Application of morchella mycelium extract

Abstract

The invention discloses application of a morchella mycelium extract, and belongs to the field of natural products. The morchella mycelium extract mainly comprises alpha-glucan, the weight-average molecular weight of the morchella mycelium extract ranges from 10000 kDa to 15000 kDa, the number-average molecular weight of the morchella mycelium extract ranges from 10000 kDa to 12000 kDa, the polydispersity index of the morchella mycelium extract ranges from 1.0 to 1.3, and the molar percentage of glucose is not lower than 99%. The preparation method comprises the steps of ultrasonic-assisted extraction, alcohol precipitation, enzymolysis, dialysis and ethanol gradient purification. The morchella mycelium extract disclosed by the invention has an effect of regulating intestinal flora, specifically, the morchella mycelium extract is used for improving the abundance of Enterococcus, Citrobacter and Bacillus species and reducing the abundance of Clostridium and Ruminococcus species, and compared with inulin, the morchella mycelium extract is more easily utilized by the intestinal flora of a human body and is quickly decomposed to generate short-chain fatty acid.

Description

Technical Field

[0001] This invention relates to the field of natural products. More specifically, this invention relates to the application of a morel mycelium extract. Background Technology

[0002] Morel mushrooms ( Morchella esculenta Morel mushrooms, a rare edible and medicinal fungus, are rich in polysaccharides, amino acids, vitamins, minerals, and other active ingredients. They possess various physiological functions, including antioxidant, immunomodulatory, and antitumor effects, and have broad application prospects in the food and pharmaceutical fields. However, current research on morel mushrooms largely focuses on the nutritional composition analysis of the fruiting body, extraction of active substances, and verification of single physiological functions, with relatively little research on its mycelial extracts. In fact, compared to the fruiting body, morel mycelium has core advantages such as a shorter growth cycle, stable and controllable yield, higher content of active ingredients, and better bioavailability, making it more suitable for industrial-scale production and applications in food, health products, and biomedicine. Crucially, existing research has not yet explored the regulatory effects of morel mycelial extracts on the gut microbiota, resulting in its potential medicinal value not being fully explored. Meanwhile, existing methods for regulating gut microbiota have limitations. For example, probiotic regulation suffers from weak survival rates, low intestinal colonization rates, and significant individual variability. While the use of polysaccharides as prebiotics to regulate gut microbiota has been extensively studied, it still faces the following technical biases or challenges: 1. Conventional prebiotics (such as inulin) have low molecular weights or wide distributions, limiting their utilization speed and efficiency by gut microbiota and easily leading to significant individual variability and gastrointestinal issues; 2. Most naturally derived polysaccharides have complex structures and non-uniform molecular weights, resulting in unstable biological activity and unclear mechanisms of action, making standardized and targeted regulation difficult. Therefore, there is an urgent need to develop novel, efficient, and safe gut microbiota regulators. Summary of the Invention

[0003] The purpose of this invention is to provide an application of morel mycelium extract to at least solve the above-mentioned problems.

[0004] To achieve the objectives and other advantages of this invention, an application of morel mycelial extract in the preparation of products with intestinal flora regulation functions is provided. The morel mycelial extract is mainly composed of α-glucan, wherein the weight-average molecular weight (Mw) of the α-glucan is 10,000 kDa to 15,000 kDa, the number-average molecular weight (Mn) is 10,000 kDa to 12,000 kDa, the polydispersity index (Mw / Mn) is 1.0-1.3, and the glucose molar percentage is not less than 99%. The intestinal flora regulation function specifically involves improving… Enterococcus , Citrobacter and Bacillus Species abundance of genera, decreased Clostridium andRuminococcus species abundance; The preparation method of the Morel mycelium extract comprises the following steps: Step one, after the Morel spores are activated, they are inoculated into PDB liquid medium for liquid fermentation, and the liquid fermentation conditions are as follows: temperature 23-27℃, stirring speed 130-170rpm, aeration amount 0.9-1.1vvm, and fermentation time 3-4 days, and then the mycelium is obtained by filtration; Step two, the mycelium is vacuum freeze-dried and crushed to obtain mycelium powder; Step three, the mycelium powder is mixed with water, and ultrasonic-assisted extraction is performed, the ultrasonic power is 250-350W, the temperature is 70-90℃, and the extraction time is 1-3 hours, and then the supernatant is collected by centrifugation; Step four, after the supernatant is concentrated, 4-5 times the volume of anhydrous ethanol is added for alcohol precipitation, and then the precipitate is collected by centrifugation; Step five, after the precipitate is redissolved, papain is added for enzymatic hydrolysis to remove proteins; Step six, the solution after enzymatic hydrolysis is dialyzed in a dialysis bag with a molecular weight of 3-4kD for 2-3 days to remove small molecular impurities; Step seven, anhydrous ethanol is added to the dialyzed solution to make the ethanol volume concentration in the system 15%-25%, and the supernatant is collected by centrifugation after precipitation at 4℃ overnight, and then anhydrous ethanol is continuously added to the supernatant to make the ethanol volume concentration in the system 35%-45%, and the precipitate is collected by centrifugation after precipitation at 4℃ overnight; Step eight, the precipitate is vacuum freeze-dried to obtain the Morel mycelium extract.

[0005] Preferably, in step one, the activation treatment comprises inoculating the Morel spores into 150ml PDB liquid medium, and then culturing at 25℃ and 150rpm for 3 days on a shaking table, and then crushing the obtained mycelial ball for subsequent liquid fermentation.

[0006] Preferably, in step three, the mass-volume ratio of the mycelium powder to water is 1:30, and after mixing by stirring, the mixture is left standing for 30min.

[0007] Preferably, in step three, after the first ultrasonic-assisted extraction, water is added to the precipitate obtained after centrifugation again for the second ultrasonic-assisted extraction, and then the supernatant is collected by centrifugation, and the supernatants of the two times are combined for subsequent alcohol precipitation.

[0008] Preferably, in step five, the amount of papain is 0.1% of the weight of the mycelium powder, the enzymatic hydrolysis temperature is 60℃, and the time is 30min, and after the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling water bath for 5min.

[0009] The present application at least comprises the following advantages: Firstly, the present application provides a preparation method of Morel mycelium extract with stable process, complete active ingredient and high extraction efficiency, realizes efficient utilization of Morel mycelium resources, and the prepared extract has a glucose purity of more than 99%, and presents a combination of ultra-high molecular weight (Mw 10000-15000 kDa) and extremely narrow molecular weight distribution (polydispersity index Mw / Mn 1.0-1.3).

[0010] Secondly, compared with existing probiotics and prebiotics (inulin), the Morel mycelium extract of the present application can significantly promote the generation of short-chain fatty acids in the intestinal tract, and can further optimize the intestinal flora structure by enriching a variety of beneficial bacteria and reducing the abundance of potential harmful bacteria, and has no problem of weak survival ability of probiotics, is suitable for a wider population, and has more stable regulation effect. Other advantages, objects and features of the present application will be embodied in part by the following description, and will be understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is an ion chromatogram of monosaccharide standard and Morel mycelium extract; Figure 2 is an absolute molecular weight analysis diagram of Morel mycelium extract; Figure 3 is an infrared absorption curve of Morel mycelium extract; Figure 4 is a total ion flow diagram of partial acid hydrolysis of Morel mycelium extract; Figure 5 is a secondary mass spectrum diagram of partial acid hydrolysis of Morel mycelium extract; Figure 6 is a total short-chain fatty acid content change diagram of in vitro fecal bacteria fermentation; Figure 7 is a columnar diagram of intestinal flora composition at genus level of in vitro fecal bacteria fermentation. DETAILED DESCRIPTION

[0012] The present application will be further described in detail below in combination with examples and drawings, so that those skilled in the art can implement the present application according to the description.

[0013] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0014] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials mentioned are commercially available. The core of this invention lies in the specific preparation process of the morel mycelial extract and its achieved uses, rather than relying on a specific source of morel strains. Any morel (Morchella esculenta) strain capable of producing mycelium through liquid fermentation is suitable for this invention. The strains mentioned are those that can be routinely obtained by those skilled in the art from public culture collection centers (such as CGMCC, CCTCC, ACCC, etc.) or commercial culture companies, or from commercially available morel (Morchella esculenta) strains. Morchella esculenta The fruiting bodies are obtained by separation and purification from raw materials.

[0015] Example 1: A method for preparing morel mycelium extract includes the following steps: (a) Liquid fermentation culture of morel mycelium Strain selection: Commercially available morel (Morchella esculenta) fruiting bodies were used as raw materials, and the strains were obtained through tissue isolation, purification, and culture on PDA medium.

[0016] Activation of the strain: First, activate the morel mushrooms ( Morchella esculenta The bacterial strain was activated and cultured on PDA medium until it covered the entire plate. Then, three morel mushroom spawn blocks (obtained by punching holes with a 5mm diameter punch) were inoculated into 150ml of PDB liquid medium and cultured on a shaker at 25℃ and 150 rpm for 3 days. The mycelial balls were then broken up using a sterilized pulverizer for liquid fermentation.

[0017] Fermentation process parameters: The activated morel mushroom strain was inoculated into PDB fermentation medium at an inoculation rate of 5% (v / v) and liquid fermentation was carried out for 3 days at a temperature of 25℃, a stirring speed of 150 rpm and an aeration rate of 1.0 vvm. (II) Preparation of Morel Mycelium Extract Mycelium collection and pretreatment: The fermentation broth was filtered through a 100-mesh filter to separate morel mycelium. It was washed 2-3 times with deionized water to remove residual culture medium, and then dried in a vacuum freeze dryer for 48 hours. After being pulverized, it was passed through an 80-mesh sieve to obtain morel mycelium powder. Ultrasonic-assisted extraction: accurately weigh 13 g of Morchella esculenta powder, dissolve in 390 ml (1:30) distilled water, stir and stand for 30 min. Ultrasonic extraction at 300 W, 80°C for 2 hours, cool and centrifuge (5000 rpm, 15 min) to take the supernatant. Add 390 ml (1:30) distilled water to the precipitate and repeat the extraction once. Combine the supernatants and concentrate to 1 / 5 of the extraction volume by rotary evaporation. Add 4 times the volume of anhydrous ethanol to the concentrated solution, precipitate at 4°C overnight, centrifuge (5000 rpm, 15 min) to take the precipitate. Redissolve the precipitate in 156 ml water, add papain, and enzymatically digest at 60°C for 30 min (enzyme dosage: 0.1% based on the weight of mycelium powder). Inactivate the enzyme in a boiling water bath for 5 min, quickly cool to room temperature, and centrifuge at 5000 r / min. Dialysis bag (3.5 kD) dialysis of the supernatant for 2 days.

[0018] Purification and drying: add a certain amount of anhydrous ethanol to the extraction solution after dialysis to make the ethanol volume concentration of the solution 20%, precipitate at 4°C overnight, centrifuge at 5000 rpm (4°C) for 15 min, take the supernatant, add a certain amount of anhydrous ethanol to make the ethanol volume concentration of the solution 40%, precipitate at 4°C overnight, centrifuge at 5000 rpm (4°C) for 15 min, redissolve the precipitate in 300 ml water, freeze-dry (temperature -80 to -60°C, vacuum degree 10-20 Pa) to prepare a powder extract, i.e. Morchella esculenta mycelium extract 0.560 g, yield 4.31%.

[0019] Experimental Example 1: (1) Monosaccharide composition of Morchella esculenta mycelium extract Experimental method: take a clean chromatographic bottle, weigh an appropriate amount of Morchella esculenta mycelium extract sample, add 1 ml of 2M TFA acid solution, heat at 121°C for 2 hours. Blow dry with nitrogen. Add 99.99% methanol for washing, then blow dry again, repeat the methanol washing 2-3 times. Dissolve with sterile water and transfer to a chromatographic bottle for testing.

[0020] The chromatography system used was a Thermo ICS 5000+ ion chromatography system, which used an electrochemical detector to analyze and detect monosaccharide components. A Dionex™ CarboPac™ PA20 liquid chromatography column was used; the sample size was 5 μl. The mobile phase A (H2O), mobile phase B (0.1M NaOH), and mobile phase C (0.1M NaOH, 0.2M NaAc) had a flow rate of 0.5ml / min; the column temperature was 30℃; the elution gradient was 0min A phase / B phase / C phase (95:5:0, V / V), 26min A phase / B phase / C phase (85:5:10, V / V), 42min A phase / B phase / C phase (85:5:10, V / V), 42.1min A phase / B phase / C phase (60:0:40, V / V), 52min A phase / B phase / C phase (60:40:0, V / V), 52.1min A phase / B phase / C phase (95:5:0, V / V), 60min A phase / B phase / C phase (95:5:0, V / V).

[0021] The experimental results are shown in Table 1. Figure 1 As can be seen from Table 1, the monosaccharide composition of the Morel mycelium extract (MEP40) was arabinose, galactose, glucose, and mannose, and the molar ratio of their contents was 0.10%:0.18%:99.20%:0.53%. It can be seen that most of the extract was glucose, and the extract was a glucan.

[0022] (2) Absolute molecular weight of the Morel mycelium extract Experimental method: The differential detector was used to detect the concentration information of the Morel mycelium extract sample according to its refractive intensity, and the multi-angle laser light scattering instrument was used to detect the light scattering information of the macromolecule, and the molecular weight corresponding to the component was calculated according to the Mark-Houwink equation.

[0023] The experimental results are shown in Table 2. Figure 2 The red line represents the multi-angle laser light scattering signal (i.e. LS, unit: V), and the scattering light intensity is proportional to the molecular size and molecular weight of the substance; the blue line represents the differential signal (i.e. RI, unit: RIU), and the response value depends on the change in the refractive index of the effluent after the column, and is related to the type, concentration, and molecular weight of the substance; the black line is the molecular weight fitted from the two signals. The inorganic salt phase around 37min is the solvent peak of the mobile phase. The chromatography data was processed using software ASTRA 6.1. Figure 2 The absolute molecular weight analysis chart of the sample (with the detected retention time (Time, min) as the horizontal coordinate and the molar mass (g / mol) as the vertical coordinate) is shown in Table 3. According to the chart, the number average molecular mass Mn was 11097.312 kDa, the weight average molecular weight Mw was 13476.076 kDa, and the polydispersity index Mw / Mn was 1.214.

[0024] (3) Infrared absorption spectrum of Morchella mycelium extract Experimental method: A small amount of Morchella mycelium extract sample was mixed with 200 mg of potassium bromide, pressed into a 1 mm thick sheet, and then detected on the machine. Scanning analysis was performed using a Nicolet iZ-10 Fourier transform infrared spectrometer with a resolution of 4.00 cm -1 -1, a scanning range of 4000-450 cm -1 , and a scanning number of 32. The sampling gain was 8.0; the moving mirror speed was 0.4747; the aperture was 80.00; the DTGS KBr detector; the KBr beam splitter; and the infrared light source.

[0025] The experimental results are shown in Figure 3 . The following several strong absorption peaks can be observed. The absorption peak at 3274.24 cm -1 is the O-H stretching vibration absorption peak, which is a characteristic peak of sugars. The absorption peak at 2923.89 cm -1 belongs to C-H stretching vibration. There is an absorption peak at 1077.68 cm -1 , which belongs to C-O stretching vibration. There is an absorption peak at 758.62 cm -1 , which belongs to α-substitution.

[0026] (4) Partial acid hydrolysis of Morchella mycelium extract Experimental method: The Morchella mycelium extract sample was dissolved in dimethyl sulfoxide (DMSO), and iodomethane was added in the DMSO / NaOH system for methylation reaction. After complete methylation, 2 mol / L trifluoroacetic acid (TFA) was used for hydrolysis at 121°C for 1.5 hours, and after reduction with sodium borodeuteride (NaBD4), acetylation was performed at 100°C for 2.5 hours with acetic anhydride. The acetylated product was dissolved in chloroform, and gas chromatography-mass spectrometry (GC-MS) was used for analysis. An Agilent 6890A-5977B GC-MS system was used, equipped with a BPX70 chromatographic column.

[0027] The initial temperature for mass spectrometry analysis was 140°C, maintained for 2 min, then increased to 230°C at a rate of 3°C / min, and maintained for 3 min. The scanning mode was full scan (SCAN), and the mass range (m / z) was 50-350.

[0028] The peak position of each methylation product was finally identified by ion fragments in mass spectrometry and relative retention time in gas chromatography.

[0029] The experimental results are shown in Figure 4 and Figure 5 . According to the characteristic fragments after methylation of polysaccharides, it is inferred that it contains 1,4 glycosidic bonds.

[0030] (5) NMR scanning of the Morchella mycelium extract Experimental method: Take an appropriate amount of Morchella mycelium extract and dissolve it in D2O to prepare a solution with a concentration of 40 mg / mL or more. Transfer the dissolved solution to a NMR tube and add 0.5 mL. Place the NMR tube in the NMR spectrometer and scan the one-dimensional 1 H spectrum, 13 C spectrum, DEPT135, two-dimensional COSY, HSQC, TOCSY, NOESY and HMBC spectrum.

[0031] The experimental results are shown in Table 1. It can be seen that the glycosidic bond type of the Morchella mycelium extract is →4)-α-D-Glcp-(1→.

[0032] Table 1 Chemical shifts of each sugar residue in the Morchella mycelium extract 1 H and 13 C (6) In vitro fecal bacteria fermentation experiment Experimental method: The preparation method of the basic medium is as follows: dissolve 0.01 g of magnesium sulfate heptahydrate (MgSO4·7H2O), 0.04 g of potassium dihydrogen phosphate (KH2PO4), 0.04 g of dipotassium hydrogen phosphate (K2HPO4), 2.0 g of sodium bicarbonate (NaHCO3), 0.01 g of calcium chloride hexahydrate (CaCl2·6H2O), 0.1 g of sodium chloride (NaCl), 2.0 g of proteose peptone, 0.5 g of cholate, 2.0 g of yeast extract, 10 μL of vitamin K1, 0.02 g of hemin, 0.5 g of cysteine-HCl, 2.0 mL of Tween-80 (Tween 80) and 0.01 g of resazurin in 1 L of distilled water.

[0033] The fecal samples came from 6 healthy individuals, including 3 males and 3 females, aged between 20-30 years old. These individuals had a balanced diet and had not used antibiotics or prophylactic drugs in the past three months. The fecal samples were quickly diluted with sterile modified saline solution (containing 9.0 g / L of sodium chloride and 0.5 g / L of cysteine-HCl) to prepare a 10% fecal suspension. After homogenization, the supernatant was obtained by centrifugation as the final human fecal bacteria inoculum.

[0034] The supernatant was added to a bottle, and then mixed with 9.0 mL of sterile basal medium containing 50.0 mg of Morchella mycelium extract, which was the MEP40 group. The mixture was incubated at 37°C, and the anaerobic environment of fecal fermentation was maintained throughout the process by an anaerobic incubator. The anaerobic environment was composed of 5% hydrogen (H2), 5% carbon dioxide (CO2), and 90% nitrogen (N2). The basal medium was used as a blank control group (BLK group), and the basal medium containing 50.0 mg of inulin was used as a positive control group (INL group), both of which were incubated under the same conditions. Fermentation samples were collected at 6 hours, 12 hours, and 24 hours of incubation for subsequent analysis.

[0035] The content of short-chain fatty acids (SCFAs) was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) equipped with an electrospray ionization detector (ESI). The brief steps are as follows: 100 mL of acetonitrile was added to 50 mL of fermentation supernatant, and low-temperature ultrasonic extraction was performed at 5°C and 40 kHz for 30 min, followed by centrifugation at 4°C and 15,000 rpm for 15 min. The supernatant was taken, and 20 μL of 3N PH⋅HCl (200 mM) solution and 20 μL of EDC⋅HCl (120 mM) solution (containing 6% pyridine) were added in sequence, and derivatization was performed at 40°C for 30 min. After derivatization, the mixture was diluted to 750 μL with 50% acetonitrile aqueous solution for analysis. The chromatographic separation used a Waters BEH C18 column, and the ultra-high performance liquid chromatography elution conditions were set as follows: 0-2 minutes, mobile phase B ratio 10%; 2-11 minutes, mobile phase B ratio from 10% to 55%; 11-12 minutes, mobile phase B ratio from 55% to 95%; 12-13 minutes, mobile phase B ratio remained 95%; 13-13.1 minutes, mobile phase B ratio from 95% to 10%; 13.1-16 minutes, mobile phase B ratio remained 10%. The injection volume was 2 μL, and the flow rate was 0.35 mL / min. Mass spectrometry data were collected in negative ion multiple reaction monitoring mode (MRM), and the content of short-chain fatty acids was finally calculated by standard curve.

[0036] Fecal bacteria were collected by centrifugation and stored at -80°C. DNA was extracted using a DNA extraction kit. The quality and concentration of the DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer, and then stored at -80°C for later use. 16S rRNA gene sequencing was completed by Shanghai Meiji Biomedical Technology Co., Ltd. The brief steps are as follows: the V3-V4 region was amplified by PCR with primers 338F and primers 806R. The PCR products were recovered from a 2% agarose gel. The purified amplicons were mixed in equal molar amounts, and double-end sequencing was performed on an Illumina Nextseq2000 sequencing platform.

[0037] The experimental results are shown in Figure 6 and Figure 7 . Figure 6 For the change in the total short-chain fatty acid content in the in vitro fecal bacteria fermentation experiment, it can be seen that after 12 hours of fermentation, the total short-chain fatty acid content (1.35 x 10 5 ± 23274.52 ng / ml) of the sample group was significantly higher than that of the blank control group (0.19 x 10 5 ± 2095.40 ng / ml) and the positive control group (inulin, 0.40 x 10 5 ± 9108.83 ng / ml); after 24 hours of fermentation, the total short-chain fatty acid content in each group further increased, among which the sample group was 2.15 x 10 5 ± 18418.09 ng / ml, the blank control group was 0.33 x 10 5 ± 2744.65 ng / ml, and the positive control group was 0.81 x 10 5 ± 4133.34 ng / ml. As a typical prebiotic, inulin has been confirmed by a large number of studies to be efficiently utilized by various dominant intestinal flora in the human body, and to stably produce short-chain fatty acids after fermentation, and its acid production mode and flora regulation effect are clear, which indicates that the sample group is more easily metabolized by intestinal flora than inulin and can more efficiently promote the generation of short-chain fatty acids. Figure 7 For the change in the composition of the intestinal flora in the in vitro fecal bacteria fermentation experiment, after 12 hours of fermentation, the sample group showed a significant flora optimization effect: compared with the blank control group, the abundance of beneficial bacteria genera Enterococcus , Citrobacter , Bacillus significantly increased, while the abundance of potentially harmful bacteria genera Clostridium , Ruminococcus significantly decreased; compared with the positive control group (inulin), Citrobacter , Bacillus the abundance of the two types of beneficial bacteria genera further significantly increased, indicating that the sample group is superior to inulin in enriching specific beneficial bacteria. Among them, EnterococcusSome strains of the genus can produce bacteriocin "enterocin", which can effectively inhibit pathogenic bacteria such as Listeria and Staphylococcus aureus; Citrobacter As a human intestinal symbiotic bacteria, some strains of the genus can decompose complex nutrients and improve the efficiency of nutrient absorption. Bacillus The genus has the core benefits of regulating intestinal flora balance and enhancing immune function, and some strains also have the effects of antioxidant and maintaining blood circulation health. Clostridium Some strains of the genus can produce toxins, which can easily cause food poisoning and cause abdominal pain, diarrhea and other discomforts. Ruminococcus Some strains of the genus can produce inflammatory polysaccharides, induce dendritic cells to secrete inflammatory cytokines such as tumor necrosis factor, and then induce inflammatory reactions.

[0038] In summary, the method of the present application can extract an alpha-glucan from the Morchella mycelium, which has a number average molecular weight Mn of 11097.312 kDa, a weight average molecular weight Mw of 13476.076 kDa, a polydispersity index Mw / Mn of 1.214, and a glucose molar percentage of 99.20%. The specific narrow-distribution ultrahigh molecular weight structure of the alpha-glucan is more easily utilized by the human intestinal flora than inulin, rapidly decomposes to produce short-chain fatty acids, and has a unique directional intestinal flora regulation function.

[0039] Comparative Example 1: A preparation method of Morchella mycelium extract, the main steps are the same as those of Example 1, except that in the purification and drying step, anhydrous ethanol is directly added to the dialyzed extract to make the ethanol volume concentration in the system reach 40%, and the precipitate is collected after 4℃ precipitation overnight and 5000 rpm (4℃) centrifugation for 15 min. The subsequent treatment is the same as that of Example 1. Morchella mycelium extract 0.715g, yield 5.50%.

[0040] Comparative Example 2: A preparation method of Morchella mycelium extract, the main steps are the same as those of Example 1, except that in the purification and drying step, anhydrous ethanol is directly added to the dialyzed extract to make the ethanol volume concentration in the system reach 40%, and the precipitate is collected after 4℃ precipitation overnight and 5000 rpm (4℃) centrifugation for 15 min. The subsequent treatment is the same as that of Example 1. Morchella mycelium extract 0.715g, yield 5.50%.

[0041] Comparative Example 3: A preparation method of the Morel mycelium extract, the main steps of which are the same as those of Example 1, except that in the purification and drying step, anhydrous ethanol is added to the dialyzed extract to make the volume concentration of ethanol in the system 20%, and the supernatant is collected after precipitation at 4°C overnight and centrifugation at 5000 rpm (4°C) for 15 min. Then anhydrous ethanol is continuously added to the supernatant to make the volume concentration of ethanol in the system 60%, and the precipitate is collected after precipitation at 4°C overnight and centrifugation at 5000 rpm (4°C) for 15 min, and the subsequent processing is the same as that of Example 1. The Morel mycelium extract 0.652 g is obtained, and the yield is 5.02%.

[0042] Experimental Example 2: The concentration information of the Morel mycelium extract samples prepared in Comparative Examples 1 to 3 is detected by a differential detector according to the refractive intensity thereof, and the light scattering information of macromolecules is detected by a multi-angle laser light scattering instrument, and the molecular weight of the components is calculated according to the Mark-Houwink equation. The results are shown in Table 2.

[0043] Table 2 Influence of different purification methods on the yield of extract, weight average molecular weight and polydispersity index As can be seen from Table 2, the one-time alcohol precipitation (Comparative Example 1) has a slightly higher yield, but the molecular weight distribution of the obtained product is significantly widened (Mw / Mn = 1.651), which proves that the one-time alcohol precipitation cannot effectively separate and purify the target active components, and a large amount of impurity polysaccharides with different molecular weights will be introduced. The first-stage ethanol concentration is too low (Comparative Example 2), which cannot effectively remove some interfering components, resulting in a wide molecular weight distribution of the final product and affecting the specificity of its bioavailability. The second-stage ethanol concentration is too high (Comparative Example 3), which will co-precipitate a large amount of polysaccharide components with low molecular weight, no activity or low activity, resulting in a sharp drop in the molecular weight of the product and a wide distribution again.

[0044] Further, the Morel mycelium extract samples prepared in Comparative Examples 1 to 3 are subjected to in vitro fecal bacteria fermentation experiment, and the fermentation samples are collected at 6 hours, 12 hours and 24 hours of culture, respectively, and the content of short-chain fatty acids (SCFAs) in the fermentation samples is determined by ultra-high performance liquid chromatography-tandem mass spectrometry. The results are shown in Table 3.

[0045] Table 3 Influence of different extracts on the total short-chain fatty acid content (×10 5 ng / mL) in in vitro fecal bacteria fermentation Note: The data is represented by mean ± standard deviation (n = 3) From Table 3, firstly, the content of short-chain fatty acids of all extract groups increased with the extension of fermentation time, showing time dependence, in which the MEP40 group maintained the highest content at each time point. Secondly, the molecular weight and its distribution had a decisive influence on the activity. The MEP40 group had the highest weight average molecular weight and the narrowest polydispersity index, which showed a significant metabolic starting advantage at the early stage of fermentation (6 hours), far exceeding the content of inulin and other comparative groups, and further expanded the advantage at 12 hours and 24 hours. In contrast, the comparative extract groups (Comparative Example 1 to Comparative Example 3) with lower molecular weight or wider distribution had different degrees of weakening in the ability to promote the generation of short-chain fatty acids, which confirmed that high molecular weight and narrow distribution were the key structural features to exert the best prebiotic activity.

[0046] In summary, the specific purification step of "taking supernatant with 15%-25% ethanol in the first stage and taking precipitate with 35%-45% ethanol in the second stage" defined in the present application is an indispensable key step to obtain the narrow-distribution, high-molecular-weight, and high-activity α-glucan, and the Morel mycelium extract with the characteristics of narrow distribution and ultra-high molecular weight has the optimal efficiency in promoting the production of short-chain fatty acids by intestinal flora.

[0047] The number of devices and the scale of processing described herein are used to simplify the description of the present application. The applications, modifications, and variations of the Morel mycelium extract of the present application are obvious to those skilled in the art.

[0048] Although the embodiments of the present application have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. Use of a Morel mycelium extract for the preparation of a product having a modulating effect on the functioning of the intestinal flora, characterized in that, The Morchella mycelium extract is mainly alpha-glucan, the weight average molecular weight Mw of the alpha-glucan is 10000 kDa to 15000 kDa, the number average molecular weight Mn is 10000 kDa to 12000 kDa, the polydispersity index Mw / Mn is 1.0-1.3, and the glucose molar percentage is not less than 99%; the regulation of intestinal flora function is specifically to increase Enterococcus 、 Citrobacter and Bacillus abundance of the genus, reduce Clostridium and Ruminococcus abundance of the genus; The preparation method of the Morel mycelium extract comprises the following steps: Step one, after the Morel spores are activated, the spores are inoculated into PDB liquid medium for liquid fermentation, and the liquid fermentation conditions are as follows: temperature 23-27 DEG C, stirring speed 130-170 rpm, ventilation amount 0.9-1.1 vvm, and fermentation time 3-4 days, and then the mycelium is obtained by filtration; Step two, the mycelium is vacuum freeze-dried and crushed to obtain mycelium powder; Step three, the mycelium powder is mixed with water, and ultrasonic assisted extraction is carried out, the ultrasonic power is 250-350 W, the temperature is 70-90 DEG C, the extraction time is 1-3 hours, and the supernatant is collected by centrifugation; Step four, after the supernatant is concentrated, 4-5 times concentrated liquid volume of anhydrous ethanol is added for alcohol precipitation, and the precipitate is collected by centrifugation; Step five, after the precipitate is redissolved, papain is added for enzymatic hydrolysis treatment to remove protein; Step six, the solution after enzymolysis is dialyzed for 2-3 days with a dialysis bag with a molecular weight of 3-4 kD to remove small molecular impurities; Step seven, anhydrous ethanol is added to the dialyzed solution, the volume concentration of ethanol in the system is 15%-25%, and the supernatant is collected by centrifugation after precipitation at 4 DEG C overnight; anhydrous ethanol is continuously added to the supernatant to make the volume concentration of ethanol in the system 35%-45%, and the precipitate is collected by centrifugation after precipitation at 4 DEG C overnight; Step eight, the precipitate is vacuum freeze-dried to obtain the Morel mycelium extract.

2. Use according to claim 1, wherein In step one, the activation treatment comprises the following steps: the Morel spores are inoculated into 150 ml PDB liquid medium, and the mycelium ball is crushed after being cultured at 25 DEG C and 150 rpm for 3 days in a shaking bed, and then used for subsequent liquid fermentation.

3. The use according to claim 1, wherein In step three, the mass / volume ratio of the mycelium powder and water is 1:30, and after stirring and mixing, the mixture is placed for 30 min.

4. The use according to claim 3, wherein the compound is ###00002### In step three, after the first ultrasonic assisted extraction, water is added to the precipitate obtained after centrifugation again for the second ultrasonic assisted extraction, the supernatant is collected by centrifugation, and the supernatants of the two times are combined for subsequent alcohol precipitation.

5. The use according to claim 3, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. In step five, the amount of papain is 0.1% of the weight of the mycelium powder, the enzymolysis temperature is 60 DEG C, and the enzymolysis time is 30 min, and the enzyme is inactivated by boiling water bath for 5 min after enzymolysis.

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