Fermentation preparation method of clavulanic acid

Abstract

The invention discloses a clavulanic acid fermentation preparation method, and belongs to the technical field of biological medicines. Comprising the following steps: preparing a fermentation culture medium by adopting a composite nitrogen source instead of a single nitrogen source, inoculating a streptomyces clavuliformis seed solution to the fermentation culture medium for culturing, supplementing different material supplementing modes according to different fermentation stages in the fermentation process, and collecting a fermentation product to obtain clavulanic acid, a nitrogen source in the fermentation culture medium comprises 10-20g / L of soybean cake powder, 5-10g / L of peanut protein powder and 2-5g / L of peptone; the supplementary materials comprise supplementary materials A, B, C and D; the supplementary material A comprises glycerol, the supplementary material B comprises corn oil, the supplementary material C comprises proteolysis liquid, and the supplementary material D comprises threonine and glutamic acid. According to the method, the clavulanic acid fermentation cost can be reduced, the viscosity in the clavulanic acid fermentation process is reduced, the oxygen dissolving condition in the process is improved, the mass transfer efficiency of fermentation liquor is improved, thallus aging is delayed, the fermentation time is prolonged, the fermentation titer is improved, generation of waste such as fungus residues is reduced, the economic value is increased, and the environmental protection pressure is relieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fermentation preparation method for clavulanic acid. Background Technology

[0002] Clavulanic acid (CA), also known as clavulanic acid, is a naturally occurring β-lactamase inhibitor produced by *Streptomyces clavuligerus*. It irreversibly binds to β-lactamases, thus inhibiting bacterial degradation of the β-lactam ring. β-lactam antibiotics, such as cephalosporins and penicillins, are the most widely used antibiotics in clinical practice. However, the use of these antibiotics is significantly affected by the drug resistance of pathogens. The main reason for pathogen resistance is the production of β-lactamases, which inactivate β-lactam antibiotics by disrupting the β-lactam ring. Because clavulanic acid irreversibly binds to the serine active site of β-lactamases, it protects the antibacterial activity of penicillins and cephalosporins against resistant bacteria.

[0003] In the prior art, clavulanic acid fermentation is carried out using *Streptomyces roximate* as the starting strain. The inventors of this application recognize that the amount of nitrogen source added in the fermentation medium is an important factor affecting the viscosity of the clavulanic acid fermentation process. In the actual fermentation process, soybean meal powder is used as the only nitrogen source and glycerol is used as the only supplementary nutrient source. This results in problems such as high viscosity of the fermentation broth, difficulty in controlling dissolved oxygen, low initial titer, premature cell aging, small increase in titer in the later stage of fermentation, and low titer yield per unit cell. Summary of the Invention

[0004] According to one embodiment of the present invention, the objective is to provide a method for preparing clavulanic acid by fermentation. By reducing the amount of nitrogen source added to the fermentation medium, replacing a single nitrogen source with a composite nitrogen source and adjusting the proportion of nitrogen source substances, and by adding different feeds at each stage of clavulanic acid fermentation according to the growth and metabolic needs of the microorganisms, the relationship between cell growth and metabolism during fermentation is balanced. This reduces the viscosity of the clavulanic acid fermentation process, improves dissolved oxygen conditions during fermentation, increases the mass transfer efficiency of the fermentation broth, delays cell aging, extends the fermentation cycle, improves fermentation potency, significantly reduces production costs, and reduces the generation of waste such as mycelium, thereby increasing economic value and alleviating environmental pressure.

[0005] The above objective can be achieved through the following technical solutions: According to one aspect of the present invention, a method for preparing clavulanic acid by fermentation is provided, comprising: preparing a fermentation medium using a composite nitrogen source instead of a single nitrogen source; inoculating the seed culture of *Streptomyces rod-shapedis* into the fermentation medium for fermentation culture; and adding different feeds according to different fermentation stages during the fermentation process; and collecting the fermentation products to obtain clavulanic acid; wherein the composite nitrogen source in the fermentation medium comprises: 10-20 g / L soybean meal, 5-10 g / L peanut protein powder, and 2-5 g / L peptone; the feeds comprise feed A, feed B, feed C, and feed D; wherein feed A comprises glycerol; feed B comprises corn oil; feed C comprises protease hydrolysate; and feed D comprises threonine and glutamic acid.

[0006] Preferably, during the fermentation process, different feeds are added according to different fermentation stages, including: feed A is added when fermentation reaches the first time point; feed B is added when fermentation reaches the second time point; feed C is added when fermentation reaches the third time point; feed D is added when fermentation reaches the fourth time point; and feeds B, C, and D are stopped when fermentation reaches the fifth time point, while feed A is added until the end of fermentation.

[0007] Furthermore, the fermentation culture reaching the first time point refers to a point within 13–17 hours of fermentation culture. The fermentation culture reaching the second time point refers to a point within 48–52 hours of fermentation culture. The fermentation culture reaching the third time point refers to a point within 68–72 hours of fermentation culture. The fermentation culture reaching the fourth time point refers to a point within 78–82 hours of fermentation culture. The fermentation culture reaching the fifth time point refers to a point within 98–102 hours of fermentation culture.

[0008] Preferably, after 15 hours of fermentation, feed A is added; after 50 hours of fermentation, feed B is added; after 70 hours of fermentation, feed C is added; after 80 hours of fermentation, feed D is added; after 100 hours of fermentation, feed B, feed C, and feed D are stopped, while feed A is added until the end of fermentation.

[0009] Preferably, during the replenishment of each feed, samples are taken at preset time intervals to test the nutrient content in the fermentation broth, and the nutrient content in the fermentation broth is controlled based on the monitoring results. Further, the preset time is 6–10 hours. Further, the glycerol content in the fermentation broth is controlled to be 2.0–8.0 g / L; the corn oil clear layer content in the fermentation broth is controlled to be ≤2.0%; the amino nitrogen content in the fermentation broth is controlled to be ≤0.8 g / L; the threonine concentration in the fermentation broth is controlled to be ≤1.0 g / L; and the glutamic acid concentration in the fermentation broth is controlled to be ≤1.0 g / L.

[0010] Preferably, in the feed, the glycerol is an aqueous solution of glycerol with a concentration of 400-500 g / L; the corn oil is pure corn oil; the concentration of the protease hydrolysate is 250-350 g / L; and the threonine and glutamic acid are prepared aqueous solutions, wherein the content of threonine is 1.0-6.0 g / L and the content of glutamic acid is 1.0-6.0 g / L.

[0011] Preferably, the protease hydrolysate is prepared by adding alkaline protease to a protein aqueous solution of soybean protein. Further, the preparation includes: preparing a 25%–35% protein aqueous solution of soybean protein; adding 0.05%–0.15% alkaline protease to the protein aqueous solution, adjusting the pH to 9.5–10.5, rapidly heating to 55–65°C, maintaining the reaction for 25–35 minutes, rapidly heating to 105–115°C under steam sterilization conditions, and then cooling to 25°C to obtain the protease hydrolysate.

[0012] Preferably, the fermentation process also includes: controlling the dissolved oxygen content to ≥30% throughout the fermentation process by adjusting the stirring speed; wherein, during the 36h to 100h fermentation stage, the stirring speed is 520 to 560 rpm.

[0013] Preferably, the fermentation culture medium, using deionized water as the medium, further includes: starch: 5-20 g / L; corn oil: 12-17 g / L; NaH2PO4: 2.6-2.9 g / L; FeCl3·6H2O: 0.04-0.06 g / L; MgCl2·6H2O: 0.7-0.9 g / L; ZnCl2: 0.010-0.014 g / L; MnSO4: 0.004-0.006 g / L; and defoamer: 0.5-1.5 g / L.

[0014] Preferably, the seed culture of Streptomyces rotaforme is inoculated into the fermentation medium at an inoculation rate of 10-20% (V / V) for fermentation culture.

[0015] Preferably, during the fermentation process, the pH of the fermentation broth is controlled to be 6.40–6.80 by adding ammonia water, and the fermentation is terminated after 120–130 hours.

[0016] Preferably, the method further includes: inoculating a glycerol cryopreservation solution of Streptomyces lanceolata into a slant culture medium for activation culture and preparing a spore suspension; inoculating the spore suspension into a shake flask seed culture medium and culturing it on a self-controlled constant temperature shaker to obtain a shake flask seed solution; and inoculating the shake flask seed solution into a seed tank culture medium for culture to obtain the seed solution of Streptomyces lanceolata.

[0017] Preferably, the slant culture medium uses deionized water as the 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; the pH is 7.00-7.40.

[0018] Preferably, the shake flask seed culture medium, using deionized water as the medium, comprises: 8-12 g / L soybean meal, 3-7 g / L yeast extract, and 13-17 g / L glycerol; with a pH of 6.80-7.10.

[0019] Preferably, the seed tank culture medium uses deionized water as the medium and contains: 18-22 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 defoamer; the pH is 6.90-7.10.

[0020] Beneficial effects: According to one embodiment of the present invention, the provided method for preparing clavulanic acid by fermentation changes the way nitrogen source is added. Instead of adding the conventional substrate all at once, the method improves by adding only the basic nitrogen source sufficient for cell growth to the basal culture medium. This is combined with a staged and precise addition of nitrogen source during the fermentation process. By reducing the amount of nitrogen source added to the fermentation medium, using a compound nitrogen source instead of a single nitrogen source, and adjusting the proportion of nitrogen source substances, it is beneficial for rapid cell growth. While ensuring the rapid establishment of a high-density cell population, it can solve problems such as increased viscosity of the fermentation broth and difficulty in controlling dissolved oxygen caused by excessive addition of soybean meal during fermentation. When the cell population reaches a certain density, different feeds are added according to the growth and metabolic needs of the strain during fermentation, according to different fermentation stages. Strict control of the timing of each feed addition balances the relationship between cell growth and metabolism at each stage of fermentation, which helps create a state of nutritional stress, forcing the metabolic flow to shift from growth to product synthesis.

[0021] This invention employs a method of reducing the nitrogen source content in the culture medium and supplementing the nitrogen source in batches for the preparation of clavulanic acid. The limiting nitrogen source relieves the inhibition of secondary metabolism, while a continuous low-level carbon source supply provides energy and precursors for product synthesis, promoting the synthesis of secondary metabolites. It also maintains a suitable osmotic pressure, balances heat production and dissipation during fermentation, more stably controls dissolved oxygen (DO) and pH, and reduces the accumulation of harmful byproducts. Ultimately, this invention achieves the goals of improving the cell metabolic environment, reducing fermentation viscosity, improving dissolved oxygen levels, increasing the mass transfer efficiency of the fermentation broth, delaying cell aging, extending the product synthesis period, significantly increasing the total yield of clavulanic acid, reducing production costs, and minimizing mycelial waste.

[0022] Compared with the prior art, the embodiments of the present invention also have the following advantages: 1) Compared with the existing method of using excessive nitrogen source (soybean meal powder) as the nitrogen source for fermentation substrate, this invention reduces the amount of nitrogen source used in fermentation substrate (i.e. fermentation culture medium) by about 20% to 50% while ensuring normal cell growth in the early stage of fermentation. By reducing the nitrogen source in fermentation substrate culture medium, the viscosity of the fermentation process is reduced and the mass transfer efficiency is improved.

[0023] 2) Compared with existing single-nitrogen source formulations for culture media, this invention uses a composite nitrogen source, combining slow-release nitrogen sources with fast-acting nitrogen sources. Furthermore, soybean meal and peanut protein powder are used as slow-release nitrogen sources, and peptone is used as a fast-acting nitrogen source. This better meets the nitrogen source requirements of the cells in the early stage of growth, maintains rapid cell growth, and quickly establishes high-density cells. At the same time, it solves the problem of increased fermentation broth viscosity caused by adding large amounts of soybean meal during fermentation, thereby improving the cell growth rate. The compound ratio of the above nitrogen sources is further optimized, resulting in a significant increase in cell growth rate in the early stage of fermentation, earlier potency synthesis time, rapid start-up of clavulanic acid production, and a significant increase in unit cell potency.

[0024] 3) Based on reducing the nitrogen source in the substrate culture medium, and according to the nutritional requirements of the microorganisms at different growth stages, the feed is divided into four types of feed containing different nutrients: A, B, C, and D. During the fermentation process, the above feeds are added in a targeted manner according to different fermentation stages, which extends the production cycle of the microorganisms and improves the final potency level. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is actually illustrative only and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are all within the protection scope of the present invention.

[0026] As described above, the inventors of this application recognize that there are related technologies for the production of clavulanic acid by using Streptomyces rotaforme as the starting strain. However, excessive addition of soybean meal leads to problems such as high viscosity of the fermentation liquid, difficulty in controlling dissolved oxygen during the fermentation process, low initial fermentation titer, small increase in titer in the later fermentation, premature cell aging, and low efficiency of unit cell titer synthesis.

[0027] To address these identified problems, a fermentation preparation method for clavulanic acid is provided through further research and improvement. The method includes: preparing a fermentation medium using a composite nitrogen source instead of a single nitrogen source; inoculating the seed culture of *Streptomyces lanceolata* into the fermentation medium for fermentation; adding different feeds according to different fermentation stages during the fermentation process; and collecting the fermentation products to obtain clavulanic acid. The method further optimizes the nitrogen source ratio by reducing the amount of nitrogen source added to the fermentation medium and adjusting the nitrogen source in the substrate to include soybean meal, peanut protein powder, and peptone, with the ratio being 10–20 g / L soybean meal, 5–10 g / L peanut protein powder, and 2–5 g / L peptone. This is achieved by reducing the amount of nitrogen source added to the substrate. Based on the nitrogen source of the culture medium, and according to the nutrient requirements of the microbial cells at different growth stages, the feed is divided into four types: A, B, C, and D, each containing different nutrients. These different feeds containing specific nutrients are added at different stages of clavulanic acid fermentation. This balances the relationship between microbial growth and metabolism during fermentation, accelerates the microbial growth rate in the early stages of fermentation, reduces the viscosity of the clavulanic acid fermentation process, improves the mass transfer efficiency in the fermentation broth, increases the initial potency of clavulanic acid synthesis, prolongs the product synthesis period, increases the synthesis rate of clavulanic acid potency per unit cell, improves the potency of clavulanic acid in tank fermentation, reduces fermentation production costs, reduces the output of mycelium and other waste, improves economic benefits, and alleviates environmental pressure.

[0028] To supplement specific nutrients during fermentation to maintain cell growth and product synthesis, the feed is divided into four types (A, B, C, and D) containing different nutrients during staged feeding. Feed A includes glycerol, which directly provides a key precursor (glyceraldehyde-3-phosphate) required for clavulanic acid synthesis. Preferably, the glycerol is prepared as an aqueous solution with a concentration of 400–500 g / L. Feed B includes corn oil, which is slowly hydrolyzed into glycerol and free fatty acids within the cells, providing a more stable and sustained carbon source for cell growth and metabolism, and also acting as an antifoaming agent during fermentation. Preferably, the corn oil is added in pure corn oil form. Feed C includes protease hydrolysate, prepared from soybean protein through enzymatic hydrolysis, forming peptides and amino acids, which facilitates the rapid replenishment of nutrients required for cell metabolism. Preferably, the protease hydrolysate is prepared as a solution with a concentration of 250–350 g / L. Feed D includes threonine and glutamic acid. Threonine and glutamic acid provide the necessary carbon skeleton and nitrogen atoms for clavulanic acid metabolism, and may affect the expression and activity of enzyme systems involved in clavulanic acid synthesis. Preferably, threonine and glutamic acid are prepared as an aqueous solution, wherein the content of threonine is 1.0–6.0 g / L and the content of glutamic acid is 1.0–6.0 g / L.

[0029] In some preferred embodiments, while reducing the nitrogen source in the substrate culture medium, the timing of each feed addition and cessation of feed addition is strictly controlled to meet the different nutrient requirements at different fermentation stages. This includes: adding feed A at the first fermentation time (a certain time between 13 and 17 hours); adding feed B at the second fermentation time (a certain time between 48 and 52 hours); adding feed C at the third fermentation time (a certain time between 68 and 72 hours); adding feed D at the fourth fermentation time (a certain time between 78 and 82 hours); and ceasing the addition of feeds B, C, and D at the fifth fermentation time (a certain time between 98 and 102 hours), while continuing to add feed A until the end of fermentation.

[0030] More preferably, after 15 hours of fermentation, feed A is added; after 50 hours of fermentation, feed B is added; after 70 hours of fermentation, feed C is added; after 80 hours of fermentation, feed D is added; after 100 hours of fermentation, feed B, feed C, and feed D are stopped, while feed A is added until the end of fermentation.

[0031] When fermentation reaches the first time, for example, 15 hours, feed A is added. The carbon source in the fermentation substrate is mainly starch and corn oil, which are slowly utilized by the cells. Glycerol can be used as a carbon source that is quickly utilized by the cells, and it can also directly provide the key precursor (glyceraldehyde-3-phosphate) required for clavulanic acid synthesis. Therefore, adding feed A containing glycerol at an earlier time, such as 15 hours, is beneficial to the rapid growth of the cells and the synthesis of products.

[0032] When fermentation reaches the second time point, for example, 50 hours, feed B should be added. At this time, the corn oil is consumed in large quantities, and the microbial metabolism is vigorous, producing a lot of foam. Adding corn oil can provide a stable and long-lasting carbon source supply and reduce the impact of foam production on the fermentation process. Therefore, feed B containing corn oil should be added at this time.

[0033] When fermentation reaches the third time point, for example, 70 hours, feed C is added. Since the nitrogen source in the optimized fermentation medium is reduced by 20%–50%, it cannot support the entire fermentation process and needs to be replenished before the nitrogen source is depleted. At this time, protease hydrolysate is added, which not only helps maintain cell growth but also effectively provides the precursor amino acids needed for clavulanic acid metabolism, thereby increasing the clavulanic acid potency. Therefore, feed C containing protease hydrolysate is added at this point. Furthermore, the protease hydrolysate is preferably prepared by adding alkaline protease to a protein aqueous solution made from soybean protein. After enzymatic hydrolysis, soybean protein forms peptides and amino acids of different molecular weights, which can improve the clavulanic acid potency.

[0034] When fermentation reaches the fourth time point, e.g., 80 hours, feed D is introduced to supplement threonine and glutamate. This regulates metabolic flux during the rapid synthesis of clavulanic acid, directing more carbon sources towards the clavulanic acid synthesis pathway and indirectly increasing the supply of clavulanic acid precursors, thereby enhancing clavulanic acid potency. Threonine provides the necessary carbon skeleton and nitrogen atoms for clavulanic acid metabolism. By inhibiting aspartate kinase through feedback, it regulates metabolic flux, reducing carbon flow to the methionine branch and directing more carbon sources towards the clavulanic acid synthesis pathway, indirectly increasing the supply of clavulanic acid precursors. Glutamate is an organic nitrogen source easily absorbed and utilized by microorganisms. It provides essential nitrogen for cell growth, reproduction, and the synthesis of various enzymes and proteins. In many microbial secondary metabolic processes, the metabolic level of glutamate is related to global nitrogen metabolism regulation and may affect the expression and activity of enzymes involved in clavulanic acid synthesis, thus enhancing clavulanic acid potency. Therefore, feed D, containing threonine and glutamate, is introduced at this time.

[0035] When fermentation reaches the fifth time point, such as 100 hours, the addition of feed BCD is stopped because after 100 hours of fermentation, the respiration rate of the cells weakens, the oxygen consumption rate of the cells decreases significantly, some cells undergo autolysis, the demand for nutrients decreases significantly, and the growth of antibiotic yield slows down or even decreases. If nutrients are continued to be added, it will be wasteful, and the unconsumed nutrients will affect the metabolism of the cells. Therefore, at this time, all feeds except glycerol are stopped.

[0036] In some preferred embodiments, during the replenishment of various materials, samples are taken at preset time intervals to detect the nutrient content in the fermentation broth. Based on the monitoring results, the nutrient content in the fermentation broth is controlled. The preset time interval is 6–10 hours, for example, 8 hours. The nutrients include glycerol, corn oil, amino nitrogen, and amino acids.

[0037] Furthermore, the glycerol content in the fermentation broth is controlled to be 2.0–8.0 g / L. Excessive glycerol concentration triggers the "carbon metabolism repression" mechanism of the production strain, causing the cells to preferentially utilize glycerol for rapid growth and energy metabolism. This process inhibits the expression of genes and enzymes related to clavulanic acid synthesis, leading to the closure or severe inhibition of the clavulanic acid biosynthesis pathway and a sharp decrease in fermentation units. Therefore, the glycerol content in the fermentation broth is controlled within this range.

[0038] Furthermore, the content of the clear corn oil layer in the fermentation broth should be controlled to ≤2.0%. Oil utilization is a highly oxygen-intensive process. Excessive oil will cause the microbial metabolism to rapidly shift to the highly oxygen-consuming oil decomposition pathway, reducing the dissolved oxygen in the fermentation broth, resulting in impaired microbial growth and a reduced product synthesis rate. Therefore, the content of the clear corn oil layer in the fermentation broth must be strictly controlled within this range during the fermentation process.

[0039] Furthermore, the amino nitrogen content in the fermentation broth should be controlled to ≤0.8 g / L. Excessive amino nitrogen content leads to excessive cell growth, prompting the production strain to engage in vigorous primary metabolism. A high-nitrogen environment inhibits the activity or gene expression of key enzymes responsible for clavulanic acid synthesis, diverting metabolic flow towards the synthesis of the cell's own proteins and nucleic acids, rather than clavulanic acid synthesis. Therefore, the amino nitrogen content must be strictly controlled within the aforementioned range during the fermentation process.

[0040] Furthermore, the concentrations of threonine and glutamate in the fermentation broth were controlled to be ≤1.0 g / L. Clavulanic acid synthesis is closely linked to primary metabolism; excessive amino acids may inhibit the gene expression of key synthases and suppress their activity. High amino acid concentrations can also be toxic to the production strain, thus affecting cell growth and clavulanic acid synthesis. Therefore, the concentrations of threonine and glutamate were controlled within this range during the fermentation process.

[0041] In some preferred embodiments, the fermentation process further includes: controlling the dissolved oxygen level to ≥30% throughout the fermentation process by adjusting the stirring speed; specifically, after 36 hours of fermentation (e.g., from 36 to 100 hours), a lower stirring speed, such as a third stirring speed of 520-560 rpm (e.g., 550 rpm), can control the dissolved oxygen level at 40%-50%. In contrast, in existing related technologies, due to the high viscosity of the fermentation broth, the stirring speed needs to be controlled at 650 rpm to maintain the dissolved oxygen level at 30%-40%. Therefore, compared with existing technologies, this application also has advantages such as reduced energy consumption, cost savings, increased economic benefits, reduced foam generation, reduced defoamer dosage, reduced equipment wear, and precise control of dissolved oxygen and cell metabolism.

[0042] Specifically, during the 0-35h fermentation stage, the microbial growth is vigorous and the dissolved oxygen decreases, so the stirring speed is increased from the first stirring speed of 300-320 rpm to the second stirring speed of 680-700 rpm; during the 36h-100h fermentation stage, as the dissolved oxygen increases, the stirring speed is decreased from the second stirring speed to the third stirring speed of 520-560 rpm, such as 550 rpm; from 100h to the discharge stage, as the dissolved oxygen increases, the stirring speed can be further decreased to the fourth stirring speed of 300-510 rpm.

[0043] In some embodiments, the provided method for fermenting and preparing clavulanic acid further includes: preparing a seed culture of *Streptomyces lanceolata*. The fermentation production of clavulanic acid using *Streptomyces lanceolata* as the production strain specifically includes the following steps: Step S1: Preparation of slant culture: Take a glycerol cryovial of Streptomyces rotaforme, inoculate the glycerol cryovial of Streptomyces rotaforme into a slant culture medium for activation culture, and prepare a spore suspension.

[0044] Further, the inoculum volume is 0.1 mL to 0.2 mL, the activation culture temperature is 25℃ to 27℃, the relative humidity is 40% to 60%, and the activation culture is carried out for 12 to 14 days. The culture medium surface is covered with mycelial growth and grayish-green spores are produced. Take the cultured slant culture, add an appropriate amount of sterile water, and wash off the mycelial growth to prepare a spore suspension. The slant culture medium, using deionized water as the medium, 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, adjust the pH to 7.00–7.40 using 20% ​​sodium hydroxide solution, for example, to 7.20. Heat to melt the agar, pour into 25 mm × 200 mm test tubes, sterilize, and then tilt and place at room temperature until solidified. This is then used as the slant culture medium.

[0045] Step S2, preparation of shake flask seed culture: The spore suspension prepared in step S1 is inoculated into shake flask seed culture medium and placed on a self-controlled constant temperature shaker for culture to obtain shake flask seed culture.

[0046] Furthermore, the inoculum size is 0.5 mL to 1 mL. The culture temperature is 27℃ to 29℃, the shaker speed is 200 to 250 rpm, and the culture time is 64 to 72 hours. Shake flask specifications: Erlenmeyer flask, 500 mL volume, with 100 mL of culture medium. The shake flask seed culture medium, using deionized water as the medium, contains: 8 to 12 g / L soybean meal, 3 to 7 g / L yeast extract, and 13 to 17 g / L glycerol. After preparation, the pH of the culture medium is adjusted to 6.80 to 7.10 using 20% ​​sodium hydroxide solution.

[0047] Step S3, preparation of seed culture: The seed culture prepared in step S2 is inoculated into the seed tank culture medium to obtain the seed culture.

[0048] Furthermore, the inoculum size is 0.5%–1.5% (V / V). The culture temperature is 27℃–29℃, the stirring speed is 200–500 rpm, the aeration rate is 0.4 vvm–0.8 vvm (air flow rate / culture medium volume / min), and the tank pressure is 0.05–0.06 MPa, with a culture time of 30–40 hours. The seed tank culture medium, using deionized water as the medium, contains: 18–22 g / L soybean meal, 8–12 g / L starch, 8–12 g / L yeast extract, 1.3–1.7 g / L NaH2PO4, and 1–3 g / L antifoaming agent. After preparation, the pH of the culture medium is adjusted to 6.90–7.10 using a 20% sodium hydroxide solution.

[0049] Step S4, fermenter culture: The seed culture prepared in step S3 is inoculated into the optimized fermentation medium for fermentation culture. Different feeds are added at different cycles during the fermentation process, and the fermentation products are collected to obtain clavulanic acid.

[0050] The fermentation medium, using deionized water as the medium, contains: 10–20 g / L soybean meal, 5–10 g / L peanut protein powder, 2–5 g / L peptone, 5–20 g / L 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.

[0051] Furthermore, the fermentation process was carried out in a 50L fermenter with an inoculation ratio of 10-20% (V / V) and a culture medium volume of 36L. During the fermentation process, the fermentation temperature was 25℃-28℃, the stirring speed was 300-700 rpm, the aeration rate was 0.6-1.0 vvm (air flow rate / culture medium volume / minute), and the tank pressure was 0.05-0.08 MPa.

[0052] During the fermentation process, ensure that the dissolved oxygen (DO) level is ≥30%. Stirring speed control: From 0 to 35 hours, as cell growth is vigorous and dissolved oxygen decreases, gradually increase the stirring speed from 300 rpm to 700 rpm; from 36 to 100 hours, as dissolved oxygen increases, gradually decrease the stirring speed from 700 rpm to 550 rpm; from 101 hours until the discharge stage, as dissolved oxygen increases, reduce the stirring speed to 500 rpm.

[0053] During fermentation, different feeding methods are used to supplement the nutrient content in the fermentation broth at different fermentation stages. During this process, samples are taken at preset time intervals to monitor the nutrient content, and the nutrient content is controlled based on the monitoring results. Specifically, this includes: adding feed A after 15 hours of fermentation; adding feed B after 50 hours; adding feed C after 70 hours; adding feed D after 80 hours; and stopping feed B, C, and D after 100 hours. Feed A is added continuously from the beginning until the end of fermentation, meaning feed A is added throughout the entire fermentation process.

[0054] During the feeding process, samples are taken at preset time intervals to test the nutrient content in the fermentation broth, and the nutrient content in the fermentation broth is controlled based on the monitoring results. Specifically, the glycerol content in the fermentation broth is controlled to be 2.0–8.0 g / L; the corn oil clear layer content in the fermentation broth is controlled to be ≤2.0%; the amino nitrogen content in the fermentation broth is controlled to be ≤0.8 g / L; the threonine concentration in the fermentation broth is controlled to be ≤1.0 g / L; and the glutamic acid concentration in the fermentation broth is controlled to be ≤1.0 g / L.

[0055] In addition, during the fermentation process, the pH of the fermentation broth was adjusted by adding 20% ​​ammonia water, maintaining the pH range at 6.40–6.80. Fermentation was terminated after 120–130 hours of cultivation.

[0056] Further, feed A is an aqueous glycerol solution with a concentration of 400-500 g / L. Feed B is pure corn oil. Feed D is an aqueous solution prepared from threonine and glutamic acid, with threonine content of 1-6 g / L and glutamic acid content of 1-6 g / L. Feed C is a protease hydrolysate with a concentration of 250-350 g / L; preparation method: prepare a 25%-35%, for example, 30% aqueous solution of soybean protein; add 0.05%-0.15%, for example, 0.1% alkaline protease to the aqueous solution, adjust the pH to 9.5-10.5, rapidly heat to 55-65°C, maintain the reaction for 25-35 minutes, rapidly heat to 105-115°C under steam sterilization conditions, and cool to 25°C to obtain the protease hydrolysate.

[0057] The technical solution of the present invention will be described in more detail below with reference to specific embodiments and their effects.

[0058] The strain used in the following examples is *Streptomyces lanceolata*, specifically *Streptomyces lanceolata* KNR1, which is derived from the American Type Culture Collection (ATCC) with accession number ATCC27064.

[0059] Example 1 Step 1: Preparation of spore suspension Preparation of slant culture medium: Using deionized water as the medium, prepare the slant culture medium with the following proportions: malt extract 10 g / L, yeast extract 8 g / L, glucose 5 g / L, agar 25 g / L. Adjust the pH to 7.20 using 20% ​​sodium hydroxide solution. Prepare 200 mL of the culture medium, heat to melt, stir well, and pour into 20 test tubes (25 mm × 200 mm). After sterilization, tilt the tubes at a 25° angle and allow them to solidify to form slant culture medium for later use.

[0060] Take a rod-shaped Streptomyces glycerol cryovial, and after thawing, take 0.2 mL and spread it evenly on the surface of the slant. Place it in a constant temperature and humidity incubator at 26℃ and 40-60% relative humidity for 12 days. The surface of the culture medium will be covered with mycelial moss and gray-green spores will be produced. Add 8 mL of sterile water to wash off the mycelial moss and prepare a spore suspension.

[0061] Step 2: Preparation of Seed Culture in Shake Flasks Shake-flask seed culture medium: Using deionized water as the medium, a shake-flask seed culture medium was prepared, containing: 10 g / L soybean meal, 5 g / L yeast extract, and 15 g / L glycerol. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution, and 200 mL of the medium was prepared. This medium was then divided into two 500 mL Erlenmeyer flasks, 100 mL each, and sterilized for later use.

[0062] Take 0.5 mL of the spore suspension from step 1 and inoculate it into the seed culture medium in a shake flask. Place the shake flask in a self-controlled constant temperature shaker at 28℃ and 230 rpm for 68 hours.

[0063] Step 3: Seed culture Seed tank culture medium: The seed tank culture medium, prepared using deionized water, comprises: 20 g / L soybean meal, 10 g / L starch, 10 g / L yeast extract, 1.5 g / L NaH2PO4, and 1.5 g / L defoamer. The pH is adjusted to 6.90 using a 20% sodium hydroxide solution, resulting in a final volume of 9 liters.

[0064] 45 mL of the shake flask seed culture prepared in step 2 above (mycelial concentration: 20%~22%) was inoculated into the seed tank culture medium. The culture was carried out at 28℃, with a stirring speed of 300 rpm, an aeration rate of 0.5 VVM (air flow rate / culture medium volume / minute), and a tank pressure of 0.05 MPa for 33 hours.

[0065] Step 4: Fermentation tank cultivation 4.3L of the seed culture (mycelial concentration: 20%–22%) prepared in step 3 of the above-mentioned seed tank was inoculated into the fermentation tank culture medium. The stirring speed was gradually increased from 300 rpm to 700 rpm from 0 to 35 h, gradually decreased from 700 rpm to 550 rpm from 36 h to 100 h, and further decreased to 450 rpm from 101 h to 130 h. Fermentation was carried out under the conditions of 0.8 vvm (air flow rate / culture medium volume / min) and tank pressure of 0.07 MPa. During the culture process, the dissolved oxygen (DO) was maintained at ≥30%, and the pH was controlled at 6.60 after automatic ammonia water control. The fermentation was terminated after 130 h.

[0066] Fermentation medium preparation: Using deionized water as the medium, the fermentation tank culture medium was prepared, containing: 10 g / L soybean meal powder, 5 g / L peanut protein powder, 2 g / L peptone, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution. The actual volume after defoaming was 33 liters.

[0067] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a 300 g / L protease hydrolysate. Preparation: Soy protein is prepared into a 30% aqueous solution; 0.1% alkaline protease is added to the aqueous solution, the pH is adjusted to 10.0, the temperature is rapidly increased to 60°C, the reaction is maintained for 30 minutes, and then rapidly increased to 110°C under steam sterilization conditions, followed by cooling to 25°C to obtain the protease hydrolysate. Feed D is an aqueous solution of threonine and glutamic acid, with a threonine content of 1 g / L and a glutamic acid content of 1 g / L.

[0068] Feeding control during fermentation: Feed A is introduced after 15 hours of fermentation, and is added throughout the entire fermentation process, maintaining the glycerol content in the fermentation broth at 2 g / L. Feed B is introduced after 50 hours of fermentation, maintaining the corn oil clear layer in the fermentation broth at ≤2%. Feed C is introduced after 70 hours of fermentation, maintaining the amino nitrogen content in the fermentation broth at ≤0.3 g / L. Feed D is introduced after 80 hours of fermentation, maintaining the threonine content and glutamic acid content in the fermentation broth at ≤0.4 g / L. Feeding of feeds B, C, and D is stopped after 100 hours of fermentation.

[0069] During fermentation, the dissolved oxygen content in the fermentation broth was controlled above 30%, and the dissolved oxygen content was monitored using a dissolved oxygen electrode. The pH of the fermentation broth was controlled at 6.60 by adding 20% ​​ammonia solution, and the fermentation process lasted 130 hours.

[0070] The process parameters are detected as follows: PMV determination method: Take 10 mL of bacterial culture and centrifuge at 4500 rpm / min for 20 minutes. Calculate the proportion of precipitate in the centrifuge tube, and record each division as 2%.

[0071] Method for determining bacterial cell weight: Take 10 mL of bacterial solution and centrifuge at 10000 rpm / min for 20 minutes. Discard the supernatant, blot the liquid on the centrifuge tube wall with filter paper, and weigh it on a balance. The unit is g / 10 mL.

[0072] Method for determining the content of clear oil layer in corn oil in fermentation broth: Take 10 mL of bacterial solution and centrifuge at 4500 rpm / min for 20 minutes. Calculate the proportion of clear oil layer in the centrifuge tube, and record each division as 2%.

[0073] Viscosity testing method: Take 50 mL of fermentation broth and use a digital viscometer (DV-C) to measure the viscosity of the fermentation broth. During the measurement, use a No. 62 rotor at 30% speed. Submerge the rotor with the fermentation broth and read the viscosity value after the data stabilizes.

[0074] Methods for detecting clavulanic acid potency and impurity G content: 1.5 mL of sample was added to a 2 mL centrifuge tube and centrifuged at 12000 rpm for 10 minutes to separate the bacterial cells and supernatant. The supernatant was filtered through a 0.22 μm microporous membrane. The contents of clavulanic acid and impurity G in the fermentation broth supernatant were detected using an Agilent HPLC system 1260. The chromatographic column was a Thermo-C18 equipped with a UV detector. The mobile phase was tetrabutylammonium hydrogen sulfate:acetonitrile = 100:10, and the column temperature was 33℃.

[0075] Glycerol content detection method: Add 1.5 mL of sample to a 2 mL centrifuge tube, centrifuge at 12000 rpm for 10 minutes to separate the bacterial cells and supernatant. Filter the supernatant through a 0.22 μm microporous membrane. Detect glycerol in the fermentation broth supernatant using an Agilent HPLC system 1260. The chromatographic column was Ultimate-XB, equipped with a RID detector. The mobile phase was acetonitrile:water = 90:10, and the column temperature was 30℃.

[0076] Amino nitrogen detection method: Take 5.0 mL of sample and put it into a digestion tube. Distill it on a nitrogen analyzer until the ammonia is completely distilled off. Remove the receiving bottle and titrate with 0.1 mol / L hydrochloric acid solution until the endpoint is pink. Formula: Ammonia nitrogen concentration (g / L) = (V sample – V blank) × 1.4 / 5.

[0077] Methods for detecting threonine and glutamic acid content: 1.5 mL of sample was added to a 2 mL centrifuge tube and centrifuged at 12000 rpm for 10 minutes to separate the bacterial cells and supernatant. The supernatant was filtered through a 0.22 μm microporous membrane, and the threonine and glutamic acid content in the fermentation broth supernatant was detected using an Agilent HPLC system 1260. The chromatographic column was an InfinityLab PorosheL HPH-C18, and the mobile phase was A: 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, with the pH adjusted to 8.2 using hydrochloric acid; B: methanol: acetonitrile: water = 45:45:10 (V:V:V). The detector was VWD, 338 nm, and the column temperature was 40℃.

[0078] The difference between Examples 2-6 and Example 1 is that the fermentation medium preparation, feed preparation and feed control during fermentation are carried out in step 4. Other undescribed contents are the same as in Example 1.

[0079] Example 2 Fermentation medium preparation: Using deionized water as the medium, the fermentation tank culture medium was prepared, containing: 12 g / L soybean meal, 7 g / L peanut protein powder, 4 g / L peptone, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution. The actual volume after defoaming was 33 liters.

[0080] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a protein hydrolysate with a concentration of 300 g / L; Method: Prepare a 30% aqueous solution of soybean protein; add 0.1% alkaline protease to the aqueous solution, adjust the pH to 10.0, rapidly heat to 60°C, maintain the reaction for 30 minutes, rapidly heat to 110°C under steam sterilization conditions, and cool to 25°C to obtain the protein hydrolysate. Feed D is an aqueous solution of threonine and glutamic acid, with a threonine content of 2 g / L and a glutamic acid content of 2 g / L.

[0081] Feeding control during fermentation: Feed A is introduced after 15 hours of fermentation, and is added throughout the entire fermentation process, maintaining the glycerol content in the fermentation broth at 3 g / L. Feed B is introduced after 50 hours of fermentation, maintaining the corn oil clear layer in the fermentation broth at ≤2%. Feed C is introduced after 70 hours of fermentation, maintaining the amino nitrogen content in the fermentation broth at ≤0.4 g / L. Feed D is introduced after 80 hours of fermentation, maintaining the threonine content and glutamic acid content in the fermentation broth at ≤0.50 g / L. Feeding of feeds B, C, and D is stopped after 100 hours of fermentation.

[0082] Example 3 Fermentation medium preparation: Using deionized water as the medium, the fermentation tank culture medium was prepared, containing: 14 g / L soybean meal, 8 g / L peanut protein powder, 5 g / L peptone, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution. The actual volume after defoaming was 33 liters.

[0083] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a 300 g / L protease hydrolysate; method: Prepare a 30% aqueous solution of soybean protein; add 0.1% alkaline protease to the aqueous solution, adjust the pH to 10.0, rapidly heat to 60°C, maintain the reaction for 30 minutes, rapidly heat to 110°C under steam sterilization conditions, and cool to 25°C to obtain the protease hydrolysate. Feed D is an aqueous solution of threonine and glutamic acid, wherein the threonine content is 3 g / L and the glutamic acid content is 3 g / L.

[0084] Feeding control during fermentation: Feed A is introduced after 15 hours of fermentation, and is added throughout the entire fermentation process, maintaining the glycerol content in the fermentation broth at 4 g / L. Feed B is introduced after 50 hours of fermentation, maintaining the corn oil clear layer in the fermentation broth at ≤2%. Feed C is introduced after 70 hours of fermentation, maintaining the amino nitrogen content in the fermentation broth at ≤0.5 g / L. Feed D is introduced after 80 hours of fermentation, maintaining the threonine content and glutamic acid content in the fermentation broth at ≤0.60 g / L. Feeding of feeds B, C, and D is stopped after 100 hours of fermentation.

[0085] Example 4 Fermentation medium preparation: Using deionized water as the medium, the fermentation tank culture medium was prepared, containing: 16 g / L soybean meal, 9 g / L peanut protein powder, 5 g / L peptone, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution. The actual volume after defoaming was 33 liters.

[0086] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a protein hydrolysate with a concentration of 300 g / L; Method: Prepare a 30% aqueous solution of soybean protein; add 0.1% alkaline protease to the aqueous solution, adjust the pH to 10.0, rapidly heat to 60°C, maintain the reaction for 30 minutes, rapidly heat to 110°C under steam sterilization conditions, and cool to 25°C to obtain the protein hydrolysate. Feed D is an aqueous solution of threonine and glutamic acid, wherein the threonine content is 4 g / L and the glutamic acid content is 4 g / L.

[0087] Feeding control during fermentation: Feed A was introduced after 15 hours of fermentation, and was added throughout the entire fermentation process, maintaining the glycerol content in the fermentation broth at 5 g / L. After 50 hours, feed B was introduced, maintaining the corn oil clear layer in the fermentation broth at ≤2%. After 70 hours, feed C was introduced, maintaining the amino nitrogen content in the fermentation broth at ≤0.6 g / L. After 80 hours, feed D was introduced, maintaining the threonine and glutamic acid content in the fermentation broth at ≤0.70 g / L. Feeding of feeds B, C, and D was stopped after 100 hours of fermentation. Dissolved oxygen was maintained above 30%, pH was 6.60, and the fermentation period was 130 hours.

[0088] Example 5 Fermentation medium preparation: Using deionized water as the medium, the fermentation tank culture medium was prepared, containing: 18 g / L soybean meal, 8 g / L peanut protein powder, 5 g / L peptone, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution. The actual volume after defoaming was 33 liters.

[0089] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a protein hydrolysate with a concentration of 300 g / L; Method: Prepare a 30% aqueous solution of soybean protein; add 0.1% alkaline protease to the aqueous solution, adjust the pH to 10.0, rapidly heat to 60°C, maintain the reaction for 30 minutes, rapidly heat to 110°C under steam sterilization conditions, and cool to 25°C to obtain the protein hydrolysate. Feed D is an aqueous solution of threonine and glutamic acid, wherein the threonine content is 5 g / L and the glutamic acid content is 5 g / L.

[0090] Feeding control during fermentation: Feed A is introduced after 15 hours of fermentation, and is added throughout the entire fermentation process, maintaining the glycerol content in the fermentation broth at 6 g / L. Feed B is introduced after 50 hours of fermentation, maintaining the corn oil clear layer in the fermentation broth at ≤2%. Feed C is introduced after 70 hours of fermentation, maintaining the amino nitrogen content in the fermentation broth at ≤0.7 g / L. Feed D is introduced after 80 hours of fermentation, maintaining the threonine content and glutamic acid content in the fermentation broth at ≤0.80 g / L. Feeding of feeds B, C, and D is stopped after 100 hours of fermentation.

[0091] Example 6 Fermentation medium preparation: After optimization, the fermentation medium was prepared using deionized water as the medium, and included the following: 20 g / L soybean meal, 10 g / L peanut protein powder, 5 g / L peptone, 10 g / L starch, 15 g / L vegetable oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution, and the actual volume after defoaming was 33 liters.

[0092] Feed preparation: Feed A is a 450 g / L aqueous solution of glycerol. Feed B is pure corn oil. Feed C is a protein hydrolysate with a concentration of 300 g / L; the method is as follows: soybean protein is prepared into a 30% aqueous solution; 0.1% alkaline protease is added to the aqueous solution, the pH is adjusted to 10.0, the temperature is rapidly increased to 60°C, the reaction is maintained for 30 minutes, and then the temperature is rapidly increased to 110°C under steam sterilization conditions, and then decreased to 25°C to obtain the protein hydrolysate. Feed D is an aqueous solution prepared from threonine and glutamic acid, wherein the threonine content is 6 g / L and the glutamic acid content is 6 g / L.

[0093] Feeding control during fermentation: After 15 hours of fermentation, feed A was introduced, and it was added throughout the fermentation process, with the glycerol content in the fermentation broth controlled at 7 g / L. After 50 hours of fermentation, feed B was introduced, with the corn oil clear layer in the fermentation broth controlled at ≤2%. After 70 hours of fermentation, feed C was introduced, with the amino nitrogen content in the fermentation broth controlled at ≤0.8 g / L. After 80 hours of fermentation, feed D was introduced, with the threonine content in the fermentation broth controlled at ≤0.90 g / L and the glutamic acid content in the fermentation broth controlled at ≤0.90 g / L. After 100 hours of fermentation, feeds B, C, and D were discontinued.

[0094] Comparison Examples The difference from Example 1 is that step 4 involves fermentation tank cultivation, fermentation medium preparation, feed preparation, and feed control during the fermentation process.

[0095] Fermentation in a fermenter: During the period of 36h to 100h, the stirring speed was gradually reduced from 700rpm to 650rpm, and all other aspects were the same as in Example 1.

[0096] Fermentation medium preparation: Using deionized water as the medium, a fermentation tank medium was prepared, consisting of: 40 g / L soybean meal, 10 g / L starch, 15 g / L corn oil, 2.7 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 1.0 g / L defoamer. The pH was adjusted to 6.90 using 20% ​​sodium hydroxide solution, and the actual volume after defoaming was 33 liters.

[0097] Feed preparation: Feed A is a glycerol aqueous solution with a concentration of 450 g / L.

[0098] Feed control during fermentation: Feed A is introduced after 15 hours of fermentation. The glycerol content is controlled within the range of 6-8 g / L before 80 hours and 2-6 g / L after 80 hours. Feed A is added continuously throughout the fermentation process, starting after 15 hours of fermentation. The glycerol content is controlled within the range of ≤8 g / L before 80 hours and ≤6 g / L after 80 hours.

[0099] The experimental results of the examples and control examples are shown in the table below. According to the clavulanic acid fermentation method provided by this invention, after 18 hours of fermentation, the viscosity was slightly increased by about 20% compared to the control example; and the PMV increased significantly, with the PMV of this invention increasing by about 188%, while that of the control example increased by 30%. The PMV of this invention reached its maximum at 42 hours, while that of the control example reached its maximum at 66 hours, indicating that this invention has a significant advantage for cell growth. After 50 hours of fermentation, the viscosity reached its maximum value, and the viscosity of this invention decreased by about 22% compared to the control example. After 130 hours of fermentation, the viscosity of the control example remained at a high level, but it had decreased significantly compared to 90 hours. In contrast, the viscosity of this invention decreased after 130 hours of fermentation, but the decrease was less than that of the control example, about 9%, indicating that the cell aging rate of this invention was lower than that of the control example, and the strain maintained high activity in the later stages of fermentation.

[0100] After fermentation for 130 hours, the PMV of this invention was lower than that of the control example by about 41%. The lower PMV is beneficial to improve the efficiency of filtration and extraction, shorten the filtration and extraction time, and reduce the amount of waste such as mycelium generated, which improves economic benefits and reduces environmental pressure.

[0101] After 58 hours of fermentation, the clavulanic acid potency of this invention was significantly improved by approximately 67% compared to the control example, indicating that the initial potency synthesis rate of clavulanic acid in this invention was significantly higher than that in the control example. After 90 hours of fermentation, the potency increase of this invention remained at 1000 μg / mL compared to the control example. After 130 hours of fermentation, the potency increase of this invention was still relatively high. The potency growth rate of this invention from 90 to 130 hours was 60 μg / mL / h, while that of the control example was 49 μg / mL / h. Furthermore, the potency of this invention at 130 hours after fermentation was approximately 20% higher than that of the control example. Compared to the control example, the potency synthesized per unit cell weight was approximately 1.23 times higher, indicating that this invention, compared to the control example, not only improved the initial fermentation potency of clavulanic acid but also improved the potency synthesis rate and the potency level synthesized per unit cell weight in the later stages of fermentation, resulting in a significant improvement in potency upon fermentation.

[0102] After 58 hours of fermentation, the content of impurity G in this invention was significantly increased by approximately 52% compared to the control example. This is because the cell metabolism of this invention is vigorous and the titer synthesis rate is high at 58 hours. After 90 hours of fermentation, the content of impurity G in this invention was lower than that in the control example. After 130 hours of fermentation, the content of impurity G in this invention decreased significantly by approximately 48%, while the content of impurity G in the control example continued to increase by approximately 35%. The reason why the content of impurity G in this invention can be reduced from 90 to 130 hours is mainly due to the reduction of nitrogen source in the fermentation medium substrate by approximately 20% to 50%, the replacement of a single nitrogen source with a compound nitrogen source, and the precise control of feeding in the later stage of fermentation.

[0103] In summary, by reducing the amount of nitrogen source added to the fermentation medium, replacing the single nitrogen source with a compound nitrogen source, and adjusting the proportion of nitrogen source substances, different feeding is carried out at each stage of clavulanic acid fermentation according to the growth and metabolic needs of the strain, so as to balance the relationship between cell growth and metabolism during fermentation. That is, the clavulanic acid fermentation preparation method provided by this invention can significantly reduce the viscosity of the clavulanic acid fermentation process, improve the dissolved oxygen conditions during fermentation, increase the mass transfer efficiency of the fermentation broth, significantly increase the initial potency synthesis rate of clavulanic acid, delay cell aging, extend the fermentation cycle, improve fermentation potency, greatly reduce production costs, and reduce the generation of waste such as mycelium.

[0104] The description of this invention is given for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for preparing clavulanic acid by fermentation, characterized in that, include: A fermentation medium was prepared using a composite nitrogen source instead of a single nitrogen source. The seed culture of Streptomyces rod-shapedis was inoculated into the fermentation medium for fermentation culture. During the fermentation process, different feeds were added according to different fermentation stages. Clavulanic acid was obtained by collecting the fermentation products. The complex nitrogen source in the fermentation medium includes: 10-20 g / L soybean meal, 5-10 g / L peanut protein powder, and 2-5 g / L peptone. The feed includes feed A, feed B, feed C and feed D; wherein feed A includes glycerol; feed B includes corn oil; feed C includes protease hydrolysate; and feed D includes threonine and glutamic acid.

2. The method for preparing clavulanic acid by fermentation according to claim 1, characterized in that, The steps described above, which involve adding different feeds according to different fermentation stages, include: adding feed A when fermentation reaches the first time point; adding feed B when fermentation reaches the second time point; adding feed C when fermentation reaches the third time point; adding feed D when fermentation reaches the fourth time point; and stopping adding feeds B, C, and D when fermentation reaches the fifth time point, while adding feed A until fermentation ends. The first time refers to a moment between 13 and 17 hours, the second time refers to a moment between 48 and 52 hours, the third time refers to a moment between 68 and 72 hours, the fourth time refers to a moment between 78 and 82 hours, and the fifth time refers to a moment between 98 and 102 hours.

3. The method for preparing clavulanic acid by fermentation according to claim 1, characterized in that, The steps described above, which involve adding different feeds according to different fermentation stages, include: adding feed A after 15 hours of fermentation; adding feed B after 50 hours of fermentation; adding feed C after 70 hours of fermentation; adding feed D after 80 hours of fermentation; and stopping adding feeds B, C, and D after 100 hours of fermentation, while adding feed A until the end of fermentation.

4. The method for preparing clavulanic acid by fermentation according to claim 2 or 3, characterized in that, During the process of adding various materials, samples are taken at preset intervals to test the content of nutrients in the fermentation broth, and the content of the nutrients in the fermentation broth is controlled according to the monitoring results. The preset time is 6-10 hours; the glycerol content in the fermentation broth is controlled to be 2.0-8.0 g / L; the corn oil clear oil layer content in the fermentation broth is controlled to be ≤2.0%; the amino nitrogen content in the fermentation broth is controlled to be ≤0.8 g / L; the threonine concentration in the fermentation broth is controlled to be ≤1.0 g / L; and the glutamic acid concentration in the fermentation broth is controlled to be ≤1.0 g / L.

5. The method for preparing clavulanic acid by fermentation according to claim 2 or 3, characterized in that, In the feed, the glycerol is an aqueous solution of glycerol with a concentration of 400-500 g / L; the corn oil is pure corn oil; the concentration of the protease hydrolysate is 250-350 g / L; the threonine and glutamic acid are prepared aqueous solutions, wherein the content of threonine is 1.0-6.0 g / L and the content of glutamic acid is 1.0-6.0 g / L.

6. The method for preparing clavulanic acid by fermentation according to claim 5, characterized in that, The protease hydrolysate is prepared by adding alkaline protease to a protein aqueous solution of soybean protein. Specifically, the process includes: preparing a 25%–35% protein aqueous solution of soybean protein; adding 0.05%–0.15% alkaline protease to the protein aqueous solution; adjusting the pH to 9.5–10.5; rapidly heating to 55–65°C; maintaining the reaction for 25–35 minutes; rapidly heating to 105–115°C under steam sterilization conditions; and cooling to 25°C to obtain the protease hydrolysate.

7. The method for preparing clavulanic acid by fermentation according to claim 2 or 3, characterized in that, The fermentation process also includes: controlling the dissolved oxygen level to ≥30% throughout the fermentation process by adjusting the stirring speed; specifically, during the 36h to 100h fermentation stage, the stirring speed is 520 to 560 rpm. And / or, the fermentation medium, using deionized water as the medium, further includes: starch: 5-20 g / L; corn oil: 12-17 g / L; NaH2PO4: 2.6-2.9 g / L; FeCl3·6H2O: 0.04-0.06 g / L; MgCl2·6H2O: 0.7-0.9 g / L; ZnCl2: 0.010-0.014 g / L; MnSO4: 0.004-0.006 g / L; and defoamer: 0.5-1.5 g / L.

8. The method for preparing clavulanic acid by fermentation according to claim 1, characterized in that, The seed culture of Streptomyces roxburghii was inoculated into the fermentation medium at an inoculation rate of 10-20% (V / V) for fermentation culture. And / or, during the fermentation process, the pH of the fermentation broth is controlled to be 6.40–6.80 by adding ammonia water, and fermentation is terminated after 120–130 hours.

9. The method for preparing clavulanic acid by fermentation according to any one of claims 1-8, characterized in that, Also includes: Glyceryl succinate was inoculated into slant culture medium for activation culture and spore suspension was prepared. The spore suspension was inoculated into a shake flask seed culture medium and placed on a self-controlled constant temperature shaker for culture to obtain a shake flask seed solution. The shake flask seed solution was inoculated into a seed tank culture medium and cultured to obtain the seed solution of the Streptomyces rotaforme.

10. The method for preparing clavulanic acid by fermentation according to claim 9, characterized in that, The slant culture medium, using deionized water as the medium, contains: 8-12 g / L malt extract, 6-10 g / L yeast extract, 3-7 g / L glucose, and 23-27 g / L agar; the pH is 7.00-7.

40. The shake-flask seed culture medium, using deionized water as the medium, contains: 8-12 g / L soybean meal, 3-7 g / L yeast extract, and 13-17 g / L glycerol; pH is 6.80-7.

10. The seed tank culture medium, using deionized water as the medium, contains: 18-22 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 defoamer; the pH is 6.90-7.10.

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