Wild phellinus igniarius SH-27 sporocarp polyphenol extract and application thereof
Purification of wild Mulberry SH-27 fruiting polyphenols by ultrasonic combined with polyamide resin has solved the problem of high cost and low efficiency of existing polyphenol extraction methods, and achieved efficient and economical polyphenol extraction and purification, which is suitable for functional food and drug development.
Patent Information
- Application Number
- CN202510770506.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-09-02
AI Technical Summary
The existing polyphenol extraction methods are costly and inefficient, unable to be used in industrial use, and have limited solvent selection, making it difficult to meet the needs of functional foods.
Ultrasonic combined polyamide resin was used to purify the fruit polyphenols of wild Mulberry SH-27. The content of mycelium and fruit polyphenols was increased by optimizing the liquid culture medium, and the hydrogen bonding ability of the polyamide resin was used for separation and purification.
It improves the extraction rate and purity of polyphenols, shortens the purification process, reduces costs, is suitable for industrial production, and provides efficient polyphenol extracts for functional food and drug development.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of bioengineering, and particularly relates to a polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27 and an application thereof. Background Art
[0002] Phellinus igniarius, a rare medicinal fungus, is named for its yellow-brown fruiting body. It's mentioned in ancient texts such as the Compendium of Materia Medica and the Shennong's Classic of Materia Medica. With rising living standards and a growing interest in healthy living, medicinal and edible substances have become a focus, and the development of functional foods has become a trend. Phellinus igniarius, which contains a variety of bioactive substances and exhibits a variety of beneficial biological activities, has gradually gained public attention. Research has shown that the strain of Phellinus igniarius best reflects the characteristics of "Pellinus igniarius." Modern pharmacological research has shown that Phellinus igniarius is rich in various active substances that can enhance immunity, exhibit antioxidant and anti-inflammatory properties, inhibit cancer cells, and reduce uric acid levels. Polyphenols are one of the most important active substances in Phellinus igniarius, exhibiting significant anti-tumor and antioxidant properties.
[0003] Currently, polyphenol extraction methods include Soxhlet extraction, organic solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction, and supercritical fluid extraction. However, practical applications often present challenges such as high cost, low efficiency, limited solvent selection, and inability to industrialize. Polyamides, a general term for polymers with repeating amide groups (CO-NH) in their backbone, effectively bind to polyphenols through hydrogen bonds, exhibiting excellent chemical stability and mechanical strength. They offer advantages such as excellent separation efficiency, reusability, and cost savings, making them highly suitable for industrial applications.
[0004] Therefore, polyamide resin can be used to effectively extract and purify total polyphenols from Phellinus igniarius, providing an effective and cost-saving method for producing functional foods. Summary of the Invention
[0005] In view of the deficiencies in the prior art, the present invention provides a polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27 and its application.
[0006] The technical solutions of the present invention are as follows: A polyphenol extract of wild Phellinus igniarius SH-27 fruiting body, the extract is extracted from the fruiting body of wild Phellinus igniarius SH-27 preserved in Guangdong Province Microbial Culture Collection Center, the wild Phellinus igniarius SH-27 is a wild trichome porifera ( Inonotus hispidus ), the deposit number is GDMCC No: 65865.
[0007] The preparation method of the polyphenol extract of the wild Phellinus igniarius SH-27 fruiting body comprises the following steps: (1) The fruiting bodies of wild Phellinus igniarius SH-27 were dried and crushed, and the fruiting body powder was mixed with 60% (v / v) ethanol solution and ultrasonically extracted to obtain an alcohol extract; (2) The alcohol extract was subjected to polyamide resin chromatography, and the eluate was collected, concentrated, and freeze-dried to obtain the polyphenol extract of the fruiting bodies of wild Phellinus igniarius SH-27.
[0008] According to a preferred embodiment of the present invention, the method for culturing the wild Phellinus igniarius SH-27 fruiting body in step (1) comprises the following steps: The cultivation medium was placed in a spawn bag, which was tied tightly and sterilized. The mycelial balls of wild Phellinus igniarius SH-27 were inoculated into the spawn bag. The spawn was cultured in a dark environment at 24-30°C and a relative humidity of 60-70% for 10-14 days. Then, the fruiting bodies of wild Phellinus igniarius SH-27 were obtained by continuing to culture under the same conditions for 30-40 days.
[0009] Further preferably, the cultivation medium is composed of: 50-80 parts by mass of poplar wood chips, 10-30 parts by mass of wheat bran, 0.5-1 parts by mass of yeast extract, 0.5-1 parts by mass of calcium oxide, 1-2 parts by mass of calcium sulfate, and 0.5-1 parts by mass of baomazi peel powder; the moisture content of the cultivation medium is 55-65%; the poplar wood chips are sieved with 20-30 mesh, and the baomazi peel powder is sieved with 60-80 mesh.
[0010] Further preferably, the method for culturing the mycelial pellets of the wild Phellinus igniarius SH-27 comprises the following steps: The wild SH-27 strain of Phellinus igniarius was inoculated onto a PDA solid culture medium plate and cultured at 24-30°C until a round bacterial lawn grew. The bacterial lawn was inoculated into a PDA liquid culture medium and cultured at 24-30°C and 100-200 rpm for 10-14 days until a mycelial ball grew.
[0011] Further preferably, the components of the liquid culture medium are: glucose 15~35g / L, yeast extract 0.5~3.5g / L, MgSO4 0.5~3.5g / L, KH2PO4 0.5~3.5g / L, vitamin B1 0.01~0.2g / L, the solvent is water, and the pH is 6.5~7.5.
[0012] Preferably, according to the present invention, in step (1), the wild Morinda igniarius SH-27 fruiting body is crushed and passed through a 60-80 mesh sieve, and the fruiting body powder is mixed with ethanol at a material-liquid ratio of 1:(20-40) (m / v, g / mL).
[0013] Preferably, according to the present invention, the ultrasonic extraction conditions in step (1) are: 40-50°C, 100-150 W, and ultrasonic time of 30-60 min.
[0014] According to the present invention, preferably, the polyamide resin chromatography in step (2) comprises the following steps: a. Pretreatment: First, clean the dirt on the resin surface with distilled water, then soak the resin in 95% (v / v) ethanol solution for 24-36 hours; then, wash the resin with deionized water until there is no alcohol smell; then, soak the cleaned resin in 6% (v / v) HCl solution for 5-6 hours, and rinse with deionized water until the pH is neutral; then, soak it in 6% (v / v) NaOH solution for 5-6 hours, and rinse with deionized water until the pH is neutral; finally, soak the treated resin in distilled water for later use; b. Column: The resin bed filling height is 18-20 cm, the column flow rate is set to 2-6 BV / h, and the sample volume is 3-5 BV; c. Elution: Use 50~70% (v / v) ethanol solution for elution, set the elution flow rate to 2~6BV / h, and the desorption volume to 5~7 BV.
[0015] The application of the above-mentioned wild Phellinus igniarius SH-27 fruiting body polyphenol extract in the preparation of antioxidant products.
[0016] Application of the polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27 in the preparation of functional food or medicine for treating or preventing colitis.
[0017] Beneficial effects: 1) The wild Morinda igniarius SH-27 used in the present invention is a rare medicinal and edible fungus rich in active ingredients. The polyphenol content of its liquid cultured mycelium pellet is 29.74 mg / g, the triterpenoid content is 3.52 mg / g, the polysaccharide content is 88.61 mg / g, and the flavonoid content is 5.14 mg / g. By optimizing the liquid culture medium, the polyphenol content of its mycelium pellet can reach 46.98 mg / g, and the polyphenol content of its cultivated fruiting body can reach 77.32 mg / g. The polyphenol extract of its fruiting body has great medicinal potential in the treatment and intervention of antioxidant, anti-inflammatory, anti-cancer, anti-tumor, diabetes and hypertension.
[0018] 2) The present invention uses ultrasound combined with polyamide resin to purify phenolic substances from the fruiting bodies of wild Morinda ignia SH-27, which effectively shortens the purification process, improves the separation and purification efficiency, and has excellent separation effect. Ultrasonic-assisted extraction effectively improves the extraction rate of total polyphenols from the fruiting bodies of wild Morinda ignia SH-27. In addition, polyamide resin has a good adsorption effect on phenolic substances, is green and non-toxic, and can be used repeatedly, making it suitable for industrial production. The advantage of the present invention is that it integrates a variety of separation and purification methods, making the entire extraction process more efficient and economical. The polyphenol purity of the polyphenol extract from the fruiting bodies of wild Morinda ignia SH-27 can reach 446.9 mg / g, providing an effective theoretical basis for the research and application of Morinda ignia phenolic substances in industrial production, especially in functional foods. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is the mycelial morphology of wild Phellinus igniarius SH-27; Figure 2 This is the morphological picture of the fruiting body of wild Phellinus igniarius SH-27; Figure 3 This is the dynamic adsorption curve of polyamide resin at different flow rates; Figure 4 This is the desorption curve of polyamide resin in ethanol solutions with different concentrations; Figure 5 This is the dynamic desorption curve of polyamide resin at different flow rates; Figure 6 This is the chromatogram of the polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27. DETAILED DESCRIPTION
[0020] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.
[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available materials and reagents unless otherwise specified.
[0022] Example 1 Isolation, Screening and Identification of Wild Phellinus igniarius (1) Wild Phellinus igniarius fruiting bodies were collected from the century-old ancient mulberry garden in Xiajin, Shandong Province, China. Under sterile conditions, the surface was disinfected by wiping with 75% (v / v) alcohol cotton. The alcohol cotton should not be too wet, and no alcohol or cotton wool should remain on the fruiting bodies after wiping. The blade was burned with an alcohol lamp to disinfect it, and then the blade was used to cut along the fiber of the fruiting body. After cutting, the blade was burned again, and a square was cut in the center of the cut surface with the blade to facilitate picking up with tweezers. The cut fruiting body tissue was picked up with pointed tweezers burned with an alcohol lamp; (2) Preparation of mother culture medium: After sterilizing the plate at 121°C under high temperature and high pressure, pour the prepared and sterilized PDA solid culture medium onto the clean bench and wait for it to solidify; The PDA solid culture medium comprises the following components: 12 g / L potato extract powder, 20 g / L glucose, 15 g / L agar, water as solvent, and natural pH.
[0023] (3) Preparation of mother culture: Transfer the fruiting body tissue separated in step (1) to the PDA solid culture medium in step (2) and culture in a 30°C constant temperature box in the dark; (4) Preparation of original seed: When the mycelium has grown but other bacteria have not yet grown, pick the tip of the mycelium and place it on the PDA solid culture medium, and culture it in a 30℃ constant temperature box in the dark.
[0024] This strain grows slowly. After about three weeks of culture, the colony diameter is about 7~9cm. In the early stage of growth, white velvety hyphae grow. As the hyphae grow, they gradually change from white to yellow-brown, but the fresh hyphae at the edge of the colony are still white. Figure 1 As shown, this is the daily storage form of the strain. Mycelium or fungus moss can be picked from this strain in subsequent experiments.
[0025] (5) Expansion culture: Pick the hyphae from the edge of the colony of the original culture for liquid culture, culture in a shake flask at 24°C and 100 rpm for 10-14 days to obtain mycelial balls; The liquid culture medium is PDB liquid culture medium, which comprises 12 g / L potato extract powder, 20 g / L glucose, water as solvent, and natural pH.
[0026] (6) Identification of fungal species: The mycelial pellets obtained in step (5) were ground with liquid nitrogen, and the sample genome was extracted using a plant genomic DNA extraction kit. ITS-PCR amplification was performed using universal primers for the fungal internal transcribed spacer region, ITS1 / ITS4, synthesized by Shanghai Sangon Biotechnology Co., Ltd.; The primers are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4:5'-TCCTCCGCTTATTGATATGC-3', The ITS-PCR experiment was performed with a system of 50 μL, as shown in Table 1.
[0027] Table 1
[0028] The reaction conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. The amplified products were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.
[0029] The above ITS sequences were BLASTed using the NCBI database and found to be Inonotus hispidus The clone SH1 similarity was as high as 99%, and it was identified as a strain of Porifera scalypifolia ( Inonotus hispidus ) and named SH-27. It was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on February 10, 2025. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65865.
[0030] The wild mulberry fungus SH-27 screened by the present invention belongs to the family of Coriolus, the genus of Microporus, and has different shapes and colors, black or yellow-brown, with a cap in the shape of a horseshoe, and solitary fruiting bodies. The surface of some sample fruiting bodies is smooth, while the surface of some sample fruiting bodies has rough objects. The mycelium morphology is as follows: Figure 1 As shown, the fruiting body morphology is as follows Figure 2 The ITS sequence of the wild mulberry fungus SH-27 is shown in SEQ ID NO.1.
[0031] Example 2 Liquid Culture of Wild Phellinus Igneus SH-27 An alcohol lamp was lit on a sterile clean bench to create a local aseptic operating environment. Mycelial moss was picked from the mycelium of wild Phellinus igneus SH-27 and inoculated into the optimized culture medium for liquid culture. The culture was shaken at 24°C and 100 rpm for 10-14 days to obtain mycelial balls.
[0032] The optimized culture medium components are: glucose 30.1 g / L, yeast extract 3.15 g / L, MgSO4 3.1 g / L, KH2PO4 1.1 g / L, vitamin B1 0.1 g / L, solvent is water, pH 7.2.
[0033] Example 3 Determination method of active ingredient content of wild Phellinus igniarius SH-27 1. Determination of polyphenol content (1) Preparation of standard curve Accurately pipette 1 mL of the gallic acid standard stock solution (200 mg / L) into a 10 mL volumetric flask and dilute to volume with 70% ethanol to obtain the gallic acid working solution. Then, pipette 0, 0.2, 0.4, 0.6, 1.0, and 1.5 mL of the gallic acid working solution into separate 10 mL volumetric flasks. Add 2.5 mL of Folin-phenol reagent and shake well. Add 2.5 mL of 15% Na₂CO₃ solution, dilute to the mark with water, and shake well. Incubate in a 40°C water bath for 60 minutes, then allow to cool for 20 minutes. Measure the absorbance at 760 nm. Plot a standard curve with concentration as the horizontal axis and absorbance as the vertical axis.
[0034] (2) Detection of polyphenols in samples The mycelial pellets obtained in step (5) of Example 1 or the liquid culture of Example 2 were dried at 60°C and then ground into powder. 0.4 g of mycelial powder was accurately weighed and added with 4 mL of 70% ethanol. After ultrasonic extraction at 55°C for 1 h, the mixture was centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The residue was extracted twice with 3 mL of 70% ethanol solution under the same extraction conditions as above. The supernatants from the three extractions were combined and diluted to 10 mL with 70% ethanol to obtain the ethanol extract sample solution for the determination of polyphenols, triterpenes, and flavonoids.
[0035] (3) Sample testing Pipette 1.0 mL of the alcohol extract into a 10 mL stoppered colorimetric tube. Add 2.5 mL of Folin-phenol reagent and shake well. Add 2.5 mL of 15% Na₂CO₃ solution, bring to volume with water, and shake well. Incubate in a 40°C water bath for 60 minutes, allow to cool for 20 minutes, and measure the absorbance at 760 nm. Calculate the total polyphenol concentration in each sample using the standard curve.
[0036] 2. Determination of polysaccharide content The extraction of polysaccharides was carried out according to the method of GB / T 15672-2009 for the determination of total sugar content in edible fungi. (1) Preparation of glucose standard curve To create a glucose standard curve: Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of a glucose standard solution (100 mg / L) into a 10 mL stoppered colorimetric tube. Fill to 1.0 mL with distilled water. Add 1.0 mL of 5% (v / v) phenol solution to each solution. Quickly add 5.0 mL of concentrated sulfuric acid and allow to react for 10 minutes. Then, place the colorimetric tube in a 30°C water bath for 20 minutes. Measure the absorbance of a certain amount of the reaction solution at 490 nm. Based on the measured values, plot a standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.
[0037] (2) Detection of polysaccharides in samples The mycelial pellets obtained from the liquid culture in step (5) of Example 1 were dried at 60°C and ground into powder. 0.25 g of the crushed mycelial powder was accurately weighed and added to 50 mL of distilled water. The mixture was then boiled in a water bath for 3 h. A group containing only distilled water and no mycelial powder was also boiled in a water bath for 3 h as a blank control. After the boiling water bath, the mixture was filtered, and the filtrate obtained was used as the sample for the intracellular polysaccharide content test.
[0038] (3) Sample testing Sample polysaccharide content determination: Accurately pipette 0.1 mL of each diluted sample (to ensure accurate experimental results, dilute the liquid to an absorbance between 0.2 and 0.8) into a 10 mL stoppered colorimetric tube. Fill to 1.0 mL with distilled water. Add 1.0 mL of 5% (v / v) phenol solution to the tube. Quickly add 5 mL of concentrated sulfuric acid, allow to react for 10 minutes, and place the tube in a 30°C water bath for 20 minutes. Measure absorbance using the same method as the standard curve. Perform three replicates for each sample. Use a blank control sample to zero the spectrophotometer. Calculate the polysaccharide content of each sample based on the standard curve.
[0039] 3. Determination of triterpenoid content (1) Standard curve: Weigh the oleanolic acid standard and add anhydrous ethanol to prepare a 0.2 mg / mL standard solution. Accurately measure 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the standard solution into centrifuge tubes. Evaporate the solvent. Accurately add 0.2 mL of freshly prepared 0.05 g / mL vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid. Shake well. Heat in a 70°C water bath for 15 minutes, cool in an ice bath for 5 minutes, add 4 mL of ethyl acetate, shake well, and use the vanillin-glacial acetic acid and ethyl acetate mixture as a blank. Measure the absorbance of the standard at 560 nm to construct a standard curve.
[0040] (2) Sample testing Take 1 mL of the ethanol extract sample, add 0.2 mL of freshly prepared 0.05 g / mL vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid, shake well, heat in a 70°C water bath for 15 minutes, cool in an ice bath for 5 minutes, add 4 mL of ethyl acetate, shake well, and measure the absorbance. Calculate the triterpenoid content in each sample using the standard curve.
[0041] 4. Determination of flavonoid content (1) Standard curve: Accurately weigh 10 mg of rutin standard, dried to a constant weight, dissolve in anhydrous ethanol, and adjust to volume in a 50 mL volumetric flask. Pipette 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the 0.2 g / L rutin standard solution into a test tube and add anhydrous ethanol to make the volume 1.5 mL. Sequentially add 0.1 mL of 100 g / L aluminum nitrate solution and 0.1 mL of 98 g / L potassium acetate solution, and make up to 5 mL with water. Let the solution stand for 1 hour. Measure the absorbance at 420 nm using 30% (v / v) ethanol as a blank to construct a standard curve.
[0042] (2) Sample testing Take 1.5 mL of the alcohol extract sample and add 0.1 mL of 100 g / L aluminum nitrate solution and 0.1 mL of 98 g / L potassium acetate solution, respectively. Make up to 5 mL with water. Prepare three replicates for each sample and let it stand for 1 hour. Measure the absorbance using the same method as the standard curve. Calculate the flavonoid content in each sample using the regression equation of the standard curve.
[0043] The wild mulberry fungus SH-27 screened by the present invention has a liquid culture mycelial pellet with a polyphenol content of 29.74 mg / g, a polysaccharide content of 88.61 mg / g, a triterpene content of 3.52 mg / g, and a flavonoid content of 5.14 mg / g. By optimizing the liquid culture medium, the polyphenol content of the mycelial pellet can reach 46.98 mg / g.
[0044] Example 4 Preparation of wild Phellinus igniarius SH-27 fruiting bodies 1. Preparation of cultivation medium The cultivation medium used is granular poplar wood chips. The cultivation medium formula is 77% poplar wood chips, 20% wheat bran, 1% yeast extract, 0.5% calcium oxide, 1% calcium sulfate, and 0.5% tamarind bark powder; the tamarind bark powder is sieved through a 60-mesh screen.
[0045] First, pick fresh, mold-free granular poplar chips, pass them through a 20-mesh sieve to remove wood blocks and other debris, weigh the poplar chips according to the formula, then put the weighed yeast extract, calcium oxide, calcium sulfate and baomazipi powder into water, add the poplar chips, stir evenly, adjust the moisture content of the cultivation medium to 60%, and then put it into the culture bag. The culture bag is a high-temperature resistant polypropylene bag (specifications 17cm×33cm×0.05cm). The bag should be moderately tight when filling, then tie the bag tightly, and use high-pressure steam sterilization at 121°C for 2 hours.
[0046] 2. Cultivation, inoculation and fungus culture On a clean bench, inoculate wild Phellinus igniarius SH-27 mycelial pellets into spawn bags, inoculating 3-5 pellets per bag. Immediately move the inoculated bags to the incubation room and place them on a culture rack for growth. Cultivate at 24°C, 70% relative humidity, and shade from light. Regularly inspect for bacterial contamination and mycelial growth during incubation. Any contamination should be removed promptly to prevent spread. After 10-14 days of incubation, mycelium should cover the entire surface of the culture medium and penetrate deep into the culturing medium. Continue incubation under the same conditions, and Phellinus igniarius fruiting bodies will emerge in approximately 30-40 days.
[0047] The cultured wild Phellinus igniarius SH-27 fruiting bodies were freeze-dried and ground into powder, and the polyphenol content of the fruiting bodies was measured to be 77.32 mg / g.
[0048] Example 5 Preparation method of polyphenol extract from wild Phellinus igniarius SH-27 fruiting body A. Pick the fresh wild Phellinus igniarius SH-27 fruiting bodies obtained by cultivation in Example 4, place them in an oven at 60°C to dry the moisture contained therein, then use a high-speed grinder to grind them, pass them through a 60-mesh sieve, use 60% (v / v) ethanol solution as a solvent, mix the fruiting body powder with ethanol at a solid-liquid ratio of 1:40 (m / v, g / mL), and perform ultrasonic extraction at 45°C, 100 W, 60 min to obtain an alcohol extract.
[0049] B. Centrifuge the alcohol extract at 8000 rpm for 10 min, collect the supernatant, concentrate by rotary evaporation and then freeze-dry to obtain the crude total polyphenol extract.
[0050] C. The crude extract is dissolved in water to prepare a crude extract, which is subjected to polyamide resin chromatography. The eluate is collected, concentrated, and freeze-dried to obtain a purified total polyphenol product, i.e., a polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27.
[0051] The process of polyamide resin pretreatment and elution includes the following steps: a. Pretreatment: First, clean the resin surface with distilled water to remove dirt. Then, soak the resin in 95% (v / v) ethanol for 24 hours to remove surface preservatives and impurities. Next, rinse the resin with deionized water until the alcohol odor is gone. Then, soak the cleaned resin in 6% (v / v) HCl for 5 hours and rinse with deionized water until the pH is neutral. Then, soak the resin in 6% (v / v) NaOH for 5 hours and rinse with deionized water until the pH is neutral. Finally, soak the treated resin in distilled water until ready for use.
[0052] b. Column: Wet packing was used to perform dynamic adsorption in a chromatography column (1.0 cm × 30 cm) filled with polyamide resin. The resin bed was filled to a height of 20 cm. A portion of distilled water was first pumped in to remove air and make the resin layer more compact. The crude extract was then slowly loaded onto the column. The dynamic adsorption curve of the polyamide resin at different flow rates (e.g. Figure 3 As the sample flowed through the column, the polyamide resin's adsorption rate for polyphenols gradually decreased, and adsorption capacity was inversely proportional to the flow rate. With an initial sample concentration of 0.32 mg / mL, leakage points occurred at 5, 4, and 3 BV for 2, 4, and 6 BV / h, respectively. While a sample flow rate of 2 BV / h increased the contact time between the polyamide resin and the polyphenols, it was time-consuming and reduced purification efficiency. Considering both adsorption effectiveness and production efficiency, a flow rate of 4 BV / h and a sample loading volume of 4 BV were set.
[0053] c. Elution: According to the desorption of polyamide resin in ethanol solutions of different concentrations (such as Figure 4 As shown in Figure 2), 60% (v / v) ethanol solution was selected for elution. According to the dynamic desorption curve of polyamide resin at different flow rates (as shown in Figure 2), the desorption rate of polyamide resin was 100%. Figure 5 As shown in the figure), set the flow rate to 4 BV / h, the desorption volume to 6.5 BV, and collect the eluate.
[0054] The polyphenol content of the crude extract and purified total polyphenols of wild Phellinus igniarius was determined according to the polyphenol content determination method in Example 2. After purification with polyamide resin, the polyphenol purity of the polyphenol extract of wild Phellinus igniarius SH-27 fruiting body increased from 77.32 mg / g to 446.9 mg / g, an increase of 5.78 times.
[0055] Example 6 Composition analysis of purified substances The phenolic components of the polyphenol extract of the wild Phellinus igniarius SH-27 fruiting body prepared in Example 5 were determined using ultra-performance liquid chromatography-mass spectrometry (UPLC-Q-TOF-MS) under the following conditions: 1. Chromatographic conditions: Chromatographic column: BEH C18 column; flow rate: 0.3 mL / min; column temperature: 25°C; mobile phase: 0.1% formic acid (A) and acetonitrile (B); injection volume: 5 μL; gradient elution; Elution procedure: 1) From 0 to 12 min, the mobile phase B content was increased from 10% to 30%; 2) From 12 to 30 min, increase the mobile phase B from 30% to 85%; 3) After maintaining 85% mobile phase B for 3 minutes, return the column to the initial conditions and allow the column to remain in equilibrium for 5 minutes.
[0056] 2. Mass spectrometry conditions: ESI ion source (positive and negative modes); spray voltage: -4.5 kV; gas temperature: 550 °C; scan range: 100-1200 m / z; 800-1000 m / z 3. Experimental results: A total of 16 phenolic compounds were detected in the purified product, and the chromatograms are shown in Figure 2. Figure 6 Specific data are shown in Table 2.
[0057] Table 2. UPLC-Q-TOF-MS identification results of polyphenols in the purified products
[0058] As shown in Table 2, wild SH-27 fungus is rich in various active ingredients. For example, syringaldehyde exhibits potent antioxidant and antimicrobial activity and can be used as a lead compound in drug development for the development of healthcare and therapeutic drugs. Obovatol can regulate the body's immune function, inhibit the release of inflammatory mediators, and inhibit a variety of bacteria and viruses. It also scavenges free radicals and reduces oxidative stress damage, showing great potential for the development of new drugs. Therefore, wild SH-27 fungus is expected to play a key role in the development of innovative drugs and functional foods, making greater contributions to human health.
Claims
1. A polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27, characterized in that The extract is obtained by extracting the fruiting body of wild mulberry fungus SH-27 preserved in the Guangdong Provincial Microbial Culture Collection Center. The wild mulberry fungus SH-27 is a thick hair pore fungus ( Inonotus hispidus ), the deposit number is GDMCC No: 65865.
2. The method for preparing the polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27 according to claim 1, characterized in that: The steps include: (1) The fruiting bodies of wild Phellinus igniarius SH-27 were dried and crushed, and the fruiting body powder was mixed with a 60% by volume ethanol solution and subjected to ultrasonic extraction to obtain an alcohol extract; (2) the alcohol extract was subjected to polyamide resin chromatography, and the eluate was collected, concentrated, and freeze-dried to obtain a polyphenol extract of the fruiting bodies of wild Phellinus igniarius SH-27.
3. The preparation method according to claim 2, wherein The method for culturing the wild Phellinus igniarius SH-27 fruiting body in step (1) comprises the following steps: The culture medium is placed in a spawn bag, the bag is tied tightly and sterilized, the mycelial pellets of wild Phellinus igniarius SH-27 are inoculated into the spawn bag, and the spawn is cultured at 24-30°C and a relative humidity of 60-70% under shade for 10-14 days, and then cultured under the same conditions for 30-40 days to obtain the fruiting bodies of wild Phellinus igniarius SH-27; Further preferably, the cultivation medium is composed of: 50-80 parts by mass of poplar wood chips, 10-30 parts by mass of wheat bran, 0.5-1 parts by mass of yeast extract, 0.5-1 parts by mass of calcium oxide, 1-2 parts by mass of calcium sulfate, and 0.5-1 parts by mass of baomazi peel powder; the moisture content of the cultivation medium is 55-65%; the poplar wood chips are sieved with 20-30 mesh, and the baomazi peel powder is sieved with 60-80 mesh.
4. The preparation method according to claim 3, wherein The method for culturing wild mulberry igniarius SH-27 mycelial pellets comprises the following steps: The wild SH-27 strain of Phellinus igniarius was inoculated onto a PDA solid culture plate and cultured at 24-30°C until a circular lawn was grown. The lawn was then inoculated onto a PDA liquid culture medium and cultured at 24-30°C and 100-200 rpm for 10-14 days until a mycelial ball was formed. Further preferably, the components of the liquid culture medium are: glucose 15-35 g / L, yeast extract 0.5-3.5 g / L, MgSO4 0.5-3.5 g / L, KH2PO4 0.5-3.5 g / L, vitamin B1 0.01-0.2 g / L, the solvent is water, and the pH is 6.5-7.
5.
5. The preparation method according to claim 2, wherein In step (1), the wild mulberry fungus SH-27 fruiting body is crushed and then passed through a 60-80 mesh sieve.
6. The preparation method according to claim 2, wherein In step (1), the fruiting body powder is mixed with ethanol at a mass-to-volume ratio of 1:(20-40), with the unit being g / mL.
7. The preparation method according to claim 2, wherein The ultrasonic extraction conditions in step (1) are: 40-50°C, 100-150 W, and ultrasonic time of 30-60 min.
8. The preparation method according to claim 2, wherein The polyamide resin chromatography comprises the following steps: a. Pretreatment: First, clean the dirt on the resin surface with distilled water, then soak the resin in a 95% by volume ethanol solution for 24-36 hours; then, wash the resin with deionized water until there is no alcohol smell; then, soak the cleaned resin in a 6% by volume HCl solution for 5-6 hours, and rinse with deionized water until the pH is neutral; then, soak it in a 6% by volume NaOH solution for 5-6 hours, and rinse with deionized water until the pH is neutral; finally, soak the treated resin in distilled water for later use; b. Column: The resin bed filling height is 18-20 cm, the column flow rate is set to 2-6 BV / h, and the sample volume is 3-5 BV; c. Elution: Use 50~70% ethanol solution by volume for elution, set the elution flow rate to 2~6BV / h, and the desorption volume to 5~7 BV.
9. Use of the polyphenol extract from the fruiting body of wild Phellinus igniarius SH-27 according to claim 1 in the preparation of antioxidant products.
10. Use of the wild Phellinus igniarius SH-27 fruiting body polyphenol extract according to claim 1 in the preparation of functional foods or medicines for treating or preventing colitis.