阅读说明：本技术 一种高堆积密度的l-缬氨酸及其制备方法和其应用 (High-bulk-density L-valine and preparation method and application thereof ) 是由 马祥亮 刘树蓬 刘磊 于 2020-06-10 设计创作，主要内容包括：本发明涉及一种高堆积密度的L-缬氨酸及其制备方法和其应用,包括下述步骤,向待纯化的L-缬氨酸溶液中加入式Ⅰ所示结构的助晶剂,将其均匀混合后,浓缩,析晶,分离,收集固体,干燥,即得,其中,R含有一个羧基。本发明制得了高堆积密度的L-缬氨酸晶体,显著提高了L-缬氨酸的质量和收率,降低其成本。(The invention relates toA process for preparing high-stacking-density L-valine includes such steps as adding crystallizing aid with structure shown in formula I to L-valine solution to be purified, mixing, concentrating, crystallizing, separating, collecting solid, drying, wherein R contains a carboxyl group. The invention prepares the L-valine crystal with high bulk density, obviously improves the quality and yield of the L-valine and reduces the cost thereof.)

1. A high-efficiency purification method of L-valine comprises the following steps of adding a crystallization promoter with a structure shown in a formula I into an L-valine solution to be purified, uniformly mixing, concentrating, crystallizing, separating, collecting solids, and drying to obtain the L-valine,

wherein R contains a carboxyl group.

2. The method of claim 1, wherein R comprises at least two linear methylene structures.

3. The method according to any one of claims 1-2, wherein the crystallization promoter is selected from any one of aspartic acid, adipic acid, glutamic acid, or a combination thereof.

4. The method of any one of claims 1-3, wherein the seeding agent: the molar ratio of L-valine is 1:270-1:70, preferably 1:250-1: 120.

5. The process according to any one of claims 1 to 4, wherein the L-valine solution to be purified has a pH of from 3.0 to 5.0, preferably from 3.5 to 4.5.

6. The process according to any one of claims 1 to 5, wherein the crystals of L-valine produced have a bulk density of 0.60g/mL or more, preferably 0.80g/mL or more.

7. A high-bulk density L-valine crystal having a bulk density of 0.60g/mL or more, preferably 0.75g/mL or more.

8. L-valine crystals according to claim 7, characterized in that the purity of the L-valine crystals is 99.0% or more, preferably 99.7% or more.

9. Crystals of L-valine according to any one of claims 7 to 8, characterized in that the crystals of L-valine produced have a water content of not more than 0.13%, preferably not more than 0.19%.

10. Use of the L-valine crystal produced by the process according to any one of claims 1 to 6 or the L-valine crystal according to any one of claims 7 to 8 for producing any one of foods, food additives, pharmaceuticals, pharmaceutical intermediates, pharmaceutical raw materials, nutritional supplements, amino acids, feeds, and feed additives.

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to L-valine with high bulk density, a preparation method and application thereof.

Background

The method for producing L-valine by microbial fermentation has the advantages of bio-based raw materials, high-efficiency conversion, mild reaction, environmental protection, economy and the like, and is widely applied to the industry. CN110643547A discloses a Brevibacterium flavum producing L-valine and a method for producing L-valine by using the same. However, the valine crystal prepared by the existing method has the defects of low bulk density, low purity, no contribution to production, packaging and transportation, high cost and the like. For this reason, it is required to develop a more efficient purification method for L-valine crystals.

Disclosure of Invention

The invention aims to provide a high-efficiency L-valine purification method which comprises the following steps of adding a crystallization aid with a structure shown in a formula I into an L-valine solution to be purified, uniformly mixing the crystallization aid with the L-valine solution, concentrating, crystallizing, separating, collecting solids, drying to obtain the L-valine,

wherein R contains a carboxyl group.

In a preferred embodiment of the present invention, R has at least two or more linear methylene structures.

In a preferable technical scheme of the invention, the L-valine solution to be purified is selected from any one of an L-valine fermentation clear liquid prepared after pretreatment and a solution prepared from an L-valine solid to be purified.

In the preferable technical scheme of the invention, the pretreatment comprises the steps of degerming, desalting, removing mixed acid and concentrating.

In a preferred embodiment of the present invention, the concentration of the solution of L-valine to be purified may be any concentration as long as the crystallization operation is performed by adding a crystallization aid thereto to obtain crystalline particles of L-valine.

In the preferred technical scheme of the invention, the concentration of the L-valine solution to be purified is more than or equal to 40g/L, preferably more than or equal to 50g/L, and further preferably more than or equal to 60 g/L.

In a preferred embodiment of the present invention, the crystallization assistant is selected from any one of aspartic acid, adipic acid, glutamic acid, or a combination thereof.

In the preferred technical scheme of the invention, the crystallization assistant is aspartic acid, preferably L-aspartic acid.

In the preferable technical scheme of the invention, the crystallization assistant is glutamic acid, and preferably L-glutamic acid.

In the preferred technical scheme of the invention, the crystallization assistant is adipic acid, and preferably L-2-aminoadipic acid.

In a preferred technical scheme of the invention, the crystallization assistant agent comprises: the molar ratio of L-valine is 1:270 to 1:70, preferably 1:250 to 1: 120.

In a preferred embodiment of the invention, the pH of the L-valine solution to be purified is from 3.0 to 5.0, preferably from 3.5 to 4.5.

In a preferred embodiment of the present invention, the acid used for adjusting the pH of the solution is selected from any one of hydrochloric acid, phosphoric acid, acetic acid, or a combination thereof.

In a preferred embodiment of the present invention, the pH of the L-valine solution to be purified is adjusted to an acid concentration of 0.5 to 6mol/L, more preferably 1 to 4 mol/L.

In a preferred embodiment of the present invention, the concentration is selected from any one of vacuum concentration, membrane concentration, and atmospheric concentration, or a combination thereof.

In the preferred technical scheme of the invention, the concentration temperature is 40-90 ℃, preferably 50-70 ℃, and most preferably 55-65 ℃.

In a preferred embodiment of the present invention, the concentrated solution is selected from a saturated solution of L-valine and a supersaturated solution of L-valine.

In a preferred embodiment of the present invention, the sedimentation ratio of the concentrated solution is not less than 60%, and most preferably not less than 80%.

In the preferred technical scheme of the invention, the prepared concentrated solution is naturally cooled to normal temperature, and preferably stands for crystallization for 1-8 hours, and more preferably stands for crystallization for 2-5 hours.

In the preferred technical scheme of the invention, the crystallization is cooling crystallization, preferably cooling standing crystallization, and more preferably gradient cooling standing crystallization.

In the preferred technical scheme of the invention, the gradient cooling frequency is not less than 2.

In the preferred technical scheme of the invention, the first temperature reduction condition of gradient temperature reduction is that the temperature of the crystal system is reduced to 35-55 ℃ within 0.5-3h, and the temperature is kept for 1-10 h; preferably, the temperature is reduced to 40-50 ℃ within 1.0-2.5h, and the temperature is kept for 2-5 h.

In the preferred technical scheme of the invention, the second temperature reduction condition of gradient temperature reduction is that the temperature of the crystal system is reduced to 5-35 ℃ within 0.5-24h, and the temperature is kept for 0.5-4 h; preferably, the temperature is reduced to 10-30 ℃ within 1.0-2.5h, and the temperature is kept for 1-12 h; more preferably 1.2-2.0h, cooling to 15-25 deg.C, and maintaining for 1.5-6 h.

In a preferred technical scheme of the invention, the temperature reduction is selected from any one of natural cooling and forced cooling or a combination thereof.

In the preferred technical scheme of the invention, the forced cooling adopts a cooling medium to realize forced cooling on the crystallization system.

In a preferred embodiment of the present invention, the cooling medium is selected from any one of condensed water, ice water, ethanol, and ethylene glycol, or a combination thereof.

In a preferred embodiment of the present invention, the separation is selected from any one of filtration, centrifugation, and membrane treatment, or a combination thereof.

In the preferable technical scheme of the invention, after uniformly mixing the separated concentrated mother liquor with the L-valine solution to be purified, adding the required amount of crystallization assistant agent, and concentrating again to prepare the L-valine crystal, wherein the volume ratio of the concentrated mother liquor to the treated fermentation clear liquid is 1: 3-1: 5.

in the preferable technical scheme of the invention, the separated concentrated mother liquor is concentrated again until the sedimentation ratio is 50-60%, and then is kept stand for crystallization to prepare the L-valine crystal of the invention.

In the preferred technical scheme of the invention, the concentration mother liquor obtained by separation is concentrated for no less than 2 times.

In a preferred technical scheme of the invention, the collected solid is dried after being washed, and preferably, the solvent used for washing is water.

In a preferred embodiment of the present invention, the drying is selected from any one of vacuum drying, reduced pressure drying, atmospheric drying, spray drying, and boiling drying, or a combination thereof.

In the preferred technical scheme of the invention, the drying temperature is 25-80 ℃, preferably 35-70 ℃, and more preferably 40-60 ℃.

In the preferred technical scheme of the invention, the bulk density of the prepared L-valine crystal is more than or equal to 0.60g/mL, preferably more than or equal to 0.80 g/mL.

In a preferred embodiment of the present invention, the purity of the produced L-valine crystals is not less than 99.0%, preferably not less than 99.5%, more preferably not less than 99.7%.

In a preferred embodiment of the present invention, the water content of the produced L-valine crystals is not more than 0.13%, preferably not more than 0.19%.

Another object of the present invention is to provide a high bulk density L-valine crystal having a bulk density of 0.60g/mL or more, preferably 0.75g/mL or more.

In the preferred technical scheme of the invention, the purity of the L-valine crystal is more than or equal to 99.0 percent, preferably more than or equal to 99.5 percent, and more preferably more than or equal to 99.7 percent.

In a preferred embodiment of the present invention, the water content of the produced L-valine crystals is not more than 0.13%, preferably not more than 0.19%.

Another object of the present invention is to provide use of the L-valine crystal of the present invention for producing any one of foods, food additives, pharmaceuticals, pharmaceutical intermediates, pharmaceutical raw materials, nutritional supplements, amino acid drugs, feeds, and feed additives.

In a preferred technical scheme of the invention, the feed is selected from any one of chicken feed, duck feed, goose feed, pig feed and fish feed.

In a preferred technical scheme of the invention, the feed additive is selected from any one of chicken feed additives, duck feed additives, goose feed additives, pig feed additives and fish feed additives.

Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.

Unless otherwise stated, the present invention examined the bulk density, purity, water content and hygroscopicity of L-valine by the following methods:

the method for detecting the bulk density of the L-valine crystal comprises the following steps: the powder tap density instrument XF-20 is adopted for detection, the amplitude is 3-30 mm, and the tap is performed for 250 times.

The purity detection method of the L-valine crystal comprises the following steps: the detection was carried out by high performance liquid chromatography disclosed in reference 1 (Takara Ohwei et al, quantitative analysis of branched chain amino acids by high performance liquid chromatography, Biotechnology communication, vol. 20, No. 4 of 2009).

A method for detecting the water content and hygroscopicity of L-valine: the measurement was carried out by a method disclosed in document 2 (Zhongshiping, research on the moisture absorption principle and moisture absorption measurement method of a granular seasoning, Chinese seasoning, No. 4 of 2009).

The crystal form of L-valine was observed by a microscope.

Sedimentation ratio of L-valine solution: and standing the concentrated L-valine solution for layering to obtain the precipitate accounting for the total volume of the concentrated solution.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the crystallization assistant is added into the L-valine solution to be purified, and the L-valine crystal with high bulk density, high purity and low water content is prepared by optimizing the crystallization and purification conditions, so that the quality and the yield of the L-valine are obviously improved, and the cost of the L-valine is reduced.

2. The purification method has the advantages of simple and convenient operation, obviously shortened production period, obviously reduced cost, environmental protection, less energy consumption, contribution to industrial production and the like.

Drawings

FIG. 1 is an appearance diagram of a scale-like crystal of L-valine obtained in comparative example 1;

FIG. 2 is an appearance diagram of granular crystals of L-valine obtained in example 5;

FIG. 3 microscopic image of granular crystals of L-valine obtained in example 5.

Detailed Description

The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

Reference example 1Preparation of L-valine fermentation supernatant to be purified

The L-valine solution to be purified described in reference example 1 was L-valine fermentation supernatant to be purified, which was prepared by a method comprising the steps of:

(1) collecting 3m L-valine fermentation liquid prepared by fermentation method in Anhuihua Hengshengshi GmbH³Adding concentrated sulfuric acid to adjust the pH value of the fermentation liquor to 5.5, adding polyaluminium chloride which is 4 percent of the weight of the fermentation liquor under the stirring condition, stirring for 15 minutes, naturally standing for 30 minutes, starting stirring again when a large amount of floccules are generated and begin to settle, adding prepared 0.15 percent cationic polyacrylamide, continuously stirring for 20 minutes, naturally standing for 2 hours, and separating out supernatant after the solution is layered;

(2) adding diatomite which is 1.0 percent of the weight of the supernatant into the supernatant, continuously stirring for 30 minutes, performing filter pressing treatment through a plate-and-frame filter press, and collecting the filtered supernatant;

(3) adding 1.0% of activated carbon into the filtered clear liquid, stirring and decoloring for 45 minutes at 60 ℃, and performing filter pressing treatment by using a plate-and-frame filter press again to obtain a second filtered clear liquid;

(4) pumping the clear liquid obtained by the second filtration into a technical chromatographic column, removing inorganic salt and part of the mixed acid in the fermentation liquid to obtain an L-valine clear liquid, wherein the treatment temperature of the technical chromatographic column is 30 ℃;

(5) removing residual micromolecular impurities and organic pigments in the L-valine clear liquid by using a nanofiltration membrane (the specification is 800 daltons in molecular weight cut-off), and preparing the L-valine fermented clear liquid to be purified with the concentration of 49.7 g/L.

Comparative example 1 and examples 1 to 5Purification method of L-valine crystals

The purification method of L-valine crystals as described in comparative example 1 and examples 1 to 5, comprising the steps of:

6000ml of the L-valine fermentation supernatant to be purified obtained in reference example 1 was measured, and the solution was divided into six equal parts and used as the L-valine solution to be purified in comparative example 1 and examples 1 to 5, respectively (see Table 1).

Adding concentrated sulfuric acid into the L-valine solution to be purified of comparative example 1 and examples 1-5, adjusting the pH value to 4, respectively adding a crystallization assistant according to the table 1, stirring, uniformly mixing, concentrating the mixed solution in vacuum at 65 ℃ until the sedimentation ratio is 75%, naturally cooling to normal temperature, standing for 3h, centrifugally separating crystals, washing, and drying to obtain the L-valine crystals.

The L-valine crystals obtained in examples 1 to 5 and comparative example 1 were examined for appearance, purity and bulk density according to the method of the present invention. The results are shown in Table 1.

TABLE 1

Test example 1Water content detection of L-valine crystals

The water content of the L-valine crystals obtained in example 5 and comparative example 1 was examined according to the method of the present invention. The results are shown in Table 2.

TABLE 2

Reference example 2Preparation of L-valine fermentation supernatant to be purified

The L-valine solution to be purified described in referential example 2 was an L-valine fermentation supernatant to be purified, which was prepared by a process comprising the steps of:

(1) collecting 3m L-valine fermentation liquid prepared by fermentation method in Anhuihua Hengshengshi GmbH³Adding concentrated sulfuric acid to adjust the pH value of the fermentation liquor to 5.5, adding polyaluminium chloride which is 4 percent of the weight of the fermentation liquor under the stirring condition, stirring for 15 minutes, naturally standing for 30 minutes, starting stirring again when a large amount of floccules are generated and begin to settle, adding prepared 0.15 percent cationic polyacrylamide, continuously stirring for 20 minutes, naturally standing for 2 hours, and separating out supernatant after the solution is layered;

(2) adding diatomite which is 1.0 percent of the weight of the supernatant into the supernatant, continuously stirring for 30 minutes, performing filter pressing treatment through a plate-and-frame filter press, and collecting the filtered supernatant;

(3) adding 1.0% of activated carbon into the filtered clear liquid, stirring and decoloring for 45 minutes at 60 ℃, and performing filter pressing treatment by using a plate-and-frame filter press again to obtain a second filtered clear liquid;

(4) pumping the clear liquid obtained by the second filtration into a technical chromatographic column, removing inorganic salt and part of the mixed acid in the fermentation liquid to obtain an L-valine clear liquid, wherein the treatment temperature of the technical chromatographic column is 30 ℃;

(5) removing residual micromolecular impurities and organic pigments in the L-valine clear liquid by using a nanofiltration membrane (the specification is 800 daltons in molecular weight cut-off), and preparing the L-valine fermented clear liquid to be purified with the concentration of 56.8 g/L.

Examples 6 to 14Purification method of L-valine crystals

The purification process for L-valine crystallization described in examples 6 to 14, comprising the steps of:

9000ml of the L-valine fermented supernatant to be purified, which had been obtained in reference example 2, was measured and divided into nine equal parts, which were used as the L-valine solution to be purified in examples 6 to 14, respectively (see Table 3).

Adjusting the pH value of the L-valine solution to be purified according to the table 3, adding L-2-aminoadipic acid, stirring, uniformly mixing, concentrating the mixed solution in vacuum at 65 ℃ until the sedimentation ratio is 80%, naturally cooling to normal temperature, standing for 3h, centrifugally separating crystals, washing, and drying to obtain the L-valine crystals.

The L-valine crystals obtained in examples 6 to 14 were examined for appearance, purity and bulk density according to the method of the present invention. The results are shown in Table 3.

TABLE 3

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.