Preparation method and application of cowpea fat transfer protein microcapsule

Abstract

The invention discloses a preparation method and application of a cowpea fat transfer protein microcapsule, and the preparation method comprises the following steps: step 1, connecting a base sequence of a parent to a pGAPZ alpha A carrier to obtain a recombinant plasmid, and transforming the recombinant plasmid into escherichia coli DH5alpha; 2, inoculating the escherichia coli DH5 alpha into an LB culture medium in a monoclonal manner, and extracting plasmids; 3, carrying out enzyme digestion and purification to obtain high-concentration linearized plasmids; 4, electrically transferring the linearized plasmids into pichia pastoris competent cells, and coating the cells on a bleomycin YPD solid plate for culturing; 5, selecting single colonies, and further screening, culturing and purifying to obtain the cowpea fat transfer protein; step 6, mixing the cowpea fat transfer protein with sodium alginate, and wrapping chitosan to obtain cowpea fat transfer protein microcapsules; the preparation method is easy to purify and low in production cost, and can be used for foods, dietary supplements and the like; and the cowpea fat transfer protein microcapsule prepared after modification is relatively high in stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering protein microcapsule technology, and particularly relates to a preparation method and application of cowpea lipid transfer protein microcapsules. Background Art

[0002] Lipid-transfer proteins (LTPs) are a kind of soluble plant polypeptides, which can be divided into two categories, LTP1 and LTP2 according to their sizes. The former is composed of about 91 amino acids (about 9 kDa); the latter is about 70 amino acids (about 7 kDa). LTP1 has various physiological functions. For example, crops into which the LTP1 gene is transferred can resist diseases caused by bacteria and fungi; the use of LTP1 can reduce the generation of foam and help improve the quality of beer; LTP1 can also be used as a drug carrier, etc.

[0003] Recently, it has been found that cowpea lipid transfer protein (belonging to LTP1) can inhibit the activities of human and insect α-amylase, which expands its functions to the fields of human disease prevention (such as preventing obesity and diabetes) and crop pest control. Most of the reported α-amylase inhibitors are extracted from plants at present. For example, the commercially available α-amylase inhibitor, the natural extract of white kidney bean - phaseolin, not only has a high cost in its extraction process, but also involves the use of harmful solvents, and the extracted phaseolin has low activity and contains anti-nutritional factors. These factors limit its large-scale application in real life; most of the reported α-amylase inhibitors in the literature are prepared by traditional heating methods, organic solvent precipitation or inorganic salt precipitation and other methods, and these methods have problems such as high cost, a large amount of chemical residues, long cycle, and high pollution. In addition, during actual application, α-amylase inhibitors have extremely poor tolerance to the low pH environment during digestion and are easily degraded and inactivated by pepsin. Summary of the Invention

[0004] In view of the above problems, the present invention provides a preparation method and application of cowpea lipid transfer protein microcapsules, and uses the cowpea lipid transfer protein microcapsules as an α-amylase inhibitor to solve the problems of large environmental pollution, high cost, long cycle during the acquisition of α-amylase inhibitors, and easy inactivation during actual application.

[0005] The present invention adopts the following technical solutions: A preparation method of cowpea lipid transfer protein microcapsules, comprising:

[0006] Step 1: Connect the base sequence of the parent to the pGAPZαA vector to obtain a recombinant plasmid, and transform the recombinant plasmid into Escherichia coli DH5α;

[0007] Step 2: Inoculate the Escherichia coli DH5α monoclonal into an LB medium and extract the plasmid;

[0008] Step 3: Digest and purify the plasmid to obtain a high-concentration linearized plasmid;

[0009] Step 4: Electroporate the linearized plasmid into Pichia pastoris competent cells, and spread the cells on a bleomycin YPD solid plate for culture;

[0010] Step 5: Pick single colonies for further screening, culture, and purification to obtain cowpea lipid transfer protein;

[0011] Step 6: Mix the cowpea lipid transfer protein with sodium alginate and coat it with chitosan to obtain cowpea lipid transfer protein microcapsules.

[0012] Furthermore, the base sequence of the parent is:

[0013] ATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAG.

[0014] Furthermore, the base sequence of the parent is:

[0015] ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGGTGGAGGTCCCCCCCCCCCATTA.

[0016] Furthermore, the base sequence of the parent is as follows:

[0017] ATGGGCGGTGGTGGAGGCGAAGAGGAAGAGGAGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。

[0018] Furthermore, the base sequence of the parent is as follows:

[0019] TTGAGAAGTCTAACCTTGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGTTGAGAAGTCTAACCTTG。

[0020] Furthermore, the base sequence of the parent is as follows:

[0021] ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。

[0022] Furthermore, the base sequence of the cowpea lipid transfer protein is as follows:

[0023] ATGACCTGTGGACAAGTTCAGGGTAATCTGGCTCAGTGTATCGGATTTCTTCAGAACGGTGGAATTGTTCCACCTGCTTGTTGTAACGGTGTCAAGAACATTCTGAACTCTGCTAGAACCACTGCTGACAGAAGAGGTATCTGCTCTTGTCTGAAATCCGCTGCAGGTGCTGTTAGAGGTTTGAATCCATCCAATGCTCAGGCTTTGCCAGGTAAATGCGGTGTTAACATACCTTGGAAGATTTCTAGTTCTACCAACTGTAACACTATCAACTAG。

[0024] Furthermore, the LB medium in step 2 is LB medium containing 25 μg / mL bleomycin;

[0025] The culture conditions for the Escherichia coli DH5α monoclonal in step 2 are: overnight culture at 37 °C for 12 - 16 h.

[0026] Furthermore, the inoculation amount of the seed liquid during culture in step 5 is 5%.

[0027] An application of a cowpea lipid transfer protein microcapsule for preparing a dietary supplement for preventing obesity and diabetes.

[0028] The beneficial effects of the present invention are:

[0029] The preparation method of the present invention is easy to purify, has low production cost, and can be used in foods, dietary supplements, etc.; moreover, the prepared cowpea lipid transfer protein microcapsules have relatively high stability after being modified;

[0030] The average inhibition rate of the cowpea lipid transfer protein of the present invention on the activity of porcine pancreatic amylase reaches more than 90%, and the stability of the cowpea lipid transfer protein is significantly improved by adding anti-pepsin cleavage fragments and by preparing protein microcapsules. Description of the Drawings

[0031] Figure 1 The Pichia pastoris strain secreting and expressing cowpea lipid transfer protein screened in the examples of the present invention;

[0032] Figure 2 The SDS-PAGE electrophoresis pattern of the supernatant of the 72-hour fermentation broth of cowpea lipid transfer protein in the examples of the present invention;

[0033] Figure 3 The inhibition rate and remaining enzyme activity of cowpea lipid transfer protein LTP-WT on the activity of porcine pancreatic amylase in the examples of the present invention;

[0034] Figure 4 The prediction of the interaction between cowpea lipid transfer protein and porcine pancreatic amylase in the examples of the present invention;

[0035] Figure 5 The inhibition rate of different cowpea lipid transfer proteins on the activity of porcine pancreatic amylase after being treated with simulated gastric juice in the examples of the present invention;

[0036] Figure 6 The inhibition rate of LTP-D protein microcapsules on the activity of porcine pancreatic amylase after being treated with simulated gastric juice in the examples of the present invention. Detailed Description of the Invention

[0037] The present invention will be described in detail below in conjunction with the drawings and specific embodiments.

[0038] The present invention discloses a method for preparing cowpea lipid transfer protein microcapsules, including:

[0039] Step 1: Connect the base sequence of the parent to the pGAPZαA vector to obtain a recombinant plasmid, and transform the recombinant plasmid into Escherichia coli DH5α;

[0040] Step 2: Inoculate the monoclonal of Escherichia coli DH5α into an LB medium, and extract the plasmid;

[0041] Step 3: Digest the plasmid and purify it to obtain a high-concentration linearized plasmid;

[0042] Step 4: Electroporate the linearized plasmid into Pichia pastoris competent cells, and spread the cells on a bleomycin YPD solid plate for culture;

[0043] Step 5: Pick single colonies for further screening, culture, and purification to obtain cowpea lipid transfer protein;

[0044] Step 6: Mix the cowpea lipid transfer protein with sodium alginate and coat it with chitosan to obtain cowpea lipid transfer protein microcapsules.

[0045] Among them, the LB medium in Step 2 is LB medium containing 25 μg / mL bleomycin, and the culture conditions for Escherichia coli DH5α monoclonal are: overnight culture at 37°C for 12 - 16 h.

[0046] Among them, in Step 4, it is spread on a bleomycin YPD solid plate containing 300 μg / mL and cultured upside down at 30°C until single colonies grow out.

[0047] Among them, in Step 5, the inoculation amount of the seed liquid during culture is 5%.

[0048] Among them, the base sequence of the cowpea lipid transfer protein is shown in Sequence Listing 1, specifically:

[0049] ATGACCTGTGGACAAGTTCAGGGTAATCTGGCTCAGTGTATCGGATTTCTTCAGAACGGTGGAATTGTTCCACCTGCTTGTTGTAACGGTGTCAAGAACATTCTGAACTCTGCTAGAACCACTGCTGACAGAAGAGGTATCTGCTCTTGTCTGAAATCCGCTGCAGGTGCTGTTAGAGGTTTGAATCCATCCAATGCTCAGGCTTTGCCAGGTAAATGCGGTGTTAACATACCTTGGAAGATTTCTAGTTCTACCAACTGTAACACTATCAACTAG.

[0050] The cowpea lipid transfer protein is optimized according to the yeast codon preference. The parental base sequence named LTP-WT is shown in Sequence Listing 2, specifically:

[0051] ATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAG。

[0052] Taking advantage of the advantages of low production cost, no production of toxic factors, and wide application in the food and pharmaceutical fields by fermenting with the probiotic Pichia pastoris.

[0053] Proline-rich modules were added to the amino and carboxyl termini of LTP-WT and named LTP-A. The base sequence of LTP-A is shown in Sequence Listing 3, specifically:

[0054] ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGGTGGAGGTCCCCCCCCCCCATTA。

[0055] A polyglycine-polyglutamic acid chimera was added to the amino and carboxyl termini of LTP-WT and named LTP-B. The base sequence of LTP-B is shown in Sequence Listing 4, specifically:

[0056] ATGGGCGGTGGTGGAGGCGAAGAGGAAGAGGAGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。

[0057] Pepsin inhibitor-derived peptides were added to the amino terminus and carboxyl terminus of LTP-WT and named LTP-C. The base sequence of LTP-C is shown in Sequence Listing 5, specifically:

[0058] TTGAGAAGTCTAACCTTGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGTTGAGAAGTCTAACCTTG。

[0059] Adding an anti-pepsin cleavage module to the sequence of cowpea lipid transfer protein optimized by yeast codon preference can improve its stability in practical applications.

[0060] A proline-rich module and a polyglycine-polyglutamic acid chimera (double-terminal synergistic protection fragment) were added to the amino terminus and carboxyl terminus of LTP-WT respectively and named LTP-D. The base sequence of LTP-D is shown in Sequence Listing 6, specifically:

[0061] ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。

[0062] The present invention also discloses the use of a cowpea lipid transfer protein microcapsule, which is used as a dietary supplement and for preparing a dietary supplement for preventing obesity and diabetes.

[0063] Examples

[0064] Step 1: The base sequences of LTP-WT, LTP-A, LTP-B, LTP-C, and LTP-D parents were respectively optimized according to the yeast codon preference to obtain base sequences, and the base sequences were ligated to the pGAPZαA vector to obtain recombinant plasmids, and the recombinant plasmids were transformed into Escherichia coli DH5α.

[0065] Obtain a Pichia pastoris strain that can secrete and express cowpea (Vigna unguiculata, Vu) lipid transfer protein (LTP). The required reagents and materials are as follows: Pichia pastoris GS115, Escherichia coli DH5α, expression plasmid pGAPZαA. YPD medium formula: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L. LB medium formula: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0066] The base sequence was ligated to the vector pGAPZαA through the EcoRⅠ and XbaⅠ restriction sites to construct the recombinant plasmid pGAPZαA-Vu-LTP, and the plasmid with correct sequencing was transformed into Escherichia coli DH5α.

[0067] Step 2: Inoculate the Escherichia coli DH5α monoclonal into LB medium and extract the plasmid.

[0068] The monoclonal Escherichia coli DH5α containing the recombinant plasmid pGAPZαA-Vu-LTP was inoculated into LB medium containing 25 μg / mL bleomycin and cultured overnight at 37 °C for 12 - 16 h. The plasmid was extracted and the concentration of plasmid DNA was measured. Subsequently, the restriction enzyme Avr II was added and digested at 37 °C for 3 h. Nucleic acid electrophoresis was used to verify the complete digestion of the plasmid. The digested plasmid was added to 1 mL of phenol-chloroform and mixed well, centrifuged at 10000 rpm for 3 min. The upper layer liquid was carefully aspirated and transferred to a clean 1.5 mL centrifuge tube. 1 mL of absolute ethanol was added, centrifuged at 10000 rpm for 3 min, and the supernatant was discarded. 1 mL of 70% ethanol was added, centrifuged at 10000 rpm for 3 min, and the supernatant was discarded. This step was repeated three times. The lid was opened to volatilize the residual ethanol, and 10 μL of ultrapure water was taken to resuspend, and the DNA concentration was measured to obtain a purified and high-concentration linearized plasmid.

[0069] Step 3: Digest and purify the plasmid to obtain a high-concentration linearized plasmid. The specific purification method is as follows:

[0070] The above digested plasmid was added to 1 mL of phenol-chloroform and mixed well, centrifuged at 10000 rpm for 3 min. The upper layer liquid was carefully aspirated and transferred to a clean 1.5 mL centrifuge tube. 1 mL of absolute ethanol was added, centrifuged at 10000 rpm for 3 min, and the supernatant was discarded. 1 mL of 70% ethanol was added, centrifuged at 10000 rpm for 3 min, and the supernatant was discarded. This step was repeated three times. The lid was opened to volatilize the residual ethanol, and 10 μL of ultrapure water was taken to resuspend, and the DNA concentration was measured to obtain a purified and high-concentration linearized plasmid.

[0071] Step 4: Electroporate the linearized plasmid into Pichia pastoris competent cells, and spread the cells on a bleomycin YPD solid plate for culture.

[0072] Step 5: Pick single colonies for further screening, culture, and purification to obtain cowpea lipid transfer protein.

[0073] Among them, the single colonies screened in step 5 were picked onto a bleomycin YPD solid containing 500 μg / mL for further screening, and cultured at 30 °C in an inverted position until single colonies grew. Subsequently, the single colonies were inoculated into a bleomycin liquid YPD medium containing 500 μg / mL, cultured at 30 °C and 250 rpm for 24 h to obtain a seed solution. The seed solution was inoculated into a liquid YPD medium at an inoculation amount of 5%, cultured at 30 °C and 250 rpm for 3 days, centrifuged at 10000 rpm for 10 min, and the supernatant was collected and transferred to ice. Ammonium sulfate was added while stirring to make its saturation in the solution reach 80%, and it was left standing at low temperature for more than 4 h. Centrifuged at 4 °C and 10000 rpm for 5 min, and the supernatant was discarded. The precipitate was resuspended with 1 mL of 50 mM PBS buffer at pH 7.4, and the cowpea lipid transfer protein was obtained after desalting, as Figure 2The SDS-PAGE electrophoresis pattern of the supernatant of the fermented liquid of cowpea lipid transfer protein for 72 h is shown. In the figure, M represents the protein Marker; 1-4 respectively represent 4 screened strains, from Figure 2 it can be seen that cowpea lipid transfer protein was successfully obtained in Pichia pastoris.

[0074] Detection of the inhibitory effect of cowpea lipid transfer protein on porcine pancreatic amylase activity

[0075] Prepare glucose standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Take 6 centrifuge tubes and add 100 μL of glucose standard solutions with different concentrations and 100 μL of DNS color reagent respectively. After boiling water bath for 5 min, add water to make the volume up to 1 mL. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 540 nm and make a glucose standard curve.

[0076] Prepare 1% starch solution and 10 mg / mL porcine pancreatic amylase solution. Take 25 μL of each prepared cowpea lipid transfer protein and 12.5 μL of porcine pancreatic amylase solution into centrifuge tubes, label them in sequence, and incubate at 37 °C for 15 min. Similarly, mix porcine pancreatic amylase solution and PBS buffer (pH 7.4) as a control. Add 50 μL of the prepared starch solution to the above centrifuge tubes, keep at 25 °C for 30 min, add 100 μL of DNS color developing solution to the above centrifuge tubes, boil water bath for 5 min, add ultrapure water to make the volume up to 1 mL, use an ELISA reader to detect the absorbance at 540 nm, and then calculate the remaining activity of porcine pancreatic amylase according to the drawn standard curve. In this example, 40 strains were obtained through screening, as Figure 1 shown; further detect the remaining enzyme activity and inhibition rate of cowpea lipid transfer protein on porcine pancreatic amylase activity as Figure 3 shown. The specific data are shown in Table 1. It can be seen from Table 1 that cowpea lipid transfer protein has a high inhibitory activity on porcine pancreatic amylase.

[0077] Table 1

[0078]

[0079]

[0080] The predicted interaction between cowpea lipid transfer protein and porcine pancreatic amylase using AlphaFold3 is shown as Figure 4 shown. In the figure, light blue represents cowpea lipid transfer protein and purple represents porcine pancreatic amylase. From Figure 4 it can be seen that the mechanism of action of cowpea lipid transfer protein in inhibiting porcine pancreatic amylase is to block its substrate pocket, making the substrate unable to bind to the enzyme.

[0081] Tolerance experiment of cowpea lipid transfer protein to simulated gastric juice

[0082] Five cowpea lipid transfer proteins (LTP-WT, LTP-A, LTP-B, LTP-C, LTP-D) were separately purified and adjusted to the same concentration.

[0083] 200 μL of each cowpea lipid transfer protein was taken, pretreated with simulated gastric juice (SL66003, Coolaber, Beijing) and divided into two groups: the control group, added with 200 μL of PBS buffer (pH 7.4) to maintain the original activity; the simulated gastric juice treatment group, added with 200 μL of simulated gastric juice. Then it was incubated in a water bath at 37 °C for 30 min. After the incubation, 600 μL of PBS buffer (pH 7.4) was immediately added to adjust the pH back to neutral and terminate the reaction.

[0084] The inhibition rate of the above-treated proteins on the activity of porcine pancreatic amylase was detected. As Figure 5 shown, the inhibition rates of LTP-WT, LTP-A, LTP-B, LTP-C, LTP-D after treatment with simulated gastric juice were 16.2%, 50.28%, 45.96%, 42.54%, 68.88% respectively, indicating that LTP-D could retain the highest inhibition on the activity of porcine pancreatic amylase after treatment with simulated gastric juice.

[0085] Step 6: Mix the cowpea lipid transfer protein with sodium alginate and coat it with chitosan to obtain cowpea lipid transfer protein microcapsules.

[0086] Prepare a sodium alginate solution with a concentration of 1.5%. After dissolving at room temperature, let it stand for 2 h to remove air bubbles. Then mix the cowpea lipid transfer protein (LTP-D) solution with the sodium alginate solution at a volume ratio of 1:1 and stir at a low speed to ensure uniform dispersion of the protein.

[0087] Prepare a 0.1 M CaCl2 solution, place it on a stirring device, stir at a constant temperature of 37 °C, and slowly drip the protein-sodium alginate mixture in the form of uniform small drops into the continuously stirred CaCl2 solution to prepare gel beads. After the dripping is completed, continue to stand in the CaCl2 solution for 1 h to ensure sufficient cross-linking. Then collect the formed calcium alginate microspheres with a fine sieve and gently wash them with cold PBS buffer several times to remove unreacted CaCl2 and residual impurities.

[0088] Chitosan secondary coating: Slowly add chitosan powder to a 1% dilute acetic acid solution to make the final concentration of chitosan 1%. Slowly stir with a magnetic stirrer until completely dissolved. Then adjust the pH to 6.0 and let it stand at room temperature for 1 h to make the solution uniform and transparent. Uniformly disperse the washed calcium alginate microspheres in the chitosan solution, and gently stir and mix to ensure that the surface of each microsphere is fully in contact with the chitosan solution. React the microspheres in the chitosan solution for 60 minutes to form a dense coating on the surface of the microspheres by electrostatic adsorption of chitosan. After the reaction, gently wash the coated microspheres several times with an appropriate amount of PBS buffer to remove free chitosan and stabilize the coating structure. After washing, uniformly disperse the microspheres in PBS buffer for standby, and the vigna unguiculata lipid transfer protein microcapsules can be obtained.

[0089] Take 1 mL of the prepared vigna unguiculata lipid transfer protein microcapsules for the simulated gastric juice tolerance experiment, as Figure 6 shown. The average inhibition rate of 3 replicates in the control group was 95.4%; the average inhibition rate of 3 replicates in the simulated gastric juice treatment group was 79.7%; this indicates that the vigna unguiculata lipid transfer protein microcapsules improved the tolerance of vigna unguiculata lipid transfer protein to simulated gastric juice.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing cowpea lipid transfer protein microcapsules, characterized in that, Including: Step 1: Connect the base sequence of the parent to the pGAPZαA vector to obtain a recombinant plasmid, and transform the recombinant plasmid into Escherichia coli DH5α; Step 2: Inoculate the monoclonal of the Escherichia coli DH5α into an LB medium and extract the plasmid; Step 3: Digest the plasmid with enzymes and purify it to obtain a high-concentration linearized plasmid; Step 4: Electrotransform the linearized plasmid into Pichia pastoris competent cells, and coat the cells on a bleomycin YPD solid plate for culture; Step 5: Pick single colonies for further screening, culture and purification to obtain cowpea lipid transfer protein; Step 6: Mix the cowpea lipid transfer protein with sodium alginate and coat it with chitosan to obtain cowpea lipid transfer protein microcapsules.

2. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 1, characterized in that, The base sequence of the parent is: ATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAG.

3. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 2, characterized in that, The base sequence of the parent is: ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGGTGGAGGTCCCCCCCCCCCATTA.

4. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 2, characterized in that, The base sequence of the parent is: ATGGGCGGTGGTGGAGGCGAAGAGGAAGAGGAGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。 5. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 2, characterized in that, The base sequence of the parent is as follows: TTGAGAAGTCTAACCTTGATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGTTGAGAAGTCTAACCTTG。 6. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 2, characterized in that, The base sequence of the parent is as follows: ATGGCTCCACCACCACCTCCCATGACATGCGGACAAGTTCAAGGTAATTTAGCTCAATGTATAGGTTTCTTGCAAAATGGTGGTATCGTACCACCTGCTTGCTGTAACGGTGTCAAGAATATCTTGAATTCAGCACGTACTACTGCAGATAGAAGGGGCATTTGTTCCTGTCTTAAATCCGCTGCAGGCGCTGTGAGAGGATTGAATCCTTCCAATGCTCAGGCCTTACCAGGCAAATGTGGTGTTAACATACCCTGGAAAATCTCTTCCTCCACGAACTGTAACACTATTAACTAGGAAGAGGAGGAGGAAGGCGGTGGTGGTGGT。 7. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 1, characterized in that The base sequence of the cowpea lipid transfer protein is as follows: ATGACCTGTGGACAAGTTCAGGGTAATCTGGCTCAGTGTATCGGATTTCTTCAGAACGGTGGAATTGTTCCACCTGCTTGTTGTAACGGTGTCAAGAACATTCTGAACTCTGCTAGAACCACTGCTGACAGAAGAGGTATCTGCTCTTGTCTGAAATCCGCTGCAGGTGCTGTTAGAGGTTTGAATCCATCCAATGCTCAGGCTTTGCCAGGTAAATGCGGTGTTAACATACCTTGGAAGATTTCTAGTTCTACCAACTGTAACACTATCAACTAG。 8. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 1, characterized in that: The LB medium in step 2 is an LB medium containing 25 μg / mL bleomycin; The culture conditions for the Escherichia coli DH5α monoclonal in step 2 are: overnight culture at 37°C for 12 - 16 h.

9. The preparation method of a cowpea lipid transfer protein microcapsule according to claim 1, wherein, The inoculation amount of the seed liquid during culture in step 5 is 5%.

10. An application of a cowpea lipid transfer protein microcapsule for preparing a dietary supplement for preventing obesity and diabetes.

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