CN116218747A - Streptomyces avermitilis fermentation medium based on biomass and fermentation method - Google Patents
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Abstract
The invention relates to a fermentation medium and a fermentation method of streptomyces avermitilis based on biomass, wherein the fermentation medium comprises 30-32 g/L of soybean cake powder, 172-182 g/L of starch, 10-12 g/L of yeast powder, 0.2-0.3 g/L of ammonium sulfate, 1-2 g/L of light calcium carbonate, 4-5 g/L of defoamer, 0.2-0.3 g/L of solid alkali, and the addition amount of amylase is 0.025% of the weight of starch, and the balance of water. The invention optimizes the fermentation medium and the fermentation condition by two indexes of biomass and fermentation titer, greatly reduces the gap between the level of industrial production and laboratory level, and improves the titer level of industrial production.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a biomass-based streptomyces avermitilis fermentation medium and a fermentation method.
Background
The avermectin is a multicomponent biological pesticide and miticide and medical intermediate produced by fermentation of streptomyces avermitilis. The fermentation production level has been greatly improved through 20 years of strain improvement, formula and control process optimization. However, at present, whether laboratory or industrial production is performed, the fermentation titer is an index as long as the fermentation method and fermentation medium are optimized for the purpose of the titer; however, through many studies of laboratory and industrial level fermentation titers versus biomass by the present company inventors, it was found that: due to different purposes of laboratory and industrial production, different control conditions exist, and as shown in fig. 1-2, a certain difference exists between fermentation levels, for example: the biomass in the laboratory is above 52g/L, and the fermentation titer is up to 8000 mug/mL; and the maximum biomass of the industrial production tank is 48g/L, and the fermentation titer is about 7000 mug/mL. These phenomena show that there may be a certain correlation between biomass and fermentation titer, so developing a fermentation medium and a fermentation method based on the biomass for Streptomyces avermitilis is crucial to reduce the gap between industrial production and laboratory level and improve the titer level of industrial production by finding the balance point between biomass and fermentation titer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a biomass-based streptomyces avermitilis fermentation medium and a fermentation method thereof. The fermentation medium and the fermentation condition are optimized by using two indexes of biomass and fermentation titer, so that the gap between the industrial production and laboratory level is greatly reduced, and the titer level of the industrial production is improved.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the avermectin streptomycete fermentation medium based on biomass is obtained by formula adjustment based on two indexes of biomass and fermentation titer, and comprises the following components: soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, defoamer, solid alkali and amylase.
Further, the amounts of the raw materials are as follows: 30-32 g/L of soybean cake powder, 172-182 g/L of starch, 10-12 g/L of yeast powder, 0.2-0.3 g/L of ammonium sulfate, 1-2 g/L of light calcium carbonate, 4-5 g/L of defoamer, 0.2-0.3 g/L of solid alkali, and the addition amount of amylase is 0.025% of the weight of starch, and the balance of water.
Further, the addition amount of soybean cake powder 32g/L, starch 182g/L, yeast powder 10g/L, ammonium sulfate 0.3g/L, light calcium carbonate 2g/L, defoamer 4g/L, solid alkali 0.2g/L and amylase is 0.025% of the weight of starch, and the balance is water.
A preparation method of the streptomyces avermitilis fermentation medium,
adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, a defoaming agent, solid alkali and amylase into water, stirring and dissolving, fixing the volume, saccharifying for 35-40 min at 90 ℃, and sterilizing for later use.
Further, the sterilization adopts moist heat sterilization for 30min at 120-122 ℃.
Further, the defoamer adopts one or more of foaming, corn oil and soybean oil.
The fermentation method of the streptomyces avermitilis is obtained by performing parameter adjustment on the basis of two indexes of biomass and fermentation titer, and comprises the following steps of:
inoculating the seed liquid into the fermentation medium for fermentation culture for 350-360h at a time according to the inoculation amount of 8-10%, and placing the seed liquid into a tank; the stirring frequency is controlled during the fermentation culture.
Further, during the fermentation period of 0-10 hours, the stirring frequency is controlled to be 0Hz;
during the fermentation period of 11-100 h, controlling the stirring frequency to be 45Hz;
controlling the stirring frequency to be 40Hz during the fermentation period of 101-200 hours;
during the fermentation period of 201-300 h, the stirring frequency is controlled to be 37Hz;
during the period from 301h of fermentation to tank discharge, the stirring frequency is controlled to be 35Hz.
Further, the temperature is controlled to be 27-28 ℃ in the fermentation process, and the air quantity is controlled to be 0.8-1 m in the fermentation process 3 /(h·m 3 )。
Further, the fermentation method of the seed liquid comprises the following steps: inoculating spore suspension for sterilizationCooling to 29 deg.c in seed culture medium at 28-29 deg.c and air flow rate of 0.8-1 m 3 /(h·m 3 ) Fermenting and culturing for 50-60 h;
the seed culture medium comprises the following raw materials in concentration:
25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract and 0.025g/L of cobalt chloride.
Further, when the fermentation tank is placed, the biomass of the fermentation tank is more than 50g.L -1 Fermentation titer is greater than 7500 mug.mL -1 。
Compared with the prior art, the invention has the following beneficial effects:
1. the invention uses soybean cake powder and yeast powder as slow-acting nitrogen sources and ammonium sulfate as quick-acting nitrogen sources to meet the demands of different periods of bacterial growth and propagation of streptomyces avermitilis and synthetic products, and uses starch as a carbon source to not only meet the demands of bacterial growth and propagation, but also provide energy for the middle-later period biosynthesis of streptomyces avermitilis to synthesize avermectin; the light calcium carbonate is used for neutralizing organic acid generated in the fermentation process and stabilizing the pH value of fermentation liquor; the solid alkali is used for adjusting the PH of the culture medium before sterilization; the defoamer is used for defoaming in the culture medium sterilization process and the strain fermentation culture process, so as to prevent material escape and night escape; the amylase has the function of hydrolyzing starch into micromolecular glucose, so that the streptomyces avermitilis can be conveniently absorbed and utilized.
2. Compared with the traditional industrial fermentation medium, the improvement of the fermentation culture is more beneficial to the increase of biomass and the increase of viscosity of fermentation liquor, and the invention improves the stirring frequency according to the rheological property of the fermentation liquor, increases the residence time of air in the fermentation liquor by strictly controlling the stirring frequency, ensures the transfer of dissolved oxygen and energy and quality in the fermentation process, is beneficial to the conversion process of streptomyces avermitilis from primary metabolism to secondary metabolism, and ensures the growth and propagation of streptomyces avermitilis and the production of secondary metabolites.
The invention breaks through the optimization of the fermentation medium and the fermentation condition by using one index of the fermentation titer in the traditional technology, creatively optimizes the fermentation medium and the fermentation condition by using two indexes of biomass and the fermentation titer, obtains the optimal fermentation medium formula and fermentation condition parameters, greatly reduces the gap between the industrial production and the laboratory level, and improves the titer level of the industrial production.
Drawings
FIG. 1 is a graph of biomass level versus fermentation titer level at the time of tank discharge of a fermenter under laboratory conditions;
FIG. 2 is a graph of biomass level versus fermentation titer level for a fermenter under commercial conditions;
FIG. 3 is a schematic structural view of a fermenter;
FIG. 4 is a graph showing biomass versus fermentation titer during fermentation for example 1 and comparative example 1.
Detailed Description
The present invention will be described in further detail with reference to examples.
The fermentation tanks shown in FIG. 3 are used for fermentation in the examples and comparative examples of the present invention;
the raw materials used in each of the examples and comparative examples of the present invention are commercially available unless otherwise specified.
Example 1
The avermectin fermenting process with biomass and fermenting titer includes the following steps:
1) By 12m 3 Seed pot
2) A seed culture medium comprising:
25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract, 0.025g/L of cobalt chloride and 8m of volume after digestion 3 The method comprises the steps of carrying out a first treatment on the surface of the Sterilizing for later use;
3) Culturing: inoculating the spore suspension into a seed culture medium which is sterilized and cooled to 29 ℃, culturing at 28-29 ℃ and air quantity of 0.8-1 m 3 /(h·m 3 ) Fermenting and culturing for 50-60h under the condition, microscopic examination of mycelium into balls is good, no contamination of mixed bacteria exists, and cooling and culturing are carried out for standby.
1) By 120m 3 A fermentation tank;
2) A fermentation medium comprising the following concentrations of the respective raw materials: 32g/L of soybean cake powder, 182g/L of starch, 10g/L of yeast powder, 0.3g/L of ammonium sulfate, 2g/L of light calcium carbonate, 4g/L of foam, 0.2g/L of solid alkali and amylase: starch=0.025%.
3) Preparation of fermentation medium:
by volume 92m 3 Adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, foam, solid alkali and amylase into water after calculating the material amount, stirring and dissolving, fixing the volume, saccharifying at 90 ℃ for 40min, and sterilizing at 120-122 ℃ for 30min under damp heat for later use
4) Fermentation culture:
transferring the seed liquid prepared in the step 1 into a fermentation tank which is sterilized and cooled to 28 ℃, and controlling the temperature to 27-28 ℃ and the air quantity to be 0.5-0.8 m 3 /(h·m 3 ) Fermenting and culturing for 352 hours under the condition, and placing the fermentation and culturing in a tank, wherein the stirring frequency is strictly controlled in the fermentation and culturing process, the fermentation is not performed for 0-10 hours, and the stirring frequency is controlled to be 45Hz during the fermentation for 11-100 hours; the stirring frequency is controlled to be 40Hz during the fermentation period of 101-200 h, the stirring frequency is controlled to be 37Hz during the fermentation period of 201-300 h, and the stirring frequency is controlled to be 35Hz during the fermentation period of 301 h-tank discharging.
Fermentation titers and biomass were determined every 24 hours after 50 hours of fermentation. The fermentation titer and biomass trend with time of incubation are shown in table 3 and fig. 4; the data show that 52 hours biomass 36.5g/L,196 hours up to 47.8g/L, has been equivalent to the biomass of the traditional medium when it was placed in the tank (350 hours). This is because the nitrogen source soybean cake powder concentration of the culture medium is high, and in order to ensure transfer of dissolved oxygen in the fermentation process, the stirring frequency is increased up to 45Hz. Under the fermentation method, the titer is steadily increased in the whole culture process, and the titer is 8327 mug/mL when the culture is placed in a tank for 352 hours, so that the laboratory level is reached.
Example 2
The avermectin fermenting process with biomass and fermenting titer includes the following steps:
1) By 12m 3 Seed pot
2) A seed culture medium comprising:
25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract, 0.025g/L of cobalt chloride and 8m of volume after digestion 3 The method comprises the steps of carrying out a first treatment on the surface of the Sterilizing for later use;
3) Culturing: inoculating the spore suspension into a seed culture medium which is sterilized and cooled to 29 ℃, culturing at 28-29 ℃ and air quantity of 0.8-1 m 3 /(h·m 3 ) Fermenting and culturing for 50-60h under the condition, microscopic examination of mycelium into balls is good, no contamination of mixed bacteria exists, and cooling and culturing are carried out for standby.
1) By 120m 3 A fermentation tank;
2) A fermentation medium comprising the following concentrations of the respective raw materials: 32g/L of soybean cake powder, 178g/L of starch, 10g/L of yeast powder, 0.3g/L of ammonium sulfate, 2g/L of light calcium carbonate, 4g/L of foam, 0.2g/L of solid alkali and amylase: starch=0.025%.
3) Preparation of fermentation medium:
by volume 92m 3 Adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, foam, solid alkali and amylase into water after calculating the material amount, stirring and dissolving, fixing the volume, saccharifying at 90 ℃ for 40min, and sterilizing at 120-122 ℃ for 30min under damp heat for later use
4) Fermentation culture:
transferring the seed liquid prepared in the step 1 into a fermentation tank which is sterilized and cooled to 28 ℃, and controlling the temperature to 27-28 ℃ and the air quantity to be 0.5-0.8 m 3 /(h·m 3 ) Fermenting and culturing for 352 hours under the condition, and placing the fermentation and culturing in a tank, wherein the stirring frequency is strictly controlled in the fermentation and culturing process, the fermentation is not performed for 0-10 hours, and the stirring frequency is controlled to be 45Hz during the fermentation for 11-100 hours; the stirring frequency is controlled to be 40Hz during the fermentation period of 101-200 h, the stirring frequency is controlled to be 37Hz during the fermentation period of 201-300 h, and the stirring frequency is controlled to be 35Hz during the fermentation period of 301 h-tank discharging.
Fermentation titers and biomass were determined every 24 hours after 50 hours of fermentation. The fermentation titers and biomass changes with time of culture are shown in Table 1. The data show that the tank release titers are lower than in example 1, although the biomass is comparable. The nitrogen source of the culture medium is the same as that of example 1, but the carbon source is less, so the biomass is basically not different; and because the starch content is lower, the streptomyces avermitilis lacks sufficient energy supply in the later fermentation period (314 hours), the synthesis rate of the avermectin is small, the potency is increased slowly, the can release potency is 384 mug/mL lower than that of the embodiment 1, but the potency is 943 mug/mL higher than that of the traditional technology.
TABLE 1 Table of biomass and fermentation titers over time of cultivation
Example 3
The avermectin fermenting process with biomass and fermenting titer includes the following steps:
1) By 12m 3 Seed pot
2) A seed culture medium comprising:
25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract, 0.025g/L of cobalt chloride and 8m of volume after digestion 3 The method comprises the steps of carrying out a first treatment on the surface of the Sterilizing for later use;
3) Culturing: inoculating the spore suspension into a seed culture medium which is sterilized and cooled to 29 ℃, culturing at 28-29 ℃ and air quantity of 0.8-1 m 3 /(h·m 3 ) Fermenting and culturing for 50-60h under the condition, microscopic examination of mycelium into balls is good, no contamination of mixed bacteria exists, and cooling and culturing are carried out for standby.
1) By 120m 3 A fermentation tank;
2) A fermentation medium comprising the following concentrations of the respective raw materials: 30g/L of soybean cake powder, 178g/L of starch, 10g/L of yeast powder, 0.3g/L of ammonium sulfate, 2g/L of light calcium carbonate, 4g/L of foam, 0.2g/L of solid alkali and amylase: starch=0.025%.
3) Preparation of fermentation medium:
by volume 92m 3 Adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, foam, solid alkali and amylase into water after calculating the material amount, stirring and dissolving, fixing the volume, saccharifying at 90 ℃ for 40min, and sterilizing at 120-122 ℃ for 30min under damp heat for later use
4) Fermentation culture:
transferring the seed liquid prepared in the step 1 into a fermentation tank which is sterilized and cooled to 28 ℃, and controlling the temperature to 27-28 ℃ and the air quantity to be 0.5-0.8 m 3 /(h·m 3 ) Fermenting and culturing for 352 hours under the condition, and placing the fermentation and culturing in a tank, wherein the stirring frequency is strictly controlled in the fermentation and culturing process, the fermentation is not performed for 0-10 hours, and the stirring frequency is controlled to be 45Hz during the fermentation for 11-100 hours; the stirring frequency is controlled to be 40Hz during the fermentation period of 101-200 h, the stirring frequency is controlled to be 37Hz during the fermentation period of 201-300 h, and the stirring frequency is controlled to be 35Hz during the fermentation period of 301 h-tank discharging.
Fermentation titers and biomass were determined every 24 hours after 50 hours of fermentation. The fermentation titers and biomass changes with time of culture are shown in Table 2. The data shows that, in comparison with example 1, the stepwise control was carried out despite the constant stirring frequency, but the tank release biomass was only 50.6g/L, and the fermentation titer was 7523. Mu.g/mL. Both biomass and pot titer were reduced compared to example 1 due to the reduced nitrogen source (soybean meal) in the medium and reduced avermectin biomass. Comprehensive examples 1 and 3, the biomass of streptomyces avermitilis is the basis and precondition for fermentation potency, which is the main body for fermentative synthesis of avermectin, and high biomass is a necessary but insufficient condition for high potency.
TABLE 2 Table of biomass and fermentation titers over time of cultivation
Comparative example 1
The avermectin fermenting process with fermentation titer as index includes the following steps:
1) By 12m 3 A seed tank;
2) A seed culture medium comprising: 25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract, 0.025g/L of cobalt chloride and 8m of volume after digestion 3 。
3) Fermentation culture: inoculating the spore suspension into seed culture medium sterilized and cooled to 29 deg.c, and air flow rate of 0.8-1 m at 28-29 deg.c 3 /(h·m 3 ) Fermenting and culturing for 50-60h under the condition, microscopic examination of mycelium into balls is good, no contamination of mixed bacteria exists, and cooling and culturing are carried out for standby.
1) By 120m 3 Fermentation tank
2) A fermentation medium comprising: 27g/L of soybean cake powder, 170g/L of starch, 10g/L of yeast powder, 0.3g/L of ammonium sulfate, 2g/L of light calcium carbonate, 4g/L of foam, 0.2g/L of solid alkali and amylase: starch=0.025%.
3) Preparation of fermentation medium:
by volume 92m 3 Adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, foam, solid alkali and amylase into water after calculating the material amount, stirring and dissolving, fixing the volume, saccharifying for 40min at 90 ℃, and carrying out damp-heat sterilization for 30min at 120-122 ℃ for later use;
4) Fermentation culture
Transferring the seed liquid prepared in the step 1 into a fermentation tank which is sterilized and cooled to 28 ℃, and controlling the temperature to 27-28 ℃ and the air quantity to be 0.5-0.8 m 3 /(h·m 3 ) Fermenting and culturing for 352 hours under the condition, wherein the stirring frequency is strictly controlled in the fermentation process, and the stirring frequency is controlled to be 0Hz during the fermentation and culturing for 0-10 hours; during the fermentation culture for 11-100 h, the stirring frequency is controlled to be 40Hz; the stirring frequency is controlled to be 38Hz during the fermentation culture period of 101-200 h, the stirring frequency is controlled to be 36Hz during the fermentation culture period of 201-300 h, and the stirring frequency is controlled to be 34Hz during the fermentation culture period of 301 h-tank discharging.
Effect example 1
In the fermentation of example 1 and comparative example 1, fermentation titer and biomass were measured every 24 hours after 50 hours of fermentation. The fermentation titers and biomass trends over time for example 1 and comparative example 1 are shown in Table 3 and FIG. 4.
TABLE 3 Table 3
From comparison of the data of example 1 and comparative example 1, it is known that: fermenting and culturing for 52 hours, wherein the biomass is 36.5g/L; the biomass reaches 47.8g/L after 196 hours, which is equivalent to the biomass of the tank discharge under the traditional fermentation formula; when the fermenter of example 1 was placed (352 hours), the biomass was 54.3g/L and the titer was 8327. Mu.g/mL, which reached laboratory levels far higher than the biomass 47.7g/L and the titer was 7136. Mu.g/mL when the fermenter of comparative example 1 was placed. The biomass increase in this example means an increase in the number of Streptomyces avermitilis mycelia per unit volume; after the stirring frequency is improved, the detention time of air in the fermentation broth is increased, dissolved oxygen is ensured, and the avermectin can be rapidly and continuously synthesized by the streptomyces avermitilis strain, so that the tank release titer is improved, and the difference between industrial production and laboratory level is reduced.
The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (10)
1. The avermectin fermentation medium based on biomass is characterized by being obtained by formula adjustment based on two indexes of biomass and fermentation titer, and comprises the following components: soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, defoamer, solid alkali and amylase.
2. A biomass-based Streptomyces avermitilis fermentation medium according to claim 1,
30-32 g/L of soybean cake powder, 172-182 g/L of starch, 10-12 g/L of yeast powder, 0.2-0.3 g/L of ammonium sulfate, 1-2 g/L of light calcium carbonate, 4-5 g/L of defoamer, 0.2-0.3 g/L of solid alkali, and the addition amount of amylase is 0.025% of the weight of starch, and the balance of water.
3. A biomass-based Streptomyces avermitilis fermentation medium according to claim 2,
32g/L of soybean cake powder, 182g/L of starch, 10g/L of yeast powder, 0.3g/L of ammonium sulfate, 2g/L of light calcium carbonate, 4g/L of defoamer, 0.2g/L of solid alkali and the balance of water, wherein the addition amount of amylase is 0.025% of the weight of the starch.
4. A process for preparing a fermentation medium of Streptomyces avermitilis as claimed in any one of claims 1 to 3,
adding soybean cake powder, starch, yeast powder, ammonium sulfate, light calcium carbonate, a defoaming agent, solid alkali and amylase into water, stirring and dissolving, fixing the volume, saccharifying for 35-40 min at 90 ℃, and sterilizing for later use.
5. A process for preparing a fermentation medium for Streptomyces avermitilis as claimed in claim 4, wherein,
the sterilization adopts moist heat sterilization for 30min at 120-122 ℃.
6. The fermentation method of the streptomyces avermitilis is characterized by comprising the following steps of:
inoculating the seed solution into the fermentation medium according to any one of claims 1-3 for fermentation culture for 350-360h at a time according to the inoculation amount of 8-10%, and placing the seed solution into a tank; the stirring frequency is controlled during the fermentation culture.
7. The fermentation process of Streptomyces avermitilis according to claim 6, wherein,
controlling stirring frequency to be 0Hz during fermentation for 0-10 h;
during the fermentation period of 11-100 h, controlling the stirring frequency to be 45Hz;
controlling the stirring frequency to be 40Hz during the fermentation period of 101-200 hours;
during the fermentation period of 201-300 h, the stirring frequency is controlled to be 37Hz;
during the period from 301h of fermentation to tank discharge, the stirring frequency is controlled to be 35Hz.
8. The fermentation process of Streptomyces avermitilis according to claim 6, wherein,
the temperature is controlled to be 27-28 ℃ in the fermentation process, and the air quantity is controlled to be 0.8-1 m in the fermentation process 3 /(h·m 3 )。
9. The fermentation process of Streptomyces avermitilis according to claim 6, wherein,
the fermentation method of the seed liquid comprises the following steps: inoculating the spore suspension into a seed culture medium which is sterilized and cooled to 29 ℃, and controlling the temperature to 28-29 ℃ and the air quantity to 0.8-1 m 3 /(h·m 3 ) Fermenting and culturing for 50-60 h;
the seed culture medium comprises the following raw materials in concentration:
25g/L of starch, 6g/L of soybean cake powder, 6g/L of peanut cake powder, 3g/L of yeast extract and 0.025g/L of cobalt chloride.
10. The fermentation method of Streptomyces avermitilis according to claim 6, wherein the biomass of the fermentation tank is greater than 50g.L -1 Fermentation titer is greater than 7500 mug.mL -1 。
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