A fermentation medium for monensin production, its preparation method and application

Abstract

This invention provides a fermentation medium for monensin production, its preparation method, and its application, relating to the field of microbial fermentation technology. The fermentation medium comprises the following components at the following mass concentrations: carbon source 28-55 g / L, nitrogen source 10-20 g / L, and eucommia ulmoides 1-3 g / L. The fermentation medium of this invention uses simple raw materials, and the added eucommia ulmoides not only regulates bacterial lipid metabolism but also provides more fatty acid precursors for monensin synthesis, thereby increasing the monensin synthesis rate. Shake-incubator fermentation using the fermentation medium of this invention significantly enhances the potency of monensin, demonstrating significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a fermentation medium for producing monensin, its preparation method, and its application. Background Technology

[0002] Monensin is produced by *Streptomyces cinnamon* ( Streptomyces cinnamonensis This is a class of polyether ionocarrier antibiotics produced by [the company name is missing]. It is widely used to prevent and treat coccidiosis in chickens, lambs, calves, and rabbits, and to promote the growth of ruminants. It has the functions of controlling the proportion of volatile fatty acids in the rumen, reducing the degradation of protein in the rumen, reducing the consumption of dry matter in feed, improving the utilization rate of nutrients, and increasing the energy utilization rate of animals.

[0003] Monensin has shown good inhibitory activity against various types of tumor cells and is considered a potential antitumor agent. Biofermentation is the main method for producing monensin, and numerous studies have focused on optimizing the fermentation medium formulation to increase monensin yield. The fermentation medium mainly contains glucose, maltose, soybean meal, yeast powder, and inorganic salts. However, the monensin obtained using this fermentation medium has a low potency, affecting the monensin synthesis rate and reducing the overall yield.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] This invention provides a fermentation medium for producing monensin, its preparation method, and its application. Using the fermentation medium of this invention for shaker fermentation can significantly increase the yield of monensin in the fermentation broth.

[0006] In a first aspect, the present invention provides a fermentation medium for producing monensin, comprising the following components at mass concentrations: 28-55 g / L of carbon source, 10-20 g / L of nitrogen source, and 1-3 g / L of eucommia ulmoides extract; wherein the carbon source comprises propylene glycol, lard, glucose, and soybean oil; The mass ratio of eucommia ulmoides to propylene glycol is 1:1~12; The fermentation medium also includes glyceryl monooleate at a mass concentration of 8-10 g / L, the mass ratio of lard to glyceryl monooleate is 1:1-2, and the mass ratio of eucommia ulmoides extract to lard is 1:1.6-8. The nitrogen source includes one or more of soybean meal powder and yeast powder; The eucommia extract is an extract of Eucommia ulmoides containing chlorogenic acid, flavonoids and total sugars; the chlorogenic acid content in the eucommia extract is ≥3.5% by mass; the flavonoid content in the eucommia extract is ≥8.0% by mass; and the total sugar content in the eucommia extract, calculated as glucose, is ≥20.0% by mass.

[0007] This invention has found that adding eucommia ulmoides to the fermentation medium for monensin production can regulate the lipid metabolism of the microorganisms, provide more fatty acid precursors for monensin synthesis, increase the monensin synthesis rate, and significantly improve the yield of monensin.

[0008] In the fermentation production of monensin, propylene glycol provides a key precursor, carbon skeleton, and hydroxyl groups for the synthesis of the monensin polyether ring, directly increasing the product synthesis throughput. However, excessive propylene glycol not only inhibits mycelial growth and causes metabolic overflow, but also easily leads to membrane damage and ROS accumulation. This invention found that when propylene glycol is used in combination with eucommia ulmoides extract, the aforementioned adverse effects of propylene glycol can be effectively suppressed. Moreover, when the mass ratio of eucommia ulmoides extract to propylene glycol is controlled within the range of 1:1 to 12, the inhibitory effect is more significant, and the yield of monensin can be significantly increased.

[0009] Eucommia ulmoides extract contains various active ingredients such as chlorogenic acid, flavonoids, and polysaccharides. During fermentation, eucommia ulmoides not only inhibits lateral metabolism (such as fatty acid and cholesterol synthesis) and promotes carbon flow to monensin synthesis, but also enhances the activities of SOD, POD, and CAT, and reduces oxidative stress. This reduces membrane damage and ROS accumulation caused by propylene glycol, protects mycelia and synthases, increases the expression of key enzymes in polyether ring synthesis (such as PKS and cyclase), and prolongs the high-yield period. Furthermore, it reduces propylene glycol leakage, improves precursor utilization, and reduces byproducts. Therefore, the synergistic effect of both significantly increases the yield of monensin.

[0010] In the fermentation medium of the present invention, propylene glycol is added to participate in the primary metabolism of Streptomyces cinnamon. The pyruvate produced is catalyzed by a dehydrogenase complex to generate acetyl-CoA. Acetyl-CoA is not only a key substrate of the tricarboxylic acid cycle, but also one of the core precursors for monensin synthesis, providing more prerequisite substances for monensin synthesis and further improving the yield of monensin.

[0011] Glyceryl monooleate (GMO) provides a fast-acting carbon source, while lard provides a long-acting carbon source, slowly releasing saturated or monounsaturated fatty acids to provide a stable carbon flow; the two complement each other in carbon supply. This invention found that, under the emulsifying, dispersing, and interfacial regulation effects of GMO, the degradation of lipid carbon sources in lard by *Streptomyces cinnamon* can be enhanced, releasing more fatty acids and glycerol metabolic intermediates, providing a core material basis for the generation of key precursors in monensin biosynthesis; and when the mass ratio of lard to GMO is controlled within the aforementioned range, the yield of monensin can be further improved.

[0012] The addition of lard can easily lead to low dissolved oxygen, high surface tension, and numerous byproducts, as well as membrane damage and ROS accumulation. This invention has found that when lard is used in combination with eucommia ulmoides extract, the aforementioned adverse effects of lard can be effectively suppressed. Furthermore, when the mass ratio of the two is controlled within the aforementioned range, the inhibitory effect is more pronounced, significantly increasing the yield of monensin.

[0013] During fermentation, eucommia ulmoides not only inhibits lateral metabolism and promotes carbon flow to monensin synthesis, but also enhances the activities of SOD, POD, and CAT, and reduces oxidative stress. This reduces membrane damage and ROS accumulation caused by lard, protects mycelia and synthases, increases the expression of key enzymes in polyether ring synthesis (such as PKS and cyclases), prolongs the high-yield period, and reduces lard leakage, improves carbon source utilization, reduces byproducts, and improves dissolved oxygen. Eucommia ulmoides upregulates the expression of key enzymes in polyether ring synthesis (such as PKS and cyclases), and GMOs and lard synergistically increase unit yield. Eucommia ulmoides also alleviates excessive growth inhibition caused by oil. Therefore, the synergistic effect of these three factors significantly increases monensin yield.

[0014] Preferably, the mass concentration of propylene glycol in the fermentation medium is 3~12 g / L.

[0015] Preferably, the mass concentration of lard in the fermentation medium is 5-8 g / L.

[0016] Preferably, the glucose concentration in the fermentation medium is 10-15 g / L, and the soybean oil concentration is 10-20 g / L. Under the emulsifying and dispersing effects of monooleate glycerol and the interfacial regulation, the lipid carbon source in the soybean oil can be better degraded by *Streptomyces cinnamon*, releasing more fatty acids and glycerol metabolic intermediates. This provides a core material basis for the generation of key precursors in the biosynthesis of monensin, thereby increasing the yield of monensin.

[0017] In some optional embodiments of the present invention, the carbon source is composed of propylene glycol, lard, glucose and soybean oil. In the fermentation medium, the mass concentration of propylene glycol is 3-12 g / L, the mass concentration of lard is 5-8 g / L, the mass concentration of glucose is 10-15 g / L, and the mass concentration of soybean oil is 10-20 g / L.

[0018] In some optional embodiments of the present invention, the nitrogen source is composed of soybean meal and yeast powder. In the fermentation culture medium, the mass concentration of soybean meal is 8-15 g / L and the mass concentration of yeast powder is 2-5 g / L.

[0019] Furthermore, the fermentation medium also includes a buffer with a mass concentration of 5-8 g / L and minerals with a mass concentration of 3.5-5 g / L; the buffer includes one or more of ammonium dihydrogen phosphate and calcium carbonate, and the minerals include one or more of potassium sulfate and sodium chloride. The minerals synergistically regulate the ion balance of the fermentation system, while the buffer stabilizes the pH and microbial enzyme activity of the fermentation system. Together, they provide a more suitable environment for fermentation, helping to increase the yield of monensin.

[0020] Preferably, the buffer is composed of ammonium dihydrogen phosphate and calcium carbonate, wherein the mass concentration of ammonium dihydrogen phosphate in the fermentation medium is 0.15-0.2 g / L and the mass concentration of calcium carbonate is 5-7 g / L.

[0021] Preferably, the mineral is composed of potassium sulfate and sodium chloride, wherein the mass concentration of potassium sulfate in the fermentation medium is 1.5-2 g / L and the mass concentration of sodium chloride is 2-3 g / L.

[0022] Furthermore, the fermentation medium also includes an activator with a mass concentration of 1-2 g / L, the activator including manganese sulfate.

[0023] The activator is a substance that can target and activate key enzymes related to monensin synthesis, thereby further increasing the yield of monensin.

[0024] Preferably, the activator is manganese sulfate, which can target and activate key enzymes related to monensin synthesis, and the mass concentration of manganese sulfate in the fermentation medium is 1-2 g / L.

[0025] Furthermore, the fermentation medium also includes a growth factor with a mass concentration of 5-8 g / L, the growth factor including corn extract.

[0026] Preferably, the growth factor is corn syrup, and the mass concentration of corn syrup in the fermentation medium is 5-8 g / L. Specific amino acids (such as valine and isoleucine) contained in corn syrup can provide trace amounts of growth factors and can also serve as precursors for the synthesis of side chains in monensin, thereby increasing the yield of monensin.

[0027] In some optional embodiments of the present invention, the fermentation culture medium comprises the following components at the following mass concentrations: glucose 10-15 g / L, soybean meal 8-15 g / L, yeast powder 2-5 g / L, potassium sulfate 1.5-2 g / L, ammonium dihydrogen phosphate 0.15-0.2 g / L, manganese sulfate 1-2 g / L, soybean oil 10-20 g / L, lard 5-8 g / L, calcium carbonate 5-7 g / L, corn syrup 5-8 g / L, sodium chloride 2-3 g / L, eucommia ulmoides extract 1-3 g / L, propylene glycol 3-12 g / L, and glyceryl monooleate 8-10 g / L. The balance is water.

[0028] By employing specific mass concentrations of components in the aforementioned fermentation medium, the synergistic effects of these components on enhancing monensin production are achieved. Specifically, glucose, corn syrup, soybean meal, yeast powder, and eucommia ulmoides serve as core nutrient sources, ensuring energy supply for the microbial growth period and supporting cell structure construction. Buffers and minerals provide a more suitable environment for monensin synthesis, while eucommia ulmoides, propylene glycol, corn syrup, soybean oil, and lard provide more precursors for monensin synthesis, significantly increasing the synthesis rate. Furthermore, the emulsifying and dispersing effects of monooleate glycerides and the activating effect of activators further promote monensin synthesis, resulting in a fermentation formula with synergistic nutrient supply, strong pH stability, and high monensin synthesis efficiency, possessing significant industrial application value.

[0029] In one optional embodiment of the present invention, the fermentation culture medium comprises the following components in mass concentrations: glucose 13 g / L, soybean meal powder 10 g / L, yeast powder 3 g / L, potassium sulfate 1.8 g / L, ammonium dihydrogen phosphate 0.15 g / L, manganese sulfate 1.5 g / L, soybean oil 15 g / L, lard 6.5 g / L, calcium carbonate 6.5 g / L, corn syrup 6.5 g / L, sodium chloride 2.5 g / L, eucommia ulmoides extract 2 g / L, propylene glycol 7.5 g / L, glyceryl monooleate 9 g / L, with the remainder being water.

[0030] Preferably, the pH of the fermentation medium is 7.0 to 7.2.

[0031] A second aspect of the present invention provides a method for preparing a fermentation medium for producing monensin as described above, comprising the following steps: (1) First, dissolve glucose, eucommia ulmoides, propylene glycol, nitrogen source, buffer, activator and growth factor in water to obtain mixture A; (2) After heating the lard, it is mixed with soybean oil and glyceryl monooleate to obtain mixture B; (3) After mixing mixture A and mixture B, adjust the pH to 6.8-7.0, and then add calcium carbonate and mix.

[0032] A third aspect of the present invention provides a method for producing monensin, comprising the following steps: inoculating the fermentation seed liquid of *Streptomyces cinnamon* into the fermentation medium as described above or the fermentation medium prepared by the preparation method as described above at an inoculation amount of 8-12% by volume, and carrying out fermentation culture to increase the yield of monensin. The fermentation conditions include a temperature of 35±0.5℃ and a time of 168~192h.

[0033] Preferably, the inoculation amount is 10%.

[0034] In this invention, the *Streptomyces cinnamon* used for fermentation to produce monensin can be a conventional *Streptomyces cinnamon* with the corresponding function. In one optional embodiment of this invention, the *Streptomyces cinnamon* ( Streptomyces cinnamonensis The strain ATCC 15413 was selected as the starting strain for monensin fermentation. This strain is publicly available. Other Streptomyces cinnamon that can ferment and produce monensin are also suitable for this invention.

[0035] Furthermore, the method for preparing the fermentation seed liquid includes: inoculating *Streptomyces cinnamon* onto a slant culture medium for slant culture to obtain activated *Streptomyces cinnamon*, and then inoculating it onto a seed culture medium for seed culture.

[0036] Furthermore, the conditions for the slant culture include: a temperature of 32±0.5℃ and a time of 144~168h.

[0037] Furthermore, the conditions for seed culture include: a culture temperature of 32±0.5℃, a shaking speed of 220~250rpm, and a culture time of 22~24h.

[0038] Furthermore, the seed culture medium comprises the following components at the following mass concentrations: glucose 5-8 g / L, soybean meal 5-10 g / L, yeast powder 3-8 g / L, calcium carbonate 1-2 g / L, with the balance being water, and a pH of 6.8-7.0.

[0039] The beneficial effects of the fermentation culture medium for monensin production, its preparation method, and its application provided by this invention are as follows: The fermentation culture medium of this invention uses simple raw materials, and the added eucommia ulmoides not only regulates the lipid metabolism of the microorganisms, but also provides more fatty acid precursors for the synthesis of monensin, thereby increasing the synthesis rate of monensin, significantly improving the potency of monensin, and increasing the yield of monensin. It has important industrial application value. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The raw material information in the following examples and comparative examples is shown in Table 1.

[0043]

[0044] Beef extract: biological reagent, specification BR250g, purchased from Aoboxing; Peptone: Biological reagent, specification BR250g, purchased from Aoboxing; Glyceryl monooleate: analytical grade, AR98%, purchased from Yuanfeng Chemical / Ron Reagent; Propylene glycol: analytical grade, AR 500ml, purchased from Hengxing Reagent; Sodium chloride: analytical grade, AR 500g, purchased from Hengxing Reagent; Potassium sulfate: analytical grade, AR 500g, purchased from Hengxing Reagent; Ammonium dihydrogen phosphate: analytical grade, AR 500g specification, purchased from Tianjin Damao; Manganese sulfate: analytical grade, AR 500g, purchased from Hengxing Reagent; Calcium carbonate: analytical grade, AR 500g specification, purchased from Hengxing Reagent; Streptomyces cinnamon: strain preservation number CPCC 240245, other preservation center number ATCC 15413.

[0045] Example 1 A fermentation medium for producing monensin comprises the following components in the indicated mass concentrations: glucose 13 g / L, soybean meal 10 g / L, yeast extract 3 g / L, potassium sulfate 1.8 g / L, ammonium dihydrogen phosphate 0.15 g / L, manganese sulfate 1.5 g / L, soybean oil 15 g / L, lard 6.5 g / L, calcium carbonate 6.5 g / L, corn syrup 6.5 g / L, sodium chloride 2.5 g / L, eucommia ulmoides 2 g / L, propylene glycol 7.5 g / L, glyceryl monooleate 9 g / L, with the balance being water, and a pH of 7.0.

[0046] This embodiment also provides a method for preparing the above-mentioned fermentation medium. The method for preparing 1L of fermentation medium comprises the following steps: (1) First, dissolve glucose, soybean meal powder, yeast powder, potassium sulfate, ammonium dihydrogen phosphate, corn syrup, sodium chloride, manganese sulfate, eucommia ulmoides and propylene glycol in 500 mL of water and stir until completely dissolved to obtain mixture A.

[0047] (2) Put the lard into a beaker and heat it in a warm water bath until it becomes completely liquid. When the liquid lard cools down to 50°C, pour in the soybean oil and stir with a spoon for 1 minute until the two are evenly blended without obvious layering. Then slowly add the monooleic glycerol while stirring to make the monooleic glycerol evenly dispersed in the oil phase to obtain mixture B.

[0048] (3) Mix mixture A and mixture B, add water to make up to 1L, stir for 5min to mix thoroughly, then adjust the pH to 6.80 with 10% NaOH, then add calcium carbonate and stir until completely and uniformly mixed to obtain a fermentation medium with pH 7.0.

[0049] This embodiment also provides a method for producing monensin, wherein a 10% (volume concentration) inoculum of *Streptomyces cinnamon* fermentation seed liquid is inoculated into the fermentation medium for fermentation culture to increase monensin yield. The specific steps are as follows: (a) Dispense the above fermentation medium into 500mL shake flasks, with a filling volume of 100mL, and autoclave at 121℃ for 30min.

[0050] (b) After cooling to 30°C, inoculate with Streptomyces cinnamon seed liquid (10% inoculation amount), place in a shaker and incubate at 35°C and 250r / min for 192h to obtain fermentation broth.

[0051] The seed culture medium consists of the following components at the following mass concentrations: glucose 6.0 g / L, soybean meal 5.0 g / L, yeast extract 3.5 g / L, calcium carbonate 1.2 g / L, with the balance being water, and a pH of 6.8. The preparation method is as follows: First, dissolve the glucose, soybean meal, and yeast extract in 1 L of water and stir until completely dissolved; then adjust the pH to 6.6 with 10% NaOH, and finally add calcium carbonate and stir until completely homogeneous to obtain the seed culture medium with a pH of 6.8.

[0052] The preparation method of the fermentation seed culture is as follows: *Streptomyces cinnamon* was inoculated onto a slant culture medium (the slant culture medium composition was: glucose 10.0 g / L, peptone 5.0 g / L, beef extract 2.0 g / L, dipotassium hydrogen phosphate 3.0 g / L, agar 20.0 g / L, dissolved in 1 L of water) and cultured at 32℃ for 168 h to obtain activated strains; then, the seed culture medium was dispensed into 500 mL shake flasks, with a volume of 100 mL, and autoclaved at 121℃ for 30 min; finally, the activated strains were added at 1.0 cm... 2 The inoculum was inoculated into the seed culture medium at a rate of / L, and cultured at 32℃, 40% humidity, and 250r / min for 24h to obtain a fermentation seed liquid with a bacterial concentration of 32% and a pH of 6.76. Microscopic examination showed no contaminants.

[0053] Examples 2-16, Comparative Examples 1-4 Examples 2-16 and Comparative Examples 1-4 are basically the same as Example 1, except that the raw material content of the fermentation medium is different, and the raw material formula is shown in Tables 2 and 3.

[0054] The preparation method of the fermentation medium is basically the same as that in Example 1. The difference is that, depending on the increase or decrease of the raw material composition, the corresponding raw materials and / or steps in the preparation method of Example 1 can be reduced or increased. This will not be elaborated further here.

[0055] The pH of the fermentation medium prepared in the above examples and comparative examples was 7.0.

[0056]

[0057]

[0058] Experimental Example Based on the fermentation medium of Comparative Example 1, the effect of fermentation mediums prepared with different concentrations of eucommia ulmoides on monensin yield was studied. The concentrations of eucommia ulmoides in the fermentation medium were 0 g / L, 0.3 g / L, 0.65 g / L, 1 g / L, 2 g / L, 3 g / L, and 6.5 g / L. The results are shown in Table 4. Table 4. Effects of different concentrations of eucommia ulmoides on monensin yield

[0059] As shown in Tables 2 and 3, the monensin potency of Examples 1-16 is significantly higher than that of Comparative Examples 1-4, especially the potency of Examples 1-9, indicating that fermentation using the specific fermentation medium of the present invention can significantly increase the yield of monensin.

[0060] As shown in Table 4, when the concentration of eucommia ulmoides is between 1 and 3 g / L, it has a better effect on improving the potency of monensin.

[0061] Further analysis of the results in Example 9, Comparative Example 2, and Table 4 (Eucomycin concentrations of 0 g / L and 2 g / L) in Table 3 shows that in the system containing propylene glycol and glyceryl monooleate, adding 2 g / L of eucomycin can increase the potency by 12000 μg / mL, while in the system without propylene glycol and glyceryl monooleate, adding 2 g / L of eucomycin only increases the potency by 6125 μg / mL. This indicates that in the system containing propylene glycol and glyceryl monooleate, eucomycin has a more significant effect on increasing the yield of monensin, suggesting a synergistic effect between eucomycin, propylene glycol, and glyceryl monooleate.

[0062] In the embodiments, comparative examples, and experimental examples of this invention, the detection method for determining the yield of monensin in the fermentation broth using high-performance liquid chromatography after fermentation is based on the "Veterinary Drug Quality Standards 2017 Edition," and the specific operation is as follows: 1. Reagents and Solutions 1.1 Mobile phase: methanol-water-glacial acetic acid (volume ratio 94:6:0.1), shake well, filter through a 0.45μm filter membrane, and sonicate for 20 min.

[0063] 1.2 Derivatization reagent: Methanol-sulfuric acid-vanillin was filtered through a 0.45 μm filter membrane and sonicated for 20 min. The preparation method of methanol-sulfuric acid-vanillin was as follows: 2.0 mL of sulfuric acid was slowly added to 95 mL of methanol along the wall of a beaker, mixed well, cooled, and then 3.0 g of vanillin was added.

[0064] 1.3 Instruments 1.3.1 Shimadzu high performance liquid chromatograph.

[0065] 1.3.2 Mobile phase filter.

[0066] 1.3.3 Filter membrane 0.45μm.

[0067] 1.3.4 Microsyringe 100μL.

[0068] 1.3.5 Sample vials.

[0069] 1.3.6 Analyze the balance.

[0070] 1.4 Chromatographic conditions 1.4.1 Chromatographic column: Octadecylsilane-bonded silica gel is used as the packing material.

[0071] 1.4.2 Post-column reaction coil: 2 mL.

[0072] 1.4.3 Derivatization reaction temperature: 98℃.

[0073] 1.4.4 Detection wavelength: 520nm.

[0074] 1.4.5 Mobile phase flow rate: 0.7 mL / min.

[0075] 1.4.6 Derivatization reagent flow rate: 0.7 mL / min.

[0076] 1.4.7 Injection volume: 20 μL.

[0077] 1.5 Sample Preparation Accurately weigh an appropriate amount of this product (approximately equivalent to 10 mg of monensin), place it in a 50 mL volumetric flask, add 40 mL of methanol solution, sonicate for 60 min, dilute to the mark with methanol to prepare a solution containing approximately 0.2 mg of monensin per mL, and shake well; filter, accurately inject 20 µL into the liquid chromatograph for isocratic elution, and record the chromatogram. Separately, accurately weigh an appropriate amount of monensin reference standard and determine it using the same method.

[0078] 1.6 Calculation The peak area is calculated using the following formula:

[0079] In the formula: i represents monensin component A, B, or C / D, respectively; A 供（i） These represent the peak areas of monensin components A, B, and C / D in the test sample, respectively. A 对（A） The peak area of ​​monensin A in the reference standard; W 对 W is the weight of the reference standard. 供 The weight of the test sample; Q 对（A） This represents the percentage content of monensin A in the reference standard. P 对 The content of monensin in the reference standard (unit / mg); D 对 This is the dilution factor of the reference standard; D 供 This is the dilution factor of the test sample.

[0080] Monensin potency (P) = A + 0.28B + 1.5C / D.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermentation medium for the production of monensin, characterized in that, The components include the following mass concentrations: carbon source 28~55 g / L, nitrogen source 10~20 g / L, and eucommia ulmoides 1~3 g / L; The carbon source includes propylene glycol, lard, glucose, and soybean oil; The mass ratio of eucommia ulmoides to propylene glycol is 1:1~12; The fermentation medium also includes glyceryl monooleate at a mass concentration of 8-10 g / L, the mass ratio of lard to glyceryl monooleate is 1:1-2, and the mass ratio of eucommia ulmoides extract to lard is 1:1.6-8. The nitrogen source includes one or more of soybean meal powder and yeast powder; The eucommia extract is an extract of Eucommia ulmoides containing chlorogenic acid, flavonoids, and total sugars; the chlorogenic acid content in the eucommia extract is ≥3.5% by mass; the flavonoid content in the eucommia extract is ≥8.0% by mass. The total sugar content in the eucommia ulmoides extract is ≥20.0% based on glucose.

2. The fermentation medium for production of monensin according to claim 1, characterized by, The fermentation medium further includes: a buffer with a mass concentration of 5-8 g / L and minerals with a mass concentration of 3.5-5 g / L; the buffer includes one or more of ammonium dihydrogen phosphate and calcium carbonate, and the minerals include one or more of potassium sulfate and sodium chloride.

3. The fermentation medium for production of monensin according to claim 2, characterized by, The fermentation medium also includes an activator with a mass concentration of 1-2 g / L, the activator including manganese sulfate.

4. The fermentation medium for producing monensin according to claim 3, characterized in that, The fermentation medium also includes a growth factor with a mass concentration of 5-8 g / L, and the growth factor includes corn extract.

5. The fermentation medium for producing monensin according to claim 4, characterized in that, The fermentation medium comprises the following components at the following mass concentrations: glucose 10–15 g / L, soybean meal 8–15 g / L, yeast powder 2–5 g / L, potassium sulfate 1.5–2 g / L, ammonium dihydrogen phosphate 0.15–0.2 g / L, manganese sulfate 1–2 g / L, soybean oil 10–20 g / L, lard 5–8 g / L, calcium carbonate 5–7 g / L, corn syrup 5–8 g / L, sodium chloride 2–3 g / L, eucommia ulmoides 1–3 g / L, propylene glycol 3–12 g / L, and glyceryl monooleate 8–10 g / L.

6. The method for preparing the fermentation medium for monensin production according to claim 4 or 5, comprising the following steps: (1) First, dissolve glucose, eucommia ulmoides, propylene glycol, nitrogen source, buffer, activator and growth factor in water to obtain mixture A; (2) After heating the lard, it is mixed with soybean oil and glyceryl monooleate to obtain mixture B; (3) After mixing mixture A and mixture B, adjust the pH to 6.8-7.0, and then add calcium carbonate and mix.

7. A method for producing monensin, characterized in that, The method includes the following steps: inoculating the fermentation seed liquid of Streptomyces cinnamon into the fermentation medium according to any one of claims 1 to 5 or the fermentation medium prepared by the preparation method according to claim 6 at an inoculation amount of 8 to 12% by volume, and carrying out fermentation culture to increase the yield of monensin. The fermentation conditions include a temperature of 35±0.5℃ and a time of 168~192h.

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