A white-backed auricularia polysaccharide, a preparation method and application thereof

Abstract

The application provides white-backed wooly ear mushroom polysaccharide and a preparation method and application thereof, relates to the technical field of polysaccharide, and the white-backed wooly ear mushroom polysaccharide is a polymer composed of rhamnose, arabinose, mannose, glucose and galactose, the molar ratio of the 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. The white-backed wooly ear mushroom polysaccharide is obtained by water extraction, alcohol precipitation and 300000 Da-350000 Da dialysis bag dialysis of white-backed wooly ear mushroom powder and Sepacryl S-400 chromatographic column elution. Research finds that the white-backed wooly ear mushroom polysaccharide has a significant AGEs inhibiting effect and can inhibit tumor cell growth; can effectively reduce the generation of AGEs in the production process of baked foods, especially cookies; the cookies prepared by using the white-backed wooly ear mushroom polysaccharide have low AGEs content and better quality; the white-backed wooly ear mushroom polysaccharide is obtained by extraction, separation and purification from white-backed wooly ear mushroom, is a natural extract, and has good safety.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide technology, and more specifically, to a polysaccharide from white-backed hairy fungus, its preparation method, and its application. Background Technology

[0002] Advanced glycation end products (AGEs) are mainly generated through Maillard reactions, lipid oxidation, or polyol degradation. Studies have 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 poses potential health risks.

[0003] During food processing, the Maillard reaction produces fluorescent advanced glycation end products (AGEs), especially in common baked goods such as bread, cakes, shortbread and puff pastry, cream puffs, and cookies, which are high in sugar and fat. These foods are prone to Maillard reactions, sugar degradation, and fat oxidation during baking. The Maillard reaction in carbohydrates and amino acids in proteins produces reactive carbonyl compounds such as glyoxal, acetone aldehyde, and 3-deoxyglucone. These carbonyl compounds are highly reactive and easily induce protein cross-linking and glycosylation, readily generating advanced glycation end products (AGEs) that are harmful to human health. Therefore, inhibiting the formation of AGEs during cookie production is particularly important.

[0004] Chinese invention patent CN 109806276 A discloses the application of lentinan in the preparation of drugs for preventing diabetic complications. It discloses a naturally extracted lentinan with AGEs-inhibiting effects to reduce dependence caused by synthetic AGEs inhibitors such as aminoguanidine. Therefore, it is necessary to develop more natural polysaccharides to enrich the range of natural polysaccharides that inhibit AGEs.

[0005] White-backed hairy wood ear (Auricularia polytricha), belonging to the class Basidiomycetes of the phylum Basidiomycota, order Tremellae, family Auricularialae, and genus Auricularia, is a newly introduced wood ear variety distributed throughout China in recent years. It is rich in nutrients, has a crisp and smooth texture, and contains abundant polysaccharides. Studies have shown that polysaccharides from Auricularia polytricha (APP) have effects such as enhancing immunity, anti-tumor activity, anticoagulation, antithrombosis, and lowering blood lipids. Current research on APP polysaccharides mainly focuses on extraction and bioactivity. To date, no application of APP polysaccharides in inhibiting AGEs (Advanced Glycation End Products) during the production of baked goods has been found. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned technical problems and provide a polysaccharide from Auricularia auricula-judae, which has a significant inhibitory effect on AGEs. Another purpose of this invention is to provide a method for preparing the polysaccharide from Auricularia auricula-judae. A further purpose of this invention is to provide its application in inhibiting AGEs or as an AGEs inhibitor, particularly in the preparation of baked goods such as cookies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A polysaccharide from Auricularia auricula-judae, wherein the polysaccharide is a polymer composed of rhamnose, arabinose, mannose, glucose, and galactose, wherein 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] This invention has discovered a novel polysaccharide from Auricularia auricula-judae, which exhibits significant inhibitory effects on AGEs.

[0010] This invention provides a method for preparing polysaccharides from white-backed hairy fungus, comprising the following steps:

[0011] S1. Mix white-backed hairy fungus powder with water to dissolve the polysaccharides in the white-backed hairy fungus in the water, and then add ethanol to precipitate the polysaccharides to obtain crude polysaccharides from white-backed hairy fungus.

[0012] S2. Dissolve the crude polysaccharide of Auricularia auricula-judae in water to form a mixture of water and crude polysaccharide of Auricularia auricula-judae. Dialyze the mixture of water and crude polysaccharide of Auricularia auricula-judae using a dialysis bag with a molecular weight cutoff of 300,000 Da to 350,000 Da. Elute the dialysate with a Sepacryl S-400 chromatographic column to obtain polysaccharide of Auricularia auricula-judae.

[0013] Further, the preparation method of the crude polysaccharide of white-backed hairy fungus in step S1 is as follows: mix white-backed hairy fungus powder with water at a material-to-liquid ratio of 1:10-20 g / mL, and extract at 60-80℃ for 6-10 h to dissolve the polysaccharide in the white-backed hairy fungus in the water. Centrifuge at 4000-6000 r / min for 10-30 min, take the supernatant, and then add 2-5 times the volume of 90-100% ethanol to precipitate the polysaccharide and obtain the crude polysaccharide of white-backed hairy fungus.

[0014] Furthermore, before dialysis of the mixture of water and crude polysaccharide from white-backed wood ear fungus described in step S2, the proteins contained therein need to be removed. The method for removing proteins is to add 1 / 6 to 1 / 2 volume of Sevage solution, mix and shake for 20-40 minutes, then let stand for 10-30 minutes and collect the supernatant. The supernatant is then rinsed with nitrogen to remove n-butanol from the Sevage solution.

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

[0016] Furthermore, the dialysis described in step S2 uses distilled water for dialysis for 36–96 hours.

[0017] Furthermore, the polysaccharide from the white-backed wood ear fungus is a polysaccharide that has been pre-eluted.

[0018] This invention seeks to protect the use of the above-mentioned white-backed hairy fungus polysaccharide or the white-backed hairy fungus polysaccharide prepared by the above method in the preparation of antitumor drugs.

[0019] This invention seeks to protect the use of the above-mentioned white-backed hairy fungus polysaccharide or the white-backed hairy fungus polysaccharide prepared by the above method as an additive to inhibit AGEs.

[0020] Furthermore, the application involves adding the above-mentioned white-backed wood ear polysaccharide or the white-backed wood ear polysaccharide prepared by the above method to the raw materials of baked goods, and then baking them.

[0021] Furthermore, the baked goods are cookies.

[0022] This invention provides a cookie, which is prepared by mixing and baking the following ingredients in parts by weight:

[0023] 80-120 portions of low-gluten flour;

[0024] 30-50 parts butter;

[0025] 20-45 parts powdered sugar;

[0026] 8-20 parts of polysaccharide from white-backed hairy fungus;

[0027] 9-18 parts skim milk powder;

[0028] 30-55 parts egg liquid.

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

[0030] (1) The present invention provides a polysaccharide of white-backed hairy fungus, which has significant inhibitory effects on AGEs and tumor cells; it can effectively reduce the generation of late glycosylation end products during the production of baked goods and has anti-tumor applications.

[0031] (2) The polysaccharide of white-backed hairy fungus obtained by the present invention through extraction, separation and purification is a natural extract with good safety.

[0032] (3) The polysaccharide of white-backed hairy fungus of the present invention can be used to prepare baked goods. Cookies prepared using the polysaccharide of white-backed hairy fungus of the present invention have low AGEs content and better quality. Attached Figure Description

[0033] Figure 1 Elution curves of Sepacryl S-400 column for the purification of polysaccharides from Auricularia auricula-judae.

[0034] Figure 2 The high-performance liquid chromatogram shows the composition of polysaccharide APP-a monosaccharide in Auricularia auricula-judae.

[0035] Figure 3 This is a molecular weight distribution diagram of APP-a polysaccharide from Auricularia auricula-judae.

[0036] Figure 4 This is a molecular weight distribution diagram of polysaccharide APP-b from Auricularia auricula-judae.

[0037] Figure 5 Infrared spectrum of polysaccharide APP-a from Auricularia auricula-judae.

[0038] Figure 6 The infrared spectrum of polysaccharide APP-b from white-backed hairy fungus. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] The raw materials, instruments, and statistical data in this embodiment of the invention are described below:

[0041] In this embodiment of the invention, the white-backed hairy fungus was commercially available, and the reagents used, such as glucose, phenol, concentrated sulfuric acid, and ethanol, were all of analytical grade.

[0042] The instruments and equipment used in this invention are: HH-4 digital display constant temperature water bath; RE-52A rotary evaporator; DJ-04 pulverizer; LD4-ZA centrifuge; 752 UV-Vis 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. Data are expressed as mean ± SD. Statistical analysis was performed using t-tests or ANOVA. A p-value < 0.05 was considered statistically significant. Furthermore, in the following embodiments, unless otherwise specified, all reagents, raw materials, and instruments used in this invention are well-known in the art, but this does not limit the implementation of the invention. Other well-known reagents and equipment can be applied to the implementation of the following embodiments of this invention.

[0044] Example 1: Extraction of crude polysaccharides from Auricularia auricula-judae

[0045] The white-backed hairy fungus was cleaned and dried at 60℃ for 12 hours. Then it was crushed and deionized water was added at a material-to-liquid ratio of 1:15 (g / mL). It was extracted at 70℃ for 8 hours and then centrifuged (5000 r / min, 20 min) to obtain the supernatant. Four times the volume of 95% ethanol was added to the supernatant and precipitated at 4℃ for 24 hours. The precipitate was obtained by centrifugation (7000 r / min, 10 min) and then freeze-dried to obtain the crude polysaccharide APP from the white-backed hairy fungus.

[0046] Example 2: Isolation and purification of polysaccharides from Auricularia auricula-judae

[0047] Dissolve 1g of crude polysaccharide APP from *Auricularia auricula-judae* in 500mL of ultrapure water, then add 1 / 4 volume of Sevage solution (n-butanol and chloroform (1:4, v / v)), mix and shake for 30min, then let stand for 20min and collect the supernatant. Wash with nitrogen to remove n-butanol. Place the supernatant (with n-butanol removed) in a dialysis bag (molecular weight cutoff 250,000 Da) and dialyze with distilled water for 72h. Load the dialysate onto a Sepacryl S-400 column (1.6cm × 50cm), using distilled water as the eluent. Collect the eluent at a flow rate of 0.2mL / min using an automatic fractionating collector (2mL per tube). Detect OD using the phenol-sulfuric acid method. 490 Elution curves were plotted, and based on these curves, two polysaccharide fractions, APP-a and APP-b, were collected. The eluent from fraction APP-a was freeze-dried to obtain the polysaccharide APP-a from *Auricularia auricula-judae* (see attached image). Figure 1Meanwhile, this study found that other chromatographic columns, such as Sephadex G200 (1.5cm×80cm), Sephadex G100 (2.0cm×50cm), DEAE Sepharose FastFlow (1.5cm×80cm), and DEAE sepharose CL6B (2.0cm×50cm), could not yield the polysaccharide APP-a from Auricularia auricula-judae. The main reason is that different chromatographic columns use different packing materials (ion exchange resins), resulting in different separation principles. Ion exchange chromatography is a separation technique based on the difference in charge between ions and their interaction with the stationary phase (ion exchange resin). It achieves selective separation of different ions through the reversible exchange between the ion exchange resin and ions in the sample, such as the DEAE Sepharose Fast Flow (1.5cm×80cm) and DEAE sepharose CL6B (2.0cm×50cm) ion exchange columns. Gel filtration chromatography is a technique that utilizes the molecular sieve effect of particles with a porous network structure to elute and separate components in the sample based on differences in their relative molecular mass. Examples include Sephadex G200 (1.5cm × 80cm) and Sephadex G100 (2.0cm × 50cm) gel filtration columns. Therefore, different columns will result in different polysaccharide components being separated from the crude polysaccharide APP in *Auricularia auricula-judae*. This example demonstrates, through accidental research, that the target polysaccharide can be obtained using a Sephadex S-400 column.

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

[0049] Monosaccharide composition determination: 10 mg of *Auricularia auricula-judae* polysaccharide APP-a was placed in a stoppered tube, 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution was added, the tube was sealed under vacuum, and hydrolyzed at 121℃ for 1 h. After removing excess TFA from the hydrolysate, it was dried under vacuum. The glyconitrile acetate derivatization method was used: 10 mg of hydroxylamine hydrochloride, an appropriate amount of inositol (internal standard), and 0.5 mL of pyridine were added. The mixture was heated at 90℃ for 30 min, then cooled to room temperature. 0.5 mL of acetic anhydride was added, and the reaction was continued at 90℃ for another 30 min for acetylation. The reaction products were directly analyzed by gas chromatography (GC). The monosaccharide types were qualitatively determined based on the retention time, and the ratio of each monosaccharide was determined based on the peak area ratio.

[0050] Chromatographic conditions: OV1701 flexible quartz capillary column (Φ0.32mm×30mm) was used, N2 was used as the carrier gas, the flow rate was 1.5mL / min, FID flame ionization detector was used, the vaporization chamber temperature was 260℃, the detector temperature was 250℃, and the temperature program was used: the initial temperature was 150℃, the residence time was 1 min, the temperature was increased to 190℃ at 10℃ / min, the residence time was 1 min, the temperature was increased to 240℃ at 3℃ / min, and the residence time was 20 min.

[0051] Relative molecular mass determination: Dextran standards with relative molecular masses of 6100, 26290, 84000, 158000, and 291000 were successively injected. Retention times (TR) were recorded by HPLC. A standard curve was plotted with TR on the x-axis and lgM on the y-axis, and the regression equation was obtained. 10 μL of the test samples APP-a and APP-b were injected. Based on the obtained TR, the relative molecular mass of the polysaccharides was calculated using the regression equation. Chromatographic column: Waters Ullrallydrogel™ Linear (Φ7.8 mm × 300 mm); detector: Waters 2410 differential refractive index detector; mobile phase: 0.1 mol / L NaNO3; flow rate: 0.9 mL / min; column temperature: 45 °C.

[0052] Infrared spectroscopy determination: 2 mg each of polysaccharides APP-a and APP-b from *Auricularia auricula-judae* and 200 mg of KBr were mixed, ground, and compressed into tablets. Measurements were performed using a Thermo Scientific Nicolet iS20 Fourier transform infrared spectrometer with a scanning range of 4000-4000 cm⁻¹. -1 The resolution is 4cm. -1 .

[0053] like Figure 2 As shown, the monosaccharide composition of the polysaccharide APP-a in Auricularia auricula-judae is rhamnose, arabinose, mannose, glucose, and galactose, with a molar ratio of 5.30:6.10:9.35:3.20:4.70.

[0054] like Figure 3 As shown, the relative molecular mass of the polysaccharide APP-a from *Auricularia auricula-judae* is 357926 Da. (The last sentence appears to be incomplete and possibly refers to a different topic.) Figure 4 As shown, the relative molecular mass of polysaccharide APP-b from white-backed hairy fungus is 254086 Da.

[0055] like Figure 5 As shown, the infrared spectrum of polysaccharide APP-a from Auricularia auricula-judae can be observed at 3393 cm⁻¹. -1 There is a broad peak, which is caused by the stretching vibration of -OH; 2938 cm⁻¹ -1 The weak peak is a typical absorption peak characteristic of -CH, at 1625 cm⁻¹. -1The absorption peak is caused by CO vibration and has the characteristic absorption peak of polysaccharides.

[0056] like Figure 6 As shown, the infrared spectrum of polysaccharide APP-b from Auricularia auricula-judae can be observed at 3420 cm⁻¹. -1 There is a broad peak nearby, which is caused by the stretching vibration of -OH; 2930 cm⁻¹ -1 The nearby peaks are attributed to the asymmetric stretching vibrations of -CH2-; 1620 cm⁻¹ -1 The strong peak nearby is a typical absorption peak characteristic of carbonyl groups, at 1099 cm⁻¹. -1 The nearby absorption peaks are attributed to the stretching vibrations of the pyran ring. Infrared analysis results indicate that the polysaccharide APP-b from *Auricularia auricula-judae* is a pyran-type polysaccharide.

[0057] Example 4: Cookie Preparation

[0058] (1) Mixing raw materials and shaping the dough: Weigh 100 parts of low-gluten flour, 35 parts of butter, 25 parts of powdered sugar, 10 parts of white-backed wood ear polysaccharide APP-a, 11 parts of skim milk powder, and 35 parts of egg liquid. Add them to a mixer and mix well. Use a mold to shape the dough into a dough.

[0059] (2) Baking: The dough is baked for 20 minutes at an upper heat temperature of 170℃ and a lower heat temperature of 110℃, and then cooled at room temperature to obtain cookie products.

[0060] Example 5: Preparation of Cookies

[0061] (1) Mixing raw materials and shaping the dough: Weigh 100 parts of low-gluten flour, 40 parts of butter, 30 parts of powdered sugar, 12 parts of white-backed wood ear polysaccharide APP-a, 13 parts of skim milk powder, and 40 parts of egg liquid. Add them to a mixer and mix well. Use a mold to shape the dough into a dough.

[0062] (2) Baking: The dough is baked for 20 minutes at an upper heat temperature of 170℃ and a lower heat temperature of 110℃, and then cooled at room temperature to obtain cookie products.

[0063] Example 6: Preparation of Cookies

[0064] (1) Mixing raw materials and shaping dough: Weigh 100 parts of low-gluten flour, 45 parts of butter, 35 parts of powdered sugar, 15 parts of white-backed wood ear polysaccharide APP-a, 16 parts of skim milk powder, and 40 parts of egg liquid. Add them to a mixer and mix well. Use a mold to shape the dough into dough.

[0065] (2) Baking: The dough is baked for 20 minutes at an upper heat temperature of 170℃ and a lower heat temperature of 110℃, and then cooled at room temperature to obtain cookie products.

[0066] Example 7: Preparation of Cookies

[0067] (1) Mixing raw materials and shaping the dough: Weigh 100 parts of low-gluten flour, 35 parts of butter, 25 parts of powdered sugar, 25 parts of white-backed wood ear polysaccharide APP-a, 11 parts of skim milk powder, and 35 parts of egg liquid (increase the mass of white-backed wood ear polysaccharide APP-a compared to Example 4), add them to the mixer and mix well, and shape the dough into dough using a mold.

[0068] (2) Baking: The dough is baked for 20 minutes at an upper heat temperature of 170℃ and a lower heat temperature of 110℃, and then cooled at room temperature to obtain cookie products.

[0069] Example 8: Preparation of Cookies

[0070] (1) Mixing raw materials and shaping the dough: Weigh 100 parts of low-gluten flour, 35 parts of butter, 25 parts of powdered sugar, 5 parts of white-backed wood ear polysaccharide APP-a, 11 parts of skim milk powder, and 35 parts of egg liquid (compared to Example 4, the mass of white-backed wood ear polysaccharide APP-a is reduced), add them to the mixer and mix well, and shape the dough into dough using a mold.

[0071] (2) Baking: The dough is baked for 20 minutes at an upper heat temperature of 170℃ and a lower heat temperature of 110℃, and then cooled at room temperature to obtain cookie products.

[0072] Example 9: Preparation of Cookies (equal amounts of white-backed black fungus polysaccharide APP-b)

[0073] The difference between Example 9 and Example 4 is that an equal amount of white-backed wood ear fungus polysaccharide APP-b was added to the cookie dough raw material, while all other aspects were the same as in Example 4.

[0074] Example 10: Preparation of Cookies (equal amounts of white-backed black fungus polysaccharide APP-b)

[0075] The difference between Example 10 and Example 5 is that an equal amount of white-backed wood ear fungus polysaccharide APP-b was added to the cookie dough raw material, while all other aspects were 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 the cookie dough raw material does not contain white-backed wood ear fungus polysaccharide APP-a, while all other aspects 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 the cookie dough raw material does not contain white-backed wood ear fungus polysaccharide APP-a, while all other aspects 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 the cookie dough raw material does not contain white-backed wood ear fungus polysaccharide APP-a, while all other aspects are the same as in Example 6.

[0082] Test Example 1

[0083] 1. Experimental Methods

[0084] 1.1 Cell Culture

[0085] Jurkat (human T-lymphocytic leukemia cell line) tumor cells were cultured routinely in RPMI 1640 medium with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged every other day to adjust their growth status. Cells in the logarithmic growth phase with good growth were collected, and the cell density was adjusted to 1×10⁻⁶. 8 The experiment was conducted at / mL.

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

[0087] Jurkat cells in logarithmic growth phase were seeded at 100 μL per well in 96-well plastic culture plates and cultured statically for 24 h. The control group received no APP-a or APP-b, while the sample groups received APP-a and APP-b in each well, with final concentrations of 5, 10, and 15 μg / mL, respectively. Three parallel wells were set for each concentration. The culture plates were then transferred to a CO2 incubator and cultured at 37°C, 5% CO2, and saturated humidity for 48 h. Then, 20 μL of MTT reagent was added to each well, with a final concentration of 400 mg / L. After further incubation at 37°C for 5 h, the culture was terminated, the supernatant was discarded, and 200 μL of LDMSO was added to each well. The plates were gently shaken for 15 min, and the absorbance (OD) values ​​of each well in the control and sample groups were measured at 570 nm using a microplate reader. The average OD value of each group was taken as the average of the three wells in each group. The inhibition rate of Jurkat cells was calculated using the following formula.

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

[0089] 2. Experimental Results

[0090] The inhibitory effects of different concentrations of APP-a and APP-b on the growth of Jurkat cells are shown in Table 1. Table 1 shows that APP-a significantly inhibited the proliferation of Jurkat tumor cells, with the inhibition rate increasing from 26.64% to 62.61%. The effect showed a dose-dependent increasing trend with increasing polysaccharide concentration, and was significantly different from the negative control group (p<0.05). Compared with APP-b, APP-a had a stronger inhibitory effect on the proliferation of Jurkat tumor cells, with a significant difference (p<0.05), indicating a statistically significant difference.

[0091] Table 1. Effects of APP-a and APP-b on Jurkat cell proliferation (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. Testing Method

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

[0097] (2) Sensory evaluation of cookies

[0098] The panel consisted of 10 members, of all genders and with no history of allergies, who were willing to objectively evaluate the product. The cookies were placed on a white porcelain plate and observed under natural light for their shape, color, and texture, as well as their taste and mouthfeel. The cookie samples were randomly arranged, and between each two sets of cookie evaluations, the evaluators were required to clean their mouths with purified water. A sensory evaluation form was created based on the specific sensory requirements for the cookies, as shown in Table 2.

[0099] Table 2 Sensory Evaluation Criteria

[0100]

[0101]

[0102] 2. Test Results

[0103] The results of the determination of fluorescent AGEs in cookies are shown in Table 3. As can be seen from Table 3, using the method of this invention, the polysaccharide APP-a from *Auricularia auricula-judae* was prepared by separation and purification. Adding *Auricularia auricula-judae* polysaccharide APP-a to the cookie dough raw materials to produce cookies significantly reduced the content of late-stage glycosylation end products in the cookies (p < 0.05) compared to comparative examples 1, 2, and 3 (p < 0.05). Furthermore, the cookies exhibited better morphology, color, taste, texture, and structure (p < 0.05), indicating that A... PP-a significantly reduced the content of late-stage glycosylation end products in cookies. However, adding too much APP-a (Example 7) or too little APP-a (Example 8) to cookies could reduce the content of late-stage glycosylation end products to some extent (p < 0.05), but the sensory scores of the cookies were significantly lower than those of Examples 4, 5, and 6 (p < 0.05), indicating that adding too much or too little APP-a during cookie production deteriorated the shape, color, taste, texture, and structure of the cookies. Adding APP-b (Examples 9 and 10) to cookies did not reduce the content of late-stage glycosylation end products, 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 could not reduce the content of late-stage glycosylation end products in cookies and negatively impacted the shape, color, taste, texture, and structure of the cookies.

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

[0105]

[0106]

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

[0108] Finally, it should be noted that the above embodiments 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A polysaccharide from white-backed hairy fungus, characterized in that, The polysaccharide from *Auricularia auricula-judae* is a polymer composed of rhamnose, arabinose, mannose, glucose, and galactose, with a molar ratio of 5.30:6.10:9.35:3.20:4.70 and a relative molecular weight of 357,926 Da.

2. The method for preparing polysaccharide from white-backed Auricularia auricula-judae according to claim 1, characterized in that, Includes the following steps: S1. Mix white-backed hairy fungus powder with water to dissolve the polysaccharides in the white-backed hairy fungus in the water, and then add ethanol to precipitate the polysaccharides to obtain crude polysaccharides from white-backed hairy fungus. S2. Dissolve the crude polysaccharide of Auricularia auricula-judae in water to form a mixture of water and crude polysaccharide of Auricularia auricula-judae. Dialyze the mixture of water and crude polysaccharide of Auricularia auricula-judae using a dialysis bag with a molecular weight cutoff of 300,000 Da to 350,000 Da. Elute the dialysate with a Sepacryl S-400 chromatographic column to obtain polysaccharide of Auricularia auricula-judae.

3. The method for preparing polysaccharide from white-backed hairy fungus according to claim 2, characterized in that, The preparation method of the crude polysaccharide of Auricularia auricula-judae in step S1 is as follows: Auricularia auricula-judae powder and water are mixed at a material-to-liquid ratio of 1:10~20 g / mL, and extracted at 60~80℃ for 6-10 h to dissolve the polysaccharide in the water. After centrifugation at 4000~6000 r / min for 10-30 min, the supernatant is collected, and then 2-5 times the volume of 90~100% ethanol is added to precipitate the polysaccharide, thus obtaining the crude polysaccharide of Auricularia auricula-judae.

4. The method for preparing polysaccharide from white-backed hairy fungus according to claim 2, characterized in that, Before dialysis of the mixture of water and crude polysaccharide from white-backed wood ear fungus described in step S2, it is necessary to remove the protein contained therein. The method for removing the protein is to add 1 / 6 to 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. The supernatant is then rinsed with nitrogen to remove n-butanol from the Sevage solution.

5. The method for preparing polysaccharide from white-backed hairy fungus according to claim 2, characterized in that, The dialysis described in step S2 involves dialysis with distilled water for 36 to 96 hours.

6. The use of the polysaccharide of Auricularia auricula-judae as described in claim 1 or the polysaccharide of Auricularia auricula-judae prepared by the method described in any one of claims 2-5 in the preparation of a drug for treating human T-lymphocytic leukemia.

7. The application of the polysaccharide of Auricularia auricula-judae according to claim 1 or the polysaccharide of Auricularia auricula-judae prepared by any one of claims 2-5 in the preparation of additives for inhibiting AGEs.

8. The application according to claim 7, characterized in that, Add the white-backed wood ear polysaccharide of claim 1 or the white-backed wood ear polysaccharide prepared by any one of claims 2-5 to the raw materials of baked goods, and then bake them.

9. The application according to claim 8, characterized in that, The baked goods mentioned are cookies.

10. A cookie, characterized in that, The cookies are made by mixing and baking the following ingredients in parts by weight: 80-120 portions of low-gluten flour; 30-50 parts butter; 20-45 parts powdered sugar; 8-20 parts of polysaccharide from white-backed hairy wood ear fungus; 9-18 parts skim milk powder; 30-55 parts egg liquid; The white-backed hairy fungus polysaccharide is the white-backed hairy fungus polysaccharide according to claim 1 or the white-backed hairy fungus polysaccharide prepared by the method according to any one of claims 2 to 5.

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