Preparation process of food-grade soluble soybean polysaccharide

Abstract

The present invention relates to the technical field of polysaccharides, and particularly to a preparation process of food-grade soluble soybean polysaccharide. The process comprises the following steps: preparing a template molecule solution with a high-purity soluble soybean polysaccharide standard product, dissolving acrylic acid and ethylene glycol dimethacrylate in toluene, adding the template molecule solution and an ammonium bicarbonate porogen, and mixing uniformly by ultrasonic treatment to obtain a molecularly imprinted polymer. Pretreating soybean meal with a sodium hydroxide solution, filtering, adjusting the pH with hydrochloric acid to precipitate soybean polysaccharide, collecting by centrifugation, and dissolving to obtain a crude product solution. Loading the molecularly imprinted polymer into a chromatographic column, introducing the crude product solution, collecting the eluate after elution, and spray-drying to obtain the food-grade soluble soybean polysaccharide. The process of using the molecularly imprinted polymer to prepare the food-grade soluble soybean polysaccharide according to the present invention can specifically recognize and bind the soluble soybean polysaccharide, effectively remove impurities that are difficult to separate by traditional methods, and thus greatly improve the purity of the soybean polysaccharide.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and particularly relates to a preparation process of food-grade soluble soybean polysaccharide. Background Art

[0002] Soluble Soybean Polysaccharide (SSPS) is a type of functional polysaccharide extracted from soybeans and has wide applications in the food industry. Due to its good emulsifying property, stability, thickening property, and prebiotic characteristics, it is used in fields such as beverages, dairy products, and baked foods, and can effectively improve food quality and extend the shelf life. Traditional preparation technologies of soluble soybean polysaccharide mainly include water extraction method, enzymatic hydrolysis method, and membrane separation method, etc.

[0003] The water extraction method is the most basic extraction method. By mixing soybean raw materials with water, under certain temperature and time conditions, the soluble soybean polysaccharide is dissolved in water, and then products are obtained through steps such as filtration, concentration, and drying. This method has a simple process and low cost, but has problems of low extraction rate and low product purity. A large amount of impurities such as soybean protein and oligosaccharides are difficult to effectively remove, affecting the performance of the product in high-end food applications, and the subsequent separation and purification are difficult and costly.

[0004] The enzymatic hydrolysis method uses specific enzymes to hydrolyze soybean raw materials to break the cell wall structure and promote the release of soluble soybean polysaccharide. Compared with the water extraction method, the enzymatic hydrolysis method can improve the extraction rate and product purity. However, the selection and use conditions of enzymes are relatively harsh, and the enzyme activities of different sources and batches vary greatly, resulting in unstable product quality. Moreover, the cost of enzymes is high, increasing the overall production cost and limiting large-scale industrial applications.

[0005] The membrane separation method separates soybean extract by membranes with different pore sizes, and separates soluble soybean polysaccharide from impurities according to molecular size. This method can effectively remove macromolecular impurities and some small molecules, improve product purity, and the operation process is relatively mild, with little impact on the polysaccharide structure and activity. However, the price of membranes is expensive, and they are prone to pollution and blockage, requiring frequent cleaning and replacement, increasing equipment investment and operating costs. At the same time, part of the polysaccharide is retained and lost during the membrane separation process, reducing the final yield. Summary of the Invention

[0006] In order to overcome the above prior art problems, the present invention provides a preparation process of food-grade soluble soybean polysaccharide. Compared with traditional preparation technologies, the process of using molecularly imprinted polymers to prepare food-grade soluble soybean polysaccharide in the present invention can specifically recognize and bind soluble soybean polysaccharide, effectively remove impurities that are difficult to separate by traditional methods, and thus greatly improve the purity of soybean polysaccharide.

[0007] To achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0008] A preparation process of food-grade soluble soybean polysaccharide, comprising the following steps:

[0009] 1) Crush soybean meal, soak the crushed soybean meal in an ethanol solution to remove oil and impurities, then perform filtration separation, wash the filter residue with water until neutral, and then dry it to obtain pretreated soybean meal;

[0010] 2) Take a high-purity soluble soybean polysaccharide standard as a template molecule, dissolve the template molecule in deionized water to prepare a template molecule solution;

[0011] 3) Add acrylic acid and ethylene glycol dimethacrylate to a toluene solvent, stir and dissolve to obtain a mixed solution, add the template molecule solution and ammonium bicarbonate porogen to the mixed solution, ultrasonically oscillate and disperse evenly, then add azobisisobutyronitrile initiator, under nitrogen protection, heat and react, and then wash to remove the template molecule and unreacted substances to obtain a molecularly imprinted polymer;

[0012] 4) Add the pretreated soybean meal to a sodium hydroxide solution, stir and extract, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to acidic to precipitate soybean polysaccharide, collect the precipitate by centrifugation, and then dissolve the precipitate to obtain a crude soybean polysaccharide solution;

[0013] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, then use an elution solution for elution, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

[0014] Traditional methods for preparing food-grade soluble polysaccharides mainly include hot water extraction, acid-base extraction, enzymatic hydrolysis, etc. Hot water extraction is simple to operate and has a relatively low cost, but it has problems such as low extraction rate, long extraction time, and high energy consumption, and high temperature may destroy the biological activity of polysaccharides. Although acid-base extraction can improve the extraction rate, the acid-base conditions are harsh, which easily leads to changes in the polysaccharide structure. Subsequently, cumbersome neutralization and impurity removal steps are required, introducing the risk of chemical residues. Enzymatic hydrolysis is relatively mild and highly specific, but the enzyme cost is high, the reaction conditions are strict, and the enzymatic hydrolysis products are complex, making the separation and purification difficult. The present invention uses molecularly imprinted polymer extraction technology to prepare food-grade soluble polysaccharides. Molecularly imprinted polymers are polymers with specific recognition sites for target polysaccharides formed through molecular imprinting technology using the target soybean polysaccharide molecule as a template under the action of specific functional monomers, cross-linking agents, and initiators. During the preparation process, the template molecule and the functional monomer form a complex through covalent or non-covalent interactions. After the cross-linking agent polymerizes, the complex is fixed. After removing the template molecule, cavities complementary to the spatial structure and binding sites of the target polysaccharide molecule are left in the polymer, thus achieving specific recognition and separation of the target polysaccharide. Compared with traditional preparation techniques, the process for preparing food-grade soluble soybean polysaccharides based on molecularly imprinted polymers in the present invention can specifically recognize and bind soluble soybean polysaccharides, significantly improve the product purity, and effectively remove impurities that are difficult to separate by traditional methods, such as proteins and oligosaccharides. It has high selectivity, can avoid complex multi-step separation and purification processes, simplify the process flow, and reduce production costs. At the same time, the process conditions are mild, have little impact on the structure and functional properties of soluble polysaccharides, can better retain their biological activity, and meet the needs of the food industry for high-quality and highly active polysaccharide products.

[0015] Preferably, in step 2), the mass concentration of the template molecule solution is 2.0 - 5.0 wt%.

[0016] Preferably, in step 3), the preparation method of the ammonium bicarbonate porogen includes the following steps:

[0017] Add ammonium bicarbonate and gelatin to deionized water and stir to dissolve to obtain a mixed solution; add Span 80 to liquid paraffin and stir evenly to obtain a stable oil-phase solution;

[0018] Slowly drop the mixed solution into the oil-phase solution, continuously stir to make the system form a stable water-in-oil emulsion. Under stirring conditions, slowly drop glutaraldehyde solution into the water-in-oil emulsion for cross-linking reaction, and then through centrifugal separation, washing, and drying, the product is obtained.

[0019] In the technical solution of the present invention, as described above, ammonium bicarbonate is used as a porogen. The gas generated by the decomposition of the porogen can form pores inside the polymer, increasing the specific surface area of the polymer, which is beneficial to the diffusion of template molecules and the binding of recognition sites, and improving the adsorption and recognition performance of the molecularly imprinted polymer. Although ammonium bicarbonate can achieve the purpose of pore formation, due to its too fast decomposition and uneven dispersion, the pore structure of the polymer is not ideal, which limits the performance of the molecularly imprinted polymer. To solve this technical problem, the present invention uniformly coats a layer of gelatin polymer on the outer surface of ammonium bicarbonate through a specific coating process. In the polymerization reaction system, the gelatin polymer plays a role of a physical barrier, delaying the contact between ammonium bicarbonate and other substances in the reaction system, thereby slowing down its decomposition rate. At the same time, the hydrophilicity of gelatin enables the coated ammonium bicarbonate to be better dispersed in the polymerization system. With the help of the intermolecular interaction, it is evenly distributed throughout the system, ensuring the stable and orderly progress of the pore formation process.

[0020] Preferably, the addition amount of the ammonium bicarbonate porogen is 3.0 - 6.0 wt% of the mass of acrylic acid.

[0021] In order to form a sufficient pore structure in the molecularly imprinted polymer and promote the diffusion of template molecules and the binding of recognition sites, a sufficient amount of ammonium bicarbonate porogen must be added during the preparation of the molecularly imprinted polymer. Therefore, the present invention controls the addition amount of the ammonium bicarbonate porogen to be at least 3.0 wt% of the mass of acrylic acid. However, when the addition amount of the ammonium bicarbonate porogen is controlled to be greater than 3.0 wt% of the mass of acrylic acid, a problem of relatively high impurity content appears in the soy polysaccharide prepared by this process of the present invention. After research by the team of the present invention, it is found that this is closely related to the addition amount of ammonium bicarbonate. When the ammonium bicarbonate is added in excess, that is, the addition amount of the ammonium bicarbonate porogen is higher than 6.0 wt% of the mass of acrylic acid, a small amount of undecomposed ammonium bicarbonate and ammonia gas remain in the molecularly imprinted polymer. The ammonia gas combines with the acidic substances in the soy polysaccharide to form ammonium salts, thereby causing a decrease in the purity of the soy polysaccharide. Therefore, the present invention also controls the addition amount of the ammonium bicarbonate porogen to be lower than 6.0 wt% of the mass of acrylic acid.

[0022] Preferably, in step 3), the heating reaction temperature is 60 - 70 °C, and the reaction time is 4 - 6 h.

[0023] Preferably, in step 4), the mass concentration of the sodium hydroxide solution is 0.2 - 0.5 wt%.

[0024] Preferably, in step 4), hydrochloric acid is added to the filtrate to adjust the pH value to 4.0 - 4.5.

[0025] Preferably, in step 5), the elution solution is an ethanol solution of sodium chloride.

[0026] The present invention has the following beneficial effects:

[0027] 1) The process of using molecularly imprinted polymers to prepare food-grade soluble soybean polysaccharides can specifically recognize and bind soluble soybean polysaccharides, effectively removing impurities that are difficult to separate by traditional methods, thereby greatly improving the purity of soybean polysaccharides;

[0028] 2) Avoid complex multi-step separation and purification processes, simplify the process flow, reduce production costs. At the same time, the process conditions are mild, with little impact on the structure and functional properties of soluble soybean polysaccharides, and can better retain their biological activities, meeting the needs of the food industry for high-quality and highly active polysaccharide products. Detailed implementation manners

[0029] The following further clearly and detailedly describes the present invention in combination with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are usually only a part of the embodiments of the present invention, rather than all the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available or raw materials that can be obtained by those skilled in the art; unless otherwise specified, the methods used in the embodiments of the present invention are all methods mastered by those skilled in the art.

[0031] Example 1: A preparation process of food-grade soluble soybean polysaccharides includes the following steps:

[0032] 1) Crush soybean meal, and soak the crushed soybean meal in an ethanol solution with a volume fraction of 70%, stir and extract at 50°C for 3 h, then filter and separate, wash the filter residue with water until neutral, and then dry at 40°C to constant weight to obtain pretreated soybean meal;

[0033] 2) Take a high-purity soluble soybean polysaccharide standard as the template molecule, dissolve the template molecule in deionized water, and prepare a template molecule solution with a mass concentration of 4.0 wt%;

[0034] 3) Add acrylic acid monomer to toluene solvent according to the mass-volume ratio of 1 g / 100 mL, and then add ethylene glycol dimethacrylate crosslinking agent, and the addition amount of ethylene glycol dimethacrylate is 25 wt% of the acrylic acid monomer,

[0035] Stir and dissolve to obtain a mixed solution. Add the template molecule solution and ammonium bicarbonate porogen to the mixed solution. The molar ratio of the template molecule to the acrylic acid monomer is 1:5, and the addition amount of ammonium bicarbonate porogen is 5.0 wt% of the mass of the acrylic acid monomer. Disperse evenly by ultrasonic oscillation, then add azobisisobutyronitrile initiator, and the addition amount of azobisisobutyronitrile is 0.1 wt% of the mass of the acrylic acid monomer. Under nitrogen protection, heat and react. The heating reaction temperature is 65 °C, and the reaction time is 5 h. Then use a solvent (volume ratio of methanol: acetic acid = 9:1) to wash away the template molecules and unreacted substances to obtain a molecularly imprinted polymer;

[0036] 4) Add the pretreated soybean meal to a sodium hydroxide solution with a mass concentration of 0.4 wt% according to the mass-to-volume ratio of 1 g / 30 mL, stir and extract at 60 °C for 3 h, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to 4.5 to precipitate soybean polysaccharide, collect the precipitate by centrifugation, and then add the precipitate to deionized water according to the mass-to-volume ratio of 1 g / 80 mL and stir to dissolve to obtain a crude soybean polysaccharide solution;

[0037] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, control the flow rate at 10 mL / min, then elute with an ethanol solution of 0.2 mol / L sodium chloride, collect the eluate, and spray-dry the eluate to obtain a food-grade soluble soybean polysaccharide.

[0038] The preparation method of the ammonium bicarbonate porogen includes the following steps:

[0039] Add 3 g of ammonium bicarbonate and 10 g of gelatin to 500 mL of deionized water and stir to dissolve to obtain a mixed solution; add Span 80 to liquid paraffin and stir evenly to obtain an oil-phase solution with a mass concentration of 3%;

[0040] Slowly drop the mixed solution into the oil-phase solution, continuously stir to form a stable water-in-oil emulsion in the system. Under stirring conditions, slowly drop 3 mL of glutaraldehyde solution into the water-in-oil emulsion for cross-linking reaction for 10 h, and then obtain the product through centrifugation, washing and drying.

[0041] Example 2: A preparation process of food-grade soluble soybean polysaccharide, including the following steps:

[0042] 1) Crush the soybean meal, soak the crushed soybean meal in an ethanol solution with a volume fraction of 70%, stir and extract at 50 °C for 3 h, then filter and separate, wash the filter residue with water until neutral, and then dry at 40 °C to constant weight to obtain pretreated soybean meal;

[0043] 2) Take the high-purity soluble soybean polysaccharide standard as the template molecule, dissolve the template molecule in deionized water, and prepare a template molecule solution with a mass concentration of 2.5 wt%.

[0044] 3) Add acrylic acid monomer to toluene solvent according to the mass-volume ratio of 1 g / 100 mL, and then add ethylene glycol dimethacrylate crosslinker. The addition amount of ethylene glycol dimethacrylate is 25 wt% of the acrylic acid monomer.

[0045] Stir and dissolve to obtain a mixed solution. Add the template molecule solution and ammonium bicarbonate porogen to the mixed solution. The molar ratio of the template molecule to the acrylic acid monomer is 1:5, and the addition amount of ammonium bicarbonate porogen is 4.0 wt% of the mass of the acrylic acid monomer. Ultrasonically oscillate and disperse evenly, then add azobisisobutyronitrile initiator. The addition amount of azobisisobutyronitrile is 0.1 wt% of the mass of the acrylic acid monomer. Under nitrogen protection, heat and react. The heating reaction temperature is 65 °C, and the reaction time is 5 h. Then use a solvent (volume ratio of methanol: acetic acid = 9:1) to wash and remove the template molecule and unreacted substances to obtain a molecularly imprinted polymer.

[0046] 4) Add pretreated soybean meal to a sodium hydroxide solution with a mass concentration of 0.3 wt% according to the mass-volume ratio of 1 g / 30 mL, stir and extract at 60 °C for 3 h, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to 4.0 to precipitate soybean polysaccharide, collect the precipitate by centrifugation, and then add the precipitate to deionized water according to the mass-volume ratio of 1 g / 80 mL and stir to dissolve to obtain a crude soybean polysaccharide solution.

[0047] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, control the flow rate at 10 mL / min, then use an ethanol solution of 0.2 mol / L sodium chloride for elution, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

[0048] The preparation method of ammonium bicarbonate porogen includes the following steps:

[0049] Add 3 g of ammonium bicarbonate and 10 g of gelatin to 500 mL of deionized water, stir and dissolve to obtain a mixed solution; add Span 80 to liquid paraffin, stir evenly to obtain an oil-phase solution with a mass concentration of 3%.

[0050] Slowly drop the mixed solution into the oil-phase solution, continuously stir to form a stable water-in-oil emulsion in the system. Under stirring conditions, slowly drop 3 mL of glutaraldehyde solution into the water-in-oil emulsion for cross-linking reaction for 10 h, and then perform centrifugation, washing and drying to obtain it.

[0051] Example 3: A preparation process of food-grade soluble soybean polysaccharide, comprising the following steps:

[0052] 1) Crush soybean meal, soak the crushed soybean meal in an ethanol solution with a volume fraction of 70%, stir and extract at 50 °C for 3 h, then filter and separate, wash the filter residue with water until neutral, and then dry at 40 °C to constant weight to obtain pretreated soybean meal;

[0053] 2) Take a high-purity soluble soybean polysaccharide standard as a template molecule, dissolve the template molecule in deionized water, and prepare a template molecule solution with a mass concentration of 3.5 wt%;

[0054] 3) Add acrylic acid monomer to toluene solvent according to the mass-volume ratio of 1 g / 100 mL, then add ethylene glycol dimethacrylate cross-linking agent, and the addition amount of ethylene glycol dimethacrylate is 25 wt% of the acrylic acid monomer,

[0055] Stir and dissolve to obtain a mixed solution. Add the template molecule solution and ammonium bicarbonate porogen to the mixed solution. The molar ratio of the template molecule to the acrylic acid monomer is 1:5, and the addition amount of ammonium bicarbonate porogen is 4.5 wt% of the mass of the acrylic acid monomer. Ultrasonically oscillate and disperse evenly, then add azobisisobutyronitrile initiator, and the addition amount of azobisisobutyronitrile is 0.1 wt% of the mass of the acrylic acid monomer. Under nitrogen protection, heat and react. The heating reaction temperature is 65 °C, and the reaction time is 5 h. Then use a solvent (volume ratio of methanol: acetic acid = 9:1) to wash and remove the template molecule and unreacted substances to obtain a molecularly imprinted polymer;

[0056] 4) Add the pretreated soybean meal to a sodium hydroxide solution with a mass concentration of 0.3 wt% according to the mass-volume ratio of 1 g / 30 mL, stir and extract at 60 °C for 3 h, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to 4.0 to precipitate soybean polysaccharide, collect the precipitate by centrifugation, and then add the precipitate to deionized water according to the mass-volume ratio of 1 g / 80 mL and stir to dissolve to obtain a crude soybean polysaccharide solution;

[0057] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, control the flow rate at 10 mL / min, then elute with an ethanol solution of 0.2 mol / L sodium chloride, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

[0058] The preparation method of ammonium bicarbonate porogen comprises the following steps:

[0059] 3 g of ammonium bicarbonate and 10 g of gelatin were added to 500 mL of deionized water and stirred until dissolved to obtain a mixed solution; Span 80 was added to liquid paraffin and stirred evenly to obtain an oil-phase solution with a mass concentration of 3%;

[0060] The mixed solution was slowly added dropwise to the oil-phase solution, and continuous stirring was carried out to form a stable water-in-oil emulsion in the system. Under stirring conditions, 3 mL of glutaraldehyde solution was slowly added dropwise to the water-in-oil emulsion for a cross-linking reaction for 10 h, and then centrifugal separation, washing, and drying were carried out to obtain the product.

[0061] Example 4: A preparation process of food-grade soluble soybean polysaccharide, comprising the following steps:

[0062] 1) The soybean meal was pulverized, and the pulverized soybean meal was soaked in an ethanol solution with a volume fraction of 70% and stirred and extracted at 50 °C for 3 h, and then filtered and separated. The filter residue was washed with water until neutral, and then dried to constant weight at 40 °C to obtain pretreated soybean meal;

[0063] 2) A high-purity soluble soybean polysaccharide standard was taken as the template molecule, and the template molecule was dissolved in deionized water to prepare a template molecule solution with a mass concentration of 5.0 wt%;

[0064] 3) Acrylic acid monomer was added to toluene solvent according to the mass-volume ratio of 1 g / 100 mL, and then ethylene glycol dimethacrylate cross-linking agent was added. The addition amount of ethylene glycol dimethacrylate was 25 wt% of the acrylic acid monomer.

[0065] Stir and dissolve to obtain a mixed solution. The template molecule solution and ammonium bicarbonate porogen were added to the mixed solution. The molar ratio of the template molecule to the acrylic acid monomer was 1:5, and the addition amount of ammonium bicarbonate porogen was 6.0 wt% of the mass of the acrylic acid monomer. After ultrasonic oscillation and dispersion to be uniform, then azobisisobutyronitrile initiator was added. The addition amount of azobisisobutyronitrile was 0.1 wt% of the mass of the acrylic acid monomer. Under nitrogen protection, heating reaction was carried out. The heating reaction temperature was 70 °C, and the reaction time was 6 h. Then, the template molecule and unreacted substances were removed by washing with a solvent (volume ratio of methanol: acetic acid = 9:1) to obtain a molecularly imprinted polymer;

[0066] 4) The pretreated soybean meal was added to a sodium hydroxide solution with a mass concentration of 0.5 wt% according to the mass-volume ratio of 1 g / 30 mL, and stirred and extracted at 60 °C for 3 h. After filtration, the filtrate was obtained. Hydrochloric acid was added to the filtrate to adjust the pH value to 4.5 to precipitate soybean polysaccharide. The precipitate was collected by centrifugal separation, and then the precipitate was added to deionized water according to the mass-volume ratio of 1 g / 80 mL and stirred until dissolved to obtain a crude soybean polysaccharide solution;

[0067] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, control the flow rate at 10 mL / min, then elute with an ethanol solution of 0.2 mol / L sodium chloride, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

[0068] The preparation method of the ammonium bicarbonate porogen includes the following steps:

[0069] Add 3 g of ammonium bicarbonate and 10 g of gelatin to 500 mL of deionized water and stir to dissolve to obtain a mixed solution; add Span 80 to liquid paraffin and stir evenly to obtain an oil-phase solution with a mass concentration of 3%;

[0070] Slowly drop the mixed solution into the oil-phase solution, continuously stir to form a stable water-in-oil emulsion in the system. Under stirring conditions, slowly drop 3 mL of glutaraldehyde solution into the water-in-oil emulsion for cross-linking reaction for 10 h, and then perform centrifugal separation, washing and drying to obtain it.

[0071] Example 5: A preparation process of food-grade soluble soybean polysaccharide, including the following steps:

[0072] 1) Crush soybean meal, soak the crushed soybean meal in an ethanol solution with a volume fraction of 70%, stir and extract at 50 °C for 3 h, then perform filtration separation, wash the filter residue with water until neutral, and then dry at 40 °C to constant weight to obtain pretreated soybean meal;

[0073] 2) Take a high-purity soluble soybean polysaccharide standard as a template molecule, dissolve the template molecule in deionized water to prepare a template molecule solution with a mass concentration of 2.0 wt%;

[0074] 3) Add acrylic acid monomer to toluene solvent according to the mass-volume ratio of 1 g / 100 mL, then add ethylene glycol dimethacrylate cross-linking agent, and the addition amount of ethylene glycol dimethacrylate is 25 wt% of the acrylic acid monomer.

[0075] Stir and dissolve to obtain a mixed solution. Add the template molecule solution and ammonium bicarbonate porogen to the mixed solution. The molar ratio of the template molecule to the acrylic acid monomer is 1:5, and the addition amount of ammonium bicarbonate porogen is 3.0 wt% of the mass of the acrylic acid monomer. Ultrasonically oscillate and disperse evenly, then add azobisisobutyronitrile initiator, and the addition amount of azobisisobutyronitrile is 0.1 wt% of the mass of the acrylic acid monomer. Under nitrogen protection, heat and react. The heating reaction temperature is 60 °C, and the reaction time is 4 h. Then use a solvent (volume ratio of methanol: acetic acid = 9:1) to wash away the template molecule and unreacted substances to obtain the molecularly imprinted polymer;

[0076] 4) Add the pretreated soybean meal to the sodium hydroxide solution with a mass concentration of 0.2 wt% according to the mass-volume ratio of 1 g / 30 mL, stir and extract at 60 °C for 3 h, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to 4.0 to precipitate the soybean polysaccharide, collect the precipitate by centrifugation, and then add the precipitate to deionized water according to the mass-volume ratio of 1 g / 80 mL and stir to dissolve to obtain a crude soybean polysaccharide solution;

[0077] 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, control the flow rate at 10 mL / min, and then elute with an ethanol solution of 0.2 mol / L sodium chloride, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

[0078] The preparation method of the ammonium bicarbonate porogen includes the following steps:

[0079] Dissolve 3 g of ammonium bicarbonate and 10 g of gelatin in 500 mL of deionized water to obtain a mixed solution; add Span 80 to liquid paraffin and stir evenly to obtain an oil-phase solution with a mass concentration of 3%;

[0080] Slowly drip the mixed solution into the oil-phase solution, continuously stir to form a stable water-in-oil emulsion in the system, and slowly drip 3 mL of glutaraldehyde solution into the water-in-oil emulsion under stirring for a cross-linking reaction for 10 h, and then perform centrifugation, washing and drying to obtain it.

[0081] Comparative Example 1

[0082] The difference between Comparative Example 1 and Example 1 is that:

[0083] Replace the porogen with ordinary ammonium bicarbonate;

[0084] The remaining operation steps are the same as those in Example 1.

[0085] Comparative Example 2

[0086] The difference between Comparative Example 2 and Example 5 is that:

[0087] The addition amount of the ammonium bicarbonate porogen is 2.0 wt% of the mass of the acrylic monomer;

[0088] The remaining operation steps are the same as those in Example 5.

[0089] Comparative Example 3

[0090] The difference between Comparative Example 3 and Example 4 is that:

[0091] The addition amount of the ammonium bicarbonate porogen is 7.0 wt% of the mass of the acrylic monomer

[0092] The remaining operation steps are the same as those in Example 4.

[0093] Comparative Example 4

[0094] The difference between Comparative Example 4 and Example 4 is that:

[0095] The addition amount of ammonium bicarbonate pore-forming agent is 8.0 wt% of the mass of acrylic acid monomer

[0096] The remaining operation steps are the same as those in Example 4.

[0097] Performance test

[0098] The purity of soybean polysaccharide in the examples and comparative examples was measured by gas chromatography: The soybean polysaccharide sample was hydrolyzed to degrade the polysaccharide into monosaccharides, and then the monosaccharides were derivatized to make them volatile. The gas chromatograph was turned on, and a suitable chromatographic column and detection conditions were selected. A standard curve was established with a monosaccharide standard of known purity. The derivatized sample was injected into the gas chromatograph, and qualitative and quantitative analyses were performed based on the retention time and peak area of each monosaccharide to calculate the content of various monosaccharides in the sample, and then the purity of soybean polysaccharide was determined.

[0099] The content of ammonium salt in soybean polysaccharide was determined by Nessler's reagent colorimetry: Under alkaline conditions, ammonium ions react with Nessler's reagent to form a yellow-brown complex. Within a certain concentration range, the color depth is proportional to the ammonium salt content, and the ammonium salt content can be determined by colorimetry. Nessler's reagent, potassium sodium tartrate solution, ammonium standard stock solution, ammonium standard working solution, etc. were prepared. A series of different volumes of ammonium standard working solution were taken and placed in colorimetric tubes, water was added to a certain volume, potassium sodium tartrate solution and Nessler's reagent were added, and after color development, the absorbance was measured at a wavelength of 420 nm with a spectrophotometer. A standard curve was plotted with ammonium content as the abscissa and absorbance as the ordinate.

[0100] An appropriate amount of soybean polysaccharide sample was weighed, dissolved in water and filtered. A certain amount of the filtrate was placed in a colorimetric tube, and color development and absorbance measurement were carried out according to the steps for drawing the standard curve. The ammonium content was obtained from the standard curve according to the absorbance of the sample, and the ammonium salt content in the sample was calculated.

[0101] Determination of ash content: First, the crucible was washed and dried, then placed in a muffle furnace and burned to constant weight. A certain amount of soybean polysaccharide sample was accurately weighed into the crucible, charred gently on an electric furnace until there was no smoke, and then placed in a muffle furnace and burned to constant weight at a specified temperature of 550 °C. After cooling, it was taken out, placed in a desiccator and cooled to room temperature, and the mass of the ash was weighed. The ash content (%) was measured according to the following formula.

[0102] Ash content (%) = (ash mass / sample mass) × 100%.

[0103] Protein content determination: Weigh an appropriate amount of the soybean polysaccharide sample accurately into a Kjeldahl flask, add copper sulfate, potassium sulfate, and concentrated sulfuric acid, and heat and digest on a digestion furnace until the solution is clear and transparent. After cooling, transfer the digested solution to a nitrogen analyzer, add sodium hydroxide solution for distillation, absorb the distillate with boric acid solution, titrate to the end point with a standard hydrochloric acid solution, and record the volume of the standard hydrochloric acid solution.

[0104] Protein content (%) = (V × c × 0.014 × F) / m × 100%;

[0105] Wherein, V is the volume of the standard hydrochloric acid solution consumed in titration, c is the concentration of the standard hydrochloric acid solution, 0.014 is the millimolar mass of nitrogen, F is the protein conversion factor 6.25, and m is the sample mass.

[0106]

[0107] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A preparation process of food-grade soluble soybean polysaccharide, characterized in that, It includes the following steps: 1) Crush the soybean meal, soak the crushed soybean meal in an ethanol solution to remove grease and impurities, then filter and separate, wash the filter residue with water until neutral, and then dry to obtain pretreated soybean meal; 2) Take a high-purity soluble soybean polysaccharide standard as the template molecule, dissolve the template molecule in deionized water to prepare a template molecule solution; 3) Add acrylic acid and ethylene glycol dimethacrylate to a toluene solvent, stir and dissolve to obtain a mixed solution. Add the template molecule solution and ammonium bicarbonate porogen to the mixed solution. The addition amount of the ammonium bicarbonate porogen is 3.0 - 6.0 wt% of the mass of acrylic acid. Ultrasonically oscillate and disperse evenly, then add azobisisobutyronitrile initiator, and under nitrogen protection, heat and react. Then wash to remove the template molecule and unreacted substances to obtain a molecularly imprinted polymer; The preparation method of the ammonium bicarbonate porogen includes the following steps: Add ammonium bicarbonate and gelatin to deionized water and stir to dissolve to obtain a mixed solution; add span 80 to liquid paraffin and stir evenly to obtain a stable oil-phase solution; Slowly drip the mixed solution into the oil-phase solution, continuously stir to make the system form a stable water-in-oil emulsion. Under stirring conditions, slowly drip glutaraldehyde solution into the water-in-oil emulsion for cross-linking reaction, and then perform centrifugal separation, washing and drying to obtain it; 4) Add the pretreated soybean meal to a sodium hydroxide solution, stir and extract, filter to obtain a filtrate, add hydrochloric acid to the filtrate to adjust the pH value to acidic to precipitate soybean polysaccharide, collect the precipitate by centrifugal separation, and then dissolve the precipitate to obtain a crude soybean polysaccharide solution; 5) Pack the molecularly imprinted polymer into a chromatographic column, then introduce the crude soybean polysaccharide solution into the chromatographic column, then use an elution solution for elution, collect the eluate, and spray-dry the eluate to obtain food-grade soluble soybean polysaccharide.

2. The preparation process of a food-grade soluble soybean polysaccharide according to claim 1, wherein, In step 2), the mass concentration of the template molecule solution is 2.0 - 5.0 wt%.

3. The preparation process of a food-grade soluble soybean polysaccharide according to claim 1, characterized in that, In step 3), the heating reaction temperature is 60 - 70 °C, and the reaction time is 4 - 6 h.

4. The preparation process of a food-grade soluble soybean polysaccharide according to claim 1, characterized in that, In step 4), the mass concentration of the sodium hydroxide solution is 0.2 - 0.5 wt%.

5. The preparation process of a food-grade soluble soybean polysaccharide according to claim 1, wherein, In step 4), add hydrochloric acid to the filtrate to adjust the pH value to 4.0 - 4.

5.

6. The preparation process of a food-grade soluble soybean polysaccharide according to claim 1, characterized in that, In step 5), the elution solution is an ethanol solution of sodium chloride.

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