High-emulsification-activity gluten protein based on irradiation modification and preparation method thereof
By treating wheat gluten protein with alkaline protease hydrolysis and intermittent gamma-ray irradiation combined with an ozone-deuterium-enriched water system, the problems of emulsification activity and stability were solved, and efficient emulsification effect and adhesive performance improvement were achieved.
Patent Information
- Application Number
- CN202510928732.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-07
- Publication Date
- 2025-10-17
AI Technical Summary
The emulsifying activity of existing irradiated modified wheat gluten protein has limited improvement, poor emulsification stability, and continuous irradiation can easily lead to protein aggregation, affecting the effect.
Wheat gluten was subjected to limited hydrolysis by alkaline protease, combined with intermittent γ-ray irradiation and ozone-deuterium-enriched water system. By generating high concentrations of free radicals and directional release of OD radicals, the irradiation conditions were optimized to enhance the emulsifying activity and stability.
It significantly improves the emulsifying activity and emulsion stability of gluten protein, reduces protein aggregation, forms nano-level uniform emulsion, and improves the stability and adhesion of adhesives.
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Figure CN120796423A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of wheat protein processing, in particular to a high-emulsifying-activity gluten protein based on irradiation modification and a preparation method thereof. BACKGROUND
[0002] Wheat gluten protein, i.e., wheat gluten, is a byproduct in the process of wheat starch processing, and has wide applications in the fields of adhesives, food, feed, biodegradable materials, etc. For example, in adhesives, the addition of high-emulsifying gluten can improve the stability and adhesion of the adhesives. At present, the methods for modifying wheat gluten to improve its emulsifying activity include physical methods, enzymatic methods and chemical methods. Among them, the common physical methods include heat treatment and extrusion treatment, which can change the structure of protein molecules and improve the functional properties of gluten; the common types of chemical methods include pH shift and glycosylation, which have good modification effect on gluten and are simple in reaction; the enzymatic method refers to the low-level hydrolysis of protein by protease to change the structure and functional properties of protein. These common heat, pH shift and enzymatic modification methods have problems of long processing time, high energy consumption, introduction of chemical reagents and high cost, etc.
[0003] Irradiation modification treatment belongs to a physical method, and among them, gamma ray irradiation is a technology using ionizing radiation, which has the advantages of strong penetration, low energy consumption, no pollution and suitability for industrial production, and is widely used at present. According to relevant research (reference literature "Research on the Influence of Gamma Ray Irradiation on the Structure and Functional Properties of Wheat Gluten", Shen Huishan, July 2, 2024), gamma ray irradiation treatment destroys the disulfide bond of gluten, changes its secondary structure, reduces the content of alpha-helix and increases the content of beta-fold, and changes the tertiary structure of gluten, making tryptophan and tyrosine more tend to "exposed state", thereby improving the surface hydrophobicity of gluten, and thus changing the functional properties (water holding capacity, oil holding capacity, emulsifying activity and solubility) of gluten to different degrees. Compared with unirradiated gluten, when the irradiation dose is 15 kGy, the water holding capacity, oil holding capacity, solubility and emulsifying activity of the gluten increase the most, indicating that appropriate dose of irradiation treatment can improve the functional properties of gluten.
[0004] However, the irradiation modification treatment of wheat gluten in this study has the following problems: (1) the continuous irradiation method is easy to cause protein aggregation under the effect of heat, affecting the emulsification effect; (2) the emulsifying activity is improved by a limited range, and the particle size of the emulsion after treatment is large, resulting in poor stability of the emulsion. SUMMARY
[0005] The application aims to provide a high-emulsifying-activity gluten protein based on irradiation modification and a preparation method thereof, which solves the problems of limited emulsifying activity improvement and poor emulsifying stability of the existing irradiation modified gluten protein.
[0006] The application achieves the above-mentioned purposes through the following technical solutions.
[0007] The application provides a preparation method of a high-emulsifying-activity gluten protein based on irradiation modification, which comprises the following steps:
[0008] S1, pretreatment
[0009] The wheat gluten protein is dispersed in 6-10 times the mass of deionized water, and stirring treatment is performed to form a uniform suspension, the pH of the suspension is adjusted to 8-9 by using a NaOH solution, and the suspension is subjected to enzymatic hydrolysis treatment by using an alkaline protease; after the enzymatic hydrolysis treatment, the suspension is subjected to centrifugal treatment, the supernatant is collected, and the protein primary powder is obtained by freeze-drying.
[0010] S2, irradiation treatment
[0011] The protein primary powder is dissolved in 6-10 times the mass of an irradiation solvent to obtain a protein dispersion, a cobalt-60 gamma ray is used as an irradiation source to irradiate the protein dispersion, the irradiation dose is 5-6 kGy, and intermittent irradiation is adopted.
[0012] S3, post-treatment
[0013] The protein dispersion after irradiation is subjected to centrifugal treatment, the supernatant is collected, and the high-emulsifying-activity gluten protein is obtained by freeze-drying.
[0014] Further improvement lies in that in step S1, the stirring treatment refers to magnetic stirring at a temperature of 24-28 DEG C and a rotating speed of 250-350 rpm for 25-35 min.
[0015] Further improvement lies in that in step S1, the enzymatic hydrolysis treatment refers to that the suspension is warmed to 48-52 DEG C and kept warm, 2-2.5% of the mass of the wheat gluten protein of alkaline protease is added, the enzymatic hydrolysis treatment is performed for 60-80 min, the enzyme is inactivated by heating in a boiling water bath for 15-20 min, and the temperature is cooled to room temperature.
[0016] Further improvement lies in that in step S2, the irradiation solvent is deionized water.
[0017] Further improvement lies in that in step S2, the irradiation solvent is an ozone-deuterium-rich water system, the ozone-deuterium-rich water system mainly comprises deuterium-rich water, the dissolved concentration of ozone in the system is 15-25 ppm, the pH of the system is 6.0-7.0, and the temperature is 4-10 DEG C.
[0018] Further improvement lies in that in step S2, the ozone-d2O water system is added in the following way: after the protein primary powder is dissolved in d2O to obtain a suspension, the suspension is placed in a container and O3 / N2 mixed gas is introduced, the volume ratio of O3 in the mixed gas is 5-10%, the flow rate is controlled at 0.4-0.6 L / min, and the circulation aeration is performed for 20-30 min.
[0019] Further improvement lies in that when the ozone-d2O water system is used as the irradiation solvent, after the irradiation is completed, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution is repeated for 3-4 times to obtain a protein dispersion liquid without d2O residue.
[0020] Further improvement lies in that in step S2, the intermittent irradiation refers to that 2 min is interval after each irradiation for 1 min, and the irradiation dose rate is controlled at 0.15-0.25 kGy / s during each irradiation for 1 min.
[0021] Further improvement lies in that in steps S1 and S3, the centrifugal treatment is performed at a speed of 6000-8000 rpm for 12-15 min, and the freeze-drying is performed at a temperature of-50 ℃ for 18-36 h.
[0022] The application further provides a high emulsifying activity gluten protein based on irradiation modification, which is prepared by the above preparation method.
[0023] The application has the following beneficial effects:
[0024] (1) The application first performs limited hydrolysis on the gluten protein by using a protease to produce smaller peptide segments, so as to increase the sensitivity to irradiation, improve the irradiation efficiency, and help to reduce the emulsion particle size in the later stage and improve the emulsion stability; then, the application adopts an intermittent irradiation treatment mode, the high instantaneous dose rate during irradiation can produce high-concentration free radicals, the intermittent period is beneficial to free radical reactions and provides heat dissipation and uniform temperature conditions, reduces the protein aggregation caused by heat effect, and improves the emulsification effect, and when the application is applied to an adhesive, the stability and adhesion of the adhesive can be improved.
[0025] (2) In the preferred embodiment, the application also uses an ozone-d2O water system as the irradiation solvent, which can realize the directional release of ·OD free radicals by coupling the active oxygen produced by ozone decomposition with the radiolysis reaction of d2O (i.e., O3+d2O→O2+d2O2, d2O2+γ rays→2·OD), plays a desensitization role, and prolongs the irradiation time, which, in cooperation with the intermittent irradiation treatment mode, effectively improves the irradiation effect. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 It is a microstructure diagram of the protein emulsion of Example 5 of the application;
[0027] Figure 2 Figure for particle size distribution results of Examples 1-6 of the present application;
[0028] Figure 3 Figure for particle size distribution results of Examples 2 and 5 and Comparative Examples 1-4 of the present application;
[0029] Figure 4 Figure for zeta potential results of each group. DETAILED DESCRIPTION
[0030] It is necessary to point out here that the following detailed description is only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.
[0031] I. Main materials
[0032] Wheat gluten: purchased from Henan Jinyuan Grain and Oil Co., Ltd.;
[0033] Alkaline protease: purchased from Beijing Solabio Technology Co., Ltd.;
[0034] Deuterium water: CAS No. 7789-20-0, purchased from Shanghai Maier Biotech Co., Ltd. The deuterium water is diluted 3 times with deionized water to obtain deuterium-rich water.
[0035] II. Implementation experiment
[0036] Example 1
[0037] A preparation method of high emulsification activity gluten protein based on irradiation modification, the steps comprising:
[0038] S1, pretreatment
[0039] The wheat gluten was dispersed in 6 times the mass of deionized water, and under the conditions of temperature 24℃ and rotation speed 250rpm, the magnetic stirring was carried out for 35min to form a uniform suspension. The pH of the suspension was adjusted to 8 with NaOH solution, and then the suspension was subjected to enzymatic treatment with alkaline protease: the suspension was heated to 48℃ and kept, 2% of the mass of the wheat gluten was added as alkaline protease, and the enzymatic treatment was carried out for 80min. Then the enzyme was inactivated by heating in a boiling water bath for 15min, and cooled to room temperature. After the enzymatic treatment, the suspension was subjected to centrifugal treatment (rotation speed 6000rpm, time 15min), and the supernatant was collected. After freeze-drying (temperature -50℃, time 18h), the protein primary powder was obtained;
[0040] S2, irradiation treatment
[0041] The protein primary powder is dissolved in 6 times of deionized water to obtain a protein dispersion liquid, and the protein dispersion liquid is irradiated by taking a cobalt-60 γ ray as an irradiation source, the irradiation dose is 5 kGy, and the intermittent irradiation is adopted, that is, after each irradiation for 1 min, the interval is 2 min, and the irradiation dose rate in each irradiation for 1 min is controlled to be 0.15 kGy / s (the intermittent irradiation can be realized by adopting the mode of conveying belt circulation conveying and batch switching irradiation, so that the operation can be simplified and the efficiency can be improved);
[0042] S3, post-processing
[0043] The irradiated protein dispersion liquid is centrifuged (at a speed of 6000 rpm for 15 min), the supernatant is collected, and is freeze-dried (at a temperature of -50°C for 18 h) to obtain the high-emulsifying-activity wheat gluten protein.
[0044] Example 2
[0045] A preparation method of a high-emulsifying-activity wheat gluten protein based on irradiation modification, comprising the following steps:
[0046] S1, pretreatment
[0047] The wheat gluten protein is dispersed in 8 times of deionized water, and is magnetically stirred at a temperature of 26°C and a speed of 300 rpm for 30 min to form a uniform suspension liquid, the pH of the suspension liquid is adjusted to 8.5 by using a NaOH solution, and the suspension liquid is subjected to enzymatic hydrolysis treatment by using an alkaline protease, that is, the suspension liquid is heated to 50°C and is kept, 2.2% of the alkaline protease based on the mass of the wheat gluten protein is added, and the enzymatic hydrolysis treatment is performed for 70 min, then the suspension liquid is heated in a boiling water bath for 16 min to inactivate the enzyme, and is cooled to room temperature, and the suspension liquid after the enzymatic hydrolysis treatment is subjected to centrifugal treatment (at a speed of 7000 rpm for 13 min), the supernatant is collected, and is freeze-dried (at a temperature of -50°C for 24 h) to obtain the protein primary powder;
[0048] S2, irradiation treatment
[0049] The protein primary powder is dissolved in 8 times of deionized water to obtain a protein dispersion liquid, and the protein dispersion liquid is irradiated by taking a cobalt-60 γ ray as an irradiation source, the irradiation dose is 5.5 kGy, and the intermittent irradiation is adopted, that is, after each irradiation for 1 min, the interval is 2 min, and the irradiation dose rate in each irradiation for 1 min is controlled to be 0.2 kGy / s;
[0050] S3, post-processing
[0051] The irradiated protein dispersion liquid is centrifuged (at a speed of 7000 rpm for 13 min), the supernatant is collected, and is freeze-dried (at a temperature of -50°C for 24 h) to obtain the high-emulsifying-activity wheat gluten protein.
[0052] Example 3
[0053] A preparation method of high emulsifying activity gluten protein based on irradiation modification, comprising the following steps:
[0054] S1, pretreatment
[0055] The wheat gluten protein was dispersed in 10 times the mass of deionized water, and a uniform suspension was formed under the conditions of a temperature of 28℃ and a rotation speed of 350 rpm for 25 min of magnetic stirring. The pH of the suspension was adjusted to 9 using a NaOH solution, and the suspension was subjected to enzymatic treatment using alkaline protease. Specifically, the suspension was warmed to 52℃ and held, 2.5% of the mass of the wheat gluten protein was added as alkaline protease, and the enzymatic treatment was performed for 60 min. The enzyme was then inactivated by heating in a boiling water bath for 20 min, and the suspension was cooled to room temperature. After the enzymatic treatment, the suspension was subjected to centrifugal treatment (at a rotation speed of 8000 rpm for 12 min), and the supernatant was collected. The protein primary powder was obtained by freeze-drying (at a temperature of -50℃ for 36 h).
[0056] S2, irradiation treatment
[0057] The protein primary powder was dissolved in 10 times the mass of deionized water to obtain a protein dispersion. A cobalt-60 gamma ray was used as the irradiation source to irradiate the protein dispersion. The irradiation dose was 6 kGy, and the irradiation was intermittent, i.e., after each 1 min of irradiation, there was a 2 min interval, and the irradiation dose rate during each 1 min of irradiation was controlled to be 0.25 kGy / s.
[0058] S3, post-treatment
[0059] The irradiated protein dispersion was subjected to centrifugal treatment (at a rotation speed of 8000 rpm for 12 min), and the supernatant was collected. The high emulsifying activity gluten protein was obtained by freeze-drying (at a temperature of -50℃ for 36 h).
[0060] Example 4
[0061] A preparation method of high emulsifying activity gluten protein based on irradiation modification, comprising the following steps:
[0062] S1, pretreatment
[0063] The wheat gluten protein is dispersed in 8 times mass of deionized water, and a uniform suspension is formed under the conditions of a temperature of 26°C and a rotating speed of 300 rpm by magnetic stirring for 30 min. The pH of the suspension is adjusted to 8.5 by using a NaOH solution, and the suspension is subjected to enzymatic hydrolysis treatment by using an alkaline protease, namely, the suspension is warmed to 50°C and kept, 2.2% of the mass of the wheat gluten protein is added as the alkaline protease, and the enzymatic hydrolysis treatment is performed for 70 min. The enzyme is inactivated by heating in a boiling water bath for 16 min, and the suspension is cooled to room temperature. The suspension is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min) after the enzymatic hydrolysis treatment, the supernatant is collected, and protein primary powder is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0064] S2, irradiation treatment
[0065] The protein primary powder is dissolved in 8 times mass of an ozone-deuterium-rich water system to obtain a protein dispersion. Specifically, the protein primary powder is first dissolved in deuterium-rich water to obtain a suspension, the suspension is then placed in a container and O3 / N2 mixed gas is introduced, the volume ratio of O3 in the mixed gas is 5%, the flow rate is controlled at 0.4 L / min, and the suspension is subjected to cyclic aeration for 20 min to make the dissolved concentration of ozone 15 ppm. In addition, the pH of the system is adjusted to 6.0, and the temperature is controlled at 4°C.
[0066] The protein dispersion is irradiated by using a cobalt-60 gamma ray as an irradiation source, and the irradiation dose is 5.5 kGy. The irradiation is intermittent, namely, after irradiation for 1 min, the interval is 2 min, and the irradiation dose rate is controlled at 0.2 kGy / s during each irradiation for 1 min. After the irradiation is completed, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution is repeated for 3-4 times to obtain a protein dispersion without residual deuterium water.
[0067] S3, post-treatment
[0068] The protein dispersion after irradiation is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), and the supernatant is collected. The protein dispersion is freeze-dried (at a temperature of -50°C for 24 h) to obtain the high-emulsifying-activity gluten protein.
[0069] Example 5
[0070] A preparation method of a high-emulsifying-activity gluten protein based on irradiation modification, the steps comprising:
[0071] S1, pretreatment
[0072] The wheat gluten protein is dispersed in 8 times mass of deionized water, and a uniform suspension is formed under the conditions of a temperature of 26°C and a rotating speed of 300 rpm by magnetic stirring for 30 min. The pH of the suspension is adjusted to 8.5 by using a NaOH solution, and the suspension is subjected to enzymatic hydrolysis treatment by using an alkaline protease, namely, the suspension is warmed to 50°C and kept, 2.2% of the mass of the wheat gluten protein is added as the alkaline protease, and the enzymatic hydrolysis treatment is performed for 70 min. The enzyme is inactivated by heating in a boiling water bath for 16 min, and the suspension is cooled to room temperature. The suspension is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min) after the enzymatic hydrolysis treatment, the supernatant is collected, and protein primary powder is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0073] S2, irradiation treatment
[0074] The protein primary powder is dissolved in 8 times mass of an ozone-deuterium-rich water system to obtain a protein dispersion. Specifically, the protein primary powder is first dissolved in deuterium-rich water to obtain a suspension, the suspension is placed in a container, and O3 / N2 mixed gas is introduced, the volume ratio of O3 in the mixed gas is 8%, the flow rate is controlled to be 0.5 L / min, and the suspension is subjected to cyclic aeration for 25 min to make the dissolved concentration of ozone be 20 ppm. In addition, the pH of the system is adjusted to 6.5, and the temperature is 6°C.
[0075] The protein dispersion is irradiated by using a cobalt-60 gamma ray as an irradiation source, the irradiation dose is 5.5 kGy, and the irradiation is intermittent, namely, after irradiation for 1 min, the interval is 2 min, and the irradiation dose rate is controlled to be 0.2 kGy / s during the irradiation for 1 min each time. After the irradiation is completed, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution is repeated for 3-4 times to obtain a protein dispersion without residual deuterium water.
[0076] S3, post-treatment
[0077] The protein dispersion after the irradiation is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), the supernatant is collected, and the high emulsification activity gluten protein is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0078] Example 6
[0079] A preparation method of a high emulsification activity gluten protein based on irradiation modification, the steps comprising:
[0080] S1, pretreatment
[0081] The wheat gluten protein is dispersed in 8 times mass of deionized water, and a uniform suspension is formed under the conditions of a temperature of 26 DEG C and a rotating speed of 300 rpm by magnetic stirring for 30 min. The pH of the suspension is adjusted to 8.5 by using a NaOH solution, and the suspension is subjected to enzymatic hydrolysis by using an alkaline protease, namely, the suspension is heated to 50 DEG C and kept, 2.2% of the mass of the wheat gluten protein is added as the alkaline protease, and the enzymatic hydrolysis is performed for 70 min. The enzyme is inactivated by using a boiling water bath for 16 min, and the suspension is cooled to room temperature. The suspension is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min) after the enzymatic hydrolysis, the supernatant is collected, and the protein primary powder is obtained by freeze-drying (at a temperature of -50 DEG C for 24 h).
[0082] S2, irradiation treatment
[0083] The protein primary powder is dissolved in 8 times mass of an ozone-deuterium-rich water system to obtain a protein dispersion. Specifically, the protein primary powder is dissolved in deuterium-rich water to obtain a suspension, the suspension is placed in a container, and O3 / N2 mixed gas is introduced, the volume ratio of O3 in the mixed gas is 10%, the flow rate is controlled to be 0.6 L / min, and the suspension is subjected to cyclic aeration for 30 min to make the dissolved concentration of ozone be 25 ppm. In addition, the pH of the system is adjusted to 7.0, and the temperature is 10 DEG C.
[0084] Cobalt-60 gamma rays are used as an irradiation source to irradiate the protein dispersion, the irradiation dose is 5.5 kGy, and the irradiation is intermittent, namely, after irradiation for 1 min, the interval is 2 min, and the irradiation dose rate is controlled to be 0.2 kGy / s during the irradiation for 1 min each time. After the irradiation, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution is repeated for 3-4 times to obtain the protein dispersion without residual deuterium water.
[0085] S3, post-treatment
[0086] The protein dispersion after irradiation is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), the supernatant is collected, and the high emulsification activity gluten protein is obtained by freeze-drying (at a temperature of -50 DEG C for 24 h).
[0087] Comparative Example 1
[0088] A preparation method of a high emulsification activity gluten protein based on irradiation modification, the steps comprising:
[0089] S1, pretreatment
[0090] The wheat gluten protein is dispersed in 8 times mass of deionized water, and a uniform suspension is formed under the condition of magnetic stirring at a temperature of 26°C and a rotating speed of 300 rpm for 30 min. The pH of the suspension is adjusted to 8.5 by using a NaOH solution, and the suspension is subjected to enzymatic hydrolysis treatment by using an alkaline protease, namely, the suspension is warmed to 50°C and kept, 2.2% of the mass of the wheat gluten protein of alkaline protease is added, and the enzymatic hydrolysis treatment is performed for 70 min. The enzyme is inactivated by using a boiling water bath for 16 min, and the suspension is cooled to room temperature. The suspension after the enzymatic hydrolysis treatment is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), and the supernatant is collected. The protein primary powder is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0091] S2, irradiation treatment
[0092] The protein primary powder is dissolved in 8 times mass of deionized water to obtain a protein dispersion. The protein dispersion is irradiated by using a cobalt-60 gamma ray as an irradiation source. The irradiation dose is 5.5 kGy, and the irradiation is continuous. The irradiation dose rate is controlled to be 0.2 kGy / s.
[0093] S3, post-treatment
[0094] The protein dispersion after the irradiation is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), and the supernatant is collected. The high emulsifying activity gluten protein is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0095] Comparative Example 2
[0096] A preparation method of a high emulsifying activity gluten protein based on irradiation modification, the steps comprising:
[0097] S1, irradiation treatment
[0098] The wheat gluten protein powder is dissolved in 8 times mass of deionized water to obtain a protein dispersion. The protein dispersion is irradiated by using a cobalt-60 gamma ray as an irradiation source. The irradiation dose is 5.5 kGy, and the irradiation is intermittent. Namely, after irradiation for 1 min, an interval of 2 min is provided, and the irradiation dose rate is controlled to be 0.2 kGy / s during each irradiation for 1 min.
[0099] S2, post-treatment
[0100] The protein dispersion after the irradiation is subjected to centrifugal treatment (at a rotating speed of 7000 rpm for 13 min), and the supernatant is collected. The high emulsifying activity gluten protein is obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0101] Comparative Example 3
[0102] A preparation method of high emulsifying activity wheat protein based on irradiation modification, comprising the following steps:
[0103] S1, pretreatment
[0104] The wheat gluten protein was dispersed in 8 times the mass of deionized water, and a uniform suspension was formed under the conditions of a temperature of 26°C and a rotation speed of 300 rpm for 30 min of magnetic stirring. The pH of the suspension was adjusted to 8.5 using a NaOH solution, and the suspension was subjected to enzymatic treatment using alkaline protease. Specifically, the suspension was warmed to 50°C and held, 2.2% of the mass of the wheat gluten protein was added as alkaline protease, and the enzymatic treatment was performed for 70 min. The enzyme was then inactivated by heating in a boiling water bath for 16 min, and the suspension was cooled to room temperature. After the enzymatic treatment, the suspension was subjected to centrifugal treatment (at a rotation speed of 7000 rpm for 13 min), and the supernatant was collected. The protein primary powder was obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0105] S2, irradiation treatment
[0106] The protein primary powder was dissolved in 8 times the mass of deuterium-rich water to obtain a protein dispersion. The pH of the system was adjusted to 6.5, and the temperature was adjusted to 6°C.
[0107] The protein dispersion was irradiated using a cobalt-60 gamma ray as the irradiation source, with an irradiation dose of 5.5 kGy. The irradiation was intermittent, with a 2 min interval after each 1 min of irradiation. The irradiation dose rate was controlled at 0.2 kGy / s during each 1 min of irradiation. After the irradiation, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution was repeated 3-4 times to obtain a protein dispersion without residual deuterium water.
[0108] S3, post-treatment
[0109] The irradiated protein dispersion was subjected to centrifugal treatment (at a rotation speed of 7000 rpm for 13 min), and the supernatant was collected. The high emulsifying activity wheat protein was obtained by freeze-drying (at a temperature of -50°C for 24 h).
[0110] Comparative Example 4
[0111] A preparation method of high emulsifying activity wheat protein based on irradiation modification, comprising the following steps:
[0112] S1, pretreatment
[0113] The wheat gluten protein was dispersed in 8 times the mass of deionized water, and a uniform suspension was formed under the conditions of a temperature of 26°C and a rotation speed of 300 rpm for 30 min of magnetic stirring. The pH of the suspension was adjusted to 8.5 using a NaOH solution, and the suspension was subjected to enzymatic hydrolysis using alkaline protease. Specifically, the suspension was warmed to 50°C, 2.2% of the mass of the wheat gluten protein was added as alkaline protease, and the enzymatic hydrolysis was performed for 70 min. The enzyme was then inactivated by heating in a boiling water bath for 16 min, and the suspension was cooled to room temperature. After the enzymatic hydrolysis, the suspension was subjected to centrifugal treatment (at a rotation speed of 7000 rpm for 13 min), and the supernatant was collected. The supernatant was freeze-dried (at a temperature of -50°C for 24 h) to obtain a protein primary powder.
[0114] S2, irradiation treatment
[0115] The protein primary powder was dissolved in 8 times the mass of deionized water to obtain a suspension, and the suspension was placed in a container and subjected to aeration with O3 / N2 mixed gas. The O3 volume fraction in the mixed gas was 8%, the flow rate was controlled at 0.5 L / min, and the suspension was subjected to cyclic aeration for 25 min to achieve an ozone solubility of 20 ppm. In addition, the pH of the system was adjusted to 6.5, and the temperature was adjusted to 6°C.
[0116] Cobalt-60 gamma rays were used as the irradiation source to irradiate the protein dispersion. The irradiation dose was 5.5 kGy, and intermittent irradiation was used, i.e., a 2-min interval was provided after each 1-min irradiation, and the irradiation dose rate was controlled at 0.2 kGy / s during each 1-min irradiation. After the irradiation, the operation of ultrafiltration membrane ultrafiltration and deionized water redissolution was repeated 3-4 times to obtain a protein dispersion without residual deuterium water.
[0117] S3, post-treatment
[0118] The irradiated protein dispersion was subjected to centrifugal treatment (at a rotation speed of 7000 rpm for 13 min), and the supernatant was collected. The supernatant was freeze-dried (at a temperature of -50°C for 24 h) to obtain the high-emulsifying-activity gluten protein.
[0119] III. Performance test
[0120] (1) Emulsifying activity
[0121] The emulsifying activity and emulsifying stability were determined by turbidity method. The gluten protein samples prepared in Examples 1-6 and Comparative Examples 1-4 were each prepared into a 10 mg / mL gluten protein hydrolysate as an aqueous phase using deionized water. After stirring at 800 rpm at room temperature for 2 h, the mixture was allowed to stand at 4°C overnight. Soybean oil was used as an oil phase, and the oil and water were mixed at a volume ratio of 1:4. The mixture was treated in a high-speed shearing homogenizer at 10000 r / min for 2 min to obtain a protein emulsion.
[0122] First, to verify the successful preparation of the emulsion, using Example 5 as an example, laser confocal microscopy (LSCM) was used to observe the microstructure of the protein emulsion of Example 5. Second, 50 μL of each group of protein emulsion prepared above was taken from the bottom with a pipette gun, 5 mL of 0.1% SDS solution was added, and the absorbance at 500 nm was measured by ultraviolet spectrophotometer (UV-1800PC) at 0 min (A0) and 10 min (A10) after the emulsion was prepared, and the emulsification activity index (EAI) and emulsification stability index (ESI) were calculated, and the calculation formula is as follows: 10
[0123]
[0124] In the formula, c is the protein concentration (0.01 g / mL), D is the dilution factor (1000), φ is the oil phase volume fraction (20%), A0and A 10 are the absorbances at 0 min and 10 min.
[0125] (2) Particle size and zeta potential
[0126] The particle size distribution and zeta potential were determined by Zetasizer Nano ZS particle size potential analyzer. The specific process is as follows: each group of protein emulsion prepared above was diluted with deionized water according to the ratio of 1:100, the measurement temperature was set to 25°C, the solute refractive index and deionized water refractive index were set to 1.450 and 1.330 respectively, each sample was measured 3 times, and the average value was taken.
[0127] (3) Surface hydrophobicity
[0128] The protein samples prepared in Examples 1-6 and Comparative Examples 1-4 were diluted with 0.01 M phosphate buffer (pH 7.0) to make the protein mass concentration 0.02-0.10 mg / mL, and then 8.0 mmol / L 1-anilino-8-naphthalenesulfonic acid solution was prepared with 0.01 M phosphate buffer (pH 7.0). 20 μL of 1-anilino-8-naphthalenesulfonic acid solution was added to 4 mL of sample solution, mixed well, and placed in the dark for 15 min, then shaken well and the fluorescence intensity was quickly measured. The excitation wavelength and emission wavelength were set to 390 nm and 470 nm respectively, and the excitation and emission slit width were both 5 nm. The initial slope of the fluorescence intensity versus protein mass concentration graph was used to characterize the surface hydrophobicity of the protein.
[0129] IV. Results analysis
[0130] (1) Emulsification activity
[0131] As Figure 1 The microstructure of the protein emulsion of Example 5 is shown, wherein the oil phase is dyed bright by Nile red, and it can be seen that the droplet distribution is stable, the particle size is small and uniform, and there is no obvious flocculation phenomenon, forming a stable emulsion system.
[0132] As shown in Table 1, the emulsification activity index (EAI) and emulsion stability index (ESI) of each group are shown, and it can be seen that the overall emulsification activity and emulsion stability of Examples 1-3 of the present application are maintained at a relatively optimal level. For example, the emulsification activity index (EAI) of Example 2 is 69.5 m 2 / g, and the emulsion stability index (ESI) is 77.1 min. Examples 3-6 further improve the emulsification activity index (EAI) and emulsion stability index (ESI) by adjusting the irradiation solvent from deionized water to an ozone-d 2 O system based on Example 2. For example, compared with Example 2, the emulsification activity index (EAI) of Example 5 is increased by 16.0%, and the emulsion stability index (ESI) is increased by 18.7%, which is very significant. Comparative Example 1 replaces the intermittent irradiation mode with continuous irradiation based on Example 2, and the total irradiation dose remains unchanged, but the emulsification activity index (EAI) and emulsion stability index (ESI) decrease slightly; Comparative Example 2 cancels the enzymatic pretreatment process based on Example 2, resulting in a significant decrease in emulsification activity index (EAI) and emulsion stability index (ESI), especially in emulsion stability; Comparative Example 3 removes dissolved ozone from the irradiation solvent based on Example 5, leaving only d 2 O, resulting in a significant decrease in emulsification activity index (EAI) and emulsion stability index (ESI); Comparative Example 4 removes d 2 O from the irradiation solvent based on Example 5, leaving only dissolved ozone, resulting in a significant decrease in emulsification activity index (EAI) and emulsion stability index (ESI), and the level is comparable to that of Example 2, indicating that the addition of dissolved ozone alone has no obvious promoting effect, and it needs to be used in cooperation with d 2 O to play a promoting effect.
[0133] Table 1: EAI and ESI results of each group
[0134]
[0135] (2) Particle size and zeta potential
[0136] As Figure 2 shown in the particle size distribution comparison chart of Examples 1-6 of the present application, it can be seen that the particle sizes of Examples 1-6 are all at the nanometer level, and the particle size distribution of Examples 4-6 is more concentrated than that of Example 2, and the average particle size is significantly smaller. The particle size and distribution of the emulsion show the distribution state of the emulsion system, and the smaller the diameter of the emulsion droplet, the better the stability of the emulsion; as Figure 3As shown in the figure, which is a comparison of particle size distribution between Example 2 and Example 5 and Comparative Examples 1-4, it can be seen that the particle sizes of Comparative Examples 1 and 2 are different degrees of increase compared with Example 2, especially Comparative Example 2; the particle sizes of Comparative Examples 3 and 4 are also different degrees of increase compared with Example 5.
[0137] As shown in the figure, which is a comparison of particle size distribution between Example 2 and Example 5 and Comparative Examples 1-4, it can be seen that the particle sizes of Comparative Examples 1 and 2 are different degrees of increase compared with Example 2, especially Comparative Example 2; the particle sizes of Comparative Examples 3 and 4 are also different degrees of increase compared with Example 5. Figure 4 As shown in the figure, which is a comparison of particle size distribution between Example 2 and Example 5 and Comparative Examples 1-4, it can be seen that the particle sizes of Comparative Examples 1 and 2 are different degrees of increase compared with Example 2, especially Comparative Example 2; the particle sizes of Comparative Examples 3 and 4 are also different degrees of increase compared with Example 5.
[0138] (3) Surface hydrophobicity
[0139] As shown in Table 2, which is the surface hydrophobicity index result of each group, it can be seen that the surface hydrophobicity index of Examples 1-6 of the present application is high, and the hydrophobicity is excellent, especially the surface hydrophobicity index of Examples 4-6 is higher than that of Example 2, the surface hydrophobicity index of Comparative Examples 1 and 2 is lower than that of Example 2, and the surface hydrophobicity index of Comparative Examples 3 and 4 is lower than that of Example 5. The surface hydrophobicity of protein is an important indicator of the number of protein surface hydrophobicity groups connected with the external polar water environment, and is closely related to the emulsifying properties of protein. Generally speaking, the higher the surface hydrophobicity, the better the emulsifying properties and emulsion stability.
[0140] Table 2: Surface hydrophobicity index of each group
[0141]
[0142] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the present patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.
Claims
1. A method for preparing irradiation-modified high emulsifying active gluten protein, characterized in that the steps include: S1. Preprocessing Wheat gluten protein is dispersed in 6-10 times its mass of deionized water, stirred to form a uniform suspension, the pH of the suspension is adjusted to 8-9 with NaOH solution, and the suspension is enzymatically hydrolyzed with alkaline protease. After enzymatic hydrolysis, the suspension is centrifuged, the supernatant is collected, and freeze-dried to obtain protein powder; S2. Irradiation treatment The protein powder is dissolved in 6-10 times the mass of the irradiation solvent to obtain a protein dispersion, and the protein dispersion is irradiated with cobalt-60 gamma rays as an irradiation source at a dose of 5-6 kGy, and intermittent irradiation is adopted; S3, post-processing The irradiated protein dispersion is taken, centrifuged, the supernatant is collected, and freeze-dried to obtain the high emulsifying activity gluten protein.
2. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In step S1, the stirring treatment refers to: magnetic stirring at a temperature of 24-28° C. and a rotation speed of 250-350 rpm for 25-35 minutes.
3. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In step S1, the enzymatic hydrolysis treatment refers to: heating the suspension to 48-52°C and keeping it warm, adding alkaline protease accounting for 2-2.5% of the quality of wheat gluten protein, enzymatic hydrolysis for 60-80 minutes, then heating in a boiling water bath for 15-20 minutes to inactivate the enzyme, and cooling to room temperature.
4. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In step S2, the irradiation solvent is deionized water.
5. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In step S2, the irradiation solvent is an ozone-deuterium-enriched water system, the main component of the ozone-deuterium-enriched water system is deuterium-enriched water, the dissolved ozone concentration is 15-25 ppm, the system pH is 6.0-7.0, and the temperature is 4-10°C.
6. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 5, characterized in that: In step S2, the ozone is added to the ozone-deuterium-enriched water system as follows: after the protein powder is dissolved in deuterium-enriched water to obtain a suspension, the suspension is placed in a container and an O3 / N2 mixed gas is introduced, wherein the volume proportion of O3 in the mixed gas is 5-10%, the flow rate is controlled at 0.4-0.6 L / min, and the aeration is circulated for 20-30 minutes.
7. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 6, characterized in that: When an ozone-deuterium-enriched water system is used as the irradiation solvent, ultrafiltration with an ultrafiltration membrane and re-dissolution with deionized water are repeated 3-4 times after the irradiation to obtain a protein dispersion free of residual deuterium water.
8. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In step S2, the intermittent irradiation means that each 1-minute irradiation is followed by a 2-minute interval, and the irradiation dose rate during each 1-minute irradiation is controlled within a range of 0.15-0.25 kGy / s.
9. The method for preparing a gluten protein with high emulsifying activity based on radiation modification according to claim 1, characterized in that: In steps S1 and S3, the rotation speed of the centrifugal treatment is 6000-8000 rpm, the time is 12-15 minutes, and the temperature of the freeze-drying is -50°C, and the time is 18-36 hours.
10. A gluten protein with high emulsification activity based on radiation modification, characterized in that: The high emulsifying activity gluten protein is prepared by the preparation method according to any one of claims 1 to 9.