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

Abstract

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC₅₀ value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC₅₀ = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.

Keywords:

• Casein hydrolysate
• Plastein reaction
• Alcalase
• Proline
• ACE-inhibitory activity
• Solvent fractionation

INTRODUCTION

Extensive scientific evidence has been provided for the existence of biologically active peptides derived from food proteins that have beneficial effects on human health. Among these bioactive peptides, angiotensin converting enzyme (ACE)−inhibitory peptides are receiving special attention as hypertension becomes more prevalent and important in many countries.[1,2] ACE−inhibitory peptides have been found in many food protein sources,[3,4] among which milk proteins are a particularly good source of bioactive peptides.[5] By in vitro digestion, casein hydrolysates with ACE−inhibitory activity were obtained from casein preparations[6,7] or individual components of casein,[8 –10] showing that casein was a good source of ACE−inhibitory peptides. Pripp et al.[11] studied the structure–activity relationship of ACE−inhibitory peptides derived from milk proteins and concluded that increased hydrophobicity of the amino acid and absence of positive charge in the C−terminal position of the peptides enhanced the activity. Proline, tryptophane, tyrosine, and phenylalanine were found to be the most effective at the ultimate C-terminal position.[12] Higher ACE−inhibitory activities of the peptides, such as IPP, VPP,[13] LPYPP,[14] VYPFPGPIPNSLPQNIPP, and LVYPFPGPIPNSLPQNIPP,[15] indicated the importance of proline in these peptides.

The plastein reaction is a classic enzyme reaction and has been used for many purposes. For example, Yamashita et al.[16] found that, when the mixture of soybean protein hydrolysate and estered L−methionine was incubated with papain, the methionine was incorporated into the peptides. The plastein reaction was also used to remove the bitterness of protein hydrolysates,[17] reduce the content of some amino acids (e.g., phenylalanine) for dietetic applications,[18] or modify functional properties of the proteins (e.g., solubility).[19] In recent years, the plastein reaction has been used to modify some protein hydrolysates to improve their antioxidant,[20] ACE−inhibitory,[21 –23] or radical scavenging[24] activities in vitro.

Although ACE-inhibitory peptides are generated from enzymatic hydrolysis of protein substrates, further hydrolysis might cause damage to structures and activities. Some peptides were found to be susceptible to enzymatic digestion.[25,26] Whether a protein hydrolysate modified by the plastein reaction is also sensitive to proteases is unknown. In other reported works, organic solvents were used to enhance immunological activity of egg yolk extract[27] or ACE-inhibitory activity of a fermented soybean food extract.[28] The two results showed the potential of an organic solvent to separate protein hydrolysates with higher activity. Based on these mentioned facts, a casein hydrolysate was prepared by alcalase-catalyzed hydrolysis of casein and modified by alcalase-catalyzed plastein reaction in the presence of extrinsic proline in the present work. The modified hydrolysate was then fractionated by organic solvents or digested by some proteases. The ACE-inhibitory activities in vitro of the resulted hydrolysates were evaluated and compared. The objective of the present study was to investigate the impacts of the plastein reaction and solvent fractionation on the activity and enzymatic resistance of the prepared casein hydrolysate.

MATERIALS AND METHODS

Materials and Chemicals

Rabbit lung acetone powder, caseinate, trypsin (40 kU/g), and N-(3-[2-furyl]acryloyl)-L-phenylalanylglycylglycine (FAPGG) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Alcalase (100 kU/mL) was purchased from Novozymes Biotechnology Co., Ltd. (Tianjin, China). Pepsin (30 kU/g) was purchased from Hui Shi Biochem. Reagent Co. (Shanghai, China). Papain (28 kU/g) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other reagents used were of analytical grade. Highly purified water prepared with Milli-Q PLUS (Millipore Corporation, New York, NY, USA) was used for the preparation of all buffers and solutions.

Preparation and Modification of Casein Hydrolysate

Casein hydrolysate was prepared with the conditions of Zhao and Li[21] but with an alcalase addition of 1 kU/g proteins. Caseinate solution of 10% (w/v) was hydrolyzed for 1–8 h, heated at 90°C for 15 min, cooled to room temperature, and centrifuged at 3000 × g for 15 min. The supernatant (casein hydrolysate) was collected, lyophilized, stored at −20°C, and evaluated for its degree of hydrolysis and ACE-inhibitory activity in vitro. The hydrolysate with the maximum activity was bulk prepared, lyophilized, and used as the substrate of the plastein reaction.

Optimal plastein reaction conditions were accomplished by employing the response surface methodology with a central composite design. An experimental design consisting of 20 runs and three independent variables, including alcalase addition (i.e., E/S ratio, kU/g peptides), reaction temperature (°C), and substrate concentration (%, w/v) at five levels, was applied. The detailed design is listed in Table 1. The lyophilized hydrolysate was dissolved in water (containing different amounts of alcalase to give different E/S ratios) to give the selected substrate concentration. Then, some proline was added to give a fixed level of 0.4 mol/mol free amino groups (on the basis of the hydrolysate). The mixture was kept at the designed temperature for 6 h with continuous stirring, heated at 90°C for 15 min, cooled to room temperature, and analyzed for the content of free amino groups. The decrease of free amino groups of the modified hydrolysate was calculated by subtracting the content of free amino groups of the modified hydrolysate from that of the original hydrolysate, expressed as μmol/g peptides and taken as the dependent variable (i.e., the response) to select suitable conditions.

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

With the selected conditions, four modified hydrolysates were prepared by applying reaction time of 1, 3, 6, and 9 h, respectively, and evaluated for ACE-inhibitory activities and content of free amino groups. The modified hydrolysate with the maximum activity was bulk prepared, lyophilized, and subjected to solvent fractionation and further enzymatic hydrolysis.

Solvent Fractionation of Casein Hydrolysate or Modified Hydrolysate

The modified hydrolysate with the maximum activity or the original hydrolysate was mixed well with either of the ethanol- or methanol-water solvents (in volume proportions of 3:7, 5:5, or 7:3) at room temperature to give a ratio of 1 g peptides/mL. The mixture was centrifuged at 9000× g for 30 min to obtain supernatant and precipitate fractions (the fractionated hydrolysates). The fractionated hydrolysates were lyophilized, reconstituted in water, and analyzed for peptide recovery, content of free amino groups, and ACE-inhibitory activities. Pure solvent water was also used to fractionate the modified hydrolysate with the same conditions, and the obtained two fractions were subjected to the same assaying as above.

Enzymatic Hydrolysis of the Modified or Fractionated Casein Hydrolysate

The modified hydrolysate of the maximum activity and its two fractionated hydrolysates by ethanol-water of 7:3 (v/v) were reconstituted in water to a peptide content of 10% (w/v). A portion of the prepared solutions was adjusted to either of the selected pHs by adding a few drops of 2 mol/L NaOH or HCl solution. The further hydrolysis of these hydrolysates was triggered by adding papain (2 kU/g peptides, pH 6.5, 45°C), alcalase (1 kU/g peptides, pH 8.0, 55°C), trypsin (2 kU/g peptides, pH 8.0, 37°C), or pepsin (4 kU/g peptides, pH 2, 37°C), and lasted for 10 or 30 min, respectively. For the blank sample (zero time), the protease added was preheated at 90°C for 15 min. After the hydrolysis, all 24 samples were heated at 90°C for 15 min and assayed for the content of free amino groups and the ACE-inhibitory activities.

Chemical Analyses or Activity Assays

Protease activity was assayed with the protocol described by Sarath et al.[29] Protein or peptide content of all samples was determined by multiplying the nitrogen content from the Kjeldahl procedure[30] by a factor of 6.38. The content of free amino groups of the hydrolysate was assayed by the method of Church et al.[31] and used to calculate the degree of hydrolysis (DH) with the method suggested by Adler-Nissen.[32] ACE-inhibitory activity in vitro of all samples at different concentrations was assayed by the method of Murray et al.[33] with FAPGG as the substrate, while the extract of rabbit lung acetone powder was used as the ACE source. IC₅₀ value of the selected hydrolysate was estimated as the reported method of Shalaby et al.[34]

Statistical Analysis

All trials were carried out in triplicate. All data were expressed as means and standard deviations. Differences between the means of multiple groups were analyzed by one-way analysis of variance (ANOVA) with Duncan's multiple range tests. Design Expert 7.0 software (Stat-Ease Inc., Minneapolis, MN, USA) was used in the central composite design. Microsoft Excel 2003 software (Microsoft Corporation, Redmond, WA, USA) and SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) were used to analyze or report the data.

RESULTS AND DISCUSSION

Preparation of Casein Hydrolysate

ACE-inhibitory activities of eight casein hydrolysates prepared over a hydrolysis period of 8 h were evaluated at a final peptide content of 50 μg/mL and are shown in Fig. 1, together with the hydrolysis extent (DH) of the hydrolysates. As the hydrolysis progressed from 0 to 6 h, the DH and activity of the prepared hydrolysate was enhanced from 0 and 5.8% to 10.9 and 48.3%, respectively. Hydrolysis time longer than 6 h led to the hydrolysate little increased DH but lower activity. The hydrolysate with a DH of 10.9% had the maximum activity (IC₅₀ = 52.6 μg/mL) and was selected as the substrate of the plastein reaction.

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.)

When rapeseed proteins were hydrolyzed by alcalase for 30 min, the hydrolysate showed higher ACE-inhibitory activity.[35] Wu and Ding[36] had prepared a soybean protein hydrolysate by alcalase with an IC₅₀ value of 65 μg/mL. The casein hydrolysate prepared in the present study showed an increasing trend in the activity when hydrolysis time increased from 0 to 6 h; after that, a decreasing trend in the activity was observed. This result shared similarity to the findings of Mao et al.,[37] who hydrolyzed yak (Bos grunniens) casein by alcalase.

Modification of Casein Hydrolysate in the Presence of Extrinsic Proline

Response surface methodology was applied to select the optimal plastein reaction conditions. Figure 2 shows the practical results and the effects of substrate concentrate, reaction temperature, and alcalase addition (i.e., E/S ratio) on the decrease of free amino groups of the modified hydrolysate (i.e., the response). A detailed discussion for Fig. 2 is not given here. Besides the fixed proline addition (0.4 mol/mol free amino groups) optimized from previous work (not discussed here), the suitable conditions were optimized as follows: substrate concentration of 50% (w/v), E/S ratio of 3.1 kU/g peptides, and reaction temperature of 25°C. With these conditions, the predicted response was 117.0 μmol/g peptides but the actual one was 112.1 μmol/g peptides. Plastein reaction usually takes place at a higher substrate concentration,[38,39] and lower reaction temperature is beneficial as the reaction is exothermic.[17] The substrate concentration and reaction temperature selected in the present study were consistent to the mentioned conclusions.

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.)

Four modified hydrolysates were prepared with a reaction time of 1, 3, 6, or 9 h, respectively. The evaluation results (Table 2) show that the four modified hydrolysates were different in reaction extent (i.e., contents of free amino groups), and had higher activities than the original hydrolysates (45.8–76.4% versus 27.8%), particularly for MCH6 (IC₅₀ = 13.0 μg/mL). This result indicates that the alcalase-catalyzed plastein reaction in the presence of extrinsic proline was capable of improving ACE inhibition of the casein hydrolysate. Therefore, MCH6 with the maximum activity was subjected to solvent fractionation and further hydrolysis in vitro.

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.

Influence of Solvent Fractionation on ACE-Inhibitory Activity

Three ethanol-water (E-W) or methanol-water (M-W) solvents were applied to fractionate the prepared MCH6 and obtain corresponding fractionated products (supernatant or precipitate fractions). The data in Table 3 show that the solvent fractionation led to the two fractions different in the content of free amino groups and activities. Fractionation solvents with higher ethanol or methanol content (i.e., 7:3 E-W or M-E, v/v) had lower polarity and extracted less soluble fractions from the MCH6. The supernatant fractions obtained had higher activity than the precipitate fractions (81.2% versus 69.4% or 79.3% versus 68.8%). When less ethanol or methanol was added into water, the applied solvents (i.e., 3:7 E-W or M-E, v/v) had higher polarity and extracted more soluble fractions. The supernatant fractions obtained had slightly higher activity than the precipitate fractions (76.8% versus 74.6% or 76.8% versus 75.4%). Fractionation of the original hydrolysate by the two solvents of lower polarity (7:3 E-W or M-W, v/v) also led to the supernatant (or precipitate) fractions higher (or lower) activity. If pure solvent water of the highest polarity was used to fractionate the MCH6, the result was opposite to the result mentioned above. The supernatant fractions of the MCH6 by water exhibited lower activity than the precipitate fractions. These results indicate that an organic solvent-water-based system with suitable polarity might be applicable to separate physically the modified or original hydrolysate with higher activity, and ethanol was more effective than methanol.

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%.

ACE-inhibitory peptides are usually rich in Pro, Leu, Ile, and aromatic amino acids,[12,40,41] such as PLPLL and VPP from milk proteins[37,42] and DLP from soybean proteins.[43] Zhang et al.[44] used resins to separate the peptides with a higher content of hydrophobic amino acids, which led to the peptides’ higher ACE-inhibitory activity. Ethanol or methanol has lower polarity than water. In the present work, the applied fractionation solvents with lower polarity than water could extract the peptides with lower polarity (i.e., more hydrophobic amino acids), or precipitate the peptides with higher polarity (i.e., more hydrophilic amino acids) from the MCH6. Thus, the soluble fractions obtained had higher activity while the precipitate factions had lower activity. The formed peptides with lower polarity during plastein reaction were insoluble in water (a classic result of plastein reaction); therefore, the precipitate fractions of MCH6 by water exhibited logically higher activity.

Influence of Further Enzymatic Hydrolysis on ACE-Inhibitory Activity

The sensitivity of the MCH6 and its fractionated products (FMCH6) to enzymatic hydrolysis was examined in vitro. After hydrolysis of the MCH6 or the FMCH6 by one of the selected four proteases, the activities and the hydrolysis extent of the resulted 24 digests were evaluated and are given in Table 4. Each resulting digest had higher content of free amino groups than the corresponding substrate, showing that the MCH6 and the FMCH6 were hydrolyzed. The residual activities of the digests were from 22.0 to 54.0%, less than that of the MCH6 (76.4%) or the FMCH6 (81.2% for supernatant, 69.4% for precipitates), and were clearly related to the protease and hydrolysis time applied. The result indicates that further hydrolysis of the hydrolysates led to the activities impaired. Among the 24 digests, 2 were from the FMCH6 hydrolyzed by alcalase for 30 min. Their residual activities were 22.0 and 24.7%, less than the original hydrolysate (27.8%). Other digests had residual activities from 33.8 to 54.0%, higher than the original hydrolysate, indicating that the alcalase-catalyzed plastein reaction might be capable of enhancing enzymatic resistance of casein hydrolysate in ACE inhibition.

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).

Among the four proteases studied, alcalase caused the strongest damage to the activities of the MCH6 and the FMCH6, as the resulted digests by alcalase had the lowest activities. It is difficult to distinguish the different influences of other proteases. In comparison with the FMCH6, the MCH6 was more resistant to enzymatic hydrolysis, as the resulted digests from it had higher residual activities. This fact reveals that solvent fractionation could not improve the enzymatic resistance of the modified hydrolysate, although such treatment might enhance the activity.

Gómez-Ruiz et al.[25] found that the ACE-inhibitory activities of two peptides isolated from Manchego cheese were decreased after enzymatic digestion. Three peptides, YAEERYPIL, RADHPFL,[45] and LVYPFPGPIPNSLPQNIPP,[26] showed susceptibility to pepsin or pancreatic extract with decreased activities. In the work of Tavares et al.,[46] two peptides, DKVGINY and KGYGGVSL, also exhibited decreased activities upon enzymatic digestion. The mentioned results supported the present result that enzymatic hydrolysis of the modified or fractionated casein hydrolysate had a negative effect on the activity. Alcalase has higher specificity mainly for hydrophobic amino acids,[47] thus inducing more hydrophobic amino acids released from the MCH6 or the FMCH6 (together with higher hydrolysis extent, as shown in Table 4); consequently, the obtained digests had lower activities.

Resistance of ACE-inhibitory peptides to physiological digestion is an important and interesting scientific issue. The present study shows that the plastein reaction could give casein hydrolysate an improved ACE-inhibition and better enzymatic resistance. More works are needed to reveal the details about peptide composition, relationship between residual ACE-inhibition, and protease or hydrolysis profiles.

CONCLUSION

In vitro ACE-inhibitory activity of a casein hydrolysate prepared by alcalase was enhanced from 27.8 to 76.4% by applying an alcalase-catalyzed plastein reaction in the presence of proline addition, leading to the IC₅₀ value decreasing from 52.6 to 13.0 μg/mL. Solvent fractionation of the modified hydrolysate by ethanol- or methanol-water solvent with a ratio of 7:3 (v/v) led to the soluble (or precipitate) fractions higher (lower) activity. The modified or fractionated hydrolysate showed sensitivity in ACE inhibition to the enzymatic hydrolysis by four proteases, but 22 out of 24 resulted digests had residual activities higher than the original casein hydrolysate. It was the plastein reaction that conferred casein hydrolysate enzymatic resistance in ACE inhibition.

ACKNOWLEDGMENTS

This work was funded by the Innovative Research Team of Higher Education of Heilongjiang Province (Project No. 2010td11) and the National Natural Science Foundation of China (Project Nos. 30972132 and 31140009). The authors wish to thank the anonymous reviewers and the editors for their valuable work and suggestions to this article.