Method of production of casamino asids

FIELD: bioengineering; genetic engineering; medicine; methods of production casamino acids.

SUBSTANCE: the invention is pertaining to the field of bioengineering, genetic engineering, medicine, in particular, to the methods of production of components for nutrient mediums from hydrolysates of animal protein. The invention offers the method of production of casamino acids by the method of the gel permeation chromatography of the hydrolyzed crude acid casein with the contents of the general nitrogen - 0.7-0.95 g in 100 ml of the solution and concentration - 6-10 % on Sefadex G-15, eluating by a distilled water of fractions of an eluate, selection of the active fractions of an eluate by a spectophotometery of portions of the eluate (D254), evaporation of the active fractions under vacuum at the temperature of no more than 55°C. The method allows to simplify the process of production of casamino acids, to reduce its cost and also to obtain casamino acids possessing the high growth- stimulating activity.

EFFECT: the invention ensures simplification of the process of production of casamino acids, reduction of its cost and also production of casamino acids possessing the high growth- stimulating activity.

2 cl, 2 dwg, 1 tbl, 1 ex

 

The invention relates to biotechnology, genetic engineering and medicine, in particular to methods of producing components for nutrient media of animal protein hydrolyzate.

A known method of separation of active fractions from enzymatically hydrolyzed casein by gel-chromatography on Sephadex G-25 and drying by sublimation (Ibrahim Salam A. Hydrolysis by trypsin Kappa casein of cow's milk to obtain growth stimulants Bifidobakterium longum. Chem. Environ, Technol. Lebensm. - 1994. - 16, No. 3-4. Pp.69-72).

Also known is a method of obtaining Kazimirovich acids by separating the active fractions, including dual extraction dry cleaned, acid-hydrolyzed casein ethanol, distribution chromatography on paper, gel-penetrating chromatography on Sephadex G-15, then G-10, crystallization by evaporation in vacuum (Tcarr, Williams and other isolation and Purification of substances, substituting nicotinic acid, hydrolyzed casein. Journal of Dairy industry, T, No. 4, April 1983. S-756).

The disadvantages of the known methods is the complexity of the technological process and its use for analytical purposes only.

A method of obtaining Kazimirovich acids by separating the active fractions of acid-hydrolyzed casein by gel-chromatography with subsequent evaporation e is enta, moreover, gel-chromatography is subjected to a solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g in 100 ml solution (EN 95112551, epubl, prototype)

This method of obtaining Kazimirovich acids characterized by its simplicity and low cost, however, the functional activity of the obtained product, depending on the characteristics of the original product, type of eluent, brand cephadex, as well as the mode of evaporation of eluent is not stable, and in some cases quite low.

The problem solved by the claimed invention is to develop a simple and cheap method of obtaining Kazimirovich acids with high growth stimulating activity.

Put in the invention the problem is solved in the following way.

Method for obtaining Kazimirovich acids, namely, that the solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g per 100 ml of solution and concentration of the acid-hydrolyzed casein 6-10% is subjected to gel chromatography on Sephadex G-15, elution with distilled water followed by selection of the active fractions of the eluate and evaporation under vacuum.

A solution of the crude acid-hydrolyzed casein in distilled water with a concentration of 6-10% and the total nitrogen content of 0.7 to 0.95 g in 100 ml RA is down is a mixture of protein and fractions of other impurities, which by gel-chromatography on Sephadex G-15 can be divided into discrete protein fractions and fractions of other impurities, so the solution of acid-hydrolyzed casein as the original product to receive Kazimirovich acids, does not require pre-treatment by re-peated extraction, re-filter, which greatly simplifies the technology of obtaining Kazimirovich acids and reduces the cost of the final product.

The selection of the active fractions of the eluate corresponding to Kazarinova acids, produced by spectrophotometry portions of the eluate (D254), since the chromatographic column is configured with the complete absence of light absorption at λ254 nmreceive portions of the eluate.

The parameters characterizing the initial solution of the crude acid-hydrolyzed casein and appropriate 6-10% concentration of the total nitrogen content of 0.7 to 0.95 g in 100 ml of solution are optimal.

Experimental data confirm that the increase in the content of total nitrogen in the source solution and the increase in the concentration of acid-hydrolyzed casein in it, make it sticky and unsuitable for the separation into fractions by gel-chromatography, reducing the same concentration and nitrogen content reduces the output Kazimirovich acids.

Evaporation of portions of the eluate provided the active fractions of acid-hydrolyzed casein, produce at the temperature of the eluate no more than 55°because the temperature rise affects the functional activity of the final product, in particular, leads to lower growth stimulating properties Kazimirovich acids. It is known that cephalexih different brands can be obtained different results.

Brand Sephadex G-15, using distilled water as eluent, and the original solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g per 100 ml of solution and concentration of 6-10% allow exactly this combination of characteristics to obtain portions of the eluate containing the maximum number of amino acids and peptides. These results were obtained by experiment.

The invention is illustrated in the drawings.

1 shows a diagram of an installation for implementing this method, figure 2 is a chromatogram of the separation of the eluate fractions.

The installation for carrying out the proposed method is a sealed system including a flow indicator eluent And the receiver pressure gauge, metering chamber D, chromatographic column E, the collector of fractions F, G spectrophotometer, timer fractions I, Transcriber N, which are technologically connected.

1,2,3,4,5,6,7 and 8 - valve, by which the presence or absence of technologically the links between modules And, ...N setup for performance stages of the claimed process of obtaining Kazimirovich acids.

The method of obtaining Kazimirovich acids include the following:

gel chromatography of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g per 100 ml of solution and concentration of 6-10% on Sephadex G-15;

- elution with distilled water fractions of the eluate;

- selection of the active fractions of the eluate by spectrophotometry portions of the eluate (D254);

- evaporation active fractions of the eluate under vacuum and at a temperature of not more than 55°C.

The claimed method is as follows. In the metering chamber D is fed under pressure a solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g 100 ml and the concentration of 6-10% and by eluting solution - distilled water - displace it in a chromatographic column that is the Indication and selection of the active fractions of the eluate is done using spectrophotometry receive portions of the eluate. The manifold F produces automatic sampling fractions, the spectrophotometer is G - continuous recording of the optical density of the portions of the eluate, and the recorder N draws a chromatogram of the separation of the eluate fractions. Select the tube with portions of the eluate corresponding to the active fractions identified by the absorption of light at trago a specific wavelength, which are then subjected to drying under vacuum.

An example of a specific implementation.

For gel chromatography used a glass chromatographic column, which was filled with Sephadex G-15.

As an eluting solvent used distilled water (pH1-2Oc=7,0). The column was considered ready to work with the complete absence of light absorption at λ254 nmreceive portions of the eluate.

A solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g per 100 ml of solution and concentration of 6-10% served in the metering chamber D through an eluting solvent is distilled water under a pressure of 0.5 kgf/cm2displace it in a chromatographic column, which provides the eluent flow 6 ml/min

The elution of the active fractions of the eluate produced in the above mode, using spectrophotometry receive portions of the eluate (D254) to build a chromatogram of the separation of the eluate fractions (figure 2).

Containing the active fraction portion of the eluate, identified by the absorption of light in λ254 nmwere combined and then dried under vacuum at a temperature not exceeding 55°C.

The yield of dried active fractions of the eluate is Kazimirovich acids was 90%. Kasuminome acid is a homogeneous white powder C is the ETA. The composition of a mixture of amino acids and peptides with a molecular weight of up to 2200 D, the presence of which is due to growth-stimulating activity Kazimirovich acids. The total nitrogen content of 12%, amino nitrogen - 0.7 percent.

Stimulating growth of the quality of the received Kazimirovich acids was determined by introducing them to the composition of the nutrient medium and growing the test cultures of microorganisms.

The ability to stimulate the growth of microorganisms (auxotrophic mutants of E. coli) was estimated by the values of optical density (D540-560 nm) culture of cells in nutrient media M-9 with the content Kazimirovich acids that make up 1%, obtained by the claimed method, compared with the values of optical density (D540-560 nm) cell culture in a nutrient medium M-9 with 1% Kazimirovich acids firms "Difco", because the method of obtaining kazeminejad acids prototype, depending on the specific conditions of its implementation allows to obtain a final product with unstable characteristics, while kasuminome acid company "Difco" are characterized by very high growth stimulating activity. Therefore, the comparative characteristic growth stimulating ability Kazimirovich acids, obtained by the claimed method, and Kazimirovich acids firms "Difco" may indicate the task - the development of the e simple and cheap method of obtaining Kazimirovich acids, high growth-stimulating activity.

Measurement of the optical density of the nutrient medium were performed on fotoelektrokalorimetry at a wavelength of 540-560 nm in a cell with a layer thickness of 10 mm

According to the law of the Bouguer-Lambert-Bera expressing the relationship between the transmittance of the solution and its concentration, the higher the concentration of the cell culture organisms in a nutrient medium, the higher its growth-stimulating activity.

Comparative characteristics of growth-stimulating activity Kazimirovich acids, obtained by the claimed method, and Kazimirovich acids firms "Difco" is presented in the table.

The claimed method of obtaining Kazimirovich acids characterized by its simplicity and low cost of the final product with high functional activity and allows you to get kasuminome acid not only for analysis but also to establish industrial production, for example, enterprises producing hydrolyzed casein acid, which, bypassing the stage ion-exchange treatment, immediately subjected to gel chromatography.

1. The method of obtaining Kazimirovich acids by separating the active fraction from the solution of the crude acid-hydrolyzed casein with a total nitrogen content of 0.7 to 0.95 g in 100 ml solution by gel-chromatography with subsequent you what Arianism eluent, characterized in that the gel chromatography is carried out on Sephadex G-15 using as eluent distilled water, the concentration of the crude acid-hydrolyzed casein in solution take 6-10%, and evaporation of eluent is carried out at a temperature of not more than 55°C.

2. The method according to claim 1, characterized in that the separation of active fractions produced by spectrophotometry portions of the eluate (D254).



 

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