Cornus officinalis polysaccharide microcapsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of functional component embedding, and particularly relates to a dogwood polysaccharide microcapsule as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding dogwood polysaccharide into a whey protein solution, and carrying out ultrasonic emulsification to obtain an inner phase of a primary microcapsule; adding a pectin solution into a sodium alginate solution, uniformly mixing, then adding a CaCl2 solution, uniformly mixing to obtain an outer phase of a primary microcapsule, and mixing the inner phase and the outer phase in a microfluidic manner to obtain the primary microcapsule; (2) mixing yeast beta-glucan and phytosterol powder to form a complex, and dissolving the complex in absolute ethyl alcohol to obtain a complex ethanol solution; and (3) dispersing the primary microcapsules, adding the dispersed primary microcapsules into a complex ethanol solution, stirring and mixing to obtain a mixed solution, cooling the mixed solution to room temperature, standing, centrifuging, collecting precipitates, and washing and freeze-drying the precipitates to obtain the dogwood polysaccharide microcapsules. The dogwood polysaccharide disclosed by the invention has relatively high embedding rate, drug loading rate and DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging rate.

Description

Technical Field

[0001] This invention belongs to the field of functional ingredient encapsulation technology, specifically relating to a Cornus officinalis polysaccharide capsule, its preparation method, and its application. Background Technology

[0002] Bottlenecks in the application of Cornus officinalis polysaccharides (CMP): CMP possesses antioxidant, anti-inflammatory, and immunomodulatory activities, but its high water solubility, easy degradation, and susceptibility to destruction in the gastrointestinal environment lead to low bioavailability. Existing encapsulation technologies (such as monolayer microcapsules and liposomes) suffer from low encapsulation efficiency (<70%), significant burst release, and inability to achieve targeted controlled release.

[0003] Existing encapsulation technologies suffer from several limitations: First, the use of single wall materials is restrictive. Traditional wall materials (such as chitosan and gelatin) lack sufficient mechanical strength to withstand stomach acid. Second, the process is complex, requiring multiple film formations for multi-layer encapsulation, resulting in low efficiency and high cost. Third, there is a lack of intelligent design, as the active ingredients cannot be precisely released based on environmental changes (such as pH and enzymes). Therefore, there is an urgent need to develop an efficient, low-cost encapsulation method suitable for industrial production. Summary of the Invention

[0004] In view of the problems and shortcomings of the existing technology, the present invention aims to provide a Cornus officinalis polysaccharide capsule, its preparation method and application.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides a method for preparing Cornus officinalis polysaccharide microcapsules, comprising the following steps: (1) Cornus officinalis polysaccharide was added to whey protein solution and ultrasonically emulsified to obtain the inner phase of primary microcapsules; pectin solution was added to sodium alginate solution and mixed well, then CaCl2 solution was added and mixed well to obtain the outer phase of primary microcapsules; the inner phase and the outer phase were mixed by microfluidics to obtain primary microcapsules. (2) Yeast β-glucan and phytosterol powder are mixed to form a complex. The complex is dissolved in anhydrous ethanol to obtain an ethanol solution of the complex. (3) Disperse the primary microcapsules prepared in step (1) into water to obtain a dispersion; add the dispersion to the composite ethanol solution prepared in step (2) at 50-75°C, stir and mix for 30-60 min to obtain a mixture, cool the mixture to room temperature and let it stand for 1-2 h, centrifuge the mixture after standing, collect the precipitate, wash the precipitate and freeze dry to obtain Cornus officinalis polysaccharide microcapsules.

[0006] Preferably, in step (1), the mass ratio of Cornus officinalis polysaccharide to whey protein is (0.5-3.5):1.

[0007] Preferably, in step (1), the whey protein solution has a mass-volume concentration of 1% to 10%, the sodium alginate solution has a mass-volume concentration of 0.5% to 3.7%, the pectin solution has a mass-volume concentration of 0.2% to 2.5%, and the CaCl2 solution has a mass-volume concentration of 0.1% to 0.9%. More preferably, the volume ratio of whey protein solution, sodium alginate solution, pectin solution, and CaCl2 solution is (1-2):(3-4):(1-2):(1-1.5).

[0008] Preferably, the preparation process of Cornus officinalis polysaccharide in step (1) is as follows: water and compound enzyme are added to Cornus officinalis powder, and enzymatic hydrolysis is performed at 25-75℃ to obtain enzymatic hydrolysate. After the enzyme is inactivated by enzymatic hydrolysate, the supernatant is collected by centrifugation. The supernatant is concentrated and subjected to alcohol precipitation treatment to collect the precipitate. The precipitate is purified and dried to obtain Cornus officinalis polysaccharide.

[0009] More preferably, the compound enzyme is a mixture of cellulase and pectinase, with the amount of cellulase added being 0.5% to 1.5% of the mass of Cornus officinalis powder, the amount of pectinase added being 0.3% to 0.8% of the mass of Cornus officinalis powder, the enzymatic hydrolysis pH being 1.5 to 8.5, and the enzymatic hydrolysis time being 0.5 to 5 hours; the ratio of Cornus officinalis powder to water is 1:(5 to 30).

[0010] More preferably, the alcohol precipitation process employs a gradient concentration of ethanol for stepwise precipitation, with ethanol concentration gradients of 40%, 60%, and 80%; purification is performed using membrane separation-chromatography, with a molecular cutoff of 10 kDa for membrane separation, using DEAE-52 column chromatography (eluting with 0.1-0.3 M NaCl); the drying temperature is 35–45 °C, the vacuum degree is -0.095 to -0.08 MPa, and the drying time is 8–12 h.

[0011] Preferably, in step (2), the mass ratio of yeast β-glucan to phytosterol powder is (1-3):(0.5-2.5).

[0012] Preferably, the stirring temperature in step (2) is 40-80℃, the stirring speed is 300-500 rpm, and the stirring time is 1-2h.

[0013] Preferably, the ethanol solution of the complex in step (2) is stored at 40–80°C.

[0014] Preferably, in step (3), the mass ratio of the primary microcapsule to the complex is 1:(1-5), and the mass-volume concentration of the complex ethanol solution is 2%-7%.

[0015] Preferably, the primary microcapsules dispersed in step (3) are added to the ethanol solution of the complex at a dropping rate of 5 to 10 mL / min and a stirring speed of 100 to 500 rpm.

[0016] Preferably, the primary microcapsule dispersion process in step (3) is as follows: add the primary microcapsules to water, stir magnetically for 5 to 15 minutes, and then use an ultrasonic cell disruptor to sonicate for 5 to 10 minutes at a power of 100 to 400 W.

[0017] Preferably, during microfluidic mixing in step (1), the internal phase flow rate is 0.1 mL / min, the external phase flow rate is 0.3 mL / min, the microfluidic mixing temperature is 20–30 °C, and the mixing time is 30–90 s.

[0018] Preferably, in step (1) ultrasonic emulsification, the ultrasonic power is 20-70 W and the time is 2-8 min; in step (3) precipitation washing process, the process is: wash twice with anhydrous ethanol and then wash 1-2 times with water; in step (3) freeze drying temperature is -90℃ to -70℃ and time is 12-24 h.

[0019] The second aspect of the present invention provides a Cornus officinalis polysaccharide microcapsule prepared using the preparation method described in the first aspect.

[0020] The third aspect of this invention provides the application of the Cornus officinalis polysaccharide microcapsules described in the second aspect in food or health products.

[0021] Compared with the prior art, the present invention has the following advantages: (1) The primary encapsulation layer of this application adopts a triple composite wall material of sodium alginate (SA), whey protein (WPI), and pectin. A dense network structure is formed through ionic cross-linking (Ca²⁺) and heat treatment, which enhances mechanical stability. Sodium alginate provides pH responsiveness, whey protein enhances emulsification, and pectin delays burst release, thereby improving the overall encapsulation efficiency.

[0022] (2) The secondary encapsulation layer (yeast β-glucan-phytosterol) can enhance the loading of lipid-soluble components: Phytosterol itself is lipid-soluble and binds to the hydrophobic regions of primary particles (such as the hydrophobic groups of whey protein) through hydrophobic interactions, thereby improving the encapsulation efficiency of lipid-soluble functional components; forming a barrier layer: After ethanol evaporates, β-glucan (hydrophilic) and phytosterol (hydrophobic) form an amphiphilic composite membrane, preventing the core material (cornus polysaccharide) from diffusing into the aqueous environment, while promoting its release in the lipid phase, which is suitable for oil-water dual-phase systems; the immunomodulatory effect of β-glucan and the anti-inflammatory properties of phytosterol may form a functional synergy with cornus polysaccharide to enhance the overall efficacy.

[0023] (3) The primary encapsulation layer of this application constructs a physical barrier through a double-layer structure, which solves the problems of Cornus officinalis polysaccharide (CMP) being easily degraded by gastric acid, having poor thermal stability, and having a fragile molecular chain. At the same time, it avoids the loss of active ingredients during processing, storage and in vivo transport. It achieves synergistic effects: The secondary encapsulation layer introduces yeast β-glucan and phytosterols to form a multi-dimensional synergistic effect with the antihypertensive activity of CMP, thereby expanding the health value of the product.

[0024] (4) The primary embedding layer of this application adopts microfluidic technology to achieve gradient coating. The gradient coating of the inner layer (WPI-CMP complex) and the outer layer (SA-Pectin) is realized simultaneously through the microfluidic chip, which shortens the process time and has the technical advantages of high coating uniformity and efficient and controllable preparation process.

[0025] (5) This application solves the problem of "difficulty in stably loading hydrophobic components onto the surface of hydrophilic particles" by loading hydrophobic complexes with ethanol solutions. The yeast β-glucan-phytosterol complex is adsorbed onto the surface of primary microcapsules through hydrophobic interactions (the hydrophobic groups of phytosterols combine with the hydrophobic groups on the surface of primary microparticles), avoiding the rapid addition of primary microcapsule suspension that would cause a sudden increase in local ethanol concentration and trigger aggregation.

[0026] (6) The activity retention effect of Cornus officinalis polysaccharide in this application is ≥95%, which is significantly better than traditional heat drying and effectively avoids the decline in efficacy caused by molecular chain breakage; Encapsulation and loading effect: Encapsulation rate ≥90%, drug loading rate is stable and there is no residual curing agent (the primary coating surface is clean); Functional synergy effect: DPPH free radical scavenging ability is improved compared with uncoated CMP. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer and more explicit, the invention is further described in detail below through embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] (I) Effect of the ratio of Cornus officinalis polysaccharide to whey protein on the encapsulation effect of Cornus officinalis polysaccharide Example 1 A method for preparing Cornus officinalis polysaccharide microcapsules, the specific steps of which are as follows: (1) The dried Cornus officinalis was pulverized and passed through a 60-mesh sieve to obtain Cornus officinalis powder. 500 mL of deionized water and a compound enzyme were added to 100 g of Cornus officinalis powder. The mixture was enzymatically hydrolyzed at pH 1.5 and 25℃ for 0.5 hours to obtain the hydrolysate. After inactivating the enzyme, the supernatant was collected by centrifugation. The compound enzyme was a mixture of vitamin B complex and pectinase. The amount of vitamin B complex added was 0.5% of the mass of Cornus officinalis powder, and the amount of pectinase added was 0.3% of the mass of Cornus officinalis powder. (2) Concentrate the supernatant prepared in step (1) to 1 / 5 of the original volume, add ethanol to the final concentrations of 40%, 60% and 80% in sequence, precipitate the polysaccharide step by step, and collect the precipitate by centrifugation. (3) Membrane separation-chromatographic purification: Ultrafiltration membrane (10 kDa) retains large molecular polysaccharides, and the permeate is subjected to DEAE-52 column chromatography (elution with 0.1-0.3M NaCl) to collect the target component; (4) The purified polysaccharide solution in step (3) is vacuum dried at a temperature of 40°C and a vacuum degree of -0.06 MPa for 10 hours to preserve the bioactivity of the polysaccharide (activity retention rate ≥95%) and avoid molecular chain breakage caused by traditional heat drying to obtain Cornus officinalis polysaccharide. (5) Weigh 2g of sodium alginate and add it to 400mL of deionized water to prepare sodium alginate with a concentration of 0.5% w / v; weigh 5g of whey protein and add it to 500mL of deionized water to prepare whey protein with a concentration of 1% w / v; weigh 1g of pectin and add it to 500mL of deionized water to prepare pectin with a concentration of 0.2% w / v; adjust the pH of each solution to 5.5. (6) Weigh 3.5g of Cornus officinalis polysaccharide from step (4), and 100mL of whey protein solution from step (5). Mix the Cornus officinalis polysaccharide and whey protein solution, and ultrasonically emulsify at 50 W for 5 min to prepare the inner phase of the primary microcapsules. Mix 350mL of sodium alginate solution from step (5) with 150mL of pectin solution, and add 120mL of CaCl2 solution with a concentration of 0.5% w / v. Crosslinking time: 18 min (at a constant temperature of 25℃) to prepare the outer phase of the primary microcapsules. At an inner phase flow rate of 0.1mL / min, an outer phase flow rate of 0.3mL / min, a temperature of 25℃, and a mixing time of 10s, the inner and outer phases are mixed in a microfluidic manner to obtain the primary microcapsules. The surface of the primary microcapsules is clean (without residual curing agent), and the diameter is 100-500μm (to facilitate the uniform adhesion of the secondary encapsulation layer). (7) Weigh 3g of yeast β-glucan and 2g of phytosterol to form a complex. Add an appropriate amount of anhydrous ethanol and stir to disperse (250 rpm, 1 min). Add anhydrous ethanol to make up to 100 mL to obtain a complex mixture with a total concentration of 5% w / v. Transfer the mixture to a constant temperature water bath stirrer, set the temperature to 60℃, stir at 300 rpm for 1 h, centrifuge at 3000 rpm for 5 min. The supernatant is completely dissolved when there is no residue. Prepare the complex ethanol solution. After dissolution, keep the temperature at 60℃ to avoid precipitation of the complex due to cooling. (8) Weigh 1.25 g of the primary microcapsules prepared in step (6) and add them to 80 mL of deionized water for dispersion treatment. After stirring magnetically for 10 min, use a 300 W ultrasonic cell disruptor to sonicate for 5 min to obtain a primary microcapsule suspension. Add the primary microcapsule suspension dropwise to the composite ethanol solution prepared in step (7) (dropping rate 5 mL / min, stirring while adding, maintaining a speed of 300 rpm). After the dropwise addition is completed, continue stirring at 60 ℃ for 30 min to obtain a mixture. (9) The mixture was slowly cooled to room temperature for 1 hour to allow the complex to solidify further on the surface of the microparticles and enhance the bonding stability. The cooled mixture was transferred to a centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was discarded and the precipitate was collected. The precipitate was washed twice with a small amount of anhydrous ethanol (to remove the free complex remaining on the surface) and then washed once with deionized water (to remove the residual ethanol). The washed secondary microparticles were transferred to a freeze-drying bottle and freeze-dried at -80℃ for 12 hours to obtain Cornus officinalis polysaccharide microcapsules.

[0029] Example 2 The process is basically the same as in Example 1, except that in step (6), 1g of Cornus officinalis polysaccharide is weighed.

[0030] Example 3 The process is basically the same as in Example 1, except that in step (6), the mass of Cornus officinalis polysaccharide is 2 g.

[0031] Example 4 It is basically the same as Example 1, except that in step (6), the mass of Cornus officinalis polysaccharide is 3 g.

[0032] Comparative Example 1 The comparative example is basically the same as Example 1, except that the mass of Cornus officinalis polysaccharide in step (6) is 4 g.

[0033] Comparative Example 2 The comparative example is basically the same as Example 1, except that the mass of Cornus officinalis polysaccharide in step (6) is 0.25g.

[0034] Performance testing: 1. Detection methods for encapsulation efficiency and drug loading rate of Cornus officinalis polysaccharide microcapsules Sample pretreatment: The prepared Cornus officinalis polysaccharide microcapsule sample was dried to constant weight in a 60℃ drying oven and cooled to room temperature for later use. 2.0 g of the dried microcapsule sample was accurately weighed and placed in a 100 mL stoppered conical flask. 50.0 mL of 0.2 mol / L sodium citrate solution was added, and the flask was sealed and placed in a constant temperature water bath shaker. The temperature was set to 50℃ and the shaking frequency to 150 r / min for 6 h (to ensure complete dissolution of the wall material and full release of the core material). After shaking, the solution was transferred to a centrifuge tube and centrifuged at 4000 r / min for 10 min (room temperature). The supernatant was collected and filtered through a 0.45 μm aqueous filter membrane (to remove a small amount of undissolved wall material particles). The filtrate was collected as the sample solution for testing. Blank control group treatment: Blank microcapsules were prepared according to steps (6) to (9) of Example 1. Cornus officinalis polysaccharide was not added in step (6). Other processes were the same as in Example 1 to obtain blank microcapsules. 2.0 g of blank microcapsules were accurately weighed and the above steps were followed. The filtrate was collected as a blank control solution.

[0035] Polysaccharide content detection: Accurately weigh 10.0 mg of Cornus officinalis polysaccharide standard, place it in a 100 mL volumetric flask, add 80 mL of deionized water, sonicate for 10 min (300 W power), cool, and dilute to the mark to obtain a 100 μg / mL standard stock solution; Preparation of standard series solutions: Pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.5 mL of the standard stock solution into 25 mL stoppered colorimetric tubes, and add deionized water to 2.0 mL; Colorimetric reaction: Add 1.0 mL of 5% phenol solution (weigh 5 g of phenol, dissolve in deionized water and dilute to 100 mL) to each colorimetric tube, shake well, and quickly add 5.0 mL of concentrated sulfuric acid, shake well immediately; Place the colorimetric tube in a 40℃ constant temperature water bath for 30 min, remove and cool to room temperature; Use blank reagent (2.0 mL deionized water + 1.0 mL 5% phenol solution) Using phenol + 5.0 mL concentrated sulfuric acid as a reference, the absorbance (A) of each standard series solution was measured at a wavelength of 490 nm. Standard curve fitting: A standard curve was plotted with polysaccharide concentration (C, μg / mL) on the x-axis and absorbance (A) on the y-axis. Linear regression analysis was performed to obtain the regression equation A = aC + b, R²≥0.995.

[0036] Determination of polysaccharide content in the sample test solution: Take 1.0 mL of the sample test solution and place it in a 25 mL stoppered colorimetric tube, then add deionized water to a final volume of 2.0 mL; perform the colorimetric reaction and absorbance measurement, and record the absorbance of the sample (sample A); Determination of blank control solution: Take 1.0 mL of blank control solution, follow the above steps, and record the absorbance of the blank (blank A); Calculation of polysaccharide concentration: Calculate the actual concentration of polysaccharides in the sample test solution according to the regression equation.

[0037] Cactual = (Asample - Aempty - b) / a.

[0038] (1) Calculation of actual encapsulated polysaccharide amount (ρ1, mg): ρ1 = Creal × V × f; Where: Cactual: the actual concentration of polysaccharides in the sample test solution (μg / mL), calculated from the standard curve; V: the total dilution volume during sample processing (mL), which is 50 mL (the volume of sodium citrate solution) here; f: the additional dilution factor.

[0039] (2) Calculation of theoretical encapsulated polysaccharide amount (ρ2, mg): ρ2 = m×w; In the formula: m: total mass of Cornus officinalis polysaccharides added during microcapsule preparation (mg); w: theoretical yield (%) during microcapsule preparation, w = (actual total mass of microcapsules / theoretical total mass of microcapsules) × 100%, theoretical total mass of microcapsules = mass of wall material added + mass of polysaccharides added.

[0040] (3) Total mass of microcapsules (ρ3, mg): That is, the mass of the microcapsules after sample pretreatment and drying to constant weight (2000.0 mg, accurate weighing).

[0041] (4) Calculation of encapsulation rate (EE, %): EE = (ρ1 / ρ2) ×100% (Core indicator: reflects the proportion of the core material embedded in the wall material; the higher the percentage, the better the embedding effect). (5) Calculation of drug loading rate (DL, %): DL = (ρ1 / ρ3) ×100% (Core indicator: reflects the actual amount of core material contained in a unit mass of microcapsules, and embodies the drug loading capacity of microcapsules).

[0042] 2. Determination of DPPH free radical scavenging ability Weigh 0.5000 g of the dried microcapsule powder using an analytical balance. Slowly add the powder to an appropriate amount of preheated deionized water (300–40°C) while stirring with a magnetic stirrer (300–500 r / min) for 10–15 min. If a small amount of insoluble matter remains, place the mixture in an ultrasonic cleaner (300 W, water temperature ≤40°C) and sonicate for 5–10 min to promote wall material dissolution and core material release (avoiding high temperature from damaging the antioxidant activity of the core material). Transfer the evenly dispersed solution to a 100 mL volumetric flask. Wash the beaker 3–4 times with deionized water, transferring all the washings into the volumetric flask. Finally, add solvent to bring the volume to the mark and mix well. This yields a microcapsule solution with a dry matter concentration of 5.0 mg / mL.

[0043] Prepare a 0.5 mmol / L DPPH solution using anhydrous ethanol. Mix the microcapsule solution and DPPH solution at a 1:1 volume ratio (v / v), react at room temperature in the dark for 30 min, and then measure the absorbance at 517 nm, denoted as A1. Repeat the 1:1 mixture of anhydrous ethanol and microcapsule solution, denoted as A2; and repeat the 1:1 mixture of anhydrous ethanol and DPPH solution, denoted as A0. Each group was measured three times, and the average value was taken. The calculation formula is as follows: In the formula: A1 is the absorbance value with the sample added; A2 is the absorbance value of the sample solution without DPPH added; A0 is the absorbance value without the sample added. The performance data results of Examples 1-4 and Comparative Examples 1 and 2 are shown in Table 1.

[0044] Table 1 Performance data of Examples 1-4 and Comparative Examples 1 and 2 As shown in Table 1, the encapsulation efficiency is high. The molecular structure of saturated whey protein with molecular interactions contains hydrophobic domains and hydrophilic groups. Cornus officinalis polysaccharides (mainly glucan and arabinoyl xylan) are water-soluble polysaccharides. The two form a complex system through hydrophobic interactions, hydrogen bonds, and electrostatic attraction. In Example 1, the mass ratio of Cornus officinalis polysaccharide core material to inner phase wall material (1g whey protein) was 3.5:1. The polysaccharide molecules completely covered the hydrophobic sites of whey protein, forming a stable "protein-polysaccharide" composite micelle. After ultrasonic emulsification, the inner phase (polysaccharide-whey protein) was uniformly dispersed, with no free polysaccharide precipitation. In Comparative Example 1, the amount of core material exceeded the maximum embedding capacity of the wall material, and some polysaccharides could not be encapsulated by whey protein. Furthermore, the external phase cross-linking network developed pores due to overload, resulting in a low actual embedding rate. In Examples 2, 3, 4, and Comparative Example 2, the amount of core material was insufficient, the polysaccharides in the inner phase system were unevenly dispersed, and some wall material did not bind with the polysaccharides to form ineffective embedding, resulting in embedding rates and drug loading rates lower than in Example 1.

[0045] Drug loading rate: Example 1 has the highest encapsulation rate, and the amount of polysaccharide used is in the optimal range of "encapsulation efficiency - core material amount": it avoids insufficient drug loading rate due to too little core material, and does not have the "encapsulation saturation effect" due to excessive core material (excessive core material cannot be encapsulated, but dilutes the proportion of effective ingredients in the microcapsules). DPPH free radical scavenging rate: Example 1 showed the highest drug loading rate, the highest amount of active polysaccharides per unit mass of microcapsules, and the highest concentration of effective components reacting with DPPH free radicals. Comparative Example 1, due to overload and rupture of the primary microcapsule structure, resulted in uneven adhesion of the secondary encapsulation layer, making the polysaccharides easily oxidized and degraded, leading to a low scavenging rate. Comparative Example 2, due to insufficient core material and insufficient effective components, also had a low scavenging rate and failed to demonstrate the antioxidant activity advantage of polysaccharides. Examples 2, 3, and 4, due to insufficient polysaccharide usage, had low active ingredient content per unit mass of microcapsules, and their scavenging rates increased gradually with increasing polysaccharide usage, but were all lower than that of Example 1.

[0046] (II) Effect of wall material dosage on the encapsulation effect of Cornus officinalis polysaccharides Based on the above experimental results, the mass ratio of Cornus officinalis polysaccharide to whey protein was selected as 3.5:1 to continue the single-factor experiment on the wall material usage ratio.

[0047] Example 5 It is basically the same as Example 1, except that in step (6), 300 mL of sodium alginate solution and 100 mL of pectin solution are mixed and 100 mL of CaCl2 solution is added.

[0048] Example 6 It is basically the same as Example 1, except that in step (6), 400 mL of sodium alginate solution and 200 mL of pectin solution are mixed and 150 mL of CaCl2 solution is added.

[0049] Comparative Example 3 The comparative example is basically the same as Example 1, except that in step (6), 250 mL of sodium alginate solution and 90 mL of pectin solution are mixed and 90 mL of CaCl2 solution is added.

[0050] Comparative Example 4 The comparative example is basically the same as Example 1, except that in step (6), 450 mL of sodium alginate solution and 250 mL of pectin solution are mixed and 180 mL of CaCl2 solution is added.

[0051] The performance testing method is the same as above. The performance data of Examples 5-6 and Comparative Examples 3 and 4 are shown in Table 2.

[0052] Table 2 Performance data of Examples 5-6 and Comparative Examples 3 and 4 As shown in Table 2, in Example 5, the amount of sodium alginate and pectin reached the lower limit of the optimal range, and the total amount of wall material could completely encapsulate the inner phase polysaccharide. However, the gel network density was slightly lower than that in Example 1, and a small amount of polysaccharide leaked from the gaps in the wall material, resulting in a slight decrease in the encapsulation rate and drug loading rate. The amount of CaCl2 met the basic cross-linking requirements, the wall material structure was stable, and the active groups of polysaccharide were not damaged. Therefore, the DPPH free radical scavenging rate remained above 87%. During microfluidic mixing, the volumes of the inner and outer phases were matched, the mixing uniformity was good, and there was no problem of insufficient wall material in some areas.

[0053] In Example 6, the dosage of sodium alginate and pectin reached the upper limit of the optimal range, resulting in sufficient total wall material, a denser gel network, and stronger polysaccharide encapsulation ability than in Example 5, thus achieving higher encapsulation and drug loading rates. The dosage of CaCl2 was at the upper limit of the optimal range, with a moderate degree of cross-linking, avoiding both excessively loose wall material and excessive cross-linking (which would damage the polysaccharide structure). The DPPH scavenging rate reached 88.1%. With the increased dosage of the external phase wall material, the microcapsule surface became smoother (without residual curing agent), allowing for better adhesion of the secondary encapsulation (yeast β-glucan + phytosterols), indirectly improving the protective effect on the polysaccharide and further reducing activity loss.

[0054] In Comparative Example 3, insufficient amounts of sodium alginate and pectin wall materials prevented the formation of a complete external gel network. Some Cornus officinalis polysaccharides remained unencapsulated and were lost with the supernatant during centrifugation, leading to a significant decrease in encapsulation and drug loading rates. Insufficient CaCl2 content resulted in incomplete ionic cross-linking between sodium alginate and pectin, leading to a loose wall material structure with high porosity. This not only caused polysaccharide leakage but also exposed the unleashed polysaccharides to an oxidizing environment, damaging active groups (such as phenolic hydroxyl groups) and reducing DPPH free radical scavenging rates. During microfluidic mixing, insufficient external wall material prevented uniform encapsulation of the internal phase, causing some microcapsules to develop "holes," further exacerbating polysaccharide leakage and activity loss.

[0055] In Comparative Example 4, excessive use of sodium alginate and pectin increased the viscosity of the external phase, making it difficult for the internal and external phases to disperse evenly during microfluidic mixing. This resulted in wall material aggregation in some areas, and some internal polysaccharides were not effectively encapsulated, leading to a decrease in encapsulation and drug loading rates. Excessive cross-linking damaged the structure: excessive CaCl2 usage caused the sodium alginate-pectin gel network to become overly dense, leaking some polysaccharides. It also damaged the molecular chain structure of Cornus officinalis polysaccharides, leading to a reduction in the number of active groups and a decrease in DPPH free radical scavenging rate. Excessively thick wall material hindered activity: even if some polysaccharides were successfully encapsulated, an excessively thick wall material would hinder the contact between polysaccharides and free radicals, reducing their antioxidant activity and further lowering the scavenging rate.

[0056] (III) Effect of the mass ratio of primary microcapsules to complex on the encapsulation effect of Cornus officinalis polysaccharides Based on the above experimental results, the volume ratio of whey protein solution, sodium alginate solution, pectin solution and calcium chloride solution was selected as 1:3.5:1.5:1.2, and the single-factor experiment on the mass ratio of primary microcapsules to complex was continued.

[0057] Example 7 It is basically the same as Example 1, except that in step (8), 2.5 g of primary microcapsules are weighed.

[0058] Example 8 It is basically the same as Example 1, except that in step (8), 1.67g of primary microcapsules are weighed.

[0059] Example 9 It is basically the same as Example 1, except that in step (8), 1g of primary microcapsules is weighed.

[0060] Comparative Example 5 It is basically the same as Example 1, except that in step (8), 10g of primary microcapsules are weighed.

[0061] Comparative Example 6 It is basically the same as Example 1, except that in step (8), 0.83g of primary microcapsules are weighed.

[0062] The performance testing method is the same as above. The performance data of Examples 7-9 and Comparative Examples 5 and 6 are shown in Table 3.

[0063] Table 3 Performance data of Examples 7-9 and Comparative Examples 3 and 4 As shown in Table 2, Example 1 exhibits the best performance for the following reasons: In Example 1, the primary microcapsules and the complex are fully combined in the optimal ratio, forming a dense double-coating layer (sodium alginate-pectin-Ca²⁺ cross-linked layer + secondary complex layer) without any exposed areas. This not only minimizes the loss of polysaccharides during washing and centrifugation, making the actual amount of encapsulated polysaccharides close to the theoretical value, but also eliminates "ineffective mass redundancy," achieving peak drug loading. Simultaneously, the double barrier effectively isolates the polysaccharides from oxidation and damage caused by oxygen and light. During freeze-drying (-80℃, 12h), the coating layer structure remains stable without shrinkage or cracking, resulting in a polysaccharide activity retention rate ≥98% and a final DPPH free radical scavenging rate of 89.70%.

[0064] In Example 7, the complex was relatively insufficient, failing to form a continuous and dense secondary encapsulation layer, resulting in inadequate pore filling. Furthermore, the primary microcapsule dispersion concentration was close to the aggregation critical value, and a small number of small aggregates remained after ultrasonic treatment, forming an "uncovered core" within the microcapsules. Aggregation also caused mutual obstruction of the microcapsule surface active groups, reducing effective binding sites and further decreasing coverage efficiency. The exposed polysaccharides were easily dissolved or physically detached during centrifugation and ethanol washing, and cracks formed in the primary wall material at the aggregation interface, leading to additional polysaccharide leakage and ultimately reducing polysaccharide retention. The relatively insufficient complex in Example 7 prevented the formation of a complete "water-soluble + lipid-soluble" full-spectrum synergistic antioxidant network with all polysaccharides. The amount of yeast β-glucan was insufficient to activate all polysaccharide activity, and the limited total amount of phytosterols made it difficult to fully scavenge lipid-soluble free radicals, weakening the synergistic antioxidant effect and ultimately reducing the DPPH free radical scavenging rate.

[0065] In Example 8, the complex was relatively insufficient, and the secondary encapsulation layer contained micropores, failing to completely seal the primary microcapsules. The internal polysaccharide slightly leaked through these pores and was dissolved and lost by anhydrous ethanol during washing, resulting in a decrease in drug loading. Due to the insufficient complex and the porosity of the secondary encapsulation layer in Example 8, the polysaccharide underwent slight oxidation, leading to a slight decrease in activity retention.

[0066] In Example 9, the insufficient amount of primary microcapsules resulted in a relative excess of the complex. The excess portion could not be completely anchored to the microcapsule surface and easily aggregated in solution to form free particles, reducing the effective amount of the complex actually involved in encapsulation. Furthermore, the stacked structure formed by self-aggregation resulted in a loose and porous encapsulation layer, poor sealing, and increased polysaccharide leakage. Simultaneously, during centrifugation, the aggregates separated from the microcapsules, and the attached polysaccharides were discarded with the supernatant, further exacerbating the loss. In Example 9, due to the locally excessively thick encapsulation layer formed by the self-aggregation of the complex, mass transfer within the microcapsules was hindered. Some polysaccharides were damaged during drying due to internal pressure changes, reducing the activity retention rate to 90%–92%, and consequently weakening the antioxidant activity.

[0067] In Comparative Example 5, the amount of primary microcapsules was excessive, the complex was severely insufficient, the coating layer was incomplete and contained numerous pores, and microcapsule aggregation caused internal wall material to crack under pressure. A large amount of polysaccharide was lost through direct dissolution due to exposure and through double leakage via pores and cracks, resulting in a significantly increased total loss rate, a sharp drop in encapsulation efficiency, and a simultaneous and substantial decline in drug loading. In Comparative Example 5, the coating layer was incomplete, and the polysaccharides were directly exposed to the environment, resulting in severe oxidative degradation and an activity retention rate of only about 80%. Furthermore, the insufficient complex prevented the formation of a synergistic effect, relying solely on the polysaccharide's own antioxidant capacity, leading to a significant decline in DPPH free radical scavenging rate.

[0068] In Comparative Example 6, the excessive complex led to supersaturation in the ethanol solution, resulting in severe self-agglomeration. The formed coating layer contained cracks and aggregates, failing to achieve effective sealing. Excessive polysaccharide leakage occurred through the cracks in the primary microcapsules, and some polysaccharide was carried away during the separation of the aggregates from the microcapsules, ultimately resulting in the lowest polysaccharide retention and the lowest drug loading rate. In Comparative Example 6, the excessive aggregated complex increased the permeability of the coating layer, exacerbating polysaccharide oxidative degradation. Furthermore, the residual free complex carried away some active polysaccharides during washing, reducing both the drug loading rate and further weakening antioxidant activity, ultimately resulting in the lowest DPPH free radical scavenging rate.

[0069] (iv) Effect of coating layer on the encapsulation effect of Cornus officinalis polysaccharides Comparative Example 7 Comparative Example 7 consists of primary microcapsules prepared in steps (1) to (6) of Example 1, without secondary encapsulation.

[0070] The performance testing method is the same as above, and the performance data of Comparative Example 7 is shown in Table 4.

[0071] Table 4 Performance data of Comparative Example 7 Comparative Example 7 only underwent primary coating (without yeast β-glucan-phytosterol secondary loading). The core difference resulted in all three indicators being significantly lower than those of Example 1: Encapsulation rate: Due to the lack of the barrier effect of the secondary coating layer, some polysaccharides leaked out during subsequent processing, leading to a decrease in encapsulation rate; Drug loading rate: Without the binding of lipophilic components from the secondary loading, the drug loading rate relied solely on the coating capacity of the primary wall material, resulting in a decrease in drug loading rate; DPPH free radical scavenging rate: Lacking the synergistic antioxidant effect of yeast β-glucan and phytosterols, the scavenging rate relied solely on the activity of Cornus officinalis polysaccharide itself, thus verifying the functional synergistic value of the secondary coating layer.

Claims

1. A preparation method of Cornus officinalis polysaccharide microcapsules, characterized in that, The method comprises the following steps: (1) adding the cornus officinalis polysaccharide to the whey protein solution, ultrasonic emulsification, and obtaining an inner phase of the primary microcapsule; adding the pectin solution to the sodium alginate solution, mixing, and then adding the CaCl2 solution, mixing, and obtaining an outer phase of the primary microcapsule, and mixing the inner phase and the outer phase through microfluidic mixing to obtain the primary microcapsule; (2) mixing the yeast beta-glucan and the phytosterol powder to form a complex, dissolving the complex in anhydrous ethanol to obtain a complex ethanol solution; (3) dispersing the primary microcapsule prepared in step (1) into water to obtain a dispersion liquid; adding the dispersion liquid to the complex ethanol solution prepared in step (2) at 50-75 DEG C, stirring and mixing for 30-60 min to obtain a mixed liquid, cooling the mixed liquid to room temperature, and then standing for 1-2 h, centrifuging the mixed liquid after standing, collecting the precipitate, and washing and freeze-drying the precipitate to obtain the cornus officinalis polysaccharide microcapsule.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of the cornus officinalis polysaccharide to the whey protein is (0.5-3):

1.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass / volume concentration of the whey protein solution is 1%-10%, the mass / volume concentration of the sodium alginate solution is 0.5%-3.7%, the mass / volume concentration of the pectin solution is 0.2%-2.5%, and the mass / volume concentration of the CaCl2 solution is 0.2%-1.0%.

4. The production method according to claim 3, characterized by, In step (1), the volume ratio of the whey protein solution to the sodium alginate solution to the pectin solution to the CaCl2 solution is (1-2):(3-4):(1-2):(1-1.5).

5. The preparation method according to claim 4, characterized in that, In step (3), the mass ratio of the primary microcapsule to the complex is 1:(1-5), and the mass / volume concentration of the complex ethanol solution is 2%-7%.

6. The method of claim 1, wherein, In step (2), the mass ratio of the yeast beta-glucan to the phytosterol powder is (1-3.5):(0.5-2.5).

7. The production method according to claim 1, characterized by, In step (1), the flow rate of the inner phase is 0.05-0.15 mL / min, the flow rate of the outer phase is 0.2-0.4 mL / min, and the temperature of the microfluidic mixing is 20-30 DEG C.

8. The production method according to claim 7, characterized by, In step (1), the ultrasonic power is 20-70 W, and the time is 2-8 min; in step (3), the washing process of the precipitate is as follows: using anhydrous ethanol for washing twice, and then using water for washing 1-2 times; the temperature of the freeze-drying in step (3) is -90 DEG C to -70 DEG C, and the time is 12-24 h.

9. The cornus officinalis polysaccharide microcapsule prepared by the preparation method in any one of claims 1-8.

10. The cornus officinalis polysaccharide microcapsule in claim 9 is used in food or health products.

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