A fermentation medium and a preparation method of gentamicin C1a

Abstract

The present invention discloses a fermentation medium and a preparation method of gentamicin C1a. The preparation method comprises the following steps: inoculating Micromonospora purpurea CICC11015 into the fermentation medium of the present invention to obtain a fermentation broth, and obtaining a solution containing gentamicin C1a after fermentation; the volume ratio of Micromonospora purpurea to the fermentation medium is (1-9):9. The fermentation medium provided by the present invention has the advantage of highly efficient fermentation of gentamicin C1a. When the preparation method of gentamicin C1a of the present invention uses Micromonospora purpurea for fermentation, the fermentation titer of gentamicin C1a is improved; after adding a strong acidic cation exchange resin, the fermentation titer of gentamicin C1a can be further improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial engineering, and particularly to a fermentation medium and a preparation method of gentamicin C1a. Background Art

[0002] The "Pharmacopoeia of the People's Republic of China" stipulates that the C components of gentamicin include four main components: C1, C1a, C2, and C2a. Among them, gentamicin C1a not only has strong antibacterial activity itself but also can be used as the parent nucleus precursor of etimicin, a domestic first-class semi-synthetic new drug. Therefore, its market demand is relatively urgent. At present, the strains for industrial production of gentamicin C1a are mostly obtained through mutagenesis or modification in genetically engineered bacteria. Optimization of the preparation method also includes optimization of the medium ratio and feeding mode, but the fermentation unit in current industrial production is generally low.

[0003] CN106801076A (Cheng Guozheng et al.) discloses that by adjusting the components and ratios of the seed medium and the fermentation medium, and at the same time adjusting the feeding mode and dosage, and controlling various parameters comprehensively, the yield of gentamicin C1a is increased by 43%, but its yield is only 1000 ug / mL.

[0004] CN110358718A (Hong Wenrong) discloses that by knocking out the key gene genK to block the metabolic flow in the direction of gentamicin C1, and then constructing a new species of genL micromonospora purpurea engineering bacterium Gb1098, the final titer reaches 1098 ug / mL.

[0005] CN106498011A (Yang Liang et al.) uses chemical mutagenesis or microwave mutagenesis to obtain a new strain with gentamicin C1a as the main component, and optimizes the components of the seed and fermentation media. However, the titer of gentamicin C1a is not improved well finally.

[0006] Due to the poor fermentation titer of gentamicin C1a in the prior art, it is necessary to develop a fermentation method of gentamicin C1a with a higher fermentation titer. Summary of the Invention

[0007] Aiming at the technical defect that the fermentation titer of gentamicin C1 in the prior art is not high, the present invention provides a fermentation medium and a preparation method of gentamicin C1a. Among them, the fermentation medium provided by the present invention has the advantage of highly efficient fermentation of gentamicin C1a. When the preparation method of gentamicin C1a of the present invention uses micromonospora purpurea for fermentation, the fermentation titer of gentamicin C1a is improved; after adding a strong acid cation exchange resin, the fermentation titer of gentamicin C1a can be further improved.

[0008] To solve the above technical problems, one of the technical solutions provided by the present invention is: a fermentation medium for fermenting Micromonospora purpurea to produce gentamicin C1a, and the fermentation medium comprises the following components:

[0009] Corn starch 1 - 5, corn flour 1 - 5, cold-rolled soybean powder 1 - 5, feather meal 0.5 - 3, (NH4)2SO4 0.05 - 0.2, KNO3 0.005 - 0.03, CaCO3 0.5 - 2, CoCl2 0.001 - 0.05, CaCl2 0.02 - 0.15, amylase 0.0005 - 0.003 and antifoaming agent 0.02 - 0.1, and the content unit of the components is g / 100 mL;

[0010] The pH of the fermentation medium is 7.5 - 8.5.

[0011] In a preferred embodiment, the fermentation medium comprises the following components: corn starch 2.5, corn flour 2.0, cold-rolled soybean powder 2.0, feather meal 1.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001 and antifoaming agent 0.05, and the content unit of the components is g / 100 mL.

[0012] In a preferred embodiment, the pH of the fermentation medium is 8.0.

[0013] In a preferred embodiment, the Micromonospora purpurea is Micromonospora purpurea CICC11015.

[0014] To solve the above technical problems, another technical solution provided by the present invention is: a preparation method of gentamicin C1a, and the preparation method comprises the following steps: inoculating Micromonospora purpurea CICC11015 into the fermentation medium as described in Technical Solution One to obtain a fermentation broth, and fermenting to obtain a solution containing gentamicin C1a; the volume ratio (L:L) of the Micromonospora purpurea to the fermentation medium is (1 - 9):9, for example, 6:9.

[0015] Preferably, the fermentation is carried out in the presence of a strongly acidic cation exchange resin. Preferably, the strongly acidic cation exchange resin is the strongly acidic cation exchange resin 001*8 of Shanghai Huazhen Technology Co., Ltd., and the resin particle diameter of the strongly acidic cation exchange resin is 0.4 - 0.6 mm. More preferably, the strongly acidic cation exchange resin is added to the fermentation broth 12 - 36 h after the start of the fermentation, preferably added to the fermentation broth 24 h after the start of the fermentation. Further preferably, the volume ratio (L:L) of the strongly acidic cation exchange resin to the fermentation medium is (0.2 - 0.4):15, such as 0.3:15.

[0016] More preferably, in the fermentation, the specific conditions include:

[0017] The fermentation is carried out in a fermenter, and the aeration rate during fermentation is not less than 1.0 L·L -1 min -1 , the tank pressure is not less than 0.03 MPa, the dissolved oxygen is more than 30%, and the temperature is 35 - 39 °C;

[0018] And / or, stirring is maintained during the fermentation, and the stirring speed is 200 - 400 r / min; the rotation speed is preferably set as 250 r / min for 0 - 2 h and 300 r / min after 2 h, more preferably 250 r / min for 0 - 2 h and 300 r / min after 2 h;

[0019] And / or, in the fermentation, the total sugar in the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by adding a diluent medium, and at the same time, water is added to control the total volume of the fermentation broth to remain unchanged.

[0020] And / or, the fermentation time is 110 - 120 h.

[0021] In a preferred embodiment of Technical Solution 2, the aeration rate is 1.2 - 1.4 L·L -1 ·min -1 , the tank pressure is 0.05 - 0.06 MPa, and the temperature is 37 °C.

[0022] In a preferred embodiment of Technical Solution 2, when the fermentation proceeds to 24 h, the total sugar in the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by feeding a diluent medium, and the feeding rate of the fed-batch is 0.041 L / h - 0.075 L / h.

[0023] In a preferred embodiment of Technical Solution 2, it is characterized in that the thin medium comprises the following components: corn starch 5 - 10, corn flour 4 - 8, cold-rolled soybean powder 4 - 8, feather meal 1 - 5, (NH4)2SO4 0.05 - 0.2, KNO3 0.005 - 0.03, CaCO3 0.5 - 2, CoCl2 0.001 - 0.05, CaCl2 0.02 - 0.15, amylase 0.0005 - 0.003, and antifoaming agent 0.02 - 0.1, and the content unit of the components is g / 100mL; the pH of the thin medium is 7.5 - 8.5. Preferably, the thin medium comprises the following components: corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, and antifoaming agent 0.05, and the content unit of the components is g / 100mL; the pH of the thin medium is 8.0.

[0024] In a preferred embodiment of Technical Solution 2, the preparation method further includes performing seed liquid culture on Micromonospora purpurea before the fermentation.

[0025] In a preferred embodiment of Technical Solution 2, the preparation method comprises the following steps: adding 6L of the seed liquid of Micromonospora purpurea CICC11015 with a cell content of 25% of Micromonospora purpurea into a fermenter containing 9L of the above fermentation medium for fermentation; when the fermentation reaches 24h, adding the strong acidic cation exchange resin 001*8 of Shanghai Huazhen Technology Co., Ltd., with a resin particle diameter of 0.4 - 0.6mm, to obtain a solution containing gentamicin C1a;

[0026] During the fermentation, the specific conditions are as follows:

[0027] (1) The ventilation rate is 1.2L·L -1 ·min -1 , the tank pressure is 0.06MPa, the dissolved oxygen is above 30%, and the temperature is 37°C;

[0028] (2) Stirring is maintained during the fermentation, and the stirring speed is set as follows: 0 - 2h: 250r / min, after 2h: 300r / min;

[0029] (3) When the fermentation proceeds to 24 h, the total sugar in the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by fed-batch addition. The feeding rate of the fed-batch addition is 0.045 L / h - 0.065 L / h. The diluent medium for the fed-batch addition comprises the following components: corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001 and antifoaming agent 0.05. The content unit of the components is g / 100 mL. The pH of the diluent medium is 8.0;

[0030] (4) The fermentation time is 120 h.

[0031] To solve the above technical problems, the third technical solution provided by the present invention is: the application of the fermentation medium as described in Technical Solution 1 or the strongly acidic cation exchange resin 001*8 of Shanghai Huazhen Technology Co., Ltd. in the fermentation of gentamicin C1a. The resin particle diameter of the strongly acidic cation exchange resin 001*8 is preferably 0.4 - 0.6 mm.

[0032] In the present invention, before the experiment, the resin can also be treated by a conventional method in the art. In a preferred embodiment, before the experiment, the resin is treated in the following manner: the resin is rinsed with 2M HCl at a rate of 0.5 BV / h until the pH of the effluent is below 1.0, and the amount used is about 3 BV. Then, it is rinsed with water until the pH is above 4.0 for standby.

[0033] In the present invention, the method for purifying gentamicin C1a from the solution containing gentamicin C1a is conventional in the art.

[0034] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0035] The reagents and raw materials used in the present invention are all commercially available.

[0036] The positive and progressive effects of the present invention are as follows:

[0037] The fermentation medium of the present invention optimizes the components, and a high fermentation titer of gentamicin C1a can be obtained. The preparation method of gentamicin C1a of the present invention improves the fermentation titer of gentamicin C1a when using Micromonospora purpurea for fermentation. After adding the strongly acidic cation exchange resin, the fermentation titer of gentamicin C1a can be further increased, up to 1850 μg / ml at most, while ensuring a high recovery rate of the resin. It is more conducive to industrial production. Description of the Drawings

[0038] Figure 1 For the exchange effects of strong acid resins with different crosslinking degrees;

[0039] Figure 2 For the exchange effects of strong acid resins with different particle diameters. Specific implementation mode

[0040] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0041] Experimental materials

[0042] 1. Instruments

[0043]

[0044]

[0045] 2. Reagents

[0046] Reagent Specification Manufacturer Glucose Food grade Xiwang Sugar Industry Co., Ltd. Corn starch Industrial grade Shanghai Yuanju Biotechnology Co., Ltd. Corn flour Industrial grade Qingdao Kerry Culture Medium Co., Ltd. Cold-rolled soybean powder Industrial grade Qingdao Kerry Culture Medium Co., Ltd. Feather meal Industrial grade Qingdao Kerry Culture Medium Co., Ltd. Calcium carbonate Analytical reagent Sinopharm Reagent Calcium chloride Analytical reagent Sinopharm Reagent Cobalt chloride Analytical reagent Sinopharm Reagent Sodium chloride Analytical reagent Sinopharm Reagent Ammonium sulfate Analytical reagent Sinopharm Reagent Potassium dihydrogen phosphate Analytical reagent Sinopharm Reagent Magnesium sulfate heptahydrate Analytical reagent Sinopharm Reagent Potassium nitrate Analytical reagent Sinopharm Reagent Soluble starch Industrial grade Sinopharm Reagent L-Asparagine Analytical reagent Sinopharm Reagent Peptone Industrial grade Shanghai Liangyou Haishi Oil Industry Co., Ltd. Bran Feed grade Commercially available Defoamer Industrial grade Shandong Fengying Environmental Protection Materials Co., Ltd. Amylase Industrial grade Beijing Kewibo Biotechnology Co., Ltd. Agar powder Industrial grade Sinopharm Reagent

[0047] 3. Bacterial strains

[0048] The bacterial strain used in the present invention is Micromonospora purpurea CICC11015, purchased from the China Center of Industrial Culture Collection (CICC).

[0049] 4. Resins

[0050] Strong acid cation exchange resins: 001*6, 001*7, 001*8, 001*9 and 001*10 resins, with a particle size of 0.2 - 1.2 mm, all purchased from Shanghai Huazhen Technology Co., Ltd.

[0051] 5. Culture media

[0052] Culture medium 1: Slant culture medium (g / 100 mL): corn starch 1.0, KNO3 0.1, K2HPO3 0.03, MgSO4·7H20 0.05, sodium chloride 0.05, L-asparagine 0.002, CaCO3 0.1, wheat bran 1.8, agar powder 1.4; pH 7.5, sterilized at 121 °C for 25 min.

[0053] Culture medium 2: Liquid seed culture medium (g / 100 mL): soluble starch 1.0, corn flour 2.0, glucose 0.1, cold-rolled soybean powder 2.0, KNO3 0.05, CaCO3; pH 7.0, sterilized at 121 °C for 25 min.

[0054] Medium 3: Shake flask fermentation medium (g / 100 mL): glucose 0.5, corn starch 2.0, corn flour 3.5, KNO3 0.05, cold-rolled soybean powder: 2.6, peptone 1.0, CaCO3 0.7, CoCl2 0.002, (NH4)2SO4 0.1; pH 8.0, sterilized at 121°C for 25 min.

[0055] Medium 4: Seed tank medium (g / 100mL): starch 1.0, corn flour 2.0, glucose 0.1, cold-rolled soybean powder 1.5, peptone 0.5, (NH4)2SO4 0.015, KNO3 0.05, CaCO3 0.6, defoamer 0.05; pH 7.0, sterilize at 121℃ for 30min.

[0056] Culture medium 5: Fermentation tank medium (g / 100mL): corn starch 2.5, corn flour 2.0, cold-rolled soybean powder 2.0, peptone 1.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, amylase 0.001, defoamer 0.05; pH 8.0, sterilization at 121℃ for 30min.

[0057] Medium VI: Dilute medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, peptone 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, amylase 0.001, defoamer 0.05; pH 8.0, sterilize at 121°C for 30 min.

[0058] Other experimental methods

[0059] 1.HPLC detection method

[0060] (1) Preparation of standard products

[0061] Accurately weigh the gentamicin C1a standard and dissolve it in distilled water to prepare a solution with a concentration of 1000 μg / ml. Store in a refrigerator at 4°C and dilute it before use.

[0062] (2) Preparation of OPA derivatization reagent

[0063] Accurately weigh 1 g of o-phthalaldehyde, dissolve it in 5 ml of methanol, add 2 ml of thioglycolic acid, and finally add 95 ml of 0.4 mol / l boric acid (adjust the pH to 10.4 with 45% sodium hydroxide) and mix well. Then adjust the pH to 10.4 with 45% sodium hydroxide and store in a refrigerator away from light.

[0064] (3) Sample processing

[0065] Dilute the sample with deionized water to a concentration of about 1000 μg / ml, and then carry out the derivatization reaction. After the reaction, use a pipette to take 1 ml and transfer it into a 1.5 ml EP tube, centrifuge (13000×g) for 5 min, and take the supernatant for HPLC detection.

[0066] (4) Derivatization reaction

[0067] Accurately measure 0.25 ml of the sample into a 20 ml test tube, add 3.75 ml of distilled water and 1 ml of OPA derivatization reagent, mix well, then add 5 ml of isopropanol, mix thoroughly and seal. After heating in a water bath at 60 °C for 15 min, place it in ice water to terminate the reaction.

[0068] (5) Chromatographic conditions

[0069] Chromatographic column: Navi C18 column (Suzhou Navi Biotechnology Co., Ltd.) (5 μm, 4.6×250 mm), mobile phase: sodium heptanesulfonate aqueous solution (5 g / L, 200 ml) - glacial acetic acid (50 ml) - methanol (750 ml), flow rate: 1 ml / min, column temperature: 40 °C, injection volume: 20 μL, detection wavelength: 330 nm.

[0070] 2. Resin and fermentation broth treatment methods

[0071] (1) Resin treatment

[0072] Rinse the resin with 2M HCl at a rate of 0.5 BV / h (BV is the bed volume, the volume of the resin packed in the column) until the pH of the effluent is below 1.0, with a consumption of about 3 BV, and then rinse with water until the pH is above 4.0 for standby.

[0073] (2) Fermentation broth treatment and titer detection

[0074] Fermentation broth without resin added: Take an appropriate amount of fermentation broth, adjust the pH to 1.5 - 2.0 with 9 mol / L sulfuric acid. After 1 h, take 1 ml of the fermentation broth treatment solution, centrifuge (13000×g) for 5 min, and take the supernatant for HPLC detection to determine the concentration of gentamicin C1a. The obtained concentration is the titer of the fermentation broth.

[0075] Fermentation broth with resin added: Separate the resin and fermentation broth using a 100-mesh vibrating sieve. After washing the residual fermentation broth with tap water, add 1.5M sodium hydroxide solution with the same volume as the taken fermentation broth, shake and soak for about 15 h. Take 0.5 ml of the supernatant of the soaking solution, add 0.5 ml of 1M hydrochloric acid for neutralization. Then, use HPLC detection method to determine the concentration of gentamicin C1a in the acid-neutralized solution of the resin soak and the fermentation supernatant respectively. Multiply the concentration of the acid-neutralized solution by 2 and add it to the residual concentration in the fermentation supernatant to obtain the total titer of the fermentation broth.

[0076] 4. Detection of total sugar and reducing sugar: Fehling's reagent method.

[0077] 5. Detection of free amino nitrogen: formaldehyde oxidation assay.

[0078] Example 1 Investigation of Resin Crosslinking Degree

[0079] Take 20 ml of each treated resin and add it to 3000 ml of fermentation broth (containing 1000 μg / mL of gentamicin C1a). Static adsorption with stirring was performed for 6 hours. The residual units in the fermentation broth were measured, and the exchange capacity was calculated according to Equation 1. The adsorption-saturated resin was eluted with 3M concentrated ammonia for 10 hours, and then evaporated and concentrated to remove ammonia. The resin desorption rate was calculated according to Equation 2.

[0080] Formula 1: Exchange capacity (g / ml) = (I1-I2)*V1 / V2

[0081] Formula 2: Desorption rate = I3*V3 / [(I1-I2)*V1]

[0082] Note: I1 is the unit of fermentation broth before exchange (g), I2 is the unit of fermentation broth residue after exchange (g), V1 is the volume of fermentation broth (ml), V2 is the volume of resin (ml), I3 is the unit of eluent after deamination (μg / mL), V3 is the volume of eluent after deamination (ml).

[0083] Depend on Figure 1 It can be seen that 001*9 has the largest exchange capacity, followed by 001*8. The desorption rate decreases with the increase of cross-linking degree. Therefore, 001*8 is selected as the optimal enrichment resin in the fermentation broth after comprehensive consideration.

[0084] Example 2 Investigation of resin particle diameter

[0085] 001*8 resins with different particle diameters of 0.2-0.4mm, 0.4-0.6mm, 0.6-0.8mm, 0.8-1.0mm, and 1.0-1.2mm were selected to investigate their exchange capacity and desorption rate. The resin recovery vibration screen was 100 mesh. Figure 2 The data shows that the resin particle diameter of 0.4-0.6mm is more suitable, and the desorption rate and exchange capacity are more ideal. As the resin particle diameter decreases, the surface area becomes larger and larger, and the resin exchange capacity also becomes larger and larger. However, when the resin diameter is lower than 0.4mm, although the resin diameter is small, it is easy to pass through the resin separator vibration screen, resulting in a significant reduction in the resin recovery rate, which directly affects the desorption rate. As the resin diameter increases, the desorption rate increases slightly, but the exchange capacity decreases significantly. Figure 2 Therefore, the final choice is 001*8 resin with a resin diameter of 0.4-0.6mm.

[0086] Example 3 Gentamicin C1a Basic Fermentation Method

[0087] Inoculate the glycerol tube bacterial suspension into the slant medium containing Medium 1, and incubate at 37°C for 7 - 8 days. Cut the agar blocks and transfer them into Medium 2 with a liquid volume of 100 ml / 750 ml, and shake culture at 37°C and 240 r / min for 48 h to obtain the seed liquid of Medium 2.

[0088] Transfer 70 ml of the seed liquid of Medium 2 to a 25 L first-stage seed tank with a liquid volume of 15 L of Medium 4. Adjust the rotation speed: 0 - 18 h: 250 r / min, 18 - 30 h: 300 r / min, 30 - 36 h: 350 r / min, 36 - 48 h: 375 r / min; aeration rate (VVM) 1.0 L·L -1 ·min -1 , tank pressure 0.05 MPa, control the dissolved oxygen above 30%, and culture at 37°C for 48 h to obtain the seed liquid of Medium 4 with a cell content of 25%.

[0089] Transfer 6 L of the well-cultured seed liquid of Medium 4 to a 25 L fermenter with a liquid volume of 9 L of the fermentation medium (Medium 5). Adjust the rotation speed: 0 - 2 h: 250 r / min, 2 - 4 h: 300 r / min, 4 - 6 h: 350 r / min, 6 - 120 h: 400 r / min; aeration rate (VVM) 1.0 L·L -1 min -1 , tank pressure 0.03 MPa, control the dissolved oxygen above 30%, and ferment and culture at 37°C for 110 - 120 h. During the fermentation process, control the total sugar to be greater than 0.5 g / 100 ml and the amino nitrogen to be greater than 20 mg / 100 ml by batchwise supplementing Medium 6, and at the same time control the total amount of the fermentation broth to remain basically unchanged by adding water. The total feeding amount during the fermentation process is 4.4 L, and the titer of gentamicin C1a in the fermenter is 1.0 g / L.

[0090] Example 4 Optimization of Fermentation Medium

[0091] On the basis of Example 3, with other conditions remaining unchanged, the formulation of the fermentation medium (Medium Five) (g / 100 mL) was changed to: corn starch 2.5, corn flour 2.0, cold-rolled soybean powder 2.0, feather meal 1.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. The thin medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. During the fermentation process, the total sugar was controlled to be greater than 0.5 g / 100 ml and the amino nitrogen was greater than 20 mg / 100 ml by batch addition of the thin medium, and at the same time, water was added to keep the total volume of the fermentation broth basically unchanged. The total amount of supplementary feed during the fermentation process was 4.4 L, and the titer of gentamicin C1a in the fermenter was 1.08 g / L.

[0092] Optimization of the Fermentation Medium in Example 5

[0093] On the basis of Example 4, with other conditions remaining unchanged, the formulation of the fermentation medium (Medium Five) (g / 100 mL) was changed to: corn starch 2.5, corn flour 2.0, cold-rolled soybean powder 2.0, feather meal 1.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. The thin medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. During the fermentation process, the total sugar was controlled to be greater than 0.5 g / 100 ml and the amino nitrogen was greater than 20 mg / 100 ml by batch addition of the thin medium, and at the same time, water was added to keep the total volume of the fermentation broth basically unchanged. The total amount of supplementary feed during the fermentation process was 4.4 L, and the titer of gentamicin C1a in the fermenter was 1.2 g / L.

[0094] Fermentation Method of Gentamicin C1a with Resin Added in Example 6

[0095] On the basis of Example 5, when fermentation reached 24 h, 300 ml of activated and sterilized 001*8 resin was added to the fermentation broth. During the fermentation process, the thin medium was added in batches to control the total sugar to be greater than 0.5 g / 100 ml and the amino nitrogen to be greater than 20 mg / 100 ml. At the same time, water was added to keep the liquid level basically unchanged. The total supplementary feeding amount of this process was higher than that of Example 1, being 6.6 L, and the titer of gentamicin C1a in the fermenter was 1.58 g / L.

[0096] After discharging the fermenter, 100-mesh sieve was used for resin recovery. According to the formula: resin recovery rate (%) = 100% * volume of recovered resin / volume of input resin, the resin recovery rate was calculated to be 70%.

[0097] Optimization of Fermenter Control Parameters in Example 7

[0098] On the basis of Example 6, the rotation speed was adjusted as follows: 250 r / min for 0 - 2 h, 300 r / min for 2 - 4 h, and 350 r / min for 4 - 120 h, and other fermentation conditions remained unchanged. Then the total amount of thin medium added during the fermentation process was 5 L, the titer of gentamicin C1a in the fermenter was 1.43 g / L, and the calculated resin recovery rate was 87%.

[0099] Optimization of Fermenter Control Parameters in Example 8

[0100] On the basis of Example 6, the rotation speed was adjusted as follows: 250 r / min for 0 - 2 h, 300 r / min for 2 - 120 h, and other fermentation conditions remained unchanged. Then the total amount of thin medium added during the fermentation process was 5 L, the titer of gentamicin C1a in the fermenter was 1.2 g / L, and the calculated resin recovery rate was 95%.

[0101] Optimization of Fermenter Control Parameters in Example 9

[0102] On the basis of Example 8, the aeration rate was increased to (VVM) 1.2 L·L -1 ·min -1 , the tank pressure was 0.03 MPa, and other fermentation conditions remained unchanged. Then the total amount of thin medium added during the fermentation process was 6 L, the titer of gentamicin C1a in the fermenter was 1.33 g / L, and the calculated resin recovery rate was 95%.

[0103] Optimization of Fermenter Control Parameters in Example 10

[0104] On the basis of Example 9, the aeration rate was increased to (VVM) 1.2 L·L -1 ·min -1 , the tank pressure was increased to 0.05 MPa, and other fermentation conditions remained unchanged. Then the total amount of thin medium added during the fermentation process was 6 L, the titer of gentamicin C1a in the fermenter was 1.40 g / L, and the calculated resin recovery rate was 94.5%.

[0105] Example 11 Optimization of Fermenter Control Parameters

[0106] On the basis of Example 10, the aeration rate was increased to (VVM) 1.2 L·L -1 ·min -1 , the tank pressure was increased to 0.06 MPa, and other fermentation conditions remained unchanged. The total amount of supplementary thin medium added during the fermentation process was 6 L, the titer of gentamicin C1a in the fermenter was 1.45 g / L, and the calculated resin recovery rate was 94.5%.

[0107] Example 12 Optimization of Fermenter Control Parameters

[0108] On the basis of Example 10, the aeration rate was increased to (VVM) 1.4 L·L -1 ·min -1 , the tank pressure was increased to 0.06 MPa, and other fermentation conditions remained unchanged. The total amount of supplementary thin medium added during the fermentation process was 6 L, the titer of gentamicin C1a in the fermenter was 1.43 g / L, and the calculated resin recovery rate was 94.5%.

[0109] Example 13 Optimization of Feeding Method

[0110] On the basis of Example 11, the fed-batch feeding method was used instead of the original batch feeding. After 24 h of fermentation, thin medium was fed for 120 h. The thin medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. The feeding rate was 0.045 L / h, and other fermentation conditions remained unchanged. The total amount of supplementary thin medium added during the fermentation process was 4.32 L, the titer of gentamicin C1a in the fermenter was 1.55 g / L, and the calculated resin recovery rate was 95%.

[0111] Example 14 Optimization of Feeding Method

[0112] On the basis of Example 11, the fed-batch feeding method was adopted instead of the original batch feeding. After 24 h of fermentation, the thin medium was fed until 120 h. The thin medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. The feeding rate was 0.055 L / h, and other fermentation conditions remained unchanged. The total amount of the thin medium added during the fermentation process was 5.28 L, the titer of gentamicin C1a in the fermenter was 1.85 g / L, and the calculated resin recovery rate was 95%.

[0113] Optimization of the feeding method in Example 15

[0114] On the basis of Example 11, the fed-batch feeding method was adopted instead of the original batch feeding. After 24 h of fermentation, the thin medium was fed until 120 h. The thin medium (g / 100 mL): corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, antifoaming agent 0.05; pH 8.0, sterilized at 121 °C for 30 min. The feeding rate was 0.065 L / h, and other fermentation conditions remained unchanged. The total amount of the thin medium added during the fermentation process was 6.24 L, the titer of gentamicin C1a in the fermenter was 1.48 g / L, and the calculated resin recovery rate was 94.5%.

[0115] Conclusion

[0116] The present invention provides a fermentation process route that not only saves raw material costs but also significantly improves the fermentation titer of gentamicin C1a. In the fermentation process of gentamicin C1a, the input of 001*8 resin into the fermentation broth can effectively relieve the feedback inhibition of gentamicin C1a on its biosynthesis process, promote the metabolism of the producing bacteria to proceed in the direction of synthesizing gentamicin C1a, and greatly improve the fermentation titer of gentamicin C1a. As can be seen from Example 15, in the fermentation process of gentamicin C1a, the input of 001*8 resin, combined with the optimization of the medium, the control parameters of the fermenter and the feeding process, while reducing the stirring speed of the fermenter, increasing the air volume and tank pressure, and adopting the fed-batch feeding technology to add the thin medium, can further significantly improve the fermentation titer, increase the effective utilization rate of the fermentation medium, and at the same time ensure a high resin recovery rate.

Claims

1. A fermentation medium for fermenting Micromonospora purpurea Micromonospora purpurea to produce gentamicin C1a, characterized in that The described fermentation medium comprises the following components: Corn starch 1 - 5, corn flour 1 - 5, cold-rolled soybean powder 1 - 5, feather meal 0.5 - 3, (NH4)2SO4 0.05 - 0.2, KNO3 0.005 - 0.03, CaCO3 0.5 - 2, CoCl2 0.001 - 0.05, CaCl2 0.02 - 0.15, amylase 0.0005 - 0.003, and antifoaming agent 0.02 - 0.1, where the content unit of the components is g / 100 mL; The pH of the described fermentation medium is 7.5 - 8.

5.

2. The fermentation medium according to claim 1, characterized in that, The described fermentation medium comprises the following components: corn starch 2.5, corn flour 2.0, cold-rolled soybean powder 2.0, feather meal 1.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001, and antifoaming agent 0.05, where the content unit of the components is g / 100 mL; And / or, the pH of the described fermentation medium is 8.

0.

3. A method for preparing gentamicin C1a, characterized in that, The preparation method includes the following steps: inoculating Micromonospora purpurea ( Micromonospora purpurea ) CICC11015 into the fermentation medium as described in Claim 1 or 2 to obtain a fermentation broth, and obtaining a solution containing gentamicin C1a after fermentation; The volume ratio of the described Micromonospora purpurea to the fermentation medium is (1 - 9):

9.

4. The preparation method according to claim 3, characterized in that, The volume ratio of the described Micromonospora purpurea to the fermentation medium is 6:

9.

5. The preparation method according to claim 3, characterized in that, The described fermentation is carried out in the presence of a strong acidic cation exchange resin.

6. The preparation method according to claim 5, characterized in that, The described strong acidic cation exchange resin is the strong acidic cation exchange resin 001*8 of Shanghai Huazhen Technology Co., Ltd., and / or the resin particle diameter of the described strong acidic cation exchange resin is 0.4 - 0.6 mm.

7. The preparation method according to claim 6, characterized in that, The described strong acidic cation exchange resin is added to the fermentation broth 12 - 36 h after the start of the fermentation.

8. The preparation method according to claim 7, characterized in that, The described strong acidic cation exchange resin is added to the fermentation broth 24 h after the start of the fermentation.

9. The preparation method according to claim 7, characterized in that, The volume ratio of the described strong acidic cation exchange resin to the fermentation broth is (0.2 - 0.4):

15.

10. The preparation method according to claim 9, characterized in that, The volume ratio of the described strong acidic cation exchange resin to the fermentation broth is 0.3:

15.

11. The preparation method according to claim 3, characterized in that The fermentation is carried out in a fermenter, and the aeration volume during fermentation is controlled to be not less than 1.0 L·L -1 min -1 , the tank pressure is not less than 0.03 MPa, the dissolved oxygen is more than 30%, and the temperature is 35 - 39 °C; And / or, stirring is maintained during the fermentation, and the stirring speed is 200 - 400 r / min; And / or, during the fermentation, the total sugar in the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by supplementing the diluent medium, and at the same time, water is added to control the total volume of the fermentation broth to remain unchanged; And / or, the fermentation time is 110 - 120 h.

12. The preparation method according to claim 11, wherein, The speed is set as follows: 0 - 2 h: 250 r / min, after 2 h: 300 - 350 r / min.

13. The preparation method according to claim 12, wherein, The speed is set as follows: 0 - 2 h: 250 r / min, after 2 h: 300 r / min.

14. The preparation method according to claim 11, wherein, The ventilation volume is 1.2 - 1.4 L·L -1 ·min -1 , the tank pressure is 0.05 - 0.06 MPa, and the temperature is 37°C.

15. The preparation method according to claim 14, characterized in that, When the fermentation proceeds to 24 h, the total sugar of the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by feeding a diluted medium, and the feeding rate of the fed-batch is 0.041 L / h - 0.075 L / h.

16. The preparation method according to any one of claims 11-15, characterized in that, The diluted medium comprises the following components: corn starch 5 - 10, corn flour 4 - 8, cold-rolled soybean powder 4 - 8, feather meal 1 - 5, (NH4)2SO4 0.05 - 0.2, KNO3 0.005 - 0.03, CaCO3 0.5 - 2, CoCl2 0.001 - 0.05, CaCl2 0.02 - 0.15, amylase 0.0005 - 0.003 and antifoaming agent 0.02 - 0.1, and the content unit of the components is g / 100 mL; the pH of the diluted medium is 7.5 - 8.

5.

17. The preparation method according to claim 16, wherein, The diluted medium comprises the following components: corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001 and antifoaming agent 0.05, and the content unit of the components is g / 100 mL; the pH of the diluted medium is 8.

0.

18. The preparation method according to any one of claims 3-15, characterized in that, The preparation method further includes culturing a seed solution of Micromonospora purpurea before the fermentation.

19. The preparation method according to claim 3, wherein The preparation method comprises the following steps: adding 6 L of a seed solution of Micromonospora purpurea ( Micromonospora purpurea ) CICC 11015 with a cell content of 25% of Micromonospora purpurea into a fermenter containing 9 L of the fermentation medium as described in claim 2 for fermentation; adding strongly acidic cation exchange resin 001*8 from Shanghai Huazhen Technology Co., Ltd. with a resin particle diameter of 0.4 - 0.6 mm to the fermentation broth at 24 h of fermentation to obtain a solution containing gentamicin C1a; In the fermentation, the specific conditions are as follows: (1) The ventilation volume is 1.2 L·L -1 ·min -1 , the tank pressure is 0.06 MPa, the dissolved oxygen is more than 30%, and the temperature is 37 °C; (2) Stirring is maintained during the fermentation, and the stirring speed is set at 250 r / min for 0 - 2 h and 300 r / min after 2 h; (3) When the fermentation proceeds to 24 h, the total sugar of the fermentation broth is controlled to be greater than 0.5 g / 100 ml and the amino nitrogen is greater than 20 mg / 100 ml by fed-batch, and the feeding rate of the fed-batch is 0.045 L / h - 0.065 L / h; the diluted medium for the fed-batch comprises the following components: corn starch 7.5, corn flour 6.0, cold-rolled soybean powder 6.0, feather meal 3.0, (NH4)2SO4 0.08, KNO3 0.017, CaCO3 0.9, CoCl2 0.002, CaCl2 0.05, amylase 0.001 and antifoaming agent 0.05, and the content unit of the components is g / 100 mL; the pH of the diluted medium is 8.0; (4) The fermentation time is 120 h.

20. Use of the fermentation medium according to claim 1 or 2 in the fermentation of gentamicin C1a, characterized in that, The bacteria used for fermentation are Micromonospora purpurea ( Micromonospora purpurea Micromonospora purpurea ) CICC11015.

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