Method for inhibiting advanced glycosylation end product in cookie production process
By adding the polysaccharide FVSP2b of enoki mushrooms to cookies, the problem of more AGEs generation in cookies is solved, effectively inhibiting the generation of AGEs, and reducing the content of fluorescent AGEs in cookies.
Patent Information
- Application Number
- CN202510481175.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-17
- Publication Date
- 2025-07-08
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
During the cookie production process, the production of late glycosylated terminal products (AGEs) is more common, resulting in potential health hazards. The utilization of polysaccharides under the enoki mushrooms has failed to effectively inhibit the production of AGEs.
The enoki mushroom pulp polysaccharide FVSP2b was added to the cookie production. The obtained enoki mushroom pulp polysaccharide FVSP2b was characterized by a relative molecular mass of 236712Da. The monosaccharide composition was rhamnosaccharide, arabinose, xylose, mannose, glucose and galactose molar ratio was 11.8:52.3:42.8:1:1.03:45.9. It was added to the cookie body to inhibit protein crosslinking reaction.
Effectively inhibit the generation of glycosylation terminal products in the middle and late stages of cookies, reduce the content of fluorescent AGEs in cookies, and achieve a safe and effective inhibitory effect of AGEs.
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Figure CN120266884A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of comprehensive utilization of agricultural product wastes, and particularly to the separation and purification of polysaccharide FVSP2b from the waste of Flammulina velutipes and a method for inhibiting advanced glycation end products in the production of cookies. Background Art
[0002] Advanced glycation end products (AGEs) are mainly generated through the Maillard reaction, lipid oxidation reaction or polyol degradation pathway. Research shows that AGEs are closely related to the pathogenesis of various chronic diseases such as diabetes, atherosclerosis, renal failure, etc., and their large accumulation in the body has potential harmful effects on human health.
[0003] During the food processing process, the Maillard reaction will produce fluorescent AGEs. Cookies are high in sugar and fat, and are prone to various reactions such as the Maillard reaction, sugar degradation, and oil oxidation during the baking process. The oil undergoes a series of reactions such as decomposition, polymerization, oxidation, and condensation at high temperatures. The Maillard reaction occurs between the sugar and the amino acids in the protein, generating reactive carbonyl compounds such as glyoxal, methylglyoxal, and 3-deoxyglucosone. These carbonyl compounds have high reactivity and are prone to protein cross-linking and glycosylation, easily generating advanced glycation end products AGEs, which are harmful to the human body. Therefore, how to inhibit the generation of AGEs during the production of cookies is particularly important.
[0004] During the processing of Flammulina velutipes, waste materials such as stipes, caps, and deformed mushrooms are produced, and most of them are directly discarded without treatment, which not only pollutes the environment but also causes great waste. Currently, the research on polysaccharides from the waste of Flammulina velutipes (FVSP) mainly focuses on extraction methods such as ultrasonic-microwave assisted and microwave, decolorization, and antibacterial activity research. So far, no application of the polysaccharide from the waste of Flammulina velutipes in inhibiting AGEs during the production of cookies has been found. Flammulina velutipes scraps ,FVSP) mainly focuses on the research of extraction methods such as ultrasonic-microwave assisted and microwave, decolorization, and antibacterial activity research. So far, no application of the polysaccharide from the waste of Flammulina velutipes in inhibiting AGEs during the production of cookies has been found. Summary of the Invention
[0005] In order to solve the problem of the relatively large generation of advanced glycation end products AGEs during the production of cookies, aiming at the current situation that the waste of Flammulina velutipes is rich in polysaccharides but not effectively utilized, the polysaccharide FVSP2b extracted and purified from the waste of Flammulina velutipes is added to the cookies, which can effectively inhibit the reaction between the carbonyl groups in the sugar and the protein, peptide, and amino acids in the cookie dough, inhibit protein carbonylation, reduce protein cross-linking, and thus effectively inhibit the advanced glycation end products in the cookies.
[0006] To achieve the above object, the technical solution of the present invention is: a method for inhibiting advanced glycation end products in cookie production, wherein polysaccharide FVSP2b from Flammulina velutipes waste is added to the cookie dough. The structural characteristics of the polysaccharide FVSP2b from Flammulina velutipes waste are as follows: the relative molecular mass is 236712 Da, and its monosaccharide composition is rhamnose, arabinose, xylose, mannose, glucose, and galactose, with a molar ratio of 11.8:52.3:42.8:1:1.03:45.9.
[0007] Preferably, adding the polysaccharide FVSP2b from Flammulina velutipes waste to the production of cookies includes the following steps; (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of low-gluten flour, (40 - 60) parts of butter, (25 - 40) parts of powdered sugar, (15 - 30) parts of polysaccharide FVSP2b from Flammulina velutipes waste, and (20 - 40) parts of egg liquid, add them to a blender and mix well by stirring. Then use a mold to form the dough into cookie dough.
[0008] (2) Baking: Bake the cookie dough at an upper fire temperature of 180 °C and a lower fire temperature of 120 °C for 15 minutes, and then cool it at room temperature to obtain the cookie product of the present invention.
[0009] Preferably, in step (1), 100 parts of low-gluten flour, 45 parts of butter, 30 parts of powdered sugar, 19 parts of polysaccharide FVSP2b from Flammulina velutipes waste, and 30 parts of egg liquid The method for separating and purifying the polysaccharide FVSP2b from Flammulina velutipes waste includes the following steps; (1) Extraction of crude polysaccharide from Flammulina velutipes waste Wash the Flammulina velutipes waste, dry it at 70 °C for 24 hours, then crush it, add deionized water according to a solid-liquid ratio of 1:20 (g / mL), extract at 80 °C for 6 hours, then centrifuge (10000 r / min) for 15 minutes to obtain the supernatant. Concentrate the supernatant under reduced pressure at 50 °C for 12 hours, add four times the volume of 95% ethanol to the concentrated solution, precipitate at 4 °C for 24 hours, centrifuge to obtain the precipitate, and freeze-dry the precipitate to obtain the crude polysaccharide FVSP from Flammulina velutipes waste.
[0010] (2) Separation and purification of the polysaccharide FVSP2b from Flammulina velutipes waste The crude polysaccharide FVSP from the waste of Flammulina velutipes was dissolved in distilled water to prepare a solution with a concentration of 10 mg / mL, which was loaded onto a DEAE-52 cellulose ion exchange column. First, it was eluted with 0.1 mol / L sodium acetate buffer solution, and then eluted with a 0 - 1.5 mol / L NaCl gradient solution at a flow rate of 2 mL / min. It was automatically collected, 10 mL per tube. The OD490 was detected by the phenol-sulfuric acid method, and an elution curve was plotted. According to the elution curve, three polysaccharide components FVSP1, FVSP2, and FVSP3 were collected. The eluate of the FVSP2 component was selected and loaded onto a Sepharose CL-6B gel filtration column, and eluted with 0.01 mol / L NaCl eluent at a flow rate of 2 mL / min. It was automatically collected, 10 mL per tube. The OD 490 was detected by the phenol-sulfuric acid method to plot an elution curve; according to the elution curve, two elution peaks FVSP2a and FVSP2b were obtained, and the eluate corresponding to the main peak FVSP2b with the largest peak area was selectively collected. The eluate was dialyzed through a dialysis bag for 24 h, and the dialyzed eluate was freeze-dried to obtain the Flammulina velutipes waste polysaccharide FVSP2b of the present invention.
[0011] Preferably, in the step (2), the dialysis of the eluate is carried out using a dialysis bag with a cut-off molecular weight of 5000 Da.
[0012] Preferably, the chromatographic columns used for loading in the step (2) are a DEAE-52 cellulose ion exchange column and a Sepharose CL-6B gel filtration column.
[0013] The beneficial technical effects of the present invention: (1) The waste of Flammulina velutipes was effectively utilized, and the Flammulina velutipes waste polysaccharide FVSP2b of the present invention was obtained through extraction, separation, and purification. It is a natural extract with good safety.
[0014] (2) The Flammulina velutipes waste polysaccharide FVSP2b of the present invention can effectively reduce the generation of advanced glycation end products during the production of cookies. Description of the Drawings
[0015] The present invention will be further described in detail below with reference to the drawings and specific embodiments.
[0016] Figure 1 It is the elution curve of the DEAE-52 cellulose ion exchange column for purifying the polysaccharide from the waste of Flammulina velutipes.
[0017] Figure 2 It is the Sepharose CL-6B elution curve of the Flammulina velutipes waste polysaccharide component FVSP2.
[0018] Figure 3It is the high performance liquid chromatography chart of the monosaccharide composition of Flammulina velutipes waste polysaccharide FVSP2b.
[0019] Figure 4 It is the molecular weight distribution chart of Flammulina velutipes waste polysaccharide FVSP2b.
[0020] Figure 5 It is the infrared spectrum chart of Flammulina velutipes waste polysaccharide FVSP2b. Specific embodiments
[0021] The embodiments of the present invention use Flammulina velutipes waste as raw material.
[0022] The reagents used in the embodiments of the present invention: glucose, phenol, concentrated sulfuric acid, ethanol, etc. are all of analytical grade.
[0023] 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 type ultraviolet-visible spectrophotometer; Nicolet iS20 Fourier transform infrared spectrometer; ALPHA 1-4 / 2-4 LSC freeze dryer; GC-14A gas chromatograph. All experiments are done in triplicate, and the data are expressed as mean ± SD. The statistical analysis of the data uses t-test or ANOVA analysis, and p < 0.05 is considered to have statistical differences. In addition, in the following embodiments, unless otherwise specified, all reagents, raw materials and instruments selected in the present invention are well-known and selected in the art, but do 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.
[0024] The Flammulina velutipes waste is cleaned, dried at 70 °C for 24 h, then crushed, deionized water is added according to the solid-liquid ratio of 1:20 (g / mL), extracted at 80 °C for 6 h, then centrifuged (10000 r / min) for 15 min to take the supernatant. The supernatant is concentrated under reduced pressure at 50 °C for 12 h. Four times the volume of 95% ethanol is added to the concentrated solution, and precipitated at 4 °C for 24 h. The precipitate is obtained by centrifugation, and the Flammulina velutipes waste crude polysaccharide FVSP is obtained after freeze-drying the precipitate.
[0025] The Flammulina velutipes waste crude polysaccharide FVSP is dissolved in distilled water to prepare a solution with a concentration of 10 mg / mL and loaded onto a DEAE-52 cellulose ion exchange column. First, it is eluted with 0.1 mol / L sodium acetate buffer solution, and then eluted with a 0-1.5 mol / L NaCl gradient solution at a flow rate of 2 mL / min. Automatically collect, 10 mL per tube, detect OD490 by phenol-sulfuric acid method, draw an elution curve. According to the elution curve, three polysaccharide components FVSP1, FVSP2, FVSP3 are collected (attached Figure 1), Select the FVSP2 component eluent, load it onto a Sepharose CL-6B gel filtration column, and elute it with a 0.01 mol / L NaCl eluent at a flow rate of 2 mL / min. Automatically collect 10 mL per tube and detect the OD by the phenol-sulfuric acid method. 490 , Plot the elution curve; according to the elution curve, obtain two elution peaks, FVSP2a and FVSP2b, selectively collect the eluent corresponding to the main peak FVSP2b with the largest peak area, dialyze the eluent through a dialysis bag for 24 h, and freeze-dry the dialyzed eluent to obtain the Flammulina velutipes waste polysaccharide FVSP2b of the present invention (Appended Figure 2 ).
[0026] Determination of monosaccharide composition: Take 10 mg of the Flammulina velutipes waste polysaccharide FVSP2b sample in a stoppered tube, add 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution, seal the tube under vacuum, and hydrolyze it at 121 °C for 1 h. After removing the excess TFA from the hydrolyzate, dry it under vacuum. Adopt 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 it at 90 °C for 30 min, take it out and cool it 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). Qualitatively judge the types of monosaccharides according to the retention time of the monosaccharides, and determine the proportional relationship between the monosaccharides according to the ratio of the peak areas.
[0027] Chromatographic conditions: Use an OV1701 elastic quartz capillary column (Φ0.32 mm × 30 m), the carrier gas is N2, the flow rate is 1.5 mL / min, the FID hydrogen flame detector, the vaporization chamber temperature is 260 °C, the detector temperature is 250 °C, and adopt a programmed temperature rise: the initial temperature is 150 °C, stay for 1 min, rise to 190 °C at a rate of 10 °C / min, stay for 1 min, and rise to 240 °C at a rate of 3 °C / min, and stay for 20 min.
[0028] Determination of relative molecular mass: Inject the standard Dextrans with relative molecular masses of 6100, 26290, 84000, 158000, and 291000 successively, record the retention time TR by HPLC, plot the standard curve with TR as the abscissa and LgM as the ordinate, and obtain the regression equation. Inject 20 μL of the sample to be measured, FVSP2b, and calculate the relative molecular mass of the polysaccharide through the regression equation according to the obtained TR. Chromatographic column: Waters UllrallydrogelTM Linear (Φ7.8 mm × 300 mm), detector: Waters2410 differential refractive index detector, use 0.1 mol / L NaNO3 as the mobile phase, the flow rate is 0.9 mL / min, and the column temperature is 45 °C.
[0029] Infrared spectroscopy determination: 1 mg of polysaccharide FVSP2b from Flammulina velutipes waste and 100 mg of KBr were mixed and ground into a tablet, and then measured using a Nicolet iS20 Fourier transform infrared spectrometer from Thermo Scientific. The scanning range was 4000 - 400 cm -1 , and the resolution was 4 cm -1 .
[0030] As Figure 3 shown, the monosaccharide composition of polysaccharide FVSP2b from Flammulina velutipes waste was rhamnose, arabinose, xylose, mannose, glucose, and galactose, and the molar ratio was 11.8:52.3:42.8:1:1.03:45.9.
[0031] As Figure 4 shown, the relative molecular mass of polysaccharide FVSP2b from Flammulina velutipes waste was 236712 Da.
[0032] As Figure 5 shown, from the infrared spectrogram of polysaccharide FVSP2b from Flammulina velutipes waste, a broad peak was observed at 3404.61 cm -1 , which was caused by the stretching vibration of -OH; the weak peak at 2923.74 cm -1 was a typical absorption peak of -CH, and the absorption peak at 1037.25 cm −1 indicated a pyranose structure. The band at 889.29 cm −1 was attributed to the pyranose structure in the polysaccharide, indicating that the sugar was a pyranose.
[0033] (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of low-gluten flour, 45 parts of butter, 27 parts of powdered sugar, 18 parts of polysaccharide FVSP2b from Flammulina velutipes waste, and 25 parts of egg liquid, add them to a blender and mix well by stirring. Then, use a mold to form the dough into a cookie dough.
[0034] (2) Baking: Bake the cookie dough at an upper fire temperature of 180 °C and a lower fire temperature of 120 °C for 15 min, and then cool it at room temperature to obtain the cookie product of the present invention.
[0035] (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of low-gluten flour, 50 parts of butter, 30 parts of powdered sugar, 23 parts of polysaccharide FVSP2b from Flammulina velutipes waste, and 30 parts of egg liquid, add them to a blender and mix well by stirring. Then, use a mold to form the dough into a cookie dough.
[0036] (2) Baking: Bake the cookie dough at an upper fire temperature of 180 °C and a lower fire temperature of 120 °C for 15 min, and then cool it at room temperature to obtain the cookie product of the present invention.
[0037] (1)Mixing of raw materials and forming of cookie dough: Weigh 100 parts of low-gluten flour, 55 parts of butter, 35 parts of powdered sugar, 26 parts of polysaccharide FVSP2b from the waste of Flammulina velutipes, and 35 parts of egg liquid, add them to a blender and mix well by stirring. Then use a mold to form the dough into cookie blanks.
[0038] (2)Baking: Bake the cookie blanks at an upper fire temperature of 180 °C and a lower fire temperature of 120 °C for 15 minutes, and then cool them at room temperature to obtain the cookie products of the present invention.
[0039] The difference between Comparative Example 1 and Example 4 is that: polysaccharide FVSP2b from the waste of Flammulina velutipes is not added to the raw materials of the cookie blank, and the others are the same as in Example 4.
[0040] The difference between Comparative Example 1 and Example 5 is that: polysaccharide FVSP2b from the waste of Flammulina velutipes is not added to the raw materials of the cookie blank, and the others are the same as in Example 5.
[0041] The difference between Comparative Example 1 and Example 6 is that: polysaccharide FVSP2b from the waste of Flammulina velutipes is not added to the raw materials of the cookie blank, and the others are the same as in Example 6.
[0042] Determination of fluorescent AGEs in cookies Weigh 4 g of the crushed cookie sample, add 200 mL of phosphate buffer solution (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 at an excitation wavelength of 350 nm and an emission wavelength of 440 nm using a fluorescence spectrophotometer. The fluorescence intensity is expressed in AU / mg, indicating the content of fluorescent AGEs in the sample, that is, the content of fluorescent AGEs per milligram of cookies. Conduct parallel tests three times, and the results are shown in Table 1: Table 1 Item Content of fluorescent AGEs (AU / mg) Comparative Example 1 1328±5.28a Comparative Example 2 1311±4.85a Comparative Example 3 1295±5.12a Example 4 759±3.87b Example 5 766±4.26b Example 6 753±3.59b Note: Different letters indicate significant statistical differences between different groups (p < 0.05).
[0043] As can be seen from Table 1, by using the method of the present invention, polysaccharide FVSP2b from the waste of Flammulina velutipes is prepared by separation and purification, and adding polysaccharide FVSP2b from the waste of Flammulina velutipes to the raw materials of the cookie blank to produce cookies can significantly reduce the content of advanced glycation end products in cookies (p < 0.05).
[0044] The above embodiments do not limit the present invention in any way. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A method for inhibiting advanced glycation end products during the production process of cookies, characterized in that: Flammulina velutipes offcuts polysaccharide FVSP2b is added to the cookie dough. The structural characteristics of the Flammulina velutipes offcuts polysaccharide FVSP2b are as follows: the relative molecular mass is 236712 Da, and its monosaccharide composition is rhamnose, arabinose, xylose, mannose, glucose, and galactose, with a molar ratio of 11.8:52.3:42.8:1:1.03:45.
9.
2. A method for inhibiting advanced glycation end products during the production of cookies according to claim 1, characterized in that: Adding Flammulina velutipes offcuts polysaccharide FVSP2b to the production of cookies includes the following steps; (1) Mixing raw materials and forming cookie dough: Weigh 100 parts of low-gluten flour, (40 - 60) parts of butter, (25 - 40) parts of powdered sugar, (15 - 30) parts of Flammulina velutipes offcuts polysaccharide FVSP2b, and (20 - 40) parts of egg liquid, add them to a blender and mix and beat evenly, and use a mold to form the dough into cookie dough; (2) Baking: Bake the cookie dough at an upper fire temperature of 180 °C and a lower fire temperature of 120 °C for 15 min, and cool it at room temperature to obtain the cookie product of the present invention.
3. The method for separating and purifying Flammulina velutipes waste polysaccharide FVSP2b is characterized in that: Includes the following steps; (1) Extraction of Flammulina velutipes offcuts crude polysaccharide Wash the Flammulina velutipes offcuts, dry them at 70 °C for 24 h, then crush them, add deionized water according to a solid-liquid ratio of 1:20 (g / mL), extract at 80 °C for 6 h, then centrifuge (10000 r / min) for 15 min to obtain the supernatant. Concentrate the supernatant under reduced pressure at 50 °C for 12 h, add four times the volume of 95% ethanol to the concentrated solution, precipitate at 4 °C for 24 h, centrifuge to obtain the precipitate, and freeze-dry the precipitate to obtain the Flammulina velutipes offcuts crude polysaccharide FVSP; (2) Isolation and purification of Flammulina velutipes offcuts polysaccharide FVSP2b The crude polysaccharide FVSP from the waste of Flammulina velutipes was dissolved in distilled water to prepare a solution with a concentration of 10 mg / mL, which was loaded onto a DEAE-52 cellulose ion exchange column. First, it was eluted with 0.1 mol / L sodium acetate buffer solution, and then eluted with a 0-1.5 mol / L NaCl gradient solution at a flow rate of 2 mL / min. It was automatically collected, 10 mL per tube, and the OD490 was detected by the phenol-sulfuric acid method to draw an elution curve. According to the elution curve, three polysaccharide components FVSP1, FVSP2, and FVSP3 were collected. The eluate of the FVSP2 component was selected and loaded onto a Sepharose CL-6B gel filtration column, and eluted with 0.01 mol / L NaCl eluent at a flow rate of 2 mL / min. It was automatically collected, 10 mL per tube, and the OD was detected by the phenol-sulfuric acid method 490 , and an elution curve was drawn; According to the elution curve, two elution peaks FVSP2a and FVSP2b are obtained. Selectively collect the eluate corresponding to the main peak FVSP2b with the largest peak area. Dialyze the eluate with a dialysis bag for 24 h, and freeze-dry the dialyzed eluate to obtain the Flammulina velutipes offcuts polysaccharide FVSP2b of the present invention.
4. The isolation and purification of the polysaccharide FVSP2b from the waste materials of Flammulina velutipes according to claim 3, characterized in that: In the step (2), the eluate is dialyzed using a dialysis bag with a cut-off molecular weight of 5000 Da.
5. The separation and purification of the polysaccharide FVSP2b from the waste materials of Flammulina velutipes according to claim 3, characterized in that: In the step (2), the chromatographic columns for sample loading are DEAE-52 cellulose ion exchange column and Sepharose CL-6B gel filtration column.