Polysaccharide substance encapsulated resveratrol compound as well as preparation method and application thereof

Abstract

The invention relates to a polysaccharide substance encapsulated resveratrol compound and a preparation method and application thereof.The compound comprises resveratrol and a polysaccharide substance encapsulating the resveratrol, the polysaccharide substance is linear polysaccharide, and the resveratrol can induce polysaccharide chains to expand so that the resveratrol and the polysaccharide chains can be preliminarily combined through hydrogen-bond interaction; the preparation method comprises the following steps: by taking resveratrol as a raw material, and reacting under the condition that the pH value is 6.0-9.0, double effects of carrier (polysaccharide substance) self-enhancement and object (resveratrol) stabilization are realized, the antioxidant activity of polysaccharide is enhanced through nanocrystallization modification, and the retention rate and the stability of resveratrol are improved through encapsulation.

Description

Technical Field

[0001] This invention relates to the field of encapsulation complex technology, specifically to a polysaccharide-encapsulated resveratrol complex, its preparation method, and its application. Background Technology

[0002] Hydrophobic bioactive substances (such as resveratrol) have poor water solubility and weak environmental stability (they are easily degraded by heat, light and ions), which limits their application in food.

[0003] To address the aforementioned issues, Chinese invention patent application CN201910556255.9 (publication number CN112120945 A) discloses a "Water-in-Water Composition with High Resveratrol Content," which comprises: an internal phase containing a polyol polymer with a molecular weight range of 200 to 600 g / mol and resveratrol, wherein the weight ratio of the polyol polymer to resveratrol is equal to or greater than 10:1, and the content of the polyol polymer is less than 55% by weight and the content of resveratrol is up to 5% by weight based on the total weight of the composition; and an external phase containing a natural high molecular weight polysaccharide polymer with a molecular weight range of 2×10⁵ to 5×10⁵ g / mol, and the content of the natural high molecular weight polysaccharide polymer is 1% to 10% by weight based on the total weight of the composition. This patent improves the stability and water solubility of resveratrol through the polyol polymer and the high molecular weight polysaccharide polymer.

[0004] The outer phase (natural high molecular weight polysaccharide polymer) in this patent is only used as a "protection / delivery tool" to improve the solubility, stability or bioavailability of resveratrol, without enhancing the function of the outer phase itself. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a polysaccharide-encapsulated resveratrol complex in light of the current state of the art. The polysaccharide can not only improve the performance of resveratrol, but also improve the performance of the polysaccharide itself in the process.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned polysaccharide-encapsulated resveratrol complex, in view of the current state of the prior art.

[0007] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned polysaccharide material encapsulating resveratrol complex, in light of the current state of the prior art.

[0008] The technical solution adopted by the present invention to solve the first technical problem mentioned above is: a polysaccharide-encapsulated resveratrol complex, characterized in that: it includes resveratrol and a polysaccharide encapsulating resveratrol, wherein the polysaccharide is a linear polysaccharide, and resveratrol can induce the polysaccharide chain to unfold so that the two are initially combined through hydrogen bonding, and then reacted under pH 6.0-9.0 conditions.

[0009] Preferably, the linear polysaccharide is extracted from Dendrobium officinale. As a medicinal and edible raw material, Dendrobium officinale's polysaccharide (DOP) has excellent antioxidant, immunomodulatory and other biological activities, and is thermally stable and has good film-forming properties, making it a potential natural bioactive substance encapsulation carrier.

[0010] The complex of this invention has good biocompatibility, and the polysaccharides are derived from natural medicinal and edible raw materials, with no toxic side effects and high biological safety, making it suitable for the field of functional foods.

[0011] Preferably, the linear polysaccharide is at least one of the following: stem polysaccharide extracted from Dendrobium officinale stems, leaf polysaccharide extracted from Dendrobium officinale leaves, and flower polysaccharide extracted from Dendrobium officinale flowers.

[0012] This method achieves high utilization of Dendrobium officinale polysaccharides, reduces resource waste, and incorporates Dendrobium officinale leaf and flower by-products into polysaccharide extraction raw materials. Stem polysaccharides (DOSP), leaf polysaccharides (DOLP), and flower polysaccharides (DOFP) all have good free radical scavenging capabilities and have great potential for developing antioxidant functional foods, enabling comprehensive utilization of the entire plant resource.

[0013] Among them, DOFP (flower polysaccharide) has the best encapsulation stability due to its high galacturonic acid content and branched structure, which provides more hydrogen bond sites and forms more hydrogen bonds with resveratrol.

[0014] In the above scheme, the method for extracting the linear polysaccharide is as follows:

[0015] (1) Raw material pretreatment: The stems, leaves and flowers of Dendrobium officinale are dried to constant weight, crushed and sieved to obtain stem powder, leaf powder and flower powder. The stem powder, leaf powder and flower powder are collectively referred to as powder. The treatment methods of each powder are as follows.

[0016] (2) Pigment removal: Add ethanol solution to the powder and soak in the dark until the solution color changes from light pink to colorless. Then, separate the solid and liquid and collect the solid precipitate.

[0017] (3) Extraction: Add deionized water and compound enzyme to the solid precipitate from step (2), and heat in a constant temperature water bath at 55-60 ℃ for 65-70 min. Then, separate the solid and liquid and collect the supernatant. The mass ratio of solid precipitate to deionized water is 1:10-1:20, and the addition ratio of compound enzyme to solid precipitate is 300-1500 U / g. The compound enzyme is composed of pectinase, cellulase and papain. The addition ratio of pectinase to solid precipitate is 100-500 U / g, the addition ratio of cellulase to solid precipitate is 100-500 U / g, and the addition ratio of papain to solid precipitate is 100-500 U / g.

[0018] (4) Alcohol precipitation and aging: Add anhydrous ethanol to the supernatant of step (3), let it stand at 2-4 ℃ until no more solid precipitate is formed in the anhydrous ethanol, and then stop. The solid and liquid are separated to obtain the precipitate. The precipitate is freeze-dried to obtain powdered polysaccharide.

[0019] The technical solution adopted by the present invention to solve the second technical problem mentioned above is: a method for preparing a polysaccharide-encapsulated resveratrol complex according to claim 1, characterized in that it includes the following steps:

[0020] Step 1: Dissolve polysaccharides in deionized water to prepare a polysaccharide stock solution with a concentration of 1-5 mg / mL; dissolve resveratrol in ethanol solution to prepare a resveratrol stock solution with a concentration of 1.00-1.50 mg / mL.

[0021] Step 2: Under stirring conditions, the resveratrol stock solution is added dropwise to the polysaccharide stock solution at a volume ratio of 1:10-1:12. During this process, resveratrol induces the polysaccharide chain to unfold, so that the two are initially combined through hydrogen bonding. Then, the pH of the system is adjusted to 6.0-9.0, and the mixture is stirred at 35-38 °C for 1-1.5 h to form a mixture.

[0022] Step 3: Perform solid-liquid separation on the mixture. The resulting clear liquid is a polysaccharide-encapsulated resveratrol complex solution, which is further freeze-dried to obtain solid nanoparticles.

[0023] The technical solution adopted by the present invention to solve the third technical problem mentioned above is: an application of the above-mentioned polysaccharide-encapsulated resveratrol complex, characterized in that: the polysaccharide-encapsulated resveratrol complex can be consumed as an antioxidant functional food.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] (1) The core objective of this invention is to achieve the dual effects of "carrier (polysaccharide) self-enhancement + guest (resveratrol) stabilization". It enhances the antioxidant activity of the polysaccharide itself through nano-modification and improves the retention rate and stability of resveratrol through encapsulation, breaking through the limitation of "single guest protection" in existing patents. The specific mechanism is as follows:

[0026] Polysaccharide self-enhancing mechanism: Without resveratrol, polysaccharides exist in a linear aggregated state (particle size > 350 nm), and their active groups (hydroxyl groups) are encapsulated, limiting their antioxidant activity. The addition of resveratrol acts as a "molecular bridge," inducing the dispersion and folding of polysaccharide chains through hydrogen bonds, forming nanoparticles (particle size < 300 nm) under pH induction, exposing more active hydroxyl groups. At the same time, the synergistic effect of polysaccharides and resveratrol (superposition of free radical scavenging sites) makes the antioxidant activity of the complex significantly higher than that of pure polysaccharides and free resveratrol (DPPH / ABTS scavenging rate > 60%). In other words, the "resveratrol-triggered-pH-induced synergistic nano-sizing" technology is adopted: using polysaccharides as raw materials, resveratrol (polyphenol) is first added, and its phenolic hydroxyl groups form preliminary hydrogen bonds with the polysaccharide hydroxyl groups. Then, the pH of the system is adjusted to between 6 and 9 to trigger the conformational change of the polysaccharide molecular chain (from linear chain extension to folded cross-linking). Nanoparticles are formed through non-covalent interactions (mainly hydrogen bonds), and finally a composite system of "polysaccharide nano-sizing self-reinforcement + resveratrol stable encapsulation" is obtained.

[0027] The stabilization mechanism of resveratrol: The nano-sized polysaccharides form a dense network structure, encapsulating resveratrol inside and building a physical barrier; at the same time, the high thermal stability of the polysaccharides (degradation temperature > 300℃) and the pH-induced cross-linking structure effectively resist the damage of resveratrol to heat, ultraviolet rays, ion changes and storage process. The resveratrol encapsulation rate is > 89.65%, which solves the pain points of low encapsulation rate and insufficient stability of existing technologies.

[0028] As can be seen from the above, this invention achieves a two-way synergy of "guest activating carrier + carrier protecting guest": the phenolic hydroxyl groups of resveratrol form hydrogen bonds with the hydroxyl groups of polysaccharides, which not only induces the nano-sizing of polysaccharides to enhance their antioxidant activity (DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) / ABTS (2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid)) scavenging rate >60%, which is better than pure polysaccharides), but also significantly improves the tolerance of resveratrol to thermal stress (retention rate >75% after treatment at 60~80 ℃), ultraviolet irradiation (retention rate >75%), ion changes (small particle size fluctuation after treatment with 5~20mM NaCl) and storage conditions (retention rate >84% after 7 days of storage at 4 ℃), thus solving the problem of easy degradation of resveratrol.

[0029] The preparation process of this invention is simple, using a pH-driven method, requiring no complex equipment, with mild reaction conditions (37 ℃, pH 8.0), low production cost, and suitable for industrial production. Attached Figure Description

[0030] Figure 1 The particle size (Figure A) and zeta potential (Figure B) of the complex in Example 1 of the present invention are shown.

[0031] Figure 2 The XRD pattern of the complex in Example 1 of this invention;

[0032] Figure A shows the XRD pattern of DOSP-Res; Figure B shows the XRD pattern of DOLP-Res; and Figure C shows the XRD pattern of DOFP-Res.

[0033] Figure 3 This is a scanning electron microscope image of the complex in Example 1 of the present invention;

[0034] DOSP, DOLP, and DOFP are the stem polysaccharide, leaf polysaccharide, and flower polysaccharide of Dendrobium officinale, respectively.

[0035] DOSP-Res, DOLP-Res, and DOFP-Res are complexes formed by stem polysaccharides, leaf polysaccharides, and flower polysaccharides of Dendrobium officinale and resveratrol, respectively.

[0036] In the first column, the resveratrol stock solution concentration corresponding to DOSP, DOLP, and DOFP is 0;

[0037] In the second column, the resveratrol stock solution concentration corresponding to DOSP-Res1, DOLP-Res1, and DOFP-Res1 is 1.00.

[0038] In the third column, the resveratrol stock solution concentration corresponding to DOSP-Res2, DOLP-Res2, and DOFP-Res2 is 1.25.

[0039] In the fourth column, the resveratrol stock solution concentration corresponding to DOSP-Res3, DOLP-Res3, and DOFP-Res3 is 1.50.

[0040] Figure 4 The retention rate of resveratrol in the heat-treated composite in Example 1 of this invention;

[0041] Figure 5 The retention rate of resveratrol in the UV-treated composite in Example 1 of this invention;

[0042] Figure 6 The particle size of the NaCl-treated complex in Example 1 of this invention;

[0043] Figure 7 The particle size (Figure A) and resveratrol retention rate (Figure B) of the complex in Example 1 of the present invention after storage at 4°C for 7 days are shown.

[0044] Figure 8 The figures show the DPPH radical scavenging rate (Figure A), ABTS radical scavenging rate (Figure B), and total reducing power (Figure C) of the complex in Example 1 of this invention.

[0045] Figure 9 The scavenging rates of ABTS radicals (Figure A) and DPPH radicals (Figure B) and the total reducing power (Figure C) of Dendrobium officinale polysaccharides (including stem polysaccharides, leaf polysaccharides and flower polysaccharides).

[0046] Figure 10 The effects of polysaccharide concentration (Figure A), resveratrol concentration (Figure B), and pH (Figure C) on nanoparticle size;

[0047] Figure 11 The effect of different amounts of compound enzyme added on the concentration of extracted linear polysaccharides;

[0048] Figure 12 The effect of different material-to-liquid ratios on the concentration of extracted linear polysaccharides. Detailed Implementation

[0049] Unless otherwise specified, all reagents, materials, and equipment used in the examples are commercially available.

[0050] Example 1

[0051] The polysaccharide-encapsulated resveratrol (Res) complex in this embodiment includes resveratrol and a polysaccharide encapsulating the resveratrol. The polysaccharide is a linear polysaccharide extracted from Dendrobium officinale, and can be at least one of the following: stem polysaccharide extracted from Dendrobium officinale stems, leaf polysaccharide extracted from Dendrobium officinale leaves, and flower polysaccharide extracted from Dendrobium officinale flowers. In this embodiment, stem polysaccharide, leaf polysaccharide, and flower polysaccharide were all extracted.

[0052] The extraction method for linear polysaccharides is as follows:

[0053] (1) Raw material pretreatment: The stems, leaves and flowers of Dendrobium officinale were dried at 60℃ to constant weight, pulverized and passed through a 0.25 mm sieve to obtain stem powder, leaf powder and flower powder respectively. The stem powder, leaf powder and flower powder are collectively referred to as powder. The treatment methods of each powder are as follows to obtain stem polysaccharide, leaf polysaccharide and flower polysaccharide respectively.

[0054] (2) Pigment removal: Add 150 mL of 80% ethanol solution to 10 g of powder and soak in the dark for 12 h. The solution color changes from light pink to colorless. Then, after solid-liquid separation, collect the solid precipitate. The solid-liquid separation method in this embodiment is to centrifuge at 5000 r / min for 10 min.

[0055] (3) Extraction: Add 150 mL of deionized water and 9000 U of compound enzyme to the solid precipitate from step (2). The compound enzyme consists of 3000 U of pectinase, 3000 U of cellulase and 3000 U of papain. Incubate in a constant temperature water bath at 58 °C and 100 r / min for 66 min. Then, separate the solid and liquid (by centrifugation) and collect the supernatant. Multiple extractions can be performed as needed. The supernatants from multiple extractions are combined.

[0056] (4) Alcohol precipitation and aging: Add three times the volume of anhydrous ethanol to the supernatant of step (3), let stand at 4 ℃ for 12 h, and no more solid precipitate is generated in the anhydrous ethanol. Separate the solid and liquid (filter) to obtain the precipitate, freeze dry at -50 ℃ for 24 h to obtain powdered linear polysaccharide.

[0057] Following the extraction method described above, 2.112 g of stem polysaccharide (DOSP) powder (extraction rate 21.12%) was finally obtained; 2.309 g of leaf polysaccharide (DOLP) powder (extraction rate 23.09%) and 1.377 g of flower polysaccharide (DOFP) powder (extraction rate 13.77%) were obtained.

[0058] The extraction rates, compositions, and molecular weights of stem polysaccharide powder, leaf polysaccharide powder, and flower polysaccharide powder are shown in Table 1 below.

[0059] Table 1

[0060]

[0061] Note: Abbreviations: Man, mannose; Glc, glucose; Gal, galactose; Ara, arabinose; Xyl, xylose; Rha, rhamnose; Gal-UA, galacturonic acid; Fuc, fuc; Mw: weight-average molecular weight. ND: not determined. Different lowercase letters (ac) in the same row indicate significant differences between data (p < 0.05).

[0062] like Figure 9As shown, the stem polysaccharide, leaf polysaccharide, and flower polysaccharide solutions prepared in this invention all exhibit high DPPH / ABTS free radical scavenging rates and strong total reducing power, indicating that *Dendrobium officinale* polysaccharides possess excellent antioxidant capabilities. The differences in antioxidant activity among *Dendrobium officinale* polysaccharides from different sources are related to their different monosaccharide compositions. In particular, the presence of abundant flavonoid active substances and galacturonic acid groups in *Dendrobium officinale* flower polysaccharide (DOFP) contributes to its outstanding antioxidant activity.

[0063] Example 2

[0064] The stem polysaccharide powder, leaf polysaccharide powder, and flower polysaccharide powder prepared in Example 1 were reacted with resveratrol to prepare a polysaccharide-encapsulated resveratrol complex. This example included multiple parallel experiments, and the methods of each experiment included the following steps:

[0065] Step 1: Dissolve 0.04 g of polysaccharide powder in 10 mL of deionized water to prepare a polysaccharide stock solution with a concentration of 4 mg / mL; dissolve the set amount of resveratrol in 10 mL of 80% ethanol to prepare a resveratrol stock solution with a set concentration.

[0066] Step 2: Under stirring conditions of 300 r / min, 1 mL of resveratrol stock solution was added dropwise to 10 mL of each polysaccharide stock solution to induce polysaccharide chain expansion. The two were initially combined through hydrogen bonding. Then, the pH of the mixture was adjusted to 8.0 with 0.1 mol / L NaOH and the mixture was sealed and magnetically stirred at 37 ℃ for 1 h to form a mixture.

[0067] Step 3: Centrifuge the mixture at 10000 r / min for 15 min to separate the solid and liquid components, discard the precipitate, and the resulting clear liquid is the Dendrobium officinale polysaccharide-based (DOPs-Res) composite nanoparticle solution, which is a polysaccharide-encapsulated resveratrol complex solution. Further freeze-dry to obtain solid nanoparticles.

[0068] Following the above method, 0.01, 0.0125, and 0.015 g of resveratrol were dissolved in 10 mL of 80% ethanol to prepare resveratrol stock solutions with concentrations of 1.00, 1.25, and 1.50 mg / mL, respectively. These solutions were then reacted with stem polysaccharide stock solutions. The resveratrol encapsulation efficiency of the complexes (DOSP-Res) prepared with different concentrations of resveratrol is shown in the second row of Table 2.

[0069] 0.01, 0.0125, and 0.015 g of resveratrol were dissolved in 10 mL of 80% ethanol to prepare resveratrol stock solutions with concentrations of 1.00, 1.25, and 1.50 mg / mL, respectively. These solutions were then reacted with leaf polysaccharide stock solutions. The resveratrol encapsulation efficiency of the complexes (DOLP-Res) prepared with different concentrations of resveratrol is shown in the third row of Table 2.

[0070] 0.01, 0.0125, and 0.015 g of resveratrol were dissolved in 10 mL of 80% ethanol to prepare resveratrol stock solutions with concentrations of 1.00, 1.25, and 1.50 mg / mL, respectively. These solutions were then reacted with flower polysaccharide stock solutions. The resveratrol encapsulation efficiency of the complexes (DOFP-Res) prepared with different concentrations of resveratrol is shown in the fourth row of Table 2.

[0071] Pure polysaccharide powder was used as a control group.

[0072] Table 2. Resveratrol encapsulation efficiency of complexes prepared with different concentrations of resveratrol:

[0073]

[0074] Note: The resveratrol concentrations in the prepared samples were 1.00, 1.25, and 1.50 mg / mL, labeled as 1, 2, and 3, respectively. Different lowercase letters (ab) in the same row indicate significant differences between data (p < 0.05), while ab indicates no difference between the data and the corresponding data of a and b.

[0075] like Figure 1 As shown in Table 2, the composites (DOPs-Res nanoparticles) prepared by the pH-driven method of this invention all exhibit small particle sizes: less than 300 nm, and have high Res encapsulation efficiency: greater than 89.65%.

[0076] like Figure 2 As shown, no Res characteristic crystal peaks were observed in the DOPs-Res nanoparticles prepared in this invention, indicating that the polysaccharide has successfully encapsulated resveratrol inside, and the nanoparticle structure has been formed.

[0077] like Figure 3As shown, the three polysaccharides DOPs in this invention all exhibit a dendritic structure and are linear polysaccharides. When a low concentration of Res is added to form composite particles, each DOPs-Res sample transforms from rod-shaped to spherical particles, and the size decreases significantly. After the addition of a high concentration of Res, the DOFP-Res particles, along with the other two particles (DOSP-Res and DOLP-Res), no longer exhibit a single spherical structure. Instead, they form a dense multilayer network structure due to cross-linking between particles. This may be because the composite particles in this invention are prepared under pH 8 conditions. This alkaline condition may alter the conformation of the polysaccharide chains, causing them to extend and expose more uronic acid and hydroxyl sites, increasing the chance of hydrogen bonding with the phenolic hydroxyl groups of Res, thus leading to the formation of a denser structure. No crystalline structure was observed on the surface of the nanoparticles prepared at different Res concentrations, indicating that resveratrol has been embedded within the polysaccharide chain structure.

[0078] like Figure 4-7 As shown, in this invention, after being placed at 60, 70, and 80°C for 1 h, the retention rates of resveratrol were 39.91%, 38.37%, and 33.98%, respectively, with a loss rate exceeding 60%, demonstrating the poor thermal stability of resveratrol. However, after encapsulating it within Dendrobium officinale polysaccharides to form composite nanoparticles, the retention rate of resveratrol in each DOPs-Res particle exceeded 40%. Among them, the DOFP-Res particles showed better protection for resveratrol, maintaining a retention rate of over 75% even after being subjected to different high-temperature treatments.

[0079] like Figure 5 As shown, the DOFP-Res particles in this invention retain at least 75.24% of Res after being irradiated with ultraviolet light, which is significantly better than DOSP-Res (42.47%) and DOLP-Res (45.24%).

[0080] like Figure 6 As shown, in this invention, the particle size of DOFP-Res particles decreased with increasing sodium ion treatment concentration, while the particle size of DOSP-Res and DOLP-Res particles showed an increasing trend, indicating that DOFP-Res particles have stronger stability. After 7 days of storage, the particle size of DOSP-Res particles increased the most, ranging from 62.70 to 85.13 nm; the particle size of DOLP-Res particles increased the second most, ranging from 36.27 to 55.40 nm; while the particle size of DOFP-Res particles was the smallest, ranging from only 10.47 to 38.87 nm. This indicates that the nanoparticles prepared from Dendrobium officinale flower polysaccharide DOFP have the best storage stability.

[0081] like Figure 8As shown in Figure A, the DPPH free radical scavenging rate of aqueous solutions of Res at different concentrations was only 7.83%-10.50%. This is because Res has low solubility in the aqueous phase, making it difficult for it to fully exert its biological activity. However, different Dendrobium officinale polysaccharides, DOSP, DOLP, and DOFP, all exhibited better DPPH free radical scavenging ability, with scavenging rates of 21.70%, 32.85%, and 55.26%, respectively. This indicates that, unlike common polysaccharides (such as pectin, carrageenan, and sodium alginate, which have almost no free radical scavenging ability), Dendrobium officinale polysaccharides possess a large number of flavonoid active substances and galacturonic acid groups, which endow them with good antioxidant activity.

[0082] Compared with single polysaccharide DOPs and free Res, the nano-sized DOPs-Res particles exhibited higher DPPH free radical scavenging rates, all exceeding 60%. This invention achieves pH-induced nano-sizing of Dendrobium officinale polysaccharides triggered by resveratrol, simultaneously enhancing polysaccharide self-activation and stabilizing resveratrol encapsulation.

[0083] The composite nanoparticles of this invention possess both "self-enhanced antioxidant activity" and "stable delivery of active ingredients," and can be directly applied to functional foods, health products, and other fields. They can be used as natural antioxidants (without the need for additional active ingredients) and as active carriers to encapsulate resveratrol, thus having a wider range of applications.

[0084] The reasons for this are twofold. First, the hydrophilic polysaccharides bind to the hydrophobic Res molecules via hydrogen bonds, effectively improving the water solubility of Res and enabling it to disperse uniformly and remain stable in the aqueous phase, thereby maximizing its antioxidant activity. Second, Dendrobium officinale polysaccharides themselves possess excellent free radical scavenging capabilities, which can synergistically interact with the Res molecules encapsulated within, thus significantly enhancing the antioxidant capacity of the nanoparticles. The trends in ABTS free radical scavenging rate (Figure B) and total reducing power (Figure C) for each sample are similar to the DPPH scavenging rate results.

[0085] Comparison of granule samples prepared from polysaccharides from different sources (stems, leaves, and flowers) revealed that the DOFP-Res granules of this invention exhibited the highest DPPH radical scavenging rate, ABTS radical scavenging rate, and total reducing power, indicating their significant potential for the development of antioxidant functional foods.

[0086] Example 3

[0087] Following the method for preparing polysaccharide-encapsulated resveratrol complexes as described in Example 2, several parallel experiments were conducted in this example:

[0088] See Figure 10Table A shows the effect of different polysaccharide concentrations on the particle size of the final complex (in these experiments, the concentration of resveratrol stock solution was fixed at 1.00 mg / mL, the pH value was 8.0, and the concentrations of polysaccharide stock solution were adjusted to 1, 2, 3, 4, and 5 mg / mL, respectively, with other parameters being the same as those in the preparation method in Example 2).

[0089] See Figure 10 Table B shows the effect of different resveratrol concentrations on the particle size of the final complex (in these experiments, the concentration of the polysaccharide stock solution was fixed at 4.00 mg / mL, the pH value was 8.0, and the concentration of the resveratrol stock solution was adjusted to 0.50, 0.75, 1.00, 1.25, and 1.50 mg / mL, respectively, with other parameters being the same as those in the preparation method in Example 2).

[0090] See Figure 10 Table C shows the effect of adjusting the reaction system to different pH values ​​on the particle size of the final complex (in these experiments, the concentration of the polysaccharide stock solution was 4.00 mg / mL, the concentration of the resveratrol stock solution was 1.00 mg / mL, the pH values ​​were adjusted to 6, 7, 8, and 9, and other parameters were the same as those in the preparation method in Example 2).

[0091] from Figure 10 In the analysis, the particle size of each DOP (control) sample was greater than 350 nm, indicating that single polysaccharide molecules readily aggregate to form polymers with larger particle sizes. However, when DOP (Dendrobium officinale polysaccharide, which can be one of stem, leaf, or flower polysaccharides) was combined with Res to prepare nanoparticles, their particle sizes were significantly smaller than those of the corresponding polysaccharide solutions. This clearly demonstrates that the addition of Res effectively inhibits the aggregation behavior between polysaccharide molecules. Resveratrol, as a natural phenolic compound, is rich in phenolic hydroxyl groups in its molecular structure. It can bind to anionic polysaccharides (DOP) through electrostatic repulsion and remain stably present in the system, while also improving the water solubility of resveratrol itself. Specifically, when the polysaccharide concentration was 4.00 mg / mL, the pH was 8.0, and the resveratrol concentration was 1-1.25 mg / mL, the nanoparticles formed by the combination of each polysaccharide and resveratrol had lower particle sizes, indicating that the particles prepared under these conditions had better stability.

[0092] In addition, following the linear polysaccharide extraction method of Example 1, the applicant also conducted multiple parallel experiments with different amounts of the compound enzyme, see [link to relevant documentation]. Figure 11 As shown in the figure, different lowercase letters in "ad" indicate that the data have significant differences (p<0.05), while the same letter indicates that there are no significant differences.

[0093] from Figure 11 The results show that as the amount of compound enzyme added increases, the polysaccharide extraction rate first increases and then decreases. The polysaccharide extraction rate reaches its highest level when the amount of compound enzyme activity added is 900 U / g (i.e., the amount of each of the three enzymes is 300 U / g).

[0094] In addition, following the linear polysaccharide extraction method of Example 1, the applicant conducted multiple parallel experiments with different solid-liquid ratios (solid-liquid ratio) for the solid precipitate to deionized water in step (3), see [link to relevant documentation]. Figure 12 As shown in the figure, different lowercase letters in "ad" indicate that the data have significant differences (p<0.05), while the same letter indicates that there are no significant differences.

[0095] from Figure 12 The results show that as the material-to-liquid ratio increases, the polysaccharide extraction rate first increases and then decreases, with the highest extraction rate observed when the material-to-liquid ratio is 1:15 g / mL.

[0096] To distinguish between different groups of samples, different uppercase and lowercase letters are used. For example, in Figure 1, AB represents the difference in data between control, 1, 2, and 3 samples for DOSP-Res samples; A'-B' represents the difference in data between control, 1, 2, and 3 samples for DOLP-Res samples; and ab represents the difference in data between control, 1, 2, and 3 samples for DOFP-Res samples. Figure 4 , 6 In Figure 7, the different letters AG, A'-G', and ag represent significant differences between the data in the same color bar chart (p<0.05). Figure 8 The different letters AD, A'-D', ad, and a'-d' indicate that there are significant differences (p<0.05) between the Res, DOSP-Res, DOLP-Res, and DOFP-Res sample groups (control, 1, 2, and 3 samples).

[0097] In the above embodiments, the pectinase, cellulase and papain were manufactured by Shanghai Yuanye Biotechnology Co., Ltd., and the resveratrol was manufactured by Shanghai Mailin Biotechnology Co., Ltd.

Claims

1. A polysaccharide-encapsulated resveratrol complex, characterized in that: It includes resveratrol and polysaccharides encapsulating resveratrol, wherein the polysaccharides are linear polysaccharides. Resveratrol can induce the unfolding of polysaccharide chains, so that the two are initially combined through hydrogen bonding, and then reacted under pH 6.0-9.0 conditions.

2. The polysaccharide-encapsulated resveratrol complex according to claim 1, characterized in that: The linear polysaccharide was extracted from Dendrobium officinale.

3. The polysaccharide-encapsulated resveratrol complex according to claim 2, characterized in that: The linear polysaccharide is at least one of the following: stem polysaccharide extracted from Dendrobium officinale stem, leaf polysaccharide extracted from Dendrobium officinale leaf, and flower polysaccharide extracted from Dendrobium officinale flower.

4. The polysaccharide-encapsulated resveratrol complex according to claim 3, characterized in that: The extraction method of the linear polysaccharide is as follows: (1) Raw material pretreatment: The stems, leaves and flowers of Dendrobium officinale are dried to constant weight, crushed and sieved to obtain stem powder, leaf powder and flower powder. The stem powder, leaf powder and flower powder are collectively referred to as powder. The treatment methods of each powder are as follows. (2) Pigment removal: Add ethanol solution to the powder and soak in the dark until the solution color changes from light pink to colorless. Then, separate the solid and liquid and collect the solid precipitate. (3) Extraction: Add deionized water and compound enzyme to the solid precipitate from step (2), and heat in a constant temperature water bath at 55-60 ℃ for 65-70 min. Then, separate the solid and liquid and collect the supernatant. The mass ratio of solid precipitate to deionized water is 1:10-1:20, and the addition ratio of compound enzyme to solid precipitate is 300-1500 U / g. The compound enzyme is composed of pectinase, cellulase and papain. The addition ratio of pectinase to solid precipitate is 100-500 U / g, the addition ratio of cellulase to solid precipitate is 100-500 U / g, and the addition ratio of papain to solid precipitate is 100-500 U / g. (4) Alcohol precipitation and aging: Add anhydrous ethanol to the supernatant of step (3), let it stand at 2-4 ℃ until no more solid precipitate is formed in the anhydrous ethanol, and then stop. The solid and liquid are separated to obtain the precipitate. The precipitate is freeze-dried to obtain powdered polysaccharide.

5. A method for preparing a polysaccharide-encapsulated resveratrol complex according to any one of claims 1 to 4, characterized in that: Includes the following steps: Step 1: Dissolve polysaccharides in deionized water to prepare a polysaccharide stock solution with a concentration of 1-5 mg / mL; dissolve resveratrol in ethanol solution to prepare a resveratrol stock solution with a concentration of 1.00-1.50 mg / mL. Step 2: Under stirring conditions, the resveratrol stock solution is added dropwise to the polysaccharide stock solution at a volume ratio of 1:10-1:

12. During this process, resveratrol induces the polysaccharide chain to unfold, so that the two are initially combined through hydrogen bonding. Then, the pH of the system is adjusted to 6.0-9.0, and the mixture is stirred at 35-38 °C for 1-1.5 h to form a mixture. Step 3: Perform solid-liquid separation on the mixture. The resulting clear liquid is a polysaccharide-encapsulated resveratrol complex solution, which is further freeze-dried to obtain solid nanoparticles.

6. The application of a polysaccharide-encapsulated resveratrol complex according to any one of claims 1 to 4, characterized in that: This polysaccharide-encapsulated resveratrol complex can be consumed as an antioxidant functional food.

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