A soybean polypeptide, and a preparation method and application thereof

Abstract

The application relates to a soybean polypeptide and a preparation method and application thereof. The preparation method of the soybean polypeptide comprises the following steps: performing an enzymolysis reaction on soybean protein isolate by using a protease, centrifuging and collecting supernatant, performing ultrasonic treatment on the supernatant, and obtaining the soybean polypeptide. The preparation method of the soybean polypeptide utilizes the protease to perform the enzymolysis on the soybean protein isolate, can efficiently degrade the soybean protein, performs the ultrasonic treatment on the supernatant, can further promote the degradation of a beta-conglycin alpha subunit anti-enzymolysis peptide segment, and obtains a high-quality product which is clear and transparent.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a soybean polypeptide, its preparation method, and its application. Background Technology

[0002] Soybeans are rich in nutrients, containing many essential amino acids for humans and animals. They also contain bioactive substances such as soy isoflavones, saponins, soy phospholipids, and vitamins, as well as trace elements such as calcium, iron, zinc, and selenium. After processing, soybeans can provide humans with a variety of soy products, such as soybean oil, soy milk, various types of tofu, and soy sauce. Currently, soybeans have a wide range of applications, including in the food, biological, and pharmaceutical fields. However, soybeans contain anti-nutritional factors, which make them very difficult to degrade, thus limiting their widespread use.

[0003] Anti-nutritional factors in soybean protein mainly consist of four types of globulins, which can be classified into four main components according to their sedimentation coefficient (s): 2S, 7S, 11S, and 15S. Among them, β-conglycinin (7S) and β-glycinin (11S) are the main antigenic proteins, accounting for approximately 30% and 40% of the total soybean protein, respectively. These two are the main causes of allergic reactions. β-Conglycinin (7S) is one of the main storage proteins in soybeans and is a heat-stable antigenic protein. It is a trimer composed of α', α, and β subunits, with molecular masses of 76 kDa, 72 kDa, and 52-54 kDa, respectively. The α' and α subunits are the most antigenic.

[0004] Currently, there are various methods for degrading antigenic proteins, such as physical treatment, enzymatic hydrolysis, microbial fermentation, and microbial-enzyme co-fermentation. Microbial fermentation can effectively degrade anti-nutritional factors in soybeans, but microbial fermentation alone is not sufficient to completely degrade soybean antigenic proteins in soybean meal. Microbial-enzyme co-fermentation can effectively reduce soybean antigenic proteins. For example, CN106538867A describes a method for preparing low-molecular-weight soybean peptides with health benefits. Soybean meal is ultra-finely pulverized, hydrolyzed once with a complex enzyme of cellulase, pectinase, endo-β-glucanase, and phytase, and then hydrolyzed a second time with neutral protease to obtain secondary hydrolyzed soybean meal. Then, using the secondary hydrolyzed soybean meal as a substrate, solid-state fermentation with Clostridium butyricum is carried out to obtain soybean peptides. However, there is currently little research in this direction, and further research is needed. Also, because antigenic proteins are difficult to degrade, the supernatant after centrifugation is prone to becoming turbid after the degradation of soybean proteins.

[0005] In summary, this invention provides a method for preparing soybean peptides that can efficiently degrade soybean protein and obtain high-quality products, which has good social and economic benefits. Summary of the Invention

[0006] In view of the shortcomings of existing technologies and practical needs, the present invention provides a soybean polypeptide, its preparation method and application. The method can efficiently degrade soybean protein and obtain a clear and transparent high-quality product.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing soybean polypeptides, the method comprising:

[0009] Soy protein isolate (SPI) was enzymatically hydrolyzed using a protease, centrifuged, and the supernatant was collected. The supernatant was then sonicated to obtain soybean polypeptides.

[0010] In this invention, soy protein isolate is enzymatically hydrolyzed using proteases, which can efficiently degrade soy protein. Ultrasonic treatment of the supernatant can further promote the degradation of the β-congammaglobulin α subunit anti-enzymatic peptide, resulting in a clear and transparent high-quality product.

[0011] Preferably, the enzymatic hydrolysis reaction includes mixing soy protein isolate with a buffer solution and adding a protease to carry out the enzymatic hydrolysis reaction.

[0012] Preferably, the protease comprises a protease secreted by Bacillus sp. 8A6.

[0013] In this invention, the protease secreted by Bacillus sp. 8A6 is used to enzymatically hydrolyze soybean protein isolate, which can efficiently degrade soybean protein and effectively increase the variety of short peptides and the content of free amino acids in soybean polypeptides.

[0014] Preferably, the buffer solution comprises Na2CO3 and NaHCO3.

[0015] Preferably, the Na2CO3 content in the buffer solution is 0.02~0.06 mol / L, including but not limited to 0.03 mol / L, 0.04 mol / L or 0.05 mol / L.

[0016] Preferably, the NaHCO3 content in the buffer solution is 0.02~0.06 mol / L, including but not limited to 0.03 mol / L, 0.04 mol / L or 0.05 mol / L.

[0017] Preferably, the pH of the buffer solution is 6 to 10, including but not limited to 6.2, 6.4, 6.8, 7, 7.5, 8 or 9, and preferably 8 to 9.

[0018] Preferably, the enzyme activity of the protease secreted by Bacillus sp. 8A6 is 5000~30000 U / mL, including but not limited to 5100 U / mL, 5200 U / mL, 5300 U / mL, 5500 U / mL, 6000 U / mL, 6500 U / mL, 8000 U / mL, 10000 U / mL, 15000 U / mL, 18000 U / mL, 20000 U / mL, 21000 U / mL, 22000 U / mL, 25000 U / mL, 26000 U / mL, 28000 U / mL or 29000 U / mL.

[0019] Preferably, the temperature of the enzymatic hydrolysis reaction is 30~80℃, including but not limited to 31℃, 32℃, 35℃, 36℃, 38℃, 40℃, 50℃, 60℃, 65℃, 70℃, 72℃, 74℃, 76℃ or 78℃, and preferably 60~70℃.

[0020] Preferably, the enzymatic hydrolysis reaction is carried out under stirring conditions at a speed of 100~300 rpm, including but not limited to 101 rpm, 102 rpm, 105 rpm, 108 rpm, 110 rpm, 150 rpm, 200 rpm, 220 rpm, 240 rpm, 260 rpm or 280 rpm, and preferably 200~250 rpm.

[0021] Preferably, the enzymatic hydrolysis reaction time is 0.5-5 h, including but not limited to 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.6 h, 4 h, 4.2 h, 4.4 h, 4.6 h or 4.8 h, and preferably 1-2 h.

[0022] Preferably, the ultrasonic treatment time is 1 s to 5 min, including but not limited to 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 10 s, 50 s, 1 min, 2 min, 3 min or 4 min, preferably 5 s to 4 min.

[0023] Preferably, the method for preparing soybean polypeptides further includes the step of preparing a protease secreted by Bacillus sp. 8A6.

[0024] Preferably, the method for preparing the protease secreted by Bacillus sp. 8A6 includes:

[0025] Bacillus sp. 8A6 was inoculated into a culture medium and cultured. The supernatant was collected to obtain the protease.

[0026] Preferably, the method for preparing the protease secreted by Bacillus sp. 8A6 includes:

[0027] Bacillus sp. 8A6 was inoculated into seed culture medium for seed culture, and then the bacterial cells in the seed culture medium were inoculated into enzyme production medium for enzyme production culture. The supernatant was then collected to obtain the protease.

[0028] As a preferred technical solution, the method for preparing the soybean polypeptide includes:

[0029] Soy protein isolate was mixed with buffer solution, and protease secreted by Bacillus sp. 8A6 was added. The mixture was subjected to enzymatic hydrolysis at 30-80℃ and 100-300 rpm for 0.5-5 h. The mixture was centrifuged and the supernatant was collected. The supernatant was then sonicated for 1 s-5 min to obtain soybean polypeptides.

[0030] According to the present invention, electrophoresis technology is used to analyze the degradation of soy protein isolate.

[0031] Preferably, the electrophoresis voltage is 100~120 V, including but not limited to 102 V, 104 V, 106 V, 108 V, 110 V, 115 V, 118 V or 119 V.

[0032] Preferably, the electrophoresis time is 80-100 min, including but not limited to 82 min, 84 min, 86 min, 90 min, 92 min, 95 min or 99 min.

[0033] In a second aspect, the present invention provides a soybean polypeptide, which is prepared by the soybean polypeptide preparation method described in the first aspect.

[0034] Thirdly, the application of soybean polypeptides as described in the second aspect in the preparation of feed or food.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) In this invention, the use of protease to enzymatically hydrolyze soybean protein isolate can efficiently degrade soybean protein. The ultrasonic treatment of the supernatant can further promote the degradation of the β-congglobulin α subunit anti-enzymatic peptide, resulting in a clear and transparent high-quality product.

[0037] (2) In this invention, the protease secreted by Bacillus sp. 8A6 is used to enzymatically hydrolyze soybean protein isolate, which can efficiently degrade soybean protein and effectively increase the types of short peptides and the content of free amino acids in soybean polypeptides. Attached Figure Description

[0038] Figure 1 Electrophoresis image of soybean protein hydrolysate supernatant;

[0039] Figure 2 This is a diagram of a soybean polypeptide solution. Detailed Implementation

[0040] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] In this embodiment of the invention, soy protein isolate was purchased from Shanghai Maclean Biotechnology Co., Ltd., protease NY100 was purchased from Amano Enzyme Products Co., Ltd. (Japan), protease NS37071 was purchased from Novozymes Biotechnology Co., Ltd., and BCA kit (Pierce) was used. TM BCA Protein Assay Kit, Ultrasonic Homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0043] The method for preparing the protease secreted by Bacillus sp. 8A6 includes the following steps:

[0044] (1) Inoculate Bacillus sp. 8A6 in 5 mL of LB medium and culture at 37℃ and 200 rpm for 20 h. Take 200 μL of the above culture solution and inoculate it into 50 mL of LB medium. Culture at 37℃ and 200 rpm for 12 h. The OD value is 4.8. Pour it into a centrifuge tube and centrifuge at 6000 rpm for 10 min. Discard the supernatant, add 25 mL of sterile PBS, and vortex the bacteria again to obtain the bacterial solution.

[0045] (2) Prepare a culture medium according to the ratio of 0.75 g / L NaCl, 1.75 g / L K2HPO4, and 10 mM MOPS, and adjust the pH to 9. Pour 50 mL of the above culture medium into a shake flask, add chicken feathers with a mass concentration of 5%, and sterilize in an autoclave (121℃, 20 min) to obtain the enzyme-producing culture medium. Take 2 mL of the bacterial solution obtained in step (1) and inoculate it into the above enzyme-producing culture medium, and add sterile trace elements (0.25 g / L MgSO4·7H2O, 0.055 g / L CaCl2, 0.010 g / L FeSO4·7H2O, 0.005 g / L ZnSO4·7H2O). Incubate at 37℃ and 200 rpm for 20 h, centrifuge at 4℃ and 12000 rpm for 10 min, collect the supernatant, and obtain the protease (BS8A6) secreted by Bacillus sp. 8A6.

[0046] Example 1

[0047] 1 g of soy protein isolate (SPI) was dissolved in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), and 2 mL of 26600 U / mL proteinase BS8A6 was added. The mixture was incubated at 60℃ and 250 rpm for 1 h, centrifuged, and the supernatant was collected. The protein content was measured using a BCA kit, and the remaining solid was dried and weighed. The results are shown in Table 1. The supernatant was then sonicated for 5 s (100% power).

[0048] Example 2

[0049] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 10 s (100% power).

[0050] Example 3

[0051] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 15 s (100% power).

[0052] Example 4

[0053] Dissolve 1 g of soy protein isolate (SPI) in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 30 s (100% power).

[0054] Example 5

[0055] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 1 min (100% power).

[0056] Example 6

[0057] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 2 min (100% power).

[0058] Example 7

[0059] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 3 min (100% power).

[0060] Example 8

[0061] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 4 min (100% power).

[0062] Example 9

[0063] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 5 min (100% power).

[0064] Example 10

[0065] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 5 min (power 40%).

[0066] Example 11

[0067] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 5 min (power 60%).

[0068] Example 12

[0069] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, collect the supernatant, and then sonicate the supernatant for 5 min (power 80%).

[0070] Comparative Example 1

[0071] Dissolve 1 g of soy protein isolate SPI in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), add 2 mL of 26600 U / mL proteinase BS8A6, incubate at 60℃ and 250 rpm for 1 h, centrifuge, and collect the supernatant.

[0072] Comparative Example 2

[0073] 1 g of soy protein isolate (SPI) was dissolved in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), and 2 mL of 26600 U / mL proteinase NY100 was added. The mixture was incubated at 60℃ and 250 rpm for 1 h, centrifuged, and the supernatant was collected. The protein content was measured using a BCA kit, and the remaining solid was dried and weighed. The results are shown in Table 1.

[0074] Comparative Example 3

[0075] 1 g of soy protein isolate (SPI) was dissolved in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), and 2 mL of 26600 U / mL protease NS37071 was added. The mixture was incubated at 60℃ and 250 rpm for 1 h, centrifuged, and the supernatant was collected. The protein content was measured using a BCA kit, and the remaining solid was dried and weighed. The results are shown in Table 1.

[0076] Comparative Example 4

[0077] 1 g of soy protein isolate (SPI) was dissolved in buffer (containing 0.05 mol / L Na2CO3 and 0.05 mol / L NaHCO3, pH=9), 2 mL of buffer was added, and the mixture was incubated at 60℃ and 250 rpm for 1 h. After centrifugation, the supernatant was collected, and the protein content was measured using a BCA kit. The remaining solid was dried and weighed. The results are shown in Table 1.

[0078] Experimental Example 1

[0079] This experimental case analyzes the degradation of soy protein isolate.

[0080] Table 1

[0081] experimental group Protein content (µg / mL) Solid dry weight (g) Example 1 43.49 0.14 Comparative Example 2 27.08 0.17 Comparative Example 3 22.92 0.33 Comparative Example 4 27.24 0.68

[0082] As shown in Table 1, Example 1 utilized protease secreted by Bacillus sp. 8A6 for enzymatic hydrolysis, while Comparative Examples 2 and 3 used protease NY100 and protease 37071, respectively. The protein content of the supernatant in Example 1 was 43.49 µg / mL, which was significantly higher than that in Comparative Examples 2 and 3. Furthermore, analysis of the remaining solid dry weight showed that the remaining solid dry weight in Example 1 was significantly lower than that in Comparative Examples 2 and 3. In summary, this indicates that the protease secreted by Bacillus sp. 8A6 can efficiently degrade soy protein isolate.

[0083] The supernatant collected after the protease degraded SPI was subjected to electrophoresis (100 V, 80 min), and the results are as follows. Figure 1As shown, some large protein molecules were not completely degraded. The undegraded bands were identified by gel strip analysis. First, the gel strips were enzymatically digested and desalted, and then mass spectrometry was performed. The identification results showed that the amino acid sequence was as shown in SED ID NO.1. It can be seen that the undegraded protein molecule is β-conglycinin.

[0084] SED ID NO.1:

[0085] .

[0086] Comparative analysis of the solution states of the soybean polypeptides obtained in this invention yielded the following results: Figure 2 As shown, the present invention can effectively degrade β-conglycinin by ultrasonic treatment of the supernatant after SPI enzymatic hydrolysis, thereby clarifying the soybean polypeptide solution.

[0087] In summary, this invention utilizes the protease secreted by Bacillus sp. 8A6 for enzymatic hydrolysis combined with ultrasonic treatment, which can make the supernatant of degraded soybean protein transparent. Furthermore, the protein content of the degraded soybean protein is significantly higher than that of some commercially available enzyme-degraded soybean protein, demonstrating good development prospects and potential for industrial application.

[0088] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention. SEQUENCE LISTING <110> Institute of Process Engineering, Chinese Academy of Sciences <120> A soybean polypeptide, its preparation method and application <130> 20210611 <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 605 <212> PRT <213> Artificial sequence <400> 1 Met Met Arg Ala Arg Phe Pro Leu Leu Leu Leu Gly Leu Val Phe Leu 1 5 10 15 Ala Ser Val Ser Val Ser Phe Gly Ile Ala Tyr Trp Glu Lys Glu Asn 20 25 30 Pro Lys His Asn Lys Cys Leu Gln Ser Cys Asn Ser Glu Arg Asp Ser 35 40 45 Tyr Arg Asn Gln Ala Cys His Ala Arg Cys Asn Leu Leu Lys Val Glu 50 55 60 Lys Glu Glu Cys Glu Glu Gly Glu Ile Pro Arg Pro Arg Pro Arg Pro 65 70 75 80 Gln His Pro Glu Arg Glu Pro Gln Gln Pro Gly Glu Lys Glu Glu Asp 85 90 95 Glu Asp Glu Gln Pro Arg Pro Ile Pro Phe Pro Arg Pro Gln Pro Arg 100 105 110 Gln Glu Glu Glu His Glu Gln Arg Glu Glu Gln Glu Trp Pro Arg Lys 115 120 125 Glu Glu Lys Arg Gly Glu Lys Gly Ser Glu Glu Glu Asp Glu Asp Glu 130 135 140 Asp Glu Glu Gln Asp Glu Arg Gln Phe Pro Phe Pro Arg Pro Pro His 145 150 155 160 Gln Lys Glu Glu Arg Lys Gln Glu Glu Asp Glu Asp Glu Glu Gln Gln 165 170 175 Arg Glu Ser Glu Glu Ser Glu Asp Ser Glu Leu Arg Arg His Lys Asn 180 185 190 Lys Asn Pro Phe Leu Phe Gly Ser Asn Arg Phe Glu Thr Leu Phe Lys 195 200 205 Asn Gln Tyr Gly Arg Ile Arg Val Leu Gln Arg Phe Asn Gln Arg Ser 210 215 220 Pro Gln Leu Gln Asn Leu Arg Asp Tyr Arg Ile Leu Glu Phe Asn Ser 225 230 235 240 Lys Pro Asn Thr Leu Leu Leu Pro Asn His Ala Asp Ala Asp Tyr Leu 245 250 255 Ile Val Ile Leu Asn Gly Thr Ala Ile Leu Ser Leu Val Asn Asn Asp 260 265 270 Asp Arg Asp Ser Tyr Arg Leu Gln Ser Gly Asp Ala Leu Arg Val Pro 275 280 285 Ser Gly Thr Thr Tyr Tyr Val Val Asn Pro Asp Asn Asn Glu Asn Leu 290 295 300 Arg Leu Ile Thr Leu Ala Ile Pro Val Asn Lys Pro Gly Arg Phe Glu 305 310 315 320 Ser Phe Phe Leu Ser Ser Thr Glu Ala Gln Gln Ser Tyr Leu Gln Gly 325 330 335 Phe Ser Arg Asn Ile Leu Glu Ala Ser Tyr Asp Thr Lys Phe Glu Glu 340 345 350 Ile Asn Lys Val Leu Phe Ser Arg Glu Glu Gly Gln Gln Gln Gly Glu 355 360 365 Gln Arg Leu Gln Glu Ser Val Ile Val Glu Ile Ser Lys Glu Gln Ile 370 375 380 Arg Ala Leu Ser Lys Arg Ala Lys Ser Ser Ser Arg Lys Thr Ile Ser 385 390 395 400 Ser Glu Asp Lys Pro Phe Asn Leu Arg Ser Arg Asp Pro Ile Tyr Ser 405 410 415 Asn Lys Leu Gly Lys Phe Phe Glu Ile Thr Pro Glu Lys Asn Pro Gln 420 425 430 Leu Arg Asp Leu Asp Ile Phe Leu Ser Ile Val Asp Met Asn Glu Gly 435 440 445 Ala Leu Leu Leu Pro His Phe Asn Ser Lys Ala Ile Val Ile Leu Val 450 455 460 Ile Asn Glu Gly Asp Ala Asn Ile Glu Leu Val Gly Leu Lys Glu Gln 465 470 475 480 Gln Gln Glu Gln Gln Gln Glu Glu Gln Pro Leu Glu Val Arg Lys Tyr 485 490 495 Arg Ala Glu Leu Ser Glu Gln Asp Ile Phe Val Ile Pro Ala Gly Tyr 500 505 510 Pro Val Val Val Asn Ala Thr Ser Asn Leu Asn Phe Phe Ala Ile Gly 515 520 525 Ile Asn Ala Glu Asn Asn Gln Arg Asn Phe Leu Ala Gly Ser Gln Asp 530 535 540 Asn Val Ile Ser Gln Ile Pro Ser Gln Val Gln Glu Leu Ala Phe Pro 545 550 555 560 Gly Ser Ala Gln Ala Val Glu Lys Leu Leu Lys Asn Gln Arg Glu Ser 565 570 575 Tyr Phe Val Asp Ala Gln Pro Lys Lys Lys Glu Glu Gly Asn Lys Gly 580 585 590 Arg Lys Gly Pro Leu Ser Ser Ile Leu Arg Ala Phe Tyr 595 600 605

Claims

1. A method for preparing soybean polypeptides, characterized in that, The method for preparing the soybean polypeptide includes: Soy protein isolate was enzymatically hydrolyzed using a protease, the supernatant was collected by centrifugation, and the supernatant was subjected to ultrasonic treatment to obtain soybean polypeptides. The enzymatic hydrolysis reaction includes mixing soy protein isolate with a buffer solution, adding a protease to carry out the enzymatic hydrolysis reaction, wherein the pH of the buffer solution is 8-9, and the temperature of the enzymatic hydrolysis reaction is 60-70℃. The proteases include those secreted by Bacillus sp. 8A6; The method for preparing the protease secreted by Bacillus sp. 8A6 includes the following steps: Bacillus sp. 8A6 was inoculated into seed culture medium for seed culture, and then the bacterial cells in the seed culture medium were inoculated into enzyme production medium for enzyme production culture. The supernatant was then collected to obtain the protease. The preparation method of the enzyme-producing culture medium includes: preparing the culture medium according to the ratio of 0.75 g / L NaCl, 1.75 g / L K2HPO4, and 10 mM MOPS, adjusting the pH to 9, pouring 50 mL of the above culture medium into a shake flask, adding chicken feathers with a mass concentration of 5%, sterilizing, and obtaining the enzyme-producing culture medium.

2. The method for preparing soybean polypeptides according to claim 1, characterized in that, The buffer solution includes Na2CO3 and NaHCO3.

3. The method for preparing soybean polypeptides according to claim 1, characterized in that, The enzyme activity of the protease is 5000~30000 U / mL.

4. The method for preparing soybean polypeptides according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out under stirring conditions at a speed of 100-300 rpm; The enzymatic hydrolysis reaction takes 0.5 to 5 hours.

5. The method for preparing soybean polypeptides according to claim 4, characterized in that, The enzymatic hydrolysis reaction is carried out under stirring conditions at a speed of 200-250 rpm; The enzymatic hydrolysis reaction takes 1-2 hours.

6. The method for preparing soybean polypeptides according to any one of claims 1-5, characterized in that, The duration of the ultrasonic treatment is 1 second to 5 minutes.

7. The method for preparing soybean polypeptides according to claim 6, characterized in that, The duration of the ultrasonic treatment is 5 seconds to 4 minutes.

8. The method for preparing soybean polypeptides according to any one of claims 1-5, characterized in that, The method for preparing the soybean polypeptide includes: Soy protein isolate was mixed with buffer solution, and protease secreted by Bacillus sp. 8A6 was added. The mixture was subjected to enzymatic hydrolysis at 60-70°C and 100-300 rpm for 0.5-5 h. The mixture was centrifuged and the supernatant was collected. The supernatant was then sonicated for 1 s-5 min to obtain soybean peptides.

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