Fermentation method of clavulanic acid

Abstract

The invention provides a fermentation method of clavulanic acid, and relates to the technical field of biological pharmacy. The method comprises the following steps: inoculating streptomyces clavuliformis into a shake flask seed culture medium, and carrying out shake flask seed culture; inoculating the cultured shake flask seeds to a seed culture medium, and performing seed tank culture; the method comprises the following steps: inoculating a strain into a fermentation culture medium, culturing in a fermentation tank, centrifugally separating the obtained fermentation liquid, and separating and purifying the supernatant liquid to obtain clavulanic acid; wherein in the 20th hour of fermentation, glycerol is supplemented; supplementing the mixed supplementary material at the 40th hour of fermentation; the mixed supplementary material comprises L-glutamic acid, magnesium chloride, dipotassium phosphate, glacial acetic acid and potassium hydroxide. According to the method disclosed by the invention, the glycerol and the mixed supplementary material are supplemented at a specific time point in the fermentation process of the clavulanic acid, so that the fine regulation and control of the fermentation process are realized, and the technical effect of remarkably improving the titer of the clavulanic acid is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a clavulanic acid fermentation method. Background Art

[0002] β-lactam antibiotics are widely used in clinical practice because of their strong antibacterial activity against both Gram-positive and Gram-negative bacteria. However, long-term use of these antibiotics can easily lead to bacterial resistance. The main mechanism by which pathogens resist β-lactam antibiotics is the production of β-lactamase, an enzyme that can decompose the β-lactam ring of β-lactam antibiotics, thereby inactivating the drug.

[0003] Clavulanic acid, also known as clavulanic acid, is a natural β-lactamase inhibitor produced by Streptomyces clavuligerus. The molecular structure of clavulanic acid consists of a fused bicyclic structure consisting of a β-lactam ring and an oxazole ring, and its structural formula is as follows:

[0004]

[0005] Clavulanic acid is a strong and broad-spectrum β-lactamase inhibitor that can bind to β-lactamase irreversibly, thereby inhibiting the bacterial decomposition of the β-lactam ring. It has antibacterial activity against both Gram-positive and Gram-negative bacteria. However, due to its relatively weak antibacterial activity, clavulanic acid is usually used in combination with other antibiotics that are sensitive to β-lactamase.

[0006] The prior art discloses a method for producing clavulanic acid by fermentation of Streptomyces clavuliferus (publication number, CN101280329A), which uses Streptomyces clavuliferus as the starting bacteria and mainly includes the steps of preparing slant strains, preparing seed liquid, and controlling the fermentation process. The present invention uses cottonseed powder and starch as the main nitrogen source and carbon source of the fermentation medium and reduces the production cost of clavulanic acid by regulating the fermentation process. However, the titer of the above method is 5.4 g / L; there are disadvantages of low final titer and high fermentation cost.

[0007] Therefore, there is an urgent need for a fermentation method for clavulanic acid with high final potency but low fermentation cost. Summary of the invention

[0008] In view of the above problems, the present invention provides a clavulanic acid fermentation method to solve at least one problem existing in the prior art.

[0009] To achieve the above object, the present invention provides a clavulanic acid fermentation method, which comprises inoculating Streptomyces clavuligerus into a shake flask seed culture medium to perform shake flask seed culture;

[0010] Take the cultured shake flask seeds and inoculate them into the seed culture medium for seed tank culture;

[0011] The strain is inoculated into a fermentation medium, cultured in a fermenter, and then the obtained fermentation liquid is centrifuged and separated, and the upper clear liquid is separated and purified to obtain clavulanic acid; wherein glycerol is added at the 20th hour of fermentation; and mixed feed is added at the 40th hour of fermentation;

[0012] The mixed feed includes L-glutamic acid, magnesium chloride, dipotassium hydrogen phosphate, glacial acetic acid and potassium hydroxide.

[0013] Further, the preferred method is that the conditions for the shake flask seed culture are to carry out the shake flask seed culture at a pH value of 6.5 to 7.1 and a temperature of 28° C. for 60 to 68 hours;

[0014] The conditions for carrying out the seed tank culture are: carrying out the seed tank culture for 25 to 35 hours at an initial pH value of 7.0 and a temperature of 28°C;

[0015] The fermentation tank culture was carried out at a pH of 6.7 to 7.0 and a temperature of 24 to 26° C. for 120 to 130 hours.

[0016] Furthermore, a preferred method is that the mass percentages of the mixed feed are: L-glutamic acid 0.5%-1.5%, magnesium chloride 0.06%-0.12%, dipotassium hydrogen phosphate 0.6%-1%, glacial acetic acid 2.5%-10%, and potassium hydroxide 1%-2.5%.

[0017] Further, the preferred method is that the mass percentage of the mixed feed is,

[0018] L-glutamic acid 0.6%-1.2%, magnesium chloride 0.08%-0.1%, dipotassium hydrogen phosphate 0.7%-0.8%, glacial acetic acid 5%-7.5%, potassium hydroxide 1.5%-2%.

[0019] Furthermore, a preferred method is that the mass percentages of the mixed feed are: 1% L-glutamic acid, 0.1% magnesium chloride, 0.8% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

[0020] Further, a preferred method is that the fermentation medium comprises:

[0021] Deionized water is used as the medium, with 34 g / L soybean protein concentrate, 16 g / L vegetable oil, 2.8 g / L NaH2PO4, 0.05 g / L FeCl3·6H2O, 0.8 g / L MgCl2·6H2O, 0.012 g / L ZnCl2, 0.005 g / L MnSO4 and 1 g / L defoaming agent.

[0022] Furthermore, a preferred method is that in the step of adding mixed feed at the 40th hour of fermentation, the feed rate is 48 g / hour.

[0023] Furthermore, in a preferred method, the strain of Streptomyces clavuligerus is Streptomyces clavuligerus KNR1.

[0024] Furthermore, a preferred method is that, when the fermentation is continued for 20 hours, in the step of adding glycerol, a glycerol solution with a concentration of 25% is added, and the addition rate is controlled according to the residual glycerol in the fermentation broth to maintain the set residual glycerol in the fermentation broth.

[0025] Furthermore, a preferred method is that, during the step of fermentation culture for 120 to 130 hours, the method further comprises adding ammonia water to control the pH value to 7.0.

[0026] The beneficial effects of the present invention are as follows: the fermentation method of clavulanic acid provided by the present invention not only ensures the quality of clavulanic acid and reduces the production cost by improving the fermentation process, but also can achieve a potency of 6400 μg / ml; and has broad industrial application prospects. DETAILED DESCRIPTION

[0027] The present invention will be described in more detail with reference to the following examples. However, the protection scope of the present invention is not limited to the following examples.

[0028] The strain is Streptomyces clavuligerus KNR1, which is from the American Type Culture Collection (ATCC) with the accession number of ATCC27064.

[0029] Among the various experimental supplies mentioned in this article (including but not limited to: chemical reagents, biological products, cells, organisms, instruments, etc.), for those that are special or difficult to obtain, the manufacturers, references or detailed preparation methods have been indicated in the article; those not otherwise specified are conventional experimental supplies and can be easily obtained by various means (such as purchase, self-preparation, etc.) before the date of this application.

[0030] The present invention provides a clavulanic acid fermentation method, which comprises the following steps:

[0031] S110, inoculating the Streptomyces clavuligerus into a shake flask seed culture medium, and performing shake flask seed culture at a pH value of 6.5 to 7.1 and a temperature of 28° C. for 60 to 68 hours;

[0032] S120, taking the cultured shake flask seeds and inoculating them into the seed culture medium, and culturing them in a seed tank for 25 to 35 hours at an initial pH of 7.0 and a temperature of 28° C.;

[0033] S130, inoculating the strain into a fermentation medium, and culturing in a fermenter for 120 to 130 hours at a pH of 6.7 to 7.0 and a temperature of 24 to 26° C., wherein ammonia water is added to control the pH to 7.0.

[0034] Fermentation medium: 34 g / L soybean protein concentrate, 16 g / L vegetable oil, 2.8 g / L NaH2PO4, 0.05 g / L FeCl3·6H2O, 0.8 g / L MgCl2·6H2O, 0.012 g / L ZnCl2, 0.005 g / L MnSO4, 1 g / L defoamer. The soybean protein concentrate may be soybean meal. The vegetable oil may be one or more of soybean oil, corn oil or cottonseed oil. The defoamer may be PPG2000.

[0035] Among them, at the 20th hour of fermentation, a glycerol solution with a concentration of 25% was added, and the feeding rate was controlled according to the residual glycerol in the fermentation broth to maintain the set residual glycerol in the fermentation broth; at the 40th hour of fermentation, mixed feed was added at a feeding rate of 48 g / hour.

[0036] The mixed feed comprises (by mass percentage): 0.5%-1.5% L-glutamic acid, 0.06%-0.12% magnesium chloride, 0.6%-1% dipotassium hydrogen phosphate, 2.5%-10% glacial acetic acid, and 1%-2.5% potassium hydroxide.

[0037] It should be noted that adding amino acids can promote the production of clavulanic acid by Streptomyces clavuliferus; adding dipotassium hydrogen phosphate can supplement phosphorus and promote the metabolism of Streptomyces clavuliferus; adding magnesium chloride can alleviate the degradation of clavulanic acid; adding glacial acetic acid provides energy for the growth of Streptomyces clavuliferus; adding potassium hydroxide is to adjust pH and provide potassium ions to maintain the ionization balance of Streptomyces clavuliferus.

[0038] S140, centrifuging the obtained fermentation broth, taking the upper clear liquid for separation and purification to obtain clavulanic acid.

[0039] The present invention achieves fine control of the fermentation process by adding glycerol and mixed feed at specific time points during the clavulanic acid fermentation process, and the obtained clavulanic acid solution has a titer greater than 5552 μg / ml, which significantly increases the yield of clavulanic acid. The mycelium concentration of the fermentation broth is greater than 47%, indicating that the method can effectively promote the growth of rod-shaped Streptomyces, thereby increasing biomass and providing more bioactive substances for the synthesis of clavulanic acid. By accurately controlling the mass percentage of each component in the mixed feed, especially when the mass percentages of L-glutamic acid, magnesium chloride, dipotassium hydrogen phosphate, glacial acetic acid and potassium hydroxide are optimized to 1%, 0.1%, 0.8%, 7.5% and 2% respectively, the titer of the clavulanic acid solution can reach 6406 μg / ml; the technical effect of improving the clavulanic acid fermentation efficiency is achieved. In summary, the fermentation method of the present invention not only promotes the growth and metabolism of Streptomyces clavuligerus, but also increases the titer of clavulanic acid and the mycelium concentration through precise feeding strategy and optimized feeding formula, as well as by providing necessary nutrients and regulating the growth environment, thereby achieving efficient and stable clavulanic acid production.

[0040] The method for determining the mycelium concentration of a fermentation broth comprises: taking out 10 mL of a sample from the fermentation broth and placing it in a centrifuge tube. Centrifuging the sample at a speed of 4000 r / min for 20 minutes. Calculating the ratio of the solid volume to the total volume of the fermentation broth after centrifugation, and using the ratio as the mycelium concentration.

[0041] The method for determining the potency of the obtained clavulanic acid by HPLC comprises the following specific steps: chromatographic column: using an ODS C18 chromatographic column with a size of 4.6×100 mm and a particle size of 3.5 μm. Preparation of mobile phase: weighing 3.40 g of tetrabutylamine hydrogen sulfate and dissolving it in 1000 ml of water. Using 0.5 mol / L sodium hydroxide solution to adjust the pH value to 5.95-6.05. Adding 130 ml of acetonitrile, filtering with a 0.45 μm microporous filter membrane, the obtained filtrate is the mobile phase. Preparation of diluent: weighing 3.12 g of sodium dihydrogen phosphate and dissolving it in 1000 ml of water. Using 0.5 mol / L sodium hydroxide to adjust the pH to 5.40. Adding 60 ml of acetonitrile, filtering with a 0.45 μm microporous filter membrane, the obtained filtrate is the diluent. Chromatographic conditions: flow rate: 1.0 mL / min; UV detection wavelength: 210 nm; column temperature: 33° C.; injection volume: 20 μL. Purity calculation: The purity of clavulanic acid is calculated as the percentage of the clavulanic acid liquid phase peak area to the total area.

[0042] Example 1

[0043] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0044] Fermentation method:

[0045] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0046] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0047] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0048] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0049] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0050] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0051] The mixed feed contains (by mass percentage): 0.5% L-glutamic acid, 0.06% magnesium chloride, 0.6% dipotassium hydrogen phosphate, 2.5% glacial acetic acid, and 1% potassium hydroxide.

[0052] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0053] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0054] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 1.

[0055] Table 1 Clavulanic acid titer and mycelium concentration test results of Example 1

[0056]

[0057] Example 2

[0058] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0059] Fermentation method:

[0060] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 60 hours.

[0061] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0062] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0063] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0064] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0065] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 125 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0066] The mixed feed contains (by mass percentage): 0.8% L-glutamic acid, 0.08% magnesium chloride, 0.8% dipotassium hydrogen phosphate, 5% glacial acetic acid, and 1.5% potassium hydroxide.

[0067] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0068] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0069] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 2.

[0070] Table 2 Clavulanic acid titer and mycelium concentration test results of Example 2

[0071]

[0072] Example 3

[0073] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0074] Fermentation method:

[0075] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 68 hours.

[0076] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0077] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0078] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 35 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0079] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0080] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0081] The mixed feed contains (by mass percentage): 1% L-glutamic acid, 0.1% magnesium chloride, 1% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

[0082] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0083] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0084] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 3.

[0085] Table 3 Clavulanic acid titer and mycelium concentration test results of Example 3

[0086]

[0087] Example 4

[0088] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0089] Fermentation method:

[0090] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0091] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0092] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0093] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 28 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5 VVM and a tank pressure of 0.05 MPa.

[0094] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0095] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0096] The mixed feed contains (by mass percentage): 1.2% L-glutamic acid, 0.12% magnesium chloride, 1% dipotassium hydrogen phosphate, 10% glacial acetic acid, and 2.5% potassium hydroxide.

[0097] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0098] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0099] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 4.

[0100] Table 4 Clavulanic acid titer and mycelium concentration test results of Example 4

[0101]

[0102] Example 5

[0103] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0104] Fermentation method:

[0105] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0106] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0107] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0108] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0109] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0110] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0111] The mixed feed contains (by mass percentage): 1.5% L-glutamic acid, 0.12% magnesium chloride, 1% dipotassium hydrogen phosphate, 10% glacial acetic acid, and 2.5% potassium hydroxide.

[0112] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0113] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0114] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 5.

[0115] Table 5 Clavulanic acid titer and mycelium concentration test results of Example 5

[0116]

[0117] Example 6

[0118] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0119] Fermentation method:

[0120] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0121] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0122] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0123] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0124] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0125] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0126] The mixed feed contains (by mass percentage): 1% L-glutamic acid, 0.1% magnesium chloride, 0.8% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

[0127] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0128] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0129] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 6.

[0130] Table 6 Clavulanic acid titer and mycelium concentration test results of Example 6

[0131]

[0132] Example 7

[0133] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0134] Fermentation method:

[0135] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0136] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0137] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0138] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0139] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0140] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0141] The mixed feed contains (by mass percentage): 0.8% L-glutamic acid, 0.1% magnesium chloride, 1% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

[0142] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0143] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0144] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 7.

[0145] Table 7 Clavulanic acid titer and mycelium concentration test results of Example 7

[0146]

[0147]

[0148] Example 8

[0149] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0150] Fermentation method:

[0151] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 60 hours.

[0152] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0153] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0154] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 25 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0155] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0156] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 130 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 40th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0157] The mixed feed contains (by mass percentage): 1% L-glutamic acid, 0.1% magnesium chloride, 0.8% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

[0158] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0159] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0160] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 8.

[0161] Table 8 Clavulanic acid titer and mycelium concentration test results of Example 8

[0162]

[0163]

[0164] It can be seen from the observation that, by adding glycerol at 20 hours and mixed feed at 40 hours during the fermentation process, the titer of the obtained clavulanic acid solution is greater than 5552 μg / ml, and the mycelium concentration of the fermentation broth is greater than 47%. When the mass percentage of the feed is limited to the optimal value, L-glutamic acid 1%, magnesium chloride 0.1%, dipotassium hydrogen phosphate 0.8%, glacial acetic acid 7.5%, potassium hydroxide 2%, the titer of the obtained clavulanic acid solution reaches 6454 μg / ml.

[0165] Comparative Example 1

[0166] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0167] Fermentation method:

[0168] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0169] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0170] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0171] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0172] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0173] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at 20 hours, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0174] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0175] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0176] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 8.

[0177] Table 9 Clavulanic acid titer and mycelium concentration test results of Comparative Example 1

[0178]

[0179] Except for adding glycerol as a supplementary material during the fermentation process, Comparative Example 1 did not use the mixed feed described in the present invention. In addition, the other implementation conditions of Comparative Example 1 are completely consistent with those of Example 1. By observing the data in Table 9, it can be seen that the titer of the clavulanic acid solution obtained in Comparative Example 1 is only 5178 μg / ml; while the titer of the clavulanic acid solution obtained in Example 1 is greater than 5552 μg / ml. In other words, compared with Example 1, the titer of the clavulanic acid solution in Comparative Example 1 is lower, which indicates that the use of mixed feed (such as the mixed feed containing L-glutamic acid, magnesium chloride, dipotassium hydrogen phosphate, glacial acetic acid and potassium hydroxide described in Example 1) during the fermentation process helps to increase the titer of clavulanic acid.

[0180] Comparative Example 2

[0181] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0182] Fermentation method:

[0183] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0184] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0185] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0186] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0187] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0188] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 20th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0189] The mixed feed contains (by mass percentage): 1.5% L-glutamic acid, 0.12% magnesium chloride, 1% dipotassium hydrogen phosphate, 10% glacial acetic acid, and 2.5% potassium hydroxide.

[0190] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0191] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0192] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 9.

[0193] Table 10 Clavulanic acid titer and mycelium concentration test results of comparative example 2

[0194]

[0195] In addition to adding glycerol and mixed feed as supplementary materials during the fermentation process, the time for adding mixed feed in Comparative Example 2 is the 20th hour of fermentation. In addition, the other implementation conditions of Comparative Example 2 are completely consistent with those of Example 5. By observing the data in Table 10, it can be seen that the titer of the clavulanic acid solution obtained in Comparative Example 2 is only 5265 μg / ml; while the titer of the clavulanic acid solution obtained in Example 5 is greater than 6149 μg / ml. In other words, compared with Example 5, the titer of the clavulanic acid solution in Comparative Example 2 is lower, which shows that accurately controlling the addition time of mixed feed during the fermentation process is helpful to improve the titer of clavulanic acid.

[0196] Comparative Example 3

[0197] Preparation of fermentation medium: Fermentation medium was prepared according to the following formula: soybean cake powder 34 g / L, corn oil 16 g / L, NaH2PO4 2.8 g / L, FeCl3·6H2O 0.05 g / L, MgCl2·6H2O 0.8 g / L, ZnCl2 0.012 g / L, MnSO4 0.005 g / L, and defoaming agent 1 g / L.

[0198] Fermentation method:

[0199] Seed culture: S110. Take the clavulose Streptomyces strain KNR1, inoculate it into the shake flask seed culture medium, and culture it at 28° C. and 260 rpm for 64 hours.

[0200] The slant culture medium is composed of 15 g / L malt extract, 15 g / L yeast extract, 12 g / L glucose and 25 g / L agar.

[0201] The shake flask seed culture medium is composed of: 18 g / L soybean cake powder, 8 g / L yeast extract, and 15 g / L glycerol, and the pH is adjusted to 7.00.

[0202] Seed expansion culture: S120, take 10 ml (0.03% volume ratio) of the seeds cultured in step S110 and inoculate them into a seed tank, and culture them for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM and a tank pressure of 0.05MPa.

[0203] Among them, the main components of the seed tank culture medium are: 20g / L soybean cake powder, 20g / L corn starch, 8g / L yeast powder, 1g / L defoaming agent, and the pH is adjusted to 7.00.

[0204] Fermentation process: S130, 4.2L (13.125% by volume) of the seeds cultured in step S120 are inoculated into a fermentation tank, and the fermentation is carried out for 120 hours at a temperature of 25°C, a stirring speed of 150-600 rpm, a ventilation volume of 0.3-1.3VVM, and a tank pressure of 0.05MPa, while the pH is controlled to be 7.00. During the fermentation process, glycerol is added at the 20th hour, mixed feed is added at the 30th hour, and ammonia water is used to control the pH of the fermentation liquid to be maintained at 7.00.

[0205] The mixed feed contains (by mass percentage): 1.5% L-glutamic acid, 0.12% magnesium chloride, 1% dipotassium hydrogen phosphate, 10% glacial acetic acid, and 2.5% potassium hydroxide.

[0206] S140. After the fermentation is completed, the obtained fermentation liquid is centrifuged at a speed of 4000 rpm for 20 minutes, and the obtained supernatant is the clavulanic acid solution.

[0207] The mycelium concentration and potency of the fermentation broth and clavulanic acid solution were determined respectively.

[0208] The mycelium concentration, clavulanic acid titer and clavulanic acid purity were tested, and the results are shown in Table 10.

[0209] Table 11 Clavulanic acid titer and mycelium concentration test results of comparative example 3

[0210]

[0211] In addition to adding glycerol and mixed feed as supplementary materials during the fermentation process, the time for adding mixed feed in Comparative Example 3 is the 30th hour of fermentation. In addition, the other implementation conditions of Comparative Example 3 are completely consistent with those of Example 5. By observing the data in Table 11, it can be seen that the titer of the clavulanic acid solution obtained in Comparative Example 3 is only 5475 μg / ml; while the titer of the clavulanic acid solution obtained in Example 5 is greater than 6149 μg / ml. In other words, compared with Example 5, the titer of the clavulanic acid solution in Comparative Example 3 is lower, which shows that accurately controlling the addition time of mixed feed during the fermentation process is helpful to improve the titer of clavulanic acid.

[0212] In summary, the fermentation method of clavulanic acid provided by the present invention not only promotes the growth and metabolism of Streptomyces clavuligerus, but also improves the titer of clavulanic acid and the mycelium concentration through a precise feeding strategy and an optimized feeding formula, as well as by providing necessary nutrients and adjusting the growth environment, thereby achieving the technical effect of efficient and stable clavulanic acid production.

[0213] Although the present invention has been described in detail above by general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications and improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A clavulanic acid fermentation method, characterized in that the method include: Inoculating the Streptomyces clavuligerus into a shake flask seed culture medium to perform shake flask seed culture; Take the cultured shake flask seeds and inoculate them into the seed culture medium for seed tank culture; The strain is inoculated into a fermentation medium, cultured in a fermenter, and then the obtained fermentation liquid is centrifuged and separated, and the upper clear liquid is separated and purified to obtain clavulanic acid; wherein glycerol is added at the 20th hour of fermentation; and mixed feed is added at the 40th hour of fermentation; The mixed feed includes L-glutamic acid, magnesium chloride, dipotassium hydrogen phosphate, glacial acetic acid and potassium hydroxide.

2. The clavulanic acid fermentation method according to claim 1, characterized in that: The conditions for the shake flask seed culture are: pH 6.5 to 7.1, temperature 28° C., and the shake flask seed culture for 60 to 68 hours; The conditions for carrying out the seed tank culture are: carrying out the seed tank culture for 25 to 35 hours at an initial pH value of 7.0 and a temperature of 28°C; The fermentation tank culture was carried out at a pH of 6.7 to 7.0 and a temperature of 24 to 26° C. for 120 to 130 hours.

3. The clavulanic acid fermentation method according to claim 1, characterized in that: The mass percentages of the mixed feed are: 0.5%-1.5% L-glutamic acid, 0.06%-0.12% magnesium chloride, 0.6%-1% dipotassium hydrogen phosphate, 2.5%-10% glacial acetic acid, and 1%-2.5% potassium hydroxide.

4. The clavulanic acid fermentation method according to claim 3, characterized in that: The mass percentage of the mixed feed is, L-glutamic acid 0.6%-1.2%, magnesium chloride 0.08%-0.1%, dipotassium hydrogen phosphate 0.7%-0.8%, glacial acetic acid 5%-7.5%, potassium hydroxide 1.5%-2%.

5. The clavulanic acid fermentation method according to claim 4, characterized in that: The mass percentages of the mixed feed are: 1% L-glutamic acid, 0.1% magnesium chloride, 0.8% dipotassium hydrogen phosphate, 7.5% glacial acetic acid, and 2% potassium hydroxide.

6. The clavulanic acid fermentation method according to claim 1, characterized in that: The fermentation medium comprises: Deionized water is used as the medium, with 34 g / L soybean protein concentrate, 16 g / L vegetable oil, 2.8 g / L NaH2PO4, 0.05 g / L FeCl3·6H2O, 0.8 g / L MgCl2·6H2O, 0.012 g / L ZnCl2, 0.005 g / L MnSO4 and 1 g / L defoaming agent.

7. The clavulanic acid fermentation method according to claim 1, characterized in that: At the 40th hour of fermentation, in the step of adding mixed feed, the feeding rate was 48 g / hour.

8. The clavulanic acid fermentation method according to claim 1, characterized in that: The strain of Streptomyces clavuligerus is Streptomyces clavuligerus KNR1.

9. The clavulanic acid fermentation method according to claim 1, characterized in that: At the 20th hour of fermentation, in the step of adding glycerol, a glycerol solution with a concentration of 25% is added, and the addition rate is controlled according to the residual glycerol in the fermentation broth to maintain the set residual glycerol in the fermentation broth.

10. The clavulanic acid fermentation method according to claim 1, characterized in that: During the step of fermentation culture for 120 to 130 hours, aqueous ammonia is added to control the pH to 7.0.

Images