Antioxidant capacity of soybean protein isolate hydrolyzed with protease produced by Aspergillus 3 . 084 in vitro

Aspergillus, being known as a protease producing fungi, is used in Chinese and Korean traditional fermentation. In this paper, Aspergillus 3.084, one of the most common fungi in China fermentation industry, was employed to produce protease and hydrolyze soybean protein, and then the optimum extraction condition of protease was determined by the antioxidant activities of protease extraction solution. The protease was extracted from the culture medium, and performed in hydrolyzing soybean protein isolation. Extract condition of protease, hydrolysis ability of proteases of Aspergillus in hydrolyzing soybean protein isolation and the antioxidant ability of hydrolysate were investigated by detecting enzyme activity, degree of hydrolysis, scavenging activities of OH∙, O2∙ and DPPH∙. Maximum protease activity was obtained at 60 min, pH 7.5 and 32°C in extraction step. Optimum protease hydrolyzing time, E/S, temperature and pH for degree of hydrolysis and scavenging activities was 3 h, 6000, 37°C and 7.5, respectively.


INTRODUCTION
Soybean is a well-known source of protein with high protein and peptide content.Peptides made from soybean exhibit variety of functional properties, including immunomodulatory, antioxidant, anticancer (Lin et al., 2013).Fermentation, one of the oldest food-processing techniques, can improve the antioxidant properties of soybean through various biochemical transformations.
Aspergillus 3.084 is a common fungi in Chinese fermentation industry, it secretes amylase, glucoamylase, α-amylase, alkaline protease, acid protease and neutral protease.The most familiar product made by Aspergillus is soybean paste, and the antioxidant capacity of soybean paste is given by fermentation process.This process also improved the properties of soybean by biochemical transformations including proteolysis, saccharification, esterification, etc (Rhyu and Kim, 2011).Soybean paste produced generally by two steps is a traditional seasoning in China and Korean, first step was pretreatment by steaming and shaping, fermentation was followed by microorganisms (Lee et al., 2014).In the beginning of fermentation processing, starter named qu in China and meju in Korean was added into pretreated soybean, which contained the microorganisms, including Bacillus, Aspergillus, Scopulariopsis, Cladosporium, Mucor, Lichtheimia, Rhizopus and Penicillium (Jung et al., 2014).Microorganisms have been identified in Korean meju, and Aspergillus was the main microorganism in china soybean paste (Wardhani et al., 2013).Soybean hydrolysates, such as amino acids and peptides, enriched the taste, flavor, even the antioxidant capacity of soybean paste.
The antioxidant capacity test included hydroxyl free radical (OH•) scavenging activity, 1,1-diphenyl-2-picrylhydrazylfree radical (DPPH•) scavenging activity and superoxide anion (O2• -) scavenging activity.Superoxide radical is known to be harmful to cellular components as a precursor of more reactive oxidative species; it is also documented to play an important role in the peroxidation of lipids (Monod et al., 1991).Therefore, the scavenging activity on superoxide radicals is a way to investigate the antioxidant activity.Free radical is one of the factors that induce the pathogenesis of a wide range of age-related degenerative diseases through the oxidative modification of DNA, proteins and vital molecules (Malathi and Chakraborty, 1991;Cohen, 1973).DPPH, a stable radical-generating reagent, was used in the present study for primary screening of the antiradical activities (Yokozawa et al., 1998;Kalaskar et al., 2014).The main antioxidant properties was mainly given by the peptides, which was hydrolyzed by enzyme and acid.Protease secreted by Aspergillus 3.084 was employed to hydrolyze the soybean protein isolate, and the antioxidant properties of peptides, the enzymatic hydrolysate, was studied in this paper.Effects of temperature, pH value and ratio of enzyme/soybean protein powder ([E/S]) on the antioxidant properties of peptides hydrolyzed by extracted protease was investigated.

Protease preparation
Aspergillus 3.084, obtained from Shandong wife food group co., LTD, Zibo, Shandong, was used in this study.The culture media were prepared with 20 g soybean meal, 20 g wheat bran and 10 g flour in 250 ml erlenmeyer flasks and soaked with 50 g distilled water.Flask was mixed thoroughly and all flasks were autoclaved at 121°C for 20 min, cooled to room temperature, inoculated with 1 ml of conidial inoculums (1 × 10 9 ), and then incubated at 32°C for 72 h (Belmessikh et al., 2013).

Optimization of extract condition of protease
Fermented substrate (10 g) was milled and mixed with 50 ml 0.1 mol/L pH7.5 phosphate buffer and homogenized by ultrasound for 1 hour at 37°C to extract the enzyme.
Extraction condition under different pH, temperature and time was optimized.Protease activity of the enzyme solution was analyzed by the method described by Lowry, enzyme-activity was micrograms of tyrosine produced by hydrolyzing the casein within one minute.
A, tyrosine (μg) T, time (min) N, dilution ratio Total protein content of the extracted solution was measured by using coomassie brilliant blue staining with bovine serum albumin as a protein standard (de Castro and Sato, 2014).

Peptide preparation
Hydrolysis reaction was carried out under different temperature (27,32,37,42, 47 o C), hydrolysis time (1, 2, 3, 4 and 5 h), pH (3.5, 5.5, 7.2, 9 and 10.5) and ratio of enzyme/soybean protein ([E/S], 1000, 2000, 4000, 6000 and 8000U per gram soybean protein).The original temperature, hydrolysis time, pH and protease activity were set at 37°C, 2 h, 7.2 and 25 U/ml.After the hydrolysis reaction, solution was kept at 95°C for 3 min to inactivate the protease and cooled as the experimental group.Reaction solution (10 ml) was adjusted to pH 8.2 by 10 mol/L NaOH or 10 mol/L HCl and diluted to 20 ml for the determination of the degree of hydrolysis and, 10 reaction solution was adjusted to pH 7.0 and diluted following the same step as above-mentioned for the other tests [14].Mixture of enzyme and soybean protein was kept at 95°C for 3 min to inactivate the protease, which was unhydrolyzed solution and used for the control group.

Determination of the degree of hydrolysis (DH) [15-17]
Distilled water (10 ml) was mixed with 10 ml reaction solution, and then 20 ml 36.5% formaldehyde solution (adjusted to pH 8.2 with 0.1 mol/L NaOH) was added (Castillo et al., 1962;Gould and Frantz, 1945;Mehler, 1957).The mixture solution was titrated to pH 9.2 by 0.1 mol/L NaOH, titration volume was V1.Unhydrolyzed solution 10 ml (mixture of enzyme and soybean protein) was used for control, titration volume of which was V2.The degree of protein hydrolysis was calculated as follows

Superoxide anion (O2• -) scavenging activity
All experiments were performed at 25°C, the assay medium (0.05 mol/L TRIS buffer, pH 8.2) 4.5 ml mixed with 100 μl hydrolyzed solution and 400 μl 2.5 mmol/L pyrogallol, stopped reaction 4 min later with 0.1 ml 10 mol/L HCl and recorded at 325 nm.Also, 100 μl distilled water was used in blank group, and 100 μl unhydrolyzed solution replaced hydrolyzed solution in control group.
The scavenging percentage of hydrolyzed solution on O2• - was calculated according to the following equation.

Protease preparation
Aspergillus 3.084 grew on potato agar medium with yellow mature spores in the middle part of fungi colony (Figure 1A).When Aspergillus grow and secreted the enzyme, medium beside the Aspergillus became more transparent for culture medium contained 1% casein and enzyme hydrolyzed casein to make this vitrification.

Optimization of extract condition of protease
Fermentation production was extraction with different condition included time, temperature and pH to investigate the optimal extract condition, and 60 min, pH 7.5 and 32°C were performed in this step (Figure 3).

DH and antioxidant activity under different E/S
DH increased following the increase of E/S (decrease of substrate concentration), even the same trend appeared in DPPH• scavenging activity of hydrolyzed soybean protein.DPPH• scavenging activity was less steep than DH, the values were between 13 to 19.5.Small molecular weight proteins and peptides generated following the E/S increase, which had higher DPPH• scavenging activity  (Figure 4).O2• -and OH• scavenging activities declined, and the trend of O2• -scavenging activity was slower than OH• scavenging activity.Soybean protein hydrolyzed by protease to small molecular weight protein and peptide, more of that were produced in low substrate concentration (high E/S).This result means that small molecular weight proteins and peptides had stronger O2• -and OH• scavenging ability (Figure 4).

DH and antioxidant activity under different reaction time
The DH and antioxidant activities of hydrolyzate solution were monitored every 1 h up to 5 h by mixture of soybean protein and proteases solution at 6000 E/S.Curves of DH, DPPH• scavenging activity, O2• -scavenging activity and OH• scavenging activity showed rising trend following reaction time (Ortiz and An, 2000), which suggested that scavenging activities increased with the DH (Figure 5).B.P. Lamsal depicted that the subunits of β-conglycinins, and acidic and basic subunits of glycinin, almost entirely disappeared hydrolysate at 2% DH of soybean protein isolation (Lamsal et al., 2007), and then small molecular weight peptides and amino acids were obtained.Among those hydrolyzed peptides and amino acids, several kinds of them in soybean hydrolysate had antioxidant activity which was obtained from the hydrolyzed bitter peptides.

DH antioxidant activity under different temperature
Those detected indexes had the same trend following the increase of temperature (Figure 6).Low DH was detected in high temperature for the induction of protease inactivation and protein denaturation, and antioxidant activities were also decreased with the DH.Antioxidant activities were lower in low temperature than high, coincided with trend of DH.

DH and antioxidant activity under different pH
Extracted enzyme solution contained many kinds of proteases with the optimum reaction pH of this solution was 7.5.Antioxidant activities varied following the change of DH, and low values measured at two extreme points (Figure 7).Acid protease included in solution had optimum  action pH at or nearly 3 with its lower hydrolysis ability than alkaline protease at its optimum pH 10.5.Middle protease also had enzyme activity at pH 9 and 5.5, which contributed to the high antioxidant activities.Even the DH at pH 3 was more than double times at pH 7.5 and 9 nearly double times at pH 5.5, there was no so much difference between the oxidant activities at pH 3 and pH 5.5, 7.5 and 9.  other two, alkaline and acid protease could not reach the same hydrolysis ability even in the optimal pH.

CONCLUSIONS
Action of different kind of protease on soybean protein was different[20], Aspergillus production contained many kinds of proteases which had different extracting properties, hydrolyzing abilities [10].Longer extracting time give the solution higher enzyme activity following the increasing dissolved enzyme, and enzyme activity reached the top point at pH 7.5 and 37°C, This result indicated that the middle protease played the most important part in enzyme activity of this solution.
Optimum reaction time, E/S, temperature and pH was 3 h, 6000, 37°C and 7.5, following the DH, antioxidant activities of this solution reached the maximum point at the same reaction conditions.Soybean proteins hydrolyzed by limited or controlled enzyme could provide ingredients with desired peptides and functionalities (Lamsal et al., 2007), Aspergillus 3.084 with multifarious proteases used in traditional fermentation technology contributed to the peculiar flavor of production composed by different hydrolyzed ingredients (Han et al., 2001;Zhao et al., 2006;Zhao et al., 2009).Rising trend of DH was slower after 3 h hydrolysis than before.It reached the top point at 5 h within given time interval, and longer time enhanced the DH and facilitated the peptides production.Higher DH and antioxidant properties were obtained in industrial manufacture for it had longer fermentation time.

Figure
Figure 1B was fermented substrate, fungi covered the surface and grow inside the substrate.Optical density of tyrosine in the enzyme activity detection of extracted solution and the linear trend line of OD and tyrosine concentration (correlation between OD and tyrosine concentration) was Figure 2, the enzyme activity of solution was obtained.

Figure 2 .
Figure 2. Standard curve of OD and tyrosine concentration.