Flavor-improved pleurotus eryngii protein and preparation method thereof

Abstract

The invention discloses flavor-improved pleurotus eryngii protein and a preparation method thereof, and belongs to the technical field of food processing. The method comprises the following steps: (1) crushing and sieving dried pleurotus eryngii to obtain pleurotus eryngii powder; (2) carrying out degreasing treatment on the pleurotus eryngii powder by using normal hexane so as to remove fat-derived peculiar smell precursor substances; (3) mixing the degreased pleurotus eryngii powder with water, extracting protein under an alkaline condition, and centrifuging to obtain supernate; (4) adjusting the pH value of the supernate to be neutral, adding glutaminase (PG) to carry out an enzymolysis reaction, and modifying a protein structure through a deamidation effect; and (5) inactivating the enzymatic hydrolysate, adjusting to an isoelectric point to precipitate protein, washing, neutralizing and drying to obtain a final product. The method has the remarkable advantages that a deamidase modification process is innovatively integrated into a protein extraction link, and structural modification, flavor improvement and extraction and purification are integrally completed through the synergistic effect of alkali extraction, acid precipitation and enzymolysis. The method effectively overcomes the defects that the inherent peculiar smell of the traditional pleurotus eryngii protein is obvious, and the processing performance (such as water solubility) is poor, the obtained product is pure in flavor, the functional characteristics are remarkably improved, and a new way is opened up for high-value application of the pleurotus eryngii protein in various healthy foods.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for deep processing of edible fungal protein, particularly a method that combines alkaline extraction, acid precipitation, and enzyme treatment to improve the physicochemical properties and flavor characteristics of king oyster mushroom protein. Background Technology

[0002] The resource pressures brought about by rapid global population growth, the limitations of traditional plant protein sources, and the market's continued pursuit of nutritious and healthy diets have jointly driven the urgent demand for novel alternative protein resources. Against this backdrop, microbial-derived proteins, especially fungal proteins, are increasingly becoming a focus of research and industrialization as a sustainable and nutrient-rich emerging option. Among them, edible fungal proteins, due to their unique nutritional value and environmentally friendly characteristics, demonstrate enormous development potential.

[0003] King oyster mushrooms (Pleurotus eryngii), as a representative edible fungus, are not only nutritionally complete and have a unique texture, but are also rich in protein, dietary fiber, various vitamins, and minerals. The essential amino acid composition of its protein is balanced, superior to most plant proteins, making it a high-quality plant protein source. In the context of increasingly popular healthy eating and sustainable development concepts, king oyster mushroom protein, with its low-fat, low-calorie, and easily digestible characteristics, has the potential to replace traditional animal protein. Furthermore, this protein exhibits excellent emulsifying properties, water-holding capacity, and thermal stability in food processing, further enhancing its feasibility in practical applications.

[0004] However, the practical application of king oyster mushroom protein still faces significant technical bottlenecks, particularly its inherent unpleasant flavor (such as the typical "mushroom taste"), which severely restricts consumer acceptance and its promotion in high-quality foods. Currently, methods for improving protein flavor mainly include chemical, physical, and biological methods. Chemical methods, such as organic solvent defatting and pH adjustment, have some effect, but they carry the risk of solvent residue or are inefficient. Physical methods, such as heat treatment or flavor masking, easily lead to the destruction of heat-sensitive components or only achieve flavor coverage rather than fundamental removal. Enzymatic treatment technology in biological methods is considered a more promising solution due to its mild reaction conditions, high specificity, and alignment with clean label trends.

[0005] Therefore, in view of the above-mentioned defects in the existing technology, there is an urgent need in the field to develop a green and efficient method that can simultaneously improve flavor during the protein extraction stage, so as to overcome the flavor limitations of king oyster mushroom protein in food applications and promote its further development and application in high value-added products. Summary of the Invention

[0006] This invention addresses the limitation of the application of king oyster mushroom protein due to its inherent unpleasant flavor, and discloses a green and efficient method for simultaneously improving flavor during protein extraction. This method aims to effectively remove off-flavors, enhance flavor quality, and simplify the production process through integrated process steps, thereby laying the foundation for the high-value application of king oyster mushroom protein in high-quality foods.

[0007] A method for improving the flavor of king oyster mushroom protein, characterized by comprising the following steps:

[0008] (1) Raw material pretreatment: Dried king oyster mushrooms are crushed and sieved to obtain king oyster mushroom powder;

[0009] (2) Degreasing treatment: The king oyster mushroom powder is degreased using an organic solvent and then dried to obtain degreased king oyster mushroom powder;

[0010] (3) Alkali extraction: The defatted king oyster mushroom powder is mixed with water for hydration, the pH of the slurry is adjusted to 8.0-10.0 with alkali solution, the mixture is stirred and extracted, then centrifuged and the supernatant is collected;

[0011] (4) Enzymatic modification: Adjust the pH of the supernatant to 6.0-8.0, then add PG, and carry out the enzymatic hydrolysis reaction at 40-50℃ for 0-24h. After the reaction is completed, perform heat inactivation treatment.

[0012] (5) Isoelectric point precipitation: Adjust the pH of the enzyme-inactivated system to the isoelectric point of Pleurotus eryngii protein with acid solution, let it stand to allow the protein to precipitate fully, and then centrifuge to collect the protein precipitate.

[0013] (6) Post-processing: After washing the protein precipitate, it is redissolved and the pH is adjusted to neutral. Finally, it is dried to obtain the flavor-improved king oyster mushroom protein.

[0014] Preferably, in step (1), the sieving is done through an 80-mesh sieve.

[0015] Preferably, in step (2), the organic solvent is n-hexane, the ratio of king oyster mushroom powder to n-hexane is 1:3 (g / mL), and the degreasing treatment is repeated 2-3 times.

[0016] Preferably, in step (3), the ratio of defatted king oyster mushroom powder to water is 1:10 (g / mL); the alkaline solution is a 2M NaOH solution, and the pH is adjusted to 9.0; the stirring extraction time is 2-4 hours.

[0017] Preferably, in step (4), the pH of the supernatant is adjusted to 7.0 using 2M NaOH solution; the amount of PG added is 0.2% of the protein substrate mass (i.e., 1:500); the temperature of the enzymatic hydrolysis reaction is 45°C; and the heat inactivation treatment is maintained at 80°C for 5-10 minutes.

[0018] Preferably, the enzyme activity of PG in step (4) is 100 U / g.

[0019] Preferably, in step (5), the acid solution is a 2M HCl solution, the pH is adjusted to 4.0, and the standing time is 2h.

[0020] Preferably, in step (6), the drying is spray drying.

[0021] This invention has the following advantages:

[0022] The core advantage of this invention lies in its innovative integration of flavor enhancement into the protein extraction process, creating a highly efficient and synergistic green processing technology. This method effectively removes precursors of fat-soluble off-flavor substances through organic solvent defatting pretreatment. Subsequently, during alkaline protein extraction, PG is introduced for specific catalysis, significantly degrading aldehydes and alcohols that cause unpleasant flavors. Furthermore, enzymatic modification alters the protein conformation, reducing the release of volatile off-flavor compounds, thus achieving a fundamental improvement in flavor. This integrated process avoids the additional steps and costs associated with traditional post-modification methods, simplifies the production process, and utilizes a predominantly aqueous reaction, ensuring mild, safe, and residue-free conditions. The resulting king oyster mushroom protein product not only boasts a pure flavor and significantly reduced off-flavors, but its solubility and other functional properties are also simultaneously optimized, providing solid technical support for expanding its application in high-quality health foods. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a graph showing the degree of deamidation of Pleurotus eryngii protein under different enzymatic hydrolysis conditions provided in Test Example 1 of the present invention;

[0025] Figure 2 Solubility diagram of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of the present invention;

[0026] Figure 3 This is a diagram showing the surface hydrophobicity of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of the present invention;

[0027] Figure 4 The potential (A) and particle size (B) of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of the present invention are shown.

[0028] Figure 5 Fourier transform infrared spectra (A) and secondary structures (B) of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of the present invention;

[0029] Figure 6 Fluorescence spectra of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of this invention;

[0030] Figure 7 The electronic nose diagram (A) and correlation diagram (B) of Pleurotus eryngii protein at different enzymatic hydrolysis times provided in Test Example 1 of the present invention are shown.

[0031] Figure 8 This is a graph showing the changes in the content of different flavor compounds in Pleurotus eryngii protein under different enzymatic hydrolysis times provided in Test Example 1 of the present invention;

[0032] Figure 9 This is a graph showing the binding rate of Pleurotus eryngii protein to dodecylaldehyde at different enzymatic hydrolysis times provided in Test Example 1 of this invention;

[0033] Figure 10 The corrected Stern-Volmer curves show the interaction between Pleurotus eryngii protein and dodecyl aldehyde at different enzymatic hydrolysis times provided in Test Example 1 of this invention. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0035] Protein-PG (EC 3.5.1.44, PG) specifically catalyzes the deamidation of glutamine (Gln) residues in proteins or peptides, converting them into negatively charged glutamate (Glu) residues. This reaction does not trigger peptide bond cleavage and effectively increases the net negative charge on the protein molecule's surface, thereby regulating its electrostatic interactions. This process further improves the physicochemical properties of proteins, such as significantly increasing solubility and reducing particle size; simultaneously, changes in charge and conformation also affect the protein's ability to bind to undesirable volatile flavor compounds. Therefore, PG-based deamidation can serve as an effective strategy for improving protein flavor quality.

[0036] (1) Raw material pretreatment: Dried king oyster mushrooms are crushed and sieved to obtain king oyster mushroom powder;

[0037] (2) Degreasing treatment: The king oyster mushroom powder is degreased using an organic solvent and then dried to obtain degreased king oyster mushroom powder;

[0038] (3) Alkali extraction: The defatted king oyster mushroom powder is mixed with water for hydration, the pH of the slurry is adjusted to 8.0-10.0 with alkali solution, the mixture is stirred and extracted, then centrifuged and the supernatant is collected;

[0039] (4) Enzymatic modification: Adjust the pH of the supernatant to 6.0-8.0, then add PG, and carry out the enzymatic hydrolysis reaction at 40-50℃ for 0-24h. After the reaction is completed, perform heat inactivation treatment.

[0040] (5) Isoelectric point precipitation: Adjust the pH of the enzyme-inactivated system to the isoelectric point of Pleurotus eryngii protein with acid solution, let it stand to allow the protein to precipitate fully, and then centrifuge to collect the protein precipitate.

[0041] (6) Post-processing: After washing the protein precipitate, it is redissolved and the pH is adjusted to neutral. Finally, it is dried to obtain the flavor-improved king oyster mushroom protein.

[0042] The method for improving the flavor of Pleurotus eryngii protein proposed in this invention involves simultaneous deamidation modification of Pleurotus eryngii protein during the extraction process using PG. In this reaction, the Pleurotus eryngii protein, a natural protein obtained through alkaline extraction, has limited original flavor-binding sites on its molecular surface, resulting in insufficient binding capacity for undesirable flavor compounds and a higher proportion of free off-flavor components. The addition of PG, through specific deamidation, exposes more active sites on the protein molecules and alters their surface charge distribution and spatial conformation, thereby significantly enhancing the binding capacity between the protein and flavor compounds. The modified Pleurotus eryngii protein can more effectively bind and immobilize undesirable volatile compounds in the system, reducing their release during processing and consumption, ultimately achieving a significant improvement in product flavor. This protein shows good application potential in plant-based foods, high-protein beverages, and other foods with high flavor requirements.

[0043] In one embodiment of the present invention, in step (1), king oyster mushroom powder is obtained by crushing and passing through an 80-mesh sieve.

[0044] In one embodiment of the present invention, in step (2), the ratio of the king oyster mushroom powder to n-hexane is 1:3.

[0045] In one embodiment of the present invention, in step (3), the ratio of defatted king oyster mushroom powder to deionized water is 1:10 (g / mL).

[0046] In one embodiment of the present invention, in step (3), the stirring is specifically mechanical stirring with a rotation speed of 500 rpm and a stirring time of 1 hour.

[0047] In one embodiment of the present invention, in step (3), the pH is adjusted to 9.0 using an alkaline solution.

[0048] In one embodiment of the present invention, in step (3), the alkaline solution is a 2M NaOH solution.

[0049] In one embodiment of the present invention, in step (3), the stirring speed is 300 rpm and the extraction time is 2-4 h.

[0050] In one embodiment of the present invention, in step (4), PG is used to simultaneously modify the protein of king oyster mushroom, which helps to change the protein conformation and expose more flavor binding sites.

[0051] In one embodiment of the present invention, in step (4), the enzyme activity of the PG is 100 U / g.

[0052] In one embodiment of the present invention, in step (4), the amount of PG added is 1:500 (i.e. 0.2%) of the protein substrate mass.

[0053] In one embodiment of the present invention, in step (4), the enzymatic hydrolysis reaction is carried out by isothermal oscillation.

[0054] In one embodiment of the present invention, in step (4), the enzymatic hydrolysis reaction is carried out at 45°C and the oscillation speed is 100 rpm.

[0055] In one embodiment of the present invention, in step (4), the enzymatic hydrolysis reaction takes 0-24 hours.

[0056] In one embodiment of the present invention, in step (5), the pH is adjusted to the isoelectric point using acid to precipitate the protein.

[0057] In one embodiment of the present invention, in step (5), the acid solution is a 2M HCl solution.

[0058] In one embodiment of the present invention, in step (5), the pH of the isoelectric point precipitate is 3.5-4.0.

[0059] In one embodiment of the present invention, the settling time in step (5) is 2 hours.

[0060] In one embodiment of the present invention, in step (6), the product is dried by spray drying.

[0061] In one embodiment of the present invention, in step (6), the inlet temperature of the spray dryer is 180°C and the outlet temperature is 80°C.

[0062] The present invention will now be described in detail with reference to the embodiments and the accompanying drawings.

[0063] Example 1 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0064] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0065] (2) Slowly add 2M NaOH solution dropwise while stirring continuously to precisely adjust the pH of the mixed solution to 9.0, and continue stirring for 2 hours for extraction;

[0066] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0067] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0068] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 4 hours;

[0069] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0070] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0071] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0072] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder.

[0073] Example 2 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0074] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0075] (2) Slowly add 2M NaOH solution dropwise while stirring continuously to precisely adjust the pH of the mixed solution to 9.0, and continue stirring for 2 hours for extraction;

[0076] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0077] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0078] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 8 hours;

[0079] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0080] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0081] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0082] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder.

[0083] Example 3 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0084] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0085] (2) Slowly add 2M NaOH solution dropwise while stirring continuously to precisely adjust the pH of the mixed solution to 9.0, and continue stirring for 2 hours for extraction;

[0086] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0087] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0088] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 12 h;

[0089] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0090] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0091] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0092] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder. Example 4 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0093] Example 4 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0094] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0095] (2) Slowly add 2M NaOH solution while stirring continuously to precisely adjust the pH of the mixed slurry to 9.0, and continue stirring for 3 hours for extraction;

[0096] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0097] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0098] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 16 h;

[0099] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0100] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 4.0, and let stand for 2 hours;

[0101] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0102] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder.

[0103] Example 5 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0104] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0105] (2) Slowly add 2M NaOH solution dropwise while stirring continuously to precisely adjust the pH of the mixed solution to 9.0, and continue stirring for 2 hours for extraction;

[0106] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0107] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0108] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 20 h;

[0109] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0110] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0111] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0112] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder.

[0113] Example 6 The extraction of protein from king oyster mushrooms and the simultaneous enzymatic flavor improvement include the following steps:

[0114] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0115] (2) Slowly add 2M NaOH solution dropwise while stirring continuously to precisely adjust the pH of the mixed solution to 9.0, and continue stirring for 2 hours for extraction;

[0116] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0117] (4) Under magnetic stirring, the pH of the crude protein extract was precisely adjusted to 7.0 with 2M NaOH solution, and PG (100U / g enzyme activity) was added at a ratio of 1:500 of protein substrate mass.

[0118] (5) Transfer the reaction system to a 45℃ constant temperature water bath shaker and carry out the enzymatic hydrolysis reaction at 100 rpm for 24 h;

[0119] (6) Heat the reaction solution in an 80°C water bath for 10 minutes to completely inactivate the enzyme;

[0120] (7) Cool the enzyme-inactivated reaction solution to room temperature, slowly add 2M HCl solution dropwise with stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0121] (8) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0122] (9) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain flavor-improved king oyster mushroom protein powder.

[0123] Comparative Example 1 Traditional alkaline extraction and acid precipitation method (without enzymatic hydrolysis)

[0124] (1) Mix 10g of defatted king oyster mushroom powder with 100mL of deionized water and stir with a mechanical stirrer at 500rpm for 1h to fully hydrate it.

[0125] (2) Add 2M NaOH solution slowly with continuous stirring to precisely adjust the pH of the mixed solution to 9.0. Place the system in a 45℃ constant temperature water bath and continue stirring for 3 hours to extract.

[0126] (3) After extraction, centrifuge at 4℃ and 8000rpm for 20min and collect the supernatant;

[0127] (4) Slowly add 2M HCl solution dropwise while stirring to adjust the pH to 3.9, and let stand for 2 hours;

[0128] (5) Centrifuge at 4℃ and 8000rpm for 15min, collect the protein precipitate, and wash it three times with deionized water.

[0129] (6) The protein precipitate was redispersed, the pH was adjusted to 7.0 with 2M NaOH solution, and dried using a spray dryer to obtain king oyster mushroom protein powder.

[0130] Test Example 1

[0131] 1. Analysis of the degree of deamidation of Pleurotus eryngii protein

[0132] Reagent A (phenol-sodium nitroprusside solution) is 2.50g phenol and 0.012g sodium nitroprusside dissolved in deionized water and diluted to 250mL. Reagent B (alkaline hypochlorite solution) is 1.25g NaOH dissolved in deionized water, 2.1mL sodium hypochlorite added, and diluted to 250mL. 10μL of the Pleurotus ostreatus protein (denoted as PEP) prepared in Examples 1-6 was added to 2.5mL of Reagent A and 2.5mL of Reagent B, and vortexed to mix. The mixture was reacted at 37℃ for 20min, and the absorbance was measured at 625nm. The sample (0.5g) was dissolved in 10mL HCl (3mol / L) and heated at 110℃ for 3h to determine the ammonia produced by complete deamidation. The degree of deamidation was calculated according to the following formula (1):

[0133]

[0134] Appendix Figure 1 The results showed that the degree of deamidation of Pleurotus eryngii protein continuously increased during the 0-16 h hydrolysis period, after which the increase slowed down and plateaued. Meanwhile, we found that the degree of deamidation in untreated Pleurotus eryngii protein did not change significantly over time. The slower deamidation in the later stages was due to the reduced number of glutamine residues in the protein, and the fact that the removed NH3 in solution could act as a competitive inhibitor, reducing the reactivity of glutamine residues. Given that the degree of deamidation did not change significantly between 16 and 24 h, subsequent experiments in this study will be conducted with a reaction time of 0-20 h.

[0135] 2. Solubility analysis of Pleurotus eryngii protein

[0136] Take the Pepta oyster mushroom protein (denoted as PEP) obtained in Examples 1-5 respectively and use it in phosphate buffer (0.2 mol / L). -1 Dilute to 10 mg / mL (pH 7.0), vortex for 30 min, centrifuge (1000 g, 30 min, 4 °C), collect the supernatant, and determine the protein content in the supernatant using a BCA protein assay kit (Beijing Sora Biotechnology Co., Ltd., Beijing, China), with bovine serum albumin as the standard. Protein solubility is expressed as the percentage of protein in the collected supernatant relative to the total protein content.

[0137] Appendix Figure 2 The results showed that the solubility of Pleurotus eryngii protein increased proportionally with PG treatment time. This phenomenon was mainly attributed to deamidation inducing the conversion of the amide group of the protein to a carboxyl group, increasing the charge, promoting protein unfolding, and enhancing the interaction between the protein and water, thereby improving solubility. Furthermore, deamidation can reduce intermolecular hydrogen bonds, thereby increasing molecular flexibility and promoting protein hydration.

[0138] 3. Analysis of the surface hydrophobicity (H0) of Pleurotus ostreatus protein

[0139] Using 8-aniline-1-naphthalenesulfonic acid (ANS) as a fluorescent probe, PEP protein (prepared in Examples 1-5) was diluted with deionized water to a concentration of 0.05-0.25 mg / mL. 1 mL of the diluted sample solution was mixed with 5 μL of ANS and reacted in the dark for 20 min. The emission wavelength range was 400-600 nm, the excitation wavelength was 390 nm, the slit width was 2.5 nm, and the voltage was 500 V. The scan rate was set to 1200 nm / min. Fluorescence intensity and protein concentration were fitted using linear regression. The slope of the curve represents the surface hydrophobicity of the sample.

[0140] Appendix Figure 3 The results indicate that H0 is a key indicator for characterizing protein conformation, reflecting the abundance of its surface hydrophobic groups and its ability to interact with hydrophobic molecules. PG treatment significantly increased the H0 value of Pleurotus eryngii protein (p<0.05), from 4500.85 at 0 h to 9284.15 at 20 h. This may be because deamidation alters the tertiary structure of Pleurotus eryngii protein, exposing internal hydrophobic groups and thus increasing surface hydrophobicity, which creates favorable conditions for its binding with small hydrophobic molecules. Furthermore, during deamidation, the protein loses an amino group and gains a strongly hydrophilic carboxyl group, thereby increasing its overall hydrophilicity and water solubility.

[0141] 4. Analysis of Zeta potential and particle size of Pleurotus eryngii protein

[0142] The zeta potential and particle size of the samples were determined using a nanoparticle size and zeta potential analyzer (BeNano 90Zeta, Dandong Better Instruments Co., Ltd., China). Peptoprotein (PEP) from Examples 1-5 was used to prepare 1.0 mg / mL solutions with deionized water. Before potential measurement, the solutions were centrifuged (12000 g, 3 min, 4 °C) to remove insoluble fractions. The experiments were conducted at room temperature (25 °C) in triplicate.

[0143] Appendix Figure 4 The results indicate that the zeta potential is commonly used to characterize the charge density and electrostatic repulsion of proteins, which are related to their solution stability. After PG modification, the zeta potential of Pleurotus eryngii protein increased from -19.50 mV (0 h) to -28.38 mV (20 h). PG catalyzes the deamidation of glutamine residues in the protein into negatively charged glutamate residues, thereby increasing the molecular surface charge. Furthermore, the increase in surface charge leads to an increase in the electrostatic repulsion between protein molecules, which is beneficial to improving its solution stability.

[0144] Appendix Figure 4 The results showed that the particle size of Pleurotus eryngii protein decreased significantly with increasing deamidation degree (p < 0.05), indicating that the particle size of Pleurotus eryngii protein became smaller and more uniformly distributed after deamidation treatment. This is attributed to the conversion of amide groups to carboxyl groups during deamidation, which enhances the electrostatic repulsion between molecules, avoids protein aggregation, and thus leads to a reduction in particle size.

[0145] 5. FTIR and secondary structure

[0146] The structure of the samples was determined using an IS50 Fourier transform infrared spectrometer (VECTOR 22, Bruker Instruments, USA). Before testing, 1 mg of Pleurotus eryngii protein (PEP) prepared in Examples 1-5 and 150 mg of potassium bromide powder were ground together until they adhered to the wall of the sample, and then pressed into transparent thin films. The detection wavelength was 400-4000 cm⁻¹. -1 The secondary structure was determined by fitting the 1700-1600 cm⁻¹ infrared spectrum using OMNIC software. -1 Calculated.

[0147] Appendix Figure 5 The results indicate that the structural and conformational changes of Pleurotus eryngii proteins before and after PG-catalyzed deamidation were assessed using FTIR. In the FTIR spectra, the amide I band (1600-1700 cm⁻¹) was observed. -1 The primary source of these vibrations is the stretching vibration of the C=O bond, and its peak position and shape directly reflect the main chain conformation and hydrogen bond pattern of the protein's secondary structure. The spectra of the native PEP group and the deamidated group are similar, indicating that PG modification did not alter the protein's primary structure. However, some subtle differences can still be observed. We found that after deamidation treatment, amide I underwent a blue shift, a change stemming from the conversion of the amide group to the carboxyl group during deamidation. Specifically, when the C=O bond no longer participates in the hydrogen bond network between peptide bonds, but instead exists in a free state or in new hydrogen bond forms (such as carboxyl self-association hydrogen bonds or hydrogen bonds formed with water molecules), the electron cloud density of the C=O bond decreases, the bond energy increases, and thus the vibrational frequency rises.

[0148] Appendix Figure 5The results showed that with increasing deamidation, the α-helix proportion of *Pleurotus eryngii* protein increased from 6.02% to 28.13%, while the β-sheet proportion decreased from 47.20% to 26.57%. This is because glutamine residues in the protein form a β-sheet structure with repeating sequences through intramolecular and intermolecular hydrogen bonds. When PG-catalyzed deamidation occurs, the intermolecular charge of the protein increases, enhancing the electrostatic repulsion between β-sheet structures and disrupting the hydrogen bond network that maintains its stability, thus leading to a decrease in β-sheet content. The β-sheet structure is relatively stable, while the α-helix structure is "cyclic" and relatively flexible. Therefore, deamidation promotes protein unfolding, forming a more extended and flexible structure. This conformational change helps expose the binding sites originally embedded within the protein, potentially enhancing its binding ability to flavor compounds.

[0149] 6. Intrinsic fluorescence spectrum

[0150] The tertiary structure of the samples was determined using a fluorescence spectrophotometer (F-7100, Hitachi, Japan). 1 mg of Pleurotus eryngii protein (PEP) prepared in Examples 1-5 was diluted to 0.2 mg / mL with phosphate buffer (0.01 M, pH 7.0). The emission spectrum was set to 290-400 nm, the excitation wavelength to 290 nm, and the excitation and emission slits to 5 nm.

[0151] Appendix Figure 6 The results indicate that tryptophan, phenylalanine, and tyrosine residues in protein molecules produce intrinsic fluorescence, the intensity of which is influenced by the polarity of the residue microenvironment. Therefore, changes in fluorescence intensity can be used to infer changes in the polarity of the environment surrounding tryptophan, phenylalanine, and tyrosine residues, thus indirectly reflecting changes in tertiary structure. The fluorescence intensity of *Pleurotus eryngii* protein increases with increasing deamidation, indicating a conformational change and internal structural unfolding. This phenomenon is attributed to the introduction of hydrophilic carboxyl groups during PG deamidation, which alters the hydrophilicity of the amino acid microenvironment, inducing protein conformational unfolding and exposing more hidden polar groups. This is consistent with the observed changes in surface hydrophobicity and solubility. The novel protein modified with PG deamidation exhibits high surface hydrophobicity and solubility, making it a promising candidate for an ideal carrier for delivering hydrophobic bioactive compounds or flavor substances.

[0152] 7. Electronic nose (E-nose)

[0153] E-nose analysis was performed using PEN3 (AIRSENSE, Germany). 100 mg of the Pleurotus eryngii protein (PEP) prepared in Examples 1-5 was mixed thoroughly with 10 mL of distilled water in a 20 mL headspace vial and incubated at 50 °C for 30 min. The program was set to a 180 s wash time, a gas flow rate of 200 mL / min, and a 180 s measurement time.

[0154] Appendix Figure 7 The results showed that the W2S, W3S, and W5S sensors had high response values ​​in PEP-0h. The W2S, W3S, W5S, and W5C sensors mainly responded to volatile substances such as alcohols, aldehydes, ketones, alkanes, and nitrogen oxides. These compounds combine to form unpleasant flavors. Among them, aldehydes and alcohols (W2S), key contributors to unpleasant flavors, showed a decreasing trend after deamidation. This may be because deamidation alters the protein structure, enhancing its binding ability with volatile compounds, thereby reducing the release of off-odor compounds. To analyze the potential correlation between proteins with different degrees of deamidation and volatile compounds, principal component analysis (PCA) was performed. The results showed that the cumulative variance contribution rates of PC1 and PC2 for Pleurotus eryngii proteins were both greater than 90%, indicating that the model can fully reflect the overall odor information of the sample. Pleurotus eryngii proteins with different degrees of deamidation showed significant separation, indicating that the difference in deamidation had a significant impact on the volatile characteristics of the sample.

[0155] 8. Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS)

[0156] The content of volatile flavor compounds in the protein was determined using HS-SPME-GC-MS (GCMS-QP2010Ultral, Shimadzu, Japan). 0.5 g of Pleurotus eryngii protein (PEP) prepared in Examples 1-5 was dissolved in 4.5 g of NaCl (20%, w / v) in a 20 ml headspace vial, and 1 μL of cyclohexanone (1 ml / L) was added as an internal standard. Before testing, the CAR / DVB / PDMS SPME fiber tip (50 / 30 μm; Sigma-Aldrich, St. Louis, MO, USA) was placed in the GC inlet at 250 °C for 30 min. The sample was incubated at 60 °C for 15 min, followed by headspace extraction using an aged SPME fiber tip for 25 min. Desorption was performed at 250 °C for 5 min. Volatile compounds were then analyzed by GC-MS using an Rtx-5MS capillary column (30 m × 0.25 mm × 0.25 μm). After maintaining the temperature at 40 °C for 3 min, the temperature was increased to 150 °C at a rate of 4 °C / min and held for 4 min, and finally increased to 250 °C at a rate of 8 °C / min and held for 3 min. The helium flow rate was 1.0 mL / min. The split ratio was 15:1. GC-MS data were analyzed and quantified using the NIST17 library and internal standard method, and retention indices were calculated using n-alkanes (C7-C40). Experiments were performed in triplicate.

[0157] Table 1 shows that 64 volatile compounds were identified in the protein of *Pleurotus eryngii*. Analysis revealed that the undeamidated protein group of *Pleurotus eryngii* contained large amounts of aldehydes and alcohols, particularly hexanal, nonanal, benzaldehyde, pentanal, 2-hexylethanol, and 1-octen-3-ol. These compounds are associated with unpleasant flavors such as green, grassy, ​​mushroom, earthy, and plastic notes, and are considered major contributors to undesirable volatile compounds. Aldehydes and alcohols are involved in lipid oxidation, have low thresholds, and are key factors in producing unpleasant flavors. (See attached table.) Figure 8 The results showed that deamidation treatment effectively reduced the content of off-flavor compounds in king oyster mushroom protein. This was because the deamidation treatment altered the protein conformation, exposing internal hydrophobic regions and changing the surface charge distribution. This improved the binding affinity and interaction between the protein and volatile flavor molecules, thereby reducing the content of unwanted flavor substances.

[0158] Table 1. Content of volatile flavor compounds in king oyster mushroom protein

[0159]

[0160]

[0161]

[0162]

[0163] Note: Different lowercase letters represent significant differences (p < 0.05) across the cross-section.

[0164] 10. Odor Activity Value (OAV)

[0165] To assess the contribution of flavor compounds in undeamidated and deamidated protein samples, an odor activity value (OAV) was introduced, calculated according to the following formula (2).

[0166]

[0167] Where C is the concentration of the compound, and T is the threshold of the compound.

[0168] As shown in Table 2, the most abundant OAV (Odor Ability Value) in *Pleurotus eryngii* protein was found in 23 compounds, with benzaldehyde, nonanal, dodecanal, 1-octen-3-ol, and linalool exhibiting the highest OAV values. Aldehydes and alcohols, particularly decanal, nonanal, 1-octen-3-ol, pentanal, and n-hexanol, are typical off-flavor compounds, considered major contributors to beany, grassy, ​​and fatty odors. Furthermore, we observed a decreasing OAV value with increasing deamidation, indicating that deamidation treatment positively reduces the contribution of off-flavor compounds to the protein. This phenomenon may be related to the conformational changes in the protein induced by deamidation, which in turn affect its binding ability with flavor compounds, ultimately leading to a decrease in the contribution of off-flavor compounds to the protein.

[0169] Table 2. Volatile flavor compounds with OAV ≥ 1 in King Oyster Mushroom Protein

[0170]

[0171]

[0172] Note: " / " is not recognized.

[0173] 11. The binding affinity between proteins and flavor compounds

[0174] Take the Pepta oyster mushroom protein (denoted as PEP) prepared in Example 4 and Comparative Example 1 respectively, and prepare a 2% (w / v) protein solution with 0.01M PBS (pH 7.2). Stir at room temperature for 2 hours. Dissolve dodecyl aldehyde in phosphate buffer / methanol (19:1, v / v) mixed solvent to prepare a 5mM stock solution and vortex thoroughly. Take 1.8 mL of protein solution and 0.2 mL of flavor stock solution, place them in a 20 mL headspace bottle and vortex thoroughly. Experimental group: The final protein concentration is 1.8% (w / v), and the final concentrations of flavor compounds are 0.2 mM or 1 mM respectively. Control group: Use an equal volume of PBS solution instead of protein solution (determined by the HS-SPME-GC-MS method described in 8, and calculate the binding rate using the following formula (3):

[0175]

[0176] Appendix Figure 9 The results showed that the binding rate of Pleurotus eryngii protein to dodecyl aldehyde exhibited a significant concentration-dependent effect. As the concentration of flavor compounds increased within a certain range, the percentage of protein binding to flavor compounds gradually increased. This phenomenon may be attributed to the fact that more flavor molecules enter the protein's internal structure and occupy its binding sites at higher concentrations. The results indicate that deamidation significantly alters the flavor-binding capacity of the protein. Compared to the untreated group, deamidated Pleurotus eryngii protein consistently showed higher flavor-binding affinity. This suggests that the treatment effectively enhances the protein's ability to adsorb flavor compounds. This is because deamidation alters the protein's conformation, making its structure more flexible, thereby affecting the characteristics of the binding sites. Furthermore, deamidated proteins enhance geometric complementarity and physical interactions (adsorption and polymerization) with flavor compounds, thus improving the protein's binding capacity to flavor compounds.

[0177] 12. Fluorescence spectrum

[0178] Sample solutions were prepared according to Section 11, with the final concentration of flavor compounds ranging from 0.2 to 1 mM. Fluorescence spectroscopy analysis was performed after reacting the samples at 298 K, 303 K, and 310 K for 40 min. Here, 298 K corresponds to room temperature, 303 K represents the optimal temperature for human taste perception, and 310 K reflects human oral temperature. Spectroscopic scans were performed in the emission wavelength range of 280–500 nm, with a rate scan rate of 240 nm / s. The excitation wavelength was maintained at 290 nm, and the slit width for both excitation and emission wavelengths was 5 nm. Background subtraction was performed using a protein-free flavor solution with an equal concentration of protein-free flavor compounds to eliminate potential fluorescence quenching caused by internal filtering effects.

[0179] To gain a deeper understanding of the interaction between flavor compounds and proteins, the Stern-Volmer equation was used to analyze the quenching mechanism of flavor compounds and proteins.

[0180]

[0181] F and F0 are the fluorescence intensities of proteins containing and without flavor compounds, respectively. Kq is the quenching rate constant, τ0 is the lifetime of the fluorophore in the absence of quenching groups, and [Q] is the concentration of the flavor compound, K... SV It is the Stern-Volmer quenching constant.

[0182] The classic linear Stern-Volmer equation is based on a single uniform site model, where the quencher binds uniformly to a class of fluorescent groups to form a non-fluorescent complex. However, in real systems, fluorescence quenching usually originates from both collision (dynamic quenching) and ground-state complex formation (static quenching) mechanisms. To analyze the contributions of these two mechanisms, an improved Stern-Volmer equation is used, as shown in the following formula (5):

[0183]

[0184] Where K D and K S denoted by and , respectively, the dynamic and static quenching constants. This second-order polynomial equation resolves the upward curvature observed with increasing quencher group concentration ([Q]), which deviates from linearity when both quenching mechanisms coexist. The model interprets fluorescence data by simultaneously quantifying the contributions of dynamic and static quenching, as validated in recent studies.

[0185] The binding parameters (binding sites and constants) between proteins and flavor compounds are determined by the following formula (6). The molecular interactions between proteins and flavor compounds are represented by Van't Hoff equations (7) and (8).

[0186]

[0187] ln Ka=-ΔH / RT+ΔS / R(7)

[0188] ΔG=-RT ln Ka=ΔH-TΔS(8)

[0189] Appendix Figure 10The results show the corrected Stern-Volmer curves of the interaction between *Pleurotus eryngii* protein and dodecylaldehyde. The Stern-Volmer plots all exhibit an upward bend at high concentrations, deviating from a linear relationship. This nonlinear behavior indicates the coexistence of dynamic and static quenching mechanisms. Table 3 lists the numerical values ​​of the correlation constants between *Pleurotus eryngii* protein and dodecylaldehyde at different temperatures. It can be observed that as the temperature increases, K... SV K D and K S The decreasing value indicates that the binding of Pleurotus eryngii protein and dodecanal is primarily quenched by a static quenching mechanism. In the static quenching small molecule-protein binding model, the binding constant (Ka) represents the binding affinity between the protein and the corresponding flavor compound, and the number of binding sites (n) represents the number of binding sites between the protein and the corresponding flavor compound. As shown in Table 3, the Ka value of the Pleurotus eryngii deamide group was higher than that of the non-deamide group, indicating that the deamided protein has enhanced binding ability with flavor. After deamide treatment, the n value increased significantly, indicating that the number of binding sites increased in the deamide group. The above results reveal the molecular mechanism by which deamide improves the flavor characteristics of proteins. PG deamide catalyzes the conversion of the amide group on the side chain of glutamine residues to a glutamic acid carboxyl group. This modification induces protein conformational rearrangement, leading to the exposure of internal hydrophobic regions and embedded flavor binding sites. Increased site accessibility significantly enhances the binding affinity of proteins to volatile flavor ligands, strengthens their retention, and thus improves the overall flavor perception characteristics through a flavor sustained-release effect.

[0190] As shown in Table 3, all ΔG values ​​are negative, confirming that the reaction between *Pleurotus eryngii* protein and dodecylaldehyde is spontaneous. The ΔG value of the deamidated group is lower than that of the non-deamidated group, indicating that the deamidated protein can form more stable complexes with the corresponding flavor compounds, which is consistent with the Ka value results. Specific interactions can be obtained by calculating ΔH and ΔS. Specifically, hydrophobic interactions occur when ΔH>0 and ΔS>0; van der Waals interactions and hydrogen bonds occur when ΔH<0 and ΔS<0; and electrostatic interactions occur when ΔH<0 and ΔS>0. Analysis shows that both ΔH and ΔS values ​​for *Pleurotus eryngii* protein and dodecylaldehyde are negative, indicating that van der Waals forces and hydrogen bonds are the main forces in these two complexes. Table 1. Content of volatile flavor compounds in *Pleurotus eryngii* protein.

[0191] Table 3. Interaction and thermodynamic parameters between Pleurotus ostreatus protein and dodecylaldehyde

[0192]

Claims

1. A method for improving the flavor of king oyster mushroom protein, characterized in that, Includes the following steps: (1) Crush and sieve dried king oyster mushrooms to obtain king oyster mushroom powder; (2) The king oyster mushroom powder was degreased using an organic solvent to obtain degreased king oyster mushroom powder; (3) Mix defatted king oyster mushroom powder with water and hydrate. Adjust the pH to 8.0-10.0 with alkali solution, perform alkali extraction, centrifuge, and collect the supernatant. (4) Adjust the pH of the supernatant to 6.0-8.0, add PG to carry out enzymatic hydrolysis, and inactivate the enzyme after the reaction is completed; (5) Adjust the pH of the enzyme-inactivated system to the isoelectric point of Pleurotus eryngii protein, let it stand, and centrifuge to obtain protein precipitate. (6) The protein precipitate is washed, neutralized and dried to obtain flavor-improved king oyster mushroom protein.

2. The method according to claim 1, characterized in that, In step (1), the sieving is done through an 80-mesh sieve.

3. The method according to claim 1, characterized in that, In step (2), the organic solvent is n-hexane; in the degreasing treatment, the mass-to-volume ratio of king oyster mushroom powder to n-hexane is 1:3, and the degreasing treatment is repeated 2-3 times.

4. The method according to claim 1, characterized in that, In step (3), the mass ratio of defatted king oyster mushroom powder to water is 1:10; the alkaline solution is a sodium hydroxide solution, and the pH is adjusted to 9.

0. The stirring time for the alkali extraction is 2-4 hours.

5. The method according to claim 1, characterized in that, In step (3), the alkaline solution is a sodium hydroxide solution with a concentration of 2 mol / L.

6. The method according to claim 1, characterized in that, In step (4), the pH of the supernatant is adjusted to 7.0 using sodium hydroxide solution; the amount of PG added is 1:500 of the protein substrate mass; the enzymatic hydrolysis reaction is carried out at 45℃ for 0-24 hours; and the enzyme inactivation is carried out at 80℃. Inactivate enzymes by heating.

7. The method according to claim 1, characterized in that, In step (4), the enzyme activity of the PG is 100 U / g.

8. The method according to claim 1, characterized in that, In step (5), the pH of the system is adjusted to 4.0 using a hydrochloric acid solution with a concentration of 2 mol / L, and then left to stand for 2 hours.

9. The method according to claim 1, characterized in that, In step (6), the neutralization is achieved by using sodium hydroxide solution to adjust the washed protein precipitate to neutral; the drying is achieved by spray drying.

10. A type of Pleurotus eryngii protein prepared by the method according to any one of claims 1 to 7, characterized in that, The protein has a deamidation degree of greater than 14%, a particle size of less than 165 nm, and a binding rate with flavor substances of greater than 30%.

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