CN111334440A - Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide - Google Patents
Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide Download PDFInfo
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Abstract
The invention discloses rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide, wherein the application method comprises the following steps: inoculating Rhizopus oryzae CH5 spore in Ganoderma sinensis powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, and oven drying the fermented product to obtain fermented Ganoderma sinensis powder; decolorizing the fermented Ganoderma sinensis powder to obtain decolorized Ganoderma sinensis powder; extracting the decolorized Ganoderma sinensis powder with water, and concentrating to obtain concentrated solution; precipitating the concentrated solution with ethanol and vacuum drying to obtain crude polysaccharide of Ganoderma. Rhizopus oryzae CH5 is separated from microorganism enrichment culture of Ganoderma sinense, and excellent strain obtained by screening is moderately grown in Ganoderma sinense powder to generate polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure of Ganoderma sinense, promote dissociation of soluble polysaccharide in binding state, and significantly improve extraction yield of Ganoderma sinense polysaccharide. Under the optimized technological conditions, the extraction yield of the ganoderma sinensis polysaccharide is 5.92 percent, and is improved by over 35 percent compared with the extraction method without fermentation.
Description
(I) technical field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for improving extraction yield of ganoderma sinensis polysaccharide by adopting a microbial fermentation method.
(II) background of the invention
Ganoderma sinensis (Ganoderma sinense) is also called black Ganoderma and XUANZHI, also called Chinese Ganoderma and Ganoderma Mesona, and is fungus of Ganoderma of Polyporaceae of Basidiomycetes. Ganoderma sinensis is one of the famous and precious traditional Chinese medicines, Ganoderma lucidum, recorded in compendium of materia medica: the purple ganoderma is a woody ganoderma, and has sweet, warm and nontoxic smell; mainly treats deafness, benefits joints, keeps spirit, benefits vital essence and energy, strengthens bones and muscles, has good color, is taken for a long time, is light and not old and prolongs life. The ganoderma lucidum, one of ganoderma lucidum in ganoderma sinensis of genus ganoderma, recorded in the first pharmacopoeia of China in 2015 edition, is sweet and neutral in nature and flavor, and enters heart, lung, liver and kidney meridians. Has the functions of invigorating qi, tranquilizing, relieving cough and asthma, and is mainly used. Restlessness of heart-mind, insomnia, palpitation, cough and asthma due to lung deficiency, asthenia, shortness of breath, poor appetite.
Modern science proves that ganoderma sinensis has wide pharmacological action and various health care values, more than 150 components are separated from ganoderma sinensis at present, the main physiological active substances of ganoderma sinensis comprise polysaccharides, triterpenes, grease, proteins, alkaloids, sterols and the like, wherein polysaccharides are particularly concerned, and the ganoderma sinensis polysaccharide products have the effects of resisting tumors, regulating immunity, resisting oxidation, resisting aging, reducing blood sugar and blood fat and the like, so the ganoderma sinensis polysaccharide products are favored by people. At present, Ganoderma lucidum polysaccharide tablets are sold in the market, have the effects of nourishing and strengthening, nourishing the heart and soothing the nerves, and are used for treating neurasthenia, leukopenia and thrombocytopenia, ionizing radiation and occupational hematopoietic injury, leukopenia of tumor patients after radiotherapy or chemotherapy and the like.
At present, ganoderma lucidum polysaccharide is mainly extracted from ganoderma lucidum fruiting bodies, people make more researches on extraction technology of ganoderma lucidum polysaccharide, and extraction methods such as a hot water extraction method, an ultrasonic method and a supercritical method are available at present. Although some advanced techniques greatly improve the yield of polysaccharides, some methods can destroy the structure of the polysaccharides and reduce their physiological activity. In order to further improve the extraction yield of the ganoderma sinensis polysaccharide, the invention improves the traditional hot water extraction process of the polysaccharide and adds microbial fermentation pretreatment, thereby improving the extraction yield of the ganoderma sinensis polysaccharide.
Disclosure of the invention
The invention aims to provide a novel microbial strain Rhizopus oryzae (Rhizopus oryzae) CH5 and application thereof in extraction of Ganoderma sinensis polysaccharide, and the yield of Ganoderma sinensis polysaccharide can be remarkably improved.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain Rhizopus oryzae (Rhizopus oryzae) CH5, which is preserved in Guangdong province microorganism strain preservation center with the preservation number: GDMCC No. 60990, preservation date 2020, 3 months and 27 days, address: building No. 59, building No. 5 of Jie No. 100 of the first Lianzhou city, Guangdong province; a zip code 510070.
The rhizopus oryzae CH5 is an excellent strain obtained by separating and screening a microorganism enrichment culture of ganoderma sinensis. The morphological characteristics of the rhizopus oryzae CH5 are as follows: culturing on potato agar plate culture medium at 28 deg.C to obtain white fine villus colony, and gradually turning into grey brown after 1 day; the rhizoid is developed and brown; the cyst stalks grow from creeping hypha at the positions of the pseudorhizomes, are upright or slightly bent, and are clustered by 3-5 branches; the sporangium is spherical or elliptical, dark brown, and most conidia are elliptical, with diameter of 7.0-10.0 μm, and brown. A photograph of a colony of Rhizopus oryzae CH5 strain cultured on PDA plate medium at 28 deg.C for 3d is shown in figure 1. The nucleotide sequence of rDNA ribose internal transcribed spacer (rDNA-ITS) of Rhizopus oryzae CH5 is shown in SEQ ID NO. 1.
The invention also provides an application of the rhizopus oryzae CH5 in extraction of ganoderma sinensis polysaccharide, and the application method comprises the following steps: inoculating Rhizopus oryzae CH5 spore in Ganoderma sinensis powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, and oven drying (preferably 85 deg.C) to obtain fermented Ganoderma sinensis powder; decolorizing the fermented Ganoderma sinensis powder to obtain decolorized Ganoderma sinensis powder; extracting the decolorized Ganoderma sinensis powder with water, and concentrating to obtain concentrated solution; precipitating the concentrated solution with ethanol and vacuum drying to obtain crude polysaccharide of Ganoderma.
Furthermore, the volume of the sterile water is 1.5-2.0 mL/g calculated by the weight of the Ganoderma sinensis powder, and the Ganoderma sinensis powder is obtained by crushing Ganoderma sinensis and sieving with a 40-mesh sieve.
Further, the inoculation amount of the rhizopus oryzae CH5 spores is 3-5 × 10 by weight of ganoderma sinensis powder7Per gram.
Further, the Rhizopus oryzae CH5 spore is added in the form of spore solution, and the spore solution is prepared by inoculating Rhizopus oryzae CH5 with PDA plate culture medium, culturing at 28-30 deg.C for 2-3 d, adding sterile water into the plate culture, suspending the spore with inoculating loop to obtain spore solution, preferably transferring the spore solution into sterile test tube, and adjusting spore concentration to 3-5 × 10 with sterile water8one/mL. The final concentration composition of the PDA plate culture medium (potato sucrose agar culture medium) is as follows: 200g/L of potato (filtering to obtain juice after boiling for 30 min), 20g/L of cane sugar, 20g/L of agar, and a solvent which is tap water, wherein the pH value is natural (6.5 is actually measured).
Further, the decoloring method comprises the following steps: adding acetone into fermented Ganoderma powder, decolorizing at 50 deg.C under oscillation at 100r/min for 1-2 times (preferably 2 hr each time), and drying at 50 deg.C to constant weight to obtain decolorized Ganoderma powder; the volume dosage of the acetone is 5-10 mL/g calculated by the weight of the Ganoderma sinensis before fermentation.
Further, the preparation method of the concentrated solution comprises the following steps: adding deionized water into decolorized Ganoderma powder, stirring, leaching in 90-95 deg.C constant temperature water bath for 2-4 hr, filtering while hot, and concentrating the filtrate at 65 deg.C under reduced pressure to 1/4-1/5 of original volume to obtain concentrated solution; the volume dosage of the deionized water is 15-20 mL/g based on the weight of the Ganoderma sinensis before fermentation.
Further, the method for carrying out alcohol precipitation and vacuum drying on the concentrated solution comprises the following steps: adding 3-4 times volume of 95% ethanol into the concentrated solution, standing at room temperature for precipitation for 10-15 h, vacuum filtering with Buchner funnel, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain crude polysaccharide of Ganoderma.
Compared with the prior art, the invention has the following beneficial effects: the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of Ganoderma sinensis polysaccharide decolorization water. Rhizopus oryzae CH5 is separated from microorganism enrichment culture of Ganoderma sinense, and excellent strain obtained by screening is moderately grown in Ganoderma sinense powder to generate polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure of Ganoderma sinense, promote dissociation of soluble polysaccharide in binding state, and significantly improve extraction yield of Ganoderma sinense polysaccharide. Under the optimized technological conditions, the extraction yield of the ganoderma sinensis polysaccharide is 5.92 percent, and is improved by over 35 percent compared with the extraction method without fermentation.
(IV) description of the drawings
FIG. 1 colony morphology of Rhizopus oryzae CH 5.
FIG. 2 is a standard curve for measuring polysaccharide by phenol-sulfuric acid method.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the purple lucid Ganoderma in the embodiment of the invention is fruiting body of fungus purple lucid Ganoderma (Ganoderma sinense), and the biological classification position is (refer to Mycobank, http:// www.mycobank.org): kingdom Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricales (Agaricamycetes), Polyporales (Polyporales), Ganodermataceae (Ganoderma), and Ganoderma (Ganoderma).
The room temperature of the invention is 25-30 ℃.
Example 1 isolation, screening and identification of fermentation strains
1. Strain CH5 isolation and screening
(1) Adding 20g of Ganoderma sinensis powder crushed and sieved with 40 mesh sieve into 250-mL triangular flask, adding 30mL of sterile water for wetting, culturing at 28 deg.C for 4d, diluting the enriched culture of overgrown mold with sterile water to 1 × 106Coating the mixture on a PDA plate culture medium after doubling, culturing at the constant temperature of 28 ℃ for 3 days, selecting mould bacterial colonies with different colors and forms, transferring the mould bacterial colonies to a fresh PDA culture medium, culturing at the constant temperature of 28 ℃ for 3 days to obtain 7 pure culture strains, adding 5mL of sterile water into fresh plate cultures of the strains with the numbers of CH 1-CH 7.7 respectively, stirring by using an inoculating loop to suspend spores, transferring the spore liquid to a sterile test tube, and adjusting the concentration of the spores to be 3 × 10 by using the sterile water8one/mL.
The plate culture medium is a potato sucrose agar culture medium (PDA) and is prepared according to the following components and methods: cleaning potato, peeling, cutting into small pieces, weighing 200g, adding 1000mL of tap water, boiling for 30min, filtering with 4 layers of gauze to remove residues, adding the filtrate to 1000mL, adding 20g of sucrose and 20g of agar, naturally measuring pH (actually measured to be 6.5), heating until the agar is dissolved, placing in a triangular flask, sterilizing at 121 ℃ by high-pressure steam for 15min, pouring into a sterile culture dish with the diameter of 9cm, and cooling for later use.
(2) Sterilizing in 250-mL triangular flask at 160 deg.C for 2 hr, adding 10g of pulverized Ganoderma sinensis powder sieved with 40 mesh sieve, adding 15mL of sterile water, inoculating 1mL of spore solution of above strains, and stirring. Tying the triangular flask with eight layers of gauze, culturing at 28 deg.C for 3d, oven drying at 85 deg.C to constant weight to obtain fermented Ganoderma powder.
(3) And (3) adding 100mL of acetone into all the fermented ganoderma sinensis powder obtained in the step (2), oscillating at 50 ℃ and 100r/min for 2h, filtering by using a Buchner funnel to remove the acetone, and drying the ganoderma sinensis powder at 50 ℃ to constant weight to obtain the decolored ganoderma sinensis powder.
(4) Adding 150mL of deionized water into the all decolorized Ganoderma sinensis powder obtained in step (3), shaking uniformly, placing in a constant temperature water bath kettle at 85 ℃, leaching for 2h, and shaking by hand once every 15 min. After water extraction is finished, suction-filtering by using a Buchner funnel while the water is hot, concentrating the filtrate at 65 ℃ under reduced pressure to 30mL, adding 90mL of 95% ethanol, standing and precipitating for 10h, suction-filtering by using the Buchner funnel, collecting polysaccharide precipitate, drying at 60 ℃ in vacuum for 10h to obtain crude polysaccharide of the ganoderma sinensis, weighing, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
Under the same conditions, when the non-inoculated spore liquid is used as an unfermented control, the extraction yield of the polysaccharide after the ganoderma sinensis powder is fermented by different strains is shown in table 1.
TABLE 1 extraction yield of polysaccharide from Ganoderma sinensis fermented with different strains
As can be seen from the data in Table 1, after the Ganoderma sinensis is fermented by the strain CH5, compared with the unfermented control, the extraction yield of polysaccharide is improved to the highest extent, which reaches 15.3%.
The content of the ganoderma sinensis polysaccharide is determined by adopting a phenol-sulfuric acid method, and the specific method comprises the following steps: preparing the crude polysaccharide of Ganoderma sinensis into 0.1mg/mL aqueous solution as sample solution. Sucking 2mL of sample solution into a 25mL colorimetric tube, adding 1mL of 6% phenol aqueous solution, shaking uniformly, quickly dropwise adding 5mL of concentrated sulfuric acid, standing at room temperature for 10min after shaking uniformly, heating in a boiling water bath for 15min, and cooling to room temperature with running water. Absorbance A was measured with 2mL of distilled water as a reference for the same treatment as a blank490. Determination of A of samples of aqueous glucose solutions of different concentrations (g/L) in the same way490Plotting the glucose concentration-A490And (3) obtaining a regression equation by using a standard curve (figure 2), and calculating and measuring the polysaccharide content in the crude polysaccharide sample of the ganoderma sinensis by using the regression equation.
2. Identification of Strain CH5
Culturing Rhizopus oryzae CH5 on potato agar plate culture medium at 28 deg.C to obtain white fine villus at the early stage of colony, and gradually turning into grey brown after 1 day; the rhizoid is developed and brown; the cyst stalks grow from creeping hypha at the positions of the pseudorhizomes, are upright or slightly bent, and are clustered by 3-5 branches; the sporangium is spherical or elliptical, dark brown, and most conidia are elliptical, with diameter of 7.0-10.0 μm, and brown. A photograph of a colony of Rhizopus oryzae CH5 strain cultured on PDA plate medium at 28 deg.C for 3d is shown in figure 1.
The strain CH5 is handed to the Biotechnology engineering (Shanghai) limited company for identification, the nucleotide sequence of rDNA ribose in vivo transcription spacer region (rDNA-ITS) is determined to be shown as SEQ ID NO.1, and the biological classification position of the strain CH5 is determined according to the strain CH5 colony characteristics and rDNA-ITS nucleotide sequence alignment (refer to Mycobank, http:// www.mycobank.org): kingdom fungoides (Fungi), phylum mucor (mucomyceta), class Mucorales (mucomycetes), order Mucorales (Mucorales), family Rhizopus (Rhizopus), genus Rhizopus (Rhizopus), Rhizopus oryzae (Rhizopus oryzae). Namely, the strain is Rhizopus oryzae (Rhizopus oryzae) CH5, is preserved in Guangdong province microorganism strain preservation center, and has the preservation number: GDMCC No. 60990, preservation date 2020, 3 months and 27 days.
Example 2 application of Rhizopus oryzae CH5 to extraction of Ganoderma sinensis polysaccharide
(1) Preparation of Rhizopus oryzae CH5 spore solution by inoculating Rhizopus oryzae CH5 plate colony stored in refrigerator at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5mL sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring the spore solution into sterile test tube, adjusting spore concentration to 4 × 10 with sterile water8Per mL; the components and the preparation method of the PDA plate culture medium are the same as those of the example 1;
(2) and (3) carrying out dry heat sterilization on a 500-mL triangular flask at 160 ℃ for 2h, adding 20g of the purple lucid ganoderma powder which is crushed and sieved by a 40-mesh sieve, adding 30mL of sterile water, inoculating 2mL of the rhizopus oryzae CH5 spore suspension prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, culturing at 28 deg.C for 3d, oven drying at 85 deg.C to constant weight to obtain fermented Ganoderma powder.
(3) And (3) adding 100mL of acetone into all the fermented ganoderma sinensis powder obtained in the step (2), oscillating at 50 ℃ for 2h at 100r/min, filtering by using a Buchner funnel to remove the acetone, repeating the operations (adding the acetone, oscillating and filtering) for 1 time, and drying at 50 ℃ to constant weight to obtain the decolored ganoderma sinensis powder.
(4) And (4) adding 300mL of deionized water into all the decolorized Ganoderma sinensis powder obtained in the step (3), uniformly stirring, placing in a constant-temperature water bath kettle at 90 ℃, leaching for 3h, and shaking by hand once at intervals of 15 min. The filtrate was filtered through a Buchner funnel while hot, and concentrated to 75mL under reduced pressure at 65 ℃ to obtain a concentrated solution.
(5) And (4) adding 300mL of 95% ethanol into all the concentrated solution obtained in the step (4), standing and precipitating at room temperature for 12h, then carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying at 60 ℃ for 10h to obtain crude polysaccharide of the ganoderma sinensis, weighing, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 1.76g of crude ganoderma sinensis polysaccharide with the polysaccharide content of 65.6 percent is obtained, namely 1.15g of ganoderma sinensis polysaccharide is obtained, the extraction yield is 5.75 percent, and the product is light brown powder.
Example 3 application of Rhizopus oryzae CH5 to extraction of Ganoderma sinensis polysaccharide
(1) Preparation of rhizopus oryzae CH5 spore liquid: inoculating Rhizopus oryzae CH5 (freeze-dried tube or plate colony) in fresh PDA plate culture medium, culturing at 28 deg.C for 3 days, and adding 5mL sterile waterIn plate culture, spore suspension is obtained by inoculating with inoculating loop, transferring spore solution into sterile test tube, and adjusting spore concentration to 5 × 10 with sterile water8Per mL; the components and the preparation method of the PDA plate culture medium are the same as those of the example 1;
(2) and (3) carrying out dry heat sterilization on a 2-L triangular flask at 160 ℃ for 2h, adding 50g of the purple lucid ganoderma powder which is crushed and sieved by a 40-mesh sieve, adding 100mL of sterile water, inoculating 5mL of the rhizopus oryzae CH5 spore suspension prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, culturing at 30 deg.C for 2d, oven drying at 85 deg.C to constant weight to obtain fermented Ganoderma powder.
(3) Adding 250mL of acetone into the whole fermented ganoderma sinensis powder obtained in the step (2), oscillating at 50 ℃ and 100r/min for 2h, filtering by using a Buchner funnel to remove the acetone, repeating the operation for 1 time (same as the example 2), and drying at 50 ℃ to constant weight to obtain the decolored ganoderma sinensis powder.
(4) And (4) adding 1000mL of deionized water into all the decolorized Ganoderma sinensis powder obtained in the step (3), uniformly stirring, placing in a constant-temperature water bath kettle at 95 ℃, leaching for 4h, shaking by hand once at intervals of 15min, filtering by a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 200mL to obtain a concentrated solution.
(5) And (4) adding 800mL of 95% ethanol into all the concentrated solution obtained in the step (4), standing and precipitating at room temperature for 15h, then carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying at 60 ℃ for 10h to obtain crude polysaccharide of the ganoderma sinensis, weighing, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 4.74g of crude ganoderma sinensis polysaccharide is obtained, the polysaccharide content is 64.1%, 3.04g of ganoderma sinensis polysaccharide is obtained, the extraction yield is 6.08%, and the product is light brown powder.
Example 4 application of Rhizopus oryzae CH5 to extraction of Ganoderma sinensis polysaccharide
(1) Preparation of Rhizopus oryzae CH5 spore solution by inoculating low-temperature preserved Rhizopus oryzae CH5 (lyophilized tube or plate colony) into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5mL sterile water into the plate culture, suspending spores by using inoculating ring to obtain Rhizopus oryzae CH5 spore suspension, adjusting spore concentration to 5 × 10 with sterile water8Per mL; the components of the PDA plate culture medium and the preparation methodThe same as example 1;
(2) fermentation: and (3) carrying out dry heat sterilization on a 5-L triangular flask at 160 ℃ for 2h, adding 100g of the purple lucid ganoderma powder which is crushed and sieved by a 40-mesh sieve, adding 200mL of sterile water, inoculating 10mL of the rhizopus oryzae CH5 spore suspension prepared in the step (1), and uniformly stirring. Tying the triangular flask with eight layers of gauze, culturing at 30 deg.C for 2d, oven drying at 85 deg.C to constant weight to obtain fermented Ganoderma powder.
(3) Adding 500mL of acetone into the whole fermented ganoderma sinensis powder obtained in the step (2), oscillating at 50 ℃ and 100r/min for 2h, filtering by using a Buchner funnel to remove the acetone, repeating the operation for 1 time (same as the example 2), and drying the ganoderma sinensis powder at 50 ℃ to constant weight to obtain the decolored ganoderma sinensis powder.
(4) Adding 2000mL of deionized water into the whole decolorized Ganoderma sinensis powder obtained in the step (3), uniformly stirring, placing in a constant-temperature water bath kettle at 95 ℃, leaching for 4h, shaking by hand once at intervals of 15min, filtering by a hot Buchner funnel, and concentrating the filtrate at 65 ℃ under reduced pressure to 400 mL.
(5) And (4) adding 1600mL of 95% ethanol into all the concentrated solution obtained in the step (4), standing and precipitating at room temperature for 15h, then carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying at 60 ℃ for 10h to obtain crude ganoderma sinensis polysaccharide, weighing, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 9.56g of crude ganoderma sinensis polysaccharide with the polysaccharide content of 63.9 percent is obtained, 6.11g of ganoderma sinensis polysaccharide is obtained, the extraction yield is 6.11 percent, and the product is light brown powder.
If the fermentation process of the step (2) in the embodiment is not performed, other steps are performed according to the method in the embodiment, 7.03g of crude polysaccharide of Ganoderma sinensis can be obtained by extracting 100g of Ganoderma sinensis powder, the polysaccharide content is 64.2%, 4.51g of Ganoderma sinensis polysaccharide can be obtained, and the extraction yield is 4.51%. As can be seen, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic water treatment of the ganoderma sinensis polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the ganoderma sinensis polysaccharide can be improved by 35.4%.
Sequence listing
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Claims (9)
1. Rhizopus oryzae (Rhizopus oryzae) CH5, deposited at the Guangdong province center for the culture Collection of microorganisms with the deposit number: GDMCC No. 60990, preservation date 2020, 3 months and 27 days, address: building No. 59, building No. 5 of Jie No. 100 of the first Lianzhou city, Guangdong province; a zip code 510070.
2. An application of Rhizopus oryzae CH5 in extraction of Ganoderma sinensis polysaccharide as claimed in claim 1.
3. The application according to claim 2, characterized in that the method of application is: inoculating Rhizopus oryzae CH5 spore in Ganoderma sinensis powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, and oven drying the fermented product to obtain fermented Ganoderma sinensis powder; decolorizing the fermented Ganoderma sinensis powder to obtain decolorized Ganoderma sinensis powder; extracting the decolorized Ganoderma sinensis powder with water, and concentrating to obtain concentrated solution; precipitating the concentrated solution with ethanol and vacuum drying to obtain crude polysaccharide of Ganoderma.
4. The use according to claim 3, wherein the sterile water is added in a volume of 1.5-2.5 mL/g based on the weight of Ganoderma sinensis powder.
5. The use as claimed in claim 3, wherein the Rhizopus oryzae CH5 spore inoculum size is 3-5 × 107Per gram.
6. The use according to claim 3, wherein the Rhizopus oryzae CH5 is inoculated in the form of a Rhizopus oryzae CH5 spore liquid, and the Rhizopus oryzae CH5 spore liquid is prepared as follows: inoculating Rhizopus oryzae CH5 in PDA plate culture medium, culturing at 28-30 deg.C for 2-3 days, adding sterile water, inoculating loop, and stirring to suspend spore to obtain Rhizopus oryzae CH5 spore solution; the final concentration composition of the PDA plate culture medium is as follows: 200g/L of potato, 20g/L of cane sugar, 20g/L of agar and a natural pH value, wherein the solvent is tap water.
7. Use according to claim 3, characterized in that the bleaching process is: adding acetone into fermented Ganoderma powder, decolorizing at 50 deg.C and 100r/min under oscillation for 1-2 times, and drying at 50 deg.C to constant weight to obtain decolorized Ganoderma powder; the volume dosage of the acetone is 5-10 mL/g calculated by the weight of the Ganoderma sinensis before fermentation.
8. The use according to claim 3, wherein the concentrate is prepared by a process comprising: adding deionized water into decolorized Ganoderma powder, stirring, leaching in 90-95 deg.C constant temperature water bath for 2-4 hr, filtering while hot, and concentrating the filtrate at 65 deg.C under reduced pressure to 1/4-1/5 of original volume to obtain concentrated solution; the volume dosage of the deionized water is 15-20 mL/g based on the weight of the Ganoderma sinensis before fermentation.
9. The use as claimed in claim 3, wherein the method of precipitating the concentrate with ethanol and vacuum drying comprises: adding 3-4 times volume of 95% ethanol into the concentrated solution, standing at room temperature for precipitation for 10-15 h, vacuum filtering, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain crude polysaccharide of Ganoderma.
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