CN110846233B - Strain of endophytic aspergillus of liriope hubeiensis and application thereof - Google Patents

Abstract

The invention discloses a strain of endophytic Aspergillus of ophiopogon japonicus in Hubei province and application thereof, wherein the strain of endophytic Aspergillus of ophiopogon japonicus in Hubei province is Aspergillus sp (Aspergillus sp.) fungus EF029 with a preservation number of CCTCC No: m2018781, with a preservation time of 11 months and 12 days in 2018. The strain of the endophytic aspergillus of the Hubei radix ophiopogonis provided by the invention can be applied to preparation of the Hubei radix ophiopogonis polysaccharide, so that the pollution to the environment caused by chemical synthesis in the existing preparation method of the Hubei radix ophiopogonis polysaccharide is avoided, various adverse factors in planting and production of Hubei radix ophiopogonis medicinal materials can be avoided, the strain is an important microorganism for searching new resources of the Hubei radix ophiopogonis polysaccharide, and the strain has a high application value.

Description

Strain of endophytic aspergillus of liriope hubeiensis and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a strain of endophytic aspergillus of ophiopogon japonicus in Hubei province and application thereof.

Background

The Hubei radix ophiopogonis polysaccharide is one of main active ingredients of a national geographical sign product and Hubei tract land Chinese medicinal plant-Hubei radix ophiopogonis Liriope spicata var. The Hubei ophiopogon root polysaccharide has pharmacological effects of resisting myocardial ischemia, resisting thrombosis, resisting anoxia, resisting aging, reducing blood sugar and the like, has positive effects on the aspects of improving the immune system of an organism, enhancing the gastrointestinal motility and the like, and has an anti-tumor effect according to latest research.

The traditional Hubei radix ophiopogonis polysaccharide production is mainly obtained by directly separating Hubei radix ophiopogonis root tuber extract. However, in the actual planting production of ophiopogon japonicus in Hubei, factors such as "relatively small planting area due to continuous cropping obstacle", "greatly influenced by climatic conditions", "long root tuber production period", "high extraction cost of polysaccharides" exist, and the like, so that the market release of ophiopogon japonicus polysaccharides in Hubei is limited.

Disclosure of Invention

The invention mainly aims to provide a strain of endophytic aspergillus of radix ophiopogonis in Hubei and application thereof, and aims to solve the problem that market release of radix ophiopogonis polysaccharide in Hubei is limited due to various adverse factors in planting and production of radix ophiopogonis in Hubei.

In order to achieve the purpose, the invention provides a strain of endophytic Aspergillus of ophiopogon japonicus in Hubei, wherein the strain of endophytic Aspergillus of ophiopogon japonicus in Hubei is Aspergillus sp (Aspergillus sp.) fungus EF029 with a preservation number of CCTCC No: m2018781, with a preservation time of 11 months and 12 days in 2018.

Alternatively, the strain of aspergillus endophytic bacteria of ophiopogon japonicus in north Hu as described above is obtained by the following screening method:

soaking cleaned fresh radix Ophiopogonis tuber in 75% (V/V) ethanol for 2min, and rinsing with sterile water; soaking the Hubei radix ophiopogonis tuber roots for 2min by using a sodium hypochlorite solution with the effective chlorine content of 4-6%, and finally rinsing and dipping the Hubei radix ophiopogonis tuber roots in sterile water;

cutting two brown stain parts of the treated Hubei radix ophiopogonis root tuber under the aseptic condition, cutting into small tissue slices of 0.5cm multiplied by 0.5cm, placing on a PDA culture medium (containing ampicillin with the final concentration of 50ug/mL), planting 2 slices in each dish, sealing the culture dish by a parafiLm sealing film, and placing at the constant temperature of 28 ℃ for culture;

picking up yellow green loose top hyphae of the colony growing on the periphery of the root tuber slice on the PDA culture medium, transferring the top hyphae to a fresh PDA culture medium, and performing purification culture for many times until only a single pure colony grows, namely Aspergillus sp (Aspergillus sp.) bacterium EF 029.

The strain of the endophytic aspergillus of the Hubei radix ophiopogonis obtained by the screening method can be used for producing the Hubei radix ophiopogonis polysaccharide through liquid fermentation. The Hubei radix ophiopogonis polysaccharide in the prior art is mainly obtained by directly separating Hubei radix ophiopogonis root tuber extract, and the strain of the endophytic aspergillus of Hubei radix ophiopogonis provided by the invention can be applied to preparation of the Hubei radix ophiopogonis polysaccharide, so that the pollution to the environment caused by chemical synthesis in the existing method for preparing the Hubei radix ophiopogonis polysaccharide is avoided, various adverse factors in the planting and production of Hubei radix ophiopogonis medicinal materials can be avoided, the strain is an important microorganism for searching new resources of the Hubei radix ophiopogonis polysaccharide, and the strain has a great application value.

The invention also provides an application of the strain of the endophytic aspergillus of the liriope Hubei, and the polysaccharide of the liriope Hubei is prepared by adopting the strain of the endophytic aspergillus of the liriope Hubei through liquid fermentation.

Optionally, the 18S rRNA gene sequence of the aspergillus endophytic strain of ophiopogon japonicus in north Hu is as shown in SEQ ID NO: 1 is shown.

Optionally, the preparation method of the Hubei radix ophiopogonis polysaccharide comprises the following steps:

taking the Aspergillus sp EF029, picking a small amount of hyphae with an inoculating needle under the aseptic condition, inoculating into a sterilized PDA culture medium test tube, and performing activation culture at 28 ℃ for 72 hours;

taking the activated and cultured strain, transferring the strain into a sterilized liquid potato seed culture medium under an aseptic condition, and carrying out shaking culture at the temperature of 28 ℃ and at the speed of 180rpm for 72 hours to obtain seeds;

respectively filling the prepared liquid fermentation culture medium into 250mL triangular flasks, wherein each flask is about 100mL, sterilizing, and cooling for later use; inoculating seeds according to the inoculation amount of 10% under the aseptic condition, and performing shaking culture at the temperature of 28 ℃ and the rpm of 180 for 7 days;

after fermentation, placing the fermented solid-liquid mixture in an ultra-low temperature refrigerator for quick freezing into a solid state, and performing low-temperature freeze drying treatment by using a small freeze dryer to obtain a dried fermented product;

grinding the strain fermentation product freeze-dried at low temperature into powder, respectively adding 100mL of 95% ethanol repeatedly for three times, washing the ground material, and drying;

adding distilled water into the dried sample according to a ratio of 1: 10(g/mL), carrying out water bath at 80 ℃ for 2h, filtering, and repeating the operation on the filter residue for 1 time;

mixing extractive solutions, centrifuging, collecting supernatant, and dialyzing for 48 hr;

after dialysis, concentrating under reduced pressure to obtain extract, and then carrying out alcohol precipitation on the extract by using 100mL of 95% ethanol;

and washing the alcohol precipitation product with absolute ethyl alcohol, acetone and ethyl ether in sequence, and drying to obtain the Hubei ophiopogon japonicus polysaccharide.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a morphological diagram of Aspergillus EF029 on PDA medium in example 1 of the present invention;

FIG. 2 is a positive control HPLC assay of acid hydrolyzed monosaccharides from polysaccharides extracted from Ophiopogon japonicus blocks;

FIG. 3 is a HPLC chromatogram of acid-hydrolyzed monosaccharides from polysaccharide extracts produced by the bacteria of example 2 of the present invention;

FIG. 4 shows OH and O of polysaccharide extracts produced by the bacteria in example 2 of the present invention₂ ^-DPPH, ABTS radical scavenging activity.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments.

It should be noted that those whose specific conditions are not specified in the examples were performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The existing Hubei radix ophiopogonis polysaccharide production is mainly obtained by directly separating Hubei radix ophiopogonis root tuber extract. However, in the actual planting production of ophiopogon japonicus in Hubei, factors such as "relatively small planting area due to continuous cropping obstacle", "greatly influenced by climatic conditions", "long root tuber production period", "high extraction cost of polysaccharides" exist, and the like, so that the market release of ophiopogon japonicus polysaccharides in Hubei is limited.

In view of the above, the invention provides a strain of Aspergillus endophytic in ophiopogon japonicus in Hubei, wherein the strain of Aspergillus endophytic in ophiopogon japonicus in Hubei is Aspergillus (Aspergillus sp.) EF029, and the preservation number is CCTCC No: m2018781, with a preservation time of 11 months and 12 days in 2018. Specifically, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is China, Wuhan university, and the specific address is within eight paths of Wuhan university 299 in Wuhan district, Wuhan City, Hubei province. The Aspergillus EF029 is dark yellow in the middle and white in the edge, and a single colony is circular.

The results of morphology and 18S rRNA gene sequence alignment of Aspergillus (Aspergillus sp.) EF029 are as follows:

culturing Aspergillus (Aspergillus sp.) EF029 in potato glucose agar (PDA) culture medium at 28 deg.C, wherein colony is yellow green, loose, and flat, as shown in FIG. 1; (ii) a

The 18S rRNA gene sequence of Aspergillus (Aspergillus sp.) EF029 of the present invention is shown in SEQ ID NO: 1 is shown in the specification; the sequencing results were aligned to the NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi). Homology is 100% with Aspergillus sp, HM 590656.1. Thus, the strain is of the genus Aspergillus (Aspergillus sp.) and the strain is designated Aspergillus EF 029.

The screening method of the strain of the Aspergillus endophytic bacteria in the Hubei Ophiopogon japonicus comprises the following steps:

step S10, soaking the cleaned fresh Hubei radix ophiopogonis tuber in 75% (V/V) ethanol for 2min, and rinsing with sterile water; soaking the Hubei radix ophiopogonis tuber roots for 2min by using a sodium hypochlorite solution with the effective chlorine content of 4-6%, and finally rinsing and dipping the Hubei radix ophiopogonis tuber roots in sterile water;

specifically, a large amount of tap water is used for fully cleaning soil on the surfaces of fresh roots of the liriope spicata in Hubei province, then the fresh roots of the liriope spicata are put into a clean beaker, deionized water is added, and the beaker is placed in an ultrasonic cleaner for repeated cleaning until the water after cleaning becomes extremely clear; after the water on the surface of the tuber root of ophiopogon japonicus in north of dry lake is dipped by sterile filter paper, the treatment is carried out according to the following steps: soaking root tuber in 75% (V/V) ethanol for 2min, and rinsing with sterile water for 3 times; soaking in sodium hypochlorite solution with effective chlorine content of 4-6% for 2min, continuously rinsing with a large amount of sterile water for 4 times, and dipping in water with sterile filter paper.

Step S20, cutting two ends of the browned parts of the Hubei dwarf lilyturf tuber under the aseptic condition, cutting the browned parts into small tissue slices of 0.5cm multiplied by 0.5cm, placing the small tissue slices on a PDA culture medium (containing ampicillin with the final concentration of 50ug/mL), implanting 2 slices into each dish, sealing the culture dish by a parafiniL sealing film, and placing the culture dish at the constant temperature of 28 ℃;

specifically, the surface sterilized Hubei dwarf lilyturf tuber sample is taken, two brown stain parts of the tuber are cut off under the aseptic condition, the rest part is cut into small tissue slices (transverse slices and longitudinal slices) of 0.5cm multiplied by 0.5cm by a dissecting blade, the small tissue slices are cut as thin as possible so as to be fully contacted with the surface of a culture medium, the nutrient can be absorbed by fungi from the culture medium, the small tissue slices are placed on a PDA culture medium (containing ampicillin with the final concentration of 50ug/ml), the small tissue slices are lightly pressed and pressed by tweezers uniformly to be tightly attached to a flat plate, 2 slices are planted in each dish, five times are arranged, and finally the culture dish is sealed by a parafilm and is placed at the constant temperature of 28 ℃ for culture.

S30, picking up top hyphae of a yellow green, loose and flat colony growing on the periphery of a root tuber slice on the PDA culture medium, and transferring the top hyphae to a fresh PDA culture medium for multiple times of purification culture until only a single pure colony grows, namely Aspergillus sp (Aspergillus sp.) bacterium EF 029.

Specifically, observing the growth condition of fungi of a sample every day, and growing hyphae around the small piece of the root tuber tissue after 5-7 days to form bacterial colonies with different colors, shapes and the like; picking up top hyphae of yellow green, loose and flat colony growing on the periphery of a root tuber slice on the PDA culture medium, transferring the top hyphae to a fresh PDA culture medium, carrying out purification culture for many times until only a single pure colony grows, and carrying out molecular biology classification identification to finally obtain Aspergillus EF029, which is classified and named Aspergillus sp with the preservation number of CCTCC No: and M2018781.

According to the invention, Aspergillus sp (Aspergillus sp) EF029 is obtained by the screening method, and Hubei ophiopogon japonicus polysaccharide can be produced by liquid fermentation. The Hubei radix ophiopogonis polysaccharide in the prior art is mainly obtained by directly separating Hubei radix ophiopogonis root tuber extract, and the strain of the endophytic aspergillus of Hubei radix ophiopogonis provided by the invention can be applied to preparation of the Hubei radix ophiopogonis polysaccharide, so that the pollution to the environment caused by chemical synthesis in the existing method for preparing the Hubei radix ophiopogonis polysaccharide is avoided, various adverse factors in the planting and production of Hubei radix ophiopogonis medicinal materials can be avoided, the strain is an important microorganism for searching new resources of the Hubei radix ophiopogonis polysaccharide, and the strain has a great application value.

Furthermore, the invention also provides an application of the strain of the Aspergillus endophytic bacteria in the Hubei radix ophiopogonis, and the Hubei radix ophiopogonis polysaccharide is prepared by adopting the strain of the Aspergillus endophytic bacteria in the Hubei radix ophiopogonis through liquid fermentation.

The preparation method of the Hubei ophiopogon japonicus polysaccharide comprises the following steps:

a10, taking the Aspergillus EF029, picking a small amount of hyphae with an inoculating needle under the aseptic condition, inoculating into a sterilized PDA culture medium test tube, and performing activation culture at 28 ℃ for 72 hours;

step A20, taking the activated and cultured strain, transferring the strain into a sterilized liquid potato seed culture medium under the aseptic condition, and carrying out shaking culture at 180rpm at 28 ℃ for 72 hours to obtain seeds;

step A30, respectively filling the prepared liquid fermentation culture medium into 250mL triangular flasks, wherein each flask is about 100mL, sterilizing, and cooling for later use; inoculating seeds according to the inoculation amount of 10% under the aseptic condition, and performing shaking culture at the temperature of 28 ℃ and the rpm of 180 for 7 days;

step A40, after fermentation, placing the fermented solid-liquid mixture in an ultra-low temperature refrigerator for quick freezing into a solid state, and then carrying out low-temperature freeze drying treatment by using a small freeze dryer to obtain a dried fermented product;

step A50, grinding the strain fermentation product freeze-dried at low temperature into powder, respectively adding 100mL 95% ethanol for three times, washing the ground material, and drying;

step A60, adding distilled water into the dried sample according to the ratio of 1: 10(g/mL), carrying out water bath at 80 ℃ for 2h, filtering, and repeating the operation for 1 time on filter residues;

step A70, combining the extracting solutions, centrifuging, and taking the supernatant for dialysis for 48 h;

step A80, after dialysis, concentrating under reduced pressure to obtain extract, and then carrying out alcohol precipitation on the extract by using 100mL 95% ethanol;

and step A90, washing the alcohol precipitation product by absolute ethyl alcohol, acetone and ether in sequence, and drying to obtain the Hubei ophiopogon root polysaccharide.

A specific example of fermentation preparation of Hubei radix Ophiopogonis polysaccharide is given below:

taking the Aspergillus sp EF029, picking a small amount of hyphae with an inoculating needle under the aseptic condition, inoculating into a sterilized PDA culture medium test tube, and performing activation culture at 28 ℃ for 72 hours; taking the activated and cultured strain, transferring the strain into a sterilized liquid potato seed culture medium under an aseptic condition, and carrying out shaking culture at the temperature of 28 ℃ and at the speed of 180rpm for 72 hours to obtain seeds; respectively filling the prepared liquid fermentation culture medium into triangular flasks of 250ml, wherein each flask is about 100ml, sterilizing, and cooling for later use; inoculating seeds according to the inoculation amount of 10% under the aseptic condition, and performing shaking culture at the temperature of 28 ℃ and the rpm of 180 for 7 days; after fermentation, placing the fermented solid-liquid mixture in an ultra-low temperature refrigerator for quick freezing into a solid state, and performing low-temperature freeze drying treatment by using a small freeze dryer to obtain a dried fermented product; grinding the strain fermentation product freeze-dried at low temperature into powder, respectively adding 100ml of 95% ethanol repeatedly for three times, washing the ground product, and oven-drying; adding distilled water into the dried sample according to a ratio of 1: 10(g/mL), carrying out water bath at 80 ℃ for 2h, filtering, and repeating the operation on the filter residue for 1 time; mixing extractive solutions, centrifuging, collecting supernatant, and dialyzing for 48 hr; concentrating under reduced pressure after dialysis to obtain extract, and precipitating with 100ml 95% ethanol; and washing the alcohol precipitation product with absolute ethyl alcohol, acetone and ethyl ether in sequence, and drying to obtain the Hubei ophiopogon japonicus polysaccharide.

The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.

Example 1 screening of Hubei Ophiopogon japonicus polysaccharide producing strains

(1) Fully cleaning soil on the surfaces of fresh roots of the liriope spicata in Hubei by using a large amount of tap water, then filling the fresh roots of the liriope spicata in a clean beaker, adding deionized water, and repeatedly cleaning the beaker in an ultrasonic cleaner until the water after cleaning becomes extremely clear; after the water on the surface of the tuber root of ophiopogon japonicus in north of dry lake is dipped by sterile filter paper, the treatment is carried out according to the following steps: soaking root tuber in 75% (V/V) ethanol for 2min, and rinsing with sterile water for 3 times; soaking in sodium hypochlorite solution with effective chlorine content of 4-6% for 2min, continuously rinsing with a large amount of sterile water for 4 times, and dipping in dry water with sterile filter paper;

(2) taking the radix ophiopogonis tuber sample with the sterilized surface, shearing brown stain parts at two ends of the tuber under the aseptic condition, cutting the rest part into small tissue slices (transverse slices and longitudinal slices) of 0.5cm multiplied by 0.5cm by using a dissecting blade, cutting the small tissue slices as thin as possible so that the small tissue slices can be fully contacted with the surface of a culture medium, facilitating the absorption of fungi from the culture medium, placing the small tissue slices on a PDA culture medium (containing ampicillin with the final concentration of 50ug/ml), lightly pressing the small tissue slices with forceps uniformly to ensure that the small tissue slices are tightly attached to a flat plate, planting 2 slices in each dish, setting five times of repetition, sealing the culture dish by using a parafilm sealing membrane, and placing the culture dish at the constant temperature of 28 ℃ for culture;

(3) observing the growth condition of fungi in the sample every day, and growing hyphae around the small piece of the root tuber tissue after 5-7 days to form bacterial colonies with different colors, shapes and the like; randomly picking hypha tips of the different colonies, transferring the hypha tips into a fresh PDA culture medium, carrying out purification culture for many times until only a single pure colony grows, respectively carrying out numbering and molecular biology classification identification, and finally obtaining the Aspergillus EF 029.

Example 2 preparation of Hubei Ophiopogon japonicus polysaccharides

Taking the Aspergillus EF029, picking a small amount of hyphae by using an inoculating needle under the aseptic condition, inoculating into a sterilized PDA culture medium test tube, and performing activated culture at 28 ℃ for 72 hours; taking the activated and cultured strain, transferring the strain into a sterilized liquid potato seed culture medium under an aseptic condition, and carrying out shaking culture at the temperature of 28 ℃ and at the speed of 180rpm for 72 hours to obtain seeds; respectively filling the prepared liquid fermentation culture medium into triangular flasks of 250ml, wherein each flask is about 100ml, sterilizing, and cooling for later use; inoculating seeds according to the inoculation amount of 10% under the aseptic condition, and performing shaking culture at the temperature of 28 ℃ and the rpm of 180 for 7 days; after fermentation, placing the fermented solid-liquid mixture in an ultra-low temperature refrigerator for quick freezing into a solid state, and performing low-temperature freeze drying treatment by using a small freeze dryer to obtain a dried fermented product; grinding the strain fermentation product freeze-dried at low temperature into powder, respectively adding 100ml of 95% ethanol repeatedly for three times, washing the ground product, and oven-drying; adding distilled water into the dried sample according to a ratio of 1: 10(g/mL), carrying out water bath at 80 ℃ for 2h, filtering, and repeating the operation on the filter residue for 1 time; mixing extractive solutions, centrifuging, collecting supernatant, and dialyzing for 48 hr; concentrating under reduced pressure after dialysis to obtain extract, and then precipitating with 100ml 95% ethanol; and washing the alcohol precipitation product with absolute ethyl alcohol, acetone and ethyl ether in sequence, and drying to obtain the Hubei ophiopogon japonicus polysaccharide.

Example 3 Strain identification

(1) Morphological characteristic identification of strain culture medium

The Aspergillus EF029 obtained in example 1 was streaked on a PDA medium plate, cultured at 28 ℃ for 24 hours, and observed for characteristics such as colony shape, size, color, and the like. Wherein, the PDA culture medium also comprises ampicillin with the concentration of 50 ug/ml.

The observation result is shown in figure 1, the colony of the aspergillus EF029 is yellow green, loose and flat.

(2) 18S rRNA sequencing of Aspergillus EF029

The 18S rRNA gene sequence of Aspergillus EF029 is shown as SEQ ID NO.1, and the sequencing result is subjected to sequence alignment at NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi). Homology is 100% with Aspergillus sp, HM 590656.1.

(3) Color reaction of fermentation product

The following two color reaction methods are adopted for detection:

(a) the naphthol-concentrated sulfuric acid reaction method comprises the following steps: 200ul of fermentation product solution sample is dissolved in 400ul of 15% 1-naphthol, and 100ul of concentrated sulfuric acid is added, and the sample is positive in bluish purple.

(b) The anthrone-concentrated sulfuric acid reaction method comprises the following steps: 400ul of fermentation product solution sample was added with 300ul of anthrone sulfuric acid solution, and the sample was positive in blue-green color.

The two reaction results of the polysaccharide extract produced by the Aspergillus EF029 strain are positive. I.e. indicating that the Aspergillus EF029 fermentation is capable of producing polysaccharides.

(4) Thin layer chromatography detection (TLC)

20mg of the polysaccharide extract produced by the Aspergillus EF029 strain is dissolved by adding 5ml of pure water, then 0.25ml of 12mol/L HCL is added, and the mixture is placed in an environment with the temperature of 120-126 ℃ for reaction for 4 hours. Neutralizing, dialyzing, and diluting to 10ml to obtain acidolysis solution.

Performing thin layer chromatography on acidolysis solution of polysaccharide extract produced by Aspergillus EF029 by using acidolysis product of polysaccharide extracted from Hubei radix Ophiopogonis tuber as positive control. The developing solvent is n-butyl alcohol: anhydrous ethanol: glacial acetic acid: pure water is 2:3:1:1(V/V/V/V/V), the developer is prepared by weighing 4g diphenylamine, 4ml aniline and 20ml 85% phosphoric acid dissolved in 200ml acetone, and heating in an oven at 105 ℃ for 3min until clear red spots appear.

The acidolysis monosaccharide of the polysaccharide extract produced by the Aspergillus EF029 strain has a similar Rf value to that of the positive control acidolysis monosaccharide. Namely, the Aspergillus EF029 fermentation can produce Hubei ophiopogon japonicus polysaccharide.

(5) High performance liquid chromatography detection (HPLC)

20mg of the polysaccharide extract produced by the Aspergillus EF029 strain is dissolved by adding 5ml of pure water, then 0.25ml of 12mol/L HCL is added, and the mixture is placed in an environment with the temperature of 120-126 ℃ for reaction for 4 hours. After neutralization and dialysis, the solution was diluted to 10 ml.

Placing 1mL of the acidolysis product of the polysaccharide extract produced by the Aspergillus EF029 strain in a centrifuge tube, adding 1mL of 0.5mol/mL PMP-methanol solution and 0.3mol/L NaOH solution, swirling for 1min, cooling to room temperature in 70 ℃ water bath for 60min, neutralizing with 1mL of 0.3mol/LHCL solution, extracting with 2mL of chloroform for 3 times, combining water layers, filtering with a water system filter membrane, and performing ultrasonic treatment for 0.5h before use. Meanwhile, the acidolysis product of the polysaccharide extracted from the Hubei radix ophiopogonis tuber is taken, the operation steps are repeated, and a positive control sample is prepared.

HPLC chromatographic conditions: shimadzu LC-20 high performance liquid chromatograph (detector: SPD-M20A, column oven: CTO-20A, vacuum degasser DGU-20A3, chromatographic column Inert Sustain C18); utilizing acetonitrile: isocratic elution with phosphate buffer (pH 5.0) 20:80 at a flow rate of 1 mL/min; spectral data acquisition channel: ch1(250 nm); the sample volume is 10 mu L; recording data for 30 min; signal strength is in units of mAU.

FIG. 2 is a positive control HPLC detection chart of acid hydrolysis monosaccharide of polysaccharide extracted from Hubei radix Ophiopogonis block, and FIG. 3 is a positive control HPLC detection chart of acid hydrolysis monosaccharide of polysaccharide extract produced by bacteria in example 2 of the present invention. The map comparison shows that Aspergillus EF029 can produce Hubei ophiopogon japonicus polysaccharide by fermentation.

(6) Fermentation product antioxidant assay

The alpha-deoxyribose method, the pyrogallol autoxidation method, the DPPH method and the ABTS method are adopted to respectively test the scavenging activity of polysaccharide extracts produced by Fungal bacteria EF028 on OH, O2-, DPPH and ABTS free radicals.

FIG. 4 shows the scavenging activity of polysaccharide extracts of Aspergillus EF029 on OH, O2-, DPPH, and ABTS free radicals. As can be seen from FIG. 4, the polysaccharide extract produced by Aspergillus EF029 has a certain in vitro scavenging activity on four free radicals (DPPH, O2-, OH and ABTS), and the antioxidant ability is positively correlated with the concentration.

In conclusion, the bacterial colony of the aspergillus EF029 provided by the invention is yellow green, loose and flat; homology of 100% to Aspergillus sp, HM 590656.1; the two reaction results of the polysaccharide extract produced by the Aspergillus EF029 strain are positive; the acidolysis monosaccharide of the polysaccharide extract produced by the Aspergillus EF029 has a similar Rf value with the positive control acidolysis monosaccharide, and the HPLC comparison map of the acidolysis product of the polysaccharide extract produced by the Aspergillus EF029 with the positive control Hubei ophiopogon japonicus polysaccharide shows that the Aspergillus EF029 can produce Hubei ophiopogon japonicus polysaccharide by fermentation; the Hubei ophiopogon japonicus polysaccharide produced by fermentation has certain in vitro removal activity, which shows that the Aspergillus EF029 has good performance and can be widely applied to production.

The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.

SEQUENCE LISTING

<110> Hubei academy of culture and management

<120> bacterial strain of endophytic aspergillus of liriope spicata in Hubei province and application thereof

<130> 2019.12.12

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1558

<212> DNA

<213> Aspergillus

<400> 1

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aaacctcgac ttcggaaggg gtgtatttat tagataaaaa accaatgccc ttcggggctc 180

cttggtgatt cataataact taacgaatcg catggccttg cgccggcgat ggttcattca 240

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acggggaatt agggttcgat tccggagagg gagcctgaga aacggctacc acatccaagg 360

aaggcagcag gcgcgcaaat tacccaatcc cgacacgggg aggtagtgac aataaatact 420

gatacggggc tcttttgggt ctcgtaattg gaatgagtac aatctaaatc ccttaacgag 480

gaacaattgg agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta 540

tattaaagtt gttgcagtta aaaagctcgt agttgaacct tgggtctggc tggccggtcc 600

gcctcaccgc gagtactggt ccggctggac ctttccttct ggggaacctc atggccttca 660

ctggctgtgg ggggaaccag gacttttact gtgaaaaaat tagagtgttc aaagcaggcc 720

tttgctcgaa tacattagca tggaataata gaataggacg tgcggttcta ttttgttggt 780

ttctaggacc gccgtaatga ttaataggga tagtcggggg cgtcagtatt cagctgtcag 840

aggtgaaatt cttggatttg ctgaagacta actactgcga aagcattcgc caaggatgtt 900

ttcattaatc agggaacgaa agttagggga tcgaagacga tcagataccg tcgtagtctt 960

aaccataaac tatgccgact agggatcggg cggtgtttct atgatgaccc gctcggcacc 1020

ttacgagaaa tcaaagtttt tgggttctgg ggggagtatg gtcgcaaggc tgaaacttaa 1080

agaaattgac ggaagggcac cacaaggcgt ggagcctgcg gcttaatttg actcaacacg 1140

gggaaactca ccaggtccag acaaaataag gattgacaga ttgagagctc tttcttgatc 1200

ttttggatgg tggtgcatgg ccgttcttag ttggtggagt gatttgtctg cttaattgcg 1260

ataacgaacg agacctcggc ccttaaatag cccggtccgc gtttgcgggc cgctggcttc 1320

ttagggggac tatcggctca agccgatgga agtgcgcggc aataacaggt ctgtgatgcc 1380

cttagatgtt ctgggccgca cgcgcgctac actgacaggg ccagcgagta catcaccttg 1440

gccgagaggt ccgggtaatc ttgttaaacc ctgtcgtgct ggggatagag cattgcaatt 1500

attgctcttc aacgaggaat gcctagtagg cacgagtcat cagctcgtgc cgattact 1558

Claims (2)

1. The strain of the endophytic Aspergillus of the ophiopogon japonicus in Hubei is Aspergillus (Aspergillus sp.) fungus EF029 with the preservation number of CCTCC No: m2018781, with a preservation time of 11 months and 12 days in 2018.

2. The use of the strain of Aspergillus endophytic bacteria of Ophiopogon Hubei of claim 1 in preparing polysaccharides from Ophiopogon Hubei, wherein the polysaccharides from Ophiopogon Hubei are prepared by liquid fermentation.

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