PaPer 450 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: egg yolk proteins, Cucurbita ficifolia protease, hydrolysis, antioxidant, ACE-inhibitory activity, immunostimulating activity - Biological activity of egg-yolk protein By-product hydrolysates oBtained with the use of non-commercial plant protease A. ZAmbrowicZ*, E. EckErt, m. PokorA, A. DąbrowskA, m. sZołtysik, ł. bobAk, t. trZisZkA and J. chrZAnowskA Wrocław University of Environmental and Life Sciences, Department of Animal Product Technology and Quality Management, Chełmońskiego 37/41, 51-630 Wrocław, Poland *Corresponding author: Tel. +48 71 320 7773, email: aleksandra.zambrowicz@up.wroc.pl AbstrAct Enzymatic hydrolysis leads to improved functional and biological properties of protein by-prod- ucts, which can be further used as nutraceuticals and protein ingredients for food applications. the present study evaluated AcE-inhibitory, antioxidant and immunostimulating activities in hydrolysates of egg-yolk protein by-product (YP), generated during industrial process of delipidation of yolk. the protein substrate was hydrolyzed using non-commercial protease from Asian pump- kin (Cucurbita ficifolia). the reaction was conducted in 0.1 M tris-Hcl buffer (pH 8.0) at tempera- ture of 37°c for 4 hours using different enzyme doses (100-1000 U/mg of substrate). the protein degradation was monitored by the determination of the degree of hydrolysis (DH), release of free amino groups (FAG) and by rP-HPLc. In the obtained hydrolysates we also evaluated biological activities. It was shown that the highest DH of substrate (46.6%) was obtained after 4h of reaction at the highest amount of enzyme. this hydrolysate exhibited antioxidant activity, including ferric ion reducing (FrAP) (56.41 μg Fe2+/mg), ferric ion chelating (695.76 μg Fe2+/mg) and DPPH free radical scavenging (0.89 μmol trolox eq /mg) as well as AcE-inhibitory (Ic 50 =837.75 μg/mL) activi- ties. the research showed improved biological properties of enzymatically modified YP by-product. Ital. J. Food Sci., vol. 27 - 2015 451 IntroDUctIon nowadays, the identification of bioactive food components, which can provide health bene- fits is one of the objectives of scientific research worldwide. special attention is given to bioactive peptides due to their role in the prevention of numerous diseases (sHArMA and rAnA, 2011). these peptides, released via the enzymatic hy- drolysis of food proteins reveal numerous bio- logical activities: antioxidant, antihypertensive, antimicrobial, antidiabetic, opioid and immu- nostimulating. these may have positive effects the cardiovascular, nervous, immune or diges- tive systems of the body (MInE and KovAKs-no- LAn, 2006; cHAY PAK tInG et al., 2011; YU et al., 2011; PoKorA et al., 2014). the egg is recognized as a very valuable source of proteins for human nutrition, as well as pro- teins, which may be precursors of peptides with biological activity (MInE and KovAKs-noLAn, 2006; YU et al., 2011; ZHIPEnG et al., 2011). AcE-inhibitory peptides are one of the best char- acterized peptides derived from eggs. the hydro- lysis of ovalbumin, the main protein of egg white, conducted by gastrointestinal enzymes, results in the release of several AcE-inhibitory peptides (MIGUEL et al., 2004; MIGUEL et al., 2007). the effectiveness of these peptides was validated in tests in vivo conducted on spontaneously hyper- tensive rats (MIGUEL et al., 2007). Antihyperten- sive activity was also demonstrated by some pep- tides released from egg-white protein treated by alcalase (LIU et al., 2010; ZHIPEnG et al., 2011). As a result of peptide purification from those hy- drolysates, AcE-inhibitory peptides: rvPsL and QIGLF were obtained (LIU et al., 2010; ZHIPEnG et al., 2011). It was also shown that egg is a rich source of proteins in which sequence numerous anti- oxidant peptides are encrypted. Phosphopep- tides derived from egg phosvitin having molec- ular masses 1-3 kDa exert a strong ability to in- hibit the oxidation of linoleic acid, to scavenge DPPH free radicals and to chelate iron ions (II) (XU et al., 2007). Egg-yolk hydrolysates com- posed of peptides with a molecular weight low- er than 1 kDa obtained with the use of protein- ase from bacillus ssp., also exhibited antioxi- dant capacities. superoxide-scavenging activity and suppression of discoloration by β-carotene have also been observed (sAKAnAKA and tAcHI- bAnA, 2006). Egg yolk peptides obtained during alkalase and protease n digestion of delipidat- ed egg yolk proteins were found to boost the systemic antioxidant status in the blood by in- creasing the GsH concentration in red blood cells (Young, Fan and Mine, 2010). It has been demonstrated that the consumption of egg yolk protein hydrolysates with antioxidative prop- erties leads to the inhibition of tumor cell pro- liferation in the colon (IsHIKAwA et al., 2009). some peptides derived from egg proteins can act as immune modulators and may be used as nutraceuticals for the prevention or treatment of lifestyle dependent diseases. Immunomodu- latory peptides may exhibit anti-inflammatory activity by decreasing the production of pro-in- flammatory cytokines (MAttsbY-bALtZEr et al., 1996; cross and GILL, 2000; MInE and KovAcs noLAn, 2006). Egg yolk peptides significantly reduce pro-inflammatory cytokine, IL-8, in the caco-2 cell line (YoUnG and MInE, 2010). Fur- thermore, immunostimulatory activity, assayed as the ability to enhance the capacity of phago- cytic cells in mice, was present in ovalbumin hy- drolysates prepared by gastrointestinal enzymes (bIZIULEvIčIUs et al., 2005). bioactive peptides can be also released from protein by-products generated during isolation of biologically active substances naturally occur- ring in egg. one such protein waste is a by-prod- uct of lysozyme and cystatin extraction from hen egg white by ethanol method (soKołowsKA et al., 2007). our previous studies showed that this by-product, which itself exhibits poor function- al properties, can be a rich source of AcE-inhib- itory and antioxidative peptides (PoKorA et al., 2013; 2014; ZAMbrowIcZ et al., 2013). Attention is also drawn to egg yolk as a source of substanc- es, which may find wide application in the pre- vention and treatment of various medical condi- tions. Egg yolk is mainly used for the extraction of valuable phospholipids such as lecithin, which is more valuable than plant-derived lecithin due to the specific chemical composition. the main by-products of this process are partially dena- tured and defatted egg yolk proteins in the form of insoluble granule fractions (sIEPKA et al., 2010). the preparation of bioactive peptides by en- zymatic hydrolysis of proteinaceous by-prod- ucts could become an interesting method of waste disposal if the process was cost- effective. therefore cheap and effective enzymes for this process are preferred. Plant serine protease iso- lated from Cucurbita ficifolia pulp used in this study exhibits strong proteolytic properties and is a relatively cheap proteolytic enzyme (ILLAnEs et al., 1985; cUrotto et al., 1988). the aim of this study was the enzymatic hy- drolysis of a by-product of egg yolk phospholip- id isolation, in order to obtain hydrolysates with antioxidant, AcE-inhibitory and immunostimu- latory activities. MAtErIALs AnD MEtHoDs Substrate Eggs from 40-45 weeks old Lohman brown laying hens (housed in a bedding system) were stored at 4°c for 1 week. the eggs were automat- ically broken and their macroscopic parts were separated on an industrial scale. Phospholipids were extracted from the egg yolks (siepka et al., 452 Ital. J. Food Sci., vol. 27 - 2015 2010).  Defatted granules, a by-product of phos- pholipid extraction from the egg yolk, were lyo- philized and stored frozen until used. Enzyme non-commercially available protease from C. ficifolia was isolated according to the procedure described by DrYjAńsKI and wILUsZ (1990). serine protease was obtained by extraction of the homogenized pumpkin pulp separated from the solids by centrifugation (5000 G, 20 min, 4°c). to the supernatant, ammonium sulfate was added to 50% saturation, and allowed to stand for 24 hours, and then centrifuged (9600 rev/min, 30 min). the resulting precipitate (the enzyme preparation) was desalted by dialysis for 12 hours using distilled water (4°c), and then 0.02 M of phosphate buffer at pH 6.0. Determination of proteolytic activity of protease from C. ficifolia Proteolytic activity was determined by reac- tion with 1% casein as a substrate (bDH, Ltd., England) at pH 8.3 (KUnItZ, 1945). the sub- strate with the enzyme was incubated for 10 min at 37°c. the reaction was stopped by the addi- tion of 5% trichloroacetic acid (tcA). the sam- ples were then centrifuged, and the absorbance of supernatants were measured at λ=280 nm. one unit of enzymatic activity (U) was defined as the amount of enzyme giving an increase in absorbance of 0.1 at 280 nm under reaction conditions. Determination of protein content total protein content (n x 6.25) in insoluble substrate was determined using the Kjeldahl method. Protein content in hydrolysates and peptide fractions was determined by the meth- od of LowrY et al. (1951). Enzymatic hydrolysis YP hydrolysis was carried out according to a modified method of Zambrowicz et al. (2013a). 1% substrate suspension in 0.1 M tris-Hcl buf- fer (pH 8.0) was hydrolyzed at 37o c for 4 hours using C. ficifolia protease at doses of 100, 200, 400 and 1000 U of active enzyme applied on 1 mg of YP substrate. the reaction was ended by heating the mixture at 100ºc for 15 min. the hydrolysates were cooled, centrifuged (5500 G, 10 min, 10°c), then the supernatants were lyo- philized and stored at 4°c until used. The degree of hydrolysis the degree of hydrolysis (DH %) was deter- mined as the percentage ratio of protein sol- uble in 10% trichloroacetic acid (tcA) to total protein (sPELLMAn, 2003). tcA was added to the hydrolysates (1:1) and after 1 h of incuba- tion at 4°c the samples were centrifuged (4500 G, 15 min, 20°c). the concentration of the tri- chloroacetic acid-soluble product in the super- natant was measured spectrophotometrical- ly and calculated from the following equation: DH (%) = (mg soluble protein after hydrolysis / mL ÷ mg soluble protein before hydrolysis/mL) × 100% The content of free amino acid groups the content of free amino acid groups (FAG) (μmol/g) was determined by using trinitroben- zene sulfonic acid (tnbs, sigma) according to a modified method by Kuchroo et al. (1983). Reversed-phase high-performance liquid chromatography Peptide profiles of hydrolysates were moni- tored by reversed-phase high-performance liq- uid chromatography (rP-HPLc). separation was performed using a Zorbax XDb-c 18 Agilent col- umn (1.8 mm × 50 mm). the operation condi- tions were as follows: injection volume: 50 μL; mobile phase A – 0.1% tFA in water; mobile phase b – 0.1% tFA in acetonitrile, column temperature: 30°c. Flow rate: 1mL/ min. Analysis time and gradient conditions can be found in drawings. the ab- sorbance of eluent was monitored at λ=230 nm. Determination of ACE-inhibitory activity AcE (Ec 3.4.15.1) inhibitory activity was mea- sured spectrophotometrically according to the method described by MIGUEL et al. (2004) with some modifications. A hydrolysate solution (40 µL) mixed with a Hippuryl-His-Leu (HHL) sub- strate solution (5 mmol/L in 100 mmol/L potas- sium phosphate containing 300 mmol/L sodium chloride, pH 8.3) was preincubated at 37°c for 5 min, and the reaction was initiated by adding 20 µL (2 mU) of AcE solution, and then incu- bated for 30 min at the same temperature. the enzymatic reaction was terminated by the addi- tion of 150 µL of 1 M Hcl. the liberated hippu- ric acid was extracted using 1 mL of ethyl ace- tate and vigorously shaking, 750 µL of the upper layer was transferred into a test tube and evap- orated under vacuum. the hippuric acid left in the tubes was re-dissolved in 800 µl of distilled water. the content of hippuric acid was deter- mined spectrophotometrically at λ=228 nm. All samples were tested in 3 replications. In- hibition activity was calculated using the follow- ing equation: Inhibitory activity (%) = = ((Ac – As) / (Ac – Ab)) ×100 Ital. J. Food Sci., vol. 27 - 2015 453 where Ac is the absorbance of the buffer (con- trol), As is the absorbance of the reaction mix- ture (sample), Ab is the absorbance when the stop solution was added before the reaction oc- curred (blank). the Ic 50 value was defined as the concentra- tion of peptides in µg/mL required to reduce 50% of AcE activity, which was determined by analysis of AcE inhibition (%) versus peptide concentration. Determination of antioxidant activity as the ability to scavenge of DPPH free radicals Antioxidant activity was determined by a modified method of Yen and chen (1995) as the ability to scavenge of DPPH (2,2-di(4-tert-oc- tylphenyl)-1-picrylhydrazyl) free radicals in an aqueous solution of peptides. Absorbance mea- surements were made at λ=517 nm after 30 min incubation. the antioxidant activity of the ana- lyzed peptides was determined on the basis of the standard curve prepared for trolox equivalent. Determination of antioxidant activity by FRAP method Antioxidant activity was determined as the ability to reduce the oxidation of iron Fe(III) to Fe(II) ions in a reaction with tPtZ (2,3,5-triphen- yltetrazoliumchloride). the absorbance was mea- sured at λ=593 nm. the concentration of Fe2+ ions was determined on the basis of the stan- dard curve for known Feso 4 solutions (bEnZIE and strAIn, 1996). Determination of iron Fe(II) ion chelation chelation of iron ions was determined by col- orimetric measurement of the quantity of Fe(II) not bound to the peptides in a reaction mix- ture with ferrozine (3-(2-pyridyl)-5,6-diphe- nyl-1,2,4-triazine-p,p′-disulfonic acid monoso- dium salt hydrate) (Xu et al., 2007). Absorbance measurement was made at λ=562 nm. the abil- ity to chelate iron ions was determined on the basis of the standard curve for a Fecl 2 solution. Determination of immunostimulatory activity Immunostimulatory activity of the cytokine se- cretion in human whole blood was determined at the Department of Immunochemistry, Insti- tute of Immunology and Experimental therapy, Polish Academy of sciences in wrocław (Poland). cytokine secretion was induced according to the procedure described by InGLot et al. (1996). blood samples from at least 10 donors were col- lected in syringes containing sodium heparin. within 1 h after collection, the blood was dilut- ed  10-times  with rPMI 1640 medium supple- mented with penicillin/streptomycin, L-gluta- mine and 2% fetal bovine serum. 1 ml portions of the cell suspension were distributed in two 24-well flat-bottomed tissue culture plates. to the cell suspension of whole human blood (1 mL sample) the hydrolysates were added at 1.0, 10 and 100 μg. As a reference, the positive lipopoly- saccharide inducer of E. coli at a concentration of 4 mg/mL was used. control wells contain- ing non-treated  blood cell samples were used to measure the spontaneous production of cy- tokines (negative control). the plates were incu- bated for 22 h at 37°c in a 5% co 2 atmosphere. After incubation, the plates were centrifuged at 200 G for 15 min at room temperature. the su- pernatants were collected and used for determi- nation of the cytokines. IL-6 and IL-10 were de- termined by microplate enzyme-linked  immu- nosorbent assay using commercially available sets from becton Dickinson (Franklin Lakes, nj, UsA) according to the procedure recommended by the manufacturer. Statistical analysis All experiments were carried out in triplicates. the data obtained were subjected to multi-fac- tor variance analysis (AnovA), followed by the Duncan’s multiple range test to determine the significant difference between sample at p<0.05 level using the statistica v.9.0. the results of immunostimulating activity were considered significant by a non-paramet- ric wilcoxon test at p≤0.05 (*) and 0.05≤ p≤ 0.1 (**) versus control (untreated cells). rEsULts AnD DIscUssIon Enzymatic hydrolysis Egg yolk protein preparation (YP), as a by-product of lecithin extraction, was treat- ed by a non-commercial serine protease isolat- ed from C. ficifolia in order to evaluate antiox- idant, AcE-inhibitory and immunostimulatory properties. the progress of hydrolysis was monitored by determining the degree of hydrolysis (DH) (%) (Fig. 1), the increase in the concentration of free amino groups (FAG) (Fig. 2), and by rP-HPLc peptide profile analysis (Fig. 3). DH depended on the enzyme dose and reaction time. DH in- creased slowly in the first 0.5 h, followed by a faster rate of increase up to 4 h, indicating that the maximum cleavage of proteins occurred in the last hour of hydrolysis. Dissimilar kinet- ics of protein substrate degradation with vari- ous proteolytic enzymes has been observed by other authors (ottE et al., 1998; ZAMbrowIcZ et al., 2012). typically, enzymatic hydrolysis is most extensive during the first 30 minutes and then slows down, indicating a maximum of pro- tein degradation in the first hour of hydrolysis. 454 Ital. J. Food Sci., vol. 27 - 2015 Fig. 1 - Effect of time on the degree of hydrolysis (DH) during hydrolysis of YP by protease from C. ficifolia. a-c means fol- lowed by the same letter do not differ significantly (p≤0.05). Fig. 2 - Effect of time on the free amino groups contents (FAG) during hydrolysis of YP by protease from C. ficifolia. Fig. 3 - Peptide profiles (rP-HPLc) of YP protein hydrolysates obtained after using: A. 100 U/mg, b. 200 U/mg, c. 400 U/ mg and D. 1000 U/mg of protease from C. ficifolia. Probably, the hydrolysis process with the use of protease from C. ficifolia proceeded according to the “one-by-one” mechanism. During the initial stage of the reaction (determining the overall lev- el of hydrolysis) it is necessary to partially unfold native protein molecules. As a result, the pro- tein loses its stability, more peptide bonds are exposed on the outside of the molecule (inter- mediate products) and the enzyme has access to the hydrolyzed peptide bond. In a further step, intermediate products are very rapidly degrad- ed to small peptides (KUnst, 2003). the use of the lowest dose of enzyme (100 U/ mg) resulted in a nearly 15% DH of YP. the in- crease of the dose of C. ficifolia proteinase to 400 U/mg did not exert any significant impact on DH rate, whereas the addition of the enzyme at 1000 U/mg resulted in a DH value of more than 46% after 4 hours of digestion. Analysis of the FAG concentration of the obtained hydrolysates con- firmed the above results. the greatest increase in concentration was observed during the long time of hydrolysis. the application of enzyme at doses from 100 to 400 U/mg resulted in simi- lar levels of increases of FAG from 2255.20 to 2325.74 µM Gly/g. the most intensive increase of FAG (4525.1 µM Gly/g) occurred during the 4-hour reaction using 1000 U/mg of protease. Ital. J. Food Sci., vol. 27 - 2015 455 Protein-peptide profiles obtained by rP-HPLc technique demonstrate the extent of YP hydro- lysis (Fig. 3). they were identified with both a longer retention time (3-7 min) specific for the more hydrophobic peptides, and peaks with a short retention time (0.5-1 min) typical for hy- drophilic peptides. the wide distribution of deg- radation products indicates that each of the hy- drolysates is composed of peptides with different hydrophobic properties, which may impact the biological activity of the obtained hydrolysates. the results described above indicate that ser- ine protease from C. ficifolia is an effective en- zyme in hydrolyzing egg yolk protein by-product. Previously, the proteolytic properties of this en- zyme had been tested on casein, a protein from corn gluten, and egg white proteins (ILLAnEs et al., 1985; cUrotto et al., 1989; PoKorA et al., 2014). The biological activities of YP enzymatic hydrolysates the antioxidant activity of YP hydrolysates was studied in terms of the scavenging effect on DPPH radicals, ferric reducing power (FrAP), and iron chelating activity (table 1). the enzymatic treatment of YP leads to an in- crease in DPPH free radical scavenging activi- ty in the hydrolysates. ELIAs et al. (2008) ex- plained that the antioxidant activity of the hy- drolysates/peptides is the result of the proteolyt- ic action of the enzyme. the specific amino-acid sequence of peptides and their changed physical properties allow exposing the amino acid resi- dues and their action as electron donors. As a result of these reactions, the peptides combine with radicals and form stable complexes, which inhibit oxidation processes. the various YP hy- drolysates showed different potencies in scav- enging DPPH radicals. the results indicated no direct relationship between DH and the values of DPPH free radical scavenging activity. the highest DPPH scavenging potency was shown by the hydrolysate obtained after 4 hours deg- radation with an enzyme dose of 1000 U/mg (0.89 μmol trolox eq /mg) (table 1). the signifi- cant level of DPPH free radical scavenging ac- tivity (0.63 μmol trolox eq /mg) was also observed for the hydrolysate obtained with the use of 200 U/mg protease after 0.5 hour digestion. In pre- vious works, YP protein by-product was treat- ed with pepsin and neutrase leading to final hy- drolysates with DH values: 45.3% and 27.6%, respectively (ZAMbrowIcZ et al., 2014; PoKo- rA et al., 2013). Peptic hydrolysate and hydro- lysate obtained by neutrase showed DPPH free radical scavenging activity values: 0.5 troloxeq/ mg and 0.44 µM trolox eq /mg, respectively (ZAM- browIcZ et al., 2014; PoKorA et al., 2013). It may be explained that the potency of YP-hydro- lysates to scavenge DPPH free radical depends more on the specificity of the enzyme than on the degree of hydrolysis (DH). our results also indicate that YP is a better source for peptides exhibiting DPPH scavenging activity than other protein waste, such as by-products of lysozyme and cystatin isolation from egg white. Egg white protein by-product hydrolysates obtained with trypsin and neutrase exhibited free radical scav- enging activity up to 0.21 and 0.17 µmol trolox- eq /mg, respectively (ZAMbrowIcZ et al., 2013). Previously, serine protease C. ficifolia was used by DąbrowsKA et al. (2013) to evaluate the antioxidant activity of bovine casein. casein hy- drolysates possessed a different ability to scav- enge of DPPH radicals (from 0.06 to 2.21 µmol trolox eq /mg), depending on enzyme dose and re- action time. However, the DPPH scavenging po- tency of many of them was at the same level as the YP hydrolysates. In most of the hydrolysates obtained with different doses of protease, the fer- ric reducing ability was increasing gradually with the time of hydrolysis. the only exception was the 0.5 h hydrolysate obtained with the enzyme dose of 200 U/mg, which possessed significant- ly higher ferric reducing activity than the prod- ucts obtained during long time of degradation (more than 30 minutes). A maximum value of this activity reached 56.41 μg Fe2+/mg, for the 4 h hydrolysate obtained with the participation of 1000 U/mg of the enzyme (table 1). the ferric reducing activity of the 4h hydrolysate increased with the increased doses of the enzyme. the ap- plication of protease at 100, 400 and 1000 U/ mg resulted in hydrolysates 3.44, 5.0 and 5.47 times more potent than YP, respectively. on the other hand, hydrolysates exerted more than 3 times lower ferric reducing activity in compar- ison to the YP hydrolysate prepared with neu- trase (177.35 μg Fe2+ /mg) (Pokora et al., 2013). this results gives an indication that unconven- tional protease from C. ficifolia is characterized by a lesser ability to release peptides with fer- ric reducing activity from YP than commercially available neutrase. An increase in chelating ac- tivity was also observed as a result of progress in hydrolysis. the highest chelating activity was obtained in hydrolysates with DH above 35 %. YP degraded with participation of 1000 U/mg protease during 3 and 4 hour reactions exhib- ited ferrous ion chelating activity at 692.49 and 695.76 μg Fe2+/mg, respectively (table 1). sig- nificant ferric chelating power was also shown by hydrolysates in which DH ranged from 15% to 20%. similar results were obtained by tor- res-Fuentes et al. (2011), who analyzed the anti- oxidant properties of plant protein hydrolysates in terms of their ability to complex iron ions. numerous antihypertensive peptides (eg. ovo- kinin, ovokinin 2-7, rADHP, YPI, DLIn) derived from egg white proteins by enzymatic hydrolysis have been characterized (MIGUEL et al., 2004; LIU et al., 2010). Interest has been aroused in AcE- inhibitory peptides generated from egg yolk pro- teins, because they have not been described as a 456 Ital. J. Food Sci., vol. 27 - 2015 source of peptide inhibitors of AcE as much. the hydrolysates obtained in this work exhibited var- ious abilities to inhibit the AcE enzyme (table 1). the hydrolysates of DH lower than 10% did not exert any AcE inhibitory activity. the most ac- tive inhibitor of AcE (Ic 50 =467.5 μg/mL) was the hydrolysate obtained by an enzyme dose of 1000 U/mg after 4-hours digestion. whole egg yolk in native form as a potential source of AcE-inhibi- tory peptides was tested by YoU and wU (2011). the level of this activity (Ic 50 ) for hydrolysates prepared with the use of gastrointenstinal (pep- sin, pancreatin) and microbial (thermolysin, al- kalase) proteases ranged from 133.4 µg/mL to 210.2 µg/mL (YoU and wU, 2011). such signif- icant differences in the level of AcE-inhibitory activity may result from the fact that in the pres- ent study we used a by-product, denatured pro- tein of the yolk granular fraction. Denaturation has a significant impact on the physico-chemi- cal and biological properties of proteins. the results indicate that protein by-prod- uct obtained from the isolation of phospholip- ids from hen egg yolk may be a better source of AcE-inhibitory peptides than other protein by- product from the isolation of cystatin and ly- sozyme from egg white. the peptic hydrolysate (DH: 38.3%) of this protein preparation exhib- ited an activity of Ic 50 =643.1 μg/mL (ZAMbro- wIcZ et al., 2013). recently, we indicated that serine protease from C. ficifolia may be effective in the conversion of this protein by-product to a value added product with AcE-inhibitory activi- table 1 - biological activities of YP hydrolysates obtained with using serine protease from C. ficifolia. All data were expressed as mean values (mean±sD, n=3). values sharing the same letter at the same enzyme dose and test group are not significant- ly different at p<0.05. Enzyme dose Time of hydrolysis DPPH scavenging Ferric reducing Ferrous ion-chelating ACE inhibitory [U/mg] activity ability (FRAP) activity activity [IC 50 ] [µM Trolox eq /mg] [µg Fe2+/mg] [µg Fe2+/mg] [µg/ml] 0 substrate 0.15±0.01a 10.3±0.16a 376.2±18.80a nda 100 0.5 0.28±0.03a 33.26±1.38a 497.10±8.22b ndc 1 0.32±0.03b 36.44±1.62b 466.26±3.89c ndc 3 0.18±0.02c 48.78±1.20c 474.25±4.01d 968.5±17.25b 4 0.42±0.02b 35.40±0.67d 574.38±13.67a 837.75±15.25a 200 0.5 0.63±0.02a 46.14±1.33a 460.72±11.74b ndc 1 0.20±0.03c 35.53±1.21a 507.59±2.88d ndc 3 0.28±0.01d 37.11±0.73b 553.75±13.79c 890.75±11.25b 4 0.27±0.01b 44.68±1.66b 626.16±6.56a 777.0±14.25a 400 0.5 0.20±0.03ab 38.91±1.41a 468.70±12.07d ndc 1 0.21±0.03a 36.77±1.20a 513.73±3.81c ndc 3 0.20±0.04a 35.44±1.30ab 573.56±3.77b 803.12±10.25b 4 0.26±0.02a 51.46±0.78ab 625.86±7.11a 718.75±4.5a 1000 0.5 0.22±0.02a 21.71±1.37d 642.32±2.83d 657.75±4.5a 1 0.25±0.02c 27.78±1.75c 654.79±5.78b 650.0±4.5a 3 0.43±0.03b 43.33±1.06b 692.49±1.86c 554.75±7.75a 4 0.89±0.03a 56.41±1.13a 695.76±14.91a 467.5±6.0a ty (PoKorA et al., 2014). the 50% inhibition of AcE was obtained with the presence of 9071.7 μg/mL of the hydrolysate. studies have shown a high linear correlation between DPPH free radical scavenging activity and immune activity with a positive correlation coefficient of 0.96 (HE et al., 2014). therefore hydrolysates with the highest DPPH free radi- cal scavenging potency were also evaluated in terms of their immunostimulatory properties. It was assessed as the results of cytokines IL-10 and IL-6 induction by hydrolysates in whole hu- man blood cell cultures (ex vivo) (Fig. 4). 4-hour hydrolysate obtained with the use of 1000 U/ mg of C. ficifolia protease appeared to be slight inducer. the use of 100 µg/ mL of protease re- sulted in a low increase the concentration of IL- 6, which reached the value: 3.05 ng/ mL (Fig. 4 A). However, the results were not statistical- ly significant compared to the positive control (LP). Extremely different cytokine inducing ac- tivity was exerted by the yolkin, naturally oc- curring in egg yolk. Yolkin is a mixture consist- ing of several peptides of an apparent molecu- lar weight of 1 to 35 kDa, produced as a result of vitellogenin II hydrolysis by cathepsins during the formation of an egg. Its constituent peptides were found to be efficient inducers of IL-1β, IL-6 and IL-10 secretion. A complex at a concentra- tion of 100 µg/mL showed almost the same ac- tivity as the LPs-treated control in stimulating cytokine production. (PoLAnowsKI et al., 2013). the biological activity of enzymatic hydrolysates Ital. J. Food Sci., vol. 27 - 2015 457 is determined by the protein sequence (type and location of amino acid residues) as well as by the specifity of the enzyme (cLEMEntE, 2000; PArK et al., 2001). these two crucial factors are responsible for the disparate level of immuno- stimulatory activity of YP-hydrolysates. concLUsIons the effect of enzymatic modification of an egg yolk protein preparation (YP), obtained as a by- product of phospholipid extraction, on its bio- logical properties were evaluated. the hydroly- sis process of YP was performed with the use of noncommercial serine protease from C. fici- folia. the most effective degradation of YP was noticed under the conditions: enzyme dose 1000 U/ mg and duration 4 h, when a significant de- gree of hydrolysis (46.6%) was obtained. Enzy- matic hydrolysis of YP provided hydrolysates/ peptides exhibiting antioxidant and AcE-inhibi- tory activities.  the YP hydrolysates showed sig- nificant antioxidant and degree of hydrolysis-de- pendent AcE-inhibitory activity. the 4-hour hy- drolysate obtained with the highest amount of enzyme (1000 U/mg) showed the highest bio- logical activity among the tested hydrolysates. It exhibited ferric ion reducing potential (FrAP) (56.41 μg Fe2+/mg), ferric ion chelating activity (695.76 μg Fe2+/mg), DPPH free radical scaveng- ing activity (0.89 μmol trolox eq /mg) and AcE- in- hibitory (467.5 μg/mL) activity. novel biological effects of egg-yolk protein by-product hydroly- sates was shown. 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