Agrocybe cylindracea solid state fermentation wheat aleurone layer polysaccharide with anti-oxidation effect and preparation method thereof

Abstract

The invention belongs to the technical field of food biotechnology and functional polysaccharide, and particularly relates to agrocybe cylindracea solid-state fermentation wheat aleurone layer polysaccharide with an anti-oxidation effect and a preparation method of the agrocybe cylindracea solid-state fermentation wheat aleurone layer polysaccharide. The method comprises the following steps: carrying out solid-state fermentation on a wheat aleurone layer by utilizing agrocybe cylindracea, carrying out crude extraction on polysaccharide by utilizing an ultrasonic-assisted hot water extraction method, and purifying the crude polysaccharide through anion exchange column chromatography to obtain the purified wheat aleurone layer polysaccharide. The polysaccharide is composed of fucose, arabinose, galactose, glucose, xylose and mannose, the molar percentages of the fucose, the arabinose, the galactose, the glucose, the xylose and the mannose are respectively 0.77%, 29.69%, 4.88%, 3.09%, 57.70% and 3.87%, and the average molecular weight is 6.00 * 10 <-7.00 > * 10 < Da >, preferably 6.39 * 10 < Da >. The prepared fermented wheat aleurone layer polysaccharide can be used for improving oxidative stress.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and functional polysaccharide technology, specifically to a polysaccharide from solid-state fermented wheat aleurone layer of *Agrocybe aegerita* with antioxidant properties and its preparation method. Background Technology

[0002] Oxidative stress is a key factor contributing to aging and various chronic diseases such as Alzheimer's and diabetes. The search for highly effective and safe natural antioxidants is currently a hot research topic. Polysaccharides have attracted much attention due to their diverse biological activities and good safety profile; however, their biological activity is highly dependent on their specific structural characteristics, such as monosaccharide composition, molecular weight, and glycosidic bond configuration.

[0003] The aleurone layer, the innermost layer of wheat bran, is rich in dietary fiber and bioactive components such as arabinoxylan and β-glucan. However, in traditional milling processes, the aleurone layer is often discarded along with the bran, resulting in resource waste. The complex cell wall structure of the aleurone layer leads to low yields and unsatisfactory polysaccharide extraction when extracted directly. Current extraction methods mostly rely on physical cell disruption or chemical solvents, which have drawbacks such as high energy consumption, potential damage to the natural polysaccharide structure, or solvent residue.

[0004] Solid-state fermentation is an environmentally friendly bioprocessing technology that utilizes the growth, metabolism, and secreted enzyme systems of microorganisms to gently and directionally degrade and transform substrates, releasing and modifying active ingredients. *Agrocybe aegerita*, a fungus used for both medicinal and edible purposes, secretes abundant lignocellulose-degrading enzymes such as cellulase and hemicellulase, which are beneficial for the release and biotransformation of polysaccharides from wheat aleurone layers. However, there are currently no reports on using *Agrocybe aegerita* solid-state fermentation technology specifically for preparing wheat aleurone layer polysaccharides with specific structures and excellent antioxidant activity. Summary of the Invention

[0005] Based on the shortcomings of existing technologies, the purpose of this invention is to develop a green and efficient method for preparing polysaccharides, and to obtain a wheat aleurone layer polysaccharide with novel structural features and significant antioxidant activity.

[0006] This invention provides a polysaccharide from the solid-state fermentation of wheat aleurone layer of *Agrocybe aegerita* and its preparation method. The polysaccharide is composed of fucose, arabinose, galactose, glucose, xylose, and mannose, with molar percentages of 0.77%, 29.69%, 4.88%, 3.09%, 57.70%, and 3.87%, respectively, and an average molecular weight of 6.00 × 10⁻⁶. 3 - 7.00×10 3 Da, preferably 6.39 × 10 3 Da has the structure shown in equation (1): .

[0007] The specific technical solution for preparing the polysaccharide from the solid-state fermentation of wheat aleurone layer of *Agrocybe aegerita* is as follows: (1) Wheat grains are first dehulled to 4-8% using a dehulling machine, and then separated into aleurone layers using traditional milling methods; (2) The wheat aleurone layer and deionized water were mixed evenly in a ratio of 1:1 to 1:2 and sterilized at 121℃ for 20 min to obtain the solid fermentation medium of wheat aleurone layer. (3) Under sterile conditions, the activated tea tree mushroom strain was inoculated into the wheat aleurone layer solid fermentation medium prepared in step (2), and cultured at 26°C in the dark for 9-18 days to obtain the fermentation substrate; (4) After fermentation, under aseptic conditions, deionized water is added to the fermentation substrate obtained in step (3) to carry out enzymatic hydrolysis reaction and obtain enzymatic hydrolysis mixture; (5) The enzymatic hydrolysis mixture obtained in step (4) is subjected to ultrasonic-assisted hot water extraction, and the extract is collected by centrifugation. The above steps are repeated to extract the filter residue after centrifugation again. The supernatants obtained from the two centrifugations are combined to obtain the polysaccharide extract. (6) The combined extract obtained in step (5) is subjected to starch removal and protein removal treatment in sequence. Then, 3-4 times the volume of ethanol is added to the extract obtained after treatment for alcohol precipitation. The precipitate is collected and freeze-dried to obtain crude polysaccharide. (7) The crude polysaccharide obtained in step (6) is reconstituted, purified by anion exchange chromatography column, and the eluent of the target component is collected. After concentration, dialysis and freeze-drying, the purified tea tree mushroom solid fermentation wheat aleurone layer polysaccharide is obtained.

[0008] As a further description of the above technical solution, the method for preparing the wheat aleurone layer in step (1) is as follows: a certain amount of cleaned wheat grains are weighed, 2% of the weight of the wheat grains is added to water for pre-peeling conditioning, the mixture is quickly mixed evenly, and after standing for 30 seconds, it is sent into the peeling combination machine. By controlling the tightness of the valve, wheat grains with a peeling rate of 4-8% are obtained. Then, the peeled wheat grains are processed by the traditional milling method, and the bran component obtained is the wheat aleurone layer.

[0009] As a further description of the above technical solution, the method for obtaining the activated tea tree mushroom strain in step (3) is as follows: inoculate the tea tree mushroom into a PDA solid plate culture medium and culture it under a constant temperature of 26°C until the mycelium completely covers the surface of the plate. After activation and subculturing for more than 3 generations, it can be used.

[0010] As a further description of the above technical solution, the step (3) of inoculating the tea tree mushroom strain into the wheat aleurone layer solid fermentation medium is as follows: select activated tea tree mushroom strains in good growth condition, punch holes at the edge of the mycelium to obtain a 15mm diameter mycelium block, pick up the mycelium block with an inoculation needle, and inoculate the mycelium downwards evenly onto the aleurone layer culture medium, inoculating 5 mycelium blocks per 100g of culture medium.

[0011] As a further description of the above technical solution, the warm enzymatic hydrolysis reaction step in step (4) is to add deionized water to the fermentation substrate obtained in step (3) at a ratio of 1:5-1:15 (w / v), mix evenly, and then transfer it to a soymilk maker and stir for 40 s to make the sample into a homogenous paste.

[0012] As a further description of the above technical solution, the incubation enzymatic hydrolysis reaction in step (4) is carried out at a temperature of 40-60℃ for 24 hours, with a natural pH.

[0013] As a further description of the above technical solution, the conditions for ultrasonic-assisted hot water extraction in step (5) are: ultrasonic extraction for 1 h at a power of 50-200 W in a water bath at 40-60℃, with continuous stirring during the extraction process.

[0014] As a further description of the above technical solution, the method for removing starch and protein in step (6) is as follows: a small amount of calcium chloride (to improve the heat resistance of the enzyme) is added to the polysaccharide extract, the pH of the extract is adjusted to 6.0 with 1 mol / L HCl, the temperature is raised to 70℃ and then 0.6% (w / v) of high-temperature α-amylase is added, the reaction is stirred at a constant temperature for 3 h, then the temperature is lowered to 40℃, the pH of the extract is adjusted to 4.6 with 1 mol / L HCl, 0.48% (w / v) of saccharifying enzyme is added, and the mixture is incubated at 40℃ overnight to remove starch; then the pH is adjusted back to 7.0 with 1 mol / L NaOH, 0.3% (w / v) of alkaline protease is added, and the mixture is reacted at 50℃ for 3 h to remove protein; finally, the enzyme is inactivated by boiling water bath for 10 min, and the supernatant is obtained by centrifugation.

[0015] As a further description of the above technical solution, the specific method for polysaccharide separation and purification in step (7) is as follows: the crude polysaccharide obtained in step (6) is dissolved in water at a ratio of 1:100 (w / v), filtered through a 0.45 μm filter membrane, and then uniformly loaded onto an anion exchange chromatography column. Gradient elution is performed sequentially with 0, 0.1, 0.2, 0.3, and 0.5 mol / L NaCl solutions. The eluent of the target component is collected, and finally concentrated under reduced pressure and freeze-dried to obtain the purified polysaccharide from the solid-state fermentation of wheat aleurone layer of *Agrocybe aegerita*.

[0016] Preferably, the wheat grain dehulling rate in step (1) is 6%.

[0017] Preferably, the mass ratio of wheat aleurone powder to water in step (2) is 1:1.5, and the pH is natural.

[0018] Preferably, the solid-state fermentation time in step (3) is 12 days.

[0019] Preferably, the mass ratio of the fermentation substrate to deionized water in step (4) is 1:10, and the temperature of the enzymatic hydrolysis reaction in the warm bath is 50°C.

[0020] Preferably, the ultrasonic extraction conditions in step (5) are 50°C and 100 W.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least the following: (1) For the first time, tea tree mushrooms were used to carry out solid-state fermentation of wheat aleurone layer. The process was green and mild, and the polysaccharide extraction rate was significantly improved compared with unfermented raw materials. (2) A novel wheat aleurone layer polysaccharide with a low molecular weight, composed of fucose, arabinose, galactose, glucose, xylose and mannose, was obtained. Its purity is high, reaching more than 83%, and the impurity content is low. (3) The solid-state fermented wheat aleurone layer polysaccharide prepared by the present invention exhibits significant antioxidant activity in cell models, which can effectively alleviate oxidative stress damage and has the potential to be developed into a natural antioxidant. Attached Figure Description

[0022] Figure 1 This is the anion exchange column elution curve of the polysaccharide layer in the solid-state fermentation of wheat aleurone from *Agrocybe aegerita* in this embodiment of the invention; Figure 2 This is an ion chromatogram of polysaccharides from the solid-state fermentation of wheat aleurone layer in an embodiment of the present invention; Figure 3 This is a gel permeation chromatogram of polysaccharides from the solid-state fermentation of wheat aleurone layer in an embodiment of the present invention; Figure 4 This is the microstructure of the polysaccharide in the solid-state fermentation layer of wheat aleurone from *Agrocybe aegerita* in this embodiment of the invention. Figure 5 This invention illustrates the effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on the survival rate of HepG2 cells. Figure 6 This invention illustrates the effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on H2O2-induced survival of HepG2 cells. Figure 7 This invention illustrates the effect of solid-state fermentation of wheat aleurone layer polysaccharides from *Agrocybe aegerita* on intracellular mitochondrial membrane potential levels. Figure 8This invention illustrates the effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on H2O2-induced intracellular ROS levels in HepG2 cells. Figure 9 This invention illustrates the effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on H2O2-induced intracellular MDA content in HepG2 cells. Figure 10 This invention relates to the effect of polysaccharides from solid-state fermented wheat aleurone layer of *Agrocybe aegerita* on H2O2-induced intracellular antioxidant enzyme activity in HepG2 cells. Detailed Implementation

[0023] To better understand the present invention, the present invention will be further described and illustrated below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0024] Unless otherwise specified, all reagents used in the following examples are commercially available.

[0025] Example 1: Preparation of a polysaccharide from a solid-state fermented wheat aleurone layer of *Agrocybe aegerita* (tea tree mushroom). (1) Weigh the cleaned wheat grains, add 2% water by weight of wheat for pre-peeling conditioning, mix quickly and evenly, let stand for 30 seconds and then send to the peeling combination machine. By controlling the tightness of the valve, wheat grains with a peeling rate of 6% are obtained. Then, the peeled wheat grains are processed by traditional milling methods to obtain the bran component, which is the wheat aleurone layer.

[0026] (2) Weigh 100 g of wheat aleurone layer and mix it evenly with 150 g of deionized water. The pH is natural and there are no other auxiliary materials or additives. Sterilize at 121℃ for 20 min to obtain solid fermentation medium for wheat aleurone layer.

[0027] (3) Under sterile conditions, select activated tea tree mushroom strains in good growth condition, punch holes at the edge of the mycelium to obtain mycelial blocks with a diameter of 15 mm, pick up the mycelial blocks with an inoculation needle, and inoculate the mycelium downwards evenly on the aleurone layer culture medium. Inoculate 5 mycelial blocks for every 100 g of fermentation substrate, and culture at 26℃ for 12 days to obtain fermentation substrate.

[0028] (4) After fermentation, under sterile conditions, 2.5 kg of deionized water was added to 250 g of fermentation substrate, mixed evenly, and then transferred to a soymilk maker and stirred for 40 s to make the sample homogenous. The obtained sample was then placed in a constant temperature shaker (50℃, 150 r / min) and the endogenous extracellular enzymes produced by the tea tree mushroom during fermentation were used for incubation and enzymatic hydrolysis. The pH was natural throughout the process, which lasted for 24 h to obtain the enzymatic hydrolysate mixture.

[0029] (5) The obtained enzymatic fermentation substrate was ultrasonically extracted for 60 min at 100 W in a 50℃ water bath. During the extraction process, the mixture was continuously stirred, and the extract was collected by centrifugation. The above steps were repeated to extract the filter residue after centrifugation, and the polysaccharide extracts obtained from the two centrifugations were combined.

[0030] (6) Add 0.6 g of calcium chloride (to improve the heat resistance of the enzyme) to 5000 mL of extract, adjust the pH of the extract to 6.0 with 1 mol / L HCl, heat to 70℃ and add 30 mL of high-temperature α-amylase, stir at constant temperature for 3 h, then cool to 40℃, adjust the pH of the extract to 4.6 with 1 mol / L HCl, add 24 mL of saccharifying enzyme, and incubate at 40℃ overnight to remove starch; then adjust the pH back to 7.0 with 1 mol / L NaOH, add 15 mL of alkaline protease, and react at 50℃ for 3 h to remove protein; finally, inactivate the enzyme in a boiling water bath for 10 min, centrifuge and collect the supernatant. Then add 4 times the volume of ethanol to the treated extract for alcohol precipitation, collect the precipitate, and freeze-dry to obtain crude polysaccharide.

[0031] (7) Dissolve 0.1 g of crude polysaccharide in 10 mL of deionized water, filter through a 0.45 μm filter membrane, and load the sample onto an anion exchange chromatography column at a constant speed. Elute with gradients of 0, 0.1, 0.2, 0.3 and 0.5 mol / L NaCl solutions, respectively. Combine the polysaccharide eluents under the same NaCl elution gradient, concentrate under reduced pressure and freeze dry to obtain polysaccharide from the solid-state fermentation of wheat aleurone layer of mushroom.

[0032] Experimental Example 1: Isolation and purification of polysaccharides from wheat aleurone layer of solid-state fermented *Agrocybe aegerita* Following the method in step (7) of Example 1, the crude polysaccharide obtained in step (6) of Example 1 was separated using a DEAE-52 anion exchange column. The elution curve is shown below. Figure 1 As shown. Three main elution fractions were obtained, among which the neutral polysaccharide (FALP-1) eluted with 0 M NaCl solution had the highest yield and symmetrical peak shape, and was used for subsequent experiments.

[0033] Experiment Example 2: Structural Characterization of Polysaccharides from Wheat Aleurone Layers Fermented from Tea Tree Mushrooms (1) Monosaccharide composition of polysaccharides in wheat aleurone layer from solid-state fermentation of tea tree mushroom like Figure 2 As shown, FALP-1 is composed of fucose, arabinose, galactose, glucose, xylose, and mannose, with molar percentages of 0.77%, 29.69%, 4.88%, 3.09%, 57.70%, and 3.87%, respectively. Xylose and arabinose are the main monosaccharides, accounting for over 60%. The A / X value of 0.51 indicates that FALP-1 is a moderately branched polysaccharide with good solubility.

[0034] (2) Molecular weight of polysaccharides in solid-state fermented wheat aleurone layer of *Tea Tree Mushroom* Gel permeation chromatography (GPC) Figure 3 The single symmetrical peak indicates that FALP-1 is a homogeneous component with an average molecular weight of 6.39 × 10³ Da and a polydispersity index (Mw / Mn) of 2.09.

[0035] (3) Methylation analysis of polysaccharides in wheat aleurone layer from solid-state fermentation of Agrocybe aegerita. As shown in Table 1, FALP-1 contains 56.0% xylose residues, of which 5.2% are terminal residues and 33.5% are unsubstituted residues. Based on these results, FALP-1 is preliminarily identified as a 1,4-linked xylose compound. p Xyl 1,3,4-connected p Xyl connected to 1,2,3,4- p The main chain is composed of arabinoxylan. The arabinose residues are primarily t-Arabinose. f 1,2-Ara f 1,3-Ara f 1,5-Ara f 1,2,5-Ara f and 1,2,3,5-Ara f The FALP-1 residues were present in the form of arabinose, accounting for 27.4% of the total sugar residues, indicating that FALP-1 was mainly replaced by arabinose residues. In addition, small amounts of fucose (0.352%), galactose (5.276%), glucose (6.280%) and mannose (4.680%) residues were also detected.

[0036] (4) NMR analysis of polysaccharides from solid-state fermentation of wheat aleurone layer of *Agrocybe aegerita* FALP-1 1 The 1H NMR spectrum showed eight terminal hydrogen signal peaks at δH values ​​of 5.27, 5.23, 5.19, 4.63, 4.59, 4.59, 4.48, and 4.46. The corresponding peaks were determined using the correlation signals in the HSQC spectrum. 13 The relevant carbon signals in the C10 NMR spectra were δC 108.14, 108.66, 91.96, 99.83, 101.79, 96.45, 101.59, and 101.17. Based on these signals, the sugar residue fragments were numbered A, B, C, D, E, F, G, and H (see Table 2). Combined with... 1 H NMR, 13 C NMR, 1 H- 1 H COSY、 1 H-13 C HMBC, 1 H- 13 Based on the C HSQC spectrum, AH is inferred to be t-α-L-Ara. f t-α-L-Ara f α-D-Glc p 1,2,3,4-β-D-Xyl p t-β-D-Xyl p t-β-D-Glc p 1,4-β-D-Xyl p 1,3,4-β-D-Xyl p .

[0037] Based on the NMR spectrum and methylation results, FALP-1 was determined to be in the form of →1)-β-D-Xyl p -(4→1)-β-D-Xyl p -(4→ is the main sugar chain structure, with t-Ara at the C2 and C3 positions.) f Substitution. The specific molecular structure is shown in formula (1).

[0038] (5) Morphological and structural analysis of polysaccharides in wheat aleurone layer from solid-state fermentation of Agrocybe aegerita. Figure 4 shows a scanning electron microscope image of FALP-1, revealing a honeycomb-like porous framework with dense pores, accompanied by a small number of irregular blocky and rod-shaped structures. Further magnification to 10000× exposes a rough morphology on the polysaccharide surface, specifically characterized by dense pits and raised structures.

[0039] Experiment Example 3: Antioxidant Activity Test of Polysaccharides from Solid-State Fermented Wheat Aleurone Layer of *Agrocybe aegerita* (1) The toxic effect of solid-state fermented wheat aleurone layer polysaccharide on HepG2 cells of Agrocybe aegerita. The cell viability of cells co-cultured with culture media containing different concentrations of FALP-1 was determined using the CCK-8 assay, as shown in Figure 5. Within the concentration range of 25–400 μg / mL, the cell viability was significantly higher than that of the control group, demonstrating a clear cell growth-promoting effect. When the FALP-1 concentration exceeded 200 μg / mL, the cell viability began to decrease. These results indicate that FALP-1 is not toxic to cells within this concentration range.

[0040] (2) Protective effect of solid-state fermented wheat aleurone layer polysaccharide on H2O2-induced oxidative damage in HepG2 cells. As shown in Figure 6, compared with the H2O2-induced oxidative damage model group, low, medium, and high doses of FALP-1 significantly improved cell survival rates, reaching 51.73%, 61.54%, and 71.40%, respectively. The results indicate that within the concentration range of 25–400 μg / mL, the protective effect of FALP-1 against oxidative damage increases in a dose-dependent manner with increasing concentration.

[0041] (3) Effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on H2O2-induced intracellular mitochondrial membrane potential in HepG2 cells External stimuli generate highly reactive free radicals within cells, damaging mitochondria and altering their membrane potential. JC-1 is a cationic lipid fluorescent dye used to detect changes in mitochondrial transmembrane potential. Its principle is based on changes in the mitochondrial membrane potential-dependent aggregation state. JC-1 can cross the normal cell membrane, accumulating intracellularly as monomers and emitting green fluorescence. In normal healthy cells, the mitochondrial membrane potential is polar; JC-1 is rapidly taken up into the mitochondria in a polarity-dependent manner, forming polymers and emitting red fluorescence.

[0042] Under a fluorescence microscope, normal cells appear highly red and green under a two-color filter, and yellow-green under the same filter. As shown in Figure 7, the cells in the oxidative damage model group showed weak yellow-green fluorescence, indicating a decrease in mitochondrial membrane potential. However, after treatment with FALP-1, the cells in the low, medium, and high dose polysaccharide treatment groups showed enhanced yellow-green fluorescence and significantly increased mitochondrial membrane potential, indicating that FALP-1 can effectively alleviate cellular oxidative damage.

[0043] (4) Effect of solid-state fermentation of wheat aleurone layer polysaccharide from *Agrocybe aegerita* on H2O2-induced intracellular ROS levels in HepG2 cells ROS (Reactive OS) are involved in cell growth, proliferation, development, differentiation, senescence, apoptosis, and many physiological and pathological processes. Generally, elevated intracellular ROS is a marker of apoptosis. Excessive ROS levels may lead to oxidative damage, resulting in biomembrane damage or other diseases. As shown in Figure 8, compared with the blank control group, the ROS content in the H2O2-induced oxidative damage model group increased by 75.89%. After treatment with 25, 100, and 400 μg / mL FALP-1, the intracellular ROS content decreased in a dose-dependent manner, with the high-dose group showing a decrease to 110.99%, close to the 106.93% in the positive control group. These results indicate that FALP-1 can effectively inhibit excessive ROS accumulation, thus playing a protective role against H2O2-induced oxidative damage.

[0044] (5) Effect of solid-state fermentation of wheat aleurone layer polysaccharide on H2O2-induced intracellular MDA content in HepG2 cells Free radicals attack unsaturated fatty acids in cell membranes, triggering a chain reaction of lipid peroxidation, with MDA being a stable product of this process. Therefore, elevated MDA levels directly reflect the degree of lipid peroxidation damage to the cell membrane and can serve as an important indicator for assessing cellular oxidative stress. As shown in Figure 9, compared to the blank control group, the MDA content in the H2O2-induced oxidative damage model group was significantly increased. After treatment with FALP-1 at concentrations of 25, 100, and 400 μg / mL, the intracellular MDA content decreased to varying degrees, with the high-dose group (400 μg / mL) showing a decrease to 5.38 nmol / L. These results indicate that H2O2 successfully induced lipid peroxidation in HepG2 cells, while FALP-1 treatment effectively inhibited MDA production, demonstrating that FALP-1 can effectively alleviate cellular oxidative stress damage.

[0045] (6) Effect of solid-state fermentation of wheat aleurone layer polysaccharide on H2O2-induced intracellular antioxidant enzyme activity in HepG2 cells Besides inhibiting the increase of lipid peroxidation products, enhancing the activity of antioxidant enzymes in the antioxidant defense system is also an important measure to protect cells. The protective effect of FALP-1 against oxidative stress in cells was studied by measuring the activities of SOD, CAT, and GSH-Px. The results, shown in Figure 10, indicate that the activities of all three enzymes in the model group were significantly reduced compared to the control group, suggesting that H2O2 disrupts the cellular antioxidant defense system. Cells treated with FALP-1 showed a dose-dependent increase in enzyme activity, with the effect of 400 μg / mL FALP-1 treatment being similar to that of the positive control group.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions or modifications made by those skilled in the art within the spirit and principle of the present invention without any creative effort should be included within the protection scope of the present invention.

Claims

1. A polysaccharide from solid-state fermented wheat aleurone layer of *Agrocybe aegerita*, characterized in that, It is composed of fucose, arabinose, galactose, glucose, xylose, and mannose, with molar percentages of 0.77%, 29.69%, 4.88%, 3.09%, 57.70%, and 3.87%, respectively, and an average molecular weight of 6.00 × 10⁻⁶. 3 - 7.00×10 3 Da has the structure shown in equation (1): 。 2. The method for preparing the polysaccharide from the solid-state fermentation of *Agrocybe aegerita* wheat aleurone layer according to claim 1, characterized in that, Includes the following steps: (1) Wheat grains are first dehulled to 4-8% using a dehulling machine, and then separated into aleurone layers using traditional milling methods; (2) Mix the wheat aleurone layer obtained in step (1) with deionized water at a mass ratio of 1:1-1:2, keep the pH at a natural level, and sterilize at high temperature to prepare a solid fermentation culture medium. (3) Under aseptic conditions, the activated tea tree mushroom strain was inoculated into the solid fermentation medium prepared in step (2) and cultured at 26°C in the dark for 9-18 days to obtain the fermentation substrate; (4) After fermentation, under aseptic conditions, deionized water is added to the fermentation substrate obtained in step (3) to carry out enzymatic hydrolysis reaction and obtain enzymatic hydrolysis mixture; (5) The enzymatic hydrolysis mixture obtained in step (4) is subjected to ultrasonic-assisted hot water extraction, centrifuged, and the extract is collected. The above steps are repeated to extract the filter residue after centrifugation. The supernatant obtained from the two centrifugations is the polysaccharide extract. (6) The extract obtained in step (5) is subjected to starch removal and protein removal treatment in sequence. Then, 3-4 times the volume of ethanol is added to the extract for alcohol precipitation. The precipitate is collected and freeze-dried to obtain crude polysaccharide. (7) The crude polysaccharide obtained in step (6) is reconstituted, purified by anion exchange chromatography column, and the eluent of the target component is collected. After concentration, dialysis and freeze-drying, the solid fermentation wheat aleurone layer polysaccharide of mushroom is obtained.

3. The preparation method according to claim 2, characterized in that, The preparation method of the wheat aleurone layer in step (1) is as follows: Weigh a certain amount of cleaned wheat grains, add water to condition the wheat grains before dehulling, mix quickly and evenly, let stand and then send them into the dehulling combination machine. By controlling the tightness of the valve, wheat grains with a dehulling rate of 4-8% are obtained. Then, the dehulled wheat grains are processed by the traditional milling method, and the bran component obtained is the wheat aleurone layer.

4. The preparation method according to claim 2, characterized in that, The activation of the strain mentioned in step (3) refers to inoculating the tea tree mushroom into a PDA solid plate culture medium and culturing it at a constant temperature of 26°C until the mycelium completely covers the surface of the plate. After activation and subculturing for more than 3 generations, it can be used.

5. The preparation method according to claim 2, characterized in that, The method for inoculating the tea tree mushroom strain in step (3) is as follows: Select a well-grown activated tea tree mushroom strain, punch holes at the edge of the mycelium to obtain a mycelial block, pick up the mycelial block with an inoculation needle, and inoculate the mycelium downwards evenly onto the aleurone layer culture medium.

6. The preparation method according to claim 2, characterized in that, The incubation enzymatic hydrolysis reaction in step (4) involves adding deionized water to the fermented aleurone layer matrix obtained in step (3) at a weight-to-volume ratio of 1:5 to 1:15, mixing evenly, and then transferring the mixture to a soymilk maker for stirring to make the sample homogenous.

7. The preparation method according to claim 2, characterized in that, In step (4), the fermentation substrate is subjected to a warm bath enzymatic hydrolysis reaction at a temperature of 40-60℃ for 24 hours, with a natural pH.

8. The preparation method according to claim 2, characterized in that, The polysaccharide extraction method in step (5) is to extract the enzymatic fermentation substrate obtained in step (3) in hot water at 40-60℃ using ultrasonic extraction at a power of 50-200 W for 1 h, with continuous stirring during the extraction process.

9. The preparation method according to claim 2, characterized in that, The polysaccharide separation and purification method in step (7) is to dissolve the crude polysaccharide obtained in step (6) in water at a ratio of 1:100, filter it through a 0.45 μm filter membrane, and then load it onto an anion exchange chromatography column at a uniform speed. The column is then eluted sequentially with NaCl solutions of 0, 0.1, 0.2, 0.3, and 0.5 mol / L. The eluent of the target component is collected, and finally concentrated under reduced pressure and freeze-dried to obtain the purified polysaccharide from the solid-state fermentation of wheat aleurone layer of *Agrocybe aegerita*.

10. The use of the solid-state fermented wheat aleurone layer polysaccharide of *Agrocybe aegerita* according to claim 1, or the solid-state fermented wheat aleurone layer polysaccharide of *Agrocybe aegerita* prepared by any one of claims 2-9, in the preparation of products for improving oxidative stress.

Images