Preparation method for monascus polysaccharide

Abstract

The present invention relates to a preparation method for a monascus polysaccharide, comprising: mycelium preparation: activating a monascus strain, then carrying out inoculation to obtain a monascus seed solution, further fermenting the monascus seed solution, collecting mycelia, and drying the mycelia for later use, wherein the monascus strain is monascus pilosus, and the strain number of monascus pilosus is CICC 5045; crude polysaccharide extraction: mixing the dried mycelia with alkaline liquor, then carrying out leaching to obtain a leaching solution, carrying out ultrafiltration concentration on the leaching solution to obtain a concentrated solution, and using a sevag reagent to carry out deproteinization on the concentrated solution so as to obtain a monascus crude polysaccharide; preliminary purification: using a DEAE-52 cellulose chromatography column to carry out preliminary separation and purification on the monascus crude polysaccharide so as to obtain a polysaccharide solution; and secondary purification: using a Sephadex G-100 glucan gel chromatography column to further carry out separation and purification on the polysaccharide solution so as to obtain a monascus polysaccharide product. The purity of the monascus polysaccharide in the product prepared by the present invention reaches 95.23±3.66%, and the product has good economic value.

Description

A method for preparing Monascus polysaccharide Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and in particular to a method for preparing monascus polysaccharide. Background Art

[0002] Red yeast rice polysaccharide (Monascus koji polysaccharide) is a fungal polysaccharide with no toxic side effects. It is commonly used as a raw material in foods and health supplements. Internationally, fungal polysaccharides are also known as "biological response modifiers," or BRMs. Studies have shown that the active ingredients in red yeast rice polysaccharide have a certain effect on heterologous, homologous, and even hereditary tumors. Furthermore, it has antibacterial, antiviral, and anti-agglomeration properties.

[0003] Existing research on Monascus has mostly focused on secondary metabolites such as pigments, γ-aminobutyric acid, and lovastatin, while less research has focused on the biological activity of Monascus polysaccharides metabolized by Monascus and how to achieve high-yield fermentation of Monascus polysaccharides. In the prior art, Monascus polysaccharides are mostly cultured in solid-state culture, which has a long culture cycle and is easily contaminated by bacteria, leading to production failure. Furthermore, solid-state fermentation is mainly manual, with complex steps, high labor intensity, and long cycles. The process also has low quality stability, making it unsuitable for large-scale production, and the resulting product contains low levels of Monascus polysaccharides.

[0004] In recent years, some researchers have made some progress in the production technology of red yeast rice polysaccharides by developing different red yeast rice strains. For example, Chinese patent application number CN106929428A discloses a method and product for producing red yeast rice polysaccharides by liquid fermentation. The method discloses a mutagenized strain of red yeast rice (Monascus anka Yang YZ2001). The method also discloses a method and product for producing red yeast rice polysaccharides by liquid fermentation using the mutagenized strain of red yeast rice. The resulting red yeast rice mycelium extract has a red yeast rice polysaccharide content of up to 95-99%. This method is suitable for industrial production and the mycelium extract obtained has a high content of red yeast rice polysaccharides. However, this method is only applicable to the mutagenized strain of Monascus anka, with a biological deposit number of CGMCC 8167, has a single source of raw materials, and is not applicable to other red yeast rice strains. Therefore, it is urgent to develop more processing methods that can be industrialized, improve production efficiency, and produce red yeast rice polysaccharide products with higher purity.

[0005] Summary of the Invention

[0006] The technical problem solved by the present invention is to provide a method for preparing Monascus mycelium polysaccharide, which can improve production efficiency and produce a Monascus polysaccharide product with higher purity and can be industrially processed and produced.

[0007] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0008] A method for preparing Monascus polysaccharide, comprising:

[0009] Mycelium preparation: The Monascus purpureus strain is activated and inoculated to obtain a Monascus seed solution, which is further fermented and then the mycelium is collected and dried for later use; the Monascus purpureus strain is Monascus pilosus, strain number CICC 5045;

[0010] Extraction of crude polysaccharide: Mix the dried mycelium with alkali solution and extract to obtain an extract, which is then concentrated by ultrafiltration to obtain a concentrate. The ultrafiltration temperature is 20-30°C and the rotor speed is 7500-8500 rpm. The concentrate is deproteinized using Sevag reagent to obtain Monascus crude polysaccharide.

[0011] Preliminary purification: The crude Monascus polysaccharide was preliminarily separated and purified using a DEAE-52 cellulose chromatography column to obtain a polysaccharide solution;

[0012] Secondary purification: using Sephadex G-100 dextran gel chromatography column to further separate and purify the polysaccharide solution to obtain red yeast polysaccharide product.

[0013] Furthermore, in the mycelium preparation step, the Monascus seed solution was prepared at a concentration of 1×10 5 ~1×10 7 mg / mL Monascus spore suspension.

[0014] Furthermore, in the mycelium preparation step, the Monascus seed liquid is added to the culture medium, and then fermented in a constant temperature shaker at 28-32° C. and 120-180 rpm in the dark for 48-72 hours.

[0015] Furthermore, the volume ratio of the Monascus seed liquid to the culture medium is 10-20:100; the culture medium includes: 40-60 g / L glucose, 5-10 g / L peptone, 0.5-1 g / L MgSO4, 3-5 g / L yeast powder, and 1.0-1.5 g / L KH2PO4.

[0016] Furthermore, in the crude polysaccharide extraction step, the alkali solution is a NaOH solution, the concentration of the NaOH solution is 0.15-0.25M, the material-liquid ratio of the dried mycelium to the NaOH solution is 1:25-35; the extraction temperature is 90-95°C, and the extraction time is 2-3h.

[0017] Furthermore, in the crude polysaccharide extraction step, the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cut-off of 10k MWCO.

[0018] Furthermore, in the crude polysaccharide extraction step, the method of deproteinizing with sevag reagent is as follows: the concentrate and sevag reagent are mixed in a volume ratio of 1:3 to 5, the supernatant is obtained by centrifugation, the supernatant is heated at a constant temperature of 65 to 75°C for 2.5 to 3.5 hours, and then 4 to 5 times the volume of anhydrous ethanol is added to the supernatant. After mixing, the mixture is allowed to stand at 3 to 5°C for 10 to 14 hours, the precipitate is obtained by centrifugation, and the crude Monascus polysaccharide is obtained by dissolving it in pure water and freeze-drying it.

[0019] Furthermore, in the preliminary purification step, the crude Monascus polysaccharide was prepared into a polysaccharide solution with a concentration of 4.5-5.5 mg / mL. After the polysaccharide solution was loaded, it was gradient eluted with 0, 0.1, 0.3, 0.5, and 0.7 M NaCl solutions at a flow rate of 1 mL / min, and the polysaccharide solution was detected and collected.

[0020] Furthermore, during the secondary purification process, the polysaccharide solution obtained by preliminary purification on a DEAE-52 cellulose chromatography column was prepared into a solution with a concentration of 4.5 to 5.5 mg / mL. After loading on a Sephadex G-100 dextran gel chromatography column, it was eluted with deionized water at a flow rate of 0.4 to 0.6 mL / min, and the red yeast polysaccharide product was detected and collected.

[0021] Furthermore, the purity of the red yeast rice polysaccharide in the red yeast rice polysaccharide product is 95.23±3.66%.

[0022] Furthermore, in the mycelium preparation step, after fermentation is completed, high-temperature sterilization time is 15 to 25 minutes, the temperature is 121° C., and the pressure is 0 to 0.25 MPa.

[0023] Beneficial Effects: The method for preparing monascus polysaccharide effectively prevents bacterial contamination, the production of mycotoxins, and impurity metabolites, while efficiently conducting liquid fermentation. A unique extraction and purification process yields high-purity monascus polysaccharide. The purity of the final product can reach 95.23±3.66%, demonstrating excellent economic value. Furthermore, the yield and purity are consistent across batches, making it suitable for mass production.

[0024] The method for preparing monascus polysaccharide of the present invention uses a combination of alkali extraction and ultrafiltration concentration after fermentation of monascus pilosus with the strain number CICC 5045 to achieve rapid extraction of polysaccharide from the mycelia of monascus pilosus. This effectively improves the extraction efficiency of polysaccharide from the mycelia of monascus pilosus while preliminarily removing macromolecular impurities. Subsequent preliminary purification and secondary purification are further combined to further remove impurities, thereby preparing a monascus polysaccharide product with high purity, effectively increasing the purity of the polysaccharide from the mycelia of monascus pilosus from 36.86% to 98.65%. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments.

[0026] Example 1

[0027] The method for preparing Monascus polysaccharide comprises:

[0028] Mycelium preparation:

[0029] The Monascus strains were activated and transferred to PDA plate medium. Sterile water was added, mycelia were scraped and filtered with sterile cotton to prepare a concentration of 1×10 6 The Monascus spore suspension is 100 mg / mL. The Monascus species is Monascus pilosus, with the strain number CICC 5045, purchased from the China Industrial Microbiology Culture Collection Center.

[0030] Monascus seed liquid was added to the culture medium at a volume ratio of 15% of the culture medium volume, and then fermented in a light-protected shaker at 30°C and 150 rpm for 54 hours. The volume ratio of the Monascus seed liquid to the culture medium was 15:100. The culture medium contained 50 g / L glucose, 8 g / L peptone, 0.8 g / L MgSO4, 4 g / L yeast extract, and 1.2 g / L KH2PO4.

[0031] After fermentation is complete, high-temperature sterilization is performed to terminate the fermentation. The high-temperature sterilization time is 20 minutes, the temperature is 121°C, and the pressure is 0.25 MPa. After high-temperature sterilization, the mycelium is collected, dried at a constant temperature of 60°C, crushed and passed through a 60-mesh sieve, and stored for later use.

[0032] Crude polysaccharide extraction:

[0033] The dried mycelium was mixed with 0.2M NaOH solution at a material-liquid ratio of 1:30, and placed in a 95°C water bath for 3 hours. After the water bath, the mixture was centrifuged at 4°C and 8000 rpm for 10 minutes to collect the supernatant; the supernatant was intercepted and concentrated using a 10k MWCO ultrafiltration tube at 20°C and 8000 r / min to obtain a concentrate; the concentrate and sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube at a volume ratio of 1:4, mixed evenly, centrifuged at 4°C and 8000 rpm for 3 minutes, and the supernatant was taken; the supernatant was placed in a 70°C water bath for 3 hours, 4 times the volume of anhydrous ethanol was added, mixed evenly, and placed at 4°C for 12 hours; centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected; the precipitate was dissolved in ultrapure water and vacuum freeze-dried to obtain Monascus crude polysaccharide;

[0034] Initial purification:

[0035] The DEAE-52 cellulose chromatography column was pretreated as follows: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h, floating particles were removed, and the column was filtered; the column was soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, filtered, and set aside.

[0036] Mount a chromatography column (1.6 × 30 cm) vertically, add 1 / 3 of its volume to the column with deionized water, open the lower outlet, and uniformly add cellulose slurry to the column wall until the slurry settles to 2 / 3 of the column volume. Stop loading the column. Connect the upper end of the column to a constant flow pump, open the lower outlet, and flush with deionized water at a flow rate of 1 mL / min for 12 hours before use.

[0037] The crude Monascus polysaccharide was prepared into a polysaccharide solution with a concentration of 5 mg / mL. After the polysaccharide solution was loaded on a DEAE-52 cellulose chromatography column, it was gradient eluted with 0, 0.1, 0.3, 0.5, and 0.7 M NaCl solutions at a flow rate of 1 mL / min. Twenty tubes were collected for each gradient, and each tube was collected for 10 to 12 minutes. The sugar content of each tube of solution was detected by the sulfuric acid-phenol method. The best peak segment was selected according to the absorbance at 490 nm to collect samples. The samples with the same gradient peak segment were combined and freeze-dried to prepare a 5 mg / mL polysaccharide solution.

[0038] Secondary purification

[0039] Sephadex G-100 gel chromatography column pretreatment: Add 1 g of gel to 30 mL of deionized water and allow to swell for 24 hours to remove suspended particles. Degas the swollen gel and stir thoroughly. Drain the gel into the column using a glass rod, taking care to avoid bubbles and fractures. After the gel settles, equilibrate it with deionized water for 24 hours before use.

[0040] A 5 mg / mL polysaccharide solution, initially purified using a DEAE-52 cellulose chromatography column, was added to the wall of a Sephadex G-100 dextran gel chromatography column. The column wall was washed with a small amount of deionized water and eluted with deionized water at a flow rate of 0.5 mL / min. Each tube was collected for 10 minutes, and a total of 20 tubes were collected. The sugar content of each tube of solution was tested using the sulfuric acid-phenol method. Samples were collected from the optimal peak segment based on the absorbance at 490 nm. Samples with the same gradient peak segment were combined and freeze-dried to obtain the red yeast polysaccharide product. After testing, the mycelial polysaccharide purity in the red yeast polysaccharide product was 98.65%.

[0041] Comparative Example 1

[0042] The method for preparing Monascus polysaccharide comprises:

[0043] Mycelium preparation:

[0044] The Monascus strains were activated and transferred to PDA plate medium. Sterile water was added, mycelia were scraped and filtered with sterile cotton to prepare a concentration of 1×10 6 The Monascus spore suspension is 100 mg / mL. The Monascus species is Monascus pilosus, with the strain number CICC 5045, purchased from the China Industrial Microbiology Culture Collection Center.

[0045] 15% by volume of Monascus seed liquid was added to a culture medium, followed by fermentation in a light-protected shaker at 30°C and 150 rpm for 54 hours. The volume ratio of the Monascus seed liquid to the culture medium was 15:100. The culture medium contained 50 g / L glucose, 8 g / L peptone, 0.8 g / L MgSO₄, 4 g / L yeast extract, and 1.2 g / L KH₂PO₄.

[0046] After fermentation is complete, high-temperature sterilization is performed to terminate the fermentation. The high-temperature sterilization time is 20 minutes, the temperature is 121°C, and the pressure is 0.25 MPa. After high-temperature sterilization, the mycelium is collected, dried at a constant temperature of 60°C, crushed and passed through a 60-mesh sieve, and stored for later use.

[0047] Crude polysaccharide extraction:

[0048] The dried mycelium was mixed evenly with 0.2M NaOH solution in a material-liquid ratio of 1:30, and placed in a 95℃ water bath for 3 hours. After the water bath, the mixture was centrifuged at 4℃ and 8000rpm for 10 minutes, and the supernatant was collected; the supernatant was intercepted and concentrated using a 10k MWCO ultrafiltration tube at 10℃ and 2000r / min to obtain a concentrate; the concentrate and sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube in a volume ratio of 1:4, mixed evenly, centrifuged at 4℃ and 8000rpm for 3 minutes, and the supernatant was taken; the supernatant was placed in a 70℃ water bath for 3 hours, 4 times the volume of anhydrous ethanol was added, mixed evenly, and placed at 4℃ for 12 hours; centrifuged at 8000rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected; after dissolving the precipitate in ultrapure water, vacuum freeze-dried, and the crude Monascus polysaccharide was obtained;

[0049] Initial purification:

[0050] The DEAE-52 cellulose chromatography column was pretreated as follows: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h, floating particles were removed, and the column was filtered; the column was soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, filtered, and set aside.

[0051] Mount a chromatography column (1.6 × 30 cm) vertically, add 1 / 3 of its volume to the column with deionized water, open the lower outlet, and uniformly add cellulose slurry to the column wall until the slurry settles to 2 / 3 of the column volume. Stop loading the column. Connect the upper end of the column to a constant flow pump, open the lower outlet, and flush with deionized water at a flow rate of 1 mL / min for 12 hours before use.

[0052] The crude Monascus polysaccharide was prepared into a polysaccharide solution with a concentration of 5.0 mg / mL. After the polysaccharide solution was loaded on a DEAE-52 cellulose chromatography column, it was gradient eluted with 0, 0.1, 0.3, 0.5, and 0.7 M NaCl solutions at a flow rate of 1 mL / min. Twenty tubes were collected for each gradient, and each tube was collected for 10 to 12 minutes. The sugar content of each tube of solution was detected by the sulfuric acid-phenol method. The best peak segment was selected according to the absorbance at 490 nm to collect samples. The samples with the same gradient peak segment were combined and freeze-dried to prepare a 5 mg / mL polysaccharide solution.

[0053] Secondary purification:

[0054] Sephadex G-100 gel chromatography column pretreatment: Add 1 g of gel to 30 mL of deionized water and allow to swell for 24 hours to remove suspended particles. Degas the swollen gel and stir thoroughly. Drain the gel into the column using a glass rod, taking care to avoid bubbles and fractures. After the gel settles, equilibrate it with deionized water for 24 hours before use.

[0055] A 5 mg / mL polysaccharide solution that had been preliminarily purified by a DEAE-52 cellulose chromatography column was added along the column wall of a Sephadex G-100 dextran gel chromatography column, and the column wall was washed with a small amount of deionized water. The deionized water was eluted at a flow rate of 0.5 mL / min, and each tube was collected for 10 minutes, with a total of 20 tubes collected. The sugar content of each tube of solution was detected by the sulfuric acid-phenol method. The best peak segment was selected based on the absorbance at 490 nm to collect the sample. The samples of the same gradient peak segment were combined and freeze-dried to obtain the red yeast polysaccharide product. In this control example, ultrafiltration concentration was not used within the parameter range to remove impurities. After testing, the purity of the red yeast polysaccharide in the red yeast polysaccharide product was 67.75%, and the purity of the mycelial polysaccharide in the red yeast polysaccharide product was 67.75%.

[0056] Comparative Example 2

[0057] The method for preparing Monascus polysaccharide comprises:

[0058] The Monascus strains were activated and transferred to PDA plate medium. Sterile water was added, mycelia were scraped and filtered with sterile cotton to prepare a concentration of 1×10 6 The Monascus spore suspension is 100 mg / mL. The Monascus species is Monascus pilosus, with the strain number CICC 5045, purchased from the China Industrial Microbiology Culture Collection Center.

[0059] 15% by volume of Monascus seed liquid was added to a culture medium, followed by fermentation in a light-protected shaker at 30°C and 150 rpm for 54 hours. The volume ratio of the Monascus seed liquid to the culture medium was 15:100. The culture medium contained 50 g / L glucose, 8 g / L peptone, 0.8 g / L MgSO₄, 4 g / L yeast extract, and 1.2 g / L KH₂PO₄.

[0060] After fermentation is complete, high-temperature sterilization is performed to terminate the fermentation. The high-temperature sterilization time is 20 minutes, the temperature is 121°C, and the pressure is 0.25 MPa. After high-temperature sterilization, the mycelium is collected, dried at a constant temperature of 60°C, crushed and passed through a 60-mesh sieve, and stored for later use.

[0061] Crude polysaccharide extraction:

[0062] The dried mycelium was mixed evenly with 0.2M NaOH solution in a material-liquid ratio of 1:30, and placed in a 95°C water bath for 3 hours. After the water bath ended, the mixture was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected; the supernatant and sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube in a volume ratio of 1:4, mixed evenly, and centrifuged at 4°C and 8000 rpm for 3 minutes, and the supernatant was taken; the supernatant was placed in a 70°C water bath for 3 hours, 4 times the volume of anhydrous ethanol was added, mixed evenly, and placed at 4°C for 12 hours; centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected; after dissolving the precipitate in ultrapure water, vacuum freeze-dried, and the crude Monascus polysaccharide was obtained;

[0063] Initial purification:

[0064] The DEAE-52 cellulose chromatography column was pretreated as follows: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h, floating particles were removed, and the column was filtered; the column was soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; the column was soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, filtered, and set aside.

[0065] Mount a chromatography column (1.6 × 30 cm) vertically, add 1 / 3 of its volume to the column with deionized water, open the lower outlet, and uniformly add cellulose slurry to the column wall until the slurry settles to 2 / 3 of the column volume. Stop loading the column. Connect the upper end of the column to a constant flow pump, open the lower outlet, and flush with deionized water at a flow rate of 1 mL / min for 12 hours before use.

[0066] The crude Monascus polysaccharide was prepared into a polysaccharide solution with a concentration of 5 mg / mL. After the polysaccharide solution was loaded on a DEAE-52 cellulose chromatography column, it was gradient eluted with 0, 0.1, 0.3, 0.5, and 0.7 M NaCl solutions at a flow rate of 1 mL / min. Twenty tubes were collected for each gradient, and each tube was collected for 10 to 12 minutes. The sugar content of each tube of solution was detected by the sulfuric acid-phenol method. The best peak segment was selected according to the absorbance at 490 nm to collect samples. The samples with the same gradient peak segment were combined and freeze-dried to prepare a 5 mg / mL polysaccharide solution.

[0067] Secondary purification:

[0068] Sephadex G-100 gel chromatography column pretreatment: Add 1 g of gel to 30 mL of deionized water and allow to swell for 24 hours to remove suspended particles. Degas the swollen gel and stir thoroughly. Drain the gel into the column using a glass rod, taking care to avoid bubbles and fractures. After the gel settles, equilibrate it with deionized water for 24 hours before use.

[0069] A 5 mg / mL polysaccharide solution that had been initially purified by a DEAE-52 cellulose chromatography column was added along the column wall of a Sephadex G-100 dextran gel chromatography column, and the column wall was washed with a small amount of deionized water. The deionized water was eluted at a flow rate of 0.5 mL / min, and each tube was collected for 10 minutes, with a total of 20 tubes collected. The sugar content of each tube of solution was detected by the sulfuric acid-phenol method. The best peak segment was selected based on the absorbance at 490 nm to collect the sample. The samples of the same gradient peak segment were combined and freeze-dried to obtain a red yeast polysaccharide product. In this control example, ultrafiltration concentration was not used to remove impurities. After testing, the purity of the red yeast polysaccharide in the red yeast polysaccharide product was 45.43%.

[0070] Comparative Example 3

[0071] The method for preparing Monascus polysaccharide comprises:

[0072] Mycelium preparation:

[0073] The Monascus strains were activated and transferred to PDA plate medium. Sterile water was added, mycelia were scraped and filtered with sterile cotton to prepare a concentration of 1×10 6The Monascus spore suspension is 100 mg / mL. The Monascus species is Monascus pilosus, with the strain number CICC 5045, purchased from the China Industrial Microbiology Culture Collection Center.

[0074] 15% by volume of Monascus seed liquid was added to a culture medium, followed by fermentation in a light-protected shaker at 30°C and 150 rpm for 54 hours. The volume ratio of the Monascus seed liquid to the culture medium was 15:100. The culture medium contained 50 g / L glucose, 8 g / L peptone, 0.8 g / L MgSO₄, 4 g / L yeast extract, and 1.2 g / L KH₂PO₄.

[0075] After fermentation is complete, high-temperature sterilization is performed to terminate the fermentation. The high-temperature sterilization time is 20 minutes, the temperature is 121°C, and the pressure is 0.25 MPa. After high-temperature sterilization, the mycelium is collected, dried at a constant temperature of 60°C, crushed and passed through a 60-mesh sieve, and stored for later use.

[0076] Crude polysaccharide extraction:

[0077] The dried mycelium was mixed evenly with 0.2M NaOH solution in a material-liquid ratio of 1:30, and placed in a 95°C water bath for 3 hours. After the water bath, the mixture was centrifuged at 4°C and 8000rpm for 10 minutes, and the supernatant was collected; the supernatant and sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube in a volume ratio of 1:4, mixed evenly, centrifuged at 4°C and 8000rpm for 3 minutes, and the supernatant was taken; the supernatant was placed in a 70°C water bath for 3 hours, 4 times the volume of anhydrous ethanol was added, mixed evenly, and placed at 4°C for 12 hours; centrifuged at 8000rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected; after dissolving the precipitate in ultrapure water, vacuum freeze-dried to obtain Monascus crude polysaccharide; in this control example, ultrafiltration concentration and secondary purification technology were not used. After testing, the purity of Monascus polysaccharide in the Monascus polysaccharide product was 36.86%.

[0078] Comparative Example 4

[0079] This comparative example used Monascus pilosus Zheng YZ101, with biological deposit number CGMCC 8167, and the liquid fermentation technology described in CN106929428A to produce a monascus polysaccharide product. Testing showed that the purity of the monascus polysaccharide in the product was 5-6%. This indicates that the technology used in CN106929428A is not suitable for the production of monascus polysaccharides from other Monascus species.

[0080] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications are possible without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Monascus polysaccharide, characterized in that: include: Preparation of mycelium: activating the Monascus species and inoculating it to obtain Monascus seed liquid, further fermenting the Monascus seed liquid, collecting mycelium, and drying it for later use; the Monascus species is Monascus pilosus, and the species number is CICC 5045; Extraction of crude polysaccharide: Mix the dried mycelium with alkali solution and extract to obtain an extract, and ultrafilter and concentrate the extract to obtain a concentrated solution. The ultrafiltration and concentration temperature is 20-30°C, and the rotor speed during ultrafiltration is 7500-8500r / min. After deproteinizing the concentrated solution with sevag reagent, the Monascus crude polysaccharide is obtained; Preliminary purification: Use DEAE-52 cellulose chromatography column to perform preliminary separation and purification of Monascus crude polysaccharide to obtain polysaccharide solution; Secondary purification: The polysaccharide solution is further separated and purified using a Sephadex G-100 dextran gel chromatography column to obtain a red yeast polysaccharide product.

2. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the mycelium preparation step, the Monascus seed solution was prepared at a concentration of 1×10 5 ~1×10 7 mg / mL of Monascus spore suspension, add Monascus seed liquid to the culture medium, and then ferment in a constant temperature shaker at 28-32°C and 120-180rpm in the dark for 48-72h.

3. The method for preparing Monascus polysaccharide according to claim 3, characterized in that: The volume ratio of the Monascus seed liquid to the culture medium is 10-20:100; the culture medium comprises: 40-60 g / L glucose, 5-10 g / L peptone, 0.5-1 g / L MgSO4, 3-5 g / L yeast powder, and 1.0-1.5 g / L KH2PO4.

4. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the crude polysaccharide extraction step, the alkali solution is a NaOH solution, the concentration of the NaOH solution is 0.15-0.25M, the solid-liquid ratio of the dried mycelium to the NaOH solution is 1:25-35; the leaching temperature is 90-95°C, and the leaching time is 2-3h.

5. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the crude polysaccharide extraction step, the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 10k MWCO.

6. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the crude polysaccharide extraction step, the method of deproteinizing with sevag reagent is as follows: the concentrated solution and sevag reagent are mixed in a volume ratio of 1:3-5, the supernatant is taken after centrifugation, the supernatant is heated at a constant temperature of 65-75°C for 2.5-3.5 hours, and then 4-5 times the volume of anhydrous ethanol is added to the supernatant, mixed, and allowed to stand at 3-5°C for 10-14 hours. The precipitate is taken after centrifugation, dissolved in pure water, and freeze-dried to obtain Monascus crude polysaccharide.

7. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the preliminary purification step, the crude Monascus polysaccharide was prepared into a polysaccharide solution with a concentration of 4.5-5.5 mg / mL. After the polysaccharide solution was loaded, it was gradient eluted with 0, 0.1, 0.3, 0.5, and 0.7 M NaCl solutions at a flow rate of 1 mL / min, and the polysaccharide solution was detected and collected.

8. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: During the secondary purification process, the polysaccharide solution obtained by preliminary purification with a DEAE-52 cellulose chromatography column was prepared into a solution with a concentration of 4.5 to 5.5 mg / mL. After loading on a Sephadex G-100 dextran gel chromatography column, it was eluted with deionized water at a flow rate of 0.4 to 0.6 mL / min, and the red yeast polysaccharide product was detected and collected.

9. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: The purity of the monascus polysaccharide in the monascus polysaccharide product is 95.23±3.66%.

10. The method for preparing Monascus polysaccharide according to claim 1, characterized in that: In the mycelium preparation step, after fermentation is completed, the high temperature sterilization time is 15 to 25 minutes, the temperature is 121° C., and the pressure is 0 to 0.25 MPa.