Rhizopus arrhizus ds6-14 and application thereof in extracting polysaccharide from radix codonopsis

Abstract

The application discloses a Rhizopus arrhizus strain DS6-14 and application thereof in extraction of Codonopsis pilosula polysaccharide. The Rhizopus arrhizus strain DS6-14 is preserved in the Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 63063, and a preservation date of December 20, 2022. The strain is obtained by targeted separation and screening in view of the ability of hydrolyzing cell walls of Codonopsis pilosula. Before polysaccharide is extracted from Codonopsis pilosula, fermentation pretreatment of the Rhizopus arrhizus strain DS6-14 is added, the strain is moderately grown in Codonopsis pilosula powder added with sucrose, and a plurality of hydrolytic enzymes are generated. Then, the fermented Codonopsis pilosula powder is hydrolyzed by adding water and keeping warm, and substances such as cellulose, hemicellulose and pectin in the cell wall are hydrolyzed, which is helpful to the dissolution of the combined Codonopsis pilosula polysaccharide in ultrasonic water extraction, so that the extraction yield of the polysaccharide can be significantly improved. Compared with a conventional method without fermentation pretreatment, the extraction yield of the Codonopsis pilosula polysaccharide is increased by 46.7%.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a strain of Rhizopus DS6-14 and its application in the extraction of Codonopsis pilosula polysaccharides. (II) Background Technology

[0002] Codonopsis radix is ​​the dried root of Codonopsis pilosula, Codonopsis pilosula var. modesta, or Codonopsis tangshen, all belonging to the Campanulaceae family. Codonopsis radix is ​​a traditional wild plant used both as medicine and food. As a Chinese medicinal herb, it has effects such as enhancing hematopoietic function, regulating blood pressure, protecting the gastrointestinal tract, enhancing immunity, anti-oxidation, anti-tumor, anti-fatigue, protecting nerves, antibacterial, anti-inflammatory, and lowering blood lipids. Clinically, it can be used to prevent and treat hyperlipidemia, hypotension, hematopoietic dysfunction, acute altitude sickness, and dysfunctional uterine bleeding. As a food ingredient, it has effects such as tonifying the middle energizer, replenishing qi, strengthening the spleen and lungs. Codonopsis pilosula mainly contains sterols, glycosides, polysaccharides, alkaloids, and triterpenoids. Among them, Codonopsis pilosula polysaccharide is one of the main active ingredients of Codonopsis pilosula. It has antioxidant, anti-aging, anti-cancer, anti-inflammatory, antiviral, immune-enhancing, diabetes treatment, platelet aggregation inhibition, and central nervous system depression effects. Therefore, the market prospects for developing medicines and health foods with Codonopsis pilosula polysaccharide as the main ingredient are broad.

[0003] Currently, there are numerous research reports on extraction methods for Codonopsis pilosula polysaccharides, with the basic method being hot water extraction followed by ethanol precipitation (referred to as "water extraction and ethanol precipitation"). Based on hot water extraction, there are extraction methods employing auxiliary measures such as ultrasound, microwave, and enzymatic hydrolysis. Conventional hot water extraction has the advantages of simple operation and low cost, but it suffers from disadvantages such as long extraction time, multiple extraction cycles, and low extraction efficiency. Ultrasonic and microwave extraction methods, while simple to operate and short in time, are energy-intensive and costly. Enzyme-assisted extraction utilizes cellulase, pectinase, or a combination of enzymes to decompose cellulose, hemicellulose, pectin, and other substances in the cell walls and intercellular matrix of Codonopsis pilosula through enzymatic reactions. This loosens and breaks down the cell walls, reducing mass transfer resistance and thus increasing the extraction rate of Codonopsis pilosula polysaccharides. It offers advantages such as mild conditions, short extraction time, low energy consumption, low cost, and better preservation of the original medicinal efficacy of the active ingredients.

[0004] Some studies have used enzymatic hydrolysis to assist in the extraction of Codonopsis pilosula polysaccharides. For example, Yue Xianwen used cellulase to assist in the extraction of Codonopsis pilosula polysaccharides, which increased the yield by 23.92% compared with the non-enzymatic extraction method. However, the amount of cellulase added was only 2.4% of the extraction system, which is obviously a large amount of enzyme [Yue Xianwen. Optimization of enzymatic hydrolysis extraction process of Codonopsis pilosula. Heilongjiang Medicine, 2011, 24(5):743-744.]. In the enzymatic hydrolysis-assisted extraction method of plant active ingredients, if the amount of enzyme used is large, it will undoubtedly increase the extraction cost. If the economic benefits brought by the increased polysaccharide extraction yield are not as good as the cost brought by the enzyme and the enzymatic hydrolysis operation, then the process has no value for industrial application.

[0005] In nature, plant debris and decay are decomposed by microorganisms. These microorganisms produce various enzymes that break down plant tissues during their growth, including cellulase, hemicellulase, ligninase, and pectinase. Therefore, if Codonopsis pilosula is pretreated by direct microbial fermentation, as long as the growth level is well controlled to achieve enzyme production that breaks down the cell walls of Codonopsis pilosula without breaking down soluble polysaccharides, thereby promoting the dissolution of Codonopsis pilosula polysaccharides, the extraction yield can be improved.

[0006] To improve the extraction yield of Codonopsis pilosula polysaccharides, this invention improves the conventional water extraction and alcohol precipitation extraction method by adding a microbial fermentation pretreatment step, which can significantly increase the extraction yield of Codonopsis pilosula polysaccharides. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a novel microbial strain—Rhizopus arrhizus DS6-14—and its application in the extraction of polysaccharides from Codonopsis pilosula. After fermentation by this strain, the polysaccharide extraction yield of Codonopsis pilosula can be significantly improved.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a novel microbial strain—Rhizopus arrhizus DS6-14, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No:63063, deposit date December 20, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode 510070.

[0010] The *Rhizopus oligorhizoides* DS6-14 described in this invention is an excellent strain isolated from microbial enrichment cultures of *Codonopsis pilosula* and obtained through screening. The morphological characteristics of *Rhizopus oligorhizoides* DS6-14 are as follows: When cultured on potato dextrose agar (PDA) plates at 28°C, the colonies are initially grayish-white and fluffy, turning gray after 2 days. The mycelial layer is relatively thick and loose, with long hyphae and gray spores on the surface. The reverse side of the colony is grayish-white with no pigment diffusion. Creeping hyphae and rhizoids are underdeveloped, with few and short rhizoids. Sporangiophores are mostly curved, rarely solitary, and often form umbrella-shaped aggregates, growing directly from the hyphae without corresponding to the rhizoids. Sporangia are spherical or nearly spherical, dark brownish-gray. Sporangiospores are elliptical or polygonal, with a diameter of 4–7 μm. A photograph of *Rhizopus oligorhizoides* DS6-14 colonies cultured on PDA plates at 28°C for 3 days is shown below. Figure 1 .

[0011] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Rhizopus DS6-14 is shown in SEQ ID NO.1.

[0012] The present invention also provides an application of the aforementioned Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides. The method of application is as follows: (1) adding Rhizopus DS6-14 spore liquid to Codonopsis pilosula powder, stirring evenly, and fermenting at 28°C for 60–64 h to obtain Codonopsis pilosula fermentation product; (2) adding deionized water to Codonopsis pilosula fermentation product, stirring evenly, enzymatically hydrolyzing at 30–35°C for 4–8 h, and then filtering and concentrating after ultrasonic water extraction to obtain Codonopsis pilosula water extract concentrate; (3) adding ethanol to Codonopsis pilosula water extract concentrate to precipitate polysaccharides, and then washing the precipitate with ethanol and vacuum drying to obtain Codonopsis pilosula polysaccharides.

[0013] Furthermore, the Codonopsis powder in step (1) is the dried root of the plant Codonopsis pilosula, which is pulverized and passed through an 80-mesh sieve.

[0014] Further, the preparation method of Rhizopus oligosporus DS6-14 spore solution in step (1) is as follows: Low-temperature preserved Rhizopus oligosporus DS6-14 spores are inoculated onto potato dextrose agar (PDA) plates and cultured at 28℃ for 56–72 h. Then, sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain the spore solution. Preferably, the spore concentration of the spore solution is adjusted to 5 × 10⁻⁶ spores using sterile sucrose aqueous solution. 6 –10×10 6The sterile sucrose aqueous solution has a sucrose concentration of 20–30 g / L and is sterilized by autoclaving at 115°C for 20 min. The PDA plate culture medium is a commercially available potato dextrose agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., prepared with tap water at a concentration of 46 g / L, with natural pH, and sterilized by autoclaving at 121°C for 20 min.

[0015] Furthermore, the volume of the Rhizopus DS6-14 spore solution used in step (1) is 1.5–2.0 mL / g based on the mass of Codonopsis pilosula powder.

[0016] Further, the preparation method of the Codonopsis pilosula water extract concentrate in step (2) is as follows: deionized water is added to the Codonopsis pilosula fermentation product, and after stirring evenly, it is enzymatically hydrolyzed at 30–35℃ for 4–8 hours. Then, it is transferred to an ultrasonic cleaner at a water temperature of 90–95℃ and ultrasonically extracted at 100–150W for 1.5–2.0 hours. After the ultrasonic water extraction is completed, it is filtered through a Buchner funnel while hot, and the filtrate is concentrated under reduced pressure at 60℃ and -0.1Mpa to 1 / 25–1 / 20 of the original volume to obtain the Codonopsis pilosula water extract concentrate. The volume of deionized water added is 20–25 mL / g based on the mass of Codonopsis pilosula powder before fermentation.

[0017] Further, the preparation method of Codonopsis pilosula polysaccharide in step (3) is as follows: add 4-5 times the volume of anhydrous ethanol to the water extract concentrate of Codonopsis pilosula to make the ethanol volume fraction of the system reach 80%-83.3%, let it stand at 4℃ for 12-16h, centrifuge at 8000r / min for 5-10min, collect the precipitate, add 1-2mL / g of anhydrous ethanol (by weight of raw Codonopsis pilosula powder) to wash once, centrifuge again, and dry the precipitate under vacuum at 50℃ and -0.1Mpa to constant weight to obtain Codonopsis pilosula polysaccharide.

[0018] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: This invention provides a novel microbial strain, *Rhizopus oligosporus* DS6-14, which was specifically isolated and screened to hydrolyze the cell walls of *Codonopsis pilosula*. *Rhizopus oligosporus* DS6-14 grows moderately in *Codonopsis pilosula* powder with added sucrose, producing various hydrolytic enzymes. Subsequently, the fermented *Codonopsis pilosula* powder is incubated with water for enzymatic hydrolysis, hydrolyzing substances such as cellulose, hemicellulose, and pectin in the cell walls. This facilitates the dissolution of bound *Codonopsis pilosula* polysaccharides during ultrasonic water extraction, thereby significantly improving the polysaccharide extraction yield. Compared with conventional methods without fermentation pretreatment, the extraction yield of *Codonopsis pilosula* polysaccharides is increased by 46.7%. (iv) Description of the attached drawings

[0019] Figure 1 Photographs of the colony morphology of Rhizopus DS6-14 cultured on a PDA at 28°C for 3 days.

[0020] Figure 2This is the standard curve for the determination of polysaccharides using the phenol-sulfuric acid method.

[0021] Figure 3 This is the standard curve for glucose determination using the DNS method. (V) Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0023] The Codonopsis pilosula mentioned in this embodiment of the invention is the dried root of Codonopsis pilosula, a plant of the Campanulaceae family. Codonopsis pilosula powder is the fine powder obtained by crushing the dried Codonopsis pilosula and passing it through an 80-mesh sieve.

[0024] Example 1: Isolation and screening of microbial strains for fermented Codonopsis pilosula

[0025] The microbial strains for fermenting Codonopsis pilosula were isolated and screened according to the following steps:

[0026] (1) Add 5g of Codonopsis pilosula powder to a 50-mL Erlenmeyer flask, then add 10mL of sterile physiological saline to moisten the mixture, and incubate at 28℃ for 72h. Dilute the enriched culture contaminated with mold with sterile physiological saline at 1×10⁻⁶. -6 1×10 -7 1×10 -8 After dilution, 0.1 mL of the diluted solution was spread onto potato dextrose agar (PDA) plates and incubated at 28°C for 48 h. Mold colonies with different colors and morphologies were picked and transferred to fresh PDA plates and incubated at 28°C for 72 h to obtain 10 pure culture strains. The strain numbers are shown in Table 1.

[0027] (2) 10 mL of sterile sucrose solution was added to each of the 10 strains' fresh plate cultures. The spores were suspended by stirring with an inoculation loop. The spore solutions were then transferred to sterile test tubes, and the spore concentration was adjusted with sterile sucrose solution to ensure that the spore solutions of different strains were at a concentration of 5 × 10⁻⁶. 6 –10×10 6 The spore concentration of each strain was between [number] spores / mL to obtain spore suspensions. The concentration of the sterile sucrose aqueous solution was 30 g / L, and it was sterilized by high-pressure steam at 115°C for 20 min.

[0028] (3) Add 1g of Codonopsis pilosula powder to 10 50-mL Erlenmeyer flasks that have been dry heat sterilized at 160℃ for 2h, and then add 1.5mL of the mold spore liquid prepared in step (2) (the volume of the liquid is 1.5mL / g based on the mass of Codonopsis pilosula powder). After stirring evenly, seal the Erlenmeyer flasks with 8 layers of gauze and incubate at 28℃ for 64h to obtain Codonopsis pilosula ferment.

[0029] (4) Take all the Codonopsis pilosula powder fermented by each strain in step (3), add 25 mL of deionized water [material-to-liquid ratio of 1:25 (g:mL)], stir evenly, and enzymatically hydrolyze in a 35℃ water bath for 4 h. Then transfer to an ultrasonic cleaner at 95℃ and ultrasonically extract at 100W for 2 h. After ultrasonic water extraction, centrifuge at 8000 r / min for 5 min, take 1 mL of supernatant into a 10-mL centrifuge tube, add 5 mL of anhydrous ethanol (ethanol volume fraction of the solution is 83.3%), shake thoroughly, and let stand at 4℃ for 12 h. Centrifuge at 8000 r / min for 5 min, discard the supernatant, add 5 mL of deionized water to dissolve, and determine the soluble polysaccharide content in the extract using the phenol-sulfuric acid method.

[0030] Starting from step (3), 1.5 mL of sterile sucrose solution (concentration 30 g / L) was added to 1 g of Codonopsis pilosula powder to serve as a blank fermentation control without inoculation with mold; following the method in step (4), 1 g of Codonopsis pilosula was added to 25 mL of deionized water to directly extract polysaccharides to serve as a non-fermentation extraction control. The polysaccharide extraction yields of Codonopsis pilosula fermented with different strains and the control are shown in Table 1.

[0031] Table 1. Polysaccharide extraction yields from Codonopsis pilosula fermented with different strains and the control group.

[0032]

[0033] As shown in Table 1, the blank fermentation control, which added sterile sucrose aqueous solution but was not inoculated with mold, showed no significant difference in polysaccharide extraction yield compared to the unfermented control due to almost no mold growth. Most strains did not significantly improve polysaccharide extraction yield after fermentation of *Codonopsis pilosula*; in fact, some strains, such as DS1, DS3, and DS7, even significantly reduced the polysaccharide yield. This indicates species selectivity in the microbial strains used to ferment *Codonopsis pilosula* to improve polysaccharide extraction yield. After fermentation with strain DS-6, the polysaccharide extraction yield was 21.3%, a 27.5% increase compared to the 16.7% of the unfermented control. Therefore, strain DS-6 was selected as the fermentation strain for improving the polysaccharide extraction yield of *Codonopsis pilosula* in this invention.

[0034] The PDA plate culture medium is a ready-made potato dextrose agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd. It is prepared with tap water at a concentration of 46 g / L, with natural pH. It is placed in an Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by high-pressure steam at 121℃ for 20 min. Before solidification, it is poured into sterile petri dishes with a diameter of 9 cm, 15–20 mL per dish.

[0035] The content of Codonopsis pilosula polysaccharides was determined using the phenol-sulfuric acid method. Specifically, the Codonopsis pilosula polysaccharide extract was diluted with deionized water at an appropriate ratio (to estimate that the polysaccharide concentration in the sample was within the range of the standard curve). A sample solution with a concentration of 0.2 mg / mL was prepared from the solid Codonopsis pilosula polysaccharide extract using deionized water. 1 mL of the sample solution was pipetted into a 10 mL stoppered tube, 1 mL of 5% (v / v) phenol aqueous solution was added, and the mixture was shaken well. Then, 5 mL of concentrated sulfuric acid (98% (w / v)) was quickly added, shaken well, and heated in a boiling water bath for 15 min, followed by cooling to room temperature. Using 1 mL of deionized water as a blank control, the absorbance (A) at a wavelength of 490 nm was measured. 490 A was determined in glucose samples of different concentrations using the same method. 490 Plot glucose concentration—A 490 Standard curve ( Figure 2 The regression equation was obtained, and the polysaccharide content in the Codonopsis pilosula polysaccharide sample was calculated and determined from the regression equation.

[0036] The yield of Codonopsis pilosula polysaccharide extraction is calculated using the following formula:

[0037]

[0038] Example 2: Secondary screening of microbial strains for fermenting Codonopsis pilosula

[0039] Single spores of strain DS6 were isolated again to screen for strains with better fermentation performance. The specific method is as follows:

[0040] (1) Preparation of spore suspension: After culturing strain DS-6 on PDA slant medium at 28℃ for 48 h, 5 mL of sterile physiological saline was added. The spores were then suspended by stirring with an inoculation loop and transferred to an Erlenmeyer flask containing 45 mL of sterile physiological saline (with approximately 50 glass beads). The flask was shaken for 15 min. The spore suspension was filtered to remove mycelia (the neck of the Erlenmeyer funnel was plugged with a small amount of absorbent cotton). The spore suspension was diluted with sterile physiological saline to 1×10⁻⁶. -6 1×10 -7 1×10 -8 After dilution, 0.1 mL of spore solution at different dilutions was transferred and spread onto PDA agar plates. The plates were then inverted and incubated at 28°C for 48 hours. Single colonies from the PDA plates were transferred to fresh PDA agar plates, resulting in 35 bacterial strains. The strain numbers are shown in Table 2.

[0041] (2) Screening of strains: 10 mL of sterile physiological saline was added to fresh plate cultures of each strain after 72 h of incubation at 28 °C. The spores were suspended by stirring with an inoculation loop to obtain the spore liquid of each strain. 2 mL of the spore liquid of each strain was inoculated into 50 mL of enzyme-producing medium and cultured at 28 °C and 200 r / min for 72 h. The fermentation broth was then filtered using a Buchner funnel, and the filtrate (i.e., crude enzyme solution) was collected. The cellulase activity of the fermentation filtrate of each strain was measured. Thirteen strains with relatively higher enzyme production activity than the original strain DS6 were selected. The spore liquid of these 13 strains was then used to inoculate Codonopsis pilosula powder for fermentation according to the method in Example 1. Polysaccharides were extracted by ultrasonic water extraction and alcohol precipitation. The polysaccharide extraction yields of the re-screened strains fermenting Codonopsis pilosula and the control are shown in Table 2.

[0042] Table 2. Polysaccharide extraction yield of fermented Codonopsis pilosula and control strains after secondary screening.

[0043]

[0044]

[0045] As shown in Table 2, among the 13 strains screened, strain DS6-14 had a cellulase activity of 65.6 U / mL, which was 16.7% higher than the 56.2 U / mL of the original strain DS-6. After fermenting Codonopsis pilosula powder with this strain, the polysaccharide extraction rate was 24.4%, which was 14.6% higher than the 21.3% of the wild strain DS-6 and 46.1% higher than the 16.8% of the unfermented extraction control.

[0046] The enzyme-producing culture medium consists of: 40 g / L soluble starch, 6 g / L peptone, 4 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L CaCl2, and 0.1 g / L FeSO4·7H2O, with tap water as the solvent and a pH of 6.0. 50 mL of the enzyme-producing culture medium is placed in a 250 mL Erlenmeyer flask, sealed with eight layers of gauze, and sterilized by autoclaving at 121°C for 20 min.

[0047] The cellulase activity assay was performed as follows: 1.5 mL of 10 g / L sodium carboxymethyl cellulose solution (pH 6.0, prepared with 0.2 mol / L phosphate buffer) and 0.5 mL of crude enzyme solution were added to 10 mL graduated test tubes. The tubes were incubated in a 50°C water bath for 30 min, then 3 mL of DNS reagent was added, and the mixture was boiled for 5 min. After cooling under running water, deionized water was added to bring the volume to 10 mL, and the mixture was shaken well. A crude enzyme solution inactivated by boiling at 100°C for 10 min was used as a reference. The absorbance (A) was measured at 540 nm using a spectrophotometer. 540 ). From the glucose standard curve ( Figure 3Calculate the glucose concentration in the sample, and then calculate the cellulase activity (U / mL). Cellulase activity is defined as the amount of enzyme required per minute to hydrolyze sodium carboxymethyl cellulose to produce 1 μg of glucose under pH 6.0 and 50℃ conditions; one unit of activity (U) is defined as this amount of enzyme.

[0048] Cellulase activity is calculated according to the following formula (1).

[0049]

[0050] In formula (1), C: glucose concentration (μg / mL) calculated from the standard curve; V1: enzyme reaction system volume, i.e. 2mL; T: reaction time, i.e. 30min; V2: crude enzyme solution volume, i.e. 0.5mL.

[0051] Construction of the glucose standard curve: Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of 1 mg / mL standard glucose aqueous solution to seven 10-mL test tubes, respectively. Then add 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, and 0.8 mL of pH 6.0, 0.2 mol / L phosphate buffer to each tube, respectively. Add 3.0 mL of DNS solution to each tube. Boil the mixture in a water bath for 5 minutes, cool under running water, and then dilute to 10 mL with deionized water. Shake well and measure A using a spectrophotometer. 540 Plotting glucose concentration on the x-axis, A 540 Plot a standard curve for the ordinate ( Figure 3 ).

[0052] Preparation of DNS reagent: Add 6.3g of 3,5-dinitrosalicylic acid and 262mL of 2mol / L NaOH aqueous solution to 500mL of hot aqueous solution containing 182g of sodium tartrate, then add 5g of redistilled phenol and 5g of sodium sulfite, stir to dissolve, cool, and add deionized water to make up to 1L. Store in a brown bottle and use after 7 days.

[0053] Example 3: Classification and identification of strain DS6-14

[0054] Strawberry strain DS6-14 was streaked onto PDA plates and cultured at 28°C. Initially, colonies were grayish-white and fluffy, turning gray after 2 days. The mycelial layer was thick and loose, with relatively long hyphae and gray spores on the surface. The reverse side of the colony was grayish-white with no pigment diffusion. Creeping hyphae and rhizoids were underdeveloped, with few and short rhizoids. Sporangiophores were mostly curved, rarely solitary, and often formed umbrella-like aggregates, growing directly from the hyphae without corresponding to rhizoids. Sporangia were spherical or nearly spherical, dark brownish-gray. Sporangiospores were elliptical or polygonal, 4–7 μm in diameter. A photograph of DS6-14 colonies cultured on PDA plates at 28°C for 3 days is shown below. Figure 1 .

[0055] The rDNA-ITS nucleotide sequence of strain DS6-14 was determined as shown in SEQ ID NO.1. This sequence underwent BLAST alignment at NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) and showed 99.28% homology with the rDNA-ITS sequence of the known typical Rhizopus arrhizus strain CBS112.07. Based on the colony morphology characteristics of strain DS6-14 and the rDNA-ITS nucleotide sequence alignment results, the biological taxonomic position of strain DS6-14 can be determined as follows (refer to Mycobank, http: / / www.mycobank.org): Kingdom Fungi, Phylum Mucoromycota, Class Mucoromycetes, Order Mucorales, Family Mucoraceae, Genus Rhizopus, Rhizopus arrhizus.

[0056] The rDNA-ITS nucleotide sequence of strain DS6-14 is as follows:

[0057] .

[0058] In summary, strain DS6 was isolated from the microbial enrichment of Codonopsis pilosula powder. After a second isolation and purification, strain DS6-14, namely Rhizopus arrhizus DS6-14, was obtained through screening. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No:63063, deposit date December 20, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China.

[0059] Example 4: Application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides

[0060] The application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides can be carried out according to the following steps:

[0061] (1) Freeze-dried Rhizopus DS6-14 spore powder was inoculated onto fresh PDA agar plates and incubated at 28℃ for 72 h. 10 mL of sterile sucrose aqueous solution was added to the petri dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was transferred to sterile test tubes, and the spore concentration was adjusted to 8.75 × 10⁻⁶ spores with sterile sucrose aqueous solution.6 The concentration of Rhizopus spores was 30 g / mL, yielding a Rhizopus DS6-14 spore suspension. The composition and preparation method of the PDA plate culture medium were the same as in Example 1; the concentration of the sterile sucrose aqueous solution was 30 g / L, and it was sterilized by high-pressure steam at 115°C for 20 min.

[0062] (2) Add 5g of Codonopsis pilosula powder to a 100-mL Erlenmeyer flask that has been sterilized by dry heat at 160℃ for 2 hours, and then add 7.5mL of Rhizopus DS6-14 spore solution prepared in step (1) (the volume of which is 1.5mL / g based on the mass of Codonopsis pilosula powder), and stir well. Seal the Erlenmeyer flask with 8 layers of gauze and incubate at 28℃ for 64 hours to obtain Codonopsis pilosula ferment.

[0063] (3) Transfer all the Codonopsis pilosula fermentation material from step (2) into a 250-mL beaker, add 125mL of deionized water [material-to-liquid ratio of 1:25 (g:mL)], stir well, and enzymatically hydrolyze in a 35℃ water bath for 4 hours. Then, transfer the Erlenmeyer flask to an ultrasonic cleaner at 95℃ and ultrasonically extract at 100W for 2 hours. Filter the extract using a Buchner funnel while it is still hot, and concentrate all the filtrate under reduced pressure at 60℃ and –0.1Mpa to 5mL (1 / 25 of the original filtrate volume) to obtain the Codonopsis pilosula aqueous extract concentrate.

[0064] (4) Add 25 mL of anhydrous ethanol (5 times the volume of the concentrate, the ethanol volume fraction of the system is 83.3%) to all the Codonopsis pilosula water extract concentrate obtained in step (3), let it stand at 4℃ for 12 h, centrifuge at 8000 r / min for 5 min, discard the supernatant, add 10 mL of anhydrous ethanol (2.0 mL / g based on the mass of raw Codonopsis pilosula powder) to the precipitate and wash it once, centrifuge again, and dry the precipitate under vacuum at 50℃ and –0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0065] Following the steps above, 1.87g of polysaccharide extract was obtained from 5g of Codonopsis pilosula, with a polysaccharide content of 65.7%, which yielded 1.23g of Codonopsis pilosula polysaccharide, representing an extraction yield of 24.6%.

[0066] Comparative Example 1: Extraction of Codonopsis pilosula polysaccharides using conventional water extraction and alcohol precipitation method (compared with Example 4)

[0067] (1) 5g of Codonopsis pilosula powder was placed in a 250-mL beaker, and 125mL of deionized water was added [the ratio of material to liquid was 1:25 (g:mL)]. After stirring evenly, the mixture was kept warm in a 35℃ water bath for 4 hours. Then, the Erlenmeyer flask was transferred to an ultrasonic cleaner at 95℃ and ultrasonically extracted at 100W for 2 hours. The mixture was filtered through a Buchner funnel while still hot. All the filtrate was concentrated under reduced pressure at 60℃ and –0.1Mpa to 5mL (1 / 25 of the original filtrate volume) to obtain the Codonopsis pilosula water extract concentrate.

[0068] (2) Add 25 mL of anhydrous ethanol (5 times the volume of the concentrate, the ethanol volume fraction of the system is 83.3%) to all the Codonopsis pilosula water extract concentrate obtained in step (1), let it stand for 12 h at 4 ℃, centrifuge at 8000 r / min for 5 min, discard the supernatant, add 10 mL of anhydrous ethanol (2.0 mL / g based on the mass of raw Codonopsis pilosula powder) to the precipitate and wash it once, centrifuge again, and dry the precipitate under vacuum at 50 ℃ and -0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0069] Following the steps above, 1.53g of polysaccharide extract was obtained from 5g of Codonopsis pilosula, with a polysaccharide content of 56.4%, resulting in 0.863g of Codonopsis pilosula polysaccharide and an extraction yield of 17.3%.

[0070] Comparing the results of Example 4 and Comparative Example 1, it can be seen that: before the ultrasonic water extraction of polysaccharides from Codonopsis pilosula, the addition of Rhizopus DS6-14 fermentation pretreatment increased the polysaccharide extraction yield from 17.3% without fermentation pretreatment to 24.6%, an increase of 42.2%.

[0071] Example 5: Application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides

[0072] The application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides can be carried out according to the following steps:

[0073] (1) PDA plate spores of Rhizopus oligosporus DS6-14 stored at 4℃ were inoculated onto fresh PDA plate medium and cultured at 28℃ for 64 h. 10 mL of sterile sucrose aqueous solution was added to the culture dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was transferred to sterile test tubes, and the spore concentration was adjusted to 7.05 × 10⁻⁶ using sterile sucrose aqueous solution. 6 The concentration of Rhizopus spores was 25 g / L, yielding a Rhizopus DS6-14 spore suspension. The composition and preparation method of the PDA plate culture medium were the same as in Example 1; the concentration of the sterile sucrose aqueous solution was 25 g / L, and it was sterilized by high-pressure steam at 115°C for 20 min.

[0074] (2) 10g of Codonopsis pilosula powder was placed in a 100-mL Erlenmeyer flask that had been sterilized by dry heat at 160℃ for 2 hours. Then, 18mL of Rhizopus DS6-14 spore solution prepared in step (1) was added (the volume was 1.8mL / g based on the weight of Codonopsis pilosula powder), and the mixture was stirred evenly. The Erlenmeyer flask was sealed with 8 layers of gauze and cultured at 28℃ for 62 hours to obtain Codonopsis pilosula ferment.

[0075] (3) Transfer all the Codonopsis pilosula fermentation material from step (2) into a 500-mL beaker, add 250mL of deionized water [material-to-liquid ratio of 1:25 (g:mL)], stir evenly, and enzymatically hydrolyze in a 30℃ water bath for 6 hours. Then, transfer the Erlenmeyer flask to an ultrasonic cleaner at 90℃ and ultrasonically extract at 125W for 1.5 hours. Filter the extract while hot using a Buchner funnel, and concentrate all the filtrate under reduced pressure at 60℃ and –0.1Mpa to 10mL (1 / 25 of the original filtrate volume) to obtain the Codonopsis pilosula aqueous extract concentrate.

[0076] (4) Add 40 mL of anhydrous ethanol (4 times the volume of the concentrate, with an ethanol volume fraction of 80%) to all the Codonopsis pilosula aqueous extract obtained in step (3). After standing for 14 h at 4 °C, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw Codonopsis pilosula powder) to the precipitate and wash once. Centrifuge again, and dry the precipitate under vacuum at 50 °C and –0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0077] Following the steps above, 3.64g of polysaccharide extract was obtained from 10g of Codonopsis pilosula, with a polysaccharide content of 64.3%, resulting in 2.34g of Codonopsis pilosula polysaccharide and an extraction yield of 23.4%.

[0078] Comparative Example 2: Extraction of Codonopsis pilosula polysaccharides using conventional water extraction and alcohol precipitation method (compared with Example 5)

[0079] (1) 10g of Codonopsis pilosula powder was placed in a 500-mL beaker, and 250mL of deionized water was added [the ratio of material to liquid was 1:25 (g:mL)]. After stirring evenly, the mixture was kept warm in a 30℃ water bath for 6 hours. Then, the Erlenmeyer flask was transferred to an ultrasonic cleaner at 90℃ and ultrasonically extracted at 125W for 1.5 hours. The mixture was filtered through a Buchner funnel while still hot. All the filtrate was concentrated under reduced pressure at 60℃ and –0.1Mpa to 10mL (1 / 25 of the original filtrate volume) to obtain the Codonopsis pilosula water extract concentrate.

[0080] (2) Add 40 mL of anhydrous ethanol (4 times the volume of the concentrate, the ethanol volume fraction of the system is 80%) to all the Codonopsis pilosula water extract concentrate obtained in step (1). After standing for 14 h at 4 °C, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 15 mL of anhydrous ethanol (1.5 mL / g based on the mass of the raw Codonopsis pilosula powder) to wash once, centrifuge again, and dry the precipitate under vacuum at 50 °C and –0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0081] Following the steps above, 2.99g of polysaccharide extract was obtained from 10g of Codonopsis pilosula, with a polysaccharide content of 55.2%, resulting in 1.65g of Codonopsis pilosula polysaccharide and an extraction yield of 16.5%.

[0082] Comparing the results of Example 5 and Comparative Example 2, it can be seen that: before the ultrasonic water extraction of polysaccharides from Codonopsis pilosula, the addition of Rhizopus DS6-14 fermentation pretreatment increased the polysaccharide extraction yield from 16.5% without fermentation pretreatment to 23.4%, an increase of 41.8%.

[0083] Example 6: Application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides

[0084] The application of Rhizopus DS6-14 in the extraction of Codonopsis pilosula polysaccharides can be carried out according to the following steps:

[0085] (1) PDA plate spores of Rhizopus oligosporus DS6-14 stored at 4℃ were inoculated onto fresh PDA plate medium and cultured at 28℃ for 56 h. 10 mL of sterile sucrose aqueous solution was added to the culture dish, and the spores were suspended by stirring with an inoculation loop. The spore suspension was transferred to sterile test tubes, and the spore concentration was adjusted to 8.10 × 10⁻⁶ using sterile sucrose aqueous solution. 6 The PDA plate culture medium composition and preparation method are the same as in Example 1; the concentration of the sterile sucrose aqueous solution is 20 g / L, and it is sterilized by high-pressure steam at 115°C for 20 min.

[0086] (2) 25g of Codonopsis pilosula powder was placed in a 150-mL Erlenmeyer flask that had been sterilized by dry heat at 160℃ for 2 hours. 50mL of Rhizopus DS6-14 spore solution prepared in step (1) was added (the volume was 20mL / g based on the weight of the Codonopsis pilosula powder), and the mixture was stirred evenly. The Erlenmeyer flask was sealed with 8 layers of gauze and incubated at 28℃ for 60 hours to obtain the Codonopsis pilosula ferment.

[0087] (3) Transfer all the Codonopsis pilosula fermentation material from step (2) into a 1-L beaker, add 500mL of deionized water [material-to-liquid ratio of 1:20 (g:mL)], stir evenly, and enzymatically hydrolyze in a 30℃ water bath for 8 hours. Then, transfer the Erlenmeyer flask to an ultrasonic cleaner at 90℃ and ultrasonically extract at 150W for 1.5 hours. Filter the extract using a Buchner funnel while it is still hot, and concentrate all the filtrate under reduced pressure at 60℃ and –0.1Mpa to 25mL (1 / 20 of the original filtrate volume) to obtain the Codonopsis pilosula aqueous extract concentrate.

[0088] (4) Add 100 mL of anhydrous ethanol (4 times the volume of the concentrate, with an ethanol volume fraction of 80%) to all the Codonopsis pilosula aqueous extract obtained in step (3). After standing for 16 h at 4 °C, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 25 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw Codonopsis pilosula powder) to the precipitate and wash once. Centrifuge again, and dry the precipitate under vacuum at 50 °C and –0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0089] Following the steps described above, 9.38g of polysaccharide extract was obtained from 25g of Codonopsis pilosula, with a polysaccharide content of 66.1%, yielding 6.20g of Codonopsis pilosula polysaccharide, representing an extraction yield of 24.8%.

[0090] Comparative Example 3: Extraction of Codonopsis pilosula polysaccharides using conventional water extraction and alcohol precipitation method (compared with Example 6)

[0091] (1) 25g of Codonopsis pilosula powder was placed in a 1-L beaker, and 500mL of deionized water was added [the ratio of material to liquid was 1:20 (g:mL)]. After stirring evenly, the mixture was kept warm in a 30℃ water bath for 8 hours. Then, the Erlenmeyer flask was transferred to an ultrasonic cleaner at 90℃ and ultrasonically extracted at 150W for 1.5 hours. The mixture was filtered through a Buchner funnel while still hot. All the filtrate was concentrated under reduced pressure at 60℃ and -0.1Mpa to 25mL (1 / 20 of the original filtrate volume) to obtain the Codonopsis pilosula water extract concentrate.

[0092] (2) Add 100 mL of anhydrous ethanol (4 times the volume of the concentrate, with an ethanol volume fraction of 80%) to all the Codonopsis pilosula aqueous extract obtained in step (1). After standing for 16 h at 4 °C, centrifuge at 8000 r / min for 10 min, discard the supernatant, add 25 mL of anhydrous ethanol (1.0 mL / g based on the mass of the raw Codonopsis pilosula powder) to wash once, centrifuge again, and dry the precipitate under vacuum at 50 °C and –0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide extract.

[0093] Following the steps described above, 7.61g of polysaccharide extract was obtained from 25g of Codonopsis pilosula, with a polysaccharide content of 55.6%, yielding 4.23g of Codonopsis pilosula polysaccharide, representing an extraction yield of 16.9%.

[0094] Comparing the results of Example 6 and Comparative Example 3, it can be seen that: before the ultrasonic water extraction of polysaccharides from Codonopsis pilosula, the addition of Rhizopus DS6-14 fermentation pretreatment increased the polysaccharide extraction yield from 16.9% without fermentation pretreatment to 24.8%, an increase of 46.7%.

Claims

1. Rhizopus septemlobus ( Rhizopus arrhizus DS6-14, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 63063, deposited on December 20, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode 510070.

2. The application of Rhizopus DS6-14 as described in claim 1 in the extraction of Codonopsis pilosula polysaccharides, characterized in that, The method of application is as follows: (1) Add the spore liquid of Rhizopus DS6-14 to Codonopsis powder, stir evenly, and ferment at 28℃ for 60–64 h to obtain Codonopsis fermentation product; (2) Add deionized water to Codonopsis fermentation product, stir evenly, and enzymatically hydrolyze at 30–35℃ for 4–8 h, then filter and concentrate after ultrasonic water extraction to obtain Codonopsis water extract concentrate; (3) Add ethanol to Codonopsis water extract concentrate to precipitate polysaccharides, and after washing the precipitate with ethanol and vacuum drying, obtain Codonopsis polysaccharides.

3. The application as described in claim 2, characterized in that, The Codonopsis powder in step (1) is the dried root of Codonopsis pilosula, which is then pulverized and passed through an 80-mesh sieve.

4. The application as described in claim 2, characterized in that, In step (1), the volume of Rhizopus DS6-14 spore liquid used is 1.5–2.0 mL / g based on the mass of Codonopsis pilosula powder.

5. The application as described in claim 2 or 4, characterized in that, The preparation method of Rhizopus DS6-14 spore solution in step (1) is as follows: Rhizopus DS6-14 spores preserved at low temperature are inoculated on potato dextrose agar plates and cultured at 28℃ for 56–72 h. Then, sterile sucrose aqueous solution is added to the culture, and the spores are suspended by stirring with an inoculation loop to obtain spore solution.

6. The application as described in claim 5, characterized in that, The concentration of the sterile sucrose aqueous solution is 20–30 g / L, and the concentration of the spore solution is 5 × 10⁻⁶ g / L. 6 –10×10 6 per mL.

7. The application as described in claim 2, characterized in that, The preparation method of the Codonopsis pilosula water extract concentrate in step (2) is as follows: add deionized water to the Codonopsis pilosula fermentation product, stir evenly, and then enzymatically hydrolyze at 30–35℃ for 4–8 h. Then transfer it to an ultrasonic cleaner at 90–95℃ and ultrasonically extract at 100–150 W for 1.5–2.0 h. After ultrasonic water extraction, filter it with a Buchner funnel while it is hot, and concentrate the filtrate under reduced pressure at 60℃ and -0.1 MPa to 1 / 25–1 / 20 of the original volume to obtain the Codonopsis pilosula water extract concentrate.

8. The application as described in claim 2 or 7, characterized in that, The volume of deionized water added is 20–25 mL / g based on the mass of the raw Codonopsis pilosula powder.

9. The application as described in claim 2, characterized in that, The preparation method of Codonopsis pilosula polysaccharide in step (3) is as follows: add 4-5 times the volume of anhydrous ethanol to the water extract concentrate of Codonopsis pilosula to make the ethanol volume fraction of the system reach 80%-83.3%. After standing at 4℃ for 12-16 h, centrifuge at 8000 r / min for 5-10 min, collect the precipitate, add 1-2 mL / g of anhydrous ethanol (by weight of raw Codonopsis pilosula powder) to wash once, centrifuge again, and dry the precipitate under vacuum at 50℃ and -0.1 MPa to constant weight to obtain Codonopsis pilosula polysaccharide.

Images