Modification of Bioactive Properties in Food Protein Hydrolysates by Alcalase and Trypsin

Abstract Background  Protein hydrolysates are the fragments of proteins that form during the hydrolysis with promised bioactive properties. Enzymatic hydrolysis produces bioactive peptides with specific amino acid profile. Among all the properties, angiotensin-I converting enzyme (ACE-I) inhibitory activity of the protein hydrolysates attained maximum attention to combat life-threatening disease like hypertension. Methodology  In the present study 10 protein-rich food sources (> 20% protein content on dry basis) were used to prepare protein hydrolysates using alcalase and trypsin to investigate the effect of enzymes on ACE-I inhibitory and antioxidant activity. Results  Among all the sources, the highest degree of hydrolysis was observed in sardine (62.89%) followed by soybean (61.24%) when hydrolyzed by alcalase. Kidney pea exhibited highest ACE-I inhibitory activity with IC 50 value of 0.517 and 0.521 when hydrolyzed using both trypsin and alcalase, respectively. All the sources selected showed nonsignificant changes in ACE-I inhibitory activity between alcalase and trypsin ( p  < 0.05) except Bengal gram and chicken. Antioxidant activity was higher in alcalase hydrolyzed egg yolk (43.08%). Conclusion  Among the sources, all animal proteins hydrolyzed using alcalase exhibited significantly higher ( p  < 0.05) antioxidant activity than trypsin. However, protein hydrolysates prepared from sardine using alcalase would yield bioactive peptides with higher ACE-I inhibitory and antioxidant activity. Therefore, use of alcalase as a substitution for trypsin would be economical for large-scale production. Henceforth, food protein hydrolyzed by alcalase can be used as a functional ingredient for the development of functional or nutraceutical foods to combat lifestyle diseases.


Introduction
Proteins are large biomolecules comprised of chains of amino acids.It is also an important macronutrient in a diet which is required for overall growth and development of the body.Bioactive peptides are the small fraction of a protein which are composed of 2 to 20 amino acids which are encrypted within the protein and released during hydrolysis of protein. 1 Protein hydrolysates are the biostimulants that can be defined as the fragments of protein which contain enormous quantity of bioactive peptides which are made up of single amino acids to oligo-and polypeptides. 2Hydrolysis of proteins can be achieved by digestion of proteins using different proteolytic enzymes obtained from natural digestive enzymes such as pepsin, trypsin, and chymotrypsin or from microorganisms such as alcalase, neutrase, and flavourzyme. 35][6][7][8] Therefore, use of such protein hydrolysates from food sources can serve as a natural remedy for the treatment or controlling of lifestyle diseases.
Hypertension is a coronary heart disease which is characterized by the constant raise in blood pressure.Angiotensin-I converting enzyme (ACE-I) plays a vital role in the regulation of blood pressure in the human body.ACE-I regulates regular blood pressure by converting the inactive decapeptide angiotensin-I into an active form angiotensin-II which is a potent vasoconstrictor. 9The excessive production of ACE-I can lead to raise in blood pressure and can lead to hypertension.Hypertension is a coronary heart disorder condition in which the systolic blood pressure is above 180 mm Hg.Henceforth, inhibition of ACE-I is a preliminary action to control high blood pressure.Synthetic ACE-I inhibitors such as captopril, enalapril, lisinopril, and such others are available in the market.Extensive consumption of synthetic ACE-I inhibitors has proven to cause side effects such as cough, dizziness, headache, taste disturbance, visual disorders, dry mouth, insomnia, tiredness, and depression. 1 Therefore, use of natural compound with minimum or no side effects and high ACE-I inhibitory activity can be an alternative to synthetic drugs.The side effects posed by synthetic ACE-I inhibitors have made people look toward natural remedies as a cure for the disease.Thus, protein hydrolysates extracted by enzymatic hydrolysis can be a suitable alternative for the synthetic drugs as the specificity of enzymes will allow peptide production and simplify the identification of bioactive peptides. 10Trypsin is a classical proteolytic enzyme chiefly used in protein hydrolysis, but the enzyme may not be ideal for the hydrolysis of all types of protein due to its substrate specificity, presence of inhibitors, high cost, and it is also reported to have allergic and can carry animal-borne diseases.Researchers and industry professionals are looking for an alternative protease with different bioactive properties based on the specific requirements and their applications, the use of which does not comply the requirement for large-scale production of protein hydrolysates. 11Therefore, there is indeed an alternative for trypsin for industrial production of protein hydrolysates without compromising the bioactive properties of the hydrolysates.The study by Yathisha et al 8 reported that the protein hydrolysates prepared using alcalase exhibited higher bioactive properties than that of the other proteolytic enzymes such as papain and flavourzyme.Therefore, in the present study, two different enzymes, alcalase and trypsin, were used to prepare hydrolysates from protein-rich food sources (plant and animal) available locally.Hydrolysis of plant and animal sources with a proteolytic enzyme can produce protein hydrolysates but the specific amino acid which is responsible for demonstrating the ACE-I inhibitory activity may vary. 1 On the other hand, hydrolysis of plant and animal proteins has its own pros and cons in terms of breakdown of protein during hydrolysis (such as protease inhibitors, lower degree of hydrolysis (DH), and interference of fat), digestibility, and bioavailability of the protein hydrolysates.
Therefore, the purpose of the study was to investigate the ability of these enzymes to produce protein hydrolysates with better antihypertensive activity.The outcome of the study can facilitate the economic way of larger scale production of protein hydrolysates for developing nutraceutical products to combat hypertension.

Reagents
The chemicals required for the study including 1,1-diphenyl-2 picrylhydrazyl (DPPH), ACE-I from rabbit lung (1 U/mL), and the ACE synthetic substrate hippuryl-l-histidyl l-leucine (HHL) and proteolytic enzymes such as alcalase (Bacillus licheniformis) and trypsin (bovine pancreas) were purchased from Sigma-Aldrich.All the other chemicals such as sodium hydroxide, sodium chloride, L-ascorbic acid, and other chemicals used in the study were of analytical grade.

Preparation of Protein Hydrolysates
The protein hydrolysates from selected sources were prepared as per the method described by Yathisha et al, 8 with slight modifications.The selected food sources (50 g) in 500 mL distilled water were ground finely using a mixer grinder.Animal sources were ground using meat mincer (Panasonic MK-MG1500) to get a homogenate mixture.The obtained homogenate was heated at 90°C for 20 minutes to inactivate endogenous enzymes followed by rapid cooling.
The homogenized samples were adjusted to pH 8.5 using 0.1 N NaOH and kept in water bath at 55°C followed by the addition of 1% (w/v) alcalase.Similarly, the pH was adjusted to 8.0 and kept in water bath at 55°C with 1% (w/w) of trypsin.Hydrolysis was performed for 4 hours for maximum production of protein hydrolysates.The pH was maintained at 8.5 and 8.0 throughout the hydrolysis using 0.1 N NaOH.After the completion of hydrolysis time, the temperature was raised to 90°C for 20 minutes to terminate the hydrolysis by inactivating the enzymes and cooled to 4°C.
Further, both hydrolysates (alcalase and trypsin) were centrifuged at 8,000 Â g for 30 minutes at 4°C using cold centrifuge (Eppendorf Germany 5810R).The supernatant collected was lyophilized (ZIRBUS Sublimator VaCo 2) at a pressure of 0.200 mbar.The lyophilized protein hydrolysates were stored at -20°C and reconstituted in distilled water prior to every analysis.

Determination of Degree of Hydrolysis
DH refers to the extent of free peptides released into the solution by cleaving the peptide bond.The DH was measured by estimating the soluble nitrogen using trichloroacetic acid (TCA) method as described by Yathisha et al. 8 Briefly, up to 950 µL of protein hydrolysates (5 mg/mL [50 µl of 10% TCA]) was added and incubated at room temperature for 15 minutes followed by centrifugation at 5,000 revolutions per minute for 10 minutes.The supernatant was estimated for the protein content and the DH was calculated using the formula: DH% ¼ (10% TCA soluble protein / Total protein) Â 100

Proximate Analysis
Moisture, protein, fat, and ash content of protein hydrolysates from both alcalase and trypsin were estimated using the methods of AOAC. 12Moisture was estimated gravimetrically using hot air oven (Rotek Instruments-RHO-24HNS-1813) by heating at 105°C.Protein was estimated using total nitrogen by Kjeldahl digestion and distillation unit (Borosil India 100KID000006).Fat was estimated using Soxhlet apparatus (Rotek Instruments-RHMS-60-1807) using petroleum ether and total ash by combustion (at 550°C) using Muffle furnace (Rotek Instruments RMFH-4-1804).

Measurement of ACE-I Inhibitory Activity
ACE-I inhibitory assay was performed to evaluate the effectiveness of alcalase over trypsin.Briefly, 50 µL of protein hydrolysates were added to 150 µL of 5 mM/L HHL in 50 mM of sodium borate buffer (pH 8.3).The mixture was incubated at 37°C for 5 minutes followed by the addition of 50 µL standard ACE-I (0.1 U/mL).The reaction mixture was again incubated at 37°C for 1 hour.The reaction was terminated by adding 250 µL of 1 N HCl.The liberated hippuric acid was extracted by adding 500 µL of ethyl acetate and evaporated.The residue was redissolved in 3 mL of 1 M NaCl and the absorbance was determined at 228 nm using ultraviolet (UV) spectrophotometer (Eppendorf, Model MJEA129893). 13

Determination of Antioxidant Activity
DPPH radical scavenging activity of the protein hydrolysates was estimated to check the antioxidant potential of the protein hydrolysates.Briefly, 2 mL of 0.16 mM/L of DPPH solution in methanol was added to the test tube containing 100 µL of protein hydrolysates (1 mg/mL concentration) and the mixture was made up to 2 mL with distilled water.The mixture was incubated at room temperature for 30 minutes at dark.The absorbance of the solution was measured at 517 nm using UV spectrophotometer (Eppendorf, Model MJEA129893). 14avenging activity (%)

Statistical Analysis
All data represent the mean value AE standard deviation of three independent measurements.The comparison between two groups was performed using two-way analysis of variance.GraphPad prism software version 8.3.0 was used to analyze the differences and statistical significance at p < 0.05.

Preparation of Protein Hydrolysates
The present study aimed to investigate the application of proteolytic enzyme alcalase for a large-scale production of protein hydrolysates as an alternative to trypsin.A comparative study was performed to prepare protein hydrolysates from different food sources using both trypsin and alcalase to see the action of enzymes on the protein sources.The product yield from all the sources is given in ►Table 1. Modification of Bioactive Properties in Food Protein Hydrolysates Jogi et al.
Kidney pea and cashew showed higher product yield when hydrolyzed with both trypsin and alcalase.Similarly, in animal source egg white and mackerel showed higher product yield among others.

Proximate Composition of Protein Hydrolysates
Nutritional analysis of obtained protein hydrolysates was performed to estimate the chemical composition of the hydrolysates.The proximate analysis of protein hydrolysates from both plant and animal sources are given in ►Table 2. The moisture content of all the samples were found to be > 15%.
Protein content in the hydrolysates of plant and animal sources from alcalase and trypsin hydrolyzed food sources were ranged between 25.81 to 61.28% and 30.69 to 62.19%, respectively.The maximum protein content among the plant sources was found in green gram protein hydrolysate (61.28%) and sardine (65.04%) from animal source using alcalase.

Degree of Hydrolysis Influenced by Proteolytic Enzymes
DH can be defined as the ratio of cleaved peptide bond to the total number of peptide bond in the substance. 13In the present study, protein hydrolysates extracted from alcalase, and trypsin were analyzed for their DH% (►Fig.1A and B).Among all the protein hydrolysates, alcalase extracted hydrolysates from soybean and sardine showed the highest DH% of 61.24 and 62.89%, respectively.Whereas trypsin extracted hydrolysates from kidney pea and egg white could only exhibit maximum of 42.91 and 54.43% of DH, respectively.

Higher Degree of Hydrolysis Exhibited Improved ACE-I Inhibitory Activity
In the current study, protein hydrolysates obtained from both plant and animal sources using alcalase and trypsin were assessed for its ACE-I inhibitory activity.►Table 3 shows the IC 50 values of protein hydrolysates.The ACE-I inhibitory activity of alcalase hydrolyzed sources ranged between 0.52 and 0.61 mg/mL.Among which kidney pea exhibited 50% inhibitory activity at 0.517 mg/mL followed by egg white and green gram (IC 50 value 0.5278 and 0.5227 mg/mL, respectively, p > 0.05).Similarly, trypsin hydrolyzed kidney pea exhibited 50% inhibitory activity at 0.5214 mg/mL followed by sardine and egg white (IC 50 value 0.5221 and 0.5265 mg/mL, respectively).However, Bengal gram dal and chicken protein hydrolysates showed significantly different ACE-I inhibitory activity between trypsin and alcalase.In due course, food sources hydrolyzed with alcalase can corroborate with the trypsin in producing protein hydrolysates exhibiting ACE-I inhibitory activity.Similar results were also observed in free radical scavenging activity of protein hydrolysates.

Alcalase-Mediated Protein Hydrolysates in Animal Sources Shows Greater Free Radical Scavenging (DPPH) Activity
Among all the hydrolysates prepared, egg yolk hydrolyzed using alcalase showed higher antioxidant activity (43.08%) followed by chicken (39.48%).Whereas hydrolysates from Modification of Bioactive Properties in Food Protein Hydrolysates Jogi et al.
green gram using alcalase showed the least (5.96%).On the other hand, higher antioxidant activity was observed in egg white (25.31%) and the least was found in cashew (9.74%) when hydrolyzed with trypsin.The antioxidant activity of all the sources is given in ►Table 4.Among the plant sources, antioxidant activity of both alcalase and trypsin hydrolyzed sources were found to be nonsignificant except green gram (p > 0.05).Besides, alcalase hydrolyzed animal sources also exhibited significantly higher antioxidant activity than trypsin (p < 0.05).

Discussion
Protein hydrolysates are the mixture of bioactive peptides ranging from poly-, oligo-, tri-, and dipeptides which are proven for its therapeutic application against various lifestyle disorders.Formation of protein hydrolysates comprises many techniques such as alkali, acid, enzymatic, and thermal hydrolysis.Among which enzymatic hydrolysis has its own advantage in terms of producing specific bioactive peptides exhibiting bioactive properties. 1 Each proteolytic enzyme has its specific cleaving point on a polypeptide chain under provided conditions for the hydrolysis such as pH, temperature, time, and enzyme-substrate concentration. 9Trypsin is a pancreatic serine protease that commonly serves as a digestive enzyme specifically cleaving peptide bonds at the carboxyl side of the arginine and lysine residues. 15Despite of its high efficiency, trypsin has shown certain negative impact on hydrolysis of protein.7][18] The presence of trypsin inhibitor can affect the action on source protein during hydrolysis.On the other hand, usage of digestive enzyme which needs to be extracted from body organs requires additional cost for industrial application.Therefore, researchers are looking for an alternative economical proteolytic enzyme for industrial application. 19lcalase is a serine endopeptidase produced by fermentation by B. licheniformis. 20Alcalase is observed to have high specificity compared with trypsin for aromatic (Phe, Trp, Tyr), sulfur containing (Met), acidic (Glu), basic (Lys),  ¼ 3).Values between the enzymes that do not share similar letters in the superscript are significantly different at p < 0.05.Values within the sources that do not share similar letters in the superscript are significantly different at p < 0.05 as determined using two-way analysis of variance (ANOVA).Note: Values are represented as triplicates of mean AE standard deviation (SD) (N ¼ 3).Values between the enzymes that do not share similar letters in the superscript are significantly different at p < 0.05.Values within the sources that do not share similar letters in the superscript are significantly different at p < 0.05 as determined using two-way analysis of variance (ANOVA).
obtained was analyzed further.The higher protein content in the hydrolysates observed could be a result of solubilization of protein during hydrolysis and removal of solid nonprotein residues, chiefly separation of fat layer by centrifugation. 9However, alcalase hydrolyzed proteins exhibited significantly higher (p < 0.05) protein content than trypsin hydrolyzed proteins except for kidney pea and sardine.Similar results were reported earlier by Rawdkuen et al 22 in which change in the protein content was observed when the same source was hydrolyzed using two different proteolytic enzymes (crude papain and Calotropis proteases).However, hydrolysates possessing peptides with specific amino acids and their position makes significant changes in the bioactive properties they possess. 1 The protein content in the hydrolysates is extremely influenced by the extrinsic and intrinsic factors such as type of enzymes, condition during hydrolysis, and type of protein and their amino acid composition, respectively.Additionally, the type of enzyme (exopeptidase or endopeptidase) used for the hydrolysis has an impact on the protein content and DH of the hydrolysates. 1he fat content in the raw materials also can interrupt proteolytic hydrolysis so as the bioactive properties.Among the plant protein hydrolysates, soybean possessed higher fat content when hydrolyzed with both trypsin and alcalase (0.71 and 15.41%, respectively).Similarly, among the animal sources mackerel showed 23.15 and 18.0% of fat when hydrolyzed with alcalase and trypsin, respectively.In this study, all the protein hydrolysates possessed higher protein content when the fat content was lower.This could be because higher fat content in the source can restrict the hydrolysis process, thereby resulting in lesser protein content.The presence of fat in the protein hydrolysates is also reported to have negative effect on the shelf life of the protein hydrolysates which described that the protein hydrolysates from Nile tilapia was observed to develop fishy odor, flavor, and lipid oxidation during storage. 23he action of proteolytic enzymes on the source proteins are key feature during the preparation of protein hydrolysates and they are termed as DH. 24In the present study, both plant and animal sources exhibited significantly higher (p < 0.05) DH% when hydrolyzed with alcalase except egg white.The high DH% also corroborate with the study reported by Daliri et al and Baharuddin et al, 25,26 in which DH of soybean protein hydrolysates and eel protein hydrolysates showed 55 and 69%, respectively.Our results also corroborate with previous studies performed by Yathisha et al and Karimi et al, 8,27 where they found alcalase hydrolyzed protein hydrolysates exhibited higher DH% than the trypsin hydrolyzed proteins.Several other studies also revealed that alcalase hydrolyzed food sources such as okara, egg yolk, and black soldier fly exhibited high DH %. [28][29][30] Several methods are employed to determine the DH % in protein hydrolysates.But then, TCA methods generally show higher DH for the protein hydrolysates when compared with pH stat and formol titration method. 31With respect to all the above-mentioned factors essential for an efficient DH%, our study observed a higher efficiency in hydrolysis by alcalase as compared with trypsin.The occurrence of high DH% directly influences the bioactive properties of the protein hydrolysates. 8,32,33he excess synthesis of ACE-I is a key enzyme responsible for constant raise in blood pressure.Therefore, inhibition of ACE-I can be a first-line therapy for hypertension.In our study, both plant and animal sources exhibited ACE-I inhibitory activity (►Table 3).These results are in line with the findings of Ambigaipalan et al and Hanafi et al. 13,34 In the present study, the ACE-I inhibitory activity of plant protein hydrolysates prepared with both alcalase, and trypsin was found to be nonsignificant (p > 0.05) except for Bengal gram and soybean.On the other hand, all the animal sources showed significant difference in the ACE-I inhibitory activity between the two proteolytic enzymes (p < 0.05).However, not much difference was observed among the sources hydrolyzed with the same proteolytic enzymes.Previous reports also suggest that protein hydrolysates prepared using alcalase shows higher ACE-I inhibitory and antioxidant activity than trypsin, papain, and flavourzyme. 8,35These results shed light on the efficiency of alcalase enzymes in producing bioactive peptides with high ACE-I inhibitory activity.The higher ACE-I inhibitory activity shown by alcalase might be because of the wide range of amino acid recognition capability of alcalase than any other enzymes used for hydrolysis. 20ACE-I inhibitory activity of unhydrolyzed Moringa oleifera seed protein hydrolysates showed negative ACE-I inhibitory activity.The study articulates that the enzymatic hydrolysis is responsible for the release of biologically active peptides with potent ACE inhibitory properties. 36However, the type of source protein used for the hydrolysis plays a key intrinsic factor to generate bioactive peptide with specific amino acid profile exhibiting potent ACE-I inhibitory activity.These results endorse the use of alcalase for the production of protein hydrolysates with high ACE-I inhibitory activity either from plant or animal sources.Protein hydrolysates from plant and animal sources are also assessed for their free radical scavenging activity.Free radical scavenging activity of a biomolecule donates hydrogen atom to exert antioxidant activity.DPPH assay is an extensively employed technique for the quantitative assessment of antioxidant effectiveness of compounds as free radical scavengers or hydrogen donors. 6In our study, it was observed that alcalase hydrolyzed animal sources exhibited significantly higher antioxidant activity than trypsin.These results are also supported by the study of Shazly et al 37 in which hydrolysates from bovine and buffalo caseins hydrolyzed using alcalase exhibited higher antioxidant activity than trypsin.Other studies demonstrating antioxidant activity by protein hydrolysates used black soldier fly, cricket fly, housefly larvae etc. 30,38,39 The bioactive mechanisms of protein hydrolysates are not well understood and only a few studies have attempted relating to the structure-activity relationship. 38Nevertheless, to exhibit highest antioxidant activity, the presence of amino acids in the peptides plays an important role in exhibiting antioxidant activity.Henceforth, no single antioxidant mechanism can represent the overall antioxidant activity of the peptides. 37,38However, the antioxidant activity of protein hydrolysates from both plant and animal sources used in this study are lousy when compared with other naturally occurring antioxidant agents.However, further evaluation of its potential in combating oxidation was not performed.Nevertheless, use of alcalase for the preparation of protein hydrolysates can yield hydrolysates with higher antioxidant activity than trypsin.

Conclusion
Protein hydrolysates obtained from food sources showed ACE-I inhibitory and antioxidant activity.The ACE-I inhibitory activity of alcalase and trypsin hydrolyzed food sources were found to be nonsignificant except for Bengal gram.Whereas antioxidant activity of animal sources hydrolyzed using alcalase exhibited significantly higher antioxidant activity.The results suggest that good protein source like kidney pea or sardine fish will be a better choice for the production of protein hydrolysates.The selection of alcalase enzymes over trypsin would be economically beneficial for the production of protein hydrolysates with high ACE-I inhibitory activity for a large-scale production.However, modification in the hydrolysis process to eliminate fat will be a noble way to prepare protein hydrolysates with high DH% and bioactive property.Therefore, protein hydrolysates from kidney pea or sardine fish prepared using alcalase will be a suitable option for the development of nutraceutical and functional foods with antihypertensive activity.However, the ability of protein hydrolysates to resist gastrointestinal digestion and reaching the targeted organ to exhibit expected bioactive properties is still a challenge.
Protein hydrolysates obtained from food sources showed ACE-I inhibitory and antioxidant activity.The ACE-I inhibitory activity of alcalase and trypsin hydrolyzed food sources were found to be nonsignificant except for Bengal gram.Whereas antioxidant activity of animal sources hydrolyzed using alcalase exhibited significantly higher antioxidant activity.The results suggest that good protein source like kidney pea or sardine fish will be a better choice for the production of protein hydrolysates with bioactive properties.The preference of alcalase over trypsin can generate low molecular weight peptides which intend to have high ACE-I inhibitory activity and can also be economically beneficial for the production of protein hydrolysates on a large scale.However, the ability of protein hydrolysates to resist gastrointestinal digestion and reaching the targeted organ to exhibit expected bioactive properties is still a challenge.

Table 1
Product yield of plant and animal protein hydrolysates obtained from alcalase and trypsin Journal of Health and Allied Sciences NU © 2024.The Author(s).

Table 2
Proximate composition of plant and animal protein hydrolysates obtained from alcalase and trypsin Sl. no Food sources Note: Values are represented as triplicates of mean AE standard deviation (SD).Mean value with different alphabets in the superscript between the columns in the same group indicates significant difference (p < 0.05) determined using one-way analysis of variance (ANOVA).Journal of Health and Allied Sciences NU © 2024.The Author(s).

Table 3
ACE-I inhibitory activity of protein hydrolysates extracted using alcalase and trypsin Abbreviation: ACE-I, angiotensin-I converting enzyme.Note: Values are represented as triplicates of mean AE standard deviation (SD) (N

Table 4
Free radical scavenging activity of protein hydrolysates extracted using alcalase and trypsin