Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Figures & data

Table 1  Reaction conditions and their levels studied by the response surface methodology

	Levels
Independent variables	−α	−1	0	+1	+α
E/S ratio (kU/g peptides)	1.32	2	3	4	4.68
Reaction temperature (°C)	13.2	20	30	40	46.8
Substrate concentration (w/v, %)	23.2	30	40	50	56.8

Figure 1 ACE-inhibitory activity and degree of hydrolysis (DH) of casein hydrolysate prepared over a hydrolysis period of 8 h. The column chart is for ACE-inhibitory activity and the graph chart is for DH. The final peptide concentration used in the assay system was fixed at 50 μg/mL. Different capital letters above the columns (or below the graph) indicate that one-way ANOVA of the data is significantly different (P < 0.01). (Color figure available online.)

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

Table 2  Content of free amino groups and ACE-inhibitory activities of four modified casein hydrolysates with different plastein reaction times

Hydrolysate ^a	Reaction time (h)	CFAG ^b (μmol/g peptides)	ACEI ^c (%)	IC50 ^d (μg/mL)
CH	0	1780.9 ± 6.9	27.8 ± 1.8 A	52.6
MCH1	1	1700.7 ± 7.3	50.3 ± 2.0 C	NA
MCH3	3	1642.6 ± 6.7	50.2 ± 2.3 C	NA
MCH6	6	1593.3 ± 6.3	76.4 ± 1.8 D	13.0
MCH9	9	1627.8 ± 6.1	45.8 ± 1.6 B	NA
^aCH: casein hydrolysate; MCH: modified casein hydrolysate.
^bContent of free amino groups.
^cACEI: ACE-inhibitory activity. The final peptide concentration used in the assay system was 25 μg/mL. Different capital letters after the values in the same column indicate that one-way ANOVA of the data (expressed as means ± standard deviations) is significantly different (P < 0.05).
^dNA: Not assayed.

Table 3  Effects of ethanol- and methanol-water solvent fractionation on ACE-inhibitory activity (ACEI) of a modified casein hydrolysate (MCH) and casein hydrolysate (CH) ^a

Hydrolysate	Solvent ^b	Fraction	Peptide recovery (%)	ACEI ^c (%)
MCH6 ^d	3:7 E-W	Supernatant	64.4 ± 0.8	76.8 ± 0.4
		Precipitate	33.6 ± 0.7	74.6 ± 0.7
	5:5 E-W	Supernatant	54.3 ± 0.7	77.6 ± 0.9
		Precipitate	44.1 ± 0.5	73.7 ± 0.9
	7:3 E-W	Supernatant	44.6 ± 0.8	81.2 ± 0.8
		Precipitate	55.1 ± 0.7	69.4 ± 1.0
	3:7 M-W	Supernatant	65.4 ± 0.5	76.8 ± 0.7
		Precipitate	34.0 ± 0.6	75.4 ± 0.5
	5:5 M-W	Supernatant	51.5 ± 0.6	78.2 ± 0.8
		Precipitate	48.4 ± 0.7	74.5 ± 0.6
	7:3 M-W	Supernatant	45.4 ± 0.5	79.3 ± 0.7
		Precipitate	54.6 ± 0.6	68.8 ± 0.6
	W	Supernatant	72.6 ± 0.7	64.6 ± 1.2
		Precipitate	24.0 ± 0.6	79.5 ± 0.9
CH	7:3 E-W	Supernatant	78.2 ± 0.5	34.0 ± 2.3
		Precipitate	17.6 ± 0.4	16.7 ± 1.7
	7:3 M-W	Supernatant	80.4 ± 0.4	33.3 ± 2.3
		Precipitate	17.5 ± 0.3	17.4 ± 1.4
^aAll analysis results are expressed as means ± standard deviations.
^bE: ethanol; M: methanol; W: water. The given ratios are the volume ratios of ethanol or methanol to water.
^cACE-inhibitory activity was assayed at a final peptide concentration of 25 μg/mL.
^dMCH6, modified casein hydrolysate with a reaction time of 6 h. The ACEI of the MCH6 or the CH at 25 μg/mL was 76.4% or 27.8%.

Table 4  Effect of further hydrolysis in vitro on ACE-inhibitory activities of a modified casein hydrolysate or its two fractionated products

Hydrolysate ^a	Protease ^b	Hydrolysis time (min)	Increase in free amino groups ^c (μmol/g)	ACEI ^d (%)
MCH6	Alcalase	10	61.8 ± 5.8	44.2 ± 2.1
		30	125.9 ± 8.3	35.6 ± 2.0
	Papain	10	59.8 ± 4.8	54.0 ± 2.1
		30	80.0 ± 7.1	42.0 ± 1.9
	Trypsin	10	38.5 ± 5.6	48.1 ± 1.7
		30	47.8 ± 5.6	42.6 ± 2.4
	Pepsin	10	52.0 ± 6.4	35.4 ± 2.0
		30	79.2 ± 7.4	33.8 ± 2.0
FMCH6 (7:3 E-W, supernatant fractions)	Alcalase	10	61.3 ± 6.0	33.9 ± 1.5
		30	130.3 ± 5.3	22.0 ± 1.8
	Papain	10	38.5 ± 5.6	42.8 ± 1.8
		30	88.5 ± 5.7	32.3 ± 1.8
	Trypsin	10	35.3 ± 5.6	46.0 ± 1.9
		30	46.8 ± 5.1	39.0 ± 1.9
	Pepsin	10	30.8 ± 5.4	46.3 ± 1.5
		30	56.0 ± 5.3	38.6 ± 1.7
FMCH6 (7:3 E-W, precipitate fractions)	Alcalase	10	44.6 ± 4.8	34.8 ± 2.1
		30	116.1 ± 5.9	24.7 ± 1.9
	Papain	10	20.5 ± 4.7	47.4 ± 2.2
		30	33.2 ± 4.4	42.2 ± 1.7
	Trypsin	10	44.1 ± 4.9	47.6 ± 2.4
		30	80.0 ± 4.6	39.8 ± 1.9
	Pepsin	10	28.6 ± 6.4	43.6 ± 2.0
		30	87.9 ± 6.6	37.9 ± 2.2
^aMCH6, the modified casein hydrolysate with reaction time of 6 h; FMCH, fractionated product of MCH6.
^bAlcalase, trypsin, papain or pepsin addition were 1, 2, 2 or 4 kU/g peptides, respectively.
^cThe data equal to subtracting the content of free amino groups of the MCH 6 or FMCH 6 after hydrolysis from that before hydrolysis, and are expressed as means ± standard deviations.
^dACE-inhibitory activity is expressed as mean ± standard deviation. The final peptide concentration used in the assay was 25 μg/mL. ACE-inhibitory activities of MCH6 and casein hydrolysate at 25 μg/mL were 76.4% and 27.8%, while that of two fractionated products of MCH 6 (FMCH6) were 81.2% (supernatant) and 69.4% (precipitate).