CN110804558B - Endophytic penicillium strain of ophiopogon japonicas and application thereof - Google Patents

Endophytic penicillium strain of ophiopogon japonicas and application thereof Download PDF

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CN110804558B
CN110804558B CN201911296804.XA CN201911296804A CN110804558B CN 110804558 B CN110804558 B CN 110804558B CN 201911296804 A CN201911296804 A CN 201911296804A CN 110804558 B CN110804558 B CN 110804558B
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余海忠
郑炜炜
张得祥
田文清
于博
王海燕
黄升谋
阎华�
孙永林
李云捷
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Hubei University of Arts and Science
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Abstract

The invention discloses an endophytic Penicillium sp strain of ophiopogon japonicus and application thereof, wherein the endophytic Penicillium sp strain of ophiopogon japonicus is Penicillium EF010 with a preservation number of CCTCC NO of M2018778 and a preservation time of 11 months and 12 days in 2018. The endophytic Penicillium sp.train EF010 provided by the invention is obtained by separating and screening fresh ophiopogon japonicus plant root tuber through the steps of culturing, screening, purifying and the like, and can be metabolized by itself to generate the Hubei ophiopogon japonicus polysaccharide, so that the Hubei ophiopogon japonicus polysaccharide can be prepared by microbial liquid fermentation.

Description

Endophytic penicillium strain of ophiopogon japonicas and application thereof
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a paecilomyces endophytic strain of ophiopogon japonicus and application thereof.
Background
The Hubei radix ophiopogonis polysaccharide is one of main active ingredients of a national geographical sign product and a Hubei tract Chinese medicinal plant-Xiang radix ophiopogonis, namely Hubei radix ophiopogonis Liriope spicata var. prolifera Y.T.Ma tuberous root, and research shows that: the Hubei ophiopogon root polysaccharide has pharmacological effects in aspects of myocardial ischemia resistance, thrombosis resistance, hypoxia tolerance, aging resistance, blood sugar reduction and the like, and has positive effects on aspects of improving the immune system of an organism, improving the gastrointestinal motility and the like, and the latest research shows that the Hubei ophiopogon root polysaccharide also has an anti-tumor effect.
The existing Hubei radix ophiopogonis polysaccharide production is mainly obtained by directly separating root tuber extract. However, in the actual production of the ophiopogon japonicus polysaccharide in Hubei, the problems of relatively small planting area caused by continuous cropping obstacles, large influence caused by climatic conditions, long root tuber production period, high extraction cost of the ophiopogon japonicus polysaccharide in Hubei and the like exist, so that the market of the ophiopogon japonicus polysaccharide in Hubei is limited. Therefore, a preparation method of Hubei ophiopogon japonicus polysaccharide with less interference factors and stable yield is needed.
Disclosure of Invention
The invention mainly aims to provide a penicillium strain produced from ophiopogon japonicus and application thereof, and aims to provide a penicillium strain capable of stably producing large quantities of Hubei ophiopogon japonicus polysaccharide.
In order to achieve the purpose, the invention utilizes the dwarf lilyturf tuber produced in GAP base of the European temple Zhenwei dwarf lilyturf tuber in Xianyang province in Hubei to separate and purify a Penicillium sp.strain EF010 from living root tubers. The endophytic penicillium of ophiopogon has been preserved in China center for type culture Collection (CCTCC for short), and the preservation addresses are as follows: wuhan, Wuhan university, classified as Penicillium sp.strain EF010, with the preservation number of CCTCC NO: M2018778, and the preservation time of 2018, 11 months and 12 days.
Optionally, the 18R sRNA gene sequence of the penicillium stipiti endophytic strain is as shown in SEQ id no: 1 is shown.
The invention also provides a method for separating and purifying the Penicillium sp.strain EF010, which comprises the following steps:
cleaning fresh radix ophiopogonis tuber, sequentially and respectively soaking the fresh radix ophiopogonis tuber in 70% ethanol and 4% -6% sodium hypochlorite solution by volume fraction, and removing surface water to obtain the clean radix ophiopogonis tuber, wherein the soaking time is 1-3 min;
removing brown stain parts from the clean ophiopogon root tuber, cutting the clean ophiopogon root tuber into slices, placing the slices on a PDA culture medium, and performing sealed culture at 25-30 ℃ for 5-7 days until hyphae grow on the periphery of the slices to form a plurality of bacterial colonies with different colors and shapes, wherein the PDA culture medium also comprises ampicillin with the final concentration of 50 mug/mL;
selecting hypha tips of the colonies with green middle and white pilus at the edges from the multiple colonies, transferring the hypha tips to a new PDA culture medium, and performing purification culture to obtain Penicillium sp.strain EF 010.
The invention also provides an application of the above-mentioned endophytic Penicillium of ophiopogon japonicus in preparing Hubei ophiopogon japonicus polysaccharide, and the Hubei ophiopogon japonicus polysaccharide is prepared by using the Penicillium sp.strain EF010 through liquid fermentation.
Optionally, the step of preparing ophiopogonpolysaccharide from Penicillium sp.train) EF010 by liquid fermentation comprises:
inoculating Penicillium sp (EF 010) to a PDA (PDA) culture medium under aseptic conditions, and performing activated culture at 28 deg.C for 72h to obtain activated strain;
inoculating the activated strain to a potato liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 72 hours to obtain a seed solution;
inoculating the seed solution into a liquid fermentation culture medium according to the inoculation amount of 10%, and performing shake culture at 28 ℃ and 180rpm for 7 days to obtain a fermentation mixture;
freeze-drying the fermented mixture, pulverizing into powder, washing with 95% ethanol, and oven-drying to obtain dry powder;
extracting the dry powder with hot water, concentrating into extract, and precipitating with ethanol to obtain precipitate;
and washing the precipitate with absolute ethyl alcohol, acetone and diethyl ether in sequence, and then drying to obtain the Hubei ophiopogon japonicus polysaccharide.
Optionally, the formula of the liquid fermentation medium is: 200g of potato and 20g of glucose were added per 1000mL of distilled water.
The endophytic Penicillium sp.train EF010 provided by the invention is obtained by separating and screening fresh ophiopogon japonicus plant root tuber through the steps of culturing, screening, purifying and the like, and can be metabolized by itself to generate the Hubei ophiopogon japonicus polysaccharide, so that the Hubei ophiopogon japonicus polysaccharide can be prepared by microbial liquid fermentation.
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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 Penicillium EF010 on PDA medium in example 1 of the present invention;
FIG. 2 is a HPLC detection result chart of the acid hydrolysis solution of Hubei radix Ophiopogonis polysaccharide extracted from positive control radix Ophiopogonis;
FIG. 3 is a HPLC analysis result chart of an acid hydrolyzed solution of a polysaccharide extract derived from Penicillium EF010 obtained in example 2;
FIG. 4 shows Penicillium bacteria obtained in example 2Polysaccharide extract pair OH and O produced by EF010 bacteria2 -And DPPH and ABTS free radical scavenging activity test result chart.
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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The existing Hubei radix ophiopogonis polysaccharide production is mainly obtained by directly separating root tuber extract. However, in the actual production of the ophiopogon root, the problems of relatively small planting area caused by continuous cropping obstacles, large influence of climatic conditions, long root tuber production period, high extraction cost of the ophiopogon root polysaccharide in Hubei and the like exist, so that the market of the ophiopogon root polysaccharide in Hubei is limited.
In view of this, the invention utilizes the dwarf lilyturf tuber (liliope spicata var. prolifera Y.T.Ma) produced by GAP base of the European temple Zhenwang in Xiangyang city, Hubei to separate and purify a strain of Penicillium sp.strain EF010 from the living root tuber thereof. The endophytic penicillium of ophiopogon has been preserved in China center for type culture Collection (CCTCC for short), and the preservation addresses are as follows: wuhan, Wuhan university, classified as Penicillium sp.strain EF010, with the preservation number of CCTCC NO: M2018778, and the preservation time of 2018, 11 months and 12 days.
The Penicillium EF010 of the present invention was cultured on Potato Dextrose Agar (PDA) medium at 28 ℃ to obtain colonies having green middle and white pili at the edges, as shown in FIG. 1.
The Penicillium EF010 is identified to belong to Penicillium (Penicillium sp) through an 18S rRNA gene sequence, and the gene sequence is shown as SEQ ID NO: 1, the sequencing results were aligned to the NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi) with 99% homology to Penicillium sp, KF 954542.1.
The endophytic penicillium EF010 provided by the invention is obtained by separating and screening fresh radix ophiopogonis tuber through the steps of culturing, screening, purifying and the like, and can be metabolized by itself to generate Hubei radix ophiopogonis polysaccharide, so that the Hubei radix ophiopogonis polysaccharide can be prepared by microbial liquid fermentation.
The invention also provides a method for screening the penicillium EF010, which comprises the following steps:
step S10, cleaning fresh radix ophiopogonis tuber, soaking the fresh radix ophiopogonis tuber in 70% ethanol and 4% -6% sodium hypochlorite solution in volume fraction, and removing surface water to obtain the clean radix ophiopogonis tuber, wherein the soaking time is 1-3 min.
One embodiment of step S10 is given below: fully cleaning soil on the surfaces of tuberous roots of fresh ophiopogon japonicus plants by using a large amount of tap water, then filling the cleaned soil into a clean beaker, adding deionized water, and repeatedly cleaning the beaker in an ultrasonic cleaner until the cleaned water becomes clear; dipping water on the surface of the tuber of ophiopogon japonicus in north of dry lake by using sterile filter paper, soaking the tuber in 75% ethanol by volume for 2min, and rinsing with sterile water for 3 times; and soaking the root tuber of the dwarf lilyturf tuber in a sodium hypochlorite solution with the effective chlorine content of 4-6% for 2min, continuously rinsing the root tuber of the dwarf lilyturf tuber for 4 times by using a large amount of sterile water, and dipping the root tuber of the dwarf lilyturf tuber in dry water by using sterile filter paper to obtain the clean tuber root of the dwarf lilyturf tuber.
And S20, removing brown stain parts from the clean radix ophiopogonis roots, cutting the clean radix ophiopogonis roots into slices, placing the slices on a PDA culture medium, performing sealed culture at 25-30 ℃ for 5-7 days until hyphae grow on the periphery of the slices to form a plurality of colonies with different colors and shapes, wherein the PDA culture medium further comprises ampicillin with the concentration of 50 mug/mL.
One embodiment of step S20 is given below: taking the clean radix ophiopogonis tuber, shearing brown stain parts at two ends of the tuber under aseptic condition, cutting the rest part into 0.5cm × 0.5cm tissue small pieces (transverse slices or longitudinal slices) by using a dissecting blade, cutting the tissue small pieces as thin as possible so that the tissue small pieces can be fully contacted with the surface of a culture medium, facilitating the absorption of fungi from the culture medium, placing the tissue small pieces on a PDA culture medium (containing ampicillin with a final concentration of 50 μ g/mL), uniformly lightly pressing the tissue small pieces by using tweezers to enable the tissue small pieces to be tightly attached to a flat plate, planting 2 pieces in each dish, setting five times of repetition, sealing the culture dish by using a parafilm sealing membrane, placing the culture dish at 28 ℃ for 5-7 days, and growing hyphae at the periphery of the tuber slice to form a plurality of bacterial colonies with different colors and shapes.
And step S30, selecting hypha tips of the bacterial colonies with green middle and white pilus at the edges from the plurality of bacterial colonies, transferring the hypha tips to a new PDA culture medium for purification and culture, and obtaining the penicillium EF 010.
One embodiment of step S30 is given below: observing the growth condition of fungi in a sample every day, growing hyphae around a root tuber slice after 5-7 days to form a plurality of bacterial colonies with different colors and shapes, selecting hypha tips of the bacterial colonies, transferring the hypha tips to a fresh PDA culture medium, respectively carrying out purification culture for a plurality of times until only a single pure bacterial colony grows out, then selecting the bacterial colony with green middle and white pilus at the edge, selecting the hypha tip, transferring the hypha tip to the new PDA culture medium, and carrying out purification culture, namely separating and purifying the penicillium EF 010. The Penicillium EF010 was identified as Penicillium (Penicillium sp) by molecular biology.
The invention further provides an application of the above-mentioned endophytic penicillium of radix ophiopogonis in preparation of Hubei ophiopogon japonicus polysaccharide, and the Hubei ophiopogon japonicus polysaccharide is prepared by using the penicillium EF010 through liquid fermentation.
In a specific embodiment of the present invention, the step of preparing the liriope spicata polysaccharide by liquid fermentation using the penicillium EF010 includes:
step a, inoculating penicillium EF010 to a PDA culture medium under aseptic condition, and performing activated culture at 28 ℃ for 72 hours to obtain activated strains;
b, inoculating the activated strain to a potato liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 72 hours to obtain a seed solution;
step c, inoculating the seed liquid into a liquid fermentation culture medium according to the inoculation amount of 10%, and performing shake culture at 28 ℃ and 180rpm for 7 days to obtain a fermentation mixture;
step d, freeze-drying the fermentation mixture, then crushing the fermentation mixture into powder, washing the powder with 95% ethanol, and drying the powder to obtain dry powder;
step e, carrying out hot water extraction on the dry powder, concentrating the dry powder into an extract, and carrying out alcohol precipitation treatment to obtain a precipitate;
and f, washing the precipitate by using absolute ethyl alcohol, acetone and ether in sequence, and then drying to obtain the Hubei ophiopogon japonicus polysaccharide.
Wherein the formula of the liquid fermentation medium adopted in the step c is as follows: 200g of potato and 20g of glucose were added per 1000mL of distilled water.
A specific example of fermentation preparation of Hubei radix Ophiopogonis polysaccharide is given below: taking penicillium EF010, picking a small amount of hypha by using an inoculating needle under the aseptic condition, inoculating into a sterilized PDA culture medium test tube, and carrying out activated culture at 28 ℃ for 72 hours; taking the activated and cultured strain, transferring into sterilized potato liquid culture medium (PDA) under aseptic condition, and performing shake culture at 28 deg.C and 180rpm for 72 hr to obtain seed solution; respectively filling the prepared liquid fermentation culture medium (200 g of potatoes and 20g of glucose are added in every 1000mL of distilled water) into 250mL triangular flasks, wherein each flask is about 100mL, sterilizing, and cooling for later use; then inoculating the seed solution into a liquid fermentation culture medium according to the inoculation amount of 10% under the aseptic condition, and carrying out shake culture at 180rpm at 28 ℃ for 7 days; after fermentation, placing the solid-liquid mixture obtained by fermentation 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 fermentation product; grinding the strain fermentation product freeze-dried at low temperature into powder, adding 100mL of ethanol with volume fraction of 95%, repeating for three times, washing the ground material, and drying; adding distilled water into the dried product according to the solid-to-liquid ratio of 1: 10(g/mL), performing water bath at 80 ℃ for 2h, filtering, and repeating the above operation for 1 time to extract the filter residue for the second time; then mixing the extracting solutions, centrifuging, and taking the supernatant for dialysis for 48 h; collecting trapped fluid after dialysis, carrying out reduced pressure concentration to prepare an extract, carrying out alcohol precipitation treatment on the extract by using 100mL of ethanol with the volume fraction of 95%, and collecting precipitate obtained by the alcohol precipitation treatment; washing the obtained precipitate with anhydrous ethanol, acetone and diethyl ether, and drying to obtain Hubei radix Ophiopogonis polysaccharide.
The penicillium EF010 is used for preparing the Hubei ophiopogon japonicus polysaccharide in a liquid fermentation mode, the Hubei ophiopogon japonicus polysaccharide can be rapidly and industrially produced in large quantities, the pollution of the Hubei ophiopogon japonicus polysaccharide prepared by a chemical synthesis method to the environment can be avoided, and the method for preparing the Hubei ophiopogon japonicus polysaccharide is more environment-friendly.
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 Penicillium EF010
(1) Fully cleaning soil on the surfaces of tuberous roots of fresh ophiopogon japonicus plants by using a large amount of tap water, then filling the cleaned soil into a clean beaker, adding deionized water, and repeatedly cleaning the beaker in an ultrasonic cleaner until the cleaned water becomes clear; dipping water on the surface of the tuber root of the dry ophiopogon root with sterile filter paper, soaking the tuber root with 75 percent ethanol by volume for 2min, and rinsing with sterile water for 3 times; and soaking the root tuber of the dwarf lilyturf tuber in a sodium hypochlorite solution with the effective chlorine content of 4-6% for 2min, continuously rinsing the root tuber of the dwarf lilyturf tuber for 4 times by using a large amount of sterile water, and dipping the root tuber of the dwarf lilyturf tuber in dry water by using sterile filter paper to obtain the clean tuber root of the dwarf lilyturf tuber.
(2) Taking the clean ophiopogon japonicus, cutting brown stain parts at two ends of the root tuber under aseptic condition, cutting the rest part into small tissue slices (transverse slices or longitudinal slices) of 0.5cm multiplied by 0.5cm by using a dissecting blade, cutting the tissue slices as thin as possible so that the tissue slices can be fully contacted with the surface of a culture medium, facilitating the absorption of fungi from the culture medium, placing the tissue slices on a PDA culture medium (containing ampicillin with a final concentration of 50 mu g/mL), slightly and slightly pressing the tissue slices with tweezers uniformly to make the tissue slices 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, and placing the culture dish at the constant temperature of 28 ℃ for 5-7 days.
(3) Observing the growth condition of fungi in a sample every day, growing hyphae around a root tuber slice after 5-7 days to form a plurality of bacterial colonies with different colors and shapes, selecting hypha tips of the bacterial colonies, transferring the hypha tips to a fresh PDA culture medium, respectively carrying out purification culture for a plurality of times until only a single pure bacterial colony grows out, then selecting the bacterial colony with green middle and white pilus at the edge, selecting the hypha tip, transferring the hypha tip to the new PDA culture medium, and carrying out purification culture, namely screening out penicillium EF 010.
Colony morphology and 18S rRNA gene sequence identification were performed on the Penicillium strain EF010 selected in example 1, and the method and results were as follows:
the selected Penicillium EF010 was cultured in Potato Dextrose Agar (PDA) medium at 28 ℃ to obtain colonies having green middle and white pili at the edges, and the growth characteristics are shown in FIG. 1.
The Penicillium EF010 is identified to belong to Penicillium (Penicillium sp) through an 18S rRNA gene sequence, and the gene sequence is shown as SEQ ID NO: 1, the sequencing results were aligned to the NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi) with 99% homology to Penicillium sp, KF 954542.1.
Example 2 liquid fermentation of Penicillium EF010
(1) Taking the penicillium EF010 screened in the embodiment, 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 carrying out activated culture at 28 ℃ for 72 hours; taking the activated and cultured strain, transferring into sterilized potato liquid culture medium (PDA) under aseptic condition, and performing shake culture at 28 deg.C and 180rpm for 72 hr to obtain seed solution; respectively filling the prepared liquid fermentation culture medium (200 g of potatoes and 20g of glucose are added in every 1000mL of distilled water) into 250mL triangular flasks, wherein each flask is about 100mL, sterilizing, and cooling for later use; then inoculating the seed solution into a liquid fermentation culture medium according to the inoculation amount of 10% under the aseptic condition, and carrying out shake culture at 180rpm at 28 ℃ for 7 days; after fermentation is finished, a solid-liquid mixed fermentation product is obtained;
(2) quickly freezing the fermentation product obtained in the step (1) in an ultra-low temperature refrigerator to be a solid state, and then carrying out low-temperature freeze drying treatment by using a small freeze dryer to obtain a dried fermentation product; grinding the strain fermentation product freeze-dried at low temperature into powder, adding 100mL of ethanol with volume fraction of 95%, repeating for three times, washing the ground material, and drying; adding distilled water into the dried product according to the solid-to-liquid ratio of 1: 10(g/mL), performing water bath at 80 ℃ for 2h, filtering, and repeating the above operation for 1 time to extract the filter residue for the second time; then mixing the extracting solutions, centrifuging, and taking the supernatant for dialysis for 48 h; collecting trapped fluid after dialysis, carrying out reduced pressure concentration to prepare an extract, carrying out alcohol precipitation treatment on the extract by using 100mL of ethanol with the volume fraction of 95%, and collecting precipitate obtained by the alcohol precipitation treatment; washing the obtained precipitate with anhydrous alcohol, acetone and diethyl ether, and drying to obtain polysaccharide extract of Penicillium EF 010.
The polysaccharide extract produced by the penicillium EF010 strain prepared in example 2 was tested for its related properties, and the following methods and results were obtained:
(1) color reaction of fungus polysaccharide extract
The following two color reaction methods are adopted for detection:
(a) the naphthol-concentrated sulfuric acid reaction method comprises the following steps: 200 mu L of a solution of a polysaccharide extract produced by Penicillium EF010 in a sample was dissolved in 400 mu L of 15% 1-naphthol, and 100 mu L of concentrated sulfuric acid was added thereto, which was positive bluish purple.
(b) The anthrone-concentrated sulfuric acid reaction method comprises the following steps: 400. mu.L of a sample of a polysaccharide extract produced by Penicillium EF010 was added to 300. mu.L of an anthrone sulfuric acid solution, and the sample was positive in blue-green color.
The two color reaction results of the polysaccharide extracts produced by the penicillium EF010 bacteria are positive, which shows that the penicillium EF010 bacteria provided by the invention can produce polysaccharide through fermentation.
(2) Thin layer chromatography detection (TLC)
Taking 20mg of the polysaccharide-produced extract of the penicillium EF010 strain prepared in the example 2, adding 5mL of pure water for dissolving, adding 0.25mL of 12mol/L HCL, then placing the mixture in an environment with the temperature of 120-126 ℃ for reaction for 4h, neutralizing after the reaction is finished, dialyzing, and diluting to 10mL to prepare the acidolysis solution of the polysaccharide-produced extract of the penicillium EF010 strain.
Performing thin layer chromatography on acidolysis solution of polysaccharide extract produced by Penicillium EF010 with acidolysis product of Hubei radix Ophiopogonis polysaccharide extracted from 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 4g diphenylamine, 4mL aniline and 20mL 85% phosphoric acid solution are measured and dissolved in 200mL acetone, and the mixture is heated in an oven at 105 ℃ for 3min until clear red spots appear.
The positive control of the TCL detection result of the acidolysis product of the polysaccharide extracts produced by the Hubei ophiopogon japonicus polysaccharide and the penicillium EF010 bacteria is as follows: the acidolysis monosaccharide of the polysaccharide extract produced by the penicillium EF010 bacteria has similar Rf value with the acidolysis monosaccharide of the positive control, which indicates that the penicillium EF010 can be fermented to produce the Hubei ophiopogon japonicus polysaccharide.
(3) High performance liquid chromatography detection (HPLC)
Taking 20mg of the polysaccharide-produced extract of the penicillium EF010 strain prepared in example 2, adding 5mL of pure water for dissolving, adding 0.25mL of 12mol/L HCL, reacting at 120-126 ℃ for 4 hours, neutralizing after the reaction is finished, dialyzing, and diluting to 10mL to prepare the acidolysis solution of the polysaccharide-produced extract of the penicillium EF010 strain.
Taking acidolysis product of Hubei radix Ophiopogonis polysaccharide extracted from radix Ophiopogonis, as positive control, placing 1mL of acidolysis solution of the prepared polysaccharide extract produced by Penicillium EF010 into a centrifuge tube, adding 1mL, 0.5mol/mL of PMP-methanol solution and 0.3mol/L of NaOH solution, vortex for 1min, cooling to room temperature in 70 deg.C water bath for 60min, neutralizing with 1mL of 0.3mol/L of HCL solution, extracting with 2mL of chloroform for 3 times, mixing water layers, filtering with water system filter membrane, and performing ultrasonic treatment for 0.5h before use.
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 result diagram of an acidolysis solution of Hubei Ophiopogon japonicus polysaccharide, FIG. 3 is a positive control HPLC detection result diagram of an acidolysis solution of a polysaccharide extract produced by Penicillium EF010, and a comparison of FIG. 2 and FIG. 3 shows that the Penicillium EF010 provided by the present invention can produce Hubei Ophiopogon japonicus polysaccharide through fermentation. (4) Measurement of antioxidation of fungus polysaccharide extract
Respectively testing the polysaccharide extracts produced by the penicillium EF010 bacteria on OH and O by adopting an alpha-deoxyribose method, a pyrogallol autoxidation method, a DPPH method and an ABTS method2 -DPPH, ABTS radical scavenging activity.
FIG. 4 shows the polysaccharide pair OH, O of Penicillium EF0102 -And DPPH and ABTS free radical scavenging activity test result chart. As can be seen from FIG. 4, the polysaccharide extract produced by fermentation of Penicillium EF010 of the present invention is resistant to four kinds of free radicals (DPPH, O)2 -OH, ABTS) has certain in vitro scavenging activity, and the antioxidant capacity is positively correlated with the concentration.
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, improvement and the like 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> a kind of bacterial strain of Xiangyang ophiopogon root endophytic penicillium and its application
<130> 20191213
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1564
<212> DNA
<213> Penicillium sp
<400> 1
gagtgcagtt agtatagcac tcttttactg tgaactgcga atggctcatt aaatcagtta 60
tcgtttattt gatagtaccc tactacatgg atacctgtgg taattctaga gctaatacat 120
gcgcaaaacc ccgacttcgg aaggggtgta tttattagat aaaaaaccaa tgcccttcgg 180
ggctccttgg tgattcataa taacttcacg aatcgcatgg ccttgcgccg gcgatggttc 240
attcaaattt ctgccctatc aactttcgat ggtaggatag tggcctacca tggtggcaac 300
gggtaacggg gaattagggt tcgattccgg agagggagcc tgagaaacgg ctaccacatc 360
caaggaaggc agcaggcgcg caaattaccc aatcccgata cggggaggta gtgacaataa 420
atactgatac agggctcttt tgggtcttgt aattggaatg agaacaatct aaatccctta 480
acgaggaaca attggagggc aagtctggtg ccagcagccg cggtaattcc agctccaata 540
gcgtatatta aagttgttgc agttaaaaag ctcgtagttg aaccttgggc ccgtcctgcc 600
ggtccgcctc accgcgagta ctggtccgga tgggcctttc tttctgggga atcccatggc 660
cttcactggc tgtggcgggg aaccaggact tttactgtga aaaaattaga gtgttcaaag 720
caggcctttg ctcggataca ttagcatgga ataatagaat aggacgtgcg gttctatttt 780
gttggtttct aggaccgccg taatgattaa tagggatagt cgggggcgtc agtattcagc 840
tgtcagaggt gaaattcttg gatttgctga agactaacta ctgcgaaagc attcgccaag 900
gatgttttca ttaatcaggg aacgaaagtt aggggatcga agacgatcag ataccgtcgt 960
agtcttaacc ataaactatg ccgactaggg atcgggcggg gtttctatga tgacccgctc 1020
ggcaccttac gagaaatcaa agtttttggg ttctgggggg agtatggtcg caaggctgaa 1080
acttaaagaa attgacggaa gggcaccaca aggcgtggag cctgcggctt aatttgactc 1140
aacacgggga aactcaccag gtccagacaa aataaggatt gacagattga gagctctttc 1200
ttgatctttt ggatggtggt gcatggccgt tcttagttgg tggagtgatt tgtctgctta 1260
attgcgataa cgaacgagac ctcggccctt aaatagcccg gtccgcgttt gcgggccgct 1320
ggcttcttag ggggactatc ggctcaagcc gatggaagta cgtggcaata acaggtctgt 1380
gatgccctta gatgttctgg gccgcacgcg cgctacactg acagggccag cgagtacatc 1440
accttggccg agaggtctgg gtaatcttgt taaaccctgt cgtgctgggg atagagcatt 1500
gcaattattg ctcttcaacg aggaatgcct agtaggcacg agtcatcagc tcgtgccgat 1560
tact 1564

Claims (2)

1. The endophytic Penicillium sp strain of ophiopogon japonicas is Penicillium sp (EF 010) with the preservation number of CCTCC NO of M2018778 and the preservation time of 11 months and 12 days in 2018.
2. The use of the endophytic penicillium of lilyturf of claim 1 in the preparation of polysaccharide of liriope hupehensis, wherein the polysaccharide of liriope hupehensis is prepared by liquid fermentation using the endophytic penicillium of lilyturf of claim 1.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2703000A1 (en) * 2011-04-25 2014-03-05 Shanghai Zhangjiang Engineering Research Center of Modern Preparation Technology of Traditional Chinese Medicine Application of dwarf lilyturf tuber polysaccharide extract in preparation of dietary supplement, health food or medicine with the function of weight loss
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2703000A1 (en) * 2011-04-25 2014-03-05 Shanghai Zhangjiang Engineering Research Center of Modern Preparation Technology of Traditional Chinese Medicine Application of dwarf lilyturf tuber polysaccharide extract in preparation of dietary supplement, health food or medicine with the function of weight loss
CN107723247A (en) * 2017-10-18 2018-02-23 湖北文理学院 It is a kind of to assist raw cladosporium category fungi and the application in steroid saponin is prepared in the tuber of dwarf lilyturf
CN107723248A (en) * 2017-10-18 2018-02-23 湖北文理学院 It is a kind of to assist raw penicillium fungi and the application in steroid saponin is prepared in the tuber of dwarf lilyturf

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Title
药用植物内生真菌研究进展;华永丽等;《世界科学技术-中医药现代化》;20080815;第10卷(第4期);第105-111页 *
襄麦冬多糖及皂苷体外活性研究;陈哲等;《食品工业科技》;20180731;第39卷(第13期);第7-12页 *

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