Preparation method of high-emulsibility soybean peptide aggregate

Abstract

A preparation method of a high-emulsibility soybean peptide aggregate belongs to the field of soybean protein deep processing, and comprises the following steps: (1) preparing soybean protein isolate, (2) preparing a soybean peptide aggregate, and (3) preparing the soybean peptide aggregate modified by L-arginine after hydrodynamic cavitation; the method has the advantages of being simple in process, safe to operate and the like, the soybean peptide aggregate is homogenized through hydrodynamic cavitation, then L-arginine is used for modification, the product has high emulsibility, and the application of the soybean peptide aggregate prepared into an emulsifier in the functional food industry can be expanded.

Description

[0001] The invention relates to the technical field of soybean deep processing, and mainly relates to a method for preparing soybean peptide aggregates (SPA) with enhanced emulsification properties by utilizing hydrodynamic cavitation combined with arginine modification. Background Art

[0002] As a natural emulsifier, plant protein is of irreplaceable importance in the food system. Compared with synthetic emulsifiers, plant protein has a wide range of sources, and has both nutritional functionality and biocompatibility, which meets the "clean label" and "reduction" requirements of modern food. In the food field, plant protein-based emulsifiers have a wide range of applications, such as plant-based dairy products (such as soy milk, vegetable cream), and acidic beverages (such as fruit juice milk drinks). In addition, plant protein emulsifiers can also be used as a delivery carrier for active ingredients (such as polyphenols and fat-soluble vitamins) to achieve targeted delivery and sustained release of nutrients. Its application not only optimizes food texture and stability, but also meets consumers' demand for healthy and sustainable food.

[0003] Among various plant proteins, soy protein is an ideal choice, offering high nutritional value and functional properties. However, soy protein typically exists in a globular structure, which limits its functional properties to a certain extent. Enzymatic hydrolysis can modulate the exposure of internal hydrophobic groups to improve functional properties. During the enzymatic hydrolysis of soybeans, peptides and proteins inevitably interact and aggregate, ultimately forming SPAs. In the industrial production of soy peptides, due to their poor functionality, these aggregates are often discarded or used as raw materials for animal feed, significantly reducing the utilization rate of soy protein.

[0004] Currently, single protein modification methods mainly include chemical modification, physical modification, enzymatic modification, and genetic engineering modification. However, single modification still has limitations in improving protein functional properties. A combined physical-chemical modification method is a more efficient modification method that is more suitable for industrial production. Among them, hydrodynamic cavitation treatment is a new, green, and convenient physical technology with great application prospects in the field of food processing. During the cavitation process, by generating instantaneous high pressure, high temperature, strong shear and impact forces in the liquid, it can cause changes in the molecular structure of the protein, increase the exposure of its emulsifying sites, and improve its solubility. At the same time, chemical modification methods utilize active groups such as arginine's rich amino and guanidine groups to bind to proteins through electrostatic interactions, hydrogen bonds, etc., changing their surface charge distribution and chemical composition, which has the potential to significantly improve the emulsification properties of SPA.

[0005] In summary, in order to significantly enhance the emulsifying properties of SPA, prepare a SPA emulsion with excellent performance, and achieve efficient utilization of soy peptide resources, the present invention is dedicated to overcoming the shortcomings of existing SPA modification technologies. SPA is modified using a method combining hydrodynamic cavitation with arginine modification, resulting in a modified SPA with excellent emulsifying properties that can be used to prepare food emulsifiers. Summary of the Invention

[0006] To address the above problems, the present invention modifies SPA through hydrodynamic cavitation and arginine modification. The modified SPA has high emulsification properties and can be applied to emulsions and functional foods, which has great potential value.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] A method for preparing a highly emulsifiable SPA, characterized in that the method comprises the following steps:

[0009] (1) First, fresh soybeans were ground into powder, mixed with n-hexane at a ratio of 1:3 (w / v), and stirred at room temperature for 2 h for three defatting steps. Second, defatted soybean powder was mixed with deionized water at a ratio of 1:10 (w / v), and the pH value was adjusted to 8.5 with 2 mol / L NaOH solution. After stirring at room temperature for 2 h, the mixture was centrifuged at 9000 rpm for 30 min. The supernatant was taken and the pH value was adjusted to 4.5 with 2 mol / L HCl solution. After standing for 2 h, the mixture was centrifuged at 6000 rpm for 20 min to obtain protein precipitate. Finally, the protein precipitate was dissolved in deionized water, the pH value of the protein was adjusted to 7.0 with 2 mol / L NaOH solution, and the protein solution was freeze-dried to obtain powdered SPI. (2) The SPI powder prepared above was dissolved in distilled water at a mass fraction of 0.5-5% (w / v), magnetically stirred at 25°C until completely dissolved, the pH of the solution was adjusted to 8.5 with 2 mol / L NaOH, and alkaline protease was added at a substrate concentration of 0.5-2% (w / v) for enzymatic hydrolysis at 50°C for 2-12 hours. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 10 minutes, and the solution was quickly cooled to room temperature with ice water, the pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 minutes. After centrifugation, the precipitate was washed three times with water and freeze-dried to obtain SPA. (3) The SPA and L-arginine were dissolved and poured into a cavitation jet machine for hydrodynamic cavitation treatment. The cavitation jet treatment parameters were a pressure of 100-120 MPa and a treatment time of 5-15 minutes. The ratio of arginine to SPI is 1:8-12.

[0010] The method for preparing a SPA emulsion using hydrodynamic cavitation combined with arginine modification to enhance emulsification according to claim 1 is characterized in that the preferred addition amount of SPI powder is 4%, the preferred addition amount of alkaline protease is 1%, and the preferred enzymatic hydrolysis time is 8 hours.

[0011] The method for preparing a SPA emulsion using hydrodynamic cavitation combined with arginine modification to enhance emulsification according to claim 1 is characterized in that: the hydrodynamic cavitation equipment used in step (3) includes a feed tank, a peristaltic pump, and a hydrodynamic cavitation generator, wherein the feed port of the hydrodynamic cavitation generator is connected to the peristaltic pump, and the discharge port is connected to the feed tank, and the discharge port and the feed port are provided with temperature detectors. The sample enters the hydrodynamic cavitation generator from the feed tank through the peristaltic pump for cavitation treatment and then flows back to the feed tank. After multiple cycles, the aggregate is modified.

[0012] The method for preparing a SPA emulsion using hydrodynamic cavitation combined with arginine modification to enhance emulsification according to claim 1 is characterized in that: the preferred conditions are an L-arginine structure, and the volume ratio of the amino acid to the insoluble aggregate solution is 1:9. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The overall process route diagram of the present invention is

[0014] Figure 2 The curves showing the effects of different treatments on the particle size potential of insoluble peptide aggregates

[0015] Figure 3 This is the curve showing the effect of the ratio of L-arginine to insoluble aggregates on the emulsification activity and emulsification stability of the aggregates.

[0016] Figure 4 The curve showing the effect of hydrodynamic cavitation equipment parameters on aggregate solubility DETAILED DESCRIPTION

[0017] For a better understanding of the present invention, the present invention is described in further detail below with reference to examples and drawings.

[0018] The detection method of the present invention comprises:

[0019] 1. Determination of particle size and zeta potential: The sample was diluted with deionized water to a mass concentration of 0.1 mg / mL, and then the average particle size and zeta potential of the sample were measured using a Malvern nanoparticle size potential analyzer.

[0020] 2. Solubility Determination: Take 5 mL of the treated sample solution and determine the total protein content (Kjeldahl method). Centrifuge at 8000 rpm for 20 minutes. Collect the supernatant and determine the protein content using the Kjeldahl method. Protein solubility = protein content in supernatant / total protein content × 100%.

[0021] 3. Stability Determination: Dispersions of various samples were prepared by dissolving them in deionized water (1%, m / v). 2 mL of soybean oil was added to 8 mL of the dispersion and homogenized using a high-speed shear at 10,000 rpm for 2 minutes to form an emulsion. Immediately thereafter, 40 μL of the emulsion was diluted 200-fold with 0.1% sodium dodecyl sulfate (SDS) solution. The absorbance was measured at 500 nm using SDS as a blank. After the emulsion rested for 30 minutes, the same procedure was repeated to measure the absorbance.

[0022]

[0023]

[0024] Where A0 and A 30 are the absorbance of the emulsion at 0 min and 30 min, respectively; C is the concentration of the sample before emulsion formation (g / mL); is the volume fraction of the oil phase in the emulsion; D is the sample dilution multiple

[0025] Implementation Case 1: (1) First, fresh soybeans were ground into powder, mixed with n-hexane at a ratio of 1:3 (w / v), and stirred at room temperature for 2 h for three defatting steps. Second, defatted soybean powder was mixed with deionized water at a ratio of 1:10 (w / v), and the pH value was adjusted to 8.5 with 2 mol / L NaOH solution. After stirring at room temperature for 2 h, the mixture was centrifuged at 9000 rpm for 30 min. The supernatant was taken and the pH value was adjusted to 4.5 with 2 mol / L HCl solution. After standing for 2 h, the mixture was centrifuged at 6000 rpm for 20 min to obtain protein precipitate. Finally, the protein precipitate was dissolved in deionized water, the pH value of the protein was adjusted to 7.0 with 2 mol / L NaOH solution, and the protein solution was freeze-dried to obtain powdered SPI. (2) The SPI powder prepared above was dissolved in distilled water at a mass fraction of 4% (w / v), magnetically stirred at 25°C until completely dissolved, and the pH of the solution was adjusted to 8.5 with 2 mol / L NaOH. Alkaline protease was added at a substrate concentration of 1% (w / v) and enzymatically hydrolyzed at 50°C for 8 h. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 10 min, and the solution was quickly cooled to room temperature with ice water, the pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 min. After centrifugation, the precipitate was washed three times with water and freeze-dried to obtain SPA. (3) The SPA and arginine were mixed in a ratio of 9:1 and introduced into a cavitation jet machine for hydrodynamic cavitation treatment. The cavitation jet treatment parameters were a pressure of 110 MPa and a treatment time of 10 min.

[0026] Implementation Case 2: (1) First, fresh soybeans were ground into powder, mixed with n-hexane at a ratio of 1:3 (w / v), and stirred at room temperature for 2 h for three defatting steps. Second, defatted soybean powder was mixed with deionized water at a ratio of 1:10 (w / v), and the pH value was adjusted to 8.5 with 2 mol / L NaOH solution. After stirring at room temperature for 2 h, the mixture was centrifuged at 9000 rpm for 30 min. The supernatant was taken and the pH value was adjusted to 4.5 with 2 mol / L HCl solution. After standing for 2 h, the mixture was centrifuged at 6000 rpm for 20 min to obtain protein precipitate. Finally, the protein precipitate was dissolved in deionized water, the pH value of the protein was adjusted to 7.0 with 2 mol / L NaOH solution, and the protein solution was freeze-dried to obtain powdered SPI. (2) The SPI powder prepared above was dissolved in distilled water at a mass fraction of 4% (w / v), magnetically stirred at 25°C until completely dissolved, and the pH of the solution was adjusted to 8.5 with 2 mol / L NaOH. Alkaline protease was added at a substrate concentration of 1% (w / v) and enzymatically hydrolyzed at 50°C for 8 h. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 10 min, and the solution was quickly cooled to room temperature with ice water, the pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 min. After centrifugation, the precipitate was washed three times with water and freeze-dried to obtain SPA. (3) The L-arginine and SPA were taken in a ratio of 1:12 and poured into a cavitation jet machine for hydrodynamic cavitation treatment. The cavitation jet treatment parameters were a pressure of 110 MPa and a treatment time of 10 min. Implementation Case 3:

[0027] (1) First, fresh soybeans were ground into powder, mixed with n-hexane at a ratio of 1:3 (w / v), and stirred at room temperature for 2 h for three defatting steps. Second, defatted soybean powder was mixed with deionized water at a ratio of 1:10 (w / v), and the pH value was adjusted to 8.5 with 2 mol / L NaOH solution. After stirring at room temperature for 2 h, the mixture was centrifuged at 9000 rpm for 30 min. The supernatant was taken and the pH value was adjusted to 4.5 with 2 mol / L HCl solution. After standing for 2 h, the mixture was centrifuged at 6000 rpm for 20 min to obtain protein precipitate. Finally, the protein precipitate was dissolved in deionized water, the pH value of the protein was adjusted to 7.0 with 2 mol / L NaOH solution, and the protein solution was freeze-dried to obtain powdered SPI. (2) The SPI powder prepared above was dissolved in distilled water at a mass fraction of 4% (w / v), magnetically stirred at 25°C until completely dissolved, the pH of the solution was adjusted to 8.5 with 2 mol / L NaOH, and alkaline protease was added at a substrate concentration of 1% (w / v) for enzymatic hydrolysis at 50°C for 8 h. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 10 min, and the solution was quickly cooled to room temperature with ice water, the pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 min. After centrifugation, the precipitate was washed three times with water and freeze-dried to obtain SPA. (3) The L-arginine solution and SPA were poured into a cavitation jet machine at a ratio of 1:9 and subjected to hydrodynamic cavitation treatment. The cavitation jet treatment parameters were a pressure of 110 MPa and a treatment time of 15 min.

[0028] Table 1 Factor level coding table. Examples 4 to 9 are compared with Example 1, with the substrate concentration, enzyme addition amount and enzymolysis time changed, while the other parameters remain unchanged, specifically: Table 1

[0029] pass Figure 3 The results show that the particle size, Zeta potential and turbidity value of the SPA prepared under the conditions of SPI concentration of 4%, enzyme addition amount of 1% and enzymatic hydrolysis time of 8 h are relatively small; in addition, compared with Examples 4 to 9, the emulsion activity and stability under the conditions of Example 1 are the best.

[0030] Table 2 Factor level coding table. Examples 10 to 15 are compared with Example 2, except that the ratio of insoluble peptide aggregates to arginine is changed, while the other parameters remain unchanged, specifically: Table 2

[0031] pass Figure 4The results show that the SPA prepared when the ratio of arginine to insoluble peptide aggregates is 1:9 has the highest emulsification activity and emulsification stability index; in addition, compared with Examples 2, 11 to 13, the conditions under Example 10 are the best.

[0032] Table 3 Factor level coding table. Examples 4 to 16 are compared with Example 3, in which the pressure and treatment time of the hydraulic cavitation machine are changed, and the other parameters remain unchanged, specifically: Table 3

[0033] pass Figure 4 The results show that the SPA was processed under the conditions of 110 MPa pressure and 10 min. In addition, compared with Examples 14 to 17, the effect under the conditions of Example 3 was the best.

[0034] Based on the above-mentioned specific embodiments and experimental analysis, it can be seen that the use of hydrodynamic cavitation combined with arginine modification is a highly effective method for preparing highly emulsifying SPAs. Furthermore, experimental comparisons show that the preferred experimental conditions of the present invention are: 4% SPI mass fraction, 1% enzyme addition, 8 hours of enzymatic hydrolysis time, a ratio of L-arginine to insoluble peptide aggregates, a hydrodynamic cavitation pressure of 110 MPa, and a hydrodynamic ...

[0035] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing highly emulsifiable soybean peptide aggregates (SPA), characterized in that: The method comprises the following steps: (1) First, fresh soybeans were ground into powder, mixed with n-hexane at a ratio of 1:3 (w / v), and stirred at room temperature for 2 hours for three defatting steps. Secondly, defatted soybean powder was mixed with deionized water at a ratio of 1:10 (w / v), and the pH value was adjusted to 8.5 with 2mol / L NaOH solution. After stirring at room temperature for 2 hours, the mixture was centrifuged at 9000 rpm for 30 minutes. The supernatant was taken and the pH value was adjusted to 4.5 with 2mol / L HCl solution. After standing for 2 hours, the mixture was centrifuged at 6000 rpm for 20 minutes to obtain protein precipitate. Finally, the protein precipitate was dissolved in deionized water, the pH value of the protein was adjusted to 7.0 with 2mol / L NaOH solution, and the protein solution was freeze-dried to obtain powdered SPI. (2) The SPI powder prepared above was dissolved in distilled water at a mass fraction of 0.5-5% (w / v), and magnetically stirred at 25°C until completely dissolved. The pH of the solution was adjusted to 8.5 with 2 mol / L NaOH, and alkaline protease was added at a substrate concentration of 0.5-2% (w / v) for enzymatic hydrolysis at 50°C for 2-12 hours. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 10 minutes, and the solution was rapidly cooled to room temperature with ice water. The pH was adjusted to 7.0, and the solution was centrifuged at 8000 rpm for 20 minutes. After centrifugation, the precipitate was washed three times with water and freeze-dried to obtain SPA. (3) Dissolve the above SPA and L-arginine in a cavitation jet machine and perform hydrodynamic cavitation treatment. The cavitation jet treatment parameters are a pressure of 100-120 MPa and a treatment time of 5-15 minutes. The ratio of arginine to SPI is 1:8-12.

2. The method for preparing a highly emulsifiable SPA according to claim 1, characterized in that: The preferred addition amount of SPI powder is 4%, the preferred addition amount of alkaline protease is 1%, and the preferred enzymolysis time is 8 hours.

3. The method for preparing a highly emulsifiable SPA according to claim 1, characterized in that: The hydrodynamic cavitation equipment described in step (3) includes a feed tank, a peristaltic pump, and a hydrodynamic cavitation generator. The feed port of the hydrodynamic cavitation generator is connected to the peristaltic pump, and the discharge port is connected to the feed tank. The discharge port and the feed port are equipped with temperature detectors. The sample enters the hydrodynamic cavitation generator from the feed tank through the peristaltic pump for cavitation treatment and then flows back to the feed tank. The optimal treatment parameters are a pressure of 110 MPa and a treatment time of 10 minutes.

4. The method for preparing a highly emulsifiable SPA according to claim 1, characterized in that The optimal condition is that the volume ratio of L-arginine to SPA solution is 1:9.

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