Method for preparing barley leaf protein with high foaming stability by using sonochemistry-assisted alkaline process and application of barley leaf protein

Abstract

The invention relates to a method for preparing barley leaf protein with high foaming stability by using a sonochemistry-assisted alkaline method and application of the barley leaf protein. The preparation scheme comprises the following steps: firstly, optimizing alkali extraction and acid precipitation conditions through a single-factor experiment; secondly, combining ultrasonic treatment, extracting, centrifuging and collecting supernate; then, adjusting the pH value of the supernate to an isoelectric point of the supernate, and carrying out acid precipitation for 24 hours; and after the acid precipitation is completed, adjusting the pH to redissolve the protein, and then dialyzing and freeze-drying to finally obtain the barley leaf protein. Compared with a pure alkali extraction and acid precipitation method, the method has the advantages that the Zeta potential absolute value of the barley leaf protein prepared by the method is less than 25mV, the particle size is greater than 2900nm, the fluorescence intensity exceeds 290, and the foaming stability is improved to 27%. The method is environment-friendly and efficient, can extract novel vegetable protein with high foaming stability from barley leaves, is suitable for making products such as food, beverage and ice cream with rich foam, and effectively improves the taste and quality of the products.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method for extracting high-foaming stability barley leaf protein by using sound chemistry assisted alkali method and its application, and belongs to the field of food-derived leaf protein extraction and functional property research. BACKGROUND

[0002] Barley (Hordeum vulgare) is an important cereal crop widely planted around the world. During its growth and harvesting, a large amount of by-products, barley leaves, are produced. These leaves are widely present in many temperate agricultural regions around the world, although they do not have a long history of direct use like some traditional medicinal plants or specialty economic crops. They are an objective resource in agricultural production. With the increasing emphasis on resource recycling and sustainable development in modern agriculture, and the deepening exploration of new plant protein sources in food technology, the value of barley leaves is being reevaluated. Currently, the utilization of barley leaves is still in the exploratory stage, and people not only want to preserve their possible traditional application value, but also strive to develop their high-value active ingredients. However, to truly realize their high-value utilization, a key challenge is how to efficiently and high-quality extract proteins from them.

[0003] Barley leaves have a considerable protein content and a balanced amino acid composition, which makes them a potential source of high-quality food protein, making them a focus of research. However, the reality is that the cell wall structure of barley leaves is quite tough, like a natural barrier, making it difficult and inefficient to extract proteins directly from them. Moreover, there are naturally occurring anti-nutritional factors such as polyphenols and lignin in barley leaves, as well as pigments such as chlorophyll. These substances not only interfere with the extraction process of proteins, but also affect the color, flavor, and functional properties of the final protein product, such as imparting a green color, bitter taste, and possibly weakening its emulsifying and solubility properties. It is particularly worth noting that the foaming properties of proteins are a key functional indicator for their widespread application in the food industry, such as making foam foods, beverages, and ice cream. However, existing extraction methods often fail to effectively preserve or enhance this property. Therefore, how to overcome these obstacles and develop more optimized extraction techniques to obtain barley leaf proteins with white color, good flavor, and excellent functional properties (especially foaming properties) is a technical problem that needs to be solved.

[0004] Currently, the common methods for extracting proteins from plant materials include alkali extraction and acid precipitation, enzymatic hydrolysis, etc. Among them, (1) alkali extraction and acid precipitation is widely used due to its relatively simple operation and low cost. The principle of this method is to take advantage of the high solubility of proteins at a specific pH (usually alkaline) to dissolve them from plant cells, and then precipitate the proteins by adjusting the pH to the isoelectric point, thereby achieving extraction. However, although the traditional alkali extraction and acid precipitation method is relatively simple in operation, it is not perfect. It can cause partial protein denaturation and inactivation during the extraction process, and puts higher requirements on the subsequent purification steps. In addition, the extraction efficiency is also deeply affected by various factors such as the processing method of the raw material, the pH of the extraction solution, the temperature, the time, and the solid-liquid ratio, which means that these conditions need to be finely optimized to obtain ideal protein yield and quality.

[0005] To overcome the limitations of traditional alkali extraction, existing research has introduced various auxiliary technologies, among which sonication-assisted technology has attracted attention due to its unique physical effects. (2) Sonication-assisted refers to the use of cavitation effects generated by ultrasonic waves to assist the extraction process. Ultrasonic waves can generate cavitation effects when propagating in a liquid, forming tiny bubbles that release a large amount of energy during growth and collapse, which can destroy plant cell walls and membrane structures, making it easier for proteins to be released from cells, increasing the contact area between the solvent and the raw material, and promoting the release and dissolution of proteins, thereby improving extraction efficiency. (3) In addition, the non-thermal effects of ultrasonic waves may help maintain the natural conformation of proteins or improve their unfolding degree, exposing more hydrophobic groups, thereby improving the functional properties of proteins, such as increasing their solubility, foaming ability, and foam stability. Although sonication-assisted alkali extraction technology has been applied in the extraction of other plant proteins, there is still a lack of research on the systematic optimization and preparation of barley leaf proteins using sonication-assisted alkali extraction, especially for barley leaf proteins with high foaming stability. (Comprehensive Reviews in Food Science and Food Safety, 2021, 20(2): 1457-80.)(Journal of Food Process Engineering, 2024, 47(10): e14758.)

[0006] Therefore, there is an urgent need to develop a preparation method that can effectively combine the advantages of ultrasonic cavitation and alkali extraction, improve the extraction rate of barley leaf proteins, and significantly enhance their foaming stability. Based on the above background, the present invention provides a sonication-assisted alkali extraction process, which aims to provide a new technical solution for obtaining high-quality, high-extraction-rate, and high-foaming-stability barley leaf proteins, and to open up new avenues for their widespread application in the food industry. SUMMARY

[0007] The present application is directed to the limitations of conventional preparation techniques, and proposes a method for preparing high-foaming stability barley leaf protein using acoustic chemistry assisted alkali method. Specifically, the present application first optimizes the key parameters of the traditional alkali extraction and acid precipitation method, including solid-liquid ratio, extraction pH, extraction temperature and extraction time, through systematic single factor experiment. This step aims to determine the basic extraction condition range that can obtain higher protein yield under the condition of no ultrasonic assistance, and to provide a benchmark for the subsequent ultrasonic cavitation assisted step.

[0008] The present application is implemented by the following technical solutions:

[0009] The present application provides a method for preparing high-foaming stability barley leaf protein using acoustic chemistry assisted alkali method, comprising the following steps:

[0010] Step 1) Mix the barley leaf powder according to a certain solid-liquid ratio, stir, and obtain a barley leaf aqueous solution;

[0011] Step 2) Alkali extraction of the barley leaf aqueous solution obtained in step 1);

[0012] Step 3) Centrifuge and collect the supernatant of the protein extraction solution obtained in step 2) under the condition of 4-25℃;

[0013] Step 4) Adjust the pH of the supernatant obtained in step 3) to pH 3.8-4.5 for acid precipitation;

[0014] Step 5) Adjust the pH of the solution obtained in step 4) to 6-8 to resolubilize the protein;

[0015] Step 6) Freeze the protein solution obtained in step 5) under the condition of -80--70℃, and then vacuum dry to obtain barley leaf protein;

[0016] Before step 6), the protein solution obtained in step 5) needs to be dialyzed for 24-48 hours;

[0017] Further, ultrasonic treatment is applied during the extraction process of step 1) and step 2).

[0018] Further, the solid-liquid ratio of the barley leaf powder and distilled water in step 1) is 1:10-1:30 (g / mL).

[0019] Further, the pH of the barley leaf extraction solution in step 2) is 9.0-12.0.

[0020] Further, the extraction temperature of the barley leaf in step 2) is 40-55℃.

[0021] Further, the extraction time of the barley leaves in step 2) is 60-120 minutes.

[0022] Further, the centrifugal condition in step 3) is 7000-9000 rpm, 5-15 min; further, the centrifugal condition is 8000 rpm, 10 min.

[0023] Further, steps 3), 4), and 5) are carried out at 4-25℃; the dialysis is carried out at 4℃.

[0024] Further, the ultrasonic treatment is carried out in the alkali extraction process in step 2), the fixed frequency is 40 kHz, and the ultrasonic treatment time is 60-120 minutes.

[0025] The application further provides a barley leaf protein prepared by the above method.

[0026] Further, the barley leaf protein has higher protein content, the protein content can reach 43%; the foam stability is significantly higher; the foam stability value is increased to 27%; the absolute value of Zeta potential of the barley leaf protein is less than 25 mV, the protein particle size is greater than 2900 nm; and the fluorescence spectrum intensity is greater than 290.

[0027] The application further provides an application of the barley leaf protein in preparation of food and medicine, in particular, as an additive with excellent foam stability.

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

[0029] (1) The application improves the extraction efficiency of the barley leaf protein; compared with the barley leaf protein prepared by the simple alkali extraction and acid precipitation method without ultrasonic assistance, the barley leaf protein prepared by the method of the application has higher protein extraction rate (up to 32%).

[0030] (2) The application significantly improves the foam stability of the obtained protein; compared with the barley leaf protein prepared by the simple alkali extraction and acid precipitation method without ultrasonic assistance, the foam stability value of the barley leaf protein prepared by the method of the application is increased to 27%.

[0031] (3) Compared with the barley leaf protein prepared by the simple alkali extraction and acid precipitation method without ultrasonic assistance, the absolute value of Zeta potential of the barley leaf protein prepared by the method of the application is smaller (less than 25 mV).

[0032] (4) Compared with the barley leaf protein prepared by the simple alkali extraction and acid precipitation method without ultrasonic assistance, the particle size of the barley leaf protein prepared by the method of the application is larger (greater than 2900 nm).

[0033] (5) Compared with the barley leaf protein prepared by the simple alkali extraction and acid precipitation method without the use of ultrasonic assistance, the barley leaf protein prepared by the method has greater fluorescence intensity (greater than 290).

[0034] (6) The ultrasonic treatment is synchronously performed in the alkali extraction process, the cavitation effect and mechanical shearing force generated by the ultrasonic wave are used to more effectively destroy the tough cell wall structure of the barley leaf, promote the dissolution of the protein from the cell interior into the extraction liquid, and possibly change the secondary or tertiary structure of the protein, so that the subsequent functional properties, in particular the foaming stability, are improved.

[0035] (7) The ultrasonic frequency adopted in the method is fixed as 40 kHz, and the treatment time is controlled in the range of 60-120 minutes, which is a preferred interval selected on the basis of experiments and capable of significantly improving the foaming stability and taking into account the energy consumption and efficiency.

[0036] (8) The barley leaf protein prepared by the method provides a new type of plant protein source with excellent performance for the food and drug industries.

[0037] (9) The preparation of the barley leaf protein by the method helps to improve the economic value of the barley leaf. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Results of the influence of the pH values of the comparative examples 1-4 on the extraction rate of the barley leaf protein.

[0039] Figure 2 Results of the influence of the solid-liquid ratios of the comparative examples 5-7 on the extraction rate of the barley leaf protein.

[0040] Figure 3 Results of the influence of the extraction time of the comparative examples 8-10 on the extraction rate of the barley leaf protein.

[0041] Figure 4 Results of the influence of the extraction temperature of the comparative examples 11-13 on the extraction rate of the barley leaf protein.

[0042] Figure 5 Results of the influence of the ultrasonic extraction time of the examples 1-3 on the extraction rate of the barley leaf protein.

[0043] Figure 6 Results of the influence of the extraction rate of the barley leaf protein of the examples 1, 4 and 5.

[0044] Figure 7 Results of the content and extraction rate of the barley leaf protein prepared by the comparative example 15 and the example 1.

[0045] Figure 8 Results of the foaming stability and stability of the barley leaf protein prepared by the comparative example 15 and the example 1.

[0046] Figure 9 Zeta potential and particle size determination results of barley leaf protein prepared for Comparative Example 15 and Example 1.

[0047] Figure 10 Fluorescence spectrum determination results of barley leaf protein prepared for Comparative Example 15 and Example 1. DETAILED DESCRIPTION

[0048] The specific implementation of the present application is further illustrated below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present application are not limited thereto. It should be noted that if the following processes are not specifically detailed, they can be implemented or understood by those skilled in the art with reference to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be obtained by commercial purchase.

[0049] Comparative Examples 1-4

[0050] (1) Four portions of barley leaf raw material powder were accurately weighed, each with a mass of 2.00 grams. Distilled water was quantitatively added to each portion of powder according to a solid-liquid ratio of 1:10 (g / mL) to prepare an extraction solution;

[0051] (2) The pH values of the four extraction solutions were precisely adjusted to 9.0, 10, 11, and 12, respectively, using 1M NaOH;

[0052] (3) Each extraction solution was placed in a 45°C constant-temperature water bath shaker for constant-temperature vibration extraction, with a duration of 2 hours;

[0053] (4) After the extraction was completed, the supernatant was collected by centrifugation at a speed of 8000 rpm for 10 minutes in a 4°C environment;

[0054] (5) The supernatant obtained in step (4) was taken and its pH value was precisely adjusted to 4.5 using 1M HCl;

[0055] (6) After the adjustment was completed, the solution was placed in a 4°C environment for 24h acid precipitation;

[0056] (7) After the acid precipitation was completed, the precipitated protein was collected by centrifugation again for 10 minutes (8000 rpm, 4°C);

[0057] (8) An equal volume of distilled water was added to step (1), and 1M NaOH was slowly stirred and added dropwise to adjust the pH to 7.0 to resuspend the protein;

[0058] (9) The concentrated protein solution was loaded into a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0059] (10) After dialysis, the protein solution was transferred to a plate and placed in a -80°C ultra-low temperature freezer. After freezing, the sample was freeze-dried for 24 hours until the sample was completely dried, and finally the protein powder was obtained.

[0060] Comparative Examples 5-7

[0061] (1) Three portions of barley leaf raw powder were accurately weighed, each with a mass of 2.00 grams. According to the solid-liquid ratio of 1:10, 1:20 and 1:30 (g / mL), distilled water was quantitatively added to each portion of powder to prepare the extract;

[0062] (2) 1M NaOH was used to accurately adjust the pH of the three extracts to 12.0, respectively;

[0063] (3) Each extract was placed in a 45°C constant temperature water bath shaker for constant temperature oscillation extraction, with a duration of 2 hours;

[0064] (4) After extraction, centrifugation was performed at 8000 rpm for 10 minutes in a 4°C environment, and the supernatant was collected;

[0065] (5) The supernatant obtained in step (4) was taken and its pH was accurately adjusted to 4.5 using 1M HCl;

[0066] (6) After adjustment, the solution was placed in a 4°C environment for 24 hours of acid precipitation;

[0067] (7) After acid precipitation, the precipitated protein was collected by centrifugation again for 10 minutes (8000 rpm, 4°C);

[0068] (8) An equal volume of distilled water was added to step (1), and 1M NaOH was added dropwise while stirring to adjust the pH to 7.0 to resuspend the protein;

[0069] (9) The concentrated protein solution was placed in a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0070] (10) After dialysis, the protein solution was transferred to a plate and placed in a -80°C ultra-low temperature freezer. After freezing, the sample was freeze-dried for 24 hours until the sample was completely dried, and finally the protein powder was obtained.

[0071] Comparative Examples 8-10

[0072] (1) Three portions of barley leaf raw powder were accurately weighed, each with a mass of 2.00 grams. According to the solid-liquid ratio of 1:30 (g / mL), distilled water was quantitatively added to each portion of powder to prepare the extract;

[0073] (2) Using 1M NaOH, the pH value of each of the four extracts was precisely adjusted to 12.0;

[0074] (3) Each extract was placed in a 45°C constant temperature water bath shaker and subjected to constant temperature oscillation extraction for 60, 90 and 120 minutes;

[0075] (4) After the extraction was completed, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes in a 4°C environment;

[0076] (5) The supernatant obtained in step (4) was taken and its pH value was precisely adjusted to 4.5 using 1M HCl;

[0077] (6) After the adjustment was completed, the solution was placed in a 4°C environment and allowed to stand for 24 hours for acid precipitation;

[0078] (7) After the acid precipitation was completed, the precipitated protein was collected by centrifugation for 10 minutes (8000 rpm, 4°C);

[0079] (8) An equal volume of distilled water was added to step (1), and 1M NaOH was slowly added dropwise to adjust the pH to 7.0 to resuspend the protein;

[0080] (9) The concentrated protein solution was loaded into a dialysis bag and placed in a 4°C refrigerator for dialysis for 48 hours;

[0081] (10) After the dialysis was completed, the protein solution was transferred to a plate and placed in a -80°C ultra-low temperature freezer for freezing. The sample was then subjected to freeze-drying treatment for 24 hours until the sample was completely dried, and finally a protein powder was obtained.

[0082] Comparative Examples 11-14

[0083] (1) Four portions of barley leaf raw powder were accurately weighed, each with a mass of 2.00 grams. Distilled water was added to each portion of powder according to a solid-liquid ratio of 1:30 (g / mL) to prepare an extract;

[0084] (2) Using 1M NaOH, the pH value of each of the four extracts was precisely adjusted to 12.0;

[0085] (3) Each extract was placed in a 40°C, 45°C, 50°C and 55°C constant temperature water bath shaker and subjected to constant temperature oscillation extraction for 90 minutes;

[0086] (4) After the extraction was completed, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes in a 4°C environment;

[0087] (5) The supernatant obtained in step (4) was taken and its pH value was precisely adjusted to 4.5 using 1M HCl;

[0088] (6) After the adjustment, the solution was placed at 4°C for 24 hours of acid precipitation;

[0089] (7) After the acid precipitation, the precipitated protein was collected by centrifugation at 8000 rpm for 10 minutes at 4°C;

[0090] (8) An equal volume of distilled water was added to the solution, and 1M NaOH was added dropwise while stirring slowly to adjust the pH to 7.0 to resuspend the protein;

[0091] (9) The concentrated protein solution was placed in a dialysis bag and dialyzed in a refrigerator at 4°C for 48 hours;

[0092] (10) After dialysis, the protein solution was transferred to a plate and frozen in a ultra-low temperature freezer at -80°C. The sample was freeze-dried for 24 hours until it was completely dry, and finally a protein powder was obtained.

[0093] Comparative Example 15

[0094] (1) Extraction of barley leaf protein by alkali extraction and acid precipitation

[0095] 1) 2 grams of barley leaf powder was accurately weighed, and distilled water was added to the powder according to a solid-liquid ratio of 1:30 (g / mL) to prepare an extraction solution;

[0096] 2) The pH of the four extraction solutions was adjusted to 12.0 using 1M NaOH, respectively;

[0097] 3) Each extraction solution was placed in a constant temperature water bath shaker at 50°C for constant temperature oscillation extraction, and the duration was 90 minutes;

[0098] 4) After the extraction, the supernatant was collected by centrifugation at 8000 rpm for 10 minutes at 4°C;

[0099] 5) The supernatant obtained in step (4) was taken and its pH was adjusted to 4.5 using 1M HCl;

[0100] 6) After the adjustment, the solution was placed at 4°C for 24 hours of acid precipitation;

[0101] 7) After the acid precipitation, the precipitated protein was collected by centrifugation at 8000 rpm for 10 minutes at 4°C;

[0102] 8) An equal volume of distilled water was added to the solution, and 1M NaOH was added dropwise while stirring slowly to adjust the pH to 7.0 to resuspend the protein;

[0103] 9) The concentrated protein solution was loaded into a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0104] 10) After dialysis, the protein solution was transferred to a plate and placed in a -80°C ultra-low temperature freezer. After freezing, the sample was subjected to freeze-drying treatment for 24 hours until the sample was completely dried, and finally the protein powder was obtained.

[0105] (2) Foaming ability and foaming stability of barley leaf protein

[0106] 20 mg of barley leaf protein powder was mixed with 4 mL of distilled water, and the protein solution was stirred with a homogenizer at 10000 rpm for 2 min to absorb air and generate foam. The volume of the protein solution before homogenization was recorded as V0, and the volume of the protein solution containing foam after homogenization was recorded as V f . After the foaming protein solution was placed at 23°C for 30 min, the final volume of the protein solution containing foam was recorded as V t . FC (foaming activity index) and FS (foaming stability index) were calculated according to the following formula:

[0107]

[0108] Wherein, V0 is the initial volume of the protein solution before homogenization (mL), V f is the volume of the protein solution containing foam after homogenization (mL); V t is the volume of the protein solution containing foam after 30 min of standing (mL).

[0109] (3) Measurement of zeta potential and particle size of barley leaf protein

[0110] The barley leaf protein powder was prepared into a 1 mg / mL protein solution with deionized water, and the Zeta potential value and particle size of the barley leaf protein obtained by the method described in Comparative Example 15 were measured using a particle size analyzer to analyze the effect of different extraction methods on the zeta potential and particle size of the barley leaf protein, and further analyze the correlation between them and the foaming stability of the protein.

[0111] (4) Fluorescence measurement of barley leaf protein

[0112] The fluorescence intensity of the barley leaf protein prepared by the method described in Example 1 was measured using a fluorescence spectrometer to analyze the correlation between the fluorescence intensity of the protein obtained by different extraction methods and its foaming stability.

[0113] Example 1

[0114] (1) A method for preparing barley leaf protein with high foaming stability by using sound chemistry assisted alkali method

[0115] 1) 2.00 grams of barley leaf raw material powder was weighed out, and distilled water was added to the powder according to a solid-liquid ratio of 1:30 (g / mL) to prepare an extraction liquid;

[0116] 2) The pH of the extraction liquid was adjusted to 12.0 using 1M NaOH, and then placed in a constant-temperature water bath shaker at 50°C, and ultrasonic waves with a fixed frequency of 40kH were applied for constant-temperature oscillation extraction, with a duration of 90 minutes;

[0117] 3) After the extraction was completed, centrifugation was performed at a speed of 8000 rpm in a 4°C environment for 10 minutes, and the supernatant was collected;

[0118] 4) The supernatant was adjusted to a pH of 4.5 using 1M HCl, and acid precipitation was performed by standing at 4°C for 24 hours;

[0119] 5) After the acid precipitation was completed, centrifugation was again performed for 10 minutes (8000 rpm, 4°C), and the precipitated protein was collected;

[0120] 6) An equal volume of distilled water was added, stirring was performed slowly, and 1M NaOH was added dropwise to adjust the pH to 7.0 to resuspend the protein;

[0121] 7) The concentrated protein solution was loaded into a dialysis bag, and dialysis was performed in a 4°C refrigerator for 48 hours;

[0122] 8) After the dialysis was completed, the protein solution was transferred to a plate, and after being frozen in a -80°C ultra-low temperature refrigerator, the sample was subjected to freeze-drying treatment for 24 hours until the sample was completely dried, and finally a protein powder was obtained.

[0123] Example 2

[0124] 1) 2.00 grams of barley leaf raw material powder was weighed out, and distilled water was added to the powder according to a solid-liquid ratio of 1:30 (g / mL) to prepare an extraction liquid;

[0125] 2) The pH of the extraction liquid was adjusted to 12.0 using 1M NaOH, and then placed in a constant-temperature water bath shaker at 50°C, and ultrasonic waves with a fixed frequency of 40kH were applied for constant-temperature oscillation extraction, with a duration of 60 minutes;

[0126] 3) After the extraction was completed, centrifugation was performed at a speed of 7000 rpm in a 4°C environment for 10 minutes, and the supernatant was collected;

[0127] 4) The supernatant was adjusted to a pH of 4.5 using 1M HCl, and acid precipitation was performed by standing at 4°C for 48 hours

[0128] 5) After the acid precipitation was completed, centrifugation was again performed for 10 minutes (8000 rpm, 4°C), and the precipitated protein was collected;

[0129] 6) Add an equal volume of distilled water to step (1), stir slowly, and add 1M NaOH dropwise to adjust the pH to 7.0 to re-dissolve the protein;

[0130] 7) The concentrated protein solution is placed in a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0131] 8) After dialysis, the protein solution is transferred to a plate and frozen in a -80°C ultra-low temperature freezer. The sample is then freeze-dried for 24 hours until the sample is completely dry, and finally a protein powder is obtained.

[0132] Example 3

[0133] 1) 2.00 grams of barley leaf raw powder is weighed, and distilled water is added to the powder according to a solid-liquid ratio of 1:30 (g / mL) to prepare an extraction solution;

[0134] 2) The pH of the extraction solution is adjusted to 12.0 using 1M NaOH, and then placed in a 50°C constant temperature water bath shaker, and ultrasonic vibration extraction is carried out at a fixed frequency of 40kH for 120 minutes;

[0135] 3) After extraction, centrifugation is performed at 8000 rpm for 10 minutes at 4°C to collect the supernatant;

[0136] 4) The pH of the supernatant is adjusted to 4.5 using 1M HCl, and acid precipitation is carried out at 4°C for 24 hours;

[0137] 5) After acid precipitation, centrifugation is performed again for 10 minutes (8000 rpm, 4°C) to collect the precipitated protein;

[0138] 6) Add an equal volume of distilled water to step (1), stir slowly, and add 1M NaOH dropwise to adjust the pH to 7.0 to re-dissolve the protein;

[0139] 7) The concentrated protein solution is placed in a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0140] 8) After dialysis, the protein solution is transferred to a plate and frozen in a -80°C ultra-low temperature freezer. The sample is then freeze-dried for 24 hours until the sample is completely dry, and finally a protein powder is obtained.

[0141] Example 4

[0142] 1) 2.00 grams of barley leaf raw powder is weighed, and distilled water is added to the powder according to a solid-liquid ratio of 1:20 (g / mL) to prepare an extraction solution;

[0143] 2) Adjust the pH of the extract to 9.0 with 1M NaOH, then place it in a constant temperature water bath shaker at 55°C, and apply ultrasonic waves with a fixed frequency of 40kH for constant temperature oscillation extraction, with a duration of 120 minutes;

[0144] 3) After the extraction is completed, centrifuge at 7000 rpm in an environment of 25°C for 5 minutes, and collect the supernatant;

[0145] 4) Adjust the pH of the supernatant to 3.8 with 1M HCl, and stand for 48 hours at 25°C for acid precipitation;

[0146] 5) After the acid precipitation is completed, centrifuge again for 5 minutes (7000 rpm, 25°C), and collect the precipitated protein;

[0147] 6) Add an equal volume of distilled water from step (1), stir slowly, and add 1M NaOH dropwise to adjust the pH to 8.0 to resuspend the protein;

[0148] 7) Place the concentrated protein solution in a dialysis bag, and dialyze it in a 4°C refrigerator for 24 hours;

[0149] 8) After the dialysis is completed, transfer the protein solution to a plate, freeze it in a -70°C ultra-low temperature refrigerator, and then perform freeze-drying treatment on the sample for 24 hours until the sample is completely dried, and finally obtain a protein powder.

[0150] Example 5

[0151] 1) Weigh 2.00 grams of barley leaf raw powder, and add distilled water to the powder according to a solid-liquid ratio of 1:10 (g / mL) to prepare an extract;

[0152] 2) Adjust the pH of the extract to 11.0 with 1M NaOH, then place it in a constant temperature water bath shaker at 40°C, and apply ultrasonic waves with a fixed frequency of 40kH for constant temperature oscillation extraction, with a duration of 60 minutes;

[0153] 3) After the extraction is completed, centrifuge at 9000 rpm in an environment of 4°C for 15 minutes, and collect the supernatant;

[0154] 4) Adjust the pH of the supernatant to 4.5 with 1M HCl, and stand for 24 hours at 4°C for acid precipitation;

[0155] 5) After the acid precipitation is completed, centrifuge again for 15 minutes (9000 rpm, 4°C), and collect the precipitated protein;

[0156] 6) Add an equal volume of distilled water from step (1), stir slowly, and add 1M NaOH dropwise to adjust the pH to 6.0 to resuspend the protein;

[0157] 7) The concentrated protein solution was loaded into a dialysis bag and dialyzed in a 4°C refrigerator for 48 hours;

[0158] 8) After dialysis, the protein solution was transferred to a plate and frozen in a -80°C ultra-low temperature refrigerator. The sample was then freeze-dried for 24 hours until the sample was completely dried, and finally a protein powder was obtained.

[0159] (2) Foaming ability and foam stability of barley leaf protein

[0160] 20 mg of barley leaf protein powder was mixed with 4 mL of distilled water, and the protein solution was stirred with a homogenizer at 10000 rpm for 2 min to absorb air and generate foam. The volume of the protein solution before homogenization was recorded as V0, and the volume of the protein solution containing foam after homogenization was recorded as V f . After the foaming protein solution was placed at 23°C for 30 min, the final volume of the protein solution containing foam was recorded as V t . FC (foaming activity index) and FS (foaming stability index) were calculated according to the following formula:

[0161]

[0162] wherein V0 is the initial volume of the protein solution before homogenization (mL), V f is the volume of the protein solution containing foam after homogenization (mL); and V t is the volume of the protein solution containing foam after 30 min of standing (mL).

[0163] (3) Measurement of zeta potential and particle size of barley leaf protein

[0164] The barley leaf protein powder was prepared into a 1 mg / mL protein solution with deionized water, and the zeta potential value and particle size of the barley leaf protein obtained by the method described in Comparative Example 15 were measured using a particle size analyzer to analyze the effect of different extraction methods on the zeta potential and particle size of the barley leaf protein, and further analyze the correlation between the zeta potential and particle size and the foaming stability of the protein.

[0165] (4) Fluorescence measurement of barley leaf protein

[0166] The fluorescence intensity of the barley leaf protein prepared by the method described in Example 1 was measured using a fluorescence spectrometer to analyze the effect of different extraction methods on the tertiary structure of the barley leaf protein.

[0167] Experimental results

[0168] Figure 1The results of the effect of pH on the extraction efficiency of barley leaf protein for Comparative Examples 1-4. The extraction efficiency of barley leaf protein increases with increasing pH and reaches a maximum at pH 12. This indicates that alkaline conditions are more favorable for the extraction of barley leaf protein, which may be related to the solubility and stability of proteins at different pH. In a high pH environment, the charge distribution on the protein molecule changes, which may make it easier for them to separate from the cell wall or other biological matrix, thereby increasing the extraction amount. In addition, high pH may also help to break down the cell wall structure, making the internal protein more easily extracted. Therefore, when extracting barley leaf protein, it is recommended to maintain a high alkaline environment, i.e. pH 12.

[0169] Figure 2 The results of the effect of solid-liquid ratio on the extraction efficiency of barley leaf protein for Comparative Examples 5-7. The method used in this invention shows that the extraction efficiency of barley leaf protein is positively correlated with the solid-liquid ratio, i.e. the larger the solid-liquid ratio, the higher the extraction rate. This trend may be related to the contact area and mass transfer efficiency between the solvent and the raw material. A higher solid-liquid ratio means more solvent can come into contact with the raw material, thereby increasing the solubility of the protein and the extraction efficiency. Therefore, it is recommended to use a solid-liquid ratio of 1:30 (g / mL) during the extraction of barley leaf protein.

[0170] Figure 3 The results of the effect of extraction time on the extraction efficiency of barley leaf protein for Comparative Examples 8-10. The method used in this invention shows that the extraction efficiency of barley leaf protein changes with time in a nonlinear growth pattern, i.e. rapid initial rise followed by stabilization. This trend can be attributed to the dynamic relationship between protein solubility rate and extraction system saturation equilibrium. In the initial stage, the high solubility of proteins promotes their rapid release and dissolution, resulting in a significant increase in extraction rate. However, as time goes on, the easily soluble proteins are gradually depleted, and the remaining proteins are difficult to further dissolve due to structural stability or chemical inertness, limiting the continuous improvement of extraction rate, and finally reaching a plateau. Therefore, in order to obtain a higher extraction rate of barley leaf protein in a shorter time and reduce energy consumption costs, it is recommended to choose an extraction time of 90 minutes.

[0171] Figure 4The results of the effect of extraction temperature on the extraction rate of barley leaf protein for Comparative Examples 11-13. The method used in the present application shows that the extraction rate of barley leaf protein increases with the increase of extraction temperature, and then tends to be stable. This phenomenon is closely related to the thermal stability of protein and the possible activity of enzymatic reaction in the system. Within the appropriate temperature range, the increase of temperature can effectively promote the dissolution process of protein molecules and may accelerate the rate of related enzymatic reactions, thereby improving the extraction efficiency. However, when the temperature exceeds a certain critical threshold, the protein molecules may undergo irreversible denaturation or structural degradation, which not only limits the further growth of the extraction rate, but also may cause the extraction rate to decrease. Therefore, in order to obtain a higher extraction rate of barley leaf protein and economy, it is recommended to choose an extraction temperature of about 50°C.

[0172] Figure 5 The results of the effect of ultrasonic extraction time on the extraction rate of barley leaf protein for Example 2. The method used in the present application shows that under the condition of fixed frequency, the extraction rate of barley leaf protein increases significantly with the extension of ultrasonic treatment time, and then tends to be stable. This phenomenon is closely related to the physical mechanism of ultrasonic waves, especially the cavitation effect and the mechanical vibration on the cell wall breaking. In the initial stage, with the increase of ultrasonic time, the cell wall structure is gradually and more effectively destroyed, which greatly promotes the release and dissolution of intracellular protein, resulting in a continuous increase of the extraction rate. However, when the degree of cell wall breakage reaches saturation, i.e. most of the accessible protein has been released, further extension of the ultrasonic time has little effect on the extraction rate. Therefore, considering the extraction efficiency, energy consumption and equipment durability, etc., under the condition of fixed frequency, it is recommended to set the ultrasonic extraction time to 60 minutes.

[0173] Figure 6 The results of the effect of barley leaf protein extraction rate for Examples 1, 4 and 5. Through experimental verification of the centrifugation conditions, acid precipitation conditions, dialysis and freezing conditions in the examples, it can be seen from the experimental results that the experimental conditions adopted in Example 1 significantly improve the extraction rate of barley leaf protein, and the protein content is 43%. Therefore, compared with the above examples, in order to improve the extraction rate of barley leaf, the preferred experimental conditions of the present application are: centrifugation at 8000 rpm for 10 min at 4°C; acid precipitation pH is 4.5, time is 24 h, and protein resolubilization pH is 7; protein dialysis time is 48 h, and finally frozen at -80°C ultra-low temperature refrigerator.

[0174] Figure 7The results of the protein content and extraction rate of the barley leaf protein prepared in Comparative Example 15 and Example 1. The protein extraction rate results show that the protein content and extraction rate of the protein prepared in Example 1 (ultrasound-assisted alkaline extraction) are significantly higher than those of Comparative Example 15 (alkaline extraction), which can better extract the protein in the barley leaf. The method used in the present application has obvious advantages in the extraction of barley leaf protein, not only improving the extraction rate, but also ensuring the stability of the extraction process.

[0175] Figure 8 The results of the foamability and stability of the barley leaf protein prepared in Comparative Example 15 and Example 1. From the foam photos, it can be seen that there are differences in the foaming of the barley leaf protein prepared in Comparative Example 15 (alkaline extraction, left) and Example 1 (ultrasound-assisted alkaline extraction, right): the color of the barley leaf protein foaming sample prepared in Comparative Example 15 is darker, while the color of the barley leaf protein foaming sample prepared in Example 1 is lighter. This may imply differences in composition or concentration. In addition, the foam stability of the barley leaf protein prepared in Comparative Example 15 and Example 1 was investigated, and it was found that the foam stability of the barley leaf protein prepared in Example 1 was better, which may be due to its unique preparation process or composition. This property is of great significance for the application of barley leaf protein in the food industry, especially in products that require good foam stability, such as baked goods, beverages, ice cream, etc.

[0176] Figure 9 The results of the Zeta potential and particle size determination of the barley leaf protein prepared in Comparative Example 15 and Example 1. Zeta potential is the electric potential at the slip plane near the surface of the particle, which can reflect the charge state and electrostatic repulsion of the particle surface; particle size reflects the size of the particles in the solution. From the Zeta potential results, it can be seen that the absolute value of the Zeta potential of the barley leaf protein prepared in Example 1 (ultrasound-assisted alkaline extraction) is 19.8±0.48 mV, which is smaller than the absolute value of the Zeta potential of the barley leaf protein prepared in Comparative Example 15 (alkaline extraction), and the decrease in the absolute value of the potential means that the electrostatic repulsion between protein molecules decreases, which may lead to easier interaction between adsorbed protein aggregates at close range, even forming a network structure, thereby strengthening the mechanical strength and elasticity of the interface film, making it more difficult to break and enhancing the foam stability. The particle size results show that the particle size of the barley leaf protein prepared in Example 1 (ultrasound-assisted alkaline extraction) is 2930.6±20.3 nm; which is much larger than the particle size of the barley leaf protein prepared in Comparative Example 15 (alkaline extraction), and the larger particle size means that the individual protein molecules have a larger effective contact surface area or a more complex structure, although the diffusion of large particles is slow; but once it reaches the interface, the larger size or complex structure may enable it to more effectively "spread" on the foaming surface, forming a thicker and denser interface layer. This thick protective layer can better resist external disturbances, thereby improving the foam stability. This is consistent with the results of the foamability and stability of the barley leaf protein prepared in Comparative Example 15 and Example 1. Figure 7The foaming stability of the barley leaf protein prepared by the embodiment 1 (ultrasound-assisted subtractive extraction) was significantly higher than that of the barley leaf protein prepared by the comparative example 15 (alkali extraction).

[0177] Figure 10 The fluorescence measurement results of the barley leaf proteins prepared by the comparative example 15 and the embodiment 1. The natural amino acid residues contained in the protein, tryptophan (Trp) and tyrosine (Tyr), are important sources of protein fluorescence, and tryptophan becomes the main contributor to protein fluorescence because it has a stronger fluorescence intensity than tyrosine. The tertiary structure of the protein, i.e., its three-dimensional folding state, directly determines the microenvironment in which tryptophan is located, and thus affects its fluorescence properties. The fluorescence spectrum results show that the fluorescence peaks of the barley leaf proteins prepared by the embodiment 1 and the comparative example 15 are distributed between 340-360 nm, and the fluorescence intensity of the embodiment 1 (ultrasound-assisted subtractive extraction) is 295.7±4.9, which is much higher than that of the comparative example 15 (alkali extraction). The stronger the surface hydrophobicity of the corresponding barley leaf protein, the more the barley leaf protein prepared by the embodiment 1 tends to and more firmly adsorbs to the gas-liquid interface, forming a more compact, more ordered and stronger network structure interface film, thus it can more effectively resist the denaturation and rupture of the liquid film, and can significantly improve the foaming stability of the protein. This is consistent with the results of the foaming stability test. Figure 7 The foaming stability of the barley leaf protein prepared by the embodiment 1 (ultrasound-assisted subtractive extraction) was significantly higher than that of the barley leaf protein prepared by the comparative example 15 (alkali extraction).

[0178] The method protected by the patent is an innovative barley leaf protein extraction technology, the superiority of which has been verified by a series of experimental data. First, the higher pH condition adopted in the extraction stage of the method significantly improves the extraction rate of barley leaf protein. This phenomenon can be attributed to the increase in solubility of proteins in alkaline environment and the improvement of their structural stability. Secondly, the optimized solid-liquid ratio further enhances the mass transfer efficiency, so that a larger proportion of proteins can be effectively dissolved. In addition, through fine regulation of extraction time and temperature, the method ensures high extraction rate while effectively avoiding protein denaturation or degradation due to excessive processing, thereby realizing the economy and efficiency of the extraction process. The introduction of ultrasonic assistance technology is another significant feature of the method. Ultrasonic treatment at a specific frequency not only effectively destroys the plant cell wall structure, promoting the release of intracellular proteins, but also experimental results show that this technology significantly improves the extraction efficiency of proteins, reduces the absolute value of Zeta potential (19.8±0.48mV), increases the particle size (2930.6±20.3nm), increases the fluorescence intensity (295.7±4.9), and optimizes its functional properties, specifically the significant improvement of foam stability by 27%. The experimental results clearly show that the barley leaf protein sample prepared according to Example 1 has better foam stability than the control sample extracted by traditional alkali method. This finding provides important scientific basis for expanding the diversified application of barley leaf protein in the food industry.

[0179] In summary, the technical method protected by the present application has outstanding advantages in extracting barley leaf protein. The method not only effectively improves the yield and purity of the target protein, but also significantly improves its functional properties, especially the key indicators such as foam stability. These technical advantages make it show important application prospects in the food industry, and are expected to provide key technical support for establishing an efficient barley leaf protein industry system.

[0180] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A method for preparing high foaming stability barley leaf protein using a soni-chemically assisted alkali method, characterized by; The method comprises the following steps: Step 1) mixing barley leaf powder with distilled water at a certain solid-liquid ratio, stirring to obtain a barley leaf water solution; Step 2) alkali extraction of the barley leaf water solution obtained in step 1); Step 3) centrifugation of the protein extract obtained in step 2) under the condition of 4-25℃ and collection of the supernatant; Step 4) acid precipitation of the supernatant obtained in step 3) by adjusting the pH to 3.8-4.5; Step 5) protein re-dissolution by adjusting the pH of the solution obtained in step 4) to 6-8; Step 6) vacuum drying after freezing the protein solution obtained in step 5) under the condition of -80--70℃, thereby obtaining barley leaf protein; Ultrasonic treatment is applied during the extraction process of step 1) and step 2).

2. The method for preparing high-foaming stability barley leaf protein using a soni-chemically assisted alkali method according to claim 1, characterized in that, The protein solution obtained in step 5) is also dialyzed for 24-48 hours before step 6).

3. The method for preparing high foaming stability barley leaf protein using alkali method with the aid of sonochemistry according to claim 1, characterized in that, The solid-liquid ratio of the barley leaf powder and distilled water in step 1) is 1:10-1:30 in g / mL.

4. The method for preparing high foaming stability barley leaf protein using alkali process with the aid of sonochemistry according to claim 1, characterized in that, The pH of the alkali extraction in step 2) is 9.0-12.0; the temperature of the alkali extraction is 40-55℃.

5. The method for preparing high foaming stability barley leaf protein using alkali process with the aid of sonochemistry according to claim 1, characterized in that, The time of the alkali extraction in step 2) is 60-120 minutes.

6. The method for preparing high foaming stability barley leaf protein using alkali process with the aid of sonochemistry according to claim 1, characterized in that, The centrifugation condition in step 3) is 7000-9000rpm, 5-15min.

7. The method for preparing high foaming stability barley leaf protein using alkali process with the aid of sonochemistry according to claim 1, characterized in that, The steps of 3), 4), 5) are carried out under the condition of 4-25℃.

8. The process for preparing high foaming stability barley leaf protein using alkali method with the aid of sonochemistry as claimed in any one of claims 1 to 7, wherein, The ultrasonic treatment is carried out during the alkali extraction in step 2) with a fixed frequency of 40kHz and an ultrasonic treatment time of 60-120 minutes.

9. The barley leaf protein as set forth in claim 8, characterized by The protein content is higher, which can reach 43%; the foaming stability is significantly higher; The foaming stability value is increased to 27%; The absolute value of Zeta potential is less than 25mV, and the particle size is greater than 2900nm; The fluorescence intensity is greater than 290.

10. The use of the barley leaf protein in claim 9 in the preparation of food and medicine, particularly as an additive with excellent foaming stability.

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