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SDS-PAGE analysis of a-casein, b-casein, k-casein, and b-lactoglobulin after heating at 90 1C and pH 2.0 for 0–48 h.
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Amyloid-like fibrils are studied because of their significance in understanding pathogenesis and creating functional materials. Amyloid-like fibrils have been studied by heating globular proteins at acidic conditions. In the present study, intrinsically disordered α-, β-, and κ-caseins were studied to form amyloid-like fibrils at pH 2.0 and 90 ºC....
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... by several para- meters such as pH and temperature that determine the kinetics of acid hydrolysis, as well as nucleation and growth which are a function of both hydrolysate structures and environment conditions. 15,37 To understand the rate-determining step dur- ing fibril formation, SDS-PAGE was used to monitor acid hydrolysis during heating (Fig. 2). Hydrolysis of proteins to peptides smaller than 10 kDa mostly completed in the first 12 h, with b-casein being the slowest. The hydrolysis kinetics quantified from SDS-PAGE after fitting the model in eqn (5) is shown in Fig. 3, and the estimated rate constants are presented in Table 2 Therefore, a more hydrophobic protein is ...
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... The TEM micrographs show that both UG and 2G κ-CN B are present as curly and rugged surface linear aggregates, which aligns with Chun et al. (2012) who observed thicker fibrils with a rugged surface using unreduced forms of κ-CN, while Ecroyd et al. (2008), Ecroyd et al. (2010), Leonil et al. (2008), and Pan and Zhong (2015) observed substantially longer fibrils with a smooth surface using reduced forms of κ-CN. Although disulphide bond reduction does not significantly affect the secondary and tertiary structure of κ-CN (Farrell et al., 2003), it is believed that the ability of κ-CN to form oligomers, and the subsequent propensity for amyloid fibril formation, is influenced by changes in the number of disulphide bonds links of cysteine 11 and cysteine 88 (Hewa Nadugala et al., 2022;Rasmussen et al., 1992). ...
In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0–28.7 nm range for a 1 mg mL⁻¹ solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22–0.44 mg mL⁻¹. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.
... Finally, heating whole casein at temperatures below [41] and above [105] those applied in the present study does not lead to well-ordered casein fibrils. While the formation of co-aggregates, or even homogeneous fibrils, during heat treatment and/ or acidification of milk is possible [86,[106][107][108], it is apparent from our experiments that the amyloidogenic caseins impede each other's fibril assembly, that is, heterotypic interactions overcome homotypic interactions (leading to dysfunctional amyloid fibrils) to produce functional oligomeric assemblies (i.e., the casein micelle). Avoidance of amyloid fibril formation in the mammary gland appears therefore to have been a strong driver for the diversification of caseins as it has other protein families [109]. ...
Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, αS2- and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (αS1 and β) as well as β-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow αS1- and β-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, αS1- and β-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel β-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel β-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of β-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly; in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins diversified partly to avoid dysfunctional amyloid fibril formation.
... Expired nonfat yogurt, which is normally thrown away, is a potential source of a raw material for producing carbon nanonetworks since the yogurt contains casein micelle and whey protein. Moreover, carbon from b-casein, j-casein, and b-lactoglobulin (whey protein) could turn into nanowires under acidic thermal treatment [13]. It will be interesting if these nanowire structures are preserved even after carbonization of hydrothermally treated yogurt. ...
The dye-sensitized solar cells (DSSCs) were assembled using counter electrodes (CEs) constructed of carbonized yogurt/PEDOT:PSS composites and evaluated for their efficiency. The influences of two different crucible materials used during the pyrolysis of hydrothermally treated nonfat yogurt on the structures and properties of derived carbons were compared. Carbons denoted by Aa and Ab were pyrolyzed on an aluminum alloy boat and an alumina one, respectively. Hydrophobic carbon/nano-Whitlockite (WH) composites were obtained for both crucible materials. The carbon features are primarily made up of nanocarbons, micronized carbon spheres, and carbyne nanowires. Aa and Ab have surface areas (SBET) of 1060 and 289.7 m² g⁻¹, respectively. Moreover, the Aa carbon has a lower degree of oxidation than the Ab sample. The electrocatalytic activity of Aa carbon is higher than that of Ab carbon but Aa is less hydrophobic than Ab. Whitelockites were also prepared from hydrothermal synthesis and oxidation of Aa and Ab samples. Notably, the electrocatalytic activities of Whitlockites/PEDOT:PSS are as good as that of Pt but it is still lower than Aa/PEDOT:PSS and Ab/PEDOT:PSS electrodes. The DSSC with an Aa/PEDOT:PSS CE has a slightly higher efficiency (7.40%) than the DSSC with an Ab/PEDOT:PSS CE (7.05%) and the one with a Pt CE (7.01%) because Aa DSSC has a slightly higher fill factor (FF) and short-circuit current density (Jsc). Note that the Whitelockites/PEDOT: PSS can not be used as counter electrodes since these films swell excessively, resulting in a short circuit. Carbon in Aa and Ab plays a very important role in preventing such swelling incidence.
... The development of microscopy, and spectroscopy techniques, has improved knowledge about the properties of casein micelles properties by means of image modeling and molecular dynamics (Michael Byler et al., 1988;Horne, 2006;Moitzi et al., 2011;Pan & Zhong, 2015;Yang et al., 2015). For decades, different spectroscopy techniques (X-ray diffraction, nuclear magnetic resonance, Fourier transform infrared (FTIR), and Raman spectroscopy) have been widely useful to elucidate the molecular structure of proteins and evaluate conformational changes produced by their processing and variations of pH (Horne, 2002;Wang et al., 2017). ...
This study aimed to evaluate the influence of pH changes on morphometric parameters of casein micelles and a general overview of their conformational structure through microscopy techniques, Raman spectroscopy and multivariate analysis. It was found that casein micelles morphology and protein secondary structure depend strongly upon pH. The changes of arithmetic average roughness (Ra), size, and shape of casein micelles at different pH are properly characterized by atomic force and cryo-transmission electron microscopy. Morphometric changes of casein micelles were correlated correctly with folding and unfolding of casein molecules as evaluated by Raman spectroscopy when the pH was varied. The novelty of this contribution consists in demonstrating that there is a close structure-functionality relationship between the morphometric parameters of proteins and their secondary structure. Knowledge about casein micelles can help improve their use of its diverse applications.
... Amorphous aggregates form clumps without any defined secondary structures, while the ordered aggregates usually form fibrillar morphology containing extensive beta-sheet secondary structures (Chiti & Dobson, 2006;Colvin et al., 2016). Under in vitro conditions, several milk proteins are reported to form amyloid fibrils and display typical amyloid characteristics (Lambrecht et al., 2019;Pan & Zhong, 2015;Thorn et al., 2021;Thorn, Ecroyd, & Carver, 2009). Generally, amyloids are associated with many debilitating diseases like Alzheimer's and Parkinson's diseases, cataracts, and diabetes type II (Pan & Zhong, 2015). ...
... Under in vitro conditions, several milk proteins are reported to form amyloid fibrils and display typical amyloid characteristics (Lambrecht et al., 2019;Pan & Zhong, 2015;Thorn et al., 2021;Thorn, Ecroyd, & Carver, 2009). Generally, amyloids are associated with many debilitating diseases like Alzheimer's and Parkinson's diseases, cataracts, and diabetes type II (Pan & Zhong, 2015). Therefore, the presence of amyloid-like structures in foods has always been a serious concern due to their possible cross seeding and transmissibility. ...
Cottage cheese, extensively consumed worldwide, contains coagulated milk protein (casein), produced through boiling and acidification of milk. Casein forms amyloid or amyloid-like structures at high temperatures and low pH. Due to the similarities in the preparation of casein amyloids and cottage cheese, we hypothesized the presence of amyloid or amyloid-like protein aggregates in cottage cheese. To examine this hypothesis, cottage cheese was prepared from cow (Bos indicus) milk and isolated amyloids through a water extraction method. The isolated protein aggregates displayed typical characteristics of amyloids, such as a bathochromic shift in the wavelength of maximum absorption (λmax) of Congo red (CR), high thioflavin T (ThT) binding, increased surface hydrophobicity, and high β-sheet structure. However, they did not show antibacterial activity and toxic properties against erythrocytes. Our study revealed that the heat-treatment and subsequent acidification during cottage cheese preparation lead to the formation of non-toxic amyloid-like aggregates.
... Fibril formation is uncommon in liquid milk, although some evidence suggests the possible existence of very short (7-10 nm) fibril-like structures in bovine casein micelles (Lencki, 2007). Amyloid fibril formation has been documented for κ-CN (Farrell et al., 2003;Thorn et al., 2005;Leonil et al., 2008), for α S2 -CN (Thorn et al., 2008), and to some extent for isolated β-CN (Pan and Zhong, 2015). The latter, however, formed fibrils only under nonphysiological conditions at pH 2 and 90°C over 30 h. ...
The effects of varying the concentration of pentasodium triphosphate (PP) emulsifying salt [0, 0.6, 1.2, 1.5, and 1.8%, plus 0.9% of a mixture of citric acid (CA) and disodium phosphate (DSP) to adjust cheese pH to 5.85] on rheological, textural, physicochemical, and microstructural properties were studied in a processed cheese model system containing ~20% micellar casein concentrate, ~20% sunflower oil, and ~59% water. Special emphasis was placed on the unique casein fibrils recently described in a comparable processed cheese model system. Our results show that during processing (90°C, 17.37 rpm over 270 min) the apparent viscosity increased more and faster for formulations containing higher concentrations of PP, in analogy to the so-called creaming reaction, a general thickening of the molten cheese mass with prolonged processing. We found that 1.2% PP (plus 0.9% CA-DSP) appeared to be the threshold for the creaming reaction to take place. With increasing PP concentrations, cheese hardness increased in a sigmoidal fashion, and insoluble (protein-bound) calcium concentration decreased exponentially. Light micrographs of samples taken at the end of processing indicated initially large and dense casein aggregates within the matrix that disappeared with higher levels of PP, in parallel with the development of a finer emulsion. With transmission electron microscopy analysis on the same samples, the highly complex restructuring of the casein matrix was evident; casein fibrils had formed de novo at the periphery of the loosening casein aggregates. With higher levels of PP, amorphous areas were observed in place of the dense casein aggregates that appeared progressively void of protein, whereas fibril concentration increased throughout the rest of the matrix. Fibrils progressively attached to the surface of fat globules, thereby emulsifying them. Reverse-phase HPLC analysis of insoluble and soluble fractions indicated κ-casein to be the most likely constituent of the newly formed fibrils. The results of this study suggest that PP induced a concentration-dependent dissociation of caseins (through increased calcium chelation) and further led to their spatial separation. In essence, their chaperone activity was hindered, which resulted in amorphous aggregation on the one hand and fibril formation on the other.
... This localization occurs as consequence of specific interactions between the hydrophobic domains of α s1 -, α s2 -and β-caseins and their phosphoserine residues that bind colloidal calcium phosphate (Huppertz, Fox, & Kelly, 2018). The disordered and amphiphilic nature of caseins allows different degrees of oligomerization, and is permissive of the formation of micelles or fibrils by single caseins even in the absence of other members of the family (Pan & Zhong, 2015;Wijaya et al., 2020). These characteristics make caseins desirable hydrocolloids, and useful in the design of bio-films and for biomedical purposes (Khodaverdi et al., 2019;Picchio et al., 2018). ...
... This protein is therefore susceptible to destabilization by both oxidation to give these species, and reducing agents that remove them (C. Holt et al., 2019;Pan & Zhong, 2015). To gain a better understanding of the oligomers generated by protein oxidation, the shape and size of these structures was examined using TEM. ...
The co-existence of proteins, lipids and riboflavin (RF) in milk together with the harsh conditions encountered during processing (e.g. high temperatures, light exposure) results in oxidative damage. Proteins represent ∼30% of the dry mass of milk, with caseins accounting for ∼80% (28 g L⁻¹). Due to their high abundance and amphiphilic nature, caseins are targets for both hydrophilic and lipophilic oxidants. Although caseins are key milk components, and highly abundant, most previous work has employed non-biological dilute solutions. In this work we have investigated oxidative modification of αs-, β- and κ-caseins elicited by AAPH-derived oxygenated radicals, or RF-mediated photo-oxidation, at both low and high protein concentrations, to determine whether and how oxidative damage and resulting structural modifications are modulated by the protein concentration. The data obtained demonstrate that the pathways leading to casein modification are dependent on both the protein concentration and the oxidant employed. AAPH-mediated oxidation was more efficient than RF-induced photo-oxidation, in respect to the number of moles of amino acid side-chains consumed per mole of oxidant generated, the extent of damage detected by SDS-PAGE, and immunoblot detection of oxidation products. Quantification of amino acid consumption and product generation, using UPLC and LC/MS, demonstrates the occurrence of short chain reactions, with the chain-length dependent on the protein concentration. LC/MS peptide mass mapping analyses provide data on the sites of modification. Molecular crowding, arising from high casein concentrations and casein-casein interactions, therefore favors the occurrence of radical chain events that enhance the extent of protein oxidative damage.
... Fibril formation has been demonstrated for native and reduced forms of κ-CN (Farrell et al., 2003;Thorn et al., 2005Thorn et al., , 2008Lee et al., 2019), α S2 -CN (Thorn et al., 2008), and, to some degree, β-CN, although not under physiological conditions (Pan and Zhong, 2015). Fibril formation could not be induced for α S1 -CN (Thorn et al., 2008). ...
The “creaming reaction,” a general thickening of the molten cheese mass during the manufacture of processed cheese, which is often seen to occur in a stepwise fashion, affects the viscosity and texture of the finished product. Thus, this phenomenon is of critical importance for the processed cheese industry, yet mechanisms underlying the structure formation in this surprisingly complex and dynamic food system are only poorly understood. Using a model system consisting of micellar casein concentrate, vegetable oil, water, and a mixture of melting salts, we followed the characteristic viscosity profile with its primary and secondary increase over time. A rheometer equipped with a custom-made cup geometry was used, which served as a mini-reaction vessel to simulate the conditions during the manufacture of processed cheese. The mixture was subjected to constant heat (90°C) and stirring (7.93 rpm), comparable to processed cheese cooking, for up to 410 min. At specific time points, samples were taken, and the micro- and ultrastructure was investigated with light and transmission electron microscopy. Results from our extensive study uncovered the following key steps: (1) a decrease in fat globule size with concomitant increase in the number of fat globules, which were also more evenly distributed; (2) a progressive separation of the casein matrix into fibrillogenic and nonfibrillogenic fractions; (3) formation of fibrils and their higher-order structuring followed by their partial degradation; and (4) increasing interactions of the fibrils with the fat globule surface leading to a higher degree of emulsification. Of these different observations, results indicate that after the caseins dissociated under the influence of the melting salts, protein–protein interactions were the primary driver of the structure formation and thus contributed to the initial viscosity increase. Fat globules were involved in the structure formation at later time points. Therefore, fat–protein interactions in addition to continued protein–protein interactions were assumed to contribute to the secondary viscosity increase. An updated processed cheese creaming model is presented. The use of the term “texturization” instead of “creaming” is proposed.
... Later, the same fibrils were mixed with silica precursor tetraethyl orthosilicate, and fibril-silica core-shell nanostructures with stiffness up to beyond ∼20 GPa (similar Young's moduli as many metal alloys and inorganic materials) were made [101]. In fact, many proteins can be reconstructed into this particular structure under certain conditions, including many sustainably sourced proteins, such as casein, haemoglobin (from meat industry waste), crystallins (from fish industry waste), and soy proteins [94,[102][103][104][105]. However, compared with whey protein fibrils, less research has focused on these alternative nanofibril sources as biomaterials. ...
Research into the development of sustainable biomaterials is increasing in both interest and global importance due to the increasing demand for materials with decreased environmental impact. This research field utilises natural, renewable resources to develop innovative biomaterials. The development of sustainable biomaterials encompasses the entire material life cycle, from desirable traits, and environmental impact from production through to recycling or disposal. The main objective of this review is to provide a comprehensive definition of sustainable biomaterials and to give an overview of the use of natural proteins in biomaterial development. Proteins such as collagen, gelatin, keratin, and silk, are biocompatible, biodegradable, and may form materials with varying properties. Proteins, therefore, provide an intriguing source of biomaterials for numerous applications, including additive manufacturing, nanotechnology, and tissue engineering. We give an insight into current research and future directions in each of these areas, to expand knowledge on the capabilities of sustainably sourced proteins as advanced biomaterials.
... The published literature on the influence of salts or pH conditions on structure formation in concentrated protein systems during TMP is scarce, whereas numerous studies on structure formation in diluted protein dispersions (<10% protein concentration) have been reported (Loveday et al., 2010;Loveday et al., 2011;Pan & Zhong, 2015). ...
Increasing awareness of inefficient meat production and its future impact on global food security has led the food industry to look for a sustainable approach. Meat products have superior sensorial perception, because of their molecular composition and fibrous structure. Current understanding in the science of food structuring has enabled the utilization of alternative or nonmeat protein ingredients to create novel structured matrices that could resemble the textural functionality of real meat. The physicochemical and structural changes that occur in concentrated protein systems during thermomechanical processing lead to the creation of a fibrous or layered meat‐like texture. Phase transitions in concentrated protein systems during protein‒protein, protein‒polysaccharide, protein‒lipid, and protein‒water interactions significantly influence the texture and the overall sensory quality of meat analogs. This review summarizes the roles of raw materials (moisture, protein type and concentration, lipids, polysaccharides, and air) and processing parameters (temperature, pH, and shear) in modulating the behavior of the protein phase during the restructuring process (structure‒function‒process relationship). The big challenge for the food industry is to manufacture concept‐based (such as beef‐like, chicken‐like, etc.) meat analogs with controlled structural attributes. This information will be useful in developing superior meat analogs that fulfill consumer expectations when replacing meat in their diet.
