Antioxidant activity of arrowhead protein hydrolysates produced by a novel multi-frequency S-type ultrasound-assisted enzymolysis

: Effects of multi-frequency S-type ultrasound (MFSU) assisted arrowhead protein (AP) hydrolysis on the antioxidant activity of its hydrolysates were studied. The results showed the DPPH• and ABTS•+ scavenging activity of hydrolysates obtained with dual frequency ultrasound (20/40 kHz) was 63.61% and 65.11%, respectively, and was higher than that noted for hydrolysates acquired with assistance o f other mode (single and triple frequency ultrasound). Compared with hydrolysates without ultrasonic treatment, products of AP hydrolysis assisted by dual frequency ultrasound (20/40 kHz) could significantly alleviate oxidative stress induced by H 2 O 2 in RAW 264.7 cells, mainly embodied in improving the survival rate and increasing the activity of antioxidant enzymes (CAT and SOD). Taken together, these results showed that MFSU-assisted enzymatic treatment can significantly improve the antioxidant activity of AP hydrolysates. Thus, the development of the novel MFSU could lay a foundation for assisting the protein enzymolysis in food and pharmaceutical industries.

in RAW 264.7 cells, mainly embodied in improving the survival rate and increasing the activity of antioxidant enzymes (CAT and SOD). Taken together, these results showed that MFSU-assisted enzymatic treatment can significantly improve the antioxidant activity of AP hydrolysates. Thus, the development of the novel MFSU could lay a foundation for assisting the protein enzymolysis in food and pharmaceutical industries.

Arrowhead protein extraction
The method of AP extraction was following the previous method (Zhu et al. 2006) with minor modification. The arrowhead powder was dispersed into distilled water (1:20), and the pH of the mixture was adjusted to 9.5 by using 1 M NaOH. Stirring continuously for 4 hours, then the mixture solution was centrifuged at 4,500g for thirty minutes. The supernatant was adjusted the pH to 4.0 with 1.0 mol/L HCl to precipitate the protein. The precipitates were dispersed in distilled water, and its pH was adjusted to 7 by using 1 M HCl and lyophilized for further analysis. The protein content of precipitate was 91.6% according to the Kjeldahl method.

MFSU-assisted and traditional enzymolysis
The MFSU device diagram is shown in Figure S1A, which was produced by Jiangsu University and mainly composed with two parts (ultrasonic generator, and circulation pump). In addition, Figure S1B-D showed the diagrams with mechanism of action of the MFSU device. Ultrasonic waves can produce cavitation bubbles in the process of spread, and the cavitation bubble will be changed as the change of ultrasonic frequency.
The experimental conditions were carried out according to the following methods: An aliquot (1L) of AP solution (3 g/L) was prepared and hydrolyzed with 10 mL alcalase (activity 23,400 U/mL) at pH 9.0 and 50 ℃ . The MFSU-assisted enzymatic hydrolysis of protein solution was carried out under the three frequency mode (Single frequency, double frequency, and triple frequency) as follow: enzymolysis time of 120 min, ultrasonic power of 100 W. After completion, the enzyme in solution need be inactivated in a water bath (100℃) for 10 min and cooled it down to room temperature. The hydrolysis solution was adjusted pH to neutral by using 1 M HCl and lyophilized for further analysis. The control was used the same manner, except the sample was replaced by distilled water. All the experiments were carried out in triplicate.

ABTS•+ radical scavenging capacity assay
The The control was used the same manner, except that the samples were replaced by distilled water. The entire experiments were conducted in triplicate.

Cell culture
RAW 264.7 cell line was obtained from American Type Culture Collection (ATCC; Arlington, USA). Cells were generally cultured in DMEM medium. It is worth noting that the medium contains 100 units/mL penicillin and 100 g/mL streptomycin. The condition of cell culture was at atmosphere of 5% CO 2 and the temperature of 37 ℃ . Cell viability rate (%) = (experimental absorbance/ control absorbance)×100

Antioxidant enzyme activity
The antioxidant enzyme activity was measured by the SOD assay kit and the Catalase (CAT) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The experimental method refers to the instruction manual of the kit. Briefly, CAT activity was determined by the production of formaldehyde produced by the reaction of the sample with methanol and H 2 O 2 . The absorbance was measured at a wavelength of 540 nm by using a microplate reader. In addition, the determination of SOD activity was based on the ability of the sample to inhibit O 2produced by the xanthine-xanthine oxidase system. One unit of SOD activity was defined as the amount that reduces the absorbance at 450 nm by 50%.

Statistical analysis
All experiments were performed in triplicate. Data were expressed as means ± SD.
The statistical analyses were performed for multiple comparison analysis with Duncan's tests by using SPSS 18.0 software (SPSS Inc. Chicago, IL, USA). p<0.05 was considered as statistically significant.