阅读说明：本技术 一种唾液酸水合晶体的制备方法及其产品和应用 (Preparation method of sialic acid hydrated crystal, product and application thereof ) 是由 陈祥松 王刚 袁丽霞 朱薇薇 吴金勇 王煜 费贤春 李翔宇 孙立洁 王纪 姚建铭 于 2019-09-25 设计创作，主要内容包括：本发明提供一种唾液酸水合晶体的制备方法及其产品和应用。所述制备方法为：将唾液酸原料调节至酸性环境,温度调节至20℃以下,并在搅拌下进行结晶,析出所述唾液酸水合晶体。该制备方法流程简单,易操作；避免了产物大量附着于容器内壁的弊端,适合大规模的工业化生产；获得的晶体稳定性好,不易失去结晶水；同时,能够实现结晶的均一性,含水量符合水合晶体的比例,晶体颗粒的粒径为微米级别,避免了后续对晶体进行粉碎时导致的晶体中结晶水不稳定问题；另外,在保证上述有益的效果的前提下,制备得到的水合晶体纯度依然高达98％以上。(The invention provides a preparation method of sialic acid hydrated crystal, a product and application thereof. The preparation method comprises the following steps: adjusting the sialic acid raw material to an acidic environment, adjusting the temperature to be below 20 ℃, and crystallizing under stirring to separate out the sialic acid hydrated crystal. The preparation method has simple flow and easy operation; the defect that a large amount of products are attached to the inner wall of the container is avoided, and the method is suitable for large-scale industrial production; the obtained crystal has good stability and is not easy to lose crystal water; meanwhile, the uniformity of crystallization can be realized, the water content accords with the proportion of hydrated crystals, and the particle size of crystal particles is in a micron level, so that the problem of unstable crystal water in the crystals caused by the subsequent crushing of the crystals is avoided; in addition, on the premise of ensuring the beneficial effects, the purity of the prepared hydrated crystal is still as high as more than 98%.)

1. A preparation method of sialic acid hydrated crystal is characterized by comprising the following steps: adjusting the sialic acid raw material to an acidic environment, adjusting the temperature to be below 20 ℃, and crystallizing under stirring to separate out the sialic acid hydrated crystal.

2. The method of claim 1, wherein the acidic environment is a pH of 0.2 to 3.5.

3. The method of claim 1 or 2, wherein the rate of adjustment of the acidic environment is: the pH change per 1min does not exceed 0.1.

4. The production method according to any one of claims 1 to 3, wherein the temperature adjustment means adjusting the temperature to 0 to 10 ℃.

5. The method of any one of claims 1-4, wherein the rate of temperature adjustment is: the temperature is changed by 1 ℃ for 10-15 min.

6. The method of any one of claims 1 to 5, wherein the stirring speed is 1 to 100rpm, preferably 1 to 30 rpm.

7. The process according to any one of claims 1 to 6, wherein the crystallization time is from 12 to 48 hours;

preferably, the preparation method further comprises pretreating the sialic acid raw material, wherein the pretreatment operation comprises the following steps: preparing sialic acid raw material into an aqueous solution containing 50-600g/L sialic acid and filtering;

preferably, the sialic acid source comprises an N-acetylneuraminic acid source;

preferably, the N-acetylneuraminic acid raw material comprises an N-acetylneuraminic acid raw material obtained by fermentation, cubilose extraction, animal milk extraction, chemical synthesis or bio-enzyme catalysis;

preferably, said sialic acid hydrated crystals are subjected to a post-treatment after precipitation, said post-treatment being performed by: filtering, washing and drying the sialic acid hydrated crystal;

preferably, the washing comprises washing with ethanol, methanol or isopropanol;

preferably, the temperature of the drying is not more than 120 ℃.

8. The method of any one of claims 1-7, comprising the steps of:

(1) preparing sialic acid raw material into an aqueous solution containing 50-600g/L sialic acid and filtering;

(2) adjusting the pH value of the filtrate obtained in the step (1) to 0.2-3.5, wherein the pH change value is not more than 0.1 every 1min, the temperature is adjusted to 0-10 ℃, and the time is 10-15min when the temperature changes by 1 ℃; stirring at the speed of 1-100rpm, crystallizing for 12-48h, and separating out sialic acid hydrated crystal;

(3) filtering the sialic acid hydrated crystal obtained in the step (2), washing with absolute ethyl alcohol, and drying at the temperature of not more than 120 ℃.

9. A hydrated crystal of sialic acid produced by the production method according to any one of claims 1 to 8.

10. Use of the hydrated crystalline sialic acid as claimed in claim 9 in a food product, health product or pharmaceutical product.

Technical Field

The invention belongs to the technical field of biochemical engineering, particularly relates to a preparation method of a crystal, a product and application thereof, and particularly relates to a preparation method of a sialic acid hydrated crystal, a product and application thereof.

Background

Sialic Acid (SA) is a type of monosaccharide or glycan commonly found in nature, is mainly present in vertebrate bodies, is usually combined on cell surfaces in the form of polymers and complexes, and plays an important role in brain development, immunoregulation, antibiosis and antivirus, antioxidation and whitening, intestinal flora colonization and the like. SA is synthesized by the liver in the human body, forms an activated CMP-SA form in the nucleus, forms a glycocomplex in the golgi apparatus, is secreted extracellularly to form oligosaccharides, glycoproteins, mucins, gangliosides, etc., and is bound to the cell surface, or is dissociated, or is secreted into milk, to exert various functions and actions. The edible bird's nest, milk, egg and other foods contain relatively rich SA, wherein the SA accounts for 5-11% of the dry weight of the bird's nest. Some microorganisms can also produce sialic acid, such as Escherichia coli K1, K92, K235, and so forth, and SA can be obtained by fermentation. SA in humans, cubilose and escherichia coli, are a class of SA, N-acetylneuraminic acid (NeuAc) or its homopolymer (PSA).

NeuAc is the only sialic acid synthesized by normal human body, and the molecule has pyranose structure and molecular formula C₁₁H₁₉NO₉The full name is 5-amino-3, 5-dideoxy-D-glycerol-D-galactononulose. The molecular weight of NeuAc is 309.3, the NeuAc has a carboxyl group in the molecule, is an acidic amino sugar, is very soluble in water but not easily soluble in organic solvents such as ethanol, and is acidic in aqueous solution, and the pH of 2% aqueous solution is about 1.8-2.3. The NeuAc hydrate contains 2 crystal waters and has a molecular formula of C₁₁H₁₉NO₉·2H₂O, molecular weight 345.3, hydrate being some of the precursors of drug synthesis.

At present, sialic acid is mainly used as a monomer (SA) of sialic acid, but the market demand of SA hydrate is also great. There are some prior art reports on the preparation of SA hydrates. For example, CN109180749A discloses a method for preparing high-purity N-acetylneuraminic acid hydrate by supersaturation crystallization, which is to prepare N-acetylneuraminic acid hydrate by standing and crystallizing supersaturated aqueous solution of N-acetylneuraminic acid at low temperature. After drying detection, the purity of the N-acetylneuraminic acid hydrate is at least 99 percent, and the N-acetylneuraminic acid hydrate can meet the requirements of the N-acetylneuraminic acid hydrate in the fields of food, health care, medicine and the like.

However, most of the methods adopted in the prior art are still standing and crystallizing to obtain hydrated crystals, and because a large amount of products are attached to the inner wall of a container, the method is not suitable for large-scale industrial production; while crystallization under stirring partially avoids the problem of wall attachment, process control does not achieve uniformity of crystallization and crystal water is easily lost. There is therefore a need for better methods for the preparation of hydrated crystals.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a preparation method of a crystal, a product and application thereof, in particular to a preparation method of a sialic acid hydrated crystal, a product and application thereof.

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

in one aspect, the present invention provides a preparation method of a sialic acid hydrated crystal, the preparation method comprising: adjusting the sialic acid raw material to an acidic environment, adjusting the temperature to be below 20 ℃, and crystallizing under stirring to separate out the sialic acid hydrated crystal.

The method for preparing the hydrated crystal of the sialic acid adopts a mode of regulating the acidity and the temperature simultaneously under the stirring state for crystallization, avoids the defect that a large amount of products are attached to the inner wall of a container, and is suitable for large-scale industrial production; the obtained crystal has good stability and is not easy to lose crystal water; meanwhile, the uniformity of crystallization can be realized, the water content accords with the proportion of hydrated crystals, and the particle size of crystal particles is in a micron level, so that the problem of unstable crystal water in the crystals caused by the subsequent crushing of the crystals is avoided; in addition, on the premise of ensuring the beneficial effects, the purity of the prepared hydrated crystal is still as high as more than 98%.

It is well known to those skilled in the art that the manner of static crystallization inevitably causes a large amount of crystallized product to adhere to the inner wall of the crystallization tank, and the crystallized mass is large and the hardness is high, and the size of the crystallized particles is generally above 0.1cm, which makes it necessary to take a lot of time to transfer the product from the wall of the tank and subject the product to a pulverization operation, which may seriously affect the stability of the crystal water. The invention carries out crystallization under non-static conditions (stirring state), so that the product is not adhered to the wall of the container; in order to overcome the problems that the crystallization is not uniform and the crystal water is easy to lose under the stirring condition, the sialic acid raw material is regulated to the acid environment, the temperature is regulated to be below 20 ℃, the stability of the crystallization is realized, and the precipitated crystal particles are fine and do not need to be crushed.

Preferably, the acidic environment is pH 0.2-3.5, for example, pH 0.2, pH 0.4, pH 0.5, pH 0.7, pH 0.8, pH 1.0, pH 1.2, pH 1.5, pH 1.8, pH 3.0, pH 3.2 or pH 3.5, and the like, and the points in other intervals are not described herein.

The pH value of the preparation environment related to the invention is preferably controlled within the range of 0.2-3.5, and if the pH value is higher than the range, sialic acid can not be normally crystallized; if the pH is less than this range, the crystal may be broken, the crystal may be unstable, and impurities may increase.

Preferably, the rate of adjustment of the acidic environment is: the pH does not change more than 0.1 per 1min, e.g., the pH changes 0.01, 0.02, 0.04, 0.05, 0.07, 0.08, 0.09, or 0.1 per 1min, etc.

The pH value change per 1min is not more than 0.1, which means that the pH value increase or decrease per 1min is not more than 0.1. The adjusting rate is a key process influencing the crystallization effect of the invention, and only under the condition, the prepared crystal is more stable, the crystal water is less prone to losing, the crystal grains are more uniform, and the purity of the crystal is higher.

Preferably, the temperature regulation means regulating the temperature to 0-10 ℃, such as 0 ℃, 1 ℃, 2 ℃, 3 ℃, 4 ℃,5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃.

The temperature of the preparation environment related to the invention is preferably controlled within the range of 0-10 ℃, and if the temperature value is higher than the range, sialic acid can not be normally crystallized; if the temperature is lower than this range, the crystal may be broken, the crystal may be unstable, and impurities may increase.

Preferably, the rate of temperature adjustment is: the time duration is 10-15min for each 1 ℃ change of temperature, for example, the time duration consumed for each 1 ℃ change of temperature is 10min, 11min, 12min, 13min, 14min or 15min, etc.

The time duration of 10-15min when the temperature changes by 1 ℃ means that the time duration of 10-15min when the temperature increases or decreases by 1 ℃. The adjusting rate is a key process influencing the crystallization effect of the invention, and only under the condition, the prepared crystal is more stable, the crystal water is less prone to losing, the crystal grains are more uniform, and the purity of the crystal is higher.

Preferably, the stirring speed is 1-100rpm, such as 1rpm, 2rpm, 5rpm, 10rpm, 15rpm, 20rpm, 25rpm, 30rpm, 35rpm, 40rpm, 50rpm, 60rpm, 70rpm, 100rpm, or the like, preferably 1-30 rpm.

The stirring speed is a key process influencing the crystallization effect of the invention, and if the stirring speed exceeds the numerical range, the stability of a crystallization system is deteriorated, the growth and the structure of crystals are influenced, and the stability of hydrated crystals is influenced.

Preferably, the crystallization time is 12-48h, such as 12h, 15h, 18h, 20h, 24h, 27h, 30h, 32h, 35h, 38h, 40h, 42h, 44h, 45h, 46h, 47h or 48h, and the like.

Preferably, the preparation method further comprises pretreating the sialic acid raw material, wherein the pretreatment operation comprises the following steps: sialic acid raw material is formulated into an aqueous solution containing 50-600g/L sialic acid (e.g., 50g/L, 60g/L, 80g/L, 100g/L, 150g/L, 200g/L, 250g/L, 300g/L, 350g/L, 400g/L, 500g/L, or 600g/L, etc.) and filtered. Filtration is to remove insoluble impurities.

Preferably, before the sialic acid raw material is prepared into an aqueous solution, the raw material may be subjected to a series of pretreatment operations such as cell removal, desalting, hydrolysis, decolorization, or protein removal.

Preferably, the sialic acid source comprises an N-acetylneuraminic acid source.

Preferably, the N-acetylneuraminic acid raw material comprises an N-acetylneuraminic acid raw material obtained by fermentation, cubilose extraction, animal milk extraction, chemical synthesis or bio-enzyme catalysis. The N-acetylneuraminic acid starting material obtained by fermentation is, for example, a sialic acid-rich Escherichia coli fermentation broth.

Preferably, said sialic acid hydrated crystals are subjected to a post-treatment after precipitation, said post-treatment being performed by: filtering, washing and drying the sialic acid hydrated crystal.

Preferably, the washing comprises washing with ethanol, methanol or isopropanol.

Preferably, the temperature of the drying is not more than 120 ℃, such as 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃ or 120 ℃ and the like. If the temperature exceeds 120 ℃, sialic acid is partially decomposed or discoloration is accelerated.

As a preferred technical scheme of the invention, the preparation method comprises the following steps:

(1) preparing sialic acid raw material into an aqueous solution containing 50-600g/L sialic acid and filtering;

(2) adjusting the pH value of the filtrate obtained in the step (1) to 0.2-3.5, wherein the pH change value is not more than 0.1 every 1min, the temperature is adjusted to 0-10 ℃, and the time is 10-15min when the temperature changes by 1 ℃; stirring at the speed of 1-100rpm, crystallizing for 12-48h, and separating out sialic acid hydrated crystal;

(3) filtering the sialic acid hydrated crystal obtained in the step (2), washing with absolute ethyl alcohol, and drying at the temperature of not more than 120 ℃.

It should be noted here that it is well known to those skilled in the art that when the pH is higher than 5.5 or the temperature is higher than 30 ℃, there is no great influence on crystallization, and therefore, for time and cost reasons, if the pH of the solution is higher than 5.5 in step (1), the pH is quickly adjusted to approach 5.5; if the temperature of the solution is higher than 30 ℃, quickly reducing the temperature to be close to 30 ℃, and then carrying out the step (2).

In another aspect, the present invention provides a sialic acid hydrated crystal prepared by the preparation method as described above. The sialic acid hydrated crystal has high purity, good stability and good uniformity, and is not adhered to the inner wall of a container.

In a further aspect, the present invention provides a use of the hydrated crystal of sialic acid as described above in food products, health products or pharmaceutical products.

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

the preparation method has simple process and easy operation; the defect that a large amount of products are attached to the inner wall of the container is avoided, and the method is suitable for large-scale industrial production; the obtained crystal has good stability and is not easy to lose crystal water; meanwhile, the uniformity of crystallization can be realized, the water content accords with the proportion of hydrated crystals, and the particle size of crystal particles is in a micron level, so that the problem of unstable crystal water in the crystals caused by the subsequent crushing of the crystals is avoided; in addition, on the premise of ensuring the beneficial effects, the purity of the prepared hydrated crystal is still as high as more than 98%.

Drawings

FIG. 1 is a diagram of a crystal of sialic acid hydrate prepared in example 1;

FIG. 2 is a diagram showing a crystal of sialic acid hydrate prepared in comparative example 1.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The starting material sialic acid-rich Escherichia coli fermentation broth referred to in the following examples was obtained by fermentation methods which have been disclosed in the prior art, for example, the method disclosed in "fermentation Medium for polysialic acid, production method for polysialic acid and polysialic acid preparation" patent 201810458924.4. Wherein the removal of bacteria, salt, hydrolysis, decolorization, and protein are also performed according to conventional procedures known to those skilled in the art, for example, the procedure disclosed in patent 201811364040.9 "a method for preparing N-acetylneuraminic acid by separation and purification from polysialic acid-containing material".

The following examples relate to the determination of sialic acid content in the product as follows: Q/WZG 0001S-2019 appendix B, or GB/T30636-2014.

The following examples relate to the determination of the moisture content in the product by: GB 5009.3.