Preparation of ACE Inhibitory Peptides from Mytilus coruscus Hydrolysate Using Uniform Design

The angiotensin-I-converting enzyme (ACE) inhibitory peptides from mussel, Mytilus coruscus, were investigated and the variable factors, protease concentration, hydrolysis time, pH, and temperature, were optimized using Uniform Design, a new statistical experimental method. The results proved that the hydrolysate of alkali proteases had high ACE-inhibitory activity, especially the alkali protease E1. Optimization by Uniform Design showed that the best hydrolysis conditions for preparation of ACE-inhibitory peptides from Mytilus coruscus were protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, and Temperature at 59.5°C, respectively. The verification experiments under optimum conditions showed that the ACE-inhibitory activity (91.3%) were agreed closely with the predicted activity of 90.7%. The amino acid composition analysis of Mytilus coruscus ACE-inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid.


Introduction
About 30% of Americans are suffering hypertension and risk of cardiovascular disease development as an independent factor [1]. Hypertension is one of the most frequent chronic diseases and the incidence of this disease was increased in recent years. is disease affected about 65% of 65-75-yearold people in Western developed countries and its incidence was increased with age [2]. Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) is one of the metalloproteases and zinc is neccessary for its activity [3]. ACE cleaves dipeptides from oligopeptide's carboxylic terminus, which plays important physiological role in blood pressure regulation [4]. Functional foods, containing ACE inhibitory peptides, may control blood pressure moderately. Many ACE inhibitory peptides in vitro have been isolated from various food derived proteins hydrolysate, such as milk [5], seed protein [6], blue mussel protein [7], bovine blood plasma [8], casein [9][10][11], zein [12], sardine [13], and tuna muscle [14]. ACE inhibitors have also been isolated from fermented foods, such as yoghourt [15], soy sauce [16], and soybean [17].
Mytilus coruscus is one of the most important bivalves in both Chinese aquaculture and Chinese market [18].
Like other marine animals, some biactive peptides have been reported from Mytilus mussel protein, such as Mytilus inhibitory peptides [19], antimicrobial peptides [20], and anticoagulant peptide [21]. In addition, an ACE inhibitory peptide has been puri�ed by chromatography method and identi�ed from blue mussel sauce [7]. However, there was no report to obtain ACE inhibitory peptides from Mytilus coruscus mussel protein hydrolysate.
Uniform Design method, a new experimental technique, is established together by Fang [22]. One of the most important advantages of the Uniform Design is that many factors and levels can be desined simulataneously. Uniform Design offers many convenient experimental tables [23]. But, unlike orthogonal design, the largest possible amount of levels for each factor can be allowed in Uniform Design, and so much so that the number of levels sometimes can be equal to the number of experiment runs [24]. As a statistical and experiment design technique, Uniform Design method has been successfully used for many experiments, especially in optimizing processes [23,25,26]. In

Preparation of Hydrolysates. Mussels, Mytilus coruscus,
were washed with water to remove salt and other materials. e mussels were �lleted and defatted with petroleum ether at 50 ∘ C by re�ux extraction. en the mussels were minced and mixed with distilled water (ratio of 1 : 10). e mixture was homogenate and then was boiled for 10 minutes to inactive the inner protease. en the mixture was digested by �ve proteases at designed conditions, respectively. e pH of the reaction mixture was maintained stably by addition of either 1 N NaOH or HCl. en, the mixture was incubated at 90 ∘ C for 10 min to terminate the reaction. Aer centrifugation (12,000 ×g, 4 ∘ C) for 10 min, the supernatant of the hydrolysate was collected for test the ACE inhibitory activity.

Determination of ACE Inhibitory
Activity. e ACE inhibitory activity was determined by Wang et al. method [27] with slight modi�cations. All samples were diluted to the same protein content (1.0 mg/mL), which was determined by Biuret assay method [28]. Sample solution (10 L) and ACE solution (50 units/mL, 30 L) were mixed together. Aer the mixture being preincubated at 37 ∘ C for 5 min, 50 L 7.6 mmol/L HHL substrate solution, which was solved in 50 mM sodium borate buffer and 6.8 mM NaCl at pH 8.3, was added. e mixture was incubated at 37 ∘ C for 25 min. e reaction was terminated aer addition of 10 L of 20% tri�uoroacetic acid (TFA). e solution was �ltrated through 0.22 m membrane. e hippuric acid liberated by ACE was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) on an Inertsil ODS C 18 (4.6 mm × 300 mm, 5 m) column. e mobile phase was 30% methanol, which contained 0.1% TFA and 0.05% acetic acid. e �ow rate was 1.0 mL/min. e U� detection wavelength was 228 nm. e ACE inhibitory activity was obtained from peak area and expressed as percent.

Choice of Protease.
Five kinds of proteases were used for hydrolysis at their labeled optimum temperature and pH (shown in Table 1). And the protease was added at 50 U per mL mixture and the hydrolysis time was �xed at 4.0 h. en ACE inhibitory activity of hydrolysate was determined.  (7 4 ) was applied to determine the optimum hydrolysis conditions for obtaining ACE inhibitory activity peptides from Mytilus coruscus. e combination effects of independent variables 1 (protease concentration, U/mL), 2 (hydrolysis time, h), 3 (hydrolysis pH), and 4 (hydrolysis temperature, ∘ C) at 7 variation levels in the hydrolysis process were shown in Table  2. A total of 21 combinations (three replicates) for four factors were chosen according to Uniform Design table. e actual values were also shown in Table 2. e responses functions (Y) were ACE inhibitory activity. ese values were related to the variables by a second-order polynomial (1) below: where is the predicted response. and are the independent variables. 0 , , , and were the regression coefficients.
e signi�cance was evaluated by Student�s t-test. e actual values were compared with model predictions. e optimum hydrolysis conditions were veri�ed by additional triplicate experiments under these conditions. 2.6. Amino Acid Composition Analysis. e amino acid analyses were conducted by the method of Noreen and Salim [29]. Brie�y, the 10 mL of the sample was hydrolyzed under vacuum by addition of 10 mL concentrated HCl at 110 ∘ C for 24 h. When the free amino acids were analyzed, the sample did not hydrolyzed by HCl. Amino acids were analyzed in a Shimadzu HPLC system by separation in an ion-exchange column and post-column reaction with ninhydrin.

Statistical
Analysis. Data were expressed as means ± standard deviation of triplicate. A probability value of 0.05 was considered signi�cantly.

Results and Discussion
3.1. Choice of Protease. ACE inhibitory peptides generally were short peptides and enzymatic hydrolysis of food derived protein was one of important measures to obtain ACE inhibitory peptides. A lot of ACE inhibitory peptides had been reported from food derived proteins hydrolysates. In this investigation, �ve kinds of commercial proteases, including three alkali proteases, one neutral protease, and one acid protease, were chosen to obtain ACE inhibitory   Figure 1.
From the results, it was shown that the hydrolysate produced by alkali protease E1 had the highest ACE inhibitory activity. In addition, alkali proteases (E1, E2 and E3) were more effective for hydrolysis Mytilus coruscus mussel protein to obtain ACE inhibitory peptides than other two proteases (E4 and E5). erefore, alkali protease E1 was chosen to next experiments to optimize hydrolysis conditions for producing ACE inhibitory peptides from Mytilus coruscus.

Data Analysis of Uniform Design.
A regression analysis was conducted to �t a mathematical model to the experimental data. e results of regression analysis were summarized (Table 2) e statistical analysis indicated the predicted model was adequate, possessing signi�cant value ( 0.047 0.05 and satisfactory values of the regression coefficient 2 ( 2 0.9712 for the response. e high regression coefficient make clear that the experimental values of the ACE inhibitory activity agreed with predicted values, which meant that the predicted model seemed to reasonably represent the observed values. e largest relative error of predicted value was less than 5% shown in Table 2. e signi�cance was tested by Student's t-test and value in Table 3. It was shown that temperature, pH, and protease added quantity affected signi�cantly the ACE inhibitory activity of hydrolysates.
3.3. �eri�cation ��periments. en the optimum hydrolysis conditions of protease E1 and the prediction ACE inhibitory activity were obtained by (2). e optimum hydrolysis conditions of protease E1 were protease concentration (  inhibitory activity was 90.7% at optimum hydrolysis conditions. Under this optimum hydrolysis conditions, other three veri�cation experiments were conducted and the average actual ACE inhibitory activity was 91.3%, which was in agreement with the predicted values of 90.7%.

Amino Acid
Composition. e compositions of free amino acid and amino acid in peptides of the ACE inhibitory peptides from Mytilus coruscus were determined and the results were shown in Table 4. From the results, it was seen that the ACE inhibitory peptides solution had only a few free amino acid content, not equal with amino acid in peptides. e peptides, not amino acid, might contributed to high activity. e ACE inhibitory peptides had high percent of glutamic acid, taking 0.578 mmol/g, which could improve the breath ability of brain cell and be favorable to the expulsion of ammonia in brain and regulation of body metabolism, and these phases could impact the blood pressure directly. Also the ACE inhibitory peptides had high percent of lysine, leucine, glycine, and aspartic acid. ese amino acids might play crucial role in the inhibitory  [30] reported that dipeptides having hydrophobic amino acids such as valine (Val) and isoleucine (Ile) at the amino temerninus have higher ACE inhibitory activities. e amino acids, such as lysine, leucine, glycine, aspartic acid, and glutamic acid, were key constitutes with tall frequency appeared among many reported ACE inhibitory peptides [31][32][33][34][35].

Conclusions
Alkali protease was a good choice for hydrolyzing Mytilus coruscus protein for producing ACE inhibitory peptides. e factors, including protease concentration, hydrolysis time, hydrolysis pH, and temperature, affected the ACE inhibitory peptides of hydrolysates. Uniform Design was chosen to investigate the effects of preceding variables on ACE inhibitory activity. And the best hydrolysis conditions of alkali protease E1 optimized by Uniform Design were protease concentration of 36.0 U/mL, hydrolysis time of 2.7 hours, pH 8.2, temperature at 59.5 ∘ C. e optimal predicted ACE inhibitory activity of 90.7% was obtained at the optimum conditions. e experimental activity (91.3%) under optimized conditions was agreed closely with the predicted activity. e amino acid composition analysis of the ACE inhibitory peptides proved that it had high percent of lysine, leucine, glycine, aspartic acid, and glutamic acid. It was suggested that the ACE inhibitory peptides derived from Mytilus coruscus could be utilized to develop nutraceuticals and pharmaceuticals.