A kind of monodisperse type 3 resistant starch nanoparticles with uniform particle size and a preparation method thereof
By improving the purity of short-chain dextran and self-assembly in water, the problems of uneven particle size and aggregation of type 3-type resistant starch nanoparticles are solved, and nanoparticles with monodispersity, uniform particle size and good colloidal stability are achieved. They are suitable for the food industry and have a safe, non-toxic and simple preparation process.
Patent Information
- Application Number
- CN202210684021.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-06-16
- Publication Date
- 2025-06-24
- Estimated Expiration
- 2042-06-16
AI Technical Summary
In the prior art, the preparation of type 3 resistant starch nanoparticles has problems of uneven particle size and aggregation, which affects the sensory quality of the food. The use of surfactants and polyelectrolytes will increase the preparation cost and is not suitable for practical applications in the food industry.
By increasing the purity of short-chain dextran and self-assembled in water, monodisperse type 3 resistant starch nanoparticles with uniform particle size were prepared. This method does not require any chemical modifications and crosslinking agents, and reversible self-assembly of the particles is achieved by heating and cooling.
The type 3-type resistant starch nanoparticles with monodispersity, uniform particle size and good colloidal stability are achieved, and the stability is maintained over a long time and a wide pH range, and does not affect the sensory quality of the food. It has the advantages of safety, non-toxicity and simple preparation technology.
Smart Images
Figure CN114874467B_ABST
Abstract
Description
Technical Field:
[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a monodisperse type 3 resistant starch nanoparticle with uniform particle size and a preparation method thereof. Background Art:
[0002] Starch is a dietary carbohydrate and one of the most important nutritional energy sources, accounting for more than half of the total calories consumed in the human diet. However, because it is rapidly digested by enzymes in the gastrointestinal tract, it causes a rapid increase in blood glucose levels, thereby leading to the occurrence of various chronic diseases, such as obesity, type 2 diabetes, etc. Starch is composed of semi-crystalline polymers, having both amorphous regions and crystalline regions, and the ratio of the two determines their physical properties and digestibility.
[0003] Resistant starch (RS) is starch that cannot be absorbed by healthy individuals and is a prebiotic carbohydrate that can promote the growth of beneficial gut flora, thereby reducing the risk of colon cancer. RS has 5 types, among which type 3 resistant starch (RS3) is a retrograded starch produced by cooling gelatinized starch, with a dense structure, higher thermal stability than other RS, low digestibility, and characteristics such as low cost, biocompatibility, biodegradability, and non-toxicity, having great application prospects in the food industry.
[0004] In the prior art, there are still some disadvantages and deficiencies in the preparation of RSNP (type 3 resistant starch nanoparticles). For example, when short-chain glucan (SCG) self-assembles without being controlled by thermodynamic parameters, it usually forms RSNP with highly polydisperse and aggregated shapes, which will affect the sensory quality when dispersed in food as powder or colloid. Common methods to overcome starch granule aggregation include using surfactants and polyelectrolytes. Letona et al. [Letona C.A.M., Luo K., Jeong K.-B., et al. Effect of Lecithin on the Spontaneous Crystallization of Enzymatically Synthesized Short-Chain Amylose Molecules into Spherical Microparticles[J]. Polymers, 2019, 11(2):264.] prepared amylose polymer microparticles in the presence of lecithin. The principle is that lecithin forms uniform amylose microparticles through the steric stabilization and diffusion-limited growth effect of growing granules. Luo et al. [Luo K., Lee D.-H., Adra H.J., et al. Synthesis of monodisperse starch microparticles through molecular rearrangement of short-chain glucans from natural waxy maize starch[J]. Carbohydrate Polymers, 2019, 218:261-268.] used chitosan as a steric stabilizer to regulate the nucleation process, which significantly reduced the undesirable aggregation in the nucleation and growth stages, thus producing highly monodisperse microparticles. However, using these surfactants will increase the preparation cost of type 3 resistant starch nanoparticles, and the steric stabilization effect of polyelectrolytes is generally affected by the pH value of the reaction solution, which is not suitable for practical applications in the food industry. Summary of the Invention:
[0005] To solve the disadvantages and deficiencies in the preparation of type 3 resistant starch in the prior art, the present invention provides a method for preparing highly uniform and monodisperse type 3 resistant starch nanoparticles (RSNP). By increasing the purity of short-chain glucan (SCG), high-purity SCG is self-assembled in water to prepare uniform and monodisperse RSNP.
[0006] To achieve the above object, the present invention is realized through the following technical solutions. A method for preparing monodisperse type 3 resistant starch nanoparticles with a uniform particle size includes the following steps:
[0007] (1) Prepare crude starch granule RSMP by the self-assembly reaction of preparing SCG through starch debranching treatment.
[0008] (2) Gelatinize the self-assembled crude starch granule RSMP obtained in step (1); dissolve SCG through the gelatinization treatment of RSMP.
[0009] (3) After cooling the gelatinized solution in step (2) to room temperature, centrifuge and filter it to obtain a high-purity short-chain glucan SCG solution; among them, SCG self-assembly is induced during the cooling process. After centrifugation and filtration, the incompletely debranched macromolecular dextrin generated during the starch debranching process is removed to obtain a high-purity short-chain glucan SCG solution. Since the incompletely debranched macromolecular dextrin in the self-assembly reaction solution is the main factor inducing starch granule aggregation, after this step of treatment, the content of incompletely debranched macromolecular dextrin in the high-purity short-chain glucan SCG is low, and its self-assembly reaction can overcome granule aggregation.
[0010] (4) Cool and incubate the solution in step (3) to obtain monodisperse type 3 resistant starch nanoparticles RSNP with a uniform particle size. RSNP is formed by the hydrogen bond interaction between and within SCG molecules, and this interaction can proceed reversibly under thermodynamic control. Therefore, the reversible self-assembly of RSNP can be achieved by heating and cooling. This method does not require any chemical modification and cross-linking agent, and can simply, effectively and environmentally generate RSNP with a certain particle size and a uniform particle size distribution.
[0011] Furthermore, the preparation of the self-assembled crude starch granule RSMP in step (1) includes the following steps:
[0012] (1-1) Weigh starch, add sodium acetate buffer solution, and mix evenly; among them, sodium acetate can improve the stability of pullulanase.
[0013] (1-2) Heat and gelatinize the mixed solution; use the gelatinization reaction to destroy the starch crystal structure, and then improve the debranching efficiency of pullulanase on amylopectin.
[0014] (1-3) After the gelatinized solution is cooled, add 2-10 ASPU / mL of pullulanase to it, and incubate it in a water bath at 40-60 °C with shaking for 12-48 h; use pullulanase to prepare short-chain glucan SCG by debranching amylopectin.
[0015] (1-4) Incubate the mixed solution in (1-3) at 4 °C for 12-24 h to induce the self-assembly of RSMP.
[0016] Furthermore, in step (1-1), the mass concentration of starch is 1-10% (w / v), and the concentration of sodium acetate buffer solution is 20-100 mM. By regulating this parameter, the debranching efficiency of pullulanase on amylopectin can be improved.
[0017] Furthermore, in step (1-2), microwave heating is used for gelatinization.
[0018] Furthermore, in step (2), microwave heating is used for gelatinization. Compared with the conventional water bath heating treatment method, the gelatinization reaction rate is faster.
[0019] Furthermore, in step (3), centrifugation is carried out under the condition of 8000g-10000g, and filtration is carried out through a membrane with a pore size of 0.20μm-0.45μm.
[0020] Furthermore, the solution in step (4) is incubated at 4°C for 12-24 h to obtain the finished product. Among them, 4°C is the optimal temperature for SCG self-assembly.
[0021] A kind of monodisperse type 3 resistant starch nanoparticles with uniform particle size prepared by the above method, which are highly uniform spheres, and the average diameter is concentrated in the range of 100nm-200nm.
[0022] The beneficial effects of the present invention are as follows: on the basis of the existing technology, through the purification treatment of SCG in the debranched starch solution, the present invention prepares the retrograded resistant starch nanoparticles (RSNP) with monodispersity, uniform particle size and good colloidal stability. The prepared RSNP maintains good stability in a relatively long time and a wide pH range, and does not use surfactants, polyelectrolytes, cross-linking agents, etc., and does not require any chemical modification. It has the advantages of safety, non-toxicity, simple preparation process, etc., and can be used as an auxiliary material for food, medicine and cosmetics, thereby improving the physical and chemical properties and quality of products, and has great application potential in cosmetics, drug delivery, food industry and other aspects. Description of the drawings:
[0023] Figure 1 It is the SEM image of RSNP prepared by centrifugation / filtration treatment and RSMP prepared without centrifugation / filtration treatment.
[0024] Figure 2 It is the particle size distribution histogram of RSNP and RSMP.
[0025] Figure 3 It is the photo of RSNP and RSMP dispersed in deionized water within 120 minutes.
[0026] Figure 4 It is the surface charge of RSNP under different pH (2-10) conditions.
[0027] Figure 5It is the sedimentation photos of RSNP under different pH conditions (pH = 2, 4, 6, 8, 10) for 24 hours.
[0028] Figure 6 It is the hydrolysis rate (a) of NS, RSMP and RSNP at different in vitro digestion times and the contents (b) of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). Specific implementation manners:
[0029] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. 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 protection scope of the present invention.
[0030] Example 1:
[0031] A preparation method of monodisperse type 3 resistant starch nanoparticles with uniform particle size, comprising the following steps:
[0032] (1) Prepare self-assembled crude starch particles RSMP:
[0033] (1-1) Weigh starch, add sodium acetate buffer solution, and mix evenly; wherein, the mass concentration of starch is 1% (w / v), and the concentration of sodium acetate buffer solution is 20 mM.
[0034] (1-2) Heat the mixed solution in a microwave oven for 2 - 10 min for gelatinization;
[0035] (1-3) After the gelatinized solution is cooled to 50 °C, add pullulanase to it to make the final concentration 2 ASPU / mL, and incubate in a water bath with oscillation. Among them, the set condition parameters are: temperature is 60 °C; shaker rotation speed is 100 rpm; incubation time is 48 h.
[0036] (1-4) Incubate the mixed solution in (1-3) at 4 °C for 24 h to induce the self-assembly of RSMP.
[0037] (2) Wash the self-assembled RSMP obtained in step (1) with distilled-deionized water, and then heat it in a microwave oven to gelatinize it;
[0038] (3) After the gelatinized solution in step (2) is cooled to room temperature, centrifuge the gelatinized solution at 10000 g for 10 minutes, and filter it through a 0.45 μm membrane to improve the purity of SCG in the gelatinized solution.
[0039] (4) Cool and incubate the solution in step (3), place it at 4 °C for 24 h to induce the self-assembly of RSNP.
[0040] Example 2:
[0041] A preparation method of monodisperse type 3 resistant starch nanoparticles with uniform particle size, comprising the following steps:
[0042] (1) Prepare self-assembled crude starch particles RSMP:
[0043] (1-2) Weigh starch, add sodium acetate buffer solution, and mix evenly; wherein, the mass concentration of starch is 4% (w / v), and the concentration of sodium acetate buffer solution is 50 mM.
[0044] (1-2) Heat the mixed solution in a microwave oven for 2 min for gelatinization;
[0045] (1-3) After the gelatinized solution is cooled to 40 °C, add pullulanase to it to make the final concentration 6 ASPU / mL, and incubate it in a water bath with oscillation. Among them, the set condition parameters are: temperature is 50 °C; shaker rotation speed is 100 rpm; incubation time is 48 h.
[0046] (1-4) Incubate the mixed solution in (1-3) at 4 °C for 18 h to induce the self-assembly of RSMP.
[0047] (2) Wash the self-assembled RSMP obtained in step (1) with distilled-deionized water, and then heat it in a microwave oven to gelatinize it;
[0048] (3) After the gelatinized solution in step (2) is cooled to room temperature, centrifuge the gelatinized solution at 8000 g for 10 minutes, and filter it through a 0.45 μm membrane to improve the purity of SCG in the gelatinized solution.
[0049] (4) Cool and incubate the solution in step (3), place it at 4 °C for 18 h to induce the self-assembly of RSNP.
[0050] Example 3:
[0051] A preparation method of monodisperse type 3 resistant starch nanoparticles with uniform particle size, comprising the following steps:
[0052] (1) Prepare self-assembled crude starch particles RSMP:
[0053] (1-3) Weigh starch, add sodium acetate buffer solution, and mix evenly; wherein, the mass concentration of starch is 8% (w / v), and the concentration of sodium acetate buffer solution is 100 mM.
[0054] (1-2) Heat the mixed solution in a microwave oven for 10 min for gelatinization;
[0055] After the gelatinized solution in (1-3) was cooled to 60 °C, pullulanase was added thereto to a final concentration of 10 ASPU / mL, and the mixture was incubated with shaking in a water bath. Among them, the set condition parameters were: temperature was 40 °C; shaker rotation speed was 100 rpm; incubation time was 24 h.
[0056] (1-4) The mixture in (1-3) was incubated at 4 °C for 12 h to induce self-assembly of RSMP.
[0057] (2) The self-assembled RSMP obtained in step (1) was washed with distilled-deionized water and then heated in a microwave oven to gelatinize it;
[0058] (3) After the gelatinized solution in step (2) was cooled to room temperature, the gelatinized solution was centrifuged at 10,000 g for 10 minutes and filtered through a 0.20 μm membrane to improve the purity of SCG in the gelatinized solution.
[0059] (4) The solution in step (3) was cooled and incubated at 4 °C for 12 h to induce self-assembly of RSNP.
[0060] Effect verification:
[0061] The surfaces of the RSNP and SRMP samples were sputter-coated with gold, and the morphology and surface topography of the samples were observed with a high-resolution scanning electron microscope at an acceleration voltage of 5 kV. The diameters of at least 100 particles in the SEM images were measured to estimate the particle size distribution histogram ( Figure 2 ). It can be clearly seen from the scanning electron microscope that, compared with the RSMP prepared without centrifugation / filtration treatment ( Figure 1 , ii), the RSNP prepared after centrifugation / filtration treatment ( Figure 1 , i) has better particle size uniformity, indicating that improving the purity of SCG by centrifugation / filtration treatment can effectively solve the problems of self-assembled resistant starch particle aggregation and uneven particle size; and RSNP is a highly uniform spherical shape with a narrow size distribution, and the average diameter is concentrated around 150 nm, while RSMP is a highly aggregated non-uniform morphology with a wide size distribution and an average diameter of more than 2 μm, which also shows that the quality of RSNP after centrifugation / filtration treatment is better. Figure 1
[0062] To further study the dispersion stability of RSNP and RSMP in an aqueous environment, the sedimentation behavior of RSNP and RSMP during 120 minutes was studied, as Figure 3 shown. RSNP has higher dispersion stability than RSMP and does not sink within 120 minutes, while RSMP shows sedimentation at 60 minutes.
[0063] Figure 4 The surface charge of RSNP at different pH values (2-10) is as Figure 4As shown. Whether in acidic, neutral or alkaline conditions, the surface charge of RSNP is negative in aqueous medium, and the surface charge density increases with the increase of pH value. The sedimentation behavior of RSNP under acidic, neutral and alkaline conditions is as Figure 5 shown. It was found that in acidic medium, due to the low surface charge density of RSNP, it is more likely to aggregate and the sedimentation rate is faster. While RSNP has strong negative charges and good colloidal stability in alkaline and neutral environments, indicating that this nanoparticle can be applied to complex food systems. In vitro digestion experiments showed that compared with starch raw materials (NS) and RSMP, RSNP has higher anti-digestive properties.
Claims
1. A preparation method of monodisperse type 3 resistant starch nanoparticles with uniform particle size, characterized in that It includes the following steps: (1) Prepare self-assembled rough starch particles RSMP, including the following steps: (1-1) Weigh starch, add sodium acetate buffer solution, and mix evenly; (1-2) Heat the mixed solution for gelatinization; (1-3) After the gelatinized solution cools, add pullulanase to it and incubate by heating in a water bath; (1-4) Incubate the mixture in (1-3) at low temperature to induce the self-assembly of RSMP; (2) Gelatinize the self-assembled rough starch particles RSMP obtained in step (1); (3) After cooling the gelatinized solution in step (2) to room temperature, centrifuge and filter through a membrane to obtain a high-purity short-chain dextran SCG solution; (4) Cool and incubate the solution in step (3) to obtain monodisperse type 3 resistant starch nanoparticles RSNP with a uniform particle size.
2. The preparation method of the monodisperse type 3 resistant starch nanoparticles with uniform particle size according to claim 1, characterized in that: In step (1-1), the mass concentration w / v of starch is 1-10%, and the concentration of sodium acetate buffer solution is 20-100 mM.
3. The preparation method of the monodisperse type 3 resistant starch nanoparticles with uniform particle size according to claim 1, characterized in that: In step (1-2), microwave heating is used for gelatinization.
4. The preparation method of the monodisperse type 3 resistant starch nanoparticles with uniform particle size according to claim 1, characterized in that: In step (2), microwave heating is used for gelatinization.
5. The preparation method of the monodisperse type 3 resistant starch nanoparticles with uniform particle size according to claim 1, characterized in that: Step (3) is to centrifuge under the condition of 8000g - 10000g and filter through a membrane with 0.20μm - 0.45μm to obtain high-purity short straight-chain dextran.
6. The preparation method of the monodisperse type 3 resistant starch nanoparticles with uniform particle size according to claim 1, characterized in that: The solution in step (4) is placed at 4°C for incubation for 12 - 24h to obtain the finished product.
7. A monodisperse type 3 resistant starch nanoparticle with uniform particle size prepared by any of the methods according to claims 1-6, characterized in that: It is a highly uniform sphere with an average diameter of 100nm - 200nm.