CN112391432B - Fermentation medium and fermentation method for producing pingyangmycin - Google Patents
Fermentation medium and fermentation method for producing pingyangmycin Download PDFInfo
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Abstract
The invention discloses a fermentation medium and a fermentation method for producing pingyangmycin. The fermentation medium comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium. The fermentation culture medium disclosed by the invention is used for fermentation of pingyangmycin, so that the yield of pingyangmycin can be greatly improved (the yield can be 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), the fermentation cost is obviously reduced, and a good foundation is laid for industrial production of pingyangmycin.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and particularly relates to a fermentation medium and a fermentation method for producing pingyangmycin.
Background
Influence of organic nitrogen source on fermentation component change of bleomycin in the third pharmaceutical factory (corn poppy 24733, longevity, plum.)]Microbiological report, 1980,7 (4): 14-17.) was obtained in 1976The fact that the fermentation titer of the pingyangmycin can be obviously improved by adding the corn steep liquor is pointed out, and the research finds that the fermentation titer of the pingyangmycin is related to the existence of spermidine in the corn steep liquor. The conditions influencing the fermentation of pingyangmycin are studied by a shaking test, and the fermentation conditions are 0.5 percent of glucose, 7 percent of maltose, 0.3 percent of NaCl0.3 percent of NaNO 3 0.2％，KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％、CuSO 4 Shake flask fermentations were run in 0.01%, ph6.5, 150ml/750ml Erlenmeyer flask conditions and indicated that the formation of component A5 was promoted during the fermentation by feeding corn steep liquor (1.5% in the 48 th hour at the beginning of the fermentation followed by 1.5% every 24 hours) to make A5 the major component, increasing from 14.8% to 56.8%, regardless of the strain used. In addition, researches show that in other organic nitrogen source proportioning experiments in the basic culture medium, other formulas are unchanged, corn steep liquor is supplemented under the conditions of 2.5% of peanut cake powder and 2.5% of cicada pupa powder, and the yield can be improved by 3 times. The pingyangmycin detection method in the document adopts thin layer chromatography, and compared with the existing liquid phase detection method, the thin layer chromatography method has low accuracy, larger interference and small referential significance of the pingyangmycin fermentation titer.
In precursor experiments, it was found by the inventors of the present invention that the addition of spermidine at the beginning of fermentation resulted in 100% yield of bleomycin-producing bacteria, whereas the addition of spermidine hydrochloride after sterile filtration of spermidine hydrochloride prior to inoculation to the fermentation medium before inoculation gave a 466% increase in A5 over the control. And the main component is A5. However, the precursor used in the method is expensive and has high production cost, so that the production requirement cannot be met.
Disclosure of Invention
The invention aims to overcome the defects of high production cost, difficult expanded production and the like of fermentation bleomycin in the prior art and provides a fermentation culture medium and a fermentation method for producing bleomycin.
The invention adopts the following technical scheme to solve the technical problems:
the invention provides a fermentation medium for producing pingyangmycin, which comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9 percent, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium.
Preferably, the corn steep liquor content is 1.2-2.0%, such as 1.4%, 1.6%, or 1.8%; the percentage is the mass percentage of the fermentation medium.
Wherein, the content of the soybean cake powder is preferably 1.1 to 3.5%, more preferably 1.7 to 2.9%, and further more preferably 1.9 to 2.7%, such as 2.1%, 2.3% or 2.5%; the percentage is the mass percentage of the fermentation medium.
The soybean cake powder is preferably cold-pressed soybean cake powder.
The nitrogen source as described above preferably further comprises one or more selected from the group consisting of cottonseed meal, peanut meal, albumen powder, feather meal, bran, medium temperature soybean meal, yeast powder and hot-pressed soybean meal, preferably peanut meal and yeast powder.
Wherein, the content of the peanut cake is preferably 0.6-2.4%, preferably 0.8-1.6%, such as 1.0%, 1.2% or 1.4%, and the percentage is the mass percentage of the fermentation medium.
Preferably, the total content of the peanut cake and the cold-pressed soybean cake powder is 3.5%, and the percentage is the mass percentage of the fermentation medium.
In the present invention, the content of the carbon source may be conventional in the art; preferably, the content of the carbon source is 3.8-9.8%, and the percentage is the mass percentage of the fermentation medium.
The carbon source preferably comprises glucose and starch, and the content of the glucose is 0.3-0.8%, preferably 0.5%; the content of the starch is 3.5-9%, preferably 6%, and the percentage is the mass percentage of the fermentation medium; the starch is preferably alpha-amylase liquefied starch or alpha-amylase gelatinized starch.
The inorganic salt described in the present invention preferably includes NaNO 3 、KH 2 PO 4 、ZnSO 4 ·7H 2 O and CuSO 4 ·5H 2 O; wherein:
the NaNO 3 The content of (C) is preferably 0.2%, and the KH is 2 PO 4 The content of (b) is preferably 0.1%, and the ZnSO 4 ·7H 2 The content of O is preferably 0.05%, and the CuSO is 4 ·5H 2 The content of O is preferably 0.01 percent, and the percentage is the mass percentage of the fermentation medium.
In a preferred embodiment of the invention, the fermentation medium comprises 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), naNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 0.05% of O and CuSO 4 ·5H 2 0.01 percent of O, wherein the percentage is the mass percentage of the fermentation medium.
The invention provides an application of the fermentation medium in fermentation production of pingyangmycin.
The invention provides a fermentation method of pingyangmycin, which comprises the following steps:
(1) Inoculating seed solution of Streptomyces pingyangensis (Streptomyces pingyangensis n.sp) into the fermentation culture medium, and performing liquid fermentation culture to obtain fermentation broth;
(2) Separating the pingyangmycin from the fermentation liquor.
The streptomyces verticillata sun-exposed variety described in the step (1) is preferably the streptomyces verticillata sun-exposed variety with the number of CPCC 200554.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the fermentation of the pingyangmycin by using the fermentation culture medium can greatly improve the yield of the pingyangmycin, the titer can be improved to 32.11 mu g/ml from the original 13 mu g/ml (namely, the yield can reach 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), and simultaneously, the fermentation cost is obviously reduced, thereby laying a good foundation for the industrial production of the pingyangmycin.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.
First, experimental material
1. Instrument for measuring the position of a moving object
2. Reagent
3. Bacterial strain
The invention adopts the strain Streptomyces pingyangensis n.sp purchased from China pharmaceutical microorganism strain preservation management center (CPCC) and the strain number is CPCC 200554.
4. Culture medium
A first culture medium: slant medium (g/L): soluble starch (20 g), KNO 3 (1g)，K 2 HPO 4 (0.5g)，MgSO 4 ·7H 2 O(0.5g)，NaCl(0.5g)，FeSO 4 ·7H 2 O (0.01 g), corn steep liquor (0.5 g), agar 20g, pH value is 7.4-7.6
And (2) culture medium II: seed fermentation culture medium: 3% of corn steep liquor, 2% of soybean cake meal, 0.2% of pomegranate flower yeast powder, 1% of glucose, 1% of maltodextrin and KH 2 PO 4 0.1％，ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％；pH 6.5。
And (3) culture medium III: basic fermentation medium: 0.75% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of pomegranate flower yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％；KH 2 PO 4 0.1％,ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％；pH 6.5。
Second, the method
1. Culture method
The invention comprises the following processes:
and (3) sterilization conditions: 121 ℃ for 20min
Under the aseptic condition, a freeze-dried tube in which the altemaria verticillata sun-exposed variety is preserved is inoculated into an eggplant bottle filled with a slant culture medium by using a sterilized inoculating needle for culture, the light white thalli on the surface can be seen on the next day of slant culture, the color deepens on the third day or the fourth day, a small amount of white spore is seen to generate, the culture is continued until a large amount of white spores can be seen on the surface of the culture medium, and the culture medium matrix is changed into ginger yellow due to the generation of the altemaria verticillata sun-exposed variety pigment. The slant can be used for passage or inoculation.
Preparing a seed culture medium according to a seed culture medium formula, slightly scratching a slant culture medium with spores by using a sterilization inoculating shovel under an aseptic condition, digging a slant culture medium which is as thin as possible and is full of spores and about 0.8cm multiplied by 1.5cm, inoculating the slant culture medium into the killed seed culture medium, shaking at the temperature of 240rpm for 24-28 hours at 29 ℃, changing the color of a culture in a shaking bottle from dark brown to yellow turbid substances, shaking, having a flowing sense, having high viscosity and being uniformly and smoothly adhered to the wall. The hyphae are in a ball shape and spread to the periphery, the hyphae are long and free of foreign bacteria, and the pH value is 6.9-7.0.
Inoculating the cultured shake flask seeds into a sterilized fermentation culture medium, wherein the inoculation amount is 3ml/35ml, the shaking table is 29 ℃, the rotation speed is 240rpm, and the days are 8-9. The fermentation shake flask becomes thick and deepens color in about 24 hours, a layer of white spore layer appears on the upper wall of the shake flask in 3-4 days, the viscosity of the shake flask is not reduced in 8-9 days, microscopic examination shows that hyphae are broken more, dense vacuoles are generated in the hyphae, the hyphae are thin and weak, and the pH value is 7.0-8.0 in the later stage of secondary metabolism.
In the invention, the conditions except the nitrogen source in the three formulas of the culture medium are not changed, and multiple shake flask fermentation experiments are carried out by increasing the types of the nitrogen source and adjusting the proportion and the total amount of the corresponding nitrogen source, so that the fermentation culture medium formula with higher yield is finally obtained.
2. Detection method
The fermentation broth was centrifuged at 12000rpm, the supernatant was collected, methanol was added for precipitation of protein impurities according to the ratio of the supernatant to methanol of 1:3, the supernatant was collected by centrifugation at 12000rpm, and the fermentation unit was measured by HPLC.
1) Mobile phase: mobile phase a = sodium hexane sulfonate 7.53g/L, disodium EDTA 3.72g/L, acetic acid 0.08mol/L, pH adjusted to 4.3 with pure ammonia water, mobile phase B = methanol-acetonitrile (70-30)
2) Liquid phase conditions: a chromatographic column: agilent Eclipse Plus C18 (4.6 mm. Times.250mm, 5 μm);
the flow rate is 1.0mL/min; injecting 20 mu L of sample; the detection wavelength is 254nm; the column temperature is 40 ℃; gradient elution.
Unless otherwise specified, "%" referred to in the following examples is a mass percentage of a specific component based on the total mass of the medium.
The starch (alpha-amylase liquefaction) in the following examples can be replaced by alpha-amylase gelatinized starch, and the effect of the culture medium containing the starch and the starch is basically the same under the condition that other components are kept unchanged.
Example 1
Taking the basic fermentation medium (the third culture medium) as a reference, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 0.75% to 1%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 1
| Culture medium III | Example 1 | |
| Corn steep liquor dosage (%) | 0.75 | 1 |
| Relative potency | 1.00 | 1.04 |
The optimized formula comprises: 1% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 0.05% of O and CuSO 4 ·5H 2 O 0.01％。
The titer obtained by fermentation using the above medium III was 13. Mu.g/ml.
Example 2
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1% to 1.2%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 2
| Corn steep liquor dosage (%) | 1 | 1.2 |
| Relative potency | 1.00 | 1.12 |
The optimized formula comprises: 1.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％；KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％。
Example 3
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.2% to 1.4%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 3
| Corn steep liquor dosage (%) | 1.2 | 1.4 |
| Relative potency | 1 | 1.09 |
The optimized formula is as follows: corn steep liquor 1.4%, cold-pressed yellow3.5% of bean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％。
Example 4
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.4% to 1.6%, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 4
| Corn steep liquor dosage (%) | 1.4 | 1.6 |
| Relative potency | 1 | 1.18 |
The optimized formula comprises: 1.6 percent of corn steep liquor, 3.5 percent of cold-pressed soybean cake powder, 0.2 percent of yeast powder, 0.5 percent of glucose, 6 percent of starch (alpha-amylase liquefaction), naNO 3 0.2％；KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％。
Example 5
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.6 percent to 1.8 percent, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid chromatography.
TABLE 5
| Corn steep liquor dosage (%) | 1.6 | 1.8 |
| Relative potency | 1 | 1.12 |
The optimized formula is as follows: 1.8% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％；KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％、CuSO 4 ·5H 2 O 0.01％。
Example 6
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.8% to 2.0%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 6
| Corn steep liquor dosage (%) | 1.8 | 2.0 |
| Relative potency | 1 | 1.17 |
The optimized formula is as follows: 2.0 percent of corn steep liquor, 3.5 percent of cold-pressed soybean cake powder, 0.2 percent of yeast powder, 0.5 percent of glucose, 6 percent of starch (alpha-amylase liquefaction), naNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％、CuSO 4 ·5H 2 O 0.01％。
Example 7
Taking a basic culture medium formula as a reference, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 2.0 percent to 2.2 percent, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid chromatography.
TABLE 7
| Corn steep liquor dosage (%) | 2.0 | 2.2 |
| Relative potency | 1 | 1.13 |
The optimized formula is as follows: 2.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose and starch (alpha-starch)Enzymatic liquefaction) 6% NaNO 3 0.2％；KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％。
Example 8
Taking the optimized culture medium formula as a reference, under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, under the condition that the contents of corn steep liquor and the pomegranate flower yeast powder are unchanged, the content of cold-pressed soybean cake powder in the basic culture medium is changed from 3.5 percent to 2.5 percent, the rest 1 percent of cold-pressed soybean cake powder is subjected to substitution experiments by adopting cottonseed powder, peanut cake powder, pomegranate flower yeast powder, protein powder, feather meal, bran, medium-temperature soybean cake powder and hot-pressed soybean cake powder, and the mixture is subjected to shake flask fermentation for 8 days and a high-efficiency liquid phase fermentation unit is adopted.
TABLE 8
The optimized formula is as follows: 2.2% of corn steep liquor, 2.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％；CuSO 4 ·5H 2 O 0.01％。
Example 9
Taking the optimized culture medium formula as a reference, performing an experiment for adjusting the content ratio of the cold-pressed soybean cake powder to the peanut cake powder under the condition that the total content of the cold-pressed soybean cake powder and the peanut cake powder is 3.5% under the condition that the components such as carbon source, inorganic salt, durian yeast powder and the like are kept unchanged, performing shake-flask fermentation for 8 days, and measuring a fermentation unit by using a high performance liquid phase.
TABLE 9
| Peanut cake powder (%) | 0.6 | 0.8 | 1.0 | 1.2 | 1.4 | 1.6 | 1.8 | 2.0 | 2.2 | 2.4 |
| Relative potency | 0.97 | 0.98 | 1.00 | 1.01 | 1.03 | 0.98 | 0.95 | 0.92 | 0.86 | 0.78 |
According to the results of the above experiments, the optimized formula is as follows: 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 O 0.05％、CuSO 4 ·5H 2 O 0.01％。
The titer obtained after the final fermentation using this optimized formulation was 32.11. Mu.g/ml.
Claims (4)
1. The fermentation culture medium for producing pingyangmycin is characterized by comprising 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of durian yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of alpha-amylase liquefied starch, and NaNO 3 0.2％、KH 2 PO 4 0.1％、ZnSO 4 ·7H 2 0.05% of O and CuSO 4 ·5H 2 And O is 0.01 percent, and the percent is the mass percent of the fermentation medium.
2. Use of a fermentation medium according to claim 1 for the fermentative production of pingyangmycin.
3. A fermentation method of pingyangmycin is characterized by comprising the following steps:
(1) Streptomyces verticillatus Pingyang variant (Streptomyces pingyangensis n.sp) Inoculating the seed solution into the fermentation medium of claim 1 to perform liquid fermentation culture to obtain a fermentation solution;
(2) Separating pingyangmycin from the fermentation liquor.
4. The fermentation process of claim 3, wherein the Streptomyces verticillatus Pingyang variety is Streptomyces verticillatus Pingyang variety numbered CPCC 200554.
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| CN102747115A (en) * | 2011-04-19 | 2012-10-24 | 上海医药工业研究院 | Fermentation medium for fermentation production of antibiotic calicheamicin gamma1 I, fermentation method suitable for the fermentation medium and use of the fermentation medium |
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