LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Properties of low-fat Cheddar cheese prepared from bovine-camel milk blends: Chemical composition, microstructure, rheology, and volatile compounds.

Making cheese from camel milk (CM) presents various challenges due to its different physicochemical properties compared with bovine milk (BM). In this study, we investigated the chemical composition, proteolysis, meltability, oiling off, texture profile, color, microstructure, and rheological properties of low-fat Cheddar cheese (LFCC) prepared from BM-CM blends. LFCC was produced from BM or BM supplemented with 15% CM (CM15) and 30% CM (CM30), and analyzed after 14, 60, 120, and 180 d of ripening at 8°C. Except for salt content, no significant differences were observed among LFCC from BM, CM15, and CM30. The addition of CM increased the meltability and oiling off in the resulting cheese throughout storage. With respect to color properties, after melting, LFCC CM30 showed lower L* values than LFCC made from BM and CM15, and a* and b* values were higher than those of BM and CM15 samples. LFCC from CM30 also exhibited lower hardness compared with the other cheeses. Moreover, LFCC made from BM showed a rough granular surface, but cheese samples made from BM-CM blends exhibited a smooth surface. The rheological parameters, including storage modulus, loss modulus, and loss tangent, varied among cheese treatments. The determined acetoin and short-chain volatile acids (C2-C6) in LFCC were affected by the use of CM, because CM15 showed significantly higher amounts than BM and CM30, respectively. The detailed interactions between BM and CM in the cheese matrix should be further investigated.

Soluble Salts in Processed Cheese Prepared with Citrate- and Phosphate-Based Calcium Sequestering Salts.

In this study, the protein and salts distribution (Ca, P, Na and Mg) in processed cheese (PC) samples prepared with 180 or 360 mEq/kg of the calcium sequestering salts (CSS) disodium phosphate (DSP), disodium pyrophosphate (DSPP), sodium hexametaphosphate (SHMP) and trisodium citrate (TSC) was studied. For this purpose, a water-soluble extract (WSE) of PC samples was prepared. All PC samples contained 45-46% moisture, 26-27% fat and 20-21% protein and had a pH of 5.2 or 5.7. Ultracentrifugation slightly reduced the protein content of the WSE of PC, indicating that most protein in the WSE was non-sedimentable. At equal concentration of CSS, the protein content of the WSE was higher for PC at pH 5.7 compared to PC at pH 5.2. Approximately 55-85% of the Ca and P in the WSE of samples was 10 kDa-permeable for PC prepared with DSPP and SHMP. This suggests that the formation of non-permeable Ca-polyphosphate-casein complexes. For PC prepared with TSC, >90% of Ca in the WSE was 10 kDa-permeable, indicating that micellar disruption arises from sequestration of micellar Ca. These results indicate that the WSE method is an appropriate method to understand how salts present in PC are distributed. However, the WSE and ultracentrifugal supernatant of the WSE can include both soluble and protein-associated salts. Therefore, determining levels of salts in 10 kDa permeate of ultracentrifugal supernatant of the WSE is most appropriate.

Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation.

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5 and 28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50 percent of the protein content of every 0.5 d colostrum sample, which reduced to ≤3 percent in mature milk. In parallel, we analyzed IgG captured from the bovine milk samples to characterize its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation; data are available via ProteomeXchange with identifier PXD037755. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf's immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.

Milk proteins: Processing, gastric coagulation, amino acid availability and muscle protein synthesis.

It is well-known that the postprandial muscle protein synthetic response to protein ingestion is regulated on various levels, including dietary protein digestion and amino acid (AA) absorption, splanchnic AA retention, the availability of dietary protein-derived AA in the circulation, delivery of AA to the muscle, uptake of AA by the muscle, and intramuscular signaling. AA availability after consumption of dairy products is primarily determined by the rate of gastric emptying of milk proteins, which is mainly linked to coagulation of milk proteins in the stomach. Caseins form gastric coagula, which make their gastric emptying and subsequent postprandial aminoacidemia notably slower than that of whey proteins. Only recently, the role of processing, food structure, preservation and matrix on coagulation herein has been getting attention. In this review we describe various processes, that affect gastric coagulation of caseins and therewith control gastric emptying, such as the conversion to caseinate, heat treatment in the presence of whey proteins, conversion to stirred yoghurt and enzymatic hydrolysis. Modulating product characteristics by processing can be very useful to steer the gastric behavior of protein, and the subsequent digestion and AA absorption and muscle anabolic response to maintain or increase muscle mass.

Effect of whey protein isolate addition on set-type camel milk yogurt: Rheological properties and biological activities of the bioaccessible fraction.

The manufacture of camel milk (CM) yogurt has been associated with several challenges, such as the weak structure and watery texture, thereby decreasing its acceptability. Therefore, this study aimed to investigate the effect of whey protein isolate (WPI) addition on the health-promoting benefits, texture profile, and rheological properties of CM yogurt after 1 and 15 d of storage. Yogurt was prepared from CM supplemented with 0, 3, and 5% of WPI and compared with bovine milk yogurt. The results show that the water holding capacity was affected by WPI addition representing 31.3%, 56.8%, 64.7%, and 45.1% for yogurt from CM containing 0, 3 or 5% WPI, and bovine milk yogurt, respectively, after 15 d. The addition of WPI increased yogurt hardness, adhesiveness, and decreased the resilience. CM yogurt without WPI showed lower apparent viscosity, storage modulus, and loss modulus values compared with other samples. The supplementation of CM with WPI improved the rheological properties of the obtained yogurt. Furthermore, the antioxidant activities of yogurt before and after in vitro digestion varied among yogurt treatments, which significantly increased after digestion except the superoxide anion scavenging and lipid oxidation inhibition. After in vitro digestion at d 1, the superoxide anion scavenging of the 4 yogurt treatments respectively decreased from 83.7%, 83.0%, 79.1%, and 87.4% to 36.7%, 38.3%, 44.6%, and 41.3%. The inhibition of α-amylase and α-glucosidase, angiotensin-converting enzyme inhibition, cholesterol removal, and degree of hydrolysis exhibited different values before and after in vitro digestion.

Influence of pH on Heat-Induced Changes in Skim Milk Containing Various Levels of Micellar Calcium Phosphate.

The present study investigated the effect of micellar calcium phosphate (MCP) content and pH of skim milk on heat-induced changes in skim milk. Four MCP-adjusted samples, ranging from 67 to 113% of the original MCP content, were heated (90 °C for 10 min) at different pH values (6.3, 6.6, 6.9, and 7.2), followed by determining changes in particle size, turbidity, protein distribution, and structure. The results demonstrate a strong effect of MCP level and pH on heat-induced changes in milk, with the MCP67 samples revealing the greatest thermal stability. Specifically, decreasing MCP content by 33% (MCP67) led to a smaller increase in non-sedimentable κ-casein and a lower decrease in αs2-casein concentrations after heating compared to other samples. Lower MCP content resulted in a moderate rise in the average particle size and turbidity, along with lower loading of ß-turn structural component after heating at low pH (pH 6.3). Notably, MCP113 exhibited instability upon heating, with increased particle size, turbidity, and a significant decrease in non-sedimentable αs2-casein concentration, along with a slight increase in non-sedimentable κ-casein concentration. The FTIR results also revealed higher loading of intermolecular ß-sheet, ß-turn, and random coil structures, as well as lower loading of α-helix and ß-sheet structures in MCP-enhanced skim milk samples. This suggests significant changes in the secondary structure of milk protein and greater formation of larger aggregates.

A Review on the Effect of Calcium Sequestering Salts on Casein Micelles: From Model Milk Protein Systems to Processed Cheese.

Phosphates and citrates are calcium sequestering salts (CSS) most commonly used in the manufacture of processed cheese, either singly or in mixtures. Caseins are the main structure forming elements in processed cheese. Calcium sequestering salts decrease the concentration of free calcium ions by sequestering calcium from the aqueous phase and dissociates the casein micelles into small clusters by altering the calcium equilibrium, thereby resulting in enhanced hydration and voluminosity of the micelles. Several researchers have studied milk protein systems such as rennet casein, milk protein concentrate, skim milk powder, and micellar casein concentrate to elucidate the influence of calcium sequestering salts on (para-)casein micelles. This review paper provides an overview of the effects of calcium sequestering salts on the properties of casein micelles and consequently the physico-chemical, textural, functional, and sensorial attributes of processed cheese. A lack of proper understanding of the mechanisms underlying the action of calcium sequestering salts on the processed cheese characteristics increases the risk of failed production, leading to the waste of resources and unacceptable sensorial, appearance, and textural attributes, which adversely affect the financial side of processors and customer expectations.

Determination of Minerals in Soft and Hard Cheese Varieties by ICP-OES: A Comparison of Digestion Methods.

For sample preparation prior to mineral analysis, microwave digestion (~2 h) is quicker and requires lower acid volume as compared to dry (6-8 h) and wet digestion (4-5 h). However, microwave digestion had not yet been compared systematically with dry and wet digestion for different cheese matrices. In this work, the three digestion methods were compared for measuring major (Ca, K, Mg, Na and P) and trace minerals (Cu, Fe, Mn and Zn) in cheese samples using inductively coupled plasma optical emission spectrometry (ICP-OES). The study involved nine different cheese samples with moisture content varying from 32 to 81% and a standard reference material (skim milk powder). For the standard reference material, the relative standard deviation was lowest for microwave digestion (0.2-3.7%) followed by dry (0.2-6.7%) and wet digestion (0.4-7.6%). Overall, for major minerals in cheese, strong correlation was observed between the microwave and the dry and wet digestion methods (R2 = 0.971-0.999), and Bland-Altman plots showed best method agreement (lowest bias), indicating the comparability of all three digestion methods. A lower correlation coefficient, higher limits of agreement and higher bias of minor minerals indicate possibilities of measurement error.

Low-moisture part-skim mozzarella cheese made from blends of camel and bovine milk: Gross composition, proteolysis, functionality, microstructure, and rheological properties.

Camel (CM) milk is used in variety of ways; however, it has inferior gelling properties compared with bovine milk (BM). In this study, we aimed to investigate the physicochemical, functional, microstructural, and rheological properties of low-moisture part-skim (LMPS) mozzarella cheese, made from BM, or BM mixed with 15% CM (CM15%) or 30% CM (CM30%), at various time points (up to 60 d) of storage at 4°C after manufacture. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% had high moisture and total Ca contents, but lower soluble Ca content. Compared with BM cheese, CM15% and CM30% LMPS mozzarella cheese exhibited higher proteolysis rates during storage. Adding CM affected the color properties of LMPS mozzarella cheese manufactured from mixed milk. Scanning electron microscopy images showed that the microstructure of CM15% and CM30% cheeses had smooth surfaces, whereas the BM cheese microstructures were rough with granulated surfaces. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% showed significantly lower hardness and chewiness, but higher stringiness than BM cheese. Compared with BM cheese, CM15% and CM30% cheeses showed lower tan δ levels during temperature surges, suggesting that the addition of CM increased the meltability of LMPS mozzarella cheese during temperature increases. Camel milk addition affected the physicochemical, microstructural, and rheological properties of LMPS mozzarella cheese.

Low-fat akawi cheese made from bovine-camel milk blends: Rheological properties and microstructural characteristics.

Camel milk (CM) can be used as an ingredient to produce various dairy products but it forms weak rennet-induced and acid-induced gels compared with bovine milk (BM). Therefore, in this study, we aimed to investigate the effect of blending bovine milk with camel milk on the physicochemical, rheological (amplitude sweep and frequency sweep), and microstructural properties of low-fat akawi (LFA) cheese. The cheeses were made of BM only or BM blended with 15% (CM15%) or 30% (CM30%) camel milk and stored at 4°C for 28 d. The viscoelastic properties as a function of temperature were assessed. The LFA cheeses made from blended milks had higher moisture, total Ca, and soluble Ca contents, and had higher pH 4.6-water-soluble nitrogen compared with those made from BM. Analysis by scanning electron microscopy demonstrated that the microstructures formed in BM cheese were rough with granular surfaces, whereas those in blended milk cheeses had smooth surfaces. Hardness was lower for LFA cheeses made from blended milk than for those made from BM only. The LFA cheeses demonstrated viscoelastic behavior in a linear viscoelastic range from 0.1 to 1.0% strain. The storage modulus (G') was lower in LFA cheese made from BM over a range of frequencies. Adding CM reduced the resistance of LFA cheeses to flow as temperature increased. Blended cheeses exhibited lower complex viscosity values than BM cheeses during temperature increases. Thus, the addition of camel milk improved the rheological properties of LFA cheese.

Biological activities of the bioaccessible compounds after in vitro digestion of low-fat Akawi cheese made from blends of bovine and camel milk.

The objective of this study was to assess protein degradation and biological activities of the water-soluble extract (WSE) and the 10 kDa permeable and nonpermeable fractions of in vitro digesta of low-fat Akawi cheese made from blends (100:0, 85:15, or 70:30) of bovine milk and camel milk and ripened for 28 d. Biological activities, such as antioxidant activities, amylase and glucosidase inhibition, angiotensin-converting enzyme inhibition, and antiproliferative of the WSE, and the 10 kDa permeable and nonpermeable fraction of the digesta were assessed. To identify the nature of the bioaccessible compounds, untargeted metabolomic analysis was carried out by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Higher o-phthaldialdehyde absorbances were observed in cheeses made of bovine-camel milk blends compared with cheese from bovine milk only. The WSE from these blends also exhibited higher angiotensin-converting enzyme inhibitory effects and higher antiproliferative effects than from bovine milk. The results from this study suggest that the use of blends of camel milk and bovine milk can modulate biological activities of low-fat Akawi cheese.

Structural Changes of ß-Casein Induced by Temperature and pH Analysed by Nuclear Magnetic Resonance, Fourier-Transform Infrared Spectroscopy, and Chemometrics.

This study investigated structural changes in ß-casein as a function of temperature (4 and 20 °C) and pH (5.9 and 7.0). For this purpose, nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy were used, in conjunction with chemometric analysis. Both temperature and pH had strongly affected the secondary structure of ß-casein, with most affected regions involving random coils and α-helical structures. The α-helical structures showed great pH sensitivity by decreasing at 20 °C and diminishing completely at 4 °C when pH was increased from 5.9 to 7.0. The decrease in α-helix was likely related to the greater presence of random coils at pH 7.0, which was not observed at pH 5.9 at either temperature. The changes in secondary structure components were linked to decreased hydrophobic interactions at lower temperature and increasing pH. The most prominent change of the α-helix took place when the pH was adjusted to 7.0 and the temperature set at 4 °C, which confirms the disruption of the hydrogen bonds and weakening of hydrophobic interactions in the system. The findings can assist in establishing the structural behaviour of the ß-casein under conditions that apply as important for solubility and production of ß-casein.

Invited review: Heat stability of milk and concentrated milk: Past, present, and future research objectives.

The ability of milk and concentrated milk to withstand a defined heat treatment without noticeable changes such as flocculation of protein is commonly denoted as heat stability. A heat treatment that exceeds the heat stability limit of milk or concentrated milk, which has a much lower heat stability, may result in undesired changes, such as separation of milk fat, grittiness, sediment formation, and phase separation. Most laboratory-scale batch heating methods were developed in the early 20th century to simulate commercial sterilization, and these methods have since been standardized. Heat stability studies have been motivated by different objectives during that time, addressing different processing issues and targets in the framework of available technology, legislation, and consumer demand. Although milk hygiene has improved during the last couple of decades, rendering milk less sensitive to coagulation, different standard methods suffered from poor comparability of results, even when comparing results for the same milk sample, indicating that unknown procedural steps affect heat stability. The prediction of heat stability of concentrated milk from the heat stability results of the corresponding unconcentrated milk for rapid quality testing purposes has been difficult, mainly due to different experimental conditions. The objective of this study is to review literature on heat stability, starting from studies in the early 20th century, to summarize the vast number of studies on compositional aspects of milk affecting heat stability, and to lead the way to the most recent work related to compositional changes in concentrates produced by membrane concentration and fractionation, respectively. Particular attention is paid to early and most recent developments and findings, such as the application of kinetic models to predict and limit protein aggregation to assess and describe heat stability as a temperature-time-total milk solids continuum.

A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk.

The white appearance of skim milk is due to strong light scattering by colloidal particles called casein micelles. Bovine casein micelles comprise expressed proteins from four casein genes together with significant fractions of the total calcium, inorganic phosphate, magnesium and citrate ions in the milk. Thus, the milk salts are partitioned between the casein micelles, where they are mostly in the form of nanoclusters of an amorphous calcium phosphate sequestered by caseins through their phosphorylated residues, with the remainder in the continuous phase. Previously, a salt partition calculation was made assuming that the nanoclusters are sequestered only by short, highly phosphorylated casein sequences, sometimes called phosphate centres. Three of the four caseins have a proportion of their phosphorylated residues in either one or two phosphate centres and these were proposed to react with the nanoclusters equally and independently. An improved model of the partition of caseins and salts in milk is described in which all the phosphorylated residues in competent caseins act together to bind to and sequester the nanoclusters. The new model has been applied to results from a recent study of variation in salt and casein composition in the milk of individual cows. Compared to the previous model, it provides better agreement with experiment of the partition of caseins between free and bound states and equally good results for the partition of milk salts. In addition, new calculations are presented for the charge on individual caseins in their bound and free states.

Lactose in dairy ingredients: Effect on processing and storage stability.

Lactose is the main carbohydrate in the milk of most species. It is present in virtually all dry dairy ingredients, with levels ranging from <2% (e.g., caseinates, milk protein isolates) to 100% in lactose powders. The presence of lactose has a strong effect on ingredient processing and stability. Lactose can negatively influence powder properties and lead to undesirable effects, such as the stickiness of powder resulting in fouling during drying, or caking and related phenomena during storage. In addition, being a reducing carbohydrate, lactose can also participate in the Maillard reaction with free amino groups of proteins, peptides, and free AA. In this review, the influence of the presence (or absence) of lactose on physiochemical properties of dairy ingredients is reviewed, with particular emphasis on behavior during processing and storage. Particularly important features in this respect are whether lactose is in the (glassy) amorphous phase or in the crystalline phase, which is strongly affected by precrystallization conditions (e.g., in lactose, permeate, and whey powders) and by drying conditions. Furthermore, the moisture content and water activity of the ingredients are important parameters to consider, as they determine both mobility and reactivity, influencing Maillard reactions and concomitant browning, the crystallization of amorphous lactose during storage of dairy ingredients, glass transitions temperatures, and associated stickiness and caking phenomena. For the stickiness and caking, a crucial aspect to take into account is powder particle surface composition in relation to the bulk powder. Lactose is typically underrepresented at the powder surface, as a result of which deviations between observed lactose-induced caking and stickiness temperatures, and determined glass transition temperatures arise. By considering lactose as an integral part of ingredient composition along with all other compositional and environmental properties, lactose behavior in dairy ingredients can be understood, controlled, and optimized. Routes to achieve this are outlined in this review paper.

Influence of Heating Temperature and pH on Acid Gelation of Micellar Calcium Phosphate-Adjusted Skim Milk.

Micellar calcium phosphate (MCP) plays an important role in maintaining the structure and stability of the casein micelle and its properties during processing. The objective of this study was to investigate how heating (10 min at 80 or 90 °C) at different pH levels (6.3, 6.6, 6.9, or 7.2) impacted the acid-induced gelation of MCP-adjusted milk, containing 67 (MCP67), 100 (MCP100), or 113 (MCP113) % of the original MCP content. The unheated sample MCP100 at pH 6.6 was considered the control. pH acidification to pH 4.5 at 30 °C was achieved with glucono delta-lactone while monitoring viscoelastic behaviour by small-amplitude oscillatory rheology. The partitioning of calcium and proteins between colloidal and soluble phases was also examined. In MCP-depleted skim milk samples, the concentrations of non-sedimentable caseins and whey proteins were higher compared to the control and MCP-enriched skim milk samples. The influence of MCP adjustment on gelation was dependent on pH. Acid gels from sample MCP67 exhibited the highest storage modulus (G'). At other pH levels, MCP100 resulted in the greatest G'. The pH of MCP-adjusted skim milk also impacted the gel properties after heating. Overall, this study highlights the substantial impact of MCP content on the acid gelation of milk, with a pronounced dependency of the MCP adjustment effect on pH variations.

Dairy Matrix Effects: Physicochemical Properties Underlying a Multifaceted Paradigm.

When food products are often considered only as a source of individual nutrients or a collection of nutrients, this overlooks the importance of interactions between nutrients, but also interactions between nutrients and other constituents of food, i.e., the product matrix. This product matrix, which can be defined as 'The components of the product, their interactions, their structural organization within the product and the resultant physicochemical properties of the product', plays a critical role in determining important product properties, such as product stability, sensory properties and nutritional and health outcomes. Such matrix effects can be defined as 'the functional outcome of specific component(s) as part of a specific product matrix'. In this article, dairy matrix effects are reviewed, with particular emphasis on the nutrition and health impact of dairy products. Such matrix effects are critical in explaining many effects of milk and dairy products on human nutrition and health that cannot be explained solely based on nutrient composition. Examples hereof include the low glycemic responses of milk and dairy products, the positive impact on dental health, the controlled amino acid absorption and the absence of CVD risk despite the presence of saturated fatty acids. Particularly, the changes occurring in the stomach, including, e.g., coagulation of casein micelles and creaming of aggregated fat globules, play a critical role in determining the kinetics of nutrient release and absorption.

Effect of pH and Shear on Heat-Induced Changes in Milk Protein Concentrate Suspensions.

The effect of shear on heat-induced changes in milk protein concentrate suspensions was examined at different pH levels, revealing novel insights into micellar dissociation and protein aggregation dynamics. Milk protein concentrate suspensions, adjusted to pH of 6.1, 6.4, 6.8, or 7.5, underwent combined heat (90 °C for 5 min or 121 °C for 2.6 min) and shear (0, 100, or 1000 s-1) treatment. The fragmentation of protein aggregates induced by shear was evident in the control MPC suspensions at pH 6.8, irrespective of the temperature. At pH 7.5, shear increased the heat-induced micellar dissociation. This effect was particularly pronounced at 121 °C and 1000 s-1, resulting in reduced particle size and an elevated concentration of κ-casein (κ-CN) in the non-sedimentable phase. At pH 6.1 or 6.4, shear effects were dependent on sample pH, thereby modifying electrostatic interactions and the extent of whey protein association with the micelles. At pH 6.1, shear promoted heat-induced aggregation, evidenced by an increase in particle size and a significant decline in both whey proteins and caseins in the non-sedimentable phase. At pH 6.4, shear-induced fragmentation of aggregates was observed, prominently due to comparatively higher electrostatic repulsions and fewer protein interactions. The influence of shear on heat-induced changes was considerably impacted by initial pH.

Effect of Protein Content on Heat Stability of Reconstituted Milk Protein Concentrate under Controlled Shearing.

Milk protein concentrates (MPCs) possess significant potential for diverse applications in the food industry. However, their heat stability may be a limitation to achieving optimal functional performance. Shearing, an inherent process in food manufacturing, can also influence the functionality of proteins. The aim of this research was to examine the heat stability of reconstituted MPCs prepared at two protein concentrations (4% and 8% w/w protein) when subjected to varying levels of shearing (100, 1000, or 1500 s-1) during heating at 90 °C for 5 min or 121 °C for 2.6 min. While the impact of shear was relatively minor at 4% protein, it was more pronounced in 8% protein MPC suspensions, leading to a considerable decline in heat stability. An increase in protein concentration to 8% amplified protein interactions, intensified by shearing. This, in turn, resulted in comparatively higher aggregation at elevated temperatures and subsequently reduced the heat stability of the reconstituted MPCs.