阅读说明：本技术 一种红腰豆酶解多肽及其促进创面愈合的用途 (Red kidney bean enzymolysis polypeptide and application thereof in promoting wound healing ) 是由 鲍会梅 曹兴玉 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种红腰豆酶解多肽及其促进创面愈合的用途。本发明提供了一种来源于红腰豆的酶解多肽,采用的酶为碱性蛋白酶,其分子量为20～30kDa,该酶解多肽可以有效促进人皮肤成纤维细胞的增殖,因此具有促进创面愈合的作用,具有开发制备成促进创面愈合的药物或者化妆品的前景。(The invention discloses a red kidney bean enzymolysis polypeptide and application thereof in promoting wound healing. The invention provides an enzymolysis polypeptide from red kidney beans, the adopted enzyme is alkaline protease, the molecular weight of the alkaline protease is 20-30 kDa, and the enzymolysis polypeptide can effectively promote the proliferation of human skin fibroblasts, so that the enzymolysis polypeptide has the effect of promoting wound healing and has the prospect of developing and preparing medicines or cosmetics for promoting wound healing.)

1. A red kidney bean enzymolysis polypeptide is characterized in that: is an enzymolysis polypeptide with the molecular weight of 20-30 kDa in the red kidney beans.

2. The anacardic polypeptide of claim 1, wherein: the enzyme is an alkaline protease.

3. The anacardic polypeptide of claim 2, which is prepared by the following steps:

alkali extraction and acid precipitation extraction of total protein: taking a proper amount of kidney bean powder, adding a proper amount of water, mixing to prepare a mixed solution, adjusting the pH of the mixed solution to 10.2 by using a sodium hydroxide solution, extracting in a water bath, centrifuging to obtain a supernatant, adjusting the pH to 5.5 by using a hydrochloric acid solution, centrifuging, collecting precipitate, and washing the precipitate with deionized water;

alkaline protease enzymolysis: weighing a proper amount of total protein, adding a proper amount of water to prepare a mixed solution, adjusting the pH of the mixed solution to 8.5 by using a sodium hydroxide solution, controlling the temperature to be 50 ℃, adding alkaline protease for enzymolysis, inactivating enzyme in a boiling water bath after the enzymolysis is finished, and centrifuging to obtain a supernatant;

and (3) ultrafiltration: and (3) carrying out ultrafiltration classification by using a small tangential flow ultrafiltration system according to the instruction, sequentially separating the supernate by ultrafiltration membranes with molecular weights of 20 kDa and 30kDa, collecting components with molecular weights of 20 kDa to 30kDa, and freeze-drying.

4. The anacardic polypeptide of claim 3, wherein: the centrifugation condition in the step of extracting total protein by alkali extraction and acid precipitation is 4500r/min rotation speed centrifugation for 20 min.

5. The anacardic polypeptide of claim 3, wherein: the enzyme adding amount in the alkaline protease enzymolysis step is 5000U/g calculated by substrate.

6. The anacardic polypeptide of claim 3, wherein: the enzymolysis time in the enzymolysis step of the alkaline protease is 120min, and the pH is measured and adjusted to 8.5 every 10min in the enzymolysis process.

7. The anacardic polypeptide of claim 3, wherein: the centrifugation condition in the alkaline protease enzymolysis step is 12000r/min for 10 min.

8. Use of the anacardium procumbens enzymatic polypeptide of claims 1-7 for preparing a medicament or a cosmetic for promoting human skin fibroblast proliferation.

9. Use of the anacardium procumbens enzymatic polypeptide of claims 1-7 for preparing a medicament or a cosmetic for promoting wound healing.

Technical Field

The invention belongs to the field of food chemistry, and particularly relates to a red kidney bean enzymolysis polypeptide and application thereof in promoting wound healing.

Background

The kidney beans (Phaseolus vulgaris Linn) are one of beans rich in nutrition, contain abundant vitamins A, B, C and E, and also contain abundant antioxidants, proteins, dietary fibers and various nutrients such as iron, magnesium, phosphorus and the like, and have the effects of enriching blood, enhancing immunity, helping cell repair, preventing aging and the like.

At present, polysaccharides are mostly studied for high molecular weight chemical components in the red kidney beans. The prior art shows that the polysaccharide in the red kidney beans is a main active component of the red kidney beans, and the polysaccharide is a high molecular compound with various bioactive functions and has various pharmacological activity characteristics, such as characteristics of delaying senescence, regulating immune cells, resisting viruses, oxidation, fatigue, tumors, thrombus, reducing blood sugar and blood fat and the like (reference document: rien and the like, ultrasonic-assisted enzymatic extraction process optimization, journal of agricultural engineering, volume 31, 15, 8/2015).

Proteins are also a very important class of ingredients in foods, especially seed foods. However, the protein component in food has a high molecular weight and is difficult to be directly absorbed and utilized by the human body.

The enzymolysis is an important method for researching and utilizing protein, and the polypeptide formed after enzymolysis overcomes the defect of overlarge molecular weight of the protein and has good absorption and utilization effects. However, it is undeniable that there is great uncertainty about whether the polypeptide has activity after enzymolysis due to uncertainty of enzymolysis, which increases the research difficulty of researchers.

The invention is especially provided and created in order to fully utilize the resource advantages of the red kidney beans and dig up the commercial value of the red kidney beans.

Disclosure of Invention

The invention aims to provide a red kidney bean enzymolysis polypeptide and application thereof in promoting wound healing.

The technical scheme of the invention is as follows:

an enzymolysis polypeptide of red kidney beans is an enzymolysis polypeptide with the molecular weight of 20-30 kDa in red kidney beans.

Further, the enzyme is an alkaline protease.

Furthermore, the red kidney bean enzymolysis polypeptide is prepared by the following steps:

alkali extraction and acid precipitation extraction of total protein: taking a proper amount of kidney bean powder, adding a proper amount of water, mixing to prepare a mixed solution, adjusting the pH of the mixed solution to 10.2 by using a sodium hydroxide solution, extracting in a water bath, centrifuging to obtain a supernatant, adjusting the pH to 5.5 by using a hydrochloric acid solution, centrifuging, collecting precipitate, and washing the precipitate with deionized water;

alkaline protease enzymolysis: weighing a proper amount of total protein, adding a proper amount of water to prepare a mixed solution, adjusting the pH of the mixed solution to 8.5 by using a sodium hydroxide solution, controlling the temperature to be 50 ℃, adding alkaline protease for enzymolysis, inactivating enzyme in a boiling water bath after the enzymolysis is finished, and centrifuging to obtain a supernatant;

and (3) ultrafiltration: and (3) carrying out ultrafiltration classification by using a small tangential flow ultrafiltration system according to the instruction, sequentially separating the supernate by ultrafiltration membranes with molecular weights of 20 kDa and 30kDa, collecting components with molecular weights of 20 kDa to 30kDa, and freeze-drying.

More specifically, the centrifugation condition in the step of extracting total protein by alkali extraction and acid precipitation is 4500r/min rotation speed centrifugation for 20 min.

More specifically, the amount of the enzyme added in the alkaline protease hydrolysis step is 5000U/g based on the substrate.

More specifically, the enzymolysis time in the alkaline protease enzymolysis step is 120min, and the pH is measured and adjusted to 8.5 every 10min during the enzymolysis.

More particularly, the centrifugation condition in the alkaline protease enzymolysis step is 12000r/min for 10 min.

The application of the anacardium enzymatic polypeptide in preparing medicines or cosmetics for promoting human skin fibroblast proliferation.

The application of the red kidney bean enzymolysis polypeptide in preparing a medicine or a cosmetic for promoting wound healing.

The invention has the technical effects that:

the invention provides an enzymolysis polypeptide from red kidney beans, the adopted enzyme is alkaline protease, the molecular weight of the alkaline protease is 20-30 kDa, and the enzymolysis polypeptide can effectively promote the proliferation of human skin fibroblasts, so that the enzymolysis polypeptide has the effect of promoting wound healing and has the prospect of developing and preparing medicines or cosmetics for promoting wound healing.

Drawings

FIG. 1 is a bar graph comparing the proliferation activity of cells in each group.

Detailed Description

Example 1:

1. raw materials, instruments and reagents

Drying and crushing mature red kidney bean seeds, and sieving the crushed red kidney bean seeds with a 60-mesh sieve for later use.

Labscale TFF System Small tangential flow ultrafiltration System from Millipore, USA.

The water is deionized water, and the alkaline protease is purchased from Beijing Solaibao technology.

2. Preparation method

Alkali extraction and acid precipitation extraction of total protein: taking a proper amount of kidney bean powder, adding water according to a material-liquid ratio of 1:10(g: mL) to prepare a mixed solution, adjusting the pH of the mixed solution to 10.2 by using 1mol/L sodium hydroxide solution, extracting for 3h in a water bath at 60 ℃, centrifuging at the rotating speed of 4500r/min for 20min to take supernatant, adjusting the pH to 5.5 by using 1mol/L hydrochloric acid solution, centrifuging at the rotating speed of 4500r/min for 20min to collect precipitate, and washing the precipitate with deionized water for 3 times.

Alkaline protease enzymolysis: weighing a proper amount of total protein, adding water according to a material-liquid ratio of 1:10(g: mL) to mix the total protein and the water to prepare a mixed solution, adjusting the pH of the mixed solution to 8.5 by using 1mol/L sodium hydroxide solution, controlling the temperature to be 50 ℃, adding alkaline protease to carry out enzymolysis, wherein the enzyme adding amount is 5000U/g calculated by a substrate, the enzymolysis time is 120min, and measuring the pH and adjusting the pH to 8.5 every 10min in the enzymolysis process. Inactivating enzyme in boiling water bath for 10min after enzymolysis, centrifuging at 12000r/min for 10min, and collecting supernatant.

And (3) ultrafiltration: and (2) carrying out ultrafiltration classification by using a small tangential flow ultrafiltration system according to the specification, separating the supernate by sequentially passing through ultrafiltration membranes with molecular weights of 10 kDa, 20 kDa and 30kDa, sequentially preparing components with molecular weights of less than or equal to 10 kDa, 10-20 kDa, 20-30 kDa and more than or equal to 30kDa, and respectively freeze-drying the components with molecular weights of 10-20 kDa and 20-30 kDa to obtain the enzymolysis polypeptide A and the enzymolysis polypeptide B.

The enzymatically hydrolyzed polypeptide A and the enzymatically hydrolyzed polypeptide B were stored at 4 ℃ and used for the activity test in example 2.

Example 2:

1. experimental methods

Will be paired withHuman Skin Fibroblasts (HSFs) in several growth stages were digested and resuspended in 1X 10 DMEM medium⁵Suspension at 1X 10⁴And each well is inoculated in a 96-well plate, each well is 100 mu L and is divided into a polypeptide A low-concentration group, a polypeptide A high-concentration group, a polypeptide B low-concentration group, a polypeptide B high-concentration group and a control group, after the culture is carried out for 24 hours, the polypeptide A low-concentration group and the polypeptide A high-concentration group are respectively replaced by DMEM culture media containing 100 and 200 mu g/mL enzymolysis polypeptide A for continuous culture, the polypeptide B low-concentration group and the polypeptide B high-concentration group are respectively replaced by DMEM culture media containing 100 and 200 mu g/mL enzymolysis polypeptide B for continuous culture, and the control group is replaced by fresh DMEM culture media for continuous culture. Continuously culturing for 48h, removing the culture solution, washing with PBS for 2-3 times, adding 100 μ L of MTT solution with concentration of 0.5mg/L into each well, incubating for 4h, carefully removing the liquid in each well, adding 100 μ L of DMSO into each well, oscillating for 10min, and measuring the light absorption OD of each well under 490nm wavelength of an enzyme-labeling instrument_490nmCell proliferation activity was calculated according to the formula:

cell proliferation Activity ═ Polypeptides OD_490nmControl group OD_490nm×100％。

2. Results of the experiment

The proliferation activity of each group of cells is shown in table 1 and figure 1, the proliferation promoting activity of 100 and 200 mug/mL enzymolysis polypeptide A on HSFs is not obvious, the proliferation promoting activity of 100 and 200 mug/mL enzymolysis polypeptide B on HSFs can effectively promote the proliferation of the HSFs, and the dose effect can be seen.

TABLE 1 cell proliferation Activity of each group

As known to those skilled in the art, human dermal fibroblasts are an important component of human skin cells, and dermal fibroblasts are the main repairing cells involved in wound repair, and whether the function of dermal fibroblasts is normal or not directly affects the generation of granulation tissue of wound and the healing of wound (reference: He Xiu Juan et al, research progress of dermal fibroblasts in wound healing, China J. Damage and repair, Vol. 16, No. 1 of 2021). Research results show that the enzymolysis polypeptide B provided by the invention can effectively promote the proliferation of human skin fibroblasts, so that the enzymolysis polypeptide B has the effect of promoting wound healing and has the prospect of developing and preparing medicines or cosmetics for promoting wound healing.