Method for extracting amomum villosum polysaccharide through microbial fermentation and application of amomum villosum polysaccharide in preparation of antioxidative and hypoglycemic drugs

Abstract

The invention discloses an extraction method of fructus amomi polysaccharide through microbial fermentation. The extraction method comprises the following steps: 1) drying fructus amomi, powdering and sieving; 2) mixing fructus amomi powder and water according to a material-liquid ratio of 1: (15-20), respectively inoculating bacillus subtilis (CICC21095) and aspergillus niger (ATCC16404), uniformly shaking, and culturing in a shaking table; (3) carrying out centrifugal solid-liquid separation on the fermentation liquor obtained in the step (2), discarding solids, and collecting supernate; (4) removing protein by an isoelectric point method, adjusting the pH to 3 by using hydrochloric acid, and centrifuging when floccules appear; 5) adjusting the pH value of the liquid to be neutral, and adding macroporous resin to dynamically adsorb pigment substances; 6) adding absolute ethyl alcohol, and carrying out alcohol precipitation; and 7) collecting the precipitate, redissolving, dialyzing, collecting the dialyzed solution, and freeze-drying to obtain the purified fructus amomi polysaccharide. According to the method, the extraction rate is remarkably increased, and the fructus amomi extracted by the extraction preparation method has higher extraction rate and has certain oxidation resistance and in-vitro blood glucose reduction effect.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of Chinese herbal medicines, and in particular to a method for extracting Amomum villosum polysaccharide by microbial fermentation and application thereof in preparing antioxidant and hypoglycemic drugs. Background Art

[0002] Amomum villosum Lour. is a traditional medicinal plant widely distributed in southern my country, particularly in Guangdong and Guangxi, where it is used as both a medicinal and edible resource. Its seeds are rich in a variety of active ingredients, including volatile oils, polysaccharides, and flavonoids. They possess medicinal properties such as invigorating the spleen and appetite, regulating qi, and alleviating pain, earning them a prominent position in Traditional Chinese Medicine. In recent years, with the increasing attention paid to the development of natural medicines and functional foods, polysaccharides from Amomum villosum have become a research hotspot due to their unique biological activities. As important natural macromolecules, polysaccharides have been widely demonstrated to possess significant antioxidant, immunomodulatory, anti-tumor, and anti-inflammatory properties. Current research on Amomum villosum Lour. focuses primarily on its volatile oil components and their pharmacological effects, while the extraction process, physicochemical properties, biological activities, and potential health applications of polysaccharides from Amomum villosum Lour. are still in their infancy. The activity of polysaccharides from different plant sources is closely related to their molecular structure. Molecular weight, monosaccharide composition, glycosidic bond type, and branching pattern significantly influence their biological functions. Therefore, the isolation, purification, structural characterization and activity evaluation of Amomum villosum polysaccharides will not only help deepen the understanding of the medicinal value of Amomum villosum, but also provide a theoretical basis for the development and utilization of functional foods and medicines.

[0003] At present, the research on Amomum villosum polysaccharide is still in the primary stage, and particularly in extraction process, there are many deficiencies in the prior art. Traditional polysaccharide extraction methods, such as hot water extraction method, alcohol precipitation method etc., although simple to operate, have the problems such as low extraction yield, high impurity content, long extraction time. For example, hot water extraction method requires long-term high temperature treatment, which not only causes the polysaccharide part to degrade and reduce its biological activity, but also may dissolve some impurities, affecting the purity of the polysaccharide. In addition, alcohol precipitation method, while removing impurities, also can cause the loss of part polysaccharide, further reduces the extraction yield. The limitations of these traditional methods have seriously restricted the industrial production and application of Amomum villosum polysaccharide.

[0004] Furthermore, while modern extraction techniques such as ultrasound-assisted extraction, microwave-assisted extraction, and enzymatic extraction have improved extraction efficiency to a certain extent, these methods still face challenges when applied to the extraction of polysaccharides from Amomum villosum. For example, while ultrasound-assisted extraction can shorten extraction time, the high intensity of ultrasound waves can damage the structure of polysaccharides, affecting their biological activity. Enzymatic extraction, while offering the advantages of strong selectivity and mild conditions, is subject to high enzyme costs and the susceptibility of enzyme activity to environmental factors, limiting its large-scale application.

[0005] In summary, existing polysaccharide extraction technologies from Amomum villosum have problems such as low extraction rate, high impurity content, complex process, and high cost. These problems have seriously hindered the development and utilization of polysaccharides from Amomum villosum in the fields of functional foods and medicine. Therefore, developing an efficient, environmentally friendly, low-cost extraction method that can improve the extraction rate of polysaccharides from Amomum villosum is of great significance for fully utilizing Amomum villosum resources and promoting its application in related fields. Summary of the Invention

[0006] One purpose of the present invention is to provide a method for extracting Amomum villosum polysaccharide by microbial fermentation, specifically using Aspergillus niger and Bacillus subtilis to extract Amomum villosum polysaccharide by composite fermentation, which has high extraction rate, low cost and is suitable for industrial production.

[0007] Furthermore, the present invention also provides the use of the Amomum villosum polysaccharide prepared by the above extraction method in the preparation of antioxidant and hypoglycemic drugs.

[0008] The technical solution adopted in the present invention is:

[0009] A method for extracting Amomum villosum polysaccharide by microbial fermentation comprises the following steps:

[0010] 1) Dry the Amomum villosum, grind into powder, and sieve;

[0011] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:15-20, inoculate with Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404), shake well, and culture in a shaker;

[0012] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0013] 4) Deproteinize by isoelectric point method, adjust pH to 3 with hydrochloric acid, and centrifuge until flocs appear;

[0014] 5) Adjusting the pH of the liquid to neutral, and adding macroporous resin to dynamically adsorb the pigment;

[0015] 6) adding anhydrous ethanol and precipitating;

[0016] 7) Collect the precipitated material, redissolve it, dialyze it, and collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0017] Wherein, the total inoculation amount of Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) in step 2) is 5 wt%.

[0018] Wherein, the volume ratio of the Bacillus subtilis (CICC21095) to the Aspergillus niger (ATCC16404) is (1-3): (1-3).

[0019] The inoculation conditions of step 2) are as follows: mix Amomum villosum powder and water at a material-liquid ratio of 1:15, and act at 120-130° C. and 0.1 MPa for 20 minutes before inoculation.

[0020] The step 1) is as follows: drying the Amomum villosum in an oven at 50° C. for 48 hours, grinding the powder with a Chinese medicinal material grinder, and passing the powder through a 60-mesh sieve.

[0021] Wherein, the reagent for adjusting pH in step (4) is 10 wt% hydrochloric acid.

[0022] Wherein, the reagent for adjusting pH in step (5) is 10wt% sodium hydroxide solution.

[0023] Wherein, the macroporous resin in step (5) comes from Reda Henghui, and its model is macroporous resin AB-8.

[0024] Wherein, the volume ratio of the depigmented liquid to ethanol in step (6) is 1:4.

[0025] Wherein, in the step (1), the dialysis is performed to cut off the substance with a molecular weight of 3000 Da, and the Amomum villosum polysaccharide with a molecular weight above 3000 Da is collected.

[0026] Furthermore, the present invention also provides the use of the Amomum villosum polysaccharide prepared by the above method in the preparation of antioxidant and hypoglycemic drugs.

[0027] Compared with the prior art, the extraction method of Amomum villosum polysaccharide by microbial fermentation provided by the present invention comprises the following steps: 1) drying Amomum villosum, beating powder, and sieving; 2) mixing Amomum villosum powder and water with a material-liquid ratio of 1:15-20, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) respectively, shaking and placing in a shaking table for cultivation; 3) the fermentation broth obtained in step 2) is subjected to solid-liquid separation by centrifugation, the solid is discarded, and the supernatant is collected; 4) protein is removed by the isoelectric point method, the pH is adjusted to 3 with hydrochloric acid, floccules appear, and centrifugation is performed; 5) the liquid pH is adjusted to neutral, and macroporous resin is added to dynamically adsorb pigment substances; 6) anhydrous ethanol is added, and alcohol precipitation is performed; 7) the precipitated material is collected and redissolved, dialyzed, and the dialyzed solution is collected and freeze-dried to obtain the purified Amomum villosum polysaccharide. The extraction rate of the present invention is significantly increased, and the Amomum villosum extracted by the extraction and preparation method has a higher extraction rate and has certain antioxidant capacity and hypoglycemic effects in vitro;

[0028] The present invention provides a method for extracting Amomum villosum polysaccharide with a high extraction rate and suitable for industrial mass production, which is used to solve the problems of low efficiency and high cost in the current polysaccharide extraction process and unsuitable for mass extraction and preparation of Amomum villosum polysaccharide. The polysaccharide extracted by this method has a unique molecular weight and monosaccharide composition. The polysaccharide molecular weight is about 3000Da, and the monosaccharide composition is composed of mannose, rhamnose, glucose and arabinose, wherein the glucose content is the highest. The present invention adopts anhydrous ethanol precipitation, isoelectric point method deproteinization, and structural characterization by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) and other technologies to characterize the monosaccharide composition, molecular weight and microstructure of the Amomum villosum polysaccharide. Finally, its hypoglycemic activity is evaluated in combination with in vitro antioxidant and amylase glycosidase inhibitory effects. By deeply revealing the structure and function relationship of the Amomum villosum polysaccharide, a new scientific basis is provided for the high-value utilization of the plant resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a comparison of the polysaccharide extraction rates of Amomum villosum in the embodiment and the comparative example;

[0030] Figure 2 is the infrared spectrum of the monosaccharide standard;

[0031] Figure 3 This is the monosaccharide composition diagram of polysaccharides;

[0032] Figure 4 This is the infrared spectrum of Amomum villosum polysaccharide;

[0033] Figure 5 This is the SEM image of Amomum villosum polysaccharide;

[0034] Figure 6 This is the result of DPPH free radical scavenging ability of Amomum villosum polysaccharide;

[0035] Figure 7 This is the result of the hydroxyl radical scavenging ability of Amomum villosum polysaccharide;

[0036] Figure 8 This is the result of the determination of the iron ion reducing ability of Amomum villosum polysaccharide;

[0037] Figure 9 The inhibitory effect of Amomum villosum polysaccharide on α-amylase;

[0038] Figure 10 This is the inhibitory effect of Amomum villosum polysaccharide on α-glucosidase. DETAILED DESCRIPTION

[0039] In order to better demonstrate the technical solutions and beneficial effects of the present invention, the technical solutions of the present invention are further described below in conjunction with embodiments, but this does not limit the scope of protection of the present invention.

[0040] Ingredients

[0041] Spring Amomum villosum: JD.com, Yangchun, Guangdong, 20250217

[0042] Bacillus subtilis: CICC21095, laboratory reserved

[0043] Papain: M009, Hongrun Baoshun Technology Co., Ltd.

[0044] Pectinase: M011, Hongrun Baoshun Technology Co., Ltd.

[0045] Cellulase: M012, Hongrun Baoshun Technology Co., Ltd.

[0046] Yeast ATCC9763, laboratory-retained

[0047] Aspergillus niger: ATCC16404, laboratory-retained

[0048] Pediococcus pentosaceus: ATCC8041, JD.com

[0049] Lactobacillus plantarum: ATTCC14917, laboratory-retained

[0050] Lactobacillus fermentum: ATCC14931, laboratory-retained

[0051] Macroporous resin: Macroporous resin AB-8, Ruida Henghui Jingdong

[0052] Dialysis membrane: MD44-3000, Beekman

[0053] The remaining materials were commercially available.

[0054] Example 1

[0055] This embodiment provides a method for extracting Amomum villosum polysaccharide by microbial fermentation, which has antioxidant and hypoglycemic activities and is suitable for industrial mass production, comprising the following steps:

[0056] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0057] 2) Mixing Amomum villosum powder and water at a material-liquid ratio of 1:15, incubating at 120-130°C and 0.1 MPa for 20 minutes, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) at a total inoculum size of 5 wt%, with a mass ratio of 1:1 between Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404), shaking well, and incubating in a shaker at 30°C for 16 hours;

[0058] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0059] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0060] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0061] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0062] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0063] Example 2

[0064] This embodiment provides a method for extracting Amomum villosum polysaccharide by microbial fermentation, which has antioxidant and hypoglycemic activities and is suitable for industrial mass production, comprising the following steps:

[0065] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0066] 2) Mixing Amomum villosum powder and water at a material-liquid ratio of 1:20, incubating at 120-130°C and 0.1 MPa for 20 minutes, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) at a total inoculum size of 5 wt%, with a mass ratio of Bacillus subtilis (CICC21095) to Aspergillus niger (ATCC16404) of 1:2, shaking, and incubating in a shaker at 30°C for 16 hours;

[0067] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0068] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0069] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0070] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0071] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0072] Example 3

[0073] This embodiment provides a method for extracting Amomum villosum polysaccharide by microbial fermentation, which has antioxidant and hypoglycemic activities and is suitable for industrial mass production, comprising the following steps:

[0074] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0075] 2) Mixing Amomum villosum powder and water at a material-liquid ratio of 1:18, incubating at 120-130°C and 0.1 MPa for 20 minutes, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) at a total inoculum size of 5 wt%, with a mass ratio of Bacillus subtilis (CICC21095) to Aspergillus niger (ATCC16404) of 1:3, shaking well, and incubating in a shaker at 30°C for 16 hours;

[0076] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0077] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0078] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0079] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0080] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0081] Example 4

[0082] This embodiment provides a method for extracting Amomum villosum polysaccharide by microbial fermentation, which has antioxidant and hypoglycemic activities and is suitable for industrial mass production, comprising the following steps:

[0083] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0084] 2) Mixing Amomum villosum powder and water at a material-liquid ratio of 1:18, incubating at 120-130°C and 0.1 MPa for 20 minutes, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) at a total inoculum size of 5 wt%, with a mass ratio of Bacillus subtilis (CICC21095) to Aspergillus niger (ATCC16404) of 2:1, shaking, and incubating in a shaker at 30°C for 16 hours;

[0085] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0086] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0087] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0088] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0089] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0090] Example 5

[0091] This embodiment provides a method for extracting Amomum villosum polysaccharide by microbial fermentation, which has antioxidant and hypoglycemic activities and is suitable for industrial mass production, comprising the following steps:

[0092] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0093] 2) Mixing Amomum villosum powder and water at a material-liquid ratio of 1:18, incubating at 120-130°C and 0.1 MPa for 20 minutes, inoculating Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) at a total inoculum size of 5 wt%, with a mass ratio of Bacillus subtilis (CICC21095) to Aspergillus niger (ATCC16404) of 3:1, shaking, and incubating in a shaker at 30°C for 16 hours;

[0094] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0095] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0096] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0097] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0098] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0099] Comparative Example 1

[0100] This comparative example discloses a method for extracting polysaccharides from Amomum villosum using an ultrasound-assisted enzymatic extraction method, and the specific steps are as follows:

[0101] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0102] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:18, and treat with an ultrasonic cell disruptor at 40% power for 30 minutes. Then, add papain, pectinase, and cellulase at a ratio of 1:1:1, with the enzyme dosage of 0.5%. Place in a 50°C water bath, and perform enzymatic hydrolysis for 6 hours. Then, inactivate the enzymes at 85°C for 10 minutes.

[0103] 3) The mixed solution obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0104] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0105] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0106] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0107] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0108] Comparative Example 2

[0109] This comparative example discloses a high-pressure and high-heat extraction method for Amomum villosum polysaccharide, which specifically comprises the following steps:

[0110] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0111] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:18 and react at 121°C and 0.1 MPa for 15-30 minutes;

[0112] 3) The mixed solution obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0113] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0114] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0115] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0116] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0117] Comparative Example 3

[0118] This comparative example discloses a method for extracting polysaccharides from Amomum villosum by using a yeast fermentation extraction method, and the specific steps are as follows:

[0119] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0120] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:18, incubate at 121°C, 120-130°C, and 0.1 MPa for 20 minutes, inoculate with yeast ATCC9763 at a rate of 5 wt%, shake well, and culture in a shaker at 37°C for 16 hours.

[0121] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0122] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0123] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0124] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0125] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0126] Comparative Example 4

[0127] This comparative example discloses a method for extracting Amomum villosum polysaccharide by fermentation with Aspergillus niger, which comprises the following steps:

[0128] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0129] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:15-20, incubate at 121° C., 120-130° C., and 0.1 MPa for 20 minutes, inoculate Aspergillus niger ATCC16404 at a 5 wt % inoculum, shake well, and culture in a shaker at 37° C. for 16 hours.

[0130] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0131] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0132] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0133] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0134] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0135] Comparative Example 5

[0136] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0137] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:18, incubate at 121°C, 120-130°C, 0.1 MPa for 20 minutes, inoculate with Pediococcus pentosaceus ATCC8041 at a 5 wt% inoculum, shake well, and incubate in a shaker at 37°C for 12 hours;

[0138] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0139] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0140] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0141] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0142] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0143] Comparative Example 6

[0144] This comparative example discloses a method for extracting polysaccharides from Amomum villosum using a Lactobacillus plantarum fermentation extraction method, and the specific steps are as follows:

[0145] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0146] 2) Mix spring Amomum villosum powder and water at a material-liquid ratio of 1:15-20, incubate at 121° C., 120-130° C., 0.1 MPa for 20 minutes, inoculate Lactobacillus plantarum ATTCC14917 at a 5 wt% inoculum, shake well, and culture in a shaker at 37° C. for 12 hours;

[0147] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0148] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0149] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0150] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0151] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0152] Comparative Example 7

[0153] This comparative example discloses a method for extracting polysaccharides from Amomum villosum using a Bacillus fermentation extraction method, and the specific steps are as follows:

[0154] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0155] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:15-20, incubate at 120-130°C and 0.1 MPa for 20 minutes, inoculate with Bacillus CICC21095 at a 5 wt% inoculum, shake well, and incubate in a shaker at 30°C for 16 hours.

[0156] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0157] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0158] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0159] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0160] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0161] Comparative Example 8

[0162] This comparative example discloses a method for extracting polysaccharides from Amomum villosum using a Lactobacillus fermentum fermentation extraction method, and the specific steps are as follows:

[0163] 1) Dry Amomum villosum thoroughly in a 50°C oven for 48 h, grind into powder using a Chinese herbal medicine grinder, and sieve through a 60-mesh sieve;

[0164] 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:18, incubate at 121°C, 120-130°C, 0.1 MPa for 20 minutes, inoculate with Lactobacillus fermentum ATCC14931 at a 5 wt% inoculum, shake well, and incubate in a shaker at 37°C for 12 hours;

[0165] 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0166] 4) removing proteins by isoelectric point method, adjusting pH to 3 with 10 wt % hydrochloric acid, and centrifuging until flocs appear;

[0167] 5) adjusting the pH of the liquid to neutral with 10 wt% sodium hydroxide, and adding macroporous resin to statically adsorb the pigment;

[0168] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the depigmented liquid to ethanol is 1:4, and precipitate with alcohol for 12 hours;

[0169] 7) Collect the precipitated material, redissolve it, dialyze it, and retain the material with a molecular weight greater than 3000 Da. Collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

[0170] Verification experiment

[0171] 1. The detection method of Amomum villosum polysaccharide is as follows:

[0172] (1) Preparation of glucose standard curve

[0173] Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of a 100 mg / mL glucose standard solution into colorimetric tubes and add water to 1 mL. Then, add 1.0 mL of 5% phenol solution to each colorimetric tube, followed by 5.0 mL of concentrated sulfuric acid. Shake thoroughly, let stand for 10 minutes, then place in a 30°C water bath for 20 minutes. Measure the absorbance at a wavelength of 490 nm using a blank as a control. Plot a standard curve with absorbance as the ordinate and glucose content as the abscissa to obtain a regression equation.

[0174] (2) Determination of polysaccharide content in samples

[0175] Take 1 mL of the prepared sample aqueous solution and place it in a 10 mL dry colorimetric tube. The rest of the operation is the same as (1). Measure the absorbance at a wavelength of 490 nm.

[0176] (3) Calculation of polysaccharide content in Amomum villosum

[0177] Polysaccharide content (%) = m / W × dilution factor × 100%

[0178] m: Glucose content calculated according to the regression equation (mg)

[0179] w: sample dry weight (mg)

[0180] 2. Molecular Weight Determination

[0181] First, the polysaccharide (5 mg) was heated and dissolved in NaNO3 (0.1 M) (100°C). Then, 100 μL of the polysaccharide supernatant was obtained after centrifugation (14,000 rpm, 10 min). The solution was detected by gel permeation chromatography connected to an RI detector and a multi-angle laser light scattering detector. Three chromatographic columns (Ohpak SB-805 / SB-804 / SB-803HQ) were used in combination, with the mobile phase being 0.1 M NaNO3 and the flow rate being 0.4 mL / min. Finally, the analysis was performed using ASTRA6.1 software.

[0182] 3. Monosaccharide composition analysis

[0183] First, the polysaccharide sample (5 mg) was dissolved in a TFA solution (1 mL) at 121°C for 2 h. Then, the polysaccharide acid hydrolyzate was dried using a nitrogen blower. Next, methanol was added to the dried polysaccharide sample, and then blown dry. This process was repeated 3 times. Finally, the polysaccharide sample was dissolved in sterile water and transferred to a chromatographic bottle for detection. 100 mg of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid and glucuronic acid were added to sterile water in turn, quantitatively dissolved in a 10 mL volumetric flask, and prepared into a mixed standard solution. The mixed standard solution was gradiently diluted to 1 / 100, 5 / 100, 10 / 1000, 20 / 1000, 30 / 100, 40 / 100, 50 / 100, 60 / 100 to obtain a standard working solution. Finally, the diluted solution was transferred to a chromatographic bottle for detection. The results of the monosaccharide standard are shown in Figure 2 , polysaccharide monosaccharide composition see Figure 3 .

[0184] 4. Infrared spectroscopy analysis

[0185] The polysaccharide was vacuum dried for 48 h, mixed with potassium bromide at a ratio of 1:150, ground, and pressed into tablets. The absorption spectrum was scanned by Fourier transform infrared spectrometer at 400-4000 cm -1 The results are shown in Figure 4 .

[0186] 5. Scanning Electron Microscope Analysis

[0187] The freeze-dried Amomum villosum polysaccharide powder was sprayed with gold and observed under appropriate magnification. Figure 5 .

[0188] 6. Antioxidant Capacity Determination

[0189] (1) DPPH free radical scavenging ability determination

[0190] Take 500 μL of sample solution (2, 4, 6, 8, 10 mg / ml) and mix it with 500 μL of anhydrous ethanol and 125 μL of 0.02% DPPH mixture. Incubate in the dark for 25 minutes and measure the absorbance at 510 nm. C As a positive control. The results are shown in Figure 6 .

[0191] DPPH free radical scavenging rate = (1-(A1-A2) / A0) × 100

[0192] Where:

[0193] A0 is the absorbance of a mixture of 500 μL of sample solution, 500 μL of anhydrous ethanol, and 125 μL of 0.02% DPPH.

[0194] A1 is the absorbance value of 500 μL sample solution and 625 μL anhydrous ethanol.

[0195] A2 is the absorbance of a mixture of 500 μL H2O, 500 μL anhydrous ethanol, and 125 μL 0.02% DPPH.

[0196] (2) Hydroxyl radical scavenging ability

[0197] Take 500 μL of sample solution (2, 4, 6, 8, 10 mg / ml) and mix it with 500 μL of 9 mmol / L FeSO4 and 500 μL of salicylic acid-50% ethanol (9 mmol / L). Add 500 μL of 0.03% H2O2 and mix thoroughly. Incubate in a 37°C water bath for 1 hour. Let it stand at room temperature for 10 minutes and measure the absorbance at 510 nm. C As a positive control. The results are shown in Figure 7 .

[0198] Hydroxyl radical scavenging rate = (1-(A1-A2) / A0) × 100

[0199] Where:

[0200] A0 is the absorbance of a mixture of 500 μL of sample solution, 500 μL of 9 mmol / L FeSO4, and 500 μL of salicylic acid-50% ethanol (9 mmol / L).

[0201] A1 is the absorbance of a mixture of 500 μL H2O, 500 μL 9 mmol / L FeSO4, and 500 μL salicylic acid-50% ethanol (9 mmol / L).

[0202] A2 is the absorbance of 500 μL of sample solution and 500 μL of 9 mmol / L FeSO4 and 50% ethanol solution.

[0203] (3) Reducing ability determination

[0204] Take 500μL of sample solution (2, 4, 6, 8, 10mg / ml), 500μL of PBS buffer solution (pH=6.6) and 500μL of 1% K3[Fe(CN)6] solution and mix them. After 20 minutes in a 50℃ water bath, add 500μL of 10% trichloroacetic acid and mix them. Take 500μL of the mixed solution, add 500μL of H2O and 100μL of 0.1% FeCl3, let it stand at room temperature for 10 minutes, and measure the absorbance at 700nm. VC is used as a positive control. By comparing the absorbance of the sample and VC, the reducing ability of the polysaccharide is determined. The results are shown in Figure 8 .

[0205] 7. In vitro hypoglycemic activity

[0206] (1) Determination of α-amylase activity inhibition rate

[0207] Prepare DNS solution in a brown bottle and use after one week. First, prepare PBS at pH 6.8 and 0.1 mol / L. Then, use PBS to prepare a 5% starch solution, a 20 U / mL amylase solution, and solutions of purified polysaccharide fractions at 2, 4, 6, 8, and 10 mg / mL, as well as the positive drug acarbose. First, add 300 μL of polysaccharide solution and 400 μL of enzyme solution to the sample tube A1. Add equal volumes of water and enzyme solution to the blank group A0. Add equal volumes of polysaccharide solution and buffer at the corresponding concentrations to the control group A2. Incubate in a biochemical incubator at 37°C for 10 minutes. Then, add 300 μL of soluble starch solution to all tubes, shake, and incubate in a biochemical incubator at 37°C for 15 minutes. Then, add 2 mL of DNS solution to each tube for color development. Inactivate the enzyme on an induction cooker for 10 minutes. Finally, add PBS to 10 mL. Measure absorbance at 540 nm. Calculate the α-amylase activity inhibition rate using the following formula. The results are shown in Figure 9 .

[0208] α-amylase inhibition rate = (1-(A1-A2) / A0) × 100

[0209] (2) Determination of α-glucosidase activity inhibition rate

[0210] Use PBS (pH 6.8, 0.1 mol / L) to prepare 2.5 mmol / L PNPG solution, 0.25 U / mL α-glucosidase solution, 2, 4, 6, 8, 10 mg / ml polysaccharide purified component solution and positive drug acarbose solution, and then perform spot reaction in a 96-well plate. Add 50 μL of polysaccharide solution and 50 μL of enzyme solution to sample group A1, add equal volumes of buffer and enzyme solution to blank group A0, and add equal volumes of polysaccharide solution and buffer of corresponding concentration to control group A2. Then, place them in a biochemical incubator and incubate at 37°C for 10 minutes, then add 50 μL of PNPG solution, shake the well plate to mix, and place it in a biochemical incubator and incubate at 37°C for 30 minutes. Measure the absorbance at 405 nm, and perform 3 parallel experiments. Calculate the α-glucosidase activity inhibition rate according to the following formula. The results are shown in Figure 10 .

[0211] α-glucosidase inhibition rate = (1-(A1-A2) / A0) × 100

[0212] Comparison of polysaccharide extraction rates from Amomum villosum

[0213] Calculate the extraction rate of polysaccharide, polysaccharide extraction rate = (V*C*N*10 -3) / M*100% where V: constant volume (mL), C: concentration (μg / mL), N: dilution factor, M: sample mass (mg), respectively, the extraction rates of Ophiopogon japonicus polysaccharides of the embodiment and the comparative example were measured and calculated, as shown in Table 1, and the obtained Figure 1 , where different letters represent significant differences.

[0214] Table 1 Comparison of extraction rates between the embodiment and the comparative example

[0215]

[0216]

[0217] It can be seen that the extraction rate of Amomum villosum in the embodiment is significantly higher than that in the comparative example.

[0218] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting polysaccharides from Amomum villosum by microbial fermentation, characterized in that The following steps are involved: 1) Dry the Amomum villosum, grind into powder, and sieve; 2) Mix Amomum villosum powder and water at a material-liquid ratio of 1:15-20, inoculate with Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404), shake well, and culture in a shaker; 3) The fermentation broth obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected; 4) Deproteinize by isoelectric point method, adjust pH to 3 with hydrochloric acid, and centrifuge until flocs appear; 5) Adjusting the pH of the liquid to neutral, and adding macroporous resin to dynamically adsorb the pigment; 6) adding anhydrous ethanol and precipitating; 7) Collect the precipitated material, redissolve it, dialyze it, and collect the dialyzed solution and freeze-dry it to obtain the purified Amomum villosum polysaccharide.

2. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: In the step 2), the total inoculation amount of Bacillus subtilis (CICC21095) and Aspergillus niger (ATCC16404) is 5 wt %.

3. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 2, wherein: The volume ratio of the Bacillus subtilis (CICC21095) to the Aspergillus niger (ATCC16404) is (1-3): (1-3).

4. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 3, wherein: The inoculation conditions of step 2) are as follows: mix Amomum villosum powder and water at a material-liquid ratio of 1:15, react at 120-130° C. and 0.1 MPa for 20 minutes, and then perform inoculation.

5. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: Specifically, the step 1) comprises the following steps: drying the Amomum villosum in an oven at 50° C. for 48 hours, grinding the powder with a Chinese medicinal material grinder, and passing the powder through a 60-mesh sieve.

6. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: The reagent for adjusting pH in step (4) is 10 wt% hydrochloric acid.

7. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: The reagent for adjusting pH in step (5) is 10 wt % sodium hydroxide solution.

8. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: The macroporous resin in step (5) comes from Reda Henghui, and its model is macroporous resin AB-8.

9. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: In the step (6), the volume ratio of the depigmented liquid to ethanol is 1:

4.

10. The method for extracting polysaccharide from Amomum villosum by microbial fermentation according to claim 1, wherein: In the step (1), the dialysis is performed to cut off substances with a molecular weight of 3000 Da, and the Amomum villosum polysaccharides above 3000 Da are collected.

11. Use of the Amomum villosum polysaccharide prepared by the extraction method according to any one of claims 1 to 10 in the preparation of antioxidant and hypoglycemic drugs.

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