Method for improving extraction yield of total flavonoids of pleurotus eryngii by utilizing rhizopus oryzae XB4-16

Abstract

The invention discloses a method for improving the extraction yield of total flavonoids of pleurotus eryngii by utilizing rhizopus oryzae XB4-16. According to the method, microbial fermentation pretreatment is added before ultrasonic extraction of the total flavonoids of pleurotus eryngii with an ethanol water solution. The used fermentation strain rhizopus oryzae XB4-16 is obtained by purposeful screening and mutation breeding for hydrolyzing the cell wall of the pleurotus eryngii. The rhizopus oryzae XB4-16 moderately grows in the pleurotus eryngii powder added with cane sugar to generate various hydrolases, then water is added into the fermented pleurotus eryngii powder for heat preservation, substances such as cellulose in cell walls are hydrolyzed, and flavone in a combined state can be dissolved out during ultrasonic extraction, so that the extraction yield of the total flavone can be remarkably improved. Compared with an extraction method without fermentation pretreatment, the method has the advantages that microbial fermentation pretreatment is added before ultrasonic extraction of the total flavonoids of the pleurotus eryngii with the ethanol aqueous solution, and the extraction yield of the total flavonoids of the pleurotus eryngii is increased by 35.4%.

Description

(I) Technical field

[0001] The invention belongs to the field of microbial fermentation applications, and in particular relates to a method for improving the extraction rate of total flavonoids from Pleurotus eryngii by utilizing Rhizopus oryzae XB4-16. (II) Background technology

[0002] King oyster mushroom is the fruiting body of the fungus Pleurotuseryngii of the family Pleurotaceae of the phylum Basidiomycota, also known as Pleurotus eryngii or snow mushroom. King oyster mushroom is named because of its thick flesh, crisp texture, almond fragrance and abalone-like taste. King oyster mushroom is native to Japan, and later gradually distributed in the Mediterranean region of Europe, the Middle East, North Africa and parts of Asia. There are also wild king oyster mushrooms distributed in Sichuan, Qinghai and Xinjiang in my country. Since the first successful artificial cultivation in 1958, King oyster mushroom has been artificially cultivated in various countries around the world, and large-scale cultivation is carried out in Taiwan, Fujian Province, Jiangsu Province, Zhejiang Province and Shanghai in my country.

[0003] Pleurotus eryngii contains rich nutrients, including polysaccharides, proteins, peptides, fats, flavonoids, vitamins, sterols, etc., so it is not only nutritious and delicious, but also has a variety of medicinal and health benefits, such as lowering blood lipids and blood pressure, enhancing immunity, beautifying and nourishing the skin, etc. It also has the effect of regulating blood sugar for diabetic patients. In recent years, flavonoids in edible fungi have attracted much attention. They have good antioxidant, immune enhancement, anti-infection, antibacterial, antiviral, anti-allergic and other biological activities. As a natural substance, it has high safety and can be widely used in food, medicine and health products. In 2023, the annual output of Pleurotus eryngii in my country has reached more than 2 million tons, and it is growing at a rate of more than 20% per year, facing a situation of oversupply. Pleurotus eryngii has a high content of flavonoids. Extracting flavonoids from Pleurotus eryngii and applying them in the food field has broad prospects, and can also promote the development of Pleurotus eryngii cultivation industry.

[0004] At present, there have been many research reports on the extraction of total flavonoids from Pleurotus eryngii. In the reported studies, the main method for extracting total flavonoids is ethanol-water extraction. Conventional ethanol-water extraction is simple to operate, but the extraction yield is often not high. On the basis of ethanol-water extraction, ultrasonic and microwave treatment can promote the dissolution of flavonoids and improve the extraction yield to a certain extent. Ultrasonic-assisted extraction has low equipment requirements and will not significantly increase the extraction cost, but the effect of improving the extraction yield is not significant enough; microwave-assisted extraction has a significant effect on improving the extraction yield, but the internal thermal effect often leads to the degradation of some flavonoids, which greatly reduces the activity of the extracted total flavonoids.

[0005] In recent years, enzymatic hydrolysis technology has been widely used in the extraction of natural active substances. This method has the advantages of mild conditions, little damage to the structure of active ingredients, and can significantly improve the extraction rate of products. Among the enzymatic hydrolysis-assisted extraction methods, cellulase is the most commonly used, followed by pectinase and protease. The cell wall composition of plants or edible fungi includes substances such as cellulose, hemicellulose, lignin and pectin, so the use of a single enzyme has limited effect on improving the extraction rate of products. Many studies use multiple enzymes (complex enzymes) at the same time. If the amount of enzyme used is large, it will undoubtedly increase the extraction cost.

[0006] At present, commercialized cellulases, hemicelluloses and proteases are all produced by microbial fermentation. Therefore, if a certain microorganism is used to directly ferment the raw materials to be extracted, the microorganism will produce a variety of hydrolases during its growth, which can hydrolyze substances such as cellulose, pectin and protein in the cell walls of the raw materials, thereby facilitating the release of flavonoids in the cells. Of course, microorganisms may also produce enzymes to decompose flavonoids, but suitable fermentation strains can be selected and the degree of fermentation can be controlled to achieve enzymatic damage to the cell walls, but not yet to produce enzymes to degrade flavonoids, thereby significantly improving the total flavonoid extraction rate. In addition, compared with enzymatic extraction, microbial fermentation-assisted extraction saves the cost of enzymes.

[0007] In order to overcome the problem of low total flavonoids extraction rate from Pleurotus eryngii, the present invention performs microbial fermentation pretreatment on Pleurotus eryngii and then uses ethanol aqueous solution ultrasonic extraction to significantly improve the extraction rate of total flavonoids from Pleurotus eryngii. (III) Summary of the invention

[0008] The invention aims to provide a method for improving the extraction rate of total flavonoids from Pleurotus eryngii by using Rhizopus oryzae XB4-16. The invention screens a new microorganism strain, Rhizopus oryzae XB4-16, and after Pleurotus eryngii is fermented by the strain and then ultrasonically extracted with an ethanol aqueous solution, the extraction rate of total flavonoids is significantly improved.

[0009] The technical solution adopted by the present invention is:

[0010] In order to improve the extraction rate of total flavonoids from Pleurotus eryngii, the present invention screened and obtained a new microbial strain, Rhizopus oryzae XB4-16, which was preserved in Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No: 65283 and a preservation date of October 24, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0011] The Rhizopus oryzae XB4-16 of the present invention is separated from the microorganism enrichment culture of Pleurotus eryngii, and is obtained through screening and mutagenesis breeding. Its morphological characteristics are as follows: when cultured on a potato dextrose agar (PDA) plate medium at 28°C, the colony is grayish white and fluffy at the beginning, gradually turns gray after 24 hours, the mycelium layer is thick and loose, and can cover the culture dish in 48 hours, with gray spores on the surface, and the back of the colony is grayish white. The creeping hyphae and rhizoids are obvious; the sporangial stalks are upright or slightly curved, 2 to 4 strains are bundled, opposite to the rhizoids, and mostly form umbrella-shaped aggregations; the sporangium is spherical or nearly spherical, light black; the sporangium spores are elliptical or spherical, uneven in size, and 3 to 6 μm in diameter.

[0012] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Rhizopus oryzae XB4-16 is shown in SEQ ID NO.1.

[0013] The invention provides a method for improving the extraction rate of total flavonoids of Pleurotus eryngii by using Rhizopus oryzae XB4-16. The method comprises the following steps: (1) adding spore liquid of Rhizopus oryzae XB4-16 to Pleurotus eryngii powder, stirring evenly and fermenting at 28-30 DEG C for 48-56 hours to obtain Pleurotus eryngii fermented product; (2) adding deionized water to the Pleurotus eryngii fermented product, stirring evenly and keeping warm at 35-40 DEG C for 2-4 hours, adding anhydrous ethanol, extracting by ultrasonic and filtering to obtain Pleurotus eryngii extract; (3) evaporating the Pleurotus eryngii extract to dryness under reduced pressure, dissolving in anhydrous ethanol and drying to obtain Pleurotus eryngii total flavonoids extract.

[0014] Furthermore, the Pleurotus eryngii in step (1) is a mature fruiting body of the artificially cultivated Pleurotus eryngii. The Pleurotus eryngii powder is a fine powder obtained by slicing fresh Pleurotus eryngii, drying at 85°C to a constant weight, and then crushing it to pass through a 60-mesh sieve.

[0015] Furthermore, the preparation method of the spore liquid of Rhizopus oryzae XB4-16 in the step (1) is as follows: the spores of Rhizopus oryzae XB4-16 stored at low temperature are inoculated on a potato dextrose agar (PDA) plate culture medium, and after culturing at 28° C. for 36 to 48 hours, a sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain a spore liquid; preferably, the spore liquid is adjusted to a spore concentration of 4×10 6 ~7×10 6The final concentration of the PDA plate medium is as follows: 150-200 g / L of potato (cut into cubes with a side length of 1 cm, boiled in water for 20 minutes, and the residue is removed and the juice is retained), 20 g / L of sucrose, 1-2 g / L of MgSO4·7H2O, the solvent is tap water, pH 6.0-6.5, and sterilized at 121°C with high pressure steam for 15 minutes. The sucrose concentration in the sterile sucrose aqueous solution is 50 g / L, and sterilized at 115°C with high pressure steam for 20 minutes.

[0016] Furthermore, the volume dosage of the spore liquid of Aspergillus oryzae XB4-16 in the step (1) is 1.0 to 1.2 mL / g based on the mass of Pleurotus eryngii powder.

[0017] Furthermore, the preparation method of the Pleurotus eryngii extract in step (2) is as follows: deionized water is added to the Pleurotus eryngii fermentation product, stirred evenly and kept warm at 35-40°C for 2-4h, then anhydrous ethanol is added, and the product is transferred to an ultrasonic cleaning machine at a water temperature of 50-60°C, and ultrasonic extraction is performed at 100-200W for 20-30min. After the ultrasonic extraction is completed, the product is filtered with a Buchner funnel, and the filtrate is collected to obtain the Pleurotus eryngii extract. The volume amount of the deionized water added is 6-7mL / g based on the mass of the raw material Pleurotus eryngii powder; the volume amount of the anhydrous ethanol added is 1.5-2.0 times the volume of the deionized water.

[0018] Furthermore, the preparation method of the total flavonoid extract of Pleurotus eryngii in the step (3) is as follows: the Pleurotus eryngii extract is distilled at 45°C and -0.1MPa until no liquid flows out, anhydrous ethanol is added in a volume of 0.4-1.0mL / g based on the mass of the raw material Pleurotus eryngii powder, and after sufficient shaking, the mixture is centrifuged at 5000-8000r / min for 5-10min, and the supernatant is transferred to a clean culture dish and vacuum dried at 50°C and -0.1MPa to constant weight to obtain the total flavonoid extract.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that the present invention adds microbial fermentation pretreatment before ultrasonic extraction of total flavonoids from Pleurotus eryngii with ethanol aqueous solution. The fermentation strain Rhizopus oryzae XB4-16 used is obtained by purposeful screening and mutagenesis breeding for the ability to hydrolyze the cell wall of Pleurotus eryngii. Rhizopus oryzae XB4-16 grows moderately in Pleurotus eryngii powder added with sucrose, produces a variety of hydrolases, and then the fermented Pleurotus eryngii powder is added with water for heat preservation, and the cellulose and other substances in the cell wall are hydrolyzed, which helps the bound flavonoids to dissolve during ultrasonic extraction, thereby significantly improving the total flavonoid extraction rate. Compared with the extraction method that does not use fermentation pretreatment, the present invention adds microbial fermentation pretreatment before ultrasonic extraction of total flavonoids from Pleurotus eryngii with ethanol aqueous solution, and the total flavonoid extraction rate of Pleurotus eryngii is increased by 35.4%. (IV) Description of the drawings

[0020] Figure 1 , standard curve of total flavonoids determined by spectrophotometry (with rutin as standard).

[0021] Figure 2 , standard curve for determining glucose concentration by DNS method.

[0022] Figure 3 , photos of the colony morphology of Rhizopus oryzae XB4-16 cultured on PDA at 28℃ for 36h. (V) Specific implementation methods

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0024] The raw materials, instruments and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials, instruments and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0025] In the embodiment of the present invention, the Pleurotus eryngii is a mature fruiting body of the artificially cultivated Pleurotus eryngii. The Pleurotus eryngii powder is a fine powder obtained by slicing fresh Pleurotus eryngii, drying at 85°C to a constant weight, and then crushing and passing through a 60-mesh sieve.

[0026] In the embodiment of the present invention, the final concentration of the PDA plate medium is as follows: 200 g / L potato (cut into cubes with a side length of 1 cm, boiled in water for 20 min, and the residue is removed and the juice is retained), 20 g / L sucrose, 1 g / L MgSO4·7H2O, the solvent is tap water, pH 6.0-6.5, and sterilized at 121°C with high pressure steam for 15 min. Pour into a sterile culture dish with a diameter of 9 cm before solidification, 15-20 mL per dish.

[0027] In the embodiment of the present invention, the sucrose concentration of the sterile sucrose aqueous solution is 50 g / L, and it is sterilized by high-pressure steam at 115° C. for 20 min; the sterile physiological saline is a 0.9% NaCl aqueous solution, and it is sterilized by high-pressure steam at 121° C. for 15 min.

[0028] In the embodiment of the present invention, the total flavonoid content is determined by spectrophotometry, and the specific method is as follows: the total flavonoid extract is diluted with a 60% ethanol aqueous solution (hereinafter referred to as "60% ethanol") by an appropriate multiple so that the concentration of total flavonoids in the sample to be tested is within the determination range of the standard curve; the solid total flavonoid extract is dissolved with 60% ethanol and diluted by an appropriate multiple to be the sample to be tested. Pipette 1 mL of the sample solution to be tested into a 25-mL colorimetric tube, add 1 mL of a 5% NaNO2 aqueous solution by mass concentration, let stand for 6 minutes, add 1 mL of a 10% Al(NO3)3 aqueous solution by mass concentration, let stand for 6 minutes, add 10 mL of a 4% NaOH aqueous solution by mass concentration, and then dilute to 25 mL with a 60% ethanol aqueous solution. Shake well and let stand for 10 minutes, and measure the absorbance (A) at a wavelength of 510 nm with a spectrophotometer. 510 ), the total flavonoids concentration in the sample was calculated using the rutin standard curve, and the total flavonoids mass was obtained by multiplying the concentration by the volume of the measured sample.

[0029] Drawing of rutin standard curve: Prepare 0.25g / L rutin standard solution with 60% ethanol. Take 2, 3, 4, 5, 6, 7mL of rutin standard solution in 25-mL colorimetric tubes, add 1mL of 5% NaNO2 aqueous solution, let stand for 6min, add 1mL of 10% Al(NO3)3 aqueous solution, let stand for 6min, add 10mL of 4% NaOH aqueous solution, and then dilute to 25mL with 60% ethanol, shake well and let stand for 10min, and measure the absorbance (A) at a wavelength of 510nm with a spectrophotometer. 510 ). With rutin concentration as the horizontal axis, A 510 Draw a standard curve for the ordinate ( Figure 1 ).

[0030] The total flavonoids extraction rate from Pleurotus eryngii is calculated according to Formula 1:

[0031]

[0032] Example 1: Isolation and screening of Pleurotus eryngii fermentation strains

[0033] The microbial strain for fermenting Pleurotus eryngii is isolated and screened according to the following steps:

[0034] (1) The naturally dried Pleurotus eryngii was crushed and passed through a 60-mesh sieve. 5 g was taken into a 150-mL conical flask, and 6 mL of sterile sucrose aqueous solution was added and stirred evenly. The mixture was cultured at 28°C for 72 h. The enriched culture full of mold was diluted with sterile saline to 1×10 -5 , 1×10 -6 , 1×10 -7After dilution was repeated for 2 times, 0.1 mL of the dilution solution was spread on PDA plate culture medium and cultured at 28 °C for 36 h. Fungal colonies with different colors and morphologies were picked and transferred to fresh PDA plate culture medium and cultured at 28 °C for 48 h to obtain 9 pure culture strains. The numbers of the strains are shown in Table 1.

[0035] (2) Add 10 mL of sterile sucrose aqueous solution to each of the 9 fresh plate cultures, stir with an inoculating loop to suspend the spores, transfer the spore solution to a sterile test tube, and adjust the spore concentration with sterile sucrose aqueous solution so that the spore solution of different strains is between 4 × 10 6 ~7×10 6 / mL to obtain the spore liquid of each strain.

[0036] (3) Add 5 g of Pleurotus eryngii powder to 9 150-mL triangular flasks sterilized by dry heat at 160°C for 2 h, and then add 6 mL of each mold spore solution prepared in step (2) (the volume dosage is 1.2 mL / g based on the mass of Pleurotus eryngii powder), and stir evenly. The triangular flasks are sealed with 8 layers of gauze and cultured at 28°C for 72 h to obtain Pleurotus eryngii fermentation products.

[0037] (4) 35 mL of deionized water (7 mL / g of the raw material Pleurotus eryngii powder) was added to all the fermented products of Pleurotus eryngii fermented by each strain in step (3), stirred evenly, and kept warm in a 35°C water bath for 4 h, and then 65 mL of anhydrous ethanol [the volume of ethanol is 1.86 times that of deionized water, the solid-liquid ratio of the extraction system is 1:20 (g:mL), and the volume fraction of ethanol is 65%] was added, stirred evenly, and ultrasonic extraction was performed at 100W for 30 min in an ultrasonic cleaner at 60°C. After the ultrasonic extraction was completed, the filtrate was filtered with a Buchner funnel, and the total flavonoid content in the filtrate was determined by spectrophotometry.

[0038] At the same time, the following three controls were performed: ① blank fermentation control: in step (3), 6 mL of sterile sucrose aqueous solution was added to 5 g of Pleurotus eryngii powder, and the mixture was cultured at 28°C for 72 h, and then the total flavonoids were extracted according to step (4); ② enzymatic extraction control: 35 mL of 250 U / mL cellulase phosphate buffer (pH 6.0, 0.2 mol / L) was added to 5 g of Pleurotus eryngii powder, and the mixture was incubated at 35°C for 4 h, and then the total flavonoids were extracted according to the subsequent operation of step (4); ③ direct extraction control: 35 mL of deionized water was added to 5 g of Pleurotus eryngii powder, and the mixture was incubated at 35°C for 4 h, and then the total flavonoids were extracted according to the subsequent operation of step (4).

[0039] Table 1 Total flavonoids extraction rate of Pleurotus eryngii fermented by different strains and control

[0040]

[0041] As can be seen from the data in Table 1, the blank fermentation control with sterile sucrose aqueous solution but without mold spores has no significant difference in the total flavonoid extraction rate (2.59%) and the direct extraction control (2.57%) because there is basically no mold growth. After the Pleurotus eryngii powder is fermented by different mold strains, the total flavonoid extraction rate is quite different, indicating that the microbial strain used to ferment Pleurotus eryngii to improve the total flavonoid extraction rate has species selectivity. After Pleurotus eryngii is fermented by strain XB4, the total flavonoid extraction rate is 3.02%, which is 17.5% higher than the direct extraction control (2.57%), and is also significantly better than the enzymatic extraction control (2.81%), so the present invention selects strain XB4 as the initial fermentation strain to improve the total flavonoid extraction rate of Pleurotus eryngii.

[0042] Example 2: Classification and Identification of Strain XB4

[0043] Strain XB4 was streaked on PDA plate culture medium and cultured at 28°C. The colonies were initially off-white and fluffy, and gradually turned gray after 24 hours. The mycelium layer was thick and loose, and the culture dish was fully grown in 48 hours. There were gray spores on the surface, and the back of the colonies was off-white. The creeping hyphae and rhizoids were obvious; the sporangial stalks were upright or slightly curved, 2 to 4 plants in bundles, opposite the rhizoids, and mostly formed umbrella-shaped aggregates; the sporangium was spherical or nearly spherical, light black; the sporangiospores were elliptical or spherical, uneven in size, and 3 to 6 μm in diameter. The colony photos of strain XB4 cultured on PDA plate culture medium at 28°C for 36 hours are shown in Figure 3 .

[0044] The rDNA-ITS nucleotide sequence of strain XB4 was measured as shown in SEQ ID NO.1. The sequence was compared by BLAST (Database: Standard databases, Limit to: Sequences from type material) in NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov). The two strains with homology greater than 99% were Rhizopus oryzae NRRL 1469 (99.82%) and Rhizopus sexualis var. americanus NRRL 2626 (99.64%). The colony morphology of strain XB4 is more consistent with that of Rhizopus oryzae, so the biological classification position of strain XB4 can be determined as follows (refer to Mycobank, http: / / www.mycobank.org): Fungi, Mucoromycota, Mucoromycetes, Mucorales, Mucoraceae, Rhizopus, Rhizopus oryzae.

[0045] The rDNA-ITS nucleotide sequence of the strain XB4 is:

[0046] .

[0047] Example 3: Mutation breeding of microbial strains for fermenting Pleurotus eryngii

[0048] The strain XB4 was subjected to mutation breeding to screen strains with better fermentation performance. The specific method is as follows:

[0049] (1) Preparation of spore solution: After strain XB4 was activated and cultured on PDA plate medium at 28°C for 36 hours, 10 mL of sterile saline was added and stirred with an inoculation loop to suspend the spores. 1 mL of spore solution was transferred to a conical flask containing 50 mL of sterile saline (with 20 to 30 glass beads) and shaken at room temperature for 15 minutes. The spore solution was filtered to remove the hyphae (a small ball of fluffy cotton was plugged at the bottom of the triangular funnel), and the spores in the spore solution were counted under a microscope using a hemocytometer. The spores were appropriately diluted with sterile saline to adjust the number of spores to 1.48 × 10 7 Pieces / mL.

[0050] (2) Mutagenesis: Under red light illumination, take 1.5 mL of the above spore suspension and place it in a 6 cm diameter culture dish. The culture dishes are placed on a magnetic stirrer and irradiated for 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 min at a distance of 30 cm from a 15W UV lamp preheated for 30 min. After appropriate dilution of the irradiated spore solution, take 0.1 mL and apply it to a PDA plate medium containing 10 g / L sodium deoxycholate. In the same way, dilute the spore solution that has not been irradiated with ultraviolet light and apply it to the plate as a control to calculate the lethality. The inoculated PDA plate is wrapped with black cloth and inverted at 30°C for 36 h. The colonies on the plate are counted and the lethality is calculated.

[0051] (3) Screening: The colonies on the PDA plate with a lethality rate of more than 90% were picked and transferred to a fresh PDA plate culture medium to obtain 44 strains. Each strain was inoculated into a fresh culture on a PDA plate and cultured at 28°C for 48 hours, and 10 mL of sterile physiological saline was added respectively. The spores were suspended by stirring with an inoculation loop to obtain the spore liquid of each strain. 2 mL of the spore liquid of each strain was taken and inoculated into 50 mL of enzyme production culture medium. After shaking culture at 28°C and 180 r / min for 48 hours, the fermentation liquid was filtered with a Buchner funnel, and the filtrate was collected. The cellulase activity of the fermentation filtrate of each strain was first determined. The top 10 strains with enzyme production activity higher than that of the original strain XB4 were selected, and then the spore liquid of these 10 strains was inoculated with Pleurotus eryngii powder for fermentation according to the method of Example 1. The total flavonoids were extracted by ultrasonic extraction of ethanol aqueous solution. The total flavonoid extraction yield of Pleurotus eryngii fermented by the mutant strains and the control is shown in Table 2.

[0052] Table 2 Total flavonoids extraction rate of Pleurotus eryngii fermented by mutant strains and control

[0053]

[0054] It can be seen from the data in Table 2 that among the 10 mutant strains, the strain numbered XB4-16 has a cellulase activity of 76.5U / mL, which is 35.2% higher than the 56.6U / mL of the original strain XB4. After fermenting Pleurotus eryngii powder with this strain, the total flavonoid extraction rate is 3.43%, which is 13.6% higher than the 3.02% of the wild strain XB4 and 33.5% higher than the 2.57% of the direct extraction control.

[0055] In summary, strain XB4 was isolated from the microbial enrichment of Pleurotus eryngii powder and identified as Rhizopus oryzae. After strain XB4 was induced by ultraviolet light, strain XB4-16 with higher cellulose production activity was screened, namely Rhizopus oryzae XB4-16, which is deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCCNo:65283, the deposit date October 24, 2024, and the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0056] The enzyme production culture medium is composed of: 50g / L wheat bran, 4g / L peptone, 6g / L (NH4)2SO4, 2g / L KH2PO4, 1g / L MgSO4·7H2O, 0.5g / L CaCl2, and the solvent is tap water with a pH value of 6.0. 50mL of the enzyme production culture medium is placed in a 250-mL triangular bottle, which is tied with 8 layers of gauze and sterilized with high-pressure steam at 121°C for 20 minutes.

[0057] The cellulase activity assay is as follows: 1.5 mL of 10 g / L sodium carboxymethyl cellulose solution (pH 6.0, 0.2 mol / L phosphate buffer) and 0.5 mL of crude enzyme solution are added to a 10-mL graduated test tube, respectively, and the mixture is kept warm in a 50°C water bath for 30 min. Then, 3 mL of DNS reagent is added, and the mixture is boiled for 5 min. After cooling with running water, the mixture is fixed to 10 mL with deionized water and shaken well. The crude enzyme solution inactivated by boiling at 100°C for 10 min is used as a reference and the absorbance (A) at a wavelength of 540 nm is measured by a spectrophotometer. 540 ), based on the glucose standard curve ( Figure 2 ) Calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Definition of cellulase activity: Under the conditions of pH 6.0 and 50°C, the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 μmol of glucose per minute is 1 enzyme activity unit (U).

[0058] The cellulase activity was calculated according to formula 2.

[0059]

[0060] In formula 2, C: glucose concentration (μmol / mL) calculated from the standard curve; V1: volume of enzyme reaction system, i.e. 2 mL; T: reaction time, i.e. 30 min; V2: volume of crude enzyme solution, i.e. 0.5 mL.

[0061] Plotting of glucose standard curve: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of standard glucose solution with a concentration of 1 g / L were added to 7 10-mL graduated test tubes, respectively. Then, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer were added, and 3.0 mL of DNS solution was added to each tube. The mixture was boiled in a boiling water bath for 5 min, cooled in running water, and then diluted to 10 mL with deionized water. The mixture was shaken and measured with a spectrophotometer. 540 , with glucose concentration as the horizontal axis, A 540 Draw a standard curve for the ordinate ( Figure 2 ).

[0062] Preparation of DNS reagent: Add 6.3g of 3,5-dinitrosalicylic acid and 262mL of 2mol / L NaOH aqueous solution to 500mL of hot aqueous solution containing 182g of sodium tartrate, then add 5g of redistilled phenol and 5g of sodium sulfite, stir to dissolve, cool, add deionized water to make up to 1L, store in a brown bottle, and use after 7 days.

[0063] Example 4: Comparison of the application of Rhizopus oryzae XB4-16 and other Rhizopus oryzae strains in extracting total flavonoids from Pleurotus eryngii

[0064] Compare the total flavonoids extraction yield of Rhizopus oryzae XB4-16 and other three strains of Rhizopus oryzae in Pleurotus eryngii. 6 ~7×10 6 After the spore liquid was inoculated with Pleurotus eryngii powder and fermented, the total flavonoids were extracted by ultrasonic extraction of ethanol aqueous solution. The total flavonoid extraction rates of Pleurotus eryngii fermented with different Rhizopus oryzae strains and the control are shown in Table 3.

[0065] Table 3 Total flavonoids extraction yield of Pleurotus eryngii fermented with different Rhizopus oryzae strains and control

[0066]

[0067] From the data in Table 3, it can be seen that after Pleurotus eryngii was fermented with Rhizopus oryzae GDMCC NO 3.128, GDMCC NO 3.163 and GDMCC NO 3.529 strains, the extraction rate of total flavonoids was improved to a certain extent compared with the direct extraction control, but the degree of improvement was far less than that of XB4 strain and XB4-16 strain, indicating that the Rhizopus oryzae XB4 separated from the microbial enrichment culture of Pleurotus eryngii and the XB4-16 strain obtained by mutagenesis thereof were applied to the fermentation of Pleurotus eryngii, and the effect of improving the extraction rate of total flavonoids was significantly better than that of other Rhizopus oryzae strains.

[0068] Rhizopus oryzae strains GDMCC NO 3.128, GDMCC NO 3.163 and GDMCC NO 3.529 were all from Guangdong Provincial Microbial Culture Collection Center.

[0069] Example 5: Application of Rhizopus oryzae XB4-16 in the extraction of total flavonoids from Pleurotus eryngii

[0070] Rhizopus oryzae XB4-16 is used to extract total flavonoids from Pleurotus eryngii. The following steps can be followed:

[0071] (1) Slant spores of Rhizopus oryzae XB4-16 stored in a 4°C refrigerator were inoculated on fresh PDA plate culture medium and cultured at 28°C for 48 h. 10 mL of sterile sucrose aqueous solution was added to the plate culture and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 6.75 × 10 6 / mL, and obtain the spore liquid of Rhizopus oryzae XB4-16.

[0072] (2) 10 g of Pleurotus eryngii powder was sterilized by dry heat at 160° C. for 2 h in a 150-mL conical flask, and 12 mL of the spore solution of Rhizopus oryzae XB4-16 prepared in step (1) was added (the volume dosage was 1.2 mL / g based on the mass of Pleurotus eryngii powder). The mixture was stirred evenly, and the conical flask was sealed with 8 layers of gauze. The mixture was cultured at 28° C. for 56 h to obtain Pleurotus eryngii fermentation product.

[0073] (3) All the fermented materials of Pleurotus eryngii in step (2) were transferred into a 250-mL beaker, 70 mL of deionized water (7 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after stirring evenly, the mixture was kept warm in a 35° C. water bath for 4 h, and then 130 mL of anhydrous ethanol was added [the volume of ethanol was 1.86 times that of deionized water, the solid-liquid ratio of the extraction system was 1:20 (g:mL), and the volume fraction of ethanol was 65%]. After shaking evenly, the mixture was transferred into an ultrasonic cleaner at a water temperature of 60° C., and ultrasonic extraction was performed at 100 W for 30 min. The mixture was filtered with a Buchner funnel, and the filtrate was collected to obtain a Pleurotus eryngii extract.

[0074] (4) The entire filtrate prepared in step (3) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 20 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient shaking, it was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0075] According to the above steps, 1.73 g of total flavonoids extract was extracted from 10 g of Pleurotus eryngii, and the total flavonoids content was 20.1%, that is, 3.48 g of total flavonoids of Pleurotus eryngii were obtained, and the extraction yield was 3.48%.

[0076] Comparative Example 1: Ultrasonic Extraction of Pleurotus eryngii Total Flavonoids with Conventional Ethanol-Water Solution (Compared with Example 4)

[0077] (1) 10 g of Pleurotus eryngii powder was placed in a 250-mL beaker, and 70 mL of deionized water (7 mL / g based on the mass of the raw Pleurotus eryngii powder) was added. After stirring evenly, the mixture was kept in a 35°C water bath for 4 h, and then 130 mL of anhydrous ethanol [1.86 times the volume of deionized water, the solid-liquid ratio of the extraction system was 1:20 (g:mL), and the volume fraction of ethanol was 65%] was added. After shaking evenly, the mixture was transferred to an ultrasonic cleaner at a water temperature of 60°C, and ultrasonic extraction was performed at 100 W for 30 min. The filtrate was collected by Buchner funnel to obtain Pleurotus eryngii extract.

[0078] (2) The entire filtrate prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 10 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient oscillation, the mixture was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0079] According to the above steps, 1.42 g of total flavonoids extract was extracted from 10 g of Pleurotus eryngii, and the total flavonoids content was 18.5%, that is, 0.263 g of total flavonoids of Pleurotus eryngii was obtained, and the extraction yield was 2.63%.

[0080] By comparing the results of Example 5 and Comparative Example 1, it can be seen that before ultrasonic extraction of total flavonoids from Pleurotus eryngii with ethanol aqueous solution, fermentation pretreatment with Rhizopus oryzae XB4-16 was added, and the total flavonoid extraction rate was increased from 2.63% without fermentation pretreatment to 3.48%, an increase of 32.3%.

[0081] Example 6: Application of Rhizopus oryzae XB4-16 in the extraction of total flavonoids from Pleurotus eryngii

[0082] Rhizopus oryzae XB4-16 is used to extract total flavonoids from Pleurotus eryngii. The following steps can be followed:

[0083] (1) Slant spores of Rhizopus oryzae XB4-16 stored in a refrigerator at 4°C were inoculated on fresh PDA plate culture medium and cultured at 28°C for 42 h. 10 mL of sterile sucrose water was added to the plate culture and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 5.62 × 10 6 / mL, and obtain the spore liquid of Rhizopus oryzae XB4-16.

[0084] (2) 20 g of Pleurotus eryngii powder was sterilized by dry heat at 160°C for 2 h in a 250-mL conical flask, and 22 mL of the spore solution of Rhizopus oryzae XB4-16 prepared in step (1) was added (the volume dosage was 1.1 mL / g based on the mass of Pleurotus eryngii powder). The mixture was stirred evenly, and the conical flask was sealed with 8 layers of gauze. The mixture was cultured at 30°C for 52 h to obtain Pleurotus eryngii fermentation product.

[0085] (3) All the fermented materials of Pleurotus eryngii in step (2) were transferred into a 500-mL beaker, 130 mL of deionized water (6.5 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after stirring evenly, the mixture was kept warm in a water bath at 37.5° C. for 3 h, and then 220 mL of anhydrous ethanol [1.69 times the volume of deionized water, a solid-liquid ratio of the extraction system of 1:17.5 (g:mL), and a volume fraction of ethanol of 63%] was added. After shaking evenly, the mixture was transferred into an ultrasonic cleaner at a water temperature of 55° C., and ultrasonic extraction was performed at 150 W for 25 min. The mixture was filtered with a Buchner funnel, and the filtrate was collected to obtain a Pleurotus eryngii extract.

[0086] (4) All the filtrate prepared in step (3) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 15 mL of anhydrous ethanol (0.75 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0087] According to the above steps, 3.55 g of total flavonoids extract was extracted from 20 g of Pleurotus eryngii, and the total flavonoids content was 19.8%, that is, 0.703 g of total flavonoids of Pleurotus eryngii was obtained, and the extraction yield was 3.52%.

[0088] Comparative Example 2: Ultrasonic Extraction of Pleurotus eryngii Total Flavonoids with Conventional Ethanol-Water Solution (Compared with Example 5)

[0089] (1) 20 g of Pleurotus eryngii powder was placed in a 500-mL beaker, and 130 mL of deionized water (6.5 mL / g based on the mass of the raw Pleurotus eryngii powder) was added. After stirring evenly, the mixture was kept in a 37.5°C water bath for 3 h, and then 220 mL of anhydrous ethanol [1.69 times the volume of deionized water, a solid-liquid ratio of 1:17.5 (g:mL) for the extraction system, and a volume fraction of ethanol of 63%] was added. After shaking evenly, the mixture was transferred to an ultrasonic cleaner at a water temperature of 55°C, and ultrasonic extraction was performed at 150 W for 25 min. The mixture was filtered using a Buchner funnel, and the filtrate was collected to obtain a Pleurotus eryngii extract.

[0090] (2) All the filtrate prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 15 mL of anhydrous ethanol (0.75 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0091] According to the above steps, 2.94 g of total flavonoids extract was extracted from 20 g of Pleurotus eryngii, and the total flavonoids content was 17.7%, that is, 0.520 g of total flavonoids of Pleurotus eryngii was obtained, and the extraction yield was 2.60%.

[0092] By comparing the results of Example 6 and Comparative Example 2, it can be seen that before ultrasonic extraction of total flavonoids from Pleurotus eryngii with ethanol aqueous solution, fermentation pretreatment with Rhizopus oryzae XB4-16 was added, and the total flavonoid extraction rate was increased from 2.60% without fermentation pretreatment to 3.52%, an increase of 35.4%.

[0093] Example 7: Application of Rhizopus oryzae XB4-16 in the extraction of total flavonoids from Pleurotus eryngii

[0094] Rhizopus oryzae XB4-16 is used to extract total flavonoids from Pleurotus eryngii. The following steps can be followed:

[0095] (1) Slant spores of Rhizopus oryzae XB4-16 stored in a refrigerator at 4°C were inoculated on fresh PDA plate culture medium and cultured at 28°C for 36 h. 10 mL of sterile sucrose water was added to the plate culture and stirred with an inoculation loop to suspend the spores. The spore solution was transferred to a sterile test tube and the spore concentration was adjusted to 4.37 × 10 6 / mL, and obtain the spore liquid of Rhizopus oryzae XB4-16.

[0096] (2) 50 g of Pleurotus eryngii powder was sterilized by dry heat at 160° C. for 2 h in a 500-mL conical flask, and 50 mL of the spore solution of Rhizopus oryzae XB4-16 prepared in step (1) (the volume dosage was 1 mL / g based on the mass of Pleurotus eryngii powder) was added. The mixture was stirred evenly, and the conical flask was sealed with 8 layers of gauze. The mixture was cultured at 30° C. for 36 h to obtain Pleurotus eryngii fermentation product.

[0097] (3) All the fermented materials of Pleurotus eryngii in step (2) were transferred into a 1-L beaker, 300 mL of deionized water (6 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after stirring evenly, the mixture was kept warm in a 40°C water bath for 2 h, and then 450 mL of anhydrous ethanol [1.5 times the volume of deionized water, the solid-liquid ratio of the extraction system was 1:15 (g:mL), and the volume fraction of ethanol was 60%] was added. After shaking evenly, the mixture was transferred into an ultrasonic cleaner at a water temperature of 50°C, and ultrasonic extraction was performed at 200 W for 20 min. The mixture was filtered with a Buchner funnel, and the filtrate was collected to obtain a Pleurotus eryngii extract.

[0098] (4) The entire filtrate prepared in step (3) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 20 mL of anhydrous ethanol (0.4 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient oscillation, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0099] According to the above steps, 8.78 g of total flavonoids extract was extracted from 50 g of Pleurotus eryngii, and the total flavonoids content was 19.3%, that is, 1.69 g of total flavonoids of Pleurotus eryngii were obtained, and the extraction yield was 3.38%.

[0100] Comparative Example 3: Ultrasonic Extraction of Pleurotus eryngii Total Flavonoids with Conventional Ethanol-Water Solution (Compared with Example 6)

[0101] (1) 50 g of Pleurotus eryngii powder was placed in a 1-L beaker, and 300 mL of deionized water (6 mL / g based on the mass of the raw Pleurotus eryngii powder) was added. After stirring evenly, the mixture was kept in a 40°C water bath for 2 h, and then 450 mL of anhydrous ethanol [1.5 times the volume of deionized water, extraction system solid-liquid ratio 1:15 (g:mL), ethanol volume fraction 60%] was added. After oscillation evenly, the mixture was transferred to an ultrasonic cleaner at a water temperature of 50°C, and ultrasonic extraction was performed at 200 W for 20 min. The filtrate was collected by Buchner funnel to obtain Pleurotus eryngii extract.

[0102] (2) All the filtrate prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 20 mL of anhydrous ethanol (0.4 mL / g based on the mass of the raw material Pleurotus eryngii powder) was added, and after sufficient shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to constant weight to obtain a total flavonoid extract.

[0103] According to the above steps, 7.23 g of total flavonoids extract was extracted from 50 g of Pleurotus eryngii, and the total flavonoids content was 17.5%, that is, 1.26 g of total flavonoids of Pleurotus eryngii were obtained, and the extraction yield was 2.52%.

[0104] By comparing the results of Example 7 and Comparative Example 3, it can be seen that before ultrasonic extraction of total flavonoids from Pleurotus eryngii with ethanol aqueous solution, fermentation pretreatment with Rhizopus oryzae XB4-16 was added, and the total flavonoid extraction rate was increased from 2.52% without fermentation pretreatment to 3.38%, an increase of 34.1%.

[0105] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change, enlargement and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for improving the extraction rate of total flavonoids from Pleurotus eryngii using Rhizopus oryzae XB4-16, characterized in that: The method comprises the following steps: (1) adding spore liquid of Rhizopus oryzae XB4-16 to Pleurotus eryngii powder, stirring evenly, and fermenting at 28-30°C for 48-56 hours to obtain Pleurotus eryngii fermented product; the Rhizopus oryzae XB4-16 has a preservation number of GDMCC No: 65283; (2) adding deionized water to the Pleurotus eryngii fermented product, stirring evenly, and keeping the temperature at 35-40°C for 2-4 hours, then adding anhydrous ethanol, and filtering after ultrasonic extraction to obtain a Pleurotus eryngii extract; (3) evaporating the Pleurotus eryngii extract to dryness under reduced pressure, dissolving in anhydrous ethanol, and drying to obtain a Pleurotus eryngii total flavonoid extract.

2. The method according to claim 1, characterized in that The Pleurotus eryngii powder described in step (1) is obtained by slicing fresh Pleurotus eryngii, drying at 85° C. to constant weight, and then crushing and passing through a 60-mesh sieve to obtain fine powder.

3. The method according to claim 1, characterized in that The preparation method of the spore liquid of Rhizopus oryzae XB4-16 in the step (1) is as follows: the spores of Rhizopus oryzae XB4-16 stored at low temperature are inoculated on a PDA plate culture medium, cultured at 28° C. for 36 to 48 hours, a sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain a spore liquid.

4. The method according to claim 1, characterized in that The spore solution was adjusted to a spore concentration of 4 × 10 6 ~7×10 6 / mL, and the sucrose concentration in the sterile sucrose aqueous solution is 50g / L.

5. The method according to claim 1, characterized in that The volume dosage of the spore liquid of Aspergillus oryzae XB4-16 in the step (1) is 1.0-1.2 mL / g based on the mass of Pleurotus eryngii powder.

6. The method according to claim 1, characterized in that The preparation method of the Pleurotus eryngii extract in the step (2) is as follows: deionized water is added to the Pleurotus eryngii fermentation product, the product is stirred evenly and kept warm at 35-40°C for 2-4 hours, anhydrous ethanol is added, the product is placed in an ultrasonic cleaning machine at a water temperature of 50-60°C, ultrasonic extraction is performed at 100-200W for 20-30 minutes, and after the ultrasonic extraction is completed, the product is filtered with a Buchner funnel, the filtrate is collected, and the Pleurotus eryngii extract is obtained.

7. The method according to claim 6, characterized in that The volumetric amount of the deionized water added is 6-7 mL / g based on the mass of the raw material Pleurotus eryngii powder; the volumetric amount of the anhydrous ethanol added is 1.5-2.0 times the volume of the deionized water.

8. The method according to claim 1, characterized in that The preparation method of the total flavonoid extract of Pleurotus eryngii in step (3) is as follows: distill the Pleurotus eryngii extract at 45°C and -0.1MPa until no liquid flows out, add anhydrous ethanol with a volume of 0.4-1.0mL / g based on the mass of the raw material Pleurotus eryngii powder, shake thoroughly, and centrifuge at 5000-8000r / min for 5-10min, transfer the supernatant into a clean culture dish, and vacuum dry at 50°C and -0.1MPa to constant weight to obtain the total flavonoid extract.

9. A Rhizopus oryzae XB4-16 for improving the extraction rate of total flavonoids from Pleurotus eryngii according to claim 1, deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No: 65283, a deposit date of October 24, 2024, and an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

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