Preparation method and application of silybum marianum seed protein with high alpha helical structure content

Abstract

The invention discloses a preparation method and application of silybum marianum seed protein with high alpha helical structure content. The method comprises the following steps: mixing silybum marianum seed powder with a reverse micelle solution by adopting a reverse micelle system formed by a surfactant in water, carrying out pre-extraction and back-extraction operations, then carrying out rotary evaporation concentration and dialysis treatment, and finally obtaining powdery silybum marianum alpha-helical structure protein through a freeze-drying technology. According to the method, the protein extraction efficiency and purity are remarkably improved, the extracted protein is more uniform in particle size distribution, better in solubility and not prone to aggregation, and the oil holding capacity, foamability and foam stability of the protein are remarkably improved. A new technical route is provided for extraction and preparation of the alpha-helical structure protein in the plant source grain protein, a high-quality novel plant protein resource is provided for the industries such as food, medicine and cosmetics, and the method has wide application prospects and market potential.

Description

Technical Field

[0001] The invention belongs to the technical field of food-derived protein extraction, preparation and activity, and particularly relates to a preparation method of milk thistle seed protein with high alpha helical structure content and application thereof. Background Art

[0002] The development and utilization of new plant-based food raw materials have received widespread attention. These new resources include not only traditional crops and wild plants, but also new plant varieties improved or cultivated through modern biotechnology. The purpose of developing these plant-based food raw materials is to provide richer, healthier and more sustainable food options to meet the growing population's demand for nutritious and healthy foods. They can not only supplement traditional foods, but also have the potential to solve the problem of nutritional deficiencies in specific populations, reduce the risk of chronic diseases, and reduce the impact on the environment, promoting the green and sustainable development of the food industry. In particular, milk thistle, as a plant rich in nutritional value and multiple biological activities, the research and development of its functional components is particularly important.

[0003] α-helical proteins are a class of proteins with specific spatial structures. They not only provide the necessary structural stability and mechanical strength for proteins, but also participate in many biological processes, such as enzyme catalysis and signal transduction, which are essential for maintaining life activities and health. These proteins play a key role in drug development, disease research, food safety, agricultural applications, and materials science. Their functional diversity and biological activity make them an important focus of scientific research and technological innovation. However, traditional protein extraction methods, such as acid-base extraction and salting out, have problems such as low extraction efficiency, serious environmental pollution, and large activity loss, and it is often difficult to maintain the natural structure and activity of α-helical proteins, which to a certain extent limits their application in multiple fields. Therefore, developing a method that can efficiently and greenly extract and maintain the activity of α-helical proteins is crucial to promoting their in-depth application in the fields of food, medicine, and health products. Milk thistle seeds contain a large amount of protein, and its potential value encourages researchers to explore more efficient extraction technologies. The present invention is based on this demand and proposes an innovative extraction method.

[0004] In recent years, reverse micelle technology has attracted much attention in the field of protein extraction due to its unique advantages. This technology can effectively extract target proteins from plant raw materials while maintaining the integrity of their structure and function by forming a nanoscale reverse micelle structure in a microemulsion. Compared with traditional extraction methods, reverse micelle technology has higher extraction efficiency, better selectivity and milder operating conditions, thereby reducing the loss of active ingredients and environmental pollution. The present invention aims to solve the following technical problems: improve the extraction efficiency of α-helical structure protein in milk thistle seeds and realize industrial-scale production; optimize the extraction process, reduce the damage to the protein structure and activity, and maintain its biological activity; reduce environmental pollution during the extraction process and realize a green and sustainable production method. Therefore, the rapid extraction method of milk thistle α-helical structure protein based on reverse micelle technology provided by the present invention can not only effectively extract the α-helical structure protein in milk thistle seeds, but also provide an efficient, environmentally friendly and sustainable solution for related industries, thereby promoting the development and application of new plant-based resource food raw materials. Summary of the invention

[0005] The present invention aims at the deficiencies of the existing preparation technology and proposes a method for preparing milk thistle seed protein with high alpha helix content. The method uses milk thistle seed as raw material, extracts alpha helix protein in milk thistle seed through pretreatment, and has the characteristics of high extraction efficiency, high purity and strong product structure specificity.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0007] The preparation method of the present invention comprises:

[0008] performing pre-extraction to extract α-helical structural proteins in milk thistle seeds to obtain a supernatant;

[0009] Performing back extraction, the supernatant is mixed with the separation phase solution to further purify the extracted α-helical structure protein;

[0010] The extract was concentrated by rotary evaporation to remove excess reverse micelle solution;

[0011] Remove small molecule impurities by dialysis;

[0012] The powdered milk thistle α-helical protein was obtained by freeze drying.

[0013] Specifically, a method for preparing milk thistle seed protein with high alpha-helix content comprises the following steps:

[0014] (1) Pre-extraction of milk thistle seed protein: Milk thistle seed powder and extraction solution (i.e., reverse micelle solution containing surfactant) are mixed in a certain proportion, extracted in a low temperature environment for a certain period of time, and the supernatant is collected.

[0015] In step (1), the milk thistle seed powder refers to a milk thistle seed component that is dried at 50-60° C. to a constant weight and has a particle size of ≤355 μm after crushing.

[0016] In step (1), the pre-extraction solution is a mixture of a surfactant solution and a buffer solution in a ratio of 1:10-1:30, and the milk thistle seeds and the extraction solution are mixed in a ratio of 1:10-30 (g / mL).

[0017] Further optimized, in the surfactant solution, the surfactant is composed of one or more of sodium dodecyl sulfate, sodium dioctyl succinate sulfonate, and sodium alkylbenzene sulfonate, the solvent is composed of one or more of n-octane, n-hexanol, and 2,2,4-trimethylpentane, and the concentration of the surfactant is 0.1 to 1 mol / L;

[0018] Further optimized, the buffer is composed of one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, the inorganic salt dissolution concentration is 0.5-2.5 mol / L, the phosphate buffer concentration is 20-100 mmol / L, and the pH value is 7.0-8.0;

[0019] Further optimized, in step (1), the low temperature environment is 20-40°C; the extraction time is 0.5-2h, and the supernatant is the upper clear liquid obtained after centrifugation at 4000-6000×g for 10-30min.

[0020] (2) Re-extraction of milk thistle seed protein: the supernatant obtained in step (1) is mixed with a certain volume of the separated phase solution, extracted again in a low temperature environment for a certain period of time, and after centrifugation, the interface layer and the lower aqueous phase liquid are collected as the prepared protein solution;

[0021] In step (2), the separation phase solution is an inorganic salt (one or more of NaCl, KCl, Na2SO4, K2SO4) dissolved in a phosphate buffer (composed of one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate) with a concentration of 20 to 100 mmol / L, the inorganic salt dissolution concentration is 0.5 to 2.5 mol / L, and the pH value is 7.0 to 8.0;

[0022] In step (2), the mixing ratio of the supernatant to the separated phase solution is 1:3 to 3:1.

[0023] In step (1), the low temperature environment is 20-40°C; the extraction time is 0.5-2h, and the interface layer and the lower aqueous phase liquid are the white solid in the middle layer of the two phases and the lower liquid obtained after centrifugation at 4000-6000×g for 10-30min.

[0024] (3) Purification and preparation of milk thistle seed protein: The protein solution obtained in step (2) is concentrated by rotary evaporation at 40-60° C., desalted, and vacuum freeze-dried to obtain milk thistle seed α-helical protein.

[0025] In step (3), the desalting refers to removing small molecules by membrane permeation or ultrafiltration, with the upper limit of the molecular weight being 1000 to 3000 Da. During the rotary evaporation concentration process, the volume is concentrated to 1 / 3 to 1 / 5 of the original volume.

[0026] The alpha helical protein of milk thistle seeds prepared by any of the methods described above has an extraction efficiency that is more than 20% higher than that of the traditional alkali extraction and acid precipitation method, and a purity that is more than 15% higher. The alpha helical protein accounts for more than 90% of the total protein, and its protein particle size distribution is more uniform (122-396nm), solubility is better and it is not easy to aggregate, and its oil retention, foaming properties and foam stability are significantly improved.

[0027] The low-temperature repeated reverse phase extraction method adopted by the present invention can extract milk thistle seed protein in a targeted manner, promote the dissolution of alpha helical structure, and improve protein extraction efficiency; the low-temperature repeated reverse phase extraction method has a high extraction rate, the solvent can be recycled, and at the same time, the prepared milk thistle seed functional protein with high alpha helical structure content.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] 1. The present invention adopts a low-temperature repeated reverse phase extraction method, does not involve severe physical and chemical treatment, has a simple and mild preparation method, and the solvent can be recycled and reused multiple times, which is suitable for large-scale production and efficient preparation of milk thistle seed alpha helical protein.

[0030] 2. The method for preparing α-helical protein from milk thistle seeds provided by the present invention enriches the α-helical protein components from milk thistle seeds through surface active adsorption and reverse phase extraction, thereby significantly increasing the α-helical structure content.

[0031] 3. Compared with the traditional alkaline extraction and acid precipitation method and ultrasound-assisted alkaline method, the method of the present invention has significantly improved the protein extraction efficiency and purity (by more than 20% and 15% respectively), especially the proportion of α-helical structure protein increased from 45-50% in the alkaline extraction method or the alkaline extraction plus ultrasound-assisted method to more than 90% in the reverse micelle method. The extracted protein particle size distribution is more uniform (122-396nm), the solubility is better and it is not easy to aggregate, which significantly improves its oil retention, foaming property and foam stability.

[0032] 4. The silybum seed α-helical protein prepared by the method provided by the present invention has a low degree of structural destruction, a high protein activity retention rate, good oil retention, foaming and foam stability, and extremely strong processing characteristics, thereby increasing the added value of silybum seed protein, thereby expanding the application scope of high α-helical structure content proteins in the food industry, providing high-quality new plant protein resources for industries such as food, medicine and cosmetics, and having broad application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The SDS-PAGE electrophoresis bands of the alpha helical protein of milk thistle seeds prepared in Example 1 and Comparative Example 1 and Comparative Example 2;

[0034] Figure 2 The circular dichroism results of the alpha helical protein of milk thistle seeds prepared in Example 1 and Comparative Example 1 and Comparative Example 2;

[0035] Figure 3 The particle size measurement results of the alpha helical protein of milk thistle seeds prepared in Example 1 and Comparative Examples 1 and 2;

[0036] Figure 4 The endogenous fluorescence measurement results of the alpha helical protein of milk thistle seeds prepared in Example 1 and Comparative Example 1 and Comparative Example 2;

[0037] Figure 5 The water-holding and oil-holding capacity test results of the alpha helical protein of milk thistle seeds prepared in Example 1 and Comparative Example 1 and Comparative Example 2; Figure 6 These are the foaming property and foam stability test results of the milk thistle seed alpha helical protein prepared in Example 1 and Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0038] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0039] Comparative Example 1

[0040] Mix milk thistle seed powder with distilled water at a material-water ratio of 1:10, adjust the pH to 9.0 with 1M NaOH, shake at 45℃ for 2h, centrifuge at 8000rpm for 10min, and collect the supernatant. Repeat the above steps for re-extraction once. Combine the supernatants, adjust the pH to 4.0 with 1M HCl. Centrifuge at 8000rpm for 10min and collect the precipitate. After the precipitate is redissolved, add 1M NaOH to the solution, adjust the pH to 7.0, concentrate by rotary evaporation at 55℃, desalt for 48h, freeze-dry for 48h, and collect milk thistle seed protein.

[0041] Comparative Example 2

[0042] Mix milk thistle seed powder with distilled water at a material-water ratio of 1:10, adjust the pH to 9.0 with 1M NaOH, perform ultrasonic extraction at 45℃ for 2h, centrifuge at 8000rpm for 10min, and collect the supernatant. Repeat the above steps for extraction once. Combine the supernatants, adjust the pH to 4.0 with 1M HCl. Centrifuge at 8000rpm for 10min, and collect the precipitate. After the precipitate is redissolved, add 1M NaOH to the solution, adjust the pH to 7.0, concentrate by rotary evaporation at 55℃, desalt for 48h, and freeze-dry for 48h.

[0043] Example 1

[0044] Use 2,2,4-trimethylpentane solution to prepare 0.18M sodium dioctyl succinate sulfonate solution, add 0.05M KCl-50mM PBS at a ratio of 18:1, and stir at 37°C for 30 minutes to obtain the pre-extraction solution. Mix the milk thistle seed powder with the pre-extraction solution at a ratio of 1:15. Stir at 37°C for 60 minutes, centrifuge at 5500×g for 15 minutes, and collect the supernatant. Mix the supernatant with the separation phase solution at a ratio of 1:1, stir at 37°C for 60 minutes, centrifuge at 5500×g for 15 minutes, and collect the interface layer and the lower aqueous phase liquid. Concentrate the solution by rotary evaporation at 55°C, desalt for 48 hours, and freeze-dry for 48 hours.

[0045] Example 2

[0046] Prepare 0.18M sodium alkylbenzene sulfonate solution with n-hexane, add 0.01M KCl-20mM PBS at a ratio of 10:1, and stir at 25°C for 120min to obtain the pre-extraction solution. Mix the milk thistle seed powder with the pre-extraction solution at a ratio of 1:10. Stir at 25°C for 60min, centrifuge at 6000×g for 10min, and collect the supernatant. Mix the supernatant with the separation phase solution at a ratio of 1:3, stir at 25°C for 60min, centrifuge at 6000×g for 10min, and collect the interface layer and the lower aqueous phase liquid. Concentrate the solution by rotary evaporation at 40°C, desalt for 48h, and freeze-dry for 48h.

[0047] Example 3

[0048] Use n-octane to prepare 0.2M sodium dodecyl sulfate solution, add 0.1M KCl-50mM PBS at a ratio of 30:1, and stir at 40℃ for 60min to obtain the pre-extraction solution. Mix the milk thistle seed powder with the pre-extraction solution at a ratio of 1:10. Stir at 40℃ for 20min, centrifuge at 4000×g for 30min, and collect the supernatant. Mix the supernatant with the separation phase solution at a ratio of 1:1, stir at 40℃ for 60min, centrifuge at 4000×g for 30min, and collect the interface layer and the lower aqueous phase liquid. Concentrate the solution by rotary evaporation at 60℃, desalt for 48h, and freeze-dry for 48h.

[0049] Effect verification example 1: SDS-PAGE analysis of milk thistle seed protein

[0050] The alpha helical protein of milk thistle seeds prepared by the methods described in Example 1, Comparative Example 2 and Comparative Example 3 was subjected to SDS=PAGE electrophoresis analysis. The mass fraction of acrylamide in the separation gel was 12%, and the mass fraction in the concentrated gel was 5%. The protein sample concentration was 2 mg / mL, and the protein sample was mixed with 5× protein loading buffer at a volume of 4:1, heated in a boiling water bath for 3 to 5 min, taken out and cooled, centrifuged at 10000 rpm for 15 s, and the supernatant was taken for standby use. The constant voltage of the electrophoresis process was 85 V. After the electrophoresis was completed, the gel was stained in 0.125% Coomassie Brilliant Blue R-250 for 4 h, and decolorized in distilled water containing 10% acetic acid and 30% methanol until the protein bands were clear. The molecular weight range of the standard protein was 6.5 to 200 kDa, and the gel image was collected using a gel imaging system. The protein concentration was detected by the BCA method.

[0051] Effect verification example 2: Secondary structure analysis of milk thistle seed protein

[0052] The absorbance values ​​of the alpha helical protein of milk thistle seeds prepared by the methods described in Example 1, Comparative Example 2 and Comparative Example 3 were measured in the range of 190-260 nm using a circular dichroism spectrometer to analyze the effects of different extraction methods on the content of the alpha helical structure in the protein. Before the measurement, the protein solution was diluted with deionized water to a protein concentration of 0.2 mg / mL, the curve measured by distilled water was used as the baseline, and the measurement temperature was 25°C.

[0053] Effect verification example 3: Particle size analysis of milk thistle seed protein

[0054] The method described in Example 1, Comparative Example 2 and Comparative Example 3 was used to prepare milk thistle seed protein for particle size distribution analysis. Before measurement, the milk thistle seed α-helical protein solution was diluted with deionized water to a protein concentration of 1 mg / mL. The laser was 4mW He-Ne (633nm), the scattering angle was 173°, and the test temperature was 25°C.

[0055] Effect verification example 4: Analysis of the tertiary structure of milk thistle seed protein

[0056] The endogenous fluorescence intensity of the alpha helical protein of milk thistle seeds prepared by the methods described in Example 1, Comparative Example 2 and Comparative Example 3 at a wavelength of 300-440 nm was analyzed to analyze the effects of different extraction methods on the tertiary structure of the alpha helical protein of milk thistle seeds. Before measurement, the protein solution was diluted with deionized water to a protein concentration of 0.5 mg / mL. The "Wavelength scan" mode was selected, the measurement temperature was 25°C, the voltage was 550V, the excitation wavelength was 280nm, the scanning range was 300-440nm, the scanning was performed at a speed of 240nm / min, and the slit width was fixed at 5nm.

[0057] Effect verification example 5: Water and oil holding capacity test of milk thistle seed protein

[0058] 100 mg of the milk thistle seed protein prepared by the methods described in Example 1, Comparative Example 2 and Comparative Example 3 was accurately weighed, mixed with 1000 g of deionized water or soybean oil, vortexed for 10 seconds and allowed to stand for 30 minutes, centrifuged at 8000 g for 10 minutes, and the supernatant was discarded. The centrifuge tube was placed at 45°, and the weight was weighed after draining for 20 minutes. The water-holding capacity and oil-holding capacity (g / g) of the milk thistle seed protein prepared in Example 1 were expressed as the ratio of the weight difference before and after draining to the weight of the dry sample.

[0059] Effect verification example 6: Testing of foaming properties and foam stability of milk thistle seed protein

[0060] The foaming property (FE) and foam stability (FS) of the milk thistle seed protein prepared by the method described in Example 1, Comparative Example 2 and Comparative Example 3 are determined as follows: a certain volume of protein solution (0.5% w / v) is placed in a graduated test tube and homogenized at 15000 rpm for 2 min using a homogenizer. FE and FS are calculated according to the following formula:

[0061] FE(%)=(V T -V0) / Vox 100

[0062] Among them, V T is the volume of the foam solution after homogenization, and V0 is the volume of the foam solution before homogenization.

[0063] FS(%)=(V t -V0) / Vax 100

[0064] Among them, V t V0 is the volume of the foam solution after homogenization for 30 min, and V0 is the volume of the foam solution before homogenization.

[0065] Experimental results analysis:

[0066] Figure 1The electrophoresis bands of the milk thistle seed protein prepared by the inventive method and the traditional method in Example 1 and Comparative Example 1 and Comparative Example 2. In contrast, the milk thistle seed protein obtained by the method of Example 1 has a richer number of bands on the gel, with clear band morphology and good separation. This shows that the protein obtained by this method has high abundance and purity, and proteins of different molecular weights are effectively separated. The method used in the present invention utilizes a surfactant to form a micellar system in an aqueous solution, thereby achieving the extraction and purification of protein. The method has good selectivity, can selectively extract specific proteins, and can effectively reduce the mixing of impurities.

[0067] Figure 2 The circular dichroism spectra and secondary structure ratios of the alpha helical protein of milk thistle seeds prepared by the inventive method and the traditional method in Example 1 and Comparative Example 1 and Comparative Example 2. The circular dichroism spectrum results of milk thistle seed protein show that the alpha helical structure content of the milk thistle seed protein prepared by the inventive method is significantly increased to more than 90%, and the ultrasound and alkaline extraction methods cause the alpha helical structure of the milk thistle seed protein to change, the secondary structure is loose, and the activity is reduced.

[0068] Figure 3 The particle size distribution of the silybum seed α-helical protein prepared by the inventive method and the traditional method in Example 1 and Comparative Example 1 and Comparative Example 2. Different methods lead to significant differences in the particle size distribution of silybum seed protein. The silybum seed α-protein obtained by the method of Example 1 has a larger particle size and a more concentrated distribution, indicating that the silybum seed α-helical protein prepared by the method of the present invention has a more uniform particle size, a smaller surface energy difference, and the protein is not easy to aggregate, and the solubility and stability of the protein are better.

[0069] Figure 4 The endogenous fluorescence spectra of the alpha helical protein of milk thistle seeds prepared by the inventive method and the traditional method in Example 1 and Comparative Example 1 and Comparative Example 2. The hydrophobic amino acids phenylalanine (Phe), tyrosine (Tyrosine, Tyr) or tryptophan (Trp) all have strong fluorescence quantum yields, and the position of the Trp residue among the three hydrophobic amino acids is closest to the outside of the protein. Therefore, the changes in the tertiary structure of the protein can be determined by monitoring the fluorescence intensity and maximum emission wavelength (λmax) of the Trp residue. The λmax of the Trp residue can reflect the changes in the polarity of the microenvironment in which it is located. When the Trp residue is more exposed to the solvent, the λmax red shift is greater. Regardless of the excitation wavelength, when the fluorescence of the fluorescence spectrum is enhanced, it indicates that the hydrophobic amino acid is deeper into the protein molecule; otherwise, it indicates that the hydrophobic amino acid is closer to the surface of the protein molecule. The results show that the α-helical protein of milk thistle seeds prepared by the method of the present invention has the strongest fluorescence intensity, the non-polarity of the microenvironment where the Trp residue is located is enhanced, the protein is folded or converged in the tertiary structure, and tryptophan is deeply buried in the hydrophobic core.

[0070] Figure 5 The results of water-holding and oil-holding determination of the silybum seed α-helical protein prepared in Example 1 and Comparative Examples 1 and 2 are shown. The water-holding of the silybum seed protein obtained by the method of Example 1 is relatively poor, about 0.15%, and the water-holding of the protein obtained by Comparative Examples 1 and 2 is about 2.5% and 1.9%, respectively. The results of oil-holding showed that the oil-holding of the silybum seed proteins obtained by the three methods was relatively good, ranging between 4.2% and 5.3%, among which the silybum seed protein obtained in Comparative Example 2 had the best oil-holding of 5.3%. Based on the water-holding and oil-holding results, it was found that the low-temperature repeated reverse micelle technology treatment enhanced the surface hydrophobicity of the silybum seed protein.

[0071] Figure 6 The foaming property and foam stability test results of the silybum seed α-helical protein prepared in Example 1 and Comparative Examples 1 and 2 are shown. In food processing, the foaming property of protein is an important property that affects the texture, taste and stability of food. In comparison, the silybum seed protein obtained by the method of Example 1 has better foaming property. The protein (0.5% solution) obtained by Example 1 has a foaming property of up to 42% and a stability of about 38%. The foaming property of the protein obtained by Comparative Examples 1 and 2 is between 2% and 3%, and the stability is less than 2%. Compared with Comparative Examples 1 and 2, the foaming property and stability of the silybum seed protein with a high α-helical structure content prepared in Example 1 are more than 20 times higher. The foaming property of protein mainly depends on the structure and properties of protein. The silybum seed protein obtained in Example 1 has higher foaming property because it may have a longer amphiphilic α-helical structure, which enables it to form stable foam at the gas-liquid interface.

[0072] In summary, the low-temperature repeated reverse phase extraction method prepared by the method of the present invention is simple, mild, green and efficient, and the degree of destruction of the α-helical protein structure of milk thistle seeds is low, the protein activity retention rate is high, and it has good oil retention, foaming and foam stability, and has extremely strong processing characteristics, thereby improving the added value of milk thistle seed protein, and further expanding the application scope of proteins with high α-helical structure content in the food industry.

[0073] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing milk thistle seed protein with high α-helix structure content, characterized in that: The following steps are involved: (1) mixing milk thistle seed powder with an extraction solution, extracting in a low temperature environment, and collecting the supernatant; (2) mixing the supernatant obtained in step (1) with the separated phase solution, extracting again in a low temperature environment, and collecting the interface layer and the lower aqueous phase liquid after centrifugation as the prepared protein solution; (3) The protein solution obtained in step (2) is concentrated by rotary evaporation, desalted, and vacuum freeze-dried to obtain milk thistle seed α-helical protein.

2. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: The extraction solution in step (1) is a mixture of a surfactant solution and a buffer solution in a ratio of 1:10-1:30, and the mixing ratio of the milk thistle seeds to the extraction solution is 1:10-30 (g / mL).

3. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: The low temperature environment is 20-40° C.; the extraction time is 0.5-2 hours; and the supernatant is the upper clear liquid obtained after centrifugation at 4000-6000×g for 10-30 minutes.

4. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 2, characterized in that: The surfactant is a nonionic surfactant.

5. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: In step (1), the mixing ratio of the milk thistle seeds to the extraction solution is 1:10-30 (g / mL); and in step (2), the mixing ratio of the supernatant to the separation phase solution is 1:3-3:

1.

6. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: During the rotary evaporation concentration process, the volume is concentrated to 1 / 3 to 1 / 5 of the original volume.

7. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: In the desalting process, a dialysis membrane with a molecular weight cut-off range of 1000 to 10000 Da is used.

8. The method for preparing a milk thistle seed protein with a high α-helix structure content according to claim 1, characterized in that: The extraction efficiency is increased by more than 20% compared with the traditional alkaline extraction and acid precipitation method, the purity is increased by more than 15%, and the proportion of α-helical structure protein reaches more than 90%.

9. A milk thistle seed protein with a high α-helix structure content obtained by the preparation method according to any one of claims 1 to 8.

10. The use of a milk thistle seed protein with a high α-helix structure content as claimed in claim 9 in preparing food, characterized in that: The protein has the properties of oil retention, stability, foaming and foam stability during food processing; the milk thistle protein with high alpha-helix structure content can also be used as a functional food additive to enhance the protein content and nutritional value of food.

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