Effect and Mechanism of Moist-Heat Phosphorylation on Improving the Thermal Stability of Whey Protein

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.