Method for improving solubility by coordination of walnut meal protein isolate and metal polyphenol network

Abstract

The invention discloses a method for improving solubility by coordinating walnut meal protein isolate with a metal polyphenol network. The method comprises the following steps: preparing a metal polyphenol network conjugate and forming a ternary graft of the conjugate and walnut protein isolate, the metal polyphenol network conjugate is formed by coordination chelation of caffeic acid and zinc ions; the formation of the ternary graft comprises the step of forming the hydrophilic ternary graft by the caffeic acid-zinc ion conjugate and different proportions of walnut protein isolate under pH cycle induction, so that the zinc-rich walnut protein isolate with remarkably improved solubility is obtained. According to the method, the solubility of the walnut meal protein can be improved to 70.88% from 30.71%, the steps are simple, the safety is high, the period is short, and the method has important significance in expanding the application of the walnut meal protein in the field of food processing.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing of plant proteins, and particularly relates to a method for improving the solubility of walnut meal isolated protein coordinated with a metal polyphenol network. Background Art

[0002] With the development of the global economy and the advancement of urbanization, animal protein has gradually failed to meet people's diverse dietary needs, and the utilization of plant protein resources has therefore attracted much attention. Plant protein is not only renewable and environmentally friendly, but its rich nutritional value also makes it one of the most popular sources of protein. Walnut, also known as walnut, belongs to the genus Juglans of the Juglandaceae family. It contains about 18-24% protein and rich essential amino acids. It is an important source of plant protein, and China is the country with the largest walnut production in the world. Walnut meal is one of the main by-products of walnut oil processing, with a protein content of more than 50%. However, due to the low utilization rate of walnut meal, it is often discarded or used only as animal feed, resulting in a great waste of protein resources. Studies have shown that walnut protein has multiple physiological functions such as antioxidant, hypotensive, anti-fatigue, and anti-diabetes. Its main components include gluten, alcohol protein, globulin and albumin, of which gluten accounts for more than 70%, showing great commercial value and application potential. However, the walnut protein extracted by the existing technology has poor solubility, which limits its application in the food industry.

[0003] At present, there have been studies on changing the physicochemical properties of walnut protein by physical and chemical modification to improve its solubility. Patent CN 107232388 A discloses a preparation method for optimizing the processing characteristics of walnut protein, which uses alkali dissolution-acid precipitation technology to extract isolated protein from defatted walnut meal powder, and then uses different ultrasonic treatment methods to modify it. This process not only improves the solubility and emulsification of walnut protein, but also retains its nutritional value, but involves problems such as expensive equipment costs. Patent CN 115669794 A discloses a method for improving the solubility of rice bran protein, which uses ultrasound combined with ultrahigh pressure to treat rice bran to obtain modified rice bran protein with improved solubility. However, it brings higher energy consumption, increases industrial production costs and environmental impact. Patent CN 110643661 A discloses a method for rapid separation and purification of walnut protein from walnuts, the method comprising the following steps: firstly, grinding the walnuts into powder, defatting them with petroleum ether, then extracting the protein with alcohol solution and enzymatic hydrolysis technology, then purifying the protein with supercritical fluid extraction and macroporous resin adsorption, and finally obtaining the protein by elution, concentration and drying. However, this process may result in solvent residues due to the use of petroleum ether, and the process is complicated and costly.

[0004] Therefore, in order to solve the above technical problems, a new technical solution needs to be proposed to solve the problem, especially to provide a method for improving the solubility of walnut meal protein isolate coordinated with metal polyphenol network. Summary of the invention

[0005] The invention aims to solve the technical problems of low walnut protein separation rate and high cost in the existing technical solutions and provides a method for improving the solubility of walnut meal isolated protein coordinated with a metal polyphenol network.

[0006] To achieve the above object, the following technical solution is provided: a method for improving the solubility of walnut meal protein isolate by coordinating a metal polyphenol network, comprising the following steps:

[0007] S1. Caffeic acid-Zn 2+ Preparation of stock solution

[0008] Dissolve caffeic acid and zinc chloride in PBS buffer, mix them, stir for 5 min at room temperature and stabilize the pH of the system at 10.0 to form caffeic acid-Zn 2+ Metal polyphenol network;

[0009] S2. Extraction of walnut protein

[0010] The defatted walnut meal powder was mixed with water at a ratio of 1:15 (w / v), and the walnut protein isolate was obtained by pH cycle induced alkaline dissolution-acid precipitation method;

[0011] S3. Walnut protein isolate and caffeic acid-Zn 2+ Complex preparation

[0012] S31. The walnut protein isolate was resuspended in PBS buffer to prepare a 10% (w / v) walnut protein solution;

[0013] S32. Caffeic acid-Zn 2+ The metal polyphenol network solution and the walnut protein solution are mixed in proportion to prepare a ternary graft;

[0014] S33. After dialysis to remove unbound polyphenols, vacuum freeze-drying was performed to obtain the complex walnut protein isolate.

[0015] Preferably, the caffeic acid and Zn 2+ The mixture was dissolved in buffer at a mass ratio of 2:1, and the final concentration of caffeic acid was 0.5 mg / mL.

[0016] Preferably, the alkali dissolution-acid precipitation method in step S2 comprises the following steps:

[0017] (1) Defatted walnut meal powder and water were mixed at a ratio of 1:15 (w / v), the pH value was adjusted to 10.0 using a 6 mol / L NaOH solution, and magnetic stirring was performed at a temperature of 55° C. for 2.0 h. After stirring, centrifugation was performed at a speed of 4,000 r / min and a centrifugation time of 20 min to obtain the supernatant after centrifugation;

[0018] (2) Adjust the pH to 4.5 with 6 mol / L HCl, and repeat the above stirring and centrifugation steps;

[0019] (3) After centrifugation, the precipitate is resuspended with a small amount of distilled water and the pH is adjusted to neutral. Walnut protein isolate can be obtained by vacuum freeze drying for 48 hours.

[0020] Preferably, in step S31, the buffer is 10 mMol / L PBS buffer with a pH of 10.0, which can form more caffeic acid-Zn 2+ Complex and promote the dissolution of walnut protein isolate.

[0021] Preferably, in step S32, the caffeic acid-Zn 2+ The mass ratio of metal polyphenol network to walnut protein isolate was 1:10, which was proven to form a more stable ternary graft.

[0022] Preferably, in step S33, the walnut protein isolate-caffeic acid-Zn 2+ The solution was dialyzed using a 7 kDa dialysis bag for 36 h and the water was changed at least five times to remove free caffeic acid.

[0023] Beneficial effects:

[0024] (1) The present invention prepares ternary grafts of walnut protein isolate based on metal polyphenol network self-assembly, which greatly improves the solubility of walnut protein isolate (up to 70.88%), and there are no related patent reports on the preparation of high-solubility proteins using the same type of metal polyphenol network. In addition, the preparation method provided by the present invention introduces the supplement of zinc ions, which can expand the application of walnut protein in the food industry.

[0025] (2) Zinc, as an indispensable trace element for the human body, can participate in the physiological metabolic process of the body. However, zinc from natural sources easily forms insoluble precipitates, thereby reducing the body's absorption of zinc. Therefore, the method provided by the present invention significantly improves the solubility of walnut protein isolate and gives it the zinc ion supplementation property, and supplements the zinc ions required by the human body by combining the metal polyphenol network with the pH cycle induction modification of the walnut protein isolate.

[0026] (3) The preparation method provided by the present invention has a simple process flow, mild conditions, does not require the intervention of large or high-end equipment, can greatly reduce energy consumption and does not produce side reactions, thereby reducing production costs and is suitable for industrial production. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0030] Caffeic acid-Zn in the technical solution of the present invention 2+ The order of the steps of preparing the storage solution and extracting the walnut protein can be interchanged.

[0031] The specific steps of this embodiment are as follows:

[0032] Embodiment 1:

[0033] (1) Extraction of walnut protein: Walnut protein isolate was extracted by alkali dissolution-acid precipitation method. Defatted walnut meal powder was mixed with water at a ratio of 1:15 (w / v). The pH value was adjusted to 10.0 using 6 mol / L NaOH solution. The mixture was magnetically stirred at 55°C for 2.0 h. After stirring, the mixture was centrifuged at a speed of 4,000 r / min for 20 min. The supernatant was obtained and the pH value was adjusted to 4.5 using 6 mol / L HCl. The above stirring and centrifugation steps were repeated. The precipitate after centrifugation was resuspended with a small amount of distilled water and the pH value was adjusted to neutral. Walnut protein isolate was obtained by vacuum freeze drying for 48 h.

[0034] (2) Caffeic acid-Zn 2+ Preparation of stock solution: Use phosphate buffered saline (PBS, 10 mMol / L, pH 10.0) to prepare a 0.5 mg / mL caffeic acid solution. 2+ Add a certain amount of Zn at a mass ratio of 2:1 2+The mixed solution was rapidly stirred at room temperature for 5 min while maintaining the pH at 10.0, and then stored at 4°C for future use.

[0035] (3) Walnut protein isolate-caffeic acid-Zn 2+ Preparation of complex: Walnut protein isolate was resuspended in 10 mMol / L, pH 10.0 PBS buffer to prepare a 10% (w / v) walnut protein solution. 2+ Add caffeic acid-Zn in a ratio of 5:1 2+ The storage solution was reacted at 25°C for 2 hours and the pH was stabilized at 10.0. After that, the solution pH was adjusted to 7.0 and dialyzed for 36 hours using a 7kDa dialysis bag and the water was changed at least five times to remove free caffeic acid. Finally, the dialyzed solution was freeze-dried at a temperature of -50°C and a vacuum degree of 2Pa to obtain walnut protein powder.

[0036] Embodiment 2:

[0037] The difference from Example 1 is that: Walnut protein isolate: caffeic acid-Zn 2+ The ratio is adjusted to 8:1. The rest of the steps are the same.

[0038] Embodiment 3:

[0039] The difference from Example 1 is that: Walnut protein isolate: caffeic acid-Zn 2+ The ratio is adjusted to 10:1. The rest of the steps are the same.

[0040] Embodiment 4:

[0041] The difference from Example 1 is that: Walnut protein isolate: caffeic acid-Zn 2+ The ratio is adjusted to 12:1. The rest of the steps are the same.

[0042] Embodiment 5:

[0043] The difference from Example 1 is that: Walnut protein isolate: caffeic acid-Zn 2+ The ratio is adjusted to 15:1. The rest of the steps are the same.

[0044] Comparative Example 1:

[0045] (1) Extraction of walnut protein: Walnut protein isolate was extracted by alkali dissolution-acid precipitation method. Defatted walnut meal powder was mixed with water at a ratio of 1:15 (w / v). The pH value was adjusted to 10.0 using 6 mol / L NaOH solution. The mixture was magnetically stirred at 55°C for 2.0 h. After stirring, the mixture was centrifuged at a speed of 4,000 r / min for 20 min. The supernatant was obtained and the pH value was adjusted to 4.5 using 6 mol / L HCl. The above stirring and centrifugation steps were repeated. The precipitate after centrifugation was resuspended with a small amount of distilled water and the pH value was adjusted to neutral. Walnut protein isolate was obtained by vacuum freeze drying for 48 h.

[0046] (2) Preparation of walnut protein isolate solution: Walnut protein isolate was resuspended in 10 mM, pH 10.0 PBS buffer to prepare a 10% (w / v) walnut protein solution. It was then reacted at 25°C for 2 h and the pH was stabilized at 10.0. The solution pH was then adjusted to 7.0 and dialyzed for 36 h using a 7 kDa dialysis bag and the water was changed at least five times. Finally, the dialyzed solution was freeze-dried at a temperature of -50°C and a vacuum of 2 Pa to obtain walnut protein powder.

[0047] Comparative Example 2:

[0048] (1) Extraction of walnut protein: Walnut protein isolate was extracted by alkali dissolution-acid precipitation method. Defatted walnut meal powder was mixed with water at a ratio of 1:15 (w / v). The pH value was adjusted to 10.0 using 6 mol / L NaOH solution. The mixture was magnetically stirred at 55°C for 2.0 h. After stirring, the mixture was centrifuged at a speed of 4,000 r / min for 20 min. The supernatant was obtained and the pH value was adjusted to 4.5 using 6 mol / L HCl. The above stirring and centrifugation steps were repeated. The precipitate after centrifugation was resuspended with a small amount of distilled water and the pH value was adjusted to neutral. Walnut protein isolate was obtained by vacuum freeze drying for 48 h.

[0049] (2) Preparation of caffeic acid stock solution: A 0.5 mg / mL caffeic acid solution was prepared using phosphate buffered saline (PBS, 10 mM, pH 10.0), and then stored at 4°C until use.

[0050] (3) Preparation of walnut protein isolate-caffeic acid complex: Take walnut protein isolate and resuspend it in 10mM, pH10.0 PBS buffer to prepare a 10% (w / v) walnut protein solution. Then, add caffeic acid storage solution at a ratio of 10:1 of walnut protein isolate: caffeic acid, react at 25°C for 2 hours, and stabilize the pH at 10.0. After adjusting the solution pH to 7.0, dialyze it for 36 hours using a 7kDa dialysis bag and change the water at least five times to remove free caffeic acid. Finally, freeze-dry the dialyzed solution at a temperature of -50°C and a vacuum degree of 2Pa to obtain walnut protein powder, which is used to compare caffeic acid or caffeic acid-Zn with the example. 2+ Effect of complex addition on the solubility of walnut protein isolate.

[0051] Caffeic acid-Zn 2+ Addition ratio Protein solubility (%) Surface hydrophobicity (H0)

[0052]

[0053] Table 1 shows the solubility and surface hydrophobicity of walnut protein isolate of the embodiment and comparative example. From the data comparison in the table, it can be found that the walnut protein isolate extracted by alkali dissolution and acid precipitation has the lowest solubility, only 30.71%, and the surface hydrophobicity can reach 730.33. After adding caffeic acid to walnut protein isolate, its solubility increased significantly to 45.31%. When caffeic acid-Zn was added in a ratio of 10:1, the solubility of walnut protein isolate increased significantly to 45.31%. 2+ After the metal network, the two are coordinated and chelated with walnut protein isolate, and the solubility is significantly increased to 70.88%, and the surface hydrophobicity is reduced to 436.33, indicating that caffeic acid and zinc ions can form a more stable and soluble ternary graft with walnut protein isolate than adding caffeic acid alone. In summary, the solubility of the walnut protein powder obtained by ternary grafting is significantly enhanced, and the zinc ion supplement is given to it, which can be better applied to the field of food deep processing and improve the nutritional value of food.

[0054] The solubility and surface hydrophobicity determination methods are as follows:

[0055] (1) Walnut protein solubility determination method

[0056] Take 1g of walnut protein isolate or walnut protein complex and resuspend it in 100mL of PBS buffer with a pH value of 7. After standing at room temperature for 1h, centrifuge at 4,000g for 20min at 25℃. Use BCA protein concentration determination kit to detect the protein content in the supernatant. The protein solubility is calculated using the following formula:

[0057]

[0058] (2) Determination of Surface Hydrophobicity of Walnut Protein

[0059] The hydrophobicity of the protein surface was determined using the hydrophobic fluorescent probe ANS. The walnut protein sample was prepared into a 0.5 mg / mL test sample with PBS buffer. Then 400 μL of the sample was mixed with 10 μL of 8 mM ANS stock solution. After each sample was reacted in the dark for 2 minutes, the fluorescence emission spectrum was collected in the range of 370 to 680 nm, and the excitation wavelength was set to 365 nm. The initial slope of the fluorescence intensity and protein concentration was calculated as an indicator of surface hydrophobicity.

[0060] In summary, the present invention provides a preparation method that significantly improves the solubility of walnut protein isolate, while giving the protein the characteristic of zinc ion supplementation. Compared with the existing chemical and enzymatic methods for preparing walnut protein isolate, there are disadvantages such as side reactions or high costs. The preparation method provided by the present invention only needs to add walnut protein isolate, caffeic acid and zinc ions respectively according to the step ratio to obtain walnut protein isolate with high solubility. The operation is simple, low energy consumption, economic cost saving, and industrial feasibility.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the solubility of walnut meal protein isolate coordinated with a metal polyphenol network, characterized in that: The following steps are involved: S1. Caffeic acid-Zn 2+ Preparation of stock solution Dissolve caffeic acid and zinc chloride in PBS buffer, mix them, stir for 5 min at room temperature and stabilize the pH of the system at 10.0 to form caffeic acid-Zn 2+ Metal polyphenol network; S2. Extraction of walnut protein The defatted walnut meal powder was mixed with water at a ratio of 1:15 (w / v), and the walnut protein isolate was obtained by pH cycle induced alkaline dissolution-acid precipitation method; S3. Walnut protein isolate and caffeic acid-Zn 2+ Complex preparation S31. The walnut protein isolate was resuspended in PBS buffer to prepare a 10% (w / v) walnut protein solution; S32. Caffeic acid-Zn 2+ The metal polyphenol network solution and the walnut protein solution are mixed in proportion to prepare a ternary graft; S33. After dialysis to remove unbound polyphenols, vacuum freeze-drying was performed to obtain the complex walnut protein isolate.

2. The method according to claim 1, characterized in that: The caffeic acid and Zn 2+ The mixture was dissolved in buffer at a mass ratio of 2:1, and the final concentration of caffeic acid was 0.5 mg / mL.

3. The method according to claim 1, characterized in that: The alkali dissolution-acid precipitation method in step S2 comprises the following steps: (1) Defatted walnut meal powder and water were mixed at a ratio of 1:15 (w / v), the pH value was adjusted to 10.0 using a 6 mol / L NaOH solution, and magnetic stirring was performed at a temperature of 55° C. for 2.0 h. After stirring, centrifugation was performed at a speed of 4,000 r / min and a centrifugation time of 20 min to obtain the supernatant after centrifugation; (2) Adjust the pH to 4.5 with 6 mol / L HCl, and repeat the above stirring and centrifugation steps; (3) After centrifugation, the precipitate is resuspended with a small amount of distilled water and the pH is adjusted to neutral. Walnut protein isolate can be obtained by vacuum freeze drying for 48 hours.

4. The method according to claim 1, characterized in that: In step S31, the buffer solution is 10 mMol / L PBS buffer solution with a pH of 10.0, which can form more caffeic acid-Zn 2+ Complex and promote the dissolution of walnut protein isolate.

5. The method according to claim 1, characterized in that: In step S32, the caffeic acid-Zn 2+ The mass ratio of metal polyphenol network to walnut protein isolate was 1:

10.

6. The method according to claim 1, characterized in that: In the step of S33, the walnut protein isolate-caffeic acid-Zn 2+ The solution was dialyzed using a 7 kDa dialysis bag for 36 h and the water was changed at least five times to remove free caffeic acid.

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