Methods for improving the fermentation yield of PF1022A

Abstract

The present invention provides a method for improving the fermentation yield of PF1022A, comprising using a Roselinia strain producing PF1022A as a production strain and carrying out aerobic fermentation in a fermentation medium, the fermentation medium comprising an assimilable carbon source, an assimilable nitrogen source and inorganic salts, the assimilable carbon source containing solid malt extract. The technical solution provided by the present invention is simple and easy to implement, which improves the production efficiency of PF1022A, greatly improves the yield of PF1022A, reduces production costs, and facilitates the large-scale industrial production of PF1022A.

Description

[Technical field]

[0001] The present invention relates to the technical field of industrial microbial fermentation, and in particular to a method for improving the fermentation yield of PF1022A. [Background technology]

[0002] Emodepside is a semi-synthetic derivative of PF1022A, which has cidal effects against invertebrates such as nematodes and roundworms. Bayer Animal Health GmbH has developed combinations of emodepside and praziquantel (Profender) and emodepside and toltrazuril (Procox) for use as anthelmintics in cats and dogs, respectively. Profender (combined with praziquantel) has been marketed in the United States since 2007. Bayer has collaborated with DNDi to develop Emodepside (a human drug) for the treatment of onchocerciasis "river blindness," which began phase 1 trials in healthy volunteers in 2016, completed a single ascending dose study in 2017, and a multiple ascending dose study at the end of 2018.

[0003] As a precursor raw material for the synthesis of emodepside, PF1022A has a very promising market prospect and has attracted much attention. At present, there are solid-phase synthesis and biosynthesis methods for the synthesis of PF1022A. The solid-phase synthesis method is not suitable for large-scale production due to its cumbersome steps and complicated process. In addition, there are few reports on the fermentation production of PF1022A, and the yield of PF1022A is low. In response to this situation, our company selected and bred a brand new strain of PF1022A-producing bacteria, which is specifically disclosed in CN201711276671.0. Although this strain has advantages such as high productivity and high genetic stability, the disclosed fermentation method still has problems such as insufficient fermentation yield and high fermentation cost, which is not suitable for large-scale industrialized production. Summary of the Invention

[0004] The present invention provides a method for improving the fermentation yield of PF1022A, comprising using a Roselinia strain producing PF1022A as a production strain and performing aerobic fermentation in a fermentation medium comprising an assimilable carbon source, an assimilable nitrogen source and inorganic salts, the assimilable carbon source containing solid malt extract.

[0005] In a preferred embodiment, the content of the solid malt extract in the fermentation medium is 70 to 200 g / L, more preferably 100 to 200 g / L, even more preferably 140 to 200 g / L, and most preferably 160 g / L.

[0006] In a preferred embodiment, the assimilable carbon source further comprises a carbon source selected from one of glucose, maltodextrin, maltose, soybean oil, soluble starch, corn starch, or a combination of any two or more of the foregoing.

[0007] In a preferred embodiment, said assimilable carbon source further comprises soybean oil and maltodextrin, preferably soybean oil.

[0008] In a preferred embodiment, the assimilable nitrogen source is selected from one of yeast powder, cottonseed cake powder, wheat germ powder, yeast extract powder, soybean cake powder, peanut cake powder, or a combination of any two or more of the above.

[0009] In a preferred embodiment, the assimilable nitrogen source is a combination of yeast flour and cottonseed cake flour.

[0010] In a preferred embodiment, the inorganic salt is selected from one or a combination of any two or more of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, diammonium hydrogen sulfate, potassium chloride, sodium chloride, magnesium chloride, nickel chloride, calcium carbonate, and calcium chloride.

[0011] In a preferred embodiment, the inorganic salt is a combination of calcium carbonate, sodium chloride and magnesium sulfate.

[0012] In a preferred embodiment, the fermentation medium contains 75 to 210 g / L of an assimilable carbon source, 15 to 40 g / L of an assimilable nitrogen source, and 6 to 10 g / L of an inorganic salt, preferably, the fermentation medium contains 105 to 210 g / L of an assimilable carbon source, 15 to 40 g / L of an assimilable nitrogen source, and 6 to 10 g / L of an inorganic salt, and more preferably, the fermentation medium contains 145 to 200 g / L of an assimilable carbon source, 15 to 40 g / L of an assimilable nitrogen source, and 6 to 10 g / L of an inorganic salt.

[0013] In a preferred embodiment, the fermentation medium comprises solid malt extract, soybean oil, cottonseed cake powder, yeast powder, sodium chloride, magnesium sulfate, and calcium carbonate.

[0014] In a preferred embodiment, in the fermentation medium, soybean oil is 5-10 g / L, yeast powder is 5-10 g / L, cottonseed cake powder is 10-30 g / L, sodium chloride is 2-3 g / L, magnesium sulfate is 2-3 g / L, and calcium carbonate is 2-4 g / L.

[0015] In a preferred embodiment, the fermentation medium contains 70-200 g / L solid malt extract, preferably 100-200 g / L, more preferably 140-200 g / L, most preferably 160 g / L solid malt extract, 10 g / L soybean oil, 25 g / L cottonseed cake powder, 10 g / L yeast powder, 2 g / L sodium chloride, 2 g / L magnesium sulfate, and 2 g / L calcium carbonate.

[0016] In a preferred embodiment, the culture temperature of the fermentation is 22 to 28°C, preferably 24 to 26°C, more preferably 25°C, and the culture time of the fermentation is 6 to 10 days, preferably 7 to 10 days, and most preferably 9 days.

[0017] In a preferred embodiment, the Rosellinia strain producing PF1022A is Rosellinia sp. HS-NF-1412Z having the deposit number CGMCC NO. 13893.

[0018] In the present invention, "g / L" refers to the weight of the relevant substance added to 1 L of fermentation medium.

[0019] In the course of research, the inventor has unexpectedly discovered through many experiments that when the fermentation medium contains solid malt extract as a carbon source (especially within a certain range), the fermentation yield of PF1022A can be significantly improved (the fermentation yield can reach up to 6000 μg / mL or more), but when it does not contain solid malt extract as a carbon source, or when the carbon source is other substances (such as corn starch, sucrose, maltose, etc.), this effect cannot be obtained. The technical solution provided by the present invention is simple and easy to implement, which can improve the production efficiency of PF1022A, greatly improve the yield of PF1022A, reduce production costs, and facilitate the large-scale industrial production of PF1022A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The technical solutions in the embodiments of the present invention are described below, but the described embodiments are only a part of the present invention. The following comparative examples and examples are used to explain the present invention, but are not used to limit the scope of the present invention. The experimental methods in the following examples that do not show specific conditions are selected according to conventional methods and conditions, or product instructions.

[0021] In the present invention, the strain producing PF1022A is HS-NF-1412Z, and the strain used in the examples of the present invention is Rosellinia sp. HS-NF-1412Z, which is deposited at the Center for Ordinary Microorganisms of the China Microbial Species Depository under the accession number CGMCC NO. 13893 and disclosed in CN201711276671.0. Various materials and reagents described in the present invention are commonly used in this field and can be obtained through conventional commercial channels.

[0022] High-performance liquid chromatography detection method for PF1022A: The chromatography column used was an Elite C18 silica gel column (5 μm, 4.6 × 250 mm). Separation was performed using acetonitrile:water (volume ratio 80:20) as the mobile phase at a flow rate of 1.0 mL / min, UV wavelength of 218 nm, injection volume of 20 μL, and column temperature of 30°C. The retention time of PF1022A was approximately 10.5 minutes, and the runtime was 1.9 times that of PF1022A.

[0023] Preparation Example 1 (1) Preparation of slant medium: Potato dextrose agar (PDA) medium was used for the slant: 200g of potatoes were cut into small pieces, 1000mL of water was added and boiled for 30 minutes, potato debris was removed by filtration, water was added to the filtrate to make 1000mL, 20g of dextrose and 18g of agar were added, the pH was adjusted to natural pH, sterilized at 121℃ for 20 minutes, cooled to about 55℃, poured into a flat plate, and cooled to solidify. The frozen strain with the number HS-NF-1412Z was inoculated into the slant medium and cultivated at 25℃±1℃ for 6 days.

[0024] (2) Preparation of seed medium: The seed medium per liter contained 20 g glucose, 20 g soluble corn starch, 15 g yeast extract powder, 20 g cottonseed cake powder, 2 g sodium chloride, 2 g magnesium sulfate, and 2 g calcium carbonate, and was adjusted to 1000 mL with water and pH 6.0. 25 mL was filled into a 250 mL shaking Erlenmeyer flask and sterilized at 121°C for 20 minutes.

[0025] Seed culture method: Freshly growing Rosellinia sp. HS-NF-1412Z strain was inoculated into the seed culture medium and cultured at a shaking table speed of 250 r / min at 25°C ± 1°C for 80 hours.

[0026] Control Example 1 Carbon source containing corn starch (the content of corn starch in the fermentation medium was 120 g / L) The seed culture liquid prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (1 L basis) contained 120 g of corn starch to which 0.024 g of amylase for gelatinization had been added in advance, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and water was added to make it 1000 mL, the pH was adjusted to 6.0, and 25 mL was filled into a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. for 9 days.

[0027] PF1022A in the fermentation broth was detected according to the following method (the detection method for PF1022A in Comparative Examples 2 to 4 and Examples 1 to 9 was the same as this): (1) The fermentation liquid was diluted 5-fold with anhydrous methanol and mixed well. (2) Sonication was performed for 30 minutes, and the sonicated fermentation liquid was filtered. (3) The supernatant was removed and filtered through a 0.22 μm organic phase needle filter membrane. (4) The filtered liquid was detected by HPLC, and the fermentation unit of PF1022A was found to be 3290 μg / mL.

[0028] Control Example 2: Carbon source containing maltodextrin (the maltodextrin content in the fermentation medium was 120 g / L) The seed culture liquid prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 120 g of maltodextrin, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 3112 μg / mL when the fermentation liquid was detected by HPLC.

[0029] Control Example 3: Carbon source containing maltose (the maltose content in the fermentation medium was 120 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 120 g of maltose, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL in a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 3213 μg / mL when the fermentation liquid was detected by HPLC.

[0030] Control Example 4 Carbon source containing sucrose (the sucrose content in the fermentation medium was 120 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 120 g sucrose, 10 g soybean oil, 25 g cottonseed cake powder, 10 g yeast powder, 2 g sodium chloride, 2 g magnesium sulfate, and 2 g calcium carbonate, and was made 1000 mL with water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 623 μg / mL when the fermentation liquid was detected by HPLC.

[0031] Example 1 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 120 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 120 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 5540 μg / mL when the fermentation liquid was detected by HPLC.

[0032] Example 2 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 70 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 70 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was adjusted to 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL of a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 4112 μg / mL when the fermentation liquid was detected by HPLC.

[0033] Example 3 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 100 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 100 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 4905 μg / mL when the fermentation liquid was detected by HPLC.

[0034] Example 4 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 140 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 140 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 5870 μg / mL when the fermentation liquid was detected by HPLC.

[0035] Example 5 Carbon source containing solid malt extract The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 140 g of solid malt extract, 10 g of soybean oil, 25 g of soybean cake powder, 10 g of yeast extract powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was adjusted to 1000 mL by adding water and adjusting the pH to 6.0. 25 mL was filled into a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. for 9 days. The fermentation liquid was detected by HPLC, and the yield of PF1022A was 5562 μg / mL.

[0036] Example 6 Carbon Source Containing Solid Malt Extract The seed culture liquid prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 140 g of solid malt extract, 40 g of maltodextrin, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 5412 μg / mL when the fermentation liquid was detected by HPLC.

[0037] Example 7 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 160 g / L) The seed culture prepared in Preparation Example 1 was inoculated into the fermentation medium. The fermentation medium (based on 1 L) contained 160 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and water was added to make 1000 mL, pH was adjusted to 6.0, and 25 mL was filled using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The temperature was 25 ° C and the shaking culture was performed at 250 r / min on a shaking table for 9 days, and the content of PF1022A in the fermentation liquid cultured for different culture times (6 to 10 days) was detected respectively. The results of the fermentation unit of PF1022A measured by HPLC are shown in Table 1. When the fermentation culture time was 6 to 10 days, the yield of PF1022A was higher, especially the highest yield of PF1022A on the 9th day was 6148 μg / mL.

[0038] [Table 1]

[0039] Example 8 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 180 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 180 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 5900 μg / mL when the fermentation liquid was detected by HPLC.

[0040] Example 9 Carbon source containing solid malt extract (content of solid malt extract in fermentation medium was 200 g / L) The seed culture prepared in Preparation Example 1 was inoculated into a fermentation medium. The fermentation medium (based on 1 L) contained 200 g of solid malt extract, 10 g of soybean oil, 25 g of cottonseed cake powder, 10 g of yeast powder, 2 g of sodium chloride, 2 g of magnesium sulfate, and 2 g of calcium carbonate, and was made 1000 mL by adding water, adjusted to pH 6.0, and filled with 25 mL using a 250 mL shaking Erlenmeyer flask and sterilized at 121 ° C for 20 minutes. The fermentation liquid was obtained by shaking culture at 250 r / min on a shaking table at a temperature of 25 ° C. The yield of PF1022A was 5743 μg / mL when the fermentation liquid was detected by HPLC.

Claims

1. A method for improving the fermentation yield of PF1022A, comprising using a Roselinia strain producing PF1022A as a production strain and performing aerobic fermentation in a fermentation medium, the fermentation medium comprising an assimilable carbon source, an assimilable nitrogen source and inorganic salts, the assimilable carbon source containing solid malt extract.

2. 2. The method according to claim 1, characterized in that the content of said solid malt extract in the fermentation medium is between 70 and 200 g / L, even more preferably between 100 and 200 g / L, even more preferably between 140 and 200 g / L, and most preferably 160 g / L.

3. 3. The method of claim 1 or 2, wherein the assimilable carbon source further comprises one selected from the group consisting of glucose, maltodextrin, maltose, soybean oil, soluble starch, corn starch, or a combination of any two or more of the above.

4. 4. The method according to any one of claims 1 to 3, characterized in that the assimilable nitrogen source is selected from one of the following: yeast powder, cottonseed cake powder, wheat germ powder, yeast extract powder, soybean cake powder, peanut cake powder, or a combination of any two or more of the above, preferably a combination of yeast powder and cottonseed cake powder.

5. 5. The method according to claim 1, wherein the inorganic salt is selected from one or a combination of any two or more of zinc sulfate, magnesium sulfate, ferrous sulfate, copper sulfate, manganese sulfate, diammonium hydrogen sulfate, potassium chloride, sodium chloride, magnesium chloride, nickel chloride, calcium carbonate, calcium chloride, and preferably a combination of calcium carbonate, sodium chloride and magnesium sulfate.

6. The method according to any one of claims 1 to 5, wherein the fermentation medium contains 75 to 210 g / L of an assimilable carbon source, 15 to 40 g / L of an assimilable nitrogen source, and 6 to 10 g / L of inorganic salts.

7. 7. The method of any one of claims 1 to 6, wherein the fermentation medium comprises solid malt extract, soybean oil, cottonseed cake powder, yeast powder, sodium chloride, magnesium sulfate, and calcium carbonate.

8. 8. The method of claim 7, wherein the soybean oil is 5-10 g / L, the yeast powder is 5-10 g / L, the cottonseed cake powder is 10-30 g / L, the sodium chloride is 2-3 g / L, the magnesium sulfate is 2-3 g / L, and the calcium carbonate is 2-4 g / L.

9. The method according to any one of claims 1 to 8, characterized in that the fermentation incubation temperature is 22 to 28°C, preferably 24 to 26°C, more preferably 25°C, and the fermentation incubation time is 6 to 10 days, preferably 7 to 10 days, most preferably 9 days.

10. The method according to any one of claims 1 to 9, wherein the Rosellinia strain producing PF1022A is Rosellinia sp. HS-NF-1412Z having the deposit number CGMCC NO. 13893.

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