CN109295149B - Method for increasing aureomycin yield by supplementing fresh spore liquid during seed transfer - Google Patents
Method for increasing aureomycin yield by supplementing fresh spore liquid during seed transfer Download PDFInfo
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- CN109295149B CN109295149B CN201811057041.9A CN201811057041A CN109295149B CN 109295149 B CN109295149 B CN 109295149B CN 201811057041 A CN201811057041 A CN 201811057041A CN 109295149 B CN109295149 B CN 109295149B
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Abstract
The invention belongs to the technical field of antibiotic preparation by microbial fermentation, and particularly relates to a patent application of a method for improving the yield of aureomycin. The method comprises the following steps: fermenting to prepare seed liquid, supplementing fresh spore liquid into the seed liquid, fermenting to prepare aureomycin and the like. In the application, the inventor adds a fresh spore liquid mode during the seed transferring, so that the mycelium quantity in the early and middle stages of fermentation is effectively improved, the mycelium quantity is still higher in the later stage of fermentation, the stabilization period is prolonged by delaying the integral decay rate of the mycelium, and the integral fermentation efficiency is effectively improved. Preliminary detection results show that after the process is optimized, the fermentation level of the aureomycin can be improved by about 10 percent, and the aureomycin has better practical application prospect, thereby having better practical value and popularization and application significance.
Description
Technical Field
The invention belongs to the technical field of antibiotic preparation by microbial fermentation, and particularly relates to a patent application of a method for improving the yield of aureomycin.
Background
The antibiotic is an indispensable additive in the large-scale feeding of poultry and livestock, and the aureomycin is a commonly used antibiotic type. Aureomycin is a secondary metabolite of streptomyces aureofaciens, so the prior aureomycin antibiotic is mainly prepared by the processes of seed culture of streptomyces aureofaciens, fermentation culture, mixing fermentation liquor after fermentation with calcium carbonate, plate-frame filtration to obtain wet filter cakes, drying and crushing the wet filter cakes and the like.
With the increasing scale and concentration of poultry and livestock, the demand for aureomycin in the field of feed is increasing. In the prior art, in order to improve the fermentation efficiency of aureomycin, generally speaking, a more fundamental technical idea is to obtain a high-yield strain by screening in modes of strain mutagenesis, strain screening and the like, but the method is time-consuming and labor-consuming, and has great limitation because the induction or screening effect is unpredictable. The direct way of improving the fermentation efficiency of aureomycin is to optimize the culture medium in the fermentation process or improve and adjust the fermentation process, so as to improve the fermentation efficiency. In terms of culture medium optimization, due to the fact that types of related nutrient components are various and the production cost is limited, the improvement difficulty is often large, and the effect is not obvious enough sometimes, and therefore the practical production process is limited more. In the aspect of improving a specific fermentation process, the improvement difficulty is high due to more process flows, but the effect is easily and directly reflected after the process flows are improved, so the improvement of the fermentation process flows is still the mainstream in terms of improving and improving the fermentation effect.
Disclosure of Invention
The method is realized by adding fresh spore liquid during seed transferring, and then improving the hypha concentration in a fermentation tank, and belongs to further optimization of the fermentation process flow.
The technical solution adopted in the present application is detailed as follows.
A method for increasing fermentation yield of aureomycin by supplementing fresh spore liquid during seed transfer comprises the following steps:
(1) fermentation preparation of seed liquid
Under aseptic conditions, adding sterile distilled water (about 30mL per bottle generally) into a culture bottle containing spores, scraping the spores by using a sporulation shovel, and pouring the spore liquid into a sterilized inoculation bottle to obtain a fresh spore liquid;
specifically, when the seed solution is prepared, inoculating 0.0015% volume ratio inoculum size of fresh spore solution into a sterilized seed culture medium, keeping the pressure of a tank at 0.040-0.060 Mpa at 26-33 ℃, and culturing for 19-25 hours to obtain the seed solution;
the seed culture medium is a liquid culture medium, and in each liter of culture medium, 20-40 g of soybean cake powder, 30-50 g of corn starch, 8-14 g of yeast powder, 1-2 g of sodium chloride, 2-4 g of ammonium sulfate, 3-5 g of calcium carbonate, 0.2-0.4 g of magnesium sulfate, 0.3-0.5 g of monopotassium phosphate and 0.1-0.2 g of soybean oil are added;
after the seed liquid is prepared, adding fresh spore liquid and mixing uniformly; in terms of volume ratio, the adding amount of the fresh spore liquid is not more than 18% of the inoculation amount during the preparation of the seed liquid, and preferably, the adding amount of the fresh spore liquid is 5-15% of the inoculation amount during the preparation of the seed liquid, namely, if the inoculating amount of the fresh spore liquid during the preparation of the seed liquid is 100mL, 5-15 mL of fresh spore liquid is supplemented at the moment; under further preferable conditions, the adding amount of the fresh spore liquid is 10 percent of the inoculation amount when the seed liquid is prepared;
(2) fermentation preparation of aureomycin
Inoculating the seed solution added with the fresh spore solution in the step (1) into a fermentation tank, and fermenting according to the prior art to prepare aureomycin; specifically, the method comprises the following steps:
transferring seeds (inoculating the seed liquid added with the fresh spore liquid in the step (1)) to a fermentation culture medium, and controlling and adjusting the viscosity of the fermentation culture medium to be 1-35 seconds;
the fermentation medium is a liquid medium, and each liter of the fermentation medium contains 20-25 g of peanut cake powder, 6-15 g of soybean cake powder, 100-120 g of corn starch, 1-5 g of yeast powder, 0.1-0.5 g of amylase, 2-3 g of sodium chloride, 5-8 g of ammonium sulfate, 5-10 g of calcium carbonate, 0.1-0.3 g of magnesium sulfate and 0.1-0.2 g of soybean oil;
in the fermentation process, controlling the pressure of a fermentation tank to be 0.01-0.04 Mpa and the culture temperature to be 28-34 ℃; controlling the fermentation temperature to be 29 +/-3 ℃ for fermentation culture after the hyphae grow over the fermentation culture medium;
in the fermentation culture process, if the pH value of the fermentation culture medium is reduced to about 5.7-6.0, introducing ammonia gas or adding ammonia water, and adjusting and controlling the pH value in the fermentation process to be stabilized at about 5.9 +/-0.5;
after the seed liquid added with the fresh spore liquid in the step (1) is transferred to a fermentation medium, the total fermentation period is controlled to be 90-124 hours;
and after fermentation is finished, performing titer determination and evaluation, and sending the fermentation liquor to an extraction workshop for extraction to further prepare the aureomycin.
After research is carried out on the growth process of the streptomyces aureofaciens, the inventor thinks that in the fermentation process, the streptomyces aureofaciens hyphae needs to go through the processes of a delay period, a logarithmic growth period, a stabilization period, a decay period and the like, the production rate of the aureomycin is highest when the hyphae is in the stabilization period, the hyphae starts to crack after the decay period, and the rate of the aureomycin is rapidly reduced at the moment. Aiming at the characteristics, how to properly prolong the stationary phase, delay the decay phase or increase the number of viable bacteria in the decay phase also becomes a main way for improving the fermentation efficiency of the aureomycin.
In the application, the inventor adds a fresh spore liquid mode during the seed transferring, so that the mycelium quantity in the early and middle stages of fermentation is effectively improved, the mycelium quantity is still higher in the later stage of fermentation, the stabilization period is prolonged by delaying the integral decay rate of the mycelium, and the integral fermentation efficiency is effectively improved. Preliminary detection results show that after the process is optimized, the fermentation level of the aureomycin can be improved by about 10 percent, and the aureomycin has better practical application prospect, thereby having better practical value and popularization and application significance.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
During the fermentation, the relevant production process is carried out on the actual production line of the applicant, specifically, 8m for preparing the seed liquid3The seed tank for production adopts 60m for actually preparing aureomycin3In the fermentation tank for production, an online detection method is adopted for temperature detection and monitoring, a mode of combining online monitoring and offline detection is respectively adopted for pH detection as a final detection result, and a mode of combining a chemical method and a high performance liquid method is adopted for titer detection as a final detection result; in addition, the preparation process of the concrete aureomycin semi-finished product belongs to the prior art, so that the process conditions are not described in detail in part regardless of the application.
Comparative example
For comparison, this example is first summarized below in terms of the fermentative preparation of aureomycin as employed in the applicant's prior art.
(1) Fermentation preparation of seed liquid
Under aseptic conditions, adding sterile distilled water (about 30mL per bottle generally) into a culture bottle containing spores, scraping the spores by using a sporulation shovel, and pouring the spore liquid into a sterilized inoculation bottle to obtain a fresh spore liquid;
when the seed solution is prepared specifically, inoculating 0.0015% volume ratio inoculum size of fresh spore solution into sterilized seed culture medium, maintaining the pressure of the tank at about 0.050Mpa at 32 ℃, and culturing for 24 hours to obtain seed solution;
the seed culture medium is a liquid culture medium, and each liter of the culture medium comprises 30g of soybean cake powder, 40g of corn starch, 11g of yeast powder, 1.5g of sodium chloride, 3g of ammonium sulfate, 4g of calcium carbonate, 0.3g of magnesium sulfate, 0.4g of monopotassium phosphate and 0.15g of soybean oil;
(2) fermentation preparation of aureomycin
Inoculating the seed liquid obtained in the step (1) into a fermentation tank, and fermenting according to the prior art to prepare aureomycin; specifically, the method comprises the following steps:
after the seeds are transferred to a fermentation medium, controlling and adjusting the viscosity of the fermentation medium to be about 15 seconds;
the fermentation medium is a liquid culture medium, and each liter of the fermentation medium contains 20g of peanut cake powder, 12g of soybean cake powder, 120g of corn starch, 1g of yeast powder, 0.5g of amylase, 2g of sodium chloride, 8g of ammonium sulfate, 5g of calcium carbonate, 0.15g of magnesium sulfate and 0.1g of soybean oil;
in the fermentation process, the pressure of the fermentation tank is controlled to be about 0.05Mpa, and the culture temperature is controlled to be about 32 ℃; controlling the fermentation temperature to be about 29 ℃ after the hyphae grow over the fermentation medium for fermentation culture;
in the fermentation culture process, if the pH value of the fermentation culture medium is reduced to about 5.9, introducing ammonia gas, and adjusting and controlling the pH value in the fermentation process to be stabilized at about 5.9 +/-0.5;
after the seeds in the step (1) are transferred to a fermentation medium, controlling the total fermentation period to be 110 hours;
and after fermentation is finished, performing titer determination and evaluation, and sending the fermentation liquor to an extraction workshop for extraction to further prepare the aureomycin.
Example 1
The method for increasing the fermentation yield of aureomycin by supplementing fresh spore liquid during the seed transfer provided by the embodiment has the specific operation process basically the same as that of the control example, and is only adjusted as follows:
when the seed solution is prepared by fermentation in the step (1), adding fresh spore solution and uniformly mixing the fresh spore solution after the seed solution is prepared, wherein the adding amount of the spore solution is 5% of the amount of the spore suspension inoculated into the seed tank (namely, the adding amount of the spore in the original seed culture medium is 100ml, and then additionally adding 5ml of fresh spore solution); the seed solution added with the fresh spore liquid is inoculated into a fermentation culture medium for preparing the aureomycin.
The bacterial concentration, the fermentation broth tank-placing titer and the yield in the fermentation process of different fresh spore liquid adding ratios and the existing control examples are respectively measured and counted, and the results are shown in the following tables 1 and 2.
The following statistical data are the measurement statistical results of a specific certain batch (that is, in actual operation, when the technical scheme in this embodiment is adopted, the operation is performed in comparison with the batch setting, and in the case of different production batches, the statistical aspects of the data results are slightly different).
TABLE 1 statistics of the results of the concentration measurements at different fermentation cycles
TABLE 2 fermentation broth tank discharge titer, yield results
Analysis of the data in the table shows that, for the bacterial concentration index in the fermentation process, after the fresh spore solution is supplemented, the bacterial count can be better ensured in the early and middle stages of fermentation, the hypha fading time is effectively delayed, and the titer and the final yield are reflected, namely, the titer (improved by about 9.3%) and the final yield (improved by about 2.4%) of the fermentation liquor are effectively improved by supplementing the fresh spore solution, and a better application effect is shown.
Example 2
Referring to example 1, only the ratio of the amount of spore liquid added in this example to 10% of the amount of spore suspension inoculated into the seed tank was adjusted (i.e., the amount of spore added to the stock seed medium was 100ml, and 10ml of fresh spore liquid was additionally added).
The concentrations of the bacteria, the titer of the fermentation liquid in the tank and the yield of the fermentation liquid in the control and the example after a certain batch of actual experiments are respectively measured and counted, and the results are shown in the following tables 3 and 4.
TABLE 3 statistics of the results of the concentration measurements at different fermentation cycles
TABLE 4 fermentation broth tank discharge titer, yield results
Analysis of the data in the table shows that, regarding the bacterial concentration index in the fermentation process, after 10% of fresh spore liquid is added, compared with the addition ratio in example 1, the effect of maintaining the bacterial number in the early and middle stages of fermentation is better, and the titer (improved by about 9.7%) and the final yield (improved by about 3.0%) of the fermentation liquid are improved in terms of reaction to the titer and the final yield.
Example 3
Referring to example 1, the ratio of the amount of spore liquid added in this example to 15% of the amount of spore suspension inoculated into the seed tank was adjusted (i.e., the amount of spore added to the stock seed medium was 100ml, and 15ml of fresh spore liquid was additionally added).
The concentrations of the bacteria, the titer of the fermentation liquid in the tank and the yield of the fermentation liquid in the control and the examples after a certain batch of actual experiments are respectively measured and counted, and the results are shown in the following tables 5 and 6.
TABLE 5 statistics of the results of the concentration measurements at different fermentation cycles
TABLE 6 fermentation broth tank discharge titer, yield results
Analysis of the data in the table above shows that, regarding the bacterial concentration index in the fermentation process, after 15% of fresh spore liquid is supplemented, compared with the addition ratio in examples 1 and 2, the effect of maintaining the number of bacteria in the early stage and the middle stage of fermentation is better, but because the hyphae are too dense, the number of bacteria in the later stage of fermentation is rapidly reduced inversely, the titer and the final yield are reflected, and the effect of improving the titer (by about 2.9%) and the final yield (by about 1.8%) of the fermentation liquid is obviously inferior to the improvement effect of examples 1 and 2.
Example 4
Further, referring to example 1, the amount of spore liquid added in this example was adjusted to 20% of the amount of spore suspension inoculated into the seed tank (i.e., the amount of spores added to the stock seed medium was 100ml, and then 20ml of fresh spore liquid was additionally added).
The concentrations of the bacteria, the titer of the fermentation liquid in the tank and the yield of the fermentation liquid in the control and the examples after a certain batch of actual experiments are respectively measured and counted, and the results are shown in the following tables 7 and 8.
TABLE 7 statistics of the results of the concentration measurements at different fermentation cycles
TABLE 8 fermentation broth tank discharge titer, yield results
Analysis of the data in the table shows that, regarding the bacterial concentration index in the fermentation process, after 20% of fresh spore liquid is supplemented, compared with the addition ratio in examples 1, 2 and 3, the bacterial count in the early stage of fermentation keeps better effect, but because the hyphae are too dense, the bacterial count is rapidly reduced in the middle and later stages of fermentation, and the titer and final yield of the fermentation liquid are reduced to a certain extent in the aspect of reaction to the titer and final yield, so that the addition amount of the fresh spore liquid obviously has certain requirements.
Claims (6)
1. A method for increasing fermentation yield of aureomycin by supplementing fresh spore liquid during seed transfer is characterized by comprising the following steps:
(1) fermentation preparation of seed liquid
Under the aseptic condition, adding aseptic distilled water into a culture bottle containing spores, scraping the spores, and then pouring the spore liquid into a sterilized inoculation bottle to obtain fresh spore liquid;
specifically, when the seed solution is prepared, fresh spore solution is inoculated into a sterilized seed culture medium according to the volume ratio of 0.0015%, the temperature is 26-33 ℃, the pressure of a tank is kept at 0.040-0.060 Mpa, and the seed solution is cultured for 19-25 hours;
after the seed liquid is prepared, adding fresh spore liquid and mixing uniformly; the adding amount of the fresh spore liquid is 5-15% of the inoculation amount in the preparation of the seed liquid in terms of volume ratio;
(2) fermentation preparation of aureomycin
Inoculating the seed solution added with the fresh spore solution in the step (1) into a fermentation tank, and fermenting to prepare aureomycin;
the process for preparing the aureomycin by fermentation specifically comprises the following steps:
after the seeds are transferred to a fermentation medium, controlling and adjusting the viscosity of the fermentation medium to be 1-35 seconds;
in the fermentation process, controlling the pressure of a fermentation tank to be 0.01-0.04 Mpa and the culture temperature to be 28-34 ℃; controlling the fermentation temperature to be 29 +/-3 ℃ for fermentation culture after the hyphae grow over the fermentation culture medium;
in the fermentation culture process, if the pH value of the fermentation culture medium is reduced to a range of 5.7-6.0, introducing ammonia gas or adding ammonia water, and adjusting and controlling the pH value in the fermentation process to be stable at 5.9 +/-0.5;
after the seed liquid added with the fresh spore liquid in the step (1) is transferred to a fermentation medium, the total fermentation period is controlled to be 90-124 hours;
and after fermentation is finished, sending the fermentation liquor to an extraction workshop for extraction, and further preparing the aureomycin.
2. The method for increasing fermentation yield of aureomycin according to claim 1 by supplementing fresh spore liquid during seed transfer, wherein in step (1), the seed culture medium is a liquid culture medium, and each liter of the culture medium contains 20-40 g of soybean cake powder, 30-50 g of corn starch, 8-14 g of yeast powder, 1-2 g of sodium chloride, 2-4 g of ammonium sulfate, 3-5 g of calcium carbonate, 0.2-0.4 g of magnesium sulfate, 0.3-0.5 g of potassium dihydrogen phosphate and 0.1-0.2 g of soybean oil.
3. The method for increasing fermentation yield of aureomycin according to claim 2 by adding fresh spore liquid during seed culture, wherein the seed culture medium contains 30g of soybean cake powder, 40g of corn starch, 11g of yeast powder, 1.5g of sodium chloride, 3g of ammonium sulfate, 4g of calcium carbonate, 0.3g of magnesium sulfate, 0.4g of potassium dihydrogen phosphate and 0.15g of soybean oil per liter of the culture medium.
4. The method for improving fermentation yield of aureomycin according to claim 1, by supplementing fresh spore liquid during the transfer, wherein the fresh spore liquid is added in a proportion of 10% of the inoculum size during the preparation of the seed liquid by volume ratio.
5. The method for improving fermentation yield of aureomycin by supplementing fresh spore liquid during seed transfer according to claim 1, characterized in that the fermentation medium is a liquid medium, and each liter of the fermentation medium contains 20-25 g of peanut cake powder, 6-15 g of soybean cake powder, 100-120 g of corn starch, 1-5 g of yeast powder, 0.1-0.5 g of amylase, 2-3 g of sodium chloride, 5-8 g of ammonium sulfate, 5-10 g of calcium carbonate, 0.1-0.3 g of magnesium sulfate and 0.1-0.2 g of soybean oil.
6. The method for increasing fermentation yield of aureomycin according to claim 5 by adding fresh spore liquid during the transfer of the seed, wherein the fermentation medium contains 20g of peanut meal, 12g of soybean meal, 120g of corn starch, 1g of yeast powder, 0.5g of amylase, 2g of sodium chloride, 8g of ammonium sulfate, 5g of calcium carbonate, 0.15g of magnesium sulfate and 0.1g of soybean oil per liter of the fermentation medium.
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| GB1357538A (en) * | 1970-11-03 | 1974-06-26 | Prodotti Antibiotici Spa | New antibiotic |
| TR24186A (en) * | 1988-04-11 | 1991-05-30 | Monsanto Co | YOENTEM TO INCREASE THE ACTIVITY OF POISONOUS POISONS |
| CN1542137A (en) * | 2003-11-04 | 2004-11-03 | 福建师范大学 | Technology for reducing the fermentation period of chlorotetracycline |
| CN103074245A (en) * | 2012-08-22 | 2013-05-01 | 驻马店华中正大有限公司 | Method for inoculation by adopting streptomyces aureofaciens freezing shake flask hypha to replace spore |
| CN103614445B (en) * | 2013-03-29 | 2015-05-27 | 驻马店华中正大有限公司 | A fermentation production method for aureomycin by utilizing mycoprotein in place of a portion of yeast powder |
| CN104988103A (en) * | 2015-08-07 | 2015-10-21 | 金河生物科技股份有限公司 | Aureomycin strain breeding culture medium and strain breeding method |
| CN105063155B (en) * | 2015-09-25 | 2018-09-11 | 驻马店华中正大有限公司 | Ferment of DM culture medium and the Ferment of DM production method for utilizing the culture medium |
| CN106047977A (en) * | 2016-08-22 | 2016-10-26 | 浦城正大生化有限公司 | Chlortetracycline fermentation production method using refined protein powder |
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