CN115583997B - Penicillium chrysogenum inactivated mycelium polysaccharide and extraction method and application thereof - Google Patents
Penicillium chrysogenum inactivated mycelium polysaccharide and extraction method and application thereof Download PDFInfo
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- CN115583997B CN115583997B CN202211047440.3A CN202211047440A CN115583997B CN 115583997 B CN115583997 B CN 115583997B CN 202211047440 A CN202211047440 A CN 202211047440A CN 115583997 B CN115583997 B CN 115583997B
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- inactivated
- penicillium chrysogenum
- mycelium
- polysaccharide
- precipitate
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
- C12R2001/82—Penicillium chrysogenum
Abstract
The invention discloses a penicillium chrysogenum inactivated mycelium polysaccharide, an extraction method and application thereof, relates to the field of penicillium, and aims at solving the problem that the application of the penicillium chrysogenum inactivated mycelium polysaccharide is restricted because specific active ingredients in the penicillium chrysogenum inactivated mycelium polysaccharide cannot be separated. The invention exactly discloses a structure and a preparation method of the penicillium chrysogenum inactivated mycelium polysaccharide, which comprises ethanol precipitation, enzymolysis protein removal, gel chromatographic column separation and purification, infrared spectrum, high performance liquid chromatography and nuclear magnetic resonance analysis structure, the prepared penicillium chrysogenum inactivated mycelium polysaccharide has better effect on the prevention and treatment of tobacco mosaic virus, the prepared penicillium chrysogenum inactivated mycelium polysaccharide enables active ingredients of the penicillium chrysogenum inactivated mycelium to be more definite, the preparation method is simple, pharmacology is clearer, the basis and the method are provided for developing natural antiviral preparations, and the invention has wide applicability in the field of natural product plant disease resistance.
Description
Technical Field
The invention relates to the field of penicillium, in particular to a penicillium chrysogenum inactivated mycelium polysaccharide, an extraction method and application thereof.
Background
The polysaccharide has various activities of immunoregulation, anti-tumor, anti-infection, blood lipid reduction, blood sugar reduction, anti-aging, antivirus and the like, and the antiviral activity of the fungal polysaccharide is paid more attention to by more researchers in recent years, and the fungal polysaccharide achieves the aim of preventing and treating plant virus diseases by inducing plant resistance, stimulating plant immunity, inhibiting virus infection replication, preventing and in vitro passivating virus diseases, for example, oyster mushroom polysaccharide, flammulina velutipes polysaccharide, lentinan and the like can prevent and treat plant viruses.
The penicillium chrysogenum inactivated mycelium polysaccharide is a residual byproduct of penicillin production by penicillium fermentation, contains 90 percent of organic substances, 7%N, 1 percent of P and 2%K, can induce plants to express related proteins of disease course, has a certain prevention and treatment effect on melon verticillium wilt, watermelon fusarium wilt, cotton fusarium wilt, tobacco black shank, tobacco brown spot and tobacco mosaic disease, researchers separate and purify active ingredients of the penicillium chrysogenum inactivated mycelium polysaccharide by utilizing molecular sieve chromatography and ion exchange chromatography, but do not obtain better separation results, and only presumes that the active ingredients of the penicillium chrysogenum inactivated mycelium polysaccharide can be protein or polypeptide by enzymolysis experiments, however, the research shows that the root irrigation is carried out by using the water extract of the penicillium chrysogenum inactivated mycelium polysaccharide, the prevention effect on cotton fusarium wilt can reach 51-77%, the prevention effect on cotton verticillium wilt is also obvious, and the application of the penicillium chrysogenum inactivated mycelium polysaccharide to crops such as grapes, apples, potatoes, onions and tomatoes can be improved by further using the part with the molecular weight larger than 2KD in the water extract of the penicillium chrysogenum inactivated mycelium polysaccharide, although the penicillium chrysogenum inactivated mycelium polysaccharide has good prevention effect on various diseases of various crops, the specific active ingredients cannot be separated, the structure of the specific active ingredients cannot be clarified, and the application of the penicillium chrysogenum inactivated mycelium polysaccharide is restricted.
Tobacco mosaic virus (tobacco mosaic virus, TMV) is regarded as cancer of tobacco, sustainable development of tobacco production is threatened, at present, measures such as breeding disease-resistant varieties, applying chemical pesticides and comprehensive control management are mainly adopted for TMV control, but in actual production, the breeding period of disease-resistant varieties is long, the planting adaptability is not ideal, chemical control causes environmental pollution, the aim of effectively controlling TMV cannot be fundamentally achieved by agricultural measures, after pathogenic organisms, plant extracts, non-biochemical reagents and the like are found by people in the 20 th century, biochemical preparations for inducing plant to obtain systemic resistance to resist diseases are paid attention to, and proteins, polypeptides, plant protection factors, polysaccharides and the like are developed and applied in production.
Penicillium belongs to fungi, and mycelia are rich in a large amount of polysaccharide, and at present, no literature report and patent for extracting the polysaccharide of the inactivated mycelia of the penicillium chrysogenum exist, so that a polysaccharide of the inactivated mycelia of the penicillium chrysogenum and a preparation method thereof are needed to solve the problems.
Disclosure of Invention
Object of the invention
In view of the above, the invention aims to provide a penicillium chrysogenum inactivated mycelium polysaccharide, and an extraction method and application thereof, so as to realize analysis, extraction and application of specific active components in the penicillium chrysogenum inactivated mycelium polysaccharide.
(II) technical scheme
In order to achieve the technical aim, the invention provides a penicillium chrysogenum inactivated mycelium polysaccharide:
the preparation method comprises the following raw materials: the penicillium chrysogenum inactivated mycelium polysaccharide consists of mannose, galactose, glucose and rhamnose, wherein the molecular connection mode of the penicillium chrysogenum inactivated mycelium polysaccharide is an A segment-B segment-C segment, and the molecular structures of the segment A segment, the segment B segment and the segment C segment are as follows:
preferably, the total sugar content of the penicillium chrysogenum inactivated mycelium polysaccharide is 100%.
Preferably, the penicillium chrysogenum inactivated mycelium polysaccharide is a neutral sugar.
Preferably, the molecular weight of the penicillium chrysogenum inactivated mycelium polysaccharide is 19.5kDa.
The invention also provides an extraction method of the penicillium chrysogenum inactivated mycelium polysaccharide, which comprises the following steps:
step one: soaking Penicillium mycelium oven dried at high temperature in 1.6L distilled water per 100g Penicillium mycelium overnight, decocting at 100deg.C for 2-4 hr, filtering with gauze to obtain supernatant, adding distilled water with the same volume as that used in soaking, continuously decocting for 2-4 hr, filtering with gauze to obtain supernatant, mixing the two extractive solutions, concentrating to 1/20 of original volume, centrifuging at 4000rpm for 15min, discarding precipitate, adding 95% ethanol to concentrate until final concentration of ethanol is 80%, standing overnight, centrifuging at 4000rpm for 15min the next day, collecting precipitate, and freeze drying;
step two: weighing the precipitate obtained in the first step, dissolving the crude precipitate according to the proportion of adding 400mL of distilled water into each 100g, adding 95% ethanol after the precipitate is fully dissolved until the final concentration of the ethanol is 60%, precipitating the precipitate with 4 ℃ ethanol for 1.5-3h, centrifuging at 4000rpm for 15min, collecting the precipitate, washing with 95% ethanol twice, washing with absolute ethanol once, and freeze-drying the precipitate;
step three: weighing the precipitate obtained in the second step, adding a proper amount of distilled water for dissolving until the concentration of a substrate is 10mg/mL, adding trypsin according to the mass ratio of the enzyme bottom to be 4% for enzymolysis, reacting for 1h at 30-40 ℃, boiling for 10min at 100 ℃ to inactivate enzymes, centrifuging at 4000rpm for 15min, taking the supernatant, and freeze-drying overnight;
dissolving the precipitate obtained by freeze drying in the step three with distilled water of 10 times of mass, fully stirring at room temperature until the precipitate is completely dissolved, putting the sample solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 4-6 times, each time for 3 hours, collecting the solution in the dialysis bag, and freeze drying;
step five: dissolving the precipitate obtained in the step four by using distilled water with the mass being 0.5 times that of the precipitate, fully stirring at room temperature until the precipitate is completely dissolved, loading the solution to a gel chromatographic column filled with the solution completely in balance, eluting the gel chromatographic column by using 0.15M NaCl eluent at the flow rate of 0.4mL/min for 15 min/tube, sequentially eluting the solution according to the order of the molecular weight from large to small, measuring the sugar content of the sample in each test tube by using a phenol-sulfuric acid method, and collecting the sample until no sugar is detected to obtain two stages of DPS and DPS-1 solutions;
step six: placing DPS solution into dialysis bag with a retention of 3.5kDa, dialyzing in distilled water for 2-4 times, each for 3 hr, collecting solution in the dialysis bag, lyophilizing to obtain active polysaccharide of mycelium polysaccharide of Penicillium chrysogenum, and storing in a dryer.
Preferably, the time of the two boiling and extracting steps in the first step is 3 hours optimally; the optimal glycol sedimentation time in the step is 2h; the optimal reaction temperature in the third step is 37 ℃; in the fourth step, the optimal dialysis times are 5 times; in the sixth step, the dialysis frequency is preferably 3 times.
The invention also provides application of the penicillium chrysogenum inactivated mycelium polysaccharide in preventing and treating tobacco mosaic virus.
Preferably, the concentration of the penicillium chrysogenum inactivated mycelium polysaccharide for preventing tobacco mosaic virus infection caused by heart leaf smoke is 100 mug/mL.
Preferably, the concentration of the penicillium chrysogenum inactivated mycelium polysaccharide for inhibiting tobacco mosaic virus diffusion in the cultivation K326 is 100 mug/mL.
From the above technical scheme, the application has the following beneficial effects:
1: the invention provides the penicillium chrysogenum inactivated mycelium polysaccharide, and the extraction method and the application thereof, so that the active ingredients of the penicillium chrysogenum inactivated mycelium are more definite and the pharmacology is more clear.
2: the invention ensures that the method for inactivating mycelium polysaccharide by the chrysogenum is more accurate and the drug effect is easier to control in the field of preventing and controlling plant diseases, and provides an active ingredient with development prospect for preventing and controlling tobacco diseases.
Detailed Description
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses.
Example 1
The penicillium chrysogenum inactivated mycelium polysaccharide consists of mannose, galactose, glucose and rhamnose, wherein the molecular connection mode of the penicillium chrysogenum inactivated mycelium polysaccharide is an A segment-B segment-C segment, and the molecular structures of the segments A segment, B segment and C segment are as follows:
example two
An extraction method of a penicillium chrysogenum inactivated mycelium polysaccharide comprises the following steps:
step one: taking 500g of high-temperature dried penicillium mycelium, adding 8L of distilled water into 100g of yellow-producing penicillium inactivated mycelium polysaccharide according to the proportion of adding 1.6L of distilled water, soaking overnight, boiling and extracting at 100 ℃ for 2-4h the next day, filtering with gauze to keep supernatant, adding 8L of distilled water into residues, continuously boiling and extracting for 2-4h, filtering with gauze to keep supernatant, mixing the two extracts, concentrating to 800mL, centrifuging at 4000rpm for 15min, discarding the precipitate, adding 95% ethanol into the concentrate until the final concentration of the ethanol is 80%, standing overnight, centrifuging at 4000rpm for 15min the next day, and collecting the precipitate;
step two: weighing 100g of the precipitate obtained in the step one, adding 400mL of distilled water to dissolve the precipitate, adding 95% ethanol until the final concentration of the ethanol is 60%, precipitating with ethanol at 4 ℃ for 1.5-3h, centrifuging at 4000rpm for 15min, collecting the precipitate, washing twice with 200mL of 95% ethanol, washing once with 200mL of absolute ethanol, and freeze-drying the precipitate;
step three: weighing 30g of the precipitate obtained in the second step, adding 3L of distilled water to dissolve the precipitate, diluting the substrate concentration to 10mg/mL, adding trypsin according to the mass ratio of 4% of the enzyme bottom to perform enzymolysis, reacting for 1h at 30-40 ℃, boiling for 10min at 100 ℃ to inactivate the enzyme, centrifuging at 4000rpm for 15min, taking the supernatant, and freeze-drying overnight;
step four: weighing 20g of the precipitate obtained in the third step, dissolving with 200mL of distilled water, fully stirring at room temperature until the precipitate is completely dissolved, filling the sample solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 4-6 times, each time for 3 hours, collecting the solution in the dialysis bag, and freeze-drying;
step five: weighing the precipitate obtained in the step four to obtain 18g, dissolving the precipitate with 9mL of distilled water, fully stirring the solution at room temperature until the solution is completely dissolved, loading the solution to a gel chromatographic column sephadex G-50 (2.6 cm multiplied by 100 cm) filled with the gel chromatographic column sephadex G-50 completely balanced, eluting the solution with 0.15M NaCl eluent, and measuring the sugar content of a sample in each test tube by using a phenol-sulfuric acid method according to the sequence of the molecular weight from large to small at the flow rate of 0.4mL/min and 15 min/tube, taking the absorption value at 490nm as the ordinate as the curve, determining the collecting position, and collecting the solution until no sugar is detected to obtain two stages of DPS and DPS-1 solutions;
step six: placing DPS solution into dialysis bag with a retention of 3.5kDa, dialyzing in distilled water for 2-4 times, each for 3 hr, collecting solution in the dialysis bag, lyophilizing to obtain active polysaccharide of mycelium polysaccharide of Penicillium chrysogenum, and storing in a dryer.
Example III
An extraction method of a penicillium chrysogenum inactivated mycelium polysaccharide comprises the following steps:
step one: taking 500g of high-temperature dried penicillium mycelium, adding 8L of distilled water into 100g of yellow-producing penicillium inactivated mycelium polysaccharide according to the proportion of adding 1.6L of distilled water, soaking overnight, boiling and extracting for 2 hours at 100 ℃ the next day, filtering and retaining supernatant by using gauze, adding 8L of distilled water into residues, continuously boiling and extracting for 2 hours, filtering and retaining supernatant by using gauze, combining the two extracts, concentrating the volume to 800mL, centrifuging at 4000rpm for 15min, discarding the precipitate, adding 95% ethanol into the concentrate until the final concentration of the ethanol is 80%, standing overnight, centrifuging at 4000rpm for 15min the next day, and collecting the precipitate;
step two: weighing 100g of the precipitate obtained in the first step, adding 400mL of distilled water to dissolve the precipitate, adding 95% ethanol until the final concentration of the ethanol is 60%, precipitating with ethanol at 4 ℃ for 1.5h, centrifuging at 4000rpm for 15min, collecting the precipitate, washing twice with 200mL of 95% ethanol, washing once with 200mL of absolute ethanol, and freeze-drying the precipitate;
step three: weighing 30g of the precipitate obtained in the second step, adding 3L of distilled water to dissolve the precipitate, diluting the substrate concentration to 10mg/mL, adding trypsin according to the mass ratio of 4% of the enzyme bottom to perform enzymolysis, reacting for 1h at 30 ℃, boiling for 10min at 100 ℃ to inactivate the enzyme, centrifuging at 4000rpm for 15min, taking the supernatant, and freeze-drying overnight;
step four: weighing 20g of the precipitate obtained in the third step, dissolving with 200mL of distilled water, fully stirring at room temperature until the precipitate is completely dissolved, filling the sample solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 4 times, each time for 3 hours, collecting the solution in the dialysis bag, and freeze-drying;
step five: weighing the precipitate obtained in the step four to obtain 18g, dissolving the precipitate with 9mL of distilled water, fully stirring the solution at room temperature until the solution is completely dissolved, loading the solution to a gel chromatographic column sephadex G-50 (2.6 cm multiplied by 100 cm) filled with the gel chromatographic column sephadex G-50 completely balanced, eluting the solution with 0.15M NaCl eluent, and measuring the sugar content of a sample in each test tube by using a phenol-sulfuric acid method according to the sequence of the molecular weight from large to small at the flow rate of 0.4mL/min and 15 min/tube, taking the absorption value at 490nm as the ordinate as the curve, determining the collecting position, and collecting the solution until no sugar is detected to obtain two stages of DPS and DPS-1 solutions;
step six: and (3) placing DPS solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 2 times and 3 hours each time, collecting solution in the dialysis bag, freeze-drying to obtain the active polysaccharide DPS of the penicillium chrysogenum inactivated mycelium polysaccharide, and storing in a dryer for later use.
Example IV
An extraction method of a penicillium chrysogenum inactivated mycelium polysaccharide comprises the following steps:
step one: taking 500g of high-temperature dried penicillium mycelium, adding 8L of distilled water into 100g of yellow-producing penicillium inactivated mycelium polysaccharide according to the proportion of adding 1.6L of distilled water, soaking overnight, boiling and extracting for 3 hours at 100 ℃ the next day, filtering and retaining supernatant by using gauze, adding 8L of distilled water into residues, continuously boiling and extracting for 3 hours, filtering and retaining supernatant by using gauze, combining the two extracts, concentrating the volume to 800mL, centrifuging at 4000rpm for 15min, discarding the precipitate, adding 95% ethanol into the concentrate until the final concentration of the ethanol is 80%, standing overnight, centrifuging at 4000rpm for 15min the next day, and collecting the precipitate;
step two: weighing 100g of the precipitate obtained in the first step, adding 400mL of distilled water to dissolve the precipitate, adding 95% ethanol until the final concentration of the ethanol is 60%, precipitating with ethanol at 4 ℃ for 2h, centrifuging at 4000rpm for 15min, collecting the precipitate, washing twice with 200mL of 95% ethanol, washing once with 200mL of absolute ethanol, and freeze-drying the precipitate;
step three: weighing 30g of the precipitate obtained in the second step, adding 3L of distilled water to dissolve the precipitate, diluting the substrate concentration to 10mg/mL, adding trypsin according to the mass ratio of 4% of the enzyme bottom to perform enzymolysis, reacting for 1h at 37 ℃, boiling for 10min at 100 ℃ to inactivate the enzyme, centrifuging at 4000rpm for 15min, taking the supernatant, and freeze-drying overnight;
step four: weighing 20g of the precipitate obtained in the third step, dissolving with 200mL of distilled water, fully stirring at room temperature until the precipitate is completely dissolved, filling the sample solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 5 times, each time for 3 hours, collecting the solution in the dialysis bag, and freeze-drying;
step five: weighing the precipitate obtained in the step four to obtain 18g, dissolving the precipitate with 9mL of distilled water, fully stirring the solution at room temperature until the solution is completely dissolved, loading the solution to a gel chromatographic column sephadex G-50 (2.6 cm multiplied by 100 cm) filled with the gel chromatographic column sephadex G-50 completely balanced, eluting the solution with 0.15M NaCl eluent, and measuring the sugar content of a sample in each test tube by using a phenol-sulfuric acid method according to the sequence of the molecular weight from large to small at the flow rate of 0.4mL/min and 15 min/tube, taking the absorption value at 490nm as the ordinate as the curve, determining the collecting position, and collecting the solution until no sugar is detected to obtain two stages of DPS and DPS-1 solutions;
step six: and (3) placing DPS solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 3 times and 3 hours each time, collecting solution in the dialysis bag, freeze-drying to obtain the active polysaccharide DPS of the penicillium chrysogenum inactivated mycelium polysaccharide, and storing in a dryer for later use.
And comparing and analyzing the inactivated mycelium polysaccharides of the chrysogenum prepared in the second to fourth embodiments to obtain the best extraction effect of the inactivated mycelium polysaccharides of the chrysogenum obtained by processing in the fourth embodiment.
Example five
A method for extracting penicillium chrysogenum inactivated mycelium polysaccharide comprises the following steps of:
(1) The sugar content is measured by using a phenol sulfuric acid method, and 0.1g/L standard glucose is prepared by taking glucose as a standard substanceAbsorbing 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL and 0.6mL of glucose solution in glass test tubes respectively by a pipette, adding distilled water to supplement 1.0mL, repeating three samples at each concentration, adding 0.5mL of 6% phenol reagent and 2.5mL of concentrated sulfuric acid into each test tube respectively, rapidly and uniformly oscillating, cooling to room temperature, measuring absorbance A at lambda 490nm, taking absorbance A as an ordinate, and sugar content C as an abscissa, obtaining a standard curve A=0.01336C and R 2 =0.9985, preparing a dried sample into a 0.05g/L sample solution, taking 1mL of the sample solution, performing color development according to the method, measuring absorbance, taking the absorbance into a standard curve, and calculating the sugar content of the sample;
(2) The protein content is measured by using a Bio-Rad protein kit, bovine serum albumin is used as a standard substance, absorbance A at lambda 595nm is used as an ordinate, and bovine serum albumin mass C is used as an abscissa, so that a standard curve A=0.0113C and R are obtained 2 Preparing a dry sample into a sample solution of 0.5g/L, taking 1mL, operating according to a method of a Bio-Rad protein kit, measuring the absorbance, carrying into a standard curve, and calculating the protein content in the sample;
(3) The ash content is measured by a firing method, 200mg polysaccharide samples are weighed and placed in a crucible which is fired to constant weight, each polysaccharide sample is made into two groups of parallel, the crucible is transferred into a muffle furnace, the crucible is fired for 300min at 550 ℃, the crucible is cooled and weighed, the firing is repeated for multiple times, and the ash content in the polysaccharide is obtained when the difference of the front mass and the rear mass is not more than 0.001 g;
(4) Preparing DPS solution with the concentration of 0.5mg/mL, taking distilled water as a blank, placing the DPS solution in a 1cm quartz cuvette, and performing ultraviolet-visible full scanning at the wavelength of 190nm-1000nm, wherein the DPS has no absorption peak after the wavelength of 240nm and 400nm, which indicates that the DPS does not contain nucleic acid and pigment;
(5) The chemical composition of the polysaccharide sample extracted by the extraction method is 94.6 percent of polysaccharide, 2.4 percent of protein and 3.6 percent of ash.
Example six
The extraction method of the penicillium chrysogenum inactivated mycelium polysaccharide comprises the following steps of:
(1) The standard curve y= -0.1938x+0.9977 was drawn using blue dextran as a standard, an LC-10Avp high performance liquid chromatograph system manufactured by Shimadzu corporation, japan, RID-10A parallax refractive detector, a TSK-GelG-3000PWXL stainless steel chromatographic column (7.8X100 mm), a mobile phase of 0.2MNaCl aqueous solution at a column temperature of 35℃and a flow rate of 0.6 mL/min. Dissolving a dried polysaccharide sample in 0.2MNaCl to prepare a solution with the concentration of 5mg/mL, filtering, taking 20 mu L of the solution, and injecting the solution into a high performance liquid chromatography for analysis;
(2) The polysaccharide extracted by the extraction method has the molecular weight of 19.5kDa and shows uniform and symmetrical peaks.
Example seven
A method for extracting the polysaccharide of the inactivated mycelium of the chrysogenum comprises the steps of:
(1) Weighing polysaccharide sample 2mg, adding 1mL of anhydrous methanol solution containing 1M hydrochloric acid, filling N2, sealing a tube, hydrolyzing at 80 ℃ for 16 hours, drying by air pump, adding 1mL of 2M trifluoroacetic acid, hydrolyzing at 120 ℃ for 1 hour, adding a small amount of absolute ethyl alcohol, drying in water bath at 60 ℃, repeating for 3 times, and completely steaming to remove the trifluoroacetic acid. Adding 0.5mL of PMP reagent and 0.5mL of 0.3M NaOH solution into a dried sample obtained after complete acid hydrolysis, taking 0.1mL of the dried sample into a small centrifuge tube after the sample is fully dissolved, carrying out water bath at 70 ℃ for 30min, adding 0.05mL of 0.3M hydrochloric acid solution and 0.05mL of ultrapure water, fully mixing, adding 0.9mL of chloroform, extracting the residual PMP reagent after mixing, sucking the chloroform layer, reserving a water layer, centrifuging at 120000rpm for 3min, repeating for three times, and filtering with a 0.22 mu M filter membrane for later use;
(2) Feeding the prepared sample in the step (1) into a WatersHPLC system with an E2695 pump, a 2998PDA ultraviolet detector and an Agilenteclipse XDB-C18 chromatographic column (4.6X1250 mm), wherein the volume ratio is PBS (0.1M, pH 7.0), acetonitrile=82.5:17.5 is taken as a mobile phase, the flow rate is 1.0mL/min, the feeding amount is 10 mu L, and the detection wavelength is 245nm, and detecting;
(3) The polysaccharide DPS extracted by the extraction method is a galactomannan, mainly comprising mannose (Man) and galactose (Gal), and also contains a small amount of neutral polysaccharide of glucose (Glc) and rhamnose (Rha).
Example eight
The invention discloses a method for extracting a penicillium chrysogenum inactivated mycelium polysaccharide, which comprises the following steps of:
(1) Grinding a proper amount of polysaccharide powder and KBr into fine powder, pressing into transparent sheets, taking the transparent sheets pressed by KBr as blank control, placing the pressed transparent sheets into an infrared spectrum acquisition box for signal acquisition, analyzing the spectrogram, wherein DPS has an-OH telescopic vibration absorption peak at 3257cm < -1 >, 2936cm < -1 > has a C-H telescopic vibration absorption peak, the absorption peak in the region is also a characteristic sugar peak, 1407cm < -1 >, 1214cm < -1 > are absorption peaks caused by C-H angular vibration, 1058cm < -1 > is an absorption peak caused by C-O telescopic vibration, and 812cm < -1 > represents that DPS has an alpha glycosidic bond.
(2) Methylation of sugar-like: dissolving 20mg of a dried DPS sample in 2mL of dimethyl sulfoxide, filling N2 for sealing, magnetically stirring until a sugar sample is dissolved, weighing 80mg of solid NaOH and 2mL of dimethyl sulfoxide for mixing, filling N2 for sealing, fully stirring to form a uniform NaOH-DMSO suspension, adding the NaOH-DMSO suspension into the dissolved sugar sample, filling N2 for sealing, magnetically stirring for 1h, adding 4mL of methyl iodide, magnetically stirring in a dark place, placing in ice water for reaction for 30min, stopping the reaction with 2mL of distilled water, dialyzing the product for 24h with 3.5KDa dialysis bag flowing water, dialyzing with distilled water for 24h, changing water once every 4h, concentrating the dialyzate to 10mL, adding equal volume of dichloromethane, magnetically stirring, extracting for 30min, standing for layering, taking a dichloromethane layer, repeatedly extracting three times, merging the extracts, washing with distilled water for 4 times, sucking, collecting methylated polysaccharide, blowing off dichloromethane by an air pump, drying the sample, adding 1mL of distilled water for redissolving the sample, and drying the water layer in vacuum;
b. hydrolysis of polysaccharide: adding 85% formic acid 1mL into the dried methylated sugar sample, sealing, hydrolyzing at 100 ℃ for 4 hours, adding a methanol air pump to blow dry to remove formic acid until the pH=7, adding 2M trifluoroacetic acid solution 2mL, hydrolyzing at 100 ℃ for 6 hours, adding absolute ethanol air pump to blow dry to remove trifluoroacetic acid until the pH=7;
c. reduction of polysaccharide: the sugar sample was dissolved in 2mL of distilled water, and 70mg of NaBH was added 4 Stirring at room temperatureStirring, reducing for 24h, adding appropriate amount of strong acid type cation exchange resin, magnetically stirring for 2h, repeating for several times until pH=7, sucking out the solution, removing resin, adding methanol into the filtrate, drying with air pump to remove boric acid to neutrality, and vacuum drying.
d. Acetylation of polysaccharide: adding 0.5mL of acetic anhydride and 0.5mL of anhydrous pyridine respectively, filling N2, sealing, reacting for 2 hours at 100 ℃, repeatedly adding the anhydrous ethanol, drying by an air pump to remove the acetic anhydride, extracting by using dichloromethane, filtering by using a filter membrane, and performing GC-MS analysis;
DPS has various connection modes, and the main chain of DPS is supposed to be mainly composed of 1,6-Manp,1,6-Galf and 1,4-Galf, and partial substitution is carried out on Man and Gal, the substitution degree is 15.2%, wherein the O-3 position of 1,6-Manp and the O-2 position of 1,6-Galf are connected with side chains, the side chain structure mainly exists in the forms of 1,4-Glcp, t-Rhap, t-Galf and t-Manp, and in addition, the side chain structure of 1,4-Manp can be possibly also connected with branches at the O-2 position of a small amount of Man.
(3) Oxidation of periodic acid: preparation of 30mM NaIO 4 Diluting with 15mM, diluting with distilled water to 0mM, 3mM, 6mM, 9mM, 12mM, 15mM, repeating each of the concentrations three times, measuring absorbance at 223nm, drawing standard curve with NaIO4 concentration as abscissa and absorbance as ordinate, weighing DPS sample 50mg, dissolving small amount of distilled water in 50mL volumetric flask, adding 25mL 30mM NaIO 4 Distilled water is fixed to 50ml and NaIO 4 The final concentration is 15mM, tin foil is wrapped to avoid light, dark place reaction is carried out at 4 ℃, 0.1mL is taken when 0, 6, 12, 24, 48, 60, 72, 84 and 96 hours are carried out, distilled water is used for dilution 250 times, distilled water is used as blank control, the absorbance value at the wavelength of 223nm is measured, 2mL of reaction liquid in each time period is taken, 1 drop of bromocresol purple is added as an indicator, 0.005N NaOH solution is used for titration, the formic acid yield is calculated, 0.25mL of glycol is added into the reaction system to stop the reaction, the running water and distilled water are dialyzed for 24 hours respectively, the concentration is carried out to 10mL, and 70mgNaBH is added 4 The reaction was carried out overnight, the next day was carried out with 50% acetic acid to neutralize ph=7, each of the running water and distilled water was dialyzed for 24 hours, 1/3 of the reaction sample was dried and then analyzed by Gas Chromatography (GC), the amount of periodic acid consumed (mmol) = (concentration of periodic acid before reaction-concentration of periodic acid after reaction) ×reaction volume, formic acid yield (mmol) =(NaOH concentration. Times. Volume for titration/2 mL). Times.reaction volume, consumption of periodic acid was 0.245mmol in terms of periodic acid by 50mgDPS in the periodic acid oxidation reaction experiment, and 0.0682mmol of formic acid was co-produced, thus indicating consumption of periodic acid>2 Xformic acid yield, the DPS is presumed to have the presence of 1-and/or 1-6 hexoses, and only 1-2 or 1-2, 6, 1-4, 6 hexoses which consume the periodic acid to produce no formic acid, while there are several types of linkage bonds which cannot be oxidized by periodic acid such as: 1- & gt 3,6, 1- & gt 2,3, 1- & gt 2,4, 1- & gt 3,4, 1- & gt 2,3,4, and DPS mainly contains Man and ethylene glycol as a result of gas chromatography analysis, which shows that a fragment which is not oxidized by periodic acid in the DPS has a monosaccharide composition of mannose, and the mannose is in two connection modes of 1- & gt 3,6 and 1- & gt 2, 4.
(4) 13C-NMR analysis: weighing 20mgDPS, dissolving in 0.5. 0.5mLD 2 In O, the mixture was stirred at room temperature and dissolved well, and 13C-NMR spectroscopic analysis was performed on a Varian INOVA600 nuclear magnetic resonance spectrometer, and based on the galactomannan carbon spectrum chemical shift, it was assumed that mannose, galactose and glucose were present in DPS → 6) - α -DManp- (1 → 3, 6) - α -D-Manp- (1 → 6) - β -D-Galf- (1 → and → 4) - β -D-Glcp- (1- > glycosidic bond).
Specifically, the neutral polysaccharide DPS is a galactomannan mainly composed of mannose (Man) and galactose (Gal), and further contains a small amount of glucose (Glc) and rhamnose (Rha), the main chain mainly consists of 1,6-Manp,1,6-Galp and 1,4-Galp, and partial substitution occurs on both Mans and Gals with a substitution degree of 15.2%, wherein the O-3 position of 1,6-Manp and the O-2 position of 1,6-Galf are linked with side chains, the side chain structures mainly exist in the form of 1,4-Glcp, t-Rhap, t-Galf and t-Manp, and further, the side chain structures of 1,4-Manp and branches are linked at the O-2 position of a small amount of Mans.
Example nine
The invention discloses application of a penicillium chrysogenum inactivated mycelium polysaccharide, and application of the penicillium chrysogenum inactivated mycelium polysaccharide extracted by the invention in preventing and treating Tobacco Mosaic Virus (TMV):
(1) Tobacco variety heart leaf tobacco (Nicotiana glutinosa), selecting tobacco plants with consistent growth for test when 5-6 true leaves are cultivated in a greenhouse;
(2) Diluting DPS with deionized water to 15 mug/mL, 25 mug/mL, 50 mug/mL, 100 mug/mL and 110 mug/mL, respectively smearing left half leaf of tobacco, right half leaf of tobacco with deionized water, each half leaf with 100ul polysaccharide, each tobacco with 3 leaves, each 3 cigarettes with 3 parallel treatments with 3 concentrations, respectively, after the polysaccharide is smeared for 3d, inoculating TMV with a friction inoculation method, wherein the TMV concentration is 0.35mg/mL;
(3) Counting the number of dead spots of tobacco leaves at the 5d after inoculation, and calculating TMV dead spot inhibition rate, wherein the dead spot inhibition rate is = (the number of dead spots of the right half leaf control-the number of dead spots of the left half leaf treatment)/the number of dead spots of the right half leaf control multiplied by 100%;
(4) As a result, the DPS with different concentrations has the inhibition rates of 50.49%, 73.06%, 80.74%, 87.83% and 90.33% on TMV, and the polysaccharide prepared by the method has obvious effect on preventing and controlling TMV of tobacco, and the DPS has the most reasonable concentration of 100 mug/mL in preventing TMV according to the principle of economy and high efficiency.
Examples ten
The invention discloses application of a penicillium chrysogenum inactivated mycelium polysaccharide, and application of the penicillium chrysogenum inactivated mycelium polysaccharide extracted by the invention in preventing and treating K326 Tobacco Mosaic Virus (TMV):
(1) Tobacco cultivar K326 (Nicotiana tabacum K) 326, when grown in greenhouse to 5-6 true leaves, selecting consistently growing tobacco plants for testing;
(2) Diluting DPS to 100 mug/mL by deionized water, respectively smearing K326 4 th, 5 th and 6 th leaves, smearing 200 mug polysaccharide on each leaf, respectively smearing K326 4 th, 5 th and 6 th leaves by a control group by deionized water instead of polysaccharide, smearing 3 leaves on each cigarette, respectively treating 3 cigarettes, and carrying out parallel treatment on each cigarette with 3 concentrations; after smearing for 3d, inoculating TMV virus to the leaves of the DPS treatment group and the water treatment group by using a friction inoculation method, wherein the TMV concentration is 0.35mg/mL, and taking two groups of top leaves of tobacco plants for TMV virus detection respectively at 1d, 2d, 3d, 4d, 5d and 7d after inoculation;
(3) Preparation of a Standard plasmid: a pair of specific primers TMV-CP-F (5'-ACGACTGCCGAAACGTTAGA-3') and TMV-CP-R are designed according to the nucleotide sequence of tobacco mosaic virus registered in GenBank (accession number is AJ 239099)5'-CAAGTTGCAGGACCAGAGGT-3') extracting total RNA of infected tobacco leaves according to the product specification of TriPureIsolationReagent, synthesizing a cDNA first strand according to One-StepgDNARemoval reverse transcription kit, carrying out PCR amplification by using a primer TMV-CP-F, TMV-CP-R, and carrying out a PCR reaction system: cDNA 5. Mu.L, 10 XEasyTaqBuffer 5. Mu.L, 2.5mM dNTPs 1. Mu. L, TMV-CP-F1. Mu. L, TMV-CP-R1. Mu. L, easyTaqDNA polymerase 0.5. Mu.L, supplemented with ddH 2 O to a total volume of 50. Mu.L, PCR reaction conditions: 94℃1min,56℃40s,72℃1min,30 cycles; finally, the mixture is extended for 10min at 72 ℃; the PCR amplified product is connected to pEASY-T1Simple carrier, colibacillus is transformed, plasmid is extracted, sequencing is verified to be correct, then the recombinant plasmid pEASY-TMVcp of TMVcp gene is obtained, nano drop2000 is used to measure that the concentration of pEASY-TMVcp is 49.6 ng/. Mu.L, A260/A280 ratio is 1.88, and the copy number is 1.13 multiplied by 10 10 copies/μL；
(4) Standard curve establishment: performing 10-time serial gradient dilution on the recombinant plasmid pEASY-TMVcp obtained in the step (3), taking plasmids with concentration differences of 10-2, 10-3, 10-4, 10-5 and 10-6 and a blank control as TMVcp gene detection positive standards, performing fluorescence real-time quantitative RT-PCR according to a TransStartTipGrenqPCRSuperMix reagent instruction book, performing 40-cycle amplification on a Quantum studio6 fluorescence PCR instrument according to a standard curve equation of Y= 3.043X, wherein the reaction system is TipMix12.5 mu L, referenceDyeII0.5 mu L, TMV-CP-F0.26 mu mol/L, TMV-CP-R0.26 mu mol/L, cDNA mu L, adding RNAse-freeH2O to a total volume of 25 mu L, performing presoaking 30s at 94 ℃, denaturing 5s at 56 ℃, annealing for 30s at 72 ℃, collecting fluorescence, and performing 40-cycle amplification on the Quantum studio6 fluorescence instrument to obtain a standard curve equation of Y= 31.046-52X, wherein the amplification efficiency is 104%, and the correlation coefficient is 0.998;
(5) Detection of TMV virions of DPS treated K326 tobacco strain: RNA extraction and reverse transcription were performed on the sample of (2) according to the method of (3), amplification was performed according to the reaction system and procedure of (4), and the copy number of cp gene in the leaves of the water treatment group was detected to be 1.33X10 after 7 days of TMV inoculation 7 copies/. Mu.L, copy number of cp gene in DPS treatment group leaf was 3.18X10 6 The inhibition of TMV in K326 by DPS was 76.04% for copies/. Mu.L.
The exemplary implementation of the solution proposed by the present disclosure has been described in detail hereinabove with reference to the preferred embodiments, however, it will be understood by those skilled in the art that various modifications and adaptations can be made to the specific embodiments described above and that various combinations of the technical features, structures proposed by the present disclosure can be made without departing from the scope of the present disclosure, which is defined by the appended claims.
Claims (8)
1. The penicillium chrysogenum inactivated mycelium polysaccharide is characterized by comprising mannose, galactose, glucose and rhamnose, wherein the molecular connection mode of the penicillium chrysogenum inactivated mycelium polysaccharide is an A segment-B segment-C segment, and the molecular structures of the A segment, the B segment and the C segment are as follows:
the extraction method comprises the following steps:
step one: soaking Penicillium mycelium oven dried at high temperature in 1.6L distilled water per 100g Penicillium mycelium overnight, decocting at 100deg.C for 2-4 hr, filtering with gauze to obtain supernatant, adding distilled water with the same volume as that used in soaking, continuously decocting for 2-4 hr, filtering with gauze to obtain supernatant, mixing the two extractive solutions, concentrating to 1/20 of original volume, centrifuging at 4000rpm for 15min, discarding precipitate, adding 95% ethanol to concentrate until final concentration of ethanol is 80%, standing overnight, centrifuging at 4000rpm for 15min the next day, collecting precipitate, and freeze drying;
step two: weighing the precipitate obtained in the first step, dissolving the crude precipitate according to the proportion of adding 400mL of distilled water into each 100g, adding 95% ethanol after the precipitate is fully dissolved until the final concentration of the ethanol is 60%, precipitating the precipitate with 4 ℃ ethanol for 1.5-3h, centrifuging at 4000rpm for 15min, collecting the precipitate, washing with 95% ethanol twice, washing with absolute ethanol once, and freeze-drying the precipitate;
step three: weighing the precipitate obtained in the second step, adding a proper amount of distilled water for dissolving until the concentration of a substrate is 10mg/mL, adding trypsin according to the mass ratio of the enzyme bottom to be 4% for enzymolysis, reacting for 1h at 30-40 ℃, boiling for 10min at 100 ℃ to inactivate enzymes, centrifuging at 4000rpm for 15min, taking the supernatant, and freeze-drying overnight;
dissolving the precipitate obtained by freeze drying in the step three with distilled water of 10 times of mass, fully stirring at room temperature until the precipitate is completely dissolved, putting the sample solution into a dialysis bag with a retention amount of 3.5kDa, dialyzing in distilled water for 4-6 times, each time for 3 hours, collecting the solution in the dialysis bag, and freeze drying;
step five: dissolving the precipitate obtained in the step four by using distilled water with the mass being 0.5 times that of the precipitate, fully stirring at room temperature until the precipitate is completely dissolved, loading the solution to a gel chromatographic column filled with the solution completely in balance, eluting the gel chromatographic column by using 0.15M NaCl eluent at the flow rate of 0.4mL/min for 15 min/tube, sequentially eluting the solution according to the order of the molecular weight from large to small, measuring the sugar content of the sample in each test tube by using a phenol-sulfuric acid method, and collecting the sample until no sugar is detected to obtain two stages of DPS and DPS-1 solutions;
step six: placing DPS solution into dialysis bag with a retention of 3.5kDa, dialyzing in distilled water for 2-4 times, each for 3 hr, collecting solution in the dialysis bag, lyophilizing to obtain active polysaccharide of mycelium polysaccharide of Penicillium chrysogenum, and storing in a dryer.
2. The penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, wherein the total sugar content of the penicillium chrysogenum inactivated mycelium polysaccharide is 100%.
3. The penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, wherein the penicillium chrysogenum inactivated mycelium polysaccharide is a neutral sugar.
4. The penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, wherein the molecular weight of the penicillium chrysogenum inactivated mycelium polysaccharide is 19.5KDa.
5. The inactivated mycelium polysaccharide of penicillium chrysogenum according to claim 1, wherein the time of the two boiling-off steps in the first step is 3 hours; the glycol sedimentation time in the step is 2h; the reaction temperature in the third step is 37 ℃; in the fourth step, the dialysis times are 5 times; and in the step six, the dialysis times are 3 times.
6. An application of a penicillium chrysogenum inactivated mycelium polysaccharide is characterized in that: the use of a penicillium chrysogenum inactivated mycelium polysaccharide as claimed in claim 1 for the control of tobacco mosaic virus.
7. The use of a penicillium chrysogenum inactivated mycelium polysaccharide according to claim 6, wherein: the concentration of the penicillium chrysogenum inactivated mycelium polysaccharide for preventing tobacco mosaic virus infection caused by heart leaf smoke is 100 mug/mL.
8. The use of a penicillium chrysogenum inactivated mycelium polysaccharide according to claim 6, wherein: the concentration of the penicillium chrysogenum inactivated mycelium polysaccharide for inhibiting tobacco mosaic virus diffusion in cultivated tobacco K326 is 100 mug/mL.
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