Method for preparing clavulanic acid by biological fermentation method

Abstract

The invention belongs to the technical field of biological medicines, and discloses a method for preparing clavulanic acid by a biological fermentation method. The method comprises the following steps: inoculating a seed bacterial liquid of streptomyces clavuliformis into a fermentation culture medium for fermentation culture, and collecting a fermentation product to obtain clavulanic acid, wherein in the fermentation culture process, an amylase enzymolysis solution is supplemented, the total sugar concentration range of fermentation liquor is maintained to be 6 g / L-12 g / L, and the amylase enzymolysis solution is obtained by performing enzymolysis and liquefaction on starch through alpha-amylase. By adopting the method, the fermentation titer can be improved, and the production cost of the clavulanic acid can be reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology and relates to a new method for preparing clavulanic acid by biological fermentation, specifically, a method for using amylase hydrolysis solution instead of glycerol feed for fermentation production of clavulanic acid. Background Art

[0002] The widespread use of β-lactam antibiotics such as penicillin, cephalosporin, and cephamycin has led to bacterial resistance. Clavulanic acid, also known as clavulanic acid, is a natural β-lactamase inhibitory antibiotic produced by Streptomyces clavuligerus. It can irreversibly bind to β-lactamase, thereby inhibiting bacterial decomposition of the β-lactam ring. Since clavulanic acid has very weak antibacterial activity, it is more often used in combination with other antibiotics that are sensitive to β-lactamase to inhibit β-lactamase, thereby restoring the antibacterial activity of penicillins and cephalosporins against resistant bacteria.

[0003] The molecular structure of clavulanic acid is as follows:

[0004]

[0005] At present, the industrial production of clavulanic acid at home and abroad is prepared by fermentation of Streptomyces clavulosus. Literature reports that one of the precursors of clavulanic acid biosynthesis comes from glycerol molecules. Therefore, in the current clavulanic acid fermentation production, the fermentation medium uses glycerol as the main feed, which is large in amount and high in unit price, and thus the production cost is high. Moreover, during the fermentation process, the detection cost of glycerol is high, and the method for detecting glycerol is a kit or HPLC; among them, the kit is expensive, the test result has a large deviation, and the credibility is not high; and HPLC, its equipment is expensive and the operation requirements are high. Furthermore, the inventors of the present application also noticed that the fermentation process stability is not very good when glycerol is used as a feed, and the fermentation titer is low (generally lower than 5.0 g / L, or even lower than 4.0 g / L).

[0006] It can be seen that the above-mentioned problems still exist in the current process of producing clavulanic acid by fermentation using glycerol as a carbon source and feed supplement. Summary of the invention

[0007] According to one embodiment of the present invention, the purpose is to provide a method for preparing clavulanic acid by biological fermentation, so as to solve the problems of poor fermentation process stability, low fermentation titer and high production cost in the prior art.

[0008] The above purpose can be achieved by implementing the following technical solutions:

[0009] According to one aspect of the present invention, a method for preparing clavulanic acid by a biological fermentation method provided by the present invention comprises: step 4, inoculating a seed culture of Streptomyces clavuligerus into a fermentation medium for fermentation culture, collecting the fermentation product, and obtaining clavulanic acid; wherein, during the fermentation culture, a starch enzymatic hydrolysis solution is added to maintain the total sugar concentration of the fermentation liquid in the range of 6 g / L to 12 g / L; the starch enzymatic hydrolysis solution is obtained by enzymatically hydrolyzing starch with α-amylase to liquefy it;

[0010] The fermentation medium uses deionized water as a medium and contains: 30-44 g / L soybean meal, 5-20 g / L starch, 12-17 g / L vegetable oil, 2.6-2.9 g / L NaH2PO4, 0.04-0.06 g / L FeCl3·6H2O, 0.7-0.9 g / L MgCl2·6H2O, 0.010-0.014 g / L ZnCl2, 0.004-0.006 g / L MnSO4, and 0.5-1.5 g / L defoaming agent.

[0011] Optionally, the method for preparing the starch enzymatic hydrolysis solution comprises: preparing starch into a 30% to 35% starch aqueous solution; adding 0.1 to 0.2% α-amylase to the starch aqueous solution, rapidly heating the solution to 110 to 120° C. under steam sterilization conditions, maintaining the reaction for 30 to 40 minutes, and rapidly cooling the solution to below 90° C. to obtain the starch enzymatic hydrolysis solution.

[0012] Optionally, the starch used in the method for preparing the starch enzymatic hydrolysis solution is one of corn starch, wheat starch and potato starch.

[0013] Preferably, the starch used in the method for preparing the starch enzymatic hydrolysis solution is corn starch.

[0014] Optionally, when adding the starch enzymatic solution, a step-by-step decreasing addition method is adopted, including: in the first stage, 43 to 65 g of starch enzymatic solution is added per hour; in the second stage, 23 to 50 g of starch enzymatic solution is added per hour; in the third stage, the addition of starch enzymatic solution is stopped.

[0015] Optionally, the first stage is from the time when the starch enzymatic solution is added to 80 hours of fermentation culture; the third stage is 0 to 4 hours before the end of fermentation; the second stage is the fermentation culture time period between the first stage and the third stage; the time when the starch enzymatic solution is added is 14 to 18 hours of fermentation culture.

[0016] Optionally, during the fermentation culture process of step 4, the inoculation amount is 10% to 20%; the fermentation culture temperature is 25°C to 27°C, the stirring speed is 300 rpm to 700 rpm, the ventilation volume is 0.6vvm to 1.0vvm, the tank pressure is 0.05MPa to 0.08MPa, and the dissolved oxygen value DO is ensured to be not less than 20% during the fermentation culture process; and the pH of the fermentation liquid is controlled at 6.7 to 7.10 during the fermentation culture process.

[0017] Optionally, the method further comprises: step 3, inoculating the shaking flask seed liquid of the streptomyces clavuligerus into the seed tank culture medium and culturing for 30 to 40 hours to obtain the seed bacterial liquid.

[0018] Furthermore, the seed tank culture medium uses deionized water as a medium and contains: 9-22 g / L soybean cake powder, 8-12 g / L starch, 8-12 g / L yeast powder, 1.3-1.7 g / L NaH2PO4, and 1-3 g / L defoaming agent; and the pH of the seed tank culture medium is adjusted to 6.90-7.10.

[0019] Furthermore, in step 3, the inoculation amount is 0.3% to 1.5% (V / V)%, the temperature is 27°C to 29°C, the stirring speed is 200rpm to 500rpm, the ventilation volume is 0.4vvm to 0.8vvm, the tank pressure is 0.05Mpa to 0.06Mpa, and the dissolved oxygen value DO is ensured to be not less than 30% during the culture process.

[0020] Optionally, in the fermentation medium and the seed tank medium, the starch is one of corn starch, wheat starch and potato starch; and the vegetable oil is soybean oil or corn oil.

[0021] Optionally, the method further comprises: step 1, inoculating the rod-shaped Streptomyces into a slant solid culture medium and culturing for 12 to 14 days to prepare a spore suspension. Further, the slant seed culture medium uses deionized water as a medium and contains: 8 to 12 g / L malt extract, 6 to 10 g / L yeast extract, 3 to 7 g / L glucose, and 23 to 27 g / L agar.

[0022] Optionally, the method further comprises: step 2, inoculating the spore suspension into a shake flask seed culture medium and culturing for 64 to 72 hours to obtain a shake flask seed solution. Further, the shake flask seed culture medium uses deionized water as a medium and contains: 8 to 12 g / L bean cake powder, 3 to 7 g / L yeast extract, and 13 to 17 g / L glycerol.

[0023] Beneficial effects: According to one embodiment of the present invention, starch and oil are used as the main carbon sources during the fermentation culture process, the starch enzymatic solution obtained by liquefying starch by α-amylase is used as feed, and the feeding control strategy of the fermentation process is further optimized, the fermentation process is stabilized, the fermentation titer is improved, and the clavulanic acid fermentation titer can reach more than 5000 μg / mL; the quality of clavulanic acid is improved, and the content of impurity G is reduced to less than 50 μg / mL; the cost of raw materials is greatly reduced, and the production cost is reduced. The method for preparing clavulanic acid by biological fermentation of the present invention has broad application prospects. DETAILED DESCRIPTION

[0024] The research process and inventive concept of the present invention are described below: The inventors of the present application noticed that clavulanic acid is a secondary metabolite of Streptomyces clavuli, and can only be continuously biosynthesized under strict substrate restriction regulation. However, since Streptomyces clavuli itself lacks receptors for monosaccharides to enter the cell, Streptomyces clavuli cannot efficiently utilize monosaccharides, showing slow bacterial cell growth and almost no product clavulanic acid concentration. Based on this, and in order to reduce the production cost of clavulanic acid, the inventors of the present application studied starch such as corn starch, wheat starch, and potato starch, and found that the starch enzymatic solution component formed by starch hydrolysis by α-amylase is a polysaccharide, and then the inventors of the present application thought of the inventive concept of using starch enzymatic solution to replace glycerol for the preparation of clavulanic acid.

[0025] The method for preparing clavulanic acid by biological fermentation of the present invention comprises the following steps: inoculating a seed culture of clavulanic streptomyces into a fermentation medium for fermentation and liquefying starch such as corn starch, wheat starch, potato starch, etc. by alpha-amylase to obtain a starch hydrolyzed solution (after sterilization) as feed for clavulanic acid fermentation production; further optimizing the feed regulation strategy during the fermentation process, stabilizing the fermentation process, improving the fermentation titer, reducing the production cost, and improving the quality of clavulanic acid.

[0026] In addition, the process of fermentation production of clavulanic acid is a very complicated process. The inventors of the present application are committed to the process research of fermentation production of clavulanic acid. In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail in combination with a specific embodiment. The embodiment includes the preparation of slant strains, the preparation of shake flask seed liquid, the preparation of seed liquid and fermentation culture, etc. The process of fermentation production of clavulanic acid is further studied, and the composition and culture process of the culture medium in each step and the interaction between them are studied.

[0027] In the present invention, the strain of the clavulose Streptomyces used is not limited, as long as it can produce clavulanic acid by fermentation. For example, the clavulose Streptomyces can be derived from the American Type Culture Collection (ATCC), with the accession number: ATCC27064, obtained by mutagenesis and breeding by our company, with the strain number KA-3.

[0028] According to an optional embodiment of the present invention, a method for preparing clavulanic acid by biological fermentation method is provided, which specifically comprises the following steps:

[0029] Step 1, preparation of slant bacterial strain: take a frozen glycerol tube of clavulose Streptomyces, inoculate the frozen glycerol solution of clavulose Streptomyces into a test tube slant culture medium for activation culture to prepare a spore suspension.

[0030] The inoculation volume can be 0.1 mL to 0.2 mL. The activation culture temperature is 25°C to 27°C, preferably 26°C, and the relative humidity is 40% to 60%. The activation culture is carried out for 12 to 14 days, and the surface of the culture medium is covered with bacterial fetuses and produces gray-green spores. Take the cultured slant strain, wash off the bacterial moss by adding an appropriate amount of sterile water, and prepare a spore suspension.

[0031] The slant seed culture medium uses deionized water as a medium and contains: 8-12 g / L malt extract, 6-10 g / L yeast extract, 3-7 g / L glucose, and 23-27 g / L agar. After the culture medium is prepared, a 20% sodium hydroxide solution is used to adjust the pH to 7.00-7.40, for example, to 7.20. After the agar is heated to melt, it is placed in a 20 mm*200 mm test tube, sterilized, and placed at an angle of 25 degrees to solidify to form a slant culture medium for use.

[0032] Step 2, preparation of shake flask seeds: inoculate the spore suspension prepared in step 1 into a shake flask seed culture medium, place the culture medium in a rotary reciprocating shaker, and obtain a shake flask seed solution.

[0033] The inoculation volume can be 0.5 ml to 1 ml. The culture temperature is 27 to 29° C., preferably 28° C., the shaking speed is 270 to 290 rpm, preferably 280 rpm, and the culture is carried out for 64 to 72 hours.

[0034] The shake flask seed culture medium uses deionized water as a medium and contains: 8-12 g / L bean cake powder, 3-7 g / L yeast extract, and 13-17 g / L glycerol. After the culture medium is prepared, a 20% sodium hydroxide solution is used to adjust the pH to 6.80-7.10.

[0035] Step 3, preparation of seed solution: inoculate the shake flask seed solution cultured in step 2 into the seed tank culture medium to obtain seed bacterial solution.

[0036] The inoculation amount is 0.3% to 1.5% (V / V), for example, 1.0% (V / V). The specific operation is, for example, to inoculate the shake flask seed solution cultured in step 2 into the culture medium of the seed tank.

[0037] The culture temperature is 27°C to 29°C, preferably 28°C, the stirring speed is 200 to 500 rpm, the ventilation volume is 0.4 vvm to 0.8 vvm (air flow / culture medium volume / minute), and the tank pressure is 0.05 to 0.06 MPa, and the culture is carried out for 30 to 40 hours.

[0038] The culture medium of the seed tank uses deionized water as the medium, and contains: 18-22 g / L soybean meal, 8-12 g / L corn starch, 8-12 g / L yeast powder, 1.3-1.7 g / L NaH2PO4, and 1-3 g / L defoamer. After the culture medium is prepared, a 20% sodium hydroxide solution is used to adjust the pH to 6.90-7.10.

[0039] Step 4, fermentation tank culture: inoculate the seed bacterial liquid cultured in the seed tank in step 3 into the fermentation medium for fermentation culture, add starch enzymatic solution, collect the fermentation product, and obtain clavulanic acid, wherein the inoculation ratio is 10-20% (V / V).

[0040] During the fermentation culture process, the fermentation culture temperature is 25°C to 27°C, preferably 26°C, the stirring speed is 300 to 700 rpm, the ventilation volume is 0.6 to 1.0 vvm (air flow / culture medium volume / minute), and the tank pressure is 0.05 to 0.08 MPa.

[0041] The fermentation medium, with deionized water as the medium, contains: 30-44 g / L soybean meal, 5-20 g / L corn starch, 12-17 g / L corn oil, 2.6-2.9 g / L NaH2PO4, 0.04-0.06 g / L FeCl3·6H2O, 0.7-0.9 g / L MgCl2·6H2O, 0.010-0.014 g / L ZnCl2, 0.004-0.006 g / L MnSO4, and 0.5-1.5 g / L defoamer. Furthermore, the defoamer in the culture medium of the present invention is preferably polypropylene glycol-PPG2000. In addition, during the fermentation culture process, the pH of the fermentation liquid is controlled at 6.90-7.10 by adding ammonia water with a concentration of 20%.

[0042] In the fermentation medium of the present application, starch and oil are used as carbon sources, and the ratio of the two is controlled so that the amount of starch added is close to 1:1. Since starch and oil have different metabolic pathways, the metabolites and the functions they play are different; oil can provide energy for bacterial growth through β-oxidation, and the product generates acetyl coenzyme A, which can be used for TCA cycle, amino acid metabolism, etc.; in addition, the addition of vegetable oils such as soybean oil or corn oil in the present application also plays a defoaming role; based on the influence of factors such as the growth needs of bacterial strains, the inventors of the present application have found that the carbon source in the current clavulanic acid fermentation medium cannot be all starch. Further research has found that by using the above-mentioned content of starch and oil as carbon sources and adding starch enzymatic hydrolysis solution during the fermentation culture process, the carbon source cost is reduced, the growth needs of bacterial strains are met, and the fermentation process is stabilized, and the fermentation titer is improved.

[0043] In the present invention, during the fermentation and culture process, what is added is a starch enzymatic solution, which is an incompletely hydrolyzed starch solution obtained by liquefying starch by α-amylase. Starch is a long-chain carbon source, which is not easily utilized by clavulanic acid bacteria. The clavulanic acid bacteria can only utilize the starch in the culture medium when it grows to a certain stage and can produce starch hydrolyzing enzymes, providing energy for strain production, and the utilization rate is low, which affects strain growth and production. The present invention hydrolyzes starch by α-amylase, cuts the starch chain into polysaccharide carbon sources (mixed carbon sources) such as dextrin, oligosaccharides and a small amount of maltose and glucose, which is more conducive to the utilization of clavulanic acid bacteria; secondly, the polysaccharide carbon source can reduce the inhibitory effect of carbon source concentration on the synthesis of clavulanic acid through a sustained release effect; moreover, the starch unit price is cheap, the fermentation process detection method is simple and easy to learn, and the final production titer is not affected. The inventors of the present application further realized that glycerol is a short-chain carbon source and is easily utilized by the strain. However, if the amount of glycerol added is insufficient, it will affect the synthesis of clavulanic acid by the strain; if the glycerol content exceeds 10g / L, it will inhibit the synthesis of clavulanic acid, and the use of glycerol will accelerate the aging of the strain. Compared with glycerol, the α-amylase hydrolysis in the present invention obtains a mixed carbon source. After adding the starch hydrolysis solution, it has been verified experimentally that the amount used is small, the strain ages slowly, and it will not affect the synthesis of clavulanic acid by the strain.

[0044] Furthermore, by adding starch enzymatic solution, the total sugar concentration of the fermentation liquid is maintained in the range of 6g / L to 12g / L. By adding starch enzymatic solution, the total sugar concentration of the fermentation liquid is controlled within the above range, so that the titer synthesis is high and the impurities are low; if it is lower than this range, the titer synthesis may be affected; and if the concentration is higher than 12g / L, the impurities will increase.

[0045] In addition, the preparation method of the starch enzymatic solution comprises the following steps: using starch to prepare a 30% to 35% starch aqueous solution, adding 0.1 to 0.2% α-amylase (Novozymes Fungamyl800L), using steam to quickly heat up to 115°C, maintaining the reaction for 30 to 40 minutes, and the rapid heating is to increase the enzyme reaction temperature in a short time, shorten the non-reaction time, and improve efficiency; then quickly cool down to below 90°C for standby use, and quickly cool down to reduce the enzyme activity as soon as possible after the end of the enzyme reaction to prevent the enzymatic hydrolysis from completely generating glucose. Preferably, the corn starch enzymatic solution used for the feed is prepared by enzymatic hydrolysis and liquefaction of corn starch by α-amylase using the above-mentioned preparation method. Corn starch is cheap and easy to obtain, and α-amylase has a high degree of specificity and hydrolyzes 1-4 glycosidic bonds. After enzymatic hydrolysis, amylose with different degrees of polymerization is formed, which is an incomplete hydrolysis product of starch, such as maltodextrin. Clavulanic acid bacteria have low utilization rate of long-chain carbon sources, and cannot utilize glucose due to lack of carriers for transporting glucose in cells. However, oligosaccharides obtained after enzymatic hydrolysis of corn starch by α-amylase contain carbon chains of different lengths, which can meet the growth and production requirements of bacteria and avoid the drawback that clavulanic acid bacteria cannot utilize glucose. In addition, considering the price and the availability of products, corn starch is preferably used as the raw material in the present invention, and other starches can be used in shake flask experiments and utilized by clavulanic acid bacteria.

[0046] Furthermore, when adding starch enzymatic solution, a stage-by-stage decreasing addition method is adopted. Specifically, it includes: in the first stage, 43-65g of starch enzymatic solution is added per hour, for example, 45g, 50g, 55g, 60g, 65g, etc.; in the second stage, 23-50g of starch enzymatic solution is added per hour, for example, 25g, 30g, 35g, 40g, etc., and for example, when the addition in the first stage is not less than 45g, 45g, 45g, 50g, etc. can be added in the second stage; in the third stage, the addition of starch enzymatic solution is stopped. As the metabolic capacity of the strain weakens, the carbon source addition is gradually reduced (i.e., the stage-by-stage decreasing addition method). On the one hand, the total sugar content of the fermentation broth can be controlled more efficiently to avoid the increase of total sugar and the impact on strain metabolism or the increase of impurities; on the other hand, unnecessary waste is also avoided.

[0047] Furthermore, the first stage is from the time when the starch enzymolysis solution is added to the fermentation culture for 80 hours, wherein the time when the starch enzymolysis solution is added is 14 to 18 hours of fermentation culture. The third stage is 0 to 4 hours before the end of fermentation, for example, the feeding is stopped 1 hour before the end of fermentation, the feeding is stopped 2 hours before the end of fermentation, and the feeding is stopped 4 hours before the end of fermentation. The second stage is the fermentation culture period between the first stage and the third stage. The total fermentation culture time is 120 to 140 hours. By clearly adding enzymolysis solution for 14 to 18 hours in the early stage of fermentation, the primary growth of the strain is promoted and the conversion of the strain from primary metabolism to secondary metabolism is accelerated; in the second stage, the strain growth enters the late stage of the steady period, the metabolic capacity is weakened, and the feeding is appropriately reduced to avoid the accumulation of total sugar; the third stage is the end of fermentation, the strain metabolism is weak, and the feeding is stopped to consume the residual carbon source in the fermentation broth.

[0048] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments. In the following embodiments:

[0049] Determination of mycelium concentration: Take 10 mL of fermentation broth in a centrifuge tube, centrifuge at 3500 r / min for 15 min, and calculate the ratio of solid volume to the total volume of fermentation broth.

[0050] Clavulanic acid potency determination: HPLC method was used, chromatographic column: ODS C18 4.6×100mm, 3.5μm; mobile phase: 3.40g tetrabutylammonium hydrogen sulfate was dissolved in 1000mL water, 0.5mol / L sodium hydroxide solution was used to adjust the pH value to 5.95-6.05, 130ml acetonitrile was added, and the solution was filtered with a 0.45μm microporous membrane, and the filtrate obtained was the mobile phase; diluent: 3.12g sodium dihydrogen phosphate was weighed, dissolved in 1000mL water, 0.5mol / L sodium hydroxide was used to adjust the pH value to 5.40, 60mL acetonitrile was added, and the solution was filtered with a 0.45μm microporous membrane, and the filtrate obtained was the diluent; flow rate: 1.0mL / min, UV detection wavelength: 210nm, column temperature: 33℃, injection volume 20μL.

[0051] Example 1

[0052] The composition of the fermentation tank culture medium is: water as the medium, soybean cake powder 34g / L, corn starch 20g / L, corn oil 16g / L, NaH2PO42.8g / L, FeCl3·6H2O 0.05g / L, MgCl2·6H2O 0.8g / L, ZnCl20.012g / L, MnSO40.005 g / L, PPG20001g / L.

[0053] Preparation of starch enzymatic solution: 30% starch aqueous solution was prepared with corn starch, 0.1% α-amylase (Novozymes Fungamyl 800L) was added, the temperature was quickly raised to 115° C. using steam, the reaction was maintained for 35 minutes, and the temperature was quickly lowered to below 90° C. for standby use.

[0054] Step 1: Preparation of slant cultures

[0055] Preparation of slant culture medium: Use deionized water as medium to prepare slant culture medium, the proportion is: malt extract 10g / L, yeast extract 8g / L, glucose 5g / L, agar 25g / L, use 20% sodium hydroxide solution to adjust pH to 7.20. Prepare 200ml of culture medium, heat and melt, stir evenly, and put into 20 20mm*200mm test tubes, sterilize, tilt 25°, and wait for solidification to make slant culture medium for use.

[0056] Take a glycerol cryovial of rod-shaped Streptomyces, wait for it to be thawed, take 0.2 ml and evenly spread it on the surface of the slope, place it in a constant temperature and humidity incubator, culture it for 14 days under the conditions of temperature 26°C and relative humidity 40-60%, the surface of the culture medium is covered with fungi and gray-green spores are produced, add 5 ml of sterile water to make a spore suspension.

[0057] Step 2: Preparation of shake flask seeds

[0058] Preparation of shake flask seed culture medium: use deionized water as the medium, 10 g / L soybean cake powder, 5 g / L yeast extract, 15 g / L glycerol, use 20% sodium hydroxide solution to adjust the pH to 7.00, sterilize and set aside.

[0059] Take 0.6 ml of the spore suspension in step 1 and inoculate it into the seed culture medium of the shake flask, and place it in an automatic constant temperature shaker for cultivation at a temperature of 28° C. and a rotation speed of 29 rpm for 64 hours.

[0060] Step 3: Cultivation of seed tanks

[0061] Preparation of seed tank culture medium: deionized water as the medium, soybean cake powder 20g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 1.0g / L, pH 7.00, and the volume after digestion is 6 liters.

[0062] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 30 hours at a temperature of 28°C, a stirring speed of 100 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0063] Step 4: Cultivation of secondary seeds

[0064] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 12g / L, corn starch 12g / L, FeCl3·6H2O 0.008g / L, ZnCl20.004g / L, MnSO40.004 g / L, PPG20001.1g / L, and pH is 7.00.

[0065] The first-level seed liquid cultured in the above step 3 was inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 17 hours at a temperature of 28°C, a stirring speed of 180 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0066] Step 5: Fermentation

[0067] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13% (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 500 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0068] Addition of starch enzymatic solution: When the fermentation reaches 16 hours, start adding the prepared starch enzymatic solution. From 16 hours to 80 hours, add 45 g per hour. From 80 hours to 120 hours, add 25 g per hour.

[0069] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 120 hours.

[0070] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0071] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 1 below.

[0072] Table 1 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 1.

[0073]

[0074] Example 2

[0075] Fermentation medium: deionized water as the medium, soybean cake powder 30g / L, corn starch 18g / L, corn oil 14g / L, NaH2PO42.6g / L, FeCl3·6H2O 0.05g / L, MgCl2·6H2O 0.8g / L, ZnCl20.012g / L, MnSO40.005 g / L, PPG20001.2g / L.

[0076] Preparation of starch enzymatic solution: prepare 32% starch solution, add 0.13% α-amylase, react at 115°C for 33 minutes, quickly cool to below 90°C, and set aside.

[0077] Step 1: Preparation of slant cultures

[0078] The composition of the slant seed culture medium is: deionized water as the medium, 12 g / L malt extract, 9 g / L yeast extract, 4 g / L glucose, 24 g / L agar, and pH is 7.00.

[0079] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.15 ml, and culture for 13 days at a temperature of 28° C. and a relative humidity of 40-60%.

[0080] Step 2: Preparation of shake flask seeds

[0081] The composition of the shake flask seed culture medium is: deionized water as the medium, 9 g / L soybean cake powder, 6 g / L yeast extract, and 14 g / L glycerol; the pH is 7.00.

[0082] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.7 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 52 hours.

[0083] Step 3: Cultivation of primary seeds

[0084] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 11g / L, corn starch 9g / L, yeast powder 10g / L, NaH2PO4 1.6g / L, PPG2000 2.3g / L, and pH is 6.95.

[0085] The shake flask seed liquid cultured in step 2 above was inoculated into the culture medium of the primary seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 28 hours at a temperature of 28°C, a stirring speed of 90 rpm, a ventilation volume of 0.4VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0086] Step 4: Cultivation of secondary seeds

[0087] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 12g / L, corn starch 9g / L, FeCl3·6H2O 0.008g / L, ZnCl20.004g / L, MnSO40.004 g / L, PPG20001.1 g / L, and pH is 7.00.

[0088] The first-level seed liquid cultured in the above step 3 was inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 17 hours at a temperature of 28°C, a stirring speed of 180 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0089] Step 5: Fermentation

[0090] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13% (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 26°C, a stirring speed of 450 rpm, a ventilation volume of 0.7VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0091] Addition of starch enzymatic solution: When the fermentation reaches 15 hours, start adding the prepared starch enzymatic solution. From 15 hours to 80 hours, add 43 g per hour. From 80 hours to 120 hours, add 23 g per hour.

[0092] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 123 hours.

[0093] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0094] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 2 below.

[0095] Table 2 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 2.

[0096]

[0097]

[0098] Example 3

[0099] Fermentation medium composition: (prepared according to the actual volume of 32 liters after digestion), deionized water as the medium, soybean cake powder 32 g / L, corn starch 19 g / L, corn oil 17 g / L, NaH2PO4 2.7 g / L, FeCl3·6H2O 0.06 g / L, MgCl2·6H2O 0.7 g / L, ZnCl2 0.013 g / L, MnSO4 0.006 g / L, PPG2000 1.1 g / L.

[0100] Preparation conditions of starch enzymatic solution: prepare 34% starch solution, add 0.16% α-amylase, react at 117°C for 30 minutes, quickly cool to below 90°C, and set aside.

[0101] Fermentation for the production of clavulanic acid:

[0102] Step 1: Preparation of slant cultures

[0103] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 7g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0104] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.18 ml, and culture for 12 days at a temperature of 27° C. and a relative humidity of 50-60%.

[0105] Step 2: Preparation of shake flask seeds

[0106] The composition of the shake flask seed culture medium is: deionized water as the medium, 10 g / L soybean cake powder, 7 g / L yeast extract, and 13 g / L glycerol; the pH is 6.98.

[0107] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0108] Step 3: Cultivation of primary seeds

[0109] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0110] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0111] Step 4: Cultivation of secondary seeds

[0112] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0113] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0114] Step 5: Fermentation

[0115] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium according to the inoculation ratio of 13 (v / v) mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0116] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0117] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0118] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0119] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 3 below.

[0120] Table 3 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 3.

[0121]

[0122]

[0123] Example 4

[0124] Fermentation medium composition: deionized water as the medium, soybean cake powder 32g / L, corn starch 19g / L, corn oil 17g / L, NaH2PO4 2.7g / L, FeCl3·6H2O 0.06g / L, MgCl2·6H2O 0.7g / L, ZnCl2 0.013g / L, MnSO4 0.006g / L, PPG2000 1.1g / L.

[0125] Preparation conditions of starch enzymatic solution: prepare 35% starch solution, add 0.20% α-amylase, react at 120°C for 30 minutes, quickly cool to below 90°C, and set aside.

[0126] Fermentation for the production of clavulanic acid:

[0127] Step 1: Preparation of slant cultures

[0128] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 9g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0129] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.2 ml, and culture for 12 days at a temperature of 28° C. and a relative humidity of 50-60%.

[0130] Step 2: Preparation of shake flask seeds

[0131] The composition of the shake flask seed culture medium: deionized water as the medium, soybean cake powder 10g / L, yeast extract 7g / L, glycerol 13g / L; pH 7.00.

[0132] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0133] Step 3: Cultivation of primary seeds

[0134] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0135] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0136] Step 4: Cultivation of secondary seeds

[0137] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0138] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0139] Step 5: Fermentation

[0140] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium according to the inoculation ratio of 13 (v / v) mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0141] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0142] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0143] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0144] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 4 below.

[0145] Table 4 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 4.

[0146]

[0147] Example 5

[0148] Fermentation medium composition: deionized water as the medium, soybean cake powder 32g / L, corn starch 19g / L, corn oil 17g / L, NaH2PO4 2.7g / L, FeCl3·6H2O 0.06g / L, MgCl2·6H2O 0.7g / L, ZnCl2 0.013g / L, MnSO4 0.006g / L, PPG2000 1.1g / L.

[0149] Preparation conditions of starch enzymatic solution: prepare 35% starch solution, add 0.15% α-amylase, react at 120°C for 30 minutes, quickly cool to below 90°C, and set aside.

[0150] Fermentation for the production of clavulanic acid:

[0151] Step 1: Preparation of slant cultures

[0152] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 9g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0153] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.2 ml, and culture for 12 days at a temperature of 28° C. and a relative humidity of 50-60%.

[0154] Step 2: Preparation of shake flask seeds

[0155] The composition of the shake flask seed culture medium: deionized water as the medium, soybean cake powder 10g / L, yeast extract 7g / L, glycerol 13g / L; pH 7.00.

[0156] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0157] Step 3: Cultivation of primary seeds

[0158] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0159] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0160] Step 4: Cultivation of secondary seeds

[0161] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0162] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0163] Step 5: Fermentation

[0164] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13 (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0165] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0166] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0167] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0168] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 5 below.

[0169] Table 5 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 5.

[0170]

[0171] Example 6

[0172] Fermentation medium composition: deionized water as the medium, soybean cake powder 32g / L, corn starch 19g / L, corn oil 17g / L, NaH2PO4 2.7g / L, FeCl3·6H2O 0.06g / L, MgCl2·6H2O 0.7g / L, ZnCl2 0.013g / L, MnSO4 0.006g / L, PPG2000 1.1g / L.

[0173] Preparation conditions of starch enzymatic solution: prepare 35% starch solution, add 0.10% α-amylase, react at 110°C for 40 minutes, quickly cool to below 90°C, and set aside.

[0174] Fermentation for the production of clavulanic acid:

[0175] Step 1: Preparation of slant cultures

[0176] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 9g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0177] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.2 ml, and culture for 12 days at a temperature of 28° C. and a relative humidity of 50-60%.

[0178] Step 2: Preparation of shake flask seeds

[0179] The composition of the shake flask seed culture medium: deionized water as the medium, soybean cake powder 10g / L, yeast extract 7g / L, glycerol 13g / L; pH 7.00.

[0180] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0181] Step 3: Cultivation of primary seeds

[0182] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0183] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0184] Step 4: Cultivation of secondary seeds

[0185] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0186] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0187] Step 5: Fermentation

[0188] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13 (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0189] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0190] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0191] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0192] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 6 below.

[0193] Table 6 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 6.

[0194]

[0195] Example 7

[0196] Fermentation medium composition: deionized water as the medium, soybean cake powder 32g / L, corn starch 19g / L, corn oil 17g / L, NaH2PO4 2.7g / L, FeCl3·6H2O 0.06g / L, MgCl2·6H2O 0.7g / L, ZnCl2 0.013g / L, MnSO4 0.006g / L, PPG2000 1.1g / L.

[0197] Preparation conditions of starch enzymatic solution: prepare 35% starch solution, add 0.15% α-amylase, react at 110°C for 30 minutes, quickly cool to below 90°C, and set aside.

[0198] Fermentation for the production of clavulanic acid:

[0199] Step 1: Preparation of slant cultures

[0200] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 9g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0201] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.2 ml, and culture for 12 days at a temperature of 28° C. and a relative humidity of 50-60%.

[0202] Step 2: Preparation of shake flask seeds

[0203] The composition of the shake flask seed culture medium: deionized water as the medium, soybean cake powder 10g / L, yeast extract 7g / L, glycerol 13g / L; pH 7.00.

[0204] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0205] Step 3: Cultivation of primary seeds

[0206] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0207] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0208] Step 4: Cultivation of secondary seeds

[0209] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0210] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0211] Step 5: Fermentation

[0212] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13 (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0213] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0214] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0215] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0216] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 7 below.

[0217] Table 7 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 7.

[0218]

[0219] Example 8

[0220] Fermentation medium composition: deionized water as the medium, soybean cake powder 32g / L, corn starch 19g / L, corn oil 17g / L, NaH2PO4 2.7g / L, FeCl3·6H2O 0.06g / L, MgCl2·6H2O 0.7g / L, ZnCl2 0.013g / L, MnSO4 0.006g / L, PPG2000 1.1g / L.

[0221] Preparation conditions of starch enzymatic solution: prepare 30% starch solution, add 0.20% α-amylase, react at 110°C for 40 minutes, quickly cool to below 90°C, and set aside.

[0222] Fermentation for the production of clavulanic acid:

[0223] Step 1: Preparation of slant cultures

[0224] The composition of the slant seed culture medium is: deionized water as the medium, malt extract 11g / L, yeast extract 9g / L, glucose 7g / L, agar 23g / L, and pH is 7.05.

[0225] Take the frozen and preserved rod-shaped Streptomyces strains for slant culture activation, inoculate each slant with: 0.2 ml, and culture for 12 days at a temperature of 28° C. and a relative humidity of 50-60%.

[0226] Step 2: Preparation of shake flask seeds

[0227] The composition of the shake flask seed culture medium: deionized water as the medium, soybean cake powder 10g / L, yeast extract 7g / L, glycerol 13g / L; pH 7.00.

[0228] Take the slant cultured in step 1, wash the bacterial moss with sterile water to obtain a bacterial suspension, inoculate 0.6 ml of the suspension into the shake flask seed culture medium, and culture at a temperature of 28°C and a shaker speed of 250 rpm for 53 hours.

[0229] Step 3: Cultivation of primary seeds

[0230] The composition of the first-level seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, yeast powder 10g / L, NaH2PO4 1.5g / L, PPG2000 2.2g / L, and pH 7.00.

[0231] The shake flask seed liquid cultured in the above step 2 was inoculated into the culture medium of the first-level seed tank at an inoculation ratio of 0.3% (v / v) (mycelium concentration: 18%-20%), and cultured for 29 hours at a temperature of 29°C, a stirring speed of 95 rpm, a ventilation volume of 0.5VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0232] Step 4: Cultivation of secondary seeds

[0233] The composition of the secondary seed tank culture medium is: deionized water as the medium, soybean cake powder 9g / L, corn starch 10g / L, FeCl3·6H2O 0.007g / L, ZnCl20.003g / L, MnSO40.005g / L, PPG20001.2 g / L, and pH is 7.05.

[0234] The first-level seed liquid cultured in the above step 3 is inoculated into the culture medium of the second-level seed tank at an inoculation ratio of 10% (v / v) (mycelium concentration: 16%-20%), and cultured for 16 hours at a temperature of 29°C, a stirring speed of 170 rpm, a ventilation volume of 0.6VVM (volume / volume / minute), and a tank pressure of 0.05MPa.

[0235] Step 5: Fermentation

[0236] The secondary seed liquid cultured in the seed tank in step 4 is inoculated into the fermentation tank culture medium at an inoculation ratio of 13 (v / v) (mycelium concentration: 16%-20%), and fermentation culture is carried out at a temperature of 25°C, a stirring speed of 550 rpm, a ventilation volume of 0.9VVM (volume / volume / minute), a tank pressure of 0.05MPa, and a pH of 7.00.

[0237] Addition of starch enzymatic solution: After 14 hours of fermentation, start adding the prepared starch hydrolyzate. From 14 hours to 80 hours, add 48 g per hour. From 80 hours to 120 hours, add 27 g per hour.

[0238] During the fermentation process, 20% ammonia water was used to control the pH of the fermentation liquid to 7.00. The fermentation was cultured for 124 hours.

[0239] After the fermentation is completed, the obtained fermentation liquid is centrifuged at 4000 rpm for 20 min, and the obtained supernatant is the clavulanic acid solution.

[0240] At the same time, according to the above method, mycelial concentration, clavulanic acid titer and clavulanic acid impurity G were detected. The results are shown in Table 8 below.

[0241] Table 8 Mycelium concentration, clavulanic acid titer and clavulanic acid impurity G detection results of Example 8.

[0242]

[0243] According to the culture medium and culture method provided by the present invention, the fermentation titer can reach 5000-6000 μg / mL, the impurity G content is less than 50 μg / mL (as shown in Examples 4 to 8, it can be less than 40 μg / mL), and the impurity content is reduced by about 30% compared with the fermentation method using glycerol as feed. The use of starch hydrolyzate for clavulanic acid fermentation feed provided by the present invention not only does not affect the fermentation titer of clavulanic acid, but also greatly reduces the feed cost.

[0244] In summary, the clavulanic acid fermentation medium and the corresponding culture method provided by the present invention have broad application prospects.

[0245] The description of the present invention is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the invention to the disclosed form. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for particular uses.

Claims

1. A method for preparing clavulanic acid by biological fermentation, characterized in that: include: Step 4, inoculating the seed culture liquid of Streptomyces clavuligerus into a fermentation medium for fermentation culture, and collecting the fermentation product to obtain clavulanic acid; During the fermentation process, starch enzymatic hydrolysis solution is added to maintain the total sugar concentration of the fermentation liquid in the range of 6 g / L to 12 g / L; the starch enzymatic hydrolysis solution is obtained by liquefying starch with α-amylase; The fermentation medium uses deionized water as a medium and contains: 30-44 g / L soybean meal, 5-20 g / L starch, 12-17 g / L vegetable oil, 2.6-2.9 g / L NaH2PO4, 0.04-0.06 g / L FeCl3·6H2O, 0.7-0.9 g / L MgCl2·6H2O, 0.010-0.014 g / L ZnCl2, 0.004-0.006 g / L MnSO4, and 0.5-1.5 g / L defoaming agent.

2. The method for preparing clavulanic acid by biological fermentation according to claim 1, characterized in that: The preparation method of the starch enzymatic hydrolysis solution comprises: preparing starch into a 30% to 35% starch aqueous solution; adding 0.1 to 0.2% α-amylase to the starch aqueous solution, rapidly heating the solution to 110 to 120° C. under steam sterilization conditions, maintaining the reaction for 30 to 40 minutes, and rapidly cooling the solution to below 90° C. to obtain the starch enzymatic hydrolysis solution.

3. The method for preparing clavulanic acid by biological fermentation according to claim 2, characterized in that: The starch used in the method for preparing the starch enzymatic hydrolysis solution is one of corn starch, wheat starch and potato starch.

4. The method for preparing clavulanic acid by biological fermentation according to claim 2, characterized in that: The starch used in the method for preparing the starch enzymatic hydrolysis solution is corn starch.

5. The method for preparing clavulanic acid by biological fermentation according to any one of claims 1 to 4, characterized in that: When adding starch enzymatic solution, a stage-by-stage decreasing addition method is adopted, including: in the first stage, 43-65g of starch enzymatic solution is added per hour; in the second stage, 23-50g of starch enzymatic solution is added per hour; in the third stage, the addition of starch enzymatic solution is stopped.

6. The method for preparing clavulanic acid by biological fermentation according to claim 5, characterized in that: The first stage is from the time when the starch enzymatic solution is added to 80 hours of fermentation culture; the third stage is 0 to 4 hours before the end of fermentation; the second stage is the fermentation culture time period between the first stage and the third stage; the time when the starch enzymatic solution is added is 14 to 18 hours of fermentation culture.

7. The method for preparing clavulanic acid by biological fermentation according to claim 1, characterized in that: Step 4, during the fermentation culture process, the inoculation amount is 10% to 20%; The fermentation culture temperature is 25°C to 27°C, the stirring speed is 300 rpm to 700 rpm, the ventilation volume is 0.6vvm to 1.0vvm, the tank pressure is 0.05MPa to 0.08MPa, and the dissolved oxygen value DO is ensured to be not less than 20% during the fermentation culture process; During the fermentation process, the pH of the fermentation liquid was controlled at 6.7-7.

10.

8. The method for preparing clavulanic acid by biological fermentation according to claim 1, characterized in that: The invention also includes: step 3, inoculating the shaking flask seed liquid of the streptomyces clavuligerus into the seed tank culture medium and culturing for 30 to 40 hours to obtain the seed bacterial liquid; The seed tank culture medium uses deionized water as a medium, and contains: 9-22 g / L soybean meal, 8-12 g / L starch, 8-12 g / L yeast powder, 1.3-1.7 g / L NaH2PO4, and 1-3 g / L defoaming agent; and the pH of the seed tank culture medium is adjusted to 6.90-7.10; The inoculation amount is 0.3% to 1.5% (V / V)%, the temperature is 27°C to 29°C, the stirring speed is 200rpm to 500rpm, the ventilation volume is 0.4vvm to 0.8vvm, the tank pressure is 0.05Mpa to 0.06Mpa, and the dissolved oxygen value DO is ensured to be not less than 30% during the culture process.

9. The method for preparing clavulanic acid by biological fermentation according to claim 8, characterized in that: In the fermentation medium and the seed tank medium, the starch is one of corn starch, wheat starch and potato starch; and the vegetable oil is soybean oil or corn oil.

10. The method for preparing clavulanic acid by biological fermentation according to claim 9, characterized in that: The method also includes: step 1, inoculating the rod-shaped Streptomyces into a slant solid culture medium and culturing for 12 to 14 days to prepare a spore suspension; wherein the slant seed culture medium uses deionized water as a medium and contains: 8 to 12 g / L malt extract, 6 to 10 g / L yeast extract, 3 to 7 g / L glucose, and 23 to 27 g / L agar; Step 2, inoculating the spore suspension into a shake flask seed culture medium and culturing for 64 to 72 hours to obtain a shake flask seed solution; wherein the shake flask seed culture medium uses deionized water as a medium and contains: 8 to 12 g / L bean cake powder, 3 to 7 g / L yeast extract, and 13 to 17 g / L glycerol.

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