Acetylated flammulina velutipes polysaccharide as well as preparation method and application thereof
By modifying the enoki polysaccharide with strong antioxidant and AGEs inhibitor effects, acetylated enoki polysaccharide with strong antioxidant and AGEs inhibitor effects was prepared, which solved the problem of AGEs generation in baked foods and improved food safety and added value.
Patent Information
- Application Number
- CN202311698433.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-12
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art is difficult to effectively solve the problem of late glycosylated terminal products (AGEs) generated during baked food processing, affecting food safety and human health.
By modifying the enoki polysaccharide with acetylation, acetylated enoki polysaccharide (Ac-FVP-0.6-c) was prepared. This product not only increases the added value of enoki, but also has significant antioxidant effects and AGEs inhibitor effects.
Acetylated enoki mushroom polysaccharides have performed well in the field of food antioxidants, and effectively inhibit the generation of AGEs in the production of baked food cookies and improve food safety.
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Figure CN119661730A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a natural ingredient extraction technology, and in particular to an acetylated Flammulina velutipes polysaccharide and a preparation method and application thereof. Background Art
[0002] Advanced Glycation End Products (AGEs) are one of the products of the Maillard reaction. In the food processing industry, glycosylation reactions are often used to improve the flavor, color and taste of food. However, food systems or food proteins can also form AGEs that are harmful to the human body during the glycosylation process. Studies have found that long-term intake of foods rich in AGEs will lead to the accumulation of AGEs in the body. AGEs are potentially harmful to the human body and are closely related to diabetes, kidney disease, atherosclerosis, cardiovascular disease and Alzheimer's disease. Adding AGEs inhibitors to reduce the formation of AGEs during food thermal processing is one of the effective methods to improve food safety and promote human health. AGEs inhibitors include synthetic and natural compounds. Among them, natural AGEs inhibitors from plants have become a research hotspot at home and abroad due to their advantages such as many types, good effects and few side effects. AGEs cause food safety problems in the process of baked food processing, which are generated in the processing system. Therefore, it is of great significance to develop key control technologies such as AGEs blocking and inhibition in the process of baked food processing and further build a global control model and technical system for the safe manufacturing of baked food.
[0003] Flammulina velutipes, also known as mushroom, winter mushroom, mushroom, money mushroom, etc., belongs to the order Agaricales, family Tricholomataceae, and genus Money Mushroom. Flammulina velutipes polysaccharide (Flammulina velutipes polysaccharide, FVP) is one of the important active ingredients in Flammulina velutipes. Liu Xiaomin et al. optimized the hot water extraction process of Flammulina velutipes polysaccharide using response surface methodology. Luo Wenfeng et al. used enzymatic pretreatment-microwave-assisted extraction of Flammulina velutipes polysaccharide and optimized the extraction process using response surface methodology. Pigments in Flammulina velutipes polysaccharide can be removed by adsorption of activated carbon and macroporous resin. Macroporous resin has the advantages of high efficiency and high purification rate. For proteins in Flammulina velutipes polysaccharide, the main removal methods currently include Sevag method, trichlorotrifluoroethane method, etc.
[0004] Flammulina velutipes polysaccharides can improve the ability to scavenge free radicals, reduce the damage of free radicals to cell membranes, prevent lipid peroxidation, and can enhance the ability of the liver to scavenge free radicals by inducing the activity of liver drug metabolizing enzymes, and have significant antioxidant effects. Liu Xiaoxiao et al. found that Flammulina velutipes fruiting body polysaccharide FVPB1 can activate mouse T cells and has a good immunomodulatory effect. Meng et al. found that Flammulina velutipes polysaccharides can significantly reduce the mitotic index of liver cancer cells in vitro by directly adding Flammulina velutipes polysaccharides to tumor cell culture medium. Su Anxiang et al. found that Flammulina velutipes proteoglycan 1-1 can effectively inhibit the inflammatory response of the co-culture model of human colon adenocarcinoma cells (Caco-2) and macrophages (RAW264.7) induced by lipopolysaccharide. Li Yifang et al. found that Flammulina velutipes polysaccharides have a certain protective effect on CCl4-induced acute liver injury, which can significantly reduce the ALT and AST activities in the serum of mice with CCl4-induced acute liver injury, increase the activity of superoxide dismutase in liver homogenate, and reduce the content of malondialdehyde. Tian Xuewen et al. found that Flammulina velutipes polysaccharides have significant anti-fatigue effects, can effectively increase the activity of lactate dehydrogenase in rat blood, and further improve the speed of removing blood lactate in rats during the recovery period after exercise. Huang Qiong et al. used the paper diffusion method to study the antibacterial effect of Flammulina velutipes polysaccharides on 8 kinds of bacteria and fungi, and found that the inhibitory effect of Flammulina velutipes polysaccharides on fungi was stronger than that on bacteria.
[0005] In summary, the current research on Flammulina velutipes polysaccharides mainly focuses on the extraction, preliminary purification and biological activity of crude polysaccharides, but there are few reports on the purification, structural modification and activity evaluation of single components of Flammulina velutipes polysaccharides. Therefore, the present invention studies and develops an acetylated modified product of a single polysaccharide component of Flammulina velutipes and its new use, which is of great significance for improving the added value of Flammulina velutipes, developing acetylated Flammulina velutipes polysaccharides as a new antioxidant and an inhibitor of late glycosylation end products in the production process of baked food cookies. Summary of the invention
[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide an acetylated Flammulina velutipes polysaccharide to increase the added value of Flammulina velutipes, and also to provide a new food antioxidant and glycosylation end product inhibitor for the food field.
[0007] The technical problem that the present invention needs to solve is to provide a method for preparing the acetylated Flammulina velutipes polysaccharide.
[0008] Another technical problem to be solved by the present invention is to provide the application of the acetylated Flammulina velutipes polysaccharide in the field of food antioxidants.
[0009] The final technical problem to be solved by the present invention is to provide the application of the acetylated Flammulina velutipes polysaccharide as a glycosylation terminal product inhibitor in the processing of baked foods.
[0010] A method for preparing acetylated Flammulina velutipes polysaccharide comprises the following steps: (1) Extraction of crude polysaccharides: Wash, dry and crush Flammulina velutipes, add water at a solid-liquid ratio of 1g:50mL-1g:20mL, extract at 40℃-100℃ for 40min-120min, and centrifuge to obtain a supernatant; concentrate under reduced pressure at 30℃, add five times the volume of anhydrous ethanol to the concentrate, let stand at 4℃ for 48h, and then centrifuge at 5000r / min for 20min, collect the precipitate, and freeze-dry the precipitate for 48h to obtain crude Flammulina velutipes polysaccharides; (2) Purification of crude polysaccharides: The crude polysaccharides of Flammulina velutipes were prepared into a solution with a concentration of 10 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize for 5 times, and then dialyzed for 48 h in a dialysis bag. The dialysate was freeze-dried to obtain the initially purified polysaccharides of Flammulina velutipes. (3) Separation of Flammulina velutipes polysaccharide components: The purified polysaccharide was prepared into a solution with a concentration of 5 mg / mL, loaded onto a DEAE-Cellulose 52 ion column (2.5 cm × 45 cm), and eluted with deionized water, 0.1 mol / L, 0.3 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl solutions in sequence at a flow rate of 2 mL / min. The eluate was automatically collected at a concentration of 5 mL / tube, and the OD was detected by the phenol-sulfuric acid method. 490 Then, an elution curve was drawn, and the eluate corresponding to the main peak with the largest peak area was selectively collected, that is, the eluate corresponding to the 0.6 mol / L NaCl solution was combined, and the eluate was dialyzed with deionized water for 24 h, and freeze-dried to obtain the primary purified product FVP-0.6 of the Flammulina velutipes polysaccharide component; Weigh 5 g of the first purification of Flammulina velutipes polysaccharide component FVP-0.6, dissolve it in 200 mL of deionized water, load it onto a Sephadex G-200 gel column (1.5 cm × 100 cm), elute with deionized water at a flow rate of 2 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, three elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected. The eluate was dialyzed with deionized water for 24 hours and freeze-dried to obtain the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide, which was used as a polysaccharide substrate for further acetylation modification. The molecular weight of the homogeneous monosaccharide component FVP-0.6-c in the Flammulina velutipes is 357486Da, and its monosaccharide composition is arabinose, xylose, mannose, glucose and galactose in a molar ratio of 6.02:1.00:1.09:1.71:3.25; (4) Acetylation modification of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide: Weigh 0.3 g of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide, add it to 15 mL of distilled water, and stir to dissolve it at room temperature; adjust the pH value of the solution to 9.0 with 0.5 mol / L NaOH solution, add 1-6 mL of acetic anhydride, and react at 50-80 ° C for 1-5 h. After the reaction is completed, adjust the pH to 7 with 5 mol / L HCl solution, place the reaction solution in a dialysis bag, and dialyze it with deionized water for 24 h. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c, i.e., the final product, with an acetylation degree of substitution of 0.277-0.463.
[0011] Preferably, in step (1), the liquid ratio of Flammulina velutipes to water is 1 g:30 mL.
[0012] Preferably, in step (1), the supernatant is obtained by extraction at 70° C. for 80 min and then centrifugation.
[0013] Preferably, in step (2), the dialysis is performed through a dialysis bag, and the molecular weight cut-off of the dialysis bag is 3000Da.
[0014] Preferably, in step (3), the dialysis of the eluate is performed using a dialysis bag having a molecular weight cutoff of 5000 Da.
[0015] Preferably, in step (4), the amount of acetic anhydride added is 3.3 mL, the reaction time is 3.1 h, and the reaction temperature is 64.4°C.
[0016] Preferably, in step (4), the acetylation degree of substitution of the acetylated Flammulina velutipes polysaccharide is 0.415.
[0017] The acetylated Flammulina velutipes polysaccharide prepared by the above-mentioned method for preparing acetylated Flammulina velutipes polysaccharide is within the protection scope of the present invention.
[0018] The application of the above-mentioned acetylated Flammulina velutipes polysaccharide in the field of food antioxidants is within the protection scope of the present invention.
[0019] The use of the above-mentioned acetylated Flammulina velutipes polysaccharide as a glycosylation terminal product inhibitor in the processing of baked foods is within the protection scope of the present invention.
[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The Flammulina velutipes polysaccharide component product of the present invention has significantly enhanced antioxidant activity after acetylation modification, and has strong inhibitory activity on late glycosylation end products in the production process of baked food cookies. It can be widely used in the field of food additives as an antioxidant and an inhibitor of late glycosylation end products in the production process of baked food cookies. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The DEAE-Cellulose 52 elution curve of the purified Flammulina velutipes polysaccharide of the present invention is shown.
[0022] Figure 2 This is the Sephadex G-200 elution curve of the Flammulina velutipes polysaccharide component FVP-0.6 of the present invention.
[0023] Figure 3 The high performance liquid chromatogram of the monosaccharide composition of the Flammulina velutipes polysaccharide component FVP-0.6-c of the present invention.
[0024] Figure 4 This is a molecular weight distribution diagram of the Flammulina velutipes polysaccharide component FVP-0.6-c of the present invention.
[0025] Figure 5 This is an ultraviolet scanning image of the Flammulina velutipes polysaccharide component FVP-0.6-c of the present invention.
[0026] Figure 6 This is the infrared spectrum of the Flammulina velutipes polysaccharide component FVP-0.6-c of the present invention.
[0027] Figure 7 This is a diagram showing the scavenging effect of the Flammulina velutipes polysaccharide component FVP-0.6-c and the acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c of the present invention on hydroxyl radicals.
[0028] Figure 8 This is a diagram showing the scavenging effect of the Flammulina velutipes polysaccharide component FVP-0.6-c and the acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c on DPPH free radicals.
[0029] Fig. 9 This is a diagram showing the scavenging effect of the Flammulina velutipes polysaccharide component FVP-0.6-c and the acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c on superoxide anion radicals. DETAILED DESCRIPTION
[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.
[0031] The present invention adopts fresh golden needle mushroom.
[0032] The reagents used in the present invention: acetic anhydride, 1,1-diphenyl-2-picrylhydrazyl / DPPH, tris(hydroxymethylaminomethane) / Tris (Shanghai Kaiyang Biotechnology Co., Ltd.); anhydrous ethanol, sodium hydroxide, methanol, n-butanol, hydrogen peroxide, ferrous sulfate, hydrochloric acid, salicylic acid, pyrogallol, etc. are all domestic analytical reagents.
[0033] Instruments and equipment used in the present invention: DJ-04 pulverizer, HHS-11-2 water bath, CR22GII high-speed refrigerated centrifuge, Alpha 1-2 LD freeze dryer, RE-52A rotary evaporator, SHB-B95 circulating water multi-purpose vacuum pump, EL-800 enzyme-linked detector, DF-101S constant temperature heating magnetic stirrer, Nicolet Nexus Fourier transform infrared spectrometer, 752 UV-visible spectrophotometer, UV2102 PCS UV scanner, GC-14A gas chromatograph, Waters 600 high pressure liquid gas spectrometer. 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. p <0.05 and p <0.01 was considered statistically significant.
[0034] In addition, in the following examples, unless otherwise specified, all reagents, raw materials and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.
[0035] Example 1: Extraction and purification of crude polysaccharides from Flammulina velutipes The Flammulina velutipes was washed, dried and crushed, and water was added at a solid-liquid ratio of 1:20 (g / mL). The extraction was performed at 50°C for 110 minutes. After the extraction, the supernatant was centrifuged and concentrated under reduced pressure at 30°C. After concentration, five times the volume of anhydrous ethanol was added to the concentrate, and the concentrate was allowed to stand at 4°C for 48 hours, and then centrifuged (5000r / min, 20min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain the crude polysaccharide of Flammulina velutipes. The crude polysaccharide was prepared into a solution with a concentration of 10 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 5 times, and then dialyzed through a dialysis bag (molecular weight cutoff 3000Da) for 48 hours. The dialyzate was freeze-dried to obtain the initially purified purified polysaccharide of Flammulina velutipes.
[0036] Example 2: Extraction and purification of crude polysaccharides from Flammulina velutipes The Flammulina velutipes was washed, dried and crushed, and water was added at a solid-liquid ratio of 1:30 (g / mL). The extraction was performed at 60°C for 90 minutes. After the extraction, the supernatant was centrifuged and concentrated under reduced pressure at 30°C. After concentration, five times the volume of anhydrous ethanol was added to the concentrate, and the concentrate was allowed to stand at 4°C for 48 hours, and then centrifuged (5000r / min, 20min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain the crude polysaccharide of Flammulina velutipes. The crude polysaccharide was prepared into a solution with a concentration of 10 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 5 times, and then dialyzed through a dialysis bag (molecular weight cutoff 3000Da) for 48 hours. The dialyzate was freeze-dried to obtain the initially purified purified polysaccharide of Flammulina velutipes.
[0037] Example 3: Extraction and purification of crude polysaccharides from Flammulina velutipes The Flammulina velutipes was washed, dried and crushed, and water was added at a solid-liquid ratio of 1:40 (g / mL). The extraction was performed at 70°C for 80 minutes. After the extraction, the supernatant was centrifuged and concentrated under reduced pressure at 30°C. After concentration, five times the volume of anhydrous ethanol was added to the concentrate, and the concentrate was allowed to stand at 4°C for 48 hours. Then, the precipitate was collected after centrifugation (5000r / min, 20min). The precipitate was freeze-dried for 48 hours to obtain the crude polysaccharide of Flammulina velutipes. The crude polysaccharide was prepared into a solution with a concentration of 10 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 5 times, and then dialyzed through a dialysis bag (molecular weight cutoff 3000Da) for 48 hours. The dialyzate was freeze-dried to obtain the initially purified purified polysaccharide of Flammulina velutipes.
[0038] Example 4: Extraction and purification of crude polysaccharides from Flammulina velutipes The Flammulina velutipes was washed, dried and crushed, and water was added at a solid-liquid ratio of 1:50 (g / mL). The extraction was performed at 90°C for 60 minutes. After the extraction, the supernatant was centrifuged and concentrated under reduced pressure at 30°C. After concentration, five times the volume of anhydrous ethanol was added to the concentrate, and the concentrate was allowed to stand at 4°C for 48 hours, and then centrifuged (5000r / min, 20min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain the crude polysaccharide of Flammulina velutipes. The crude polysaccharide was prepared into a solution with a concentration of 10 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 5 times, and then dialyzed through a dialysis bag (molecular weight cutoff 3000Da) for 48 hours. The dialyzate was freeze-dried to obtain the initially purified purified polysaccharide of Flammulina velutipes.
[0039] Example 5: Separation of Flammulina velutipes polysaccharide components The purified polysaccharide was prepared into a solution with a concentration of 5 mg / mL, loaded onto a DEAE-Cellulose 52 ion column (2.5 cm×45 cm), and eluted with deionized water, 0.1 mol / L, 0.3 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl solutions in sequence, with an elution flow rate of 2 mL / min. 5 mL / tube of eluate was automatically collected, and an elution curve was drawn after OD490 was detected by the phenol-sulfuric acid method. The eluate corresponding to the main peak with the largest peak area was selectively collected, that is, the eluate corresponding to the 0.6 mol / L NaCl solution was combined, and the eluate was dialyzed with deionized water for 24 h, and freeze-dried to obtain the primary purified product of Flammulina velutipes polysaccharide component FVP-0.6( Figure 1 ); Weigh 5 g of the first purification of Flammulina velutipes polysaccharide component FVP-0.6, dissolve it in 200 mL of deionized water, load it onto a Sephadex G-200 gel column (1.5 cm × 100 cm), elute with deionized water at a flow rate of 2 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, three elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected. The eluate was dialyzed with deionized water for 24 h and freeze-dried to obtain the uniform monosaccharide component FVP-0.6-c ( Figure 2 ), that is, used as a polysaccharide substrate for further acetylation modification.
[0040] Example 6: Structural characteristics of Flammulina velutipes polysaccharide component FVP-0.6-c Determination of monosaccharide composition: 10 mg of the sample of the Flammulina velutipes polysaccharide component FVP-0.6-c was placed in a stoppered tube, 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution was added, the tube was sealed in vacuum, and then hydrolyzed at 121°C for 1 h. After removing the excess TFA from the hydrolyzate, it was vacuum dried. The sugar nitrile 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, heated at 90°C for 30 min, then taken out and cooled to room temperature, 0.5 mL of acetic anhydride was added, and the reaction was continued at 90°C for 30 min for acetylation. The reaction product was directly analyzed by gas chromatography (GC), and the type of monosaccharide was qualitatively determined according to the retention time of the monosaccharide, and the proportional relationship between the monosaccharides was determined according to the ratio of the peak areas.
[0041] Chromatographic conditions: OV1701 elastic quartz capillary column (Φ0.32mm×30m), N2 carrier gas, flow rate 1.5mL / min, FID hydrogen flame detector, vaporization chamber temperature 260℃, detector temperature 250℃, programmed temperature rise: starting temperature 150℃, dwell for 1min, increase to 190℃ at 10℃ / min, dwell for 1min, increase to 240℃ at 3℃ / min, dwell for 20min.
[0042] Relative molecular mass determination: Standard Dextran with relative molecular mass of 6100, 26290, 84000, 158000 and 291000 were injected successively, and the retention time TR was recorded by HPLC. The standard curve was drawn with TR as the horizontal axis and LgM as the vertical axis to obtain the regression equation. 20μL of the sample VBLP-3b to be tested was injected, and the relative molecular mass of the polysaccharide was calculated by the regression equation based on the obtained TR. Chromatographic column: Waters UllrallydrogelTM Linear (Φ7.8mm×300mm), detector: Waters2410 differential refractometer, with 0.1mol / L NaNO 3 The mobile phase was 0.9 mL / min and the column temperature was 45 °C.
[0043] Ultraviolet spectroscopic determination: The ultraviolet spectroscopic determination of the Flammulina velutipes polysaccharide component FVP-0.6-c was carried out using a UV2102 PCS ultraviolet scanner from Shanghai Precision Instrument Co., Ltd., with a scanning range of 190nm-900nm.
[0044] Infrared spectroscopic determination: 1 mg of Flammulina velutipes polysaccharide component FVP-0.6-c and 100 mg of KBr were mixed, ground and pressed into tablets. Thermo Electron's Nicolet Nexus Fourier transform infrared spectrometer was used for determination with a scanning range of 4000-400 cm -1 , resolution 4cm -1 .
[0045] like Figure 3 As shown, the monosaccharides of Flammulina velutipes polysaccharide component FVP-0.6-c are mainly composed of arabinose, xylose, mannose, glucose and galactose, with a molar ratio of 6.02:1.00:1.09:1.71:3.25.
[0046] like Figure 4 As shown, the relative molecular mass of Flammulina velutipes polysaccharide component FVP-0.6-c is 357486Da.
[0047] like Figure 5 As shown, from the UV spectrum of the Flammulina velutipes polysaccharide component FVP-0.6-c, it can be observed that there is no absorption peak around 280nm, indicating that FVP-0.6-c does not contain protein after purification.
[0048] like Figure 6 As shown in the infrared spectrum of the Flammulina velutipes polysaccharide component FVP-0.6-c, it can be observed that the -1 There is a broad peak near 3000cm, which is caused by the stretching vibration of -OH; -1The nearby peaks are assigned to -CH 2 -Asymmetric stretching vibration; 1629cm -1 The strong peak nearby is a typical carbonyl absorption peak, 1020cm -1 The absorption peaks near the pyranose ring belong to the stretching vibration. The infrared analysis results show that FVP-0.6-c conforms to the structural characteristics of pyranose polysaccharides.
[0049] Example 7: Acetylation modification of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide Weigh 0.3 g of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide, add it to 15 mL of distilled water, and stir to dissolve at room temperature. After adjusting the pH value of the solution to 9.0 with 0.5 mol / L NaOH solution, add 3.3 mL of acetic anhydride, react at 64.4 ° C for 3.1 hours, and after the reaction is completed, adjust the pH to 7 with 5 mol / L HCl solution, place the reaction solution in a dialysis bag, and dialyze with deionized water for 24 hours. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c, whose acetylation degree of substitution is 0.415.
[0050] Example 8: Antioxidant effect of acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c Determination of hydroxyl radical scavenging rate: Prepare different concentrations of Flammulina velutipes polysaccharide FVP-0.6-c and acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c sample solutions, add 9.0 mmol / L FeSO 4 , 1.0 mL of 9.0 mmol / L salicylic acid-ethanol solution, shake well, then add 2.0 mL of acetylated pumpkin polysaccharide solutions of different concentrations, and after fully mixing, add 1 mL of 9.0 mmol / L H 2 O 2 , react in a 37°C water bath for 30 min, and measure the absorbance A at a wavelength of 510 nm using distilled water as a reference. 2 The blank control group was replaced with the same volume of distilled water. The OH removal rate was calculated according to the following formula and the measurement was repeated 3 times.
[0051] Clearance rate (%) = [A 0 - (A 2 -A 1 )] / A 0 ×100 Where: A 2 —Absorbance measured by polysaccharide samples of different concentrations; A 1 —Absorbance measured by using distilled water instead of salicylic acid; A 0 —Absorbance measured with distilled water instead of polysaccharide at different concentrations.
[0052] Determination of DPPH free radical scavenging rate: Take 2 mL of Flammulina velutipes polysaccharide FVP-0.6-c and acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c sample solutions of different concentrations, place them in different test tubes, add 2 mL of DPPH solution (0.1 mmol / L), mix well, react at 25°C in the dark for 30 min, and measure the absorbance A at 517 nm. 2 The blank control group was replaced by distilled water instead of polysaccharide sample, and the absorbance was measured as A 1 The scavenging rate of DPPH free radicals was calculated according to the following formula and the measurement was repeated 3 times.
[0053] Clearance rate (%) = [A 0 - (A 2 -A 1 )] / A 0 ×100 Where: A 2 —Absorbance measured by polysaccharide samples of different concentrations; A 1 —Absorbance measured by using distilled water instead of salicylic acid; A 0 —Absorbance measured with distilled water instead of polysaccharide at different concentrations.
[0054] Determination of superoxide anion radical scavenging rate: 4.5 mL of 0.05 mol / L Tris-HCl buffer solution with pH 8.2, 1 mL of different concentrations of Flammulina velutipes polysaccharide FVP-0.6-c and acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c sample solution and 3.2 mL of distilled water were placed in a test tube, mixed and preheated in a 25°C water bath for 20 min, and 0.3 mL of 7 mmol / L pyrogallol solution was immediately added. After the liquid was fully shaken, it was placed in a 25°C water bath for accurate reaction for 3 min, and the reaction was immediately terminated with 1 drop of 10 mol / L concentrated hydrochloric acid. The absorbance at a wavelength of 325 nm was measured as A. Calculate O according to the following formula 2 - Clearance rate was measured 3 times.
[0055] Clearance rate (%) = (A 0 -A) / A 0 ×100 Where: A—absorbance measured by polysaccharide samples of different concentrations; A 0 —Absorbance measured using distilled water instead of polysaccharide sample The scavenging effects of FVP-0.6-c and Ac-FVP-0.6-c on hydroxyl radicals, DPPH radicals and superoxide anion radicals were shown in Figure 2. Figure 7 , 8As shown in Figures 9 and 10, within the selected mass concentration range, the scavenging effects of FVP-0.6-c and Ac-FVP-0.6-c on hydroxyl radicals, DPPH radicals and superoxide anion radicals increased with increasing concentrations. After acetylation modification, the antioxidant activity of Ac-FVP-0.6-c was significantly higher than that of FVP-0.6-c, indicating that acetylation modification can enhance the antioxidant activity of FVP-0.6-c.
[0056] Example 9: Anti-glycosylation effect of acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c in the production process of baked food cookies 1. Preparation of Cookies The baking percentage was adopted, in which the weight of low-gluten flour was 100%, acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c solution (concentrations were 15%, 20%, 25%, 30%), butter, powdered sugar, and eggs were added according to the experimental proportion based on low-gluten flour. The basic formula was: 100% low-gluten flour, 50% butter, 35% powdered sugar, 20% acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c solution of different concentrations, and 30% egg liquid. The preparation process was as follows: soften butter → beat → add egg liquid and powdered sugar → beat → add low-gluten flour → mix well → add acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c solution → knead dough → transfer dough into piping bag → squeeze and shape → bake → cool → finished product. The baking temperature was 170℃ on the upper fire, 160℃ on the lower fire, and the time was 20min.
[0057] 2. Determination of fluorescent AGEs content 1 mg of cookie was crushed and added with 5 mL of methanol, then stored at -80°C for 2 h, centrifuged at 5000 r / min for 40 min, 0.5 mL of supernatant was taken, and the fluorescence value was measured at λex / λem=340 nm / 465 nm. Phosphate buffer solution was used instead of sample as control, and each group of samples was repeated 3 times.
[0058] The effect of acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c on fluorescent AGEs in the cookie production process is shown in Table 1. It can be seen from Table 1 that compared with the control group, with the increase in the concentration of acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c solution, the amount of fluorescent AGEs generated in the cookies was significantly reduced (p < 0.05 or p < 0.01). Acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c can inhibit the generation of fluorescent AGEs in the cookie production process, which may be because Ac-FVP-0.6-c has a strong antioxidant effect, thereby blocking the pathway of synthesizing AGEs initiated by free radicals through antioxidant action.
[0059] Table 1 Effect of acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c on fluorescent AGEs in cookie production
[0060] Note: Different lowercase letters indicate significant differences among different groups (p < 0.05 or p < 0.01).
[0061] The above results show that acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c has a strong antioxidant effect, which blocks the pathway of AGE synthesis initiated by free radicals through antioxidant action. It can be used in the food field as an antioxidant and an inhibitor of late glycation end products in the production process of baked food cookies.
[0062] The above-mentioned embodiments are merely descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the present invention.
Claims
1. A method for preparing acetylated Flammulina velutipes polysaccharide, characterized in that: The steps include: (1) Extraction of crude polysaccharides: Wash, dry and crush Flammulina velutipes, add water according to the solid-liquid ratio of 1 g:50 mL to 1 g:20 mL, extract at 40°C to 100°C for 40 min to 120 min, and centrifuge to obtain the supernatant; concentrate under reduced pressure at 30°C, add five times the volume of anhydrous ethanol to the concentrate, let it stand, and then collect the precipitate after centrifugation. The precipitate is freeze-dried to obtain crude Flammulina velutipes polysaccharides; (2) Purification of crude polysaccharides: The crude polysaccharides of Flammulina velutipes were prepared into a solution with a concentration of 10 mg / mL, and the solution was deproteinized by adding Sevag reagent for 5 times, and then dialyzed for 48 hours through a dialysis bag. The Sevag reagent was a solution of n-butanol: chloroform = 1:
4. The dialysate was freeze-dried to obtain the preliminarily purified polysaccharides of Flammulina velutipes. (3) Separation of Flammulina velutipes polysaccharide components: The purified polysaccharide of Flammulina velutipes was prepared into a solution with a concentration of 5 mg / mL, loaded onto an ion exchange column, and eluted with deionized water, 0.1 mol / L, 0.3 mol / L, 0.6 mol / L, and 0.8 mol / L NaCl solutions in sequence at a flow rate of 2 mL / min. The eluate was automatically collected at a concentration of 5 mL / tube, and the OD was detected by the phenol-sulfuric acid method. 490 Then, an elution curve was drawn, and the eluate corresponding to the main peak with the largest peak area was selectively collected, that is, the eluate corresponding to the 0.6 mol / L NaCl solution was combined, and the eluate was dialyzed with deionized water for 12 to 60 hours, and freeze-dried to obtain the primary purified product FVP-0.6 of the Flammulina velutipes polysaccharide component; Weigh 5 g of the first purification of Flammulina velutipes polysaccharide component FVP-0.6, dissolve it in 200 mL of deionized water, load it onto a Sephadex G-200 gel column, elute with deionized water at a flow rate of 2 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, three elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected. The eluate was dialyzed with deionized water for 24 hours and freeze-dried to obtain the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide, which was used as a polysaccharide substrate for further acetylation modification. The molecular weight of the homogeneous monosaccharide component FVP-0.6-c of the Flammulina velutipes polysaccharide is 357486Da, and its monosaccharide composition is arabinose, xylose, mannose, glucose and galactose in a molar ratio of 6.02:1.00:1.09:1.71:3.25; (4) Acetylation modification of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide: Weigh 0.3 g of the uniform monosaccharide component FVP-0.6-c of Flammulina velutipes polysaccharide, add it to 15 mL of distilled water, and stir to dissolve it at room temperature; adjust the pH value of the solution to 9.0 with NaOH solution, add 1-6 mL of acetic anhydride, and react at 50-80 ° C for 1-5 h. After the reaction is completed, adjust the pH to 7 with 5 mol / L HCl solution, place the reaction solution in a dialysis bag, and dialyze it with deionized water for 24 h. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated Flammulina velutipes polysaccharide Ac-FVP-0.6-c, i.e., the final product, with an acetylation degree of substitution of 0.277-0.
463.
2. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (1), the liquid ratio of Flammulina velutipes to water is 1 g:30 mL.
3. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (1), the supernatant is obtained by extraction at 70° C. for 80 min and centrifugation.
4. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (3), the dialysis of the eluate is performed using a dialysis bag, and the molecular weight cut-off of the dialysis bag is 5000Da.
5. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (3), the ion exchange column is a DEAE-Cellulose 52 ion exchange column, and the gel chromatography column is a Sephadex G-200 gel chromatography column.
6. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (4), the amount of acetic anhydride added was 3.3 mL, the reaction time was 3.1 h, and the reaction temperature was 64.4 °C.
7. The method for preparing acetylated Flammulina velutipes polysaccharide according to claim 1, characterized in that: In step (4), the acetylation degree of substitution of the acetylated Flammulina velutipes polysaccharide is 0.
415.
8. Acetylated Flammulina velutipes polysaccharide prepared by the method for preparing acetylated Flammulina velutipes polysaccharide according to any one of claims 1 to 7.
9. Use of the acetylated Flammulina velutipes polysaccharide according to claim 8 in the field of food antioxidants.
10. Use of the acetylated Flammulina velutipes polysaccharide according to claim 9 as a glycosylation end product inhibitor in the processing of baked foods.