Antioxidant and antidiabetic activities of peptides isolated from a hydrolysate of an egg-yolk protein by-product prepared with a proteinase from Asian pumpkin (Cucurbita ficifolia)

Abstract

Bioactive peptides derived from food have been increasingly popular due to their therapeutic properties. Of particular importance are peptides with a multidirectional activity that can be used in the treatment and prevention of diet-related diseases. This paper attempts to utilize a by-product of phospholipid extraction from egg yolk as a source of peptides with a broad spectrum of biological activity. In addition, in this research we used a non-commercial enzyme obtained from Asian pumpkin, which has not been sufficiently researched in terms of its ability to release biopeptides from food proteins. In the present study the biological properties of peptides, derived from egg-yolk protein by-products (YP) remaining after phospholipid extraction, and their four synthetic analogs were investigated with regard to their antioxidant (radical scavenging capacity, Fe²⁺ chelating effect, reducing power (FRAP)) and antidiabetic (α-glucosidase and DPP-IV inhibitory activities) properties. One of them, with the sequence LAPSLPGKPKPD, exhibited the highest antioxidant activity (free radical scavenging activity (6.03 μM Trolox_eq per mg protein); FRAP (296.07 μg Fe²⁺ per mg protein)). This peptide also revealed the strongest DPP-IV (361.5 μmol L⁻¹) and α-glucosidase (1065.6 μmol L⁻¹) inhibitory activities, a novel multifunctional effect for peptides from an egg-yolk hydrolysate.

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1 Introduction

In the past two decades, researchers have focused on the search for food-derived peptides, amino acid sequences with specific biological activity.^1,2 Interest in biopeptides is associated with their high therapeutic potential from the wide spectrum of action in vivo, including antioxidant, antibacterial, antihypertensive, immuno-modulatory or opiate functions. The various properties of these peptides make it possible to use them not only in the treatment, prevention and alleviation of various diseases, but also in extending the life of food products. For this reason, they are extremely attractive to both the food and biopharmaceutical industries.^1,2 Although biopeptides are usually produced in vitro via chemical synthesis or enzymatic hydrolysis, many studies are using molecular biology methods to develop synthetic genes for the delivery and expression of biopeptides or their precursors in microbial cells.^3,4

Despite these effort, enzymatic hydrolysis conducted with the use of various enzymes of microbial, plant and animal origin, is still the main process for obtaining bioactive peptides from food products.^5,6 The application of enzymes increases the cost of the process which is why cheap sources of them as well as a simple procedure of isolation are preferred. One of example of such enzyme is non-conventional serine proteinase from Cucurbita ficifolia. The enzyme constitutes about 15% of the total protein extracted from the pumpkin pulp. The proteinase has a molecular weight of about 60 kDa and is relatively stable under different temperature conditions. It exhibits strong proteolytic properties toward food proteins of animal and plant origin.^7,8 Antioxidant peptides are the most commonly occurring bioactive peptides in food. They demonstrate activity through multiple pathways, including scavenging of free radicals, chelating pro-oxidative transition metal ions, reducing hydroperoxides and inactivating reactive oxygen species.⁹ Thanks to these abilities, peptides play an important role in the inhibition of oxidative processes which lead to the formation of highly reactive and harmful radicals, responsible for cancer, coronary heart disease and Alzheimer's disease, amongst others.^5,10 Due to the close relationship between oxidative stress and the listed diseases, control of oxidative stress would seem to be an effective strategy in the prevention of such diseases.

Nowadays, diabetes mellitus (DM) is a serious disease affecting a significant part of populations worldwide. DM is characterized by hyperglycemia as a result of peripheral insulin resistance inefficiently compensated by pancreatic b-cell insulin secretion.¹¹ The most effective therapy for DM is to maintain the optimal blood glucose level. α-Glucosidase, a membrane-bound carbohydrase present in the epithelium of the small intestine, works to facilitate the absorption of glucose by the small intestine by catalyzing the hydrolytic cleavage of oligosaccharides and disaccharides into absorbable monosaccharides. Dipeptidyl-peptidase-IV (DPP-IV) participates in the degradation of the glucagon-like peptide-1 (7–36) amide (antihyperglycemic hormone), which stimulates glucose-dependent insulin secretion in pancreatic β-cells.^12,13

α-Glucosidase and dipeptidyl-peptidase-IV (DPP-IV) inhibitors are considered to be an effective strategy for the control of DM type 2.^12,13 There are several known natural α-glucosidase and DPP-IV inhibitors including food protein hydrolysates/peptides.^14–16 However, there is little information regarding the hydrolysis of food proteins and antidiabetic activity of those generated peptides and the sequence of the peptides in relationship with antidiabetic activity.

Bioactive peptides with multiple functions may be important in the prevention of diet-related diseases, which have been shown to have a number of common development and progression factors.

In the present study, we investigated the biological activity of a hydrolysate of egg yolk protein by-product obtained with the use of proteinase from C. ficifolia, isolated peptide fractions and synthetic peptides. The priority of the experiments was to validate the antioxidant and antidiabetic activities of these novel egg-yolk derived peptides.

2 Materials and methods

Piperidine solution, diethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrozine, α-glucosidase from Saccharomyces cerevisiae, P-nitro phenyl glucopyranoside (pNPG), dipeptidyl-peptidase (DPP-IV) from porcine kidney, Gly-Pro–p-nitroaniline were purchased from Sigma-Aldrich. O-(1H-6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-fluoroborate (TCTU), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA) and Wang resins were obtained from Iris Biotech GmbH, dimethylformamide (DMF) was purchased from Carl Roth GmbH + Co. KG, 1-hydroxybenzotriazole (HOBt) was purchased from GL Biochem, triisopropylsilane (TIS) was purchased from Alfa Aesar and 2,4,6-tripyridyl-s-triazine (TPTZ) was purchased from Fluka. All other reagents were chemically grade.

2.1. Substrate

The 40–45 weeks old laying hens of Lohman brown line were housed in a bedding system. Eggs were automatically broken out, and macroscopic parts of eggs were separated on industry scale. Egg yolk protein preparation (YP), a by-product of phospholipid extraction using ethanol was used as a substrate.¹⁷

2.2. Enzyme

Non-commercially available proteinase from C. ficifolia fruit pulp was isolated following the procedure described for seeds by Dryjański, Otlewski and Wilusz.¹⁸

2.3. Determination of protein content

Protein content in hydrolysates, peptide fractions and synthetic peptides was determined by the Whitaker and Granum method.¹⁹

2.4. Enzymatic hydrolysis

YP hydrolysis was carried out according to modified method of Zambrowicz et al.²⁰ 1% substrate suspension in 0.1 M Tris–HCl buffer (pH 8.0) was hydrolyzed at 37 °C for 4 hours using C. ficifolia protease at enzyme: substrate ratio E/S (w/w) = 1 : 7.52. The reaction was terminated by heating the mixture at 100 °C for 15 min. The hydrolysate was cooled, centrifuged (5500 × g, 10 min, 10 °C), then the supernatant was lyophilized and stored at 4 °C until used. A detailed description of the procedure of peptide isolation has been described in our previous work in which the ACE-inhibitory property of the YP-derived peptides was examined.²¹

2.5. Chemical synthesis of the peptides

The peptides RASDPLLSV, RNDDLNYIQ, LAPSLPGKPKPD and AGTTCLFTPLALPYDYSH were chemically synthetized by prof. Szewczuk's team from the Faculty of Chemistry, University of Wroclaw.

Synthesis of specific peptides was carried out manually in a syringe reactor (BRAUN Inject, Germany). The preloaded Wang resins (0.58–0.79 mM g⁻¹) (Iris Biotech GmbH) were used for the synthesis of fully protected peptides. Fmoc-protecting group (9-fluorenylmethyloxycarbonyl) was removed with 25% piperidine solution (Sigma-Aldrich) in DMF (3 and 17 minutes) (Carl Roth GmbH + Co. KG). Amino acid coupling reaction was carried out using DMF as a solvent with the use of 3 equivalents TCTU (Iris Biotech GmbH) as the coupling reagent, 3 equivalents of HOBt (GL Biochem (Shanghai) Ltd.), and 6 equivalents of DIEA (Iris Biotech GmbH) as additives. The reaction was carried out for 150 min. Peptides were cleaved from the resin simultaneously with the side chain deprotection using a mixture of TFA (Iris Biotech GmbH)/TIS (Alfa Aesar)/H₂O (95 : 2.5 : 2.5, v/v/v). The reaction was carried out for 120 minutes. Then the solution was transferred into cold diethyl ether (Sigma-Aldrich). Crude residue was collected, dissolved in water, lyophilized, and purified by reversed-phase high-performance liquid chromatography. All peptides were obtained as their trifluoroacetate salts.

2.6. Analysis of chemically synthesized peptides using HR-ESI-MS

Analysis of chemically synthesized peptides was carried out by HR-ESI-MS mass spectrometer (FT-ICR Apex-Qe Ultra 7 T, Bruker Daltonics, Bremen, Germany) equipped with standard electrospray ion source. The instrument was operated in the positive-ion mode. The instrument was calibrated using TuneMix™ mixture (Bruker Daltonics, Bremen, Germany). The solutions used for the measurement were CH₃CN/H₂O/HCOOH (50 : 50 : 0.1, v/v/v), with the m/z range of 100 to 1800.

2.7. Determination of antioxidant activity as the ability to scavenge DPPH free radicals

The antioxidant activity of the obtained hydrolysates was assessed on the basis of the radical scavenging effect of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH (Sigma, D21140-0)) – free radical activity according to Yen and Chen with minor modifications.²² The tested samples were dissolved in water to a final volume of 1 mL and mixed with 1 mL of ethanol (98%). The reaction was started by adding 0.5 mL of 0.3 M DPPH in ethanol. The mixtures were left for 30 minutes at room temperature and the absorbance of the resulting solutions was measured at 517 nm. For calibration, aqueous solutions of known Trolox concentrations ranging from 2 to 20 μg (able to scavenge 500 μL of 0.3 mM DPPH radical solution) were used. Radical scavenging activity of the peptides was expressed as μM trolox_eq per mg protein.

2.8. FRAP method

The FRAP method (Ferric Reducing Antioxidant Power) was used to determine the antioxidant capacity of hydrolysates according to Benzie and Strain.²³ 3 mL of FRAP working solution (300 mM acetate buffer pH 3.6; 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) (Fluka, 93285) and 20 mM FeCl₃·6H₂O (10 : 1 : 1 v/v)) was mixed with 1 mL of the sample. After 10 min of reaction, the absorbance was measured at λ = 593 nm. An aqueous solution of known Fe(II) concentration was used for calibration (in the range from 100 to 1000 μg). Results were expressed as μg Fe²⁺ per mg protein.

2.9. Determination of Fe(II) ion chelation

Chelation of iron ions by hydrolysates was estimated by the method of Xu et al., with modifications.²⁴ A 250 μL sample was mixed with 1250 μL H₂O and 110 μL 1 mM FeCl₂. After 2 min, 1 mL of 500 μM ferrozine (Sigma, 160601) aqueous solution was added and the mixture was allowed to react for 10 minutes. The absorbance of ferrous iron–ferrozine complex was measured spectrophotometrically at λ = 562 nm. A known concentration of FeCl₂ (0–20 μg) was used to generate a standard curve and the ability to chelate iron ions was expressed as μg Fe²⁺ per mg protein.

2.10. α-Glucosidase-inhibitory activity

The α-glucosidase-inhibition assay was performed according to Yu et al., where α-glucosidase from Saccharomyces cerevisiae (Sigma, G0660) hydrolyzes the substrate – p-nitro phenyl glucopyranoside (pNPG) (Sigma, N1377) and the produced p-nitrophenol can be measured spectrophotometrically.²⁵ Five μL of the α-glucosidase solution (10 U mL⁻¹, in 0.1 M potassium phosphate buffer, pH 6.8) was premixed with 10 μL of the sample solution at different concentrations (in 10% DMSO) in 620 μL of 0.1 M potassium phosphate buffer (pH 6.8). Following incubation at 37.5 °C for 20 min, 10 μL of p-nitro phenyl glucopyranoside (pNPG, 10 mM) as substrate was added to the mixture to start the reaction and was then incubated at 37.5 °C for 30 minutes, followed by addition of 650 μL of 1 M Na₂CO₃ solution to terminate the reaction. The amount of released product (p-nitro phenol) was measured at λ = 410 nm. All samples were tested in 3 replications. The inhibition activity was calculated using the following equation:

Inhibition activity (%) = [(A − B)/A] × 100%,

where A is the reaction blank, of which the mixture contained the same volume of the buffer solution instead of the α-glucosidase inhibitor sample; B is the reaction in the presence of both α-glucosidase and its inhibitor. The IC₅₀ value was estimated from a dose response curve of an inhibitor versus the enzyme activity.

2.11. Dipeptidyl-peptidase-IV (DPP-IV)-inhibitory activity

DPP-IV-inhibitory activity (IC₅₀) was determined using a modified method of Lacroix and Li-Chan.¹³ DPP-IV from porcine kidney was purchased from Sigma (D7052). The lyophilized peptide fractions were re-suspended in 0.1 M Tris–HCl buffer, pH 8.0. The test sample (25 μL) was preincubated with an equal volume of the substrate Gly-Pro–p-nitroaniline (1.6 mM) at 37 °C for 10 min. Afterwards, 50 μL of DPP-IV (0.01 U mL⁻¹, in 0.1 M Tris–HCln buffer, pH 8.0) was added and the mixture was incubated at 37 °C for 60 min. The reaction was stopped by the addition of 100 μL of 1 M sodium acetate buffer, pH 4.0. The released p-nitroaniline as a hydrolysis product was measured at λ = 405 nm. All samples were tested in 3 replications. The inhibition activity and IC₅₀ values were calculated just as at 2.10.

2.12. Statistical analysis

Hydrolysate was prepared in two independent batches. The biological activity measurements of hydrolysates as well as various fractions obtained in the purification process were done in triplicate for each batch. The data obtained were subjected to multi-factor variance analysis (ANOVA), followed by a Duncan multiple range test to determine the significant difference between sample at p < 0.05 level using Statistica v.9.0 software.

3 Results and discussion

3.1. The biological activities of the enzymatic hydrolysate of YP

Serine proteinase from C. ficifolia fruit pulp, due to their high proteolytic activity as well as the simple procedure of its isolation was selected. Hydrolysis of egg yolk protein by-product (YP) was conducted for four hours (E/S (w/w) = 1 : 7.52).

The antioxidant activity of the YP hydrolysate was studied in terms of its scavenging effect on DPPH radicals, reducing power (FRAP), and iron chelating activity. The analyzed hydrolysate possessed strong ferric reducing capacity (56.41 μg Fe²⁺ per mg protein) and chelating activity (695.76 μg Fe²⁺ per mg protein). The obtained hydrolysate also showed significant potency in scavenging DPPH radicals (0.42 μM Trolox_eq per mg protein). The evaluated antioxidant activity of the hydrolysate is the result of the specific activity of the enzyme, which releases antioxidant peptides from the protein molecules. However, the protein's antioxidant activity is limited by its tertiary structure.⁹ In previous work we evaluated the biological properties of egg-yolk protein by-product left during the course of phospholipid isolation by treating a commercial neutrase. The scavenging capacity, ferric reducing power and chelating capacity were observed for the hydrolysate at the level: 0.44 μM Trolox_eq per mg protein, 177.35 μg Fe²⁺ per mg protein and 549.87 μg Fe²⁺ per mg protein, respectively.²⁶ The results indicated that unconventional serine proteinase from C. ficifolia is characterized by better ability to release metal chelating peptides from YP by-product than neutrase.

Importantly, we previously indicated that this hydrolysate also exhibited a significant ACE inhibitory activity (IC₅₀ = 482.5 μg mL⁻¹).²¹ Finally, it is very important to determine the cytotoxic activity of potential nutraceuticals or/and pharmacological substances to establish their safety and proper impact on the body. In our previous work we also tested the hydrolysate in terms of cytotoxicity and/or proliferation activity; it showed no cytotoxic activity on human and animal cell lines, which makes it a very useful multifunctional peptide preparation.²¹

3.2. The antioxidant activity of peptide fractions isolated from the YP hydrolysate

The compiled peptide isolation procedure included: ultrafiltration (cut-off 5 kDa) (samples: retentate, permeate); size-exclusion chromatography (samples: F1–F4) and RP-HPLC (samples: SF 2.1; 2.2; 4.1–4.4; 4.4.1; 4.4.2). The 4 h-hydrolysate of YP was ultrafiltered through a cellulose membrane (MWCO 30 kDa). Then the obtained permeate (fraction with molecular weight <30 kDa) was ultrafiltered on a 5 kDa cut-off membrane in order to isolate and purify the biopeptides. The peptide fractions with molecular weights lower than 5 kDa exhibited significant antioxidant properties (Table 1). The ability of this fraction to scavenge DPPH free radicals and to reduce the oxidation of iron(III) reached 3.63 μM Trolox_eq per mg protein and 70.83 μg Fe²⁺ per mg protein, respectively. This is in the line with results obtained by Park et al., who demonstrated that egg yolk protein hydrolysates, which are a mixture of peptides with MW lower than 5 kDa, exhibited antioxidant activity, and were the most effective in the inhibition of lipid oxidation.²⁷ Moreover, Katayama et al., showed that peptides with MW ranging between 1 and 3 kDa obtained from phosvitin hydrolysates were effective in protecting against oxidative stress in human intestinal epithelial cells in vitro due to their strong antioxidant properties.²⁸

Table 1 Biological activities of the fractions separated by ultrafiltration gel permeation and reverse-phase chromatography of 4 h-egg-yolk protein hydrolysate treated by serine proteinase from C. ficifolia. All data were expressed as mean values (n = 3). Values sharing the same letter at the same purification level and test group are not significantly different at p < 0.05

Isolation step	Sample	DPPH scavenging activity [μM Troloxeq per mg protein]	Ferric reducing ability (FRAP) [μg Fe^2+ per mg protein]	Ferrous ion-chelating activity [μg Fe^2+ per mg protein]
Starting material	4 h YP hydrolysate	0.42a	56.41a	695.76a
Ultrafiltration cut-off 5 kDa	Retentate	2.2a	41.59a	0.42a
	Permeate	3.63b	70.83b	440.68b
HPLC-gel	F 1	3.31c	140.66a	53.66a
	F 2	2.00a	158.94a	401.73c
	F 3	2.20ab	141.59a	78.42b
	F 4	2.35b	149.67a	440.68d
RP-HPLC	SF 2.1	2.23a	237.20a	122.97c
	SF 2.2(VVSGPYIVY LLGAVASMGALLCAP)	3.85e	252.61ab	100.25a
	SF 4.1	1.87c	158.02b	50.32b
	SF 4.2	1.55b	233.65a	90.77a
	SF 4.3(RASDPLLSV RNDDLNYIQ)	2.38a	257.51a	202.78d
	SF 4.4	3.19d	239.83a	387.93e
Rechromatography	SF 4.4.1(LAPSLPGKPKPD AGTTCLFTPLALPYDYSH)	3.24b	291.15a	345.04b
	SF 4.4.2(ITMIAPSAF)	2.97a	281.9a	231.43a
Synthetic analogs of peptides	RASDPLLSV (P1)	4.71c	140.84c	26.36a
	RNDDLNYIQ (P2)	2.28a	99.22b	196.96b
	LAPSLPGKPKPD (P3)	6.03d	296.07d	179.33b
	AGTTCLFTPLALPYDYSH (P4)	2.59b	28.37a	27.6a

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It was observed that ultrafiltration led to a decrease in the chelating activity of iron ions. These results are not consistent with other literature data. For example, Jiang and Mine and Feng and Mine obtained phosphopeptides with a molecular weight between 1–3 kDa, which showed a greater ability to chelate selected metal ions than the initial phosvitin hydrolysates.^29,30

The obtained permeate was subjected to gel filtration chromatography on a Zorbax GF-250 column. The molecular filtration process allowed the isolation of four fractions, which were collected, concentrated by lyophilization and then evaluated for antioxidant properties (Table 1).

Among the four fractions, fraction 4 possessed the highest ferrous chelating activity. However, the level of those activity was the same as for the permeate (F of MW < 5 kDa). All of collected fractions were characterized by an almost two fold increase in reducing power (FRAP) compared to the chromatographed material. Interestingly, fraction no 1 possessed the strongest scavenging ability (3.31 μM Trolox_eq per mg protein) and at the same time had the lowest reducing power and chelating activity.

It was observed no direct interdependence between molecular weights of peptides eluted from GF-250 column and their antioxidant activity (Table 1).

The proposed procedure of biopeptide isolation, which included ultrafiltration and then gel filtration chromatography in the first step, was more efficient compared to the fractionation process of egg yolk hydrolysates proposed by You and Wu.³¹ As a result of using ion-exchange chromatography, they obtained peptide fractions that possessed no significantly higher antioxidant activity than the initial hydrolysates.

In our study, fractions 2 and 4 were further purified by RP-HPLC to obtain two (2.1; 2.2) and four (4.1–4.4) subfractions, respectively.

Evidently, the highest DPPH free radical scavenging activity was reached by fraction 2.2 at 3.85 μM Trolox_eq per mg protein (Table 1). The ability of this fraction to reduce the oxidation of iron ions reached a value of 252.6 μg Fe²⁺ per mg protein.

The strongest ability to reduce the oxidation of iron ions (257.5 μg Fe²⁺ per mg protein) was estimated for fraction 4.3 (Table 1). On the other hand, subfraction 4.4 demonstrated the highest iron ion chelating ability (387.9 μg Fe²⁺ per mg protein). It should also be noted that in all the cases, chelating activity significantly had decreased compared to the output subfraction. Fraction 4.4 was a mixture of peptides; therefore chromatography was repeated on a Zorbax XDB C18 column. The procedure produced two fractions: 4.4.1 and 4.4.2., which were characterized by a significantly high reducing activity (FRAP) (4.4.1 – 291.2 μg Fe²⁺ per mg protein, 4.4.2 – 281.9 μg Fe²⁺ per mg protein).

The mass spectrometry analysis, referred to in detail in Eckert et al.,²¹ indicated that almost all of the isolated peptide fractions were heterogenous, and peptides consisting from 9 to 18 amino acid residues were found (Table 1). A total of 7 egg yolk-derived peptides were identified, with the following sequences: VVSGPYIVY; LLGAVASMGALLCAP; RASDPLLSV; RNDDLNYIQ; LAPSLPGKPKPD AGTTCLFTPLALPYDYSH and ITMIAPSAF (Table 1). So far, long chain antioxidant peptides have been isolated and characterized from a variety of source. These include peptides from plants such as barley glutelin (Gln-Lys-Pro-Phe-Pro-Gln-Gln-Pro-Pro-Phe) and peptides from animals such as the oyster (His-Leu-Arg-Gln-Glu-Glu-Lys-Glu-Glu-Val-Thr-Val-Gly-Ser-Leu-Lys).^9,32

3.3. Biological activity of synthetic analogues of YP-derived peptides

In order to clearly confirm which peptide is responsible for which biological activity and to understand their relationship of activity and sequence, a chemical synthesis of the chosen peptides was carried out. The effectiveness of this process was analyzed using HR-ESIMS. As a result, the pure peptides: RASDPLLSV (m/z[M + H]⁺ 957.531); RNDDLNYIQ (m/z [M + H]⁺ 1150.542); LAPSLPGKPKPD (m/z [M + H]⁺ 1219.701) and AGTTCLFTPLALPYDYSH (m/z [M + 2H]²⁺ 985.484) were obtained (Fig. 1).

Fig. 1 The mass spectra (HR-ESI-MS) of the synthetic peptides: (a) RASDPLLSV; (b) RNDDLNYIQ; (c) LAPSLPGKPKPD and (d) AGTTCLFTPLALPYDYSH.

The chemically synthesized peptides were evaluated in terms of antioxidant activity (Table 1). The strongest antioxidant properties were exhibited by the peptide with the sequence: LAPSLPGKPKPD, with an ability to scavenge DPPH free radicals of 6.03 mmol Trolox_eq per mg protein. RASDPLLSV also showed a strong DPPH scavenging activity, 21.89% lower (Fig. 2).

Fig. 2 Antidiabetic activity of synthetic analogs of peptides isolated from 4 h-egg-yolk protein hydrolysate treated by serine proteinase from C. ficifolia: RASDPLLSV (P1); RNDDLNYIQ (P2); LAPSLPGKPKPD (P3); AGTTCLFTPLALPYDYSH (P4); n.d. means no activity.

The hydrophobic property of the peptide plays an important role in antioxidant activity, especially in the scavenging of free radicals. Numerous studies have indicated that the presence of hydrophobic amino acid residues such as: A, V, L, I, P, M or W, in their sequence, determine strong antioxidant properties.^33–36 Both the analysed peptides contained in their sequence several hydrophobic amino acids such as Ala, Leu and Pro. Furthermore, peptide: LAPSLPGKPKPD isolated from YP hydrolysate, had leucine at the N-terminus. This localization of leucine in the sequence probably enhances the antioxidant properties of the peptide. According to Guo et al., the high antioxidant activity results from the location of L, V, I, A and K residues at the N terminus of the peptide chain.³⁷ Peptide RASDPLLSV held a 2 times higher antiradical activity than the RNDDLNYIQ peptide (composed of mostly hydrophilic amino acids, resulting in a lower antioxidant activity). On the other hand peptide RNDDLNYIQ possessed the strongest Fe²⁺ chelating potency, which may be explained by the presence of two asparagine and one glutamine residue in that sequence. The carbonyl groups of these amino acid residues bind ferrous ions.³⁸ These results are in accordance with the study conducted by Lv et al., who isolated two iron-chelating peptides from soybean hydrolysate prepared with Protease M. These peptides were characterized by high Glu and Asp content.³⁹

The ability to chelate divalent cations is related to the presence of branched chain amino acids such as E, Q, L, R, D, H, T in the sequence of the peptide. The side chains of these amino acids can react with metal ions and thereby inactivate their pro-oxidative activity.⁴⁰

Moreover, Elias et al., point out that the chelating activity of the peptides is crucially dependent on the location of these amino acids in the sequence.³⁴ It is particularly advantageous if they are located at the ends of the peptide chains. The YP derived peptide RNDDLNYIQ had R at the C-terminus and Q at the N-terminus.

There are several known natural α-glucosidase inhibitors (including acarbose, voglibose and miglitol) and DPP-IV inhibitors (including Diprotins A and B, isolated from culture filtrates of Bacillus cereus BMF673-RF1).^14,15 More recently, food protein hydrolysates have also been tested for those inhibitory abilities. For example, products after the peptic treatment of whey proteins exert DPP-IV inhibitory activity.¹³ In addition; ovalbumin enzymatic hydrolysate has been shown to have α-glucosidase inhibitory ability.^16,25

To show the multifunctional properties of the YP derived peptides, the antidiabetic activity, as reflected in α-glucosidase and DPP-IV inhibitory activities, was determined (Fig. 2). The peptides: RASDPLLSV, RNDDLNYIQ and LAPSLPGKPKPD were characterized by a significant DPP-IV inhibitory activity (IC₅₀) in the range from 361.5 to 426.25 μM. LAPSLPGKPKPD also exerted a α-glucosidase inhibitory activity: IC₅₀ = 1065.6 μM. Recently, You et al., isolated peptides with α-glucosidase inhibitory activity from egg white hydrolysate prepared with alkalase.²⁵ The IC₅₀ values of the peptides RVPSLM and TPSPR were 23.07 and 40.02 μM, respectively. More recently they described eight novel antidiabetic peptides from ovalbumin, among which the most potent peptide: KLPGF had α-glucosidase inhibitory activity at IC₅₀ = 59.5 μM.¹⁶ You can see that the YP derived peptide: LAPSLPGKPKPD and the peptides described by You et al. share common characteristics: the presence of P, S and L residues in their sequence. One could suppose that the high level of α-glucosidase inhibition is due to the presence of the R residue on the C or N terminus of the peptide chain.²⁵ The lower α-glucosidase inhibition by the LAPSLPGKPKPD peptide compared to these aforementioned peptides may be explained by the lack of an R residue in the peptide sequence.

Some studies have reported that bioactive peptides possessed DPP-IV inhibitory activity. Two bioactive peptides, Ile-Pro-Ala and Val-Ala-Gly-Thr-Trp-Tyr, derived from β-lactoglobulin hydrolyzed by proteinase K and trypsin, showed IC₅₀ values of 49 and 174 μM, respectively, against DPP-IV “in vitro”.^41,42 Li-Chan et al., also successfully isolated two peptides, Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala from Atlantic salmon skin gelatin, which showed inhibitory effects on DPP-IV with IC₅₀ values of 49.6 and 41.9 μM, respectively.⁴³

According to the report of studies, DPP-IV inhibitory peptides consisted of at least one Pro and mostly as the penultimate N-terminal residue.^41–43 This is in line with our results in which peptide: LAPSLPGKPKPD with the strongest DPP-IV inhibitory properties contained four residues of proline, one of which is located in position 3 at the N terminus of the peptide chain. Diprotins A (Ile-Pro-Ile) and B (Val-Pro-Leu), isolated from culture filtrates of Bacillus cereus BMF673-RF1 exhibit the DPP-IV inhibitory activity with IC₅₀ values of 3.2 and 16.8 μM, respectively.⁴⁴ The results showed that peptide: LAPSLPGKPKPD obtained in this study showed much lower DPP-IV inhibitory activity than peptides mentioned above, which may be determined by the peptide length. DPP-IV inhibitory activity of bioactive peptides may be also determined by the two N-terminal amino acid sequence of X-Pro, where X is the hydrophobic amino acid.⁴⁴ Interestingly this peptide exerted a strong DPPH free radical scavenging activity at the same time.

Conclusion

Results showed that phospholipid extraction of an egg yolk protein by-product may indeed be a novel source of peptides with multiple biological activity. Antioxidant (radical scavenging) capacity, Fe²⁺ chelating effect, reducing power (FRAP) and antidiabetic (α-glucosidase and DPP-IV inhibitory activities) properties were tested. Almost all of the peptides showed antioxidant and DPP-IV inhibitory activity, while peptide LAPSLPGKPKPD exhibited antioxidant, DPP-IV and α-glucosidase inhibitory activities. The presented method of producing new peptide preparations proved to be advantageous thanks to the combination of desirable properties of these peptides, the use of protein waste (defatted egg yolk granules) from an industrial process, as well as a cheap enzyme source (Asian pumpkin).

Conflict of interest

All the authors who have taken part in this study declare that they have nothing to disclose regarding competing interests, or funding from industry with respect to this manuscript.

Acknowledgements

Project “Innovative technologies in the production of bio-preparations based on new generation eggs,” Innovative Economy Operational Programme Priority 1.3.1, thematic area “Bio”, co-financed by the European Union through the European Regional Development Fund within the Innovative Economy Operational Programme, 2007–2013. This project was supported by the Wroclaw Centre of Biotechnology programme, The Leading National Research Centre (KNOW), 2014–2018.

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