A process for extracting polysaccharide based on dandelion residue fermentation

Abstract

This invention relates to the field of biopolysaccharide extraction technology, and discloses a process for extracting polysaccharides based on the fermentation of dandelion residue. The process includes: inoculating Bacillus subtilis into a culture medium and activating it to obtain an activated bacterial solution; mixing dandelion residue with water and sterilizing to obtain a fermentation culture medium; inoculating the activated bacterial solution into the fermentation culture medium, fermenting, centrifuging, concentrating, adding anhydrous ethanol to precipitate and redissolve, obtaining a crude polysaccharide reconstituted solution; deproteinizing with Sevag reagent, dialysis, and drying to obtain crude polysaccharide from the fermented dandelion residue; and eluting and purifying sequentially through a cellulose chromatography column and a dextran gel chromatography column to obtain purified dandelion polysaccharide components. This invention improves the extraction rate by controlling the fermentation temperature and inoculum volume ratio to balance the polysaccharide release and consumption process; it purifies and targets the separation of acidic polysaccharide components, retaining the natural triple helix conformation and uronic acid groups, providing hydrogen protons to neutralize free radicals and enhance antioxidant enzyme activity, thereby improving cellular oxidative damage.

Description

Technical Field

[0001] This invention relates to the field of biopolysaccharide extraction technology, specifically a process for extracting polysaccharides based on the fermentation of dandelion residue. Background Technology

[0002] The dandelion residue produced after processing and utilization contains polysaccharide components. Current technologies for extracting polysaccharides from dandelion residue employ simple solvent extraction or microbial fermentation without controlled fermentation parameters. In existing fermentation extraction operations, the lack of strict control over fermentation temperature and inoculum volume ratio makes it impossible to balance the biochemical reactions of microbial growth, extracellular enzyme accumulation, and polysaccharide release. Microorganisms with excessively long growth times over-consume the dissolved polysaccharides as a carbon source, resulting in a low polysaccharide extraction rate.

[0003] Current purification procedures lack targeted elution steps for acidic polysaccharides, failing to completely eliminate nucleic acid and free protein impurities. Existing crude extraction and purification processes easily disrupt the natural triple helix conformation and highly branched skeletal structure of polysaccharides, causing the loss of uronic acid groups within the polysaccharide molecular backbone. Due to the unwinding of the polysaccharide conformation and the shedding of acidic groups, the final extract's ability to provide hydrogen protons to neutralize free hydroxyl radicals is diminished.

[0004] The structurally damaged extract cannot participate in regulating the intracellular antioxidant defense mechanism, cannot enhance the activity of catalase and glutathione peroxidase, and cannot prevent the accumulation of malondialdehyde, a lipid peroxidation product in cells. As a result, the prepared dandelion polysaccharide does not have the ability to improve the oxidative stress damage state of bovine mammary epithelial cells.

[0005] Therefore, this invention proposes a process for extracting polysaccharides based on the fermentation of dandelion residue to overcome the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a process for extracting polysaccharides from dandelion residue through fermentation, which solves the problems of low extraction rate of polysaccharides from dandelion residue and loss of natural spatial conformation and antioxidant bioactivity of the extracted products.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a process for extracting polysaccharides based on the fermentation of dandelion residue, comprising the following steps: Bacillus subtilis was inoculated into an activation medium and activated twice to obtain an activated bacterial solution; Dandelion residue was mixed with water at a weight-to-volume ratio of 1:20 and then autoclaved to obtain a fermentation medium. The activated bacterial solution was inoculated into the fermentation medium at an inoculation volume ratio of 1% to 10% and fermented at a fermentation temperature of 27℃ to 37℃ for 6 hours to 30 hours to obtain a fermentation mixture. The fermentation mixture was centrifuged at 6500 r / min for 10 min to obtain the fermentation supernatant. The fermentation supernatant was concentrated under vacuum at 55°C to one-quarter of its original volume to obtain the fermentation concentrate. Add four times the volume of anhydrous ethanol to the fermentation concentrate and refrigerate overnight at 4°C to obtain a polysaccharide precipitation mixture. The polysaccharide precipitation mixture was centrifuged to collect the precipitate, and the precipitate was redissolved in pure water to obtain a crude polysaccharide redissolved solution. The Sevag reagent is obtained by mixing n-butanol and chloroform in a volume ratio of 1:4. Sevag reagent and crude polysaccharide complex solution were mixed at a volume ratio of 1:4 and repeatedly extracted to remove protein until no protein precipitate layer was obtained. The upper aqueous layer was then separated by centrifugation to obtain the polysaccharide aqueous phase. The polysaccharide aqueous phase was concentrated by rotary evaporation and desalted by dialysis using a dialysis bag, followed by freeze drying to obtain crude polysaccharide from fermented dandelion residue; The crude polysaccharide from fermented dandelion residue was dissolved in pure water and loaded into a DEAE-52 cellulose chromatography column. Step elution was performed sequentially with distilled water, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution and 0.6 mol / L NaCl solution at a flow rate of 1 mL / min. The target polysaccharide eluent corresponding to the 0.4 mol / L NaCl solution was collected to obtain the first chromatography eluent. The first chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was reconstituted with pure water and loaded into a G-75 dextran gel chromatography column. The column was eluted with pure water at a flow rate of 0.5 mL / min, and the eluent fraction of a single symmetrical target polysaccharide was collected to obtain the second chromatography eluent. The second chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain the refined dandelion polysaccharide fraction.

[0008] As a preferred technical solution, the activated bacterial solution is inoculated into the fermentation medium at a volume ratio of 5% and fermented at a fermentation temperature of 33°C for 24 hours to obtain a fermentation mixture.

[0009] Bacillus subtilis grows and multiplies in the fermentation medium, secreting extracellular enzymes. These enzymes degrade the cell walls of dandelion residue, promoting the release of polysaccharides from the dandelion residue cells into the fermentation mixture. Controlling the fermentation environmental parameters balances the processes of microbial growth, extracellular enzyme accumulation, and polysaccharide release, avoiding excessive polysaccharide consumption and yielding a product containing total sugars and a high proportion of uronic acids, free of protein impurities.

[0010] Protein removal combined with a stepwise elution step in anion exchange chromatography eliminated nucleic acid and free protein impurities. Elution with 0.4 mol / L NaCl solution targeted the separation of acidic polysaccharide components. The refined dandelion polysaccharide fraction obtained retained its natural triple helix conformation and highly branched skeletal structure.

[0011] The molecular backbone of refined dandelion polysaccharide contains uronic acid groups and a triple helix structure, which can provide hydrogen protons to neutralize free hydroxyl radicals and block the oxidation chain reaction of hydrogen peroxide. In a cellular testing environment, refined dandelion polysaccharide can directly neutralize reactive oxygen species (ROS) that enter the cell, reducing the damage of ROS to cell membrane lipid molecules, protecting the integrity of the cell membrane structure, and preventing the leakage of intracellular lactate dehydrogenase. Simultaneously, refined dandelion polysaccharide molecules participate in regulating intracellular antioxidant defense mechanisms, increasing the activity of catalase and glutathione peroxidase, accelerating the decomposition of intracellular hydrogen peroxide and lipid peroxides, reducing malondialdehyde (MDA) accumulation, and demonstrating the ability to improve the oxidative stress damage state of bovine mammary epithelial cells.

[0012] This invention provides a process for extracting polysaccharides from dandelion residue through fermentation. It has the following beneficial effects: 1. This invention controls the fermentation environment parameters of Bacillus subtilis, inoculates the activated bacterial solution into the fermentation medium at a volume ratio of 5%, and ferments at 33°C for 24 hours. This balances the biochemical reaction processes of microbial growth, extracellular enzyme accumulation, and polysaccharide release, avoids excessive consumption of polysaccharides as a carbon source, improves the extraction rate of polysaccharides from dandelion residue, and prepares a product containing total sugar and uronic acid components.

[0013] 2. This invention utilizes Sevag reagent to repeatedly extract and deproteinize crude polysaccharide solution, followed by loading the crude polysaccharide from fermented dandelion residue onto a DEAE-52 cellulose chromatography column and collecting the target polysaccharide elution fraction corresponding to 0.4 mol / L NaCl solution. This process removes nucleic acid and free protein impurities, and targets and separates the acidic polysaccharide component. As a result, the refined dandelion polysaccharide component retains the natural triple helix spatial conformation and highly branched skeletal characteristics.

[0014] 3. This invention obtains refined dandelion polysaccharide components through a combined fermentation extraction step and a chromatography purification step. The uronic acid groups and triple helix structure retained in the molecular skeleton provide hydrogen protons to neutralize free hydroxyl radicals. At the same time, the refined dandelion polysaccharide components participate in regulating the intracellular antioxidant defense mechanism, enhance the activity of catalase and glutathione peroxidase, reduce the accumulation of malondialdehyde in cells, and have the ability to improve the oxidative stress damage state of bovine mammary epithelial cells. Attached Figure Description

[0015] Figure 1 This is the glucose standard curve of the present invention.

[0016] Figure 2 This is the standard curve of uronic acid in this invention.

[0017] Figure 3 This is the protein standard curve of the present invention.

[0018] Figure 4 This is the experimental spectrum of dandelion polysaccharide Congo red of the present invention.

[0019] Figure 5 This is the ultraviolet spectrum of dandelion polysaccharide of the present invention.

[0020] Figure 6 This is the iodine-potassium iodide absorption spectrum of dandelion polysaccharide of the present invention.

[0021] Figure 7 This is the spectrum of the β-elimination reaction of dandelion polysaccharide in this invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0023] Examples 1-3: Example 1: This embodiment provides a process for extracting polysaccharides based on the fermentation of dandelion residue, including the following steps: Bacillus subtilis was inoculated into an activation medium and activated twice to obtain an activated bacterial solution; Dandelion residue was mixed with water at a weight-to-volume ratio of 1:20 and then autoclaved to obtain a fermentation medium. The activated bacterial solution was inoculated into the fermentation medium at a ratio of 5% by volume and fermented at 33°C for 24 hours to obtain a fermentation mixture. The fermentation mixture was centrifuged at 6500 r / min for 10 min to obtain the fermentation supernatant. The fermentation supernatant was concentrated under vacuum at 55°C to one-quarter of its original volume to obtain the fermentation concentrate. Add four times the volume of anhydrous ethanol to the fermentation concentrate and refrigerate overnight at 4°C to obtain a polysaccharide precipitation mixture. The polysaccharide precipitation mixture was centrifuged to collect the precipitate, and the precipitate was redissolved in pure water to obtain a crude polysaccharide redissolved solution. The Sevag reagent is obtained by mixing n-butanol and chloroform in a volume ratio of 1:4. Sevag reagent and crude polysaccharide complex solution were mixed at a volume ratio of 1:4 and repeatedly extracted to remove protein until no protein precipitate layer was obtained. The upper aqueous layer was then separated by centrifugation to obtain the polysaccharide aqueous phase. The polysaccharide aqueous phase was concentrated by rotary evaporation and desalted by dialysis using a dialysis bag, followed by freeze drying to obtain crude polysaccharide from fermented dandelion residue; The crude polysaccharide from fermented dandelion residue was dissolved in pure water and loaded into a DEAE-52 cellulose chromatography column. Step elution was performed sequentially with distilled water, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution and 0.6 mol / L NaCl solution at a flow rate of 1 mL / min. The target polysaccharide eluent corresponding to the 0.4 mol / L NaCl solution was collected to obtain the first chromatography eluent. The first chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was reconstituted with pure water and loaded into a G-75 dextran gel chromatography column. The column was eluted with pure water at a flow rate of 0.5 mL / min, and the eluent fraction of a single symmetrical target polysaccharide was collected to obtain the second chromatography eluent. The second chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain the refined dandelion polysaccharide fraction.

[0024] Example 2: This embodiment provides a process for extracting polysaccharides based on the fermentation of dandelion residue, including the following steps: Bacillus subtilis was inoculated into an activation medium and activated twice to obtain an activated bacterial solution; Dandelion residue was mixed with water at a weight-to-volume ratio of 1:20 and then autoclaved to obtain a fermentation medium. The activated bacterial solution was inoculated into the fermentation medium at a ratio of 10% of the inoculum volume and fermented at 37°C for 30 hours to obtain the fermentation mixture. The fermentation mixture was centrifuged at 6500 r / min for 10 min to obtain the fermentation supernatant. The fermentation supernatant was concentrated under vacuum at 55°C to one-quarter of its original volume to obtain the fermentation concentrate. Add four times the volume of anhydrous ethanol to the fermentation concentrate and refrigerate overnight at 4°C to obtain a polysaccharide precipitation mixture. The polysaccharide precipitation mixture was centrifuged to collect the precipitate, and the precipitate was redissolved in pure water to obtain a crude polysaccharide redissolved solution. The Sevag reagent is obtained by mixing n-butanol and chloroform in a volume ratio of 1:4. Sevag reagent and crude polysaccharide complex solution were mixed at a volume ratio of 1:4 and repeatedly extracted to remove protein until no protein precipitate layer was obtained. The upper aqueous layer was then separated by centrifugation to obtain the polysaccharide aqueous phase. The polysaccharide aqueous phase was concentrated by rotary evaporation and desalted by dialysis using a dialysis bag, followed by freeze drying to obtain crude polysaccharide from fermented dandelion residue; The crude polysaccharide from fermented dandelion residue was dissolved in pure water and loaded into a DEAE-52 cellulose chromatography column. Step elution was performed sequentially with distilled water, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution and 0.6 mol / L NaCl solution at a flow rate of 1 mL / min. The target polysaccharide eluent corresponding to the 0.4 mol / L NaCl solution was collected to obtain the first chromatography eluent. The first chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was reconstituted with pure water and loaded into a G-75 dextran gel chromatography column. The column was eluted with pure water at a flow rate of 0.5 mL / min, and the eluent fraction of a single symmetrical target polysaccharide was collected to obtain the second chromatography eluent. The second chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain the refined dandelion polysaccharide fraction.

[0025] Example 3: This embodiment provides a process for extracting polysaccharides based on the fermentation of dandelion residue, including the following steps: Bacillus subtilis was inoculated into an activation medium and activated twice to obtain an activated bacterial solution; Dandelion residue was mixed with water at a weight-to-volume ratio of 1:20 and then autoclaved to obtain a fermentation medium. The activated bacterial solution was inoculated into the fermentation medium at a volume ratio of 1% and fermented at 27°C for 6 hours to obtain the fermentation mixture. The fermentation mixture was centrifuged at 6500 r / min for 10 min to obtain the fermentation supernatant. The fermentation supernatant was concentrated under vacuum at 55°C to one-quarter of its original volume to obtain the fermentation concentrate. Add four times the volume of anhydrous ethanol to the fermentation concentrate and refrigerate overnight at 4°C to obtain a polysaccharide precipitation mixture. The polysaccharide precipitation mixture was centrifuged to collect the precipitate, and the precipitate was redissolved in pure water to obtain a crude polysaccharide redissolved solution. The Sevag reagent is obtained by mixing n-butanol and chloroform in a volume ratio of 1:4. Sevag reagent and crude polysaccharide complex solution were mixed at a volume ratio of 1:4 and repeatedly extracted to remove protein until no protein precipitate layer was obtained. The upper aqueous layer was then separated by centrifugation to obtain the polysaccharide aqueous phase. The polysaccharide aqueous phase was concentrated by rotary evaporation and desalted by dialysis using a dialysis bag, followed by freeze drying to obtain crude polysaccharide from fermented dandelion residue; The crude polysaccharide from fermented dandelion residue was dissolved in pure water and loaded into a DEAE-52 cellulose chromatography column. Step elution was performed sequentially with distilled water, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution and 0.6 mol / L NaCl solution at a flow rate of 1 mL / min. The target polysaccharide eluent corresponding to the 0.4 mol / L NaCl solution was collected to obtain the first chromatography eluent. The first chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain a preliminarily purified polysaccharide. The preliminarily purified polysaccharide was reconstituted with pure water and loaded into a G-75 dextran gel chromatography column. The column was eluted with pure water at a flow rate of 0.5 mL / min, and the eluent fraction of a single symmetrical target polysaccharide was collected to obtain the second chromatography eluent. The second chromatography eluent was desalted and concentrated using a dialysis bag, and then freeze-dried to obtain the refined dandelion polysaccharide fraction.

[0026] Comparative Examples 1-3: Comparative Example 1: Compared with Example 1, the difference is that the fermentation temperature is replaced with 37°C and the fermentation time is replaced with 30h, while the rest are the same.

[0027] Comparative Example 2: Compared with Example 1, the difference is that the inoculation volume ratio of the activated bacterial solution into the fermentation medium is replaced with 3%, while the rest are the same.

[0028] Comparative Example 3: Compared with Example 1, the only difference is that the fermentation temperature is replaced with 30°C, otherwise they are the same.

[0029] Test Examples 1-4: Test Example 1: Accurately weigh glucose to constant weight, add distilled water to prepare glucose standard stock solution, pipette different volumes of glucose standard stock solution into glass test tubes, add distilled water to make up to volume, add 5% phenol solution and concentrated sulfuric acid, heat in a water bath to constant temperature, cool to room temperature, measure absorbance at 490 nm and plot total sugar standard curve. The purified dandelion polysaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were weighed and prepared into polysaccharide sample solutions. The absorbance of the polysaccharide sample solutions at 490 nm was measured using the same steps as those used to plot the total sugar standard curve. The total sugar mass fraction was calculated in conjunction with the total sugar standard curve.

[0030] Accurately weigh galacturonic acid to constant weight, add distilled water to prepare galacturonic acid standard stock solution, pipette different volumes of galacturonic acid standard stock solution into stoppered glass test tubes, add distilled water to make up to volume, add concentrated sulfuric acid under ice-water bath conditions, heat in boiling water bath to constant temperature, cool to room temperature, add 0.1% carbazole anhydrous ethanol solution and react in the dark, measure the absorbance value at 530 nm and plot the galacturonic acid standard curve; The purified dandelion polysaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were weighed and prepared into polysaccharide sample solutions. The absorbance of the polysaccharide sample solutions at 530 nm was measured using the same steps as those used to plot the uronic acid standard curve. The mass fraction of uronic acid was calculated by combining the uronic acid standard curve.

[0031] Accurately weigh bovine serum albumin and prepare a protein standard stock solution with distilled water. Pipette different volumes of the protein standard stock solution into glass test tubes, add distilled water to make up to the volume, add Coomassie Brilliant Blue G-250 staining solution, shake well and let stand to react. Measure the absorbance value at 595 nm to plot a protein standard curve. Weigh the purified dandelion polysaccharide groups obtained in Examples 1 to 3 and Comparative Examples 1 to 3 to prepare polysaccharide sample solutions. Measure the absorbance value of the polysaccharide sample solutions at 595 nm using the same steps as plotting the protein standard curve. Calculate the protein mass fraction based on the protein standard curve.

[0032] Fermentation supernatants from Examples 1 to 3 and Comparative Examples 1 to 3 were collected. The polysaccharide concentration in the fermentation supernatant was determined by the phenol-sulfuric acid method. The polysaccharide extraction rate was calculated based on the relationship between the polysaccharide concentration in the fermentation supernatant and the volume of the fermentation medium.

[0033] Table 1. Polysaccharide extraction rate and physicochemical property test data in each example and comparative example

[0034] The test results are as follows: See attached document Figure 1 , attached Figure 1 The horizontal axis represents glucose concentration in mg / mL, and the vertical axis represents absorbance at 490 nm.

[0035] See attached document Figure 2 , attached Figure 2 The horizontal axis represents the uronic acid concentration in mg / mL, and the vertical axis represents the absorbance value at 530 nm.

[0036] See attached document Figure 3 , attached Figure 3 The horizontal axis represents protein concentration in μg / mL, and the vertical axis represents absorbance at 595 nm.

[0037] Based on the data in Table 1 and the appendix Figure 1 Appendix Figure 2 and attached Figure 3 The content shows that by measuring the absorbance values ​​of each embodiment and comparative example and substituting them into the appendix... Figure 1 Appendix Figure 2 and attached Figure 3 The standard curve linear regression equation was calculated. In Example 1, fermentation conditions of 33℃, 24h and 5% inoculum were used. The polysaccharide extraction rate reached 1.42%, the uronic acid mass fraction reached 51.34%, and the protein mass fraction was 0.

[0038] Bacillus subtilis grows and multiplies in the fermentation medium and secretes extracellular enzymes. These extracellular enzymes degrade the cell walls of dandelion residues, promoting the dissolution of polysaccharides from inside the dandelion residue cells into the fermentation mixture.

[0039] Comparative Example 1 changed the fermentation time and temperature; Comparative Example 2 changed the inoculum volume ratio; and Comparative Example 3 changed the fermentation temperature. The resulting polysaccharide extraction rate and uronic acid mass fraction were all lower than those calculated in Example 1. A fermentation temperature deviation of 33℃ altered the enzymatic reaction rate of Bacillus subtilis; a fermentation time deviation of 24 hours caused the microorganisms to consume the already dissolved polysaccharides as a carbon source; and an inoculum volume ratio deviation of 5% resulted in insufficient enzyme concentration or excessively rapid nutrient consumption.

[0040] The combination of conditions in Example 1 balanced the processes of microbial growth, extracellular enzyme accumulation, and polysaccharide release, avoiding excessive consumption of polysaccharides and obtaining a refined dandelion polysaccharide component containing total sugars and uronic acid without protein impurities.

[0041] Test Example 2: Accurately weigh the refined dandelion polysaccharide components obtained in Examples 1 to 3 and Comparative Examples 1 to 3, add them to distilled water to prepare a polysaccharide solution, add Congo red solution to the polysaccharide solution and shake well, add sodium hydroxide solution dropwise to adjust the final concentration of sodium hydroxide in the mixed reaction solution to different concentration gradients between 0 mol / L and 0.5 mol / L, let it stand at room temperature for 10 min, and scan and record the maximum absorption wavelength of the mixed reaction solution in the wavelength range of 400 nm to 600 nm.

[0042] Accurately weigh the refined dandelion polysaccharide components obtained in Examples 1 to 3 and Comparative Examples 1 to 3, add them to distilled water to prepare a polysaccharide solution, place the polysaccharide solution in a quartz cuvette, and use a UV-Vis spectrophotometer to perform a full wavelength scan in the wavelength range of 200 nm to 400 nm, and record the absorbance values ​​of the polysaccharide solution at 260 nm and 280 nm.

[0043] Accurately weigh the refined dandelion polysaccharide components obtained in Examples 1 to 3 and Comparative Examples 1 to 3, add them to distilled water to prepare a polysaccharide solution, add iodine-potassium iodide reagent to the polysaccharide solution and react under light-protected conditions, use a spectrophotometer to perform a full wavelength scan in the wavelength range of 300 nm to 800 nm, and record the absorbance value at each wavelength.

[0044] Accurately weigh the refined dandelion polysaccharide components obtained in Examples 1 to 3 and Comparative Examples 1 to 3, add them to distilled water to prepare a polysaccharide solution, add an equal volume of 0.2 mol / L sodium hydroxide solution to the polysaccharide solution, place it in a 45°C constant temperature water bath and heat it to react. Scan the wavelength range of 220 nm to 400 nm using a spectrophotometer and record the change in absorbance value of the mixed reaction solution at 240 nm.

[0045] Table 2. Polysaccharide structure test data in each embodiment and comparative example

[0046] The test results are as follows: See attached document Figure 4 , attached Figure 4 The horizontal axis represents the concentration of sodium hydroxide (NaOH) in mol / L, and the vertical axis represents the maximum absorption wavelength in nm. In the figure, the solid lines marked with squares represent the polysaccharide fraction eluted with distilled water in the first chromatography eluent; the solid lines marked with circles represent the polysaccharide fraction eluted with 0.2 mol / L NaCl solution in the first chromatography eluent; the solid lines marked with triangles represent the purified dandelion polysaccharide fraction obtained in Example 1; and the solid lines marked with diamonds represent the blank control mixture.

[0047] See attached document Figure 5 , attached Figure 5 The horizontal axis represents wavelength in nm, and the vertical axis represents absorbance. The solid line in the figure represents the purified dandelion polysaccharide component obtained in Example 1.

[0048] See attached document Figure 6 , attached Figure 6 The horizontal axis represents wavelength in nm, and the vertical axis represents absorbance. The black solid line in the figure represents the purified dandelion polysaccharide component obtained in Example 1.

[0049] See attached document Figure 7 , attached Figure 7 The horizontal axis represents wavelength in nm, and the vertical axis represents absorbance. The solid line in the figure represents the purified dandelion polysaccharide obtained in Example 1, and the dashed line represents the reaction product of the purified dandelion polysaccharide obtained in Example 1 mixed with sodium hydroxide solution and heated.

[0050] Based on the data in Table 2 and Figures 4 to 7 The content can be obtained in Figure 4 In Example 1, the maximum absorption wavelength of the mixed reaction solution red-shifted and increased to 528.3 nm when the sodium hydroxide concentration reached 0.1 mol / L. With further increases in sodium hydroxide concentration, the maximum absorption wavelength showed a decreasing trend. The red-shift in absorption wavelength is caused by the formation of a complex between polysaccharide molecules and Congo red molecules in a low-concentration alkaline environment. In a high-concentration alkaline environment, the hydrogen bonds within the polysaccharide molecules break, and the polysaccharide spatial conformation unwinds into a random single-chain coiled state, resulting in a decrease in absorption wavelength. This demonstrates that the purified dandelion polysaccharide component obtained in Example 1 possesses a triple-helix spatial structure.

[0051] exist Figure 5 As shown in Table 2, the polysaccharide solutions of Examples 1 to 3 did not show absorption peaks at wavelengths of 260 nm and 280 nm, and the absorbance values ​​were close to 0. The nucleic acid substances had a characteristic absorption peak at 260 nm, and the protein substances had a characteristic absorption peak at 280 nm. The presence of nucleic acid and free protein impurities was eliminated by combining the extraction and deproteinization and chromatography elution steps.

[0052] exist Figure 6 In Example 1, the polysaccharide solution exhibited an absorption peak near 350 nm but no absorption peak at 565 nm. Sugars with long linear chains, when combined with iodine-potassium iodide reagent, would produce an absorption peak at 565 nm. The purified dandelion polysaccharide component of Example 1 has a highly branched molecular skeleton and lacks long linear chains.

[0053] exist Figure 7In Example 1, after the polysaccharide solution was heated with sodium hydroxide solution, the absorbance at 240 nm did not show an increasing trend. Glycoproteins containing —O-glycopeptide bonds undergo β-elimination under alkaline conditions, leading to an increase in the absorbance of the product at 240 nm. The purified dandelion polysaccharide fraction obtained in Example 1 does not contain bound —O-glycopeptide bonds and is a pure carbohydrate. Combining the fermentation conditions of Bacillus subtilis with the step-elution steps of the chromatography column, the obtained purified dandelion polysaccharide fraction retained its natural triple helix conformation and highly branched skeletal characteristics.

[0054] Test Example 3: Accurately weigh the refined dandelion polysaccharide components obtained in Examples 1 to 3 and Comparative Examples 1 to 3, the crude polysaccharide from fermented dandelion residue obtained in Example 1, and vitamin C. Add distilled water to prepare polysaccharide sample solutions and control solutions with concentration gradients ranging from 0.2 mg / mL to 1.0 mg / mL. Place quantitative amounts of the polysaccharide sample solutions and control solutions into glass test tubes, add ferrous sulfate solution and salicylic acid-ethanol solution, mix thoroughly, add hydrogen peroxide solution to initiate the reaction, and heat in a 37°C water bath for 30 min. Measure the absorbance of the reaction mixture at 510 nm and calculate the hydroxyl radical scavenging rate by combining the absorbance of the blank control group.

[0055] Accurately weigh potassium persulfate powder and ABTS powder to prepare a mixed solution. Allow the solution to react at room temperature and in the dark for 12-16 hours to generate the ABTS free radical working solution. Dilute the ABTS free radical working solution with anhydrous ethanol until the absorbance at 734 nm is approximately 0.7. Add quantitative amounts of polysaccharide sample solution and control solution to the diluted ABTS free radical working solution. Allow the solution to react at room temperature and in the dark for 6 minutes. Measure the absorbance of the reaction mixture at 734 nm and calculate the ABTS free radical scavenging rate by combining the absorbance of the blank control group.

[0056] Table 3. In vitro antioxidant activity test data of polysaccharides in each example and comparative example.

[0057] The test results are as follows: According to the data in Table 3, when the sample concentration increased from 0.5 mg / mL to 1.0 mg / mL, the hydroxyl radical scavenging rate and ABTS radical scavenging rate of all groups showed an increasing trend.

[0058] In Table 3, when the sample concentration reached 1.0 mg / mL, the scavenging rate of hydroxyl radicals of the refined dandelion polysaccharide obtained in Example 1 reached 89.24%, which was higher than the scavenging rate of hydroxyl radicals of the crude polysaccharide from fermented dandelion residue obtained in Example 1, and also higher than the refined dandelion polysaccharide obtained in Examples 2, 3 and all comparative examples.

[0059] The molecular skeleton of refined dandelion polysaccharide contains uronic acid groups and a triple helix structure. It contains a large number of exposed hydroxyl groups, which can provide hydrogen protons to neutralize free hydroxyl radicals and block the oxidation chain reaction of hydrogen peroxide.

[0060] In Table 3, when the sample concentration reached 1.0 mg / mL, the crude polysaccharide from fermented dandelion residue obtained in Example 1 achieved a 94.62% scavenging rate of ABTS free radicals, which was higher than that of the purified dandelion polysaccharide obtained after anion exchange chromatography. Before elution separation, the crude polysaccharide from fermented dandelion residue retained polyphenolic impurities accumulated during fermentation. These polyphenols formed a spatial binding state with the polysaccharide molecules, and this binding provided electrons to reduce ABTS free radical molecules.

[0061] Comparative Example 1 changed the fermentation time and temperature; Comparative Example 2 changed the inoculum volume ratio; and Comparative Example 3 changed the fermentation temperature. These changes in fermentation conditions altered the metabolic pathways of Bacillus subtilis, leading to a decrease in the uronic acid content within the dissolved polysaccharide or a loosening of the polysaccharide's triple helix structure. This resulted in a weakened ability of the purified dandelion polysaccharide obtained in the comparative examples to donate electrons or hydrogen protons, causing the free radical scavenging rate to be lower than the test value in Example 1. The specific combination of fermentation parameters used in Example 1, combined with the subsequent purification process, preserved the high uronic acid content and natural spatial configuration of the polysaccharide, obtaining a target product with both hydroxyl radical scavenging capabilities.

[0062] Test Example 4: Bovine mammary epithelial cells were seeded into culture plates and cultured. The purified dandelion polysaccharide groups obtained in Examples 1 to 3 and Comparative Examples 1 to 3 were fractionated to prepare polysaccharide culture solutions with concentration gradients and added to the wells for incubation for 24 h. CCK-8 reagent was added to the wells and incubated in the dark. Cell viability was calculated by measuring absorbance at 450 nm, and the polysaccharide intervention concentration was screened and determined to be 125 μg / mL.

[0063] Bovine mammary epithelial cells were seeded into culture plates and incubated with medium containing 800 μmol / L hydrogen peroxide to establish an oxidative damage cell model.

[0064] A blank control group, an oxidative damage model group, and a polysaccharide intervention group were established. The blank control group was added to conventional culture medium, the oxidative damage model group was added to culture medium containing 800 μmol / L hydrogen peroxide, and the polysaccharide intervention group was first incubated with culture medium containing 125 μg / mL of purified dandelion polysaccharide fraction obtained from various examples and comparative examples, and then incubated with culture medium containing 800 μmol / L hydrogen peroxide.

[0065] Cell culture supernatants and cell lysates were collected from each group. Lactate dehydrogenase leakage in the cell culture supernatant was determined using the corresponding biochemical assay kits. Simultaneously, malondialdehyde (MDA) content, catalase activity, and glutathione peroxidase activity in the cell lysates were measured.

[0066] Table 4. Biochemical index test data of bovine mammary epithelial cells in each group

[0067] The test results are as follows: According to the data in Table 4, hydrogen peroxide induces oxidative stress damage in bovine mammary epithelial cells, leading to increased cell membrane permeability, leakage of intracellular lactate dehydrogenase into the cell culture supernatant, accumulation of malondialdehyde (MDA), a lipid peroxidation product, and decreased activity of antioxidant enzymes, including catalase and glutathione peroxidase. The biochemical indicators in the polysaccharide intervention group all approached those in the blank control group.

[0068] In Table 4, the lactate dehydrogenase leakage test value of the polysaccharide intervention group in Example 1 decreased to 84.6 U / L, the malondialdehyde content test value decreased to 3.18 nmol / mgprot, while the catalase activity increased to 28.4 U / mgprot and the glutathione peroxidase activity increased to 97.5 U / mgprot.

[0069] The refined dandelion polysaccharide obtained in Example 1 retains the natural triple helix structure and is rich in uronic acid groups, which can neutralize reactive oxygen free radicals that enter the cell, reduce the attack and damage of reactive oxygen on cell membrane lipid molecules, protect the integrity of the cell membrane structure, and prevent intracellular lactate dehydrogenase from leaking out.

[0070] The refined components of dandelion polysaccharides participate in regulating the antioxidant defense mechanism in cells, enhancing the activity of catalase and glutathione peroxidase, accelerating the decomposition of hydrogen peroxide and lipid peroxides in cells, and reducing the accumulation of malondialdehyde.

[0071] Comparative Example 1 changed the fermentation time and temperature; Comparative Example 2 changed the inoculum volume ratio; and Comparative Example 3 changed the fermentation temperature. These changes in fermentation conditions altered the spatial configuration and acidic group content of the obtained polysaccharides, weakening the ability of the refined polysaccharide fraction to neutralize free radicals. Consequently, the protective effect of each comparative group against oxidative stress-damaged cells was weaker than that in Example 1. The refined dandelion polysaccharide fraction obtained in Example 1, through controlled fermentation environmental parameters and chromatography purification, demonstrated the ability to improve the oxidative stress-damaged state of bovine mammary epithelial cells.

Claims

1. A process for extracting polysaccharides based on the fermentation of dandelion residue, characterized in that, Includes the following steps: Bacillus subtilis was inoculated into an activation medium and activated twice to obtain an activated bacterial solution; The dandelion residue was mixed with water and autoclaved to obtain the fermentation medium. The activated bacterial solution was inoculated into the fermentation medium at an inoculation volume ratio of 1-10%, and fermented at a fermentation temperature of 27-37°C for 6-30 hours to obtain a fermentation mixture. The fermentation mixture was centrifuged to obtain the fermentation supernatant; The fermentation supernatant was concentrated under vacuum to obtain a concentrated fermentation liquid; Add four times the volume of anhydrous ethanol to the fermentation concentrate and refrigerate overnight at 4°C to obtain a polysaccharide precipitation mixture. The polysaccharide precipitation mixture was centrifuged to collect the precipitate.

2. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 1, characterized in that: When preparing the fermentation medium, the dandelion residue is mixed with water at a weight-to-volume ratio of 1:

20.

3. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 1, characterized in that: The centrifugation of the fermentation mixture is performed as follows: Centrifuge at 6500 r / min for 10 min.

4. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 1, characterized in that: The operation of vacuum concentration of the fermentation supernatant is as follows: vacuum concentration is carried out at 55°C to one-quarter of the original volume of the fermentation supernatant.

5. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 1, characterized in that, After collecting the precipitate, the following deproteinization process is also included: The precipitate was redissolved in pure water to obtain a crude polysaccharide redissolved solution; Sevag reagent is obtained by mixing n-butanol and chloroform. The Sevag reagent is then mixed with the crude polysaccharide complex solution and repeatedly extracted to remove proteins until no protein precipitate layer is obtained. The upper aqueous layer is then separated by centrifugation to obtain the polysaccharide aqueous phase.

6. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 5, characterized in that: When preparing the Sevag reagent, the n-butanol and chloroform are mixed at a volume ratio of 1:4; During the extraction and deproteinization process, the Sevag reagent and the crude polysaccharide complex solution are mixed at a volume ratio of 1:

4.

7. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 6, characterized in that, After obtaining the polysaccharide aqueous phase, the method further includes the following steps: The polysaccharide aqueous phase was concentrated by rotary evaporation and then desalted by dialysis using a dialysis bag before freeze-drying to obtain crude polysaccharide from fermented dandelion residue.

8. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 7, characterized in that, After obtaining the crude polysaccharide from the fermented dandelion residue, the following first chromatography step is also included: The crude polysaccharide from the fermented dandelion residue was dissolved in pure water and loaded into a DEAE-52 cellulose chromatography column. Elution was performed stepwise with elution buffer, and the corresponding target polysaccharide eluent was collected to obtain the first chromatography eluent. The first chromatographic eluent was dialyzed and concentrated using a dialysis bag, and then freeze-dried to obtain a preliminarily purified polysaccharide.

9. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 8, characterized in that: The specific operation of the step elution is as follows: step elution is performed sequentially using distilled water, 0.2 mol / L NaCl solution, 0.4 mol / L NaCl solution and 0.6 mol / L NaCl solution at a flow rate of 1 mL / min, and the target polysaccharide eluent corresponding to the 0.4 mol / L NaCl solution is collected.

10. The process for extracting polysaccharides based on dandelion residue fermentation according to claim 8, characterized in that, After obtaining the preliminarily purified polysaccharide, the following second chromatography step is also included: The preliminarily purified polysaccharide was reconstituted with pure water and loaded into a G-75 dextran gel chromatography column. It was eluted with pure water at a flow rate of 0.5 mL / min, and the eluent fraction of a single symmetrical target polysaccharide was collected to obtain the second chromatography eluent. The second chromatographic eluent was dialyzed and concentrated using a dialysis bag, and then freeze-dried to obtain the refined dandelion polysaccharide component.

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