Walnut protein coffee foaming milk as well as preparation method and application thereof

Abstract

The invention relates to the technical field of coffee foaming milk, in particular to walnut protein coffee foaming milk as well as a preparation method and application thereof. The walnut protein coffee foaming milk comprises modified walnut protein and milk. The method comprises the following steps: mixing walnut protein powder with water, carrying out ultrasonic treatment, adding alkaline protease, carrying out enzymolysis, carrying out enzyme deactivation, cooling, and carrying out freeze drying to obtain the modified walnut protein. Through ultrasonic-alkaline protease synergistic modification, the foaming performance of the walnut protein is remarkably enhanced; the method is simple in process, green and low-carbon, a certain theoretical basis is provided for further extending walnut protein in food innovation and deep processing, a certain thought is provided for solving walnut protein resources, and the method has important economic and social significance in promoting walnut kernel industry development.

Description

Technical Field

[0001] The invention relates to the technical field of coffee foaming milk, in particular to a walnut protein coffee foaming milk and a preparation method and application thereof. Background Art

[0002] Walnuts are widely accepted by consumers for their diverse nutrients and bioactive ingredients, including essential fatty acids, fiber, gamma-tocopherol, folic acid, minerals, and plant protein. Their protein contains eight essential amino acids, and all other amino acids meet or exceed the standards recommended by the FAO / WHO (2013). However, walnut protein's poor solubility, emulsification, and foaming properties limit its application in the food processing industry.

[0003] Coffee is one of the world's most popular beverages. Due to its unique sensory characteristics, it can be enjoyed in various styles, such as latte, cappuccino, and mocha. Cappuccino is typically made by adding milk foam to coffee. The appearance, texture, taste, and aroma release of the foam on top are key indicators of consumer perception of the final product's overall quality. Cappuccino requires high milk content; not all dairy products are suitable for production. It requires dairy products with excellent foaming properties, fast foaming speed, and white, fine foam. Furthermore, the ideal foam should have small, uniform bubbles and be stable for a long time. Cow's milk is commonly used as a foaming milk for cappuccino because of its excellent foaming properties. While extensive research has been conducted on the foaming properties of milk, its foaming properties are easily affected by various factors, including its protein and fat content, cow breed, origin, processing conditions, and foaming method, resulting in either poor or no foaming. However, there are currently no reports on the application of modified walnut protein in coffee foaming milk. Summary of the Invention

[0004] Based on the above content, the purpose of the present invention is to provide a walnut protein coffee foaming milk and its preparation method and application.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a method for modifying walnut protein, comprising the following steps:

[0007] The walnut protein powder is mixed with water and then ultrasonically treated, and then alkaline protease is added for enzymolysis, and the enzyme is inactivated, followed by cooling and freeze-drying to obtain modified walnut protein.

[0008] In a preferred embodiment of the present invention, the mass volume ratio of the walnut protein powder to water is 1 g:mL; the power of the ultrasonic treatment is 400-800 W, the time is 15 min, and the ultrasonic treatment is performed for 5 s with a rest period of 5 s.

[0009] In a preferred embodiment of the present invention, the addition amount of the alkaline protease is 0.07% to 0.3% of the mass of the walnut protein powder; the enzymatic hydrolysis conditions are: pH 9.0, temperature 50° C., and enzymatic hydrolysis for 2 hours.

[0010] The second technical solution of the present invention is a modified walnut protein prepared according to the above-mentioned walnut protein modification method.

[0011] The third technical solution of the present invention is a walnut protein coffee foaming milk, which includes the above-mentioned modified walnut protein and milk.

[0012] A fourth technical solution of the present invention is a method for preparing the above-mentioned walnut protein coffee foamed milk, comprising the following steps:

[0013] The modified walnut protein is mixed with water to obtain a walnut protein liquid; the walnut protein liquid is mixed with milk to obtain the walnut protein coffee foaming milk.

[0014] In a preferred embodiment of the present invention, the mass concentration of the walnut protein liquid is 10%.

[0015] In a preferred embodiment of the present invention, the volume fraction of the walnut protein liquid in the walnut protein coffee foam is 2% to 14%.

[0016] The fifth technical solution of the present invention is the use of the above-mentioned modified walnut protein or the above-mentioned walnut protein coffee foaming milk in preparing coffee liquid.

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

[0018] The present invention significantly enhances the foaming performance of walnut protein through ultrasound-alkaline protease synergistic modification; the process of the present invention is simple, green and low-carbon, and provides a certain theoretical basis for further extending the use of walnut protein in food innovation and deep processing, and provides a certain idea for solving the problem of walnut protein resources, which has important economic and social significance for promoting the development of the walnut kernel industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 These are SEM photos of walnut protein powder and modified walnut proteins prepared in Example 1 and Comparative Examples 1-4; among them, A is walnut protein powder, B is Comparative Example 1, C is Comparative Example 2, D is Comparative Example 3, E is Comparative Example 4, and F is Example 1.

[0021] Figure 2 The SDS-PAGE and subunit contents of the modified walnut proteins prepared in Example 1 and Comparative Examples 1-4; wherein A is SDS-PAGE and B is subunit content.

[0022] Figure 3 FTIR spectra of the modified walnut proteins prepared in Example 1 and Comparative Examples 1-4.

[0023] Figure 4 The particle size distribution of the modified walnut protein prepared in Example 1 and Comparative Examples 1-4.

[0024] Figure 5 The foam microstructure and bubble number of the modified walnut protein prepared in Example 1 and Comparative Examples 1-4; A is the foam microstructure, and B is the bubble number.

[0025] Figure 6 The foam microstructure (A), number of bubbles (B) and average bubble diameter (C) of the walnut protein coffee foamed milk in Example 2.

[0026] Figure 7 This is the digestibility of the walnut protein coffee foamed milk in Example 2.

[0027] Figure 8 This is a picture of the finished product of the walnut protein coffee foaming milk and coffee mixed in Example 2.

[0028] Figure 9 This is the particle size distribution of the walnut protein coffee foaming milk and coffee mixture in Example 2.

[0029] Figure 10 It is the zeta potential of the mixture of walnut protein coffee foaming milk and coffee in Example 2. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The first aspect of the present invention provides a method for modifying walnut protein, comprising the following steps:

[0036] The walnut protein powder is mixed with water and then ultrasonically treated, and then alkaline protease is added for enzymolysis, and the enzyme is inactivated, followed by cooling and freeze-drying to obtain modified walnut protein.

[0037] In a preferred embodiment of the present invention, the mass volume ratio of the walnut protein powder to water is 1 g:mL; the power of the ultrasonic treatment is 400-800 W, the time is 15 min, and the ultrasonic treatment is performed for 5 s with a rest period of 5 s.

[0038] In the embodiment of the present invention, the ultrasonic device adopts an ultrasonic cell disruptor (JY92-IIN, Shanghai Jipu Electronic Technology Co., Ltd.). When in use, the ultrasonic mode is set to 09 and the speed lever is set to Φ03.

[0039] With increasing ultrasonic power, the solubility and foaming properties of walnut protein initially increase and then decrease. This increase in solubility and foaming may be attributed to the fact that ultrasound disrupts the physical bonds between protein molecules, reduces aggregate formation, and promotes interactions between protein and water molecules. It may also induce structural changes, affecting the chemical properties of surface residues and altering their solubility. Foaming stability decreases with further increases in ultrasonic power. This is because foams formed by partially expanded protein are more stable than those formed by fully expanded protein. When fully expanded, the resulting foam is prone to collapse due to the further expansion of air, thus reducing the protein's stability at the interface. Therefore, excessive ultrasonic power stretches the protein structure and increases solubility, which is detrimental to foam stability. The principle of solubility is the same as above.

[0040] The enhanced solubility, foaming properties, and foaming stability of walnut protein after alkaline protease treatment may be due to the fact that alkaline protease can specifically hydrolyze peptide bonds in proteins, resulting in partial degradation of the protein molecules. This partial degradation disrupts the protein structure, exposing hydrophobic groups that were originally encapsulated internally, and increasing the flexibility and solubility of the protein molecules. As the amount of enzyme added increases, the solubility and foaming stability of walnut protein increase, then level off. Considering both the effect and cost, the optimal alkaline protease addition level is 0.10%.

[0041] In a preferred embodiment of the present invention, the addition amount of the alkaline protease is 0.07% to 0.3% of the mass of the walnut protein powder; the enzymatic hydrolysis conditions are: pH 9.0, temperature 50° C., and enzymatic hydrolysis for 2 hours.

[0042] The second aspect of the present invention provides a modified walnut protein prepared according to the above-mentioned walnut protein modification method.

[0043] A third aspect of the present invention provides a walnut protein coffee foamed milk comprising the modified walnut protein and milk.

[0044] A fourth aspect of the present invention provides a method for preparing the above-mentioned walnut protein coffee foamed milk, comprising the following steps:

[0045] The modified walnut protein is mixed with water to obtain a walnut protein liquid; the walnut protein liquid is mixed with milk to obtain the walnut protein coffee foaming milk.

[0046] In a preferred embodiment of the present invention, the mass concentration of the walnut protein liquid is 10%.

[0047] In a preferred embodiment of the present invention, the volume fraction of the walnut protein liquid in the walnut protein coffee foam is 2% to 14%.

[0048] The fifth aspect of the present invention provides the use of the modified walnut protein or the walnut protein coffee foaming milk in preparing coffee liquid.

[0049] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0050] The walnut protein powder used in the examples of the present invention has a purity of 83.80% and was purchased from Liaoning Changbaixianzi Biotechnology Co., Ltd.

[0051] The alkaline protease used in the examples of the present invention was analytically pure and purchased from Solebao Biotechnology Co., Ltd.

[0052] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1 (Ultrasound-assisted alkaline protease treatment)

[0054] 5 g of walnut protein powder was mixed with 100 mL of ultrapure water and magnetically stirred for 30 minutes. The mixture was then ultrasonicated for 15 minutes under the conditions of an ultrasonic power of 600 W and an ultrasonic interval of 5 seconds (5 seconds interval for every 5 seconds of ultrasonication); after the ultrasonication, 0.10% of alkaline protease by weight of the walnut protein powder was added, and the pH was adjusted to 9.0. The mixture was enzymatically hydrolyzed at a temperature of 50°C (water bath) for 2 hours; after the enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath for 10 minutes, and the mixture was then cooled to room temperature and freeze-dried to obtain modified walnut protein.

[0055] Comparative Example 1 (Ultrahigh Pressure Treatment)

[0056] 20g of walnut protein was vacuum-sealed in a polyethylene bag and placed in a pressurized chamber of an ultrahigh-pressure device. Using tap water as the pressure transmission medium, the modified walnut protein was obtained by treating it at room temperature and an ultrahigh-pressure pressure of 188 MPa for 14 minutes.

[0057] Based on Comparative Example 1, the present invention uses single-factor experimental design and response surface optimization experimental design to determine that when the pressure, time and treatment amount of walnut protein during ultrahigh pressure treatment are the parameters recorded in Comparative Example 1, it is the optimal process, and the solubility of the prepared modified walnut protein is 18.91%.

[0058] Comparative Example 2 (ultrasonic treatment)

[0059] Walnut protein powder was mixed with water to prepare a walnut protein solution with a mass concentration of 5%, and magnetically stirred at room temperature for 30 minutes. Ultrasonic treatment was performed with a power of 600 W for 15 minutes (<30°C), with an ultrasonic treatment period of 5 seconds and a rest period of 5 seconds. After the ultrasonic treatment, the solution was freeze-dried in a vacuum freeze dryer to obtain modified walnut protein, which was then placed in a refrigerator at 4°C for testing.

[0060] Based on Comparative Example 2, the present invention tested the effects of different ultrasonic powers of 0, 200, 400, 600, 800, and 1000 W on the solubility, foaming properties, and foaming stability of walnut protein. The results showed that 600 W was the optimal ultrasonic power; the solubility, foaming properties, and foaming stability of the modified walnut protein prepared in Comparative Example 2 were 12.15%, 49.33%, and 55.39%, respectively.

[0061] Comparative Example 3 (alkaline enzyme treatment)

[0062] Walnut protein powder was mixed with water to prepare a walnut protein solution with a mass concentration of 5%, and magnetic stirring was performed at room temperature for 30 minutes. Alkaline protease (enzyme activity 200,000 U / g) accounting for 0.10% of the mass of walnut protein powder was added and enzymatic hydrolysis was carried out at 50°C and pH 9.0 for 2 hours. The enzyme was inactivated in a boiling water bath for 10 minutes, cooled to room temperature, and then freeze-dried in a vacuum freeze dryer to obtain modified walnut protein, which was stored in a 4°C refrigerator and subsequently analyzed.

[0063] Based on Comparative Example 3, the present invention tested the effects of varying the amount of alkaline protease added (0, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%) on the solubility, foaming properties, and foaming stability of walnut protein. The results showed that 0.1% was the optimal amount of alkaline protease added; the solubility, foaming properties, and foaming stability of the modified walnut protein prepared in Comparative Example 3 were 31.11%, 82.67%, and 90.32%, respectively.

[0064] The foaming property showed a trend of first increasing, then decreasing, and then increasing again with the increase of the amount of alkaline protease added. When the enzyme addition amount was 0.10%, the foaming property reached the maximum value, increasing from 32.67% to 82.67%.

[0065] Comparative Example 4 (Ultrahigh Pressure Synergistic Alkaline Protease Treatment)

[0066] Take 20g of walnut protein powder, vacuum-seal it in a polyethylene bag, and then place it in a pressurized cabin of an ultrahigh pressure device. Using tap water as a pressure transmission medium, treat it at room temperature and an ultrahigh pressure treatment pressure of 188MPa for 14 minutes. Then, mix the ultrahigh pressure treated walnut protein powder with water to prepare a walnut protein solution with a mass concentration of 5%. Add alkaline protease accounting for 0.10% of the mass of the ultrahigh pressure treated walnut protein powder, adjust the pH to 9.0, and enzymolyze it at a temperature of 50°C (water bath) for 2 hours; after the enzymolysis is completed, the enzyme is inactivated in a boiling water bath for 10 minutes, then cooled to room temperature, and freeze-dried to obtain modified walnut protein.

[0067] Test Example 1

[0068] The modified walnut proteins prepared in Example 1 and Comparative Examples 1-4 were tested as follows:

[0069] 1. Imaging observation of the microstructure of modified walnut protein

[0070] The surface microstructure of walnut protein was observed using an environmental scanning electron microscope (SEM). Freeze-dried walnut protein was fixed to a sample stage using conductive adhesive and then gold-sputtered using an ion sputtering device under vacuum. SEM images were collected at magnifications of 500, 2000, and 4000x, and multiple views were observed and photographed.

[0071] The microstructure of different walnut proteins was observed by scanning electron microscopy. Figure 1 As shown by Figure 1 It can be seen that ultrahigh pressure treatment, ultrasonic treatment, alkaline protease treatment, ultrahigh pressure combined with alkaline protease treatment and ultrasonic combined with alkaline protease treatment have an impact on the microstructure of walnut protein. Figure 1 In Figure A, we can see that the walnut protein in the Control group (raw walnut protein powder) presents a spherical structure, and at different magnifications, it presents more sparse and porous spherical structures. Figure 1 As can be seen in Figure B, compared with the Control group, the walnut protein of group H (ultrahigh pressure treatment, comparative example 1) presents an irregular flaky structure. At 2000 and 4000 magnifications, it can be clearly seen that the surface of the protein aggregate is not smooth, uneven, and there are some holes, and there are protruding globular protein particles on the surface. Compared with the Control group, group U (ultrasound treatment, comparative example 2) ( Figure 1 Middle C) retains the original spherical shape and morphology of walnut protein. At a magnification of 4000, it can be seen that there are many small holes on the surface and protruding globular protein particles. Compared with the Control group, Group E (alkaline protease treatment, comparative example 3) ( Figure 1 The surface of walnut protein in D) is relatively flat and smooth and has a compact structure. HE group (ultra-high pressure synergistic alkaline protease treatment, comparative example 4) ( Figure 1E) and UE group (ultrasound synergistic alkaline protease treatment, Example 1) ( Figure 1 The surface structure of group F) is a rough lamellar structure, and there are more globular protein particles than in the treated samples of group E.

[0072] 2. Determination of modified walnut protein by SDS-PAGE gel electrophoresis

[0073] The concentration of the separation gel is 12%, the concentration of the stacking gel is 5%, and the walnut protein solution (concentration of 0.5 mg / mL) is mixed with the loading buffer (0.08 mol / LTris-HCl buffer, 1% SDS, 2% β-mercaptoethanol, 5% glycerol, 0.025% bromophenol blue) at a ratio of 4:1. Vortex for 30 minutes, boil at 100°C for 5 minutes, cool to room temperature, centrifuge at 8000r / min for 10 minutes, take 30μL of supernatant and 3μL of standard protein (Maker) and add them to the stacking gel tank, perform electrophoresis at a voltage of 80V (5%), and perform electrophoresis at a voltage of 120V (12%). After electrophoresis, the gel is stained with Coomassie Brilliant Blue Quick Staining Solution and repeatedly destained with ultrapure water. Use Gel Doc TM The obtained gel was scanned using XR gel imaging system (Bio-Rad, CA, USA).

[0074] The present invention obtains the SDS-PAGE spectrum of walnut protein samples with different modification methods by gel electrophoresis ( Figure 2 The figure shows that the molecular weight of walnut protein is primarily distributed in the 8-68 kDa range. The Control, H, and U groups exhibited distinct bands at 8-13 kDa, 13-20 kDa, and 30-40 kDa, with a lighter band at 55-68 kDa. In contrast, the E, HE, and UE groups exhibited distinct bands at 8-20 kDa, with a lighter band at 20-68 kDa. The molecular weights of the two major subunits of walnut protein are 13-20 kDa and 30-40 kDa, and these two major bands did not differ significantly between the different treatment groups.

[0075] The changes in subunit content were obtained by density analysis of gel electrophoresis patterns. Figure 2 As shown in Figure B, compared with the control group, the protein subunit content of groups H and U changed little; however, the subunit content of groups E, HE, and UE changed significantly, with the content of subunits in the 55-68 kDa and 30-40 kDa ranges significantly decreasing by 7.65%, 8.20%, 9.00%, 4.75%, 5.38%, and 5.43%, respectively. In contrast, the content of subunits in the 13-20 kDa and 8-13 kDa ranges increased significantly by 30.67%, 30.67%, 31.39%, 28.90%, 30.09%, and 30.22%, respectively.

[0076] 3. Determination of modified walnut protein by Fourier infrared spectroscopy

[0077] After using KBr pelleting, the walnut protein sample was -1 Fourier transform infrared spectroscopy is performed within the wavelength range. The secondary structure information of proteins is mainly concentrated in the 1600-1700 cm -1 PeakFitv 4.12 software was used to perform curve fitting within this range and calculate the percentage of secondary structure.

[0078] Modification of protein structure usually leads to changes in its secondary and tertiary structures. Fourier transform infrared spectroscopy (FTIR) is a common method for analyzing protein secondary structure. Proteins have several characteristic absorption bands in the infrared spectral region: 3200-3600 cm -1 -OH stretching vibration, 2800-3000cm -1 The stretching vibration of -CH, amide I band (1600-1700cm -1 ) corresponds to the stretching vibration of C=O in the peptide bond, the amide II band (1500-1600 cm -1 ) mainly corresponds to the stretching vibration of C≡N and the bending vibration of NH in the amino group, and the amide III band (1200-1300cm -1 ) corresponds to the stretching vibration of C≡N and the deformation vibration of NH in the amino group. FTIR spectra of walnut protein treated with different methods are as follows Figure 3 As shown in the figure, it can be observed that the total FTIR chromatograms of walnut protein obtained by different treatment methods are similar, with the peak at 3321.61 cm -1 The broad peak at is caused by the stretching vibration of -OH. The peak intensities of the H, U, and UE groups shift toward lower wavenumbers, and the peak shapes are broader than those of the control group, indicating stronger hydrogen bonding between these three treatment groups. The characteristic peak of the amide I band can reflect changes in the secondary structure of the protein. Compared with the control group, the absorption peak at amide II in the different treatment groups has a blue shift, and the characteristic peak at amide I has also undergone slight changes.

[0079] Since different secondary structures have strong absorption of infrared radiation at different wave numbers, by analyzing the amide I band of walnut protein, the corresponding relationship between the secondary structure and the sub-peak in the fitting diagram is as follows: β-Sheet is characterized by a wave number range of 1612-1640 cm -1 and 1689-1698cm -1 α-Helix corresponds to 1650-1660 cm -1 The characteristic of Randoncoil is the wave number between 1640-1649cm -1Range: β-Turn from 1660-1688cm -1 The wavenumber between them reflects the difference. The spectra of the characteristic peaks of the amide I band were analyzed by fitting the walnut protein with different treatment methods, and the percentage of protein secondary structure was calculated. The results showed that compared with the control group, the β-sheet content of the different treatment groups showed a downward trend, while the β-turn content showed an increasing trend.

[0080] 4. Determination of modified walnut protein circular dichroism

[0081] Walnut proteins with different treatment methods were prepared into solutions with a concentration of 0.5 mg / mL using ultrapure water and placed at 25°C for 3 h. The circular dichroism properties of the samples were measured using a circular dichroism spectrometer with a scanning wavelength of 190-250 nm, a scanning rate of 100 nm / min, a response temperature of 25°C, a resolution of 0.5 nm, and a sensitivity of 100 mdeg / cm.

[0082] Through the CD spectra of walnut proteins treated with different methods, it can be observed that the CD spectra of all walnut proteins treated with different methods have similar shapes compared with the control group. All samples have a broad negative band in the range of 200-230nm, and the CD peaks of samples in different treatment groups are blue-shifted compared with those in the control group. This indicates that the secondary structure of walnut protein has been changed under the action of ultrahigh pressure, ultrasound, alkaline protease, ultrahigh pressure synergistic alkaline protease and ultrasound synergistic alkaline protease, which is consistent with the results of FTIR spectrum and the proportion of protein secondary structure.

[0083] 5. UV spectrum scanning of modified walnut protein

[0084] Prepare a 0.2 mg / mL walnut protein solution in ultrapure water. Measure the absorbance of the sample solution at a wavelength of 225-400 nm using ultrapure water as a reference solution.

[0085] UV spectroscopy revealed no significant differences in the absorption peak positions of the modified walnut protein samples obtained by the different treatment methods. However, the absorption intensity of the H, U, E, HE, and UE groups increased, particularly for the walnut protein samples treated with E, HE, and UE. Walnut protein exhibits a characteristic absorption peak near 280 nm due to the presence of tryptophan, tyrosine, and phenylalanine. The different treatments increased the protein's absorption at 280 nm, indicating that the different modification methods exposed the aromatic amino acids hidden within the walnut protein.

[0086] 6. Fluorescence spectrum scanning of modified walnut protein

[0087] A 0.2 mg / mL walnut protein solution was prepared in ultrapure water, and the intrinsic fluorescence spectrum of the protein was measured using a microplate reader. The slit width was set to 10 nm, the scan speed was 200 nm / min, the excitation wavelength was 280 nm, and the scan range was 310-460 nm.

[0088] Measuring the fluorescence spectrum of samples can further reflect changes in protein structure. Changes in environmental polarity can cause changes in the fluorescence spectra emitted by tryptophan, tyrosine, and phenylalanine residues, which is a common characterization technique for evaluating protein tertiary structure. At an excitation wavelength of 280 nm, the emission spectra of modified walnut proteins obtained by different treatment methods were measured. The maximum emission wavelength of all protein samples was around 335 nm. Compared with the control group, the maximum emission wavelength of the different treatment groups remained almost unchanged. Except for the decrease in fluorescence intensity in the U group, the fluorescence intensity of the other treatment groups increased. This suggests that ultrasonic treatment may promote the binding of chromophobic groups to other side chain amino acids, thereby quenching the fluorescence intensity.

[0089] 7. Determination of modified walnut protein by X-ray diffraction

[0090] The walnut protein samples with different treatment methods were evenly spread in the sample pool and then placed on the XRD measurement conditions: tungsten filament lamp wavelength of 0.15406nm, power of 1600W (40kV×40mA), scanning rate of 5° / min, and scanning range of 5~80°.

[0091] X-ray diffraction patterns obtained by different modification methods showed that all walnut proteins displayed a diffraction peak near 20° or 21°. Compared with the control group, the different modification methods had little effect on the position of the walnut protein diffraction peak, and the intensity was slightly enhanced, which may be due to the increase in the crystallization area. These results show that after ultrahigh pressure treatment, ultrasound treatment, alkaline protease treatment, ultrahigh pressure combined with alkaline protease treatment, and ultrasound combined with alkaline protease treatment, no significant changes were found in the crystallization area of ​​walnut protein.

[0092] 8. Determination of particle size of modified walnut protein

[0093] Walnut protein samples were measured for particle size using a Mastersizer 2000 laser particle size analyzer. The material had a refractive index of 1.334 and was dispersed in water. The sample was diluted to 1 mg / mL with ultrapure water and equilibrated at 25°C for 120 seconds before testing.

[0094] The particle size distribution of protein affects functional properties, including solubility. The particle size of walnut protein treated by different methods was measured using a laser particle size distribution analyzer and displayed in the form of a particle size distribution graph. Figure 4As shown, the control, UHP, and ultrasound-treated groups exhibited trimodal distribution curves within the 0.3-560 μm range, indicating that the untreated, UHP, and ultrasound-treated proteins not only had a wide range of particle size distributions but also had significant variations in powder size. Compared to the control, the UHP and ultrasound-treated protein particle size distribution peaks shifted slightly to the left. Meanwhile, alkaline protease treatment, UHP combined with alkaline protease treatment, and ultrasound combined with alkaline protease treatment exhibited unimodal distributions with narrower particle size distributions, resulting in smaller and more uniform protein particle sizes.

[0095] 9. Determination of contact angle of modified walnut protein

[0096] The sitting drop method was used to measure the static water contact angle of walnut protein. The walnut protein powder was pressed into a thin circular sheet, and 6 μL of distilled water was carefully dropped on the surface of the sheet sample using an automatic pipette to capture a stable image.

[0097] The larger the contact angle, the stronger the hydrophobicity. The contact angle can be used to define the interfacial wettability of proteins and can also directly reflect the hydrophilicity and hydrophobicity of materials. Compared with the control group, the contact angles of the different treatment groups decreased. Among them, the contact angles of the E, HE, and UE groups decreased significantly, from 87.45° to 57.84°, 55.74°, and 57.96°, respectively; the H and U groups decreased less, by 12.59° and 7.72°, respectively. This indicates that under the action of ultrahigh pressure, ultrasound, alkaline protease, ultrahigh pressure assisted alkaline protease, and ultrasound assisted alkaline protease, the amino acid residues on the surface of walnut protein are transformed from hydrophobic to hydrophilic, resulting in a decrease in the hydrophobicity and an increase in the hydrophilicity of the walnut protein surface.

[0098] 10. Determination of surface hydrophobicity of modified walnut protein

[0099] Dissolve the sample in pH 7 phosphate buffer and prepare and dilute protein solutions at concentrations of 0.05, 0.15, 0.25, 0.35, and 0.45 mg / mL. For measurement, add 10 μL of 8 mM ANS solution to 2 mL of the test solution, mix thoroughly by vortexing, and incubate in the dark for 15 minutes. Fluorescence intensity is then measured using a microplate reader. Excitation wavelength: 390 nm, emission wavelength: 470 nm, slit width: 5 nm. Plot the protein concentration against the fluorescence intensity. The initial slope of the curve represents the surface hydrophobicity (H0).

[0100] Surface hydrophobicity, a measure of the amount of hydrophobic groups on the surface of protein molecules in contact with polar water, influences protein structure and function. Compared to the control group, surface hydrophobicity increased significantly in all treatment groups except for the U and E groups, with the UE group showing the greatest increase of 5100.67 au. This may be due to the significant reduction in particle size caused by high-intensity ultrasonic cavitation and cleavage by alkaline protease, which weakens the nonpolar bond interactions between amino acid residues on the particle surface and leads to increased hydrophobicity.

[0101] 11. Determination of free sulfhydryl groups in modified walnut protein

[0102] Stir 10 mg / mL walnut protein for 30 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C, and collect the supernatant. Ellman's reagent (DNTB dissolved in Tris-glycine buffer, 4 mg / mL) needs to be prepared immediately before use. Then, take 1 mL of the supernatant, 5 mL of Tris-glycine buffer, and 100 μL of Ellman's reagent and quickly mix and stir. After standing for 15 minutes, measure the absorbance at 412 nm. The calculation formula is as follows:

[0103] Where: 73.53 = 106 / 13600, 13600 is the molar extinction coefficient of Ellman's reagent; D is the dilution factor; C is the protein concentration, mg / mL.

[0104] The presence of sulfhydryl groups affects the formation of intra- and intermolecular covalent bonds, and changes in sulfhydryl content can reflect the degree of protein denaturation. Compared with the control group, the free sulfhydryl content in groups H and U did not change significantly (P>0.05). Conversely, the free sulfhydryl content in groups E, HE, and UE increased significantly (P<0.05), consistent with the SDS-PAGE results. No new covalent bonds were generated in walnut protein during ultrahigh pressure and ultrasound treatment, whereas new covalent bonds were generated during treatment with alkaline protease, ultrahigh pressure combined with alkaline protease, and ultrasound combined with alkaline protease.

[0105] 12. Determination of thermal stability of modified walnut protein

[0106] Place 8 mg of sample in an aluminum crucible and seal it with a capping machine. Use an empty aluminum crucible as a blank control. Determine the thermal stability of the protein using a differential scanning calorimeter. Parameters: temperature scan range: 30–200°C; heating rate: 20°C / min; nitrogen flow rate: 20 mL / min.

[0107] DSC is a common method for determining the thermal stability of proteins, which reflects the changes in molecular structure and conformation. Different thermal properties including onset temperature (T0), glass transition temperature (T g), denaturation temperature (T d ) and thermal enthalpy (ΔH) are shown in Table 1, where denaturation temperature (Td) represents thermal stability and thermal enthalpy reflects the degree of protein molecular aggregation. Compared with the Control group, T0 of the H and UE groups increased, while that of the U and HE groups decreased. g In general, except for the H and UE groups, the U, E and HE groups all decreased, among which the HE group had the lowest temperature (87.80±1.98)℃. d ) changed very little, and there was no significant difference among H, U, E and UE groups (P>0.05). d The ΔH of walnut protein in the U, E, HE, and UE groups decreased compared with the Control group, with the E group having the lowest ΔH (193.80±3.54) J / g.

[0108] Table 1

[0109]

[0110] Note: Different lowercase letters indicate significant differences (P<0.05).

[0111] 13. Determination of amino acid composition of modified walnut protein

[0112] Accurately weigh 10 mg of solid sample and add it to the hydrolysis tube. Add 10 mL of 1:1 hydrochloric acid (about 6 mol / L), blow nitrogen into the tube for 30 seconds and seal it. Place it in an oil bath at 110°C for hydrolysis for 22 to 24 hours. After the hydrolysis is completed, cool it to room temperature, filter it with a 0.45μm membrane into a 50mL volumetric flask and make up the volume. Pipette 2 mL of the sample after making up the volume into a 50mm diameter weighing bottle and heat it at 85°C on a flat plate for deacidification twice. Until a small amount of solid or stain remains at the bottom of the bottle, add 1 mL of sodium citrate buffer to fully dissolve it, filter it through a 0.22μm filter, and determine it using a fully automatic amino acid analyzer.

[0113] The results show that the modification methods of Example 1 and Comparative Examples 1-4 did not affect the amino acid composition of walnut protein, but affected the amino acid content.

[0114] 14. Effect of modification on the solubility of walnut protein

[0115] Prepare a bovine serum albumin standard solution with a concentration of 0.1 mg / mL. Take 0, 200, 400, 600, 800 and 1000 μL of the standard solution and add them to the EP tube respectively, and use ultrapure water to make up to a total volume of 1000 μL. Then add 5 mL of Coomassie Brilliant Blue solution to each EP tube and mix well. Let it stand at room temperature in the dark for 10 minutes. Use ELX808 microplate reader to measure the absorbance of the solution at a wavelength of 595 nm. Draw a standard curve with the concentration of the standard solution as the horizontal axis (x) and the absorbance value as the vertical axis (y). The resulting linear regression equation is y = 4.0579x + 0.0042, R 2 =0.999, the results showed that BSA showed a good linear relationship in the concentration range of 0-0.1 mg / mL.

[0116] 1% (w / v) walnut protein before and after modification was magnetically stirred at room temperature for 30 min, centrifuged at 8000 rpm for 15 min at 4°C, and the supernatant was collected. The protein content of the supernatant was determined by the Coomassie brilliant blue method. The protein solubility (S) was calculated as follows:

[0117]

[0118] Where: S-solubility; W2-supernatant protein content (mg / mL); W1-sample protein content (mg / mL).

[0119] Solubility is an important indicator of protein denaturation and aggregation, reflecting the functional characteristics and application potential of proteins. The solubility of proteins obtained by different modification methods showed an upward trend. The solubility of the E, HE, and UE groups was significantly higher than that of the U, H, and Control groups, reaching 37.22%, 37.35%, and 37.76%, respectively. The UE group had the highest solubility, increasing by 26.59% compared to the Control group. This indicates that the E, HE, and UE groups improved the solubility of walnut protein better than the H and U groups.

[0120] 15. Effect of modification on the foaming properties of walnut protein

[0121] Weigh 0.5g of walnut protein before and after modification, add 50mL of ultrapure water, and stir magnetically at room temperature for 30 minutes. Then, shear the 50mL protein solution in a high-speed homogenizer at 10,000 rpm for 1 minute, then transfer it to a 100mL graduated cylinder. Measure the foam volume at 0 and 30 minutes. Calculate foaming properties and foam stability using the following formula:

[0122]

[0123] Where: FC-foaming property; FS-foaming stability; V0-initial solution volume (mL); V-foam volume at 0 min after shearing (mL); V 30 - Foam volume 30 min after shearing (mL).

[0124] Foaming properties refer to the ability to form a strong film between the gas and liquid, thereby binding and stabilizing a large number of bubbles. These properties were assessed using foaming capacity (FC) and foam stability (FS). Compared with the control group, foaming capacity increased significantly in all treatment groups except group H (P < 0.05), with the increases in groups E, HE, and UE being particularly significant (P < 0.01). These values ​​were 81.33%, 85.33%, and 96.67%, respectively, representing increases of 46.67%, 50.67%, and 62.01%, respectively, compared to the control group. Foam stability decreased in groups H and U, while significantly increased in groups E, HE, and UE, reaching 90.16%, 95.31%, and 93.79%, respectively, representing increases of 30.57%, 35.72%, and 34.20%, respectively, compared to the control group.

[0125] 16. Effect of modification on walnut protein foam

[0126] Protein foams were prepared using the method described in "15. Effect of Modification on the Foaming Properties of Walnut Protein." A small amount of foam was then placed on a glass slide and the slide was placed under an inverted fluorescence microscope to observe the sample's microstructure. The eyepiece magnification was 10x, and the objective lens magnification was 4x. Images of the foam were taken at different time intervals between 0 and 30 minutes. Images were analyzed using Image J software, and the "Analyze Particles" tool within the software was used to quantify the number of bubbles.

[0127] The stable interface structure of bubbles is related to their number; at the same time, the instability process of foam can be studied by changing the number of bubbles to further explain the foaming property and foam stability of protein samples. Figure 5 The state of walnut protein foam obtained by different modification methods at 0, 15, and 30 minutes is shown by observing it under a fluorescence inverted microscope. Figure 5 In Figure A, we can see that the protein bubbles obtained by different modification methods are all close to spherical. Compared with the control group, the bubble size of group H was uneven at 0 min, resulting in strong light transmittance of the bubble and a darker visual field. The bubble size of the ultrasonic treatment group and the control group was not much different. At 0 min, the bubble size of groups E, HE, and UE was relatively uniform. At the same time, it was found that the foam of the control group, group H, and group U changed most significantly from 0 min to 30 min, with the bubbles in the visual field gradually increasing in size, while the foam of groups E, HE, and UE changed less during this time period.

[0128] from Figure 5 As shown in Figure B, the number of bubbles obtained by each modification method was the highest at 0 min throughout the entire time period, with the UE group reaching the maximum number (423), indicating that the control group had the best foaming properties. Over time, the number of bubbles in all groups decreased, likely due to the fusion or disproportionation of bubbles during the 0-30 min timeframe. Fusion occurs when two or more bubbles merge to form a larger bubble. Conversely, disproportionation occurs when bubbles migrate from smaller bubbles to larger ones, a process called disproportionation. This causes the small bubbles to gradually disappear while the larger bubbles continue to grow, resulting in a decrease in the number of bubbles. Furthermore, the H, E, HE, and UE groups experienced the largest decrease in number between 0 and 15 min, with the H group decreasing from 227 to 62, while the E, HE, and UE groups decreased by 202, 270, and 251, respectively, indicating that these four groups experienced the greatest degree of fusion or disproportionation during this timeframe. Conversely, the decrease was smaller between 15 and 30 min, indicating that the foam stability was better during this period, consistent with the foam stability results previously reported.

[0129] 17. Effect of modification on the emulsification properties of walnut protein

[0130] A 1% (w / v) solution of walnut protein before and after modification was magnetically stirred at room temperature for 30 minutes. 15 mL was mixed with 5 mL of soybean oil and sheared in a high-speed homogenizer for 1 minute. 50 μL of the emulsion at 0 and 10 minutes were taken and mixed with 5 mL of 0.1% SDS solution. The absorbance was measured at 500 nm using 0.1% SDS solution as the reference solution. The calculation formula is as follows:

[0131]

[0132]

[0133] Where: EA-emulsification; ES-emulsification stability; A0-initial emulsion absorbance; C-sample concentration, g / mL; - the proportion of oil phase in the emulsion; N- dilution multiple; A 10 -Absorbance of emulsion after 10 minutes

[0134] The emulsifying properties of proteins are very important for many food applications. They refer to the adsorption capacity of proteins at the water-oil interface. The emulsifying properties of proteins are usually evaluated by emulsifying capacity and emulsifying stability, which are used to measure the ability of proteins to form emulsions and describe their stability, respectively. The present invention found that compared with the Control group, H, UE, E, HE, and UE can improve emulsification, which is consistent with the solubility results. In addition, the UE group has the best emulsification and the largest increase in emulsification is 4.74m2 / g, which is 4.65 times that of the control group. It was also found that the emulsion stability of groups E, HE, and UE was significantly higher than that of groups H and U. This may be because ultrahigh pressure and ultrasonic treatment can cause protein aggregation and folding, thereby reducing the emulsion stability. Among them, group E had the highest emulsion stability of 3.35min.

[0135] 18. Effect of modification on water holding capacity of walnut protein

[0136] Weigh 2g of walnut protein sample into a 50mL centrifuge tube, add 40mL of ultrapure water, vortex to mix thoroughly, let stand at room temperature for 30 minutes, centrifuge at 8000r / min at 4℃ for 15 minutes, remove the supernatant, invert on filter paper for 1 minute, and reweigh the centrifuge tube. Repeat the measurement three times for each sample. Calculate according to the following formula:

[0137]

[0138] Where: W is water holding capacity; m1 is the weight of the centrifuge tube and sample (g); m0 is the weight of the dry sample (g); m2 is the weight of the centrifuge tube and sample pellet after centrifugation (g).

[0139] Water holding capacity is an important characteristic of food, especially protein. Water holding capacity represents the water binding capacity of protein, which can indicate the texture and viscosity of protein. During the production and processing, the use of ingredients with good water holding capacity can significantly improve the water retention capacity of the product, thereby having a positive impact on the quality, taste and storage stability of the product. The water holding capacity of the control group was 3.11g / g. After modification by different methods, the water holding capacity decreased to 2.96, 2.91, 0.99, 0.97, and 0.96g / g, respectively. Among them, the ultrasound-assisted alkaline protease treatment group had the lowest water holding capacity, which decreased by 2.15g / g.

[0140] 19. Effect of modification on oil retention of walnut protein

[0141] Weigh 0.5 g of walnut protein, add 5 mL of soybean oil, vortex to mix thoroughly, let stand at room temperature for 30 minutes, and centrifuge at 8000 rpm for 15 minutes at 4°C. Remove the supernatant, invert on filter paper for 1 minute, and weigh the centrifuge tube again. Calculate according to the following formula:

[0142]

[0143] Where: O-oil holding capacity; X1-weight of centrifuge tube and sample (g); X0-weight of dry sample (g); X2-weight of centrifuge tube and sample pellet after centrifugation (g).

[0144] The oil-holding capacity of proteins refers to the ability of their side chains to adsorb oil. Specifically, certain side chains in protein molecules can interact with oil, thereby absorbing and retaining oil. This property is particularly important in food processing as it affects the functional performance of proteins in oil-containing systems, such as emulsifying and stabilizing properties. The oil-holding capacity of the control group was 3.44 g / g. When treated with ultrasonic waves in combination with alkaline protease, the oil-holding capacity increased from 3.44 g / g to 3.92 g / g, indicating that ultrasonic waves and enzyme treatment can expose non-polar groups in the protein conformation, thus increasing the oil absorption rate. In addition, except for the ultra-high pressure treatment group where the oil-holding capacity showed no significant difference from the control group (P > 0.05), the oil-holding capacity of other treatment groups decreased, by 0.42, 0.54, and 0.80 g / g respectively.

[0145] 20. Effects of Modification on the Static Rheological Properties of Walnut Proteins

[0146] Apparent viscosity is a measure that describes the intermolecular friction of a liquid when subjected to an external force. The apparent viscosity of the samples decreased with the increase in shear rate, showing the characteristic of shear thinning, indicating that the samples were all non-Newtonian fluids. At a shear rate of 0.01 S -1 ~1000 S -1 range, the apparent viscosities from high to low were the UE group, HE group, E group, U group, Control group, and H group. At a shear rate of 0.01 S -1 When, at a shear rate of 0.01 S -1 When, the apparent viscosities of walnut proteins treated by the UE group and HE group were 4.16 times and 4.83 times that of the Control group respectively, indicating that these two modifications had the best effect on enhancing the viscosity of walnut proteins. In addition, the apparent viscosity of the H group was less than that of the Control group, indicating that ultra-high pressure treatment could reduce the viscosity of walnut proteins.

[0147] Since the protein samples conformed to the flow law of pseudoplastic fluids, the consistency index (K) and flow behavior index (n) of walnut protein samples were fitted based on the power-law equation. An increase in the K value indicates an increase in the consistency of the protein sample, while an increase in the n value indicates the degree of deviation of the sample from Newtonian fluids. For shear-thinning fluids: 0 < n < 1, approaching 0 indicates a high degree of non-Newtonian shear-thinning behavior. As can be seen from the figure, the K values of the UK group, HE group, and E group were significantly higher than that of the Control group, and the n values of other treatment groups were higher than that of the Control group except for the H group.

[0148] The shear stress of walnut proteins treated by different methods was affected by the shear rate, at 0.01 - 1000 S -1Within the range, with the increase of shear rate, the shear stress of walnut protein solutions obtained by different treatment methods showed an increasing trend, and the shear stress of different walnut proteins increased in the order of H group, Control group, U group, E group, HE group and UE group, indicating that the cavitation effect of ultrasound, the shearing effect of alkaline protease, and the interaction between ultra-high pressure extrusion and alkaline protease shearing exposed the hydrophobic groups inside the walnut protein, aggregated the protein molecules, enhanced the intermolecular friction of the protein during the flow process, and thus led to an increase in shear stress.

[0149] 21. Effect of modification on dynamic rheological properties of walnut protein

[0150] The storage modulus (G'), also known as the elastic modulus, is related to the elasticity of the foam, while the loss modulus (G") is the response value of viscosity. The loss angle tanδ (G" / G') is a factor that measures damping. When tanδ is greater than 1, the sample tends to be liquid, and when tanδ is less than 1, the sample maintains elasticity to a greater extent. Except for the HE group, the G′ value of walnut protein in other treatment groups first increases and then decreases with increasing frequency, while the G" value of walnut protein with different treatment methods increases with increasing frequency, indicating that G" is frequency-dependent. Except for the H group, the G' of walnut protein in other different treatment groups is always higher than G" in the frequency range of 1-100 rad / s, indicating that the sample is mainly elastically deformed. The E, HE and UE groups significantly increased the elastic modulus, thereby forming a more stable and lasting foam. The H group significantly reduced the elastic modulus, making it difficult to form a stable foam.

[0151] The G" viscosity increased in the U, E, HE, and UE groups, but decreased in the H group. This indicates that ultrasound, alkaline protease, ultrahigh pressure-assisted alkaline protease treatment, and ultrasound-assisted alkaline protease treatment significantly enhanced the structural integrity of walnut protein. The main reason is that the interaction between the composite treatments strengthened covalent bonds, such as disulfide bonds, and increased the elastic solidity of the solution. In the frequency range of 1-100 rad / s, the protein samples in the E, HE, and UE groups had similar G' and G" curves, and similar tanδ values. In the frequency range of 100-1000 rad / s, the G' and G" curves of the HE group showed an opposite trend to those of the E and UE groups, so the change trend of tanδ was also opposite.

[0152] 22. Effect of modification on thixotropy of walnut protein

[0153] Thixotropy describes the process of rebuilding the molecular structure after the reversible path is destroyed by shear. The three-stage shear rate cycle method was used to evaluate the thixotropic recovery properties of different walnut proteins. The results showed the trend of the change in the viscosity balance of walnut proteins treated with different methods during the 3-ITT period. Under low shear speed in the initial stage, the viscosity hardly changed; when the shear rate suddenly increased in the second stage, the strong shearing effect changed the structural arrangement of the walnut protein solution droplets, and the viscosity dropped sharply; when the shear rate in the third stage dropped back to the initial value, the viscosity quickly returned to the initial state. In the first and third stages, the walnut protein solution was at a low shear rate (0.1s -1 ) showed high viscosity under different treatment conditions, and thixotropic recoveries exceeded 70%, indicating that walnut protein obtained by different treatment methods still has good recovery properties. The Control group and Group H had the highest recoveries, at 122.97% and 130.89%, respectively. The reason for the viscosity recovery in thixotropic properties is that the shear reduction and the reconstruction of the network structure in the solution gradually establish the molecular structure and restore the viscosity.

[0154] The above results show that the solubility, emulsification, foaming and bubble number of the modified walnut protein are significantly improved, among which the ultrasound-assisted alkaline protease treatment has the best effect, which are 37.76%, 4.74%, 96.67% and 423 respectively.

[0155] Example 2

[0156] The modified walnut protein prepared in Example 1 was mixed with water to prepare a walnut protein liquid with a mass concentration of 10%; 5 mL of the above walnut protein liquid was mixed evenly with 45 mL of pure milk to obtain a walnut protein coffee foaming milk with a walnut protein liquid volume fraction of 10%. The walnut protein coffee foaming milk was heated to 60°C, whipped with an electric stirrer at 12000r for 1 minute, poured into a 250 mL graduated cylinder, and the foam volume was measured at 0 min, 15 min, and 30 min respectively. The foaming property and foaming stability were calculated according to the following formula:

[0157]

[0158] Where: FC-foamability; FS-foaming stability; V0-initial solution volume (mL); V-foam volume at 0 min after whipping (mL); V 15 - Foam volume 15 min after whipping (mL); V 30 - Foam volume (mL) 30 min after whipping.

[0159] The present invention also adjusts the volume fraction of walnut protein liquid in the walnut protein coffee foaming milk to 0, 2%, 6%, and 14% on the basis of Example 2 (that is, the only difference from Example 2 is that the volume fractions of walnut protein liquid in the walnut protein coffee foaming milk are 0, 2%, 6%, and 14%, respectively). The walnut protein coffee foaming milk prepared with different volume fractions of walnut protein liquid is respectively marked as WPM0 (pure milk, that is, the volume fraction of walnut protein liquid is 0), WPM2% (the volume fraction of walnut protein liquid is 2%), WPM6% (the volume fraction of walnut protein liquid is 6%), WPM10% (the volume fraction of walnut protein liquid is 10%), and WPM14% (the volume fraction of walnut protein liquid is 14%).

[0160] Test Example 2

[0161] The coffee foaming milk prepared in Example 2 was tested as follows:

[0162] 1. Effect of walnut protein addition on the foaming properties of foamed milk

[0163] The foaming properties of WPM0, WPM2%, WPM6%, WPM10% and WPM14% were 176.00%, 181.33%, 234.67%, 282.67% and 240.00% respectively. With the increase of the amount of walnut protein liquid added, the foaming properties first increased and then decreased. When the addition amount of walnut protein liquid was 10%, the foaming property of the mixed liquid reached the maximum value, which was significantly increased by 106.67% compared with WPM0 (P < 0.05). The foam stability after 15 minutes of standing after foaming also showed an initial increase followed by a decrease with increasing walnut protein liquid addition. The foam stability reached its maximum value (95.59%) with 2% WPM, indicating that only 4.41% of the liquid precipitated after 15 minutes of standing at room temperature. There was no significant change in the foam stability of the control group and the 14% WPM foam after 15 minutes of standing (P>0.05), indicating that the proportion of liquid precipitated after 15 minutes of standing in both groups was similar. Meanwhile, the foam stability of the 6% and 10% WPM foams significantly increased compared to the control group (P<0.05), increasing by 2.88% and 0.94%, respectively. The foam stability after 30 minutes of standing after 15 minutes showed no significant change compared to the control group, except for the 2% and 14% WPM foams (P>0.05). In summary, the 10% WPM foam addition exhibited the best foaming properties.

[0164] 2. Effect of walnut protein addition amount and time on foaming milk foam

[0165] The foam structure of different walnut protein coffee foams at 0, 15, and 30 minutes Figure 6As shown in Figure A, the foamed milk foam is the most uniform and has the largest number at 0 min. As time goes by, the average bubble size gradually increases and the number gradually decreases. This is most obvious in the WPM0 and WPM14% groups. The changes in the number and average size of bubbles in the remaining groups from 0 to 30 min are relatively small, indicating that the bubbles in the WPM0 and WP14% groups experience instability such as coalescence and drainage after a period of time, resulting in an increase in bubble size and a decrease in bubble number. In addition, the figure shows that the foam size of the WPM10% group is relatively uniform from 0 to 30 min.

[0166] Ideal coffee-specific frothed milk foam should have uniform bubble size and a dense texture. A uniform bubble diameter distribution provides better foam stability. Generally, larger bubble sizes are associated with lower foam stability, while smaller bubble sizes exhibit greater stability. Therefore, smaller foam sizes tend to be more stable. Figure 6 Middle B is the number of bubbles in different walnut protein coffee foamers. It can be seen from the figure that at 0 min, the number of bubbles in different groups is the largest, which are 228, 226, 217, 254 and 195 respectively; with the extension of time, the number of bubbles in all groups is gradually decreasing, among which the WPM0 group has the largest decrease in the range of 0-15 min and 15-30 min, from 228 to 180 and 180 to 113; on the contrary, the number of bubbles in the WPM10% group has a relatively constant change range in the range of 0-30 min, decreasing from 254 to 206 and 206 to 154, with a decrease of 48 and 52 in each interval respectively. Figure 6Figure C shows the average bubble diameters of different walnut protein coffee foams. The figure shows that the average diameter of each group gradually increases over time. The WPM0 group has the largest average diameter at different times, at 860.00, 956.67, and 1333.33 μm, respectively; while the WPM2% group has the smallest average diameter within this time range, at 666.67, 770.00, and 940.00 μm, respectively. Furthermore, the foam size of different walnut protein coffee foams changes differently over time. For the WPM0 and WPM2% groups, the change is small from 0 to 15 minutes, while the change amplitude is larger from 15 to 30 minutes. The WPM0 group expands from 956.67 μm to 1333.33 μm, an increase of 376.66 μm; the WPM2% group increases from 770.00 μm to 940.00 μm, an increase of 170.00 μm. Conversely, the WPM14% group showed a significant change from 740.00 μm to 936.67 μm between 0 and 15 minutes, expanding by 196.67 μm. The WPM6% and WPM10% groups changed at roughly the same rate throughout the entire time range, indicating that WPM6% and WPM10% have good foaming stability and exhibit minimal changes over a longer period of time, which is consistent with the results of the foaming properties of foamed milk mentioned above.

[0167] 3. Effect of walnut protein addition on the digestibility of foamed milk

[0168] The digestibility of protein is an important indicator to measure the degree of its decomposition in the body. A higher digestibility indicates that more protein can be effectively degraded, thereby improving the body's absorption and utilization of it. A higher digestibility means that more protein components can be broken down into absorbable small molecules, such as amino acids and small peptides, during the digestion process and fully utilized by the body. Since foamed milk stays in the mouth for a short time, oral digestion was not performed in this experiment. Figure 7 As shown in the results, during the gastric digestion stage, the digestibility of the foamed milk with 6% walnut protein solution was the highest (40.06%). There was no significant difference in digestibility between WPM6%, WPM10%, and WPM14% and the control group (P>0.05). The digestibility of the foamed milk with 2% addition was the lowest (33.19%), indicating that the foamed milk with 6% addition was easier to digest in the stomach than other foamed milks, while the 2% addition was the most difficult to digest. During the intestinal digestion stage, as the amount of walnut protein added increased, the protein digestibility showed an upward and then downward trend. When the addition amount was 10%, the digestibility reached the maximum (82.72%). As the addition amount continued to increase, the digestibility decreased. This shows that the appropriate amount of walnut protein can promote protein digestibility. When added in excess, walnut protein reacts with milk protein to produce indigestible substances, which leads to a decrease in the in vitro digestibility of protein.

[0169] 4. Effect diagram of mixing different walnut protein coffee foams with coffee

[0170] Figure 8 The finished products of different walnut protein coffee foams mixed with coffee are shown. All finished products were prepared using an espresso machine. Due to technical limitations, the milk foam was not very beautiful, and there were certain differences in the walnut protein foams with different addition amounts. As can be seen from the figure, compared with WPM0, the foam of the different foams changed less within the time period of 0 to 30 minutes. Among them, the WPM2% and WPM10% groups were the most dense and had smaller foam size, which could maintain good stability. Compared with the control group, the foam size of the WPM6% and WPM14% groups was too large and the foam distribution was uneven, affecting the aesthetics of the final product. The figure shows that the foam state of the WPM10% group was most similar to that of the control group.

[0171] 5. Effect of walnut protein addition on sensory evaluation of foamed milk and coffee mixture

[0172] WPMC0 (pure milk: coffee liquid = 36:150, v / v), WPMC2% (2% walnut protein coffee foaming milk: coffee liquid = 36:150, v / v), WPMC6% (6% walnut protein coffee foaming milk: coffee liquid = 36:150, v / v), WPMC10% (10% walnut protein coffee foaming milk: coffee liquid = 36:150, v / v) and WPMC14% (14% walnut protein coffee foaming milk: coffee liquid = 36:150, v / v).

[0173] Sensory evaluation of different walnut protein coffee foams mixed with coffee showed that the overall sensory scores of the WPMC0, WPMC2%, WPMC6%, WPMC10%, and WPMC14% groups were 70.67, 71.22, 74.11, 75.11, and 73.33, respectively. The coffee foam with a 10% walnut protein solution scored the highest overall sensory score. The WPMC10% group achieved the highest color score (26.11 points), showing a pure, uniform milk coffee color. The WPMC0, WPMC2%, WPMC6%, and WPMC14% groups all had similar color scores, with the first two groups showing a slightly darker milky white color and the last two groups showing a slightly darker coffee color. The aroma scores of walnut protein coffee foams with different walnut protein addition levels were similar, all exhibiting a balanced aroma of coffee and milk. The WPMC 6% group achieved the highest aroma score (15.67 points), while the WPMC 14% group had the lowest (14.78 points). The taste scores of the WPMC 14%, WPMC 10%, and WPMC 6% groups were similar, all exhibiting a harmonious blend of coffee and milk flavors, with a balanced bittersweet flavor. The WPMC 14% group had the highest score of all walnut protein coffee foams (18.78 points), while the WPMC 2% group had the lowest score (17.22 points), indicating a stronger milk flavor than coffee. The texture scores of all walnut protein coffee foams were similar, all exhibiting uniformity and no precipitation. The WPMC10% group had the highest overall preference score (7.00 points), and the WPMC0 group had the lowest score (5.89 points). The WPMC0 group was a mixture of pure milk and coffee. From the perspective of overall acceptance score, the WPMC10% score was more ideal.

[0174] 6. Effect of walnut protein addition on the electronic tongue of foamed milk coffee mixture

[0175] The SA-402B Plus electronic tongue is equipped with six sensors for sourness, bitterness, astringency, umami, saltiness, and sweetness. By measuring the response values ​​of different walnut protein coffee foam milks mixed with coffee on these six sensors, radar analysis maps were created. This method visually displays the characteristics of each mixture across various taste dimensions, facilitating comparison and analysis of flavor differences. The maps show that the sourness sensor response values ​​for the different mixtures were less than 0, indicating that this flavor had little effect on the different mixtures. The response values ​​were ranked as follows: WPMC0 > C > WPMC2% > WPMC6% > WPMC10% > WPMC14%. The WPMC0 group had the highest sweetness sensor response value (1.11); the C group had the lowest response value (-3.86), indicating that the C group had a lower sugar content and was less sensitive to sweetness. The sweetness response values ​​for the remaining groups ranked as WPMC2% > WPMC6% > WPMC14% > WPMC10%. The response values ​​of group C were the highest among the seven tastes: bitterness, astringency, umami, saltiness, umami, bitter aftertaste, astringency aftertaste and umami aftertaste, which were 13.37, 8.55, 14.46, 18.35, 7.77, 4.71 and 8.08 respectively. Among them, saltiness was the most obvious, indicating that the coffee liquid may contain relatively high sodium ions (Na + ) or other ions that can cause salty perception. In terms of umami characteristics, WPMC14% had the highest response value, followed by WPMC10%; in terms of mouthfeel, WPMC0 had the highest response value, followed by WPMC10%, and WPMC14% had the lowest; this shows that WPMC10% has a significant advantage in maintaining umami, while WPMC14% is the worst. In terms of bitterness, aftertaste and astringency, there was no significant difference among the WPMC0, WPMC2%, WPMC6%, WPMC10% and WPMC14% groups. This may be because the bitter flavor components are relatively stable after adding different foaming milks to the coffee liquid. Among them, WPMC10% has the lowest response value in bitterness characteristics, and is at a medium level in aftertaste and astringency characteristics. In summary, WPMC10% has better flavor characteristics, showing advantages in reducing sourness, bitterness and astringency, and maintaining flavor coordination. It has both coffee flavor and milk flavor, which is consistent with the sensory evaluation results mentioned above, and has the highest overall acceptance.

[0176] 7. Effect of walnut protein addition on the electronic nose of foaming milk coffee mixture

[0177] The WPMC2% group was more sensitive to nitrogen oxides than the other groups, while the WPMC14% group was the least sensitive. The flavor characteristics of hydrogen compounds were the most obvious in the WPMC14% group, while the WPMC10% group was the least obvious.

[0178] 8. Analysis of volatile components in the mixture of walnut protein coffee foam and coffee liquid

[0179] HS-GC-MS was used to analyze and identify the volatile compounds in coffee liquid, pure milk, 10% walnut protein coffee foam (10% walnut protein liquid: pure milk, v / v), a pure milk coffee mixture, and a mixture of 10% walnut protein coffee foam and coffee. Compounds with matching degrees greater than 500 and 1000 were screened to identify the types of volatile compounds in the samples and to quantify the volatile compounds. The results showed that Group C (coffee liquid) was rich in volatile compounds, with 46 volatile compounds detected and qualitatively identified. These 46 compounds primarily included 9 ketones, 7 esters, 6 furans, 6 pyrazines, 4 alcohols, and 14 other compounds. This is consistent with the results of the electronic nose analysis mentioned above. The volatile compounds in Group C were mainly ketones, esters, furans, and pyrazines, which is consistent with the basic distribution of volatile compounds in coffee. There was little difference in the types and contents of volatile substances in the WPM0 (pure milk) and WPM10% (10% walnut protein solution: pure milk, v / v) groups, which were ketones, alcohols, acids and other substances respectively; the volatile compounds in WPM0 and WPM10% were mainly ketones, namely 4-octanone, 2-octen-4-one and 3-methyl-2-heptanone, which are often used in fruit flavors because of their fruity, sweet and grassy aromas; 2,3-dimethylbutane-2,3-diol was detected in WPM10% compared with WPM0, giving it a slightly sweet and woody aroma. The types of volatile substances in the WPMC0 (pure milk: coffee liquid = 36:150, v / v) and WPMC10% (10% walnut protein coffee foaming milk: coffee liquid = 36:150, v / v) groups were slightly different, but were richer than those in the WPM0 and WPM10% groups, with 14 more compounds added. This indicates that after mixing with coffee liquid, the volatile substances of the two solutions were combined, thereby increasing the types of volatile substances in WPMC0 and WPMC10%; WPM The contents of ketones, esters, pyrazines and alcohols in C10% are all higher than those in WPMC0. Among ketones, 4-octanone and 1-hydroxy-2-propanone are detected in WPMC10%, giving it a creamy and caramelized sweet aroma compared to WPMC0. The methyl acetate content in WPMC10% is higher, giving it a richer and sweeter fruity aroma. Among furans and pyrazines, 5-methylfurfural and 2,6-dimethylpyrazine are detected in WPMC10%, giving it a caramelized, spicy and nutty aroma.

[0180] In order to further analyze the changes in the types of volatile compounds in the C, WPM0, WPM10%, WPMC0 and WPMC10% groups, a stacking diagram was made according to the analysis data of volatile substances in the mixture of coffee and foamed milk. The results showed the distribution of ketones, esters, furans, pyrazines and other compounds in the C, WPM0, WPM10%, WPMC0 and WPMC10% groups. According to the types of compounds measured, it can be seen that the composition of compounds contained in the WPM0 group and the WPM10% group is similar, but the quantity is different. There are 3 ketones in both WPM0 and WPM10%, but there are 8 other substances in WPM0, while there are 7 other substances and 1 alcohol in WPM10%. This shows that the addition of 10% walnut protein solution has little effect on the overall volatile substances of foamed milk. The compound composition of the WPMC0 and WPMC10% groups was slightly different from that of the C group, but the difference in quantity was large. It was found that WPMC0 and WPMC10% combined the types of compounds of C, WPM0 and WPM10%, but the quantity was reduced. In addition, no furan compounds were detected in the WPMC0 group, indicating that a reaction occurred after the milk coffee was mixed, causing the disappearance of such compounds or the disappearance of such compounds due to their low content.

[0181] 9. Particle size analysis of walnut protein coffee foam

[0182] The average particle size of the sample is expressed as D(4,3). The smaller the average particle size, the smaller the protein particles and fat globules in the product, the more uniform the distribution, and the better the product stability. Figure 9 As shown in the figure, the particle sizes of coffee liquid, walnut protein milk, and walnut protein milk coffee show an overall normal distribution. Except for Group C, the distribution trends of the other four groups of samples are basically the same, and the particle distribution of these systems is relatively uniform. Group C has the largest average particle size D(4,3), while the average particle sizes of WPM0, WPM10%, WPMC0, and WPMC10% do not differ significantly (P>0.05). This indicates that the stability of the foamed milk with the addition of 10% walnut protein liquid is similar to that of the control group, making it an ideal coffee foamed milk. The stability of the walnut protein milk coffee is similar to that of milk coffee, and the final finished liquid is ideal.

[0183] 10. Zeta-potential analysis of walnut protein coffee foam

[0184] Zeta potential is one of the important evaluation indicators of colloidal stability; the stability of milk is closely related to Zeta potential. Under normal circumstances, the surface of casein micelles in milk is negatively charged, and its Zeta potential ranges from -10mV to -30mV. When the absolute value reaches about 25mV, the system is considered to be relatively stable. Figure 10 As shown in the figure, C, WPM0, and WPM10% all carry negative charges, with potentials of -13.00mV, -26.63mV, and -24.60mV, respectively. When C, WPM0, and WPM10% are mixed, the absolute value of the potential of the WPMC0 group decreases, while the absolute value of the potential of the WPMC10% group reaches a maximum of 26.80mV.

[0185] The above results show that when the walnut protein liquid volume fraction is 10% (v / v), the foaming property, foam stability, bubble number, digestibility and sensory evaluation of the walnut protein coffee foamed milk are all maximum, which are 282.67%, 84.43%, 254, 82.72% and 75.11 points respectively.

[0186] This study uses walnut protein as raw material, optimizes the optimal conditions for processing walnut protein using different modification methods, and studies the structural, rheological, and functional properties of walnut protein treated with different modification methods, as well as the development of walnut protein coffee foam. The following conclusions are drawn:

[0187] (1) The optimal process for ultrahigh pressure treatment of walnut protein was as follows: treatment time of 14 min, ultrahigh pressure treatment pressure of 188 MPa, and walnut protein treatment amount of 20 g. Under these conditions, the solubility was 18.91%. The optimal power for ultrasonic treatment of walnut protein was 600 W. Under this condition, the solubility, foaming property and foam stability were 12.15%, 49.33% and 55.39%, respectively. The optimal enzyme addition amount for alkaline enzyme treatment of walnut protein was 0.10%. Under this condition, the solubility, foaming property and foam stability were 31.11%, 82.67% and 90.32%, respectively. The final modification methods and conditions were ultra-high pressure treatment (treatment time of 14 min, treatment pressure of 188 MPa and treatment volume of 20 g), ultrasonic treatment (5% walnut protein solution, power of 600 W and time of 15 min), alkaline protease treatment (walnut protein concentration of 5%, temperature of 50°C, pH of 9.0 and enzymatic hydrolysis time of 2 h), ultra-high pressure synergistic alkaline protease treatment (treatment time of 14 min, pressure of 188 MPa, walnut protein treatment volume of 20 g, 5% walnut protein concentration, alkaline protease addition of 0.10%, temperature of 50°C, pH of 9.0 and enzymatic hydrolysis time of 2 h), and ultrasonic synergistic alkaline protease treatment (walnut protein concentration of 5%, ultrasonic power of 600 W, ultrasonic time of 15 min, alkaline protease addition of 0.10%, temperature of 50°C, pH of 9.0 and enzymatic hydrolysis time of 2 h).

[0188] (2) Scanning electron microscopy results showed that the untreated walnut protein showed a loose and porous spherical structure; after ultrahigh pressure treatment, it showed an irregular and compact flake structure; after ultrasonic treatment, the walnut protein showed a smooth, compact and porous spherical structure; the walnut protein treated by alkaline protease, ultrahigh pressure combined with alkaline protease, and ultrasonic combined with alkaline protease showed a smooth and compact flake structure with protruding globular protein particles on the surface. SDS-PAGE gel electrophoresis analysis results showed that the protein subunit content of the ultrahigh pressure treatment group and the ultrasonic treatment group changed little in the range of 8-68kDa; the proportion of subunits in the 55-68kDa and 30-40kDa groups treated by alkaline protease, ultrahigh pressure combined with alkaline protease, and ultrasonic combined with alkaline protease decreased significantly, by 7.65%, 8.20%, 9.00%, 4.75%, 5.38%, and 5.43%, respectively. In contrast, the contents of the 13-20 kDa and 8-13 kDa subunits increased significantly, reaching 30.67%, 30.67%, 31.39%, 28.90%, 30.09%, and 30.22%, respectively. Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed changes in the secondary structure of the modified walnut protein. The β-sheet content decreased, while the β-turn content increased. The β-sheet content was lowest at 34.99% in the ultrasound-assisted alkaline protease treatment group, while the β-turn content was highest at 27.33%. Compared with the untreated group, the UV absorbance of the modified walnut protein increased, reaching a maximum of 0.42 in the ultrahigh pressure-assisted alkaline protease treatment group. Fluorescence intensity was enhanced in all groups except the ultrasound-treated group. Furthermore, ultrahigh pressure and ultrasound modification resulted in a trimodal distribution of walnut protein particle size, while alkaline protease, ultrahigh pressure-assisted alkaline protease, and ultrasound-assisted alkaline protease modifications exhibited a bimodal distribution, with no significant effect on the potential. The contact angle of walnut protein decreased after modification, with the lowest contact angle reaching 55.74° after ultrahigh pressure and alkaline protease treatment. Except for ultrahigh pressure and ultrasound treatment, the surface hydrophobicity and free thiol groups of walnut protein treated with other modification methods increased. The highest surface hydrophobicity reached 25192.67 after ultrasound and alkaline protease treatment, and the highest free thiol group reached 4.14 μmoL / g after alkaline protease treatment. Modification had no effect on the crystallinity and amino acid composition of walnut protein, and had no effect on the denaturation temperature (T d ) had little effect; except for the ultrahigh pressure group, the thermal enthalpy (ΔH) decreased in all other groups, with the lowest value of 193.80 J / g in the alkaline protease treatment.

[0189] (3) The solubility, emulsification, foaming and bubble number of the modified walnut protein were significantly improved, among which the ultrasound-assisted alkaline protease treatment had the best effect, which were 37.76%, 4.74%, 96.67% and 423 respectively. The emulsification stability and foaming stability of the modified walnut protein were significantly improved except for ultra-high pressure treatment and ultrasound treatment. The emulsification stability of alkaline protease treatment was the highest at 3.35min, and the foaming stability of ultra-high pressure assisted alkaline protease treatment was the highest at 95.31%. The water holding capacity showed a downward trend, with the ultrasound-assisted alkaline protease treatment group having the lowest value of 0.96g / g. The oil holding capacity showed a downward trend except for the ultrasound-assisted alkaline protease treatment group, with the ultra-high pressure assisted alkaline protease treatment group having the lowest value of 2.65g / g.

[0190] (4) The apparent viscosity of the modified walnut protein solution decreases with increasing shear rate, and it is a shear-thinning pseudoplastic fluid with certain thixotropic properties, and the thixotropic recovery rate is greater than 70%. The dynamic viscoelastic behavior of the modified walnut protein solution shows that, except for the ultrahigh pressure treatment group, the storage modulus (G') of the protein solution of other modified groups is always greater than the loss modulus (G"), and the solution foam appears solid. In addition, ultrasound, alkaline protease, ultrahigh pressure synergistic alkaline protease and ultrasound synergistic protease treatment increase the elasticity of the solution foam and improve the foam stability.

[0191] (5) Using pure milk as raw material, coffee foam containing 0% to 14% walnut protein (walnut protein solution: milk, v / v) was prepared. When the walnut protein solution was added at a concentration of 10% (v / v), the foaming property, foam stability, bubble number, digestibility, and sensory evaluation all reached their maximum values, namely 282.67%, 84.43%, 254, 82.72%, and 75.11 points, respectively. Electronic tongue and electronic nose tests of C (coffee solution), WPM0 (pure milk), WPM10% (10% walnut protein solution: pure milk, v / v), WPMC0 (pure milk: coffee solution = 36:150, v / v), and WPMC10% (10% walnut protein coffee foam: coffee solution = 36:150, v / v) revealed significant flavor differences between the samples. Further HS-GC-MS analysis revealed that WPM10% contained 2,3-dimethylbutane-2,3-diol compared to WPM0, contributing to its slightly sweet and woody aroma. WPMC10% contained higher levels of ketones, esters, pyrazines, and alcohols than WPMC0. Among the ketones, WPMC10% contained more 4-octanone and 1-hydroxy-2-propanone than WPMC0, contributing to its creamy and caramelized sweet aromas. WPMC10% also had a higher level of methyl acetate, resulting in a richer and sweeter fruity aroma. Among the furans and pyrazines, WPMC10% contained 5-methylfurfural and 2,6-dimethylpyrazine, contributing to its caramelized, spicy, and nutty aromas. By measuring the particle size and Zeta-potential of C, WPM0, WPM10%, WPMC0 and WPMC10%, it was found that there was no significant difference in the particle size of WPM0 and WPM10%; while the absolute value of the Zeta-potential of WPM10% was smaller than that of WPM0, but still around 25mV, indicating that WPM10% had better stability.

[0192] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A walnut protein modification method, characterized in that: The following steps are involved: The walnut protein powder is mixed with water and then ultrasonically treated, and then alkaline protease is added for enzymolysis, and the enzyme is inactivated, followed by cooling and freeze-drying to obtain modified walnut protein.

2. The walnut protein modification method according to claim 1, characterized in that The mass volume ratio of the walnut protein powder to water is 1 g:mL; the power of the ultrasonic treatment is 400-800 W, the time is 15 minutes, and the ultrasonic treatment is performed for 5 seconds with a rest period of 5 seconds.

3. The walnut protein modification method according to claim 1, characterized in that The addition amount of the alkaline protease is 0.07% to 0.3% of the mass of the walnut protein powder; the enzymatic hydrolysis conditions are: pH 9.0, temperature 50° C., and enzymatic hydrolysis for 2 hours.

4. Modified walnut protein obtained according to the walnut protein modification method according to any one of claims 1 to 3.

5. A walnut protein coffee foaming milk, characterized in that: The invention comprises the modified walnut protein according to claim 4 and milk.

6. A method for preparing the walnut protein coffee foamed milk according to claim 5, characterized in that: The following steps are involved: The modified walnut protein is mixed with water to obtain a walnut protein liquid; the walnut protein liquid is mixed with milk to obtain the walnut protein coffee foaming milk.

7. The preparation method according to claim 6, characterized in that The mass concentration of the walnut protein liquid is 10%.

8. The preparation method according to claim 6, characterized in that The volume fraction of the walnut protein liquid in the walnut protein coffee foaming milk is 2% to 14%.

9. Use of the modified walnut protein according to claim 4 or the walnut protein coffee foaming emulsion according to claim 5 in preparing coffee liquid.

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