CN113604522A

CN113604522A - Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder

Info

Publication number: CN113604522A
Application number: CN202110882872.5A
Authority: CN
Inventors: 黄丽; 林晖翔; 滕建文; 夏宁; 韦保耀; 邱思绮
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-11-05
Anticipated expiration: 2041-08-02
Also published as: CN113604522B

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binders, wherein the penicillium D306 strain is preserved in China Center for Type Culture Collection (CCTCC) at 29 months 4 in 2021, and the preservation number is CCTCC NO: M2021487. The extracellular polysaccharide produced by the D306 strain has good bile acid binding capacity, and the half-inhibitory concentration IC50 value of the prepared bile acid binding agent is 20.075mg/ml, so that the extracellular polysaccharide has good application prospects in the aspects of prevention, treatment and the like of hyperlipidemia.

Description

Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binders.

Background

At present, hyperlipidemia has become the source of various diseases, and seriously affects human health. When the cholesterol in blood is too high, hyperlipidemia is caused and cardiovascular diseases are caused. Because the main metabolic pathway of the cholesterol is the conversion of the cholesterol into the bile acid in the liver, the bile acid can be irreversibly combined with the bile acid through the bile acid chelating agent, the liver and intestine circulation of the bile acid is blocked, the discharge of the bile acid is promoted, and the accelerated conversion of the cholesterol into the bile acid is caused; in addition, when the intestinal canal absorbs exogenous cholesterol, the bile acid needs to be emulsified, and the bile acid combined by the chelating agent is discharged along with the feces, so that the digestion and absorption of the cholesterol from the intestinal canal are influenced, and the aim of reducing the cholesterol level in the liver and the plasma is fulfilled. The most common medicine for treating the diseases is cholestyramine clinically, but the side effect is great, the most common adverse reaction is constipation, obvious gastrointestinal adverse reaction also influences the absorption of various vitamins and some acidic medicines and the like. Therefore, it is necessary to develop natural, effective drugs with less side effects on human body.

Exopolysaccharides are secondary metabolites that are secreted extracellularly by microorganisms during growth metabolism. The extracellular polysaccharide is an active macromolecule, has physiological effects of antioxidation, anti-tumor, immunoregulation, anticoagulation and the like on human bodies, has the characteristics of low production cost, short production period and easy separation, and becomes one of research hotspots in the fields of food science, natural medicines and the like.

PIGEON R M et al [ PIGEON R M, CUESTAAP, GIL LILAND S E. binding of free bile acids by cells of yogurt starter culture bacteria [ J ]. J Dairy Sci,2002, 85(11): 2705-. The extracellular polysaccharide plays an important role in metabolic processes such as combination, absorption, excretion and the like of cholesterol, and the combination of the extracellular polysaccharide and free bile acid causes that the bile acid cannot be absorbed by intestinal tracts, thereby inhibiting the progress of enterohepatic circulation. However, no report that exopolysaccharide produced by Penicillium (Penicillium gerundense) D306 strain has bile acid binding ability exists at present, so that a new bile acid binding agent is sought, which is beneficial to the development of natural, effective and small-side-effect medicines for human bodies.

Disclosure of Invention

The invention aims to make up the defects in the prior art, and provides an application of a Penicillium (Penicillium gerundense) D306 strain in exopolysaccharide production. The penicillium D306 strain has the characteristic of producing extracellular polysaccharide, and the extracellular polysaccharide has bile acid binding capacity, provides a new direction for developing a medicament for preventing and treating hyperlipemia, and is a very potential strain.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of a Penicillium (Penicillium gerundense) D306 strain in exopolysaccharide production, wherein the Penicillium (Penicillium gerundense) D306 strain is preserved in China Center for Type Culture Collection (CCTCC) at 29 months 4 in 2021, and the preservation number is CCTCC NO: M2021487.

Preferably, the penicillium D306 strain is subjected to conventional fermentation, and the supernatant contains exopolysaccharides after fermentation.

The strain Penicillium gerundense D306 belongs to the phylum Ascomycota, Ascomycetes, Eurotiales.

The Penicillium (Penicillium gerundense) D306 strain for producing the extracellular polysaccharide is obtained by fermenting tea leaves in the pile fermentation process of Liubao tea enterprises in Sterculia City of Guangxi Zhuang autonomous region. The colony in the CA culture medium is round, gray on the front side, compact in texture, free of exudate and gray on the back side; the colony on the CYA culture medium is oval, gray on the front side, loose in texture, free of exudate and gray on the back side; the hyphae are spaced under a microscope, the wall of the spore stalk is smooth, the brood branches are more recurrent, and the conidia are round or round-like and smooth.

It is another object of the present invention to provide the use of the strain Penicillium sp (Penicillium gerundense) D306 for the preparation of bile acid binder exopolysaccharides. The specific technical scheme is as follows:

the application of a Penicillium (Penicillium gerundense) D306 strain in the preparation of bile acid binder extracellular polysaccharide is disclosed, wherein the Penicillium (Penicillium gerundense) D306 strain is preserved in China center for type culture Collection (with the address of Wuhan, Wuhan university) at 29/4 in 2021, and the preservation number is CCTCC NO: M2021487.

The invention also provides bile acid binder extracellular polysaccharide prepared from the Penicillium (Penicillium gerundense) D306 strain.

The purpose is realized by the following technical scheme:

a method for preparing bile acid binder exopolysaccharide by using Penicillium (Penicillium gerundense) D306 strain, comprising the following steps:

s1, fermenting the penicillium D306 strain with the preservation number of CCTCC NO: M2021487, and filtering the fermentation product to obtain fermentation liquor;

s2, centrifuging the fermentation liquor obtained in the step S1 to remove thalli, taking supernate and extracting polysaccharide by an alcohol precipitation method;

s3, redissolving the polysaccharide in the S2, removing protein of the solution by a Sevag method, taking supernatant, precipitating with ethanol, extracting the polysaccharide, redissolving and dialyzing the product, and carrying out vacuum freeze drying on the obtained solution to obtain the bile acid binder extracellular polysaccharide which is marked as bile acid binder EPS-D306.

Preferably, the fermentation conditions in step S1 are: PDB culture medium is selected as the culture medium, the fermentation temperature is 28 ℃, the rotating speed of a shaking table is 120r/min, and the fermentation time is 168 hours.

Preferably, the centrifugal rotation speed in the step S2 is 6000r/min, and the centrifugation is 20 min.

Preferably, the volume ratio of ethanol to fermentation liquor in the alcohol precipitation method in the step S2 is 3:1, and the alcohol precipitation temperature is 4 ℃.

Preferably, the volume ratio of the n-butanol to the chloroform solution in the Sevag method in the step S3 is 1:4, and the reagent is magnetically stirred for 20 min; the cut-off molecular weight of the dialysis bag used for dialysis is 8000Da-12000 Da, the dialysis condition is that 100 times volume of distilled water is added for dialysis, the dialysis bag is replaced every 4 hours, and the dialysis time is 48 hours.

The invention identifies and analyzes the components and molecular weight of the bile acid binder extracellular polysaccharide prepared from the Penicillium (Penicillium gerundense) D306 strain.

Preferably, the bile acid binder extracellular polysaccharide component contains 76.14% of total sugar, 21.63% of uronic acid, and no protein; the molecular weight consisted of 8.88% 1092.677kDa and 91.12% 13.187 kDa; the monosaccharide consists of Man, GluA, Rha and Gal, and the molar ratio is 0.35: 1.16: 0.09: 52.46.

the invention also aims to provide application of the bile acid binder extracellular polysaccharide prepared from the Penicillium (Penicillium gerundense) D306 strain in medicines for preventing and treating cardiovascular diseases caused by hyperlipidemia and hyperlipemia.

The bile acid binder extracellular polysaccharide produced by the penicillium D306 strain has the ability of being combined with bile acid, the half-inhibitory concentration IC50 value is 20.075mg/ml, the cholesterol level can be effectively reduced, and the bile acid binder extracellular polysaccharide is an active ingredient which has potential application in preventing and treating hyperlipidaemia.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the Penicillium (Penicillium gerundense) D306 strain provided by the invention can produce extracellular polysaccharide with bile acid binding capacity, and the half-inhibitory concentration IC50 value of the bile acid binder prepared by using the Penicillium (Penicillium gerundense) D306 strain is 20.075mg/ml, so that the liver and intestine circulation of bile acid can be hindered, the discharge of the bile acid can be promoted, the accelerated conversion of cholesterol into bile acid can be caused, and the cholesterol level in vivo can be reduced; the extracellular polysaccharide is natural and effective, has small side effect on human body, provides a new idea for developing a medicine for preventing and treating hyperlipemia, and has good application prospect.

Drawings

FIG. 1 is a microscopic view of the D306 strain.

FIG. 2 is a phylogenetic tree diagram of the D306 strain.

FIG. 3 is a UV spectrum of extracellular polysaccharide EPS-D306 of D306 strain.

FIG. 4 is an infrared spectrum of extracellular polysaccharide EPS-D306 of the D306 strain.

FIG. 5 is a gel chromatogram of extracellular polysaccharide EPS-D306 of strain D306.

FIG. 6 is a diagram of the monosaccharide composition of exopolysaccharide of strain D306; wherein a is different monosaccharide standards, and b is EPS-D306.

FIG. 7 shows bile acid binding ability of exopolysaccharide of strain D306.

Detailed Description

The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.

Example 1 isolation, culture and characterization of Penicillium D306 Strain

1. Strain isolation and purification

(1) Collecting bacterial colonies: weighing 2g of each tea sample (the tea is fermented in the pile fermentation process of Liupao tea enterprises in Sterculia City of Guangxi Zhuang autonomous region), adding 20mL of sterile water into a blue-mouth bottle after being cut into pieces, and oscillating for 30min at the speed of 80r/min to ensure that microorganisms attached to the tea fall off.

(2) And (3) flora culture: diluting 1mL of the above bacterial solution to 10^-1、10^-2、10^-3、10^-4、 10^-5、10^-6The bacterial suspension with the gradient concentration is added into a culture dish after 200 mu L of the bacterial suspension is sucked, the bacterial suspension is evenly coated by a disposable coating rod and is placed in an incubator at 28 ℃ and 37 ℃ for 2 to 3 days.

(3) Strain purification: picking the white hairy colony on the coating culture dish, carrying out streak culture by using a PDA plate, marking, and repeatedly purifying for more than 4 times until a single strain is obtained, wherein the strain is named as a D306 strain.

2. And (3) morphological observation of the strain: the purified strain was inoculated to CA and CYA media and cultured at 28 ℃ for 3 to 7 days. Observing the front and back sides of the two culture dishes, describing the characteristics of colony color, growth speed, exudate and the like, and identifying according to 'fungal identification handbook'.

(1) The D306 strain is in a CA culture medium, and the colony is circular, gray in the front, compact in texture, free of exudates and gray in the back.

(2) The D306 strain is in CYA culture medium, the colony is oval, the front side is grey, the texture is loose, no exudate exists, and the back side is grey.

(3) And (4) microscopic observation: dropping a drop of lactophenol cotton blue staining solution staining agent at the center of the glass slide, clamping a small amount of mycelia in the staining solution by using forceps, picking out the mycelia, covering a cover glass, and observing by using 10, 20 and 40 times of lens.

D306 belongs to the genus Penicillium, is blue after being dyed with a gossypol lactate staining solution, and can be observed under a microscope that hyphae have intervals, the spore stalk wall is smooth, the broom-shaped branches are multi-recurrent, and conidia are round or round-like and smooth. (the microscopic view is shown in FIG. 1).

3. And (3) molecular identification:

(1) extraction of DNA: the single colony after purification was inoculated to ISP2 medium and cultured in an incubator at 37 ℃ for 12 to 24 hours. 50 μ L of the sterilized resin solution was put in an eight-connected tube, and a very small portion of the cells were picked with a toothpick and ground in the eight-connected tube for 1 min. The eight tubes are kept for 10min at the temperature of 100 ℃ in a metal bath, and the supernatant fluid is taken as the template DNA by centrifugation.

(2) And (3) PCR reaction: PCR reaction System for preparing fungi:

TABLE 1 PCR reaction System

mu.L of the PCR reaction system of Table 1 above was uniformly distributed into eight sterilized tubes, and 0.5. mu.L of template DNA was added to each well. Primer 27F: AGTTTGATCMTGGCTCAG, primer 1492R: GGTTACCTTGTTACGACTT.

PCR amplification conditions: pre-denaturation at 95 ℃ for 8 min; denaturation at 94 deg.C for 50s, annealing at 55 deg.C for 45s, extension at 72 deg.C for 1.5min, 31 cycles, and final extension at 72 deg.C for 10min, and storing at-20 deg.C for use.

(3) Detecting the PCR amplification result: preparing an agarose solution with the mass fraction of 1%, heating and dissolving the agarose solution in a microwave oven, and adding a Gold view I staining agent. The agarose solution was slowly poured into the electrophoresis apparatus mold, and a comb was inserted to form a small hole, and the comb was taken out after the agarose was cooled to form a gel. The 1 XTAE electrophoresis buffer was slowly poured over the agarose gel surface. Pipette 2.5. mu.L of PCR product into the gel well using a pipette gun. Electrophoresis was carried out at 110V and 100mA for 25 min. The gel imager was used to observe whether the PCR amplified bands were clear, to check whether the DNA amplification was successful.

(4) Sequencing a PCR product: and (3) transporting qualified PCR products to Meiji Co Ltd at low temperature for sequence determination, wherein the sequencing result is shown as SEQ ID NO. 1.

(5) Blast alignment: and selecting a proper sequence from the sequencing result, comparing the nucleotide sequence in Blast, selecting a strain with higher homology, establishing an evolutionary tree by adopting MEGAX software, and determining the species of the separated strain.

Phylogenetic analysis was performed by sequence alignment and the D306 strain belongs to the Penicillium (Penicillium gerundense) and the evolutionary tree is shown in FIG. 2.

Example 2 extraction and composition determination of exopolysaccharides from Penicillium D306 strains

1. Extraction and purification of extracellular polysaccharide

(1) Strain activation: the strain of example 1 was inoculated into PDA medium and cultured at 28 ℃ for 72 hours.

(2) Strain culture: in the place where the culture medium of the penicillium grows most densely, a small piece is taken out by a puncher and inoculated into PDB culture medium, and fermentation is carried out for 168 hours under the conditions of 28 ℃ and 120 r/min.

(3) Crude extraction of extracellular polysaccharide: the Penicillium fermentation liquid is first vacuum filtered to eliminate most of thallus, the filtrate is centrifuged at 6000r/min for 20min, and the supernatant is added with 3 times volume of 95% ethanol and maintained at 4 deg.c overnight. Centrifuging to collect precipitate, air drying the precipitate, adding distilled water with a certain volume to melt, adding 95% ethanol with 3 times volume, and standing at 4 deg.C overnight. Collecting the precipitate, and air drying to obtain the crude extracellular polysaccharide extract of penicillium.

(4) And (3) purifying extracellular polysaccharide: adding distilled water into the crude extract of the exopolysaccharide to dissolve the crude extract of the exopolysaccharide to prepare a 1 percent sugar solution, and removing protein by a Sevag method. Mixing n-butanol with distilled water at equal volume, performing ultrasonic treatment for 10min, collecting the upper layer solution as water saturated n-butanol solution, and adding chloroform 4 times the volume of the n-butanol solution to obtain Sevag reagent. Adding Sevag reagent with volume one third of that of 1% sugar solution, stirring with a magnetic stirrer for 20min, pouring into a separating funnel, standing until obvious layering, removing lower organic reagent and middle proteopectin, and collecting upper sugar solution. The protein removal operation was repeated until no opalescent precipitate was present. The organic reagent was then removed by rotary evaporation at 60 ℃, three volumes of 95% ethanol were added and the precipitate was collected by centrifugation overnight in a refrigerator at 4 ℃. Dissolving the precipitate with appropriate amount of distilled water, loading into dialysis bag with molecular weight cutoff of 8000Da-12000 Da, dialyzing in 100 times volume of distilled water, changing water every 4 hr, and dialyzing for 48 hr. After dialysis, the retentate was freeze-dried to obtain extracellular polysaccharide of strain D306, which was designated EPS-D306.

2. EPS-D306 extracellular polysaccharide component determination

Determination of the dry matter content of the fermentation liquor: the weight of the dish after constant weight was recorded (m1), 1mL of fermentation supernatant was added and the weight was recorded (m 2). The mixture was dried in a 110 ℃ forced air drying cabinet to a constant weight and the mass (m3) was recorded. The dry matter content (D) per 1mL of fermentation supernatant was calculated as follows:

(1) and (3) total sugar content determination: prepare 0.1mg/mL standard glucose mother liquor. 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6mL of standard glucose mother liquor distilled water is sucked to be constant volume of 2 mL. Respectively adding 1mL of phenol solution, rapidly adding 5mL of concentrated sulfuric acid, shaking, standing for 10min, and reacting at 25 deg.C for 20 min. 200. mu.L of the reaction solution was transferred to a 96-well microplate, and the absorbance was measured at 490 nm. The linear regression equation of the standard curve is that y is 10.69x-0.0096, and R2 is 0.9966. y is absorbance and x is glucose concentration (mg/mL). 2mL of sample to be tested is taken, and the rest operations are performed under the same conditions as the standard curve. And (5) bringing the light absorption value of the sample to be detected into a standard curve to obtain the total sugar concentration. The total sugar content of the fermentation broth is calculated according to the following formula:

c2 is the total sugar concentration (mg/mL) of the sample to be tested of the strain, C1 is the total sugar concentration (mg/mL) of the sample to be tested of distilled water, A is the dilution factor, and D is the dry matter content (g/mL) of the sample to be tested of the strain.

(2) Measurement of uronic acid content: preparing 200 mu g/mL standard furfural acid mother liquor, taking a certain amount of standard furfural acid mother liquor, and supplementing distilled water to 0.5mL to obtain 0, 15, 30, 45, 60, 75, 90, 105, 120, 135 and 150 mu g/mL series standard solutions. The 0.5mL series of standard solutions was placed in 10mL EP tubes, the EP tubes were placed on crushed ice, 3mL of 0.025mol/L sodium tetraborate-sulfuric acid reagent was added to each tube, the tube cap was closed, the tube was gently shaken, then placed in a boiling water bath and heated for 10min, and removed to cool to room temperature. 0.1mL of 0.125% carbazole-ethanol reagent was added to each tube, the tube was shaken again, the cap was closed, heated in a boiling water bath for 15min, and cooled to room temperature. Absorbance was measured at 530nm with the aspiration of 200. mu.L of the reaction solution. The linear regression of the galacturonic acid standard curve is y ═ 0.0044x-0.0238, R²0.9933. y is absorbance and x is galacturonic acid concentration (. mu.g/mL). 0.5mL of sample to be tested is taken, and the rest operations are performed according to the standard curve. The uronic acid content of the strain fermentation liquor is calculated according to the following formula:

wherein C2 is the uronic acid concentration (mg/mL) of the strain test sample, C1 is the uronic acid concentration (mg/mL) of the blank medium test sample, A is the dilution factor, and D is the dry matter content (g/mL) of the strain test sample.

(3) Protein content determination: the protein reagent was prepared by dissolving 100mg of Coomassie Brilliant blue G-250 in 50mL of 95% ethanol and adding 100mL of 85% (w/v) phosphoric acid to the solution. The resulting solution was diluted to a final volume of 1000 mL. The final concentrations of the reagents were 0.01% (w/v) Coomassie Brilliant blue G-250, 4.7% (w/v) ethanol and 8.5% (w/v) phosphoric acid.

The resulting mixture was prepared into 100. mu.g/mL bovine serum albumin standard solution. 0, 0.1, 0.2, 0.3, 0.4, 0.5mL of the standard solution was aspirated, 0.5mL of distilled water was made up, 2.5mL of the protein reagent was added, and vortexed and mixed well. After reacting for 15min, absorbance at 595nm was measured using a 96-well microplate. The concentration of the protein and the corresponding absorbance are plotted as a standard curve for the determination of the protein in an unknown sample. The linear regression of the standard curve for the protein was 0.0033x +0.0183, R²0.9936. y is absorbance and x is protein concentration (. mu.g/mL). Diluting exopolysaccharide extract with distilled water, adding 0.5mL, adding 2.5mL Coomassie brilliant blue solution, reacting for 15min, and performing colorimetry under the same conditions. The protein content of the crude exopolysaccharide extract is calculated according to the following formula:

c2 is the protein concentration (mg/mL) of the test sample of the strain, C1 is the protein concentration (mg/mL) of the blank distilled water, A is the dilution factor, and D is the mass (g) of the test sample.

The results of the experiment show that EPS-D306 has 76.14% of total sugar content, 21.63% of uronic acid content, and no protein.

Example 3 characterization of the exopolysaccharide Structure produced by the Penicillium D306 Strain

1. Ultraviolet spectral analysis

Exopolysaccharide samples were dissolved in distilled water and adjusted to the appropriate concentration, followed by ultraviolet spectroscopy (UV) scanning analysis at a wavelength in the range of 200nm to 400 nm.

The UV spectrum scan shows that there is no absorption peak at 260nm-280nm, indicating no protein or nucleic acid.

2. Infrared spectroscopic analysis

The exopolysaccharide sample 3mg and the potassium bromide solid powder 270mg are weighed, mixed and ground until the sample particles have no crystalline lens, and then made into uniform thin slices by a tablet machine. Scanning with a Fourier transform infrared spectrometer with a scanning range of 4000cm^-1-400cm^-1。

Infrared Spectrum Scan as shown in FIG. 4 at 3426.69cm^-1The appearance of a strong absorption peak can be attributed to the stretching vibration peak of an O-H bond, and the existence of hydroxyl is indicated; 2936.65cm^-1The peak at (A) is-CH on the sugar chain₂Or the stretching vibration peak of the C-H bond of-CH. 1642.70cm^-1The peaks near (a) are due to the presence of bound water. 1420.64cm^-1The peaks are probably characteristic of carboxyl groups or carboxylate salts, indicating that the exopolysaccharides are all acidic polysaccharides. The polysaccharide content is 1240.23cm^-1There is an absorption peak, which is the stretching vibration peak of S ═ O, indicating a sulfated polysaccharide. 1100cm^-1-1010cm^-1There were 3 absorption peaks representing the presence of a pyran ring and 2 absorption peaks representing the presence of a furan ring. The results demonstrated that all 6 polysaccharides had only 2 peaks, indicating furan rings. At 1087.52 cm^-1Absorption peaks at the ether bond C-O-C and hydroxyl of the pyran ring; the polysaccharide content is 1010.68cm^-1Is C-O stretching vibration peak; 928.11cm^-1Peaks indicate that the polysaccharide may contain mannose; at 890cm^-1And 812 cm^-1No absorption peak, and at 850cm^-1The peak appears from the left and the right, which shows that the alpha-glycosidic bond is contained. At 762.98cm^-1The absorption peak is caused by the symmetric stretching vibration of the alpha-pyran ring.

3. Determination of molecular weight

The molecular weight of exopolysaccharides is determined by Gel Permeation Chromatography (GPC). Exopolysaccharide and series dextran markers (580, 29330, 10730, 31420, 63350, 143400, 280500, 841700, 2560000 and 7500000Da) were dissolved in ultrapure water to prepare a solution of 1 mg/mL.

Chromatographic conditions are as follows: waters GPC 1515 model, equipped with differential refractometer detector, column: the water Ultrahydrogel 500column (7.8 × 300mm), the water Ultrahydrogel 250column (7.8 × 300mm) and the water Ultrahydrogel 120column (7.8 × 300mm) are connected in series for combination. Mobile phase: ultrapure water; flow rate: 1.0 mL/min; column temperature: 30 ℃; sample introduction amount: 40 μ L.

Calculating to obtain R according to the standard curve of the glucan standard product, wherein the obtained standard curve is-0.5409 x +12.424²The linear range is better when the value is 0.9985. FIG. 5 is a GPC chart of exopolysaccharide in a strain, and the retention time of exopolysaccharide is substituted into a standard curve to obtain the molecular weight, as shown in Table 2.

TABLE 2 molecular weight of exopolysaccharides

The results showed that EPS-D306 is a heterogeneous polysaccharide with molecular weights of 1092.677kDa (8.88%), 13.187kDa (91.12%).

4. Monosaccharide composition analysis

And (3) measuring the monosaccharide composition of the exopolysaccharide by adopting a PMP pre-column derivatization method.

Hydrolysis of polysaccharide: placing 0.0500g extracellular polysaccharide powder in hydrolysis tube, adding 1mL HCl (3.0mol/L), sealing, mixing, hydrolyzing at 110 deg.C for 4-6 hr, cooling, adjusting pH to neutral with 3.0mol/L NaOH, and absorbing 400 μ L for derivatization.

Formulation of single and gradient mixed standards (1) single standard: each monosaccharide control (arabinose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, fucose and rhamnose) was dissolved in ultrapure water at a concentration of 2 mmol/L. (2) Mixing the standard: monosaccharide controls were co-dissolved in ultrapure water to give a concentration of 1.6mg/mL per standard, and then diluted to 6 concentrations of gradient mix of 1.6, 1.2, 1.0, 0.8, 0.4, 0.2 mg/mL.

And (3) monosaccharide derivation: taking a standard solution (single standard and gradient mixed standard) and 400 mu L of hydrolyzed polysaccharide sample, adding 400 mu L of PMP-methanol solution of 0.5mol/L and NaOH of 0.3mol/L respectively, mixing uniformly, and carrying out water bath reaction at 70 ℃ for 100 min. Then, 500. mu.L of 0.3mol/L HCl was added thereto, and PMP reagent was extracted with chloroform for 3 times, 5mL each, the chloroform layer was discarded, the aqueous layer was centrifuged, and 1mL of the supernatant was filtered through a 0.45 μm organic filter and analyzed by High Performance Liquid Chromatography (HPLC).

Analysis was performed using a Waters 2984LC HPLC. Chromatographic conditions are as follows: waters C18 column (4.6X 150mm, 5 μm); acetonitrile-0.02 mol/L ammonium acetate solution (17: 83) is used as a mobile phase chromatographic condition; the column temperature is 30 ℃; the flow rate is 1.0 mL/min; the detection wavelength is 250nm, and the sample injection amount is 10 mu L.

Experimental results As shown in FIG. 6, the extracellular polysaccharide of EPS-D306 is composed of Man, GluA, Rha and Gal, and the molar ratio is 0.35: 1.16: 0.09: 52.46 (1-mannose, 2-glucuronic acid, 3-galacturonic acid, 4-rhamnose, 5-glucose, 6-galactose, 7-arabinose, 8-fucose, x-unknown components, PMP-PMP derivatizing agent).

Example 4 determination of the exopolysaccharide bile acid Activity binding Capacity of Penicillium D306 Strain

The prepared bile acid binder EPS-D306 was formulated into a solution at a concentration of 10mg/mL-110 mg/mL and binding capacity was determined by a bile acid test kit.

The bile acid test kit is purchased from Nanjing institute of bioengineering, Inc.

Bile acid working solution: glycocholic acid (9mmol/L), chenodeoxycholic acid (9mmol/L), glycodeoxycholic acid (9mmol/L), taurocholic acid (4.5mmol/L), taurodeoxycholic acid (4.5mmol/L) were dissolved in phosphate buffer solution at pH 6.8, i.e., prepared as 36mmol/L bile acid stock solution. The 36mmol/L stock solution was stored in a refrigerator at-20 ℃ and diluted to 0.72. mu. mol/mL bile acid working solution before use.

1mL of the prepared bile acid binder exopolysaccharide EPS-D306 solution or cholestyramine with the same concentration as the exopolysaccharide is put into a 15X 150mm glass test tube, 1mL of 0.01mol/L HCl is added, and the acid digestion of the stomach is simulated for 2 hours at 37 ℃ and 120 r/min. The pH of the sample was adjusted to 7-7.5 with 0.2mol/L NaOH. To each test sample, 4mL of bile acid working solution (0.72 μmol/mL) and 5mL of pancreatin (10mg/mL in phosphate buffer pH 6.8) were added, simulating in vitro intestinal digestion at 37 ℃ for 2 hours at 120 r/min. Centrifuging the mixture at 6000r/min for 20min, and storing the supernatant at-20 deg.C. Distilled water was used as a negative control.

And (4) detecting the content of bile acid in the supernatant according to the determination method of the total bile acid kit. The formula for bile acid content is:

wherein, the enzyme-linked immunosorbent assay reads the absorbance A at 405nm₀3min later the absorbance A was read₁，ΔA＝A₁-A₀. The Δ A measurement is the Δ A value of the sample, the Δ A calibration is the Δ A value of the calibrator, and the concentration of the bile acid calibrator is 50 μmol/L.

The determination result is shown in figure 7, the bile acid binder EPS-D306 shows activity in the concentration range of 10mg/mL-110 mg/mL, and the half-inhibitory concentration IC50 value is 20.075mg/mL, which indicates that the bile acid binder extracellular polysaccharide prepared by the D306 strain has good application prospect in the aspects of prevention and treatment of hyperlipidemia and the like.

Sequence listing

<110> Guangxi university

<120> exopolysaccharide-producing penicillium D306 strain and application thereof in preparation of bile acid binder

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acgcagcgaa atgcgataag taatgtgaat tgcagaattc agtgaatcat cgagtctttg 240

aacgcacatt gcgccccctg gtattccggg gggcatgcct gtccgagcgt cattgctgcc 300

ctcaagcccg gcttgtgtgt tgggcgccgt ccccccgggg acgggcccga aaggcagcgg 360

cggcaccgcg tccggtcctc gagcgtatgg ggctctgtca cccgctctgc aggcccggcc 420

ggcgccagcc gaccccctca accctttttt ttttttcagg ttgacctcgg atcaggtagg 480

gat 483

Claims

1. The application of the strain of Penicillium (Penicillium gerundense) D306 in the production of exopolysaccharides is characterized in that the strain of Penicillium (Penicillium gerundense) D306 is preserved in China center for type culture Collection at 29.4.2021 with the preservation number of CCTCC NO: M2021487.

2. The application of the Penicillium (Penicillium gerundense) D306 strain in exopolysaccharide production according to claim 1, wherein the Penicillium D306 strain is subjected to conventional fermentation, and the supernatant contains exopolysaccharide after the fermentation.

3. The application of the Penicillium (Penicillium gerundense) D306 strain in the preparation of bile acid binder exopolysaccharide is characterized in that the Penicillium (Penicillium gerundense) D306 strain is preserved in China center for type culture Collection at 2021, 4-29 months with the preservation number of CCTCC NO: M2021487.

4. A bile acid binder exopolysaccharide prepared from the Penicillium gerundense D306 strain according to claim 3.

5. The Penicillium (Penicillium gerundense) D306 strain according to claim 4, wherein the total sugar content of the extracellular polysaccharide is 76.14%, the uronic acid content of the extracellular polysaccharide is 21.63%, and the extracellular polysaccharide is free of protein; the molecular weight consisted of 8.88% 1092.677kDa and 91.12% 13.187 kDa; the monosaccharide consists of Man, GluA, Rha and Gal, and the molar ratio is 0.35: 1.16: 0.09: 52.46.

6. the use of bile acid binder exopolysaccharide prepared from Penicillium (Penicillium gerundense) D306 strain according to claim 4 in the preparation of a medicament for preventing and treating hyperlipidemia and cardiovascular disease caused by hyperlipidemia.