A method for recovering l-histidine from an l-carnosine mother liquor

Abstract

The application belongs to the technical field of biological medicine, and particularly discloses a method for preparing L-histidine from L-carnosine mother liquor. The method mainly comprises the following steps: A. vacuum concentrating the L-carnosine mother liquor into a mother liquor concentrate, adding a strong acid, a strong base or an amide hydrolase into the mother liquor concentrate to hydrolyze the peptide component into a hydrolysate mainly containing amino acids; B. removing the acid, the base or the enzyme from the hydrolysate, and then concentrating; after the concentration, sample loading column chromatography is performed; C. sequentially using purified water and gradient concentration ammonia water for elution, collecting the eluate, and using HPLC for tracking detection of the purity of L-histidine in the eluate, and collecting the eluate with the purity of L-histidine of 94% to 99%; D. combining the eluate, vacuum concentrating the eluate into a solution with the weight concentration of L-histidine of 30 to 40%, and adding simple alcohol for crystallization; E. filtering the precipitated L-histidine crystals, washing the filter cake with simple alcohol, drying by suction and drying, and then the L-histidine crystals can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a method for recovering and preparing L-histidine from L-carnosine mother liquor. Background Technology

[0002] L-histidine is a semi-essential amino acid in the human body, participating in important physiological processes such as growth and development, antioxidation, and immune regulation. It is an important medicinal amino acid, used in intravenous and oral amino acid preparations for the treatment of heart disease, anemia, and gastrointestinal ulcers. L-carnosine is a chain-like dipeptide compound formed by the dehydration condensation of L-histidine and β-alanine. As a novel and highly effective antioxidant, L-carnosine is widely used in the food, pharmaceutical, and cosmetic industries, with a very broad application scope and market prospect.

[0003] Currently, industrial production of carnosine generally uses β-alanine and L-histidine as raw materials, employing either fully chemical or semi-enzymatic methods. Due to the inherent complexity of organic synthesis reactions and the diversity of products, the key raw material L-histidine cannot be completely converted into L-carnosine. Regardless of the production process, more than 40% of L-histidine cannot enter the final product as L-carnosine, but instead enters the L-carnosine production mother liquor as various impurities such as dipeptides and tripeptides. If the mother liquor is not recycled, the high proportion of residual L-histidine will enter the ecological environment as solid and liquid waste, which not only seriously wastes resources and increases environmental treatment costs, but also causes environmental pollution problems.

[0004] Therefore, effectively disposing of the large amount of mother liquor generated during the L-carnosine production process and recycling the high-value-added component L-histidine remaining in the mother liquor as much as possible can not only achieve resource recovery and reduce production costs, but also significantly reduce pollution and emissions, resulting in significant economic and social benefits. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low raw material conversion rate and low recovery rate in the L-carnosine synthesis process, and to provide a method for recovering and preparing L-histidine from L-carnosine mother liquor. The production process is simple and the raw material recovery rate is high, thereby achieving the goals of reducing production costs, recovering resources, and reducing the emission of waste.

[0006] The composition of L-carnosine mother liquor is complex. In addition to a large amount of alcohol solvent, it also contains peptides such as dipeptides and tripeptides, as well as solid components such as L-histidine, β-alanine, and salts. The alcohol solvent can be recovered and reused by distillation. However, the remaining valuable peptide components, histidine, and alanine are difficult to recover by simple physical methods. Chemical methods are required to hydrolyze the various peptide components with large molecular weights and unknown composition into free amino acids with smaller molecular weights for recycling.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing L-histidine from L-carnosine mother liquor includes the following steps:

[0009] A. Vacuum concentrate the L-carnosine mother liquor into a concentrated mother liquor, and add strong acid, strong base or amide hydrolase to the concentrated mother liquor to hydrolyze the peptide components into a hydrolysate mainly containing amino acids.

[0010] B. After removing acid, alkali or enzymes from the hydrolysate, concentrate it; after concentration, add the sample for column chromatography;

[0011] C. Elute sequentially with purified water and ammonia water of gradient concentrations, collect the eluent, and use HPLC to monitor the purity of L-histidine in the eluent. Collect the eluent with a purity of 94% to 99% for L-histidine.

[0012] D. Combine the eluents, concentrate under vacuum to a solution with an L-histidine weight concentration of 30-40%, and add a simple alcohol to crystallize;

[0013] E. Filter the precipitated L-histidine crystals, wash the filter cake with a simple alcohol, vacuum dry and then dry it.

[0014] In step A of this invention, the volume of the mother liquor concentrate is 14% to 30% of the L-carnosine mother liquor.

[0015] In step A of this invention, the strong acid is any one of hydrochloric acid and sulfuric acid, and the strong base is any one of sodium hydroxide, potassium hydroxide, and concentrated ammonia.

[0016] In step A of this invention, the hydrolysis temperature is 40–90°C, and the hydrolysis time is 3–5 hours.

[0017] In step B of this invention, the chromatography column used for sample loading and chromatography has a specification of 5cm×500mm and is made of fully activated strong acidic cation exchange resin.

[0018] In step C of this invention, when eluting with ammonia solution of gradient concentration, the concentration of the ammonia solution is 1%, and the elution flow rate is 150 ml / h.

[0019] In steps C and D of this invention, the pH of the eluent is 5 to 10.

[0020] In steps D and E of this invention, the simple alcohol is any one of methanol, ethanol, isopropanol, and butanol. Further, ethanol is preferred as the simple alcohol.

[0021] The L-histidine recovered from the L-carnosine mother liquor in this technical solution meets the raw material standards for L-carnosine, with a recovery rate of approximately 15% per batch. The L-carnosine process has been validated to produce L-carnosine that meets quality standards, thus enabling circular production.

[0022] The advantages and beneficial effects of this invention are as follows:

[0023] This invention can recover more than 98% L-histidine from L-carnosine mother liquor, achieving 100% material recovery, reducing costs, and creating a competitive advantage compared to traditional processes. It saves costs, generates economic benefits, and is environmentally friendly and easy to operate. Attached Figure Description

[0024] Figure 1 A process route diagram for the recovery and preparation of L-histidine from L-carnosine mother liquor. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0026] The specific embodiment describes the process route for recovering and preparing L-histidine from L-carnosine mother liquor as follows: Figure 1 As shown.

[0027] Example 1

[0028] 1000ml of L-carnosine stock solution was vacuum concentrated to a volume of 250ml, 20g of solid sodium hydroxide was added, the temperature was raised to 80-85℃, and hydrolysis was carried out for 4 hours (HPLC analysis showed that it contained about 30g of L-histidine). The pH value was adjusted to 2.0 with dilute HCl.

[0029] Take another 550g of strong acid cation exchange resin, fully activate it, and pack it into a 5cm×500mm chromatography column. Add the hydrolysate to the column, elute with 800ml of purified water, and then elute with 3000ml of 1% ammonia solution, controlling the elution rate at 150ml / h.

[0030] The eluent with pH values ​​of 6.5–9.5 was collected, and the purity of L-histidine in the eluent was monitored by HPLC. The eluents with L-histidine purity of 94%–99% were combined and concentrated under vacuum to a solution with an L-histidine weight concentration of approximately 35%. Ethanol was added to adjust the concentration to 80%, and L-histidine crystals precipitated. The crystals were filtered, washed with 50 ml of ethanol, and dried to obtain 21 g of white L-histidine crystals. The yield was calculated to be 70%, and the purity was 99.5%.

[0031] L-histidine can be used to produce L-carnosine by refeeding the feedstock. After the mother liquor is evaporated to dryness and ethanol is removed, it can be reused for the next batch of column elution.

[0032] Example 2

[0033] 1000ml of L-carnosine stock solution was vacuum concentrated to 140ml, 100g of concentrated hydrochloric acid was added, the temperature was raised to 90℃, and hydrolysis was carried out for 4 hours (HPLC analysis showed that it contained about 30g of L-histidine). The pH value was adjusted to 2.0 with ammonia water for later use.

[0034] Take another 550g of strong acid cation exchange resin, activate it, and pack it into a 5cm×500mm chromatography column. Add the hydrolysate to the column, elute with 800ml of purified water, and then elute with 3000ml of 1% ammonia solution, controlling the elution rate at 150ml / h.

[0035] The eluent with pH values ​​ranging from 6.0 to 9.5 was collected, and the purity of L-histidine in the eluent was monitored by HPLC. Eluents with L-histidine purity ranging from 94% to 99% were combined. The solution was concentrated under vacuum to a weight concentration of approximately 35% L-histidine, and then adjusted to 80% concentration with ethanol. L-histidine crystals precipitated, were filtered, washed with 50 ml of ethanol, and dried to obtain 24.4 g of white L-histidine crystals. The yield was calculated to be 81.3%, and the purity was 99.1%.

[0036] Example 3

[0037] 1000ml of L-carnosine stock solution was vacuum concentrated to approximately 140ml. 30g of amide hydrolase solution was added, and the temperature was adjusted to 40℃ for 4 hours of hydrolysis. During the hydrolysis process, the pH was adjusted to 8.5-9.0 with ammonia. After the hydrolysis reaction was completed, the enzyme was removed by filtration, and the pH was adjusted to 2.0 with hydrochloric acid for later use.

[0038] Take another 560g of strong acid cation exchange resin, activate it, and pack it into a 5cm×500mm chromatography column. Add the hydrolysate to the column, elute with 800ml of purified water, and then elute with 3000ml of 1% ammonia solution, controlling the elution rate at 150ml / h.

[0039] The eluent with pH values ​​ranging from 5.5 to 9.5 was collected, and the purity of L-histidine in the eluent was monitored by HPLC. Eluents with L-histidine purity ranging from 94% to 99% were combined. The solution was concentrated under vacuum to a weight concentration of approximately 35% L-histidine, and then adjusted to 80% concentration with ethanol. L-histidine crystals precipitated, were filtered, washed with 50 ml of ethanol, and dried to obtain 23.6 g of white L-histidine crystals. The yield was calculated to be 78.66%, and the purity was above 98.5%.

Claims

1. A method for recovering L-histidine from L-carnosine mother liquor, characterized in that... Specifically, the steps include the following: A. Vacuum concentrate the L-carnosine mother liquor into a concentrated mother liquor, and add strong acid, strong base or amide hydrolase to the concentrated mother liquor to hydrolyze the peptide components into a hydrolysate mainly containing amino acids. B. After removing acid, alkali or enzymes from the hydrolysate, concentrate it; after concentration, add the sample for column chromatography; C. Elute sequentially with purified water and ammonia water of gradient concentrations, collect the eluent, and use HPLC to monitor the purity of L-histidine in the eluent. Collect the eluent with L-histidine purity of 94% to 99%. D. Combine the eluents, concentrate under vacuum to a solution with an L-histidine weight concentration of 30-40%, and add a simple alcohol to crystallize; E. Filter the precipitated L-histidine crystals, wash the filter cake with a simple alcohol, dry it under vacuum and then dry it. In step A, the strong acid is any one of hydrochloric acid and sulfuric acid; the strong base is any one of sodium hydroxide, potassium hydroxide, and concentrated ammonia. In step B, the chromatography column used for sample loading and column chromatography has a specification of 5cm×500mm and the material is a fully activated strong acidic cation exchange resin. In steps C and D, the pH of the eluent is 5 to 10; In steps D and E, the simple alcohol is any one of methanol, ethanol, isopropanol, and butanol.

2. The method for recovering L-histidine from L-carnosine mother liquor according to claim 1, characterized in that... In step A, the volume of the concentrated mother liquor is 14% to 30% of the L-carnosine mother liquor.

3. The method for recovering L-histidine from L-carnosine mother liquor according to claim 1, characterized in that... In step A, the hydrolysis temperature is 40–90°C, and the hydrolysis time is 3–5 hours.

4. The method for recovering L-histidine from L-carnosine mother liquor according to claim 1, characterized in that... In step C, the concentration of the ammonia solution is 1%, and the elution flow rate is 150 ml / h.

5. The method for recovering L-histidine from L-carnosine mother liquor according to claim 1, characterized in that... The simple alcohol is ethanol.

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