Preparation method of ball-milling soybean protein / chlorogenic acid covalent complex stable nanoemulsion

Abstract

The application provides a preparation method of a ball-milling soybean protein / chlorogenic acid covalent compound stable nanoemulsion. Soybean protein can stabilize an emulsion system. However, the natural functional properties of natural soybean protein are not prominent enough, which limits the development and application of products to a certain extent. Ball-milling modification is a new protein physical modification method, which can significantly change the physicochemical properties of proteins and improve the functional properties of proteins. In the application, ball-milling technology and polyphenol covalent combination technology are combined, the ball-milling time is designed, and the content of chlorogenic acid is optimized, so that the nanoemulsion is prepared. The nanoemulsion provided in the application has good stability of enhanced emulsifying property and antioxidant activity, and provides a certain theoretical basis and reference for developing a food-grade emulsion carrying system with high antioxidant property.

Description

Technical Field

[0001] This invention pertains to soybean protein processing technology, specifically a method for preparing nanoemulsions. Background Technology

[0002] Oil-in-water (O / W) nanoemulsions are generally prepared using a top-down high-energy method. This involves utilizing the strong forces generated by mechanical devices such as microfluidics and high-pressure homogenizers to mix the oil and water phases into tiny droplets in the presence of an emulsifier. The size of these droplets depends on the homogenization method, operating conditions, and the composition of the delivery system. Soy protein possesses an amphiphilic structure, which can reduce oil-water interfacial tension and form a protective film, stabilizing the emulsion system. However, the natural functional properties of soy protein are not sufficiently prominent, limiting product development and application to some extent. Therefore, modifying proteins using specific techniques to obtain specialized protein products with high emulsifying properties is of great significance.

[0003] Ball milling is an emerging physical modification method for proteins, capable of significantly altering their physicochemical properties and enhancing their functional properties. This method utilizes mechanical energy input to process proteins, causing changes in their molecular structure and chemical bonds, thereby modifying their properties. Liang Yingjie et al. (2023) found that ball milling can alter the secondary structure of pumpkin seed protein, exposing more aromatic amino acid residues, reducing particle size, and improving solubility. Mehnaza Manzoor et al. (2022) found that ball milling significantly reduces the particle size of apple seed protein, improving its solubility and emulsifying properties. Gu Yinghui et al. (2022) found that ball milling significantly improves the surface hydrophobicity and zeta potential of peanut protein; with increasing milling time, the molecular particle size of peanut protein gradually decreases, while the content of β-turns and random coils increases, improving emulsifying performance.

[0004] Covalent binding of polyphenols can affect the functional properties of proteins. Sun et al. (2020) used a free radical grafting method to covalently graft catechin (CT) and chlorogenic acid (CA) onto egg white protein (EWP), respectively, which lowered the isoelectric point of EWP and improved DPPH free radical scavenging activity and Fe3+. + Reducing ability. Liu et al. (2015) successfully prepared three protein-polyphenol covalent complexes using free radical grafting: lactoferrin (LF)-epigallocatechin gallate (EGCG), lactoferrin-CA, and lactoferrin-gallic acid (GA), which significantly improved the solubility and emulsifying properties of LF.

[0005] Chlorogenic acid is a natural hydroxycinnamic acid compound with strong antioxidant, anti-inflammatory, anticancer, antihypertensive, and antilipidemic effects. Studies by Chen Yichun et al. (2019) have found that after proteins covalently bind to chlorogenic acid, polyphenols can act as H+ ions. + Donors terminate free radical chain reactions, thereby improving the antioxidant properties of protein-chlorogenic acid covalent complexes. Therefore, developing ball-milled soybean protein-chlorogenic acid covalent complex emulsions is a feasible approach to improve their stability and antioxidant properties. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing a ball-milled soybean protein / chlorogenic acid covalent complex stabilized nanoemulsion. This invention combines ball milling technology with polyphenol covalent bonding technology, optimizing the chlorogenic acid content by designing the ball milling time, thus obtaining the nanoemulsion. The nanoemulsion proposed in this invention exhibits good stability with enhanced emulsifying and antioxidant activity, providing a theoretical basis and reference for developing food-grade emulsion delivery systems with high antioxidant properties.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions: 1. Investigating the effect of ball milling on the structure and emulsifying properties of soy protein isolate; 2. Preparation of a binary covalent complex of soy protein isolate and CA (chlorogenic acid); 3. Preparation of a stable O / W nanoemulsion of soy protein isolate complex.

[0008] The method for stabilizing nanoemulsions by ball milling a complex of soy protein isolate and chlorogenic acid includes the following steps:

[0009] (1) Ball milling treatment of soy protein isolate: A planetary ball mill was used with a rotation speed of 260 r / min and a ball-to-material ratio of 8:1. The treatment time was 20, 40, 60 and 80 min respectively.

[0010] (2) The effects of ball milling on the structure and emulsifying properties of soy protein isolate were investigated, and the optimal ball milling time was determined to be 60 min.

[0011] (3) Disperse the ball-milled SPI in deionized water, stir magnetically for 2 hours, and leave the solution at 4°C overnight to allow it to fully hydrate.

[0012] (4) Centrifuge the dispersion at 8000 rpm for 20 min and take the supernatant. Mix it with an equal volume of chlorogenic acid solutions of different concentrations (0.2 mmol / L, 0.5 mmol / L, 0.8 mmol / L).

[0013] (5) Adjust the pH of the solution to 9.0 using 0.1 mol / L NaOH. Stir continuously for 12 h under alkaline and oxygen-containing conditions, and then use HCl to adjust the pH of the solution back to 7.4.

[0014] (6) After dialysis with a dialysis bag with a molecular weight of 3500 kDa for 48 h, a binary covalent complex was obtained.

[0015] (7) The binary complex was characterized and the optimal binding concentration of CA was determined to be 0.5 mmol / L.

[0016] (8) The complex solution and soybean oil were mixed at a ratio of 9:1 (v / v) and homogenized by an IKA homogenizer (16,000 rpm, 2 min) to obtain a crude emulsion.

[0017] (9) O / W nanoemulsion was obtained by homogenizing five times at a homogenization pressure of 90 MPa using a high-pressure homogenizer.

[0018] This invention employs a method that uses ball milling to process soybean protein isolate and combine it with chlorogenic acid to prepare a nanoemulsion, thereby improving the antioxidant and stability properties of the food-grade emulsion delivery system. Using soybean protein isolate as the raw material, a planetary ball mill is used to grind the soy protein isolate. The ground protein then covalently binds with chlorogenic acid under alkaline induction to prepare a binary covalent complex. Using the prepared binary complex as an emulsifier, the oil and aqueous phases are mixed, and after coarse homogenization to obtain a crude emulsion, high-pressure homogenization yields a stable nanoemulsion. The production process employed in this invention is simple, environmentally friendly, and conducive to industrial production, broadening the application of soybean protein isolate in the food industry; it also produces a protein-polyphenol composite nanoemulsion with strong stability and high antioxidant properties. Attached Figure Description

[0019] Figure 1 This is a roadmap of the method of the present invention.

[0020] Figure 2 The effect of ball milling time on the emulsifying properties of soy protein isolate Detailed Implementation

[0021] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0022] Implementation Case 1

[0023] I. Ball milling of soy protein isolate: A planetary ball mill was used, with a speed of 260 r / min and a ball-to-material ratio of 8:1 for 60 min.

[0024] II. Binding of ball-milled protein with chlorogenic acid: An equal volume of ball-milled soy protein isolate (2 mg / mL) and chlorogenic acid solution (0.5 mmol / L) was mixed. The pH of the solution was adjusted to 9.0 using 0.1 mol / L NaOH. The mixture was stirred continuously for 12 h under alkaline and oxygen-containing conditions, and then the pH was adjusted back to 7.4 using 0.1 mol / L HCl. Dialysis using a dialysis bag with a molecular weight of 3500 kDa for 48 h yielded a binary covalent complex.

[0025] III. Preparation of nanoemulsions from binary composites: The composite solution was mixed with soybean oil at a ratio of 9:1 (v / v), and first homogenized using an IKA homogenizer (16,000 rpm, 2 min) to obtain a crude emulsion; then, it was homogenized 5 times using a high-pressure homogenizer at a homogenization pressure of 90 MPa to obtain an O / W nanoemulsion.

[0026] Implementation Case 2

[0027] I. Ball milling of soy protein isolate: A planetary ball mill was used, with a speed of 260 r / min and a ball-to-material ratio of 8:1 for 60 min.

[0028] II. Preparation of nanoemulsion: Soy protein isolate solution and soybean oil were mixed at a ratio of 9:1 (v / v) and homogenized first using an IKA homogenizer (16,000 rpm, 2 min) to obtain a crude emulsion; then, the mixture was homogenized 5 times using a high-pressure homogenizer at a homogenization pressure of 90 MPa to obtain an O / W nanoemulsion.

[0029] Implementation Case 3

[0030] I. Conjugation of Soy Protein Isolate with Chlorogenic Acid: Equal volumes of soy protein isolate solution (2 mg / mL) and chlorogenic acid solution (0.5 mmol / L) were mixed. The pH of the solution was adjusted to 9.0 using 0.1 mol / L NaOH. The mixture was stirred continuously for 12 h under alkaline conditions and in the presence of oxygen, and then the pH was adjusted back to 7.4 using 0.1 mol / L HCl. After dialysis using a dialysis bag with a molecular weight of 3500 kDa for 48 h, a binary covalent complex was obtained.

[0031] II. Preparation of nanoemulsions from binary composites: The composite solution was mixed with soybean oil at a ratio of 9:1 (v / v), and first homogenized using an IKA homogenizer (16,000 rpm, 2 min) to obtain a crude emulsion; then, it was homogenized 5 times using a high-pressure homogenizer at a homogenization pressure of 90 MPa to obtain an O / W nanoemulsion.

Claims

1. A method for preparing high-functionality soy protein isolate, characterized in that... The SPI extracted from soybean meal using the alkali dissolution and acid precipitation method is controlled in the final stage of freeze-drying through a dynamic synergistic strategy of "freezing temperature-freezing time". This is achieved through the following steps:

1. Dissolve defatted soybean meal in deionized water at a ratio of 1:10 and stir with a magnetic stirrer for 2 hours. Adjust the pH of the mixed solution to 8 with NaOH solution (2 mol / L) and stir for 1 hour. Centrifuge at 4℃, 7155g (8000r) for 30 minutes and collect the supernatant.

2. Adjust the pH of the supernatant to 4.5 with HCl solution (2 mol / L) and let it stand for 1 hour until the protein precipitates. After removing part of the supernatant, centrifuge at 7155g (8000r) at 4℃ for 15 minutes and collect the precipitate.

3. Wash the precipitate with deionized water, neutralize it with NaOH solution (2 mol / L) to pH=7.0, and centrifuge it again (4℃, 7155g, 10min). Then pour it into a dialysis bag and dialyze for 48h. IV. The dialyzed protein solutions were frozen at -20, -40 and -80°C for 1, 3 and 5 days, respectively. Finally, the solutions were freeze-dried to obtain SPI and packaged in sealed bags. They were stored at 4°C for later use in detecting the corresponding indicators.

2. The preparation method of a high-functional soy protein isolate according to claim 1, the core of which is step four: the final stage of freeze-drying is controlled by a dynamic synergistic strategy of "freezing temperature-freezing time", the protein solution is placed in an environment of -20, -40 and -80℃ for 1, 3 and 5 days respectively, the freezing temperature is strictly controlled to be constant and the freezing time is precise, and finally the freeze-dried SPI is stored in a sealed bag.

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