Disclosure of Invention
The invention provides a preparation method of a fermentation liquid rich in ergonovine, which can obtain the fermentation liquid with high titer and high components and is suitable for large-scale fermentation production of the ergonovine.
The preparation method comprises the following steps: inoculating a seed bacterial liquid of the ergot bacteria into a liquid fermentation culture medium, and performing shake culture to obtain ergometrine fermentation liquid; wherein the liquid fermentation medium comprises (consists of):
80-120 g/L of glucose, 20-60 g/L of maltodextrin, 4-8 g/L of yeast powder, 50-80 g/L of soybean meal and 0.7-1.5 g/L of magnesium sulfate.
The fermentation direction of the ergot can be controlled by adopting the fermentation medium, so that the ergot can be fermented in large quantity to produce the ergometrine. The concentration of the ergometrine in the obtained fermentation liquor can reach more than 3000ug/ml, and the ergometrine has high quality, no toxicity, high proportion of main components and no high component impurities as seen by a liquid phase detection atlas.
Preferably, the liquid fermentation medium provided by the invention consists of the following components:
80-90 g/L of glucose, 25-35 g/L of maltodextrin, 5-6 g/L of yeast powder, 50-60g/L of soybean meal and 0.8-1.2g/L of magnesium sulfate. By adopting the components, the quality of the ergometrine and the stability of the fermentation process can be further improved.
The fungus age of the seed bacterial liquid is controlled to be 48-60 hours, the concentration of the bacteria is 25-30%, the feed liquid is not viscous, and the activity is vigorous; can be quickly adapted and grown when entering a fermentation tank.
The inoculation amount of the ergot bacteria liquid inoculated into the liquid fermentation culture medium is controlled to be 8 +/-3 percent, the range is higher than the range, the seeds grow very fast after entering a fermentation tank, the control is difficult, the fermentation liquid is easy to be sticky and thickened, and the unit is low; when the content is lower than the range, the growth period in the early stage after entering a fermentation tank is longer, the unit amplification in the later stage after entering the metabolic phase is slow, and hyphae are aged and autolyzed.
The pH value of the fermentation medium is preferably controlled to be 7.0-7.8, and the fermentation medium is more suitable for the fermentation growth of the ergot bacteria.
In the preparation method, the fermentation temperature is preferably controlled to be 28-32 ℃, hyphae grow slowly due to too low temperature, early-stage unit growth is slow after metabolism delay, and unit growth is stopped in later stage; too high a temperature may cause premature aging of the mycelia, and the mycelia begin to autolyze before reaching the can-releasing period, while the product components may be reduced by about 10%, thereby failing to achieve the desired effect.
More preferably, the tank pressure is kept at 0.03-0.06 mpa, and the ventilation is kept at 1: 0.5-1: 1; controlling the rotation speed of a shaking table to be 150-200 rpm, and culturing for 400-480 h; by optimizing the parameters, more ideal fermentation products can be obtained.
The above-mentioned ergometrine fermentation broth can be extracted and purified according to the conventional procedures in the art, and is not particularly limited herein.
The ergot bacteria seed bacterial liquid can be prepared by a conventional method, is not particularly limited, and only provides a preferable preparation method, which comprises the following steps:
1) inoculating ergot strain into a shake flask for culture to prepare shake flask seed liquid;
2) inoculating the shake flask seed liquid into a seed fermentation culture medium (the inoculation amount is 3-5%), and culturing to obtain a seed bacterial liquid; wherein the seed fermentation medium comprises:
20-80g/L of glucose, 10-20g/L of maltodextrin, 1-5g/L of cottonseed cake powder, 2-8g/L of yeast powder and 0.5-1.5g/L of magnesium sulfate;
preferably, the culture conditions are: culturing at 28-32 ℃, under the pressure of 0.03-0.06 Mpa in a seed tank, with the aeration ratio of 1: 0.5-1: 1 and the rotation speed of 100-200 r/min for 96-144 h; the carbon source and the nitrogen source used by the liquid fermentation medium are close to those of the fermentation tank, so that the seeds can adapt to the environment in the fermentation tank as soon as possible after entering the fermentation tank and enter a product metabolism stage in advance; meanwhile, the culture medium can also meet the requirement of normal growth of seeds and can provide sufficient living ergot bacteria.
The fermentation method of the invention also comprises a sterilization operation on the culture medium, and the method can be carried out according to the conventional method by a person skilled in the art, and the invention provides a more preferable sterilization method, which comprises the following steps:
and sterilizing the seed fermentation culture medium and the liquid fermentation culture medium at the temperature of 119-123 ℃ for 30 min.
The preparation method provided by the invention has the advantages of low cost of required materials, simple and convenient operation, proper conditions and stable result, and the fermentation liquor prepared by the method has the content of ergonovine over 3000ug/ml, and has the advantages of high liquid chromatography component and no higher component impurities.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The sterilization operation is carried out at the sterilization temperature of 119-123 ℃ for 30 min.
The concentration of ergometrine described below was measured by HPLC.
The seed tank bacterial liquid adopted in the following embodiments is prepared by the following method:
1) inoculating Clavicipides into a shake flask for culture to prepare shake flask seed liquid;
2) a seed fermentation medium is prepared in a seed fermentation tank, wherein the seed tank is 500L, and the charging amount is 300L. The seed fermentation medium comprises the following components: 18Kg of glucose, 4.5Kg of maltodextrin, 0.9Kg of cottonseed cake powder, 1.2Kg of yeast powder, 0.21Kg of magnesium sulfate and the balance of water; wherein the initial pH of the seed fermentation medium is 7.3;
3) and sterilizing the seed fermentation culture medium, wherein the initial pH of the sterilized seed fermentation culture medium is 7.2.
4) Inoculating the shake flask seed liquid into a seed fermentation culture medium for culture, wherein the inoculation volume percentage is 3-5%, the culture temperature is 28-32 ℃, the seed tank pressure is 0.03-0.06 mpa, the aeration ratio is 1:0.5, the rotating speed is 110 rpm, and culturing is carried out for 100h to obtain the seed tank bacterial liquid.
Example 1
The embodiment provides a preparation method of a fermentation liquid rich in ergometrine, which specifically comprises the following steps:
1) preparing a liquid fermentation culture medium in a liquid fermentation tank, wherein the fermentation tank is 5000L, the charging amount is 3000L, and the fermentation liquid culture medium comprises the following components in percentage by weight: 350Kg of glucose, 90Kg of maltodextrin, 23Kg of yeast powder, 150Kg of soybean meal powder, 3Kg of magnesium sulfate and the balance of water; wherein the initial pH of the liquid fermentation medium is 7.4.
2) And sterilizing the liquid fermentation culture medium, wherein the initial pH value of the sterilized liquid fermentation culture medium is 7.3.
3) Aseptically transferring the seed tank bacterial liquid into a liquid fermentation culture medium, wherein the inoculation volume percentage is 8%, the fermentation temperature is 28-32 ℃, the tank pressure is 0.03-0.06 mpa, the ventilation volume is 1:0.8, the rotating speed is 180 r/min, and the culture is 440 h.
The ergometrine fermentation liquor with the titer of 3325ug/ml is obtained.
Example 2
The embodiment provides a preparation method of a fermentation liquid rich in ergometrine, which specifically comprises the following steps:
1) preparing a liquid fermentation culture medium in a liquid fermentation tank, wherein the fermentation tank is 5000L, the charging amount is 3000L, and the fermentation liquid culture medium comprises the following components in percentage by weight: 250Kg of glucose, 90Kg of maltodextrin, 15Kg of yeast powder, 150Kg of soybean meal powder, 3Kg of magnesium sulfate and the balance of water; wherein the initial pH of the liquid fermentation medium is 7.8;
2) and sterilizing the liquid fermentation culture medium, wherein the initial pH value of the sterilized liquid fermentation culture medium is 7.5.
3) Aseptically transferring the seed tank bacterial liquid into a liquid fermentation culture medium, wherein the inoculation volume percentage is 5%, the fermentation temperature is 28-32 ℃, the tank pressure is 0.03-0.06 mpa, the ventilation volume is 1:0.8, the rotating speed is 180 r/min, and the culture is 432 h.
Placing in a tank to obtain ergometrine fermentation liquor with the titer of 3126 ug/ml.
Example 3
The embodiment provides a preparation method of a fermentation liquid rich in ergometrine, which specifically comprises the following steps:
1) preparing a liquid fermentation culture medium in a liquid fermentation tank, wherein the fermentation tank is 5000L, the charging amount is 3000L, and the fermentation liquid culture medium comprises the following components in percentage by weight: 300Kg of glucose, 180Kg of maltodextrin, 24Kg of yeast powder, 200Kg of soybean meal powder, 4Kg of magnesium sulfate and the balance of water; wherein the initial pH of the liquid fermentation medium is 7.6;
2) and sterilizing the liquid fermentation culture medium, wherein the initial pH value of the sterilized liquid fermentation culture medium is 7.2.
3) Aseptically transferring the seed tank bacterial liquid into a liquid fermentation culture medium, wherein the inoculation volume percentage is 6%, the fermentation temperature is 28-32 ℃, the tank pressure is 0.03-0.06 mpa, the ventilation volume is 1:1, the rotating speed is 180 r/min, and the culture is 456 h.
Placing in a tank to obtain the ergometrine fermentation liquor with the titer of 3323 ug/ml.
Example 4
The embodiment provides a preparation method of a fermentation liquid rich in ergometrine, which specifically comprises the following steps:
1) preparing a liquid fermentation culture medium in a liquid fermentation tank, wherein the fermentation tank is 5000L, the charging amount is 3000L, and the fermentation liquid culture medium comprises the following components in percentage by weight: 275Kg of glucose, 150Kg of maltodextrin, 12Kg of yeast powder, 225Kg of soybean meal, 3Kg of magnesium sulfate and the balance of water; wherein the initial pH of the liquid fermentation medium is 7.5;
2) and sterilizing the liquid fermentation culture medium, wherein the initial pH value of the sterilized liquid fermentation culture medium is 7.2.
3) Aseptically transferring the seed tank bacterial liquid into a liquid fermentation culture medium, wherein the inoculation volume percentage is 10%, the fermentation temperature is 28-32 ℃, the tank pressure is 0.03-0.06 mpa, the ventilation volume is 1:0.6, the rotating speed is 180 r/min, and the culture is carried out for 430 h.
Placing in a tank to obtain ergometrine fermentation liquor with the titer of 3196 ug/ml.
Comparative example 1
This comparative example provides a process for the preparation of a fermentation broth of ergometrine, differing from example 2 only in that,
the soybean meal powder was replaced with soybean meal, and the culture procedure was the same as that described in example 2.
After the seeds enter a fermentation tank, the growth speed is relatively high in the early stage, hypha metabolism is performed in the early stage, unit growth is normal, but when the unit reaches about 2000 in the middle and later stages, the fluctuation becomes slow, and in addition, the main components begin to reduce and the impurity components rise from the liquid phase map.
Comparative example 2
This comparative example provides a process for the preparation of a fermentation broth of ergometrine, differing from example 2 only in that,
the glucose was replaced with sucrose and the culture procedure was as described in example 2.
After the seeds enter a fermentation tank, the seeds are basically in a growth stagnation state in the early stage, and grow until the third day, and enter the slow metabolism; the starting unit and the unit amplitude are relatively low; after 406h of culture, the unit is 1876 and the composition is not ideal.
Comparative example 3
This comparative example provides a process for the preparation of a fermentation broth of ergometrine, differing from example 2 only in that,
the fermentation liquid culture medium comprises the following components in percentage by weight: 250Kg of glucose, 90Kg of maltodextrin, 150Kg of yeast powder, 15Kg of soybean meal, 3Kg of magnesium sulfate and the balance of water; wherein the initial pH of the liquid fermentation medium is 7.0;
after the seeds enter a fermentation tank, hyphae grow crazy, and the culture medium is sticky and has high concentration; no ergonovine units were detected.
Test example 1
Non-toxicity test
And (3) carrying out toxicological detection: the virus control of Luji (No. 2011020).
The fermentation broth provided in examples 1-4, MTD > 10000mg/kg (bw) in rat (female, male) acute oral toxicology test; in an observation period of 14 days, no obvious pathological change, no obvious acute toxic reaction and no animal death are observed in gross disease examination of animals, and the animals are sacrificed at the end of the experiment and are subjected to gross dissection, so that no obvious abnormality is observed in each organ. The result shows that the fermentation liquor belongs to non-toxic grade, and the quality standard is as follows: meets Q/LYC 01-2010.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.