Erythromycin lactobionate raw material and production process of erythromycin lactobionate for injection

Abstract

The invention discloses an erythromycin lactobionate raw material and a production process of erythromycin lactobionate for injection, which greatly increases the output value of Saccharopolyspora erythraea strains by improving a co-culture system and utilizing the synergistic metabolism between the two strains of Escherichia coli BL21 and Saccharopolyspora erythraea, and improves the yield of the Saccharopolyspora erythraea. Wherein a metabolite of the escherichia coli BL21 and peanut oil in a plate culture medium have a synergistic effect, and the growth time of a saccharopolyspora erythraea strain can be effectively shortened and the titer of erythromycin can be improved through a gene transfer effect. Meanwhile, the erythromycin lactobionate obtained through a microbe co-culture means can remarkably reduce the toxic effect on mouse livers, liver injuries caused by antibiotics are effectively relieved, and the erythromycin lactobionate is superior to traditional erythromycin lactobionate.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and relates to a high-output-value erythromycin lactobionate raw material and a production process of erythromycin lactobionate for injection, which can significantly reduce the toxic effect on mouse liver and effectively alleviate liver damage caused by antibiotics. Background Art

[0002] Erythromycin is a macrolide antibiotic with a huge market application. It is generally obtained through erythromycin. Its molecular formula is C 37 H 67 O 13 It has stable chemical properties, is easily soluble in organic matter, slightly soluble in water, and has an antibacterial spectrum similar to that of penicillin. Its mechanism of action is mainly to bind to the 50S subunit of the ribonucleoprotein body, thereby inhibiting peptidyl transferase, affecting the translocation process of the ribonucleoprotein body, affecting the growth rate of the peptide chain, and inhibiting the synthesis of bacterial proteins. It is an antibacterial agent. Common clinical adverse reactions are mostly gastrointestinal reactions, such as nausea, stomach pain, diarrhea, oral pain, vomiting, and loss of appetite.

[0003] Among them, erythromycin lactobionate is the product of a 1:1 reaction between erythromycin and lactobionic acid. It belongs to the first generation of macrolide antibiotics. It is mainly used clinically to produce antibacterial effects by synthesizing proteins. It can treat infections caused by sensitive Legionella, Chlamydia, Mycoplasma, etc. Studies have shown that oils have a significant effect on the biosynthesis of erythromycin, and adding oils to erythromycin culture medium can effectively increase the titer of erythromycin. In addition, a common method for improving the titer of microbial culture is the co-culture system. Joint co-culture refers to the simultaneous inoculation of two or more microorganisms into the same system for cultivation. This culture method contains complex metabolic relationships, often accompanied by effects such as gene transfer and synergistic metabolism, but one of the relationships will be dominant. This method can be used to improve the quality of microorganisms and discover new substances. Summary of the invention

[0004] The present invention relates to a production process of erythromycin lactobionate raw material and erythromycin lactobionate for injection, and the present invention is achieved through the following technical scheme:

[0005] In a first aspect, the present invention provides a method for preparing an erythromycin-Escherichia coli co-culture composite high-yield strain, characterized in that: the specific method is: weigh the following substances of each concentration: starch 30-50g / L, dextrin 20-40g / L, corn steep liquor 20-40g / L, malt extract 20-40g / L, soybean powder cake 10-20g / L, agar 10-15g / L, glucose 3-6g / L, yeast extract 3-6g / L, ammonium sulfate 1.4-1.7g / L, ammonium nitrate 1.0-1.4g / L, calcium carbonate 2- 5g / L, sodium chloride 1-2g / L, peanut oil 2-5g / L, take 100mL of the composite plate culture medium prepared by the above formula, add 10mL of sterile water, use sterile operation to inoculate erythromycin and BL21 Escherichia coli 1:1 on the plate culture medium, culture in a constant temperature chamber at 31°C for 5-7 days, scrape and wash the cultured fresh spores in a 250mL triangular flask with glass beads, place it in a shaker with a shaking speed of 250r / min for 20min, take it out and filter to remove the supernatant to obtain the erythromycin-Escherichia coli co-cultured composite high-yield strain.

[0006] In a second aspect, the present invention provides a production process of erythromycin lactobionate raw material, comprising the following steps:

[0007] S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: weigh the following substances at different concentrations: starch 30-50 g / L, dextrin 20-40 g / L, corn steep liquor 20-40 g / L, malt extract 20-40 g / L, soybean powder cake 10-20 g / L, agar 10-15 g / L, glucose 3-6 g / L, yeast extract 3-6 g / L, ammonium sulfate 1.4-1.7 g / L, ammonium nitrate 1.0-1.4 g / L, calcium carbonate 2-5 g / L, sodium chloride 1 -2g / L, peanut oil 2-5g / L, take 100mL of the composite plate medium prepared by the above formula and add 10mL of sterile water, use aseptic operation to inoculate erythromycin and BL21 Escherichia coli 1:1 on the plate medium, culture in a constant temperature room at 31°C for 5-7 days, scrape and wash the cultured fresh spores in a 250mL triangular flask with glass beads, place it in a shaker at a shaking speed of 250r / min for 20min, and take it out to obtain the erythromycin-Escherichia coli composite strain fermentation liquid;

[0008] S2. Crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: Weigh 10-20 mL of erythromycin-Escherichia coli composite strain fermentation broth, centrifuge to remove bacterial cells and hyphae, adsorb using HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 mL, and then use 1000 mL of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is kept at 4°C under sterile conditions for use;

[0009] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: The crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2 was eluted with a solution having a content of methanol:chloroform = 1:15 and then subjected to thin layer chromatography analysis. After combining the color components, methanol was used for elution, and the chromatography analysis process was repeated twice to obtain the co-cultured erythromycin fermentation broth;

[0010] S4. Preparation of erythromycin lactobionate: Weigh 100-200 mL of the co-cultured erythromycin prepared in S3 and 20-40 mL of sterile water, mix them evenly, take 150-300 mL of commercial lactobionic acid with a purity of 97%, and add it dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C. The reaction end point is when the reaction liquid is clear and the pH is 6.9. After constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained.

[0011] Preferably: the S1 weighs 30 g / L starch, 20 g / L dextrin, 20 g / L corn steep liquor, 20 g / L malt extract, 10 g / L soybean powder, 10 g / L agar, 3 g / L glucose, 3 g / L yeast extract, 1.4 g / L ammonium sulfate, 1.0 g / L ammonium nitrate, 2 g / L calcium carbonate, 1 g / L sodium chloride, 2 g / L peanut oil, and the total composite plate medium is 100 mL, and sterile water is 10 mL;

[0012] Preferably: the erythromycin-Escherichia coli composite strain fermentation liquid weighed in S2 is 10 mL;

[0013] Preferably: the ratio of the methanol and chloroform solution weighed in S3 is 1:15;

[0014] Preferably: 100 mL of the co-cultured erythromycin prepared by S3 and 20 mL of sterile water are weighed and mixed evenly in S4, and 150 mL of commercial lactobionic acid with a purity of 97% is used;

[0015] Preferably, the commercial lactobionic acid weighed in S4 is added dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C.

[0016] In a third aspect, the present invention also provides a process for producing erythromycin lactobionate for injection using the erythromycin lactobionate raw material prepared above.

[0017] Preferably: a production process of erythromycin lactobionate for injection, the specific method is: add 500mL of water for injection and 500mL of 0.06g / mL mannitol injection solution into a batching pot, then use a stainless steel barrel to take 500mL of water for injection, add 175g of erythromycin lactobionate, 8.4g of aspartic acid and 21g of famotidine and stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600mL, then add 0.05% of needle-use activated carbon to the liquid medicine and stir for 15 minutes, decarbonize and filter, add water for injection to 2100mL, stir evenly, perform terminal sterilization filtration with a 0.22um pore size folded filter element, sterilization filtration is carried out under laminar flow in a Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is divided into 350 bottles and sealed in cleaned and sterilized glass bottles to obtain erythromycin lactobionate for injection.

[0018] The invention is beneficial in that:

[0019] 1. The erythromycin lactobionate prepared by the present invention is erythromycin co-cultured with Saccharopolyspora erythrae and Escherichia coli BL21. By improving the co-culture system and utilizing the synergistic metabolism between the two strains, not only can the output value of the Saccharopolyspora erythrae strain be greatly increased, but also the metabolites of Escherichia coli BL21 have a synergistic effect with the peanut oil in the plate culture medium required by Saccharopolyspora erythrae, and the growth time of the strain can be effectively shortened through gene transfer, thereby improving the potency of erythromycin.

[0020] 2. The erythromycin lactobionate obtained by the microbial co-culture method of the present invention can significantly reduce the toxic effect on the mouse liver and effectively alleviate the liver damage caused by antibiotics, which is superior to the traditional erythromycin lactobionate.

[0021] 3. The preparation process of erythromycin lactobionate prepared by the present invention is simple, suitable for batch production, and has excellent development prospects and medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the high performance liquid chromatography apparatus.

[0023] Figure 2 Graphs showing plasma biochemical indices of liver function in Examples 1-2 of the present invention and Comparative Examples 1-7.

[0024] Figure 3 It is a data diagram of the total potency of erythromycin of Examples 1-4 of the present invention and Comparative Examples 1-13.

[0025] Figure 4 It is a comparison chart of erythromycin A and erythromycin total potency data of Examples 1-4 of the present invention and Comparative Examples 1-13.

[0026] Figure 5 It is a graph of erythromycin content of Examples 1-4 of the present invention and Comparative Examples 1-13. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0028] Erythromycin Lactobionate Injection

[0029] Example 1

[0030] This embodiment provides a production process for preparing erythromycin lactobionate raw material, comprising the following steps:

[0031] S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: weigh the following substances of each concentration as follows: 30 g / L starch, 20 g / L dextrin, 20 g / L corn steep liquor, 20 g / L malt extract, 10 g / L soybean powder cake, 10 g / L agar, 3 g / L glucose, 3 g / L yeast extract, 1.4 g / L ammonium sulfate, 1.0 g / L ammonium nitrate, 2 g / L calcium carbonate, 1 g / L sodium chloride, and 2 g / L peanut oil, take 100 mL of the composite plate medium configured with the above formula, add 10 mL of sterile water, use aseptic operation to inoculate erythromycin and BL21 Escherichia coli 1:1 on the plate medium, culture in a constant temperature chamber at 31°C for 5 days, scrape and wash the cultured fresh spores in a 250 mL triangular flask with glass beads, place it in a shaker at a shaking speed of 250 r / min for 20 min, and then take it out to obtain the erythromycin-Escherichia coli composite strain fermentation liquid;

[0032] The specific preparation method of 1L composite plate medium is as follows: weigh the following materials: 30g starch, 20g dextrin, 20g corn steep liquor, 20g malt extract, 10g soybean powder cake, 10g agar, 3g glucose, 3g yeast extract, 1.4g ammonium sulfate, 1.0g ammonium nitrate, 2g calcium carbonate, 1g sodium chloride, 2g peanut oil, add 900ml deionized water, stir and heat to boil until completely dissolved, then adjust the volume to 1L, and sterilize at 121℃ for 15min. After sterilization, pour every 100ml into a sterile plate to make 100mL composite plate medium for later use;

[0033] S2. Crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: Weigh 10 mL of erythromycin-Escherichia coli composite strain fermentation broth, centrifuge to remove bacterial cells and hyphae, adsorb using HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 mL, and then use 1000 mL of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is kept at 4°C under sterile conditions for use;

[0034] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: The crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2 was eluted with a solution having a content of methanol:chloroform = 1:15 and then subjected to thin layer chromatography analysis. After combining the color components, methanol was used for elution, and the chromatography analysis process was repeated twice to obtain the co-cultured erythromycin fermentation broth;

[0035] S4. Preparation of erythromycin lactobionate: Weigh 100 mL of the co-cultured erythromycin prepared in S3 and 20 mL of sterile water, mix them evenly, take 150 mL of commercial lactobionic acid with a purity of 97%, and add it dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5° C. The reaction end point is when the reaction solution is clear and the pH is 6.9. After constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained;

[0036] S5. Production process of erythromycin lactobionate for injection: add 500mL of water for injection and 500mL of 0.06g / mL mannitol injection solution into a batching pot, then use a stainless steel bucket to take 500mL of water for injection, add 175g of erythromycin lactobionate, 8.4g of aspartic acid and 21g of famotidine and stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600mL, then add 0.05% of needle-grade activated carbon to the liquid medicine and stir for 15 minutes, decarbonize and filter, add water for injection to 2100mL, stir evenly, perform terminal sterilization filtration with a 0.22um pore size folded filter element, sterilization filtration is carried out under laminar flow in a Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is divided into 350 bottles and sealed in cleaned and sterilized glass bottles to obtain erythromycin lactobionate for injection.

[0037] Comparative Example 1: In step S1, except that BL21 Escherichia coli was replaced with DH5a Escherichia coli, the other parameters were the same as those in Example 1.

[0038] Comparative Example 2: In step S1, except that BL21 Escherichia coli was replaced with JM109 Escherichia coli, the other parameters were the same as those in Example 1.

[0039] Comparative Example 3: In step S1, except that BL21 Escherichia coli was replaced with TOP10 Escherichia coli, other parameters were the same as those in Example 1.

[0040] Comparative Example 4: In step S1, except that BL21 Escherichia coli was replaced by HB101 strain Escherichia coli, the other parameters were the same as those in Example 1.

[0041] Note: The erythraea and BL21 Escherichia coli required for the examples in this experiment were purchased from Shanghai Yiyan Biotechnology Co., Ltd., and the DH5a Escherichia coli, JM109 Escherichia coli, TOP10 Escherichia coli, and HB101 strain Escherichia coli required for the comparative example were purchased from the Microbiology Laboratory of the Institute of Agricultural Quality Standards and Testing Technology, Fujian Academy of Agricultural Sciences.

[0042] Example 2

[0043] This embodiment provides a production process for preparing erythromycin lactobionate raw material, comprising the following steps:

[0044] S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: Weigh the following substances at different concentrations: 35 g / L starch, 25 g / L dextrin, 25 g / L corn steep liquor, 25 g / L malt extract, 13 g / L soybean powder, 12 g / L agar, 4 g / L glucose, 4 g / L yeast extract, 1.5 g / L ammonium sulfate, 1.2 g / L ammonium nitrate, 3 g / L calcium carbonate, 1.3 g / L sodium chloride, 3 g / L peanut oil, and take 100 mL of the composite plate medium prepared by the above formula was added with 10 mL of sterile water, and erythromycin and BL21 Escherichia coli were inoculated on the plate medium at a ratio of 1:1 using aseptic operation, and cultured in a constant temperature room at 31° C. for 6 days, and the cultured fresh spores were scraped and placed in a 250 mL triangular flask with glass beads, and placed in a shaker at a shaking speed of 250 r / min for 20 minutes, and after taking out, the erythromycin-Escherichia coli co-cultured composite high-yield strain was obtained;

[0045] Among them, the specific configuration method of 1L composite plate culture medium is as follows: weigh the following substances: 35g starch, 25g dextrin, 25g corn steep liquor, 25g malt extract, 13g soybean powder cake, 12g agar, 4g glucose, 4g yeast extract, 1.5g ammonium sulfate, 1.2g ammonium nitrate, 3g calcium carbonate, 1.3g sodium chloride, and 3g peanut oil, add 900ml deionized water, stir, heat and boil until completely dissolved, then adjust the volume to 1L, and sterilize at 121℃ under high pressure for 15min. After sterilization, pour every 100 ml into a sterile plate to prepare 100 ml of composite plate culture medium for standby use; S2, crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: weigh 13 ml of erythromycin-Escherichia coli composite strain fermentation liquid, centrifuge to remove bacterial cells and hyphae, adsorb using HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 ml, and then use 1000 ml of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is set aside under sterile conditions at 4 ° C;

[0046] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: The crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2 was eluted with a solution having a content of methanol:chloroform = 1:15 and then subjected to thin layer chromatography analysis. After combining the color components, methanol was used for elution, and the chromatography analysis process was repeated twice to obtain the co-cultured erythromycin fermentation broth;

[0047] S4. Preparation of erythromycin lactobionate: Weigh 130 mL of the co-cultured erythromycin prepared in S3 and 25 mL of sterile water and mix them evenly; take 195 mL of commercial lactobionic acid with a purity of 97% and add it dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C; the reaction end point is when the reaction solution is clear and the pH is 6.9; after constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained;

[0048] S5. Production process of erythromycin lactobionate for injection: add 500mL of water for injection and 500mL of 0.06g / mL mannitol injection solution into a batching pot, then use a stainless steel bucket to take 500mL of water for injection, add 175g of erythromycin lactobionate, 8.4g of aspartic acid and 21g of famotidine and stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600mL, then add 0.05% of needle-grade activated carbon to the liquid medicine and stir for 15 minutes, decarbonize and filter, add water for injection to 2100mL, stir evenly, perform terminal sterilization filtration with a 0.22um pore size folded filter element, sterilization filtration is carried out under laminar flow in a Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is divided into 350 bottles and sealed in cleaned and sterilized glass bottles to obtain erythromycin lactobionate for injection.

[0049] Comparative Example 5: In step S1, except that BL21 Escherichia coli was replaced with derived Escherichia coli BL21 (DE3), the other parameters were the same as those in Example 2.

[0050] Comparative Example 6: In step S1, except that BL21 Escherichia coli was replaced with derived Escherichia coli BL21 (AI), the other parameters were the same as those in Example 2.

[0051] Comparative Example 7: In step S1, except that BL21 Escherichia coli was replaced with derived Escherichia coli BL21 (DE3) pLysS, the other parameters were the same as those in Example 2.

[0052] Note: The erythraea and BL21 Escherichia coli required for the examples in this experiment were purchased from Shanghai Yiyan Biotechnology Co., Ltd., and the Escherichia coli BL21 (DE3), Escherichia coli BL21 (AI), and Escherichia coli BL21 (DE3) pLysS required for the comparative examples were purchased from the Microbiology Laboratory of the Institute of Agricultural Quality Standards and Testing Technology, Fujian Academy of Agricultural Sciences.

[0053] Liver damage caused by antibiotics is a common adverse drug reaction in clinical practice. Various data show that erythromycin can cause abnormal changes in various liver biochemical indicators. This experiment selected 110 specific pathogen-free all-male BALB / c mice, all purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., with a mass of 20-25g. They were randomly divided into 11 groups, each with 10 animals. Two control groups and nine experimental groups composed of erythromycin lactobionate cultivated with different schemes were established. The 9 experimental groups were injected with erythromycin lactobionate injection prepared by Examples 1-2 of the present invention and Comparative Examples 1-7. The specific experimental process is as follows:

[0054] Each mouse was injected with normal saline every day. Control group 1 was not injected with erythromycin lactobionate, and control group 2 was injected with traditional erythromycin lactobionate produced by Hunan Kelun Pharmaceutical Co., Ltd. The experimental groups were injected with 250 mg / kg erythromycin lactobionate by intraperitoneal injection on days 1, 3, and 7, respectively. After the last administration, each group was fasted for 12 hours, bled through the femoral artery, and the plasma samples were tested for liver function plasma biochemical indicators. The specific results are shown in Tables 1 and Figure 2 .

[0055] Table 1 Table of plasma biochemical indicators of liver function in the control group and experimental group

[0056]

[0057] In Table 1 and Figure 2By observing the plasma biochemical indicators of liver function in the data, it can be seen that compared with the control group 1, the three plasma liver function biochemical indicators showed significant differences in alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Compared with Examples 1-2, the triglyceride content of Comparative Examples 1-7 increased significantly, and the difference was statistically significant, while there was no significant numerical difference in the alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase content between Examples 1-2 and Control Group 1, which shows that the erythromycin lactobionate cultured in the examples can basically avoid damage to the liver of mice. At the same time, compared with the indicators of liver function of mice injected with traditional erythromycin lactobionate in the control group 2, it can be seen that the various indicators of Examples 1-2 are obviously at a lower level, indicating that the erythromycin lactobionate prepared by co-culturing BL21 Escherichia coli with erythromycin can significantly reduce the toxic effect on the liver of mice compared with traditional erythromycin lactobionate. This indicates that whether BL21 E. coli is replaced with other E. coli strains DH5a, JM109, TOP10, HB101, or BL21 E. coli derivatives BL21(DE3), BL21(AI), BL21(DE3)pLysS, it is impossible to avoid the damage of erythromycin to mouse liver metabolism. The data prove that only the erythromycin lactobionate obtained by the co-culture model of BL21 E. coli and Saccharopolyspora erythrae can significantly reduce the toxic effect on mouse liver, which has great clinical application value.

[0058] Example 3

[0059] This embodiment provides a production process for preparing erythromycin lactobionate raw material, comprising the following steps:

[0060] S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: weigh the following substances at different concentrations: starch 45 g / L, dextrin 35 g / L, corn steep liquor 35 g / L, malt extract 35 g / L, soybean powder 17 g / L, agar 14 g / L, glucose 5 g / L, yeast extract 5 g / L, ammonium sulfate 0.6 g / L, ammonium nitrate 1.3 g / L, calcium carbonate 4 g / L, sodium chloride 1.7 g / L, peanut oil 4 g / L, and take 100 mL of the composite plate medium prepared by the above formula was added with 10 mL of sterile water, and erythromycin and BL21 Escherichia coli were inoculated on the plate medium at a ratio of 1:1 using aseptic operation, and cultured in a constant temperature room at 31° C. for 6 days, and the cultured fresh spores were scraped and placed in a 250 mL triangular flask with glass beads, and placed in a shaker at a shaking speed of 250 r / min for 20 minutes, and after taking out, the erythromycin-Escherichia coli co-cultured composite high-yield strain was obtained;

[0061] Among them, the specific configuration method of 1L composite plate culture medium is as follows: weigh the following substances: 45g starch, 35g dextrin, 35g corn steep liquor, 35g malt extract, 17g soybean powder cake, 14g agar, 5g glucose, 5g yeast extract, 0.6g ammonium sulfate, 1.3g ammonium nitrate, 4g calcium carbonate, 1.7g sodium chloride, and 4g peanut oil, add 900ml deionized water, stir, heat and boil until completely dissolved, then adjust the volume to 1L, and sterilize at 121℃ under high pressure for 15min. After sterilization, pour every 100 ml into a sterile plate to prepare 100 ml of composite plate culture medium for later use; S2, crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: weigh 17 ml of erythromycin-Escherichia coli composite strain fermentation liquid, centrifuge to remove bacterial cells and hyphae, adsorb with HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 ml, and then use 1000 ml of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is set aside under sterile conditions at 4 ° C;

[0062] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: The crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2 was eluted with a solution having a content of methanol:chloroform = 1:15 and then subjected to thin layer chromatography analysis. After combining the color components, methanol was used for elution, and the chromatography analysis process was repeated twice to obtain the co-cultured erythromycin fermentation broth;

[0063] S4. Preparation of erythromycin lactobionate: Weigh 170 mL of the co-cultured erythromycin prepared in S3 and 35 mL of sterile water, mix them evenly, take 255 mL of commercial lactobionic acid with a purity of 97%, and add it dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C. The reaction end point is when the reaction solution is clear and the pH is 6.9. After constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained;

[0064] S5. Production process of erythromycin lactobionate for injection: add 500mL of water for injection and 500mL of 0.06g / mL mannitol injection solution into a batching pot, then use a stainless steel bucket to take 500mL of water for injection, add 175g of erythromycin lactobionate, 8.4g of aspartic acid and 21g of famotidine and stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600mL, then add 0.05% of needle-grade activated carbon to the liquid medicine and stir for 15 minutes, decarbonize and filter, add water for injection to 2100mL, stir evenly, perform terminal sterilization filtration with a 0.22um pore size folded filter element, sterilization filtration is carried out under laminar flow in a Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is divided into 350 bottles and sealed in cleaned and sterilized glass bottles to obtain erythromycin lactobionate for injection.

[0065] Comparative Example 8: In step S1, except that the peanut oil in the composite plate culture medium was replaced with olive oil of the same concentration, the other parameters were the same as those in Example 3.

[0066] Comparative Example 9: In step S1, except that the peanut oil in the composite plate culture medium was replaced with soybean oil of the same concentration, the other parameters were the same as those in Example 3.

[0067] Comparative Example 10: In step S1, except that the peanut oil in the composite plate culture medium was replaced with rapeseed oil of the same concentration, the other parameters were the same as those in Example 3.

[0068] Note: The Saccharopolyspora erythraea and BL21 Escherichia coli required in this experiment were purchased from Shanghai Yiyan Biotechnology Co., Ltd.

[0069] Example 4

[0070] This embodiment provides a production process for preparing erythromycin lactobionate raw material, comprising the following steps:

[0071] S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: weigh the following substances of each concentration: 50 g / L starch, 40 g / L dextrin, 40 g / L corn steep liquor, 40 g / L malt extract, 20 g / L soybean powder, 15 g / L agar, 6 g / L glucose, 6 g / L yeast extract, 1.7 g / L ammonium sulfate, 1.4 g / L ammonium nitrate, 5 g / L calcium carbonate, 2 g / L sodium chloride, 5 g / L peanut oil, take 100 mL of the composite plate medium prepared by the above formula, add 10 mL of sterile water, use aseptic operation to inoculate erythromycin and BL21 Escherichia coli 1:1 on the plate medium, culture in a constant temperature room at 31°C for 7 days, scrape and wash the cultured fresh spores in a 250 mL triangular flask with glass beads, place it in a shaker at a shaking speed of 250 r / min for 20 min, and take it out to obtain the erythromycin-Escherichia coli co-culture composite high-yield strain;

[0072] Among them, the specific configuration method of 1L composite plate culture medium is as follows: weigh the following substances: 50g starch, 40g dextrin, 40g corn steep liquor, 40g malt extract, 20g soybean powder cake, 15g agar, 6g glucose, 6g yeast extract, 1.7g ammonium sulfate, 1.4g ammonium nitrate, 5g calcium carbonate, 2g sodium chloride, and 5g peanut oil, add 900ml deionized water, stir, heat and boil until completely dissolved, then adjust the volume to 1L, and sterilize at 121℃ under high pressure for 15min. After sterilization, pour every 100 ml into a sterile plate to prepare 100 ml of composite plate culture medium for later use; S2, crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: weigh 20 ml of erythromycin-Escherichia coli composite strain fermentation liquid, centrifuge to remove bacterial cells and hyphae, adsorb with HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 ml, and then use 1000 ml of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is set aside under sterile conditions at 4 ° C;

[0073] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: The crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2 was eluted with a solution having a content of methanol:chloroform = 1:15 and then subjected to thin layer chromatography analysis. After combining the color components, methanol was used for elution, and the chromatography analysis process was repeated twice to obtain the co-cultured erythromycin fermentation broth;

[0074] S4. Preparation of erythromycin lactobionate: Weigh 200 mL of the co-cultured erythromycin prepared in S3 and 40 mL of sterile water, mix them evenly, take 300 mL of commercial lactobionic acid with a purity of 97%, and add it dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5° C. The reaction end point is when the reaction solution is clear and the pH is 6.9. After constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained;

[0075] S5. Production process of erythromycin lactobionate for injection: add 500mL of water for injection and 500mL of 0.06g / mL mannitol injection solution into a batching pot, then use a stainless steel bucket to take 500mL of water for injection, add 175g of erythromycin lactobionate, 8.4g of aspartic acid and 21g of famotidine and stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600mL, then add 0.05% of needle-grade activated carbon to the liquid medicine and stir for 15 minutes, decarbonize and filter, add water for injection to 2100mL, stir evenly, perform terminal sterilization filtration with a 0.22um pore size folded filter element, sterilization filtration is carried out under laminar flow in a Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is divided into 350 bottles and sealed in cleaned and sterilized glass bottles to obtain erythromycin lactobionate for injection.

[0076] Comparative Example 11: In step S1, except that BL21 Escherichia coli was not added, the other parameters were the same as those of Example 4.

[0077] Comparative Example 12: In step S1, except that the inoculation ratio of Saccharopolyspora erythraea and BL21 Escherichia coli was changed to 1:2, the other parameters were the same as those in Example 4.

[0078] Comparative Example 13: In step S1, except that the inoculation ratio of Saccharopolyspora erythraea and BL21 Escherichia coli was changed to 2:1, the other parameters were the same as those in Example 4.

[0079] Note: The Saccharopolyspora erythraea and BL21 Escherichia coli required in this experiment were purchased from Shanghai Yiyan Biotechnology Co., Ltd.

[0080] (1) Determination of the chemical potency of erythromycin in co-culture by sulfuric acid hydrolysis method

[0081] This method is based on the property that erythromycin turns yellow after hydrolysis with sulfuric acid. The maximum absorption value can be reached by measuring the absorbance at 483nm. The following are the specific experimental steps.

[0082] First, 5 mL of the separated, purified, co-cultured erythromycin fermentation broth prepared in all S3 steps of Examples 1-4 and Comparative Examples 1-11 were accurately weighed, respectively, and placed in 100 mL volumetric flasks, 20 mL of anhydrous ethanol was added, and the mixture was shaken to dissolve, and then water was added to dilute the mixture to a fixed volume, and then the fermentation broth was filtered and diluted, and the filtrate was discarded. 2 mL of the mixed solution was accurately measured and placed in a 50 mL volumetric flask, 12 mL of a 5 mol / L sulfuric acid solution was added, and the mixture was placed in a water bath and heated for 7 min, maintaining the temperature at 80° C., and immediately taken out and cooled to room temperature in a water bath environment, and the absorption value was measured at 483 nm. Each sample was measured three times and the average value was taken. The test results are shown in Tables 2 and 3. Figure 3 .

[0083] Table 2 Data table of total potency of erythromycin determined by sulfuric acid hydrolysis method

[0084]

[0085]

[0086] From Table 2 and Figure 3 The data show that the total potency of erythromycin in Examples 1-4 is all above 7000 U·mL -1 Above, this shows that there is a synergistic effect in the preparation of erythromycin lactobionate by the co-cultivation mode of BL21 Escherichia coli and red Saccharopolyspora. In general, erythromycin will have a weak inhibitory effect on Escherichia coli, but Escherichia coli BL21 may have a strong resistance to erythromycin, and the inhibitory effect can be ignored, and this resistance will produce special metabolites to promote red Saccharopolyspora to produce erythromycin, and other Escherichia coli cannot produce this effect. And there is a synergistic effect between the metabolites produced by Escherichia coli BL21 and the peanut oil in the plate culture medium required for erythromycin, and the growth time of the strain can be effectively shortened by gene transfer, and the erythromycin titer can be improved. Comparing the total titer of erythromycin in Examples 1-4, it can be found that the total titer of Example 1 is the highest and the fluctuation is smaller and more stable. By observing the data of Comparative Examples 1-4, it is found that the titer of Example 1 cannot be achieved by replacing the BL21 Escherichia coli strain with different types of DH5a Escherichia coli strains, JM109 Escherichia coli strains, TOP10 Escherichia coli strains and HB101 Escherichia coli strains, indicating that the strain most preferably co-cultured with Saccharopolyspora erythrae is the BL21 Escherichia coli strain. By observing the data of Comparative Examples 8-10, it can be found that the special metabolites produced by Escherichia coli only have a synergistic effect with the peanut oil in the plate culture medium required for erythromycin, and the effect with soybean oil, olive oil and rapeseed oil is not obvious. In addition, the data of Comparative Examples 11-13 can also be concluded that whether it is a single culture of erythromycin or a change in the inoculation ratio of Escherichia coli BL21 and erythromycin, the total titer of the embodiment cannot be achieved.

[0087] (2) Determination of chemical potency of erythromycin A by high performance liquid chromatography

[0088] The high performance liquid chromatography method for the determination of erythromycin A is simple and quick, with good stability and data reproducibility. This method uses a Waters-e2695 high performance liquid chromatograph, such as Figure 1 As shown, the stationary phase used is a C18 column, the mobile phase is acetonitrile and 0.05mol / L potassium dihydrogen phosphate solution, the substance volume ratio is 32:68, the column flow rate is 1mL / min, and the detection wavelength is 210nm. Under the above conditions, 25mg of erythromycin separated, purified and co-cultured prepared in all S3 steps of Examples 1-4 and Comparative Examples 1-11 are placed in a 50mL volumetric flask, and the mobile phase solution is added to dilute the volume. After shaking, 20μL is added to the high performance liquid chromatograph, the retention time is 6.5min, and the separation degree with the magazine peak is kept greater than 1.5, and the chromatogram is recorded. Then the peak area is calculated using the external standard method, and the average value is taken after each sample is repeated three times to obtain the content of erythromycin A, and the potency of erythromycin A is calculated. The calculation formula is erythromycin A potency = erythromycin A content / erythromycin chemical total potency × 100%. The test data are shown in Table 3 and Figure 4-5 .

[0089] Table 3 HPLC determination of erythromycin A potency data and content

[0090] Test samples Erythromycin A content / % <![CDATA[Erythromycin A titer / U·mL -1 > Example 1 97.0 7180 Example 2 96.8 7133 Example 3 96.8 7154 Example 4 96.7 6980 Comparative Example 1 56.1 3166 Comparative Example 2 57.2 3082 Comparative Example 3 59.3 3051 Comparative Example 4 65.6 3616 Comparative Example 5 66.5 3285 Comparative Example 6 66.1 3370 Comparative Example 7 59.0 3309 Comparative Example 8 63.3 3019 Comparative Example 9 63.9 3571 Comparative Example 10 64.6 3308 Comparative Example 11 71.0 3619 Comparative Example 12 61.2 3126 Comparative Example 13 60.7 3077

[0091] It can be seen from the data in Table 3 that the erythromycin A content of Examples 1-4 is stable at more than 95%, and the erythromycin A titer is very high, which indicates that in the preparation material of erythromycin lactobionate, there is a synergistic metabolism between the two strains of Escherichia coli BL21 and the co-cultured red Saccharopolyspora, which can effectively shorten the growth time of the strain and significantly increase the erythromycin content. Observing the data of Comparative Examples 5-7, it is found that the erythromycin content of Example 2 cannot be reached by replacing the BL21 Escherichia coli strain with the derivative strains of the same genus BL21 (DE3) Escherichia coli strain, BL21 (AI) Escherichia coli strain, and BL21 (DE3) pLysS Escherichia coli strain, indicating that the synergistic effect of red Saccharopolyspora and BL21 Escherichia coli strain is the strongest.

[0092] The above-mentioned embodiments only express the specific implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present invention. It should be pointed out that, for those skilled in the art, without departing from the concept of the present invention, any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an erythromycin-Escherichia coli co-culture composite high-yield strain, characterized in that: The specific method is as follows: weigh the following substances of each concentration: starch 30-50g / L, dextrin 20-40g / L, corn steep liquor 20-40g / L, malt extract 20-40g / L, soybean powder 10-20g / L, agar 10-15g / L, glucose 3-6g / L, yeast extract 3-6g / L, ammonium sulfate 1.4-1.7g / L, ammonium nitrate 1.0-1.4g / L, calcium carbonate 2-5g / L, sodium chloride 1-2g / L, peanut oil 2-5g / L, take 100mL of the composite plate culture medium prepared by the above formula and add 10mL of sterile water, use aseptic operation to inoculate erythromycin and BL21 Escherichia coli in a 1:1 ratio on the plate culture medium, culture in a constant temperature chamber at 31°C for 5-7 days, scrape and wash the cultured fresh spores in a 250mL triangular flask with glass beads, place it in a shaker at a shaking speed of 250r / min for 20min, take it out and filter to remove the supernatant to obtain the erythromycin-Escherichia coli co-cultured composite high-yield strain.

2. A production process for erythromycin lactobionate raw material, characterized in that: The following steps are involved: S1. Preparation of erythromycin-Escherichia coli co-culture composite high-yield strain: weigh the following substances at different concentrations: starch 30-50 g / L, dextrin 20-40 g / L, corn steep liquor 20-40 g / L, malt extract 20-40 g / L, soybean powder cake 10-20 g / L, agar 10-15 g / L, glucose 3-6 g / L, yeast extract 3-6 g / L, ammonium sulfate 1.4-1.7 g / L, ammonium nitrate 1.0-1.4 g / L, calcium carbonate 2-5 g / L, sodium chloride 1 -2g / L, peanut oil 2-5g / L, take 100mL of the composite plate medium prepared by the above formula and add 10mL of sterile water, use aseptic operation to inoculate erythromycin and BL21 Escherichia coli 1:1 on the plate medium, culture in a constant temperature room at 31°C for 5-7 days, scrape and wash the cultured fresh spores in a 250mL triangular flask with glass beads, place it in a shaker at a shaking speed of 250r / min for 20min, and take it out to obtain the erythromycin-Escherichia coli composite strain fermentation product; S2. Crude extraction of erythromycin-Escherichia coli co-cultured composite high-yield strain: weigh 10-20 mL of erythromycin-Escherichia coli composite strain fermentation product, centrifuge to remove bacterial cells and hyphae, adsorb using HP-20 macroporous adsorption resin, and elute with ethanol repeatedly for 5 times, collect the eluate and add it to a spherical vacuum decompression concentrator until it is dry, use deionized water to make the volume 1000 mL, and then use 1000 mL of ethyl acetate solution to extract 3 times, and then reduce the pressure and concentrate again to obtain a crude extract, which is kept at 4°C under sterile conditions for use; S3. Isolation and purification of the co-cultured erythromycin fermentation broth: taking the crude extract of the erythromycin-Escherichia coli co-cultured composite high-yield strain prepared in S2, eluting with a solution having a content of methanol:chloroform = 1:15 and then performing thin layer chromatography analysis, combining the color components, eluting with methanol, and repeating the chromatography analysis process twice to obtain the co-cultured erythromycin fermentation broth; S4. Preparation of erythromycin lactobionate: Weigh 100-200 mL of the co-cultured erythromycin fermentation broth prepared in S3 and 20-40 mL of sterile water, mix them evenly, take 150-300 mL of commercial lactobionic acid with a purity of 97%, add the commercial lactobionic acid dropwise into the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C, and the reaction end point is when the reaction liquid is clear and the pH is 6.

9. After constant volume and filtration, high-titer and stable erythromycin lactobionate can be obtained.

3. The production process of erythromycin lactobionate raw material according to claim 2, characterized in that: The S1 weighed starch is 30g / L, dextrin is 20g / L, corn steep liquor is 20g / L, malt extract is 20g / L, soybean powder cake is 10g / L, agar is 10g / L, glucose is 3g / L, yeast extract is 3g / L, ammonium sulfate is 1.4g / L, ammonium nitrate is 1.0g / L, calcium carbonate is 2g / L, sodium chloride is 1g / L, peanut oil is 2g / L, and the total composite plate culture medium is 100mL.

4. The production process of erythromycin lactobionate raw material according to claim 3, characterized in that: The erythromycin-Escherichia coli composite strain fermentation liquid weighed by S2 is 10 mL.

5. The production process of erythromycin lactobionate raw material according to claim 4, characterized in that: The ratio of the methanol and chloroform solution weighed in S3 is 1:

15.

6. A raw material for preparing erythromycin lactobionate according to claim 5, characterized in that: The S4 weighs 100 mL of the co-cultured erythromycin prepared by S3, 20 mL of sterile water and mixes them evenly, and 150 mL of commercial lactobionic acid with a purity of 97%.

7. The production process of erythromycin lactobionate raw material according to claim 2, characterized in that: The crude extraction adsorption resin of the erythromycin-Escherichia coli co-cultured composite high-yield strain weighed by S2 is HP-20 macroporous adsorption resin.

8. The production process of erythromycin lactobionate raw material according to claim 2, characterized in that: The commercial lactobionic acid weighed in S4 is added dropwise to the erythromycin suspension at a rate of 300 mL / min under a constant temperature of 5°C.

9. A process for producing erythromycin lactobionate for injection using the erythromycin lactobionate raw material prepared according to any one of claims 2 to 8.

10. The production process of erythromycin lactobionate for injection according to claim 9, characterized in that: Add 500 mL of water for injection and 500 mL of 0.06 g / mL mannitol injection solution into a batching pot, then use a stainless steel bucket to take 500 mL of water for injection, add 175 g of erythromycin lactobionate, 8.4 g of aspartic acid and 21 g of famotidine, stir to dissolve, add the dissolved liquid into the batching pot, adjust the pH value to 4.7-5.5, add water for injection to 1600 mL, then add 0.05% of needle-use activated carbon into the liquid medicine, stir for 15 minutes, decarbonize and filter, add water for injection to 2100 mL, stir evenly, perform terminal sterilization filtration with a 0.22 um pore size folded filter element, perform sterilization filtration under laminar flow in a Class A clean area, strictly implement aseptic operation system, divide the sterilization filtration into 350 bottles, put them into cleaned and sterilized glass bottles and seal them, and obtain erythromycin lactobionate for injection.

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