阅读说明：本技术 一种降低单一相关杂质含量的腺苷提取方法 (Adenosine extraction method for reducing content of single related impurities ) 是由 李静 张孟涛 李闯 封浪 刘晓东 刘永超 于 2019-09-12 设计创作，主要内容包括：本发明涉及一种降低单一相关杂质含量的腺苷提取方法,属于生物工程技术领域,所述方法是在发酵结束后,发酵液过膜进行固液分离,得到含有腺苷的清液；向所得清液中加入絮凝剂A缓慢搅拌至出现白色絮状物,然后再加入絮凝剂B缓慢搅拌至出现蛋白絮状物时静置,得料液；向所得料液中加入活性炭进行预脱色,然后过板框；经减压蒸发浓缩后养晶、降温结晶、离心、粗品重溶脱色、降温结晶、洗涤、干燥后,即可得腺苷晶体。所述方法,通过加入絮凝剂和采用预脱的方式降低腺苷成品中单一相关杂质含量,从而提高产品收率。(The invention relates to an adenosine extraction method for reducing the content of single related impurities, which belongs to the technical field of bioengineering, and the method comprises the following steps of performing solid-liquid separation on fermentation liquor through a membrane after the fermentation is finished to obtain a clear liquid containing adenosine; adding a flocculating agent A into the obtained clear liquid, slowly stirring until white floccules appear, then adding a flocculating agent B, slowly stirring until protein floccules appear, and standing to obtain feed liquid; adding active carbon into the obtained feed liquid for pre-decoloring, and then passing through a plate frame; after decompression evaporation and concentration, crystal growing, cooling crystallization, centrifugation, crude product redissolution and decoloration, cooling crystallization, washing and drying, the adenosine crystal can be obtained. According to the method, the content of single related impurities in an adenosine finished product is reduced by adding a flocculating agent and adopting a pre-removing mode, so that the product yield is improved.)

1. An adenosine extraction method for reducing the content of single related impurities is characterized in that: the method comprises the following steps:

step one, after fermentation is finished, carrying out solid-liquid separation on fermentation liquor through a membrane to obtain a clear liquid containing adenosine;

step two, adding a flocculating agent A into the clear liquid obtained in the step one, slowly stirring until white floccules appear, then adding a flocculating agent B, slowly stirring until protein floccules appear, standing for 30min, and obtaining feed liquid; the flocculant A is aluminum sulfate, the addition amount of the flocculant A is 2g/L, and the addition amount of the flocculant B is polyacrylamide, and the addition amount of the flocculant B is 0.1 g/L;

step three, adding 3% of activated carbon into the feed liquid obtained in the step two for pre-decoloring, wherein the adding amount of the activated carbon is 30 g/L, the pre-decoloring temperature is 90 ℃, the time is 30min ~ 1h, and then passing through a plate frame;

and step four, carrying out reduced pressure evaporation and concentration, then growing crystals, cooling and crystallizing, centrifuging, re-dissolving and decoloring a crude product, cooling and crystallizing, washing and drying to obtain the adenosine crystals.

2. The method for extracting adenosine according to claim 1, wherein the adenosine is extracted from the plant by using a single relevant impurity: and step one, solid-liquid separation is carried out by adopting a ceramic membrane or adopting an ultrafiltration membrane to filter fermentation liquor.

3. The method for extracting adenosine according to claim 2, wherein the adenosine is extracted from the plant with a reduced content of single related impurities, and the method comprises the following steps: the membrane flux of the ultrafiltration membrane is 15m³H to 20m³/h。

4. The method for extracting adenosine according to claim 1, wherein the adenosine is extracted from the plant by using a single relevant impurity: and step four, the evaporation concentration magnesium is 12.

5. The method for extracting adenosine according to claim 1, wherein the adenosine is extracted from the plant by using a single relevant impurity: and step four, the cooling crystallization is to stir and grow the crystal for 1 hour at the temperature of below 10 ℃.

6. The method for extracting adenosine according to claim 1, wherein the adenosine is extracted from the plant by using a single relevant impurity: and fourthly, the concentration of re-dissolution and decoloration is 70g/L, the temperature is 90 ℃, and the time is 1 h.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an adenosine extraction method for reducing the content of single related impurities.

Background

Adenosine is an endogenous purine nucleotide, and in the cardiovascular context, adenosine is a reliable physiological regulator. Clinically adenosine is effectively used for treatment as a vasodilator, and also as an antiarrhythmic drug. The greatest advantage is the short plasma half-life. Adenosine, as a versatile drug substance, has important value both in clinical applications and as a precursor for ATP production. At present, the domestic production of adenosine is mainly an enzymatic method or a chemical method, and the cost is higher. Along with the continuous expansion of the action range of adenosine, the market demand is also continuously increased, the production method of adenosine mainly comprises a chemical synthesis method, an enzymatic method and a fermentation method, and the production of adenosine at home at present mainly comprises the chemical method and the enzymatic method, so that the cost is high, the pollution is serious, and the popularization and the application are severely limited. The adenosine produced by the fermentation method has the advantages of mild reaction conditions, low cost, greenness, cleanness, environmental protection and the like.

At present, the requirement on the quality standard of raw material medicines is stricter and single related impurities are an important index in the adenosine finished product detection standard, but the content of the index of a finished product can be reduced by only using a small amount of activated carbon in a decoloring link in the existing adenosine extraction process in an auxiliary manner, and a clear single link is not provided for reducing the index content of the finished product, so that the adenosine is produced by using a fermentation method under the condition of low carbon and environmental protection, and the optimization of the adenosine extraction process for further improving the yield and quality of the adenosine has important significance.

Disclosure of Invention

In order to solve the problems of poor extraction quality and low yield of adenosine produced by a fermentation method in the prior art, the invention aims to provide an adenosine extraction method for reducing the content of single related impurities, and the content of the single related impurities in an adenosine finished product is reduced by adding a flocculating agent and adopting a pre-removing mode, so that the product yield is improved.

In order to achieve the purpose, the invention adopts the specific scheme that:

an adenosine extraction method for reducing the content of single related impurities comprises the following steps:

step one, after fermentation is finished, carrying out solid-liquid separation on fermentation liquor through a membrane to obtain a clear liquid containing adenosine;

step two, adding a flocculating agent A into the clear liquid obtained in the step one, slowly stirring until white floccules appear, then adding a flocculating agent B, slowly stirring until protein floccules appear, standing for 30min, and obtaining feed liquid; the flocculant A is aluminum sulfate, the addition amount of the flocculant A is 2g/L, and the addition amount of the flocculant B is polyacrylamide, and the addition amount of the flocculant B is 0.1 g/L;

step three, adding 3% of activated carbon into the feed liquid obtained in the step two for pre-decoloring, wherein the adding amount of the activated carbon is 30 g/L, the pre-decoloring temperature is 90 ℃, the time is 30min ~ 1h, and then passing through a plate frame;

and step four, carrying out reduced pressure evaporation and concentration, then growing crystals, cooling and crystallizing, centrifuging, re-dissolving and decoloring a crude product, cooling and crystallizing, washing and drying to obtain the adenosine crystals.

As a further optimization of the scheme, the solid-liquid separation in the step one is carried out by adopting a ceramic membrane or an ultrafiltration membrane to filter fermentation liquor. More closely, the membrane flux of the ultrafiltration membrane is 15m³H to 20m³/h。

As a further optimization of the above scheme, the evaporation concentration magnesium in the fourth step is 12.

As a further optimization of the scheme, in the step four, the temperature reduction crystallization is to stir the crystal for 1 hour at the temperature of below 10 ℃.

As a further optimization of the scheme, the concentration of the redissolution decoloration in the step four is 70g/L, the temperature is 90 ℃, and the time is 1 h.

Has the advantages that:

the invention flocculates the ceramic clear liquid, not only further reduces the protein content in the fermented clear liquid, but also removes the flocculated protein and can absorb part of impurities in the clear liquid after pre-removing treatment.

Drawings

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.