A fermenting medium for kasugamycin and a method for fermenting kasugamycin
By optimizing the fermentation medium formula and fermentation parameters of kasugamycin, using soybean oil and enzymatically hydrolyzed maltodextrin as carbon sources, and combining a targeted feeding strategy, the problems of low potency and high viscosity in traditional kasugamycin fermentation were solved, resulting in a significant improvement in bacterial concentration and potency, making it suitable for industrial production.
Patent Information
- Application Number
- CN202610801421.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-04
- Publication Date
- 2026-07-10
AI Technical Summary
Traditional kasugamycin fermentation processes suffer from problems such as low fermentation potency, short cell growth cycle, short cytokine production period, high fermentation broth viscosity, and poor dissolved oxygen efficiency, making it difficult to improve the level of industrial production of kasugamycin.
The fermentation medium formula for kasugamycin was optimized by using soybean oil and enzymatically hydrolyzed maltodextrin as carbon sources, combined with the addition of soybean meal and inositol in the middle and late stages. Fermentation parameters such as temperature, pH, tank pressure, aeration ratio and stirring speed were controlled, and the feed medium was added in a targeted manner.
It significantly improved the fermentation potency of kasugamycin, increasing the bacterial concentration by 16.04% and the potency from 14,500 u/ml to 19,600 u/ml. It also prolonged the production period, reduced the viscosity of the fermentation broth, improved dissolved oxygen capacity, and reduced the use of defoamers, making it suitable for large-scale industrial production.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of kasugamycin production technology, specifically relating to a kasugamycin fermentation culture medium and a kasugamycin fermentation method. Background Technology
[0002] Kasugamycin is an aminoglycoside agricultural antibiotic produced by the fermentation of Streptomyces simonii. It has the characteristics of strong systemic activity, broad antibacterial spectrum, and safety to crops. It has good control effect on a variety of fungal and bacterial diseases such as rice blast, tomato leaf mold, and beet brown spot. It is an environmentally friendly biological pesticide widely used in agricultural production.
[0003] Currently, the industrial production of kasugamycin mainly adopts microbial fermentation. However, traditional fermentation processes generally suffer from problems such as low fermentation potency, short cell growth cycle, short product production period, high viscosity of fermentation broth, and poor dissolved oxygen efficiency.
[0004] Traditional culture medium formulations often rely on a single, fast-acting carbon and nitrogen source, leading to excessively rapid cell growth in the early stages and premature senescence in the later stages, resulting in an imbalance in carbon and nitrogen metabolism and limiting product synthesis efficiency. Simultaneously, the large amount of foam generated during fermentation necessitates the addition of substantial amounts of antifoaming agents, which can inhibit cell metabolism. Furthermore, maltodextrin, without enzymatic hydrolysis, is difficult to utilize efficiently. An improperly proportioned soybean oil cannot effectively control viscosity and energy supply. The lack of targeted supplementation of key precursors such as inositol further hinders significant improvements in overall fermentation levels, impacting the synthesis and accumulation of kasugamycin and restricting its industrial production. Summary of the Invention
[0005] The purpose of this invention is to provide a kasugamycin fermentation culture medium and a kasugamycin fermentation method, in order to solve the problem mentioned in the background art that the existing kasugamycin fermentation synthesis level is poor, which restricts the industrial production of kasugamycin.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] A kasugamycin fermentation medium comprises a basal fermentation medium and a fed culture medium. By volume percentage, the basal fermentation medium consists of the following components: 5-8% soybean meal, 0.1-0.5% monosodium glutamate, 0.26-0.5% potassium dihydrogen phosphate, 0.25-0.5% corn steep liquor, 7-9% soybean oil, and 1-3% maltodextrin. The fed culture medium contains: 0.5-1.6% soybean meal and 0.1-0.3% inositol.
[0008] Preferably, the fermentation basal culture medium formula is: 8% soybean meal, 0.2% sodium glutamate, 0.4% potassium dihydrogen phosphate, 0.4% corn steep liquor, 8.0% soybean oil, and 2% maltodextrin.
[0009] Preferably, the feed culture medium is formulated as follows: 1.0% soybean meal powder and 0.2% inositol.
[0010] A method for fermenting kasugamycin includes the following steps:
[0011] S1. Culture medium preparation: Prepare the fermentation basal culture medium and feed culture medium according to the proportions;
[0012] The maltodextrin needs to undergo enzymatic pretreatment: after dissolving in water, heat to 58-60℃, add 1.5-2.5% saccharifying enzyme, stir and maintain for 2-6 hours, then heat to 85-90℃ and add the remaining materials for sterilization, and adjust the pH to 6.7-6.8.
[0013] The pH of the basal fermentation medium was adjusted to 6.7-6.8 using liquid ammonia, while the pH of the fed medium was left at its natural state.
[0014] S2. Sterilization: Both the fermentation basal medium and the fed medium were sterilized at 121-125℃ under pressure for 30 minutes.
[0015] S3. Inoculation: After sterilization, cool to 28-29℃ and inoculate with *Streptomyces simonii* at a rate of 5 vol%-15 vol%.
[0016] S4. Fermentation culture: control the temperature at 28-29℃, the tank pressure at 0.05MPa, the aeration ratio at 1:0.5-1:1.2, the stirring speed at 100-300rpm, the pH at 6.7-6.8, and the fermentation cycle at 200±24h.
[0017] S5. Feeding: When the cell concentration increases slowly or stops increasing after 90-100 hours of fermentation, start feeding the culture medium and continue feeding for 50 hours.
[0018] S6 Collection: After fermentation is complete, collect the fermentation products.
[0019] Preferably, in the maltodextrin pretreatment, the constant temperature stirring time at 58-60℃ is 5 hours, and the amount of saccharifying enzyme added is 2.0%.
[0020] Compared with the prior art, the beneficial effects of the present invention are:
[0021] This invention optimizes the basal culture medium formula, specifically including the ratio of soybean oil to maltodextrin and the enzymatic pretreatment of maltodextrin, and combines the addition of soybean meal and inositol in the middle and late stages, thereby increasing the fermentation potency of kasugamycin from approximately 14,500 u / ml in the traditional process to over 19,600 u / ml, effectively increasing the potency.
[0022] This invention uses soybean oil and enzymatically hydrolyzed maltodextrin as delayed-release carbon sources. The triglycerides in soybean oil are gradually broken down by the microorganisms into fatty acids and glycerol, providing a continuous energy source for mycelial growth. The enzymatically hydrolyzed maltodextrin can be rapidly utilized by the microorganisms and continuously provide a carbon skeleton over a long period. The synergistic effect of these two sources effectively improves mycelial vitality, slows down the aging rate of the microorganisms in the later stages of fermentation, thereby extending the synthesis cycle of kasugamycin and increasing the yield per unit volume.
[0023] This method avoids the problem of increased fermentation broth viscosity and impaired dissolved oxygen transfer caused by an improper carbon-to-nitrogen ratio in traditional fermentation processes. It significantly reduces fermentation broth viscosity, improves oxygen supply, and promotes cell growth and the synthesis of secondary metabolites. It also reduces the amount of defoamer used, minimizing its negative impact on cell metabolism.
[0024] Furthermore, all parameters described in this invention (temperature, pH, tank pressure, aeration ratio, stirring speed, feeding timing, etc.) are within the controllable range of conventional industrial fermentation equipment, making operation simple, reproducible, and easy to scale up from laboratory scale to industrial production. Detailed Implementation
[0025] The technical solutions 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0026] Example 1:
[0027] A method for fermenting and culturing kasugamycin includes the following steps:
[0028] Culture medium preparation: Fermentation basal culture medium (volume percentage): soybean meal 8%, sodium glutamate 0.2%, potassium dihydrogen phosphate 0.4%, corn steep liquor 0.4%, soybean oil 8.0%, maltodextrin 2%.
[0029] Maltodextrin pretreatment: Add water to the tank, add maltodextrin and stir until completely dissolved, heat to 58~60℃, add 2.0% saccharifying enzyme, maintain 58~60℃ and stir for 5 hours, then heat to 85~90℃, add the remaining materials and mix, make up to volume with tap water, and adjust the pH to 6.7~6.8 with liquid ammonia.
[0030] Prepare the supplemental culture medium (volume percentage): 1.0% soybean meal, 0.2% inositol, and bring to a final volume with tap water. pH is natural.
[0031] Sterilization: Both the fermentation basal medium and the feed medium were sterilized at 121~125℃ under pressure for 30 minutes.
[0032] Inoculation: After sterilization, cool to 28~29℃ and inoculate with Streptomyces simonii at a volume of 10 vol.
[0033] Fermentation culture: Control conditions: temperature 28~29℃, tank pressure 0.05MPa, aeration ratio 1:0.8, stirring speed 200rpm, pH 6.7~6.8 (adjusted with liquid ammonia), fermentation cycle 200h.
[0034] Feeding operation: When the cell concentration growth slows down after 96 hours of fermentation, feed medium is added continuously for 50 hours and then feeding is stopped.
[0035] Fermentation results: After fermentation, the kasugamycin potency reached 19600 u / ml, which is 35% higher than the 14500 u / ml of the traditional process.
[0036] Example 2: (Maltoidin enzymatic hydrolysis control example)
[0037] Based on the principle of no supplemental culture medium, the basic soybean oil content was 7.0%. Except for the maltodextrin, which was not enzymatically pretreated, the other formulations and process parameters were the same as in Example 1.
[0038] Fermentation results: highest bacterial concentration 35.5%, titer 14500u / ml after 200h.
[0039] The experimental data are as follows:
[0040] Experiment on enzymatic hydrolysis and non-enzymatic hydrolysis of maltodextrin:
[0041] No enzymatic hydrolysis Enzymatic hydrolysis highest bacterial concentration 35.5% 41.2% Bacterial concentration difference +16.05% Potency of stopping the can (u / ml) 200h-14500 200h-15050 Valence Difference Rate +3.79%
[0042] Experiments showed that enzymatic hydrolysis of maltodextrin helped increase the bacterial cell count and potency. The bacterial concentration increased from 35.5% to 41.2%, an increase of 16.05%, and the potency increased from 14500u / ml to 15050u / ml, an increase of 3.79%.
[0043] The maltodextrin hydrolyzed by the present invention can be rapidly utilized by the microorganisms and can continuously provide carbon skeleton over a long period of time. The principle is that the saccharifying enzyme can gradually convert the glucose units at the end of the oligosaccharide chain in the maltodextrin into small glucose molecules, which are more easily absorbed and utilized by the microorganisms.
[0044] The amount of saccharifying enzyme added directly affects the release rate and concentration of glucose. If the amount is too high, it can easily cause glucose inhibition and metabolic overflow, inhibiting bacterial growth. If the amount is too low, the glucose supply will be insufficient, resulting in slow growth and reduced yield of bacteria due to lack of carbon source.
[0045] If the enzymatic hydrolysis time is too short, the maltodextrin will not be completely hydrolyzed, resulting in low utilization, high viscosity of the liquid, low nutrient supply efficiency, difficulty in filtration in the later stage, and high unit consumption. If the enzymatic hydrolysis time is too long, the enzyme activity will decrease, the Maillard reaction will occur, the color will darken, the light transmittance will decrease, by-products will be produced, and it will be easy to be contaminated by bacteria.
[0046] This invention specifies that after dissolving in water, the temperature should be raised to 58-60℃, and 1.5-2.5% of saccharifying enzyme should be added, followed by stirring for 2-6 hours. To verify the experimental effects regarding the amount of saccharifying enzyme added and the enzymatic hydrolysis time, the following experiment was designed:
[0047] Experiment on the addition of saccharifying enzyme: "Normal" is the control group without enzymatic hydrolysis. Maltodextrin with a concentration of 20% was prepared, and 1.5%, 2.0%, and 2.5% of saccharifying enzyme were added respectively. Enzymatic hydrolysis was carried out at 58-60℃ for 4 hours.
[0048] normal 1.5% 2.0% 2.5% Dissolved oxygen range 5-30% 10-50% 15-50% 10-40% 200h potency u / ml 14400 14750 14900 14800
[0049] The above experiments show that the amount of saccharifying enzyme added is directly proportional to dissolved oxygen and potency. The solution with 1.5% saccharifying enzyme is significantly more viscous than the solution with 2.5% saccharifying enzyme. According to the experimental data, the optimal effect is achieved when the saccharifying enzyme is added at 2% after 4 hours of enzymatic hydrolysis. This increases dissolved oxygen and potency from 14400u / ml to 14900u / ml, an increase of 3.47%.
[0050] Maltodextrin enzymatic hydrolysis time experiment: "Normal" was the control group without enzymatic hydrolysis. A 20% concentration of maltodextrin was prepared and enzymatically hydrolyzed at 58-60℃ for 3, 4, 5, and 6 hours respectively, with 2% saccharifying enzyme added in each case.
[0051] normal 3 hours 4 hours 5 hours 6 hours Dissolved oxygen range 0-25% 10-50% 5-30% 10-40% 5-45% 200h potency u / ml 14560 14720 14980 15100 15200 Aseptic condition normal normal normal normal Contamination
[0052] The above experiments show that the enzymatic hydrolysis time is directly proportional to dissolved oxygen and potency. The longer the enzymatic hydrolysis time, the higher the potency. After 6 hours of reaction, the liquid color became significantly darker and had a slight sour taste. Microscopic examination showed a significant increase in miscellaneous bacteria. In summary, the optimal effect was achieved with 2% saccharifying enzyme and 5 hours of enzymatic hydrolysis, which increased dissolved oxygen and potency from 14560 u / ml to 15100 u / ml, an increase of 3.71%.
[0053] Example 3: (Optimization of Soybean Oil Addition)
[0054] Based on the principle of no supplemental culture medium, the soybean oil content in the basal culture medium was set at 7%, 7.5%, 8%, and 8.5%, respectively, and the remaining formulas and processes were the same as in Example 1.
[0055] Soybean oil dosage test
[0056] 7% soybean oil 7.5% soybean oil 8% soybean oil 8.5% soybean oil highest bacterial concentration 40.4% 41.6% 45.6% 45.9% Bacterial concentration difference +2.97% +12.87% +13.61% Potency of stopping the can (u / ml) 200h-15100 200h-15600 200h-16090 200h-16100 Valence Difference Rate +3.31% +6.55% +6.62%
[0057] Experiments showed that increasing the amount of soybean oil in the base material was positively effective for cell growth and titer increase. Based on the principle of reducing costs and increasing efficiency, 8.0% soybean oil was the most suitable amount. The bacterial concentration increased from 40.4% to 45.6%, an increase of 12.87%, and the titer increased from 15100u / ml to 16090u / ml, an increase of 6.55%.
[0058] Example 4: (Optimization of Feeding Formula)
[0059] "Normal" refers to a control without supplementation. The soybean meal powder in the supplemented culture medium was set as follows: 0.7% in Scheme A, 1.0% in Scheme B, and 1.5% in Scheme C. Inositol was 0.2% in all cases. The rest of the process was the same as in Example 1.
[0060] normal Option A Option B Option C Potency of stopping the can (u / ml) 200h-16200 200h-18400 200h-19500 200h-19550 Valence difference rate +13.58% +20.37% +20.67%
[0061] The experiment shows that the fed-batch process is positively effective for cell growth and product synthesis. Considering the need to reduce raw material costs, scheme B has the best effect, which is 1.0% soybean meal and 0.2% inositol. The potency increased from 16200u / ml to 19500u / ml, an increase of 20.37%.
[0062] Example 5: (Optimization of Inositol Dosage)
[0063] "Normal" refers to the control group without replenishment. The replenishment of soybean meal powder is fixed at 1.0%, and the inositol content is 0.15% in Scheme D, 0.2% inositol in Scheme E, and 0.25% inositol in Scheme F. The remaining processes are the same as in Example 1.
[0064] normal Option D Option E Option F Potency of stopping the can (u / ml) 200h-16100 200h-17200 200h-19600 200h-18800 Valence Difference Rate +6.83% +21.73% +16.77%
[0065] The experiment showed that the feeding process was positively effective for cell growth and product synthesis. Scheme E had the best effect, which was 1.0% soybean meal and 0.2% inositol. The potency increased from 16100u / ml to 19600u / ml, an increase of 21.73%.
[0066] The final conclusion drawn from the above two sets of experiments is that the optimal feed ratio is 1.0% soybean meal powder and 0.2% inositol.
[0067] In summary, this invention solves the problems of low bacterial concentration, short kasugamycin production period, high fermentation viscosity, poor dissolved oxygen, and low potency in traditional processes by optimizing culture medium composition, maltodextrin enzymatic hydrolysis pretreatment, soybean oil ratio, and targeted feeding strategy. It significantly improves the industrial production level of kasugamycin. Compared with traditional processes, this invention can increase bacterial concentration by 16.04% and kasugamycin potency from 14500 u / ml to 19600 u / ml, an increase of 35%. At the same time, it reduces fermentation broth viscosity, improves dissolved oxygen, reduces the use of defoaming agents, maintains carbon-nitrogen balance, and prolongs the kasugamycin production period. The process is stable, cost-controllable, and suitable for large-scale industrial production, with significant economic benefits and application value.
[0068] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.
[0069] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A kasugamycin fermentation medium, characterized in that, It consists of a basal fermentation medium and a supplemental fermentation medium. By volume percentage, the basal fermentation medium consists of the following components: 5-8% soybean meal, 0.1-0.5% sodium glutamate, 0.26-0.5% potassium dihydrogen phosphate, 0.25-0.5% corn steep liquor, 7-9% soybean oil, and 1-3% maltodextrin. The supplemental fermentation medium consists of the following components: 0.5-1.6% soybean meal and 0.1-0.3% inositol.
2. The kasugamycin fermentation medium according to claim 1, characterized in that, The fermentation basal culture medium formula is as follows: 8% soybean meal, 0.2% sodium glutamate, 0.4% potassium dihydrogen phosphate, 0.4% corn steep liquor, 8.0% soybean oil, and 2% maltodextrin.
3. The kasugamycin fermentation medium according to claim 1, characterized in that, The feed culture medium formula is: 1.0% soybean meal powder and 0.2% inositol.
4. A fermentation method for kasugamycin, characterized in that, The fermentation medium containing kasugamycin according to any one of claims 1-3 comprises the following steps: S1. Culture medium preparation: Prepare the fermentation basal culture medium and feed culture medium according to the proportions; The maltodextrin needs to undergo enzymatic pretreatment: after dissolving in water, heat to 58-60℃, add 1.5-2.5% saccharifying enzyme, stir and maintain for 2-6 hours, then heat to 85-90℃ and add the remaining materials for sterilization, and adjust the pH to 6.7-6.
8. The pH of the basal fermentation medium was adjusted to 6.7-6.8 using liquid ammonia, while the pH of the fed medium was left at its natural state. S2. Sterilization: Both the fermentation basal medium and the fed medium were sterilized at 121-125℃ under pressure for 30 minutes. S3. Inoculation: After sterilization, cool to 28-29℃ and inoculate with *Streptomyces simonii* at a rate of 5 vol%-15 vol%. S4. Fermentation culture: control the temperature at 28-29℃, the tank pressure at 0.05MPa, the aeration ratio at 1:0.5-1:1.2, the stirring speed at 100-300rpm, the pH at 6.7-6.8, and the fermentation cycle at 200±24h. S5. Feeding: When the cell concentration increases slowly or stops increasing after 90-100 hours of fermentation, start feeding the culture medium and continue feeding for 50 hours. S6 Collection: After fermentation is complete, collect the fermentation products.
5. The fermentation method for kasugamycin according to claim 4, characterized in that, In the maltodextrin pretreatment, the constant temperature stirring time at 58-60℃ is 5 hours, and the amount of saccharifying enzyme added is 2.0%.