European Food Research and Technology

, Volume 229, Issue 5, pp 795–805

An approach to improve ACE-inhibitory activity of casein hydrolysates with plastein reaction catalyzed by Alcalase

Authors

    • Key Laboratory of Dairy Science, Ministry of EducationNortheast Agricultural University
  • Ya-Yun Li
    • Key Laboratory of Dairy Science, Ministry of EducationNortheast Agricultural University
Original Paper

DOI: 10.1007/s00217-009-1110-4

Cite this article as:
Zhao, X. & Li, Y. Eur Food Res Technol (2009) 229: 795. doi:10.1007/s00217-009-1110-4

Abstract

The preparation method of casein hydrolysates with high ACE-inhibitory activity was studied by Alcalase-catalyzed hydrolysis coupled with plastein reaction. Casein hydrolysates with an IC50 value of about 47 μg mL−1 were first prepared by hydrolysis of casein with Alcalase and then modified with plastein reaction catalyzed by the same enzyme. The impacts of four reaction conditions on plastein reaction of casein hydrolysates were studied, and then optimal conditions were determined using response surface methodology with the decrease of free amino groups in the reaction mixture as response. When the concentration of casein hydrolysates was fixed at 35% by weight, the maximum decrease of free amino groups in the reaction mixture of 181.8 μmol g−1 proteins was obtained. The optimum conditions for the above decrease were found to be an E/S ratio of 7.7 kU g−1 proteins, reaction temperature of 42.7 °C and reaction time of 6 h. Analysis results showed that ACE-inhibitory activity of casein hydrolysates prepared could be improved significantly by plastein reaction. When casein hydrolysates were modified by plastein reaction, with a decrease of free amino groups in the mixture of about 154.7 μmol g−1 proteins and 181.8 μmol g−1 proteins, their IC50 values could be decreased to 0.6 and 0.5 μg mL−1.

Keywords

CaseinHydrolysisACE-inhibitory activityPlastein reactionAlcalase

Introduction

Hypertension, estimated to affect one-third of the Western population, is a risk factor for cardiovascular disease and stroke [16]. The currently used synthetic drugs for the treatment of hypertension, e.g., captopril and enalapril, have certain side effects such as coughing, skin discomfort and, in particular, excessively low blood pressure [8]. Natural ACE inhibitors such as bioactive peptides, as alternatives to synthetic drugs, have attracted attention of both food and medical scientists. ACE-inhibitory peptides have been found in many food protein sources [14, 15, 20, 29], and milk proteins are considered to be particularly good sources of bioactive peptides [16]. Enzymatic hydrolysis now has become widely used in biotechnological process to improve the functional properties of milk proteins or to obtain milk protein hydrolysates with some bioactivities [10, 13, 22, 30]. Recently, increasing attention is focused on milk protein [6, 21, 25]. To obtain peptides with high ACE-inhibitory activity, lot of work has been done using enzymatic hydrolysis in vitro.

Peptides with high ACE-inhibitory activity can be prepared through proper selection of the proteins and enzymes that give rise to a high yield of the bioactive peptides. In a screening study hydrolyzing nine milk protein preparations with five bacterial and digestive enzymes, it was found that thermolysin was a very good enzyme for the release of ACE-inhibitory peptides from four casein substrates and five whey protein substrates. The IC50 values for thermolysin hydrolysates of caseins and whey proteins were in the range of 90–400 μg mL−1 and 45–83 μg mL−1, respectively, with purified α-lactalbumin giving the highest inhibitory activity (IC50 = 45 μg mL−1) [26]. In another study hydrolyzing yak milk casein with six commercial proteases, it was found that the hydrolysates obtained by Neutrase from bacillus amyloliquefaciens showed the highest ACE-inhibitory activity (IC50 = 0.38 mg mL−1) [12]. It was also a good approach to obtain peptides with high ACE-inhibitory activity by separating and purifying these peptides from protein hydrolysates [18, 27, 31]. Two ACE-inhibitory peptides IPP and VPP, with highest antihypertensive activity in human and IC50 values of 5 and 9 μmol L−1, respectively, had been obtained from the commercial fermented milk products or milk protein hydrolysates [8, 24]. Another method to improve ACE-inhibitory activity of milk protein hydrolysates was to optimize hydrolysis conditions [9]. If ACE-inhibitory peptides were obtained just by hydrolyzing protein substrates, their amino acid sequences would be coincident with the primary structure of the proteins substrates they were derived from. It was shown that when β-casein was hydrolyzed by thermolysin, two potent ACE-inhibitory peptides isolated were identified as f58-76 and f59-76 of β-casein A2 [27]. Generally, it is impossible to obtain ACE-inhibitory peptides that have different amino acid sequences from protein substrates when protein substrates are enzymatically hydrolyzed.

Although the plastein reaction was first described almost 100 years ago, it remains at the stage of an intellectual curiosity and there is continuing argument about the precise reaction mechanism. There were three different mechanisms in early thoughts, which were classified as condensation [40], transpeptidation [5] and physical forces [1]. But recent work suggested that several pathways might play a role in the plastein reaction simultaneously [34]. Plastein reaction had been used for removing the bitterness of protein hydrolysates [35], enhancing the nutritional value of proteins deficient in essential amino acids (e.g., methionine, tryptophane), reducing the content of some amino acids (e.g., phenylalanine) for dietetic applications [2, 41] and modifying the functional properties of proteins [1, 37, 38]. It is not yet possible to predict the products of plastein reaction except in general terms. However, if condensation and transpeptidation do exist in plastein reaction of protein hydrolysates, some new peptides that have different amino acid sequences form raw proteins might be generated, which might lead to the modification in their ACE-inhibitory activity. There exists a need to reveal whether plastein reaction might have influence on the ACE-inhibitory activity of protein hydrolysates.

The objective of the present work was to show the improvement of plastein reaction catalyzed by Alcalase on ACE-inhibitory activity of casein hydrolysates. Casein hydrolysates with a relatively high ACE-inhibitory activity was prepared by the enzymatic hydrolysis of Alcalase, an alkaline protease from Bacillus subtilis, and then used as substrate of plastein reaction catalyzed by the same enzyme. The response surface methodology was applied to optimize the conditions of plastein reaction, including enzyme to substrate (E/S) ratio, reaction temperature and time. ACE-inhibitory activity of casein hydrolysates and five modified casein hydrolysates were analyzed with the spectrophotometric method. The results showed that plastein reaction might be an effective approach to improve ACE-inhibitory activity of casein hydrolysates.

Materials and methods

Materials and chemicals

Casein, N-(3-[2-furyl]acryloyl)-l-phenylalanylglycylglycine (FAPGG) and rabbit lung acetone powder were purchased from Sigma-Aldrich Co.(St. Louis, USA). Alcalase with activity of 130,000 U/g was purchased from Pangbo Biochem. Inc. (Nanning, China). Captopril (purity >99.0%) was purchased from Fluka. Other reagents used were reagent-grade chemicals. Highly purified water prepared with Milli-Q PLUS (Millipore Corporation) was used for the preparation of all buffers and solutions.

Preparation of casein hydrolysates

Casein solution (10% w/w on protein basis) was made by dispersion of approximately 11.1 g of casein powder in 100 g water and kept at 4 °C overnight for rehydration. Casein solution was heated in a water bath at 90 °C for 20 min and cooled to hydrolysis temperature, 55 °C. The pH of the solution was adjusted to 8.5 by adding a few drops of 1 mol L−1 NaOH. The Alcalase solution (0.38 g mL−1 water) was prepared immediately prior to use. After withdrawal of a 5 mL sample (zero-time sample), the hydrolysis process was started by the addition of 2 mL of Alcalase solution to the remaining casein solution (giving approximately 10,000 units (U) g−1 proteins) and mixing well. The reaction mixture was kept at 55 °C, and 5 mL samples were withdrawn after 2, 4, 6, 8, 10 and 12 h of hydrolysis, respectively. All samples were heated to 90 °C for 15 min to inactivate the enzyme. After cooling to room temperature, all samples were centrifuged at 11,150×g for 10 min, and the supernatant was used for further analysis as described below.

Determination of the degree of hydrolysis

The OPA (o-pthaldialdehyde) method [4, 33] with some modifications was used to determine the free amino groups in the reaction mixture and to calculate the degree of hydrolysis (DH) of casein hydrolysates. The OPA reagent was prepared by combining the following reagents along with water to a final volume of 100 mL: 75 mL 200 mmol L−1 sodium borate buffer (pH 9.5), 5 mL 400 g SDS L−1, 80 mg OPA (in 1 mL methanol) and 400 μL β-mercaptoethanol. The reagent was prepared daily and protected from light. The OPA assay was carried out by the addition of 3 mL casein hydrolysates (or standard) to 3 mL of the OPA reagent. The absorbance of this solution was measured at 340 nm with a UV spectrophotometer (UV-2401PC, Shimadzu, Japan) and taken after 5 min. l-leucine (12–36 μg mL−1) was used as standard. Nitrogen content of casein hydrolysates was determined by the Kjeldahl procedure and multiplied by 6.38 to give the protein contents [11].

DH of casein hydrolysates was calculated as % DH = (h/htot) × 100, where h was the number of broken peptide bonds per unit weight, and htot was the total number of bonds per unit weight, which equal to 8.2 meq g−1 proteins.

Determination of ACE-inhibitory activity

ACE activity was measured by a spectrophotometric method described in literature [23] with FAPGG as substrate and extract of rabbit lung acetone powder as the ACE source. The reaction mixture contained 100 μL of casein hydrolysates dissolved in deionized water in a concentration range from 0 to 20 mg mL−1, 500 μL of 1.6 mmol L−1 FAPGG in 100 mmol L−1 sodium borate buffer (pH 8.3) with 300 mmol L−1 NaCl, and 300 μL 10× diluted rabbit lung acetone extract in 100 mmol L−1 sodium borate buffer (pH 8.3) containing 5% (v/v) glycerol. ACE extract was added to initiate the reaction. The reaction was terminated by the addition of 100 μL of 100 mmol L−1 EDTA after 30 min incubation at 37 °C. The EDTA was added immediately before ACE extract in zero-time control assays. The decrease in absorbance at 340 nm was determined in triplicate over a 30 min incubation period and was taken as a measure of the ACE activity. A control sample containing 100 μL of deionized water instead of casein hydrolysates was assayed in quadruplicate. The ACE inhibition (%) was calculated as [1 − (ΔAinhibitorAcontrol)] × 100%, where ΔAinhibitor and ΔAcontrol were the decrease in absorbance at 340 nm of the sample with casein hydrolysates and of the control sample, respectively.

The concentration of selected casein hydrolysates needed to inhibit the ACE by 50% (IC50) under these conditions was determined by assaying variously diluted casein hydrolysates and plotting the ACE inhibition percentage as a function of peptide concentration [32]. Captopril, a synthetic ACE inhibitor, was used as a positive control with IC50 equal to 5.2 nmol L−1.

Casein hydrolysates modified by plastein reaction

On the basis of the analysis results above, casein hydrolysates with the highest ACE-inhibitory activity was prepared, lyophilized and stored at −20 °C. Later, it was used as the substrate for plastein reaction.

Different amounts of lyophilized casein hydrolysates were mixed with 5.0 mL H2O (containing different amounts of Alcalase to give a fixed E/S ratio of 7.5 kU g−1 proteins) to give a final concentration of 25, 30, 35, 40 and 45% by weight. All mixtures were kept at 35 °C for 5 h, heated to 90 °C and kept for 15 min to inactivate the enzyme. All mixtures were cooled to room temperature and diluted properly for analyzing free amino groups in the mixture. The decrease of free amino groups in the mixture was expressed as μmol –NH2 g−1 proteins and used to show the extent of plastein reaction.

A similar approach was used to study the influence of E/S ratio (by changing the concentration of Alcalase), reaction temperature (by keeping the mixture at different temperature) and reaction time (by choosing different reaction time) on the decrease of free amino groups in the mixture by single factor trial.

Optimal design of plastein reaction

Optimal conditions of plastein reaction were accomplished by employing the response surface methodology (RSM) with a central composite design (CCD). In the experimental design E/S ratio (kU g−1 proteins) (X1), reaction temperature (X2) and reaction time (X3) were chosen as independent variables. An experimental design consisting of 20 runs and three independent variables at five different levels was applied in our study. The detailed design is listed in Tables 1 and 2, and all experiments were carried out in triplicate. The decrease of free amino groups in the reaction mixture was taken as the dependent variable or response (Y). The second-order polynomial coefficients were calculated and analyzed using the Design Expert software (Version 7.0). Second-degree polynomials, Eq. 1, which includes all interaction terms, were used to predict the response:
$$ y = \beta_{0} + \Upsigma_{i = 1}^{3} \beta_{i} x_{i} + \Upsigma_{i = 1}^{3} \beta_{ii} x_{i}^{2} + \Upsigma_{i = 1}^{2} \Upsigma_{j = i + 1}^{3} \beta_{ij} x_{i} x_{j} $$
(1)
where y was the dependent variable (the decrease of free amino groups in the mixture); β0, βi, βii and βij were coefficients estimated by the model, and xi, xj were the levels of the independent variables. They represent the linear, quadratic and cross-product effects of the X1, X2 and X3 factors on the response, respectively. The fitted polynomial equation was then expressed in the form of three-dimensional surface plots to visualize the relationship between the response and experimental levels of each of the variables and to deduce the optimal reaction conditions. The combination of different optimized variables, which yielded the maximum response, was determined in an attempt to verify the validity of the model. Subsequently, an additional confirmation experiment was conducted to verify the validity of the statistical experimental strategies.
Table 1

Range of values for the response surface methodology

Independent variables

Levels

−α

−1

0

+1

E/S ratio (kU g−1 proteins)

5

6

7.5

9

10

Reaction temperature ( °C)

10

18

30

42

50

Reaction time (h)

0.5

2.3

5

7.7

9.5

Table 2

Experimental design used in RSM studies by using three independent variables with six center points showing observed decrease of free amino groups in the mixture (Y)

Run order

Actual levels of independent variables

Y

X1

X2

X3

1

7.5

30

5.0

167.19

2

9.0

18

7.7

133.75

3

9.0

18

2.3

129.57

4

5.0

30

5.0

125.39

5

7.5

30

5.0

163.01

6

6.0

42

2.3

133.75

7

7.5

50

5.0

163.01

8

6.0

18

2.3

112.85

9

6.0

18

7.7

129.57

10

7.5

30

5.0

162.80

11

7.5

30

5.0

165.10

12

6.0

42

7.7

154.65

13

10.0

30

5.0

133.75

14

7.5

30

5.0

167.19

15

7.5

10

5.0

129.57

16

7.5

30

5.0

166.35

17

7.5

30

9.5

154.65

18

7.5

30

0.5

133.75

19

9.0

42

7.7

150.47

20

9.0

42

2.3

152.56

Results

Preparation of casein hydrolysates

Alcalase was selected in our study to prepare casein hydrolysates. It was found that the intact casein preparations also had ability to inhibit ACE by 6% at a concentration of 50 μg mL−1. The ACE-inhibitory activities of casein hydrolysates obtained over a 12 h period of hydrolysis were evaluated at concentration of 50 μg mL−1 and is shown in Fig. 1. As hydrolysis progressed during the first 6 h, the DH of casein hydrolysates increased from 0 to 11.2% and ACE-inhibitory activity of casein hydrolysates increased from 5.9 to 51.5%. Thereafter, the DH changed a little and ACE-inhibitory activity of casein hydrolysates showed a trend to decrease. ACE-inhibitory activity of casein hydrolysates with DH of 11.2% appeared the highest (IC50 value 47 μg mL−1), which made us select this product as the substrate for plastein reaction. It could also be seen from Fig. 1 that the ACE-inhibitory activity of casein hydrolysates prepared correlated with its DH.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1110-4/MediaObjects/217_2009_1110_Fig1_HTML.gif
Fig. 1

Angiotensin-converting enzyme (ACE) inhibitory activity (%, mean) and DH of casein hydrolysate obtained at different hydrolysis times. The final concentration of casein or casein hydrolysates for ACE-inhibitory activity assay was 50 μg mL−1on the basis of protein. Column chart is for ACE-inhibitory activity and graph chart for DH

Influence of concentration of casein hydrolysates, E/S ratio, reaction temperature and time on the plastein reaction of casein hydrolysates

The influences of the concentration of casein hydrolysates on the extent of plastein reaction were determined at 35 °C, E/S ratio of 7.5 kU g−1 proteins and reaction time of 5 h, and are shown in Fig. 2a. The increase in substrates’ concentration resulted in much decrease of the free amino groups in the mixture. Unfortunately, it was found that the application here of plastein reaction to casein hydrolysates with a concentration of more than 40% by weight would lead to modified products that took the physical form of thixotropic gels. The efficiency of the enzyme would be hindered for the reaction mixture would be too viscous. If the concentration of casein hydrolysates was too low, such as 25% by weight, increase of total free amino groups in the mixture (about 33.2 μmol g−1 proteins) would occur, which suggested further hydrolysis of casein hydrolysates into smaller peptides indeed. Considering all the above factors, the concentration of casein hydrolysates of 35% by weight was a suitable selection for plastein reaction in a later study.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1110-4/MediaObjects/217_2009_1110_Fig2_HTML.gif
Fig. 2

Effect of concentration of casein hydrolysate (a), temperature (b), E/S ratio (c) and time (d) on plastein reaction

As shown in Fig. 2b, the more Alcalase was added to the reaction mixture, the higher was the decease in the free amino groups of the reaction mixture. Considering the cost of reaction, the center point for E/S ratio was selected at 7.5 kU g−1 proteins, with a step change of 1.5 kU g−1 proteins in optimal design (as shown in Table 1).

When samples of casein hydrolysates (35% by weight) were incubated with Alcalase at temperature from 10 to 50 °C for 5 h at E/S ratio of 7.5 kU g−1 proteins, the reaction results are shown in Fig. 2c. It could be seen that the higher the reaction temperature, the higher was the decrease of free amino groups in the reaction mixture. The highest decrease of free amino groups in mixture, 163.0 μmol g−1 proteins, appeared at the highest reaction temperature (50 °C). Based on the consideration of the heat stability of Alcalase, 30 °C was chosen as the center point with 12 °C as a step change to determine optimal reaction temperature of Alcalase in the optimal design (as shown in Table 1).

There was a rapid, progressive decrease of free amino groups in the reaction mixture as plastein reaction progressed from the beginning to 6 h. When the plastein reaction progressed for 6 h, the decrease of free amino groups in the mixture was up to 150.5 μmol g−1 proteins. Thereafter, the variation in the extent of free amino groups in the mixture declined clearly (as shown in Fig. 2d). Therefore, reaction time of 5 h was chosen as the center point, with 2.7 h as a step change in optimal design (Table 1).

Optimization of plastein reaction for casein hydrolysates

Model fitting

The experimental design and results of central composite design are presented in Table 2. Response results were analyzed using Design Expert 7.0 and are shown in Tables 3 and 4. The probability p value was very low (p < 0.0001), indicating the significance of the model. The ANOVA also showed that there was a non-significant (p > 0.05) lack of fit, which further validated the model. The value of the determination coefficient (R2 = 0.9896) indicated a high correlation between the experimentally observed and predicted values, showing the degree of precision with which the treatments were compared. The value of adjusted determination coefficient (RAdj2 = 0.9803) was also high enough to advocate for a high significance of the model. Finally, the lower value of coefficient of variation (CV = 1.66%) indicated that the experiments were precise and reliable. The Design Expert 7.0 was then used to find out the quadratic mathematical model. After eliminating the non-significant parameters (p > 0.1) from the model, we obtained the final model given as Eq. 2.
$$ Y = - 333.972 + 95.247X_{1} + 4.147X_{2} + 21.104X_{3} - 1.097X_{1} X_{3} - 5.736 \, X_{1}^{2} - 0.049 \, X_{2}^{2} - 1.061X_{3}^{2} $$
(2)
Table 3

ANOVA response for linear, quadratic and interactive effect of variables used in the model

Model term

Coefficient estimate

p Value

Intercept

−333.972

 

X1

95.247

0.0003

X2

4.147

<0.0001

X3

21.104

<0.0001

X1 × X1

−5.736

<0.0001

X2 × X2

−0.049

<0.0001

X3 × X3

−1.061

<0.0001

X1 × X2

−0.044a

0.3829

X1 × X3

−1.097

0.0004

X2 × X3

−0.008a

0.7672

aCoefficients with p values greater than 0.10 indicate that they are not significant

Table 4

ANOVA for response surface quadratic model

Source

Sum of squares

Degree of freedom

Mean squares

F value

p Value

 

Model

5620.82

9

624.54

105.88

<0.0001

Significant

Residual

58.99

10

5.90

   

Lack of fit

39.21

5

7.84

1.98

0.2353

Not significant

Pure error

19.78

5

3.96

   

Corrected total

5679.81

19

    

Optimal conditions of plastein reaction

To assess further the effect of independent variables on the decrease of free amino groups in the reaction mixture, three-dimensional response surfaces plots were generated from the regression equation by keeping one variable at zero level and changing other two variables with different combinations. The response surface, whose regression coefficients are given in Table 3, is shown in Fig. 3. Figure 3a shows the effect of E/S ratio and reaction temperature on the decrease of free amino groups in the mixture (time at the central of its level); quadratic effect for both variables was observed, though reaction temperature had greater influence on the response. The decrease of free amino groups in the mixture increased with the increase of reaction temperature. Also, higher values of the decrease of free amino groups could be found when the E/S ratio took place between 6.7 and 8.2 kU g−1 proteins. Figure 3b shows the effect of E/S ratio and reaction time (temperature at the central of its level); quadratic effect of E/S ratio and reaction time on the response could be noticed. The decrease of free amino groups in the mixture increased with the increase of reaction time. In Fig. 3c, the quadratic effect of both variables, reaction temperature and time (E/S ratio at the central of its level), is also presented.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1110-4/MediaObjects/217_2009_1110_Fig3_HTML.gif
Fig. 3

Response surface graphs for the decrease of free amino groups in the reaction mixture as a function of: (a) E/S ratio and reaction temperature (reaction time at the central of its level), (b) E/S ratio and reaction time (reaction temperature at the central of its level), (c) reaction temperature and time (E/S ratio at the central of its level)

These results have shown that the response surface had a maximum point within the experimental range of the independent variables. The stationary point (maximum) of the fitted model was found by deriving first derivatives of the function (3), as follows:
$$ \begin{aligned} 4. 1 4 7- 0.0 9 8X_{ 2} & = 0 \\ 9 5. 2 4 7- 1.0 9 7X_{ 3} - 1 1. 4 7 2X_{ 1} & = 0 \\ 2 1. 10 4- 1.0 9 7X_{ 1} - 2. 1 2 2X_{ 3} & = 0. \\ \end{aligned} $$
(3)
The system of linear Eq. 3 was solved and the following results were obtained: X1 = 7.7, X2 = 42.7 and X3 = 6. The calculated values (X1, X2 and X3) correspond to the values of the independent variables for the maximum value of the response, the decrease of free amino groups in the mixture as an indicator of reaction extent. The optimal conditions for the plastein reaction of casein hydrolysates are listed in Table 5. It was calculated that with these optimal conditions, the decrease of free amino groups in the mixture was 185.7 μmol g−1 proteins.
Table 5

Optimal conditions of casein hydrolysates for plastein reaction catalyzed by Alcalase, obtained applying RSM

Independent variables

Optimal conditions

E/S ratio (kU g−1 proteins)

7.7

Temperature ( °C)

42.7

Time (h)

6

To confirm the applicability of the model designed, confirmation runs using the calculated levels of the variables were carried out. The results from two parallel trials were that the decrease of free amino groups in the mixture was 179.7 μmol g−1 proteins and 183.9 μmol g−1 proteins. Although the mean value (181.8 μmol g−1 proteins) was lower than the calculated value (185.7 μmol g−1 proteins), there was no significant difference (p < 0.05). This result also indicated that the second-order polynomial model [Eq. 2] could be used to predict the decrease of free amino groups in the mixture of casein hydrolysates during plastein reaction.

ACE-inhibitory activity of plastein-modified casein hydrolysates with different reaction extents

Casein hydrolysates modified by plastein reaction with different reaction extents were prepared with an optimal ratio of E/S and reaction temperature, but different reaction time. The actual decrease of free amino groups in the mixture ranged from 83.6 μmol g−1 proteins to 181.8 μmol g−1 proteins. The ACE-inhibitory activity and IC50 value of plastein-modified casein hydrolysates (PMCH) prepared were evaluated and the results are shown in Table 6. It appeared that the higher the decrease of free amino groups of PMCH, the higher was its ACE-inhibitory activity. If casein hydrolysates were modified to a reaction extent, such that the decrease of free amino groups was more than 155 μmol g−1 proteins, IC50 value would be as low as 0.6 μg mL−1, while IC50 value of the original casein hydrolysates would be as high as 47 μg mL−1. This indicated that plastein reaction catalyzed by Alcalase was an effective method to improve ACE-inhibitory activity of casein hydrolysates.
Table 6

Influence of plastein reaction on ACE-inhibitory activity of casein hydrolysates

Samples

Decrement of free amino groups (μmol g−1 proteins)

ACE-inhibitory activity (%)a

IC50 (μg mL−1)

Casein hydrolysates

0

20.6

47

PMCH 1

83.6

22.1

35

PMCH 2

129.6

42.7

24

PMCH 3

142.1

60.3

5

PMCH 4

154.7

94.1

0.6

PMCH 5

181.8

95.6

0.5

PMCH Plastein modified casein hydrolysates

aThe final concentration of casein hydrolysates or PMCHs for ACE-inhibitory activity assay was 10 μg mL−1 on the basis of protein

Discussions

Preparation of casein hydrolysates

Many studies have confirmed that the ACE-inhibitory activity of peptides was correlated with their primary structures. Peptide inhibitors of ACE are expected to exhibit good binding to the active site of ACE. Structure–activity correlations among different peptide inhibitors of ACE indicate that binding to ACE is strongly influenced by the C-terminal tripeptide sequence of the peptide inhibitors. It seems that hydrophobic amino acids at each of the three C-terminal positions are favorable [14, 16]. Cheung et al. [3] found that proline, tryptophane, tyrosine and phenylalanine were most effective at the ultimate C-terminal position, proline binding exceptionally well to ACE. Casein is a good protein substrate to prepare ACE-inhibitory peptides, because it is rich in proline, tryptophane, tyrosine and phenylalanine, which give casein hydrolysates more opportunity to have these amino acids at the C-terminal position. Alcalase, used as a catalyst in our study, is a good candidate for the preparation of casein hydrolysates, because it contains endoprotease with broad specificity. Alcalase was confirmed to have a high specificity for aromatic and aliphatic amino acids [7]. Therefore, hydrolysis of casein with Alcalase makes it possible to prepare casein hydrolysates with good ACE-inhibitory activity. In our study, casein hydrolysates with DH of 11.2% appeared to have the highest ACE-inhibitory activity (Fig. 1) and its IC50 value was 47 μg mL−1. The study by Otte et al. [26] had also observed similar findings with respect to hydrolysis of sodium caseinate with thermolysin, and the IC50 value of sodium caseinate hydrolysates was 95 μg mL−1. Miguel et al. [21] also studied the hydrolysis of bovine casein with pepsin, and the IC50 value of casein hydrolysates prepared was 52.8 μg mL−1. The ACE-inhibitory activity of casein hydrolysates prepared in our study was close to these studies.

The hydrolysis of casein tended to slow down during 8–12 h of hydrolysis. In contrast to the time course of hydrolysis, the ACE-inhibitory activity of casein hydrolysates prepared increased rapidly from the beginning to 6 h of hydrolysis, remained at a plateau during the next 6–8 h of hydrolysis, then decreased during 8–12 h (Fig. 1). The study by Mao et al. [18] had similar findings with respect to the time course of hydrolysis of yak milk casein with Alcalase. Besides milk protein, in vitro hydrolysis of other food proteins also revealed a similar pattern, in which peptides produced during the initial stages of hydrolysis had a greater ACE-inhibitory activity than peptides produced during the later stages of hydrolysis [19, 36].

Influence of concentration of casein hydrolysates, E/S ratio, reaction temperature and time on the extent of plastein reaction

When highly concentrated solutions of protein hydrolysates were incubated with proteases, water-insoluble and gel-forming products may be formed, as reported by Sukan and Andrews [37, 38]. The extent of plastein reaction could be evaluated from the yield of water-insoluble products in the gel or thixotropic solutions [39], but condensation or transpeptidation that occurred in the plastein reaction cannot be reflected directly by this expression. In our study, the decrease of free amino groups in the reaction mixture was used to reflect the extent of plastein reaction of casein hydrolysates, which indeed was an indicator of condensation. The decrease of free amino groups in the mixture of plastein reaction would be dependent on reaction conditions, such as substrate concentration, reaction temperature and reaction time. When the concentration of casein hydrolysates was too low, hydrolysis might be the predominating reaction. When the concentration of casein hydrolysates was too high, catalytic efficiency of Alcalase was restricted because the solution was very viscous and much of it in the form of gel. Sukan and Andrews [37] had reported that the formation of plastein products was at a maximum when the concentration of protein hydrolysates was in the region of about 20–40% by weight, and fell sharply both above and below that range. The range of substrate concentration was from 25 to 45% by weight in our study, and the increase in the concentration of substrates resulted in a great decrease of free amino groups in the mixture. Plastein reaction took place usually at a higher substrate concentration (from 30 to 50% by weight) [17, 28]. In our study, the decrease of free amino groups in the reaction mixture occurred when the concentration of casein hydrolysates was in the region of 30–45% by weight, which were similar to these reports.

Although the precise reaction mechanism for plastein reaction is doubtful, it has been shown unequivocally that there is a requirement for an active proteinase to catalyze the reaction [1]. Alcalase was used in our study to catalyze the plastein reaction of casein hydrolysates. This is because there is always potential for further hydrolysis to occur at the same time as the synthesis reaction; but if the most susceptible peptide bonds have already been cleaved during hydrolysis of casein, this will be a less important factor than when an enzyme with different peptide bond specificity is used [37]. The analysis results showed that the more the Alcalase added to casein hydrolysates, the higher is the decease of free amino groups in the reaction mixture (see Fig. 2b). This result was caused as more condensation occurred between the peptides as more Alcalase was added.

The decease of free amino groups in the reaction mixture increased with rising reaction temperature from 10 to 50 °C (Fig. 2c). This result was consistent with literature trends, although different expressions for the extent of plastein reaction were employed in other reaction systems [37]. The range of reaction temperature was restricted by the optimal catalytic temperature of the enzyme used. Therefore, higher reaction temperature might not be a suitable selection. With regard to the influence of reaction time on the extent of plastein reaction, the reaction temperature must be considered. If the reaction temperature was too high, although the initial rate of plastein reaction was very rapid, the reaction might be soon stopped and the overall result would be much lower than at low temperature for long reaction time [37]. In our study, influence of reaction time on the extent of plastein reaction was investigated at a fixed temperature (Fig. 2d). There was a rapid, progressive decrease of free amino groups in the reaction mixture at the initial stage of reaction (0–6 h), which then tended to slow down (6–24 h). This result was consistent with the study of Williams et al. (2001).

The impact of plastein reaction on the ACE-inhibitory activity of casein hydrolysates

Significant improvement in ACE-inhibitory activity of casein hydrolysates could be obtained by plastein reaction catalyzed by Alcalase, as shown in Table 6. Five plastein-modified casein hydrolysates (PMCH), PMCH 1 to PMCH 5 (different in the extent of plastein reaction) all had higher ACE-inhibitory activity than the original casein hydrolysates, especially when PMCH was prepared with a greater extent of plastein reaction (e.g., the decrease of free amino groups in the mixture was greater than 142 μmol g−1 proteins). Significant increases in ACE-inhibitory activity as shown in Table 6 could not be the cause of prolonging the hydrolysis time (see the results in Fig. 1). The possible reason might be related to the formation of new peptides during plastein reaction of casein hydrolysates. Based on the fact that a decrease of free amino groups occurred in the reaction mixture, there would be some new peptides generated in PMCH, because the decrease of free amino groups in the reaction mixture was the direct reflection of peptide condensation. The more the modification reaction occurred (the more decrease of free amino groups in the reaction mixture), the greater was the number of new peptides formed.

Miguel et al. [21] had investigated the potential ACE-inhibitory activity of a pepsin-hydrolyzed bovine casein. A fraction of bovine casein hydrolysates with molecular mass lower than 3,000 Da was separated, and their IC50 values were 52.8 and 5.5 μg mL−1, respectively. Also, when sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4, the crude peptide fractions of hydrolysates from bovine had the highest ACE-inhibitory activity (IC50 = 16.2 μg mL−1) [22]. When the highest decrease of free amino groups in the reaction mixture (181.8 μmol g−1 proteins) was achieved in our study, the product, PMCH 5, had the highest ACE-inhibitory activity (IC50 = 0.5 μg mL−1). Because PMCH 5 was derived directly from a new preparation method, enzymatic hydrolysis of casein coupled with modification of plastein reaction and without any separation, its strong ACE-inhibitory activity suggested that this approach was superior to traditional enzymatic hydrolysis for the preparation of ACE-inhibitory peptides. This result might also present possible application of plastein reaction to improve ACE-inhibitory activity of other protein hydrolysates.

Although the decrease of free amino groups in the reaction mixture could not indicate that transpeptidation occurred during plastein reaction, it could not be ignored as a previous study had confirmed that several pathways (condensation, transpeptidation and physical forces) might exist in the plastein reaction simultaneously [17, 34]. The detailed reaction that occurs in casein hydrolysates and its impact on the composition of peptides during plastein reaction needs to be studied more in future.

Conclusions

Casein was hydrolyzed by Alcalase to obtain casein hydrolysates with ACE-inhibitory activity and then the prepared hydrolysates were subjected to plastein reaction catalyzed by the same enzyme. When casein was hydrolyzed with Alcalase to DH of 11.2%, the casein hydrolysates had the highest ACE-inhibitory activity with IC50 value of 47 μg mL−1. Reaction conditions such as substrate concentration, E/S ratio, reaction temperature and time all had influence on plastein reaction of casein hydrolysates and were determined. When the concentration of casein hydrolysates was fixed at 35% by weight, the optimal conditions for plastein reaction were studied using response surface methodology with the decrease of free amino groups in the reaction mixture as response, which showed that E/S ratio was 7.7 kU g−1 proteins, reaction temperature was 42.7 °C and reaction time was 6 h. Analysis results showed that ACE-inhibitory activity of modified casein hydrolysates prepared was improved by plastein reaction. When the decrease of free amino groups in the reaction mixture was more than 142.1 μmol g−1 proteins, the IC50 value of modified casein hydrolysates was 5 μg mL−1. If the casein hydrolysates prepared with the decrease of free amino groups in the mixture was about 154.7 or 181.8 μmol g−1 proteins, the corresponding IC50 value might be decreased to 0.6 or 0.5 μg mL−1. The results showed that the modification of casein hydrolysates with plastein reaction improved their ACE-inhibitory activity clearly. The possible application of plastein reaction to improve ACE-inhibitory activity of other protein hydrolysates exists.

Acknowledgments

This work was supported by the National High Technology Research and Development Program (“863” Program) of China (No. 2006AA10Z324).

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© Springer-Verlag 2009