Modification of Soybean Protein Hydrolysates by Alcalase-Catalyzed Plastein Reaction and the ACE-Inhibitory Activity of the Modified Product In Vitro

Figures & data

Table 1 Range of values for the response surface methodology

	Levels
Independent variables	−1.682	−1	0	1	1.682
E/S ratio (U/g peptides)	147.73	250.00	400.00	550.00	652.27
Substrate concentration (%)	19.82	28.00	40.00	52.00	60.18
Reaction time (h)	0.64	2.00	4.00	6.00	7.36

Figure 1 ACE-inhibitory activity in vitro and degree of hydrolysis of soybean protein hydrolysates prepared with different hydrolysis times. The concentration of soybean protein hydrolysates for ACE-inhibitory activity assay was 1 mg/mL on a peptide basis. The bars were for the ACE-inhibitory activity of the hydrolysates and the solid line was for degree of hydrolysis. Different capital letters A to E (or lowercase letters a to g) above the columns (or below the line) indicate that one-way ANOVA of means obtained are significantly different (P < 0.05).

Table 2 ANOVA response for linear, quadratic, and interactive effect of variables used in the model. ^a

Model term	p-Value ^b
X 1	0.0566
X 2	<0.0001
X 3	0.0026
X 1 X 2	0.7764
X 1 X 3	0.6375
X 2 X 3	0.5887
X 1 ^2	0.8680
X 2 ^2	<0.0001
X 3 ^2	0.0014
^a X 1, X 2, and X 3 represent E/S ratio, substrate concentration and reaction time, respectively.
^bCoefficients with p-values greater than 0.05 indicate that they are not significant.

Figure 2 Response surface graphs for the decreased amount of free amino groups of the modified products of plastein reaction as a function of: (a) E/S ratio and substrate concentration (reaction time at the central of its level), (b) substrate concentration and reaction time (E/S ratio at the central of its level), and (c) E/S ratio and reaction time (substrate concentration at the central of its level). (Color figure available online.)

Table 3 The ACE-inhibitory activities of soybean protein hydrolysates (SPH) and eight modified products (MP) of plastein reaction in vitro

Sample	Reaction time (h)	Decreased amount of free amino groups (μmol/g peptides)	IC50 ^a (mg/mL)
SPH	—	—	1.45
MP 1	1	53.50	1.04
MP 2	2	80.25	0.90
MP 3	3	84.70	0.74
MP 4	4	84.70	0.70
MP 5	5	75.79	0.64
MP 6	6	57.96	0.76
MP 7	8	46.81	0.92
MP 8	10	44.58	1.11
^aThe values represent the solution concentration of the samples needed to inhibit ACE by 50% under assaying conditions.

Figure 3 Analysis of the standards (a), soybean protein hydrolysates (SPH) and three modified products (MP3, MP5, and MP8) (b) by size exclusion chromatography in an AKTA Explorer 100 equipped with a Superdex-75 column. The analysis was performed at a flow rate of 0.5 mL/min with 0.1 mol/L Na₂HPO₄–0.1 mol/L NaOH buffer (pH 12) and monitored at 280 nm. The DH of SPH was 16.6% and the decrease amount of free amino groups of MP3, MP5, and MP8 were 84.70, 75.79, and 44.58 μmol/g peptides, respectively. The standards were bovine serum albumin (66.2 kDa), cytochrome c (12.4 kDa), insulin (5.7 kDa), oxidized L-glutathione (0.6 kDa), and L-tyrosine (0.2 kDa), and appeared as peaks A to E in Fig. 3a, respectively. (Color figure available online.)