Effects of simultaneous dual-frequency divergent ultrasound-assisted extraction on the structure, thermal and antioxidant properties of protein from Chlorella pyrenoidosa

The aim of this study was to investigate the effects of simultaneous dual-frequency divergent ultrasound-assisted extraction (SDFUE) on the yield, structure, thermal and antioxidant activities of protein from Chlorella pyrenoidosa (CPP). Based on single factor experiments, the extraction conditions were optimized by response surface methodology (RSM). Under optimal conditions, ultrasonic power of 360 W, extraction temperature of 45 degrees C, 3% (w/v) alkali concentration and ultrasonic time of 20 min, the maximal extraction rate of CPP was 52.36 +/- 0.31%, significantly higher than that of hot water extraction (HWE). In addition, the amino acid composition, the secondary and tertiary structure of CPP were significantly changed due to the unfolding of protein molecules and the exposure of hydrophobic groups. Moreover, the thermal property of CPP was more stable, the denaturation temperature and the heat required for denaturation were increased. Besides, the antioxidant properties of CPP (6 mg/mL) extracted by SDFUE showed that, the maximum 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was 86.96 +/- 0.33%, maximum 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was 59.07 +/- 0.31%, maximum Fe2+ chelating capacity was 34.37 +/- 0.43%, and the maximum 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) radical scavenging activity at pH 4.5 was 66.24 +/- 0.98%, which were all improved obviously compared with that of HWE, PTIO center dot trapping assay demonstrated that electron transfer (ET) was mainly involved in the antioxidant process of CPP. These findings revealed that ultrasound-assisted extraction not only increased the yield of CPP, but also enhanced its thermal and antioxidant properties, which was related to the changes of CPP structure. This work might break the traditional single-frequency ultrasonic extraction model and provide a reference for the utilization of ultrasonic extraction in the field of Chlorella pyrenoidosa as bioactive materials.