A purified polysaccharide of Trichoderma tricholoma and its preparation method and application

Abstract

The present invention belongs to the field of fungal polysaccharides, and discloses a purified polysaccharide of Pleurotus eryngii, and a preparation method and application thereof. The molar ratio of monosaccharide composition in the polysaccharide of Pleurotus eryngii is fucose: mannose: glucose: galactose=1.00:1.20:3.54:1.88. The polysaccharide is obtained by extracting crude polysaccharide from the fruiting body of Pleurotus eryngii by hot water, and then purifying the crude polysaccharide. Experiments have shown that the purified polysaccharide has the effects of anti-oxidation, enhancing immunity, and regulating blood sugar, and can be used for the development of health foods and functional foods, thereby improving the utilization value of Pleurotus eryngii.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal polysaccharide application, and particularly relates to a purified polysaccharide of Trichoderma tricholoma and a preparation method and application thereof. Background Art

[0002] Hairy mushroom, also known as golden mushroom and roadside mushroom, belongs to the species of Lyophyllum fumosum, Lyophyllaceae, Agaricales, and is mainly produced in the northeast of Daxinganling in my country.

[0003] As a natural active substance, fungal polysaccharides have multiple activities such as anti-tumor, antioxidant, anticoagulant, and immunomodulatory, and are safe and non-toxic. They have broad potential for development as functional foods or health products in the future. The extraction method of fungal polysaccharides will affect the yield of fungal polysaccharides, and will also affect the structure and activity of the extracted polysaccharides. The extracted crude fungal polysaccharides often contain impurities such as proteins and pigments, and different methods must be used to remove them before purified polysaccharide components can be obtained. The structure of polysaccharides will also affect the active functions of polysaccharides. On the basis of correctly analyzing the polysaccharide structure, we can better understand the structure-activity relationship of polysaccharides.

[0004] Liang Xiufeng and Hu Haibing reported the artificial cultivation method of Maojian mushroom in "Technology of Maojian mushroom under forest" (Liang Xiufeng, Li Hongtao et al. 2019) and "Technology of Maojian mushroom indoor cultivation" (Hu Haibing, Liang Xiufeng et al. 2020), respectively, which improved the yield of Maojian mushroom to a certain extent. Luo Wenzhe reported in "Effects of Maojian mushroom crude polysaccharide on P53 expression in H22 tumor-bearing mice" (Luo Wenzhe, Lv Dongxia et al. 2016) and "Effects of Maojian mushroom crude polysaccharide on TNF-α and IL-2 expression in H22 liver cancer mice" (Luo Wenzhe, Wang Jianjie et al. 2015) that Maojian mushroom polysaccharide has anti-tumor effects, and its mechanism may be related to the regulation of cytokine expression.

[0005] It can be seen that the existing research has not carried out a purification step for the crude polysaccharide of Trichoderma, lacks research on the fine structure of the purified polysaccharide of Trichoderma, and has not evaluated the antioxidant, hypoglycemic, immune regulatory and other activities of Trichoderma polysaccharide. Summary of the invention

[0006] The purpose of the present invention is to more reasonably utilize the resources of Trichoderma tricholoma, provide a method for purifying crude polysaccharide of Trichoderma tricholoma, select LFP-I with the highest yield and more concentrated molecular weight from multiple purified components for structure and activity research, and provide application ideas of the Trichoderma tricholoma polysaccharide.

[0007] The specific plan is:

[0008] The present invention provides a purified polysaccharide of Trichoderma tricholoma LFP-I. The purified polysaccharide of Trichoderma tricholoma LFP-I is a heteropolysaccharide composed of fucose Fucp: mannose Manp: glucose Glcp: galactose Galp=1.00:1.20:3.54:1.88, and the structural fragment is as follows:

[0009]

[0010] R1 / R2 represents that this position is connected to two groups, R1 and R2, respectively, where R1 is -α-D-Glcp; R2 is -α-Manp-(3→1)-α-D-Manp-(3→1)-α-L-Fucp.

[0011] Furthermore, the weight average molecular weight of the purified Trichoderma lucidum polysaccharide LFP-I is 8000-30000Da.

[0012] Furthermore, the weight average molecular weight of the purified Agaricus tricholoma polysaccharide LFP-I is 13288Da.

[0013] The present invention also provides a method for preparing the above-mentioned Trichoderma tricholoma purified polysaccharide LFP-I, comprising the following steps:

[0014] (1) The fruiting bodies of Pleurotus eryngii are taken and ground into powder, and a polysaccharide extract is obtained by hot water extraction. The polysaccharide extract is decolorized, deproteinized, and precipitated with alcohol to obtain crude polysaccharide LFPs of Pleurotus eryngii;

[0015] (2) eluting the LFPs obtained in step (1) through an ion exchange chromatography column and collecting the eluate;

[0016] (3) the eluate is concentrated, dialyzed, and freeze-dried to obtain the purified polysaccharide LFP-I of Trichoderma tricholoma;

[0017] In step (2), the ion exchange chromatography column is filled with DEAE-52;

[0018] In step (2), the eluent is a NaCl solution with a concentration of 0-0.2 mol / L.

[0019] Furthermore, in step (1), the temperature of hot water extraction is 60-100° C., the extraction time is 100-140 min; the ratio of the Maojian mushroom fruiting body powder to water is 1 g: 10-30 mL;

[0020] In step (1), the macroporous resin D354FD is used for decolorization, and the macroporous resin and the polysaccharide extract are mixed in a volume ratio of 1:1, and the decolorization is carried out at 50±10°C with stirring for 3±1h;

[0021] In step (1), sevage reagent is used for deproteinization, and the sevage reagent is a mixed solution of n-butanol and chloroform in a volume ratio of 1:4.

[0022] Furthermore, in step (1), the hot water extraction temperature is 70° C., the extraction time is 120 min, and the ratio of the Maojian mushroom fruiting body powder to water is 1 g: 25 mL;

[0023] In step (1), the volume ratio of sevage reagent to polysaccharide extract is 1:4.

[0024] Furthermore, in step (1), the alcohol precipitation is performed by mixing the polysaccharide extract with 95% ethanol in a volume ratio of 1:4, and collecting the precipitate;

[0025] In step (3), a dialysis bag of 5000-8000Da is used for dialysis.

[0026] The present invention also provides the use of the above-mentioned purified polysaccharide LFP-I of Trichoderma lucidum in the preparation of food and / or health products for anti-oxidation, blood sugar regulation and immunity enhancement.

[0027] The polysaccharide component can be used alone or in combination with other nutritional ingredients.

[0028] The physical and chemical properties of the above polysaccharide components were measured, and the total sugar content of LFPs and LFP-I was 84.38% and 80.16%, respectively, the protein content was 5.11% and 0.55%, respectively, and the uronic acid content was 1.95% and 1.23%, respectively.

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] The inventors of the present invention separated and purified the hairy mushroom fruiting body to obtain the hairy mushroom purified polysaccharide component LFP-I, analyzed its primary structure, determined the monosaccharide composition and molecular weight, and inferred its glycosidic bond connection mode. Through in vitro antioxidant experiments, it was determined that the component has strong antioxidant ability, can effectively reduce the activity of α-amylase and α-glucosidase to regulate blood sugar, and through cell morphology observation and cytokine secretion detection, it was determined that the component has a stimulating and activating effect on the immune response of macrophages, and can be used in the development of health foods and functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the elution curve of crude polysaccharide from Trichoderma quinata.

[0032] Figure 2 This is the UV scanning spectrum of LFP-I.

[0033] Figure 3 This is the GPC standard spectrum.

[0034] Figure 4 This is the GPC spectrum of LFP-I.

[0035] Figure 5 FT-IR spectrum of LFP-I.

[0036] Figure 6 This is the GC standard spectrum.

[0037] Figure 7 This is the GC spectrum of LFP-I.

[0038] Figure 8 Total ion current of LFP-I.

[0039] Fig. 9 This is the 1H NMR spectrum of LFP-I.

[0040] Fig.10 This is the 13C NMR spectrum of LFP-I.

[0041] Fig.11 This is the HSQC spectrum of LFP-I.

[0042] Fig.12 This is the HMBC spectrum of LFP-I.

[0043] Fig.13 This is the NOESY spectrum of LFP-I.

[0044] Fig.14 This is the SEM image of LFP-I.

[0045] Fig.15 The cell morphology of RAW264.7 mouse macrophages was shown under the action of different concentrations of LFP-I.

[0046] Fig.16 The morphology of RAW264.7 mouse macrophages not treated with LFP-I.

[0047] Fig.17 The effect of different concentrations of LFP-I on the release of TNF-α cytokine.

[0048] Fig.18 is the inhibition rate of α-glucosidase by different concentrations of LFP-I.

[0049] Fig.19 is the inhibition rate of α-amylase by different concentrations of LFP-I.

[0050] Fig. 20 FRAP values ​​of different concentrations of LFP-I. DETAILED DESCRIPTION

[0051] The present invention is further explained below in conjunction with examples and drawings, but the present invention has more embodiments. If the specific operating conditions are not indicated in the examples, they are carried out according to the conventional or manufacturer's recommended methods. If the manufacturer of reagents and instruments is not indicated, they can be obtained from commercial or other supplier channels.

[0052] Example 1 Extraction and purification of Trichoderma polysaccharide LFP-I

[0053] 1. Weigh 100g of dried Maojian mushroom fruiting bodies, crush them into powder with a grinder, and then pass through a 60-mesh sieve. Mix the fruiting body powder with distilled water at a volume ratio of 1:20 (g:mL), and heat in a water bath at 70℃ for 120min. Filter out the residue in the extract and concentrate the supernatant to 200mL. Mix the supernatant with the pre-soaked macroporous resin D354FD at a volume ratio of 1:1, and decolorize by heating in a water bath at 50℃ for 3h. Filter the macroporous resin from the decolorized crude extract, and use sevage reagent to centrifuge and remove the protein. The supernatant after protein removal is concentrated and mixed with four times the volume of 95% ethanol. Collect the precipitate and dry it to obtain Maojian mushroom crude polysaccharide LFPs.

[0054] 2. Soak the DEAE-52 filler in advance. Slowly and evenly introduce the filler into the chromatography column, and tap the column with an ear bulb to compact and level the filler. Turn on the constant flow pump to the maximum flow rate to degas the filler. After degassing, redissolve the Maojian mushroom polysaccharide LFPs to 8 mg / mL, spread the polysaccharide solution on the filler, adjust the flow rate to 5-6s / drop, and use 0, 0.025, 0.05, 0.1, and 0.2 mol / L NaCl solutions as elution solutions, collect the eluents and determine the total sugar content, and draw the elution curve as shown in the figure. Figure 1 As shown. Among them, the components eluted with 0.025 mol / L NaCl solution had low content and were difficult to enrich. The purified components LFP-I, LFP-II, LFP-III, and LFP-IV were eluted with 0, 0.05, 0.1, and 0.2 mol / L NaCl solutions, respectively, with yields of 15.29%, 5.54%, 11.45%, and 8.2%, respectively. The eluate was dialyzed with distilled water and lyophilized to obtain purified LFP-I.

[0055] 3. Soak G-100 dextran gel in advance. Load the column and load the sample in the same way as DEAE-52 filler. Use the phenol-sulfuric acid method to detect LFP-I. The sugar content curve obtained is a single symmetrical peak, indicating that the purity of LFP-I is high.

[0056] Example 2 Identification of the physicochemical properties of Trichoderma polysaccharide LFP-I

[0057] 1. The total sugar content was determined to be 84.25% using the phenol-sulfuric acid method.

[0058] 2. The protein content was determined to be 0.55% using the BCA protein quantification kit.

[0059] 3. The uronic acid content was determined to be 1.23% using the carbazole sulfate method.

[0060] 4. Scan the sample solution using a UV full-band scanner in the range of 200-600nm (e.g. Figure 2 With wavelength as the horizontal axis and OD value as the vertical axis, the full-band scanning curve is plotted to have a small signal peak near 260-280nm, indicating that it may contain a small amount of nucleic acid and polypeptide components.

[0061] Example 3 Structural Identification of Pleurotus eryngii Polysaccharide LFP-I

[0062] 1. Determine molecular weight

[0063] 3 mg of Agrocybe quinoa polysaccharide LFP-I sample was dissolved in 1 mL of 20 mmol / L potassium dihydrogen phosphate solution. The instrument used was Thermo Ultimate 3000 high performance liquid chromatography, the detector was RefractoMax 521 differential detector, the gel column was Ultrahydrogel 1000 (7.8×300mm) and Ultrahydrogel 500 (7.8×300mm) in series, and the guard column was Ultrahydrogel (6×40mm). The mobile phase used 0.02 mol / L potassium dihydrogen phosphate buffer solution, the injection volume was 20 μL, the flow rate was constant at 0.8 mL / min, the single sample was set to run for 35 minutes, and the column oven was controlled at 35°C. HPGPC analysis was carried out under these conditions and compared with the standard results. Figure 3 For comparison, the LFP-I sample test results are as follows Figure 4 As shown, the weight average molecular weight of LFP-I was 13288 Da.

[0064] 2. Fourier infrared spectroscopy analysis

[0065] Weigh 2 mg of LFP-I, mix with appropriate amount of KBr powder and grind, and then evenly distribute it on the sample table by tableting. -1 Infrared scanning is performed within the range, and the results are as follows Figure 5 The molecule is at 3445cm -1 There is a strong and broad absorption peak of -OH stretching vibration at 2928cm -1 The absorption peak at 1640 cm is the CH stretching vibration peak of the H–C=O group and CH2. -1 The peak at is the absorption peak of the symmetric deformation vibration of the carbonyl C=O of the polysaccharide. The above characteristic absorption peaks indicate that LFP-I is a polysaccharide substance.

[0066] 3. Gas phase analysis of monosaccharide composition

[0067] First, LFP-I was subjected to acid hydrolysis and acetylation, and then detected by gas chromatography (GC) and compared with monosaccharide standards ( Figure 6 ) peak analysis of LFP-I monosaccharide composition ( Figure 7 ). The main monosaccharide composition and molar ratio of LFP-I were obtained by area normalization method: Fucose Fucp: Mannose Manp: Glucose Glcp: Galactose Galp = 1.00: 1.20: 3.54: 1.88.

[0068] 4. Methylation GC-MS analysis

[0069] The individual sugar components of the polysaccharide were derivatized into partially methylated polyacyl alcohol acetates (PMAAs), which were then analyzed and quantified using gas chromatography-mass spectrometry (GC-MS). The total ion chromatogram of LFP-I was analyzed by comparing it with the MASSBANK database ( Figure 8 ) and ion fragments, and it was found that LFP-I was mainly composed of seven sugar residues: T-Fucp, T-Glcp, T-Manp, 1,3-Manp, 1,6-Galp, 1,6-Glcp, and 1,2,6-Galp, with molar percentages of 15.24%, 8.33%, 4.62%, 10.50%, 8.77%, 40.74%, and 11.78%, respectively.

[0070] 5. Nuclear Magnetic Resonance Analysis

[0071] Weigh 60 mg of LFP-I, dissolve it in 1 mL of D2O, freeze dry it in a freeze dryer, and repeatedly dissolve it in D2O and freeze dry it 3 times. After repeated freeze-thaw cycles, fully dissolve the sample in 1 mL of D2O again, and carefully transfer the supernatant to a 5 mm NMR tube to ensure that the solution height in the sample tube is greater than 3.5 cm. Determine the 1H, 13C, HSQC, HMBC, and NOESY spectra of the polysaccharide.

[0072] 1H spectrum Fig. 9 , which shows 5 chemical signals (δ5.33, δ4.99, δ4.91, δ4.70, δ4.46ppm),

[0073] 1C spectrum Fig.10 , where 6 chemical signals were displayed (δ102.93ppm, δ102.20ppm, δ101.48ppm, δ99.72ppm, δ98.03ppm and δ97.87ppm).

[0074] In the HSQC spectrum of LFP-I ( Fig.11) Six signal cross peaks appeared in the sample. The positions are δ5.33 / 99.72, δ4.99 / 101.48, δ4.91 / 97.87, δ4.99 / 97.87, δ4.46 / 102.93 and δ1.16 / 15.66, which are respectively attributed to T-α-L-Fucp, T-α-D-Glcp, 1,3-α-D-Manp, 1,6-β-D-Glcp, 1,2,6-α-D-Galp, and δ1.16 / 15.66 is the characteristic signal of fucose H6 / C6. The chemical shifts obtained are shown in Table 1.

[0075] Table 1 NMR chemical shifts of LFP-I

[0076]

[0077] Combined with HMBC spectrum ( Fig.12 ) and NOESY spectra ( Fig.13 ), the δ4.99 / 66.54 signal appears in the HMBC diagram, indicating the presence of B 1-6 E connection or 6E 1-6 E1 repeat structure. Similarly, it can be inferred that there are also δ4.99 / 78.00, δ4.46 / 66.54, δ3.78 / 102.93, and δ3.55 / 97.87 signals in LFP-I, indicating the presence of B1 / E 1-2 E, D 1-6 E, D 6-1 D, C 3-1 The δ5.33 / 3.55 signal appears in the NOESY spectrum, indicating that H1 of residue A is coupled to H3 of residue C. 1-3 C connection mode. Similarly, based on the presence of NOESY, we can infer that there are also δ4.99 / 3.55, δ4.46 / 3.55, δ4.91 / 3.78, δ4.99 / 4.99, and δ3.78 / 4.99 signals in LFP-I, and we can infer that there is B1 / E in LFP-I. 1-3 C, D 1-3 C, C 1-6 D, B1 / E 1-1 E / 1B, D 6-1 B / 1E connection mode. Finally, the structural fragment of LFP-I is deduced as follows:

[0078]

[0079] 6. Scanning electron microscopy analysis

[0080] Use tweezers to pick up a small amount of LFP-I and carefully stick it to the sample stage covered with conductive glue. Use a vacuum sputtering device to coat the conductive layer, and then observe the surface morphology of the mushroom polysaccharide at different magnifications of 200 times at an accelerating voltage of 10kV. The images obtained are as follows Fig.14 As shown in Figure 2, LFP-I has a loose and porous network structure with tight connections and soft texture.

[0081] Example 4 Activity Identification of Pleurotus eryngii Polysaccharide LFP-I

[0082] 1. Identification of the immunoreactivity of LFP-I

[0083] Dilute the RAW264.7 cells in the logarithmic growth phase to a density of 5 × 10 5 The cells were cultured for 24 h in complete medium (DMEM basal medium + double antibody), then the original medium was discarded, different concentrations of LFP-I were added and cultured for 24 h. The morphological changes of RAW264.7 mouse macrophages were observed with an Eclipse Ti inverted microscope. Fig.15 As shown, the cell morphology after culturing in new complete medium for 24 h without adding LFP-I is as follows Fig.16 As shown in the figure, the comparison of the two groups of pictures shows that the cells grow pseudopodia under the stimulation of LFP-I and become activated, and the stimulation effect increases with the increase of concentration.

[0084] When macrophages are activated, the expression of tumor necrosis factor TNF-α also shows an increasing trend. The increase in TNF-α can in turn induce the proliferation of other immune cells such as T cells and B cells, and ultimately complete the benign promotion of the immune response. According to the instructions of Huamei Bio's mouse tumor necrosis factor TNF-α ELISA kit, complete the steps of adding samples, incubation, washing, and color development. Detect the release of TNF-α after adding LFP-I, and get the results as follows Fig.17 As shown in Figure 2, as the concentration of LFP-I increases, the amount of cytokine released increases, that is, macrophages are activated. It can be inferred that LFP-I can stimulate macrophages and enhance immune response.

[0085] 2. Identification of hypoglycemic activity of LFP-I

[0086] Add 20 μL of 1U / mL α-glucosidase solution, 80 μL of PBS solution and 10 μL of LFP-I solution of different concentrations to each well of the 96-well plate, react in a 37°C incubator for 30 min, then add 50 μL of pNPG solution and mix well, and continue to react in a 37°C incubator for 60 min. Take out the 96-well plate, add 100 μL of 1 mol / L Na2CO3 solution to terminate the hydrolysis reaction, and measure its A 405 .

[0087] Add 100 μL of 5 U / mL α-amylase solution and 50 μL of different concentrations of LFP-I solution to each well of a 96-well plate, react in a 37°C incubator for 10 min, then add 50 μL of 1% starch solution and mix evenly, continue to react in a 37°C incubator for 10 min, and finally add 100 μL of 10 mg / mL dinitrosalicylic acid solution to measure A 540 .

[0088] The inhibitory effects of different concentrations of LFP-I on α-glucosidase and α-amylase were calculated respectively. The background group was replaced with an equal amount of PBS solution instead of the α-glucosidase solution and α-amylase solution, and the blank group was replaced with an equal amount of PBS instead of the sample. The results are shown in Fig.18 and Fig.19 As shown in the figure, as the concentration of LFP-I increases, the inhibitory effect on the two enzymes is enhanced and reaches a high level of more than 70%, indicating that LFP-I can reduce enzyme activity and delay starch digestion, thereby effectively regulating blood sugar levels after starchy foods.

[0089] 3. Identification of the antioxidant activity of LFP-I

[0090] Antioxidants remove free radicals by giving away electrons through reduction. The stronger the reducing power, the stronger the antioxidant property. 3+ Can be reduced to Fe by antioxidants 2+ , and then form a blue tripyridine triazine complex with TPTZ to detect the total antioxidant capacity of LFP-I. Pipette 180 μL of TPTZ working solution into a 96-well plate, then add 20 μL of 1-5 mg / mL LFP-I solution, mix thoroughly, and react at room temperature in the dark for 10 minutes. Determine A 595 , and then calculate the FRAP value of each sample based on the standard curve. The results are as follows Fig. 20 As shown, as the concentration of LFP-I increases, the FRAP value increases, and the antioxidant capacity increases.

[0091] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A purified polysaccharide of Trichoderma tricholoma LFP-I, characterized in that: The purified polysaccharide LFP-I of Trichoderma tricholoma is a heteropolysaccharide composed of fucose Fucp: mannose Manp: glucose Glcp: galactose Galp=1.00:1.20:3.54:1.88, and the structural fragment is shown below: ； R1 / R2 represents that this position is connected to two groups, R1 and R2, respectively, where R1 is -α-D-Glcp; R2 is -α-Manp-(3→1)-α-D-Manp-(3→1)-α-L-Fucp.

2. The purified polysaccharide LFP-I from Trichoderma tricholoma according to claim 1, characterized in that: The weight average molecular weight of the purified Trichoderma lucidum polysaccharide LFP-I is 13288 Da.

3. A method for preparing the purified polysaccharide LFP-I from Trichoderma tricholoma according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) The fruiting bodies of Pleurotus eryngii were taken and ground into powder, and then the polysaccharide extract was obtained by hot water extraction. The polysaccharide extract was decolorized, deproteinized, and precipitated with alcohol to obtain the crude polysaccharide LFPs of Pleurotus eryngii. (2) eluting the LFPs obtained in step (1) through an ion exchange chromatography column and collecting the eluate; (3) The eluate is concentrated, dialyzed, and freeze-dried to obtain the purified polysaccharide LFP-I from Trichoderma tricholoma; In step (2), the ion exchange chromatography column is filled with DEAE-52; In step (2), the eluent is a NaCl solution with a concentration of 0-0.2 mol / L.

4. The preparation method according to claim 3, characterized in that: In step (1), the hot water extraction temperature is 60-100° C., the extraction time is 100-140 min; the ratio of the Maojian mushroom fruiting body powder to water is 1 g: 10-30 mL; In step (1), the macroporous resin D354FD is used for decolorization, and the macroporous resin and the polysaccharide extract are mixed in a volume ratio of 1:1, and the decolorization is carried out at 50±10°C with stirring for 3±1h; In step (1), sevage reagent is used for deproteinization, and the sevage reagent is a mixed solution of n-butanol and chloroform in a volume ratio of 1:

4.

5. The preparation method according to claim 4, characterized in that: In step (1), the hot water extraction temperature is 70°C, the extraction time is 120 min, and the ratio of the Maojian mushroom fruiting body powder to water is 1 g:25 mL; In step (1), the volume ratio of sevage reagent to polysaccharide extract is 1:

4.

6. The preparation method according to claim 5, characterized in that: In step (1), the alcohol precipitation is performed by mixing the polysaccharide extract with 95% ethanol in a volume ratio of 1:4, and collecting the precipitate; In step (3), a dialysis bag of 5000-8000Da is used for dialysis.

7. Use of the purified Trichoderma tricholoma polysaccharide LFP-I according to any one of claims 1 to 2 in the preparation of foods and / or health products for anti-oxidation, blood sugar regulation and immunity enhancement.

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