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Aqueous enzymatic extraction (AEE) can simultaneously separate oil and protein. However, a stable O/W emulsion is present in the AEE process, which is not favorable for extracting oils. This study optimized the use of heptanoic and octanoic acids for demulsification in aqueous enzymatic extraction. The optimal condition for demulsification, including a fatty acid ratio of 1:3 (heptanoic acid to octanoic acid) with 1.00% addition, a reaction time of 40 min, a temperature of 70 °C, and a solid-to-liquid ratio of 1:5, resulted in a demulsification rate of 97.95% ± 0.03%. After demulsification, the particle size of the peanut emulsion increased, while the absolute potential value and conductivity decreased. The type and content of proteins decreased, and the tertiary structure also changed, with tryptophan residues buried within the proteins, shifting the system from a polar to nonpolar environment. The microstructure of the emulsion changed and the emulsion transformed into W/O. To summarize, composite fatty acid had a significant effect on the demulsification of emulsion.
