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

Abstract

The present invention belongs to the technical field of food colloids, and specifically relates to a preparation method and application of soybean polysaccharide nanoparticles with high emulsification and stability. The method uses soybean polysaccharide as a raw material for ball milling pretreatment, then uses octenylsuccinic anhydride as a modifier, and adds the two to a mixed solvent system of 1-ethyl-3-methylimidazolium acetate ionic liquid and water for esterification modification reaction; then uses ethanol as an anti-solvent to precipitate it to make it nano-sized, reduce pressure and concentrate, and vacuum dry to obtain the product esterified soybean polysaccharide nanoparticles. This preparation method not only significantly improves the emulsification of soybean polysaccharide, making its emulsification reach 6.06 m² / g, but also achieves an emulsification stability value of 6.48 within 10 minutes, showing excellent emulsification stability. At the same time, the method also successfully improves the degree of substitution of soybean polysaccharide, 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 particularly relates to a preparation method and application of soybean polysaccharide nanoparticles with high emulsification and stability. Background Art

[0002] Soybean polysaccharide is a natural, green, and safe emulsifier with a wide variety of raw materials. It rapidly adsorbs to the surface of oil droplets, where its sugar chains extend into the aqueous phase, forming a thick hydration film around the droplets, which stabilizes them through steric hindrance. However, compared to widely used, established emulsifiers, soybean polysaccharide's emulsifying properties and emulsion stability are inferior, particularly in oil / water (O / W) emulsion systems, where its stability is poor.

[0003] It is necessary to improve its emulsification and emulsion 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 their amphiphilicity is an important direction for improving emulsification performance. Generally, long-chain (C8-C18) esters have a high degree of substitution, and their expected effect on improving emulsification and emulsion stability is better. Octenyl succinic anhydride (OSA for short) is a long-chain esterifying agent that 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, if octenyl succinic anhydride is used as a modifier for soybean polysaccharides, it will face some challenges. Because soybean polysaccharides have a highly branched structure and long-chain olefin esterification agents (i.e., octenyl succinic anhydride) are insoluble in aqueous media, this makes it 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 properties, their emulsification stability still needs to be further improved. In order to improve the emulsification stability of polysaccharides, physical or chemical means 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 together determine the nucleation rate and growth rate of the crystal nucleus.

[0007] For soybean polysaccharides, since they are a heteropolysaccharide with numerous 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 antisolvent precipitation method, this technology is not applicable to soybean polysaccharides. In addition, although patent documents report a method for preparing insoluble soybean polysaccharide nanoparticles, this method uses the highly toxic reagent sodium azide, which poses a significant safety risk. Summary of the Invention

[0008] In order to solve the above-mentioned technical problems, the present invention provides a method for preparing soybean polysaccharide nanoparticles with high emulsification and stability. To address 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 modifies the soybean polysaccharide in an ionic liquid-water mixed system with octenyl succinic anhydride as a modifier to improve its emulsification; and further reduces the size of the modified polysaccharide particles through 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 and pretreated 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. The product was freeze-dried and passed through a 100-mesh sieve.

[0012] (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. The esterification modification reaction was carried out in a water bath at 30-40° C., and an alkali solution was added every 5 minutes during the reaction 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. After dialysis for 48 hours, the solution was freeze-dried to obtain a crude sample of octenyl succinic anhydride-soybean polysaccharide.

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

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

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

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

[0017] The above aqueous solution was slowly added dropwise to ethanol at room temperature at a flow rate of 15-25 μL / s, wherein the volume ratio of the aqueous phase to the ethanol was 1:18-25. While adding dropwise, the mixture was stirred at 900 rpm for 180 minutes, and then concentrated under reduced pressure.

[0018] When the reduced pressure concentration is completed to 2 / 3 of the original volume, water is added to make up to the original volume, and the reduced pressure concentration is repeated 3 times and then vacuum dried 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, making the esterification process easier based on the above steps.

[0020] In step (2), the raw materials are pre-treated by ball milling according to the method of step (1), and then modified by esterification mediated by ionic liquid, 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 polysaccharide is 1:7. The ionic liquid used is 1-ethyl-3-methylimidazolium acetate, which 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, the 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 antisolvent precipitation method is used, with ethanol as the antisolvent. The antisolvent precipitation method is a method that utilizes the phase separation effect between the solvent and the non-solvent to precipitate the target substance in the non-solvent and form nanospheres. 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 octenylsuccinic anhydride to soybean polysaccharide is 1:7.

[0024] Preferably, in (3), the volume ratio of the aqueous phase to the 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] The esterified soybean polysaccharide nanoparticles prepared by the above preparation method are added to water and stirred in a water bath at 35-45°C to fully dissolve them to a concentration of 1-3wt%; then the oil phase is added at a volume ratio of 3:1 between water and oil, and treated twice with dynamic high-pressure microfluidization at 40 MPa 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. In the presence of an ionic liquid medium, the soybean polysaccharides are esterified with octenyl succinic anhydride to increase the degree of substitution and enhance the emulsification of the polysaccharide. 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 their emulsification stability. After applying it to Pickering emulsion, the droplet size is concentrated between 0.8-6.5 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, which has a smaller and more uniform particle size and a higher encapsulation efficiency for the loaded substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of single factor experiment on soybean polysaccharide esterification are shown in Figure 2.

[0034] Figure 2 This is a process flow chart for 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 Comparison chart of emulsification and emulsion 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 The particle size comparison chart 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 emulsifying properties of different esterified soybean polysaccharides;

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

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

[0043] Figure 11 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 will be further explained in conjunction with specific embodiments.

[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: reaction time, temperature, pH, and the ratio of esterifying agent to soybean polysaccharide. The optimal preparation process for esterified polysaccharides was determined through single-factor and orthogonal experiments, using the degree of substitution as the evaluation indicator.

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

[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. 0.2 mol / L lithium chloride-dimethyl sulfoxide solution was used as a ball milling aid. The mass volume ratio of soybean polysaccharide to 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] 10 g of ball-milled soybean polysaccharide (SSPS) was dispersed in 90 mL of a 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixture. Octenylsuccinic anhydride (OSA, a modifier) ​​was added at a ratio of OSA:SSPS = 1:7. The solution was adjusted to different pH values ​​(7.5, 8, 8.5, 9, and 9.5). Esterification was performed in a water bath at 40°C with stirring. 0.1 mol / L NaOH was added every 5 minutes to maintain the pH. The reaction was maintained for 40 minutes. After completion of the reaction, the pH of the solution was adjusted to 6.0. The crude OSA-SSPS sample was dialyzed for 48 hours using a 500 Da dialysis bag and freeze-dried.

[0051] The substitution degree of the crude OSA-SSPS sample was tested. 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 its 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 polysaccharide was freeze-dried and passed through a 100-mesh sieve.

[0058] 10 g of pretreated soybean polysaccharide (SSPS) was dispersed in 90 mL of a 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixture. Octenylsuccinic anhydride was added at a ratio of OSA:SSPS = 1:7, and the pH of the solution was adjusted to 8.5. Esterification was carried out in a water bath at 40°C with stirring. 0.1 mol / L NaOH was added every 5 minutes to maintain the pH of the solution. The reaction was maintained for 25, 30, 35, 40, and 45 minutes. After completion of the reaction, the pH of the solution was adjusted to 6.0. The crude OSA-SSPS sample was dialyzed for 48 hours using a 500 Da dialysis bag and freeze-dried.

[0059] The substitution degree of the crude OSA-SSPS sample was tested. 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 crude OSA-SSPS 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 polysaccharide was freeze-dried and passed through a 100-mesh sieve.

[0066] 10 g of pretreated soybean polysaccharide (SSPS) was dispersed in 90 mL of a 1-ethyl-3-methylimidazolium acetate / water (wt 1:1) mixture. Octenylsuccinic anhydride was added at a ratio of OSA:SSPS of 1:7. The pH of the SSPS solution was adjusted to 8.5. Esterification was performed in a water bath with stirring at different temperatures (30°C, 35°C, 40°C, 45°C, and 50°C). 0.1 mol / L NaOH was added every 5 minutes to maintain the pH of the solution. The reaction was maintained for 40 minutes. After completion of the reaction, the pH of the solution was adjusted to 6.0. The crude OSA-SSPS sample was dialyzed for 48 hours using a 500 Da dialysis bag and freeze-dried.

[0067] The substitution degree of the crude OSA-SSPS sample was tested. 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 substitution degree 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 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] 10 g of pretreated soybean polysaccharide was dispersed in 90 mL of a 1-ethyl-3-methylimidazolium acetate / water (wt:1) mixture. Octenylsuccinic anhydride (OSA) was added at varying ratios (OSA:SSPS = 0:1, 1:5, 1:6, 1:7, and 1:8). The pH of the solution was adjusted to 8.5. Esterification was performed in a 40°C water bath with stirring. 0.1 mol / L NaOH was added every 5 minutes to maintain the pH. The reaction was maintained for 40 minutes. After completion of the reaction, the pH of the solution was adjusted to 6.0. The crude OSA-SSPS sample was dialyzed for 48 hours using a 500 Da dialysis bag and freeze-dried.

[0075] The OSA-SSPS crude sample obtained in the above steps was subjected to the degree of substitution test. The results are as follows: 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 substitution degree 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] Based on the F value of variance analysis, the order of influence on the degree of substitution of esterified soybean polysaccharides was determined to be pH > time > temperature > ratio. According to post hoc tests, the highest value was the optimal level, namely A3, B3, C2, and D3, which were the best solutions, namely T = 35°C, t = 35 min, pH = 8.5, and 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 these conditions was 0.0860%, which was much higher than the single result level of single factor and orthogonal test, 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 octenylsuccinic anhydride-soybean polysaccharide (OSA-SSPS) is measured by titration. The specific measurement steps are as follows:

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

[0099] (2) Add 50 mL of isopropyl alcohol 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 no chloride ions can be detected with 0.1 mol / L AgNO3 solution;

[0101] (4) The washed residue was dried at 40°C for 24 hours and then passed through a 200-mesh nylon sieve;

[0102] (5) Under stirring, 1.0000 g of the sieved residue was dispersed in 50 mL of deionized water until completely dissolved. The solution was titrated with 0.1 mol / L standard NaOH solution using phenolphthalein as an indicator. 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] Wherein, 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) Dissolve 0.5 g of OSA-SSPS (prepared by the optimized solution in Example 1) in 100 mL of water, add a rotor to a beaker, and stir until completely dissolved;

[0111] (2) Using a constant flow pump and a fine needle, slowly add the solution dropwise to 20 volumes of ethanol at a flow rate of 15-25 μL / s. Stir while adding the solution dropwise. Stir at 900 rpm for 180 min at room temperature and then concentrate 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 three times. Finally, vacuum drying was performed to obtain the product esterified polysaccharide nanoparticles.

[0113] After preparing esterified polysaccharide nanoparticles using the antisolvent precipitation method described above, dynamic light scattering (DLS) was used to measure the particle size of the three polysaccharides (SSPS, OSA-SSPS, and OSA-SSPS-NP). DLS measurements were performed using an Anton Paar Particle Analyzer Litesizer 500. Water, with a refractive index of 1.333, was used as the solvent. 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 and the average value was calculated.

[0114] Result analysis: Figure 4 As shown, the particle size distribution of SSPS is mainly between 200nm and 5000nm; that of OSA-SSPS is mainly between 400nm and 12000nm, indicating that the particle size increases after esterification. After further preparation into nanoparticles (OSA-SSPS-NP), the particle size decreases and concentrates 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] The emulsification activity index (EAI) is a method for evaluating emulsification properties. Take 0.15g of OSA-SSPS-NP, SSPS or OSA-SSPS freeze-dried sample and stir it in 15mL of pure water in a 40℃ water bath until it is fully dissolved. Add 5mL of soybean oil and homogenize it 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), using 0.1SDS as a blank control. Emulsification properties are calculated 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 of standing 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. 0.1% SDS solution was used as a 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] Results: Compared with ordinary polysaccharides, the emulsification and emulsion stability of the esterified polysaccharide linked to octenylsuccinic anhydride were significantly improved. The emulsion stability of the esterified polysaccharide nanoparticles prepared by the antisolvent precipitation method was significantly improved compared with the esterified polysaccharide.

[0125] Comparison of polysaccharide infrared spectra in Example 4

[0126] Fourier transform infrared spectroscopy was used to analyze the chemical bonds and molecular structures of the three samples (SSPS, OSA-SSPS, and OSA-SSPS-NP). The polysaccharide samples were dried in an infrared dryer for 2 hours. Using the potassium bromide (KBr) pelleting method, the samples were thoroughly mixed with KBr at a ratio of 1%, ground, and pelleted. The pellets were then placed on an infrared spectrometer for analysis. The scanning wavenumber range was 400–4000 cm -1 , with a resolution of 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 the C=C double bond, 1420cm -1 ~1200cm -1 The peak of CH is the variable angle vibration, 1044.9 cm -1 The absorption peaks at 1732.7cm are the stretching vibration peaks of ether CO, which are the characteristic absorption peaks of sugars. 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 are connected through an ester bond.

[0128] Example 5: Particle size comparison of polysaccharide emulsions

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

[0130] (2) Direct observation under a light microscope: dilute the prepared emulsion 20 times, take 10 μL and drop it onto 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, selecting 200 times magnification for observation.

[0131] Result analysis: Figure 6 Particle size diagram and Figure 7 It can be seen from the light microscopy photos that the particle size of the emulsion prepared by SSPS is the largest, mainly concentrated between 55.36-61.61μm, while 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, and citric anhydride were selected to esterify soybean polysaccharides, and the differences in their emulsifying properties were compared.

[0134] Result analysis: Figure 8As shown in the figure, the emulsifying properties of different soybean polysaccharide esters are as follows: 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 agent for improving the emulsifying 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, including ball milling, ultrasound, microwave, and ultrahigh pressure, were used to pretreat soybean polysaccharides, followed by octenylsuccinic anhydride esterification and substitution, and the degree of substitution of soybean polysaccharides was compared. 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 polysaccharide to auxiliary agent was 10 mg:1 mL, the ball-to-material ratio was 3:1, and the ball milling time was 5-8 hours; the ultrasonic pretreatment conditions were 300 W power, 25 ° C temperature, and treatment time of 30 minutes; the microwave pretreatment conditions were 400 W power, 30 minutes; and the ultrahigh pressure pretreatment conditions were 350 MPa at 5 minutes.

[0139] Result analysis: Figure 9 As shown in the figure, 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 each other, 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 preprocessing 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] Dissolve OSA-SSPS in water, add a rotor to a beaker to dissolve it, and prepare a sugar solution with a concentration of 10 mg / mL. Use ethanol, methanol, and isopropanol as anti-solvents, respectively. Use the method for preparing soybean polysaccharide nanoparticles in Example 2, use a constant flow pump, connect a fine needle to slowly add the solution to 20 times the anti-solvent, with a flow rate of 15-25 μL / s, and stir while adding dropwise. After stirring at room temperature for 180 minutes at a speed of 900 rpm, 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 microparticles.

[0144] Using the above method, sugar solutions with concentrations of 10, 15, and 20 mg / mL were prepared, and 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: Figure 10 As shown in the figure, 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 particle size range of >1μm, which does not meet the requirements.

[0146] like Figure 11 As shown in the figure, 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 showed 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 the raw material and pretreated by ball milling. The ball-to-material mass ratio was 3:

1. 0.2 mol / L lithium chloride-dimethyl sulfoxide solution was used as the 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. 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. After dialysis for 48 hours, the mixture was 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 1g: (9-10)mL The mass ratio of the octenylsuccinic anhydride to soybean polysaccharide is 1:4-8; (3) taking the crude sample of octenylsuccinic anhydride-soybean polysaccharide obtained in (2), stirring it to completely dissolve it in water, and obtaining an octenylsuccinic anhydride-soybean polysaccharide aqueous solution with a concentration of 0.3-0.6%; The above aqueous solution was slowly added dropwise to ethanol at room temperature at a flow rate of 15-25 μL / s, wherein the volume ratio of the aqueous phase to the ethanol was 1:18-25. While adding dropwise, the solution was stirred at 900 rpm for 180 min, and then concentrated 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 repeated 3 times and then vacuum dried 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 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: (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, wherein: (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 preparing Pickering emulsion, characterized in that: The application mainly includes the following steps: Take the esterified soybean polysaccharide nanoparticles of claim 1, add them to water, stir and fully dissolve them in a water bath at 35-45°C to a concentration of 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 40 MPa to obtain a Pickering emulsion.

6. The use according to claim 5, characterized in that The oil phase is soybean oil.

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