Ganoderma lucidum as well as solid-state fermentation process and application thereof in biological feed conversion of distiller's grains

Abstract

The invention discloses ganoderma lucidum, a solid-state fermentation process thereof and application of the ganoderma lucidum in biological feed conversion of distiller's grains. The ganoderma lucidum strain is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO: 66895, the preservation date is August 29, 2025, and the strain has extremely strong adaptability and degradation capability under the harsh conditions of complex fermentation substrate components, strong acidity, high fiber, high lignin and the like, and can effectively break a compact physicochemical barrier. And the nutritional and functional values of the fermented product are remarkably improved, the true protein content improvement rate is up to 39.39%, the soluble dietary fiber content improvement rate is up to 270.72%, and bioactive substances such as ganoderan and the like are successfully enriched. According to the invention, high-pollution wastes are efficiently converted into feed raw materials with high nutrition and functionality, so that the method has great economic and environment-friendly significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of Ganoderma lucidum, a solid-state fermentation process thereof and application thereof in conversion of distiller's grains into biological feed. BACKGROUND

[0002] In China, the supply of traditional protein feed resources such as soybean meal and fish meal is unstable and their prices fluctuate, which has restricted the sustainable development of the livestock industry. Therefore, the development of new and sustainable local protein feed resources has become a key issue that needs to be broken through in the industry.

[0003] The distiller's grains, a byproduct of the liquor-making industry, amount to tens of millions of tons per year, and its centralized disposal is a great pressure. For example, the leading enterprises such as Yibin Wuliangye, the fresh distiller's grains have high water content and chemical oxygen demand, and strong acidity. If they are directly discharged or left in the open air, they will cause water eutrophication, soil acidification and release of foul-smelling gases, posing a serious threat to the environment. The Luzhou-flavor distiller's grains are rich in nutrients such as protein, starch and minerals, but the rice hull accounts for more than 1 / 3 of the total, and the high content of lignocellulose forms a dense network structure, which seriously hinders the release and animal digestion and utilization of nutrients, and has low direct feeding value. The common treatment methods (such as physical pretreatment, chemical pretreatment and direct feeding after drying) cannot fundamentally solve the structural bottleneck, and have high energy consumption and low added value. Therefore, it is of great significance to realize the efficient and high-value utilization of distiller's grains as feed, for environmental protection and feed resource development.

[0004] Biotechnology has great potential in resource recycling and waste treatment. Microbial solid-state fermentation technology shows unique advantages due to its environmental friendliness and low cost. In the aspect of lignocellulose degradation, white-rot fungi are the only microbial group known to efficiently degrade lignin in nature. The secreted laccase and manganese peroxidase can open the structure of lignin, thereby greatly improving the accessibility of microorganisms and enzymes to the substrate. Ganoderma lucidum, as a white-rot fungus with medicinal and lignocellulose-degrading properties, not only has a complete lignocellulose-degrading enzyme system, but also can synthesize bioactive substances such as ganoderma polysaccharides and triterpenoids with immunomodulatory and antioxidant functions during the metabolic process, which has unique potential in the development of biological feed. Ganoderma lucidum

[0005] ​However, the application of existing white-rot fungi has obvious limitations: on the one hand, most strains have low enzyme activity and unstable degradation efficiency, and research is mostly focused on macro changes in components after fermentation, lacking of analyzing the degradation potential from the genomic level and deep understanding of the micro mechanism of multi-enzyme synergistic deconstruction of substrates, leading to lack of systematic guidance for process optimization; on the other hand, existing schemes mostly focus on single target degradation or nutritional component improvement, and have not been systematically applied to liquor vinasse fermentation by using Ganoderma lucidum to achieve the dual goals of efficient conversion of waste and development of feed resources. SUMMARY

[0006] Based on the above problems, the present application proposes to use Ganoderma lucidum to carry out solid-state fermentation on liquor vinasse, screen strains with high yield of lignocellulose-degrading enzymes, combine genomic analysis to determine the degradation potential, and explore the effect of fermenting fresh Wuliangye liquor vinasse, aiming to effectively deconstruct liquor vinasse and simultaneously achieve efficient biological treatment and high-value conversion of liquor vinasse, providing an innovative path for the resource utilization of industrial by-products.

[0007] The purpose of the present application is achieved by the following measures: A Ganoderma lucidum strain (Ganoderma lucidum 2G1-6) Ganoderma lucidum , preserved in the Guangdong Microbial Culture Collection Center, located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, with the preservation number GDMCC NO: 66895 and the preservation date August 29, 2025. The strain belongs to white-rot fungi and is named 2G1-6.

[0008] Efficient lignocellulose-degrading ability and efficient secondary metabolite (such as ganoderma polysaccharide) synthesis ability are often difficult to balance because the metabolic resources of the strain are limited. However, the Ganoderma lucidum 2G1-6 of the present application simultaneously exhibits high efficiency in both aspects, achieving the synergy of waste degradation, nutrient value-added, and function-endowed.

[0009] The above-mentioned Ganoderma lucidum in the application of treating liquor vinasse and / or preparing biological feed.

[0010] A method for treating liquor vinasse and / or preparing biological feed, which uses the above-mentioned Ganoderma lucidum to ferment liquor vinasse. Specifically, the method includes the following steps: Inoculate Ganoderma lucidum 2G1-6 on a solid culture medium and cultivate at 30°C under inversion, then take out the whole plate after the mycelium covers the entire plate, and cut the mycelium of the whole plate into blocks; Take liquor vinasse, adjust the moisture content to 70% and the pH to 6.0 with an inorganic salt solution, and sterilize at 121°C for 20 min; Inoculate the liquor vinasse with the blocks and cultivate at 30°C for 5-30 days.

[0011] A feed raw material prepared by the above-mentioned method.

[0012] An animal feed comprising the feed material described above.

[0013] The present application has the following beneficial effects: 1. The present application efficiently realizes the biological treatment and structure breaking of distiller's grains. The Ganoderma lucidum strain 2G1-6 provided by the present application can directly perform solid-state fermentation with distiller's grains as substrate, and has strong adaptability and degradation capacity. For example, in the face of the high-difficulty challenge of Luzhou-flavor distiller's grains, which has strong acidity, high fiber and high lignin, and the dense physical and chemical barrier formed by the cross-linking of fiber and lignin, which seriously hinders the growth and degradation of conventional microorganisms, the strain can grow vigorously and efficiently ferment in this harsh environment, has strong acid resistance and decomposition capacity for complex substrates, and the physical structure of the distiller's grains fermented by the strain becomes rough and loose from flat and dense, the fiber is broken and disordered, and the lignin and hemicellulose in the chemical structure are significantly degraded, thus fundamentally solving the core technical problem of low direct feeding value of distiller's grains caused by the physical and chemical barrier.

[0014] 2. The present application significantly improves the feed application value of the treatment product. The present application not only treats waste, but also converts it into high-value feed raw materials. After being treated by the strain and method of the present application, the nutritional value of the product is greatly improved: the true protein increase rate can be as high as 39.39%, and the increase of soluble dietary fiber is as high as 270.72%. This shows that the present application not only degrades anti-nutritional factors, but also significantly improves the nutritional composition and digestion and utilization rate of the product through the synthesis of bacterial protein and the transformation of complex carbohydrates, so that it becomes a high-quality protein feed raw material.

[0015] 3. The present application endows the treatment product with unique biological activity function. In addition to the improvement of nutritional ingredients, the present application successfully enriches the treatment product with bioactive substances such as Ganoderma lucidum polysaccharide (45-60 mg / g), total phenol (13-18 mg / g) and flavonoids (2-5 mg / g) with immune regulation and antioxidant functions through fermentation of Ganoderma lucidum. This makes the product feed raw material not only have high nutritional value, but also have the potential to improve the health level of animals, and has a unique market competitive advantage.

[0016] 4. The present application provides an industrialization path with both environmental protection and economic benefits. The present application directly uses high-pollution distiller's grains as the only fermentation substrate, and the process flow is simple and the conditions are mild, which effectively reduces the environmental governance cost, successfully converts an industrial by-product into a high-value biological feed raw material, realizes the recycling of waste resources, and provides a strain resource with significant competitive advantage and reliable process support for solving the shortage of feed resources and environmental pressure in China. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 . COG functional annotation of Ganoderma lucidum 2G1-6.

[0018] Figure 2 . KEGG functional annotation of Ganoderma lucidum 2G1-6.

[0019] Figure 3 . CAZyme annotation of Ganoderma lucidum 2G1-6.

[0020] Figure 4 . KEGG terpenoid backbone biosynthesis pathway of Ganoderma lucidum 2G1-6.

[0021] Figure 5 . KEGG amino sugar and nucleotide sugar metabolism pathway of Ganoderma lucidum 2G1-6.

[0022] Figure 6 . SEM images of fermented distiller's grains by Ganoderma lucidum 2G1-6: A. SEM micrograph of unfermented distiller's grains; B. SEM micrograph of distiller's grains fermented by Ganoderma lucidum 2G1-6 for 10 days; C. SEM micrograph of distiller's grains fermented by Ganoderma lucidum 2G1-6 for 20 days; D. SEM micrograph of distiller's grains fermented by Ganoderma lucidum 2G1-6 for 30 days.

[0023] Figure 7 . FTIR analysis of fermented distiller's grains by Ganoderma lucidum 2G1-6.

[0024] Figure 8 . XRD analysis of fermented distiller's grains by Ganoderma lucidum 2G1-6. DETAILED DESCRIPTION

[0025] The following examples of the experiment are further elaboration of the present application, not the limitation of the present application. The specific experimental conditions and methods are not specified in the following examples, and the experimental methods without specific conditions in the following examples are selected according to the conventional methods and conditions, or according to the instructions of the commodity.

[0026] In the present application, the various materials and reagents are the materials and reagents commonly used in the art, and can be obtained through conventional commercial channels.

[0027] Example 1, strain screening, identification and genome analysis

[0028] 1.1 Preliminary screening of white rot fungi The sample was collected from rotten branches and fungi in Yunnan. The surface of the collected fruiting body was disinfected with 75% alcohol, the fruiting body was torn open, and the yellow bean-sized tissue at the junction of the handle cover was taken with a scalpel and placed on a PDA plate. The plate was cultured in a 30°C constant temperature incubator until the mycelium was full. The mycelium was transferred several times until the mycelium in the plate became pure white. The white mycelium was inoculated into PDA medium, and when the mycelium covered the plate, the fungus cake was taken with a puncher and inoculated into MEA medium containing 0.04% guaiacol (malt powder 30 g / L, peptone 5 g / L, glucose 20 g / L, streptomycin sulfate 20 g / L, o-phenylphenol 0.06 g / L, agar 25 g / L), and cultured at 30°C for 5-6 days. The strain producing red-brown oxidation ring was the primary screening of white rot fungi.

[0029] 1.2 Rescreening of white rot fungi and molecular identification The method for measuring the activity of laccase was as follows: 50 μL of enzyme solution was mixed with 950 μL of citric acid-disodium hydrogen phosphate buffer to prepare 1 mmol / L ABTS solution. 1 mL of ABTS solution was placed in another centrifuge tube and preheated at 30°C for 3 min. Then the two solutions were mixed in one centrifuge tube. The mixture was stored at 30°C for 10 min, and the change in absorbance at 420 nm was measured by an enzyme marker. The unit of laccase activity (U) was defined as the amount of enzyme that oxidized 1 μmol of ABTS per minute.

[0030] The method for measuring the activity of cellulase was as follows: 80 μL of carboxymethyl cellulose sodium solution was added to a 1.5 mL centrifuge tube and preheated in a 37°C electric heating constant temperature water bath for 5 min. 80 μL of appropriately diluted enzyme solution was added, and citric acid-disodium hydrogen phosphate buffer (pH 5.5) was added to the blank control. The reaction was carried out at 37°C for 30 min, 200 μL of DNS solution was added to the centrifuge tube to terminate the reaction, and the metal bath was heated at 100°C for 5 min. The sample was cooled to room temperature with tap water, and the volume was adjusted to 1 mL with distilled water. The absorbance of the sample was measured at 540 nm by an enzyme marker, and the enzyme activity was calculated. The activity of cellulase was defined as: the amount of enzyme required to degrade 1 μmol of reducing sugar per minute at 37°C and pH 5.5.

[0031] The method for measuring xylanase activity is as follows: 0.8% xylan solution is prepared with citric acid-disodium hydrogen phosphate buffer at pH 5.5, 80 μL of the xylan solution is added to a 1.5 mL centrifuge tube, and the tube is preheated in a 37°C electric heating water bath for 5 min. 80 μL of the enzyme solution is added after appropriate dilution, and the blank control is added with citric acid-disodium hydrogen phosphate buffer (pH 5.5). The reaction is carried out at 37°C for 30 min, 200 μL of DNS solution is added to the centrifuge tube to terminate the reaction, the metal bath is cooled to room temperature with tap water, and the volume is adjusted to 1 mL with distilled water. The absorbance of the sample is measured at 540 nm wavelength with an enzyme marker, and the enzyme activity is calculated. The xylanase activity is defined as follows: the amount of enzyme required to degrade 1 μmol of reducing sugar per minute under the conditions of 37°C and pH 5.5 is 1 unit (U).

[0032] The strain with a large oxidation ring is inoculated on a PDA plate, and after the mycelium grows over the plate, a spore suspension is prepared by washing the plate with normal saline. The plate is inoculated with 5% inoculation amount in PDB medium, and the seed liquid is prepared by culturing at 30°C and 220 rpm for 2 days. The enzyme production medium (rice straw 5 g, wheat bran 5 g, (NH4)2SO4 3 g, yeast powder 2 g, and distilled water to 1 L) is inoculated with 10% inoculation amount, and the fermentation is carried out at 30°C and 220 rpm for 4 days. After centrifugation, the supernatant is detected for the activities of laccase, cellulase and xylanase. A strain producing red-brown oxidation ring is screened, which is named 2G1-6. After induction by the enzyme production medium, the activities of laccase, cellulase and xylanase are 2.88, 2.52 and 3.65 U / mL, respectively. The genomic DNA is extracted, and 18S rDNA primers (primer (NS1-F 5'-GTATCATATGCTTGTCTC-3', NS6-R 5'-GCATCACAGACCTGTTATTGCCTC-3') are used for amplification. The amplified product is sequenced and identified as Ganoderma lucidum. Ganoderma lucidum ).

[0033] 1.3 Genome analysis of Ganoderma lucidum 2G1-6 Ganoderma lucidum 2G1-6 was inoculated onto PDA solid medium and cultured upside down at 30°C until the hyphae covered the plate. Sterile physiological saline was added to the plate to prepare a suspension, which was then inoculated onto PDB medium and cultured at 28°C and 220 rpm for 2-3 days. The cells were centrifuged at 4°C and 5,000 rpm for 5 min, the supernatant was discarded, and the cells were retained. This process was repeated multiple times until sufficient cells were collected. The whole genome of the collected Ganoderma lucidum 2G1-6 cells was extracted using a fungal whole genome DNA extraction kit (Omega, USA). Sequencing was performed using a third-generation sequencing system (IIllumina PE). After sequencing and gene prediction, the results were aligned with databases (NR, Swiss-Prot, Pfam, COG, GO, CAZy) for functional annotation. Statistical analysis was performed on genes encoding carbohydrate-active enzymes (CAZymes) and probiotics in the Ganoderma lucidum 2G1-6 genome.

[0034] COG annotation classification statistics, such as Figure 1 As shown, the genes were divided into 24 functions. The function with the most genes was general function prediction only (475 genes), carbohydrate transport and metabolism (425 genes), followed by lipid transport and metabolism (322 genes), translation, ribosome structure and biosynthesis (291 genes), coenzyme transport and metabolism (288 genes), energy production and conversion (282 genes), and synthesis, transport and metabolism of secondary metabolites (281 genes). COG annotation results indicate that Ganoderma lucidum 2G1-6 has a strong function in carbohydrate transport and metabolism.

[0035] KEGG annotation classification statistics, such as Figure 2 As shown, KEGG annotated 9743 genes, which were assigned to 48 secondary signaling pathways in 6 primary signaling pathways. The primary signaling pathway with the most genes was metabolism, with 4058 genes. The secondary signaling pathways with the most annotated genes mainly included global and overview maps (1609), transport and catabolism (527), signal transduction (492), and carbohydrate metabolism (490).

[0036] like Figure 3 As shown, a total of 441 CAZymes were identified in the genome of Ganoderma lucidum 2G1-6, including 8 polysaccharide lyases (2%), 196 glycoside hydrolases (44%), 60 carbohydrate esterases (13%), 3 carbohydrate binding modules (1%), 61 glycosyltransferases (14%), and 113 auxiliary activities (26%), among which glycoside hydrolases were the most abundant.

[0037] Triterpenoids are one of the main active substances in Ganoderma lucidum, which are synthesized through the mevalonate pathway. In the terpenoid backbone biosynthesis (map00900) pathway of Ganoderma lucidum 2G1-6, there are a total of 98 genes Figure 4 The process of catalyzing the synthesis of mannans from ergosterol downstream of the mevalonate pathway is completed by cytochrome P450 family (CYPs), which contains various complex and unclear oxidation, reduction and acylation modification reactions. As shown in Table 1, in the genome of Ganoderma lucidum 2G1-6, 159 genes encoding CYPs were annotated. Exopolysaccharide is another important bioactive compound in Ganoderma lucidum, and its biosynthesis includes the synthesis of nucleotide-activated sugars, the connection and modification of sugar chains, and the secretion to the extracellular space. In the KEGG pathway of amino sugar and nucleotide sugar metabolism (map00520) of the genome of Ganoderma lucidum 2G1-6, genes related to the synthesis of UDP-Glc (glucose), UDP-L-Arb (arabinose), UDP-D-Xyl (xylose), UDP-GlcA (glucuronic acid), UDP-Gal (galactose), GDP-man (mannose), and GDP-D-Rha4N (rhamnose) were found Figure 5 The above results show that Ganoderma lucidum 2G1-6 has great potential in the synthesis of triterpenoids (ganoderic acids) and exopolysaccharides.

[0038]

[0039] Example 2, Enzyme production characteristics of Ganoderma lucidum 2G1-6 and its effect on fermentation of distiller's grains 1. Characteristics of Ganoderma lucidum 2G1-6 solid-state fermentation for producing lignocellulose-degrading enzymes

[0040] Method for measuring enzyme activity in solid-state fermentation substrate: Take 2 g of solid-state substrate after solid-state fermentation of Ganoderma lucidum 2G1-6, add an appropriate amount of distilled water, shake at 28°C, 200 rpm for 1 h, centrifuge at 4°C, 5,000 rpm for 10 min, collect the supernatant, and obtain the crude enzyme solution. The laccase, cellulase and xylanase activities are measured by the method in Example 1. At the same time, weigh 1 g of solid-state substrate in an electric thermostatic oven until the weight is constant, then weigh, calculate the moisture content, and calculate the enzyme activity (U / g) in 1 g of dry substrate.

[0041] The Ganoderma lucidum strain 2G1-6 was inoculated on PDA solid culture medium, and was taken out after being cultured at 30°C for 5 days. 20 mL of sterile normal saline was added to the solid culture medium to prepare a spore suspension. The solid-state fermentation enzyme production medium was prepared by using 60% wheat bran, 40% soybean meal, 0.60% (HN4)2SO4, 0.80% CuSO4·5H2O, an initial pH of 7.0, and a water content of 67.5%. The spore suspension was inoculated at a ratio of 12.5% (v / w), and was cultured in a constant-temperature incubator at 33°C for 9 days. After the fermentation was completed, the activities of laccase, cellulase, and xylanase in the medium were detected. The results showed that after the solid-state fermentation of the Ganoderma lucidum strain 2G1-6, the activities of laccase, cellulase, and xylanase were 960.21, 314.31, and 570.83 U / g, respectively, and had high activities.

[0042] 2. Effect of solid-state fermentation of the Ganoderma lucidum strain 2G1-6 on distiller's grains

[0043] Fresh distiller's grains from the production line of Wuliangye Distillery were used as the only component of the fermentation medium, and were inoculated with the Ganoderma lucidum strain 2G1-6 for fermentation. The distiller's grains had a complex composition and were extremely difficult to process. Specifically, the water content was higher than 56%, the pH value was in the acidic range of 3.5-4.0, the rice hull accounted for more than one-third, the neutral detergent fiber content was 40-43%, the acid detergent fiber content was 35-38%, and the lignin content was 11-13%. The cellulose and lignin were cross-linked with each other to form a dense barrier, which seriously hindered the contact and degradation of microorganisms and enzymes.

[0044] The treatment method is as follows: Ganoderma lucidum 2G1-6 is inoculated on PDA solid medium and cultured at 30°C under inversion, and after the mycelium grows over the entire plate (90 mm), the mycelium of the entire plate is cut into several 5 mm x 5 mm size blocks. 42 portions of distiller's grains, each 30 g (calculated as dry matter), are weighed, and an inorganic salt solution (20 g of proteose peptone, 20 g of (NH4)2SO4, 4 g of CuSO4·5H2O, 1.5 g of KH2PO4, 3 g of MgSO4, 0.3 g of CaCl2, 2 mL of Tween-80, distilled water to 1 L, and 1 mL of trace element mixture (containing 0.005 g of FeSO4, 0.002 g of MnSO4, and 0.0016 g of ZnSO4·7H2O per mL)) is used to adjust the moisture content to 70% and the pH to 6.0, and high-pressure sterilization is performed at 121°C for 20 min. Each portion of distiller's grains is inoculated with 1 / 8 of a 5 mm x 5 mm size block, and static culture is performed at 30°C, with 6 replicates for each fermentation stage. Every 5 days, 6 distiller's grain samples are randomly taken out, of which 3 are used for determination of lignocellulose-degrading enzyme activity during fermentation, and the other 3 are dried in a 65°C air-drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.) to constant weight for subsequent determination of dry matter loss and related nutritional components (crude protein, true protein, acid-soluble protein, and soluble dietary fiber).

[0045] 2.1 Changes in lignocellulose-degrading enzyme activity, nutritional components, and active substances of Ganoderma lucidum 2G1-6 during solid-state fermentation of distiller's grains

[0046] The determination method of β-glucosidase activity is as follows: 100 µL of PNPG and 100 µL of crude enzyme solution are added to a 1.5 mL centrifuge tube containing 5 mmol / L of p-nitrophenol-β-D-cellobioside (PNPG) prepared using a citric acid-disodium hydrogen phosphate buffer with a pH of 5.0, and the blank control group only contains PNPG. The reaction is performed in a 40°C electric heating constant-temperature water bath for 30 min, and then 100 µL of 1 mol / L Na2CO3 solution is immediately added to terminate the reaction. Finally, 100 µL of crude enzyme solution is added to the blank control group, and 200 µL of the sample is taken for determination of absorbance at a wavelength of 410 nm using an enzyme marker, and enzyme activity is calculated. The activity of β-glucosidase is defined as the amount of enzyme required to release 1 µmol of p-nitrophenol from the substrate per minute at 40°C and pH 5.0 (U).

[0047] Total phenol determination method: the sample was extracted with 50%-70% methanol or ethanol (50°C, 30 min), centrifuged (4,000 rpm, 10 min) to take the supernatant. Repeat extraction 2 times, combine the supernatant, and dilute to a certain volume (such as 25 mL). 20 μL of sample extract (or standard solution) 100 μL of 10-fold diluted Folin-Ciocalteu reagent (freshly prepared), room temperature, light protection, 5 min. Add 150 μL of 7% Na2CO3 solution, mix well. Incubate at 50°C for 10 min (or room temperature for 60 min). After cooling to room temperature, the absorbance at 765 nm was measured by a microplate reader (blank wells were solvent controls). The standard curve was prepared using gallic acid standards (0-100 μg / mL), and the absorbance at 765 nm was measured by a microplate reader. The linear equation was fitted. The total phenol content was calculated according to the standard curve.

[0048] The content of flavonoids was determined according to the instructions of the kit (Solabio, BC1335).

[0049] Ganoderma lucidum polysaccharide determination method: the sample (0.5 g) was mixed with distilled water (5 mL), then 20 mL of anhydrous ethanol was added, and ultrasonic extraction was performed for 30 min, followed by centrifugation (4,000 rpm, 5 min). The precipitate was washed with 80% ethanol solution, and distilled water was added at a ratio of 1:20. After boiling in a water bath for 2 h, it was cooled, filtered, and the supernatant was transferred to a 100 mL volumetric flask and diluted to volume. The extract (1 mL), phenol solution (1 mL, 5%) and concentrated sulfuric acid (5 mL) were mixed and allowed to stand at room temperature for 20 min. The absorbance was measured at 490 nm. A standard curve was prepared using glucose as a standard. The polysaccharide content was calculated according to the standard curve.

[0050]

[0051] Samples at different fermentation time points were taken, and the enzyme activities of laccase, cellulase, xylanase and β-glucosidase were determined. The results are shown in Table 2. During the fermentation process, the enzyme activities of laccase, cellulase, xylanase and β-glucosidase all showed a trend of first increasing and then fluctuating. Among them, the laccase activity reached a peak value (231.23 U / g) on the 15th day; the activities of cellulase, xylanase and β-glucosidase all reached peak values on the 20th day, which were 13.31, 538.81 and 24921.75 U / g, respectively. After 20 days of fermentation, the enzyme activities showed a fluctuating or decreasing trend.

[0052] Meanwhile, the contents of crude protein, true protein, acid-soluble protein and soluble dietary fiber in the distiller's grains fermented for different time were determined, and the results were shown in Table 3. After the fermentation by the Ganoderma lucidum strain 2G1-6, the contents of crude protein, true protein, acid-soluble protein and soluble dietary fiber in the distiller's grains were significantly improved. Among them, the highest increase rate of true protein reached the peak value (39.39%) at the 10th day of fermentation, and then gradually decreased with the extension of fermentation time; the increase rate of crude protein reached the highest value (27.45%) at the 30th day of fermentation; while the contents of acid-soluble protein and soluble dietary fiber continuously increased with the extension of fermentation time, and the highest increase rates were 41.94% and 270.72%, respectively. The changes of the contents of total phenol, flavonoids and Ganoderma lucidum polysaccharide in the distiller's grains after the fermentation by Ganoderma lucidum were determined. The results showed that the contents of total phenol, flavonoids and Ganoderma lucidum polysaccharide in the distiller's grains after the fermentation were 13-18, 2-5 and 45-60 mg / g, respectively.

[0053]

[0054] The above results showed that the Ganoderma lucidum strain 2G1-6 secreted various high-activity lignocellulose-degrading enzymes and active substances during the fermentation of the distiller's grains, significantly improved the contents of protein and soluble dietary fiber in the distiller's grains, and effectively improved the nutritional composition thereof. Therefore, the strain had significant application potential in the conversion of the distiller's grains into high-nutritional-value feed.

[0055] 2.2 Multi-dimensional characterization of the effect of the Ganoderma lucidum strain 2G1-6 on the lignocellulose structure of the distiller's grains subjected to solid-state fermentation

[0056] The untreated and fermented distiller's grains by Ganoderma lucidum for different time were dried, and then subjected to scanning electron microscopy (S-3400N, Hitachi, Japan), Fourier transform infrared spectrometer (Nicolet iN10, Thermo Fisher Scientific, USA), X-ray diffractometer (X Pert Powder, Netherlands, PANalytical) and thermal gravimetric analyzer (TGA2, Switzerland, Mettler) for the analysis of the physicochemical properties of the samples.

[0057] The samples for scanning electron microscopy (SEM) analysis were taken at a magnification of 1,000 x after gold plating. The scanning electron microscope could directly observe the changes in the physical surface structure of the materials. The results were shown in Figure 6 A, the surface of the distiller's grains before fermentation was smooth and flat, dense, the fiber bundle was neat, regular, and stretched naturally. After the fermentation by Ganoderma lucidum (6B, 6C and 6D), the integrity of the sample surface was partially destroyed, the surface was rough, the structure was loose, the fabric was loose, and the fibers were broken and disordered. Figure 6

[0058] ​The samples for Fourier transform infrared spectroscopy (FTIR) analysis were prepared by KBr method and scanned by Fourier transform infrared spectroscopy at a resolution of 400-4,000 cm −1 The results are shown in Figure 7 The vibration signals of functional groups of the distiller's grains fermented by the red ganoderma changed, part of the lignin and hemicellulose were removed, and the exposure area and proportion of cellulose were increased.

[0059] The samples for X-ray diffraction (XRD) analysis were analyzed under the following conditions: 2θ of 10°-80°, scanning speed of 10° min -1 , step of 0.02°, accelerating voltage of 40 kV, current of 40 mV, and crystallinity was calculated according to the diffraction intensity. The results are shown in Figure 8 Compared with the distiller's grains before fermentation by the red ganoderma, the diffraction peak intensity related to the crystalline and amorphous regions of cellulose changed after fermentation by 2G1-6, and the degree of change increased with the extension of fermentation time. The above changes indicated that the fermentation by 2G1-6 removed the amorphous structure of the distiller's grains, decomposed the amorphous components such as lignin and non-cellulosic polysaccharides, and increased the exposure degree of cellulose, which was more conducive to the degradation of cellulose.

[0060] The above multi-dimensional structure characterization consistently indicated that the strain 2G1-6 of the red ganoderma could efficiently destroy the physical and chemical structures of lignocellulose in the distiller's grains, and significantly degrade the components of lignin, hemicellulose and cellulose. This strain not only greatly improved the bioconversion efficiency of the distiller's grains, but also effectively promoted the resource development of the distiller's grains into high-value protein feed.

Claims

1. A strain of Ganoderma lucidum, characterized in that: It is preserved in Guangdong Microbial Culture Collection Center with the preservation number of GDMCC NO: 66895 and the preservation date of August 29, 2025.

2. The application of the Ganoderma lucidum strain in treating liquor dregs and / or preparing biological feed according to claim 1.

3. A method of treating white spirit lees and / or producing bio-feed, characterized in that: The Ganoderma lucidum strain according to claim 1 is used for solid-state fermentation of liquor dregs.

4. The method of claim 3, wherein: The method comprises the following steps: The Ganoderma lucidum strain according to claim 1 is inoculated on a solid culture medium and cultured at 30°C in an inverted manner. After the mycelium grows over the entire culture dish, the mycelium of the entire culture dish is cut into blocks. Liquor dregs are taken and adjusted to a water content of 70% and a pH of 6.0 by using an inorganic salt solution. The liquor dregs are sterilized at 121°C for 20 min. The liquor dregs are inoculated with the blocks and cultured at 30°C for 5-30 days.

5. A feed raw material prepared by the method according to claim 3 or 4.

6. An animal feed, characterized by: It comprises the feed raw material according to claim 5.

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