Angelica keiskei bioactive peptide and preparation method thereof

Abstract

The invention discloses an angelica keiskei bioactive peptide and a preparation method thereof. The angelica keiskei bioactive peptide is prepared by carefully selecting fresh leaves and roots of angelica keiskei, treating by adopting a bacillus subtilis and lactobacillus composite fermentation method, and separating and purifying by adopting a modern extraction process. The angelica keiskei bioactive peptide can effectively remove sediments on blood vessel walls, expand blood vessels, reduce peripheral resistance, improve blood circulation and play a role in reducing blood pressure; the blood fat such as serum cholesterol can be reduced, and the auxiliary treatment effect on diabetes mellitus such as hyperglycemia is achieved.

Description

Technical Field

[0001] The invention relates to the field of health care products, and in particular to a keiskei leaf bioactive peptide and a preparation method thereof. Background Art

[0002] Bioactive peptides (BAPs) are specific protein fragments composed of 2-20 amino acid residues, which are generally obtained by in vitro hydrolysis, in vivo gastrointestinal digestion or microbial fermentation. The biological activity of BAPs is related to their amino acid composition, sequence and structure. They can be directly digested and absorbed by the human intestine and enter the circulatory system in a complete form, thus having high bioavailability. Compared with traditional chemical drugs, BAPs have greater tissue affinity and specificity, and fewer side effects, so they have great application prospects in the prevention and treatment of many diseases.

[0003] The sources of plant-derived BAPs include beans, grains, nuts, fruits and vegetables. Compared with animal sources, plant-derived BAPs are abundant, simple to prepare and low in cost, and have a good development trend. In recent years, the various biological activities of plant-derived BAPs have been continuously explored, including lowering blood pressure, anti-oxidation, antibacterial, hypoglycemic and anti-cancer. Related studies have pointed out that plant-derived BAPs can be used as drug ingredients to treat chronic metabolic diseases such as diabetes and hypertension. They have high edible and medicinal value and can meet the various needs of human health.

[0004] Leaf protein is a high-quality concentrate prepared from fresh plant stems and leaves by squeezing juice, separating protein from the liquid, and concentrating and drying. Plant leaf protein is a "functional protein" and is mainly composed of matrix proteins in chloroplasts and cytoplasmic proteins with good solubility in the cytoplasm. Plant leaf protein is one of the largest renewable protein resources in the world. Plant-derived protein is nutritionally balanced and does not contain cholesterol. There are many types of protein-rich plants in my country, with a wide range of sources, a large planting area, and low costs. Fully developing and utilizing its potential value has important economic and social significance.

[0005] Angelica keiskei Koidz. is a perennial large herb belonging to the Apiaceae family. The stems and leaves contain yellow sap, and the stems are erect and branched. The basal leaves are clustered, with long petioles, and the base is enlarged and clasping the stem, with shallow or deep lobes. The pinnates are ovate or broadly ovate, with sharp tips and fine serrations, and smooth and hairless on both sides. The leaves on the stems gradually become smaller. The inflorescence is compound and covered with short hairs. There is no involucre, and the bracts are several and broadly linear. There are many small flowers, which are creamy yellow. The fruit is oblong and slightly flat. The flowering period is from May to October, and the fruiting period is from September to December. Angelica keiskei Koidz. is named for its strong vitality that if you pick the leaves today, new leaves will emerge tomorrow, so it is called Angelica keiskei Koidz.

[0006] There are significant records of tomorrow leaf in traditional medicinal herbs. In the Compendium of Materia Medica by Li Shizhen, a famous herbalist in the late Ming Dynasty, the Yamato Materia Medica of the sixth year of Hoei in Japan, and the Heman Sancai Tuhui by the doctor Terajima Ryo of the second year of Shotoku in Japan, tomorrow leaf is highly praised. It is also the longevity fairy grass that Qin Shihuang ordered Xu Fu to find. Modern research shows that tomorrow leaf has an improvement effect on high blood pressure, low blood pressure, insomnia, menopausal disorders, gastrointestinal diseases, asthma, palpitations, arteriosclerosis, diabetes, gastric cancer, angina pectoris, energy loss, physical strength, liver cirrhosis, rheumatism, sciatica, nasal empyema, shoulder stiffness, constipation, etc. Summary of the invention

[0007] In view of this, the present invention provides a Angelica keiskei bioactive peptide and a preparation method thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A bioactive peptide from Angelica keiskei leaf is prepared by the following process:

[0010] The first step is to pick fresh leaves and roots of Angelica keiskei, remove impurities, rinse with clean water, dry and crush, and then place in a polypropylene bag for high pressure sterilization to obtain Angelica keiskei powder;

[0011] The second step is to inoculate the composite strains of Bacillus subtilis and Lactobacillus, add an appropriate amount of water, and perform solid-state fermentation. After the fermentation is completed, the mixture is transported to a drying tower through a pipeline for drying and crushing to obtain fermented powder.

[0012] The third step is to dissolve the fermented powder in a NaOH solution, heat and extract, and centrifuge to obtain the supernatant; then add acetic acid overnight and centrifuge to obtain a precipitate;

[0013] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0014] In some embodiments, the weight ratio of the leaves and roots of Angelica keiskei in the first step is 1-5:1; the drying temperature is 50° C., and the pulverization is pulverized into 50 meshes.

[0015] In some embodiments, the high pressure sterilization in the first step is carried out at 0.3 MPa and 121° C. for 20 minutes.

[0016] In some embodiments, the content of Bacillus subtilis in the composite bacteria in the second step is 10 6 -10 7 cfu / g, and the added amount is 1-5% of the weight of the akebia powder.

[0017] In some embodiments, the content of the lactobacillus in the composite bacteria in the second step is 106 -10 7 cfu / g, and the added amount is 1-5% of the weight of the akebia powder.

[0018] In some embodiments, the addition of water in the second step makes the moisture content of the Angelica keiskei powder reach 35-45%.

[0019] In some embodiments, the solid-state fermentation conditions in the second step are sealed and fermented at 30-35°C for 5-7 days; the temperature in the drying tower is 50°C.

[0020] In some of the embodiments, the content of NaOH in the NaOH solution in the third step is 1 wt %; the material-liquid ratio is 1:10-20.

[0021] In some embodiments, the heating temperature in the third step is 50-60° C.; the extraction time is 1-5 hours.

[0022] In some of the embodiments, acetic acid is added in the third step to make the pH of the system be 4.0-4.5.

[0023] The beneficial effects of the technical solution provided by the embodiment of the present invention are as follows: the Angelica keiskei bioactive peptide obtained by the present invention is obtained by selecting fresh Angelica keiskei leaves and roots, treating them by a composite fermentation method of Bacillus subtilis and Lactobacillus, and then separating and purifying them by a modern extraction process. The Angelica keiskei bioactive peptide can effectively remove vascular wall deposits, dilate blood vessels, reduce peripheral resistance, improve blood circulation, and have a blood pressure lowering effect; it can also reduce blood lipids such as serum cholesterol, and has an auxiliary therapeutic effect on diabetes such as hyperglycemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for describing the embodiments or the prior art are briefly introduced below.

[0025] Figure 1 The appearance of Angelica keiskei powder in Example 1 before and after fermentation is shown. DETAILED DESCRIPTION

[0026] The present invention discloses a kind of Angelica keiskei bioactive peptide and its preparation method. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0027] The Angelica keiskei bioactive peptide provided by the invention and the raw materials and reagents used in the preparation method thereof can be purchased from the market.

[0028] The present invention will be further described below in conjunction with embodiments:

[0029] Example 1

[0030] A bioactive peptide from Angelica keiskei leaf is prepared by the following process:

[0031] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 1:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0032] The second step is to inoculate a composite strain of Bacillus subtilis and Lactobacillus, wherein the bacterial content of Bacillus subtilis in the composite strain is 10 6 cfu / g, the addition amount is 5% of the weight of the Angelica keiskei powder; the lactobacillus content is 10 6 cfu / g, the addition amount is 5% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 35%, seal it, and ferment it in the solid state at 35°C for 5 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0033] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:20; heat and extract at 50°C for 5 hours, and centrifuge to obtain the supernatant; then add acetic acid to adjust the pH of the system to 4.0; overnight, centrifuge to obtain a precipitate;

[0034] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0035] Example 2

[0036] A bioactive peptide from Angelica keiskei leaf is prepared by the following process:

[0037] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 5:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0038] The second step is to inoculate a composite strain of Bacillus subtilis and Lactobacillus, wherein the bacterial content of Bacillus subtilis in the composite strain is 10 7 cfu / g, the addition amount is 1% of the weight of the Angelica keiskei powder; the lactobacillus content is 10 7cfu / g, the addition amount is 1% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 45%, seal it, and ferment it in the solid state at 30°C for 7 days; after the fermentation is completed, transport it into a drying tower through a pipeline, dry it at 50°C, and crush it to obtain fermented powder;

[0039] The third step is to dissolve the fermented powder in a 1wt% NaOH solution with a solid-liquid ratio of 1:10; heat and extract at 60°C for 1 hour, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.5; overnight, centrifuge and obtain a precipitate;

[0040] The fourth step is to vacuum freeze-dry the precipitate to obtain the Angelica keiskei bioactive peptide.

[0041] Example 3

[0042] A bioactive peptide from Angelica keiskei is prepared by the following process:

[0043] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 3:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0044] The second step is to inoculate a composite strain of Bacillus subtilis and Lactobacillus, wherein the bacterial content of Bacillus subtilis in the composite strain is 5×10 6 cfu / g, the addition amount is 3% of the weight of the angelica leaf powder; the lactobacillus content is 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 40%, seal it, and ferment it in the solid state at 32°C for 6 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0045] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0046] The fourth step is to vacuum freeze-dry the precipitate to obtain the Angelica keiskei bioactive peptide.

[0047] Comparative Example 1

[0048] A bioactive peptide from Angelica keiskei is prepared by the following process:

[0049] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 3:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0050] The second step is to inoculate Bacillus subtilis strains, wherein the Bacillus subtilis strain contains 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 40%, seal it, and ferment it in the solid state at 32°C for 6 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0051] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0052] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0053] Comparative Example 2

[0054] A bioactive peptide from Angelica keiskei leaf is prepared by the following process:

[0055] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 3:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0056] The second step is to inoculate the lactobacillus strain, wherein the lactobacillus strain contains 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 40%, seal it, and ferment it in the solid state at 32°C for 6 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0057] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0058] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0059] Comparative Example 3

[0060] A bioactive peptide from Angelica keiskei is prepared by the following process:

[0061] The first step is to pick fresh leaves and roots of Angelica keiskei, wherein the weight ratio of leaves to roots is 3:1; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, and then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0062] The second step is to transport the product into a drying tower through a pipeline and dry it at 50°C;

[0063] The third step is to dissolve the Angelica keiskei powder in a 1 wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0064] The fourth step is to vacuum freeze-dry the precipitate to obtain the Angelica keiskei bioactive peptide.

[0065] Test 1

[0066] The polypeptide content of the tomorrow leaf bioactive peptides obtained in Examples 1-3 and Comparative Examples 1-3 was determined. The determination method was based on the literature "Optimization of preparation process of Cardamine violaceum oligopeptides and study of structural characteristics and oxidative activity [J]. Food and Machinery, 2024, 40(01): 189-196.", and the test results are shown in Table 1.

[0067] Table 1 Content of bioactive peptides in Angelica keiskei

[0068] Test Group Peptide content / % Example 1 78.40±1.02 Example 2 74.39±0.28 Example 3 79.98±0.01 Comparative Example 1 51.27±0.11 Comparative Example 2 53.94±0.04 Comparative Example 3 46.44±0.22

[0069] As can be seen from Table 1, the Angelica keiskei bioactive peptides prepared in the embodiment of the present invention have a higher peptide content.

[0070] Test 2

[0071] In order to investigate the effect of the Angelica keiskei bioactive peptide prepared by the present invention on diabetes, the test was conducted by referring to the method described in the literature "Zang Yanqing, Chuang Yingying, Wang Changyuan, et al. Regulatory effect of quinoa polysaccharide on glucose and lipid metabolism in type 2 diabetic mice [J / OL]. Food Industry Science and Technology, 1-13." The preferred embodiment 3 was used as the test object, and comparative examples 4-5 were set.

[0072] Comparative Example 4

[0073] A bioactive peptide from Angelica keiskei is prepared by the following process:

[0074] The first step is to pick fresh Angelica keiskei leaves; remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0075] The second step is to inoculate a composite strain of Bacillus subtilis and Lactobacillus, wherein the bacterial content of Bacillus subtilis in the composite strain is 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; the lactobacillus content is 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 40%, seal it, and ferment it in the solid state at 32°C for 6 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0076] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0077] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0078] Comparative Example 5

[0079] A bioactive peptide from Angelica keiskei leaf is prepared by the following process:

[0080] The first step is to pick fresh roots of Angelica keiskei, remove impurities, rinse with clean water, dry at 50°C, and crush to 50 mesh, then place in a polypropylene bag and sterilize under high pressure at 0.3MPa and 121°C for 20 minutes to obtain Angelica keiskei powder;

[0081] The second step is to inoculate a composite strain of Bacillus subtilis and Lactobacillus, wherein the bacterial content of Bacillus subtilis in the composite strain is 5×10 6 cfu / g, the addition amount is 3% of the weight of the angelica leaf powder; the lactobacillus content is 5×10 6 cfu / g, the addition amount is 3% of the weight of the Angelica keiskei powder; add an appropriate amount of water to make the moisture content of the Angelica keiskei powder reach 40%, seal it, and ferment it in the solid state at 32°C for 6 days; after the fermentation is completed, transport it into a drying tower through a pipeline and dry it at 50°C, and crush it to obtain fermented powder;

[0082] The third step is to dissolve the fermented powder in 1wt% NaOH solution with a solid-liquid ratio of 1:15; heat and extract at 55°C for 3 hours, centrifuge and take the supernatant; then add acetic acid to adjust the pH of the system to 4.2; centrifuge overnight to obtain a precipitate;

[0083] The fourth step is to vacuum freeze-dry the precipitate to obtain the tomorrow leaf bioactive peptide.

[0084] Basic test steps: 40 old rats with SPF-grade high-fat diet and streptozotocin-induced diabetes model were randomly divided into 4 groups, 10 rats in each group. The blank control group was fed with basic feed, and the other groups were fed with feed mixed with 10wt% Angelica keiskei bioactive peptides for 4 consecutive weeks. Blood was collected from the eyeballs, and the total cholesterol-TC value and triglyceride-TG value were determined using a kit. The specific test results are shown in Table 2.

[0085] Table 2 Effects of Angelica keiskei bioactive peptides on blood lipid indexes in diabetic mice

[0086] Test Group TC(mmol / L) TG (mmol / L) Example 3 2.14±0.16 0.72±0.07 Comparative Example 4 3.15±0.28 0.89±0.17 Comparative Example 5 3.05±0.02 0.93±0.22 Blank control group 3.56±0.13 0.98±0.05

[0087] As can be seen from Table 2, the Angelica keiskei bioactive peptides prepared in the examples of the present invention can improve dyslipidemia in elderly rats with a diabetic model.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bioactive peptide from Angelica keiskei, characterized in that: Prepared by the following process: The first step is to pick fresh leaves and roots of Angelica keiskei, remove impurities, rinse with clean water, dry and crush, and then place in a polypropylene bag for high pressure sterilization to obtain Angelica keiskei powder; The second step is to inoculate the composite strains of Bacillus subtilis and Lactobacillus, add an appropriate amount of water, and perform solid-state fermentation. After the fermentation is completed, the mixture is transported to a drying tower through a pipeline for drying and crushing to obtain fermented powder. The third step is to dissolve the fermented powder in a NaOH solution, heat and extract, and centrifuge to obtain the supernatant; then add acetic acid overnight and centrifuge to obtain a precipitate; The fourth step is to vacuum freeze-dry the precipitate to obtain the Angelica keiskei bioactive peptide.

2. The Angelica keiskei bioactive peptide according to claim 1, characterized in that In the first step, the weight ratio of the leaves and roots of Angelica keiskei is 1-5:1; the drying temperature is 50° C., and the pulverization is pulverized into 50 meshes.

3. The Angelica keiskei bioactive peptide according to claim 1, characterized in that The high pressure sterilization described in the first step is carried out at 0.3 MPa and 121° C. for 20 minutes.

4. The Angelica keiskei bioactive peptide according to claim 1, characterized in that The bacterial content of Bacillus subtilis in the composite bacteria in the second step is 10 6 -10 7 cfu / g, and the added amount is 1-5% of the weight of the akebia powder.

5. The Angelica keiskei bioactive peptide according to claim 1, characterized in that The bacterial content of the lactobacillus in the composite bacteria in the second step is 10 6 -10 7 cfu / g, and the added amount is 1-5% of the weight of the akebia powder.

6. The Angelica keiskei bioactive peptide according to claim 1, characterized in that The addition of water in the second step brings the moisture content of the ashitaba powder to 35-45%.

7. The Angelica keiskei bioactive peptide according to claim 1, characterized in that: The solid-state fermentation conditions in the second step are sealed and fermented at 30-35°C for 5-7 days; the temperature in the drying tower is 50°C.

8. The Angelica keiskei bioactive peptide according to claim 1, characterized in that: The content of NaOH in the NaOH solution in the third step is 1wt%; the material-liquid ratio is 1:10-20.

9. The Angelica keiskei bioactive peptide according to claim 1, characterized in that: The heating temperature in the third step is 50-60° C.; the extraction time is 1-5 hours.

10. The Angelica keiskei bioactive peptide according to claim 1, characterized in that: In the third step, acetic acid is added to make the pH of the system 4.0-4.5.

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