Morchella enzymolysis polysaccharide and preparation method thereof

Abstract

The invention discloses morchella enzymolysis polysaccharide and a preparation method thereof. The preparation method of the morchella esculenta enzymolysis polysaccharide comprises the following steps: carrying out enzymolysis on morchella esculenta polysaccharide by adopting glucamylase, and sequentially carrying out inactivation, centrifugation, dialysis, alcohol precipitation and freeze drying on the obtained enzymolysis product to obtain the morchella esculenta enzymolysis polysaccharide, the enzymolysis conditions are as follows: the enzymolysis temperature is 60-65 DEG C, the pH value is 4.3-4.7, the enzyme addition amount is 45-55 U / mL, and the enzymolysis time is 2.5-3.5 hours. The morchella esculenta enzymolysis polysaccharide disclosed by the invention has a good pancreatic lipase inhibition effect, and has oxidation resistance superior to that of morchella esculenta polysaccharide; in addition, the morchella enzymolysis polysaccharide disclosed by the invention can also regulate lipid metabolism disorder, regulate intestinal flora and prevent liver injury, oxidative stress and inflammation.

Description

Technical Field

[0001] The present invention relates to a Morchella enzyme-digested polysaccharide and a preparation method thereof, belonging to the technical field of biomedicine. Background Art

[0002] Polysaccharides are a class of natural macromolecular compounds with various biological activities and have broad application prospects in the fields of food, medicine, etc. The biological activities of polysaccharides are closely related to factors such as their molecular weight, structure, chemical composition, etc. Research shows that moderate degradation of polysaccharides can reduce their molecular weight and improve biological activities, especially lipid-lowering activities. However, there are relatively few studies on the polysaccharides of Morchella importuna and their degradation products at present, and the understanding of their lipid-lowering mechanisms is not deep enough.

[0003] As a fungus with both medicinal and edible properties, Morchella importuna has functions such as promoting defecation and improving metabolism, and its polysaccharide component is considered to be one of the main biological active components. However, the molecular weight of Morchella importuna polysaccharides is relatively large, which may limit their absorption and utilization in the body, thereby affecting the exertion of their biological activities. Therefore, moderately degrading Morchella importuna polysaccharides, improving their lipid-lowering activities, and deeply studying their action mechanisms are of great significance for developing new lipid-lowering drugs or dietary supplements. Summary of the Invention

[0004] The purpose of the present invention is to provide a Morchella enzyme-digested polysaccharide and a preparation method thereof. The Morchella enzyme-digested polysaccharide has a good inhibitory effect on pancreatic lipase and has an antioxidant ability superior to that of Morchella importuna polysaccharides; in addition, the Morchella enzyme-digested polysaccharide can also regulate lipid metabolism disorders, regulate the intestinal flora, and prevent liver damage, oxidative stress, and inflammation.

[0005] The preparation method of the Morchella enzyme-digested polysaccharide provided by the present invention includes the following steps:

[0006] Using glucoamylase to enzymatically digest Morchella importuna polysaccharides, and the obtained enzymatic hydrolysis products are successively inactivated, centrifuged, dialyzed, ethanol-precipitated, and freeze-dried to obtain Morchella enzyme-digested polysaccharides.

[0007] Wherein, the conditions for the enzymatic hydrolysis are:

[0008] The enzymatic hydrolysis temperature is 60 - 65 °C, the pH is 4.3 - 4.7, the enzyme addition amount is 45 - 55 U / mL, and the enzymatic hydrolysis time is 2.5 - 3.5 h;

[0009] Preferably: the enzymatic hydrolysis temperature is 65 °C, the pH is 4.5, the enzyme addition amount is 50 U / mL, and the enzymatic hydrolysis time is 3 h.

[0010] Preferably, the Morchella importuna polysaccharides are prepared by a hot water extraction method;

[0011] The conditions of the hot water extraction method are as follows:

[0012] The solid-liquid ratio of Morchella fruiting body powder to water is 1:30, and the extraction is carried out at 75-85° C. for 1.5-3 hours, and the extraction is repeated 2-4 times.

[0013] The pancreatic lipase inhibition rate of the Morchella enzymatic hydrolyzed polysaccharide prepared by the present invention is significantly higher than that of the original polysaccharide (MIP), reaching 64.72% and 42.42% respectively at 2.5 mg / mL; the total sugar content of the Morchella enzymatic hydrolyzed polysaccharide is reduced, both polysaccharides contain more sulfate groups, and EMIP is slightly higher than MIP.

[0014] The results of scanning electron microscopy, molecular weight and particle size all proved that degradation caused Morchella polysaccharide particles to become smaller. The Mw values ​​of MIP were 3.98×10 5 Da and 2.15×10 4 Da, the Mw of EMIP is 2.08×10 4 Da; the particle size results of the two were MIP (255.16nm) and EMIP (164.32nm). HPIC results showed that both MIP and EMIP were composed of GlcN, Gal, Glc, and Man, with molar ratios of 0.023:0.136:0.668:0.173 and 0.046:0.398:0.085:0.471, respectively. It can be seen that degradation did not change the monosaccharide types of Morchella polysaccharide, but significantly changed its molar ratio.

[0015] In addition, MIP and EMIP have similar UV spectra, thermogravimetric and Congo red curves, indicating that the degradation did not destroy the main structure of Morchella polysaccharide. However, in the FT-IR spectrum analysis, EMIP showed a strong -1 The β-glycosidic bond signal that does not appear in MIP appeared at the site, indicating that degradation changed the functional groups of MIP.

[0016] In the in vitro antioxidant test, EMIP showed antioxidant capacity superior to that of MIP, which indicates that degradation can not only improve the pancreatic lipase inhibitory ability of Morchella polysaccharides, but also enhance its antioxidant capacity. These changes may be related to the changes in the molecular weight, structure and chemical composition of polysaccharides.

[0017] The present invention also studies the regulatory effects of Morchella enzymatic polysaccharides on lipid metabolism disorders, liver damage and oxidative stress, inflammation and intestinal permeability:

[0018] (1) Both MIP and EMIP can improve the changes in body weight, liver index, and fasting blood glucose levels in mice caused by a high-fat diet. By detecting the TC, TG, LDL-C, and HDL-C levels in the serum and liver of mice, it was found that EMIP exhibited stronger lipid-lowering ability than MIP. High-dose EMIP reduced serum and liver TG by 63.62% and 66.86% respectively, restoring them to normal levels. Effective reduction of liver lipid deposition was also observed in the liver H&E sections, indicating that EMIP has good ability to improve lipid metabolism disorders.

[0019] (2) By detecting the contents of TBA, ALT, and AST in the serum and liver of mice, the protective effects of MIP and EMIP on liver injury in mice were evaluated. The results showed that polysaccharides at different doses could improve liver injury in mice, and EMIP had a better effect on reducing the levels of AST and TBA.

[0020] (3) By detecting the levels of SOD, MDA, GSH-Px, and CAT in liver tissues, the effects of MIP and EMIP on the oxidative stress state of the mouse liver were explored. It was found that both polysaccharides could significantly increase the activity of antioxidant enzymes in the body and reduce the content of lipid peroxides. EMIP made a more prominent contribution in enhancing the activities of SOD and CAT and reducing the content of MDA, while MIP had a better effect on increasing the activity of GSH-Px.

[0021] (4) ELISA kits were used to detect the contents of LPS, TNF-α, and IL-6 in the serum of mice. The results showed that MIP and EMIP could improve the inflammatory state of mice, reduce intestinal permeability, and showed a dose-dependent manner. Subsequently, fluorescence quantitative PCR was used to detect the gene expression levels of ZO-1, Occludin, and Claudin-1 in the colon tissues of mice, further indicating that Morchella polysaccharides have a good effect on repairing the intestinal barrier.

[0022] The present invention further studied the effects of enzymatically hydrolyzed Morchella polysaccharides on the intestinal flora and liver metabolites of high-fat diet mice:

[0023] (1) Different doses of MIP and EMIP did not significantly improve the intestinal flora diversity and richness in high-fat diet mice, but could alleviate lipid metabolism disorders in high-fat diet mice by enriching Lactobacillus and inhibiting Dubosiella.

[0024] (2) MIP intervention significantly affected 39 metabolites, while EMIP caused significant changes in 236 metabolites. Both could reverse the trends of increased LPG 16:2 and decreased 8 - aminooctanoic acid and NADPH caused by a high - fat diet. Among them, 8 - aminooctanoic acid is a potential biomarker for lipid metabolism disorders, and its activity has not been reported yet. In addition, EMIP had a significant impact on the contents of various LPGs and LPCs, and had a more extensive physiological significance for the regulation of liver metabolites.

[0025] (3) Through heatmap and Spearman correlation analysis, the effects of MIP and EMIP on the contents of 38 differential metabolites and the potential associations between these metabolites and differential flora and physiological indicators were explored. It was speculated that MIP and EMIP might relieve lipid disorders, liver injury, oxidative stress, and inflammatory responses in mice by changing the intestinal microbial composition and affecting liver metabolic activities.

[0026] (4) KEGG pathway enrichment analysis showed that the differential metabolites between EMIP and MC were mainly enriched in 20 pathways such as aldosterone synthesis and secretion, synaptic vesicle cycle, amino acid synthesis and metabolism, indicating that EMIP could improve various physiological activities of mice. Description of the Drawings

[0027] Figure 1 are the results of the inhibitory rate of pancreatic lipase after different enzyme hydrolyses of Morchella polysaccharide.

[0028] Figure 2 are the results of the inhibitory rate of pancreatic lipase of Morchella polysaccharide at different enzymatic hydrolysis temperatures (A), enzymatic hydrolysis pH (B), enzyme addition amount (C), and enzymatic hydrolysis time (D).

[0029] Figure 3 are the response surface plots (A, B, C) and contour plots (D, E, F) of the interaction of enzymatic hydrolysis conditions on the inhibitory rate of pancreatic lipase.

[0030] Figure 4 are the pancreatic lipase inhibitory abilities of MIP and EMIP.

[0031] Figure 5 are the scanning electron microscopy results of MIP (A, B) and EMIP (C, D) at 3000× and 10000×.

[0032] Figure 6 are the ultraviolet (A) and infrared (B) spectral analyses of MIP and EMIP.

[0033] Figure 7 are the high - performance ion chromatogram (A), high - performance gel chromatogram (B), particle size distribution curve (C), thermogravimetric curve (D), and Congo red (E) of MIP and EMIP.

[0034] Figure 8 are the DPPH (A), ·OH (B), ABTS (C) radical scavenging abilities and total antioxidant capacity (D) of MIP and EMIP.

[0035] Figure 9 are the effects of MIP and EMIP on the body weight (A), weight gain (B), liver index (C) and fasting blood glucose (D) of high-fat diet mice.

[0036] Figure 10 are the effects of MIP and EMIP on the contents of serum TC (A), TG (B), LDL-C (C), HDL-C (D) and the ratios of TC / HDL-C (E), LDL-C / HDL-C (F) in high-fat diet mice.

[0037] Figure 11 are the effects of MIP and EMIP on the contents of liver TC (A), TG (B), LDL-C (C) and HDL-C (D) in high-fat diet mice.

[0038] Figure 12 are the pathological changes (H&E, 100× and 200×) of the liver in high-fat diet mice after MIP and EMIP intervention.

[0039] Figure 13 are the effects of MIP and EMIP on the contents of serum ALT (A), AST (B), TBA (C), and liver ALT (D), AST (E), TBA (F), SOD (G), MDA (H), GSH-Px (I) and CAT (J) in high-fat diet mice.

[0040] Figure 14 are the effects of MIP and EMIP on the contents of serum LPS (A), TNF-α (B), IL-6 (C) and the gene expression levels of colon ZO-1 (D), Occludin (E), Claudin-1 (F) in high-fat diet mice.

[0041] Figure 15 are the effects of MIP and EMIP intervention on the intestinal microbial diversity of high-fat diet mice (n = 3): petal diagram (A), α-diversity (B), principal component analysis (C).

[0042] Figure 16 are the effects of MIP and EMIP intervention on the intestinal microbial abundance of high-fat diet mice (n = 3): phylum level (A), genus level (B).

[0043] Figure 17 are the microbial differential contribution (A) at the genus level between different groups and the MC group and the LDA distribution bar chart (B).

[0044] Figure 18 Effects of H-MIP and H-EMIP on liver metabolites in high-fat diet mice (n = 5): PCA (A); OPLS-DA (B).

[0045] Figure 19 Volcano plots of metabolites in MC.vs.NC (A), H-MIP.vs.MC (B), and H-EMIP.vs.MC (C); ROC curve of 8-aminooctanoic acid (D).

[0046] Figure 20 Heat map of potential metabolic biomarkers.

[0047] Figure 21 Spearman correlation analysis of metabolites with physiological indices and gut microbiota.

[0048] Figure 22 Enrichment maps of KEGG metabolic pathways: MC.vs.NC (A), H-MIP.vs.MC (B), H-EMIP.vs.MC. Specific implementation manners

[0049] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0050] The materials, reagents, etc. used in the following examples can all be obtained from commercial channels unless otherwise specified.

[0051] Example 1: Preparation of enzymatically hydrolyzed polysaccharide from Morchella

[0052] The fruiting bodies of Morchella importuna were provided by the Edible Fungi Center of Shanxi Agricultural University.

[0053] α-Amylase, β-amylase, glucoamylase, and Triton X-100 were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; cellulase was purchased from Beijing Bio-ATech Co., Ltd.; pancreatic lipase was purchased from Sigma-Aldrich, USA.

[0054] I. Extraction of Morchella polysaccharide (MIP)

[0055] The Morchella esculenta fruit bodies were placed in an oven at 50 °C until constant weight, ultramicro pulverized and passed through a 100-mesh sieve. 1 g of the powder was weighed and extracted at 80 °C for 2 h at a solid-liquid ratio of 1:30. The extraction was repeated three times, and the supernatant was concentrated by centrifugation at 6000 r / min for 10 min. After deproteinization with zinc acetate-potassium ferrocyanide, it was centrifuged at 6000 r / min for 10 min, alcohol-precipitated overnight and then centrifuged at 6000 r / min for 10 min. The precipitate was redissolved and dialyzed against running water (3500 Da), and then freeze-dried to obtain MIP (total carbohydrate content: 62.60%).

[0056] II. Preparation of enzymatically hydrolyzed polysaccharide from Morchella esculenta (EMIP)

[0057] 1. Single factor experiment

[0058] The 20 mg / mL pancreatic lipase solution was prepared with 0.2 mmol / L phosphate buffer (PBS) at pH 8.0, and the 0.8 mg / mL p-nitrophenyl laurate was prepared with 5 mmol / L anhydrous sodium acetate solution (pH 5.0, containing 1% Triton X-100). In a 1.5 mL centrifuge tube, 200 μL of the 2 mg / mL polysaccharide solution, 100 μL of PBS, 100 μL of the pancreatic lipase solution were added in sequence. After incubation in a metal bath at 37 °C for 30 min, 200 μL of the p-nitrophenyl laurate substrate solution was added, and after incubation at 37 °C for 60 min, the absorbance was measured at 405 nm.

[0059]

[0060] Where: A1: Absorbance value of 200 μL of polysaccharide, 100 μL of PBS, 100 μL of pancreatic lipase and 200 μL of substrate

[0061] A2: Absorbance value of 200 μL of polysaccharide, 100 μL of PBS, 100 μL of distilled water and 200 μL of substrate

[0062] A3: Absorbance value of 300 μL of PBS, 100 μL of pancreatic lipase and 200 μL of substrate

[0063] A4: Absorbance value of 300 μL of PBS, 100 μL of distilled water and 200 μL of substrate

[0064] In the present invention, α-amylase, β-amylase, glucoamylase and cellulase were used to hydrolyze MIP under their respective optimal conditions. The enzyme concentration was 40 U / mL, the MIP concentration was 4 mg / mL. After 3 h of enzymatic hydrolysis, it was inactivated at 100 °C for 15 min. After centrifugation to obtain the supernatant, dialysis and alcohol precipitation were carried out, followed by freeze-drying. For the enzymes screened according to the pancreatic lipase inhibition rate, the control variable method was used to set different enzymatic hydrolysis temperatures (35 °C, 45 °C, 55 °C, 65 °C, 75 °C), different enzymatic hydrolysis pH values (3.5, 4.0, 4.5, 5.0, 5.5), different enzyme addition amounts (30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL) and different enzymatic hydrolysis times (2.0 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h) to determine the optimal enzymatic hydrolysis conditions.

[0065] 1) Determination of enzymes

[0066] The inhibitory effects of the products obtained by hydrolyzing MIP with various enzymes under the optimal pH and temperature conditions on pancreatic lipase are as Figure 1 shown. From high to low, they are glucoamylase (51.80%), cellulase (48.56%), β-amylase (46.72%), α-amylase (44.30%) and without enzyme (40.38%). This shows that enzymatic hydrolysis can improve the pancreatic lipase inhibitory ability of Morchella polysaccharide. Among them, glucoamylase has the best effect. Therefore, glucoamylase was selected for the subsequent single-factor experiments.

[0067] 2) Determination of enzymatic hydrolysis temperature

[0068] As Figure 2 shown, under the conditions of pH 4.5, glucoamylase concentration of 40 U / mL, and temperatures of 35 °C, 45 °C, 55 °C, 65 °C, and 75 °C respectively, the inhibitory effects of the products of each group on pancreatic lipase showed a trend of first increasing and then decreasing with the increase of temperature. This is because before 65 °C, with the increase of temperature, the movement rates of Morchella polysaccharide and glucoamylase molecules increased, the effective collision frequency between the two increased, and the degradation effect was strengthened accordingly. When the temperature increased to 75 °C, it exceeded the tolerance range of glucoamylase, and the enzyme molecular bonds began to break, and the polysaccharide degradation efficiency also decreased rapidly. Therefore, in the subsequent experiments, the enzymatic hydrolysis temperature was considered to be set at 65 °C.

[0069] 3) Determination of enzymatic hydrolysis pH

[0070] Too high or too low pH will affect the dissociation of active groups on the enzyme molecule, affect the binding efficiency of the enzyme. In addition, it will also change the conformation of the enzyme or even inactivate it. The optimal pH range of glucoamylase is around 4.0 - 4.5. As Figure 2As shown, when the pH is between 3.5 and 4.5, the inhibitory rate of Morchella enzymolysis polysaccharide on pancreatic lipase increases steadily, indicating that the enzymolysis reaction becomes gradually sufficient. However, as the pH value further increases, the inhibitory effect weakens significantly. This may be because it exceeds the optimal pH range of glucoamylase, resulting in a decrease in enzyme activity and affecting the degradation efficiency. Therefore, it is considered to select pH 4.5 as the enzymolysis condition in subsequent experiments.

[0071] 4) Determination of enzyme addition amount

[0072] From Figure 2 It can be seen that as the enzyme addition amount increases, the inhibitory activity of the polysaccharide on pancreatic lipase also increases. However, after exceeding 50 U / mL, the inhibitory rate begins to decline significantly. This may be because the increase in enzyme concentration leads to excessive degradation of the polysaccharide, with too small a molecular weight to form an active polymer structure, reducing the polysaccharide activity. Therefore, the addition amount of glucoamylase is determined to be 50 U / mL.

[0073] 5) Determination of enzymolysis time

[0074] See Figure 2 , when the enzymolysis time is less than 3 h, the polysaccharide cannot reach the best inhibitory activity, which may be due to insufficient degradation. After exceeding 3 h, the inhibitory effect shows a downward trend, indicating that it is most appropriate to select 3 h as the enzymolysis time. From the above results, it can be seen that the change in enzymolysis time has little effect on the inhibitory activity of the polysaccharide. Therefore, three factors, namely enzymolysis temperature, enzymolysis pH, and enzyme addition amount, are selected for the subsequent response surface optimization experiment.

[0075] 2. Response surface experiment

[0076] According to the results of the single-factor experiment, with the inhibitory rate of pancreatic lipase (Y) as the response index, enzymolysis temperature (A), enzymolysis pH (B), and enzyme addition amount (C) as the response variables, the Box-Behnken experiment is used to optimize the degradation conditions of MIP by response surface. The detailed data are shown in Table 1, and EMIP prepared under the optimal degradation process is obtained.

[0077] Table 1 Variables and levels in Box-Behnken design

[0078]

[0079]

[0080] 1) Model fitting

[0081] According to the results of the single-factor experiment, Design Expert software is used to perform response surface analysis on the hydrolysis conditions of glucoamylase, and the operation data are shown in Table 2.

[0082] Table 2 Analysis of variance table for the inhibitory rate of pancreatic lipase

[0083]

[0084] 2) Response surface analysis

[0085] The correlation between the dependent variable and the independent variables can be intuitively reflected by 3D response surface plots and 2D contour plots( Figure 3 ). By observing the 3D response surface plots( Figure 3 Figures A, B, and C therein), it can be seen that the pancreatic lipase inhibition rate first increases and then decreases with the increase of any two of these factors. Corresponding to the 2D contour plots( Figure 3 Figures D, E, and F therein), it can be seen that the interaction between pH and temperature and enzyme addition amount is relatively strong. The optimal conditions predicted by the response surface are that the glucoamylase addition amount is 50.42 U / mL, the temperature is 64.96 °C, the pH is 4.46, and the pancreatic lipase inhibition rate is 60.9673%. For the convenience of actual experimental operation, the predicted optimal conditions are modified to an enzyme addition amount of 50 U / mL, a temperature of 65 °C, and a pH of 4.5. Under these conditions, the result of the verification test is that the pancreatic lipase inhibition rate is 59.81%.

[0086] 3) In vitro pancreatic lipase inhibitory activities of MIP and EMIP

[0087] As Figure 4 , after hydrolysis by glucoamylase, the inhibitory ability of Morchella polysaccharide against pancreatic lipase has been significantly improved. In the concentration range of 0.5 - 2.5 mg / mL, the inhibition rate of EMIP is always higher than that of MIP, and it shows concentration dependence. When the concentration is 0.5 mg / mL, the pancreatic lipase inhibition rates of MIP and EMIP are 16.74% and 37.52% respectively, and the inhibitory effect of EMIP is 2.24 times that of MIP. Therefore, enzymatic hydrolysis can effectively improve the inhibitory ability of MIP against pancreatic lipase.

[0088] 3. Determination of chemical components of MIP and EMIP

[0089] The total carbohydrate, protein, uronic acid, reducing sugar, sulfate group, and total polyphenol contents in MIP and EMIP were determined by the phenol - sulfuric acid method, Coomassie brilliant blue method, sulfuric acid - carbazole method, DNS method, barium chloride - gelatin turbidimetry method, and Folin - Ciocalteu method.

[0090] The results of the chemical components of MIP and EMIP are shown in Table 3. After degradation by glucoamylase, the total carbohydrate, protein, and total phenol contents of MIP decreased, while the reducing sugar and sulfate group contents increased. This may be due to the cleavage of glycosidic bonds, which causes changes in the molecular conformation and exposes the sulfate groups. Some studies have found that the components with better obesity inhibitory effects in fucoidan contain more sulfate groups. The increase in sulfate groups may be one of the reasons for the stronger pancreatic lipase inhibitory ability of EMIP.

[0091] Table 3 Chemical Compositions of MIP and EMIP

[0092] Sample MIP EMIP Carbohydrate content (%) <![CDATA[62.60% ± 1.89 a > <![CDATA[41.11% ± 1.68 b > Protein content (%) <![CDATA[1.81% ± 0.03 a > <![CDATA[1.02% ± 0.05 b > Uronic acid content (%) NA NA Reducing sugar content (%) <![CDATA[4.25% ± 0.05 b > <![CDATA[5.11% ± 0.02 a > Sulfate content (%) <![CDATA[21.79% ± 0.46 b > <![CDATA[23.01% ± 0.20 a > Total phenol content (%) <![CDATA[7.21% ± 0.11 a > <![CDATA[5.11% ± 0.19 b >

[0093] 4. Structural Characterization of MIP and EMIP

[0094] 1) Scanning Electron Microscope

[0095] Take appropriate amounts of dry MIP and EMIP powders and place them on the sample stage. After coating with gold powder using a vacuum sputtering coater, observe the morphological characteristics of the samples through a scanning electron microscope.

[0096] Observe the microtopographies of MIP and EMIP at 3000× and 10000× using a scanning electron microscope ( Figure 5 ). MIP presents as reticular fragments of different sizes, with an irregular internal structure, loose arrangement, and large pores, while EMIP consists of smooth flakes and irregular small particles, which are closely packed and evenly distributed, indicating that after degradation, the polysaccharide molecular chains are damaged, and the particle size decreases and becomes more uniform.

[0097] 2) Ultraviolet and Infrared Spectrum Scans

[0098] Prepare 0.5 mg / mL solutions of MIP and EMIP, and use a UV-visible spectrophotometer to detect the UV-visible absorption spectra of the samples in the range of 190 - 800 nm; treat the MIP and EMIP powders by the KBr tablet method, and scan the infrared absorption spectra of the samples in the range of 4000 - 500 cm -1 using a spectrometer.

[0099] The results of the ultraviolet spectrum scan are shown in Figure 6 Figure A. There are no obvious absorption peaks at 260 nm and 280 nm for both MIP and EMIP, indicating no nucleic acid and protein residues. Moreover, the absorbance values of EMIP at these two wavelengths are lower than those of MIP, indicating that degradation can reduce the nucleic acid and protein contents, which is consistent with the changing trend of the protein content in 2.3.5.

[0100] The results of the FT-IR spectrum scan are shown in Figure 6 Figure B. The absorption peak near 3400 cm -1 is caused by the OH stretching vibration on the polysaccharide molecule, and the peak at 2930 cm -1 represents the stretching vibration peak of C-H; the peak observed at 1652 cm -1 is not only attributed to the water adsorbed in the sample but also the stretching vibration peak of C=O. The absorption peak at 1400 cm -1 is the characteristic peak of the symmetric stretching vibration of the carboxyl group, while the peaks at 1030 cm -1 and 1066 cm-1 The absorption peak at -1 indicates the presence of pyranose rings in MIP and EMIP. Signals of α-glycosidic bonds appear at 814 cm -1 for both polysaccharides, but the signal of β-glycosidic bond at 890 cm -1 only appears in the EMIP spectrum.

[0101] 3) Determination of monosaccharide composition

[0102] Add 2 mL of 3M TFA to 5 mg of polysaccharide sample, hydrolyze at 120 °C for 3 h, then dry. Add 5 mL of water, vortex to mix evenly, dilute ten times, centrifuge at 12000 rpm for 5 min, and take the supernatant for IC analysis. NaOH (15 mM) is used as the eluent, flow rate: 0.3 mL / min; injection volume: 25 μL; column temperature: 30 °C.

[0103] As shown in Table 4 and Figure 7 Figure A in

[0104] MIP and EMIP are both composed of glucosamine hydrochloride, galactose, glucose and mannose, with molar ratios of 0.023∶0.136∶0.668∶0.173 and 0.046∶0.398∶0.085∶0.471 respectively. Glucoamylase hydrolysis increases the contents of galactose and mannose and decreases the glucose content, but does not affect the types of monosaccharides.

[0105] The molecular weight distribution characteristics of MIP and EMIP were analyzed by HPGPC, and the particle sizes of MIP and EMIP solutions were measured using a nanoparticle size analyzer.

[0106] Enzymatic hydrolysis can break the glycosidic bonds of polysaccharides and reduce the molecular weight. It can be seen from Table 4 that MIP has two main peaks, with weight-average molecular weights of 3.98×105 Da (27.876%) and 2.15×104 Da (72.124%) respectively. After degradation, EMIP has only one peak, with a weight-average molecular weight of 2.08×104 Da, and the Mw / Mn value is close to 1. And Figure 7 As shown in Figure B in Figure 7 EMIP shows a single-peak symmetric state (the peak of the mobile phase is at 45.5 min), indicating that the molecular weight distribution is relatively uniform. As shown in Table 4 and

[0107] 6、Thermogravimetric determination

[0108] Accurately weigh 5 mg of the sample and place it in a crucible. Set the heating rate to 10 °C / min, and use a thermogravimetric analyzer to analyze the thermal stability of the sample during the heating process from 25 °C to 600 °C.

[0109] The thermograms of MIP and EMIP are shown in Figure 7 Figure D in it. There are two main weight loss processes in the TGA curves of the two polysaccharides. First, there is a slight mass loss between 30 - 100 °C, which belongs to the bound water and free water in the polysaccharide; secondly, the mass drops rapidly in the range of 250 - 400 °C, and the weight loss rate is nearly 60%, which is due to the large amount of degradation of the polysaccharide at high temperature. After that, the temperature continues to rise, but the mass loss of the polysaccharide begins to enter a flat stage.

[0110] 6. Congo red test

[0111] Using the Congo red solution without polysaccharide sample as the experimental control, the triple helix structures of MIP and EMIP were determined.

[0112] Figure 7 Figure E in it shows the maximum absorption wavelength of the polysaccharide-Congo red solution after mixing with different concentrations of NaOH solution. Congo red can bind to polysaccharides containing triple helix conformations, causing a red shift in the maximum absorption wavelength of the solution. The red shift phenomenon appears in both polysaccharides in the figure, indicating that they both have triple helix structures and the degradation does not cause the disappearance of the triple helix structure.

[0113] Table 4 Monosaccharide composition, molecular weight and particle size of MIP and EMIP

[0114]

[0115] Example 2. Determination of in vitro antioxidant activities of MIP and EMIP

[0116] Prepare MIP and EMIP solutions with different concentrations (0.25, 0.5, 1.0, 2.0, 4.0 mg / mL). Using VC as the positive control, determine the DPPH free radical scavenging ability and hydroxyl free radical scavenging ability; use the ABTS detection kit and the total antioxidant capacity (T-AOC) detection kit (Beijing Solarbio Science & Technology Co., Ltd.) to determine the ABTS free radical scavenging ability and total antioxidant capacity.

[0117] Data analysis was performed using Design Expert 8.0.6 Trial, Excel and SPSS Statistics 26. Different lowercase letters indicate statistical significance between groups (p < 0.05).

[0118] 1. DPPH free radical scavenging ability

[0119] From Figure 8As can be seen from Figure A, at different concentration gradients, the DPPH radical scavenging ability of EMIP is stronger than that of MIP, and both are positively correlated with the concentration. The IC50 values of MIP and EMIP are 0.306 and 0.258 mg / mL, respectively, which are much lower than those of ginseng polysaccharide GPS-1A (12.45 mg / mL) and degraded ginseng polysaccharide DGPS-1A (2.32 mg / mL), indicating that MIP and EMIP can be used as natural antioxidants, and EMIP has a better effect.

[0120] 2. Hydroxyl radical scavenging ability

[0121] Hydroxyl radicals are considered to be the most active oxygen radicals, which can easily penetrate cell membranes and cause oxidative damage to cells. As can be seen from Figure 8 Figure B, MIP and EMIP have different scavenging abilities for hydroxyl radicals (IC50 values are 0.368 and 0.268 mg / mL, respectively), and the results are the same as the trend of DPPH radical scavenging rate. When the concentration is 0.5 and 1 mg / mL, the difference in scavenging ability between the two polysaccharides is relatively large, which are 15.71% and 16.60% respectively. Wu et al. found that the enzymatic hydrolysis products of Auricularia auricula polysaccharide (AAP) have improved hydroxyl radical scavenging ability, which is similar to the results of the present invention, indicating that enzymatic hydrolysis can be an effective method to improve the antioxidant activity of polysaccharides.

[0122] 3. ABTS radical scavenging ability

[0123] The ABTS radical scavenging abilities of MIP and EMIP are shown in Figure 8 Figure C. With the increase of concentration, the scavenging abilities of MIP and EMIP also increase. After calculation, the IC50 values of MIP and EMIP are 0.424 mg / mL and 0.281 mg / mL, respectively. At low concentrations, there is a significant difference in the scavenging abilities of the two. When the concentration is 1 mg / mL, the scavenging rate of MIP reaches 75.56%, and the scavenging rate of EMIP is 87.59% at this time, which is higher than that of Boletus luridus polysaccharide (21.17%) at the same concentration, indicating that MIP and EMIP have good ABTS radical scavenging abilities.

[0124] 4. Total antioxidant capacity

[0125] As can be seen from Figure 8 Figure D, in the concentration range of 0.25 - 4 mg / mL, the total antioxidant capacities of MIP and EMIP are positively correlated with the concentration. Although within the test concentration range, the T-AOC of MIP and EMIP is always lower than that of ascorbic acid, EMIP still shows a higher total antioxidant capacity than MIP.

[0126] Based on the above four antioxidant capacity tests, it was found that the degraded Morchella polysaccharide exhibited more excellent antioxidant activity than before. The stronger antioxidant capacity of MIP may be related to the decrease in molecular weight, the increase in the contents of sulfate groups and galactose. There is a close relationship between the molecular weight, monosaccharide molar ratio, functional groups and sulfate group content of polysaccharides and their antioxidant activity.

[0127] Example 3: Regulation of lipid metabolism disorder by enzymatically hydrolyzed Morchella polysaccharide

[0128] Experimental animals: 60 6-week-old SPF-grade male C57BL / 6J mice were provided by Beijing Spearf Bio-Technology Co., Ltd., with the license number SCXK(Jing)2019-0010.

[0129] Standard feed, high-fat feed (D12492) (Beijing Spearf Bio-Technology Co., Ltd.); TC, TG, HDL-C, LDL-C, AST, ALT, TBA, CAT, GSH-Px, SOD, MDA kits (Nanjing Jiancheng Bioengineering Institute); LPS, TNF-α, IL-6 ELISA kits (Wuhan Fine Biotech Co., Ltd.); hematoxylin-eosin staining solution, neutral resin (Beijing Solarbio Science & Technology Co., Ltd.); RNAiso Plus (Takara Bio Inc., Japan); HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novoprotein Co., Ltd.).

[0130] 1. Experimental method

[0131] 1) Establishment of experimental animal model

[0132] After acclimating 60 male C57BL / 6J mice for one week, they were randomly divided into 6 groups (n = 10), namely the normal diet group (NC group), the high-fat diet group (MC), the low-dose Morchella polysaccharide group (L-MIP) and high-dose group (H-MIP), the enzymatically hydrolyzed Morchella polysaccharide low-dose group (L-EMIP) and high-dose group (H-EMIP). During the following experiment, except for the NC group which was given normal diet, the mice in the other groups were provided with high-fat diet. At the same time, the mice were gavaged daily according to their body weights. The NC group and the MC group were gavaged with normal saline; the L-MIP group and the H-MIP group were gavaged with MIP solution at 100 mg / kg and 400 mg / kg respectively; the L-EMIP group and the H-EMIP group were gavaged with EMIP solution at 100 mg / kg and 400 mg / kg respectively, and gavage was continued for 8 weeks. The body weights of the mice were monitored and recorded once a week. After the experiment, the mice were fasted for 12 h and then blood was collected from the orbital cavity. After the blood was allowed to stand for a period of time, it was centrifuged (3000 r / min, 15 min) to obtain the supernatant, which was stored in a -80 °C refrigerator. The remaining organs and feces were collected and stored for future use according to the corresponding operations.

[0133] 2) Determination of mouse body weight, liver, kidney, spleen indices and fasting blood glucose value

[0134] During the feeding period, the body weight was monitored once a week. The complete livers of the mice were collected, rinsed with pre-cooled normal saline and then dried with sterile filter paper, and the liver index was calculated after weighing. Liver index (%) = liver weight (g) / mouse weight (g) × 100%. Before the end of the experiment, the fasting blood glucose of the mice (fasted for 12 h) was detected using a blood glucose meter.

[0135] 3) Determination of mouse serum biochemical indices

[0136] The contents of TC, TG, HDL-C, LDL-C, ALT, AST, and TBA in the serum were detected using kits from Nanjing Jiancheng, and the levels of LPS, TNF-α, and IL-6 factors in the serum were detected using ELISA kits.

[0137] 4) Determination of mouse liver biochemical indices

[0138] Using kits from Nanjing Jiancheng, the contents of TC, TG, HDL-C, LDL-C, ALT, AST, SOD, MDA, GSH-Px, CAT, and TBA in the liver tissue were detected.

[0139] 5) H&E staining of the liver

[0140] Mouse liver tissues were taken, fixed in 4% paraformaldehyde for 48 h, and the paraffin section and H&E staining procedures were performed according to the method of Guo Dongdong et al.

[0141] 6) RT-qPCR analysis of the expression of related genes in colon tissues

[0142] Total RNA was extracted from the tissues according to the instructions of the RNAiso Plus kit. The total RNA was reverse-transcribed into cDNA using the reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) and stored at -20 °C for later use. Using a real-time fluorescence quantitative PCR instrument, with β-actin as the internal reference gene, the expression of ZO-1, Occludin, and Claudin-1 genes in colon tissues was quantified. The details of the primer sequences are shown in Table 5.

[0143] Table 5 RT-qPCR primer sequences

[0144]

[0145]

[0146] 7) Data statistics

[0147] Excel was used to draw charts, and SPSS software was used for significance analysis. Different lowercase letters indicate statistical significance between groups (p < 0.05).

[0148] 2. Experimental results

[0149] 1) Effects of MIP and E-MIP on the body weight, liver, kidney, spleen indices, and fasting blood glucose of mice

[0150] As Figure 9 shown in Figure A below, before the start of the experiment, the body weights of the mice in each group were similar. After 8 weeks of feeding, the body weights of all groups showed an upward trend. The body weight gain in the MC group was significantly higher than that in other groups. Although the polysaccharide group also used a high-fat diet as the daily ration, the process of weight gain was similar to that in the NC group, indicating that Morchella polysaccharide has a good effect on reducing weight gain caused by a high-fat diet. From Figure 9 Figure B below, it can be seen that the weight gain amounts of each group during the experiment were NC (5.57 ± 0.74 bg), MC (8.04 ± 0.71 a g), L-MIP (5.19 ± 0.81 bc g), H-MIP (4.85 ± 0.49 c g), L-EMIP (4.92 ± 0.61 bc g), and H-EMIP (4.90 ± 0.72 bc g), respectively. The body weight gain of the mice in the MC group was significantly higher than that in other groups (p < 0.05). Except for the high-dose MIP group, there was no significant difference between other polysaccharide groups and the normal group, indicating that the high-dose MIP has the best effect on reducing body weight.

[0151] The effects of Morchella polysaccharide on the organ indices of high-fat diet mice are shown in Figure 9In Figure C, a high-fat diet increased the proportion of the liver in mice, and intragastric administration of polysaccharide could significantly restore the liver state of mice. From Figure 9 As can be seen from Figure D, the intervention of polysaccharide significantly reduced the fasting blood glucose index of high-fat diet mice, and EMIP had a better effect than MIP.

[0152] 2) Effects of MIP and E-MIP on improving serum lipid levels in high-fat diet mice

[0153] After 8 weeks of feeding with a high-fat diet, the amount of TC in the serum of mice increased from 4.39 mM to 5.97 mM, and the intervention of Morchella polysaccharide significantly reduced the content of TC in the serum of mice ( Figure 10 Figure A); a high-fat diet increased the contents of TG and LDL-C in the serum of mice to 1.68 times and 2.28 times that of the normal group (NC) respectively, and intragastric administration of Morchella polysaccharide could significantly improve this pathological change. Regarding the reduction of TG, the effect of EMIP was more prominent, and it achieved the effect of the high dose of MIP even at a low dose ( Figure 10 Figure B); but for the reduction of LDL-C content, the effect of MIP was a little better ( Figure 10 Figure C). From Figure 10 As can be seen from Figure D, a high-fat diet significantly reduced the serum HDL-C level in mice, while MIP and EMIP significantly increased the level of HDL-C, and showed a dose-dependent manner, and the effect of EMIP was better.

[0154] As Figure 10 shown in Figures E and F, with the increase of polysaccharide concentration, the ratios of TC / HDL-C and LDL-C / HDL-C both decreased significantly, and the effect of EMIP was significantly better than that of MIP. Considering the above blood lipid indexes, the lipid-lowering effect of EMIP is stronger than that of MIP.

[0155] 3) Effects of MIP and E-MIP on improving liver lipid levels and fat accumulation in high-fat diet mice

[0156] From Figure 11 it can be seen that the contents of TC, TG, and LDL-C in the liver of mice in the MC group were significantly higher than those in the NC group, indicating that a high-fat diet caused serious lipid deposition in the liver of mice. Intake of MIP and EMIP could relieve the symptoms of high-fat diet mice, and polysaccharide could also increase the level of HDL-C in mice and relieve the disorder of liver lipid metabolism.

[0157] Liver H&E sections are as Figure 12As shown, a high-fat diet can cause a large accumulation of lipids in the body, and the liver's inability to promptly transport the excess lipids will result in fat deposition in the liver tissue. It can be observed from the figure that there are a large number of lipid droplets in the livers of the mice in the MC group, the arrangement of hepatocytes is disordered, the cell nucleus is squeezed to one side by the enlarged fat and atrophies or dissolves, and extensive inflammatory infiltration can be seen, indicating that the liver fat deposition and inflammatory response caused by the high-fat diet are very serious. By ingesting different doses of polysaccharides, the liver fat deposition in mice can be improved to varying degrees. The low-dose MIP reduces the lipid droplets, but the inflammatory infiltration is not much improved, while the high-dose MIP effectively reduces the lipid droplets and alleviates the inflammatory infiltration; the effect of L-EMIP in reducing lipid droplets is similar to that of H-MIP, and the hepatic plates are radial. There are almost no lipid droplets in the livers of the mice intervened with high-dose EMIP, the edges of the hepatocytes are clear, and it is close to the normal group. The above results indicate that EMIP can effectively reduce the liver fat deposition in high-fat diet mice.

[0158] 4) Effects of MIP and E-MIP on improving liver injury in high-fat diet mice

[0159] As Figure 13 shown, a high-fat diet significantly increased the levels of ALT, AST, and TBA in the serum and liver of mice (p < 0.05), but EMIP inhibited the changes brought about by the high-fat diet in mice to varying degrees, and at the same concentration, EMIP showed a stronger inhibitory ability than MIP.

[0160] 5) Effects of MIP and E-MIP on improving liver oxidative stress in high-fat diet mice

[0161] As Figure 13 shown, a high-fat diet led to a significant increase in the MDA level in the livers of mice, while reducing the activities of antioxidant enzymes SOD, GSH-Px, and CAT. MIP and EMIP can enhance the antioxidant capacity of the livers of mice by increasing the levels of antioxidant enzymes and reducing the MDA content, showing significant differences compared with the MC group (p < 0.05).

[0162] 6) Effects of MIP and E-MIP on improving inflammation and intestinal permeability in high-fat diet mice

[0163] As Figure 14 shown, ingesting MIP and EMIP can reduce the contents of LPS, TNF-α, and IL-6 in the serum, improve the LPS leakage and inflammatory state caused by the high-fat diet. At the same time, EMIP can also significantly increase the mRNA expression of intestinal tight junction proteins ZO-1, Occludin, and Claudin-1, protect the intestinal mucosal barrier, and maintain the integrity of intestinal function.

[0164] Example 4. Effects of enzymatically hydrolyzed polysaccharides from Morchella esculenta on intestinal flora and liver metabolites in high-fat diet mice

[0165] Use the mouse feces and liver tissues collected in Example 3.

[0166] The fecal DNA extraction kit was purchased from Beijing Novogene Bioinformatics Technology Co., Ltd.; the mass spectrometer was purchased from ThermoFisher Q Exactive TM HF / Q Exactive TM HF-X; the chromatograph was purchased from Thermo Fisher Vanquish UHPLC, Thermo Fisher Hypesil Gold column; the chromatographic column (100×2.1 mm, 1.9 μm).

[0167] 1. Test method

[0168] 1) Intestinal flora analysis

[0169] Extract the total DNA in mouse feces using a fecal DNA extraction kit, amplify the V3-V4 region of the 16S rRNA gene with reference to the method of Liu et al., and sequence the obtained amplification products using NovaSeq6000.

[0170] 2) Liver untargeted metabolomics analysis

[0171] Extract the metabolites in mouse liver tissues, detect the liver metabolites by LC-MS, select Hypersil Gold column (C18) for the chromatographic column, and select 40 °C and 0.2 mL / min for the column temperature and flow rate respectively. In the positive ion mode, mobile phase A uses 0.1% formic acid and mobile phase B uses methanol; in the negative ion mode, mobile phase A uses 5 mM ammonium acetate and mobile phase B uses methanol; the scanning range is selected as m / z 100-1500; the settings of the ESI source are as follows: Spray Voltage: 3.5 kV; Sheath gas flow rate: 35 psi; Aux Gas flow rate: 10 L / min; Capillary Temp: 320 °C; S-lens RF level: 60; Aux gas heater temp: 350 °C.

[0172] 3) Data statistics

[0173] The 16S was annotated for species using the QIIME2 software, with the Silva 138.1 database. Analyses such as abundance calculation, Alpha diversity calculation, and petal plots were performed on the obtained ASVs to obtain information on species richness and evenness within the samples, information on shared and unique ASVs among different groups, etc. On the other hand, PCA was used to explore the differences in community structure among different groups. To further explore the differences in community structure among grouped samples, statistical analysis methods such as Simper and LEfSe were selected to perform a significance test on the species composition and community structure of the grouped samples.

[0174] The original metabolite data files were imported into the CD3.3 library search software for screening, and compared with the mzCloud, mzVault, and Masslist databases to obtain the identification and relative quantification results of metabolites. The data processing part was based on the Linux operating system (CentOS version 6.6) and the software R and Python. The KEGG database was used to annotate metabolites. In the multivariate statistical analysis part, metaX was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), and the VIP value of each metabolite was obtained. In the univariate analysis part, the statistical significance (P value) of each metabolite between the two groups was calculated based on the t-test, and the fold change (FC value) of the metabolite between the two groups was calculated. By combining the three parameters of the VIP value, log2(FoldChange), and -log10(p value) of the metabolite, differential metabolites were screened. The obtained differential metabolites were respectively subjected to hierarchical clustering analysis, correlation analysis, and KEGG pathway enrichment analysis.

[0175] 2. Experimental results

[0176] 1) Effects of MIP and EMIP on the diversity of mouse gut microbiota

[0177] Figure 15 The petal plot in Figure A shows the overlap of OTUs among groups. There are 60 shared OTUs, 362 unique OTUs in the NC group, 131 in the MC group, 76 in the L-MIP group, 86 in the H-MIP group, 89 in the L-EMIP group, and 89 in the H-EMIP group. Alpha diversity can characterize the abundance and evenness of gut microbiota. The Chao 1 index represents the number of OUTs in the sample; the Shannon index represents the species richness in the sample; the Simpson index represents the species distribution evenness in the sample. As Figure 15 shown in Figure B, a high-fat diet reduced the richness and diversity of mouse gut microbiota, but the intervention with Morchella polysaccharide did not show an obvious improvement effect. Beta diversity can intuitively reflect the differences in species composition among different samples. From the PCoA plot ( Figure 15It can be seen from Figure C in the Chinese version that there is a significant separation between the NC group and the MC group, and the groups of Morchella polysaccharides are distributed between the NC group and the MC group, indicating that the intervention of Morchella polysaccharides has a certain impact on the changes in the microbial community structure caused by a high-fat diet. Therefore, further research can be carried out from the perspective of gut microbiota.

[0178] 2) Effects of MIP and EMIP on the composition of the gut microbiota in mice

[0179] Figure 16 Figure shows the species abundance composition of the gut microbiota in mice at the phylum level (A) and genus level (B). At the phylum level, the microbial communities in each group are mainly composed of Firmicutes, Verrucomicrobia, Bacteroidetes, Deferribacteres, and Actinobacteria, which account for more than 99.3% of the gut microbiota. Since a high-fat diet can increase the F / B ratio in the gut microbiota, similar results also occurred in this experiment, and the supplementation of high-dose EMIP can reverse this change.

[0180] Next, the species difference analysis between each group and the MC group at the genus level was carried out. From Figure 17 Figure A in the Chinese version, the two genera with the largest contribution to the difference (contribution > 0.2) were selected, namely Akkermansia and Dubosiella; Figure 17 From Figure B in the Chinese version, 2 families and 2 genera with significant differences between groups (LDA > 4) were selected, namely Lactobacilaceae, Lachnospiraceae, Lactobacillus, and Blautia. Among them, Lactobacillus belongs to Lactobacilaceae, and Blautia belongs to Lachnospiraceae. Many strains in the genus Lactobacillus play important roles in reducing lipid accumulation, alleviating oxidative stress, preventing intestinal inflammation, and lowering blood sugar. From Figure 17In Figure B, it can be observed that Morchella polysaccharide significantly increased the relative abundance of Lactobacillus, which were 14.18% in the L-MIP group, 10.05% in the H-MIP group, 3.07% in the L-EMIP group, and 11.84% in the H-EMIP group, respectively. While the relative abundance of Lactobacillus in the MC group was only 0.29%. Wu et al. reported that Panax japonicus Torr. polysaccharide could improve the intestinal flora disorder induced by high-fat diet, mainly by reducing the abundances of Turicibacter, Dubosiella, and Staphylococcus and increasing the abundances of Bacteroides, Blautia, and Lactobacillus. And the study by Xu et al. showed that tartary buckwheat flavonoids alleviated the intestinal flora imbalance caused by high-fat diet by increasing the relative abundances of Dubosiella and Bacteroidetes. The association between Dubosiella and high-fat diet is still unclear. In this experiment, it was found that the relative abundance of Dubosiella in the MC group was increased compared with that in the NC group, and the intake of Morchella polysaccharide could regulate its abundance. The increase in the abundances of Akkermansia and Blautia is considered to be of great significance in reducing inflammation, enhancing immunity, lowering blood lipids and blood glucose. However, the Morchella intervention did not increase their abundances. Therefore, it is speculated that Morchella polysaccharide may alleviate the lipid metabolism disorder in high-fat diet mice by enriching Lactobacillus and inhibiting Dubosiella.

[0181] 3) PCA and PLS-DA Analyses of Liver Metabolites

[0182] Liver tissues of mice in the NC group, MC group, H-MIP group, and H-EMIP group were collected for non-targeted metabolomics analysis, and a total of 810 metabolites were identified. As Figure 18 , PCA plots (A) and OPLS-DA plots (B) were drawn using R language. The results of PCA and OPLS-DA were similar, both showing that the distance between the MC group and the H-MIP group was relatively close, indicating that H-MIP had no significant effect on the metabolite changes caused by high-fat diet. While the NC group and the H-EMIP group were distributed at both ends and had a good separation effect from the MC group, which indicated that there were significant differences in metabolites between them and the MC group. The QC samples were collected closely, indicating that LC-MS had good stability.

[0183] 4) Screening of Differential Liver Metabolites

[0184] Figure 19The results of differential metabolites between each group and the MC group were shown (VIP > 1, P < 0.05). Respectively, 271, 39, and 236 differential metabolites were identified between the MC group and the NC group, the H-MIP group and the MC group, and the H-EMIP group and the MC group. Among them, a high-fat diet led to a significant down-regulation of eicosapentaenoic acid (EPA), LPE(20:5), LPC(20:5-SN1), LPC(20:5), and 8-aminooctanoic acid in the livers of mice, and a significant up-regulation of estradiol-17β-D-glucuronide, LPG(18:1), LPG(16:2), and cholest-4-en-3-one. EPA is an ω-3 fatty acid that can reduce the content of TG in serum and is used to prevent cardiovascular diseases; LPE(20:5) is a lysophosphatidylethanolamine. Studies have shown that the activation of brown fat can cause a significant increase in the level of LPE(20:5) in plasma; both LPC(20:5-SN1) and LPC(20:5) belong to lysophosphatidylcholines. It has been found in lipid-lowering treatment that administration can increase the content of LPC(20:5) and its high-density lipoprotein level in the plasma of patients; 8-aminooctanoic acid is an octanoic acid with an amino group at the 8th position and belongs to medium-chain fatty acids. Multiple studies have shown that octanoic acid can improve the lipid metabolism of obese rats by promoting adipose tissue browning and increasing fat decomposition. However, the biological activity of 8-aminooctanoic acid has not been reported yet. Therefore, it can be used as a new potential biomarker waiting for further verification. The AUC value of 1 indicates its good predictive effect ( Figure 19 in Figure D). Estradiol-17β-D-glucuronide has been proven to cause cholestasis in humans; both LPG(18:1) and LPG(16:2) are lysophosphatidylglycerols. It has been reported that the decrease in plasma LPG is related to the reduction of fat mass in rats. Jin Fangsha constructed a mouse model of lipid metabolism disorder under BPA exposure and found that cholest-4-en-3-one was significantly up-regulated in lipid-disordered mice. After H-MIP intervention, the levels of LPG(16:0) and LPG(16:2) in the livers of mice were significantly decreased, and the contents of 8-aminooctanoic acid and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were significantly increased. Gavage with H-EMIP significantly reduced cholest-4-en-3-one, LPG(18:1), and LPG(16:2) in the livers of mice, and significantly increased the levels of LPC(18:2-SN1), LPC(20:5-SN1), LPC(20:5), 8-aminooctanoic acid, and NADPH. And NADPH plays an important role in the cellular antioxidant system and can scavenge ROS generated by the oxidative degradation of fat.

[0185] 5) Correlation analysis of liver differential metabolites with physiological indexes and intestinal flora

[0186] Thirty-eight metabolites most likely to be biomarkers of the effect of Morchella polysaccharide on liver lipid metabolism were screened according to significance ( Figure 20 ), and correlation analysis was performed with the significantly different flora (2 families and 4 genera) and physiological indicators in each group ( Figure 21 ). The data showed that compared with the NC group, high-fat diet upregulated 20 metabolites such as estradiol-17-β-D-glucuronide, LPG, adipic acid, cholestenone, 20-hydroxyeicosatetraenoic acid (20-HETE), acylcarnitine, and hydroxy acid in the livers of mice, and downregulated 18 metabolites such as inosine, phosphatidylcholine (PC), LPC, LPE, 20-hydroxy prostaglandin F2α (20hydroxy PGF2α), 5'-cytidine monophosphate (CMP), and hexosylceramide (HexCer 14:0; 3O / 18:1; (2OH)). After intervention with H-MIP and H-EMIP, the levels of these metabolites approached those of the normal group to varying degrees. Among them, H-MIP mainly alleviated liver oxidative stress in mice by increasing the levels of NADPH and NADH, while H-EMIP could improve lipid metabolism disorders in mice by increasing the levels of LPC, inosine, hexosylceramide, and CMP and decreasing the contents of LPG, 20-HETE, and cholestenone, and also increased the levels of NADPH and NADH, indicating that H-EMIP has more diverse effects in regulating lipid metabolism in mice.

[0187] It can be seen from Figure 21 that Blautia, Dubosiella, and Lachnospiraceae were extremely strongly positively correlated with 20-HETE, CAR 21:1, cholestenone, LPG 18:1, LPG 22:4, and LPS22:5, and extremely strongly negatively correlated with 20hydroxy PGF2α, 8-aminocaprylic acid, LPC, and LPE. Lactobacilaceae and Lactobacillus showed similar trends, being negatively correlated with hydroxy acid and LPG and positively correlated with NADH, NADPH, and LPC. Some studies have shown that Lachnospiraceae is related to the development of non-alcoholic fatty liver and diabetes; Lactobacilaceae plays an important role in preventing diabetes and obesity, regulating lipid metabolism and inflammation, etc. It is inferred that Morchella polysaccharide may affect liver metabolism and improve liver injury by changing the abundances of various gut microbiota such as Lachnospiraceae and Lactobacilaceae.

[0188] The correlation analysis was performed on the physiological indexes of mouse serum and liver and liver metabolites. It was found that hydroxy acids, CAR21:1, cholestenone, estradiol-17-β-D-glucuronide, LPC 20:2, and LPG were closely and positively correlated with TC, TG, TBA, LDL-C, ALT, AST, MDA, LPS, and TNF-α, while EPA, daidzein, and other LPCs showed negative correlations. Therefore, reducing the contents of metabolites such as hydroxy acids and LPG by supplementing EMIP may help regulate the lipid levels, reduce lipid peroxidation, and alleviate inflammatory responses in hyperlipidemic mice.

[0189] 6) KEGG metabolic pathway enrichment analysis

[0190] The screened differential metabolites were subjected to KEGG enrichment analysis, and the results were as Figure 22As shown. The main differential metabolic pathways between the MC group and the NC group are sulfur metabolism, ferroptosis, primary bile acid biosynthesis, arachidonic acid metabolism, and biosynthesis of unsaturated fatty acids. Ferroptosis is a death process driven by iron-dependent phospholipid peroxidation in cells and plays a key role in various disease processes. The regulation of biological lipid metabolism is inseparable from the regulation of ferroptosis. Primary bile acid synthesis, arachidonic acid metabolism, and unsaturated fatty acid synthesis are important metabolic processes in the glycolipid metabolism of organisms. Disorders of bile acid secretion can affect the digestion and absorption of lipids; arachidonic acid metabolites (such as prostaglandin G2, E2, D2, etc.) are related to inflammation, thrombosis, and atherosclerosis; unsaturated fatty acids such as EPA and docosapentaenoic acid (DPA) play important roles in promoting endothelial cell migration, reducing inflammation, and decreasing the triglyceride content in the blood. H-MIP mainly affects the calcium signaling pathway and amino acid metabolism in mice; the metabolites affected by H-EMIP are mainly enriched in pathways such as aldosterone synthesis and secretion, synaptic vesicle cycle, amino acid synthesis and metabolism, etc. An increase in aldosterone content is positively correlated with the risk of atherosclerosis and can also lead to an aggravation of inflammatory infiltration. Extracellular vesicles play a key role in cell-to-cell communication. The lipid composition of vesicles can lead to their dysfunction. A high-fat diet promotes metabolic processes such as inflammation, insulin resistance, and obesity by affecting the structure and function of vesicles. Amino acids play important roles in lipid metabolism. Glycine inhibits the production of inflammatory cytokines by regulating genes related to adipose tissue storage and energy burning, improves the inflammatory condition in mice, and can also reduce fat accumulation and adipocyte hypertrophy in rats; threonine can lower blood lipids and inhibit fat increase, regulating lipid metabolism, while deficiencies in threonine and lysine can lead to fatty liver and various metabolic disorders. The results show that EMIP can affect lipid metabolism in mice by regulating the synthesis and metabolism of various amino acids, hormones, etc. and cell signal transduction.

Claims

1. A preparation method of enzymatically hydrolyzed Morchella polysaccharide, comprising the following steps: Enzymatically hydrolyze Morchella polysaccharide with glucoamylase, and the obtained enzymolysis product is successively inactivated, centrifuged, dialyzed, ethanol-precipitated, and freeze-dried to obtain enzymatically hydrolyzed Morchella polysaccharide.

2. The preparation method according to claim 1, wherein: The conditions for the enzymatic hydrolysis are as follows: The enzymatic hydrolysis temperature is 60 - 65 °C, the pH is 4.3 - 4.7, the enzyme addition amount is 45 - 55 U / mL, and the enzymatic hydrolysis time is 2.5 - 3.5 h.

3. The preparation method according to claim 1 or 2, characterized in that: The Morchella polysaccharide is prepared by a hot water extraction method.

4. The preparation method according to claim 3, characterized in that: The conditions of the hot water extraction method are as follows: The material-liquid ratio of Morchella fruticosa powder to water is 1:30, and it is extracted at 75 - 85 °C for 1.5 - 3 hours, and the extraction is repeated 2 - 4 times.

5. The enzymatically hydrolyzed Morchella polysaccharide prepared by the method according to any one of claims 1 - 4.

6. The application of the enzymatically hydrolyzed Morchella polysaccharide according to claim 5 in the preparation of antioxidant products.

7. An antioxidant product, the active ingredient of which is the enzymatically hydrolyzed Morchella polysaccharide according to claim 5.

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