RU2428047C1 - Method of producing whey protein hydrolysate with high degree of hydrolysis and whey protein hydrolysate with high degree of hydrolysis - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to dairy industry. The method involves ultra and diafiltration of a whey protein solution prepared from a dry whey protein concentrate and bringing the whey protein concentrate solution obtained after ultra and diafiltration to weight ratio of dry substances equal to 4.8-5%, pasteurisation thereof followed by enzymatic hydrolysis using a substrate enzyme in ratio of 5 wt %, ultra-filtration of the obtained crude hydrolysate on membranes with carrying capacity on molecular weight of 5 kDa and 2 kDa, while constantly returning the obtained concentrate to ultrafiltration. The obtained first run filtrate is further purified through ultrafiltration on membranes with carrying capacity on molecular weight of 5 kDa and 2 kDa, while constantly returning the obtained concentrate to ultrafiltration, and the obtained second run filtrate which contains purified hydrolysate is concentrated vai reverse osmosis and then thickened, pasteurised and dried. The obtained hydrolysate is characterised by that it is delactosed and over 75% of the protein material is concentrated in fractions of low-molecular peptides in molecular weight range of 0.5-2 kDa.

EFFECT: group of inventions enables to increase biological and nutrition values, as well as improve organoleptic properties and quality of the obtained hydrolysate with possibility of its use in hypoallergic mixtures for functional purposes.

2 cl, 5 tbl, 5 ex, 5 dwg

Description

The invention relates to the dairy industry, in particular to the production of purified whey protein hydrolysates with a high degree of hydrolysis, used in the production of a wide range of hypoallergenic food products, including for children and adults suffering from severe forms of food allergy.

In the foreign patent literature, methods for producing milk protein hydrolysates (including whey) using proteases of microbial (fungal) origin are known.

A known method of producing a hydrolyzate of milk (casein or whey) protein, including carrying out hydrolysis of the protein, clarification of the resulting hydrolyzate and drying (see US No. 6589574, NCI 426/34, 08.07.2003). The hydrolysis is carried out in one stage using enzymes from the fungi Aspergyllus flavus, A. japonicus, A. niger or A. awamori. The process is carried out in a 10% protein solution at a pH of 7.6-8, maintaining the pH by adding alkali, at a temperature of 40 ° C for no more than 2 hours. The enzyme is then inactivated by heating at 65 ° C for 30 minutes, the hydrolyzate is clarified by centrifugation and dried by spray drying. According to the authors, the degree of hydrolysis of the product can reach 45-50%. The disadvantages of this method is an analog of the claimed process is the following:

- during the hydrolysis, it is mandatory to maintain the pH value with an alkali solution. The consequence of this is that additional amounts of sodium ions get into the product, which cannot be removed from the hydrolyzate using the technological approaches claimed in the method. This leads to the fact that this hydrolyzate may be unsuitable for use in baby and specialized foods (too salty);

- additionally, a stage of relatively long 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;

- hydrolysis is carried out for a fixed time (up to 2 hours), while the quality of the resulting hydrolyzate is not monitored. The claimed degree of hydrolysis of 40-50% is not confirmed by tools. It would be preferable to carry out hydrolysis under the control of the molecular weight distribution and residual antigenicity.

A known method of producing a hydrolyzate of milk protein (casein), including the hydrolysis of protein (see US No. 6372282, NKI 426/656, 04/16/2002). The method uses enzyme preparations Flavourzyme and Sumizyme RTM from Aspergillus orhizae in combination with recombinant amino or carboxypeptidases for hydrolysis in one stage casein. Processing a 10% solution of the substrate is carried out at a temperature of 55 ° C, pH 5.5, the total content of enzymes up to 2% by weight of the substrate. The advantage of this analogue over the previous one is the use of an enzyme preparation with pronounced exopeptidase activity, which allows to achieve a higher degree of hydrolysis. However, the proposed method also has disadvantages comparable to the foregoing. It:

- the progress of hydrolysis is monitored by the amount of free amino acids released. This parameter is of little use in the preparation of hydrolysates intended for use in hypoallergenic products, since a) it does not say anything about the preservation of unsplit antigenic protein structures in the hydrolyzate; b) in itself, the high content of free amino acids in the hydrolyzate is an undesirable effect, since the osmolarity increases and the taste of the product worsens, that is, its quality deteriorates;

- at the end of the hydrolysis, ultrafiltration is not used to clean the hydrolyzate, as a result of which the final product is contaminated with materials of the enzyme preparation, which may have allergenic properties and, therefore, worsen the taste of the product;

- during the process, recombinant (that is, genetically modified) sources of enzyme preparations that are not approved for use in the food industry are used).

Closest to the technical solution to the claimed method is a method for producing an enzymatic hydrolyzate of whey proteins with a high degree of hydrolysis, comprising ultra and diafiltration of a solution of whey proteins, pasteurization of the obtained concentrate, its enzymatic hydrolysis, ultrafiltration of the obtained crude hydrolyzate with separation into a filtrate and concentrate, temperature processing of the obtained the filtrate and its drying (see US No. 5112812, NKI 514/21, 05/12/92). It proposes a process for producing a whey protein hydrolyzate suitable for the preparation of infant hypoallergenic mixtures containing predominantly peptides with a molecular weight (mol. M.) Of up to 6 kDa (kilodaltons), while almost all of these peptides have a molecular weight of 2.0 kDa up to 2.5 kDa Proteolysis is carried out in two stages. In the first step of the process, proteolysis of a 5% whey protein solution is carried out with 2% by weight of an enzyme from Aspergillus orhizae (a drug under the brand name Rhozyme 41 PC). The process is carried out at 50 ° C and pH 8.0 in the mode of pH-statirovanie (the addition of NaOH) to achieve a degree of hydrolysis of 9%. After this, the mixture is ultrafiltered through a 6 kDa membrane and the retentate (comprising 55-60% of the total protein by weight) is re-hydrolyzed using the bacterial Alkalase protease (liquid preparation) in an amount of 0.5% by volume of the mixture. Hydrolysis is carried out at pH 7.5 and a temperature of 50 ° C until a degree of hydrolysis of 12% is reached, after which ultrafiltration is carried out. The final yield of protein nitrogen in the filtrate (permeate) is 75-80%.

The disadvantages of this method is the following:

- the need for pH-stating leads to the receipt in the final product of additional quantities of sodium ions, which cannot be removed from the hydrolyzate in the framework of the claimed process. As a result, the hydrolyzate becomes very salty and its use in the composition of infant formula is difficult. Together, this factor leads to a deterioration in organoleptic properties, and hence the quality of the hydrolyzate;

- the claimed process leads to the formation of large quantities of free amino acids that enter the final product and cannot be removed from the hydrolyzate in the framework of the claimed technological process. The consequence of this is a decrease in the biological value of the hydrolyzate, a deterioration in its taste, an increase in its osmolarity and, therefore, a deterioration in its quality;

- at the same time, the antigenicity of the hydrolyzate obtained in the framework of this method and containing peptides mainly with a molecular weight of 2-2.5 kDa is not low enough for use for patients with severe forms of food allergy;

- the use of the inventive two-stage scheme of proteolysis complicates the process and, therefore, increases the cost of the product;

- during the hydrolysis, the molecular weight distribution and residual antigenicity, that is, key parameters that reliably determine the quality of the product, are not controlled.

The closest technical solution to the claimed hydrolyzate with a high degree of hydrolysis is a hypoallergenic and practically lactose-free composition containing peptides that are formed as a result of hydrolysis of casein-free (milk) whey, in which the composition is practically lactose-free, and peptides formed from serum have a maximum molecular weight of about 6000 Daltons, with almost all of these peptides having a molecular weight between 2000 and 2500 Daltons (see US No. 5112812 , NCI 514/21, 05/12/92). This composition is obtained according to the above prototype method and relates to hydrolysates with a high degree of hydrolysis.

The disadvantage of this hydrolyzate is its low antigenicity, which does not allow its use in products for patients with severe forms of food allergy (to cow's milk protein).

As mentioned earlier, the need for pH-statization leads to the receipt in the hydrolyzate of additional quantities of sodium ions, which cannot be removed from it in the framework of the claimed technological process. As a result, the hydrolyzate becomes very salty and its use in the composition of infant formula is difficult. This hydrolyzate is characterized as practically lactose free. But in cases with severe forms of lactase deficiency, its use in infant formula is undesirable.

Together, all of the above leads to a decrease in biological, nutritional value, organoleptic properties and quality of the described hydrolyzate.

The technical result of the claimed invention on a method for producing a whey protein hydrolyzate with a high degree of hydrolysis is to increase the biological, nutritional value, organoleptic properties and quality of the obtained hydrolyzate with a high degree of hydrolysis with the possibility of its use in hypoallergenic mixtures of functional purpose.

The technical result is achieved in that in a method for producing an enzymatic hydrolyzate of whey proteins with a high degree of hydrolysis, comprising ultra and diafiltration of a solution of whey proteins, pasteurization of the resulting solution of a whey protein concentrate, its enzymatic hydrolysis, ultrafiltration of the obtained crude hydrolyzate with separation into a filtrate and a hydrolyzate concentrate, temperature processing the obtained filtrate and drying it, a solution is used as a solution of whey proteins, prepared from a dry whey protein concentrate, and the enzymatic hydrolysis is fed a solution of whey protein concentrate after ultra and diafiltration, brought to a mass fraction of solids of 4.8-5.0%, and hydrolysis is carried out at an enzyme-substrate ratio of 5%, ultrafiltration of the crude hydrolyzate carried out on membranes with a throughput of mol. m. 5 kDa and 2 kDa, with a constant return of the resulting concentrate to ultrafiltration, after which the resulting filtrate is further purified by ultrafiltration on membranes with a throughput of mol. m. 5 kDa and 2 kDa, with a constant return of the resulting concentrate to ultrafiltration, and the obtained filtrate of the second run containing purified hydrolyzate is concentrated by reverse osmosis to a mass fraction of solids of 10-15% before temperature treatment, then it is concentrated to a mass fraction of solids of 43 -45%, and pasteurization is used as a heat treatment.

The claimed technical result is achieved by combining the following factors:

It was experimentally found that carrying out enzymatic hydrolysis of a solution of whey protein concentrate with a mass fraction of solids of 4-5% at an enzyme-substrate ratio of 5% allows to obtain a hydrolyzate with a higher degree of cleavage of the protein component, respectively, with lower antigenicity, within the framework of the proposed method means with increased biological and nutritional value.

With a decrease in the enzyme-substrate ratio below 5%, the hydrolysis depth decreases and the loss of protein substrate during subsequent ultrafiltration increases unacceptably. With an increase in the enzyme-substrate ratio above 5%, the accumulation of free amino acids in the hydrolyzate increases, increasing the osmolarity of the product and worsening its taste, which leads to a decrease in the organoleptic properties and quality of the hydrolyzate. In addition, the consumption of the enzyme preparation is unacceptably increased, which leads to a rise in the cost of the product.

It was experimentally established that ultrafiltration of the crude hydrolyzate on membranes with a molar capacity of m. 5 kDa and 2 kDa upon repeated return of the obtained crude hydrolyzate concentrate to ultrafiltration and additional ultrafiltration of the crude hydrolyzate filtrate on membranes with a throughput of mol. m. 5 kDa and 2 kDa with repeated return of the resulting concentrate to ultrafiltration allows for maximum purification of the resulting hydrolyzate with its maximum yield from the initial amount of protein substrate. If the hydrolyzate is not returned for repeated ultrafiltration, then we obtain a lower quality hydrolyzate with insufficiently low antigenicity.

The applied scheme of two-stage ultrafiltration of 5 and 2 kDa is more selective than that used in the prototype ultrafiltration through 6 kDa membranes, which leads to better cleaning of the hydrolyzate from antigenic structures and therefore improves the quality of the product. The use of membranes with a throughput below 2 kDa leads to a decrease in the yield of the finished product and to an increase in the mass fraction of free amino acids in it, that is, correspondingly, to a rise in the cost of the product and a decrease in its quality. The use of membranes with a throughput of more than 5 kDa leads to an unacceptable increase in the residual antigenicity of the product, which makes it impossible to use it in hypoallergenic mixtures.

The advantage of the proposed method in comparison with the prototype is the absence of the need for pH-stating, since due to the optimally selected technology, the decrease in pH during hydrolysis due to the release of free amino acids goes up to values not lower than pH 6.0 ± 0.2, which corresponds to the optimum pH used enzyme preparation. The enzyme preparation Flavozyme (Flavourzyme) used has a high complex of both exo- and endopeptidase activity, which allows for deep hydrolysis of the protein in one stage. It does not require maintaining a constant pH (pH-statization) by constantly adding alkali, that is, significantly less salts enter the final product than in the prototype, which improves the organoleptic properties of the resulting hydrolyzate, and hence its quality. The specificity of the enzyme preparation used in the inventive method is such that when it acts on the substrate, a relatively smaller amount of free aromatic amino acids and their short peptides with a bitter taste are formed. As a result, the palatability of the resulting product improves.

Pasteurization is necessary to ensure microbiological parameters of the product that meet hygienic requirements in accordance with SanPiN 2.3.2.1078-01, clause 3.3.5. When reducing the time and (or) pasteurization temperature below the proposed values, the risk of the development of microorganisms in the product increases, which leads to a deterioration in the quality and safety of the product. With increasing temperature and (or) pasteurization time in excess of the proposed values, the loss of essential amino acids increases, which leads to a decrease in biological value and, therefore, the quality of the product. At the same time, the formation of adverse unwanted impurities caused by the reactions of free amino acids in the product between themselves and with residual amounts of lactose, including Mayar compounds, melanoidins, and caramelization products, is also increasing. These impurities give the product an undesirable color, that is, impair its quality.

Carrying out reverse osmosis allows you to concentrate the product before spray drying to acceptable concentrations. If reverse osmosis is not carried out, then a too dilute solution (with a high water content) would be supplied for drying, which would drastically slow down the drying process, increase its energy intensity, increase the cost of the product and lead to a deterioration in its solubility, i.e. the quality of the product.

The inventive method allows to obtain a lactose-free hydrolyzate (and in the prototype - almost lactose-free). This is achieved by the fact that a solution prepared from a dry whey protein concentrate is used as a whey protein solution.

In contrast to the prototype in the claimed invention, the degree of removal (elimination) of undigested protein and antigenic structures from the product is controlled by more sensitive and specific methods of high-pressure liquid chromatography and enzyme-linked immunosorbent assay, which are adequate to the task. This allows you to get a guaranteed product of higher quality, with a lower content of unsplit proteins and allergens, compared with the prototype method, where these control methods are not used.

The claimed combination of features of the proposed method allows to obtain an enzymatic hydrolyzate of whey proteins with a high degree of hydrolysis with high biological and nutritional value, improved quality and organoleptic properties. This makes it possible to use it in hypoallergenic mixtures for patients with severe forms of food allergy.

Deviation from the claimed features to a greater or lesser extent leads to a decrease in the nutritional and biological value of the resulting hydrolyzate, deterioration in its quality and organoleptic properties.

The technical result of the claimed invention on a hydrolyzate with a high degree of hydrolysis obtained by the claimed method is also to increase the biological, nutritional value, organoleptic properties and quality of the resulting hydrolyzate with the possibility of its use in hypoallergenic mixtures of functional purpose.

The technical result is achieved by the fact that in a hydrolyzate with a high degree of hydrolysis, more than 75% of the protein material is concentrated in the fraction of low molecular weight peptides in the range of molecular masses of 0.5-2 kDa, the content of peptides with a range of molecular masses of 2-4.5 kDa does not exceed 18% , the content of peptides with a molecular mass of more than 4.5 kDa does not exceed 7%, and the residual antigenicity of cow's milk proteins does not exceed 1 · 10 ^-5 by weight of the protein component. The inventive hydrolyzate obtained by the claimed method.

The claimed technical result is achieved by combining the following factors:

- in the inventive enzymatic hydrolyzate of more than 75% of the protein material is concentrated in the fraction of low molecular weight peptides in the range of molecular weights of 0.5-2 kDa, which allows to achieve lower residual antigenicity (in comparison with the prototype), not exceeding 1-10 ^-5 by weight of protein component. Due to this, the hydrolyzate as a protein component can be used to obtain products for the therapeutic nutrition of children and adults, with severe forms of food allergies and intolerance to cow's milk proteins;

- due to hydrolysis without pH-stating, the inventive hydrolyzate contains fewer salts, which improves its taste characteristics in comparison with the prototype and favors its use in infant formulas;

- the claimed hydrolyzate is lactose-free and can be used in infant formula for patients with severe forms of lactase deficiency;

All of the above proves in comparison with the prototype increased biological and nutritional value, organoleptic properties, the quality of the claimed hydrolyzate.

A method of obtaining an enzymatic hydrolyzate of whey proteins with a high degree of hydrolysis is as follows.

To produce a whey protein hydrolyzate with a high degree of hydrolysis, a dry whey protein concentrate (KSB) is dissolved in a continuous circulation mode in drinking water with a temperature of 43 ÷ 47 ° С until a solution with a mass fraction of solids of 2 ÷ 3% is obtained.

The resulting solution of whey protein concentrate (KSB whey protein solution) is cooled to a temperature of 6 ÷ 8 ° C and left to swell for 3 ÷ 3.5 hours with constant stirring. Then determine the purity group. If the solution needs to be cleaned, it is heated to a temperature of 38 ÷ 42 ° C and cleaned on a clarifier. A solution of whey protein KSB is pasteurized at a temperature of 70-74 ° C with a soak of 14 ÷ 18 s, cooled to a temperature of 50 ÷ 55 ° C and sent for ultra- and diafiltration to completely remove lactose.

The ultrafiltration and diafiltration of the obtained KSB whey protein solution is carried out on an ultrafiltration unit with a membrane capacity of 20 kDa (kilodaltons) at a temperature of 48 ÷ 52 ° С, pH 6.5 ÷ 7.0 and pressure 1 , 5 ÷ 6 bar to obtain a concentrate containing a high molecular weight fraction of KSB (Navy KSB), and a filtrate containing a low molecular weight fraction of KSB-lactose.

Ultrafiltration concentration of a solution of whey protein KSB is carried out until the solids content in the resulting concentrate is 17 ÷ 20%. To increase the output of the Navy KSB during ultrafiltration, diafiltration water with a temperature of 48 ÷ 52 ° C is fed into the installation. The diafiltration process is considered complete when the mass fraction of solids in the filtrate is 0%. When the solids content in the KSB concentrate reaches 17 ÷ 20%, the latter is sent to the fermentation tank, in which the concentrate is diluted with process water to a mass fraction of solids of 4.8 ÷ 5.0%.

The resulting solution is pasteurized at a temperature of 70 ÷ 74 ° C with a holding time of 14 ÷ 18 s and cooled to a temperature of 50 ÷ 52 ° C.

To obtain a whey protein hydrolyzate with a deep degree of hydrolysis, the industrial ferment preparation Flavozyme 500 MG (Flavozyme 500 MG) from Novozymes (Denmark) is used.

The enzyme is pre-dissolved in drinking water with a temperature of 35 ÷ 45 ° C until a solution is formed with a mass fraction of solids of 9 ÷ 10% at an enzyme-substrate ratio of 5% (5% of the mass fraction of protein in KSB) and introduced with constant stirring into enzymatic hydrolysis of 4.8 ÷ 5.0% solution of KSB concentrate. Enzymatic hydrolysis is carried out at a temperature of 48 ÷ 52 ° C with constant stirring. The enzyme-substrate ratio is 5% per dry protein, the pH during hydrolysis decreases from 6.8 ÷ 7.2 to 5.8 ÷ 6.2 due to the release of free amino acids.

The duration of hydrolysis is 16–22 hours (until the distribution of peptides by molecular mass ceases to change, and the mass fraction of whey antigens in the protein component of the product becomes less than 10 ^–3 ).

The obtained crude whey protein hydrolyzate (GSB) is sent for ultrafiltration, which is carried out in a unit with a membrane capacity of 5 kDa and 2 kDa to obtain a concentrate containing a high molecular weight fraction of the GSB (Navy GSB) and a filtrate containing a low molecular weight fraction of the GSB (NSF GSB).

To increase the yield of the low molecular weight fraction of HSB, the resulting HSB concentrate is returned to ultrafiltration on 5 kDa and 2 kDa membranes, pre-mixed with the crude hydrolyzate.

To increase the yield of the low molecular weight fraction of the HSB contained in the filtrate, softened process water is added to the concentrate at the end of the ultrafiltration process.

The supply of GSB concentrate for ultrafiltration is stopped when the so-called "dead volume" of the high molecular weight fraction of the concentrate is reached (the concentrate becomes a jelly-like mass that is not diluted with water).

To ensure a more complete purification of the hydrolyzate from large peptides with increased antigenicity, the obtained filtrate of the first run is additionally fed to an ultrafiltration unit with a throughput of 5 and 2 kDa membranes with separation into the filtrate and concentrate of the second run. To increase the yield of the low molecular weight fraction, the obtained concentrate of the second run is returned to ultrafiltration through 5 and 2 kDa membranes, mixed with the filtrate of the first run.

The obtained filtrate of the second run, containing a low molecular weight fraction of HSB - a purified hydrolyzate, is sent to a reverse osmosis (OO) unit for preliminary thickening - concentration. The pressure at the outlet of the OO installation is 35–40 bar, the temperature of the concentrated solution is 30–35 ° C.

The filtrate of the second run, containing the purified hydrolyzate, is concentrated on an OO unit until the solids content in the resulting concentrate is 10-15%.

The filtrate obtained by thickening at the OO installation is sent to a drain, the concentrate containing the purified hydrolyzate, hereinafter referred to as the "hydrolyzate", is sent to the concentrate storage tank.

Then, the condensation of the concentrate with OO containing the purified hydrolyzate is continued on a vacuum-evaporation plant to a mass fraction of solids of 43 ÷ 45%.

The condensed hydrolyzate is pasteurized, for example, at a temperature of 76 ÷ 80 ° C with a holding time of 45 ÷ 75 s and sent to the drying, which is carried out on a spray dryer in the following modes:

- air temperature at the entrance to the drying tower - 170 ÷ 180 ° C

- air temperature at the outlet of the drying tower - 75 ÷ 85 ° C.

Mass fraction of solids in the final product 95%.

During the drying process, the hydrolyzate enters the installer, where the powder is cooled, collected in containers (big bags) and sent to intermediate storage, where it can be stored for no more than 10 months. The product yield is 60-65%.

Example 1

Dry whey protein concentrate in an amount of 2202 kg is dissolved in continuous circulation in drinking water at a temperature of 45 ° C until a solution with a mass fraction of solids (DM) of 2.5% (solution pH 6.91 units) is obtained. The resulting solution was cooled to a temperature of 8 ° C and allowed to swell for 3.5 hours with constant stirring, then the solution was pasteurized at a temperature of 72 ° C for 16 s, cooled to a temperature of 52 ° C and sent for ultrafiltration and diafiltration.

Ultra- and diafiltration is carried out on an ultrafiltration unit with polysulfone membranes UF DHT 20-63 38/3 0FF, the throughput of which is 20 kD by molecular weight. The whey protein solution is divided into a concentrate containing Navy KSB, and a filtrate containing NMP KSB (lactose).

To increase the output of the Navy KSB in the process of ultrafiltration processing, diafiltration water heated to a temperature of 50 ° C is fed into the installation. The diafiltration water flow is about 1,500 l / h. The filtrate during the UV treatment is sent to the drain. The remains of the NFM and Navy from the installation are pushed with process water until the solids content in the filtrate and the concentrate is 0.01%.

Get 10951 l of concentrate with a mass fraction of solids of 18.5% and a pH of 6.8 units, which is then diluted with process water to a solids content of 5% in solution, pasteurized at a temperature of 72 ° C for 16 s and cooled to a hydrolysis temperature of 52 ° C.

Enzymatic hydrolysis of the obtained 5% solution of Navy KSB (40520 L) is carried out using the industrial enzyme preparation Flavozyme 500 MG (Flavozyme 500 MG) from Novozymes (Denmark) obtained from Aspergillus oryzae strain (with an enzyme-substrate ratio of 5% ( 5% by weight of dry protein in KSB - 88 kg) for 20 hours.

The enzyme preparation is pre-dissolved in 792 liters of drinking cold water to obtain a solution with a mass fraction of solids of 10%, which, with constant stirring, is added to the above-prepared solution of the Navy KSB. Enzymatic hydrolysis is carried out at a temperature of 52 ° C with constant monitoring of the temperature for 20 hours without pH-statirovanie ..

After 10 and 20 hours of hydrolysis, samples of the crude hydrolyzate are taken (sample 1 and sample 2, respectively) and examined by high pressure exclusion chromatography (determination of mass-molecular distribution). The data are presented in table 1.

Figure 1 and figure 2 presents the chromatogram of the crude hydrolyzate after 10 and 20 hours, respectively. The ordinate axis is the optical density at a wavelength of 280 nm (relative units), the abscissa axis is the molecular mass (kDaltons). The mass fraction of whey antigens of samples 1 and 2 is determined by the method of inhibition of indirect enzyme-linked immunosorbent assay (see table 2). The pH in samples 1 and 2 was 5.86 and 5.82 units. respectively.

Upon completion of the hydrolysis, the resulting crude hydrolyzate is sent for ultrafiltration on polysulfone membranes with a throughput of 5 and 2 kDa connected in parallel (use UF AES5-B-6338S / 30 & UF AE10-B-6338/30 membranes). The hydrolyzate is divided into a concentrate containing Navy and a filtrate containing NMP.

To increase the yield of NMF at the end of the ultrafiltration process, softened process water is added to the concentrate, heated to 50 ° C. The residual NMF from the installation is pushed with water to the content of CB in the filtrate of 0.001%. Get 38089 l of the filtrate of the first run with a solids content of 5%. A sample is taken in which the mass-molecular distribution of the peptides by fractions is determined by high pressure size exclusion chromatography (sample 3). The data are presented in table 1. Figure 3 shows the exclusion chromatogram of sample 3. Determination of the residual antigenicity of sample 3 is carried out by the method of inhibition of indirect solid-phase analysis. The data are presented in table 2.

During filtration, the resulting Navy is constantly returned to the tank with the crude hydrolyzate, where the Navy is mixed with the latter and fed to ultrafiltration (to optimize the process and increase the yield of the NMP), and at the end of the treatment, it is sent to the drainage.

The filtrate of the first run is additionally passed through an ultrafiltration unit with a membrane capacity of 5 and 2 kDa with separation into the filtrate and concentrate of the second run. This allows for a more complete purification of the hydrolyzate from large peptides with increased antigenicity.

To increase the yield of NMF at the end of the ultrafiltration process, softened process water heated to 50 ° C is fed into the concentrate of the second run. The residual NMF from the installation is pushed with water to the content of CB in the filtrate of 0.001%. Get about 32760 l of the filtrate of the second run with a solids content of 5%. A sample is taken in which the mass-molecular distribution of the peptides by fractions is determined by high pressure size exclusion chromatography (sample 4). The data are presented in table 1. Figure 4 shows the exclusion chromatogram of sample 4. The mass fraction of milk antigens of sample 4 is determined by the method of inhibition of indirect solid-phase analysis. The data are presented in table 2. Sample pH 4 - 5.95 units.

To increase the yield of NMF during ultrafiltration, the resulting concentrate (Navy) of the second run is returned to the filtrate tank after the first run, where the concentrate of the second run is mixed with the filtrate of the first run and fed to ultrafiltration. At the end of the process, the concentrate of the second run remains in the UV unit and then washed off with a washing solution.

The filtrate of the second run containing the purified hydrolyzate is sent to a reverse osmosis unit for preliminary thickening. Thickening modes: the pressure at the outlet of the installation is 36 bar, the temperature of the concentrated solution is 30 ° C. Polysulfone membranes of the 40-RO-SF384 OS type with a membrane capacity of 1 kDa are used.

The filtrate of the second run is concentrated to a solids content in the concentrate, leaving the OO installation of 15%. The filtrate with OO is sent to the drainage. The concentrate, hereinafter referred to as the "hydrolyzate", is cooled to a temperature of 4 ° C, collected in an intermediate container for subsequent thickening. The remaining concentrate from the installation is pushed with process water to a solids content of 0%. Get 10265 l of hydrolyzate with a mass fraction of solids of 15%.

Subsequent thickening is carried out on a vacuum-stripping unit until the solids content in the hydrolyzate is 44%. The thickened mixture is pasteurized at a temperature of 78 ° C for 60 seconds and sent to a drying unit.

The thickened hydrolyzate is dried in a spray dryer under the following conditions: air temperature at the inlet to the drying tower 176 ° C, air temperature at the outlet of the drying tower 85 ° C.

Get 1431 kg of hydrolyzate with a solids content of 96.5%. The hydrolyzate yield is 64.9%.

The obtained hydrolyzate is examined for dry matter content, total nitrogen, molecular weight distribution (MMP), residual antigenicity, osmolarity, ash, active acidity (sample 5). Data on MMP are presented in table 1, the mass fraction of whey antigens is in table 2, the remaining characteristics of the hydrolyzate are presented in table 3. Figure 5 shows the exclusion chromatogram of sample 5.

Table 1 No. The range of molecular masses, kDa The content of the fraction (weight integration of the chromatogram),% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 one More than 275 6.9 4.1 0 0 0 2 275-126 0.9 0.5 0 0 0 3 126-72.4 0.8 0.9 0 0 0 four 72.4-22.4 8.8 5.5 0 0 0 5 22.4-10.2 1.8 1.7 0 0 0 6 10.2-4.5 14,4 8.4 5.1 4.9 6.1 7 4,5-1,7 12.7 12,4 12,2 13.3 13.5 8 Less than 1.7 53.6 66.5 82.7 81.8 80,4

table 2 No. Sample Name Mass fraction of whey antigens in the protein component of the product The rate of decrease in antigenicity in comparison with undigested whey protein, times one Sample 1 1.6 · 10 ^-3 600 2 Sample 2 1.7 · 10 ^-4 5800 3 Sample 3 1,0 · 10 ^-5 96000 four Sample 4 9.0 · 10 ^-7 1,200,000 5 Sample 5 1 · 10 ^-7 1,000,000

Table 3 No. Name of the characteristics of the hydrolyzate Characteristic Value one Total nitrogen,% 12.3 2 Solids content,% 96.5 3 Ash% 5.2 four Active acidity, units pH 5.84-6.72 5 Osmolarity, mOsm / kg H ₂ O up to 700

As can be seen from table 1 in the hydrolyzate, the maximum content of peptides is concentrated in the molecular weight range of less than 1.7 kDa, which characterizes this product as a hydrolyzate with a high degree of hydrolysis. Analysis of the residual antigenicity of this hydrolyzate showed a decrease of antigenicity by a factor of 1,000,000 (Table 2), which allows it to be used as part of hypoallergenic medicinal products. The hydrolyzate dissolves in warm drinking water with the formation of a turbid solution with a small amount of sediment (up to 40% by weight of solids). Odor and taste - specific, consistency - fine powder, color - cream.

Example 2. The process is carried out analogously to example 1, only the solids content in the initial solution of whey proteins is 2%, and after ultra- and diafiltration at the installation with a membrane capacity of 20 kDa, the concentrate with a mass fraction of solids of 20% and enzymatic hydrolysis serves a whey protein concentrate with a mass fraction of solids of 4.8%. The concentration of the filtrate of the second run on the reverse osmosis installation is carried out to a solids content of 12% in concentrate. Get 1365 kg of hydrolyzate with a solids content of 97.0%. The molecular weight distribution of the obtained dry hydrolyzate is shown in table 4.

Table 4 No. The range of molecular masses, kDa The content of the fraction (weight integration of the chromatogram),% one 10.2-4.5 6.0 2 4,5-1,7 16,0 3 Less than 1.7 78.0

The residual antigenicity of the dry product was 7.5-10 ^-6 by weight of protein, which allows the use of a hydrolyzate to create substitutes for human milk for the therapeutic nutrition of children with severe forms of food allergies. Nitrogen - 12.4%, ash - 3.2%. The hydrolyzate yield is 62%.

Example 3. The process is carried out analogously to example 1, only the solids content in the initial solution of whey proteins is 3%, and after ultra- and diafiltration at the installation with a membrane capacity of 20 kDa, a concentrate with a mass fraction of solids of 17% is obtained, enzymatic hydrolysis serves a whey protein concentrate with a mass fraction of solids of 4.9%. Concentrated filtrate of the second run on the reverse osmosis installation lead to a solids content of 15% in concentrate. Subsequent thickening is carried out to a solids content of 45% in the hydrolyzate.

Get 1390 kg of hydrolyzate with a solids content of 97.0%. The hydrolyzate yield is 63%. The molecular weight distribution of the obtained dry hydrolyzate is shown in table 5.

Table 5 No. The range of molecular masses, kDa The content of the fraction (weight integration of the chromatogram),% one 10.2-4.5 5.5 2 4,5-1,7 12.5 3 Less than 1.7 81.0

The residual antigenicity of the dry product was 2 · 10 ^-7 by weight of protein, which makes it possible to use the hydrolyzate as a protein component of FFA for the therapeutic nutrition of children with severe forms of food allergies. Nitrogen - 12.3%, ash - 4.3%.

Example 4. The process is carried out analogously to example 1, only the enzyme-substrate ratio is 4.5% by weight of protein. The residual antigenicity of the obtained product was 5 · 10 ^-5 by weight of protein, this indicator of residual antigenicity is characteristic of hydrolysates with an average degree of hydrolysis, which means that the above hydrolysis mode should be considered unsuitable.

Example 5. The process is carried out analogously to example 2, only the process of purification of the hydrolyzate after proteolysis on membranes with a throughput of 2 and 5 kD lead in one stage. Get 1000 kg of dry hydrolyzate with residual antigenicity of 2.8 · 10 ^-5 by weight of protein. This hydrolyzate is not sufficiently purified, with a higher antigenicity, which does not allow it to be used for medicinal purposes by patients with a severe form of food allergy.

The inventive method allows to obtain a lactose-free enzymatic hydrolyzate with a high degree of hydrolysis, which can be used in the design of products for children of the first year and older with food allergies of various etiologies, with malabsorption syndrome, lactase deficiency, galactosemia, hypotrophy of the II-III degrees, as well as in the postoperative period with bowel surgery.

The inventive method allows to obtain a hydrolyzate with low antigenicity, but lower osmolarity and better taste than a mixture of amino acids.

The inventive hydrolyzate contains the minimum possible amount of inorganic salts (in the process of hydrolysis, inorganic salts are not introduced, since it is not necessary to maintain the optimum pH under the given conditions of hydrolysis).

The resulting hydrolyzate (due to the low content of inorganic salts) is optimally suitable for further nano-processing if the task is to obtain a product with improved organoleptic properties and a peptide-amino acid mixture with even lower residual antigenicity.

The use of a single enzyme preparation greatly simplifies and reduces the cost of the production process.

Claims (2)

1. A method of producing a whey protein hydrolyzate with a high degree of hydrolysis, comprising ultra and diafiltration of a whey protein solution, pasteurization of the obtained whey protein concentrate solution, its enzymatic hydrolysis, ultrafiltration of the obtained crude hydrolyzate with separation into the filtrate and hydrolyzate concentrate, temperature processing of the obtained filtrate and its drying characterized in that a solution prepared from a dry concentrate with whey proteins, and a solution of whey protein concentrate after ultra and diafiltration, brought to a mass fraction of solids of 4.8-5%, is fed to enzymatic hydrolysis, with hydrolysis at an enzyme-substrate ratio of 5% by weight, ultrafiltration of the crude hydrolyzate is carried out on membranes with throughput with a molecular weight of 5 kDa and 2 kDa with a constant return of the resulting concentrate to ultrafiltration, after which the resulting filtrate is further purified by ultrafiltration on membranes with a throughput with a molecular weight of 5 kDa and 2 kDa with a constant return of the resulting concentrate to ultrafiltration, and the obtained filtrate of the second run containing purified hydrolyzate is concentrated by reverse osmosis to a mass fraction of solids of 10-15% before temperature treatment, then it is concentrated to a mass fraction of dry substances 43-45%. 2. A hydrolyzate of whey proteins with a high degree of hydrolysis obtained by the method of claim 1, characterized in that it is lactose-free and that more than 75% of the protein material is concentrated in the fraction of low molecular weight peptides in the molecular weight range of 0.5-2 kDa, the content of peptides with the molecular weight range of 2-4.5 kDa does not exceed 18%, the content of peptides with a molecular mass of more than 4.5 kDa does not exceed 7%, and the residual antigenicity of cow's milk proteins does not exceed 1 · 10 ^-5 by weight of the protein component.

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