Preparation method of a whey protein isolate-propolis complex

Abstract

The present invention provides a method for preparing a whey protein isolate-propolis complex. It provides that commercially available whey protein isolate is dissolved in distilled water to obtain a 10 mg / mL whey protein isolate solution, ascorbic acid and hydrogen peroxide solution are added, after reacting for 2 hours, the pH value is adjusted to 7.5, a propolis solution is added, and the reaction is carried out for 20 minutes; after standing, dialysis, and freeze-drying, the whey protein isolate-propolis complex is obtained. The present invention uses whey protein isolate derived from food, which is safer than polymers. The prepared whey protein isolate-propolis complex has good water solubility and good stability, effectively solves the problem of poor water solubility of propolis, and retains the good antioxidant biological activity of propolis.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein derivatives, and relates to a protein complex and a preparation method thereof, in particular to a preparation method of a whey protein-propolis complex. Technical Background

[0002] Propolis is a solid gum with an aromatic odor formed by worker bees collecting plant buds or resins and mixing them with their mandibular glands and wax glands. The composition of propolis is complex, and the main substance categories include polyphenolic substances (flavonoids, phenolic acids, tannins, etc.), terpenoids, and fatty acids. Among them, the polyphenolic substances in propolis have been proven to have good antibacterial, anti-inflammatory, anti-tumor, hypoglycemic, liver-protecting, obesity-relieving, and immune-regulating effects.

[0003] Since most of the polyphenolic substances in propolis are soluble in organic solvents such as ethanol and insoluble in water, the propolis products that can be purchased on the market mainly include propolis liquid preparations, propolis capsules, and propolis tablets (such as CN111000881B, a soft capsule of Baccharis dracunculifolia type propolis rich in flavonoids and its preparation method; CN110025685B, a liquid preparation of a composition containing propolis). The propolis liquid preparation uses different concentrations of alcohol as a solvent, has a strong unpleasant odor, is highly irritating, and has a poor sensory experience. At the same time, due to the irritation of ethanol, it will also have a stimulating effect on the gastrointestinal tract, limiting its application. A large amount of other substances (such as vegetable oil and cyclodextrin, etc.) are added to the propolis capsules and propolis tablets for processing, and the absorption effect is poor. Therefore, the preparation of a water-soluble propolis preparation is one of the hotspots in the current research on propolis product processing.

[0004] At present, the methods for preparing water-soluble propolis preparations are as follows: (1) using water-extracted propolis as raw material; (2) preparing a propolis nano-solution; (3) grafting a polymer with propolis to prepare a soluble substance. Since the solubility of propolis in water is very small, when using water to extract propolis, the extraction efficiency is low and the production cost is increased. In the preparation process of the propolis nano-solution, organic reagents such as ethanol, acetone or methanol are required (such as a preparation method of propolis nanoparticles, a preparation method, an application and a preparation method of its freeze-dried powder in CN110604747B; a preparation method of nano-propolis solution with the publication number of CN102743416A), which destroys the effective active ingredients of propolis; in addition, organic solvents have a certain stimulating effect on the human digestive tract. Application No. CN 113332453 A provides a preparation method of water-soluble propolis nanoparticles. The preparation method is to quickly add an alkaline solution of propolis to an acidic solution containing a polymer cross-linking agent under stirring, magnetically stir for 2 hours, then centrifuge to separate to obtain a solid product, and obtain water-soluble propolis nanoparticles after washing with water 3 times. The disadvantage of this method is that the polymer used is a polymer cross-linking agent (polyvinyl alcohol, polyvinylpyrrolidone, poloxamer 188), which limits its scope of use.

[0005] Protein is a kind of biological macromolecule substance with a certain spatial structure formed by the dehydration condensation of amino acids and through coiling and folding, and it is one of the nutrients that the human body must ingest every day. Research shows that proteins can form protein-polyphenol complexes through covalent interactions, and the complexes have good stability and water solubility. Using hydroxyl radicals generated by redox pairs (ascorbic acid and hydrogen peroxide) to radicalize proteins and then complex with polyphenols under the action of covalent bond forces, this method is called the free radical grafting method, which has the advantages of simplicity and rapidity. At present, there is no report on using this technology to solve the problem of poor water solubility of propolis. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problem of poor water solubility of existing propolis, and provide a preparation method of a whey protein isolate-propolis complex. This method uses a protein from a food source as a raw material, and the prepared complex has good water solubility, good stability, and excellent antioxidant performance.

[0007] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0008] (1) Prepare radicalized whey protein isolate;

[0009] (2) Mix the radicalized whey protein isolate in step (1) with propolis, and ultrasonicate or shake for 20 minutes to obtain a whey protein isolate-propolis complex solution;

[0010] (3) The protein-propolis complex solution obtained in step (2) is allowed to stand, dialyzed, and freeze-dried to obtain whey protein isolate-propolis complex powder.

[0011] Further, in step (1), the method for preparing radicalized whey protein isolate is to dissolve commercially available whey protein isolate in distilled water to obtain a 10 mg / mL whey protein isolate solution, add 0.1 g of ascorbic acid and 2 mM hydrogen peroxide, and react for 2 hours.

[0012] Further, in step (2), the ratio of whey protein isolate to propolis is 20:1, and the ultrasonic conditions are: 25 °C, 270 W, 20 minutes, pulse time 2 s, intermittent time 2 s.

[0013] Further, in step (3), the standing time is 24 hours; the dialysis conditions are 3500 Da, and distilled water is changed every 6 h for 48 hours of dialysis; the whey protein isolate-propolis complex powder obtained after freeze-drying is light yellow particles.

[0014] In the said step (3), the solubility of the water-soluble protein-propolis complex in water is greater than 95%.

[0015] In the said step (3), the average particle size of the water-soluble protein-propolis complex is 150 - 300 nm, and the zeta potential at pH = 7 is -20 mV - -30 mV.

[0016] In the preparation method of the present invention, taking advantage of the characteristic that proteins and polyphenols can form protein-polyphenol complexes under the action of redox pairs, whey protein isolate-propolis complexes are prepared. The present invention uses whey protein isolate derived from food, which is safer compared to polymers. The prepared whey protein isolate-propolis complexes have good water solubility, effectively solve the problem of poor water solubility of propolis, and retain the good antioxidant biological activity of propolis. Description of the Drawings

[0017] Figure 1 Antioxidant properties of whey protein isolate, Example 1 and Example 2.

[0018] Figure 2 Water solubility of whey protein isolate, Example 1 and Example 2.

[0019] Figure 3 Electron micrographs of whey protein isolate, Example 1 and Example 2. Specific Embodiment Methods

[0020] The following further elaborates on the present invention with reference to the drawings. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the reagents used in the examples are all of analytical grade.

[0021] Example 1: Preparation method of a whey protein isolate-propolis complex

[0022] Specifically, it includes the following steps:

[0023] (1) Dissolve whey protein isolate at a concentration of 10 mg / mL in 40 mL of distilled water, add 0.1 g of ascorbic acid and 2 mM of hydrogen peroxide, and react for 2 hours to obtain radicalized whey protein isolate;

[0024] (2) Add 0.5 mL of 40 mg / mL propolis alcohol solution to the radicalized whey protein isolate, and react with shaking at 25 °C for 20 minutes to obtain a whey protein isolate-propolis solution;

[0025] (3) After standing the whey protein isolate-propolis solution obtained in (2) for 24 hours, dialyze it against 3500 Da for 48 hours, and freeze-dry to obtain whey protein isolate-propolis complex 1.

[0026] Example 2: Preparation method of a whey protein isolate-propolis complex

[0027] Specifically, it includes the following steps:

[0028] (1) Dissolve whey protein isolate at a concentration of 10 mg / mL in 40 mL of distilled water, add 0.1 g of ascorbic acid and 2 mM of hydrogen peroxide, and react for 2 hours to obtain radicalized whey protein isolate;

[0029] (2) Add 0.5 mL of 40 mg / mL propolis alcohol solution to the radicalized whey protein isolate, and perform ultrasonic treatment at 25 °C and 270 W for 20 minutes to obtain an ultrasonicated whey protein isolate-propolis solution;

[0030] (3) After standing the ultrasonicated whey protein isolate-propolis solution obtained in (2) for 24 hours, dialyze it against 3500 Da for 48 hours, and freeze-dry to obtain whey protein isolate-propolis complex 2.

[0031] Example 3: Determination of the total flavonoid content of the whey protein isolate-propolis complex

[0032] Take 100 μL of 20 mg / mL whey protein isolate-propolis complex solution, place it in a 1.5 mL centrifuge tube, add 440 μL of absolute ethanol, mix well, then add 30 μL of 5% sodium nitrate solution, shake well, let stand for 6 minutes, add 30 μL of 10% aluminum nitrate solution and shake well; let stand for 6 minutes, add 400 μL of 4% sodium hydroxide, shake well, let stand for 15 minutes. Using ethanol as the blank, measure the absorbance value of the reaction solution at 510 nm. Draw a standard curve based on the absorbance of rutin solutions with different concentrations, and calculate the total flavonoid content in the whey protein isolate-propolis complex. The total flavonoid content of the whey protein isolate-propolis complex in Example 1 is 7.73 ± 0.27 mg rutin equivalent / g, and the total flavonoid content of the whey protein isolate-propolis complex in Example 2 is 9.89 ± 0.56 mg rutin equivalent / g

[0033] Example 4: Determination of the total phenolic content of whey protein isolate-propolis complex

[0034] Take 90 μL of 20 mg / mL whey protein isolate-propolis complex solution, add 600 μL of Folin-Ciocalteu reagent, mix well, let stand in the dark at room temperature for 1 hour, add 54 μL of 7.5% sodium carbonate solution, mix well, let stand in the dark at room temperature for 15 minutes, measure the absorbance value of the supernatant at 760 nm, and draw a standard curve based on the absorbance of gallic acid solutions with different concentrations, and calculate the total phenolic content in the whey protein isolate-propolis complex. The total phenolic content of the whey protein isolate-propolis complex in Example 1 is 2.14 ± 0.02 gallic acid equivalent / g, and the total phenolic content of the whey protein isolate-propolis complex in Example 2 is 2.68 ± 0.08 mg gallic acid equivalent / g

[0035] Example 5: Evaluation of the antioxidant performance of whey protein isolate-propolis complex by ABTS method

[0036] Accurately weigh 37.845 mg of potassium persulfate and dissolve it in 1 mL of pure water to obtain Solution A. Weigh 192.038 mg of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and dissolve it in 20 mL of pure water to obtain Solution B. Mix 176 μL of Solution A and 10 mL of Solution B, and let it stand for 16 hours to obtain the ABTS stock solution. Dilute the ABTS stock solution so that the absorbance of the ABTS working solution is in the range of 0.7 - 0.9 to obtain the ABTS working solution. Accurately pipette 20 μL of different concentrations of the test sample solution at 10 mg / mL into a 96-well plate, add 180 μL of the ABTS working solution to each well, mix well, and read the absorbance value at 765 nm after reacting for 5 min. Draw a standard curve based on the water-soluble vitamin E at different concentrations and the absorbance, and calculate the antioxidant performance of the whey protein isolate-propolis complex. The ABTS radical scavenging ability of the whey protein isolate-propolis complex in Example 1 was 140.90 ± 2.76 mmol trolox / g, and the ABTS radical scavenging ability of the whey protein isolate-propolis complex in Example 2 was 160.43 ± 1.08 mmol trolox / g( Figure 1 ).

[0037] Example 6: Evaluation of the antioxidant performance of the whey protein isolate-propolis complex by the FRAP method

[0038] The antioxidant performance of the whey protein isolate-propolis complex was measured using a total antioxidant capacity detection kit (FRAP method) (Shanghai Beyotime Biotechnology Co., Ltd.), and the sample concentration was 10 mg / mL. The ability of the whey protein isolate-propolis nanocomplex in Example 1 to reduce Fe 3+ was 67.52 ± 1.24 mM Fe 2+ / g, and the ability of the whey protein isolate-propolis complex in Example 2 to reduce Fe 3+ was 72.63 ± 2.61 mM Fe 2+ / g( Figure 2 ).

[0039] Example 7: Particle size and zeta potential of the whey protein isolate-propolis complex

[0040] The average particle size of the prepared whey protein isolate-propolis complex was 150 - 300 nm, and the zeta potential at pH = 7 was -20 mV - -30 mV, with good stability.

[0041] Example 8: Determination of the solubility of the whey protein isolate-propolis complex

[0042] The whey protein isolate-propolis complex was dissolved in water at a concentration of 1.0 mg / mL, the pH was adjusted to 2.0 - 10.0, and it was magnetically stirred for 1 hour. After calibrating the pH, it was centrifuged at 5000 rpm for 20 minutes, and the protein content in the supernatant was determined by the Coomassie Brilliant Blue method. The ratio of the protein concentration in the supernatant to the total protein concentration was the protein solubility. The results are shown in Figure 3 . The solubility of the whey protein isolate-propolis complex in Example 1 at pH = 7 was 96.8 ± 1.05%, and the solubility of the whey protein isolate-propolis complex in Example 2 at pH = 7 was 97.8 ± 1.1%.

[0043] Conclusion: The whey protein isolate-propolis complex was prepared by the free radical method. The raw materials are economical and safe, the method is simple and feasible, effectively solving the problem of poor water solubility of propolis. The obtained whey protein isolate-propolis complex has a solubility above 95% at pH = 7, good stability, and good antioxidant performance.

[0044] Although the above embodiments have described the present invention in detail, the protection scope of the present invention is not limited thereto. Modifications that do not deviate from the core of the present invention can also be made to the solutions of the present invention, and they all belong to the protection scope of the present invention.

Claims

1. A method for preparing a whey protein isolate-propolis complex, characterized in that, Prepared by the following steps: (1) Radicalization of whey protein isolate; the radicalization of whey protein isolate is to dissolve 0.4 g of whey protein isolate in 40 mL of distilled water to obtain a 10 mg / mL whey protein isolate solution, add 0.1 g of ascorbic acid and 2 mM hydrogen peroxide, and react for 2 hours to obtain radicalized whey protein isolate; (2) Mix the radicalized whey protein isolate obtained in step (1) with propolis, and ultrasonicate or shake for 20 minutes to obtain a whey protein isolate-propolis complex solution; the weight ratio of propolis to whey protein isolate is 1:20, the ultrasonic temperature is 25 °C, the ultrasonic power is 270 W, the pulse time is 2 s, and the intermittent time is 2 s; (3) Let the protein-propolis complex solution obtained in step (2) stand, dialyze, and lyophilize to obtain a whey protein isolate-propolis complex powder. The standing time is 24 hours, the dialysis condition is 3500 Da, the liquid is changed every 6 hours, and dialysis is carried out for 48 hours. The whey protein isolate-propolis complex powder obtained after lyophilization is a slightly yellow granule.

2. The preparation method according to claim 1, characterized in that, The solubility of the water-soluble whey protein isolate-propolis complex in step (3) in water is greater than 95%.

3. The preparation method according to claim 1, characterized in that, The average particle size of the water-soluble whey protein isolate-propolis complex in step (3) is 150 - 300 nm, and the zeta potential at pH = 7 is -20 mV - -30 mV.

Images