Schizophyllum and use thereof

Abstract

This invention discloses a *Schizophyllum commune* strain and its applications, belonging to the field of fermentation technology. The strain number is *Schizophyllum commune* 125, which was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41699. This invention provides a strain capable of utilizing cellulose and hemicellulose to produce glucose, xylose, arabinose, and other substances, and also capable of decomposing lignin. This strain can also utilize the sugars produced from these cellulose and hemicellulose components, as well as the nutrients in the distiller's grains themselves, as carbon and nitrogen sources for its own growth and high protein production. It is a protein source with excellent protein content and quality, laying the foundation for the development of novel feed additives and the full utilization of renewable resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation, in particular to a schizophyllum commune and application thereof. BACKGROUND

[0002] The solid brewing waste of the liquor industry, i.e. distiller's grains, is also known as distiller's grains or discarded grains, and is the solid material remaining after the brewing process. Generally, about 3-4 tons of fresh distiller's grains are produced for every ton of liquor. The distiller's grains inherit the organic components such as proteins, starches, cellulose and fats in the brewing raw materials, and has the value of resource utilization. On the other hand, the distiller's grains have high water content and microbial content, and are prone to spoilage during storage, and will produce harmful gases and high-concentration organic leachate during stacking. If not properly handled or disposed of in a timely manner, it will pose a risk of environmental pollution.

[0003] Therefore, harmless and resourceful utilization of distiller's grains is of great significance for reducing carbon emissions in the basin, maintaining the health of the ecological environment and realizing the recycling of material resources. Studies have shown that distiller's grains have good resource utilization potential and can be used in the fields of biological feed, organic fertilizer, functional material, energy production, extraction of active substances, preparation of raw materials and secondary brewing.

[0004] As a solid byproduct in the production process of liquor grains, the physicochemical properties and composition of liquor grains are closely related to factors such as the type of grain used, the brewing process and the storage time. The water content of fresh distiller's grains and dry distiller's grains differs greatly, with a difference of more than 58.0%. In the dry matter composition of distiller's grains, the contents of cellulose, hemicellulose, crude protein, crude starch and ash differ by more than 15.0%, the content of lignin differs by about 10.0%, and the contents of hemicellulose and crude fat differ by about 7.0%, which may be caused by the following three reasons:

[0005] ①Brewing raw materials. Different brands of liquor use different raw materials, such as sorghum, rice, millet and wheat, and the contents of components such as starch, protein, fat, cellulose and lignin in these raw materials differ.

[0006] ②Brewing process. Different process conditions, such as fermentation times, turning times and microbial species, will result in different decomposition and utilization rates of the components of the grain raw materials.

[0007] ③Storage time. With the passage of storage time, water in the distiller's grains will diffuse into the environment, and microorganisms will continue to decompose the remaining organic matter.

[0008] Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. Among them, cellulose, hemicellulose and lignin are cross-linked together, hemicellulose and lignin are filled between the microfibers formed by cellulose macromolecules, and lignin can inhibit the degradation of cellulose and hemicellulose by microorganisms. This protective effect makes it difficult for enzymes to contact the surface of cellulose. Therefore, a strain that can open the three-element structure is needed to first decompose part of the lignin, and then hydrolyze the cellulose and hemicellulose into glucose and other monosaccharides for subsequent conversion production. SUMMARY

[0009] The purpose of the present application is to provide a kind of schizophyllum and its application, using schizophyllum to degrade cellulose, hemicellulose and lignin in distiller's grains, produce glucose, xylose, arabinose and the like, using the produced glucose, xylose, arabinose and the like as carbon source for schizophyllum growth, schizophyllum converts non-protein nitrogen in distiller's grains into protein of the strain itself, and secretes polysaccharide and some amino acids and the like.

[0010] To achieve the above purpose, on the one hand, the present application provides a kind of schizophyllum, the strain number of schizophyllum is Schizophyllum commune 125, which was preserved in China General Microbiological Culture Collection Center on December 13, 2024, and the preservation number is CGMCC No.41699.

[0011] On the other hand, the present application provides a kind of schizophyllum as described above in the application of liquor distiller's grains, and the aerobic solid fermentation of schizophyllum is carried out with liquor distiller's grains as substrate, which comprises the following steps:

[0012] S1, preparation of primary starter: inoculate schizophyllum in primary culture medium with bran as substrate, culture temperature 32.5 DEG C, air relative humidity 80%, culture until each gram of dry starter contains laccase ≥0.18 μ / g, cellulase ≥21.15 μ / g, filter paper enzyme ≥0.89 μ / g, xylanase ≥47.24 μ / g, and manganese peroxidase ≥0.022 μ / g;

[0013] S2, preparation of secondary starter: inoculate the primary starter in the secondary culture medium containing 50% pretreated distiller's grains at an inoculation amount of 10-15%, and ferment in the shallow tray fermentation area for 4 days;

[0014] S3, culture of Daqu: inoculate the secondary starter in the tertiary culture medium containing 70% pretreated distiller's grains at an inoculation amount of 10-15%, and ferment in the shallow tray fermentation area for 5 days.

[0015] Preferably, in S1, the component ratio of the primary culture medium is as follows: 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.

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

[0017] Preferably, in S2, the loading amount of the shallow plate fermentation area per unit space is 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 component ratio of the tertiary culture medium is 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 bran 13%, and the pH value of the tertiary culture medium is 5.9-6.2.

[0019] Preferably, in S3, the loading amount of the shallow plate fermentation area per unit space is 3.0-3.5 kg / m 3 .

[0020] Preferably, the pretreatment of the distiller's grains includes adjusting the pH value of the distiller's grains to 5.9-6.2 by using 3.5% ammonia water and adjusting the moisture content of the distiller's grains to 50-55% by using bran and corn bran.

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

[0022] In another aspect, the present application provides the use of the product of the above-mentioned aerobic solid-state fermentation of Schizophyllum with white spirit distiller's grains as the substrate in the preparation of feed.

[0023] Therefore, the Schizophyllum and the use thereof have the following beneficial effects:

[0024] (1) First isolation and identification of a strain of Schizophyllum commune 125, which has cellulose, hemicellulose, lignin enzyme activity; using Schizophyllum commune 125 to degrade cellulose, hemicellulose and lignin in distiller's grains, producing glucose, xylose, arabinose, etc., to produce sugar as carbon source for Schizophyllum commune 125 growth; at the same time, Schizophyllum commune 125 makes full use of the nutrients and non-protein nitrogen in distiller's grains, and converts the non-protein nitrogen in distiller's grains into the protein of the fungus itself, and secretes polysaccharides and amino acids and other substances.

[0025] (2) In the past, chemical reagents such as acid and alkali treatment methods are used to treat distiller's grains, which can decompose a large amount of cellulose and hemicellulose to produce glucose, xylose, arabinose, etc., but will produce many inhibitors such as furfural and acetic acid, which will inhibit the growth of the strain, and the pretreatment will also increase the risk of secondary pollution, resulting in increased treatment cost; most of the current cellulase is pure enzyme, and the effect of direct treatment of natural lignocellulose is poor, and the price of commercial cellulase is expensive, which increases the production steps and production cost, and is not conducive to large-scale production.

[0026] (3) The present application uses Schizophyllum commune 125 to treat distiller's grains by segmented fermentation, which converts biomass raw materials into monosaccharides that can be utilized 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; thin-layer aerobic solid-state fermentation is used to convert non-protein nitrogen into true protein, which improves the protein quality of the fermentation product, and also digests 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 evaporates a large amount of water in the raw material, which reduces a large amount of energy consumption for subsequent drying.

[0027] The technical solutions of the present application will be further described below through the accompanying drawings and examples. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments of the present application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 The electron microscope image of Schizophyllum commune 125;

[0030] Figure 2 The change of the diameter of Schizophyllum commune 125 in different environmental temperatures within 2-5 days;

[0031] Figure 3 The changes in bacterial diameter of Schizophyllum commune 125 over 2-5 days at different pH levels;

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

[0033] Figure 5 The growth of Schizophyllum commune 125 under optimal temperature, pH and nutrient conditions;

[0034] Figure 6 The changes in bacterial diameter of Schizophyllum commune 125 over 2-5 days under different glucose addition levels;

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

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

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

[0038] Figure 10 The presence of drug resistance genes in Schizophyllum commune 125;

[0039] Figure 11 Analysis of polysaccharide composition in the secretions of Schizophyllum commune 125;

[0040] Figure 12 Blood agar plates for Schizophyllum commune 125 and E. coil K88, where A is E. coil K88 and B is Schizophyllum commune 125;

[0041] Figure 13 The growth of Schizophyllum commune 125 on different plates is shown. Among them, A is CMC-Na plate, B is hemicellulose plate, C is guaiacol plate, D is aniline blue plate, and E is ferulic acid esterase plate.

[0042] Figure 14 The fermentation process of Schizophyllum commune 125 using distiller's grains as a substrate;

[0043] Figure 15 Images of Schizophyllum commune 125 and fermented lees from Schizophyllum commune 125 are shown. In the images, A is the shape of the Schizophyllum commune plate, B is an electron micrograph of the untreated lees, C is an image of the lees after fermentation treatment with Schizophyllum commune, and D is an electron micrograph of the lees after fermentation treatment with Schizophyllum commune. Detailed Implementation

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

[0045] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0047] Example 1

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

[0049] Add 90 mL of 0.85% physiological saline to a 250 mL Erlenmeyer flask, add glass beads, sterilize at 121 °C for 20 min, and cool. Add 10 g of soil containing mushroom roots, shake at 220 rpm for 3 min at 30 °C, let stand for 10 min, and determine the colony count using a hemocytometer. Dilute to 50-100 CFU / mL using YPD medium (peptone: 2%, yeast extract: 1%, glucose: 2%), and culture in MISS Cell droplet microfluidic for 3 days. After culture, use a plate containing Congo red and carboxymethyl cellulose sodium (CMC-Na) as the sole carbon source for directional screening, using the appearance of a clear zone as an indicator. Isolate, purify, and identify a strain of *Schizophyllum commune*.

[0050] The strain number (Schizophyllum commune 125) was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.41699.

[0051] Experimental testing:

[0052] (1) Electron micrograph of Schizophyllum commune 125 is shown below. Figure 1 As shown.

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

[0054] ① Schizophyllum commune 125 was inoculated into PDA medium (0.3% potato extract, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol), and the changes in bacterial diameter of Schizophyllum commune 125 were observed over 2-5 days at different ambient temperatures. The initial temperature was 22.5℃, and the temperature was increased by 2.5℃ each time, with the highest temperature being 40℃.

[0055] Changes in bacterial diameter as follows Figure 2 As shown, it can be seen that the best growth of Schizophyllum commune 125 was achieved at an ambient temperature of 32.5℃, and the colony diameter reached 72.83 mm on the 5th day.

[0056] ② Schizophyllum commune 125 was inoculated into PDA medium (0.3% potato extract, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol). PDA medium with unadjusted pH (pH 5.6) served as the control. The pH of PDA medium in the experimental group was adjusted to 4.0, 5.0, 6.0, 7.0, and 8.0, respectively. The changes in the diameter of Schizophyllum commune 125 were observed over 2-5 days at different pH levels.

[0057] Changes in bacterial diameter as follows Figure 3 As shown, the growth rate of *Schizophyllum commune* 125 was higher than that of the control group at pH 6.0-7.0. Considering the cost of pH adjustment, pH 6.0 was determined to be the optimal pH for culturing *Schizophyllum commune* 125.

[0058] ③ Schizophyllum commune 125 was inoculated into PDA medium (0.3% potato extract, 2% glucose, 1.5% agar powder, 0.01% chloramphenicol). The experimental groups were supplemented with 0.01%, 0.05%, 0.10%, 0.50%, and 1% MnCl2, respectively, with no MnCl2 added as the control. The changes in the diameter of Schizophyllum commune 125 were observed over 2-5 days.

[0059] Changes in bacterial diameter as follows Figure 4 As shown, the addition of MnCl2 at 0.01% promotes the growth of Schizophyllum commune 125.

[0060] ④ Schizophyllum commune 125 was inoculated into PDA liquid medium (0.3% potato extract, 2% glucose, 0.01% chloramphenicol, 0.01% MnCl2). The pH of the PDA liquid medium was 6.0, and the ambient temperature was 32.5℃. The absorbance of the liquid medium at 600 nm was measured. The results are as follows. Figure 5 As shown, Schizophyllum commune 125 entered the plateau phase after 72 hours.

[0061] ⑤ Schizophyllum commune 125 was inoculated into PDA medium (0.3% potato extract, 1.5% agar powder, 0.01% chloramphenicol, 0.01% MnCl2). The experimental groups were supplemented with 5%, 15%, 25%, 35%, 45%, and 50% glucose in PDA medium, respectively, while the control group was supplemented with 2% glucose. The changes in the diameter of Schizophyllum commune 125 were observed over 2-5 days.

[0062] Changes in bacterial diameter as follows Figure 6 As shown, the growth rate was faster at a glucose concentration of 5% compared to the control group, while glucose concentrations of 15%-50% inhibited the growth of the strain.

[0063] ⑥ Using PDA medium as the basal medium (control), with a carbon source addition of 2%, different carbon sources (starch, sucrose, maltose, lactose) were introduced, and the changes in the diameter of *Schizophyllum commune* 125 were observed over 2-5 days. The changes in bacterial diameter are shown in the figure below. Figure 7 As shown, except for the lactose group, whose growth rate was lower than that of the control group, the other experimental groups were not significantly different from the control.

[0064] ⑦ Using PDA medium as the basal medium (control), with a nitrogen source addition of 2%, different nitrogen sources (soybean peptone powder, beef extract, yeast powder, tryptone) were introduced, and the changes in the diameter of *Schizophyllum commune* 125 were observed over 2-5 days. The changes in bacterial diameter are shown in the figure below. Figure 8 As shown in the figure, the growth rate of each group is lower than that of the control group.

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

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

[0067] The CARD database integrates information on resistance genes, their products, and phenotypes in the genome, allowing for the screening of specific genes exhibiting antibiotic resistance. For example... Figure 10 As shown, the *Schizophyllum coli* 125 genome annotated a total of 140 antibiotic resistance-related genes, mainly involving three resistance mechanisms: antibiotic efflux, antibiotic inactivation, and antibiotic target alteration. The most prevalent resistance category is tetracycline antibiotics, with 36 resistance genes (19 in total), predominantly tetB (27.7%). These genes primarily encode membrane-associated proteins that pump tetracycline out of the cell, reducing intracellular concentration and protecting intracellular ribosomes, thus leading to resistance. The second most common resistance category is disinfectant and antiseptic resistance genes, with 22 genes (3 in total), predominantly *Escherichia coli* fabG mutations conferring resistance to triclosan. Fluoroquinolone antibiotics and rifampicin antibiotics also have a relatively high number of resistance genes, with 18 and 17 genes respectively, accounting for 12.9% and 12.1%.

[0068] (4) Evaluation of polysaccharides secreted by Schizophyllum commune 125:

[0069] The monosaccharide and uronic acid composition of the polysaccharide was analyzed by derivatization with 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 thoroughly and diluted to 10 mL methanol), followed by high performance liquid chromatography (HPLC, C18 column). The specific method is as follows:

[0070] Schizophyllum commune was cultured on YPD medium at 30°C and 150 rpm for 7 days. The supernatant was collected by centrifugation at 1000 rpm for 5 min, and the polysaccharides secreted by Schizophyllum commune were detected.

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

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

[0073] The standard consists of 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 commune 125:

[0076] E. coil K88 and Schizophyllum commune 125 were streaked onto blood agar plates and incubated at 37℃ and 32.5℃ for 12h and 72h respectively. The presence of a clear zone was used to determine whether the bacteria were safe.

[0077] The results are as follows Figure 12 As shown, the positive control group E. coil K88 colonies had obvious hemolytic zones, while the colonies of Schizophyllum commune 125 did not have hemolytic zones, indicating that Schizophyllum commune 125 does not have hemolytic activity.

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

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

[0080] Hemicellulose plate preparation: 1% hemicellulose, 0.12% disodium hydrogen phosphate, 0.09% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.05% acid-hydrolyzed casein, 0.02% Congo red, 1.5% agar powder. Sterilize at 115℃ for 20 minutes, then turn the plates over when the temperature drops to 60℃.

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

[0082] Aniline blue plate preparation: 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℃ for 20 minutes, and then turn the plates over after the temperature drops to 60℃.

[0083] Ferulic acid esterase plate preparation: 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 extract, 2% agar powder, 8 mL of 1.5% ethyl ferulic acid solution, sterilize at 115℃ for 30 min, and then deplate at 60℃.

[0084] Schizophyllum 125 was inoculated into the above-mentioned plates and cultured, and the results are as follows: Figure 13 As shown.

[0085] A clear halo was observed around the Schizophyllum commune 125 colonies growing on CMC-Na plates, hemicellulose plates, and ferulic acid esterase plates, proving that Schizophyllum commune can secrete cellulase, hemicellulase, and ferulic acid esterase (FAE). The brown color of the guaiacol plate proves that it can produce laccase (Lac), and the fading of the blue color of the aniline blue plate proves that it can produce lignin peroxidases (LiP) and manganese peroxidases (MnP).

[0086] ② Cellulase breaks down β-1,4-glucan chains (cellulose); xylanase hydrolyzes xylan in hemicellulose; filter paper enzyme (exocellulase) synergistically enhances the degradation efficiency of crystalline cellulose with cellulase; laccase and manganese peroxidase oxidize and decompose lignin, releasing it from its encapsulation of cellulose and promoting polysaccharide exposure. These enzymes work together to achieve efficient decomposition of lignocellulose. Therefore, the activities of cellulase, xylanase, filter paper enzyme, laccase, and manganese peroxidase in the culture were measured in the experiment.

[0087] Schizophyllum 125 was cultured using a solid-state culture method. The solid culture medium consisted of: 100% wheat bran, 5% molasses, 2% ammonium sulfate, 0.5% magnesium sulfate, 0.1% Tween-80, 1% superphosphate, and 0.5% potassium chloride. The pH of the solid culture medium was 5.9-6.2. After five days of culture, the culture was air-dried, pulverized, and passed through a 60-mesh sieve. The activities of cellulase, xylanase, and filter paper enzyme were detected using a national standard spectrophotometer.

[0088] The ABTS method was used to detect laccase activity. Enzyme activity is defined as the amount of enzyme required to oxidize 1 nmol of substrate ABTS per minute per liter of culture medium, which is one unit of enzyme activity.

[0089] The activity of manganese peroxidase was detected using the Solarbio reagent kit. Enzyme activity is defined as the amount of enzyme required to oxidize 1 nmol of guaiacol per minute per milliliter of culture medium, which is one unit of enzyme activity.

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

[0091] Table 1 Enzyme activity of Schizophyllum commune 125

[0092]

[0093] Example 2

[0094] The *Schizophyllum commune* 125 strain screened in Example 1 was used for fermentation with distiller's grains as the substrate. The fermentation process included four stages (excluding seed preparation and drying): preparation of primary starter culture, pretreatment of distiller's grains, preparation of secondary starter culture, and preparation of large starter culture. The fermentation process flow is as follows: Figure 14 As shown.

[0095] S1, Preparation of primary seed culture.

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

[0097] The Erlenmeyer flask starter culture is prepared under strict pure culture conditions, with fermentation enzyme activity as the key indicator. The substrate is 100% wheat bran, which has strong inducing ability and can bring the fermentation enzyme activity to a high level.

[0098] Sterilization: The moisture content of the koji is 40%, and it is dispensed into 500mL Erlenmeyer flasks, with 40g (wet weight) per flask. Sterilize at 121℃ for 40 minutes. Cool to 30℃ before inoculation.

[0099] Scrape the *Schizophyllum commune* 125 bacterial growth with sterile water to disperse the spores. Count the spores using a hemocytometer to ensure that the number of spores per mL of bacterial suspension is ≥10. 7 Each bottle of starter culture is inoculated with 4 mL of spore suspension.

[0100] Incubation: Incubate for 5 days in a constant temperature incubator. Maintain the incubation temperature at 32.5℃ and the humidity at 80%.

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

[0102] S2. Pretreatment of distiller's grains: The pH is adjusted to 5.9-6.2 using ammonia water, and the moisture content of the distiller's grains is reduced to 50-55% by mixing wheat bran and corn husks with the distiller's grains.

[0103] S3, preparation of secondary seed culture.

[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 5.9-6.2.

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

[0106] Sterilization: The moisture content of the koji is 50-55%. The koji is dispensed into shallow trays and kept at 121℃ for 40 minutes in an autoclave. After cooling to below 32.5℃, it is inoculated.

[0107] Inoculation: Select first-grade koji that is growing well, free from contamination by other microorganisms, and has high enzyme activity. Inoculate at a rate of 10%. Mix the koji and culture medium thoroughly and evenly, and send them to a shallow tray fermentation bed for fermentation via conveyor belt.

[0108] The curing capacity per unit space in the curing room is 2.0-2.5 kg / m². 3 The air temperature is maintained at 30-35℃. The relative humidity of the air in the fermentation room is adjusted to 85-90%, and the entire fermentation process takes 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 husks 13%, pH value 5.9-6.2.

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

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

[0113] Inoculation: Take 100 kg of well-cultured, high-enzyme-activity, and uncontaminated secondary koji (mature secondary koji has a moisture content of about 45%), inoculate at a rate of 10%, break it up with a machine, mix it evenly with the koji material, spread it out, and carry out aerobic solid-state fermentation for 5 days.

[0114] Cultivation: The material loading in the fermentation chamber should be controlled at 3.0-3.5 kg / m². 3 During the 72-hour cultivation period, the growth and reproduction of *Schizophyllum commune* reaches its peak, resulting in significant energy consumption. Simultaneously, due to high heat production and substantial water evaporation, attention must be paid to cooling, heat dissipation, and moisture loss. After another 48 hours of aerobic solid-state fermentation, the fermented product can be used directly in feed formulation, or it can be air-dried before use in feed formulation.

[0115] Airflow drying: After fermentation, the moisture content of the starter culture is about 30%-35%. Airflow is used to reduce the moisture content to below 10% before sample testing.

[0116] like Figure 15 The image shows the morphology of *Schizophyllum commune* 125 and electron micrographs of the fermented mash before and after fermentation, as well as an image of the mash after fermentation. Before fermentation, the mash surface was smooth and structurally intact; after fermentation, the mash became rough, with pores and deposits, indicating that secreted cellulases and xylanases degraded the cell wall and may have produced polysaccharides such as β-glucan. Lignin was oxidized by laccase / manganese peroxidase, resulting in a loose substrate structure. The results confirm that *Schizophyllum commune* 125 can effectively decompose lignocellulose and is suitable for biomass conversion.

[0117] The main indicators of the *Schizophyllum commune* 125 treatment group and the control group (without *Schizophyllum commune* inoculation) were compared and analyzed as shown in Table 2 below:

[0118] Table 2 Comparison and Analysis of Key Indicators

[0119]

[0120]

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

[0122] Non-protein nitrogen and true protein: Non-protein nitrogen decreased significantly from 0.95g to 0.55g, a decrease of 42.1%; the quality of true protein increased significantly from 4.74g to 5.43g, an increase of 14.6%; the change in 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 reduction of 23.3%, and ADF (acid detergent fiber) decreased from 20.35% to 16.34%, a reduction of 19.7%, indicating that the fermentation treatment effectively degraded the fiber components.

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

[0125] The changes in amino acid content in the fermented mash of Schizophyllum commune 125 are shown in Table 3:

[0126] Table 3. Changes in amino acid content in fermented distiller's grains

[0127]

[0128]

[0129] Table 3 shows that shallow-pan fermentation significantly increases the amino acid content, with the highest increase in lysine at 46.48%, followed by tryptophan at 30.95%, and then arginine at 24.48%.

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

Claims

1. A type of schizophyllum commune ( Schizophyllum commune ), characterized by: The strain number of Schizophyllum commune is Schizophyllum commune Accession number 125 was deposited on December 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41699.

2. The application of *Schizophyllum commune* as described in claim 1 in the treatment of baijiu (Chinese liquor) lees, characterized in that, Aerobic solid-state fermentation of *Schizophyllum commune* using baijiu lees as substrate includes the following steps: S1. Preparation of primary starter culture: Schizophyllum commune was inoculated into primary culture medium with wheat bran as substrate and cultured at 32.5℃ and 80% relative humidity until each gram of dry starter culture contained laccase ≥0.18μ / g, cellulase ≥21.15μ / g, filter paper enzyme ≥0.89μ / g, xylanase ≥47.24μ / g, and manganese peroxidase ≥0.022μ / g. S2. Preparation of secondary starter culture: The primary starter culture is inoculated into a secondary culture medium containing 50% pretreated distiller's grains at an inoculation rate of 10-15%, and fermented in a shallow fermentation zone for 4 days; S3, Daqu cultivation: The secondary koji is inoculated into the tertiary culture medium containing 70% pretreated lees at an inoculation rate of 10-15%, and fermented in the shallow fermentation zone for 5 days.

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

2.

4. The application according to claim 2, characterized in that, In S2, the composition of the secondary culture medium, based on dry matter, is 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 application according to claim 2, characterized in that: In S2, the koji loading per unit space in the shallow fermentation zone is 2.0-2.5 kg / m². 3 The air temperature is 30-35℃ and the relative humidity is 85-90%.

6. The application according to claim 2, characterized in that, In S3, the composition of the tertiary culture medium, on a dry matter basis, is 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 application according to claim 2, characterized in that: In S3, the koji loading per unit space in the shallow fermentation zone is 3.0-3.5 kg / m². 3 .

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

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