Fusarium graminearum AY2-19 and its application in extracting total flavonoids from Artemisia argyi

Abstract

The present invention discloses a strain of Fusarium graminearum (Fusarium graminearum) AY2-19 and its application in extracting total flavonoids from mugwort. Fusarium graminearum AY2-19 is deposited in Guangdong Province Microbial Culture Collection Center, with deposit number GDMCC No: 63062 and deposit date December 20, 2022. Before extracting total flavonoids from mugwort ethanol ultrasonic method, the crude enzyme solution prepared by fermentation of Fusarium graminearum AY2-19 is added. Because the crude enzyme solution contains a variety of hydrolases, it is possible to synergistically hydrolyze substances such as cellulose, hemicellulose and pectin in mugwort, damage the cell wall, contribute to the dissolution of flavonoids, and significantly improve the total flavonoid extraction yield. The method for extracting total flavonoids from mugwort by microbial enzymolysis assisted by the present invention is more directly compared with the use of ethanol ultrasonic extraction method, and the yield of total flavonoids extracted from mugwort can be increased by 25.2%.

Description

(1) Technical field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a strain of Fusarium graminearum AY2-19 and an application thereof in extracting total flavonoids from mugwort leaves. (2) Background technology

[0002] Artemisia argyi (Artemisia argyi L. et Vant.), a plant of the genus Artemisia in the Asteraceae family, is a commonly used traditional Chinese medicinal material in my country and has been included in all editions of the Chinese Pharmacopoeia (known as Artemisiae Argyi Folium). Its properties are pungent, bitter, warm, and slightly toxic. It enters the liver, spleen, and kidney meridians and has the effects of warming the meridians to stop bleeding, dispelling cold and relieving pain, and externally removing dampness and relieving itching. It is used to treat hematemesis, epistaxis, metrorrhagia, menorrhagia, menorrhagia, lower abdominal pain, irregular menstruation, and uterine coldness and infertility. It is also used externally to treat pruritus. Vinegar and charcoal-based mugwort warm the meridians to stop bleeding and is used to treat bleeding caused by deficiency-cold. Modern pharmacological studies have shown that artemisia argyi and its extracts have multiple pharmacological effects, including antibacterial, antiviral, antioxidant, hepatoprotective and choleretic, hemostatic, anticoagulant, anti-inflammatory, antiallergic, immunomodulatory, and anticancer properties. Artemisia argyi contains a variety of physiologically active components, including volatile oils, flavonoids, triterpenes, sesquiterpenes, eucalyptone, and polysaccharides. In recent years, flavonoids have been widely used in the pharmaceutical and food industries due to their pharmacological effects such as lowering blood lipids, anti-thrombosis, antioxidant, anti-aging and immune enhancement, and are of great development value. The flavonoid content in mugwort is relatively high, making it a good raw material for extracting flavonoids.

[0003] At present, there are many reports on the extraction of total flavonoids from Artemisia argyi leaves, and the main method is ethanol extraction. Conventional ethanol extraction is simple to operate, but the extraction yield is often not high. The reflux method can significantly improve the extraction yield, but it has high energy consumption and a complicated process. On the basis of the ethanol extraction method, ultrasonic, microwave or high-pressure treatment can promote the dissolution of total flavonoids and improve the extraction yield to a certain extent. The most reported method is ethanol ultrasonic extraction, which has the advantage that ultrasonic treatment does not increase the complexity of the extraction process or significantly increase production costs.

[0004] In recent years, there have been numerous reports on the application of enzymatic hydrolysis technology in the extraction of total flavonoids from plants. Enzymatic hydrolysis extraction methods employ mild conditions, minimally disrupt the structure of flavonoids, and can significantly improve extraction yields. For example, Ning Na et al. used a 2:1 ratio of cellulase and pectinase to hydrolyze mugwort leaves, achieving a maximum flavonoid yield of 12.79% [Ning Na, Han Jianjun. Optimization of enzymatic hydrolysis-assisted ultrasonic extraction of total flavonoids from mugwort leaves using response surface methodology, CN111358821A, April 17, 2020]. Plant cell walls are composed of substances such as cellulose, hemicellulose, lignin, and pectin. Using a single enzyme often has little effect on improving extraction yield; using several enzymes simultaneously to assist extraction is generally superior to using a single enzyme alone. However, the use of multiple enzymes undoubtedly increases extraction costs.

[0005] Microorganisms, especially some molds and actinomycetes, can produce a variety of hydrolytic enzymes that decompose plant tissues, including cellulases, hemicellulases, ligninases, and pectinases. Therefore, if a certain microorganism is cultured under appropriate conditions, the fermentation broth contains a large amount of hydrolases, and the fermentation broth (crude enzyme solution) is used to directly hydrolyze plant materials. These enzymes can synergistically decompose cell wall components, thereby facilitating the release of active ingredients. The extraction yield can be significantly improved, and importantly, the cost can be significantly reduced compared to using pure enzymes.

[0006] In order to improve the extraction rate of total flavonoids from mugwort, the invention uses crude enzyme solution prepared by microbial fermentation to hydrolyze mugwort, and then uses ethanol ultrasonic method to extract total flavonoids, which can greatly improve the extraction rate. (3) Summary of the invention

[0007] The present invention aims to provide a new microbial strain, Fusarium graminearum AY2-19, and its use in extracting total flavonoids from mugwort leaves. After mugwort leaves are enzymatically hydrolyzed with a crude enzyme solution prepared by fermentation with the strain, the total flavonoid extraction rate can be significantly improved.

[0008] The technical solution adopted in the present invention is:

[0009] The present invention provides a new microbial strain - Fusarium graminearum (Fusarium graminearum) AY2-19, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 63062 and a deposit date of December 20, 2022. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code: 510070.

[0010] The Fusarium graminearum AY2-19 described in the present invention is an excellent strain isolated from the microbial enrichment culture of mugwort leaves and obtained through screening and mutagenesis. The colony morphology characteristics of the Fusarium graminearum AY2-19 are as follows: when cultured on potato dextrose agar (PDA) plate culture medium at 28°C, the initial colonies are white and flocculent. After 3 days, the color of the colonies slightly deepens, the mycelium layer is thicker, there is no spore powder on the surface, and there is no pigment diffusion on the back. Two types of conidia can be observed under an optical microscope: large conidia and small conidia. Large conidia are sickle-shaped or crescent-shaped, with 3-5 septa and a size of 4-5×25-40μm; small conidia are fewer, oval or elliptical, and a size of 2.5-4×4.5-1μm. A photo of the colony of Fusarium graminearum AY2-19 streaked on a PDA plate culture medium and cultured at 28°C for 3 days is shown in Figure 1 .

[0011] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Fusarium graminearum AY2-19 is shown in SEQ ID NO.1.

[0012] The present invention also provides an application of the Fusarium graminearum AY2-19 in extracting total flavonoids from mugwort, and the application method is as follows: (1) filtering the fermentation liquid obtained by culturing the Fusarium graminearum AY2-19, and collecting the filtrate as a crude enzyme liquid; (2) mixing the crude enzyme liquid with mugwort powder, and performing enzymatic hydrolysis at 26-30° C. for 4-6 hours to obtain a mugwort hydrolyzate; (3) adding deionized water and anhydrous ethanol to the mugwort hydrolyzate, performing ultrasonic extraction, and filtering to obtain a total flavonoid extract; (4) evaporating the total flavonoid extract to dryness under reduced pressure, dissolving it in anhydrous ethanol, and vacuum drying it to obtain a total flavonoid extract.

[0013] Furthermore, the method for preparing the crude enzyme solution in step (1) is as follows: inoculating Fusarium graminearum AY2-19 spores into an enzyme-producing culture medium, culturing the culture medium at 30°C and 180-200 r / min for 64-72 hours, filtering the fermentation liquid, and obtaining the filtrate as the crude enzyme solution; the final concentration composition of the enzyme-producing culture medium is: wheat bran 40-50 g / L, peptone 8-10 g / L, NaNO3 5-6 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.25-0.5 g / L, CaCl2 0.4-0.5 g / L, the solvent is tap water, and the pH value is 6.0-6.5.

[0014] Furthermore, the enzyme production culture medium preferably comprises: 50 g / L wheat bran, 10 g / L peptone, 5 g / L NaNO3, 2 g / L KH2PO4, 0.25 g / L MgSO4·7H2O, 0.5 g / L CaCl2, the solvent is tap water, and the pH is 6.0.

[0015] Before the enzyme production culture, the Fusarium graminearum AY2-19 of the present invention needs to be cultured on a plate culture medium to produce spores, and then the spores are suspended in physiological saline to obtain a Fusarium graminearum AY2-19 spore liquid, which is inoculated into the enzyme production culture medium at a volume fraction of 3%-5% for culture. The specific enzyme production culture method is as follows:

[0016] ① Preparation of spore liquid: Fusarium graminearum AY2-19 was inoculated into potato dextrose agar (PDA) plate culture medium and cultured at 28°C for 64–72 hours to obtain a plate culture; sterile physiological saline was added to the plate culture, and the spores were suspended by stirring with an inoculating loop to obtain a spore liquid of Fusarium graminearum AY2-19; ​​the PDA plate culture medium was a finished potato dextrose agar medium purchased from Qingdao Haibo Biotechnology Co., Ltd., prepared with tap water at a concentration of 46 g / L, with a natural pH, and sterilized by high-pressure steam at 121°C for 20 minutes.

[0017] ② Enzyme production culture: Use the spore liquid of Fusarium graminearum AY2-19 prepared in step ①, inoculate it with an enzyme production culture medium at a volume fraction of 3%-5%, and culture it at 30°C and 180-200r / min with shaking for 64-72h to obtain a fermentation liquid with a dry cell concentration of 3.67-3.84g / L and a cellulase activity of 34.2-35.8U / mL.

[0018] Furthermore, the volume of the crude enzyme solution in step (2) is 4-8 mL / g based on the mass of the mugwort powder [i.e., the material-enzyme ratio is 1:4-1:8 (g:mL)]; the mugwort powder is fresh mugwort that is washed with tap water, dried at 85°C, crushed and then sieved through a 40-mesh sieve.

[0019] Furthermore, the preparation method of the total flavonoid extract in step (3) is as follows: after the enzymatic hydrolysis of the mugwort leaves is completed, deionized water and anhydrous ethanol are added to the mugwort leaves enzymatic hydrolyzate to adjust the material-liquid ratio to 1:20-1:25 (g:mL) and the ethanol volume fraction to 50%-60%. After stirring evenly, the mixture is extracted in an ultrasonic cleaning machine at a water temperature of 80-85°C and a power of 150W for 30-45 minutes. After the ultrasonic alcohol extraction is completed, the mixture is filtered while hot to obtain the total flavonoid extract.

[0020] Furthermore, the method for recovering total flavonoids from the total flavonoid extract in step (4) is as follows: the total flavonoid extract is distilled at 45° C. and –0.1 MPa until no liquid flows out, anhydrous ethanol is added in a volume of 1–2 mL / g based on the mass of the raw material mugwort leaves, the mixture is fully shaken and centrifuged at 8000 r / min for 5–10 min, the supernatant is transferred to a clean culture dish, and vacuum dried at 50° C. and –0.1 MPa to obtain a total flavonoid extract.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that before the ethanol ultrasonic extraction of total flavonoids from mugwort leaves, enzymatic hydrolysis of crude enzyme liquid prepared by microbial fermentation is added. The enzyme-producing microorganism Fusarium graminearum AY2-19 used is purposefully separated and screened for its ability to hydrolyze the cell walls of mugwort leaves, and is obtained through mutagenesis. The crude enzyme liquid prepared by enzyme-producing fermentation contains a variety of hydrolases that can synergistically hydrolyze substances such as cellulose, hemicellulose and pectin in mugwort leaves, causing cell wall damage, which helps to dissolve flavonoids and significantly improve the extraction rate of total flavonoids. The method for extracting total flavonoids from mugwort leaves assisted by microbial enzymatic hydrolysis provided by the present invention can increase the extraction rate of total flavonoids from mugwort leaves by 25.2% compared with the direct use of ethanol ultrasonic extraction method. (IV) Description of the accompanying drawings

[0022] Figure 1 This is a photo of the colonies of Fusarium graminearum AY2-19 cultured on PDA at 28°C for 3 days.

[0023] Figure 2 The standard curve for the spectrophotometric determination of total flavonoids was used.

[0024] Figure 3 This is the standard curve for determining glucose using the DNS method. (V) Specific implementation methods

[0025] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] The mugwort leaf of the present invention is fresh leaves of Artemisia argyi, a plant of the genus Artemisia in the Asteraceae family, which are washed with tap water and then dried at 85° C. The mugwort leaf is produced in Anyang City, Henan Province. The mugwort leaf powder is fine powder obtained by crushing the dried mugwort leaf and passing it through a 40-mesh sieve.

[0027] Example 1: Isolation and screening of enzyme-producing strains

[0028] The microbial strains producing enzymes for enzymatic hydrolysis of mugwort leaves are isolated and screened according to the following steps:

[0029] (1) Add 5 g of mugwort powder to a 250-mL conical flask, add 7.5 mL of sterile saline, stir evenly, and culture at 28°C for 72 h. Dilute the enriched culture full of mold with sterile saline to 1×10 -6 , 1×10 -7 , 1×10 -8 After 2 times, 0.1 mL of the dilution was respectively applied to potato dextrose agar (PDA) plate medium and cultured at 28 °C for 48 h. Fungal colonies with different colors and morphologies were picked and transferred to fresh PDA plate medium and cultured at 28 °C for 72 h. Eight pure culture strains were obtained. The strain numbers are shown in Table 1.

[0030] (2) Add 10 mL of sterile saline to the fresh plate culture of each of the eight strains and stir with an inoculating loop to suspend the spores to obtain the spore liquid of each strain.

[0031] (3) 1.5 mL of the spore solution of each strain prepared in step (2) was inoculated into 50 mL of enzyme production culture medium (the inoculation amount was 3% by volume). After culturing for 72 h at 30°C and 180 r / min of shaking, all the fermentation liquid was filtered using a Buchner funnel, and the collected filtrate was the crude enzyme solution.

[0032] (4) 0.5 g of mugwort powder was added to each of 8 50-mL centrifuge tubes, and 4 mL of the crude enzyme solution of each strain prepared in step (3) was added [i.e., the feed-enzyme ratio was 1:8 (g:mL)], and the mixture was stirred evenly and enzymatically hydrolyzed in a 30°C water bath for 6 h to obtain mugwort hydrolysate.

[0033] (5) 1 mL of deionized water and 5 mL of anhydrous ethanol (system material-liquid ratio 1:20 (g:mL), ethanol volume fraction 50%) were added to all the mugwort hydrolysates obtained by enzymatic hydrolysis with the crude enzyme solution of each strain in step (4), respectively. After shaking, the mixture was extracted in an ultrasonic cleaning machine at a water temperature of 80°C and a power of 150W for 30 min. The mixture was filtered through a Buchner funnel while still hot, and the filtrate was collected and the total flavonoid content was determined by spectrophotometry.

[0034] Starting from step (4), 4 mL of uninoculated enzyme-producing culture medium was used instead of the crude enzyme solution as a non-enzymatic control; 4 mL of 2000 U / mL cellulase activity in phosphate buffer (pH 6.0, 0.2 mol / L) was used instead of the crude enzyme solution as a cellulase enzymatic control. The total flavonoid extraction yields of mugwort leaves enzymatically digested with the crude enzyme solutions of different strains and the control are shown in Table 1.

[0035] Table 1 Total flavonoids extraction rate of Artemisia argyi leaf enzymatically hydrolyzed by different strains of crude enzyme solution and control

[0036]

[0037] As can be seen from the data in Table 1, after the wormwood leaf is hydrolyzed with the crude enzyme solution prepared by fermentation of AY2 strain, the total flavonoids extraction yield is 7.75%, which is 8.70% higher than the control without hydrolysis (7.13%). The wormwood leaf hydrolyzed with cellulase can also improve the total flavonoids extraction yield (7.46%), which is 4.63% higher than the control without hydrolysis, but is far inferior to the crude enzyme solution prepared by fermentation of AY2 strain. After the wormwood leaf is hydrolyzed with the crude enzyme solution of some microbial strains, the total flavonoids extraction yield does not improve, and even significantly decreases, such as the AY6 strain. This shows that the microbial strains used for enzymatic hydrolysis of wormwood leaf to improve the total flavonoids extraction yield are selective, requiring not only the ability to produce enzymes to hydrolyze wormwood leaf cell wall components, but also the inability to produce enzymes to degrade flavonoids. The present invention selects the AY2 strain as the enzyme-producing strain to improve the total flavonoids extraction yield of wormwood leaf.

[0038] The PDA plate medium is a finished potato dextrose agar medium purchased from Qingdao Haibo Biotechnology Co., Ltd. It is prepared with tap water at a concentration of 46 g / L, with a natural pH, sterilized by high-pressure steam at 121°C for 20 minutes, and poured into a 9 cm diameter sterile culture dish before solidification, with 15–20 mL per dish.

[0039] The final concentration composition and preparation method of the enzyme production culture medium are as follows: 50 g / L wheat bran, 10 g / L peptone, 35 g / L NaNO, 2 g / L KH2PO4, 0.25 g / L MgSO4·7H2O, and 0.5 g / L CaCl2. The solvent is tap water with a pH of 6.0. A 250-mL conical flask is filled with 50 mL of the enzyme production culture medium, the flask is tied with 8 layers of gauze, and the flask is sterilized with high-pressure steam at 121°C for 20 minutes.

[0040] The total flavonoid content is determined by spectrophotometry, and the specific method is: the total flavonoid extract is diluted with a 60% ethanol aqueous solution (hereinafter referred to as "60% ethanol") by appropriate multiples 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 appropriate multiples to be the sample to be tested. 1 mL of the sample solution to be tested is taken into a 25-mL colorimetric tube, 1 mL of a 5% sodium nitrite (NaNO2) aqueous solution is added, and the solution is allowed to stand for 6 minutes. 1 mL of a 10% aluminum nitrate [Al(NO3)3] aqueous solution is added, and the solution is allowed to stand for 6 minutes. 10 mL of a 4% sodium hydroxide (NaOH) aqueous solution is added, and the volume is made up to 25 mL with a 60% ethanol aqueous solution. After shaking, the solution is allowed to stand for 10 minutes. The absorbance at a wavelength of 510 nm is measured using a spectrophotometer (A 510 ), the total flavonoids concentration in the sample was calculated from the rutin standard curve, and then the total flavonoids mass was obtained by multiplying the concentration by the volume of the measured sample.

[0041] Drawing of rutin standard curve: Prepare 0.25 g / L rutin standard solution with 60% ethanol. Take 2, 3, 4, 5, 6, and 7 mL of rutin standard solution respectively in 25-mL colorimetric tubes, add 1 mL of 5% sodium nitrite (NaNO2) aqueous solution by mass concentration, let it stand for 6 minutes, add 1 mL of 10% aluminum nitrate [Al(NO3)3] aqueous solution by mass concentration, let it stand for 6 minutes, add 10 mL of 4% sodium hydroxide (NaOH) aqueous solution by mass concentration, and then dilute to 25 mL with 60% ethanol. Shake well and let it stand for 10 minutes. Measure the absorbance (A) at a wavelength of 510 nm using a spectrophotometer. 510 ). With rutin concentration as the horizontal axis, A 510 Draw a standard curve for the vertical axis ( Figure 2 ).

[0042] The yield of total flavonoids extracted from Artemisia argyi is calculated according to formula (1):

[0043]

[0044] Example 2: Mutagenesis of fermentation strain AY2

[0045] The strain AY2 was subjected to mutation breeding to screen strains with excellent fermentation performance. The specific method is as follows:

[0046] (1) Preparation of spore solution: After strain AY2 was activated and cultured on PDA plate medium at 28°C for 64 hours, 5 mL of sterile saline was added and stirred with an inoculating loop to suspend the spores. 1 mL of spore solution was transferred to a conical flask containing 50 mL of sterile saline (with 20–30 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). The spores in the spore solution were counted under a microscope using a hemocytometer. Appropriate dilutions were made with sterile saline to adjust the spore count to 1.32 × 10 7 pieces / mL.

[0047] (2) Mutagenesis: Under red light illumination, take 1.5 mL of the above spore solution and a sterile paper clip and place them in 6 culture dishes with a diameter of 6 cm. The culture dishes are placed on magnetic stirrers and irradiated at a distance of 30 cm from a 15W UV lamp that has been preheated for 30 minutes for 1, 2, 3, 4, 5, and 6 minutes. Take 0.5 mL of the spore solution after the above irradiation treatment, dilute it appropriately, and transfer 0.1 mL to spread on PDA plate culture medium. In the same way, dilute the spore solution that has not been irradiated with UV light and spread it on the plate as a control to calculate the lethality. The inoculated PDA plate is wrapped with black cloth and inverted at 28℃ for 48 hours. The colonies on the plate are counted and the lethality is calculated.

[0048] (3) Screening: Pick colonies on PDA plates with a lethality rate of more than 90% and transfer them to fresh PDA plate culture medium to obtain 40 strains. 10 mL of sterile physiological saline was added to the fresh plate culture of each strain after culturing at 28°C for 72 hours, and the spores were suspended by stirring with an inoculating loop to obtain the spore liquid of each strain. 1.5 mL of the spore liquid of each strain was taken and inoculated into 50 mL of enzyme production culture medium. After 72 hours of shaking culture at 30°C and 200 r / min, the fermentation liquid was filtered with a Buchner funnel, and the filtrate (i.e., crude enzyme liquid) was collected to determine the cellulase activity of the fermentation filtrate of each strain. 10 strains with a relatively higher enzyme activity than that of the wild strain were selected. According to the method of Example 1, the crude enzyme liquid fermented by these 10 strains was used to enzymatically hydrolyze mugwort leaves, and the total flavonoids were extracted by ethanol ultrasonication. The total flavonoids extraction yield of mugwort leaves enzymatically hydrolyzed by the crude enzyme liquid of the mutant strain and the control is shown in Table 2.

[0049] Table 2 Total flavonoids extraction rate of mugwort leaves hydrolyzed by crude enzyme solution of mutant strains and control

[0050]

[0051] As can be seen from the data in Table 2, among the 10 strains screened, the strain numbered AY2-19 had a fermentation cellulase activity of 35.4 U / mL, which was 32.6% higher than the 26.7 U / mL of the wild strain AY2. The crude enzyme solution prepared by fermentation with this strain hydrolyzed mugwort leaves, resulting in a total flavonoid extraction rate of 8.78%, a 13.3% increase over the 7.75% of the wild strain AY2, and a 23.1% increase over the 7.13% of the unhydrolyzed control. Therefore, the present invention selected the AY2-19 strain as an enzyme-producing strain for improving the total flavonoid extraction rate of mugwort leaves.

[0052] The final concentration composition and preparation method of the enzyme production culture medium are the same as those in Example 1.

[0053] The cellulase activity assay was performed by adding 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 to a 10-mL graduated test tube, respectively, and incubating in a 50°C water bath for 30 minutes. Then, 3 mL of DNS reagent was added and boiled for 5 minutes. After cooling with running water, the volume was made up to 10 mL with deionized water and shaken well. The crude enzyme solution inactivated by boiling at 100°C for 10 minutes was subjected to the same treatment as a reference, and the absorbance (A) at a wavelength of 540 nm was measured using a spectrophotometer. 540 ), by the glucose standard curve ( Figure 3 ) to calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Definition of cellulase activity: At pH 6.0 and 50°C, the amount of enzyme required to hydrolyze sodium carboxymethyl cellulose to produce 1 μg of glucose per minute is 1 enzyme activity unit (U).

[0054] The cellulase activity was calculated according to formula (2).

[0055]

[0056] In formula (2), C is the glucose concentration (μg / mL) calculated from the standard curve; V1 is the volume of the enzyme reaction system, i.e., 2 mL; T is the reaction time, i.e., 30 min; and V2 is the volume of the crude enzyme solution, i.e., 0.5 mL.

[0057] Preparation of glucose standard curve: 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of standard glucose solution with a concentration of 1 mg / mL were added to 7 10-mL graduated test tubes, respectively. Then, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, and 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer were added to each tube, 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 the A value was measured on a spectrophotometer. 540 , with glucose concentration as the horizontal axis, A 540 Draw a standard curve for the vertical axis ( Figure 3 ).

[0058] 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.

[0059] Example 3: Classification and Identification of Strain AY2-19

[0060] Strain AY2-19 was streaked onto potato agar plate medium and cultured at 28°C. The initial colonies were white and flocculent. After 3 days, the color of the colonies darkened slightly, the mycelium layer was thicker, there was no spore powder on the surface, and no pigment spread on the back. Under an optical microscope, two types of conidia can be observed: large conidia and small conidia. Large conidia are sickle-shaped or crescent-shaped, with 3-5 septa and a size of 4-5×25-40μm; small conidia are fewer, oval or elliptical, and a size of 2.5-4×4.5-1μm. A photo of the colonies of Fusarium graminearum AY2-19 streaked onto PDA plate medium and cultured at 28°C for 3 days is shown in Figure 1 .

[0061] The rDNA-ITS nucleotide sequence of strain AY2-19 was determined to be shown in SEQ ID NO. 1. BLAST comparison of the sequence at NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) showed greater than 99% homology with 77 strains of Sporidiobolus and Sporobolomyces. Obviously, strain AY2-19 is not a yeast. Its colony and conidia morphology are consistent with the characteristics of Fusarium. Its rDNA-ITS is consistent with a strain of Fusarium graminearum (Accession No. ON705448) showed 99.45% homology with the rDNA-ITS sequence of strain AY2-19, so the biological classification position of strain AY2-19 can be determined as follows (refer to Mycobank, http: / / www.mycobank.org): Fungi, Ascomycota, Pezizomycotina, Sordariomycetes, Hypocreomycetidae, Hypocreales, Nectriaceae, Fusarium, Fusarium graminearum.

[0062] The rDNA-ITS nucleotide sequence of the strain AY2-19 is:

[0063] .

[0064] In summary, strain AY2 was isolated from the microbial enrichment of mugwort powder, and after ultraviolet mutagenesis, strain AY2-19 was screened and obtained, namely Fusarium graminearum AY2-19. The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 63062 and the deposit date December 20, 2022. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0065] Example 4: Application of Enzymatic Hydrolysis-Assisted Extraction of Total Flavonoids from Artemisia argyi Using Fusarium graminearum AY2-19

[0066] The extraction of total flavonoids from mugwort leaves using enzymatic hydrolysis assisted by Fusarium graminearum AY2-19 can be performed as follows:

[0067] (1) Spore powder of Fusarium graminearum AY2-19 stored in a freeze-dried tube was inoculated onto a fresh PDA plate culture medium and cultured at 28°C for 72 h. 10 mL of sterile physiological saline was added to the plate culture medium and stirred with an inoculating loop to suspend the spores to obtain a spore solution of Fusarium graminearum AY2-19. The components and preparation method of the PDA plate culture medium were the same as those in Example 1.

[0068] (2) 3 mL of the spore solution prepared in step (1) was inoculated into 100 mL of enzyme production medium (inoculation volume was 3% by volume), and cultured at 30°C and 180 rpm for 72 h to obtain a fermentation liquid with a dry cell concentration of 3.75 g / L. The entire fermentation liquid was filtered using a Buchner funnel, and the collected filtrate was the crude enzyme liquid, with a cellulase activity of 34.7 U / mL. The final concentration composition of the enzyme production medium was the same as in Example 1. 100 mL of the enzyme production medium was placed in a 250-mL conical flask, the mouth of the flask was sealed with 8 layers of gauze, and sterilized with high-pressure steam at 121°C for 20 min.

[0069] (3) 5 g of mugwort powder was placed in a 250-mL conical flask, and 40 mL of the crude enzyme solution prepared in step (2) [i.e., the feed-enzyme ratio was 1:8 (g:mL)] was added. After stirring evenly, the mixture was enzymolyzed in a 30°C water bath for 6 h to obtain mugwort hydrolysate.

[0070] (4) Add 10 mL of deionized water and 50 mL of anhydrous ethanol (material-liquid ratio 1:20 (g:mL), ethanol volume fraction 50%) to the entire mugwort hydrolysate from step (3). Shake well and extract in an ultrasonic cleaner at 80°C and 150W for 30 min. After ultrasonic alcohol extraction, filter the solution through a hot Buchner funnel to obtain a total flavonoid extract.

[0071] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 10 mL of anhydrous ethanol (2 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, 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 obtain a total flavonoid extract.

[0072] According to the above steps, 1.39 g of total flavonoid extract was obtained, with a total flavonoid content of 31.7%, that is, 0.441 g of total flavonoids was obtained, and the extraction yield was 8.82%.

[0073] Comparative Example 1: Extraction of Total Flavonoids from Artemisia argyi without Enzymolysis (Compared with Example 4)

[0074] (1) 5 g of mugwort leaf powder was added to 100 mL of 50% ethanol (1:20 g / mL) in a 250 mL Erlenmeyer flask. The mixture was shaken and extracted in an ultrasonic bath at 80°C and 150 W for 30 min. After ultrasonic extraction, the mixture was filtered through a hot Buchner funnel to obtain the total flavonoid extract.

[0075] (2) The total flavonoid extract 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 (2 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, 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 dried under vacuum at 50°C and -0.1 MPa to obtain a total flavonoid extract.

[0076] According to the above steps, 1.21 g of total flavonoid extract was obtained, with a total flavonoid content of 29.6%, that is, 0.358 g of total flavonoids was obtained, and the extraction yield was 7.16%.

[0077] Comparing the results of Example 4 and Comparative Example 1, it can be seen that before extracting total flavonoids from mugwort, enzymatic hydrolysis of crude enzyme solution prepared by fermentation of Fusarium graminearum AY2-19 was added, and the total flavonoid extraction rate increased from 7.16% to 8.82%, an increase of 23.2%.

[0078] Example 5: Application of Enzymatic Hydrolysis-Assisted Extraction of Total Flavonoids from Artemisia argyi Using Fusarium graminearum AY2-19

[0079] The enzymatic extraction of total flavonoids from mugwort leaves using Fusarium graminearum AY2-19 can be performed by following the steps below:

[0080] (1) Spores of a PDA colony of Fusarium graminearum AY2-19 stored at 4°C were inoculated onto fresh PDA plate culture medium and cultured at 28°C for 68 h. 10 mL of sterile saline was added to the plate culture and the spores were agitated with an inoculating loop to suspend the spores to obtain a spore solution of Fusarium graminearum AY2-19. The components and preparation method of the PDA plate culture medium were the same as those in Example 1.

[0081] (2) 4 mL of the spore solution prepared in step (1) was inoculated into 100 mL of enzyme production medium (inoculation volume was 4% by volume), and cultured at 30°C and 200 rpm for 68 h to obtain a fermentation broth with a dry cell concentration of 3.84 g / L. The entire fermentation broth was filtered using a Buchner funnel, and the collected filtrate was the crude enzyme solution. The cellulase activity of the filtrate was 35.8 U / mL. The final concentration composition and preparation method of the enzyme production medium were the same as those in Example 4.

[0082] (3) 10 g of mugwort powder was placed in a 250-mL conical flask, and 60 mL of the crude enzyme solution prepared in step (2) [i.e., the feed-enzyme ratio was 1:6 (g:mL)] was added. After stirring evenly, the mixture was enzymolyzed in a 28°C water bath for 5 h to obtain mugwort hydrolyzate.

[0083] (4) Add 40 mL of deionized water and 150 mL of anhydrous ethanol (system material-liquid ratio 1:25 (g:mL), ethanol volume fraction 60%) to the entire mugwort leaf hydrolysate from step (3). Shake well and extract in an ultrasonic cleaner at 85°C and 150 W for 40 min. After ultrasonic alcohol extraction, filter through a hot Buchner funnel to obtain a total flavonoid extract.

[0084] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 8 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to obtain a total flavonoid extract.

[0085] According to the above steps, 2.67 g of total flavonoid extract was obtained, with a total flavonoid content of 33.5%, that is, 0.894 g of total flavonoids was obtained, and the extraction yield was 8.94%.

[0086] Comparative Example 2: Extraction of Total Flavonoids from Artemisia argyi without Enzymolysis (Compared with Example 5)

[0087] (1) 10 g of mugwort leaf powder was added to 250 mL of 60% ethanol in a 500 mL Erlenmeyer flask (material-liquid ratio 1:25 (g:mL)). After shaking, the mixture was extracted in an ultrasonic cleaner at 85°C and 150 W for 40 min. After ultrasonic extraction, the mixture was filtered through a hot Buchner funnel to obtain the total flavonoid extract.

[0088] (2) The total flavonoid extract 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 (1.5 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 8 min. The supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to obtain a total flavonoid extract.

[0089] According to the above steps, 2.35 g of total flavonoid extract was obtained, with a total flavonoid content of 30.4%, that is, 0.714 g of total flavonoids was obtained, and the extraction yield was 7.14%.

[0090] Comparing the results of Example 5 and Comparative Example 2, it can be seen that before extracting total flavonoids from mugwort, enzymatic hydrolysis of the crude enzyme solution prepared by fermentation of Fusarium graminearum AY2-19 was added, and the total flavonoid extraction rate increased from 7.14% to 8.94%, an increase of 25.2%.

[0091] Example 6: Application of Enzymatic Hydrolysis-Assisted Extraction of Total Flavonoids from Artemisia argyi Using Fusarium graminearum AY2-19

[0092] The enzymatic extraction of total flavonoids from mugwort leaves using Fusarium graminearum AY2-19 can be performed by following the steps below:

[0093] (1) Spores of a PDA colony of Fusarium graminearum AY2-19 stored at 4°C were inoculated onto fresh PDA plate culture medium and cultured at 28°C for 64 h. 10 mL of sterile physiological saline was added to the plate culture and the spores were agitated with an inoculating loop to suspend the spores to obtain a spore solution of Fusarium graminearum AY2-19. The components and preparation method of the PDA plate culture medium were the same as those in Example 1.

[0094] (2) 5 mL of the spore solution prepared in step (1) was inoculated into 100 mL of enzyme production medium (inoculation volume was 5% by volume), and cultured at 30°C and 200 rpm for 64 h to obtain a fermentation broth with a dry cell concentration of 3.67 g / L. The entire fermentation broth was filtered using a Buchner funnel, and the collected filtrate was the crude enzyme solution. The cellulase activity of the filtrate was 34.2 U / mL. The final concentration composition and preparation method of the enzyme production medium were the same as those in Example 4.

[0095] (3) 20 g of mugwort powder was placed in a 500-mL conical flask, and 80 mL of the crude enzyme solution prepared in step (2) [i.e., the feed:enzyme ratio was 1:4 (g:mL)] was added. After stirring evenly, the mixture was enzymolyzed in a 26°C water bath for 4 h to obtain mugwort hydrolysate.

[0096] (4) Add 120 mL of deionized water and 300 mL of anhydrous ethanol (system material-liquid ratio 1:25 (g:mL), ethanol volume fraction 60%) to the entire mugwort leaf hydrolysate from step (3). Shake well and extract in an ultrasonic cleaner at 85°C and 150W for 45 minutes. After ultrasonic alcohol extraction, filter with a hot Buchner funnel to obtain a total flavonoid extract.

[0097] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out, 20 mL of anhydrous ethanol (1 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 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 obtain a total flavonoid extract.

[0098] According to the above steps, 5.42 g of total flavonoid extract was obtained, with a total flavonoid content of 32.7%, that is, 1.77 g of total flavonoids was obtained, and the extraction yield was 8.85%.

[0099] Comparative Example 3: Extraction of Total Flavonoids from Artemisia argyi without Enzymolysis (Compared with Example 6)

[0100] (1) 20 g of mugwort leaf powder was added to 500 mL of 60% ethanol in a 500 mL Erlenmeyer flask (material-liquid ratio 1:25 (g:mL)). After shaking, the mixture was extracted in an ultrasonic cleaner at 85°C and 150 W for 45 min. After the ultrasonic extraction, the mixture was filtered through a hot Buchner funnel to obtain the total flavonoid extract.

[0101] (2) The total flavonoid extract 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 (1 mL / g based on the mass of the raw mugwort powder) was added, and after sufficient shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 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 obtain a total flavonoid extract.

[0102] According to the above steps, 4.67 g of total flavonoid extract was obtained, with a total flavonoid content of 30.8%, that is, 1.44 g of total flavonoids was obtained, and the extraction yield was 7.20%.

[0103] Comparing the results of Example 6 and Comparative Example 3, it can be seen that before extracting total flavonoids from mugwort, enzymatic hydrolysis of the crude enzyme solution prepared by fermentation of Fusarium graminearum AY2-19 was added, and the total flavonoid extraction rate was increased from 7.20% to 8.85%, an increase of 22.9%.

Claims

1. Fusarium graminearum ( Fusarium graminearum )AY2-19, deposited in Guangdong Provincial Microbiological Culture Collection Center, deposit number GDMCC No: 63062, deposit date December 20, 2022, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

2. A use of the Fusarium graminearum AY2-19 according to claim 1 in extracting total flavonoids from mugwort.

3. The use according to claim 2, characterized in that The application method is as follows: (1) inoculating spores of Fusarium graminearum AY2-19 into an enzyme-producing culture medium, culturing at 30°C and 180-200 r / min for 64-72 h, filtering the fermentation liquid, and collecting the filtrate as a crude enzyme liquid; the final concentration composition of the enzyme-producing culture medium is: wheat bran 40-50 g / L, peptone 8-10 g / L, NaNO3 5-6 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.25-0.5 g / L, CaCl2 0.4-0.5 g / L, the solvent is tap water, pH 6.0-6.5; (2) mixing the crude enzyme liquid with mugwort powder, and enzymolyzing the culture medium at 26-30°C for 4-6 h. h, to obtain mugwort hydrolysate; (3) after adding deionized water and anhydrous ethanol to the mugwort hydrolysate, ultrasonic extraction was performed, and filtration was performed to obtain a total flavonoid extract; (4) the total flavonoid extract was distilled at 45°C and -0.1 MPa until no liquid flowed out, and anhydrous ethanol with a volume of 1-2 mL / g based on the mass of the raw mugwort was added, and after sufficient shaking, the supernatant was centrifuged at 8000 r / min for 5-10 min, and the supernatant was transferred to a clean culture dish and vacuum dried at 50°C and -0.1 MPa to obtain a total flavonoid extract.

4. The use according to claim 3, characterized in that The final concentration composition of the enzyme production medium is: 50 g / L wheat bran, 10 g / L peptone, 5 g / L NaNO3, 2 g / L KH2PO4, 0.25 g / L MgSO4·7H2O, 0.5 g / L CaCl2, the solvent is tap water, and the pH is 6.

0.

5. The use according to claim 3, characterized in that Before the enzyme production culture, the Fusarium graminearum AY2-19 is first cultured on a plate culture medium to produce spores, and then the spores are suspended in physiological saline to obtain a Fusarium graminearum AY2-19 spore liquid, which is inoculated into the enzyme production culture medium at a volume fraction of 3%-5% for culture. The spore liquid preparation method is as follows: inoculating Fusarium graminearum AY2-19 on a potato dextrose agar plate culture medium, culturing at 28°C for 64-72 hours to obtain a plate culture; adding sterile physiological saline to the plate culture, stirring with an inoculation loop to suspend the spores, and obtaining the Fusarium graminearum AY2-19 spore liquid.

6. The use according to claim 3, characterized in that The mugwort powder in step (2) is fresh mugwort that is washed with tap water, dried at 85°C, crushed and then sieved through a 40-mesh sieve to form a fine powder.

7. The use according to claim 3, characterized in that The volume of the crude enzyme solution in step (2) is 4–8 mL / g based on the mass of mugwort powder.

8. The use according to claim 3, characterized in that The preparation method of the total flavonoids extract in step (3) is as follows: after the enzymatic hydrolysis of mugwort leaves is completed, deionized water and anhydrous ethanol are added to the mugwort leaf hydrolysate to make the material-liquid ratio of the system reach 1g:20 mL-1g:25 mL, and the volume fraction of ethanol reaches 50%-60%. After stirring evenly, the mixture is extracted in an ultrasonic cleaning machine with a water temperature of 80-85°C and a power of 150 W for 30-45 min; after the ultrasonic alcohol extraction is completed, the mixture is filtered while hot to obtain the total flavonoids extract.

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