Schizophyllum commune and application thereof

Abstract

The invention discloses schizophyllum commune and application thereof, and belongs to the technical field of fermentation. The strain number of the schizophyllum commune is Schizophyllum commune 125, and the schizophyllum commune is preserved in the China General Microbiological Culture Collection Center on December 13, 2024, and the preservation number is CGMCC No.41699. The schizophyllum commune is preserved in the China General Microbiological Culture Collection Center on December 13, 2024. The invention provides a strain capable of producing glucose, xylose, arabinose and other substances by using cellulose and hemicellulose, decomposing lignin, and providing self-growth and high-yield protein by using sugar produced by the cellulose and hemicellulose and nutrient substances of vinasse as a carbon source and a nitrogen source. The strain is a protein source with excellent protein content and quality, and lays a foundation for development of novel feed additives and full utilization of renewable resources.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation, in particular to a Schizophyllum sp. and an application thereof. Background Art

[0002] The solid brewing waste of the liquor industry, lees, also known as fermented grains or discarded lees, is the solid material left over from the brewing process. Generally speaking, every ton of liquor produced produces approximately 3-4 tons of fresh lees. Lees contain organic components such as protein, starch, cellulose, and fat from the brewing raw materials, offering valuable resource utilization. However, lees are high in moisture and microbial content, making them susceptible to spoilage during storage. Stacking can generate harmful gases and high concentrations of organic leachate, posing an environmental risk if not properly handled or disposed of promptly.

[0003] Therefore, the harmless treatment, volume reduction, and resource utilization of distiller's grains are crucial for reducing carbon emissions in watersheds, maintaining ecological health, and achieving material resource recycling. Research has shown that distiller's grains have significant potential for resource utilization, including biofeed, organic fertilizer production, functional material production, energy generation, extraction of active substances, raw material preparation, and secondary brewing.

[0004] As a solid byproduct of the baijiu lees production process, its physical and chemical properties and composition are closely related to factors such as the type of grain used, brewing process, and storage time. The moisture content of fresh and dried lees varies significantly, exceeding 58.0%. Within the dry matter composition of lees, the contents of cellulose, hemicellulose, crude protein, crude starch, and ash vary by more than 15.0%. The upper and lower limits of lignin content vary by approximately 10.0%. The contents of hemicellulose and crude fat each vary by approximately 7.0%. This may be due to the following three reasons:

[0005] ① Brewing raw materials. Different brands of liquor use different raw materials, such as sorghum, rice, millet, wheat, etc. The content of starch, protein, fat, cellulose, lignin and other components in these raw materials varies to a certain extent.

[0006] ② Brewing process. Different process conditions, such as the number of fermentations, the number of turnings, and the type of microorganisms, will result in different decomposition, conversion, and utilization rates of the various components of the grain raw materials.

[0007] ③ Storage time: As storage time goes by, the moisture in the lees will diffuse into the environment, and microorganisms will continue to decompose the remaining organic matter.

[0008] Lignocellulose is primarily composed of cellulose, hemicellulose, and lignin. Cellulose, hemicellulose, and lignin are cross-linked, with hemicellulose and lignin filling the spaces between the microfibrils formed by the cellulose macromolecules. Lignin inhibits microbial degradation of cellulose and hemicellulose, making it difficult for enzymes to reach the cellulose surface. Therefore, a strain capable of opening this three-component structure is needed to first decompose some of the lignin and then hydrolyze the cellulose and hemicellulose into simple sugars like glucose for subsequent conversion and production. Summary of the Invention

[0009] The present invention aims to provide a schizophyllum fungus and its application, wherein the schizophyllum fungus is used to degrade cellulose, hemicellulose and lignin in distiller's grains to produce glucose, xylose, arabinose and the like, and the produced glucose, xylose, arabinose and the like are used as carbon sources for the growth of the schizophyllum fungus. The schizophyllum fungus utilizes the non-protein nitrogen of the distiller's grains to convert it into protein in the fungus itself, and secretes substances such as polysaccharides and some amino acids.

[0010] To achieve the above-mentioned purpose, on the one hand, the present invention provides a Schizophyllum commune, the strain number of which is Schizophyllum commune 125, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 13, 2024, with a deposit number of CGMCC No.41699.

[0011] In another aspect, the present invention provides a method for using the above-mentioned Schizophyllum sp. in the treatment of liquor lees, wherein the liquor lees are used as a substrate for aerobic solid-state fermentation of the Schizophyllum sp., comprising the following steps:

[0012] S1. Preparation of primary seed koji: inoculate Schizophyllum sp. into a primary culture medium with bran as substrate, and culture at 32.5°C and 80% relative humidity until the content of laccase ≥ 0.18 μg / g, cellulase ≥ 21.15 μg / g, filter paper enzyme ≥ 0.89 μg / g, xylanase ≥ 47.24 μg / g, and manganese peroxidase ≥ 0.022 μg / g per gram of dry koji is achieved;

[0013] S2. Preparation of secondary seed koji: inoculate the primary seed koji at a rate of 10-15% into the secondary culture medium containing 50% pre-treated distiller's grains, and ferment in a shallow tray fermentation area for 4 days;

[0014] S3. Daqu culture: inoculate the secondary seed koji at a rate of 10-15% into the tertiary culture medium containing 70% pre-treated distiller's grains, and ferment in a shallow tray fermentation area for 5 days.

[0015] Preferably, in S1, the composition ratio of the primary culture medium is calculated on a dry matter basis as follows: bran 100%, molasses 5%, ammonium sulfate 2%, magnesium sulfate 0.5%, Tween-80 0.1%, superphosphate 1%, and potassium chloride 0.5%, and the pH value of the primary culture medium with bran as the substrate is 5.9-6.2.

[0016] Preferably, in S2, the composition ratio of the secondary culture medium is calculated on a dry matter basis as follows: 0.5% magnesium sulfate, 0.5% potassium chloride, 2.5% ammonia water, 1.25% soybean meal, 27.0% wheat bran, 18% corn husk, 50% distiller's grains, and 3.75% molasses, and the pH value of the secondary culture medium is 5.9-6.2.

[0017] Preferably, in S2, the fermentation area has a unit space capacity of 2.0-2.5 kg / m 3 , the air temperature is 30-35℃, and the relative humidity of the air is 85-90%.

[0018] Preferably, in S3, the composition ratio of the tertiary culture medium is calculated on a dry matter basis as follows: magnesium sulfate 0.5%, potassium chloride 1.3%, ammonia water 2.5%, manganese chloride 0.01%, soybean meal 2.0%, molasses 5.0%, wheat bran 15.0%, distiller's grains 70%, corn husk 13%, and the pH value of the tertiary culture medium is 5.9-6.2.

[0019] Preferably, in S3, the fermentation area has a unit space capacity of 3.0-3.5 kg / m 3 .

[0020] Preferably, the pretreatment of the vinasse comprises: adjusting the pH of the vinasse to 5.9-6.2 using 3.5% ammonia water, and adjusting the moisture content of the vinasse to 50-55% using bran and corn husk.

[0021] In another aspect, the present invention provides a use of the above-mentioned Schizophyllum sp. in feed preparation.

[0022] On the other hand, the present invention provides a use of the product of the aerobic solid-state fermentation of Schizophyllum using white wine lees as a substrate in feed preparation.

[0023] Therefore, the present invention provides a Schizophyllum sp. and its application, which have the following beneficial effects:

[0024] (1) A strain of Schizophyllum commune 125 was isolated and identified for the first time. The strain has enzyme activities for cellulase, hemicellulose and lignin degradation. Schizophyllum commune 125 was used to degrade cellulose, hemicellulose and lignin in the lees to produce glucose, xylose, arabinose and other sugars, and the produced sugars were used as carbon sources for the growth of Schizophyllum commune 125. At the same time, Schizophyllum commune 125 fully utilized the nutrients and non-protein nitrogen in the lees, converted the non-protein nitrogen in the lees into protein in the bacteria itself, and secreted substances such as polysaccharides and amino acids.

[0025] (2) In the past, chemical reagents such as acid and alkali treatment were used to treat wine lees. Although these methods can decompose cellulose and hemicellulose to produce glucose, xylose, arabinose, etc. in large quantities, they will produce many inhibitors, such as furfural, acetic acid and other substances. These substances will inhibit the growth of strains. In addition, pretreatment will increase the risk of secondary pollution, resulting in increased treatment costs. Most of the current cellulases are pure enzymes, which have poor effects on directly treating natural wood cellulose. In addition, commercial cellulases are expensive, which increases the production steps and increases the production cost, making it unfavorable for large-scale production.

[0026] (3) The present invention uses Schizophyllum 125 to ferment distiller's grains in a segmented manner, converting the biomass raw materials into monosaccharides that can be used by microorganisms for their own growth; no other strains are added during the fermentation process, and there is no antagonism between strains, which is more conducive to later production; a thin layer is used for aerobic solid-state fermentation to convert non-protein nitrogen into true protein, thereby improving the protein quality of the fermentation product, and at the same time digesting soluble compounds (starch, residual sugar), cellulose, organic acids, etc. in the material, reducing the viscosity of the material, and providing favorable conditions for subsequent material drying; the metabolic heat generated by aerobic fermentation is used to evaporate a large amount of water in the raw material, thereby reducing a large amount of energy consumption for subsequent drying.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is an electron microscope image of Schizophyllum 125;

[0030] Figure 2 The changes in the diameter of Schizophyllum 125 at different ambient temperatures within 2-5 days;

[0031] Figure 3 The changes in the bacterial diameter of Schizophyllum 125 at different pH values ​​within 2-5 days;

[0032] Figure 4 The changes in bacterial diameter of Schizophyllum 125 within 2-5 days under different MnCl2 addition amounts;

[0033] Figure 5 The growth of Schizophyllum 125 under the optimum temperature, pH and nutrient elements;

[0034] Figure 6 The changes in the bacterial diameter of Schizophyllum 125 within 2-5 days under different glucose addition amounts;

[0035] Figure 7 The changes in the bacterial diameter of Schizophyllum 125 under different carbon sources within 2-5 days;

[0036] Figure 8 The changes in the diameter of Schizophyllum 125 under different nitrogen sources within 2-5 days;

[0037] Figure 9 The distribution of carbohydrate-active enzymes in different families in Schizophyllum 125 is shown;

[0038] Figure 10 The status of drug-resistant genes carried by Schizophyllum sp. 125;

[0039] Figure 11 To analyze the polysaccharide composition of the secretion products of Schizophyllum 125;

[0040] Figure 12 Blood agar plates for Schizophyllum 125 and E.coilK88, where A is E.coilK88 and B is Schizophyllum 125;

[0041] Figure 13 The growth of Schizophyllum 125 on different plates, where A is a CMC-Na plate, B is a hemicellulose plate, C is a guaiacol plate, D is an aniline blue plate, and E is a ferulic acid esterase plate;

[0042] Figure 14 The fermentation process of Schizophyllum 125 using wine lees as substrate;

[0043] Figure 15 The shapes of Schizophyllum 125 and images of distiller's grains fermented by Schizophyllum 125 are shown, among which A is the shape of a Schizophyllum flat plate, B is an electron microscope image of untreated distiller's grains, C is an image of distiller's grains after Schizophyllum fermentation treatment, and D is an electron microscope image of distiller's grains after Schizophyllum fermentation treatment. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0045] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0046] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0047] Example 1

[0048] Isolation, identification and preservation of Schizophyllum commune 125.

[0049] 90 mL of 0.85% saline was added to a 250 mL Erlenmeyer flask, along with glass beads. The flask was sterilized at 121°C for 20 minutes and cooled. 10 g of soil with mushroom roots was added, and the mixture was shaken at 220 rpm for 3 minutes at 30°C. The mixture was allowed to stand for 10 minutes, and the colony count was determined using a hemocytometer. The culture was diluted to a concentration of 50-100 cfu / mL using YPD (peptone: 2%, yeast powder: 1%, glucose: 2%) medium, and cultured in a MISS cell droplet microfluidizer for 3 days. After the culture was completed, targeted screening was performed using plates containing sodium carboxymethylcellulose (CMC-Na) containing Congo red as the sole carbon source. The presence of a clearing zone was used as an indicator for the isolation, purification, and identification of a strain of Schizophyllum commune.

[0050] The strain number is (Schizophyllum commune 125), and it was deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2024, with the deposit number CGMCC No.41699.

[0051] Experimental testing:

[0052] (1) Electron microscopic image of Schizophyllum 125 Figure 1 shown.

[0053] (2) Detection of physiological and biochemical indicators of Schizophyllum 125:

[0054] ① Inoculate Schizophyllum 125 into PDA medium (0.3% potato extract powder, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol) and observe changes in the diameter of Schizophyllum 125 at different temperatures over 2-5 days. The starting temperature was 22.5°C, and the temperature was increased by 2.5°C each time, reaching a maximum of 40°C.

[0055] Changes in bacterial diameter Figure 2 As shown, it can be seen that when the ambient temperature is 32.5℃, Schizophyllum 125 grows best and the colony diameter reaches 72.83mm on the 5th day.

[0056] ② The Schizophyllum 125 was inoculated into PDA medium (0.3% potato extract powder, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol), and the PDA medium without pH adjustment (pH 5.6) was used as the control. The pH of the PDA medium in the experimental group was adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and the changes in the bacterial diameter of Schizophyllum 125 at different pH values ​​within 2-5 days were observed.

[0057] Changes in bacterial diameter Figure 3 As shown in the figure, it can be seen that when the pH is 6.0-7.0, the growth rate of Schizophyllum 125 is higher than that of the control group. Further considering the cost of pH adjustment, pH 6.0 is the optimal pH for the culture of Schizophyllum 125.

[0058] ③Schizophyllum 125 was inoculated into PDA medium (0.3% potato extract powder, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol), and 0.01%, 0.05%, 0.10%, 0.50%, and 1% MnCl2 were added to the experimental groups, respectively. The control group was set without adding MnCl2, and the changes in the bacterial diameter of Schizophyllum 125 were observed within 2-5 days.

[0059] Changes in bacterial diameter Figure 4 As shown in the figure, it can be seen that when the addition amount of MnCl2 is 0.01%, it has a promoting effect on the growth of Schizophyllum 125.

[0060] ④ Inoculate Schizophyllum 125 into PDA liquid culture medium (0.3% potato extract powder, 2% glucose, 0.01% chloramphenicol, 0.01% MnCl2), the pH of the PDA liquid culture medium is 6.0, the ambient temperature is 32.5℃, and the absorbance of the liquid culture medium at 600nm is measured. Figure 5 As shown, Schizophyllum 125 entered the plateau phase after 72 h.

[0061] ⑤ Inoculate Schizophyllum 125 into PDA medium (0.3% potato extract powder, 1.5% agar powder, 0.01% chloramphenicol, 0.01% MnCl2). Add 5%, 15%, 25%, 35%, 45%, and 50% glucose to the PDA medium of the experimental group, and add 2% glucose to the PDA medium of the control group. Observe the changes in the bacterial diameter of Schizophyllum 125 within 2-5 days.

[0062] Changes in bacterial diameter Figure 6 As shown, it can be seen that when the glucose concentration is 5%, the growth rate is faster than that of the control group, and when the glucose concentration is 15%-50%, it has an inhibitory effect on the growth of the strain.

[0063] ⑥ Using PDA medium as the base medium (control), adding 2% carbon source, changing different carbon sources (starch, sucrose, maltose, lactose), and observing the changes in the bacterial diameter of Schizophyllum 125 within 2-5 days. Figure 7 As shown, it can be seen that except for the lactose group, the growth rate was lower than that of the control group, and the other experimental groups had no significant differences from the control.

[0064] ⑦ Using PDA medium as the base medium (control), the nitrogen source addition amount was 2%, and different nitrogen sources (soybean peptone powder, beef extract, yeast powder, tryptone) were replaced to observe the changes in the bacterial diameter of Schizophyllum 125 within 2-5 days. Figure 8 As shown, it can be seen that the growth rate of each group was lower than that of the control group.

[0065] (3) Whole genome sequencing of Schizophyllum 125:

[0066] like Figure 9 As shown, a total of 371 carbohydrate-active enzymes (CAZymes) were identified in Schizophyllum 125, including 182 members of the GH family, 50 members of the GT family, 9 members of the CBM family, 42 members of the CE family, 73 members of the AA family, and 15 members of the PL family. Currently, the GH family is the catalytic enzyme that contributes most to lignocellulose degradation, and the degradation capacity of Schizophyllum can be simply judged by the number of GH family members. Within the GH family, genes encoding GH16 (25) are the most numerous, followed by GH18 (14). All members of the AA family belong to the glucose-methanol-choline (GMC) family of oxidoreductases, which assist glycoside hydrolases (GHs) in degrading lignocellulose. The AA family in the Schizophyllum genome contains the most genes encoding AA9 (19). Studies have shown that the AA9 family LPMO can degrade crystalline cellulose by oxidatively cleaving glycosidic bonds. Research on the AA9 family is of great significance for biomass conversion.

[0067] The CARD database integrates the information of resistance genes, their products and phenotypes in the genome, and can screen out specific genes with antibiotic resistance. Figure 10 As shown, the genome of Schizophyllum coli 125 has a total of 140 antibiotic resistance-related genes annotated, mainly involving three resistance mechanisms: antibiotic efflux, antibiotic inactivation, and antibiotic target alteration. The most common resistance category is tetracycline antibiotics, with a total of 36 resistance genes, 19 of which are mainly tetB (27.7%). This type of gene mainly encodes membrane-associated proteins that pump tetracycline out of the cell, reducing the intracellular concentration and protecting the ribosomes, thereby conferring resistance. The second largest group is disinfecting agents and antiseptics resistance genes, with a total of 22 genes, 3 of which are mainly Escherichia coli fabG mutations conferring resistance to triclosan. There were also relatively many drug-resistant genes for fluoroquinolone antibiotics and rifampicin antibiotics, 18 and 17 respectively, accounting for 12.9% and 12.1%.

[0068] (4) Evaluation of polysaccharide secretion by Schizophyllum 125:

[0069] The monosaccharide and uronic acid composition of the polysaccharide was analyzed by high performance liquid chromatography (HPLC, C18 separation column) using a methanol solution of p-aminobenzoic acid (0.7 g p-aminobenzoic acid, 0.1 g sodium cyanoborohydride, and 1 g glacial acetic acid, mixed evenly and fixed to volume with 10 mL of methanol). The specific method is as follows:

[0070] Schizophyllum was cultured in YPD medium at 30°C and 150 rpm for 7 days, and the supernatant was collected by centrifugation at 1000 rpm for 5 min to detect the polysaccharide secreted by Schizophyllum.

[0071] Monosaccharide standards: Weigh 10 mg of monosaccharide standard and dissolve in 10 mL of deionized water to prepare a 1000 ppm solution.

[0072] Weigh 50 mg of sample (monosaccharide, polysaccharide) into a 10 mL centrifuge tube, add 4 mL of 2 mol / L trifluoroacetic acid (TFA), and sonicate to dissolve. Seal the tube, oven-hydrolyze at 110°C for 4 h, and concentrate to dryness under vacuum. Wash with HPLC methanol and blow dry with nitrogen twice to remove the TFA. Dissolve in 400 μL of pure water, sonicate for 25 min, and gently vortex to mix.

[0073] Standard composition: approximately 50 mg each of D-xylose, L-rhamnose, D-mannose, L-arabinose, D-galactose, D-glucose, D-galacturonic acid, and D-glucuronic acid.

[0074] The results are as follows Figure 11 As shown, 125 can secrete galactose, mannose, glucose, ribose, arabinose, xylose, and fucose.

[0075] (5) Biosafety evaluation of Schizophyllum 125:

[0076] Blood agar plates were streaked with E.coil K88 and Schizophyllum 125, and cultured at 37°C and 32.5°C for 12 h and 72 h, respectively, to see if there was a transparent zone to determine whether it was safe.

[0077] The results are as follows Figure 12 As shown, there was an obvious hemolytic zone around the colonies of E.coilK88 in the positive control group, while there was no hemolytic zone around the colonies of Schizophyllum 125, indicating that Schizophyllum 125 had no hemolytic activity.

[0078] (6) Determination of enzyme activity of Schizophyllum 125:

[0079] ① Configuration of CMC-Na plate: CMC-Na 1%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.03%, ammonium sulfate 0.14%, yeast powder 0.02%, agar powder 1.6%, Congo red 0.02%. Sterilize at 121℃ for 20min, then pour the plate after the temperature drops to 60℃.

[0080] Hemicellulose plate configuration: hemicellulose 1%, disodium hydrogen phosphate 0.12%, potassium dihydrogen phosphate 0.09%, magnesium sulfate 0.05%, acid hydrolyzed casein 0.05%, Congo red 0.02%, agar powder 1.5%, sterilize at 115℃ for 20min, then drop the temperature to 60℃ and pour the plate.

[0081] Guaiacol plate configuration: 0.2% potassium dihydrogen phosphate, 0.03% magnesium sulfate, 0.14% ammonium sulfate, 0.02% yeast powder, 1.6% agar powder, 0.01% 10% guaiacol (1 g guaiacol dissolved in 10% mL ethanol to make a 10% stock solution).

[0082] Aniline blue plate configuration: 0.1% lignin, 0.5% ammonium sulfate, 0.02% yeast powder, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 0.005% aniline blue, 2% agar powder. Sterilize at 121°C for 20 min, and pour the plate after the temperature drops to 60°C.

[0083] Ferulic acid esterase plate configuration: 0.1% ammonium sulfate, 0.05% magnesium sulfate, 0.02% potassium dihydrogen phosphate, 0.03% calcium chloride, 0.001% ferrous sulfate, 0.025% peptone, 0.05% yeast powder, 2% agar powder, 8 mL of 1.5% ethyl ferulate solution, sterilize at 115°C for 30 min, then cool to 60°C and pour the plate.

[0084] The Schizophyllum 125 was inoculated into the above plates and cultured. The results were as follows: Figure 13 shown.

[0085] Obvious transparent circles can be observed around the colonies of Schizophyllum 125 grown on CMC-Na plates, hemicellulose plates, and ferulic acid esterase plates, proving that Schizophyllum can secrete cellulase, hemicellulase, and ferulic acid esterase (FAE). The brown color of the guaiacol plates proves that it can produce laccase (Laccase, Lac), and the fading of the blue color of the aniline blue plates proves that it can produce lignin peroxidases (Lignin peroxidases, LiP) and manganese peroxidases (Man-ganese peroxidases, MnP).

[0086] ② Cellulase decomposes β-1,4-glucan chains (cellulose); xylanase hydrolyzes xylan in hemicellulose; filter paper enzyme (exocellulase) collaborates with cellulase to improve the efficiency of crystalline cellulose degradation; laccase and manganese peroxidase oxidatively decompose lignin, removing its coating on cellulose and promoting the exposure of polysaccharides. These enzymes work together to achieve efficient decomposition of lignocellulose. Therefore, when designing the experiment, the activities of cellulase, xylanase, filter paper enzyme, laccase, and manganese peroxidase in the culture medium were measured.

[0087] The solid culture method is used to culture Schizophyllum 125. The components and proportions of the solid culture medium are as follows: 100% bran, 5% molasses, 2% ammonium sulfate, 0.5% magnesium sulfate, 0.1% Tween-80, 1% superphosphate, and 0.5% potassium chloride. The pH value of the solid culture medium is 5.9-6.2. The culture is cultured for five days, and an air-dried sample is prepared from the culture, which is crushed and passed through a 60-mesh sieve. The activities of cellulase, xylanase, and filter paper enzyme in the sample are detected using a national standard spectrophotometer method.

[0088] The ABTS method was used to detect the activity of laccase. The definition of enzyme activity was: the amount of enzyme required to oxidize 1 nmol of substrate ABTS per minute per liter of culture medium was one unit of enzyme activity.

[0089] The activity of manganese peroxidase was determined using a Solebro kit. The definition of enzyme activity was: the amount of enzyme required to oxidize 1 nmol of guaiacol per minute per milliliter of culture medium was one unit of enzyme activity.

[0090] The activities of cellulase, xylanase, filter paper enzyme, laccase and manganese peroxidase are shown in Table 1 below:

[0091] Table 1 Enzyme activity of Schizophyllum 125

[0092]

[0093] Example 2

[0094] The fermentation process of the strain 125 selected in Example 1 with distiller's grains as substrate includes four stages (excluding seed preparation and drying): preparation of the first-level seed koji, pretreatment of the distiller's grains, preparation of the second-level seed koji, and preparation of the large koji. Figure 14 shown.

[0095] S1. Preparation of first-grade seed koji.

[0096] Culture medium (dry matter basis): bran 100%, molasses 5%, ammonium sulfate 2%, magnesium sulfate 0.5%, Tween-80 0.1%, superphosphate 1%, potassium chloride 0.5%, pH 5.9-6.2.

[0097] The triangular bottle koji is prepared under a strict pure strain culture environment, with fermentation enzyme activity as the key indicator. The substrate is 100% bran. Bran has a strong induction ability and can make the fermentation enzyme activity reach a higher level.

[0098] Sterilization: The moisture content of the koji is 40%. Dispense into 500 mL Erlenmeyer flasks, 40 g (wet weight) per flask. Sterilize in an autoclave at 121°C for 40 minutes. Cool to 30°C before inoculation.

[0099] Use sterile water to scrape the schizophyllum 125 bacterial lawn and disperse the spores into the sterile water. Use a hemocytometer to count the spores to ensure that the number of spores per mL of bacterial suspension is ≥10 7 Inoculate 4 mL of spore suspension into each bottle of koji material.

[0100] Cultivation: Cultivate in a constant temperature incubator for 5 days. The temperature is controlled at 32.5°C and the humidity is controlled at 80%.

[0101] Indicators: Each gram of dry koji contains laccase ≥ 0.18μ / g, cellulase ≥ 21.15μ / g, filter paper enzyme ≥ 0.89μ / g, xylanase ≥ 47.24μ / g, and manganese peroxidase ≥ 0.022μ / g.

[0102] S2. Pretreatment of distiller's grains: Use ammonia water to adjust the pH to 5.9-6.2, and use bran, corn husks and distiller's grains to reduce the moisture content to 50-55%.

[0103] S3. Preparation of secondary seed koji.

[0104] Culture medium (dry matter basis): magnesium sulfate 0.5%, potassium chloride 0.5%, ammonia water 2.5%, soybean meal 1.25%, wheat bran 27.0%, corn husk 18%, distiller's grains 50%, molasses 3.75%, pH value is 5.9-6.2.

[0105] The cultivation of the secondary seed koji is carried out under relatively strict pure culture conditions. The composition of the culture medium is between that of the primary seed koji and the large koji material, playing a transitional role, effectively balancing the enhancement of fermentation activity and the requirements of stable bacterial growth, and optimizing the fermentation process.

[0106] Disinfection and sterilization: The moisture content of the koji is 50-55%. The koji is divided into shallow dishes, kept at 121℃ in a sterilizer for 40 minutes, and then cooled to below 32.5℃ for inoculation.

[0107] Inoculation: Select a first-class starter koji with good growth, no bacterial contamination and high enzyme activity. Mix the starter koji and culture medium thoroughly according to the inoculation amount of 10%, and send it to the shallow tray fermentation bed via a conveyor belt for fermentation.

[0108] The bending load per unit space in the bending room is 2.0-2.5kg / m 3 The air temperature was maintained at 30-35°C. The relative humidity of the air in the koji room was adjusted to 85-90% for the entire culturing process of 4 days.

[0109] S3. Daqu cultivation.

[0110] Culture medium (dry matter basis): magnesium sulfate 0.5%, potassium chloride 1.3%, ammonia water 2.5%, manganese chloride 0.01%, soybean meal 2.0%, molasses 5.0%, wheat bran 15.0%, distiller's grains 70%, corn husk 13%, pH 5.9-6.2.

[0111] The culture medium is rich in organic nitrogen, which can shorten the fermentation time, reduce cost investment and equipment requirements, and effectively improve the overall production efficiency.

[0112] Sterilization: The water content of the culture medium is 50% to 55%. High-temperature, high-pressure saturated steam is directly introduced into the sterilization tank to rapidly heat the culture medium to 100°C for 10-15 minutes of sterilization. After sterilization, the culture medium is transferred to a shallow tray fermentation bed via a conveyor device for cooling and leveling. When the temperature drops below 35°C, it is ready for inoculation.

[0113] Inoculation: Take 100 kg of well-cultured, high enzyme activity, and free of bacterial contamination secondary koji (the moisture content of mature secondary koji is about 45%), inoculate it at a 10% inoculation rate, break it up by machine, stir it with the large koji material, mix it evenly, and flatten it for 5 days of aerobic solid-state fermentation.

[0114] Cultivation: The material load in the fermentation room is controlled at 3.0-3.5 kg / m 3 After 72 hours of incubation, the Schizophyllum reaches its peak growth and reproduction, consuming significant amounts of energy. Simultaneously, due to high heat production, a significant amount of water evaporates, so attention should be paid to cooling and dissipating heat and preventing water loss. After another 48 hours of aerobic solid-state fermentation, the fermented product can be used directly in feed preparation or can be air-dried before use.

[0115] Airflow drying: After fermentation, the moisture content of Daqu is around 30%-35%. Airflow is used to reduce the moisture content to below 10% for sample testing.

[0116] like Figure 15 Shown are the shape of Schizophyllum 125, electron micrographs of distiller's grains before and after fermentation, and images of the distiller's grains after fermentation. Before fermentation, the distiller's grains had a smooth surface and intact structure. After fermentation, the grains became rough, with holes and attachments. This indicates that secreted cellulases and xylanases degraded the cell wall, potentially producing polysaccharides such as β-glucan. Lignin was oxidized by laccases and manganese peroxidases, resulting in a loose substrate structure. These results confirm that Schizophyllum 125 can effectively degrade lignocellulose and is suitable for biomass conversion.

[0117] The comparative analysis of the main indicators of the Schizophyllum 125-treated group and the control group (not inoculated with Schizophyllum) is shown in Table 2 below:

[0118] Table 2 Comparative analysis of main indicators

[0119]

[0120]

[0121] Crude protein content: 5.98g in the treated group was 5.1% higher than that in the control group (5.69g), indicating that the fermentation treatment with Schizophyllum 125 effectively increased the protein content.

[0122] Non-protein nitrogen and true protein: Non-protein nitrogen was significantly reduced from 0.95g to 0.55g, a decrease of 42.1%; the mass of true protein was significantly increased from 4.74g to 5.43g, an increase of 14.6%; the true protein content increased from 17.27% to 21.70%, indicating that the fermentation process effectively converted non-protein nitrogen into true protein.

[0123] Fiber content: NDF (neutral detergent fiber) decreased from 40.82% to 31.32%, a decrease of 23.3%, and ADF (acid detergent fiber) decreased from 20.35% to 16.34%, a decrease of 19.7%, indicating that the fermentation treatment effectively degraded the fiber components.

[0124] Changes in dry matter: Dry weight decreased from 27.45g to 25.02g, a decrease of 8.9%, which may be the result of part of the matter being decomposed and utilized by microorganisms.

[0125] The changes in amino acid content in the fermentation lees of Schizophyllum 125 are shown in Table 3:

[0126] Table 3 Changes in amino acid content in fermented lees

[0127]

[0128]

[0129] From Table 3, we can see that the amino acid content of shallow tray fermentation increased the most, with lysine increasing the most by 46.48%, followed by tryptophan increasing by 30.95%, and then arginine increasing by 24.48%.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A Schizophyllum sp., characterized in that: The strain number of Schizophyllum is Schizophyllum commune125, and it was deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2024, with the deposit number CGMCCNo.41699.

2. An application of the Schizophyllum sp. according to claim 1 in the treatment of liquor lees, characterized in that: Aerobic solid-state fermentation of Schizophyllum using liquor lees as substrate comprises the following steps: S1. Preparation of primary seed koji: Schizophyllum sp. is inoculated into a primary culture medium with bran as the substrate, and cultured at 32.5°C and 80% relative humidity until the content of laccase ≥ 0.18 μg / g, cellulase ≥ 21.15 μg / g, filter paper enzyme ≥ 0.89 μg / g, xylanase ≥ 47.24 μg / g, and manganese peroxidase ≥ 0.022 μg / g per gram of dry koji is achieved; S2. Preparation of secondary seed koji: inoculate the primary seed koji at a rate of 10-15% into the secondary culture medium containing 50% pre-treated distiller's grains, and ferment in a shallow tray fermentation area for 4 days; S3. Daqu culture: inoculate the secondary seed koji at a rate of 10-15% into the tertiary culture medium containing 70% pre-treated distiller's grains, and ferment in a shallow tray fermentation area for 5 days.

3. The use according to claim 2, characterized in that In S1, the composition ratio of the primary culture medium is as follows on a dry matter basis: bran 100%, molasses 5%, ammonium sulfate 2%, magnesium sulfate 0.5%, Tween-80 0.1%, superphosphate 1%, potassium chloride 0.5%, and the pH value of the primary culture medium with bran as the substrate is 5.9-6.

2.

4. The use according to claim 2, characterized in that In S2, the composition ratio of the secondary culture medium is calculated on a dry matter basis as follows: magnesium sulfate 0.5%, potassium chloride 0.5%, ammonia water 2.5%, soybean meal 1.25%, wheat bran 27.0%, corn husk 18%, distiller's grains 50%, molasses 3.75%, and the pH value of the secondary culture medium is 5.9-6.

2.

5. The use according to claim 2, characterized in that: In S2, the fermentation area has a unit space capacity of 2.0-2.5 kg / m 3 , the air temperature is 30-35℃, and the relative humidity of the air is 85-90%.

6. The use according to claim 2, characterized in that In S3, the composition ratio of the tertiary culture medium is calculated on a dry matter basis as follows: magnesium sulfate 0.5%, potassium chloride 1.3%, ammonia water 2.5%, manganese chloride 0.01%, soybean meal 2.0%, molasses 5.0%, wheat bran 15.0%, distiller's grains 70%, corn husk 13%, and the pH value of the tertiary culture medium is 5.9-6.

2.

7. The use according to claim 2, characterized in that: In S3, the fermentation area has a unit space capacity of 3.0-3.5 kg / m 3 .

8. The use according to claim 2, characterized in that The pretreatment of the lees includes: adjusting the pH of the lees to 5.9-6.2 using 3.5% ammonia water, and adjusting the moisture content of the lees to 50-55% using bran and corn husks.

9. Use of the Schizophyllum sp. according to claim 1 in feed preparation.

10. Use of a product of aerobic solid-state fermentation of Schizophyllum using white wine lees as a substrate according to any one of claims 2 to 8 in feed preparation.

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