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![Schematic illustration of the casein micelle structure, incorporated calcium phosphate nanoclusters (grey) with their attached caseins (red), the surface-located κ-casein (green), and hydrophobically bound mobile β-casein (blue). Individual components are not to scale. Reproduced with permission from [3], copyright by The Royal Society of Chemistry, 2011.](profile/Norbert-Raak/publication/323129231/figure/fig1/AS:593303800139777@1518466057890/Schematic-illustration-of-the-casein-micelle-structure-incorporated-calcium-phosphate.png)
Schematic illustration of the casein micelle structure, incorporated calcium phosphate nanoclusters (grey) with their attached caseins (red), the surface-located κ-casein (green), and hydrophobically bound mobile β-casein (blue). Individual components are not to scale. Reproduced with permission from [3], copyright by The Royal Society of Chemistry, 2011.
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![Table 1 . Characteristics of main casein types in bovine milk [17].](profile/Norbert-Raak/publication/323129231/figure/tbl1/AS:631599477559347@1527596458633/Characteristics-of-main-casein-types-in-bovine-milk-17_Q320.jpg)
Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size...
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... colloidal stability of casein micelles in aqueous systems is ensured by steric repulsion of the so-called "hairy layer", which corresponds to the glycosylated, hydrophilic C-termini of κ-casein located on the micelle surface [6][7][8]. A schematic illustration of the casein micelle structure, as suggested by Dalgleish [3], is shown in Figure 1. In spite of decades of research, structure of casein micelles and type of interactions between caseins are still a topic of controversial discussion (e.g., [9][10][11] vs. [12,13]. ...
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... many cases, the results were linked to the size of the generated casein oligomers and polymers, but size determination by separation techniques is still challenging, mainly because of insufficient resolution and the unavailability of suitable standard substances for molar mass determination. In this review we provide an overview on prominent cross-linking reactions (Section 2), followed by a discussion of two size separation techniques that are commonly applied for the characterisation of cross-linked casein: gel electrophoresis (Section 3) Figure 1. Schematic illustration of the casein micelle structure, incorporated calcium phosphate nanoclusters (grey) with their attached caseins (red), the surface-located κ-casein (green), and hydrophobically bound mobile β-casein (blue). ...
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... separation efficiency depends strongly on column material. Taking non-micellar casein cross-linked by TGase as example, Figure 10a compares chromatograms obtained from Superdex 200 and Superose 6, exhibiting fractionation ranges from 10 to 600 and 5 to 5000 kg/mol for globular proteins, respectively. Superdex 200 consequently results in a narrower elution of fractions and better separation in the low molar mass region, i.e., monomers and dimers, whereas Superose 6 reveals an additional fraction at ~9-12 mL, which coelutes with the largest polymers at ~7.5 mL when using Superdex 200. ...
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... separation efficiency depends strongly on column material. Taking non-micellar casein cross-linked by TGase as example, Figure 10a compares chromatograms obtained from Superdex 200 and Superose 6, exhibiting fractionation ranges from 10 to 600 and 5 to 5000 kg/mol for globular proteins, respectively. Superdex 200 consequently results in a narrower elution of fractions and better separation in the low molar mass region, i.e., monomers and dimers, whereas Superose 6 reveals an additional fraction at ~9-12 mL, which coelutes with the largest polymers at ~7.5 mL when using Superdex 200. ...
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... contrast to gel electrophoresis, different casein types elute together in a single peak and cannot be clearly discriminated. However, when injecting standards of different casein types slightly different elution following the order β-casein < α S1 -casein < κ-casein can be seen (Figure 10b), which is in accordance to their respective molar mass (Table 1) and allows at least rough hints on their different polymerisation velocity [148]. Using Superdex 200, the monomer peak is clearly shifted to higher elution volumes with higher cross-linking of non-micellar casein by TGase (Figure 10a), which suggests the presence of remaining κ-casein, as also observed by SDS-PAGE (see Section 3.3). ...
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... when injecting standards of different casein types slightly different elution following the order β-casein < α S1 -casein < κ-casein can be seen (Figure 10b), which is in accordance to their respective molar mass (Table 1) and allows at least rough hints on their different polymerisation velocity [148]. Using Superdex 200, the monomer peak is clearly shifted to higher elution volumes with higher cross-linking of non-micellar casein by TGase (Figure 10a), which suggests the presence of remaining κ-casein, as also observed by SDS-PAGE (see Section 3.3). This shift, however, is less pronounced when using Superose 6 because the broader fractionation range results in a narrower elution of monomers. ...
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... of the heterogeneity of caseins and heteropolymers resulting from cross-linking, peaks of polymeric fractions in size exclusion chromatograms are broader than respective homopolymers and may also vary in their position depending on cross-linking intensity. Figure 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. ...
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... 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. It is thus safe to presume that the dimer peak of acid casein is shifting because dimers containing β-casein are further cross-linked whereas dimers with κ-casein remain and/or are formed in later stages of cross-linking. ...
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... 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. It is thus safe to presume that the dimer peak of acid casein is shifting because dimers containing β-casein are further cross-linked whereas dimers with κ-casein remain and/or are formed in later stages of cross-linking. ...
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... 2018, 5, x FOR PEER REVIEW 13 of 34 In contrast to gel electrophoresis, different casein types elute together in a single peak and cannot be clearly discriminated. However, when injecting standards of different casein types slightly different elution following the order β-casein < αS1-casein < κ-casein can be seen (Figure 10b), which is in accordance to their respective molar mass (Table 1) and allows at least rough hints on their different polymerisation velocity [148]. Using Superdex 200, the monomer peak is clearly shifted to higher elution volumes with higher cross-linking of non-micellar casein by TGase (Figure 10a), which suggests the presence of remaining κ-casein, as also observed by SDS-PAGE (see Section 3.3). ...
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... when injecting standards of different casein types slightly different elution following the order β-casein < αS1-casein < κ-casein can be seen (Figure 10b), which is in accordance to their respective molar mass (Table 1) and allows at least rough hints on their different polymerisation velocity [148]. Using Superdex 200, the monomer peak is clearly shifted to higher elution volumes with higher cross-linking of non-micellar casein by TGase (Figure 10a), which suggests the presence of remaining κ-casein, as also observed by SDS-PAGE (see Section 3.3). This shift, however, is less pronounced when using Superose 6 because the broader fractionation range results in a narrower elution of monomers. ...
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... of the heterogeneity of caseins and heteropolymers resulting from cross-linking, peaks of polymeric fractions in size exclusion chromatograms are broader than respective homopolymers and may also vary in their position depending on cross-linking intensity. Figure 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. ...
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... 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. It is thus safe to presume that the dimer peak of acid casein is shifting because dimers containing β-casein are further cross-linked whereas dimers with κ-casein remain and/or are formed in later stages of cross-linking. ...
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... 11a depicts non-micellar acid casein cross-linked by TGase: the maximum of the dimer peak is shifted from ~9.85 mL to ~10.12 mL with ongoing cross-linking. The position of this peak, however, is not changing when dimers are comprised either of only β-casein (Figure 11b) or of only κ-casein (Figure 11c), and they can be distinguished by their elution volumes of ~10.6 mL and ~11.4 mL, respectively. It is thus safe to presume that the dimer peak of acid casein is shifting because dimers containing β-casein are further cross-linked whereas dimers with κ-casein remain and/or are formed in later stages of cross-linking. ...
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... limitation is that whey proteins may be less effectively separated from caseins. In case of both Superdex 200 and Superose 6, β-lactoglobulin coelutes with the monomers while α-lactalbumin can be separated to some extent (Figure 10c). For quantitative analysis of casein polymerisation in milk, whey proteins should be removed from the sample prior to SEC. ...
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... to gel electrophoresis, the most simple application of SEC under denaturing and reducing conditions is for verification that polymerisation occurred, e.g., [54,116,133,134,147]. In several studies, Bönisch et al. used SEC to investigate the action and inactivation of an inhibitor of Figure 11. Sections of typical size exclusion chromatograms (Superdex 200 column) of cross-linked (a) casein in phosphate buffer (CN-PB); (b) β-casein; and (c) κ-casein depicting monomers (~12 mL) and dimers (~10 mL) (originally presented in [94]; see also Appendix A.4). ...
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... limitation is that whey proteins may be less effectively separated from caseins. In case of both Superdex 200 and Superose 6, β-lactoglobulin coelutes with the monomers while α-lactalbumin can be separated to some extent (Figure 10c). For quantitative analysis of casein polymerisation in milk, whey proteins should be removed from the sample prior to SEC. ...
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... the polymerisation degree (sometimes also referred to as oligomerisation degree) was calculated in several studies from size exclusion chromatograms by relating peak areas of cross-linked caseins to the entire sample area [30,32,44,47,48,53,78,[134][135][136][146][147][148]. Because reference casein samples often reveal a low level of polymerised casein (see Figures 9 and 10), the polymerisation degree after cross-linking was also expressed as the difference between cross-linked samples and reference [137][138][139]144,145]. A different definition of polymerisation degree was given by Monogioudi et al. [91] for β-casein cross-linked by tyrosinase: they used SEC in combination with multi-angle laser light scattering (MALS) for molar mass determination (see also Section 6) and calculated the ratio of average molar mass to molar mass of monomeric β-casein. ...
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... external cross-flow is applied orthogonally to the channel flow, and separates the analysed molecules with respect to their diffusivity [165]. The procedure of a typical AF4 separation approach is shown in Figure 12. During the first two steps (i.e., injection and focusing) the eluent enters the channel from the inlet and outlet port, and permeates the ultra-filtration membrane. ...
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... external cross-flow is applied orthogonally to the channel flow, and separates the analysed molecules with respect to their diffusivity [165]. The procedure of a typical AF4 separation approach is shown in Figure 12. During the first two steps (i.e., injection and focusing) the eluent enters the channel from the inlet and outlet port, and permeates the ultra-filtration membrane. ...
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... kB is the Boltzmann constant, T is the temperature and η is the viscosity of the eluent. By combining the Stokes-Einstein equation with the retention theory [169] Rh can be obtained from the elution time: Figure 12. Schematic illustration of the separation mechanism using asymmetrical flow field flow fractionation. ...
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... an alternative to the conventional AF4 represents the frit-inlet channel (FI-AF4), which reduces the concentration of the sample near the ultra-filtration membrane. The channel design allows by-passing the focusing step, and the samples are hydrodynamically relaxed by a compressing action of an additional flow that enters the channel from a small permeable frit placed close to the injection port ( Figure 13). After the sample components have passed the frit inlet flow area and reached a ready state equilibrium, the separation process takes place in the same way as the elution step for the conventional AF4. ...
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... applied AF4 for the investigation of casein micelles over the entire size range. Figure 14 depicts the molar mass and size distributions, with peak 3 representing the casein micelles. ...
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... applied AF4 for the investigation of casein micelles over the entire size range. Figure 14 depicts the molar mass and size distributions, with peak 3 representing the casein micelles. Molar mass and radius of gyration were determined by MALS detector, whereas Rh was calculated from the elution time (see Section 5.1, Equation (3)). ...
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... authors firstly performed SAXS and osmometry measurements, with the latter contradicting results published in literature. Therefore, AF4 was performed, resulting in the plot shown in Figure 15. Over the whole fractogram, Rg ranged from approx. ...
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... authors firstly performed SAXS and osmometry measurements, with the latter contradicting results published in literature. Therefore, AF4 was performed, resulting in the plot shown in Figure 15. Over the whole fractogram, R g ranged from approx. ...
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... is possible because the light scattering intensity is in direct relation with the molar mass. In this regard, the combination of AF4 or SEC with scattering techniques (see Figure 16) is a powerful tool for comprehensive characterisation of a wide range of polymers [194][195][196][197]. In MALS experiments, the intensity of light scattered by an analyte is measured at different angles (θ). ...
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... MALS experiments, the intensity of light scattered by an analyte is measured at different angles (θ). For a dilute sample solution, and assuming a vertically polarised incident light, the intensity (I) of scattered light is proportional to the properties of the sample solution according to the Zimm equation [198]: Figure 15. Fractograms of β-casein assemblies obtained by asymmetrical flow field flow fractionation together with the distribution of the molar mass and radius of gyration (R g ). ...
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... is possible because the light scattering intensity is in direct relation with the molar mass. In this regard, the combination of AF4 or SEC with scattering techniques (see Figure 16) is a powerful tool for comprehensive characterisation of a wide range of polymers [194][195][196][197]. Separations ...
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... is possible because the light scattering intensity is in direct relation with the molar mass. In this regard, the combination of AF4 or SEC with scattering techniques (see Figure 16) is a powerful tool for comprehensive characterisation of a wide range of polymers [194][195][196][197]. In MALS experiments, the intensity of light scattered by an analyte is measured at different angles (θ). ...
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... et al. [188] calculated the apparent densities ρ and the R g /R h ratio from the molar mass and radii distributions in order to describe the shape of casein micelles (Figure 17). From the data, the authors found a different tendency of ρ h and ρ g as a function of the R g , suggesting large differences in conformation over the entire size distribution. ...
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... to now little has been reported about appropriate standard substances for molar mass calibration of casein polymers. Therefore, absolute molar mass determination will be of increased Figure 17. (a) Apparent density calculated from the radius of gyration (R g ; ) and the hydrodynamic radius (R h ; ) vs. R g , and (b) R g /R h ( ) and differential mass distribution (-) vs. R g for casein micelles in milk. ...
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Milk protein concentrates (MPC) are widespread food ingredients, due to their high protein-to-solids ratio, nutritional value, and technological properties, which can be tailored to various end uses. Interest has been given to modifying such ingredients through enzymatic cross-linking by microbial transglutaminase. However, no systematic studies on...
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... In particular, these are devices for horizontal and vertical electrophoresis, electrophoresis in PAG tubes and plates. Currently, various modifications of devices for vertical electrophoresis in PAG plates are most often used [21]. There are many varieties of such devices, which are designed for the analytical version of various electrophoretic systems. ...
The object of research is a Stadier-type apparatus for analytical electrophoresis of proteins. In the milk proteins research, in addition, there is a need to carry out serial express analyzes of their various groups, as well as the isolation of individual homogeneous fractions. The dimensions of working chambers for analytical, express, and micro preparative electrophoresis of caseins and milk whey proteins were proposed to solve this task. For each type of electrophoresis, different chambers and formers are used without changing the design of the apparatus. The apparatus is suitable for electrophoretic systems used for the analysis of milk proteins. Analysis of casein in the anodic system of a homogeneous polyacrylamide gel in the presence of urea allows identification of the main fractions: αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, αS2-CN-13P, β-CN-5P, ϰ-CN-1P and three β-casein fragments f(29-209), f(106-209) and f(108-209). Express electrophoresis in the presence of urea reveals four fractions of caseins: αS1-CN, αS2-CN, β-CN, and ϰ-CN. The analysis of whey proteins in the Davis native disc electrophoresis system allows identification of β-Lg A, β-Lg B, α-La, BSA fractions, and a group of immunoglobulin fractions. The express electrophoregram differs by a common band A and B variants of β-Lg. Due to an adequate selection of electrophoretic systems, it is possible to identify semi-quantitatively all the main fractions of milk proteins under analytical or express mode. The adapted apparatus also makes it possible to conduct micro preparative electrophoresis and obtain the main fractions of milk proteins. In this case, the yield of electrophoretically pure proteins is: β-CN-5P (23±5 %), β-Lg (A+B) (27±6 %), α-La (11±3 %), and purified groups of αS1-CN-8P+αS1-CN-9P (25±6 %), αS2-CN-(10-13P) (6±1.5 %) and ϰ-CN-1P (7±2 %). The apparatus could be used at enterprises producing dairy protein products
... For identifying these crosslinked formations, commonly used techniques are gel electrophoresis (GE), size exclusion chromatography (SEC), and isopeptide content quantification (ICQ). GE allows identification of casein molecules' crosslinks, such as aS1-, aS2-, and κ-casein, whereas high-MW polymers with over 250 kDa cannot go through the gel [31]. The quantification of monomer conversions to dimer, trimer, and oligomer is identified by SEC, which provides an estimation of the polymerization degree (PD), also considered the crosslinking degree. ...
Stabilization and reusability of enzyme transglutaminase (TGM) are important goals for the enzymatic process since immobilizing TGM plays an important role in different technologies and industries. TGM can be used in many applications. In the food industry, it plays a role as a protein-modifying enzyme, while, in biotechnology and pharmaceutical applications, it is used in mediated bioconjugation due to its extraordinary crosslinking ability. TGMs (EC 2.3.2.13) are enzymes that catalyze the formation of a covalent bond between a free amino group of protein-bound or peptide-bound lysine, which acts as an acyl acceptor, and the γ-carboxamide group of protein-bound or peptide-bound glutamine, which acts as an acyl donor. This results in the modification of proteins through either intramolecular or intermolecular crosslinking, which improves the use of the respective proteins significantly.
... The protein profiles of bovine milk caseins, as a substrate for films production, and casein films are presented on Figure 4A. The identification of those proteins and peptides resulting from their degradation was performed using previously published results of other authors as a reference [46][47][48]. The comparison of protein profiles of casein films was performed. ...
... Panel (A) presents the protein profile of bovine milk casein fraction (CNs) that serves as a reference for the casein films' protein profiles (1-4; 1 -4 ). On line 1, representing the Cas sample protein profile, the known bovine milk caseins and their dimers, trimers and oligo-, polymers as well as known caseins degradation products are marked according to [46][47][48]. Panel (B) represents the casein films protein profiles. Lines 1-4 represent, respectively, unaged Cas, 0.10% MEL, 0.25% MEL and 0.50% MEL sample protein profiles. ...
... Panel A presents the protein profile of bovine milk casein fraction (CNs) that serves as reference for casein films protein profiles (1-4; 1 -4 ). On line 1 representing the Cas sample protein profile the known bovine milk caseins and their dimers, trimers and oligo-, polymers as well es known caseins degradation products are marked according to [45][46][47]. Panel B represents the casein films protein profiles. Lines 1-4 represents respectively unaged: Cas, 0.10% MEL, 0.25% MEL and 0.50% MEL sample protein profiles. ...
Petroleum-based polymer food packaging is causing increasing concern. Their biopolymer alternatives should have some added value to compete with them and push them out of the market. This article presents new information related to the effects of melanin on casein films and their protection against artificial UV aging. Casein films were modified with melanin as an active additive and then subjected to artificial aging using UV radiation to evaluate its effect on the preservation of the films’ properties. The films were tested for hydrological (moisture content and water solubility), mechanical, barrier against UV-Vis radiation, colorimetric, and antioxidant properties, and the content of free amino acids and sulfhydryl and disulfide groups were checked before and after aging. Melanin influenced the preservation of mechanical properties of the films (elongation at break increased by no more than 20% for melanin-modified samples compared to more than 50% increase for the control sample), better UV barrier properties, increased antioxidant properties (two-fold higher scavenging of DPPH radicals by films modified with the highest melanin content compared to unmodified films before aging, and four times higher scavenging of DPPH radicals after aging). In addition, the presence of melanin had protective properties for sulfhydryl bonds and proteins (the increase in free amino acids after aging for melanin-modified films was not statistically significant), and it also had the effect of increasing the abundance of bands corresponding to oligomers and polymers in electrophoretic separation. The results indicate that melanin has UV-protecting properties on casein films, and it can be assumed that the obtained casein films modified with melanin could potentially find application as food packaging or edible coatings.
... Additionally to R h , the R g was determined, which is defined by the mass distribution within a particle and is thus, in case of a sphere, smaller than its geometric radius. The ratio of R g to R h characterises the shape of a particle, with 0.775 corresponding to ideal solid spheres and 1-2 to linear random coils [57]. Neither R g nor R g /R h were significantly affected by the concentration and milieu exchange, indicating that there was no irreversible change in the overall shape of the casein micelles. ...
The impact of membrane concentration with different milieu exchange conditions on the structural changes of casein micelles was investigated by studying their dissociation as well as their internal structure using small-angle X-ray scattering (SAXS). Micellar casein concentrates were prepared using ultrafiltration of milk (< 10 °C) followed by microfiltration and diafiltration (DF) of the resulting 2 × milk protein concentrate (< 10 °C) using different DF media (demineralised water vs. ultrafiltration permeate) and DF modes (continuous at 2 × concentration vs. discontinuous at 4 × concentration). All concentrates showed a higher degree of casein micelle dissociation when DF was conducted with water compared to ultrafiltration permeate. SAXS data were evaluated using a model describing the scattering on an absolute scale, and using protein and mineral concentrations as constraints. The results showed changes in the internal structure of the casein micelles at intermediate length scales for the concentrates prepared using water as DF medium, linked to the partial dissociation of caseins from the colloidal phase to the dispersion phase. In contrast, the casein micelles, in spite of their concentrated state, retained their structural integrity better when DF was conducted with ultrafiltration permeate. These results clearly demonstrate that maintaining the ionic equilibrium during membrane filtration of skim milk is critical to preserve the original structure of the protein particles in skim milk. This has important consequences in developing processes aiming at tailoring the technological properties of micellar casein concentrates.
... Asymmetrical Flow Field Flow Fractionation (AF4) is a powerful separation technique that allows the separation of a wide range of molecule sizes (1-1000 nm) with the advantage of the lack of a stationary phase. The lack of the stationary phase allows the use of nondenaturing buffers that enable the investigation of the molecules under their native state (Raak, Abbate, Lederer, Rohm, & Jaros, 2018). In the case of the casein micelles, this technique has been applied to study the casein micelle structure (Glantz, Håkansson, Lindmark Månsson, Paulsson, & Nilsson, 2010;Lie-Piang, Leeman, Castro, Börjesson, & Nilsson, 2021a, 2021bLoiseleux et al., 2018). ...
... The molecular weight of pure αs-CN was 25.620 KD and 26 KD, respectively, compared with the molecular weight of the standard protein in Ladder. The results agree with [29] and [30] who found that αs-CN was migrated to less than 26.8 KD in SDS-PAGE. ...
... Trypsin was used in the hydrolysis immediately after different proteolytic enzymes to increase the DH of casein from 44.5 to 72%. The high hydrolysis rates after purification of casein from sheep milk indicated that αs-CN and β-CN have low molecular weight peptides obtained after hydrolysis according to the structure of αs-CN, their biochemical components of amino acids and sequences, in addition to the type and concentration of enzymes used in the analysis, and the increase of αs-casein decomposers by pepsin and trypsin in sheep [30]. ...
Casein is a biologically and physiologically complex fluid that contains proteins, water, fat, lactose, minerals, and vitamins as its principal constituents that might support the treatment of human body. Isolation of casein using urea and salts was used. Then, casein was partially purified using DEAE-Cellulose and Sephadex-G-75. The hydrolysis degree was estimated after incubation with pepsin, trypsin, and a mixture of them. αs-CN was partially purified with one peak after DEAE-cellulose ion exchange and there is also one peak that appeared after the Sephadex G-25 gel filtration technique. The maximum hydrolysis was gained after 8 hours of incubation with hydrolyzing enzymes. αH hydrolysates of αs-casein showed that the inhibition rate reached about 67% after 8 hours of incubation with a mixture of pepsin and trypsin with a hydrolysis concentration of 0.114 μmol. Results showed that casein extracted from sheep milk can be highly purified using both ion exchange and gel filtration chromatography. Hydrolysis of αs-CN produces low molecular weight protein using pepsin and trypsin and a mixture of them. The αs-CN has a high inhibition rate of the angiotensin-converting enzyme (ACE).
... Thus, the molecular weight standards used in SEC with spectrophotometric detection do not directly indicate molecular weight of cross-linked products not behaving like Stokes-Einstein spheres. As reported SEC-MALS or gel electrophoresis can be used to get more accurate values (Raak et al., 2018). Reducing SDS-PAGE indicated cross-linked β-Lg ranging from dimeric (~38 kDa) to larger than dodecameric (>240 kDa, which is the size cutoff of the SDS-PAGE stated by the manufacturer). ...
There is a dogma within whey protein modification, which dictates the necessity of pretreatment to enzymatic cross-linking of β-lactoglobulin (β-Lg). Here microbial transglutaminase (MTG) cross-linked whey proteins and β-Lg effectively in 50 mM NaHCO3, pH 8.5, without pretreatment. Cross-linked β-Lg spanned 18 to >240 kDa, where 6 of 9 glutamines reacted with 8 of 15 lysines. The initial isopeptide bond formation caused loss of β-Lg native structure with t1/2 = 3 h, while the polymerization occurred with t1/2 = 10 h. Further, cross-linking effects on protein carbohydrate interaction have been overlooked, leaving a gap in understanding of these complex food matrices. Complexation with alginate showed that β-Lg cross-linking decreased onset of particle formation, hydrodynamic diameter, stoichiometry (β-Lg/alginate) and dissociation constant. The complexation was favored at higher temperatures (40°C), suggesting that hydrophobic interactions were important. Thus, β-Lg was cross-linked without pretreatment and the resulting polymers gave rise to altered complexation with alginate.
... Crosslinking the proteins with transglutaminase (TGase, EC 2.3.2.13) is one method to modify the functionality and structural properties of proteins such as caseins and WP. Transglutaminase catalyzes the acyl transfer reaction between the protein-bound glutaminyl residues and the primary amines (Folk and Finlayson, 1977;Chen and Hsieh, 2016;Raak et al., 2018). Transglutaminase has the potential to modify the physical properties of caseins by covalently crosslinking the caseins and thereby reducing the steric stability of κ-CN. ...
The amount of intact casein provided by dairy ingredients is a critical parameter in dairy-based imitation mozzarella cheese (IMC) formulation because it has a significant effect on unmelted textural parameters such as hardness. From a functionality perspective, rennet casein (RCN) is the preferred ingredient. Milk protein concentrate (MPC) and micellar casein concentrate (MCC) cannot provide the required functionality due to the higher steric stability of casein micelle. However, the use of transglutaminase (TGase) has the potential to modify the surface properties of MPC and MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC powders on the unmelted textural properties of IMC and compare them with IMC made using commercially available RCN. Additionally, we studied the degree of crosslinking by TGase in MPC and MCC retentates using capillary gel electrophoresis. Three lots of MCC and MPC retentate were produced from pasteurized skim milk via microfiltration and ultrafiltration, respectively, and randomly assigned to 1 of 3 treatments: no TGase (control); low TGase: 0.3 units/g of protein; and high TGase: 3.0 units/g of protein, followed by inactivation of enzyme (72°C for 10 min), and spray drying. Each MCC, MPC, and RCN was then used to formulate IMC that was standardized to 21% fat, 1% salt, 48% moisture, and 20% protein. The IMC were manufactured by blending, mixing, and heating ingredients (4.0 kg) in a twin-screw cooker. The capillary gel electrophoresis analysis showed extensive inter- and intramolecular crosslinking. The IMC formulation using the highest TGase level in MCC or MPC did not form an emulsion because of extensive crosslinking. In MPC with a high level of TGase, whey protein and casein crosslinking were observed. In contrast, crosslinking and hydrolysis of proteins were observed in MCC. The IMC made from MCC powder had significantly higher texture profile analysis hardness compared with the corresponding MPC powder. Further, many-to-one (multiple) comparisons using the Dunnett test showed no significant differences between IMC made using RCN and treatment powders in hardness. Our results demonstrated that TGase treatment causes crosslinking hydrolysis of MCC and MPC at higher TGase levels, and MPC and MCC have the potential to be used as ingredients in IMC applications.
... Actually, SDS-PAGE is widely used as it does not require expensive equipment. However, large protein aggregates fail to migrate into the gel and the analysis only provides a semi-quantitative estimation [28,29]. Recent applications reported by literature support the reliability of CZE under reducing conditions in the separation of both native and denatured whey proteins [25,30,31], although the incomplete solubilization of large aggregates may also represent a limitation with this technique, as discussed below. ...
Increasing awareness of balanced diet benefits is boosting the demand for high-protein food and beverages. Sports supplements are often preferred over traditional protein sources to meet the appropriate dietary intake since they are widely available on the market as stable ready-to-eat products. However, the protein components may vary depending on both sources and processing conditions. The protein fraction of five commercial sports supplements was characterized and compared with that of typical industrial ingredients, i.e., whey protein concentrates and isolates and whey powder. The capillary electrophoresis profiles and the amino acid patterns indicated that, in some cases, the protein was extensively glycosylated and the supplemented amino acids did not correspond to those declared on the label by manufacturers. The evaluation by confocal laser scanning microscopy evidenced the presence of large aggregates mainly enforced by covalent crosslinks. The obtained findings suggest that, beside composition figures, provisions regarding sports supplements should also consider quality aspects, and mandatory batch testing of these products would provide more reliable information to sport dieticians.
... However, these variations in purification fold and specific enzyme activity purified by gel filtration chromatography may depend on the microbial strains and substrate [46]. Gel filtration chromatography referred to as size exclusion chromatography is a chromatography technique that allows for the separation of macromolecules based on their hydrodynamic size, whereby the smaller molecules can access the higher number of pores and stay inside the column longer, while the larger molecules elute earlier because they can only penetrate larger pores [53]. ...
Pectinase enzymes are important industrial enzymes having considerable applications in several industries, especially in food processing. Pectinases contribute 25% of global food enzyme sales. Therefore, the demand for a commercial enzyme with desirable characteristics and low production costs has become one of the great targets. Hence, this study aims to produce exo-polygalacturonase (exo-PG) using local fungal isolate Penicillium oxalicum AUMC 4153 by utilizing sugar beet manufacturing waste (sugar beet pulp) as a sole raw carbon source under shaken submerged fermentation, which is purified and characterized to optimize enzyme biochemical properties for industrial application. The purity of the obtained exo-PG was increased by about 28-fold, and the final enzyme yield was 57%. The partially purified enzyme was active at a broad range of temperatures (30–60 °C). The optimum temperature and pH for the purified exo-PG activity were 50 °C and pH 5. The enzyme was stable at a range of pH 3 to 6 and temperature 30–50 °C for 210 min. The values for Km and Vmax were 0.67 mg/mL, with polygalacturonic acid as substrate and 6.13 µmole galacturonic acid/min/mg protein, respectively. It can be concluded that purified exo-PG production by P. oxalicum grown on sugar beet waste is a promising effective method for useful applications.
