whey protein concentrates (35% proteins) and electrodialyzed whey powders (12% proteins). Reliable characterization of hydration required a combination of methods.     

Microparticulation of mixtures of whey protein and inulin

This study investigated the effect of the presence of inulin, in the range 0 - 3.81 g/100g, on the physical and microstructural properties of microparticulated whey protein concentrates (MWPC) and powders. Substitution of lactose with inulin increased levels of whey protein denaturation during microparticulation. The increased denaturation levels were correlated with reductions in lactose and calcium content of the microparticulated solutions, which increased aggregate size and solution viscosity post-processing. In conclusion, it appears possible to successfully manufacture a low-calorie microparticulated whey protein based fat replacer using the dietary fibre inulin as the carbohydrate source.