Preparation method and application of peony seed protein with high surface hydrophobicity

Abstract

The invention discloses a preparation method and application of peony seed protein with high surface hydrophobicity. The method comprises the following steps: washing and drying peony seeds, carrying out superfine grinding treatment, removing grease through a squeezing method, mixing peony seed meal with distilled water according to a certain proportion, carrying out homogenization pretreatment, adopting an ultrasonic physical field superposition auxiliary alkali extraction method, and carrying out operations of multiple times of acid precipitation, redissolution, centrifugation, dialysis and the like, so as to obtain the peony seed meal. And finally freeze-drying to obtain powdery peony seed protein. The peony seed protein obtained by the method has high surface hydrophobicity, the foam property of the protein is remarkably improved, and the protein yield is also remarkably increased. A new technical route is provided for extraction and preparation of protein with high surface hydrophobicity in plant source grain protein, high-quality novel plant protein resources are provided for 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 and application of peony seed protein with high surface hydrophobicity. Background Art

[0002] With the rapid development of the peony seed oil industry, a significant amount of peony meal, a byproduct of peony seed oil production, is produced. This meal, rich in protein (typically 18.41-25.40%), represents a potential new source of plant protein with high nutritional value and functional properties. Effectively utilizing peony seed protein not only enhances the economic value of the peony industry but also addresses the need for sustainable development and reduces resource waste.

[0003] The surface hydrophobicity of proteins is one of their important functional properties and is closely related to their interfacial behaviors (such as emulsification, foaming properties, foam stability, gelling properties, and binding ability with non-polar substances). High surface hydrophobicity generally means that proteins have stronger adsorption and stabilization capabilities at oil-water or air-water interfaces. This has significant advantages for the development of emulsion-based foods (such as salad dressings and beverages), foaming products (such as desserts and plant-based creams), edible films, encapsulation carriers, and certain industrial applications (such as adhesives and biomaterials).

[0004] However, existing peony seed proteins or their conventional extracts generally have the problem of low surface hydrophobicity. This is mainly due to: 1) the structural characteristics of peony seed protein itself. Its natural conformation may encapsulate the hydrophobic groups (Journal of Colloid and Interface Science, 293 (2006) 263-269,) 2) the relatively mild pH value, low temperature treatment or drying method during traditional extraction processes (such as alkali dissolution and acid precipitation) during which the protein hydrophobic groups are not fully exposed or are buried. This low surface hydrophobicity seriously limits the application potential of peony seed protein in the above-mentioned high-value-added functional foods and industrial fields, making it difficult for it to effectively play an interfacial stabilization role.

[0005] To improve the functional properties of plant proteins, researchers have developed a variety of chemical (e.g., acid, alkali, acylation, phosphorylation) and enzymatic modification techniques. Although these methods can improve hydrophobicity to a certain extent, they are often accompanied by obvious limitations: chemical modification may introduce reagent residues, posing safety and regulatory risks, and the harsh reaction conditions may lead to protein nutritional loss or unpleasant flavors; enzymatic modification is costly and highly specific, and the process control is complex (Food Chemistry: X, 23 (2024) 101540). Therefore, there is an urgent need to develop an efficient, safe, and economically feasible method specifically for improving the surface hydrophobicity of peony seed proteins to fully unleash their functional potential.

[0006] In summary, addressing the key issues limiting peony seed protein's high-value applications, such as low resource utilization and insufficient surface hydrophobicity of natural and conventional extracts, there is an urgent need for an innovative preparation method that can significantly and controllably enhance the surface hydrophobicity of peony seed protein. This method should balance high efficiency, safety (avoiding harmful chemical reagents), process simplicity, and preservation of the protein's nutritional value. Summary of the Invention

[0007] In view of the shortcomings of existing preparation technologies, the present invention proposes a preparation method of peony seed protein with high surface hydrophobicity.

[0008] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0009] The preparation method of the present invention comprises:

[0010] Pretreatment is performed to obtain peony seed powder with smaller pore size to increase its surface area in contact with the alkaline environment;

[0011] Ultrasound-assisted treatment was performed to disrupt the peony seed cell structure and further promote its exposure to an alkaline environment;

[0012] Perform multiple acid precipitation treatments to further purify the protein;

[0013] Remove small molecule impurities by dialysis;

[0014] The high surface hydrophobicity protein from peony seeds was obtained in powder form by freeze-drying.

[0015] Specifically, the present invention provides a method for preparing peony seed protein with high surface hydrophobicity, comprising the following steps:

[0016] (1) Defatting: The ultrafinely crushed peony seed powder is defatted by pressing;

[0017] (2) Pretreatment: Mix the defatted peony seed powder with distilled water and homogenize the mixture using a homogenizer;

[0018] (3) Extraction: extracting protein from the homogenized mixture obtained in step (2) under a low-temperature alkaline environment while simultaneously superimposing an ultrasonic physical field, and collecting the supernatant after extraction;

[0019] (4) Re-extraction: collecting the precipitate after extraction, extracting the protein under a low-temperature alkaline environment with an ultrasonic physical field, collecting the supernatant after extraction, and combining the supernatants obtained in step (3) and step (4);

[0020] (5) Acid precipitation and re-dissolution: Adjust the pH to the isoelectric point of the protein and precipitate the protein with acid for 8-12 hours. Collect the precipitated protein and adjust the pH to 7.0-7.2 for re-dissolution.

[0021] Desalting: The protein solution obtained in step (5) is dialyzed for desalting and then freeze-dried in a vacuum to obtain peony seed protein with high surface hydrophobicity.

[0022] Furthermore, step (4) is repeated 3 to 5 times; and step (5) is repeated 2 to 4 times.

[0023] Furthermore, the peony seed powder in step (1) refers to a component with a particle size of ≤355 μm after crushing; the protein content of the peony seed powder is ≥20%; and the oil content of the peony seed powder after defatting is ≤8%.

[0024] Furthermore, in step (2), the homogenization speed is 8000-20000 rpm, the time is 2-10 min, and the material-liquid ratio of peony seed powder to distilled water is 1:10-30 (g / mL).

[0025] Furthermore, in step (3) and step (4), the low temperature environment is 30-50° C.; the alkaline environment is pH 9-12; the frequency of the ultrasonic physical field is 40-50 kHz; the extraction time is 0.5-2 h; and the supernatant is the upper clear liquid obtained after centrifugation at 4000-6000×g for 10-30 min.

[0026] Furthermore, in step (5), the isoelectric point of the protein is pH = 3.9 to 4.5.

[0027] Furthermore, the desalting in step (6) refers to the removal of small molecular substances using membrane permeation or ultrafiltration, with the upper limit of the removal molecular weight being 1000 to 3000 Da.

[0028] Furthermore, the peony seed high surface hydrophobic protein prepared by any of the above methods has an extraction rate of 80.8%-84.7%, a surface hydrophobicity index H0 of more than 20878, an improvement rate of more than 44%, and significantly improves its foaming properties.

[0029] The present invention also provides the use of a peony seed protein with high surface hydrophobicity prepared by the preparation method in preparing food.

[0030] Furthermore, the protein has high surface hydrophobicity and foaming property greater than or equal to 70% and foam stability greater than or equal to 30% during food processing.

[0031] Furthermore, the protein can also be used as a functional food additive to enhance the foaming performance and nutritional value of food.

[0032] The present invention adopts a homogenization pretreatment and ultrasonic physical field superposition assisted alkaline method to extract protein, which can improve protein extraction efficiency, reduce the use of chemical solvents, and obtain peony seed functional protein with high surface hydrophobicity in a green and efficient manner.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] (1) The present invention adopts a homogenization pretreatment and ultrasonic physical field superposition method, does not involve chemical treatment, and the preparation method is simple, green and pollution-free, and is suitable for large-scale production and efficient preparation of peony seed protein with high surface hydrophobicity of peony seeds.

[0035] (2) Compared with traditional alkaline extraction, acid precipitation and enzymatic extraction, the method of the present invention has high protein extraction efficiency (extraction rate 80.8% to 84.7%), and the surface hydrophobicity of the protein is improved by more than 136%. It has good solubility and is not easy to aggregate, which significantly improves foaming properties and foam stability.

[0036] (3) The peony seed protein prepared by the method provided by the present invention has strong surface hydrophobicity and good foaming properties, and has extremely strong processing characteristics, which greatly expands the application breadth of peony seed protein in the food industry, increases the added value of peony seed protein, and opens up a new way for the efficient utilization of peony seed meal. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the results of determining the extraction rates of the peony seed proteins prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0038] Figure 2 These are the surface hydrophobicity measurement results of the peony seed proteins prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0039] Figure 3 These are the endogenous fluorescence measurement results of the peony seed proteins prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3.

[0040] Figure 4 These are the foaming property and foam stability test results of the peony seed proteins prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3. DETAILED DESCRIPTION

[0041] The specific implementation of the present invention is further described below with reference to 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 any processes not specifically described 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.

[0042] According to an embodiment of the present invention, the steps for obtaining defatted peony seed meal are as follows:

[0043] The peony seeds are ultrafinely ground to obtain a component with a particle size of ≤355 μm, which is peony seed powder. The peony seed powder is defatted by a pressing method. The protein content of the peony seed powder is ≥20%; and the oil content of the defatted peony seed powder is ≤8%.

[0044] Comparative Example 1

[0045] 1) Peony Seed Protein Extraction

[0046] Defatted peony seed meal was mixed with distilled water at a mass-to-volume ratio of 1:30 (g / mL). The pH was adjusted to 5.0 with 1M HCl solution. Cellulase (0.1% (mg / g) of the defatted peony seed meal) was then added. Enzymatic hydrolysis was performed at 50°C with shaking for 1 hour. Lipase (0.1% (mg / g) of the defatted peony seed meal) was then added and extracted with shaking for 1 hour. The mixture was adjusted to pH 12.0 with 1M NaOH and extracted in a waterbath at 45°C for 1 hour. The supernatant was collected after centrifugation at 8000 rpm for 10 minutes and the pH was adjusted to 4.2 with 1M HCl. The precipitate was collected after centrifugation at 8000 rpm for 10 minutes. The precipitated protein was reconstituted in 50 mL of distilled water, and the pH was adjusted to 7.0 with 1M NaOH. The protein was then magnetically stirred for 1 hour. The peony seed protein was then dialyzed for desalination for 48 hours and collected.

[0047] 2) Determination of surface hydrophobicity of peony seed protein

[0048] The surface hydrophobicity (H0) of proteins was determined using the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS). The supernatant of the centrifuged sample solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with 10 mM phosphate buffer (pH 7.0). An 8 mmol / L ANS solution was prepared in 10 mM phosphate buffer (pH 7.0). 1 μL of ANS solution and 200 μL of each sample solution were added to a 96-well plate, mixed thoroughly, and allowed to stand in a dark environment for 30 minutes. The fluorescence intensity of each protein sample was measured using a BioTek multi-function microplate reader with an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 3 nm. A graph was plotted with fluorescence intensity against protein concentration. The initial slope of the curve was the surface hydrophobicity (H0) of the protein sample.

[0049] 3) Tertiary structure analysis of peony seed protein

[0050] The intrinsic fluorescence intensity of the peony seed protein prepared in Comparative Example 1 at a wavelength of 300-440 nm was analyzed to analyze the effects of different extraction methods on the tertiary structure of the peony seed protein. 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, with a measurement temperature of 25°C, a voltage of 550 V, an excitation wavelength of 280 nm, a scanning range of 300-440 nm, and a scanning speed of 240 nm / min. The slit width was fixed at 5 nm.

[0051] 4) Foaming and foam stability testing of peony seed protein

[0052] The foaming properties (FE) and foam stability (FS) of the peony seed protein prepared in Comparative Example 1 were determined as follows: 10 mL of a 0.5% (g / mL) protein solution was placed in a graduated test tube and homogenized at 10,000 rpm for 2 minutes. FE and FS were calculated using the following formula:

[0053] FE(%)=(V T -V0) / V0x 100

[0054] Among them, V T is the volume of the foam solution after homogenization, and V0 is the volume of the foam solution before homogenization.

[0055] FS(%)=(V t -V0) / V0x 100

[0056] Among them, V t is the volume of the foam solution after homogenization for 30 min, and V0 is the volume of the foam solution before homogenization.

[0057] Comparative Example 2

[0058] 1) Peony Seed Protein Extraction

[0059] According to the mass volume ratio g / mL, defatted peony seed meal was mixed with distilled water at a material-water ratio of 1:30, the pH was adjusted to 9.0 with 1M NaOH, shaken at 40°C for 2h, and centrifuged at 6000rpm for 10min to collect the supernatant. The precipitate was collected, and the pH was adjusted to 9.0 with 1M NaOH, shaken at 40°C for 2h, and centrifuged at 6000rpm for 10min to collect the supernatant. The supernatants were combined and the pH was adjusted to 4.2 with 1M HCl. Centrifuged at 6000rpm for 10min and the precipitate was collected. The precipitated protein was redissolved in 50mL distilled water, adjusted to pH 7.0 with 1M NaOH, and magnetically stirred for 1h. Desalted by dialysis for 48h, freeze-dried for 48h, and the peony seed protein was collected.

[0060] 2) Determination of surface hydrophobicity of peony seed protein

[0061] The surface hydrophobicity (H0) of proteins was determined using the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS). The supernatant of the centrifuged sample solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with 10 mM phosphate buffer (pH 7.0). An 8 mmol / L ANS solution was prepared in 10 mM phosphate buffer (pH 7.0). 1 μL of ANS solution and 200 μL of each sample solution were added to a 96-well plate, mixed thoroughly, and allowed to stand in a dark environment for 30 minutes. The fluorescence intensity of each protein sample was measured using a BioTek multi-function microplate reader with an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 3 nm. A graph was plotted with fluorescence intensity against protein concentration. The initial slope of the curve was the surface hydrophobicity (H0) of the protein sample.

[0062] 3) Tertiary structure analysis of peony seed protein

[0063] The intrinsic fluorescence intensity of the peony seed protein prepared in Comparative Example 2 at a wavelength of 300-440 nm was analyzed to analyze the effects of different extraction methods on the tertiary structure of the peony seed protein. 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, with a measurement temperature of 25°C, a voltage of 550 V, an excitation wavelength of 280 nm, a scanning range of 300-440 nm, and a scanning speed of 240 nm / min. The slit width was fixed at 5 nm.

[0064] 4) Foaming and foam stability testing of peony seed protein

[0065] The foaming properties (FE) and foam stability (FS) of the peony seed protein prepared in Comparative Example 2 were determined as follows: 10 mL of a 0.5% (g / mL) protein solution was placed in a graduated test tube and homogenized at 10,000 rpm for 2 minutes. FE and FS were calculated using the following formula:

[0066] FE(%)=(V T -V0) / V0x 100

[0067] Among them, V T is the volume of the foam solution after homogenization, and V0 is the volume of the foam solution before homogenization.

[0068] FS(%)=(V t -V0) / V0x 100

[0069] Among them, V t is the volume of the foam solution after homogenization for 30 min, and V0 is the volume of the foam solution before homogenization.

[0070] Comparative Example 3

[0071] 1) Peony Seed Protein Extraction

[0072] According to the mass volume ratio g / mL, defatted peony seed meal was mixed with distilled water at a material-water ratio of 1:30, the pH was adjusted to 12.0 with 1M NaOH, shaken at 40°C for 2h, centrifuged at 6000rpm for 10min, and the supernatant was collected. The precipitate was collected, and the pH was adjusted to 12.0 with 1M NaOH, shaken at 40°C for 2h, centrifuged at 6000rpm for 10min, and the supernatant was collected. The supernatants were combined and the pH was adjusted to 4.2 with 1M HCl. Centrifuged at 6000rpm for 10min and the precipitate was collected. The precipitated protein was re-dissolved in 50mL of distilled water, and the pH was adjusted to 7.0 with 1M NaOH. The mixture was magnetically stirred for 1h. The peony seed protein was dialyzed for desalination for 48h, freeze-dried for 48h, and collected.

[0073] 2) Determination of surface hydrophobicity of peony seed protein

[0074] The surface hydrophobicity (H0) of proteins was determined using the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS). The supernatant of the centrifuged sample solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with 10 mM phosphate buffer (pH 7.0). An 8 mmol / L ANS solution was prepared in 10 mM phosphate buffer (pH 7.0). 1 μL of ANS solution and 200 μL of each sample solution were added to a 96-well plate, mixed thoroughly, and allowed to stand in a dark environment for 30 minutes. The fluorescence intensity of each protein sample was measured using a BioTek multi-function microplate reader with an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 3 nm. A graph was plotted with fluorescence intensity against protein concentration. The initial slope of the curve was the surface hydrophobicity (H0) of the protein sample.

[0075] 3) Tertiary structure analysis of peony seed protein

[0076] The intrinsic fluorescence intensity of the peony seed protein prepared in 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 peony seed protein. 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, with a measurement temperature of 25°C, a voltage of 550 V, an excitation wavelength of 280 nm, a scanning range of 300-440 nm, and a scanning speed of 240 nm / min. The slit width was fixed at 5 nm.

[0077] 4) Foaming and foam stability testing of peony seed protein

[0078] The foaming properties (FE) and foam stability (FS) of the peony seed protein prepared in Comparative Example 3 were determined as follows: 10 mL of a 0.5% (g / mL) protein solution was placed in a graduated test tube and homogenized at 10,000 rpm for 2 minutes. FE and FS were calculated using the following formula:

[0079] FE(%)=(V T -V0) / V0x 100

[0080] Among them, V T is the volume of the foam solution after homogenization, and V0 is the volume of the foam solution before homogenization.

[0081] FS(%)=(V t -V0) / V0x 100

[0082] Among them, V t is the volume of the foam solution after homogenization for 30 min, and V0 is the volume of the foam solution before homogenization.

[0083] 1) Peony Seed Protein Extraction

[0084] Example 1

[0085] Defatted peony seed meal was mixed with distilled water at a material-water ratio of 1:30 (g / mL), homogenized at 10,000 rpm for 3 min, and then adjusted to pH 12.0 with 1 M NaOH. The mixture was extracted with ultrasound (40 kHz) at 45 °C for 2 h, and the supernatant was collected after centrifugation at 6,000 × g for 10 min.

[0086] The following steps were repeated three times: the precipitate was collected, the pH was adjusted to 12.0 with 1 M NaOH, shaken at 45 °C for 2 h, centrifuged at 6000 × g for 10 min, and the supernatant was collected and combined;

[0087] Repeat the following steps three times: adjust the pH to 4.2, acid-precipitate the protein for 8 h, collect the precipitated protein in 50 mL of distilled water, adjust the pH to 7.0 with 1 M NaOH, and re-dissolve under magnetic stirring for 1 h;

[0088] The reconstituted protein solution was dialyzed with a 3000Da dialysis bag for 48 hours and then freeze-dried for 48 hours to obtain the target protein.

[0089] Example 2

[0090] Defatted peony seed meal was mixed with distilled water at a material-water ratio of 1:20 (g / mL), homogenized at 20,000 rpm for 2 min, and then adjusted to pH 10.0 with 1 M NaOH. The mixture was extracted by ultrasonication (45 kHz) at 30 °C for 1 h, and the supernatant was collected after centrifugation at 4,000 × g for 30 min.

[0091] The following steps were repeated 4 times: the precipitate was collected, the pH was adjusted to 10.0 using 1 M NaOH, ultrasonic extraction (45 kHz) was performed at 30° C. for 1 h, and the supernatant was collected after centrifugation at 4000 × g for 30 min. The supernatants were combined.

[0092] Repeat the following steps twice: adjust the pH to 3.9, acid-precipitate the protein for 10 h, then re-dissolve the precipitated protein in 50 mL of distilled water, adjust the pH to 7.1 with 1 M NaOH, and magnetically stir for 1 h.

[0093] The reconstituted protein solution was dialyzed with a 2000Da dialysis bag for 48 hours and then freeze-dried for 48 hours to obtain the target protein.

[0094] Example 3

[0095] Defatted peony seed meal was mixed with distilled water at a material-water ratio of 1:10 (g / mL), homogenized at 8000 rpm for 10 min, and then adjusted to pH 9.0 with 1 M NaOH. The mixture was extracted with ultrasound (50 kHz) at 50°C for 0.5 h, and centrifuged at 5000 × g for 20 min, after which the supernatant was collected.

[0096] The following steps were repeated 5 times: the precipitate was collected, the pH was adjusted to 9.0 using 1 M NaOH, ultrasonic extraction (50 kHz) was performed at 50° C. for 3 h, and the supernatant was collected after centrifugation at 5000 × g for 20 min. The supernatants were combined.

[0097] Repeat the following steps 4 times: adjust the pH to 4.5, acid precipitate the protein for 12 h, add 1 M NaOH to 50 mL of distilled water to adjust the pH to 7.2, and magnetically stir for 1 h to redissolve the precipitated protein;

[0098] The reconstituted protein solution was dialyzed with a 1000Da dialysis bag for 48 hours and then freeze-dried for 48 hours to obtain the target protein.

[0099] 2) Determination of surface hydrophobicity of peony seed protein

[0100] The surface hydrophobicity (H0) of proteins was determined using the fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS). The supernatant of the centrifuged sample solution was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with 10 mM phosphate buffer (pH 7.0). An 8 mmol / L ANS solution was prepared in 10 mM phosphate buffer (pH 7.0). 1 μL of ANS solution and 200 μL of each sample solution were added to a 96-well plate, mixed thoroughly, and allowed to stand in a dark environment for 30 minutes. The fluorescence intensity of each protein sample was measured using a BioTek multi-function microplate reader with an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 3 nm. A graph was plotted with fluorescence intensity against protein concentration. The initial slope of the curve was the surface hydrophobicity (H0) of the protein sample.

[0101] 3) Tertiary structure analysis of peony seed protein

[0102] The endogenous fluorescence intensity of the peony seed protein prepared in Example 1, Example 2, and Example 3 at a wavelength of 300-440 nm was analyzed to analyze the effect of different extraction methods on the tertiary structure of the peony seed protein. 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, with a measurement temperature of 25°C, a voltage of 550 V, an excitation wavelength of 280 nm, a scanning range of 300-440 nm, and a scanning speed of 240 nm / min. The slit width was fixed at 5 nm.

[0103] 4) Foaming and foam stability testing of peony seed protein

[0104] The foaming properties (FE) and foam stability (FS) of the peony seed proteins prepared in Examples 1, 2, and 3 were determined as follows: 10 mL of a 0.5% (g / mL) protein solution was placed in a graduated test tube and homogenized at 10,000 rpm for 2 minutes. FE and FS were calculated using the following formula:

[0105] FE(%)=(V T -V0) / V0x 100

[0106] Among them, V T is the volume of the foam solution after homogenization, and V0 is the volume of the foam solution before homogenization.

[0107] FS(%)=(V t -V0) / V0x 100

[0108] Among them, V t is the volume of the foam solution after homogenization for 30 min, and V0 is the volume of the foam solution before homogenization.

[0109] Experimental results

[0110] Figure 1 The results of the determination of the extraction rate of peony seed protein prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, and Comparative Example 3 are shown. Protein extraction rate and purity are decisive indicators for evaluating the effectiveness of protein extraction methods: the extraction rate directly reflects the efficiency and economy of raw material utilization, and determines the feasibility of industrialization. The extraction rates of Example 1, Example 2, and Example 3 are 84.7%, 82.4%, and 80.8%, respectively. Except for Comparative Example 2, whose extraction rate is significantly lower than that of the other three groups, the protein extraction rates of the other groups remain at a high level. The extraction rate of Example 1 is increased by 53.4% ​​relative to that of Comparative Example 2. The reason for this phenomenon may be that the ultrasonic cavitation effect enhances the penetration of alkali solution into peony seed cells, promotes the dissolution of peony seed protein, and thus increases the extraction rate.

[0111] Figure 2The surface hydrophobicity measurement results of the peony seed protein prepared in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3. Surface hydrophobicity is generally used to indicate the extent to which hydrophobic amino acids buried in protein molecules are exposed to the protein surface through protein unfolding and denaturation, and can be used to characterize changes in protein conformation. Surface hydrophobicity directly determines the interfacial adsorption capacity of the protein. High hydrophobicity significantly improves foaming and emulsifying properties, thereby expanding its application in food processing (such as plant-based foams, emulsions) and drug delivery. The surface hydrophobicity indexes of Example 1, Example 2, and Example 3 are 23812, 21941, and 20878, respectively. Relative to the peony seed protein obtained in Comparative Example 1, Comparative Example 2, and Comparative Example 3, the surface hydrophobicity index improvement rate of the peony seed protein obtained in Example 1, Example 2, and Example 3 ranges from 44% to 131%. The reason for this phenomenon may be that the microjets generated by the ultrasonic cavitation effect instantly destroy the hydrophobic core barrier of the protein's tertiary structure, and the high electrostatic repulsion of the strong alkaline environment of pH 12 is simultaneously superimposed, prompting the protein to completely unfold from the globular conformation; the two synergistically expose deeply buried tryptophan / phenylalanine hydrophobic residues and avoid the incomplete unfolding of traditional alkaline extraction or the random cutting defects of enzymatic hydrolysis, thereby maximizing the exposure of the hydrophobic area and thus significantly improving its interfacial functional activity.

[0112] Figure 3 The endogenous fluorescence spectra of peony seed proteins prepared by the inventive method and the traditional method in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3 are shown. The hydrophobic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) all have high fluorescence quantum yields, wherein the Trp residue is located at the outermost side of the protein. By monitoring the fluorescence intensity and maximum emission wavelength (λmax) of the Trp residue, the changes in the tertiary structure of the protein can be characterized: the red shift of λmax indicates that the polarity of the Trp microenvironment is enhanced (the residue is solvated and exposed), and the fluorescence enhancement reflects that the hydrophobic amino acid is embedded in the protein; otherwise, it indicates that it is close to the surface. The high surface hydrophobic peony seed protein obtained in the embodiment of the present invention shows the strongest fluorescence intensity, confirming that the hydrophobic amino acid is deeply buried in the hydrophobic core, but the red shift of λmax indicates that the polarity of the Trp microenvironment is enhanced, which indicates that the tertiary structure of the protein may be in a semi-folded state, while moderately exposing the hydrophobic residues to enhance surface activity, while maintaining fluorescence efficiency through the dynamic hydrophobic core.

[0113] Figure 4The foaming properties and foam stability test results of the high surface hydrophobic peony seed protein prepared in Example 1, Example 2, Example 3 and Comparative Examples 1, Comparative Example 2, and Comparative Example 3 are shown. Protein foaming property is a key functional characteristic in food processing, which is related to texture, taste and stability. In comparison, the peony seed protein obtained by the method of Example 1 has better foaming property. The protein obtained by Example 1 has a foaming property of up to 73% and a stability of about 31% at 0.5% (g / mL). The foaming property of Example 2 is 71% and the stability is about 30%. The foaming property of Example 3 is 70% and the stability is about 33%. The foaming property of the protein obtained in Comparative Examples 1, 2, and 3 is between 5% and 36%, and the stability is below 18%. Compared with Comparative Examples 1, 2, and 3, the foaming property and stability of the high surface hydrophobic peony seed protein prepared in Examples 1, 2, and 3 are improved by up to 100%. The foaming property of the protein mainly depends on the structure and properties of the protein. The peony seed protein obtained in Example 1, Example 2, and Example 3 has higher foaming properties, which may be attributed to its high surface hydrophobicity, which enables the protein to be quickly adsorbed to the air-liquid interface, significantly reducing the surface tension to improve the foaming efficiency; at the same time, the hydrophobic group forms a strong interfacial film through intermolecular interaction, enhancing the foam stability.

[0114] In summary, the present invention utilizes ultrasound-assisted alkaline extraction to prepare peony seed protein, combining the advantages of a simple process, low cost, and efficient extraction. The resulting protein maintains its native conformational integrity, exhibits significantly enhanced surface hydrophobicity, and possesses excellent foaming ability. This significantly expands the application of peony seed protein in the food industry and provides innovative raw material support for the development of high-value-added plant protein products.

[0115] The above embodiments are preferred implementations of the present invention, but the implementations 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing peony seed protein with high surface hydrophobicity, characterized in that: The following steps are involved: (1) Defatting: The ultrafinely crushed peony seed powder is defatted by pressing; (2) Pretreatment: Mix the defatted peony seed powder with distilled water and homogenize the mixture using a homogenizer; (3) Extraction: extract the protein from the homogenized mixture obtained in step (2) by superimposing an ultrasonic physical field under a low-temperature alkaline environment, and collect the supernatant after extraction; (4) Re-extraction: collecting the precipitate after extraction, extracting the protein under a low-temperature alkaline environment with an ultrasonic physical field, collecting the supernatant after extraction, and combining the supernatants obtained in step (3) and step (4); (5) Acid precipitation and re-dissolution: Adjust the pH to the isoelectric point of the protein and precipitate the protein with acid for 8-12 hours. Collect the precipitated protein and adjust the pH to 7.0-7.2 for re-dissolution. (6) Desalting: The protein solution obtained in step (5) is dialyzed for desalination and vacuum freeze-dried to obtain peony seed protein with high surface hydrophobicity.

2. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The step (4) is repeated 3 to 5 times; the step (5) is repeated 2 to 4 times.

3. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The peony seed powder of step (1) is crushed to obtain a component with a particle size of ≤355 μm; the protein content of the peony seed powder is ≥20%; and the oil content of the peony seed powder after defatting is ≤8%.

4. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The homogenization speed in step (2) is 8000-20000 rpm, and the time is 2-10 min; the material-liquid ratio of the peony seed powder to the distilled water is 1:10-30 in terms of mass-to-volume ratio g / mL.

5. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The low-temperature environment in step (3) and step (4) is 30-50° C.; the alkaline environment is at a pH of 9-12; the frequency of the ultrasonic physical field is 40-50 kHz, and the extraction time is 0.5-2 h; and the supernatant is the upper clear liquid obtained after centrifugation at 4000-6000×g for 10-30 min.

6. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The isoelectric point of the protein in step (5) is pH = 3.9 to 4.

5.

7. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The desalination in step (6) refers to the removal of small molecular substances using membrane permeation or ultrafiltration methods, and the upper limit of the removal molecular weight is 1000 to 3000 Da.

8. The method for preparing a peony seed protein with high surface hydrophobicity according to claim 1, characterized in that: The extraction rate is 80.8% to 84.7%, and the surface hydrophobicity index H0 is above 20878.

9. Use of a peony seed protein with high surface hydrophobicity prepared by the preparation method according to any one of claims 1 to 8 in preparing food.

10. The use of a peony seed protein with high surface hydrophobicity in food preparation according to claim 9, characterized in that: The protein has high surface hydrophobicity during food processing, a foaming property greater than or equal to 70%, and a foam stability greater than or equal to 30%.

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