Preparation method of peanut protein polysaccharide composite nanoparticles loaded with selenomethionine
By preparing core-shell structured nanoparticles composed of peanut protein and polysaccharides, the instability problem of plant protein beverages was solved, achieving the dual functions of stabilizer and nutrient delivery, thereby improving the stability and health value of the beverages.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LIAONING UNIVERSITY
- Filing Date
- 2026-03-30
- Publication Date
- 2026-06-09
AI Technical Summary
Existing plant protein beverages are prone to physical instability during storage and transportation, such as fat floating, protein precipitation, and system stratification. The use of traditional stabilizers affects the flavor of the product and is not stable in the long term.
By combining peanut protein with polysaccharides, nanoparticles with a core-shell structure are prepared. Utilizing the emulsifying properties of proteins and the electrostatic stabilizing effect of polysaccharides, selenomethionine is loaded as a stabilizer and nutrient delivery carrier to form a highly efficient stabilizer to improve the long-term stability of beverages.
It significantly improves the long-term physical stability of complex food systems such as plant protein beverages, enhances nutrient delivery capabilities, and imparts health benefits to products, providing efficient stabilizer and carrier functions.
Smart Images
Figure CN122162947A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of functional food and nanotechnology, specifically relating to a method for preparing peanut protein polysaccharide composite nanoparticles loaded with selenomethionine. Background Technology
[0002] Plant-based protein beverages, such as peanut milk, soy milk, and almond milk, are popular among consumers due to their rich nutrition and unique flavor. However, these beverages are complex thermodynamically unstable systems, mainly composed of proteins, fats, and water. During storage and transportation, due to gravity and interparticle interactions, they are prone to physical instability phenomena such as fat floating, protein precipitation, and system stratification. This not only seriously affects the sensory quality of the product but also greatly limits its shelf life.
[0003] To address this technical challenge, existing technologies typically employ the addition of small-molecule emulsifiers and high-molecular-weight hydrocolloids as composite stabilizers. Small-molecule emulsifiers emulsify fat droplets by reducing the interfacial tension between oil and water, while hydrocolloids thicken the entire system to delay particle settling and buoyancy. However, this traditional stabilization method still presents several challenges: for example, excessive stabilizer dosage may affect the original flavor and texture of the product, resulting in an undesirable "gelatinous" feel; furthermore, the long-term stability of complex beverage systems containing multiple ingredients still needs improvement. Therefore, developing a novel stabilizer that requires less dosage, is more efficient, and is itself a nutrient component has become a research hotspot in this field.
[0004] On the other hand, peanut protein, as a high-quality plant protein resource, is widely available and inexpensive. It possesses certain emulsifying and gelling properties, making it an ideal substrate for constructing novel food ingredients. However, single peanut protein particles exhibit limited stability when faced with pH fluctuations, ionic strength changes, and heat treatment in complex food systems, making them unsuitable for direct application as efficient stabilizers. Therefore, developing peanut protein into a stable functional ingredient by combining it with polysaccharides—allowing the polysaccharides to act as a protective shell—while simultaneously loading selenomethionine internally to enhance its functionality, has significant theoretical and market value.
[0005] Based on the aforementioned background, this invention prepares a core-shell structured nanoparticle by compounding peanut protein with food-grade polysaccharides. This nanoparticle not only utilizes its protein core to exert excellent emulsifying properties but also provides strong steric and electrostatic stabilization through its polysaccharide shell, thus serving as a highly efficient stabilizer to improve the long-term stability of plant-based protein beverages. Furthermore, this nanoparticle can also act as a carrier to load the functional nutrient selenomethionine, providing additional health benefits to the product while achieving stabilization. Summary of the Invention
[0006] This invention aims to prepare a high-performance, core-shell structured composite nanoparticle by combining peanut protein isolate with a complex polysaccharide (low-methoxyl pectin, sodium alginate) and introducing selenomethionine. The ingenious design of this nanoparticle integrates the emulsifying properties of proteins with the stabilizing effects of polysaccharides. It not only serves as a novel and highly efficient stabilizer, significantly improving the long-term physical stability of complex food systems such as plant-based protein beverages and preventing stratification and sedimentation, but also greatly enhances its ability as a nutrient delivery carrier, providing additional health benefits to the products. This invention shows broad application prospects in multiple fields such as functional foods, fortification, and the beverage industry, providing new ideas and technical support for the high-value utilization of plant proteins and the development of nanodelivery systems.
[0007] The technical solution adopted in this invention is:
[0008] A method for preparing peanut protein polysaccharide composite nanoparticles loaded with selenomethionine, characterized by comprising the following steps:
[0009] Step 1: Accurately weigh peanut protein isolate, selenomethionine, low-methoxyl pectin, and sodium alginate as the core and shell materials for constructing composite nanoparticles. The selection of these components is based on their respective excellent physicochemical properties and biological functions. Peanut protein isolate, as the protein matrix, has good biocompatibility and emulsifying properties, making it an ideal carrier for constructing nanoparticles. Selenomethionine, as an organic selenium source, has higher bioavailability and lower toxicity compared to inorganic selenium, making it a core functional component for nutritional fortification. Low-methoxyl pectin and sodium alginate serve as the composite polysaccharide shell. Both of these food-grade polysaccharides carry a negative charge, allowing them to bind to proteins through electrostatic interactions and providing excellent colloidal stability through their steric hindrance effect.
[0010] Step 2: Peanut protein isolate and selenomethionine are fully hydrated separately in PBS buffer at a specific pH. Through precisely controlled addition and coordination reactions, selenomethionine molecules are stably loaded onto the protein, forming the positively charged peanut protein isolate-selenomethionine core solution required for subsequent self-assembly. This step ensures efficient loading and uniform distribution of selenium.
[0011] The reaction was carried out in 0.01 M PBS buffer at pH 5.0 to enable the formed PPI-Se cores to carry a positive charge, laying the foundation for subsequent electrostatic self-assembly.
[0012] Step 3: Dissolve low-methoxyl pectin and sodium alginate thoroughly in PBS buffer at a specific pH, stir magnetically for 1 h, and finally sonicate at 300 W for 3 min. This step aims to prepare a clear, homogeneous, and strongly negatively charged composite polysaccharide solution—a low-methoxyl pectin-sodium alginate shell solution—to provide a high-quality "shell" material for the subsequent coating process.
[0013] The polysaccharide solution was prepared in 0.01 M PBS buffer at pH 6.0 to ensure that the carboxyl groups on the low-methoxyl pectin and sodium alginate molecular chains were fully dissociated, so that the resulting shell solution carried a strong negative charge.
[0014] Step 4: The positively charged peanut protein isolate-selenomethionine core solution is slowly added dropwise at a constant rate to the negatively charged low-methoxyl pectin-sodium alginate shell solution under vigorous magnetic stirring. This step utilizes the electrostatic attraction between polyelectrolytes, the principle of "opposites attract," to drive the spontaneous binding of the protein core and polysaccharide shell, forming a regular core-shell structure. Precise control of the dropping rate and stirring intensity is key to avoiding uncontrolled particle aggregation and obtaining a uniformly dispersed nanoscale system.
[0015] Step 5: The formed nanoparticle suspension was further matured by magnetic stirring under mild conditions for 1 h to promote the formation of secondary forces such as hydrogen bonds between protein and polysaccharide molecules, thereby further enhancing the structural stability of the complex. Subsequently, the purified final product was obtained by centrifugation at 3000 rpm for 15 min and the supernatant was collected.
[0016] Step 6: Collect the purified composite nanoparticles. If long-term storage or subsequent structural characterization is required, freeze-dry them to obtain easily stored composite nanoparticle powder.
[0017] Furthermore, in the above preparation method, in step 1), the mass ratio of peanut protein isolate to selenomethionine is 25:1.
[0018] Furthermore, in the above preparation method, step 1), the conditions for the coordination reaction are: the coordination reaction is carried out under magnetic stirring at 25°C for 4 h.
[0019] Furthermore, in the above preparation method, in step 1), the dialysis uses a 10000 Da dialysis bag, and the dialysis time is 56 h.
[0020] Furthermore, in the above preparation method, in step 2), the mass ratio of the amount of low-methoxyl pectin to the amount of sodium alginate is 1:1.
[0021] Furthermore, in the above preparation method, in step 3), the volume ratio of peanut protein isolate-selenomethionine kernel solution to low-methoxyl pectin-sodium alginate shell solution is 1:5, 1:4, 1:3, 1:2 or 1:1.
[0022] Preferably, in step 3), the volume ratio of peanut protein isolate-selenomethionine kernel solution to low-methoxyl pectin-sodium alginate shell solution is 1:3.
[0023] Furthermore, in the above preparation method, step 3), the constant rate is 1.5 mL / min.
[0024] The beneficial effects of this invention are:
[0025] 1. Excellent stability and biocompatibility: By constructing a unique core-shell structure, the composite polysaccharide shell provides strong electrostatic and steric hindrance protection for the inner peanut protein isolate-selenomethionine core, which makes the prepared nanoparticles have extremely high dispersion stability in the aqueous phase and can effectively resist aggregation and precipitation caused by environmental changes.
[0026] 2. Highly efficient nutrient delivery capability: The nanoparticles prepared by this invention can efficiently encapsulate selenomethionine in the core, and the polysaccharide shell can protect it from damage by gastric acid and other environments, which is expected to achieve targeted and controllable release in the intestinal environment, thereby significantly improving the bioavailability of selenium.
[0027] 3. Wide range of applications: These selenium-loaded nanoparticles can be used as a novel and efficient functional food ingredient or stabilizer in various liquid or solid foods, such as plant protein drinks, yogurt, and nutrition bars, to develop selenium-enriched functional foods, and have broad market prospects.
[0028] 4. The preparation method is mild and controllable, with industrialization potential: The preparation method adopted in this invention is carried out under mild physicochemical conditions throughout, avoiding the use of organic solvents and the damage caused by extreme conditions, thus maximizing the preservation of the natural activity of various biomolecules. The process flow is clear, the parameters are controllable, and the repeatability is good, providing a solid theoretical foundation and technical support for the large-scale production and practical application of this functional nanoparticle. Attached Figure Description
[0029] Figure 1 This is a process flow diagram of the preparation process of peanut protein polysaccharide composite nanoparticles loaded with selenomethionine provided by the present invention.
[0030] Figure 2 It is PDI of peanut protein polysaccharide composite nanoparticles loaded with selenomethionine in different raw material ratios.
[0031] Figure 3The Z-average particle size is that of peanut protein polysaccharide composite nanoparticles loaded with selenomethionine in different raw material ratios.
[0032] Figure 4 The zeta potentials are obtained from peanut protein polysaccharide composite nanoparticles loaded with selenomethionine in different raw material ratios. Detailed Implementation
[0033] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.
[0034] It should be understood that terms such as “having,” “comprising,” and “including” as used in this invention do not imply the presence or addition of one or more other elements or combinations thereof.
[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0036] Example 1
[0037] (a) Preparation
[0038] like Figure 1 As shown in the figure, this embodiment provides a method for preparing peanut protein polysaccharide composite nanoparticles loaded with selenomethionine, which mainly includes the following steps:
[0039] 1) First, accurately weigh 250 mg of peanut protein isolate and 10 mg of selenomethionine. Hydrate the peanut protein isolate and selenomethionine separately in 0.01 M PBS buffer (pH 5.0) to form homogeneous protein and selenomethionine solutions. Then, under gentle stirring, slowly add the selenomethionine solution dropwise to the protein solution and perform a coordination reaction at 25°C with magnetic stirring for 4 h. Finally, purify the solution by dialyzing through a 10000 Da dialysis bag for 56 h to obtain a positively charged peanut protein isolate-selenomethionine core solution.
[0040] 2) Accurately weigh 100 mg of low-methoxyl pectin and 100 mg of sodium alginate and dissolve them in 0.01 M PBS buffer at pH 6.0. Mix the solutions by magnetic stirring for 1 h and then sonicate them in a 300 W bath for 3 min to obtain a clear, homogeneous, negatively charged low-methoxyl pectin-sodium alginate shell solution.
[0041] 3) Under vigorous magnetic stirring, the peanut protein isolate-selenomethionine kernel solution obtained in step 1) was slowly added dropwise to the low methoxy pectin-sodium alginate shell solution obtained in step 2) at a rate of 1.5 mL / min in ratios of 1:5, 1:4, 1:3, 1:2, and 1:1, respectively.
[0042] 4) After the addition is complete, continue magnetic stirring for 1 hour to mature the particles and make the particle structure more stable. Collect the supernatant by centrifugation at 3000 rpm for 15 minutes to obtain the final peanut protein polysaccharide composite nanoparticles loaded with selenomethionine.
[0043] (II) Performance Measurement
[0044] Determination of PDI of nanoparticles: Suspensions of composite nanoparticles with different raw material ratios were diluted to suitable concentrations with 0.01 M PBS buffer. The PDI values of each group of samples were determined using a laser particle size analyzer under constant temperature conditions of 25℃ via dynamic light scattering. Each sample was measured in triplicate, and the average value was taken.
[0045] The results are as follows Figure 2 The study found that the PDI value was highest when the raw material ratio was 1:5. This is likely because the polysaccharide concentration was relatively high, and excessive polysaccharide molecules in the solution may form independent micelles or become entangled. Simultaneously, some polysaccharides also coated the peanut protein isolate-selenomethionine core, resulting in a diverse particle type, wide particle size distribution, and poor uniformity throughout the system. When the raw material ratio was 1:3, the PDI value reached its lowest point. This indicates that the peanut protein isolate-selenomethionine core and the low-methoxyl pectin-sodium alginate shell reached their optimal interaction state. Polysaccharide molecules could efficiently and uniformly coat the protein core surface, forming dense, uniformly sized core-shell nanoparticles. As the polysaccharide ratio decreased, the nanoparticles might not completely cover the protein core due to insufficient coating, leading to partial particle exposure or bridging aggregation, thus causing the PDI value to rise again.
[0046] Determination of Z-mean particle size of nanoparticles: Suspensions of composite nanoparticles with different raw material ratios were diluted to suitable concentrations with 0.01 M PBS buffer. The Z-mean particle size of each group of samples was determined using a laser particle size analyzer under constant temperature conditions of 25℃ via dynamic light scattering. Each sample was measured in triplicate, and the average value was taken.
[0047] The results are as follows Figure 3When the raw material ratio is 1:5, i.e., when the polysaccharide coating is relatively excessive, the Z-mean particle size of the system is the largest. This may be because, while the excess long-chain polysaccharide molecules coat the core particles, their excess chain segments connect multiple core particles together through "bridging flocculation," forming larger aggregates, thus resulting in a larger measured average particle size. When the raw material ratio is 1:3, the Z-mean particle size decreases sharply, indicating that as the amount of polysaccharide decreases, the bridging flocculation phenomenon weakens, and the polysaccharide molecules tend to coat individual peanut protein isolate-selenomethionine cores, forming more compact and independent core-shell structured particles. At a ratio of 1:3, the ratio of the two reaches its optimal state, resulting in the smallest nanoparticle size. When the raw material ratio is further adjusted from 1:3 to 1:2, and then to 1:1, i.e., when the polysaccharide coating is relatively insufficient, the Z-mean particle size begins to increase again. This may be because the amount of polysaccharide is insufficient to completely cover all PPI / Se core surfaces, resulting in some protein surfaces being exposed. These incompletely coated particles aggregate due to weakened hydrophobic interactions or electrostatic attraction, which also leads to an increase in average particle size.
[0048] Determination of Zeta potential of nanoparticles: Suspensions of composite nanoparticles with different raw material ratios were diluted to suitable concentrations with 0.01 M PBS buffer. The Zeta potential values of each group of samples were measured using a laser particle size analyzer under constant temperature conditions of 25℃ via laser Doppler velocimetry. Each sample was measured in triplicate, and the average value was taken.
[0049] The results are as follows Figure 4 All five groups of raw material ratios produced composite nanoparticles exhibited negative charge, with high Zeta potential values. Statistical analysis showed no significant difference in Zeta potential values among the groups. This result indicates that the method of this invention can stably prepare nanoparticles with high electrostatic stability within a raw material ratio range of 1:5 to 1:1. Combining this Zeta potential result with the aforementioned results regarding Z-particle size and PDI shows that although the system maintains charge stability over a wide ratio range, the optimal physical morphology is unique. Only at a raw material ratio of 1:3 do the nanoparticles simultaneously exhibit the smallest Z-average particle size and the lowest PDI value.
[0050] Determination of selenium encapsulation efficiency in nanoparticles: A suspension of composite nanoparticles prepared at a raw material ratio of 1:3 was divided into two parts: one part was used as the total selenium sample for subsequent analysis; the other part was used to separate the free selenium sample. The sample used for free selenium separation was placed in a 10000 Da ultrafiltration centrifuge tube and centrifuged in a high-speed refrigerated centrifuge (5000 g, 4 ℃, 30 min). The filtrate passing through the ultrafiltration membrane was collected, representing the fraction containing free selenium. The total selenium sample and the free selenium sample were pretreated separately, and then the selenium concentration of both was accurately determined by inductively coupled plasma mass spectrometry (ICP-MS).
[0051]
[0052] Table 1 Selenium content of composite nanoparticles
[0053] Sample Name Content (μg / L) RSD / % Total selenium 14.67±0.09 0.64 Free selenium 4.27±0.05 1.37
[0054] The results are shown in Table 1, and the precision of the determination method was evaluated. The relative standard deviation (RSD) for the total selenium content determination was 0.64%, and the RSD for the free selenium content determination was 1.37%. Both RSD values were far below the threshold required for validation by conventional methods, indicating that the inductively coupled plasma mass spectrometry method used in this determination has high precision and the results are accurate and reliable. The results of this embodiment clearly demonstrate that, under the optimal process conditions determined in this invention, the prepared composite nanoparticles have a highly efficient encapsulation ability for the active substance selenomethionine, with an encapsulation rate reaching 70.89%. This result verifies that the ideal physical structure observed in the aforementioned embodiments can indeed be transformed into excellent functional properties. From the perspective of functional realization, it reaffirms the advancement and effectiveness of the 1:3 raw material ratio as the core technical solution of this invention, proving that this invention can be used to prepare nanodelivery systems with high drug loading.
Claims
1. A method for preparing peanut protein polysaccharide composite nanoparticles loaded with selenomethionine, characterized in that, Includes the following steps: 1) Peanut protein isolate and selenomethionine were fully hydrated in PBS buffer, and the mixture was magnetically stirred to carry out the coordination reaction. Finally, the mixture was purified by dialysis through a 10000 Da dialysis bag to obtain a positively charged peanut protein isolate-selenomethionine core solution. 2) Dissolve low-methoxyl pectin and sodium alginate in PBS buffer in sequence, stir magnetically for 1 h, and finally sonicate in a 300 W bath for 3 min to obtain a clear, homogeneous, negatively charged low-methoxyl pectin-sodium alginate shell solution. 3) Under vigorous magnetic stirring, the peanut protein isolate-selenomethionine kernel solution obtained in step 1) is slowly added dropwise at a constant rate to the low methoxy pectin-sodium alginate shell solution obtained in step 2); 4) After the addition is complete, continue magnetic stirring for 1 hour to mix and make the particle structure more stable. Collect the supernatant by centrifugation at 3000 rpm for 15 minutes to obtain the final peanut protein polysaccharide composite nanoparticles loaded with selenomethionine.
2. The preparation method according to claim 1, characterized in that, In step 1), the mass ratio of peanut protein isolate to selenomethionine is 25:
1.
3. The preparation method according to claim 1, characterized in that, In step 1), the PBS buffer is a 0.01 M PBS buffer with pH 5.
0.
4. The preparation method according to claim 1, characterized in that, In step 1), the conditions for the coordination reaction are: the coordination reaction is carried out at 25°C for 4 h.
5. The preparation method according to claim 1, characterized in that, In step 1), the dialysis time is 56 h.
6. The preparation method according to claim 1, characterized in that, In step 2), the mass ratio of the amount of low-methoxyl pectin to the amount of sodium alginate is 1:
1.
7. The preparation method according to claim 1, characterized in that, In step 2), the PBS buffer is a 0.01 M PBS buffer with pH 6.
0.
8. The preparation method according to claim 1, characterized in that, In step 3), the volume ratio of peanut protein isolate-selenomethionine kernel solution to low-methoxyl pectin-sodium alginate shell solution is 1:5, 1:4, 1:3, 1:2 or 1:
1.
9. The preparation method according to claim 8, characterized in that, In step 3), the volume ratio of peanut protein isolate-selenomethionine kernel solution to low-methoxyl pectin-sodium alginate shell solution is 1:
3.
10. The preparation method according to claim 1, characterized in that, In step 3), the constant rate is 1.5 mL / min.