Method for regulating the fermentation of macrolide antibiotics

Abstract

The present invention is a method for regulating the fermentation of macrolide antibiotics. It solves the problem that the fermentation process of the traditional method is not easy to control, the energy consumption is high, and the yield of the target product is low. It includes the following steps: in the primary metabolic stage of macrolide antibiotic producing bacteria, the culture temperature is controlled at 27.0-30.0°C according to normal requirements, and the mycelium grows rapidly; after the macrolide antibiotic producing bacteria complete the logarithmic growth period, the fermentation culture temperature is reduced to 18.0-27.0°C to promote the large-scale synthesis of the target product, and the macrolide antibiotic is an antibiotic and its homologues produced by fermentation using the PKS biosynthetic pathway. The process of the present invention is simple and easy to operate, and is very suitable for commercial production.

Description

Technical Field

[0001] The invention relates to the technical field of microbial medicines, and in particular to a method for regulating the fermentation of macrolide antibiotics based on culture temperature regulation. Background Art

[0002] Macrolide antibiotics such as Sirolimus, Tacrolimus, Doramectin, Spinosyn and Kitasamycin are all polyketide substances in structure, and the biosynthesis process of this type of polyketide structure also shows similar rules. Polyketide synthase (PKS) is a multi-enzyme complex that catalyzes the formation of polyketide compounds, catalyzing the synthesis of the basic skeleton with a long carbon chain structure formed by the polymerization of low-level fatty acids of macrolide antibiotics. The starting unit of polyketide synthesis and the short-chain fatty acid units required for carbon chain extension, such as acetic acid and propionic acid, have been accumulated in the primary metabolism stage of microorganisms. In the middle and late stages of fermentation, if the fermentation temperature is maintained at a high level, the microorganisms will quickly consume the raw materials such as sugars, oils or amino acids that produce short-chain fatty acid units, forming waste and loss, and cannot continuously meet the needs of product synthesis. At the same time, higher carbon source consumption will produce a large amount of organic acid, causing the pH and dissolved oxygen of the fermentation liquid to drop rapidly. After the carbon source is exhausted, the pH value will rise rapidly again, which will make the fermentation process difficult to control. Summary of the invention

[0003] The present inventor aims to provide a method for regulating the fermentation of macrolide antibiotics, which has the advantages of simple process, easy control, low energy consumption, improved equipment utilization, reduced consumption of nutrients, and significantly increased yield of target products.

[0004] The present invention is achieved in that:

[0005] The method for regulating and controlling the fermentation of macrolide antibiotics comprises the following steps:

[0006] In the primary metabolic stage of macrolide antibiotic-producing bacteria, the culture temperature is controlled at 27.0-30.0°C according to normal requirements, and the mycelium grows rapidly; after the macrolide antibiotic-producing bacteria complete the bacterial growth period, the fermentation culture temperature is lowered to 18.0-27.0°C to promote the large-scale synthesis of the target product. The macrolide antibiotics are antibiotics and their homologues produced by fermentation using the PKS biosynthesis pathway.

[0007] After the macrolide antibiotic-producing bacteria complete the bacterial growth period, the fermentation culture temperature is lowered for at least two periods, and the fermentation culture temperature in each period is different, first high and then low.

[0008] The macrolide antibiotics include Sirolimus, Tacrolimus, Avermectin, Spinosyn and Kitasamycin.

[0009] The macrolide antibiotic producing bacteria completes the bacterial growth phase 15-96 hours after the start of fermentation.

[0010] After the macrolide antibiotic-producing bacteria complete the bacterial growth period, i.e., 15-96 hours, the fermentation culture temperature is reduced from 27.0-30.0°C to 22.0-26.0°C to promote the large-scale synthesis of the target product.

[0011] After the macrolide antibiotic-producing bacteria complete the logarithmic growth phase, i.e., 15-96 hours, the fermentation culture temperature is reduced from 27.0-30.0°C to 23.0-25.0°C to promote the large-scale synthesis of the target product.

[0012] The entire fermentation cycle is 1-4 days longer than the traditional fermentation cycle.

[0013] The method for regulating the biosynthesis and fermentation of macrolide antibiotics comprises the following steps:

[0014] In the primary metabolic stage of macrolide antibiotic-producing bacteria, the culture temperature is controlled according to traditional requirements and the mycelium grows rapidly; after the macrolide antibiotic-producing bacteria complete the bacterial growth period, that is, 15-96 hours, the fermentation culture temperature is lowered by 3-8°C to promote the large-scale synthesis of the target product. The macrolide antibiotic is an antibiotic produced by fermentation using the PKS biosynthesis pathway and its homologues.

[0015] The macrolide antibiotic is erythromycin.

[0016] The advantages of the present invention are as follows:

[0017] The present invention adopts a two-stage temperature control strategy. In the primary metabolic stage of macrolide antibiotic producing bacteria, the culture temperature is controlled according to normal requirements and the mycelium grows rapidly. After the macrolide antibiotic producing bacteria completes the bacterial growth period, the fermentation culture temperature is significantly reduced to promote the large-scale synthesis of the target product.

[0018] The present invention significantly reduces the fermentation culture temperature after the macrolide antibiotic producing bacteria complete the logarithmic growth phase, and unexpectedly finds that the pH value and dissolved oxygen of the fermentation liquid change steadily during the fermentation process, which is easy to control, and the target product yield is significantly improved. By implementing a cooling process, the equipment utilization rate is improved, the consumption of nutrients is reduced, and energy conservation and emission reduction are achieved. The method is simple and easy to operate, and is very suitable for commercial production. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described in further detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The present invention encompasses all possible alternatives, improvements and equivalents within the scope of the claims. The specific techniques or conditions not specified in the following examples are all conventional techniques or conditions, or are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions.

[0020] Embodiment 1:

[0021] Species: Streptomyces avermitilis Streptomyces avermitilis SIIA-1502

[0022] Fermentation medium ratio:

[0023]

[0024] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, add amylase (0.5% of the weight of starch) and steam heat, gradually raise the temperature to 85℃, keep warm and stir for 60 minutes to fully liquefy the starch. Then put the above ingredients into the batching tank and add water to the fixed volume, stir for 15 minutes and measure the pH, the pH requirement is 6.9, raise the temperature, and when the temperature reaches 121℃, start sterilization for 30 minutes.

[0025] Fermentation volume: 6000L;

[0026] Inoculum size: 10 eggplant bottles;

[0027] Stirring speed: 80-220rpm;

[0028] Air flow: 1:0.5-0.8vvm;

[0029] Cultivation period: 315 hours.

[0030] In the first 80 hours of fermentation of avermectin-producing bacteria, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 29.0°C; from 80 hours to 200 hours, the culture temperature is controlled at 25.0°C; from 200 hours to 315 hours, the culture temperature is controlled at 27.0°C.

[0031] In the present invention, the content of avermectin is determined by HPLC high performance liquid phase detection method, as follows.

[0032] Analytical column: C18 column, 4.6mm×250mm×5um, column temperature: 30℃; isocratic elution, mobile phase: methanol: water: acetonitrile (8:1:1); flow rate: 1.2mL / min; detection wavelength: 245nm; injection volume: 10µL. The content of avermectin was calculated according to the area normalization method.

[0033] Take 2 ml of fermentation broth, add 10 ml of ethanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the avermectin content shows that the potency of avermectin at 315 hours of fermentation is 8.8 g / L.

[0034] Comparative Example 1:

[0035] Species: Streptomyces avermitilis Streptomyces avermitilis SIIA-1502

[0036] Fermentation medium ratio:

[0037]

[0038] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, add amylase (0.5% of the weight of starch) and steam heat, gradually raise the temperature to 85℃, keep warm and stir for 60 minutes to fully liquefy the starch. Then put the above ingredients into the batching tank and add water to the fixed volume, stir for 15 minutes and measure the pH, the pH requirement is 6.9, raise the temperature, and when the temperature reaches 121℃, start sterilization for 30 minutes.

[0039] Fermentation volume: 6000L;

[0040] Inoculum size: 10 eggplant bottles;

[0041] Stirring speed: 80-220rpm;

[0042] Air flow: 1:0.5-0.8vvm;

[0043] Cultivation period: 285 hours.

[0044] During the fermentation process of avermectin-producing bacteria, the culture temperature was controlled at 29°C.

[0045] In the present invention, the content of avermectin is determined by HPLC high performance liquid phase detection method, as follows.

[0046] Analytical column: C18 column, 4.6mm×250mm×5um, column temperature: 29℃; isocratic elution, mobile phase: methanol: water: acetonitrile (8:1:1); flow rate: 1.2mL / min; detection wavelength: 245nm; injection volume: 10µL. The content of avermectin was calculated according to the area normalization method.

[0047] Take 2 ml of fermentation broth, add 10 ml of ethanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the avermectin content shows that the potency of avermectin at 315 hours of fermentation is 5.1 g / L.

[0048] Embodiment 2:

[0049] Bacterial species: Actinomycetes SIIA-1602 ( Actinoplanes SIIA-1602

[0050] Fermentation medium ratio:

[0051]

[0052] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, steam heating, gradually raise the temperature to 70℃, keep warm and stir for 10 minutes to make the starch fully gelatinized. Then put the above ingredients into the batching tank and add water to the fixed volume. After stirring for 15 minutes, measure the pH. The pH requirement is 6.9. Raise the temperature. When the temperature reaches 121℃, start sterilization for 30 minutes.

[0053] Fermentation volume: 6000L;

[0054] Inoculation amount: 8%;

[0055] Stirring speed: 80-220rpm;

[0056] Air flow: 1:0.5-0.8vvm;

[0057] Cultivation period: 220 hours.

[0058] In the early stage of sirolimus-producing bacteria fermentation, 0-60 hours, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 28.0°C; from 80 hours to 220 hours, the culture temperature is controlled at 23.0°C.

[0059] In the present invention, the content of sirolimus is determined by HPLC high performance liquid phase detection method, specifically as follows:

[0060] Take 3 ml of fermentation broth, add 7 ml of acetone and mix well, ultrasonicate for 30 minutes, centrifuge at 12000 rpm, and take the supernatant for HPLC analysis. Chromatographic conditions: C18 (250 mm×4.6 mm, 5μm) chromatographic column, mobile phase methanol: acetonitrile: water = 60:25:30 (v:v:v), column temperature 50℃, flow rate 1.0 mL / min, detection wavelength 277 nm, injection volume 10µl. The potency of sirolimus at 220 hours of fermentation was 1.98 g / L.

[0061] Comparative Example 2:

[0062] Bacterial species: Actinomycetes SIIA-1602 ( Actinoplanes SIIA-1602

[0063] Fermentation medium ratio:

[0064]

[0065] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, steam heating, gradually raise the temperature to 75℃, keep warm and stir for 20 minutes to make the starch fully gelatinized. Then put the above ingredients into the batching tank and add water to the fixed volume. After stirring for 15 minutes, measure the pH, the pH requirement is 6.9, raise the temperature, and when the temperature reaches 121℃, start sterilization for 30 minutes.

[0066] Fermentation volume: 6000L;

[0067] Inoculation amount: 8%;

[0068] Stirring speed: 80-220rpm;

[0069] Air flow: 1:0.5-0.8vvm;

[0070] Cultivation period: 192 hours.

[0071] The culture temperature was controlled at 28.0°C during the fermentation process of sirolimus-producing bacteria.

[0072] In the present invention, the content of sirolimus is determined by HPLC high performance liquid phase detection method, specifically as follows:

[0073] Take 3 ml of fermentation broth, add 7 ml of acetone and mix well, ultrasonicate for 30 minutes, centrifuge at 12000 rpm, and take the supernatant for HPLC analysis. Chromatographic conditions: C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase methanol: acetonitrile: water = 60:25:30 (v:v:v), column temperature 50 ° C, flow rate 1.0 mL / min, detection wavelength 277 nm, injection volume 10 µl. The potency of sirolimus at 192 hours of fermentation was 1.25 g / L.

[0074] Embodiment 3:

[0075] Bacterial species: New strain of Streptomyces truncatula SIIA-9818 ( Streptomyces qianfoensis sp.nov. SIIA-9818

[0076] Fermentation medium ratio:

[0077]

[0078] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, steam heating, gradually raise the temperature to 70℃, keep warm and stir for 10 minutes to make the starch fully gelatinized. Then put the above ingredients into the batching tank and add water to the fixed volume. After stirring for 15 minutes, measure the pH. The pH requirement is 6.9. Raise the temperature. When the temperature reaches 121℃, start sterilization for 30 minutes.

[0079] Fermentation volume: 6000L;

[0080] Inoculation amount: 8%;

[0081] Stirring speed: 80-220rpm;

[0082] Air flow: 1:0.8-1.0vvm;

[0083] Cultivation period: 220 hours.

[0084] In the early stage of tacrolimus-producing bacteria fermentation, 0-48 hours, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 28.5°C; from 80 hours to 220 hours, the culture temperature is controlled at 22.0°C.

[0085] In the present invention, the content of tacrolimus is determined by HPLC high performance liquid phase detection method, specifically as follows:

[0086] Take 3 ml of fermentation broth, add 7 ml of ethanol and mix well, ultrasonicate for 30 minutes, centrifuge at 12000 rpm, and take the supernatant for HPLC analysis. Chromatographic conditions: C8 (250 mm×4.6 mm, 5μm) chromatographic column, mobile phase V (isopropanol) ∶ V (water) = 35∶65, column temperature 50℃, flow rate 1.0 mL / min, detection wavelength 220 nm, injection volume 10μl. The potency of tacrolimus at the 220th hour of fermentation was 1.13 g / L.

[0087] Comparative Example 3:

[0088] Bacterial species: Actinomycetes SIIA-1602 ( Actinoplanes SIIA-1602

[0089] Fermentation medium ratio:

[0090]

[0091] Weigh the starch accurately according to the specified ratio and put it into the fermentation batching tank (add appropriate amount of water in advance), start stirring, steam heating, gradually raise the temperature to 75℃, keep warm and stir for 20 minutes to make the starch fully gelatinized. Then put the above ingredients into the batching tank and add water to the fixed volume. After stirring for 15 minutes, measure the pH, the pH requirement is 6.9, raise the temperature, and when the temperature reaches 121℃, start sterilization for 30 minutes.

[0092] Fermentation volume: 6000L;

[0093] Inoculation amount: 8%;

[0094] Stirring speed: 80-220rpm;

[0095] Air flow: 1:0.8-1.0vvm;

[0096] Cultivation period: 192 hours.

[0097] The culture temperature was controlled at 28.5°C during the fermentation process of tacrolimus-producing bacteria.

[0098] In the present invention, the content of tacrolimus is determined by HPLC high performance liquid phase detection method, specifically as follows:

[0099] Take 3 ml of fermentation broth, add 7 ml of ethanol and mix well, ultrasonicate for 30 minutes, centrifuge at 12000 rpm, and take the supernatant for HPLC analysis. Chromatographic conditions: C8 (250 mm×4.6 mm, 5μm) chromatographic column, mobile phase V (isopropanol) ∶ V (water) = 35∶65, column temperature 50℃, flow rate 1.0 mL / min, detection wavelength 220 nm, injection volume 10μl. The potency of tacrolimus at 192 hours of fermentation was 0.65 g / L.

[0100] Embodiment 4:

[0101] Bacterial species: Saccharopolyspora spinosa SIIA 1802 ( S. spinosa SIIA 1802)

[0102] Fermentation medium ratio:

[0103]

[0104] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank and add water to the fixed volume. Measure the pH after stirring for 15 minutes. The pH requirement is 7.2. Raise the temperature. When the temperature reaches 121°C, start sterilization for 35 minutes.

[0105] Fermentation volume: 6000L;

[0106] Inoculation amount: 10%;

[0107] Culture temperature: 28.0-30.0℃;

[0108] Stirring speed: 80-220rpm;

[0109] Air flow: 1:0.5-0.8vvm;

[0110] Cultivation period: 360 hours.

[0111] In the early stage of the fermentation of spinosad-producing bacteria, 0-96 hours, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 30.0°C; from 96 hours to 360 hours, the culture temperature is controlled at 26.0°C.

[0112] Take 1.0 ml of fermentation broth, add 10 ml of ethanol, shake for 15 minutes, centrifuge at high speed to obtain the supernatant, and analyze the content of spinosad by HPLC. The chromatographic column specifications are ZORBAX SB-C18 (5 μm, 4.6 mm×150 mm); column temperature: room temperature; mobile phase: methanol / acetonitrile / 2% ammonium acetate solution (volume ratio 45 / 45 / 10); flow rate: 1.5 mL / min; sample volume: 10 μL; detection wavelength: 250 nm. The response peak areas of spinosad A and D are calculated by the analysis software provided by the detection system, and the content of spinosad in the sample can be obtained by comparing with the standard.

[0113] Take 1.0 ml of the fermentation liquid, add 10 ml of ethanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the content of spinosad showed that the titer of spinosad at the 360th hour of fermentation was 1.82 g / L.

[0114] Comparative Example 4:

[0115] Bacterial species: Saccharopolyspora spinosa SIIA 1802 ( S. spinosa SIIA 1802)

[0116] Fermentation medium ratio:

[0117]

[0118] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank and add water to the fixed volume. Measure the pH after stirring for 15 minutes. The pH requirement is 7.2. Raise the temperature. When the temperature reaches 121°C, start sterilization for 35 minutes.

[0119] Fermentation volume: 6000L

[0120] Inoculation amount: 10%

[0121] Culture temperature: 28.0-30.0℃

[0122] Stirring speed: 80-220rpm

[0123] Air flow: 1:0.7-1.1vvm

[0124] Cultivation period: 320 hours

[0125] The culture temperature was controlled at 30.0°C during the fermentation process of tacrolimus-producing bacteria.

[0126] Take 1.0 ml of fermentation broth, add 10 ml of ethanol, shake for 15 minutes, centrifuge at high speed to obtain the supernatant, and analyze the content of spinosad by HPLC. The chromatographic column specifications are ZORBAX SB-C18 (5 μm, 4.6 mm×150 mm); column temperature: room temperature; mobile phase: methanol / acetonitrile / 2% ammonium acetate solution (volume ratio 45 / 45 / 10); flow rate: 1.5 mL / min; sample volume: 10 μL; detection wavelength: 250 nm. The response peak areas of spinosad A and D are calculated by the analysis software provided by the detection system, and the content of spinosad in the sample can be obtained by comparing with the standard. Take 1.0 ml of fermentation broth, add 10 ml of ethanol, shake for 15 minutes, centrifuge at high speed to obtain the supernatant, and analyze the content of spinosad by HPLC. The titer of spinosad at the 320th hour of fermentation is 1.05 g / L.

[0127] Embodiment 5:

[0128] Bacterial species: Streptomyces kitasato NBRC 13686

[0129] Fermentation medium ratio:

[0130]

[0131] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank and add water to the fixed volume. Measure the pH after stirring for 15 minutes. The pH requirement is 7.2. Raise the temperature. When the temperature reaches 121°C, start sterilization for 35 minutes.

[0132] Fermentation volume: 6000L;

[0133] Inoculation amount: 10%;

[0134] Culture temperature: 28.0-30.0℃;

[0135] Stirring speed: 80-220rpm;

[0136] Air flow: 1:0.8-1.0vvm;

[0137] Cultivation period: 116 hours.

[0138] Add 0.4% n-propanol every 10-20 hours (add when the bacterial concentration is greater than 20%).

[0139] In the early stage of fermentation of the columnar leucomycin producing bacteria, 0-15 hours, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 28.0°C; from 15 hours to 116 hours, the culture temperature is controlled at 24.0°C.

[0140] HPLC was used to analyze the content of leucopeniac. Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; 0.1 mol / L ammonium acetate solution (pH 4.5 adjusted by phosphoric acid)-methanol-acetonitrile (40:55:5) was used as mobile phase; column temperature was 60°C; detection wavelength was 231 nm. 10 μl of the reference solution (1) was injected into the liquid chromatograph. The recorded chromatogram should be consistent with the standard spectrum. The peak order of the main components of leucopeniac was A9, A8, A7, A6, A5, A4, A1, A3, and A13. According to the external standard method, based on the peak area of ​​leucopeniac A5, kitasamycin A5 should be 35% to 70%, A4 should be 5% to 25%, and A1 and A13 should both be 3% to 15%; the sum of the main components A9, A8, A7, A6, A5, A4, A1, A3, and A13 should not be less than 85%. The response peak area of ​​each component is calculated by the analysis software provided by the detection system, and the content of leucopenia in the sample can be obtained by comparing it with the standard.

[0141] Take 1.0 ml of the fermentation liquid, add 10 ml of ethanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the content of leukomycin in the column crystal showed that the titer at the 116th hour of fermentation was 11.22 g / L.

[0142] Comparative Example 5:

[0143] Bacterial species: Streptomyces kitasato NBRC 13686

[0144] Fermentation medium ratio:

[0145]

[0146] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank and add water to the fixed volume. Measure the pH after stirring for 15 minutes. The pH requirement is 7.2. Raise the temperature. When the temperature reaches 121°C, start sterilization for 35 minutes.

[0147] Fermentation volume: 6000L;

[0148] Inoculation amount: 10%;

[0149] Culture temperature: 28.0℃;

[0150] Stirring speed: 80-220rpm;

[0151] Air flow: 1:0.8-1.0vvm;

[0152] Cultivation period: 92 hours.

[0153] Add 0.4% n-propanol every 15-20 hours (add when the bacterial concentration is greater than 20%).

[0154] The culture temperature was controlled at 28°C.

[0155] HPLC was used to analyze the content of leucopeniac. Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler; 0.1 mol / L ammonium acetate solution (pH 4.5 adjusted by phosphoric acid)-methanol-acetonitrile (40:55:5) was used as mobile phase; column temperature was 60°C; detection wavelength was 231 nm. 10 μl of the reference solution (1) was injected into the liquid chromatograph. The recorded chromatogram should be consistent with the standard spectrum. The peak order of the main components of leucopeniac was A9, A8, A7, A6, A5, A4, A1, A3, and A13. According to the external standard method, based on the peak area of ​​leucopeniac A5, kitasamycin A5 should be 35% to 70%, A4 should be 5% to 25%, and A1 and A13 should both be 3% to 15%; the sum of the main components A9, A8, A7, A6, A5, A4, A1, A3, and A13 should not be less than 85%. The response peak area of ​​each component is calculated by the analysis software provided by the detection system, and the content of leucopenia in the sample can be obtained by comparing it with the standard.

[0156] Take 1.0 ml of the fermentation liquid, add 10 ml of ethanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the content of leucopeniain in the column crystal showed that the titer at the 92nd hour of fermentation was 8.02 g / L.

[0157] Embodiment 6:

[0158] Bacterial species: Streptomyces erythromycin SIIA1201 ( Streptomyces erythreus SIIA 1201)

[0159] Fermentation medium ratio:

[0160]

[0161] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank and add water to the fixed volume. Measure the pH after stirring for 15 minutes. The pH requirement is 6.2. Raise the temperature. When the temperature reaches 121°C, start sterilization for 35 minutes.

[0162] Fermentation volume: 600L;

[0163] Inoculation amount: 10%;

[0164] Stirring speed: 80-220rpm;

[0165] Air flow: 1:0.8-1.0vvm;

[0166] Cultivation period: 230 hours.

[0167] Add 0.4% n-propanol after 20 hours, soybean oil after 25 hours, and start adding sugar when the pH is higher than 6.9.

[0168] In the early stage of erythromycin-producing bacteria fermentation, 0-20 hours, the mycelium is rapidly grown through the basic culture medium, and the culture temperature is controlled at 34.0°C; from 20 hours to 230 hours, the culture temperature is controlled at 28.0°C.

[0169] HPLC was used to analyze the content of erythromycin. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; phosphate solution (8.7 g of potassium dihydrogen phosphate, 1000 ml of water, and the pH value was adjusted to 8.2 with phosphoric acid)-acetonitrile (40:60) was used as mobile phase; the flow rate was 0.8-1.0 ml per minute; the column temperature was 35°C; and the wavelength was 215 nm. 10 μl of the reference solution (1) was injected into the liquid chromatograph. The recorded chromatogram should be consistent with the standard spectrum. The peaks of erythromycin C, erythromycin A, impurity 1, erythromycin B, and erythromycin enol ether were eluted in the order of peaks. The content of erythromycin A in the test sample was calculated by the peak area according to the external standard method. The response peak area of ​​each component was calculated by the analysis software provided by the detection system, and the content of erythromycin in the sample was obtained by comparing it with the standard.

[0170] Take 1.0 ml of the fermentation liquid, add 10 ml of methanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the erythromycin content shows that the titer at the 230th hour of fermentation is 11.05 g / L.

[0171] Comparative Example 6:

[0172] Bacterial species: Streptomyces erythromycin SIIA1201 ( Streptomyces erythreus SIIA 1201)

[0173] Fermentation medium ratio:

[0174]

[0175] Accurately weigh the above ingredients according to the prescribed ratio and put them into the mixing tank, add water to the fixed volume, stir for 15 minutes and measure the pH. The pH requirement is 6.2. Raise the temperature and start sterilization for 35 minutes when the temperature reaches 121°C.

[0176] Fermentation volume: 600L;

[0177] Inoculation amount: 10%;

[0178] Culture temperature: 34.0℃;

[0179] Stirring speed: 80-220rpm;

[0180] Air flow: 1:0.8-1.0vvm;

[0181] Cultivation period: 200 hours.

[0182] Add 0.4% n-propanol after 20 hours, soybean oil after 25 hours, and start adding sugar when the pH is higher than 6.9.

[0183] HPLC was used to analyze the content of erythromycin. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; phosphate solution (8.7 g of potassium dihydrogen phosphate, 1000 ml of water, and the pH value was adjusted to 8.2 with phosphoric acid)-acetonitrile (40:60) was used as mobile phase; the flow rate was 0.8-1.0 ml per minute; the column temperature was 35°C; and the wavelength was 215 nm. 10 μl of the reference solution (1) was injected into the liquid chromatograph. The recorded chromatogram should be consistent with the standard spectrum. The peaks of erythromycin C, erythromycin A, impurity 1, erythromycin B, and erythromycin enol ether were eluted in the order of peaks. The content of erythromycin A in the test sample was calculated by the peak area according to the external standard method. The response peak area of ​​each component was calculated by the analysis software provided by the detection system, and the content of erythromycin in the sample was obtained by comparing it with the standard.

[0184] Take 1.0 ml of the fermentation liquid, add 10 ml of methanol, shake for 15 minutes, and centrifuge at high speed to obtain the supernatant. HPLC analysis of the erythromycin content shows that the titer at the 200th hour of fermentation is 7.82 g / L.

[0185] According to the embodiments 1-6 of the present invention, a two-stage temperature control strategy is adopted, the primary metabolic stage of macrolide antibiotics producing bacteria, in the primary metabolic stage of macrolide antibiotics producing bacteria, the culture temperature is controlled according to normal requirements, and the mycelium grows rapidly; after the macrolide antibiotic producing bacteria complete the logarithmic growth period, the fermentation culture temperature is significantly reduced to promote the large-scale synthesis of the target product. The method according to the present invention can promote the significant increase in the output of macrolide antibiotics, the fermentation process is easy to control, and the biosynthesis is maintained for a longer time, the equipment utilization rate is improved, the consumption of nutrients is reduced, and energy conservation and emission reduction are achieved. In comparative examples 1-6, the fermentation stage maintains the fermentation temperature to fluctuate within a small range, and the pH value is unstable in the middle and late stages of fermentation, which is manifested as a low pH value in the middle stage, a slow increase in titer, a rapid rise in the pH value in the late stage, and a stagnation in titer growth, which is more than 30% lower than the fermentation unit of the embodiment in which the temperature is lowered.

[0186] It can be seen that the method according to the present invention can promote the synthesis of macrolide antibiotics in the fermentation process and maintain a rapid synthesis rate for a long time. The method is simple and easy to operate, and is very suitable for commercial production.

[0187] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for regulating the fermentation of macrolide antibiotics, characterized in that: The macrolide antibiotics include Sirolimus, Tacrolimus, Avermectin, Spinosyn and Kitasamycin, and include the following steps: In the primary metabolic stage of macrolide antibiotic-producing bacteria, the culture temperature is controlled at 27.0-30.0°C according to normal requirements, the mycelium grows rapidly, and the macrolide antibiotic-producing bacteria complete the bacterial growth period 15-96 hours after the start of fermentation. After that, the fermentation culture temperature is reduced from 27.0-30.0°C to 22.0-27.0°C, wherein Avermectin is fermented for 80-200 hours, the culture temperature is controlled at 25.0°C, and for 200-315 hours, the culture temperature is controlled at 27.0°C. The entire fermentation cycle is extended by 1-4 days compared with the traditional fermentation cycle, thereby promoting the large-scale synthesis of the target product.

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