Preparation method and application of high-absorptivity tea polyphenol microcapsule

Abstract

The invention belongs to the technical field of microcapsules, and particularly relates to a preparation method and application of a high-absorptivity tea polyphenol microcapsule. Chitosan and tea polyphenol are used as core layer materials, and shaddock peel protein is used as a shell layer material to prepare the microcapsule. According to the invention, protein-polysaccharide with high stability and good absorption effect is screened and optimized as a wall material to compound and embed the activity of the tea polyphenol, so that the prepared tea polyphenol microcapsule not only has higher stability after being digested by intestines and stomach in vivo, but also can improve the cell absorption rate of the tea polyphenol; the bioavailability of the tea polyphenol in a human body can be effectively improved. Besides, experiments prove that the tea polyphenol microcapsule provided by the invention has relatively high stability in application of different beverages, can effectively promote popularization and application of tea polyphenol products, and has relatively high market value.

Description

Technical Field

[0001] The invention belongs to the technical field of microcapsules, and in particular relates to a preparation method and application of tea polyphenol microcapsules with high absorption rate. Background Art

[0002] Green Tea Polyphenolsphenols (GTP) is the general name for polyphenols in tea. Tea polyphenols can be divided into flavanols, 4-hydroxyflavanols, anthocyanins, flavonoids, flavonols and phenolic acids. Flavanols are the main type of tea polyphenols, and catechins are the main type of flavanols. Tea polyphenols have active hydroxyl hydrogen in their molecular structure, which can terminate the chain reaction of free radicals, remove superoxide anion free radicals in the body, and prevent and delay the auto-oxidative decomposition of unsaturated fatty acids. Therefore, tea polyphenols have the effects of anti-oxidation, scavenging free radicals, enhancing immunity, lowering blood sugar, lowering blood lipids, etc., and are widely used in the food field.

[0003] However, tea polyphenols have the defects of poor stability and low system compatibility during application. Under the influence of factors such as light, temperature, pH, metal ions, etc., tea polyphenols are easily oxidized into o-quinones and diphenylphenol quinone substances, making them very easy to degrade during storage and processing. In addition, tea polyphenols are difficult to dissolve in the matrix of other products. If tea polyphenols are directly applied to the product system, they will be incompatible, which greatly limits the use of tea polyphenols.

[0004] Microencapsulation technology refers to the technology of using natural or synthetic polymer materials to encapsulate dispersed solid, liquid, and gaseous substances to form tiny particles with semi-permeable or sealed capsules. In recent years, microencapsulation technology has been widely used in food industry production to change the form of substances, protect sensitive ingredients, isolate active substances, reduce volatility, mix incompatible ingredients, and reduce the toxicity of certain chemical additives. At present, the use of microencapsulation technology to embed tea polyphenols can not only improve the stability of tea polyphenols, but also improve the system compatibility of tea polyphenols, solve the problem of the lack of special products for tea polyphenols, and promote the application of tea polyphenols.

[0005] Patent document CN103330213A discloses a tea polyphenol fat-soluble microcapsule and a preparation method thereof. The core material of the tea polyphenol fat-soluble microcapsule is composed of tea polyphenol and reduced glutathione, and the wall material of the tea polyphenol fat-soluble microcapsule is one or more of gum arabic, dextrin, corn syrup, ethyl cellulose, methyl cellulose, and hydroxypropyl methylcellulose phthalate. The prepared tea polyphenol fat-soluble microcapsule has the characteristics of fine average particle size and slow release of tea polyphenol, which greatly improves the antioxidant property and absorption efficiency of tea polyphenol.

[0006] Patent document CN118320739A discloses a core-shell microcapsule loaded with EGCG and its preparation method and application. The core-shell microcapsule loaded with EGCG uses grapefruit peel pectin and EGCG as core layer materials, zein as shell material, and uses coaxial electrospray technology to prepare core-shell microcapsules loaded with EGCG. The microcapsule can effectively improve the stability of EGCG in high temperature, neutral and alkaline environments.

[0007] Patent document CN111528270A discloses an antioxidant tea polyphenol microcapsule and a preparation method thereof. The tea polyphenol microcapsule is composed of tea polyphenol, gelatin and chitosan. The preparation method is as follows: ingredients; take 0.2 mol / L acetic acid-sodium acetate buffer solution, dissolve gelatin and chitosan in the acetic acid-sodium acetate buffer solution, dissolve tea polyphenol in the chitosan solution to prepare a tea polyphenol-chitosan solution; drip the tea polyphenol-chitosan solution into the gelatin solution, stir and heat at a temperature of 50-70°C for 15-30 minutes, centrifuge the tea polyphenol-gelatin-chitosan nanosuspension, precipitate, remove the supernatant, and freeze-dry to obtain the obtained tea polyphenol microcapsule. The prepared tea polyphenol microcapsule has high stability and sustained-release properties.

[0008] However, the current tea polyphenol microcapsule products mainly focus on the research of product stability and system compatibility, and there is less research on the absorption rate and bioavailability of the human body, which cannot guarantee the efficacy of tea polyphenol microcapsules in the human body. Therefore, researching and developing a tea polyphenol microcapsule with a high absorption rate is still a problem that needs to be solved urgently. Summary of the invention

[0009] In order to solve the defects of the prior art, the purpose of the present invention is to provide a method for preparing tea polyphenol microcapsules with high absorption rate. The present invention uses chitosan and tea polyphenol as core layer materials and uses naringin as shell layer material to prepare microcapsules. After a large number of exploratory experiments, the inventor screened and optimized protein-polysaccharide with high stability and good absorption effect as wall material composite to embed tea polyphenol activity, so that the prepared tea polyphenol microcapsules not only have high stability after gastrointestinal digestion in the body, but also can improve the cellular absorption rate of tea polyphenols, which can effectively improve the bioavailability of tea polyphenols in the human body.

[0010] The present invention provides a method for preparing tea polyphenol microcapsules with high absorption rate, comprising the following steps:

[0011] Step S1, drying and crushing the pomelo peel to obtain pomelo peel powder, mixing the pomelo peel powder with deionized water to obtain a pomelo peel powder solution, adjusting the pH to 10-12, extracting in a water bath at a temperature of 45-55° C. for 1-2 hours, centrifuging, taking the supernatant, adjusting the pH to 7, extracting in a water bath at a temperature of 45-55° C. for 20-40 minutes, centrifuging, taking the supernatant, adjusting the pH to 3-4, centrifuging, taking the solid, pre-freezing at -20° C., and vacuum freeze-drying to obtain pomelo peel protein;

[0012] Step S2, dissolving tea powder in phosphate buffer to obtain a tea solution, adding pectin cellulose complex enzyme to the tea solution for enzymolysis, then performing ultrasonic treatment, centrifugation, suction filtration, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0013] Step S3, dissolving the naringin obtained in step S1 in water to prepare a naringin protein aqueous solution with a mass concentration of 0.5-2%, magnetically stirring for 50-80 minutes, centrifuging, taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0014] Step S4, dissolving chitosan in water to prepare a chitosan aqueous solution with a mass concentration of 0.5-2%, stirring magnetically for 1-3 hours, then adding tea polyphenols, stirring in a water bath at a temperature of 45-55° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0015] Step S5, uniformly mix the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, adjust the pH to 6±0.1 to obtain a mixed solution III, add sodium benzoate to the mixed solution III, then perform ultrasonic treatment, precool and then freeze-dry to obtain.

[0016] Furthermore, the pH adjuster in step S1 is a 0.1 mol / L NaOH solution or a 0.1 mol / L HCl solution.

[0017] Furthermore, in the step S1, the material-liquid ratio of the pomelo peel powder to the deionized water is 1 g: (12-16) mL, and the centrifugal condition in the step S1 is: centrifugation for 8-15 min at a rotation speed of 4000-5000 r / min.

[0018] Furthermore, the pH value of the phosphate buffer in step S2 is 3.5, the added amount of the pectin-cellulose complex enzyme is 1.5% w / v of the tea solution, the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is 2:1, and the enzymolysis time is 4 to 5 hours.

[0019] Furthermore, the ultrasonic treatment conditions in step S2 are: ultrasonic treatment at a power of 500-700 W and a temperature of 50-70° C. for 25-35 min.

[0020] Furthermore, the centrifugation conditions in step S2 are: centrifugation at a speed of 2500-3500 r / min and a temperature of 18-25° C. for 4-6 min.

[0021] Furthermore, the centrifugation conditions in step S3 are: centrifugation at a rotation speed of 7500-8500 r / min and a temperature of 4° C. for 15-25 min.

[0022] Furthermore, the chitosan in step S4 is MacLean 850124 chitosan quaternary ammonium salt with a degree of substitution of 92%; and the mass concentration of the tea polyphenols in the chitosan aqueous solution is 0.4-0.6%.

[0023] Furthermore, in step S4, the volume ratio of the mixed solution I to the mixed solution II is 1:1.

[0024] Furthermore, the ultrasonic treatment conditions in step S4 are: the power density is 15-20 W / cm 3 , 5s on, 5s off, ultrasonic treatment for 4 to 6 minutes.

[0025] In addition, the present invention also provides the use of the tea polyphenol microcapsules prepared by the method for preparing the tea polyphenol microcapsules with high absorption rate in the preparation of foods or health products.

[0026] At present, the prior art generally uses microencapsulation technology to embed tea polyphenols to improve the stability of tea polyphenols. The embedding material of the microcapsule directly affects the stability of the microcapsules of tea polyphenols in the body, especially the stability after gastrointestinal digestion, and also affects the cellular absorption rate of tea polyphenols. In order to better improve the absorption rate of tea polyphenols in the human body, the inventors proposed the idea of ​​using protein-polysaccharide as a wall material to embed the active ingredients of tea polyphenols. During the experiment, the inventors found that embedding tea polyphenols with protein-polysaccharide as a wall material can better improve the stability of tea polyphenols in the human body, especially improve the stability of tea polyphenols after gastrointestinal digestion, which can ensure that tea polyphenols play a better role in the human body.

[0027] In addition, the inventors found in in-depth research that different types of proteins and polysaccharides affect the absorption rate of tea polyphenol microcapsules in the human body. The inventors unexpectedly found in the experiment that microcapsules were made by ultrasound at a pH of about 6 using chitosan and tea polyphenols at a specific concentration as the core layer material and naringin at a specific concentration as the shell layer material. The dynamic simulated gastrointestinal digestion experiment found that the tea polyphenol microcapsules were relatively stable in the simulated stomach and slowly released tea polyphenols in the simulated intestine. At the same time, the Caco-2 cell absorption experiment found that the tea polyphenol microcapsules had a high cell absorption rate, which can effectively solve the problem of low absorption rate of tea polyphenols in the human body.

[0028] Furthermore, the inventor applied the tea polyphenol microcapsules to different beverages. When the tea polyphenol microcapsules were applied to grapefruit juice beverages, the tea polyphenols had a high stability in the grapefruit juice beverages, especially when stored at 4°C, the loss rate of the tea polyphenols was the lowest. When the tea polyphenol microcapsules were applied to milk, after heating at 95°C for 1 hour, the tea polyphenols retention rate was still above 90%, indicating that the tea polyphenol microcapsules provided by the present invention have high stability in the application of different beverages, and can promote the promotion and application of tea polyphenol products.

[0029] In summary, compared with the prior art, the method for preparing high-absorption tea polyphenol microcapsules provided by the present invention has the following advantages:

[0030] (1) The method for preparing tea polyphenol microcapsules with high absorption rate provided by the present invention has the advantages of simple preparation process, abundant raw materials, convenient operation and high repeatability, and is suitable for large-scale promotion and application.

[0031] (2) The tea polyphenol microcapsules prepared by the method for preparing tea polyphenol microcapsules with high absorption rate provided by the present invention have the advantages of high in vivo stability and high absorption rate, and are a relatively ideal preparation process for tea polyphenol microcapsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The scanning electron micrograph of the tea polyphenol microcapsules prepared in Example 2, wherein: (a): naringin;

[0033] (b): Tea polyphenols; (c): Chitosan; (d): Tea polyphenols microcapsules.

[0034] Figure 2 This is the Fourier transform infrared spectrum of the tea polyphenol microcapsules prepared in Example 2.

[0035] Figure 3 CD images of naringin and microcapsules.

[0036] Figure 4 This is the thermogravimetric analysis diagram of the tea polyphenols microcapsules prepared in Example 2.

[0037] Figure 5 This is a graph showing the relationship between tea polyphenols retention rate and time under different storage conditions.

[0038] Figure 6 This is a graph showing the relationship between the retention rate of tea polyphenols and time after dynamic simulated gastrointestinal digestion.

[0039] Figure 7 The figure shows the results of cytotoxicity assay.

[0040] Figure 8 This is a graph showing the tea polyphenol retention rate of tea polyphenol microcapsules in grapefruit juice under different storage conditions.

[0041] Fig. 9 This is the tea polyphenol retention rate of tea polyphenol microcapsules in milk after heating at 95℃ for 1h. DETAILED DESCRIPTION

[0042] The present invention is further described below by describing specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention. The materials involved in the present invention are all food grade and can be obtained by commercial or conventional technical means in the art.

[0043] Example 1: A method for preparing tea polyphenol microcapsules with high absorption rate

[0044] Step S1, drying and crushing the pomelo peel, passing through a 100-mesh sieve to obtain pomelo peel powder, taking 50 g of the pomelo peel powder and mixing it with 650 mL of deionized water to obtain a pomelo peel powder solution, using a 0.1 mol / L NaOH solution to adjust the pH of the pomelo peel powder solution to 10, extracting it in a water bath at a temperature of 45° C. for 2 h, centrifuging it at a speed of 4000 r / min for 12 min, taking the supernatant, adjusting the pH of the supernatant to 7 with a 0.1 mol / L HCl solution, extracting it in a water bath at a temperature of 45° C. for 35 min, centrifuging it at a speed of 4000 r / min for 12 min, taking the supernatant, adjusting the pH of the supernatant to 4 with a 0.1 mol / L HCl solution, and centrifuging it at a speed of 4000 r / min.

[0045] r / min for 12 min, take the solid, pre-freeze at -20°C, and vacuum freeze-dry to obtain naringin;

[0046] Step S2, dissolving tea powder in a phosphate buffer with a pH of 3.5 to obtain a tea solution, adding pectin-cellulose complex enzyme to the tea solution for enzymatic hydrolysis for 4 hours, wherein the amount of pectin-cellulose complex enzyme added is 1.5% (w / v) of the tea solution, and the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is 2:1, then ultrasonically treating at a power of 500 W and a temperature of 70° C. for 35 minutes, centrifuging at a speed of 2500 r / min and a temperature of 22° C. for 6 minutes, filtering, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0047] Step S3, dissolving the naringin obtained in step S1 in water to prepare a naringin protein aqueous solution with a mass concentration of 1.2%, stirring with a magnetic force for 80 minutes, centrifuging for 18 minutes at a speed of 8500 r / min and a temperature of 4° C., taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0048] Step S4, dissolving chitosan in water, wherein the chitosan is MacLean 850124 chitosan quaternary ammonium salt, with a substitution degree of 92%, and preparing a chitosan aqueous solution with a mass concentration of 1.2%, stirring magnetically for 2.5 hours, then adding tea polyphenols until the concentration of the tea polyphenols reaches a mass concentration of 0.6%, stirring in a water bath at a temperature of 55° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0049] Step S5, uniformly mixing the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, wherein the volume ratio of the mixed solution I to the mixed solution II is 1:1, adjusting the pH to 6±0.1 to obtain a mixed solution III, adding 0.1% (w / v) sodium benzoate to the mixed solution III, and then heating the mixed solution III at a power density of 18 W / cm 3 , on for 5s and off for 5s, ultrasonically treat for 5min, and freeze-dry after precooling.

[0050] Example 2: A method for preparing tea polyphenol microcapsules with high absorption rate

[0051] Step S1, drying and crushing the pomelo peel, passing through a 100-mesh sieve to obtain pomelo peel powder, taking 50 g of the pomelo peel powder and mixing it with 750 mL of deionized water to obtain a pomelo peel powder solution, adjusting the pH of the pomelo peel powder solution to 11 with a 0.1 mol / L NaOH solution, extracting it in a water bath at a temperature of 50° C. for 1.5 hours, centrifuging it at a speed of 4500 r / min for 10 minutes, taking the supernatant, adjusting the pH of the supernatant to 7 with a 0.1 mol / L HCl solution, extracting it in a water bath at a temperature of 50° C. for 30 minutes, centrifuging it at a speed of 4500 r / min for 10 minutes, taking the supernatant, adjusting the pH of the supernatant to 4 with a 0.1 mol / L HCl solution, centrifuging it at a speed of 4500 r / min for 10 minutes, taking the solid, pre-freezing it at -20° C., and vacuum freeze-drying it to obtain pomelo protein;

[0052] Step S2, dissolving tea powder in a phosphate buffer with a pH of 3.5 to obtain a tea solution, adding pectin cellulose complex enzyme to the tea solution for enzymolysis for 4.5 hours, wherein the amount of pectin cellulose complex enzyme added is 1.5% (w / v) of the tea solution, and the mass ratio of pectinase to cellulase in the pectin cellulose complex enzyme is 2:1, then ultrasonically treating at a power of 600 W and a temperature of 60° C. for 30 minutes, centrifuging at a speed of 3000 r / min and a temperature of 20° C. for 5 minutes, filtering, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0053] Step S3, dissolving the naringin obtained in step S1 in water to prepare a naringin aqueous solution with a mass concentration of 1%, stirring with a magnetic force for 60 minutes, centrifuging for 20 minutes at a speed of 8000 r / min and a temperature of 4° C., taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0054] Step S4, dissolving chitosan in water, wherein the chitosan is MacLean 850124 chitosan quaternary ammonium salt, with a substitution degree of 92%, and preparing a chitosan aqueous solution with a mass concentration of 1%, stirring magnetically for 2 hours, and then adding tea polyphenols until the concentration of the tea polyphenols reaches a mass concentration of 0.5%, stirring in a water bath at a temperature of 50° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0055] Step S5, uniformly mixing the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, wherein the volume ratio of the mixed solution I to the mixed solution II is 1:1, adjusting the pH to 6±0.1 to obtain a mixed solution III, adding 0.1% (w / v) sodium benzoate to the mixed solution III, and then heating the mixed solution III at a power density of 20 W / cm 3 , on for 5s and off for 5s, ultrasonically treat for 5min, and freeze-dry after precooling.

[0056] Example 3: A method for preparing tea polyphenol microcapsules with high absorption rate

[0057] Step S1, drying and crushing the pomelo peel, passing through a 100-mesh sieve to obtain pomelo peel powder, taking 50 g of the pomelo peel powder and mixing it with 800 mL of deionized water to obtain a pomelo peel powder solution, adjusting the pH of the pomelo peel powder solution to 12 with a 0.1 mol / L NaOH solution, extracting it in a water bath at a temperature of 55° C. for 1 hour, centrifuging it at a speed of 5000 r / min for 8 minutes, taking the supernatant, adjusting the pH of the supernatant to 7 with a 0.1 mol / L HCl solution, extracting it in a water bath at a temperature of 55° C. for 25 minutes, centrifuging it at a speed of 5000 r / min for 8 minutes, taking the supernatant, adjusting the pH of the supernatant to 4 with a 0.1 mol / L HCl solution, centrifuging it at a speed of 5000 r / min for 8 minutes, taking the solid, pre-freezing it at -20° C., and vacuum freeze-drying it to obtain pomelo protein;

[0058] Step S2, dissolving tea powder in a phosphate buffer with a pH of 3.5 to obtain a tea solution, adding pectin-cellulose complex enzyme to the tea solution for enzymolysis for 5 hours, wherein the amount of pectin-cellulose complex enzyme added is 1.5% (w / v) of the tea solution, and the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is 2:1, then ultrasonically treating at a power of 700 W and a temperature of 50° C. for 25 minutes, centrifuging at a speed of 3500 r / min and a temperature of 18° C. for 4 minutes, filtering, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0059] Step S3, dissolving the naringin obtained in step S1 in water to prepare a naringin protein aqueous solution with a mass concentration of 0.8%, stirring with a magnetic force for 60 minutes, centrifuging for 22 minutes at a speed of 7500 r / min and a temperature of 4° C., taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0060] Step S4, dissolving chitosan in water, wherein the chitosan is MacLean 850124 chitosan quaternary ammonium salt, with a substitution degree of 92%, and preparing a chitosan aqueous solution with a mass concentration of 0.8%, stirring magnetically for 1.5 hours, then adding tea polyphenols until the concentration of the tea polyphenols reaches a mass concentration of 0.4%, stirring in a water bath at a temperature of 45° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0061] Step S5, uniformly mixing the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, wherein the volume ratio of the mixed solution I to the mixed solution II is 1:1, adjusting the pH to 6±0.1 to obtain a mixed solution III, adding 0.1% (w / v) sodium benzoate to the mixed solution III, and then heating the mixed solution III at a power density of 20 W / cm 3 , on for 5s and off for 5s, ultrasonically treat for 5min, and freeze-dry after precooling.

[0062] Comparative Example 1: A method for preparing tea polyphenol microcapsules with high absorption rate

[0063] Step S1, dissolving tea powder in a phosphate buffer with a pH of 3.5 to obtain a tea solution, adding pectin-cellulose complex enzyme to the tea solution for enzymolysis for 4.5 hours, wherein the amount of pectin-cellulose complex enzyme added is 1.5% (w / v) of the tea solution, and the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is 2:1, then ultrasonically treating at a power of 600 W and a temperature of 60° C. for 30 minutes, centrifuging at a speed of 3000 r / min and a temperature of 20° C. for 5 minutes, filtering, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0064] Step S2, dissolving gelatin in water to prepare a gelatin aqueous solution with a mass concentration of 1%, stirring with a magnetic force for 60 minutes, centrifuging at a speed of 8000 r / min and a temperature of 4° C. for 20 minutes, taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0065] Step S3, dissolving chitosan in water, wherein the chitosan is MacLean 850124 chitosan quaternary ammonium salt, with a substitution degree of 92%, and preparing a chitosan aqueous solution with a mass concentration of 1%, stirring magnetically for 2 hours, and then adding tea polyphenols until the concentration of the tea polyphenols reaches a mass concentration of 0.5%, stirring in a water bath at a temperature of 50° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0066] Step S4, uniformly mixing the mixed solution I obtained in step S2 and the mixed solution II obtained in step S3, wherein the volume ratio of the mixed solution I to the mixed solution II is 1:1, adjusting the pH to 6±0.1 to obtain a mixed solution III, adding 0.1% (w / v) sodium benzoate to the mixed solution III, and then heating the mixed solution III at a power density of 20 W / cm 3 , on for 5s and off for 5s, ultrasonically treat for 5min, and freeze-dry after precooling.

[0067] Comparative Example 2: A method for preparing tea polyphenol microcapsules with high absorption rate

[0068] Step S1, drying and crushing corn kernels, passing through a 100-mesh sieve to obtain corn flour, taking 50 g corn flour and mixing it with 750 mL deionized water to obtain a corn flour solution, adjusting the pH value of the corn flour solution to 11 with a 0.1 mol / L NaOH solution, extracting it in a water bath at a temperature of 50° C. for 1.5 h, centrifuging it at a speed of 4500 r / min for 10 min, taking the supernatant, adjusting the pH value of the supernatant to 7 with a 0.1 mol / L HCl solution, extracting it in a water bath at a temperature of 50° C. for 30 min, centrifuging it at a speed of 4500 r / min for 10 min, taking the supernatant, adjusting the pH value of the supernatant to 4 with a 0.1 mol / L HCl solution, centrifuging it at a speed of 4500 r / min for 10 min, taking the solid, pre-freezing it at -20° C., and vacuum freeze-drying it to obtain zein;

[0069] Step S2, dissolving tea powder in a phosphate buffer with a pH of 3.5 to obtain a tea solution, adding pectin cellulose complex enzyme to the tea solution for enzymolysis for 4.5 hours, wherein the amount of pectin cellulose complex enzyme added is 1.5% (w / v) of the tea solution, and the mass ratio of pectinase to cellulase in the pectin cellulose complex enzyme is 2:1, then ultrasonically treating at a power of 600 W and a temperature of 60° C. for 30 minutes, centrifuging at a speed of 3000 r / min and a temperature of 20° C. for 5 minutes, filtering, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols;

[0070] Step S3, dissolving the corn protein obtained in step S1 in water to prepare a corn protein aqueous solution with a mass concentration of 1%, stirring with a magnetic force for 60 minutes, centrifuging for 20 minutes at a speed of 8000 r / min and a temperature of 4° C., taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I;

[0071] Step S4, dissolving chitosan in water, wherein the chitosan is MacLean 850124 chitosan quaternary ammonium salt, with a substitution degree of 92%, and preparing a chitosan aqueous solution with a mass concentration of 1%, stirring magnetically for 2 hours, and then adding tea polyphenols until the concentration of the tea polyphenols reaches a mass concentration of 0.5%, stirring in a water bath at a temperature of 50° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II;

[0072] Step S5, uniformly mixing the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, wherein the volume ratio of the mixed solution I to the mixed solution II is 1:1, adjusting the pH to 6±0.1 to obtain a mixed solution III, adding 0.1% (w / v) sodium benzoate to the mixed solution III, and then heating the mixed solution III at a power density of 20 W / cm 3 , on for 5s and off for 5s, ultrasonically treat for 5min, and freeze-dry after precooling.

[0073] Experimental Example 1: Experiment on the determination of the embedding rate of tea polyphenols microcapsules

[0074] 1. Experimental methods:

[0075] Determine the extraction rate of citronella protein obtained in step S1 of Example 2, the extraction rate of tea polyphenols obtained in step S2, and the embedding rate of tea polyphenol microcapsules obtained in step S5. Wherein: the embedding rate is determined by: preparing 10 mL of 1.0 mg / mL tea polyphenol standard solution, taking 0.4, 0.8, 1.2, 1.6, and 2.0 mL respectively and placing them in a 25 mL volumetric flask, adding 5 mL of ferrous tartrate solution, and diluting to the scale with a phosphate buffer solution of pH 7.5. After 10 minutes, the absorbance is measured at 540 nm to draw a standard curve. Weigh 1 g of tea polyphenol microcapsules, crush and shake, ultrasonically dissolve for 20 minutes, stand, take 1 mL of supernatant, add 5 mL of ferrous tartrate solution, dilute to 25 mL with a phosphate buffer solution of pH 7.5, and measure the absorbance at 540 nm after 10 minutes. The content of tea polyphenols is obtained by comparing the standard curve, and the embedding rate is calculated by formula 1.

[0076] Formula 1:

[0077] 2. Experimental results:

[0078] In Example 2, the extraction rate of citronella protein obtained in step S1 is 15.07±0.25%; the extraction rate of tea polyphenols obtained in step S2 is 10.37±0.21%; the standard curve of tea polyphenol microcapsules obtained in step S5 is y=2.808x+0.0012, R 2 =0.9994, and the embedding rate was 43.04±4.86%.

[0079] Experimental Example 2: Structural Characterization of Tea Polyphenols Microcapsules

[0080] 1. Experimental methods:

[0081] The tea polyphenol microcapsules prepared in Example 2 were subjected to scanning electron microscopy analysis, particle size range, dispersion index, potential analysis, Fourier transform infrared spectroscopy analysis and circular dichroism analysis.

[0082] 1.1 Scanning electron microscopy analysis:

[0083] Trace amounts of naringenin, tea polyphenols, chitosan, and tea polyphenols microcapsules were directly adhered to the conductive adhesive and gold was sprayed for 45 seconds using a Quorum SC7620 sputtering coater at a current of 10 mA. The sample morphology was then photographed using a TESCAN MIRA LMS scanning electron microscope at an accelerating voltage of 3 kV and a SE2 secondary electron detector.

[0084] 1.2. Particle size range, dispersion index, and potential analysis:

[0085] After the naringin, tea polyphenols, chitosan and tea polyphenols microcapsules were diluted to appropriate times, the particle size distribution, dispersion index (PDI) and potential (Zeta) of the particles were measured using a nanoparticle size and Zeta potential analyzer (DLS).

[0086] 1.3. Fourier transform infrared spectroscopy analysis:

[0087] In a dry environment, place the ATR accessory in the optical path of the spectrometer, scan the air background, use a dropper to drop 1 drop of liquid naringenin, tea polyphenols, chitosan, and tea polyphenols microcapsules on the crystal surface of the ATR accessory, and then collect the infrared spectrum of the sample with a resolution of 4cm -1 , the scanning times are 32 times, and the test wave number range is 400~4000cm -1 .

[0088] 1.4 Circular dichroism analysis:

[0089] At room temperature, take 0.4 mL of 0.1 mg / mL sample solution in a quartz cuvette with a width of 1 mm, scan wavelength of 190-400 nm, and response time of 2 s. Ultrapure water was used as blank under the same conditions. The CD spectrum is the average of 3 measurements.

[0090] 2. Experimental results:

[0091] 2.1、Scanning electron microscopy analysis results are as follows Figure 1 shown.

[0092] Figure 1 The following is a scanning electron microscope image of the tea polyphenol microcapsules prepared in Example 2, wherein: (a): naringin; (b): tea polyphenols; (c): chitosan; (d): tea polyphenol microcapsules. Figure 1 (d) The scanning electron microscope image of the tea polyphenol microcapsules shows that the tea polyphenol microcapsules are spherical in shape, with no cracks or holes on the surface, and have a good molding effect.

[0093] 2.2. Particle size range, dispersity index, and potential analysis results are shown in Table 1.

[0094] Table 1 Particle size range, dispersion index and potential of samples

[0095] sample Particle size range nm Dispersion Index Potential mV Naringin 342.0～553.1 0.188±0.072 -31.43±0.66 Tea polyphenols 40.32～106.6 0.878±0.213 -3.63±0.75 Chitosan 357.6～624.2 0.872±0.123 +62.9±0.92 Tea polyphenols microcapsules 538.5～668.6 0.326±0.022 +65.86±0.37

[0096] As shown in Table 1, the larger the PDI value, the wider the molecular weight distribution; the smaller the PDI value, the more uniform the molecular weight distribution. Negatively charged naringin and tea polyphenols and positively charged chitosan combine to form more stable microcapsules.

[0097] 2.3、The results of Fourier transform infrared spectroscopy analysis are as follows Figure 2 shown.

[0098] Figure 2 The Fourier transform infrared spectrum of the tea polyphenol microcapsules prepared in Example 2 is shown in FIG. Figure 2 It can be seen that the microcapsules encapsulating and carrying tea polyphenols have characteristic vibration peaks of tea polyphenols in many places, indicating that tea polyphenols have been successfully encapsulated in the microcapsule structure.

[0099] 2.4. Circular dichroism analysis results Figure 3 And as shown in Table 2. Among them: Figure 3 Figure 2 is the CD diagram of naringenin and microcapsules. Table 3 is the relative content of protein secondary structure.

[0100] Table 2 Relative content of protein secondary structure (band range 190~240nm)

[0101]

[0102] Depend on Figure 3 As shown in Table 2, the samples containing protein components (naringin, microcapsules) were subjected to circular dichroism detection. When naringin and chitosan were used to encapsulate tea polyphenols into microcapsules, the CD spectrum characteristics of the samples changed significantly, indicating that naringin was successfully combined with chitosan and tea polyphenols.

[0103] Experimental Example 3: Evaluation of Tea Polyphenols Microcapsule Encapsulation Effect

[0104] 1. Experimental methods:

[0105] The tea polyphenol microcapsules prepared in Example 2 were subjected to simultaneous thermal analysis, microcapsule storage stability determination and microcapsule digestion stability determination.

[0106] 1.1. Analysis by synchronous thermal analyzer:

[0107] A synchronous thermal analyzer was used. After the instrument was automatically zeroed, 8 mg of the sample to be tested was accurately weighed, the N2 flow rate was controlled to 20 mL / min, the heating rate was 10°C / min, the temperature was raised from 30°C to 500°C, and the thermogravimetric curve of the sample was measured.

[0108] 1.2. Determination of storage stability of microcapsules:

[0109] The microcapsules were stored at 4°C, 20°C, and 35°C for 30 days, and the retention rate of tea polyphenols in the microcapsules was determined every 5 days. The retention rate was determined as follows: 10 mL of 1.0 mg / mL tea polyphenol standard solution was prepared, 0.4, 0.8, 1.2, 1.6, and 2.0 mL were taken and placed in a 25 mL volumetric flask, 5 mL of ferrous tartrate solution was added, and the solution was diluted to scale with a pH 7.5 phosphate buffer solution. After 10 minutes, the absorbance was measured at 540 nm to draw a standard curve. 1 g of tea polyphenol microcapsules was weighed, crushed and shaken, ultrasonically dissolved for 20 minutes, and allowed to stand. 1 mL of the supernatant was taken, 5 mL of ferrous tartrate solution was added, and the solution was diluted to 25 mL with a pH 7.5 phosphate buffer solution. After 10 minutes, the absorbance was measured at 540 nm. The content of tea polyphenols was obtained by comparing with the standard curve, and the tea polyphenols retention rate was calculated by formula 2. Three parallel tests were performed, and the percentage of tea polyphenols content in the samples before treatment was used as an indicator to compare the changes in the tea polyphenols retention rate after treatment for different time periods under different storage conditions.

[0110] Formula 2:

[0111] 1.3. Determination of digestion stability of microcapsules:

[0112] Prepare simulated gastric fluid (taking 250 mL as an example): take 246.3 mL of gastric electrolyte stock solution, add 125 μL of CaCl2(H2O)2 solution, adjust the pH to 1.58 (about 6 mL) with 6 mol / L HCl, add pepsin at a ratio of 800 U / mL for a final concentration of pepsin, and filter with a 0.22 μm sterile filter membrane for sterilization after it is fully dissolved.

[0113] Preparation of simulated intestinal fluid (500 mL as an example): Take 491.5 mL of intestinal electrolyte stock solution, add 1000 μL of CaCl2(H2O)2, adjust the pH to 6.8 (about 2 mL) with 6 mol / L HCl, add trypsin and bile salts in a ratio of 200 U / mL and 20 mmol / L respectively, and filter with a 0.22 μm sterile filter membrane to sterilize after fully dissolving.

[0114] Before the experiment, turn on the heating switch to preheat the temperature of the equipment and the simulated digestive fluid to 37°C. Inject 200mL of sample from the injection funnel within 1min, and then turn on the equipment for continuous gastrointestinal digestion. When digested for 30, 60, 90, and 120min, take 5mL of digested chyme from the gastric and duodenal sampling ports respectively, centrifuge and take the supernatant to measure its retention rate. The method for determining the retention rate of tea polyphenols is shown in 1.2.

[0115] 2. Experimental results:

[0116] 2.1. The results of the simultaneous thermal analysis are as follows Figure 4 shown.

[0117] Figure 4 Thermogravimetric analysis of tea polyphenol microcapsules prepared in Example 2. Figure 4 It can be seen that the main weight loss stage of naringin is at 232-306℃, the main weight loss stage of tea polyphenols is at 201-249℃, the main weight loss stage of chitosan is at 258-275℃, and the main weight loss stage of microcapsules is at 253-304℃. At 500℃, the residual amounts of naringin, tea polyphenols, chitosan and microcapsules are 29.42%, 49.59%, 15.46% and 37.31% respectively, which shows that the heat resistance of tea polyphenols has been improved, further indicating the success of microcapsule embedding.

[0118] 2.2. Microcapsule storage stability test results Figure 5 shown.

[0119] Figure 5 The graph below shows the relationship between the retention rate of tea polyphenols and time under different storage conditions. Figure 5 It can be seen that the retention rate of tea polyphenols in the embedded microcapsule system was significantly improved during storage. Storage at 4°C and 20°C was more beneficial to the stability of the microcapsules, and the stability of the microcapsules decreased with increasing temperature.

[0120] 2.3. Results of microcapsule digestion stability test Figure 6 shown.

[0121] Figure 6 The figure is the relationship between the retention rate of tea polyphenols and time after dynamic simulated gastrointestinal digestion. Figure 6 It can be seen that the encapsulation technology reduces the digestibility of tea polyphenols in the stomach, and the microcapsule structure effectively protects the tea polyphenols. It is also concluded that the microcapsules are relatively stable in the simulated stomach and will slowly release tea polyphenols in the simulated intestine.

[0122] Experimental Example 4: Caco-2 Cell Experiment

[0123] 1. Caco-2 cell culture:

[0124] After Caco-2 cells were revived in a 37°C water bath, they were cultured in a glucose-containing DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 U / mL streptomycin, and 100 U / mL penicillin. They were then cultured in a cell culture incubator with 5% CO2, 37°C, and saturated humidity.

[0125] 2. Cytotoxicity assay:

[0126] The tea polyphenol microcapsule samples prepared in Example 2 were prepared into different concentrations of 100, 200, 400, 800, and 1000 μg / mL and incubated with Caco-2 cells (1×10 6 / mL) and continue to cultivate for 24h. Blank microcapsules without tea polyphenols were used to characterize the toxicity of the carrier material. An equal volume of culture medium was used as a control group. After removing the cell supernatant, the cell viability was determined by the MTT method (20μL MTT solution was added to each well, cultured for another 4h, 150μL DMSO was added and shaken for 1min, and the absorbance at a wavelength of 490nm was measured with an enzyme reader) and calculated as follows.

[0127] Formula 3:

[0128] 3. Establishment of in vitro small intestinal Caco-2 model:

[0129] Use DMEM with 10% fetal bovine serum as the culture medium for Caco-2 cells, and culture them in an incubator at 37°C and 5% CO2. When the cells grow to 80% to 90% in the bottle, discard the old culture medium, add 0.25% trypsin (containing 0.02% EDTA) solution for digestion, make a cell suspension with DMEM culture medium, and inoculate it on the filter membrane of a 12-well Transwell plate. Add 0.5mL of cell suspension and 1.5mL of DMEM culture medium to the apical side (AP) and basolateral side (BL) of the filter membrane, respectively. Change the culture medium after 24 hours, change the medium every other day for the first 7 days, and then change the medium every day for 21 days.

[0130] 4. Determination of cell absorption rate:

[0131] When the Caco-2 cell layer is formed and stabilized, the culture medium on the AP and B sides is aspirated and slowly rinsed twice with 37°C HBSS solution. Subsequently, HBSS (37)°C with pH 6.0 is added to the AP and HBSS (37°C) with pH 7.4 is added to the BL, respectively, and incubated at 37°C for 15 minutes. After incubation, the HBSS (37)°C in the well plate is removed. Microcapsules and free tea polyphenols diluted in HBSS are added to the upper chamber as the supply solution, and 2.5 mL of blank HBSS is added to the lower chamber as the receiving solution. All well plates are incubated in a 37°C incubator, and the receiving solution is collected after 3 hours. The collected solution is centrifuged to obtain the supernatant. Before and after sample collection, the transepielectrical resistance (TEER) of the cells is measured using a microporous ERS-2 resistor (Millipore, USA). Calculation of TEER value of Caco-2 cell monolayer:

[0132] Formula 4: TEER value (Ω·cm 2) = (Caco-2 cell resistance - blank membrane resistance) × membrane area (cm 2 )

[0133] The absorbance of the microcapsule solution with a concentration gradient dissolved in HBSS solution was measured at 540 nm, and a standard curve was drawn. The Caco-2 cell absorption rate of the microcapsule was then calculated using the formula.

[0134] Formula 5:

[0135] 5. Experimental results:

[0136] 5.1. Cytotoxicity assay results Figure 7 shown.

[0137] Figure 7 The results of cytotoxicity assay. Figure 7 It can be seen that when the concentration of empty microcapsules and tea polyphenols microcapsules is lower than 400 μg / mL, the cell viability is lower than 80%, so a microcapsule concentration of 400 μg / mL was selected for the cell absorption experiment.

[0138] 5.2. The results of cell absorption rate determination are shown in Table 3.

[0139] Table 3 Absorption rate of microcapsules on Caco-2 cells

[0140]

[0141] The obtained standard curve at 540 nm after the microcapsule components were dissolved in HBSS solution was y=0.7952x+0.0091(R 2 =0.99879). It can be seen from Table 3 that the TEER values ​​measured before and after the experiment are similar, indicating that the Caco-2 monolayer was not damaged during the experimental operation, and the absorption rate of tea polyphenols after embedding was significantly improved.

[0142] Experimental Example 5: Application of Tea Polyphenols Microcapsules in Different Beverages

[0143] 1. Experimental methods:

[0144] 40 mg of tea polyphenol microcapsules prepared in Example 2 were added to 100 g of grapefruit juice (double grapefruit juice compound fruit juice beverage produced by Meizhou Zhenbao Golden Pomelo Industrial Co., Ltd., item number: 6972210152333) and 100 g of milk (pure milk produced by Inner Mongolia Yili Industrial Group Co., Ltd., item number: 6907992502052), and the storage stability of the microcapsule grapefruit juice and the heating stability of the microcapsule milk were measured, wherein: the method for measuring the tea polyphenol retention rate is as shown in 1.2 of Experimental Example 3.

[0145] 2. Experimental results:

[0146] 2.1. Storage stability results of microencapsulated grapefruit juice Figure 8 shown.

[0147] Figure 8 is the retention rate of microcapsules in grapefruit juice under different storage conditions. Figure 8 It can be seen that the most suitable storage condition for applying microcapsules to grapefruit juice beverages is storage at 4°C away from light, and it is not suitable for storage under natural light outdoors.

[0148] 2.2. The results of heating stability of microencapsulated milk are as follows Fig. 9 shown.

[0149] Fig. 9 is the retention rate of microcapsules in milk before and after heating. Fig. 9 It can be seen that after heating at 95°C for 1h, the retention rate of tea polyphenols is still 90.98±8.37%, indicating that tea polyphenols microcapsules have the potential to be used in dairy products. They can be sterilized at high temperatures and can also remain stable during the regular heating time of beverages.

[0150] Experimental Example 6: Comparative Experiment on Absorption Rate of Microcapsules

[0151] 1. Experimental methods:

[0152] The tea polyphenol microcapsules prepared in Example 2, Comparative Example 1 and Comparative Example 2 were tested using the cell absorption rate determination method described in Example 4, and tea polyphenol was used as the control group.

[0153] 2. Experimental results:

[0154] The experimental results are shown in Table 4.

[0155] Table 4 Comparative experimental data of cell absorption rate of microcapsules

[0156] Group Absorption rate (%) Control group 8.829±0.013 Example 2 41.72±0.016 Comparative Example 1 30.67±0.057 Comparative Example 2 38.97±0.015

[0157] It can be seen from Table 4 that the tea polyphenol microcapsules provided by the present invention have a high absorption rate in the body and can effectively improve the bioavailability of tea polyphenols in the human body.

Claims

1. A method for preparing tea polyphenol microcapsules with high absorption rate, characterized in that: The following steps are involved: Step S1, drying and crushing the pomelo peel to obtain pomelo peel powder, mixing the pomelo peel powder with deionized water to obtain a pomelo peel powder solution, adjusting the pH to 10-12, extracting in a water bath at a temperature of 45-55° C. for 1-2 hours, centrifuging, taking the supernatant, adjusting the pH to 7, extracting in a water bath at a temperature of 45-55° C. for 20-40 minutes, centrifuging, taking the supernatant, adjusting the pH to 3-4, centrifuging, taking the solid, pre-freezing at -20° C., and vacuum freeze-drying to obtain pomelo peel protein; Step S2, dissolving tea powder in phosphate buffer to obtain a tea solution, adding pectin cellulose complex enzyme to the tea solution for enzymolysis, then performing ultrasonic treatment, centrifugation, suction filtration, taking the supernatant, distilling under reduced pressure, and freeze-drying to obtain tea polyphenols; Step S3, dissolving the naringin obtained in step S1 in water to prepare a naringin protein aqueous solution with a mass concentration of 0.5-2%, magnetically stirring for 50-80 minutes, centrifuging, taking the supernatant, and standing at 4° C. overnight to obtain a mixed solution I; Step S4, dissolving chitosan in water to prepare a chitosan aqueous solution with a mass concentration of 0.5-2%, stirring magnetically for 1-3 hours, adding tea polyphenols, stirring in a water bath at a temperature of 45-55° C. until completely dissolved, and standing at 4° C. overnight to obtain a mixed solution II; Step S5, uniformly mix the mixed solution I obtained in step S3 and the mixed solution II obtained in step S4, adjust the pH to 6±0.1 to obtain a mixed solution III, add sodium benzoate to the mixed solution III, then perform ultrasonic treatment, precool and then freeze-dry to obtain.

2. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The material-liquid ratio of the pomelo peel powder to the deionized water in the step S1 is 1 g: (12-16) mL, and the centrifugal condition in the step S1 is: centrifugation at a speed of 4000-5000 r / min for 8-15 min.

3. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The pH value of the phosphate buffer in step S2 is 3.5, the added amount of the pectin-cellulose complex enzyme is 1.5% w / v of the tea solution, the mass ratio of pectinase to cellulase in the pectin-cellulose complex enzyme is 2:1, and the enzymolysis time is 4 to 5 hours.

4. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The ultrasonic treatment conditions in step S2 are: ultrasonic treatment at a power of 500-700 W and a temperature of 50-70° C. for 25-35 min.

5. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The centrifugation conditions in step S2 are: centrifugation at a speed of 2500-3500 r / min and a temperature of 18-25° C. for 4-6 minutes.

6. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The centrifugation conditions in step S3 are: centrifugation at a rotation speed of 7500-8500 r / min and a temperature of 4° C. for 15-25 min.

7. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The chitosan in step S4 is chitosan quaternary ammonium salt with a substitution degree of 92%; the mass concentration of the tea polyphenols in the chitosan aqueous solution is 0.4-0.6%.

8. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: In step S4, the volume ratio of the mixed solution I to the mixed solution II is 1:

1.

9. The method for preparing tea polyphenol microcapsules with high absorption rate according to claim 1, characterized in that: The ultrasonic treatment conditions in step S4 are: power density of 15-20 W / cm 3 , 5s on, 5s off, ultrasonic treatment for 4 to 6 minutes.

10. Use of the tea polyphenol microcapsules prepared by the method for preparing tea polyphenol microcapsules with high absorption rate according to any one of claims 1 to 9 in preparing food or health products.

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