Production process of erythromycin lactobionate raw material and erythromycin lactobionate for injection

Abstract

The present invention discloses a production process of erythromycin lactobionate raw material and erythromycin lactobionate for injection. By improving the co-culture system and utilizing the synergistic metabolism between two strains, namely Escherichia coli BL21 and Saccharopolyspora erythraea, the output value of Saccharopolyspora erythraea strain is significantly increased. Among them, there is a synergistic effect between the metabolite of Escherichia coli BL21 and peanut oil in the plate medium, and through gene transfer, the growth time of Saccharopolyspora erythraea strain can be effectively shortened and the erythromycin titer can be improved. At the same time, the erythromycin lactobionate obtained by the means of microbial co-culture in the present invention can significantly reduce the toxic effect on the liver of mice and effectively relieve the liver injury caused by antibiotics, which is superior to traditional erythromycin lactobionate.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and relates to a raw material of erythromycin lactobionate with high output value, significantly reduced toxic effects on the livers of mice, and an effective alleviation of liver injury caused by antibiotics, as well as a production process of injectable erythromycin lactobionate. Background Art

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

[0003] Among them, erythromycin lactobionate is the product of the 1:1 reaction of erythromycin and lactobionic acid. It belongs to the first-generation macrolide antibiotics and is mainly used clinically to inhibit protein synthesis to produce a bacteriostatic effect and can treat infections caused by sensitive Legionella, Chlamydia, Mycoplasma, etc. Studies have shown that oils have a significant impact on the biosynthesis of erythromycin. Adding oils to the erythromycin culture medium can effectively improve the erythromycin titer. In addition, common methods to improve the titer of microbial culture also include the co-culture system. Co-culture refers to inoculating two or more microorganisms into the same system for cultivation at the same time. This cultivation method contains complex metabolic relationships and is often accompanied by effects such as gene transfer and co-metabolism. However, one of the relationships will be in a dominant position, and the quality of microorganisms can be improved and new substances can be discovered through this method. Summary of the Invention

[0004] The present invention relates to a raw material of erythromycin lactobionate and a production process of injectable erythromycin lactobionate, and the present invention is realized through the following technical solutions:

[0005] In the first aspect, the present invention provides a method for preparing a co-cultured composite high-yield strain of erythromycin and Escherichia coli, which is characterized in that the specific method is as follows: Weigh the following substances at the following concentrations: 30-50 g / L of starch, 20-40 g / L of dextrin, 20-40 g / L of corn steep liquor, 20-40 g / L of malt extract, 10-20 g / L of soybean cake powder, 10-15 g / L of agar, 3-6 g / L of glucose, 3-6 g / L of yeast extract, 1.4-1.7 g / L of ammonium sulfate, 1.0-1.4 g / L of ammonium nitrate, 2-5 g / L of calcium carbonate, 1-2 g / L of sodium chloride, and 2-5 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula and adding 10 mL of sterile water, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 in a ratio of 1:1 onto the plate medium using aseptic operation, and culture in a constant temperature room at 31 °C for 5-7 days. Scrape and wash the cultured fresh spores into a 250 mL Erlenmeyer flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and after taking it out, filter to remove the supernatant to obtain the co-cultured composite high-yield strain of erythromycin and Escherichia coli.

[0006] In the second aspect, the present invention provides a production process for lactobionate erythromycin raw materials, including the following steps:

[0007] S1. Preparation of the fermentation broth of the co-cultured composite high-yield strain of erythromycin and Escherichia coli: Weigh the following substances at the following concentrations: 30-50 g / L of starch, 20-40 g / L of dextrin, 20-40 g / L of corn steep liquor, 20-40 g / L of malt extract, 10-20 g / L of soybean cake powder, 10-15 g / L of agar, 3-6 g / L of glucose, 3-6 g / L of yeast extract, 1.4-1.7 g / L of ammonium sulfate, 1.0-1.4 g / L of ammonium nitrate, 2-5 g / L of calcium carbonate, 1-2 g / L of sodium chloride, and 2-5 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula and adding 10 mL of sterile water, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 in a ratio of 1:1 onto the plate medium using aseptic operation, and culture in a constant temperature room at 31 °C for 5-7 days. Scrape and wash the cultured fresh spores into a 250 mL Erlenmeyer flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and after taking it out, the fermentation broth of the composite strain of erythromycin and Escherichia coli is obtained;

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

[0009] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli prepared in S2 and elute it with a solution with the content of methanol: chloroform = 1:15, and then conduct thin-layer chromatography analysis. After combining the colored components, elute with methanol, and repeat the chromatography analysis process 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 the constant temperature condition of 5°C. When the reaction solution becomes clear and the pH is 6.9, it is the end point of the reaction. After making up the volume and filtering, high-potency and stable erythromycin lactobionate can be obtained.

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

[0012] Preferably: The fermentation broth of the erythromycin - Escherichia coli co-cultured strain weighed in S2 is 10 mL;

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

[0014] Preferably: The co-cultured erythromycin prepared in S3 weighed in S4 is 100 mL and 20 mL of sterile water are mixed evenly, 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 the constant temperature condition of 5°C.

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

[0017] Preferably: A production process for erythromycin lactobionate for injection, the specific method being: Add 500 mL of injection water and 500 mL of mannitol injection solution at 0.06 g / mL to a batching pot, then use a stainless steel bucket to take 500 mL of injection water, add 175 g of erythromycin lactobionate, 8.4 g of aspartic acid, and 21 g of famotidine and stir to dissolve them. Add the dissolved liquid to the batching pot, adjust the pH value to 4.7 - 5.5, make up the injection water to 1600 mL, then add 0.05% (by mass) of activated carbon for injection to the liquid medicine and stir for 15 minutes, filter to remove carbon, make up the injection water to 2100 mL, stir evenly, and perform terminal sterilization filtration with a folded filter element with a pore size of 0.22 μm. The sterilization filtration is carried out under laminar flow in a Class A clean area, strictly implementing the aseptic operation system. Divide the liquid medicine after sterilization filtration into 350 bottles and seal them in washed and sterilized glass bottles, thus obtaining an erythromycin lactobionate for injection.

[0018] Advantages of the present invention:

[0019] 1. The erythromycin lactobionate prepared by the present invention selects erythromycin co-cultured from Saccharopolyspora erythraea 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 erythraea strain be greatly increased, but there is a synergistic effect between the metabolite of Escherichia coli BL21 and the peanut oil in the plate culture medium required by Saccharopolyspora erythraea. Through gene transfer, the growth time of the strain can be effectively shortened and the erythromycin titer can be increased.

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

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

[0022] Figure 1 It is a schematic diagram of a high-performance liquid chromatograph device.

[0023] Figure 2 It is a graph of plasma biochemical indexes of liver function for Examples 1 - 2 and Comparative Examples 1 - 7 of the present invention.

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

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

[0026] Figure 5 It is a chart of the erythromycin content of Examples 1-4 and Comparative Examples 1-13 of the present invention. Detailed implementation manners

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

[0028] Erythromycin Lactobionate Injection Example 1

[0029] This example provides a production process for preparing raw materials of erythromycin lactobionate, including the following steps:

[0030] S1. Preparation of fermentation broth of the co-cultured composite high-yield strain of erythromycin - Escherichia coli: Weigh the following substances at the following concentrations: 30 g / L of starch, 20 g / L of dextrin, 20 g / L of corn steep liquor, 20 g / L of malt extract, 10 g / L of soybean cake powder, 10 g / L of agar, 3 g / L of glucose, 3 g / L of yeast extract, 1.4 g / L of ammonium sulfate, 1.0 g / L of ammonium nitrate, 2 g / L of calcium carbonate, 1 g / L of sodium chloride, 2 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula and adding 10 mL of sterile water, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 at a ratio of 1:1 onto the plate medium using aseptic operation, and culture in a constant temperature room at 31 °C for 5 days. Scrape and wash the cultured fresh spores into a 250 mL triangular flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and the fermentation broth of the erythromycin - Escherichia coli composite strain can be obtained after taking it out;

[0031] Among them, the specific preparation method of 1 L of the composite plate medium is: Weigh the following substances: 30 g of starch, 20 g of dextrin, 20 g of corn steep liquor, 20 g of malt extract, 10 g of soybean cake powder, 10 g of agar, 3 g of glucose, 3 g of yeast extract, 1.4 g of ammonium sulfate, 1.0 g of ammonium nitrate, 2 g of calcium carbonate, 1 g of sodium chloride, 2 g of peanut oil. Add 900 ml of deionized water, stir and heat to boiling until completely dissolved, then make up the volume to 1 L, and sterilize at 121 °C for 15 min. After sterilization, pour 100 ml into a sterile petri dish to make 100 mL of the composite plate medium for standby;

[0032] S2. Crude extraction of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli: Weigh 10 mL of the fermentation broth of the erythromycin - Escherichia coli co-cultured strain, centrifuge to remove the bacteria and mycelia, adsorb with HP-20 macroporous adsorption resin, elute repeatedly 5 times with ethanol, collect the eluate and add it to a spherical vacuum concentrator until it is in a dry state, make up the volume to 1000 mL with deionized water, then extract 3 times with 1000 mL of ethyl acetate solution, and concentrate under reduced pressure again to obtain the crude extract, which is reserved under sterile conditions at 4°C;

[0033] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli prepared in S2 and elute it with a solution with a content of methanol: chloroform = 1:15, and then perform thin-layer chromatography analysis. After combining the colored components, elute with methanol, and continue to repeat the chromatography analysis process twice to obtain the co-cultured erythromycin fermentation broth;

[0034] 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 speed of 300 mL / min under the constant temperature condition of 5°C. When the reaction solution becomes clear and the pH is 6.9, it is the end point of the reaction. After making up the volume and filtering, high-potency and stable erythromycin lactobionate can be obtained;

[0035] S5. Production process of erythromycin lactobionate for injection: Add 500 mL of injection water and 500 mL of mannitol injection solution with a concentration of 0.06 g / mL to the batching pot. Then use a stainless steel bucket to take 500 mL of injection water, add 175 g of erythromycin lactobionate, 8.4 g of aspartic acid and 21 g of famotidine, stir to dissolve them, add the dissolved liquid to the batching pot, adjust the pH value to 4.7 - 5.5, make up the volume with injection water to 1600 mL, then add 0.05% (mass fraction) of needle-use activated carbon to the liquid medicine, stir for 15 minutes, filter to remove carbon, make up the volume with injection water to 2100 mL, stir evenly, and perform terminal sterilization filtration with a folded filter element with a pore size of 0.22 um. The sterilization filtration is carried out under the laminar flow in the Class A clean area, and the aseptic operation system is strictly implemented. The liquid medicine after sterilization filtration is filled into 350 cleaned and sterilized glass bottles and sealed to obtain a kind of erythromycin lactobionate for injection.

[0036] Comparative Example 1: In step S1, except that Escherichia coli BL21 is changed to Escherichia coli DH5a, the other parameters are the same as those in Example 1.

[0037] Comparative Example 2: In step S1, except that Escherichia coli BL21 is changed to Escherichia coli JM109, the other parameters are the same as those in Example 1.

[0038] Comparative Example 3: In step S1, except that Escherichia coli BL21 was changed to Escherichia coli TOP10, the other parameters were the same as those in Example 1.

[0039] Comparative Example 4: In step S1, except that Escherichia coli BL21 was changed to Escherichia coli HB101, the other parameters were the same as those in Example 1.

[0040] Note: In this experiment, the Saccharopolyspora erythraea and Escherichia coli BL21 required in the examples were both purchased from Shanghai Yiyan Biotechnology Co., Ltd. The Escherichia coli DH5a, Escherichia coli JM109, Escherichia coli TOP10, and Escherichia coli HB101 required in the comparative examples were all purchased from the Microbial Laboratory of the Institute of Agricultural Quality Standards and Testing Technology, Fujian Academy of Agricultural Sciences.

[0041] Example 2

[0042] This example provides a production process for preparing raw materials of erythromycin lactobionate, including the following steps:

[0043] S1: Preparation of the fermentation broth of the co-cultured composite high-yield strain of erythromycin-Escherichia coli: Weigh the following substances at the following concentrations: 35 g / L of starch, 25 g / L of dextrin, 25 g / L of corn steep liquor, 25 g / L of malt extract, 13 g / L of soybean cake powder, 12 g / L of agar, 4 g / L of glucose, 4 g / L of yeast extract, 1.5 g / L of ammonium sulfate, 1.2 g / L of ammonium nitrate, 3 g / L of calcium carbonate, 1.3 g / L of sodium chloride, and 3 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula and adding 10 mL of sterile water, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 on the plate medium at a ratio of 1:1 using aseptic operation, and culture in a constant temperature room at 31 °C for 6 days. Scrape and wash the cultured fresh spores into a 250 mL triangular flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and then the fermentation broth of the co-cultured composite high-yield strain of erythromycin-Escherichia coli is obtained;

[0044] Among them, the specific preparation method of 1 L of the composite plate medium is: Weigh the following substances: 35 g of starch, 25 g of dextrin, 25 g of corn steep liquor, 25 g of malt extract, 13 g of soybean cake powder, 12 g of agar, 4 g of glucose, 4 g of yeast extract, 1.5 g of ammonium sulfate, 1.2 g of ammonium nitrate, 3 g of calcium carbonate, 1.3 g of sodium chloride, and 3 g of peanut oil. Add 900 ml of deionized water, stir and heat to boiling until completely dissolved, then make up the volume to 1 L, and sterilize at 121 °C for 15 min. After sterilization, pour 100 ml of the sterile medium into a sterile petri dish to make 100 mL of the composite plate medium for standby;

[0045] S2. Crude extraction of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli: Weigh 13 mL of the fermentation broth of the erythromycin - Escherichia coli co-cultured strain, centrifuge to remove bacteria and mycelia, adsorb with HP-20 macroporous adsorption resin, elute repeatedly 5 times with ethanol, collect the eluate and add it to a spherical vacuum concentrator until it is in a dry state, make up the volume to 1000 mL with deionized water, then extract 3 times with 1000 mL of ethyl acetate solution, and concentrate under reduced pressure again to obtain the crude extract, which is reserved under sterile conditions at 4°C;

[0046] S3. Isolation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli prepared in S2 and elute it with a solution with a content of methanol: chloroform = 1:15, and then perform thin-layer chromatography analysis. After combining the colored components, elute with methanol, and continue to repeat the chromatography analysis process 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, 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 the constant temperature condition of 5°C. When the reaction solution becomes clear and the pH is 6.9, it is the end point of the reaction. After making up the volume and filtering, high-potency and stable erythromycin lactobionate can be obtained;

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

[0049] Comparative Example 5: In step S1, except that Escherichia coli BL21 is changed to derivative Escherichia coli BL21(DE3), the other parameters are the same as those in Example 2.

[0050] Comparative Example 6: In step S1, except that Escherichia coli BL21 is changed to derivative Escherichia coli BL21(AI), the other parameters are the same as those in Example 2.

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

[0052] Note: In this experiment, Saccharopolyspora erythraea and Escherichia coli BL21 required in the examples were both purchased from Shanghai Yiyan Biotechnology Co., Ltd. Escherichia coli BL21(DE3), Escherichia coli BL21(AI), and Escherichia coli BL21(DE3)pLysS required in the comparative examples were all purchased from the Microbial Laboratory of the Institute of Agro - quality Standards and Testing Technology, Fujian Academy of Agricultural Sciences.

[0053] Liver injury caused by antibiotics is a relatively common adverse drug reaction in clinical practice. A variety of data indicate that erythromycin can cause abnormal changes in multiple liver biochemical indexes. In this experiment, 110 specific - pathogen - free male BALB / c mice, all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a mass in the range of 20 - 25 g, were randomly divided into 11 groups, with 10 animals in each group. Two control groups and nine experimental groups composed of erythromycin lactobionate cultivated by different schemes were established. The 9 experimental groups were respectively injected with the erythromycin lactobionate injection prepared in Examples 1 - 2 and Comparative Examples 1 - 7 of the present invention. The specific experimental process is as follows:

[0054] Each mouse was injected with normal saline every day. Control group 1 was not given an injection of erythromycin lactobionate, and control group 2 was given traditional erythromycin lactobionate for injection produced by Hunan Kelun Pharmaceutical Co., Ltd. The experimental groups were respectively given continuous intraperitoneal injection of 250 mg / kg erythromycin lactobionate on the 1st, 3rd, and 7th days. After the last administration in each group, the animals were fasted for 12 hours, sacrificed by bleeding from the femoral artery, and the plasma specimens were tested for liver function plasma biochemical indexes. The specific results are shown in Table 1 and Figure 2 。

[0055] Table 1: Plasma biochemical indexes of liver function in the control group and the experimental groups

[0056]

[0057] In Table 1 and Figure 2From the plasma biochemical indexes of liver function observed in the data, it can be seen that compared with Control Group 1, significant differences occurred in three plasma liver function biochemical indexes, namely alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Compared with Examples 1-2, the triglyceride content in Comparative Examples 1-7 showed a significant increase, and the difference was statistically significant. However, there was no significant numerical difference in the alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase contents between Examples 1-2 and Control Group 1. This indicates that the erythromycin lactobionate cultured in the examples can basically avoid damaging the liver of mice. At the same time, from the indexes of the liver function of the mice injected with traditional erythromycin lactobionate in Control Group 2, it can be seen that each index of Examples 1-2 was significantly at a lower level, indicating that the erythromycin lactobionate prepared by co-culturing Escherichia coli BL21 and erythromycin can significantly reduce the toxic effect on the liver of mice compared with traditional erythromycin lactobionate. It shows that whether Escherichia coli BL21 is replaced with other Escherichia coli strains such as DH5a, JM109, TOP10, HB101, or the derivative strains of Escherichia coli BL21, namely BL21(DE3), BL21(AI), BL21(DE3)pLysS, the damage of erythromycin to the liver metabolism of mice cannot be avoided. The data prove that only the erythromycin lactobionate obtained by using the co-culture mode of Escherichia coli BL21 and Saccharopolyspora erythraea can significantly reduce the toxic effect on the liver of mice and has great clinical application value.

[0058] Example 3

[0059] This example provides a production process for preparing the raw material of erythromycin lactobionate, including the following steps:

[0060] S1. Preparation of the fermentation broth of the co-cultured composite high-yield strain of erythromycin-Escherichia coli: Weigh the following substances at each concentration: 45 g / L of starch, 35 g / L of dextrin, 35 g / L of corn steep liquor, 35 g / L of malt extract, 17 g / L of soybean cake powder, 14 g / L of agar, 5 g / L of glucose, 5 g / L of yeast extract, 0.6 g / L of ammonium sulfate, 1.3 g / L of ammonium nitrate, 4 g / L of calcium carbonate, 1.7 g / L of sodium chloride, and 4 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula and adding 10 mL of sterile water, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 on the plate medium at a ratio of 1:1 using aseptic operation, and culture them in a constant temperature room at 31°C for 6 days. Scrape and wash the cultured fresh spores into a 250 mL triangular flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and the fermentation broth of the co-cultured composite high-yield strain of erythromycin-Escherichia coli can be obtained after taking it out;

[0061] Among them, the specific preparation method of 1L of complex plate medium is as follows: Weigh the following substances: 45 g of starch, 35 g of dextrin, 35 g of corn steep liquor, 35 g of malt extract, 17 g of soybean cake powder, 14 g of agar, 5 g of glucose, 5 g of yeast extract, 0.6 g of ammonium sulfate, 1.3 g of ammonium nitrate, 4 g of calcium carbonate, 1.7 g of sodium chloride, and 4 g of peanut oil. Add 900 ml of deionized water, stir, heat, and boil until completely dissolved, then make up the volume to 1L. Autoclave at 121 °C for 15 min. After sterilization, pour 100 ml into a sterile petri dish for each to prepare 100 mL of complex plate medium for standby;

[0062] S2. Crude extraction of the fermentation broth of the co-cultured high-yield erythromycin-Escherichia coli strain: Weigh 17 mL of the fermentation broth of the erythromycin-Escherichia coli complex strain, centrifuge to remove the bacteria and mycelia, adsorb with HP-20 macroporous adsorption resin, elute repeatedly 5 times with ethanol, collect the eluate and add it to a spherical vacuum concentrator until it is in a dry state, make up the volume to 1000 mL with deionized water, then extract 3 times with 1000 mL of ethyl acetate solution, and concentrate under reduced pressure again to obtain the crude extract, which is kept for standby under sterile conditions at 4 °C;

[0063] S3. Separation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the fermentation broth of the co-cultured high-yield erythromycin-Escherichia coli strain prepared in S2 and elute it with a solution with the content of methanol: chloroform = 1:15, and then perform thin-layer chromatography analysis. After combining the colored components, elute with methanol and continue to repeat the chromatography analysis process twice to obtain the co-cultured erythromycin fermentation broth;

[0064] 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 the constant temperature condition of 5 °C. When the reaction solution becomes clear and the pH is 6.9, it is the end point of the reaction. After making up the volume and filtering, high-potency and stable erythromycin lactobionate can be obtained;

[0065] S5. Production process of erythromycin lactobionate for injection: Add 500 mL of injection water and 500 mL of mannitol injection solution with a concentration of 0.06 g / mL into the batching pot. Then, use a stainless steel bucket to take 500 mL of injection water, add 175 g of erythromycin lactobionate, 8.4 g of aspartic acid, and 21 g of famotidine, and stir to dissolve them. Add the dissolved liquid into the batching pot, adjust the pH value to 4.7 - 5.5, make up the injection water to 1600 mL. Then, add 0.05% (mass fraction) of activated carbon for injection into the liquid medicine, stir for 15 minutes, filter to remove carbon, make up the injection water to 2100 mL, stir evenly, and perform terminal sterilization filtration with a folded filter element with a pore size of 0.22 μm. The sterilization filtration is carried out under the laminar flow in the Class A clean area, and the aseptic operation system is strictly implemented. Divide the liquid medicine after sterilization filtration into 350 bottles and seal them in the washed and sterilized glass bottles to obtain a kind of erythromycin lactobionate for injection.

[0066] Comparative Example 8: In step S1, except that the peanut oil in the complex plate medium is changed to olive oil with the same concentration, the other parameters are the same as those in Example 3.

[0067] Comparative Example 9: In step S1, except that the peanut oil in the complex plate medium is changed to soybean oil with the same concentration, the other parameters are the same as those in Example 3.

[0068] Comparative Example 10: In step S1, except that the peanut oil in the complex plate medium is changed to rapeseed oil with the same concentration, the other parameters are the same as those in Example 3.

[0069] Note: In this experiment, the Saccharopolyspora erythraea and Escherichia coli BL21 required in the examples were both purchased from Shanghai Yiyan Biotechnology Co., Ltd.

[0070] Example 4

[0071] This example provides a production process for preparing raw materials of erythromycin lactobionate, including the following steps:

[0072] S1. Preparation of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli: Weigh the following substances at the following concentrations: 50 g / L of starch, 40 g / L of dextrin, 40 g / L of corn steep liquor, 40 g / L of malt extract, 20 g / L of soybean cake powder, 15 g / L of agar, 6 g / L of glucose, 6 g / L of yeast extract, 1.7 g / L of ammonium sulfate, 1.4 g / L of ammonium nitrate, 5 g / L of calcium carbonate, 2 g / L of sodium chloride, and 5 g / L of peanut oil. After taking 100 mL of the composite plate medium prepared according to the above formula, add 10 mL of sterile water. Using aseptic operation, inoculate Saccharopolyspora erythraea and Escherichia coli BL21 on the plate medium at a ratio of 1:1, and culture them in a constant temperature room at 31°C for 7 days. Scrape and wash the cultured fresh spores into a 250 mL Erlenmeyer flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and then the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli can be obtained after taking it out;

[0073] Among them, the specific preparation method of 1 L of the composite plate medium is: Weigh the following substances: 50 g of starch, 40 g of dextrin, 40 g of corn steep liquor, 40 g of malt extract, 20 g of soybean cake powder, 15 g of agar, 6 g of glucose, 6 g of yeast extract, 1.7 g of ammonium sulfate, 1.4 g of ammonium nitrate, 5 g of calcium carbonate, 2 g of sodium chloride, and 5 g of peanut oil. Add 900 ml of deionized water, stir and heat to boiling until completely dissolved, then make up the volume to 1 L, and sterilize it at 121°C for 15 min. After sterilization, pour 100 ml into a sterile petri dish to make 100 mL of the composite plate medium for standby;

[0074] S2. Crude extraction of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli: Weigh 20 mL of the fermentation broth of the co-cultured strain of erythromycin - Escherichia coli, centrifuge to remove the bacteria and mycelia, adsorb it with HP-20 macroporous adsorption resin, elute it 5 times repeatedly with ethanol, collect the eluate and add it to a spherical vacuum concentrator until it is in a dry state, make up the volume to 1000 mL with deionized water, then extract it 3 times with 1000 mL of ethyl acetate solution, and concentrate it under reduced pressure again to obtain the crude extract, which is kept for standby under sterile conditions at 4°C;

[0075] S3. Separation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the fermentation broth of the co-cultured high-yielding strain of erythromycin - Escherichia coli prepared in S2, elute it with a solution with the content of methanol: chloroform = 1:15, and then perform thin-layer chromatography analysis. After combining the colored components, elute it with methanol, and continue to repeat the chromatography analysis process twice to obtain the co-cultured erythromycin fermentation broth;

[0076] S4. Preparation of erythromycin lactobionate: Weigh 200 mL of the co-cultured erythromycin obtained 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 the constant temperature condition of 5°C. When the reaction solution becomes clear and the pH is 6.9, it is the end point of the reaction. After volume fixation and filtration, highly potent and stable erythromycin lactobionate can be obtained.

[0077] S5. Production process of erythromycin lactobionate for injection: Add 500 mL of injection water and 500 mL of mannitol injection solution with a concentration of 0.06 g / mL into the batching pot. Then, use a stainless steel bucket to take 500 mL of injection water, add 175 g of erythromycin lactobionate, 8.4 g of aspartic acid, and 21 g of famotidine, stir to dissolve them. Add the dissolved liquid into the batching pot, adjust the pH value to 4.7 - 5.5, supplement injection water to 1600 mL. Then, add 0.05% (mass fraction) of activated carbon for injection into the liquid medicine, stir for 15 minutes, filter to remove carbon, supplement injection water to 2100 mL, stir evenly, and perform terminal sterilization filtration with a folded filter element with a pore size of 0.22 μm. The sterilization filtration is carried out under the laminar flow in the Class A clean area, and the aseptic operation system is strictly implemented. Fill the sterilized and filtered liquid medicine into 350 cleaned and sterilized glass bottles and seal them to obtain an erythromycin lactobionate for injection.

[0078] Comparative Example 11: In step S1, except for not adding Escherichia coli BL21, the other parameters are the same as those in Example 4.

[0079] Comparative Example 12: In step S1, except for changing the inoculation ratio of Saccharopolyspora erythraea and Escherichia coli BL21 to 1:2, the other parameters are the same as those in Example 4.

[0080] Comparative Example 13: In step S1, except for changing the inoculation ratio of Saccharopolyspora erythraea and Escherichia coli BL21 to 2:1, the other parameters are the same as those in Example 4.

[0081] Note: In this experiment, Saccharopolyspora erythraea and Escherichia coli BL21 required in the examples were both purchased from Shanghai Yiyan Biotechnology Co., Ltd.

[0082] (1) Determination of the chemical potency of co-cultured erythromycin by sulfuric acid hydrolysis method

[0083] Based on the property that erythromycin shows a yellow color after hydrolysis reaction with sulfuric acid, the absorbance can reach the maximum absorption value at 483 nm. The following are the specific experimental steps.

[0084] First, accurately weigh 5 mL of the separated and purified co-cultured erythromycin fermentation broth prepared in step S3 of Examples 1-4 and Comparative Examples 1-11 respectively, place them in 100 mL volumetric flasks, add 20 mL of absolute ethanol, shake to dissolve, then dilute to the mark with water, and then filter and dilute the fermentation broth. Discard the filter residue. Accurately measure 2 mL of the mixed solution and place it in a 50 mL volumetric flask. Add 12 mL of 5 mol / L sulfuric acid solution, place it in a water bath and heat for 7 min, keep the temperature at 80 °C, immediately take it out and cool it to room temperature in a water bath environment, measure the absorbance at 483 nm, repeat the measurement three times for each sample and then take the average value. The test results are shown in Table 2 and Figure 3 .

[0085] Table 2 Data table for the determination of the total potency of erythromycin by sulfuric acid hydrolysis method

[0086]

[0087] From Table 2 and Figure 3 the data, it can be seen that the total potency of erythromycin in Examples 1-4 is all above 7000 U·mL -1 . This shows that there is a synergistic effect in the preparation of erythromycin lactobionate by the co-culture method of Escherichia coli BL21 and Saccharopolyspora erythraea. Generally speaking, erythromycin has 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. Moreover, this resistance effect will produce special metabolites to promote Saccharopolyspora erythraea 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 peanut oil in the plate medium required for erythromycin. Through gene transfer, the growth time of the strain can be effectively shortened and the erythromycin potency can be improved. Comparing the total potency of erythromycin in Examples 1-4, it can be found that the total potency of Example 1 is the highest and the fluctuation is smaller and more stable. Observing the data of Comparative Examples 1-4, it is found that replacing the Escherichia coli BL21 strain with different types of Escherichia coli DH5a strain, Escherichia coli JM109 strain, Escherichia coli TOP10 strain and Escherichia coli HB101 strain cannot reach the potency of Example 1, indicating that the most preferred strain for co-culturing with Saccharopolyspora erythraea is the Escherichia coli BL21 strain. 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 peanut oil in the plate medium required for erythromycin, and the effects with soybean oil, olive oil and rapeseed oil are not obvious. In addition, from the data of Comparative Examples 11-13, it can also be concluded that whether it is the single culture of erythromycin or changing the inoculation ratio of Escherichia coli BL21 and erythromycin, the total potency of the examples cannot be achieved.

[0088] (2) Determination of the chemical potency of erythromycin A by high performance liquid chromatography

[0089] The operation of determining erythromycin A by high performance liquid chromatography is simple and fast, with good stability and data reproducibility. This method uses a Waters-e2695 high performance liquid chromatograph. As Figure 1 shown, the stationary phase used is a C18 column, the mobile phase is acetonitrile and a 0.05 mol / L potassium dihydrogen phosphate solution, with a volume ratio of 32:68. The flow rate through the column is 1 mL / min, and the detection wavelength is 210 nm. Under the above conditions, 25 mg of the separated and purified co-cultured erythromycin prepared in all S3 steps of Examples 1-4 and Comparative Examples 1-11 was taken, placed in a 50 mL volumetric flask, diluted and fixed with the mobile phase solution. After shaking well, 20 μL was taken and added to the high performance liquid chromatograph. The retention time was 6.5 min, and the resolution from the impurity peak was maintained at greater than 1.5. The chromatogram was recorded. Then, the external standard method was used to calculate the peak area. After each sample was measured three times and the average value was taken, the content of erythromycin A was obtained, and the titer of erythromycin A was calculated. The calculation formula is erythromycin A titer = erythromycin A content / total chemical titer of erythromycin × 100%. The test data are shown in Table 3 and Figures 4 - 5 .

[0090] Table 3 Data and content table for determining the titer of erythromycin A by high performance liquid chromatography

[0091]

[0092] It can be seen from the data in Table 3 that the content of erythromycin A in Examples 1-4 is stable above 95%, and the titer of erythromycin A is very high. This shows that in the preparation materials of erythromycin lactobionate, there is a synergistic metabolic effect between the two strains of Escherichia coli BL21 and co-cultured Saccharopolyspora erythraea, which can effectively shorten the growth time of the strains and significantly increase the content of erythromycin. Observing the data of Comparative Examples 5-7, it was found that replacing the Escherichia coli BL21 strain with its derivative homologous strains Escherichia coli BL21(DE3) strain, Escherichia coli BL21(AI) strain, and Escherichia coli BL21(DE3)pLysS strain could not reach the erythromycin content of Example 2, indicating that the synergistic effect between Saccharopolyspora erythraea and Escherichia coli BL21 strain is the strongest.

[0093] The above embodiments only represent the specific implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A production process for erythromycin lactobionate raw materials, characterized in that: It includes the following steps: S1. Preparation of the erythromycin-Escherichia coli composite strain fermentate: First, prepare a composite plate medium with the following formula: starch 30 - 50 g / L, dextrin 20 - 40 g / L, corn steep liquor 20 - 40 g / L, malt extract 20 - 40 g / L, soybean cake powder 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 - 2 g / L, peanut oil 2 - 5 g / L, and the balance is deionized water. Take 100 mL of the composite plate medium prepared according to the above formula, add 10 mL of sterile water, and inoculate Saccharopolyspora erythraea and Escherichia coli BL21 at a ratio of 1:1 onto the composite plate medium added with sterile water under aseptic operation. Cultivate in a constant temperature room at 31 °C for 5 - 7 days, scrape and wash the cultured fresh spores into a 250 mL triangular flask with glass beads, place it in a shaker with a shaking speed of 250 r / min for 20 min, and the erythromycin-Escherichia coli composite strain fermentate can be obtained after taking it out; S2. Coarse extraction of the erythromycin-Escherichia coli composite strain fermentate: Weigh 10 - 20 mL of the erythromycin-Escherichia coli composite strain fermentate, centrifuge to remove bacteria and mycelia, adsorb it with HP-20 macroporous adsorption resin, elute it repeatedly 5 times with ethanol, collect the eluate and add it to a spherical vacuum concentration device until it is in a dry state, make up the volume to 1000 mL with deionized water, then extract it 3 times with 1000 mL of ethyl acetate solution, and concentrate it under reduced pressure again to obtain a crude extract, and reserve it under aseptic conditions at 4 °C; S3. Separation and purification of the co-cultured erythromycin fermentation broth: Take the crude extract of the erythromycin-Escherichia coli composite strain fermentate prepared in S2, elute it with a solution with the content of methanol:chloroform = 1:15 and then perform thin-layer chromatography analysis. After combining the colored components, elute it with methanol, and continue to repeat the chromatography analysis process twice to obtain the co-cultured erythromycin fermentation broth; S4. Preparation of the raw material 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%, and drip the commercial lactobionic acid into the erythromycin suspension at a speed of 300 mL / min under the constant temperature condition of 5 °C. When the reaction solution is clarified and the pH is 6.9, it is the reaction end point. After making up the volume and filtering, the raw material of erythromycin lactobionate can be obtained.

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

3. The production process of the erythromycin lactobionate raw material according to claim 2, characterized in that: The fermented product of the erythromycin-Escherichia coli composite strain weighed in S2 is 10 mL.

4. The production process of an erythromycin lactobionate raw material according to claim 3, characterized in that: The co-cultured erythromycin prepared in S3 weighed in S4 is 100 mL, the sterile water is 20 mL, and they are mixed evenly, and the commercial lactobionic acid with a purity of 97% is 150 mL.

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