CN116790699A - Preparation method of protein hydrolysate for treating acute alcoholic liver injury cell repair - Google Patents
Preparation method of protein hydrolysate for treating acute alcoholic liver injury cell repair Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Abstract
The application relates to a preparation method of a protein hydrolysate for treating acute alcohol liver injury cell repair, which comprises the following steps: preparing a black bean protein hydrolysate by enzymolysis of black bean protein peptide, wherein a peptide segment with the molecular weight of 2014-355 Da is taken as a main component, and is 45.53-49.12%, a peptide segment with the molecular weight of less than 355Da is taken as 36.85-39.98%, the OH clearance is 28.15-32.40%, and the enzymolysis time is 70-90min, so that the black bean protein hydrolysate containing black bean peptide and amino acid is obtained; regulating pH of black bean protein hydrolysate to 7-9; lyophilizing the hydrolysate with pH value regulated to obtain lyophilized powder; adding the freeze-dried powder into a buffer solution to prepare a solution, so as to obtain a black bean peptide solution, wherein the content of black bean protein hydrolysate in the solution is 0.05-0.2mg/mL; the black bean protein hydrolysate with concentration of 0.05-0.2mg/mL can improve oxidation resistance, relieve oxidative stress, and inhibit alcohol-induced hepatic cell injury.
Description
Technical Field
The application relates to the technical field of biomedical science, in particular to a preparation method of a protein hydrolysate for treating acute alcohol liver injury cell repair.
Background
Alcohol is one of the mechanisms responsible for liver injury, and intake of large amounts of alcohol can lead to oxidative stress and inflammatory reactions, resulting in liver injury. Therefore, the liver protection problem is more and more important, and liver injury is increasingly serious for more and more people.
It is currently believed that the mechanism of alcohol-induced liver injury is the activation of large amounts of reactive oxygen species, which ultimately lead to liver cell membrane damage and cell death, and thus reducing ROS levels is effective in protecting tissues from oxidative stress.
Many scholars are trying to develop natural effective non-toxic liver protection products, which are not of a small nutritional value as natural cereal proteins, black bean proteins. The black bean protein hydrolysate has extremely high antioxidant activity and very strong free radical quenching capacity and alcohol dehydrogenase activating capacity, so that the product capable of treating acute alcohol liver injury cell repair is prepared and has important significance.
Disclosure of Invention
In order to realize the blood purification of tumors caused by immunodeficiency and reduce the toxic and side effects of medicines, the application provides a preparation method of a protein hydrolysate for treating acute alcohol liver injury cell repair.
In order to solve the problems, the following technical scheme is provided:
the preparation method of the protein hydrolysate for treating acute alcohol liver injury cell repair comprises the following steps:
a. preparing a black bean protein hydrolysate by enzymolysis of black bean protein peptide, wherein a peptide segment with the molecular weight of 2014-355 Da is taken as a main component, and is 45.53-49.12%, a peptide segment with the molecular weight of less than 355Da is taken as 36.85-39.98%, the OH clearance is 28.15-32.40%, and the enzymolysis time is 70-90min, so that the black bean protein hydrolysate containing black bean peptide and amino acid is obtained;
b. regulating pH of black bean protein hydrolysate to 7-9;
c. lyophilizing the hydrolysate with pH value regulated to obtain lyophilized powder;
d. adding the freeze-dried powder into a buffer solution to prepare a solution, and obtaining the black bean peptide solution, wherein the content of black bean protein hydrolysate in the solution is 0.05-0.2mg/mL.
In the step a, 5-7 parts of black bean peptide and 3-5 parts of enzyme are added into 70-100 parts of water according to the mass ratio, so as to obtain the black bean protein hydrolysate before enzymolysis.
The enzymolysis temperature is 50-60 ℃, and the enzyme adopted is black bean protease.
In said step c, a lyophilizer is used as the device for the lyophilization process.
The black bean peptide solution is adopted to prepare freeze-dried powder at the temperature of-40 to-70 ℃ by a freeze dryer.
The pH value of the black bean peptide solution is regulated by acid and alkali, the used solution is NaOH and HCL, and the concentration of the NaOH and the HCL is 0.1mol/L.
The buffer solution is PBS buffer solution.
By adopting the scheme, the method has the following advantages:
the black bean protein hydrolysate with concentration of 0.05-0.2mg/mL can improve oxidation resistance, relieve oxidative stress, and inhibit alcohol-induced hepatic cell injury.
Drawings
FIG. 1 is a graph showing experimental results of screening alcohol concentration by CCK-8 method;
FIG. 2 is a graph showing experimental results of the degree of damage of LO2 cells by different concentrations of alcohol under a microscope;
FIG. 3 is a graph showing the experimental results of the CCK-8 method for verifying whether the black bean protein hydrolysate has toxicity;
FIG. 4 is a graph showing the experimental results of the degree of repair of LO2 cells by black bean protein hydrolysate under a microscope;
FIG. 5 is a graph of experimental results of the effect of black bean protein hydrolysate on LO2 cell reactive oxygen species;
FIG. 6 is a graph of experimental results of the effect of black bean protein hydrolysate on LO2 intracellular AST content;
FIG. 7 is a graph of experimental results of the effect of black bean protein hydrolysate on LO2 intracellular ALT content;
FIG. 8 is a graph showing experimental results of the effect of black bean protein hydrolysate on the SOD content in LO2 cells;
FIG. 9 is a graph of experimental results of the effect of black bean protein hydrolysate on the intracellular GSH content of LO 2.
Detailed Description
The embodiment aims at providing a preparation method of a protein hydrolysate for treating acute alcoholic liver injury cell repair, which comprises the following steps:
example 1
Adding 5 parts of black bean peptide and 3 parts of enzyme into 100 parts of water according to the mass ratio, and carrying out enzymolysis on the black bean peptide at 50 ℃ for 70min to obtain a black bean protein hydrolysate containing the black bean peptide and the amino acid; secondly, regulating the pH value of the black bean protein hydrolysate to 8; and (5) performing freeze-drying treatment on the hydrolysate with the pH value adjusted to obtain freeze-dried powder. Then, adding the freeze-dried powder into PBS buffer solution to prepare a solution, and obtaining black bean peptide solution, wherein the content of black bean protein hydrolysate in the prepared black bean peptide solution is 0.05mg/mL, and the clearance of OH is 29.25%.
Example 2
Adding 6 parts of black bean peptide and 4 parts of enzyme into 100 parts of water according to the mass ratio, and carrying out enzymolysis on the black bean peptide at 50 ℃ for 80 minutes to obtain black bean protein hydrolysate containing the black bean peptide and amino acid; secondly, regulating the pH value of the black bean protein hydrolysate to 8; and (5) performing freeze-drying treatment on the hydrolysate with the pH value adjusted to obtain freeze-dried powder. And then, adding the freeze-dried powder into PBS buffer solution to prepare a solution, so as to obtain a black bean peptide solution, wherein the content of black bean protein hydrolysate in the black bean peptide solution is 0.15mg/mL, the clearance rate of OH is 32.40%, the ROS clearance rate of cells reaches the maximum, and meanwhile, the growth state of cells under the action of the black bean protein hydrolysate of 0.15mg/mL is good, the growth form is fusiform, the adherence is good, the contour is clear, and few cells die.
Example 3
Adding 7 parts of black bean peptide and 5 parts of enzyme into 100 parts of water according to the mass ratio, and carrying out enzymolysis on the black bean peptide at 50 ℃ for 80 minutes to obtain a black bean protein hydrolysate containing the black bean peptide and the amino acid; secondly, regulating the pH value of the black bean protein hydrolysate to 8; and (5) performing freeze-drying treatment on the hydrolysate with the pH value adjusted to obtain freeze-dried powder. And then, adding the freeze-dried powder into PBS buffer solution to prepare a solution, so as to obtain a black bean peptide solution, wherein the content of black bean protein hydrolysate in the black bean peptide solution is 0.2mg/mL, the clearance rate of OH is 30.15%, and the clearance capacity of cells is slightly reduced.
In examples 1-3, the black bean peptide solution was lyophilized powder prepared at-50deg.C using a lyophilizer; the pH value of the black bean peptide solution is regulated by acid and alkali, the used solution is NaOH and HCL, and the concentration of the NaOH and the HCL is 0.1mol/L.
Comparative example 1
Adding 4 parts of black bean peptide and 2 parts of enzyme into 100 parts of water according to the mass ratio, and carrying out enzymolysis on the black bean peptide at 50 ℃ for 70min to obtain a black bean protein hydrolysate containing the black bean peptide and the amino acid; secondly, regulating the pH value of the black bean protein hydrolysate to 8; and (5) performing freeze-drying treatment on the hydrolysate with the pH value adjusted to obtain freeze-dried powder. Then, the freeze-dried powder is added into PBS buffer solution to prepare a solution, so as to obtain black bean peptide solution, wherein the content of black bean protein hydrolysate in the prepared black bean peptide solution is 0.025mg/mL, the clearance rate of OH is 18.29%, and the clearance capacity of cells is greatly reduced.
Comparative example 2
Adding 8 parts of black bean peptide and 6 parts of enzyme into 100 parts of water according to the mass ratio, and carrying out enzymolysis on the black bean peptide at 50 ℃ for 80 minutes to obtain a black bean protein hydrolysate containing the black bean peptide and the amino acid; secondly, regulating the pH value of the black bean protein hydrolysate to 8; and (5) performing freeze-drying treatment on the hydrolysate with the pH value adjusted to obtain freeze-dried powder. Then, the freeze-dried powder is added into PBS buffer solution to prepare a solution, so as to obtain black bean peptide solution, wherein the content of black bean protein hydrolysate in the prepared black bean peptide solution is 0.25mg/mL, the clearance rate of OH is 19.72%, and the clearance capacity of cells is greatly reduced.
In comparative examples 1-2, the black bean peptide solution was lyophilized powder prepared at-50 ℃ using a lyophilizer; the pH value of the black bean peptide solution is regulated by acid and alkali, the used solution is NaOH and HCL, and the concentration of the NaOH and the HCL is 0.1mol/L.
As is clear from the comparison between examples 1-3 and comparative examples 1-2, the black soybean protein hydrolysate having a concentration of 0.05-0.2mg/mL has a good OH scavenging effect, and the OH scavenging effect can be optimized at a concentration of 0.15 mg/mL.
In order to test that the concentration of the black bean protein hydrolysate is 0.05-0.2mg/mL and the black bean protein hydrolysate has the function of treating acute alcohol liver injury cell repair, the following experiment is established:
1. experimental procedure
Firstly, determining the alcohol concentration and the non-toxic effect of the black bean peptide solution on LO2 cells, and then establishing a cell experiment to determine the repair degree of the concentration of the black bean protein hydrolysate in the black bean peptide solution on the LO2 cells.
2. Preparation of experiments
Rodents are selected as modeling animals, such as rats and mice, as most alcoholic liver injury animal model studies. The big and small mice are experimental animals used conventionally, but are not suitable for experimental study of blood perfusion due to small weight and small total blood volume of the whole body. Adult New Zealand rabbits are also experimental animals which are used conventionally, and no research report on an alcoholic liver injury model of the New Zealand rabbits exists in the past. Meanwhile, the weight of the medicine is large, and the medicine can be used for blood perfusion treatment.
In summary, the application takes New Zealand rabbits as research objects to design and prepare an animal model for treating alcoholic liver injury by blood perfusion.
3. Experimental results
1. Screening for alcohol effect concentration
(1) Screening of alcohol concentration by CCK-8 method
As shown in FIG. 1, the liver cells of New Zealand rabbits were induced with alcohol concentrations of 0%, 0.1%, 0.3%, 0.6%, 1.2%, 2.4%, 5%, and 10%, respectively, for 24 hours.
And selecting the optimal alcohol concentration to establish an alcoholic liver cell injury model according to the CCK-8 experimental result. LO2 cell viability was reduced to varying degrees (P < 0.01) for the prognosis of alcohol trunk, and the results are shown in figure 1. When the alcohol concentration is 2.4%, the cell survival rate is about 54%, and when the alcohol concentration is higher than 2.4%, the cell survival rate is too low and is far lower than 50%, which is unfavorable for establishing a cell model, so that the optimal concentration of the alcohol is 2.4%.
(2) The results of microscopic observation of the degree of LO2 cell damage caused by different concentrations of alcohol after the hepatic cells were induced for 24 hours with different concentrations of alcohol are shown in fig. 2.
The cell growth state under the action of 0-0.6% alcohol can be obviously observed under a microscope, and the cell is in a fusiform shape and has complete cell membranes. After 24 hours of alcohol with the concentration of 1.2%, the cell number is slightly reduced, but the cell state is not obviously changed, and the cell state is still in a fusiform shape and has poor adherence. When the alcohol concentration is more than 2.4%, the cell membrane is damaged, a large number of cells die, the number of cells is obviously reduced, and the state of complete breakage is obviously reached.
The experimental result of CCK-8 and the observed cell state are combined to obtain the optimal alcohol-induced injury concentration of 2.4%.
2. Toxicity test of Black Soy protein hydrolysate
The CCK-8 method is used to verify whether the black bean protein hydrolysate has toxicity, and the result is shown in figure 3.
Experimental results show that when the concentration of the black bean protein hydrolysate in the solution is 0.025-0.2mg/mL, the black bean protein hydrolysate is nontoxic, and the cell survival rate (P is less than 0.01) can be obviously improved, so that the concentration can be selected for subsequent experiments.
3. Microscope observation of the degree of repair of LO2 cells by black bean protein hydrolysate
As shown in fig. 4, the cell morphology was observed, the cell boundary of the model group was blurred, the cell membrane was ruptured, the cell nucleus was severely damaged, and a large number of cells were apoptotic. After 24 hours of the effect of the black bean protein hydrolysate with the concentration of 0.05-0.1mg/mL, the survival number of the cells is gradually increased along with the increase of the concentration, but a large amount of cells still die. The black soybean protein hydrolysate with the concentration of 0.125-0.2mg/mL has good cell growth state, shuttle-shaped growth form, good adhesion, clear outline and few cell death.
In conclusion, the black bean protein hydrolysate can recover alcohol-induced liver cell injury and has a good protective effect on the growth of liver cells.
4. Effect of black bean protein hydrolysate on LO2 cell active oxygen
The results of the ROS content change after 24h treatment of cells with black bean protein hydrolysate (concentration 0.025-0.2 mg/mL) are shown in FIG. 5.
After the black bean protein hydrolysate is treated on the induced LO2 cells for 24 hours, the ROS content in each group of cells is reduced to different degrees, the scavenging capacity is increased to different degrees, when the concentration of the black bean peptide is 0.15mg/mL, the ROS scavenging rate of the cells is maximum, no obvious difference is caused between the black bean peptide concentration and a control group, and when the concentration is higher than 0.15mg/mL, the scavenging capacity of the cells is slightly reduced, which is possibly related to the osmotic pressure of the cells, the extracellular concentration is too high, the plasma wall separation can be caused, the content of active oxygen in the cells is increased, and the growth of the cells is greatly influenced.
5. Effect of Black Soy protein hydrolysate on LO2 intracellular AST, ALT, GSH and SOD content
After the black bean protein hydrolysate is adopted to act for 24 hours, the 2.4% alcohol is continuously induced for 24 hours, and the contents of LO2 cells AST, ALT, GSH and SOD are measured.
ALT and AST are indicators of the extent of hepatic cell damage. Alcohol-induced LO2 cells were treated with black bean protein hydrolysate (concentration 0.025-0.2 mg/mL), as shown in fig. 6, 7, with significantly elevated ALT and AST levels (P < 0.01) compared to control in model group; ALT and AST levels of cells after 24h induction were not significantly different (P > 0.05) from the control group using the black bean protein hydrolysate dose group at a concentration of 0.15 mg/mL. Compared to the model group, LO2 intracellular ALT and AST levels induced by alcohol were significantly reduced by black bean protein hydrolysate treatment (P < 0.01).
In conclusion, the black bean protein hydrolysate can improve oxidation resistance and relieve oxidative stress, so that alcohol-induced hepatocyte injury is inhibited.
Superoxide dismutase (SOD) can reflect the deoxidized free radical capacity of the organism and plays an important role in organism balance; glutathione (GSH) can prevent the human body from being damaged by oxidation, and is closely related to the oxidation resistance of the human body. As shown in fig. 8, 9, the GSH and SOD content of the model group was extremely significantly reduced (P < 0.01) compared to the control group; GSH and SOD levels were significantly elevated in the black bean protein hydrolysate (concentration 0.05-0.2 mg/mL) treated group compared to the model group.
The experimental result proves that the black bean protein hydrolysate (the concentration is 0.05-0.2 mg/mL) has the function of improving the content of the antioxidant enzyme, thereby protecting the ethanol-induced liver cell injury.
While the application has been described in connection with specific embodiments, it will be apparent to those skilled in the art that the description is intended to be illustrative and not limiting in scope. Various modifications and alterations of this application will occur to those skilled in the art in light of the spirit and principles of this application, and such modifications and alterations are also within the scope of this application.
Claims (7)
1. The preparation method of the protein hydrolysate for treating acute alcohol liver injury cell repair is characterized by comprising the following steps:
a. preparing a black bean protein hydrolysate by enzymolysis of black bean protein peptide, wherein a peptide segment with the molecular weight of 2014-355 Da is taken as a main component, and is 45.53-49.12%, a peptide segment with the molecular weight of less than 355Da is taken as 36.85-39.98%, the OH clearance is 28.15-32.40%, and the enzymolysis time is 70-90min, so that the black bean protein hydrolysate containing black bean peptide and amino acid is obtained;
b. regulating pH of black bean protein hydrolysate to 7-9;
c. lyophilizing the hydrolysate with pH value regulated to obtain lyophilized powder;
d. adding the freeze-dried powder into a buffer solution to prepare a solution, and obtaining the black bean peptide solution, wherein the content of black bean protein hydrolysate in the solution is 0.05-0.2mg/mL.
2. The method for preparing the protein hydrolysate for treating acute alcohol liver injury cell repair according to claim 1, wherein in the step a, 5-7 parts of black bean peptide and 3-5 parts of enzyme are added into 70-100 parts of water according to a mass ratio to obtain the black bean protein hydrolysate before enzymolysis.
3. The method for preparing the protein hydrolysate for treating acute alcohol liver injury cell repair according to claim 1 or 2, wherein the enzymolysis temperature is 50-60 ℃, and the adopted enzyme is black bean protease.
4. The method for preparing a protein hydrolysate for treating acute alcohol liver injury cell repair according to claim 1 wherein in the step c, a freeze dryer is used as a freeze drying treatment device.
5. The method of claim 4, wherein the black bean peptide solution is lyophilized to a temperature of-40 to-70 ℃.
6. The method for preparing the protein hydrolysate for treating acute alcohol liver injury cell repair according to claim 1, wherein the pH value of the black bean peptide solution is regulated by acid and alkali, the used solution is NaOH and HCL, and the concentration of the NaOH and the HCL is 0.1mol/L.
7. The method of claim 1, wherein the buffer is PBS buffer.
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