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.