CN109207536B

CN109207536B - Method for increasing content of milbemycin A3 in milbemycin fermentation product

Info

Publication number: CN109207536B
Application number: CN201711295635.9A
Authority: CN
Inventors: 赵子刚; 张葵
Original assignee: CHONGQING DAXIN PHARMACEUTICAL CO LTD; Peking University Founder Group Co Ltd; PKU Healthcare Industry Group
Current assignee: CHONGQING DAXIN PHARMACEUTICAL CO LTD; New Founder Holdings Development Co ltd; Peking University Medical Management Co ltd
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2021-12-03
Anticipated expiration: 2037-12-08
Also published as: CN109207536A

Abstract

The invention discloses a method for increasing the content of milbemycin A3 in a milbemycin fermentation product, which comprises the following steps: the method comprises the steps of slant strain culture, liquid seed culture, liquid fermentation culture and the like, wherein a supplemented sucrose solution is fed into fermentation liquor in the fermentation culture process, particularly after fermentation is carried out for 48 hours, a sodium acetate solution with the specificity of 0.05 percent of precursor is intermittently or continuously supplemented into the fermentation liquor every day, so that the content of A3 in a fermentation product is obviously improved, and the fermentation yield of the milbemycins is also improved.

Description

Method for increasing content of milbemycin A3 in milbemycin fermentation product

Technical Field

The invention relates to a fermentation process of milbemycins, belongs to the technical field of biological pharmacy, and particularly relates to a method for producing milbemycins by using a microbial liquid submerged fermentation technology.

Background

Milbemycins (milbemycins) are mixtures of a series of sixteen-membered macrolide antibiotics produced by fermentation of Streptomyces hygroscopicus (Streptomyces hygroscopicus). Most of more than 20 components of the milbemycins have broad-spectrum control activity on agricultural pests, such as aphids, mites, tentacles fulvidraco, intestinal parasites and other parasites which harm crops and livestock, and particularly have higher control effect on various mites. The milbemycins have the characteristics of strong action on pests, small dosage, safety to people and livestock, no environmental pollution, only killing the pests, no killing of natural enemies of the pests and no easy generation of drug resistance, and are a promising broad-spectrum, high-efficiency, novel and non-cross-resistance biological pesticide.

In the milbemycins, because the milbemycins A3 and A4 have strong biological activity and high yield, the commercialization of the milbemycins is performed around the milbemycins A3 and A4. Besides the wide use of the mixture of milbemycins A3, A4 as acaricides, the semisynthetic oximation products of milbemycins A3, A4, and Milbemycin oxime (Milbemycin oxime, CAS number: 129496-10-2), which is a novel macrocyclic lactone anthelmintic, has good effect on killing endoparasites and ectoparasites, and has good effect on controlling and preventing most common pet parasitic diseases. The milbemycin oxime is recognized as a broad-spectrum, efficient and safe anthelmintic especially due to the characteristics of low toxicity and high efficiency, and is especially safe for some dogs sensitive to ivermectin.

The milbemycin oxime is formed by mixing milbemycin oxime A3 and milbemycin oxime A4 according to a certain proportion, and the content of the milbemycin oxime A3+ A4 is required to be not less than 95%, wherein the proportion of A3 and A4 is (20 +/-5): (80. + -.5). The structural formulas of the milbemycin oxime A3 and A4 are shown as the following formulas:

milbemycin Oxime A3(Milbemycin Oxime A3): r is CH₃

Milbemycin Oxime a4(Milbemycin Oxime a 4): r is CH₂CH₃

In the industrial production of milbemycins, Streptomyces hygroscopicus (Streptomyces hygroscopicus) is mainly used for fermentation production, and in the fermentation process, milbemycins A3 and A4 are produced by thalli simultaneously and exist in a fermentation liquid in the form of a mixture. Because the structures and properties of A3 and A4 are very similar, only the substituents on C-25 are different and are methyl and ethyl respectively, and the separation of single components of A3 and A4 from fermentation liquor is very difficult, at present, the A3 and A4 are generally regarded as the same component in industry, and are extracted and separated from the fermentation liquor together, and then subjected to a series of semi-synthesis steps such as oxidation, oximation and the like, and finally separated and purified to obtain the finished product of the milbemycin oxime. This requires that the ratio of milbemycins A3 to A4 in the fermentation broth must be exactly (20. + -.5): (80 +/-5) so that the ratio of the finally obtained milbemycin oxime A3 to A4 can meet the requirement.

At present, a problem is encountered in domestic milbemycin oxime fermentation production, the total fermentation unit of milbemycin continuously increases with the continuous fermentation process of each batch of fermentation, but the fermentation unit of A3 in the middle and later stages of fermentation relatively slowly increases, so that the proportion of A3/A4 also continuously decreases from about 1:3 in the middle stage of fermentation to about 1:7 before tank placement, and the requirement of a final product is about 1:4, so the milbemycin oxime is often placed in the tank in advance when the fermentation unit is still low. The difficulty in production needs to find a method which can specifically improve the yield of A3 without affecting the yield of A4, so that the yield of milbemycin can be improved on the whole, the difficulty in the subsequent semi-synthesis production of milbemycin can be greatly reduced, and the production cost is greatly reduced.

According to the analysis of 400MHz 13C nuclear magnetic resonance spectrum of the alpha 2, alpha 4 and D components of the milbemycins, the carbon on the glycoside skeleton is derived from 7 acetate and 5 propionate, and the structure of the glycoside skeleton is completely the same as that of the abamectin. Therefore, it was concluded from the avermectin synthetic pathway (H Ikeda, S Omura. control of avermectin biosynthesis in Streptomyces avermitilis for the selective production of a usesul component, Chemiform, 1996,27(1):549-62) that the methyl substituent at C-25 of the milbemycin A3 component was derived from acetate metabolism and the ethyl substituent at C-25 of the A4 component was derived from propionate metabolism.

Disclosure of Invention

On the basis of the existing technology for producing the milbemycins by fermentation, the invention solves the technical problem of slow increase of the output of the milbemycins A3 in the middle and later stages of the fermentation by adding a proper amount of specific precursor sodium acetate in the fermentation process, and provides a process method capable of specifically increasing the fermentation output of the milbemycins A3, thereby improving the proportion of A3 in the milbemycins fermentation products.

The inventor finds that the milbemycins are secondary metabolites of streptomyces hygroscopicus, the specific ligands in the structure of the milbemycins A3 are derived from acetate, and the specific ligands in the structure of the milbemycins A4 are derived from propionate, so that the acetate is a specific precursor substance for the biosynthesis of the milbemycins A3, and the addition of acetate in the fermentation broth can increase the synthesis of the milbemycins A3 without affecting the synthesis of A4, so that the yield of A3 can be increased by a method of adding a proper amount of sodium acetate in the fermentation broth, and the yield of A4 is not increased, and the aim of improving the proportion of A3/A4 is fulfilled. The inventor proves through experiments that the yield of the milbemycin A3 can be improved in a small degree by adding a proper amount of sodium acetate in a fermentation formula, and meanwhile, the yield of the milbemycin A4 is not influenced basically.

On the basis of the research, the addition of a proper amount of acetate in the fermentation medium of the milbemycins is presumed to save the time and nutrition required by the bacteria for synthesizing acetate by themselves, so that the yield of A3 is improved, but experiments show that the effect of improving the yield of A3 is not obvious by adopting a measure of adding acetate in the fermentation basic medium, after the fermentation period is 48 hours, the primary metabolism is basically completed, and the acetate is supplemented when the secondary metabolism occupies the main position, so that the effect is better, and particularly, the effect of supplementing each time every day or continuously feeding is better.

On the basis of the research, the fermentation yield and the fermentation proportion of the milbemycin A3 are remarkably improved by the following technical means:

(1) slant culture of strain: inoculating Streptomyces hygroscopicus strain to slant culture medium, and culturing at 27-29 deg.C and 20-60% humidity for 10-12 days to obtain mature slant strain. The formula of the slant culture medium comprises 0.4% of sucrose, 0.1% of skimmed milk powder, 0.2% of yeast powder, 0.1% of peptone and 2.0% of agar.

(2) Liquid seed culture: scraping a proper amount of spores from mature slant strains, inoculating the spores into a liquid seed culture medium, and culturing for 40-48 hours at the temperature of 27-29 ℃ and the stirring speed of 100-. The formula of the liquid seed culture medium comprises 2 percent of sucrose, 0.1 percent of skim milk powder, 0.5 percent of soybean cake powder, 0.5 percent of peptone and 0.1 percent of calcium carbonate.

(3) Liquid fermentation culture: inoculating mature seed liquid into a fermentation culture medium according to the inoculation amount of 4-12%, fermenting and culturing at the temperature of 27-29 ℃ under the conditions of stirring speed of 100-. The fermentation is finished after 240-288h fermentation, and the fermentation broth containing the milbemycin is obtained. The formula of the liquid fermentation medium is as follows: 8 percent of cane sugar, 1.0 percent of skim milk powder, 1.0 percent of soybean cake powder, 1.0 percent of peptone, 0.5 percent of dipotassium hydrogen phosphate, 0.1 percent of ferrous sulfate, 0.01 percent of zinc sulfate, 0.05 percent of copper sulfate, 0.05 percent of sodium molybdate and 0.5 percent of calcium carbonate.

(4) Fermentation precursor supplement: after fermentation for 48 hours, 0.05 percent of sodium acetate is supplemented into the fermentation liquor every day, the sodium acetate is prepared into a solution with the concentration of 5 percent, and after sterilization, the sodium acetate solution is supplemented into a fermentation tank every day according to 1 percent of the volume of the fermentation liquor.

The technical scheme of the invention is as follows:

a method for increasing the content of milbemycin A3 in milbemycin fermentation product comprises adding a certain amount of acetate into the fermentation liquid during fermentation.

Further, the acetate comprises one or more of sodium acetate, potassium acetate, ammonium acetate, zinc acetate and the like, and is preferably sodium acetate.

Further, the acetate is added in the form of a solution; the concentration of the acetate solution used is generally 0.5% to 20% (mass fraction); preferably a 5% strength sodium acetate solution.

Further, at 48h of fermentation, the fermentation broth is supplemented daily with a certain amount of acetate (preferably sodium acetate) solution until the end of the fermentation.

Further, the amount of acetate to be fed to the fermentation broth per day is 0.01 to 0.1% (referring to the mass fraction of the fed acetate in the fermentation broth), preferably 0.05%.

Further, the acetate is supplemented to the fermentation broth in an intermittent manner once a day or in a continuous fed-batch manner.

Further, when the total sugar concentration in the fermentation broth is reduced to below 2.0% (mass fraction, the same applies hereinafter), the sucrose solution is added to maintain the total sugar concentration in the fermentation broth between 1.5 and 2.5%.

Further, after fermenting for 150h to 120-.

Further, sucrose supplementation is stopped 12h before the tank is placed (fermentation is finished).

Further, fermentation was terminated after 240-288h fermentation.

Further, the fermentation conditions include: the temperature is 27-29 ℃, the stirring speed is 100-.

In general, fermentation can be carried out using microorganisms that produce milbemycins as are conventional in the art, such as Streptomyces hygroscopicus (Streptomyces hygroscopicus).

On the basis of the common knowledge in the field, the above preferred conditions can be combined with each other to obtain the preferred embodiments of the invention.

Specifically, the method for increasing the proportion of the milbemycin A3 in the milbemycin fermentation product comprises the following steps:

1) slant culture of bacterial

Inoculating Streptomyces hygroscopicus (Streptomyces hygroscopicus) strain to a slant culture medium, and culturing at 27-29 deg.C and 20-60% humidity for 10-12 days to obtain mature slant strain;

the formula of the slant culture medium is as follows: 0.4 percent of sucrose, 0.1 percent of skim milk powder, 0.2 percent of yeast powder, 0.1 percent of peptone and 2.0 percent of agar.

2) Liquid seed culture

Scraping a proper amount of spores from a mature slant strain, inoculating the spores into a liquid seed culture medium, and performing aeration culture for 40-48 hours under the conditions of the temperature of 27-29 ℃, the stirring speed of 100-;

or further carrying out amplification culture on the mature seed liquid again according to the same method;

the formula of the liquid seed culture medium is as follows: 2% of cane sugar, 0.1% of skim milk powder, 0.5% of soybean cake powder, 0.5% of peptone and 0.1% of calcium carbonate;

typically, the seed liquid is inoculated in an amount of 1% to 8%.

3) Liquid fermentation culture

Inoculating mature seed liquid into a fermentation culture medium according to the inoculation amount of 4-12%, and performing aeration fermentation culture at the temperature of 27-29 ℃ and the stirring rotation speed of 100-;

the formula of the liquid fermentation medium is as follows: 8 percent of cane sugar, 1.0 percent of skim milk powder, 1.0 percent of soybean cake powder, 1.0 percent of peptone, 0.5 percent of dipotassium hydrogen phosphate, 0.1 percent of ferrous sulfate, 0.01 percent of zinc sulfate, 0.05 percent of copper sulfate, 0.05 percent of sodium molybdate and 0.5 percent of calcium carbonate;

4) when the fermentation time is 48 hours, sodium acetate solution with the concentration of 5 percent is supplemented into the fermentation liquor every day, the amount of the sodium acetate supplemented into the fermentation liquor every day is 0.01-0.1 percent (referring to the mass fraction of the supplemented sodium acetate in the fermentation liquor), and the preferred amount is 0.05 percent until the end of the fermentation;

the sodium acetate solution is supplemented intermittently once a day or continuously fed;

5) sampling and detecting the total sugar concentration in the fermentation liquid after fermenting for 150h after 120-f, and when the total sugar concentration is reduced to be below 2.0%, starting to supplement the sucrose solution, maintaining the total sugar concentration in the fermentation liquid to be between 1.5 and 2.5%, stopping supplementing sugar 12h before the fermentation is placed in a tank, and ending the fermentation after fermenting for 288 h.

The invention also comprises a fermentation product prepared by the method.

The invention has the beneficial effects that:

the invention provides a method for specifically improving the fermentation yield of milbemycin A3, and basically has no influence on the yield of milbemycin A4, so that the proportion of a milbemycin A3 component in a final product is improved. The process method solves the problem that the fermentation yield of the milbemycins A3 in the middle and later stages of the traditional milbemycins fermentation is slowly increased, so that the milbemycins A3 and A4 can be synchronously increased, the proportion of the milbemycins A3 in the fermentation product in the later stage of the fermentation is improved, the fermentation period is prolonged, and the fermentation yield of the milbemycins is obviously improved.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Slant culture was used as follows: inoculating Streptomyces hygroscopicus strain to slant culture medium, and culturing at 27-29 deg.C and 20-60% humidity for 10-12 days to obtain mature slant strain. The formula of the slant culture medium is as follows: 0.4 percent of sucrose, 0.1 percent of skim milk powder, 0.2 percent of yeast powder, 0.1 percent of peptone and 2.0 percent of agar.

The following formula of the liquid seed culture medium is as follows: 2 percent of cane sugar, 0.1 percent of skim milk powder, 0.5 percent of soybean cake powder, 0.5 percent of peptone and 0.1 percent of calcium carbonate.

The following formula of the liquid fermentation medium is as follows: 8 percent of cane sugar, 1.0 percent of skim milk powder, 1.0 percent of soybean cake powder, 1.0 percent of peptone, 0.5 percent of dipotassium hydrogen phosphate, 0.1 percent of ferrous sulfate, 0.01 percent of zinc sulfate, 0.05 percent of copper sulfate, 0.05 percent of sodium molybdate and 0.5 percent of calcium carbonate.

Example 1

(1) Taking the slant of the grown strain, scraping spores on the slant of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shake flask with the capacity of 50ml, and culturing the inoculated seed shake flask on a shaking table with the rotating speed of 180rpm at 28 ℃ for 48 h.

(2) Inoculating the cultured shake flask seeds to a 5L glass fermentation tank according to the inoculation amount of 8%, wherein the loading amount of the fermentation tank is 2.5L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring speed in the fermentation process.

(3) When the fermentation culture is carried out for 48 hours, the sterilized sodium acetate solution with the concentration of 5% is supplemented once a day, 25ml is supplemented each time, namely, the sodium acetate is supplemented once when the fermentation is carried out for 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours and 216 hours.

(4) When the fermentation culture is carried out for 150 hours, the sucrose supplement solution is fed in, the sucrose concentration in the fermentation liquor is controlled to be 1.5-2.5% by adjusting the sugar supplement speed, and the sugar supplement is stopped when the time is 228 hours.

(5) And (3) finishing the culture when the fermentation culture lasts for 240 hours, taking the fermentation liquor for detection, wherein the content of the total milbemycins in the fermentation liquor is 1.88g/L, the content of the milbemycins A3 is 0.37g/L, and the content of A3: a4 ═ 1: 4.08.

and taking the fermentation liquor of the same batch without being supplemented with sodium acetate as a reference, wherein the content of the total milbemycins in the fermentation liquor is 1.79g/L when the fermentation liquor is cultured for 240h, the content of the milbemycins A3 is 0.25g/L, A3: a4 ═ 1: 6.16.

the fermentation process for supplementing sodium acetate in the batch can improve the yield of the milbemycin A3 by about 48 percent, and the ratio of A3 to A4 is from 1: about 6, improvement by 1: and 4, the requirement of the final product of the milbemycin oxime is met, and the production cost of the milbemycin oxime is greatly reduced.

Example 2

(2) Inoculating the cultured shake flask seeds to a 10L stainless steel seed tank according to the inoculation amount of 1%, wherein the loading amount of the seed tank is 5.0L, the culture condition of the seed tank is that the tank temperature is 28 ℃, the ventilation volume is 1.0vvm, the stirring speed is 100rpm, the Dissolved Oxygen (DO) value on the seed tank is controlled to be more than or equal to 30% by increasing the stirring speed in the culture process, and the culture time of the seed tank is 24 h.

(3) Inoculating the seed liquid of the cultured seeding tank to a 50L fermentation tank according to the inoculation amount of 8%, wherein the loading amount of the fermentation tank is 30L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring rotating speed is 100rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring rotating speed in the culture process.

(4) When the fermentation culture is carried out for 48 hours, the sterilized sodium acetate solution with the concentration of 5 percent is supplemented, the supplement amount is 300 ml/day by using a constant-speed flow supplement manner, and the sodium acetate supplement is stopped at 288 hours.

(5) When the fermentation culture is carried out for 150 hours, the sucrose supplement solution is fed in, the sucrose concentration in the fermentation liquor is controlled to be 1.5-2.5% by adjusting the sugar supplement speed, and the sugar supplement is stopped when 276 hours.

(6) And (3) ending the culture when the fermentation culture lasts for 288h, taking the fermentation liquor for detection, wherein the content of the milbemycins in the fermentation liquor is 2.17g/L, the content of the milbemycins A3 is 0.42g/L, and the content of the milbemycins A3: a4 ═ 1: 4.17.

and taking the fermentation liquor of the same batch without being supplemented with sodium acetate as a reference, wherein the content of the total milbemycins in the fermentation liquor is 2.03g/L when the fermentation liquor is cultured for 288h, the content of the milbemycins A3 is 0.26g/L, A3: a4 ═ 1: 6.81.

the fermentation process for supplementing sodium acetate in the batch can improve the yield of the milbemycin A3 by about 62 percent, and the ratio of A3 to A4 is from 1: about 7, improvement by 1: about 4, the requirements of the final product of the milbemycin oxime are met, single components of A3 and A4 do not need to be separated, and the production difficulty and the production cost of the milbemycin oxime are greatly reduced.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for increasing the content of milbemycin A3 in a milbemycin fermentation product is characterized in that when the fermentation is carried out for 48 hours, a certain amount of acetate is supplemented into the fermentation liquor every day until the fermentation is finished; the amount of acetate added to the fermentation broth is 0.01-0.1% per day;

when the total sugar concentration in the fermentation liquor is reduced to below 2.0 percent, adding sucrose solution, and maintaining the total sugar concentration in the fermentation liquor between 1.5 and 2.5 percent;

the fermentation strain is Streptomyces hygroscopicus (Streptomyces hygroscopicus).

2. The method of claim 1, wherein the acetate salt comprises one or more of sodium acetate, potassium acetate, ammonium acetate, and zinc acetate.

3. The method of claim 2, wherein the acetate is added as a solution.

4. A process according to claim 3, characterized in that the acetate solution is used in a concentration of 0.5% to 20%.

5. A method according to claim 3, characterized in that the acetate solution used is a 5% strength sodium acetate solution.

6. The method according to any one of claims 1 to 5, wherein the amount of acetate fed to the fermentation broth per day is 0.05%.

7. The process according to any one of claims 1 to 5, wherein the acetate is fed to the fermentation broth in an intermittent manner once a day or in a continuous fed-batch manner.

8. The method according to any one of claims 1 to 5, wherein the total sugar concentration in the fermentation broth is measured after fermentation for 150 hours and the sucrose solution is started to be added when the total sugar concentration in the fermentation broth is reduced to below 2.0%, so as to maintain the total sugar concentration in the fermentation broth between 1.5 and 2.5%.

9. The method of claim 8, wherein sucrose supplementation is stopped 12 hours before fermentation is completed.

10. The method of any one of claims 1 to 5, wherein the fermentation conditions comprise: the temperature is 27-29 ℃, the stirring speed is 100-; and/or ending the fermentation after 240-288h of fermentation.

11. The method of claim 1, comprising the steps of:

1) slant culture of bacterial

the formula of the slant culture medium is as follows: 0.4% of cane sugar, 0.1% of skim milk powder, 0.2% of yeast powder, 0.1% of peptone and 2.0% of agar;

2) liquid seed culture

Scraping a proper amount of spores from a mature slant strain, inoculating the spores into a liquid seed culture medium, and performing aeration culture for 40-48 hours under the conditions of the temperature of 27-29 ℃, the stirring speed of 100-; or further carrying out amplification culture on the mature seed liquid again according to the same method;

3) liquid fermentation culture

Inoculating mature seed liquid into a fermentation culture medium according to the inoculation amount of 4-12%, and performing aeration fermentation culture at the temperature of 27-29 ℃ and the stirring rotation speed of 100-600rpm, wherein the aeration rate is 0.4-1.2vvm, and the dissolved oxygen value on a fermentation tank is controlled to be more than or equal to 30%;

4) when the fermentation is carried out for 48 hours, sodium acetate solution with the concentration of 5 percent is supplemented into the fermentation liquor every day, and the mass fraction of the sodium acetate supplemented into the fermentation liquor every day is 0.05 percent until the fermentation is finished;

12. A fermentation product produced by the method of any one of claims 1 to 11.