CN114668783A - Application of lactobacillus plantarum strain WKA86 in preparing liver protection/antioxidation/prevention and treatment of alcoholic liver injury medicines - Google Patents
Application of lactobacillus plantarum strain WKA86 in preparing liver protection/antioxidation/prevention and treatment of alcoholic liver injury medicines Download PDFInfo
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- CN114668783A CN114668783A CN202210595501.3A CN202210595501A CN114668783A CN 114668783 A CN114668783 A CN 114668783A CN 202210595501 A CN202210595501 A CN 202210595501A CN 114668783 A CN114668783 A CN 114668783A
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- wka86
- lactobacillus plantarum
- liver
- plantarum
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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Abstract
The invention discloses application of lactobacillus plantarum WKA86 in preparing a liver protection/antioxidation/alcoholic liver injury prevention and treatment drug, and belongs to the technical field of microorganisms. The preservation number of the strain WKA86 is CCTCC NO: m2022193. The invention also provides application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury. The strain WKA86 has strong acetaldehyde dehydrogenase production capacity, gastrointestinal tract tolerance and intestinal epithelial cell adhesion capacity, can reduce the inflammatory reaction of Kupper cells induced by ethanol, reduce the content of inflammatory factors in the liver of a drinking mouse, obviously improve liver injury caused by the ethanol, has the liver protection function, and can be widely applied to food or medicines.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to application of a lactobacillus plantarum strain WKA86 in preparing a liver-protecting/anti-oxidation/alcoholic liver injury preventing and treating drug.
Background
Alcoholic liver injury (ALD) is a series of liver injury lesions mainly caused by long-term heavy drinking, and the scope of ALD includes fatty liver, liver inflammation and necrosis, further progressing into liver fibrosis and hepatocyte necrosis, and liver failure. Continuous high levels of alcohol intake can lead to alcoholic liver disease, with ALD being one of the most common diseases that severely compromise public health worldwide and showing an increasing trend year by year. In China, the consumption of alcohol is increasing day by day, and liver injury diseases caused by alcohol are raised to be the second largest liver diseases after viral hepatitis.
The probiotics can be fermented in the intestinal tract to generate lactic acid, hydrogen peroxide, antibiotics and the like, and has obvious effect on preventing and treating gastrointestinal diseases; the low pH environment of the vagina is maintained, and the invasion of exogenous pathogens is prevented; has the beneficial effects of preventing cancer, delaying senility and the like. The regulation of intestinal flora, the protection of intestinal barrier, and the immune function of probiotics have been widely reported. Recent researches show that some probiotics can improve liver injury by improving intestinal permeability, regulating intestinal flora and the like, and have the effect of protecting the liver. While probiotics are often subjected to various environmental stresses in product production and the gastrointestinal tract, ethanol stress is one of the important challenges for their survival in patients with alcoholic liver injury. Ethanol permeating into cell membrane can change membrane structure and destroy membrane integrity, thereby inhibiting membrane function, possibly causing reduction of thallus specificity adhesion capability, and influencing the probiotic function.
Therefore, there is a need in the art to develop more new strains that are ethanol tolerant.
Disclosure of Invention
The invention does not solve the technical problems, and finds that the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The strain WKA86 has excellent ethanol resistance and high ethanol tolerance, and can be used for brewing and producing fermented fruit and vegetable juice, fermented milk, health wine and vinegar. The strain can grow in the environment of high-concentration ethanol, can obviously improve alcoholic liver injury, and has the function of protecting the liver.
The technical scheme of the invention is as follows:
the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a liver protection and/or antioxidation and/or alcoholic liver injury prevention and treatment medicine.
The liver protection means: reducing the content of TNF-alpha in Kupffer cells.
The antioxidant is as follows: producing an antioxidant enzyme;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
The prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
preferably, the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
preferably, the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
The medicine comprises a medicinal effective component; the preservation number is CCTCC NO: one of M2022193Strain of Lactobacillus plantarum: (Lactiplantibacillus plantarum) The strain WKA86 is used as a drug effect component;
preferably, the drug is selected from: one or two or more of liver protecting medicine, antioxidant medicine, and medicine for preventing and treating alcoholic liver injury
Preferably, the medicament further comprises: a pharmaceutical excipient;
preferably, the dosage form of the drug is selected from: one or more of powder, tablet, liquid and capsule.
The preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury.
The food product comprises an active ingredient; the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (R) ((R))Lactiplantibacillus plantarum) Strain WKA86 as active ingredient;
preferably, the food product further comprises: edible auxiliary materials;
preferably, the food product is selected from: one or more than two of health food, fermented dairy product, fermented bean product, fermented fruit and vegetable product, fermented meat product, fermented beverage, probiotic starter, probiotic solid beverage, fermented wine product and fermented vinegar product.
The liver protection means: reducing the content of TNF-alpha in Kupffer cells;
preferably, the antioxidant means: producing an antioxidant enzyme;
preferably, the prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
The antioxidant is as follows: producing an antioxidant enzyme;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
The prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
Under the condition that patent laws of some countries or regions allow, the invention also requests to protect the preservation number of CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The strain WKA86 can be used for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury.
The invention aims to provide an ethanol-resistant lactobacillus plantarum, which is preserved in the China general microbiological culture Collection center with the preservation number: CCTCC NO: m2022193
The invention provides a high ethanol tolerance microbial inoculum, and the lactobacillus plantarum can survive well in 17% ethanol concentration and has higher ethanol tolerance.
The invention provides lactobacillus plantarum for producing acetaldehyde dehydrogenase, and the enzyme activity is 58.34U/ml.
The invention provides lactobacillus plantarum with antioxidant capacity, wherein the contents of superoxide dismutase (SOD) and Glutathione (GSH) are respectively as follows: 122.76U/ml and 145.66U/ml.
The invention provides a microbial inoculum for remarkably reducing the content of ethanol-induced inflammatory factors, which is characterized in that lactobacillus plantarum remarkably inhibits ethanol-induced Kupper cells from generating TNF-alpha.
The invention provides lactobacillus plantarum for improving alcoholic liver injury and application thereof, which can remarkably enhance the oxidation resistance of mice drinking wine, and remarkably reduce the ALT and AST contents in serum and the triglyceride content in liver.
The lactobacillus plantarum can obviously improve the levels of antioxidant factors SOD and GSH-Px of mice drinking alcohol.
The lactobacillus plantarum can obviously reduce the contents of IL-6, IL-17, TNF-alpha and IL-1 beta in the liver of a drinking mouse.
The invention provides lactobacillus plantarum with high ethanol resistance and application thereof in improving alcoholic liver injury. The strain has strong acetaldehyde dehydrogenase producing capacity, gastrointestinal tract tolerance and intestinal epithelial cell adhesion capacity, can reduce the inflammatory reaction of Kupper cells induced by ethanol, reduce the content of inflammatory factors in the liver of a drinking mouse, obviously improve liver injury caused by the ethanol, has the liver protecting function, and can be widely applied to health-care food, health-care products or medicines.
Lactobacillus plantarum of the present invention: (Lactiplantibacillus plantarum) The accession information for strain WKA86 is as follows:
the preservation number is: CCTCC NO: m2022193;
and (3) classification and naming: lactobacillus plantarum;
latin name:Lactiplantibacillus plantarum；
the preservation unit: china center for type culture Collection;
the address of the depository: university of china, wuhan;
the preservation date is as follows: 3, month and 4 days 2022.
Drawings
FIG. 1 is a diagram showing the tolerance of various Lactobacillus strains to alcohol in Experimental example 1 of the present invention.
FIG. 2 is a diagram showing tolerance of Lactobacillus plantarum in Experimental example 1 of the present invention to alcohol of various concentrations, wherein A is the growth OD of each strain with 12% alcohol concentration, B is the growth OD of each strain with 17% alcohol concentration, C is the growth OD of each strain with 19% alcohol concentration, and D is the growth OD of each strain with 21% alcohol concentration.
FIG. 3 is a graph showing the growth of A4 strain in Experimental example 1 in MRS medium containing different concentrations of alcohol; the alcohol concentrations are 12%, 17%, 19% and 21% from left to right respectively; the hatched area in the photograph of the medium indicates that there is a strain growing, and the more hatched the more densely grown strain is, the higher the OD value is.
FIG. 4 is a graph showing the effect of Lactobacillus plantarum on bile salt tolerance in Experimental example 5 of the present invention.
Detailed Description
The contents and effects of the present invention will be described in further detail with reference to specific examples and experimental examples, but the scope of the present invention is not limited thereto.
Sources of biological material
Experimental example 7 Kupffer cells used were extracted from mouse livers.
Experimental examples 7 and 8 the mice used were purchased from the animal center of university of agriculture in huazhong.
The preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a medicament for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury.
In some embodiments, the liver protection refers to: reducing the content of TNF-alpha in Kupffer cells.
In specific embodiments, the antioxidant means: producing an antioxidant enzyme;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
In specific embodiments, the preventing or treating alcoholic liver injury is selected from the group consisting of: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
preferably, the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
preferably, the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
In a more specific embodiment, the medicament includes a pharmaceutically effective ingredient; the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The strain WKA86 is used as the drug effect component;
preferably, the drug is selected from: one or two or more of liver protecting medicine, antioxidant medicine, and medicine for preventing and treating alcoholic liver injury
Preferably, the medicament further comprises: a pharmaceutical excipient;
in more specific embodiments, the pharmaceutical excipient is selected from the group consisting of: solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retardants, and the like.
According to the content of the present invention, due to different requirements in practical production and application, in combination with conventional technical means in the field of pharmaceutical preparation (e.g., encyclopedia of pharmaceutical technology, pharmaceutical preparation technology, etc.), a person skilled in the art can select and mix the above pharmaceutical excipients, and add the CCTCC NO: m2022193 Lactobacillus plantarum (Lactiplantibacillus plantarum) The strain WKA86 can be made into different dosage forms, such as powder, tablet, injection, oral liquid, etc.
Preferably, the dosage form of the drug is selected from: one or more of powder, tablet, liquid and capsule.
Food use of group 2 strain WKA86
The embodiment of the group provides a preservation number of CCTCC NO: m2022193A strain of Lactobacillus plantarumLactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury.
In some embodiments, the food product comprises an active ingredient; the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (R) ((R))Lactiplantibacillus plantarum) The strain WKA86 is used as an active ingredient;
preferably, the food product further comprises: edible auxiliary materials;
in other embodiments, the dietary supplement is selected from the group consisting of: bleaching agents, preservatives, antioxidants, coloring agents, sweeteners, acidulants, flavoring agents, color fixatives, and the like.
According to the content of the present invention, due to different requirements in practical production applications, in combination with conventional technical means in the field of food production and processing technology (e.g. general treatise on food production, encyclopedia of food and food production, food processing technology, etc.), one skilled in the art can select and mix the above edible auxiliary materials, and add CCTCC NO: m2022193 Lactobacillus plantarum (Lactiplantibacillus plantarum) The strain WKA86 can be made into different dosage forms, such as powder, tablet, injection, oral liquid, etc.
Preferably, the food product is selected from: one or more than two of health food, fermented dairy product, fermented bean product, fermented fruit and vegetable product, fermented meat product, fermented beverage, probiotic starter, probiotic solid beverage, fermented wine product and fermented vinegar product.
Preferably, the food product is selected from: one or more than two of health food, fermented dairy product, fermented bean product, fermented fruit and vegetable product, fermented meat product, fermented beverage, probiotic starter, probiotic solid beverage, fermented wine product and fermented vinegar product.
In specific embodiments, the liver protection refers to: reducing the content of TNF-alpha in Kupffer cells;
preferably, the antioxidant means: producing an antioxidant enzyme;
preferably, the prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
In other embodiments, the antioxidant means: producing an antioxidant enzyme;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
In specific embodiments, the preventing or treating alcoholic liver injury is selected from the group consisting of: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
The embodiment of the group provides a preservation number of CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The strain WKA86 can be used for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury.
In specific embodiments, the liver protection means: reducing the content of TNF-alpha in Kupffer cells;
preferably, the antioxidant means: producing an antioxidant enzyme;
preferably, the prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the content of antioxidase in the drinking mouse;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
In other embodiments, the antioxidant means: producing an antioxidant enzyme;
preferably, the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
In specific embodiments, the preventing or treating alcoholic liver injury is selected from the group consisting of: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
preferably, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
Experimental example 1 screening and identification of ethanol-resistant Lactobacillus plantarum
(1) Collecting a stool sample of a healthy university student, taking 0.5g to 4.5mL of sterile physiological saline, fully oscillating and dispersing the sample, taking 100 mu L of the sample for gradient dilution, selecting a proper gradient to be uniformly coated on an MRS agar plate, placing the MRS agar plate on an anaerobic condition, culturing for 36-48 h at 37 ℃, selecting a single bacterial colony, carrying out gram staining, observing the morphological characteristics of the bacterial colony by microscopic examination, and primarily screening the bacterial strain suspected of lactobacillus. The strain is subjected to repeated streak purification culture on an MRS solid culture medium to obtain a lactobacillus purified strain, and the purified strain is preserved in a glycerin pipe at the temperature of minus 80 ℃.
MRS medium formula (g/L): 10 parts of peptone, 5 parts of beef extract powder, 5 parts of yeast powder, 20 parts of glucose, 5 parts of anhydrous sodium acetate, 2 parts of diammonium hydrogen citrate, 801 parts of tween and K parts of2 HPO 4 2、MgSO4 0.2、MnSO40.05, pH6.5, autoclaving at 121 deg.C for 20min, and adding 1.5% agar powder into solid MRS culture medium.
(2) Ethanol tolerance screening experiment
Inoculating lactobacillus plantarum into an MRS culture medium containing liquid L-cysteine hydrochloride for activation, culturing to a logarithmic growth phase, inoculating into MRS containing L-cysteine hydrochloride with the final ethanol concentration of 7% according to the inoculation amount of 2%, inoculating 250ul of the culture medium into a 96-well plate, culturing at 37 ℃, and measuring the growth curve of the lactobacillus plantarum by using a bioscreen full-automatic growth curve measuring instrument in an experiment, wherein the experimental result is shown in figure 1.
As can be seen from FIG. 1, of the 6 L.plantarum strains, L.plantarum strains A1, A4 and A5 were better in growth, and OD600nm reached 3.79, 5.05 and 4.31, respectively. The results show that A1, A4 and A5 have better ethanol tolerance.
(3) Tolerance of lactobacillus plantarum to alcohol of different concentrations
Inoculating lactobacillus plantarum into a liquid MRS culture medium containing L-cysteine hydrochloride for activation, culturing to a logarithmic growth phase, preparing MRS containing L-cysteine hydrochloride with different ethanol final concentrations (7%, 12%, 17%, 19%, 21%), taking 250 mu L of the culture medium to be inoculated into a 96-well plate, inoculating three strains of lactobacillus plantarum A1, A4 and A5 according to the inoculation amount of 2%, culturing at 37 ℃, and determining the growth curve of the lactobacillus plantarum by using a bioscreen full-automatic growth curve determinator in an experiment, wherein the experimental result is shown in figure 2. The experiment was controlled with LGG, a well-established commercial strain with probiotic capacity.
As can be seen from FIG. 2, the growth OD of Lactobacillus plantarum A4 was highest at an alcohol concentration of 12%, reaching 3.26. The OD values of A4 and A5 were all better at 17% alcohol concentration. At an alcohol concentration of 19% and 21%, the OD growth of A4 was higher than that of the other two plants. When the alcohol concentration is 17%, the A4 grows well, and the OD value reaches 0.439; whereas at 19% and 21% alcohol concentrations, only 0.26 and 0.23, the A4 strain grew in a high ethanol concentration environment, although the growth was inhibited by the high ethanol concentration (FIG. 3).
Experimental example 2 identification experiment of 16SrDNA of ethanol-tolerant Lactobacillus plantarum
Culturing the screened target strain A4, collecting thalli, extracting genome DNA, and performing PCR by adopting a universal primer 27F: AGAGTTTGATCCTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT amplifies 16SrDNA fragment, detects PCR amplification product by agarose gel electrophoresis, and sequences the PCR product. Wherein the PCR reaction system comprises: 10 XBuffer 10 uL, 10 MdNTP2 uL, 1 uL of each of the upper and lower primers, 2 uL of DNA template, 0.5 uL of Taq enzyme, ddH2O34 μ L. Pre-denaturation at 95 ℃ for 10 min; then 35 cycles of 94 ℃ 30s, 60 ℃ 30s and 72 ℃ 1min, and after completion, 72 ℃ extension is carried out for 5 min. And (3) detecting the PCR product through gel electrophoresis, and then sending the PCR product to Wuhan Jinrui bioengineering company Limited for sequencing. Comparing the identified gene sequences in NCBI database by using BLAST tool, determining the strain to be lactobacillus plantarum according to the identification result of molecular biology, naming the strain to be WKA86, and sending the deposit information as follows:
the preservation number is: CCTCC NO: m2022193;
and (3) classification and naming: lactobacillus plantarum;
latin name:Lactiplantibacillus plantarum；
the preservation unit: china center for type culture Collection;
the address of the depository: university of china, wuhan;
the preservation date is as follows: 3, month and 4 days 2022.
Experimental example 3 measurement of acetaldehyde dehydrogenase-producing ability and antioxidant ability of Lactobacillus plantarum WKA86
Inoculating lactobacillus plantarum into an MRS culture medium containing 1% (V/V) acetaldehyde according to the inoculation amount of 2%, culturing for 12h at 37 ℃, collecting thalli, washing for 2 times by PBS, then resuspending by a BindingBuffer extracting solution with the pH of 7.4, carrying out cell disruption by an ultrasonic disruptor, centrifuging for 20min at 4 ℃ and 1000g, removing supernatant, carrying out enzyme activity determination according to the steps of an acetaldehyde dehydrogenase kit, and determining the absorbance value at 450nm by using an enzyme-labeling instrument in an experiment.
Activating and culturing lactobacillus plantarum to logarithmic growth phase, centrifugally collecting thalli, washing twice by PBS, then suspending, carrying out cell disruption by using an ultrasonic disruptor, centrifuging for 20min at 4 ℃ at 1000g, removing supernatant, processing according to the steps of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) kits, then respectively measuring absorbance values at 420nm and 450nm, and calculating the SOD and GSH production capacity of the strain.
TABLE 1 results of acetaldehyde dehydrogenase, SOD and GSH-Px production by Lactobacillus plantarum WKA86
Ethanol is oxidized into acetaldehyde in vivo, acetaldehyde can cause damage to the organism, acetaldehyde can be oxidized into nonhazardous acetic acid through acetaldehyde dehydrogenase, most people do not have enough acetaldehyde dehydrogenase to oxidize acetaldehyde when drinking, therefore, the lactobacillus plantarum has strong capacity of producing acetaldehyde dehydrogenase, and can relieve discomfort symptoms caused by acetaldehyde stimulation. Alcohol metabolism induces a surge of Reactive Oxygen Species (ROS), which triggers hepatic oxidative stress, causing oxidative damage to hepatocyte DNA, proteins, and peroxidation of lipids, thereby damaging hepatocytes. The SOD and CSH can directly remove free radicals, and the lactobacillus plantarum can generate a large amount of SOD and CSH active enzymes and effectively remove the free radicals.
Experimental example 4 gastrointestinal tolerance assay of Lactobacillus plantarum WKA86
Preparing artificial gastric juice: preparing PBS solution, adding 0.3% pepsin, adjusting pH value to 2.5 with 1mol/LHCL, fully dissolving, and filtering and sterilizing with 0.22 μm microporous membrane for use.
Preparing artificial intestinal juice: dissolving 0.1% trypsin in the prepared PBS solution, adding 0.3% fel bovis Seu Bubali powder, adjusting pH to 8.0 with 0.1mol/LNaOH, and filtering with 0.22 μm microporous membrane for sterilization.
Activating the strain to be detected for 2 generations, and adjusting the concentration of the bacterial liquid to 108CFU/mL. And (3) centrifuging 1mL of bacterial suspension, collecting thalli, respectively inoculating 1mL of prepared artificial gastric juice and artificial intestinal juice, uniformly mixing, digesting at 37 ℃, simultaneously respectively taking 0h and 3h of digestive juice to detect the number of viable bacteria, and calculating the survival rate, wherein the result is shown in table 2. Wherein the survival rate of the strain (%) = Nt/N0X 100%, wherein N0The number of viable bacteria (CFU/mL) of the strain 0h was shown, and Nt was the number of viable bacteria (CFU/mL) of the strain 3 h.
TABLE 2 Lactobacillus plantarum gastrointestinal tolerance assay
The results are shown in table 2, the survival rates of lactobacillus plantarum WKA86 and lactobacillus plantarum WKA86 are 91.85% after digestion of gastric juice for 3 hours and 93.85% after digestion of intestinal juice for 3 hours, which indicates that lactobacillus plantarum WKA86 has strong gastric acid and intestinal juice resistance.
Experimental example 5 Lactobacillus plantarum WKA86 bile salt tolerance determination
In the experiment, a bioscreen full-automatic growth curve determinator is used for determining the growth curve of the lactobacillus plantarum WKA86, MRS liquid culture medium (containing 0.2% of sodium thioglycolate and 0.1% of bovine bile salt) containing L-cysteine hydrochloride and containing bovine bile salt is inoculated into a 96-well plate by 250ul of the culture medium, the strain at the end of a culture period is inoculated according to 1% of inoculation amount, and the MRS liquid culture medium (containing 0.2% of sodium thioglycolate) without the bovine bile salt is used as a blank control. The length of the lag phase is taken as the evaluation standard of the bile salt tolerance of the strain, and the lag phase refers to the difference of time required by the absorbance increase of 0.3 unit between the strains in the experimental group and the blank group.
As shown in FIG. 4, in 0.1% of bovine bile salts, the Lactobacillus plantarum lag phase was much lower than that of LGG, compared to LGG, and had better bile salt tolerance.
Experimental example 6 measurement of cell adhesion of Lactobacillus plantarum WKA86
Inoculating Lactobacillus plantarum WKA86 in MRS liquid culture medium according to the inoculation amount of 2%, culturing at 37 ℃ for 12h, centrifugally collecting thallus, washing with PBS for 3 times, suspending the thallus in DMEM culture solution without double antibody, and adjusting the concentration of the suspension to 108CFU/mL. Adding 5ml of complete culture medium (containing 10% fetal calf serum, 1% penicillin and streptomycin solution) into a cell culture flask, incubating at 37 ℃ in a 5% CO2 constant temperature incubator, changing the culture medium 1 time per day, and adding 0.2% digestive juice (pancreas is fused at 70% -80%) after the cells are in good stateenzyme-EDTA) were subjected to digestion passage.
Adjusting the concentration of the digested HT-29 cells to 105cell/mL, 1mL per well in 5% CO in 12-well cell culture plates2Incubating in an incubator with a concentration until the cells grow to a monolayer, washing twice with sterile PBS, wherein 1 hole is digested with pancreatin, and counting the cells with a blood counting chamber; respectively adding 1mL of lactobacillus plantarum WKA86 and control bacterium LGG bacterial suspension into 2 holes, and adding 5% CO2After incubation for 2h at 37 ℃ in an incubator, washing cells for 5 times by using sterile PBS, removing non-adhered bacterial suspension, adding 0.2mL of pancreatin-EDTA buffer solution into each hole to digest the cells for 5min, adding 0.8mL of PBS after digestion, blowing and beating uniformly, taking bacterial liquid to dilute and count viable bacteria, and performing 3 parallel experiments. Lactobacillus rhamnosus LGG is a commercial strain recognized in the industry to have better adhesiveness, and the lactobacillus rhamnosus LGG is used as a control strain for measurement.
TABLE 3 measurement of the adhesion of Lactobacillus plantarum WKA86 to HT-29
As can be seen from Table 3, LGG is used as a control strain, and the Lactobacillus plantarum WKA86 has strong adhesion capability and obvious advantages.
Experimental example 7 Effect of Lactobacillus plantarum WKA86 on ethanol-induced Kupffer cells
Kupffer cell culture: taking out liver cells from mouse liver by collagenase perfusion, taking liver cell suspension (suspended in RPMI1640 culture medium containing 2mM glutamine and 0.1mg/mL gentamicin without serum) and centrifuging for 2min at 50g, centrifuging for 10min at 300g of obtained supernatant, suspending the obtained precipitate in the RPMI1640 culture medium, incubating for 2h, and obtaining cells attached to the wall, namely Kupffer cells.
Experiments Kupffer cells (1X 10) were treated with 0.4mM ethanol solution6cells/well) were processed, adding 1X 105CFU/mL Lactobacillus plantarum WKA86 was used for mediation, and after 90min of culture, the tissue or culture cell supernatant was transferred to 96-well ELISA plates. Determination of the cytokine TNF-Alpha content, blank group was Kupffer cells without treatment, ethanol treated group was ethanol induced Kupffer cells.
TABLE 4 Effect of Lactobacillus plantarum WKA86 on TNF- α in Kupffer cells
Ethanol treatment significantly induced the expression of TNF-alpha. However, after the lactobacillus plantarum WKA86 stem prognosis, the TNF- α content in Kupffer cells was significantly reduced. This indicates that lactobacillus plantarum WKA86 can significantly inhibit ethanol-induced TNF- α expression.
Experimental example 8 Effect of Lactobacillus plantarum WKA86 on alcohol-induced liver injury mice
(1) Establishment of animal model
Clean male ICR mice (6 weeks old, 22g + -2 g body weight) were acclimatized for one week and then divided into 4 groups of 15 mice each for the experiment. Each mouse was marked and gavage was performed at 9 points daily in strict fixed order. The alcohol treatment group was first gavaged with 200 μ L of LPBS at 1h intervals, and then 1 gavage was performed with 200 μ L of 60% ethanol each time. The blank group was gavaged with 200. mu.L of PBS buffer twice daily at 1h intervals. Lactobacillus plantarum WKA86 intervenes in the alcohol treatment low dose group: first gavage 1X 107CFU/d, interval 1h, then gavage 200 μ L with 60% ethanol, medium dose group: 2X 108CFU/d, high dose group: 1X 1010CFU/d, the remaining experimental steps were consistent with the low dose group. Feeding food and water freely for 4 weeks, fasting for 12h on the last day of experiment, weighing, collecting eyeball blood, dislocating and killing mouse, collecting liver, weighing, and storing in-80 deg.C ultra-low temperature refrigerator for use.
(2) Measurement of physiological and biochemical indexes
And (3) centrifuging the blood sample at 3500r/min and 4 ℃ for 10min, and measuring the activities of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum of the mouse and the contents of SOD and GSH-Px oxidative stress indexes in liver homogenate according to the steps of a kit specification. 500 μ L of liver homogenate was thoroughly mixed in 4mL of chloroform-methanol (volume ratio 2: 1) lipid extraction buffer, and centrifuged at 8000r/min overnight at 4 ℃ for 10 min. The lower lipid-containing organic phase was transferred to an EP tube and dried, and the TG content was determined using a Triglyceride (TG) assay kit.
TABLE 5 influence of Lactobacillus plantarum WKA86 on ALT, AST, SOD, GSH-Px, TG in drinkable mice
ALT and AST are mainly present in various tissues including liver, and when liver is damaged, transaminase enters blood, resulting in elevation of ALT and AST levels in serum. As can be seen from the above table, the Lactobacillus plantarum WKA86 provided by the invention can obviously reduce the AST and ALT levels in the serum of mice drunk under low dose, medium dose and high dose, and is dose-dependent. When the lactobacillus plantarum WKA86 is used in a gastric lavage high dose, ALT and AST in serum basically recover normal levels, which shows that the lactobacillus plantarum WKA86 has the functions of remarkably alleviating hangover, protecting liver cells and reducing the damage of alcohol to the liver cells.
Ethanol and metabolites thereof can generate a large amount of free radicals to damage organs such as liver, and the like, while in the invention, the drinking mouse intervened by the lactobacillus plantarum WKA86 generates SOD and GSH-Px enzyme active substances which can effectively remove the free radicals, thereby achieving the effects of relieving alcoholism and protecting liver. Alcohol damage to the liver is also reflected in the accumulation of fat. Alcohol exposure increased TG levels in the mouse liver, resulting in fat accumulation in the mouse. After lactobacillus plantarum WKA86 is used for prognosis, TG level in the liver of a drinking mouse is obviously reduced, and the function of reducing fat and protecting the liver is achieved.
(3) Determination of inflammatory factor index in mouse liver
The contents of IL-6, IL-17, IL-1 beta and TNF-alpha are determined according to the specification of the enzyme linked immunosorbent assay kit. 0.2g of liver tissue was taken, homogenized by adding PBS buffer, and then centrifuged at 12000rpm for 10min at 4 ℃ to separate the liver tissue supernatant.
TABLE 6 Effect of Lactobacillus plantarum on inflammatory factors in mouse liver cells
As can be seen from Table 6 above, excessive drinking caused significant increases in the levels of inflammatory factors including IL-6, IL-17, IL-1 β, TNF- α in the mouse liver, indicating that chronic alcohol exposure caused an inflammatory response in the mouse liver. And through the probiotic dry prognosis, the high-level IL-6, IL-17, IL-1 beta and TNF-alpha in the liver of the drinking mouse are all obviously reduced (p is less than 0.05), and the level of inflammatory factors in the liver is basically recovered to the normal level after the high-dose lactobacillus plantarum WKA86 is perfused.
SEQUENCE LISTING
<110> Mikang Probiotics (Suzhou) GmbH
Application of lactobacillus plantarum strain WKA86 in preparation of liver protection/antioxidation/prevention and treatment of alcoholic liver injury medicines
<130> P220496/WKY
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal primer 27F
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Universal primer 1492R
<400> 2
ggttaccttg ttacgactt 19
Claims (10)
1. The preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a medicament for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury.
2. The preservation number of claim 1 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a medicament for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury is characterized in that the liver protection refers to: reducing the content of TNF-alpha in Kupffer cells.
3. The deposit number of claim 1 or 2 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a liver-protecting and/or anti-oxidation and/or alcoholic liver injury preventing and treating medicine is characterized in that the anti-oxidation means: producing an antioxidant enzyme;
and/or the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
4. The preservation number of claim 3 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a medicament for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury is characterized in that the medicament for preventing and treating alcoholic liver injury is selected from the following medicaments: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
and/or, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
and/or, the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
and/or the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
5. The culture medium according to any one of claims 1, 2 and 4, wherein the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (R) ((R))Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing a medicament for protecting liver and/or resisting oxidation and/or preventing and treating alcoholic liver injury is characterized in that the medicament comprises medicinal effective components; the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The strain WKA86 is used as a drug effect component;
and/or, the drug is selected from: one or two or more of liver protecting medicine, antioxidant medicine, and medicine for preventing and treating alcoholic liver injury
And/or, the medicament further comprises: a pharmaceutical excipient;
and/or, the dosage form of the medicament is selected from: one or more of powder, tablet, liquid and capsule.
6. The preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury.
7. The preservation number of claim 6 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (R) ((R))Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury is characterized in that the food comprises active ingredients; the preservation number is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) Strain WKA86 as active ingredient;
and/or, the food product further comprises: edible auxiliary materials;
and/or, the food product is selected from: one or more than two of health food, fermented dairy product, fermented bean product, fermented fruit and vegetable product, fermented meat product, fermented beverage, probiotic starter, probiotic solid beverage, fermented wine product and fermented vinegar product.
8. The preservation number of claim 6 or 7 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury is characterized in that the liver protection refers to: reducing the content of TNF-alpha in Kupffer cells;
and/or, the antioxidant means: producing an antioxidant enzyme;
and/or, the prevention and treatment of alcoholic liver injury is selected from: inhibiting the expression level of TNF-alpha in Kupffer cells induced by ethanol, reducing the transaminase content and TG content in a drinking mouse, reducing the level of inflammatory factors in the drinking mouse, and improving the antioxidant enzyme content in the drinking mouse;
and/or the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
9. The preservation number of claim 6 or 7 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (M2022193)Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury is characterized in that the oxidation resistance refers to: generating an antioxidant enzyme;
and/or the antioxidant enzyme is selected from one or two or three of superoxide dismutase, glutathione peroxidase and acetaldehyde dehydrogenase.
10. The preservation number of claim 9 is CCTCC NO: M2022193A strain of Lactobacillus plantarum (R) ((R))Lactiplantibacillus plantarum) The application of the strain WKA86 in preparing health-care food for protecting liver and/or resisting oxidation and/or preventing alcoholic liver injury is characterized in that the strain WKA86 is used for preventing and treating alcoholic liver injury and is selected from the following components: inhibiting expression level of TNF-alpha in Kupffer cell induced by ethanol, reducing transaminase content and TG content in mice drinking alcohol, reducing inflammatory factor level in mice drinking alcohol, and increasing in mice drinking alcoholThe antioxidant enzyme content of (a);
and/or, the transaminase is selected from: alanine aminotransferase and/or aspartate aminotransferase;
the inflammatory factor is selected from: one or more of IL-6, IL-17, IL-1 beta and TNF-alpha;
the antioxidant enzyme is selected from: superoxide dismutase and/or glutathione peroxidase.
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| CN117448178A (en) * | 2023-10-31 | 2024-01-26 | 湖北嫦娥生物股份有限公司 | Aspergillus purpureus CEWL18 and application thereof in preparation of liver protection products |
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