Onion neutral polysaccharide YC-W and preparation method thereof

Abstract

The invention discloses onion neutral polysaccharide YC-W and a preparation method thereof, the onion neutral polysaccharide YC-W is a single polysaccharide formed by polymerization of galactose, has a triple helical structure, has a molecular weight Mw of 2.3 * 10 < 4 > Da, takes 1, 4-Galp as a skeleton, t-Galp and 1, 4, 6-Galp as short-chain branches, and 1, 6-Galp and 1, 2, 4-Galp as terminal residues, and the polymerization degree n is approximately equal to 13 through molecular weight calculation. After the process is optimized, the onion neutral polysaccharide YC-W with higher extraction rate and better purity can be obtained, meanwhile, the specific structure of the onion neutral polysaccharide YC-W is also provided, more technical theoretical support is provided for the onion neutral polysaccharide YC-W, and the obtained onion neutral polysaccharide YC-W is simple in structure, convenient to modify the structure and discuss the structure-function relationship of the onion neutral polysaccharide YC-W, and has broad application prospects. The method is of great significance to subsequent polysaccharide research and product development.

Description

Technical Field

[0001] The present invention relates to, in particular to, an onion neutral polysaccharide YC-W and a preparation method thereof. Background Art

[0002] Onion (Allium cepa L.), also known as bulb onion, round onion, jade onion, Dutch onion, leather teeth, etc., is a perennial herbaceous plant of the genus Allium (Allium L.) of the Liliaceae family (Liliaceae). According to its skin color, it is divided into three types: white skin, purple skin and yellow skin. There are many kinds of onion products, such as dehydrated onion food, onion sauce and onion oil. The onion usually refers to the common onion, which has a strong aroma. Onions are native to Central Asia or West Asia. Now there are many different varieties and they are grown all over the world. The global average annual onion production area is about 3.8 million hectares, and the annual output is about 98 million tons. It is a food raw material with a large amount of consumption worldwide. Onions are cultivated in a large area in my country, with a wide range. They are cultivated in all parts of the north and south. Not only is the planting area vast, but the output is also huge. Stir-frying, or as an ingredient, is also a well-known condiment used to enhance the flavor and color of various foods. As a green food, onion contains a variety of rich bioactive substances, such as sulfide, flavonoids, phenylpropanol, steroidal saponin, prostaglandin, phenylpropanol, nitride, onion oleoresin, phenolic compounds, etc. Among them, onion polysaccharide is one of the main active ingredients of onion. Due to its strong reducing power, it has many health functions such as anti-oxidation, antibacterial, anti-cancer, anti-tumor, lowering blood lipids, lowering blood sugar, stabilizing blood pressure, delaying aging, and improving immunity.

[0003] At present, the research contents of onion polysaccharides at home and abroad are almost the same. There are a lot of research on the extraction and separation process, functional food and medicinal use of onion polysaccharides at home and abroad; some studies on the effects of different extraction methods and solvents, extraction sequence on polysaccharide structure and biological activity, some studies on the development of onion polysaccharide research on different separation methods, some studies on the effects of structural characterization on the activity of onion polysaccharides, and some studies on the anti-inflammatory, antibacterial, hypoglycemic and hypolipidemic aspects of onion polysaccharides; some studies on solid beverages made of onion polysaccharides, effervescent tablets, etc., and the use of polysaccharides as oral liquid medicine.

[0004] For example, the Chinese patent number CN 113980150 A discloses a method for extracting onion polysaccharides, a method for preparing onion polysaccharide derivatives and their applications, which increases the extraction rate and purity of polysaccharides, but the disadvantage is that the primary structure and advanced structure of the extracted polysaccharides are not analyzed, and there is a lack of experimental basis for subsequent research on the relationship between its structure and activity. Another example is the Chinese patent application number CN 108117606 A, which discloses an extraction process for crude onion polysaccharide extracts, which increases the extraction rate and morphological application of crude polysaccharides, and can be used as food gelatin, but the disadvantage is also that there is no clear analysis of the structure, lack of scientific basis for subsequent development, and the purity needs to be improved. Summary of the invention

[0005] Based on this, the present invention provides an onion neutral polysaccharide YC-W and a preparation method thereof, and the specific technical scheme is as follows:

[0006] A neutral onion polysaccharide YC-W, wherein the neutral onion polysaccharide YC-W is a single polysaccharide polymerized from galactose, has a triple helix structure, a molecular weight Mw of 2.3×104Da, has 1,4-Galp as a skeleton, t-Galp and 1,4,6-Galp as short branches, 1,6-Galp and 1,2,4-Galp as terminal residues, and a degree of polymerization n≈13 calculated by molecular weight. The specific structural formula is as follows:

[0007]

[0008] In addition, the present invention also provides a method for preparing onion neutral polysaccharide YC-W, the preparation method comprising the following steps:

[0009] The onion is pretreated, and then distilled water is added, and the onion is boiled and filtered. The onion is boiled and filtered several times, and the filtrate is collected after each filtration.

[0010] The filtrate is concentrated under reduced pressure, and then subjected to a first centrifugation treatment to obtain a supernatant;

[0011] Add 95% ethanol by mass to the supernatant, mix well, place the mixture at 4°C for precipitation reaction for 8h to 12h, perform a second centrifugation to obtain an alcohol precipitate, and perform a first freeze-drying to obtain crude onion polysaccharide;

[0012] The onion polysaccharide was added to distilled water to dissolve, and a solution with a concentration of 20 mg / mL was prepared. An organic reagent was added for extraction, and the extraction was repeated several times until no protein layer appeared.

[0013] The upper layer solution was transferred to a rotating bottle for rotary evaporation to remove the organic reagent, and then ethanol with a mass percentage concentration of 95% was added, and centrifuged for a third time at 4°C for 8h to 12h to obtain a precipitate, and distilled water was added again to dissolve it, and the precipitate was transferred to a rotating bottle again for rotary evaporation, and then freeze-dried for a second time to obtain onion crude polysaccharide without protein;

[0014] The onion crude polysaccharide after protein removal was subjected to depigmentation, cellulose removal and dialysis treatment, and then freeze-dried for the third time to obtain onion neutral polysaccharide YC-W.

[0015] Furthermore, the pretreatment is to cut the fresh onion into small pieces, then dry them at 50°C to 65°C, and then grind them.

[0016] Furthermore, the crushed onion was mixed with distilled water at a solid-liquid ratio of 1:(15-20), and the mixture was steamed in an autoclave at 105° C. for 90 minutes.

[0017] Furthermore, the first centrifugal treatment, the second centrifugal treatment and the third centrifugal treatment are all performed at 5000 r / min to 8000 r / min for 15 min to 20 min.

[0018] Furthermore, the organic reagent is chloroform and n-butanol in a volume ratio of (4-7):1.

[0019] Furthermore, the depigmentation treatment is as follows: dissolving the onion crude polysaccharide from which protein has been removed in distilled water, adding ammonia water to adjust the pH to 8.0, then adding a 30% H2O2 solution accounting for 6% of the volume of the polysaccharide solution to remove water-soluble pigments, and then reacting for 2h to 3h in a water bath at 60°C to 65°C until the color of the solution becomes significantly lighter, and then centrifuging at a speed of 5000r / min to 8000r / min for 5min, taking the supernatant for reduced pressure concentration, and then freeze-drying for 48h to obtain deproteinized and depigmented onion polysaccharide.

[0020] Furthermore, the cellulose removal treatment is as follows: the deproteinized and depigmented onion polysaccharide is dissolved in distilled water, centrifuged at a speed of 15000 r / min to 18000 r / min for 15 minutes, and after removing the insoluble matter, it is added to a chromatography column, and a series of NaCl solutions with increasing concentrations are used, gradually increasing from 0.1 mol / L to 0.5 mol / L, for gradient elution treatment, and the flow rate is controlled at 1 mL / min.

[0021] Furthermore, the time for the first freeze-drying, the second freeze-drying and the third freeze-drying is 48 hours.

[0022] Furthermore, the dialysis treatment time is 48h.

[0023] After process optimization in the above scheme, onion neutral polysaccharide YC-W with higher extraction rate and better purity can be obtained. At the same time, the specific structure of onion neutral polysaccharide YC-W is also provided, providing more technical theoretical support for onion neutral polysaccharide YC-W. The obtained onion neutral polysaccharide YC-W has a simple structure and is easy to modify. Exploring its structure-activity relationship is of great significance to subsequent polysaccharide research and product development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the elution curve of the DEAE-52 cellulose anion exchange column chromatography of the crude onion polysaccharide in Example 4;

[0025] Figure 2 This is a schematic diagram of the elution results of the Sephacryl-S200 gel chromatography column of onion neutral polysaccharide YC-W in Example 5;

[0026] Figure 3 This is a schematic diagram of the total sugar content of onion neutral polysaccharide YC-W in Example 6;

[0027] Figure 4 This is a schematic diagram of the protein content of onion neutral polysaccharide YC-W in Example 7;

[0028] Figure 5 This is a schematic diagram of the uronic acid content of onion neutral polysaccharide YC-W in Example 8;

[0029] Figure 6 This is a schematic diagram of the ultraviolet absorption of onion neutral polysaccharide YC-W in Example 9;

[0030] Figure 7 This is a schematic diagram of the electron microscope analysis of the onion neutral polysaccharide YC-W in Example 10 at a magnification of 1000 times;

[0031] Figure 8 This is a schematic diagram of the electron microscope analysis of the onion neutral polysaccharide YC-W in Example 10 at a magnification of 5000 times;

[0032] Fig. 9 This is a schematic diagram of infrared spectrum analysis of onion neutral polysaccharide YC-W in Example 11;

[0033] Fig.10 This is a schematic diagram of the change in the maximum absorption wavelength of onion neutral polysaccharide YC-W and Congo red reagent in Example 12;

[0034] Fig.11 Schematic diagram of the LogMp-T calibration curve of onion neutral polysaccharide YC-W in Example 13;

[0035] Fig.12 Schematic diagram of the LogMw-T calibration curve of onion neutral polysaccharide YC-W in Example 13;

[0036] Fig.13 Schematic diagram of the LogMn-RT calibration curve of onion neutral polysaccharide YC-W in Example 13;

[0037] Fig.14 This is a schematic diagram of the HPGPC chromatogram of onion neutral polysaccharide YC-W in Example 13;

[0038] Fig.15 This is the chromatogram of the monosaccharide composition standard;

[0039] Fig.16 This is a sample chromatogram of the monosaccharide composition of onion neutral polysaccharide YC-W in Example 14;

[0040] Fig.17 This is a schematic diagram of the total ion flow of onion neutral polysaccharide YC-W in Example 15;

[0041] Fig.18 is the 1H NMR spectrum of the onion neutral polysaccharide YC-W in Example 16;

[0042] Fig.19 is the 13C NMR spectrum of the onion neutral polysaccharide YC-W in Example 16;

[0043] Fig. 20 is the 1H-1H COSY spectrum of the onion neutral polysaccharide YC-W in Example 16;

[0044] Fig.21 is the HMBC spectrum of the onion neutral polysaccharide YC-W in Example 16;

[0045] Fig. 22 is the HSQC spectrum of onion neutral polysaccharide YC-W in Example 16; DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0047] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0048] Embodiment 1:

[0049] 1 kg of fresh onion is cut into small pieces, and then placed in a constant temperature drying oven at 50°C, dried to a fluffy state, and then the onion slices are crushed, and then distilled water is added at a solid-liquid ratio of 1:20, heated and boiled at 105°C in an autoclave for 90 minutes, and the filter residue is retained after filtration, and the boiling operation is repeated three times; at the same time, the filtrate is collected and concentrated to 1 / 2 of the original volume under reduced pressure at 60°C, and the concentrated solution is placed in a centrifuge and centrifuged at a speed of 5000r / min for 15 minutes. Subsequently, the clear liquid is sucked from the upper layer with a pipette, and 95% ethanol by mass percentage is added thereto, and its volume is 3 times that of the clear liquid taken, and then the mixed solution is placed at 4°C for precipitation reaction, left to stand for 10 hours or overnight, and centrifuged again at a speed of 5000r / min for 10 minutes, and the upper clear liquid and precipitate are removed after centrifugation, and the precipitate is freeze-dried under vacuum conditions to obtain onion crude polysaccharide.

[0050] Embodiment 2:

[0051] Extraction: Mix chloroform and n-butanol in a volume ratio of 4:1 to prepare Sevage reagent, and store it in a brown bottle for later use; accurately weigh an appropriate amount of onion crude polysaccharide using an analytical balance, dissolve it with distilled water, and prepare a solution with a concentration of 20 mg / mL, then pour it into a separatory funnel, shake it sufficiently, and extract it with Sevage reagent, and let it stand until obvious stratification is observed. At this time, separate and remove the lower protein layer and organic layer, and repeat the above operation 5-6 times until no protein layer (i.e., milky white layer) appears;

[0052] Alcohol precipitation: pour the upper polysaccharide layer from the upper outlet of the separatory funnel and transfer it to a spinner flask, and perform rotary evaporation at 35°C to remove the organic reagent. When the dripping rate of the distillate becomes too slow, take out the spinner flask and add 3 times the volume of 95% ethanol of the concentrate, then put the solution in a refrigerator at 4°C overnight to allow the polysaccharide to precipitate;

[0053] Freeze drying: centrifuge the sample solution after alcohol precipitation at a speed of 5000r / min for 15min, ensuring that the mass difference between each tube does not exceed 0.2g; after centrifugation, take the precipitate sample of the lower layer, add water to dissolve it, and transfer it to the spin bottle again; perform rotary evaporation at 40°C to remove the ethanol, and then pour the solution evenly into the culture dish, ensuring that the thickness does not exceed 1cm, and put it into a vacuum freeze dryer for 48h to obtain the onion crude polysaccharide after protein removal.

[0054] Embodiment 3:

[0055] Ammonia water was added to the deproteinized crude onion polysaccharide solution to adjust the pH to 8.0, and then 30% H2O2 solution accounting for 6% of the volume of the crude onion polysaccharide solution was added to remove some water-soluble pigments therein. The solution was reacted for 2 hours in a water bath at 60°C until the color of the solution became significantly lighter, and then centrifuged at a speed of 5000r / min for 5 minutes. The supernatant was taken and then concentrated under reduced pressure, and then freeze-dried for 48 hours to obtain deproteinized and depigmented onion polysaccharide.

[0056] Embodiment 4:

[0057] Column activation: prepare about 300mL of swollen DEAE fiber powder solution, filter out the 20% ethanol aqueous solution, soak in 0.5mol / L NaOH solution for 2h, wash with distilled water until the droplets are neutral, filter again to dryness, soak in 0.5mol / L HCl solution for 2h, then wash thoroughly with distilled water until the droplets are neutral, and set aside. After activation, add an appropriate amount of distilled water to the DEAE cellulose, stir evenly, use a glass rod to drain into a sand core chromatography column with a diameter of about 2cm, balance with 5 times the volume of distilled water, and close the outlet when the liquid surface is tangent to the column surface;

[0058] Add sample: dissolve 200 mg of deproteinized and depigmented onion polysaccharide in 10 mL of distilled water, centrifuge at 15000 r / min for 15 min to remove precipitate or insoluble matter, and then slowly drip the supernatant into the column along the wall;

[0059] Elution process: After the supernatant drips into the column, continue to discharge the solution to fill the sample in the chromatography column. Use a series of NaCl solutions with increasing concentrations, gradually increasing from 0.1mol / L to 0.5mol / L, to perform a gradient elution process, and control the flow rate at 1mL / min. Use a penicillin bottle to collect the eluent, collect 10mL in each bottle, and the elution volume of each mobile phase is 500mL;

[0060] Phenol-sulfuric acid analysis: Take 60μL of sample solution in the ELISA plate, add 30μL of 6% phenol solution, mix well, and then add 150μL of concentrated sulfuric acid to react. When the reaction is completely completed or the solution is cooled naturally, measure the absorbance at a wavelength of 490nm. The absorbance value measured in each bottle is used as the ordinate and the number of vials is used as the abscissa to draw a curve of the onion polysaccharide solution on the DEAE-52 cellulose anion exchange column, as shown in the figure. Figure 1 As shown, Figure 1 The figure is a schematic diagram of the elution curve of DEAE-52 cellulose anion exchange column chromatography of crude onion polysaccharide, and the corresponding solutions are collected and combined according to the curve;

[0061] Sample processing: The collected components were subjected to rotary evaporation, dialyzed for 48 hours, and then filtered to remove insoluble impurities such as fibers. The sample solution was concentrated by rotary evaporation using a rotary flask and collected in a vial, and finally vacuum-freeze-dried for 48 hours to obtain white onion neutral polysaccharide YC-W.

[0062] Embodiment 5:

[0063] Activation and cleaning: Refill the column, then add 25mL of 0.5mol / L NaOH solution and clean it at a flow rate of 0.25mL / min to remove proteins. Rinse the column with 250mL of distilled water until the liquid surface is tangent to the column surface, replace it with 50mL of 30% isopropanol for cleaning to eliminate lipid impurities, and finally rinse the column with 200mL of distilled water again for standby use. If the column efficiency is weakened, it can be cleaned with 0.5mol / L NaOH and HCL, and then balanced with 4 times the column volume of distilled water.

[0064] Add sample: Separate the polysaccharide component YC-W through a DEAE fiber chromatography column, and then accurately weigh 10 mg of the component. Then dissolve 10 mg of the polysaccharide component in 1 mL of distilled water, filter through a 0.45 μm membrane, and prepare a 10 mg / mL polysaccharide solution. Slowly drip this solution into the Sephacryl-S200 gel chromatography column along the wall. Once the liquid surface is tangent to the column surface, immediately add 250 mL of distilled water as the mobile phase, and control the flow rate to 16 to 20 seconds per drop. Collect 2 mL of solution in each vial until all are collected.

[0065] Detection and purification: In order to track and determine the polysaccharide content in each tube of collected solution, the phenol-sulfuric acid method was used to draw a graph, in which the horizontal axis represents the number of collected tubes and the vertical axis represents the corresponding absorbance, thereby obtaining the elution curve of the polyacrylamide dextran gel Sephacryl-S200 column of the polysaccharide solution. According to this curve, the corresponding eluate was collected and combined. Subsequently, the combined solution was concentrated by rotary evaporation at 60°C and filled into a vial. Finally, a vacuum freeze dryer was used to perform freeze drying at -40°C to obtain onion neutral polysaccharides YC-W, which were labeled as YC-W. Figure 2 As shown, Figure 2 Schematic diagram of the Sephacryl-S200 gel chromatography column chromatography elution results of YC-W.

[0066] Embodiment 6:

[0067] Onion neutral polysaccharide YC-W was determined by the phenol-sulfuric acid method.

[0068] 0.1mg / mL glucose standard solution: Take 1mg of glucose, dissolve it in water, and make up to 10mL.

[0069] Preparation of 0.1 mg / mL sample solution: Accurately weigh 1 mg of onion neutral polysaccharide YC-W, using the same method as above.

[0070] Preparation of 6% phenol solution: Heat phenol at 60°C until the solid is dissolved, pipette 500 μL of phenol solution into a volumetric flask, and dilute to 10 mL with water.

[0071] Glucose standard curve drawing: Prepare glucose standard solutions with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL, take 60 μL of each standard solution in the ELISA plate, add 30 μL of 5% phenol solution, mix well, and add 150 μL of concentrated sulfuric acid. Place in a 40°C water bath for 15 minutes, then let it cool naturally to room temperature. At the same time, set up a control experiment, measure the absorbance at 490 nm, and draw the glucose standard curve with absorbance as the ordinate and concentration as the abscissa.

[0072] Sample content determination: 60 μL of sample solution was transferred into the ELISA plate, followed by 30 μL of 6% phenol solution and shaken thoroughly. Then 150 μL of concentrated sulfuric acid was added to each sample. The reaction process needs to be carried out in a 40°C water bath for 15 minutes and cooled naturally. With the blank group as a reference, the absorbance value was measured at 490 nm, and the measurement was repeated three times. The total sugar content was calculated according to the standard curve.

[0073]

[0074] C: concentration of polysaccharide after substituting into the formula (mg / mL);

[0075] V: volume of polysaccharide extract (mL);

[0076] M: sample weight (mg).

[0077] Embodiment 7:

[0078] Coomassie Brilliant Blue Solution: Dissolve 10 mg of Coomassie Brilliant Blue powder in 5 mL of 95% ethanol and 10 mL of 85% phosphoric acid, and dilute to 100 mL. Store in a brown bottle at 4°C for later use.

[0079] 0.1mg / mL BSA solution: Dissolve 10mg BSA in water and make up to 100mL.

[0080] Preparation of 1 mg / mL sample solution: Weigh 1 mg of YC-W in EP tubes. Then add 1 mL of distilled water to each EP tube to obtain the sample solution.

[0081] Standard curve drawing: Dilute the mother solution into standard solutions with concentrations of 0.01mg / mL, 0.02mg / mL, 0.04mg / mL, 0.06mg / mL, 0.08mg / mL, and 0.1mg / mL, respectively. Pipette 30μL of standard solution into the ELISA plate, and then add 150μL of Coomassie Brilliant Blue solution to mix. Place the sample to be tested for 2-5min, and measure the absorbance at a wavelength of 595nm with a mixture of pure water and Coomassie Brilliant Blue solution as a reference. Three sets of parallel experiments should be set for each concentration of sample to ensure accuracy, and the standard curve should be drawn with concentration as the horizontal axis and absorbance as the vertical axis.

[0082] Sample determination: Take 30μL of sample solution into the ELISA plate, add 150μL of Coomassie Brilliant Blue solution, react fully, and after standing for 2-5 minutes, measure the absorbance at 595nm with the mixture of pure water and Coomassie Brilliant Blue solution as reference. Three parallel experiments should be set up for each group of samples to ensure accuracy.

[0083]

[0084] C: protein concentration after substituting into the formula (mg / mL);

[0085] V: volume of protein extract (mL);

[0086] M: sample weight (mg).

[0087] Embodiment 8:

[0088] The uronic acid content was determined by the sulfuric acid-carbazole method.

[0089] Sodium tetraborate-sulfuric acid solution: Weigh 0.4788 g of sodium tetraborate, dissolve it in concentrated sulfuric acid, and make up to 100 mL.

[0090] Carbazole solution: Weigh 50 mg of carbazole, dissolve it in 95% ethanol, and make up to 50 mL.

[0091] 0.1mg / mL galacturonic acid solution: Take 10mg of galacturonic acid, dissolve it in water, and make up to 100mL.

[0092] Drawing of uronic acid standard curve: Take 0.0, 0.05mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL of uronic acid standard solution, dilute to 0.5mL with water, add 2.5mL of sodium tetraborate-sulfuric acid solution, shake in an ice water bath, heat in a boiling water bath for 20min, and let it cool naturally to room temperature. Then add 0.1mL of carbazole to each test tube and shake it again. Then let the test tube stand for 2 hours and measure the absorbance at 523nm. Draw the uronic acid standard curve with absorbance as the ordinate and uronic acid concentration as the abscissa.

[0093] Determination of uronic acid content in samples: Take 0.1 mg / mL polysaccharide YC-W and calculate the uronic acid content according to the standard curve.

[0094]

[0095] C: concentration of uronic acid after substituting into the formula (mg / mL);

[0096] V: volume of uronic acid extract (mL);

[0097] M: sample weight (mg).

[0098] The test results of Examples 6 to 8 are shown in Table 1 below.

[0099] Table 1: Chemical composition

[0100] sample Total sugar content Protein Content Uric acid content YC-W 88.58±1.85％ Not detected Not detected

[0101] From the data in Table 1, combined with Figure 3 to Figure 5 Analysis shows that the onion neutral polysaccharide YC-W of the present application has a high purity.

[0102] Embodiment 9:

[0103] Take 2 mg of YC-W separated by DEAE ion exchange column chromatography, dissolve it in 1 mL of distilled water to prepare a solution with a concentration of 2.0 mg / mL, and prepare distilled water as a blank control. Then use a UV spectrophotometer to scan the solution at all wavelengths within the wavelength range of 200-400 nm, and analyze the UV scanning spectrum to determine whether the onion neutral polysaccharide YC-W contains protein and nucleic acid components. Further observation revealed that the YC-W polysaccharide did not contain nucleic acids and proteins after purification. In addition, after DEAE chromatography, the purification effect was better, which also confirmed that the protein in the onion polysaccharide mainly exists in a free form, and salt can elute and remove some of the protein. Figure 6 As shown, Figure 6 Schematic diagram of UV absorption of onion neutral polysaccharide YC-W.

[0104] Embodiment 10:

[0105] Electron microscopic analysis of onion neutral polysaccharide YC-W:

[0106] The neutral polysaccharide YC-W sample of onion was vacuum-plated with gold, and the surface structure was observed under a scanning electron microscope at magnifications of 1000 and 5000 times. Figure 7 When magnified 1000 times, it can be observed that the surface is a loose porous structure with a flaky and layered structure and many hollow areas. Figure 8When observing the details at a magnification of 5000 times, it can be seen that the surface has multiple holes arranged in an interlaced manner, and the surface is smooth with small pad-like solids attached.

[0107] Embodiment 11:

[0108] Infrared spectrum analysis of onion neutral polysaccharide YC-W:

[0109] The measurement was performed using a Fourier transform micro-infrared spectrometer equipped with an ATR sampler. Weigh 2 mg of dried onion purified polysaccharide component YC-W, and then place these samples in the center of the sample tray of the ATR infrared spectrometer, and set the scanning range to 500-4000 cm-1. In order to ensure the accuracy of the results, this operation was repeated 3 times, and the corresponding infrared spectrum was finally obtained after instrument processing.

[0110] The results of the peaks after infrared spectroscopy microanalysis are as follows Fig. 9 As shown. It can be seen that for the peak of YC-W, the peak at 3308cm-1 is relatively wide and not sharp, and there is a large non-concentrated area, which is usually attributed to the stretching vibration of hydroxyl (-OH), indicating that there are a small number of hydroxyl groups in the polysaccharide; 2899cm-1, a broad and weak absorption peak, may correspond to the stretching vibration of methyl or methylene (CH), indicating that there is a (CH) structure on the polysaccharide skeleton; 1655cm-1, a weak but concentrated single peak, is attributed to the conjugated C=C bond, or the asymmetric stretching vibration of carboxylate (COO-); 1421cm-1 and 1357cm-1, broad absorption peaks, may be attributed to the bending vibration of methyl (CH) or the symmetric stretching vibration of carboxylate (COO-); 11 20cm-1: shoulder peak, which may represent the asymmetric vibration of glycosidic bond (COC), indicating that the polysaccharide contains glycosidic bond structure; 1044cm-1, a sharp absorption peak was observed, which may be attributed to the stretching vibration of phosphate (P=O) or caused by the stretching vibration of CO bond, indicating that there may be CO structure in phosphate bond or sugar ring; 888cm-1 may be attributed to CH angle vibration of pyranose ring, indicating that the polysaccharide contains pyranose ring; 760cm-1, 699cm-1, 657cm-1, 608cm-1, 538cm-1, 501cm-1, 477cm-1, 440cm-1, these lower frequency peaks may represent ring structure or side chain vibration in polysaccharide.

[0111] The peaks of YC-W showed typical characteristics of polysaccharides, such as the presence of hydroxyl groups, glycosidic bonds, and pyranose rings. The aggregation at the asymmetric vibration peaks of glycosidic bonds may indicate that their glycosidic bond structures are more complex and require further structural analysis.

[0112] Embodiment 12:

[0113] Congo red test of onion neutral polysaccharide YC-W:

[0114] 5.0 mg of onion neutral polysaccharide YC-W was accurately weighed and dissolved in 2.0 mL of distilled water. Then, 2.0 mL of 80 μmol / L Congo red reagent was added to the solution and mixed thoroughly. Then, 1.0 mol / L NaOH solution was added to the solution to make the final concentrations 0, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L, respectively. After gently shaking and standing for 5 minutes, ultraviolet spectroscopy analysis was performed under the condition of a wavelength range of 200-800 nm, and the maximum absorption wavelength of the solution at different NaOH concentrations was recorded. For the convenience of comparison, a blank control experiment was performed, that is, distilled water was used instead of the polysaccharide solution to perform the same operation, and the ultraviolet spectral characteristics of the polysaccharide components at different NaOH concentrations were accurately evaluated.

[0115] When a polysaccharide with a triple helix structure combines with Congo red to form a complex, the maximum absorption wavelength of the solution significantly red-shifts, that is, increases; and when the helical structure is destroyed, the absorption wavelength decreases.

[0116] Fig.10 It shows how the maximum absorption wavelength of the complex solution of polysaccharide YC-W and Congo red reagent changes under different concentrations of NaOH solution. By observing the chart, it can be clearly seen that when the concentration of the NaOH solution changes in the range of 0 to 0.2 mol / L, the wavelength of polysaccharide YC-W will increase, showing a red shift phenomenon of the absorption wavelength, and reaching the maximum wavelength at 0.2 mol / L. In the range of 0.2-0.5 mol / L, the maximum wavelength decreases with the decrease in concentration, but the final wavelength is still higher than that of the reference, indicating that a complex is formed, but then the conformation is destroyed and decreases. Therefore, polysaccharide YC-W has a triple helical structure.

[0117] The wavelength of the reference Congo red reagent is always decreasing. Different concentrations of NaOH affect the structural stability of the acid dye, which shows that there is a strong interaction between the Congo red reagent and NaOH. This interaction may lead to changes in the molecular structure of the Congo red reagent, thereby affecting its light absorption properties.

[0118] Embodiment 13:

[0119] Molecular weight determination of onion neutral polysaccharide YC-W:

[0120] Establishment of molecular weight calibration curve: Dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards) were accurately weighed, and 0.05M NaCl solution was added to prepare 5 mg / mL dextran standard solution, which was filtered through a 0.22 μm microporous filter membrane for later use. The HPGPC method was used with high performance gel permeation chromatography tandem columns for detection, and linear regression was performed with the logarithm of the relative molecular mass of the standard as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the calibration curve.

[0121] The logarithmic value of the relative molecular mass of the standard was used as the ordinate and the retention time (T) of the corresponding chromatographic peak was used as the abscissa to perform linear regression to obtain the calibration curves of LogMp-T (peak molecular weight), LogMw-T (weight average molecular weight), and LogMn-T (number average molecular weight), as shown in the figure. Figure 11 to Figure 13 As shown below:

[0122] LogMp-T calibration curve equation: Log Mp = -0.1748T + 10.7296, R 2 =0.9901

[0123] LogMw-T calibration curve equation: Log Mw = -0.1837T + 11.1410, R 2 =0.9928

[0124] LogMn-T calibration curve equation: Log Mn = -0.1745T + 10.6380, R 2 =0.9926;

[0125] Accurately weigh 5 mg of the sample, add 1 mL of 0.05 M NaCl solution to the sample to prepare a 5 mg / mL test sample solution, centrifuge at 8000 rpm for 10 min, take the supernatant and filter it with a 0.22 μm microporous filter membrane, and then transfer the sample to a 2 mL injection bottle for use.

[0126] The molecular weight of each sample was calculated based on the standard calibration curve. The results are shown in Table 2 below.

[0127] Table 2: Molecular weight

[0128]

[0129]

[0130] Embodiment 14:

[0131] Analysis of monosaccharide composition of onion neutral polysaccharide YC-W:

[0132] Weigh 5 mg of each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride monosaccharides, and 10 mg of fucose in sequence, dissolve them and use a volumetric flask to make a standard stock solution of 10 mL. Dilute them to the fixed volume and filter them through a 0.22 μm microporous membrane before placing them in a sample injection bottle.

[0133] Take a clean chromatographic bottle, accurately weigh 5 mg (± 0.05 mg) of YC-W sample, add 1 mL 2M TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 3 mL methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add 5 mL sterile water to dissolve, and transfer to the chromatographic bottle for testing.

[0134] Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolyzate into a stoppered conical bottom centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution, vortex mix well and react in a 70°C water bath for 1 h. After the reaction is completed, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make up to 1 mL.

[0135] ThermoU3000 liquid chromatography system, the chromatographic column is ZORBAX EclipseXDB-C18, the mobile phase is acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, 2M NaOH to adjust the pH to 6.8) isocratic elution, the volume ratio of acetonitrile to phosphate buffer is 17:83, the flow rate is 0.8 mL / min, the column temperature is 30°C, the detection wavelength is 250 nm, and the injection volume is 10 μl.

[0136] The project uses external standard method for quantification and establishes a standard curve by preparing standard samples of different concentrations.

[0137] The information of the standard curves of monosaccharide standards is summarized in Table 3.

[0138] Table 3: Monosaccharide standard sample information

[0139]

[0140]

[0141] Combination Fig.16 The analysis showed that the onion neutral polysaccharide YC-W was a single polysaccharide composed only of galactose.

[0142] Embodiment 15:

[0143] The methylation analysis of onion neutral polysaccharide YC-W is shown in Table 4 below.

[0144] Table 4: Methylation analysis results of onion neutral polysaccharide YC-W

[0145]

[0146] The methylation results showed that YC-0 was based on 1,4-Galp as the main chain, t-Galp and 1,4,6-Galp as the side chains, 1,6-Galp and 1,2,4-Galp as the terminal residues, and the degree of polymerization n≈13 was calculated by molecular weight.

[0147] Embodiment 16:

[0148] YC-W NMR analysis of neutral polysaccharide in onion:

[0149] 30 mg of purified polysaccharide components SDLP-1, SDLP-3a and SDLP-3b from dried miracle fruit leaves were weighed and fully dissolved in 0.5 mL D2O. One-dimensional NMR (1H-NMR, 13C-NMR) and two-dimensional NMR analysis were performed using a Bruker Avance 400MHz NMR spectrometer at 20°C. The one-dimensional NMR analysis is shown in Tables 5 and 6 below.

[0150] Table 5: 1H NMR chemical shifts (δ) of sugar residues in onion neutral polysaccharide YC-W

[0151]

[0152] Table 6: 13C NMR chemical shifts (δ) of sugar residues in onion neutral polysaccharide YC-W

[0153]

[0154] From Table 5 and Table 6, combined with Figure 18 to Figure 22 The analysis showed that the molecular weight of onion neutral polysaccharide YC-W was 2.3×104Da. It was a single polysaccharide composed of galactose and had a triple helix structure. The methylation analysis showed that 1,4-Galp was the backbone, t-Galp and 1,4,6-Galp were short branches, and 1,6-Galp and 1,2,4-Galp were terminal residues. The degree of polymerization was calculated by molecular weight, and n≈13. Infrared spectroscopy analysis showed the typical characteristics of polysaccharides, such as the presence of hydroxyl groups, glycosidic bonds, and pyranose rings. Nuclear magnetic resonance analysis showed that YC-W had β-glycosidic bonds, and the analytical results were consistent with the monosaccharide composition and methylation analysis.

[0155] The specific structural formula is as follows:

[0156]

[0157] In summary, the present application can obtain onion neutral polysaccharide YC-W with high purity, and also provides the specific structure of onion neutral polysaccharide YC-W, providing more technical theoretical support for onion neutral polysaccharide YC-W. The obtained onion neutral polysaccharide YC-W has a simple structure and is convenient for structural modification. The exploration of its structure-activity relationship is of great significance for subsequent polysaccharide research and product development.

[0158] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An onion neutral polysaccharide YC-W, characterized in that: The onion neutral polysaccharide YC-W is a single polysaccharide polymerized from galactose, has a triple helix structure, a molecular weight Mw of 2.3×104Da, 1,4-Galp as a skeleton, t-Galp and 1,4,6-Galp as short branches, 1,6-Galp and 1,2,4-Galp as terminal residues, and a degree of polymerization n≈13 calculated by molecular weight. The specific structural formula is as follows:

2. A method for preparing onion neutral polysaccharide YC-W, characterized in that: The preparation method comprises the following steps: The onion is pretreated, and then distilled water is added, and the onion is boiled and filtered. The onion is boiled and filtered several times, and the filtrate is collected after each filtration. The filtrate is concentrated under reduced pressure, and then subjected to a first centrifugation treatment to obtain a supernatant; Add 95% ethanol by mass to the supernatant, mix well, place the mixture at 4°C for precipitation reaction for 8h to 12h, perform a second centrifugation to obtain an alcohol precipitate, and perform a first freeze-drying to obtain crude onion polysaccharide; The onion polysaccharide was added to distilled water to dissolve, and a solution with a concentration of 20 mg / mL was prepared. An organic reagent was added for extraction, and the extraction was repeated several times until no protein layer appeared. The upper layer solution was transferred to a rotating bottle for rotary evaporation to remove the organic reagent, and then ethanol with a mass percentage concentration of 95% was added, and centrifuged for a third time at 4°C for 8h to 12h to obtain a precipitate, and distilled water was added again to dissolve it, and the precipitate was transferred to a rotating bottle again for rotary evaporation, and then freeze-dried for a second time to obtain onion crude polysaccharide without protein; The onion crude polysaccharide after protein removal was subjected to depigmentation, cellulose removal and dialysis treatment, and then freeze-dried for the third time to obtain onion neutral polysaccharide YC-W.

3. The preparation method according to claim 2, characterized in that: The pretreatment is to cut the fresh onion into small pieces, dry them at 50°C to 65°C, and then grind them.

4. The preparation method according to claim 3, characterized in that: The crushed onion was mixed with distilled water at a solid-liquid ratio of 1:(15-20), and the mixture was steamed in an autoclave at 105°C for 90 minutes.

5. The preparation method according to claim 2, characterized in that: The first centrifugal treatment, the second centrifugal treatment and the third centrifugal treatment are all carried out at 5000 r / min to 8000 r / min for 15 min to 20 min.

6. The preparation method according to claim 2, characterized in that: The organic reagent is chloroform and n-butanol in a volume ratio of (4-7):

1.

7. The preparation method according to claim 2, characterized in that: The depigmentation treatment comprises the following steps: dissolving the onion crude polysaccharide after protein removal in distilled water, adding ammonia water to adjust the pH value to 8.0, then adding 30% H2O2 solution accounting for 6% of the volume of the polysaccharide solution to remove water-soluble pigments, then reacting for 2h to 3h in a water bath at 60°C to 65°C until the color of the solution becomes obviously lighter, then centrifuging for 5min at a speed of 5000r / min to 8000r / min, taking the supernatant for reduced pressure concentration, and then freeze-drying for 48h to obtain the deproteinized and depigmented onion polysaccharide.

8. The preparation method according to claim 7, characterized in that: The cellulose removal treatment is as follows: the deproteinized and depigmented onion polysaccharide is dissolved in distilled water, centrifuged at a speed of 15000 r / min to 18000 r / min for 15 minutes, and after removing insoluble matter, it is added to a chromatography column, and a series of NaCl solutions with increasing concentrations, gradually increasing from 0.1 mol / L to 0.5 mol / L, are used for gradient elution treatment, and the flow rate is controlled at 1 mL / min.

9. The preparation method according to claim 8, characterized in that: The time for the first freeze-drying, the second freeze-drying and the third freeze-drying is 48 hours.

10. The preparation method according to claim 1, characterized in that: The dialysis treatment time is 48h.

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