IntroductionMeat is a source of complete protein in the humandiet. Meat proteins have a high nutritional valueand therefore contribute to the normal physiologicalstatus. They are converted into various formsduring cooking, processing, and digestion. Forexample, bioactive peptides obtained from meatproteinsthrough an enzymatic reaction can helpmaintain the immune status of the human body. Not onlymuscle meat is a good source of protein and bioactivepeptides, but offal is as well. Therefore, meat by-productshave increasingly been studied and used to producefunctional ingredients and bioactive peptides [1–6].547Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554Consumers are becoming increasingly aware ofthe immune-boosting properties of bioactive peptidesderived from dietary proteins. Bioactive peptides aresmall fragments of dietary proteins, mostly consistingof 2–20 amino acid residues. They can be ligandsand therefore have many targets in the human body,such as the immune, cardiovascular, digestive, andendocrine systems [7–11].Hydrolysis is the main process of isolating peptides.Many studies confirm that the proteins of meat byproductsare hydrolyzed by proteolytic enzymesunder controlled conditions, enabling bioactive peptidesto form. The main proteolytic enzymes that canbe used to hydrolyze proteins are pepsin, trypsin,chymotrypsin, corolase, papain, as well as enzymesof microbial origin, such as Neutrase from Bacillusamyloliquefaciens and Alcalase from Bacillus licheniformis[12–15].The efficacy and safety of bioactive peptides mustbe proven by clinical studies on living organisms beforethey can be approved for use by regulatory andsupervisory authorities [16–18].Some food-derived peptides have antioxidant,immunomodulatory, antihypertensive, anticancerous,anti-inflammatory, antimicrobial, hypocholesterolemic,intestine-modulatory, antidiabetic, opioid, and metalchelatingproperties [19–23]. Their biological activitymainly depends on their amino acid composition,sequence, length, and charge [24, 25].Currently, hundreds of peptides with differentbiological action have been identified and isolated fromvarious food sources, including milk, whey, eggs, fish,rice, soybeans, peanuts, chickpeas, corn, and algae [24].However, only a few of them are marketed as functionalnutraceutical products. For example, bioactive peptidesderived from milk and fish are more commonly usedas food ingredients than peptides from other foodsources. Table 1 presents some bioactive peptidesobtained from various protein sources and lists theirproperties [26].Antioxidant peptides usually contain hydrophobicamino acids and residues of histidine, phenylalanine,tryptophan, or tyrosine.Thus, the food industry needs to develop technologiesfor isolating bioactive peptides from by-products,including meat by-products.We aimed to develop a technology for obtainingpeptides from poultry by-products and study thebioactivity of some peptide sequences.To achieve this aim, we set to:– select enzymes and develop a scheme for hydrolyzinga homogeneous mass of chicken skin;– develop two alternative schemes for producing bioactivepeptides through the hydrolysis of poultry by-products;– conduct an electron microscopy of the obtainedhydrolysates;– determine the main physicochemical parameters ofthe hydrolysates; and– perform a comparative analysis of the peptides.Study objects and methodsWe studied broiler chicken skin in a homogeneousstate. First, we selected proteolytic enzymes andhydrolysis conditions that could increase the bioactivityand yield of hydrolysates. In particular, we chose aTable 1. Some bioactive peptides and their propertiesТаблица 1. Некоторые биоактивные пептиды и их свойстваProtein source Peptide sequence Bioactive propertiesCow’s milk whey lactoferrin Glu-Asn-Leu-Pro-Glu-Lys-Ala-Asp-Arg-Asp-Gln-Tyr-Glu-Leu Osteoblast-proliferatingBeans (Phaseolus vulgari) Gly-Leu-Thr-Ser-Lys, Leu-Ser-Gly-Asn-Lys, Gly-Glu-Gly-Ser-Gly-Ala,Met-Pro-Ala-Cys-Gly-Ser-Ser, and Met-Thr-Glu-Glu-TyrAnticancerousSoy Met-Ile-Thr-Leu-Ala-Ile-Pro-Val-Asn-Lys-Pro-Gly-Arg ImmunomodulatoryChicken egglysozymeVal-Ala-Trp-Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp, Trp-Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp, Ala-Trp-Ile-Arg-Gly-Cys-Arg-Leu, Trp-Ile-Arg-Gly-Cys-Arg-Leu, and Ile-Arg-Gly-Cys-Arg-LeuAntioxidantEgg white Ala-Glu-Glu-Arg-Tyr-Pro, Asp-Glu-Asp-Thr-Gln-Ala-Met-Pro, Pro-Val-Asp-Glu-Asn-Asp-Glu-Gly, Gln-Pro-Ser-Ser-Val-Asp-Ser-Gln-Thr-Ala-Met, and Glu-Glu-Arg-Tyr-ProAntioxidantCasein Tyr-Gln-Lys-Phe-Pro-Gln-Tyr-Leu-Gln-Tyr AntihypertensiveEgg yolk Ile-Arg-Trp and Ile-Gln-Trp AntidiabeticTuna Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser-Asn-Trp-Ser-Pro-Pro-Lys-Try-Lys-Asp-Thr-ProAntihypertensiveFreshwater clam(Corbicula fluminea)Val-Lys-Pro and Val-Lys-Lys HypocholesterolemicSoy Tyr-Val-Val-Asn-Pro-Asp-Asn-Asp-Glu-Asn and Tyr-Val-Val-Asn-Pro-Asp-Asn-Asn-Glu-AsnHypocholesterolemic548Voroshilin R.A. et al. Food Processing: Techniques and Technology. 2022;52(3):545–554pepsin produced by Meito Sangyo Co. (Japan) for ourexperiments as a potentially more suitable enzyme forobtaining peptides from poultry by-products (Table 2).As can be seen in Table 2, the enzyme has highproteolytic properties and is activated at temperaturesthat are favorable for protein structures.Table 2. Characteristics of the enzyme used in the studyТаблица 2. Характеристика фермента, используемого в исследовани иIndicator DescriptionComposition Pepsin based on Rhyzomucor miehei (CAS: 9001-92-7)Origin MicrobialAssumed splitting Phe1 + Val, Gln4 + His, Glu13 + Ala, Ala14 + Leu, Leu15 + Tyr,Tyr16 + Leu, Gly23 + Phe, Phe24 + Phe, and Phe25Form, color White powderActivity, units per 1 g At least 300 000Activation temperature, °С 30 ± 2Producer Meito Sangyo Co., Ltd., JapanFigure 1. Flow chart for producing bioactive peptides from poul try by-products using hydrolysis (two variants)Рисунок 1. Схема производства биоактивных пептидов с применение м гидролиза (в двух вариациях) из вторичных сырьевыхресурсов переработки птицыHomogeneous mass of chicken skinHydrolysis parameters:Hydrolysis 1:Enzyme: pepsin30 units per 100 g of materialTime: 12 ht = 28 ± 2 оСpH 3.0 ± 0.2Enzyme neutralization with weak alkali and inactivationat t = 45 ± 2 оСFractionationby centrifugation at 3000 rpmAqueous extraction of the protein fractionat t = 70 ± 5 °СConcentration by ultrafiltrationat a controlled pressure of 3.0 barSpray dryingat 90°C and a solution supply rate of 3.0 ± 0.2 mL/minProtein hydrolysates (bioactive peptides)from poultry by-productsHydrolysis 2:Enzyme: pepsin45 units per 100 g of materialTime: 12 ht = 28 ± 2 оСpH 3.0 ± 0.2549Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554Enzymatic hydrolysis was carried out in vitro. MM-5laboratory magnetic stirrers were used to ensure auniform treatment of the materials with an enzymesolution throughout the experiment (100 rpm, 28 ± 2°C).The acidity was maintained with 1 M hydrochloric acid.The hydrolysates were dried in a B-290 Mini SprayDryer (Buchi, Sweden) at 100 ± 2°C and at a solutionsupply rate of 3.5–4.0 mL/min. The dried hydrolysateswere microphotographed with a JEOL JED-2300electronic microscope (Japan). The mass fractionof protein was determined on a RapidN Elementarnitrogen (protein) analyzer. This analyzer uses theDumas method that involves combusting the samplesand registering total nitrogen on a thermal conductivitydetector. Molecular weight distribution was performedby polyacrylamide gel electrophoresis in the presenceof an anionic detergent (sodium dodecyl sulfate).The amino acid sequence of the peptides was determinedby matrix-activated laser desorption/ionization on aMALDI Biotyper (Bruker). The molecular weightwas calculated by using the Peptide Mass Calculator.The bioactivity of the peptides was assessed in silicousing the PeptideRanker online server that rankspeptides by the predicted probability of their bioactivity.The structure of the peptides was modeled by using thePepDraw online tool.The experiments were performed at the Departmentof Animal Origin Food Technology and the Scientificand Educational Center of the Research and InnovationDepartment, Kemerovo State University.Results and discussionThe flow chart for producing bioactive peptides frompoultry by-products is presented in Fig. 1. It includestwo sets of parameters for enzymatic hydrolysis of ahomogeneous mass of chicken skin.Chicken skin was homogenized with a laboratoryhomogenizer. The homogeneous mass was hydrolyzedwith pepsin in two variations during 12 h at 28 ± 2°C.The enzyme was then thermally inactivated at 45 ± 2°Cand neutralized with a weak alkali to pH 7.0 ± 2. Next,the hydrolysate was fractionated into protein and fatparts using a CM 6 M Multi centrifuge at 3000 rpm.The protein part was then subjected to thermal waterextraction in order to dissolve the protein fractions at70 ± 5°C. The resulting solution was filtered using aMFU-R-45-300 laboratory ultrafiltration unit (Russia)at a controlled pressure of 3.0 bar and a differenceof 0.2–0.5 kgf/cm2 in the discharge and returnheaders. The protein part of the solution went into theretentate, while the water and minerals went into thepermeate. Further, protein hydrolysate samples wereobtained by spray drying at 90 ± 2°C and a solutionsupply rate of 3.0 ± 0.2 mL/min. The samples were thenmicrophotographed with an electronic microscope(magnified 500 times), as can be seen in Fig. 2.First, we evaluated the color and particle size ofthe hydrolysates. As we can see in Fig. 2, the samplesdiffered in color, with sample 1 having a darker creamycolor and sample 2 having a whitish color. Also, thehydrolysates differed in particle size, although theywere dried under the same conditions. In particular,sample 2 had a more finely dispersed structure,which can be explained by a deeper hydrolysis of thissample.Next, we determined the main physicochemicalparameters of the hydrolysates for further studies(Table 3).As can be seen in Table 3, the samples had nosignificant differences in physicochemical parameters.Since the hydrolysates had a high protein content(over 90%), they can be classified as a high-proteinproduct.Next, we analyzed the distribution of proteinfractions by polyacrylamide gel electrophoresis in theFigure 2. Protein hydrolysates from poultry by-products:a – Sample 1 based on pepsin with an activity of 30 unitsper 100 g of material; b – Sample 2 based on pepsin withan activity of 45 units per 100 g of materialРисунок 2. Белковые гидролизаты из вторичных сырьевыхресурсов переработки птицы: a – образец № 1, полученныйс применением пепсина активностью 30 ед. на 100 г сырья;b – образец № 2, полученный с применением пепсинаактивностью 45 ед. на 100 г сырьяTable 3. Physicochemical parameters of the hydrolysatesТаблица 3. Основные физико-химические показателигидролизатовParameters, % Sample 1 Sample 2Protein 90.7 ± 0.1 91.4 ± 0.2Fat 0.60 ± 0.03 0.40 ± 0.06Moisture 8.1 ± 0.1 7.8 ± 0.2Ash 0.60 ± 0.04 0.40 ± 0.03a b550Voroshilin R.A. et al. Food Processing: Techniques and Technology. 2022;52(3):545–554presence of an anionic detergent, sodium dodecyl sulfate.Table 4 shows the molecular weight distribution of thehydrolysates.As can be seen in Table 4, most peptide fractionshad a molecular weight below 20 kDa, especially thosein sample 2. Thus, we can assume that the hydrolysatescontained peptides with bioactive properties, especiallysample 2.Then, we determined the amino acid sequence ofthe hydrolysates under study (Table 5).According to Table 5, sample 1 had a chain of 41 peptides,while sample 2 was represented by 27 peptides.We can also see that the first and the second variantsof hydrolysis, which used pepsin with lower and higheractivity, respectively (Fig. 1), split the protein into14 and 8 fragments of amino acids (peptide sequences),respectively.Next, we used online resources to determinethe molecular weight and bioactivity of individualpeptide sequences, as well as modelled their structure(Tables 6 and 7).The PeptideRanker service has a threshold valueof 0.5 for peptides’ bioactive properties, i.e. any peptidewith an estimated value above 0.5 is ranked asbioactive. However, literature shows that using a higherthreshold, particularly 0.8, reduces the number offalse positive results. Therefore, we assessed thebioactive properties of peptides based on a thresholdbioactivity value of 0.8 and the maximumvalue of 1.According to Table 6, the FD peptides (38–39 inthe sequence) and the NW peptides (40–41 in thesequence) had greater bioactivity values of 0.922094and 0.934148, respectively. Their structure showsaromatic rings which are mainly represented byphenylalanine and tryptophan.As can be seen in Table 7, sample 2 had threepeptide sequences with high bioactive properties. Theywere the CYG peptides (25–27 in the sequence), theGHG peptides (13–15), and the AYG peptides (22–24),with bioactivity values of 0.947378, 0.839383, and0.815664, respectively. Structurally, these bioactivepeptides had aromatic rings represented by the aromaticα-amino acid of tyrosine.We found no correlation between the bioactivityvalues of the peptides and their molecular weight.Table 4. Molecular weight distribution of the hydrolysatesТаблица 4. Молекулярно-массовое распределение образцов гидролиз атовMolecular weight, kDa Distribution of peptide fractions by molecular weight, %Sample 1 Sample 2200 1.06376 2.194861150 4.905115 2.328694100 2.186618 4.00734285 14.60372 0.94830260 6.369427 3.00168250 4.813185 0.77623140 3.224112 1.69776730 0.965264 2.01131825 1.267319 5.76628920 4.392935 11.56317Less than 20 56.20855 65.70435Table 5. Amino acid sequence of the hydrolysatesТаблица 5. Аминокислотная последовательность исследуемых гидрол изатовParameter Sample 1 Sample 2Amino acid sequence in aone-letter code*PILG/PILV/ILA/ILG/ILV/ILT/PSV/GVS/HGL/HVI/SVP/SV/FD/NWILKH/PSPV/PSVP/GHG/SPV/SVP/AYG/CYGPeptide chain length 41 27Isoelectric point (pI) 6.05 8.84* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine;L – leucine; M – methionine; N – asparagine; P – proline; Q – g lutamine; R – arginine; S – serine; T – threonine; V – valine; W – tryptophan;Y –tyrosine.* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин;I – изолейцин; K – лизин; L – лейцин; M – метионин; N – аспа рагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин;V – валин; W – триптофан; Y – тирозин.551Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554Table 6. Individual peptide sequences for hydrolysate Sample 1Таблица 6. Характеристика отдельных пептидных последовательност ей гидролизата образца № 1The number of peptidein the chainPeptide sequencein a one-letter code*Molecular weight,g/molBioactivity Peptide structure1–4 PILG 398 0.4926745–8 PILV 440 0.2915079–11 ILA 315 0.23744312–14 ILG 301 0.48096515–17 ILV 343 0.12338118–20 ILT 345 0.15422221–23 PSV 301 0.16645924–26 GVS 261 0.10953327–29 HGL 325 0.44678330–32 HVI 367 0.088748633–35 SVP 301 0.19437336–37 SV 204 0.052321838–39 FD 280 0.92209440–41 NW 318 0.934148* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine; L – leucine; M –methionine; N – asparagine; P – proline; Q – glutamine; R – arg inine; S – serine; T – threonine; V – valine; W – tryptophan; Y –tyrosine.* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин; I – изолейцин; K – лизин;L – лейцин; M – метионин; N – аспарагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин; V – валин; W – триптофан; Y – тирозин.552Voroshilin R.A. et al. Food Processing: Techniques and Technology. 2022;52(3):545–554products. Then, we evaluated the bioactive propertiesof the peptides obtained. The hydrolysate sampleobtained with a lower pepsin activity (30 units per 100 gof material) had greater bioactivity values in the FD andNW peptides (0.922094 and 0.934148, respectively).The sample obtained with a higher pepsin activity(45 units per 100 g of material) had higher bioactivityin the CYG, GHG, and AYG peptides (0.947378 units,0.839383, and 0.815664, respectively). However, thesedata obtained in silico need to be confirmed by in vitroexperiments for further work. Such experiments willdetermine their reliability and identify specific bioactiveproperties of the peptides.We should note that the detection of more bioactivepeptides by the in silico method should be confirmedby further in vitro experiments with variousmodifications of peptides. Based on literature, wecan assume that some peptide sequences obtained inour study may have antimicrobial and antioxidantactivity due to the presence of proline and leucinethat have such properties [26]. Further in vitroexperiments can determine the reliability of ourresults and identify specific bioactive properties of thestudied peptides.ConclusionBased on the research results, we designed a flowchart for obtaining hydrolysates from poultry by-Table 7. Individual peptide sequences for hydrolysate sample 2Таблица 7. Характеристика отдельных пептидных последовательност ей гидролизата опытного образца № 2The number of peptidein the chainPeptide sequencein a one-letter code*Molecular weight,g/molBioactivity Peptide structure1–4 ILKH 509 0.1627115–8 PSPV 398 0.3906689–12 PSVP 398 0.32722913–15 GHG 269 0.83938316–18 SPV 301 0.22256219–21 SVP 301 0.19437322–24 AYG 309 0.81566425–27 CYG 341 0.947378* A – alanine; C – cysteine; D – aspartic acid; E – glutamic acid; F – phenylalanine; G – glycine; H – histidine; I – isoleucine; K – lysine;L – leucine; M – methionine; N – asparagine; P – proline; Q – g lutamine; R – arginine; S – serine; T – threonine; V – valine; W – tryptophan;Y –tyrosine.* A – аланин; C – цистеин; D – аспарагиновая кислота; E – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин;I – изолейцин; K – лизин; L – лейцин; M – метионин; N – аспа рагин; P – пролин; Q – глутамин; R – аргинин; S – серин; T – треонин;V – валин; W – триптофан; Y – тирозин.553Ворошилин Р. А. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 545–554ContributionThe authors were equally involved in writingthe manuscript and are equally responsible forplagiarism.Conflict of interestThe authors declare that there is no conflict of interestregarding the publication of this article.Критерии авторстваАвторы были в равной степени вовлечены в на-писание рукописи и несут равную ответственностьза плагиат.Конфликт интересовАвторы заявляют, что конфликта интересовнет.