CN116178579A - Black skin termitomyces albuminosus polysaccharide and preparation method and application thereof - Google Patents
Black skin termitomyces albuminosus polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN116178579A CN116178579A CN202310134592.5A CN202310134592A CN116178579A CN 116178579 A CN116178579 A CN 116178579A CN 202310134592 A CN202310134592 A CN 202310134592A CN 116178579 A CN116178579 A CN 116178579A
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- China
- Prior art keywords
- polysaccharide
- termitomyces albuminosus
- orps
- black
- albuminosus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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- A61P3/04—Anorexiants; Antiobesity agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a black termitomyces albuminosus polysaccharide and a preparation method and application thereof, wherein the black termitomyces albuminosus polysaccharide is taken as a raw material, is subjected to degreasing, ultrasonic treatment, water boiling extraction, deproteinization, decoloring treatment, DEAE cellulose DE-52 chromatographic column separation, and propylene glucan gel S-400HR gel column purification to obtain single black termitomyces albuminosus polysaccharide OrPs-1, the monosaccharide composition of the single black termitomyces albuminosus polysaccharide OrPs-1 is mannose, glucose and xylose are in molar ratio of 10.16:74.16:10.5:3.18, and the black termitomyces albuminosus polysaccharide OrPs-1 prepared through extraction conditions under mild conditions and simple operation steps can regulate intestinal flora disorder caused by diet, change the composition of intestinal flora, enable the intestinal flora imbalance in vivo to be normalized, further regulate obesity caused by the intestinal flora disorder caused by diet, and can prepare natural auxiliary foods, medicines and health care products for losing weight and regulating the intestinal flora without side effects.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to black skin termitomyces albuminosus polysaccharide, and a preparation method and application thereof
Background
With the improvement of the living standard of substances, people often prefer to eat and drink with high fat, high sugar and high calorie, and the high fat diet is one of important causes of diseases such as obesity, diabetes and the like. In recent years, researches show that some foods and medicines rich in polysaccharide have better effect in reducing blood sugar and blood fat. Studies have shown that the intestinal flora can control fat metabolism, induce systemic chronic low-grade inflammation, induce obesity and insulin resistance. The dietary structure is an important factor in determining the composition of the intestinal flora. Maintaining intestinal microecological balance is essential for maintaining human health. The existing microecological regulator is mainly live bacteria preparations such as bifidobacterium, lactobacillus and the like, but the live bacteria preparations have the problems that the quantity of live bacteria is difficult to ensure, the stability is poor and the like. In recent years, researches show that some Chinese herbal medicines have the effect of supporting normal flora growth, play a role in prebiotics, and are ideal microecological regulators. Compared with probiotics, the traditional Chinese medicine microecological regulator has a plurality of advantages, such as strong stability, long effective period, lower price and the like, and the technical problem of keeping the viable bacteria is solved. Especially, the traditional Chinese medicine not only supports normal flora growth, but also can improve the immune function of the organism, thereby achieving the purposes of regulating yin and yang, strengthening body resistance and eliminating pathogenic factors.
The black skin termitomyces albuminosus Oudemansiella raphanipies is one of fungi used as both medicine and food, and is also a traditional Chinese medicinal macro fungus. He has the characteristics of fresh, tender and mellow taste, tender meat, unique taste, rich nutrition and the like. Research shows that the mushroom polysaccharide can regulate the balance of intestinal microbial community to promote health. The mushroom polysaccharide can be used as a prebiotic to reduce the proliferation of pathogens by increasing the growth of probiotics in the intestinal tract.
The invention patent with publication number of CN110627919B, named as 'an intestinal tract prebiotic black termitomyces albuminosus polysaccharide ORP-1, and a preparation method and application thereof', discloses that the novel intestinal tract prebiotic black termitomyces albuminosus polysaccharide ORP-1 is obtained by adopting alcohol precipitation extraction, DEAE-52 cellulose chromatographic column separation and superdex-75 molecular sieve gel chromatographic column purification of black termitomyces albuminosus as raw materials, and has the defects that: the prepared polysaccharide has large molecular weight and poor effect, and the step-by-step alcohol precipitation method has complicated steps and is not easy to operate.
Disclosure of Invention
The invention aims to provide the black termitomyces albuminosus polysaccharide and the preparation method and application thereof, and the black termitomyces albuminosus polysaccharide OrPs-1 prepared by the extraction conditions under mild conditions and simple operation steps can regulate intestinal flora disturbance caused by diet, change the composition of the intestinal flora, lead the intestinal flora unbalanced in vivo to be normal, further regulate obesity caused by the intestinal flora disturbance caused by diet, and can prepare auxiliary foods, medicines and health care products for reducing weight and regulating the intestinal flora naturally without side effects.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the molecular weight of the black termitomyces albuminosus polysaccharide OrPs-1 is 6742Da, the total sugar content of the black termitomyces albuminosus polysaccharide OrPs-1 is 98.9%, and the monosaccharide composition of the black termitomyces albuminosus polysaccharide is mannose, glucose, xylose, arabinose=12.16:74.16:10.5:3.18 according to the molar ratio.
Further, the preparation method of the black skin termitomyces albuminosus polysaccharide is characterized by comprising the following specific steps of:
1) Taking black skin termitomyces albuminosus as a raw material, degreasing, carrying out ultrasonic treatment, carrying out water boiling extraction, deproteinizing and decoloring to obtain a crude extract;
2) Separating the crude extract obtained in the step 1) by adopting a DEAE cellulose DE-52 chromatographic column to obtain crude polysaccharides OrPs of Collybia albuminosa;
3) Purifying the black skin termitomyces albuminosus polysaccharide OrPs obtained in the step 2) by adopting a propylene glucan gel S-400HR gel column to obtain single black skin termitomyces albuminosus polysaccharide OrPs-1.
Further, the specific method for obtaining the crude extract in the step 1) is as follows:
degreasing: drying and crushing black skin termitomyces albuminosus fruiting body, sieving with 18 mesh sieve to obtain black skin termitomyces albuminosus fruiting body powder, placing the black skin termitomyces albuminosus fruiting body powder into a 200 mesh nylon mesh bag, fastening the mesh bag mouth, and soaking in a container containing 95% ethanol for degreasing to obtain degreased product;
and (3) ultrasonic treatment: adding deionized water 20-30 times of the degreased material, and performing ultrasonic treatment at room temperature under 300-600W for 30-60min to obtain an ultrasonic treated material;
decocting and extracting: placing the ultrasonic treated material in a water decoction extraction container, extracting at 75-98deg.C for 3-5 hr, filtering, repeatedly extracting the residue for 2-4 times, collecting filtrate, and concentrating to obtain concentrate;
deproteinization: repeatedly freezing and thawing the concentrate in a refrigerator at-80deg.C for 2-4 times, thawing, centrifuging to remove protein, collecting supernatant, collecting 500ml supernatant, adding absolute ethanol under stirring to obtain ethanol with final concentration of 70-80%, standing at room temperature for 24 hr, filtering, collecting precipitate, lyophilizing, and pulverizing to obtain polysaccharide powder;
decoloring: adding 5g of polysaccharide powder into 5-10 times of deionized water for dissolving, centrifuging, retaining supernatant, and decolorizing the supernatant with macroporous resin to obtain crude extract.
Further, the ultrasonic power is 400W, and the ultrasonic treatment time is 45min.
Further, the specific method for obtaining the crude polysaccharides OrPs of Collybia albuminosa in the step 2) is as follows:
taking 10mL of the crude extract in the step 1), separating by DEAE cellulose DE-52, eluting by deionized water at the flow rate of 2mL/min, collecting the eluent, concentrating by rotary evaporation, and freeze-drying to obtain the black skin termitomyces albuminosus polysaccharide OrPs.
Further, the specific method for obtaining the single Collybia albuminosa polysaccharide OrPs-1 in the step 3) is as follows:
taking 20mg of the Collybia albuminosa polysaccharide OrPs in the step 2), dissolving in 3mL of deionized water, centrifuging at 6000 rpm for 6min, taking supernatant, placing the supernatant on a propylene sephadex S-400HR gel column, purifying by adopting 0.2M ammonium bicarbonate eluent with the flow rate of 0.13mL/min, collecting the eluent, removing ammonium bicarbonate by rotary evaporation, concentrating, and freeze-drying to obtain single Collybia albuminosa polysaccharide OrPs-1.
Further, the monosaccharide composition of the collybia albuminosa polysaccharide OrPs is mannose, galactose, xylose, arabinose, fucose=13.39:35.65:38.07:0.58:1.94:10.38 according to the molar ratio.
Further, the black skin termitomyces albuminosus polysaccharide OrPs-1 is applied to the preparation of medicines, foods and health care products for reducing weight and regulating intestinal flora disorder.
Due to the adoption of the technical scheme, the invention has the following advantages:
1. the invention takes black skin termitomyces albuminosus as raw material, and single black skin termitomyces albuminosus polysaccharide OrPs-1 is obtained through degreasing, ultrasonic treatment, water boiling extraction, deproteinization, decoloring treatment, DEAE cellulose DE-52 chromatographic column separation, and propylene sephadex S-400HR gel column purification, and the purity is high at 98.9%.
2. The preparation method is simple, the reaction condition is mild, the obtained black termitomyces albuminosus polysaccharide OrPs-1 can regulate intestinal flora disturbance caused by diet, can obviously change the composition of the intestinal flora, enables the intestinal flora unbalanced in vivo to be normal, further regulates obesity caused by the intestinal flora disturbance caused by diet, and can be used as a natural auxiliary food for losing weight and regulating the intestinal flora without side effects.
3. The black termitomyces albuminosus polysaccharide OrPs-1 prepared by the invention can reduce the level of LPS and inflammatory factors MCP-1, TNF-alpha and IL-6 in serum of HFD mice, and has anti-inflammatory effect.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.
Drawings
The drawings of the present invention are described as follows:
FIG. 1 shows the composition of the intestinal flora of mice of each experimental group at the portal classification level.
FIG. 2 shows the composition of the intestinal flora of mice of each experimental group at the genus level.
FIG. 3 shows a differential analysis of the intestinal flora composition at the genus level for mice of each experimental group.
FIG. 4 shows the levels of LPS, inflammatory factors MCP-1, TNF- α and IL-6 in the serum of mice from the experimental group.
FIG. 5 is a molecular weight measurement of OrPs-1.
FIG. 6 is a map of the monosaccharide group of OrPs-1.
Detailed Description
The present invention will be further described with reference to the drawings and examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and all techniques realized based on the above description of the present invention are within the scope of the present invention.
Example 1: preparation of Collybia albuminosa polysaccharide OrPs-1
The method comprises the following specific steps:
1) Degreasing: drying and crushing black skin termitomyces albuminosus fruiting body, sieving with 18 mesh sieve to obtain black skin termitomyces albuminosus fruiting body powder, filling the black skin termitomyces albuminosus fruiting body powder into a 200 mesh nylon mesh bag, and soaking and degreasing in a container containing 95% ethanol to obtain degreased product;
and (3) ultrasonic treatment: adding 100g of degreased material into 20-30 times of deionized water, and performing ultrasonic 400W treatment at normal temperature for 45min to obtain an ultrasonic treated material;
decocting and extracting: placing the ultrasonic treated material in a water decoction extraction container, extracting at 75-98deg.C for 3-5 hr, filtering, repeatedly extracting the residue for 2-4 times, collecting filtrate, and concentrating to obtain concentrate;
deproteinization: repeatedly freezing and thawing the concentrate in a refrigerator at-80deg.C for 2-4 times, thawing, centrifuging to remove protein, collecting supernatant, collecting 500ml supernatant, adding absolute ethanol under stirring to obtain ethanol final concentration of 70-80%, standing at room temperature for 24 hr, precipitating with ethanol, filtering, collecting precipitate, lyophilizing, and pulverizing to obtain polysaccharide powder;
decoloring: adding 5g of polysaccharide powder into 5-10 times of deionized water for dissolution, centrifuging, retaining supernatant, and decolorizing the supernatant with macroporous resin to obtain crude extract;
2) Taking 10-20ml of the crude extract in the step 1), separating by DEAE cellulose DE-52, eluting by deionized water, and collecting eluent, concentrating by rotary evaporation, and freeze-drying to obtain the Collybia velutipes polysaccharide OrPs.
3) Dissolving 20mg of the Collybia albuminosa polysaccharide OrPs in the step 2) in 3ml of deionized water, centrifuging at 6000 rpm for 6min, placing the supernatant on a propylene sephadex S-400HR gel column, purifying by adopting 0.2M ammonium bicarbonate eluent with the flow rate of 0.13ml/min, collecting the eluent, removing ammonium bicarbonate by rotary evaporation, concentrating, and freeze-drying to obtain single Collybia albuminosa polysaccharide OrPs-1.
Example 2: analysis of Collybia Albuminosa polysaccharide OrPs-1
1. Experimental materials
The Collybia albuminosa polysaccharide OrPs-1 prepared under the conditions of example 1.
2. Experimental method
Polysaccharide content determination: measuring the total sugar content in the extracted crude polysaccharide by a sulfuric acid-phenol method;
average molecular weight determination: detecting the average molecular weight of the termitomyces albuminosus polysaccharide by adopting HPGPC;
monosaccharide composition determination: after hydrolysis by trifluoroacetic acid and derivatization by PMP, the measurement was carried out by HPLC.
3. Experimental results
The analysis shows that the total sugar content of the black termitomyces albuminosus polysaccharide OrPs-1 is 98.9%, the average molecular weight of the black termitomyces albuminosus polysaccharide OrPs-1 is 6742Da (shown in figure 5), and the monosaccharide composition of the black termitomyces albuminosus polysaccharide OrPs-1 comprises mannose, glucose, xylose, arabinose=12.16:74.16:10.5:3.18 (shown in figure 6).
Example 3: effect of Collybia Albuminosa polysaccharide OrPs-1 on composition and content of intestinal flora of mice
1. Experimental materials
1) The black termitomyces albuminosus polysaccharide OrPs-1 prepared under the conditions of example 1;
2) 32C 57BL/6 mice, SPF grade, 18-22 g, male: SCXK (Beijing) 2019-0008, a biological technology Co., ltd., beijing, fukang;
3) Mice maintained feed: jiangsu province cooperative medical bioengineering limited liability company gavage (2019) 01008, 60% high-fat feed: beijing feed certificate (2019) 06076, the biological sciences of Fukang, beijing, and details are shown in Table 1.
Table 1 feed function with different fat content
2. Experimental method
1) Mouse experiment: after the tested C57BL/6 mice enter the experimental animal center of southwest medical university, 3-4 mice/box, the relative humidity is 20-25 ℃ and is 60+/-10 percent, and the time is 12 hours: lighting intermittently for 12 hours; and (3) drinking water which is distilled water prepared by experimental animal centers is taken freely. After one week of adaptive feeding, the groups were randomly and evenly distributed according to Body Weight (BW): normal group (n=8), model group (n=8), black termitomyces albuminosus polysaccharide OrPs-1, positive control group (n=8), details are shown in table 2;
after the experiment is started, the normal group is given with maintenance feed, the other groups are given with high-fat purified feed, and gastric lavage administration is started at the same time, the normal group and the model group are given with distilled water, the other groups are given with corresponding medicines, and the gastric lavage amount is 200mg/kg/day for 40 days; observing the hair color and mental state of the mice every day, recording the feed intake, recording the weight of the mice every 6 days, collecting fresh feces of the mice on the 40 th day, pre-freezing in liquid nitrogen, and transferring to a refrigerator at-80 ℃ for preservation, wherein the feces of the rectum are used for sequencing, and the feces of the small intestine, the colon and the cecum are used for measuring the content of short chain fatty acids;
2) Microbial flora structure and diversity analysis: genomic DNA extraction was performed according to manufacturer' S protocol, amplification was performed using V3-V4 hypervariable region of 16S ribosomal RNA gene, wherein polymerase chain amplification of 16S rDNA domain was performed using 16S rDNA primer of V3-V4 region, high throughput sequencing by shanghai metai biomedical science and technology company was used to determine DNA sequence of sample, all sequences were demultiplexed and mass filtered using QIIME (version 1.9.1), and then data analysis was performed;
3) Short chain fatty acid content determination: fecal samples (30 mg) were placed in a grind tube and 500 μl of water containing 0.5% phosphoric acid was added. Adding n-butanol containing internal standard 2-ethylbutyric acid with concentration of 10 μg/mL, extracting, performing low temperature ultrasonic treatment for 10min, centrifuging at 13000×g at 4deg.C for 5min, and filtering with 0.22 μm filter membrane. Analysis was performed on a 8890B-5977B GC/MSD instrument (Agilent technologies Co.). A high pressure FFAP capillary column (30 m 0.25mm 0.25 μm; agilent J & W Scientific, folsom, calif., USA) was used to carry high purity helium gas at a flow rate of 1.0mL/min and an inlet temperature of 260 ℃. The injection volume was 1. Mu.L, the lysis ratio was 10:1, and the solvent delay time was 2.5min. The initial temperature of the column chamber is 80 ℃; the temperature was raised to 120℃at 40℃per minute, then to 200℃at 10℃per minute, and finally maintained at 230℃for 3 minutes;
4) The levels of LPS, inflammatory factors MCP-1, TNF- α and IL-6 in serum were determined using the Elisa kit.
TABLE 2 animal Experimental raw Material List of Collybia Albuminosa polysaccharide OrPs-1
3. Experimental results
Table 3 results of comparison
1) Mouse experiment:
as can be seen from table 3, after 40 days of feeding, the model group mice had the most weight gain, and had constituted an obesity model as compared with the normal group. Compared with the model group, the increase of the body weight of the experimental group is obviously reduced, and the experimental group has no obvious difference from the positive control group, and compared with the normal group, the SCFAs in the feces of the model group are obviously reduced; the amounts of acetic acid, propionic acid, butyric acid and valeric acid in the stool of the experimental group were significantly increased compared to the model group. The contents of acetic acid, propionic acid, butyric acid and valeric acid in the experimental group are slightly higher than those in the positive control group, no obvious difference exists, and the content of SCFAs in intestinal tracts of obese mice is obviously improved by the black termitomyces albuminosus polysaccharide OrPs-1 in the experimental group as a whole;
2) Microbial flora structure and diversity analysis:
as shown in fig. 1, the intestinal flora of mice is mainly composed of 7 phyla, namely Firmicutes, bacteroides (bacterioides), campylobacter (campylobacter), proteus (deglycobacter), actinomycetes (actylobacter), verrucomicrobios (verrucomicrobios), and deironides (Firmicutes), with bacteroides and Firmicutes being dominant flora. On the door level, compared with a normal group, the model group bacteroides and campylobacter are reduced, and the firmicutes and the proteus are increased; compared with the model group, the Proteus phylum and the actinomycete phylum of the experimental group are slightly reduced, and the thick-wall phylum and the bacteroides phylum are slightly increased; compared with the model group, the positive control group has fewer Proteus, and the Bacteroides and actinomycetes are increased; compared with a positive control group, the bacteroides, actinomycetes and wart microbacteria of the experimental group are reduced, and the firmicutes and the proteus are increased;
as shown in fig. 2 and 3, the differential flora between the model group and the normal group is mainly Faecalibaculum, blautia; the main differential flora between the experimental group and the positive control group is Blautia, dubosiella; faecalibaculum, blautia (fig. 2) appears for all three groups except the model group; model group Dubosiella, norank _f __ Muribaculaceae, lachnospiraceae _nk4a136_group decreased, faecalibaculum, unclassified _f __ lachnospirace increased compared to normal group (fig. 3A); faecalibaculum, dubosiella, norank _f __ Desulfovinbrionaceae was reduced in the experimental group compared to the model group; unclassified_f __ Lachnospiraceae, blautia (fig. 3B); the positive control group Faecalibaculum, unclassified _f_ Lachnospiraceae, norank _f_Desulfovinationaceae decreased, dubosiella, norank _f_Eubacterium_co-prostanols_group increased compared to the model group (FIG. 3C); compared to the positive control group, experimental group Dubosiella, faecalibaculum was reduced and unclassified_f_ Lachnospiraceae, blautia was increased (fig. 3D);
the intervention of OrPs-1 reduced the levels of LPS and inflammatory factors in HFD mouse serum (FIG. 4).
The above research results show that the black termitomyces albuminosus polysaccharide OrPs-1 prepared in example 1 can reduce the abundance of Faecalibaculum, which is a pro-inflammatory bacterium, possibly damaging the intestinal barrier; up-regulating the abundance of Blautia, which can prevent colonization of pathogens by producing bacteriocins, and exhibit anti-inflammatory properties and maintain glucose homeostasis by up-regulating SCFA production, thereby reducing obesity-related diseases, effectively regulating intestinal flora structure and inhibiting the production of obesity; in addition, the black skin termitomyces albuminosus polysaccharide OrPs-1 also increases the content of small molecular organic acids such as acetic acid, propionic acid, butyric acid, valeric acid and the like in the intestinal tract of a mouse, and improves the condition of regulating and controlling the energy supply mediated by SCFAs by selectively enriching the SCFAs to promote the growth of Blaustia bacteria; in conclusion, the black termitomyces albuminosus polysaccharide OrPs-1 can be used as a potential prebiotic for preventing obesity.
Finally, the following descriptions are used: the foregoing is merely a preferred experiment of the present invention, and the present invention is not limited thereto, but it is to be understood that the present invention is described in detail with reference to the foregoing embodiments, and those skilled in the art may modify the technical solutions described in the foregoing embodiments or may equally substitute some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The black termitomyces albuminosus polysaccharide is characterized in that the molecular weight of the black termitomyces albuminosus polysaccharide OrPs-1 is 6742Da, the total sugar content of the black termitomyces albuminosus polysaccharide OrPs-1 is 98.9%, and the monosaccharide composition of the black termitomyces albuminosus polysaccharide is mannose, glucose, xylose, arabinose=12.16:74.16:10.5:3.18.
2. A process for preparing a termitomyces albuminosus polysaccharide according to claim 1, characterized by the specific steps of:
1) Taking black skin termitomyces albuminosus as a raw material, degreasing, carrying out ultrasonic treatment, carrying out water boiling extraction, deproteinizing and decoloring to obtain a crude extract;
2) Separating the crude extract obtained in the step 1) by adopting a DEAE cellulose DE-52 chromatographic column to obtain crude polysaccharides OrPs of Collybia albuminosa;
3) Purifying the black skin termitomyces albuminosus polysaccharide OrPs obtained in the step 2) by adopting a propylene glucan gel S-400HR gel column to obtain single black skin termitomyces albuminosus polysaccharide OrPs-1.
3. The method for preparing the black skin termitomyces albuminosus polysaccharide according to claim 2, wherein the specific method for obtaining the crude extract in step 1) is as follows:
degreasing: drying and crushing black skin termitomyces albuminosus fruiting body, sieving with 18 mesh sieve to obtain black skin termitomyces albuminosus fruiting body powder, placing the black skin termitomyces albuminosus fruiting body powder into a 200 mesh nylon mesh bag, fastening the mesh bag mouth, and soaking in a container containing 95% ethanol for degreasing to obtain degreased product;
and (3) ultrasonic treatment: adding deionized water 20-30 times of the degreased material, and performing ultrasonic treatment at room temperature under 300-600W for 30-60min to obtain an ultrasonic treated material;
decocting and extracting: placing the ultrasonic treated material in a water decoction extraction container, extracting at 75-98deg.C for 3-5 hr, filtering, repeatedly extracting the residue for 2-4 times, collecting filtrate, and concentrating to obtain concentrate;
deproteinization: repeatedly freezing and thawing the concentrate in a refrigerator at-80deg.C for 2-4 times, thawing, centrifuging to remove protein, collecting supernatant, collecting 500ml supernatant, adding absolute ethanol under stirring to obtain ethanol with final concentration of 70-80%, standing at room temperature for 24 hr, filtering, collecting precipitate, lyophilizing, and pulverizing to obtain polysaccharide powder;
decoloring: adding 5g of polysaccharide powder into 5-10 times of deionized water for dissolving, centrifuging, retaining supernatant, and decolorizing the supernatant with macroporous resin to obtain crude extract.
4. The method for preparing the black skin termitomyces albuminosus polysaccharide according to claim 3, wherein the ultrasonic power is 400W, and the ultrasonic treatment time is 45min.
5. The preparation method of the black termitomyces albuminosus polysaccharide according to claim 2, wherein the specific method for obtaining the crude black termitomyces albuminosus polysaccharide OrPs in the step 2) is as follows:
taking 10mL of the crude extract in the step 1), separating by DEAE cellulose DE-52, eluting by deionized water at the flow rate of 2mL/min, collecting the eluent, concentrating by rotary evaporation, and freeze-drying to obtain the black skin termitomyces albuminosus polysaccharide OrPs.
6. The preparation method of the black termitomyces albuminosus polysaccharide according to claim 2, wherein the specific method for obtaining single black termitomyces albuminosus polysaccharide OrPs-1 in the step 3) is as follows:
taking 20mg of the Collybia albuminosa polysaccharide OrPs in the step 2), dissolving in 3mL of deionized water, centrifuging at 6000 rpm for 6min, taking supernatant, placing the supernatant on a propylene sephadex S-400HR gel column, purifying by adopting 0.2M ammonium bicarbonate eluent with the flow rate of 0.13mL/min, collecting the eluent, removing ammonium bicarbonate by rotary evaporation, concentrating, and freeze-drying to obtain single Collybia albuminosa polysaccharide OrPs-1.
7. The preparation method of the black skin termitomyces albuminosus polysaccharide according to claim 5, wherein the monosaccharide composition of the black skin termitomyces albuminosus polysaccharide OrPs is mannose, galactose, xylose, arabinose, fucose=13.39:35.65:38.07:0.58:1.94:10.38 according to a molar ratio.
8. Use of the black termitomyces albuminosus polysaccharide OrPs-1 according to any one of claims 1-7 in the preparation of a medicament, food, health product for reducing weight and regulating intestinal flora disorders.
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|---|---|---|---|---|
| CN102702377A (en) * | 2012-06-21 | 2012-10-03 | 四川农业大学 | Termitomyces albuminosus polysaccharide separation and purification method and injection thereof |
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| CN110627919A (en) * | 2019-11-06 | 2019-12-31 | 安徽大学 | Intestinal prebiotics black skin termitomyces albuminosus polysaccharide ORP-1 and preparation method and application thereof |
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| CN102702377A (en) * | 2012-06-21 | 2012-10-03 | 四川农业大学 | Termitomyces albuminosus polysaccharide separation and purification method and injection thereof |
| CN209024434U (en) * | 2018-10-24 | 2019-06-25 | 四川农业大学 | The extract equipment of long root oudemansiella radicata polysaccharide |
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