RU2727431C1 - Production method of milk-clotting enzyme preparation of combined composition - Google Patents

Abstract

FIELD: food industry.SUBSTANCE: invention relates to dairy industry, in particular, to production of a milk-clotting enzyme preparation of combined composition. Method involves preparation of liquid preparations of a rennet and beef pepsin by certain methods, their calculation according to specified laws and mixing so that the ready liquid complex preparation contains chymosin in amount from 50 to 75 %, and the rest is pepsin.EFFECT: invention increases degree of purification of the obtained product, reduces labour and power consumption and obtains an economic effect.1 cl, 5 dwg, 3 tbl, 1 ex

Description

The invention relates to the production of a milk-clotting enzyme preparation (hereinafter referred to as MFP) of a combined composition obtained from calf abomasums and mucous membranes of cattle abomasums, and can be used in the food industry in the production of cheeses and cottage cheese.

A known method for producing rennet (Method for producing rennet: USSR author's certificate No. 642360; declared. 04/01/1977; publ. 01/15/1979; bull. No. 2), providing for grinding dry calf rennet into strips 5-8 mm wide, extraction of the enzyme 10% sodium chloride solution at 4 ° C, settling the extract for 24 hours, filtering, salting out the filtrate by adding sodium chloride in an amount of 10% by weight of the filtrate, acidifying with hydrochloric acid to pH 2.0 and settling for 24 hours, separation of the enzyme mass and its preliminary dehydration, drying of the sludge, followed by grinding and normalization of the dry enzyme.

The disadvantages of the known method include, first of all, the low quality of the obtained dry milk-clotting enzyme preparations, because it does not provide for fine purification of the enzyme extract from insoluble impurities and ballast substances of different nature. As a result, dry milk-clotting enzyme preparations obtained by this method are characterized by a high content of ballast substances (up to 4%), the presence of which leads to the appearance of bitterness and a decrease in the quality of dairy products produced with such preparations, in particular, cheeses.

The dry milk-clotting enzyme preparations obtained according to the method specified in the prototype method differ in the heterogeneity of the composition due to the different densities of the dry enzyme-containing substance and table salt used to normalize the enzyme activity. This can lead to the stratification of the structure of dry milk-clotting enzyme preparations and the obtaining of non-uniform activity inside the packaging unit during storage.

The aim of the invention is to develop a method for the production of a milk-clotting enzyme preparation of a combined composition having a higher quality, which consists in the absence of insoluble impurities and ballast substances of different nature, in the homogeneity of the structure, providing the same activity of the preparation in the entire volume and having a lower production cost than in the prototype, with the use of which you can produce high quality cheeses and cottage cheese.

The technical result of the claimed invention for a method for the production of a milk-clotting enzyme preparation of a combined composition obtained from calf abomasums and mucous membranes of cattle abomasums consists of:

- in increasing the degree of purification of the resulting milk-clotting enzyme preparation of the combined composition,

- in reducing the cost of the resulting milk-clotting enzyme preparation of the combined composition,

- in reducing labor and energy costs of production.

The specified technical result is achieved by the fact that in the method for the production of milk-clotting enzyme preparation of a combined composition, including grinding rennet, extraction of rennet with a solution of sodium chloride, separation of the liquid phase, filtration, according to the invention, liquid preparations of rennet and beef pepsin are prepared: extraction of rennet is carried out 10 -12% sodium chloride solution for 4-5 hours at a temperature of 34-40 ° C and periodic stirring, re-extraction is carried out with 1.5-2 times the mass of 10-12% sodium chloride solution, the resulting extracts are combined and acidified a solution of hydrochloric acid with a molar concentration of 1 mol / dm ³ to acidity pH 4.60-4.80 units. pH, chymosin is activated in the rennet extract at a temperature of 20-25 ° C for 16-20 h, 0.4-0.6% of filter perlite is added to the activated extract and stirred for 0.5-1 h, the resulting mixture filtered under a pressure of 0.015-0.02 MPa at a temperature of 20-25 ° C using a filter board with a pore size of 0.2 μm, the active acidity in the filtered extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ , add 0.5-0.7% sodium benzoate, the weight concentration of sodium chloride in the extract is adjusted to 17-19%; extraction of beef pepsin is carried out with a solution of hydrochloric acid with an active acidity of 1.3-1.5 units. pH for 24-48 hours at a temperature of 4-20 ° C and periodic stirring, re-extraction is carried out, the resulting extracts are combined, the extract is finely purified by microfiltration in a tangential liquid flow on ceramic filter elements with a pore size of 0.2 μm, purified pepsin extract is concentrated by ultrafiltration on polymer membranes with a cut-off threshold for molecular weight of 20 kDa at a temperature not higher than 25 ° C, active acidity in the concentrated pepsin extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ , add 0.5-0.7% sodium benzoate, the weight concentration of sodium chloride in the extract is adjusted to 16-18%; calculate the amount of introduced liquid rennet and liquid beef pepsin, mix liquid preparations of rennet and beef pepsin, obtain a milk-clotting enzyme preparation of the combined composition.

The calculation of the amount of added liquid rennet and liquid beef pepsin is carried out according to the formulas, which indicate the given composition of the complex preparation:

- calculation of the proportion of liquid rennet introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

, (1)

, (1)

where M is the proportion of the rennet preparation used in the volume of the produced batch of the preparation,%;

D is the proportion of chymosin activity in the drugs produced (for the “SG-50” preparation: D = 0.5; for the “SG-75” preparation: D = 0.75);

A _X1 - activity due to chymosin in the used batch of rennet, conv. units / cm ³ ;

A _X2 - activity due to chymosin in the used batch of pepsin, conv. units / cm ³ ;

A _P1 - activity due to pepsin in the used batch of rennet, conv. units / cm ³ ;

A _P2 - activity due to pepsin in the used batch of pepsin, conv. units / cm ³ .

- the calculation of the proportion of liquid pepsin introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

, (2)

, (2)

where R _G is the proportion of the pepsin preparation used in the volume of the produced batch of the preparation,%.

The finished liquid complex preparation contains chymosin in an amount of 50 to 75%, and the rest is pepsin.

Before concentration, the SG-50 milk-clotting enzyme preparation containing 50% chymosin and 50% pepsin is purified from ballast protein substances by microfiltration on polymeric polysulfone membranes with a cutoff threshold for molecular weight of 300 kDa.

The method of obtaining milk-clotting enzyme preparation of the combined composition is carried out as follows.

Liquid preparations of rennet and pepsin are being prepared.

To prepare a liquid preparation of rennet, dry calf rennet are crushed and poured with a 10-12% sodium chloride solution at a temperature of 36-40 ° C. The consumption of sodium chloride solution is 15-30 kg per 1 kg of dry rennet. The enzyme is extracted for 4-5 hours at a temperature of 34-40 ° C and periodic stirring. The liquid extract is separated from the abomasum using a sieve. The separated abomasums are poured with 1.5-2 times the mass of 10-12% sodium chloride solution and repeated extraction is carried out for 4-5 hours at a temperature of 34-40 ° C and periodic stirring. The obtained extracts are combined and acidified with a solution of hydrochloric acid with a molar concentration of 1 mol / dm ³ to an acidity of pH 4.60-4.80 units. pH. Chymosin is activated in the rennet extract at a temperature of 20-25 ° C for 16-20 hours. 0.4-0.6% of filter perlite is added to the activated rennet extract and stirred for 0.5-1 hours. filtered on a filter press under a pressure of 0.015-0.02 MPa at a temperature of 20-25 ° C using filter paper with a pore size of 0.2 μm. The active acidity in the filtered rennet extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide molar concentration of 1 mol / dm ³ . 0.5-0.7% sodium benzoate is added to the extract, the weight concentration of sodium chloride in the extract is adjusted to 17-19%.

To prepare a liquid preparation of pepsin, frozen blocks of mucous membranes of cattle abomasums are poured with a solution of hydrochloric acid with an active acidity of 1.3-1.5 units. pH and temperature 18-20 ° C. Consumption of hydrochloric acid solution is 3-5 dm ³ per 1 kg of mucous membrane. The enzyme is extracted for 24-48 hours at a temperature of 4-20 ° C and periodic stirring. During the extraction process, the blocks of mucous membranes are thawed. The low temperature during extraction and the low value of the active acidity of the medium ensure the protection of the extract from microbiological deterioration. The resulting extract is separated from the mucous membrane through a filter material (lavsan or 3-4 layers of gauze). For a more complete extraction of the enzyme, repeated extraction of mucous membranes is used at similar temperature and time conditions. The resulting extracts are combined. Consumption of hydrochloric acid solution during repeated extraction is 1-1.5 dm ³ per 1 kg of mucous membrane.

Fine purification of the extract from impurities is carried out using microfiltration in a tangential flow of liquid on ceramic filter elements with a pore size of 0.2 μm. To increase the specific milk-clotting activity, the purified pepsin extract is concentrated by ultrafiltration on polymer membranes with a molecular weight cut-off threshold of 20 kDa at a temperature not exceeding 25 ° C, which avoids the loss of enzyme activity. The active acidity in the concentrated pepsin extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide molar concentration of 1 mol / dm ³ . 0.5-0.7% sodium benzoate is added to the extract, the weight concentration of sodium chloride in the extract is adjusted to 16-18%.

A milk-clotting enzyme preparation of a combined composition is obtained from the obtained liquid enzyme preparations by mixing. The calculation of the amount of added liquid rennet and liquid beef pepsin is performed in accordance with formulas (1) and (2). Previously, the total milk-clotting activity and the enzymatic composition of rennet and pepsin extracts are determined according to GOST 34353-2017.

Example.

A preparation of liquid rennet is prepared. 2.0 kg of dry abomasums are cut into strips 1-2 cm wide. Crushed abomasums are poured into 60 kg of 10% sodium chloride solution at 38 ° C and the enzyme is extracted with periodic stirring for 5 hours. The extraction temperature is 34 ° C. The liquid extract is separated from the abomasum using a sieve. The mass of the 1st extract is 44.2 kg. After the first extraction, 13.2 kg of wet rennet remain. 19.8 kg of 10% sodium chloride solution with a temperature of 38 ° C are added to the separated rennet and the enzyme is re-extracted with periodic stirring for 4.0 hours. The extraction temperature is 38 ° C. The liquid extract is separated from the abomasum and the second extract weighing 14.2 kg is obtained. The resulting extracts are combined and acidified with a solution of hydrochloric acid with a molar concentration of 1 mol / dm ³ to obtain an acidity of 4.62 units. pH. The activation of chymosin in the rennet extract is carried out at a temperature of 22 ° C for 18 hours. An activated crude extract is obtained in an amount of 58.4 kg. 0.3 kg of filtering perlite is added to the activated extract, stirred for 1 hour and filtered on a frame filter press under a pressure of 0.015-0.02 MPa at a temperature of 22 ° C using a filter board with a pore size of 0.2 μm. 48.3 kg of a purified optically clear extract are obtained. The purified extract is deoxidized by introducing a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ until an active acidity of 5.8 units is obtained. pH. Dry table salt is added to the obtained extract to obtain a weight concentration of NaCl in the extract of 17.5%. To suppress microbiological processes, 0.5% sodium benzoate is added to the extract. The activity of liquid rennet is 12500 conventional units / cm ³ . The proportion of chymosin activity is 95%, the proportion of pepsin activity is 5%.

Prepare a preparation of liquid beef pepsin. Frozen mucous membranes of cattle in the amount of 19.4 kg are poured with 96 dm ³ of a hydrochloric acid solution with an active acidity of 1.4 units. pH at 20 ° C. After thawing the mucous membrane to accelerate the extraction of the enzyme, the extracted mixture is stirred. Extraction and activation are carried out simultaneously. The duration of the extraction is 24 hours. During the extraction, the temperature of the extractive mixture increases from 4 ° C to 20 ° C. After completion of the extraction, the extract (supernatant) is separated through a filter material (lavsan or 3-4 layers of gauze). 86 kg of liquid extract are obtained. The mucous membrane of cattle abomasum remaining after extraction is mixed with 30 dm ³ of hydrochloric acid solution with active acidity of 1.5 units. pH and re-extraction is carried out for 24 hours. After re-extraction, 27 kg of liquid extract are obtained. The resulting extracts are combined. The extract is purified from impurities by microfiltration in a tangential flow of liquid on ceramic filter elements with a pore size of 0.2 μm. Get 102 kg of purified transparent extract with an activity of 1800 conventional units / cm ³ . The purified extract is concentrated in an ultrafiltration unit equipped with polymer membranes with a molecular weight cutoff of 20 kDa. During the concentration process, the temperature of the concentrated extract is maintained at no higher than 25 ° C to prevent inactivation of the enzyme. 15 kg of concentrated pepsin extract with an activity of 11000 conventional units / cm ^{3 are} obtained. The concentrated extract is deacidified by introducing a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ to obtain an active acidity of 5.6 units. pH. Dry table salt is added to the obtained extract to obtain a weight concentration of NaCl in the extract of 17.0%. To suppress microbiological processes, 0.5% sodium benzoate is added to the extract. The activity of liquid beef pepsin is 9500 conventional units / cm ³ . The proportion of chymosin activity is 20%, the proportion of pepsin activity is 80%.

From the obtained liquid enzyme preparations, complex preparations with a given enzyme composition are prepared by mixing:

- "SG-75" with 75% chymosin and 25% pepsin;

- "SG-50" with 50% chymosin and 50% pepsin.

The calculation of the proportion of liquid rennet introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

,

,

where M is the proportion of the rennet preparation used in the volume of the produced batch of the preparation,%;

D is the proportion of chymosin activity in the drugs produced (for the “SG-50” preparation: D = 0.5; for the “SG-75” preparation: D = 0.75);

A _X1 - activity due to chymosin in the used batch of rennet, conv. units / cm ³ ;

A _X2 - activity due to chymosin in the used batch of pepsin, conv. units / cm ³ ;

A _P1 - activity due to pepsin in the used batch of rennet, conv. units / cm ³ ;

A _P2 - activity due to pepsin in the used batch of pepsin, conv. units / cm ³ .

The calculation of the proportion of liquid pepsin introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

,

,

where R _G is the proportion of the pepsin preparation used in the volume of the produced batch of the preparation,%.

In accordance with the calculation formulas, 10.38 kg of liquid rennet and 7.5 kg of liquid beef pepsin are mixed to obtain a rennet-beef enzyme preparation with a chymosin content of 75% and a pepsin of 25%. Receive 17.88 kg of complex preparation "SG-75" with a total milk-clotting activity of 11000 conventional units / cm ³ , with a share of chymosin activity of 77% and a share of pepsin activity of 23%. To obtain complex preparations "SG-75" with an activity above 20,000 conventional units / cm ³ , the complex preparation "SG-75" is concentrated by ultrafiltration on polymeric membranes made of polysulfone with a cut-off threshold for molecular weight of 20 kDa until a concentration factor of 3 is reached. A concentrated liquid enzymatic milk-clotting preparation "SG-75" with a total milk-clotting activity of 30,000 conventional units / cm ^{3 is} obtained. The enzymatic composition of the concentrated preparation "SG-75" remains unchanged.

In accordance with the calculation formulas, 3.1 kg of liquid rennet and 7.5 kg of liquid beef pepsin are mixed to obtain a rennet-beef enzyme preparation with a chymosin content of 50% and a pepsin of 50%. Get 10.6 kg of complex preparation "SG-50" with a total milk-clotting activity of 10500 conventional units / cm ³ with a share of activity of chymosin 53% and pepsin 47%. To obtain complex preparations "SG-50" with an activity higher than 20,000 conventional units / cm ³ , the complex preparation "SG-50" is concentrated by ultrafiltration. The preparation "SG-50" contains a large amount of water-soluble protein substances (mainly collagens) and is characterized by high viscosity. Before concentration, the preparation "SG-50" is purified from ballast protein substances by microfiltration on polymer membranes made of polysulfone with a cut-off threshold for molecular weight of 300 kDa. After removal of ballast proteins, the enzyme preparation is concentrated on polymeric membranes made of polysulfone with a cut-off threshold for molecular weight of 20 kDa until a concentration factor of 3 is reached. Concentrated liquid enzyme milk-clotting preparation "SG-50" with an activity of 28000 conventional units / cm ^{3 is} obtained. The enzymatic composition of the concentrated preparation "SG-50" remains unchanged.

The obtained liquid milk-clotting enzyme preparations of the combined composition are used for the production of cheeses and cottage cheese.

To determine the effect of the type of milk-clotting drug used on the quality of cheeses, comparative production of Dutch bar cheese with a mass fraction of fat in dry matter of 45% according to GOST R 52972-2008 with the following types of milk-clotting enzyme preparations were carried out:

1) dry preparation of rennet brand "SF-90" (JSC "Moscow plant of rennet") with a chymosin activity share of 90% and a pepsin activity share of 10%;

2) liquid milk-clotting enzyme preparation of the SG-75 brand;

3) liquid milk-clotting enzyme preparation of the SG-50 brand.

At the stage of making cheeses in a cheese-making bath, no differences were found in the duration of the clotting time of the milk mixture, which practically did not differ in the variants and was in the range of 35 ± 2 minutes. There was also no significant difference in the overall duration of the grain bath. Grain processing time, by operations, was:

- kneading until the 2nd heating - 30 ± 2 min

- kneading after the second heating - 25 ± 2 min.

The developed control and experimental samples of Holland cheese were ripened at a temperature of 12-14 ° C for 45 days, after which the mature cheeses were sent to storage at a temperature of 2-4 ° C for 30 days. Sampling for the analysis of physicochemical and organoleptic characteristics of cheeses was carried out after the completion of the stages of maturation and storage.

During ripening, the physicochemical and organoleptic characteristics of the cheeses were monitored. The moisture content and active acidity of the cheese mass in cheeses produced with different types of MPP are given in table. 1.

Table 1. - Moisture content and active acidity of the cheese mass in cheeses produced with different types of milk-clotting enzyme preparations

Variant of the drug used to make cheese Moisture content, % Active acidity of the cheese mass, units pH In fresh cheese after pressing In mature cheese after 30 days of storage In fresh cheese after pressing In mature cheese after 30 days of storage "SF-90" 49.6 41.8 5.71 5.11 "SG-75" 47.8 41.2 5.64 5.16 "SG-50" 46.4 40.8 5.80 5.20

As follows from the data table. 1, the mass fraction of moisture, as well as the active acidity in the cheeses produced using different types of MPP, did not differ significantly. Therefore, the influence of the physicochemical characteristics of cheeses on the ripening process could be excluded.

The organoleptic indicators of mature cheeses developed with different types of milk-clotting enzyme preparations, after 30 days of storage, are presented in table. 2.

Table 2. - Organoleptic characteristics of cheeses made with different types of milk-clotting enzyme preparations

Variant of the drug used to make cheese Taste and smell Consistency "SF-90" Moderately pronounced cheesy taste, slightly sour taste Good (slightly tight, elastic) "SG-75" Pronounced cheesy taste without extraneous flavors Good (slightly tight, elastic) "SG-50" Pronounced cheesy taste without extraneous flavors Good (slightly tight, elastic)

From the results of the organoleptic evaluation presented in table. 2, it follows that the samples of Dutch bar cheese of 45% fat content, produced using different milk-clotting preparations, did not differ significantly from each other, and at the same time had a high quality. There was no negative effect of the use of liquid milk-clotting enzyme preparations "SG-50" and "SG-75" containing beef pepsin for the production of cheeses on the taste properties of the cheeses after maturation and storage.

Also, the nature of proteolysis in mature cheeses after 30 days of storage was investigated. The nature of proteolysis was assessed by the molecular weight distribution of water-soluble nitrogen fractions contained in aqueous extracts from cheese mass. Determination of the molecular weight distribution of proteolysis products in cheeses was carried out according to the method developed at VNIIMS for determining the molecular weight distribution of hydrolysis products of milk proteins, modified for cheeses. Equipment for the implementation of the gel filtration method included a Superose 12 10/300 GL column (Amersham Pharmacia, Sweden), a P-500 precision plunger pump, an LKB Uvicord-III UV detector with a measurement unit and a control unit, an LKB 2066 recorder (LKB devices, Sweden) and a manual injector for sample injection of our own design.

The diagrams of the molecular weight distribution of water-soluble proteolysis products in cheeses produced using different types of milk-clotting enzyme preparations are shown in Fig. 2, 3, 4 and 5.

The relative content of individual water-soluble nitrogenous fractions in cheeses made with different types of milk-clotting enzyme preparations is given in table. 3. For comparison, in table. 3 also shows the relative content of water-soluble nitrogenous fractions in Holland cheese produced with Naturen Stamix 1150.

Table 3. - The relative content of individual water-soluble nitrogenous fractions in cheeses made with different types of milk-clotting enzyme preparations

Variant of the drug used to make cheese Molecular weight of nitrogenous fraction, kDa more than 20 5 - 20 15 0.5 - 1 less than 0.5 "SF-90" 22% 21% 32% 15% eleven% "SG-75" 22% 18% 29% 17% fourteen% "SG-50" 22% 22% 27% sixteen% 12% "Naturen Stamix" 29% 21% 27% 15% 7%

As follows from the data table. 3, with an increase in the relative content of beef pepsin in milk-clotting enzyme preparations (10% in SF-90, 25% in SG-75, 50% in SG-50), among the water-soluble products of proteolysis in cheeses produced with no noticeable differences are observed with these drugs.

Thus, the use of liquid milk-clotting enzyme preparations "SG-75" and "SG-50" containing beef pepsin for the production of cheeses does not have a noticeable effect on the composition of proteolysis products in mature cheeses and makes it possible to obtain cheeses with quality, at the level of cheeses produced with rennet preparation.

Brief Description of Drawings

Figure 1 shows the appearance of the MPP: "SG-75" developed by VNIIMS (in the form of a concentrate and in the form of a working solution), a solution of the preparation "Naturen Stamix 1150 NB" (Chr. Hansen A / S, Denmark) and a solution of a dry preparation of rennet brand "SF-90", obtained by traditional technology (JSC "Moscow rennet plant", Moscow).

Figure 2 shows a diagram of the molecular weight distribution of water-soluble products of proteolysis in mature Dutch cheese after 30 days of storage, produced using a dry preparation of rennet brand "SF-90".

The figure 3 shows a diagram of the molecular weight distribution of water-soluble products of proteolysis in mature Dutch cheese after 30 days of storage, produced using a liquid enzymatic milk-coagulating preparation brand "SG-75".

Figure 4 shows a diagram of the molecular weight distribution of water-soluble products of proteolysis in mature Dutch cheese after 30 days of storage, produced using a liquid enzymatic milk-coagulating preparation of the brand "SG-50".

Figure 5 shows a diagram of the molecular weight distribution of water-soluble products of proteolysis in mature Dutch cheese after 30 days of storage, produced using the enzyme milk coagulant preparation "Naturen Stamix 1150 NB" (Chr. Hansen A / S, Denmark).

Claims (13)

1. A method for the production of a milk-clotting enzyme preparation of a combined composition, including grinding rennet, extraction of rennet with a solution of sodium chloride, separation of the liquid phase, filtration, characterized in that liquid preparations of rennet and beef pepsin are prepared: the extraction of rennet is carried out with 10-12% sodium chloride solution for 4-5 hours at a temperature of 34-40 ° C and periodic stirring, re-extraction is carried out with a 1.5-2 times mass of 10-12% sodium chloride solution, the resulting extracts are combined and acidified with a solution of molar hydrochloric acid concentration of 1 mol / dm ³ to acidity pH 4.60-4.80 units. pH, chymosin is activated in the rennet extract at a temperature of 20-25 ° C for 16-20 h, 0.4-0.6% of filter perlite is added to the activated extract and stirred for 0.5-1 h, the resulting mixture filtered under a pressure of 0.015-0.02 MPa at a temperature of 20-25 ° C using a filter board with a pore size of 0.2 μm, the active acidity in the filtered extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ , add 0.5-0.7% sodium benzoate, the weight concentration of sodium chloride in the extract is adjusted to 17-19%; extraction of beef pepsin is carried out with a solution of hydrochloric acid with an active acidity of 1.3-1.5 units. pH for 24-48 hours at a temperature of 4-20 ° C and periodic stirring, re-extraction is carried out, the resulting extracts are combined, the extract is finely purified by microfiltration in a tangential liquid flow on ceramic filter elements with a pore size of 0.2 μm, purified pepsin extract is concentrated by ultrafiltration on polymer membranes with a cut-off threshold for molecular weight of 20 kDa at a temperature not higher than 25 ° C, active acidity in the concentrated pepsin extract is set at 5.6-5.9 units. pH by adding a solution of sodium hydroxide with a molar concentration of 1 mol / dm ³ , add 0.5-0.7% sodium benzoate, the weight concentration of sodium chloride in the extract is adjusted to 16-18%; calculate the amount of added liquid rennet and liquid beef pepsin according to the formulas, which indicate the given composition of the complex preparation: - calculation of the proportion of liquid rennet introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

, where M is the proportion of the rennet preparation used in the volume of the produced batch of the preparation,%; D is the proportion of chymosin activity in the drugs produced (for the “SG-50” preparation: D = 0.5; for the “SG-75” preparation: D = 0.75); A _X1 - activity due to chymosin in the used batch of rennet, conv. units / cm ³ ; A _X2 - activity due to chymosin in the used batch of pepsin, conv. units / cm ³ ; A _P1 - activity due to pepsin in the used batch of rennet, conv. units / cm ³ ; A _P2 - activity due to pepsin in the used batch of pepsin, conv. units / cm ³ ; - the calculation of the proportion of liquid pepsin introduced into the mixture to obtain a drug with a given ratio of milk-clotting activity of chymosin and pepsin is carried out according to the formula:

, where R _G is the proportion of the pepsin preparation used in the volume of the produced batch of the preparation,%; liquid preparations of rennet and beef pepsin are mixed, a milk-clotting enzyme preparation of a combined composition is obtained, while the finished liquid complex preparation contains chymosin in an amount of 50 to 75%, and the rest is pepsin. 2. The method according to claim 1, characterized in that before concentrating the milk-clotting enzyme preparation of the SG-50 brand, containing 50% chymosin and 50% pepsin, is purified from ballast protein substances by microfiltration on polymeric membranes made of polysulfone with a cutoff threshold for molecular mass 300 kDa.

Images