Production of Angiotensin-I-Converting Enzyme inhibitory peptide from goat milk casein : optimization conditions of complex protease hydrolysate by response surface methodology and purification

Production of Angiotensin-I-Converting Enzyme inhibitory peptide from goat milk casein: optimization conditions of complex protease hydrolysate by response surface methodology and purification Li Chen1*, Juan Wang2, Guowei Shu2*, He Chen2 1College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, China, 2School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, China R E G U L A R A R T I C L E


INTRODUCTION
Hypertension which leads to serious medical and lifestylerelated problem has become a worldwide cardiovascular disease.The peptides which capable of controlling blood pressure are identified as a strong inhibitor of angiotensin-I-converting enzyme (ACE, peptidyldipeptide hydrolase, E. C. 3.4.15.1).This inhibitor acts in the rennin-angiotensin system transforming angiotensin I type into II type, then deactivates the depressor bradykinin to lose catalytic function (He et al., 2004;Kong et al., 2011).Therefore, the application of ACE-inhibitory peptides in clinical treatment and function food process is becoming popular globally.
Goat is the most important cultured farm animal in Shaanxi, China.The production and consumption of goat milk and dairy products are increasing all over the world.The goat milk protein have advantage in digestive ability over Food-derived Angiotensin-I-Converting Enzyme (ACE)-inhibitory peptides have safety advantages over synthetic peptides.The application of complex enzymatic (alcalase and trypsin) in producing such peptides from goat milk casein seldom be focused.In this study, the pH, complex protease ratio (CPR) and enzyme to substrate ratio (E/S) were optimized by Response surface methodology (RSM).The optimized conditions were: pH 8.4, CPR 1:1, and E/S 8.5%.In these conditions, the ACE-inhibitory activity of the obtained hydrolysates reached 91.99%.The response model was qualified to predict the reaction optimization.Hydrolysate fragments were purified consecutively.A fraction G 2-2a exhibited highest ACE-inhibitory activity 93.50% with IC 50 value of 72.14 μg/mL.bovine milk protein, and it seldom bring harmful effects to human being (Montalbano et al., 2016;Lee et al., 2005;Jiang et al., 2007).The most abundant proteins in goat milk are caseins.Previous researches have used single enzyme to hydrolyze milk protein, but the application of combined enzymes to hydrolysis is scarce (Espejo-Carpio et al., 2013;Miguel et al., 2008, Minervini et al., 2003).Response surface methodology (RSM) has advantage to classical one-variablea-time optimization, and therefore is largely applied to optimize the analytical procedures.The experimental design should fit an adequate mathematical function and could evaluate the quality of the fitted model (Kalil et al., 2000).The Box-Behnken design (B-B design) is more efficient and economical to build the response surface mode.
The objective of this study was to optimize the complex enzymatic hydrolysis condition for ACE-inhibitory peptides from goat milk casein by RSMwith the aid of B-B design and Plackett-Burman Design (P-B).The P-B investigates a number of independent variables and determines which variables affect the performance greatly.

Enzymatic hydrolysis
Goat' casein was prepared by our lab.Hydrolysis of casein was measured as the method of Adamson and Reynolds (1996).Casein was hydrolyzed under the substrate concentration of 5% and E/S of 5%.The pH value of the hydrolysis mixture was adjusted with addition of 0.1 mol/L NaOH by pH-Stat (Metrohm Ltd., Herisan, Switzerland).During the reaction, samples were removed after 30,60,90,120,150,180,210,240,270,300,330, and 360 min, and the reaction was ended by heating at 90 °C for 15 min.The reaction mixture were adjusted to pH 3.5, centrifuged for 15 min at the speed of 5000 rpm, then collected under pH 8.3 for ACE-inhibitory activity determination.

Determination of ACE-inhibitory activity
The ACE-inhibitory activity was calculated according to the method of Cushman and Cheung (1971).A sample solution (100 μL) was incubated with 200 μL substrate (5 mM hippuryl-L-histidyl-L-leucine (HHL) dissolved in 0.1 M Na-borate buffer (0.3 M NaCl, pH 8.3) at 37 °C for 5 min.The reaction system started with 20 μL of the ACE solution (100 mU/mL) at 37 °C for 30 min and was terminated by adding 0.25 mL of 1 M HCl.The hippurylic acid was extracted with the addition of ethyl acetate.After strong vortex, separate 5min and 30min at 120 °C in a vacuum.The residue was dissolved in 3 mL of distilled water.The absorbance was determined at 228 nm using a spectrophotometer UV5300-PC (Yuanxi, Shanghai, China).
The ACE inhibition was obtained as following (Eq.1): Which A represented the absorbance of ACE, B represented absorbance with sample and ACE, and C represented the absorbance of sample.
The ACE inhibition meant 50% of the original ACE activity (IC 50 ) was inhibited at a certain protein concentration.The degree of hydrolysis (DH) and protein concentrations were determined according to the methods of pH-Stat and the method of Lowry et al., respectively (1951).Bovine serum albumin was used as the standard in the experiment.

Separation of ACE-inhibitory peptides
Goat casein hydrolysates were separated by an ultrafiltration membrane.Three fractions under 10 kDa, 5 kDa, and 1 kDa were obtained (Millipore, Billerica, USA).

Statistical evaluation
Statistical analysis system SAS9.1 (SAS Institute INC, USA) was used for statistical evaluation.All results were expressed as the means ± SD and repeated for three times.

Screening the complex protease on goat casein hydrolysates
Enzymatic hydrolysis has been widely used in the production of ACE-inhibitory peptides.It has a clear advantage in less protein destruction, easy controlling, and specific location hydrolysis.The process is consisting of substrate, specific enzyme and the reaction conditions.The enzymes with high frequency in hydrolysis are alcalase, pepsin, neutral protease and trypsin.Alcalase has been used for the generation of ACE-inhibitory peptides from sardine muscle, alaska pollack, jellyfish collagen (Matsui et al., 1993;Zhuang et al., 2012;Byun and Kim, 2001).Jiang et al. (2007) reported that As1398 neutral protease contributed greatly in bovine casein hydrolysis for production of ACE-inhibitory peptides.Pepsin was the best enzyme for the separation of ACE-inhibitory peptides from goat's casein (Lee et al., 2005;Bai et al., 2014).Success of single enzymatic hydrolysis in production of ACEinhibitory peptides has been reported, but the application of complex protease enzymatic hydrolysis in goat casein is scarce.
In order to find the favorable complex protease, we chose five commercial food grade proteases to digest the goat's casein (Table 3).Each enzymatic hydrolysis was carried out under their suggested optimal conditions during 6 h (Wang et al., 2010).The best ACE-inhibitory activity of each protease was listed in Table 3.The ACE-inhibitory activity values varied greatly from 74.56% to 90.00%, and top two were in alcalase and trypsin hydrolytic group (90.00% and 87.11%), respectively.It is consistent with previous researches that alcalase contributes greatly to produce ACE-inhibitory peptides (Adamson and Reynolds, 1996;Kim et al., 2001;Lee et al., 2005;Byun and Kim, 2001).Zhang et al. (2013) reported that the combined and sequential use of alcalase followed by trypsin provided high DH which was much superior to either of them.
The peptides derived from goat milk protein showed higher ACE-inhibitory activity when hydrolysed by the combination of ubtilisin and trypsin in comparison to the individual enzyme (Espejo-Carpio et al., 2013).
Trypsin has been widely used to produce ACE-inhibitory peptides from milk protein.Subtilisin, also known as alcalase has low specificity and preferentially cleaves at the C-terminal of hydrophobic residues.We compared five commercial proteases with the consideration of ACEinhibitory activity and fund-saving.Finally, we chose the combination of alcalase and trypsin to hydrolyze goat casein because of their great contribution to ACE-inhibitory activity.Protease K was abandoned due to high price, although it exhibited great ACE-inhibitory activity (86.67%).

Minimize the number of variables by P-B design
P-B design was adopted to minimize the number of independent variables through a limited number of experiments.In this study, temperature (X 1 ), pH (X 2 ), E/S (X 3 ), Goat's casein (X 5 ), complex protease ratio (CPR, v/w) (X 6 ), time (X 7 ),and two virtual factors (X 4 and X 8 ) were analyzed.Response (Y) represented the ACE-inhibitory activity covered from 65.45 to 92.73% (Table 4).Low values of the Y were obtained when the group consists of low E/S (X 3 ), low CPR (X 6 ) and/or high pH (X 2 ).These results indicated that the CPR and E/S had positive effect while the pH had negative effect.To further confirm the variables effect, the trends of response value Y with 8 variables were described in Fig. 1.When the four factors (X 3 ), (X 4 ), (X 6 ), (X 7 ) increased, the response value Y kept rising following.However, on the contrary, the response value Y dropped off when the value (X 1 ), (X 2 ) and (X 8 ) increased.Since (X 8 ) and (X 4 ) were virtual factors and excluded to the consideration.Three variables E/S (X 3 ), CPR (X 6 ) and pH (X 2 ) exhibited strong influences which were similar to the study of Zhuang, which found E/S and pH play important roles in jellyfish collagen hydrolysis (Zhuang et al., 2012).

The B-B design and RSM analysis
B-B design is the statistical experimental design for the efficient estimation of the second-order coefficients of the mathematical model.Some changes were made for B-B experiment based on the P-B design results.The value range of E/S and CPR was increased, and the range of pH decreased.A three-level-three-factor B-B design was carried out to evaluate the hydrolysis condition (Table 2).As shown in Table 5, the significance of variables and their interactions were expressed by p-values.E/S had strong effects (P < 0.05) but CPR and pH not (P > 0.05).The terms of CPR 2 , (E/S) 2 and pH 2 exhibited high significant (P < 0.05) and high F value, indicated that variables and response had no simple linear correlation.The term of pH × E/S, pH × CPR, and pH × CPR showed no significant (P > 0.05) and low F value, which demonstrated a weak mutual interaction between them.
Statistical significance of the equation is shown in Table 5.
The F value was 13.3654 which meant this mathematics model highly fitted the prediction of ACE inhibition (%) (P < 0.01).The suitability and validity of the quadratic model was evaluated by coefficient of determination (R 2 ).The values of R 2 and adjusted R 2 of the polynomial model were 96.01%and 88.83%, respectively.The R 2 value of 0.9601 indicated that more than 96.01% of the data fitted the equation (Eq.3): % Inhibition = 89.697+ 2.044pH -11.138E/S + 7.274CPR -14.395pH 2 -8.18pH × +4.093pH × CPR -11.667(E/S)-3.18E/S× CPR -41.21CPR 2 (3) The 3D response surface and 2D contour plots were depicted (Fig. 2).The ellipse obtained in contour plot suggested that Y had enhanced until X 1 , X 2 , and X 3 were at the best appropriate condition.The best hydrolysis seemed to be pH 8.4, CPR 7:7, and E/S 8.5% with Y of 92.35%.A Y value of 91.99% was obtained under this best hydrolysis condition.The combination of complex protease alcalase and trypsin was successful in hydrolyzing goat casein.

Separation of inhibitory peptides from casein
Casein hydrolysis were separated into three fractions.
The composition of amino acids has a close relationship with protein structure which maintains bioactive function (Zhao et al., 2007).So far, the peptides have hydrophobic amino acids on their C-terminal or contained branchedchain aliphatic amino acids exhibited high ACE-inhibitory activity (Wang et al., 2014;Cheung et al., 1980).Maruyama et al. (1985) discovered an ACE-inhibitory peptide with sequence of Ala-Val-Pro-Tyr-Pro-Gln-Arg from bovine milk.Lee et al (2005) found a peptide of Ala-Tyr-Phe-Tyr from goat milk.In our study, the composition of Glu, Ala, Val, Pro, Leu and Asp in purification peptides remains unclear.We speculate the characteristic amino acid composition is attributed to the local natural and human environment.Taken all the previous information together, peptides separated in our experiment might have different sequence characteristics to those peptides above, but needs more detailed identification.

CONCLUSION
The use of proteins from various types of milk as a source of anti-hypertensive peptides remains an increasing interest in recent years.In this study, the hydrolysis condition of complex trypsin and alcalase in goat's casein were investigated.In our study, the ACE-inhibitory activity was mainly dependent on the casein E/S and CPR.With the aid of RSM, the optimal conditions were pH of 8.4, CPR of 1:1, and E/S of 8.5%.The hydrolysate ACEinhibitory peptides exhibited high activity of 91.99% under the optimum condition.Fragment G 2-2a was purified consecutively.The IC 50 value of G 2-2a reached the maximum of 72.14 ± 5.52 μg/mL, and the ACE-inhibitory activity was up to 93.50%.The characterization of the amino acid sequence would help us better elucidate the mechanism of

Table 5 : Analysis of variance (ANOVA) Variance ACE-inhibitory activity F ration P value
*indicates significant difference P < 0.05; **indicates significant difference P < 0.01