A method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium

Abstract

The present invention discloses a method for producing 7-hydroxybutylphthalide by fermentation of fox dung Penicillium. The method adopts the NCC3421 strain, and specially designs the shake flask seed culture solution, the primary tank seed solution, the fermentation medium formula and the process parameters, especially by adding high-phosphorus organic nitrogen sources such as fish meal and corn syrup to the shake flask seed culture medium, shortening the spore germination time, effectively improving the reproduction vitality of the seed flask mycelium, and enabling the strain fermentation level to be efficiently expressed. In the subsequent industrial amplification link, a reasonable and operable inoculation amount inoculation technology is adopted to achieve this. The method of the present invention greatly improves the yield of 7-hydroxybutylphthalide synthesized by the NCC3421 strain fermentation, and its 2m 3 The fermentation level reached 5000‑10250 μg / mL on a fermenter scale.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing phthalide structural analogs using fungal metabolites, specifically using Penicillium foetidum ( Penicillium fox ) A method for preparing 7-hydroxybutylphthalide by fermentation. Background Art

[0002] In recent years, with the improvement of people's material living standards, cardiovascular and cerebrovascular diseases have gradually become one of the main diseases threatening modern human health, and the high incidence and mortality of stroke are at the forefront.

[0003] As a new type of drug for the treatment of ischemic stroke, 3-butylphthalide has a multi-target therapeutic approach, but its overall efficacy is not high and needs to be combined with conventional drugs such as aspirin to enhance its efficacy. In-depth research on this type of drug and its derivatives and structural analogs has become a hot topic in drug development in this field. With 3-butylphthalide as the lead compound for structural modification, closed-ring derivatives, open-ring derivatives and analogs have been obtained. Literature European Journal of Medicinal Chemistry, 2010, 45(5): 1941-1946. A series of halogen-substituted phenylpeptide ring derivatives were synthesized and their biological activities were studied. Among them, the vasodilator activity of compounds 4i (6-bromo-butylphthalide) and 4g (6-fluoro-butylphthalide) was better than that of NBP. The acute toxicity experiment in mice showed that the LD50 value of compound 4g (6-fluoro-butylphthalide) was 750.89 mg / kg, and the LD50 value of 4i (6-bromo-butylphthalide) was 1000 mg / kg, which was significantly higher than the LD50 value of NBP (592.93 mg / kg). Journal of medicinal chemistry, 2013, 56(7): 3078-3089. A series of isosorbide-NBP ring-opening derivatives were synthesized using 2-(α-hydroxy-n-pentyl)benzoic acid as the lead compound and hybridized with isosorbide, among which compound 9a had the best activity. In vitro antiplatelet aggregation test and in vivo antithrombotic test showed that the inhibitory activity of compound 9a was significantly enhanced compared with (S)-NBP and aspirin. Organic & Biomolecular Chemistry, 2011, 9(16): 5670-5681. NO donor-type NBP ring-opening derivatives were synthesized using ferulic acid, which has antioxidant and antiplatelet effects, as the linking group, among which the new compound ZJM-289 had the best activity. Compared with NBP, compound ZJM-289 had better or equivalent in vitro and in vivo antiplatelet aggregation, antithrombotic and anti-cerebral ischemia activities.

[0004] CN 106214674 A discloses three butylphthalide structural analogs isolated from a Penicillium fox feces fungus strain. In vitro and in vivo efficacy and toxicity studies have shown that 7-hydroxy-butylphthalide, 4-hydroxy-butylphthalide and 7-methoxy-butylphthalide all have antioxidant, anti-thrombotic and anti-cerebral ischemic effects. Among them, 7-hydroxy-butylphthalide has a lower dosage, more significant and excellent anti-platelet aggregation, anti-oxidative stress and anti-cerebral ischemic activity compared with butylphthalide, and has lower toxicity, that is, higher drug safety.

[0005] 7-Hydroxy-butylphthalide is a metabolite of Penicillium vulpinum, and its molecular formula is C 12 H 14 03, molecular weight is 206.0943, structural formula is as follows:

[0006]

[0007] (See Heterocycles, Vol. 48, NO. 9, 1998: 1931-1941).

[0008] Derived from the fungus Penicillium foetidum ( Penicillium fox ) showed rich natural activity. Natural Product Research. 2021 Feb;35(3):421-427 discloses the use of the endophytic fungus Penicillium fox feces ( Penicillium fox ) produced two new phthalide compounds (i.e., 3-carboxypropyl-7-hydroxyphthalide and 3-carboxypropyl-7-hydroxyphthalide methyl ester). The antibacterial activity test results showed that the compounds had Bacillus subtilis , Shigella dysenteriae and Enterobacter aerogenes Has a moderate inhibitory effect.

[0009] At present, the research on the production of phthalide analogs by fungal metabolism is quite active, especially the production of Penicillium breviscapus ( Penicillium brevicompactum ) The fermentation synthesis of mycophenolic acid with a phthalide ring structure is relatively advanced.

[0010] CN104726347A discloses a fox feces Penicillium ( Penicillium fox )NCC3421 (CGMCCNO.9094) strain and method for preparing L-7-hydroxybutylphthalide using the same. The L-7-hydroxybutylphthalide prepared by fermentation according to the invention method reaches 1600μg / mL, showing its industrial development prospects, but its production technology level is 1600μg / mL in a 50L tank, which is still a long way from the industrial level. Summary of the invention

[0011] The object of the present invention is to provide a method for preparing ... Penicillium fox ) A method for fermenting to produce high concentration 7-hydroxybutylphthalide, in order to further improve the industrial production efficiency of 7-hydroxybutylphthalide.

[0012] The object of the present invention is achieved by the following methods:

[0013] The invention provides a method for utilizing fox dung Penicillium ( Penicillium fox ) A method for producing high concentration 7-hydroxybutylphthalide by fermentation, comprising the following steps:

[0014] (a) Preparation of fox dung Penicillium ( Penicillium fox )Shake flask seed culture medium:

[0015] The fox feces Penicillium ( Penicillium fox )NCC3421 (CGMCC NO.9094) is a fermentation strain (hereinafter referred to as NCC3421 strain), and the slant culture of the strain is inoculated into a shake flask seed medium, cultured at 25°C, with a shaker speed of 220 rpm, and a culture period of 48-52 h to obtain a shake flask seed culture solution;

[0016] The composition ratio and preparation of the culture medium are as follows: dextrin 10.0~20.0g / L, sucrose 20.0~30.0g / L, low-temperature soybean cake powder 10.0~30.0g / L, peptone 5.0~15g / L, high-phosphorus organic nitrogen source 2~25.0g / L, ammonium sulfate 2.5~10.0 g / L, sodium chloride 2.5~10.0g / L, calcium carbonate 2.0~4.0g / L, add water to make the volume 1000mL, adjust the pH to 6.4~6.8 with sodium hydroxide solution, and sterilize at 121℃ for 30min;

[0017] The high-phosphorus organic nitrogen source can be selected from one or a combination of two or more of fish meal, corn syrup, gluten meal, and peanut meal, preferably fish meal, corn syrup, or a combination of the two.

[0018] The high-phosphorus organic nitrogen source described in the method of the present invention is preferably a composition of fish meal and corn syrup, wherein the amount of fish meal is 5.0-10.0 g / L and the amount of corn syrup is 8.0-20.0 g / L; the total amount of the composition is controlled at 10.0 g / L-23.0 g / L. The more preferred amount is 8.0 g / L of fish meal and 15.0 g / L of corn syrup.

[0019] (b) Preparation of fox dung Penicillium ( Penicillium fox )First-level tank seed liquid:

[0020] The shake flask seed culture solution prepared in (a) is inoculated on the primary seed culture medium at an inoculum amount of 0.3-0.5% by volume, and the culture temperature is 22-25° C., the tank stirring power is 35-40 Hz, the ventilation ratio is 1:0.8-1.5 vvm, the tank pressure is 0.05±0.01 MPa, and the culture period is 48-52 h to obtain the primary tank seed solution;

[0021] The primary seed tank culture medium is prepared according to the following method: dextrin 10.0-20 g / L, sucrose 20.0-30 g / L, low-temperature soybean cake powder 15.0-30 g / L, corn steep liquor 2.5-5.0 g / L, peptone 10.0-20 g / L, potassium dihydrogen phosphate 3.0-6.0 g / L, sodium chloride 2.5-5.0 g / L, calcium carbonate 1 g-2 / L, defoamer 1-2 mL / L, water is added to make the volume 1000 mL, the pH is adjusted to 6.4-6.8 with sodium hydroxide solution, and sterilized at 121°C for 30 min.

[0022] (c) Preparation of fox dung Penicillium ( Penicillium fox ) Fermentation broth:

[0023] The first-stage tank seed liquid prepared in step (b) is inoculated into the fermentation medium at an inoculation rate of 3-5% by volume, the fermentation temperature is 22-25° C., the fermentation culture speed is 220-250 rpm; the tank stirring power is 35-40 Hz, the ventilation ratio is 1:0.8-1.5 vvm, the tank pressure is 0.05±0.01 MPa, the dissolved oxygen concentration (hereinafter referred to as DO) is maintained at more than 20-30%, and the fermentation cycle is 120-180 h;

[0024] The fermentation medium is prepared by the following method: 0-40 g / L dextrin, 20.0-30.0 g / L sucrose, 30.0-45 g / L low-temperature soybean cake powder, 10.0-20 g / L fish meal, 16.0-32.0 g / L corn steep liquor, 3.0-6.0 g / L potassium dihydrogen phosphate, 8.0-12.0 g / L ammonium sulfate, 2.5-5.0 g / L sodium chloride, 2.0-4.0 g / L magnesium sulfate heptahydrate, 0-8.0 g / L precursor A, 0-8.0 g / L precursor B, 15.0-30 mL / L corn oil, 2.0-4.0 g / L calcium carbonate, 0-1.0 mL / L defoamer, add water to make the volume to 1000 mL, adjust the pH to 6.4-6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0025] (d) The fermentation broth prepared in (c) is separated and collected according to a conventional method to obtain a 7-hydroxybutylphthalide solution.

[0026] For example, the fermentation liquid prepared in (c) is centrifuged at 3000 rpm for 15 min, the supernatant is discarded, and the mycelium is retained; 95% ethanol is added to the mycelium, stirred and soaked for 25-30 min, centrifuged at 3000 rpm for 15 min, filtered, and the filtrate is rich in 7-hydroxybutylphthalide.

[0027] In the method of the present invention, the precursor substance A is methionine (precursor substance A1) or ethionine (precursor substance A2); the precursor substance B is a short-chain fatty alcohol or a short-chain fatty acid; the short-chain fatty alcohol can be ethanol, n-propanol, n-butanol; n-propanol and n-butanol are preferred. The short-chain fatty acid can be acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, norleucine and its sodium salt, among which norleucine and n-butyric acid are preferred.

[0028] In order to further enhance the efficient expression of the fermentation product of the strain, the present invention adds a feeding feed during the fermentation process of the above step (c), wherein the feeding feed is two components of glucose 300-450 g / L or glucose 300-450 g / L and glycerol 1-150 g / L, and the total mass of the two components is controlled at 300-600 g / L.

[0029] The preferred scheme of feeding during the fermentation process is: during the fermentation process, when the pH of the fermentation liquid rises to 7.2-7.3, start feeding, and the feeding flow rate is controlled at 2-4 L / h; when the reducing sugar in the fermentation liquid is below 1.0%, the feeding flow rate is controlled at 0.8-2 L / h; after the feeding is completed, continue fermentation for 8-12 hours.

[0030] The preferred preparation method of the shake flask seed culture medium of the method of the present invention is:

[0031] Dextrin 10.0g / L, sucrose 20.0g / L, low-temperature soybean cake powder 15.0g / L, peptone 10.0g / L, high-phosphorus organic nitrogen source 13-25.0g / L, ammonium sulfate 5.0g / L, sodium chloride 5.0g / L, calcium carbonate 2.0g / L, add water to make up to 1000mL, adjust the pH to 6.4-6.8 with 10% sodium hydroxide, and sterilize at 121℃ for 30min.

[0032] In the method of the present invention, the amount of precursor substance A added is preferably 0.5-8.0 g / L.

[0033] In the method of the present invention, the amount of the precursor substance B added is preferably 0.5-8.0 g / L.

[0034] The fish meal of the present invention can be commercially available common fish meal, such as commercially available feed-grade fish meal.

[0035] The beneficial effects of the present invention are mainly reflected in the following aspects: the present invention adopts the NCC3421 strain, and specially designs the formula and process parameters of the shake flask seed culture solution, the primary tank seed solution and the fermentation medium, especially adds high-phosphorus organic nitrogen sources such as fish meal and corn pulp to the shake flask seed culture medium, shortens the spore germination time, effectively improves the reproduction vitality of the seed flask mycelium, enables the efficient expression of the strain fermentation level, and in the subsequent industrial amplification link, adopts a reasonable and operable inoculation amount inoculation technology to achieve; adds biosynthesis precursor substances (methionine, n-butanol, etc.) to the fermentation medium, further promotes the synthesis of the target product; adds a combined carbon source flow feeding process in the fermentation process, and further enhances the efficient expression of the strain fermentation product.

[0036] The method of the invention greatly improves the yield of 7-hydroxybutylphthalide synthesized by fermentation of NCC3421 strain, and the production level can reach 5000-10000 μg / mL in a 50L tank. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Mycelial morphology of the seed culture solution in shake flasks after 48 h (×10) in Example 1 of the present invention (medium IV).

[0038] Figure 2 This is the mycelial morphology of seed culture in a shake flask with ordinary nitrogen source for 48 hours (×10).

[0039] Figure 3 For Example 2 of the present invention (culture medium )2m 3 Mycelial morphology in the fermentation tank 48 hours later (×40).

[0040] Figure 4 This is the HPLC chromatogram of 7-hydroxybutylphthalide produced in Example 6 of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific examples. The experimental materials used in the following examples are all industrial grade raw materials unless otherwise specified; the analytical chromatographic column Inertsil ODS-SP 5μm C18 4.6×250mm was purchased from Shimadzu, Japan, and the chromatographic solvent was purchased from Honeywell Trading (Shanghai) Co., Ltd.

[0042] Example 1

[0043] a. Preparation of fox feces Penicillium ( Penicillium fox ) in shake flask seed culture:

[0044] The slant culture of strain NCC3421 was inoculated into a 1000 mL Erlenmeyer flask filled with the mother bottle medium, cultured at 25°C, with a shaker speed of 220 rpm, and a culture period of 48 to 52 h to obtain the seed solution.

[0045] The seed culture medium is prepared according to the following method: 10.0 g / L dextrin, 20.0 g / L sucrose, 15.0 g / L low-temperature soybean cake powder, 10.0 g / L peptone, 0-23.0 g / L high-phosphorus organic nitrogen source, 5.0 g / L ammonium sulfate, 5.0 g / L sodium chloride, 2.0 g / L calcium carbonate, add water to make the volume 1000 mL, adjust the pH to 6.4-6.8 with 10% sodium hydroxide, and pack into Erlenmeyer bottles according to the filling volume of 200 mL, and sterilize at 121°C for 30 minutes.

[0046] In order to investigate the effect of shake flask seed culture medium on mycelium germination growth rate and the effect of shake flask seed quality on mycelium development and fermentation level in the primary seed tank, six different shake flask seed culture media are provided in this example, which are:

[0047] Medium I (common organic nitrogen source): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean meal powder 15.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 min;

[0048] Medium II (organic nitrogen source is low-amount corn steep liquor): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 15.0 g / L, corn steep liquor 8.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up, adjust pH to 6.6 with 10% sodium hydroxide, sterilize at 121℃ for 30 min;

[0049] Medium III (high corn steep liquor with organic nitrogen source): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 15.0 g / L, corn steep liquor 20.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up, adjust pH to 6.8 with 10% sodium hydroxide, sterilize at 121°C for 30 min;

[0050] Medium IV (organic nitrogen source is low-amount fish meal / high-amount corn syrup combination): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean meal powder 15.0 g / L, corn syrup 15.0 g / L, fish meal 3.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 min;

[0051] Medium V (organic nitrogen source high fish meal / low corn syrup combination): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean meal powder 15.0 g / L, corn syrup 8.0 g / L, fish meal 10.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 min;

[0052] Culture medium VI (combination of medium fish meal / high corn syrup as organic nitrogen source): dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean meal powder 15.0 g / L, corn syrup 15.0 g / L, fish meal 8.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, add water to make up to volume, adjust the pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0053] b. Preparation of fox feces Penicillium ( Penicillium fox )First-level tank seed liquid:

[0054] The shake flask seed culture solution prepared in step a is inoculated into a seed tank at an inoculation rate of 0.3% by volume, and the culture temperature is 25° C., the tank stirring power is 40 Hz, the ventilation ratio is 1:0.8-1.5 vvm, the tank pressure is 0.05±0.01 MPa, and the culture period is 48h-52h to obtain a first-level tank seed solution;

[0055] The seed tank culture medium is: dextrin 10.0g / L, sucrose 10.0g / L, low-temperature soybean cake powder 15.0g / L, corn steep liquor 5.0g / L, peptone 10.0g / L, potassium dihydrogen phosphate 3.0g / L, sodium chloride 5.0g / L, calcium carbonate 2.0g / L, defoamer 1.0mL / L, add water to make up to 1000mL, adjust the pH to 6.4 with 40% sodium hydroxide solution, and sterilize at 121℃ for 30min.

[0056] c. Preparation of 7-hydroxybutylphthalide fermentation broth synthesized by Penicillium vulpinum

[0057] The above seed liquid is inoculated into the fermentation medium in the fermenter at an inoculation rate of 3% by volume, the fermentation temperature is 25°C, and the fermentation cycle is 120h~180h; the fermenter stirring power is 40Hz, the fermentation culture speed is 220~250rpm; the ventilation ratio is 1:0.8~1.5vvm, the tank pressure is 0.05±0.01Mpa, and the DO is maintained above 20%.

[0058] Among them, the fermentation medium was prepared according to the following method: dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, water was added to make up the volume, 10% sodium hydroxide was used to adjust the pH to 6.4-6.8, and the culture was carried out at 25°C for 120 hours and then bottled.

[0059] After the fermentation, 10 mL of the fermentation broth was taken, centrifuged at 3000 rpm for 15 min, the supernatant was discarded, 10 mL of 95% ethanol was added to the mycelium, stirred and soaked for 30 min, centrifuged at 3000 rpm for 15 min, the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was tested for product content using Shimadzu high performance liquid chromatograph LC2030C.

[0060] Liquid chromatography detection conditions of L-7-hydroxybutylphthalide:

[0061] Chromatographic column: Inertsil ODS-SP 5μm C18 4.6×250mm

[0062] Mobile phase: acetonitrile / water = 60 / 40, containing 0.05% phosphoric acid; flow rate 1 mL / min; detector: UV, 210 nm.

[0063] Under the above conditions, the retention time of L-7-hydroxybutylphthalide is about 5.9 min.

[0064] The test results are shown in the following table:

[0065]

[0066] From the above results, it can be seen that the fish meal + corn steep liquor used as a composite high-phosphorus organic nitrogen source in the shake flask seed culture of the present invention significantly enhances the mycelial development of the seed flask, improves the culture quality of the first-level seed tank with low inoculation amount, and has a prominent and significant effect on improving the fermentation level of the seed flask. In particular, the combination of corn steep liquor 15.0 g / L / fish meal 8.0 g / L has a more significant effect, and its shake flask seed flask fermentation level reaches 2871 μg / mL, and the first-level seed tank fermentation level reaches 3007 μg / mL.

[0067] from Figure 1 , Figure 2 It can also be seen that, compared with the mycelia prepared from the common nitrogen source (control process), the mycelia prepared from the present invention have thick mycelia, deep methylene blue staining, and larger mycelium mass in microscopic morphology.

[0068] Example 2

[0069] The slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and cultured at 25°C and a shaker speed of 220 rpm for 52 h to obtain the shake flask seed liquid. The shake flask seeds were inoculated into the fermentation shake flask at an inoculum of 5%, and cultured at 25°C and a shaker speed of 220 rpm for 120 h and then released.

[0070] The shake flask seed culture medium is: dextrin 10.0g / L, sucrose 20.0g / L, low-temperature soybean cake powder 15.0g / L, corn steep liquor 15.0 g / L, fish meal 8g / L, peptone 10.0g / L, ammonium sulfate 5.0g / L, sodium chloride 5.0g / L, calcium carbonate 2.0g / L, add water to make up to 1000mL, adjust the pH to 6.6 with 10% sodium hydroxide, and sterilize at 121℃ for 30min.

[0071] The seed culture solution was prepared according to the method described in Example 1, and inoculated into the fermentation medium at an inoculum amount of 3% by volume; the 7-hydroxybutylphthalide fermentation solution was fermented and synthesized. The fermentation temperature was 25° C., the fermentation cycle was 120 h to 180 h, the fermentation tank stirring power was 40 Hz, the fermentation culture speed was 220 to 250 rpm, the ventilation ratio was 1:0.8 to 1.5 vvm, the tank pressure was 0.05±0.01 MPa, and the DO was maintained above 20%.

[0072] In this example, seven fermentation culture media were prepared (to verify the effect of the precursor substance A proposed in the present invention on the product 7-hydroxybutylphthalide), the fermentation bottle liquid volume was 30mL / 250mL, and the culture medium compositions were:

[0073] Culture medium I (without precursor substance A): dextrin 40.0 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0074] Culture medium II (containing low amount of precursor substance A1): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 2.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0075] Culture medium III (containing medium amount of precursor substance A1): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0076] Culture medium IV (containing high amount of precursor substance A1): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 8.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.3 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0077] Culture medium V: (containing low amount of precursor substance A2) dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, ethionine 2.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.5 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0078] Culture medium VI (containing medium amount of precursor substance A2): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, ethionine 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0079] Culture medium VII (containing high amount of precursor substance A2): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, ethionine 8.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up the volume, adjust the pH to 6.7 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0080] After the fermentation, 10 mL of the fermentation broth was taken and centrifuged at 3000 rpm for 15 min. The supernatant was discarded, 10 mL of 95% ethanol was added to the mycelium, stirred and soaked for 30 min, centrifuged at 3000 rpm for 15 min, the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was tested for product content using Shimadzu high performance liquid chromatograph LC2030C.

[0081] Liquid chromatography detection conditions of L-7-hydroxybutylphthalide:

[0082] Chromatographic column: Inertsil ODS-SP 5μm C18 4.6×250mm

[0083] Mobile phase: acetonitrile / water = 60 / 40, containing 0.05% phosphoric acid; flow rate 1 mL / min; detector: UV, 210 nm.

[0084] Under the above conditions, the retention time of L-7-hydroxybutylphthalide is about 5.9 min.

[0085] The results are as follows:

[0086]

[0087] It can be seen from the above results that the addition of precursor substance A in the fermentation culture further significantly promoted the synthesis of 7-hydroxybutylphthalide fermentation products, especially the addition of a medium amount of methionine (5.0 g / L), the fermentation level reached 5274 μg / mL.

[0088] from Figure 3It can also be seen that Example 2 of the present invention (culture medium )2m 3 The mycelium morphology in the fermentation tank at 48 hours (×40) shows that after the mycelium in the seed tank was transplanted to the fermentation tank, the mycelium developed rapidly and had vigorous metabolism.

[0089] Example 3

[0090] The slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and the shaker speed was 220 rpm at 25°C for 52 hours to obtain the shake flask seed liquid. The shake flask seeds were inoculated into the fermentation shake flask at an inoculation rate of 3%, and the shaker speed was 220 rpm at 25°C for 120 hours. The shake flask seed medium was: dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature bean cake powder 15.0 g / L, corn steep liquor 15.0 g / L, fish meal 8.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, water was added to the volume, pH was adjusted to 6.5 with 10% sodium hydroxide, and sterilized at 121°C for 30 minutes.

[0091] The seed culture solution was prepared by referring to the method described in Example 1, and inoculated into the fermentation medium at an inoculum amount of 5% by volume; the 7-hydroxybutylphthalide fermentation solution was fermented and synthesized. The fermentation temperature was 22° C., the fermentation cycle was 120 h to 180 h, the fermentation tank stirring power was 40 Hz, the fermentation culture speed was 220 to 250 rpm, the ventilation ratio was 1:0.8 to 1.5 vvm, the tank pressure was 0.05±0.01 MPa, and the DO was maintained at 30%.

[0092] In order to investigate the effect of the precursor substance B in the fermentation medium on the product 7-hydroxybutylphthalide, six kinds of fermentation medium were prepared in this example, and the fermentation bottle liquid volume was 30mL / 250mL.

[0093] The six culture media are:

[0094] Medium I (containing low-amount precursor B n-butanol): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to volume, adjust pH to 6.5 with 10% sodium hydroxide, and sterilize at 121°C for 30 min. After sterilization, add n-butanol to a final concentration of 2.0 g / L.

[0095] Medium II (containing high amount of precursor B n-butanol): dextrin 40 g / L, sucrose 20.0 g / L, low temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to 1000 mL, adjust pH to 6.4 with 10% sodium hydroxide, sterilize at 121°C for 30 min. After sterilization, add n-butanol to a final concentration of 5.0 g / L.

[0096] Culture medium III (containing low-quality precursor substance B norleucine): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, norleucine 2.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.7 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0097] Culture medium IV (containing high amount of precursor substance B norleucine): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, norleucine 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0098] Medium V (containing low amount of precursor substance B sodium butyrate): dextrin 40 g / L, sucrose 20.0 g / L, low temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, sodium butyrate 2.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0099] Medium VI (containing high amount of precursor B sodium butyrate): dextrin 40 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 30.0 g / L, fish meal 10.0 g / L, corn steep liquor 16.0 g / L, potassium dihydrogen phosphate 6.0 g / L, ammonium sulfate 8.0 g / L, methionine 5.0 g / L, sodium butyrate 5.0 g / L, sodium chloride 5.0 g / L, magnesium sulfate heptahydrate 2.0 g / L, corn oil 15.0 mL / L, calcium carbonate 4.0 g / L, add water to make up to volume, adjust the pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 min.

[0100] After the fermentation, 10 mL of the fermentation broth was taken, centrifuged at 3000 rpm for 15 min, the supernatant was discarded, 10 mL of 95% ethanol was added to the mycelium, stirred and soaked for 30 min, centrifuged at 3000 rpm for 15 min, the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was tested for product content using Shimadzu high performance liquid chromatograph LC2030C.

[0101] Liquid chromatography detection conditions of L-7-hydroxybutylphthalide:

[0102] Chromatographic column: Inertsil ODS-SP 5μm C18 4.6×250mm

[0103] Mobile phase: acetonitrile / water = 60 / 40, containing 0.05% phosphoric acid; flow rate 1 mL / min; detector: UV, 210 nm.

[0104] Under the above conditions, the retention time of L-7-hydroxybutylphthalide is about 5.9 min.

[0105] The results are as follows:

[0106]

[0107] From the above results, it can be seen that the method of the present invention further promotes the synthesis of 7-hydroxybutylphthalide fermentation products by adding a high-phosphorus organic nitrogen source and a precursor substance A in combination with the addition of a precursor substance B. The optimal combination is 5.0 g / L methionine and 2.0 g / L n-butanol, and the fermentation level reaches 6332 μg / mL.

[0108] Example 4

[0109] The slant culture of strain NCC3421 was inoculated into a shake flask seed medium, and cultured at 25°C and a shaker speed of 220 rpm for 48 h to obtain a shake flask seed solution, wherein the shake flask seed medium was: dextrin 10.0 g / L, sucrose 20.0 g / L, low-temperature soybean cake powder 15.0 g / L, corn steep liquor 15.0 g / L, fish meal 8.0 g / L, peptone 10.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 5.0 g / L, calcium carbonate 2.0 g / L, water was added to make up the volume, the pH was adjusted to 6.6 with 10% sodium hydroxide, and sterilized at 121°C for 30 min.

[0110] The shake flask seed liquid was inoculated into a first-level seed tank (500L) at an inoculation rate of 0.5%, and the volume after inoculation was 300L, the culture temperature was 25℃, the stirring power of the seed tank was 40Hz, the ventilation ratio was 1:1.0vvm, the tank pressure was 0.05±0.01Mpa, and the first-level seeds were obtained after 48h of culture. The seed tank culture medium was: dextrin 10.0g / L, sucrose 10.0g / L, low-temperature bean cake powder 15.0g / L, corn steep liquor 5.0g / L, peptone 10.0g / L, potassium dihydrogen phosphate 3.0g / L, sodium chloride 5.0g / L, calcium carbonate 2g / L, defoamer 1mL / L, water was added to the volume, pH was adjusted to 6.8 with 40% sodium hydroxide solution, and sterilized at 121℃ for 30min.

[0111] The seed solution of the first seed tank was inoculated at a volume percentage of 5% in 2m 3 Fermentation tank, volume after connection 1.2m 3 , fermentation temperature 25℃, fermentation tank stirring power 40Hz, ventilation ratio 1:0.8~1.5vvm, tank pressure 0.05±0.01Mpa, DO maintained above 20%. The fermentation tank culture medium is: sucrose 20.0g / L, low temperature soybean cake powder 30.0g / L, fish meal 10.0g / L, corn steep liquor 16.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium sulfate 8.0g / L, methionine 5.0g / L, sodium chloride 5.0g / L, magnesium sulfate heptahydrate 2.0g / L, corn oil 15.0mL / L, calcium carbonate 4.0g / L, add water to make up, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121℃ for 30min. After disinfection, add n-butanol to a final concentration of 2.0g / L.

[0112] During the fermentation process, feeding was performed (the feed was glucose, with a total addition of 300 g / L). The feeding method was as follows: when the pH of the fermentation liquid rose to 7.2, the sugar feed flow rate was started at 2 L / h. When the reducing sugar content dropped below 1.0% in the middle and late stages of fermentation, the sugar feed flow rate was adjusted down to 0.8 mL / h. Feeding was stopped 12 h before tank release, and the fermentation cycle was 168 h. The tank release fermentation unit was measured to be 8235 μg / mL.

[0113] Example 5

[0114] The slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and the shake flask seed solution was obtained by culturing at 25°C and a shaker speed of 220 rpm for 52 h. The shake flask seed medium was composed of 10.0 g / L dextrin, 20.0 g / L sucrose, 15.0 g / L low-temperature soybean cake powder, 15.0 g / L corn steep liquor, 8.0 g / L fish meal, 10.0 g / L peptone, 5.0 g / L ammonium sulfate, 5.0 g / L sodium chloride, and 2.0 g / L calcium carbonate. Water was added to make up the volume, the pH was adjusted to 6.8 with 10% sodium hydroxide, and the solution was sterilized at 121°C for 30 min.

[0115] The shake flask seed liquid was inoculated into the first-level seed tank (500L) at an inoculation rate of 0.3%, and the volume was 300L after inoculation, the culture temperature was 25℃, the stirring power of the seed tank was 40Hz, the ventilation ratio was 1:1.5vvm, the tank pressure was 0.05±0.01Mpa, and the first-level seeds were obtained after culturing for 52h. The seed tank culture medium was: dextrin 10.0g / L, sucrose 10.0g / L, low-temperature bean cake powder 15.0g / L, corn steep liquor 5.0g / L, peptone 10.0g / L, potassium dihydrogen phosphate 3.0g / L, sodium chloride 5.0g / L, calcium carbonate 2g / L, defoamer 1.0mL / L, water was added to the volume, the pH was adjusted to 6.4 with 40% sodium hydroxide solution, and sterilized at 121℃ for 30min.

[0116] The seed liquid in the first seed tank was inoculated at a volume percentage of 3% in 2m 3 Fermentation tank, volume after connection 1.2m 3 , fermentation temperature 25℃, fermentation tank stirring power 40Hz, ventilation ratio 1:0.8~1.5vvm, tank pressure 0.05±0.01Mpa, DO maintained above 20%. The fermentation tank culture medium is: sucrose 20.0g / L, low temperature soybean cake powder 30.0g / L, fish meal 10.0g / L, corn steep liquor 16.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium sulfate 8.0g / L, methionine 5.0g / L, sodium chloride 5.0g / L, magnesium sulfate heptahydrate 2.0g / L, corn oil 15.0mL / L, calcium carbonate 4.0g / L, add water to make up, adjust pH to 6.8 with 10% sodium hydroxide, and sterilize at 121℃ for 30min. After disinfection, add n-butanol to a final concentration of 2.0g / L.

[0117] During the fermentation process, feeding was performed (the feeding liquid concentration was 400 g / L glucose and 100 g / L glycerol). When the pH of the fermentation liquid rose to 7.3, the sugar feeding flow rate was started at 3 L / h. When the reducing sugar content dropped below 1.0% in the middle and late stages of fermentation, the sugar feeding flow rate was adjusted down to 1 L / h. Feeding was stopped 8 hours before tank release, and the fermentation cycle was 170 hours. The fermentation unit of tank release was measured to be 10168 μg / mL.

[0118] Example 6

[0119] The slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and the shake flask seed solution was obtained by culturing at 25°C and a shaker speed of 220 rpm for 50 h. The shake flask seed medium was composed of 10.0 g / L dextrin, 20.0 g / L sucrose, 15.0 g / L low-temperature soybean cake powder, 15.0 g / L corn steep liquor, 8.0 g / L fish meal, 10.0 g / L peptone, 5.0 g / L ammonium sulfate, 5.0 g / L sodium chloride, and 2.0 g / L calcium carbonate. Water was added to make up the volume, the pH was adjusted to 6.6 with 10% sodium hydroxide, and the solution was sterilized at 121°C for 30 min.

[0120] The shake flask seed liquid was inoculated into the first-level seed tank (500L) at an inoculation rate of 0.4%, and the volume after inoculation was 300L, the culture temperature was 25℃, the stirring power of the seed tank was 40Hz, the ventilation ratio was 1:1.2vvm, the tank pressure was 0.05±0.01Mpa, and the first-level seeds were obtained after culturing for 52h. The seed tank culture medium was: dextrin 10.0g / L, sucrose 10.0g / L, low-temperature bean cake powder 15.0g / L, corn steep liquor 5.0g / L, peptone 10.0g / L, potassium dihydrogen phosphate 3.0g / L, sodium chloride 5.0g / L, calcium carbonate 2g / L, defoamer 1.0mL / L, water was added to the volume, the pH was adjusted to 6.4 with 40% sodium hydroxide solution, and sterilized at 121℃ for 30min.

[0121] The seed solution of the first seed tank was inoculated at a volume percentage of 5% in 2m 3 Fermentation was carried out in a fermenter with a volume of 1.2 m 3 , fermentation temperature 25℃, fermentation tank stirring power 40Hz, ventilation ratio 1:0.8~1.5vvm, tank pressure 0.05±0.01Mpa, DO maintained above 20%. The fermentation tank culture medium is: sucrose 20.0g / L, low temperature soybean cake powder 30.0g / L, fish meal 10.0g / L, corn steep liquor 16.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium sulfate 8.0g / L, methionine 5.0g / L, sodium chloride 5.0g / L, magnesium sulfate heptahydrate 2.0g / L, corn oil 15.0mL / L, calcium carbonate 4.0g / L, add water to make up, adjust pH to 6.8 with 10% sodium hydroxide, and sterilize at 121℃ for 30min. After disinfection, add n-butanol to a final concentration of 2.0g / L.

[0122] During the fermentation process, feeding was performed (the feeding liquid concentration was 450g / L glucose and 150g / L glycerol). When the pH of the fermentation liquid rose to 7.3, the sugar feeding flow rate was started at 4L / h. In the middle and late stages of fermentation, the reducing sugar dropped below 1.0%, and the sugar feeding flow rate was adjusted down to 2L / h. Feeding was stopped 12h before tank release, and the fermentation cycle was 180h. The tank release fermentation unit was measured to be 10205μg / mL. The HPLC chromatogram is shown in detail. Figure 4 。

Claims

1. A method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium, characterized in that: The following steps are involved: (a) Preparation of fox dung Penicillium ( Penicillium vulpinum )Shake flask seed culture medium Penicillium fox feces ( Penicillium vulpinum )NCC3421 is a fermentation strain, and the corresponding strain collection number is CGMCC NO.9094; the slant culture of the strain was inoculated into a shake flask seed culture medium, cultured at 25°C, with a shaker speed of 220rpm, and a culture period of 48-52h to obtain a shake flask seed culture solution; wherein the composition ratio and preparation of the culture medium are: dextrin 10.0-20.0g / L, sucrose 20.0-30.0g / L, low-temperature soybean cake powder 10.0-30.0g / L, peptone 5.0-15g / L, high-phosphorus organic nitrogen source 2-25.0g / L, ammonium sulfate 2.5-10.0g / L, sodium chloride 2.5-10.0g / L, calcium carbonate 2.0-4.0g / L, water was added to make the volume 1000mL, the pH was adjusted to 6.4-6.8 with sodium hydroxide solution, and sterilized at 121°C for 30min; the high-phosphorus organic nitrogen source is one or a combination of two or more of fish meal, corn steep liquor, gluten powder, and peanut cake powder; (b) Preparation of fox dung Penicillium ( Penicillium vulpinum )First-class tank seed liquid The shake flask seed culture solution prepared in (a) is inoculated on the primary seed culture medium at an inoculum amount of 0.3-0.5% by volume, and the culture temperature is 22-25° C., the tank stirring power is 35-40 Hz, the ventilation ratio is 1:0.8-1.5 vvm, the tank pressure is 0.05±0.01 MPa, and the culture period is 48-52 h to obtain the primary tank seed solution; The primary seed tank culture medium is prepared as follows: dextrin 10.0-20 g / L, sucrose 10.0-30 g / L, low-temperature soybean cake powder 15.0-30 g / L, corn steep liquor 2.5-5.0 g / L, peptone 10.0-20 g / L, potassium dihydrogen phosphate 3.0-6.0 g / L, sodium chloride 2.5-5.0 g / L, calcium carbonate 1-2 g / L, defoamer 1-2 mL / L, water is added to make the volume 1000 mL, the pH is adjusted to 6.4-6.8 with sodium hydroxide solution, and sterilized at 121°C for 30 min; (c) Preparation of fox dung Penicillium ( Penicillium vulpinum ) Fermentation broth The first-stage tank seed liquid prepared in (b) is inoculated into the fermentation medium at an inoculation rate of 3-5% by volume, the fermentation temperature is 22-25°C, the fermentation cycle is 120h-180h, the fermentation culture speed is 220-250rpm; the tank stirring power is 35-40Hz, the ventilation ratio is 1:0.8-1.5vvm, the tank pressure is 0.05±0.01Mpa, and the dissolved oxygen concentration is maintained above 20-30%; The fermentation medium is prepared by the following method: dextrin 0-40 g / L, sucrose 20.0-30.0 g / L, low-temperature soybean cake powder 30.0-45 g / L, fish meal 10.0-20 g / L, corn steep liquor 16.0-32.0 g / L, potassium dihydrogen phosphate 3.0-6.0 g / L, ammonium sulfate 8.0-12.0 g / L, sodium chloride 2.5-5.0 g / L, magnesium sulfate heptahydrate 2.0-4.0 g / L, precursor substance A 0-8.0 g / L, precursor substance B 0-8.0 g / L, corn oil 15.0-30 mL / L, calcium carbonate 2.0-4.0 g / L, defoamer 0-1.0 mL / L, water is added to make up to 1000 mL, pH is adjusted to 6.4-6.8 with 10% sodium hydroxide, and sterilized at 121° C. for 30 min; The precursor substance A is methionine or ethionine; the precursor substance B is n-butanol, norleucine or sodium n-butyrate; (d) The fermentation broth prepared in (c) is separated and collected according to conventional methods to obtain a 7-hydroxybutylphthalide solution.

2. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 1, characterized in that: The high-phosphorus organic nitrogen source in step (a) is fish meal or corn steep liquor.

3. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 1 or 2, characterized in that: (c) During the fermentation process, feed is added, wherein the feed is 300-450 g / L glucose or 300-450 g / L glucose and 1-150 g / L glycerol.

4. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 1 or 2, characterized in that: The shake flask seed culture medium comprises: 10.0 g / L dextrin, 20.0 g / L sucrose, 15.0 g / L low-temperature bean cake powder, 10.0 g / L peptone, 13-23.0 g / L high-phosphorus organic nitrogen source, 5.0 g / L ammonium sulfate, 5.0 g / L sodium chloride, 2.0 g / L calcium carbonate, water is added to make the volume 1000 mL, the pH is adjusted to 6.4-6.8 with 10% sodium hydroxide, and sterilized at 121° C. for 30 min.

5. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 1 or 2, characterized in that: The amount of the precursor substance A added is 0.5-8.0 g / L; the amount of the precursor substance B added is 0-8.0 g / L.

6. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 1, characterized in that: The high-phosphorus organic nitrogen source in step (a) is composed of two components: fish meal and corn syrup, wherein the amount of fish meal is 5.0-10.0 g / L and the amount of corn syrup is 8.0-20.0 g / L; the total mass of the two components is controlled at 13.0 g / L-23.0 g / L.

7. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 2, characterized in that: The feeding process in the fermentation step (c) is as follows: during the fermentation process, when the pH of the fermentation liquid rises to 7.2-7.3, the feeding is started, and the feeding flow rate is controlled at 2-4 L / h; when the reducing sugar content of the fermentation liquid is below 1.0%, the feeding flow rate is controlled at 0.8-2 L / h; after the feeding is completed, the fermentation is continued for 8-12 hours.

8. The method for producing 7-hydroxybutylphthalide by fermentation of fox feces Penicillium according to claim 6, characterized in that: The high-phosphorus organic nitrogen source in step (a) is 8.0 g / L fish meal and 15.0 g / L corn steep liquor.

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