Preparation method of monascus polysaccharide

Abstract

The application discloses a preparation method of monascus polysaccharide, and belongs to the technical field of fermentation engineering.The method comprises the following steps: mycelium preparation: after activating monascus spores, monascus seed liquid is obtained, and after further fermentation of the monascus seed liquid, mycelium is collected and dried for standby; the monascus spores are monascus pilosus, and the strain number is CICC 5045; crude polysaccharide extraction: after mixing the dried mycelium with alkali liquid, leaching is carried out to obtain leaching liquid, the leaching liquid is concentrated through ultrafiltration to obtain concentrated liquid, and after removing protein from the concentrated liquid by using sevag reagent, monascus crude polysaccharide is obtained; preliminary purification: the monascus crude polysaccharide is preliminarily separated and purified by using a DEAE-52 cellulose chromatographic column to obtain a polysaccharide solution; secondary purification: the polysaccharide solution is further separated and purified by using a Sephadex G-100 dextran gel chromatographic column to obtain a monascus polysaccharide product.The purity of the monascus polysaccharide in the product prepared by the application can reach 95.23+ / -3.66%, and the product has good economic value.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a method for preparing Monascus purpureus polysaccharide. Background Technology

[0002] Red yeast rice polysaccharides are fungal polysaccharides with no toxic side effects. They are commonly used as raw materials and excipients in food and health food products. Internationally, fungal polysaccharides are also known as "biological response modifiers" (BRMs). Studies have shown that the active components of red yeast rice polysaccharides have certain effects on heterologous, homologous, and even hereditary tumors. In addition, they also have antibacterial, antiviral, and anticoagulant effects.

[0003] Existing research on Monascus purpureus has largely focused on its pigments, γ-aminobutyric acid (GABA), and lovastatin, among other secondary metabolites. However, research on the bioactivity of Monascus purpureus polysaccharides and methods for high-yield polysaccharide fermentation is relatively limited. Currently, most Monascus purpureus polysaccharide production utilizes solid-state fermentation, which has a long cultivation cycle and is susceptible to contamination by other microorganisms, leading to production failures. Furthermore, solid-state fermentation is primarily manual, involving cumbersome procedures, high labor intensity, and a long cycle, resulting in inconsistent quality and making it unsuitable for large-scale production. Additionally, the resulting products often have low Monascus purpureus polysaccharide content.

[0004] In recent years, some researchers have made progress in the production technology of red yeast polysaccharides by developing different strains of Monascus purpureus. For example, Chinese Patent CN106929428A discloses a method and product for producing red yeast polysaccharides by liquid fermentation. It discloses a mutagenic strain of Monascus anka Yang YZ2001 and a method for producing red yeast polysaccharides by liquid fermentation using this mutagenic strain. The resulting mycelial extract has a red yeast polysaccharide content as high as 95-99%. This method is suitable for industrial-scale production, and the mycelial extract has a high red yeast polysaccharide content. However, this method is only applicable to the mutagenic strain Monascus purpureus with bioaccession number CGMCC 8167, resulting in a single source of raw materials, and the method is not applicable to other Monascus strains. Therefore, it is urgent to develop more processing methods that can be industrially processed, improve production efficiency, and produce red yeast polysaccharide products with higher purity. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a method for preparing Monascus purpureus mycelial polysaccharide that can improve production efficiency, produce high-purity Monascus polysaccharide products, and can be industrially processed.

[0006] The technical problem solved by this invention is achieved by the following technical solution: A method for preparing Monascus purpureus polysaccharide, characterized in that it includes: Mycelium preparation: Monascus pilosus was activated and inoculated to obtain Monascus pilosus seed liquid. The Monascus pilosus seed liquid was further fermented and the mycelium was collected, dried and stored for later use. The Monascus pilosus strain was identified as Monascus pilosus, strain number CICC 5045. Crude polysaccharide extraction: The dried mycelium was mixed with alkaline solution and extracted to obtain an extract. The extract was then concentrated by ultrafiltration to obtain a concentrate. The temperature during ultrafiltration was 20~30℃ and the rotor speed was 7500~8500r / min. The concentrate was then deproteinized with Sevag reagent to obtain crude polysaccharide from Monascus purpureus. Preliminary purification: The crude polysaccharide from Monascus purpureus was initially separated and purified using a DEAE-52 cellulose chromatography column to obtain a polysaccharide solution; Secondary purification: The polysaccharide solution was further separated and purified using a Sephadex G-100 dextran gel chromatography column to obtain the red yeast rice polysaccharide product.

[0007] Furthermore, in the mycelium preparation step, the Monascus purpureus seed solution has a concentration of 1×10⁻⁶. 5 ~1×10 7 A suspension of Monascus purpureus spores per mL.

[0008] Furthermore, in the mycelium preparation step, Monascus seed liquid is added to the culture medium and then fermented in a constant temperature shaker at 28~32 ℃ and 120~180 rpm in the dark for 48~72 h.

[0009] Furthermore, the volume ratio of the Monascus purpureus seed liquid to the culture medium is 10~20:100; the culture medium comprises: glucose 40~60 g / L, peptone 5~10 g / L, MgSO4 0.5~1 g / L, yeast extract 3~5 g / L, and KH2PO4 1.0~1.5 g / L.

[0010] Furthermore, in the crude polysaccharide extraction step, the alkaline solution is a NaOH solution with a concentration of 0.15~0.25M, and the ratio of dried mycelium to NaOH solution is 1:25~35; the extraction temperature is 90~95℃, and the extraction time is 2~3h.

[0011] Furthermore, in the crude polysaccharide extraction step, the ultrafiltration membrane used for ultrafiltration concentration has a molecular weight cutoff of 10 kMWCO.

[0012] Further, in the crude polysaccharide extraction step, the method of deproteinizing with Sevag reagent is as follows: the concentrated solution and Sevag reagent are mixed at a volume ratio of 1:3~5, the supernatant is collected by centrifugation, the supernatant is heated at 65~75℃ for 2.5~3.5h, then 4~5 times the volume of anhydrous ethanol is added to the supernatant, mixed well, and allowed to stand at 3~5℃ for 10~14h. The precipitate is collected by centrifugation, dissolved in pure water, and freeze-dried to obtain crude polysaccharide of Monascus purpureus.

[0013] Furthermore, in the preliminary purification step, the crude polysaccharide of Monascus purpureus was prepared into a polysaccharide solution with a concentration of 4.5~5.5 mg / mL. After the polysaccharide solution was loaded, it was eluted with 0, 0.1, 0.3, 0.5 and 0.7 M NaCl solutions at a flow rate of 1 mL / min, respectively. The polysaccharide solution was then detected and collected.

[0014] Furthermore, during the secondary purification process, the polysaccharide solution obtained from the initial purification using a DEAE-52 cellulose chromatography column was prepared to a concentration of 4.5–5.5 mg / mL. After loading the solution onto a Sephadex G-100 dextran gel chromatography column, it was eluted with deionized water at a flow rate of 0.4–0.6 mL / min. The red yeast rice polysaccharide product was then detected and collected.

[0015] Furthermore, the purity of the red yeast rice polysaccharide in the product is 95.23±3.66%.

[0016] Furthermore, in the mycelium preparation step, after fermentation, the high-temperature sterilization time is 15~25 minutes, the temperature is 121℃, and the pressure is 0~0.25 MPa.

[0017] Beneficial Effects: The method for preparing Monascus purpureus polysaccharides described in this invention effectively prevents contamination by other microorganisms, the generation of mycotoxins and impurity metabolites, and efficiently carries out liquid fermentation production. Through a unique extraction and purification process, high-purity Monascus purpureus polysaccharides are obtained, with the final product achieving a purity of 95.23±3.66%, demonstrating significant economic value. Furthermore, the yield and purity remain stable across different batches, making it suitable for mass production.

[0018] The method for preparing Monascus pilosus polysaccharide described in this invention involves fermenting Monascus pilosus strain CICC 5045, followed by alkaline extraction and ultrafiltration concentration. This method achieves rapid extraction of Monascus pilosus mycelial polysaccharide, effectively improving the extraction efficiency while initially removing macromolecular impurities. Further preliminary and secondary purification further removes impurities, resulting in a high-purity Monascus pilosus polysaccharide product. The purity of the Monascus pilosus mycelial polysaccharide is effectively increased from 36.86% to 98.65%. Detailed Implementation

[0019] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0020] Example 1

[0021] The method for preparing Monascus purpureus polysaccharide includes: Mycelium preparation: The Monascus purpureus strain was activated and transferred to PDA agar plates. Sterile water was added, mycelia were scraped off, and filtered through sterile cotton to prepare a concentration of 1×10⁻⁶. 6 A suspension of Monascus pilosus spores per mL. The Monascus pilosus strain is CICC 5045 and was purchased from the China Industrial Microbial Culture Collection Center.

[0022] The Monascus purpureus seed culture was added to the culture medium at 15% of the medium volume, and then fermented in a constant temperature shaker at 30℃ and 150 rpm in the dark for 54 h. The volume ratio of the Monascus purpureus seed culture to the culture medium was 15:100; the culture medium consisted of: glucose 50 g / L, peptone 8 g / L, MgSO4 0.8 g / L, yeast extract 4 g / L, and KH2PO4 1.2 g / L.

[0023] After fermentation, the fermentation was terminated by high-temperature sterilization for 20 minutes at 121 ℃ and 0.25 MPa. The mycelium was collected after high-temperature sterilization, dried at a constant temperature of 60 ℃, pulverized through a 60-mesh sieve, and stored for later use.

[0024] Crude polysaccharide extraction: The dried mycelium was mixed with 0.2M NaOH solution at a ratio of 1:30 and placed in a 95 ℃ water bath for 3 h. After the water bath, the mixture was centrifuged at 4 ℃ and 8000 rpm for 10 min, and the supernatant was collected. The supernatant was concentrated using a 10 kWh MWCO ultrafiltration tube at 20 ℃ and 8000 rpm to obtain a concentrated solution. The concentrated solution and Sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube at a volume ratio of 1:4, mixed well, and centrifuged at 4 ℃ and 8000 rpm for 3 min. The supernatant was collected and placed in a 70 ℃ water bath for 3 h. Four times the volume of anhydrous ethanol was added, mixed well, and placed at 4 ℃ for 12 h. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The precipitate was dissolved in ultrapure water and then freeze-dried under vacuum to obtain crude polysaccharide from Monascus purpureus. Preliminary purification: Pretreatment of DEAE-52 cellulose chromatography column: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h to remove floating particles, and then filtered; it was then soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, and filtered for later use.

[0025] The chromatography column (1.6 × 30 cm) was vertically fixed, and 1 / 3 volume of deionized water was added. The bottom outlet was opened, and cellulose slurry was added to the column at a uniform rate, adhering to the column wall. The slurry level was increased to 2 / 3 volume of the column, and then the packing was stopped. The upper end of the column was connected to a constant flow pump, and the bottom outlet was opened. The column was rinsed with deionized water at a flow rate of 1 mL / min for 12 h, and then ready for use.

[0026] Crude polysaccharide from Monascus purpureus was prepared into a polysaccharide solution with a concentration of 5 mg / mL. After loading the polysaccharide solution onto a DEAE-52 cellulose chromatography column, it was eluted with gradients of 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 10–12 min. The sugar content of each solution was determined by the sulfuric acid-phenol method. The optimal peak segment was selected based on the absorbance at 490 nm for sample collection. Samples with the same gradient peak segment were combined, freeze-dried, and then prepared into a polysaccharide solution with a concentration of 5 mg / mL.

[0027] Secondary purification Sephadex G-100 dextran gel chromatography column pretreatment: The gel was added to deionized water at a ratio of 1 g: 30 mL and swollen for 24 h to remove suspended particulate matter. After swelling, the gel was degassed and stirred, and then guided into the column with a glass rod. Care was taken to avoid generating air bubbles and layer breaks during the guiding process. After the gel settled, it was equilibrated with deionized water for 24 h and then ready for use.

[0028] A 5 mg / mL polysaccharide solution, initially purified using a DEAE-52 cellulose chromatography column, was added along 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 min, for a total of 20 tubes. The sugar content of each solution was determined using the sulfuric acid-phenol method. Samples were collected based on the optimal peak range at 490 nm, and samples with the same gradient peak range were combined and freeze-dried to obtain the red yeast rice polysaccharide product. The purity of the mycelial polysaccharide in the red yeast rice polysaccharide product was found to be 98.65%.

[0029] Compare with Example 1 The method for preparing Monascus purpureus polysaccharide includes: Mycelium preparation: The Monascus purpureus strain was activated and transferred to PDA agar plates. Sterile water was added, mycelia were scraped off, and filtered through sterile cotton to prepare a concentration of 1×10⁻⁶. 6 A suspension of Monascus pilosus spores per mL. The Monascus pilosus strain is CICC 5045 and was purchased from the China Industrial Microbial Culture Collection Center.

[0030] The Monascus purpureus seed culture was added to the culture medium at 15% by volume, and then fermented in a constant temperature shaker at 30℃ and 150 rpm in the dark for 54 h. The volume ratio of the Monascus purpureus seed culture to the culture medium was 15:100; the culture medium consisted of: glucose 50 g / L, peptone 8 g / L, MgSO4 0.8 g / L, yeast extract 4 g / L, and KH2PO4 1.2 g / L.

[0031] After fermentation, the fermentation was terminated by high-temperature sterilization for 20 minutes at 121 ℃ and 0.25 MPa. The mycelium was collected after high-temperature sterilization, dried at a constant temperature of 60 ℃, pulverized through a 60-mesh sieve, and stored for later use.

[0032] Crude polysaccharide extraction: The dried mycelium was mixed with 0.2 M NaOH solution at a ratio of 1:30 and placed in a 95 ℃ water bath for 3 h. After the water bath, the mixture was centrifuged at 4 ℃ and 8000 rpm for 10 min, and the supernatant was collected. The supernatant was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kJ / MWCO at 10 ℃ and 2000 rpm to obtain a concentrated solution. The concentrated solution and Sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube at a volume ratio of 1:4, mixed well, and centrifuged at 4 ℃ and 8000 rpm for 3 min, and the supernatant was collected. The supernatant was placed in a 70 ℃ water bath for 3 h, 4 times the volume of anhydrous ethanol was added, mixed well, and placed at 4 ℃ for 12 h. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded and the precipitate was collected. The precipitate was dissolved in ultrapure water and then freeze-dried under vacuum to obtain crude polysaccharide from Monascus purpureus. Preliminary purification: Pretreatment of DEAE-52 cellulose chromatography column: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h to remove floating particles, and then filtered; it was then soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, and filtered for later use.

[0033] The chromatography column (1.6 × 30 cm) was vertically fixed, and 1 / 3 volume of deionized water was added. The bottom outlet was opened, and cellulose slurry was added to the column at a uniform rate, adhering to the column wall. The slurry level was increased to 2 / 3 volume of the column, and then the packing was stopped. The upper end of the column was connected to a constant flow pump, and the bottom outlet was opened. The column was rinsed with deionized water at a flow rate of 1 mL / min for 12 h, and then ready for use.

[0034] The crude polysaccharide from Monascus purpureus was prepared into a polysaccharide solution with a concentration of 5.0 mg / mL. After loading the polysaccharide solution onto a DEAE-52 cellulose chromatography column, it was 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 10–12 min. The sugar content of each solution was determined by the sulfuric acid-phenol method. The optimal peak segment was selected based on the absorbance at 490 nm for sample collection. Samples with the same gradient peak segment were combined, freeze-dried, and then prepared into a polysaccharide solution with a concentration of 5 mg / mL.

[0035] Secondary purification: Sephadex G-100 dextran gel chromatography column pretreatment: The gel was added to deionized water at a ratio of 1 g: 30 mL and swollen for 24 h to remove suspended particulate matter. After swelling, the gel was degassed and stirred, and then guided into the column with a glass rod. Care was taken to avoid generating air bubbles and layer breaks during the guiding process. After the gel settled, it was equilibrated with deionized water for 24 h and then ready for use.

[0036] A 5 mg / mL polysaccharide solution, initially purified using a DEAE-52 cellulose chromatography column, was added along the wall of a Sephadex G-100 dextran gel chromatography column. The column wall was washed with a small amount of deionized water, and the solution was eluted with deionized water at a flow rate of 0.5 mL / min. Each tube was collected for 10 min, for a total of 20 tubes. The sugar content of each solution was determined using the sulfuric acid-phenol method. The optimal peak range was selected based on the absorbance at 490 nm for sample collection. Samples with the same gradient peak range were combined and freeze-dried to obtain the red yeast rice polysaccharide product. In this control example, ultrafiltration concentration was not used within the parameter range to remove impurities. The purity of the red yeast rice polysaccharide in the product was determined to be 67.75% of the purity of the mycelial polysaccharide.

[0037] Compare with Example 2 The method for preparing Monascus purpureus polysaccharide includes: The Monascus purpureus strain was activated and transferred to PDA agar plates. Sterile water was added, mycelia were scraped off, and filtered through sterile cotton to prepare a concentration of 1×10⁻⁶. 6 A suspension of Monascus pilosus spores per mL. The Monascus pilosus strain is CICC 5045 and was purchased from the China Industrial Microbial Culture Collection Center.

[0038] The Monascus purpureus seed culture was added to the culture medium at 15% by volume, and then fermented in a constant temperature shaker at 30℃ and 150 rpm for 54 h in the dark. The volume ratio of the Monascus purpureus seed culture to the culture medium was 15:100; the culture medium consisted of: glucose 50 g / L, peptone 8 g / L, MgSO4 0.8 g / L, yeast extract 4 g / L, and KH2PO4 1.2 g / L.

[0039] After fermentation, the fermentation was terminated by high-temperature sterilization for 20 minutes at 121 ℃ and 0.25 MPa. The mycelium was collected after high-temperature sterilization, dried at a constant temperature of 60 ℃, pulverized through a 60-mesh sieve, and stored for later use.

[0040] Crude polysaccharide extraction: The dried mycelium was mixed with 0.2M NaOH solution at a ratio of 1:30 and placed in a 95 ℃ water bath for 3 h. After the water bath, the mixture was centrifuged at 4 ℃ and 8000 rpm for 10 min, and the supernatant was collected. The supernatant and Sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube at a volume ratio of 1:4, mixed well, and centrifuged at 4 ℃ and 8000 rpm for 3 min. The supernatant was collected and placed in a 70 ℃ water bath for 3 h. Four times the volume of anhydrous ethanol was added, mixed well, and placed at 4 ℃ for 12 h. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. After dissolving the precipitate in ultrapure water, it was freeze-dried under vacuum to obtain the crude polysaccharide of Monascus purpureus. Preliminary purification: Pretreatment of DEAE-52 cellulose chromatography column: DEAE-52 cellulose powder was soaked in deionized water at a ratio of 1:5 (g:mL) for 3 h to remove floating particles, and then filtered; it was then soaked in 0.5 M NaOH for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M HCl for 2 h, washed with deionized water until neutral, and filtered; it was then soaked in 0.5 M NaOH solution for 2 h, washed with deionized water until neutral, and filtered for later use.

[0041] The chromatography column (1.6 × 30 cm) was vertically fixed, and 1 / 3 volume of deionized water was added. The bottom outlet was opened, and cellulose slurry was added to the column at a uniform rate, adhering to the column wall. The slurry level was increased to 2 / 3 volume of the column, and then the packing was stopped. The upper end of the column was connected to a constant flow pump, and the bottom outlet was opened. The column was rinsed with deionized water at a flow rate of 1 mL / min for 12 h, and then ready for use.

[0042] The crude polysaccharide from Monascus purpureus was prepared into a polysaccharide solution with a concentration of 5 mg / mL. After loading the polysaccharide solution onto a DEAE-52 cellulose chromatography column, it was eluted with gradients of 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 10–12 min. The sugar content of each solution was determined by the sulfuric acid-phenol method. The optimal peak segment was selected based on the absorbance at 490 nm for sample collection. Samples with the same gradient peak segment were combined, freeze-dried, and then prepared into a polysaccharide solution with a concentration of 5 mg / mL.

[0043] Secondary purification: Sephadex G-100 dextran gel chromatography column pretreatment: The gel was added to deionized water at a ratio of 1 g: 30 mL and swollen for 24 h to remove suspended particulate matter. After swelling, the gel was degassed and stirred, and then guided into the column with a glass rod. Care was taken to avoid generating air bubbles and layer breaks during the guiding process. After the gel settled, it was equilibrated with deionized water for 24 h and then ready for use.

[0044] A 5 mg / mL polysaccharide solution, initially purified using a DEAE-52 cellulose chromatography column, was added along 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 min, for a total of 20 tubes. The sugar content of each solution was determined using the sulfuric acid-phenol method. The optimal peak range was selected based on the absorbance at 490 nm for sample collection. Samples with the same gradient peak range were combined and freeze-dried to obtain the red yeast rice polysaccharide product. In this control example, ultrafiltration concentration was not used to remove impurities. The purity of the red yeast rice polysaccharide in the product was determined to be 45.43%.

[0045] Compare with Example 3 The method for preparing Monascus purpureus polysaccharide includes: Mycelium preparation: The Monascus purpureus strain was activated and transferred to PDA agar plates. Sterile water was added, mycelia were scraped off, and filtered through sterile cotton to prepare a concentration of 1×10⁻⁶. 6 A suspension of Monascus pilosus spores per mL. The Monascus pilosus strain is CICC 5045 and was purchased from the China Industrial Microbial Culture Collection Center.

[0046] The Monascus purpureus seed culture was added to the culture medium at 15% by volume, and then fermented in a constant temperature shaker at 30℃ and 150 rpm in the dark for 54 h. The volume ratio of the Monascus purpureus seed culture to the culture medium was 15:100; the culture medium consisted of: glucose 50 g / L, peptone 8 g / L, MgSO4 0.8 g / L, yeast extract 4 g / L, and KH2PO4 1.2 g / L.

[0047] After fermentation, the fermentation was terminated by high-temperature sterilization for 20 minutes at 121 ℃ and 0.25 MPa. The mycelium was collected after high-temperature sterilization, dried at a constant temperature of 60 ℃, pulverized through a 60-mesh sieve, and stored for later use.

[0048] Crude polysaccharide extraction: The dried mycelium was mixed with 0.2M NaOH solution at a ratio of 1:30 and placed in a 95 ℃ water bath for 3 h. After the water bath, the mixture was centrifuged at 4 ℃ and 8000 rpm for 10 min, and the supernatant was collected. The supernatant and Sevag (chloroform: n-butanol = 4:1) reagent were added to a centrifuge tube at a volume ratio of 1:4, mixed well, and centrifuged at 4 ℃ and 8000 rpm for 3 min. The supernatant was collected and placed in a 70 ℃ water bath for 3 h. Four times the volume of anhydrous ethanol was added, mixed well, and placed at 4 ℃ for 12 h. The mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The precipitate was dissolved in ultrapure water and then freeze-dried under vacuum to obtain the crude polysaccharide of Monascus purpureus. In this control example, ultrafiltration concentration and secondary purification techniques were not used. The purity of the Monascus purpureus polysaccharide in the product was 36.86%.

[0049] Compare with Example 4 This comparative example uses *Monascus pilosus* Zheng YZ101 (biological preservation number CGMCC 8167) and employs the liquid fermentation technology mentioned in CN106929428A to obtain red yeast polysaccharide products. Testing showed that the purity of red yeast polysaccharide in the obtained product was 5-6%. This indicates that the technology used in CN106929428A is not applicable to the production of red yeast polysaccharides from other *Monascus* species.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing Monascus purpureus polysaccharide, characterized in that, include: Mycelium preparation: Monascus pilosus was activated and inoculated to obtain Monascus pilosus seed liquid. The Monascus pilosus seed liquid was further fermented and the mycelium was collected, dried and stored for later use. The Monascus pilosus strain was identified as Monascus pilosus, strain number CICC 5045. Crude polysaccharide extraction: The dried mycelium was mixed with alkaline solution and extracted to obtain an extract. The extract was then concentrated by ultrafiltration to obtain a concentrate. The ultrafiltration temperature was 20~30℃ and the rotor speed was 7500~8500r / min. The concentrate was deproteinized with Sevag reagent to obtain crude polysaccharide from Monascus purpureus. Preliminary purification: The crude polysaccharide from Monascus purpureus was initially separated and purified using a DEAE-52 cellulose chromatography column to obtain a polysaccharide solution; Secondary purification: The polysaccharide solution was further separated and purified using a Sephadex G-100 dextran gel chromatography column to obtain the red yeast rice polysaccharide product.

2. The method for preparing Monascus purpureus polysaccharide according to claim 1, characterized in that, In the mycelium preparation step, the Monascus purpureus seed solution has a concentration of 1×10⁻⁶. 5 ~1×10 7 A suspension of Monascus purpureus spores per mL was prepared by adding Monascus purpureus seed liquid to the culture medium and then fermenting it in a constant temperature shaker at 28-32 ℃ and 120-180 rpm in the dark for 48-72 h.

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

4. The method for preparing Monascus purpureus polysaccharide according to claim 1, characterized in that, In the crude polysaccharide extraction step, the alkaline solution is a NaOH solution with a concentration of 0.15~0.25M, and the ratio of dried mycelium to NaOH solution is 1:25~35; the extraction temperature is 90~95℃, and the extraction time is 2~3h.

5. The method for preparing Monascus purpureus 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 purpureus polysaccharide according to claim 1, characterized in that, In the crude polysaccharide extraction step, the deproteinization method using Sevag reagent is as follows: the concentrated solution and Sevag reagent are mixed at a volume ratio of 1:3~5, centrifuged, and the supernatant is collected. The supernatant is heated at a constant temperature of 65~75℃ for 2.5~3.5h, and then 4~5 times the volume of anhydrous ethanol is added to the supernatant. After mixing, the mixture is allowed to stand at 3~5℃ for 10~14h. After centrifugation, the precipitate is collected, dissolved in pure water, and freeze-dried to obtain crude polysaccharide from Monascus purpureus.

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

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

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

10. The method for preparing Monascus purpureus polysaccharide according to any one of claims 1 to 9, characterized in that, In the mycelium preparation step, after fermentation, the high-temperature sterilization time is 15~25 min, the temperature is 121 ℃, and the pressure is 0.25 MPa.