Complex Enzymatic Digestion of Rice Proteins and in Vitro Antioxidant Activity of the Digests

 1 Introduction

In recent years, there have been more and more reports on the safety of rice, such as "Cadmium Rice" in Hunan, where cadmium exceeded the standard, and "Wu Chang Fragrant Rice" in Heilongjiang, where flavorings were added to ordinary rice to make it into fragrant rice. These rices are discarded because they do not meet the requirements of China's food safety law for food processing raw materials, resulting in a great waste of rice resources. Rice protein is not only of high nutritional value, but also highly digestible and hypoallergenic [1]. If these "problematic rice" are enzymatically digested to prepare rice peptides, not only the excellent properties of rice protein are retained, but also rice peptides have good solubility, acid and heat stability, and can be used in non-food industries such as pharmaceuticals, cosmetics, etc. [2-3], which can provide a new way to dispose of these "problematic rice" appropriately. It can provide a new way to properly dispose of the "problematic rice" and avoid the waste of resources.

 

There are many methods to determine the in vitro activity of antioxidant peptides, and ABTS + - scavenging activity is the most widely used indirect assay, which is rapid, simple, and has a strong correlation with the biological activity of antioxidant peptides [4-6]. The principle of this method is that the antioxidant peptide is oxidized with a strong oxidant 2,2 '-azi- no-bis(3-ethyl benzothiazoline -6-sulfonic acid) diam- monium salt, ABTS. The principle of Fe2+ chelating activity is that at certain specific absorption wavelengths, the product of the color development reaction between Fe2+ and the chelator has an absorption peak; the addition of antioxidant peptide chelating Fe2+ reduces the concentration of the color development product and the absorbance value decreases [5-8]. The added antioxidant peptide chelates Fe2+ , which reduces the concentration of the color-developed product and the absorbance value[5-8] . Therefore, the in vitro antioxidant activity of rice proteolytic digests was determined by ABTS + - scavenging activity and Fe2+ chelating activity.

 

At present, there are many reports on the preparation of rice antioxidant peptides by optimizing the hydrolysis conditions of a single protease or by selecting a single enzyme from several proteases with antioxidant activity [9-15], but there are fewer studies on the preparation of antioxidant peptides by hydrolysis of rice proteins in steps using a combination of enzymes. Chanput et al. hydrolyzed rice bran protein with a combination of pepsin and trypsin, and found that the in vitro antioxidant activity of the enzyme hydrolysate was significantly higher than that of rice bran protein [16]. In this paper, after hydrolyzing rice protein with alkaline protease (Alcalase), trypsin, Protamex and Neutrase were added to hydrolyze the rice bran protein, and the peptide concentration, Fe2+ chelating activity, and ABTS + - removing activity of the hydrolysate and the enzyme were determined according to the degree of hydrolysis (DH) and the enzyme, and the better type of enzyme and enzyme were determined. According to the Degree of Hydrolysis (DH), peptide concentration, Fe2+ chelating activity and ABTS+ -clearing activity of the hydrolysate, it was possible to determine the better type of compound enzyme and enzyme hydrolysis conditions, in order to provide technical support for the large-scale production of antioxidant peptides prepared by the enzymatic method of rice protein, as well as to provide a new way of thinking about the rational use of heavy metal contaminated rice.

 

2 Materials and Methods

2.1 Materials and equipment

Rice protein (protein content 88.5%, moisture content 4.07%, ash content 2.6%; dry basis) 5 %, moisture content 4.07 %, ash content 2.6 %; dry basis) from JINNONG BIOTECHNOLOGY CO., LTD; alkaline protease, neutral protease, trypsin, and complex protease from NOVISON (CHINA) BIOTECHNOLOGY CO. BSA (bovine serum albumin), chloroform (CHCl 3), potassium persulfate (K 2S 20 8 ), ferrous chloride (FeCl 2 ), hydrochloric acid (HCl, excellent purity) were purchased from Sinopharm Chemical Reagent Co.

WFJ 720 Visible Spectrophotometer Unocal (Shanghai) Instrument Co., Ltd; JK-MSH-Pro-40WL Electric Mixer Shanghai Jingxue Scientific Instrument Co. Shanghai Anting Scientific Instrument Factory; DELTA 320 pH meter Metrol Toledo Instruments (Shanghai) Co.

 

2.2 Methodology

2.2.1 Enzymatic conditions

(1) Hydrolysis by alkaline protease

Referring to the method of Cai Guangxia et al [ 17]. The DH, peptide concentration, Fe2+ chelating activity and ABTS+ - scavenging activity of rice protein at a concentration of 7.5% (w/v) were determined at pH 9, enzymatic temperature of 50 ℃ and alkaline protease to rice protein ratio (E/S) of 48 AU/Kg for 15~180 min (15, 30, 60, 90, 120 and 180 min).

(2) Complex enzymatic hydrolysis

Under the optimal conditions of alkaline protease digestion, the temperature and pH were adjusted according to the optimal conditions of the three proteases in Table 1, and the DH, peptide concentration, Fe2+ chelating ability and ABTS+ - scavenging activity were measured after 15~180 min of digestion (15, 30, 60, 90, 120, 180 min) with the addition of the enzyme (0.5, 2.5, 4.5, w/w), respectively.

(3) Sample processing

During the enzyme digestion, 5 mL of enzyme solution was pipetted into a 10 mL centrifuge tube, 3 drops of HCl were added to destroy the enzyme (1 mol/L), and centrifuged at 8000 r/min for 10 min, and the supernatant was placed in a refrigerator for 12 h. The supernatant was then stored in the refrigerator.

 

2.2.2 Determination of DH

DH was calculated using Eq. (1) with a slight modification of Jens' method [ 18-19].

DH(% ) = B - N b - 1/α- 1/Mp - 1/Htot × 100(1)

Style:

B-NaOH solution volume, mL;

N b - equivalent concentration of NaOH solution, mol/L; M p - content of rice protein, g; Htot - total number of peptide bonds in rice protein, mmol/L; α - dissociation degree of α-amino group. α-amino group dissociation degree was calculated with reference to equation (2). Calculation.

α = (10pH-pK )/( 1 + 10pH -pK ) (2)

Style:

pH - pH value of the hydrolyzed solution; pK - dissociation constant of the amino group, pK 7.0.

 

2.2.3 Measurement of peptide concentration

The peptide concentration was determined by the bis-urea method [20]. The standard curve was drawn with the concentration of bovine serum albumin (BSA) (mg/mL) as the horizontal coordinate and the absorbance (wavelength 540 nm) as the vertical coordinate, and the curve equation was y = 0.0221x + 0.0074 (R2 = 0.9974).  0221x + 0.0074 (R2 = 0.9974).

 

2.2.4 Fe2+ chelation capacity

The method of Taheri et al[8] was followed. To 0.5 mL of sample, 3.2 mL of distilled water and 0.1 mL of FeCl2 solution (2 mmol/L) were added. 1 mL of FeCl2 solution (2 mmol/L) was added to 0.5 mL of the sample, homogenized and allowed to stand for 3 min. Then add 0.2 mL of phenanthrozine solution (5 mmol/L), mix well and react for 10 min, and then measure the absorbance at 562 nm (with deionized water as the blank group). The measurement was repeated 3 times and the average value was taken. Equation (3) was used to calculate the Fe2+ chelation rate.  chelation rate.

Fe2+ chelation rate/% = (1 - A sample/A blank ) × 100 (3)

 

2.2.5 ABTS + - radical scavenging activity

ABTS + - reserve solution: take 10 mL of ABTS + - solution (7 mmol/L) and 5 mL of K2 S2 O8 solution (7.35 mmol/L) and mix them, and leave them at room temperature for 12~16 h, protected from light, and prepare for use. The absorbance at 734 nm was 0.70. The absorbance of ABTS + - working solution: dilute the ABTS + - reserve solution with anhydrous ethanol to 0.70 at 734 nm, then add 3 mL of ABTS + - working solution to the sample and mix well, and measure the absorbance after 10 min reaction at room temperature (A sample), and replace the sample with water in the blank group (A blank). The ABTS + - removal rate was calculated using equation (4).

ABTS + - clearance/% = ( 1 - A sample/A blank ) x 100 (4)

 

3 Results and analysis

3.1 Hydrolysis by alkaline proteases

The results of hydrolysis of rice protein by alkaline protease are shown in Fig. 1 . From Fig. 1-a, it can be seen that DH has been increasing as the enzymatic hydrolysis proceeded (6 . 3%, 12.6%, 15.5%). (6.3%, 12.6%, 15.5%). During the enzymatic digestion, the peptide concentration of the digest increased by 55.9% from 23.6 mg/mL (15 min) to 15.6 mg/mL (15 min). The peptide concentration in the digest increased by 55.9% from 23.6 mg/mL (15 min) to 36.8 mg/mL (180 min). 8 mg/mL (180 min). It is possible that the alkaline protease broke the peptide bonds of rice protein and hydrolyzed the insoluble rice protein into soluble peptides and free amino acids [19, 21].

 

As can be seen from Figure 1-b, the Fe2+ chelating activity and ABTS + - scavenging activity of the enzyme solution gradually increased with the enzyme digestion. 15 min, the Fe2+ chelating activity of the enzyme solution was 3.2%, and the Fe2+ chelating activity of the enzyme solution increased by 8.2% at 90 min (29.8%).  At 15 min, the Fe2+ chelating activity was 3.2%, and at 90 min (29.8%), it increased by 8.31 times. At 15 min, the Fe2+ chelating activity was 3.2%, which increased 8.31 times at 90 min (29.8%) and 16.4 times at 180 min (55.7%). The ABTS+ -removal activity of the enzyme solution was 44.0% at 15 min and 56.0% at 90 min (68.9%), while that of the enzymatic solution was 44.0% at 15 min and 16.0% at 180 min. The ABTS + - removal activity of the enzyme solution was 44.0% (15 min), 56.6% at 90 min (68.9%) and 16.4 times at 180 min (55.7%). 6% at 90 min (68.9%), and 74.4-fold at 180 min (76.9%). (76.9%), and 74.8% at 180 min (76.9%).

 

The increase in peptide concentration of the alkaline protease digest was lower than that of ABTS + - scavenging activity and Fe2+ chelating activity, indicating that the increase in peptide concentration was the main reason for the increase in antioxidant activity of the rice protein digests, and the increase in ABTS + - scavenging activity and Fe2+ chelating activity might be caused by the type and amount of peptides generated [22-25]. In the early stage of digestion, the ABTS + - scavenging activity and Fe2+ chelating activity of rice protein digest increased with the increase of digestion time, which may be attributed to the hydrolysis of rice protein and the exposure of peptides or the production of amino acid side chains and sequences with antioxidant activity, thus showing significant ABTS + - scavenging activity; in the late stage of digestion, the increase of the ABTS + - scavenging activity increased slowly, which may be due to a decrease in the number of peptide bonds and the generation of peptides in rice protein [22-25]. The slower increase in ABTS+ -scavenging activity in the late stage of enzymatic hydrolysis could be attributed to the decrease in the number of peptide bonds in rice proteins and the slower increase in peptide content. In addition, further hydrolysis by alkaline protease affected the amino acid groups with antioxidant activity in the peptides.

 

Due to the low concentration of peptides and amino acids with Fe2+ chelating activity or the structure is not exposed, the Fe2+ chelating activity of the digested solution is low at the beginning of the digestion; with the increase of the digestion time, the Fe2+ chelating activity increases significantly, which may be due to the spatial structure and peptide bond of rice protein being destroyed, and the concentration of peptides and free amino acids (e.g. histidine) with Fe2+ chelating activity increasing or the side-chain amino acids containing COO- being exposed from the structure of rice protein. The increase in the concentration of peptides and free amino acids (e.g. histidine) with Fe2+ chelating activity, or the exposure of side-chain amino acids containing COO

During the hydrolysis process, the antioxidant activity of the hydrolysis solution increased all the time, but the trend of increase leveled off after 90 min. Considering the subsequent compound enzyme hydrolysis, 90 min was selected as the optimal enzyme hydrolysis time for alkaline protease.

 

3.2 Neutral protease hydrolysis

3.2.1 DH

From Fig. 2-a, it can be seen that DH increased rapidly with time in the early stage of enzymatic hydrolysis, and then increased steadily from 30 to 120 min, and then increased very little from 120 to 180 min, and remained almost unchanged. This may be due to the fact that neutral protease hydrolyzed the peptide bonds of rice protein, which increased the content of peptides and free amino acids in the digest solution and increased DH. At the early stage of enzymatic hydrolysis, the enzyme has more cleavage sites in rice protein, the hydrolysis speed is fast, the content of peptide and free amino acid increases rapidly, and the increase of DH is obvious; with the prolongation of time, the enzyme begins to inactivate and the number of cleavage sites in rice protein decreases, and the increase of DH is slowed down after 120 min [19, 24].

After the addition of neutral protease, although the DH increased throughout the digestion process, and the differences in the increases of the three enzyme additions were not significant (10.96%, 10.63%, and 10.90%, respectively), the DH was the smallest at 4 AU/mg for the same time. However, the lowest DH was observed at the same time when the enzyme addition was 4 AU/mg. In addition, the DH at 20 AU/mg was slightly larger than that at 36 AU/mg in the middle stage of digestion, but the difference was not significant in the late stage of digestion. It is possible that the enzyme contacted with sufficient rice protein peptide bonds at the lower enzyme dosage to increase the DH, but when the enzyme dosage was too high, part of the enzyme could not contact with the rice protein peptide bonds sufficiently, and thus the increase in DH was not significant. At the end of the enzyme digestion process, the DH was 13.87% to 14.26%, which was 10.08% to 13.17% higher than that without neutral protease.

 

3.2.2 Peptide concentration

From Fig. 2-b, it can be seen that the addition of alkaline protease hydrolysate to the

The peptide concentration increased to different degrees with different enzyme additions (4 AU/mg, 20 AU/mg, 36 AU/mg) after neutralization of the protease. The peptide concentration increased slowly in the early stage of digestion and stabilized after 120 min. At 4 AU/mg of neutral protein, the peptide concentration did not change significantly during the whole digestion process. When the enzyme addition rate was increased to 20 AU/mg, the peptide concentration at the beginning of the process was not much different from that at 4 AU/mg, and then the peptide concentration increased significantly in the middle and late stages. When the enzyme dosage was increased to 36 AU/mg, the peptide concentration increased significantly in the late stage, but it was always lower than that of the enzyme dosage of 4 or 20 AU/mg. The peptide concentration at the end of enzyme digestion was 28.8-30.2 mg/mg. The peptide concentration at the end of the digestion was 28.8-30.2 mg/mL, which was slightly lower than that of the group without neutral protease.

 

3.2.3 ABTS + - scavenging activity

As shown in Fig. 2-c, after the addition of neutral protease, the differences in ABTS + - removal activity of rice proteolytic digests with enzyme additions of 4 and 36 AU/mg were not significant and were significantly smaller than those of the 20 AU/mg group.  Clearance activity of rice protein digests with 4 AU/mg and 36 AU/mg of enzyme addition was not significant and was significantly smaller than that of the 20 AU/mg group. The ABTS + - scavenging activity of rice protein digest at 20 AU/mg increased from 69.3% (15 min) to 77.9% (18 min), an increase of 12.41%. The ABTS+-scavenging activity at the end of the digestion process ranged from 65.4% to 77.9%. The increase was -5.08% to 13.06% compared with that without neutral protease.

 

3.2.4 Fe2+ chelation activity

From Fig. 2-d, it can be seen that the Fe2+ chelating activity of rice protein digest was enhanced by the addition of neutral protease, but the magnitude of the enhancement varied significantly with different enzyme additions. The Fe2+ chelating activity increased from 20.6% (15 min) to 29.6% (15 min) with the addition of 20 AU/mg of enzyme. At 20 AU/mg, the Fe2+ chelating activity increased from 20.6% (15 min) to 29.5% (90 min), an increase of 43.20%, and the Fe2+ chelating activity was 30.0% at the end of the reaction. The Fe2+ chelating activity of the 20 AU/mg group increased significantly and gradually compared with that of the 4 or 36 AU/mg groups throughout the digestion process. The Fe2+ chelating activity at the end of the enzymatic process was 22.2-30.0%, which was slightly lower than that of the group without neutral protease.

 

3.3 Complex protease hydrolysis

3.3.1 DH

As shown in Figure 3-a, DH increased with the increase of reaction time after the addition of complex protease. At 7.5 AU/mg, the DH was always the smallest, but it was almost the same as the increase of DH at 37.5 AU/mg. When the enzyme dosage was increased to 67.5 AU/mg, the hydrolysis rate and the increase of DH were not much different from that of 7.5 AU/mg, and the DH at the end of the enzymatic hydrolysis was 14.48% (67.5 AU/mg) and 14.34% (37.5 AU/mg), respectively. The DH at the end of the enzymatic process was 13.17% to 14.48%, and the DH at the end of the enzymatic process was 14.34% (37.5 AU/mg). The DH at the end of the enzymatic process ranged from 13.17% to 14.48%, an increase of 4.5% to 14.9% compared with that without the addition of the complex protease.

 

3.3.2 Peptide concentration

As can be seen in Figure 3-b, the peptide concentrations of the three groups of enzyme additions slightly increased during the digestion process, but the differences in the increases were obvious. At 7.5 AU/mg, the peptide concentration remained unchanged with the increase of reaction time, and the increase was only 0.7% at the end of the reaction; at 37.5 AU/mg, the peptide concentration increased significantly at the early stage of the enzyme digestion, and then remained stable at the end of 30 min, with an increase of 3.3% at the end of the enzyme digestion; and the peptide concentration slightly increased at the early stage of the enzymatic process and then rapidly increased at 90-120 min after the addition of 37.5 AU/mg; the peptide concentration was slightly increased at the early stage of the enzymatic process and then rapidly increased at 90-120 min after the addition of 37.5 AU/mg. The peptide concentration increased slightly at the beginning of the enzymatic digestion, then increased rapidly from 90 to 120 min, and then increased slowly after 120 min, and finally increased by 6%. 180 min saw the highest peptide concentration of 28.4% (67.5 AU/mg) and 6.4% (67.5 AU/mg). 4% (67.5 AU/mg) at 180 min, but the peptide concentration increased rapidly from 90 to 120 min, and then slowly increased to 6% after 120 min.

Slightly lower compared to no added complex protease.

 

3.3.3 ABTS + - scavenging activity

From Figure 3-c, it can be seen that the ABTS + - removal activity was gradually enhanced after the addition of the complex protease, and the ABTS + - removal activity was obviously increased at 15-30 min, and the ABTS + - removal activity was enhanced to different degrees and fluctuated from 30 to 120 min, and the ABTS + - removal activity was gradually enhanced from 120 min to the end of the enzymatic digestion.

The increase in ABTS + - scavenging activity of rice proteolytic digests for the three groups of enzyme additions was 11 . 4% (7.5 AU/mg), 11.0% (37.5 AU/mg) and 8.0% (37.5 AU/mg). 0% (37.5 AU/mg) and 8.8% (67.5 AU/mg), respectively. 8% (67.5 AU/mg), respectively. Although there was not much difference between the increases at 7.5 AU/mg and 37.5 AU/mg, the ABTS +-removal activity of the 37.5 AU/mg group was stronger than that of the other two groups throughout the enzyme digestion process. The highest ABTS+ -removal activity of 75.5% (37.5 AU/mg) was observed at the end of the digestion process. The final increase was 9.58% compared to no complex protease.

 

3.3.4 Fe2+ chelation activity

From Fig. 3-d, it can be seen that the Fe2+ chelating activity of rice protein digest was enhanced to different degrees with the addition of complex protease to alkaline protease digest with the increase of reaction time, but the Fe2+ chelating activity and its enhancement varied significantly among the three groups of enzyme additions.

The increase in Fe2+ chelation activity for the three enzyme additions was 21 .  7% (7.5 AU/mg), 32.1% (37.5 AU/mg) and 18.5% (37.5 AU/mg), respectively. 1% (37.5 AU/mg) and 18.5% (67.5 AU/mg), respectively. 5% (67.5 AU/mg), in which the Fe2+ chelating activity was almost unchanged in the early stage of enzyme digestion at 7.5 AU/mg, and increased after 90 min, but was always lower than that of the other two enzyme additions. The Fe2+ chelating activity increased significantly when the enzyme addition rate was increased to 37.5 AU/mg, but remained almost unchanged after 120 min. When the enzyme dosage was increased to 67.5 AU/mg, the Fe2+ chelating activity did not change much from that of 37.5 AU/mg and was stable. The Fe2+ chelating activity was the strongest at the end of the enzyme digestion process, which was 42.3%, with an increase of 41.9%. 95%.

 

The peptide concentration increased slowly at 15 min after the addition of the enzyme, indicating that more oligopeptides and amino acids were produced, and the ABTS + - scavenging activity remained stable at the beginning of the enzyme digestion, and then increased slightly, indicating that oligopeptides and amino acids produced by the digested peptides enhanced the activity. The Fe2+ chelating activity of the composite protein digests with 37.5 AU/mg and 67.5 AU/mg of enzyme addition increased, which may be due to the increase in the content of newly produced oligopeptides and amino acids. The increase in Fe2+ chelating activity of the enzyme solution slowed down as the enzyme digestion progressed, probably due to the further hydrolysis of peptides into oligopeptides and free amino acids.

 

3.4 Trypsin hydrolysis

3.4.1 DH

As shown in Figure 4-a, after adding trypsin, the DH of the three enzyme groups increased with the increase of time, and the rate of increase was basically the same. When the hydrolysis time was 15-30 min, the DH increased faster, and then the growth rate slowed down. At the same hydrolysis time, DH increased significantly with the increase of enzyme addition, and at the end of the hydrolysis, DH was at the lowest level of 16.69% (6.9%). At the end of the enzymatic hydrolysis, the lowest DH was 16.69% (6.25 usp/mg) and the highest was 16.69% (6.25 usp/mg).  At the end of the enzymatic hydrolysis, DH was the lowest at 16.69% (6.25 usp/mg) and the highest at 18.65% (56.6%). The lowest DH was 16.69% (6.25 usp/mg) and the highest was 18.65% (56.25 usp/mg). 25 usp/mg) at the end of enzymatic hydrolysis, which was higher than that of neutral protease and complex protease with the same amount of enzyme addition.

 

3.4.2 Peptide concentration

As can be seen in Figure 4-b, the peptide concentration increased to a certain extent after the addition of trypsin, and the peptide concentration increased slowly from 30 to 180 min. The peptide concentration increased slowly from 30 to 180 min. At 6.25 usp/mg of trypsin, the peptide concentration increased significantly from 15 to 30 min, and then remained almost unchanged; at 31.25 usp/mg, the peptide concentration increased from 15 to 30 min, and then remained almost unchanged. 25 usp/mg, the peptide concentration increased rapidly from 15 to 30 min, and the peptide concentration was higher than that in the 6.25 usp/mg group after 30 min. The peptide concentration after 30 min was higher than that in the 6.25 usp/mg group. When the enzyme dosage was increased to 56.25 usp/mg, the increase was lower than that of the 6.25 usp/mg and 31.25 usp/mg groups. When the enzyme addition was increased to 56.25 usp/mg, the increase was lower than that of the 6.25 usp/mg and 31.25 usp/mg groups. The increase in peptide concentration was lower than that of the 6.25 usp/mg and 31.25 usp/mg groups, but the peptide concentration was always the highest among the three enzyme addition groups. The peptide concentration at the end of the digestion process ranged from 30.9 to 33.6 mg/mL, which was higher than that of the neutral protease and complex protease with the same enzyme dosage.

 

3.4.3 ABTS + - scavenging activity

From Fig. 4-c, it can be seen that the ABTS + - removal activity was enhanced under the conditions of different trypsin enzyme additions, and the difference of the removal activity of different enzyme additions was not significant, so it can be seen that the enzyme additions did not have much effect on the ABTS + - removal activity. 15 ~90 min, the ABTS + - removal activity was slightly enhanced, and the growth of ABTS + - removal activity slowed down and fluctuated from 90 ~180 min. At 56.25 usp/mg, the ABTS + - scavenging activity was slightly enhanced. At 56.25 usp/mg, the ABTS + - removal activity was always lower than that of the other two groups, and the ABTS + - removal activity of the group with 31.25 usp/mg at the beginning of the enzymatic digestion was lower than that of the other two groups. The ABTS + - removal activity of the 31.25 usp/mg group was slightly lower than that of the 56.25 usp/mg group at the beginning of the enzyme digestion. The ABTS+ -removal activity of the group with 31.25 usp/mg of enzyme was slightly lower than that of the group with 56.25 usp/mg of enzyme at the beginning of the enzyme digestion, and the opposite was true at the end. At the end of the digestion, the highest ABTS + - removal activity was 81.6%, which was the same as that of the group without trypsin addition. At the end of the digestion, the highest ABTS + - removal activity was 81.6%, which was 18.4% higher than that of the trypsin-naïve ABTS + - removal activity. The highest ABTS + - removal activity was 81.6% at the end of the digestion, which was 18.43% higher than that of ABTS + - removal activity without trypsin and higher than that of neutral protease and complex protease with the same enzyme addition.

 

3.4.4 Fe2+ chelation activity

From Fig. 4-d, it can be seen that the Fe2+ chelating activity increased gradually with the increase of reaction time after the addition of trypsin. The increase of Fe2+ chelating activity slowed down and fluctuated from 60 to 180 min. The increase of Fe2+ chelating activity was slowed down and fluctuated at 60~180 min. At 6.25 usp/mg, the Fe2+ chelating activity increased significantly, especially at 15-60 min, and then decreased. Although the increase of Fe2+ chelating activity at 6.25 usp/mg At the end of the reaction, the increase of 6.25 usp/mg was 36.7%, which was significantly larger than that of the other two groups.  7% at the end of the reaction, which was significantly larger than that of the other two groups (26.7% at 31.25 usp/mg). 25 usp/mg 时 26 . 6% at 31 . 25 usp/mg , 56 . 25 usp/mg), the Fe2+ chelating activity was always lower than that of the other two groups. The Fe2+ chelating activity was always lower than that of the other two groups. When the enzyme dosage was increased to 31.25 usp/mg, the Fe2+ chelating activity increased continuously, and the Fe2+ chelating activity of the enzyme solution was the strongest in all stages of the reaction, although the increase in Fe2+ chelating activity was not the largest compared with that of the other two enzyme dosages. The Fe2+ chelating activity of the enzyme solution was slightly lower than that of the 31.25 usp/mg group, although it increased slowly when the enzyme dosage continued to increase. 25 usp/mg until the end of the reaction. At the end of the enzyme digestion, the Fe2+ chelating activity was 82.0%~93.3%, which increased by 1.75~2.75%. 75~2.13 times, higher than that of the same enzyme addition. 13 times higher than that of neutral protease and complex protease with the same enzyme addition.

 

The DH and peptide concentrations of rice protein hydrolyzed by trypsin were higher than those of alkaline protease alone, probably because alkaline protease hydrolyzes rice protein at the carboxyl-terminal end of the peptide bond formed by hydrophobic amino acids, and hydrophobic amino acids are mainly found in the inner part of proteins, which utilize hydrophobic interactions to keep the tertiary structure of proteins stable [26]. The alkaline protease hydrolysate is then hydrolyzed with trypsin to further hydrolyze the peptide bond composed of lysine and arginine. Since the binding sites of neutral protease are mainly the carboxyl groups of hydrophobic amino acids such as tryptophan, phenylalanine, and alanine, and the sites of complex protease are mainly the hydrophobic amino acids at the end of the peptide chain, the two proteases are similar to the alkaline protease, and with the prolongation of hydrolysis by alkaline protease, the peptide bonds of the rice proteins were gradually consumed, and the enzyme digestion sites were reduced, so the hydrolysis of rice proteins with neutral protease and complex protease was not possible. Therefore, the increase in DH and peptide concentration was not obvious when hydrolyzed by neutral protease and complex protease, resulting in higher DH and peptide concentration in the digests of trypsin complex hydrolysis than neutral protease and complex protease.

 

The Fe2+ chelating activities of the digests of trypsin complex hydrolysis were higher than those of neutral protease and complex protease, probably because the higher concentration of oligopeptides and free amino acids in the digests contained more coordination atoms, which could chelate more Fe2+. In addition, the digests of trypsin complex hydrolysis contain more aspartic acid, glutamic acid, histidine, cysteine, etc., and the high Fe2+ chelating activity may be related to these specific amino acids [27, 28]. The ABTS+ scavenging activity of trypsin complex hydrolysis was significantly higher than that of neutral protease and complex protease, which may be due to the higher concentration of oligopeptides and free amino acids in the digest, which can release more antioxidant groups, and these groups reacted with ABTS+ - to make the reaction system discoloration, i.e., the ABTS+ - scavenging activity was stronger [24].

 

After combining the results of the above experiments, it was determined that the best conditions for the enzymatic hydrolysis of rice proteins to produce highly active antioxidant peptides were as follows: 7.5% (w/v) of rice protein, hydrolyzed by alkaline protease at pH 9, 50 ℃, with an enzyme addition rate of 48 AU/Kg, for 90 min; hydrolyzed by trypsin at pH 8.5, 37 ℃, with an enzyme addition rate of 31.25 usp/mg, for 30 min. The pH value of trypsin hydrolysis was 8.5, 37℃, and the enzyme addition rate was 31.25 usp/mg for 30 min.

 

4 Conclusion

Among the three proteases, trypsin, complex protease and neutral protease, the hydrolysis solution of rice protein hydrolyzed by a combination of trypsin and alkaline protease had the strongest antioxidant activity, and the Fe2+ chelating activity and ABTS+ -removal activity were significantly higher than those of the hydrolysis solution hydrolyzed by alkaline protease alone.

The better conditions for the complex enzymatic reaction were: 7.5% (w/v) rice protein, alkaline protease hydrolysis at pH 9, 50°C, enzyme addition 48 AU/Kg, 90 min; trypsin hydrolysis at pH 8.5, 37°C, enzyme addition 31.5 usp/mg, 30 min; trypsin hydrolysis at pH 8.5, 37°C, enzyme addition 31.25 usp/mg, 30 min; and alkaline protein hydrolysis at pH 9, 50°C, 48 AU/Kg. The pH value of trypsin hydrolysis was 8.5, 37℃, enzyme addition was 31.25 usp/mg, 30 min. The rice protein digests prepared under these conditions had high ABTS+ -scavenging activity and Fe2+ chelating activity, which can be used to guide the large-scale production of antioxidant peptides enzymatically prepared from rice protein.

 

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