Bioactive peptides of amaranth: isolation and determination of antioxidant and hypocholesterolemic properties

. Hydrolysis of the amaranth protein isolate (Amaranthus hypochondriacus L., Voronezh variety) protein isolate by trypsin, papain and alkalase was carried out in order to obtain biologically active peptides with antioxidant and hypocholesterolemic activities. A high degree of protein hydrolysis was shown by trypsin during the first two hours, papain between three and four hours, and alkalase after four hours of hydrolysis. The rate of cleaved protein increased from the first two hours of hydrolysis from 40%, 30% and 15% to 78%, 70% and 60% when trypsin, papain, alkalase were applied, respectively. Maximum hydrolysis of the amaranth protein was observed after 6 h of proteolysis by all enzyme preparations. The molecular weights of the hydrolysis products of the amaranth protein after 2, 4 and 6 h of hydrolysis were determined. All hydrolysates showed a high content of polypeptides with molecular masses of 33 kDa, 18-23 kDa, 52-54 kDa, 40-54 kDa, 6.5-15 kDa. Short-chain peptides with molecular masses of 624 to 2817 Da were detected in the hydrolysates after 6 h of proteolysis. It was determined by capillary electrophoresis method that all protein hydrolysates after 6 h of hydrolysis contain amino acids (tyrosine, phenylalanine, alanine, leucine, valine, proline, alanine) responsible for antioxidant activity of peptides in the studied hydrolysates. The antioxidant activity of peptides in all hydrolysates of amaranth protein isolate was determined by ABTS cation radical reduction method for 6 min. Hypocholesterolemic activity of peptides was shown in protein hydrolysates by trypsin and papain.


Introduction
Health and environmental issues are causing changes in the human diet, and plant-derived proteins are considered as a sustainable source of proteins compared to animal-derived proteins.Many foods containing physiologically active proteins, natural or processed, are rich sources of bioactive peptides [1].As the world practice shows, bioactive peptides formed by hydrolysis of plant proteins can be used as functional food ingredients in the technology of specialised and personalised products, in pharmaceuticals and cosmetics, feeds, microbiological media.
Based on the productivity, protein content and its balanced amino acid composition close to the optimal one for human nutrition, the amaranth plant, whose seeds are particularly rich in protein, can be considered as a promising source of bioactive peptides [2].Amaranth seed peptides have various functional properties and biological effects.In silico analyses have shown that the peptides most commonly found in amaranth have antihypertensive properties, especially globulin 11S, which can inhibit angiotensin-I-converting enzyme (ACE), which plays an important role in the renin-angiotensin system in blood pressure regulation [3].Peptides with anorexigenic and anti-inflammatory activities, as well as peptides containing dipeptidyl aminopeptidase (DPP) IV inhibitors have been found to be used in the treatment of diabetes mellitus, and ACE -inhibitory activity of amaranth peptides has been proven [4].In addition, antioxidant, antiproliferative, antitumour, antithrombotic, antimicrobial and immunomodulatory and other activities of amaranth peptides have been determined [5][6][7].These studies were conducted by scientists in Mexico, Argentina and Latin America, where amaranth is a widely cultivated and studied crop.However, different growing conditions, species and varieties of amaranth can differ in the amino acid composition of the protein, which affects the amino acid sequence and structure of isolated proteins and peptides and, consequently, their biological activity.Of the variety of amaranth varieties in Russia, A. hypochondriacus L. is the most balanced in terms of amino acid composition; therefore, it is used in breeding to develop grain varieties.Thus, in Voronezh region, the Voronezh variety, which has not been studied with regard to bioactive peptides, is cultivated.
The structure and properties of peptides depend on the peculiarities of enzymatic cleavage of protein substrates, optimal conditions of catalytic activity of the enzyme, its substrate and site specificity, and physical and chemical properties of the cleaved proteins.Therefore, various endo-and exopeptidases of microbial (alkalase, neutralase), plant (papain, ficin) and animal (pepsin, trypsin) origin are used to obtain enzymatic hydrolysates with specified parameters (peptide profile, biological activities) [8].Amaranth protein isolates are the main raw material for the production of bioactive peptides, but due to the peculiarities of isolate production, changes in protein structure may occur, which also affects the structure and bioactivities of peptides [9].
With all the variety of biological activities of amaranth peptides, the antioxidant and hypocholesterolemia activities of amaranth peptides deserve special attention due to the peculiarities of population nutrition.Thus, free radical damage of nucleic acids, proteins, membrane lipids and other cell macromolecules occurs in the body in case of excess free radicals and lack of antioxidants [10], and hypercholesterolemia is one of the causes of a number of cardiovascular diseases and also increases the risk of other complications such as arterial hypertension, obesity, cancer and diabetes [11].In this regard, obtaining peptides with these bioactivities from amaranth protein isolate of the Voronezh variety is an urgent task.
The aim of the work was to study the process of hydrolysis of amaranth protein isolate of Voronezhsky variety by proteases of plant, microbial and animal origin to obtain biologically active peptides with antioxidant and hypocholesterolemic activities.

Materials
Amaranth protein isolate (A.hypochondriacus L., Voronezh variety) with a protein mass fraction of 86.0 %, obtained earlier [9], was used to obtain active peptides.A set of protein markers with molecular mass from 6.5 to 54 kDa (Thermo Fisher Scientific, USA) was used for molecular weight determination.A standard mixture of amino acids (ICN, USA) was used to determine the amino acid composition of peptides.The reagents used in the work were those of Reahim (Russia), Bio-Rad (USA), Sigma (USA), AcrosOrganics (USA).
Preparation and characterisation of amaranth peptide hydrolysates Enzymatic hydrolysis was carried out in a thermostated reactor.For this purpose, solutions of amaranth protein isolate were prepared in phosphate buffer with pH 7.8 at a concentration of protein in solutions of 10.0 g/dm 3 , incubated for 60 min at 37 °C with constant stirring until complete dissolution, then enzyme preparations of trypsin, papain and alkalase were added at the rate of activity per the amount of protein in the solution.The reaction mixtures were incubated for 5 h at 37 o C. The pH level was controlled with NaOH solution of molar concentration 5 mol/dm3.At the end of hydrolysis, inactivation of enzymes in the reaction mixtures was carried out at 95 o C for 5 min.The obtained mixtures were centrifuged at 7000 rpm, the precipitate was separated and the supernatant was used for analysis.The degree of hydrolysis was assessed by the accumulation of amine azate as well as the amount of protein remaining unhydrolysed in the solution.The concentration of unhydrolysed protein was determined by the Lowry method.
The amino acid composition of peptide hydrolysates was determined by capillary electrophoresis [12].
Determination of antioxidant and hypocholesterolemic activities.
To determine the antioxidant capacity of peptide hydrolysates, we used the method of measuring ABTS-radical-reducing activity, which is based on the use of a previously obtained cation-radical based on the diammonium salt of 2,2'-azinobis [3ethylbenzthiazoline-6-sulfonic acid].ABTS˙+ is a metastable radical that can exist in solution for quite a long time; when various antiradical agents are introduced into the medium, a rapid reduction of the radical is observed.The antiradical agent used as a standard was Trolox [13], the antioxidant activity of which was taken as 100%.The reaction was monitored spectrophotometrically at λ = 734 nm: ABTS˙+-radical (blue staining of the solution) is converted to its colourless neutral form upon reduction.The ABTS˙+ reducing activity was measured based on a modified methodology described in [14].For this purpose, 0.06 cm3 of amaranth isolate protein hydrolysate solution was added to 5.94 mL of ABTS˙+ working solution.Incubation of the reaction mixture was carried out under constant stirring at 30 ºC.The optical density of the solution was measured after 1, 3, 5 min and further with a period of 5 min for 60 min.
Hypocholesterolemic activity of potentially active peptides in amaranth protein isolate hydrolysates was evaluated by their ability to inhibit pancreatic lipase and cholesterol esterase.For this purpose, enzyme solutions of lipase and cholesterol esterase were prepared with mass concentrations of 0.01 mg/cm3 and hydrolysates of amaranth protein isolate were added at the rate of 1% per volume of the reaction mixture.Lipase activity was determined by the method of Yamada and Machida [15].Olive oil emulsion was used as substrate.The reaction mixture consisted of 5 cm3 of olive oil emulsion in 2% polyvinyl alcohol solution and 4 ml of phosphate-citrate buffer (pH 7.0) and 1 cm3 of enzyme solution.Cholesterol esterase activity was determined according to the method [16].Cholesteryl -14C-oleate was used as a substrate.The amount of enzyme catalysing the formation of 1 μmol of C-oleic acid during hydrolysis of cholesteryl -14C-oleate under the described conditions for 1 h was taken as a unit of activity.
Graphs were plotted and mathematical processing of the research results was carried out in the programme "Microsoft Office Excel 2003" (Microsoft Corporation, USA).Experiments were performed in three repetitions, the average results of parallel determinations at the significance level of 0.05 were discussed.

Results and Discussion
Hydrolysis of amaranth protein isolate and characterisation of peptide hydrolysates Controlled characteristics of enzymatic hydrolysates are the degree of hydrolysis of protein substrates, peptide composition and the amount of cleaved and unhydrolysed protein.
Depending on the degree of hydrolysis, partial hydrolysates containing peptides of various lengths and a minimum amount of free amino acids and deep hydrolysates containing shortchain peptides and amino acids are distinguished [17].Since the depth of hydrolysis of protein substrates is directly proportional to the duration of the enzymatic process, the dependence of the degree of proteolysis of amaranth protein on the duration of the enzymatic reaction by trypsin, papin and alkalase was investigated.With increasing duration of hydrolysis by all enzyme preparations there was an accumulation of amine nitrogen (Table 1), which characterises the degree of protein hydrolysis (Fig. 1a), as well as an increase in the amount of cleaved protein (Fig. 1b).It was found that during the first two hours, about 40 and 30 % of protein was hydrolysed when trypsin and papain were applied, respectively, and 15 % of protein when alakalase was applied, and after 4 h of enzymatic reaction, about 78 %, 70 % and 60 % of protein was hydrolysed, respectively (Fig. 1b), as evidenced by amine nitrogen values at different time intervals of hydrolysis (Table 1).The maximum hydrolysis of amaranth protein was observed after 6 h of proteolysis by all enzyme preparations.The amount of unhydrolysed protein during hydrolysis by trypsin, papin and alkalase was 13, 20 and 30 %, respectively (Fig. 1b).
The data obtained indicate intensive hydrolysis of amaranth protein by trypsin during the first two hours, by papain between three and four hours, and by alkalase after four hours of hydrolysis.This is explained by the different substrate specificity of enzymes with respect to amaranth protein and the peculiarities of enzymatic catalysis by proteases of different classes, as well as the conformational state of the protein substrate under these conditions of hydrolysis.According to the literature [18], the 7S-globulins and 11S-subunits of amaranth protein are considered to be more susceptible to enzymatic hydrolysis, whereas the 11S-base subunit initially shows resistance to degradation but gradually degrades over time.The initial increase in the degree of hydrolysis after 2 h can be attributed to the degradation of the 7S and 11S acidic subunits, and after about 6 h of hydrolysis to the 11S basic subunits.After 6 hours of hydrolysis by all enzyme preparations, a decrease in the intensity of the enzymatic reaction was observed, as indicated by a decrease in the pH of the reaction mixtures.This effect is associated with the formation and accumulation of hydrolysis products, as well as a decrease in the activity of enzyme preparations due to the inhibitory effect of the formed amino acids.Due to the peculiarities of the course of enzymatic reactions by trypsin, papain and alkalase, the molecular masses of amaranth protein hydrolysis products obtained after 2, 4 and 6 hours of hydrolysis were determined in order to establish peptides with biological activity (Fig. 2).It was previously found that the major proteins of amaranth isolate are represented by globulins, albumin and glutelins [9].As shown in Figure 2, the protein profile of the isolate has dense prominent bands over a wide range of molecular weight from 6.5 to 54 kDa.Hydrolysis of amaranth protein isolate by trypsin, papain and alkalase showed disappearance or brightening of the major protein bands.All hydrolysates showed a high content of globulin fraction polypeptides: 11S-globulin, acidic polypeptides (33 kDa) and basic polypeptides (18-23 kDa), polypeptides (52-54 kDa) and 7S-globulin polypeptides (40-54 kDa).Polypeptides from albumin fractions (less than 18 kDa) were also detected.Hydrolysates A2 and A6 showed the presence of a weak band of polypeptides with molecular weights of 44 kDa and 18-23 kDa corresponding to 7S and 11S globulins, which are more resistant to enzymatic hydrolysis, as well as bands in the region of 6.5-15 kDa corresponding to hydrolysis products.In hydrolysate C2 no bands corresponding to the main polypeptides of 11S-globulin were detected, and the region corresponding to hydrolysis products (37 kDa and 23-26 kDa) has a higher intensity compared to other hydrolysates.Thus, on increasing the hydrolysis time from two to six hours, slight degradation of protein bands was observed after 4 hours, whereas after 6 hours of hydrolysis, significant degradation was observed, which is also confirmed by the increase in amine nitrogen value (Table 1) All the hydrolysates obtained by different enzymes had a long thick band near the end of the gel, indicating the presence of low molecular weight peptides.
The hydrolysates obtained after 6 h of hydrolysis showed peptides containing 5 to 21 amino acid residues with molecular masses ranging from 624 to 2817 Da, which may exhibit antioxidant (AOA) and hypocholesterolemic activities.According to the literature, some short-chain peptides can exhibit antioxidant properties by binding free radicals by peptides with hydrophobic terminal amino acids such as Ala (A), Pro (P), Val (V), Ile (I), Leu (L), Phe (F), Trp (W), Tyr (Y) and Met (M) [23].In addition, amino acids with aromatic residues (histidine and proline) can give electrons to charged radicals.In this regard, the amino acid profiles of peptides in hydrolysates after 6 hours of hydrolysis by proteases were determined.Table 2 shows that the amino acid profiles of peptides in all hydrolysates are similar.It is important that all protein hydrolysates contained a significant amount of such amino acids as tyrosine, phenylalanine, alanine, leucine, valine, proline, alanine, which may be responsible for the antioxidant activity of peptides in the studied hydrolysates.Study of antioxidant and hypocholesterolemic activity of the obtained peptide hydrolysates At determination of antioxidant activity (AOA) the reaction of cation-radical reduction was investigated when amaranth protein hydrolysates were introduced into the test system after 6 hours of hydrolysis by trypsin, papain and alkalase.The dependences of the degree of ABTS˙+ reduction by hydrolysates on the reaction duration were obtained (Fig. 3).
Reactions with all hydrolysates showed a rapid decrease in the amount of ABTS˙+ during the 1st min of the process; at the 4th-6th min of the reaction, a less intensive increase in the fraction of the reduced radical was observed; a slow reaction stage (up to the 30th min) followed.As can be seen in Fig. 2, the maximum degree of ABTS˙+ reduction was exhibited by the protein hydrolysate with trypsin, whereas the protein hydrolysate with alkalase was characterised by a lower level of AOA.This effect is due to the structure of its constituent peptides and its ability to bind to free oxygen [19].The hypocholesterolemic activity of peptides obtained by proteolysis with different enzymes for 6 h was investigated.The results of determining the ability of amaranth protein hydrolysates to inhibit lipase and cholesterol esterase are presented in Table 3.As shown in Table 3, the highest degree of inhibition of lipase and cholesterol esterase was observed in protein hydrolysate by papain, as well as in protein hydrolysate by trypsin.The lipase activity decreased 2.3 and 1.6 times and cholesterol esterase activity decreased 3.3 and 2.2 times, respectively.The hydrolysate produced by alkalase exhibited the lowest enzyme inhibition efficiency, which is attributed to the substrate specificity of the enzyme.However, similar hydrolysates are known to contain peptides with angiotensive activity [3], which was not investigated in this study.

Conclusions
The regularities of hydrolysis of amaranth protein isolate of Voronezh variety by trypsin, papain and alkalase were studied by estimating the depth of proteolysis at different time intervals of fermentation.A high degree of hydrolysis of the amaranth protein isolate by all proteases was found from two to six hours of hydrolysis.The amount of cleaved protein increased from 40% and 30% to 78% and 60% when trypsin and papain were added, respectively, and from 15% to 60% of protein when alakalase was added, confirming the high affinity of the enzymes used for amaranth protein.The molecular masses (from 624 Da to 54 kDa) of the hydrolysis products of amaranth protein obtained after 2, 4 and 6 hours of hydrolysis were determined, which characterise the peculiarities of hydrolysis reactions by different proteases.In the hydrolysates after 6 hours of hydrolysis short-chain peptides with moleolar masses of 624 to 2817 Da, capable of antioxidant and hypocholesterolemic activity, were found.The amino acid composition of amaranth protein isolate hydrolysates was determined, indicating the presence in the hydrolysates of such amino acids as tyrosine, phenylalanine, alanine, leucine, valine, proline, alanine, responsible for the antioxidant activity of the peptides.All hydrolyses of the amaranth protein isolate showed antioxidant activity.Hypocholesterolemic activity of peptides was found in the case of protein hydrolysis by trypsin and papain.The results obtained allow us to recommend amaranth protein isolate of Voronezh variety as a source of peptides with antioxidant and hypocholesterolemic activity, as well as for the production of peptides with other bioactivities.

Table 1 .
Amine nitrogen accumulation during proteolysis of amaranth protein

Table 2 .
Amino acid composition of amaranth protein isolate hydrolysates, proteolysed by different enzymes, g/100 g protein

Table 3 :
Inhibition of lipase and cholesterol esterase by amaranth protein isolate hydrolysates