CN111471727A - Method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method - Google Patents
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Abstract
The invention discloses a method for extracting atractylodes macrocephala polysaccharide by using a microbial fermentation method, which comprises the following steps: inoculating Rhizopus oryzae (Rhizopus oryzae) GDMCC No:60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma. The fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide. Rhizopus oryzae CH5 grows moderately in Atractylodis rhizoma powder to generate polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure tissue of Atractylodes macrocephala, promote dissociation of soluble polysaccharide in bound state, and dissolve easily during hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by more than 50%.
Description
(I) technical field
The invention belongs to the technical field of bioengineering, and particularly relates to application of a microbial fermentation technology in extraction of bighead atractylodes rhizome polysaccharide.
(II) background of the invention
The Chinese medicinal material Atractylodis Rhizoma (Atractylodes macrocephala Koizoma), also known as Rhizoma Atractylodis, Rhizoma Atractylodis Macrocephalae, Thunberg Atractylodis, herba seu radix Cirsii Japonici, and Rhizoma Dioscoreae essence, is dried rhizome of Atractylodes macrocephala Koidz of Compositae. The bighead atractylodes rhizome is a traditional Chinese medicinal material used as both medicine and food, is collected in 2015 pharmacopoeia of China, and is bitter, sweet and warm in nature and taste; spleen and stomach meridians entered; has the effects of strengthening spleen, tonifying qi, eliminating dampness, inducing diuresis, suppressing sweating and preventing miscarriage. Mainly treats spleen deficiency type anorexia, abdominal distention and diarrhea, phlegm and fluid retention dizziness and palpitation, edema, spontaneous perspiration and fetal irritability. The application of the white atractylodes rhizome is wide, and the white atractylodes rhizome is an important raw material of more than 40 Chinese patent medicine preparations besides the traditional Chinese medicine prescription; in addition, the tea drink, the wine soaking, the cooking delicacies and the like are usually used as substitute tea in folk, and are good functional health food materials.
The main chemical components of the atractylodes include atractylodes macrocephala polysaccharide, atractylenolide, atractylol, atractylone and the like, wherein the activity of the atractylodes macrocephala polysaccharide is concerned by people. Modern pharmacological studies show that the atractylodes macrocephala polysaccharide has the functions of resisting tumor, resisting oxidation, resisting hypertension, reducing blood sugar, regulating immunity and the like. One of the active ingredients of the Chinese patent medicine Yupingfeng oral liquid is the atractylodes macrocephala polysaccharide extract, which has the efficacy of strengthening the spleen and replenishing qi. As a plant functional polysaccharide, the atractylodes polysaccharide has wide application prospect in medical treatment and health care.
At present, the common extraction methods of the atractylodes macrocephala polysaccharide mainly comprise a hot water extraction method, an ultrasonic extraction method, an enzyme extraction method and the like. Different extraction processes have respective advantages and disadvantages, and the yield of the polysaccharide is greatly different. The hot water extraction method has long time consumption and low polysaccharide extraction rate; although the enzyme extraction method can obtain higher polysaccharide yield, the cost of the enzyme is high, which limits the wide application of the enzyme; the ultrasonic extraction method is characterized in that ultrasonic extraction steps are added on the basis of hot water extraction, and the extraction yield is relatively high, so that the ultrasonic extraction method has relative advantages. In order to further improve the extraction yield of the atractylodes macrocephala polysaccharide, the invention improves the process of the ultrasonic extraction method of the atractylodes macrocephala polysaccharide and adds the step of microbial fermentation pretreatment, thereby improving the extraction yield of the atractylodes macrocephala polysaccharide.
Disclosure of the invention
The invention aims to apply the microbial fermentation technology to the ultrasonic extraction of the atractylis ovata polysaccharide, thereby obviously improving the extraction yield of the atractylis ovata polysaccharide.
The technical scheme adopted by the invention is as follows:
the invention provides a method for extracting atractylodes macrocephala polysaccharide by using a microbial fermentation method, which comprises the following steps: inoculating Rhizopus oryzae (Rhizopus oryzae) GDMCC No:60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma.
Furthermore, the volume dosage of the sterile water is 2-3 m L/g based on the weight of the white atractylodes rhizome powder, and the inoculation amount of the rhizopus oryzae CH5 spores is 2-5 × 10 based on the weight of the white atractylodes rhizome powder7Per gram; the bighead atractylodes rhizome powder is obtained by crushing bighead atractylodes rhizome and then sieving the crushed bighead atractylodes rhizome with a 60-mesh sieve.
Further, the Rhizopus oryzae CH5 spore is added in the form of spore liquid, and the spore liquid is prepared by inoculating Rhizopus oryzae CH5 preserved at low temperature into PDA plate culture medium, culturing at 28-30 deg.C for 2-3 d, adding sterile water into culture dish, suspending the spore with inoculating loop to obtain spore liquid, preferably transferring the spore liquid into sterile test tube, and adjusting spore concentration to 2-5 × 10 with sterile water8The final concentration of the PDA plate culture medium (potato sucrose agar culture medium) is 200 g/L of potatoes (filtering and remaining juice after boiling for 30 min), 20 g/L of sucrose, 20 g/L of agar, tap water as a solvent and natural pH (the actual measurement shows that the pH is 6.5).
Further, the preparation method of the concentrate comprises the steps of adding deionized water into the fermented product, uniformly stirring, placing the fermented product in a constant-temperature water bath kettle at 80-90 ℃ for leaching for 3-5 h, transferring the fermented product into an ultrasonic cleaning machine at 80 ℃, ultrasonically extracting for 20-30 min at 100-200W, filtering when the ultrasonic water extraction is finished, concentrating the filtrate at 65 ℃ under reduced pressure to 1/4-1/5 of the original volume to obtain the concentrate, wherein the volume addition amount of the deionized water is 20-30 m L/g in terms of the weight of the bighead atractylodes rhizome powder before fermentation.
Further, the preparation method of the atractylodes macrocephala polysaccharide comprises the following steps: adding 3-4 times volume of 95% ethanol into the concentrate, standing at room temperature for precipitation for 10-16 h, vacuum filtering with Buchner funnel, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain Atractylodis rhizoma crude polysaccharide.
The Rhizopus oryzae (Rhizopus oryzae) CH5 strain 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.
Compared with the prior art, the invention has the following beneficial effects: the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide. Rhizopus oryzae CH5 grows moderately in Atractylodis rhizoma powder to generate polysaccharide hydrolase, which can hydrolyze insoluble polysaccharide structure tissue of Atractylodes macrocephala, promote dissociation of soluble polysaccharide in bound state, and dissolve easily during hot water ultrasonic extraction. Compared with the conventional method without fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by more than 50%.
(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 Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz) in the examples of the present invention is dried rhizome of Atractylodes macrocephala Koidz (Atractylodes macrocephala Koidz) belonging to Compositae family.
The room temperature of the invention is 25-30 ℃.
Example 1 screening and identification of fermentation strains
1. Screening of Rhizopus oryzae CH5 Strain
(1) Adding 20g of Ganoderma sinensis powder crushed and sieved with a 40 mesh sieve into a triangular flask of 250-m L, and addingWetting with 30m L sterile water, culturing at 28 deg.C for 4d, diluting the enriched culture of confluent mold with sterile water to 1 × 106Coating on PDA plate culture medium, culturing at 28 deg.C for 3 days, selecting mold colony with different colors and forms, transferring to fresh PDA culture medium, culturing at 28 deg.C for 3 days to obtain 7 pure strains (numbers are CH 1-CH 7), inoculating to fresh PDA culture medium, culturing at 28 deg.C for 3 d.7 strains in fresh plate culture, adding 5m L sterile water, stirring with inoculating loop to make spore suspension, transferring spore solution to sterile test tube, adjusting spore concentration to 2 × 10 with sterile water8And each m L.
(2) And (2) carrying out dry heat sterilization on a 250-m L triangular flask at 160 ℃ for 2h, adding 10g of rhizoma atractylodis macrocephalae powder which is crushed and sieved by a 60-mesh sieve, adding 20m L of sterile water, inoculating 1m L of spore liquid of rhizopus oryzae CH5 prepared in the step (1), uniformly stirring, tying the triangular flask with eight layers of gauze, and culturing at 28 ℃ for 2d to obtain fermented rhizoma atractylodis macrocephalae powder.
(3) Adding 200m L deionized water into all the white atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 80 ℃, leaching for 3h, shaking by hand once at intervals of 15min, then transferring a triangular flask into an ultrasonic cleaning machine at the water temperature of 80 ℃, carrying out 100W ultrasonic extraction for 20min, carrying out suction filtration by using a Buchner funnel when the ultrasonic water extraction is finished, and carrying out reduced pressure concentration on the filtrate at 65 ℃ to 50m L to obtain a concentrated solution.
(4) And (3) adding 150m of L% ethanol with concentration of 95% into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 10h, 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 bighead atractylodes rhizome, 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 taken as an unfermented control, the extraction yield of the polysaccharide after the bighead atractylodes rhizome powder is fermented by different strains is shown in table 1.
TABLE 1 polysaccharide extraction yield after fermentation of different bacterial strains on leukocytes
As can be seen from the data in Table 1, the extraction yield of polysaccharide is improved to the highest extent and reaches 42.2% compared with the non-fermented control after the white atractylodes rhizome is fermented by the strain CH5, and the strain is selected as the fermentation strain for improving the extraction yield of white atractylodes rhizome polysaccharide.
The Ganoderma is fruiting body of Ganoderma (Ganoderma sinense).
The plate culture medium is potato sucrose agar culture medium (PDA), and is prepared by cleaning potato, peeling, cutting into small pieces, weighing 200g, adding 1000m L tap water, boiling for 30min, filtering with 4 layers of gauze to remove residue, adding 1000m L filtrate, adding 20g sucrose and 20g agar, measuring pH naturally (actually measuring 6.5), heating until the agar is dissolved, placing in a triangular flask, sterilizing with high pressure steam at 121 deg.C for 15min, pouring into a sterile culture dish with diameter of 9cm, and cooling.
The content of the polysaccharide of the white atractylodes rhizome is measured by adopting a phenol-sulfuric acid method, and the specific method is that crude polysaccharide of the white atractylodes rhizome is prepared into aqueous solution with the concentration of 0.1mg/m L, the sample solution 2m L is absorbed into a 25m L colorimetric tube, 5 percent of phenol aqueous solution with the concentration of 1m L is added, 2.5m L concentrated sulfuric acid is quickly dripped after shaking up, the sample solution is placed for 10min at room temperature after shaking up, the sample solution is heated for 15min in a boiling water bath, running water is cooled to the room temperature, the same treatment that 1m L distilled water is used as blank control is taken as reference, and the490. Determination of A for samples of different glucose concentrations 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 bighead atractylodes rhizome by using the regression equation.
The extraction yield of the atractylodes macrocephala polysaccharide is calculated according to the following formula:
2. identification of Rhizopus oryzae CH5 Strain
The rhizopus oryzae CH5 strain is cultured on a potato agar plate culture medium at the temperature of 28 ℃, the colony is white fine villous in the initial stage, and gradually turns to 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 polysaccharide from Atractylodes macrocephala
(1) Preparation of Rhizopus oryzae CH5 spore liquid, inoculating Rhizopus oryzae CH5 plate colony preserved at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring the spore liquid into sterile test tube, adjusting spore concentration to 3 × 10 with sterile water8Per m L, the PDA plate culture medium components and the preparation method are the same as the example 2.
(2) And (2) carrying out dry heat sterilization on a 500-m L triangular flask at 160 ℃ for 2h, adding 20g of pulverized 60-mesh-screened rhizoma atractylodis macrocephalae powder, adding 50m L of sterile water, inoculating 2m L of the spore liquid of CH5 prepared in the step (1), uniformly stirring, tying the triangular flask with eight layers of gauze, and culturing at 30 ℃ for 2d to obtain fermented rhizoma atractylodis macrocephalae powder.
(3) Adding 400m L deionized water into all the white atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 85 ℃, leaching for 4h, shaking by hand once at intervals of 15min, then transferring a triangular flask into an ultrasonic cleaning machine with the water temperature of 80 ℃, carrying out 100W ultrasonic extraction for 30min, carrying out suction filtration by using a Buchner funnel when the ultrasonic water extraction is finished, and carrying out reduced pressure concentration on the filtrate at 65 ℃ to 100m L to obtain a concentrated solution.
(4) And (3) adding 300m of L% ethanol with concentration of 95% into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 12h, 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 bighead atractylodes rhizome, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 4.23g of crude polysaccharide of the largehead atractylodes rhizome is obtained, the content of the polysaccharide is 80.5 percent, and 3.41g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 17.1 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, and other steps are performed according to the method in this example, 20g of rhizoma atractylodis macrocephalae powder can be extracted to obtain 2.81g of rhizoma atractylodis macrocephalae crude polysaccharide with polysaccharide content of 82.2%, namely 2.31g of rhizoma atractylodis macrocephalae polysaccharide with extraction yield of 11.6%. In this embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 47.4%.
Example 3 application of rhizopus oryzae CH5 to extraction of atractylodes macrocephala polysaccharide:
(1) preparation of Rhizopus oryzae CH5 spore liquid, inoculating Rhizopus oryzae CH5 plate colony preserved at 4 deg.C into fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring the spore liquid into sterile test tube, adjusting spore concentration to 4 × 10 with sterile water8Per m L, the PDA plate medium components and the preparation method are the same as in example 1.
(2) Carrying out dry heat sterilization on a 2-L triangular flask at 160 ℃ for 2h, adding 50g of rhizoma atractylodis macrocephalae powder which is crushed and sieved by a 60-mesh sieve, adding 150m of L of sterile water, inoculating 5m of L of the rhizopus oryzae CH5 spore liquid prepared in the step (1), stirring uniformly, tying the triangular flask with eight layers of gauze, and culturing at 30 ℃ for 3d to obtain the fermented rhizoma atractylodis macrocephalae powder.
(3) Adding 1250m L deionized water into all the white atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 90 ℃, leaching for 5h, shaking by hand once at intervals of 15min, then transferring a triangular flask into an ultrasonic cleaning machine with the water temperature of 80 ℃, performing ultrasonic extraction at 200W for 30min, performing suction filtration by using a Buchner funnel when the ultrasonic water extraction is finished, and concentrating the filtrate at 65 ℃ under reduced pressure to 250m L to obtain a concentrated solution.
(4) And (3) adding 1000m of L% ethanol with concentration of 95% into the total concentrated solution obtained in the step (3), standing and precipitating for 14h at room temperature, carrying out suction filtration by using a Buchner funnel, collecting polysaccharide precipitate, carrying out vacuum drying for 10h at 60 ℃ to obtain crude polysaccharide of the bighead atractylodes rhizome, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
According to the steps, 11.5g of crude polysaccharide of the largehead atractylodes rhizome is obtained, the content of the polysaccharide is 80.8 percent, 9.29g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 18.6 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, 50g of Atractylodis rhizoma powder can be extracted to obtain 7.48g of crude polysaccharide of Atractylodis rhizoma with polysaccharide content of 81.1% according to the method in this example, to obtain 6.07g of polysaccharide of Atractylodis rhizoma with extraction yield of 12.1%. In this embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 53.0%.
Example 4 application of Rhizopus oryzae CH5 to extraction of polysaccharides from Atractylodes macrocephala
(1) Preparation of Rhizopus oryzae CH5 spore liquid, inoculating Rhizopus oryzae CH5 plate colony preserved at 4 deg.C in fresh PDA plate culture medium, culturing at 28 deg.C for 3d, adding 5m L sterile water into the plate culture, suspending spore with inoculating loop under stirring, transferring the spore liquid into sterile test tube, adjusting spore concentration to 5 × 10 with sterile water8Per m L, the PDA plate culture medium components and the preparation method are the same as the example 2.
(2) And (2) carrying out dry heat sterilization on a triangular flask of 5-L at 160 ℃ for 2h, adding 100g of pulverized 60-mesh bighead atractylodes rhizome powder, adding 300m of L sterile water, inoculating 10m of L of the spore solution of CH5 prepared in the step (1), uniformly stirring, tying the triangular flask with eight layers of gauze, and culturing at 30 ℃ for 3d to obtain fermented bighead atractylodes rhizome powder.
(3) Adding 3000m L deionized water into all the bighead atractylodes rhizome powder obtained in the step (2), uniformly stirring, placing in a constant-temperature water bath kettle at 90 ℃, leaching for 5h, shaking by hand once at intervals of 15min, then transferring a triangular flask into an ultrasonic cleaning machine with the water temperature of 80 ℃, performing ultrasonic extraction at 200W for 30min, performing suction filtration by using a Buchner funnel when the ultrasonic water extraction is finished, and concentrating the filtrate at 65 ℃ under reduced pressure to 600m L to obtain a concentrated solution.
(4) And (3) adding 2400m L of 95% ethanol into all the concentrated solution obtained in the step (3), standing and precipitating at room temperature for 16h, 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 bighead atractylodes rhizome, and measuring the polysaccharide content in the crude polysaccharide by adopting a phenol-sulfuric acid method.
22.2g of crude polysaccharide of the largehead atractylodes rhizome is obtained according to the steps, the content of the polysaccharide is 80.6 percent, 17.9g of polysaccharide of the largehead atractylodes rhizome is obtained, the extraction yield is 17.9 percent, and the product is off-white powder.
If the fermentation process of step (2) in this example is not performed, and other steps are performed according to the method in this example, 14.4g of crude polysaccharide of Atractylodis rhizoma can be obtained by extracting 100g of Atractylodis rhizoma powder, the polysaccharide content is 81.7%, and 11.8g of polysaccharide of Atractylodis rhizoma can be obtained, with the extraction yield of 11.8%. In this embodiment, the fermentation pretreatment of rhizopus oryzae CH5 is added in advance of the ultrasonic treatment of the atractylodes macrocephala polysaccharide, and compared with the conventional method without the fermentation pretreatment, the extraction yield of the atractylodes macrocephala polysaccharide can be improved by 51.7%.
Sequence listing
<110> Zhejiang tree college (Zhejiang tree university)
<120> a method for extracting atractylodes macrocephala polysaccharide by using microbial fermentation method
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Claims (6)
1. A method for extracting atractylodes macrocephala polysaccharide by using a microbial fermentation method is characterized by comprising the following steps: inoculating Rhizopus oryzae (Rhizopus oryzae) GDMCC No:60990 spore in Atractylodis rhizoma powder containing sterile water, fermenting at 28-30 deg.C for 2-3 d, ultrasonic extracting the fermented product with water, and concentrating to obtain concentrate; precipitating the concentrate with ethanol and vacuum drying to obtain crude polysaccharide of Atractylodis rhizoma.
2. The method according to claim 1, wherein the sterile water is used in an amount of 2-3 m L/g by volume based on the weight of the powder of Atractylodis rhizoma.
3. The method of claim 1, wherein the Rhizopus oryzae CH5 spore inoculum size is 2-5 × 10% by weight of Atractylodis rhizoma powder7Per gram.
4. The process as claimed in claim 1, wherein the spores of Rhizopus oryzae CH5 are added in the form of spore liquid, and the spore liquid is prepared by inoculating Rhizopus oryzae CH5 preserved at low temperature into PDA plate culture medium, culturing at 28-30 deg.C for 2-3 days, adding sterile water, and suspending the spores with inoculating loop under stirring to obtain spore liquid, wherein the PDA plate culture medium has final concentration of potato 200 g/L, sucrose 20 g/L, agar 20 g/L, and tap water as solvent, and has natural pH.
5. The method as claimed in claim 1, wherein the concentrate is prepared by adding deionized water to the fermented product, stirring, leaching in a 80-90 ℃ constant temperature water bath for 3-5 h, transferring to an ultrasonic cleaning machine with a water temperature of 80 ℃, performing ultrasonic extraction at 100W and 200W for 20-30 min, filtering while hot after the ultrasonic extraction is finished, and concentrating the filtrate at 65 ℃ under reduced pressure to 1/4-1/5 of the original volume to obtain the concentrate, wherein the volume of the deionized water is 20-30 m L/g based on the weight of the Atractylodis rhizoma powder before fermentation.
6. The method according to claim 1, wherein the preparation method of the atractylodes macrocephala polysaccharide comprises the following steps: adding 3-4 times volume of 95% ethanol into the concentrate, standing at room temperature for precipitation for 10-16 h, vacuum filtering with Buchner funnel, collecting polysaccharide precipitate, and vacuum drying at 60 deg.C to constant weight to obtain Atractylodis rhizoma crude polysaccharide.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111394258A (en) * | 2020-04-02 | 2020-07-10 | 浙江工业大学 | Rhizopus stolonifer F L-3 and application thereof in extracting pachyman |
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| CN113564212A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by using microbial fermentation method |
| CN113564212B (en) * | 2021-07-26 | 2024-01-16 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by utilizing microbial fermentation method |
| CN114452357A (en) * | 2022-01-17 | 2022-05-10 | 广西健铧健康产业有限公司 | Liupao tea-containing preparation with blood fat and blood sugar reducing effects and preparation method thereof |
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