Extraction method of traditional Chinese medicine lalang grass rhizome

Abstract

The invention relates to the technical field of enzyme engineering and biological fermentation, in particular to an extraction method of traditional Chinese medicine lalang grass rhizome. By combining the composite enzymatic hydrolysate with ultrasonic-assisted microbial fermentation, the lalang grass rhizome polysaccharide is extracted, and the problems that a traditional method is low in efficiency, insufficient in purity and damaged in activity are solved. The composite enzymatic hydrolysate is prepared by mixing enzyme digestion modified lumbrukinase (prepared by immobilized trypsin digestion modification) and neutral pectinase according to a ratio of 1: 1, and the microbial fermentation adopts a bacillus licheniformis and saccharomyces cerevisiae composite strain according to a ratio of 5: 1. Compared with a traditional method, the method has the advantages that the polysaccharide extraction rate reaches 67.36%-67.5% and is increased by about 6.5%-7%, the precision RSD is smaller than 1.5%, the product is stable within 12 h, no strong acid or strong alkali or toxic organic solvent exists, the safety standard is met, the polysaccharide activity is reserved, and the method has the industrialization prospect.

Description

Technical Field

[0001] This application relates to enzyme engineering and bio-fermentation technology, specifically to an extraction method for the traditional Chinese medicine Imperata cylindrica root. Background Technology

[0002] Imperata cylindrica root, a traditional Chinese medicinal herb, has a long history of medicinal use in my country. It is sweet and cold in nature, and enters the lung, stomach, and bladder meridians. In traditional Chinese medicine, it is often used to cool the blood and stop bleeding, clear heat and promote diuresis. Modern medical research has further confirmed that the polysaccharides abundant in Imperata cylindrica root are the core material basis for its pharmacological activity. These components show good potential in antioxidation, immune regulation, and metabolism improvement, and have high development and application value in traditional Chinese medicine preparations and health products. Therefore, how to efficiently and effectively extract polysaccharides from Imperata cylindrica root has become a key link in promoting its industrial application.

[0003] Currently, numerous studies have been conducted in the industry on extraction methods for Imperata cylindrica root polysaccharides, resulting in conventional technical routes such as water extraction and alcohol precipitation, single enzymatic hydrolysis, and traditional microbial fermentation. However, these methods still have significant limitations in practical applications and cannot meet the comprehensive requirements of industrial production for extraction efficiency, product purity, and cost control.

[0004] Water extraction and alcohol precipitation: As a traditional extraction process, its principle is to achieve separation by utilizing the property of polysaccharides to dissolve in water and precipitate in alcohol. However, this method has significant drawbacks. On the one hand, impurities such as cellulose and pectin in Imperata cylindrica root raw materials will dissolve in the aqueous phase along with the polysaccharides, leading to complex subsequent purification steps and low purity of the final product, usually only reaching 60% to 70%. On the other hand, to achieve effective extraction, long-term high-temperature heating (usually 80-100℃) is required, which not only consumes a lot of energy but may also damage the molecular structure of polysaccharides, resulting in reduced pharmacological activity. At the same time, the extraction cycle is as long as 8 to 12 hours, resulting in low production efficiency.

[0005] Single-enzyme hydrolysis: Existing technologies mostly use a single neutral pectinase or cellulase for enzymatic hydrolysis, aiming to disrupt the cell wall structure of Imperata cylindrica root and promote polysaccharide release. However, single enzymes have limited target sites and cannot fully degrade complex components in the raw material (such as the cross-linked structure of cellulose, hemicellulose, and pectin), resulting in insufficient cell wall disruption and limited polysaccharide dissolution rate, typically only 10%–15% higher than the traditional water extraction and alcohol precipitation method. Furthermore, some enzyme preparations (such as cellulase) have harsh operating conditions (requiring strict pH control of 4.0–5.0), placing high demands on the temperature and acid control capabilities of the production equipment, increasing the operational difficulty and cost of industrial production.

[0006] Traditional microbial fermentation: This method degrades raw materials through enzymes produced by microbial metabolism. However, commonly used single strains (such as Saccharomyces cerevisiae or Bacillus) have limited metabolic capacity and produce a limited variety of enzymes, making it difficult to efficiently decompose the complex carbohydrates in Imperata cylindrica root. This results in an excessively long fermentation cycle (usually 7-10 days). Furthermore, the fermentation process is prone to contamination by other microorganisms due to metabolic imbalances in the strains, affecting the purity and safety of the polysaccharide products. At the same time, traditional fermentation lacks auxiliary extraction methods, resulting in low efficiency in separating polysaccharides from the microbial metabolic system, further limiting the large-scale application of this method.

[0007] Furthermore, existing extraction technologies generally suffer from the dilemma of balancing extraction efficiency and product activity: increasing the extraction rate through high temperatures, strong acids, or strong alkalis can easily lead to polysaccharide molecular chain breakage and destruction of active groups, reducing its pharmacological value; while using mild conditions may fail to effectively overcome the cell wall barrier of the raw material, resulting in a low extraction rate. Simultaneously, some methods require the use of large amounts of organic reagents (such as ethanol and acetone) for purification, which not only increases production costs but may also leave organic reagent residues, posing safety hazards and contradicting the modern trend of "green, safe, and efficient" extraction of traditional Chinese medicine. Summary of the Invention

[0008] To address the problems of low extraction efficiency, insufficient product purity, and easily damaged polysaccharide activity in existing methods for extracting polysaccharides from Imperata cylindrica root, this application provides a method for extracting polysaccharides from Imperata cylindrica root. This method uses a combination of compound enzymatic hydrolysate and ultrasonic-assisted microbial fermentation to extract polysaccharides, effectively solving the problems of low extraction efficiency, insufficient product purity, and easily damaged polysaccharide activity.

[0009] This application provides a method for extracting the traditional Chinese medicine Imperata cylindrica root, wherein the extraction method involves combining a compound enzymatic hydrolysate with ultrasound-assisted microbial fermentation to extract polysaccharides.

[0010] Preferably, the composite enzymatic hydrolysate is a mixture of enzyme-modified lumbrokinase and neutral pectinase.

[0011] Preferably, the microbial fermentation is carried out by a complex fermentation strain.

[0012] Preferably, the compound fermentation strain is composed of Bacillus licheniformis and Saccharomyces cerevisiae fermentation strain.

[0013] Preferably, the extraction method comprises the following steps:

[0014] (1) Raw material pretreatment: Wash the Imperata cylindrica root, cut it into small sections, place it in an oven to dry until constant weight, then grind it into powder using a pulverizer and pass it through a 40-60 mesh sieve;

[0015] (2) Enzymatic hydrolysis: Adjust the pH, use a compound enzyme hydrolysate, the hydrolysis temperature is 40-50℃, the time is 4-6h, and the hydrolysis product is obtained.

[0016] (3) Enzyme inactivation: The hydrolysis product is heated at 90-95°C for 15-20 min to inactivate the enzyme, and an inactivated product is obtained;

[0017] (4) Ultrasonic-assisted microbial fermentation: The inactivated product was placed in 20kHz ultrasound for 20 minutes, the compound fermentation strain was sprayed in a specific ratio, purified water was added to adjust the moisture content to 65%, the mixture was thoroughly mixed and put into a breathing fermentation bag, sealed and fermented at 30-35℃ for 4-6 days to obtain polysaccharide extract.

[0018] (5) Polysaccharide purification and drying: 95% ethanol was slowly added to the polysaccharide extract to precipitate it. The polysaccharide solution was dialyzed with deionized water at 4°C for 48 hours. The polysaccharide solution in the dialysis bag was then transferred to a vacuum freeze dryer.

[0019] Preferably, the pH is adjusted to 6-8 in step (2).

[0020] Preferably, the compound fermentation strain is prepared in a specific ratio of Bacillus licheniformis to Saccharomyces cerevisiae fermentation strain at 5:1.

[0021] Preferably, the enzyme-modified lumbrokinase is an immobilized trypsin-modified lumbrokinase.

[0022] Preferably, the method for preparing the enzyme-modified lumbrokinase is as follows:

[0023] (1) Weigh out Taiping No. 1 earthworms, clean them, soak them in water in a beaker for 2 hours, wash them, and homogenize them at low temperature to obtain Taiping No. 1 homogenate.

[0024] (2) Add the Taiping No. 1 homogenate to PBS and benzoic acid solution at a ratio of 1:4 (w / v), allow it to autolyze for 8 hours, and centrifuge to obtain the supernatant to obtain crude lumbrokinase extract.

[0025] (3) Add ammonium sulfate to the crude extract of lumbrokinase for fractional precipitation, centrifuge at 25°C and 8000 rpm / min for 10 min to collect the precipitate, and dialyze to desalt and purify to obtain lumbrokinase dry powder;

[0026] (4) Enzyme-modified lumbrokinase: Trypsin was immobilized with chitosan to obtain immobilized trypsin. The immobilized trypsin was added to lumbrokinase and incubated in a water bath at 45°C for 1 hour. After filtration, the enzyme-modified lumbrokinase was obtained.

[0027] Preferably, the mixing ratio of the enzyme-modified lumbrokinase to the neutral pectinase is 1:1.

[0028] The beneficial effects of the embodiments in this application are as follows:

[0029] (1) Enhanced Cell Wall Disruption through Compound Enzymatic Hydrolysis: A compound enzymatic hydrolysis solution consisting of "enzyme-modified lumbrokinase (prepared by immobilized trypsin cleavage modification) + neutral pectinase" (1:1 mixture) was used. Compared with single enzymatic hydrolysis, this solution can comprehensively degrade cross-linked structures such as cellulose and pectin in the cell walls of Imperata cylindrica root. Enzyme-modified lumbrokinase can specifically destroy protein linkages, while neutral pectinase degrades pectin components. The synergistic effect of the two results in more thorough cell wall disruption. Combined with optimized enzymatic hydrolysis conditions of 40-50℃ and 4-6h (matching the stable activity range of lumbrokinase at pH 6-8), sufficient channels are created for polysaccharide dissolution, improving the polysaccharide dissolution efficiency compared to single enzymatic hydrolysis.

[0030] (2) Synergistic effect of ultrasound-microbial fermentation: Pretreatment with 20kHz ultrasound for 20min expands the interstitial spaces of the raw materials through cavitation effect, improving the infection and metabolic efficiency of the compound fermentation strain (Bacillus licheniformis: Saccharomyces cerevisiae = 5:1); during sealed fermentation at 30-35℃ for 4-6 days, the multi-enzyme system produced by the compound strain can continuously degrade residual impurities and promote polysaccharide release, while avoiding the problem of low fermentation efficiency of single strains. This synergistic process resulted in a polysaccharide extraction rate of 67.36% to 67.5%, which is about 6.5% to 7% higher than the group without enzymatic hydrolysis (63.24%), the group without fermentation (63.2%), and the group without ultrasound assistance (63.26%), and about 6.5% higher than the traditional water extraction and alcohol precipitation method (63.3%), significantly increasing the polysaccharide yield per unit of raw material.

[0031] (3) Precision tests showed that the RSD of the standard solution from Example 1 was less than 1.5% in six consecutive measurements, indicating excellent process repeatability. Stability tests showed that the absorbance of the polysaccharide solution gradually decreased from 0.274 to 0.260 within 12 hours, with minimal fluctuations, indicating strong product stability. Furthermore, the process uses only food-grade 95% ethanol as the purification reagent, avoiding strong acids, alkalis, or toxic organic solvents. The combined fermentation strains (Bacillus licheniformis CGMCC 1.1086 and Saccharomyces cerevisiae CGMCC 2.1814) are all safe strains, avoiding the risk of organic reagent residues and contamination by other microorganisms, and meeting the safety standards for raw materials of traditional Chinese medicine preparations and health products.

[0032] (4) Preservation of polysaccharide activity: The entire process adopts mild process conditions - enzymatic hydrolysis (40-50℃) and fermentation (30-35℃) avoid the damage to polysaccharide molecular chains caused by high temperature (such as the traditional water extraction and alcohol precipitation method of 80-100℃); the final vacuum freeze drying (-50℃ pre-freezing + vacuum drying) process can preserve the active groups and molecular structure integrity of polysaccharides to the greatest extent, laying the foundation for its subsequent pharmacological effects in the fields of anti-oxidation and immune regulation. Attached Figure Description

[0033] Figure 1This is a schematic diagram showing the hydrolysis temperature of lumbrokinase in Example 1 of this application;

[0034] Figure 2 This is a schematic diagram of the enzymatic hydrolysis time of lumbrokinase in Example 1 of this application. Detailed Implementation

[0035] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0036] The reagents and equipment used in the embodiments of this disclosure are all conventional and commercially available.

[0037] Preparation Example 1:

[0038] Lumbrokinase Extraction

[0039] (1) Weigh 300g of Taiping No. 1 earthworms, wash them and soak them in water in a beaker for 2 hours to remove some mud and feces from the earthworms. After washing them, homogenize them at low temperature to obtain Taiping No. 1 homogenate.

[0040] (2) Add 0.01 mol / L PBS (pH=7.8) solution to the homogenate at a ratio of 1:4 (w / v), add 1.2 g sodium benzoate, mix thoroughly, and autolyze in a water bath at 37°C for 8 h. Centrifuge the turbid liquid at 4000 rpm for 30 min using a refrigerated centrifuge, and take the supernatant to obtain crude lumbrokinase extract.

[0041] (3) Ammonium sulfate fractionation: ammonium sulfate saturation was gradually precipitated in 10% increments from 10% to 90%. The supernatant was placed in a 25°C water bath, and solid ammonium sulfate was added to adjust the saturation. After standing for 6 hours, the mixture was centrifuged at 8000 rpm and 25°C for 10 minutes using a high-speed refrigerated centrifuge. The precipitate was collected, reconstituted with deionized water, and then desalted by dialysis using a dialysis bag with a molecular weight cutoff of 1000 Da for 48 hours to obtain crude lumbrokinase extract powder, which was then freeze-dried and stored.

[0042] (4) Assay of lumbrokinase activity: Fibrinogen solution and 60℃ agarose solution were mixed at a ratio of 1:1 (vv). While stirring, an appropriate amount of thrombin solution was added. After stirring at low speed and mixing, the mixture was immediately poured into a plastic petri dish and placed horizontally at room temperature. After reacting for 1 hour, holes were punched. 10L of sample and 10L of standard were added, covered, and incubated at 37℃ for 16 hours. The two vertical diameters of the dissolution zone were measured with vernier calipers. A standard curve was plotted with the standard activity as the abscissa and the logarithm of the product of the vertical radii of the standard as the ordinate. The vertical radius of the sample was substituted to calculate the lumbrokinase activity. Three parallel tests were performed, and the average value was used. The enzyme activity of the product precipitated at a concentration of 40% to 50% ammonium sulfate saturation was 35.8% higher than that before autolysis, and the activities were 21825 U / mg and 29638 U / mg, respectively.

[0043] (5) Determination of degree of hydrolysis: The Kjeldahl method in GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food" was used for determination. The BCA method was used for protein concentration monitoring (Beyotime BCA Protein Concentration Assay Kit). The degree of protein hydrolysis (DH) of the enzymatic hydrolysis system was determined by formaldehyde titration. DH is the percentage of peptide bonds that were cleaved in the protein of Imperata cylindrica root raw material, as shown in the formula: Wherein, M is the concentration of the NaOH standard solution, in mol; V1 is the volume of 0.05 mol / L NaOH standard solution consumed in the sample titration, in mL; V2 is the volume of 0.05 mol / L NaOH standard solution consumed in the blank experiment, in mL; Cso is the total protein concentration in the Imperata cylindrica root enzymatic hydrolysate, in g / L; V is the volume of hydrolysate titrated with formaldehyde, in mL; h tot - The number of millimoles of peptide bonds in the substrate protein, taken as 7.5 mmol / g, referring to the characteristics of plant-derived proteins. mmol / g, for example... Figure 1 As shown, the optimal hydrolysis temperature is controlled at 40-50℃, and the enzymatic hydrolysis time is as follows. Figure 2 As shown, the optimal time is 4-6 hours.

[0044] (6) DEAE Sepharose Fast Flow anion exchange column purification:

[0045] 1) Sample preparation: DEAESepharose FastFlow anion exchange packing material was rinsed with deionized water until no ethanol residue or foam was present, then packed into a 16 mm diameter, 200 mm high chromatography column. The packing material was allowed to settle naturally for 4 hours. The constant flow pump speed was adjusted to a flow rate of 1.0 mL / min, and the column was eluted for 1 hour until the column height stabilized. The packing height was recorded. Several fractions with high activity were collected, dissolved in eluent, and prepared into a 5 mg / mL protein solution. The solution was then filtered through a 0.45 mm organic filter membrane, and the filtrate was collected for later use.

[0046] 2) Sample loading and elution: The DEAE Sepharose FastFlow anion exchange packing material was rinsed with deionized water until no ethanol residue or foam was present, then packed into a 16 mm diameter, 200 mm high chromatography column. The packing material was allowed to settle naturally for 4 hours. The constant flow pump speed was adjusted to a flow rate of 1.0 mL / min, and the column was eluted for 1 hour until the column height stabilized. The packing height was recorded. Several fractions with high activity were collected, dissolved in eluent, and prepared into a 5 mg / mL protein solution. The solution was then filtered through a 0.45 mm organic filter membrane, and the filtrate was collected for later use.

[0047] (7) Isolation and purification of lumbrokinase:

[0048] 1) Sample Preparation: Based on the molecular weight distribution of the active components on the ion exchange column, Sephadex G-75 was selected for further separation and purification of the active components. After Sephadex G-75 swelled overnight, it was degassed by a vacuum pump for 2 hours, and then repeatedly washed with deionized water until no foam was present before packing the column. The swollen Sephadex G-75 packing material was packed into a 1.6 x 100 cm glass chromatography column, and the packing material was allowed to settle naturally overnight. The gel column was equilibrated with PBS (pH = 7.8) at a flow rate of 0.5 mm / min. The fraction with the best lumbrokinase activity among the separated components was prepared into a 2 mg / hm solution using the mobile phase, filtered through a 0.45 μm organic filter membrane, and then used for later use.

[0049] 2) Sample loading and elution: Open the peristaltic pump outlet and wait until the liquid level drops to tangent to the gel column surface. Accurately transfer 4 μL of filtrate into the chromatography column using a pipette. After the sample solution is completely adsorbed by the packing material, elute with PBS (pH = 7.8) at a rate of 0.5 mL / min. Collect the eluent using an automatic collector, collecting one tube every 4 min, for a total of 80 tubes. After elution, measure the absorbance of the fraction at 280 nm using a UV spectrophotometer. Plot the elution curve, determine the kinase activity of the corresponding peak, and desalt using a 3 kDa ultrafiltration tube to obtain purified lumbrokinase, which is then lyophilized and stored.

[0050] (8) Preparation of liquid phase:

[0051] 1) Weigh 20 mg of purified lumbrokinase lyophilized sample, dissolve it in 300 μL of 0.1 mol / L sodium sulfate + 0.1 mol / L disodium hydrogen phosphate + 0.1 mol / L sodium dihydrogen phosphate aqueous solution, centrifuge at 20000g for 15 min at 4℃, and take the supernatant into a 2 mL autosampler bottle for HPLC detection;

[0052] 2) Chromatographic conditions: Instrument model: ThermoScitifc Ultimate 3000, Column: Tosoh

[0053] TSKgelUP-SW3000 (4.6 mm ID. * 30°C, 2 pm), mobile phase: A: 0.2 mol / L sodium sulfate aqueous solution, B: 0.2 mol / L disodium hydrogen phosphate + 0.2 mol / L sodium dihydrogen phosphate aqueous solution ratio: A:B = 1:1, 45 min isocratic elution, detector parameters: DAD detector, UV wavelength: 280 nm;

[0054] 3) C18 column separation and purification of lumbrokinase: A gradient mobile phase A: ultrapure water, B: methanol was used. The eluent was collected and excess methanol was removed before freeze-drying for later use. SDS-PAGE electrophoresis was performed. Mass spectrometry parameters were acquired and peptides were matched according to the database.

[0055] Preparation Example 2

[0056] Preparation of lumbrokinase by enzyme digestion and modification

[0057] (1) Immobilization of trypsin: Weigh 1.2g of chitosan, add 120mL of 1% acetic acid to prepare a transparent gel solution, then add 2mol / L NaOH to adjust the pH to about 5.5, add 0.1% glutaraldehyde to adjust the pH to 8.0, dry under vacuum, wash, add trypsin, stir magnetically for 30min, add glutaraldehyde, stir with an electric stirrer for 8h, filter and vacuum dry to obtain immobilized trypsin;

[0058] (2) The immobilized trypsin was added to 10 mL of the purified lumbrokinase solution from Preparation Example 1, and the solution was kept in a water bath at 45°C for 1 h. The lumbrokinase was then filtered to obtain the enzyme-modified lumbrokinase.

[0059] Preparation Example 3

[0060] pH effect on lumbrokinase activity assay

[0061] The activity of extracted lumbrokinase was tested by storing it at different pH values ​​for 24 hours. The results showed that the activity of lumbrokinase remained stable between pH 6 and 8.

[0062] Example 1

[0063] Polysaccharide Extract from Imperata cylindrica Root

[0064] (1) Preparation of experimental materials:

[0065] 1) Raw materials: 500g of fresh Imperata cylindrica root that is free from mold and insect infestation. The Imperata cylindrica root is produced in Bozhou and purchased from the Bozhou Chinese Medicine Market. It has been identified as the dried rhizome of Imperata cylindrica, a plant of the Poaceae family.

[0066] 2) Enzyme preparation: 2 mg of the enzyme-modified lumbrokinase (enzyme activity of 1050 FU / mg) prepared by the method in Example 2 and 1 mg of food-grade neutral pectinase (2000 FU / mg, Sigma-Aldrich) were hydrolyzed by a compound enzyme hydrolysate.

[0067] 3) Strains: Bacillus licheniformis (CGMCC 1.1086) and Saccharomyces cerevisiae (CGMCC 2.1814) were both purchased from the China General Microbiological Culture Collection Center. The two strains were inoculated into LB medium (Bacillus licheniformis) and YPD medium (Saccharomyces cerevisiae), respectively, and cultured at 30°C with shaking for 24 hours to obtain a bacterial suspension concentration of 10. 8 The seed culture of CFU / mL was then mixed at a volume ratio of 5:1 to obtain a compound fermentation strain.

[0068] 4) Reagents: PBS buffer (0.01 mol / L, pH=7.8), sodium benzoate (analytical grade), ammonium sulfate (analytical grade), ethanol (food grade, 95%), deionized water.

[0069] 5) Instruments: Chinese herbal medicine washing machine, cutting machine, forced-air drying oven (DHG-9240A), universal pulverizer (FW100), pH meter (PHS-3C), constant temperature water bath (HH-S4), ultrasonic extractor (KQ-500DE, frequency 20kHz), breathing fermentation bag (1000mL, with one-way exhaust valve), high-speed refrigerated centrifuge (GL-21M), vacuum freeze dryer (FD-1A-50), high performance liquid chromatograph (manufacturer: Suzhou Boxun Instrument Co., Ltd.; model: RoHS2.0), Spectrum 54 UV-Vis spectrophotometer (Shanghai Lingguang Technology Co., Ltd.).

[0070] (2) Experimental procedure:

[0071] 1) Raw material pretreatment: Place 500g of Imperata cylindrica root in a Chinese herbal medicine washing machine and rinse it three times with running water to remove surface mud and impurities; then cut it into small sections of 2-3cm using a cutting machine, place it in a forced-air drying oven, and dry it at 60℃ until constant weight (weigh it once every 2 hours, and the difference between two weighings is ≤0.5% to be considered constant weight). At this time, the dry weight of Imperata cylindrica root is about 120g; put the dried Imperata cylindrica root sections into a universal pulverizer and pulverize them. After pulverizing, pass them through a 50-mesh standard inspection sieve and collect the Imperata cylindrica root powder that passes through the 50-mesh sieve (to remove coarse particle impurities) for later use.

[0072] 2) Enzymatic hydrolysis: Weigh 50g of Imperata cylindrica root powder and place it in a 500mL Erlenmeyer flask. Add 200mL of deionized water and stir well. Adjust the pH of the system to 7.0 with 0.1mol / L NaOH solution or 0.1mol / L HCl solution. Add 10mL of compound enzymatic hydrolysis solution (a 1:1 mixture of enzyme-modified lumbrokinase and neutral pectinase) to the Erlenmeyer flask. Seal the flask and place it in a 45℃ constant temperature water bath. Stir magnetically (150rpm / min) for 5 hours. During this period, take a sample every 1 hour to monitor the pH of the system. If it deviates from 7.0±0.2, finely adjust it with the above acid and alkali solutions. Finally, the hydrolysis product is obtained.

[0073] 3) Enzyme inactivation: Transfer the conical flask containing the hydrolysis product to a 92°C constant temperature water bath and keep it at this temperature for 18 minutes to inactivate the enzyme. Stir gently throughout the process to ensure uniform temperature. After enzyme inactivation, quickly remove the conical flask and cool it to 30°C at room temperature to obtain the inactivated product.

[0074] 4) Ultrasonic-assisted microbial fermentation:

[0075] a. Transfer all the cooled inactivated products to the processing tank of the ultrasonic extractor, set the ultrasonic power to 300W and the frequency to 20kHz, and ultrasonically process for 20min. During the ultrasonic process, the system temperature is maintained at 30-35℃ by circulating water cooling.

[0076] b. After ultrasonic treatment, transfer the material to a 500mL beaker on a sterile operating table, add 15mL of compound fermentation strain (a 5:1 mixture of Bacillus licheniformis and Saccharomyces cerevisiae seed liquid), adjust the moisture content of the material to 65% with purified water (using a rapid moisture analyzer, testing once every 5mL of purified water added until the moisture content meets the standard), and stir thoroughly.

[0077] c. Pack the above mixture into a sterile respiratory fermentation bag, remove excess air from the bag, seal it, and place it in a 32℃ constant temperature incubator for static fermentation for 5 days. During the fermentation period, observe the gas expansion of the respiratory fermentation bag every day. If the pressure inside the bag is too high, open the one-way exhaust valve to release a small amount of gas to avoid the bag from breaking.

[0078] d. After fermentation, the fermentation product was removed and placed in a high-speed refrigerated centrifuge. It was centrifuged at 8000 rpm and 4℃ for 15 min, and the supernatant was collected. 50 mL of deionized water was added to the centrifuged precipitate, and the mixture was stirred to reconstitute it. The mixture was then centrifuged again (under the same conditions as above). The two supernatants were combined to obtain the crude polysaccharide extract.

[0079] 5) Polysaccharide purification and drying:

[0080] a. Slowly add 95% ethanol (ethanol to extract volume ratio 3:1) to the crude polysaccharide extract while stirring, and let stand at room temperature for 12 hours for alcohol precipitation; then centrifuge at 4℃ and 8000 rpm for 15 minutes, collect the precipitate, and wash the precipitate three times with 70% ethanol to remove residual impurities and pigments.

[0081] b. Redissolve the washed precipitate with 20 mL of deionized water, transfer it to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyze with deionized water at 4 °C for 48 h (replace the deionized water every 8 h) to remove small molecule impurities and salts.

[0082] c. After dialysis, the polysaccharide solution in the dialysis bag was transferred to a vacuum freeze dryer, pre-frozen at -50℃ for 2 hours, and then vacuum dried for 24 hours to obtain white and loose Imperata cylindrica polysaccharide powder. The powder was weighed and the extraction rate was calculated.

[0083] Example 2

[0084] Polysaccharide Extract from Imperata cylindrica Root

[0085] (1) Preparation of test materials: Same as in Example 1.

[0086] (2) Experimental procedure:

[0087] 1) Raw material pretreatment: Same as in Example 1.

[0088] 2) Enzymatic hydrolysis: Weigh 50g of Imperata cylindrica root powder and place it in a 500mL Erlenmeyer flask. Add 200mL of deionized water and stir well. Adjust the pH of the system to 6.0 with 0.1mol / L NaOH solution or 0.1mol / L HCl solution. Add 10mL of compound enzymatic hydrolysis solution (a 1:1 mixture of enzyme-modified lumbrokinase and neutral pectinase) to the Erlenmeyer flask. Seal the flask and place it in a 50℃ constant temperature water bath. Stir magnetically (150rpm / min) for 6 hours to obtain the hydrolysis product.

[0089] 3) Enzyme inactivation: Transfer the conical flask containing the hydrolysis product to a 90°C constant temperature water bath and keep it at this temperature for 15 minutes to inactivate the enzyme. During this process, stir gently to ensure that the system temperature is uniform. After the enzyme inactivation is complete, quickly remove the conical flask and cool it to 30°C at room temperature to obtain the inactivated product.

[0090] 4) Ultrasonic-assisted microbial fermentation:

[0091] a. Transfer all the cooled inactivated products to the processing tank of the ultrasonic extractor, set the ultrasonic power to 300W and the frequency to 20kHz, and ultrasonically process for 20min. During the ultrasonic process, the system temperature is maintained at 30-35℃ by circulating water cooling.

[0092] b. After ultrasonic treatment, the same as in Example 1;

[0093] c. Pack the above mixture into a sterile respiratory fermentation bag, remove excess air from the bag, seal it, and place it in a 30℃ constant temperature incubator for static fermentation for 4 days. During the fermentation period, observe the gas expansion of the respiratory fermentation bag every day. If the pressure inside the bag is too high, open the one-way exhaust valve to release a small amount of gas to avoid the bag from breaking.

[0094] d. After fermentation is complete, proceed as in Example 1.

[0095] 5) Polysaccharide purification and drying: Same as in Example 1.

[0096] Example 3

[0097] Polysaccharide Extract from Imperata cylindrica Root

[0098] (1) Preparation of test materials: Same as in Example 1.

[0099] (2) Experimental procedure:

[0100] 1) Raw material pretreatment: Same as in Example 1.

[0101] 2) Enzymatic hydrolysis: Weigh 50g of Imperata cylindrica root powder and place it in a 500mL Erlenmeyer flask. Add 200mL of deionized water and stir well. Adjust the pH of the system to 8.0 with 0.1mol / L NaOH solution or 0.1mol / L HCl solution. Add 10mL of compound enzymatic hydrolysis solution (a 1:1 mixture of enzyme-modified lumbrokinase and neutral pectinase) to the Erlenmeyer flask. Seal the flask and place it in a 40℃ constant temperature water bath. Stir magnetically (150rpm / min) for 4 hours to obtain the hydrolysis product.

[0102] 3) Enzyme inactivation: Transfer the conical flask containing the hydrolysis product to a 95°C constant temperature water bath and keep it at this temperature for 20 minutes to inactivate the enzyme. Stir gently throughout the process to ensure uniform temperature. After enzyme inactivation, quickly remove the conical flask and cool it to 30°C at room temperature to obtain the inactivated product.

[0103] 4) Ultrasonic-assisted microbial fermentation:

[0104] a. Transfer all the cooled inactivated products to the processing tank of the ultrasonic extractor, set the ultrasonic power to 300W and the frequency to 20kHz, and ultrasonically process for 20min. During the ultrasonic process, the system temperature is maintained at 30-35℃ by circulating water cooling.

[0105] b. After ultrasonic treatment, the same as in Example 1;

[0106] c. Place the above mixture into a sterile respiratory fermentation bag, remove excess air from the bag, seal it, and place it in a 35°C constant temperature incubator for static fermentation for 6 days. During the fermentation period, observe the gas expansion of the respiratory fermentation bag daily. If the pressure inside the bag is too high, open the one-way exhaust valve to release a small amount of gas to prevent the bag from rupturing; d. After fermentation, proceed as in Example 1.

[0107] 5) Polysaccharide purification and drying: Same as in Example 1.

[0108] Comparative Example 1

[0109] Polysaccharide Extract from Imperata cylindrica Root (without enzymatic hydrolysis)

[0110] (1) Preparation of experimental materials:

[0111] 1) Raw materials: Same as in Example 1;

[0112] 2) Strains: Same as in Example 1;

[0113] 3) Reagents: Same as in Example 1.

[0114] 4) Instruments: Same as in Example 1.

[0115] (2) Experimental procedure:

[0116] 1) Raw material pretreatment: Same as in Example 1.

[0117] 2) Ultrasonic-assisted microbial fermentation: Same as Example 1.

[0118] 3) Polysaccharide purification and drying: Same as in Example 1.

[0119] Comparative Example 2

[0120] Polysaccharide Extract from Imperata cylindrica Root (Non-Fermentation)

[0121] (3) Preparation of test materials:

[0122] 1) Raw materials: Same as in Example 1;

[0123] 2) Enzyme preparation: Same as in Example 1;

[0124] 3) Reagents: Same as in Example 1.

[0125] 4) Instruments: Same as in Example 1.

[0126] (4) Experimental steps:

[0127] 1) Raw material pretreatment: Same as in Example 1.

[0128] 2) Enzymatic hydrolysis: Same as in Example 1.

[0129] 3) Enzyme inactivation: Same as in Example 1.

[0130] 4) Polysaccharide purification and drying: Same as in Example 1.

[0131] Comparative Example 3

[0132] Extraction of polysaccharides from Imperata cylindrica root (without ultrasound assistance)

[0133] (1) Preparation of test materials: Same as in Example 1.

[0134] (2) Experimental procedure:

[0135] 1) Raw material pretreatment: Same as in Example 1.

[0136] 2) Enzymatic hydrolysis: Same as in Example 1.

[0137] 3) Enzyme inactivation: Same as in Example 1.

[0138] 4) Microbial fermentation:

[0139] a. Pack the above mixture into a sterile respiratory fermentation bag, remove excess air from the bag, seal it, and place it in a 32℃ constant temperature incubator for static fermentation for 5 days. During the fermentation period, observe the gas expansion of the respiratory fermentation bag every day. If the pressure inside the bag is too high, open the one-way exhaust valve to release a small amount of gas to avoid the bag from breaking.

[0140] b. After fermentation, remove the fermentation product and place it in a high-speed refrigerated centrifuge. Centrifuge at 8000 rpm and 4℃ for 15 min and collect the supernatant. Add 50 mL of deionized water to the centrifuged precipitate, stir to reconstitute, and centrifuge again (under the same conditions). Combine the two supernatants to obtain the crude polysaccharide extract.

[0141] 5) Polysaccharide purification and drying:

[0142] a. Slowly add 95% ethanol (ethanol to extract volume ratio 3:1) to the crude polysaccharide extract while stirring, and let stand at room temperature for 12 hours for alcohol precipitation; then centrifuge at 4℃ and 8000 rpm for 15 minutes, collect the precipitate, and wash the precipitate three times with 70% ethanol to remove residual impurities and pigments.

[0143] b. Redissolve the washed precipitate with 20 mL of deionized water, transfer it to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyze with deionized water at 4 °C for 48 h (replace the deionized water every 8 h) to remove small molecule impurities and salts.

[0144] c. After dialysis, the polysaccharide solution in the dialysis bag was transferred to a vacuum freeze dryer, pre-frozen at -50℃ for 2 hours, and then vacuum dried for 24 hours to obtain white and loose Imperata cylindrica polysaccharide powder. The powder was weighed and the extraction rate was calculated.

[0145] Comparative Example 4

[0146] Water extraction and alcohol precipitation

[0147] Weigh 500g of dried Imperata cylindrica root sample, add a certain volume of 95% ethanol at a mass (g): volume (mL) ratio of 1:20, and extract with ultrasound 3 times, 30min each time. Combine the extracts and concentrate to obtain its ethanol extract, weigh it, calculate the extraction rate, and store the extract in a refrigerator for later use.

[0148] Experimental Example 1

[0149] Detection of polysaccharide index in Imperata cylindrica root

[0150] (1) Determination of polysaccharide content:

[0151] 1) Phenol-sulfuric acid method for color development: Accurately measure 2.0 mL of glucose standard solution into four 25 mL colorimetric tubes, add 2.0, 4.0, 6.0, and 8.0 mL of 5% phenol solution respectively, then add 10.0 mL of concentrated sulfuric acid, shake to mix, and add to the mark. Incubate in a 50℃ water bath for 40 min, then remove and cool in cold water. Using water as a blank, scan in the range of 400–600 nm on a UV-Vis spectrophotometer and find the maximum absorption wavelength.

[0152] 2) Construction of the standard curve: Accurately transfer 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 mL of glucose standard solution into colorimetric tubes, add the same amount of phenol solution as described above, shake well, then add 10.0 mL of concentrated sulfuric acid to the mark, shake thoroughly, and incubate at 50°C in a water bath for 30 min. Simultaneously, use water as a blank and measure the absorbance value at the wavelength of maximum absorption.

[0153] 3) Determination of polysaccharide content: Weigh 10.0 mg of refined polysaccharide and dilute to 10 mL in a volumetric flask to prepare a polysaccharide stock solution of 1.0 mg / mL. Transfer 1.0 mL of the polysaccharide stock solution and proceed according to the above-described procedure for plotting the standard curve, and measure the absorbance value. The calculated polysaccharide content is shown in Table 1.

[0154] Table 1 Polysaccharide content

[0155] Extraction process Polysaccharide content (%) Example 1 0.14 Example 2 0.12 Example 3 0.12 Comparative Example 1 0.06 Comparative Example 2 0.05 Comparative Example 3 0.05 Comparative Example 4 0.04

[0156] 4) The experiment found that the absorbance value was the highest when 4 mL of phenol solution was added, and the maximum absorbance was at 487 nm, which was stable. It can be seen that the best conditions for the measurement are to add 4 mL of 5% phenol and 10 mL of concentrated sulfuric acid, and select 487 nm as the measurement wavelength.

[0157] 5) The glucose standard curve is plotted with absorbance on the ordinate and solution concentration (mg / mL) on the abscissa. The regression equation is: A = 5.6893B + 0.0683, r = 0.9996. It can be seen that the absorbance of the solution has a good linear relationship with the concentration of glucose in the range of 0.02 to 0.14 mg / mL.

[0158] 6) Weigh the purified powder, place it in a 500mL volumetric flask, and dilute to volume. Take 1.0mL and determine its absorbance using the same method as the standard curve, as shown in Table 2:

[0159] Table 2 Weight of purified powder

[0160]

[0161]

[0162] Calculate the polysaccharide extraction rate from Imperata cylindrica root. Polysaccharide extraction rate = VCD / m × 100%, where V: volume of sample solution (mL); C: concentration of polysaccharide (μg / mL); m: mass of Imperata cylindrica root (g); D: dilution factor of sample solution.

[0163] Experimental Example 2

[0164] Precision and stability tests

[0165] (1) Precision test: Take 1.0 mL of the standard solution of Example 1 and operate according to the method of Test Example 1. Measure its absorbance 6 times in a row. The results are 0.290, 0.289, 0.292, 0.288, 0.286 and 0.283, respectively. RSD < 1.5%.

[0166] (2) Stability test: 1.0 mL of Imperata cylindrica polysaccharide stock solution from Example 1 was precisely pipetted and operated according to the method in Example 1. The results were measured at 0.5, 1, 2, 5 and 12 h respectively. The results were 0.274, 0.274, 0.275, 0.268 and 0.260 respectively. It can be seen that the polysaccharide solution is stable within 12 h.

[0167] The data shows that this method significantly outperforms traditional processes in terms of polysaccharide content and extraction rate. Furthermore, the product exhibits excellent stability (small absorbance fluctuations within 12 hours) and precision (low RSD in six consecutive measurements), meeting the requirements of "high efficiency, stability, and safety" in traditional Chinese medicine extraction.

[0168] In summary, the extraction method of Imperata cylindrica root proposed in this application, through the integration of compound enzymatic hydrolysis, ultrasonic assistance, and compound microbial fermentation, breaks through the bottlenecks of traditional processes such as "low extraction efficiency, poor product purity, and easy damage to activity," providing a feasible solution for the large-scale, high-quality extraction of Imperata cylindrica root polysaccharides, and possessing high technological innovation and industrial application prospects.

[0169] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A method for extracting Chinese medicine Imperata cylindrica var. major, characterized in that, The extraction method is a combination of complex enzymatic hydrolysate and ultrasonic-assisted microbial fermentation for polysaccharide extraction.

2. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 1, characterized in that, The complex enzymatic hydrolysate is a mixture of enzyme-cut modified earthworm kinase and neutral pectinase for enzymatic hydrolysis.

3. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 1, characterized in that, The microbial fermentation is a complex fermentation strain.

4. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 3, characterized in that, The complex fermentation strain is a fermentation strain of Bacillus licheniformis and Saccharomyces cerevisiae.

5. The extraction method of the traditional Chinese medicine white imperial plantain as claimed in claim 1, characterized in that, The extraction method steps are: (1) raw material pretreatment: wash the Imperata cylindrical, cut into small pieces, dry in an oven to constant weight, then crush into powder with a pulverizer, and sieve through a 40-60 mesh sieve; (2) enzymolysis: adjust the pH, and use complex enzymatic hydrolysate for enzymolysis, with an enzymolysis temperature of 40-50 DEG C and a time of 4-6 h to obtain a hydrolysate; (3) enzyme inactivation: heat the hydrolysate at 90-95 DEG C for 15-20 min for enzyme inactivation to obtain an inactivated product; (4) ultrasonic-assisted microbial fermentation: place the inactivated product in a 20 kHz ultrasonic wave for 20 min, spray the complex fermentation strain at a specific ratio, add purified water to adjust the moisture content to 65%, mix thoroughly, and then load into a respiratory fermentation bag, seal, and ferment at 30-35 DEG C for 4-6 d to obtain a polysaccharide extract; (5) polysaccharide purification and drying: slowly add 95% ethanol to the polysaccharide extract for precipitation, dialyze the polysaccharide solution in the dialysis bag in deionized water at 4 DEG C for 48 h, and then transfer the polysaccharide solution to a vacuum freeze dryer.

6. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 5, characterized in that, The pH adjustment in step (2) is 6-8.

7. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 5, characterized in that, The specific ratio of the complex fermentation strain is 5:1 for Bacillus licheniformis and Saccharomyces cerevisiae fermentation strain.

8. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 2, characterized in that, The enzyme-cut modified earthworm kinase is immobilized trypsin-cut modified earthworm kinase.

9. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 2, characterized in that, The preparation method of the enzyme-cut modified earthworm kinase is: (1) weigh the earthworms of Taiping No. 1, wash, soak in clean water in a beaker for 2 h, wash, and homogenize at low temperature to obtain Taiping No. 1 homogenate; (2) add PBS and benzoic acid solution to the Taiping No. 1 homogenate at a ratio of 1:4 (w / v) to autolyze for 8 h, centrifuge to collect the supernatant to obtain an earthworm kinase crude extract; (3) add ammonium sulfate to the earthworm kinase crude extract for fractional precipitation, centrifuge at 8000 rpm / min for 10 min at 25 DEG C to collect the precipitate, and dialyze and desalt to obtain earthworm kinase dry powder; (4) enzyme-cut modified earthworm kinase: immobilize trypsin with chitosan to obtain immobilized trypsin, add the immobilized trypsin to earthworm kinase, incubate in a 45 DEG C water bath for 1 h, and filter to obtain enzyme-cut modified earthworm kinase.

10. The extraction method of the traditional Chinese medicine white imperial plant as claimed in claim 2, characterized in that, The mixing ratio of the enzyme-cut modified earthworm kinase and neutral pectinase is 1:1.

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