This study investigated the hydrothermal phosphorylation modification of whey protein isolate (WPI90) using mixtures of sodium hexametaphosphate (SHMP) and tetrasodium pyrophosphate decahydrate (SPP) at different mass ratios (1:1, 1:2, and 2:1) applied at varying mass concentrations (0.03, 0.06, and 0.09 g/100 mL). The secondary structure, thermal stability, solubility, foaming properties, emulsifying properties, and water-holding capacity of WPI were determined before and after phosphorylation. The results indicated that the negative charges introduced by phosphorylation altered the structural characteristics of WPI. This modification resulted in an increase in α-helix content in the secondary structure and induced noticeable microstructural changes. Phosphate addition enhanced the thermal stability and kinetic stability after heating to varying degrees; the phosphorylated WPI solutions remained clear and transparent after heating, showing a turbidity approximately 80% lower than that of unmodified WPI90. All phosphorylation modifications altered the foaming and emulsifying properties of WPI. Notably, the addition of the 2:1 mixture at 0.06 and 0.09 g/100 mL resulted in a 3- to 4-fold increase in the emulsion stability of WPI and increased the water-holding capacity from 20% to 50% and 90%, respectively, demonstrating a remarkable modification effect. This study provides a feasible approach to address the stability limitations of WPI in food processing.

Whey protein (WP), an important protein in dairy products, is rich in amino acids and bioactive peptides, and possesses high nutritional value. However, its poor thermal stability leads to issues such as protein denaturation, precipitation, and oxidation under high-temperature and high-concentration conditions, which limit its application in functional foods, dietary supplements, foods for special medical purpose, and other related fields. Therefore, enhancing the thermal stability of WP has become particularly important. This paper reviews recent advances in physical, chemical, and biological modification techniques, as well as the application of molecular chaperones in improving the thermal stability of WP, and summarizes the key indicators to evaluate its thermal stability and degree of denaturation and aggregation. It also discusses the potential application value and development prospects of thermally stabilized WP in food, pharmaceutical, and other industries.

Sodium tripolyphosphate (STPP), sodium dihydrogen phosphate (SHP), disodium hydrogen phosphate (DSP), sodium hexametaphosphate (SHMP), and tetrasodium pyrophosphate (SPP) were employed for the moist-heat phosphorylation of whey protein isolate (WPI90) under varying temperatures and pH conditions. Phosphorylated proteins with stronger heat resistance were selected to determine their solubility, free sulfhydryl content, surface hydrophobicity, secondary structure, sodium, phosphorus, and calcium contents. Furthermore, they underwent ultra-high temperature (UHT, 135 ℃) processing followed by evaluation of their thermal stability indicators, including centrifugal precipitation rate, viscosity, and particle size as well as their solubility and structure. Commercial heat-stable whey protein was used as control. The results revealed that phosphorylation at 75 ℃ led to an increase in the content of free sulfhydryl groups and a decrease in surface hydrophobicity. The incorporation of phosphates increased the sodium and phosphorus contents while reducing both soluble and total calcium contents, with SPP and SHP resulting in the lowest soluble calcium levels (< 3 mg/g). Fourier transform infrared spectroscopy (FTIR) indicated alterations in protein secondary structure, characterized by a general decrease in β-sheet content and an increase in β-turn content. Phosphorylation at 85 ℃ increased the random coil content. All phosphates except DSP enhanced the thermal stability of whey protein at pH 7.0 and different temperatures (75, 80, and 85 ℃), preventing flocculation of whey protein after UHT treatment. The SHMP-modified protein demonstrated the lowest centrifugal precipitation rate and apparent viscosity after UHT treatment, with an overall particle size below 15 µm. Moreover, phosphorylation with SPP, STPP, and SHP at 75 ℃ improved the solubility of WPI90 to different extents, while phosphorylation with SHMP did not. For both 75 and 80 ℃, SPP phosphorylation resulted in the highest solubility of WPI90. In conclusion, this study demonstrates that moist-heat phosphorylation effectively modifies the structure and functionality of whey protein while influencing the salt ion contents in the protein system. Specifically, the addition of SHMP under neutral pH conditions significantly enhances the thermal stability of whey protein, being of guiding significance for the UHT processing of whey protein concentrate.

In this study, we investigated the changes in the functional properties (meltability and fat precipitation), texture, rheological properties, and microstructure of processed cheese during emulsification (5–30 min) at different temperatures (80 and 85 ℃). The results showed that as the emulsification time increased, the meltability and fat precipitation showed a decreasing trend, and the fat precipitation at 85 ℃ was significantly higher than that at 80 ℃ during the emulsification process (P < 0.05). The gumminess and chewiness increased significantly (P < 0.05) with the increase in emulsification time, and the hardness, gumminess and chewiness at 85 ℃ were greater than those at 80 ℃. The storage modulus (G’) of processed cheese was greater than the loss modulus (G”) at the same degree of emulsification for both temperatures and both G’ and G” showed an upward trend with increasing frequency from 0.1 to 10 Hz. The microstructure of processed cheese showed that the number of fat globules was greatly reduced, the diameter was decreased, the distribution of fat globules became more uniform, the protein matrix became smoother and the cheese structure became denser at 5–15 min of emulsification. However, the microstructure of processed cheese was honeycomb-like at 20–30 min of emulsification, suggesting excessive creaming reaction. Therefore, a short emulsification time during the processing of processed cheese will result in inadequate creaming reaction; too long emulsification time will cause adverse changes in cheese texture. During the emulsification process, the protein-protein and protein-fat interactions were enhanced, thereby improving the functional properties of the product. In conclusion, the emulsification process of processed cheese is closely related to its physicochemical and functional properties and microstructure. This study provides some theoretical guidance for future process development of processed cheese production.

In this study, calcium-chelating casein hydrolytic peptide (CHP-Ca) was prepared by the chelation reaction between CHP, which was prepared from enzymatic hydrolysis of micelle casein, and calcium chloride at a mass ratio of 2:1. Five enzymes (flavourzyme, alcalase, trypsin, papain and neutral protease) were screened based on hydrolysis degree (DH) and calcium-chelating capacity. A mixture of flavourzyme and trypsin was found to be the best enzyme for the enzymatic preparation of CHP. Furthermore, response surface methodology (RSM) was used to optimize the enzyme hydrolysis conditions. The results showed that the optimal conditions were enzyme-to-substrate ratio 6000 U/g, flavourzyme-to-trypsin ratio 1:1, hydrolysis time 90 min, pH 6.9, and temperature 42 ℃. The calcium chelation capacity of the peptide prepared under these conditions was (90.46 ± 0.72) μg/mg. Finally, the structural properties and formation mechanism of CHP-Ca were investigated by X-ray diffraction (XRD), thermogravimetry (TG), zeta potential, particle size analysis and infrared (IR) spectroscopy. The results showed that CHP-Ca bound mainly by the interaction of carboxyl, amino and phosphate groups, and the zeta potential and particle size decreased after chelation, and the structure of CHP-Ca became more compact.

An emulsion gel stabilized by a mixture of whey protein and pectin (WP) and an emulsion gel stabilized by a mixture of whey protein and glucan (WG) were prepared by an acid induction method. The functional properties of WP and WG emulsion gels with dispersed phase volume fractions of 35% and 75% (called WP35, WG35, WP75, and WG75) were evaluated and compared, and the efficiency of high internal phase emulsion gels as a fat replacer in replacing fat in low-fat processed cheese was further investigated. The results showed that WP75 had the smallest mean particle size, uniform fat distribution, and a stable protein network structure. Within the frequency range of 0.1–10 Hz, both the elastic modulus (G’) and viscous modulus (G”) values of the emulsion gel samples tended to increase as the oil phase volume fraction increased. Compared with the other samples, WP75 was superior in terms of deformation resistance, thermal stability, and water-holding capacity. Addition of WP75 into processed cheese with 15% (m/m) fat reduced the oil syneresis and increased the meltability without resulting in significant differences in textural properties compared with the control group (full-fat). This study demonstrated that high internal phase emulsion gels stabilized by whey protein and pectin could be used as a fat replacer in the development of low-fat cheese products.

The enhancing effects of different pre-treatment techniques (addition of emulsifying salts, electrodialysis, and resin adsorption) on the stability of ultra-high temperature (UHT)-sterilized yak milk with standardized protein content was investigated by measurement of ethanol stability, protein precipitation rate, whey protein denaturation rate and turbidity stability index (TSI) as well as by using Fourier transform infrared spectroscopy (FTIR) and inductively coupled plasma mass spectrometry (ICP-MS). The results showed that all three pre-treatment methods significantly improved the heat stability of UHT-sterilized yak milk. Compared with the standardized control group, whey protein denaturation rate decreased by 92.02% and 90.15%, soluble calcium content by 3.94% and 13.93%, and protein precipitation rate by 11.71% and 20.40% in the samples treated by resin and electrodialysis, with an average TSI of 2.94 and 1.33, respectively. Whey protein denaturation rate decreased by 84.69%, and protein precipitation rate increased 18.39% in the emulsifying salt-treated sample. The resin-treated group had the highest absolute value of the zeta potential (22.83 mV), closely followed by the electrodialysis group (19.57 mV), suggesting that electrodialysis also improved the heat stability of UHT-sterilized yak milk. FTIR analysis revealed that after UHT treatment, both resin and electrodialysis groups showed an increase in the relative content of α-helix and a decrease in the relative content of β-sheet. In conclusion, this study demonstrates that adjusting the salt balance of yak milk can enhance its UHT stability, providing important guidance for the processing of UHT yak milk.