Bioactive peptides with ACE-I and antioxidant activity produced from milk proteolysis

Figures & data

Table 1. Characteristics of proteolytic enzymes used to hydrolyze skim milk.

Enzyme	EC No.	Type	pH	Units/mg protein	Specify	Origin
Protease	232.642.4	Neutral	7.5	3.5	Non-specific	Asperigillus oryzae
Pepsin	3.4.23.1	Acid	2	0.8	X-aromatic AA	Porcine gastric mucosa
Trypsin	3.4.21.4	Serine	7–8	≥ 250	Arg-X; Lys-X	Bovine pancrease
Papain	3.4.22.2	Cysteine	5–5.5	0.1	X-Arg; X-Lys	Papaya (Carica papaya)

Figure 1. Changes in proteolysis of skim milk hydrolyzed by proteolytic enzymes.

Figure 2. ACE-I activity of skim milk after hydrolysis with different proteases.

Table 2. DPPH radical scavenging activity of skim milk during hydrolysis with different proteases.

	Hydrolysis time (min)
	30			60
	Enzyme concentration (%)
Enzyme	0.001	0.005	0.01	0.001	0.005	0.01
Protease	63.37 ± 1.21^p	64.99 ± 0.97°	67.23 ± 1.09^m	68.90 ± 1.10^kl	79.14 ± 1.13^d	83.98 ± 0.92^a
Trypsin	61.27 ± 1.38^r	62.94 ± 1.42^q	64.99 ± 1.29°	69.20 ± 1.28^k	70.02 ± 1.22^j	75.42 ± 0.91^f
Pepsin	55.31 ± 1.41^s	67.04 ± 1.29^m	68.72 ± 1.35^l	66.29 ± 1.18^n	70.76 ± 1.15^i	73.43 ± 0.81^g
Papain	71.88 ± 0.67^h	77.74 ± 0.82^e	79.10 ± 0.71^d	78.03 ± 0.69^e	79.70 ± 0.91^c	80.91 ± 0.79^b

LSD= 0.3941

Table 3. The reducing power of skim milk during hydrolysis with different proteases.

	Hydrolysis time (min)
	30			60
	Enzyme concentration (%)
Enzyme	0.001	0.005	0.01	0.001	0.005	0.01
Protease	0.104 ± 0.01^ij	0.110 ± 0.012^hij	0.127 ± 0.015^fg	0.156 ± 0.001^de	0.169 ± 0.01^cd	0.186 ± 0.012^ab
Trypsin	0.095 ± 0.011^j	0.123 ± 0.012^gh	0.183 ± 0.015^abc	0.120 ± 0.012^gh	0.166 ± 0.013^cd	0.197 ± 0.01^a
Pepsin	0.114 ± 0.01^ghi	0.114 ± 0.01^ghi	0.119 ± 0.03^ghi	0.168 ± 0.013^cd	0.176 ± 0.011^bc	0.193 ± 0.001^a
Papain	0.097 ± 0.003^j	0.110 ± 0.01^hij	0.152 ± 0.025^e	0.145 ± 0.019^e	0.168 ± 0.013^cd	0.183 ± 0.02^abc

LSD = 0.0157

Table 4. The Fe²⁺ chelating activity of skim milk during hydrolysis with different proteases.

	Hydrolysis time (min)
	30			60
	Enzyme concentration (%)
Enzyme	0.001	0.005	0.01	0.001	0.005	0.01
Protease	64.62 ± 1.22^l	69.62 ± 1.51^k	75.02 ± 1.21^h	69.62 ± 1.01^k	75.29 ± 1.31^gh	75.29 ± 1.10^gh
Trypsin	60.96 ± 2.11^n	62.12 ± 2.32^m	62.25 ± 2.03^m	73.11 ± 1.25^j	75.56 ± 1.76^gh	75.90 ± 2.31^g
Pepsin	69.97 ± 1.65^k	70.17 ± 1.91^k	76.86 ± 1.87^f	72.42 ± 1.43^j	73.86 ± 1.87^i	82.05 ± 2.01^e
Papain	81.84 ± 1.41^e	86.00 ± 1.35^e	89.00 ± 0.98^b	84.16 ± 0.89^d	86.55 ± 1.20^c	89.89 ± 1.01^a

LSD = 0.7276

Table 5. Technological properties of skim milk^a hydrolyzed by proteolytic enzymes.

Enzyme	Concentration (%)	30 min				60 min
		pH	T.A.^b (%)	COB^c	Flavor	pH	T.A. (%)	COB	Flavor
Protease	0.001	6.57	0.23	+	Normal	6.59	0.23	+	Normal
	0.005	6.57	0.24	+	Bitter	6.55	0.25	+	Bitter
	0.01	6.48	0.26	+	Bitter	6.47	0.28	+	Bitter
Trypsin	0.001	6.61	0.19	-	Normal	6.57	0.23	-	Normal
	0.005	6.58	0.23	+	Normal	6.56	0.24	+	Normal
	0.01	6.49	0.25	+	Normal	6.48	0.26	+	Bitter
Pepsin	0.001	6.61	0.19	-	Normal	6.61	0.20	-	Normal
	0.005	6.59	0.23	+	Normal	6.57	0.23	+	Normal
	0.01	6.58	0.23	+	Normal	6.58	0.24	+	Bitter
Papain	0.001	6.62	0.18	-	Normal	6.61	0.20	-	Normal
	0.005	6.61	0.19	-	Normal	6.57	0.21	-	Normal
	0.01	6.61	0.19	-	Normal	6.48	0.21	-	Normal

^a non-hydrolyzed milk: pH = 6.64; T.A.= 0.17%.

^bT.A.: titratable acidity.

^c COB: clot-on-boiling test; (+): precipitate; (-): non-precipitate.

No change in milk color.