Aggregation Of Casein Micelles Research Articles

Exploration of dynamic interaction between β-lactoglobulin and casein micelles during UHT milk process

The protein aggregation induced by UHT treatment shortens the shelf life of UHT milk. However, the mechanism of β-Lg induced casein micelle aggregation remains unclear. Herein, the dynamic interaction between β-Lg and casein micelles during UHT processing was investigated by experimental techniques and molecular dynamics simulations. Results showed that β-Lg decreased the stability of casein micelles, increased their size and zeta potential. Raman and FTIR spectra analysis suggested that hydrogen and disulfide bonds facilitated their interaction. Cryo-TEM showed that the formation of the casein micelle/β-Lg complex involved rigid binding, flexible linking, and severe cross-linking aggregation during UHT processing. SAXS and MST demonstrated β-Lg bound to κ-casein on micelle surfaces with a dissociation constant (Kd) of 3.84 ± 1.14 μm. Molecular docking and dynamic simulations identified the interacting amino acid residues and clarified that electrostatic and van der Waals forces drove the interaction. UHT treatment increased hydrogen bonds and decreased total binding energy. The non-covalent binding promoted the formation of disulfide bonds between β-Lg and casein micelles under heat treatment. Ultimately, it was concluded that non-covalent interaction and disulfide bonding resulted in casein micelle/β-Lg aggregates. These findings provided scientific insights into protein aggregation in UHT milk.

Preparation of liquid yogurt in the presence of pectin and its formation mechanism

We had previously observed that adding pectin into milk before fermentation inhibited gelation of yogurt but did not affect the pH. Thus, this work aimed to prepare such liquid yogurt and clarify its formation mechanism. It was found that liquid yogurt was obtained in the presence of 0.10%–0.20% pectin. However, at lower or higher pectin concentrations, yogurt was gelled. Confocal laser scanning microscopy analysis demonstrated that 0.10%–0.20% pectin induced milk protein aggregating into separated particles rather than a continuous network, which explained why liquid yogurt was formed. Moreover, adding 0.10%–0.20% pectin into the casein micelle suspension induced aggregation of casein micelles at pH 6.8. After pH decreased to 4.3, casein micelles showed more aggregation but they were still separated particles, which was the same in the corresponding yogurt samples. These results suggested that pectin changed the aggregation mode of casein micelles and induced formation of liquid yogurt.

The aggregation of casein micelles induced by Ca2+ during in vitro digestion: effects on the release of loaded anthocyanins.

Colloidal calcium phosphate (CCP) confers a modifiable structure to micellar casein (MC), which endows it with potential advantages as a delivery carrier. However, it is difficult to achieve multipattern release of the core material in the intestine with MC as a single wall. In this study, we prepared an anthocyanin-casein-based delivery system utilizing MC with different freezing degrees as the wall material with the objective of achieving the controlled release of anthocyanin as the model core in the intestine. The results showed that freezing could significantly reduce the CCP level up to 50%. Static in vitro simulated digestion with the addition of exogenous Ca2+ showed that the designed delivery system exhibited low anthocyanin release (15%-35%) in the gastric tract. The pattern of release in the intestine depended on the CCP dissociation degree. High and low dissociation degrees corresponded to slow release (from 15% to 65% within 2 h) and burst release (from 35% to 90% within 5 min), respectively. WAXS/SAXS analysis revealed that exogenous serum Ca2+ inherent in simulated gastric fluid and endogenous serum Ca2+ induced by CCP dissociation was synergistically involved in the reconstitution of CCP-mediated nanoclusters and large aggregates. The freezing degree of MC determined the endogenous serum Ca2+ level, which influenced the gastric aggregation behavior of wall MC and ultimately led to a fairly different gastrointestinal release behavior of anthocyanins.

Investigating the Acid- and Enzyme-Induced Coagulation of Raw Milk Using Dielectric and Rheological Measurements

In this study, the enzyme-induced and acid coagulation of raw milk samples were investigated, and these two processes were monitored by determining the changes in dielectric and rheological behavior to see if any correlations could be found between these two properties. For the enzyme-induced experiments, 250 cm3 of raw milk sample and industrial chymosin were used, and during the acid coagulation, industrial thermophile yogurt culture was added to the same amount of milk. The apparent viscosity was monitored with a vibro viscometer, and the dielectric constant was measured in a frequency range of 200–2400 MHz with an open-ended dielectric assessment kit (DAK) connected to a vector network analyzer (VNA). Our experimental findings revealed that during both enzyme-induced and acid coagulation, two distinct phases could be found in terms of dielectric and rheological properties, and regardless of the molecular mechanisms behind the aggregation of casein micelles, a strong correlation (with R2-values ranging between 0.967 and 0.992) can be found between the extent of the apparent viscosity and dielectric constant, suggesting that dielectric measurements might be adequately used in these dairy processing technologies. These results verified the applicability of the non-destructive dielectric measurements in dairy technologies.

Heat stability of sheep's skim milk: Aggregation and interaction of proteins

Sheep's milk proteins are susceptible to heat-induced coagulation, but the protein interactions under high heat treatment have not been determined. Heat stability and protein interactions of sheep's skim milk (SSM) at pH 6.2–7.2 were examined at 140 °C. SSM had the longest heat coagulation time at pH 6.9, but became very unstable at higher or lower pH. Protein aggregates formed consisted mainly of whey proteins and κ-casein (κ-CN)-depleted casein micelles. Modification of SSM pH alters ionic calcium concentration, dissociation of caseins and electrostatic interactions, resulting in different extents of protein interactions. The extent of dissociation of κ-CN from casein micelles increased with increasing pH (from ∼6.6 to 7.0) before and after heat treatment, contributing to κ-CN-depleted casein micelle aggregation. High ionic calcium concentrations, low levels of κ-CN on casein micelles and ready dissociation of κ-CN from casein micelles may be responsible for the low heat stability of sheep's milk.

Effect of CSN3 Gene Polymorphism on the Formation of Milk Gels Induced by Physical, Chemical, and Biotechnological Factors.

During the last decade, research into genetic markers in the casein gene cluster has been actively introduced in cattle breeding programs. A special interest has been paid to the polymorphism of the CSN3 gene, responsible for the expression of the k-casein, playing a key role in protein coagulation, interaction with whey proteins, stabilization, and aggregation of casein micelles. This paper aimed to determine the effect of CSN3 genetic polymorphism on acid; rennet; acid-rennet; heat- and acid-induced as well as heat- and calcium-induced coagulation in skimmed milk; and protein-standardized milk systems (UF, NF, RO, VE). The influence of polymorphic variants of the CSN3 gene on the coagulation ability of milk proteins was assessed by the particle size of casein micelles, protein retention factor in the clot, and coagulation ability (duration of induction period, mass coagulation period, dynamic viscosity in gel point). The correlation between CSN3 gene polymorphism and protein coagulation was revealed. Milk systems obtained from CSN3 BB milk were found to have the shortest duration of coagulation, formation of better gel strength values, and increased yield compared to CSN3 AA. This study will improve the efficiency of milk processing and optimize the technology of dairy product production.

Effect of sodium alginate on the yogurt stability was dependent on the thickening effect and interaction between casein micelles and sodium alginate

The effect of sodium alginate (SA) on the yogurt stability and the related mechanisms were investigated. It was found that low-concentration SA (≤0.2 %) increased the yogurt stability, while high-concentration SA (≥0.3 %) decreased the yogurt stability. Sodium alginate increased the viscosity and viscoelasticity of yogurt and this effect was positively correlated with its concentration, suggesting that SA worked as the thickening agent in yogurt. However, addition of ≥0.3 % SA damaged the yogurt gel. These results suggested that interaction between milk protein and SA might play an important role in the yogurt stability besides the thickening effect. Addition of ≤0.2 % SA did not change the particle size of casein micelles. However, addition of ≥0.3 % SA induced aggregation of casein micelles and increased the size. And the aggregated casein micelles precipitated after 3 h storage. Isothermal titration calorimetry analysis showed that casein micelles and SA were thermodynamically incompatible. These results suggested that the interaction between casein micelles and SA induced aggregation and precipitation of casein micelles, which was critical in the destabilization of yogurt. In conclusion, the effect of SA on the yogurt stability was dependent on the thickening effect and the interaction between casein micelles and SA.

Spatial distribution of αs1-caseins and β-caseins in milk gels acidified with glucono-δ-lactone

Acid-induced destabilization of casein micelles leads to coagulation of milk which plays an important role in the formation of yogurt. The resulting protein network is formed by aggregation and reconfiguration of casein micelles, including the release of a portion of caseins from these micelles. It is so far unknown how individual αs1-and β-caseins are organized within this complex network, and how their distribution depends on yogurt composition and processing. Here, we imaged the spatial distribution of caseins using stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM). We used fluorescently tagged antibodies against αs1-and β-caseins to localize them inside glucono-δ-lactone (GDL)-acidified milk gels. We conducted quantitative skeleton analysis of STED images and showed that αs1-and β-caseins contribute with different levels of connectivity to the acid induced milk network.

Spectroscopic analysis of the effect of glycation on casein structure and aggregation and its dependence on lactose concentration

In order to study the effect of glycation on the molecular structure of casein (CN), three groups of simulated milk powders with different lactose (Lac) contents, CN:Lac = 1:1.5, 1:2, and 1:2.5 (m:m), were constructed. The glycation level and the structural changes of CN were analyzed by multiple spectra. The results showed that the interaction between Lac and CN molecules was based on the dynamic quenching of collision. A higher quenching constant, KSV, means that the 1:1.5 group showed more distinct glycation levels. In addition, CN glycation reduced the aggregation of casein micelles caused by heating treatment and repaired the structural damage of CN induced during the heating process. There was no positive correlation found between Lac content and protein glycation; thus, decreasing the Lac content of milk powder is not likely to be an effective method to prolong storage stability.

Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90–95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Biochemical, microbiological, and structural evaluations to early detect age gelation of milk caused by proteolytic activity of Pseudomonas fluorescens

Heat–stable peptidase AprX, released by Pseudomonas species in raw milk during cold storage, can cause gelation of UHT milk since it is able to split caseinomacropeptides (CMPtot) from κ-casein, so inducing aggregation of casein micelles. Identifying raw milk susceptibility to gelation would allow UHT milk manufacturers to select appropriate processing conditions or give the milk a different destination. Two approaches, i.e., detection of free CMPtot and evidence of casein aggregates, were evaluated as possible indicators for early detecting milk destabilization. With this aim, microfiltered milk was inoculated with a P. fluorescence strain and incubated at either 4 or 25 °C. The presence of CMPtot was detected using capillary electrophoresis after 96 and 24 h at the two temperatures, respectively, when milk also became heat unstable and small flocks of protein appeared. Confocal laser scanning microscopy evidenced initial aggregates of casein micelles after 48 and 24 h at 4 and 25 °C, respectively. Keeping the milk at 25 °C/24 h could be a useful condition to accelerate milk destabilization. Despite the similar timing of instability detection, presence of CMPtot was the only trait specific for AprX activity.

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.

Physicochemical properties of skim milk powder dispersions prepared with calcium-chelating sodium tripolyphosphate, trisodium citrate, and sodium hexametaphosphate

Due to health benefits of proteins, the demand for protein beverages has grown rapidly. Translucent protein drinks with neutral pH may have advantages over acidic beverages that may cause dental erosion, and skim milk powder (SMP) is an affordable protein ingredient. Dissociating casein micelles by calcium chelators is a well-known method to reduce SMP dispersion turbidity, but much is to be studied for physicochemical properties as affected by chelator type and concentration. The objective of the present study was to characterize physicochemical properties of dispersions with 5% (wt/vol) SMP after addition of 0 to 30 mM sodium tripolyphosphate, trisodium citrate, or sodium hexametaphosphate. The turbidity was decreased with increasing chelator concentration, with the lowest turbidity observed in the SMP dispersions with sodium hexametaphosphate. The smallest hydrodynamic diameter was observed at an intermediate chelator concentration, resulting from the balance of casein micelle dissociation and aggregation of dissociated caseins induced at an elevated ionic strength. Heating at 90°C for 5 min increased turbidity but lowered hydrodynamic diameter of SMP dispersions, with some exceptions. The morphology of SMP dispersions differed for each chelator and was also affected by chelator concentration and heating. Trisodium citrate was the most effective to demineralize colloidal calcium phosphate in casein micelles, but the amount of dissolved calcium was not directly correlated with the decreased turbidity, indicating different chelating mechanisms by each chelator. Analysis of serum calcium and phosphorus concentrations also suggested that the type and concentration of soluble and insoluble calcium phosphates and their partitioning in the serum and casein micelles were dynamically changed by the studied parameters to affect dispersion turbidity and structures of casein micelles. Findings from the present study may be used to formulate translucent beverages incorporating SMP and other casein micelle ingredients.

Single particle tracking as a new tool to characterise the rennet coagulation process

Aggregation of casein micelles (CMs) was followed with single-particle tracking using fluorescence microscopy. Initial concentrations of up to a mass fraction of 3% (w/w) were studied. A small fraction of the CMs was labelled with Atto 488 fluorescent dye to achieve sufficient dilution to position single micelles, despite the diffraction limit of light and to allow particle tracking without overlapping tracks. In addition, a higher fraction of CMs was labelled with a second dye (Atto 647N) to visualise in parallel the resulting CM aggregates. Distributions of hydrodynamic radii obtained from up to 2000 individual tracks were log-normally distributed around a most frequent value of rh = 114 nm. The evaluation of the particle tracking experiments during the temperature-induced coagulation of para-casein micelles indicates that the aggregation proceeds in two phases. First, the formation and steady growth of aggregates occurs, which, after reaching a certain size, abruptly form a coherent gel.

Microscopy tools for product innovation

Microscopy tools for product innovation

Biotechnological potential of a cysteine protease (CpCP3) from Calotropis procera latex for cheesemaking

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos’ morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.

Implications of kappa-casein evolutionary diversity for the self-assembly and aggregation of casein micelles.

Milk alpha-, beta- and kappa-casein proteins assemble into casein micelles in breast epithelial cells. The glycomacropeptide (GMP) tails of kappa-casein that extend from the surface of the micelle are key to assembly and aggregation. Aggregation is triggered by stomach pepsin cleavage of GMP from para-kappa-casein (PKC). While one casein micelle model emphasizes the importance of hydrophobic interactions, another focuses on polar residues. We performed an evolutionary analysis of kappa-casein primary sequence and predicted features that potentially impact on protein interactions. We noted more rapid change in the earlier period (166 to 60 Ma). Pepsin and plasmin cleavage sites were avoided in the GMP, which may partly explain its amino acid composition. Short tandem repeats have led to modest expansions of PKC, and to large GMP expansions, suggesting the GMP is less length constrained. Amino acid compositional constraints were assessed across species. Polarity and hydrophobicity properties were insufficient to explain differences between PKC and GMP. Among polar residues, threonine dominates the GMP, compared to serine, probably reflecting its preference for O-glycosylation over phosphorylation. Glutamine, enriched in the bovine PQ-rich region, is not positionally conserved in other species. Among hydrophobic residues, isoleucine is clearly preferred over leucine in the GMP, and patches of hydrophobicity are not markedly positionally conserved. PKC tyrosine and charged residues showed stronger conservation of position, suggesting a role for pi-interactions, seen in other structurally dynamic protein membraneless assemblies. Independent acquisitions of cysteines are consistent with a trend of increasing stabilization of multimers by covalent disulphide bonds, over evolutionary time. In conclusion, kappa-casein compositional and positional constraints appear to be influenced by modification preferences, protease evasion and protein–protein interactions.

Simple and Rapid Method for Separating Lactic Acid Bacteria from Commercially Prepared Yogurt.

We have reported a simple method for separating lactic acid bacteria (LAB) from yogurt. This method is based on the process of destructions and denaturation of casein micelle aggregates by vortexing, and can supply samples containing only LAB. Recovered LAB were clearly observable by microscopy, meaning that morphological changes could be directly detected at the single-cell level. This method will be a helpful tool for the analyzing various LAB, including their enzyme activity and protein expression.

РОЗРОБЛЕННЯ БІОРОЗКЛАДНОЇ ПОЛІМЕРНОЇ КОМПОЗИЦІЇ НА ОСНОВІ КАЗЕЇНУ МОЛОКА

The article sums up the results of the study of milk casein as a biopolymer, which is widely used in food industry, construction, pharmacy, etc. and on the basis of which polymer films for biodegradable materials can be obtained. The analysis draws on data on the structure of milk casein molecules as a complex globular micellar system, which is constructed by nanoscale sub-micelles, interconnected by calcium phosphate sites, with the formation of a casein-calcium phosphate complex. Sub-micelles differ in structure and function, according to which their four fractions are distinguished. The hydrophilic carboxyl groups of the glutamic, aspartic and orthophosphate acids of the κ-casein fraction provide the stabilization of casein micelles in the aqueous medium. Such structure of casein does not allow to receive film materials, they have low technological characteristics, very low durability and elasticity, high fragility. Moreover, they are easily soluble in water.
 Studies have shown that on the basis of casein it is possible to obtain polymer films, provided the use of a polymer composition, in which besides casein there is a modified polysaccharide. To obtain such a composition, carboxymethylcellulose was used, which also has biodegradable properties, however, unlike casein, it is capable of forming sufficiently strong polymer films. The polymer composition was synthesized in two steps: first, an aqueous solution of carboxymethyl cellulose was prepared, to which freshly precipitated casein with a moisture content of 20%, precipitated from skim milk, was added. The composition of the polymer composition was enriched with a number of additives serving as plasticizers, and structure-forming. The triatomic alcohol glycerol and diphenylamine were added as plasticizers too. In order to regulate the structure of the aqueous dispersion medium and prevent the processes of aggregation of casein micelles, calcium chloride solution was injected in the composition. The given polymer composition after polymerization in the air-drying process makes it possible to obtain sufficiently strong composite polymer films, the transparency of which depends on the type of plasticizer selected and the presence of CaCl2.

Spontaneous aggregation of bovine milk casein micelles: Ultra-small angle x-ray scattering and mathematical modeling

We have used Ultra Small Angle X-ray Scattering (USAXS) and mathematical models to study seemingly-spontaneous aggregation structures in two pasteurized bovine milks. Although extensive studies of casein micelles and their aggregation have been carried out, few have been done to numerically characterize submicron structures to micron-scale structures. We measured the USAXS intensity, I(q), as a function of the scattering vector magnitude, q, for commercial pasteurized skim milk and nonhomogenized whole milk at two temperatures, 7 °C and 45 °C. We observed broad peaks, reported previously to be related to casein micelles, centered at q ≈ 2 × 10−2 Å−1 and at q ≈ 9 × 10−2 Å−1. At lower q values, log I(q) displayed a behavior characteristic of aggregation manifested for a slope in the region 3–7 × 10−4 Å−1 < q < 4 × 10−3 Å−1. This behavior appeared in the absence of (a) chymosin, (b) any change in pH or CaCl2 concentration, and (c) temperature changes. We introduced a model of milk and used computer simulations to investigate consequences of casein micelles possessing surface areas lacking the water-soluble components of κ-casein proteins. These components exist to provide stability against aggregation to the casein micelles. We propose that bovine casein micelles spontaneously formed 1-dimensional aggregates.