Preparation and Application of a Betaine-based Protein Conjugate

Abstract

The present invention discloses the preparation and application of a protein conjugate based on betaine, belonging to the technical field of the preparation of functional food composite nanoparticles. The present invention provides a preparation method for preparing a protein conjugate assisted by betaine, and a protein conjugate based on betaine is prepared by a double cross-linking method using betaine and alkali treatment. The whey protein-tannic acid conjugate prepared based on betaine in the present invention has small particle size, and its antioxidant property is significantly improved, and it can be used as a new antioxidant for effectively encapsulating active ingredients.

Description

Technical Field

[0001] The invention relates to the preparation and application of a betaine-based protein conjugate, and belongs to the technical field of functional food composite nanoparticle preparation. Background Art

[0002] Protein has high nutritional value, good biocompatibility, and is biodegradable, meeting the requirements of clean labels. It is favored by scientific researchers as a carrier material. By utilizing the interaction between protein and polyphenols, the structure of the protein can be changed to improve the functional properties, thereby improving the performance of the carrier to a certain extent. Compared with non-covalent systems, the formation of covalent complexes and their effects on protein structure and functional properties are irreversible, but more stable to changes in environmental conditions, so they have a wider range of application significance in the food industry. Since covalent bonds lead to stronger and more lasting interactions, irreversible covalent bonds are more likely to be formed when constructing protein-polyphenol delivery carriers.

[0003] Tannic acid has a large number of phenolic hydroxyl structures, so it has a strong hydrogen supply capacity and can effectively scavenge oxygen free radicals. The ortho-phenolic hydroxyl groups in its pyrogallol structure are easily oxidized into quinone structures. When there is sufficient water and a higher pH value (such as pH>3.5), the oxidation reaction proceeds faster, thereby promoting the cross-linking between protein and tannic acid.

[0004] There have been many studies on using tannic acid to modify proteins to improve the performance of proteins. The article "Interaction between whey protein isolate and tannic acid improves the stability of rice oil Pickering emulsion" and the article "Effect of the combination of tannic acid and whey protein isolate on the stability of perilla essential oil antibacterial emulsion" disclose that the stability of the emulsion formed can be further improved after combining tannic acid and whey protein isolate using an alkaline method; the article "Research progress on the interaction mechanism between tannic acid and protein and its influence on the physicochemical and functional properties of proteins" discloses that the combination of tannic acid and protein can improve the solubility, emulsification and antioxidant activity of proteins. The antioxidant properties of tannic acid-protein nanoparticles prepared by the above technology still have room for further improvement.

[0005] Betaine (Betaine, abbreviated as Bet), chemically named N,N,N-trimethylglycine, has a variety of physiological functions and application fields, but it is rarely used in modified proteins. The article "Research Progress of Ultrasonic-Assisted Low Eutectic Solvent Extraction in Active Ingredient Extraction and Food Analysis Pretreatment" discloses the use of betaine to prepare low eutectic solvents to improve the extraction rate of proteins, but this technology achieves the extraction rate of betaine on proteins in the form of low eutectic solvents, and does not use betaine to modify proteins.

[0006] Therefore, studying the promoting effect of betaine on the covalent coupling of protein and tannic acid under alkaline conditions and further improving the properties of tannic acid-protein complexes has research significance and practical value. New biocoupling technologies can be developed to expand the application of protein-polyphenol covalent conjugates in the field of functional food nanodelivery. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for preparing a betaine-based protein conjugate, and a whey protein isolate-tannic acid protein conjugate is prepared by a double cross-linking method of betaine and alkali treatment. The betaine-based protein conjugate prepared by the present invention has small particles and significantly improved antioxidant properties, and can be used as a new antioxidant for effectively encapsulating active ingredients.

[0008] The present invention finds that betaine molecules contain multiple polar groups, such as hydroxyl (-OH) and imine (-N(CH3)3), which can form stable hydration with water molecules through hydrogen bonding. This provides an excellent hydrophilic environment for tannic acid, and it also has certain antioxidant properties, which not only helps the dissolution and dispersion of tannic acid, but also can act as a cross-linking agent to promote the covalent interaction between protein and tannic acid, thereby promoting the formation and stability of covalent complexes.

[0009] In the present invention, wt% refers to mass fraction.

[0010] The first object of the present invention is to provide a method for preparing a betaine-based protein conjugate, characterized in that the method comprises:

[0011] (1) mixing a whey protein isolate (WPI) aqueous solution and a betaine aqueous solution in a volume ratio of 1:0.1-1, adjusting the pH to 7-11, and mixing to obtain a mixed solution;

[0012] (2) mixing the mixed solution and a tannic acid (TA) aqueous solution in a volume ratio of 1:0.1-1 to obtain a reaction solution;

[0013] (3) The reaction solution is dialyzed and dried to obtain a betaine-based protein conjugate.

[0014] Preferably, the whey protein isolate aqueous solution and the betaine aqueous solution are mixed in a volume ratio of 1:0.5-1; the mixed solution and the tannic acid aqueous solution are mixed in a volume ratio of 1:0.5-1.

[0015] In one embodiment, the concentration of the whey protein isolate aqueous solution in step (1) is 0.5-5wt%, and the concentration of the betaine aqueous solution is 0.1-3wt%; the concentration of the tannic acid aqueous solution in step (2) is 0.1-1wt%;

[0016] Preferably, the concentration of the whey protein isolate aqueous solution is 0.5-3wt%, the concentration of the betaine aqueous solution is 0.1-2wt%, and the concentration of the tannic acid aqueous solution is 0.2-0.8wt%;

[0017] In one embodiment, the stirring and mixing in step (1) is carried out at 200 to 900 rpm for 0.5 to 3 hours.

[0018] In one embodiment, the stirring and mixing in step (2) is carried out at 200-900 rpm for 5-8 hours.

[0019] In one embodiment, the dialysis in step (3) uses a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, the dialysis fluid is a pure aqueous solution, and the dialysis time is 12 to 24 hours.

[0020] The second object of the present invention is to provide a betaine-based protein conjugate prepared by any of the above methods.

[0021] The third object of the present invention is to provide the use of the above-mentioned betaine-based protein conjugate in the preparation of food, medicine or health care products; the food includes: antioxidants, additives, emulsifiers, stabilizers, active substance embedding / delivery carriers, flavor substance embedding / delivery carriers or functional food embedding / delivery carriers; the medicine includes: poorly soluble drug embedding / delivery carriers, protein embedding / delivery carriers, vitamin embedding / delivery carriers, antibiotic embedding / delivery carriers.

[0022] In one embodiment, the active ingredients include resveratrol, curcumin, and pterostilbene.

[0023] The fourth object of the present invention is to provide a product, wherein the product contains the above-mentioned betaine-based protein conjugate; the product is a food, a medicine, a cosmetic or a health product;

[0024] In one embodiment, the food includes: antioxidants, additives, emulsifiers, stabilizers, active substance embedding / delivery carriers, flavor substance embedding / delivery carriers or functional food embedding / delivery carriers; the medicine includes: poorly soluble drug embedding / delivery carriers, protein embedding / delivery carriers, vitamin embedding / delivery carriers, antibiotic embedding / delivery carriers.

[0025] The fifth object of the present invention is to provide a method for improving the antioxidant property of whey protein isolate-tannic acid protein conjugate, wherein betaine is added during the preparation of the whey protein isolate-tannic acid protein conjugate, and the preparation method comprises:

[0026] (1) mixing a whey protein isolate aqueous solution and a betaine aqueous solution in a volume ratio of 1:0.1-1, adjusting the pH to 7-11, and mixing to obtain a mixed solution;

[0027] (2) mixing the mixed solution and the tannic acid aqueous solution in a volume ratio of 1:0.1-1 to obtain a reaction solution;

[0028] (3) dialyzing the reaction solution and drying it to obtain a whey protein isolate-tannic acid protein conjugate with improved antioxidant properties;

[0029] In one embodiment, the concentration of the whey protein isolate aqueous solution in step (1) is 0.5-5wt%, and the concentration of the betaine aqueous solution is 0.1-3wt%; the concentration of the tannic acid aqueous solution in step (2) is 0.1-1wt%;

[0030] In step (1), the mixing is carried out at 200 to 900 rpm for 0.5 to 3 hours;

[0031] The mixing in step (2) is carried out by stirring at 200 to 900 rpm for 5 to 8 hours.

[0032] In one embodiment, the dialysis in step (3) uses a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, the dialysis fluid is a pure aqueous solution, and the dialysis time is 12 to 24 hours.

[0033] The sixth object of the present invention is to provide a curcumin embedding carrier, which is prepared using the above-mentioned betaine-based protein conjugate, and the preparation method comprises:

[0034] Curcumin and anhydrous ethanol are prepared to obtain a curcumin anhydrous ethanol solution, and a betaine-based protein conjugate and water are prepared to obtain a betaine-based protein conjugate solution;

[0035] The curcumin anhydrous ethanol solution is added to the betaine-based protein conjugate solution, mixed and dried to obtain a curcumin embedding carrier.

[0036] In one embodiment, curcumin and anhydrous ethanol are prepared to obtain a curcumin anhydrous ethanol solution (concentration of 5 to 15 mg / mL), and a betaine-based protein conjugate and water are prepared to obtain a betaine-based protein conjugate solution (concentration of 80 to 120 mg / mL); the mass ratio of curcumin to the protein conjugate is 5 to 10:1, and the curcumin anhydrous ethanol solution is added dropwise to the betaine-based protein conjugate solution, stirred, and dried to obtain a curcumin embedding carrier.

[0037] The seventh object of the present invention is to provide an antioxidant comprising one or two of the above-mentioned betaine-based protein conjugate and the above-mentioned curcumin embedding carrier.

[0038] Beneficial effects of the present invention

[0039] The present invention provides a method for preparing a betaine-based protein conjugate, wherein a whey protein isolate-tannic acid protein conjugate is prepared by a double cross-linking method of betaine and alkali treatment. The betaine-based protein conjugate prepared by the present invention has small particles and significantly improved antioxidant properties, and can be used as a new antioxidant for effectively encapsulating active ingredients.

[0040] Specifically,

[0041] 1. The whey protein isolate used in the present invention is a natural biopolymer, which is low in price and environmentally friendly.

[0042] 2. The tannic acid used in the present invention belongs to the polyhydroxyflavonol compound and can undergo a strong covalent reaction with whey protein isolate.

[0043] 3. The present invention uses betaine as an effective hydrophilic cross-linking agent to quickly induce the covalent coupling of whey protein isolate and tannic acid under alkaline treatment conditions to form a betaine-based protein conjugate. The material is low-cost and environmentally friendly, the method is simple and easy to operate, and it also provides a theoretical basis for loading antioxidant and intestinal targeted active substances.

[0044] 4. The betaine-based protein conjugate prepared by the present invention has good antioxidant stability. When the concentration of the betaine-based protein conjugate is 0.5 mg / mL, the DPPH and ABTS free radical scavenging rates reach 98.46% and 98.52%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is an infrared spectra of the betaine-based protein conjugate, betaine and whey protein isolate prepared in Example 1.

[0046] Figure 2 The infrared spectra of the protein conjugates prepared in Example 1, Comparative Example 1 and Comparative Example 2 are compared.

[0047] Figure 3The free radical scavenging ability of different protein conjugates in the examples and comparative examples; wherein, implementation 1 (i.e., Example 1, 2% WPI, 0.8% Bet, 1:1 volume mixing pH9, and then adding 0.5% TA, 1:1 volume mixing), comparison 1 (i.e., comparative example 1, 1:1% WPI, 0.5% TA, 1:1 volume mixing, pH9), comparison 2 (i.e., comparative example 2, 2% WPI + 0.8% Bet, 1:1 volume mixing, pH9), comparison 3 (i.e., comparative example 2, 2% WPI + 0.8% Bet, 1:1 volume mixing, pH9), comparison 4 (i.e., comparative example 3, 2% WPI + 0.8% Bet, 1:1 volume mixing, pH9), comparison 5 (i.e., comparative example 4, 2% WPI + 0 Example 3, 2% WPI, 0.8% Bet, 1:1 volume mixing pH9, then adding 0.5% TA, 1:1 volume mixing), Comparative Example 4 (i.e. Comparative Example 4, 2% WPI+0.5% TA, 1:1 volume mixing pH9, then adding 0.8% Bet, 1:1 volume mixing), Comparative Example 5 (i.e. Comparative Example 5, 5% WPI+6% Bet, 1:1 volume mixing pH9, then adding 1% TA, 1:1 volume mixing), only TA is the same concentration of tannic acid (0.25% TA, pH9), only Bet is the same concentration of betaine (0.2% Bet, pH9), only WPI is the same concentration of whey protein isolate (0.5% WPI, pH9). DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0049] The raw materials used in the embodiment:

[0050] Whey protein isolate (90% purity) was purchased from Shanghai Prochin International Trading Co., Ltd.

[0051] Betaine was purchased from Yixing Tianshi Feed Co., Ltd., 98%;

[0052] Tannic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., 98%.

[0053] Example 1: Preparation of betaine-assisted protein conjugates

[0054] A method for preparing a betaine-based protein conjugate comprises the steps of:

[0055] (1) weighing whey protein isolate, betaine and tannic acid, dissolving them in water respectively, and stirring them fully to completely dissolve them, to prepare a 2wt% whey protein isolate aqueous solution, a 0.8wt% betaine aqueous solution and a 0.5wt% tannic acid aqueous solution;

[0056] (2) mixing the whey protein isolate aqueous solution and the betaine aqueous solution in a volume ratio of 1:1, adjusting the pH to 9.0 using a 1 M NaOH solution, and stirring at 500 rpm for 1 h to obtain a mixed solution;

[0057] (3) mixing the mixed solution obtained in step (2) and the tannic acid aqueous solution in a volume ratio of 1:1, stirring at 500 rpm for 24 hours at room temperature to allow the mixture to react fully, thereby obtaining a reaction solution;

[0058] (4) The reaction solution was poured into a dialysis bag of 8000-14000Da, the dialysis solution was replaced every 6 hours to remove free tannic acid, the dialysis was performed for 18 hours, and the lyophilization was performed to obtain a betaine-based protein conjugate (i.e., protein-tannic acid conjugated nanoparticles formed by cross-linking of betaine and alkali treatment).

[0059] Comparative Example 1: No betaine used

[0060] Based on Example 1, without adding betaine, the method comprises the following steps:

[0061] (1) weighing whey protein isolate and tannic acid, dissolving them in water respectively, and stirring them fully to dissolve them completely, to prepare a 1wt% whey protein isolate aqueous solution and a 0.5wt% tannic acid aqueous solution;

[0062] (2) mixing the whey protein isolate aqueous solution and the tannic acid aqueous solution, adjusting the pH to 9.0 using a 1M NaOH solution, and stirring for 1 hour to obtain a whey protein isolate-tannic acid aqueous solution;

[0063] (3) Add the whey protein isolate-tannic acid aqueous solution into a 8000-14000 Da dialysis bag, dialyze for 18 hours, replace the dialysate every 6 hours, and freeze-dry to obtain a protein conjugate (whey protein isolate-tannic acid nanoparticles).

[0064] Comparative Example 2: No use of tannic acid

[0065] Based on Example 1, without adding tannic acid, the method comprises the following steps:

[0066] (1) weighing whey protein isolate and betaine, dissolving them in water respectively, and stirring them thoroughly to completely dissolve them, thereby preparing a 1 wt % whey protein isolate aqueous solution and a 0.4 wt % betaine aqueous solution;

[0067] (2) mixing the whey protein isolate aqueous solution and the betaine aqueous solution in a volume ratio of 1:1, adjusting the pH to 9.0 using a 1M NaOH solution, and stirring for 1 hour to prepare a mixed solution;

[0068] (3) Stirring at room temperature for 24 hours to allow for sufficient reaction;

[0069] (4) The reaction solution was poured into a 8000-14000 Da dialysis bag, and the dialysis bag was changed every 6 hours for 18 hours. The protein conjugate (whey protein isolate-betaine particles) was prepared by freeze-drying.

[0070] Comparative Example 3: No pH adjustment

[0071] On the basis of Example 1, the pH adjustment to 9.0 in step (2) was omitted, and the remaining steps were the same as in Example 1 to prepare a protein conjugate.

[0072] Comparative Example 4: Changing the preparation order

[0073] On the basis of Example 1, the order of tannic acid and betaine is changed, comprising the steps of:

[0074] (1) weighing whey protein isolate, betaine and tannic acid, dissolving them in water respectively, and stirring them fully to completely dissolve them, to prepare a 2wt% whey protein isolate aqueous solution, a 0.8wt% betaine aqueous solution and a 0.5wt% tannic acid aqueous solution;

[0075] (2) mixing the whey protein isolate aqueous solution and the tannic acid aqueous solution in a volume ratio of 1:1, adjusting the pH to 9.0 and stirring for 1 hour to prepare a mixed solution;

[0076] (3) mixing the mixed solution prepared in step (2) and the betaine aqueous solution in a volume ratio of 1:1, stirring in an open state at room temperature for 24 hours to allow the mixture to react fully, thereby preparing a reaction solution;

[0077] (4) The reaction solution was poured into a 8000-14000 Da dialysis bag, the dialysis solution was replaced every 6 hours to remove free tannic acid, and the protein conjugate was prepared by freeze drying.

[0078] Comparative Example 5: Changing the concentration of whey protein isolate, betaine and tannic acid

[0079] On the basis of Example 1, the concentration of the whey protein isolate aqueous solution in step (1) was changed to 5wt%, the concentration of the betaine aqueous solution was changed to 6wt%, and the concentration of the tannic acid aqueous solution was changed to 1wt%. The remaining steps were consistent with Example 1 to prepare a protein conjugate.

[0080] Example 2: Performance testing of protein conjugates assisted by betaine

[0081] The protein conjugates prepared in Example 1 and Comparative Examples 1 to 5 were taken to test their properties.

[0082] 1. Infrared spectroscopy

[0083] The detection method of infrared spectrum is as follows:

[0084] The infrared spectra of the samples were measured by potassium bromide tablet method.

[0085] The frozen sample was freeze-dried into powder, mixed with potassium bromide at a mass ratio of 1:100, and then ground into a uniform powder using an agate mortar.

[0086] The scanning conditions were set as follows: spectral range 400~4000cm -1 The number of scans was 32, with a resolution of 4 cm.

[0087] Potassium bromide was used as a blank control. The spectrum acquisition of each sample was repeated three times under the same conditions.

[0088] Test results such as Figure 1 As shown, the main peak in the spectrum of whey protein isolate (WPI) is 3295.8 cm -1 、2962.1cm -1 、1646.9cm -1 and 1533.1cm -1 , representing amide I band (NH stretching, C=O stretching) and amide II band (CH stretching, NH bending and CN stretching). Two characteristic peaks were observed in betaine: at 1623.8 cm -1 The peak at 3484.7 is attributed to carbonyl stretching, and the peak at 3484.7 is attributed to OH stretching. These peaks are also present in the covalent complex, indicating that the betaine group also participates in the reaction.

[0089] like Figure 2 As shown, compared with whey protein isolate, the complex prepared in Comparative Example 2 is non-covalently bound, with a peak at amide I band (1600-1700 cm -1 The characteristic peak shifted significantly, which is related to the non-covalent interaction (such as hydrophobic interaction or hydrogen bond) between protein and polyphenol. The protein conjugates of Example 1 and Comparative Example 1 are covalently bound. In Comparative Example 1, the complex of tannic acid (TA) covalently grafted with whey protein isolate under alkaline treatment conditions was -1 Redshift 2cm -1 The absorption peak of amide I band is at 1652 cm -1 、1538cm -1 、1395cm -1 The red shift of 6cm in many places -1 , 5cm -1 , which indicates that the -NH 2 Participated in the grafting reaction, and this was caused by TA being oxidized to quinone, which reacted with nucleophilic groups (such as amino and sulfhydryl groups) in the protein to form protein dimers. Under the influence of betaine, after tannic acid was covalently grafted with whey protein isolate in combination with alkali treatment (Example 1), at 3297 cm -1 Redshift 8cm -1The absorption peak of amide I band is at 1654 cm -1 、1558cm -1 The red shift is 8 cm -1 , 20cm -1 Among them, betaine may provide a more hydrophilic environment for tannic acid due to its interaction with protein, which is more conducive to covalent interaction.

[0090] 2. Antioxidant testing

[0091] The antioxidant test method is as follows:

[0092] DPPH free radical scavenging rate: The betaine-based protein conjugate prepared in Example 1 was taken to prepare 2.5 mL of a 0.5 mg / mL betaine-based protein conjugate aqueous solution, and mixed with 1 mL of a 0.1 mM DPPH ethanol solution; the mixture was reacted in the dark for 25 min, and the absorbance was measured at 517 nm.

[0093] ABTS free radical scavenging rate: Take the betaine-based protein conjugate prepared in Example 1, prepare 1 mL of 0.5 mg / mL betaine-based protein conjugate and mix it thoroughly with 3 mL of ABTS solution (absorbance at 734 nm is 0.70±0.02), place the mixture in a dark place to react for 15 min, and measure the absorbance at 734 nm.

[0094] The calculation formula for the scavenging ability of DPPH free radical and ABTS free radical is as follows:

[0095] Free scavenging activity (%) = (A 0 -A) / A 0 )×100.

[0096] Among them A 0 represents the absorbance of distilled water (distilled water is used as the control in DPPH detection) or PBS (phosphate buffer solution PBS is used as the control in ABTS detection) as the blank control, and A represents the absorbance after the protein conjugate reaction.

[0097] Using equal amounts of tannic acid (0.25 wt%), whey protein isolate (0.5 wt%) and betaine (0.2 wt%) as controls, the test results are as follows Figure 3 As shown in Table 1, the antioxidant performance of the betaine-based protein conjugate prepared in Example 1 is significantly better than that of the same amount of single substance;

[0098] Comparison between Example 1 and Comparative Examples 1 and 2 shows that the antioxidant activity of the betaine-based protein conjugate prepared from three components is significantly better than that of the two components, and a good antioxidant effect cannot be achieved without any one of them.

[0099] When the whey protein isolate first forms a stable hydration with betaine through electrostatic interaction and hydrogen bonding, and then reacts with tannic acid at pH 9, the protein-polyphenol covalent conjugate (the betaine-based protein conjugate prepared in Example 1) formed with the assistance of betaine under alkaline conditions has significantly enhanced antioxidant properties compared to when the pH is not adjusted to alkaline conditions (Comparative Example 3), and the DPPH and ABTS free radical scavenging rates reach 98.46% and 98.52%, respectively.

[0100] Compared with Example 1 and Comparative Example 4, it can be seen that changing the preparation order will also affect the antioxidant properties of the protein conjugate; compared with Example 1 and Comparative Example 5, it can be seen that when the dosage ratio of whey protein isolate, betaine and tannic acid is not appropriate, the antioxidant properties of the protein conjugate will also be affected.

[0101] From the perspective of molecular mechanism, after betaine and protein build a stronger hydrophilic environment, the OH bond of the phenolic hydroxyl group in the tannic acid structure is directly broken to obtain hydrogen atoms, which directly combine with free radical molecules to remove free radicals and improve the antioxidant performance of the system. This also proves that polyphenols are more successfully grafted on whey protein isolate. The above results show that betaine-based protein conjugates can be used as a potential new natural antioxidant that can replace synthetic antioxidants.

[0102] Table 1 Antioxidant test

[0103] DPPH free radical scavenging rate (%) <![CDATA[ABTS + Free radical scavenging rate (%)]]> Example 1 98.46 98.52 Comparative Example 1 23.29 36.63 Comparative Example 2 25.43 57.53 Comparative Example 3 36.63 57.53 Comparative Example 4 59.68 67.25 Comparative Example 5 6.06 1.01 Tannic acid at the same concentration 25.33 33.56 Betaine at the same concentration 26.82 36.74 Same concentration of whey protein isolate 24.81 10.91

[0104] Example 3: Preparation of betaine-assisted protein conjugates

[0105] On the basis of Example 1, the step (1) was changed to use a 5wt% whey protein isolate aqueous solution, a 3wt% betaine aqueous solution and a 1wt% tannic acid aqueous solution, and the remaining steps were consistent with Example 1 to prepare a betaine-based protein conjugate.

[0106] After testing, the prepared betaine-based protein conjugate also has strong antioxidant properties, and its effect is slightly lower than that of Example 1.

[0107] Example 4: Application of protein conjugates

[0108] The betaine-based protein conjugate prepared in Example 1 (or Example 3) is used to prepare a curcumin embedding carrier, comprising the steps of:

[0109] (1) Curcumin was dissolved in anhydrous ethanol to prepare an anhydrous ethanol solution of curcumin with a concentration of 10 mg / mL, a betaine-based protein conjugate and water were taken to prepare a betaine-based protein conjugate solution with a concentration of 100 mg / mL, and the anhydrous ethanol solution of curcumin was added dropwise to the betaine-based protein conjugate solution, and stirred continuously at 500 rpm for 1 hour to fully dissolve, wherein the mass ratio of the betaine-based protein conjugate to curcumin was 10:1;

[0110] (2) After being fully dissolved, the mixture was continuously stirred with magnetic stirring at a rotation speed of 500 rpm for 30 min, and the ethanol was removed by rotary evaporation; and the mixture was freeze-dried to obtain the curcumin embedding carrier.

[0111] The results showed that the encapsulation rate of curcumin by the encapsulation carrier was as high as 93.1%, and the antioxidant DPPH free radical scavenging rate was as high as 97.3%, which had an excellent encapsulation effect.

[0112] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a betaine-based protein conjugate, characterized in that: The method comprises: (1) mixing a whey protein isolate aqueous solution and a betaine aqueous solution in a volume ratio of 1:0.1-1, adjusting the pH to 7-11, and mixing to obtain a mixed solution; (2) mixing the mixed solution and the tannic acid aqueous solution in a volume ratio of 1:0.1-1 to obtain a reaction solution; (3) dialyzing the reaction solution and drying it to obtain a betaine-based protein conjugate; The concentration of the whey protein isolate aqueous solution in step (1) is 2 wt% to 5 wt%, the concentration of the betaine aqueous solution is 0.8 wt% to 3 wt%, and the concentration of the tannic acid aqueous solution in step (2) is 0.5 wt% to 1 wt%.

2. The method according to claim 1, characterized in that In step (1), the mixing is carried out at 200-900 rpm for 0.5-3 h; and in step (2), the mixing is carried out at 200-900 rpm for 5-8 h.

3. The method according to claim 1, characterized in that The dialysis in step (3) is performed using a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, and the dialysis time is 12 to 24 h.

4. A betaine-based protein conjugate prepared by the method described in any one of claims 1 to 3.

5. Use of the betaine-based protein conjugate according to claim 4 in preparing food.

6. The use according to claim 5, characterized in that: The food comprises: an antioxidant, an emulsifier, a stabilizer, an active substance embedding / delivery carrier, a flavor substance embedding / delivery carrier or a functional food embedding / delivery carrier.

7. The use according to claim 6, characterized in that: The active substances include resveratrol, curcumin, quercetin, carotenoids, and pterostilbene.

8. Use of the betaine-based protein conjugate according to claim 4 in the preparation of a drug carrier.

9. The use according to claim 8, characterized in that: The drug carriers include: poorly soluble drug embedding / delivery carriers, protein embedding / delivery carriers, vitamin embedding / delivery carriers, and antibiotic embedding / delivery carriers.

10. Use of the betaine-based protein conjugate according to claim 4 in the preparation of health products.

11. A product, characterized in that The product contains the betaine-based protein conjugate according to claim 4; the product is a food, a drug carrier or a cosmetic.

12. The product according to claim 11, characterized in that The food comprises: an antioxidant, an emulsifier, a stabilizer, an active substance embedding / delivery carrier, a flavor substance embedding / delivery carrier or a functional food embedding / delivery carrier.

13. The product according to claim 11, characterized in that The drug carriers include: poorly soluble drug embedding / delivery carriers, protein embedding / delivery carriers, vitamin embedding / delivery carriers, and antibiotic embedding / delivery carriers.

14. A health product, characterized in that: The health product contains the betaine-based protein conjugate according to claim 4.

15. A method for improving the antioxidant activity of whey protein isolate-tannic acid protein conjugate, characterized in that: Betaine is added during the preparation of whey protein isolate-tannic acid protein conjugate, and the preparation method comprises: (1) mixing a whey protein isolate aqueous solution and a betaine aqueous solution in a volume ratio of 1:0.1-1, adjusting the pH to 7-11, and mixing to obtain a mixed solution; (2) mixing the mixed solution and the tannic acid aqueous solution in a volume ratio of 1:0.1-1 to obtain a reaction solution; (3) dialyzing the reaction solution and drying it to obtain a whey protein isolate-tannic acid protein conjugate with improved antioxidant properties; Wherein, the concentration of the whey protein isolate aqueous solution in step (1) is 2 wt%~5 wt%, and the concentration of the betaine aqueous solution is 0.8 wt%~3 wt%; the concentration of the tannic acid aqueous solution in step (2) is 0.5 wt%~1 wt%; In step (1), the mixing is carried out at 200-900 rpm for 0.5-3 h; In step (2), the mixing is carried out at 200-900 rpm for 5-8 h.

16. A curcumin embedding carrier, characterized in that: The betaine-based protein conjugate is prepared using the betaine-based protein conjugate according to claim 4, and the preparation method comprises: Curcumin and anhydrous ethanol are prepared to obtain a curcumin anhydrous ethanol solution, and a betaine-based protein conjugate and water are prepared to obtain a betaine-based protein conjugate solution; The curcumin anhydrous ethanol solution is added to the betaine-based protein conjugate solution, mixed and dried to obtain a curcumin embedding carrier.

17. An antioxidant, characterized in that Contains one or both of the betaine-based protein conjugate according to claim 4 and the curcumin embedding carrier according to claim 16.

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