CN106676150B - Method for producing natamycin by fermenting streptomyces fuscoporia - Google Patents

Method for producing natamycin by fermenting streptomyces fuscoporia Download PDF

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CN106676150B
CN106676150B CN201710205059.8A CN201710205059A CN106676150B CN 106676150 B CN106676150 B CN 106676150B CN 201710205059 A CN201710205059 A CN 201710205059A CN 106676150 B CN106676150 B CN 106676150B
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fermentation
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natamycin
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金伟
郑长春
王博
慕东
卢春玲
马艳茹
张兴灿
张玲
陈君
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Amicogen China Biopharm Co Ltd
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Shandong Lukang Pharmaceutical Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C12P19/626—Natamycin; Pimaricin; Tennecetin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention provides a method for producing natamycin by fermenting streptomyces fuscoporia, which comprises the following steps: 1) activating streptomyces limoflavus spore liquid; 2) inoculating the streptomyces flavipes spore solution activated in the step 1) into a seed culture medium for seed culture to obtain streptomyces flavipes; 3) inoculating the streptomyces fuscogongensis obtained in the step 2) into a fermentation medium for aerobic fermentation culture to obtain natamycin. The invention adopts proper culture medium proportion and adds synergist in the basic material, the growth activity of the mycelium is greatly improved under the environment of balanced carbon-nitrogen ratio and inorganic salt ions, the power condition does not need to be increased, the mycelium is more quickly adapted to the environment of the culture solution, the lag phase is shortened, and the fermentation period is shortened. The whole fermentation period is 79-82 h, the concentration of the final streptomyces fuscoporia mycelium is 44-45%, and the content of natamycin is 6.9-7 g/L.

Description

Method for producing natamycin by fermenting streptomyces fuscoporia
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation production method of streptomyces fuscoporia natamycin.
Background
Natamycin is a polyene macrolide antibiotic, and is a secondary metabolite produced by fermenting streptomyces natalensis, streptomyces fuscoporia and other bacteria of streptomyces under aerobic conditions, can inhibit the growth of yeast and filamentous fungi and the production of mycotoxin, and is widely applied to food preservation (food grade) and disease treatment (medical grade) caused by fungi. Currently, only three biological preservatives which are allowed to be used internationally are nisin, natamycin and polylysine, wherein natamycin is used as a high-efficiency, safe, non-toxic and low-dose biological preservative by more than 50 countries including the United states all over the world, becomes a widely-used food additive in the global range, and has great commercial value.
In the current natamycin production, two problems which need to be solved urgently are that the fermentation period is longer and the fermentation level is lower, the production cost is overhigh due to overlong fermentation period, and the final product quantity is less due to low fermentation level. Aiming at the two problems, the commonly adopted solution is to improve the initial power condition of fermentation, and add excessive nutrient substances, especially carbon and nitrogen sources, in the fermentation liquor, thereby shortening the fermentation period to a certain extent and improving the product level.
In the existing solution, increasing the amount of nutrient substances in the fermentation liquor can improve the fermentation level to a certain extent, but the carbon-nitrogen ratio is unbalanced due to excessive nutrient substances, and the growth activity of mycelium can be influenced in the excessive nutrient substances, so that the concentration of fermentation bacteria and the content of products are influenced; and the power conditions of fermentation initiation are increased, such as the aeration quantity is increased, the stirring is increased, and the production cost is greatly increased.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for producing natamycin by Streptomyces fushuangpai fermentation, which shortens the fermentation period and increases the natamycin yield.
In order to achieve the above object, the present invention provides a method for producing natamycin by fermentation of streptomyces fuscoporia, comprising the following steps:
1) activating streptomyces limoflavus spore liquid;
2) inoculating the streptomyces flavipes spores activated in the step 1) into a seed culture medium for seed culture to obtain streptomyces flavipes;
3) inoculating the streptomyces fuscogongensis obtained in the step 2) into a fermentation medium for aerobic fermentation culture to obtain natamycin;
the fermentation medium in the step 3) comprises: 60.0 +/-1.0 g/L of glucose, 2.0 +/-0.5 g/L of medium-temperature soybean cake powder, 2.0 +/-0.5 g/L of cottonseed cake powder, 10.0 +/-1.0 g/L of yeast powder, 15.0 +/-1.0 g/L of corn steep liquor, 0.5 +/-0.1 g/L of sodium chloride, 0.2 +/-0.1 g/L of magnesium sulfate, 10.0 +/-1.0 g/L, SCH-1 type synergist 5.0 +/-0.5 g/L of soybean oil, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water.
Preferably, the seed culture medium in step 2) comprises: 20.0 +/-1.0 g/L of glucose, 3.0 +/-0.5 g/L of yeast powder, 5.0 +/-0.5 g/L of peptone, 5.0 +/-0.5 g/L of corn steep liquor, 10.0 +/-1.0 g/L of sodium chloride, 0.5 +/-0.1 g/L of monopotassium phosphate, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water.
Preferably, the conditions of the seed culture in the step 2) are as follows: rotating speed of a shaking table: 210-230rpm, a culturing temperature of 25-30 ℃, a culturing time of 20-26 h and an initial pH value of 6.9-7.0.
Preferably, the inoculation amount of the fermentation culture in the step 3) is 7.5-8.5% of the volume of the fermentation medium in percentage by volume.
Preferably, the initial adding amount of the fermentation medium in the step 3) is 65-75% of the volume of the fermentation tank in percentage by volume.
Preferably, the conditions of the fermentation culture in the step 3) are as follows: the culture temperature is 25-30 ℃, the initial aeration is 0.25-0.35 VVM, the initial stirring speed is 140-160 rpm, the initial pH value is 6.4-6.6, and the pressure of the fermentation tank is maintained at 0.04-0.06 MPa in the whole fermentation process.
Preferably, the dissolved oxygen of the fermentation liquid in the fermentation culture process in the step 3) is above 30%.
Preferably, the pH value of the fermentation liquid in the fermentation culture process in the step 3) is 6.9-7.1.
Preferably, the concentration of the sugar in the fermentation culture process in the step 3) is 2.5-3.5% by weight.
Preferably, the SCH-1 type synergist is supplemented when the concentration of the streptomyces limosus mycelium in the fermentation broth reaches 30%.
The invention provides a method for producing natamycin by fermentation of streptomyces fushuangpao, which adopts a fermentation medium with a proper proportion and adds SCH-1 type synergist in a base material, so that the growth activity of mycelium in an environment with balanced carbon-nitrogen ratio and inorganic salt ions is greatly improved, the yield of natamycin is correspondingly improved, meanwhile, the power condition does not need to be increased, the mycelium is more quickly adapted to the environment of a culture solution, the lag phase is shortened, and the fermentation period is shortened. The whole fermentation period is 79-82 h, the final concentration of the streptomyces fuscoporia mycelium is 44-45%, the concentration of the mycelium is increased by 50% compared with that of 30% of a control group, and the content of natamycin is increased to 6.9-7 g/L from 5g/L of the control group.
Detailed Description
The invention provides a method for producing natamycin by fermenting streptomyces fuscoporia, which comprises the following steps:
1) activating streptomyces limoflavus spore liquid;
2) inoculating the streptomyces flavipes spore solution activated in the step 1) into a seed culture medium for seed culture to obtain streptomyces flavipes;
3) inoculating the streptomyces fuscogongensis obtained in the step 2) into a fermentation medium for aerobic fermentation culture to obtain natamycin;
the fermentation medium in the step 3) comprises: 60.0 +/-1.0 g/L of glucose, 2.0 +/-0.5 g/L of medium-temperature soybean cake powder, 2.0 +/-0.5 g/L of cottonseed cake powder, 10.0 +/-1.0 g/L of yeast powder, 15.0 +/-1.0 g/L of corn steep liquor, 0.5 +/-0.1 g/L of sodium chloride, 0.2 +/-0.1 g/L of magnesium sulfate, 10.0 +/-1.0 g/L, SCH-1 type synergist 5.0 +/-0.5 g/L of soybean oil, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water.
In the invention, the Streptomyces phaeoflavus spore liquid is activated. Preferably, before use, the streptomyces limoflavus spore liquid is frozen at-82 to-78 ℃. The strain and the source of the streptomyces limoflavus spore liquid are not specially limited, and the streptomyces limoflavus spore liquid can be obtained by adopting a commercially available product, preferably a strain which is preserved in China center for culture Collection of microorganisms with the strain number of CGMCC NO. 4628.
Preferably, the spore liquid is diluted before activation, and more preferably, the concentration after dilution is 10-5(ii) a Preferably using ddH2And O, diluting. Preferably, the activation is culturing the diluted spore liquid in a slant culture medium. Preferably, the slant medium comprises: glucose 10.0 + -1.0 g/L, malt extract powder 3.0 + -0.5 g/L, yeast extract powder 3.0 + -0.5 g/L, peptone 5.0 + -0.5 g/L, agar 15.0 + -1.0 g/L and balance water, morePreferably 10.0g/L glucose, 3.0g/L malt extract powder, 3.0g/L yeast extract powder, 5.0g/L peptone, 15.0 agar and the balance water.
In the present invention, the conditions for the activation are preferably: activation temperature: activating for 6-8 days at 26-30 ℃, and setting the initial pH value to be 6.9-7.1; more preferably: activation temperature: activation time was 7 days at 28 ℃ and initial pH 7.0. After activation, the slant was completely covered with spores and the color of the spores turned from white to grey.
The sources of the components of the slant culture medium are not particularly limited in the present invention, and the culture medium may be prepared by a conventional method by those skilled in the art.
In the invention, the slant culture medium is preferably used after being sterilized at 120-130 ℃ for 25-35 min. In the present invention, the sterilization temperature is more preferably 121 ℃; the sterilization time is more preferably 30 min. The apparatus for sterilizing the medium according to the present invention is not particularly limited, and any sterilization apparatus known to those skilled in the art may be used.
In the invention, the activated streptomyces flavipes spores are inoculated into a seed culture medium for seed culture to obtain the streptomyces flavipes. Preferably, the inoculation amount of the seed culture is 1.9-2.1%, and more preferably 2%. Preferably, the seed culture medium comprises: 20.0 +/-1.0 g/L of glucose, 3.0 +/-0.5 g/L of yeast powder, 5.0 +/-0.5 g/L of peptone, 5.0 +/-0.5 g/L of corn steep liquor, 10.0 +/-1.0 g/L of sodium chloride, 0.5 +/-0.1 g/L of monopotassium phosphate, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water, and more preferably: 20.0g/L of glucose, 3.0g/L of yeast powder, 5.0g/L of peptone, 5.0g/L of corn steep liquor, 10.0g/L of sodium chloride, 0.5g/L of monopotassium phosphate, 5.0g/L of calcium carbonate and the balance of water.
In the present invention, the seed culture preferably employs a shake flask culture; preferably, the initial addition amount of the seed culture medium is 9-11% of the volume of the culture container, and more preferably 10%. Preferably, the conditions of the seed culture are as follows: rotating speed of a shaking table: 210-230rpm, culture temperature: and (3) culturing at 25-30 ℃ for: 20-26 h, the initial pH value is 6.9-7.0, and the more preferable is: rotating speed of a shaking table: 220rpm, culture temperature: 28 ℃, incubation time: and (5) 24 h. Culturing until the concentration of Streptomyces fushuangpao is more than 10%.
The source of the components of the seed culture medium is not particularly limited in the invention, and the culture medium can be prepared by adopting the conventional medicine prepared by the technicians in the field.
In the invention, the seed culture medium is preferably used after being sterilized at 120-130 ℃ for 25-35 min. In the present invention, the sterilization temperature is more preferably 121 ℃; the sterilization time is more preferably 30 min. The apparatus for sterilizing the medium according to the present invention is not particularly limited, and any sterilization apparatus known to those skilled in the art may be used.
After the streptomyces fuscoporia is obtained by seed culture, the obtained streptomyces fuscoporia is inoculated into a fermentation culture medium for aerobic fermentation culture to obtain the natamycin. Preferably, the inoculation amount of the fermentation culture is 7.5-8.5% of the volume of the fermentation medium, and more preferably 8.0% in percentage by volume.
Preferably, the initial addition amount of the fermentation medium is 65-75% of the volume of the fermentation tank, and more preferably 70%. In the present invention, the fermentation medium preferably comprises: 60.0 +/-1.0 g/L of glucose, 2.0 +/-0.5 g/L of medium-temperature soybean cake powder, 2.0 +/-0.5 g/L of cottonseed cake powder, 10.0 +/-1.0 g/L of yeast powder, 15.0 +/-1.0 g/L of corn steep liquor, 0.5 +/-0.1 g/L of sodium chloride, 0.2 +/-0.1 g/L of magnesium sulfate, 10.0 +/-1.0 g/L, SCH-1 type synergist 5.0 +/-0.5 g/L of soybean oil, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water, and more preferably 60.0g/L of glucose, 2.0g/L of medium-temperature soybean cake powder, 2.0g/L of cottonseed cake powder, 10.0g/L of yeast powder, 15.0g/L of corn steep liquor, 0.5g/L of sodium chloride, 0.2g/L of soybean oil, L, SCH-1.0 g/L of yeast powder, and the balance of calcium carbonate.
Preferably, the conditions of the fermentation culture are as follows: the culture temperature is 25-30 ℃, the initial aeration is 0.25-0.35 VVM, the initial stirring speed is 140-160 rpm, the initial pH value is 6.4-6.6, the pressure of the fermentation tank is maintained to be 0.04-0.06 MPa in the whole fermentation process, more preferably, the culture temperature is 28 ℃, the initial aeration is 0.3VVM, the initial stirring speed is 150rpm, the initial pH value is 6.5, and the pressure of the fermentation tank is maintained to be 0.05MPa in the whole fermentation process.
In the present invention, after the start of fermentation culture, the dissolved oxygen amount gradually decreases as the cells adapt to the medium and the fermentation environment conditions. Preferably, the dissolved oxygen is controlled by stirring and/or aeration; preferably, the dissolved oxygen of the fermentation liquor in the fermentation culture process is more than 30 percent; preferably, the dissolved oxygen is measured by using a dissolved oxygen electrode carried by the fermentation tank. In the invention, in the fermentation culture process, the pH value is firstly increased by more than 7.0 and then gradually decreased, and when the pH value is decreased to 7.0, the pH value is adjusted to be 6.9-7.1. Preferably, the pH value is adjusted by ammonia water, and more preferably, the ammonia water with the mass fraction of 30% is adopted. Preferably, the pH is measured using a pH electrode of the fermentor itself. In the present invention, the sugar concentration is gradually decreased with the consumption of nutrients during the fermentation culture, and glucose is supplemented when the sugar concentration is decreased to 2.5%. Preferably, the concentration of glucose is 200. + -.2 g/L, more preferably 200 g/L. Preferably, the concentration of the sugar in the fermentation culture process is 2.5-3.5% by weight. Preferably, the sugar concentration is determined by the film reagent method.
In the invention, preferably, the concentration of the streptomyces limosus mycelium in the fermentation liquid is checked once every 3-5 h in the fermentation culture process, and more preferably 4 h. Preferably, the SCH-1 type synergist is supplemented when the concentration of the streptomyces fulvidraco mycelium reaches 30%. In the invention, the concentration of the SCH-1 type synergist is preferably 20.0 +/-1 g/L, and more preferably 20.0 g/L; preferably, the synergist is ddH2And (4) diluting with oxygen. Preferably, the addition amount of the SCH-1 type synergist is 0.25-0.35 ml/(h.L), namely, 0.25-0.35 ml, more preferably 0.3ml/(h.L), is added to 1L of fermentation broth per hour.
In the invention, the increase of natamycin begins to become slow along with the progress of fermentation, and the continuous production of natamycin can be promoted by adding n-butanol serving as a natamycin precursor. Preferably, when the fermentation is carried out for 58-62 h, n-propanol is added into the fermentation medium, and more preferably for 60 h. Preferably, the n-propanol is sterilized before addition; preferably, the sterilization mode is a filtration method, and preferably, the filtration method adopts a bacteria filter with the pore diameter of 0.22 um. Preferably, the addition amount of the n-propanol is 4.5-5.5 ml/L, and more preferably 5 ml/L. When the bacterial concentration is not increased any more, the fermentation is finished, and the whole fermentation period is about 80 hours.
The source of the components of the culture medium is not particularly limited, the culture medium can be prepared by adopting a medicament prepared by a person skilled in the art conventionally, and preferably, the SCH-1 type synergist is produced by a Wutong bridge biochemical plant in Yuanshan City.
In the invention, the fermentation medium, the supplemented glucose and the supplemented SCH-1 type synergist are preferably sterilized at 120-130 ℃ for 25-35 min for use. In the present invention, the sterilization temperature is more preferably 121 ℃; the sterilization time is more preferably 30 min. The apparatus for sterilizing the medium according to the present invention is not particularly limited, and any sterilization apparatus known to those skilled in the art may be used.
In the invention, the ratio of each nutrient element in the fermentation medium is proper, and the synergist is added into the base material, so that the growth activity of the mycelium in a balanced carbon-nitrogen ratio and inorganic salt ion environment is greatly improved, the power condition does not need to be increased, the mycelium is more quickly adapted to the environment of a culture solution, the lag phase is shortened, and the fermentation period is shortened; with the consumption of nutrients in the basic material, especially nitrogen source, the growth of mycelium becomes slow, at this time, SCH-1 type fermentation synergist is supplemented, and thallus is further propagated and grown; when the fermentation is carried out to a certain extent, the n-propanol which has been sterilized by filtration is added, so that the yield of natamycin in the middle and later stages of the fermentation is further improved. The whole fermentation period is about 80 hours, the concentration of the streptomyces fuscoporia mycelium is 44-45%, and the content of natamycin is 6.9-7 g/L.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Brown-yellow robe streptomycesActivation of a bacterial spore liquid: adopting ddH to the streptomyces limousii spore liquid with the preservation number of CGMCC NO.4628 frozen at-78 DEG C2Diluting with O at a dilution ratio of 10-5Spreading on a plate containing 20ml of slant culture medium sterilized at 120 deg.C for 35min, and performing activation culture at 26 deg.C for 8 days, wherein the slant culture medium comprises: 11.0g/L glucose, 2.5g/L malt extract powder, 3.5g/L yeast extract powder, 5.0g/L peptone, 16.0g/L agar and the balance water, and the initial pH value is 6.9.
Seed culture: adding 40ml of seed culture medium sterilized at 130 ℃ for 35min into a 500ml shake flask, selecting and inoculating activated streptomyces flavipes spores into the culture medium, wherein the inoculation amount is 1.9%, and culturing at 210rpm and 25 ℃ for 26h to obtain the streptomyces flavipes with the concentration of 10.5%, wherein the seed culture medium comprises: 21.0g/L glucose, 3.5g/L yeast powder, 5.5g/L peptone, 5.0g/L corn steep liquor, 9.0g/L sodium chloride, 0.4g/L potassium dihydrogen phosphate, 5.0g/L calcium carbonate and the balance of water, and the initial pH value is 6.9.
Fermentation culture: adding a fermentation medium which is 75% of the volume of the fermentation tank and sterilized at 121 ℃ for 35min into the fermentation tank, adding the obtained streptomyces flavipes into the fermentation medium by 8.5% of inoculation amount according to weight percentage, and fermenting under the conditions that the culture temperature is 25 ℃, the initial aeration is 0.35VVM, the initial stirring speed is 140rpm, and the pressure of the fermentation tank is maintained at 0.05MPa in the whole fermentation process. The fermentation medium comprises: 60.0g/L glucose, 2.3g/L medium-temperature soybean cake powder, 1.8g/L cottonseed cake powder, 11.0g/L yeast powder, 15.0g/L corn steep liquor, 0.6g/L sodium chloride, 0.3g/L magnesium sulfate, 5.0g/L soybean oil 9.0g/L, SCH-1 type synergist, 5.5g/L calcium carbonate and the balance of water, wherein the initial pH value is 6.5. And in the fermentation process, when the pH value is reduced to 7.0, adjusting the pH value by using 30% ammonia water by mass percent to make the pH value be 6.9-7.1. And (2) controlling the dissolved oxygen by stirring and/or ventilation in the fermentation process, wherein the dissolved oxygen of the fermentation liquor is more than 30%, determining the sugar concentration by a Fehling reagent method, and adding 200g/L of glucose sterilized at 121 ℃ for 35min when the sugar concentration is reduced to 2.5%, so that the sugar concentration in the fermentation culture process is 2.5-3.5% in percentage by weight. The concentration of the streptomyces limosus mycelium in the fermentation liquor is detected once every 3h, when the concentration of the streptomyces limosus mycelium reaches 30%, the synergist with the concentration of 21.0g/LSCH-1 is supplemented, and 0.25ml of synergist is supplemented every 1L of fermentation liquor every hour. When the fermentation was carried out for 58h, n-butanol was added to the fermentation medium in an amount of 5.5ml/L, which was filter-sterilized with a filter having a pore size of 0.22. mu.m. In the whole fermentation process, the dissolved oxygen is measured by adopting a dissolved oxygen electrode carried by the fermentation tank, and the pH is measured by adopting a pH electrode carried by the fermentation tank. After fermentation for 82h, the concentration of the streptomyces fuscoporia does not increase any more, the concentration is 44%, the content of natamycin is 6.9g/L, and the fermentation is finished.
Example 2
And (3) activation of streptomyces fulvorhii spore liquid: adopting ddH to the streptomyces limousii spore liquid with the preservation number of CGMCC NO.4628 frozen at the temperature of-82 DEG C2Diluting with O at a dilution ratio of 10-5Spreading on a plate containing 30ml of slant culture medium sterilized at 130 deg.C for 25min, and performing activation culture at 30 deg.C for 6 days, wherein the slant culture medium comprises: 9.0g/L glucose, 3.5g/L malt extract powder, 3.0g/L yeast extract powder, 5.5g/L peptone, 14.0g/L agar and the balance water, and the initial pH value is 7.1.
Seed culture: adding 60ml of seed culture medium sterilized at 130 ℃ for 25min into a 500ml shake flask, selecting and inoculating activated streptomyces flavipes spores into the culture medium, wherein the inoculation amount is 2.1%, and culturing at 230rpm and 28 ℃ for 20h to obtain the streptomyces flavipes with the concentration of 10.3%, wherein the seed culture medium comprises: 19.0g/L of glucose, 3.0g/L of yeast powder, 4.5g/L of peptone, 4.5g/L of corn steep liquor, 10.5g/L of sodium chloride, 0.45g/L of monopotassium phosphate, 5.5g/L of calcium carbonate and the balance of water, and the initial pH value is 7.0.
Fermentation culture: adding 65% volume of fermentation medium sterilized at 130 deg.C for 30min into the fermentation tank, adding 7.5% inoculum size of the obtained Streptomyces fushuangpao into the fermentation medium by weight percentage, fermenting at 29 deg.C with initial aeration of 0.25VVM and initial stirring speed of 160rpm, and maintaining the pressure of the fermentation tank at 0.06 MPa. The fermentation medium comprises: 61.0g/L glucose, 2.5g/L medium temperature soybean cake powder, 2.5g/L cottonseed cake powder, 11.0g/L yeast powder, 16.0g/L corn steep liquor, 0.4g/L sodium chloride, 0.3g/L magnesium sulfate, 11g/L, SCH-1 type soybean oil synergist 5.5g/L, 4.5g/L calcium carbonate and the balance of water, wherein the initial pH value is 6.6. And in the fermentation process, when the pH value is reduced to 7.0, adjusting the pH value by using ammonia water with the mass fraction of 30% to ensure that the pH value is 6.9-7.1. And (2) controlling the dissolved oxygen by stirring and/or ventilation in the fermentation process, wherein the dissolved oxygen of the fermentation liquor is more than 30%, determining the sugar concentration by a Fehling reagent method, adding 202g/L glucose with the concentration being 202g/L after sterilization at 121 ℃ for 35min when the sugar concentration is reduced to 2.5%, and controlling the sugar concentration in the fermentation culture process to be 2.5-3.5% in percentage by weight. The concentration of the streptomyces limosus mycelium in the fermentation liquor is detected once every 5h, when the concentration of the streptomyces limosus mycelium reaches 30%, the synergist with the concentration of 19.0g/LSCH-1 is supplemented, and 0.35ml of synergist is supplemented every 1L of fermentation liquor every hour. When the fermentation was carried out for 62 hours, n-butanol was added to the fermentation medium in an amount of 4.5ml/L, which was filter-sterilized with a filter having a pore size of 0.22. mu.m. In the whole fermentation process, the dissolved oxygen is measured by adopting a dissolved oxygen electrode carried by the fermentation tank, and the pH is measured by adopting a pH electrode carried by the fermentation tank. After fermenting for 79h, the concentration of the streptomyces fuscoporia does not increase any more, the concentration is 44.5 percent, the content of the natamycin is 6.9g/L, and the fermentation is finished.
Example 3
And (3) activation of streptomyces fulvorhii spore liquid: adopting ddH to freeze-store Streptomyces Lihuangpao spore liquid with preservation number of CGMCC NO.4628 at-80 DEG C2Diluting with O at a dilution ratio of 10-5Spread on a dish containing 25ml of a slant medium sterilized at 121 ℃ for 30min, and cultured at 28 ℃ for 6 days for activation culture, the slant medium comprising: 10.0g/L glucose, 3.0g/L malt extract powder, 3.0g/L yeast extract powder, 5.0g/L peptone, 15.0g/L agar and the balance water, and the initial pH value is 7.0.
Seed culture: adding 50ml of seed culture medium sterilized at 121 ℃ for 30min into a 500ml shake flask, selecting and inoculating activated streptomyces flavipes spores into the culture medium, wherein the inoculation amount is 2.0%, and culturing at 220rpm and 28 ℃ for 24h to obtain the streptomyces flavipes with the concentration of 10.4%, wherein the seed culture medium comprises: 20.0g/L of glucose, 3.0g/L of yeast powder, 5.0g/L of peptone, 5.0g/L of corn steep liquor, 10.0g/L of sodium chloride, 0.5g/L of monopotassium phosphate, 5.0g/L of calcium carbonate and the balance of water.
Fermentation culture: adding fermentation medium which is sterilized at 121 ℃ for 30min and has a volume of 70% of that of the fermentation tank into the fermentation tank, adding the obtained streptomyces limoflavus into the fermentation medium by an inoculation amount of 7.0% in percentage by weight, and fermenting under the conditions that the culture temperature is 28 ℃, the initial aeration is 0.3VVM, the initial stirring speed is 150rpm, and the pressure of the fermentation tank is maintained at 0.05MPa in the whole fermentation process. The fermentation medium comprises: 60.0g/L glucose, 2.0g/L medium-temperature soybean cake powder, 2.0g/L cottonseed cake powder, 10.0g/L yeast powder, 15.0g/L corn steep liquor, 0.5g/L sodium chloride, 0.2g/L magnesium sulfate, 10.0g/L, SCH-1 type soybean oil synergist 5.0g/L, 5.0g/L calcium carbonate and the balance of water, wherein the initial pH value is 6.5. And in the fermentation process, when the pH value is reduced to 7.0, adjusting the pH value by using ammonia water with the mass fraction of 30% to ensure that the pH value is 6.9-7.1. And (2) controlling the dissolved oxygen by stirring and/or ventilation in the fermentation process, wherein the dissolved oxygen of the fermentation liquor is more than 30%, determining the sugar concentration by a Fehling reagent method, and adding 200g/L of glucose sterilized at 121 ℃ for 35min when the sugar concentration is reduced to 2.5%, so that the sugar concentration in the fermentation culture process is 2.5-3.5% in percentage by weight. The concentration of the streptomyces limosus mycelium in the fermentation liquor is detected once every 4h, when the concentration of the streptomyces limosus mycelium reaches 30%, the synergist with the concentration of 20.0g/LSCH-1 is supplemented, and 0.3ml of synergist is supplemented every 1L of fermentation liquor every hour. When the fermentation was carried out for 60 hours, n-butanol was added to the fermentation medium at an amount of 5.0ml/L, which was subjected to filtration sterilization using a 0.22 μm pore size strainer. In the whole fermentation process, the dissolved oxygen is measured by adopting a dissolved oxygen electrode carried by the fermentation tank, and the pH is measured by adopting a pH electrode carried by the fermentation tank. After fermentation for 80h, the concentration of the streptomyces fuscoporia does not increase any more, the concentration is 45 percent, the content of natamycin is 7.0g/L, and the fermentation is finished.
Example 4
And (3) activation of streptomyces fulvorhii spore liquid: adopting ddH to freeze-store Streptomyces Lihuangpao spore liquid with preservation number of CGMCC NO.4628 at-80 DEG C2Diluting with O at a dilution ratio of 10-5Spreading on a slant surface containing 25ml of the extract after sterilizing at 121 deg.C for 30minActivated culture was carried out in a plate of culture medium at 28 ℃ for 6 days, and the slant culture medium included: 10.0g/L glucose, 3.0g/L malt extract powder, 3.0g/L yeast extract powder, 5.0g/L peptone, 15.0g/L agar and the balance water, and the initial pH value is 7.0.
Seed culture: adding 50ml of seed culture medium sterilized at 121 ℃ for 30min into a 500ml shake flask, selecting and inoculating activated streptomyces flavipes spores into the culture medium, wherein the inoculation amount is 2.0%, and culturing at 220rpm and 28 ℃ for 24h to obtain the streptomyces flavipes with the concentration of 10.4%, wherein the seed culture medium comprises: 20.0g/L of glucose, 3.0g/L of yeast powder, 5.0g/L of peptone, 5.0g/L of corn steep liquor, 10.0g/L of sodium chloride, 0.5g/L of monopotassium phosphate, 5.0g/L of calcium carbonate and the balance of water.
Fermentation culture: adding fermentation medium which is sterilized at 121 ℃ for 30min and has a volume of 70% of that of the fermentation tank into the fermentation tank, adding the obtained streptomyces limoflavus into the fermentation medium by an inoculation amount of 7.0% in percentage by weight, and fermenting under the conditions that the culture temperature is 28 ℃, the initial aeration is 0.3VVM, the initial stirring speed is 150rpm, and the pressure of the fermentation tank is maintained at 0.05MPa in the whole fermentation process. The fermentation medium comprises: 60.0g/L glucose, 2.0g/L medium-temperature soybean cake powder, 2.0g/L cottonseed cake powder, 10.0g/L yeast powder, 15.0g/L corn steep liquor, 0.5g/L sodium chloride, 0.2g/L magnesium sulfate, 10.0g/L soybean oil, 5.0g/L calcium carbonate and the balance of water, wherein the initial pH value is 6.5. And in the fermentation process, when the pH value is reduced to 7.0, adjusting the pH value by using ammonia water with the mass fraction of 30% to ensure that the pH value is 6.9-7.1. And (2) controlling the dissolved oxygen by stirring and/or ventilation in the fermentation process, wherein the dissolved oxygen of the fermentation liquor is more than 30%, determining the sugar concentration by a Fehling reagent method, and adding 200g/L of glucose sterilized at 121 ℃ for 35min when the sugar concentration is reduced to 2.5%, so that the sugar concentration in the fermentation culture process is 2.5-3.5% in percentage by weight. The concentration of the streptomyces limosus mycelium in the fermentation liquor is detected once every 4 hours. In the whole fermentation process, the dissolved oxygen is measured by adopting a dissolved oxygen electrode carried by the fermentation tank, and the pH is measured by adopting a pH electrode carried by the fermentation tank. After fermentation for 100h, the concentration of the streptomyces fuscoporia does not increase any more, the concentration is 30 percent, the content of the natamycin is 5.0g/L, and the fermentation is finished.
The method for producing natamycin by fermenting streptomyces fuscoporia provided by the application has a short fermentation period, and compared with the control group in example 4, the whole fermentation period is shortened from 100h to 79-82 h, the final concentration of streptomyces fuscoporia mycelium is 44-45%, the concentration of 30% mycelium of the control group is increased by 50%, and the content of natamycin is increased to 6.9-7 g/L from 5g/L of the control group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (9)

1. A method for producing natamycin by fermenting streptomyces fuscoporia comprises the following steps:
1) activating streptomyces limoflavus spore liquid;
2) inoculating the streptomyces flavipes spores activated in the step 1) into a seed culture medium for seed culture to obtain streptomyces flavipes;
3) inoculating the streptomyces fuscogongensis obtained in the step 2) into a fermentation medium for aerobic fermentation culture to obtain natamycin;
the fermentation medium in the step 3) is as follows: 60.0 +/-1.0 g/L of glucose, 2.0 +/-0.5 g/L of medium-temperature soybean cake powder, 2.0 +/-0.5 g/L of cottonseed cake powder, 10.0 +/-1.0 g/L of yeast powder, 15.0 +/-1.0 g/L of corn steep liquor, 0.5 +/-0.1 g/L of sodium chloride, 0.2 +/-0.1 g/L of magnesium sulfate, 10.0 +/-1.0 g/L, SCH-1 type synergist 5.0 +/-0.5 g/L of soybean oil, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water;
and in the step 3), when the concentration of the streptomyces limosus mycelium in the fermentation liquor reaches 30%, the SCH-1 type synergist is supplemented.
2. The fermentative production method according to claim 1, wherein the seed medium of step 2) comprises: 20.0 +/-1.0 g/L of glucose, 3.0 +/-0.5 g/L of yeast powder, 5.0 +/-0.5 g/L of peptone, 5.0 +/-0.5 g/L of corn steep liquor, 10.0 +/-1.0 g/L of sodium chloride, 0.5 +/-0.1 g/L of monopotassium phosphate, 5.0 +/-0.5 g/L of calcium carbonate and the balance of water.
3. The fermentative production method according to claim 1 or 2, wherein the seed culture in step 2) is performed under the following conditions: rotating speed of a shaking table: 210-230rpm, a culturing temperature of 25-30 ℃, a culturing time of 20-26 h and an initial pH value of 6.9-7.0.
4. The fermentation production method according to claim 1, wherein the inoculation amount of the fermentation culture in the step 3) is 7.5-8.5% of the volume of the fermentation medium in percentage by volume.
5. The fermentation production method of claim 1, wherein the fermentation medium is added in the step 3) in an amount of 65-75% by volume of the fermentation tank.
6. The fermentative production method according to claim 1, wherein the conditions of the fermentative culture in step 3) are: the culture temperature is 25-30 ℃, the initial aeration is 0.25-0.35 VVM, the initial stirring speed is 140-160 rpm, the initial pH value is 6.4-6.6, and the pressure of the fermentation tank is maintained at 0.04-0.06 MPa in the whole fermentation process.
7. The fermentation production method according to claim 1, wherein the dissolved oxygen content of the fermentation liquid during the fermentation culture in step 3) is 30% or more.
8. The fermentation production method according to claim 1, wherein the pH value of the fermentation liquid in the fermentation culture process in the step 3) is 6.9-7.1.
9. The fermentation production method according to claim 1, wherein the concentration of sugar in the fermentation culture in step 3) is 2.5 to 3.5% by weight.
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