Auricularia polytricha polysaccharide as well as preparation method and application thereof

Abstract

The invention provides auricularia polytricha polysaccharide and a preparation method and application thereof, and relates to the technical field of polysaccharides, the auricularia polytricha polysaccharide is a polymer composed of rhamnose, arabinose, mannose, glucose and galactose, the molar ratio of rhamnose to arabinose to mannose to glucose to galactose is 5.30: 6.10: 9.35: 3.20: 4.70, and the relative molecular weight is 357926 Da. The auricularia polytricha polysaccharide disclosed by the invention is obtained by carrying out water extraction on auricularia polytricha powder, carrying out alcohol precipitation, carrying out dialysis on the auricularia polytricha powder by using a 300000Da-350000Da dialysis bag, and carrying out Sepacryl S-400 chromatographic column elution. Research finds that the auricularia polytricha polysaccharide has a remarkable AGEs inhibition effect and can inhibit growth of tumor cells; the generation of AGEs in the production process of baked food, especially cookies, can be effectively reduced; cookies prepared from the auricularia polytricha polysaccharide are low in AGEs content and better in quality; the auricularia polytricha polysaccharide is obtained from auricularia polytricha through extraction, separation and purification, is a natural extract and has good safety.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and more particularly, to a polysaccharide from Auricularia polytricha var. mollis, a preparation method thereof, and an application thereof. Background Art

[0002] Advanced glycation end products (AGEs) are mainly generated through the Maillard reaction, lipid oxidation reaction, or polyol degradation pathway. Research has shown that AGEs are closely related to the pathogenesis of various chronic diseases such as diabetes, atherosclerosis, and renal failure, and their large accumulation in the body has a potential harmful effect on human health.

[0003] During food processing, the Maillard reaction produces fluorescent AGEs. In particular, some common baked foods such as bread, cakes, puff pastries, choux pastries, and cookies such as cookies are high in sugar and fat, and are prone to various reactions such as the Maillard reaction, sugar degradation, and lipid oxidation during baking. The Maillard reaction occurs between sugars and amino acids in proteins, generating reactive carbonyl compounds such as glyoxal, methylglyoxal, and 3-deoxyglucosone. These carbonyl compounds have high reactivity, are prone to protein cross-linking and glycosylation, and are likely to generate advanced glycation end products AGEs, which are harmful to the human body. Therefore, how to inhibit the formation of AGEs during the production of cookies is particularly important.

[0004] The Chinese invention patent with the publication number CN 109806276 A discloses the application of lentinan in the preparation of drugs for preventing diabetic complications, and discloses a natural extract of lentinan with AGEs inhibitory effect to reduce the dependence caused by synthetic AGEs inhibitors such as aminoguanidine. Therefore, it is necessary to develop more natural polysaccharides to enrich the natural polysaccharides that inhibit AGEs.

[0005] Auricularia polytricha var. mollis belongs to the Basidiomycetes class of the Fungi kingdom, the Auriculariales order, the Auriculariaceae family, and the Auricularia genus. It is a newly introduced ear mushroom variety in recent years and is distributed throughout the country. It is rich in nutrition, has a crispy and smooth texture, and contains abundant polysaccharides. According to research, the polysaccharides from Auricularia polytricha var. mollis (APP) have effects such as improving immunity, anti-tumor, anticoagulation, antithrombosis, and lipid-lowering. Currently, the research on the polysaccharides from Auricularia polytricha var. mollis mainly focuses on extraction and biological activities. So far, no application of the polysaccharides from Auricularia polytricha var. mollis in inhibiting AGEs during the production of baked foods has been found. Summary of the Invention

[0006] The object of the present invention is to solve the above technical problems and provide a polysaccharide from Auricularia polytricha var. pallida, which has a significant inhibitory effect on AGEs; another object of the present invention is to provide a preparation method of the polysaccharide from Auricularia polytricha var. pallida; still another object of the present invention is to provide its application in inhibiting AGEs or as an AGEs inhibitor, especially its application in preparing baked cookies.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A polysaccharide from Auricularia polytricha var. pallida, which is a polymer composed of rhamnose, arabinose, mannose, glucose, and galactose. The molar ratio of rhamnose, arabinose, mannose, glucose, and galactose is 5.30:6.10:9.35:3.20:4.70, and the relative molecular weight is 357926 Da.

[0009] The present invention discovers a new polysaccharide from Auricularia polytricha var. pallida, which has a significant inhibitory effect on AGEs.

[0010] The present invention provides a preparation method of the polysaccharide from Auricularia polytricha var. pallida, including the following steps:

[0011] S1. Mix Auricularia polytricha var. pallida powder with water to dissolve the polysaccharide in Auricularia polytricha var. pallida in water, and then add ethanol to precipitate the polysaccharide, obtaining the crude polysaccharide from Auricularia polytricha var. pallida.

[0012] S2. Dissolve the crude polysaccharide from Auricularia polytricha var. pallida in water to form a mixed solution of water and the crude polysaccharide from Auricularia polytricha var. pallida. Use a dialysis bag with a molecular weight cut-off of 300000 Da to 350000 Da to dialyze the mixed solution of water and the crude polysaccharide from Auricularia polytricha var. pallida. The dialysis solution is eluted through a Sepacryl S-400 chromatographic column to obtain the polysaccharide from Auricularia polytricha var. pallida.

[0013] Further, the preparation method of the crude polysaccharide from Auricularia polytricha var. pallida in step S1 is as follows: Mix Auricularia polytricha var. pallida powder with water at a solid-liquid ratio of 1:10 to 20 g / mL, and extract at 60 to 80 °C for 6 to 10 h to dissolve the polysaccharide in Auricularia polytricha var. pallida in water. Centrifuge at 4000 to 6000 r / min for 10 to 30 min, take the supernatant, and then add 2 to 5 volumes of 90 to 100% ethanol to precipitate the polysaccharide, obtaining the crude polysaccharide from Auricularia polytricha var. pallida.

[0014] Further, before dialyzing the mixed solution of water and the crude polysaccharide from Auricularia polytricha var. pallida in step S2, the protein contained therein needs to be removed. The method for removing protein is to add 1 / 6 to 1 / 2 volume of Sevage solution, mix and shake for 20 to 40 min, then let it stand for 10 to 30 min and collect the supernatant, and use nitrogen to rinse to remove the n-butanol in the Sevage solution.

[0015] Preferably, the Sevage solution is prepared from n-butanol and chloroform in a volume ratio of 1:4 to 1:5.

[0016] Further, in step S2, dialysis is performed using distilled water for 36 to 96 hours.

[0017] Further, the polysaccharide of Auricularia polytricha var. pallida is the polysaccharide eluted first.

[0018] The present invention claims the application of the above-mentioned polysaccharide of Auricularia polytricha var. pallida or the polysaccharide of Auricularia polytricha var. pallida prepared by the above method in the preparation of anti-tumor drugs.

[0019] The present invention claims the application of the above-mentioned polysaccharide of Auricularia polytricha var. pallida or the polysaccharide of Auricularia polytricha var. pallida prepared by the above method as an additive for inhibiting AGEs.

[0020] Further, the application is to add the above-mentioned polysaccharide of Auricularia polytricha var. pallida or the polysaccharide of Auricularia polytricha var. pallida prepared by the above method to the raw materials of baked foods, and then perform baking.

[0021] Further, the baked food is a cookie.

[0022] The present invention provides a cookie, which is prepared by mixing and baking the following raw materials in parts by weight, and the raw materials are:

[0023] 80 - 120 parts of cake flour;

[0024] 30 - 50 parts of butter;

[0025] 20 - 45 parts of icing sugar;

[0026] 8 - 20 parts of polysaccharide of Auricularia polytricha var. pallida;

[0027] 9 - 18 parts of skim milk powder;

[0028] 30 - 55 parts of egg liquid.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The present invention provides a polysaccharide of Auricularia polytricha var. pallida, which has a significant inhibitory effect on AGEs and tumor cells; it can effectively reduce the generation of advanced glycation end products during the production of baked foods and has anti-tumor applications.

[0031] (2) The polysaccharide of Auricularia polytricha var. pallida obtained by extraction, separation, and purification from Auricularia polytricha var. pallida in the present invention is a natural extract with good safety.

[0032] (3) The polysaccharide from Auricularia polytricha var. pallida of the present invention can be applied to the preparation of baked foods. The cookies prepared using the polysaccharide from Auricularia polytricha var. pallida of the present invention have a low AGEs content and better quality. Description of the Drawings

[0033] Figure 1 Elution curve of Sepacryl S-400 chromatographic column for the purification of polysaccharide from Auricularia polytricha var. pallida.

[0034] Figure 2 High performance liquid chromatography (HPLC) chromatogram of the monosaccharide composition of polysaccharide APP-a from Auricularia polytricha var. pallida.

[0035] Figure 3 Molecular weight distribution map of polysaccharide APP-a from Auricularia polytricha var. pallida.

[0036] Figure 4 Molecular weight distribution map of polysaccharide APP-b from Auricularia polytricha var. pallida.

[0037] Figure 5 Infrared spectrum of polysaccharide APP-a from Auricularia polytricha var. pallida.

[0038] Figure 6 Infrared spectrum of polysaccharide APP-b from Auricularia polytricha var. pallida. Detailed Embodiments

[0039] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. [[ID=tmp]] [[ID=tmp]]

[0040] The raw materials, instruments and statistical data in the embodiments of the present invention are described as follows:

[0041] In the embodiments of the present invention, Auricularia polytricha var. pallida is commercially available, and the reagents used: glucose, phenol, concentrated sulfuric acid, ethanol, etc. are all of analytical grade.

[0042] The instruments and equipment used in the present invention: HH-4 digital display constant temperature water bath; RE-52A rotary evaporator; DJ-04 crusher; LD4-ZA centrifuge; 752 ultraviolet-visible spectrophotometer; Nicolet iS20 Fourier transform infrared spectrometer; ALPHA 1-4 / 2-4LSC freeze dryer; GC-14A gas chromatograph.

[0043] Data description: All experiments were performed in triplicate, and the data were expressed as mean ± SD. The statistical analysis of the data was performed using t-test or ANOVA analysis. A p value < 0.05 was considered to indicate a statistically significant difference. Additionally, in the following examples, unless otherwise specified, all reagents, raw materials, and instruments used in the present invention are well-known and selected in the art, but this does not limit the implementation of the present invention. Some other well-known reagents and equipment in the art can also be applied to the implementation of the following embodiments of the present invention.

[0044] Example 1: Extraction of Crude Polysaccharide from Auricularia polytricha (Mont.) Sacc. var. pubescens

[0045] Auricularia polytricha (Mont.) Sacc. var. pubescens was cleaned and dried at 60 °C for 12 h, then pulverized. Deionized water was added according to a solid-liquid ratio of 1:15 (g / mL), and extraction was carried out at 70 °C for 8 h. Then, centrifugation was performed (5000 r / min, 20 min) to obtain the supernatant. Four volumes of 95% ethanol were added to the supernatant, and precipitation was carried out at 4 °C for 24 h. Centrifugation (7000 r / min, 10 min) was performed to obtain the precipitate, and the precipitate was freeze-dried to obtain the crude polysaccharide APP of Auricularia polytricha (Mont.) Sacc. var. pubescens.

[0046] Example 2: Isolation and Purification of Polysaccharide from Auricularia polytricha (Mont.) Sacc. var. pubescens

[0047] 1 g of the crude polysaccharide APP of Auricularia polytricha (Mont.) Sacc. var. pubescens was dissolved in 500 mL of ultrapure water, and then 1 / 4 volume of Sevage solution (n-butanol and chloroform (1:4, v / v)) was added. The mixture was shaken for 30 min and then allowed to stand for 20 min, after which the supernatant was collected and the n-butanol was removed by flushing with nitrogen. The supernatant from which n-butanol had been removed was placed in a dialysis bag (molecular weight cut-off 250000 Da) and dialyzed against distilled water for 72 h. The dialysate was loaded onto a Sepacryl S-400 chromatographic column (1.6 cm × 50 cm), and distilled water was used as the eluent. The eluent was collected using an automatic fraction collector at a flow rate of 0.2 mL / min and 2 mL per tube. Detection of OD was performed using the phenol-sulfuric acid method 490 , and an elution curve was plotted. According to the elution curve, two polysaccharide fractions APP-a and APP-b were collected. The eluate of the APP-a fraction was freeze-dried to obtain the polysaccharide APP-a of Auricularia polytricha (Mont.) Sacc. var. pubescens (Appendix Figure 1);Meanwhile, this study found that polysaccharide APP-a of Auricularia polytricha could not be obtained through other chromatographic columns such as Sephadex G200 (1.5 cm × 80 cm), Sephadex G100 (2.0 cm × 50 cm), DEAE Sepharose FastFlow (1.5 cm × 80 cm), DEAE sepharose CL6B (2.0 cm × 50 cm), etc. The main reason is that the column packings (ion exchange resins) of different chromatographic columns are different, and thus the separation principles are different. Ion exchange chromatography column is a separation technique based on the charge difference between ions and the interaction with the stationary phase (ion exchange resin). It realizes the selective separation of different ions through the reversible exchange between the ion exchange resin and the ions in the sample, such as DEAE Sepharose Fast Flow (1.5 cm × 80 cm) and DEAE sepharose CL6B (2.0 cm × 50 cm) ion exchange column chromatography. Gel filtration chromatography is a technique that uses the molecular sieve effect of particles with porous network structures to elute and separate according to the differences in the relative molecular weights of the components in the sample to be separated, such as Sephadex G200 (1.5 cm × 80 cm) and Sephadex G100 (2.0 cm × 50 cm) gel filtration chromatography columns. Therefore, the separation of crude polysaccharide APP of Auricularia polytricha by different chromatographic columns will result in the separation of different polysaccharide components. In this example, it was accidentally found through research that the target polysaccharide can be obtained through the Sepacryl S-400 chromatographic column.

[0048] Example 3: Structural characteristics of polysaccharides APP-a and APP-b of Auricularia polytricha

[0049] Determination of monosaccharide composition: Take 10 mg of the Auricularia polytricha polysaccharide APP-a sample in a stoppered tube, add 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution, vacuum seal the tube, and hydrolyze at 121 °C for 1 h. After removing the excess TFA from the hydrolysis solution, dry it under vacuum. Using the sugar nitrile acetate derivatization method, add 10 mg of hydroxylamine hydrochloride, an appropriate amount of inositol (internal standard), and 0.5 mL of pyridine, heat at 90 °C for 30 min, then take it out and cool to room temperature, add 0.5 mL of acetic anhydride, and continue to react at 90 °C for 30 min for acetylation. The reaction product is directly analyzed by gas chromatography (GC). The types of monosaccharides are qualitatively judged according to the retention time of the monosaccharides, and the proportional relationship between the monosaccharides is determined according to the ratio of the peak areas.

[0050] Chromatographic conditions: An OV1701 elastic quartz capillary column (Φ0.32 mm × 30 mm) was used. The carrier gas was N2 with a flow rate of 1.5 mL / min. An FID hydrogen flame detector was used. The vaporization chamber temperature was 260 °C, and the detector temperature was 250 °C. Programmed temperature elevation was adopted: the initial temperature was 150 °C, held for 1 min, heated to 190 °C at a rate of 10 °C / min, held for 1 min, then heated to 240 °C at a rate of 3 °C / min and held for 20 min.

[0051] Determination of relative molecular mass: Standard Dextrans with relative molecular masses of 6100, 26290, 84000, 158000, and 291000 were successively injected. HPLC was used to record the retention time TR. A standard curve was plotted with TR as the abscissa and LgM as the ordinate to obtain the regression equation. 10 μL of the test samples APP-a and APP-b were injected. According to the obtained TR, the relative molecular mass of the polysaccharide was calculated through the regression equation. Chromatographic column: Waters UllrallydrogelTM Linear (Φ7.8 mm × 300 mm), detector: Waters2410 differential refractive index detector, with 0.1 mol / L NaNO3 as the mobile phase, flow rate 0.9 mL / min, and column temperature 45 °C.

[0052] Infrared spectrum determination: 2 mg of polysaccharides APP-a and APP-b from Auricularia polytricha (Mont.) Sacc. var. hirsutula (Lev.) Imai and 200 mg of KBr were respectively taken, mixed, ground, and pressed into tablets. A Nicolet iS20 Fourier transform infrared spectrometer from Thermo Scientific Company was used for determination, with a scanning range of 4000 - 400 cm -1 , and the resolution was 4 cm -1 .

[0053] As Figure 2 shown, the monosaccharide composition of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hirsutula (Lev.) Imai was rhamnose, arabinose, mannose, glucose, and galactose, with a molar ratio of 5.30:6.10:9.35:3.20:4.70.

[0054] As Figure 3 shown, the relative molecular mass of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hirsutula (Lev.) Imai was 357926 Da. As Figure 4 shown, the relative molecular mass of polysaccharide APP-b from Auricularia polytricha (Mont.) Sacc. var. hirsutula (Lev.) Imai was 254086 Da.

[0055] As Figure 5 shown, from the infrared spectrum of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hirsutula (Lev.) Imai, a broad peak could be observed at 3393 cm -1 This was caused by the stretching vibration of -OH; the weak peak at 2938 cm -1 was a typical absorption peak characteristic of -CH, and the peak at 1625 cm -1The absorption peak at [specific position] is caused by C-O vibration and has the characteristic absorption peak of polysaccharide.

[0056] As Figure 6 shown, from the infrared spectrum of polysaccharide APP-b from Auricularia polytricha (Mont.) Sacc. var. hypoleuca Moller, it can be observed that there is a broad peak near 3420 cm -1 , which is caused by the stretching vibration of -OH; the peaks near 2930 cm -1 are respectively attributed to the asymmetric stretching vibration of -CH2-; the strong peak near 1620 cm -1 is the absorption peak characteristic of typical carbonyl, and the absorption peak near 1099 cm -1 is attributed to the stretching vibration of pyran ring. The infrared analysis results show that polysaccharide APP-b from Auricularia polytricha (Mont.) Sacc. var. hypoleuca Moller belongs to pyranose-type polysaccharide.

[0057] Example 4: Preparation of Cookies

[0058] (1) Mix raw materials and form cookie dough: Weigh 100 parts of low-gluten flour, 35 parts of butter, 25 parts of powdered sugar, 10 parts of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hypoleuca Moller, 11 parts of non-fat milk powder, and 35 parts of egg liquid, add them to a blender and mix well by stirring, and then use a mold to form the dough into cookie dough.

[0059] (2) Baking: Bake the cookie dough at an upper fire temperature of 170 °C and a lower fire temperature of 110 °C for 20 min, and then cool it at room temperature to obtain the cookie product.

[0060] Example 5: Preparation of Cookies

[0061] (1) Mix raw materials and form cookie dough: Weigh 100 parts of low-gluten flour, 40 parts of butter, 30 parts of powdered sugar, 12 parts of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hypoleuca Moller, 13 parts of non-fat milk powder, and 40 parts of egg liquid, add them to a blender and mix well by stirring, and then use a mold to form the dough into cookie dough.

[0062] (2) Baking: Bake the cookie dough at an upper fire temperature of 170 °C and a lower fire temperature of 110 °C for 20 min, and then cool it at room temperature to obtain the cookie product.

[0063] Example 6: Preparation of Cookies

[0064] (1) Mix raw materials and form cookie dough: Weigh 100 parts of low-gluten flour, 45 parts of butter, 35 parts of powdered sugar, 15 parts of polysaccharide APP-a from Auricularia polytricha (Mont.) Sacc. var. hypoleuca Moller, 16 parts of non-fat milk powder, and 40 parts of egg liquid, add them to a blender and mix well by stirring, and then use a mold to form the dough into cookie dough.

[0065] (2) Baking: Bake the cookie dough at an upper fire temperature of 170 °C and a lower fire temperature of 110 °C for 20 min, and then cool it at room temperature to obtain the cookie product.

[0066] Preparation of Cookies in Example 7

[0067] (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of cake flour, 35 parts of butter, 25 parts of powdered sugar, 25 parts of polysaccharide APP-a from Auricularia polytricha with white back, 11 parts of skim milk powder, and 35 parts of egg liquid (compared with Example 4, increasing the mass of polysaccharide APP-a from Auricularia polytricha with white back), add them to a blender and mix and beat evenly, and use a mold to form the dough into cookie dough.

[0068] (2) Baking: Bake the cookie dough at an upper fire temperature of 170 °C and a lower fire temperature of 110 °C for 20 minutes, and cool it at room temperature to obtain the cookie product.

[0069] Preparation of Cookies in Example 8

[0070] (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of cake flour, 35 parts of butter, 25 parts of powdered sugar, 5 parts of polysaccharide APP-a from Auricularia polytricha with white back, 11 parts of skim milk powder, and 35 parts of egg liquid (compared with Example 4, reducing the mass of polysaccharide APP-a from Auricularia polytricha with white back), add them to a blender and mix and beat evenly, and use a mold to form the dough into cookie dough.

[0071] (2) Baking: Bake the cookie dough at an upper fire temperature of 170 °C and a lower fire temperature of 110 °C for 20 minutes, and cool it at room temperature to obtain the cookie product.

[0072] Preparation of Cookies in Example 9 (equal amount of polysaccharide APP-b from Auricularia polytricha with white back)

[0073] The difference between Example 9 and Example 4 is that: an equal amount of polysaccharide APP-b from Auricularia polytricha with white back is added to the raw materials of the cookie dough, and the others are the same as in Example 4.

[0074] Preparation of Cookies in Example 10 (equal amount of polysaccharide APP-b from Auricularia polytricha with white back)

[0075] The difference between Example 10 and Example 5 is that: an equal amount of polysaccharide APP-b from Auricularia polytricha with white back is added to the raw materials of the cookie dough, and the others are the same as in Example 5.

[0076] Comparative Example 1: Preparation of Cookies

[0077] The difference between Comparative Example 1 and Example 4 is that: polysaccharide APP-a from Auricularia polytricha with white back is not added to the raw materials of the cookie dough, and the others are the same as in Example 4.

[0078] Comparative Example 2: Preparation of Cookies

[0079] The difference between Comparative Example 1 and Example 5 is that: polysaccharide APP-a from Auricularia polytricha with white back is not added to the raw materials of the cookie dough, and the others are the same as in Example 5.

[0080] Comparative Example 3: Preparation of Cookies

[0081] The difference between Comparative Example 1 and Example 6 is that white back Auricularia auricula polysaccharide APP-a is not added to the raw materials of the cookie dough, and the others are the same as in Example 6.

[0082] Test Example 1

[0083] 1. Experimental method

[0084] 1.1 Cell culture

[0085] Add calf serum with a volume fraction of 10% to RPMI1640 medium, and routinely culture Jurkat (human T lymphocyte leukemia cell line) tumor cells in a CO2 incubator at 37°C with a volume fraction of 5%. Passage the cells every other day, adjust the cell growth state, take cells in the logarithmic growth phase with good growth, and then adjust the cell density to 1×10 8 / mL for the experiment.

[0086] 1.2 Inhibitory effects of APP-a and APP-b on tumor cells

[0087] Take Jurkat cells in the logarithmic growth phase, 100 μL per well, and inoculate them into a 96-well plastic culture plate. After static culture for 24 h, do not add APP-a and APP-b to the control group, and add APP-a and APP-b to each well in the sample group respectively, so that their final concentrations are 5, 10, and 15 μg / mL respectively. Set 3 parallel wells for each concentration, then transfer the culture plate into a CO2 incubator and culture it at 37°C, 5% CO2 and saturated humidity for 48 h. Then add 20 μL of MTT reagent to each well, and its final concentration is 400 mg / L. After continuing to incubate at 37°C for 5 h, terminate the culture. After sucking and discarding the supernatant, add 200 μL of DMSO to each well, gently shake for 15 min, and measure the absorbance (OD) values of each well in the control group and the sample group at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Take the average value of 3 wells in each group as the average OD value of each group, and calculate the inhibition rate of tumor cell Jurka according to the following formula.

[0088] Cell inhibition rate (%) = (1 - OD of sample group / OD of control group) × 100

[0089] 2. Experimental results

[0090] The results of the growth inhibitory effects of APP-a and APP-b at different dose concentrations on Jurkat cells are shown in Table 1. It can be seen from Table 1 that APP-a has an obvious inhibitory effect on the proliferation of tumor cells Jurkat, and the inhibition rates increase from 26.64% to 62.61% respectively. With the increase of the polysaccharide concentration, the effect shows an increasing trend, with a dose-effect dependence, and there is a significant difference compared with the negative control group (p<0.05). Compared with APP-b, APP-a has a stronger proliferative effect on tumor cells Jurkat, with a significant difference (p<0.05), and the difference is statistically significant.

[0091] Table 1 Effects of APP-a and APP-b on the proliferation of Jurkat cells (x±s, n = 3)

[0092]

[0093] Note: Compared with the negative control group, different letters represent significant differences (p<0.05)

[0094] Test Example 2

[0095] 1. Test method

[0096] (1) Determination of fluorescent AGEs in cookies: Weigh 5 g of the crushed cookie sample, add 250 mL of phosphate buffer (50 mM, pH 7.4), stir at 37 °C for 2 h, then centrifuge at 10000 r / min for 5 min, filter, collect the filtrate, and measure its fluorescence value using a fluorescence spectrophotometer at an excitation wavelength of 350 nm and an emission wavelength of 440 nm. The fluorescence intensity is expressed as AU / mg, representing the content of fluorescent AGEs in the sample, that is, the content of fluorescent AGEs per milligram of cookies. Conduct parallel tests three times.

[0097] (2) Sensory evaluation of cookies

[0098] The panel consists of 10 panelists who are willing to objectively evaluate the product, regardless of gender and without a history of allergies. Place the cookie on a white porcelain plate and observe its shape, color, and tissue state under natural light, and taste its flavor and texture. The cookie samples are randomly arranged. Between the evaluations of every two groups of cookies, it is required that the panelists clean their mouths with pure water. Make a sensory evaluation form for the specific sensory requirements of the cookies, as shown in Table 2.

[0099] Table 2 Sensory evaluation criteria

[0100]

[0101]

[0102] 2. Test results

[0103] The determination results of fluorescent AGEs in cookies are shown in Table 3. It can be seen from Table 3 that by using the method of the present invention, the polysaccharide APP-a of Auricularia polytricha Mont. Henn. was prepared by separation and purification, and the polysaccharide APP-a of Auricularia polytricha Mont. Henn. was added to the raw materials of cookie dough to produce cookies. Compared with Comparative Examples 1, 2, and 3, Examples 4, 5, and 6 can significantly reduce the content of advanced glycation end products in cookies (p < 0.05), and the cookies have better shape, color, taste, texture, and structure (p < 0.05), indicating that APP-a can significantly reduce the content of advanced glycation end products in cookies. However, adding too much APP-a (Example 7) and too little APP-a (Example 8) in cookies can both reduce the content of advanced glycation end products in cookies to a certain extent (p < 0.05), but the sensory scores of the cookies are significantly lower than those of Examples 4, 5, and 6 (p < 0.05), indicating that adding too much or too little APP-a during the production of cookies will make the shape, color, taste, texture, and structure of the cookies worse. After adding APP-b (Examples 9 and 10) to the cookies, the content of advanced glycation end products in the cookies did not decrease, and the sensory scores of the cookies were significantly lower than those of Examples 4, 5, and 6 (p < 0.05), indicating that APP-b cannot reduce the content of advanced glycation end products in cookies and will make the shape, color, taste, texture, and structure of the cookies worse, having a negative impact.

[0104] Table 3 Content and sensory scores of fluorescent AGEs in cookies of Comparative Examples 1-3 and Examples 4-10

[0105]

[0106]

[0107] Note: Different letters indicate significant statistical differences between different groups of items (p < 0.05).

[0108] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can modify or equivalently replace the technical solutions of the present invention on the basis of understanding the solutions, without departing from the purpose and scope of the technical solutions of the present invention, and all of them should be covered by the scope of the claims of the present invention.

Claims

1. A polysaccharide from Auricularia polytricha (Mont.) Sacc. var. pubescens, characterized in that, The polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca is a polymer composed of rhamnose, arabinose, mannose, glucose, and galactose. The molar ratio of rhamnose, arabinose, mannose, glucose, and galactose is 5.30:6.10:9.35:3.20:4.70, and the relative molecular weight is 357926 Da.

2. A preparation method of polysaccharide from Auricularia polytricha (Mont.) Sacc. var. pallida (Fr.) Henn., characterized in that, It includes the following steps: S1. Mix Auricularia polytricha (Mont.) Sacc. var. hypoleuca powder with water to dissolve the polysaccharide in Auricularia polytricha (Mont.) Sacc. var. hypoleuca in water, and then add ethanol to precipitate the polysaccharide, obtaining crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca. S2. Dissolve the crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca in water to form a mixed solution of water and crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca. Use a dialysis bag with a molecular weight cut-off of 300000 Da - 350000 Da to dialyze the mixed solution of water and crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca. The dialysate is eluted through a Sepacryl S-400 chromatographic column to obtain the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca.

3. The preparation method of the Auricularia polytricha polysaccharide according to claim 2, characterized in that, The preparation method of the crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca described in step S1 is: Mix Auricularia polytricha (Mont.) Sacc. var. hypoleuca powder with water at a solid-liquid ratio of 1:10 - 20 g / mL, and extract at 60 - 80 °C for 6 - 10 h to dissolve the polysaccharide in Auricularia polytricha (Mont.) Sacc. var. hypoleuca in water. Centrifuge at 4000 - 6000 r / min for 10 - 30 min, take the supernatant, and then add 2 - 5 volumes of 90 - 100% ethanol to precipitate the polysaccharide, obtaining the crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca.

4. The preparation method of the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. pubescens (Schulz.) Henn. according to claim 2, characterized in that, Before dialyzing the mixed solution of water and crude polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca described in step S2, the protein contained therein needs to be removed. The method for removing protein is to add 1 / 6 - 1 / 2 volume of Sevage solution, mix and shake for 20 - 40 min, then let it stand for 10 - 30 min and collect the supernatant, and use nitrogen to rinse to remove the n-butanol in the Sevage solution.

5. The preparation method of the Auricularia polytricha polysaccharide according to claim 2, characterized in that, The dialysis described in step S2 is to use distilled water for dialysis for 36 - 96 h.

6. Use of the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca described in claim 1 or the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca prepared by the method described in any one of claims 2 - 6 in the preparation of anti-tumor drugs.

7. Use of the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca described in claim 1 or the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca prepared by the method described in any one of claims 2 - 6 as an additive for inhibiting AGEs.

8. The application according to claim 7, wherein Add the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca described in claim 1 or the polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca prepared by the method described in any one of claims 2 - 6 to the raw materials of baked foods, and then bake.

9. The application according to claim 8, wherein The baked food is cookies.

10. A kind of cookie, characterized in that, The cookies are prepared by mixing and baking the following raw materials in parts by weight: 80 - 120 parts of low-gluten flour; 30 - 50 parts of butter; 20 - 45 parts of powdered sugar; 8 - 20 parts of polysaccharide of Auricularia polytricha (Mont.) Sacc. var. hypoleuca; 9 - 18 parts of non-fat milk powder; 30 - 55 parts of egg liquid.

Images