Preparation process of beta-D-1, 4-galactan

Abstract

The invention discloses a preparation process of beta-D-1, 4-galactan in the technical field of galactan preparation, which comprises the following steps: preparing crude polysaccharide by using bean dregs; separating by adopting an ion exchange column chromatography, collecting solutions under different elution peaks in the elution process, concentrating, dialyzing and freeze-drying to obtain W polysaccharide; dissolving W polysaccharide in trifluoroacetic acid, and reacting and hydrolyzing under microwave radiation; after the reaction is finished, neutralizing, dialyzing, concentrating and freeze-drying to obtain WS polysaccharide; the method comprises the following steps: dissolving WS polysaccharide, centrifuging, taking supernate, loading a sample, purifying through a full-gel purification system, collecting symmetric peaks, dialyzing the collected eluent, concentrating, and freeze-drying to obtain separated and purified WS1 polysaccharide. According to the scheme, the purity level of beta-D-1, 4-galactan separated and prepared from the bean dregs can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of galactan preparation, in particular to a preparation process of beta-D-1,4-galactan. Background Art

[0002] As a by-product of soybean product processing, soybean dregs have long been used as feed or waste, but their potential nutritional and functional value is often overlooked. Okara is the residue filtered out during the production of soybean products such as soy milk or tofu. It is rich in dietary fiber, protein, fat, calcium, phosphorus, iron and other nutrients. Specifically, the dietary fiber in soybean dregs mainly includes galactan, cellulose, pectin and other components, among which galactan has attracted much attention due to its unique structure and physiological function. Galactan not only helps to improve intestinal health, promote intestinal peristalsis and prevent constipation, but also acts as a prebiotic to promote the growth of beneficial bacteria in the intestine and maintain the balance of intestinal microecology.

[0003] β-D-1,4-galactan is widely present in plant cell walls, especially in legumes. Bean dregs, as a by-product in the production process of tofu and soy milk, are rich in polysaccharides and are ideal raw materials for extracting β-D-1,4-galactan. At present, most galactans isolated from bean dregs are heteropolysaccharides, containing a considerable proportion of arabinose, galactose, glucose, galacturonic acid and other components, which to a certain extent limits their purity and application effects. Therefore, there is an urgent need for a preparation process to improve the purity and functionality of galactans. Summary of the invention

[0004] In order to solve the above problems, the object of the present invention is to provide a preparation process of β-D-1,4-galactan, which can improve the purity of galactan separated from bean dregs.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] A preparation process of β-D-1,4-galactan comprises the following steps:

[0007] The crude polysaccharide is prepared by weighing bean dregs as a raw material, mixing the bean dregs with water, and then extracting at a preset temperature; after the extraction is completed, the supernatant is separated by a centrifugal device, and amylase is added, and the extract is separated by filtering; the protein is removed by the Sevag method, and the extract is concentrated by vacuum rotary evaporation; the concentrated extract is transferred to a dialysis bag for dialysis, and then the dialyzed clear liquid is separated by centrifugation, and the dialyzed clear liquid is concentrated and freeze-dried to obtain a crude polysaccharide sample;

[0008] Polar separation is carried out by ion exchange column chromatography. During the elution process, the solution under the corresponding elution peak is collected, concentrated, dialyzed, and freeze-dried to obtain W polysaccharide;

[0009] Hydrolysis, dissolving W polysaccharide in trifluoroacetic acid, and reacting and hydrolyzing under microwave irradiation; after the reaction, neutralizing, dialysis, concentrating, and freeze-drying are performed to obtain WS polysaccharide;

[0010] Purification: After dissolving the WS polysaccharide, centrifuge and take the supernatant for sample loading, purify it through a full gel purification system, collect the symmetrical peaks, dialyze, concentrate, and freeze-dry the collected eluate to obtain the separated and purified WS1 polysaccharide.

[0011] Further, the centrifugal device comprises a centrifugal box; a cover body is arranged on the top of the centrifugal box; a first carrier is arranged at the bottom of the cover body, and the first carrier is rotatably connected to the cover body; a plurality of first motors are arranged in the inner circumference of the first carrier, and the output shaft of the first motor passes through the bottom of the first carrier; an electric slip ring is arranged on the top of the first carrier, and the electric slip ring is connected to a power source, and the electric slip ring is used to keep the first motor connected to the power source when the first carrier rotates;

[0012] A centrifugal tank is arranged in the centrifugal tank; a second carrier is arranged in the centrifugal tank, and the second carrier rotates in cooperation with the centrifugal tank; a second motor for driving the second carrier to rotate is arranged at the bottom of the centrifugal tank; a plurality of first chambers are arranged circumferentially in the second carrier, and the bottom centers of the first chambers are all connected with the second chambers; a plurality of discs are arranged at the axis center in the second chamber, and a transmission shaft is arranged at the axis center of the disc, and the transmission shaft coaxially fixes all the discs, and the discs are separated by a preset distance, and the preset distance is 2 times the thickness of the fluid boundary layer, and a plurality of drainage ports are circumferentially arranged on the discs; the top of the transmission shaft extends into the first chamber, and the top of the transmission shaft is keyed to the output shaft of the first motor; a filter is also arranged around the disc; a circulation outlet is also arranged at the top of the second chamber, and the circulation outlet is located above the edge of the disc.

[0013] Furthermore, when preparing the crude polysaccharide, the mixture was stirred and extracted at a constant temperature of 80° C. in a water bath for 4 hours.

[0014] Furthermore, when preparing crude polysaccharide, the molecular weight cutoff of the dialysis bag used is 8000Da.

[0015] Furthermore, when preparing crude polysaccharide, the dialysis bag was placed in distilled water at 4° C. and dialyzed for 72 hours.

[0016] Further, during polarity separation, the specific steps of separation by ion exchange column chromatography are as follows:

[0017] Soak the DEAE 52 filler in 0.5 mol / L hydrochloric acid for 1 hour;

[0018] After soaking, use deionized water or distilled water to wash away impurities in the DEAE 52 packing;

[0019] When eluting, use 4-5 times the volume of distilled water and continue washing until the pH value of the filler solution reaches neutral;

[0020] Before loading, the DEAE 52 packing was equilibrated with distilled water. During equilibration, the flow rate was adjusted to 5 ml / min, and distilled water was allowed to flow through the packing column for 2 hours.

[0021] Dissolve the crude polysaccharide in distilled water, heat and vortex;

[0022] After dissolution, the solution was centrifuged and the supernatant was taken as the loading solution;

[0023] Slowly add the supernatant to the equilibrated DEAE 52 column;

[0024] After loading, distilled water was used for elution and the flow rate was adjusted to 15 ml / min.

[0025] Furthermore, during elution, the elution solvent is three times the column volume of water, and the collected eluate is detected using the phenol-sulfuric acid method, the absorbance is measured at a wavelength of 490 nm using an enzyme reader, and the detected data is plotted as a scatter plot, and the corresponding eluate is collected according to the peak shape on the scatter plot.

[0026] Furthermore, during polar separation, the molecular weight cutoff of the dialysis bag used for dialysis is 3500Da.

[0027] Furthermore, during the hydrolysis, the reaction was carried out at 90° C. and a microwave power of 400 W for 3 hours.

[0028] Furthermore, during the purification process, the centrifugal treatment was performed at a speed of 12,000 rpm for 10 minutes.

[0029] Furthermore, the WS1 polysaccharide is β-D-1,4-galactan.

[0030] The above scheme has the following beneficial effects:

[0031] This scheme successfully prepared β-D-1,4-galactan with extremely high purity through a sophisticated preparation process, including extraction, enzymatic hydrolysis, protein removal, dialysis, ion exchange column chromatography, hydrolysis and full gel purification. GPC analysis showed a single symmetrical peak with a molecular weight of 5346Da, indicating that the molecular weight distribution was uniform and concentrated, and the total sugar content measured by the sulfuric acid phenol method was as high as 98.6%, and the content of galactose, the main component of monosaccharides, reached 98.2%, containing only trace amounts of glucose and arabinose, which far exceeded the purity level of galactan separated from traditional soybean dregs.

[0032] Traditionally, galactans isolated from soybean dregs are mostly heteropolysaccharides, containing a variety of sugar components, such as arabinose, galactose, glucose, galacturonic acid, etc., while the WS1 polysaccharide prepared by this process is almost entirely composed of galactose and is a high-purity homopolysaccharide. This homopolysaccharide has better characteristics in biological activity, physicochemical properties and application potential, such as higher biocompatibility, stronger biological activity or more stable physicochemical properties, providing a better basis for its application in food, medicine, cosmetics and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a schematic diagram of the process for preparing β-D-1,4-galactan.

[0034] Figure 2 This is a polarity separation scatter plot of an embodiment of a preparation process of β-D-1,4-galactan of the present invention.

[0035] Figure 3 This is a gel purification chromatogram of an embodiment of the preparation process of β-D-1,4-galactan of the present invention.

[0036] Figure 4 This is a GPC molecular weight analysis chart of a preparation process embodiment of β-D-1,4-galactan of the present invention.

[0037] Figure 5 This is a mixed standard diagram of monosaccharide components in an embodiment of the preparation process of β-D-1,4-galactan of the present invention.

[0038] Figure 6 This is a crude polysaccharide monosaccharide component analysis diagram of a preparation process example of β-D-1,4-galactan of the present invention.

[0039] Figure 7 This is a W monosaccharide component analysis diagram of a preparation process embodiment of β-D-1,4-galactan of the present invention.

[0040] Figure 8 This is a WS1 monosaccharide component analysis diagram of a preparation process embodiment of β-D-1,4-galactan of the present invention.

[0041] Fig. 9 This is an ultraviolet spectrum diagram of an embodiment of the preparation process of β-D-1,4-galactan of the present invention.

[0042] Fig.10 This is an infrared spectrum diagram of an embodiment of the preparation process of β-D-1,4-galactan of the present invention.

[0043] Fig.11This is a methylation GC-MS chromatogram of a preparation process embodiment of β-D-1,4-galactan of the present invention.

[0044] Fig.12 The H nuclear magnetic resonance spectrum is a preparation process example of β-D-1,4-galactan of the present invention.

[0045] Fig.13 The C nuclear magnetic resonance spectrum of a preparation process embodiment of β-D-1,4-galactan of the present invention.

[0046] Fig.14 The COSY nuclear magnetic resonance spectrum is a preparation process example of β-D-1,4-galactan of the present invention.

[0047] Fig.15 The present invention provides a HMBC nuclear magnetic resonance spectrum of a preparation process example of β-D-1,4-galactan.

[0048] Fig.16 The NOESY nuclear magnetic resonance spectrum is a preparation process example of β-D-1,4-galactan of the present invention.

[0049] Fig.17 The HSQC-EDITED nuclear magnetic resonance spectrum is a preparation process example of β-D-1,4-galactan of the present invention.

[0050] Fig.18 The TOCSY nuclear magnetic resonance spectrum is a preparation process example of β-D-1,4-galactan of the present invention.

[0051] Fig.19 This is a schematic diagram of the internal structure of a centrifugal device in an embodiment of a process for preparing β-D-1,4-galactan of the present invention.

[0052] Fig. 20 This is a schematic diagram of the internal structure of a chromatography column in an embodiment of a preparation process of β-D-1,4-galactan of the present invention.

[0053] The figure marks in the drawings of the specification include: 1, centrifuge box; 2, cover; 3, electric slip ring; 4, first motor; 5, first carrier; 6, second carrier; 7, first chamber; 8, transmission shaft; 9, second chamber; 10, filter screen; 11, disc; 12, drain port; 13, second motor; 14, chromatography column; 15, slot; 16, centrifuge tank. DETAILED DESCRIPTION

[0054] The following is further described in detail through specific implementation methods:

[0055] The embodiment is basically as shown in the attached Figure 1-Figure 20As shown: A preparation process of β-D-1,4-galactan comprises the following steps:

[0056] The crude polysaccharide is prepared by weighing bean dregs as a raw material, mixing the bean dregs with water, and then extracting at a preset temperature; after the extraction is completed, the supernatant is separated by a centrifugal device, and amylase is added, and the extract is separated by filtering; the protein is removed by the Sevag method, and the extract is concentrated by vacuum rotary evaporation; the concentrated extract is transferred to a dialysis bag for dialysis, and then the dialyzed clear liquid is separated by centrifugation, and the dialyzed clear liquid is concentrated and freeze-dried to obtain a crude polysaccharide sample;

[0057] Specifically, 500 g of dregs of soybean residue were selected as raw materials, and 5000 mL of distilled water was added as the extraction solvent. The extraction was carried out for 4 hours under constant temperature stirring in a water bath at 80°C. The selection of this temperature and time is intended to effectively dissolve the polysaccharide components in the dregs of soybean residue while avoiding the destruction of the polysaccharide structure by high temperature.

[0058] The extracted mixture was centrifuged at 4500 r / min for 10 minutes to separate the supernatant.

[0059] In this embodiment, the centrifugal device includes a centrifugal box 1; a cover body 2 is provided on the top of the centrifugal box 1, and the cover body 2 is rotatably connected to the centrifugal box 1 through a hinge; Fig.19 As shown, a first carrier 5 is provided at the bottom of the cover body 2. The first carrier 5 is cylindrical in shape, is embedded in the bottom of the cover body 2 and is rotatably connected to the cover body 2; a plurality of first motors 4 are circumferentially arranged in the first carrier 5, and the output shaft of the first motor 4 passes through the bottom of the first carrier 5; an electric slip ring 3 is provided at the top of the first carrier 5, and the electric slip ring 3 is connected to a power source. The electric slip ring 3 is used to keep the first motor 4 connected to the power source when the first carrier 5 rotates; in this embodiment, the fixed end of the electric slip ring 3 is welded and fixed to the cover body 2, and the rotating end of the electric slip ring 3 is welded and fixed to the first carrier 5.

[0060] A centrifugal groove 16 is provided in the centrifugal box 1; a second carrier 6 is provided in the centrifugal groove 16. In this embodiment, the second carrier 6 is cylindrical in shape, and the second carrier 6 rotates in cooperation with the centrifugal groove 16; a second motor 13 for driving the second carrier 6 to rotate is provided at the bottom of the centrifugal groove 16; a plurality of first chambers 7 are provided in the circumferential direction of the second carrier 6. In this embodiment, the position and number of the first chambers 7 correspond one-to-one to the number and position of the first motors 4, and the bottom center of the first chamber 7 is connected to the second chamber 9. In this embodiment, the second chamber 9 is located directly below the first chamber 7; a second chamber 9 is provided at the axis center. A plurality of discs 11 are provided, and the discs 11 are rotatably matched with the second chamber 9. A transmission shaft 8 is provided at the axis of the disc 11. The transmission shaft 8 coaxially welds and fixes all the discs 11. The discs 11 are spaced apart by a preset distance, and the preset distance is twice the thickness of the fluid boundary layer. A plurality of drainage ports 12 are circumferentially provided near the axis of the disc 11. The top of the transmission shaft 8 extends into the first chamber 7, and the top of the transmission shaft 8 is connected to the output shaft of the first motor 4 through a spline. A filter screen 10 is welded and fixed around the disc 11. A circulation outlet is also provided at the top of the second chamber 9, and the circulation outlet is located above the edge of the disc 11.

[0061] Specifically, after the extraction is completed, the extracted solution is poured into the centrifuge box 1 to separate the supernatant. The specific process is as follows:

[0062] After opening the cover 2, the solution to be centrifuged is poured into each first chamber 7, and the solution flows into the second chamber 9 along the first chamber 7. The cover 2 is closed, and the first motor 4 and the second motor 13 are started. The second motor 13 drives the second carrier 6 (including the first carrier 5) to rotate, and the first motor 4 drives the transmission shaft 8 to rotate, and the transmission shaft 8 drives the disc 11 to rotate. The disc 11 drives the solution in the second chamber 9 to make a spiral centripetal motion relative to the second chamber 9, and the impurities in the solution (mainly fixed impurities, such as suspended matter, etc.) are thrown onto the side wall of the second chamber 9 due to their large specific gravity, and the liquid part is discharged upward from the discharge port 12 at the axis of the disc 11 to the first chamber 7, and then flows back to the second chamber 9 from the circulation outlet, and the cycle is repeated, and the solid impurities are accumulated and precipitated on the side wall of the second chamber 9 after passing through the filter 10. This cycle process greatly improves the separation efficiency.

[0063] At the same time, since the second carrier 6 is rotating as a whole, the solution in the second chamber 9 also revolves while rotating, thereby further improving the centrifugal effect in each chamber, so that the solution therein is more fully separated under the action of centrifugal force, thereby improving the separation efficiency and the purity of the supernatant.

[0064] The supernatant is treated with amylase to remove soluble starch. Amylase can specifically decompose starch while having little effect on polysaccharides, thus ensuring the purity of the extract.

[0065] The extract after the amylase treatment needs to be further deproteinized. The Sevag method is a commonly used method for deproteinizing biological samples, which uses a mixed solution of chloroform and n-butanol to form a precipitate with the protein, thereby separating it from the solution.

[0066] The deproteinized extract was concentrated by vacuum rotary evaporation to reduce the volume of the solution for subsequent processing.

[0067] The concentrate was transferred to a dialysis bag with a molecular cutoff of 8000 and dialyzed in a large amount of 4°C distilled water for 72 hours. The purpose of dialysis is to remove small molecule impurities such as salts and oligosaccharides, while maintaining the integrity of large molecules such as polysaccharides.

[0068] The dialyzed solution was centrifuged again at 4500 r / min for 10 minutes to separate the dialyzed clear solution.

[0069] The dialyzed supernatant was concentrated and freeze-dried to prepare crude polysaccharide.

[0070] Polar separation, separation by ion exchange column chromatography, during the elution process, the solutions under different elution peaks are collected, concentrated, dialyzed, and freeze-dried to obtain W polysaccharide;

[0071] Specifically, the DEAE 52 filler was first soaked in 0.5 mol / L hydrochloric acid for 1 hour. This step was to remove impurities on the surface of the filler and to give it a positive charge in preparation for subsequent interaction with negatively charged polysaccharide molecules.

[0072] In this embodiment, DEAE 52 filler is filled into the chromatography column 14 to make a DEAE 52 filler column, wherein a plurality of slots 15 are provided on the side wall of the chromatography column 14, and the slots 15 are used to place a charged rod, and the charged rod is a glass rod (positively charged) after being rubbed with silk or a rubber rod (negatively charged) after being rubbed with fur. In specific implementation, the stable electric field generated by the charged rod around the chromatography column 14 can directly affect the molecules of the substance flowing through the DEAE 52 filler. Since different molecules of different substances respond differently to the electric field (such as charge properties, polarization degree, etc.), this electric field effect can significantly promote the separation efficiency between molecules and improve the separation accuracy and selectivity of the chromatography column 14.

[0073] Wash the hydrochloric acid and impurities in the packing with distilled water, and then elute with 4-5 times the volume of distilled water until it is neutral. Next, balance the packing with distilled water for 2 hours and adjust the flow rate to 5 ml / min.

[0074] The crude polysaccharide was dissolved in distilled water, heated and vortexed to promote dissolution, and then centrifuged at 12000 rpm to remove insoluble matter, and the supernatant was taken as the loading solution.

[0075] The treated crude polysaccharide solution was loaded onto the equilibrated DEAE 52 packing column.

[0076] First, distilled water (flow rate 15 ml / min) was used for elution, mainly to remove impurities not bound to the DEAE 52 filler and components with weaker affinity.

[0077] Then, different concentrations of NaCl solution (0.2M, 0.5M, 1.0M) and three times the column volume of distilled water were used for gradient elution. The ions in these solutions would compete with the polysaccharide molecules for binding to the charge sites on the filler, thereby eluting the polysaccharide components with different affinities one by one.

[0078] The phenol-sulfuric acid method was used for tracking detection, and the polysaccharide content in the eluate was detected by an enzyme marker at a wavelength of 490 nm.

[0079] A scatter plot was drawn based on the test results, and the elution ranges of different components were determined based on the peak shapes.

[0080] Then the desired components were collected, concentrated and dialyzed with a 3500Da dialysis bag to remove small molecule impurities and salts, and finally freeze-dried to obtain the purified polysaccharide component.

[0081] The obtained components were named W (distilled water elution component), 0.2M, 0.5M, and 1.0M (corresponding to components eluted with different NaCl concentrations).

[0082] Hydrolysis, dissolving W polysaccharide in trifluoroacetic acid, and reacting and hydrolyzing under microwave irradiation; after the reaction, neutralizing, dialysis, concentrating, and freeze-drying are performed to obtain WS polysaccharide;

[0083] Specifically, a certain amount of W polysaccharide sample was taken, dissolved in trifluoroacetic acid, and reacted for 3 hours at 90° and a microwave power of 400 W. After the reaction, it was neutralized with sodium hydroxide, dialyzed, concentrated by a rotary evaporator, and freeze-dried to obtain WS polysaccharide.

[0084] Purification: After dissolving the WS polysaccharide, centrifuge and take the supernatant for sample loading, purify it through a full gel purification system, collect the symmetrical peaks, dialyze, concentrate, and freeze-dry the collected eluate to obtain the separated and purified WS1 polysaccharide.

[0085] Specifically, an appropriate amount of WS polysaccharide to be purified is weighed, dissolved in a mobile phase, centrifuged at 12000 rpm for 10 min, the supernatant is loaded, purified by a full gel purification system, and the symmetrical peak is collected. The collected liquid is dialyzed, concentrated by a rotary evaporator, and freeze-dried to obtain WS1 polysaccharide separated and purified by a gel column, namely β-D-1,4-galactan.

[0086] The prepared WS1 polysaccharide sample was studied and analyzed as follows:

[0087] ① Molecular weight determination

[0088] Dissolution: Dissolve the WS1 polysaccharide sample in 0.05 M NaCl solution to ensure that the sample is completely dissolved and the concentration is appropriate.

[0089] Centrifugation: The dissolved sample was centrifuged at 12000 rpm for 10 minutes to remove insoluble matter or impurities, and the supernatant was taken for subsequent analysis.

[0090] Filtration: The supernatant was filtered through a 0.22 μm PVDF filter membrane to further remove tiny particles and impurities to ensure the purity of the sample solution.

[0091] Chromatographic column: Shodex-ks-805-804-802 chromatographic column was used.

[0092] Detector: Shodex differential detector (RI-102) was used.

[0093] Injection concentration: Adjust the sample solution to a concentration of 5 mg / mL for injection.

[0094] Mobile phase: 0.05 M NaCl was used as the mobile phase.

[0095] Flow rate: Set the flow rate to 0.6 mL / min and maintain a stable flow rate to obtain accurate chromatograms.

[0096] Curve drawing:

[0097] Standard pullulans with molecular weights of 1152, 11600, 23800, 48600, 80900, 148000, 273000, and 409800 were used as standards.

[0098] Under the same chromatographic conditions, the standards were analyzed one by one and the retention time of each standard was recorded.

[0099] Draw a standard curve with the logarithm of the molecular weight of the standard as the horizontal axis and the corresponding retention time as the vertical axis. Make sure that the standard curve has a good linear relationship so that it can be used to calculate the molecular weight of unknown samples later.

[0100] Under the same chromatographic conditions, the unknown polysaccharide samples were analyzed and their retention times were recorded.

[0101] Substitute the retention time of the sample into the standard curve equation, solve the corresponding molecular weight logarithm, and then convert it into molecular weight value.

[0102] ②Monosaccharide component analysis

[0103] Weighing samples: First, accurately weigh 5 mg of polysaccharide sample and place it in a high temperature resistant ampoule.

[0104] Add trifluoroacetic acid (TFA): Add 2 ml of 3M trifluoroacetic acid (TFA) solution to the ampoule. TFA is a strong acid that can effectively hydrolyze the glycosidic bonds in polysaccharides and convert them into monosaccharides.

[0105] Hydrolysis reaction: The ampoule was placed at 120°C for 2 hours for hydrolysis reaction.

[0106] After the hydrolysis reaction was completed, the hydrolyzate was transferred to a drying tube and dried using nitrogen purge.

[0107] Add 10 ml of water to the dried sample and vortex to fully dissolve the monosaccharides. Then, take 80 μL of the solution and add 920 μL of deionized water to dilute it to reduce the injection volume and improve the detection sensitivity. Finally, centrifuge the diluted solution at 12,000 rpm for 5 minutes to remove insoluble matter or impurities, and take the supernatant for IC analysis.

[0108] Ion chromatography (IC) analysis

[0109] Chromatographic column and detector: Dionex CarbopacTMPA20 (3*150) chromatographic column is used for separation, and an electrochemical detector is configured for detection. The electrochemical detector has high sensitivity and selectivity to sugar substances.

[0110] Mobile phase preparation: Prepare three mobile phases, namely A: H2O (pure water), B: 15mM NaOH (sodium hydroxide solution), C: 15mM NaOH & 100mM NaOAC (mixed solution of sodium hydroxide and sodium acetate).

[0111] Analysis conditions: set the flow rate to 0.3 ml / min, the injection volume to 5 μL, and the column temperature to 30 °C.

[0112] Quantitative analysis

[0113] Standard curve: Use monosaccharide standards of known concentration to prepare a mixed standard solution, and determine the mass of different monosaccharides by absolute quantitative method. Calculate their molar ratio based on the molar mass of monosaccharides to draw a standard curve.

[0114] Sample determination: Inject the treated sample supernatant into the chromatographic system for analysis, compare it with the standard curve based on parameters such as retention time and peak area, and calculate the content and molar ratio of various monosaccharides in the sample.

[0115] ③Infrared analysis

[0116] 2.0 mg of WS1 polysaccharide sample and 200.0 mg of potassium bromide were weighed and pressed into tablets, and a blank control was made by pressing potassium bromide powder into tablets. They were placed in a Fourier transform infrared spectrometer FT-IR650 for scanning and recording in the range of 400 to 3600 cm-1.

[0117] ④UV measurement

[0118] The sample was prepared at 0.1 mg / mL and scanned at 190-400 nm using a UV spectrophotometer.

[0119] ⑤Methylation analysis

[0120] Weigh 10 mg of polysaccharide sample and dissolve it in 1 mL of anhydrous DMSO.

[0121] Add 1 mL of dimethyl sulfoxide (DMSO) and an excess of a methylating agent (usually not a dimethyl sodium solution, but a combination of iodomethane and sodium hydroxide, or a commercial methylating agent such as Dimethyl Sulfate with Sodium Hydroxide in DMSO), shake well, and react at room temperature in the dark for 12 hours or according to the time recommended by the reagent instructions.

[0122] 1 mL of saturated potassium iodide solution was added dropwise to quench the excess methylating agent, and the reaction was continued at room temperature in the dark for 5 hours.

[0123] The reaction solution is dialyzed against flowing water (or a suitable solvent exchange method is used) to remove small molecule impurities and unreacted reagents, and then freeze-dried to obtain the methylated polysaccharide.

[0124] The infrared absorption spectrum of the methylated polysaccharide is measured by Fourier-transforminfrared spectroscopic (FT-IR). If the absorption peak of the hydroxyl group (OH) is significantly weakened or disappears, and a new absorption peak related to the methyl group (CH) appears, it indicates that the polysaccharide is successfully methylated.

[0125] Hydrolysis, reduction and acetylation of methylated polysaccharides

[0126] Hydrolysis: The methylated polysaccharide was hydrolyzed with 2 mL of 2 M trifluoroacetic acid (TFA).

[0127] Reduction: After the hydrolyzate is diluted with an appropriate amount of water, 50 mg of sodium borohydride (NaBH4) is added for reduction to convert the aldehyde group into a stable alcohol group.

[0128] Acetylation: The reduction products are further acetylated to methylated alditol acetates (PMAAs), which is usually accomplished by adding acetic anhydride and a catalyst (such as pyridine) under heating conditions.

[0129] GC-MS analysis

[0130] GC-MS analysis was performed using a RXI-5SIL capillary column (30 m, 0.25 mm, 0.25 μm) equipped with an ion trap mass spectrometer detector.

[0131] The initial temperature was set at 140°C, raised to 250°C at a rate of 3°C / min, and maintained at 250°C for 5 minutes.

[0132] The injection port temperature and the detector temperature were both set to 250 °C, and the carrier gas was helium with a flow rate of 1 mL / min.

[0133] The data obtained by GC-MS are compared with standard spectra or databases to analyze the monosaccharide composition and connection mode of the methylated polysaccharides.

[0134] ⑥Nuclear magnetic resonance imaging

[0135] Accurately weigh 50 mg of polysaccharide sample.

[0136] The sample was dissolved in 0.5 ml of heavy water (D2O, 99.9% purity). This step was to allow the active hydrogen (such as hydroxyl hydrogen) in the sample to exchange with the deuterium (D) in the heavy water.

[0137] Subsequent freeze drying removes excess heavy water but preserves the deuterium label in the sample.

[0138] The above dissolution-freeze drying steps were repeated 3 times to ensure sufficient exchange of active hydrogen.

[0139] The deuterated water exchanged sample was redissolved in 0.5 ml of deuterated water (D2O) and prepared for NMR analysis.

[0140] NMR spectrum acquisition

[0141] 1H NMR:

[0142] The measurement is carried out using an 800 MHz nuclear magnetic resonance spectrometer (such as Bruker series).

[0143] Pulse Sequence: zg30

[0144] Number of Scans: 16

[0145] Relaxation Delay: 1 second

[0146] 13C NMR:

[0147] Pulse sequence: zgpg30

[0148] Scan count: 20480

[0149] Relaxation delay: 2 seconds

[0150] DEPT135:

[0151] Pulse sequence: deptsp135

[0152] Scan times: 1024

[0153] Relaxation delay: 2 seconds

[0154] Two-dimensional NMR spectrum:

[0155] COSY (Correlation Spectroscopy): used to determine the connection relationship between adjacent hydrogen atoms.

[0156] HSQC (Heteronuclear Single Quantum Coherence): connects hydrogen atoms and the carbon atoms directly connected to them.

[0157] HMBC (Heteronuclear Multiple Bond Correlation): Provides long-range (usually 2-3 bonds) carbon-hydrogen connection information.

[0158] TOCSY (Total Correlation Spectroscopy) and NOESY (Nuclear Overhauser Effect Spectroscopy): used to analyze the spatial relationship of hydrogen atoms in complex structures.

[0159] The NMR data were processed and analyzed using MestReNova software.

[0160] ⑦Results and analysis

[0161] As attached Figure 2 As shown, from left to right, the corresponding peaks are collected: M-W1.4, M-0.2M1.9, -0.5M0.8g, M-1.0M0.6g. The yields of each component are: 4%, 7%, 2.6%, 2%.

[0162] The W polysaccharide was selected for further processing (dark background component).

[0163] A certain amount of W polysaccharide sample was taken, dissolved in trifluoroacetic acid, and reacted for 3 h at 90° and microwave power of 400 W. After the reaction, it was neutralized with sodium hydroxide, dialyzed, concentrated by rotary evaporator, and freeze-dried to obtain WS polysaccharide.

[0164] As attached Figure 3 As shown, the freeze-dried sample (WS polysaccharide) was gel purified. According to the gel purification chromatogram, the peak of 45-50min was collected as the final polysaccharide sample, which was concentrated by a rotary evaporator, dialyzed, and freeze-dried to obtain the WS1 polysaccharide sample separated and purified by a gel column, with a yield of 1.6%.

[0165] As attached Figure 4-Figure 8 As shown in Table 1, the WS1 polysaccharide structure analysis was performed, and a single symmetrical peak with a molecular weight of 5346Da was obtained by GPC analysis. The total sugar content was 98.6% measured by the sulfuric acid phenol method. The main component of monosaccharide was galactose, with a content of up to 98.2%. At the same time, trace amounts of glucose and arabinose were measured, indicating that it was a homopolysaccharide with considerable purity. Usually, the galactan separated from soybean dregs is mainly heteropolysaccharide, containing a considerable proportion of arabinose, galactose, glucose, galacturonic acid and other components.

[0166] Table 1 Monosaccharide composition

[0167]

[0168] Result analysis: In the polar separation process of okara polysaccharides, the components obtained by water elution are mainly neutral polysaccharides. This neutral polysaccharide is a biological macromolecule whose main chain is mainly composed of galactose, while monosaccharides such as arabinose and glucose are connected to the main chain as branches. When directly trying to separate and purify the neutral polysaccharide component from the mixture, it is often impossible to obtain high-purity galactan. This is because although the content of galactan in the original component is relatively high (such as 78.9% mentioned in this example), it still contains a certain amount of arabinose (6.4%) and glucose (14.7%), resulting in insufficient purity. However, compared with the report by JM Rodrigues et al. (reference A. Nakamura, H. Furuta, H. Maeda, T. Takao and Y. Nagamatsu published an article entitled "Study on the stepwise enzymatic degradation of the main chain of soybean soluble polysaccharides composed of galacturonic acid and rhamnosegalacturonic acid" on page 24 of the 36th page of the journal "Food & Function"), the galactose content of most soybean meal polysaccharides is higher at 40-50%.

[0169] In order to improve the purity of galactan, this scheme adopts partial hydrolysis. This method selectively decomposes the glycosidic bonds on the side chains, so that the branch sugar chains such as arabinose and glucose are hydrolyzed from the main chain. After such treatment, the arabinose content in the W component is significantly reduced to 0.7%, and the glucose content is also reduced to 1.1%.

[0170] After partial hydrolysis, the remaining mixture is further separated and purified using a gel purification method. This method is able to effectively separate the incompletely hydrolyzed main chain galactans from other small molecules or residual impurities based on the differences in molecular size and charge characteristics.

[0171] Through the above steps, high-purity galactan (WS1) was successfully obtained, and its galactose content was as high as 98.2%. This shows that the purity of the neutral polysaccharide component in okara polysaccharide can be significantly improved by combining partial hydrolysis and gel purification, thereby obtaining high-purity galactan.

[0172] UV analysis

[0173] As attached Fig. 9 As shown, the polysaccharide has no characteristic absorption peaks of nucleic acid (260 nm) and protein (280 nm), indicating that the polysaccharide has no interference from nucleic acid and protein.

[0174] Infrared analysis

[0175] As attached Fig.10 As shown, the absorption band at 3600-3200cm-1 is the stretching vibration absorption peak of -OH, and the absorption peak in this area is the characteristic peak of sugars. Specifically as follows: 3392cm-1 is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. There is an absorption peak at 2935cm-1, which may be attributed to CH stretching vibration. There is an absorption peak at 1643cm-1, which may be attributed to crystal water. There is an absorption peak at 1457cm-1, which may be attributed to CH angle vibration. There is an absorption peak at 1419cm-1, which may be attributed to CO stretching vibration. There are absorption peaks at 1074cm-1 and 1038cm-1, which may be attributed to the asymmetric ring stretching vibration of the pyran ring. There is an absorption peak at 886cm-1, which may be attributed to the CH angle vibration of the β-end diastereomer of the pyran ring. The detailed molecular structure was further verified by nuclear magnetic resonance detection.

[0176] Methylation analysis

[0177] As attached Fig.11 As shown in Figure 2, methylation is an indispensable method for the deduction of polysaccharide fine structure. The sample is initially methylated, hydrolyzed and reduced to obtain PMAAs, which are then subjected to GC-MS analysis. Fig.11As shown, there is only one major methylated sugar derivative with a retention time of 19.8 min and a content of 89.1%. Based on the comparison of ion fragments and retention times, it is shown that the main chain of the polysaccharide may be →4)-Galp-(1→, that is, a 1-4 connected galactomannan with a highly repeated linear structure. The terminal galactose was found, indicating that it is a small molecular weight polysaccharide. No branched sugar bonding mode was found, indicating that the galactomannan does not have branches. These findings were also confirmed by NMR spectroscopy.

[0178] NMR analysis

[0179] Attached Figure 12-Figure 18 As shown, the chemical structure of the polysaccharide was further determined using NMR 1H, 13C, HH-COSY, HSQC, HMBC, TOCSY and NOESY spectra.

[0180] The chemical structure of the polysaccharide was further determined by using NMR 1H, 13C, HH-COSY, HSQC, HMBC, TOCSY and NOESY spectra. Seven strong signal peaks appeared in the 1H spectrum, δH4.48, 4.00, 3.66, 3.62, 3.61, 3.56 and 3.51. From the integrated area, it can be inferred that each signal peak contains one proton. The TOCSY spectrum judged that these protons were in the same spin system and could be preliminarily determined to be seven protons on the sugar residue. δH3.66, 3.62, 3.56 and 3.61 overlapped and could be identified separately by the HMQC spectrum.

[0181] Six sharp, separated spectral lines δC106.7, 79.9, 76.9, 75.8, 74.3 and 63.3 appeared in the 13C spectrum, which were inferred to be the signals of six carbon atoms on the sugar residue. According to the HSQC-EDITED spectrum, δC106.7 can be judged as the terminal carbon, and the corresponding δH4.48 is the terminal proton. The chemical shift of the terminal proton is less than 4.6, indicating that it is a β configuration. δC63.3 is a methylene carbon. Combined with the HSQC spectrum, δH3.66 and 3.62 can be identified as two H on the methylene carbon. The COSY spectrum is analyzed for the 3J cross peaks of ortho-hydrogen, and the chemical shifts of H at positions 2, 3, 4, 5, and 6 are assigned. Combined with the HMQC spectrum, the corresponding carbon chemical shifts can be obtained, thereby obtaining the full assignment of the carbon-hydrogen chemical shifts of the sugar residue.

[0182] The glycosidic bond connection mode was analyzed by 3JCH long-range coupling HMBC spectrum. The HMBC spectrum has high resolution, the coupling information is clear at a glance, and there are obvious and clear H1-C4 and C1-H4 cross peaks, which can be judged that the glycosidic bond is 1-4 connected, and no other sugar residue fragment signals are displayed, indicating that the structure is a galactoside bond as the main chain, and the proportion of other branched sugar residues is very small, which is consistent with the results of monosaccharide composition and methylation analysis. At the same time, there is a long-range coupling signal of C1-H5, and there is no signal between 82 and 84, indicating that there is no furanose, confirming the presence of pyranose.

[0183] NOESY spectrum is a spatial distance spectrum, which shows clear and obvious coupling signals between 7 H, indicating that these H are close in space and are attributed to the H signals on the sugar ring, which is consistent with the above H spectrum attribution results. The carbon-hydrogen chemical shifts of polysaccharides are statistically shown in the following table:

[0184] C1 / H1:106.7 / 4.48,

[0185] C2 / H2:74.3 / 3.51,

[0186] C3 / H3:75.8 / 3.61,

[0187] C4 / H4:79.9 / 4.00,

[0188] C5 / H5:76.9 / 3.56,

[0189] C6 / H6: 63.3 / 3.66, 3.62

[0190] Comprehensive analysis showed that the molecular structure of the polysaccharide was identified as a linear galactan formed by β-D-Galp connected by 1→4 glycosidic bonds. The structure deduced by NMR was consistent with the structure of monosaccharide component analysis and methylation analysis, indicating that the deduced structure was reliable.

[0191] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and / or characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A process for preparing β-D-1,4-galactan, characterized in that: The following steps are involved: To prepare crude polysaccharide, bean dregs are weighed as raw materials, the bean dregs are mixed with water, and then extracted at a preset temperature; After the extraction is completed, the supernatant is separated by a centrifugal device, and amylase is added, and the extract is separated by filtration; the protein is removed by the Sevag method, and the extract is concentrated by vacuum rotary evaporation; the concentrated extract is transferred to a dialysis bag for dialysis, and then the dialyzed clear liquid is separated by centrifugation, and the dialyzed clear liquid is concentrated and freeze-dried to obtain a crude polysaccharide sample; Polar separation is carried out by ion exchange column chromatography. During the elution process, the solution under the corresponding elution peak is collected, concentrated, dialyzed, and freeze-dried to obtain W polysaccharide; Hydrolysis, dissolving W polysaccharide in trifluoroacetic acid, and reacting and hydrolyzing under microwave irradiation; after the reaction, neutralizing, dialysis, concentrating, and freeze-drying are performed to obtain WS polysaccharide; Purification: After dissolving the WS polysaccharide, centrifuge and take the supernatant for sample loading, purify it through a full gel purification system, collect the symmetrical peaks, dialyze, concentrate, and freeze-dry the collected eluate to obtain the separated and purified WS1 polysaccharide.

2. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: When preparing crude polysaccharide, the mixture was stirred and extracted at a constant temperature of 80°C in a water bath for 4 hours.

3. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: When preparing crude polysaccharide, the dialysis bag used has a molecular weight cutoff of 8000Da.

4. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: When preparing crude polysaccharide, the dialysis bag was placed in distilled water at 4°C and dialyzed for 72 hours.

5. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: When separating polarity, the specific steps of separation by ion exchange column chromatography are as follows: Soak the DEAE 52 filler in 0.5 mol / L hydrochloric acid for 1 hour; After soaking, use deionized water or distilled water to wash away impurities in the DEAE 52 packing; When eluting, use 4-5 times the volume of distilled water and continue washing until the pH value of the filler solution reaches neutral; Before loading, the DEAE 52 packing was equilibrated with distilled water. During equilibration, the flow rate was adjusted to 5 ml / min, and distilled water was allowed to flow through the packing column for 2 hours. Dissolve the crude polysaccharide in distilled water, heat and vortex; After dissolution, the solution was centrifuged and the supernatant was taken as the loading solution; Slowly add the supernatant to the equilibrated DEAE 52 column; After loading, distilled water was used for elution and the flow rate was adjusted to 15 ml / min.

6. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: During elution, the elution solvent is three times the column volume of water, and the collected eluate is detected using the phenol-sulfuric acid method. The absorbance is measured at a wavelength of 490 nm using an enzyme reader, and the detected data is plotted as a scatter plot. The corresponding eluate is collected based on the peak shape on the scatter plot.

7. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: In polar separation, the molecular weight cutoff of the dialysis bag used for dialysis is 3500Da.

8. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: During the hydrolysis, the reaction was carried out at 90°C and a microwave power of 400 W for 3 hours.

9. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: During the purification process, the centrifugal speed was 12000 rpm and the duration was 10 min.

10. The process for preparing β-D-1,4-galactan according to claim 1, characterized in that: The WS1 polysaccharide is β-D-1,4-galactan.

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