Aspergillus niger, microbial agent, solid-state fermentation broth, preparation method therefor and use thereof

Abstract

Provided are an Aspergillus niger, a microbial agent thereof, a solid-state fermentation broth thereof, a preparation method therefor, and a use thereof. The Aspergillus niger is Aspergillus niger AGM01, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2024768. The Aspergillus niger has a relatively high enzyme activity of β-glucosidase. By performing fermentation and cultivation on the Aspergillus niger in a solid-state fermentation medium containing a small-molecule inducer so as to obtain a solid-state fermentation broth of the Aspergillus niger, the enzyme activity of β-glucosidase in the solid-state fermentation broth of the Aspergillus niger is improved.

Description

A method for preparing and applying Aspergillus niger, its inoculant, solid-state fermentation broth, and their applications.

[0001] Related applications

[0002] This application claims priority to the earlier application No. 202411183576.6 filed with the China National Intellectual Property Administration on August 27, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of microbiology, specifically relating to Aspergillus niger, a fungal agent, a solid fermentation broth, its preparation method, and its application. Background Technology

[0004] With the continuous development of the bio-industry, more and more products are being prepared through microbial fermentation. Glucose, in particular, is a crucial energy source for microbial growth and metabolism during microbial fermentation, meaning that a large amount of glucose is consumed in this process. Currently, the glucose used in microbial fermentation mainly comes from starch hydrolysate, indicating that the demand for glucose will continue to increase with the development of the bio-industry. This could lead to increased demand for starchy grains, potentially resulting in higher food prices or food supply shortages.

[0005] Lignocellulose in agricultural and forestry waste is a potential source of glucose. Agricultural waste includes sugarcane bagasse, straw, rice straw, and fruit shells; forestry waste includes tree tops, branches, canopies, sawdust, and dead branches. Hydrolyzing lignocellulose in these wastes into glucose and xylose not only reduces reliance on starchy grains but also enables the resource utilization of these wastes, demonstrating environmental friendliness and economic sustainability. Therefore, the efficient production of lignocellulose hydrolysate is an engineering problem that needs to be solved in the bio-industry.

[0006] After nearly 20 years of research and development in my country on key enzymes in the hydrolysis of lignocellulose, namely cellulase and β-glucosidase, the enzyme activity of cellulase has met the industrial needs to a certain extent. However, the enzyme activity of β-glucosidase has not yet met the industrial needs. No major breakthroughs have been made in screening and cultivating strains that produce high levels of β-glucosidase. The enzyme activity of β-glucosidase reported in the literature is not high, mostly below 20 U / mL, especially cellobiase activity, which is even lower. Summary of the Invention

[0007] The problem with the existing technology is that corn cobs are usually used as the main carbon source to induce strains to produce β-glucosidase, and the strains are cultured through semi-solid fermentation. However, this method often results in low enzyme activity of the β-glucosidase produced, which is difficult to meet the needs of industrial production.

[0008] To address the problems existing in the prior art, the present invention provides Aspergillus niger, inoculant, solid fermentation broth, preparation method and application thereof.

[0009] Specifically, the present invention provides the following technical solution:

[0010] Technical Solution 1: A type of Aspergillus niger, characterized in that the Aspergillus niger is Aspergillus niger AGM01, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number: CCTCC NO: M 2024768.

[0011] Technical Solution 2: Aspergillus niger according to Technical Solution 1, characterized in that the ITS gene sequence of Aspergillus niger AGM01 is shown in SEQ ID NO.3.

[0012] Technical Solution 3: Aspergillus niger according to Technical Solution 1 or 2, characterized in that the Aspergillus niger AGM01 has the characteristic of producing β-glucosidase.

[0013] Technical Solution 4: Aspergillus niger according to Technical Solution 3, characterized in that the β-glucosidase activity of Aspergillus niger AGM01 is 20-30 U, wherein U refers to one enzyme activity unit of β-glucosidase, wherein one enzyme activity unit of β-glucosidase is defined as the amount of enzyme required per minute to produce 1 μmol of glucose by enzymatic hydrolysis of cellobiose in each mL of crude enzyme solution of Aspergillus niger AGM01;

[0014] The crude enzyme solution of Aspergillus niger AGM01 was prepared by the following method: Aspergillus niger was amplified and cultured to obtain Aspergillus niger spore suspension, the Aspergillus niger spore suspension was fermented and cultured to obtain fermentation broth, and then solid-liquid separation was performed to obtain supernatant, which is the crude enzyme solution of Aspergillus niger AGM01.

[0015] Technical Solution 5: Aspergillus niger according to Technical Solution 4, characterized in that the fermentation culture time is 5-7 days.

[0016] Technical Solution 6: A fermentation preparation method for Aspergillus niger inoculant, characterized in that the fermentation preparation method includes the following steps:

[0017] (a) Amplify and culture Aspergillus niger according to any one of technical solutions 1-5 to obtain a suspension of Aspergillus niger spores;

[0018] (b) The Aspergillus niger spore suspension obtained in step (a) is fermented and cultured.

[0019] Technical Solution 7: The fermentation preparation method according to Technical Solution 6, characterized in that, in step (a), the content of 10 spores per mL of Aspergillus niger spore suspension is... 6 -10 7 The spores of Aspergillus niger as described in any one of technical solutions 1-5.

[0020] Technical Solution 8: The fermentation preparation method according to Technical Solution 6 is characterized in that the culture temperature in step (a) is 26-30℃; and / or the culture temperature in step (b) is 26-32℃.

[0021] Technical Solution 9: The fermentation preparation method according to Technical Solution 6 is characterized in that the culture time in step (a) is 24-48h.

[0022] Technical Solution 10: A black mold agent, characterized in that it contains any one of Technical Solutions 1-5.

[0023] Technical Solution 11: A Aspergillus niger agent, characterized in that the Aspergillus niger agent is prepared by any one of the fermentation preparation methods in Technical Solutions 6-9.

[0024] Technical Solution 12: A solid-state fermentation broth of Aspergillus niger, characterized in that it contains Aspergillus niger as described in any one of Technical Solutions 1-5, wherein the enzyme activity of β-glucosidase in the solid-state fermentation broth of Aspergillus niger is 110-170 U, wherein U refers to one enzyme activity unit of β-glucosidase, and wherein the amount of enzyme required to produce 1 μmol of glucose per minute by enzymatic hydrolysis of cellobiose in each mL of the solid-state fermentation broth of Aspergillus niger is defined as one enzyme activity unit of β-glucosidase.

[0025] Technical Solution 13: The solid-state fermentation broth of Aspergillus niger according to Technical Solution 12 is characterized in that it is obtained by culturing the Aspergillus niger in a seed culture medium, wherein, by weight, the seed culture medium comprises: 1-30 parts glucose, 5-20 parts yeast extract, 0.1-0.4 parts magnesium sulfate, 1-4 parts potassium dihydrogen phosphate, 0.2-2 parts calcium chloride, 0.001-0.01 parts ferric sulfate, 0.001-0.01 parts manganese sulfate, 0.001-0.01 parts zinc sulfate, and 0.001-0.01 parts cobalt chloride.

[0026] Technical Solution 14: The solid fermentation broth of Aspergillus niger according to Technical Solution 13 is characterized in that the seed culture medium comprises: 5-20 parts glucose, and / or 5-15 parts yeast extract, and / or 0.2-0.35 parts magnesium sulfate, and / or 1-2 parts potassium dihydrogen phosphate, and / or 0.5-1.5 parts calcium chloride, and / or 0.001-0.008 parts ferric sulfate, and / or 0.001-0.008 parts manganese sulfate, and / or 0.001-0.008 parts zinc sulfate, and / or 0.001-0.008 parts cobalt chloride.

[0027] Technical Solution 15: The solid fermentation broth of Aspergillus niger according to Technical Solution 13 is characterized in that the yeast extract is yeast extract FM902.

[0028] Technical Solution 16: The solid fermentation broth of Aspergillus niger according to Technical Solution 14 is characterized in that the yeast extract is yeast extract FM902.

[0029] Technical Solution 17: The solid fermentation broth of Aspergillus niger according to any one of technical solutions 12-16, characterized in that the seed culture medium further includes: water.

[0030] Technical Solution 18: The solid fermentation broth of Aspergillus niger according to Technical Solution 17 is characterized in that the seed culture medium comprises: glucose at a concentration of 1-30 g / L, yeast extract at a concentration of 5-20 g / L, magnesium sulfate at a concentration of 0.1-0.4 g / L, potassium dihydrogen phosphate at a concentration of 1-4 g / L, calcium chloride at a concentration of 0.2-2 g / L, ferric sulfate at a concentration of 1-10 mg / L, manganese sulfate at a concentration of 1-10 mg / L, zinc sulfate at a concentration of 1-10 mg / L, and cobalt chloride at a concentration of 1-10 mg / L, with the remainder being water.

[0031] Technical Solution 19: The solid-state fermentation broth of Aspergillus niger according to Technical Solution 18 is characterized in that the seed culture medium comprises: a glucose concentration of 5-20 g / L, and / or a yeast extract concentration of 5-15 g / L, and / or a magnesium sulfate concentration of 0.2-0.35 g / L, and / or a potassium dihydrogen phosphate concentration of 1-2 g / L, and / or a calcium chloride concentration of 0.5-1.5 g / L, and / or a ferric sulfate concentration of 1-8 mg / L, and / or a manganese sulfate concentration of 1-8 mg / L, and / or a zinc sulfate concentration of 1-8 mg / L, and / or a cobalt chloride concentration of 1-8 mg / L, with the remainder being water.

[0032] Technical Solution 20: The solid-state fermentation broth of Aspergillus niger according to Technical Solution 12 is characterized in that it is obtained by culturing Aspergillus niger in a solid-state fermentation medium, wherein, by weight, the solid-state fermentation medium includes 1-20 parts of a small molecule inducer, wherein the small molecule inducer is one or more substances selected from the group consisting of xylose, cellobiose, lactose, sophorose, gentiobiose, xylose and substances containing sophoroside.

[0033] Technical Solution 21: The solid fermentation broth of Aspergillus niger according to Technical Solution 20 is characterized in that the small molecule inducer is xylose.

[0034] Technical Solution 22: The solid fermentation broth of Aspergillus niger according to Technical Solution 20 is characterized in that the substance containing sophoroside is kaempferol-3-O-β-D-sophoroside.

[0035] Technical Solution 23: The solid fermentation broth of Aspergillus niger according to Technical Solution 20 is characterized in that the solid fermentation culture medium further includes: a substance containing lignocellulose, ammonium sulfate, urea and calcium chloride.

[0036] Technical Solution 24: The solid fermentation broth of Aspergillus niger according to Technical Solution 23 is characterized in that, by weight, the solid fermentation culture medium comprises: 30-90 parts of a substance containing lignocellulose, 5-25 parts of ammonium sulfate, 1-15 parts of urea, 0.5-3 parts of calcium chloride and 1-20 parts of a small molecule inducer.

[0037] Technical Solution 25: The solid fermentation broth of Aspergillus niger according to Technical Solution 24 is characterized in that, by weight, the solid fermentation culture medium comprises: 30-75 parts of a substance containing lignocellulose, and / or 6-20 parts of ammonium sulfate, and / or 2-12 parts of urea, and / or 0.5-2 parts of calcium chloride, and / or 1-18 parts of a small molecule inducer.

[0038] Technical Solution 26: The solid fermentation liquid of Aspergillus niger according to Technical Solution 23 is characterized in that the substance containing lignocellulose includes one or more substances selected from the group consisting of wheat bran, straw and corn cob.

[0039] Technical Solution 27: The solid fermentation liquid of Aspergillus niger according to Technical Solution 26 is characterized in that the substance containing lignocellulose is a combination of wheat bran, straw and corn cob.

[0040] Technical Solution 28: The solid fermentation liquid of Aspergillus niger according to Technical Solution 27 is characterized in that, by weight, the substance containing lignocellulose is 10-30 parts wheat bran, 10-30 parts straw and 10-30 parts corn cob.

[0041] Technical Solution 29: The solid fermentation liquid of Aspergillus niger according to Technical Solution 28 is characterized in that, by weight, the substance containing lignocellulose is 10-25 parts wheat bran, and / or 10-25 parts straw, and / or 10-25 parts corn cob.

[0042] Technical solution 30: The solid fermentation broth of Aspergillus niger according to any one of technical solutions 20-25, characterized in that the solid fermentation culture medium further includes: water.

[0043] Technical Solution 31: The solid fermentation broth of Aspergillus niger according to Technical Solution 30 is characterized in that the solid fermentation culture medium comprises: a lignocellulose substance with a concentration of 30-90 g / L, an ammonium sulfate concentration of 5-25 g / L, a urea concentration of 1-15 g / L, a calcium chloride concentration of 0.5-3 g / L, and a small molecule inducer concentration of 1-20 g / L, with the remainder being water.

[0044] Technical Solution 32: The solid fermentation broth of Aspergillus niger according to Technical Solution 31 is characterized in that the solid fermentation culture medium comprises: a lignocellulose substance with a concentration of 30-75 g / L, and / or ammonium sulfate with a concentration of 6-20 g / L, and / or urea with a concentration of 2-12 g / L, and / or calcium chloride with a concentration of 0.5-2 g / L, and / or a small molecule inducer with a concentration of 1-18 g / L, with the remainder being water.

[0045] Technical solution 33: The solid fermentation broth of Aspergillus niger according to any one of technical solutions 26-29, characterized in that the solid fermentation culture medium further includes: water.

[0046] Technical Solution 34: The solid fermentation broth of Aspergillus niger according to Technical Solution 33 is characterized in that the solid fermentation culture medium comprises: wheat bran at a concentration of 10-30 g / L, straw at a concentration of 10-30 g / L, corn cob at a concentration of 10-30 g / L, ammonium sulfate at a concentration of 5-25 g / L, urea at a concentration of 1-15 g / L, calcium chloride at a concentration of 0.5-3 g / L, and a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water.

[0047] Technical Solution 35: The solid fermentation broth of Aspergillus niger according to Technical Solution 34 is characterized in that the solid fermentation culture medium comprises: wheat bran at a concentration of 10-25 g / L, and / or straw at a concentration of 10-25 g / L, and / or corn cob at a concentration of 10-25 g / L, and / or ammonium sulfate at a concentration of 5-25 g / L, and / or urea at a concentration of 1-15 g / L, and / or calcium chloride at a concentration of 0.5-3 g / L, and / or a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water.

[0048] Technical Solution 36. A method for preparing Aspergillus niger solid fermentation broth according to any one of technical solutions 12-35, characterized in that it includes the following steps: amplifying and culturing Aspergillus niger according to any one of technical solutions 1-5 to obtain Aspergillus niger spore suspension; inoculating the Aspergillus niger spore suspension into a seed culture medium for cultivation to obtain Aspergillus niger seed liquid; and inoculating the Aspergillus niger seed liquid into a solid fermentation culture medium for cultivation to obtain Aspergillus niger solid fermentation broth.

[0049] Technical Solution 37: The preparation method according to Technical Solution 36 is characterized by comprising the following steps:

[0050] (a) Amplify and culture Aspergillus niger according to any one of technical solutions 1-5 to obtain a suspension of Aspergillus niger spores;

[0051] (b) The Aspergillus niger spore suspension obtained in step (a) is inoculated into a seed culture medium for cultivation to obtain Aspergillus niger seed liquid;

[0052] (c) The Aspergillus niger seed liquid obtained in step (b) is inoculated into a solid fermentation medium for culture to obtain Aspergillus niger solid fermentation product;

[0053] (d) The Aspergillus niger solid fermentation product obtained in step (c) is mixed with a buffer solution, soaked, stirred and filtered to obtain crude enzyme solution;

[0054] (e) The crude enzyme solution obtained in step (d) is concentrated to obtain the concentrated solution, namely the solid fermentation broth of Aspergillus niger.

[0055] Technical Solution 38: The preparation method according to Technical Solution 37 is characterized in that, in step (a), each mL of Aspergillus niger spore suspension contains 10 6 -10 7 The spores of Aspergillus niger as described in any one of technical solutions 1-5.

[0056] Technical Solution 39: The preparation method according to Technical Solution 37 is characterized in that the culture temperature in step (a) is 26-30℃; and / or the culture temperature in step (b) is 26-30℃; and / or the culture temperature in step (c) is 27-29℃.

[0057] Technical Solution 40: The preparation method according to Technical Solution 37 is characterized in that the culture time in step (a) is 24-48h; and / or the culture time in step (b) is 24-48h; and / or the culture time in step (c) is 100-144h.

[0058] Technical solution 41: The preparation method according to technical solution 37 is characterized in that the culture rotation speed in step (b) is 150-300 rpm.

[0059] Technical solution 42: The preparation method according to technical solution 37 is characterized in that the culture pH in step (c) is 4.5-5.5.

[0060] Technical solution 43: The preparation method according to technical solution 37 is characterized in that the culture humidity in step (c) is 50-95%.

[0061] Technical Solution 44: The preparation method according to Technical Solution 37 is characterized in that, in step (d), the volume ratio of the solid fermentation product of Aspergillus niger to the buffer solution is (0.8-1.2):(6-10).

[0062] Technical solution 45: The preparation method according to technical solution 37 is characterized in that the soaking time in step (d) is 1-4 hours.

[0063] Technical solution 46: The preparation method according to technical solution 37 is characterized in that the stirring speed in step (d) is 100-300 rpm.

[0064] Technical solution 47: The preparation method according to technical solution 37 is characterized in that the stirring time in step (d) is 20-50 min.

[0065] Technical solution 48: The preparation method according to technical solution 37 is characterized in that the filtration in step (d) is plate and frame filtration.

[0066] Technical solution 49: The preparation method according to technical solution 37 is characterized in that, in step (e), the concentration is ultrafiltration concentration.

[0067] Technical Solution 50: The preparation method according to Technical Solution 49 is characterized in that the membrane for ultrafiltration concentration has a pore size of 10-60kD.

[0068] Technical solution 51: The preparation method according to technical solution 49 is characterized in that the ultrafiltration concentration time is 1-5 hours.

[0069] Technical solution 52: A solid fermentation broth of Aspergillus niger prepared by any one of technical solutions 36-51.

[0070] Technical Solution 53: A lignocellulose hydrolysate, characterized in that it is obtained by hydrolysis in a system containing any one of the following technical solutions: Aspergillus niger, or the Aspergillus niger agent described in technical solutions 10 or 11, or any one of the following technical solutions 12-35, or the Aspergillus niger solid fermentation broth described in technical solution 52.

[0071] Technical Solution 54: The lignocellulose hydrolysate according to Technical Solution 53 is characterized in that the lignocellulose is one or a combination of agricultural waste and forestry waste.

[0072] Technical Solution 55: The lignocellulose hydrolysate according to Technical Solution 54 is characterized in that the agricultural waste is one or a combination of sugarcane bagasse and straw.

[0073] Technical Solution 56: The lignocellulose hydrolysate according to any one of Technical Solutions 53-55, wherein the mass ratio of reducing sugar contained in the lignocellulose hydrolysate is more than 90%, wherein the mass ratio of reducing sugar refers to the percentage of the mass m2 of the lignocellulose raw material converted into reducing sugar by enzymatic hydrolysis relative to the total mass m1 of cellulose and hemicellulose contained in the lignocellulose raw material before hydrolysis.

[0074] Technical Solution 57: The application of Aspergillus niger as described in any one of Technical Solutions 1-5, or the Aspergillus niger agent described in Technical Solutions 10 or 11, or the Aspergillus niger solid fermentation broth as described in any one of Technical Solutions 12-35 or Technical Solution 52 in the preparation of lignocellulose hydrolysate. Attached Figure Description

[0075] Figure 1 shows the colony morphology of Aspergillus niger AGM01;

[0076] Figure 2 shows the phylogenetic tree of Aspergillus niger AGM01 constructed based on the ITS gene sequence.

[0077] Microbial strain preservation information

[0078] The Aspergillus niger AGM01 provided by this invention was deposited at the China Center for Type Culture Collection on April 24, 2024, with accession number CCTCC NO: M 2024768. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: 027-68754052.

[0079] The Asperillium niger strain Q270-C6 used in this invention embodiment was deposited on July 10, 2023, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20231261, address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. It has been published in Chinese patent application CN117143739A.

[0080] Beneficial effects of this invention:

[0081] (1) The Aspergillus niger AGM01 provided by the present invention has high β-glucosidase activity.

[0082] (2) The present invention ferments Aspergillus niger AGM01 in a solid fermentation medium containing small molecule inducers to obtain Aspergillus niger solid fermentation broth, which can improve the enzyme activity of β-glucosidase in the Aspergillus niger solid fermentation broth. Detailed Implementation

[0083] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely explained below in conjunction with specific embodiments. It should be noted that the content of the specific embodiments is only a specific implementation and explanation of the technical solutions of the present invention, and should not be construed as a limitation on the scope of protection of the present invention.

[0084] In existing technologies, corn cobs are typically used as the main carbon source to induce strains to produce β-glucosidase, which is then cultured through semi-solid fermentation. However, this method often results in low enzyme activity of the β-glucosidase produced, making it difficult to meet the needs of industrial production.

[0085] To address the problems existing in the prior art, the present invention specifically provides the following technical solution:

[0086] (1) A black Aspergillus, characterized in that the black Aspergillus is Aspergillus niger AGM01, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number: CCTCCNO: M 2024768.

[0087] (2) The Aspergillus niger according to technical solution (1) is characterized in that the ITS gene sequence of Aspergillus niger AGM01 is as shown in SEQ ID NO.3.

[0088] (3) The Aspergillus niger according to technical solution (1) or (2) is characterized in that the Aspergillus niger AGM01 has the characteristic of producing β-glucosidase; preferably, the enzyme activity of the β-glucosidase of the Aspergillus niger AGM01 is 20-30U, wherein U refers to one enzyme activity unit of β-glucosidase, wherein the amount of enzyme required to produce 1 μmol of glucose per minute by enzymatic hydrolysis of cellobiose in each mL of crude enzyme solution of Aspergillus niger AGM01 is defined as one enzyme activity unit of β-glucosidase;

[0089] The crude enzyme solution of Aspergillus niger AGM01 is prepared by the following method: Aspergillus niger is amplified and cultured to obtain Aspergillus niger spore suspension, and the Aspergillus niger spore suspension is fermented and cultured, preferably for 5-7 days, to obtain fermentation broth. Then, solid-liquid separation is performed to obtain supernatant, which is the crude enzyme solution of Aspergillus niger AGM01.

[0090] (4) A fermentation preparation method for an Aspergillus niger agent, characterized in that the fermentation preparation method includes the following steps:

[0091] (a) Amplify and culture Aspergillus niger according to any one of the technical solutions (1)-(3) to obtain a suspension of Aspergillus niger spores;

[0092] (b) The Aspergillus niger spore suspension obtained in step (a) is fermented and cultured.

[0093] (5) The fermentation preparation method according to technical solution (4) is characterized in that, in step (a), each mL of Aspergillus niger spore suspension contains 10 6 -10 7 The spores of Aspergillus niger as described in any one of the technical solutions (1)-(3);

[0094] Preferably, the culture temperature in step (a) is 26-30℃ and / or the culture time is 24-48h; and / or the fermentation culture temperature in step (b) is 26-32℃.

[0095] (6) A black mold agent, characterized in that it contains any one of the technical solutions (1)-(3) as a black mold.

[0096] (7) The Aspergillus niger agent according to technical solution (6) is characterized in that the Aspergillus niger agent is obtained by the fermentation preparation method described in technical solution (4) or (5).

[0097] (8) A solid fermentation broth of Aspergillus niger, characterized in that it contains Aspergillus niger as described in any one of technical solutions (1)-(3), wherein the enzyme activity of β-glucosidase in the solid fermentation broth of Aspergillus niger is 110-170U, wherein U refers to one enzyme activity unit of β-glucosidase, wherein the amount of enzyme required to produce 1 μmol of glucose per minute by enzymatic hydrolysis of cellobiose in each mL of the solid fermentation broth of Aspergillus niger is defined as one enzyme activity unit of β-glucosidase.

[0098] (9) The solid fermentation broth of Aspergillus niger according to technical solution (8) is characterized in that the solid fermentation broth of Aspergillus niger is prepared by a method including the following steps: amplifying and culturing Aspergillus niger in any one of technical solutions (1)-(3) to obtain Aspergillus niger spore suspension; inoculating the Aspergillus niger spore suspension into a seed culture medium for culture to obtain Aspergillus niger seed liquid; and inoculating the Aspergillus niger seed liquid into a solid fermentation culture medium for culture to obtain Aspergillus niger solid fermentation broth.

[0099] (10) The solid fermentation broth of Aspergillus niger according to technical solution (8) or (9), characterized in that, by weight, the seed culture medium comprises: 1-30 parts glucose, 5-20 parts yeast extract, 0.1-0.4 parts magnesium sulfate, 1-4 parts potassium dihydrogen phosphate, 0.2-2 parts calcium chloride, 0.001-0.01 parts ferric sulfate, 0.001-0.01 parts manganese sulfate, 0.001-0.01 parts zinc sulfate and 0.001-0.01 parts cobalt chloride;

[0100] Preferably, the seed culture medium comprises: 5-20 parts glucose, and / or 5-15 parts yeast extract, and / or 0.2-0.35 parts magnesium sulfate, and / or 1-2 parts potassium dihydrogen phosphate, and / or 0.5-1.5 parts calcium chloride, and / or 0.001-0.008 parts ferric sulfate, and / or 0.001-0.008 parts manganese sulfate, and / or 0.001-0.008 parts zinc sulfate, and / or 0.001-0.008 parts cobalt chloride.

[0101] (11) The Aspergillus niger solid fermentation broth according to any one of technical solutions (8)-(10), characterized in that the seed culture medium further comprises: water,

[0102] Preferably, the seed culture medium comprises: glucose at a concentration of 1-30 g / L, yeast extract at a concentration of 5-20 g / L, magnesium sulfate at a concentration of 0.1-0.4 g / L, potassium dihydrogen phosphate at a concentration of 1-4 g / L, calcium chloride at a concentration of 0.2-2 g / L, ferric sulfate at a concentration of 1-10 mg / L, manganese sulfate at a concentration of 1-10 mg / L, zinc sulfate at a concentration of 1-10 mg / L, and cobalt chloride at a concentration of 1-10 mg / L, with the remainder being water;

[0103] More preferably, the seed culture medium comprises: a glucose concentration of 5-20 g / L, and / or a yeast extract concentration of 5-15 g / L, and / or a magnesium sulfate concentration of 0.2-0.35 g / L, and / or a potassium dihydrogen phosphate concentration of 1-2 g / L, and / or a calcium chloride concentration of 0.5-1.5 g / L, and / or a ferric sulfate concentration of 1-8 mg / L, and / or a manganese sulfate concentration of 1-8 mg / L, and / or a zinc sulfate concentration of 1-8 mg / L, and / or a cobalt chloride concentration of 1-8 mg / L, with the remainder being water.

[0104] (12) The Aspergillus niger solid fermentation liquid according to any one of the technical solutions (8)-(11), characterized in that the yeast extract is yeast extract FM902.

[0105] (13) The solid fermentation broth of Aspergillus niger according to any one of the technical solutions (8)-(12) is characterized in that, by weight, the solid fermentation culture medium includes 1-20 parts of small molecule inducer, wherein the small molecule inducer is one or more of xylose, cellobiose, lactose, sophorose, gentiobiose, xylose and substances containing sophoroside.

[0106] Preferably, the small molecule inducer is xylose;

[0107] Preferably, the substance containing sophoroside is kaempferol-3-O-β-D-sophoroside.

[0108] (14) The solid fermentation broth of Aspergillus niger according to any one of the technical solutions (8)-(13) is characterized in that the solid fermentation culture medium further includes: a substance containing lignocellulose, ammonium sulfate, urea and calcium chloride;

[0109] Preferably, by weight, the solid fermentation culture medium comprises: 30-90 parts of a substance containing lignocellulose, 5-25 parts of ammonium sulfate, 1-15 parts of urea, 0.5-3 parts of calcium chloride, and 1-20 parts of a small molecule inducer;

[0110] More preferably, by weight, the solid fermentation medium comprises: 30-75 parts of a substance containing lignocellulose, and / or 6-20 parts of ammonium sulfate, and / or 2-12 parts of urea, and / or 0.5-2 parts of calcium chloride, and / or 1-18 parts of a small molecule inducer.

[0111] (15) The Aspergillus niger solid fermentation liquid according to any one of technical solutions (8)-(14), characterized in that the substance containing lignocellulose includes one or more of wheat bran, straw and corn cob.

[0112] Preferably, the lignocellulose-containing substance is a combination of bran, straw, and corn cob;

[0113] More preferably, by weight, the substance containing lignocellulose is 10-30 parts wheat bran, 10-30 parts straw and 10-30 parts corn cob;

[0114] Most preferably, by weight, the lignocellulose-containing substance is 10-25 parts bran, and / or 10-25 parts straw, and / or 10-25 parts corn cob.

[0115] (16) The Aspergillus niger solid fermentation broth according to any one of technical solutions (8)-(15), characterized in that the solid fermentation culture medium further comprises: water,

[0116] Preferably, the solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-90 g / L, an ammonium sulfate concentration of 5-25 g / L, a urea concentration of 1-15 g / L, a calcium chloride concentration of 0.5-3 g / L, and a small molecule inducer concentration of 1-20 g / L, with the remainder being water;

[0117] More preferably, the solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-75 g / L, an ammonium sulfate concentration of 6-20 g / L, a urea concentration of 2-12 g / L, a calcium chloride concentration of 0.5-2 g / L, and a small molecule inducer concentration of 1-18 g / L, with the remainder being water.

[0118] (17) The Aspergillus niger solid fermentation broth according to any one of technical solutions (8)-(16), characterized in that the solid fermentation culture medium further comprises: water,

[0119] Preferably, the solid-state fermentation medium comprises: wheat bran at a concentration of 10-30 g / L, straw at a concentration of 10-30 g / L, corn cob at a concentration of 10-30 g / L, ammonium sulfate at a concentration of 5-25 g / L, urea at a concentration of 1-15 g / L, calcium chloride at a concentration of 0.5-3 g / L, and a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water;

[0120] More preferably, the solid-state fermentation medium comprises: wheat bran at a concentration of 10-25 g / L, and / or straw at a concentration of 10-25 g / L, and / or corn cob at a concentration of 10-25 g / L, and / or ammonium sulfate at a concentration of 5-25 g / L, and / or urea at a concentration of 1-15 g / L, and / or calcium chloride at a concentration of 0.5-3 g / L, and / or a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water.

[0121] (18) A method for preparing solid fermentation broth of Aspergillus niger according to any one of technical solutions (8)-(17), characterized in that it includes the following steps: amplifying and culturing Aspergillus niger according to any one of technical solutions (1)-(3) to obtain Aspergillus niger spore suspension; inoculating the Aspergillus niger spore suspension into a seed culture medium for culture to obtain Aspergillus niger seed liquid; and inoculating the Aspergillus niger seed liquid into a solid fermentation culture medium for culture to obtain solid fermentation broth of Aspergillus niger.

[0122] (19) The preparation method according to technical solution (18) is characterized by comprising the following steps:

[0123] (a) The Aspergillus niger strain described in any one of technical solutions (1)-(3) is amplified and cultured to obtain an Aspergillus niger spore suspension. Preferably, each mL of the Aspergillus niger spore suspension contains 10 6 -10 7 The spores of Aspergillus niger in any one of the technical solutions (1)-(3),

[0124] (b) The Aspergillus niger spore suspension obtained in step (a) is inoculated into a seed culture medium for cultivation to obtain Aspergillus niger seed liquid;

[0125] (c) The Aspergillus niger seed liquid obtained in step (b) is inoculated into a solid fermentation medium for culture to obtain Aspergillus niger solid fermentation product;

[0126] (d) The Aspergillus niger solid fermentation product obtained in step (c) is mixed with a buffer solution, soaked, stirred and filtered to obtain crude enzyme solution;

[0127] (e) The crude enzyme solution obtained in step (d) is concentrated to obtain the concentrated solution, namely the solid fermentation broth of Aspergillus niger.

[0128] (20) The preparation method according to technical solution (18) or (19) is characterized in that, in step (a), the amplification culture temperature is 26-30℃ and / or the amplification culture time is 24-48h;

[0129] And / or in step (b), the culture temperature is 26-30℃, and / or the culture speed is 150-300 rpm, and / or the culture time is 24-48 h;

[0130] And / or in step (c), the culture temperature is 27-29℃, and / or the culture pH is 4.5-5.5, and / or the culture humidity is 50-95%, and / or the culture time is 100-144h.

[0131] (21) The preparation method according to any one of the technical solutions (18)-(20) is characterized in that, in step (d), the volume ratio of the solid fermentation product of Aspergillus niger to the buffer solution is (0.8-1.2):(6-10);

[0132] and / or soaking time is 1-4 hours;

[0133] and / or the stirring speed is 100-300 rpm; and / or the stirring time is 20-50 min;

[0134] And / or the filter is a plate and frame filter.

[0135] (22) The preparation method according to any one of the technical solutions (18)-(21) is characterized in that, in step (e), the concentration is ultrafiltration concentration; preferably, the membrane for ultrafiltration concentration has a pore size of 10-60kD and / or the ultrafiltration concentration time is 1-5h.

[0136] (23) A lignocellulose hydrolysate, characterized in that it is obtained by hydrolyzing in a system containing Aspergillus niger as described in any one of technical solutions (1)-(3) or Aspergillus niger agent as described in technical solutions (6) or (7) or Aspergillus niger solid fermentation liquid as described in any one of technical solutions (8)-(17) or Aspergillus niger solid fermentation liquid prepared by any one of technical solutions (18)-(22);

[0137] Preferably, the reducing sugar content of the lignocellulose hydrolysate is 90% or more. The reducing sugar content refers to the percentage of the mass of reducing sugar (m2) that is converted from the lignocellulose raw material to reducing sugar after enzymatic hydrolysis, relative to the total mass (m1) of cellulose and hemicellulose contained in the lignocellulose raw material before hydrolysis.

[0138] (24) The application of Aspergillus niger prepared by any one of the technical solutions (1)-(3) or the Aspergillus niger agent prepared by any one of the technical solutions (6) or (7) or the Aspergillus niger solid fermentation liquid prepared by any one of the technical solutions (8)-(17) or the preparation method prepared by any one of the technical solutions (18)-(22) in the preparation of lignocellulose hydrolysate.

[0139] (25) The application of the lignocellulose hydrolysate according to technical solution (23) or technical solution (24) is characterized in that the lignocellulose is one or a combination of agricultural waste and forestry waste; preferably, the agricultural waste is one or a combination of sugarcane bagasse and straw.

[0140] (26) A seed culture medium, characterized in that, by weight, the seed culture medium comprises: 1-30 parts glucose, 5-20 parts yeast extract, 0.1-0.4 parts magnesium sulfate, 1-4 parts potassium dihydrogen phosphate, 0.2-2 parts calcium chloride, 0.001-0.01 parts ferric sulfate, 0.001-0.01 parts manganese sulfate, 0.001-0.01 parts zinc sulfate and 0.001-0.01 parts cobalt chloride.

[0141] (27) A solid fermentation culture medium, characterized in that, by weight, the solid fermentation culture medium comprises 1-20 parts of a small molecule inducer, wherein the small molecule inducer is one or more of xylose, cellobiose, lactose, sophorose, gentiobiose, xylose and substances containing sophoroside.

[0142] To address the aforementioned technical problems, this invention also provides a first technical solution regarding culture media:

[0143] 1.1. A seed culture medium, characterized in that, by weight, the seed culture medium comprises: 1-30 parts glucose, 5-20 parts yeast extract, 0.1-0.4 parts magnesium sulfate, 1-4 parts potassium dihydrogen phosphate, 0.2-2 parts calcium chloride, 0.001-0.01 parts ferric sulfate, 0.001-0.01 parts manganese sulfate, 0.001-0.01 parts zinc sulfate, and 0.001-0.01 parts cobalt chloride;

[0144] Preferably, the seed culture medium comprises: 5-20 parts glucose, and / or 5-15 parts yeast extract, and / or 0.2-0.35 parts magnesium sulfate, and / or 1-2 parts potassium dihydrogen phosphate, and / or 0.5-1.5 parts calcium chloride, and / or 0.001-0.008 parts ferric sulfate, and / or 0.001-0.008 parts manganese sulfate, and / or 0.001-0.008 parts zinc sulfate, and / or 0.001-0.008 parts cobalt chloride.

[0145] 1.2. The seed culture medium according to technical solution 1.1, characterized in that the seed culture medium further includes: water,

[0146] Preferably, the seed culture medium comprises: glucose at a concentration of 1-30 g / L, yeast extract at a concentration of 5-20 g / L, magnesium sulfate at a concentration of 0.1-0.4 g / L, potassium dihydrogen phosphate at a concentration of 1-4 g / L, calcium chloride at a concentration of 0.2-2 g / L, ferric sulfate at a concentration of 1-10 mg / L, manganese sulfate at a concentration of 1-10 mg / L, zinc sulfate at a concentration of 1-10 mg / L, and cobalt chloride at a concentration of 1-10 mg / L, with the remainder being water;

[0147] More preferably, the seed culture medium comprises: a glucose concentration of 5-20 g / L, and / or a yeast extract concentration of 5-15 g / L, and / or a magnesium sulfate concentration of 0.2-0.35 g / L, and / or a potassium dihydrogen phosphate concentration of 1-2 g / L, and / or a calcium chloride concentration of 0.5-1.5 g / L, and / or a ferric sulfate concentration of 1-8 mg / L, and / or a manganese sulfate concentration of 1-8 mg / L, and / or a zinc sulfate concentration of 1-8 mg / L, and / or a cobalt chloride concentration of 1-8 mg / L, with the remainder being water.

[0148] 1.3. The seed culture medium according to technical solution 1.1 or 1.2, characterized in that the yeast extract is yeast extract FM902.

[0149] 1.4 The method for preparing the seed culture medium according to any one of the technical solutions 1.1-1.3 is characterized in that it is prepared by a method comprising the following steps: mixing glucose, yeast extract, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, ferric sulfate, manganese sulfate, zinc sulfate and cobalt chloride to obtain the seed culture medium.

[0150] 1.5. The preparation method according to technical solution 1.4 is characterized in that it is prepared by a method comprising the following steps: mixing glucose, yeast extract powder, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, ferric sulfate, manganese sulfate, zinc sulfate, cobalt chloride and water to obtain a seed culture medium.

[0151] 1.6. The seed culture medium of any one of technical solutions 1.1-1.3 or the seed culture medium prepared by the preparation method of technical solutions 1.4 or 1.5, characterized in that the seed culture medium is used to culture fungi to obtain fungal seed liquid; preferably, the fungus is Aspergillus niger; more preferably, the Aspergillus niger is Aspergillus niger AGM01, with the preservation number: CCTCC NO: M 2024768.

[0152] To address the aforementioned technical problems, this invention also provides a second technical solution for the culture medium:

[0153] 2.1. A solid-state fermentation culture medium, characterized in that, by weight, the solid-state fermentation culture medium comprises 1-20 parts of a small molecule inducer, wherein the small molecule inducer is one or more of xylose, cellobiose, lactose, sophorose, gentiobiose, xylose and substances containing sophoroside;

[0154] Preferably, the small molecule inducer is xylose;

[0155] Preferably, the substance containing sophoroside is kaempferol-3-O-β-D-sophoroside.

[0156] 2.2. The solid-state fermentation culture medium according to technical solution 2.1 is characterized in that the solid-state fermentation culture medium further includes: a substance containing lignocellulose, ammonium sulfate, urea and calcium chloride;

[0157] Preferably, by weight, the solid fermentation culture medium comprises: 30-90 parts of a substance containing lignocellulose, 5-25 parts of ammonium sulfate, 1-15 parts of urea, 0.5-3 parts of calcium chloride, and 1-20 parts of a small molecule inducer;

[0158] More preferably, by weight, the solid fermentation medium comprises: 30-75 parts of a substance containing lignocellulose, and / or 6-20 parts of ammonium sulfate, and / or 2-12 parts of urea, and / or 0.5-2 parts of calcium chloride, and / or 1-18 parts of a small molecule inducer.

[0159] 2.3. The solid-state fermentation culture medium according to technical solution 2.1 or 2.2, characterized in that the lignocellulose-containing substance includes one or more of wheat bran, straw, and corn cob.

[0160] Preferably, the lignocellulose-containing substance is a combination of bran, straw, and corn cob;

[0161] More preferably, by weight, the substance containing lignocellulose is 10-30 parts wheat bran, 10-30 parts straw and 10-30 parts corn cob;

[0162] Most preferably, by weight, the lignocellulose-containing substance is 10-25 parts bran, and / or 10-25 parts straw, and / or 10-25 parts corn cob.

[0163] 2.4. The solid-state fermentation medium according to any one of technical solutions 2.1-2.3, characterized in that the solid-state fermentation medium further comprises: water,

[0164] Preferably, the solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-90 g / L, an ammonium sulfate concentration of 5-25 g / L, a urea concentration of 1-15 g / L, a calcium chloride concentration of 0.5-3 g / L, and a small molecule inducer concentration of 1-20 g / L, with the remainder being water;

[0165] More preferably, the solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-75 g / L, and / or ammonium sulfate at a concentration of 6-20 g / L, and / or urea at a concentration of 2-12 g / L, and / or calcium chloride at a concentration of 0.5-2 g / L, and / or a small molecule inducer at a concentration of 1-18 g / L, with the remainder being water.

[0166] 2.5. The solid fermentation medium according to any one of technical solutions 2.1-2.4, characterized in that the solid fermentation medium comprises: wheat bran at a concentration of 10-30 g / L, straw at a concentration of 10-30 g / L, corn cob at a concentration of 10-30 g / L, ammonium sulfate at a concentration of 5-25 g / L, urea at a concentration of 1-15 g / L, calcium chloride at a concentration of 0.5-3 g / L, and a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water;

[0167] Preferably, the solid-state fermentation medium comprises: wheat bran at a concentration of 10-25 g / L, and / or straw at a concentration of 10-25 g / L, and / or corn cob at a concentration of 10-25 g / L, and / or ammonium sulfate at a concentration of 5-25 g / L, and / or urea at a concentration of 1-15 g / L, and / or calcium chloride at a concentration of 0.5-3 g / L, and / or a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water.

[0168] 2.6. The method for preparing the solid fermentation culture medium according to any one of technical solutions 2.1-2.5 is characterized in that it includes: mixing a substance containing lignocellulose, ammonium sulfate, urea, calcium chloride and a small molecule inducer to obtain a solid fermentation culture medium.

[0169] 2.7. The method for preparing solid fermentation culture medium according to technical solution 2.6 is characterized in that it includes: mixing a substance containing lignocellulose, ammonium sulfate, urea, calcium chloride, a small molecule inducer and water to obtain a solid fermentation culture medium.

[0170] 2.8. The solid fermentation culture medium of any one of technical solutions 2.1-2.5 or the solid fermentation culture medium prepared by the preparation method of technical solutions 2.6 or 2.7, characterized in that the solid fermentation culture medium is used to culture fungi to obtain fungal solid fermentation broth; preferably, the fungus is Aspergillus niger; more preferably, the Aspergillus niger is Aspergillus niger AGM01, with accession number: CCTCC NO: M 2024768.

[0171] The Aspergillus niger AGM01 provided by this invention was deposited at the China Center for Type Culture Collection on April 24, 2024, with accession number CCTCC NO: M 2024768, deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052.

[0172] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0173] The preparation of the culture media and reagents involved in the examples is shown below:

[0174] 1. Aescin solid medium (g / L): Mix 1g aescin, 2.5g ferric ammonium citrate, 2.8g ammonium sulfate, 4.0g potassium dihydrogen phosphate, 0.9g magnesium sulfate heptahydrate, 0.9g calcium chloride dihydrate, 40g corn cob juice, 20g agar and sterile water, and bring the volume to 1L with sterile water. Sterilize at 115℃ for 30min to obtain aescin solid medium.

[0175] 2. The preparation method of PDA solid culture medium is as follows:

[0176] (1) Cut 200g of potatoes into small pieces, mix with 1L of water, heat to boiling and maintain for 20-30 minutes, filter while hot with two layers of gauze and retain the filtrate, mix the filtrate with water to obtain 1L of mixture.

[0177] (2) Mix the 1L mixture obtained in step (1), 20g glucose, and 15-20g agar (crushed in advance) and heat it. After the agar is completely dissolved, add water to 1L to complete the preparation of PDA solid culture medium.

[0178] 3. 10 mmol / L cellobiose solution: Mix 3.42 g of cellobiose with distilled water and bring the volume up to 1 L with distilled water to obtain a 10 mmol / L cellobiose solution.

[0179] 4. 5.55 mmol / L glucose standard solution: Mix 0.999 g of glucose with distilled water and bring the volume to 1 L with distilled water to obtain a 5.55 mmol / L cellobiose solution.

[0180] In some specific embodiments, the raw materials used in preparing the culture medium according to the present invention include yeast extract. The yeast extract primarily exists in the culture medium as an organic nitrogen source, providing the necessary nitrogen element for microbial growth during fermentation. Common organic nitrogen sources such as yeast extract and yeast peptone decompose in the culture medium, releasing amino acids and small peptides, which then become nitrogen sources required for microbial growth. In other words, when using yeast extract as a common organic nitrogen source to prepare the culture medium, the present invention does not particularly limit its source; it can be any commercially available yeast extract or prepared using conventional methods. As long as the commercially available yeast extract has a total nitrogen content (dry weight) of ≥9.0 wt% and an amino nitrogen content (dry weight) of ≥3.0 wt%, it can be used in the present invention.

[0181] Preferably, in some specific embodiments, the yeast extract powder, by weight, further comprises: 2-3 ppm of vitamin B1, 40-42 ppm of vitamin B2, 80-82 ppm of vitamin B5, 10-12 ppm of vitamin B6, 5-7 ppm of vitamin B7, 24-27 ppm of vitamin B9, 0.1-0.4 (ug / 100g) of vitamin B12, 3015-3018 ppm of choline, 2015-2018 ppm of inositol, and 315-320.0 ppm of niacin.

[0182] More preferably, based on the weight of the yeast extract, the yeast extract further comprises: 20.2-28.3% free amino acids and 54.5-70.5% hydrolyzed amino acids.

[0183] The free amino acid content includes, based on the weight of the yeast extract, 1-1.5% free aspartic acid, 1.5-2.0% free threonine, 1.0-1.5% free serine, 3.5-4.0% free glutamic acid, 0.5-1.0% free glycine, 3.0-3.5% free alanine, 0.1-0.3% free cysteine, 1.5-2.0% free valine, 0.5-1.0% free methionine, 1.0-1.5% free isoleucine, 2.0-2.5% free leucine, 1.0-1.5% free tyrosine, 1.0-1.5% free phenylalanine, 1.0-1.5% free lysine, 0.1-0.5% free histidine, 1.0-1.5% free arginine, and 0.5-1.0% free proline.

[0184] The hydrolyzed amino acid content includes, based on the weight of the yeast extract, 6-7% hydrolyzed aspartic acid, 2-3.5% hydrolyzed threonine, 2-3.5% hydrolyzed serine, 10-15% hydrolyzed glutamic acid, 2-5% hydrolyzed glycine, 5-10% hydrolyzed alanine, 0.1-0.4% hydrolyzed cysteine, 1.5-2.0% hydrolyzed valine, 0.4-0.6% hydrolyzed methionine, 1-1.5% hydrolyzed isoleucine, 4-4.5% hydrolyzed leucine, 1.5-2.5% hydrolyzed tyrosine, 1.5-2.5% hydrolyzed phenylalanine, 4.5-5% hydrolyzed lysine, 1-1.5% hydrolyzed histidine, 3-3.5% hydrolyzed arginine, and 2-2.5% hydrolyzed proline.

[0185] Unless otherwise stated, all reagents / instruments used in the embodiments of this invention are conventional commercially available products. The sources of information for the experimental reagents used in this invention are shown in Table 1 below, and the sources of information for the experimental instruments used in this invention are shown in Table 2 below.

[0186] Table 1. Reagent Information Table

[0187] Table 2. Instrument Information Sheet

[0188] The yeast extract powder (model: FM902) used in this application embodiment was sold by Angel Yeast Co., Ltd. The chemical characteristics of this product are as follows: moisture content ≤ 6.0 wt%, total nitrogen content (dry weight) ≥ 9.0 wt%, amino nitrogen content (dry weight) ≥ 3.0 wt%, ash content ≤ 15.0 wt%, and sodium chloride content ≤ 2.0 wt%.

[0189] The trace elements contained in the yeast extract powder (model: FM902) used in the examples are as follows: vitamin B1 2.6 ppm, vitamin B2 41.6 ppm, vitamin B5 81.3 ppm, vitamin B6 11.5 ppm, vitamin B7 5.79 ppm, vitamin B9 25.1 ppm, vitamin B12 0.21 (ug / 100g), choline 3017.0 ppm, inositol 2016.0 ppm, and niacin 318.0 ppm.

[0190] The yeast extract (model: FM902) used in the examples contained 23.0% free amino acids and 59.16% hydrolyzed amino acids.

[0191] The free amino acid content is as follows, based on the weight of the yeast extract: free aspartic acid 1.3%, free threonine 1.7%, free serine 1.0%, free glutamic acid 3.5%, free glycine 0.7%, free alanine 3.2%, free cysteine ​​0.1%, free valine 1.8%, free methionine 0.5%, free isoleucine 1.3%, free leucine 2.4%, free tyrosine 1.1%, free phenylalanine 1.0%, free lysine 1.3%, free histidine 0.3%, free arginine 1.5%, and free proline 0.6%.

[0192] The hydrolyzed amino acid content is as follows: based on the weight of the yeast extract, hydrolyzed aspartic acid 6.56%, hydrolyzed threonine 2.99%, hydrolyzed serine 2.88%, hydrolyzed glutamic acid 10.75%, hydrolyzed glycine 2.94%, hydrolyzed alanine 5.10%, hydrolyzed cysteine ​​0.21%, hydrolyzed valine 1.8%, hydrolyzed methionine 0.5%, hydrolyzed isoleucine 1.3%, hydrolyzed leucine 4.32%, hydrolyzed tyrosine 2.10%, hydrolyzed phenylalanine 2.07%, hydrolyzed lysine 4.69%, hydrolyzed histidine 1.30%, hydrolyzed arginine 3.54%, and hydrolyzed proline 2.22%.

[0193] Example 1: Isolation and Identification of Strains

[0194] 10g of soil containing chicken and goose droppings collected from Zuojia Town, Changyi District, Jilin City, Jilin Province was dissolved in 50mL of sterile water to obtain a mixture. This mixture was then serially diluted, and the concentrations were taken at 10... -2 and 10 -3 The mixture was spread onto a esculin solid medium plate and incubated at 28°C for 36 hours. A single colony with a prominent black color was selected as the dominant strain, and its texture is shown in Figure 1. The single colony was then purified using the following method: The single colony was picked and incubated in PDA liquid medium at 28°C and 250 rpm for 36 hours to obtain a first-purified spore suspension. The purified spore suspension was then streaked onto a esculin solid medium plate and incubated at 29°C for 24 hours. Single colonies were then picked and purified a second time using the same method to obtain a second-purified spore suspension. This second-purified spore suspension was inoculated onto PDA solid medium and incubated at 28°C for 24 hours. Colony morphology was then observed.

[0195] A bacterial strain was obtained. The colonies were dark brown, and the conidia were spherical, black or dark brown, smooth or rough. The genomic sequence of this strain was extracted using primers ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO.1) and ITS4: 5′-TCCTCCGCTTATTGATATGC-3′ (SEQ ID NO.2). The sequence was then amplified, detected by 1% gel electrophoresis, and sequenced. Blast analysis was performed to compare the sequence with sequences on GenBank. The sequence similarity was greater than 99%, indicating the same species. The ITS gene sequence of this strain is shown in SEQ ID NO.3. The specific PCR reaction system is shown in Table 3 below, and the specific PCR reaction procedure is shown in Table 4 below.

[0196] Table 3. PCR reaction system

[0197] Table 4. PCR reaction procedure

[0198] SEQ ID NO.3:

[0199] Based on the ITS gene sequence shown in SEQ ID NO.3, a phylogenetic tree was constructed using MEGA11.0. The constructed phylogenetic tree is shown in Figure 2. As can be seen from Figure 2, this strain is most closely related to *Aspergillus niger*. Further morphological analysis and molecular identification confirmed that this bacterium is *Aspergillus niger* AGM01, which was deposited at the China Center for Type Culture Collection on April 24, 2024, with accession number CCTCC NO: M 2024768. Figure 2 shows the phylogenetic tree results for *Aspergillus niger* AGM01 based on the ITS gene sequence.

[0200] Example 2: The ability of Aspergillus niger AGM01 to produce β-glucosidase and protein

[0201] (1) Preparation of crude enzyme solution of Aspergillus niger AGM01: Take 1 mL of the secondary purified spore suspension and dilute to 10. -2 10 -3 The hemocytometer was used to observe and count the cells under a microscope at 40×10 magnification. 1 mL of a solution containing 10... 7 A spore suspension of Aspergillus niger AGM01 spores was inoculated into a shake flask and fermented at 28°C and 200 rpm to obtain a fermentation broth. The fermentation broth cultured for 7 days was centrifuged at 8000 rpm for 10 min, and the supernatant was retained, which is the crude enzyme solution of Aspergillus niger AGM01.

[0202] (2) Determination of β-glucosidase activity in crude enzyme solution of Aspergillus niger AGM01 using cellobiose as a substrate:

[0203] It should be noted that, using cellobiose as a substrate, β-glucosidase reacts with cellobiose to produce 2 molecules of glucose. The glucose is then converted into gluconic acid and hydrogen peroxide by glucose oxidase. Under the action of peroxidase, hydrogen peroxide couples and condenses reduced 4-aminoantipyrine with phenol to form a quinone compound that can be measured by a spectrophotometer.

[0204] (2.1) Determination of the absorbance of the crude enzyme solution of Aspergillus niger AGM01 at a wavelength of 505 nm: The crude enzyme solution of Aspergillus niger AGM01 obtained in step (1) was centrifuged at 10000 rpm for 5 min. 100 μL of the supernatant was added to 100 μL of 10 mmol / L cellobiose solution, and heated in a water bath at 60 °C for 10 min to obtain the reaction system. Then, 2.5 μL of the reaction system was mixed with 250 μL of the reagent from the glucose assay kit and incubated at 37 °C for 10 min. The absorbance was measured at a wavelength of 505 nm to obtain the absorbance value of the crude enzyme solution of Aspergillus niger AGM01 at a wavelength of 505 nm.

[0205] (2.2) Determine the absorbance of the crude enzyme solution of Asperillium niger Q270-C6 at a wavelength of 505 nm: The determination method is the same as step (2.1). Asperillium niger Q270-C6 has been disclosed in Chinese patent application with patent number CN117143739A. The preservation number of this strain is: CCTCC NO:M 20231261.

[0206] (2.3) Blank group: The absorbance of the inactivated Aspergillus niger AGM01 crude enzyme solution was measured at a wavelength of 505 nm. The specific process was as follows: The crude enzyme solution of Aspergillus niger AGM01 obtained in step (1) was centrifuged at 10000 rpm for 5 min. 100 μL of the supernatant was added to 100 μL of 10 mmol / L cellobiose solution, and the solution was inactivated at 80℃ for 30 min to obtain the reaction system. Then, 2.5 μL of the reaction system was mixed with 250 μL of the reagent in the glucose assay kit and incubated at 37℃ for 10 min. The absorbance was measured at a wavelength of 505 nm to obtain the absorbance value of the inactivated Aspergillus niger AGM01 crude enzyme solution at a wavelength of 505 nm.

[0207] (3) Method for calculating the enzyme activity of β-glucosidase: The absorbance values ​​of the crude enzyme solutions of Aspergillus niger obtained in steps (2.1) and (2.2) are compared with a 5.55 mmol / L glucose standard solution to calculate the glucose concentration. The glucose concentration is calculated as follows: glucose concentration = ((absorbance value of crude enzyme solution of Aspergillus niger - absorbance value of blank group) / (absorbance value of standard solution - absorbance value of blank group)) × 5.55, where 5.55 is the glucose concentration in the glucose standard solution. Then, the enzyme activity of β-glucosidase is calculated according to the enzyme activity formula, which is shown below:

[0208] Wherein, U represents the enzyme activity of β-glucosidase; A represents the glucose concentration in μmol / mL; N represents the dilution factor, in this embodiment the dilution factor is 200 times; t represents the reaction time in min; V1 represents the volume of the reaction system in mL; V2 represents the volume of the crude enzyme solution of Aspergillus niger AGM01 in mL.

[0209] The definition of one unit of β-glucosidase activity: Under conditions of 60℃ and pH 5, the amount of enzyme required per minute to enzymatically hydrolyze cellobiose to produce 1 μmol of glucose in 1 mL of crude enzyme solution of Aspergillus niger AGM01 is defined as one unit (U) of β-glucosidase activity.

[0210] Based on the method for calculating the enzyme activity of β-glucosidase in step (3), the comparison results of the enzyme activities of β-glucosidase in Aspergillus niger are shown in Table 5:

[0211] Table 5. Comparison of β-glucosidase activities in Aspergillus niger.

[0212] (3) Determination of protein content in crude enzyme solution of Aspergillus niger AGM01

[0213] 1 mL of the crude enzyme solution of Aspergillus niger AGM01 obtained in step (1) was diluted to 10 mL in a volumetric flask to obtain a diluted crude enzyme solution. The crude enzyme solution was centrifuged at 10,000 rpm for 5 min to obtain a supernatant. 5 μL of the supernatant was mixed with 250 μL of Coomassie brilliant blue dye and allowed to stand for 5 min to obtain a mixed solution. The absorbance value of the mixed solution at 595 nm was measured using an ELISA reader, and the absorbance value was compared with the protein standard curve to obtain a protein content of 1.5 mg / mL in the crude enzyme solution.

[0214] The process for constructing the protein standard curve is as follows: Prepare a gradient of protein standards (bovine serum albumin) solutions ranging from 0-60 μg / mL. Use the absorbance values ​​at 595 nm of the mixture of each protein standard and Coomassie Brilliant Blue dye as the ordinate and the corresponding protein standard concentration as the abscissa to obtain the protein standard curve: y = 0.5716x + 0.5075, R0 2 =0.9968.

[0215] Example 3

[0216] 1. Example 3.1 provides a method for preparing Aspergillus niger solid-state fermentation broth, as detailed below:

[0217] (1) Preparation of seed culture medium: Glucose, yeast extract FM902, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, ferric sulfate, manganese sulfate, zinc sulfate, cobalt chloride, and sterile water were mixed and sterilized at 115℃ for 30 min to obtain the seed culture medium. The concentration of glucose in the seed culture medium was 20 g / L, the concentration of yeast extract FM902 was 15 g / L, the concentration of magnesium sulfate was 0.3 g / L, the concentration of potassium dihydrogen phosphate was 2.5 g / L, the concentration of calcium chloride was 1.2 g / L, the concentration of ferric sulfate was 7 mg / L, the concentration of manganese sulfate was 6 mg / L, the concentration of zinc sulfate was 4 mg / L, and the concentration of cobalt chloride was 6 mg / L, with the remainder being water.

[0218] (2) Inoculate the spore suspension of Aspergillus niger AGM01 into the seed culture medium obtained in step (1) at 10% by volume, and culture it at 28℃ and 200 rpm for 36 h to obtain the seed liquid.

[0219] (3) Preparation of xylose-induced solid-state fermentation medium: Powdered wheat bran, powdered straw, powdered corn cob, ammonium sulfate, urea, calcium chloride, xylose, and sterile water were mixed and sterilized at 115℃ for 30 min to obtain solid-state fermentation medium. The concentrations of wheat bran, straw, corn cob, ammonium sulfate, urea, calcium chloride, and xylose in this solid-state fermentation medium were 20 g / L, 25 g / L, 22 g / L, 15 g / L, 10 g / L, 2.2 g / L, and 2.5 g / L, with the remainder being water.

[0220] (4) Preparation of xylose-induced crude enzyme solution: The seed culture obtained in step (2) was inoculated into the xylose-induced solid fermentation medium obtained in step (3) at a volume percentage of 5%. Fermentation was carried out at 28°C, pH=5 and humidity for 120 h to obtain solid fermentation product. Then, the solid fermentation product was mixed with phosphate buffer solution at pH=5 at a volume ratio of 1:8 and soaked for 2 h. After stirring at 120 rpm for 20 min, the mixture was filtered through a plate and frame filter to obtain xylose-induced crude enzyme solution.

[0221] (5) Preparation of xylose-induced concentrate: The xylose-induced crude enzyme solution obtained in step (4) and the concentrate obtained in step (5) are placed in a membrane with a pore size of 30 kD and a water flux of 80 L / m. 2 Ultrafiltration concentration was carried out for 2 hours under the condition of ·h to obtain xylose-induced concentrate.

[0222] 2. Comparative Example 3.1 provides a method for preparing Aspergillus niger solid-state fermentation broth, as detailed below:

[0223] Steps (1) and (2) are the same as in Example 3.1.

[0224] (3) Preparation of xylose-free induced solid-state fermentation medium: Powdered wheat bran, powdered straw, powdered corn cob, ammonium sulfate, urea, calcium chloride, xylose, and sterile water were mixed and sterilized at 115℃ for 30 min to obtain xylose-free induced solid-state fermentation medium. The concentrations of wheat bran, straw, corn cob, ammonium sulfate, urea, and calcium chloride in this xylose-free induced solid-state fermentation medium were 20 g / L, 25 g / L, 22 g / L, 15 g / L, 10 g / L, and 2.2 g / L, with the remainder being water.

[0225] (4) Preparation of xylose-free induced crude enzyme solution: The seed culture obtained in step (2) was inoculated into the xylose-free induced solid fermentation medium obtained in step (3) at a volume percentage of 5%. Fermentation was carried out at 28°C, pH=5 and humidity for 120 h to obtain solid fermentation product. Then, the solid fermentation product was mixed with phosphate buffer solution at pH=5 at a volume ratio of 1:8 and soaked for 2 h. After stirring at 120 rpm for 20 min, the mixture was filtered through a plate and frame filter to obtain xylose-free induced crude enzyme solution.

[0226] (5) Preparation of xylose-free induced concentrate: The xylose-free induced crude enzyme solution obtained in step (4) and the concentrate obtained in step (5) are placed in a membrane with a pore size of 30 kD and a water flux of 80 L / m. 2 Ultrafiltration concentration was carried out for 2 hours under the condition of ·h to obtain xylose-free concentrated solution.

[0227] Evaluation of technical effects: The xylose-induced crude enzyme solution obtained in Example 3.1, the xylose-induced concentrated solution obtained in Example 3.1, and the xylose-free concentrated solution obtained in Comparative Example 3.1 were subjected to enzyme activity determination of β-glucosidase. The specific method for determining the enzyme activity of β-glucosidase is the same as the determination method in step (2) of Example 2.

[0228] The results showed that the β-glucosidase activity in the xylose-induced crude enzyme solution was 16.8 U, and the β-glucosidase activity in the xylose-induced concentrated solution was 134.4 U. The β-glucosidase activity in the xylose-free concentrated solution was 95.6 U. Therefore, the β-glucosidase activity in the xylose-induced crude enzyme solution was increased by 40.6%.

[0229] Application example (in the preparation of lignocellulose hydrolysate)

[0230] (1) 540 kg of sugarcane bagasse with a moisture content of 50 wt% (the dry matter mass of the sugarcane bagasse is 270 kg) and 300 L of phosphate buffer solution with pH = 4.8 were mixed to obtain a mixed liquid. Then, a mixed enzyme solution was added to the mixed liquid in three portions to obtain an enzymatic hydrolysis reaction system. Based on the dry matter mass of the sugarcane bagasse, the enzymatic hydrolysis reaction system contained cellulase with an enzyme activity of 4350 U, xylanase with an enzyme activity of 69600 U, and β-glucosidase with an enzyme activity of 1740 U. Then, the mixture was hydrolyzed at 50 °C for 144 h to obtain a lignocellulose hydrolysate.

[0231] The mixed enzyme solution is prepared by mixing 0.435g cellulase, 0.696g xylanase, 12.95mL of xylose-induced concentrate obtained in step (5.1) of Example 3, and 37.5L phosphate buffer solution.

[0232] (2) The cellulose and hemicellulose content in the enzymatic hydrolysis substrate was determined by the NY-T3494-2019 method. The cellulose content was 35.4 wt%, the hemicellulose content was 20.6 wt%, and the total amount of cellulose and hemicellulose in the bagasse enzymatic hydrolysis substrate was 151.2 g (based on the dry matter mass of bagasse).

[0233] The reducing sugar content in the lignocellulose hydrolysate was determined to be 158 g / L using the GB 5009.7-2016 method, and the mass of reducing sugar in the lignocellulose hydrolysate was determined to be 138.65 g. The saccharification rate of the lignocellulose hydrolysate was determined using the saccharification rate calculation formula, and the saccharification rate of the lignocellulose hydrolysate was found to be 91.7%.

[0234] The formula for calculating the saccharification rate is: (m2 / m1)×100%, where m1 represents the mass of cellulose and hemicellulose in the sugarcane bagasse enzymatic hydrolysis substrate; and m2 represents the mass of reducing sugar obtained from the lignocellulose hydrolysis solution.

[0235] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not intended to limit the present invention. Any improvements made by those skilled in the art on this basis that do not highlight substantive features or make significant progress should fall within the protection scope of the present invention.

Claims

A type of Aspergillus niger, characterized in that, The Aspergillus niger mentioned is Aspergillus niger AGM01, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number: CCTCC NO: M2024768. Aspergillus niger according to claim 1, characterized in that, The ITS gene sequence of Aspergillus niger AGM01 is shown in SEQ ID NO.

3. Aspergillus niger according to claim 1 or 2, characterized in that, The Aspergillus niger AGM01 has the characteristic of producing β-glucosidase. Aspergillus niger according to claim 3, characterized in that, The β-glucosidase activity of Aspergillus niger AGM01 is 20-30 U, where U refers to one enzyme activity unit of β-glucosidase. One enzyme activity unit of β-glucosidase is defined as the amount of enzyme required per minute to produce 1 μmol of glucose by enzymatic hydrolysis of cellobiose in each mL of crude enzyme solution of Aspergillus niger AGM01. The crude enzyme solution of Aspergillus niger AGM01 was prepared by the following method: Aspergillus niger was amplified and cultured to obtain Aspergillus niger spore suspension, the Aspergillus niger spore suspension was fermented and cultured to obtain fermentation broth, and then solid-liquid separation was performed to obtain supernatant, which is the crude enzyme solution of Aspergillus niger AGM01. Aspergillus niger according to claim 4, characterized in that, The fermentation culture time is 5-7 days. A fermentation preparation method for an Aspergillus niger agent, characterized in that, The fermentation preparation method includes the following steps: (a) Amplify and culture Aspergillus niger according to any one of claims 1-5 to obtain a suspension of Aspergillus niger spores; (b) The Aspergillus niger spore suspension obtained in step (a) is fermented and cultured. The fermentation preparation method according to claim 6 is characterized in that, In step (a), the concentration of Aspergillus niger spores in each mL of suspension is 10 6 -10 7 The spores of Aspergillus niger according to any one of claims 1-5. The fermentation preparation method according to claim 6 is characterized in that, The culture temperature in step (a) is 26-30℃; and / or the culture temperature in step (b) is 26-32℃. The fermentation preparation method according to claim 6 is characterized in that, The incubation time in step (a) is 24-48 hours. A type of Aspergillus niger agent, characterized in that, Contains Aspergillus niger according to any one of claims 1-5. A type of Aspergillus niger agent, characterized in that, The Aspergillus niger agent is prepared by the fermentation preparation method according to any one of claims 6-9. A solid-state fermentation broth of Aspergillus niger, characterized in that, The product contains *Aspergillus niger* according to any one of claims 1-5, wherein the enzyme activity of β-glucosidase in the solid-state fermentation broth of *Aspergillus niger* is 110-170 U, wherein U refers to one enzyme activity unit of β-glucosidase, and one enzyme activity unit of β-glucosidase is defined as the amount of enzyme required per minute to produce 1 μmol of glucose by enzymatic hydrolysis of cellobiose in each mL of the solid-state fermentation broth of *Aspergillus niger*. The Aspergillus niger solid fermentation broth according to claim 12 is characterized in that, It is obtained by culturing the aforementioned Aspergillus niger in a seed culture medium, wherein, by weight, the seed culture medium comprises: 1-30 parts glucose, 5-20 parts yeast extract, 0.1-0.4 parts magnesium sulfate, 1-4 parts potassium dihydrogen phosphate, 0.2-2 parts calcium chloride, 0.001-0.01 parts ferric sulfate, 0.001-0.01 parts manganese sulfate, 0.001-0.01 parts zinc sulfate, and 0.001-0.01 parts cobalt chloride. The Aspergillus niger solid fermentation broth according to claim 13 is characterized in that, The seed culture medium comprises: 5-20 parts glucose, and / or 5-15 parts yeast extract, and / or 0.2-0.35 parts magnesium sulfate, and / or 1-2 parts potassium dihydrogen phosphate, and / or 0.5-1.5 parts calcium chloride, and / or 0.001-0.008 parts ferric sulfate, and / or 0.001-0.008 parts manganese sulfate, and / or 0.001-0.008 parts zinc sulfate, and / or 0.001-0.008 parts cobalt chloride. The Aspergillus niger solid fermentation broth according to claim 13 is characterized in that, The yeast extract is yeast extract FM902. The Aspergillus niger solid fermentation broth according to claim 14 is characterized in that, The yeast extract is yeast extract FM902. The Aspergillus niger solid fermentation broth according to any one of claims 12-16 is characterized in that, The seed culture medium also includes: water. The Aspergillus niger solid fermentation broth according to claim 17 is characterized in that, The seed culture medium comprises: glucose at a concentration of 1-30 g / L, yeast extract at a concentration of 5-20 g / L, magnesium sulfate at a concentration of 0.1-0.4 g / L, potassium dihydrogen phosphate at a concentration of 1-4 g / L, calcium chloride at a concentration of 0.2-2 g / L, ferric sulfate at a concentration of 1-10 mg / L, manganese sulfate at a concentration of 1-10 mg / L, zinc sulfate at a concentration of 1-10 mg / L, and cobalt chloride at a concentration of 1-10 mg / L, with the remainder being water. The Aspergillus niger solid fermentation broth according to claim 18 is characterized in that, The seed culture medium comprises: glucose at a concentration of 5-20 g / L, and / or yeast extract at a concentration of 5-15 g / L, and / or magnesium sulfate at a concentration of 0.2-0.35 g / L, and / or potassium dihydrogen phosphate at a concentration of 1-2 g / L, and / or calcium chloride at a concentration of 0.5-1.5 g / L, and / or ferric sulfate at a concentration of 1-8 mg / L, and / or manganese sulfate at a concentration of 1-8 mg / L, and / or zinc sulfate at a concentration of 1-8 mg / L, and / or cobalt chloride at a concentration of 1-8 mg / L, with the remainder being water. The Aspergillus niger solid fermentation broth according to claim 12 is characterized in that, It is obtained by culturing the aforementioned Aspergillus niger in a solid-state fermentation medium, wherein, by weight, the solid-state fermentation medium comprises 1-20 parts of a small molecule inducer, wherein the small molecule inducer is one or more substances selected from the group consisting of xylose, cellobiose, lactose, sophorose, gentiobiose, xylose and substances containing sophoroside. The Aspergillus niger solid fermentation broth according to claim 20 is characterized in that, The small molecule inducer is xylose. The Aspergillus niger solid fermentation broth according to claim 20 is characterized in that, The substance containing sophoroside is kaempferol-3-O-β-D-sophoroside. The Aspergillus niger solid fermentation broth according to claim 20 is characterized in that, The solid-state fermentation medium also includes: a substance containing lignocellulose, ammonium sulfate, urea, and calcium chloride. The Aspergillus niger solid fermentation broth according to claim 23 is characterized in that, By weight, the solid fermentation medium comprises: 30-90 parts of lignocellulose-containing material, 5-25 parts of ammonium sulfate, 1-15 parts of urea, 0.5-3 parts of calcium chloride, and 1-20 parts of small molecule inducer. The Aspergillus niger solid fermentation broth according to claim 24 is characterized in that, By weight, the solid fermentation medium comprises: 30-75 parts of a substance containing lignocellulose, and / or 6-20 parts of ammonium sulfate, and / or 2-12 parts of urea, and / or 0.5-2 parts of calcium chloride, and / or 1-18 parts of a small molecule inducer. The Aspergillus niger solid fermentation broth according to claim 23 is characterized in that, The lignocellulose-containing substance includes one or more substances selected from the group consisting of bran, straw and corn cob. The Aspergillus niger solid fermentation broth according to claim 26 is characterized in that, The lignocellulose-containing substance is a combination of bran, straw, and corn cob. The Aspergillus niger solid fermentation broth according to claim 27 is characterized in that, By weight, the substance containing lignocellulose is 10-30 parts bran, 10-30 parts straw and 10-30 parts corn cob. The Aspergillus niger solid fermentation broth according to claim 28 is characterized in that, By weight, the substance containing lignocellulose is 10-25 parts wheat bran, and / or 10-25 parts straw, and / or 10-25 parts corn cob. The solid-state fermentation broth of *Aspergillus niger* according to any one of claims 20-25 is characterized in that... The solid-state fermentation medium also includes water. The Aspergillus niger solid fermentation broth according to claim 30 is characterized in that, The solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-90 g / L, an ammonium sulfate concentration of 5-25 g / L, a urea concentration of 1-15 g / L, a calcium chloride concentration of 0.5-3 g / L, and a small molecule inducer concentration of 1-20 g / L, with the remainder being water. The Aspergillus niger solid fermentation broth according to claim 31 is characterized in that, The solid-state fermentation medium comprises: a lignocellulose-containing substance at a concentration of 30-75 g / L, and / or ammonium sulfate at a concentration of 6-20 g / L, and / or urea at a concentration of 2-12 g / L, and / or calcium chloride at a concentration of 0.5-2 g / L, and / or a small molecule inducer at a concentration of 1-18 g / L, with the remainder being water. The solid-state fermentation broth of *Aspergillus niger* according to any one of claims 26-29 is characterized in that... The solid-state fermentation medium also includes water. The Aspergillus niger solid fermentation broth according to claim 33 is characterized in that, The solid-state fermentation medium comprises: wheat bran at a concentration of 10-30 g / L, straw at a concentration of 10-30 g / L, corn cob at a concentration of 10-30 g / L, ammonium sulfate at a concentration of 5-25 g / L, urea at a concentration of 1-15 g / L, calcium chloride at a concentration of 0.5-3 g / L, and a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water. The Aspergillus niger solid fermentation broth according to claim 34 is characterized in that, The solid-state fermentation medium comprises: wheat bran at a concentration of 10-25 g / L, and / or straw at a concentration of 10-25 g / L, and / or corn cob at a concentration of 10-25 g / L, and / or ammonium sulfate at a concentration of 5-25 g / L, and / or urea at a concentration of 1-15 g / L, and / or calcium chloride at a concentration of 0.5-3 g / L, and / or a small molecule inducer at a concentration of 1-20 g / L, with the remainder being water. A method for preparing Aspergillus niger solid fermentation broth according to any one of claims 12-35, characterized in that, The method includes the following steps: amplifying and culturing Aspergillus niger according to any one of claims 1-5 to obtain Aspergillus niger spore suspension; inoculating the Aspergillus niger spore suspension into a seed culture medium for cultivation to obtain Aspergillus niger seed liquid; and inoculating the Aspergillus niger seed liquid into a solid fermentation culture medium for cultivation to obtain Aspergillus niger solid fermentation broth. The preparation method according to claim 36 is characterized in that, Includes the following steps: (a) Amplify and culture Aspergillus niger according to any one of claims 1-5 to obtain a suspension of Aspergillus niger spores; (b) The Aspergillus niger spore suspension obtained in step (a) is inoculated into a seed culture medium for cultivation to obtain Aspergillus niger seed liquid; (c) The Aspergillus niger seed liquid obtained in step (b) is inoculated into a solid fermentation medium for culture to obtain Aspergillus niger solid fermentation product; (d) The Aspergillus niger solid fermentation product obtained in step (c) is mixed with a buffer solution, soaked, stirred and filtered to obtain crude enzyme solution; (e) The crude enzyme solution obtained in step (d) is concentrated to obtain the concentrated solution, namely the solid fermentation broth of Aspergillus niger. The preparation method according to claim 37 is characterized in that, In step (a), each mL of Aspergillus niger spore suspension contains 10 6 -10 7 The spores of Aspergillus niger according to any one of claims 1-5. The preparation method according to claim 37 is characterized in that, The culture temperature in step (a) is 26-30℃; and / or the culture temperature in step (b) is 26-30℃; and / or the culture temperature in step (c) is 27-29℃. The preparation method according to claim 37 is characterized in that, The culture time in step (a) is 24-48h; and / or the culture time in step (b) is 24-48h; and / or the culture time in step (c) is 100-144h. The preparation method according to claim 37 is characterized in that, The culture rotation speed in step (b) is 150-300 rpm. The preparation method according to claim 37 is characterized in that, The culture pH in step (c) is 4.5-5.

5. The preparation method according to claim 37 is characterized in that, The culture humidity in step (c) is 50-95%. The preparation method according to claim 37 is characterized in that, In step (d), the volume ratio of the Aspergillus niger solid fermentation product to the buffer solution is (0.8-1.2):(6-10). The preparation method according to claim 37 is characterized in that, The soaking time in step (d) is 1-4 hours. The preparation method according to claim 37 is characterized in that, The stirring speed in step (d) is 100-300 rpm. The preparation method according to claim 37 is characterized in that, The stirring time in step (d) is 20-50 minutes. The preparation method according to claim 37 is characterized in that, The filtering in step (d) is plate and frame filtering. The preparation method according to claim 37 is characterized in that, In step (e), the concentration is ultrafiltration concentration. The preparation method according to claim 49 is characterized in that, The membrane used for ultrafiltration concentration has a pore size of 10-60 kD. The preparation method according to claim 49 is characterized in that, The ultrafiltration concentration time is 1-5 hours. A solid fermentation broth of Aspergillus niger prepared by any one of claims 36-51. A lignocellulose hydrolysate, characterized in that, It is obtained by hydrolysis in a system containing Aspergillus niger according to any one of claims 1-5, or the Aspergillus niger agent according to claim 10 or 11, or the Aspergillus niger solid fermentation broth according to any one of claims 12-35 or claim 52. The lignocellulose hydrolysate according to claim 53 is characterized in that, Lignocellulose is one or a combination of agricultural and forestry waste. The lignocellulose hydrolysate according to claim 54 is characterized in that, Agricultural waste consists of one or a combination of sugarcane bagasse and straw. According to any one of claims 53-55, the lignocellulose hydrolysate contains a reducing sugar content of 90% or more by mass, wherein... The reducing sugar mass ratio refers to the percentage of the mass of reducing sugar (m2) that is converted from the lignocellulosic raw material by enzymatic hydrolysis, relative to the total mass (m1) of cellulose and hemicellulose contained in the lignocellulosic raw material before hydrolysis. The use of Aspergillus niger according to any one of claims 1-5, or the Aspergillus niger agent according to claim 10 or 11, or the Aspergillus niger solid fermentation broth according to any one of claims 12-35 or claim 52 in the preparation of lignocellulose hydrolysate.

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