SU698980A1 - Method of preparing mixture of aminoacids - Google Patents

Description

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The present invention relates to an improved process for the preparation of a mixture of phenylalanine-free amino acids used for therapeutic nutrition.

Currently, therapeutic amino acid mixtures are prepared by acid hydrolysis of protein, in particular cattle blood or casein, with hydrochloric or sulfuric acids, followed by neutralization of the acid by one of the known methods and removal of phenylalanine from their demineralized hydrolysates by treatment with activated carbon. The content of phenylalanine on the requirements of the temporary pharmacopoeial monograph should not exceed 0.1% of the weight of dry substances.

A known method of obtaining therapeutic mixtures of amino acids by purifying a protein hydrolyzate from phenylalanine using activated carbon by heating the hydrolyzate with carbon at 75-80 ° for 1 hour.

The method of removing phenylalanine using activated carbon requires pretreatment of carbon, which consists in treating the latter with acetic or nitric acid with

followed by washing the coal of traces of acid, which causes a high consumption of water. In all cases, coal is used once, since its regeneration is long, laborious and economically unfeasible. Without such carbon pre-treatment with acids, the loss of valuable amino acids increases to 45%.

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In this connection, the closest to the proposed method according to the technical essence of the solution is a method of obtaining a therapeutic mixture of amino acids (hypofenate), by treating the blood hydrolyzate dehydrated with ananite polycondensacin and formaldehyde, which is an acidic hydrolyzate jjpOBH cattle, by acidifying it to a pH of 4.3 with acetic acid, followed by hydroLysate treated with carbon activated in a vacuum of 2J and boiling water at

5 70-75 s and the selection of the target product by drying.

The disadvantages of the prototype are.

1. Use of an acidic hydrolyzate only after its preliminary demineralization by any of the known routes, which is a separate stage of processing the hydrolyzate and often entails the need to immediately dry the demineralized hydrolyzate due to the possibility of rapid microbial contamination. 2. The loss of valuable amino acids (reduction of the yield of the target product to 40-45%) at the stage of demineralization, as well as during the processing of the demineralized hydrolyzate with coal. 3. Insufficient purification of the target product from phenylalanine and its peptides. The content of phenylalanine in the therapeutic mixture of amino acids, determined according to the method described in the prototype, exceeded 0.1% and was 0.24-0.35%, and the content of phenylalanine in peptides was up to 10%. The aim of the invention is to eliminate these disadvantages, namely: increasing the purity and yield of the target drug - a therapeutic mixture of amino acids. This goal is achieved by subjecting casein acid hydrolyzate to demineralization on macroporous polycondensation yomite, and the resulting demineralized hydrolyzate is then passed through sulfonic cation exchanger in the hydrogen form, followed by passing an aqueous solution of amino acids through the ionite of the polycondensation type. hold. process. 1. Acidic casein hydrolyzate is passed through a column, with a microporous polycondensation-type anion exchange resin in hydroxyl form, with a ratio of hydrolyzate and anion exchange resin (1: 2.2-1: 2.5) 0, followed by washing with two volumes of distilled water. 2. The hydrolyzed carbonate passed through the anion exchange resin then enters the strongly acid sulfonic cation; in the hydrogen form with subsequent washing of the cation exchanger with distilled water. 3. Desorption of amino acids from cation will be carried out with 4% ammonia solution until complete displacement of amino acids. 4. Mating the resulting aqueous ammonia solution of amino acids is carried out to dryness to more completely remove ammonia, 5. The dry amino acid residue is dissolved in distilled water to obtain 4.5-6.0% solution, 5, the resulting amino acid solution is passed through a macroporous anionite of a polycondensation type, washed from alkali with water to pH 8.9–9, in a ratio of solution and anion exchanger (1: 1.4), followed by washing with water and selecting fractions with a full UV spectrum DzBvnmG, 11, which indicates a high heat of purification from phenyl anina, 7; To obtain the desired product, the selected ninhydrin-positive amino acid fractions are dried under vacuum to dryness at temperatures above 37-42 ° C. Passing the non-demineralized hydrolyzate through a column with macroporous anion exchanger — the product of m-phenylenediamine and formalin with resorcinol in hydroxyl form — allowed not only er “to neutralize the hydrolyzate, but also to remove all pigment, humic impurities, to remove the pigment, to the hydrolyzate; its content in the hydrolyzate, as well as remove high molecular weight peptides. To separate the mixture of amino acids in the hydrolyzate from related impurities of such components of the hydrolyzate as sugars and amino sugars, as well as anions of organic acids and metal cations / in particular iron cations falling into the hydrolyzate from the apparatus, the hydrolyzate is passed through a strongly acidic sulfonic cation in hydrogen form . The more water is used for washing to atioion, the less it remains in the mixture of C1mino acids of impurities. Removal of ammonia amino acids from a water-ammonia mixture is necessary so that the purified amino acid mixture has a minimum ammonia content, before passing the mixture to an anion exchanger and the ph value of the solution does not exceed 6-7, the final removal of phenylalanine and its peptides occurred at the stage of processing the mixture amino acids when passing it through a column with macroporous anion exchanger IA - 1p in gilroksilny form. Example 1, 100 ml of hydrochloric acid hydrolyzate, casein with a pO of 0.2, the content of chlorine ions. 6.5%; chromaticity at 460 nm; 0.44; amino acid content according to amino acid anilysis, 60.07%, including phenylalanine. - 1.7%, tyrosine - 2.5% and tryptophan - 0.04%, passed through a 34x400 mm column filled with 220 ml of anion exchange resin IA-1r in hydroxyl form. The transmission rate was 6-9 ml / cm h, the anion exchanger was washed from alkali with water until pH 8.9-9.2, the amino acids were collected by fractional 25 ml in the ninhydrin-negative fraction. After finishing the transfer of the hydrolyzate, the anion exchanger was washed with 250 ml of distilled bath water, continuing to collect the amino acid fractions up to pH 1, b. The fractions containing amino acids with a pH value of not less than 1.5 were combined and the residual chlorine ion content was determined. It was 07%, which corresponded to a pK value of 3.2–3.6. The content of aromatic amino acids, which is determined by the absorption of a spectrophotometer at a wavelength of 258 nm, decreased compared to the initial 25–33 pb and the chromaticity was 0.02– 0.06 with a green light filter. The hydrolate passed through the anion exchange resin (250-300 ml) then entered the column with strongly acidic sulfonic cation exchanger (100 ml) at a rate of 0.5 MP / cm.h, the cation resin was washed with 250 - 300 ml of distilled water. The displacement of amino acids from cation exchanger was carried out with 4% aqueous ammonia, collecting ninhydrin-positive fractions. The ammonia fraction of amino acids was evaporated under vacuum until complete removal of ammonia was added to the flask with distilled water and again evaporated to dryness under vacuum, and rotated the Evaporator in a water bath at 3742 °. The dry residue of acid 6.45, 5 g was dissolved in distilled water (100 ml) and passed through a column with anion exchange resin IA-1p in hydroxyl form (28x49 mm, 140 ml) at a rate of 6-9 ml / cm.h. The anion exchanger must be washed to a pH of 8.9 to 9.0 and not absorbed (at 258 nm). After completing the passage of the amino acid solution, the anion exchanger was washed with distilled water of 250-300 ml. Amino acids were collected, compartment fractions with absorption D25 0.1. To obtain the desired product, the selected ningmdr-positive fractions with no absorption at 258 nm (except for the above values) were combined and. dried in vacuo at 37–42 in a water bath; the amino acid recovery from the initial content of casein from the hydrolyzate was 60–55%, with a phenylalanine content of less than 0.1%. Technical and economic indicators. The described method allows to significantly increase the purity of the therapeutic mixture of amino acids in the target product, the content of phenylalanine does not exceed 0.1%, which is 2 to 3 times higher than the purity of phenylalanine compared to the prototype. In addition, it is especially important that phenylalanine peptides do not remain in the mixture of amino acids obtained by the proposed method, which is proved by acid hydrolysis of the desired product and subsequent amino acid analysis of the hydrolyzate obtained. In addition, the yield of the target product from the initial content in casein hydrolytic acid is 5560%, which is 25-30% higher than in the product obtained by the prototype. The composition of the target product recovered by the described method and the product after acid hydrolysis are presented in the table.

6.56

ova acid

2.75

2.92

1.86 0.26

0.65 0.90

1.10 0.26

Claims (1)

0.60 9.52 7.50 9.50 Claims of Invention A method of obtaining a mixture of amino acids from an acidic protein hydrolyzate by demineralizing it with an anion exchanger, followed by removing phenylalanine from the hydrolyzate and drying the product under vacuum in a vacuum that differs from the purpose of improving the quality and yield of the target product as anion exchange resin is macroporous polycondensation Continued table. and the resulting demineralized hydrolyzate is then passed through sulfonic cation resin in the hydrogen form, followed by passing an aqueous solution, of amino acids through the ion exchanger of the polycondensation type. Sources of information taken into account in the examination 1.PatentPolshi 51.391, 1966, cl. DA h 2 / 04.2. USSR author's certificate No. 271720, cl. A 61 K 34/15, 1972 (prototype).