Pleurotus eryngii fruiting body water extraction residue polysaccharide as well as extraction method and application thereof

Abstract

The invention provides velvet antler mushroom sporocarp water extraction residue polysaccharide as well as an extraction method and application thereof, and belongs to the technical field of edible mushroom polysaccharide preparation. The invention provides an extraction method of water extraction residue polysaccharide of a mushroom fruiting body, which comprises the following steps: sequentially carrying out solid state fermentation, post-treatment and hot water extraction on the water extraction residue of the mushroom fruiting body to obtain an extraction product containing the water extraction residue polysaccharide of the mushroom fruiting body. According to the method disclosed by the invention, the water extraction residues of the pilose antler mushroom sporocarp are used as raw materials, so that high-value utilization of the water extraction residues of the pilose antler mushroom sporocarp can be realized. Moreover, the method can improve the yield and content of polysaccharide, and has the advantages of no chemical residue, ecological environmental protection, high production efficiency and the like. Besides, the polysaccharide prepared by the extraction method disclosed by the invention is stable under a digestion condition, and the prepared polysaccharide can improve the content of short-chain fatty acids in intestinal tracts, so that a basis is provided for application of the polysaccharide of the water-extracted residues of the fruiting bodies of the velvet antler mushrooms in new potential prebiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus polysaccharide preparation, and in particular relates to a polysaccharide from water extraction residue of Pleurotus eryngii fruiting body, and an extraction method and application thereof. Background Art

[0002] Porcupine, scientifically known as Lyophyllum decastes (Fr.) Singer, is a large fungus that can be used as both medicine and food. Its fruiting bodies are rich in nutrients and functional ingredients such as polysaccharides, polyphenols, proteins, vitamins, minerals and sterols, and have high nutritional value and special medicinal effects. The various biological activities of Porcupine are mainly derived from the polysaccharide components in its fruiting bodies. Due to differences in extraction methods and sources, Porcupine polysaccharides have complex and diverse structural characteristics, such as monosaccharide composition, molecular weight, branching degree, glycosidic bond configuration and the connection order of each sugar residue, which make the polysaccharide have a variety of pharmacological effects, including antioxidant, immune enhancement, antibacterial, anti-tumor and anti-diabetic.

[0003] At present, the extraction of velvet antler mushroom polysaccharides is mainly based on hot water extraction, and the extraction rate is low. The remaining large amount of velvet antler mushroom residue is mainly used as fertilizer or directly discarded, with low utilization rate, resulting in a great waste of resources. Summary of the invention

[0004] The purpose of the present invention is to provide a polysaccharide from water extraction residue of Pleurotus eryngii fruiting bodies and an extraction method and application thereof. The present invention uses water extraction residue of Pleurotus eryngii fruiting bodies as raw material to extract polysaccharides, has high utilization rate, and has a high extraction rate of Pleurotus eryngii polysaccharides.

[0005] The present invention provides a method for extracting polysaccharides from water extraction residues of Pleurotus eryngii fruiting bodies, comprising the following steps:

[0006] 1) inoculating Aspergillus oryzae into fermentation material for solid-state fermentation to obtain a fermentation product; the fermentation material comprises water extraction residue of Pleurotus eryngii fruiting bodies and water; the Aspergillus oryzae comprises Aspergillus oryzae powder; the inoculation amount of Aspergillus oryzae accounts for 0.03% to 0.06% of the mass fraction of the Pleurotus eryngii fruiting body residue; 2) mixing the fermentation product and water, keeping warm and standing to obtain a post-processed product; 3) subjecting the post-processed product to water extraction to obtain an extraction product containing polysaccharides from the water extraction residue of Pleurotus eryngii fruiting bodies; the water extraction temperature is 95 to 100° C.

[0007] Preferably, after obtaining the extraction product, the following steps are further included: after collecting the liquid components in the extraction product, concentrating and ethanol precipitation are performed in sequence to collect the precipitate; and the precipitate is redissolved and freeze-dried to obtain a freeze-dried product containing polysaccharides from the water extraction residue of Pleurotus eryngii fruiting bodies.

[0008] Preferably, the Aspergillus oryzae includes Japanese Aspergillus oryzae Changbai fungus or Shanghai Niang 3.042.

[0009] Preferably, in step 1), the ratio of the mass of the aqueous extraction residue of the Pleurotus eryngii fruiting body to the volume of water is 1 g: 2-3 mL; the temperature of the solid-state fermentation is 26-28° C.; and the time of the solid-state fermentation is 3-5 days.

[0010] Preferably, in step 2), the ratio of the mass of the fermentation product to the volume of water is 1 g:15 mL; the temperature of the heat preservation and standing is 44-48° C.; and the time of the heat preservation and standing is 2-3 h.

[0011] Preferably, the water extraction temperature is 95-100° C.; the water extraction process is accompanied by stirring; the number of water extractions is ≥2 times; and the time of each water extraction is 1.5-2.5 hours.

[0012] Preferably, the alcohol precipitation comprises: adding anhydrous ethanol to the concentrated concentrate until the volume concentration of ethanol in the solution is 75% to 80% for alcohol precipitation; the alcohol precipitation time is 12 to 24 hours.

[0013] The present invention also provides an extraction product obtained by the extraction method described in the above scheme.

[0014] The present invention also provides the use of the extraction product described in the above scheme in at least one of the following 1) to 4): 1) preparing prebiotics; 2) regulating the balance of intestinal microbial flora; 3) maintaining and / or improving intestinal function; 4) promoting the intestinal flora to produce short-chain fatty acids.

[0015] Preferably, the short-chain fatty acids include acetic acid and / or n-butyric acid.

[0016] The present invention provides a method for extracting polysaccharides from water-extracted residues of velvet antler mushroom fruiting bodies, comprising the following steps: inoculating Aspergillus oryzae into a fermentation material for solid-state fermentation to obtain a fermentation product; the fermentation material comprises water-extracted residues of velvet antler mushroom fruiting bodies and water; mixing the fermentation product with water, standing to obtain a post-processing product; collecting the solid components in the post-processing product, sequentially drying, crushing and hot water extraction to obtain an extraction product containing polysaccharides from water-extracted residues of velvet antler mushroom fruiting bodies. The extraction method of the present invention uses water-extracted residues of velvet antler mushroom fruiting bodies as raw materials, and can achieve high-value utilization of water-extracted residues of velvet antler mushroom fruiting bodies. In addition, the present invention adopts solid-state fermentation, post-treatment and then hot water extraction, which is conducive to increasing the extraction rate of polysaccharides from velvet antler mushroom residues, can promote the release of macromolecular polysaccharide components in velvet antler mushroom residues, can greatly improve the polysaccharide yield and content, and thus improve the utilization of polysaccharides from water-extracted residues of velvet antler mushroom fruiting bodies, and the prepared residue polysaccharides are rich in β-glucan. The extraction method has the advantages of no chemical residues, ecological environmental protection, high production efficiency, etc.

[0017] In addition, the polysaccharide prepared by the extraction method of the present invention is stable under digestion conditions, will not be significantly affected by saliva-stomach-intestinal fluid, will not be digested and decomposed by α-amylase, pepsin and pancreatic enzymes, and can be further directed to the colon to be utilized and degraded by intestinal microbial flora. Furthermore, the polysaccharide prepared by the extraction method of the present invention can increase the content of short-chain fatty acids such as acetic acid and butyric acid in the intestine. These short-chain fatty acids are not only energy sources for the host and microorganisms, but can also be used by body tissues and help maintain the stability of the intestinal environment, providing a basis for the application of polysaccharides from the water extraction residue of Pleurotus eryngii fruiting bodies in new potential prebiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 This is a comparison chart of the molecular weights of polysaccharides from the water-extracted residues of Pleurotus eryngii fruiting bodies treated with solid-state fermentation;

[0020] Figure 2 The changes of acetic acid content in the water-extracted residue of Pleurotus eryngii fruiting body treated by solid-state fermentation at 6h, 12h, 24h and 48h of fermentation respectively;

[0021] Figure 3 The change of propionic acid content in the polysaccharide of water extraction residue of Pleurotus eryngii fruiting body treated by solid-state fermentation at 6h, 12h, 24h and 48h of fermentation;

[0022] Figure 4 The change of butyric acid content in the polysaccharide of water extraction residue of Pleurotus eryngii fruiting body treated by solid-state fermentation at 6h, 12h, 24h and 48h of fermentation;

[0023] Figure 5 The change of total acid content of polysaccharides in the water extraction residue of Pleurotus eryngii fruiting body treated by solid-state fermentation at 6h, 12h, 24h and 48h of fermentation respectively. DETAILED DESCRIPTION

[0024] The invention provides a method for extracting polysaccharides from water-extracted residues of velvet antler mushroom fruiting bodies, comprising the following steps: 1) inoculating Aspergillus oryzae into fermentation material for solid-state fermentation to obtain a fermentation product; the fermentation material comprises water-extracted residues of velvet antler mushroom fruiting bodies and water; the Aspergillus oryzae comprises Aspergillus oryzae powder; the inoculation amount of Aspergillus oryzae accounts for 0.03% to 0.06% of the mass fraction of the velvet antler mushroom fruiting body residues; 2) mixing the fermentation product and water, keeping the mixture warm and standing to obtain a post-processing product; 3) subjecting the post-processing product to water extraction to obtain a polysaccharide extraction product containing the water-extracted residues of velvet antler mushroom fruiting bodies; the water extraction temperature is 95 to 100 DEG C.

[0025] The present invention adopts solid-state fermentation and post-treatment for the water extraction residue of Pleurotus eryngii fruiting body, and then performs hot water extraction, thereby improving the utilization of polysaccharides in the water extraction residue of Pleurotus eryngii fruiting body, wherein the post-treatment provides conditions for the enzyme generated by the solid-state fermentation to fully perform enzymolysis. The extraction method of the present invention can obtain high-purity and high-yield polysaccharides in the water extraction residue of Pleurotus eryngii fruiting body.

[0026] The invention firstly inoculates Aspergillus oryzae into fermentation material for solid-state fermentation to obtain a fermentation product; the fermentation material comprises water extraction residue of Pleurotus eryngii fruiting bodies and water; the Aspergillus oryzae comprises Aspergillus oryzae powder; the inoculation amount of Aspergillus oryzae accounts for 0.03% to 0.06% of the mass fraction of the Pleurotus eryngii fruiting body residue, and further is 0.05%; the inoculation amount is helpful to increase the extraction amount of polysaccharide in the water extraction residue of Pleurotus eryngii fruiting body.

[0027] In the specific implementation process of the present invention, the preparation method of the water extraction residue of Pleurotus eryngii fruiting body includes the following steps: mixing Pleurotus eryngii fruiting body powder and water, extracting, collecting the remaining residue of the extraction, and obtaining the water extraction residue of Pleurotus eryngii fruiting body; the extraction temperature is 95-100°C; the number of extractions includes 2 times; the time of each extraction is 2 hours; the particle size of the water extraction residue powder of Pleurotus eryngii fruiting body is 10 mesh to 20 mesh.

[0028] In the specific implementation process of the present invention, the fermentation material is composed of water extraction residue of Pleurotus ostreatus fruiting bodies and water; the Aspergillus oryzae includes Japanese Aspergillus oryzae Changbai fungus or Shanghai Niang 3.042; the Japanese Aspergillus oryzae Changbai fungus or Shanghai Niang 3.042 is purchased from Suzhou Shimu Xiaoyuan Ecological Agriculture Co., Ltd.; the dosage form of the Japanese Aspergillus oryzae Changbai fungus is bacterial powder, which does not need to be activated, and is inoculated according to the mass percentage of bacterial powder relative to the mass percentage of Pleurotus ostreatus fruiting body residue; the ratio of the mass of the water extraction residue of the Pleurotus ostreatus fruiting body to the volume of water is 1g:2-3mL; the temperature of the solid-state fermentation is 26-28°C; the time of the solid-state fermentation is 3-5 days.

[0029] After obtaining the fermentation product, the present invention mixes the fermentation product with water and allows the mixture to stand to obtain a post-processed product.

[0030] In the specific implementation process of the present invention, the ratio of the mass of the fermentation product to the volume of water is 1g:15mL; the temperature of the insulation and standing is 44-48°C, further 45°C; the insulation and standing time is 2-3h.

[0031] After obtaining the post-processed product, the present invention performs water extraction on the post-processed product to obtain a polysaccharide extraction product containing the water extraction residue of Pleurotus eryngii fruiting body.

[0032] In the specific implementation process of the present invention, the water extraction process is accompanied by stirring; the time of each water extraction is 1.5 to 2.5 hours; the number of water extractions is ≥2 times.

[0033] After obtaining the leaching product, the present invention further comprises collecting the liquid components in the leaching product, sequentially concentrating and alcohol precipitating, and collecting the precipitate.

[0034] In the specific implementation process of the present invention, the concentration includes rotary evaporation concentration; the conditions of the rotary evaporation concentration are: 60°C, 240mbar; based on the liquid volume before concentration being 3L, the volume of the concentrated liquid after concentration is 1-2L; collecting the liquid components in the extraction product includes centrifuging the extraction product and collecting the supernatant; the centrifugal force of the centrifugation is 4100-4500g, further 4200g; the centrifugation time is 20-30min, further 25min.

[0035] In the specific implementation process of the present invention, the alcohol precipitation includes: adding anhydrous ethanol to the concentrated concentrated solution until the volume concentration of ethanol in the solution is 75% to 80% for alcohol precipitation; the volume ratio of the anhydrous ethanol to the concentrated solution is (70 to 85): (15 to 30); the alcohol precipitation time is 12 to 24 hours; the alcohol precipitation is carried out under static conditions; the collection of precipitation includes centrifuging the product after alcohol precipitation and collecting the precipitate; the centrifugal force of the centrifugation is 3800 to 4200g; the centrifugation time is 15 to 30 minutes, and further 20 to 25 minutes.

[0036] After collecting the precipitate, the present invention further comprises re-dissolving the precipitate and then freeze-drying it to obtain a freeze-dried product containing polysaccharides from the water-extracted residue of Pleurotus eryngii fruiting bodies.

[0037] In the specific implementation process of the present invention, the reagent used for the re-dissolution is distilled water; the temperature of the re-dissolution is 65-70°C; the polysaccharide content in the polysaccharide of the water extraction residue of Pleurotus eryngii fruiting body in the freeze-dried product is 65.82%-68.6%, which is the chemical content determined by the phenol-sulfuric acid method.

[0038] The extraction method provided by the invention has the advantages of no chemical residue, ecological protection, high production efficiency, etc.

[0039] The present invention also provides an extraction product obtained by the extraction method described in the above scheme.

[0040] In the specific implementation process of the present invention, the polysaccharide of the water extraction residue of Pleurotus eryngii fruiting body is rich in β-glucan; the weight average molecular weight of the polysaccharide of the water extraction residue of Pleurotus eryngii fruiting body is 1.138×10 6 g / mol, 5.179×10 4 g / mol, 2.851×10 4 g / mol and 1.681×10 4 g / mol.

[0041] The present invention also provides the use of the extraction product described in the above scheme in at least one of the following 1) to 4): 1) preparing prebiotics; 2) regulating the balance of intestinal microbial flora; 3) maintaining and / or improving intestinal function; 4) promoting the intestinal flora to produce short-chain fatty acids.

[0042] In the specific implementation process of the present invention, the regulation of the balance of intestinal microbial flora includes preparing products for regulating the balance of intestinal microbial flora; the maintenance and / or improvement of intestinal function includes preparing products for maintaining and / or improving intestinal function; the promotion of intestinal flora to produce short-chain fatty acids includes preparing products for promoting intestinal flora to produce short-chain fatty acids; the short-chain fatty acids include acetic acid and / or butyric acid.

[0043] The polysaccharide prepared by the extraction method of the present invention is stable under digestion conditions, will not be significantly affected by saliva-stomach-intestinal fluid, will not be digested and decomposed by α-amylase, pepsin and pancreatic enzymes, and can be further directed to the colon to be utilized and degraded by intestinal microbial flora. Furthermore, the polysaccharide prepared by the extraction method of the present invention can increase the content of short-chain fatty acids such as acetic acid, propionic acid and butyric acid in the intestine. These short-chain fatty acids are not only energy sources for the host and microorganisms, but can also be utilized by body tissues and help maintain the stability of the intestinal environment, providing a basis for the application of polysaccharides from the water extraction residue of Pleurotus eryngii fruiting bodies in new potential prebiotics.

[0044] In order to further illustrate the present invention, a polysaccharide from the water extraction residue of Pleurotus eryngii fruiting body provided by the present invention and its extraction method and application are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Material

[0046] The preparation method of the water extraction residue of Pleurotus eryngii fruiting body is as follows: the Pleurotus eryngii fruiting body is crushed and mixed with water in a mass ratio (solid-liquid ratio) of 1:15, and water extraction is performed at 95-100°C for 2h, and the remaining residue is repeated twice.

[0047] Main reagents

[0048] Sulfuric acid and anhydrous ethanol were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.;

[0049] α-Amylase, pepsin, trypsin, and bile salts were purchased from Sigma-Aldrich, St. Louis, USA);

[0050] Scleroglucan was purchased from Shanghai Xibao Biotechnology Co., Ltd.;

[0051] BCA protein concentration assay kit and soluble starch were purchased from Solarbio, Beijing, China;

[0052] Yeast and mushroom β-glucan detection kits were purchased from Megazyme, Ireland;

[0053] Acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid were purchased from MackLin, Shanghai, China;

[0054] Glucose standard solution was purchased from Sigma-Aldrich, USA;

[0055] Aspergillus oryzae (Japanese Aspergillus oryzae Changbai fungus or Shanghai Brewing 3.042) was purchased from Suzhou Shimu Xiaoyuan Ecological Agriculture Co., Ltd.

[0056] 1. A method for determining polysaccharides in water-extracted residues of Pleurotus eryngii treated by solid-state fermentation, comprising the following steps:

[0057] (1) Yield of polysaccharides from water extraction residue of Pelvis antler mushroom treated by solid-state fermentation:

[0058] Polysaccharide yield (%) = freeze-dried precipitate weight / extraction raw material weight × 100%.

[0059] (2) Polysaccharide content detection:

[0060] 1 mg of polysaccharide sample of the water extraction residue of Pleurotus eryngii fruiting body treated with solid-state fermentation after freeze-drying was accurately weighed, 10 mL of distilled water was added to fully dissolve it, and the total sugar content was determined by the phenol-sulfuric acid method.

[0061] The specific method is as follows: prepare phenol solution, concentrated sulfuric acid, and 0.1 mg / mL glucose standard solution with a volume concentration of 5%. Take glucose standard solution (concentration of 0.00, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL) and 1 mL of sample solution and place them in long test tubes, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid, mix well, place in a 100°C water bath for 15 minutes, take out and cool to room temperature, take 200 μL in an ELISA plate, and measure the absorbance value at 490 nm. Draw a standard curve based on the concentration of the glucose standard and the corresponding absorbance value to calculate the total sugar content.

[0062] 2. The reducing sugar content in the polysaccharide of the water extraction residue of Pleurotus eryngii fruiting body treated by solid-state fermentation was determined according to the DNS method. The specific method is as follows:

[0063] First, prepare the DNS reagent: add 3.15g of 3,5-dinitrosalicylic acid and 131mL of 2mol / L sodium hydroxide to 250mL of hot water solution containing 91g of potassium sodium tartrate, then add 2.5g of phenol and 2.5g of sodium sulfite, stir to dissolve, cool and add water to make up to 500mL to prepare the DNS reagent. Store in a brown bottle for one week before use.

[0064] Next, prepare 1 mg / mL glucose standard solution and 1 mg / mL solid-state fermentation treated velvet mushroom fruiting body water extraction residue polysaccharide sample determination solution. Take 0, 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mL of glucose standard solution in a 25 mL volumetric flask, add water to 2 mL (the concentration of glucose standard is 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 mg / mL); take 2 mL of sample determination solution into the above 25 mL volumetric flask; then add 5 mL of DNS reagent, place in a 100 ° C boiling water bath for 5 minutes, cool, dilute to 25 mL, shake well, take 200 μL to the ELISA plate, measure the absorbance value at 520 nm, draw a standard curve based on the glucose standard concentration and the corresponding absorbance value, and calculate the reducing sugar content.

[0065] The polysaccharide content was calculated as follows: polysaccharide content (%) = total sugar content (%) - reducing sugar content (%).

[0066] Example 1

[0067] A method for extracting polysaccharides from water-extracted residues of Pleurotus eryngii fruiting bodies, comprising the following steps:

[0068] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 10-20 mesh;

[0069] 2. Inoculating Japanese Aspergillus oryzae into a fermentation material consisting of Pleurotus eryngii fruiting body residue and water for solid-state fermentation, the conditions are: the inoculation amount is 0.05% (the mass fraction of Japanese Aspergillus oryzae in the Pleurotus eryngii fruiting body residue); the material-liquid ratio is 1:2.5 (the ratio of the mass of the Pleurotus eryngii fruiting body water extraction residue to the volume of water is 1g:2.5mL), the fermentation temperature is 28°C; and the fermentation time is 5 days;

[0070] 3. After solid-state fermentation, distilled water was added for post-treatment, with a solid-liquid ratio of 1:15 (1 g:15 mL); the temperature was 45 °C; the time was 3 h;

[0071] 4. Extract the post-treated product by adding a rotor, stirring and extracting at 95°C for 2h; centrifuging (4000g / 30min), and retaining the supernatant; extract the residue once again according to the above steps, and combine the supernatant;

[0072] 5. Use a rotary evaporator (60°C, 240 mbar) to evaporate the supernatant and collect about 1-2 L of concentrated solution. Add anhydrous ethanol until the volume concentration of ethanol in the solution reaches 75%, stop adding ethanol for alcohol precipitation, let it stand for 24 hours, and then centrifuge (4200g / 30min) to retain the precipitate.

[0073] 6. The above precipitate was dissolved in water at 65°C and then freeze-dried to obtain the desired solid-state fermented Pleurotus eryngii fruiting body water extraction residue polysaccharide (Example 1). The yield and polysaccharide content were measured, and the results are shown in Table 1.

[0074] Comparative Example 1

[0075] A method for extracting polysaccharides from water-extracted residues of Pleurotus eryngii fruiting bodies, comprising the following steps:

[0076] 1. Weigh 200 g of the residual powder of the fruiting body of Pleurotus eryngii, add 3 L of water for extraction, add a rotor, set the temperature to 95 ° C, stir and extract for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for 1 time, and combine the supernatant;

[0077] 2. Use a rotary evaporator (60°C, 240 mbar) to evaporate the supernatant and collect about 1-2 L of concentrated solution. Add anhydrous ethanol until the volume concentration of ethanol in the solution reaches 75%, stop adding ethanol for alcohol precipitation, let it stand for 24 hours, and then centrifuge (4200g / 30min) to retain the precipitate.

[0078] 3. The above precipitate was dissolved in water at 65°C and then freeze-dried to obtain the desired polysaccharide from the water-extracted residue of Pleurotus eryngii fruiting body (Comparative Example 1). The yield and polysaccharide content were measured, and the results are shown in Table 1.

[0079] As can be seen from Table 1, the solid-state fermentation treatment group of water-extracted residual polysaccharides of Pleurotus eryngii fruiting bodies with stable quality (Example 1) can be obtained by the extraction method of Example 1, and its physical and chemical index results (Table 1) show that the polysaccharide yield reaches 20.45±0.1%, and the polysaccharide content reaches 67.21±1.39%. Compared with Comparative Example 1, the solid-state fermentation in Example 1 is conducive to promoting the release of polysaccharides from Pleurotus eryngii residuals, and can greatly improve the polysaccharide yield and content.

[0080] Table 1 Physical and chemical indicators of polysaccharides from water-extracted residues of Pleurotus eryngii fruiting bodies treated by solid-state fermentation

[0081] Group Yield (%) Polysaccharide content (%) Comparative Example 1 12.53±0.03 56.30±0.57 Example 1 20.45±0.1 67.21±1.39

[0082] Example 2 (the difference from Example 1 is that steps 5 and 6 are omitted)

[0083] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0084] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 10-20 mesh;

[0085] 2. Inoculating Japanese Aspergillus oryzae into a fermentation material consisting of Pleurotus eryngii fruiting body residue and water for solid-state fermentation, the conditions are: the inoculation amount is 0.05% (the mass fraction of Japanese Aspergillus oryzae in the Pleurotus eryngii fruiting body residue); the material-liquid ratio is 1:2.5 (the ratio of the mass of the Pleurotus eryngii fruiting body water extraction residue to the volume of water is 1g:2.5mL), the fermentation temperature is 28°C; and the fermentation time is 5 days;

[0086] 3. After solid-state fermentation, distilled water was added for post-treatment, with a solid-liquid ratio of 1:15; the temperature was 45°C; the time was 3h;

[0087] 4. The post-treated product was subjected to water extraction, a rotor was added, the temperature was 95°C, and the extraction was stirred for 2 hours; centrifuged (4000g / 30min), and the supernatant was retained; the residue was extracted once according to the above steps, and the supernatant was combined to determine the polysaccharide content.

[0088] Comparative Example 2 (the difference from Comparative Example 1 is that steps 2 and 3 are omitted)

[0089] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0090] 1. Weigh 200 g of the residual powder of the fruiting body of Pleurotus eryngii, add 3 L of water for extraction, add a rotor, set the temperature to 95 ° C, stir and extract for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for 1 time, combine the supernatants, and determine the polysaccharide content.

[0091] Comparative Example 3 (compared with Example 2, step 3 is omitted)

[0092] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0093] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 10-20 mesh;

[0094] 2. Inoculate Aspergillus oryzae into fermentation material consisting of Pleurotus eryngii fruiting body residue and water for solid-state fermentation, with a conditional inoculation amount of 0.05%; a material-liquid ratio of 1:2.5, a fermentation temperature of 28°C, and a fermentation time of 5 days;

[0095] 3. Weigh the product of solid-state fermentation for extraction, with a solid-liquid ratio of 1:15, add a rotor, set the temperature to 95°C, and stir for extraction for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for the residue, combine the supernatant, and determine the polysaccharide content.

[0096] Example 3 (the difference from Example 2 is that another strain - Shanghai Brewing 3.042 is used)

[0097] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0098] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 10-20 mesh;

[0099] 2. Inoculate Aspergillus oryzae (Shanghai Brewing 3.042, purchased from Suzhou Shimu Xiaoyuan Ecological Agriculture Co., Ltd.) into a fermentation material consisting of Pleurotus eryngii fruiting body residue and water for solid-state fermentation, with a conditional inoculation amount of 0.05%; a material-liquid ratio of 1:2.5, a fermentation temperature of 28°C, and a fermentation time of 5 days;

[0100] 3. After solid-state fermentation, distilled water was added for post-treatment, with a solid-liquid ratio of 1:15; the temperature was 45°C; the time was 3h;

[0101] 4. Weigh the post-treated product for water extraction, add a rotor, set the temperature to 95°C, stir and extract for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for the residue, combine the supernatant, and determine the polysaccharide content.

[0102] Comparative Example 4 (different inoculation amount compared with Example 2)

[0103] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0104] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 40-50 mesh;

[0105] 2. Inoculate Aspergillus oryzae into fermentation material consisting of Pleurotus eryngii fruiting body residue and water for solid-state fermentation, with a conditional inoculation amount of 0.02%; a material-liquid ratio of 1:2.5, a fermentation temperature of 28°C, and a fermentation time of 5 days;

[0106] 3. After solid-state fermentation, distilled water was added for post-treatment, with a solid-liquid ratio of 1:15; the temperature was 45°C; the time was 3h;

[0107] 4. Weigh the post-treated product for water extraction, add a rotor, set the temperature to 95°C, stir and extract for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for the residue, combine the supernatant, and determine the polysaccharide content.

[0108] Example 4 (different inoculation amount compared to Example 2)

[0109] A method for preparing a polysaccharide water extract from velvet antler mushroom fruiting body residues, the steps of which are as follows:

[0110] 1. Dry the residue of water extraction of Pleurotus eryngii fruiting body, crush it and sieve it through 10-20 mesh;

[0111] 2. Inoculate Aspergillus oryzae into the residue of Pleurotus eryngii fruiting body for solid-state fermentation, with a conditional inoculation amount of 0.06%; a solid-liquid ratio of 1:2.5, a fermentation temperature of 28°C, and a fermentation time of 5 days;

[0112] 3. After solid-state fermentation, distilled water was added for post-treatment, with a solid-liquid ratio of 1:15; the temperature was 45°C; the time was 3h;

[0113] 4. Weigh the post-treated product for water extraction, add a rotor, set the temperature to 95°C, stir and extract for 2 hours; centrifuge (4000g / 30min), and keep the supernatant; repeat the above steps for the residue, combine the supernatant, and determine the polysaccharide content.

[0114] Table 2 Comparison of polysaccharide content in aqueous extracts of Pleurotus eryngii fruiting body residues extracted with different parameters

[0115] Group Polysaccharide content (%) Comparative Example 2 8.24±0.86 Comparative Example 3 18.26±0.13 Comparative Example 4 16.12±0.92 Example 2 29.68±1.96 Example 3 25.23±1.31 Example 4 27.87±1.02

[0116] As can be seen from Table 2, the water extracts with stable quality obtained by the extraction methods of Examples 2 to 4 have polysaccharide contents of 29.68±1.96%, 25.23±1.31% and 27.87±1.02%, respectively. This indicates that compared with other comparative examples, the solid-state fermentation and post-treatment with a certain inoculation amount in Examples 2 to 4 are beneficial to promoting the release of polysaccharides from Pleurotus eryngii residues, and can significantly increase the polysaccharide yield.

[0117] Test Example 1

[0118] Molecular weight analysis of polysaccharides from the water extraction residue of Pleurotus eryngii fruiting bodies treated with solid-state fermentation prepared in Example 1

[0119] (1) Experimental method: High performance liquid chromatography coupled with multi-angle laser light scattering and differential refractive index detector analysis (HPSEC-MALLS-RI);

[0120] (2) Sample pretreatment: 2 mg of the polysaccharide samples of Comparative Example 1 and Example 1 were accurately weighed and dissolved in distilled water to prepare a solution with a mass concentration of 2 mg / mL. The supernatant was collected after centrifugation at 12000 g for 20 min, and filtered through a 0.45 μm aqueous microporous filter membrane for later use. The solutions were named Comparative Example 1 and Example 1, respectively.

[0121] (3) Analysis conditions: American Waters high performance liquid chromatograph (Waters 2695); chromatographic columns are TSK-GEL series G4000PWXL and G6000PWXL gel columns in series, with specifications of 7.8mm×300mm (TOSOH, Japan); column temperature: 35℃; mobile phase: 0.05mol / LNaNO3 and 4mL / L MIT antibacterial agent; flow rate: 0.5mL / min; injection volume: 100μL; detector: 2414 differential refractometer, 8-angle laser scattering instrument (Wyatt, HELEOS 8), laser detector light source wavelength is selected as 623.8nm. The refractive index increment (dn / dc) of polysaccharide in solution is calculated as 0.146mL / g;

[0122] (4) Data processing: Astra (version 6.1.1, Wyatt Technology, Santa Barbara, CA) data analysis software was used to collect and analyze light scattering data and calculate molecular weight.

[0123] The results of HPSEC-MALLS-RI analysis ( Figure 1 As shown in Table 3, the polysaccharides from the water extract residue of Pleurotus eryngii treated by solid-state fermentation in Comparative Example 1 and Example 1 mainly include three components, among which the molecular weight (M w ) changes most significantly, from 5.728×10 7 g / mol decreased to 1.138×10 6 g / mol, and the peak area of ​​this component was significantly increased, indicating that solid-state fermentation treatment can promote the release of macromolecular components, thereby increasing the yield and content of residual polysaccharides.

[0124] Table 3 Molecular weight analysis of polysaccharides from water extraction residues of Pleurotus eryngii fruiting bodies

[0125]

[0126] Test Example 2

[0127] An in vitro digestion model was used to compare the anti-digestive properties of polysaccharides from the water-extracted residues of Pleurotus eryngii fruiting bodies obtained by solid-state fermentation with different treatment methods.

[0128] In the in vitro digestion model, firstly, the total electrolyte digestive solution is prepared according to the following Table 4, and then each reagent required in the table is taken out from the prepared total electrolyte digestive solution and the volume is adjusted to 0.4L, which are salivary digestive fluid (SSF), gastric digestive fluid (SGF) and intestinal digestive fluid (SIF).

[0129] Table 4 Formulas of various digestive juices

[0130]

[0131] A. Simulate oral digestion

[0132] 1. Dissolve 20 mg of the polysaccharide sample of Example 1 in 4 mL of distilled water;

[0133] 2. Preheat the prepared simulated saliva digestion solution (SSF) in a 37°C water bath for 15 min, then take out 3.2 mL of SSF and add it to the above sample solutions, then add CaCl2 solution (0.025 mL, 0.3 M) and α-amylase solution (0.4 mL, 750 U / mL);

[0134] 3. Add distilled water to 8 mL to make the final ratio of polysaccharide solution to SSF digestion solution 1:1;

[0135] 4. The polysaccharide sample of Example 1 was then incubated in a water bath at 37°C for 5 min, 2 mL of each sample was taken at 0 min and 5 min, placed in a boiling water bath and heated for 15 min to inactivate the enzyme, and cooled to room temperature for subsequent determination of reducing sugars.

[0136] B. Simulate gastric digestion

[0137] 1. Adjust the pH of the remaining 4 mL of simulated saliva digestion solution to 3.0 with 1 mol / L hydrochloric acid solution;

[0138] 2. Preheat the prepared simulated gastric digestive fluid (SGF) in a 37°C water bath for 15 min, then take out 3.2 mL of SGF solution and add it to 4 mL of simulated saliva digestive fluid with pH adjusted, and then add CaCl2 solution (0.005 mL, 0.3 M) and pepsin solution (0.2 mL, 2000 U / mL);

[0139] 3. Add distilled water to a final volume of 8 mL;

[0140] 4. Incubate in a water bath at 37°C for 4 h. During gastric digestion, take out 1 mL, 1 mL and 2 mL of simulated gastric digestion fluid at three time points of 0, 2 and 4 h, respectively, and then heat in boiling water for 15 min to inactivate the enzyme, cool to room temperature and set aside for subsequent determination of reducing sugars.

[0141] C. Simulated intestinal digestion

[0142] 1. Adjust the pH of the remaining 4 mL simulated gastric juice to 7.0 with 1 mol / L sodium hydroxide solution;

[0143] 2. Preheat the prepared simulated intestinal digestive fluid (SIF) in a 37°C water bath for 15 min, then take out 3.2 mL of SIF solution and add it to 4 mL of simulated gastric digestive fluid with adjusted pH, and then add bile salt solution (0.1 mL, 10 mM), CaCl2 solution (0.008 mL, 0.3 M), and pancreatic enzyme solution (0.1 mL, 100 U / mL) respectively;

[0144] 3. Add distilled water to make up to 8 mL;

[0145] 4. Incubate in a 37°C water bath for 6 h. During the digestion process, take out 1 mL of simulated intestinal fluid at 0, 2, 4, and 6 h for further analysis. After sampling, heat in boiling water for 15 min to inactivate the enzyme, cool to room temperature and set aside for subsequent determination of reducing sugars.

[0146] Table 5 Comparison of reducing sugar content in water-extracted residues of Pleurotus eryngii fruiting bodies treated with solid-state fermentation before and after digestion of polysaccharides

[0147]

[0148]

[0149] The change in reducing sugar content can be one of the indicators that reflects whether the polysaccharide can be digested in the saliva-gastrointestinal tract. As can be seen from Table 5, in the simulated digestion experiment, there was no obvious change in the reducing sugar content of Example 1 before and after digestion, which can indicate that the chemical composition of the polysaccharide is stable under digestion conditions, is not significantly affected by saliva-gastric-intestinal fluid, will not be digested and decomposed by α-amylase, pepsin and pancreatic enzymes, and can be further directed to the colon to be utilized and degraded by the intestinal microbial flora.

[0150] Test Example 3

[0151] An in vitro fermentation model was used to explore the effect of polysaccharides from the water-extracted residues of Pleurotus eryngii fruiting bodies after solid-state fermentation on the production of short-chain fatty acids.

[0152] 1. Fresh stool samples were collected from 4 healthy volunteers (2 females and 2 males, aged 20-30 years) in Shanghai, who maintained a regular diet and had not received antibiotic or probiotic treatment within 3 months;

[0153] 2. Prepare fecal dilution solution: weigh 8g sodium chloride, 0.2g potassium chloride, 1.15g anhydrous disodium hydrogen phosphate, and 0.2g potassium dihydrogen phosphate in 1L of distilled water, dissolve by ultrasonic for 5min, sterilize at 121℃ for 15min, and cool for later use;

[0154] 3. Prepare a fecal suspension: Immediately mix the fecal samples of four volunteers with the cooled fecal dilution to produce a 10% (w / v) fecal suspension;

[0155] 4. Prepare basic culture medium: weigh 2.0 g of tryptone, 2.0 g of yeast powder, 0.5 g of cysteine ​​hydrochloride, 0.1 g of sodium chloride, 2.0 g of sodium bicarbonate, 0.04 g of potassium dihydrogen phosphate, 0.04 g of anhydrous dipotassium hydrogen phosphate, 0.01 g of magnesium sulfate heptahydrate, 0.01 g of calcium chloride hexahydrate, 0.02 g of hemin chloride, 0.5 g of bile salt, 2.0 mL of Tween 80, 110 μL of vitamin K1, and 0.1 g of resazurin in 1 L of distilled water, and dissolve by ultrasonication for 5 min;

[0156] 5. Grouping:

[0157] Comparative Example 1: The polysaccharide sample of Comparative Example 1 weighed at a concentration of 0.2% (W:V) was mixed with the starch sample weighed at a concentration of 0.8%;

[0158] Example 1 group: The polysaccharide sample of Example 1 weighed at a concentration of 0.2% (W:V) was mixed with the starch sample weighed at a concentration of 0.8%;

[0159] Blank control group: medium without carbon source added (basic medium);

[0160] Positive control group (starch group): 1% (W:V) starch.

[0161] 6. The four prepared culture media were adjusted to pH 7.0 with 1 mol / L HCl, sterilized at 121°C for 15 min, and then 1.0 mL of 10% (w / v) fecal suspension was added to the four culture media of 9.0 mL, placed in an anaerobic chamber at 37°C, and cultured for 0, 12, 24, and 48 h, respectively. Then, the samples after fermentation in each time period were collected and stored at -80°C for further analysis;

[0162] 7. The samples collected in each time period were centrifuged at 9000g for 10 min, 1 mL of the supernatant after centrifugation was taken out, and 100 μL of concentrated hydrochloric acid and 2 mL of anhydrous ether were added respectively. After standing for 20 min, the supernatant was centrifuged at 4000g for 5 min, and 500 μL of 1M NaOH was added to the supernatant. The supernatant was removed and 100 μL of concentrated hydrochloric acid was added to the lower precipitate for acidification, and then the precipitate was filtered through a 0.22 μm microporous membrane and tested on a Waters 600 high performance liquid chromatograph;

[0163] 8. Chromatographic conditions: Green ODS-AQ C18 Boston liquid chromatography column (250mm×4.6mm, 5μm, purchased from Guangzhou Zhike Experimental Instrument Co., Ltd.); mobile phase A is 20mmoL / L KH2PO4 buffer salt, pH adjusted to 2.8 with phosphoric acid, mobile phase B is acetonitrile, both mobile phases are filtered through a 0.22μm filter membrane to remove insoluble matter, and then ultrasonically degassed for use. Equal concentration elution is performed with 80% A mobile phase and 20% B mobile phase, flow rate is 1.0mL / min, detection wavelength: 210nm, column temperature: 30℃, injection volume 100μL;

[0164] 9. Accurately weigh 0.1 g of acetic acid, propionic acid and n-butyric acid respectively and dissolve them in 5 mL of distilled water to prepare standard stock solutions;

[0165] 10. The prepared standard stock solution was mixed and diluted with distilled water to form a series of mixed standard solutions of 2000, 1500, 1000, 750, 500 and 250 μg / mL. The series of mixed standard solutions were filtered through a 0.22 μm microporous filter membrane and injected into a high performance liquid chromatograph for detection.

[0166] Short-chain fatty acids are mainly fermented products of undigested carbohydrates by intestinal microorganisms in the colon, including acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, etc. They have the function of regulating the balance of intestinal microbial flora, maintaining and improving intestinal function. The total content of acetic acid, propionic acid and butyric acid accounts for 95% of the total short-chain fatty acid content, which plays a key role in human health.

[0167] After 24 hours of in vitro fermentation, the production of SCFAs was significantly promoted in all groups, especially in the Example 1 group, where the production of acetic acid and butyric acid was significantly higher than that of the other groups. In addition, compared with the other groups, the butyric acid production of Example 1 was the highest. After 48 hours of fermentation, the total SCFAs content of the Example 1 sample increased significantly, specifically in the increase of acetic acid and butyric acid content. These short-chain fatty acids are not only an energy source for the host and microorganisms, but can also be used by body tissues and help maintain the stability of the intestinal environment (see Figure 2 to Figure 5 ).

[0168] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting polysaccharides from water extraction residues of Pleurotus eryngii fruiting bodies, characterized in that: The following steps are involved: 1) inoculating Aspergillus oryzae into a fermentation material for solid-state fermentation to obtain a fermentation product; the fermentation material comprises a water extraction residue of Pleurotus eryngii fruiting bodies and water; the Aspergillus oryzae comprises Aspergillus oryzae powder; the inoculation amount of Aspergillus oryzae accounts for 0.03% to 0.06% of the mass fraction of the Pleurotus eryngii fruiting body residue; 2) mixing the fermentation product with water, keeping the mixture warm and allowing it to stand to obtain a post-processed product; 3) extracting the post-treatment product with water to obtain an extraction product containing polysaccharides from water extraction residues of Pleurotus eryngii fruiting bodies; the water extraction temperature is 95-100°C.

2. The extraction method according to claim 1, characterized in that After obtaining the leaching product, the method further comprises the following steps: After collecting the liquid components in the extraction product, concentration and alcohol precipitation are performed in sequence to collect the precipitate; the precipitate is redissolved and then freeze-dried to obtain a freeze-dried product containing polysaccharides from the water extraction residue of Pleurotus eryngii fruiting body.

3. The extraction method according to claim 1, characterized in that The Aspergillus oryzae includes Japanese Aspergillus oryzae Changbai fungus or Shanghai Niang 3.

042.

4. The extraction method according to claim 1 or 3, characterized in that In step 1), the ratio of the mass of the aqueous extraction residue of the Pleurotus eryngii fruiting body to the volume of water is 1 g: 2-3 mL; the temperature of the solid-state fermentation is 26-28° C.; and the time of the solid-state fermentation is 3-5 days.

5. The extraction method according to claim 1, characterized in that In step 2), the ratio of the mass of the fermentation product to the volume of water is 1 g:15 mL; the temperature of the heat preservation and standing is 44-48° C.; and the heat preservation and standing time is 2-3 h.

6. The extraction method according to claim 1, characterized in that The water extraction temperature is 95-100° C.; the water extraction process is accompanied by stirring; the number of water extractions is ≥2 times; and the time of each water extraction is 1.5-2.5 hours.

7. The extraction method according to claim 1, characterized in that The alcohol precipitation comprises: adding anhydrous ethanol to the concentrated solution until the volume concentration of ethanol in the solution is 75% to 80% for alcohol precipitation; the alcohol precipitation time is 12 to 24 hours.

8. An extraction product obtained by the extraction method according to any one of claims 1 to 7.

9. Use of the extract according to claim 8 in at least one of the following 1) to 4): 1) preparing prebiotics; 2) Regulate the balance of intestinal microflora; 3) Maintain and / or improve intestinal function; 4) Promote the production of short-chain fatty acids by intestinal flora.

10. The use according to claim 9, characterized in that: The short-chain fatty acids include acetic acid and / or n-butyric acid.

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