Ganoderma lucidum solid-state fermentation wheat peel polysaccharide with lipid-lowering activity and preparation method thereof

Abstract

This invention belongs to the field of plant polysaccharide technology, specifically relating to a Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide with lipid-lowering activity and its preparation method. The polysaccharide is composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, with molar percentages of 2.14%, 13.68%, 14.66%, 21.50%, 31.43%, 2.42%, 7.86%, and 6.31%, respectively, and an average molecular weight of 10.00×10³–15.00×10³ Da, preferably 11.05×10³ Da. This invention utilizes Ganoderma lucidum to ferment wheat pericarp in a solid state, followed by ultrasonic-assisted hot water extraction to crudely extract the polysaccharide. The crude polysaccharide is then purified by anion exchange column and dextran gel column chromatography. The resulting fermented wheat pericarp polysaccharide effectively improves lipid deposition, exhibits significant lipid-lowering activity, and has high safety.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide technology, specifically to a Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide with lipid-lowering activity and its preparation method. The polysaccharide is prepared by solid-state fermentation of wheat pericarp using specific edible fungi. Background Technology

[0002] The increasing prevalence of obesity and related metabolic disorders globally is closely linked to unhealthy dietary patterns, particularly the high intake of high-fat diets. Obesity is a global health challenge characterized by excessive fat accumulation leading to weight gain and increasing susceptibility to various chronic diseases, such as type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Disorders of lipid metabolism can lead to the accumulation of reactive oxygen species (ROS), which, in excess, trigger oxidative stress and inflammation, ultimately causing liver damage and further accelerating the development of obesity-related diseases.

[0003] Natural polysaccharides extracted from plants have attracted considerable attention due to their low toxicity and wide range of physiological benefits. Whole grains are widely considered excellent dietary components for lipid regulation, which may be related to the polysaccharides enriched in their bran. However, bran (especially wheat bran) is often used as animal feed or for biogas production due to its poor palatability. Despite its significant nutritional value and potential health benefits, it can be consumed by humans with proper processing. The bran, as the outermost layer of the bran, is particularly rich in polysaccharides, with arabinoxylan being the main component, accounting for approximately 46% of the bran's dry weight. These polysaccharides are intricately intertwined with cellulose, lignin, and phenolic compounds such as ferulic acid, forming a dense cell wall matrix. The cross-linking of phenolic acids with cell wall polymers limits the extraction and bioavailability of polysaccharides, posing a challenge to their efficient recovery and functional utilization.

[0004] Solid-state fermentation using recognized safe microorganisms is a promising sustainable biomodification technology that can improve the extraction efficiency of polysaccharides and alter their structure and bioactivity. Studies have shown that solid-state fermentation can significantly affect the molecular weight, monosaccharide composition, and glycosidic bond type of plant-derived polysaccharides, thereby enhancing their bioactivity. Filamentous fungi, especially Ganoderma lucidum, have attracted considerable attention in such applications due to their powerful lignocellulose-degrading enzyme system, which can enzymatically hydrolyze complex plant matrices, promoting the release of bound phenols and polysaccharides, and improving solubility and bioactivity.

[0005] Wheat pericarp is rich in polysaccharides. The metabolic activity and lignocellulose-degrading enzyme system secreted by Ganoderma lucidum during solid-state fermentation can effectively promote the release and biotransformation of polysaccharides in wheat pericarp. However, a systematic study is currently lacking to comprehensively elucidate the effects of Ganoderma lucidum solid-state fermentation on the structural changes and bioactivity of polysaccharides in wheat pericarp. Summary of the Invention

[0006] To address the problems of low extraction efficiency and insufficient bioactivity of wheat pericarp polysaccharides in existing technologies, the present invention aims to provide a green and efficient preparation method to obtain a novel wheat pericarp polysaccharide with significant lipid-lowering activity.

[0007] This invention provides a Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide with lipid-lowering activity and its preparation method; the Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide is composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, with molar percentages of 2.14%, 13.68%, 14.66%, 21.50%, 31.43%, 2.42%, 7.86% and 6.31%, respectively, and an average molecular weight of 10.00×10³–15.00×10³ Da, preferably 11.05×10³ Da, and has the structure shown in formula (1): .

[0008] The preparation method of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide specifically includes the following steps: (1) Wheat grains are dehulled to 4-8% using a dehulling machine, and the resulting bran component is the wheat pericarp; (2) Mix the wheat pericarp obtained in step (1) with deionized water in a ratio of 1:2 to 1:3, sterilize at 121℃ for 20 min, and prepare a solid fermentation culture medium. (3) Under aseptic conditions, the activated Ganoderma lucidum strain was inoculated into the wheat pericarp solid fermentation medium prepared in step (2) and cultured at 26°C in the dark for 5-12 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. The supernatants obtained from the two centrifugations are combined to obtain the polysaccharide extract. (6) The polysaccharide 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 treated 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 and purified by anion exchange chromatography column. The eluent of the target component is collected, concentrated, dialyzed, and freeze-dried. Then it is further purified by dextran gel chromatography. Finally, it is concentrated and freeze-dried to obtain the purified Ganoderma lucidum solid fermented wheat pericarp polysaccharide (FPP-3-a).

[0009] As a further description of the above technical solution, the method for obtaining wheat bran in step (1) is as follows: weigh a certain amount of cleaned wheat, add 2% water by weight of wheat for pre-conditioning before dehulling, mix quickly and evenly, let stand for 30 seconds and then send it into the dehulling combination machine, and obtain a bran component with a dehulling rate of 4-8% by controlling the tightness of the valve, that is, wheat bran.

[0010] As a further description of the above technical solution, the method for obtaining the activated Ganoderma lucidum strain in step (3) is as follows: Ganoderma lucidum is inoculated onto PDA solid plate culture medium and cultured 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 is used.

[0011] As a further description of the above technical solution, the step of inoculating Ganoderma lucidum into wheat pericarp fermentation medium in step (3) is as follows: select activated Ganoderma lucidum strains in good growth condition, cut a 15mm diameter piece of fungus at the edge of the colony, pick it up with an inoculation needle, so that the mycelial side is facing down, and inoculate it evenly on the surface of the wheat pericarp solid fermentation medium, inoculating 5 pieces of fungus for every 100g of medium.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] As a further description of the above technical solution, the specific method for destarching and deproteinization in step (6) is as follows: a small amount of calcium chloride (to improve the heat resistance of the enzyme) is added to the extract, the pH of the extract is adjusted to 6.0 with 1 mol / L HCl, the temperature is raised to 70℃, and high-temperature α-amylase is added at 0.6% (w / v). The mixture 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, and saccharifying enzyme is added at 0.48% (w / v). The mixture is then incubated at 40℃ overnight to remove starch. After that, the pH is adjusted back to 7.0 with 1 mol / L NaOH, and alkaline protease is added at 0.3% (w / v). The mixture is then 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 collected by centrifugation.

[0016] 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 using 0, 0.1, 0.2 and 0.3 mol / L NaCl solutions, and the elution fraction of 0.3 mol / L NaCl is collected. This fraction is loaded onto a dextran gel column and eluted with water as the mobile phase. The target polysaccharide eluent is collected, concentrated under reduced pressure and freeze-dried to obtain purified polysaccharide.

[0017] Preferably, the wheat pericarp component in step (1) has a dehulling rate of 6%.

[0018] Preferably, the mass ratio of wheat bran component to water in step (2) is 1:2.5, and the pH is natural. Preferably, the solid-state fermentation culture time in step (3) is 6 days.

[0019] Preferably, the ratio of 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 achieved by the present invention are as follows: (1) This invention is the first to use Ganoderma lucidum to carry out directional solid-state fermentation of wheat pericarp. The enzyme system secreted by fungi gently lyses the cell wall, avoiding the use of strong chemical reagents and violent physical treatments in traditional extraction processes. The process is green and mild, and the extraction rate of fermented wheat pericarp polysaccharides (12.14 g / 100 g DW) is more than 2 times higher than that of unfermented wheat pericarp raw material (5.38 g / 100 g DW).

[0022] (2) A novel polysaccharide with a well-defined structure and high purity was obtained through the above method. The polysaccharide is composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, with molar percentages of 2.14%, 13.68%, 14.66%, 21.50%, 31.43%, 2.42%, 7.86% and 6.31%, respectively. The average molecular weight is 10.00×10³–15.00×10³ Da, preferably 11.05×10³ Da. The purity can reach more than 85%, and the impurity content is low.

[0023] (3) The solid-state fermented wheat pericarp polysaccharide prepared by the present invention showed significant lipid-lowering activity in both cell and animal models. It can effectively reduce lipid accumulation in oleic acid-induced HepG2 cells and alleviate excessive weight gain in obese mice induced by high-fat diet, indicating that it has good application potential in regulating lipid metabolism disorders and related diseases. Attached Figure Description

[0024] Figure 1 The elution curves of Ganoderma lucidum solid-state fermentation wheat pericarp polysaccharide in this embodiment of the invention are shown in the anion exchange column and dextran gel column elution curves. Figure 2 This is an ion chromatogram of wheat pericarp polysaccharide obtained from solid-state fermentation of Ganoderma lucidum in an embodiment of the present invention; Figure 3 This is a gel permeation chromatogram of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide in an embodiment of the present invention; Figure 4 This is the microstructure of the Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide in an embodiment of the present invention; Figure 5 This embodiment of the invention illustrates the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on the survival rate of HepG2 cells. Figure 6 This embodiment of the invention illustrates the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on oleic acid-induced lipid accumulation in HepG2 cells. Figure 7 This invention illustrates the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on oleic acid-induced oxidative stress in HepG2 cells. Figure 8 This embodiment of the invention illustrates the effects of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on the body weight and food intake of obese mice. Figure 9 This embodiment of the invention illustrates the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on the blood lipid accumulation level in obese mice. Figure 10 This embodiment of the invention illustrates the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on lipid accumulation levels in the adipose tissue of obese mice. Figure 11 This invention relates to the effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on lipid accumulation levels in the liver tissue of obese mice. Detailed Implementation

[0025] 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.

[0026] Unless otherwise specified, all reagents used in the following examples are commercially available.

[0027] Example 1: Preparation of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide (FPP-3-a) (1) Weigh a certain amount of cleaned wheat, add 2% water by weight of wheat for pre-peeling conditioning, mix quickly and evenly, let stand for 30 seconds and then send it into the peeling combination machine. By controlling the tightness of the valve, a bran component with a peeling rate of 6% is obtained, namely wheat husk. (2) Weigh 100 g of wheat husks and mix them evenly with 250 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 wheat husk solid fermentation culture medium. (3) Under sterile conditions, select activated Ganoderma lucidum strains in good growth condition, cut a 15mm diameter piece of fungus from the edge of the colony, pick it up with an inoculation needle, and inoculate it evenly on the surface of wheat pericarp culture medium with the mycelial side facing down. Inoculate 5 pieces of fungus per 100g fermentation substrate, and culture at 26℃ in the dark for 6 days to obtain fermentation substrate. (4) After fermentation, under aseptic conditions, add 3.5 kg of deionized water to 350 g of fermentation substrate, mix well, and then transfer to a soymilk maker and stir for 40 s to make the sample homogenous. Place the sample in a constant temperature shaker (50℃, 150 r / min) and use the endogenous extracellular enzymes produced by Ganoderma lucidum during fermentation to carry out the enzymatic hydrolysis reaction. The pH is natural throughout the process, which lasts for 24 h to obtain the enzymatic hydrolysate mixture. (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 centrifuged to collect the extract. The above steps were repeated to extract the filter residue after centrifugation. The polysaccharide extracts obtained from the two centrifugations were combined. (6) Add 0.84 g of calcium chloride (to improve the heat resistance of the enzyme) to 7000 mL of extract, adjust the pH of the extract to 6.0 with 1 mol / L HCl, heat to 70℃ and add 42 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 33.6 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 21 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; (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 and 0.3 mol / L NaCl solutions, and collect the elution fraction of 0.3 mol / L NaCl. Load this fraction onto a dextran gel column and elute with water as the mobile phase. Collect the target polysaccharide eluent, concentrate under reduced pressure and freeze dry to obtain purified polysaccharide FPP-3-a.

[0028] Experimental Example 1: Isolation and Purification of Wheat Peel Polysaccharides from Ganoderma Lucidum Solid-State Fermentation 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, four elution peaks were obtained, and the corresponding eluents were processed to obtain four pre-purified fractions with yields of 16.98%, 9.75%, 16.85%, and 9.42%, and purities of 67.82%, 26.62%, 66.62%, and 25.22%, respectively. The main fraction obtained by elution with 0.3 mol / L NaCl was further purified using a Sephadex G-100 gel column, yielding a single symmetrical peak and increasing the purity to 88.71%.

[0029] Experimental Example 2: Structural Characterization of Wheat Pericarp Polysaccharide from Ganoderma Lucidum Solid-State Fermentation (1) Monosaccharide composition analysis The monosaccharide composition of FPP-3-a was determined by ion chromatography, and the results are as follows: Figure 2 As shown, FPP-3-a is composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, with molar percentages of 2.14%, 13.68%, 14.66%, 21.50%, 31.43%, 2.42%, 7.86%, and 6.31%, respectively. Among these, arabinose, galactose, glucose, and xylose account for more than 80% of the total monosaccharide content, indicating that FPP-3-a is a polysaccharide rich in arabinoxylan structure.

[0030] (2) Molecular weight determination The molecular weight of FPP-3-a was determined by high-performance gel permeation chromatography (HPLC). The chromatogram is shown below. Figure 3 As shown, FPP-3-a exhibits a single, sharp, and essentially symmetrical elution peak, indicating that it is a polysaccharide component with a relatively uniform molecular weight distribution. Based on the standard curve, its average molecular weight is calculated to be 11.05 × 10³ Da.

[0031] (3) Methylation analysis The methylation analysis results of FPP-3-a are shown in Table 1. A total of 13 partially methylated sugar alcohol acetate derivatives were identified. Among them, the terminal residues (t-Ara...) f , t-Glc p , t-Gal p , t-Rha p The highest proportion (49.07% in total) indicates that the polysaccharide structure contains a large number of short side chains. The main chain structure is mainly composed of 1,4-Xyl p (10.37%) and 1,2,3,4-Xyl p Composed of 9.87%. Based on the branching degree formula, FPP-3-a has a branching degree (DB) of 0.68, demonstrating its highly branched and complex structure, which may contribute to its improved water solubility and biological activity. .

[0032] (4) Nuclear magnetic resonance analysis The ¹H NMR spectrum of FPP-3-a showed seven terminal proton signal peaks at δH 5.20, 5.16, 5.13, 4.93, 4.51, and 4.49 ppm. By comprehensively analyzing the ¹H-¹³C HSQC, ¹H-¹H COSY, and HMBC spectra, the types and linkages of each sugar residue were determined (Table 2). Combined with the methylation analysis results, it was inferred that the backbone of FPP-3-a consists of 1,4-β-D-Xyl p Composed of 1,2,3,4-β-D-Xyl p As the branch point, terminal arabinose, galacturonic acid, and glucose side chains are attached at positions C-2 and C-3, respectively, as shown in formula (1). .

[0033] (5) Morphological structure analysis The microstructure of FPP-3-a was observed using scanning electron microscopy, and the results are as follows: Figure 4 As shown, FPP-3-a exhibits irregular sheet-like and blocky aggregates with a rough surface and a porous structure.

[0034] Experimental Example 3: In vitro lipid-lowering activity of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide (1) The toxic effect of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide on HepG2 cells The effect of FPP-3-a on the survival rate of HepG2 cells was detected using the CCK-8 assay. Figure 5As shown, FPP-3-a exhibited no toxic effect on cell viability within the concentration range of 25-400 μg / mL, and showed a certain tendency to promote cell proliferation within the range of 25-100 μg / mL. Therefore, 25, 50, and 100 μg / mL were selected as low, medium, and high doses for subsequent experiments.

[0035] (2) Effects of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on oleic acid-induced lipid accumulation in HepG2 cells Establish an oleic acid-induced hyperlipidemia model in HepG2 cells. For example... Figure 6 As shown, the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL) were significantly increased in the model group, while the level of high-density lipoprotein cholesterol (HDL) was decreased. After intervention with different concentrations of FPP-3-a, the levels of intracellular TC, TG, and LDL decreased in a dose-dependent manner, while the level of HDL increased, indicating that FPP-3-a can effectively alleviate the abnormal accumulation of lipids in hepatocytes.

[0036] (3) Effects of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on oleic acid-induced oxidative stress in HepG2 cells Intracellular antioxidant enzyme activity was detected to assess the antioxidant capacity of FPP-3-a. For example... Figure 7 As shown, oleic acid-induced reduction in the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in cells was significant. Treatment with FPP-3-a restored the activities of these three enzymes, reaching near-normal levels at a concentration of 100 μg / mL, indicating that FPP-3-a can alleviate oxidative damage in hepatocytes under degenerative conditions and improve lipid peroxidation.

[0037] Experiment Example 4: In vivo lipid-lowering activity of Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide (1) Effects of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on body weight and food intake in obese mice Establish a mouse model of obesity induced by a high-fat diet. For example... Figure 8 As shown, starting from week 10, mice were administered FPP-3-a (at doses of 100, 200, and 400 mg / kg bw) or the positive control drug lovastatin via gavage. Compared with the model group, all doses of FPP-3-a significantly inhibited excessive weight gain induced by a high-fat diet in a dose-dependent manner. There was no significant difference in daily food intake among the groups, indicating that the weight-loss effect of FPP-3-a was not due to appetite suppression.

[0038] (2) Effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on blood lipid accumulation levels in obese mice The results of detecting serum biochemical parameters in mice are as follows: Figure 9As shown, serum glucose (GLU), TC, TG, and LDL levels were significantly elevated in the model group mice, while HDL levels were decreased. FPP-3-a intervention could reverse these abnormal changes in a dose-dependent manner, with high-dose FPP-3-a (400 mg / kg·bw) showing comparable effects on reducing TC and LDL as the lovastatin group.

[0039] (3) Effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on lipid accumulation level in adipose tissue of obese mice Histological analysis of epididymal adipose tissue ( Figure 10 HE staining showed that the adipocytes in the model group were significantly larger and loosely arranged. Treatment with FPP-3-a, especially in the medium and high dose groups, significantly improved adipocyte hypertrophy. Oil Red O staining further confirmed that FPP-3-a significantly reduced lipid accumulation in adipose tissue in a dose-dependent manner.

[0040] (4) Effect of Ganoderma lucidum solid-state fermentation of wheat pericarp polysaccharide on lipid accumulation levels in liver tissue of obese mice Analysis of lipid content and morphology in liver tissue ( Figure 11 In the model group, the levels of total cholesterol (TC) and triglycerides (TG) in the liver were significantly elevated, and hepatocytes showed marked steatosis (vacuolation) and extensive lipid droplet deposition. FPP-3-a treatment dose-dependently reduced liver TC and TG levels and improved hepatic steatosis. Oil Red O staining showed that FPP-3-a treatment (especially at medium and high doses) significantly reduced the area of ​​red lipid droplets in the liver, indicating its good ability to clear hepatic lipids.

[0041] 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 solid-state fermented wheat pericarp polysaccharide from Ganoderma lucidum, characterized in that, Composed of fucose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, with an average molecular weight of 10.00×10³–15.00×10³ Da, it has the structure shown in formula (1): 。 2. The method for preparing Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide according to claim 1, characterized in that, Includes the following steps: (1) Select an appropriate dehulling rate to dehull wheat grains to obtain wheat pericarp components; (2) Mix the wheat husks obtained in step (1) with deionized water at a mass ratio of 1:2-1:3, 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 Ganoderma lucidum strain was inoculated into the solid fermentation medium prepared in step (2) and cultured at 26°C in the dark for 5-12 days to obtain the fermentation substrate; (4) After fermentation, under sterile conditions, deionized water is immediately 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 supernatants obtained from the two centrifugations are combined to obtain 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, the eluent of the target component is collected, concentrated, dialyzed, and freeze-dried, and then further purified by dextran gel chromatography. Finally, it is concentrated and freeze-dried to obtain the Ganoderma lucidum solid fermented wheat pericarp polysaccharide.

3. The preparation method according to claim 2, characterized in that, The preparation method of wheat husk in step (1) is as follows: weigh a certain amount of cleaned wheat, add water for pre-dehulling conditioning, mix quickly and evenly, let stand and then send it into the dehulling combination machine, and obtain a bran component with a dehulling rate of 4%-8% by controlling the tightness of the valve, that is, wheat husk.

4. The preparation method according to claim 2, characterized in that, The activation of the strain mentioned in step (3) refers to inoculating Ganoderma lucidum onto 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 specific operation of inoculation in step (3) is as follows: cut a 15 mm diameter piece of fungus from the edge of the activated Ganoderma lucidum colony, pick it up with an inoculation needle, so that the mycelial side is facing down, and inoculate it evenly on the surface of the wheat pericarp culture medium. Inoculate 5 pieces of fungus per 100 g of 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 wheat bran matrix obtained in step (3) at a ratio of 1:5 to 1:15 (w / v), mixing evenly, and then transferring the mixture to a soymilk maker and stirring for 40 seconds to make the sample homogenous.

7. The preparation method according to claim 2, characterized in that, The temperature for the incubation enzymatic hydrolysis reaction in step (4) is 40-60℃, the time is 24 h, and the pH is natural.

8. The preparation method according to claim 2, characterized in that, The conditions for ultrasonic-assisted hot water extraction in step (5) are as follows: ultrasonic extraction at a power of 50-200 W for 1 h in a water bath at 40-60℃, with continuous stirring during the extraction process.

9. The preparation method according to claim 2, characterized in that, 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, filtered through a 0.45 μm filter membrane, and then uniformly loaded onto an anion exchange chromatography column. Gradient elution is performed using 0, 0.1, 0.2 and 0.3 mol / L NaCl solutions, and the elution fraction of 0.3 mol / L NaCl is collected. This component was loaded onto a dextran gel column and eluted with water as the mobile phase. The eluent of the target polysaccharide was collected, concentrated under reduced pressure, and freeze-dried to obtain the purified polysaccharide.

10. The application of the Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide according to claim 1, or the Ganoderma lucidum solid-state fermented wheat pericarp polysaccharide prepared by any one of claims 2-9, in the preparation of products for improving lipid metabolism disorders.

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