A preparation method of a protein conjugate with pH sensitivity and potential colon targeting

Abstract

The application relates to a preparation method of a protein conjugate with pH sensitivity and potential colon targeting, and belongs to the technical field of functional food composite nanoparticle preparation. The conjugate is prepared by an enzyme-induced method, and the nanoparticle is prepared by a reverse solvent precipitation method. The whey protein isolate-tannic acid protein conjugate prepared by the method has extremely high grafting degree, the antioxidant property of the system is obviously improved, the encapsulation stability of the pterostilbene is high, the stability in the extreme pH environment of the gastrointestinal tract is good, and the protein conjugate has the dual characteristics of pH sensitivity and intestinal tract targeting.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a protein conjugate with pH sensitivity and potential colon targeting, and belongs to the technical field of functional food composite nanoparticles. BACKGROUND

[0002] As a plant-derived dietary phenolic compound, pterostilbene has high in vivo safety, and the two methoxyl groups in the benzene ring make the compound have higher lipophilicity and metabolic stability; meanwhile, the bioavailability of pterostilbene is 4 times that of resveratrol, and pterostilbene has better cell membrane permeability and pharmacokinetic characteristics and other pharmacological activities, can be used as a functional factor for targeted therapy, protects colon tissue from oxidative damage, relieves intestinal inflammation and other chronic diseases by regulating intestinal flora, and has a wide application prospect in the fields of food and medicine. However, pterostilbene has extremely low water solubility, is degraded or oxidized in the presence of light, heat and other environmental factors, and other defects, which greatly negatively affect the biological activity of pterostilbene, thereby limiting the development and application of pterostilbene.

[0003] Plant-derived dietary phenolic compounds are often used as functional ingredients for improving the nutritional composition and sensory characteristics of proteins. Covalent grafting of polyphenols can endow proteins with new functional properties, which is crucial for constructing a high-physicochemical stability targeted delivery system. Tannic acid (tannic acid) has a large number of phenolic hydroxyl structures, and thus has strong hydrogen-donating ability, and can effectively scavenge oxygen free radicals; the ortho-phenolic hydroxyl group in the ortho-phenol structure of tannic acid is easily oxidized into a quinone structure, and the oxidation reaction proceeds faster in the presence of enzymes, sufficient moisture and a higher pH (such as pH>3.5), thereby consuming oxygen in the environment.

[0004] In addition, tannic acid also has a variety of good physiological functions, such as astringency, antibacterial and antioxidant properties. The covalent interaction between betaine and laccase combined cross-linked tannic acid and whey protein isolate not only can reduce the surface hydrophobicity of whey protein isolate nanoparticles, but also can improve the stability and encapsulation and loading efficiency of hydrophobic active substances.

[0005] Currently, many studies use tannic acid to modify whey protein isolate. Patent CN110547442A discloses preparing nanoparticles from whey protein and tannic acid, and using the nanoparticles as Pickering emulsion stabilizers, which have good oxidative stability. Patent CN115363201B discloses preparing a liquid food for improving blood glucose using whey protein and tannic acid; and the document “Whey protein isolate and tannic acid interaction to improve the stability of rice oil Pickering emulsion” discloses using whey protein-tannic acid nanoparticles to improve the thermal stability, salt ion stability and oxidative stability of rice oil Pickering emulsion. The above-mentioned technologies only focus on the application of nanoparticles in subsequent emulsion systems, and ignore the properties of tannic acid-modified whey protein itself and its possibility as an embedding carrier.

[0006] Therefore, it is of great significance to develop the properties of whey protein isolate-tannic acid coupled nanoparticles themselves, further reduce the surface hydrophobicity thereof, and improve the stability during encapsulation and drug delivery in the intestinal tract, in order to expand the intestinal targeted delivery of hydrophobic bioactive substances. SUMMARY

[0007] To solve the above-mentioned problems, the present application provides a preparation method of protein conjugate with pH sensitivity and potential colon targeting, which adopts enzyme induction and anti-solvent precipitation method to prepare the conjugate. The whey protein isolate-tannic acid protein conjugate prepared by the present application has extremely high grafting degree, significantly improved antioxidant property, high encapsulation stability of pterostilbene, good stability in the extreme pH environment of gastrointestinal tract, and dual characteristics of pH sensitivity and intestinal targeting.

[0008] In the present application, wt% refers to mass fraction.

[0009] The first object of the present application is to provide a method for preparing protein conjugate with pH sensitivity and potential colon targeting, which comprises:

[0010] (1) mixing a whey protein isolate aqueous solution and a betaine aqueous solution to obtain a mixed solution; mixing the mixed solution and a tannic acid aqueous solution to obtain a reaction liquid;

[0011] (2) adding laccase to the reaction liquid, incubating and dialyzing to obtain a dialyzed reaction liquid; drying to obtain the protein conjugate with pH sensitivity and potential colon targeting.

[0012] In an embodiment, the concentration of the whey protein isolate aqueous solution in step (1) is 0.5-5 wt%, the concentration of the betaine aqueous solution is 0.1-3 wt%, and the concentration of the tannic acid aqueous solution is 0.1-1 wt%.

[0013] Preferably, the concentration of the whey protein isolate aqueous solution is 0.5-3 wt%, the concentration of the aqueous betaine solution is 0.1-2 wt%, and the concentration of the aqueous tannic acid solution is 0.2-0.8 wt%.

[0014] In one embodiment, the whey protein isolate aqueous solution and the aqueous betaine solution in step (1) are mixed at a volume ratio of 1:0.1-1; and the mixed solution and the aqueous tannic acid solution are mixed at a volume ratio of 1:0.1-1.

[0015] Preferably, the whey protein isolate aqueous solution and the aqueous betaine solution are mixed at a volume ratio of 1:0.5-1; and the mixed solution and the aqueous tannic acid solution are mixed at a volume ratio of 1:0.5-1.

[0016] In one embodiment, the amount of laccase added in step (2) is 30-120 U / g of whey protein isolate.

[0017] Preferably, the amount of laccase added is 50-70 U / g of whey protein isolate.

[0018] In one embodiment, the incubation in step (2) is at 30-50℃ for 8-16 h.

[0019] In one embodiment, the dialysis in step (2) is performed using a dialysis bag with a molecular weight cut-off of 8000-14000 Da, the dialysis liquid is pure water, and the dialysis time is 12-24 h.

[0020] In one embodiment, the mixing in step (1) is stirring at 200-900 rpm for 0.5-3 h.

[0021] In one embodiment, the mixing in step (2) is stirring at 200-900 rpm for 5-8 h.

[0022] A second object of the present application is to provide a protein conjugate with both pH sensitivity and potential colon targeting prepared by any of the above methods.

[0023] A third object of the present application is to provide a carrier for embedding active substances, which is prepared from the above protein conjugate with both pH sensitivity and potential colon targeting and active substances.

[0024] The preparation method comprises the steps of:

[0025] The protein conjugate with both pH sensitivity and potential colon targeting and water are mixed to prepare a reaction liquid, the active substances and the reaction liquid are mixed and dried to prepare the carrier for embedding active substances.

[0026] In one embodiment, the active substances include, but are not limited to, pterostilbene, curcumin, resveratrol, tea polyphenols, quercetin, lycopene, and astaxanthin.

[0027] In an embodiment, the drying includes, but is not limited to, evaporation drying, freeze drying.

[0028] A fourth object of the present application is to provide a method for embedding pterostilbene, which comprises preparing a reaction solution by mixing the above-mentioned protein conjugate with pH sensitivity and potential colon targeting and water, and preparing a pterostilbene anhydrous ethanol solution by mixing pterostilbene and anhydrous ethanol; adding the pterostilbene anhydrous ethanol solution into the reaction solution, mixing and drying to obtain a pterostilbene embedding carrier.

[0029] In an embodiment, the mass ratio of pterostilbene and the protein conjugate with pH sensitivity and potential colon targeting is 1:10-1:5.

[0030] In an embodiment, the pterostilbene and anhydrous ethanol are mixed to obtain a pterostilbene anhydrous ethanol solution (the concentration of pterostilbene is 5-15 mg / mL), the protein conjugate with pH sensitivity and potential colon targeting and water are mixed to obtain the protein conjugate with pH sensitivity and potential colon targeting (the concentration of pterostilbene is 80-120 mg / mL), i.e. a reaction solution; wherein the mass ratio of pterostilbene and the protein conjugate is 5-10:1, the pterostilbene anhydrous ethanol solution is added dropwise into the reaction solution, and stirring is performed at 200-700 rpm for 1-5 h, and then drying is performed to obtain a pterostilbene embedding carrier.

[0031] A fifth object of the present application is to provide the use of the above-mentioned protein conjugate with pH sensitivity and potential colon targeting or the above-mentioned embedding active substance carrier in the preparation of a pharmaceutical product.

[0032] The pharmaceutical product includes: a poorly soluble drug embedding / delivery carrier, a protein embedding / delivery carrier, a vitamin embedding / delivery carrier, and an antibiotic embedding / delivery carrier.

[0033] Advantages of the present application

[0034] To solve the above-mentioned problems, the present application provides a preparation method of a protein conjugate with pH sensitivity and potential colon targeting, which uses enzyme induction and anti-solvent precipitation method to prepare the conjugate. The protein conjugate (i.e. whey protein isolate-tannic acid protein conjugate) prepared by the present application has a very high grafting degree, a significantly improved antioxidant property, a high pterostilbene encapsulation stability, and a good stability in the extreme pH environment of the gastrointestinal tract, and has the dual characteristics of pH sensitivity and intestinal targeting.

[0035] Specifically,

[0036] (1) The whey protein isolate used in the present application is a natural biopolymer, which is low in price and green and environmentally friendly.

[0037] (2) The tannic acid used in the application belongs to polyhydroxy flavonol compounds, can have strong covalent reaction with whey protein isolate, and improves the stability thereof.

[0038] (3) The application uses pterostilbene with antioxidant and anti-inflammatory activity as a hydrophobic bioactive substance, uses whey protein isolate-tannic acid conjugate induced by laccase as a carrier, and adopts a reverse solvent precipitation method to prepare whey protein isolate-tannic acid protein conjugate loaded with pterostilbene, so that the material is low in cost and environmentally friendly, the method is simple and easy to operate, and meanwhile, a theoretical basis is provided for loading antioxidant and intestinal-targeted active substances.

[0039] (4) The protein conjugate prepared in the application has pH sensitivity and potential colon targeting, has a particle size of 97.28 nm and a polydispersity index PDI of only 0.26, has a tannic acid binding equivalent of 143.01 mg / g, a grafting efficiency of 74.4%, has good antioxidant stability, and has a DPPH free radical scavenging rate of 99.25% and an ABTS + free radical scavenging rate of 93.20%.

[0040] (5) The protein conjugate prepared in the application has pH sensitivity and potential colon targeting, has a positive effect on the encapsulation of pterostilbene and the slow release of pterostilbene in the gastrointestinal tract, and has a pterostilbene preservation rate of 43.60% in the colon at 480 min (the oral cavity to small intestine digestion time is usually 2 to 6 hours), which has great potential to effectively improve the delivery to the colon. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The particle size and polydispersity index (PDI) of the protein conjugate are shown in Table 1.

[0042] Figure 2 The tannic acid covalent binding equivalent and grafting rate of the protein conjugate are shown in Table 2.

[0043] Figure 3 The infrared spectrum of the protein conjugate is shown in Figure 2.

[0044] Figure 4 The free radical scavenging activity of the protein conjugate is shown in Table 3.

[0045] Figure 5 The release rate of the protein conjugate at different digestion times is shown in Table 4. DETAILED DESCRIPTION

[0046] The following describes preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not intended to limit the present application.

[0047] Raw materials:

[0048] Whey protein isolate was purchased from Shanghai Plotech International Trade Co., Ltd. Whey protein isolate (90% purity);

[0049] Betaine was purchased from Yixing Tianshi Feed Co., Ltd. 98%;

[0050] Tannic acid was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. 98%;

[0051] Pterostilbene was purchased from Hangzhou Ruishu Biochemical Co., Ltd. ≥99%, and all other single impurities <0.1%;

[0052] Laccase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd. 10000 U / g.

[0053] Detection method:

[0054] 1. Particle size and PDI of protein conjugate:

[0055] Dilute the protein conjugate to 0.01 mg / mL with deionized water, and use the Malvern nanoparticle size analyzer to measure the average particle size and PDI of the protein conjugate. The test temperature is 25℃, and the equilibrium time is 120s.

[0056] 2. Grafting efficiency

[0057] The total phenol content of the protein conjugate was determined by the Folin phenol method. 5 mg of tannic acid was dissolved in deionized water, diluted to 50 mL with a volumetric flask, and diluted with deionized water to form a series of standard solutions containing different A concentrations. Then, 0.5 mL of the standard solution and 2.5 mL of the Folin phenol reagent (0.2 M) were rotated and incubated at room temperature in the dark for 5 min. Add Na2CO3 solution (7.5%, g / L), after reacting at room temperature in the dark for 2 hours, measure the absorbance at 760 nm, and use water solution as a blank control, and use the same standard curve of tannic acid treated with the same method to determine the total phenol content of the sample. The concentration of whey protein isolate in the protein conjugate was fixed at 1 mg / mL, and the total phenol content of the sample was determined in the same way as the standard solution of tannic acid. The final result is expressed in tannic acid concentration (mg / g).

[0058] The grafting efficiency of tannic acid in the protein conjugate was determined by the OPA method (O-phthalaldehyde). After mixing 200 μL of sample solution with 4 mL of OPA reagent at 35℃ for 2 minutes, the absorbance of the sample was measured at 340 nm.

[0059] The grafting efficiency was calculated using the following formula:

[0060] Grafting efficiency (%) = (C / C0) x 100.

[0061] Wherein, C is the content of tannic acid in the complex and conjugate, and C0is the content of tannic acid in the reactants before dialysis.

[0062] 3. Antioxidant capacity detection

[0063] DPPH radical scavenging rate: Take the protein conjugate prepared in the examples and comparative examples, and prepare 2.5 mL of 0.5 mg / mL protein conjugate aqueous solution, mix with 1 mL of 0.1 mM DPPH ethanol solution; react in the dark for 25 min, and measure the absorbance at 517 nm.

[0064] ABTS radical scavenging rate: Take the protein conjugate prepared in the examples and comparative examples, and prepare 1 mL of 0.5 mg / mL protein conjugate aqueous solution, mix with 3 mL of ABTS solution (absorbance at 734 nm is 0.70±0.02), and place the mixture in the dark for 15 min, and measure the absorbance at 734 nm.

[0065] Radical scavenging capacity: Free radical scavenging activity (%) = (A0-A) / A0) x 100;

[0066] Wherein, A0represents the absorbance of distilled water (used as a control in DPPH detection) or PBS (used as a control in ABTS detection), and A represents the absorbance after the reaction of the protein conjugate.

[0067] 4. In vitro digestion and intestinal targeting

[0068] The in vitro release of Pte is as follows: 20 mL of sample is placed in 20 mL of simulated gastric fluid (SGF, 2 g of NaCl and 7 mL of HCl are dissolved in distilled water to dilute to 1 L, and the pH value is 1.2), and slowly stirred at 37°C for 2 h, then 40 mL of sample is taken, and 40 mL of simulated intestinal fluid (SIF, 187.5 mg of cholic acid is dissolved in 3.5 mL of pH 7.0 phosphate buffer PBS, and 0.144 g of pancreatin is added) stock solution (pH 7, 37°C) is added, and slowly stirred for 10 h, and the absorbance value of Pte at different time points is measured to simulate the conditions in the stomach and intestine, and the content of Pte extracted in the system is measured at 317 nm.

[0069] Example 1: Preparation of protein conjugate with pH sensitivity and potential colon targeting

[0070] 1. A method for preparing a protein conjugate with pH sensitivity and potential colon targeting, comprising the following steps:

[0071] (1) Weigh whey protein isolate, betaine and tannic acid, respectively, dissolve in water, fully stir to completely dissolve, and prepare 2 wt% whey protein isolate aqueous solution, 0.8 wt% betaine aqueous solution and 0.4 wt% tannic acid aqueous solution;

[0072] (2) Mix the whey protein isolate aqueous solution and the betaine aqueous solution according to a volume ratio of 1:1 to prepare a mixed solution; 1 h later, mix the mixed solution and the tannic acid aqueous solution according to a volume ratio of 1:1, stir and react for 8 h to prepare a reaction liquid;

[0073] (3) Add laccase to the reaction liquid in an amount of 60 U / g whey protein isolate, incubate at 40°C in a water bath for 16 h, and then place in a dialysis bag, dialyze at 4°C, replace the dialysis liquid every 6 h to remove free tannic acid, and dialyze for 18 h to prepare a reaction liquid containing protein conjugate (i.e. betaine-whey protein isolate-tannic acid protein conjugate) after dialysis;

[0074] (4) Dissolve pterostilbene in anhydrous ethanol (concentration of 10 mg / mL), add it dropwise to the reaction liquid after dialysis (adjust the concentration of protein conjugate in the reaction liquid to 100 mg / mL), and continuously stir at 500 rpm for 1 h to make it fully dissolved, wherein the mass ratio of betaine-based protein conjugate to curcumin is 10:1;

[0075] (5) Continue magnetic stirring at a rotation speed of 500 rpm for 30 min, remove ethanol by rotary evaporation, and freeze-dry to obtain the protein conjugate with pH sensitivity and potential colon targeting loaded with pterostilbene (i.e. whey protein isolate-tannic acid conjugate nanoparticles formed by betaine synergistic enzyme treatment cross-linking loaded with pterostilbene, referred to as protein conjugate loaded with pterostilbene).

[0076] 2. Change the preparation order

[0077] Change the preparation order of the reaction liquid in steps (1)-(2), and the steps are as follows:

[0078] (1) Weigh whey protein isolate, betaine and tannic acid, respectively, dissolve in water, fully stir to completely dissolve, and prepare 2 wt% whey protein isolate aqueous solution, 0.8 wt% betaine aqueous solution and 0.4 wt% tannic acid aqueous solution;

[0079] (2) The whey protein isolate aqueous solution and the tannic acid aqueous solution are mixed at a volume ratio of 1:1, stirred for 1 h to prepare a mixed solution; the mixed solution and the betaine aqueous solution are mixed at a volume ratio of 1:1, stirred at room temperature for 8 hours, to prepare a reaction liquid;

[0080] (3) The laccase is added to the reaction liquid at a dosage of 60 U / g of whey protein isolate, and the mixture is incubated at 40°C for 16 h, then placed in a dialysis bag, dialyzed at 4°C, and the dialysis liquid is replaced every 6 h to remove free tannic acid, and the dialysis is performed for 18 h to prepare a reaction liquid containing protein conjugates (i.e., betaine-whey protein isolate-tannic acid protein conjugates) after dialysis;

[0081] (4) The pterostilbene is dissolved in anhydrous ethanol (at a concentration of 10 mg / mL), and added dropwise to the reaction liquid after dialysis (to adjust the concentration of the protein conjugates in the reaction liquid to 100 mg / mL), and the mass ratio of the protein conjugates to the pterostilbene is 10:1, and the mixture is continuously stirred at 500 rpm for 1 h to make the pterostilbene fully dissolved;

[0082] (5) The ethanol is removed by magnetic stirring at a rotation speed of 500 rpm for 30 min, and freeze-drying to obtain the pterostilbene-loaded protein conjugates.

[0083] The results show that when the preparation sequence is changed, the embedding effect, antioxidant performance, and grafting efficiency of the prepared pterostilbene-loaded protein conjugates are all poor.

[0084] Example 2: Preparation of protein conjugates induced by alkaline treatment

[0085] A preparation method of a protein conjugate with pH sensitivity and potential colon targeting, comprising the following steps:

[0086] (1) The whey protein isolate, betaine, and tannic acid are weighed and dissolved in water, respectively, and fully stirred to completely dissolve, to prepare a 2 wt% whey protein isolate aqueous solution, a 0.8 wt% betaine aqueous solution, and a 0.4 wt% tannic acid aqueous solution;

[0087] (2) The whey protein isolate aqueous solution and the betaine aqueous solution are mixed at a volume ratio of 1:1, and 1 M NaOH is used to adjust the pH to 9.0, and then stirred at 500 rpm for 1 h to prepare a mixed solution;

[0088] (3) The mixed solution prepared in step (2) and the tannic acid aqueous solution are mixed according to a volume ratio of 1:1, stirred at room temperature at 500 rpm for 24 hours in the open air, and allowed to fully react to prepare a reaction liquid; the reaction liquid is poured into a dialysis bag with a molecular weight cut-off of 8000-14000 Da, and the dialysis liquid is replaced every 6 hours to remove free tannic acid, thereby preparing a reaction liquid containing protein conjugates (i.e., betaine-whey protein isolate-tannic acid protein conjugates) after dialysis;

[0089] (4) Pterostilbene is dissolved in anhydrous ethanol (at a concentration of 10 mg / mL), and is added dropwise to the reaction liquid after dialysis (to adjust the concentration of the protein conjugates in the reaction liquid to 100 mg / mL), and the mass ratio of the protein conjugates to pterostilbene is 10:1. The mixture is continuously stirred at 500 rpm for 1 hour to allow it to fully dissolve;

[0090] (5) The mixture is continuously stirred at 500 rpm for 30 minutes, and ethanol is removed by rotary evaporation; freeze-drying is performed to obtain pterostilbene-loaded protein conjugates (pterosilibene-loaded whey protein isolate-tannic acid covalent conjugate nanoparticles formed by betaine synergistic enzyme treatment cross-linking).

[0091] Comparative Example 1: Preparation of protein conjugates by radical induction

[0092] The protein conjugates are prepared by radical induction, and the preparation method is as follows:

[0093] (1) Whey protein isolate, betaine, and tannic acid are weighed and dissolved in water, respectively, and fully stirred to completely dissolve, to prepare a 2 wt% whey protein isolate aqueous solution, a 0.8 wt% betaine aqueous solution, and a 0.4 wt% tannic acid aqueous solution;

[0094] (2) The whey protein isolate and betaine solutions obtained in step (1) are mixed according to a volume ratio of 1:1, and stirred for 1 hour to prepare a mixed solution; 5 mol / L hydrogen peroxide and ascorbic acid are simultaneously added to the mixed solution, and the final concentrations of hydrogen peroxide and ascorbic acid in the mixed solution are 0.147 wt% and 0.25 wt%, respectively. After standing for two hours, the mixed solution is mixed with the tannic acid aqueous solution according to a volume ratio of 1:1, and stirred at room temperature for 24 hours in the open air to allow it to fully react, thereby preparing a reaction liquid;

[0095] (3) The reaction liquid is placed in a dialysis bag and dialyzed at 4°C, and the dialysis liquid is replaced every 6 hours to remove free tannic acid, thereby preparing a reaction liquid containing protein conjugates (i.e., betaine-whey protein isolate-tannic acid protein conjugates) after dialysis;

[0096] (4) The pterostilbene was dissolved in anhydrous ethanol (concentration of 10 mg / mL), and was added dropwise into the reaction solution after dialysis (adjust the concentration of protein conjugate in the reaction solution to 100 mg / mL), and the mass ratio of protein conjugate to pterostilbene was 10:1. The solution was continuously stirred at 500 rpm for 1 h to make the pterostilbene fully dissolved;

[0097] (5) The ethanol was removed by rotary evaporation under the condition of 500 rpm for 30 min. The freeze-drying was performed to obtain the protein conjugate loaded with pterostilbene (whey protein isolate-tannic acid covalent conjugate nanoparticles treated by betaine synergistic enzyme and cross-linked to form, and then loaded with pterostilbene).

[0098] Comparative Example 2: without laccase treatment

[0099] On the basis of Example 1, step (3) was omitted, that is, laccase was not added to the reaction solution, and the rest of the steps were the same as those in Example 1, to prepare the protein conjugate loaded with pterostilbene.

[0100] Comparative Example 3: changing the amount of laccase

[0101] On the basis of Example 1, the amount of laccase added in step (3) was changed to 30 U / g of whey protein isolate, and the rest of the steps were the same as those in Example 1, to prepare the protein conjugate loaded with pterostilbene.

[0102] Comparative Example 4: changing the amount of laccase

[0103] On the basis of Example 1, the amount of laccase added in step (3) was changed to 120 U / g of whey protein isolate, and the rest of the steps were the same as those in Example 1, to prepare the protein conjugate loaded with pterostilbene.

[0104] Comparative Example 5: changing the amount of laccase

[0105] On the basis of Example 1, the amount of laccase added in step (3) was changed to 240 U / g of whey protein isolate, and the rest of the steps were the same as those in Example 1, to prepare the protein conjugate loaded with pterostilbene.

[0106] Comparative Example 6: without adding tannic acid

[0107] On the basis of Example 1, tannic acid was not added, and the steps were as follows:

[0108] (1) Whey protein isolate and betaine were weighed and dissolved in water, respectively, and fully stirred to make them completely dissolved, to prepare 1 wt% whey protein isolate aqueous solution and 0.4 wt% betaine aqueous solution;

[0109] (2) The whey protein isolate aqueous solution and the betaine aqueous solution were mixed according to a volume ratio of 1:1, and stirred for 8 h to prepare a reaction solution;

[0110] (3) 60 U / g of laccase was added to the reaction solution, and it was incubated at 40°C for 16 h with open water bath. After incubation, it was placed in a dialysis bag and dialyzed at 4°C, and the dialysis solution was replaced every 6 h to remove free tannic acid, thereby preparing the reaction solution containing protein conjugates after dialysis;

[0111] (4) Pterostilbene was dissolved in anhydrous ethanol (concentration of 10 mg / mL), and it was added dropwise to the reaction solution after dialysis (the concentration of protein conjugates in the reaction solution was adjusted to 100 mg / mL), and the mass ratio of protein conjugates to pterostilbene was 10:1. It was continuously stirred at 500 rpm for 1 h to make it fully dissolved;

[0112] (5) The ethanol was removed by rotary evaporation under the condition of a rotation speed of 500 rpm and continuous magnetic stirring for 30 min. Freeze-drying was performed to obtain the protein conjugates loaded with pterostilbene.

[0113] Comparative Example 7: Without using betaine

[0114] On the basis of Example 1, no betaine was added, and the steps were as follows:

[0115] (1) Whey protein isolate and tannic acid were weighed and dissolved in water, and fully stirred to make them completely dissolved, thereby preparing a 1 wt% whey protein isolate aqueous solution and a 0.4 wt% tannic acid aqueous solution;

[0116] (2) The whey protein isolate aqueous solution and the tannic acid aqueous solution were mixed according to a volume ratio of 1:1, and stirred for 8 h to prepare a reaction solution;

[0117] (3) 60 U / g of laccase was added to the reaction solution, and it was incubated at 40°C for 16 h with open water bath. After incubation, it was placed in a dialysis bag and dialyzed at 4°C, and the dialysis solution was replaced every 6 h to remove free tannic acid, thereby preparing the reaction solution containing protein conjugates after dialysis;

[0118] (4) Pterostilbene was dissolved in anhydrous ethanol (concentration of 10 mg / mL), and it was added dropwise to the reaction solution after dialysis (the concentration of protein conjugates in the reaction solution was adjusted to 100 mg / mL), and the mass ratio of protein conjugates to pterostilbene was 10:1. It was continuously stirred at 500 rpm for 1 h to make it fully dissolved;

[0119] (5) The ethanol was removed by rotary evaporation under the condition of a rotation speed of 500 rpm and continuous magnetic stirring for 30 min. Freeze-drying was performed to obtain the protein conjugates loaded with pterostilbene.

[0120] Example 3: Performance detection

[0121] The protein conjugate loaded with tannin acid prepared by the preparation of example 1, comparative example 1~7 was detected for particle size, PDI, tannin acid binding equivalent and grafting rate, antioxidant capacity and in vitro digestion results.

[0122] 1, particle size and PDI

[0123] The results of particle size and PDI (polydispersity index) are shown in Figure 1 and table 1, the results show that, compared with other laccase concentration (comparative example 2~5), the protein conjugate loaded with tannin acid prepared by example 1 has smaller particle size (97.28 nm), more uniform dispersion, PDI is 0.26, better stability, which shows that 60 U / g of laccase can most efficiently graft tannin acid to whey protein isolate molecules, covalent coupling can significantly improve the stability of protein conjugate, and the subsequent drug encapsulation and transportation in gastrointestinal tract can be better completed.

[0124] Table 1 particle size and PDI

[0125]

[0126] 2, tannin acid binding equivalent and grafting rate

[0127] The results of tannin acid binding equivalent and grafting rate are shown in Figure 2 and table 2, the results show that, the effect of comparative example 2 is the worst, in the protein conjugate prepared without adding laccase, the phenolic hydroxyl and benzene ring of tannin acid will combine with protein by hydrophobic interaction, hydrogen bond or van der waals force, etc. Forming non-covalent complex, only the relatively weak hydrogen bond and hydrophobic interaction between tannin acid and whey protein isolate molecules, and non-covalent crosslinking belongs to reversible force, its stability is weaker than covalent interaction, so its polyphenol binding rate is lower.

[0128] The protein conjugate prepared by example 1, example 2 and comparative example 1 is covalent complex, in which the binding equivalent of tannin acid is 71.40 mg / g (comparative example 1), 75.60 mg / g (example 2) and 143.01 mg / g (example 1) respectively, which shows that covalent crosslinking can obtain higher polyphenol binding rate. Compared with alkali treatment and free radical induced covalent reaction, the covalent conjugate prepared by example 1 has the highest tannin acid binding equivalent and grafting rate, which shows that laccase can promote the covalent combination of whey protein isolate and tannin acid to a greater extent.

[0129] Table 2 tannin acid binding equivalent and grafting rate

[0130]

[0131] 3, infrared spectrum

[0132] The detection method for infrared spectroscopy is as follows:

[0133] The infrared spectrum of the samples was determined using the potassium bromide pellet method. The frozen samples were lyophilized into powder, mixed with potassium bromide at a ratio of 1:100, and then ground into a uniform powder using an agate mortar. The scanning conditions were set as follows: spectral range 400–4000 cm⁻¹. -1 The scans were performed 32 times at a resolution of 4 cm⁻¹. Potassium bromide was used as a blank control. The spectra of each sample were collected three times under the same conditions.

[0134] Test results as follows Figure 3 As shown, the main peak in the spectrum of whey protein isolate (whey protein isolate) is 3294.03 cm⁻¹. -1 2962.31 cm -1 1651.02 cm -1 and 1535.70 cm -1 , representing amide I band (NH stretching, C=O stretching) and amide II band (CH stretching, NH bending, and CN stretching), respectively. Compared with whey protein isolate, the complex prepared in Comparative Example 7 is a covalent complex of protein and tannic acid under the action of laccase, with a wavelength of 3294 cm⁻¹. -1 2962 cm -1 1649 cm -1 1535 cm -1 Multiple locations showed red or blue shifts of 2 cm. -1 0.3 cm -1 2 cm -1 33 cm -1 This indicates that the -NH2 group of the protein in the covalent complex participates in the grafting reaction. Simultaneously, laccase oxidizes the o- and p-diphenols in the tannin structure into reactive aromatic radicals and / or quinones, which then attack nucleophilic amino acid residues (-NH2) in the whey isolate chain, forming covalent conjugates via Michael addition and Schiff base reactions. Under the influence of betaine, the groups in Example 1 are respectively at 3301 cm⁻¹. -1 2963 cm -1 1651 cm -1 1537 cm -1 Multiple locations showed red or blue shifts of 7cm respectively. -1 1 cm -1 0.3 cm -1 2 cm -1 Among them, betaine may provide a more hydrophilic environment for tannic acid due to its interaction with proteins, so as to be more conducive to covalent interaction.

[0135] 4. Antioxidant capacity

[0136] The antioxidant capacity of the protein conjugate is shown in Table 2 and Table 3. Figure 4

[0137] As can be seen from Comparative Example 1, Comparative Example 6 and Comparative Example 7, under the same processing conditions, the antioxidant capacity of the protein conjugate prepared without adding tannic acid (Comparative Example 6) or without adding betaine (Comparative Example 7) is weaker;

[0138] As can be seen from Comparative Example 1, Example 2, Comparative Example 1 and Comparative Example 2, compared with the non-covalently bound protein conjugate (Comparative Example 2), the antioxidant capacity of the covalently bound protein conjugate (Comparative Example 1, Example 1 and Example 2) is significantly enhanced. It can be seen that tannic acid is combined with whey protein isolate molecules through non-polar bonds, the intermolecular force is weak, and tannic acid is easy to fall off; while enzyme treatment (Example 1), alkali treatment (Example 2) and free radical (Comparative Example 1) treatment covalently combine tannic acid and whey protein isolate, the intermolecular force is strong, and the content of tannic acid in the grafting product is higher, so the improvement effect on the antioxidant capacity of whey protein isolate is obvious.

[0139] From the perspective of molecular mechanism, the O-H bond of the phenolic hydroxyl group on the structure of tannic acid directly breaks to obtain a hydrogen atom, which directly combines with a free radical molecule to improve the antioxidant capacity of the free radical system, which also proves that the polyphenol is successfully grafted on the whey protein isolate. The above results show that the whey protein isolate-tannic acid covalent protein conjugate can be used as a potential natural new antioxidant that can replace synthetic antioxidants.

[0140] Table 3 Antioxidant capacity test

[0141]

[0142] 5. In vitro digestion and intestinal targeting effect

[0143] The in vitro digestion and intestinal targeting effect of the protein conjugate loaded with pterostilbene is shown in Table 4. Figure 5

[0144] The results show that under the action of protease, the whey protein isolate-tannic acid carrier is degraded in the digestive system, in addition, the target organ (intestine) can fully absorb the peptides and amino acids produced by hydrolysis, and the encapsulated active ingredient pterostilbene is exposed to the gastrointestinal tract and degraded. The pterostilbene of the covalently bound protein conjugate (Example 1) has stronger gastrointestinal resistance during the entire gastrointestinal digestion process.

[0145] ​​This is because compared with the non-covalent mixture (comparative example 2), the covalent conjugate (example 1, example 2, comparative example 1) has the advantages of whey protein isolate and tannic acid, protecting the encapsulated pterostilbene from being released under gastric conditions. Among them, the pterostilbene in the laccase-induced covalent complex (example 1) still has a 43.60% preservation rate even in the colon site, significantly prolonging the residence time of pterostilbene in the colon site, which helps Pte to be released in the colon site, interact with intestinal flora, and exhibit its good pharmacological activities such as cell membrane permeability and pharmacokinetic characteristics.

[0146] Example 4: Preparation of active substance embedding carrier

[0147] The method for preparing the active substance embedding carrier comprises the following steps:

[0148] (1) Weigh whey protein isolate, betaine and tannic acid, respectively dissolve them in water, fully stir to completely dissolve them, and prepare 2 wt% whey protein isolate aqueous solution, 0.8 wt% betaine aqueous solution and 0.4 wt% tannic acid aqueous solution;

[0149] (2) Mix the whey protein isolate aqueous solution and the betaine aqueous solution according to a volume ratio of 1:1 to prepare a mixed solution; 1 h later, mix the mixed solution and the tannic acid aqueous solution according to a volume ratio of 1:1, stir and react for 8 h to prepare a reaction liquid;

[0150] (3) Add laccase to the reaction liquid, the amount is 60 U / g whey protein isolate, incubate at 40℃ in a water bath for 16 h, after incubation, place it in a dialysis bag, dialyze at 4℃, replace the dialysis liquid every 6 h to remove free tannic acid, dialyze for 18 h to prepare a dialyzed reaction liquid; freeze-dry to obtain a betaine-whey protein isolate-tannic acid protein conjugate, namely an active substance embedding carrier.

[0151] It is detected that the active substance embedding carrier has good embedding and gastrointestinal slow-release effects on active substances such as curcumin and anthocyanins.

[0152] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a protein conjugate with both pH-sensitivity and potential colon targeting, characterized in that, The method comprises: (1) mixing a whey protein isolate aqueous solution and a betaine aqueous solution to obtain a mixed solution; mixing the mixed solution and a tannic acid aqueous solution to obtain a reaction liquid; (2) adding laccase to the reaction liquid, incubating and dialyzing to obtain a dialyzed reaction liquid; and drying to obtain a protein conjugate with pH sensitivity and potential colon targeting.

2. The method of claim 1, wherein, In step (1), the concentration of the whey protein isolate aqueous solution is 0.5-5 wt%, the concentration of the betaine aqueous solution is 0.1-3 wt%, and the concentration of the tannic acid aqueous solution is 0.1-1 wt%.

3. The method of claim 2, wherein, The concentration of the whey protein isolate aqueous solution is 0.5-3 wt%, the concentration of the betaine aqueous solution is 0.1-2 wt%, and the concentration of the tannic acid aqueous solution is 0.2-0.8 wt%.

4. The method of claim 1, wherein, In step (1), the whey protein isolate aqueous solution and the betaine aqueous solution are mixed at a volume ratio of 1:0.1-1; and the mixed solution and the tannic acid aqueous solution are mixed at a volume ratio of 1:0.1-1.

5. The method of claim 4, wherein, The whey protein isolate aqueous solution and the betaine aqueous solution are mixed at a volume ratio of 1:0.5-1; and the mixed solution and the tannic acid aqueous solution are mixed at a volume ratio of 1:0.5-1.

6. The method of claim 1, wherein, In step (2), the amount of laccase added is 30-120 U / g of whey protein isolate.

7. The method of claim 6, wherein, The amount of laccase added is 50-70 U / g of whey protein isolate.

8. The protein conjugate with pH sensitivity and potential colon targeting prepared by the method of any one of claims 1-7.

9. A carrier for embedding an active substance, characterized in that The protein conjugate with pH sensitivity and potential colon targeting of claim 8 and an active substance are prepared; The preparation method comprises the steps of: The protein conjugate with pH sensitivity and potential colon targeting and water are prepared to obtain a reaction liquid; the active substance and the reaction liquid are mixed and dried to prepare a carrier embedding the active substance.

10. The active-encapsulating carrier according to claim 9, wherein The active substance comprises pterostilbene, curcumin, resveratrol, tea polyphenol, quercetin, lycopene, and astaxanthin.

11. A method of embedding pterostilbene, characterized by, The protein conjugate with pH sensitivity and potential colon targeting of claim 8 and water are prepared to obtain a reaction liquid; pterostilbene and anhydrous ethanol are prepared to obtain a pterostilbene anhydrous ethanol solution; the pterostilbene anhydrous ethanol solution is added to the reaction liquid, mixed, and dried to obtain a pterostilbene embedding carrier.

12. The method of claim 11, wherein, The mass ratio of pterostilbene to the protein conjugate with pH sensitivity and potential colon targeting is 1:10-1:

5.

13. The protein conjugate with pH sensitivity and potential colon targeting of claim 8 is used for preparing a medicine.

14. Use according to claim 13, characterized in that, The medicine comprises a poorly soluble drug embedding / delivery carrier, a protein embedding / delivery carrier, a vitamin embedding / delivery carrier, and an antibiotic embedding / delivery carrier.

15. The carrier embedding the active substance of claim 9 is used for preparing a medicine.

16. The use according to claim 15, characterized in that, The medicine comprises a poorly soluble drug embedding / delivery carrier, a protein embedding / delivery carrier, a vitamin embedding / delivery carrier, and an antibiotic embedding / delivery carrier.

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