Resveratrol microcapsule and preparation method thereof

Abstract

The invention discloses a resveratrol microcapsule and a preparation method thereof. According to the resveratrol microcapsule, cross-linked protein is used as a microcapsule wall material, resveratrol is used as a microcapsule core material, and the mass ratio of the cross-linked protein to the resveratrol is (6-14): 1. According to the invention, the gluten protein and the peanut protein are used as raw materials, and the cross-linked protein prepared by an ultrasonic-assisted enzyme method is used as a wall material, so that the chemical stability, the water solubility and the bioavailability of the resveratrol can be improved. According to the method, the gluten protein and the peanut protein are used as raw materials, and an ultrasonic-assisted enzyme method is matched, so that intramolecular and intermolecular crosslinking of heterologous protein is realized, the protein forms a spatial network structure, the thermal stability and water (oil) holding capacity of the protein are improved, the emulsibility and foamability of the protein are improved, and the quality of a protein product is improved. Compared with single protein embedding, the cross-linked protein can achieve a better protection effect on the resveratrol.

Description

Technical Field

[0001] The invention belongs to the technical field of microcapsule embedding, and in particular relates to a resveratrol microcapsule and a preparation method thereof. Background Art

[0002] Resveratrol is a natural polyphenol found in red wine, grapes and peanuts, and has a variety of biological activities, such as antioxidant, cardioprotective, anti-inflammatory and anti-cancer activities. However, resveratrol has many pharmacokinetic limitations, such as resveratrol is a Class II drug in the Biopharmaceutical Classification System (BCS), which has the characteristics of poor water solubility (0.03g / L) and high permeability. Resveratrol will be absorbed by intestinal cells in large quantities after oral administration, and only a small part of the ingested resveratrol can reach the blood and body tissues. In addition, due to its complex structure and high molecular weight, the oral bioavailability of trans-resveratrol is about 12%. These poor pharmacokinetic properties, coupled with natural defects such as poor water solubility and unstable properties, make it difficult for the human body to absorb and utilize it directly after consumption. Therefore, maximizing the bioavailability of resveratrol has become a difficult problem in the development of its functional foods and health products.

[0003] Attempts have been made to improve the solubility of resveratrol by incorporating it into various nanocarriers, which are prepared using a single protein wall material or various nanocarriers, including liposomes, solid lipid nanodextrins, and polymeric micelles. However, these methods have some disadvantages, such as poor stability (such as liposomes), complex procedures (polymer nanoparticles), and high production costs (such as cyclodextrins and polymers). In recent years, resveratrol has been encapsulated in mesoporous silica nanoparticles (MSNs), which has improved its solubility and enhanced anticancer targeting. Although these new inorganic nanocarriers have been extensively studied, their production costs are high and their biodegradability is still under investigation. Therefore, for nutraceuticals that require high-dose delivery, such as resveratrol, there is an urgent need for cost-effective and high-quality delivery vehicles. Summary of the invention

[0004] The purpose of the present invention is to provide a resveratrol microcapsule and a preparation method thereof.

[0005] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a resveratrol microcapsule, which uses heterologous cross-linked plant protein as a microcapsule wall material and resveratrol as a microcapsule core material, and the mass ratio of the cross-linked protein to the resveratrol is 6 to 14:1.

[0007] Furthermore, the cross-linked protein is gluten protein and peanut protein after cross-linking reaction.

[0008] Furthermore, the mass ratio of gluten protein to peanut protein is 1-2:1-3, preferably 1:2.

[0009] In a second aspect, the present invention provides a method for preparing the resveratrol microcapsules described in the first aspect, comprising the following steps:

[0010] (1) preparing cross-linked plant protein: adding gluten protein and peanut protein into phosphate buffer to prepare a protein solution with a mass concentration of 2 to 10 g / 100 mL, adding glutamine aminotransferase, ultrasonic treatment, and vacuum freeze drying after the protein is pre-frozen to obtain a wall material;

[0011] (2) dispersing the cross-linked plant protein in deionized water to prepare a cross-linked protein solution, stirring, dissolving resveratrol in ethanol to prepare an ethanol-resveratrol solution, adding the ethanol-resveratrol solution dropwise into the cross-linked protein solution, mixing, and then stirring the mixture in the dark, and removing the ethanol by rotary evaporation to obtain a resveratrol-encapsulated complex; and then freeze-drying the sample to obtain a microcapsule powder.

[0012] Furthermore, the pH of the phosphate buffer is 6-8.

[0013] Furthermore, the mass of the transglutaminase is 0.25% to 1.5% of the sum of the mass of gluten protein and peanut protein.

[0014] Furthermore, the ultrasonic treatment conditions are: ultrasonic treatment at 25-150 W / L at 30-50° C. for 30-90 min at a 25kHz / 40kHz dual-frequency ultrasonic time ratio of 10:50-20:10 (s:s).

[0015] Furthermore, the concentration of the cross-linked protein solution is 1-2 mg / mL.

[0016] Furthermore, the concentration of the ethanol-resveratrol solution is 1-2 mg / mL.

[0017] Furthermore, in step 2), the mixture is stirred at 30-70° C. in the dark for 20-120 min.

[0018] The advantages and beneficial effects of the present invention are:

[0019] The present invention uses gluten protein and peanut protein as raw materials and adopts ultrasonic enzyme method to assist in preparing cross-linked proteins as wall materials, which can improve the chemical stability, water solubility and bioavailability of resveratrol. The present invention uses gluten protein and peanut protein as raw materials and cooperates with ultrasonic assisted enzyme method to achieve intramolecular and intermolecular cross-linking of heterologous plant proteins, modify proteins to form a spatial network structure, improve the thermal stability and water holding capacity of proteins, improve emulsification and foaming properties, and improve the quality of protein products. Compared with single protein embedding, cross-linked proteins can play a better protective role on resveratrol.

[0020] The present invention adopts ultrasound-assisted enzyme method to prepare cross-linked protein-embedded resveratrol microcapsule powder, which has the advantages of mild conditions, non-toxicity and substrate specificity, can improve the thermal stability and water (oil) holding capacity of protein, help to form a strong gel, improve the quality of protein products, and also improve emulsification and foaming properties. The protein function is improved by changing the internal structure and intermolecular forces of protein molecules. The modification effect of the present invention is significant, non-toxic and harmless, and the nutrients are retained intact. The wall material is made by using the ultrasound-assisted enzyme method of the present invention to cross-link proteins, so that the wall material can have a denser network structure, and the embedding effect of resveratrol is better and the efficiency is higher.

[0021] The present invention uses only a small amount of chemical reagents in the ultrasonic preparation process, is green and environmentally friendly, has high safety, and has a more concise and efficient process flow, which is easy to operate and easy to control. The operator only needs to set the ultrasonic parameters to perform automated operations, which reduces manual intervention and reduces the risk of operating errors. Accurate operations can be performed according to different protein raw material characteristics and product requirements to ensure the stability and repeatability of the preparation process. The preparation time is greatly shortened. At the same time, due to the low energy consumption, the energy consumption and corresponding environmental pollution are also indirectly reduced. In terms of subsequent waste treatment, it is also relatively simple, which is beneficial to environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron micrograph of resveratrol powder;

[0023] Figure 2 This is a transmission electron micrograph of peanut-gluten cross-linked protein powder;

[0024] Figure 3 This is a transmission electron microscope image of the microcapsule powder prepared by the present invention;

[0025] Figure 4 This is the infrared spectrum of microcapsule powder, resveratrol (core material), and cross-linked protein (wall material). DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0027] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0028] Example 1

[0029] This embodiment provides a method for preparing resveratrol microcapsules, comprising the following steps:

[0030] 1) gluten protein and peanut protein were mixed in a mass ratio of 1:2 and then a protein solution with a mass concentration of 8 g / 100 mL was prepared in a phosphate buffer solution at pH = 7;

[0031] 2) adding transglutaminase, the mass of which is 1.25% of the total mass of gluten protein and peanut protein;

[0032] 3) 25kHz / 40kHz dual-frequency ultrasound time ratio 10:10 (s:s) at 125W / L at 40°C for 60min;

[0033] 4) freeze-drying the cross-linked protein in vacuum;

[0034] 5) Disperse the cross-linked protein in deionized water to prepare a cross-linked protein solution with a concentration of 1 mg / mL, and stir at 37°C for 30 minutes;

[0035] 6) dissolving resveratrol in 90% ethanol to prepare a solution with a concentration of 1 mg / mL;

[0036] 7) adding the ethanol-resveratrol solution dropwise into the cross-linked protein solution, stirring at 700 rpm to mix, the mass ratio of the polymer cross-linked protein to the drug resveratrol being 12:1;

[0037] 8) The mixture was then stirred at 40°C in the dark for 60 min;

[0038] 9) evaporating ethanol under reduced pressure in a rotary evaporator at 45° C. to obtain a resveratrol-encapsulated complex;

[0039] 10) then freeze-drying the sample to obtain microcapsule powder;

[0040] 11) Dissolve 50 mg of microcapsule powder in 5 mL of ethyl acetate. According to the method of Liu Xin (Liu Xin, Deng Jiao, Li Jiexin, et al. Response surface methodology optimization of resveratrol / sodium lac microcapsules and their release performance [J]. Food Industry Science and Technology, 2019, 40(17): 121-126. DOI: 10.13386 / j.issn1002-0306.2019.17.020.), the embedding efficiency was measured to be 95.91% and the drug loading was 7.99.

[0041] Resveratrol, peanut-gluten cross-linked protein prepared in step 3), and microcapsule powder prepared in step 10) were dispersed in deionized water according to the method of Naisarg Pujara (Pujara N, Jambhrunkar S, Wong KY, et al. Enhanced colloidalstability, solubility and rapid dissolution of resveratrol by nanocomplexation with soy protein isolate [J]. Journal of Colloid and Interface Science, 2017, 488: 303-308.) and placed on a 300-mesh Cu grid coated with heavy carbon. The sample was negatively stained with 2% uranyl acetate and then analyzed at 200 kV.

[0042] Figure 1 is a transmission electron microscopy image of resveratrol. Figure 1 As shown, it can be seen that the original state of resveratrol is long strips. Figure 2 This is a transmission electron micrograph of peanut-gluten cross-linked protein. Figure 2 It can be seen that the cross-linked protein is granular. Figure 3 The transmission electron microscope image of the microcapsule prepared by the present invention is Figure 3 It can be seen that after embedding, the cross-linked protein used in the present invention embeds the long strips of resveratrol into round particles, and the round particles are closely adjacent, indicating that the cross-linked protein used in the present invention has a good embedding effect. Figure 4 The infrared spectra of microcapsule powder, resveratrol and cross-linked protein are shown in Figure 2. Figure 4 As shown in the figure, the vibration curve of the microcapsule is similar to that of the cross-linked protein, but the vibration intensity is similar to that of resveratrol, which is far less intense than that of the cross-linked protein. -1 The OH stretching absorption band of the alcohol group is shown at 1600-1700 cm -1) reflects the changes in protein C=O and a few CN, microcapsules and cross-linked proteins at 1600-1700 cm -1 The vibration amplitude is the same, both are 1659cm -1 , indicating that the outermost layer of the microcapsules is protein. This confirms that resveratrol is embedded in the cross-linked protein and maintains its original chemical form.

[0043] Example 2

[0044] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that in step 7), the mass ratio of the cross-linked protein to resveratrol is 14:1, and the rest is the same as embodiment 1.

[0045] The embedding efficiency of this embodiment was measured to be 96.57%, and the drug loading was 6.90%.

[0046] Example 3

[0047] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 40° C. in the dark for 100 min. The rest is the same as embodiment 1.

[0048] The embedding efficiency of this embodiment was measured to be 97.74%, and the drug loading was 8.15%.

[0049] Example 4

[0050] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 50° C. in the dark for 60 min. The rest is the same as embodiment 1.

[0051] The embedding efficiency of this embodiment was measured to be 94.75%, and the drug loading was 7.90%.

[0052] Example 5

[0053] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 30° C. in the dark for 80 min. The rest is the same as embodiment 1.

[0054] The embedding efficiency of this embodiment was measured to be 96.35%, and the drug loading was 8.03%.

[0055] Example 6

[0056] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 40° C. in the dark for 20 min. The rest is the same as embodiment 1.

[0057] The embedding efficiency of this embodiment was measured to be 91.72%, and the drug loading was 7.64%.

[0058] Example 7

[0059] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 40° C. in the dark for 40 min. The rest is the same as embodiment 1.

[0060] The embedding efficiency of this embodiment was measured to be 94.04%, and the drug loading was 7.84%.

[0061] Example 8

[0062] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that in step 7), the mass ratio of cross-linked protein to resveratrol is 14:1, and in step 8), the mixture is stirred at 50° C. for 80 min in the dark. The rest is the same as in embodiment 1.

[0063] The embedding efficiency of this embodiment was measured to be 96.47%, and the drug loading was 6.90%.

[0064] Example 9

[0065] This embodiment provides a method for preparing resveratrol microcapsules. The only difference between this embodiment and embodiment 1 is that step 8) is to stir the mixture at 40° C. in the dark for 80 min. The rest is the same as embodiment 1.

[0066] The embedding efficiency of this embodiment was measured to be 97.59%, and the drug loading was 8.13%.

[0067] Comparative Example 1

[0068] This comparative example provides a method for preparing resveratrol microcapsules. The only difference between this comparative example and Example 3 is that different microcapsule wall materials are used. In this comparative example, gluten protein is removed and the wall material is entirely made of a single protein, peanut protein. The rest is the same as Example 3.

[0069] The embedding efficiency of this comparative example was measured to be 14.21%, and the drug loading was 1.18%.

[0070] Comparative Example 2

[0071] This comparative example provides a method for preparing resveratrol microcapsules. The only difference between this comparative example and Example 3 is that different microcapsule wall materials are used. In this comparative example, peanut protein is removed and the wall material is made entirely of a single protein, gluten protein. The rest is the same as Example 3.

[0072] The embedding efficiency of this comparative example was measured to be 34.40%, and the drug loading was 2.87%.

[0073] Comparative Example 3

[0074] This comparative example provides a method for preparing resveratrol microcapsules. The difference between this comparative example and Example 3 is that the cross-linking method of gluten protein and peanut protein is different. In step 3) of this comparative example, the cross-linking method of gluten protein and peanut protein is only thermal cross-linking in a 40° C. water bath for 60 min, not cross-linking in an ultrasonic field. The rest is the same as Example 3.

[0075] The embedding efficiency of this comparative example was measured to be 85.13%, and the drug loading was 7.09%.

[0076] Comparative Example 4:

[0077] This comparative example provides a method for preparing resveratrol microcapsules, comprising the following steps:

[0078] 1) Weigh 2.0 g of zein powder and dissolve it in 100 mL of 85% (w / v) ethanol solution, and stir it with a magnetic stirrer at 500 r / min for 1 h to obtain a zein ethanol solution.

[0079] 2) Then, under stirring at 700 r / min, the mass ratio of zein to resveratrol was 10:1.2, and after stirring for 1 hour, the mixture was centrifuged at 3000 rpm for 10 minutes to remove insoluble impurities.

[0080] 3) Under stirring at a speed of 900 r / min, the resveratrol-zein solution was slowly dispersed into double distilled water (1.0 M HCl adjusted to a predetermined pH) at a ratio of 1:4 using a syringe to obtain a resveratrol-zein nanoparticle dispersion.

[0081] 4) Dissolve in alcohol at 45°C and remove ethanol by rotary evaporation.

[0082] 5) An equal volume of pH 4.0 distilled water was added to the volume before rotary evaporation, and the nanoparticle dispersion was stirred and dispersed (700 r / min) into the pectin solution at a ratio of 4:5, and stirring was continued for 4 minutes to obtain zein / pectin nanoparticles loaded with resveratrol.

[0083] 6) Centrifuge (3000r / min, 10min) to remove large particles, which is the desired sample.

[0084] The embedding efficiency of this comparative example was measured to be 86.37%, and the drug loading was 7.49%.

[0085] Comparative Example 5

[0086] This comparative example provides a method for preparing resveratrol microcapsules, comprising the following steps:

[0087] 1) Take 2 mL of 0.3% chitosan solution and adjust the pH to 4.0.

[0088] 2) Add resveratrol solution (equivalent to 2 mg of resveratrol).

[0089] 3) The probe was ultrasonicated for 10 seconds in an ice bath, and a sodium tripolyphosphate solution with a mass concentration of 1 g / L was added dropwise under stirring to form nanoparticles.

[0090] 4) Continue stirring for 2 h to obtain the product.

[0091] 5) The embedding efficiency was measured to be 63.3% and the drug loading was 7.6%.

[0092] According to the comparison of Examples 1-10, the resveratrol microcapsules prepared by the method of the present application can obtain a higher embedding rate and drug loading, among which the embedding rate and drug loading of Example 3 are the highest, reaching 97.74% and 8.15% respectively. This is because the cross-linked protein prepared by using gluten protein and peanut protein as raw materials and ultrasonic-assisted enzyme method, the raw materials and the preparation method cooperate together, produce a synergistic effect, realize the cross-linking within and between protein molecules, modify the protein to form a spatial network structure, improve the thermal stability and water holding capacity of the protein, improve the emulsification and foaming properties, and improve the quality of protein products. Therefore, it can play a better protective role on resveratrol, and obtain a higher embedding rate and drug loading.

[0093] By comparing Comparative Examples 1 and 2 with Example 3, it can be seen that both Comparative Examples 1 and 2 use a single protein as the wall material, lacking the synergistic effect between gluten protein and peanut protein, and the spatial three-dimensional structure and functional properties of the protein are not as good as those after cross-linking. Therefore, the embedding rate and drug loading capacity are significantly reduced.

[0094] By comparing Comparative Example 3 with Example 3, it can be seen that ultrasonic treatment will cause the liquid and dissolved gas in the system to interact to form an "acoustic cavitation" phenomenon. The cavitation bubbles will cause various physical and chemical reactions in the medium when vibrating and breaking, promote material quality and energy transmission, and improve the functional properties of protein. Since Comparative Example 3 did not undergo ultrasonic-assisted treatment, the cross-linking effect was weakened and the spatial network structure was reduced, resulting in a significant decrease in both the encapsulation rate and the drug loading capacity.

[0095] By comparing Comparative Example 4 with Example 3, it can be seen that when protein and pectin nanoparticles synergistically encapsulate resveratrol, the protein and pectin synergize without modification to form a spatial network structure, and the functional properties of the protein are not improved, resulting in a significant decrease in both the encapsulation rate and the drug loading capacity.

[0096] By comparing Comparative Example 5 with Example 3, it can be seen that when only chitosan is used to encapsulate resveratrol, the wall material chitosan does not have the high thermal stability, water (oil) holding capacity, emulsification and foaming properties of the cross-linked protein, resulting in a significant decrease in the encapsulation rate and drug loading.

[0097] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A resveratrol microcapsule, characterized in that: The cross-linked plant protein is used as the microcapsule wall material, and resveratrol is used as the microcapsule core material. The mass ratio of the cross-linked protein to the resveratrol is 6-14:

1.

2. The resveratrol microcapsule according to claim 1, characterized in that: The cross-linked protein is gluten protein and peanut protein after cross-linking reaction.

3. The resveratrol microcapsule according to claim 2, characterized in that: The mass ratio of gluten protein to peanut protein is 1-2: 1-3, preferably 1:

2.

4. The method for preparing resveratrol microcapsules according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of cross-linked protein: adding gluten protein and peanut protein into phosphate buffer to prepare a protein solution with a mass concentration of 2-10 g / 100 mL, adding glutamine aminotransferase, ultrasonic treatment, and vacuum freeze-drying after the protein is pre-frozen to obtain the wall material; (2) Dispersing the cross-linked protein in deionized water to prepare a cross-linked protein solution and stirring it; dissolving resveratrol in ethanol to prepare an ethanol-resveratrol solution; dropping the ethanol-resveratrol solution into the cross-linked protein solution and mixing it; then stirring the mixture in the dark and removing the ethanol by rotary evaporation to obtain a resveratrol-encapsulated complex; then freeze-drying the sample to obtain a microcapsule powder.

5. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The pH value of the phosphate buffer is 6-8.

6. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The mass of the glutamine aminotransferase is 0.25% to 1.5% of the sum of the mass of gluten protein and peanut protein.

7. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The ultrasonic treatment conditions are: 25kHz / 40kHz dual-frequency ultrasonic time ratio 10:50-20:10 (s:s) at 25-150W / L at 30-50°C for 30-90 min.

8. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The concentration of the cross-linked protein solution is 1 to 2 mg / mL.

9. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The concentration of the ethanol-resveratrol solution is 1 to 2 mg / mL.

10. The method for preparing resveratrol microcapsules according to claim 4, characterized in that: The mixture in step 2) is stirred at 30-70° C. in the dark for 20-120 min.

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