Sodium hyaluronate-meat protein-polyphenol non-covalent compound as well as preparation method and application thereof

Abstract

The invention discloses a sodium hyaluronate-meat protein-polyphenol non-covalent complex as well as a preparation method and application thereof, and the method comprises the following steps: dissolving meat protein in a phosphate buffer solution to prepare a meat protein solution; adjusting the pH value of the meat protein solution to 11.9-12.1, adding curcumin to obtain a first reaction solution, and carrying out hydrophobic interaction reaction to obtain a meat protein-curcumin composite solution; adjusting the pH value of the meat protein-curcumin composite solution to 6.9-7.1, adding the sodium hyaluronate solution to obtain a second reaction solution, and reacting to obtain the sodium hyaluronate-meat protein-polyphenol non-covalent compound. The curcumin induces the protein to generate structural change, and the sodium hyaluronate is selectively adsorbed on the surfaces of the protein and the curcumin to form a core-shell structure, so that the water solubility of the protein is improved; meanwhile, sodium hyaluronate is used for shielding hydrophobic sites of the curcumin, so that the embedding rate of the curcumin is remarkably increased, and the stability of the meat protein-curcumin nanoparticles is improved under the modification of the sodium hyaluronate.

Description

Technical Field

[0001] The invention belongs to the technical field of functional food processing, and specifically relates to a sodium hyaluronate-meat protein-polyphenol non-covalent complex and a preparation method and application thereof. Background Art

[0002] Due to the increasing awareness and preference of consumers for healthy diet, the addition of bioactive compounds such as curcumin to food has attracted increasing attention. Curcumin is a hydrophobic polyphenol and the main yellow phytochemical of turmeric (Curcuma longa) (Curcumaceae). Although the potential health benefits of curcumin are numerous, its low water solubility, chemical instability and low oral bioavailability limit its application during storage and food processing.

[0003] Protein and polysaccharide are useful materials for food and pharmaceutical grade due to their biodegradability, low price, non-toxicity, non-immunogenicity and non-inflammatory properties. Using protein and polysaccharide together is one of the ways to release curcumin continuously, promote intracellular absorption and avoid side effects. Meat protein contains all essential and non-essential amino acids and has a high digestibility compared to cereal, milk or bean protein.

[0004] However, a key issue that needs to be addressed is that the concentration of curcumin in the protein-polyphenol complex will induce precipitation, and the protein is very sensitive to enzymatic degradation and will be digested in the stomach, causing the protein-polyphenol nanoparticle structure to decompose before reaching the small intestine, resulting in the inability of bioactive substances to be absorbed in the small intestine, which can easily greatly reduce the utilization efficiency of polyphenols. Summary of the invention

[0005] Some anionic polysaccharides are dietary fibers that are stable to hydrolysis by enzymes in gastric and intestinal fluids. Sodium hyaluronate is a natural mucopolysaccharide that binds to specific receptors on the cell surface. It is expressed at low levels on the surface of epithelial cells, hematopoietic cells, and neural cells, but is overexpressed in many tumor cells. Therefore, these advantages of sodium hyaluronate make it and its derivatives effective delivery materials for drugs and nutritional supplements. At the same time, the addition of hydrophilic sodium hyaluronate greatly improves the solubility of the meat protein-curcumin nanocomposite solution.

[0006] Objective: To overcome the deficiencies in the prior art, the present invention provides a sodium hyaluronate-meat protein-polyphenol non-covalent complex and a preparation method and application thereof.

[0007] Technical solution: The preferred technical solution adopted by the present invention is:

[0008] According to a first aspect of the present invention, a sodium hyaluronate-meat protein-polyphenol non-covalent complex is provided.

[0009] S1, dissolving meat protein in a phosphate buffer solution to prepare a meat protein solution; the phosphate buffer solution contains NaCl with a concentration of 0.5 to 0.7 M;

[0010] S2, adjusting the pH of the meat protein solution to 11.9-12.1, adding curcumin to obtain a first reaction solution, and causing a hydrophobic reaction to obtain a meat protein-curcumin composite solution;

[0011] S3. Adjust the pH of the meat protein-curcumin composite solution to 6.9-7.1, add sodium hyaluronate solution to obtain a second reaction solution, and react to obtain a sodium hyaluronate-meat protein-polyphenol non-covalent complex; the solvent of the sodium hyaluronate solution is a phosphate buffer solution.

[0012] In some embodiments, in S1, the composition of the phosphate buffer solution is: 0.6M NaCl, 20mM NaH2PO4, 20mM Na2HPO4, pH is 7.0.

[0013] In some embodiments, in S1, the concentration of meat protein in the meat protein solution is 4-6 mg / mL, preferably 5 mg / mL.

[0014] In some embodiments, S2 satisfies at least one of the following:

[0015] In the first reaction solution, the mass ratio of meat protein to curcumin is 1:(0.01-0.06);

[0016] The concentration of curcumin in the first reaction solution is 0.05 to 0.30 mg / mL, preferably 0.15 to 0.20 mg / mL.

[0017] In some embodiments, the reaction condition in S2 is stirring reaction at room temperature, preferably stirring at 600 rpm for 30 min at room temperature; the reaction condition in S3 is stirring reaction at room temperature, preferably stirring at 400 rpm for 30 min at room temperature.

[0018] In some embodiments, S3 satisfies at least one of the following:

[0019] In the second reaction solution, the mass ratio of meat protein to sodium hyaluronate is 10:1;

[0020] The concentration of the sodium hyaluronate solution is 0.4-0.6 mg / mL, preferably 0.5 mg / mL.

[0021] In some embodiments, in S2, the pH of the meat protein solution is adjusted to 11.9-12.1 using NaOH;

[0022] In some embodiments, in S3, the pH of the meat protein-curcumin composite solution is adjusted to 6.9-7.1 using HCl.

[0023] In some embodiments, the method for extracting meat protein comprises:

[0024] (1) Process the chicken breast into minced meat, add the extracting solution for extraction, and centrifuge to obtain the precipitate;

[0025] (2) After step (1) is repeated three times, the precipitate is dissolved in KCl solution, the floating matter is removed and the precipitate is centrifuged to obtain the meat protein.

[0026] In some embodiments, the extract is prepared from cold distilled water containing 0.1M KCl, 2mM MgCl2, A solution of 20 mM KH2PO4, 20 mM K2HPO4, and 1 mM EGTA at pH 7.0;

[0027] In some embodiments, the concentration of the KCl solution is 0.1M.

[0028] In addition, according to the second aspect of the present invention, a sodium hyaluronate-meat protein-polyphenol non-covalent complex is provided, which is prepared by the preparation method.

[0029] According to a third aspect of the present invention, there is provided application of the above-mentioned sodium hyaluronate-meat protein-polyphenol non-covalent complex in functional food.

[0030] Beneficial effects: The method for preparing a sodium hyaluronate-meat protein-polyphenol non-covalent complex provided by the present invention has the following advantages: in the present invention, sodium hyaluronate is added to prepare a sodium hyaluronate-meat protein-polyphenol non-covalent complex, curcumin induces structural changes in the protein, and after the addition of sodium hyaluronate, the sodium hyaluronate is selectively adsorbed on the surface of the protein and curcumin to form a core-shell structure, thereby improving the water solubility of the protein and improving the stability of the complex system; at the same time, the addition of sodium hyaluronate effectively solves the hydrophobic aggregation precipitation induced by excessive curcumin in the meat protein polyphenol solution, the meat protein-curcumin aggregates will increase with the increase of the curcumin loading amount, and the hydrophobic cross-linking effect of curcumin will cause the aggregates to precipitate. The present invention uses sodium hyaluronate to shield the hydrophobic sites of curcumin, so that the aggregation and precipitation between the meat protein-curcumin nanoparticles mediated by curcumin are significantly reduced, and finally the embedding rate of curcumin is significantly increased. The stability of the meat protein-curcumin nanoparticles is improved under the modification of sodium hyaluronate, and the bioavailability of the active ingredients is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of protein solubility of the meat protein-polyphenol complex and the sodium hyaluronate-meat protein-polyphenol non-covalent complex in the examples;

[0032] Figure 2 It is a schematic diagram of the embedding rate of the meat protein-polyphenol complex and the sodium hyaluronate-meat protein-polyphenol non-covalent complex in the examples;

[0033] Figure 3 Schematic diagram of the Turbiscan instability index TSI measurement of the meat protein-polyphenol complex (A) and the sodium hyaluronate-meat protein-polyphenol non-covalent complex (B) in the examples. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments.

[0035] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0036] For the purpose of this specification and the appended claims, unless otherwise stated, all numbers expressing amounts, percentages or ratios and other numerical values ​​used in this specification and the appended claims are understood to be modified in all cases by the term "about". In addition, all ranges disclosed herein are inclusive and independently combinable.

[0037] The preparation method of the meat protein solution in this embodiment includes:

[0038] (1) Process the chicken breast into minced meat, add an appropriate amount of extracting solution for leaching, and centrifuge to obtain the precipitate; the extracting solution is prepared from cold distilled water and contains 0.1M KCl, 2mM MgCl2, a solution of 20 mM KH2PO4, 20 mM K2HPO4, and 1 mM EGTA at pH 7.0;

[0039] (2) After step 1 is repeated three times, the precipitate is dissolved in a KCl solution, the floating matter is removed and the precipitate is centrifuged to obtain the meat protein; the concentration of the KCl solution is 0.1 M;

[0040] (3) Dissolve the extracted meat protein in a phosphate buffer solution and hydrate overnight at 4°C to prepare a 5 mg / mL meat protein solution; the phosphate buffer solution is a buffer solution prepared from water containing 0.6 M NaCl, 20 mM NaH2PO4 and 20 mM Na2HPO4 with a pH of 7.0.

[0041] The preparation method of the sodium hyaluronate solution in this embodiment is as follows: sodium hyaluronate (100-150 wda) is dissolved in a phosphate buffer solution, and hydrated overnight at 4°C to prepare a 0.5 mg / mL sodium hyaluronate solution, wherein the phosphate buffer solution is a buffer solution prepared from water containing 0.6 M NaCl, 20 mM NaH2PO4 and 20 mM Na2HPO4 with a pH of 7.0.

[0042] Embodiment 1:

[0043] Preparation of sodium hyaluronate-meat protein-polyphenol non-covalent complex:

[0044] The pH value of 5 mg / mL meat protein solution was adjusted to 12 with 4M NaOH, and curcumin was added to make its concentration 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL. After stirring at room temperature for half an hour, the pH of the meat protein-curcumin composite solution was adjusted to 7.0 with 4M HCl, and then 0.5 mg / mL of sodium hyaluronate solution was added at a volume ratio of 1:1. After stirring at room temperature for half an hour, a sodium hyaluronate-meat protein-polyphenol non-covalent complex was obtained, and the final mass ratio of protein to sodium hyaluronate was 10:1.

[0045] Comparative Example 1: The preparation methods of meat protein solution and sodium hyaluronate solution are the same as above;

[0046] Preparation of meat protein-polyphenol complex:

[0047] The pH value of the 5 mg / mL meat protein solution was adjusted to 12 with 4M NaOH, and curcumin was added to make its concentration 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL. After stirring at room temperature for half an hour, the pH of the meat protein-curcumin complex solution was adjusted to 7.0 with 4M HCl to obtain a meat protein-polyphenol complex.

[0048] Example 2: Measurement of protein solubility in sodium hyaluronate-meat protein-polyphenol non-covalent complex

[0049] The complex was centrifuged at 10,000 g for 10 min in a high-speed refrigerated centrifuge (4°C), and the supernatant was collected. The protein concentration in the supernatant after centrifugation and the myofibrillar protein concentration before centrifugation were determined by the biuret method. Myofibrillar protein solubility (%) = supernatant protein concentration / uncentrifuged protein solution concentration*100%.

[0050] Good water solubility is a key requirement for utilizing proteins as functional ingredients in many commercial products such as sports drinks and nutritional beverages. Figure 1 As shown in , as the content of curcumin increases, the solubility of meat protein in the complex decreases, indicating that the hydrophobic force induced by curcumin causes the complex to aggregate. The protein solubility in the meat protein polyphenol polysaccharide complex is higher than that in the meat protein polyphenol group, indicating that after curcumin induces structural changes in the protein, sodium hyaluronate is added, and the polysaccharide selectively adsorbs on the surface of the protein and curcumin to form a core-shell structure, thereby effectively stabilizing the meat protein polyphenol solution and improving the water solubility of the protein.

[0051] Example 3: Measurement of curcumin embedding rate in sodium hyaluronate-meat protein-polyphenol non-covalent complex

[0052] The complexes prepared in Example 1 and Comparative Example 1 were centrifuged at 10000 g for 10 min to remove insoluble particles. The supernatant was mixed with a certain volume of ethanol and rotated in the dark to mix thoroughly. The absorbance at 419 nm was measured using an ELISA instrument, and the standard curve of curcumin-ethanol solution (R 2 =0.9997) to calculate the curcumin concentration. Encapsulation rate (%) = curcumin concentration in supernatant / curcumin concentration in composite solution.

[0053] The encapsulation rate of the nanoparticles indicates the content of curcumin in the nanoparticles. If the curcumin is not completely encapsulated in the nanoparticles, it will leak out. Therefore, the delivery ability of meat protein polyphenol polysaccharide nanoparticles with active substances as carriers was studied. Figure 2 The effect of the addition of sodium hyaluronate on the encapsulation efficiency of curcumin was evaluated. The results of the encapsulation efficiency showed that curcumin was successfully encapsulated in these nanoparticles. The addition of polysaccharide sodium hyaluronate increased the encapsulation efficiency of curcumin compared to meat protein polyphenol nanoparticles. At low curcumin concentrations, enough biopolymer nanoparticles could encapsulate curcumin. As the curcumin concentration increased, the encapsulation efficiency decreased.

[0054] Example 4: Turbiscan instability index TSI measurement of sodium hyaluronate-meat protein-polyphenol non-covalent complex solution

[0055] The colloidal dispersion stability is estimated by TSI using a vertical scanning analyzer. The sample is transferred to the sample cell (height about 44 mm) immediately after preparation. The fresh sample is immediately transferred to the sample cell and scanned at 25 °C for 15 hours. The formula for calculating the Turbiscan Instability Index (TSI) is:

[0056]

[0057] where scani is the average backscattered light intensity at each measurement (i), scani-1 is the average backscattered light intensity at the time of measurement (i-1), and H is the number of scans calculated on the sample.

[0058] Since food can be affected during production or use, it is necessary to evaluate the stability of nanoparticles. The measured TSI value reflects the effect of particle size change and sedimentation. In general, more stable systems show lower TSI values. As shown in Figure 3, when the curcumin concentration exceeds 0.15 mg / mL, the complex in the system undergoes hydrophobic aggregation and the stability decreases. However, after adding sodium hyaluronate to the meat protein-polyphenol group, the TSI value is smaller, indicating that the stability of the sodium hyaluronate-meat protein-polyphenol non-covalent complex system has been improved.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a sodium hyaluronate-meat protein-polyphenol non-covalent complex, characterized in that: include: S1. dissolving meat protein in phosphate buffer solution to prepare a meat protein solution; The phosphate buffer solution contains NaCl at a concentration of 0.5 to 0.7 M; S2, adjusting the pH of the meat protein solution to 11.9-12.1, adding curcumin to obtain a first reaction solution, and causing a hydrophobic reaction to obtain a meat protein-curcumin composite solution; S3. Adjust the pH of the meat protein-curcumin composite solution to 6.9-7.1, add sodium hyaluronate solution to obtain a second reaction solution, and react to obtain a sodium hyaluronate-meat protein-polyphenol non-covalent complex; the solvent of the sodium hyaluronate solution is a phosphate buffer solution.

2. The preparation method according to claim 1, characterized in that: In S1, the composition of the phosphate buffer solution is: 0.6M NaCl, 20mM NaH2PO4, 20mM Na2HPO4, and the pH is 7.

0.

3. The preparation method according to claim 1 or 2, characterized in that: In S1, the concentration of meat protein in the meat protein solution is 4-6 mg / mL, preferably 5 mg / mL.

4. The preparation method according to claim 1, characterized in that: S2 satisfies at least one of the following: In the first reaction solution, the mass ratio of meat protein to curcumin is 1:(0.01-0.06); The concentration of curcumin in the first reaction solution is 0.05 to 0.30 mg / mL, preferably 0.15 to 0.20 mg / mL.

5. The preparation method according to claim 1, characterized in that: S3 satisfies at least one of the following: In the second reaction solution, the mass ratio of meat protein to sodium hyaluronate is 10:1; The concentration of the sodium hyaluronate solution is 0.4-0.6 mg / mL, preferably 0.5 mg / mL.

6. The preparation method according to claim 1, characterized in that: Include at least one of the following: In S2, the pH of the meat protein solution was adjusted to 11.9-12.1 using NaOH; In S3, the pH of the meat protein-curcumin composite solution is adjusted to 6.9-7.1 using HCl.

7. The preparation method according to claim 1, characterized in that: The method for extracting meat protein comprises: (1) Process the chicken breast into minced meat, add the extracting solution for extraction, and centrifuge to obtain the precipitate; (2) After step (1) is repeated three times, the precipitate is dissolved in KCl solution, the floating matter is removed and the precipitate is centrifuged to obtain the meat protein.

8. The preparation method according to claim 7, characterized in that: At least one of the following is met: The extract is a solution prepared from cold distilled water containing 0.1M KCl, 2mM MgCl2, 20mM KH2PO4, 20mM K2HPO4 and 1mM EGTA with a pH of 7.0; The concentration of the KCl solution is 0.1M.

9. A sodium hyaluronate-meat protein-polyphenol non-covalent complex, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the sodium hyaluronate-meat protein-polyphenol non-covalent complex according to claim 9 in functional foods.

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