RU2467068C1 - Method of producing milk-clotting enzyme preparation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: raw material is prepared and homogenised. An extractant is added, said extractant being an aqueous solution acidified with hydrochloric acid to pH 1.5-2.0 with addition of 7.5-10.0% ammonium sulphate. Extraction is then carried out. The obtained mixture is settled until separation thereof into a liquid phase and a residue. The milk-clotting enzyme is separated and then processed.

EFFECT: invention reduces the time for producing the enzyme preparation, increases its specific milk-clotting activity and purity, and also reduces its loss in industrial production conditions.

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Description

The invention relates to methods for the production of enzyme preparations, mainly from raw materials of animal origin, and can be used in enterprises for the processing of animal raw materials, for example, for the preparation of milk-clotting enzyme preparations used in cheese making.

A known method of producing a milk-clotting enzyme preparation (beef pepsin), comprising preparing the raw material, homogenizing it, introducing an extractant and extracting the raw material followed by precipitation of the enzyme (see Technological instructions for the production of food beef pepsin to TU 9219-560-00419779-2000, - 2001 - С.1-7, OJSC "Moscow rennet plant"). When implementing this method, an aqueous HCl solution is used as an extractant. The product is obtained in satisfactory quality, however, even with double extraction with an aqueous HCl solution, the process of autolysis (self-digestion) of abomasum tissue develops and a large amount of mucus forms, which leads to significant loss of the enzyme in industrial production, since during filtration part of it remains in the extracted mucous mass and together disposed of with it.

There is also a known method for the production of a milk-clotting enzyme preparation, which involves preparing the feedstock, homogenizing it, introducing extractant into the homogenized raw materials, extracting, settling the resulting mixture until it is separated into a liquid phase and precipitate, isolating the milk-clotting enzyme and its subsequent processing (see RF patent No. 2392320 , IPC ⁷ C12N 9/00, claimed 04.05.2008, published 20.06.2010).

This technical solution is the closest to the claimed combination of essential features (prototype).

According to the prototype, ammonium sulfate is introduced into the extracted mass. This allows you to divide it into a liquid phase with the main part of the enzyme and sediment, followed by isolation of the enzyme. However, when implementing this method, the time to obtain the finished extract remains significant (extraction must first be performed sequentially, then salt addition and sedimentation of the mixture). In addition, at the extraction stage, prior to the introduction of ammonium sulfate, the process of autolysis has already taken place and mucus has formed, which connected part of the extractant with the enzyme contained in it and converted it into losses. For the same reason, the effect of increasing the specific milk-clotting activity from adding ammonium sulfate to the extractant is lost and the time for subsequent filtration increases.

The technical result of the present invention is the creation of such a method for the production of milk-clotting enzyme preparation, which would reduce its production time in industrial production, increase its specific milk-clotting activity and purity, as well as reduce its loss.

The technical result is achieved by the fact that in the method for the production of a milk-clotting enzyme preparation, which includes preparing the feedstock, homogenizing it, introducing an extractant into the homogenized raw material, extracting, settling the resulting mixture until it is separated into a liquid phase and precipitate, isolating the milk-clotting enzyme and its subsequent processing, according to of the invention before introducing the extractant into the homogenized feedstock, ammonium sulfate in an amount of 7.5-10% is added to the extractant.

The introduction of ammonium sulfate in the extractant in an amount of 7.5-10% before introducing the extractant into the homogenized feedstock leads to the fact that the extraction process proceeds simultaneously with the flocculation process (in this case, precipitation in the form of dense lumps) of mucus without transferring part of the extractant with the waste enzyme contained in it, which increases the specific milk-clotting activity of the extract, allows more efficient separation of the enzyme-containing extract during subsequent filtration (the extraction and filtration time are juice aschaetsya), the volume of filtrate increases and the loss of the enzyme is minimized.

The method is as follows.

Prepared in a known manner, the feedstock, for example, the mucous membranes of the stomachs of mammals, for example cattle, are homogenized in a known manner. Then, an extractant (acidified with hydrochloric acid to pH = 2.0-2.5 aqueous solution) is introduced into the homogenized feedstock, into which 7.5-10% ammonium sulfate is added before it is introduced into the homogenized feedstock. Under the influence of an extractant with ammonium sulfate, mucus formed during the process (the product of autolysis - self-digestion of abomasum tissue) flocculates (precipitates in the form of dense lumps), and mechanical impurities and high molecular weight ballast proteins precipitate. With the specified amount of ammonium sulfate, the enzyme does not precipitate, but remains in the liquid phase.

The introduction of ammonium sulfate in the extractant at a concentration of less than 7.5% does not completely precipitate mucus, mechanical impurities, and ballast proteins, and at a salt concentration of more than 10% it leads to the precipitation of part of the enzyme and its loss.

After completion of the extraction, the mixture is separated into an extract and a precipitate, using one of the known methods (filtration, centrifugation). The enzyme-containing extract is concentrated by ultrafiltration to the desired value of milk-clotting activity and further processing is carried out to the stage of commercial production (milk-clotting enzyme preparation). Sludge is disposed of.

Examples of the method for the production of milk-clotting enzyme preparation.

Example 1. Prepared homogenized raw materials from the mucous membranes of the stomachs of mammals. In parallel with this, an extractant was prepared (acidified with hydrochloric acid to a pH = 2.0-2.5 aqueous solution). Then the extractant was introduced into the prepared raw materials at a ratio of raw materials: extractant = 1: 5) (control sample of the prototype).

According to known methods, the mixture was stirred, extracted, and the specific milk coagulation activity, the volume of the obtained extract, the total yield of milk coagulation activity, optical density at a wavelength of 280 nm, and relative purity were determined in the obtained extraction mixtures.

Example 2. Prepared homogenized raw materials from the mucous membranes of the stomachs of mammals. In parallel with this, an extractant was prepared (acidified with hydrochloric acid to a pH = 2.0-2.5 aqueous solution). Before introducing the extractant into the homogenized feed, 5% ammonium sulfate was added to the extractant (less than the declared limits). Then, the extractant with added ammonium sulfate was introduced into the prepared raw material.

By known methods, the mixture was stirred, extracted and the characteristics of Example 1 were determined in the obtained extraction mixtures.

Example 3. Prepared homogenized raw materials from the mucous membranes of the stomachs of mammals. In parallel with this, an extractant was prepared (acidified with hydrochloric acid to a pH = 2.0-2.5 aqueous solution). Before introducing the extractant into the homogenized feed, 7.5% ammonium sulfate was added to the extractant (lower limit of the declared limits). Then, the extractant with added ammonium sulfate was introduced into the prepared raw material.

By known methods, the mixture was stirred, extracted and the characteristics of Example 1 were determined in the obtained extraction mixtures.

Example 4. Prepared homogenized raw materials from the mucous membranes of the stomachs of mammals. In parallel with this, an extractant was prepared (acidified with hydrochloric acid to a pH = 2.0-2.5 aqueous solution). Before introducing the extractant into the homogenized feed, 10% ammonium sulfate was added to the extractant (the upper limit of the declared limits). Then, the extractant with added ammonium sulfate was introduced into the prepared raw material.

By known methods, the mixture was stirred, extracted and the characteristics of Example 1 were determined in the obtained extraction mixtures.

Example 5. Prepared homogenized raw materials from the mucous membranes of the stomachs of mammals. In parallel with this, an extractant was prepared (acidified with hydrochloric acid to a pH = 2.0-2.5 aqueous solution). Before introducing the extractant into the homogenized feed, 12.5% ammonium sulfate was added to the extractant (exceeding the upper limit of the declared limits). Then, the extractant with added ammonium sulfate was introduced into the prepared raw material.

By known methods, the mixture was stirred, extracted and the characteristics of Example 1 were determined in the obtained extraction mixtures.

The results of examples 1-5 are presented in the Table.

Table Example No. and content (%) of ammonium sulfate in the extractant. Specific milk
total activity (conventional units / ml) of extraction mixtures The volume of extract obtained (ml) Optical density at a wavelength of 280 nm (D280) Relative purity, conventional units / D280 (% compared to control) The total yield of milk-clotting activity, conventional units (% yield compared to control) No. 1 (0%) (control) 2112 1350 19.7 107.2 (100%) 2851200 (100%) No. 2 (5%) 2206 1480 17.1 129.0 (120%) 3264880 (115%) No. 3 (7.5%) 2416 1670 16,0 151.0 (141%) 4034720 (142%) No. 4 (10%) 2538 1750 14.3 177.5 (166%) 4,441,500 (156%) No. 5 (12.5%) 2079 1760 12.9 161.2 (150%) 3659040 (128%)

The results presented in the Table indicate that the introduction of ammonium sulfate in the extractant in the range of 7.5% -10% allows you to get the extraction mixture with the highest specific milk-clotting activity; effectively separate the extract and increase the total enzyme yield, compared with the control, by 42% and 56%, respectively, and also increase the purity of the enzyme, compared with the control, by 41% and 66%, respectively.

Thus, the proposed method for the production of milk-clotting enzyme preparation can reduce the time to obtain it, increase its specific milk-clotting activity and purity, as well as reduce its loss in industrial production.

Claims (1)

A method for the production of a milk-clotting enzyme preparation, comprising preparing a feedstock, homogenizing it, introducing an extractant into a homogenized raw material, extracting, settling the resulting mixture until it is separated into a liquid phase and a precipitate, isolating a milk-clotting enzyme and its subsequent processing, characterized in that before introducing the extractant into Ammonium sulfate in the amount of 7.5-10% is added to the extractant homogenized feedstock.