Phosphate tweak turns macadamia waste protein into a powerful emulsion stabilizer

Scientists in China have found that a simple phosphate treatment can transform a little-used protein extracted from macadamia oil processing waste into a highly effective stabilizer for ultra-concentrated emulsions. The study, carried out with macadamia press cake supplied by the South Subtropical Crop Research Institute of the China Academy of Tropical Agricultural Sciences, shows that attaching phosphate groups to macadamia protein isolate raised its water solubility from roughly 14 percent to nearly 97 percent and allowed the modified protein to hold emulsions containing up to 80 percent oil stable for a full month. Native macadamia protein, by contrast, could not keep such oil-rich systems from separating.

Macadamia is a high-value nut crop, and the kernels left behind after oil extraction yield a protein-rich press cake that is often underused. The protein isolated from this by-product, known as macadamia protein isolate or MPI, has a favorable amino acid composition, but it suffers from two practical drawbacks: it dissolves poorly in water and it performs weakly at oil-water interfaces. Both problems appear to stem from the protein’s compact structure and its tendency to aggregate, which limit its usefulness in emulsion-based food applications, particularly high internal phase emulsions, or HIPEs, in which the internal oil phase exceeds 74 percent of the volume.

HIPEs are attractive targets for food technologists because their dense, gel-like architecture, high viscosity, and storage stability make them promising platforms for fat replacement, the encapsulation of bioactive compounds, and the design of functional foods. To unlock MPI for these uses, the research team turned to phosphorylation, a chemical modification in which phosphate groups are grafted onto reactive side chains of the protein. The introduction of these negatively charged groups increases the protein’s surface charge, strengthens electrostatic repulsion between molecules, and can trigger rearrangement of the protein’s higher-order structure, all of which tend to improve solubility and interfacial activity.

The researchers compared two widely used, low-cost phosphorylating agents with different molecular architectures: sodium tripolyphosphate, or STPP, a linear polyphosphate, and sodium trimetaphosphate, or STMP, a cyclic metaphosphate. Protein dispersions containing 4 percent MPI were treated with each reagent at concentrations of 2, 3, 4, 5, and 6 percent at pH 9.0 and 45 degrees Celsius for two hours, then dialyzed and freeze-dried. Phosphorus analysis showed that both reagents incorporated phosphate most effectively at 5 percent addition, where bound phosphorus reached 3.95 milligrams per gram for STPP and 3.22 milligrams per gram for STMP, compared with 0.27 milligrams per gram in the native protein. At every concentration, STPP outperformed STMP, a difference the authors attribute to the linear reagent’s greater accessibility to reactive sites on the protein.

The functional consequences were striking. Native MPI dissolved at only 14.01 percent, but solubility climbed to a maximum of 96.78 percent with 5 percent STPP and 84.57 percent with 4 percent STMP before declining again at higher reagent levels, where residual phosphate salts, electrostatic screening, and possible intermolecular bridging appear to counteract the benefit. Particle size measurements of the dispersion supernatants told a consistent story: the Z-average diameter fell from 197.9 nanometers for native MPI to 165.2 nanometers at 5 percent STPP and 165.7 nanometers at 6 percent STMP, indicating that phosphorylation broke up large aggregates into smaller, better-dispersed particles.

Charge measurements reinforced this picture. The native protein supernatant carried a zeta potential of minus 25.77 millivolts, but phosphorylation pushed the absolute value up to 70.67 millivolts for STPP-treated and 67.21 millivolts for STMP-treated samples at 5 percent reagent, reflecting a much higher density of negative charge on the protein surface. Transmission electron microscopy showed that the irregular, rough, heavily aggregated spherical particles of native MPI became smaller, more regularly shaped, and far more uniformly dispersed after moderate phosphorylation, although partial re-aggregation returned at the highest reagent dose of 6 percent.

A battery of spectroscopic techniques revealed how phosphorylation reshaped the protein at the molecular level. Intrinsic fluorescence spectra shifted from a maximum emission wavelength of 347 nanometers to about 351 nanometers after treatment, indicating that previously buried tryptophan residues moved into a more polar environment as the structure partially unfolded. Surface hydrophobicity rose from 13,450 for native MPI to peak values of 18,599 with 5 percent STPP and 19,772 with 5 percent STMP, signaling greater exposure of hydrophobic regions. Free sulfhydryl content also increased, reaching 21.28 and 21.50 micromoles per gram at 5 percent STPP and STMP respectively, while disulfide bond content fell, consistent with a loosened protein structure.

Circular dichroism spectroscopy quantified the secondary-structure changes: native MPI contained 21.85 percent alpha-helix and 26.10 percent beta-sheet, but after phosphorylation the alpha-helix and random coil fractions increased while beta-sheet and beta-turn contents dropped, pointing to a looser, more flexible conformation. Fourier transform infrared spectroscopy detected new absorption bands near 896 and 1155 wavenumbers, assigned to P-O and P=O stretching vibrations, and X-ray photoelectron spectroscopy confirmed a distinct phosphorus signal at about 133.3 electron volts along with a new carbon component at approximately 283.3 electron volts corresponding to C-O-P bonds. X-ray diffraction showed altered molecular packing, with enhanced ordering at moderate phosphorylation levels and renewed disruption at 6 percent reagent.

With 5 percent STPP-treated MPI, designated LMPI, showing the best overall performance, the team prepared HIPEs containing 75 and 80 percent macadamia oil. At 75 percent oil, emulsions stabilized by either native or modified protein formed gel-like systems that showed no oiling-off and remained stable for 30 days at 25 degrees Celsius. At 80 percent oil, however, the native protein emulsion underwent obvious phase separation, while the LMPI-stabilized emulsion showed no visible separation after the same storage period. Confocal microscopy confirmed that protein particles adsorbed at the oil-water interface in all samples, but the LMPI emulsions displayed smaller, more uniformly distributed droplets that packed closely and deformed into a continuous gel-like network.

Quantitative measurements supported the visual observations. The surface-weighted mean droplet diameter fell from 19.51 micrometers in the native protein emulsion at 75 percent oil to 11.89 and 10.97 micrometers in LMPI emulsions at 75 and 80 percent oil respectively, with the volume-weighted mean dropping from 28.03 to 20.64 and 20.32 micrometers. The absolute zeta potential of the emulsion droplets rose from 44.75 millivolts to 72.22 and 76.28 millivolts, and both the interfacial adsorption content and the protein adsorption rate were significantly higher for the modified protein, consistent with faster and more complete coverage of the enlarged interfacial area. Rheological testing showed typical shear-thinning behavior for all emulsions, and the storage modulus exceeded the loss modulus across the tested frequency range, confirming gel-like elasticity. The LMPI emulsion at 80 percent oil exhibited the highest storage modulus and the weakest frequency dependence, indicating the strongest elastic network, whereas the native protein emulsion at 75 percent oil showed the lowest modulus despite its relatively high apparent viscosity under steady shear.

The authors propose that phosphorylation improves HIPE stabilization through a linked sequence of events: introduced phosphate groups raise surface charge and hydration, reducing aggregation; conformational rearrangement exposes hydrophobic regions and sulfhydryl groups, boosting interfacial activity; and the modified protein then adsorbs more effectively at the oil-water interface, forming a denser protective layer. Enhanced electrostatic repulsion between droplets suppresses coalescence, while smaller droplets and closer packing build a stronger space-filling network. The team notes that only two internal phase fractions, 75 and 80 percent, were compared, so the exact critical point at which native protein fails remains to be determined. Even so, the findings indicate that phosphorylated macadamia protein, particularly the 5 percent STPP-treated form, is a promising plant-derived stabilizer for HIPE-based food systems and a route to adding value to nut processing by-products.

Subject of Research: Phosphorylation of macadamia protein isolate to improve solubility and high internal phase emulsion stability

Article Title: Phosphorylation of macadamia protein isolate: Unveiling the mechanism behind enhanced solubility and high internal phase emulsion stability

Article References: Du, L., Fang, M., Chu, X., Zhang, M., Wang, Z., Li, L., Li, C., Chen, J., Dai, T., & Shuai, X. (2026). Phosphorylation of macadamia protein isolate: Unveiling the mechanism behind enhanced solubility and high internal phase emulsion stability. Current Research in Food Science, Article 101608. https://doi.org/10.1016/j.crfs.2026.101608

Image Credits: AI Generated

DOI: 10.1016/j.crfs.2026.101608

Keywords: macadamia protein isolate, phosphorylation, sodium tripolyphosphate, sodium trimetaphosphate, high internal phase emulsions, protein solubility, zeta potential, interfacial adsorption, rheology, food emulsifiers, plant protein modification, food by-product valorization