Comparison of pH-dependent sonodisruption of re-assembled casein micelles by 35 and 130 kHz ultrasounds

Sonodisruption behavior of re-assembled casein micelles was compared at two ultrasound frequencies (35 and 130 kHz) by turbidity measurement and laser-diffraction based particle size analysis. Sonochemical ultrasound (130 kHz) was more effective than power ultrasound (35 kHz) in micelle disruption. This was attributed to the higher strain rates generated upon implosion of cavities, as well as the liberation of more free radicals to the surrounding medium. The higher the pH of solution, the more effective was the ultrasonic disruption due to a looser expanded assembly of particles at higher pH values. Sonochemical ultrasound decreased the consistency coefficient of casein solutions and increased their flow index except at a pH value of 6.35, while power ultrasound did not affect the flow behavior of solutions across the whole pH range.     

Temperature-dependent dynamics of bovine casein micelles in the range 10–40 °C

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10–40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10–40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.     

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.     

水牛奶酪蛋白胶束结构的荧光光谱研究

利用内源荧光团色氨酸（Trp）和外源荧光探针8-苯胺基-1-萘磺酸（8-anilino-1-naphthalenesulfonic acid,ANS）的荧光特性对水牛奶酪蛋白胶束结构进行研究。结果表明：当蛋白质质量浓度较低时,酪蛋白胶束结构变化不明显,而较高的蛋白质质量浓度会破坏其胶束结构,使其疏水基团暴露;此外,在离子强度较大、pH值较低条件下,对酪蛋白胶束结构影响较大,使酪蛋白疏水基团暴露,酪蛋白微球先膨胀后聚集并形成酪蛋白胶束。     

Effect of calcium chelators on physical changes in casein micelles in concentrated micellar casein solutions

The effect of calcium chelators on physical changes of casein micelles in concentrated micellar casein solutions was investigated by measuring calcium-ion activity, viscosity and turbidity, and performing ultracentrifugation. The highest viscosities were measured on addition of sodium hexametaphosphate (SHMP), because it cross-linked the caseins. For the weak calcium chelator disodium uridine monophosphate (Na₂UMP), physical changes in the solutions were negligible. Disodium hydrogen phosphate (Na₂HPO₄), trisodium citrate (TSC), and sodium phytate (SP) caused similar increases in viscosity, but had different effects on turbidity. The increase in viscosity was attributed to swelling of the casein micelles (i.e., increased voluminosity) at decreasing calcium-ion activity. The major decrease in turbidity was due to dissociation of the casein micelles. The extent of micellar dissociation was dependent on the type and concentration of calcium chelator. It seems that the micelles were dissociated in the order of SHMP ≥ SP > TSC > Na₂HPO₄ > Na₂UMP.     

Solubility and structure of milk powders manufactured with the addition of disodium phosphate and tetrasodium pyrophosphate mixtures

This study aimed to investigate the effect of adding binary mixtures of disodium phosphate and tetrasodium pyrophosphate during milk powder production. Overall, the addition of disodium phosphate–tetrasodium pyrophosphate caused an increase in pH and a decrease in the acidity and turbidity of reconstituted milk samples. The decrease in turbidity was attributed to either dispersion or swelling of the casein micelles. The addition of mixtures containing the lowest amounts of tetrasodium pyrophosphate considerably reduced the insolubility index, whereas mixtures containing higher levels of tetrasodium pyrophosphate exerted a detrimental effect on solubility. Interestingly, microscopic observations showed large agglomerated particles in mixtures with the highest level of tetrasodium pyrophosphate. We hypothesized that the formation of casein calcium–pyrophosphate complexes led to the higher insolubility index in this mixture.     

Observations of casein micelles in skim milk concentrate by transmission electron microscopy

The microstructure of casein micelles in ultrafiltrated (UF) skim milk concentrate at pH 6.5 and 5.8 was investigated by transmission electron microscopy using three different preparation methods. The volume fraction of the casein micelles in the UF concentrate was 62.8% (v/v) at pH 6.5 and fixation by glutaraldehyde revealed the close packing of micelles in the UF concentrate as well as a higher degree of micelle aggregation at pH 5.8. No details of the microstructure of the micellar surface or core could, however, be observed. Freeze-fracture of cryoprotected, i.e. glycerol, UF concentrate on the other hand, exposed the finer structures of the micellar core but no pH dependent differences were observed. As cryoprotection includes a dilution of the sample with glycerol, the packing of the micelles in the UF concentrate could not be observed. Undiluted UF concentrate exposed to rapid freezing using a propane jet followed by freeze-fracture exhibited development of ice crystals but rough areas on the micrographs were identified as fractured casein micelles. The micellar core appeared rougher and differences in the micellar core microstructure due to changing pH could be observed when this preparation method was used.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.     

Effects of emulsifying salts on the turbidity and calcium-phosphate-protein interactions in casein micelles

Influence of emulsifying salts (ES) on some physical properties of casein micelles was investigated. A reconstituted milk protein concentrate (MPC) solution (5% wt/wt) was used as the protein source and the effects of ES [0 to 2.0% (wt/wt)] were estimated by measuring turbidity, acid-base titration curves and amount of casein-bound Ca and inorganic P (P_i). Various ES, trisodium citrate (TSC), or sodium phosphates (ortho-, pyro-, or hexameta-) were added to MPC solution, and all samples were adjusted to pH 5.8. Acid-base buffering curves were used to observe changes in the amount and type of insoluble Ca phosphates. An increase in the concentration of TSC added to MPC solution decreased turbidity, buffering at pH ∼5 (contributed by colloidal Ca phosphate), and amount of casein-bound Ca and P_i. Addition of up to 0.7% disodium orthophosphate (DSP) did not significantly influence turbidity, buffering curves, or amount of casein-bound Ca and P_i. When higher concentrations (i.e., ≥1.0%) of DSP were added, there was a slow decrease in turbidity. With increasing concentration of added tetrasodium pyrophosphate (TSPP), turbidity and buffering at pH ∼5 decreased, and amount of casein-bound Ca and P_i increased. When small concentrations (i.e., 0.1%) of sodium hexameta-phosphate were added, effects were similar to those when TSPP were added but when higher concentrations (i.e., ≥0.5%) were added, the buffering peak shifted to a higher pH value, and amount of casein-bound Ca and P_i decreased. These results suggested that each type of ES influenced casein micelles by different mechanisms.     

Evaporative concentration of skimmed milk: Effect on casein micelle hydration,composition, and size

Understanding the effect of evaporative concentration on casein micelle composition is of high importance for milk processing. Alterations to the hydration, composition and size of casein micelles were investigated in skimmed milk evaporated to concentrations of 12–45% total solids content. The size of casein micelles was determined by dynamic light scattering, and the water content and composition determined by analysis of supernatants and pellets obtained by ultracentrifugation. The mass balance and hydration results showed that during the evaporation process, while micelles were dehydrated, water was removed preferentially from the serum. The amount of soluble casein and calcium in the serum decreased as a function of increasing solids content, indicating a shift of these components to the micelles. The formation of a small proportion of micelle aggregates at high concentrations appeared dependent on the time kept at these concentrations. Upon redilution with water, casein micelles were immediately rehydrated and aggregates were broken up in a matter of minutes. Soluble calcium and pH returned to their original state over a number of hours; however, only a small percentage of original soluble casein returned to the serum over the 5 h period investigated. These results showed that casein micelles are significantly affected by evaporative concentration and that the alterations are not completely and rapidly reversible.     

The effects of different cations on the physicochemical characteristics of casein micelles

Physicochemical characterisation of casein micelles suspended in milk ultrafiltrate and enriched with different cations (Fe, Cu, Ca, Zn and Mg) was investigated. After addition of 2.5–8.0 mmol kg⁻¹ of cations, associations of added cation, citrate, inorganic phosphate and calcium with casein micelles were observed. The order of association of cations with casein micelles was Fe³⁺ > Zn²⁺ > Ca²⁺ > Cu²⁺ > Mg²⁺. At the same time, the casein content increased in the casein micelles while the water content decreased. Changes in hydrophobicity and zeta potential of casein micelles were also determined while no variation in the average diameter was detected. In the presence of 8.0 mmol kg⁻¹ of magnesium or ferric iron, heat stability (115 °C for 30 min) of casein micelles was decreased. From these results, a mechanism of cation association with casein micelles is proposed, highlighting the determinant role of ultrafiltrable citrate and inorganic phosphate. This mechanism is discussed in relation to modifications in physicochemical characteristics of casein micelles.     

Micellar Transition State in Casein Between pH 5.5 and 5.0

pH-induced changes in casein micelles during direct acidification and bacterial fermentation of reconstituted skim milk at 20°C were monitored by scanning electron microscopy (SEM) in combination with biochemical and rheological methods. For SEM casein micelle observations, an original method of milk sample preparation with porous inorganic membranes was developed. Micrographs suggested that different stages of micellar association were related to pH and that between pH 5.5 and 5.0 casein micelles coalesced. Correlations between microstructural and biochemical changes in casein micelles, and rheological behavior of milk or gel, help to explain the different steps leading to the final protein network of the acid milk gel.     

Acidification of lactoferrin-casein micelle complexes in skim milk

Lactoferrin electrostatically bound to the casein micelles when added to milk, which caused the absolute zeta potential to decrease and the micelle size to increase. On acidification, the lactoferrin progressively dissociated from the micelles, which, at pH below ∼5.0, caused the zeta potential of the casein micelles to be the same as those in milk without added lactoferrin. Acidification caused increased levels of casein to dissociate from the casein micelles at ∼pH 5.0 as the level of added lactoferrin in the milk increased. Lactoferrin decreased the pH at which the milk gelled and caused the G′ and yield stress of the set gels to increase at low levels of added lactoferrin, decrease at intermediate levels of added lactoferrin and increase again at high levels of added lactoferrin. This unusual effect of lactoferrin on gelation was hypothesised to be due to combined effects of dissociated casein and the lower gelation pH.     

Effects of milk pH alteration on casein micelle size and gelation properties of milk

The effects of changes in pH above and below the natural pH of milk (ca 6.6) on the casein micelle size and the gelation properties of the pH adjusted and restored samples were investigated. The size of casein micelles increased at pH 7.0 and 7.5, then started to decrease above pH 8.5. It is postulated that at pH below 8.5 the casein micelles swell, while elevated pH causes their dissociation. Conversely, the size of casein micelles decreased on acidification to pH 5.5 and increased when the pH dropped below 5.5, due to the shrinkage of casein micelles at lower pH before their aggregation at pH below 5.5. In response to neutralising treated milk back to normal milk pH of 6.6, it was found that the casein micelle size of treated milk with a narrow range of pH change between 6.0 and 7.0 was reversible, while beyond this range the structure of casein micelles became irreversible. The restoration of casein micelle size was followed by the restoration of some parameters such as soluble calcium, ethanol stability, and milk whiteness. Acid-induced gelation did not change the elastic modulus, while rennet-induced gelation was affected by initial milk pH. In reference to the size of reversible range elastic modulus (G’) of acid or rennet gels made from restored milk, the sizes were similar to control milk after 6 h of gelation.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

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。     

Changes in the physico-chemical properties of casein micelles during ultrafiltration combined with diafiltration

The objective of this work was to determine if after membrane processing, the physical properties of casein micelles change. Milk was concentrated by ultrafiltration, and also subjected to various levels of diafiltration, by addition of water to the retentate. After the membrane concentration process, the retentates were diluted back to their original concentration, to study their physico-chemical properties. For better comparison, all the samples were dialyzed against the original milk, to obtain similar serum compositions. For the first time, the effect of different levels of diafiltration was studied. Diafiltration induced losses of colloidal calcium phosphate and caused changes in the turbidity parameter (1/l*) measured by light scattering, as well as in the ultrasonic properties (velocity and attenuation) of the casein micelles. When tested in a similar serum environment, the reconstituted micelles after diafiltration showed a lower susceptibility to aggregation and the rennet induced gels had a lower storage modulus than those formed with the original milk, at the same protein concentration. This work brings for the first time evidence of the differences in the physical properties of the casein micelles as a function of membrane processing history.     

pH treatment as an effective tool to select the functional and structural properties of yak milk caseins

Qula is made from yak milk after defatting, acidifying, and drying. Yak milk caseins are purified from Qula by dissolving in alkali solution. The effects of different pH treatments on the functional and structural properties of yak milk caseins were investigated. Over a broad range of pH (from 6.0 to 12.0), functional properties of yak milk caseins, including solubility, emulsifying activities, and thermal characteristics, and the structural properties, including 1-anilino-8-naphthalene-sulfonate fluorescence, turbidity and particle diameter, were evaluated. The results showed that the yak milk casein yield increased as the pH increased from 6.0 to 12.0. The solubility dramatically increased as the pH increased from 6.0 to 8.0, and decreased as the pH increased from 9.0 to 12.0. The changes in emulsifying activity were not significant. Caseins were remarkably heat stable at pH 9.0. The turbidity of the casein solution decreased rapidly as the pH increased from 6.0 to 12.0, and the results suggested that reassembled casein micelles were more compact at low pH than high pH. At pH values higher than 8.0, the yield of yak milk caseins reached more than 80%. The highest solubility was at pH 8.0, the best emulsification was at pH 10.0 and the greatest thermal stability was at pH 9.0. According to the functional characteristics of yak milk caseins, alkali conditions (pH 8.0–10.0) should be selected for optimum production. These results suggested that pH-dependent treatment could be used to modify the properties of yak milk caseins by appropriate selection of the pH level.     

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and β-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.