Auricularia auricula polysaccharide, its application and preparation method thereof

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.