# Angiotensin-I-Converting Enzyme Inhibitory Activity and Antioxidant Properties of Cryptides Derived from Natural Actomyosin ofCatla catla Using Papain

Authors: Krishnamoorthy Elavarasan; Bangalore Aswathnarayan Shamasundar
Journal: Journal of Food Quality
 (2018)

---

## Abstract

Natural actomyosin (NAM) from the freshwater fishCatla catla was extracted and hydrolyzed using papain enzyme at different enzyme-to-substrate ratios (0.5%, 1.0%, 2.0%, 5.0%, and 10%) to obtain the cryptides with different degrees of hydrolysis (DH). Derived cryptides were evaluated for bioactive properties such as angiotensin-I-converting enzyme (ACE) inhibitory activity and antioxidant properties. The pattern of hydrolysis of NAM as a function of time revealed that major protein components such as myosin and actin were hydrolyzed within 10 min of hydrolysis. The cryptides obtained with the DH of 29.4% had significantly higher ACE inhibitory activity and linoleic acid peroxidation inhibitory activity (P<0.05). A higher DPPH free radical-scavenging activity and ferric-reducing power were exhibited by the NAM cryptide mixture obtained with the DH of 17.38 and 26.2%, respectively. The natural actomyosin could be a potential precursor to produce the cryptides with therapeutical and antioxidant properties.


---

## Body

## 1. Introduction

In the last decade, the quantum of research on peptides derived from food proteins using enzymes has increased because of their health beneficial properties. Bioactive peptides or cryptides are peptide fragments that were encrypted in the primary sequences of proteins with different functions. Once they are released by hydrolysisin vivo or in vitro by proteases, cryptides confer positive health-promoting properties other than their basic nutritional role. Identifying a suitable protein source and the proteolytic enzyme for the preparation of cryptides is critical. It has been reported that hydrolysis of individual protein constituents yielded peptides with higher bioactivity than hydrolyzing the complex raw material [1]. The possible conformational changes of protein substrates and coexistence of multiple protein substrates may affect the accessibility and susceptibility of peptide bonds to proteolysis and subsequently the release of peptides of desired bioactivities [2]. This underscores the importance of using individual protein constituents during bioactive peptide preparation.Bioactive properties of the fish protein hydrolysate (mixture of peptides) prepared using processing by-products and underutilized fish species have been reported, and a few have reached the commercial market. Knowledge on critical process parameters such as major and minor protein constituents in the raw material, enzyme-to-substrate ratio, pH, temperature, and enzyme specificity is essential to produce multifunctional peptides or different peptides, each contributing to a specific function [2]. One common measure widely used during proteolysis is the degree of hydrolysis which can be used as a tool to monitor the cryptide production on commercial scale. In spite of extensive research on the fish protein hydrolysate, the studies on fish protein model systems like actomyosin are scarce, and such studies are essential to have more insight into the hydrolysis process and properties of cryptides.Studies have revealed that the proteins from aquatic sources are high-quality raw materials for the preparation of therapeutic cryptides [2]. However, studies on bioactive properties of peptides from individual protein constituents of fish are limited. Natural actomyosin referred to the actomyosin preparation contains mainly myosin and actin in association with other regulatory proteins. The actomyosin complex from fish has been well studied with reference to the functional properties particularly the gel-forming ability, an important property in fish product development [3].To date, ACE inhibitory and/or antihypertensive activity and antioxidant properties are probably the most intensively studied properties of bioactive peptides. Angiotensin-I-converting enzyme (ACE; EC 3.4.15.1) participates in the renin-angiotensin system and plays an important physiological role in regulating blood pressure. ACE is a peptidyl dipeptidase A and primarily cleaves a decapeptide (angiotensin-I) to an octapeptide (angiotensin-II) which is a potent vasoconstrictor. ACE also inactivates the dilatational function of bradykinin [4]. Therefore, inhibition of ACE activity is a major target in the prevention of hypertension.Lipid oxidation is one of the major issues in the food industry, and the end products of lipid oxidation are potentially toxic to human health, which also affect the quality of food [5]. The oxidation of lipids leads to liberation of free radicals which are highly reactive and damage the biological macromolecules such as DNA, RNA, proteins, and enzymes. As a result, it causes cancers, neurological disorders, early ageing, Parkinson’s and Alzheimer’s diseases, and rheumatic and coronary heart diseases. Peptides/protein hydrolysates derived from fish proteins have the potential to minimize the oxidation of lipids during processing and storage of foods [6]. Synthetic antioxidants do possess higher antioxidative properties than the natural counterparts, but there is a concern about their safety on long-term usage. For investigating the antioxidant activity of derived peptides, selection of right assays is highly critical. Most commonly, the antioxidant potential is assayed through different types of assays. There are assays associated with lipid peroxidation, including the thiobarbituric acid assay (TBA). Other types of assays associated with the electron or proton donation mechanism include the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assay. In the present study, the linoleic acid peroxidation model system, DPPH free radical-scavenging activity, and ferric-reducing antioxidant power were employed to evaluate the ability of peptides derived from natural actomyosin to donate the electron/proton.With this background, the present study was aimed to prepare the cryptides from the natural actomyosin from the fishCatla catla using papain and to study their bioactive properties. Papain is the most studied cysteine enzyme due its commercial importance. Papain has been used to release the bioactive peptides from various food proteins [7] and is having broader specificity towards hydrolyzing the peptide bonds. In the present study, the hydrolysis pattern of NAM by papain was profiled. The bioactive properties such as ACE inhibitory activity and antioxidant properties of cryptides were evaluated as influenced by the extent of hydrolysis.


## 2. Materials and Methods

### 2.1. Fish

Fresh water carp,Catla catla, is harvested from the fish farm in College of Fisheries, Mangalore, Karnataka Province, India. The fish was washed in chilled water, eviscerated, and beheaded. Meat was separated manually and subjected to water washing using chilled potable water (4 ± 1°C). The quantity of water used for washing was 1 : 3 (meat : water, w/v). The slurry was agitated for 3 min and allowed to settle for 7–10 min. Water was decanted and filtered through the muslin cloth. The excess water was removed manually by squeezing the mince by placing between coarse cloths. Water washing was carried out to remove the sarcoplasmic protein fractions and lipids. Water-washed meat was used to prepare the natural actomyosin (NAM).


### 2.2. Chemicals

Papain (from the latex ofCarica papaya, ≥3 U/mg), angiotensin-I-converting enzyme (ACE) (lyophilized powder from the rabbit lung), N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals, iron(II) chloride, linoleic acid, tyrosine, sodium dodecyl sulphate, acrylamide, bis-acrylamide (N,N’-methylene-bis-acrylamide), 2-mercaptoethanol, Trizma base (tris[hydroxyl methyl]aminomethane), Coomassie Blue G and protein molecular weight markers (wide range, MW 205 kDa to 65 kDa), TEMED (N,N,N1,N1-tetramethylethylenediamine), and bromophenol blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.


### 2.3. Preparation of Natural Actomyosin (NAM) fromC. catla

NAM was prepared according to the method of Chaijan et al. [8] with a slight modification. Water-washed meat of catla (100 g) was homogenized in 500 ml of chilled phosphate buffer (pH 7) containing 0.6 M KCl for 4 min using a homogenizer at 9000 rpm (ULTRA-TURRAX T25, IKA Labortechnik, Staufen, Germany). The homogenization was carried out with a short span of 20 s followed by a stoppage for 20 s. The total time for actual homogenization was 2 min. The homogenate was kept in ice for 30 min to settle and subjected to centrifugation at 9000 ×g for 30 min at 4°C in a refrigerated centrifuge (Sorvall Legend XTR centrifuge, Thermo Fisher Scientific, New Hampshire, USA). The supernatant obtained was added slowly to ninefold of chilled double-distilled water (<5°C) and allowed to stand for 1 h in an ice bath to precipitate. The pellet was collected by centrifuging at 9000 ×g for 30 min at 4°C. The pellet obtained was referred as NAM. Total nitrogen content of the NAM pellet was analyzed by the Kjeldahl method as described in AOAC (2002) and multiplied by a factor of 6.25 to quantify the protein content. NAM was stored in refrigerator and used for hydrolysis within 48 h.


### 2.4. Preparation of Cryptides from Natural Actomyosin Using Papain

The NAM pellet of 100 g (3 g of protein) was dispersed in 200 ml of chilled double-distilled water and homogenized at 9000 rpm using the homogenizer for 2 min. Papain at different concentrations was used for hydrolysis. The concentration of the enzyme was based on protein content of NAM. The concentrations of the enzyme used were 0.5%, 1.0%, 2.0%, 5.0%, and 10%. The NAM homogenate was preincubated at 50°C for 3 min prior to the addition of enzyme at different concentrations. The homogenate without papain was served as control. The reaction mixture was incubated for 1 h at 50°C, and the pH was 6.5 ± 0.2. After incubation, the reaction was terminated by keeping the mixture in a boiling water bath for 15 min. The slurry was filtered, and the supernatant obtained was referred as NAM cryptides, stored under refrigerated conditions, and used for the analysis within 48 h.


### 2.5. SDS-PAGE Profile of NAM

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the method described by Laemmli [9]. The samples (75 µg of protein) were loaded into the wells of the polyacrylamide gel (10% running and 4% stacking). The run was carried out on a constant-voltage mode of 30 V using the power pack (model PS-3000, Hofer Pharmacia Biotech Inc., Halliston, USA) till the samples reached the end of the stacking gel. Furthermore, the voltage was raised to 90 V, and the run was terminated when the dye front reached the bottom of the gel. A standard molecular weight marker of wide range was loaded into a separate well of the gel. After the run, the gel was stained in Coomassie Brilliant Blue G-250 (0.025% in 40% methanol and 7% acetic acid) for 30–40 min. The gels were destained using the acetic acid-methanol mixture (7% acetic acid and 2% methanol) till the protein bands were clearly visible. The molecular weight of the bands obtained in the sample was approximated by measuring the relative mobility of the standard protein markers.


### 2.6. Pattern of Cryptide Liberation from NAM by Papain

The NAM prepared from catla was subjected to proteolysis with papain using the following conditions: E/S ratio of 2.5 : 100, temperature of 50°C, pH of 6.5 ± 0.2, and duration of hydrolysis of 1 h. Aliquot samples were drawn from the reaction chamber and used for SDS-PAGE analysis to profile the generation of cryptides. The SDS-PAGE (10 and 15% gel) pattern was obtained after 10, 20, 30, 40, 50, and 60 min of hydrolysis. The NAM without enzyme incubated for 60 min at 50°C was used as control.


### 2.7. Monitoring the Extent of Proteolysis

#### 2.7.1. Degree of Hydrolysis

Degree of hydrolysis was calculated as the ratio ofα-amino nitrogen liberated from the NAM and total nitrogen content of NAM taken for the hydrolysis. The α-amino nitrogen was determined by formol titration according to the method as described by Taylor [10], and the total protein nitrogen was determined by the Kjeldahl method [11]. The following formula was used to calculate the degree of hydrolysis:(1)DH%=AAN×TVSWM×TN×100,where AAN is the α-amino nitrogen (mg/ml of the supernatant), TVS is the total volume of the supernatant (ml), TN is the total nitrogen content (mg/g of NAM), and WM is the weight of NAM taken for hydrolysis (g).


### 2.8. Tyrosine Measurement

The extent of hydrolysis was also monitored by measuring the liberated tyrosine. The supernatant (150µl) obtained after hydrolysis was diluted to 3 ml using double-distilled water, and the absorbance was measured at 280 nm using a double-beam UV-Vis spectrophotometer (Labomed, Inc., Los Angeles, CA, USA). A standard curve of L-tyrosine was used to quantify the liberated tyrosine from NAM and expressed as µM of tyrosine liberated/g of protein.


### 2.9. Bioactive Properties of NAM Cryptides Derived Using Papain

#### 2.9.1. Angiotensin-I-Converting Enzyme Inhibitory Activity

The angiotensin-I-converting enzyme inhibitory (ACE) activity of NAM cryptides was determined according to the method described by Raghavan and Kristinsson [12] with the modifications described by Elavarasan et al. [13]. A known concentration of NAM cryptide solution (1 mg/ml) was prepared and used for the ACE inhibition assay. ACE enzyme (100 µl of 30 mU enzyme), 200 µl of cryptide solutions, and substrate (2 ml of 0.5 mM FAPGG substrate) were mixed, and the absorbance at 340 nm was continuously monitored with a double-beam spectrophotometer in kinetic mode option. The absorbance at 340 nm was monitored for 20 min at 25°C. The slope of the curve was used to calculate the percentage of ACE inhibition. A sample containing the FAPGG substrate and the ACE enzyme was used as control. ACE inhibitory activity of NAM cryptides was calculated as follows:(2)%ofACEinhibition=1−slopeofthesamplecurveslopeofthecontrolcurve×100,where sample is the mixture of the substrate, enzyme, and hydrolysate or inhibitor, and control is the mixture of the enzyme and substrate.


### 2.10. Antioxidant Properties

#### 2.10.1. Diphenyl-1-picrylhydrazyl (DPPH) Free Radical-Scavenging Activity

The DPPH free radical-scavenging activities of NAM cryptides was determined according to the method described by Yen and Wu [14]. The solution of NAM cryptides at a known concentration (1 mg/ml) was prepared by dissolving them in double-distilled water. A known volume of 1.5 ml was added to 1.5 ml of 0.1 mM DPPH in 99.50% ethanol and mixed thoroughly by vortexing using a cyclomixer at high speed. The solution was stored at room temperature in dark for 30 min. The absorbance was measured at 517 nm using a double-beam spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. DPPH radical-scavenging activity was calculated as follows:(3)DPPHfreeradical−scavengingactivty%=1−AbssampleAbscontrol×100.Appropriate control was maintained along with double-distilled water. The analysis was carried out in triplicate.


### 2.11. Ferric-Reducing Power Assay

The ferric-reducing power of NAM cryptides was determined by the method as described by Oyaizu [15]. An aliquot of 1 ml of the sample (1 mg/ml) was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% (w/v) potassium ferric cyanide. The mixture was incubated at 50°C for 30 min, and the reaction was stopped by addition of 2.5 ml of 10% (w/v) trichloroacetic acid. Finally, 2.5 ml of solution from the mixture was drawn and mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) ferric chloride solution. The solution was incubated for 10 min, and the absorbance was measured at 700 nm using a double-beam spectrophotometer. Higher absorbance of the reaction mixture indicated higher reducing power. The test was carried out in triplicate.


### 2.12. Linoleic Acid Peroxidation Inhibition Activity

The linoleic acid peroxidation inhibition (LAPI) activity of NAM cryptides was measured according to the method described by Osawa and Namiki [16]. NAM cryptide solution at a known concentration (3 mg/ml) was mixed with 10 ml of 50 mM phosphate buffer (pH 7.0). To this, a solution of 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol was added. The total volume was then adjusted to 25 ml with distilled water. The mixture was incubated in a 30 ml assay tube with a screw cap at 40 ± 1°C for 5 days in a hot air oven. The tubes were wrapped with aluminum foil and brown paper to prevent the entry of light. The degree of oxidation of linoleic acid was measured using the ferric thiocyanate method [17]. To 0.1 ml of the reaction mixture, 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 20 mM ferrous chloride solution in 3.5% HCl were added. After 3 min of incubation, the colour was measured at 500 nm using a double-beam spectrophotometer. The phosphate buffer (50 mM; pH 7.0) served as control. The ability of NAM cryptides to inhibit the peroxide formation in linoleic acid was calculated using the following formula:(4)Lipidperoxidationinhibition%=1−AbssampleAbscontrol×100.


### 2.13. Data Analysis

Experiments were carried out in triplicates, and data were subjected to analysis of variance (ANOVA). The significant difference in mean values was analyzed using Duncan’s multiple range mean comparison test using statistics programme (SPSS.16.0 for windows, SPSS Inc., Chicago, IL).


## 2.1. Fish

Fresh water carp,Catla catla, is harvested from the fish farm in College of Fisheries, Mangalore, Karnataka Province, India. The fish was washed in chilled water, eviscerated, and beheaded. Meat was separated manually and subjected to water washing using chilled potable water (4 ± 1°C). The quantity of water used for washing was 1 : 3 (meat : water, w/v). The slurry was agitated for 3 min and allowed to settle for 7–10 min. Water was decanted and filtered through the muslin cloth. The excess water was removed manually by squeezing the mince by placing between coarse cloths. Water washing was carried out to remove the sarcoplasmic protein fractions and lipids. Water-washed meat was used to prepare the natural actomyosin (NAM).


## 2.2. Chemicals

Papain (from the latex ofCarica papaya, ≥3 U/mg), angiotensin-I-converting enzyme (ACE) (lyophilized powder from the rabbit lung), N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals, iron(II) chloride, linoleic acid, tyrosine, sodium dodecyl sulphate, acrylamide, bis-acrylamide (N,N’-methylene-bis-acrylamide), 2-mercaptoethanol, Trizma base (tris[hydroxyl methyl]aminomethane), Coomassie Blue G and protein molecular weight markers (wide range, MW 205 kDa to 65 kDa), TEMED (N,N,N1,N1-tetramethylethylenediamine), and bromophenol blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used were of analytical grade.


## 2.3. Preparation of Natural Actomyosin (NAM) fromC. catla

NAM was prepared according to the method of Chaijan et al. [8] with a slight modification. Water-washed meat of catla (100 g) was homogenized in 500 ml of chilled phosphate buffer (pH 7) containing 0.6 M KCl for 4 min using a homogenizer at 9000 rpm (ULTRA-TURRAX T25, IKA Labortechnik, Staufen, Germany). The homogenization was carried out with a short span of 20 s followed by a stoppage for 20 s. The total time for actual homogenization was 2 min. The homogenate was kept in ice for 30 min to settle and subjected to centrifugation at 9000 ×g for 30 min at 4°C in a refrigerated centrifuge (Sorvall Legend XTR centrifuge, Thermo Fisher Scientific, New Hampshire, USA). The supernatant obtained was added slowly to ninefold of chilled double-distilled water (<5°C) and allowed to stand for 1 h in an ice bath to precipitate. The pellet was collected by centrifuging at 9000 ×g for 30 min at 4°C. The pellet obtained was referred as NAM. Total nitrogen content of the NAM pellet was analyzed by the Kjeldahl method as described in AOAC (2002) and multiplied by a factor of 6.25 to quantify the protein content. NAM was stored in refrigerator and used for hydrolysis within 48 h.


## 2.4. Preparation of Cryptides from Natural Actomyosin Using Papain

The NAM pellet of 100 g (3 g of protein) was dispersed in 200 ml of chilled double-distilled water and homogenized at 9000 rpm using the homogenizer for 2 min. Papain at different concentrations was used for hydrolysis. The concentration of the enzyme was based on protein content of NAM. The concentrations of the enzyme used were 0.5%, 1.0%, 2.0%, 5.0%, and 10%. The NAM homogenate was preincubated at 50°C for 3 min prior to the addition of enzyme at different concentrations. The homogenate without papain was served as control. The reaction mixture was incubated for 1 h at 50°C, and the pH was 6.5 ± 0.2. After incubation, the reaction was terminated by keeping the mixture in a boiling water bath for 15 min. The slurry was filtered, and the supernatant obtained was referred as NAM cryptides, stored under refrigerated conditions, and used for the analysis within 48 h.


## 2.5. SDS-PAGE Profile of NAM

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the method described by Laemmli [9]. The samples (75 µg of protein) were loaded into the wells of the polyacrylamide gel (10% running and 4% stacking). The run was carried out on a constant-voltage mode of 30 V using the power pack (model PS-3000, Hofer Pharmacia Biotech Inc., Halliston, USA) till the samples reached the end of the stacking gel. Furthermore, the voltage was raised to 90 V, and the run was terminated when the dye front reached the bottom of the gel. A standard molecular weight marker of wide range was loaded into a separate well of the gel. After the run, the gel was stained in Coomassie Brilliant Blue G-250 (0.025% in 40% methanol and 7% acetic acid) for 30–40 min. The gels were destained using the acetic acid-methanol mixture (7% acetic acid and 2% methanol) till the protein bands were clearly visible. The molecular weight of the bands obtained in the sample was approximated by measuring the relative mobility of the standard protein markers.


## 2.6. Pattern of Cryptide Liberation from NAM by Papain

The NAM prepared from catla was subjected to proteolysis with papain using the following conditions: E/S ratio of 2.5 : 100, temperature of 50°C, pH of 6.5 ± 0.2, and duration of hydrolysis of 1 h. Aliquot samples were drawn from the reaction chamber and used for SDS-PAGE analysis to profile the generation of cryptides. The SDS-PAGE (10 and 15% gel) pattern was obtained after 10, 20, 30, 40, 50, and 60 min of hydrolysis. The NAM without enzyme incubated for 60 min at 50°C was used as control.


## 2.7. Monitoring the Extent of Proteolysis

### 2.7.1. Degree of Hydrolysis

Degree of hydrolysis was calculated as the ratio ofα-amino nitrogen liberated from the NAM and total nitrogen content of NAM taken for the hydrolysis. The α-amino nitrogen was determined by formol titration according to the method as described by Taylor [10], and the total protein nitrogen was determined by the Kjeldahl method [11]. The following formula was used to calculate the degree of hydrolysis:(1)DH%=AAN×TVSWM×TN×100,where AAN is the α-amino nitrogen (mg/ml of the supernatant), TVS is the total volume of the supernatant (ml), TN is the total nitrogen content (mg/g of NAM), and WM is the weight of NAM taken for hydrolysis (g).


## 2.7.1. Degree of Hydrolysis

Degree of hydrolysis was calculated as the ratio ofα-amino nitrogen liberated from the NAM and total nitrogen content of NAM taken for the hydrolysis. The α-amino nitrogen was determined by formol titration according to the method as described by Taylor [10], and the total protein nitrogen was determined by the Kjeldahl method [11]. The following formula was used to calculate the degree of hydrolysis:(1)DH%=AAN×TVSWM×TN×100,where AAN is the α-amino nitrogen (mg/ml of the supernatant), TVS is the total volume of the supernatant (ml), TN is the total nitrogen content (mg/g of NAM), and WM is the weight of NAM taken for hydrolysis (g).


## 2.8. Tyrosine Measurement

The extent of hydrolysis was also monitored by measuring the liberated tyrosine. The supernatant (150µl) obtained after hydrolysis was diluted to 3 ml using double-distilled water, and the absorbance was measured at 280 nm using a double-beam UV-Vis spectrophotometer (Labomed, Inc., Los Angeles, CA, USA). A standard curve of L-tyrosine was used to quantify the liberated tyrosine from NAM and expressed as µM of tyrosine liberated/g of protein.


## 2.9. Bioactive Properties of NAM Cryptides Derived Using Papain

### 2.9.1. Angiotensin-I-Converting Enzyme Inhibitory Activity

The angiotensin-I-converting enzyme inhibitory (ACE) activity of NAM cryptides was determined according to the method described by Raghavan and Kristinsson [12] with the modifications described by Elavarasan et al. [13]. A known concentration of NAM cryptide solution (1 mg/ml) was prepared and used for the ACE inhibition assay. ACE enzyme (100 µl of 30 mU enzyme), 200 µl of cryptide solutions, and substrate (2 ml of 0.5 mM FAPGG substrate) were mixed, and the absorbance at 340 nm was continuously monitored with a double-beam spectrophotometer in kinetic mode option. The absorbance at 340 nm was monitored for 20 min at 25°C. The slope of the curve was used to calculate the percentage of ACE inhibition. A sample containing the FAPGG substrate and the ACE enzyme was used as control. ACE inhibitory activity of NAM cryptides was calculated as follows:(2)%ofACEinhibition=1−slopeofthesamplecurveslopeofthecontrolcurve×100,where sample is the mixture of the substrate, enzyme, and hydrolysate or inhibitor, and control is the mixture of the enzyme and substrate.


## 2.9.1. Angiotensin-I-Converting Enzyme Inhibitory Activity

The angiotensin-I-converting enzyme inhibitory (ACE) activity of NAM cryptides was determined according to the method described by Raghavan and Kristinsson [12] with the modifications described by Elavarasan et al. [13]. A known concentration of NAM cryptide solution (1 mg/ml) was prepared and used for the ACE inhibition assay. ACE enzyme (100 µl of 30 mU enzyme), 200 µl of cryptide solutions, and substrate (2 ml of 0.5 mM FAPGG substrate) were mixed, and the absorbance at 340 nm was continuously monitored with a double-beam spectrophotometer in kinetic mode option. The absorbance at 340 nm was monitored for 20 min at 25°C. The slope of the curve was used to calculate the percentage of ACE inhibition. A sample containing the FAPGG substrate and the ACE enzyme was used as control. ACE inhibitory activity of NAM cryptides was calculated as follows:(2)%ofACEinhibition=1−slopeofthesamplecurveslopeofthecontrolcurve×100,where sample is the mixture of the substrate, enzyme, and hydrolysate or inhibitor, and control is the mixture of the enzyme and substrate.


## 2.10. Antioxidant Properties

### 2.10.1. Diphenyl-1-picrylhydrazyl (DPPH) Free Radical-Scavenging Activity

The DPPH free radical-scavenging activities of NAM cryptides was determined according to the method described by Yen and Wu [14]. The solution of NAM cryptides at a known concentration (1 mg/ml) was prepared by dissolving them in double-distilled water. A known volume of 1.5 ml was added to 1.5 ml of 0.1 mM DPPH in 99.50% ethanol and mixed thoroughly by vortexing using a cyclomixer at high speed. The solution was stored at room temperature in dark for 30 min. The absorbance was measured at 517 nm using a double-beam spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. DPPH radical-scavenging activity was calculated as follows:(3)DPPHfreeradical−scavengingactivty%=1−AbssampleAbscontrol×100.Appropriate control was maintained along with double-distilled water. The analysis was carried out in triplicate.


## 2.10.1. Diphenyl-1-picrylhydrazyl (DPPH) Free Radical-Scavenging Activity

The DPPH free radical-scavenging activities of NAM cryptides was determined according to the method described by Yen and Wu [14]. The solution of NAM cryptides at a known concentration (1 mg/ml) was prepared by dissolving them in double-distilled water. A known volume of 1.5 ml was added to 1.5 ml of 0.1 mM DPPH in 99.50% ethanol and mixed thoroughly by vortexing using a cyclomixer at high speed. The solution was stored at room temperature in dark for 30 min. The absorbance was measured at 517 nm using a double-beam spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity. DPPH radical-scavenging activity was calculated as follows:(3)DPPHfreeradical−scavengingactivty%=1−AbssampleAbscontrol×100.Appropriate control was maintained along with double-distilled water. The analysis was carried out in triplicate.


## 2.11. Ferric-Reducing Power Assay

The ferric-reducing power of NAM cryptides was determined by the method as described by Oyaizu [15]. An aliquot of 1 ml of the sample (1 mg/ml) was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% (w/v) potassium ferric cyanide. The mixture was incubated at 50°C for 30 min, and the reaction was stopped by addition of 2.5 ml of 10% (w/v) trichloroacetic acid. Finally, 2.5 ml of solution from the mixture was drawn and mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) ferric chloride solution. The solution was incubated for 10 min, and the absorbance was measured at 700 nm using a double-beam spectrophotometer. Higher absorbance of the reaction mixture indicated higher reducing power. The test was carried out in triplicate.


## 2.12. Linoleic Acid Peroxidation Inhibition Activity

The linoleic acid peroxidation inhibition (LAPI) activity of NAM cryptides was measured according to the method described by Osawa and Namiki [16]. NAM cryptide solution at a known concentration (3 mg/ml) was mixed with 10 ml of 50 mM phosphate buffer (pH 7.0). To this, a solution of 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol was added. The total volume was then adjusted to 25 ml with distilled water. The mixture was incubated in a 30 ml assay tube with a screw cap at 40 ± 1°C for 5 days in a hot air oven. The tubes were wrapped with aluminum foil and brown paper to prevent the entry of light. The degree of oxidation of linoleic acid was measured using the ferric thiocyanate method [17]. To 0.1 ml of the reaction mixture, 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 20 mM ferrous chloride solution in 3.5% HCl were added. After 3 min of incubation, the colour was measured at 500 nm using a double-beam spectrophotometer. The phosphate buffer (50 mM; pH 7.0) served as control. The ability of NAM cryptides to inhibit the peroxide formation in linoleic acid was calculated using the following formula:(4)Lipidperoxidationinhibition%=1−AbssampleAbscontrol×100.


## 2.13. Data Analysis

Experiments were carried out in triplicates, and data were subjected to analysis of variance (ANOVA). The significant difference in mean values was analyzed using Duncan’s multiple range mean comparison test using statistics programme (SPSS.16.0 for windows, SPSS Inc., Chicago, IL).


## 3. Results and Discussion

### 3.1. SDS-PAGE Pattern of NAM

The SDS-PAGE pattern of NAM fromC. catla is presented in Figure 1. The pattern revealed multiple bands with the prominent one being the component of 200 kDa which is the myosin heavy chain. The NAM comprises subunits of actin and myosin and other components such as tropomyosin and troponin. The SDS-PAGE pattern of actomyosin from C. catla is similar to that of actomyosin from other fish species [18, 19]. Earlier studies on purification of actomyosin reported that, along with actomyosin, other myofibrillar proteins such as the tropomyosin-troponin complex are also extracted during purification [19, 20].Figure 1
SDS-PAGE profile of natural actomyosin fromCatla catla (lane A: standard molecular weight markers; lane B: natural actomyosin preparation).


### 3.2. Pattern of Cryptide Liberation from NAM by Papain

The SDS-PAGE (10% gel) profile of cryptides released from NAM is given in Figure2. The major protein components in NAM were myosin heavy chains (MHCs), actin, tropomyosin, troponin, and myosin light chains. No degradation was found in the muscle protein profile during the incubation period of 60 min at 50°C without the addition of papain (Supplementary Figure 1). Yongsawatdigul and Park [21] reported no evidence of myosin heavy chain (MHC) or actin degradation in the actomyosin isolated from Pacific whiting in the temperature range of 20–80°C. However, the intensity of the MHC band decreased slightly as incubation time increased. In the sample where the papain was added, major muscle protein fractions including myosin heavy chains and actin were found to be cleaved within 10 min (Figure 2). The intensity of the band below the dye front increased with increasing time of proteolysis, indicating that the hydrolysis by papain resulted in the formation of low-molecular-weight cryptides. The SDS-PAGE pattern of cryptide generation in 15% gel showed intensive bands diffused in the approximate molecular weight mass region of less than 6.5 kDa (Supplementary Figure 1). A peptide chain with the approximate molecular weight mass of 26 kDa was detected in both 10 and 15% gel. Ha et al. [22] reported the stability of C-reactive protein (140 kDa), α-actinin (90 kDa), tropomyosins (35 kDa), and troponins (22 and 17.8 kDa) from topside myofibril extracts against the activity of commercial papain preparation. Crude papain has been reported to cleave the myofibrillar proteins from the chicken muscle rapidly [23]. The subsets B and C of given Supplementary Figure 1 (SDS-PAGE profile of peptides released during hydrolysis of natural actomyosin) show the hydrolysis changes in the sample where papain was not added. A peptide fraction around 26 kDa, suspected to be troponin, was found to be resistant to hydrolysis by papain. Similarly, the subset C of Supplementary Figure 1 also indicated the degradation of the major protein fractions myosin and actin within 20 min of hydrolysis reaction.Figure 2
SDS-PAGE (10% gel) pattern of natural actomyosin during hydrolysis at 50°C for 60 min by the papain enzyme (lane M: wide-range molecular weight markers; lanes 0, 10, 20, 30, 40, 50, and 60: time of hydrolysis (min); lane L: low-molecular-weight markers).


### 3.3. Hydrolysis of NAM by Papain

Natural actomyosin was hydrolyzed using papain at different enzyme-to-substrate ratios, namely, 0.5%, 1.0%, 2.5%, 5.0%, and 10.0%. The degree of hydrolysis and the liberated tyrosine content are presented in Figure3. The degree of hydrolysis and the liberated tyrosine content increased with the increase in enzyme-to-substrate ratio. There exists a good correlation between the liberated tyrosine content and degree of hydrolysis. The results indicated that lower E/S yielded a higher degree of hydrolysis. The high degree of hydrolysis at low E/S indicated that a large number of peptide bonds were hydrolyzed. Thereafter, the degree of hydrolysis increased marginally, mainly due to a decrease in available sites for cleavage. The degree of hydrolysis obtained for different E/S ratios varied from 3.5 to 29.4%. Higher degree of hydrolysis will yield more of low-molecular-weight cryptides. Papain has been reported to be more efficient in hydrolyzing the myofibrillar proteins [24]. The NAM cryptides obtained with different degrees of proteolysis were evaluated for their bioactive properties. Although papain is nonspecific in action, the preference for cleavage of the peptide bond is more between arginine and lysine. The specificity of papain for cleavage of the X-Y bond is as follows: where X is a nonspecific amino acid, but arginine and lysine are preferred; the phenylalanine-X-Y bond where residues following phenylalanine are preferred; and Y is a nonspecific amino acid residue. The protein sequences of fish species were retrieved from the UniProt database, and the presence of the number of arginine, lysine, and phenylalanine was documented. The results are presented in Table 1. The myosin heavy chain contains more number of arginine, lysine and phenylalanine. Hence, more number of peptides is expected from the myosin heavy chain.Figure 3
Hydrolysis of natural actomyosin (NAM) from the freshwater fishCatla catla using the papain enzyme at different enzyme-to-substrate ratios.Table 1
Number of preferred amino acid residues for cleavage sites in the sequence of myosin, actin, tropomyosin, and troponin.
Protein
Accession number
Total number of residues
Lysine
Arginine
Phenylalanine-X
Myosin heavy chain
UniProtKB-A8R0Q4 (A8R0Q4_HYPMO)
1933
210
99
56
Actin
UniProtKB-S4U1R3 (S4U1R3_HYPMO)
377
19
18
13
Troponin T
UniProtKB-A0A0F8B0M6 (A0A0F8B0M6_LARCR)
170
20
25
5
Troponin I
(UniProtKB-Q90366 (Q90366_CLUHA)
176
29
7
2
Troponin C
UniProtKB-B9VJM4 (B9VJM4_SINCH)
160
8
7
10
Tropomyosin
UniProtKB-A0A0D5MCW6 (A0A0D5MCW6_CTEID)
284
38
14
1
Myosin LC-1b
UniProtKB-Q90332 (Q90332_CYPCA)
193
16
6
8
Myosin LC-1a
UniProtKB-Q90331 (Q90331_CYPCA)
193
16
6
8
Myosin LC-3
UniProtKB-Q90333 (Q90333_CYPCA)
151
11
5
8
Myosin RLC
UniProtKB-Q9I892 (Q9I892_CYPCA)
169
15
5
11


### 3.4. Bioactive Properties of NAM Cryptides

#### 3.4.1. ACE Inhibitory Cryptides

ACE inhibitory activity of NAM cryptides derived using papain is presented in Figure4. ACE inhibitory activity of NAM cryptides increased with the increase in degree of hydrolysis. The results clearly indicate that the papain enzyme releases the cryptides from NAM with the sequence that can inhibit the ACE enzyme. It is well known that the biological properties of cryptides to a larger extent are influenced by their molecular structure and length, which in turn are affected by degree of hydrolysis. During hydrolysis, a wide variety of larger, medium, and smaller cryptides are generated depending on enzyme specificity. Increasing the degree of hydrolysis produces low-molecular-weight cryptides. Low MW cryptides are better ACE inhibitors than high MW cryptides [12]. Based on the specificity of the papain enzyme, we expect the peptides released to have the lysine or arginine in the C-terminal and phenylalanine in the penultimate position of peptides. A potent ACE inhibitory dipeptide V-R from the Atlantic salmon skin hydrolysate prepared using papain has been identified [25]. The theoretical search for this region in the retrieved amino acid sequences of proteins indicated that this peptide could be sourced to myosin heavy chains (238-239, 669-670, 1604-1605, 1819-1819, and 1839-1840), actin (211-212), and troponin T (38-39). This sequence is absent in myosin light chains and tropomyosin. The quantitative structure-activity relationship studies on di- and tri-ACE inhibitory peptides confirmed that the presence of amino acid residues with bulky side chains and hydrophobic side chains in the carboxyl terminal was preferred for dipeptides, while that for tripeptides, the most favorable residues were aromatic amino acids. The amino acid residues with positive charge in the middle position and hydrophobic amino acid residues in the N-terminal region were preferred [26].Figure 4
Angiotensin-I-converting enzyme (ACE) inhibitory activity of cryptides obtained from natural actomyosin with different degrees of hydrolysis at a peptide concentration of 1 mg/ml. Error bars represent the standard deviation from triplicate determinations. Different capital letters on the error bars indicate that the results are significantly different (P<0.05).


### 3.5. Antioxidant Properties of NAM Cryptides

The antioxidant properties of cryptides released from natural actomyosin by the action of papain including DPPH free radical-scavenging activity, ferric-reducing antioxidant power, and linoleic acid peroxidation inhibition are shown in Figures5(a)–5(c).Figure 5
Antioxidant properties of cryptides obtained from natural actomyosin with different degrees of hydrolysis. Error bars represent the standard deviation from triplicate determinations. Different capital letters on the error bars indicate that the results are significantly different (P<0.05). (a) DPPH free radical-scavenging activity at a peptide concentration of 1 mg/ml (DPPH FRSA); (b) ferric-reducing antioxidant power (FRAP) at a peptide concentration of 1 mg/ml; (c) linoleic acid peroxidation inhibition activity (LAPI) at a peptide concentration of 3 mg/ml.
(a)
(b)
(c)The DPPH free radical-scavenging activity of NAM cryptides prepared using papain increased with the increase in DH up to 17.38%, and a further increase in DH up to 29.4% showed no significant difference in radical-scavenging activity (Figure5(a)). An increase in the degree of hydrolysis produces greater numbers of low-molecular-weight cryptides [27]. The results obtained suggest that the NAM cryptides that were electron/proton donors could react with free radicals to convert them to more stable products. These cryptides could be useful in terminating the radical chain reaction-mediated oxidation process. The appropriate DH needs to be achieved to produce the NAM cryptides with maximum functions as radical scavengers.The FRAP of NAM cryptides increased with the increase in DH up to 26.2% and showed a significant reduction at DH 29.4% (Figure5(b)). Cryptides derived from loach protein using papain showed an increase in FRAP in the earlier stage of hydrolysis (DH-23%), and further hydrolysis (DH-33%) decreased the reducing power [28]. FRAP generally measures the reducing ability against ferric ions. Cryptides with a higher reducing power have better abilities to donate electrons.The antioxidant assays such as DPPH free radical-scavenging activity and ferric-reducing antioxidant power evaluate the antioxidant properties by different mechanisms, and different specific structural requirements are associated with each mechanism of antioxidant action [29]. This may not reflect the complex mechanism through which cryptides may act as antioxidants to retard or inhibit lipid oxidation. Therefore, the ability of NAM cryptides to retard the lipid peroxidation was investigated using a linoleic acid model system. NAM cryptides showed an increase in peroxidation inhibition activity with the increase in degree of hydrolysis of NAM (Figure 5(c)). A linear relationship between the degree of hydrolysis and the antioxidant properties of cryptides from a small yellow croaker derived by papain has been reported [30]. Loach protein cryptides prepared using papain showed maximum free radical-scavenging activity at the degree of hydrolysis of 23% [31]. A potent antioxidant cryptide L-N-K has been purified from the Sphyrna lewini muscle protein hydrolysate derived using papain [29]. The lipid peroxidation inhibition activity of peptides depends on the hydrophobic nature [32]. Five antioxidant peptides, namely, DSGVT (actin), IEAEGE (unknown), DAQEKLE (tropomyosin), EELDNALN (tropomyosin), and VPSIDDQEELM (myosin heavy chain), have been purified from the porcine myofibrillar hydrolysates prepared using papain [33]. Quantitative structure-activity relationship studies on peptides with antioxidant properties indicated that the properties of amino acids at C-terminal regions are more important than those at the N-terminal regions for antioxidant activity. Bulky hydrophobic amino acids at the C-terminal were related to the antioxidant activity of cryptides in free radical systems [34]. The amino acid composition, structure, and hydrophobicity of peptides influence the antioxidative properties. In addition to this, the molecular weight of peptides can also influence the antioxidant properties [35].


## 3.1. SDS-PAGE Pattern of NAM

The SDS-PAGE pattern of NAM fromC. catla is presented in Figure 1. The pattern revealed multiple bands with the prominent one being the component of 200 kDa which is the myosin heavy chain. The NAM comprises subunits of actin and myosin and other components such as tropomyosin and troponin. The SDS-PAGE pattern of actomyosin from C. catla is similar to that of actomyosin from other fish species [18, 19]. Earlier studies on purification of actomyosin reported that, along with actomyosin, other myofibrillar proteins such as the tropomyosin-troponin complex are also extracted during purification [19, 20].Figure 1
SDS-PAGE profile of natural actomyosin fromCatla catla (lane A: standard molecular weight markers; lane B: natural actomyosin preparation).


## 3.2. Pattern of Cryptide Liberation from NAM by Papain

The SDS-PAGE (10% gel) profile of cryptides released from NAM is given in Figure2. The major protein components in NAM were myosin heavy chains (MHCs), actin, tropomyosin, troponin, and myosin light chains. No degradation was found in the muscle protein profile during the incubation period of 60 min at 50°C without the addition of papain (Supplementary Figure 1). Yongsawatdigul and Park [21] reported no evidence of myosin heavy chain (MHC) or actin degradation in the actomyosin isolated from Pacific whiting in the temperature range of 20–80°C. However, the intensity of the MHC band decreased slightly as incubation time increased. In the sample where the papain was added, major muscle protein fractions including myosin heavy chains and actin were found to be cleaved within 10 min (Figure 2). The intensity of the band below the dye front increased with increasing time of proteolysis, indicating that the hydrolysis by papain resulted in the formation of low-molecular-weight cryptides. The SDS-PAGE pattern of cryptide generation in 15% gel showed intensive bands diffused in the approximate molecular weight mass region of less than 6.5 kDa (Supplementary Figure 1). A peptide chain with the approximate molecular weight mass of 26 kDa was detected in both 10 and 15% gel. Ha et al. [22] reported the stability of C-reactive protein (140 kDa), α-actinin (90 kDa), tropomyosins (35 kDa), and troponins (22 and 17.8 kDa) from topside myofibril extracts against the activity of commercial papain preparation. Crude papain has been reported to cleave the myofibrillar proteins from the chicken muscle rapidly [23]. The subsets B and C of given Supplementary Figure 1 (SDS-PAGE profile of peptides released during hydrolysis of natural actomyosin) show the hydrolysis changes in the sample where papain was not added. A peptide fraction around 26 kDa, suspected to be troponin, was found to be resistant to hydrolysis by papain. Similarly, the subset C of Supplementary Figure 1 also indicated the degradation of the major protein fractions myosin and actin within 20 min of hydrolysis reaction.Figure 2
SDS-PAGE (10% gel) pattern of natural actomyosin during hydrolysis at 50°C for 60 min by the papain enzyme (lane M: wide-range molecular weight markers; lanes 0, 10, 20, 30, 40, 50, and 60: time of hydrolysis (min); lane L: low-molecular-weight markers).


## 3.3. Hydrolysis of NAM by Papain

Natural actomyosin was hydrolyzed using papain at different enzyme-to-substrate ratios, namely, 0.5%, 1.0%, 2.5%, 5.0%, and 10.0%. The degree of hydrolysis and the liberated tyrosine content are presented in Figure3. The degree of hydrolysis and the liberated tyrosine content increased with the increase in enzyme-to-substrate ratio. There exists a good correlation between the liberated tyrosine content and degree of hydrolysis. The results indicated that lower E/S yielded a higher degree of hydrolysis. The high degree of hydrolysis at low E/S indicated that a large number of peptide bonds were hydrolyzed. Thereafter, the degree of hydrolysis increased marginally, mainly due to a decrease in available sites for cleavage. The degree of hydrolysis obtained for different E/S ratios varied from 3.5 to 29.4%. Higher degree of hydrolysis will yield more of low-molecular-weight cryptides. Papain has been reported to be more efficient in hydrolyzing the myofibrillar proteins [24]. The NAM cryptides obtained with different degrees of proteolysis were evaluated for their bioactive properties. Although papain is nonspecific in action, the preference for cleavage of the peptide bond is more between arginine and lysine. The specificity of papain for cleavage of the X-Y bond is as follows: where X is a nonspecific amino acid, but arginine and lysine are preferred; the phenylalanine-X-Y bond where residues following phenylalanine are preferred; and Y is a nonspecific amino acid residue. The protein sequences of fish species were retrieved from the UniProt database, and the presence of the number of arginine, lysine, and phenylalanine was documented. The results are presented in Table 1. The myosin heavy chain contains more number of arginine, lysine and phenylalanine. Hence, more number of peptides is expected from the myosin heavy chain.Figure 3
Hydrolysis of natural actomyosin (NAM) from the freshwater fishCatla catla using the papain enzyme at different enzyme-to-substrate ratios.Table 1
Number of preferred amino acid residues for cleavage sites in the sequence of myosin, actin, tropomyosin, and troponin.
Protein
Accession number
Total number of residues
Lysine
Arginine
Phenylalanine-X
Myosin heavy chain
UniProtKB-A8R0Q4 (A8R0Q4_HYPMO)
1933
210
99
56
Actin
UniProtKB-S4U1R3 (S4U1R3_HYPMO)
377
19
18
13
Troponin T
UniProtKB-A0A0F8B0M6 (A0A0F8B0M6_LARCR)
170
20
25
5
Troponin I
(UniProtKB-Q90366 (Q90366_CLUHA)
176
29
7
2
Troponin C
UniProtKB-B9VJM4 (B9VJM4_SINCH)
160
8
7
10
Tropomyosin
UniProtKB-A0A0D5MCW6 (A0A0D5MCW6_CTEID)
284
38
14
1
Myosin LC-1b
UniProtKB-Q90332 (Q90332_CYPCA)
193
16
6
8
Myosin LC-1a
UniProtKB-Q90331 (Q90331_CYPCA)
193
16
6
8
Myosin LC-3
UniProtKB-Q90333 (Q90333_CYPCA)
151
11
5
8
Myosin RLC
UniProtKB-Q9I892 (Q9I892_CYPCA)
169
15
5
11


## 3.4. Bioactive Properties of NAM Cryptides

### 3.4.1. ACE Inhibitory Cryptides

ACE inhibitory activity of NAM cryptides derived using papain is presented in Figure4. ACE inhibitory activity of NAM cryptides increased with the increase in degree of hydrolysis. The results clearly indicate that the papain enzyme releases the cryptides from NAM with the sequence that can inhibit the ACE enzyme. It is well known that the biological properties of cryptides to a larger extent are influenced by their molecular structure and length, which in turn are affected by degree of hydrolysis. During hydrolysis, a wide variety of larger, medium, and smaller cryptides are generated depending on enzyme specificity. Increasing the degree of hydrolysis produces low-molecular-weight cryptides. Low MW cryptides are better ACE inhibitors than high MW cryptides [12]. Based on the specificity of the papain enzyme, we expect the peptides released to have the lysine or arginine in the C-terminal and phenylalanine in the penultimate position of peptides. A potent ACE inhibitory dipeptide V-R from the Atlantic salmon skin hydrolysate prepared using papain has been identified [25]. The theoretical search for this region in the retrieved amino acid sequences of proteins indicated that this peptide could be sourced to myosin heavy chains (238-239, 669-670, 1604-1605, 1819-1819, and 1839-1840), actin (211-212), and troponin T (38-39). This sequence is absent in myosin light chains and tropomyosin. The quantitative structure-activity relationship studies on di- and tri-ACE inhibitory peptides confirmed that the presence of amino acid residues with bulky side chains and hydrophobic side chains in the carboxyl terminal was preferred for dipeptides, while that for tripeptides, the most favorable residues were aromatic amino acids. The amino acid residues with positive charge in the middle position and hydrophobic amino acid residues in the N-terminal region were preferred [26].Figure 4
Angiotensin-I-converting enzyme (ACE) inhibitory activity of cryptides obtained from natural actomyosin with different degrees of hydrolysis at a peptide concentration of 1 mg/ml. Error bars represent the standard deviation from triplicate determinations. Different capital letters on the error bars indicate that the results are significantly different (P<0.05).


## 3.4.1. ACE Inhibitory Cryptides

ACE inhibitory activity of NAM cryptides derived using papain is presented in Figure4. ACE inhibitory activity of NAM cryptides increased with the increase in degree of hydrolysis. The results clearly indicate that the papain enzyme releases the cryptides from NAM with the sequence that can inhibit the ACE enzyme. It is well known that the biological properties of cryptides to a larger extent are influenced by their molecular structure and length, which in turn are affected by degree of hydrolysis. During hydrolysis, a wide variety of larger, medium, and smaller cryptides are generated depending on enzyme specificity. Increasing the degree of hydrolysis produces low-molecular-weight cryptides. Low MW cryptides are better ACE inhibitors than high MW cryptides [12]. Based on the specificity of the papain enzyme, we expect the peptides released to have the lysine or arginine in the C-terminal and phenylalanine in the penultimate position of peptides. A potent ACE inhibitory dipeptide V-R from the Atlantic salmon skin hydrolysate prepared using papain has been identified [25]. The theoretical search for this region in the retrieved amino acid sequences of proteins indicated that this peptide could be sourced to myosin heavy chains (238-239, 669-670, 1604-1605, 1819-1819, and 1839-1840), actin (211-212), and troponin T (38-39). This sequence is absent in myosin light chains and tropomyosin. The quantitative structure-activity relationship studies on di- and tri-ACE inhibitory peptides confirmed that the presence of amino acid residues with bulky side chains and hydrophobic side chains in the carboxyl terminal was preferred for dipeptides, while that for tripeptides, the most favorable residues were aromatic amino acids. The amino acid residues with positive charge in the middle position and hydrophobic amino acid residues in the N-terminal region were preferred [26].Figure 4
Angiotensin-I-converting enzyme (ACE) inhibitory activity of cryptides obtained from natural actomyosin with different degrees of hydrolysis at a peptide concentration of 1 mg/ml. Error bars represent the standard deviation from triplicate determinations. Different capital letters on the error bars indicate that the results are significantly different (P<0.05).


## 3.5. Antioxidant Properties of NAM Cryptides

The antioxidant properties of cryptides released from natural actomyosin by the action of papain including DPPH free radical-scavenging activity, ferric-reducing antioxidant power, and linoleic acid peroxidation inhibition are shown in Figures5(a)–5(c).Figure 5
Antioxidant properties of cryptides obtained from natural actomyosin with different degrees of hydrolysis. Error bars represent the standard deviation from triplicate determinations. Different capital letters on the error bars indicate that the results are significantly different (P<0.05). (a) DPPH free radical-scavenging activity at a peptide concentration of 1 mg/ml (DPPH FRSA); (b) ferric-reducing antioxidant power (FRAP) at a peptide concentration of 1 mg/ml; (c) linoleic acid peroxidation inhibition activity (LAPI) at a peptide concentration of 3 mg/ml.
(a)
(b)
(c)The DPPH free radical-scavenging activity of NAM cryptides prepared using papain increased with the increase in DH up to 17.38%, and a further increase in DH up to 29.4% showed no significant difference in radical-scavenging activity (Figure5(a)). An increase in the degree of hydrolysis produces greater numbers of low-molecular-weight cryptides [27]. The results obtained suggest that the NAM cryptides that were electron/proton donors could react with free radicals to convert them to more stable products. These cryptides could be useful in terminating the radical chain reaction-mediated oxidation process. The appropriate DH needs to be achieved to produce the NAM cryptides with maximum functions as radical scavengers.The FRAP of NAM cryptides increased with the increase in DH up to 26.2% and showed a significant reduction at DH 29.4% (Figure5(b)). Cryptides derived from loach protein using papain showed an increase in FRAP in the earlier stage of hydrolysis (DH-23%), and further hydrolysis (DH-33%) decreased the reducing power [28]. FRAP generally measures the reducing ability against ferric ions. Cryptides with a higher reducing power have better abilities to donate electrons.The antioxidant assays such as DPPH free radical-scavenging activity and ferric-reducing antioxidant power evaluate the antioxidant properties by different mechanisms, and different specific structural requirements are associated with each mechanism of antioxidant action [29]. This may not reflect the complex mechanism through which cryptides may act as antioxidants to retard or inhibit lipid oxidation. Therefore, the ability of NAM cryptides to retard the lipid peroxidation was investigated using a linoleic acid model system. NAM cryptides showed an increase in peroxidation inhibition activity with the increase in degree of hydrolysis of NAM (Figure 5(c)). A linear relationship between the degree of hydrolysis and the antioxidant properties of cryptides from a small yellow croaker derived by papain has been reported [30]. Loach protein cryptides prepared using papain showed maximum free radical-scavenging activity at the degree of hydrolysis of 23% [31]. A potent antioxidant cryptide L-N-K has been purified from the Sphyrna lewini muscle protein hydrolysate derived using papain [29]. The lipid peroxidation inhibition activity of peptides depends on the hydrophobic nature [32]. Five antioxidant peptides, namely, DSGVT (actin), IEAEGE (unknown), DAQEKLE (tropomyosin), EELDNALN (tropomyosin), and VPSIDDQEELM (myosin heavy chain), have been purified from the porcine myofibrillar hydrolysates prepared using papain [33]. Quantitative structure-activity relationship studies on peptides with antioxidant properties indicated that the properties of amino acids at C-terminal regions are more important than those at the N-terminal regions for antioxidant activity. Bulky hydrophobic amino acids at the C-terminal were related to the antioxidant activity of cryptides in free radical systems [34]. The amino acid composition, structure, and hydrophobicity of peptides influence the antioxidative properties. In addition to this, the molecular weight of peptides can also influence the antioxidant properties [35].


## 4. Conclusion

The papain enzyme released the cryptides mainly from myosin and actin (as revealed by the SDS-PAGE profile) with angiotensin-I-converting enzyme inhibitory and antioxidant properties such DPPH free radical-scavenging activity, ferric-reducing antioxidant power, and linoleic acid peroxidation inhibition activity. The present study indicated that the fish actomyosin is a potential precursor for the production of therapeutic cryptides using papain hydrolysis and their health beneficial properties depend on the extent of hydrolysis. Further study is needed to identify the sequence of cryptides.



---

*Source: 9354829-2018-07-08.xml*