Preparation method and application of soybean polysaccharide nanoparticles with high emulsibility and stability

Abstract

The invention belongs to the technical field of food colloid, and particularly relates to a preparation method and application of soybean polysaccharide nanoparticles with high emulsibility and stability. The method comprises the following steps: carrying out ball-milling pretreatment on soybean polysaccharide serving as a raw material, adding the soybean polysaccharide and octenyl succinic anhydride serving as a modifier into a mixed solvent system of 1-ethyl-3-methylimidazolium acetate ionic liquid and water, and carrying out esterification modification reaction; and by taking ethanol as an anti-solvent, precipitating for nanocrystallization, and carrying out vacuum concentration and vacuum drying to obtain the esterified soybean polysaccharide nano-particles. According to the preparation method, the emulsifying property of the soybean polysaccharide is remarkably improved and can reach 6.06 m / g, the emulsifying stability value within 10 minutes can reach 6.48, excellent emulsifying stability is shown, meanwhile, the substitution degree of the soybean polysaccharide is successfully improved, and the esterification substitution degree range is 0.05-0.09.

Description

Technical Field

[0001] The invention belongs to the technical field of food colloids, and in particular relates to a preparation method and application of soybean polysaccharide nanoparticles with high emulsification and stability. Background Art

[0002] Soybean polysaccharide is a natural emulsifier with a wide range of raw materials, green and safe. It can quickly adsorb to the surface of oil droplets, and its sugar chain part extends to the water phase, thus forming a thick hydration film around the oil droplets, which keeps the oil droplets stable through steric hindrance. However, compared with the mature emulsifiers widely used on the market, soybean polysaccharide is at a disadvantage in terms of emulsification and emulsion stability, especially in O / W (oil / water) emulsion systems, where its stability performance is poor.

[0003] It is necessary to improve its emulsification and emulsification stability through modification in order to broaden its applicability and enrich its application.

[0004] Aiming at the large number of hydrophilic hydroxyl groups in soybean polysaccharides, introducing hydrophobic groups through esterification to enhance its amphiphilicity is an important direction to improve emulsification performance. Generally, the degree of substitution of long-chain (C8-C18) esters is higher, and its expected effect on improving emulsification and emulsification stability is better. Octenyl succinic anhydride (OSA for short) is a long-chain esterifying agent, which is often used in the food industry to modify starch to improve the emulsification of starch. Compared with other modifiers, octenyl succinic anhydride is greener and healthier. It has been certified by the FDA and can be used in the food industry. It is suitable for large-scale production, environmentally friendly, and cost-effective.

[0005] At present, although there are many reports on the application of octenyl succinic anhydride in the modification of polysaccharides, there are some challenges if octenyl succinic anhydride is used as a modifier for soybean polysaccharides. Because soybean polysaccharides have a highly branched structure and long-chain olefin esterification agents (i.e., octenyl succinic anhydride) are insoluble in aqueous media, it is difficult for them to effectively penetrate into the polysaccharide particles, thereby limiting the increase in the degree of substitution. In addition, the esterification process is also relatively slow. For example, the degree of substitution of esterified soybean polysaccharides disclosed in patent documents is limited to 0.0059% to 0.0206%, which is a narrow range, thereby limiting the improvement of emulsification performance.

[0006] Although esterification of polysaccharides helps to improve their emulsification, their emulsification stability still needs to be further improved. In order to improve the emulsification stability of polysaccharides, physical or chemical methods are used to modify them to form uniform nanoparticles, which has been proven to be an effective method. Antisolvent precipitation technology, as a means of preparing nanocrystals in the pharmaceutical field, is based on the difference in solubility of polysaccharides in solvents and antisolvents, and promotes their nucleation and growth by reducing the solubility of polysaccharides. The advantage of this technology is that it has a fast preparation speed and is easy to promote. However, the implementation of the solvent-antisolvent precipitation method is affected by many factors, including the type and ratio of the two-phase solvent, the concentration of the polysaccharide, the injection speed of the single-phase solvent, and the speed and intensity of stirring. These factors jointly determine the nucleation rate and growth rate of the crystal nucleus.

[0007] For soybean polysaccharide, since it is a heteropolysaccharide with many branches, ensuring that the formed nanoparticles are uniform and stable has become a control difficulty. Although patent documents disclose a process for preparing fucoidan using an anti-solvent precipitation method, this technology is not applicable to soybean polysaccharides. In addition, although patent documents report a method for preparing insoluble soybean polysaccharide nanoparticles, the method uses a highly toxic reagent, sodium azide, and therefore poses a significant safety hazard. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a method for preparing soybean polysaccharide nanoparticles with high emulsification and stability. In view of the problem of insufficient emulsification and emulsification stability of soybean polysaccharide, the present invention uses soybean polysaccharide as raw material, performs lithium chloride-dimethyl sulfoxide-assisted ball milling pretreatment, and uses octenyl succinic anhydride as a modifier in an ionic liquid-water mixed system to modify the soybean polysaccharide to improve its emulsification; and further reduces the particles of the modified polysaccharide by an anti-solvent precipitation method to improve its emulsification stability.

[0009] The technical solution of the present invention is:

[0010] The method for preparing soybean polysaccharide nanoparticles with high emulsification and stability comprises the following steps:

[0011] (1) Soybean polysaccharide was used as raw material for ball milling pretreatment, with a ball-to-material mass ratio of 3:1, 0.2 mol / L lithium chloride-dimethyl sulfoxide solution as a ball milling aid, and a material-liquid ratio of soybean polysaccharide to the aid of 10 mg:1 mL. The ball milling time was 5-8 h, freeze-dried, and passed through a 100-mesh sieve;

[0012] (2) taking the pretreated soybean polysaccharide, dispersing it in a mixed solvent system, then adding the modifier octenyl succinic anhydride to obtain a mixed solution, and adjusting the pH of the mixed solution to 8.0-8.5, stirring in a water bath at 30-40° C. to carry out esterification modification reaction, adding alkali solution every 5 minutes during the reaction to maintain the pH value of the reaction system, the reaction time is 25-35 minutes, and after the reaction is completed, adjusting the pH value of the mixed solution to 5.8-6.2, dialyzing for 48 hours and then freeze-drying to obtain a crude sample of octenyl succinic anhydride-soybean polysaccharide;

[0013] Wherein, the mass ratio of 1-ethyl-3-methylimidazolium acetate ionic liquid to water in the mixed solvent system is 1:1;

[0014] The weight-to-volume ratio of soybean polysaccharide to the mixed solvent system is 1 g: (9-10) mL

[0015] The mass ratio of octenyl succinic anhydride to soybean polysaccharide is 1:4-8;

[0016] (3) taking the crude sample of octenyl succinic anhydride-soybean polysaccharide obtained in (2), stirring it to completely dissolve it in water, and obtaining an octenyl succinic anhydride-soybean polysaccharide aqueous solution with a concentration of 0.3-0.6 wt %;

[0017] Take the above aqueous solution, slowly drop it into ethanol at room temperature at a flow rate of 15-25 μL / s, wherein the volume ratio of the aqueous phase to the ethanol is 1:18-25, and stir at a speed of 900 rpm for 180 minutes while dropping, and then concentrate under reduced pressure;

[0018] When the reduced pressure concentration is reached to 2 / 3 of the original volume, water is added to make up to the original volume, and then reduced pressure concentration is performed again. After repeating 3 times, vacuum drying is performed to obtain the product esterified soybean polysaccharide nanoparticles. The particle size of the final particles is below 1000 nm.

[0019] In step (1), LiCl / DMSO-assisted ball milling pretreatment is used. The electrostatic repulsion between chloride ions and hydroxyl protons of polysaccharides increases the rigidity of the sugar chain and forms a dendritic structure along the main chain. At the same time, the strong mechanical force of ball milling causes the polysaccharide to expose more active hydroxyl groups. Based on the above steps, the esterification process is easier to proceed.

[0020] In step (2), the raw material is pre-treated by ball milling according to the method of step (1), and then modified by ionic liquid-mediated esterification, which can increase the solubility of polysaccharides, improve the reaction rate, and increase the degree of substitution. In the modification, preferably, the modification temperature is 35°C, the pH is 8.5, the reaction time is 30 minutes, and the mass ratio of octenyl succinic anhydride to soybean polysaccharides is 1:7. The 1-ethyl-3-methylimidazolium acetate ionic liquid is "acetate ion ([OAc]- )" based ionic liquids are more effective in dissolving polysaccharides and have no Cl - The strong corrosiveness of 1-ethyl-3-methylimidazolium acetate will not cause the hydrolysis of glycosidic bonds. Therefore, 1-ethyl-3-methylimidazolium acetate ionic liquid assisted esterification can increase the solubility of polysaccharides, improve the reaction rate, and increase the degree of substitution.

[0021] In step (3), an anti-solvent precipitation method is used, with ethanol as the anti-solvent. The anti-solvent precipitation method is a method that utilizes the phase separation effect between a solvent and a non-solvent to precipitate the target substance in a non-solvent and form nano-microspheres. It has the advantages of being simple, easy to operate, and highly controllable.

[0022] Preferably, the temperature of the esterification modification reaction in (2) is 35° C., the pH is 8.5, and the time is 35 min; and the alkali solution is 0.1 mol / L sodium hydroxide solution.

[0023] Preferably, in (2), the mass ratio of octenyl succinic anhydride to soybean polysaccharide is 1:7.

[0024] Preferably, in (3), the volume ratio of the aqueous phase to ethanol is 1:20.

[0025] The application of the esterified soybean polysaccharide nanoparticles prepared by the above preparation method in the preparation of Pickering emulsion is also the key protection content of the present invention.

[0026] The application is mainly implemented through the following steps:

[0027] Take the esterified soybean polysaccharide nanoparticles prepared by the above preparation method, add them into water, stir them in a 35-45°C water bath to make them fully dissolved, and the concentration is 1-3wt%; then add the oil phase at a volume ratio of 3:1 between water and oil, and treat them twice with dynamic high-pressure microfluidization at 40MPa to obtain a Pickering emulsion.

[0028] Preferably, the oil phase is soybean oil.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention uses lithium chloride-dimethyl sulfoxide-assisted ball milling to activate soybean polysaccharides, and uses octenyl succinic anhydride to esterify soybean polysaccharides in an ionic liquid medium, thereby increasing the degree of substitution and enhancing the emulsification of polysaccharides. The esterification degree of substitution can reach 0.05-0.09%;

[0031] (2) Furthermore, the present invention reduces the size of the modified polysaccharide particles by an anti-solvent precipitation method to improve the emulsification stability. After applying it to the Pickering emulsion, the droplet size is concentrated between 0.8 and 6.5 μm; the emulsification stability and emulsification properties are significantly improved, and the emulsification property can reach 6.06 μm.2 / g, the emulsification stability value within 10min can reach 6.48;

[0032] (3) The present invention adopts dynamic high-pressure microfluidization to prepare the emulsion, the particle size of which is smaller and more uniform, and the encapsulation efficiency of the loaded substance is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the single factor test on the esterification of soybean polysaccharides;

[0034] Figure 2 The process flow chart of preparing esterified polysaccharide nanoparticles by anti-solvent precipitation method;

[0035] Figure 3 Appearance of emulsions prepared for SSPS, OSA-SSPS, and OSA-SSPS-NP;

[0036] Figure 4 This is a comparison chart of the emulsification and emulsification stability of SSPS, OSA-SSPS, and OSA-SSPS-NP;

[0037] Figure 5 Comparison of infrared spectra of SSPS, OSA-SSPS, and OSA-SSPS-NP;

[0038] Figure 6 It is a comparison chart of particle sizes of SSPS, OSA-SSPS, and OSA-SSPS-NP;

[0039] Figure 7 Comparison of light microscopy images of emulsions prepared for SSPS, OSA-SSPS, and OSA-SSPS-NP;

[0040] Figure 8 This is a comparison chart of the emulsification properties of different esterified soybean polysaccharides;

[0041] Fig. 9 The effects of different pretreatment methods on the degree of substitution of esterified soybean polysaccharides;

[0042] Fig.10 The effect of different antisolvents on the particle size of esterified soybean polysaccharide nanoparticles;

[0043] Fig.11 This is the effect of different concentrations of polysaccharide solutions on the particle size of esterified soybean polysaccharide nanoparticles. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0045] Example 1 Optimization of the preparation process of esterified soybean polysaccharide

[0046] There are four key control points in the preparation process of esterified soybean polysaccharides, namely reaction time, temperature, pH and the ratio of esterification agent to soybean polysaccharides. The optimal preparation process of esterified polysaccharides was determined by single factor and orthogonal experiments, with substitution degree as the evaluation index.

[0047] 1.1 Single factor optimization of the preparation process of esterified soybean polysaccharides

[0048] 1.1.1 Investigating the effect of different pH on esterification degree of substitution

[0049] Soybean polysaccharide was pretreated by ball milling with a ball-to-material mass ratio of 3:1, and 0.2 mol / L lithium chloride-dimethyl sulfoxide solution was used as a ball milling aid. The mass volume ratio of soybean polysaccharide to the ball milling aid was 10 mg:1 mL. The ball milling time was 5 h, and the mixture was freeze-dried and passed through a 100-mesh sieve.

[0050] 10g of ball-milled soybean polysaccharide (SSPS) was dispersed in 90mL of 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixed system, and octenyl succinic anhydride (OSA, modifier) ​​was added according to the ratio of OSA:SSPS = 1:7, and the solution was adjusted to different pH (7.5, 8, 8.5, 9, 9.5), and esterification was carried out at 40℃ under water bath stirring, and 0.1mol / L NaOH was added every 5min to maintain the pH value of the solution for esterification reaction, and the reaction time was maintained for 40min. After the reaction, the pH value of the solution was adjusted to 6.0. After 48h of dialysis in a dialysis bag with a MWCO of 500Da, the crude OSA-SSPS sample was obtained by freeze drying.

[0051] The substitution degree of the above OSA-SSPS crude sample was tested, and the results were as follows: Figure 1 , as shown in Table 1:

[0052] Table 1 Effect of different pH values ​​on degree of substitution

[0053]

[0054]

[0055] When the pH of the solution was 8.0, the substitution degree of the crude OSA-SSPS sample reached the maximum, which was 0.0804±0.0018%.

[0056] 1.1.2 Investigating the effect of different reaction times on esterification degree of substitution

[0057] Soybean polysaccharide was pretreated by ball milling, with a ball-to-material mass ratio of 3:1, and 0.2 mol / L lithium chloride-dimethyl sulfoxide was used as an auxiliary agent, the material-liquid ratio of soybean polysaccharide to the auxiliary agent was 10 mg:1 mL, the ball milling time was 5 h, and the mixture was freeze-dried and passed through a 100-mesh sieve.

[0058] 10g of pretreated soybean polysaccharide (SSPS) was dispersed in 90mL of 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixed system, octenyl succinic anhydride was added according to the ratio of OSA:SSPS=1:7, the pH of the solution was adjusted to 8.5, and esterification was carried out at 40℃ under water bath stirring, and 0.1mol / L NaOH was added every 5min to maintain the pH value of the solution for esterification reaction, and the reaction time was maintained at (25min, 30min, 35min, 40min, 45min), and the pH of the solution after the reaction was adjusted to 6.0. After 48h of dialysis in a dialysis bag with a MWCO of 500Da, the crude OSA-SSPS sample was obtained by freeze drying.

[0059] The substitution degree of the above OSA-SSPS crude sample was tested, and the results were as follows: Figure 1 , as shown in Table 2:

[0060] Table 2 Effect of different reaction times on degree of substitution

[0061]

[0062]

[0063] When the esterification reaction time was maintained at 30 min, the substitution degree of the OSA-SSPS crude sample reached the maximum, which was 0.0489±0.0034%.

[0064] 1.1.3 Investigating the effect of different temperatures on esterification degree of substitution

[0065] Soybean polysaccharide was pretreated by ball milling, with a ball-to-material mass ratio of 3:1, and 0.2 mol / L lithium chloride-dimethyl sulfoxide was used as an auxiliary agent, the material-liquid ratio of soybean polysaccharide to the auxiliary agent was 10 mg:1 mL, the ball milling time was 5 h, and the mixture was freeze-dried and passed through a 100-mesh sieve.

[0066] 10g of pretreated soybean polysaccharide (SSPS) was dispersed in 90mL of 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixed system, octenyl succinic anhydride was added according to the ratio of OSA:SSPS=1:7, pH of SSPS solution was adjusted to 8.5, esterification was carried out under different temperature conditions (30℃, 35℃, 40℃, 45℃, 50℃) in a water bath, and 0.1mol / L NaOH was added every 5min to maintain the pH value of the solution for esterification reaction, and the reaction time was maintained for 40min. After the reaction, the pH value of the solution was adjusted to 6.0. After 48h of dialysis in a dialysis bag with a MWCO of 500Da, the crude OSA-SSPS sample was obtained by freeze drying.

[0067] The substitution degree of the above OSA-SSPS crude sample was tested, and the results were as follows: Figure 1 , as shown in Table 3:

[0068] Table 3 Effect of different reaction temperatures on degree of substitution

[0069]

[0070]

[0071] When the esterification reaction temperature was maintained at 35°C, the degree of substitution of the crude OSA-SSPS sample reached a maximum of 0.0543±0.0016%.

[0072] 1.1.4 Effect of different ratios of OSA:SSPS on esterification degree of substitution

[0073] Soybean polysaccharide (SSPS) was pretreated by ball milling with a ball-to-material mass ratio of 3:1. 0.2 mol / L lithium chloride-dimethyl sulfoxide solution (LiCl / DMSO solution) was used as a ball milling aid. The material-liquid ratio of soybean polysaccharide to the ball milling aid was 10 mg:1 mL. The ball milling time was 5 h, and the mixture was lyophilized and passed through a 100-mesh sieve.

[0074] 10g of pretreated soybean polysaccharide was dispersed in 90mL of 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixed system, and octenyl succinic anhydride (OSA) was added according to different ratios (OSA:SSPS=0:1, 1:5, 1:6, 1:7, 1:8), and the pH of the solution was adjusted to 8.5. The solution was stirred in a water bath at 40℃ for esterification, and 0.1mol / L NaOH was added every 5min to maintain the pH of the solution for esterification. The reaction time was maintained for 40min, and the pH of the solution after the reaction was adjusted to 6.0. After 48h of dialysis in a dialysis bag with a MWCO of 500Da, the crude OSA-SSPS sample was obtained by freeze drying.

[0075] The OSA-SSPS crude sample obtained in the above steps was subjected to the degree of substitution determination. Figure 1 , the corresponding data are shown in Table 4 below:

[0076] Table 4 Effect of the ratio of OSA to SSPS on the degree of substitution

[0077]

[0078]

[0079] Depend on Figure 1 Combined with the data in Table 4, it can be seen that when OSA:SSPS=1:5, the substitution degree of the OSA-SSPS crude sample reaches the maximum, which is 0.0655±0.0034%.

[0080] 1.2 Orthogonal optimization of the optimal preparation process of esterified soybean polysaccharides

[0081] The optimal conditions in the single factor experiment were selected for orthogonal experiment to determine the best process for esterification of soybean polysaccharides.

[0082] Result analysis: In the single factor results, when the reaction time, temperature, reactant ratio (OSA:SSPS) and pH were 30 min, 35 °C, OSA:SSPS=1:5 and pH=8 respectively, the degree of substitution of the esterification reaction was the highest.

[0083] On the basis of single factor, the orthogonal experimental design table is as follows:

[0084] Table 5 Orthogonal experimental design table

[0085]

[0086] Table 6 Orthogonal experiment results display table

[0087]

[0088]

[0089] Table 7 Analysis of variance

[0090]

[0091] Table 8 Post hoc test

[0092]

[0093]

[0094] According to the F value of variance analysis, the order of influence on the degree of substitution of esterified soybean polysaccharide is pH> time> temperature> ratio. According to the post hoc test, the one with the largest value is the optimal level, that is, A3, B3, C2, and D3 are the best solutions, that is, T = 35℃, t = 35min, pH = 8.5, OSA: SSPS = 1:7.

[0095] The esterified soybean polysaccharide was prepared under the above conditions. It was verified that the degree of substitution of the esterified polysaccharide under this condition was 0.0860%, which was much higher than the single result level in single factor and orthogonal tests, proving that it is feasible.

[0096] 1.3 Determination of degree of substitution

[0097] In the above 1.1 and 1.2, the degree of substitution (DS) of octenyl succinic anhydride-soybean polysaccharide (OSA-SSPS) is measured by titration method, and the specific measurement steps are as follows:

[0098] (1) Weigh about 2.5 g of OSA-SSPS and disperse it in 15 mL of 2.5 mol / L hydrochloric acid-isopropanol solution by stirring for 30 min;

[0099] (2) Add 50 mL of isopropanol solution (90%, v / v) to the solution obtained in (1) while stirring for 10 min to obtain a suspension;

[0100] (3) Filter the suspension through a Buchner funnel and wash the residue with 90% isopropanol solution until 0.1 mol / L AgNO 3 No chloride ions were detected in the solution;

[0101] (4) drying the washed residue at 40°C for 24 hours and then passing it through a 200-mesh nylon sieve;

[0102] (5) Under stirring, accurately weigh 1.0000 g of the sieved residue was dispersed in 50 mL of deionized water until it was completely dissolved, and titrated with 0.1 mol / L standard NaOH solution using phenolphthalein as an indicator, and the unesterified SSPS was titrated as a control.

[0103] After the titration is complete, use the following formula for calculation:

[0104] DS=0.1624×(C×V) / W / [1-0.210×(C×V) / W]

[0105] Where, C is the concentration of the NaOH solution used, mol / L;

[0106] V is the volume of NaOH solution used, mL;

[0107] W is the mass of OSA-SSPS, g.

[0108] Example 2 Preparation of esterified polysaccharide nanoparticles (OSA-SSPS-NP)

[0109] Specific steps are as follows Figure 2 As shown in:

[0110] (1) Take 0.5 g of OSA-SSPS (prepared by the optimized solution in Example 1) and dissolve it in 100 mL of water. Add a rotor to a beaker and stir to completely dissolve it.

[0111] (2) Using a constant flow pump and a fine needle, slowly add the solution dropwise into 20 times the volume of ethanol at a flow rate of 15-25 μL / s, stirring while adding dropwise, stirring at 900 rpm at room temperature for 180 min, and then concentrating under reduced pressure;

[0112] (3) When the system solvent evaporated to 2 / 3 of the original volume, deionized water was added to make up the volume, and the operation was repeated 3 times. Finally, vacuum drying was performed to obtain the product esterified polysaccharide nanoparticles.

[0113] After preparing esterified polysaccharide nanoparticles according to the above anti-solvent precipitation method, dynamic light scattering (DLS) was used to measure the particle size of the three polysaccharides (SSPS, OSA-SSPS, OSA-SSPS-NP). DLS measurement was measured using Anton paarParticle analyzer litesizer 500. Water was selected as the solvent, and the refractive index of water was 1.333. The solution was diluted to a polysaccharide concentration of 1 mg / mL, placed in a cuvette, and measured at 25°C. Each sample was measured at least three times to take the average value.

[0114] Result analysis: Figure 4 As shown in the figure, the particle size of SSPS is mainly distributed at 200nm and 5000nm; the particle size of OSA-SSPS is mainly distributed at 400nm and 12000nm, indicating that the particle size becomes larger after esterification; after further preparation into (OSA-SSPS-NP) nanoparticles, the particle size is reduced and concentrated below 1200nm. Among them, polysaccharide particles below 1000nm account for 98.8% of the total polysaccharide.

[0115] Example 3 Analysis of polysaccharide emulsification and emulsion stability

[0116] Figure 3 This is the appearance of the prepared emulsion. Figure 4 This is a comparison chart of the emulsification and emulsion stability of the emulsion;

[0117] (1) Comparative analysis of emulsification

[0118] Emulsification activity index (EAI) is a method to evaluate emulsification. Take 0.15g of OSA-SSPS-NP, SSPS or OSA-SSPS freeze-dried sample and stir in 15mL pure water in a 40℃ water bath until fully dissolved. Add 5mL of soybean oil and homogenize twice using dynamic high-pressure microfluidizer (40MPa). Take 100μL of the bottom sample of the emulsion and add it to 10mL of 0.1% sodium dodecyl sulfate (SDS) solution. After shaking evenly, measure its absorbance at a wavelength of 500nm (A=500), and use 0.1SDS as a blank control. Calculate the emulsification according to the following formula:

[0119] Emulsification:

[0120] (2) Comparative analysis of emulsion stability

[0121] 100 μL of the emulsion bottom layer sample at 0 min and 10 min respectively were added to 10 mL of 0.1% sodium dodecyl sulfate (SDS) solution, and after shaking evenly, the absorbance (A500) was measured at a wavelength of 500 nm, with 0.1% SDS solution as the blank control.

[0122] Emulsion stability: ES = (EAI (0 min)) / ((EAI (0 min) - EAI (10 min)) × 100) Table 9 Results of emulsification and emulsion stability of SSPS, OSA-SSPS, and OSA-SSPS-NP

[0123] Classification <![CDATA[Emulsifying property (m 2 / g)]]> <![CDATA[Emulsion stability (10 -2 )]]> SSPS 1.98±0.04 2.45±0.17 OSA-SSPS 5.71±0.05 4.29±0.16 OSA-SSPS-NP 6.07±0.02 6.46±0.20

[0124] Result analysis: Compared with ordinary polysaccharides, the emulsification and emulsification stability of esterified polysaccharides linked to octenyl succinic anhydride were significantly improved. The emulsification stability of esterified polysaccharide nanoparticles prepared by anti-solvent precipitation method was significantly improved compared with esterified polysaccharides.

[0125] Example 4 Polysaccharide infrared spectrum comparison chart

[0126] The chemical bonds and molecular structures of the three samples (SSPS, OSA-SSPS, OSA-SSPS-NP) were analyzed by Fourier transform infrared spectroscopy. The polysaccharide samples were dried in an infrared dryer for 2 hours. The samples were mixed with KBr at a ratio of 1%, ground, pressed, and then placed on an infrared spectrometer for testing. The scanning wave number range was 400-4000cm -1 , resolution 4cm -1 , using DTGS detector, with air as blank, scanning 64 times per minute and taking the average value to obtain the infrared spectrum of the sample, as shown in Figure 5 shown.

[0127] Result analysis: In the infrared spectrum of SSPS, 3441.8cm -1 The broad peak at 2941.1 cm is caused by the stretching vibration of -OH. -1 The downward step peak is the absorption peak of CH stretching vibration in methylene, 1628.9 cm -1 It is the stretching vibration of C=C double bond, 1420cm -1 ~1200cm -1 The peak of CH vibration is 1044.9 cm -1 The absorption peaks of ether CO stretching vibration are at 1732.7cm. These peaks are characteristic absorption peaks of sugars. By comparing the infrared spectra of the modified samples, it can be found that OSA-SSPS and OSA-SSPS-NP have characteristic absorption peaks of sugars at 1732.7cm. -1 The stretching vibration absorption peak of the C=O ester carbonyl group of the ester appears, indicating that the octenyl succinic anhydride and the polysaccharide have been connected through an ester bond.

[0128] Example 5 Particle size comparison of polysaccharide emulsions

[0129] (1) Use a laser particle size analyzer wet method to compare the particle size of the emulsion: After the optical path is determined, add the sample until the sample concentration shows normal, and take the data with a light shielding degree of about 10 for comparison, such as Figure 6 As shown;

[0130] (2) Direct observation under light microscope: dilute the prepared emulsion 20 times, take 10 μL and drop it on a glass slide, cover it with a coverslip and allow it to be evenly dispersed by liquid surface tension, then observe the emulsion under an inverted microscope, and select 200 times magnification for observation.

[0131] Result analysis: Figure 6 Particle size diagram and Figure 7 It can be seen from the optical microscopy photos that the particle size of the emulsion prepared by SSPS is the largest, mainly concentrated between 55.36-61.61μm, and the particle size of the emulsions prepared by OSA-SSPS and OSA-SSPS-NP is smaller, mainly concentrated between 0.850-6.505μm. Among them, the particle size of the emulsion prepared by OSA-SSPS-NP is overall smaller than that of the emulsion prepared by OSA-SSPS.

[0132] Comparative Example 1 Comparison of emulsification properties of soybean polysaccharide esters substituted with different anhydrides

[0133] Based on the method of Example 1, different substituents such as octenylsuccinic anhydride, acetic anhydride, phosphoric anhydride, malic anhydride, citric anhydride, etc. were selected to esterify soybean polysaccharides, and the differences in their emulsification properties were compared.

[0134] Result analysis: Figure 8As shown in the figure, the emulsification properties of different soybean polysaccharide esters are octenyl succinate (SSPS-OSA) > phosphate (SSPS-P) > acetate (SSPS-Ac) > soybean polysaccharide (SSPS) > malate (SSPS-MA) > citrate (SSPS-CA). Therefore, octenyl succinic anhydride is a more suitable esterification substitute for improving the emulsification properties of soybean polysaccharides.

[0135] Table 10 Results of emulsification of soybean polysaccharide esters substituted with different anhydrides

[0136] Classification <![CDATA[Emulsifying property (m 2 / g)]]> SSPS 1.98±0.04 SSPS-OSA 5.71±0.05 SSPS-Ac 3.25±0.07 SSPS-P 4.12±0.13 SSPS-MA 1.02±0.02 SSPS-CA 0.79±0.08

[0137] Comparative Example 2 Comparison of the degree of substitution of soybean polysaccharide esters under different pretreatment methods

[0138] Based on the method of Example 1, four different pretreatment methods, ball milling, ultrasound, microwave, and ultra-high pressure, were used to pretreat soybean polysaccharides, and then octenyl succinic anhydride was esterified and substituted to compare the substitution degree of soybean polysaccharides. Among them, the ball milling pretreatment used 0.2 mol / L lithium chloride-dimethyl sulfoxide as an auxiliary agent, the material-liquid ratio of soybean polysaccharides to the auxiliary agent was 10 mg: 1 mL, the ball-to-material ratio was 3: 1, and the ball milling time was 5-8 h; the ultrasonic pretreatment conditions were 300 W power, 25 ° C temperature, and 30 min treatment time; the microwave pretreatment conditions were 400 W power and 30 min treatment time; the ultra-high pressure pretreatment conditions were 350 MPa at 5 min.

[0139] Result analysis: Fig. 9 As shown, a comprehensive comparison of the four physical treatment methods of ball milling, ultrasound, microwave and ultra-high pressure shows that all of them have the effect of improving the degree of substitution; compared with the others, the ball milling method has the best effect.

[0140] Table 11 Results of emulsification of soybean polysaccharide esters substituted with different anhydrides

[0141] Preprocessing method Degree of substitution No pre-processing 0.0217±0.0034 ball milling 0.0860±0.0056 Ultrasound 0.0452±0.0071 microwave 0.0614±0.0069 Ultra-high pressure 0.0375±0.0120

[0142] Comparative Example 3 Preparation of esterified soybean polysaccharide nanoparticles with different anti-solvents and different sugar solution concentrations

[0143] Take OSA-SSPS and dissolve it in water. Add a rotor to a beaker to dissolve it. Prepare a sugar solution with a concentration of 10 mg / mL. Select ethanol, methanol, and isopropanol as anti-solvents respectively. Use the method for preparing soybean polysaccharide nanoparticles in Example 2. Use a constant flow pump and a fine needle to slowly add the solution to 20 times the anti-solvent at a flow rate of 15-25 μL / s, and stir while adding. Stir at room temperature for 180 minutes at a speed of 900 rpm, then concentrate under reduced pressure and vacuum dry to obtain the product. Use dynamic light scattering (DLS) to measure the particle size of the three polysaccharides to explore the effect of the type of anti-solvent on the particle size of the esterified polysaccharide particles.

[0144] The above method was used to prepare sugar solutions with concentrations of 10, 15, and 20 mg / mL, respectively. Ethanol was selected as the anti-solvent to explore the effect of sugar solution concentration on the particle size of esterified polysaccharide microparticles.

[0145] Result analysis: Fig.10 As shown, by comparing different anti-solvents, it is clear that the nanoparticles prepared when ethanol is used as the anti-solvent have the smallest particle size and are more concentrated; the particles prepared by methanol and isopropanol both have peaks in the range of particle size > 1μm, which does not meet the requirements.

[0146] like Fig.11 As shown, by comparing sugar solutions of different concentrations, it was found that the particle size of the microparticles prepared at a concentration of 10 mg / mL was the smallest. When the concentration was further increased, the particles showed a peak shape in the range of particle size > 1 μm, which did not meet the requirements.

[0147] Comprehensive comparative studies found that the esterified soybean meal polysaccharide nanoparticles prepared with ethanol as the anti-solvent and a sugar solution of 10 mg / mL had the best effect.

Claims

1. A method for preparing soybean polysaccharide nanoparticles with high emulsification and stability, comprising the following steps: (1) Soybean polysaccharide was used as raw material and pre-treated by ball milling. The ball-to-material mass ratio was 3:

1. 0.2 mol / L lithium chloride-dimethyl sulfoxide solution was used as a ball milling aid. The material-liquid ratio of soybean polysaccharide to the aid was 10 mg:1 mL. The ball milling time was 5-8 h. After ball milling, the product was freeze-dried and passed through a 100-mesh sieve. (2) The pretreated soybean polysaccharide was dispersed in a mixed solvent system, and then the modifier octenyl succinic anhydride was added to obtain a mixed solution, and the pH of the mixed solution was adjusted to 8.0-8.5, and the esterification modification reaction was carried out in a water bath at 30-40°C with stirring. During the reaction, alkaline solution was added every 5 minutes to maintain the pH value of the reaction system. The reaction time was 25-35 minutes. After the reaction, the pH value of the mixed solution was adjusted to 5.8-6.2, and the mixture was dialyzed for 48 hours and then freeze-dried to obtain a crude sample of octenyl succinic anhydride-soybean polysaccharide; The mass ratio of 1-ethyl-3-methylimidazolium acetate ionic liquid to water in the mixed solvent system is 1:1; The weight-to-volume ratio of the soybean polysaccharide to the mixed solvent system is 1 g: (9-10) mL The mass ratio of octenyl succinic anhydride to soybean polysaccharide is 1:4-8; (3) taking the crude sample of octenyl succinic anhydride-soybean polysaccharide obtained in (2), stirring it to completely dissolve it in water, and obtaining an octenyl succinic anhydride-soybean polysaccharide aqueous solution with a concentration of 0.3-0.6%; Take the above aqueous solution, slowly drop it into ethanol at room temperature at a flow rate of 15-25 μL / s, wherein the volume ratio of the aqueous phase to the ethanol is 1:18-25, and stir at a speed of 900 rpm for 180 minutes while dropping, and then concentrate under reduced pressure; When the reduced pressure concentration was performed to 2 / 3 of the original volume, water was added to make up to the original volume, and the reduced pressure concentration was performed again. After repeating 3 times, vacuum drying was performed to obtain the product esterified soybean polysaccharide nanoparticles.

2. The method for preparing soybean polysaccharide nanoparticles with high emulsification and stability according to claim 1, characterized in that: (2) The temperature of the esterification modification reaction is 35°C, the pH is 8.5, and the reaction time is 35 min; the alkali solution is 0.1 mol / L sodium hydroxide solution.

3. The method for preparing soybean polysaccharide nanoparticles with high emulsification and stability according to claim 1, characterized in that: In (2), the mass ratio of octenylsuccinic anhydride to soybean polysaccharide is 1:

7.

4. The method for preparing soybean polysaccharide nanoparticles with high emulsification and stability according to claim 1, characterized in that: (3), the volume ratio of water phase to ethanol is 1:

20.

5. Use of the esterified soybean polysaccharide nanoparticles prepared by the preparation method of claim 1 in the preparation of Pickering emulsion, characterized in that: The application mainly includes the following steps: Take the esterified soybean polysaccharide nanoparticles in claim 1, add them into water, stir and fully dissolve them in a water bath at 35-45°C, and the concentration is 1-3wt%; then add the oil phase at a volume ratio of 3:1 of water: oil, and treat it twice with dynamic high-pressure microfluidization at 40 MPa to obtain a Pickering emulsion.

6. The use as claimed in claim 5, characterized in that The oil phase is soybean oil.

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