Stropharia rugoso-annulata protein as well as preparation method and application thereof

Abstract

The invention discloses a stropharia rugoso-annulata protein and a preparation method and application thereof, and belongs to the technical field of stropharia rugoso-annulata protein preparation. The preparation method of the stropharia rugoso-annulata protein comprises the following steps: adding water into stropharia rugoso-annulata powder, mixing, carrying out ultrasonic extraction by adopting slit ultrasonic equipment, centrifugally collecting supernate, and drying to obtain the stropharia rugoso-annulata protein, the working mode of ultrasonic extraction is a single-frequency processing mode or a multi-frequency processing mode; the frequency of the single-frequency processing mode comprises 23 kHz, 25 kHz, 28 kHz, 33 kHz or 40 kHz; the multi-frequency processing mode comprises the combination of any several frequencies in 23 kHz, 25 kHz, 28 kHz or 40 kHz. Stropharia rugoso-annulata is subjected to ultrasonic extraction by adopting slit ultrasonic equipment, alkalization treatment is not needed in the preparation process, the preparation method is simple, environmental and operation risks are reduced, and richer, safer and more efficient nutritional ingredients and functional products are provided for medicine, health care and food industries.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of Stropharia rugosoannulata protein, and particularly relates to a Stropharia rugosoannulata protein, a preparation method thereof and an application thereof. Background Art

[0002] Edible fungi and waste such as their low-value leftovers are rich in nutrients such as protein, peptides, amino acids and dietary fiber. The amino acids of the protein are complete in variety, including essential amino acids required by the human body, meeting the human body's requirements for nutrients such as protein and amino acids. Edible fungi protein and peptides have good biological activities and good targeting effects. The nutrients rich in edible fungi have broad application prospects in the fields of medicine and health care. Since edible fungi protein, peptides and dietary fiber often exist as complex mixtures in tissues or cells, the preparation of such nutritional components has become a complex and challenging task. Developing a preparation technology suitable for the nutritional components of edible fungi is the basis and key for obtaining edible fungi nutritional ingredients and related product development.

[0003] Stropharia rugosoannulata, also known as Stropharia rugoso-annulata, Stropharia rugoso-sphaerica, and Stropharia vinaceoavellanea, is rich in nutrients, containing functional components such as polysaccharides, phenols, and flavonoids, and the protein content is higher than that of general edible fungi. However, there are few studies on the extraction of Stropharia rugosoannulata protein. Compared with other proteins, Stropharia rugosoannulata protein is tightly cross-linked with cellulose. Therefore, at present, the main process for extracting Stropharia rugosoannulata protein is the alkali method for protein extraction. For example, Patent CN111642615A adopts ultrasonic-assisted alkali extraction. Ultrasonic extraction destroys the cell wall through the cavitation effect generated by high-frequency vibration to release proteins, and the alkaline condition can further dissolve or degrade components such as polysaccharides and lipids in the cell wall, enhancing the cell disruption effect, thereby improving the protein extraction rate. However, a strong alkali (pH>10) environment is likely to cause the destruction of the protein spatial structure, resulting in the loss of protein functional activity and affecting the subsequent application of the protein. In addition, acid neutralization is required later, which will not only generate a large amount of salts, affecting the protein purity, but also make the extraction steps complex, generate a large amount of waste liquid, increase the production cost, and the strong acid and strong alkali solutions used may also corrode the production equipment, increasing the environmental and operation safety risks. Therefore, it is particularly important to provide a preparation method that can not only improve the extraction rate of Stropharia rugosoannulata protein, but also simplify the extraction process, reduce the production cost, and reduce the environmental and operation safety risks. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of Stropharia rugosoannulata protein, which can improve the extraction rate of Stropharia rugosoannulata protein, improve the protein stability, and the preparation method is simple, reduce the production cost, and improve the environmental and operation safety.

[0005] Another purpose of the present invention is to provide a Stropharia rugosoannulata protein prepared by the above preparation method.

[0006] Another object of the present invention is to provide an application of the said Stropharia rugosoannulata protein in the preparation of antihypertensive, hypoglycemic or antioxidant products.

[0007] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a preparation method of Stropharia rugosoannulata protein, and the preparation method comprises the following steps:

[0009] Mix the Stropharia rugosoannulata powder with water, perform ultrasonic extraction by using a slit ultrasonic device, centrifuge to collect the supernatant, and dry to obtain the Stropharia rugosoannulata protein;

[0010] The working mode of the ultrasonic extraction is a single-frequency processing mode or a multi-frequency processing mode;

[0011] The frequencies of the single-frequency processing mode include 23 kHz, 25 kHz, 28 kHz, 33 kHz or 40 kHz; the multi-frequency processing mode includes any combination of 23 kHz, 25 kHz, 28 kHz or 40 kHz.

[0012] Preferably, the multi-frequency processing mode includes a dual-frequency processing mode, a triple-frequency processing mode or a quadruple-frequency processing mode.

[0013] Preferably, the multi-frequency processing mode is a sequential multi-frequency processing mode.

[0014] Preferably, the dual-frequency processing mode includes any of the following combinations: 23 kHz + 25 kHz; 23 kHz + 28 kHz; 23 kHz + 40 kHz; and the working time of each frequency is the same.

[0015] Preferably, the triple-frequency processing mode includes any of the following combinations: 23 kHz + 25 kHz + 28 kHz; 23 kHz + 25 kHz + 40 kHz; 23 kHz + 28 kHz + 40 kHz; and the working time of each frequency is the same.

[0016] Preferably, the quadruple-frequency processing mode includes the following combination: 23 kHz + 25 kHz + 28 kHz + 40 kHz; and the working time of each frequency is the same.

[0017] Preferably, the Stropharia rugosoannulata powder is Stropharia rugosoannulata powder with a particle size less than 100 mesh.

[0018] Preferably, the concentration of the material liquid after mixing the Stropharia rugosoannulata powder with water is 30 - 100 g / L.

[0019] Preferably, the conditions for ultrasonic extraction include: ultrasonic temperature of 20-30°C, ultrasonic time of 20-40 min, ultrasonic power density of 50-150 W / L, and ultrasonic intermittent ratio of (4-8):(1-3) s / s.

[0020] The present invention also provides a Stropharia rugosoannulata protein prepared by the above preparation method.

[0021] The present invention also provides an application of the Stropharia rugosoannulata protein in the preparation of antihypertensive, hypoglycemic or antioxidant products.

[0022] Advantages of the present invention:

[0023] The present invention uses a slit ultrasonic device to perform ultrasonic extraction on Stropharia rugosoannulata. During the preparation process, no alkalization treatment is required, and the preparation method is simple, reducing environmental and operation risks. The protein content in the Stropharia rugosoannulata protein prepared by the present invention is significantly increased compared to the protein content in the raw material, with the protein content increased by 1.9-2.7 times; the denaturation temperature of the Stropharia rugosoannulata protein is significantly increased, making the Stropharia rugosoannulata protein more stable; the Stropharia rugosoannulata protein prepared by the present invention has β-sheets and random coils as the main secondary structures, with a complete variety of amino acids, and there are more molecular numbers of the Stropharia rugosoannulata protein with molecular weights of 20-40 kDa and 40-60 kDa; the ACE inhibition IC 50 of the Stropharia rugosoannulata protein prepared by the present invention reaches 0.106-0.142 mg / mL, and the α-glucosidase inhibition IC 50 reaches 4.18-5.73 mg / mL, showing significant antihypertensive and hypoglycemic effects; the DPPH free radical scavenging antioxidant IC 50 reaches 0.203-0.462 mg / mL, and the ABTS free radical scavenging antioxidant IC 50 reaches 0.626-0.701 mg / mL, showing significant antioxidant effects.

[0024] The present invention has developed an efficient preparation technology suitable for the nutritional components of Stropharia rugosoannulata protein, which can specifically extract the protein nutritional functional substances in Stropharia rugosoannulata and its defective and leftover materials. The present invention will effectively overcome the preparation problems in the prior art and lay a solid foundation for the development of edible mushroom nutritional ingredients and related products. The implementation of the present invention will not only improve the comprehensive utilization rate of edible mushroom resources, but also promote the transformation and upgrading of the edible mushroom industry towards deep processing, providing richer, safer and more efficient nutritional ingredients and functional products for the pharmaceutical, health care and food industries. Description of the drawings

[0025] Figure 1 It is the slit-type five-frequency ultrasonic device used in Example 1; where A is the working mode diagram of the device, and B is the slit cavity of the slit-type five-frequency ultrasonic.

[0026] Figure 2 DSC spectra of Stropharia rugoso-annulata protein with different ultrasonic treatment methods in Test Example 1; where the ultrasonic frequencies of A-L are 23 kHz, 25 kHz, 28 kHz, 33 kHz, 40 kHz, (23 + 25) kHz, (23 + 28) kHz, (23 + 40) kHz, (23 + 25 + 28) kHz, (23 + 25 + 40) kHz, (23 + 28 + 40) kHz, (23 + 25 + 28 + 40) kHz respectively;

[0027] Figure 3 Far-ultraviolet CD spectra of Stropharia rugoso-annulata protein with different ultrasonic treatment methods in Test Example 1; where A is single-frequency ultrasonic treatment and B is multi-frequency ultrasonic treatment;

[0028] Figure 4 Infrared spectra of Stropharia rugoso-annulata protein with different ultrasonic treatment methods in Test Example 1; where A is single-frequency ultrasonic treatment and B is multi-frequency ultrasonic treatment;

[0029] Figure 5 Amino acid composition of the protein in the base material of Stropharia rugoso-annulata protein with single-frequency ultrasonic treatment method in Test Example 1; where the ultrasonic frequencies of A-E are 23 kHz, 25 kHz, 28 kHz, 33 kHz, 40 kHz respectively;

[0030] Figure 6 Amino acid composition of the protein in the base material of Stropharia rugoso-annulata protein with dual-frequency ultrasonic treatment method in Test Example 1; where the ultrasonic frequencies of A-C are (23 + 25) kHz, (23 + 28) kHz, (23 + 40) kHz respectively;

[0031] Figure 7 Amino acid composition of the protein in the base material of Stropharia rugoso-annulata protein with triple-frequency and quadruple-frequency ultrasonic treatment methods in Test Example 1; where the ultrasonic frequencies of A-D are (23 + 25 + 28) kHz, (23 + 25 + 40) kHz, (23 + 28 + 40) kHz, (23 + 25 + 28 + 40) kHz respectively;

[0032] Figure 8 Protein distribution in the base material of Stropharia rugoso-annulata protein with different ultrasonic treatment methods in Test Example 1, where the ultrasonic frequencies of A-L are 23 kHz, 25 kHz, 28 kHz, 33 kHz, 40 kHz, (23 + 25) kHz, (23 + 28) kHz, (23 + 40) kHz, (23 + 25 + 28) kHz, (23 + 25 + 40) kHz, (23 + 28 + 40) kHz, (23 + 25 + 28 + 40) kHz respectively. Detailed implementation manners

[0033] The present invention provides a method for preparing Stropharia rugosoannulata protein, and the preparation method comprises the following steps:

[0034] Mix the Stropharia rugosoannulata powder with water, perform ultrasonic extraction using a slit ultrasonic device, centrifuge to collect the supernatant, and dry to obtain Stropharia rugosoannulata protein;

[0035] The working mode of the ultrasonic extraction is a single-frequency processing mode or a multi-frequency processing mode;

[0036] The frequencies of the single-frequency processing mode include 23 kHz, 25 kHz, 28 kHz, 33 kHz or 40 kHz; the multi-frequency processing mode includes any combination of several frequencies among 23 kHz, 25 kHz, 28 kHz or 40 kHz.

[0037] In the present invention, the source of the Stropharia rugosoannulata powder is not particularly limited, and it can be prepared by a conventional method or obtained by purchase. As an alternative embodiment, the method for preparing the Stropharia rugosoannulata powder comprises: crushing and sieving the dried Stropharia rugosoannulata to obtain the Stropharia rugosoannulata powder; the Stropharia rugosoannulata includes Stropharia rugosoannulata fruiting bodies and / or its defective mushrooms; the drying, crushing and sieving methods can be conventionally selected according to actual needs. In the present invention, the Stropharia rugosoannulata powder is preferably a Stropharia rugosoannulata powder with a particle size less than 100 mesh, so as to increase the surface area and improve the subsequent protein extraction efficiency.

[0038] In the present invention, the concentration of the material liquid after mixing the Stropharia rugosoannulata powder with water is preferably 30-100 g / L, more preferably 40-60 g / L or 45-80 g / L or 50-60 g / L.

[0039] In the present invention, ultrasonic extraction is performed using a slit ultrasonic device, and the slit ultrasonic device can be conventionally selected according to actual needs. For example, a slit-type five-frequency ultrasonic device has five different frequencies, which are 23 kHz, 25 kHz, 28 kHz, 33 kHz, 40 kHz.

[0040] In the present invention, the frequencies of the single-frequency processing mode are preferably 23 kHz, 25 kHz, 28 kHz or 40 kHz. The multi-frequency processing mode preferably includes a dual-frequency processing mode, a triple-frequency processing mode or a quadruple-frequency processing mode.

[0041] In the present invention, the dual-frequency processing mode preferably includes any of the following combinations: 23 kHz + 25 kHz; 23 kHz + 28 kHz; 23 kHz + 40 kHz; and the working time of each frequency is the same. The dual-frequency processing mode is preferably a sequential dual-frequency processing mode; the sequential dual-frequency processing mode is preferably performed in the order of increasing frequency for ultrasonic treatment.

[0042] In the present invention, the preferred three-frequency processing modes include any one of the following combinations: 23 kHz + 25 kHz + 28 kHz; 23 kHz + 25 kHz + 40 kHz; 23 kHz + 28 kHz + 40 kHz; and the working time of each frequency is the same. The three-frequency processing mode is preferably a sequential three-frequency processing mode; and the sequential three-frequency processing mode is preferably to perform ultrasonic treatment in ascending order of frequency.

[0043] In the present invention, the preferred four-frequency processing mode includes the following combination: 23 kHz + 25 kHz + 28 kHz + 40 kHz; and the working time of each frequency is the same. The four-frequency processing mode is preferably a sequential four-frequency processing mode; and the sequential four-frequency processing mode is preferably to perform ultrasonic treatment in ascending order of frequency.

[0044] In the present invention, the temperature of the ultrasonic extraction is preferably 20 - 30 °C, more preferably 22 - 26 °C or 24 - 28 °C or 25 - 27 °C; the time of the ultrasonic extraction is preferably 20 - 40 min, more preferably 22 - 35 min or 25 - 38 min or 30 - 36 min; the ultrasonic power density of the ultrasonic extraction is preferably 50 - 150 W / L, more preferably 75 - 125 W / L or 80 - 130 W / L or 100 - 120 W / L; the ultrasonic intermittent ratio of the ultrasonic extraction is preferably (4 - 8):(1 - 3) s / s, indicating that the ultrasound works for 4 - 8 s and stops for 1 - 3 s, more preferably (5 - 7):(1.5 - 2.5) s / s or 6:2 s / s.

[0045] In the present invention, the centrifugation method and conditions can be conventionally selected according to actual needs. As an implementable mode, the rotation speed of the centrifugation is preferably 6000 - 10000 rpm, more preferably 7000 - 9000 rpm or 8000 - 8500 rpm; the time of the centrifugation is preferably 10 - 20 min, more preferably 12 - 18 min or 15 - 17 min. The supernatant is collected by centrifugation, and the supernatant is dried to obtain the protein of Stropharia rugosoannulata; the drying method can be conventionally selected according to actual needs, and it is preferably dried by freeze-drying; the temperature of the freeze-drying is preferably -80 - -60 °C, more preferably -75 - -65 °C or -70 - -73 °C; the time of the freeze-drying is preferably 36 - 60 h, more preferably 40 - 50 h or 48 - 54 h.

[0046] The present invention uses a slit ultrasonic device to perform ultrasonic extraction on Stropharia rugosoannulata. During the preparation process, no alkalization treatment is required, the preparation method is simple, and the environmental and operation risks are reduced.

[0047] The present invention also provides a protein of Stropharia rugosoannulata prepared by the above preparation method.

[0048] The protein content in the Stropharia rugosoannulata protein prepared by the preparation method of the present invention is significantly increased compared with that in the raw material, and the protein content is increased by 1.9 - 2.7 times. The present invention uses a slit ultrasonic device to extract Stropharia rugosoannulata protein by ultrasonic treatment, which significantly increases the denaturation temperature of Stropharia rugosoannulata protein, making the Stropharia rugosoannulata protein more stable. The denaturation temperature of Stropharia rugosoannulata protein is 67.92°C - 149.12°C, and the multi-frequency ultrasonic treatment promotes the production of protein derivative peptides. The Stropharia rugosoannulata protein prepared by the present invention has β-sheet and random coil as the main secondary structures. The prepared Stropharia rugosoannulata protein contains a complete variety of amino acids. Glutamic acid is the main amino acid, followed by alanine, valine, proline, etc. The molecular weights of the Stropharia rugosoannulata protein prepared by the present invention are relatively large in the ranges of 20 - 40 kDa and 40 - 60 kDa.

[0049] The present invention also provides an application of the Stropharia rugosoannulata protein in the preparation of antihypertensive, hypoglycemic or antioxidant products.

[0050] The ACE inhibition IC of the Stropharia rugosoannulata protein prepared by the present invention 50 reaches 0.106 - 0.142 mg / mL, and the α-glucosidase inhibition IC 50 reaches 4.18 - 5.73 mg / mL, showing significant antihypertensive and hypoglycemic effects. In addition, the DPPH free radical scavenging antioxidant IC of the Stropharia rugosoannulata protein prepared by the present invention 50 reaches 0.203 - 0.462 mg / mL, and the ABTS free radical scavenging antioxidant IC 50 reaches 0.626 - 0.701 mg / mL, showing significant antioxidant effects. The Stropharia rugosoannulata protein prepared by the present invention can be used to prepare antihypertensive, hypoglycemic or antioxidant products. There are no special limitations on the types of such products, and preferably include drugs or health products.

[0051] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] In the following embodiments, unless otherwise specified, all are conventional methods.

[0053] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0054] Example 1

[0055] A preparation method of Stropharia rugosoannulata protein, the steps of the preparation method are as follows:

[0056] (1) Raw material preparation: Select dry Stropharia rugosoannulata as the raw material, crush and sieve it to obtain Stropharia rugosoannulata powder with a particle size less than 100 mesh.

[0057] (2) Mix the powder of Stropharia rugoso-annulata with an appropriate amount of water. Use a slit ultrasonic device ( Figure 1 ) to extract the protein from the Stropharia rugoso-annulata material liquid. The working mode of ultrasonic extraction is the single-frequency processing mode, and the frequency of the single-frequency processing mode is 23 kHz. The ultrasonic extraction conditions are as follows: ultrasonic temperature 25 °C, ultrasonic time 30 min, ultrasonic power density 100 W / L, material liquid concentration 50 g / L, ultrasonic intermittent ratio 6:2 s / s (ultrasonic working for 6 s and stopping for 2 s). After the ultrasonic treatment, the extracted sample is centrifuged at 8000 rpm for 15 min, the supernatant is collected, and freeze-dried at -70 °C for 48 h to obtain Stropharia rugoso-annulata protein.

[0058] Example 2

[0059] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 1 is that the frequency of the single-frequency processing mode is 25 kHz.

[0060] Example 3

[0061] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 1 is that the frequency of the single-frequency processing mode is 28 kHz.

[0062] Example 4

[0063] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 1 is that the frequency of the single-frequency processing mode is 33 kHz.

[0064] Example 5

[0065] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 1 is that the frequency of the single-frequency processing mode is 40 kHz.

[0066] Example 6

[0067] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 1 is that the working mode of ultrasonic extraction is the dual-frequency processing mode, and the frequencies of the dual-frequency processing mode are combined as 23 kHz and 25 kHz, that is, (23 + 25) kHz. The ultrasonic treatment is carried out in ascending order of frequency. The working time of one-time ultrasonic is 6 s, and the working time of each frequency is 3 s, that is, after 23 kHz works for 3 s, 25 kHz works for 3 s, and then stops for 2 s, and so on in a cycle.

[0068] Example 7

[0069] A method for preparing Stropharia rugoso-annulata protein, the difference between the preparation method and Example 6 is that the frequencies of the dual-frequency processing mode are combined as 23 kHz and 28 kHz, that is, (23 + 28) kHz.

[0070] Example 8

[0071] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 6 is that the frequencies of the dual-frequency processing mode are a combination of 23 kHz and 40 kHz, that is, (23 + 40) kHz.

[0072] Example 9

[0073] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 1 is that the working mode of ultrasonic extraction is a triple-frequency processing mode, and the frequencies of the triple-frequency processing mode are a combination of 23 kHz, 25 kHz and 28 kHz, that is, (23 + 25 + 28) kHz. Ultrasonic treatment is carried out in ascending order of frequency. The working time of one ultrasonic treatment is 6 s, and the working time of each frequency is 2 s, that is, after 23 kHz works for 2 s, 25 kHz works for 2 s, 28 kHz works for 2 s, stops for 2 s, and so on in a cycle.

[0074] Example 10

[0075] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 9 is that the frequencies of the triple-frequency processing mode are a combination of 23 kHz, 25 kHz and 40 kHz, that is, (23 + 25 + 40) kHz.

[0076] Example 11

[0077] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 9 is that the frequencies of the triple-frequency processing mode are a combination of 23 kHz, 28 kHz and 40 kHz, that is, (23 + 28 + 40) kHz.

[0078] Example 12

[0079] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 1 is that the working mode of ultrasonic extraction is a quadruple-frequency processing mode, and the frequencies of the quadruple-frequency processing mode are a combination of 23 kHz, 25 kHz, 28 kHz and 40 kHz, that is, (23 + 25 + 28 + 40) kHz. Ultrasonic treatment is carried out in ascending order of frequency. The working time of one ultrasonic treatment is 6 s, and the working time of each frequency is 1.5 s, that is, after 23 kHz works for 1.5 s, 25 kHz works for 1.5 s, then 28 kHz works for 1.5 s, and finally 40 kHz works for 1.5 s, stops for 2 s, and so on in a cycle.

[0080] Example 13

[0081] A method for preparing stropharia rugosoannulata protein, the difference between the preparation method and Example 1 is as follows:

[0082] The ultrasonic extraction conditions were as follows: ultrasonic temperature 20°C, ultrasonic time 40 min, ultrasonic power density 150 W / L, solid-liquid concentration 100 g / L, ultrasonic intermittent ratio 8:3 s / s (ultrasonic working for 8 s and stopping for 3 s). After the ultrasonic treatment, the extracted sample was centrifuged at 10000 rpm for 10 min, the supernatant was collected, and freeze-dried at -80°C for 36 h to obtain the protein of Stropharia rugosoannulata.

[0083] Example 14

[0084] A preparation method of the protein of Stropharia rugosoannulata, the difference between the preparation method and Example 1 is that:

[0085] The ultrasonic extraction conditions were as follows: ultrasonic temperature 30°C, ultrasonic time 20 min, ultrasonic power density 50 W / L, solid-liquid concentration 30 g / L, ultrasonic intermittent ratio 4:1 s / s (ultrasonic working for 4 s and stopping for 1 s). After the ultrasonic treatment, the extracted sample was centrifuged at 6000 rpm for 20 min, the supernatant was collected, and freeze-dried at -60°C for 60 h to obtain the protein of Stropharia rugosoannulata.

[0086] Test Example 1 Determination of the protein content of Stropharia rugosoannulata

[0087] 1. Preparation of the protein of Stropharia rugosoannulata

[0088] The protein base materials of Stropharia rugosoannulata were prepared by using the preparation methods of Examples 1 - 12 respectively.

[0089] 2. Determination of the protein content in the protein base material of Stropharia rugosoannulata

[0090] The BCA method was used for the analysis of the protein content in the base material. Under alkaline conditions, cysteine, cystine, tryptophan, tyrosine and peptide bonds in the protein can reduce Cu 2+ to Cu + ; 2 molecules of BCA combine with Cu + to form a purple complex, which has an absorption peak at 540 - 595 nm and the strongest absorption peak at 562 nm. Weigh 0.1 g of the freeze-dried base material, add 1 mL of pure water, homogenize in an ice bath, centrifuge at 12000 rpm and 4°C for 10 min, and then take the supernatant. Take 10 μL of the supernatant sample, add 190 μL of the working solution of the kit (Protein Content Assay Kit by BCA Method, Suzhou Mengxi Biomedical Technology Co., Ltd.), incubate in an oven at 60°C for 30 min, and measure the absorbance value at 562 nm. The working solution is a mixture of reagent A and reagent B in the kit at a ratio of 50:1, and incubate in a water bath at 60°C for 30 min. Reagent A of the kit: BCA, Na 2 CO 3 、C 4 H 4 Na 2 O 6 、NaOH、NaHCO 3Reagent solution, pH 11; Kit reagent B: CuSO 4 ·5H 2 O solution.

[0091] The protein content calculation formula is:

[0092] Protein content (mg / g dry weight) = standard product mass concentration × (absorbance value of sample measurement - absorbance value of blank measurement) ÷ (absorbance value of standard product measurement - absorbance value of blank measurement) × V pure water volume ÷ W base material mass

[0093] Among them, the standard product is bovine serum albumin, the mass concentration is 0.5 mg / mL; the blank is water; V pure water volume is 1 mL; W base material mass is 0.1 g. The results are shown in Table 1.

[0094] Table 1 Protein content in the protein base material of Stropharia rugosoannulata under different ultrasonic treatment methods

[0095]

[0096]

[0097] After detection, the protein percentage content in the raw material of Stropharia rugosoannulata is 24% - 25%. It can be seen from Table 1 that through ultrasonic extraction and centrifugal impurity removal, the protein percentage content in the protein base material of Stropharia rugosoannulata has been significantly improved. The protein percentage content in the protein base material of Stropharia rugosoannulata treated with ultrasonic frequency combinations (23 + 28) kHz, (23 + 25 + 28) kHz, and (23 + 25 + 28 + 40) kHz is relatively high, exceeding 60%. Generally speaking, the protein percentage content in the protein base material of Stropharia rugosoannulata treated with multi-frequency ultrasound is higher than that in the protein base material of Stropharia rugosoannulata treated with single-frequency ultrasound.

[0098] 3. Determination of the thermal stability of Stropharia rugosoannulata protein

[0099] The differential scanning calorimetry (DSC) was used to determine the effect of ultrasonic treatment on the degree of protein denaturation. In the DSC spectrum, the temperature corresponding to the maximum peak represents the transition temperature during protein denaturation, and the denaturation temperature can reflect the thermal stability of the protein and the degree of aggregation of protein molecules. A differential scanning calorimeter (DSC3 of Mettler Toledo, Switzerland) was used to measure the protein thermal stability. Accurately weigh 10 mg of the protein-based material sample of Stropharia rugosoannulata, add it and dissolve it in 10 mmol / L phosphate buffer (pH 8.0), seal it in an aluminum crucible, and evenly spread the sample on the bottom of the crucible. Carefully wipe the sample solution on the edge of the crucible and ensure that the mass of the sample in the crucible is intact. Seal it with the crucible lid and press the crucible with a presser to ensure good sealing of the crucible. Let it stand in a 4 °C refrigerator for 12 h, then put it into the DSC tester. At a nitrogen flow rate of 50 mL / min and a pressure of 0.1 MPa, after maintaining a constant temperature of 10 °C for 3 min, the temperature was programmed to rise to 200 °C at a heating rate of 10 °C / min. Use a computer program to record the DSC curve and conduct a comparative analysis. The results are as Figure 2 shown.

[0100] It can be seen from Figure 2 the results that an obvious endothermic peak appeared in the DSC spectrum of the protein of Stropharia rugosoannulata prepared by single-frequency ultrasonic extraction ( Figure 2 A-E in it), and the denaturation temperature of the protein of Stropharia rugosoannulata was 67.92 °C - 128.51 °C. The samples prepared by ultrasonic extraction at frequencies of 23 kHz, 33 kHz, and 40 kHz significantly increased the denaturation temperature of the protein of Stropharia rugosoannulata, making the protein of Stropharia rugosoannulata more stable. Two endothermic peaks appeared in the DSC spectrum of the protein of Stropharia rugosoannulata treated with multi-frequency ultrasound ( Figure 2 F-L in it), and the denaturation temperature of the protein of Stropharia rugosoannulata was 133.44 °C - 149.12 °C. The difference in denaturation temperature between sample groups was not significant, indicating that the multi-frequency extraction had little difference in the extraction of the protein of Stropharia rugosoannulata. The endothermic peak that appeared at 41 °C - 58 °C may be due to the presence of peptide substances in the sample. The peptide reached the endothermic temperature during heating and produced an obvious absorption peak. As a protein derivative, the peptide only appeared in the samples prepared by multi-frequency ultrasonic extraction, indicating that multi-frequency ultrasound promoted the production of the protein derivative peptide.

[0101] 4. Determination of the secondary structure of the protein of Stropharia rugosoannulata

[0102] The secondary structure of the protein in the protein-based material of Stropharia rugoso-annulata was determined using a circular dichroism spectrometer. A circular dichroism spectrometer can determine the secondary structure of proteins. According to the wavelength range of circular dichroism, the far-ultraviolet region (190 - 260 nm) is the absorption range of the peptide chain, which can reflect the conformation of the protein. Weigh 5 mg of the Stropharia rugoso-annulata protein-based material sample, place it in a 50 mL centrifuge tube and grind it. Add 1 mL of pure water, mix and dissolve it, then centrifuge at 4500 r / min for 20 min. Filter the supernatant through a 0.22 μm membrane, and take 20 μL of the filtrate for on-machine analysis. Use a Chirascan V100 circular dichroism spectrometer (Applied Photophysics Ltd. (APL)), with a light path of 1 mm for the sample cell, a scanning wavelength range of 190 - 260 nm, a scanning speed of 500 nm / min, and a cumulative number of 3 times, and record the CD spectrum. Use software CDPro and CONTIN to fit and analyze the CD spectral data, and calculate the proportions of different secondary structures of the protein in the sample. The results are as Figure 3 shown in Table 2.

[0103] Table 2 Contents of the secondary structure of Stropharia rugoso-annulata protein treated with ultrasonic waves of different frequencies

[0104] Sample treatment method α-helix β-sheet β-turn Random coil 23 kHz 15.50％ 57.70％ 20.70％ 48.80％ 25 kHz 16.40％ 48.80％ 19.70％ 53.10％ 28 kHz 12.10％ 67.70％ 20.50％ 60.60％ 33 kHz 13.30％ 67.10％ 21.10％ 53.20％ 40 kHz 16.90％ 49.30％ 20.00％ 49.50％ (23 + 25) kHz 18.10％ 40.30％ 19.00％ 54.10％ (23 + 28) kHz 18.50％ 38.10％ 18.70％ 54.60％ (23 + 40) kHz 14.60％ 54.70％ 19.90％ 57.30％ (23 + 25 + 28) kHz 15.60％ 48.20％ 19.40％ 58.40％ (23 + 25 + 40) kHz 16.60％ 46.40％ 19.50％ 54.30％ (23 + 28 + 40) kHz 15.50％ 49.00％ 19.50％ 57.80％ (23 + 25 + 28 + 40) kHz 15.20％ 42.20％ 18.10％ 68.30％

[0105] It can be seen from Figure 3 the results that there are slight differences in the wavelength positions of the positive absorption peaks of Stropharia rugoso-annulata proteins treated with ultrasonic waves of different frequencies. The intensity of the negative absorption peaks of the samples treated with multi-frequency is gradually increasing compared to those treated with single-frequency, indicating that multi-frequency ultrasound has an increasing impact on the secondary structure of Stropharia rugoso-annulata proteins. β-sheet is a zigzag structure formed by parallel or antiparallel peptide chains connected by hydrogen bonds. It can be seen from the results in Table 2 that β-sheet and random coil account for relatively high proportions in all samples. Generally speaking, the proportion of β-sheet is relatively high in the proteins treated with single-frequency, while the proportion of random coil is relatively high in the proteins treated with multi-frequency.

[0106] 5. Infrared spectrum determination of Stropharia rugoso-annulata protein

[0107] Take 10 mg of the Stropharia rugoso-annulata protein-based material sample and make a tablet using the KBr tablet method, with a mass ratio of sample to KBr of 1:100. Use a Thermo Nicolet IS5 Fourier transform infrared spectrometer, with a silicon carbide rod as the light source, select a 2 cm -1 resolution mode, and scan in the range of 4000 - 400 cm -1Scanning was performed within the spectral range, with 32 sample scans, 32 background scans, a sampling gain of 1.0, a moving mirror speed of 0.6329, a DTGS KBr detector, and an XT-KBr beam splitter. The obtained spectra were processed for peak position calibration using OMNIC 9.7 analysis software (Thermo Nicolet Corporation), and Prism 9 software was used for spectrum plotting. The infrared spectrum results were analyzed to determine the types of functional groups. The results are as Figure 4 shown.

[0108] As can be seen from the Figure 4 results, the protein-based substrate sample of Stropharia rugosoannulata has infrared absorption peaks near 3400 cm -1 , 1600 cm -1 , 1400 cm -1 , and 1080 cm -1 . The information on the main chemical bonds and functional groups is consistent. The infrared absorption peak of the protein sample near 3400 cm -1 is related to the vibrations of functional groups such as hydroxyl (-OH) or amino (-NH), indicating the presence of intermolecular or intramolecular hydrogen bonds and being a characteristic absorption peak of protein molecules; the infrared absorption peak near 1600 cm -1 is attributed to the C═C skeletal vibration of aromatic compounds or the amide I band vibration of protein peptide bonds; the bending vibration of the C-H bond usually produces an absorption peak near 1400 cm -1 , which is characteristic of the methylene C-H in the protein backbone, and compounds containing hydroxyl (-OH) or amino (-NH 2 ) may also produce absorption in this region; the infrared absorption peak near 1080 cm -1 is attributed to the stretching vibration of the C-O bond in oxygen-containing functional groups. The characteristic absorption peak of protein hydrogen bonds at 3400 cm -1 is strong in the samples treated with multi-frequency ultrasound (23 + 28) kHz and (23 + 40) kHz, and the characteristic absorption peak of protein peptide bonds at 1600 cm -1 is sharp for single-frequency ultrasound at 23 kHz, 25 kHz, 28 kHz, and 33 kHz. This shows that the above ultrasonic treatment modes are beneficial for the release of Stropharia rugosoannulata protein.

[0109] 6. Analysis of the Amino Acid Composition of Stropharia rugosoannulata Protein

[0110] Sample pretreatment: Weigh 20 mg of the protein-based substrate sample of Stropharia rugoso-annulata, add 6 mL of analytical pure hydrochloric acid at 6 mol / L, and seal the tube after blowing with nitrogen for 15 min. Place the sealed sample in an oven and hydrolyze it at 110 °C for 24 h, then take out the cooling tube. Dilute the sample to 25 mL with pure water. Accurately pipette 2 mL of the diluted sample, concentrate and remove acid on a rotary evaporator at a rotary evaporation temperature of 60 °C until the sample is dry. Accurately add 2.0 mL of 0.02 mol / L hydrochloric acid, mix well on a vortex mixer, filter the sample through a 0.22-μm hydrophilic microfiltration head, and analyze it on an amino acid analyzer.

[0111] It was detected by Hitachi LA8080 fully automatic amino acid analyzer. Chromatographic column: Na + type cation exchange column (4.6 mm ID × 60 mm, 3 μm); ion exchange resin 2622, the detector is an ultraviolet-visible light detector; the color developing agent is ninhydrin buffer solution; the buffer solution system is citric acid buffer solution B1 (pH 3.2), B2 (pH 3.0), B3 (pH 4.0), B4 (pH 4.9); the buffer solution flow rate is 0.4 mL / min; the column temperature is 55 °C, and the reaction room temperature is 135 °C. The external standard method was used to determine the content of free amino acids in the sample solution. The detection wavelength for proline content is 440 nm, and the detection wavelength for other amino acids is 570 nm. Amino acid content calculation formula:

[0112] Amino acid content (%) = C * V / M * 100

[0113] where C is the concentration of the hydrolysis solution, V is the volume of the hydrolysis solution after dilution, and M is the sample mass. The results are shown in Figures 5 - 7 Table 3 - Table 5.

[0114] Table 3 Amino acid composition of protein in the protein-based substrate of Stropharia rugoso-annulata treated by single-frequency ultrasonic treatment

[0115]

[0116] Table 4 Amino acid composition of protein in the protein-based substrate of Stropharia rugoso-annulata treated by dual-frequency ultrasonic treatment

[0117]

[0118]

[0119] Table 5 Amino acid composition of protein in the protein-based substrate of Stropharia rugoso-annulata treated by triple-frequency and quadruple-frequency ultrasonic treatment

[0120]

[0121]

[0122] From Figures 5 - 7As can be seen from the results in Tables 3 - 5, there are 17 kinds of hydrolyzed amino acids in the protein of Stropharia rugosoannulata treated by ultrasound. The types of amino acids are complete. Glutamic acid is the main amino acid in all protein samples of Stropharia rugosoannulata treated by ultrasound, followed by alanine, valine, proline, etc. The total amino acid content in the protein samples of Stropharia rugosoannulata is between 10.18% - 25.69%. The amino acid content is relatively high in the proteins of Stropharia rugosoannulata treated by multi - frequency (23 + 28) kHz, (23 + 25 + 40) kHz and single - frequency 28 kHz ultrasound.

[0123] 7. Proteomic analysis of the protein base material of Stropharia rugosoannulata

[0124] Take 100 μg of the protein base material sample of Stropharia rugosoannulata extracted by ultrasound; add triethylammonium bicarbonate buffer (TEAB) to make the final concentration of TEAB 100 mM; add tris(2 - carboxyethyl)phosphine (TCEP) to make the final concentration of TCEP 10 mM, and react at 37 °C for 60 min; add iodoacetamide (IAM) to make the final concentration of IAM 40 mM, and react in the dark at room temperature for 40 min; centrifuge at 10000 g for 20 min and take the precipitate; fully dissolve the sample with 100 μL of 100 mM TEAB, add trypsin according to the ratio of enzyme:protein (m / m) = 1:50, and digest overnight at 37 °C. After trypsin digestion, take an equal amount of the sample and dry the peptide segments with a vacuum centrifugal concentrator; redissolve the dried peptide segments after enzymatic digestion with 0.1% trifluoroacetic acid (TFA) for desalting, and then dry with a vacuum concentrator; use NANO DROP ONE (Thermo Scientific) for peptide quantification by ultraviolet spectrophotometry. Dissolve an equal amount of peptide segments with the mass spectrometry loading buffer for data - independent acquisition mode DIA detection and analysis. Analytical column uPAC High Throughptu column (75 μm × 5.5 cm, Thermo, USA), chromatograph VanquishNeo (Thermo, USA), mass spectrometer Astral (Thermo, USA), chromatographic separation time 8 min, mobile phase A is an aqueous solution containing 2% acetonitrile and 0.1% formic acid, mobile phase B is an aqueous solution containing 80% acetonitrile and 0.1% formic acid, and the liquid - phase elution gradient is 0 min, 4% B; 0.1 min, 8% B; 1 min, 12.5% B; 1.1 min, 12.6% B; 3.6 min, 22.5% B; 5.8 min, 45% B; 6.4 min, 99% B; 8 min, 99% B. The detection mode is positive ion, the ion source voltage is set to 1.5 kV, and the mass spectrometry scanning range is set to 100 - 1700 m / z. Use Spectronaut TM software to perform qualitative and quantitative analysis on each sample according to the spectral library, and conduct data statistics and bioinformatics analysis on the obtained protein quantitative results. The results are as Figure 8 shown.

[0125] From Figure 8 It can be seen from the results that the number of molecules of Stropharia rugosoannulata protein with molecular weights in the ranges of 20 - 40 kDa and 40 - 60 kDa is relatively large. Among the samples extracted by single frequency, except for the 33 kHz sample, the number of proteins in the above molecular weight ranges in the other extracted samples exceeds 200 species, and in the samples extracted by multi - frequency, the number of proteins in the above molecular weight ranges exceeds 400 species.

[0126] 8. Evaluation of the antihypertensive effect of Stropharia rugosoannulata protein base material

[0127] Angiotensin - converting enzyme (ACE) plays a key role in inducing blood pressure elevation in the renin - angiotensin - aldosterone system in vivo and is a drug target for treating cardiovascular diseases such as hypertension and heart failure. The antihypertensive effect of Stropharia rugosoannulata base material was evaluated by measuring the inhibitory activity of Stropharia rugosoannulata base material on angiotensin - converting enzyme. The analysis method used the kit method (DOJINDO Tongren Chemical ACE Kit - WSTA502 kit, Shanghai Youlu Biotechnology Co., Ltd.). Accurately weigh 0.1 g of Stropharia rugosoannulata protein base material, add 50 mL of pure water to dissolve it, and gradually dilute it to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, 0.0032 mg / mL, and 0.00064 mg / mL. Take 20 μL of the sample solution, add 20 μL of the kit matrix buffer and 20 μL of the kit enzyme working solution, incubate at 37 °C for 60 min, add 200 μL of the kit indicator working solution, incubate at 25 °C for 10 min, and measure the absorbance at 450 nm. Using the sample solution mass concentration and the inhibition activity value as the abscissa and ordinate respectively to plot the inhibition curve, and calculate the mass concentration of the sample when the inhibition rate is 50% (IC 50 ). The results are shown in Table 6.

[0128] Table 6 ACE inhibition results of Stropharia rugosoannulata protein base material with different ultrasonic treatment methods

[0129] Sample treatment method <![CDATA[IC 50 (mg / mL)]]> Sample treatment method <![CDATA[IC 50 (mg / mL)]]> 23 kHz 0.114 (23 + 28) kHz 0.142 25 kHz 0.124 (23 + 40) kHz 0.113 28 kHz 0.106 (23 + 25 + 28) kHz 0.121 33 kHz 0.131 (23 + 25 + 40) kHz 0.131 40 kHz 0.141 (23 + 28 + 40) kHz 0.135 (23 + 25) kHz 0.142 (23 + 25 + 28 + 40) kHz 0.128

[0130] It can be seen from the results in Table 6 that the lowest IC of ACE inhibition 50 appears in the sample group treated at 28 kHz, which is 0.106 mg / mL; followed by the sample groups treated at 23 kHz and (23 + 40) kHz, and the IC 50 are 0.114 mg / mL and 0.113 mg / mL respectively. Generally speaking, the ACE inhibition IC of Stropharia rugosoannulata protein base material treated with different ultrasounds 50 varies little, indicating that different ultrasonic frequency combination methods have little difference in the ACE inhibition activity of Stropharia rugosoannulata protein base material.

[0131] 9. Evaluation of the Hypoglycemic Effect of the Protein-based Substrate of Stropharia rugosoannulata

[0132] α-Glucosidase is considered the main target enzyme for the prevention and treatment of type II diabetes. Inhibiting the activity of α-glucosidase is also an important way to reduce postprandial blood glucose levels through diet. Therefore, the development of new food-derived α-glucosidase inhibitors is of great significance for the dietary prevention of type II diabetes. α-Glucosidase can catalyze the hydrolysis of 4-nitrophenyl-D-glucopyranoside (PNPG) to produce p-nitrophenol (PNP) and glucose. After PNPG is hydrolyzed, PNP is released, and the change in the amount of the product is calculated by detecting the absorbance of PNP at 405 nm. When the activity of α-glucosidase is inhibited, the catalytic hydrolysis of PNPG by α-glucosidase weakens or disappears, the production of PNP decreases, and the inhibitory effect of α-glucosidase is calculated by detecting the change in the absorbance of PNP.

[0133] 200 μL of α-glucosidase solution (7 unit / mL) was mixed with 200 μL of protein-based substrates of Stropharia rugosoannulata solutions with different concentrations (the concentrations were 10 mg / mL, 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, and 0.0032 mg / mL). After reacting at 37 °C for 20 min, 200 μL of 2.5 mmol / L PNPG was added, and the reaction mixture was reacted at 37 °C for 20 min. The release amount of PNP was measured at a wavelength of 405 nm. The inhibitory curve was plotted with the mass concentration of the sample solution and the inhibitory activity value as the abscissa and ordinate, and the mass concentration of the sample when the inhibition rate was 50% (IC 50 ) was calculated from the inhibitory curve. The results are shown in Table 7.

[0134] Table 7 α-Glucosidase Inhibition Results of the Protein-based Substrate of Stropharia rugosoannulata with Different Ultrasonic Treatment Methods

[0135] Sample treatment method <![CDATA[IC 50 (mg / mL)]]> Sample treatment method <![CDATA[IC 50 (mg / mL)]]> 23 kHz 5.08 (23 + 28) kHz 5.01 25 kHz 5.39 (23 + 40) kHz 5.56 28 kHz 4.18 (23 + 25 + 28) kHz 4.41 33 kHz 5.73 (23 + 25 + 40) kHz 4.92 40 kHz 5.65 (23 + 28 + 40) kHz 4.99 (23 + 25) kHz 5.12 (23 + 25 + 28 + 40) kHz 4.69

[0136] It can be seen from the results in Table 7 that the lowest IC of α-glucosidase inhibition 50 appeared in the sample group treated at 28 kHz, and the IC 50 was 4.18 mg / mL. Generally speaking, the IC of α-glucosidase inhibition of the protein-based substrates of Stropharia rugosoannulata treated with different ultrasounds 50 differed little, indicating that different ultrasonic frequency combinations had little difference in the inhibitory activity of the protein-based substrates of Stropharia rugosoannulata on α-glucosidase.

[0137] 10. Evaluation of the Antioxidant Effect of the Protein-based Substrate of Stropharia rugosoannulata on Scavenging DPPH Free Radicals

[0138] The DPPH free radical scavenging antioxidant analysis method of the Stropharia rugosoannulata protein base material uses the kit method (Total Antioxidant Capacity DPPH Method Kit, Suzhou Mengxi Biomedical Technology Co., Ltd.). Weigh 0.1 g of the Stropharia rugosoannulata protein base material, add 1 mL of the kit extraction solution (0.2 M sodium acetate buffer solution, pH 5.0), homogenize in an ice bath, centrifuge at 12000 rpm and 4 °C for 10 min, and collect the supernatant. Take 20 μL of the supernatant sample, add 380 μL of the kit DPPH· reaction reagent, mix well by vortex, and react in the dark at room temperature for 20 min. Take 200 μL of the reaction solution to a 96-well plate, measure the absorbance at 515 nm, and calculate the DPPH free radical scavenging rate. The results are shown in Table 8.

[0139] Table 8 DPPH free radical scavenging antioxidant results of the Stropharia rugosoannulata protein base material with different ultrasonic treatment methods

[0140]

[0141]

[0142] As can be seen from the results in Table 8, the sample group with the best DPPH free radical scavenging antioxidant effect is the sample group treated at 28 kHz, and the IC 50 is 0.203 mg / mL; followed by the sample group treated at 33 kHz, and the IC 50 is 0.275 mg / mL. Generally speaking, the antioxidant activities of the single-frequency, triple-frequency, and quadruple-frequency treated sample groups are slightly better than those of the double-frequency treated sample group.

[0143] 11. Evaluation of the ABTS free radical scavenging antioxidant effect of the Stropharia rugosoannulata protein base material

[0144] The ABTS free radical scavenging antioxidant analysis method of the Stropharia rugosoannulata protein base material uses the kit method (Total Antioxidant Capacity ABTS Method Kit, Suzhou Mengxi Biomedical Technology Co., Ltd.). Weigh 0.1 g of the Stropharia rugosoannulata protein base material, add 1 mL of the kit extraction solution (100 mM, pH 7.4 potassium phosphate buffer solution), homogenize in an ice bath, centrifuge at 12000 rpm and 4 °C for 10 min, and take the supernatant. Take 10 μL of the supernatant sample, add 190 μL of the kit ABTS + working solution (MnO 2 powder plus 11 mL of ABTS solution, shake and mix well for 20 min, let stand, and take the supernatant), mix well by vortex, and react in the dark at room temperature for 10 min. Take 200 μL of the reaction solution to a 96-well plate, measure the absorbance at 734 nm, and calculate the ABTS free radical scavenging rate. The results are shown in Table 9.

[0145] Table 9 ABTS free radical scavenging antioxidant results of the Stropharia rugosoannulata protein base material with different ultrasonic treatment methods

[0146] Sample treatment method <![CDATA[IC 50 (mg / mL)]]> Sample treatment method <![CDATA[IC 50 (mg / mL)]]> 23 kHz 0.672 (23 + 28) kHz 0.673 25 kHz 0.653 (23 + 40) kHz 0.626 28 kHz 0.670 (23 + 25 + 28) kHz 0.635 33 kHz 0.698 (23 + 25 + 40) kHz 0.671 40 kHz 0.686 (23 + 28 + 40) kHz 0.627 (23 + 25) kHz 0.701 (23 + 25 + 28 + 40) kHz 0.653

[0147] As can be seen from the results in Table 9, the protein-based substrate of Stropharia rugosoannulata has the antioxidant effect of scavenging ABTS free radicals, but the antioxidant effect is lower than that of scavenging DPPH free radicals. Generally speaking, the ABTS free radical scavenging antioxidant IC 50 of the protein-based substrates of Stropharia rugosoannulata treated by different ultrasonic treatments does not differ much, indicating that different ultrasonic frequency combination methods have little difference in the ABTS free radical scavenging antioxidant activity of the protein-based substrates of Stropharia rugosoannulata.

[0148] In the protein-based substrates of Stropharia rugosoannulata prepared by different ultrasonic treatment methods of the present invention, the protein content is significantly increased compared with that in the raw materials, and the protein is increased by 1.9 - 2.7 times. The protein sample of Stropharia rugosoannulata prepared by ultrasonic treatment in the present invention significantly increases the denaturation temperature of the protein of Stropharia rugosoannulata, making the protein of Stropharia rugosoannulata more stable. The denaturation temperature of the protein of Stropharia rugosoannulata is 67.92 °C - 149.12 °C, and multi-frequency ultrasound promotes the generation of protein derivative peptides. The protein of Stropharia rugosoannulata mainly has β-sheet and random coil as its secondary structures. The amino acid types of the protein of Stropharia rugosoannulata treated by ultrasound are complete. Glutamic acid is the main amino acid in all protein samples of Stropharia rugosoannulata treated by ultrasound, followed by alanine, valine, proline, etc. The number of molecules of the protein of Stropharia rugosoannulata with a molecular weight of 20 - 40 kDa and 40 - 60 kDa is relatively large. The protein-based substrate of Stropharia rugosoannulata prepared in the present invention has good antihypertensive, hypoglycemic and antioxidant activities.

[0149] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and retouches can still be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing Stropharia officinalis protein, characterized in that: The preparation method comprises the following steps: The powder of Stropharia rugosa is mixed with water, ultrasonic extraction is performed using a slit ultrasonic device, the supernatant is collected by centrifugation, and dried to obtain Stropharia rugosa protein; The working mode of the ultrasonic extraction is a single-frequency processing mode or a multi-frequency processing mode; The frequency of the single-frequency processing mode includes 23kHz, 25kHz, 28kHz, 33kHz or 40kHz; the multi-frequency processing mode includes combining any several frequencies of 23kHz, 25kHz, 28kHz or 40kHz.

2. The preparation method according to claim 1, characterized in that: The multi-frequency processing mode includes a dual-frequency processing mode, a triple-frequency processing mode or a quad-frequency processing mode.

3. The preparation method according to claim 2, characterized in that: The multi-frequency processing mode is a sequential multi-frequency processing mode.

4. The preparation method according to claim 2, characterized in that: The dual-frequency processing mode includes any combination of the following: 23kHz+25kHz; 23kHz+28kHz; 23kHz+40kHz; each frequency has the same working time.

5. The preparation method according to claim 2, characterized in that: The three-frequency processing mode includes any combination of the following: 23kHz+25kHz+28kHz; 23kHz+25kHz+40kHz; 23kHz+28kHz+40kHz; each frequency has the same working time.

6. The preparation method according to claim 2, characterized in that: The four-frequency processing mode includes the following combination: 23kHz+25kHz+28kHz+40kHz; each frequency has the same working time.

7. The preparation method according to claim 1, characterized in that: The concentration of the liquid after the Stropharia officinalis powder is mixed with water is 30-100 g / L.

8. The preparation method according to claim 1, characterized in that: The ultrasonic extraction conditions include: ultrasonic temperature of 20-30° C., ultrasonic time of 20-40 min, ultrasonic power density of 50-150 W / L, and ultrasonic intermittent ratio of (4-8):(1-3) s / s.

9. The Stropharia officinalis protein prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Stropharia officinalis protein according to claim 9 in the preparation of blood pressure lowering, blood sugar lowering or antioxidant products.

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