Preparation method of monensin fermentation liquor

Abstract

The invention discloses a preparation method of monensin fermentation liquor, relates to the technical field of microbial fermentation, and aims to solve the problems of low titer and long period of the existing process. According to the method, streptomyces cinnamomi is taken as a strain, a culture medium with a specific formula is adopted, and fermentation is performed in three stages: in the first stage (0-30-50 hours), pH is not controlled; in the second stage, a phosphoric acid and compound oil mixed solution (accounting for 1-3% of the fermentation solution, and the compound oil contains 20-30% of peanut oil, 20-30% of corn oil and the like) is supplemented at a time; in the third stage, the pH is controlled to be 6.2-6.3, and soybean oil and sterile water are supplemented according to needs. The fermentation adopts a secondary process, the temperature is controlled at 30-33 DEG C, the dissolved oxygen is more than or equal to 35% and the like, the total duration is less than or equal to 320 hours, and the titer is more than or equal to 55000 mu g / ml. By optimizing the culture medium and precisely regulating and controlling, the element production capacity is improved, the method is suitable for industrial production of tanks of 50 L to 100 m < 3 >, the efficiency is improved, and the cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for preparing monensin fermentation broth. Background Technology

[0002] Monensin is a polyether antibiotic first isolated in 1967 from the fermentation broth of *Streptomyces cinnamonensis*. Monensin mainly consists of four acidic components, with monensin A and monensin B being present in higher proportions, while monensin C and D are present in very small amounts. Monensin A is its main active ingredient. Monensin can react with extracellular Na+. + K + Monensin binds to monovalent cations, carrying them into the cell and disrupting the osmotic pressure across the cell membrane, ultimately leading to cell death. This unique mechanism of action gives it broad-spectrum insecticidal activity and slows the development of resistance. Monensin can also significantly improve nutrient utilization in ruminants and can be used as a food additive. With its green, efficient, and safe characteristics, monensin is widely used in medicine, agriculture, and other fields, and there is strong market demand.

[0003] In recent years, domestic and international research has mainly focused on strain selection, improving mycelial morphology, and increasing dissolved oxygen. While these methods have made some progress, long-term use of a single mutagen may lead to low mutation rates and mutagenic fatigue, and the lack of precise control over the fermentation process limits the potential for potency improvement and results in long fermentation times, making it difficult to meet the dual requirements of efficiency and cost in industrial production. Therefore, there is an urgent need for a comprehensive technical solution capable of precise fermentation process control to overcome these bottlenecks, significantly improve the fermentation potency and production efficiency of monensin, and thus better meet market demands.

[0004] In recent years, domestic and international research has mainly focused on strain selection, improving mycelial morphology, increasing dissolved oxygen, and adding precursor substances. While these methods have made some progress, long-term use of a single mutagen may lead to low mutation rates and mutagenic fatigue, and the lack of precise control over the fermentation process limits the potential for potency improvement, making it difficult to meet the dual requirements of efficiency and cost in industrial production. Therefore, there is an urgent need for a comprehensive technical solution that can optimize culture medium formulation and refine fermentation process control to overcome these bottlenecks, significantly improve the fermentation potency and production efficiency of monensin, and thus better meet market demands. Summary of the Invention

[0005] In view of the above problems, the present invention is proposed to provide a method for preparing monensin fermentation broth for large-scale production that overcomes or at least partially solves the above problems, wherein the potency of monensin can reach up to 55000 μg / mL.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a method for preparing monensin fermentation broth, the method comprising the following steps: (1) Using *Streptomyces cinnamon* as the inoculum, fermentation was carried out in a culture medium with a specific composition; (2) During the first stage of fermentation, before the addition of phosphate mixture, the pH is not controlled; (3) In the second stage of fermentation, a mixture of phosphoric acid and compound oil is added once according to the pH; (4) In the third stage of fermentation, when pH < 6.2, add 10% ammonia water to adjust the pH to 6.2~6.3. If pH > 6.3, add 0.1% phosphoric acid.

[0007] In a specific embodiment, the present invention provides a method for preparing monensin fermentation broth, wherein the culture medium comprises: 100 parts by weight of water, 1-1.5 parts by weight of glucose, 1-2 parts by weight of soybean meal, 0.2-0.3 parts by weight of yeast extract, 3.5-4.5 parts by weight of soybean oil, 0.2-0.4 parts by weight of light calcium carbonate, 0.2-0.4 parts by weight of ammonium sulfate, 0.2-0.4 parts by weight of sodium sulfate, 0.01-0.03 parts by weight of potassium dihydrogen phosphate, 0.01-0.03 parts by weight of ferrous sulfate heptahydrate, 0.04-0.07 parts by weight of lard, and 0.02-0.04 parts by weight of defoamer.

[0008] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth. In step (2), the fermentation time of the first stage is 0-30~50h; in step (3), the fermentation time of the second stage is when the first stage ends; in step (4), the fermentation time of the third stage is from the beginning of the second stage to the end of the fermentation. The total fermentation time is controlled within 320h, the potency is ≥55000μg / ml, the mycelium shows vacuoles and more breaks, and the potency growth slows down.

[0009] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth. In step (3), the volume of the mixed liquid is 1-3% of the volume of the fermentation broth, wherein the compound oil accounts for 3-6% of the volume of the mixed liquid, and the phosphoric acid accounts for 0.4-1% of the volume of the mixed liquid. The compound oil is composed of peanut oil, corn oil, olive oil and flaxseed oil in a 20-30% ratio.

[0010] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth. In step (4), soybean oil needs to be added and the soybean oil content is controlled to account for 1-3% of the total volume of the fermentation broth.

[0011] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth. During the fermentation process, when the volume decreases by more than 5% due to water evaporation, or when the bacterial concentration is >55% or the dissolved oxygen is <30%, sterile water is added to account for 2%-3% of the total mass of the fermentation broth.

[0012] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth. The fermentation adopts a two-stage fermentation process, and the fermentation conditions are: temperature 30~33℃, air flow rate 0.2~1vvm, stirring speed 25~50 Hz, tank pressure 0.03~0.08MPa, and dissolved oxygen not less than 35%.

[0013] In a specific implementation, the present invention provides a method for preparing monensin fermentation broth, wherein in step (4), the pH is controlled within the range of 6.2-6.3 during the third stage of fermentation.

[0014] The second aspect of the present invention provides an industrial-scale production of monensin fermentation broth, including scale-up production of 50L fermenters, 200L fermenters and 100m³ fermenters.

[0015] Through the above technical solution, the present invention has at least the following beneficial technical effects: (1) By employing the fermentation medium and feeding process of this invention, and through the use of an optimized medium formulation, combined with a precise feeding strategy and phosphate mixture addition, the rate of cell metabolism and synthesis is effectively regulated, enhancing the mycelial production capacity in the later stages of fermentation. This process enables the final fermentation potency of monensin to reach over 55,000 u / mL, with a fermentation time of less than 320 hours. Compared with traditional monensin fermentation, this significantly shortens the production time, improving equipment utilization and production efficiency, and reducing the production cost per unit time.

[0016] (2) The addition of soybean oil, lard, and a complex fatty acid mixture consisting of peanut oil, corn oil, olive oil, and flaxseed oil to the culture medium provides a diverse range of fatty acid precursors. This helps to synthesize monensin more efficiently.

[0017] This invention is applicable to the industrial-scale fermentation production of monensin. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The diagram shows the structures of monensin A and monensin B. Detailed Implementation

[0020] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0021] The strain used in the following examples is *Streptomyces cinnamon*, and the strain used in production was sourced from the strain laboratory.

[0022] The method for preparing monensin fermentation broth provided in this application includes: A fermentation culture medium formula for monensin production, comprising: 100 parts by weight of water, 1-1.5 parts by weight of glucose, 1-2 parts by weight of soybean meal, 0.2-0.3 parts by weight of yeast extract, 3.5-4.5 parts by weight of soybean oil, 0.2-0.4 parts by weight of light calcium carbonate, 0.2-0.4 parts by weight of ammonium sulfate, 0.2-0.4 parts by weight of sodium sulfate, 0.01-0.03 parts by weight of potassium dihydrogen phosphate, 0.01-0.03 parts by weight of ferrous sulfate heptahydrate, 0.04-0.07 parts by weight of lard, and 0.02-0.04 parts by weight of defoamer.

[0023] The fed-feed method for producing monensin by fermentation using the above-mentioned culture medium is characterized by the following: the feeding is carried out by a fed-feed method during the fermentation process, and the feeding includes the addition of phosphoric acid, oil, water, defoaming agent, and pH control.

[0024] a. Phosphoric acid supplementation solution: During the fermentation process, when fermentation has lasted 45 hours, a phosphate compound oil mixture of 1-3% of the fermentation volume is added. The compound oil accounts for 3-6% of the volume, and the phosphate accounts for 0.4-1%. The compound oil is a mixture of peanut oil, corn oil, olive oil, and flaxseed oil in a 20-30% ratio.

[0025] b. Hydration: When the fermentation time is from 45 hours to the end of fermentation, if the amount of water evaporates excessively and the fermentation volume decreases excessively, sterile water can be added. If the bacterial concentration is higher than 55% or the dissolved oxygen is lower than 30%, 2 to 3 tons of water can also be added appropriately.

[0026] c. Add defoamer: A large amount of foam will be produced during the middle stage of fermentation. If necessary, add 0.1-0.5% defoamer to control it.

[0027] d. Control pH: pH does not need to be controlled before 30-50 hours of fermentation.

[0028] If the pH is less than 6.2 when the fermentation time is 50-320 h, add 10% ammonia water to adjust the pH to 6.2-6.3. If the pH is greater than 6.3, add 0.1% phosphoric acid.

[0029] e. Add soybean oil: After 80 hours of fermentation, soybean oil is added. The amount of soybean oil added is adjusted to control the soybean oil content to 1-3%.

[0030] The invention utilizes a fermentation culture medium that has been confirmed to be suitable for the fermentation production of monensin, and uses a fed-batch method as the control method. Combined with the monensin fermentation control processes published at home and abroad, the invention ultimately achieves a fermentation potency of monensin of more than 55,000 u / ml, and controls the fermentation time to within 320 h.

[0031] Example 1

[0032] This example is 100m 3 Fermentation experiment.

[0033] 100 m 3 Fermentation tank formula: 1500kg glucose, 1500kg soybean meal, 200kg yeast extract, 4000kg soybean oil, 300kg light calcium carbonate, 300kg ammonium sulfate, 300kg sodium sulfate, 20kg potassium dihydrogen phosphate, 20kg ferrous sulfate heptahydrate, 50kg lard, and 40kg defoamer.

[0034] After sterilizing and cooling the prepared fermentation medium, pressurize it with sterile air and transfer all the seed liquid from the secondary seed tank into the fermenter for cultivation. Fermentation conditions are as follows: cultivation temperature controlled at 30-35℃; air flow rate of 0.2-0.4 v / v / min during fermentation 0-30 h, and 0.3-1.0 v / v / m from 31 h to the end of fermentation; dissolved oxygen not lower than 35%; stirring speed of 25-35 Hz during fermentation 0-30 h, and 30-50 Hz from 31 h to the end of fermentation; tank pressure controlled at 0.03-0.08 MPa; microscopic examination during fermentation is required to ensure the absence of contaminating microorganisms; pH control is not necessary for the first 45 h of fermentation, and the pH is adjusted to 6.2-6.3 during fermentation 46-320 h; fermentation is stopped when the cycle is ≤320 h, the titer is ≥55000 u / ml, vacuoles appear in the mycelium, breakage increases, and the titer increase slows down, at which point the culture is removed from the fermenter.

[0035] During fermentation, a fed-batch method is used for feeding, which includes feeding phosphoric acid, oil, water, defoamer, and pH control. The specific process is as follows: First stage of fermentation: Fermentation time is about 0~45 hours, and pH is not controlled.

[0036] In the second stage of fermentation, after 45 hours of fermentation, a mixture of phosphoric acid and compound oil is added once. This mixture includes 5% compound oil and 0.8% phosphoric acid. The compound oil consists of 25% peanut oil, 25% corn oil, 25% olive oil, and 25% flaxseed oil. The total volume of the added liquid is 2% of the fermentation liquid volume.

[0037] In the third stage of fermentation, after replenishing the mixed solution, if the pH is <6.2, add 10% ammonia water to adjust the pH to 6.2~6.3; if the pH is >6.3, add 0.1% phosphoric acid.

[0038] After 80 hours of fermentation, soybean oil is added, with the soybean oil content controlled at 2%. When the soybean oil content is higher than 3%, no more soybean oil is added. When the soybean oil content is lower than 1%, soybean oil is added to account for 1% of the fermentation liquid volume.

[0039] To compensate for the water evaporated during fermentation and maintain the volume of the fermentation broth, when water evaporation causes a volume decrease of more than 5%, or the bacterial concentration is >55%, or the dissolved oxygen is <30%, add sterile water at a concentration of 2.5% of the total fermentation broth mass. When foam exceeds 1% of the total volume, add an antifoaming agent at a concentration of 0.3% of the total fermentation broth mass to eliminate the foam. Fermentation culture completed: bacterial concentration 46%, monensin titer 55872 u / ml, fermentation period 310 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0040] The relevant data in the embodiments are shown in Table 1.

[0041] Table 1 100m 3 Fermentation tank experimental data

[0042] Example 2

[0043] The fermentation medium consists of the following components: a 50 L fermenter with 35 L of water added; the medium formula is as follows: 350 g glucose, 525 g soybean meal, 85 g yeast extract, 1400 g soybean oil, 87.5 g light calcium carbonate, 105 g ammonium sulfate, 105 g sodium sulfate, 3.5 g potassium dihydrogen phosphate, 3.5 g ferrous sulfate heptahydrate, 17.5 g lard, and 10.5 g defoamer.

[0044] First, mix the soybean meal powder with 1L of water, then boil it using an induction cooker before mixing it into the culture medium.

[0045] Adjust the pH to 6.8 using 6M sodium hydroxide, and then add the light calcium carbonate from the culture medium formula.

[0046] After sterilizing and cooling the prepared fermentation medium, pressurize it with sterile air, and inoculate the seed liquid in the seed tank into the fermenter at a 10% inoculation rate for culture, for a total of two-stage fermentation.

[0047] Fermentation conditions are as follows: culture temperature controlled at 32℃; initial air flow rate of 0.2 vvm; stirring speed of 200 rpm; tank pressure controlled at 0.04 MPa; increase air flow rate and stirring according to dissolved oxygen, not lower than 35%; microscopic examination is performed during fermentation, and no contaminants are required.

[0048] The feeding strategy during fermentation is the same as in Example 1, except for the following proportion of phosphate mixture.

[0049] (2) The experimental group design is as follows: Blank group: The above experimental method was used, but without adding phosphoric acid solution.

[0050] Experimental Group 1: Using the above experimental method, add 700ml of 3% compound oil and 0.4% phosphoric acid as supplementary solution 1.

[0051] Experimental Group 2: Using the above experimental method, add 700ml of 4% compound oil and 0.6% phosphoric acid as supplementary solution 2.

[0052] Experimental Group 3: Using the above experimental method, add 700ml of 5% compound oil and 0.8% phosphoric acid to make supplement solution 3.

[0053] Experimental Group 4: Using the above experimental method, add 700ml of 6% compound oil and 1% phosphoric acid to make supplement solution 4.

[0054] After fermentation was completed, microscopic examination revealed that the mycelium staining was unclear, the mycelium was swollen and ruptured, which met the requirements for discharging into the tank.

[0055] The specific data in Example 2 are shown in Table 2.

[0056] Table 2. Mixtures of different phosphoric acid compound oils

[0057] As shown in Table 2, the potency of monensin fermentation broth was significantly improved after the addition of phosphate mixture, with supplement solution 3 being the best choice.

[0058] Example 3

[0059] This embodiment investigates the replenishment time of the phosphate-oil mixture during fermentation: (1) The fermentation method is the same as in Example 1.

[0060] (2) The experimental group design is as follows: Experimental Group 1: Using the above experimental method, a mixture of phosphate compound oil was added after 30 hours of fermentation.

[0061] Experimental Group 2: Using the above experimental method, a mixture of phosphate compound oil was added after 35 hours of fermentation.

[0062] Experimental Group 3: Using the above experimental method, a mixture of phosphate compound oil was added after 40 hours of fermentation.

[0063] Experimental Group 4: Using the above experimental method, a mixture of phosphate compound oil was added after 45 hours of fermentation.

[0064] Experimental Group 5: Using the above experimental method, a mixture of phosphate compound oil was added after 50 hours of fermentation.

[0065] After fermentation was completed, microscopic examination revealed that the mycelium staining was unclear, the mycelium was swollen and ruptured, which met the requirements for discharging into the tank.

[0066] The specific data for Example 3 are shown in Table 3.

[0067] Table 3. Replenishment fluid addition time

[0068] As shown in Table 3, the optimal time to replenish the mixture is at 45 hours.

[0069] Example 4

[0070] This embodiment explores the control of pH during fermentation.

[0071] (1) The fermentation method is the same as in Example 1.

[0072] (2) The experimental group design is as follows: Experimental Group 1: Using the above experimental methods, the pH was controlled at 6.0~6.1 after fermentation.

[0073] Experimental Group 2: Using the above experimental methods, the pH was controlled at 6.2~6.3 after fermentation.

[0074] Experimental Group 3: Using the above experimental methods, the pH was controlled at 6.4~6.5 after fermentation.

[0075] Experimental Group 4: Using the above experimental methods, the pH was controlled at 6.6~6.7 after fermentation.

[0076] Experimental Group 5: Using the above experimental methods, the pH was controlled at 6.8~6.9 after fermentation.

[0077] After fermentation was completed, microscopic examination revealed that the mycelium staining was unclear, the mycelium was swollen and ruptured, which met the requirements for discharging into the tank.

[0078] The corresponding data in Example 4 are shown in Table 4.

[0079] Table 4. Fermentation titers at different pH levels

[0080] As shown in Table 4, the optimal pH for monensin fermentation is between 6.2 and 6.3.

[0081] Example 5

[0082] This embodiment explores the pilot-scale amplification experiment from a 50L fermenter to a 200L fermenter.

[0083] Fermentation method: 200 L fermenter, liquid volume 140 L, other conditions same as in Example 1. 10% of the solution was inoculated from a 50 L seed tank into the fermenter.

[0084] Fermentation culture completed: bacterial concentration 47%, monensin titer 55112 u / ml, fermentation period 309 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0085] The specific data in Example 5 are shown in Table 5.

[0086] Table 5 Experimental data for a 200L fermenter Culture cycle (h) 21 45 69 117 165 213 261 309 pH 6.94 7.22 6.24 6.26 6.21 6.22 6.27 6.27 Total sugar (g / 100ml) 7.1 5.1 4.3 2.2 0 0 0 0 Amino nitrogen (mg / 100ml) 48 31 35 35 28 31 28 32 Bacterial concentration (%) 21 35 41 42 43 47 47 47 Monensin potency (u / ml) 0 4123 8178 16055 27966 40261 49636 55112 Comparative Example 1: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that lard is not added to the fermentation medium.

[0087] Fermentation culture completed: bacterial concentration 46%, monensin titer 43298 u / ml, fermentation period 310 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0088] The specific contents and process parameters for Comparative Example 1 are shown in Table 6.

[0089] Because the culture medium lacks lard, it lacks the fatty acids required for monensin fermentation, resulting in a lower potency.

[0090] Table 6 100m 3 Fermentation tank experimental data

[0091] Comparative Example 2: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that the pH of the fermentation medium is not controlled.

[0092] Fermentation culture completed: bacterial concentration 48%, monensin potency 37649 u / ml, fermentation period 309 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0093] The specific contents and process parameters for Comparative Example 2 are shown in Table 7.

[0094] Because the pH was not controlled during fermentation, the potency was low.

[0095] Table 7 100m 3 Fermentation tank experimental data

[0096] Comparative Example 3: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that the dissolved oxygen in the fermentation medium is controlled at around 20%.

[0097] Fermentation culture completed: bacterial concentration 46%, monensin potency 40182 u / ml, fermentation period 309 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0098] The specific contents and process parameters for Comparative Example 3 are shown in Table 8.

[0099] Because the pH was not controlled during fermentation, the potency was low.

[0100] Table 8 100m 3 Fermentation tank experimental data

[0101] Comparative Example 4: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that no soybean oil is added during the fermentation process.

[0102] Fermentation culture completed: bacterial concentration 46%, monensin potency 25182 u / ml, fermentation period 261 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0103] The specific contents and process parameters for Comparative Example 4 are shown in Table 9.

[0104] Because no soybean oil was added during the fermentation process, the nutrients in the fermentation liquid were depleted, resulting in low potency.

[0105] Table 9 100m 3 Fermentation tank experimental data Culture cycle (h) 21 45 69 117 165 213 261 pH 7.12 7.35 6.21 6.21 6.27 6.20 6.29 Total sugar (g / 100ml) 6.1 4.1 3.3 1.0 0 0 0 Amino nitrogen (mg / 100ml) 38 38 28 40 40 48 48 Bacterial concentration (%) 24 38 41 43 44 45 46 Monensin potency (u / ml) 0 4864 8901 15235 23726 24901 25182 Comparative Example 5: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that the added phosphoric acid mixture contains 50% peanut oil and 50% corn oil.

[0106] Fermentation culture completed: bacterial concentration 47%, monensin titer 44927 u / ml, fermentation period 309 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0107] The specific contents and process parameters for Comparative Example 5 are shown in Table 10.

[0108] Because the added blend of oils lacks olive oil and flaxseed oil, it lacks some precursors for the synthesis of monensin, resulting in a lower potency.

[0109] Table 10 100m 3 Fermentation tank experimental data

[0110] Comparative Example 6: Fermentation method: 100m 3 The fermentation tank method is the same as in Example 1. The difference is that the added phosphoric acid mixture contains 50% olive oil and 50% flaxseed oil.

[0111] Fermentation culture completed: bacterial concentration 47%, monensin titer 51278 u / ml, fermentation period 309 h. Microscopic examination revealed unclear mycelial staining, mycelial swelling, and lysis, meeting the requirements for tank placement.

[0112] The specific contents and process parameters for Comparative Example 6 are shown in Table 11.

[0113] Because the added blend of oils lacked peanut oil and corn oil, it lacked some precursors for the synthesis of monensin, resulting in a lower potency.

[0114] Table 11 100m 3 Fermentation tank experimental data

[0115] As shown in Table 2-4, the addition of phosphate compound oil mixture, feeding time, and precise pH control during fermentation have a decisive impact on the fermentation potency of monensin.

[0116] The synergistic effect of phosphoric acid and compound oils is crucial. As shown in Table 2, compared with the control group, the addition of phosphoric acid and compound oil mixture significantly improved the potency of monensin. Among them, supplement solution 3 showed the best effect, with a potency of 55128 u / ml, indicating that phosphoric acid and a specific ratio of compound oil (a mixture of peanut oil, corn oil, olive oil, and flaxseed oil) have a synergistic effect in providing synthetic precursors and maintaining cell metabolic activity.

[0117] Precise pH control is key to efficient synthesis: Table 4 clearly shows that controlling the pH during fermentation within the range of 6.2-6.3 yields the highest potency. Deviations from this optimal range, especially when the pH rises above 6.6, result in a sharp decrease in potency, demonstrating that this pH environment is most favorable for the glycogenotoxic metabolic activities of *Streptomyces cinnamon*.

[0118] In summary, the preparation methods of Examples 2-4 of this invention successfully achieved efficient production of monensin by optimizing the formulation of the phosphate-oil mixture, the feeding time, and the precise control of the pH during fermentation. Example 4 (200L tank) and Example 1 (100m... 3 The preparation method of the (tank) further demonstrates that the process has good scalability, and can stably achieve excellent indicators such as potency exceeding 55,000 u / mL and fermentation cycle controlled within 320 hours in large-scale production, providing a reliable technical solution for industrial production. 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for the preparation of a monensin fermentation broth, characterized in that, The method comprises the following steps: (1) using streptomyces cinnamoneus as a strain, and fermenting in a culture medium with a specific composition; (2) in the first stage of fermentation, the pH is not controlled before a phosphate and compound oil mixture is added; (3) in the second stage of fermentation, the phosphate and compound oil mixture is added at one time; (4) in the third stage of fermentation, when the pH is less than 6.2, 10% ammonia water is added to adjust the pH to 6.2-6.3, and if the pH is greater than 6.3, 0.1% phosphoric acid is added.

2. The method for preparing a monensin fermentation broth according to claim 1, characterized by, In step (1), the culture medium comprises 100 parts by mass of water, 1-1.5 parts by mass of glucose, 1-2 parts by mass of soybean cake powder, 0.2-0.3 parts by mass of yeast extract, 3.5-4.5 parts by mass of soybean oil, 0.2-0.4 parts by mass of light calcium carbonate, 0.2-0.4 parts by mass of ammonium sulfate, 0.2-0.4 parts by mass of sodium sulfate, 0.01-0.03 parts by mass of potassium dihydrogen phosphate, 0.01-0.03 parts by mass of ferrous sulfate heptahydrate, 0.04-0.07 parts by mass of lard, and 0.02-0.04 parts by mass of an antifoaming agent.

3. The method of preparing a monensin fermentation broth according to claim 1, characterized in that, In step (2), the first stage of fermentation lasts for 0-30-50 hours; in step (3), the second stage of fermentation starts at the end of the first stage; in step (4), the third stage of fermentation starts at the beginning of the second stage and ends at the end of fermentation; the total fermentation time is controlled to be less than 320 hours, the titer is greater than or equal to 55,000 μg / ml, the mycelium has increased vacuoles and breaks, and the titer growth is slow.

4. The method for preparing a monensin fermentation broth according to claim 1 or 3, characterized by, In step (3), the phosphate and compound oil mixture is added in a volume of 1-3% of the fermentation liquid, wherein the compound oil accounts for 3-6% of the volume of the mixture, and the phosphate accounts for 0.4-1% of the volume of the mixture; the compound oil is a mixture of peanut oil, corn oil, olive oil, and linseed oil in a ratio of 20-30% each.

5. The method of preparing a monensin fermentation broth according to claim 1 or 3, characterized in that, In step (4), soybean oil needs to be added, and the amount of soybean oil added is adjusted by observing the oil layer of the fermentation liquid after centrifugation to control the content of soybean oil in the fermentation liquid to be 1-3% of the total volume of the fermentation liquid.

6. The method for preparing a monensin fermentation broth according to claim 1 or 3, characterized by, During fermentation, when the volume decreases by more than 5% due to water evaporation, or the bacterial concentration is greater than 55%, or the dissolved oxygen is less than 30%, sterile water is added to account for 2-3% of the total mass of the fermentation liquid.

7. The method of preparing a monensin fermentation broth according to claim 1 or 2, characterized in that, The fermentation adopts a two-stage fermentation process, and the fermentation conditions are as follows: the temperature is 30-33 ℃, the air flow is 0.2-1 vvm, the stirring speed is 25-50 Hz, the tank pressure is 0.03-0.08 MPa, and the dissolved oxygen is not less than 35%.

8. The method of preparing a monensin fermentation broth according to claim 1, characterized in that, In step (4), in the third stage of fermentation, the pH control range is 6.2-6.

3.

9. The process for the preparation of a monensin fermentation broth according to any one of claims 1 to 8, characterized in that, The method is suitable for industrialized large-scale production, including 50 L fermentors, 200 L fermentors, and 100 m³ fermentors.

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