# US 12,760,127 B2 · Auricularia auricula polysaccharide, its application and preparation method thereof

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- Number: US 12,760,127 B2
- Document ID: US12760127B2
- Status: Granted
- Kind: B2
- Application number: 18/015,393
- Filed: 2022-09-07
- Published: 2025-07-10
- Granted: 2026-10-13
- Earliest claimed: 2022-08-31
- Assignees: Beijing Beiqing Boyu Information Technology Research Co. Ltd
- Applicants: Beijing Beiqing Boyu Information Technology Research Co. Ltd
- Inventors: Sun Sun Chan
- Web page: https://patentgrep.com/patent/US12760127B2

## CPC

- C08B 37/0003: {General processes for their isolation or fractionation, e.g. purification or extraction from biomass}
- A61K 31/715: Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61P 3/06: Antihyperlipidemics
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P 19/20: produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The invention provides an Auricularia auricula polysaccharide, its application and preparation method thereof, which belong to the technical field of improving polysaccharide. After being degreased, the Auricularia auricula is subjected to preliminary enzymolysis under the effect of helicase, is further subjected to deep enzymolysis under the effect of complex enzyme through synergetic ultrasound-assisted degradation and extraction with H2O2, and is subjected to mixed fermentation by Lactobacillus bulgaricus, Streptococcus thermophilus and Bifidobacterium longum, the obtained fermented Auricularia auricula polysaccharide is subjected to a phosphorylation reaction under the effect of a phosphorylation reagent, and after further deproteinization and decolorization, the obtained fermented Auricularia auricula polysaccharide is chelated with zinc salt to obtain a polysaccharide-zinc compound, and the amount of the extracted Auricularia auricula polysaccharide is improved. The finished product of the invention has the effects of reducing blood sugar and blood fat, regulating total cholesterol and effectively improving HDL cholesterol.

## Claims

### Claim 1

A preparation method of Auricularia auricula polysaccharide, comprising the steps of:
S1) drying and crushing Auricularia auricula, and then degreasing the Auricularia auricula by a supercritical fluid extraction process to obtain a degreased Auricularia auricula;
wherein in S1, conditions of the supercritical fluid extraction process are as follows: a flow rate of CO2 is in a range of 7-12 L/h, a pressure is in a range of 12-25 MPa, a temperature is in a range of 45-60° C., and an extraction time is in a range of 1-2 h;
S2) adding the degreased Auricularia auricula obtained in S1 into water, adding snailase, performing preliminary enzymolysis, deactivating the snailase, concentrating and drying an obtained system to obtain a preliminary enzymolysis product;
S3) adding the preliminary enzymolysis product prepared in S2 into a H2O2 solution, carrying out ultrasonic treatment, adding sodium bisulfite thereto, carrying out alcohol precipitation, centrifuging and drying to obtain an Auricularia auricula raw sugar extract;
S4) dissolving the Auricularia auricula raw sugar extract prepared in S3 in water, adding a complex enzyme thereto, conducting the secondar enzymolysis, and deactivating the complex enzyme to obtain a secondary enzymolysis extract, wherein the complex enzyme is at least one selected from the group consisting of β-glucanase, saccharifying enzyme, cellulase, pectinase, α-amylase, and α-glucosidase;
S5) respectively streaking Lactobacillus bulgaricus, Streptococcus thermophilus and Bifidobacterium longum in a Gauze's medium, and carrying out activation culture to obtain a strain seed liquid;
S6) inoculating the strain seed liquid prepared in S5 into the secondary enzymolysis extract prepared in S4, and carrying out fermentation culture, followed by concentration, alcohol precipitation and centrifugation to obtain a fermented Auricularia auricula polysaccharide;
S7) adding the fermented Auricularia auricula polysaccharide prepared in S6 into water, adding sodium sulfate and a phosphorylation reagent thereto, adjusting a pH value of an obtained mixture to 8.8-9.2, heating and subjecting the resulting mixture to reaction, dialyzing, and concentrating to obtain a phosphorylated Auricularia auricula polysaccharide solution;
S8) adding the phosphorylated Auricularia auricula polysaccharide solution obtained in S7 into a Sevag reagent, subjecting the resultant mixture to reaction under stirring, centrifuging to deproteinize the phosphorylated Auricularia auricula polysaccharide solution,
repeating the preceding deproteinization steps on the resulting liquid 1-3 times and removing the remaining Sevag reagent therein under reduced pressure to obtain a deproteinized Auricularia auricula polysaccharide;
S9) adding activated carbon and the deproteinized Auricularia auricula polysaccharide prepared in S8 into water, performing adsorption under stirring, filtering, performing alcohol precipitation on the resulting liquid, and centrifuging to obtain a refined Auricularia auricula polysaccharide;
S10) dissolving the refined Auricularia auricula polysaccharide prepared in S9 and trisodium citrate in water, adding a zinc salt, adjusting a pH value of the resulting solution to 7.2-7.5, heating and subjecting the resulted mixture to reaction under stirring, filtering, performing alcohol precipitation on the resulting liquid, centrifuging, collecting the resulting solid, and freeze-drying the resulting solid collected to obtain the Auricularia auricula polysaccharide.

### Claim 2 (depends on 1)

The preparation method according to claim 1, wherein
in S2, a mass ratio of the degreased Auricularia auricula to the snailase is in a range of 100:5 to 100:3, and the preliminary enzymolysis is performed at a temperature of 40-50° C. for 1-2 h;
in S3, a concentration of H2O2 in the H2O2 solution is in a range of 2-5 wt %; and the ultrasonic treatment is performed at a power of 1500-2000 W for 30-50 minutes; and
in S4, the complex enzyme is at least two selected from the group consisting of β-glucanase, saccharifying enzyme, cellulase, pectinase, α-amylase, and α-glucosidase; a mass ratio of the Auricularia auricula raw sugar extract to the complex enzyme is in a range of 100:7 to 100:5, and the secondary enzymolysis is performed at a temperature of 40-45° C. for 3-5 hours.

### Claim 3 (depends on 2)

The preparation method according to claim 2, wherein the complex enzyme is a compound mixture of the β-glucanase and the α-glucosidase at a mass ratio of 3:1 to 5:1.

### Claim 4 (depends on 1)

The preparation method according to claim 1, wherein
in S5, conditions of the activation culture are as follows: under a low-oxygen condition, a temperature of 40-45° C., a time of 18-24 hours, and a bacteria content of the strain seed liquid being 108-109 cfu/mL; and
in S6, inoculum sizes of the Lactobacillus bulgaricus, the Streptococcus thermophilus and the Bifidobacterium longum are respectively 3-5%, 1-3% and 1-2%, respectively; the fermentation culture is performed under conditions: the low-oxygen condition, a temperature of 40-45° C., and a time of 36-48 h;
wherein the low-oxygen condition is as follows: a content of O2 is in a range of 5 to 7%, a content of CO2 is in a range of 5 to 10%, and the remaining amount is nitrogen, where % is a volume percentage content.

### Claim 5 (depends on 1)

The preparation method according to claim 1, wherein
in S7, the phosphorylation reagent is at least two selected from the group consisting of polyphosphoric acid, sodium tripolyphosphate, sodium trimetaphosphate, pyrophosphoric acid, and phosphorus pentoxide; a mass ratio of the fermented Auricularia auricula polysaccharide, the sodium sulfate, the phosphorylation reagent, and the water is in a range of 10: 30-50:2-4:100; the resulting mixture is heated and subjected to reaction at a temperature of 70-90° C. for 3-5 hours, a dialysis bag used for the dialyzing has a pore size of 5000-15000 Da, and the dialyzing is performed for 24-48 hours;
in S8, a mass ratio of the phosphorylated Auricularia auricula polysaccharide solution to the Sevage reagent is in a range of 1:7 to 1:3; and the reaction under stirring is performed for 20-30 minutes;
in S9, a mass ratio of the deproteinized Auricularia auricula polysaccharide to the activated carbon is in a range of 100:15 to 100:12; and the adsorption under stirring is performed for 30-50 minutes; and
in S10, a mass ratio of the refined Auricularia auricula polysaccharide, the trisodium citrate, the zinc salt is in a range of 100: 5-12:22-27; the resulted mixture is heated and subjected to reaction at a temperature of 45-55° C. for 1-2 h, under stirring at a stirring speed of 300-500 r/min; and the zinc salt is at least one selected from the group consisting of zinc chloride, zinc sulfate, and zinc nitrate.

### Claim 6 (depends on 5)

The preparation method according to claim 5, wherein the phosphorylation reagent is a mixture of the sodium tripolyphosphate, the sodium trimetaphosphate, and the pyrophosphoric acid at a mass ratio of 3-7:2:0.2-0.4.

### Claim 7

The preparation method comprising the following steps:
S1) drying and crushing the Auricularia auricula, and then degreasing the Auricularia auricula by the supercritical fluid extraction process to obtain the degreased Auricularia auricula;
S2) adding 100 parts by weight of the degreased Auricularia auricula in S1 into 200 parts by weight of water, adding 3-5 parts by weight of the snailase, carrying out the preliminary enzymolysis at 40-50° C. for 1-2 hours, deactivating the snailase at 100-110° C. for 10-15 minutes, concentrating the resulting preliminary enzymolysis mixture with an organic membrane to ⅓-¼ of an original volume of the resulting preliminary enzymolysis mixture, and drying to obtain the preliminary enzymolysis product;
S3) adding 100 parts by weight of the preliminary enzymolysis product prepared in S2 into 100 parts by weight of 2-5 wt % H2O2 solution, carrying out ultrasonic treatment for 30-50 minutes at 1500-2000 W, adding 7-12 parts by weight of sodium bisulfite thereto, carrying out alcohol precipitation, centrifuging and drying to obtain the Auricularia auricula raw sugar extract;
S4) dissolving 100 parts by weight of the Auricularia auricula raw sugar extract prepared in S3 in 100 parts by weight of water, adding 5-7 parts by mass of the complex enzyme, carrying out the secondary enzymolysis at 40-45° C. for 3-5 hours, and deactivating the complex enzyme at 100-110° C. for 10-15 minutes to obtain the secondary enzymolysis extract; wherein the complex enzyme is a compound mixture of β-glucanase and α-glucosidase at a mass ratio of 3:1 to 5:1;
S5) respectively streaking the Lactobacillus bulgaricus, the Streptococcus thermophilus, and the Bifidobacterium longum in the Gauze's medium, performing activation culture at 40-45° C. for 18-24 hours under a low-oxygen condition to obtain the strain seed liquid with a bacteria content of 108-109 cfu/mL;
S6) inoculating the strain seed liquid prepared in S5 into the secondary enzymolysis extract prepared in S4, wherein inoculum sizes of the Lactobacillus bulgaricus, the Streptococcus thermophilus, and the Bifidobacterium longum are respectively 3-5%, 1-3%, and 1-2%, respectively, conducting fermentation culture at 40-45° C. for 36-48 hours under the low-oxygen condition, concentrating, performing alcohol precipitation, and centrifuging to obtain the fermented Auricularia auricula polysaccharide; wherein the low-oxygen condition is as follows: a content of O2 is in a range of 5% to 7%, a content of CO2 is in a range of 5% to 10%, and a balance is nitrogen, where % is a volume percentage content;
S7) adding 10 parts by weight of the fermented Auricularia auricula polysaccharide prepared in S6 into 100 parts by weight of water, adding 30-50 parts by weight of the sodium sulfate and 2-4 parts by weight of the phosphorylation reagent, adjusting the pH value of the obtained mixture to 8.8-9.2, heating and subjecting the resulting mixture to reaction at 70-90° C. for 3-5 hours, dialyzing for 24-48 hours by using a dialysis bag with a pore size of 5000-15000 Da, concentrating the resulting phosphorylation mixture with an organic membrane to ⅓-¼ of an original volume of the resulting phosphorylation mixture to obtain the phosphorylated Auricularia auricula polysaccharide solution; wherein the phosphorylation reagent is a mixture of sodium tripolyphosphate, sodium trimetaphosphate and pyrophosphoric acid with a mass ratio of 3-7:2:0.2-0.4;
S8) adding 10 parts by weight of the phosphorylated Auricularia auricula polysaccharide solution obtained in S7 into 30-70 parts by weight of the Sevag reagent, subjecting the resultant mixture to reaction under stirring for 20-30 min and centrifuging to deproteinize the phosphorylated Auricularia auricula polysaccharide solution,
repeating preceding deproteinization steps on the resulting liquid for 1-3 times, merging resulting liquids, and removing the solvent therein under reduced pressure to obtain the deproteinized Auricularia auricula polysaccharide;
S9) adding 12-15 parts by weight of the activated carbon and 100 parts by weight of the deproteinized Auricularia auricula polysaccharide prepared in S8 into 200 parts by weight of water, performing adsorption under stirring for 30-50 minutes, filtering, performing alcohol precipitation on the resulted liquid, and centrifuging to obtain the refined Auricularia auricula polysaccharide;
S10) dissolving 100 parts by weight of the refined Auricularia auricula polysaccharide prepared in S9 and 5-12 parts by weight of the trisodium citrate in 200 parts by weight of water, adding 22-27 parts by weight of the zinc salt, adjusting the pH value of the resulting solution to 7.2-7.5, heating the resulted mixture to 45-55° C. and subjecting the resulted mixture to reaction under stirring at 300-500 r/min for 1-2 h, filtering, performing alcohol precipitation on the resultant liquid, centrifuging, collecting the resulting solid, and freeze-drying the resulting solid collected to obtain the Auricularia auricula polysaccharide; wherein the alcohol precipitation comprises: adding absolute ethyl alcohol to the resultant liquid until an ethanol content in a resulting system is 75-85%, and subjecting the resulting system to precipitation for 12-24 hours.

### Claim 8 (depends on 1)

A method for reducing blood fat, comprising:
administrating the Auricularia auricula polysaccharide prepared by the preparation method according to claim 1 to a subject in need thereof.

### Claim 9 (depends on 1)

A method for regulating a total cholesterol, comprising:
administrating the Auricularia auricula polysaccharide prepared by the preparation method according to claim 1 to a subject in need thereof.

### Claim 10 (depends on 1)

A method for improving high density lipoprotein (HDL) cholesterol, comprising:
administrating the Auricularia auricula polysaccharide prepared by the preparation method according to claim 1 to a subject in need thereof.

### Claim 11 (depends on 1)

A method for reducing blood sugar, comprising:
administrating the Auricularia auricula polysaccharide prepared by the preparation method according to claim 1 to a subject in need thereof.

### Claim 12 (depends on 1)

A method for resisting inflammation, comprising:
administrating the Auricularia auricula polysaccharide prepared by the preparation method according to claim 1 to a subject in need thereof.