Preparation method of soybean protein with high interfacial activity

Abstract

The invention relates to the field of food processing, in particular to a method for improving interfacial activity of soybean protein. The invention aims to provide a preparation process for enabling obtained soybean protein to have high interfacial activity by improving an extraction method, which comprises the following steps: (1) mixing defatted soybean powder with water, and freeze-drying after alkali dissolution and acid precipitation to obtain corresponding soybean protein; (2) dissolving the obtained soybean protein, adjusting the pH value, adding succinic anhydride according to 1-5% of the mass of the soybean protein, and dialyzing to obtain a corresponding succinylation modified soybean protein solution; (3) adding 1% of chitosan into the succinylated soybean protein solution, mixing, staying overnight, and fully hydrating to obtain a corresponding succinylated modified soybean protein-chitosan composite liquid; and (4) centrifuging and freeze-drying to obtain the corresponding soybean protein with high interfacial activity. The natural and safe soybean protein can be rapidly produced, and the method can be used for improving emulsification in food processing and the like.

Description

Technical Field

[0001] The invention relates to the field of food processing, and in particular to a method for preparing soybean protein with high interface activity. Background Art

[0002] Soy protein is a high-quality plant protein with many commendable advantages. First, soy protein is a comprehensive protein source that is rich in essential amino acids, which are essential for normal growth and development of the human body. It is particularly rich in lysine and isoleucine, which are amino acids that are often less common in other plant proteins and are also required by the human body. Therefore, the intake of soy protein can help maintain good health and provide the nutrients needed by the body. Secondly, soy protein is a natural plant protein that is very suitable for vegetarians and people who are allergic to animal protein. Compared with animal protein, soy protein is not only widely available, but also has less impact on the environment. The production of soy protein requires fewer resources than the production of animal protein and reduces the environmental problems associated with the farming industry. However, the interfacial activity of pure soy protein is relatively low, which limits its use in some applications. For example, in the food industry, interfacial active substances are often used for functions such as emulsification, foam stabilization and solubility increase. Therefore, the low interfacial activity of soy protein may not fully meet the processing needs of some soy foods.

[0003] Succinylation is a chemical modification technique that changes the chemical properties and functions of a molecule by introducing a succinyl group into it. Succinylation is commonly used to modify proteins and peptides, and can introduce succinyl groups at specific positions of the molecule. This modification can not only change the hydrophilicity and solubility of the molecule, but also affect the biological activity, stability and interaction of the protein. Therefore, succinylation can provide some advantages to improve the interfacial activity and other important functional properties of proteins. First, succinylation helps to improve the emulsification properties of proteins. Emulsification is an important step in the manufacturing process of many foods and cosmetics. Succinylation increases the hydrophilicity of protein molecules by introducing succinyl groups, making them more soluble in the water phase and forming a more stable mixed system with the oil phase. This improved emulsification property makes succinylated proteins have a wide range of application prospects in the food industry and the manufacture of personal care products. Secondly, succinylation can enhance the foam stability of proteins. Foam refers to a thin film composed of bubbles, which is commonly found in foods and beverages, such as milk foam and beer foam. Succinylation increases the hydrophilicity and interaction ability between protein molecules, which helps to form a more stable protein foam. By increasing the stability of foam, succinylated proteins can be better used to prepare food and beverage products with rich taste and texture.

[0004] Chitosan is a polysaccharide polymer composed of glucose molecules connected by β-1,4 glycosidic bonds. It is a natural product widely found in nature and is often extracted from the exoskeleton of crustaceans. Chitosan has the advantages of being biodegradable, biocompatible, and multifunctional, making it widely used in medicine, food, environmental engineering, and other fields. At the same time, chitosan also has many advantages in improving the interfacial activity of proteins. When chitosan interacts with proteins, it can increase the hydrophilicity of the surface of protein molecules, making it easier to contact with water and show better interfacial activity. Secondly, chitosan has excellent surface activity and can form a stable molecular film at the interface. By forming a molecular film, chitosan can improve the dispersibility and stability of proteins on the surface of liquids, reduce their surface tension, and promote the interaction between proteins and other substances.

[0005] In summary, both succinylation and chitosan addition can improve the interfacial activity of soy protein. Therefore, we speculate that the combination of the two methods can achieve the best level of improvement in the interfacial activity of soy protein, which will provide more innovative solutions for products in the fields of food, pharmaceuticals and cosmetics. This method has good biocompatibility and environmental friendliness, and is of great significance for improving the quality, stability and performance of products. Future research and application development will further improve and promote the application of this method, bringing more opportunities for the development of related industries. Summary of the invention

[0006] In view of the problems existing in the prior art, one of the purposes of the present invention is to provide a method for preparing soybean protein with high interface activity.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A method for preparing soybean protein with high surface activity, characterized in that it comprises the following steps:

[0009] (1) Preparation of soy protein: defatted soy powder was mixed with water, and magnetically stirred at 400-450 rpm for 30 min. The pH was adjusted to 8.5-9.5 using 1-2 mol / L NaOH solution, and the pH was maintained at 4000-4500 rpm for 2 h with magnetic stirring. The suspension was then centrifuged at 4000 rpm for 10-15 min. The pH of the supernatant after centrifugation was adjusted to 4.5-5.5 using 1-2 mol / L HCl. The suspension was then centrifuged at 4000 rpm for 10-15 min to obtain a precipitate of soy protein, which was washed with water 2-3 times. Finally, the pH was adjusted to 7.0-7.5 using 1-2 mol / L NaOH solution. The obtained soy protein solution was freeze-dried for 12-14 h to obtain the corresponding soy protein.

[0010] (2) Preparation of succinylated soybean protein dispersion: The soybean protein in (1) was dissolved in deionized water, and the pH was adjusted to 7-8 with 1-2 mol / L NaOH to obtain a soybean protein dispersion, succinic anhydride was added at 1-5% by weight of the soybean protein, and then the mixture was reacted at room temperature (20-25°C) for 2 h, and 1-2 mol / L NaOH was used to maintain the pH at 7-8 during the reaction. The mixture was then dialyzed at 2-4°C for 24 h to obtain the corresponding succinylated soybean protein dispersion.

[0011] (3) Preparation of succinylated soybean protein-chitosan composite liquid: The succinylated soybean protein dispersion obtained in (2) was magnetically stirred at 400-450 rpm for 2 h, and chitosan was slowly added within 5 min of stirring at a rate of 1% of the soybean protein mass. After the stirring was completed, the dispersion was placed at 2-4°C for 24 h to allow it to be fully hydrated, thereby obtaining the corresponding succinylated soybean protein-chitosan composite liquid.

[0012] (4) Freezing and drying to obtain soybean protein with high interfacial activity: Using a freeze dryer, the succinylated soybean protein-chitosan complex solution in (3) is rapidly frozen at -60°C, and the temperature is maintained dry for 12-24 hours to obtain the corresponding soybean protein with high interfacial activity.

[0013] Preferably, in step (1), after defatted soy flour and water are mixed, 1-2 mol / L NaOH solution is used to adjust the pH to 8.5-9.5, 1-2 mol / L HCl is used to adjust the pH of the supernatant after centrifugation to 4.5-5.5, and after washing with water, 1-2 mol / L NaOH solution is used to adjust the pH to 7.0-7.5.

[0014] Preferably, in step (2), succinic anhydride is added at 1-5% by weight of the soy protein, and after reacting for 2 hours, the protein is dialyzed at 2-4° C. for 24 hours.

[0015] Preferably, chitosan is slowly added in accordance with 1% of the amount of soybean protein within 5 minutes of stirring in step (3), and after the stirring is completed, it is placed at 2-4° C. for 24 hours to allow it to be fully hydrated.

[0016] Preferably, in step (4), a freeze dryer is used to freeze at -60°C and keep the temperature dry for 12-24 hours.

[0017] The present invention provides soybean protein with high surface activity prepared by the above preparation method.

[0018] The present invention also provides the application of the above-mentioned high-surface-active soybean protein in food processing. Beneficial effects of the present invention:

[0019] (1) The present invention provides a method for preparing soybean protein with high surface activity. The protein powder prepared by the present invention can improve texture stability, meet the human body's demand for protein, and play an important role in improving the surface activity of foods and medicines such as emulsions and gels.

[0020] (2) In addition to describing the effect of succinylation combined with chitosan on the interfacial activity of soybean protein, the present invention also compares the effect of succinylation alone on the interfacial activity of soybean protein. This provides a more effective method for improving the interfacial activity of protein, and thus has a wide range of application prospects in the field of food processing.

[0021] (3) The present invention does not require the introduction of organic reagents, the raw materials are safe and non-toxic, and it has the advantages of simple operation and low cost, which makes it have great potential in terms of economy, environment and society. This will promote innovation and sustainable development and bring positive impact to the development of related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Attached Figure 1 It is a flow chart of an implementation scheme of the present invention;

[0024] Attached Figure 2 The results are for the particle sizes of different surface-active soy proteins;

[0025] Attached Figure 3 This is the result of the emulsifying activity of soybean proteins with different surface activities. DETAILED DESCRIPTION

[0026] For a specific embodiment of the present invention, a clear and complete description of the technical solution is provided below. It should be noted that the embodiment only represents part of the implementation method of the present invention, not all.

[0027] The determination method involved in the present invention is as follows:

[0028] 1. Determination method of particle size of different surface-active soybean proteins

[0029] The prepared soy protein raw materials with different surface activities were dissolved in deionized water, and then magnetically stirred at 400 rpm until dissolved. The particle size was measured by dynamic light scattering (DLS), and the parameters were set as follows: wavelength of 633 nm, laser at a fixed angle of 173°, and temperature maintained at 4°C. Each sample solution was measured three times and the average value was taken.

[0030] 2. Determination of emulsifying activity of soy proteins with different surface activities

[0031] Dissolve the prepared soy protein raw materials with different surface activities in deionized water, and then stir magnetically at 400 rpm until dissolved. Mix the four solutions with soybean oil in a volume ratio of 9:1 and use an IKA homogenizer to stir at a speed of 12,000 rpm for 2 minutes to obtain four emulsions. Then, draw 50 μL of the emulsion and dilute it 100 times with 0.1% (w / v) sodium dodecyl sulfate. Use the sodium dodecyl sulfate solution as a blank and measure its absorbance at 500 nm. The emulsification activity index calculation formula is as follows:

[0032]

[0033] 3. Method for determining the contact angle of soybean proteins with different surface activities

[0034] The wettability of soy protein raw materials with different interfacial activities was measured using a video optical contact angle meter. Freeze-dried soy protein powder was pressed into 5mm×13mm circular slices by a tablet press and immersed in a quartz dish filled with soybean oil. 3μL of distilled water was squeezed out from the tip of the syringe needle and dropped on the surface of the circular slice to form a droplet. After standing for 30s, the change in the droplet shape was recorded using a camera equipped with the contact angle meter, and the three-phase contact angle of the nanoparticles was calculated using the LaPlace-Young equation.

[0035] 4. Determination of interfacial tension of soy proteins with different interfacial activities

[0036] The interfacial tension between soy protein suspensions (1.0% w / v) with different surface activities and soybean oil was measured by video optical contact angle meter, and the interfacial tension was observed over time based on the hanging drop method. All tests were carried out at room temperature. The suspension was loaded into a syringe equipped with a stainless steel needle (inner diameter 1.65 mm), and then the syringe needle was inserted into a quartz dish filled with soybean oil. 30 μL of the suspension was squeezed out from the tip of the syringe to form the largest droplet in the soybean oil and the timing was started, and the final interfacial tension value of the droplet was recorded at 1800 s.

[0037] Example 1

[0038] The defatted soy flour was mixed with water, and magnetically stirred at 400 rpm for 30 min. The pH was adjusted to 8.5 using 1 mol / L NaOH solution, and the pH was maintained at 4000 rpm for 2 h with magnetic stirring. The suspension was then centrifuged at 4000 rpm for 10 min, and the pH of the supernatant after centrifugation was adjusted to 4.5 using 1 mol / L HCl. The suspension was then centrifuged at 4000 rpm for 10 min to obtain the precipitate of soy protein, which was washed twice with water, and finally washed with 1 mol / L NaOH solution. Adjust the pH to 7.0, add succinic anhydride according to 1% of the weight of soybean protein, then react at room temperature at 20°C for 2h and use 1mol / L NaOH to maintain pH7 during the reaction, then dialyze at 2°C for 24h, then stir magnetically at 400-450rmp for 2h, and slowly add chitosan according to 1% of the soybean protein within 5min of stirring, and after stirring, keep it at 2°C for 24h to fully hydrate, and the corresponding succinylated soybean protein-chitosan composite liquid can be obtained. Use a freeze dryer to quickly freeze the succinylated soybean protein-chitosan composite liquid at -60°C, keep the temperature dry for 12h, and obtain the corresponding soybean protein with high interfacial activity.

[0039] Example 2

[0040] The defatted soy flour was mixed with water, and magnetically stirred at 400 rpm for 30 min. The pH was adjusted to 8.5 using 1 mol / L NaOH solution, and the pH was maintained at 4000 rpm for 2 h with magnetic stirring. The suspension was then centrifuged at 4000 rpm for 10 min, and the pH of the supernatant after centrifugation was adjusted to 4.5 using 1 mol / L HCl. The suspension was then centrifuged at 4000 rpm for 10 min to obtain the precipitate of soy protein, which was washed twice with water, and finally washed with 1 mol / L NaOH solution. Adjust the pH to 7.0, add succinic anhydride according to 5% of the weight of soybean protein, then react at room temperature at 20°C for 2h and use 1mol / L NaOH to maintain pH7 during the reaction, then dialyze it at 2°C for 24h, then stir it magnetically at 400-450rmp for 2h, and slowly add chitosan according to 1% of the soybean protein within 5min of stirring, and after stirring, keep it at 2°C for 24h to fully hydrate it, and the corresponding succinylated soybean protein-chitosan composite liquid can be obtained. Use a freeze dryer to quickly freeze the succinylated soybean protein-chitosan composite liquid at -60°C, keep the temperature dry for 12h, and obtain the corresponding soybean protein with high interfacial activity.

[0041] Comparative Example 1

[0042] Preparation of untreated soy protein: defatted soy powder was mixed with water, and magnetically stirred at 400 rpm for 30 min. The pH was adjusted to 8.5 using 1 mol / L NaOH solution, and the pH was maintained at 4000 rpm for 2 h with magnetic stirring. The suspension was then centrifuged at 4000 rpm for 10 min, and the pH of the supernatant after centrifugation was adjusted to 4.5 using 1 mol / L HCl. The suspension was then centrifuged at 4000 rpm for 10 min to obtain a precipitate of soy protein, which was washed twice with water. Finally, the pH was adjusted to 7.0 using 1 mol / L NaOH solution. The obtained soy protein dispersion was freeze-dried for 12 h to obtain the corresponding soy protein.

[0043] Comparative Example 2

[0044] Preparation of succinylated soybean protein dispersion: defatted soybean powder was mixed with water, magnetically stirred at 400 rpm for 30 min, pH was adjusted to 8.5 with 1 mol / L NaOH solution, pH was maintained at 4000 rpm with magnetic stirring for 2 h, and then the suspension was centrifuged at 4000 rpm for 10 min, pH of the supernatant after centrifugation was adjusted to 4.5 with 1 mol / L HCl, and then centrifuged at 4000 rpm for 10 min to obtain the precipitate of soybean protein and washed with water twice, and finally pH was adjusted to 7.0 with 1 mol / L NaOH solution, succinic anhydride was added according to 1% of the weight of soybean protein, and then reacted at room temperature 20°C for 2 h and 1 mol / L NaOH was used to maintain pH 7 during the reaction, and then dialyzed at 2°C for 24 h, and then freeze-dried to obtain the corresponding succinylated soybean protein.

[0045] Results

[0046] 1. Particle size results of soy proteins with different surface activity

[0047] like Figure 2As shown, compared with comparative example 1, the particle size of comparative example 2 treated with succinylation is reduced, while the particle size of examples 1 and 2 treated with succinylation and chitosan is significantly reduced. This is related to succinylation. After succinylation, the protein structure gradually unfolds, and the negatively charged succinyl group will be introduced into the protein surface, thereby improving the electrostatic repulsion of the system and reducing the aggregation of the protein, which ultimately leads to a reduction in particle size. In addition, with the addition of positively charged chitosan, the surface charge density of the protein molecules is increased, and the coverage of the uniform charge on the protein surface increases, which further leads to a reduction in the size of the protein aggregates and increases the flexibility of the molecules. These characteristics help promote protein adsorption and swelling on the interface, and improve protein emulsification and foaming ability. Testing the particle size change of soy protein can provide a basis for explaining the changes in the interfacial activity of soy protein by succinylation and chitosan, thereby further evaluating its stability and functionality as food. These test results can guide us to better understand the application potential of modified soy protein in food due to changes in interfacial activity, and then optimize the formulation and production process to improve product quality and competitiveness.

[0048] 2. Results of emulsification activity of soy proteins with different surface activity

[0049] like Figure 3 As shown, the order of emulsifying activity from large to small is Example 2> Example 1> Comparative Example 2> Comparative Example 1, wherein Example 1 and Example 2 are greater than Comparative Example 1. The improvement in emulsifying activity can be attributed to the significant reduction in protein particle size caused by succinylation and the increase in electronegativity, surface hydrophobicity and oil holding capacity, which promotes the dispersion and adsorption of protein molecules at the water-oil interface. In addition, the addition of chitosan increases the content of aliphatic and hydrophilic groups, and the intermolecular interaction leads to the formation of a strong solubilization layer near the oil-water interface, thereby effectively improving the emulsifying properties of the emulsion. In addition, the improvement in emulsifying activity is usually related to the enhanced interfacial activity of proteins. Substances with higher interfacial activity can better reduce the surface tension of the liquid-liquid interface, so that two immiscible liquids such as oil and water can be effectively mixed and stabilized. In this way, in the emulsification system, the interfacial active substance can form a stable interfacial film between oil and water, prevent the aggregation and separation of droplets, and provide the stability of the emulsion. At the same time, this also shows that the high interfacial activity soybean protein provided by the method can better improve the soybean emulsion, thereby providing a new solution for food processing in this field.

[0050] Table 1. Contact angle results of soybean protein raw materials of Examples and Comparative Examples

[0051]

[0052]

[0053] The values ​​of the contact angles of different soy proteins are shown in Table 1. Among the contact angles of the four soy proteins, from large to small are Example 2, Example 1, Comparative Example 2, and Comparative Example 1. Compared with Comparative Example 1, the contact angles of Example 1 and Example 2 increase, which may be attributed to the succinylation opening the protein structure, causing the tertiary structure of the protein to change and partially unfold and attach to the surface to increase the negative charge. At the same time, the positively charged chitosan is adsorbed on the protein surface through non-covalent interactions, which causes the tertiary structure of the protein to change, thereby exposing the non-polar amino acid groups embedded in the protein molecular structure, thereby increasing the hydrophobicity and making the contact angles of Example 1 and Example 2 closer to 90°, which indicates that the soy protein treated with succinylation and chitosan has a better adsorption effect on the droplet surface, which makes the soy protein treated with succinylation and chitosan in the field of food processing more effectively used for droplet stabilization and emulsification, and at the same time, it shows that soybeans with high interfacial activity can provide better functionality and texture improvement for food processing.

[0054] Table 2. Interfacial tension results of soybean protein raw materials of Examples and Comparative Examples

[0055] Sample name Interfacial tension (mN / m) Comparative Example 1 9.24 Comparative Example 2 8.19 Example 1 6.57 Example 2 5.36

[0056] The final stable interfacial tension values ​​of different soy proteins are shown in Table 2. The interfacial tension values ​​are Comparative Example 1>Comparative Example 2>Example 1>Example 2, which shows that succinylation and chitosan modification make the hydrophilic / hydrophobic surface of the protein more balanced, and therefore more suitable for adsorption at the oil-water interface. This can be attributed to the fact that succinylation causes more protein to unfold, and at the same time, with the addition of chitosan, higher protein flexibility is formed, which makes it easier to adsorb and stretch on the interface, thereby increasing its interfacial tension. Because proteins often form films or colloidal particles on the interface, changing the interfacial tension, the surface activity and interfacial behavior of proteins can be evaluated by measuring the interfacial tension.

[0057] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing soybean protein with high surface activity, characterized in that: The method comprises the following steps: (1) Preparation of soy protein: defatted soy flour was mixed with water, and magnetically stirred at 400-450 rmp for 30 min. The pH was adjusted to 8.5-9.5 with 1-2 mol / L NaOH solution, and the pH was maintained at 4000-4500 rmp for 2 h with magnetic stirring. The suspension was then centrifuged at 4000 rmp for 10-15 min. The pH of the supernatant after centrifugation was adjusted to 4.5-5.5 with 1-2 mol / L HCl. The suspension was then centrifuged at 4000 rmp for 10-15 min to obtain a soy protein precipitate, which was then washed with water 2-3 times. Finally, the pH was adjusted to 7.0-7.5 with 1-2 mol / L NaOH solution. The soy protein solution was freeze-dried for 12-14 h to obtain the corresponding soy protein. (2) Preparation of succinylated soybean protein dispersion: The soybean protein in (1) is dissolved in deionized water, and the pH thereof is adjusted to 7-8 with 1-2 mol / L NaOH to obtain a soybean protein dispersion, succinic anhydride is added at 1-5% by weight of the soybean protein, and then the dispersion is reacted at room temperature (20-25°C) for 2 h, and 1-2 mol / L NaOH is used to maintain the pH at 7-8 during the reaction, and then the dispersion is dialyzed at 2-4°C for 24 h to obtain the corresponding succinylated soybean protein dispersion; (3) Preparation of succinylated soybean protein-chitosan composite liquid: The succinylated soybean protein dispersion obtained in (2) was magnetically stirred at 400-450 rpm for 2 h, and chitosan was slowly added within 5 min of stirring at a rate of 1% of the soybean protein mass. After the stirring was completed, the mixture was placed at 2-4°C for 24 h to allow it to be fully hydrated, thereby obtaining the corresponding succinylated soybean protein-chitosan composite liquid; (4) Freezing and drying to obtain soybean protein with high interfacial activity: Use a freeze dryer to quickly freeze the succinylated soybean protein-chitosan complex solution in (3) at -60°C, and keep the temperature dry for 12-24 hours to obtain the corresponding soybean protein with high interfacial activity.

2. The method for preparing a soybean protein with high surface activity according to claim 1, characterized in that: In step (1), after defatted soy flour and water are mixed, the pH is adjusted to 8.5-9.5 using 1-2 mol / L NaOH solution, the pH of the supernatant after centrifugation is adjusted to 4.5-5.5 using 1-2 mol / L HCl, and after washing, the pH is adjusted to 7.0-7.5 using 1-2 mol / L NaOH solution.

3. The method for preparing a soybean protein with high surface activity according to claim 1, characterized in that: In step (2), 1-5% of the weight of soy protein is added with succinic anhydride, and after reacting for 2 hours, the mixture is dialyzed at 2-4°C for 24 hours.

4. The method for preparing a soybean protein with high surface activity according to claim 1, characterized in that: In step (3), chitosan is slowly added in an amount of 1% of the soybean protein within 5 minutes of stirring. After stirring, the mixture is placed at 2-4°C for 24 hours to allow it to be fully hydrated.

5. The method for preparing a soybean protein with high surface activity according to claim 1, characterized in that: In step (4), freeze the mixture at -60°C using a freeze dryer and keep the temperature dry for 12-24 hours.

6. Soybean protein with high surface activity prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the preparation method of high surface active soybean protein according to claim 6 in food.

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