Isolation and identification of the angiotensin-I converting enzyme (ACE) inhibitory peptides derived from cottonseed protein: optimization of hydrolysis conditions

Figures & data

Table 1. Coded and uncoded settings of the process parameters for cottonseed protein hydrolysis, according to a central composite design

	Coded factor levels
Process parameter	−1.68(-α)	−1	0	+1	+1.68(+α)
A: Temperature (°C)	28.24	33	40	47	51.76
B: E/S (% w/w)	0.696	0.9	1.2	1.5	1.704
C: pH	5.66	6.5	7.0	7.5	8.34

Table 2. Central composite design of the independent variables and experimental values of DH (Y₁) and ACE inhibitory activity (Y₂)

	Coded levels of variables			Response variables
Run no	A	B	C	DH (%)	ACEI (%)
1	−1	−1	−1	17.06 ± 0.32	48.32 ± 0.81
2	1	−1	−1	17.45 ± 0.24	48.13 ± 0.74
3	−1	1	−1	32.65 ± 0.31	79.14 ± 1.01
4	1	1	−1	37.73 ± 0.29	82.09 ± 0.63
5	−1	−1	1	18.31 ± 0.17	52.42 ± 0.44
6	1	−1	1	18.50 ± 0.04	63.88 ± 0.62
7	−1	1	1	20.47 ± 0.43	69.45 ± 0.13
8	1	1	1	28.21 ± 0.35	83.34 ± 0.81
9	−1.68	0	0	15.11 ± 0.19	47.81 ± 0.43
10	1.68	0	0	22.45 ± 0.42	70.41 ± 0.11
11	0	−1.68	0	15.78 ± 0.26	38.04 ± 0.73
12	0	1.68	0	37.23 ± 0.31	82.14 ± 0.65
13	0	0	−1.68	35.20 ± 0.28	81.15 ± 0.34
14	0	0	1.68	25.59 ± 0.52	84.76 ± 0.47
15	0	0	0	23.84 ± 0.13	83.68 ± 0.53
16	0	0	0	21.32 ± 0.48	81.99 ± 0.22
17	0	0	0	22.06 ± 0.72	82.12 ± 1.12
18	0	0	0	22.03 ± 0.66	80.07 ± 0.88
19	0	0	0	21.97 ± 0.47	81.04 ± 0.74
20	0	0	0	20.83 ± 0.52	82.01 ± 0.39

Figure 1. Effects of hydrolysis time (a), temperature (b), pH (c) and E/S ratio (d) on the DH and ACE inhibitory activity of hydrolysate

Table 3. Analysis of variance for DH (Y₁) and ACE inhibitory activity (Y₂)

	Sum of Squares		F Value		Prob > F
Source	Y1	Y2	Y1	Y2	Y1	Y2
Model	909.61	4483.62	60.53	113.51	< 0.0001**	< 0.0001**
X1-Temperature	48.53	320.11	29.06	72.94	0.0003**	< 0.0001**
X2-E/S	514.38	2253.68	308.04	513.5	< 0.0001**	< 0.0001**
X3-pH	92.6	22.38	55.46	5.1	< 0.0001**	0.0475*
X1 X2	18.73	3.88	11.21	0.88	0.0074**	0.3694
X1 X3	0.76	63.79	0.45	14.53	0.5162	0.0034*
X2 X3	72	100.04	43.12	22.79	< 0.0001**	0.0008**
X1^2	31.74	961.6	19.01	219.1	0.0014*	< 0.0001**
X2^2	22.41	881.75	13.42	200.91	0.0044	< 0.0001**
X3^2	99.1	0.99	59.35	0.23	< 0.0001**	0.6453
Residual	16.7	43.89
Lack of Fit	11.48	36.6	2.2	5.02	0.2039	0.0505
Pure Error	5.22	7.29
Cor Total	926.31	4527.51
Std. Dev.	1.29	2.09
R-Squared	0.9820	0.9903
Adj R-Squared	0.9657	0.9816

* represents significant difference (P < 0.05), **represents extremely significant difference (P < 0.01)

Figure 2. Response surface three-dimensional plots of the effects of (a, d) temperature and E/S ratio; temperature and pH (b, e); pH and E/S ratio on the DH and ACE inhibitory activity of CPH (c, f)

Table 4. ACE inhibitory activity of FII fraction by semi-preparing RP-HPLC

Sample	Reaction time (min)	ACE inhibitory activity (%)
FII-1	15	39.33 ± 0.99 ^h
FII-2	18	91.37 ± 0.21 ^a
FII-3	23	71.97 ± 0.51 ^c
FII-4	25	61.20 ± 0.26 ^e
FII-5	27	73.33 ± 0.68 ^b
FII-6	31	59.20 ± 0.95 ^f
FII-7	32	48.27 ± 0.97 ^g
FII-8	37	70.37 ± 0.67 ^d
FII-9	51	31.80 ± 0.61 ^i
Captopril	–	98.67 ± 0.64

ACE inhibitory activities were tested at 1.0 mg/mL. Every experiment was carried out in triplicate. Data were expressed as means ± standard errors. Means with different letters (a – i) were significantly different (P < 0.05)

Figure 3. Chromatogram of FII separated by semi-preparing RP-HPLC (a) and of ACE inhibitory activity peptide (FII-2) by analytical RP-HPLC

Figure 4. Mass spectrum and analysis of the amino acid sequence of FII-2-p by MALDI-TOF-TOF