阅读说明：本技术 一种水解氨基酸的制备方法 (Preparation method of hydrolyzed amino acid ) 是由 林金新 郭小雷 黄平 于 2019-10-09 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种水解氨基酸的制备方法。称取试样,放入水解管,向水解管中加入盐酸,密封后,放入110℃保温水解,充分反应后,盐酸使样品中蛋白质的肽键断裂,从而形成氨基酸。取出样品过滤洗涤,经缓冲液稀释,上清液进行保存待测。本发明方法简单,解决了氨基酸分析的前期准备工作设备要求高的问题。(The invention relates to the technical field of biology and discloses a preparation method of hydrolyzed amino acid. Weighing a sample, putting the sample into a hydrolysis tube, adding hydrochloric acid into the hydrolysis tube, sealing, putting the tube into a 110 ℃ thermal insulation hydrolysis tube, and after full reaction, breaking peptide bonds of proteins in the sample by the hydrochloric acid to form amino acid. Taking out the sample, filtering, washing, diluting by buffer solution, and storing supernatant to be tested. The method is simple, and solves the problem of high requirement on equipment for early preparation of amino acid analysis.)

1. A method for preparing hydrolyzed amino acid, which is characterized by comprising the following steps:

s1, weighing a test sample, namely accurately weighing a hydrolysis sample according to the content of the amino acid in the protein sample, so that the content of the amino acid taken out is 10 ~ 20 mg;

s2: putting the weighed sample into a hydrolysis tube, adding hydrochloric acid into the hydrolysis tube, and screwing a sealing cover;

s3: sealing the hydrolysis tube: firstly, filling nitrogen into the hydrolysis tube, then vacuumizing the hydrolysis tube for many times, and ensuring that the hydrolysis bottle is completely free of oxygen;

s4: hydrolyzing amino acid: placing the sealed hydrolysis tube in a constant-temperature drying box at 110 ℃, standing for 24-30 hours, fully reacting the sample in the hydrolysis bottle with hydrochloric acid, and breaking peptide bonds of protein in the sample by the hydrochloric acid to form amino acid;

s5: opening the tube to take liquid: taking the hydrolysis tube out of the drying box, placing the hydrolysis tube in a shade place, naturally cooling the hydrolysis tube, and unscrewing a sealing cover of the hydrolysis tube to pour out a hydrolyzed sample after the hydrolysis box is cooled;

s6, filtering and washing: filtering the substances in the hydrolysis tube, pouring the substances into a volumetric flask, and removing the residual impurities after the hydrolysis of the sample; washing the hydrolysis tube with ionized water, filtering the washed solution with filter paper, and pouring the filtered solution into a volumetric flask;

s7: evaporating to dryness and deacidifying: taking out the materials in the volumetric flask, placing the materials on a vacuum deacidification instrument for deacidification, and removing the materials until the materials are dried, wherein a little solid or trace stains are left at the bottom of the materials;

s8: buffering and dissolving: adding a sodium citrate buffer solution with the pH value of 2.2 into the deacidified sample, and dissolving the sample;

s9: taking out the supernatant: and taking out the supernatant after the sample is diluted by the buffer solution for storage.

2. The method of claim 1, wherein: the hydrolysis tube is a long-neck glass test tube, the opening of the test tube is threaded and provided with a cover, and the wall thickness of the hydrolysis tube is larger than 0.5 mm.

3. The method according to claim 1, wherein the deacidification temperature in step S7 is 60 ℃.

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of hydrolyzed amino acid.

Background

An amino acid is a compound in which a hydrogen atom on a carbon atom of a carboxylic acid is substituted with an amino group, and the amino acid molecule contains both amino and carboxyl functional groups. Amino acids, like hydroxy acids, can be classified as α -, β -, γ -, w-amino acids according to the position of the amino group attached to the carbon chain, but the amino acids obtained after proteolysis are all α -amino acids, and only twenty, which are the basic units constituting proteins.

Proteins and peptides can be identified based on amino acid composition analysis, and amino acid analysis can be used to determine the content of proteins, peptides and amino acids, and to determine atypical amino acids that may be present in proteins and peptides. Before amino acid analysis, proteins and peptides must be hydrolyzed to single amino acids. After hydrolysis of proteins and peptides, the amino acid analysis process is the same as that used for the analysis of free amino acids in other pharmaceutical preparations.

In the existing laboratory amino acid hydrolysis process, the flow is complex and the equipment requirement is high. Such as CN104419949A, in addition to acid-base treatment, an electrolytic treatment process requires electrochemical equipment. The amino acid hydrolysis method with standardization and low equipment requirement can greatly improve the applicability.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method of hydrolyzed amino acid, which solves the problems of complicated amino acid analysis and high equipment requirement.

In order to achieve the purpose, the invention provides the following technical scheme:

a method of preparing a hydrolyzed amino acid comprising:

s1, weighing a test sample, namely accurately weighing a hydrolysis sample according to the content of the amino acid in the protein sample, so that the content of the amino acid taken out is 10 ~ 20 mg;

s2: the sample is put into a hydrolysis tube, and hydrochloric acid is added: putting the weighed sample into a hydrolysis tube, adding hydrochloric acid into the hydrolysis tube, and screwing a sealing cover;

s3: sealing the hydrolysis tube: firstly, filling nitrogen into the hydrolysis tube, then vacuumizing the hydrolysis tube for many times, and ensuring that the hydrolysis bottle is completely free of oxygen;

s4: hydrolyzing amino acid: placing the sealed hydrolysis tube in a constant-temperature drying box at 110 ℃, standing for 24-30 hours, fully reacting the sample in the hydrolysis bottle with hydrochloric acid, and breaking peptide bonds of protein in the sample by the hydrochloric acid to form amino acid;

s5: opening the tube to take liquid: taking the hydrolysis tube out of the drying box, placing the hydrolysis tube in a shade place, naturally cooling the hydrolysis tube, and unscrewing a sealing cover of the hydrolysis tube to pour out a hydrolyzed sample after the hydrolysis box is cooled;

s6, filtering and washing: filtering the substances in the hydrolysis tube, pouring the substances into a volumetric flask, and removing the residual impurities after the sample is hydrolyzed; washing the hydrolysis tube with ionized water, filtering the washed solution with filter paper, and pouring the filtered solution into a volumetric flask;

s7: evaporating to dryness and deacidifying: taking out the materials in the volumetric flask, placing the materials on a vacuum deacidification instrument for deacidification, and removing the materials until the materials are dried, wherein a little solid or trace stains are left at the bottom of the materials;

s8: buffering and dissolving: adding a sodium citrate buffer solution with the pH value of 2.2 into the deacidified sample, and dissolving the sample;

s9: taking out the supernatant: and taking out the supernatant after the sample is diluted by the buffer solution for storage.

In step S7, the deacidification temperature was set to 60 ℃.

When the sample is weighed, the amino acid content of the sample needs to be calculated. It should be noted that there is some error in the measurement when the sample concentration is too low, and there is salt deposition to clog the reaction column when the sample concentration is too high (usually more than 20 nmol/20. mu.L). Therefore, the estimation of the concentration of the sample and the selection of the proper sample weighing are of great significance to the measurement result and the normal use of the instrument.

In the process of evaporating to dryness and deacidifying, the high-concentration hydrochloric acid contained in the reacted materials is removed, so that the corrosion of the solution containing the high-concentration hydrochloric acid to a machine is avoided when the solution is detected on the machine.

Compared with the prior art, the preparation method of the hydrolyzed amino acid provided by the invention has the following beneficial effects:

1. the method is simple, has few operation steps, makes the early preparation of the hydrolyzed amino acid simple and easy to operate, does not need an electrolysis process, and solves the problem of high requirement on equipment for amino acid analysis.

2. According to the preparation method of the hydrolyzed amino acid, the hydrolysis pipe with the pipe wall larger than 0.55mm is used, the pipe explosion phenomenon possibly occurring in the hydrolysis process is avoided, and the efficiency and the safety in the hydrolysis process are improved.

3. According to the preparation method of the hydrolyzed amino acid, the high-purity hydrochloric acid contained in the hydrolyzed product is removed by deacidifying the hydrolyzed product, so that the corrosion of the machine caused by the high-concentration hydrochloric acid contained in the solution during the machine-up detection is avoided.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.