阅读说明：本技术 一种亮菌菌丝体多糖及其制备方法和抑菌用途 (Armillariella tabescens mycelium polysaccharide and its preparation method and bacteriostatic use ) 是由 陈彦 张坤峰 杨宏伟 张文娜 赵弟海 赵彩莲 陈伟 李勇 黄宇哲 陈浩 于 2021-09-28 设计创作，主要内容包括：本发明公开了一种亮菌菌丝体多糖及其制备方法和抑菌用途,亮菌菌丝体多糖PAT总糖含量为93.41％,糖醛酸含量为12.24％；单糖组成及摩尔比为甘露糖:鼠李糖:半乳糖醛酸:葡萄糖:阿拉伯糖＝2.87:6.41:9.56:2.53:0.81。本发明制备的亮菌菌丝体多糖PAT对大肠杆菌、变形杆菌、枯草芽孢杆菌和金黄色葡萄球菌生长过程均具有显著的抑制作用。亮菌菌丝体多糖PAT可作为新型天然抑菌剂,在日化和医药领域有着广泛应用前景。(The invention discloses a Armillariella tabescens mycelium polysaccharide and a preparation method and bacteriostatic application thereof, wherein the Armillariella tabescens mycelium polysaccharide PAT total sugar content is 93.41%, and uronic acid content is 12.24%; the monosaccharide composition and the molar ratio of mannose, rhamnose, galacturonic acid, glucose and arabinose are 2.87:6.41:9.56:2.53: 0.81. The Armillariella tabescens mycelium polysaccharide PAT prepared by the invention has obvious inhibition effect on growth processes of escherichia coli, proteus, bacillus subtilis and staphylococcus aureus. The Armillariella tabescens mycelium polysaccharide PAT can be used as a novel natural bacteriostatic agent, and has wide application prospect in the fields of daily chemicals and medicines.)

1. A polysaccharide of mycelia of armillaria tabescens, which is characterized in that:

the polysaccharide of mycelia of Armillariella tabescens has total sugar content of 93.41%, uronic acid content of 12.24%, and molecular weight distribution range of 2.95 × 10⁴-1.07×10⁶Da。

2. The polysaccharide of mycelia of armillaria tabescens according to claim 1, wherein:

the monosaccharide composition and the molar ratio of the polysaccharide of the mycelia of the armillaria tabescens are mannose, rhamnose, galacturonic acid, glucose, arabinose and 2.87:6.41:9.56:2.53: 0.81.

3. A method for preparing the polysaccharide of the mycelium of armillaria tabescens according to claim 1 or 2, which comprises the steps of:

step 1: 4-6g Armillariella tabescens (Scop. EX. Fr) Sing strain is inoculated in 1000mL culture medium, and is subjected to static culture at 28 ℃ for 15 days to prepare an artificial liquid static fermentation armillaria mycelium;

step 2: crushing the armillaria mycelia obtained in the step 1, adding distilled water according to the liquid-material ratio of 1g:10mL, leaching at 90 ℃ for 3 hours, repeatedly leaching for 3 times, sieving with a 80-mesh sieve, and combining the extracting solutions;

and step 3: concentrating the extract obtained in step 2 to 1/250 of the original volume, adding 95% ethanol of 4 times volume, standing at 4 deg.C for 12h, centrifuging at 4500rpm for 10-15min, and collecting precipitate;

and 4, step 4: and (3) dissolving the precipitate obtained in the step (3) with distilled water, and removing protein and decoloring by a polyamide method to prepare the armillaria mycelium polysaccharide PAT.

4. The production method according to claim 3, characterized in that:

in step 1, the culture medium is glucose-potato-MgSO₄The culture medium is prepared by the following steps:

peeling 150g potato, cutting, boiling for 30min, filtering with 6 layers of gauze, adding 25g glucose and 5g MgSO₄Adding water to 1000mL, sterilizing at 121 deg.C for 20min to obtain glucose-potato-MgSO₄And (4) a culture medium.

5. The production method according to claim 3, characterized in that:

in the step 4, the specific conditions for removing protein and decoloring by adopting a polyamide method are as follows: 80g of 100-mesh 120-mesh polyamide and 300mL of precipitate solution are mixed uniformly in a 500mL shake flask, and the mixture is shaken for 30min at the room temperature and 200rpm of a shaking table to fully adsorb protein and pigment, and then filtered, collected, concentrated and freeze-dried to obtain PAT.

6. Use of the polysaccharide of the mycelium of armillaria tabescens according to claim 1 or 2, characterized in that:

the Armillariella tabescens mycelium polysaccharide is used for preparing bacteriostatic preparations, and the bacteriostatic preparations have obvious inhibiting effect on the growth of escherichia coli, proteus, bacillus subtilis and staphylococcus aureus.

7. Use according to claim 6, characterized in that:

the Armillariella tabescens mycelium polysaccharide has obvious inhibition effect on the growth of Escherichia coli, and the minimum inhibition concentration is 0.5 mg/mL.

Technical Field

The invention belongs to the field of functional products, and particularly relates to a armillariella mycelium polysaccharide, and a preparation method and an antibacterial application thereof.

Background

Armillariella tabescens (Scop. ex. Fr) Sing, also called Armillariella pseudomonad, is a special rare dual-purpose medicinal and edible fungus in China, and is named because the mycelium of the Armillariella tabescens emits light blue fluorescence in the dark. The botanical classification belongs to Agaricales, Tricholomataceae, clitocybe, contains armillarisin, armillarisin B and other active ingredients, and is widely used for treating acute and chronic hepatitis, appendicitis, otitis media and cholecystitis. The polysaccharide is used as an important secondary metabolite generated in the growth process of armillaria tabescens, and researches show that the polysaccharide has various pharmacological actions of resisting tumors, reducing blood sugar, reducing blood fat, improving insulin resistance and the like. At present, the antibacterial action of the armillariella polysaccharide is rarely reported in documents.

Disclosure of Invention

The invention aims to provide a armillariella mycelium polysaccharide, a preparation method and an antibacterial application thereof. The invention designs and optimizes the fermentation technology, prepares the armillaria tabescens polysaccharide with specific chemical composition by a polyamide method, and finds that the armillaria tabescens polysaccharide has obvious inhibiting effect on the growth of escherichia coli, proteus, bacillus subtilis and staphylococcus aureus. The armillarisin polysaccharide prepared by the invention can be widely applied to the fields of daily chemicals and medicines, such as development of natural antibacterial products of antibacterial toothpaste, shower gel, antibacterial spray and the like, and has important market application value and development prospect.

The armillaria tabescens mycelium polysaccharide, abbreviated as PAT, has 93.41% of total sugar content and 12.24% of uronic acid content; the monosaccharide consists of mannose, rhamnose, galacturonic acid, glucose and arabinose in a molar ratio of 2.87:6.41:9.56:2.53: 0.81; the molecular weight distribution range is 2.95X 10⁴-1.07×10⁶Da。

The preparation method of the armillaria mycelium polysaccharide comprises the following steps:

step 1: peeling 150g potato, cutting, boiling for 30min, filtering with 6 layers of gauze, adding 25g glucose and 5g MgSO₄Adding water to 1000mL, sterilizing at 121 deg.C for 20min to obtain glucose-potato-MgSO₄A culture medium; 4-6g Armillariella tabescens (Scop. EX. Fr.) Sing strain (provided by Hefeicheng Biopharmaceutical Co., Ltd.) was inoculated into 1000mL of glucose-potato-MgSO₄Standing and culturing at 28 deg.C for 15 days in culture medium to obtain artificial liquid, standing and fermenting mycelia of Armillariella tabescens;

step 2: crushing the armillaria mycelia (2000g) obtained in the step 1, adding distilled water according to the liquid-material ratio of 1:10(g/mL), leaching for 3 hours at 90 ℃, repeatedly leaching for 3 times, sieving with a 80-mesh sieve, and combining the extracting solutions;

and step 3: concentrating the extract obtained in step 2 to 1/250 of the original volume, adding 95% ethanol of 4 times volume, standing at 4 deg.C for 12h, centrifuging at 4500rpm for 10-15min, and collecting precipitate;

and 4, step 4: and (3) dissolving the precipitate obtained in the step (3) with distilled water, and removing protein and decoloring by a polyamide method to prepare the polysaccharide of the mycelia of the armillaria tabescens. The conditions are as follows: 80g of 100-mesh 120-mesh polyamide and 300mL of precipitate solution are mixed uniformly in a 500mL shaking flask, and the mixture is shaken for 30min at the room temperature by 200rpm of a shaking table to fully adsorb protein and pigment, then liquid is filtered, and filtrate is collected, concentrated and freeze-dried to obtain PAT.

The application of the armillariella mycelium polysaccharide is used for preparing a bacteriostatic preparation, and the bacteriostatic preparation has a remarkable inhibitory effect on the growth of escherichia coli, proteus, bacillus subtilis and staphylococcus aureus.

According to the invention, the antibacterial efficacy of the bacillus coli, the proteus, the bacillus subtilis and the staphylococcus aureus is evaluated by respectively intervening the growth of the escherichia coli, the proteus, the bacillus subtilis and the staphylococcus aureus through the Armillaria tabescens mycelium polysaccharide PAT and measuring the diameter of an inhibition zone, the minimum inhibition concentration and a growth curve of a dry prognosis.

The 1mg/mL armillariella mycelium polysaccharide PAT has the inhibition zone diameters of 25.4mm +/-0.5, 18.2mm +/-0.9, 15.23mm +/-0.4 and 13.71mm +/-0.4 for escherichia coli, proteus, bacillus subtilis and staphylococcus aureus respectively, and the inhibition zone diameter of the same-concentration chloramphenicol is 29.5mm +/-0.5.

The Minimum inhibition concentrations (Minimum inhibition concentrations) of the lucidium mycelium polysaccharide PAT to escherichia coli, proteus, bacillus subtilis and staphylococcus aureus are respectively as follows: 0.5mg/mL, -1.0mg/mL, 4.0mg/mL, 4.0 mg/mL.

The invention has the beneficial effects that: 1. optimizing a liquid standing fermentation technology by adopting a variable control method, screening a specific culture medium, and standing for 15 days at 28 ℃ for culture to obtain a mycelium with high biological yield; 2. the polyamide method replaces the traditional Sevag reagent to quickly and effectively remove protein and pigment to purify active polysaccharide, and the Armillariella tabescens mycelium polysaccharide PAT rich in specific monosaccharides such as galacturonic acid and rhamnose is obtained.

Drawings

FIG. 1 is a standard monosaccharide composition HPLC chromatogram (. about. -solvent peak, 1-mannose, 2-rhamnose, 3-glucuronic acid, 4-galacturonic acid, 5-glucose, 6-galactose, 7-arabinose, 8-fucose).

FIG. 2 is a high performance liquid chromatogram of the composition of polysaccharide PAT monosaccharide of Armillariella tabescens mycelium (x-solvent peak, 1-mannose, 2-rhamnose, 4-galacturonic acid, 5-glucose, 7-arabinose).

FIG. 3 is relative molecular mass chromatogram of PAT, obtained from Arctium tabacum mycelium polysaccharide under conditions of Agilent 1260HPLC-ELSD and TSKgel-G6000-PWxL (7.8 mm. times.30 cm, 13 μm).

FIG. 4 is the bacteriostatic plot of the mycelia polysaccharides of armillaria tabescens against E.coli, Proteus, Bacillus subtilis and Staphylococcus aureus.

FIG. 5 is a graph showing the growth inhibition of the mycelia polysaccharides of Armillaria tabescens against Escherichia coli, Proteus, Bacillus subtilis and Staphylococcus aureus.

Detailed Description

The preparation and inhibition of bacterial growth of the present invention are described below by way of specific examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.

Example 1: fermentation technology of armillaria tabescens mycelium

Step 1: peeling 150g potato, cutting, boiling for 30min, filtering with 6 layers of gauze, adding 25g glucose and 5g MgSO₄Adding water to 1000mL, sterilizing at 121 deg.C for 20min to obtain glucose-potato-MgSO₄Culture medium, 4-6g Armillariella tabescens (Scop. EX. Fr) Sing strain is inoculated on 1000mL culture medium, and static culture is carried out for 15 days at 28 ℃, so as to prepare artificial liquid static fermentation armillaria mycelium, wherein the biomass is 19.3 g/L.

Step 2: glucose-potato-MgSO obtained in step 1₄Adding 15g of agar into the culture medium to obtain a solid culture medium, and adding 4-6g of Armillariella tabescens as a strain(Scop. EX. Fr) Sing strain is inoculated on the solid culture medium, and static culture is carried out for 15 days at 28 ℃ to obtain the artificial solid static fermentation armillaria tabescens mycelium with the biomass of 13.2 g/L.

And step 3: glucose-potato-MgSO obtained in step 1₄The liquid medium was shake-cultured at 28 ℃ for 15 days on a shaker at 180rpm to produce a manual liquid shaker fermentation mycelia of Armillariella tabescens with a biomass of 17.5 g/L.

Example 2: preparation of Armillariella tabescens mycelium polysaccharide

Step 1: peeling 150g potato, cutting, boiling for 30min, filtering with 6 layers of gauze, adding 25g glucose and 5g MgSO₄Adding water to 1000mL, sterilizing at 121 deg.C for 20min to obtain glucose-potato-MgSO₄A culture medium, wherein 4-6g of Armillariella tabescens (Scop. EX. Fr) Sing strain of the armillaria tabescens is inoculated on 1000mL of culture medium, and is subjected to static culture at 28 ℃ for 15 days to prepare an artificial liquid static fermentation armillaria tabescens mycelium;

step 2: crushing the mycelia of the armillaria tabescens obtained in the step 1 by using a traditional Chinese medicine crusher, adding distilled water according to the liquid-material ratio of 1:10, leaching for 3 hours at 90 ℃, repeatedly leaching for 3 times, sieving by using a 80-mesh sieve, and combining extracting solutions;

and step 3: concentrating the extractive solution of step 2 to 1/250 of original volume, adding 95% of 4 times volume, standing at 4 deg.C for precipitation for 12h, centrifuging at 4500rpm/min for 10-15min, and collecting precipitate;

and 4, step 4: 3g of the precipitate (CAT) obtained in the step 3 is weighed and dissolved by adding distilled water, and the armillaria mycelium polysaccharide is prepared by a polyamide method, a Sevag method and an enzyme method respectively. The polyamide method: 80g of 100-mesh 120-mesh polyamide and 300mL of the precipitation solution are mixed uniformly in a 500mL shake flask; shaking at 200rpm for 30min at room temperature to adsorb protein and pigment, filtering, collecting filtrate, concentrating, and lyophilizing to obtain PAT; sevag (n-butanol: chloroform v/v ═ 1:4), repeating the operation for 3 times, taking the supernatant, centrifuging, removing the organic reagent by rotary evaporation, and freeze-drying to obtain AT; an enzyme method comprises the following steps: adding papain into the solution after dissolving the precipitate, performing water bath at 60 deg.C for 2h, inactivating at 100 deg.C for 10min, cooling to room temperature, centrifuging, and lyophilizing to obtain EAT.

And 5: chemical composition determination of PAT, AT, EAT and CAT. The total sugar content is determined by a phenol-sulfuric acid method by taking glucose as a standard substance; protein content is determined by a Coomassie brilliant blue G-250 method by taking Bovine Serum Albumin (BSA) as a standard substance; measuring the content of uronic acid by a carbazole-sulfuric acid method; measuring polyphenol content by pyrogallic acid method, and measuring absorbance at 490nm, 595nm, 520nm, and 540 nm.

The results are shown in table 1, and compared with the chemical composition of the polysaccharide obtained by the three preparation methods, the total sugar and uronic acid content of PAT is significantly higher than that of AT and EAT; and the protein and phenol contents are obviously lower than those of AT and EAT, which shows that compared with the traditional Sevag method and enzyme method, the polyamide method has the advantages that the prepared polysaccharide has high purity and less impurities.

TABLE 1 polysaccharide chemical composition

Example 3: PAT monosaccharide composition and molecular weight determination

And step 3: and (3) analyzing the monosaccharide composition of the PAT by treating a sample by an acid hydrolysis-pre-column PMP derivatization method and analyzing and determining by a high performance liquid chromatography. PAT (5mg) was weighed out and dissolved in 5mL of 2mol/L trifluoroacetic acid, and the solution was sealed with nitrogen and subjected to oil bath at 110 ℃ for 8 hours to allow sufficient hydrolysis. And (3) rotating out the water by a rotary evaporator, adding a proper amount of deionized water, repeatedly rotating and steaming until the pH value of the solution is neutral, and adding 1mL of distilled water for later use.

And 4, step 4: adding 50 μ L of 0.5mol/LPMP methanol solution and 50 μ L of 0.3mol/LNaOH solution into the standard monosaccharide and hydrolyzed sample solution, reacting in a water bath at 70 deg.C for 30min for PMP pre-column derivatization, and neutralizing with 50 μ L0.3mol/LHCl to neutrality. The obtained product is detected by high performance liquid chromatography, and a DAD detector is selected. The HPLC column temperature was 30 ℃ and the chromatography column was a Zorbox Eclipse XDB-C18 column (4.6 mm. times.250 mm, 5 μm) with a wavelength of 245 nm. Detecting that the mobile phase A is acetonitrile and the mobile phase B is 0.05mol/L phosphate buffer solution. Time gradient elution for 0-60min, initial set to mobile phase a: mobile phase B is 17%: 83%, and finally eluting until the ratio of mobile phase A to mobile phase B is 20%: 80% and the sample amount is 10 μ L.

And 5: for miningEluting with ion water, and detecting PAT component by Agilent high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). 1mL of a 2mg/mL solution of PAT and dextran standards (T5, T12, T41, T100, T200) was prepared using a TSK Gel G6000 PWXL chromatography column (300X 7.0mm, 13 μm) with N as a carrier gas₂The gas flow rate was 2.5L/min, and the sample volume was 10. mu.L. The molecular weight range measured by taking the logarithm of the relative molecular mass (LgMw) and the retention time (Rt) of the standard as standard curves is 2.95X 10⁴-1.07×10⁶Da。

Example 4: determination of antibacterial ability of Armillariella tabescens mycelium polysaccharide

PAT, AT, EAT and CAT were evaluated for the inhibitory activity against E.coli, Proteus, B.subtilis and S.aureus.

Step 1: preparation of bacterial suspension such as Escherichia coli, Bacillus proteus, Bacillus subtilis, and Staphylococcus aureus (strain purchased from ATCC and CICC). Incubating slant culture strains in an incubator for 3 hours, inoculating an LB solid culture medium in an ultra-clean workbench, and performing static culture at a constant temperature of 37 ℃; after 12h, selecting bacterial colonies on the plate in a super clean bench, inoculating the bacterial colonies on an LB liquid culture medium, and performing shake culture at the same temperature and the rotation speed of 180rpm for 8h to prepare a primary bacterial suspension.

Step 2: diluting the original bacterial suspension prepared in the step 1 to 2.0 multiplied by 10 by sterile water⁵CFU/mL。

And step 3: the antibacterial efficacy of the samples was determined by thin-layer agarose hole diffusion. And (3) uniformly coating the bacterial liquid obtained in the step (2) on the surface of a solid LB culture medium, placing a filter paper sheet with the diameter of 6mm after sterilization on the surface of the culture medium, dropwise adding 10 mu L2.0mg/mL of sample to the filter paper sheet, culturing at 37 ℃ for 18-20h, taking equivalent chloramphenicol as a positive control, taking sterile water as a blank control, and indicating that the antibacterial activity is formed by an antibacterial ring.

The result is shown in fig. 4, the PAT has strong antibacterial ability to tested strains, the inhibition effect of the PAT on escherichia coli is optimal by observing the diameter of the antibacterial ring, and the inhibition effect has no significant difference with that of the positive drug chloramphenicol.

And 4, step 4: the microbus broth method measures the MIC of samples for E.coli, Proteus, Bacillus subtilis, and Staphylococcus aureus. Sterile ringIn the field, a 96-well sterile plate is added with 2X 10 in the 1 st well⁵CFU/mL 90 μ LMH liquid medium, adding 50 μ L LB liquid medium to the 2 nd to 11 th wells; after 10. mu.L of the 8.0 mg/mL sample solution was added to the first well, the mixture was mixed well, 50. mu.L of the mixture was pipetted into the 2 nd well, and the operation was sequentially carried out to the 10 th well, and 50. mu.L of the pipette was discarded in the 10 th well. And adding 50 mu L of bacterial liquid (without medicine) into the 11 holes to serve as a blank control, co-culturing the medicine and the bacterial suspension for 6-8h, and determining the MIC of the sample to the bacteria when the light absorption value of the plate scanning under 600nm of the microplate reader is lower than 50% of that of the control group.

As shown in Table 1, the inhibitory concentration of PAT against E.coli was 0.5mg/mL, which is the minimum, and this is consistent with the inhibition zone results.

TABLE 2 minimum inhibitory concentrations of polysaccharides

And 5: 100 μ L of a suspension containing 2X 10⁵CFU/mL of the bacterial suspension and 100. mu.L of 1/2MIC drug were added to 5mL of LB liquid medium and incubated at 37 ℃ in an incubator at 180rpm for 24 h. 0. The absorbance was measured at 600nm for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hour samples.

As shown in FIG. 5, the absorbance of the PAT group was the lowest in OD value and the highest in the right-shift tendency among all the bacteria tested, indicating that the normal growth of the bacteria was inhibited, and particularly that the growth curve of E.coli was most affected. The right shift trend of the EAT, AT and CAT growth curves is gradually reduced, and compared with PAT, the growth curves have relatively weak bacteriostatic action.