A method for extracting red clover polysaccharide by enzyme

Abstract

The present invention discloses an enzyme extraction method for red clover polysaccharide, which belongs to the field of natural product separation and extraction. The above enzyme extraction method includes: mixing red clover, water and composite enzyme and performing enzymolysis to obtain red clover crude polysaccharide; then precipitating with alcohol, dialysis and freeze drying to obtain red clover polysaccharide; the mass volume ratio of red clover to water is 1g: (30-50) mL; the dosage of composite enzyme is 0.6%-1.0% of the total mass of red clover, and the composite enzyme is a mixture of papain, cellulase and pectinase. The optimal process conditions for enzyme extraction are screened out by experiments: enzymolysis time 90min, enzyme dosage 1.0%, enzymolysis temperature 60℃, papain, cellulase and pectinase mass ratio of 7:2:2, red clover crude polysaccharide content of 8.85±0.05%, and refined red clover polysaccharide content of 28.42±0.68%. The present invention provides a theoretical basis for the extraction and preparation of red clover polysaccharide.

Description

Technical Field

[0001] The invention relates to the field of separation and extraction of natural products, in particular to an enzyme extraction method for red clover polysaccharide. Background Art

[0002] Red clover (Trifolium pratense L.) has a high crude protein content and a low neutral detergent fiber content. It is rich in biologically active substances such as isoflavones and polysaccharides and has the function of nitrogen fixation. It also plays an important role in ecological environmental governance and restoration. It is an important perennial leguminous forage and medicinal plant in temperate climate regions of the world.

[0003] Plant polysaccharides, also known as plant polysaccharides, are a class of natural high molecular polymers formed by aldose or ketose connected by glycosidic bonds. They are important macromolecules in plants. They are not only structural substances of plant cells, but also the main source of cell energy. Polysaccharides have certain biocompatibility, biodegradability and stability. They are widely used in health food and pharmaceutical industries. They have multiple biological activities such as antioxidant, anti-tumor, anticoagulant, hypoglycemic and immunomodulatory. They play an important role in the treatment of skin trauma, cartilage and arthritis. They are easy to process and modify, and have good development and application prospects in the fields of tissue engineering.

[0004] Enzyme extraction technology is widely used in the development and utilization of natural plant polysaccharides. The mild extraction conditions ensure the activity of polysaccharides. Protease, cellulase and pectinase are commonly used enzymes in enzyme extraction technology, which can improve the yield and antioxidant activity of polysaccharides. Enzyme extraction mainly uses the specificity of enzyme reaction to improve the extraction effect. It can selectively degrade the cell wall and cell membrane of plants, reduce the resistance from the cell wall and cell membrane during solvent extraction, and release the effective ingredients. The use of enzymes can also degrade some polysaccharides into smaller molecular weight fragments, which is more conducive to the separation of polysaccharides from cells. At present, the extraction methods of red clover polysaccharides include ultrasonic and hot water extraction. The red clover polysaccharide prepared by ultrasonic extraction has good antioxidant properties. Zhang et al. optimized the hot water extraction method using response surface methodology and found that red clover polysaccharide has good hypoglycemic activity and antioxidant properties. However, there are no reports on the enzyme extraction process of red clover polysaccharide and the comparison of the extraction effects of the three. Summary of the invention

[0005] The purpose of the present invention is to provide an enzyme extraction method for red clover polysaccharide to solve the problems existing in the above-mentioned prior art. By investigating four influencing factors, namely, enzymatic hydrolysis time, enzyme dosage, enzymatic hydrolysis temperature and enzyme ratio, the optimal process conditions for extracting red clover polysaccharide are optimized, providing a new theoretical basis for the extraction and preparation of red clover polysaccharide.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for extracting red clover polysaccharide by enzyme, comprising the following steps:

[0008] Mixing red clover, water and complex enzyme and performing enzymolysis to obtain red clover crude polysaccharide; alcohol precipitation, dialysis and freeze-drying of the red clover crude polysaccharide to obtain red clover polysaccharide;

[0009] The mass volume ratio of the red clover to the water is 1g: (30-50)mL; the dosage of the complex enzyme is 0.6%-1.0% of the total mass of the red clover, and the complex enzyme is a mixture of papain, cellulase and pectinase.

[0010] Preferably, the composite enzyme is a mixture of papain, cellulase and pectinase in a mass ratio of (2-15):(1-4):(1-4).

[0011] Preferably, the mass ratio of the papain, the cellulase and the pectinase is 7:2:2.

[0012] Preferably, the enzymatic hydrolysis temperature is 45-65°C and the time is 30-100 min.

[0013] Preferably, the enzymatic hydrolysis temperature is 60° C. and the time is 90 min.

[0014] Preferably, the dosage of the complex enzyme is 1.0% of the total mass of the red clover.

[0015] Preferably, the crude polysaccharide from red clover is precipitated with anhydrous ethanol; and the dialysis is performed using a 3500DA dialysis bag.

[0016] The invention also provides a red clover polysaccharide, which is prepared by the enzyme extraction method.

[0017] Preferably, the red clover polysaccharide is composed of mannose, rhamnose (Rha), glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, wherein the glucose, galactose and arabinose account for 3 / 4 of the red clover polysaccharide, and the molar percentage of glucose is the highest.

[0018] The invention also provides the application of the red clover polysaccharide in clearing DPPH and superoxide anions.

[0019] The present invention discloses the following technical effects:

[0020] The present invention studies the effects of four single factors, namely, enzymatic hydrolysis time, enzyme dosage, enzyme ratio and enzymatic hydrolysis temperature, on the extraction of red clover polysaccharide. On this basis, orthogonal test is used to optimize the enzyme extraction process conditions and screen out the best enzyme extraction process. The best enzyme extraction process conditions for red clover polysaccharide are: enzymatic hydrolysis time 90min, enzyme dosage 1.0%, enzymatic hydrolysis temperature 60℃, enzyme ratio of papain: cellulase: pectinase 7:2:2, red clover crude polysaccharide content of 8.85±0.05%, and refined polysaccharide content of 28.42±0.68%. The antioxidant activity results show that the red clover polysaccharide prepared by enzyme, ultrasonic and hot water extraction has high antioxidant activity, and the scavenging activity of superoxide anion free radicals is higher than that of the positive control Vc. The results of the analysis of the polysaccharide composition show that the red clover polysaccharide prepared by the enzyme, ultrasound and hot water extraction methods has the same composition, all composed of 8 monosaccharides, but the monosaccharide composition ratios are significantly different, among which glucose Glu, galactose Gal and arabinose Ara account for a high proportion, and the three account for about three quarters of the monosaccharide components. However, different extraction methods lead to significant differences in the proportions of these three monosaccharides. The molar percentage of Glu in the enzyme-extracted polysaccharide is the highest, which is 1.6 times that of the hot water-extracted polysaccharide. Compared with ultrasound and hot water extraction, the enzyme extraction method provided by the present invention does not use any organic reagents except ethanol. The method of the present invention has fewer steps, mild conditions, low cost, green and safe, and is most suitable for process production applications, providing a new theoretical basis for the extraction and preparation of red clover polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 represents the effect of different extraction factors on polysaccharide content; lowercase letters represent the difference levels of different levels under the same factor (P<0.05); AD represents the effect of enzymatic hydrolysis time, enzyme dosage, enzymatic hydrolysis temperature and enzyme ratio on polysaccharide content.

[0023] Figure 2 Red clover polysaccharides prepared by different extraction methods;

[0024] Figure 3 The antioxidant activity of polysaccharides extracted by different methods; A: DPPH free radical scavenging ability; B: superoxide anion free radical scavenging ability;

[0025] Figure 4This is the HPLC chromatogram of monosaccharide composition analysis; A is the mixed standard; B is the polysaccharide extracted by enzyme; C is the polysaccharide extracted by ultrasound; D is the polysaccharide extracted by hot water; 1, 2, 3, 4, 5, 6, 7, and 8 are Man, Rha, GluA, GalA, Glu, Gal, Xyl, and Ara, respectively. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Materials and methods

[0033] 1.1 Test materials

[0034] Red clover leaves were collected from plants in the early flowering stage grown in the smart greenhouse of Lanzhou University Yuzhong Campus, dried at 50°C to constant weight, crushed through a 60-mesh sieve, and set aside. Papain (800 U / mg), cellulase (50 U / mg), and pectinase (500 U / mg) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] 1.2 Test methods

[0036] 1.2.1 Single Factor Experiment

[0037] Take 1.0g of red clover powder, place it in a 100mL conical flask, extract with ultrapure water (add 30mL of ultrapure water), activate the enzyme in a water bath at 40℃ for 10min and then add it. After extraction, quickly raise the solution temperature to 90℃ and inactivate it for 10min, filter and collect the supernatant.

[0038] The effects of enzymatic hydrolysis time (30, 60, 80, 90, 100 min), enzyme dosage (0.6, 0.7, 0.8, 0.9, 1.0% of the material), enzymatic hydrolysis temperature (45, 50, 55, 60 and 65°C), and enzyme ratio (papain: cellulase: pectinase were 2:1:1, 3:1:1, 7:2:2, 15:4:4 and 4:1:1, respectively) on the extraction of red clover polysaccharide were investigated to screen the optimal extraction conditions.

[0039] 1.2.2 Orthogonal test

[0040] On the basis of the single factor experiment, A enzymatic hydrolysis time, B enzyme dosage, C enzymatic hydrolysis temperature and D enzyme ratio were selected, and the experiment was arranged according to the orthogonal design table. The factor levels are shown in Table 1, and each group of experiments was repeated 3 times.

[0041] Table 1 Orthogonal test factor level table

[0042]

[0043] 1.2.3 Determination of polysaccharide content

[0044] The polysaccharide content was determined by the phenol-sulfuric acid method. For specific methods, refer to "Shi Wenjuan. Study on the antioxidant activity of polysaccharides and flavonoids in Apocynum venetum and Trifolium pratense". The absorbance value was measured at 490 nm to calculate the polysaccharide content.

[0045] Polysaccharide content (%) = polysaccharide concentration of test solution × extraction volume / red clover sample mass × 100%

[0046] Glucose was used as the standard, and the horizontal axis was the concentration (mg·mL -1 ), the ordinate is the absorbance value (A), and its standard curve is Y=5.7364X+0.0465, R 2 =0.9991.

[0047] 1.2.4 Comparison of extraction methods of red clover polysaccharides

[0048] 1.2.4.1 Enzyme extraction

[0049] Take 50.0 g of red clover powder and extract polysaccharides according to the optimal extraction process obtained by orthogonal experiment.

[0050] 1.2.4.2 Ultrasonic extraction

[0051] Take 50.0g of red clover powder and place it in a 100mL conical flask. Refer to the method in Shi Wenjuan's "Study on the Antioxidant Activity of Polysaccharides and Flavonoids in Apocynum and Red Clover" for extraction. Use 40% anhydrous ethanol to extract, with a solid-liquid ratio of 1:30 (g·mL -1 ), ultrasonically extract at 50°C for 30 min, filter and collect the supernatant.

[0052] 1.2.4.3 Hot water extraction

[0053] Take 50.0g of red clover powder and place it in a 100mL conical flask. Extract it according to the method in Zhang HX, et al. "Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifoliumpratense L.) polysaccharides". Use ultrapure water for extraction, and the solid-liquid ratio is 1:21 (g·mL -1 ), extract in a water bath at 93°C for 95 min, filter and collect the supernatant.

[0054] 1.2.5 Refining of Red Clover Polysaccharide

[0055] Extract according to the three methods in 1.2.4 respectively, concentrate the supernatant to 1 / 4 volume under reduced pressure using a rotary evaporator (RV10 digital Flex, IKA, Germany), add anhydrous ethanol for precipitation, remove protein using the Sevage method, dialyze with a dialysis bag (3500DA, Beijing Solebow Technology Co., Ltd.) for 24 hours, and refine the solution in the dialysis bag with a macroporous adsorption resin (D101, Shanghai MacLean Biochemical Technology Co., Ltd.), concentrate under reduced pressure, and freeze-dry (Coolsafe100-4, Gene Co., Ltd.) to obtain refined polysaccharides.

[0056] 1.2.6 Determination of antioxidant activity

[0057] The scavenging activity of DPPH and superoxide anion free radicals was determined by referring to the methods in Mishra K, et al. "Estimation of antiradical properties of antioxidants using DPPH'assay: A critical review and results" and Halliwell B, et al. "The deoxyribose method: A simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals". Polysaccharide sample, positive control V C Prepare each substance to be tested with ultrapure water to a concentration of 1 mg mL -1 The stock solution was then diluted to (0.1 mg mL -1 , 0.2mg·mL -1 , 0.4mg·mL -1 , 0.5mg·mL -1 , 0.6mg·mL -1 , 0.8mg·mL -1 ) and (0.4 mg mL -1 , 0.5mg·mL -1 , 0.6mg·mL -1 , 0.8mg·mL -1 ) Different concentration gradients are set aside.

[0058] 1.2.7 Monosaccharide composition analysis

[0059] The monosaccharide composition of red clover polysaccharide was analyzed by high performance liquid chromatography (HPLC) using the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method (references: "Qiu Xian. Extraction, separation, purification and structural identification of polysaccharides from Acorus calamus"; Xu Z, et al. "Characterization and antioxidant activities of polysaccharides from the leaves of Lilium lancifolium Thunb"; Ren D, et al. "Chemical characterization of a novel polysaccharide ASKP-1fromArtemisia sphaerocephala Krasch seed and its macrophage activation via MAPK, PI3k / Akt and NF-κB signaling pathways in RAW264.7 cells"). The sample pretreatment and chromatographic conditions are as follows:

[0060] Preparation of red clover polysaccharide hydrolysis and monosaccharide standard solution: accurately weigh 5 mg of polysaccharide sample into a stoppered flask, add 2 mL of 2 mol·L -1 The polysaccharide hydrolyzate was obtained by hydrolyzing with trifluoroacetic acid at 105 °C for 6 h. After the reaction was completed, the hydrolyzate was repeatedly washed out with methanol, the hydrolyzate was decompressed and dried, and then dissolved in 0.5 mL of ultrapure water to obtain a polysaccharide hydrolyzate. 10 mg of mannose (Man), rhamnose (Rha), glucuronic acid (GluA), galacturonic acid (GalA), glucose (Glu), galactose (Gal), xylose (Xyl) and arabinose (Ara) standards were weighed respectively and prepared into 1 mg mL -1 The mixed monosaccharide standard stock solution was diluted 2, 4, 8, 16, 32, or 64 times for later use.

[0061] PMP derivatization step: Take 100 μL of polysaccharide hydrolysate or monosaccharide standard solution and add 400 μL of 0.5 mol·L - 1 PMP and 400 μL 0.3 mol·L -1 Mix well with NaOH, react in a 70℃ water bath for 1h, and add 405μL 0.3mol·L -1 The resulting solution was extracted with 2 mL of chloroform, and the extraction was repeated 3 times to obtain the PMP-labeled monosaccharide sample, which was filtered through a 0.45 μm aqueous filter membrane and then injected for analysis.

[0062] Chromatographic conditions: Agilent 1260 high performance liquid chromatograph (Agilent, USA), Eclipse Plus C18 column (4.6 mm × 250 mm, Agilent, USA), UV detector wavelength of 250 nm, column oven temperature of 30 °C, mobile phase of 0.05 mol·L -1 Phosphate buffer (pH 6.7): acetonitrile = 83:17 (v / v), flow rate was 0.8 mL / min, injection volume was 20 μL, and data acquisition time was 55 min.

[0063] 1.5 Data processing and analysis

[0064] One-way ANOVA analysis of variance was performed using IBM SPSS Statistics 26.0 statistical software, and Origin 2023 was used to draw the graphs.

[0065] 2. Results and Analysis

[0066] 2.1 Single factor screening

[0067] 2.1.1 Enzymatic hydrolysis time

[0068] Under the conditions of 0.9% enzyme dosage, 50℃ enzyme hydrolysis temperature and 3:1:1 enzyme ratio, the effect of enzyme hydrolysis time on the extraction of red clover polysaccharide was investigated. Figure 1 As shown in A, the longer the enzymatic hydrolysis time, the higher the polysaccharide content. When the enzymatic hydrolysis time is short, the polysaccharide content rises sharply, the increase tends to be gentle at 60-80min, and then rises, but there is no significant difference in the polysaccharide content between 90 and 100min. Considering the extraction efficiency and time cost, 90min is the best enzymatic hydrolysis time.

[0069] 2.1.2 Enzyme dosage

[0070] Under the conditions of 90 min of enzymatic hydrolysis time, constant enzymatic hydrolysis temperature and constant enzyme ratio, the effect of enzyme dosage on the extraction of red clover polysaccharide was investigated. Figure 1 As shown in B, the polysaccharide content of red clover increases first and then decreases with the increase of enzyme dosage. The more enzyme dosage, the higher the polysaccharide content is not necessarily. The polysaccharide content is also related to the dosage of the single enzyme. Enzymes are specific, and each single enzyme plays a different role in the extraction process. Under the same proportion, the enzyme dosage increases, and the ability of each single enzyme to decompose and extract plant tissues is different. When the enzyme dosage is 0.9%, the polysaccharide content is the highest, and then the polysaccharide content decreases. Therefore, 0.9% is the optimal enzyme dosage.

[0071] 2.1.3 Enzymatic hydrolysis temperature

[0072] Under the conditions of 90 min of enzymatic hydrolysis time, 0.9% of enzyme dosage and constant enzyme ratio, the effect of enzymatic hydrolysis temperature on the extraction of red clover polysaccharide was investigated. Figure 1 As shown in C, the polysaccharide content of red clover first increases and then decreases with the increase of enzymatic hydrolysis temperature. The polysaccharide content increases slowly at 55℃, while there is no significant difference in the polysaccharide content at 55℃, 60℃ and 65℃. The polysaccharide content decreases after 60℃, which may be due to the inhibition of enzyme activity by temperature. Therefore, 55℃ is the best enzymatic hydrolysis time.

[0073] 2.1.4 Enzyme ratio

[0074] Under the conditions of enzymatic hydrolysis time of 90 min, enzyme dosage of 0.9% and enzymatic hydrolysis temperature of 55°C, the effect of enzyme ratio on the extraction of red clover polysaccharide was investigated. Figure 1 As shown in D, the polysaccharide content of red clover increases first, then decreases, and then increases again with the increase of enzyme ratio. The enzyme ratio and enzyme dosage have roughly the same change trend on the polysaccharide content of red clover, which are interrelated and restricted to each other. When the enzyme ratio is 3:1:1, the polysaccharide content is the highest, and when the enzyme ratio is 7:2:2, the polysaccharide content is the lowest, so 3:1:1 is the best enzyme ratio.

[0075] 2.2 Orthogonal test results

[0076] As shown in Tables 2 and 3, the C (enzyme hydrolysis temperature) R value is the largest in the enzyme extraction process under different conditions, that is, the enzyme hydrolysis temperature has the greatest impact on the polysaccharide content of red clover. The three factors of enzymatic hydrolysis time, enzyme dosage, and enzymatic hydrolysis temperature have extremely significant effects on the polysaccharide content (P < 0.01), and the effect of enzyme ratio on the polysaccharide content of red clover is not significant (P > 0.05). The different levels of each factor are A 2 >A 1 >A 3 , B 3 >B 2 >B 1 , C 2 >C 1 >C 3 , D 2 >D 3 >D 1 The optimal process conditions for enzyme extraction are A 2 B 3 C 2 D 2 , that is, the enzymatic hydrolysis time is 90min, the enzyme dosage is 1.0%, the enzymatic hydrolysis temperature is 60℃, and the enzyme ratio is 7:2:2. Using this process to extract, the red clover polysaccharide content is 8.85±0.07%.

[0077] Table 2 Orthogonal design of red clover polysaccharide enzyme extraction process and polysaccharide content results

[0078]

[0079] Table 3 Results of variance analysis of polysaccharide content in red clover polysaccharide enzyme extraction process

[0080]

[0081] 2.3 Comparison of enzyme, ultrasonic and hot water extraction methods

[0082] 2.3.1. Preparation of red clover polysaccharides by different extraction methods

[0083] Red clover polysaccharides prepared by different extraction methods Figure 2 As shown, the polysaccharides purified by enzyme, ultrasonic and hot water extraction were 0.23 g, 0.14 g and 0.26 g, respectively. The polysaccharide samples were used to determine the antioxidant activity and analyze the monosaccharide composition.

[0084] 2.3.2 Ability of red clover polysaccharide to scavenge DPPH and superoxide anion free radicals

[0085] Red clover polysaccharide has antioxidant capacity. Figure 3 As shown in Figure A, the scavenging rates of red clover polysaccharides extracted by the three methods on DPPH were weaker than those of the positive control Vc. The polysaccharide concentration was lower than 0.2 mg·mL -1 The polysaccharide activity of enzyme and hot water extraction was stronger than that of ultrasonic extraction, and the polysaccharide concentration was 0.2-0.6 mg·mL -1 The scavenging rate of DPPH varied greatly between the two groups, and the scavenging rate of polysaccharides extracted by hot water was the highest. The concentration of polysaccharides extracted by ultrasound and hot water was 0.6-1.0 mg·mL -1 The scavenging rate of DPPH between the two groups tended to be gentle, while the scavenging rate of DPPH of polysaccharides extracted by enzyme increased greatly with the increase of concentration, 1 mg·mL -1 When the polysaccharides extracted by the three methods were scavenged by DPPH, the scavenging rate was close to 70%. Figure 3 As shown in Figure B, the scavenging rates of red clover polysaccharides extracted by the three methods on superoxide anions were stronger than those of the positive control Vc. The concentrations of polysaccharides extracted by ultrasound and hot water were between 0.6 and 1.0 mg mL -1 The clearance rate of superoxide anions varied greatly between the two groups. The clearance rate of superoxide anions by polysaccharides extracted by enzymes was 0.8 mg·mL -1 As the concentration of polysaccharide increases, it increases slowly at first and then rapidly. -1 When the concentration of polysaccharides was 0.6-1.0 mg·mL, the scavenging rates of polysaccharides extracted by the three methods tended to be close to 50%, and the scavenging rates of polysaccharides extracted by ultrasound and hot water on DPPH and superoxide anions showed basically the same trend. -1 The scavenging abilities of red clover polysaccharides extracted by three methods on DPPH and superoxide anions were as follows: ultrasound, hot water and enzyme extraction of polysaccharides.

[0086] 2.3.3 Monosaccharide composition analysis

[0087] like Figure 4 As shown in the figure, red clover polysaccharides are mainly composed of 8 monosaccharides, namely mannose (Man), rhamnose (Rha), glucuronic acid (GluA), galacturonic acid (GalA), glucose (Glu), galactose (Gal), xylose (Xyl), and arabinose (Ara). Among them, glucose Glu, galactose Gal, and arabinose Ara account for a high proportion, accounting for more than 71% of the monosaccharide components (Table 4). However, different extraction methods lead to significant differences in the proportions of these three monosaccharides. The molar percentage of Glu in enzyme-extracted polysaccharides is the highest, which is 1.6 times that of hot water-extracted polysaccharides. At the same time, Glu in enzyme-extracted polysaccharides is slightly higher than Gal, and the composition ratio is close to 1:1, while the proportion of Gal in ultrasonic and hot water-extracted polysaccharides is the highest, which is 1.5-1.8 times that of Glu. Ara has the lowest proportion in enzyme extraction, which is 64% of Glu, but the proportion in ultrasonic and hot water-extracted polysaccharides is higher than 20%, which is 1.1 times that of Glu. Among the three extracted polysaccharides, GluA has the lowest molar percentage, less than 4.1%.

[0088] Table 4 Monosaccharide composition of red clover

[0089]

[0090] From the results of the above examples, it can be seen that the optimal extraction process of red clover polysaccharide obtained by the present invention through single factor and orthogonal test is: enzymatic hydrolysis time 90min, enzyme dosage 1.0%, enzyme ratio 7:2:2, enzymatic hydrolysis temperature 60°C. Under these conditions, the content of purified and refined polysaccharide is 28.42±0.68%, and the polysaccharide is composed of 8 monosaccharides, with Glu accounting for the highest proportion. The polysaccharides extracted by the three methods all have high antioxidant activity, especially the scavenging activity against superoxide anions. At 1mg·mL -1 When the enzyme extraction was carried out, the scavenging activity of polysaccharides on DPPH and superoxide anions was close to that of hot water and ultrasonic extraction. The monosaccharide composition of different extraction methods was the same, but the molar percentages of each monosaccharide were different. The polysaccharides extracted by the three methods were mainly composed of monosaccharides such as Glu, Ara and Gal. Compared with ultrasonic and hot water extraction, the enzyme extraction method did not use any organic reagents except ethanol. This method has fewer steps, mild conditions, low cost, is green and safe, and is most suitable for process production applications.

[0091] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of red clover polysaccharide in the removal of DPPH and superoxide anions for non-disease treatment purposes. It is characterized in that The method for extracting red clover polysaccharide comprises the following steps: The red clover leaves, water and complex enzymes are mixed and then enzymatically hydrolyzed to obtain red clover crude polysaccharide; the red clover crude polysaccharide is subjected to alcohol precipitation, dialysis and freeze-drying to obtain red clover polysaccharide; The mass volume ratio of the red clover leaf to the water is 1 g: (30-50) mL; the dosage of the complex enzyme is 1.0% of the total mass of the red clover leaf, and the complex enzyme is a mixture of papain, cellulase and pectinase; The mass ratio of the papain, the cellulase and the pectinase is 7:2:2; The temperature of the enzymatic hydrolysis is 60° C. and the time is 90 min.

2. The use according to claim 1, It is characterized in that The crude polysaccharide of red clover is precipitated with anhydrous ethanol; and the dialysis is performed using a 3500DA dialysis bag.

3. The use according to claim 1, It is characterized in that The red clover polysaccharide is mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, wherein the glucose, galactose and arabinose account for 3 / 4 of the red clover polysaccharide, and the molar percentage of glucose is the highest.

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