Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Addition of sodium caseinate to skim milk inhibits rennet-induced aggregation of casein micelles

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl₂ prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α_s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.     

Factors affecting the clotting properties of sheep milk

A Formagraph was used to test the effects of some of the exogenous factors that can affect the processing properties of milk (pH, soluble calcium, rennet concentration, coagulation temperature), and two of the endogenous factors (protein and fat concentration), on the comparative clotting properties of sheep and cows' milk, namely renneting time (r), rate of firming (k20) and curd consistency (A30). A lower pH decreased r and k20 and increased A30 in both sheep and cows' milk. The addition of calcium chloride did not affect the clotting properties of sheep milk, but in cows' milk it decreased r and k20 and increased A30. Increasing the concentration of rennet decreased r and k20 and increased A30 for both sheep and cows' milk. Increasing the coagulation temperature from 30 to 38 °C decreased r for both sheep and cows' milk, but it decreased k20 and increased A30 only in cows' milk. Increasing the protein concentration decreased r in both sheep and cows' milk; it did not affect k20 of sheep milk, but it decreased that of cows' milk and increased A30 in both milks. Increasing the fat concentration had little effect on r and k20 in either sheep cows' milk, but it decreased A30 in both milks. In general, sheep milk had faster renneting times and rates of firming and greater curd consistency than cows' milk, and its clotting properties tended to be less affected by changes in the clotting conditions. © 2002 Society of Chemical Industry     

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50°C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4 ± 5.3 nm at 6°C, 177.4 ± 1.9 nm at 20°C, and 137.3 ± 2.7 nm at 50°C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20°C. Simulated milk ultrafiltrate seemed to give accurate results only at 20°C. Results obtained in simulated milk ultrafiltrate at 6°C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50°C, precipitation of amorphous calcium phosphate affected the DLS measurement.     

Stability of β-lactoglobulin/micellar casein mixtures on heating in simulated milk ultrafiltrate at pH 6.0

The stability of β-lactoglobulin (β-lg)/micellar casein (MC) mixtures was examined on heating at pH 6.0 in increasing levels of lactose-free simulated milk ultrafiltrate (SMUF). Heated β-lg associated with MC to form stable particles (up to 771 nm in size in SMUF × 0.5). Higher levels of SMUF induced reductions in the charge on particles, resulting in greater aggregation and precipitation. Results indicated that the nonthiol-containing α_s1- and β-casein fractions showed greater interaction with β-lg on heating than the thiol-containing fractions (α_s2-casein and κ-casein). Casein proteins and their fractions have potential application in the development of heat-stable dairy-based beverages.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

Modification to the renneting functionality of casein micelles caused by nonionic surfactants

Nonionic emulsifiers of small molecular weight such as polysorbates are widely used in dairy products. Nevertheless, the mechanism of interaction between these surfactants and milk proteins is not yet fully understood. This work investigated the effect of Tween 20 on casein micelles by studying the renneting behavior of skim milk in the presence of different amounts of surfactant. The presence of Tween accelerated both the first and second phase of renneting in skim milk. The gel obtained showed a higher elastic modulus than that of a skim milk gel, but also showed similar brittleness. By varying the size of the surfactant (Tween 20 or Tween 80) as well as the colloidal state of the proteins in solution, it was possible to demonstrate that the surfactant did not have a direct effect on the activity of the enzyme, but rather had a direct effect on the casein micelles. The effect of surfactant on the gelation point was reduced by increasing surfactant size. The presence of Tween caused an increase in the size of the micelles without affecting their stability. In addition, Tween did not alter the amount of caseins free in the serum phase. These findings can contribute to improving our ability to custom design final structures in rennet-induced gels, though further studies are needed to fully understand the mechanism at play when casein micelles are enzymatically cleaved in the presence of nonionic surfactants of small molecular weight.     

Modelling of heat stability and heat‐induced aggregation of casein micelles in concentrated skim milk using a Weibullian model

Heat stability is known to be limited in concentrated skim milk leading to severe coagulation and sediment formation during storage. It has often been found to be in conflict with the extension of the shelf life by thermal treatment. A Weibullian model was used to describe the course of coagulation of casein micelles in concentrated skim milk of different total solids and at different heating temperatures. A maximum heating temperature–time–total solids relationship was calculated based on a certain maximum allowance of sediment formation. Optimal heat treatment conditions for concentrated skim milk of different total solids content could be defined.     

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.     

酸性乳体系中果胶对酪蛋白胶粒稳定作用的研究进展

酪蛋白胶粒在牛乳中可稳定存在,但当pH降低时易发生聚集而使体系失稳。果胶可用于酸性乳饮料中作为稳定剂。在低pH值条件下,果胶吸附于酪蛋白上,由于产生静电排斥作用和空间位阻作用而使乳体系稳定。同时,乳体系中存在的自支持弱网络结构,也对体系有稳定作用。     

Estimating significant causes of variation in emulsions’ droplet size distributions obtained by the electrical sensing zone and laser low angle light scattering techniques

The causes of variation in the particle size distributions of a hot-processed full-fat dressing (system I) and a cold-processed full-fat mayonnaise (system II) have been studied using a newly developed modification of classical multivariate analysis of variance (MANOVA). The MANOVA analysis uses scores obtained from principal component analysis as input parameters. The modification also involves rules for selecting the number of components to be used, as well as a routine for testing each effect inherent in the experimental designs used. The results of the MANOVA analysis are compared with those obtained using ordinary ANOVA on the average volume, the volume mode diameter and the number mode diameter. The MANOVA gives a more consistent picture of the causes of variation in the particle size distribution than was obtained by choosing a single response such as average volume. In addition, more of the variance is frequently explained by MANOVA. For both systems, emulsifier concentration was identified as the most important variable affecting the volume frequency distributions. This most important effect, as judged from the MANOVA, was not always identified if a single response was used. The hot-processed system had large non-dispersible particles (aggregates) which seemed largely influenced by pH and protein levels. The number mode diameter was influenced by salt level; low salt concentrations gave more small particles than did high salt concentrations. The results were supplemented with microscopic investigations. Only a very small effect of heating procedure was noticed. For the cold processed system, the oil type also affected the particle size distribution.     

Developmental changes in proteins of casein micelles in goat milk using data-independent acquisition-based proteomics methods during the lactation cycle

Casein micelles (CM) play an important role in milk secretion, stability, and processing. The composition and content of milk proteins are affected by physiological factors, which have been widely investigated. However, the variation in CM proteins in goat milk throughout the lactation cycle has yet to be fully clarified. In the current study, milk samples were collected at d 1, 3, 30, 90, 150, and 240 of lactation from 15 dairy goats. The size of CM was determined using laser light scattering, and CM proteins were separated, digested, and identified using data-independent acquisition (DIA) and data-dependent acquisition (DDA)-based proteomics approaches. According to clustering and principal component analysis, protein profiles identified using DIA were similar to those identified using the DDA approach. Significant differences in the abundance of 115 proteins during the lactation cycle were identified using the DIA approach. Developmental changes in the CM proteome corresponding to lactation stages were revealed: levels of lecithin cholesterol acyltransferase, folate receptor α, and prominin 2 increased from 1 to 240 d, whereas levels of growth/differentiation factor 8, peptidoglycan-recognition protein, and 45 kDa calcium-binding protein decreased in the same period. In addition, lipoprotein lipase, glycoprotein IIIb, and α-lactalbumin levels increased from 1 to 90 d and then decreased to 240 d, which is consistent with the change in CM size. Protein–protein interaction analysis showed that fibronectin, albumin, and apolipoprotein E interacted more with other proteins at the central node. These findings indicate that changes in the CM proteome during lactation could be related to requirements of newborn development, as well as mammary gland development, and may thus contribute to elucidating the physical and chemical properties of CM.     

Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk

Colloidal calcium phosphate (CCP) plays a key role in the formation and integrity of casein (CN) micelles. However, limited information is available on the molecular weight (M_w) of CCP. Recently, we theoretically derived the M_w of CCP and the objectives of this study were to experimentally determine the M_w of CCP. We used 2 methods to prepare CCP fractions: skim milk was enzymatically digested with either trypsin or a combination of papain and proteinase enzymes to remove most CN. The CN phosphopeptides are resistant to trypsin hydrolysis. Digestion was carried out in a membrane tube that was dialyzed against the same bulk milk used in sample preparation to remove small peptides and to minimize perturbation of CCP. After digestion, the protein contents of the enzyme-treated milks were 0.92 and 0.36% for the trypsin and papain-proteinase treatments, respectively. Size-exclusion chromatography, coupled with multi-angle laser light scattering, was used to separate the CCP-phosphopeptide fraction from the digested mixture. Simulated milk ultrafiltrate was used as a mobile phase during size-exclusion chromatography separation to try to preserve the integrity of CCP. Size-exclusion chromatography peaks, which had higher Ca and P contents than the baseline, were identified as the likely fractions containing the phosphopeptide-stabilized CCP; this peak eluted with retention times of 100 to approximately 110 min for trypsinated samples. The papain-proteinase treatment caused excessive loss of CN that were needed to stabilize CCP, which resulted in no obvious peak that had elevated Ca and P contents. Debye plots at these retention times indicated that the weight-average M_w for the fraction prepared by trypsin was 17,450 g/mol. Attempts to estimate the M_w of the phosphopeptides associated with CCP using sodium dodecyl sulfate-PAGE were not successful, as we did not observe any peptide bands in these gels, presumably because of their low concentration in the isolated, unconcentrated fraction. Assuming that 4 CN phosphopeptides stabilized each CCP and if the M_w of each of these phosphopeptides was about 2,500 g/mol, then the M_w of CCP would be around 7,450 g/mol. This experimental value was close to the theoretically-derived M_w of 4,897 and 9,757 g/mol for tetrahedron and bi-pyramid shaped objects, respectively, when using the brushite form of calcium phosphate.     

Effect of ultrasonication on the properties of skim milk used in the formation of acid gels

Skim milk was ultrasonicated for times up to 30 min either with or without temperature control. Ultrasonication (US) without temperature control resulted in the generation of considerable heat, with the milk reaching  95 °C within 15 min of treatment. The whey proteins were denatured. Changes to the casein micelle size were observed, with decreases during the early stages of US and increases (because of aggregation) on prolonged treatment. Significant κ-casein dissociated from the micelles. Acid gels prepared from these ultrasonicated samples increased in firmness (final G′) up to a maximum final G′ after  15 min of US, followed by a decrease from this maximum on prolonged treatment. US with temperature control demonstrated that the denaturation of the whey proteins was entirely due to the heat generated during US, although the casein micelle size was still reduced. Acid gels prepared from ultrasonicated skim milk in which the temperature remained below the denaturation temperature of the whey proteins had low final G′, although a small increase was observed with increasing US time. Acid gels prepared from the samples that were ultrasonicated at temperatures above the denaturation temperature of the whey proteins had higher final G′, which could reach values similar to those obtained by the conventional heating of milk. The results of this study indicate that, in skim milk, most of the effect of US can be related to the heat generated from the treatment, with US itself having only a small effect on the milk when the temperatures are controlled.

Industrial relevance

The control and the manipulation of the firmness of acid skim milk gels are important in many dairy food applications such as yoghurts and some types of cheese. US is an emerging technology that could be used to process skim milk for use in acid gelled products. This study has demonstrated that acid gel firmness can be substantially manipulated when skim milk is ultrasonically treated before acidification; however, most of the effect is due to the heat generated during US treatment. As the effects of US are similar to those obtained through conventional heating processes, and as US can control spoilage microorganisms, using US under controlled temperature conditions could be an alternative to conventional heating to give desired functional properties and storage stability to milk products. However, the temperature/denaturation/aggregation would need to be carefully controlled to minimize the detrimental effects of excessive heating.     

广西水牛奶与荷斯坦牛奶工艺学特性比较

为了探索、比较广西水牛奶与荷斯坦牛奶的工艺学特性,对二者的缓冲容量、表面张力、粒度分布、稳定系数进行了比较分析。结果表明,广西水牛奶的缓冲容量和粒径分别是荷斯坦牛奶的1.1倍和2倍。在研究范围内,广西水牛奶在pH值为6.8时缓冲容量最大,温度50℃时表面张力最小,pH值为4.7时稳定系数最小;而荷斯坦牛奶在pH值为6.6时缓冲容量最大,温度为50℃时表面张力最小,pH值为4.6时稳定系数最小。广西水牛奶的粒径分布在91.28~458.7 nm之间,荷斯坦牛奶的粒径主要分布为91.28~190.1 nm之间,因此,认为两种牛奶的工艺学参数存在多方面的差异。     

Effect of hydroxypropyl methylcellulose on the textural and whipping properties of whipped cream

In this work, hydroxypropyl methylcellulose (HPMC) was added into whipped cream for improving its textural and whipping properties. By determination of the particle size distribution, a single peak for the emulsion after homogenization and two distinguishable peaks for the emulsion after whipping for 5 min were observed. With the increase of HPMC level, the average particle size (d_3,2) decreased for the emulsion after homogenization and increased for the emulsion after whipping for 5 min. Both whipping time and HPMC level showed positive effects on the partial coalescence of fat globules. The partial coalescence of whipped cream with 0.125% HPMC after whipping for 5 min reached 56.25%, significantly (P < 0.05) higher than that (4.77%) without whipping treatment. Surface protein concentration was measured to evaluate the change of protein content at the droplet interface. The results indicated that the increase of HPMC level could decrease the surface protein concentration slightly. The overrun of whipped cream slightly increased when the HPMC level increased in the range of 0.025–0.125%. Firmness, cohesiveness, consistency and viscosity of whipped cream were analysed in this work. HPMC showed a positive dose-dependent effect on all these textural properties.     

Factors that influence the coalescence stability of protein-stabilised emulsions,estimated from the proportion of oil extracted by hexane

The coalescence stability of protein-stabilised emulsions was estimated by measuring the degree to which the oil content could be extracted by hexane. The hexane extraction method is an empirical one but it correlates well with both an absolute method, such as increase in droplet size, and with another ‘accelerating’ technique, oil separation by centrifugation. Moreover, the hexane extraction method is capable of measuring coalescence stability over a wide range of instability, whereas the centrifugation method only provides information about the final stages of emulsion instability. Among the proteins studied, caseinates were generally the best stabilisers, especially at pH 6. Soya proteins gave rise to emulsions of minimal stability, whereas whey protein concentrate and blood plasma resulted in emulsions of medium stability. The coalescence instability of the protein-stabilised emulsions, viewed overall, was significantly and positively related to the droplet size, the degree of flocculation and the amount of protein in the membrane. High values of these emulsion parameters were due mainly to frequent recoalescence and bridging during emulsification. To minimise these effects emulsifying conditions creating high protein: surface area ratios should be used, as well as proteins that quickly change their conformation at an interface and have low aggregation numbers in solution.