Production of Angiotensin-I Converting Enzyme Inhibitory Hydrolysates from Egg Albumen

ACE (Angiotensin-I converting enzyme) inhibitory peptides derived from foods are thought to suppress high blood pressure by inhibiting ACE. We tried to make efficient production of the ACE inhibitory hydrolysate from egg albumen. A hydrolysate digested by neutrase presented the highest ACE inhibitory activity (IC50 value=256.35 μg/ml) and the proper proteolysis was occurred by 1.0% enzyme addition and 4 h incubation at 47°C. Antihypertensive effect of neutrase hydrolysate was investigated in spontaneously hypertensive rats (SHR, n=5). Systolic blood pressure (SBP) was decrease by 6.88% (-14.14 mmHg, p<0.05) at 3 h after oral administration of 300 mg/kg body weight, and by 13.33% (-27.72 mmHg, p<0.05) by emulsified hydrolysate. These results showed that it is very effective to utilize egg albumen as a protein source for the production of ACE inhibitory peptides. However, further studies are required to investigate the methods to increase recovery yield and the isolation of active peptide is necessary for determining its sequence responsible for ACE inhibitory activity. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 9 : 1369-1373)


INTRODUCTION
Hypertention is related to the incidence of coronary heart disease and over 90% of hypertension is known as an essential hypertension.Angiotensin-I converting enzyme (ACE) produces a hypertensive peptide hormone angiotensin-II from angiotensin-I that has an important role in hypertension (Peart, 1976).
Since ACE inhibitory peptide was first obtained from snake venom (Feria, 1965), various ACE inhibitors have been discovered from enzymatic hydrolysates of several food proteins (Maruyama and Suzuki, 1982;Miyosh et al., 1991;Kinoshita et al., 1993;Mullally et al., 1997).Egg albumen, which contains 90% of proteins on dry matter, is the good source of high quality protein and can be obtained with relatively low price.In Korea, though almost of egg albumen is allowed to be discarded, a small portion has been utilized in the production of boiled fish paste and the isolation of lysozyme.
Thus the loss of this valuable protein source should be prevented and we tried to estimate the production of ACE inhibitory hydrolysates from egg albumen.
The aim of this study was to produce egg albumen hydrolysate with ACE inhibitory activity and to identify its antihypertensive effect in vivo.

Assay for ACE inhibitory activity
ACE inhibitory activity was determined by the method described in Cushman and Cheung (1971).Briefly, ACE was prepared from ACE acetone powder (Sigma, USA) combined with 4 times of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl, stirring overnight at 4°C and centrifugation (8,000×g for 30 min).
Each 5 µl of sample solution (20 mg/ml) was preincubated with 12.5 mM hippuryl-l-histidyl-L-leucine (Hip-His-Leu, Sigma, USA) dissolved in 0.1 M sodium borate buffer (pH 8.3) at 37°C for 5 min.One hundred fifty microliter (µl) of ACE was added for incubation at 37°C for 1 h and the liberated hippuric acid was extracted with ethyl acetate (Sigma, USA).ACE inhibition value (%) was calculated by measuring the absorbances at 228 nm.IC 50 value was defined as the concentration of peptide (µg/ml) required to reduce 50% of the inhibition ACE activity.

Molecular mass distribution
Molecular mass distributions of the hydrolysates were determined by gel permeation chromatography.The TSK gel G3000 PWXL (7.8 mm×30 cm, Tosho) was fitted for HPLC system (Jasco, Japan).The mobile phase consisted of 50% acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA, Sigma).The eluate at flow rate of 0.3 ml/min was detected with UV detector at 220 nm.The hydrolysate was diluted in 0.2% mobile phase that was protein-filtered through 0.45 µm syringe filter (Whatman, England), and 50 µl of hydrolysate was applied to the column.The standards, aprotinin (6,500 Da), insulin B fragment (3,495 Da), bradykinin (1,060 Da), GSH (307 Da), and Try (181 Da), were purchased from Sigma, USA.

Measurement of systolic blood pressure
Spontaneously hypertensive male rats (SHR), 14-17 week old and 210-230 g in body weight, were obtained from Samtako Inc. (Korea).They were housed in transparent plastic cages with stainless wire lids in the animal facility of National Livestock Research Center in Korea.SHR was fed standard laboratory diet and had free access to water.Then SHR was randomly assigned to 5 rats per each group and systolic blood pressure (SBP) of SHR was measured as follows.The hydrolysate was dissolved in distilled water and then emulsified with egg yolk by using ultrasonic cleaner for 5 min.Hydrolysate or emulsified hydrolysate was orally administered in SHR by metal zonde in a volume of 0.5 ml.After SHR was kept at 45°C for 10-15 minutes, SBP was measured by tail-cuff method with Power Lab 800 (AD Instruments PtyLtd, Australia).

Statistical analysis
All results were expressed as the mean±standard errors.In the IC 50 value, significance between the groups was determined by Duncan's multiple range after ANOVA.The statistical significance for antihypertensive test was determined by Student's t-test.

RESULTS AND DISCUSSION
For the production of ACE inhibitory hydrolysates, egg albumen was hydrolyzed with several proteases.Table 1 showed the IC 50 value, molecular mass distribution and recovery yields.ACE inhibitory activity of neutrase hydrolysate was the highest with IC 50 value of 236.67 µg/ml.IC 50 ; value of protamax and summizyme MP hydrolysate was 283.73 µg/ml and 286.33 µg/ml, respectively.A hydrolysate digested by alcalase presented the lowest ACE inhibitory activity with 878.35 µg/ml.From the results, the ACE inhibitory activities of egg albumen hydrolysates were dependent on the kind of protease.Enzymatic hydrolysis plays an important role in releasing ACE inhibitory peptides from inactive form within the sequence of proteins (Mullally et al., 1997).The diverse ACE inhibitory activity of the hydrolysates was probably due to the presence of different peptide sequence (Yeum et al., 1992).Neutrase hydrolysate showed the highest ACE inhibitory activity, and was mainly consisted of small peptides less than 1,000 Da in molecular weight.Our result was similar to Hyun and Shin's (2000) report.Recovery yield of neutrase hydrolysate was 35.59%, which was the higher than that of any other hydrolysates.
Table 2 presented the effects of enzyme combinations on the production of ACE inhibitory hydrolysates.The data indicated the little difference between hydrolysates, and decreased by neutrase treatment alone.In addition, recovery yield of hydrolysates with all enzyme combination was rather lower than that of neutrase treatment alone.Hydrolysis was performed at 47°C for 24 h.Enzyme: substrate ratio=1:100 (w/w). 1)Amounts of inhibitors needed for ACE activity inhibition by 50%.
2) Values present the area within a defined molecular mass distribution, expressed as percent for total area of chromatogram at 220 nm.Decreased ACE inhibitory activity in enzyme combination was probably due to the presence of carboxyl peptidase that may cause the further degradation of ACE inhibitory peptides (Pihlanto-Leppälä et al., 2000).Table 3 indicated the effect of enzyme concentration on ACE inhibitory activity and recovery yield.Up to 5% enzyme addition, ACE inhibitory activity and recovery yield were increased.With 1.0% enzyme addition, IC 50 value of hydrolysate was 258.85 µg/ml, but there was no significant difference (p<0.05) up to 5%.The recovery yield was, however, increased by the additional amounts of enzyme.Table 4 indicated ACE inhibitory activity of the hydrolysates according to incubation time.The ACE inhibitory activity was the highest at 4 h incubation.The result implied that the highest activity of egg albumen hydrolysate could be obtained in the early stage hydrolysis, and more than 4 h incubation could not be useful for increasing ACE inhibitory activity.Matsui et al. (1993) also found that with an addition of 1 and 2% of alcalase (Bacillus licheniformis) for the hydrolysis of sardine muscle, the maximum activity was revealed within 1 h incubation in proteolysis and then leveled off.
It was concluded from all the above results that neutrase (from Bacillus subtilus) was the most adequate enzyme for high productivity of ACE inhibitory hydrolysate, and that the best hydrolysis was fulfilled in 1% enzyme addition and 4 h incubation at 47°C.
There are several reports on the antihypertensive effects of food protein in relation to the functional peptides in rat and human feeding studies (Suestna and Osajima, 1989;Sekiya et al., 1992;Yamamoto et al., 1994;Nakamura et al., 1996;Hata, et al., 1996;Eto et al., 1999).In SHR experiment, SBP were measured at 0, 3, 6 h after 150 mg/kg and 300 mg/kg oral administration.The average SBP was 206.31±11.46mmHg before oral administration.Figure 1 showed the change of SBP after administration of the neutrase hydrolysate that was decreased by 6.88% (-14.14   mmHg, p<0.05), observed in 3 h after 300 mg/kg oral administration.However the decreased SBP went back up to the initial level after 24 h (data not shown).These results suggest that ACE inhibitory peptide has a temporary antihypertensive effect by single oral administration (Nakamura et al., 1995).
Although peptides and proteins are more important for drug substance, most peptides and proteins are not stable and, therefore, not effective by oral given (Hilderbrand and Tack, 2000).To avoid destruction of peptide and protein by intestinal proteases, emulsification and microencapsulation techniques have been frequently used (Shively, 1997).Figure 2 showed an antihypertensive effect of oral administration of emulsified egg albumen hydrolysate.SBP was decreased by 13.33% (-27.72 mmHg, p<0.05) after 3 h, at a dose of 300 mg/kg hydrolysate.The decrease was larger than that of administration dissolved in distilled water without emulsification.The egg yolk itself had no effect on the antihypertention.The data have supported that the higher antihypertensive effect of ovokinin was potentiated by emulsion, and phophatidylcholine in egg yolk played an important role in the potent antihypertensive activity by helping the protection from peptidase and the improved intestinal absorption of ACE inhibitory peptides (Fujita et al., 1995).

CONCLUSION
For the production of ACE inhibitory hydrolysates, egg albumen was hydrolyzed with several proteases.The most active hydrolysate was observed by neutrase hydrolysis of egg albumen.IC 50 value of neutrase hydrolysate was 236.7 µg/ml and the best hydrolysis was fulfilled in 1.0% enzyme addition and 4 h incubation at 47°C.In measuring the antihypertensive effect of emulsified egg albumen hydrolysate, SBP was decreased by 3.33% (-27.72 mmHg, p<0.05) after 3 h, at a dose of 300 mg/kg.The decrement was larger than that of administration dissolved in distilled water without emulsification.These results showed that it is very effective to utilize egg albumen as a protein source for the production of ACE inhibitory peptides.However, further studies are required to investigate the methods to increase recovery yield and the isolation of active peptide is necessary for determining its sequence responsible for ACE inhibitory activity.Economical analysis is required for the industrial usage of egg albumen to produce ACE inhibitory hydrolysate as well.

Figure 1 .Figure 2 .
Figure1.Effects of oral administration of egg albumen hydrolysates on spontaneously hypertensive rats.The change in relative blood pressure at each time was calculated as follows: (Blood pressure-Blood pressure at 0 h)/(Blood pressure at 0 h)×100.Treatments were control (ο), 150 mg/kg (■), and 300 mg/kg of body weigh (•) and significant difference between groups was revealed in comparison to the control (* p<0.05, ** p<0.01; t-test, n=5).

Table 1 .
Recovery yields, IC 50 values and molecular weight distribution of egg albumen hydrolysates

Table 2 .
Recovery yields and IC 50 values obtained egg albumen hydrolysate with enzyme combinations a, b Within same rows, means with different superscripts are significantly different (p<0.05).

Table 3 .
Effects of enzyme concentrations on recovery yields and IC 50 values of hydrolysates

Table 4 .
Effects of hydrolysis on recovery yields and IC 50 values of hydrolysates in time dependent manner a-d Within same rows, means with different superscripts are significantly different (p<0.05).