CN115583997B - Penicillium chrysogenum inactivated mycelium polysaccharide and extraction method and application thereof - Google Patents

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

A kind of Penicillium chrysogenum inactivated mycelium polysaccharide and its extraction method and application

Technical field

The invention relates to the field of Penicillium, and in particular to an inactivated mycelium polysaccharide of Penicillium chrysogenum and its extraction method and application.

Background technique

Polysaccharides have various activities such as immunomodulation, anti-tumor, anti-infection, hypolipidemia, hypoglycemia, anti-aging and anti-virus. The anti-viral activity of fungal polysaccharides has attracted more and more researchers’ attention in recent years. Fungal polysaccharides can induce plant resistance by It can prevent and treat plant virus diseases by stimulating plant immunity, inhibiting virus infection and replication, preventing and inactivating viral diseases in vitro. For example, oyster mushroom polysaccharide, enoki mushroom polysaccharide, lentinan, etc. can prevent and treat plant viruses.

The inactivated mycelium polysaccharide of Penicillium chrysogenum is the remaining by-product of Penicillium fermentation to produce penicillin. It contains 90% organic matter, 7% N, 1% P and 2% K. The inactivated mycelium polysaccharide of Penicillium chrysogenum can be Inducing plants to express disease course-related proteins has certain control effects on melon verticillium wilt, watermelon fusarium wilt, cotton fusarium wilt, tobacco black shank, tobacco brown spot and tobacco mosaic. Researchers used molecular sieve chromatography and ion exchange chromatography The active ingredients of the polysaccharide of the inactivated mycelium of Penicillium chrysogenum were separated and purified, but no good separation results were obtained. It was only speculated through the enzymatic hydrolysis test that the active ingredient of the polysaccharide of the inactivated mycelium of Penicillium chrysogenum may be protein. Or polypeptides, but some studies have found that using an aqueous extract of inactivated mycelial polysaccharides of Penicillium chrysogenum for root irrigation can prevent cotton Fusarium wilt by 51-77%, and it also has a significant control effect on cotton Verticillium wilt. , further use the part of the polysaccharide extract of the inactivated mycelium of Penicillium chrysogenum with a molecular weight greater than 2KD to be applied to crops such as grapes, apples, potatoes, onions and tomatoes, which can improve the disease resistance of these crops to various fungal diseases. , although the inactivated mycelium polysaccharide of Penicillium chrysogenum has a good control effect on various diseases of various crops, the specific active ingredients have not been isolated and the structure of the specific active ingredients has not been clarified, making The application of inactivated mycelial polysaccharides of Penicillium chrysogenum is restricted.

Tobacco mosaic virus (TMV) is called a cancer of tobacco and has threatened the sustainable development of tobacco production. At present, the prevention and control of TMV mainly adopts measures such as cultivating disease-resistant varieties, applying chemical pesticides and comprehensive prevention and management. However, in In actual production, the breeding cycle of disease-resistant varieties is long, the planting adaptability is not ideal, chemical control causes environmental pollution, and agricultural measures cannot fundamentally achieve the purpose of effectively controlling TMV. In the 1960s, with the discovery of pathogenic organisms and plant extraction After biological and non-biochemical reagents can induce plants to acquire disease resistance, biochemical agents that induce plants to acquire systemic resistance to resist diseases have attracted much attention. Proteins, polypeptides, phytoalexins and polysaccharides have been developed and applied in production.

Penicillium is a fungus, and its mycelium is rich in polysaccharides. Currently, there are no literature reports and patents on polysaccharide extraction from inactivated mycelium of Penicillium chrysogenum. Therefore, there is an urgent need for an inactivated mycelium of Penicillium chrysogenum. Body polysaccharide polysaccharide and its preparation method are provided to solve the above problems.

Contents of the invention

(1) Purpose of invention

In view of this, the purpose of the present invention is to propose a Penicillium chrysogenum inactivated mycelial polysaccharide and its extraction method and application, so as to realize the analysis of specific active ingredients in the inactivated mycelium polysaccharide of Penicillium chrysogenum. Extraction and application.

(2) Technical solutions

In order to achieve the above technical objectives, the present invention provides a Penicillium chrysogenum inactivated mycelial polysaccharide:

It includes the following raw materials: the Penicillium chrysogenum inactivated mycelium polysaccharide is composed of mannose, galactose, glucose and rhamnose, and the molecular connection mode of the Penicillium chrysogenum inactivated mycelium polysaccharide is fragment A - B fragment-C fragment, the molecular structure of the fragment A fragment, B fragment and C fragment is as follows:

Preferably, the total sugar content of the inactivated mycelial polysaccharide of Penicillium chrysogenum is 100%.

Preferably, the polysaccharide of the inactivated mycelium of Penicillium chrysogenum is a neutral sugar.

Preferably, the molecular weight of the polysaccharide from the inactivated mycelium of Penicillium chrysogenum is 19.5KDa.

The invention also provides a method for extracting polysaccharide from the inactivated mycelium of Penicillium chrysogenum, which includes the following steps:

Step 1: Take the high-temperature dried Penicillium mycelium, add 1.6L distilled water per 100g of Penicillium mycelium, soak overnight, boil and extract at 100°C for 2-4 hours the next day, filter with gauze to retain the supernatant, and add to the residue Add the same volume of distilled water as during soaking, continue to cook for 2-4 hours, filter with gauze to retain the supernatant, combine the two extracts, concentrate the volume to 1/20 of the original volume, centrifuge at 4000 rpm for 15 minutes, discard the precipitate, and add to the concentrated solution 95% ethanol until the final concentration of ethanol is 80%, let it stand overnight, centrifuge at 4000 rpm for 15 minutes the next day, collect the precipitate, and freeze-dry;

Step 2: Weigh the precipitated material obtained in step 1, and dissolve the crude precipitated material at a rate of 400 mL of distilled water per 100 g. After the precipitated material is fully dissolved, add 95% ethanol until the final ethanol concentration is 60%, and alcohol precipitate at 4°C. 1.5-3h, centrifuge at 4000rpm for 15min, collect the precipitate, wash twice with 95% ethanol and once with absolute ethanol, and freeze-dry the precipitate;

Step 3: Weigh the precipitated material obtained in Step 2, add an appropriate amount of distilled water to dissolve until the substrate concentration is 10 mg/mL, add trypsin at a ratio of 4% to enzyme base mass ratio for enzymatic hydrolysis, and react at 30-40°C for 1 hour. Boil at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 15 minutes, take the supernatant, and freeze-dry overnight;

Step 4: Dissolve the precipitate obtained by freeze-drying in step 3 with 10 times the mass of distilled water, stir thoroughly at room temperature until it is completely dissolved, put the sample solution into a dialysis bag with a cutoff of 3.5KDa, and perform dialysis in distilled water. 4-6 times, 3 hours each time, collect the solution in the dialysis bag and freeze-dry;

Step 5: Dissolve the precipitate obtained by freeze-drying in step 4 with 0.5 times the mass of distilled water, stir thoroughly at room temperature until it is completely dissolved, load it onto a gel chromatography column that has been fully balanced, and wash with 0.15M NaCl eluent. Remove, flow rate 0.4mL/min, 15min/tube, the solution will be eluted in order from large to small molecular weight, use the phenol-sulfuric acid method to measure the sugar content of the samples in each test tube, collect until no sugar is detected, and obtain two Fractions DPS and DPS-1 solutions;

Step 6: Put the DPS solution into a dialysis bag with a cut-off capacity of 3.5KDa, perform dialysis in distilled water, dialyze 2-4 times, 3 hours each time, collect the solution in the dialysis bag, and freeze-dry to obtain Penicillium chrysogenum sterilization Live mycelium polysaccharide active polysaccharide is stored in a desiccator for later use.

Preferably, the optimal cooking and extraction time in step one is 3 hours; the optimal glycol precipitation time in step one is 2 hours; the optimal reaction temperature in step three is 37°C; the optimal number of dialysis times in step four is The optimal number of times is 5 times; the optimal number of dialysis times in step 6 is 3 times.

The invention also provides an application of the inactivated mycelium polysaccharide of Penicillium chrysogenum in preventing and treating tobacco mosaic virus.

Preferably, the concentration of the inactivated mycelium polysaccharide of Penicillium chrysogenum in preventing tobacco mosaic virus infection from Nicotiana cordifolia is 100 μg/mL.

Preferably, the concentration of the inactivated mycelium polysaccharide of Penicillium chrysogenum that inhibits the spread of tobacco mosaic virus in cultivated K326 is 100 μg/mL.

It can be seen from the above technical solutions that this application has the following beneficial effects:

1: The present invention provides a polysaccharide from the inactivated mycelium of Penicillium chrysogenum and its extraction method and application, which makes the effective ingredients of the inactivated mycelium of Penicillium chrysogenum more clear and the pharmacology clearer.

2: The present invention makes the method of inactivating the mycelium polysaccharide of Penicillium chrysogenum more accurate in the field of preventing and controlling plant diseases, and the efficacy is easier to control, and provides an active ingredient with development prospects for the prevention and treatment of tobacco diseases.

Detailed ways

The following description is merely exemplary in nature and is not intended to limit the disclosure, application, and uses.

Embodiment 1

A kind of Penicillium chrysogenum inactivated mycelium polysaccharide. The Penicillium chrysogenum inactivated mycelium polysaccharide is composed of mannose, galactose, glucose and rhamnose. The molecular connection method of the Penicillium chrysogenum inactivated mycelium polysaccharide is It is fragment A-fragment B-fragment C. The molecular structures of fragment A, B and C are as follows:

Embodiment 2

A method for extracting polysaccharides from inactivated mycelium of Penicillium chrysogenum, including the following steps:

Step 1: Take 500g of high-temperature dried Penicillium mycelium, add 8L of distilled water at the ratio of 1.6L of distilled water per 100g of inactivated mycelium polysaccharide of Penicillium chrysogenum, soak overnight, and cook and extract at 100°C for 2-4 hours the next day. Filter with gauze to retain the supernatant, add 8L distilled water to the residue, continue to cook for 2-4 hours, filter with gauze to retain the supernatant, combine the two extracts, concentrate the volume to 800 mL, centrifuge at 4000 rpm for 15 min, discard the precipitate, and add 95% to the concentrated solution. % ethanol until the final concentration of ethanol is 80%, let it stand overnight, and centrifuge at 4000 rpm for 15 min the next day to collect the precipitate;

Step 2: Weigh 100g of the precipitated material obtained in step 1, add 400 mL of distilled water to dissolve the precipitate, after the precipitate is fully dissolved, add 95% ethanol until the final concentration of ethanol is 60%, ethanol precipitation at 4°C for 1.5-3 hours, and centrifuge at 4000 rpm for 15 min. , collect the precipitate, wash it twice with 200 mL 95% ethanol, wash it once with 200 mL absolute ethanol, and freeze-dry the precipitate;

Step 3: Weigh 30g of the precipitated material obtained in step 2, add 3L of distilled water to dissolve the precipitate, dilute the substrate concentration to 10 mg/mL, add trypsin at a ratio of 4% to enzyme base mass ratio for enzymatic hydrolysis, and react at 30-40°C 1h, boil at 100℃ for 10min to inactivate the enzyme, centrifuge at 4000rpm for 15min, take the supernatant and freeze-dry overnight;

Step 4: Weigh 20g of the precipitated material obtained in step 3, dissolve it in 200 mL of distilled water, and stir thoroughly at room temperature until it is completely dissolved. Put the sample solution into a dialysis bag with a cutoff capacity of 3.5KDa, and perform dialysis in distilled water for 4 seconds. -6 times, 3 hours each time, collect the solution in the dialysis bag and freeze-dry;

Step 5: Weigh 18g of the precipitate obtained in Step 4, dissolve it in 9 mL of distilled water, stir thoroughly at room temperature until it is completely dissolved, and load it onto a fully balanced gel chromatography column sephadexG-50 (2.6cm×100cm). Elute with 0.15M NaCl eluent, flow rate 0.4mL/min, 15min/tube. The solution will be eluted in order from large to small molecular weight. Use the phenol-sulfuric acid method to measure the sugar content of the sample in each test tube. Make a curve with the elution volume as the abscissa and the absorbance value at 490nm as the ordinate, determine the collection position, collect until no sugar is detected, and obtain two fractions of DPS and DPS-1 solutions;

Step 6: Put the DPS solution into a dialysis bag with a cut-off capacity of 3.5KDa, perform dialysis in distilled water, dialyze 2-4 times, 3 hours each time, collect the solution in the dialysis bag, and freeze-dry to obtain Penicillium chrysogenum sterilization Live mycelium polysaccharide active polysaccharide is stored in a desiccator for later use.

Embodiment 3

A method for extracting polysaccharides from inactivated mycelium of Penicillium chrysogenum, including the following steps:

Step 1: Take 500g of high-temperature dried Penicillium mycelium, add 8L of distilled water at the ratio of 1.6L of distilled water per 100g of inactivated mycelial polysaccharide of Penicillium chrysogenum, soak overnight, boil and extract at 100°C for 2 hours the next day, and filter with gauze Keep the supernatant, add 8L distilled water to the residue, continue to cook for 2 hours, filter with gauze to keep the supernatant, combine the two extracts, concentrate the volume to 800 mL, centrifuge at 4000 rpm for 15 min, discard the precipitate, add 95% ethanol to the concentrated solution, until The final concentration of ethanol is 80%, let it stand overnight, and centrifuge at 4000 rpm for 15 min the next day to collect the precipitate;

Step 2: Weigh 100g of the precipitated material obtained in step 1, add 400 mL of distilled water to dissolve the precipitate, and after the precipitate is fully dissolved, add 95% ethanol until the final concentration of ethanol is 60%, ethanol sediment at 4°C for 1.5 hours, and centrifuge at 4000 rpm for 15 minutes. Collect the precipitate, wash it twice with 200 mL of 95% ethanol and once with 200 mL of absolute ethanol, and freeze-dry the precipitate;

Step 3: Weigh 30g of the precipitated material obtained in Step 2, add 3L of distilled water to dissolve the precipitate, dilute the substrate concentration to 10 mg/mL, add trypsin at a ratio of 4% to enzyme base mass ratio for enzymatic hydrolysis, and react at 30°C for 1 hour. Boil at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 15 minutes, take the supernatant, and freeze-dry overnight;

Step 4: Weigh 20g of the precipitated material obtained in Step 3, dissolve it in 200 mL of distilled water, and stir thoroughly at room temperature until it is completely dissolved. Put the sample solution into a dialysis bag with a cutoff capacity of 3.5KDa, and perform dialysis in distilled water for 4 seconds. times, 3 hours each time, collect the solution in the dialysis bag and freeze-dry;

Step 5: Weigh 18g of the precipitate obtained in Step 4, dissolve it in 9 mL of distilled water, stir thoroughly at room temperature until it is completely dissolved, and load it onto a fully balanced gel chromatography column sephadexG-50 (2.6cm×100cm). Elute with 0.15M NaCl eluent, flow rate 0.4mL/min, 15min/tube. The solution will be eluted in order from large to small molecular weight. Use the phenol-sulfuric acid method to measure the sugar content of the sample in each test tube. Make a curve with the elution volume as the abscissa and the absorbance value at 490nm as the ordinate, determine the collection position, collect until no sugar is detected, and obtain two fractions of DPS and DPS-1 solutions;

Step 6: Put the DPS solution into a dialysis bag with a cutoff capacity of 3.5KDa, perform dialysis in distilled water, dialyze twice, 3 hours each time, collect the solution in the dialysis bag, and freeze-dry to obtain inactivated Penicillium chrysogenum bacteria. Silk body polysaccharide active polysaccharide DPS is stored in a desiccator for later use.

Embodiment 4

A method for extracting polysaccharides from inactivated mycelium of Penicillium chrysogenum, including the following steps:

Step 1: Take 500g of high-temperature dried Penicillium mycelium, add 8L of distilled water at the ratio of 1.6L of distilled water per 100g of inactivated mycelial polysaccharide of Penicillium chrysogenum, soak overnight, boil and extract at 100°C for 3 hours the next day, and filter with gauze Keep the supernatant, add 8L distilled water to the residue, continue to cook for 3 hours, filter with gauze to retain the supernatant, combine the two extracts, concentrate the volume to 800 mL, centrifuge at 4000 rpm for 15 min, discard the precipitate, add 95% ethanol to the concentrated solution, until The final concentration of ethanol is 80%, let it stand overnight, and centrifuge at 4000 rpm for 15 min the next day to collect the precipitate;

Step 2: Weigh 100g of the precipitated material obtained in step 1, add 400 mL of distilled water to dissolve the precipitate, after the precipitate is fully dissolved, add 95% ethanol until the final concentration of ethanol is 60%, ethanol sediment at 4°C for 2 hours, centrifuge at 4000 rpm for 15 min, and collect Precipitate, wash twice with 200 mL of 95% ethanol, once with 200 mL of absolute ethanol, and freeze-dry the precipitate;

Step 3: Weigh 30g of the precipitated material obtained in Step 2, add 3L of distilled water to dissolve the precipitate, dilute the substrate concentration to 10 mg/mL, add trypsin at a ratio of 4% to enzyme base mass ratio for enzymatic hydrolysis, and react at 37°C for 1 hour. Boil at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 15 minutes, take the supernatant, and freeze-dry overnight;

Step 4: Weigh 20g of the precipitated material obtained in step 3, dissolve it in 200 mL of distilled water, and stir thoroughly at room temperature until it is completely dissolved. Put the sample solution into a dialysis bag with a cutoff capacity of 3.5KDa, and perform dialysis in distilled water for 5 seconds. times, 3 hours each time, collect the solution in the dialysis bag and freeze-dry;

Step 5: Weigh 18g of the precipitate obtained in Step 4, dissolve it in 9 mL of distilled water, stir thoroughly at room temperature until it is completely dissolved, and load it onto a fully balanced gel chromatography column sephadexG-50 (2.6cm×100cm). Elute with 0.15M NaCl eluent, flow rate 0.4mL/min, 15min/tube. The solution will be eluted in order from large to small molecular weight. Use the phenol-sulfuric acid method to measure the sugar content of the sample in each test tube. Make a curve with the elution volume as the abscissa and the absorbance value at 490nm as the ordinate, determine the collection position, collect until no sugar is detected, and obtain two fractions of DPS and DPS-1 solutions;

Step 6: Put the DPS solution into a dialysis bag with a cut-off capacity of 3.5KDa, perform dialysis in distilled water, dialyze 3 times, 3 hours each time, collect the solution in the dialysis bag, and freeze-dry to obtain the inactivated bacteria of Penicillium chrysogenum Silk body polysaccharide active polysaccharide DPS is stored in a desiccator for later use.

Comparative analysis was conducted on the inactivated mycelium polysaccharides of Penicillium chrysogenum prepared in Examples 2 to 4, and finally it was concluded that the extraction effect of the inactivated mycelium polysaccharide of Penicillium chrysogenum processed in Example 4 was the best.

Embodiment 5

A method for extracting polysaccharides from inactivated mycelium of Penicillium chrysogenum, and analysis of the chemical composition of the extracted inactivated mycelial polysaccharides of Penicillium chrysogenum:

(1) The sugar content is measured using the phenol sulfuric acid method. Use glucose as the standard to prepare a 0.1g/L standard glucose solution. Use a pipette to pipette 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, and 0.6 respectively. mL in a glass test tube, add distilled water to make up to 1.0mL, repeat three samples for each concentration, add 0.5mL of 6% phenol reagent and 2.5mL of concentrated sulfuric acid to each test tube, shake quickly and evenly, cool to room temperature, and adjust at λ490nm Measure the absorbance A at , with the absorbance A as the ordinate and the sugar content C as the abscissa, and get the standard curve A = 0.0136C, R ² = 0.9985. Take the dry sample and prepare it into a 0.05g/L sample solution. Take 1mL according to After color development by the above method, the absorbance is measured, brought into the standard curve, and the sugar content of the sample is calculated;

(2) The protein content was measured using the Bio-Rad protein kit, with bovine serum albumin as the standard, absorbance A at λ595nm as the ordinate, bovine serum albumin mass C as the abscissa, and the standard curve A = 0.0113C , R ² =0.9967, prepare the dry sample into a 0.5g/L sample solution, take 1mL, operate according to the method of Bio-Rad protein kit, measure the absorbance, bring it into the standard curve, and calculate the protein content in the sample;

(3) Determination of ash content by burning method. Weigh 200 mg of polysaccharide sample into a crucible that has been burned to constant weight. Make two parallel sets for each sugar sample. Transfer the crucible to a muffle furnace and heat at 550°C. Burn for 300 minutes, weigh after cooling, and repeat burning multiple times until the mass difference between the two times does not exceed 0.001g, which is regarded as constant weight, which is the ash content in the polysaccharide;

(4) Prepare a DPS solution with a concentration of 0.5 mg/mL, use distilled water as a blank, place the DPS solution in a 1cm quartz cuvette, and conduct a full UV-visible scan at a wavelength of 190nm-1000nm. DPS is at a wavelength of 240nm and 400nm. There are no absorption peaks at all, indicating that DPS does not contain nucleic acids and pigments;

(5) The chemical composition of the polysaccharide sample extracted by the extraction method of the present invention is 94.6% polysaccharide, 2.4% protein, and 3.6% ash.

Embodiment 6

A method for extracting polysaccharides from the inactivated mycelium of Penicillium chrysogenum, and determination of the molecular weight of the extracted polysaccharides from the inactivated mycelium of Penicillium chrysogenum:

(1) Use blue dextran as the standard product, adopt the LC-10Avp high performance liquid chromatography system produced by Japan Shimadzu Company, RID-10A parallax refraction detector, TSK-GelG-3000PWXL stainless steel chromatographic column (7.8×300mm), The column temperature is 35°C, the mobile phase is 0.2M NaCl aqueous solution, the flow rate is 0.6mL/min, and a standard curve is drawn y=-0.1938x+0.9977. Dissolve the dried polysaccharide sample in 0.2 M NaCl to make a solution with a concentration of 5 mg/mL. After filtering, take 20 μL of the solution and inject it into high performance liquid chromatography for analysis;

(2) The polysaccharide extracted by the extraction method of the present invention has a molecular weight of 19.5KDa and presents a uniform and symmetrical peak.

Embodiment 7

A method for extracting polysaccharide from inactivated mycelium of Penicillium chrysogenum. The monosaccharide composition of the extracted polysaccharide sample of inactivated mycelium of Penicillium chrysogenum is:

(1) Weigh 2 mg of the polysaccharide sample, add 1 mL of anhydrous methanol solution containing 1 M hydrochloric acid, fill with N2, seal the tube, hydrolyze at 80°C for 16 hours, blow dry with an air pump, add 1 mL of 2M trifluoroacetic acid, and hydrolyze at 120°C for 1 hour. Add a small amount of absolute ethanol, dry in a 60°C water bath, repeat three times, and completely evaporate the trifluoroacetic acid. Add 0.5 mL of PMP reagent and 0.5 mL of 0.3 M NaOH solution to the dry sample obtained after complete acid hydrolysis. After the sample is fully dissolved, take 0.1 mL of it into a small centrifuge tube and place in a 70°C water bath for 30 min. Add 0.3 M hydrochloric acid solution. 0.05mL and 0.05mL ultrapure water, mix thoroughly, add 0.9mL chloroform, mix and extract the remaining PMP reagent, absorb the chloroform layer, retain the water layer, centrifuge at 120000rpm for 3min, repeat three times, use 0.22 After filtration with μm membrane, set aside;

(2) Inject the sample prepared in (1) into a Waters HPLC system equipped with an E2695 pump, a 2998PDA UV detector, and an Agilent Eclipse ): Acetonitrile=82.5:17.5 is the mobile phase, the flow rate is 1.0mL/min, the injection volume is 10μL, and the detection wavelength is 245nm for detection;

(3) The polysaccharide DPS extracted by the extraction method of the present invention is a galactomannan, mainly composed of mannose (Man) and galactose (Gal), and also contains a small amount of glucose (Glc) and rhamnose (Rha). Neutral polysaccharide.

Embodiment 8

A method for extracting polysaccharide from the inactivated mycelium of Penicillium chrysogenum. Analysis of the fine structure of the polysaccharide from the inactivated mycelium of Penicillium chrysogenum extracted by the present invention:

(1) Take an appropriate amount of polysaccharide powder and KBr and put it into a mortar and grind it into fine powder, press it into a transparent sheet, use the transparent sheet pressed by KBr as a blank control, put the pressed transparent sheet into the infrared spectrum collection box for signal detection Collection and spectrum analysis: DPS has an -OH stretching vibration absorption peak at 3257cm-1 and a C-H stretching vibration absorption peak at 2936cm-1. The absorption peaks in this area are also characteristic peaks of sugars. 1407cm-1 and 1214cm-1 are C-H changing angle vibrations. The absorption peak caused by 1058cm-1 is the absorption peak caused by C-O stretching vibration, and 812cm-1 indicates that DPS has α-glycosidic bond.

(2) a. Methylation of sugar sample: Take 20 mg of dry DPS sample, dissolve it in 2 mL of dimethyl sulfoxide, seal with N2, stir magnetically until the sugar sample dissolves, weigh 80 mg of solid NaOH and 2 mL of dimethyl sulfoxide Mix with sulfoxide, seal with N2, stir thoroughly to make it become a uniform NaOH-DMSO suspension, add the NaOH-DMSO suspension to the dissolved sugar sample, seal with N2, stir magnetically for 1 hour, add 4 ml of methyl iodide, avoid Stir with a light-sealed magnetic force, place in ice water for 30 min, and stop the reaction with 2 mL of distilled water. The product is dialyzed with a 3.5 KDa dialysis bag for 24 h and distilled water for 24 h. The water is changed every 4 h. The above dialysate is concentrated to 10 mL, and an equal volume of dichloride is added. Methane, magnetically stir, extract for 30 minutes, let stand for layering, take the methylene chloride layer, repeat the extraction three times, combine the extracts, wash with distilled water 4 times, absorb the water layer, collect the methylated polysaccharide, and blow out the methylene chloride with an air pump , until the sample is dry, add 1 mL of distilled water to reconstitute the sample, and dry under vacuum;

b. Hydrolysis of polysaccharides: Add 1 mL of 85% formic acid to the above dry methylated sugar sample, seal with N2, hydrolyze at 100°C for 4 hours, add methanol and air pump to dry to remove formic acid until pH=7, then add 2M trifluoride 2 mL of acetic acid solution, hydrolyze at 100°C for 6 hours, add absolute ethanol and blow dry with air pump to remove trifluoroacetic acid to pH=7;

c. Reduction of polysaccharides: Dissolve the sugar sample in 2 mL of distilled water, add 70 mg NaBH ₄ , stir at room temperature, reduce for 24 hours, add an appropriate amount of strong acid cation exchange resin, stir magnetically for 2 hours, repeat several times until pH=7, aspirate the solution, and remove the resin. , add methanol to the filtrate, blow dry with an air pump to remove boric acid until it becomes neutral, and dry under vacuum.

d. Acetylation of polysaccharides: Add 0.5 mL each of acetic anhydride and anhydrous pyridine, seal with N2, react at 100°C for 2 hours, repeatedly add absolute ethanol and blow dry with an air pump to remove acetic anhydride, extract with dichloromethane, and filter with a membrane. GC-MS analysis;

e.DPS has a variety of connection methods. It is speculated that its main chain is mainly composed of 1,6-Manp, 1,6-Galf and 1,4-Galf, and partial substitution occurs on both Man and Gal, with a degree of substitution of 15.2%. , where 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 is mainly composed of 1,4-Glcp, t-Rhap, t-Galf and t- It exists in the form of Manp. In addition, there may be a side chain structure of 1,4-Manp, and there is a branch at the O-2 position of a small amount of Man.

(3) Periodic acid oxidation: Prepare 30mM NaIO ₄ and dilute it to 15mM, then dilute it with distilled water to 0mM, 3mM, 6mM, 9mM, 12mM, 15mM, each concentration is repeated three times, measure the absorbance value at 223nm, and use the NaIO4 concentration is the abscissa, the absorbance value is the ordinate, draw a standard curve, weigh 50mg of DPS sample, dissolve a small amount of distilled water in a 50mL volumetric flask, add 25mL of 30mM NaIO ₄ , adjust the volume to 50ml with distilled water, the final concentration of NaIO ₄ is 15mM, tin foil Wrap to protect from light and react in the dark at 4°C. Take 0.1ml at 0, 6, 12, 24, 48, 60, 72, 84, and 96 hours, dilute 250 times with distilled water, use distilled water as a blank control, and measure the absorbance value at a wavelength of 223 nm. , take 2 mL of the reaction solution in each of the above time periods, add 1 drop of bromocresol purple as an indicator, titrate with 0.005 NNaOH solution, calculate the amount of formic acid generated, add 0.25 mL of ethylene glycol to the reaction system to terminate the reaction, and add 0.25 mL of ethylene glycol to the reaction system. Dialyze for 24 hours, concentrate to 10 mL, add 70 mg NaBH ₄ for reduction overnight, neutralize with 50% acetic acid at pH=7 the next day, dialyze with running water and distilled water for 24 hours each, take 1/3 of the reaction sample and dry it for gas chromatography (GC) analysis. , the amount of periodic acid consumed (mmol) = (concentration of periodic acid before reaction - concentration of periodic acid after reaction) × reaction volume, the amount of formic acid generated (mmol) = (concentration of NaOH × volume for titration/2mL) × reaction Volume, according to the periodic acid oxidation reaction experiment, 50mg DPS consumes 0.245mmol of periodic acid and generates 0.0682mmol of formic acid. It can be seen that the consumption of periodic acid is >2×the amount of formic acid generated. It is speculated that DPS has 1→ and/or 1→6 hexoses. The existence of radicals, as well as the 1→2 or 1→2,6, 1→4, 1→4,6 hexosyl groups that only consume periodic acid without producing formic acid, and there are several connecting bonds that cannot be oxidized by periodic acid. Types such as: 1→3, 1→3,6, 1→2,3, 1→2,4, 1→3,4, 1→2,3,4. Gas chromatography analysis results show that DPS mainly contains Man and B Diol, indicating that the fragment in DPS that is not oxidized by periodic acid has a monosaccharide composition of mannose, and the mannose has two connection modes: 1→3,6 and 1→2,4.

(4) 13C-NMR analysis: Weigh 20mg DPS, dissolve it in 0.5mLD ₂ O, stir at room temperature to fully dissolve, conduct 13C-NMR spectrum analysis on a VarianINOVA600 nuclear magnetic resonance spectrometer, according to the galactomannan carbon spectrum chemical shift, It is speculated that mannose, galactose and glucose in DPS exist →6)-α-DManp-(1→, →3,6)-α-D-Manp-(1→, α-D-Manp-(1→, →6)-β-D-Galf-(1→, 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 also contains a small amount of glucose (Glc) and rhamnose (Rha). The main chain is mainly Composed of 1,6-Manp, 1,6-Galp and 1,4-Galp, and partial substitution occurs on both Man and Gal, with a degree of substitution of 15.2%, in which the O-3 position of 1,6-Manp and 1 , There is a side chain at the O-2 position of 6-Galf. The side chain structure mainly exists in the form of 1,4-Glcp, t-Rhap, t-Galf and t-Manp. In addition, there may be 1,4-Manp. The side chain structure has a branch at the O-2 position of a small amount of Man.

Embodiment 9

An application of the inactivated mycelial polysaccharide of Penicillium chrysogenum, the application of the inactivated mycelial polysaccharide of Penicillium chrysogenum extracted in the present invention in the prevention and treatment of tobacco mosaic virus (TMV):

(1) For the tobacco variety Nicotiana glutinosa, when 5-6 true leaves are cultivated in the greenhouse, tobacco plants with consistent growth are selected for testing;

(2) DPS was diluted with deionized water to 15 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 110 μg/mL, and applied to the left half of the tobacco leaves, and the right half of the leaves with deionized water, and applied to each half of the leaves. Apply 100ul of polysaccharide to 3 leaves of each tobacco plant, 3 tobacco plants in each treatment, and 3 parallel treatments of each concentration. 3 days after applying polysaccharide, inoculate TMV with friction inoculation, and the TMV concentration is 0.35 mg/mL;

(3) Count the number of dead spots on tobacco leaves on the 5th day after inoculation, and calculate the TMV dead spot inhibition rate. The dead spot inhibition rate % = (the number of control dead spots on the right half of the leaf - the number of treated dead spots on the left half of the leaf) / the right half of the leaf Control number of dead spots×100%;

(4) Results: The inhibition rates of different concentrations of DPS on TMV dead spots were 50.49%, 73.06%, 80.74%, 87.83% and 90.33%, proving that the polysaccharide prepared by the present invention has a significant effect on preventing and controlling tobacco TMV. According to the principle of economical and efficient DPS The most reasonable concentration for preventing TMV is 100 μg/mL.

Embodiment 10

An application of the inactivated mycelial polysaccharide of Penicillium chrysogenum, the application of the inactivated mycelial polysaccharide of Penicillium chrysogenum extracted in the present invention in the prevention and treatment of K326 Tobacco Mosaic Virus (TMV):

(1) Tobacco cultivar K326 (Nicotiana tabacum K326), when 5-6 true leaves are cultivated in the greenhouse, tobacco plants with consistent growth are selected for testing;

(2) DPS was diluted with deionized water to 100 μg/mL, and applied to the 4th, 5th, and 6th leaves of K326 respectively. Each leaf was smeared with 200 μg polysaccharide. In the control group, deionized water was used instead of polysaccharide to smear the 4th, 5th leaves of K326 respectively. , on 6 leaves, apply 3 leaves of each tobacco plant, 3 tobacco plants of each treatment, and 3 parallel treatments of each concentration; after applying for 3 days, use friction inoculation method to inoculate TMV virus into the DPS treatment group and water treatment group. On the leaves, the TMV concentration was 0.35 mg/mL. The top leaves of the two groups of tobacco plants were taken for TMV virus detection on 1d, 2d, 3d, 4d, 5d, and 7d after inoculation;

(3) Preparation of standard plasmid: Design a pair of specific primers TMV-CP-F (5′-ACGACTGCCGAAACGTTAGA-3′), TMV- CP-R (5′-CAAGTTGCAGGACCAGAGGT-3′), extract total RNA from susceptible tobacco leaves according to the product instructions of TriPureIsolationReagent, synthesize the first strand of cDNA according to the One-StepgDNARemoval reverse transcription kit, and use primers TMV-CP-F, TMV -CP-R performs PCR amplification, PCR reaction system: cDNA 5μL, 10×EasyTaqBuffer 5μL, 2.5mMdNTPs 1μL, TMV-CP-F1μL, TMV-CP-R1μL, EasyTaq DNA polymerase 0.5μL, supplement ddH ₂ O to a total volume of 50μL, PCR Reaction conditions: 94°C for 1 minute, 56°C for 40 seconds, 72°C for 1 minute, 30 cycles; final extension at 72°C for 10 minutes; the PCR amplification product is connected to the pEASY-T1Simple vector, transformed into E. coli, the plasmid is extracted, and the sequence is verified to be correct. The recombinant plasmid pEASY-TMVcp of the TMVcp gene, the concentration of pEASY-TMVcp measured with NanoDrop2000 is 49.6ng/μL, the A260/A280 ratio is 1.88, and the copy number is 1.13×10 ¹⁰ copies/μL;

(4) Standard curve establishment: Carry out 10-fold serial gradient dilution of the recombinant plasmid pEASY-TMVcp obtained in (3), and use plasmids with concentration differences of 10-2, 10-3, 10-4, 10-5, and 10-6. The blank control was used as a positive standard for TMVcp gene detection. Fluorescence real-time quantitative RT-PCR was performed according to the TransStartTipGreenqPCRSuperMix reagent instructions. The reaction system was TipMix 12.5μL, ReferenceDyeII0.5μL, TMV-CP-F0.26μmol/L, TMV-CP-R0.26μmol. /L, cDNA 2 μL, add RNAse-freeH2O to supplement the total volume to 25 μL, follow the program to pre-denature at 94°C for 30 seconds, denature at 94°C for 5 seconds, anneal at 56°C for 30 seconds, extend at 72°C for 30 seconds and collect fluorescence, and perform 40 cycles on the QuantStudio6 fluorescence PCR instrument After cyclic amplification, the standard curve equation was obtained as Y=31.046-3.043X, the amplification efficiency was 104%, and the correlation coefficient was 0.998;

(5) Detection of TMV virus particles in DPS-treated K326 tobacco strain: Extract the RNA and reverse-transcribe the sample from (2) according to the method in (3), and amplify it according to the reaction system and procedure in (4). Results Seven days after inoculation with TMV, the copy number of the cp gene in the leaves of the water treatment group was 1.33×10 ⁷ copies/μL, and the copy number of the cp gene in the leaves of the DPS treatment group was 3.18×10 ⁶ copies/μL. Inhibition of TMV in K326 by DPS The rate is 76.04%.

The exemplary implementations of the solutions proposed by the present disclosure have been described in detail above with reference to preferred embodiments. However, those skilled in the art can understand that, without departing from the concept of the present disclosure, the above specific embodiments can be modified. Various variations and modifications are possible, and the various technical features and structures proposed in this disclosure can be combined in various ways without exceeding the scope of protection of this disclosure, which is determined by the appended claims.

Claims (8)

1. A kind of Penicillium chrysogenum inactivated mycelium polysaccharide, it is characterized in that, the described Penicillium chrysogenum inactivated mycelium polysaccharide is composed of mannose, galactose, glucose and rhamnose, and the described Penicillium chrysogenum inactivated mycelium polysaccharide is composed of mannose, galactose, glucose and rhamnose. The connection mode of polysaccharide molecules of mold inactivated mycelium is fragment A-fragment B-fragment C. The molecular structures of fragment A, fragment B and fragment C are as follows: Its extraction method includes the following steps: Step 1: Take the high-temperature dried Penicillium mycelium, soak it overnight at the ratio of 1.6L distilled water per 100g of Penicillium mycelium, boil and extract it at 100°C for 2-4 hours the next day, filter it with gauze, retain the supernatant, and add it to the residue. Add the same volume of distilled water as during soaking, continue to cook for 2-4 hours, filter with gauze to retain the supernatant, combine the two extracts, concentrate the volume to 1/20 of the original volume, centrifuge at 4000 rpm for 15 minutes, discard the precipitate, and add to the concentrated solution 95% ethanol until the final concentration of ethanol is 80%, let it stand overnight, centrifuge at 4000 rpm for 15 minutes the next day, collect the precipitate, and freeze-dry; Step 2: Weigh the precipitated material obtained in step 1, and dissolve the crude precipitated material at a rate of 400 mL of distilled water per 100 g. After the precipitated material is fully dissolved, add 95% ethanol until the final ethanol concentration is 60%, and alcohol precipitate at 4°C. 1.5-3h, centrifuge at 4000rpm for 15min, collect the precipitate, wash twice with 95% ethanol and once with absolute ethanol, and freeze-dry the precipitate; Step 3: Weigh the precipitated material obtained in Step 2, add an appropriate amount of distilled water to dissolve until the substrate concentration is 10 mg/mL, add trypsin at a ratio of 4% to enzyme base mass ratio for enzymatic hydrolysis, and react at 30-40°C for 1 hour. Boil at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 15 minutes, take the supernatant, and freeze-dry overnight; Step 4: Dissolve the precipitate obtained by freeze-drying in step 3 with 10 times the mass of distilled water, stir thoroughly at room temperature until it is completely dissolved, put the sample solution into a dialysis bag with a cutoff of 3.5KDa, and perform dialysis in distilled water. 4-6 times, 3 hours each time, collect the solution in the dialysis bag and freeze-dry; Step 5: Dissolve the precipitate obtained by freeze-drying in step 4 with 0.5 times the mass of distilled water, stir thoroughly at room temperature until it is completely dissolved, load it onto a gel chromatography column that has been fully balanced, and wash with 0.15M NaCl eluent. Remove, flow rate 0.4mL/min, 15min/tube, the solution will be eluted in order from large to small molecular weight, use the phenol-sulfuric acid method to measure the sugar content of the samples in each test tube, collect until no sugar is detected, and obtain two Fractions DPS and DPS-1 solutions; Step 6: Put the DPS solution into a dialysis bag with a cut-off capacity of 3.5KDa, perform dialysis in distilled water, dialyze 2-4 times, 3 hours each time, collect the solution in the dialysis bag, and freeze-dry to obtain Penicillium chrysogenum sterilization Live mycelium polysaccharide active polysaccharide is stored in a desiccator for later use. 2. A kind of Penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, characterized in that the total sugar content of the Penicillium chrysogenum inactivated mycelium polysaccharide is 100%. 3. A kind of Penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, characterized in that the said Penicillium chrysogenum inactivated mycelium polysaccharide is a neutral sugar. 4. A kind of Penicillium chrysogenum inactivated mycelial polysaccharide according to claim 1, characterized in that the molecular weight of the said Penicillium chrysogenum inactivated mycelial polysaccharide is 19.5KDa. 5. A kind of Penicillium chrysogenum inactivated mycelium polysaccharide according to claim 1, characterized in that, the two cooking and extraction times in the step one are both 3h; the glycol precipitation time of the step is 2h; In step three, the reaction temperature is 37°C; in step four, the number of dialysis times is 5 times; in step six, the number of dialysis times is 3 times. 6. An application of the inactivated mycelial polysaccharide of Penicillium chrysogenum, characterized in that the application of the inactivated mycelial polysaccharide of Penicillium chrysogenum as claimed in claim 1 in the prevention and treatment of tobacco mosaic virus. 7. The application of a kind of Penicillium chrysogenum inactivated mycelium polysaccharide according to claim 6, characterized in that: the Penicillium chrysogenum inactivated mycelium polysaccharide prevents tobacco mosaic virus from being infected by Nicotiana cordifolia. The concentration is 100μg/mL. 8. The application of a kind of Penicillium chrysogenum inactivated mycelium polysaccharide according to claim 6, characterized in that: the Penicillium chrysogenum inactivated mycelium polysaccharide inhibits the spread of tobacco mosaic virus in cultivated tobacco K326 The concentration is 100μg/mL.