Pleurotus citrinopileatus polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses pleurotus citrinopileatus polysaccharide as well as a preparation method and application thereof. The pleurotus citrinopileatus polysaccharide is hydrolyzed to obtain a mixture containing glucose, galactose and glucuronic acid. Compared with common edible fungus polysaccharide, the pleurotus citrinopileatus polysaccharide provided by the invention can be used as a carbon source to be specifically utilized by parabacteroides gobi so as to promote metabolism and proliferation of the parabacteroides gobi, and can be applied to prebiotic products of parabacteroides probiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharides, and in particular relates to a polysaccharide of Pleurotus citrinopileatus and a preparation method and application thereof. Background Art

[0002] The impact of intestinal flora on human health has been widely studied and recognized, and changes in its structure and function are associated with a variety of diseases such as obesity and type II diabetes. The interaction between complex polysaccharides and intestinal flora is the main driving force for the formation of intestinal microbial community structure. Polysaccharides are one of the main effective ingredients of large edible and medicinal fungi. Because the human body lacks enzymes to degrade complex polysaccharides, these polysaccharides reach the ileum and colon without digestion and absorption, and are metabolized and utilized by intestinal microorganisms therein. Prebiotics refer to organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the health of the host. Large edible and medicinal fungi polysaccharides can be used as prebiotics to promote the proliferation of beneficial bacteria and the production of short-chain fatty acids, while inhibiting pathogens.

[0003] The functions of polysaccharides from large edible and medicinal fungi depend on their structural characteristics, such as molecular weight, branching configuration, conformation, and chemical modification. These structural characteristics are related to factors such as origin, growth environment, extraction and purification methods, etc. The high degree of polymerization of polysaccharide molecules, the variety of monosaccharides and glycosidic bonds, and the presence of branched chains make their structures have obvious heterogeneity.

[0004] The latest research reveals the important role of intestinal Parabacteroides in disease prevention and treatment. Parabacteroides distasonis has the effects of anti-cancer, prevention and treatment of obesity, prevention of weight rebound, and improvement of arthritis. Parabacteroides goldsteinii can prevent weight gain, reduce inflammation, improve chronic obstructive pulmonary disease, and reduce the pathogenicity of Helicobacter pylori. Parabacteroides merdae has the effect of improving high-fat diet-induced atherosclerosis. It can be seen that all three have the potential of probiotics and have received much attention in recent studies. Some studies have shown that Cordyceps polysaccharides can selectively enrich Parabacteroides goldsteinii. In addition, the research on polysaccharide prebiotics for Parabacteroides is very limited. Therefore, it is necessary to develop more polysaccharide substances that can be used as Parabacteroides prebiotics. Summary of the invention

[0005] In order to overcome the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide a polysaccharide of Pleurotus citrinopileatus. The second purpose of the present invention is to provide a method for preparing the polysaccharide of Pleurotus citrinopileatus. The third purpose of the present invention is to provide the application of the polysaccharide of Pleurotus citrinopileatus. In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a polysaccharide of Pleurotus citrinopileatus. The polysaccharide of Pleurotus citrinopileatus is hydrolyzed to obtain a mixture containing glucose, galactose and glucuronic acid.

[0007] Preferably, the hydrolyzate of Pleurotus citrinopileatus polysaccharide comprises 90-95 mol% of glucose, 3-7 mol% of galactose and 1-4 mol% of glucuronic acid.

[0008] More preferably, the hydrolyzate of Pleurotus citrinopileatus polysaccharide contains 91-94 mol% of glucose, 4-6 mol% of galactose and 1-3 mol% of glucuronic acid.

[0009] Preferably, the Pleurotus citrinopileatus polysaccharide is extracted from Pleurotus citrinopileatus.

[0010] Preferably, the weight average molecular weight of the polysaccharide is (5.73±0.15)×10 6 Da.

[0011] Preferably, the root mean square rotation radius of the Pleurotus citrinopileatus polysaccharide is (33.5±7.2) nm.

[0012] Preferably, the main chain structure of the Pleurotus citrinopileatus polysaccharide is (1→4)-D-glucan, and the side chain structure is (1→3 / 6)-D-glucan.

[0013] The second aspect of the present invention provides a method for preparing the Pleurotus citrinopileatus polysaccharide described in the first aspect, comprising the following steps: subjecting the Pleurotus citrinopileatus to water bath extraction, filtering and collecting the filtrate, and concentrating to obtain a concentrated solution; adding anhydrous ethanol to the concentrated solution for alcohol precipitation treatment to obtain the Pleurotus citrinopileatus polysaccharide.

[0014] Preferably, the water bath extraction is carried out at a temperature of 80 to 100° C. and for a time of 1 to 4 hours.

[0015] Preferably, the concentration process is: concentration under reduced pressure at 40-60°C.

[0016] Preferably, the citrinum citrinum and water are mixed at a solid-liquid ratio of 1:(7-13) (v / v) to obtain a mixed solution.

[0017] Preferably, the method comprises the following steps: mixing Pleurotus citrinopileatus with water, extracting by water bath, filtering, concentrating and collecting the filtrate, centrifuging the filtrate, and concentrating to obtain a concentrated solution; adding anhydrous ethanol to the concentrated solution and stirring and mixing, performing alcohol precipitation treatment, centrifuging and collecting the precipitate, and drying to obtain Pleurotus citrinopileatus polysaccharide.

[0018] Preferably, the alcohol precipitation concentration of the alcohol precipitation treatment is 20% to 90%.

[0019] More preferably, the alcohol precipitation concentration of the alcohol precipitation treatment is 35% to 45% or 75% to 85%.

[0020] Preferably, the alcohol precipitation treatment time is 8 to 20 hours.

[0021] The third aspect of the present invention provides the use of the Pleurotus citrinopileatus polysaccharide described in the first aspect in the preparation of an intestinal flora regulating product.

[0022] Preferably, the intestinal flora regulating product is an intestinal Parabacteroides prebiotic.

[0023] More preferably, the intestinal Parabacteroides prebiotic is Parabacteroides gordonii prebiotic.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a Pleurotus citrinopileatus polysaccharide. Compared with common edible fungus polysaccharides, the Pleurotus citrinopileatus polysaccharide can be specifically utilized as a carbon source by Parabacteroides gordonii, thereby promoting the metabolism and proliferation of Parabacteroides gordonii, and can be applied to prebiotic products of Parabacteroides probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth of Parabacteroides gordonii in different edible fungi polysaccharides;

[0027] Figure 2 The growth of Parabacteroides distichous in different edible fungi polysaccharides;

[0028] Figure 3 The effect of Pleurotus citrinopileatus obtained with different alcohol precipitation ratios on the growth of Parabacteroides gordonii. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0030] The experimental materials in the following examples are:

[0031] Pleurotus citrinum strain: Pleurotus citrinum (Pleurotus citrinum) laboratory collection number: plc0002, the strain was purchased from Jiangsu Tianda Edible Fungi Research Institute (original number: Pleurotus citrinum K-13), cultivation formula: 83% bagasse, 15% bran, 1% lime, 1% sucrose. After the mycelium grows for 15-20 days, it is transferred to the fruiting management, and the fruiting bodies are collected and dried after 7-15 days.

[0032] Parabacteroides gordonii and Parabacteroides dissimilar were from Beina Biotechnology. Tremella polysaccharide, Pleurotus eryngii polysaccharide and Ganoderma lucidum polysaccharide were preserved in the laboratory.

[0033] Example 1

[0034] This embodiment provides a polysaccharide of Pleurotus citrinopileatus, and the preparation method thereof is as follows:

[0035] Weigh 200 g of Pleurotus citrinopileatus fruiting bodies, add 10 times the volume of distilled water, extract with boiling water for 2 hours, filter after cooling, repeat extraction twice on the remaining filter residue, combine the filtrate, centrifuge the filtrate at 10000 rpm for 10 minutes, evaporate under reduced pressure and concentrate to a volume of about 1 L at 50°C, slowly add anhydrous ethanol to the concentrate and stir to make the final ethanol concentration (volume ratio) reach 40%, let stand overnight, centrifuge, collect the precipitate, dissolve it with a small amount of distilled water, and then perform reduced pressure rotary evaporation to remove a small amount of ethanol, and finally perform freeze-drying to obtain crude polysaccharide, that is, Pleurotus citrinopileatus polysaccharide.

[0036] Example 2

[0037] This embodiment provides a polysaccharide of Pleurotus citrinopileatus, and the preparation method thereof is as follows:

[0038] Weigh 200 g of Pleurotus citrinopileatus fruiting bodies, add 10 times the volume of distilled water, extract with boiling water for 2 hours, filter after cooling, extract the remaining filter residue twice, combine the filtrate, centrifuge the filtrate at 10000 rpm for 10 minutes, evaporate under reduced pressure and concentrate at 50°C to a volume of about 1L, then slowly add anhydrous ethanol to the concentrate and stir to make the final concentration of ethanol reach 20%, let it stand overnight, centrifuge, collect the precipitate, dissolve it with a small amount of distilled water, and then perform reduced pressure rotary evaporation to remove a small amount of ethanol, and finally freeze-dry to obtain crude polysaccharide, that is, Pleurotus citrinopileatus polysaccharide.

[0039] Example 3

[0040] This embodiment provides a polysaccharide of Pleurotus citrinopileatus, and the preparation method thereof is as follows:

[0041] Weigh 200 g of Pleurotus citrinopileatus fruiting bodies, add 10 times the volume of distilled water, extract with boiling water for 2 hours, filter after cooling, extract the remaining filter residue twice, combine the filtrate, centrifuge the filtrate at 10000 rpm for 10 minutes, evaporate under reduced pressure and concentrate at 50°C to a volume of about 1L, then slowly add anhydrous ethanol to the concentrate and stir to make the final concentration of ethanol reach 60%, let it stand overnight, centrifuge, collect the precipitate, dissolve it with a small amount of distilled water, and then perform reduced pressure rotary evaporation to remove a small amount of ethanol, and finally freeze-dry to obtain crude polysaccharide, that is, Pleurotus citrinopileatus polysaccharide.

[0042] Example 4

[0043] This embodiment provides a polysaccharide of Pleurotus citrinopileatus, and the preparation method thereof is as follows:

[0044] Weigh 200 g of Pleurotus citrinopileatus fruiting bodies, add 10 times the volume of distilled water, extract with boiling water for 2 hours, filter after cooling, extract the remaining filter residue twice, combine the filtrate, centrifuge the filtrate at 10000 rpm for 10 minutes, evaporate under reduced pressure and concentrate at 50°C to a volume of about 1L, then slowly add anhydrous ethanol to the concentrate and stir to make the final concentration of ethanol reach 80%, let it stand overnight, centrifuge, collect the precipitate, dissolve it with a small amount of distilled water, and then perform reduced pressure rotary evaporation to remove a small amount of ethanol, and finally freeze-dry to obtain crude polysaccharide, that is, Pleurotus citrinopileatus polysaccharide.

[0045] Experimental analysis

[0046] 1. Effects of edible fungus polysaccharides on the growth of Parabacteroides

[0047] Preparation of sole carbon source medium: 1% (w / v) carbon source, 100mM KH2PO4 (pH 7.2), 15mM NaCl, 8.5mM (NH4)2SO4, 4mM L-cysteine, 1.9μM hematin / 200μM L-histidine, 100μM MgCl2, 1.4μMFe2SO4, 50μM CaCl2, 1μg / mL vitamin K3, 5ng / mL vitamin B 12 The carbon sources were glucose (Glc), Pleurotus citrinum polysaccharide, Tremella fuciformis polysaccharide, Pleurotus eryngii polysaccharide, and Ganoderma lucidum polysaccharide of Example 1, respectively, and glucose was used as a positive control.

[0048] Preparation of seed solution of Parabacteroides disil and Parabacteroides gordonii: Parabacteroides disil and Parabacteroides gordonii are anaerobic bacteria, so be careful to operate in an anaerobic box. The lyophilized powder of Parabacteroides disil and Parabacteroides gordonii was inoculated into a liquid culture medium (peptone: 23.0, soluble starch: 1.0, sodium chloride: 5.0, pH: 7.3±0.2) and activated and revived overnight at 37°C. The revived bacterial solution was streaked on a solid culture medium (peptone: 23.0, soluble starch: 1.0, sodium chloride: 5.0, agar: 10.0, pH: 7.3±0.2) and incubated at 37°C overnight. The next day, a single clone was picked from the activated bacterial plate and inoculated into 2 mL of liquid culture medium, and anaerobically cultured at 37°C for 16 hours to obtain seed solution of Parabacteroides disil and Parabacteroides gordonii.

[0049] Determination of the culture curve of Parabacteroides dissimilar and Parabacteroides gordonii:

[0050] Pipette 20 μL of seed solution into 2 mL of sole carbon source medium (inoculation ratio 1:100), culture at 37°C, and measure OD every 24 hours. 600 (OD 600It refers to the absorbance at a wavelength of 600nm and is often used to indicate the bacterial concentration in bacterial liquid culture).

[0051] Figure 1 The growth of Parabacteroides gordonii in different edible fungi polysaccharides; Figure 2 The growth of Parabacteroides distichous in different edible fungi polysaccharides. Figure 1 and Figure 2 As shown in the figure, both Parabacteroides gordonii and Parabacteroides distriae can grow in the medium with glucose as the sole carbon source. However, when the carbon source is replaced with edible fungus polysaccharides, the growth of Parabacteroides distriae is significantly delayed, while Parabacteroides gordonii only grows in the medium with Pleurotus citrinum polysaccharides as the sole carbon source in Example 1, indicating that Parabacteroides gordonii can utilize Pleurotus citrinum polysaccharides, but cannot utilize Tremella polysaccharides, Pleurotus eryngii polysaccharides and Ganoderma lucidum polysaccharides.

[0052] 2. Effects of Pleurotus citrinopileatus polysaccharides obtained with different alcohol precipitation ratios on the growth of Parabacteroides gordonii

[0053] The preparation method of the sole carbon source culture medium and the experimental method used in the experiment are the same as those described above.

[0054] The results are as follows Figure 3 As shown in the figure, the polysaccharides obtained from different alcohol precipitation ratios had a significant effect on the growth of Parabacteroides gordonii as the sole carbon source, among which the polysaccharides obtained from 40% alcohol precipitation could support the growth of Parabacteroides gordonii, followed by the polysaccharides obtained from 80% alcohol precipitation. Parabacteroides gordonii had relatively weak utilization ability for the polysaccharides obtained from 20% and 60% alcohol precipitation.

[0055] Structural information

[0056] 1. Molecular weight distribution of Pleurotus citrinopileatus polysaccharide

[0057] The polysaccharide sample of Pleurotus citrinopileatus obtained by 40% alcohol precipitation was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being tested on a machine. The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatttechnology, CA, USA). Gel exclusion chromatography columns Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) were used in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min. The chromatographic data were processed using the software ASTRA 6.1 to obtain the weight average molecular weight of the polysaccharide of Pleurotus citrinopileatus, which was (5.73±0.15)×10 6 Da, the root mean square radius of rotation is (33.5±7.2)nm. The polydispersity coefficient is 8.1, indicating that the polysaccharide has a wide molecular weight distribution. The molecular weight of the most abundant component fragment is (6.91±0.15)×10 6 Da.

[0058] 2. Monosaccharide components of Pleurotus citrinopileatus polysaccharide

[0059] Solid sample extraction: Take a clean chromatographic bottle, weigh 5 mg of the polysaccharide sample of Example 1, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add sterile water to dissolve, and transfer to a chromatographic bottle for testing. The chromatographic system used is the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and an electrochemical detector is used to analyze and detect the monosaccharide components. The chromatographic column is Dionex TM CarboPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column, injection volume is 5μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 ml / min; column temperature was 30°C; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0060] Acid hydrolysis and ion chromatography analysis of its monosaccharide components revealed that Pleurotus citrinopileatus polysaccharide is composed of glucose, galactose and glucuronic acid, with molar compositions of 92.57%, 5.31% and 2.11%, respectively.

[0061] 3. Bond type analysis of Pleurotus citrinopileatus polysaccharide

[0062] Sample pretreatment: Take 2 mg of the polysaccharide sample of Example 1, add 500 μL DMSO to dissolve, add 1 mg NaOH, and incubate for 30 min. Add 50 μL iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and blow it dry with nitrogen. Add 100 μL 2M TFA and react at 121°C for 90 min. Evaporate at 30°C. Add 50 μL 2M ammonia water, 50 μL 1M NaBD4, mix, and react at room temperature for 2.5 h. Add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, and blow dry with nitrogen. Add 250 μL acetic anhydride, vortex to mix, and react at 100°C for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex mix, centrifuge, discard the aqueous phase, and repeat the water washing 3 times. Take the lower dichloromethane phase and use Agilent Technologies Inc. CA, USA 6890A-5977B gas spectrometer for detection. The chromatographic system uses an Agilent gas chromatography system (Agilent 6890A; Agilent Technologies, USA), and the chromatographic column is BPX70 (30m×0.25mm×0.25μm, SGE, Australia). The injection volume is 1 μL, the split ratio is 10:1, the carrier gas is high-purity helium, and the flow rate is 1.5 mL / min; the initial temperature of the column oven is 140°C for 2.0 min, and the temperature is raised to 230°C at 3°C / min and maintained for 3 min. The mass spectrometry system used was a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. Using the electron impact ion source (EI), the analytes were detected in full scan (SCAN) mode with a mass scan range (m / z): 50-350.

[0063] By analyzing the methylation sites of monosaccharides, the main connection modes of glucose residues were 1,4 (57.83%), 4,6 (7.80%), 1,3 (5.17%), 1,6 (4.48%), and 3,6 (4.33%), and the main connection mode of galactose residues was 1,6 (7.39%). Based on the above results, it is speculated that the main chain structure of the polysaccharide is (1→4)-D-glucan, and the branched chain structure is (1→3 / 6)-D-glucan.

[0064] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A polysaccharide of Pleurotus citrinopileatus, characterized in that: The citrinum citrinum polysaccharide is hydrolyzed to obtain a mixture containing glucose, galactose and glucuronic acid.

2. The polysaccharide of Pleurotus citrinopileatus according to claim 1, characterized in that The hydrolyzate of Pleurotus citrinopileatus polysaccharide comprises 90-95 mol % of glucose, 3-7 mol % of galactose and 1-4 mol % of glucuronic acid.

3. The polysaccharide of Pleurotus citrinopileatus according to claim 1, characterized in that The Pleurotus citrinopileatus polysaccharide is extracted from Pleurotus citrinopileatus 4. The polysaccharide of Pleurotus citrinopileatus according to claim 1, characterized in that The weight average molecular weight of the polysaccharide of Pleurotus citrinopileatus is (5.73±0.15)×10 6 Da.

5. The method for preparing the polysaccharide of Pleurotus citrinopileatus according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: extracting Pleurotus citrinopileatus in a water bath, collecting the filtrate by filtering, and concentrating to obtain a concentrated solution; and adding anhydrous ethanol to the concentrated solution for alcohol precipitation to obtain Pleurotus citrinopileatus polysaccharide.

6. The method for preparing Pleurotus citrinopileatus polysaccharide according to claim 5, characterized in that: The water bath extraction temperature is 80-100° C. and the time is 1-4 hours.

7. The method for preparing Pleurotus citrinopileatus polysaccharide according to claim 5, characterized in that: The alcohol precipitation concentration of the alcohol precipitation treatment is 20% to 90%.

8. Use of the Pleurotus citrinopileatus polysaccharide according to any one of claims 1 to 4 in the preparation of intestinal flora regulating products.

9. The use of Pleurotus citrinopileatus polysaccharide according to claim 8, characterized in that: The intestinal flora regulating product is an intestinal parabacteroides prebiotic.

10. The use of Pleurotus citrinopileatus polysaccharide according to claim 9, characterized in that: The intestinal Parabacteroides prebiotic is Parabacteroides gordonii prebiotic.

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