Chondroitin sulfate / whey protein isolate and anthocyanin nanocomposite as well as preparation method and application thereof

Abstract

The invention discloses a chondroitin sulfate / whey protein isolate anthocyanin nano-composite and a preparation method and application thereof.The preparation method comprises the steps that blueberry pomace is pre-frozen, freeze-dried and then ground into powder, blueberry pomace powder is obtained, the blueberry pomace powder and pectinase are added into a citric acid buffer solution, water bath oscillation enzymolysis is conducted, the blueberry pomace powder and pectinase are taken out for enzyme deactivation, and the chondroitin sulfate / whey protein isolate anthocyanin nano-composite is obtained. The preparation method comprises the following steps: dissolving whey protein isolate and chondroitin sulfate into water, carrying out ultrasonic treatment, centrifuging, filtering supernate, purifying and freeze-drying to obtain anthocyanin powder, dissolving whey protein isolate and chondroitin sulfate into water, regulating the pH value, dropwise adding an anthocyanin solution into a chondroitin sulfate solution, carrying out secondary stirring, dropwise adding the whey protein isolate solution, and carrying out three-time stirring, standing, centrifuging, freeze-drying and grinding to obtain the product. According to the method, the extraction rate of the anthocyanin is high, the embedding rate of the nanocomposite is high, the stability is good, and the method is green, efficient and simple.

Description

Technical Field

[0001] The invention belongs to the technical field of natural substance extraction, and relates to a construction method and application of anthocyanin and a nano-carrier, and specifically to a chondroitin sulfate / whey protein isolate@anthocyanin nano-complex and a preparation method and application thereof. Background Art

[0002] Blueberry is a small berry grown in abundance in the north. It is rich in anthocyanins and has strong antioxidant, anti-inflammatory, anti-obesity, and vision protection effects. It is widely used for fresh consumption and extraction of active ingredients. However, during the processing, due to defects in methods and technologies, a large amount of blueberry by-products are wasted, especially the blueberry pomace after juicing, which also contains a certain amount of anthocyanins and other active ingredients. If it is recycled, the comprehensive utilization of by-products can be achieved.

[0003] There are many methods for anthocyanin extraction, including traditional solvent extraction, ultrasound-assisted extraction, supercritical fluid extraction, etc. However, these methods usually use organic reagents, which have serious problems of reagent residues and pollution. The extracted anthocyanins are sensitive to factors such as light, heat, and pH, and are easily degraded and lose their activity. The main methods to improve the stability of anthocyanins are acylation, co-pigmentation, and microencapsulation. Acylation refers to the use of organic acids to replace anthocyanin glycosides through ester bonds to produce acylated anthocyanins, thereby preventing water molecules and other substances from attacking the anthocyanins themselves. However, the acylation process is complicated and difficult to control, and it cannot be produced as expected. At the same time, the acylation site and the type and number of acyl groups greatly affect the stability of anthocyanins. Co-pigmentation refers to the formation of supramolecular compounds by the combination of anthocyanins with phenolic acids and flavonoids, including intermolecular and intramolecular co-pigmentation, which can stabilize the color of anthocyanins. However, the complex formed is extremely sensitive to temperature, pH and organic solvents. The organic solvents used in the reaction process will cause the entire system to be unstable. In addition, the co-pigmentation process is exothermic, and the increase in temperature will accelerate the discoloration of anthocyanins, and the self-binding of anthocyanin molecules will form a certain competition with the co-pigmentation process. Microencapsulation refers to the technology of encapsulating substances (encapsulants or active cores) in solid, liquid or gaseous states using encapsulants (wall or shell materials). This method can provide a physical barrier for the active ingredients, prevent the invasion of adverse external factors to a certain extent, and achieve controlled release of the core material. Due to its small size, it can prolong the gastrointestinal retention time by improving the bioadhesive properties of the mucus covering the intestinal epithelium. However, although the traditional microencapsulation method is very effective, it usually requires a large amount of reagents, resulting in waste and low yield. Therefore, it is necessary to develop a green, simple and efficient anthocyanin extraction and embedding method. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite with high anthocyanin extraction rate, high nanocomposite embedding rate and good stability, simple preparation, and green environmental protection, as well as a preparation method and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0006] A method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite comprises the following steps:

[0007] (1) pre-freezing blueberry pomace, then freeze-drying, grinding into powder, sieving to obtain blueberry pomace powder, and storing at -20°C to -4°C in the dark;

[0008] (2) weighing blueberry pomace powder and pectinase and adding them to citric acid buffer, wherein the addition ratio of the blueberry pomace powder, pectinase and citric acid buffer is 1 g: 10 mg to 20 mg: 30 mL to 60 mL, shaking in a water bath at a temperature of 30° C. to 45° C. for enzymolysis, then removing and inactivating the enzyme, ultrasonicating, centrifuging and filtering the supernatant to obtain anthocyanin crude extract, purifying and freeze-drying to obtain anthocyanin powder;

[0009] (3) Dissolving whey protein isolate and chondroitin sulfate in water respectively and stirring once, storing at 0°C to -4°C overnight to fully hydrate, thereby obtaining a whey protein isolate solution and a chondroitin sulfate solution, and adjusting the pH values ​​of the obtained whey protein isolate solution and chondroitin sulfate solution to 3.0 to 3.5 respectively with acid solution; dissolving anthocyanin powder in water to obtain an anthocyanin solution, adding the anthocyanin solution dropwise to the chondroitin sulfate solution, stirring twice, and adding the whey protein isolate solution dropwise, stirring three times, standing, and centrifuging to remove free anthocyanins, taking the precipitate for freeze-drying, and then grinding and crushing, and sealing to obtain a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite.

[0010] In the above-mentioned method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite, preferably, in step (3), the mass ratio of the whey protein isolate, chondroitin sulfate and anthocyanin freeze-dried powder is 15-16:3-4:1.

[0011] The method for preparing the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex is preferably that in step (1), the pre-freezing temperature is 0°C to -20°C, the pre-freezing time is 5h to 10h, the freeze-drying temperature is -50°C to -60°C, the freeze-drying time is 24h to 48h, and the sieving is through a 60-80 mesh sieve.

[0012] The method for preparing the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex is preferably such that in step (2), the concentration of citric acid in the citric acid buffer is 0.1 mol / L to 1 mol / L, and the pH value of the citric acid buffer is 3.0 to 3.5; in step (3), the acid solution is HCl solution, and the concentration of the HCl solution is 0.5 mol / L to 1.5 mol / L.

[0013] The method for preparing the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex, preferably, in step (2), the enzymatic hydrolysis time is 50min to 60min, the enzyme inactivation temperature is 80°C to 100°C, the enzyme inactivation time is 3min to 7min, the ultrasonic power is 350W to 450W, the ultrasonic time is 20min to 40min, the centrifugal force is 8000g to 10000g, the centrifugal time is 5min to 15min, and the pore size of the filter membrane for filtering the supernatant is 0.45μm to 0.22μm.

[0014] The above-mentioned method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex, preferably, in step (3), the rotation speed of the first stirring is 300rpm~600rpm, the time of the first stirring is 3h~5h, the rotation speed of the second stirring is 300rpm~600rpm, the time of the second stirring is 1h~3h, the rotation speed of the third stirring is 300rpm~600rpm, and the time of the third stirring is 1h~2h.

[0015] The preparation method of the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex, preferably, in step (3), the standing time is 4h to 8h, the centrifugal temperature is -4°C to 0°C, the centrifugal speed is 8000rpm to 10000rpm, the centrifugal time is 5min to 15min, the freeze-drying cold trap temperature is -50°C to -60°C, the freeze-drying time is 24h to 48h, and the obtained chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex is stored at -20°C to -4°C in the dark.

[0016] As a general technical concept, the present invention also provides a chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex prepared by the method for preparing the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex.

[0017] As a general technical concept, the present invention also provides an application of the above-mentioned chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex in the field of food and cosmetics.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] The present invention firstly adopts ultrasound-assisted enzyme method to extract anthocyanins from blueberry pomace, and extracts anthocyanins by controlling different solid-liquid ratios, enzyme addition amount, enzymatic hydrolysis time, ultrasonic power and ultrasonic time, so that the anthocyanin yield can reach 5.41 mg / g, which is significantly higher than the anthocyanin extraction yield mentioned in most of the current literature.

[0020] Anthocyanin, as a natural active ingredient, has multiple physiological effects, but there are problems such as easy degradation and low biological activity. Most of the methods proposed in the prior art to improve the stability of anthocyanin have the problems of cumbersome preparation process, residual organic reagents and serious waste. Therefore, the present invention starts from the green and efficient aspects and develops an embedding method that can improve the stability and bioaccessibility of anthocyanin. The present invention selects the best wall material as whey protein isolate and chondroitin sulfate, which can be combined through electrostatic interaction, and the embedding rate reaches more than 84.87%. The nanocomposite is tested by scanning electron microscopy, atomic force microscopy, etc. The results show that chondroitin sulfate / whey protein isolate (CS / WPI) can achieve the embedding protection of anthocyanin, thereby preventing external adverse factors, including high temperature, light, and ascorbic acid from destroying the structure and functional activity of anthocyanin itself. On this basis, the applicant further studied the storage stability under different conditions. The results show that compared with free anthocyanin, the chondroitin sulfate / whey protein isolate@anthocyanin (CS / WPI@ACN) nanocomposite after embedding can achieve the effect of enhancing the stability of anthocyanin. In addition, the enhancement of the scavenging effect on free radicals shows that the CS / WPI@ACN nanocomposite can further enhance the antioxidant capacity of anthocyanins, thereby achieving the enhancement of the antioxidant and anti-inflammatory physiological effects in the body. It is proposed in the prior art that anthocyanins are sensitive to the pH in the gastrointestinal digestive environment, and their bioavailability is very low. Moreover, since anthocyanins are a highly hydrophilic bioactive substance, it is difficult for them to pass through the phospholipid bilayer on the cell membrane, so their bioaccessibility is also affected. The results of the in vitro simulated digestion experiment of the present invention show that the nanocomposite after embedding can protect anthocyanins from the influence of the neutral alkaline environment of the intestine, realize the slow release of anthocyanins, thereby prolonging the action time with the intestinal microbial flora, on the one hand, realizing the decomposition of anthocyanins by the intestinal flora, and on the other hand, anthocyanins can promote the colonization of beneficial flora and adjust the composition of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a single factor comparative experimental result diagram of anthocyanin extraction and embedding of blueberry pomace in Example 1 of the present invention, wherein A is the solid-liquid ratio, B is the enzyme addition amount, C is the enzymatic hydrolysis time, D is the ultrasonic power, E is the ultrasonic time, and F is the wall material concentration.

[0022] Figure 21 is a response surface optimization result diagram of anthocyanin extraction from blueberry pomace in Example 1 of the present invention, A is a relationship diagram between the solid-liquid ratio and the enzymatic hydrolysis time, B is a relationship diagram between the solid-liquid ratio and the ultrasonic time, and C is a relationship diagram between the enzymatic hydrolysis time and the ultrasonic time.

[0023] Figure 3 These are SEM images of the chondroitin sulfate / whey protein isolate@anthocyanin nanocomplexes of comparative samples 1-4 and Example 1 of the present invention, wherein A is comparative sample 1, B is comparative sample 2, C is comparative sample 3, D is comparative sample 4, and E and F are Example 1.

[0024] Figure 4 The atomic force microstructures of the chondroitin sulfate / whey protein isolate@anthocyanin nanocomplexes of comparative sample 3, comparative sample 4, and Example 1 of the present invention are shown in FIG. A1, B1, and C1 are two-dimensional atomic force microstructures of comparative sample 3, comparative sample 4, and Example 1, respectively; and A2, B2, and C2 are three-dimensional atomic force microstructures of comparative sample 3, comparative sample 4, and Example 1, respectively.

[0025] Figure 5 The diagrams are the changes in the antioxidant capacity of anthocyanins in the free group and the embedded group and the simulated digestion results of Example 1 of the present invention, wherein A and B are diagrams of the antioxidant capacity of DPPH and ABTS, respectively, and C is a diagram of the in vitro simulated digestion results.

[0026] Figure 6 Graphs showing the retention rates of anthocyanins in the free group and the embedded group under different storage conditions in Example 1 of the present invention, wherein A is a graph showing the embedding rate of anthocyanins under different illumination times, B is a graph showing the retention rate of anthocyanins after storage for different times in the presence of ascorbic acid, and C is a graph showing the retention rate of anthocyanins after high-temperature heating for different times. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following embodiments are all commercially available.

[0028] Example 1

[0029] A method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite of the present invention comprises the following steps:

[0030] (1) The blueberry pomace was pre-frozen at -20°C for 5 h, then freeze-dried at -56°C for 48 h, ground into powder using a high-speed grinder, passed through a 60-mesh sieve, and obtained blueberry pomace powder, which was then stored in a -20°C refrigerator away from light.

[0031] (2) Weigh blueberry pomace powder and pectinase and add them to citric acid buffer. The addition ratio of blueberry pomace powder, pectinase and citric acid buffer is 1g:15mg:42mL. The concentration of citric acid in the citric acid buffer is 0.1M. The pH of the citric acid buffer is 3.0. The enzyme is hydrolyzed in a water bath at 45°C for 57 minutes. The enzyme is removed and inactivated at 90°C for 5 minutes. Then, ultrasonic treatment is performed for 33 minutes at a power of 350W. Centrifugation is performed for 10 minutes at a centrifugal force of 10000g. The supernatant is filtered through a 0.45μm filter membrane to obtain anthocyanin crude extract. The anthocyanin crude extract is purified by AB-8 macroporous resin to obtain anthocyanin purified solution. After freeze-drying, anthocyanin powder is obtained for subsequent embedding.

[0032] In the present embodiment, the purification and freeze-drying process of anthocyanin crude extract is as follows: first, soak AB-8 macroporous resin with absolute ethanol for 24h to fully activate the resin, adopt wet method to pack the column, push a small amount of absorbent cotton into the bottom with a glass rod before packing the column, pour a little ethanol, knock the wall with an ear-washing ball, drive out the air in the absorbent cotton, and then evenly add the resin, the glass rod assists drainage, and the filler height is about 2 / 3 of the column height. Open the piston, and constantly knock the column with an ear-washing ball to ensure that the resin is packed tightly. Use distilled water to rinse until the eluent has no alcohol taste, add sample along the wall, and drain the glass rod to avoid the resin surface from being impacted and uneven. Regulate the flow rate to 1 drop / second, and elute the sample solution with 2BV of 60% ethanol solution. Rotary evaporation at 40°C to remove ethanol, the concentrate obtained is freeze-dried into powder and stored at -20°C for standby use.

[0033] (3) Dissolve whey protein isolate (WPI) and chondroitin sulfate (CS) in distilled water (prepared separately), stir at 600 rpm for 3 h, and store at 4° C. overnight to ensure sufficient hydration to obtain whey protein isolate solution and chondroitin sulfate solution, wherein the concentration of the whey protein isolate solution is 20 mg / mL, and the concentration of the chondroitin sulfate solution is 4 mg / mL, and adjust the pH values ​​of the obtained whey protein isolate solution and chondroitin sulfate solution to 3.0 with 1.5 mol / L HCl solution. Dissolve the above-obtained anthocyanin powder in distilled water to obtain an anthocyanin solution, wherein the concentration of the anthocyanin solution is 1.3 mg / mL. 50 mL of anthocyanin solution was added dropwise to 50 mL of chondroitin sulfate solution after pH adjustment, stirred at 600 rpm for 1 hour, then 50 mL of whey protein isolate solution was added dropwise, stirred at 600 rpm for 1 hour, allowed to stand for 4 hours, centrifuged at -4°C and 10,000 rpm for 10 minutes to remove free anthocyanin, and the precipitate was freeze-dried, the cold trap temperature was -56°C, and the freeze-drying time was 48 hours. The freeze-dried sample was ground and sealed to obtain a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite, and stored in a refrigerator at -20°C away from light. The chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite can be used in the fields of food and cosmetics.

[0034] Single factor comparative experiment (optimal condition exploration experiment):

[0035] 1. Effect of solid-liquid ratio on anthocyanin extraction rate

[0036] The same process as steps (1) and (2) of Example 1 above was used to investigate the effect of the solid-liquid ratio on the anthocyanin extraction rate. The solid-liquid ratio is the ratio of blueberry pomace powder to citric acid buffer, specifically 1g:20mL, 1g:30mL, 1g:40mL, 1g:50mL, 1g:60mL. In this experiment, 0.5g of blueberry pomace powder was used, and 7.5mg of pectinase was used. The mass of pectinase remained unchanged, that is, the ratios of blueberry pomace powder, pectinase, and citric acid buffer were 1g:15mg:20mL, 1g:15mg:30mL, 1g:15mg:40mL, 1g:15mg:50mL, 1g:15mg:60mL, respectively. Three parallel experiments were performed to determine the optimal solid-liquid ratio range.

[0037] 2. Effect of enzyme addition on anthocyanin extraction rate

[0038] The same process as steps (1) and (2) of Example 1 was used to investigate the effect of the amount of pectinase added on the anthocyanin extraction rate. The actual addition ratio of blueberry pomace powder, pectinase, and citric acid buffer was 0.5 g: 2.5 mg to 12.5 mg: 20 mL, wherein the amount of pectinase added was 2.5 mg, 5 mg, 7.5 mg, 10 mg, and 12.5 mg, respectively. Three parallel experiments were performed to determine the optimal enzyme addition range.

[0039] 3. Effect of enzymatic hydrolysis time on anthocyanin extraction rate

[0040] The same process as steps (1) and (2) of Example 1 above was adopted, wherein the ratio of blueberry pomace powder, pectinase, and citric acid buffer was 1 g: 15 mg: 40 mL, and the effect of enzymatic hydrolysis time on the anthocyanin extraction rate was investigated. The enzymatic hydrolysis times selected were 20 min, 40 min, 60 min, 0 min, and 100 min, respectively. Three parallel experiments were performed to determine the optimal enzymatic hydrolysis time range.

[0041] 4. Effect of ultrasonic power on anthocyanin extraction rate

[0042] The same process as steps (1) and (2) of Example 1 above was adopted, wherein the mass ratio of blueberry pomace powder, pectinase, and citric acid buffer was 1 g: 15 mg: 40 mL, and the effect of ultrasonic power on the anthocyanin extraction rate was investigated. The selected ultrasonic powers were 300 W, 350 W, 400 W, 450 W, and 500 W, respectively. Three parallel experiments were performed to determine the optimal ultrasonic power range.

[0043] 5. Effect of ultrasonic time on anthocyanin extraction rate

[0044] The same process as steps (1) and (2) of Example 1 above was adopted, wherein the mass ratio of blueberry pomace powder, pectinase, and citric acid buffer was 1 g: 15 mg: 40 mL, and the effect of ultrasonic time on the anthocyanin extraction rate was investigated. The selected ultrasonic times were 10 min, 20 min, 30 min, 40 min, and 50 min, respectively. Three parallel experiments were performed to determine the optimal ultrasonic time range.

[0045] 6. Effect of wall material concentration (chondroitin sulfate concentration) on anthocyanin embedding rate

[0046] The same process as step (3) of Example 1 was used to investigate the effect of wall material concentration on anthocyanin embedding efficiency. The concentration refers to the concentration of chondroitin sulfate solution, specifically 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL. In this experiment, the volume of whey protein isolate, chondroitin sulfate, and anthocyanin solution was kept at 50 mL, and the mass ratio of the three was 15:3:1, which remained unchanged. The concentration of chondroitin sulfate solution was changed, and the concentrations of whey protein isolate solution and anthocyanin solution were adjusted accordingly according to the mass ratio. Three parallel experiments were performed to determine the optimal wall material concentration range.

[0047] like Figure 1 As shown in A, with the increase of the solid-liquid ratio, the extraction rate of anthocyanins showed a trend of first increasing and then decreasing, and reached the maximum value when the solid-liquid ratio was 1g:40mL (i.e., the mass ratio of blueberry pomace powder, pectinase, and citric acid buffer was 1g:15mg:40mL). The reason for the analysis may be that the appropriate increase of the extractant helps to promote the diffusion of anthocyanin molecules from the cell wall to the extract, thereby increasing the diffusion rate and the leaching rate. Too much extractant will not only increase the extraction cost and cause waste, but also increase the workload, so 1g:40mL is determined to be the optimal solid-liquid ratio.

[0048] Depend on Figure 1 B It can be seen that with the increase of enzyme amount, the extraction rate of anthocyanin also shows a trend of first rising and then falling. Properly increasing the amount of enzyme added can accelerate the destruction of cell walls and promote the dissolution of anthocyanin, but excessive pectinase residue will degrade anthocyanin and lead to a decrease in extraction rate. Therefore, 7.5mg pectinase is determined to be the best addition amount, that is, the mass ratio of blueberry pomace powder, pectinase, and citric acid buffer is 1g:15mg:40mL.

[0049] Depend on Figure 1 C shows that 60 min is the best enzymatic hydrolysis time, under which the extraction rate of anthocyanins is the highest. If the enzymatic hydrolysis time is too short, the cell wall destruction effect is not strong, and the anthocyanins cannot be fully leached. Prolonging the enzymatic hydrolysis time is not conducive to the stable existence of anthocyanins, because pectinase will cause anthocyanins to degrade during the extraction process.

[0050] Depend on Figure 1 D shows that when the ultrasonic power is 400W, the extraction rate is the highest, which is significantly different from the results of ultrasonic powers of 300W and 350W (P<0.05). However, if the power is further increased, the extraction rate will decrease. The possible reason is that under the action of ultrasound, a large number of bubbles will be formed and explode rapidly, which has a cell wall breaking effect, accelerates the breakage of cell walls and cell membranes, is conducive to the penetration of solvents, and improves the extraction efficiency. However, when the ultrasonic power is too high, too many bubbles are generated, which reduces the blasting impact force, weakens the cavitation effect, and reduces the extraction rate.

[0051] Depend on Figure 1 E shows that within 10-30 minutes, the extraction rate of anthocyanins gradually increases with the extension of ultrasonic time. After 30 minutes, the extraction rate decreases significantly. The reason may be that at 30 minutes, the cell wall and cell membrane are completely broken under the action of ultrasound, and anthocyanins can be fully dissolved from the cells. Continuing to extend the ultrasonic time will decompose anthocyanins or promote the penetration and dissolution of impurities, affecting the purity of anthocyanins. Therefore, the optimal ultrasonic time is determined to be 30 minutes.

[0052] Depend on Figure 1 It can be seen from F that when the concentration of chondroitin sulfate is changed, the embedding rate of anthocyanins changes significantly. When the concentration of chondroitin sulfate is in the range of 2-4 mg / mL, anthocyanins have a higher embedding rate, while below or above this range, the embedding rate decreases significantly, especially when the concentration of chondroitin sulfate is 1 mg / mL, the embedding rate is only 69.71%, and at 5 mg / mL, the embedding rate is only 71.33%, which is significantly lower than the embedding rate (84.87%) when the concentration of chondroitin sulfate is 4 mg / mL. Therefore, the concentration of chondroitin sulfate used in the subsequent preparation study is determined to be 4 mg / mL.

[0053] Response surface analysis experiment:

[0054] According to the results of single factor test and the principle of Box-Behnken combined experimental design, three factors with significant influence on anthocyanin extraction rate were selected: solid-liquid ratio (A), enzymatic hydrolysis time (B), and ultrasonic time (C) as independent variables, with coding levels of -1, 0, and 1, and anthocyanin extraction rate (Y) as the response value. The Box-Behnken design of 3 factors and 3 levels in Design-Expert software was used to optimize the optimal extraction process parameters. Each group of experiments was repeated three times and the average value was taken. The analysis results of the response surface are shown in Figure 2. Figure 2 As shown, the experimental data were analyzed and the coefficient of determination of the quadratic regression equation R 2 =0.9977, close to 1, indicating that the model has a high degree of fit; the coefficient of variation (CV) of the regression equation is 0.4107% <10%, indicating that the model is accurate and reliable for analyzing the changes in the response surface. The three-dimensional response surface diagram can intuitively see the impact of different influencing factors and their interactions on the response value. All response surfaces are convex surfaces with openings downward, indicating that the anthocyanin extraction amount Y has a maximum value. When the ultrasonic time is 30min, the curves of the solid-liquid ratio and the enzymatic hydrolysis time are steeper ( Figure 2 A), indicating that both have a significant effect on the anthocyanin extraction, but the contour lines are close to a circle, indicating that the interaction between the two is not significant; when the enzymatic hydrolysis time is 60min, the curve of the solid-liquid ratio is steeper, while the curve of the ultrasonic time is smoother ( Figure 2B), indicating that this factor has little effect on the anthocyanin extraction amount, and the contour line is close to an ellipse, indicating that the interaction between the two has a significant effect on the response value; when the material-liquid ratio is 1g:40mL, the curve of enzymatic hydrolysis time is steeper, but the curve of ultrasonic time is smoother ( Figure 2 C), indicating that this factor has little effect on the anthocyanin extraction, while the contour line is close to an ellipse, indicating that the interaction between the two has a significant effect on the response value; the best extraction conditions optimized by the software are: solid-liquid ratio 1g:42mL (that is, the mass ratio of blueberry pomace powder, pectinase, and citric acid buffer is 1g:15mg:42mL), enzymatic hydrolysis time 57min, and ultrasonic time 33min. Three parallel experiments were carried out under this condition, and the anthocyanin yield was measured to be 5.41mg / g, which is close to the predicted value of 5.528mg / g, indicating that the experimental results fit the model well, and the parameters optimized by the response surface are accurate and reliable.

[0055] Scanning electron microscopy and atomic force microscopy characterization:

[0056] Comparative sample 1: chondroitin sulfate (CS) powder;

[0057] Comparative sample 2: whey protein isolate (WPI) powder;

[0058] Comparative sample 3: anthocyanin powder (ACN) prepared in Example 1;

[0059] Comparative sample 4: The powder sample obtained after freeze-drying of the CS / WPI complex is recorded as the CS / WPI complex freeze-dried powder; the preparation process of the CS / WPI complex is as follows: weigh 200 mg of chondroitin sulfate, add 50 mL of distilled water to prepare a 4 mg / mL solution, stir at 600 rpm for 3 hours, and store at 4 ° C overnight to ensure full hydration to obtain a chondroitin sulfate solution. Weigh 1 g of whey protein isolate, add 50 mL of distilled water to prepare a 20 mg / mL solution, stir at 600 rpm for 3 hours, and store at 4 ° C overnight to ensure full hydration to obtain a whey protein isolate solution. The pH values ​​of the obtained whey protein isolate solution and chondroitin sulfate solution were adjusted to 3.0 with 1.5 mol / L HCl solution, respectively. Subsequently, the chondroitin sulfate solution was added dropwise to the whey protein isolate solution, stirred at 600 rpm for 1 hour, freeze-dried for 48 hours, and the freeze-dried sample was ground and sealed to obtain a CS / WPI complex, which was stored in a refrigerator at -20 ° C.

[0060] Depend on Figure 3As shown, the comparative samples 1-4 and Example 1 were observed by scanning electron microscope. Chondroitin sulfate CS was agglomerated (Figure A), and the microstructures of whey protein isolate WPI (Figure B) and anthocyanin ACN (Figure C) were mostly nearly spherical. After WPI and CS were combined in the CS / WPI complex, the spherical structure of the protein was destroyed, showing a typical smooth lamellar structure (Figure D), with small crystal fragments attached to the surface. This may be due to the precipitation of some undissolved WPI molecules. Therefore, the addition of polysaccharides changed the surface shape of WPI by promoting protein aggregation. The CS / WPI@ACN nanocomposite prepared in Example 1 showed a dense block-like wrinkled structure (Figure E), and small spherical protrusions were observed under a high-power microscope (Figure F). This observation showed that ACN had been successfully embedded in the protein-polysaccharide complex.

[0061] Figure 4 The results of atomic force microscopy are shown, comparing the CAN of sample 3 ( Figure 4 A1, A2), CS / WPI of comparison sample 4 ( Figure 4 B1, B2) and CS / WPI@ACN nanocomposites of Example 1 ( Figure 4 AFM images of (C1, C2) show the two-dimensional and three-dimensional structures and surface roughness of different materials. Microstructural analysis shows that ACN, CS / WPI and CS / WPI@ACN exhibit different morphologies, with ACN particles uniformly distributed, CS / WPI having an irregular block structure, and CS / WPI@ACN in granular form. These observations suggest that ACN acts as a bridging agent and promotes the interaction between CS and WPI. The average surface roughness Ra values ​​of ACN, CS / WPI and CS / WPI@ACN are 0.21nm, 0.31nm and 0.11nm, respectively, and the root mean square roughness Rq values ​​are 0.57nm, 1.22nm and 0.26nm, respectively. The addition of ACN reduces the Rq and Ra values ​​of CS / WPI@ACN, indicating a reduction in surface roughness. Therefore, the addition of ACN allows CS and WPI to interact, resulting in a uniform and stable particle system.

[0062] The antioxidant properties before and after embedding were tested, such as Figure 5 A. Figure 5 As shown in Figure 2, when the anthocyanin concentration was 0.6 mg / mL, the scavenging rates of free anthocyanin on DPPH and ABTS free radicals were 74.55% and 82.56%, respectively, while the scavenging rates of CS / WPI@ACN on DPPH and ABTS free radicals were 91.24% and 88.79%, respectively, indicating that the CS / WPI@ACN nanocomposite can enhance the antioxidant activity of anthocyanin.

[0063] The simulated digestion process before and after embedding was tested, and the results were as follows Figure 5C. The degradation and limited bioavailability of ACN during simulated gastric digestion are mainly caused by the alkaline environment of the intestine. After the simulated oral digestion, the retention rate of free anthocyanins was 43.36%, and the retention rate of embedded anthocyanins was 44.43%, with no significant difference between the two. Within 2 hours of gastric digestion, due to the presence of digestive enzymes in the stomach, free anthocyanins were further degraded, and the retention rate further dropped to 33.18%. After embedding, the retention rate of anthocyanins was 54.87%. The increase in this value is due to the fact that the anthocyanins degraded during the oral digestion stage are free anthocyanins remaining on the surface of the complex, and a large amount of anthocyanins are embedded in the CS / WPI complex for protection. Therefore, when entering the gastric juice, the presence of digestive enzymes causes part of the CS / WPI@ACN structure to be destroyed, releasing the anthocyanins therein. However, due to the acidic environment of the gastric juice, anthocyanins can exist stably. Therefore, at this stage, the retention rate of anthocyanins is increased compared with that of free anthocyanins, and there is a significant difference. After entering the alkaline intestinal environment for 2 hours, anthocyanins will undergo further degradation, and the retention rate of free anthocyanins will drop to 23.52%, while that of embedded anthocyanins is 44.68%. The higher retention rate indicates that even after being degraded by the intestinal environment, some anthocyanins are still retained, which can achieve a sustained release effect in the intestine and further play a role in regulating intestinal flora.

[0064] like Figure 6 As shown in the figure, the stability of free anthocyanins and encapsulated anthocyanins under adverse environmental conditions was compared and analyzed. First, the effect of illumination time on anthocyanins was investigated. Free anthocyanins and CS / WPI@ACN were irradiated with ultraviolet light at room temperature to simulate illumination conditions. After a period of time, the retention rate of free anthocyanins was 9.74%, while the retention rate of encapsulated anthocyanins was 39.51% ( Figure 6 A), significantly higher than the free anthocyanin group, which shows that under strong light conditions, CS / WPI@ACN can effectively protect anthocyanins and delay the loss of anthocyanins. Since anthocyanin products are extremely sensitive to light, the cost of storage and sales is increased. Therefore, enhancing the stability of anthocyanins to light conditions is very important for the application of anthocyanin products. As an antioxidant, ascorbic acid has a very obvious degradation effect on anthocyanins, and ascorbic acid is added to most foods to enhance the flavor and functional properties of the food. Therefore, the applicant tested the retention rate of anthocyanins in the presence of ascorbic acid, and the results are as follows. Figure 6As shown in Figure B, after 7 days of testing, the retention rate of anthocyanins in the free group was 18.97%, and the retention rate of anthocyanins in the embedded group was 59.02%, which was significantly higher than that in the free group, indicating that the presence of CS / WPI can help anthocyanins resist the damage of ascorbic acid, a strong oxidant. Finally, since high-temperature sterilization and other processing processes are often involved in food processing, the applicant analyzed and determined the stability of anthocyanins under high temperature conditions of 90°C. The results are shown in Figure 2. Figure 6 As shown in Figure C, after high-temperature heating for 3 hours, the retention rate of anthocyanins in the free group was 50.14%, while the retention rate of anthocyanins in the embedded group was 64.77%. This result shows that the thermal stability of anthocyanins is enhanced after embedding.

[0065] The purified anthocyanin solution sample obtained in Example 1 was subjected to high performance liquid chromatography analysis, and the results are shown in Table 1. As can be seen from Table 1, the content of malvidin-3-O-glucoside is the highest. Compared with other existing technologies, the use of AB-8 macroporous resin to purify blueberry anthocyanins can only achieve a yield of 11.17%. The extraction method of the present invention can effectively increase the yield of malvidin-3-O-glucoside (75.71%). Studies have shown that malvidin-3-O-glucoside can bind well to NF-kB, TLR4 and MyD88 and downregulate their expression, thereby effectively preventing and treating Alzheimer's disease. However, since cyanidin-3-O-glucoside can be widely studied at present, there is still a lack of systematic research on the mechanism and therapeutic effect of malvidin-3-O-glucoside. The method of the present invention can efficiently extract malvidin-3-O-glucoside and improve its yield, which is conducive to its application in more clinical research fields.

[0066] Table 1 HPLC analysis results of the purified anthocyanin crude extract samples of Example 1

[0067]

[0068]

[0069] Comparative Example 1

[0070] The preparation methods of various nanocomposites are basically the same as those of Example 1, except that in step (3), the whey protein isolate-chondroitin sulfate combination is replaced with other combinations, and the resulting embedding rates are shown in Table 2. As can be seen from Table 2 (including the whey protein isolate-chondroitin sulfate combination of Example 1), when other combinations are used, the embedding rate is significantly reduced.

[0071] Table 2 Results of embedding efficiency of nanocomposites prepared in Example 1 and Comparative Example 1

[0072]

[0073] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite, characterized in that: The following steps are involved: (1) pre-freezing blueberry pomace, then freeze-drying, grinding into powder, sieving to obtain blueberry pomace powder, and storing at -20°C to -4°C in the dark; (2) weighing blueberry pomace powder and pectinase and adding them to citric acid buffer, wherein the addition ratio of the blueberry pomace powder, pectinase and citric acid buffer is 1 g: 10 mg to 20 mg: 30 mL to 60 mL, shaking in a water bath at a temperature of 30° C. to 45° C. for enzymolysis, then removing and inactivating the enzyme, ultrasonicating, centrifuging and filtering the supernatant to obtain anthocyanin crude extract, purifying and freeze-drying to obtain anthocyanin powder; (3) Dissolving whey protein isolate and chondroitin sulfate in water respectively and stirring once, storing at 0°C to -4°C overnight to fully hydrate, thereby obtaining a whey protein isolate solution and a chondroitin sulfate solution, and adjusting the pH values ​​of the obtained whey protein isolate solution and chondroitin sulfate solution to 3.0 to 3.5 respectively with acid solution; dissolving anthocyanin powder in water to obtain an anthocyanin solution, adding the anthocyanin solution dropwise to the chondroitin sulfate solution, stirring twice, and adding the whey protein isolate solution dropwise, stirring three times, standing, and centrifuging to remove free anthocyanins, taking the precipitate for freeze-drying, and then grinding and crushing, and sealing to obtain a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite.

2. The method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite according to claim 1, characterized in that: In step (3), the mass ratio of the whey protein isolate, chondroitin sulfate and anthocyanin freeze-dried powder is 15-16:3-4:

1.

3. The method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite according to claim 1, characterized in that: In step (1), the pre-freezing temperature is 0°C to -20°C, the pre-freezing time is 5h to 10h, the freeze-drying temperature is -50°C to -60°C, the freeze-drying time is 24h to 48h, and the sieving is through a 60-mesh to 80-mesh sieve.

4. The method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite according to claim 1, characterized in that: In step (2), the concentration of citric acid in the citric acid buffer is 0.1 mol / L to 1 mol / L, and the pH value of the citric acid buffer is 3.0 to 3.5; in step (3), the acid solution is HCl solution, and the concentration of the HCl solution is 0.5 mol / L to 1.5 mol / L.

5. The method for preparing the chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex according to any one of claims 1 to 4, characterized in that: In step (2), the enzymatic hydrolysis time is 50 min to 60 min, the enzyme inactivation temperature is 80°C to 100°C, the enzyme inactivation time is 3 min to 7 min, the ultrasonic power is 350 W to 450 W, the ultrasonic time is 20 min to 40 min, the centrifugal force is 8000 g to 10000 g, the centrifugal time is 5 min to 15 min, and the pore size of the filter membrane for supernatant filtration is 0.45 μm to 0.22 μm.

6. The method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite according to any one of claims 1 to 4, characterized in that: In step (3), the rotation speed of the first stirring is 300rpm~600rpm, the time of the first stirring is 3h~5h, the rotation speed of the second stirring is 300rpm~600rpm, the time of the second stirring is 1h~3h, the rotation speed of the third stirring is 300rpm~600rpm, and the time of the third stirring is 1h~2h.

7. The method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomposite according to any one of claims 1 to 4, characterized in that: In step (3), the standing time is 4h to 8h, the centrifugal temperature is -4°C to 0°C, the centrifugal speed is 8000rpm to 10000rpm, the centrifugal time is 5min to 15min, the freeze-drying cold trap temperature is -50°C to -60°C, the freeze-drying time is 24h to 48h, and the obtained chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex is stored at -20°C to -4°C in the dark.

8. A chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex prepared by the method for preparing a chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex according to any one of claims 1 to 7.

9. Use of the chondroitin sulfate / whey protein isolate@anthocyanin nanocomplex as claimed in claim 8 in the field of food and cosmetics.

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