Metarhizium anisopliae green pigment A and its extraction method

Abstract

The present invention is applicable to the extraction and application field of biological products, and proposes a green pigment A of Metarhizium anisopliae and an extraction method, comprising the following steps: screening Metarhizium anisopliae; expanding culture and collecting spores; pre-enzymatic hydrolysis treatment; mixing cellulase solution, snail enzyme solution and chitinase solution into a cell wall enzymatic hydrolysis solution, and performing enzymatic hydrolysis treatment; adding a phenol extractant to the spores after enzymatic hydrolysis treatment, and obtaining a green pigment through separation; obtaining the molecular formula of the green pigment A of Metarhizium anisopliae by liquid chromatography-mass spectrometry analysis, and obtaining the chemical structure of the compound by liquid nuclear magnetic resonance. In summary, the present invention obtains the pure product of the green pigment A of Metarhizium anisopliae by the method of enzymatic hydrolysis + phenol extraction, and obtains the structure of the green pigment A of Metarhizium anisopliae by liquid chromatography-mass spectrometry analysis, laying the foundation for the determination of its synthetic gene, the identification of its metabolic pathway and its commercial application.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of biological products, in particular to green pigment A of Metarhizium anisopliae and an extraction method thereof. Background Art

[0002] Metarhizium muscardine is a fungus that can infect many insects but is not infectious to mammals. It is a fungus with green spores that is widely found in nature.

[0003] Pigments are important metabolites of fungi, crucially involved in their environmental adaptability and playing a crucial role in human life. Currently, a variety of natural pigments are used in all aspects of human life, including clothing, food, cosmetics, and environmental decoration, making human life truly colorful. Numerous archaeological evidence indicates that the use of pigments as colorants has existed since ancient times. Since the discovery of synthetic pigments in the 19th century, they have been widely used in various industries due to their low production cost, ease of manufacture, and excellent coloring properties. However, as research deepens, synthetic pigments have been found to have many adverse effects on human health and the environment. Due to the many shortcomings of synthetic pigments, such as poor degradation, poor durability, and potential carcinogenicity / allergenicity, natural organic pigments have become a hot topic of research.

[0004] Natural pigments are pigments produced primarily by the metabolism of plants, animals, and microorganisms. Since ancient times, most widely used natural pigments have been extracted from plants, such as mahogany, grapes, indigo, beetroot, turmeric, madder, saffron, etc. However, extracting pigments from plants is not an efficient option. Plants have high requirements for their living environment and are heavily dependent on the season. High extraction costs and stability and solubility issues result in low extraction efficiency of plant pigments. Today, with the development of microbial culture technology, microorganisms including bacteria, fungi, and algae have been proven to be a high-quality alternative source of natural pigments. Fungi are one of the best producers of natural pigments because they can grow quickly in inexpensive media and are not affected by the external environment.

[0005] The patent with authorization announcement number CN114106069B discloses a type of green anisopliae A and green anisopliae B with the ability to inhibit glycosidase activity and an extraction method. This technology discloses a method for extracting green anisopliae A and green anisopliae B from green anisopliae, and identifies their physicochemical properties.

[0006] The patent with authorization announcement number CN112778797B discloses a method of extracting natural green pigments from Metarhizium anisopliae using formic acid. This method solves the problem that green fungal pigments from Metarhizium anisopliae cannot be extracted, and lays the foundation for further structural identification and utilization. However, this technology does not study the structure of natural green pigments. At the same time, the extractant used in this method is formic acid, which can dissolve the fungal cell walls, but the reaction is relatively strong and will introduce impurities related to cell wall components into the extract.

[0007] In addition, there are studies on the control mechanism of highly effective cypermethrin and Metarhizium against German cockroaches. Shandong Normal University, Master's thesis; Construction and functional study of recombinant Metarhizium robertsii photolyase, Zhejiang University, Master's thesis; Functional study of the C2H2 transcription factor MaVrf1 of Metarhizium arguta, Chongqing University, Master's thesis; Study on the mechanism of degeneration of Metarhizium robertsii culture caused by inactivation of vacuolar arginine export gene Vae, Zhejiang University, Doctoral thesis; Metarhizium robertsii MrAbaA affects conidial pigmentation via regulating MrPks1andMrMlac1 expression, Journal of invertebrate pathology, Volume 197, Issue, 2023, PP 107892-107892. The above papers all involve research related to Metarhizium, but none of them involve the molecular structure of the green pigment of Metarhizium robertsii. Summary of the Invention

[0008] In view of this, the present invention proposes Metarhizium green pigment A and an extraction method thereof, obtains the pure product of Metarhizium green pigment A, and solves the problem that the molecular structure of Metarhizium green pigment has not been resolved.

[0009] The technical solution of the present invention is achieved as follows:

[0010] In the first aspect, the present invention provides a green pigment A of Metarhizium anisopliae, the structural formula of which is as follows:

[0011]

[0012] In a second aspect, the present invention provides a method for extracting Metarhizium green pigment A from Metarhizium, comprising the following steps:

[0013] Step 1: Bacteria screening

[0014] Use flat culture medium to culture Metarhizium strains, and select the Metarhizium with the highest green pigment production based on the color of the colonies.

[0015] The Metarhizium species in the present invention are selected from the Metarhizium species cultured in the Anhui Provincial Key Laboratory of Microbial Control; the level of green pigment production is determined by manually judging the depth of green, and the species with the highest green pigment production is the Metarhizium with the darkest green color.

[0016] Step 2: Expanding the culture and collecting spores

[0017] Inoculate the selected Metarhizium anisopliae spores into PDA liquid medium in a shake flask culture to serve as the seed culture medium. Prepare rice culture medium and inoculate the seed culture medium into the rice culture medium for large-scale cultivation. Cultivate for 10-15 days until the rice surface is covered with green spores. Collect the spores using a spore harvester and store in a refrigerator at 4°C until needed.

[0018] Step 3: Pre-enzymatic treatment

[0019] Add enzymatic pretreatment reagent to the spores at a weight-to-volume ratio of 1g:5-7mL, stir evenly, extract at a temperature of 10-50°C and ultrasonically for 20-400 minutes, and then filter or centrifuge.

[0020] Step 4: Enzymatic treatment of cell walls

[0021] A cell wall enzymatic hydrolysis solution is prepared by mixing a cellulase solution, a snail enzyme solution and a chitinase solution in a volume ratio of (0.5-1.5): (0.5-1.5): (0.5-1.5), and performing enzymatic hydrolysis on the cell walls of the spores that have undergone pre-enzymatic hydrolysis treatment.

[0022] Since Metarhizium is a fungus, the fungal cell wall is mainly composed of chitin, cellulose and polysaccharides. The cellulase solution, snail enzyme solution and chitinase solution can be used to decompose the fungal cell wall.

[0023] Step 5 Preparation of crude extract

[0024] The spores after enzymatic hydrolysis are added with a phenol extractant at a weight-volume ratio of 1 g: 5-7 mL, stirred or extracted for 2-8 hours, filtered or centrifuged to obtain a crude pigment extract, and the crude pigment extract is evaporated under reduced pressure at a vacuum degree of -0.05-0.2 MP and a temperature of 85-95° C. to remove the extractant, and dried at -45-55° C. to obtain a crude green pigment extract.

[0025] The present invention first decomposes the cell wall through an enzymatic hydrolysis reaction, which is relatively mild. After the cell wall is enzymatically hydrolyzed, phenol is used for extraction. This can introduce fewer impurities while ensuring pigment extraction, which is beneficial to subsequent purification and structural identification.

[0026] Step 6: Separation of green pigment

[0027] The hydroxypropyl dextran gel (Sephadex LH-20) filler was swollen overnight with a methanol solution, and the floating particles on the upper layer were discarded and loaded onto the column. After rinsing for 3-5 column volumes, the pigment sample was added for elution at a flow rate of 9 seconds per drop. Fractions 1-5 were collected according to color. Fraction 1 was separated again using Sephadex LH-20 to obtain 12 mg of purified pigment. The extractant was evaporated under reduced pressure at a vacuum of -0.1 MP and a temperature of 15°C, and the product was dried at -50°C to obtain a green pigment.

[0028] Step 7 Preparation of pure Metarhizium anisopliae green pigment A

[0029] An Agilent Zorbax SB-C18 column was used, with ultrapure water as phase A and methanol as phase B. Elution procedures included isocratic elution with 27% B from 0 to 18 minutes, isocratic elution from 27% to 100% B from 18 to 20 minutes, isocratic elution from 100% B from 20 to 30 minutes, and isocratic elution from 100% to 27% B from 30 to 32 minutes. The UV detector scanned at wavelengths of 266 nm and 340 nm, collecting components eluting simultaneously at both wavelengths. The collected solution was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C to remove the extractant, and then dried at -50°C to obtain Metarhizium anisopliae green pigment A. The purity of Metarhizium anisopliae green pigment A was greater than 98%.

[0030] Step 8: Structural Identification

[0031] The molecular formula of the compound was obtained by high-resolution liquid chromatography-mass spectrometry analysis, and the chemical structure of the compound was obtained by liquid nuclear magnetic resonance. Since the purity of the compound must be above 98% when using liquid nuclear magnetic resonance for structural identification of the compound, the purity of the green pigment A of Metarhizium anisopliae in the present invention can meet the requirement.

[0032] Furthermore, in step one, the plate culture medium is PDA medium or SDAY medium, the culture temperature is 20-35°C, and the culture is carried out for 5-10 days. The selected Metarhizium can be any one of Metarhizium acridum, Metarhizium flavoviride, and Metarhizium robertsii.

[0033] Furthermore, in step 2, the shake flask culture conditions are: a shaker speed of 50-200 rpm, a temperature of 20-35° C., and a culture period of 3-10 days. The rice culture medium is soaked in warm water for 10-60 minutes and sterilized at 120° C. for 20 minutes.

[0034] Furthermore, in step 3, the enzymatic pretreatment reagent is a mixture of methanol or ethanol and ethyl acetate in a volume ratio of 1:0.2-0.6. The enzymatic pretreatment reagent is mainly used to remove fat-soluble components of spores, facilitate the full mixing of the enzymatic solution and spores, and improve the enzymatic hydrolysis efficiency.

[0035] Furthermore, in step 4, the mass concentration of the cellulase solution, snail enzyme solution, and chitinase solution is 1%; the solvent in the cellulase solution, snail enzyme solution, and chitinase solution is citric acid-sodium citrate buffer. In this step, 1% enzyme solutions are first prepared separately and then mixed before use.

[0036] Of course, the above solvent can be replaced with tris-HCl buffer or phosphate buffer, as long as the pH can be kept stable during the enzymatic hydrolysis to ensure the effect of the enzyme.

[0037] The citric acid-sodium citrate buffer solution is prepared as follows: 0.1M citric acid and sodium citrate solutions are prepared separately, and 12-15ml of 0.1M citric acid solution is mixed with 180-190ml of 0.1M sodium citrate solution to obtain a citric acid-sodium citrate buffer solution with a pH of 6-7.

[0038] The enzymatic hydrolysis treatment conditions are as follows: the reaction is carried out at 25-35°C and 75-85 rpm in a shaker for 3-5 hours. After the reaction is completed, the mixture is centrifuged at 7500-9000 rpm for 4-7 minutes.

[0039] Furthermore, in step 5, since phenol is a solid at room temperature, the extraction needs to be carried out in a water bath at a temperature of 65-80°C.

[0040] Furthermore, in step six, the eluent used for Sephadex LH-20 separation is a methanol solution containing 5% to 20% phenol.

[0041] Furthermore, in step seven, 0.1%-0.5% formic acid is added to mobile phase A and mobile phase B.

[0042] Furthermore, in step 8, the LC / MS instrument conditions were: nitrogen temperature 325°C, nitrogen flow rate 12 L / min, nebulizer pressure 35 psi; capillary voltage: 4000 V for positive ion; fragmentor voltage: 215 V for positive ion, separator voltage 60 V; mass acquisition range: 50-1000 Da for both positive and negative modes. Liquid NMR analysis was performed using deuterated dimethyl sulfoxide as the solvent.

[0043] The results of high-resolution liquid chromatography-mass spectrometry analysis showed that the mass-to-charge ratio (m / z) of the green pigment A of Metarhizium anisopliae in the positive ion mode was 565.0875 (C 18 H 13 N8O 14), the peak at 1129.1511 is [2M+H]+(C 36 H 25 N 16 O 28 ) mass-to-charge ratio; the mass-to-charge ratio (m / z) in negative ion mode was 563.0705 (C 18 H 11 N8O 14 ), the peak at 1127.1350 is [2M-H] - (C 36 H 23 N 16 O 28 ) mass-to-charge ratio. The molecular formula of green pigment A of Metarhizium anisopliae was finally determined to be C 18 H 12 N8O 14 The degree of unsaturation is 17. Its UV absorption peaks are 266nm and 340nm. Nuclear magnetic resonance analysis shows that the green pigment A of Metarhizium anisopliae has a symmetrical structure. The nuclear magnetic resonance data are shown in the table below:

[0044] Table 1 NMR data

[0045] serial number Carbon spectrum DEPT Proton spectrum COSY HMBC 1 133.7 C 2 163.6 C 3 161.6 C 4 100.8 CH 6.01(d2.0Hz) 6.12、4.79 1、2、3、5 5 79.1 <![CDATA[CH2]]> 4.87 (ddt 2.2Hz) 4.79 6 77.5 <![CDATA[CH2]]> 4.79 (ddt 2.2Hz) 4.87 5 7 179.1 C 8 142.4 C 9 116.7 CH 6.12 (dd2.4Hz) 6.01 7、8

[0046] Comprehensive LC-MS analysis and NMR analysis showed that the structure of the isolated and purified green pigment A from Metarhizium anisopliae is:

[0047]

[0048] In the present invention, the purity of green pigment A of Metarhizium anisopliae is detected by liquid chromatography-mass spectrometry, and is obtained from the peak area ratio of the target compound in the ion flow diagram (the peak area of ​​the target compound accounts for the total peak area of ​​the sample).

[0049] The green pigment A of Metarhizium anisopliae and the extraction method thereof of the present invention have the following beneficial effects compared with the prior art:

[0050] 1. The present invention adopts the method of enzymatic hydrolysis + phenol to obtain the pure product of Metarhizium green pigment A, and the purity of Metarhizium green pigment A is greater than 98%. The structure of Metarhizium green pigment A is obtained by liquid chromatography-mass spectrometry analysis, which solves the problem that the molecular structure of Metarhizium green pigment A has not been resolved.

[0051] 2. Based on the enzymatic hydrolysis + phenol method, the optimal volume ratio range of cellulase solution, snail enzyme solution, and chitinase solution in the cell wall enzymatic hydrolyzate was determined to ensure pigment extraction efficiency while controlling costs. In addition, the optimal addition range of phenol was determined to ensure pigment extraction efficiency and improve the extraction efficiency of Metarhizium anisopliae green pigment A.

[0052] 3. The determination of the molecular structure of Metarhizium anisopliae green pigment A lays the foundation for the identification of its synthetic gene, the characterization of its metabolic pathway, and its commercial application. Furthermore, Metarhizium anisopliae green pigment A is a natural pigment with excellent safety and stability far superior to plant chlorophyll. The pigment is only soluble in specific solvents, resulting in excellent stability after coloration and broad application prospects.

[0053] 4. The raw material strain of the green pigment A of Metarhizium anisopliae extracted by the present invention is Metarhizium anisopliae, which is a common insect parasite. There are commercial Metarhizium anisopliae spore products, which have low production costs and relatively simple process flow, and can be produced on a large scale industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0055] Figure 1 This is the mass spectrum in positive ion mode in Example 1 of the present invention;

[0056] Figure 2 This is the mass spectrum in negative ion mode in Example 1 of the present invention;

[0057] Figure 3 This is the ultraviolet absorption spectrum in Example 1 of the present invention;

[0058] Figure 4 is the nuclear magnetic H in Example 1 of the present invention 1 Atlas;

[0059] Figure 5 For the nuclear magnetic 13 C spectrum;

[0060] Figure 6 This is the nuclear magnetic DEPT135 spectrum in Example 1 of the present invention;

[0061] Figure 7 For the nuclear magnetic 1 H- 1 H COSY spectrum;

[0062] Figure 8 The HMBC spectrum of Example 1 of the present invention is shown below:

[0063] Figure 9 This is the NMR HSQC spectrum in Example 1 of the present invention. DETAILED DESCRIPTION

[0064] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0065] Example 1

[0066] The specific steps for extracting Metarhizium green pigment A from Metarhizium are as follows:

[0067] (1) Strain screening

[0068] The Metarhizium species were cultured using a plate culture medium at a temperature of 20°C for 5 days, and the species with the highest green pigment production were selected based on the colony color. The present invention uses Metarhizium acridum as a raw material for culture and extraction.

[0069] (2) Expanded culture and spore collection

[0070] Inoculate Metarhizium anisopliae spores into PDA liquid medium and culture in shake flasks as seed culture medium. Shake flask culture shaker speed 50 rpm, temperature 20 ℃, culture for 3 days. Prepare rice culture medium, soak the rice culture medium in warm water for 10 minutes, and sterilize it at 120 ℃ for 20 minutes. Inoculate the seed culture medium into the rice culture medium for large-scale culture, culture for 10 days until green spores no longer appear on the surface of the rice. Use a spore collector to collect the spores and store in a 4 ℃ refrigerator until they are ready for use.

[0071] (3) Impurity removal before extraction

[0072] Add enzymatic pretreatment reagent (methanol: ethyl acetate = 1:0.2) to the spores at a weight-to-volume ratio of 1 g:5 mL, stir evenly, extract at 10°C under ultrasonication for 20 minutes, and then filter or centrifuge.

[0073] (4) Enzyme treatment of cell walls

[0074] Before extraction, enzymatically hydrolyze the cell walls. Prepare a citric acid-sodium citrate buffer solution: Prepare 0.1M citric acid and sodium citrate solutions separately. Mix 12ml of the 0.1M citric acid solution with 180ml of the 0.1M sodium citrate solution to obtain a citric acid-sodium citrate buffer solution with a pH of 6. In the citric acid-sodium citrate buffer solution, prepare 1% cellulase solution, 1% snailase solution, and 1% chitinase solution. Mix the solution in a ratio of 0.5:0.5:0.5 to prepare the cell wall enzymatic hydrolysis solution. Perform the enzymatic hydrolysis reaction in a 50ml centrifuge tube and incubate at 25°C and 75 rpm on a shaker for 3 hours. After completion, centrifuge at 7500 rpm for 4 minutes.

[0075] (5) Preparation of crude extract

[0076] The spores after enzymatic hydrolysis are added with a phenol extractant at a weight-to-volume ratio of 1 g:5 mL, and the mixture is stirred and extracted in a water bath at a temperature of 65° C. for 2 hours. The mixture is filtered or centrifuged to obtain a crude pigment extract. The crude pigment extract is evaporated under reduced pressure at a vacuum degree of -0.05 MP and a temperature of 85° C. to remove the extractant, and the crude pigment extract is dried at -45° C. to obtain a crude green pigment extract.

[0077] (6) LH-20 separation of green pigment

[0078] Sephadex LH-20 filler was swollen overnight with a methanol solution, and the floating particles on the upper layer were discarded before loading onto the column. The eluent was a methanol solution with a 5% phenol content. After rinsing for 3 column volumes, the pigment sample was added for elution at a flow rate of 9 seconds per drop. Fractions 1-5 were collected according to color. Fraction 1 was again separated using Sephadex LH-20 to obtain 12 mg of purified pigment. The extractant was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C, and the product was dried at -50°C to obtain a green pigment.

[0079] (7) Preparation of pure green pigment A from Metarhizium anisopliae

[0080] An Agilent Zorbax SB-C18 column was used, with ultrapure water as phase A and methanol as phase B. 0.1% formic acid was added to both mobile phases A and B. The elution method was as follows: isocratic elution with 27% B from 0 to 18 minutes; isocratic elution from 27% to 100% B from 18 to 20 minutes; isocratic elution from 100% B from 20 to 30 minutes; and isocratic elution from 100% to 27% B from 30 to 32 minutes. The UV detector was scanned at wavelengths of 266 nm and 340 nm, and components eluting simultaneously at both wavelengths were collected. The collected solution was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C to remove the extractant, and then dried at -50°C to obtain Metarhizium anisopliae green pigment A.

[0081] (8) Structural identification

[0082] The molecular formula of the compound was determined by high-resolution liquid chromatography-mass spectrometry (LC-MS). The instrumental conditions were: nitrogen temperature 325°C, nitrogen flow rate 12 L / min, nebulizer pressure 35 psi; capillary voltage 4000 V for positive ion; fragmentor voltage 215 V for positive ion; and separator voltage 60 V. The mass range was 50-1000 Da for both positive and negative modes. Liquid NMR analysis was performed using deuterated dimethyl sulfoxide as the solvent. The resolved structure is shown below.

[0083]

[0084] The positive ion mode mass spectrum, negative ion mode mass spectrum, ultraviolet absorption spectrum, nuclear magnetic H 1 Spectrum, NMR 13 C spectrum, NMR DEPT135 spectrum, NMR 1 H- 1 H COSY spectrum, NMR HMBC spectrum, NMR HSQC spectrum see Figure 1-9 .

[0085] Example 2:

[0086] The specific steps for extracting Metarhizium green pigment A from Metarhizium are as follows:

[0087] (1) Strain screening

[0088] The Metarhizium species were cultured using a plate culture medium at a temperature of 28° C. for 6 days, and the species with the highest green pigment production were selected based on the colony color. The present invention uses Metarhizium flavoviride as a raw material for culture and extraction.

[0089] (2) Expanded culture and spore collection

[0090] Inoculate Metarhizium anisopliae spores into PDA liquid medium and culture in a shake flask as a seed culture medium. Incubate the shaker at 150 rpm and 24°C for 6 days. Prepare rice culture medium, soak the rice culture medium in warm water for 40 minutes, and sterilize it at 120°C for 20 minutes. Inoculate the seed culture medium into the rice culture medium for large-scale cultivation and culture for 13 days until green spores no longer appear on the surface of the rice. Collect the spores using a spore collector and store in a 4°C refrigerator until ready for use.

[0091] (3) Impurity removal before extraction

[0092] Add enzymatic pretreatment reagent (methanol:ethyl acetate=1:0.4) to the spores at a weight-to-volume ratio of 1g:6mL, stir evenly, extract at 40°C under ultrasonication for 100 minutes, and then filter or centrifuge.

[0093] (4) Enzyme treatment of cell walls

[0094] Before extraction, enzymatically hydrolyze the cell walls. Prepare a citric acid-sodium citrate buffer solution: Prepare 0.1M citric acid and sodium citrate solutions separately. Mix 14 ml of the 0.1M citric acid solution with 186 ml of the 0.1M sodium citrate solution to obtain a citric acid-sodium citrate buffer solution with a pH of 6.6. In the citric acid-sodium citrate buffer solution, prepare 1% cellulase solution, 1% snailase solution, and 1% chitinase solution. Mix the solution in a ratio of 1.5:1:1 to prepare the cell wall enzymatic hydrolysis solution. Perform the enzymatic hydrolysis reaction in a 50 ml centrifuge tube and shake at 30°C and 80 rpm for 3 hours. After completion, centrifuge at 8000 rpm for 5 minutes.

[0095] (5) Preparation of crude extract

[0096] The spores after enzymatic hydrolysis are added with a phenol extractant at a weight-to-volume ratio of 1 g:6 mL, and the mixture is stirred and extracted in a water bath at a temperature of 72° C. for 3 hours. The mixture is filtered or centrifuged to obtain a crude pigment extract. The crude pigment extract is evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 90° C. to remove the extractant, and the extract is dried at -50° C. to obtain a green pigment.

[0097] (6) LH-20 separation of green pigment

[0098] Sephadex LH-20 filler was swollen overnight with a methanol solution, and the floating particles on the upper layer were discarded and loaded onto the column. The eluent was a methanol solution with a 15% phenol content. After rinsing for 4 column volumes, the pigment sample was added for elution at a flow rate of 10 seconds per drop. Fractions 1-5 were collected according to color. Fraction 1 was separated again using Sephadex LH-20 to obtain 12 mg of purified pigment. The extractant was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C, and the product was dried at -50°C to obtain a green pigment.

[0099] (7) Preparation of pure green pigment A from Metarhizium anisopliae

[0100] An Agilent Zorbax SB-C18 column was used, with ultrapure water as phase A and methanol as phase B. 0.3% formic acid was added to both mobile phases A and B. The elution method was as follows: isocratic elution with 27% B from 0 to 18 minutes; isocratic elution from 27% to 100% B from 18 to 20 minutes; isocratic elution from 100% B from 20 to 30 minutes; and isocratic elution from 100% to 27% B from 30 to 32 minutes. The UV detector scanned at wavelengths of 266 nm and 340 nm, collecting components eluting simultaneously at both wavelengths. The collected solution was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C to remove the extractant, and then dried at -50°C to obtain Metarhizium anisopliae green pigment A.

[0101] (8) Structural identification

[0102] The molecular formula of the compound was determined by high-resolution liquid chromatography-mass spectrometry (LC-MS). The instrumental conditions were: nitrogen temperature 325°C, nitrogen flow rate 12 L / min, nebulizer pressure 35 psi; capillary voltage 4000 V for positive ion; fragmentor voltage 215 V for positive ion; and separator voltage 60 V. The mass range was 50-1000 Da for both positive and negative modes. Liquid NMR analysis was performed using deuterated dimethyl sulfoxide as the solvent. The resolved structure is shown below.

[0103]

[0104] Example 3:

[0105] The specific steps for extracting Metarhizium green pigment A from Metarhizium are as follows:

[0106] (1) Strain screening

[0107] The Metarhizium species were cultured using a plate culture medium at a temperature of 35°C for 10 days, and the species with the highest green pigment production were selected based on the colony color. The present invention uses Metarhizium robertsii as the material for culture and extraction.

[0108] (2) Expanded culture and spore collection

[0109] Inoculate Metarhizium anisopliae spores into PDA liquid medium and incubate in a shaker at 35°C and 200 rpm for 10 days to serve as seed culture. Prepare rice culture medium, soak the rice culture medium in warm water for 60 minutes, and sterilize it at 120°C for 20 minutes. Inoculate the seed culture medium into the rice culture medium for mass cultivation and incubate for 15 days until no green spores appear on the rice surface. Collect the spores using a spore collector and store in a 4°C refrigerator until needed.

[0110] (3) Impurity removal before extraction

[0111] Add enzymatic pretreatment reagent (methanol:ethyl acetate=1:0.6) to the spores at a weight-to-volume ratio of 1g:7mL and stir evenly. Perform ultrasonic extraction at 50°C for 400 minutes, and then filter or centrifuge.

[0112] (4) Enzyme treatment of cell walls

[0113] Before extraction, the cell walls were enzymatically hydrolyzed. To prepare a citric acid-sodium citrate buffer solution, prepare 0.1M citric acid and sodium citrate solutions separately. Mix 15ml of the 0.1M citric acid solution with 190ml of the 0.1M sodium citrate solution to obtain a citric acid-sodium citrate buffer solution with a pH of 6.6. In the citric acid-sodium citrate buffer solution, prepare 1% cellulase solution, 1% snailase solution, and 1% chitinase solution. Mix the solution in a ratio of 1.5:1:1.5 to prepare the cell wall hydrolyzate. Perform the enzymatic hydrolysis reaction in a 50ml centrifuge tube and incubate at 35°C, 85 rpm, on a shaker for 5 hours. After completion, centrifuge at 9000 rpm for 7 minutes.

[0114] (5) Preparation of crude extract

[0115] The spores after enzymatic hydrolysis were added with a phenol extractant at a weight-to-volume ratio of 1 g:7 mL, and the mixture was stirred and extracted in a water bath at a temperature of 75° C. for 3.5 hours. The mixture was filtered or centrifuged to obtain a crude pigment extract. The crude pigment extract was evaporated under reduced pressure at a vacuum degree of -0.2 MP and a temperature of 95° C. to remove the extractant, and the extractant was dried at -55° C. to obtain a crude green pigment extract.

[0116] (6) Separation of green pigment

[0117] Sephadex LH-20 filler was swollen overnight with a methanol solution, and the floating particles on the upper layer were discarded before loading onto the column. The eluent was a methanol solution with a 20% phenol content. After rinsing for 5 column volumes, the pigment sample was added for elution, and the flow rate was adjusted to 9 seconds per drop. Fractions 1-5 were collected according to color. Fraction 1 was separated again using Sephadex LH-20 to obtain 12 mg of purified pigment. The extractant was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C, and the mixture was dried at -50°C to obtain a green pigment.

[0118] (7) Preparation of pure green pigment A from Metarhizium anisopliae

[0119] An Agilent Zorbax SB-C18 column was used, with ultrapure water as phase A and methanol as phase B. 0.5% formic acid was added to both mobile phases A and B. The elution method was as follows: isocratic elution with 27% B from 0 to 18 minutes; isocratic elution from 27% to 100% B from 18 to 20 minutes; isocratic elution from 100% B from 20 to 30 minutes; and isocratic elution from 100% to 27% B from 30 to 32 minutes. The UV detector was scanned at wavelengths of 266 nm and 340 nm, and components eluting simultaneously at both wavelengths were collected. The collected solution was evaporated under reduced pressure at a vacuum of -0.1 MPa and a temperature of 15°C to remove the extractant, and then dried at -50°C to obtain Metarhizium anisopliae green pigment A.

[0120] (8) Structural identification

[0121] The compound's molecular formula was determined by high-resolution liquid chromatography-mass spectrometry (LC-MS). The instrumental conditions were: nitrogen temperature 325°C, nitrogen flow rate 12 L / min, nebulizer pressure 35 psi; capillary voltage 4000 V for positive ion; fragmentor voltage 215 V for positive ion; and separator voltage 60 V. The mass range was 50-1000 Da for both positive and negative modes. Liquid NMR analysis was performed using deuterated dimethyl sulfoxide as the solvent. The resolved structure is shown below.

[0122]

[0123] Comparative Example 1

[0124] On the basis of Example 2, in step 4, the volume ratio of cellulase solution: snail enzyme solution: chitinase solution was changed to 1.0:0.4:0.4, and other conditions were the same.

[0125] Comparative Example 2

[0126] On the basis of Example 2, in step 4, the volume ratio of cellulase solution: snail enzyme solution: chitinase solution was changed to 0.8:0.3:0.4, and other conditions were the same.

[0127] Comparative Example 3

[0128] On the basis of Example 2, in step 4, the volume ratio of cellulase solution: snail enzyme solution: chitinase solution was changed to 1.7:1.6:0.8, and other conditions were the same.

[0129] Comparative Example 4

[0130] Based on Example 2, in step 4, the volume ratio of cellulase solution: snail enzyme solution: chitinase solution was changed to 1.9:2.0:2.0, and other conditions were the same.

[0131] Comparative Example 5

[0132] Based on Example 2, in step 5, the spores after enzymatic hydrolysis were added to a phenol extractant at a weight volume ratio of 1 g:2 mL, and other conditions were the same.

[0133] Comparative Example 6

[0134] Based on Example 2, in step 5, the spores after enzymatic hydrolysis were added to a phenol extractant at a weight-to-volume ratio of 1 g:9 mL, and other conditions were the same.

[0135] Comparative Example 7

[0136] Based on Example 2, the three steps of pre-enzymatic treatment in step 3, enzymatic treatment of the cell wall in step 4, and preparation of the crude extract in step 5 were replaced with the following two steps:

[0137] Step 3: Spore removal

[0138] Add impurity remover No. 1 to the spores at a weight-volume ratio of 1 g: 0.5-5 mL, perform a first reflux extraction at a temperature of 10-50° C. for 30-600 minutes, and recover the extract for processing the next batch of samples. Add impurity remover No. 2 to the first solid portion at a weight-volume ratio of 1 g: 0.5-5 mL, perform a second reflux extraction for 30-600 minutes, and recover the extract for processing the next batch of samples. Add impurity remover No. 3 to the second solid portion at a weight-volume ratio of 1 g: 0.5-5 mL, stir evenly, perform ultrasonic extraction at a temperature of 20-90° C. for 20-400 minutes, and then filter or centrifuge, discard the filtrate or supernatant to obtain impurity-removed spores.

[0139] The formula of impurity remover No. 1 is prepared by ethyl acetate or chloroform: petroleum ether at a volume ratio of 0.1-3:3-0.1; the formula of impurity remover No. 2 is prepared by methanol or ethanol: acetone at a volume ratio of 0.1-3:3-0.1; the formula of impurity remover No. 3 is prepared by water: dimethyl sulfoxide at a volume ratio of 1:0-0.1; the filter membrane used for filtration is a 100-600 mesh filter membrane; the centrifugation conditions are: a centrifugal speed of 5000-20000 rpm and a time of 5-20 minutes;

[0140] Step 4: Preparation of crude extract

[0141] The extracted spores are added with an extractant at a weight-volume ratio of 1 g: 0.5-5 mL, stirred or ultrasonically extracted for 2-10 hours, filtered or centrifuged, and the filtrate or supernatant is the crude pigment extract. The extractant formula is: a mixed solution prepared by formic acid: ethanol at a volume ratio of 1:0-1; the filtration membrane is a 100-600 mesh filter membrane; and the centrifugation conditions are: a centrifugal speed of 5000-20000 rpm and a time of 5-20 minutes.

[0142] The other steps and conditions are the same.

[0143] In Comparative Examples 1-7, relevant experimental data are shown in Table 2.

[0144] Table 2 Relevant experimental data in Comparative Examples 1-7

[0145]

[0146] The data from Comparative Examples 1 and 2 show that the volume ratio of cellulase solution: snailase solution: chitinase solution is too small, resulting in incomplete enzymatic hydrolysis and a low pigment extraction rate. The data from Comparative Examples 3 and 4 show that the volume ratio of cellulase solution: snailase solution: chitinase solution is too large, resulting in excessively high production costs. To ensure a relatively high pigment extraction rate and an acceptable cost, the optimal volume ratio is that in Example 2.

[0147] It can be seen from the data in Comparative Example 5 that phenol has weak solubility, and too little phenol is not conducive to the full extraction of pigments, resulting in a lower pigment extraction rate and a waste of green muscardine raw materials; it can be seen from the data in Comparative Example 6 that too much phenol leads to a longer later concentration time, prolongs the extraction time of green muscardine green pigment A, reduces work efficiency, and wastes phenol reagents.

[0148] In Comparative Example 7, the formic acid-ethanol method was used to extract the pigment. Although the pigment extraction rate was high, the purity of Metarhizium anisopliae green pigment A was only 72.6%. Since the purity of Metarhizium anisopliae green pigment A must be above 98% when using liquid nuclear magnetic resonance for structural identification of the compound, this method cannot meet the requirements and the structural formula of the present invention cannot be obtained. However, the impurities introduced by the enzymatic hydrolysis and phenol addition of the present invention are reduced, and the purity of Metarhizium anisopliae green pigment A can be guaranteed to be above 98%. Therefore, the structural formula of Metarhizium anisopliae green pigment A can be finally obtained.

[0149] Comparative Example 8

[0150] On the basis of Example 2, the elution method using hydroxypropyl dextran gel (Sephadex LH-20) filler was replaced with ion exchange chromatography, and other conditions were the same.

[0151] The results showed that the green pigment could not be adsorbed on the ion column, so it could not be eluted and the green pigment could not be separated.

[0152] Comparative Example 9

[0153] On the basis of Example 2, the elution method using hydroxypropyl dextran gel (Sephadex LH-20) filler was replaced with silica gel chromatography, and other conditions were the same.

[0154] The results showed that the green pigment could be adsorbed on the ion column, but could not be eluted, and the green pigment could not be separated.

[0155] Comparative Example 10

[0156] On the basis of Example 2, the elution method using hydroxypropyl dextran gel (Sephadex LH-20) filler was replaced with solid phase extraction, and other conditions were the same.

[0157] The results showed that the green pigment could be adsorbed on the ion column, but could not be eluted, and the green pigment could not be separated.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A green pigment A of Metarhizium anisopliae, characterized in that The structural formula is as follows:

2. A method for extracting the green pigment A of Metarhizium anisopliae according to claim 1 from Metarhizium anisopliae, characterized in that: The steps include: Step 1: screening Metarhizium anisopliae; Step 2: expanding the culture and collecting the spores to obtain the spores of Metarhizium; Step 3: adding an enzymatic pretreatment reagent to the spores at a weight-to-volume ratio of 1 g:5-7 mL, stirring evenly, performing ultrasonic extraction at a temperature of 10-50° C. for 20-400 minutes, and then filtering or centrifuging; Step 4: Mixing cellulase solution, snail enzyme solution, and chitinase solution in a volume ratio of 1.5:1:1 to prepare a cell wall enzymatic hydrolysis solution, and performing enzymatic hydrolysis on the cell walls of the spores that have undergone pre-enzymatic hydrolysis treatment; wherein the mass concentrations of the cellulase solution, snail enzyme solution, and chitinase solution are all 1%; Step 5: adding phenol extractant to the spores after enzymatic hydrolysis at a weight volume ratio of 1 g: 5 to 7 mL, stirring or extracting for 2 to 8 hours, filtering or centrifuging to obtain a crude pigment extract, and distilling the crude pigment extract under reduced pressure at a vacuum degree of -0.05 to 0.2 MP and a temperature of 85 to 95° C. to remove the extractant, and drying at -45 to 55° C. to obtain a crude green pigment extract; Step 6: Separating the crude green pigment extract to obtain the green pigment; the method for separating the green pigment is as follows: swelling the hydroxypropyl dextran gel filler with a methanol solution overnight, discarding the floating particles on the upper layer and loading the column, flushing for 3-5 column volumes, adding the pigment sample for elution, and adjusting the flow rate to 9 seconds / drop; collecting according to color to collect fractions 1-5, and fraction 1 is again separated using hydroxypropyl dextran gel to obtain a purified pigment product; then evaporating the extractant under reduced pressure, and drying to obtain the green pigment; the eluent used for the hydroxypropyl dextran gel separation is a methanol solution with a phenol content of 5%-20%; Step seven: eluting the green pigment and performing reduced pressure distillation to obtain a pure product of Metarhizium anisopliae green pigment A; Step eight: obtain the compound molecular formula of green pigment A of Metarhizium anisopliae by liquid chromatography-mass spectrometry analysis, and obtain the chemical structure of the compound by liquid nuclear magnetic resonance.

3. The method for extracting Metarhizium green pigment A from Metarhizium as claimed in claim 2, characterized in that: In step one, a plate culture medium is used to culture the Metarhizium species. The plate culture medium is PDA medium or SDAY medium. The culture temperature is 20-35°C and the culture is carried out for 5-10 days. The selected Metarhizium is any one of Metarhizium locustii, Metarhizium aureum, and Metarhizium robertsii.

4. The method for extracting Metarhizium green pigment A from Metarhizium as claimed in claim 2, wherein: In step 3, the enzymatic pretreatment reagent is a mixture of methanol or ethanol and ethyl acetate in a volume ratio of 1:0.2-0.

6.

5. The method for extracting Metarhizium green pigment A from Metarhizium as claimed in claim 2, characterized in that: In step seven, the purity of Metarhizium anisopliae green pigment A is greater than 98%.

6. The method for extracting Metarhizium green pigment A from Metarhizium as claimed in claim 2, wherein: In step eight, the instrument conditions for LC-MS are as follows: nitrogen temperature 325°C, nitrogen flow rate 12 L / min, nebulizer pressure 35 psi; capillary voltage: 4000 V for positive ion; fragmentor voltage: 215 V for positive ion, separator voltage 60 V; mass acquisition range: 50-1000 Da for both negative and positive ion modes.

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