Use of morel mycelium extract

Abstract

This invention discloses the application of a morel mycelium extract, belonging to the field of natural products. The morel mycelium extract is mainly composed of α-glucan, with a weight-average molecular weight of 10,000 kDa to 15,000 kDa, a number-average molecular weight of 10,000 kDa to 12,000 kDa, a polydispersity index of 1.0-1.3, and a glucose molar percentage of not less than 99%. The preparation method includes ultrasonic-assisted extraction, alcohol precipitation, enzymatic hydrolysis, dialysis, and ethanol gradient purification. The morel mycelium extract of this invention has the effect of regulating intestinal flora, specifically by improving… Enterococcus , Citrobacter and Bacillus Species abundance of genera, decreased Clostridium and Ruminococcus It has a high species abundance and is more easily utilized by the human gut microbiota than inulin, rapidly decomposing to produce short-chain fatty acids.

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 Genus species abundance, decreased Clostridium andRuminococcus Abundance of species;

[0005] The preparation method of the Morel mycelium extract comprises the following steps:

[0006] 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.

[0007] Step two, the mycelium is vacuum freeze-dried and crushed to obtain mycelium powder.

[0008] 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.

[0009] 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.

[0010] Step five, after the precipitate is redissolved, papain is added for enzymatic treatment to remove proteins.

[0011] Step six, the solution after enzymolysis is dialyzed in a dialysis bag with a molecular weight of 3-4kD for 2-3 days to remove small molecular impurities.

[0012] 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.

[0013] Step eight, the precipitate is vacuum freeze-dried to obtain the Morel mycelium extract.

[0014] 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 balls for subsequent liquid fermentation.

[0015] Preferably, in step three, the mass-volume ratio of the mycelium powder to water is 1:30, and after stirring and mixing, the mixture is allowed to stand for 30min.

[0016] 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.

[0017] Preferably, in step five, the amount of papain used is 0.1% of the weight of the mycelium powder, the enzymolysis temperature is 60℃, and the time is 30 min. After the enzymolysis is completed, the enzyme is inactivated by boiling in water for 5 min.

[0018] The present application at least includes the following beneficial effects:

[0019] Firstly, the present application provides a preparation method of a 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).

[0020] 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, can more effectively enrich a variety of beneficial bacteria and reduce the abundance of potentially harmful bacteria, can comprehensively play an active role in optimizing the intestinal flora structure, does not have the problem of weak survival ability of probiotics, is suitable for a wider population, and has more stable regulation effect.

[0021] Other advantages, objects and features of the present application will be partly embodied by the following description, and will be partly understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is an ion chromatogram of a monosaccharide standard and a morel mycelium extract;

[0023] Figure 2 is an absolute molecular weight analysis diagram of the morel mycelium extract;

[0024] Figure 3 is an infrared absorption curve of the morel mycelium extract;

[0025] Figure 4 is a total ion flow diagram of partial acid hydrolysis of the morel mycelium extract;

[0026] Figure 5 is a secondary mass spectrum diagram of partial acid hydrolysis of the morel mycelium extract;

[0027] Figure 6 is a total short-chain fatty acid content change diagram of in-vitro fecal bacteria fermentation;

[0028] Figure 7 is a columnar diagram of intestinal flora composition at the genus level of in-vitro fecal bacteria fermentation. DETAILED DESCRIPTION

[0029] The present application will be further described in conjunction with the following examples and drawings, so that those skilled in the art can implement the present application according to the description herein.

[0030] 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.

[0031] It should be noted that the experimental methods in the following embodiments are all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial channels. The core of the present application lies in the specific preparation process of the Morchella mycelium extract and its use, rather than relying on a specific source of Morchella strain. Any strain belonging to Morchella (Morchella esculenta) and capable of producing mycelium by liquid fermentation is suitable for the present application. The strain can be routinely obtained by a person skilled in the art from a public strain preservation center (such as CGMCC, CCTCC, ACCC, etc.) or a commercial strain company, or a commercially available Morchella (Morchella esculenta) fruiting body is used as raw material to obtain the strain through isolation and purification. Morchella esculenta The fruiting body of Morchella esculenta is used as raw material to obtain the strain through tissue isolation and purification on PDA medium.

[0032] Example 1:

[0033] A preparation method of Morchella mycelium extract, comprising the following steps:

[0034] (I) Liquid fermentation culture of Morchella mycelium

[0035] Strain selection: The fruiting body of commercially available Morchella esculenta is used as raw material to obtain the strain through tissue isolation and purification on PDA medium.

[0036] Strain activation: First, the Morchella esculenta strain is activated and cultured on PDA medium until the plate is covered, then 3 pieces of Morchella esculenta strain blocks (diameter 5 mm punched by puncher) are inoculated in 150 ml PDB liquid medium, cultured at 25°C, 150 rpm for 3 days in a shaking bed. The mycelium ball is broken by a sterilized crusher and used for liquid fermentation. Morchella esculenta

[0037] Fermentation process parameters: The activated Morchella esculenta strain is inoculated into PDB fermentation medium at an inoculation amount of 5% (v / v), and liquid fermentation culture is carried out at a temperature of 25°C, a stirring speed of 150 rpm, and a ventilation amount of 1.0 vvm for 3 days.

[0038] (II) Preparation of Morchella mycelium extract

[0039] ​Mycelium collection and pretreatment: The fermentation broth was filtered through a 100-mesh sieve to separate the Morchella mycelium, which was washed 2-3 times with deionized water to remove residual culture medium, then dried in a vacuum freeze dryer for 48 hours, crushed and sieved through an 80-mesh sieve to obtain Morchella mycelium powder.

[0040] Ultrasonic-assisted extraction: 13 g of Morchella powder was accurately weighed and dissolved in 390 ml (1:30) of distilled water. After stirring, it was allowed to stand for 30 min. Ultrasonic extraction was performed at 300 W and 80°C for 2 hours. After cooling, centrifugation was performed (5000 rpm, 15 min) to obtain the upper liquid. The precipitate was extracted again with 390 ml (1:30) of distilled water, and the supernatant was combined. The solution was concentrated to 1 / 5 of the original volume by rotary evaporation, and 4 times the volume of anhydrous ethanol was added. Precipitation was performed at 4°C overnight, and centrifugation was performed (5000 rpm, 15 min) to obtain the precipitate. The precipitate was re-dissolved in 156 ml of water, and papain was added. Enzymatic hydrolysis was performed at 60°C for 30 min (enzyme dosage: 0.1% based on the weight of the mycelium powder), and the enzyme was inactivated by boiling in a water bath for 5 min. After rapid cooling to room temperature, centrifugation was performed at 5000 r / min, and the supernatant was dialyzed in a dialysis bag (3.5 kD) for 2 days.

[0041] Purification and drying: A certain amount of anhydrous ethanol was added to the dialyzed extract to make the ethanol volume concentration of the solution 20%, and precipitation was performed at 4°C overnight. Centrifugation was performed at 5000 rpm (4°C) for 15 min, and the supernatant was collected. A certain amount of anhydrous ethanol was added to make the ethanol volume concentration of the solution 40%, and precipitation was performed at 4°C overnight. Centrifugation was performed at 5000 rpm (4°C) for 15 min, and the precipitate was re-dissolved in 300 ml of water. Freeze-drying was performed (temperature -80 to -60°C, vacuum degree 10-20 Pa) to obtain a powdered extract, i.e., Morchella mycelium extract 0.560 g, with a yield of 4.31%.

[0042] Experimental Example 1:

[0043] (1) Monosaccharide composition of Morchella mycelium extract

[0044] Experimental method: A clean chromatography bottle was weighed, and an appropriate amount of Morchella mycelium extract sample was added. 1 ml of 2M TFA acid solution was added, and heating was performed at 121°C for 2 hours. Nitrogen was blown, and the solution was blown dry. 99.99% methanol was added for washing, and the solution was blown dry again. Methanol washing was repeated 2-3 times. Sterile water was added for dissolution, and the solution was transferred into a chromatography bottle for testing.

[0045] 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).

[0046] 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.

[0047] (2) Absolute molecular weight of Morel mycelium extract

[0048] 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.

[0049] The experimental results are shown in Figure 1. 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 abscissa and the molar mass (g / mol) as the ordinate). From the chart, the number average molecular mass Mn was calculated to be 11097.312 kDa, the weight average molecular weight Mw was calculated to be 13476.076 kDa, and the polydispersity index Mw / Mn was calculated to be 1.214.

[0050] (3) Infrared absorption spectrum of the Morchella mycelium extract

[0051] 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. A Nicolet iZ-10 Fourier transform infrared spectrometer was used for scanning analysis, with an instrument resolution of 4.00 cm -1 , a scanning range of 4000-450 cm -1 , a scanning number of 32 times. 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.

[0052] The experimental results are shown in Table 1. 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.

[0053] (4) Partial acid hydrolysis of the Morchella mycelium extract

[0054] 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.

[0055] 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.

[0056] The peak position of each methylation product was finally identified by ion fragments in mass spectrometry and relative retention time in gas chromatography.

[0057] The experimental results are shown in Table 2 and Table 3. Figure 4 ​Figure 5 The presence of 1,4 glycosidic bonds was deduced from the characteristic fragmentation after methylation of the polysaccharide.

[0058] (5) NMR scanning of the Morchella mycelium extract

[0059] 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.

[0060] 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→.

[0061] Table 1 Chemical shifts of each sugar residue in the Morchella mycelium extract 1 H and 13 C

[0062]

[0063] (6) In vitro fecal bacteria fermentation experiment

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 bacteriocins, "enterocins", 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 nutrient absorption efficiency. 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.

[0070] 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, a glucose molar percentage of 99.20%, and a sugar chain connected by →4)-α-D-Glcp-(1→. The specific narrow-distribution ultrahigh molecular weight structure of the alpha-glucan is more easily utilized by the human intestinal flora than inulin, quickly decomposes to produce short-chain fatty acids, and has a unique directional intestinal flora regulation function.

[0071] Comparative Example 1:

[0072] A preparation method of Morchella mycelium extract, the main steps are the same as Example 1, the difference is that in the purification and drying step, anhydrous ethanol is directly added to the dialyzed extract, the volume concentration of ethanol in the system reaches 40%, and the precipitate is collected after 4℃ precipitation overnight, 5000 rpm (4℃) centrifugation for 15 min, and subsequent processing is the same as Example 1. The Morchella mycelium extract 0.715g, the yield is 5.50%.

[0073] Comparative Example 2:

[0074] 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 added to the dialyzed extract to make the ethanol volume concentration in the system 10%, precipitated at 4°C overnight, centrifuged at 5000 rpm (4°C) for 15 min, and the supernatant was taken. Continue to add anhydrous ethanol to the supernatant to make the ethanol volume concentration in the system 40%, precipitate at 4°C overnight, centrifuge at 5000 rpm (4°C) for 15 min, collect the precipitate, and the subsequent treatment is the same as that of Example 1. Morchella mycelium extract 0.493g, yield 3.79%.

[0075] Comparative Example 3:

[0076] 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 added to the dialyzed extract to make the ethanol volume concentration in the system 10%, precipitated at 4°C overnight, centrifuged at 5000 rpm (4°C) for 15 min, and the supernatant was taken. Continue to add anhydrous ethanol to the supernatant to make the ethanol volume concentration in the system 40%, precipitate at 4°C overnight, centrifuge at 5000 rpm (4°C) for 15 min, collect the precipitate, and the subsequent treatment is the same as that of Example 1. Morchella mycelium extract 0.493g, yield 3.79%.

[0077] Experimental Example 2:

[0078] The concentration information of Morchella mycelium extract samples prepared in Comparative Examples 1 to 3 was detected by a differential detector according to its refractive intensity, and the light scattering information of macromolecules was detected by a multi-angle laser light scattering instrument, and the molecular weight of the components was calculated according to Mark Houwink equation. The results are shown in Table 2.

[0079] Table 2 Influence of different purification methods on extract yield, weight average molecular weight and polydispersity index

[0080]

[0081] As shown in Table 2, using conventional one-time alcohol precipitation (Comparative Example 1), although the yield is slightly higher, the molecular weight distribution of the obtained product is significantly widened (Mw / Mn=1.651), which proves that one-time alcohol precipitation cannot effectively separate and purify the target active component, and will introduce a large amount of impurity polysaccharides with different molecular weights. 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, affecting its biological specificity. The second-stage ethanol concentration is too high (Comparative Example 3), which will co-precipitate a large amount of low-molecular-weight, inactive or low-activity polysaccharide components, resulting in a sharp drop in product molecular weight and a wide distribution again.

[0082] Further, the Morchella mycelium extract samples prepared by Comparative Example 1 to Comparative Example 3 were subjected to in vitro fecal bacteria fermentation experiments, and fermentation samples were collected at 6 hours, 12 hours and 24 hours of culture, respectively, and the contents of short-chain fatty acids (SCFAs) in the fermentation samples were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. The results are shown in Table 3.

[0083] Table 3 Effect of different extracts on the content of total short-chain fatty acids in in vitro fecal bacteria fermentation (x 10 5 ng / mL)

[0084]

[0085] Note: The data are expressed as mean ± standard deviation (n = 3)

[0086] As can be seen from Table 3, first, the contents of short-chain fatty acids in all extract groups increased with the extension of fermentation time, showing time dependence, and the MEP40 group maintained the highest content at each time point. Second, the molecular weight and its distribution have a decisive influence on the activity. The MEP40 group has the highest weight average molecular weight and the narrowest polydispersity index, which shows a significant metabolic starting advantage at the early stage of fermentation (6 hours), with a content far exceeding inulin and other comparative groups, and further expands the advantage at 12 hours and 24 hours. In contrast, the comparative extract (Comparative Example 1 to Comparative Example 3) with lower molecular weight or wider distribution has different degrees of weakening in the ability to promote the generation of short-chain fatty acids, which confirms that high molecular weight and narrow distribution are the key structural features for optimal prebiotic activity.

[0087] In summary, the specific purification step of "taking the supernatant with 15%-25% ethanol in the first stage and taking the precipitate with 35%-45% ethanol in the second stage" defined in the present application is an indispensable key step for obtaining the narrow-distribution, high-molecular-weight, high-activity α-glucan, and the Morchella mycelium extract with narrow distribution and ultra-high molecular weight has the optimal efficiency in promoting the production of short-chain fatty acids by intestinal flora.

[0088] The number of devices and the scale of processing described herein are used to simplify the description of the present application. Applications, modifications and variations of the Morchella mycelium extract of the present application to those skilled in the art are obvious.

[0089] Although the embodiments of the present application have been disclosed as above, they are not limited only to the applications listed in the specification and 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. The application of morel mycelial extract in the preparation of products with intestinal flora regulation function, characterized in that, The morel mycelium extract is mainly composed of α-glucan, which has a weight-average molecular weight (Mw) of 10,000 kDa to 15,000 kDa, a number-average molecular weight (Mn) of 10,000 kDa to 12,000 kDa, a polydispersity index (Mw / Mn) of 1.0-1.3, and a glucose molar percentage of not less than 99%. The regulation of intestinal flora function specifically involves improving... Enterococcus , Citrobacter and Bacillus Species abundance of genera, decreased Clostridium and Ruminococcus Genus species abundance; The method for preparing the morel mycelium extract includes: Step 1: After activating the morel mushroom spawn, inoculate it into PDB liquid culture medium for liquid fermentation. The liquid fermentation conditions are: temperature 23℃-27℃, stirring speed 130-170rpm, aeration rate 0.9-1.1 vvm, fermentation time 3-4 days, filter, and obtain mycelium. Step 2: Vacuum freeze-dry and pulverize the mycelium to obtain mycelium powder; Step 3: Mix the mycelial powder with water and perform ultrasonic-assisted extraction. The ultrasonic power is 250-350W, the temperature is 70-90℃, and the extraction time is 1-3 hours. Centrifuge and collect the supernatant. Step 4: After concentrating the supernatant, add 4-5 times the volume of anhydrous ethanol for alcohol precipitation, centrifuge, and collect the precipitate. Step 5: After redissolving the precipitate, add papain for enzymatic hydrolysis to remove protein; Step 6: Dialyze the enzymatically hydrolyzed solution using a dialysis bag with a molecular weight of 3-4 kD for 2-3 days to remove small molecule impurities; Step 7: Add anhydrous ethanol to the dialysis solution to make the ethanol volume concentration in the system 15%-25%, let it precipitate overnight at 4°C, centrifuge, take the supernatant, and then add anhydrous ethanol to the supernatant to make the ethanol volume concentration in the system 35%-45%, let it precipitate overnight at 4°C, centrifuge, and collect the precipitate. Step 8: Vacuum freeze-dry the precipitate to obtain the morel mycelium extract.

2. The application as described in claim 1, characterized in that, In step one, the activation treatment includes inoculating morel mushroom inoculum into 150ml of PDB liquid culture medium, culturing it in a shaker at 25℃ and 150rpm for 3 days, and then breaking up the resulting mycelial balls for subsequent liquid fermentation.

3. The application as described in claim 1, characterized in that, In step three, the mass-to-volume ratio of mycelial powder to water is 1:

30. After stirring and mixing, let it stand for 30 minutes.

4. The application as described in claim 3, characterized in that, In step three, after the first ultrasonic-assisted extraction, water is added again to the precipitate obtained after centrifugation and mixed for a second ultrasonic-assisted extraction. After centrifugation, the supernatant is collected and the two supernatants are combined for subsequent alcohol precipitation.

5. The application as described in claim 3, characterized in that, In step five, the amount of papain used is 0.1% of the weight of the mycelium powder, the enzymatic hydrolysis temperature is 60℃ and the time is 30 min, and the enzyme is inactivated by boiling water bath for 5 min after the enzymatic hydrolysis is completed.

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