Study on the Preparation and Conjugation Mechanism of the Phosvitin-Gallic Acid Complex with an Antioxidant and Emulsifying Capability

Abstract

To develop a novel emulsifier with an antioxidant capacity, a phosvitin-gallic acid (Pv–GA) complex was prepared via a free-radical method. This emulsifier characterizes some key technologies. Changes in the molecular weight of the Pv–GA complex were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and the matrix-assisted laser desorption/ionization time of light mass spectrometry (MALDI-TOF-MS). Fourier transform infrared spectroscopy (FTIR) indicated that C=O, C–N and N–H groups were also likely to be involved in the formation of the complex. A redshift was obtained in the fluorescence spectrogram, thereby proving that the covalent combination of Pv and GA was a free radical-forming complex. The results indicated that Pv and GA were successfully conjugated. Meanwhile, the secondary structure of Pv showed significant changes after conjugation with GA. The antioxidant activity and emulsifying properties of the Pv–GA complex were studied. The antioxidant activity of the Pv–GA complex proved to be much higher than that of the Pv, via assays of the scavenging activities of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals and because of their ability to reduce power. The emulsification activity of the Pv–GA complex was also slightly higher than that of Pv. To function with the most demanding antioxidant and emulsification activities, the optimum conjugation condition was Pv (5 mg/mL) conjugated 1.5 mg/mL GA. Furthermore, the mechanism of Pv–GA conjugation was studied. This study indicated that GA could quench the inner fluorescence of Pv, and this quenching was static. There was a strong interaction between GA and Pv, which was not obviously affected by the temperature. Furthermore, several binding sites were close to 1, indicating that there was an independent class of binding sites on Pv for GA at different temperatures. The conjugation reaction was a spontaneous reaction, and the interaction forces of GA and Pv were hydrogen bonds and van der Waals force.

Fabrication and Characterization of a Microemulsion Stabilized by Integrated Phosvitin and Gallic Acid

“…21 When the migration was finished, the gel was stained in Coomassie Brilliant Blue R-250 in 0.1 mol/L aluminum mordant to identify Pv bands, followed by rinsing with distilled water, and then decolorized by an eluent. 22 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI−TOF−MS). A total of 1 μL of 1 mg/ mL Pv or Pv−GA complex solution was mixed with an equal volume of 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) and saturated sinapinic acid.…”

“…The emission spectrum of sample solution was recorded within the wavelength range of 300−500 nm with an excitation wavelength of 280 nm. 22 The slit widths were 5 nm.…”

“…Migration voltages for separating gel and stacking gel were set at 80 and 120 V, respectively . When the migration was finished, the gel was stained in Coomassie Brilliant Blue R-250 in 0.1 mol/L aluminum mordant to identify Pv bands, followed by rinsing with distilled water, and then decolorized by an eluent …”

“…A total of 0.01 mmol/L phosphate buffer with a pH value of 7.0 was used to dissolve Pv or the Pv–GA complex, separately. The emission spectrum of sample solution was recorded within the wavelength range of 300–500 nm with an excitation wavelength of 280 nm . The slit widths were 5 nm.…”