RU2531164C1 - Method for production of low hydrolysed peptide composition of milk whey proteins - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: protein solution containing fat in an amount no more than 0.05% is produced from a dry concentrate of whey protein, dry milk whey or native cheese whey. The protein solution is pasteurised, ultrafiltered at a temperature of 8-12°C through ultrafiltration membranes with a molecular weight of intercepted components equal to 1 - 5 kDa till weight fraction of dry substances in the retentate is equal to 8-18%. The milk whey retentate temperature is conditioned to 50-55°C; the initial pH value - to 7.0-7.2 by way of addition of 20% water solution of sodium hydroxide. Fermentative hydrolysis is performed with "Protamex" preparation in an amount of 2.5-4.0% of the weight of protein in the retentate at 50-55°C during 1.5 hours without pH- stating; the produced hydrolysate is pasteurised at 80-85°C during 5-10 minutes. The peptide composition has hydrolysis level equal to no more than 6%, total content of free amino acids in glutamic acid equivalents equal to no more than 4 mg/ml, decreased content of milk whey allergens i.e. α-lactalbumin, β- lactoglobulin and bovine serum albumin; the composition has antioxidant and hypotensive activity.

EFFECT: inventions group is aimed at manufacture of a product with attractive organoleptic characteristics, with antioxidant and hypotensive activity and decreased content of milk whey allergens proteins.

2 cl, 3 tbl, 2 ex

Description

The invention relates to the dairy industry, in particular to the production of low hydrolyzed peptide compositions from whey proteins with antioxidant and antihypertensive properties and attractive organoleptic characteristics used in the production of functional foodstuffs of mass consumption and specialized products for people with food intolerance to milk proteins.

Over the past decade, much attention has been paid to the development of functional food products of mass consumption and specialized products for the nutrition of key categories of people with food intolerance or allergies to certain food components, or alimentary-dependent diseases. In this case, the basis of these categories of food products, giving them the appropriate properties, are functional food ingredients. Numerous studies have shown that enzymatic hydrolysates of food proteins, including casein and whey protein hydrolysates, not only have nutritional value due to the content of irreplaceable and replaceable amino acids in easily digestible form, but also are a source of biologically active peptides involved in the regulation of various body functions including maintaining the balance of microflora of the gastrointestinal status, antioxidant and immune status, regulation of blood pressure, etc. Thus, It is obvious that to prevent the development of clinical signs of food intolerance to milk proteins, increase antioxidant status, prevent functional disorders of the cardiovascular system, the gastrointestinal tract and restore the balance of its microflora, it is necessary to develop methods for producing peptide compositions with antioxidant, hypotensive and prebiotic action.

In particular, a method is known for producing a whey protein hydrolyzate comprising mixing a whey protein product containing at least 65% protein on a dry matter basis and water to produce a suspension with a protein content of up to 20%, proteolytic hydrolysis of the suspension to a degree of hydrolysis of 15 and 35%, isolation from a mixture on an ultra- or microfiltration device with a cut-off value above 10,000 protein hydrolyzate in the form of permeate, characterized in that I undergo a suspension before proteolytic hydrolysis t heat treatment at a temperature above 60 ° C, the mixture so heated is subjected to pH adjustment with an alkaline substance, hydrolysis is carried out by a protease produced by B. licheniformis, preferably Alcalase, and / or a protease produced by B. subtilis, preferably Neutrase, by means of the non-static pH method, after isolation of the protein hydrolyzate, enzymes are inactivated (see RF Patent No. 2084172, 05.27.1992).

The disadvantage of this method is to obtain medium (degree of hydrolysis of 5-20%) and deep (degree of hydrolysis of more than 20%) hydrolyzed peptide compositions with an average peptide chain of not more than 5 amino acid residues having a low degree of bitterness, which reduces both consumer properties of hydrolysates and food products based on them. In addition, in the examples disclosing the invention, data are provided on the use of a composition of two enzyme preparations (Neutrase and Alcalase 2.4L) for the hydrolysis of whey proteins of milk, which leads to an increase in the cost of producing hydrolysates.

A known method of obtaining a partial hydrolyzate of milk proteins (with a whey protein / casein ratio from 40/60 to 80/20), including hydrolysis, concentration and drying. Hydrolysis is carried out in one stage under the action of a mixture of enzymes trypsin and chymotrypsin, while the protein is pre-thermally denatured at (70-80) ° C. Hydrolysis is carried out in a 4-6% substrate solution at an enzyme-substrate ratio of 0.6-0.8% at a temperature of (30-50) ° C for 2-6 hours. Set the initial pH of 7.5-8.0 units. and further during the process they do not pH-stat, allowing the pH to drop to a value of 6.5-6.8 units. Next, the enzyme is inactivated thermally at a temperature of (80-90) ° C, concentration and drying. The hydrolyzate is used directly in the products without further purification. According to enzyme-linked immunosorbent assay (carried out in a variant of the method of inhibition of indirect enzyme-linked immunosorbent assay) antigenicity of the hydrolyzate is reduced by 75-95% compared with the original protein (see US Patent No. 5405637, NKI 426/580, 04/11/1995).

Investigations of the molecular weight distribution by high-pressure liquid chromatography on a TSK column are carried out, which, however, were obtained in aqueous-organic media and therefore do not adequately reflect the composition of the hydrolyzate.

A similar method for producing "partial" milk protein hydrolysates is given in the patent (see US Patent No. 5589357, NKI 435 / 68.1, December 31, 1996). In contrast to the method described above, the inactivation of a mixture of trypsin + chymitrypsin enzymes in the hydrolyzate is carried out with direct steam.

The disadvantages of the above analogues are as follows:

- additionally, a stage of thermal inactivation of the enzyme is necessary, which can lead to a deterioration in the quality of the product (decrease in biological value due to the loss of some of the essential amino acids);

- immediately after the hydrolysis and inactivation of the enzymes, the hydrolyzate is dried without further purification. This leads to the fact that the composition of the product includes residual amounts of undigested protein substrate and components of enzyme preparations, which may themselves have an allergenic effect, which impairs the quality of the product and reduces the possibility of its use in hypoallergenic mixtures;

- the degree of decrease in the antigenicity of the resulting product is 75-95%, which corresponds to a decrease of 4-20 times. This is extremely insufficient for the use of this hydrolyzate in hypoallergenic food products. In the composition of hypoallergenic mixtures of prophylactic purposes, the degree of decrease in antigenic properties should be 10 ⁴ (10.000) times or more;

- during the process, the molecular weight distribution of peptides is monitored by size exclusion chromatography on a TSK G-2000 SWXL column. However, in this case, a solvent containing acetonitrile is used, which can distort the results of the analysis, namely, to lower the content of peptides with molecular masses of more than 10 kDa (kilodaltons) due to their precipitation upon dissolution in the mobile phase containing acetonitrile. It should be noted that the degree of loss of protein-peptide material depends on the concentration of acetonitrile in the mobile phase, which makes the results of the analysis uncertain.

Closest to the technical solution to the claimed method is a method for producing an enzymatic hydrolyzate of whey proteins with an average degree of hydrolysis. The method includes obtaining a protein solution, its pasteurization, enzymatic hydrolysis with pancreatin, ultra- and diafiltration of the obtained hydrolyzate with separation into a filtrate and concentrate, drying. The protein solution is obtained from a dry whey protein concentrate, and hydrolysis is carried out at an enzyme-substrate ratio of 1.5-2.5%, at a temperature of 48-54 ° C for 3-3.5 hours, with an initial pH of 7.9-8 , 3 (see RF Patent No. 2375910).

The disadvantages of the proposed method are:

- the use of pancreatin to obtain a hydrolyzate, which leads to an increase in its cost compared to hydrolysates obtained using bacterial food enzyme preparations.

- diafiltration of the resulting solution, leading to additional loss of protein.

The technical result of the claimed invention is to obtain low-hydrolyzed peptide compositions from whey proteins with attractive organoleptic characteristics (without bitterness), with antioxidant and antihypertensive activity and a low content of whey allergen proteins, which makes it possible to use them in the production of functional food products of mass consumption and specialized foods for people with food intolerance to milk proteins.

"Protamex" is a protease complex produced by microorganisms of the genus Bacillus. The activity of the enzyme preparation is at least 400 Proteolytic units per 1 g. The use of the Protamex enzyme, which is approved for use in food production and food systems, provides a pleasant taste to the finished product without bitterness, unlike the known similar products, and has a gray-beige or cream color .

The technical result is achieved in that a method for producing low hydrolyzed peptide compositions of whey proteins includes preparing a protein solution (whey purification, separation), pasteurization, ultrafiltration, enzymatic hydrolysis with Protamex and pasteurization of the hydrolyzate obtained for thermal inactivation of the enzyme preparation Protamex. Protein solution is obtained from a dry concentrate of whey protein, whey powder or use native cheese whey. When the fat content in the protein solution is more than 0.05%, it is degreased by passing through a separator. Pasteurization of the protein solution is carried out at a temperature of from 75 to 85 ° C with an exposure of 20-30 s. Ultrafiltration of the protein solution is carried out at a temperature of 8-12 ° C on ultrafiltration membranes with a molecular weight of the cut-off components from 1 to 5 kDa to a solids content in the retentate of 8-18%. Before introducing the enzyme preparation Protamex, the retentate is heated to a temperature of 50-55 ° C and its initial pH is adjusted by adding 20% aqueous sodium hydroxide solution to 7.0-7.2, and hydrolysis is carried out at a temperature of 50-55 ° C, dose (E / S) of the enzyme preparation "Protamex" 2.5-4.0% of the protein content in the retentate for 1.5 hours without pH-stating, after which the resulting hydrolyzate is pasteurized at a temperature of 80-85 ° C for 5-10 minutes .

It was experimentally established that the optimum temperature for hydrolysis is 50-55 ° C. Deviation to a lower side below 50 ° C is undesirable, as leads to an increased content of non-hydrolyzed high molecular weight protein structures remaining in the reaction mixture, which reduces the yield of the obtained product and leads to a decrease in quality and an increase in the residual allergenicity of the hydrolyzate. Deviation upwards (over 55 ° C) is also undesirable, as leads to an acceleration of the process of thermal inactivation of the enzyme preparation, which entails a decrease in the yield of the finished product. In addition, this leads to an excessive consumption of energy to maintain the temperature during the process and subsequent cooling of the finished product, which causes an increase in the cost of the product.

It was experimentally established that ultrafiltration of a protein solution on membranes with a molecular mass of cut-off components from 1 to 5 kDa allows to obtain a whey protein concentrate (retentant) with maximum yield. The use of membranes with lower porosity reduces the yield of both retentate and the final product (hydrolyzate).

Hydrolysis of the retentate with the enzyme preparation "Protamex" at an initial pH of 7.0-7.2 units, which gradually decreases during hydrolysis, eliminates the need for further pH-stating, which reduces the amount of salts entering the product, and therefore improves its quality. The initial pH of the reaction mixture is adjusted with a 20% alkali solution (sodium hydroxide).

Pasteurization of the hydrolyzate is necessary to improve its microbiological parameters, as well as thermal inactivation of the enzyme preparation "Protamex". A decrease in pasteurization temperature below the stated range of values leads to an increased risk of microbial contamination of the hydrolyzate and products based on it during their subsequent storage.

The claimed combination of features allows to obtain low-hydrolyzed peptide compositions of whey proteins with attractive organoleptic characteristics, reduced antigenicity, antioxidant and hypotensive activity. In the claimed invention, residual antigenicity is controlled by high performance liquid chromatography and enzyme immunoassay methods and is calculated as the ratio of the content of whey allergens in the hydrolyzate and retentate.

The results of determining the content of allergen proteins in whey retentates and hydrolysates are presented in Table 1. As can be seen from the data presented, the residual antigenicity of the hydrolysates in the content of bovine serum albumin does not exceed 1.5%, α-lactalbumin - 11%, milk proteins, mainly β β-lactoglobulin - 85%.

The antioxidant capacity of whey protein retentate and hydrolysates was determined by spectrophotometric TEAC method (Trolox Equivalent Antioxidant Capacity) in relation to the radical cation ABTS (di-ammonium salt of 2-azinobis- (3-ethylbenzothiazolin-6-sulfonic acid)) and spectrofluorimetric ORL method OR Absorbance Capacity) with respect to the peroxyl radical. Antioxidant capacity was expressed by the concentration of equivalents of a water-soluble vitamin E analogue, trolox. The results of determining the antioxidant capacity in whey retentates and hydrolysates are presented in table 2. It was shown that during enzymatic hydrolysis, the antioxidant capacity of hydrolysates increased 1.8-2.0 times with respect to the ABTS radical cation and 1.08-1, 57 times with respect to the peroxyl radical, which is due to the formation of antioxidant peptides and the removal of steric barriers for the interaction of residues of redox active amino acids with radicals due to the destruction of the tertiary structure of milk protein proteins oorotki. Moreover, an increase in the dose of the Protamex enzyme preparation from 2.5 to 4.0% of the protein in the retentate leads to an increase in the antioxidant capacity of the resulting hydrolyzate by 15-39% depending on the type of radical (table 2).

The antihypertensive activity of whey protein retentate and hydrolysates was determined by spectrofluorimetric method by their ability to inhibit angiotensin-1-converting enzyme (ACE), which is a key link in the rennin-angiotensin-aldosterone blood pressure regulation system. To determine the ACE inhibitory activity of hydrolysates and retentate, a substrate with internal fluorescence quenching of o-aminobenzoyl-phenylalanyl-arginyl-lysyl (dinitrophenyl) -proline trifluoroacetate was used. The concentration of hydrolysates and retentate (ml / l), at which 50% inhibition of ACE activity was observed, was taken as the value of hypotensive activity. The results of determining the antihypertensive activity in whey retentates and hydrolysates are presented in Table 2. It was shown that during enzymatic hydrolysis, the antihypertensive activity of hydrolysates increased by 3.7–5.3 times, which is due to the formation of ACE inhibitory peptides. Moreover, an increase in the dose of the Protamex enzyme preparation from 2.5 to 4.0% of the protein in the retentate leads to an increase in the hypotensive activity of the resulting hydrolyzate by 16% (table 2).

The peptides present in whey hydrolysates were identified by fractionation using preparative gel permeation chromatography followed by chromatographic analysis of the obtained fractions by reverse phase chromatography with detection by Fourier transform ion-cyclotron resonance mass spectrometry (FT-ICR-MS) and automatic search of the protein sequence database UniProt KB_SwisProt sprot_v.57.9. In the hydrolysates from whey proteins, 199 peptides from 5 to 25 amino acid residues in size were identified (Table 3), for which bovine serum albumin, β-lactoglobulin, and caseins were the main precursors. Among the identified peptides, 86 contain residues of redox active amino acids (tyrosine, tryptophan, methionine, cysteine, and histidine), which are responsible for the antioxidant properties of the corresponding peptides. Sequence analysis of the identified peptides using the BioPep database made it possible to establish the presence of 2 VKEAMAPK and EAMARK peptides from β-casein with antioxidant activity and 3 ALKAWSVAR (bovine serum albumin), AMKPWIQPK (αs ₂ -casein) SAPLR peptides in the composition of the peptide compositions interacting with a protein similar to factor 6 of ADP-ribosylation) with ACE inhibitory activity.

The qualitative advantages of the resulting hydrolysates are as follows:

- in reduced antigenicity due to the reduced content of whey allergens - α-lactalbumin, β-lactoglobulin, bovine serum albumin;

- the presence of antioxidant and antihypertensive properties;

- in attractive organoleptic characteristics, neutral taste without a taste of bitterness;

- the possibility of using in the production of functional foods of mass demand.

Due to this, hydrolysates as protein components can be used to obtain functional products for the prophylactic nutrition of children and adults prone to food allergies and intolerance to cow's milk proteins;

A method of producing peptide compositions from whey proteins is as follows.

The dry whey protein concentrate (CSB) is dissolved with constant stirring in drinking water at a temperature of (40-45) ° C, cooled and allowed to swell for 3.0-6.0 hours with stirring every 30 minutes. The resulting whey protein solution is pasteurized at a temperature of 85 ° C for 20 s, cooled and fed to enzymatic hydrolysis.

Dry whey is dissolved with constant stirring in drinking water at a temperature of (40-45) ° C, cooled and allowed to swell for 3.0-6.0 hours with stirring every 30 minutes. The resulting whey protein solution was pasteurized at a temperature of 85 ° C for 20 s, cooled and fed to ultrafiltration.

The raw cheese whey is cleaned, separated on cream separators for degreasing, pasteurized at a temperature of 75 to 85 ° C for 20-30 seconds, cooled and fed to ultrafiltration.

The whey protein solution is separated on an ultrafiltration unit with a membrane capacity of 1 to 5 kDa, a transmembrane pressure of 0.2-0.4 atm per retentate (mass fraction of solids 8-11% and filtrate.

The retentate or KSB solution is heated to a temperature of 50-55 ° C, the pH of the solution is adjusted to 7.0-7.2 by adding the required amount of a 20% aqueous solution of sodium hydroxide, after which Protamex enzyme preparation is added to the reaction mixture in a dosage of 2.5 -4.0% of the amount of protein in the reaction mixture.

Enzymatic hydrolysis of a solution of whey proteins is carried out using the enzyme preparation "Protamex" at a temperature of 50-55 ° C for 1.5 hours.

After fermentation, the hydrolysates are pasteurized at a temperature of 80-85 ° C for 5-10 minutes, followed by cooling to a temperature of 30 ± 2 ° C. The resulting liquid hydrolysates are Packed and sent for storage.

The following examples illustrate a method for preparing peptide compositions from whey proteins.

Example 1

65 kg of dry whey are dissolved with stirring in drinking water (935 kg) with a temperature of 40 ° C until a solution with a mass fraction of dry substances (protein, fat, lactose, salts) of 7% is formed, cooled to 4 ° C and allowed to swell for 3.0-6.0 hours with stirring every 30 minutes The resulting whey protein solution is pasteurized at a temperature of 85 ° C for 20 s, then cooled to a temperature of 40 ° C and served for concentration on membranes with a throughput of 1 to 5 kDa. UF DHT 20-6338 / 30FF polysulfone membranes are used.

After ultrafiltration, the obtained retentate with a mass fraction of solids of 9% is sent to enzymatic hydrolysis.

The retentate is heated to a temperature of 52-54 ° C, adjusted to pH 7.1 by adding 3.2 l of a 20% sodium hydroxide solution, after which 687.5 g of the enzyme preparation Protamex (2.5% of the amount of protein reaction mixture).

The enzymatic hydrolysis of the retentate is carried out at a temperature of 52-54 ° C for 1.5 hours.

The hydrolysates are pasteurized at a temperature of 82-84 ° C for 10 minutes, followed by cooling to a temperature of 30 ° C.

Get 167 l of the peptide composition from whey proteins with a mass fraction of solids of 10.6%, a mass fraction of total nitrogen of 0.43%, a total content of free amino acids in glutamic acid equivalents of 3.44 ± 0.23 mg / ml, a degree of hydrolysis of 4 , 91 ± 0.26%, residual antigenicity in the content of α-lactalbumin 4.6%, bovine serum albumin 0.6%, β-lactoglobulin 72.1%, with antioxidant and hypotensive activity (table 2).

Example 2

The process is carried out analogously to example 1. It is characterized in that 1100 g of the enzyme preparation "Protamex" (4.0% of the amount of protein in the reaction mixture) is added to the reaction mixture.

Get 160 l of the peptide composition from whey proteins with a mass fraction of solids of 9.9%, a mass fraction of total nitrogen of 0.44%, a total content of free amino acids in glutamic acid equivalents of 3.78 ± 0.25 mg / ml, a degree of hydrolysis of 5 , 37 ± 0.30%, residual antigenicity in the content of α-lactalbumin 10.8%, bovine serum albumin 1.3%, β-lactoglobulin 84.8%, which has antioxidant and hypotensive activity (table 2).

Table 1 Allergen protein content and residual allergenicity of whey protein retentates and hydrolysates Sample C (milk protein - αs ₁ -, αs ₂ -, β-, k-caseins and β-lactoglobulin), mg / ml, during immunoassay using the Ridascreen Fast Milk kit (R-Biopharm, Germany) Residual antigenicity,% C (α-lactalbumin), mg / ml, by immunoassay using the E91018Bo kit (Life Science Inc., USA) Residual antigenicity,% C (bovine serum albumin), mg / ml upon immunoassay with the Bovine serum albumin (BSA) assay kit (Cygnus technologies Inc., USA) Residual ntigenicity
% Obtaining a peptide composition at E / S = 2.5% Whey UV Retentate 53.07 ± 8.87 100.00 1.21 ± 0.18 100.00 0.96 ± 0.10 100.00 Whey Hydrolyzate 38.24 ± 7.60 72.06 0.055 ± 0.001 4,55 0.006 ± 0.002 0.63 Obtaining the peptide composition at E / S = 4.0% Whey UV Retentate 53.42 ± 7.78 100.00 0.87 ± 0.13 100.00 0.98 ± 0.07 100.00 Whey Hydrolyzate 45.32 ± 1.72 84.84 0.094 ± 0.006 10.80 0.013 ± 0.003 1.33

table 2 Antioxidant capacity (mM trolox equivalents) and antihypertensive activity (IC50 ml / l) of whey protein retentates and hydrolysates Sample Antioxidant capacity Antihypertensive activity (IC50) TEAC ORAC Obtaining a peptide composition at E / S = 2.5% Whey UV Retentate 4.31 ± 0.18 2.83 ± 0.18 321.8 Whey Hydrolyzate 7.77 ± 0.19 3.07 ± 0.19 86.8 Obtaining the peptide composition at E / S = 4.0% Whey UV Retentate 4.47 ± 0.15 2.72 ± 0.14 395.0 Whey Hydrolyzate E / S = 4.0% 8.95 ± 0.23 4.27 ± 0.14 74,4

Table 3 Peptide profile of whey protein hydrolysates Bos taurus precursor protein Peptide sequence Molecular Weight, Yes Acetyl-CoA carboxylase 1 Liaek 572.3533 Sodium- and chloride-dependent glycine transporter ILEAK 572.3533 Wdr12 Lek 572.3533 CE290 D1LQK 615.3592 Ras GTPase-activating protein 3 VFQSVK 706,4014 Beta-catenin-like protein 1 LLNKFTENDSEK 1436,715 Serum albumin DTHKSEIAHR 1192,595 K2CA LQDLK 615.3592 Structural maintenance of chromosomes protein 1A Qessr 605.2769 MKI67 FHA domain-interacting nucleolar phosphoprotein LLKCHFIPPEK 1323,737 Nuclear migration protein nudC INPENSK 800,4028 Adenylate cyclase type 1 LLNELFGK 932,5331

UPF0490 protein Clorf201 homolog AGFVSK 607,333 Complement c3 ADALVGK 672,3806 Protein KRI1 homolog Keilak 700,4483 Thyroglobulin GQEIPGTR 856,4403 Mitochondrial import inner membrane translocase subunit TIM50 QMIIEPTSPCLLPDPLR 1922,001 Transforming growth factor-beta-induced protein ig-h3 (Fragments) XKNLLLNHMDVLK 1564,876 Endoplasmic reticulum protein ERp29 LIEKNK 743.4541 Sarcoplasmic / endoplasmic reticulum calcium ATPase SRAAVGNK 801,4457 Alpha-s2-casein ISQRYQK 921.5032 Cytochrome with oxidase subunit 8B, mitochondrial Gwavpk 656.3646 DnaJ homolog subfamily C member 14 EPKSAR 686.3711 Rho guanine nucleotide exchange factor 10-like protein LMRVK 661.3945 Pyruvate carboxylase, mitochondrial DTQAMK 692,3163 Selenium-binding protein 1 VIQVPPK 779,4905 Palmitoyl-protein thioesterase 1 KMVEKK 777.4418 Rab GTPase-binding effector protein 2 ASLPR 542,3176 Rho GDP-dissociation inhibitor 3 EIVSGLK 744,4381 ERP27 LVDNK 587,3279 Beta-2-microglobulin IVKWDRDL 1043,576 Serum albumin ALKAWSVAR 1000,582 Ubiquitin carboxyl-terminal hydrolase isozyme L1 LGREAASR 858,4671 Mitochondrial Rho GTPase 1 IDLQK 615.3592 Serum albumin YTRKVPQVSTPTLVEVSR 2059,143 STMN1 EVLQK 615.3592 Interleukin-12 subunit alpha LLMDPK 715.3938 Beta casein Vkeamapk 872,4789 Aconitate hydratase, mitochondrial NINIVRKR 1011.63 Seizure protein 6 homolog LISSPK 643,3905 GLNA KDPNK 600,3231 UPF0407 protein C2orf39 homolog FDEITVKWEEGK 1479,725 Hexokinase-1 Gesais 562.2598 JSPR1 EPVSK 558,3013

Peroxisome assembly protein 12 OS = Bos taurus GN = PEX12 PE = 2 SV = 1 Lagvr 514,3227 ACTN4 Eamlk 590.3098 Protein THEMIS ILASEIR 800,4756 Myosin-2 VKQKLEK 871,5491 PAF Vvasr 530,3176 ADP-ribosylation factor-like protein 6-interacting protein 6 Saplr 542,3176 Fibrinogen gamma-b chain KTTMK 607,3363 Probable carboxypeptidase PM20D1 DGFIYGRGTLDNK 1454,715 Twinfilin-1 QLNYVQLEIDIK 1474,803 60S ribosomal protein L9 GTVQQADE 846.3719 Peptidyl-prolyl cis-trans isomerase FKBP1B QEVIK 615.3592 Vacuolar protein-sorting-associated protein 36 Lllaeck 685,4374 N-terminal kinase-like protein GPMKLGTR 858.4745 TM223 ALQSLSARR 1000,578 Serum albumin SLGKVGTR 816.4818 Serum albumin AEFVEVTK 921,4807 Serum albumin AEFVEVTKLVTDLTK 1691,935 Serum albumin LSQKFPKAEFVEVTK 1749,967 ZNHI1 RVLDR 657.3922 Transmembrane protein 131-like QILSITK 801,496 Abnormal spindle-like microcephaly-associated protein homolog (Fragment) AGKHQR 695.3827 Uveal autoantigen with coiled-coil domains and ankyrin repeats protein DLLQK 615.3592 UPF0453 protein C12orf44 homolog VGEKLCEK 904,4688 Glucosamine-fructoses-phosphate aminotransferase [isomerizing] 2 NIENVK 715.3864 Tax 1-binding protein 1 homolog EIADKTEK 932,4814 Allergen Bos d 2 DKIVEGGPLR 1082,608 DCE1 Anffr 653,3285 Beta-lactoglobulin LSFNPTQLEEQCHI 1657,777 Alpha-s2-casein LTEEEKNR 1017,509 Uncharacterized protein KIAA1539 homolog KGYSVPK 777,4385 Wdr12 MPLFK 650,3462 Uncharacterized potential VYEDSGIPLPADSPK 1586,783

DNA-binding protein C17orf49 homolog MYB TPAIK 528,3271 WD repeat-containing protein 92 DISMNK 706,332 DNA-directed RNA polymerases I, II, and III subunitRPABCl QSLVDMAPK 1003,501 Liga QLDIK 615.3592 Protein ariadne-1 homolog VQDKYR 807,4239 Lethal (3) malignant brain tumor-like 2 protein GYLMK 626.3098 Complement C4 (Fragments) VLREDSR 873,4668 Protein FAM92A1 MKRDDLK 904.48 RNA-binding protein 5 EGSGLGRK 802,4297 Complement factor B Vgsqyr 708.3555 MARCKS-related protein MGSQSSKAPR 1047,513 Acidic leucine-rich nuclear phosphoprotein 32 family member A Klenk 743.4541 Semaphorin-3C ICPNDTGGLR 1101,524 cGMP-dependent 3 ', 5'-cyclic phosphodiesterase MLDLMR 809.3775 Alpha-1-syntrophin MPILISK 816,4779 LysM and putative peptidoglycan-binding domain-containing protein 2 LDLQIK 728.4432 Chromodomain-helicase-DNA-binding protein 1-like 1 VDGSVRGEER 1102,537 Leucine-rich repeat-containing protein 41 KSEAK 561.3122 RAB6-interacting golgin TQAETMKLK 1048,559 COX assembly mitochondrial protein homolog TGIPTK 615.3592 Sarcoplasmic / endoplasmic reticulum calcium ATPase 1 AVNQDK 673,3395 XPO2 LLAVSK 633,4197 Sodium-driven chloride bicarbonate exchanger EYGLSYLSLR 1201,623 Serum albumin DAFLGSFLYEYSR 1566,735 TT23L ASPIR 542,3176 Coiled-coil domain-containing protein 104 KDMKIK 761.4469 Gtpir 542,3176 E3 SUMO-protein ligase RanBP2 (Fragment) OS = Bos taurus GN = RANBP2 PE = 2 SV = 2 LVDTGR 659.3602

SDF2 Eqvlk 615.3592 Flotillin-2 LKAEAYQK 949.5232 Myosin-ic SELSDK 677.3232 Glycosylation-dependent cell adhesion molecule 1 LPLSILKEK 1039,664 Cathelicidin-1 AVDQLNEQSSEPNIYR 1861,881 Alpha-1B-glycoprotein VLSPAGPEAQFELR 1512,794 Alpha-1B-glycoprotein SLLSELSDPVELRVAGS 1770,936 Ubiquitin-like domain-containing CTD phosphatase 1 Dkellk 744,4381 ERP27 QLDLK 615.3592 Leucine zipper putative tumor suppressor 2 LIPVSGKLEK 1082.67 RNA-binding protein 5 AAERREK 858,4671 Folate receptor alpha FYAENPTSGSTPQGI 1567,715 Zinc-alpha-2-glycoprotein RKWEAEAVYVQR 1533,805 Zinc-alpha-2-glycoprotein SLTRPLTVPWDPRQQAE 1993,038 Zinc-alpha-2-glycoprotein AEPLAPWSQVEGMEDWEKESALQR 2785,302 Zinc-alpha-2-glycoprotein RAEPLAPWSQVEGMEDWEKESALQR 2941,403 1 -phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-4 (Fragment) IVAQYDK 835,444 Phosphatidylcholine transfer protein SGVIRVK 757,481 Glycosylation-dependent cell adhesion molecule 1 SLFSHAFEVVKT 1363,714 LETM1 domain-containing protein 1 ALQMKALCR 1048,552 Polymeric immunoglobulin receptor AQDFQGR 820.3828 Polymeric immunoglobulin receptor KAQDFQGR 948,4777 Serum albumin IETMREK 905,464 Serum albumin FKDLGEEHFK 1248,614 Serum albumin LSQKFPKAEFVEVTKLVTDLTK 2520.42 Guanine nucleotide-binding protein G (I) / G (S) / G (O) subunit gamma-11 Gsciis 578.2734 Dynamin-l-like protein KEAADMLK 920,4637 UPF0609 protein C4orf27 homolog MVGGGAKR 790,412 Alpha-1-acid glycoprotein HAEDKLITR 1081,588 Monoacylglycerol lipase ABHD6 ELQESAAVEK 1102,551 KTEL motif-containing protein 1 MMAEVVRR 990.5103 Calcium-transporting ATPase type 2C member 1 Ylsmlr 797,4105

Drebrin-like protein SV = 1 LKSPFLQK 959,5804 Uncharacterized protein C14orf38 homolog KVKQELK 871,5491 Zinc-alpha-2-glycoprotein KWEAEAVYVQR 1377,704 Beta casein INKKIEK 871,5491 GA-binding protein subunit beta-2 DMLKMTALHWATEHHHRDVVELLIK 3022,563 Aldehyde dehydrogenase family 3 member B1 IVMTAAAK 819.4524 Roporin-1 ERSERVALSNWAELTPELLK 2340,244 Alpha-si-casein EKVNELSK 945.5131 RL10A KLNKNKK 871,5603 Glycosylation-dependent cell adhesion molecule 1 SSRQPQSQNPK 1255,627 Nicotinate phosphoribosyltransferase LIAGPDK 712,4119 CDKN2AIP N-terminal-like protein MVGGEAAAAVEELISGVR 1757,898 28S ribosomal protein S34, mitochondrial LIAELARRVR 1195,751 Dynein intermediate chain 1, axonemal LLNLVREVK 1082,681 Protein kintoun QMLDATALEAVEK 1417,712 39S ribosomal protein L47, mitochondrial MAAASLAVFCR 1138,563 Brain acid soluble protein 1 DAQDTTKPEDK 1246,568 Histone-binding protein RBBP7 EGKIVDAK 858.4811 Nuclear factor 1b-type GIPLESTDGER 1172,567 Beta casein Eamapk 645.3156 3-oxoacyl- [acy1-carrier-protein] synthase, mitochondrial VSPFFVPK 919.5168 Transforming growth factor-beta receptor-associated protein 1 Rnipk 626.3864 NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial QSPPK 555,3017 UPF0534 protein C4orf43 homolog LNLIGEK 785,4647 F-actin-capping protein subunit beta Divngl 785,4395 Melanocyte-stimulating hormone receptor MPALGSQRR 1014,539 Protein argonaute-2 VKFTK 621,385 S-adenosylmethionine synthetase isoform type-1 RSGQLPWLQPDSK 1510,789 Zinc finger CCHC domain-containing protein 12 TSIIARMSNSR 1250.64

COQ5 LLSQEK 716.4068 Uroplakin-3-like protein Flvmsdr 866,432 Folliculin FTKVDSRPKEDTQK 1677,869 Trichohyalin-like protein 1 QLPTKK 713,4436 Protein FAM92B LKDIQK 743.4541 UDP-glucose 6-dehydrogenase IAILGFAFK 978,5902 Transmembrane and coiled-coil domain-containing protein C6orf 129 homolog RSLEKEKSSLMNK 1564,824 General transcription factor II-I ALQSPKRPR 1051,625 Kinetochore protein spc25 Kenllk 743.4541 Coiled-coil domain-containing protein 105 HSWVNISR 997.5094 Sodium / potassium-transporting ATPase subunit alpha-2 Aqdilar 785,4395 GNAT2 Eaktvk 674.3963 Alpha-s2-casein AMKPWIQPK 1097,606 Electron transfer flavoprotein subunit beta LAEKEK 716.4068 ASAP1 Kpfdk 633,3486 Fibroblast growth factor 5 Lhasak 625.3547 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating MKFMIGNGK 1040,515 CO038 KFDPK 633,3486 SELT Ypdir 662.3388 Galactose-3-O-sulfotransferase 3 Lqqatk 687.3915 Factor Xlla inhibitor VYDPKG 677,3384 Zinc finger protein 350 SPQSSVVLQEK 1200,635 Succ1 LQGTR 573.3235 GMFB Vfeir 662,3752 Cleavage and polyadenylation specificity factor subunit 2 QVLLR 627.4068 Nucleotide exchange factor SIL1 LQQYR 706.3762 DNA nucleotidylexotransferase KMGTTR 708.3589 Heat shock factor-binding protein 1 IDDMSSR 822.3542 Dynamin-1 Demlr 678,3007 LisH domain-containing protein ARMC9 SMTYLK 741.3731 Uncharacterized protein KIAA1462 homolog LDGAVPAPDPR 1106,572 DnaJ homolog subfamily C Nefyk 699.3228

member 27 Calcyclin-binding protein MQQKSQRK 1048,545 Leucine-rich repeat-containing protein 28 HKNLFLNYR 1203,651

Claims (2)

1. A method of obtaining a low-hydrolyzed peptide composition from whey proteins, including obtaining a protein solution, pasteurization, ultrafiltration, enzymatic hydrolysis and pasteurization of the obtained hydrolyzate, characterized in that the protein solution with a fat content of not more than 0.05% is obtained from a dry whey protein concentrate , dry whey or native cheese whey, ultrafiltration of the protein solution is carried out at a temperature of 8-12 ° C on ultrafiltration membranes with molecular weight th cut-off components from 1 to 5 kDa to a mass fraction of solids in the retentate of 8-18%, before adding the enzyme preparation, the temperature of the whey retentate is brought to 50-55 ° C, and its initial pH value is up to 7.0-7.2 by adding a 20% aqueous solution of sodium hydroxide, enzymatic hydrolysis is carried out with the Protamex preparation at a dosage of 2.5-4.0% by weight of the protein in retentate at a temperature of 50-55 ° C for 1.5 hours without pH-stating, and the resulting hydrolyzate is pasteurized at a temperature of 80-85 ° C for 5-10 minutes. 2. The peptide composition obtained by the above method, with a degree of hydrolysis of not more than 6%, a total content of free amino acids in glutamic acid equivalents of not more than 4 mg / ml, a reduced content of whey allergens α-lactalbumin, β-lactoglobulin and bovine serum albumin, and with antioxidant and antihypertensive activity.