A method for preparing a high-emulsification glycosylated soybean protein isolate hydrolysate based on enzymatic pretreatment-ultrasonic combination

Abstract

The application discloses a method for preparing a high-emulsification glycosylated soybean protein isolate hydrolysate based on enzymatic pretreatment-ultrasonic combination, and belongs to the field of food ingredients and plant protein modification. The method first performs moderate enzymolysis on soybean protein isolate by alkaline protease, and then uses the enzymolyzed soybean protein isolate and gum arabic as raw materials for a Maillard reaction to prepare an enzymolyzed soybean protein isolate-gum arabic compound with excellent emulsification properties in an ultrasonic-assisted environment. Through the combined use of the enzymatic pretreatment and ultrasonic treatment technologies, the grafting degree of the Maillard reaction between the soybean protein isolate and the gum arabic is effectively improved, so that the emulsification activity of the soybean protein isolate and the particle size distribution structure of the emulsion are significantly improved. The method provided by the application greatly improves the functional properties of the soybean protein isolate, and has wide application potential.

Description

Technical Field

[0001] This invention relates to the fields of food ingredients and plant protein modification, and in particular to a method for preparing highly emulsifiable glycosylated soybean protein isolate hydrolysate based on a combination of enzymatic pretreatment and ultrasonication. Background Technology

[0002] Soybeans are one of the world's four major food crops, and my country plays a vital role in their cultivation and production. Soybeans are mainly used for edible oil, feed protein, and food processing, especially as a primary raw material for vegetable oils and protein meals. Due to the compact structure and low molecular flexibility of soybean protein, its emulsification properties are not ideal. Therefore, it is necessary to use physical, chemical, or biological modification techniques to improve the functional properties of soybean protein.

[0003] The Maillard reaction, also known as the carbonyl-amine reaction, mainly refers to a series of reactions between the carbonyl group of reducing sugars and the free amino groups of nitrogen-containing compounds such as amino acids, peptides, and proteins. The resulting biomolecular complexes often possess excellent emulsifying properties. However, traditional Maillard reaction methods often suffer from low grafting efficiency, long reaction times, and easy protein aggregation and denaturation. Therefore, it is necessary to develop new methods to improve the grafting efficiency of the Maillard reaction and enhance the emulsifying properties of the Maillard reaction complexes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing highly emulsifiable glycosylated soy protein isolate hydrolysate based on enzymatic pretreatment-ultrasound combined method, in order to solve the problems existing in the prior art. This method improves the grafting degree of Maillard reaction products and significantly improves the emulsifying properties of soy protein isolate.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing highly emulsifiable glycosylated soybean protein isolate hydrolysate based on a combined enzymatic pretreatment and ultrasonic treatment, comprising the following steps:

[0007] Soy protein isolate was enzymatically hydrolyzed with alkaline protease to obtain soy protein isolate hydrolysate;

[0008] The soybean protein isolate hydrolysate was mixed with gum arabic and ultrasonically treated under water bath heating to obtain a highly emulsifiable glycosylated soybean protein isolate hydrolysate.

[0009] Preferably, the mass ratio of the alkaline protease to the soy protein isolate is 1:(1000-1200).

[0010] Preferably, the enzymatic hydrolysis conditions are: hydrolysis time of 1-5 hours, hydrolysis temperature of 45-60°C, and hydrolysis pH of 8.0-9.0.

[0011] Preferably, before mixing the soybean protein isolate hydrolysate with gum arabic, the process further includes an enzyme inactivation step of incubating the soybean protein isolate hydrolysate in a boiling water bath for 10 minutes.

[0012] Preferably, the soybean protein hydrolysate is mixed with the gum arabic at a mass concentration of 2 wt% to 8 wt%.

[0013] Preferably, the temperature of the water bath is 80-90℃; the power of the ultrasound is 300-600W, and the duration is 20-30min.

[0014] Preferably, the pH value of the mixture of soybean protein hydrolysate and gum arabic is adjusted to 3.0-5.0.

[0015] Preferably, the soy protein isolate hydrolysate is mixed with gum arabic and ultrasonically treated under water bath heating to obtain a crude solution of enzymatically hydrolyzed soy protein isolate-gum arabic complex; the crude solution of enzymatically hydrolyzed soy protein isolate-gum arabic complex is purified by dialysis and freeze-dried to obtain the highly emulsifiable glycosylated soy protein isolate hydrolysate.

[0016] The present invention also provides a method for preparing highly emulsifiable glycosylated soybean protein isolate hydrolysate based on enzymatic pretreatment-ultrasound combined preparation, characterized in that it is prepared using the method described above.

[0017] The present invention discloses the following technical effects:

[0018] This invention utilizes a low concentration of alkaline protease (mass ratio: enzyme / soy protein = 1 / 1000-1200) to disrupt the tightly packed spherical structure of soy protein isolate, increasing the proportion of free amino groups. Then, using ultrasound-assisted heating, these minimally enzymatically hydrolyzed soy protein isolates undergo a Maillard reaction with gum arabic to prepare an enzymatically hydrolyzed soy protein isolate-gum arabic complex. The preparation method of this invention is both simple and efficient, and the resulting product exhibits excellent emulsifying properties. This invention measured the emulsifying effect of the above-mentioned enzymatically hydrolyzed soy protein isolate-gum arabic complex and compared it with the conventional Maillard product of the same mass of soy protein isolate-gum arabic. The results show that the method of this invention significantly improves the grafting degree of the Maillard reaction product, and the enzymatically hydrolyzed soy protein isolate-gum arabic complex prepared by combined ultrasound treatment exhibits the strongest emulsifying properties. Therefore, the method of this invention improves the grafting degree of the Maillard reaction and significantly improves the emulsifying characteristics of soy protein isolate, greatly enhancing the functional properties of soy protein isolate and possessing broad application potential. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The grafting degree test results are for the Maillard reaction complexes prepared in Example 3 and Comparative Examples 1-3;

[0021] Figure 2 The results of emulsifying activity detection for the Maillard reaction complexes prepared in Example 3 and Comparative Examples 1-3 are shown.

[0022] Figure 3 The particle size distribution of the emulsions formed from the Maillard reaction complexes prepared in Example 3 and Comparative Examples 1-3 is shown in the test results. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Example 1

[0029] (1) Preparation of soybean protein hydrolysate

[0030] Soy protein isolate was enzymatically hydrolyzed using alkaline protease. Immediately after hydrolysis, the protein was incubated in a boiling water bath for 10 minutes to inactivate the enzyme, yielding the soy protein isolate hydrolysate. The mass ratio of alkaline protease to soy protein isolate was controlled at 1:1100, the hydrolysis time at 1 hour, the hydrolysis temperature at 45°C, and the pH of the hydrolysate at 8.0. The obtained sample was dialyzed at room temperature for 24 hours, and then the soy protein isolate hydrolysate was freeze-dried and stored at low temperature.

[0031] (2) Preparation of ultrasound-assisted Maillard reaction complex

[0032] The soybean protein isolate hydrolysate and gum arabic obtained above were mixed at equal mass concentrations, the pH of the solution was adjusted, and the mixture was subjected to ultrasonic treatment in an ultrasonic cell disruptor. Then, the reaction was carried out under constant temperature to form a crude solution of enzymatically hydrolyzed soybean protein isolate-gum arabic complex. Specifically, the concentrations of both soybean protein isolate hydrolysate and gum arabic were 2 wt%, the reaction pH was 3.0, the ultrasonic power was 300 W, the reaction time was 20 min, and the reaction temperature was 80 °C.

[0033] The crude solution of enzymatically hydrolyzed soy protein isolate-gum arabic complex was dialyzed at room temperature for 24 hours, and then the sample was freeze-dried and stored at low temperature to obtain highly emulsified glycosylated soy protein isolate hydrolysate.

[0034] Example 2

[0035] (1) Preparation of soybean protein hydrolysate

[0036] Soy protein isolate was enzymatically hydrolyzed using alkaline protease. Immediately after hydrolysis, the protein was incubated in a boiling water bath for 10 minutes to inactivate the enzyme, yielding the soy protein isolate hydrolysate. The mass ratio of alkaline protease to soy protein isolate was 1:1200, the hydrolysis time was 5 hours, the hydrolysis temperature was 60°C, and the pH of the hydrolysate was 9.0. The obtained sample was dialyzed at room temperature for 24 hours, and then the soy protein isolate hydrolysate was freeze-dried and stored at low temperature.

[0037] (2) Preparation of ultrasound-assisted Maillard reaction complex

[0038] The soybean protein isolate hydrolysate and gum arabic obtained above were mixed at equal mass concentrations, the pH of the solution was adjusted, and the mixture was subjected to ultrasonic treatment in an ultrasonic cell disruptor. Then, the reaction was carried out under constant temperature to form a crude solution of enzymatically hydrolyzed soybean protein isolate-gum arabic complex. Specifically, the concentrations of both soybean protein isolate hydrolysate and gum arabic were 8 wt%, the reaction pH was 5.0, the ultrasonic power was 600 W, the reaction time was 30 min, and the reaction temperature was 90 °C.

[0039] The crude solution of enzymatically hydrolyzed soy protein isolate-gum arabic complex was dialyzed at room temperature for 24 hours, and then the sample was freeze-dried and stored at low temperature to obtain highly emulsified glycosylated soy protein isolate hydrolysate.

[0040] Example 3

[0041] (1) Preparation of soybean protein hydrolysate

[0042] Soy protein isolate was enzymatically hydrolyzed using alkaline protease. Immediately after hydrolysis, the protein was incubated in a boiling water bath for 10 minutes to inactivate the enzyme, yielding the soy protein isolate hydrolysate. The mass ratio of alkaline protease to soy protein isolate was 1:1000, the hydrolysis time was 2 hours, the hydrolysis temperature was 55°C, and the pH of the hydrolysate was 8.0. The obtained sample was dialyzed at room temperature for 24 hours, and then the soy protein isolate hydrolysate was freeze-dried and stored at low temperature.

[0043] (2) Preparation of ultrasound-assisted Maillard reaction complex

[0044] The soybean protein isolate hydrolysate and gum arabic obtained above were mixed at equal mass concentrations, the pH of the solution was adjusted, and the mixture was subjected to ultrasonic treatment in an ultrasonic cell disruptor. Then, the reaction was carried out under constant temperature to form a crude solution of enzymatically hydrolyzed soybean protein isolate-gum arabic complex. Specifically, the concentrations of both soybean protein isolate hydrolysate and gum arabic were 5 wt%, the reaction pH was 4.0, the ultrasonic power was 400 W, the reaction time was 25 min, and the reaction temperature was 90 °C.

[0045] The crude solution of enzymatically hydrolyzed soy protein isolate-gum arabic complex was dialyzed at room temperature for 24 hours, and then the sample was freeze-dried and stored at low temperature to obtain highly emulsified glycosylated soy protein isolate hydrolysate, which was named EU-MA.

[0046] Comparative Example 1

[0047] The only difference from Example 3 is that (2) only water bath heating treatment was performed, without ultrasonic treatment, and the prepared sample was named E-MA.

[0048] Comparative Example 2

[0049] The only difference from Example 3 is that step (1) is omitted, that is, the soy protein isolate is not enzymatically hydrolyzed, and the prepared sample is named U-MA.

[0050] Comparative Example 3

[0051] The only difference from Example 3 is that the soy protein isolate was neither enzymatically hydrolyzed nor sonicated. Under the same conditions, a conventional Maillard reaction complex was obtained, and the resulting sample was named MA.

[0052] The samples prepared in Example 3 and Comparative Examples 1-3 were subjected to the following performance tests:

[0053] 1. Compare the effects of different treatment methods on branch density.

[0054] Grafting degree determination method: Dissolve 80 mg of o-phthalaldehyde (OPA) reagent completely in 2 mL of methanol, then mix with 50 mL of 0.1 mol / L sodium tetraborate buffer (pH 9.7), 200 μL of β-mercaptoethanol, and 5 mL of 20% sodium dodecyl sulfate (SDS) solution (w / w). After mixing, adjust the OPA reagent to a constant volume of 100 mL using distilled water. During the measurement, add 100 μL of sample solution (10 mg / mL) to 4 mL of OPA reagent and incubate at 35 °C for 2 min. Then measure the absorbance of the sample at 340 nm using a UV-Vis spectrophotometer. Use unglycosylated protein / protein hydrolysate as a standard control sample and distilled water as a blank control. Calculate the grafting degree according to the following formula:

[0055] Grafting degree (%) = (C0 - Ct) / C0 × 100

[0056] Where C0 and C t These are the concentrations of free amino groups in unglycosylated soy protein isolate hydrolysate (or unglycosylated soy protein isolate) and glycosylated soy protein isolate hydrolysate (or glycosylated soy protein isolate) solutions, respectively, in mol / L.

[0057] See results Figure 1 The results showed that EU-MA had the highest grafting degree, indicating that the simultaneous use of enzyme treatment and sonication was more effective in promoting the Maillard reaction than either method alone. This is mainly because enzymatic treatment followed by sonication more effectively promotes the unfolding of soybean protein molecules, thereby exposing more free amino groups and enhancing the Maillard reaction.

[0058] 2. Compare the effects of different treatment methods on the emulsifying properties of the resulting complex.

[0059] Emulsifying activity was assessed by reconstituted the lyophilized sample in 8 mL of 0.02 mol phosphate buffer (pH 7.0). The protein concentration of the solution was 1 wt%, and then 0.8 mL of medium-chain triglycerides (MCT) was added. The mixture was dispersed at 10,000 rpm for 2 min using a high-speed shear mixer to form an emulsion. 150 μL of the dispersion was taken from the bottom of the container and added to 5 mL of 0.1% SDS solution. Using the SDS solution as a blank control, the absorbance was measured at 500 nm. The emulsifying activity was calculated using the following formula:

[0060] emulsifying activity

[0061] Where DF is the final dilution factor of the emulsion (33.3); C is the protein concentration (g / mL); θ is the optical path length of the cuvette (1 cm); θ is the volume fraction of the oil phase in the system (0.25); A0 is the absorbance measured when the system is left to stand.

[0062] See results Figure 2 The results showed that EU-MA exhibited the strongest emulsifying activity compared to other comparative samples, primarily due to the increased grafting degree. When gum arabic, with its numerous hydrophilic hydroxyl groups, covalently binds to soy protein isolate, the complex's ability to stabilize the water-oil interface is enhanced, leading to increased emulsifying properties. These results indicate that combined enzymatic hydrolysis and ultrasound-assisted Maillard reaction treatment can significantly improve the emulsifying activity of soy protein isolate.

[0063] 3. Compare the effects of different treatment methods on emulsion particle size distribution.

[0064] Method for determining the particle size distribution of the emulsion: The lyophilized sample was reconstituted in 8 mL of 0.02 mol phosphate buffer (pH = 7.0). The protein concentration of the solution was 1 wt%, and then 0.8 mL of medium-chain triglycerides (MCT) was added. The emulsion was formed by high-speed dispersion at 10,000 rpm / min for 2 min using a high-speed shear apparatus. The size distribution curve of oil droplets in the emulsion was measured using a Beckman particle size analyzer (LS13320, Beckman Instruments Limited, USA).

[0065] See results Figure 3 The results showed that, compared with other comparative examples, the EU-MA-stabilized emulsion had a narrower particle size distribution, exhibited a single-peak distribution, with the main peak shifted to the left, and the overall particle size of the emulsion was smaller.

[0066] In summary, by comparing Example 3 and Comparative Examples 1-3, it can be seen that the combined effect of enzymatic pretreatment and ultrasound-assisted Maillard reaction can effectively improve the grafting degree of soy protein isolate-gum arabic complex, improve the emulsifying activity of soy protein isolate, and reduce the average particle size of soy protein isolate stable emulsion.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing highly emulsifiable glycosylated soybean protein isolate hydrolysate based on enzymatic pretreatment-ultrasound combined method, characterized in that, Includes the following steps: Soy protein isolate was enzymatically hydrolyzed with alkaline protease to obtain soy protein isolate hydrolysate; The soybean protein isolate hydrolysate was mixed with gum arabic and ultrasonically treated under water bath heating to obtain a highly emulsifiable glycosylated soybean protein isolate hydrolysate. The mass ratio of the alkaline protease to the soy protein isolate is 1:(1000-1200). The conditions for enzymatic hydrolysis are: hydrolysis time of 1-5 h, hydrolysis temperature of 45-60℃, and pH value of the hydrolysis reaction of 8.0-9.

0. The soybean protein hydrolysate isolated from the gum arabic is mixed with the gum arabic at an equal mass concentration, and the total mass concentration of the mixture is 2-8 wt%. The pH of the mixture of soybean protein hydrolysate and gum arabic was adjusted to 3.0-5.

0. The temperature of the water bath is 80-90℃; the power of the ultrasound is 300-600 W, and the duration is 20-30 min.

2. The method as described in claim 1, characterized in that, Before mixing the soybean protein isolate hydrolysate with gum arabic, the process further includes an enzyme inactivation step of incubating the soybean protein isolate hydrolysate in a boiling water bath for 10 minutes.

3. The method as described in claim 1, characterized in that, The soybean protein isolate hydrolysate was mixed with gum arabic and ultrasonically treated under water bath heating to obtain a crude solution of enzymatically hydrolyzed soybean protein isolate-gum arabic complex. The crude solution of enzymatically hydrolyzed soybean protein isolate-gum arabic complex was purified by dialysis and freeze-dried to obtain the highly emulsifiable glycosylated soybean protein isolate hydrolysate.

4. A method for preparing highly emulsifiable glycosylated soybean protein isolate hydrolysate based on enzymatic pretreatment and ultrasonication, characterized in that, It is prepared by the method described in any one of claims 1-3.

Images