Method for producing echinocandin B through solid state fermentation by taking vinasse as substrate

Abstract

The invention relates to a method for producing echinocandin B through solid state fermentation by taking vinasse as a substrate, and belongs to the technical field of biological fermentation. According to the method, the industrial waste vinasse is used as a culture medium, the vinasse is crushed, the moisture content and the pH value are adjusted, then the Emerica rugulosa CPCC 400818 is inoculated for solid-state fermentation production of the echinocandin B, the method for producing the echinocandin B through solid-state fermentation is established, and recycling of the vinasse is achieved. On the basis, Trichoderma reesei is adopted to carry out biological pretreatment on the vinasse, and precursor amino acid in the synthesis process of the echinocandin B is added, so that the yield of the echinocandin B is further increased, and efficient utilization of the vinasse is realized.

Description

Technical Field

[0001] The invention relates to a method for producing echinocandin B by solid-state fermentation using vinasse as a substrate, and belongs to the technical field of biological fermentation. Background Art

[0002] Echinocandin B (ECB) is a natural cyclic lipopeptide compound with a cyclic hexapeptide core and fatty acid side chain structure. It is a key precursor for the synthesis of the clinical antifungal drug anidulafungin. However, the biosynthesis research of echinocandin natural products only made an important breakthrough in 2012. Tang Yi's research group discovered and characterized the biosynthetic gene cluster of echinocandin B in an Aspergillus pachycristatus (formerly known as Emericella rugulosa NRRL 11440) (CACHO RA, JIANG W, CHOOI YH, et al. Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440 [J]. Journal of the American Chemical Society, 2012, 134 (40): 16781-16790). In industry, Emericella rugulosa NRRL 11440 strain is also commonly used to obtain echinocandin B through fermentation, but the high production cost limits its widespread application.

[0003] Shanxi is one of the main liquor production areas in my country. The large amount of distillers grains (DGS) produced during the winemaking process is rich in various nutrients. Distillers grains are a biomass resource with great application potential. The current utilization rate of distillers grains is low, resulting in a huge waste of resources. If not handled properly, distillers grains are prone to corruption and deterioration, causing environmental pollution. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for producing echinocandin B by solid-state fermentation using distiller's grains as a substrate. The present invention utilizes distiller's grains to replace traditional culture medium to directly produce echinocandin B, which, on the one hand, realizes high-value reuse of distiller's grains and reduces resource waste, and on the other hand, can realize efficient green biosynthesis of echinocandin B, reduce the production cost of echinocandin B, and thus reduce the cost of medication for patients.

[0005] The technical solution of the present invention is as follows:

[0006] Application of distiller's grains as substrate in the production of echinocandin B by solid-state fermentation.

[0007] Preferably according to the present invention, the solid-state fermentation strain is Emericella rugulosa CPCC 400818.

[0008] A method for producing echinocandin B by solid-state fermentation using distiller's grains as a substrate comprises the following steps:

[0009] After the wine lees are crushed, the water content is adjusted to 40%-80% to obtain a culture matrix; after the culture matrix is ​​sterilized, spores of Emericella rugulosa CPCC 400818 are inoculated, and after being evenly mixed, solid-state fermentation is performed to produce echinocandin B.

[0010] Preferably according to the present invention, the vinasse is crushed and passed through a 10-50 mesh sieve.

[0011] Preferably according to the present invention, the pH of the culture matrix is ​​5.0-8.0, more preferably 6.0.

[0012] Further preferably, the moisture content and pH of the culture matrix can be simultaneously adjusted using disodium hydrogen phosphate-citrate buffer.

[0013] Preferably according to the present invention, the moisture content of the culture matrix is ​​60%, v / w, mL / g.

[0014] According to the preferred embodiment of the present invention, the inoculation amount of the spores of Emericella rugulosa CPCC 400818 is: the initial spore concentration in the culture matrix after inoculation is 1×10 4 -5×10 6 / gds; more preferably 1×10 6 pcs / gds.

[0015] Preferably according to the present invention, the fermentation temperature of the solid-state fermentation is 24°C-34°C; more preferably 28°C-30°C.

[0016] Preferably according to the present invention, the fermentation time of the solid-state fermentation is 4-9 days, more preferably 6 days.

[0017] Preferably according to the present invention, the method further comprises: pre-treating the wine lees, wherein the pre-treatment is co-culturing the wine lees with Trichoderma reesei at a temperature of 28° C.-32° C. for 9-10 days.

[0018] Further preferably, the Trichoderma reesei is Trichoderma reesei OEX9.

[0019] Preferably according to the present invention, the method further comprises: adding precursor amino acids ornithine, threonine, tyrosine and proline in the synthesis process of echinocandin B to the culture matrix.

[0020] Further preferably, the final concentrations of ornithine, threonine, tyrosine and proline in the culture matrix are all 1-3 mg / gds.

[0021] In the present invention, the optimal fermentation conditions for solid-state fermentation are as follows: the moisture content of the culture matrix is ​​60%, the pH of the culture matrix is ​​6.0, and the initial spore concentration in the culture matrix is ​​1×10 6 / gds, the fermentation temperature is 30°C, and the fermentation time is 6 days. Under the above-mentioned optimal fermentation conditions, the output of echinocandin B produced using wine lees as a substrate is 754.0μg / gds. In order to further increase the output of echinocandin B, the present invention also uses Trichoderma reesei OEX9 to pretreat wine lees, and the final output of echinocandin B reaches 1382.9μg / gds. The present invention also adds precursor amino acids in the synthesis process of echinocandin B on the basis of the pretreatment of Trichoderma reesei OEX9, and the output of echinocandin B is further increased to 1797.5μg / gds.

[0022] Beneficial effects:

[0023] 1. The present invention compares distiller's grains with other biomass raw materials and ultimately selects industrial waste distiller's grains as the culture matrix for producing echinocandin B. While fully utilizing industrial waste resources and reducing environmental pollution, it also provides cheaper and more abundant raw materials for the industrial production of echinocandin B, thereby creating greater economic benefits.

[0024] 2. This patent provides a method for producing echinocandin B by solid-state fermentation using vinasse as substrate. After the vinasse is crushed, the moisture content and pH are adjusted, and then Emericella rugulosa CPCC 400818 is inoculated to produce echinocandin B by solid-state fermentation. A method for producing echinocandin B by solid-state fermentation is established, and the recycling and reuse of vinasse is realized. On this basis, Trichoderma reesei is used to biologically pretreat the vinasse, and precursor amino acids in the synthesis process of echinocandin B are added, which further increases the yield of echinocandin B and realizes the efficient utilization of vinasse. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the standard curve of echinocandin B.

[0026] Figure 2 HPLC chromatogram of the echinocandin B standard.

[0027] Figure 3This is the biomass change curve of E. rugulosa growing in different biomass raw materials.

[0028] Figure 4 This is the biomass change curve of Penicillium chrysogenum and E. rugulosa growing in wine lees.

[0029] Figure 5 The bar graph shows the yield of echinocandin B under different culture pH conditions.

[0030] Figure 6 It is a bar chart of the yield of echinocandin B under different moisture contents.

[0031] Figure 7 It is a bar graph of the production of echinocandin B at different inoculation amounts.

[0032] Figure 8 It is a bar graph of the production of echinocandin B at different fermentation temperatures.

[0033] Fig. 9 It is a bar graph of the production of echinocandin B at different fermentation times.

[0034] Fig.10 It is a bar graph of the production of echinocandin B in different treatment groups.

[0035] Fig.11 The bar graph shows the production of echinocandin B before and after the addition of precursor amino acids. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is further described below in conjunction with the examples. The materials and reagents involved in the examples are all common commercially available products unless otherwise specified; the experimental steps involved in the examples are all routine operations in the art unless otherwise specified.

[0037] Sources of biomass raw materials:

[0038] Sugarcane bagasse can be purchased from Guangxi Yuante Agricultural Technology Co., Ltd.; wheat straw and corn cobs can be purchased from Donghai County, Lianyungang City, Jiangsu Province; vinegar dregs can be purchased from the Soy Sauce and Vinegar Sales Center in Pingshun County, Changzhi City, Shanxi Province; soy sauce dregs can be purchased from Handan Hao Soy Sauce Factory in Handan City, Hebei Province; distiller's dregs can be purchased from a local winery in Fenyang City, Shanxi Province.

[0039] Source of microbial materials:

[0040] The Emericella rugulosa strain can be purchased from the Chinese Pharmaceutical Microbiological Culture Collection, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, with a platform strain number of CPCC 400818 or the American Type Culture Collection, with a platform strain number of ATCC 58397. Both are the same strain.

[0041] Penicillium chrysogenum was purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd., platform strain number: bio-11266;

[0042] Trichoderma reesei OEX9 can be obtained according to the construction method in the literature "Engineering Trichoderma reesei for Hyperproduction of Cellulases on Glucose to Efficiently Saccharify Pretreated Corncobs", which is based on Trichoderma reesei QM9414 as the starting strain, and the promoter P tcul Overexpression of the cellulase regulator XYRI achieved high expression of cellulase. The strain can produce cellulase under the condition of glucose as carbon source, eliminating the need for inducing sugar for enzyme production.

[0043] Method for quantitative detection of echinocandin B by HPLC:

[0044] HPLC detection conditions: mobile phase A: water, mobile phase B: acetonitrile, 0.1% trifluoroacetic acid was added to mobile phases A and B respectively;

[0045] Gradient elution, mobile phase B: 0-25 min, 30%-100%; 25-30 min, 100%; 30-32 min, 100%-30%; 32-40 min, 30%; UV 227 nm detection, 10 μL injection each time, flow rate 1 mL / min.

[0046] According to the above method, the peak area of ​​the echinocandin B standard at different concentrations (0, 20, 40, 60, 80, 100 μg / mL) was determined to establish a standard curve of echinocandin B. The obtained standard curve of echinocandin B is as follows: Figure 1 As shown, the fitting equation is: y = 4.0164x + 5.2619, R 2 =0.9986, wherein the HPLC chromatogram of the echinocandin B standard is as follows Figure 2 shown.

[0047] Example 1: Determination of fermentation substrate

[0048] The biomass raw materials (sugar bagasse, wheat straw, corn cob, vinegar lees, wine lees, soy sauce lees) were crushed and passed through a 10-mesh sieve. 25.0 g of each sieved biomass raw material was placed in a 500 mL conical flask, and water was added to adjust the moisture content to 60% (v / w, mL / g). After sterilization, the same amount of E. rugulosa spore suspension was inoculated into different biomass raw materials so that the initial spore concentration after inoculation was about 1.0×10 5 After being evenly mixed, solid-state fermentation was carried out at 30°C for 5 days. The biomass of E. rugulosa was measured every day to determine the optimal fermentation substrate.

[0049] The biomass determination method is as follows: 2.5 g of fermentation sample is mixed with 15 mL of staining solution (0.5% 2,3,5-triphenyltetrazolium chloride-PBS buffer, w / v, g / L), and reacted at 37°C for 60 min; the reaction is terminated with 5.0 mL of formaldehyde; centrifuged at 4200×g for 10 min, and the supernatant is removed; the residue is washed with 15 mL of sterile water, centrifuged to remove the supernatant, and repeated twice; extracted with 25 mL of ethyl acetate for 2 h, and the absorbance of the extract is measured at 485 nm using a spectrophotometer.

[0050] The absorbance reflects the specific activity of dehydrogenase in living cells and is linearly related to the wet weight of living cells in the sample (mg / gds). Determining the specific activity of dehydrogenase in fungi can characterize the physiological activity of fungi and calculate the biomass of living cells in the tested sample.

[0051] The results of biomass changes of E. rugulosa growth are as follows Figure 3 As shown in the figure, among the six biomass raw materials, sugarcane bagasse, wheat straw and corn cobs are biomass raw materials that have not been degraded by microorganisms, and vinegar lees, distiller's grains and soy sauce lees are biomass raw materials that have been degraded by microbial fermentation. From the results of the change in biomass, it can be seen that the growth of E. rugulosa in sugarcane bagasse, wheat straw and corn cobs is limited, which may be because sugarcane bagasse, wheat straw and corn cobs have not been degraded by microorganisms, and the content of fermentable small molecule components (such as glucose) is relatively small, which in turn limits the growth of microorganisms; and the growth of E. rugulosa in vinegar lees, distiller's grains and soy sauce lees showed significant differences, among which the growth levels in vinegar lees and soy sauce lees were not much different from those in sugarcane bagasse, wheat straw and corn cobs, and the growth level in distiller's grains was much higher than that in the other biomass raw materials.

[0052] Example 2: Comparison of fermentation strains using wine lees as substrate

[0053] The lees were crushed and passed through a 10-mesh sieve. 25.0 g of the sieved lees were placed in a 500 mL conical flask, and water was added to adjust the moisture content to 60% (v / w, mL / g). After sterilization, spore suspensions of Penicillium chrysogenum and E. rugulosa were inoculated respectively so that the initial spore concentration in the culture matrix after inoculation was 1.0 × 10 5 / gds, mixed evenly, and then solid-state fermentation was carried out at 30℃ for 5 days. The biomass was measured every day to determine the most suitable fermentation strain.

[0054] The method for determining biomass is the same as in Example 1.

[0055] The biomass changes of Penicillium chrysogenum and E. rugulosa are shown in Figure 4 As shown, as a fungus, Penicillium chrysogenum's secondary metabolites also have antibacterial effects. It can be seen from the figure that compared with Penicillium chrysogenum, E. rugulosa has a shorter lag period in the growth of wine lees, can enter the logarithmic growth phase and reach the stable phase faster, indicating that E. rugulosa is more suitable for growing on wine lees.

[0056] Example 3: Optimization of fermentation conditions

[0057] The effects of pH and moisture content of culture medium, inoculation size of bacterial strains, and fermentation temperature and time on the production of echinocandin B were studied in order to optimize the fermentation process of echinocandin B.

[0058] (1) Effect of culture medium pH on the production of echinocandin B

[0059] The lees were crushed and passed through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500 mL conical flask. Disodium hydrogen phosphate-citric acid buffers of different pH values ​​were prepared and added to the lees to adjust the water content to 60% (v / w, mL / g). The pH values ​​of the obtained culture matrix were adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentration in the culture matrix after inoculation was 1.0 × 10 5 / gds, mixed evenly, and then solid-state fermentation was carried out at 30°C. The yield of echinocandin B was measured after 5 days of fermentation to determine the optimal pH.

[0060] The yield results of echinocandin B under different culture pH conditions are as follows Figure 5 As shown, the results showed that the yield of echinocandin B was higher when the pH was 5.0-8.0, and the optimal pH condition was 6.0.

[0061] (2) Effect of culture medium moisture content on the production of echinocandin B

[0062] The lees were crushed and passed through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500 mL conical flask. Sodium hydrogen phosphate-citric acid buffer with a pH of 7.0 was prepared and added to the lees to adjust the moisture content to 30%, 40%, 50%, 60%, 70%, and 80% (v / w, mL / g), respectively. The pH of the obtained culture matrix was adjusted to 7.0. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentration in the culture matrix after inoculation was 1.0×10 5 / gds, mixed evenly, and then solid-state fermentation was carried out at 30°C. The yield of echinocandin B was measured after 5 days of fermentation to determine the optimal moisture content.

[0063] The yield results of Echinocandin B under different moisture contents are as follows Figure 6 As shown, the results show that echinocandin B can be produced when the moisture content is 40%-80%, among which the optimal moisture content is 60%.

[0064] (3) Effect of inoculum size on the production of echinocandin B

[0065] The lees were crushed and passed through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500 mL conical flask. Sodium hydrogen phosphate-citric acid buffer with a pH of 7.0 was prepared and added to the lees to adjust the moisture content to 60% (v / w, mL / g). The pH of the resulting culture matrix was adjusted to 7.0. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentrations in the culture matrix after inoculation were 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 / gds, mixed evenly, and then solid-state fermentation was carried out at 30°C. The yield of echinocandin B was measured after 5 days of fermentation to determine the optimal inoculation amount.

[0066] The yield results of Echinocandin B at different inoculation amounts are as follows Figure 7 As shown, the results show that the inoculum size is 1×10 4 -5×10 6 / gds, all of them were able to produce echinocandin B, among which the best inoculation amount was 1×10 6 pcs / gds.

[0067] (4) Effect of fermentation temperature on the production of echinocandin B

[0068] The lees were crushed and passed through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500 mL conical flask. Sodium hydrogen phosphate-citric acid buffer with a pH of 7.0 was prepared and added to the lees to adjust the moisture content to 60% (v / w, mL / g). The pH of the resulting culture matrix was adjusted to 7.0. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentration in the culture matrix after inoculation was 1×10 5 / gds, and after uniform mixing, solid-state fermentation was carried out at 24°C, 26°C, 28°C, 30°C, 32°C, and 34°C, respectively. The production of echinocandin B was measured after 5 days of fermentation to determine the optimal fermentation temperature.

[0069] The yield of echinocandin B at different fermentation temperatures is shown in Figure 8 As shown, the results showed that echinocandin B could be produced when the fermentation temperature was 24℃-34℃, and the yield was better when the fermentation temperature was 28℃-30℃, among which the optimal fermentation temperature was 30℃.

[0070] (5) Evaluate the effect of fermentation time on the production of echinocandin B under the above optimal conditions

[0071] The lees were crushed and passed through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500 mL conical flask. Sodium hydrogen phosphate-citric acid buffer with a pH of 6.0 was prepared and added to the lees to adjust the moisture content to 60% (v / w, mL / g). The pH of the resulting culture matrix was adjusted to 6.0. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentration in the culture matrix after inoculation was 1×10 6 / gds, mixed evenly, and solid-state fermented at 30°C for 9 days. The production of echinocandin B was measured every day from the 4th day to determine the optimal fermentation time.

[0072] The yield of echinocandin B at different fermentation times is shown in Fig. 9 As shown in the results, with the extension of fermentation time, the production of echinocandin B gradually increased and reached a maximum of 754.0 μg / gds on the 6th day of fermentation, and then the content of echinocandin decreased slightly and then remained stable.

[0073] Example 4: Effect of vinasse pretreatment on the production of echinocandin B

[0074] A method for producing echinocandin B by solid-state fermentation comprises the following steps:

[0075] (1) inoculating Trichoderma reesei OEX9 into a basic medium, culturing at 30° C. for 2 days, and collecting Trichoderma reesei mycelium using a funnel;

[0076] The basic medium consists of 20 g / L glucose, 5.0 g / L (NH 4)2 SO 4 , 15g / L KH 2 PO 4 , 10 g / L peptone, adjust pH to 5.5 with NaOH and sterilize by autoclave, add 0.6 g / L MgSO 4 , 0.6 g / L CaCl 2 , 5.0mg / L FeSO 4 7H 2 O, 2.0 mg / L CoCl 2 ·2H 2 O, 1.6 mg / L MnSO 4 ·H 2 O, 1.4 mg / L ZnSO 4 7H 2 O and 10 mM uridine;

[0077] (2) crushing the wine lees and passing through a 10-mesh sieve, adding water to adjust the moisture content to 60% (v / w, mL / g), sterilizing, adding Trichoderma reesei mycelium according to a wet weight of about 2.5 g / 50.0 gds, mixing evenly, and pretreating in an incubator at 30° C. for 9 days;

[0078] (3) After the pretreatment, the lees were dried and crushed to pass through a 10-mesh sieve. 50.0 g of the sieved lees were placed in a 500-mL conical flask. Sodium hydrogen phosphate-citric acid buffer with a pH of 6.0 was prepared and added to the lees to adjust the moisture content to 60% (v / w, mL / g). The pH of the resulting culture matrix was adjusted to 6.0. After sterilization, the spore suspension of E. rugulosa was inoculated into the culture matrix so that the initial spore concentration in the culture matrix after inoculation was 1×10 6 / gds, mixed evenly, and then solid-state fermented at 30°C for 6 days to determine the content of echinocandin B in the fermentation matrix.

[0079] At the same time, a control group NP was set up: the wine lees without any pretreatment were fermented according to the above method;

[0080] And the control group Glu: the vinasse was taken without pretreatment with Trichoderma reesei but 1.1 g / 50.0 gds of glucose was added, and fermentation was carried out according to the above method.

[0081] The results of the production of echinocandin B in different treatment groups are shown in Fig.10As shown, the results showed that the production of echinocandin B in the control group NP was 728.4μg / gds; the production of echinocandin B in the control group Glu was 902.3μg / gds; the production of echinocandin B in the experimental group (Pre) was 1382.9μg / gds. The results showed that the production of echinocandin B can be greatly improved after pretreatment with Trichoderma reesei OEX9. The reason is that the cellulase produced by the metabolism of Trichoderma reesei OEX9 can further degrade cellulose and generate more fermentable small molecules (such as glucose). However, the production of echinocandin B in the experimental group is still much higher than that in the control group Glu, indicating that the pretreatment of Trichoderma reesei OEX9 brings some unknown promoting factors to the production of echinocandin B, which needs further experimental research.

[0082] Example 5: Effect of adding precursor amino acids on the production of echinocandin B based on lees pretreatment

[0083] A method for producing echinocandin B by solid-state fermentation comprises the following steps:

[0084] (1) inoculating Trichoderma reesei OEX9 into a basic medium, culturing at 30° C. for 2 days, and collecting Trichoderma reesei mycelium using a funnel;

[0085] The basic medium consists of 20 g / L glucose, 5.0 g / L (NH 4)2 SO 4 , 15g / L KH 2 PO 4 , 10 g / L peptone, adjust pH to 5.5 with NaOH and sterilize by autoclave, add 0.6 g / L MgSO 4 , 0.6 g / L CaCl 2 , 5.0mg / L FeSO 4 7H 2 O, 2.0 mg / L CoCl 2 ·2H 2 O, 1.6 mg / L MnSO 4 ·H 2 O, 1.4mg / L ZnSO 4 7H 2 O and 10 mM uridine;

[0086] (2) crushing the wine lees and passing through a 10-mesh sieve, adding water to adjust the moisture content to 60% (v / w, mL / g), sterilizing, adding Trichoderma reesei mycelium according to a wet weight of about 2.5 g / 50.0 gds, mixing evenly, and pretreating in an incubator at 30° C. for 9 days;

[0087] (3) After the pretreatment, the mixture was dried and crushed to pass through a 10-mesh sieve. 50.0 g of the sieved lees was placed in a 500 mL conical flask. Sodium hydrogen phosphate-citrate buffer with a pH of 6.0 was prepared and added to the lees to adjust the moisture content to 60% (v / w, mL / g). The pH of the resulting culture matrix was adjusted to 6.0. After sterilization, ornithine, threonine, tyrosine and proline were added to the culture matrix after filtration and sterilization at a final concentration of 2 mg / gds. The spore suspension of E. rugulosa was inoculated to make the initial spore concentration in the culture matrix after inoculation to be 1×10 6 / gds, mixed evenly, and then solid-state fermented at 30°C for 6 days to determine the content of echinocandin B in the fermentation matrix.

[0088] The results are as follows Fig.11 As shown in the figure, compared with the group without adding precursor amino acids (NA group), the production of echinocandin B was further increased after adding precursor amino acids (+A group), reaching 1797.5 μg / gds. Ornithine, threonine, tyrosine and proline are all precursor amino acids for the synthesis of echinocandin B. The above results show that increasing the precursor amino acids can further increase the production of echinocandin B.

Claims

1. Application of distiller's grains as substrate in the production of echinocandin B by solid-state fermentation.

2. The use according to claim 1, characterized in that The solid-state fermentation strain is Emericella rugulosa CPCC 400818.

3. A method for producing echinocandin B by solid-state fermentation using vinasse as substrate, characterized in that: The steps include: After the wine lees are crushed, the water content is adjusted to 40%-80% to obtain a culture matrix; after the culture matrix is ​​sterilized, spores of Emericella rugulosa CPCC 400818 are inoculated, and after being evenly mixed, solid-state fermentation is performed to produce echinocandin B.

4. The method according to claim 3, characterized in that The lees are crushed and passed through a 10-50 mesh sieve.

5. The method according to claim 3, characterized in that The pH of the culture matrix is ​​5.0-8.0, more preferably 6.

0.

6. The method according to claim 5, characterized in that The moisture content and pH of the culture matrix can be simultaneously adjusted using disodium hydrogen phosphate-citric acid buffer.

7. The method according to claim 3, characterized in that The water content of the culture matrix is ​​60%, v / w, mL / g.

8. The method according to claim 3, characterized in that The inoculation amount of the spores of Emericella rugulosa CPCC 400818 is: the initial spore concentration in the culture matrix after inoculation is 1×10 4 -5×10 6 / gds; more preferably 1×10 6 pcs / gds; Preferably, the fermentation temperature of the solid-state fermentation is 24°C-34°C; more preferably, 28°C-30°C; Preferably, the fermentation time of the solid-state fermentation is 4-9 days, more preferably 6 days.

9. The method according to claim 3, characterized in that The method further comprises: pre-treating the wine lees, wherein the pre-treatment is co-culturing the wine lees with Trichoderma reesei at 28° C.-32° C. for 9-10 days; Further preferably, the Trichoderma reesei is Trichoderma reesei OEX9.

10. The method according to claim 3, characterized in that The method further comprises: adding precursor amino acids ornithine, threonine, tyrosine and proline in the synthesis process of echinocandin B to the culture matrix; Further preferably, the final concentrations of ornithine, threonine, tyrosine and proline in the culture matrix are all 1-3 mg / gds.

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