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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Yak milk formed stronger rennet-induced gels if the milk contained smalled casein micelles and a higher concentration of calcium. Also casein gels could formed after a shorter incubation time if the milk contained smalled casein micelles. The objective of this study was to estimate the importance of yak casein micelle size on rennet-induced coagulation properties., Results: Three fractions of different-sized, undamaged casein micelles (Ф112.17 ± 0.83 nm, Ф207.13 ± 0.59 nm and Ф269.37 ± 2.89 nm) were obtained by ultracentrifugation. The smallest casein micelles had the highest concentrations of calcium (803.21 ± 8.49 mM), phosphate (445.52 ± 10.66 mM), and κ-casein/total casein (19.45%). Rheological analyses determined the optimal gelation times of small, medium, and large casein micelles to be 9.5 ± 0.5, 10.8 ± 0.5, and 13.3 ± 0.2 min, respectively. Higher κ-casein concentration in the small casein micelles appeared to facilitate their shorter incubation time. Both the faster caseinomacropeptide (CMP) release rate and rennet-induced aggregation rate of small casein micelles contributed to a faster change in turbidity. Furthermore, small casein micelles had the highest elastic modulus (G', 73.21 ± 4.5 Pa) 60 min after the addition of rennet. This was consistent with micro-photographs, which showed that small casein micelles could form a more homogeneous gel, which had smaller pore sizes. Trial cheese manufacture verified that yak cheese containing small casein micelles, formed curd faster and the cheese had higher texture profile analysis (TPA) values for hardness, cohesiveness, and springiness., Conclusion: This is important information for the optimization of yak cheese industrial production. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The thermal stability of donkey milk, which largely depends on the stability of casein and whey proteins, plays a critical role in the manufacture of donkey dairy products. However, the thermal stability of donkey casein micelles is poorly understood. Our study first found that donkey milk exhibited poor thermal stability, with sedimentation observed after heating at 75 °C for 10 min. Moreover, electrophoresis results indicated that donkey casein micelles were more sensitive to heat treatment than whey protein. The characteristics of donkey casein micelles were then studied to determine the thermal instability mechanism. Donkey casein micelles had a larger diameter (295 ± 11 nm) and lower absolute zeta potential (-15.4 ± 0.5 mV) than bovine casein micelles, mainly due to their very low κ-casein levels. The higher proportion (51%) of β-casein in donkey casein micelles made them less hydrated. Furthermore, <40% of calcium in donkey milk was associated with donkey casein micelles. To eliminate the influence of whey protein, micellar casein was isolated from skim donkey milk by ultracentrifugation and then redispersed in its ultrafiltrate. Transmission electron microscopy and particle size analysis showed the extensive aggregation of isolated casein micelles after thermal treatment, which confirmed the thermal instability of casein micelles observed in donkey milk. Overall, poor colloidal stability and a high‑calcium environment largely contributed to the thermal instability of donkey casein micelles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, the effect of trisodium citrate on the textural properties and microstructure of acid-induced, transglutaminase-treated micellar casein gels was investigated. Various concentrations of trisodium citrate (0 mmol/L, 10 mmol/L, 20 mmol/L, and 30 mmol/L) were added to micellar casein dispersions. After being treated with microbial transglutaminase (mTGase), all dispersions were acidified with 1.3% ( w / v ) gluconodelta-lactone (GDL) to pH 4.4⁻4.6. As the concentration of trisodium citrate increased from 0 mmol/L to 30 mmol/L, the firmness and water-holding capacity increased significantly. The final storage modulus (G′) of casein gels was positively related to the concentration of trisodium citrate prior to mTGase treatment of micellar casein dispersions. Cryo-scanning electron microscopy images indicated that more interconnected networks and smaller pores were present in the gels with higher concentrations of trisodium citrate. Overall, when micellar casein dispersions are treated with trisodium citrate prior to mTGase crosslinking, the resulted acid-induced gels are firmer and the syneresis is reduced.

It is essential for yak cheese processing to understand the rennet-induced coagulation properties of gel formation from casein micelles. We have previously discovered that yak milk requires a longer incubation time but forms stronger gels compared with cow milk. In this study, we are aiming to understand the rennet-induced coagulation properties of yak casein micelles comparing with cow casein micelles. Rheological analyses revealed that the gelling times of yak and cow casein micelles were 11.6±0.5 and 8.7±0.4min (P<0.05) respectively, but yak casein gel had a higher elastic modulus G' (6.5±0.2Pa) than cow casein gel (2.5±0.2Pa; P<0.05). This is consistent with the results obtained by micro-rheology. Confocal laser scanning microscopic images (CLSM) and cryo-scanning electron microscopic images (cryo-SEM) showed that yak casein gel was more homogeneous and had smaller pore size than cow casein gels. Yak casein micelles had higher calcium (26.00mM), phosphate (19.90mM) and β-casein (relative 32%) concentrations. In addition, yak casein micelles were larger (Z-average 218.6nm) than cow casein micelles, and contained lower κ-casein (relative 13%). By comparison with cow casein micelles, yak casein micelle composition corresponding to their micellar calcium phosphate and κ-casein content may greatly contribute to the longer coagulation time and denser gel structure. An initial slower caseinomacropeptide (CMP) release rate and the slower rate of aggregation between para-casein micelles contributed to a more homogeneous yak gel network. Higher colloidal calcium phosphate is crucial for yak casein micelle aggregation and gel firmness because sufficient colloidal calcium phosphates can firmly glue sub-micelles and links casein micelles. This study provides valuable information for yak cheese production., (Copyright © 2017. Published by Elsevier Ltd.)

Rennet-induced gelation crucially impacts cheese structure. In this study, effects of the size and stability of native fat globules on the kinetics of rennet-induced coagulation were revealed by determining the caseinomacropeptide release rate and rheological properties of milk. Moreover, the mobility and stability of fat globules during renneting was revealed using diffusing wave spectroscopy and confocal laser scanning microscopy. By use of a 2-stage gravity separation combined centrifugation scheme, native fat globules were selectively separated into small (SFG, D 4,3 = 1.87 ± 0.02 μm) and large fat globules (LFG, D 4,3 = 5.65 ± 0.03 μm). The protein and fat content of SFG and LFG milk were then standardized to 3.2 g/100 mL and 1.2 g/100 mL, respectively. The milk containing different sized globules were then subjected to renneting experiments in the laboratory. Reduction of globule size accelerated the aggregation of casein micelles during renneting, giving a shorter gelation time and earlier 1/l * change. The gel produced from LFG milk was broken due to coalescent fat globules and generated coarser gel strands compared to the finer strands formed with SFG milk. Structural differences were also confirmed with a higher final storage modulus of the curd made from SFG milk than that from the LFG. In conclusion, the size of fat globules affects the aggregation of casein micelles. Moreover, fat globule coalescence and creaming during renneting, also affects the structure of the rennet gel. A better understanding of the size of globules effect on milk gelation could lead to the development of cheese with specific properties., (© 2017 Institute of Food Technologists®.)

The effects of lactose on the changes in the composition and size of casein micelles induced by high-pressure treatment and the related mechanism of action were investigated. Dispersions of ultracentrifuged casein micelle pellets with 0-10% (w/v) lactose were subjected to high pressure (400 MPa) at 20 °C for 40 min. The results indicated that the level of non-sedimentable caseins was positively related to the amount of lactose added prior to pressure treatment, and negatively correlated to the size. A mechanism for the pressure-induced, lactose-dependent changes in the casein micelles is proposed. Lactose inhibits the hydrophobic interactions between the micellar fragments during or after pressure release, through the hydrophilic layer formed by their hydrogen bonds around the micellar fragments. In addition, lactose does not favour the association between calcium and the casein aggregates after pressure release. Due to these two functions, lactose inhibited the formation of larger micelles after pressure treatment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

Agar is widely used in yogurt manufacture to improve the stability of milk gels. However, the formation and structural properties of acid-induced casein–agar gel have not been elucidated. In this study, different acidification temperatures were used to evaluate the evolution of acid-induced casein–agar double networks and their microstructure and penetration properties. Agar (0.25%, w/w) was mixed with a micellar casein dispersion (3%, w/w), acidified with glucono-δ-lactone at 30, 37, 42, or 45 °C, respectively, and then cooled to 10 °C. Compared with casein gel alone, the casein–agar gel had a higher firmness and water holding capacity (WHC). Moreover, these acid-induced casein–agar double networks formed different structures depending on the acidification temperature. When acidified below the agar transition temperature (at 30 or 37 °C), the agar formed a weak gel prior to casein network formation that hindered the movement of casein micelles. Therefore, the casein–agar mixtures formed double networks during the acidification stage with relatively low elasticity indexes (EIs). In contrast, casein–agar mixtures acidified above the agar transition temperature (at 42 or 45 °C) formed double networks during the cooling stage that exhibited higher EIs. Cryo-scanning electron microscopy showed denser networks in casein–agar gels acidified above the agar transition temperature. Furthermore, casein–agar gels acidified above the agar transition temperature showed a significantly higher firmness and WHC than those acidified below. Overall, acidification temperature significantly affected the gelation sequence and, thereby, the formation and structural properties of the acid-induced casein–agar double network. [ABSTRACT FROM AUTHOR]

Yak caseins are special materials which has attracted more and more attention of researchers due to its increasing. Succinylation is a useful method of improving the functional properties of protein significantly. This study assessed the effects of succinylation on the functional properties of yak and cow caseins, including solubility, emulsifying property, water and oil absorption capacity, foaming property, and digestibility. Six succinylated levels of caseins were prepared by adding different amounts of succinic anhydride to caseins. The succinylated degree of yak and cow caseins was different when similar amounts of succinic anhydride were added. Succinylation improved solubility, emulsifying activity, water and oil absorption capacity, and reduced foam stability and digestibility of yak and cow native caseins. However, succinylation decreased foaming capacity and emulsion stability of cow caseins while increased them of yak caseins. The functional properties of caseins changed irregularly with increasing levels of succinylation. The findings of this study revealed that succinylation had different effects on the functional properties of different sources of caseins. The results will provide the basic data of functional properties of yak native caseins and succinylated yak caseins. [ABSTRACT FROM AUTHOR]

Summary [ABSTRACT FROM AUTHOR]

To select a kind of practical protease that can be used for the production of yak milk casein enzymatic hydrolysates with a high-immunoregulating activity, the effects of casein trypsin, pepsin, alcalase hydrolysates and their separated fractions on the lymphoproliferation activity and Interleukin-2 production of murine splenocytes in vitro were studied. Results showed that, compared with casein trypsin and pepsin hydrolysates, casein alcalase hydrolysate had higher immunoregulatory activity, degree of hydrolysis and peptide yield. The molecular weights of the separated fractions of casein hydrolysates were lower than 1,500 Da. Considering the low price and commercial availability in a large supply of alcalase, yak milk casein alcalase hydrolysate is more practical as a functional food ingredient in industry. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Ultra-instantaneous UHT (UI-UHT, >155°C, <0.1 s) treated milk exhibits higher retention of active protein than regular UHT milk. However, UI-UHT products demonstrate increased susceptibility to destabilization during storage. This study aimed at monitoring the destabilizing process of UI-UHT milk across different storage temperatures and uncovering its potential mechanisms. Compared with regular UHT treatment, ultra-instantaneous treatment markedly accelerated the milk's destabilization process. Aged gel formation occurred after 45 d of storage at 25°C, whereas creaming and sedimentation were observed after 15 d at 37°C. To elucidate the instability mechanism, measurements of plasmin activity, protein hydrolysis levels, and proteomics of the aged gel were conducted. In UI-UHT milk, plasmin activity, and protein hydrolysis levels significantly increased during storage. Excessive protein hydrolysis at 37°C resulted in sedimentation, whereas moderate hydrolysis and an increase in protein particle size at 25°C resulted in aged gel formation. Proteomics analysis results indicated that the aged gel from UI-UHT milk contained intact caseins, major whey proteins, and their derived peptides. Furthermore, specific whey proteins including albumin, lactotransferrin, enterotoxin-binding glycoprotein PP20K, and MFGM proteins were identified in the gel. Additionally, MFGM proteins in UI-UHT milk experienced considerable hydrolysis during storage, contributing to fat instability. This study lays a theoretical foundation for optimizing UI-UHT milk storage conditions to enhance the quality of liquid milk products. [Display omitted] [ABSTRACT FROM AUTHOR]

Age gelation is undesirable for direct UHT (dUHT) milk, which is closely related to protein hydrolysis. However, little information is available for the role of serum peptides during the age gelation. In this study, the composition and protein morphology of serum phase were characterized by RP-HPLC, ICP-MS and TEM. The results showed significant increases in soluble proteins, free amino acids, calcium, and phosphorus from casein micelles, indicating protein hydrolysis and peptide release into the serum phase. 23,466 peptides derived from caseins and other proteins were identified in serum phase by peptidomics. The serum peptide profiles of age gelation milk changed dramatically. Peptide fingerprinting revealed that plasmin and cathepsin contributed to the protein hydrolysis during age gelation, with a significant increase in their activity observed. 23 characteristic peptides were ultimately selected as potential indicators for age gelation. These findings provide new insights into the age gelation of UHT milk. • Casein micelles released numerous free caseins and peptides during age gelation. • The significantly increased peptides were mainly derived from β-, αs1- and αs2-casein. • Hydrolysis by plasmin and cathepsin leads to age gelation of dUHT milk. [ABSTRACT FROM AUTHOR]

Direct-steam-infusion ultra-high temperature (dUHT) technology has been paid much attention to in recent years because of its short heating time, which helps retain more nutritional ingredients and extend shelf life of dairy products. However, protein instability is an urgent problem for dUHT milk to solve. As the main component of protein, it is unclear whether and what structural changes casein micelles (CMs) undergo after dUHT treatment. In this study, CM dispersions were treated with dUHT technology (140 °C/3 s, 145 °C/3 s, 150 °C/3 s, 155 °C/3 s, and 157 °C/0.116 s), and unheated sample was taken as the control. As the temperature increased, hydrodynamic diameter of the CMs reduced from 190 nm to 156 nm, zeta potential did not change significantly, and turbidity decreased significantly. After dUHT, contents of casein, calcium, and phosphorus in the serum phase increased. Furthermore, dUHT treatment would lead to disruption of intermolecular hydrogen bonds, rearrangement of secondary structures, and unfolding of CM. The 31P NMR spectra demonstrated that colloidal calcium phosphate solubilized and electrostatic repulsion within micelles increased with dUHT treatments. Further analysis of molecular structure of CMs using small-angle X-ray scattering and a cryo-transmission electron microscope found that after dUHT treatment, CMs dissociated in the form of casein clusters (20–40 nm) and became loose and porous in structure. In conclusion, dUHT treatment destroyed the calcium bridges and increased electrostatic repulsion inside micelles, leading to dissociation of casein clusters and a smaller, looser, and more porous structure of the CMs. [Display omitted] • Hydrodynamic diameter and turbidity of CM dispersions reduced after dUHT treatment. • The content of casein, Ca, and P in the serum phase increased after dUHT treatment. • CCP solubilized and electrostatic repulsion inside CM increased after dUHT process. • The CM dissociated in the form of casein clusters after dUHT treatment. • The structure of CM became looser and more porous after dUHT treatment. [ABSTRACT FROM AUTHOR]

The functionality changes of processed cheese analogue with stretchability during refrigerated storage (0–60 d) at 4 °C were explored and the possible mechanisms were investigated. The results showed that stretchability, meltability, and free oil release of the cheese analogue decreased during refrigerated storage, and stretch length decreased from >270 cm to 130.63 ± 8.24 cm after refrigerated storage. Furthermore, the pH 4.6-soluble nitrogen fractions, soluble calcium and phosphorus, and the fraction of bound water of the cheese analogue significantly increased during refrigerated storage, while the β-casein and the fraction of free water decreased. The microstructure results showed that the whey channel disappeared while the proteins were swollen and squeezed and deformed the fat globules over refrigerated time. In conclusion, the functionality changes of the cheese analogue during refrigerated storage occurred under the compound effects of proteolysis and hydration of protein as well as dissolution of bound calcium. [ABSTRACT FROM AUTHOR]

To improve the mechanical and barrier properties of zein films, sodium caseinate (CN) was combined with zein to fabricate composite films, by achieving co-dispersion of the two proteins in a newly developed film-forming solvent mixture (60% v/v aqueous ethanol, at 65 °C). The synergistic interactions between CN and zein at various ratios in the films were evaluated. Incorporation of CN into zein-based films increased elongation at break, tensile strength and water contact angle, and decreased water vapor permeability. The film with a CN/zein ratio of 0.75:1 had the lowest water vapor permeability (2.53 ± 0.07 × 10−12 g/(m·s·Pa)), and that with a ratio of 1.25:1 had high elasticity, with an elongation at break 4.11 times that of a pure zein film. CN addition also increased the thermal stability of the composite films, such that films with a CN to zein ratio of 0.75:1 had the highest glass-transition temperature (105.3 °C). Infrared spectral analysis confirmed the high compatibility and strong interactions between CN and zein, and scanning electron microscopy indicated that the microstructure of the composite films was more compact and homogeneous than pure zein films. However, atomic force microscopy revealed that the composite films had a rougher surface. This study provides new perspectives for the development and application of protein-based films as food packaging materials. [Display omitted] • CN and zein can be well co-dispersed in hot (65 °C) 60% aqueous ethanol. • The CN/zein composite films showed improved barrier and water resistance. • Addition of CN reduced the brittleness of zein-based films. • Compact structure and rough surface of CN/zein composite films were observed. • The CN/zein ratio of 0.75 is the best for synergistic interactions of both. [ABSTRACT FROM AUTHOR]

The destabilization of water-in-oil (W/O) emulsions greatly restricts their application. Converting the inner water droplets into soft solid-like gelled particles is an appealing strategy to improve the stability of W/O emulsions. In the present communication, we propose a novel method for preparing stable W/O emulsions containing gelled water droplets. In this method, the 3% (w/v) casein dispersions containing 0.9% (w/v) glucono-δ-lactone (GDL) and soybean oil with 2% or 6% lipophilic emulsifier polyglycerol polyricinoleate (PGPR) were homogenized to form W/O emulsions. Three hours later, the GDL-induced gelation of caseins in the inner aqueous phase occurred; thus, the W/O emulsions containing gelled water droplets were formed. The stability of the emulsions was characterized by Turbiscan analysis, particle size changes and visual inspection of phase separation. It was found that the W/O emulsions containing GDL-induced casein gels in the inner aqueous phase showed higher resistance to destabilization than the non-gelled W/O emulsions. In addition, the stability of the emulsions formulated by the gelled aqueous phase and 2% PGPR was even higher than emulsions with 6% PGPR in the absence of the gelled aqueous phase. These findings suggested that this method is an effective strategy for improving the long-term stability of W/O emulsions. [ABSTRACT FROM AUTHOR]

Abstract: Casein micelles (CMs) and carrageenan (CG) mixtures were prepared in simulated milk ultrafiltrate. Rennet-induced gelation in the CM/CG mixtures was studied by accessing the kinetics of released caseinomacropeptide (CMP) and the rheological properties of CM/CG mixtures. Particle size distributions, ζ-potential and ionic strength measurements, and microstructure observations were performed to understand the underlying mechanisms. At low CG concentrations, κ-CG, which had a tail-adsorption to the CM surface, affected the second phase of renneting through repulsive interactions. In mixtures containing ι-CG and λ-CG, both the first and second phase of renneting were affected. Flat-adsorption and ring-adsorption of ι-CG and λ-CG to the CM surface, respectively, increased the negative charges and covered more enzymatic action sites, thereby decreasing the maximum concentration of released CMP, increasing coagulation time, and weakening gel strength. At high CG concentrations, free CG molecules contributed to rennet-induced gelation, especially in the presence of κ-CG and ι-CG, which significantly accelerated the second phase of renneting through the self-association of CG double helices. Our findings suggested that ι-CG and λ-CG lead to soft cheese curds and improved low-fat cheese quality. [Copyright &y& Elsevier]

Abstract: The composition, size and hydration of yak (Bos grunniens) casein micelles and the variation of these parameters during a warm and a cold period, were investigated. The concentrations of micellar calcium, magnesium and inorganic phosphorus in yak milk obtained during the cold period were higher than those obtained during the warm period, while the reverse result was observed for micellar citrate. In yak casein micelles, the relative proportion of β-casein decreased significantly from the warm period to the cold period, whereas the opposite was observed for αS1-casein, and the relative proportion of αS2- and κ-casein remained largely constant. Casein micelles in yak milk obtained during the cold period showed smaller average diameter, narrower size distribution and higher hydration. On the other hand, these parameters of casein micelles in yak milk differed significantly compared with those in cows' milk, with different contents of the components, larger average size and higher hydration. [Copyright &y& Elsevier]