CN114921361B - Lactobacillus plantarum ZY08 with effect of improving alcoholic liver injury and application thereof - Google Patents
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- CN114921361B CN114921361B CN202210410959.7A CN202210410959A CN114921361B CN 114921361 B CN114921361 B CN 114921361B CN 202210410959 A CN202210410959 A CN 202210410959A CN 114921361 B CN114921361 B CN 114921361B
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Classifications
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/385—Concentrates of non-alcoholic beverages
- A23L2/39—Dry compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides lactobacillus plantarum ZY08 with the effect of improving alcoholic liver injury and application thereof, wherein the lactobacillus plantarum (Lactobacillus plantarum) ZY08 has the preservation number of: CGMCC No.14847. The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention can regulate and control the abundance of various intestinal probiotics (blautia, lachnoclothridium and the like) in intestinal tracts, promote the synthesis of intestinal short-chain fatty acids so as to restore the intestinal barrier function of alcohol damage, and in addition, the ZY08 can inhibit the synthesis pathway of liver fatty acids to reduce the deposition of liver triglyceride and improve the condition of liver damage through an antioxidant pathway. Compared with the existing strain, the strain is superior to the existing strain in the aspects of reducing blood fat, reducing cholesterol, resisting oxidation and the like.
Description
Technical Field
The invention relates to the technical field of food microorganisms, in particular to lactobacillus plantarum (Lactobacillus plantarum) ZY08 with the effect of improving alcoholic liver injury and application thereof.
Background
At present, the research reports that alcohol is a common causative factor of chronic liver diseases in most industrialized countries, and liver is a main organ for metabolizing alcohol, so that the liver has long been regarded as a main target organ for alcohol damage. And thus alcoholic liver disease (alcoholic liver disease, ALD) has gradually become a worldwide widespread chronic liver disease. At the beginning of the ALD disease process, lipids enter hepatocytes and gradually accumulate under the induction of ethanol to form fatty liver, and if alcohol is still continuously taken, liver fibrosis and even cirrhosis may further progress. Currently, ALD has become a public health concern worldwide, however, to date there is no clinical medication for safe and effective treatment of ALD. How to effectively control ALD is an urgent problem to be solved in clinical medicine.
Lactobacillus plantarum belongs to one of lactobacillus beverages, and the theoretical lactobacillus beverage refers to bacteria which can cause lactobacillus in the whole process of fermentation of glucose or whey protein. Lactobacillus plantarum has multiple effects, wherein researches show that lactobacillus plantarum suppresses germs and adjusts the diversity of gastrointestinal microorganisms according to market competition with germs for nutrient elements, and an ecological natural barrier is generated. The metabolin lactobacillus and the germ have the effects of inhibiting the reproduction of other harmful germs in intestines and stomach, keep and ensure the best advantage composition of beneficial germs and the stability of the composition, block the colonization and invasion of pathogenic germs, inhibit the growth and development of pathogenic germs and harmful microorganism strains and the adhesion of endotoxin. Lactobacillus plantarum has the excellent effects of reducing blood cell cholesterol level and reducing the prevalence rate of cardiovascular diseases. Experiments prove that the lactobacillus has the working capacity of reducing substances and blood cell cholesterol.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum ZY08 for improving alcoholic liver injury, wherein the lactobacillus plantarum (Lactobacillus plantarum) ZY08 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at 11-1 of 2017, and the preservation unit address is: beijing, china, accession number: CGMCC No.14847. The full sequence of 16s rDNA of lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention is shown as SEQ ID No. 1.
Another object of the invention is to provide the use of said lactobacillus plantarum (Lactobacillus plantarum) ZY08 in the preparation of functional food or health care products. The functional food comprises fungus powder, yoghourt, milk powder and fermented beverage, and the health care product comprises products such as a microbial inoculum, oral liquid or solid beverage prepared from lactobacillus plantarum (Lactobacillus plantarum) ZY 08.
The functional food and the health care product are used for improving alcoholic liver injury by regulating and controlling the abundance of various intestinal probiotics (blautia, lachnoclothridium and the like) in intestinal tracts, promoting the synthesis of intestinal short-chain fatty acids and further recovering the intestinal barrier function of alcohol injury. In addition, ZY08 can inhibit the synthesis of fatty acid in liver to reduce triglyceride deposition in liver, and improve alcoholic liver injury via antioxidant way to achieve liver nourishing and protecting effects.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has a strong capacity of protecting liver injury.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capacity of improving in-vivo antioxidation.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capacity of reducing liver triglyceride.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capacity of reducing the synthesis of liver fatty acid.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capability of recovering the intestinal barrier function.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capability of promoting the synthesis of intestinal short-chain fatty acid.
The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention has the capability of improving intestinal flora. The lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention can regulate and control the abundance of various intestinal probiotics (blautia, lachnoclothridium and the like) in intestinal tracts, promote the synthesis of intestinal short-chain fatty acids, further recover the intestinal barrier function of alcoholic liver injury, inhibit the synthesis pathway of liver fatty acids, reduce the deposition of liver triglyceride, and improve the condition of liver injury through an antioxidant pathway. Compared with the existing strain, the strain is superior to the existing strain in the aspects of reducing blood fat, reducing cholesterol, resisting oxidation and the like.
Drawings
FIG. 1 shows the effect of Lactobacillus plantarum ZY08 of the present invention on glutamic pyruvic transaminase and glutamic oxaloacetic transaminase (ALT and AST) in mouse plasma.
FIG. 2 shows the effect of Lactobacillus plantarum ZY08 according to the invention on Free Fatty Acids (FFA) in mouse plasma.
FIG. 3 is a H & E staining chart of the liver section of mice by Lactobacillus plantarum ZY08 of the present invention.
FIG. 4 is a graph showing the staining of the mouse liver slice with oil red O by Lactobacillus plantarum ZY08 of the present invention.
FIG. 5 shows the effect of Lactobacillus plantarum ZY08 of the present invention on triglycerides in the liver of mice.
FIG. 6 shows the effect of Lactobacillus plantarum ZY08 of the present invention on Malondialdehyde (MDA), total superoxide dismutase (T-SOD) and Catalase (CAT) in mouse liver.
FIG. 7 shows the effect of Lactobacillus plantarum ZY08 of the present invention on fatty acid synthesis genes (Srebp 1c and FAS) in mouse liver.
FIG. 8 shows the effect of Lactobacillus plantarum ZY08 of the present invention on the zona tight junction protein genes (ZO-1 and Claudin-1) in the mouse intestinal tract.
FIG. 9 shows the effect of Lactobacillus plantarum ZY08 according to the invention on short chain fatty acids (acetic acid, propionic acid and butyric acid) in mouse faeces.
FIG. 10 shows the effect of Lactobacillus plantarum ZY08 of the present invention on the horizontal abundance of the intestinal flora in mice.
FIG. 11 shows the effect of Lactobacillus plantarum ZY08 of the present invention on the abundance of different strains of the intestinal flora level of mice.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
Example 1 screening and identification of lactobacillus plantarum ZY 08:
1. screening of Lactobacillus plantarum ZY08
1.1 sample Source
The strain used in the present invention was isolated from faeces of infants fed with healthy breast milk in Hangzhou area.
1.2 isolation and purification of Strain
About 5g of fresh fecal sample was collected with a sterile tube and immediately sent to the laboratory for strain isolation. 1g of sample is taken and put into 9mL of MRS broth culture medium, and after vortex mixing, enrichment culture is carried out for 48h at 37 ℃; then 1mL of enrichment solution is sucked in an ultra clean bench, ten times of gradient dilution is carried out by using sterile physiological saline, three dilution gradients of 10 < -6 >, 10 < -7 > and 10 < -8 > are selected, 100 mu L of each gradient bacterial solution is taken and coated on MRS agar medium, and the culture is carried out for 48 hours at 37 ℃. After the culture is finished, selecting a plate with 50-150 single colonies from an agar culture medium, picking typical colonies, carrying out repeated streak purification on an MRS agar plate until the colony morphology on the whole plate is consistent, and picking single colonies to the MRS broth culture medium for enrichment culture. The resulting strains were all cryopreserved in MRS broth medium with 40% glycerol at-80 ℃.
Identification of Lactobacillus plantarum ZY08
2.1 colony characterization
After the lactobacillus plantarum ZY08 is cultured in an MRS agar culture medium for 48 hours, the diameter is between 0.3 and 1.5mm, and the lactobacillus plantarum ZY08 is circular in colony, neat in edge, white and moist and smooth in surface.
2.2 morphology under microscope:
lactobacillus plantarum ZY08 colony smear: gram staining was positive, sporulation was absent, straight bacillus caldarius, single, paired or short chain.
2.3 16S rDNA identification
Extracting genome DNA of a target strain by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out a PCR experiment of 16S rDNA by using bacterial universal primers 27F and 1492R, and taking a PCR product to carry out agarose gel detection and photographing after the PCR amplification is finished, wherein the length of an amplified fragment is about 1.2 kbp. The PCR product was sent to Huada gene limited for sequencing, and the results were shown as SEQ ID NO.1, and BLAST sequence alignment was performed on NCBI website, which showed that the sequence was more than 99% homologous to the identified 16S rDNA sequence of Lactobacillus plantarum. And combining the sequence comparison result and the physiological and biochemical result of the strain lactobacillus plantarum ZY08 to determine that the screened lactobacillus plantarum ZY08 is lactobacillus plantarum. The strain lactobacillus plantarum (Lactobacillus plantarum) ZY08 was deposited at the China general microbiological culture Collection center, accession number: CGMCC No.14847.
Example 2 lactobacillus plantarum ZY08 ameliorates alcoholic liver injury:
experimental animals: c57BL/6 Male mice, 32, purchased from Shanghai Laek laboratory animal center, company license number: SCXK 2017-0005, which is fed into the laboratory animal center of Zhejiang university, SPF environment.
Reagent: lieber-DeCarli alcohol liquid model feed (product code TP-4030B, nantong Telofei feed technologies Co., ltd.); lieber-DeCarli control liquid feed (product code TP-4030BC, nantong Telofei feed technologies Co., ltd.); choline, vitamins (south Tongtong Tarofil feed technology Co., ltd.). Absolute ethyl alcohol (CAS-NO: 64-17-5, lichrosolv), ALT kit (cat No. C009-2 Nanjing institute of biological engineering), AST kit (cat No. C010-2 Nanjing institute of biological engineering), free fatty acid kit (cat No. A042-2-1 Nanjing institute of biological engineering), tissue triglyceride kit (cat No. E1013 Nanjing Priley Gene technologies Co.), malondial assay kit (cat No. A003-1 Nanjing institute of biological engineering), total superoxide dismutase assay kit (cat No. A001-1 Nanjing institute of biological engineering), catalase assay kit (cat No. A001-1 Nanjing institute of biological engineering)
2. The method comprises the following steps:
2.1 laboratory animal feeding
After C57BL/6 mice were acclimatized to the SPF-grade animal laboratory for one week, the 8-week-old C57BL/6 mice were randomly divided into 4 groups of 8 animals each, which were respectively a control liquid feed group+physiological saline group (PF group), an alcohol liquid feed group+physiological saline group (AF group), an alcohol liquid feed+lactobacillus plantarum E680 group (af+lactobacillus plantarum E680 group), an alcohol liquid feed+lactobacillus plantarum ZY08 group (af+lactobacillus plantarum ZY08 group).
The PF group is fed with Lieber-DeCarli control liquid feed for 4 weeks, the volume of the PF group control liquid feed is adjusted according to the feed intake of the AF group Lieber-DeCarli alcohol liquid model, and each stomach is filled with 0.2mL of physiological saline 1 time per day; lieber-DeCarli control liquid feed was administered on days 1-3, 5.5% caloric alcoholic liquid feed was administered on days 4-5, 11% caloric alcoholic liquid feed was administered on days 6-7, 22% caloric alcoholic liquid feed was administered on the second week, 27% caloric alcoholic liquid feed was administered on the third week, 32% caloric alcoholic liquid feed was administered on the fourth week, 0.2mL physiological saline was administered 1 time per stomach in AF group from the first day, 0.2mL Lactobacillus plantarum E680 (10 9 CFU/animal, solvent for physiological saline), AF+Lactobacillus plantarum ZY08 group was irrigated 1 time per day with 0.2mL Lactobacillus plantarum ZY08 (10) 9 CFU/solvent for physiological saline).
The liquid feed eaten by the mice is changed into a new formula feed every day, the weight of the mice is recorded every week, the change is detected, after feeding is finished, the mice are anesthetized by intraperitoneal injection of 1% pentobarbital, ALT, AST and FFA are measured by blood sampling of inferior vena cava, and relevant index measurement is carried out by taking liver and intestinal part tissues of the mice.
3. Feed formula
(1) Control liquid feed formulation method for PF group mice:
table 1: liquid feed formula for mice in control group (1L)
About 600ml of water is added, and after the water is stirred and dissolved completely, the water is added to a constant volume of 1L.
(2) Preparing liquid feeds with different alcohol concentrations: taking the preparation of 1 liter of liquid as an example,
table 2: liquid feed formula for mice of experimental group (1L)
About 600ml of water is added, and after the water is stirred and dissolved completely, the water is added to a constant volume of 1L.
4. Index measurement
4.1 plasma ALT assay
Taking a mouse plasma sample for direct sampling and determination, preheating matrix liquid at 37 ℃ in advance, adding 20 mu L of matrix liquid into a determination hole and 5 mu L of sample to be detected, uniformly mixing, adding 20 mu L of matrix liquid into a control hole, and incubating for 30min at 37 ℃. 20 mu L of 2, 4-dinitrophenylhydrazine solution is added to the measuring hole and the control hole respectively, 5 mu L of a sample to be measured is added to the control hole, and the mixture is uniformly mixed and incubated for 20min at 37 ℃. 200 mu L of 0.4mol/L sodium hydroxide solution is added into each hole, the mixture is uniformly mixed, the mixture is placed at room temperature for 15min, the wavelength of 510nm, the OD value of each hole is measured by an enzyme label instrument, and a standard curve is checked to obtain a corresponding ALT/GPT activity unit.
4.2 plasma AST detection
Taking a mouse plasma sample for direct sampling and determination, preheating matrix liquid at 37 ℃ in advance, adding 20 mu L of matrix liquid into a determination hole and 5 mu L of sample to be detected, uniformly mixing, adding 20 mu L of matrix liquid into a control hole, and incubating for 30min at 37 ℃. 20 mu L of 2, 4-dinitrophenylhydrazine solution is added to the measuring hole and the control hole respectively, 5 mu L of a sample to be measured is added to the control hole, and the mixture is uniformly mixed and incubated for 20min at 37 ℃. 200 mu L of 0.4mol/L sodium hydroxide solution is added into each hole, the mixture is uniformly mixed, the mixture is placed at room temperature for 15min, the wavelength of 510nm, the OD value of each hole is measured by an enzyme label instrument, and a standard curve is checked to obtain a corresponding AST/GPT activity unit.
4.3 plasma FFA detection
4. Mu.L of double distilled water was added to the blank wells, 4. Mu.L of standard was added to the calibrated wells, and 4. Mu.L of sample was added to the sample wells. 200. Mu.L of reagent one was added to the three wells. Mixing, incubating at 37 ℃ for 5min, reading an absorbance value A1, adding 50 mu L of a second reagent into the three holes respectively, mixing, incubating at 37 ℃ for 5min, reading an absorbance value A2, calculating the value of A2-A1, and calculating by two-point calibration.
4.4 liver H & E staining
Fixing fresh animal liver tissue with 4% paraformaldehyde, dehydrating with gradient alcohol after fixing, embedding paraffin after penetrating with xylene, slicing the embedded paraffin, taking 4 μm slice, H & E dyeing, dewaxing with xylene, and gradually dehydrating with ethanol: xylene (I) for 5min; xylene (II) for 5min;100% ethanol for 2min;95% ethanol for 1min;80% ethanol for 1min;75% ethanol for 1min; washing with distilled water for 2min. Hematoxylin staining for 5min, washing with water, differentiating with ethanol hydrochloride for 30s, soaking in water for 15min, and standing with eosin solution for 2-3min. Conventional dehydration, transparency and sealing sheet: 95% ethanol (I) 30s;95% ethanol (II) for 30s;100% ethanol (I) for 30s;100% ethanol (II) for 1min; xylene for 15min; and (3) sealing the sheet with neutral resin.
4.5 liver oil Red O staining
Immersing fresh tissue of animal liver into OCT (optical coherence tomography) for fixation, performing slicing after quick freezing by liquid nitrogen, performing oil red O staining on a 10 mu m slice, performing 60% isopropanol washing on the slice after washing by distilled water, performing color separation by 60% isopropanol after the staining is completed by using 60% oil red O staining solution for 10-15 min, performing hematoxylin counterstaining on cell nuclei after washing by distilled water, performing water washing for 1-3 min, performing glycerol sealing, and observing and photographing.
4.6 detection of liver TG
50mg of liver is accurately weighed, and the lysate is added in a proportion of 1mg of liver to 20 mu L of lysate. The tissue is crushed by a full-automatic rapid sample grinding instrument, then the tissue is kept stand for 10min, a proper amount of supernatant is transferred into a 1.5mL centrifuge tube, the following steps are carried out, and the rest lysate can be used for protein quantification by a BCA method. The supernatant was heated in a metal bath at 70℃for 10min. Centrifuge at 2000rpm for 5min at room temperature and collect the supernatant for TG assay. 10. Mu.L of supernatant was plated in 96-well plates and 4mM glycerol standards were diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125. Mu. Mol/L and 10. Mu.L of each dilution was plated in 96-well plates to prepare working solutions as described in Beijing plaril liquid TG kit. mu.L of working solution was added to the sample, incubated at 37℃for 15min, and OD was measured using a working wavelength of 550 nm.
4.7 Malondialdehyde (MDA) detection
Accurately weighing liver tissue according to the weight (g): volume (mL) =1: 9, adding physiological saline with the volume being 9 times, shearing tissues, preparing homogenate by using ice water bath, carrying out 3000 r/min, centrifuging for 10min, and taking supernatant, namely 10% homogenate supernatant to be measured. 0.1mL of absolute ethyl alcohol is added into a blank tube, 0.1mL of 10nmol/mL of standard substance is added into a standard tube, 0.1mL of test sample is added into a measuring tube and a control tube, 0.1mL of first reagent is added into the four tubes respectively, the four tubes are uniformly mixed, 3mL of second reagent application liquid is added into the four tubes, 1mL of third reagent application liquid is added into the blank tube, the standard tube and the measuring tube, and 1mL of 50% glacial acetic acid is added into the control tube. Mixing uniformly, incubating for 40min at 95 ℃, taking out, cooling, centrifuging for 10min at 3500-4000 r/min, taking supernatant, and measuring OD value at 532 nm. And calculating the MDA content between the groups according to a calculation formula.
4.8 detection of superoxide dismutase (T-SOD)
Taking 10% liver homogenate supernatant to be measured. 1mL of the reagent I application solution is added into the two pipes, 0.05mL of the sample is added into the measuring pipe, 0.05mL of distilled water is added into the control pipe, 0.1mL of the reagent II, reagent III and reagent IV application solutions are respectively added into the two pipes, the two pipes are fully and uniformly mixed by a vortex mixer, incubation is carried out for 40min at 37 ℃, and 2mL of the color reagent is respectively added into the two pipes. Mixing, standing at room temperature for 10min, and measuring OD value at 550 nm.
4.9 detection of Catalase (CAT)
Taking part of 10% liver homogenate according to the physiological saline to 1:9, diluting to prepare 1% liver homogenate, simultaneously measuring the concentration of the homogenate protein by using a BCA kit, preparing a substrate solution before each measurement, enabling the absorbance of the substrate solution to be between 0.5 and 0.55, and preheating the substrate solution to 25 ℃ for later use. Taking a 1cm optical path quartz cuvette, carrying out ultraviolet 240nm, and carrying out double-distilled water zeroing for standby. Taking a pretreated sample 0.02 and mL, adding the pretreated sample into the bottom of a cuvette, directly flushing 3mL of substrate solution which is preheated to 25 ℃ and has an OD of between 0.5 and 0.55 into the cuvette by using a 5mL large pipette, immediately measuring absorbance at 240nm, recording an OD1 value, taking out the cuvette, immediately measuring absorbance again when the cuvette is not taken out, and recording an OD2 value. CAT activity between groups was calculated according to the calculation formula.
4.10 Real-time quantitative fluorescence PCR (Real-time PCR)
(1) Taking out the tissue from the refrigerator at the temperature of minus 80 ℃ into an ice box, weighing about 0.02g of the tissue into an EP tube by using an electronic balance, and precooling the tissue into a centrifuge at the temperature of 4 ℃;
(2) Adding 1mL of Trizol and 3 steel balls into an EP pipe provided with a tissue block, grinding by a grinder, taking out liquid, pouring out the steel balls, and reacting for 10min at room temperature;
(3) Adding 200 mu L of chloroform into an EP tube, shaking vigorously and mixing for 30s, and standing on ice for 5-10min;
(4) Placing the centrifuge tube in a centrifuge at 4 ℃ after standing, centrifuging at 12000rpm for 15min;
(5) Aspirate the aqueous phase (supernatant) of the centrifuged sample into a new 1.5mL centrifuge tube;
(6) Adding isopropanol solution with the same volume as the centrifugally extracted solution, slightly reversing and uniformly mixing, and standing at-20 ℃ for 20min;
(7) Taking out the sample at-20deg.C, centrifuging at 12000rpm and 4deg.C for 15min;
(8) Removing supernatant after centrifugation to obtain white (or colorless transparent) precipitate, adding pre-cooled 100-300 μl of 75% ethanol prepared with DEPC water into the inner wall of the centrifuge tube, and washing for 2-3 times;
(9) Removing liquid, air-drying at room temperature for about 15min, adding pre-cooled DEPC water into the centrifuge tube for 20-50 mu L, dissolving precipitate (RNA) obtained after air-drying at the bottom, and storing in a refrigerator at-20deg.C for use;
(10) Measuring the concentration of RNA by using an ultra-micro ultraviolet spectrophotometer, recording the result and calculating the loading quantity of each group;
(11) As shown in Table 1, the corresponding reaction solution in the reverse transcription kit is added for premixing, and the sample is added for reverse transcription, and after shaking and centrifugation, the mixture is put into a PCR instrument, and the corresponding PCR reaction conditions are set in the reverse transcription kit. Placing the cDNA sample subjected to reverse transcription by a reverse transcription instrument in a refrigerator at the temperature of-20 ℃ for standby;
(13) Adding a reactant into 0.2mL fluorescence quantitative PCR octal tube, taking cDNA as a template, amplifying the experimental target gene by using a fluorescence quantitative PCR amplification instrument, and carrying out fluorescence quantitative PCR determination;
(14) The mixed reactants are subjected to shaking and uniform mixing, and after centrifugation, the eight-connecting tube is placed into a qRT-PCR reactor. Setting a PCR reaction program: pre-denaturation: 94 ℃ for 5min; denaturation: 94 ℃,30s,60 ℃,30s,72 ℃,30s,40 cycles; extension: storing at 72 deg.C, 5min,4 deg.C;
(15) The expression change of the target gene was calculated by the 2- ΔΔct method.
4.11 short chain fatty acid assay
Extruding the segmented colon slice with sterile forceps, taking out the content, and storing in a-80deg.C low-temperature storage tube. . The colon contents were diluted five times with ultrapure water and vortexed for 3 minutes. Next, the suspension was allowed to stand for 5 minutes, and then centrifuged at 5000 Xg for 20 minutes at 4 ℃. One milliliter of the supernatant was mixed with 20. Mu.L of chromatographic grade phosphoric acid, and the mixture was injected into a chromatographic bottle through a 0.45 μm membrane filter for gas chromatography. The gas chromatograph consisted of an AOC-20S autosampler and GC-2010 equipped with a flame ionization detector. Nitrogen was used as a carrier gas at a flow rate of 3ml/min. An SH stable wax high-polarity column is arranged on the gas chromatograph, the sample injection quantity is 0.2 mu L, the split injection ratio is 50, and the injection temperature is 200 ℃. Ethyl acetate was injected as a blank solvent between each sample to eliminate any memory effect. The initial column temperature was set at 80℃and held for 1 minute, then increased to 170℃at a rate of 8℃per minute, then immediately increased to 220℃at a rate of 20℃per minute and held for 4 minutes. The total time was 18.75 minutes. Finally, the content of SCFAs calculation is calibrated by an external standard method according to an SCFAstandard curve.
4.12 intestinal flora 16s rRNA sequencing analysis
Samples of the colon contents were collected for total DNA isolation and 16s rRNA high throughput sequencing techniques by the hangzhou Ming family organism. 16s rRNA was amplified in the V3-V4 region, the amplicon was purified using QIA rapid PCR purification kit, sequencing was performed by the Illumina Novaseq platform (PE 300), and the original sequence was quality controlled by UPARSE. The Operational Taxon (OTU) was constructed by binding sequences into clusters with sequence similarity greater than 97% using QIIME.
5. Experimental results:
the results of FIG. 1 show that Lactobacillus plantarum ZY08 significantly reduces the ALT and AST elevation caused by alcohol intake compared to E680. ALT, i.e. glutamic-pyruvic transaminase, mainly exists in liver cell plasma, the intracellular concentration is 1000-3000 times higher than that in serum, and only 1% of liver cells are destroyed, so that serum enzyme can be increased by one time. Therefore, glutamic pyruvic transaminase is recommended by the world health organization as the most sensitive detection index for liver function damage. ALT reduction means reduced liver damage. AST, glutamic-oxaloacetic transaminase, also known as aspartate aminotransferase. AST is mainly distributed in mitochondria of hepatocytes and is also one of indexes of sensitivity to damage of hepatocytes.
The results in fig. 2 demonstrate that lactobacillus plantarum ZY08 can significantly reduce the rise in FFA caused by alcohol intake compared to E680. Free Fatty Acids (FFA) are substances into which triglycerides are broken down. Under normal conditions, the plasma content is extremely low, the increase of free fatty acid can change the permeability of mucous membrane, so that mucous membrane is damaged, and excessive intake of free fatty acid by liver exceeds oxidation of fatty acid by liver mitochondria, so that triglyceride can be promoted to be increased, and fatty liver is aggravated.
The results in FIG. 3 show that Lactobacillus plantarum ZY08 can significantly improve the liver pathological damage process caused by alcohol intake compared with E680. Model group H & E staining showed significant aggregation of lipid droplets in hepatocytes, and ZY08 staining showed fewer lipid droplets in stem cells, improving liver pathology.
The results in fig. 4 show that lactobacillus plantarum ZY08 can significantly improve liver lipid deposition caused by alcohol intake compared to E680. Compared with a control group, the liver oil red staining result of the alcohol model group is obviously increased, ZY08 has the phenomenon of obviously improving liver lipid deposition, and the effect is more obvious compared with lactobacillus plantarum E680.
The results in fig. 5 show that lactobacillus plantarum ZY08 can significantly reduce the liver TG increase caused by alcohol intake compared with E680, the triglyceride content in the liver of mice in the alcohol group (AF) is significantly increased compared with the control group (PF), and the triglyceride content in the liver of af+lactobacillus plantarum ZY08 is significantly reduced compared with the AF group, so that lactobacillus plantarum ZY08 can be judged to be effective in reducing the triglyceride content in the liver of mice.
The results in FIG. 6 show that Lactobacillus plantarum ZY08 has a strong antioxidant capacity, and that under normal conditions, the antioxidant enzymes in the natural antioxidant defense system of the organism can act synergistically with antioxidants in the diet or medicine to scavenge peroxides. Among the most important antioxidant enzymes include superoxide dismutase (SOD) and Catalase (CAT). SOD is responsible for the differentiation of superoxide anions into hydrogen peroxide, whereas CAT can reduce hydrogen peroxide, thereby preventing the generation of highly toxic hydroxyl radicals. On the other hand, the level of MDA, a lipid peroxidation product, was examined to indirectly determine the severity of the free radical attack on the cells.
The results of FIG. 7 show that Lactobacillus plantarum ZY08 has the capacity of inhibiting the synthesis of liver triglyceride from the head, SREBP-1c is a main transcription factor of genes related to regulating the synthesis of liver fatty acid and triglyceride, the gene is closely related to the occurrence of fat toxicity generated by the excessive accumulation of triglyceride in liver cells, FAS is a target gene of SREBP-1c, the SREBP-1c and FAS are remarkably expressed and increased in an AF group, and the intervention of Lactobacillus plantarum ZY08 can remarkably inhibit the expression of the genes, so that Lactobacillus plantarum ZY08 has the capacity of inhibiting the de novo synthesis of triglyceride.
The results in FIG. 8 show that Lactobacillus plantarum ZY08 has the ability to improve the intestinal mucosal barrier, and that tight junctions are the primary means of junction between intestinal epithelial cells, and play an important role in maintaining the intestinal mucosal epithelial mechanical barrier and permeability. Tight junction proteins are important protein molecules that constitute the intestinal mucosal barrier, determine the permeability of the intestinal wall, and have a great impact on the composition and function of tight junctions. Wherein ZO-1 and Claudin1 are important factors for forming cell-cell tight connection, and the interference of lactobacillus plantarum ZY08 significantly restores the condition of reduced gene expression of ZO-1 and Claudin1 induced by alcohol.
The results in FIG. 9 show that Lactobacillus plantarum ZY08 has the ability to promote intestinal short-chain fatty acid synthesis, SCFA being the major metabolite produced by intestinal microbial fermentation. SCFA act as their metabolic end products, maintaining redox equivalents in the aerobically environment of the gut. SCFA are saturated fatty acids with 1-6 carbon atoms, with the most abundant SCFA (-95%) being acetic acid (C2), propionic acid (C3) and butyric acid (C4). The interference of lactobacillus plantarum ZY08 significantly increases the content of acetic acid, propionic acid and butyric acid in the intestinal tract.
The results in fig. 10 and 11 show that lactobacillus plantarum ZY08 has the function of regulating intestinal flora, and that the distribution bar graph represents LDA values for significantly different species, showing significantly enriched taxonomies and defined important species per group, at the phylum level, lactobacillus plantarum ZY08 is able to significantly increase the abundance of the Firmicutes flora (fig. 10). In addition, at the genus level, lactobacillus plantarum ZY08 significantly increased the abundance of the flora of the genera Blautia, oscillibacter, lachnoclostricium, intelsinimonas, lachnospiraceae NK a136 group, and christenseneella (fig. 11).
The results show that lactobacillus plantarum (Lactobacillus plantarum) ZY08 provided by the invention can improve the balance of intestinal flora by regulating and controlling the abundance of various intestinal probiotics (blautia, lachnoclothrium and the like) in the intestinal tract; and promote the synthesis of short chain fatty acids in the intestinal tract to restore the intestinal barrier function of alcohol damage. In addition, lactobacillus plantarum ZY08 can inhibit liver fatty acid synthesis pathway to reduce liver triglyceride deposition and improve alcohol damage condition to liver through antioxidant pathway.
Example 3 preparation of functional fermented yogurt Using Lactobacillus plantarum ZY08
1. The processing process flow of the yoghourt comprises the following steps:
raw material preheating, homogenizing, blending, sterilizing, cooling, preparing and inoculating, fermenting, after-ripening and refrigerating
2. Key points of operation
(1) Raw materials: 2L whole UHT sterilized milk or fresh cow milk;
(2) Preheating: placing the mixture into a container and heating to 63 ℃;
(3) Homogenizing: homogenizing in a homogenizer under 15-25 MPa), pouring the mixed solution into an iron tank, adding 100g white sugar, and sterilizing in water bath at 90deg.C for 10min;
(4) And (3) blending: adding ingredients into cow milk, and dissolving;
(5) Sterilizing: sterilizing the sweetened milk in water bath at 90deg.C for 10min;
(6) And (3) cooling: cooling sterilized cow milk to 40-50deg.C for use;
(7) Preparing a starter: lactobacillus plantarum ZY08 strain was inoculated in a tube containing sterilized skim milk (12%, w/v) under aseptic conditions and cultured at 37℃for 20 hours. The inoculation amount for each passage is 2-4% (v/v), the passage is carried out for 2-3 times to restore activity, and the culture medium is placed in a refrigerator at 4 ℃ for preservation;
(8) Inoculating and fermenting: under aseptic condition, the activated lactobacillus plantarum ZY08 is inoculated, and the inoculation amount is 2-4% (v/v). Fermenting at 42 deg.C for 6-10 hr;
(9) Post-ripening: after fermentation, placing the mixture in a refrigerator at the temperature of 4 ℃ for after-ripening for 12-24 hours;
(10) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.
Example 4 preparation of functional fermented fruit and vegetable juice Using Lactobacillus plantarum ZY08
1. Process flow of fermented fruit and vegetable juice
Raw material cleaning, flash evaporation, pulping, blending, homogenizing, sterilizing
Cooling, inoculating, sealing, fermenting, post-ripening, filling and refrigerating
2. Key points of operation
(1) Raw materials: fresh pumpkin and dragon fruit are selected;
(2) Cleaning and cutting into blocks: cleaning, peeling (removing pulp of pumpkin), and cutting into small pieces;
(3) Flash evaporation: inactivating enzyme by flash evaporation, treating at 121 ℃ and 0.5-1 min, and exhausting rapidly;
(4) Pulping: according to the proportion of pumpkin to water (weight ratio) =1:1, a proper amount of pumpkin and water are gradually put into a colloid mill for grinding, and coarse grinding and fine grinding are carried out once. Pulping the dragon fruits by a pulping machine until pulp is uniform and has no lumps;
(5) Blending and homogenizing: 15 percent of pumpkin juice, 30 percent of dragon fruit juice, regulating the content of soluble solids to 10 degrees Brix by using sucrose, adding 0.2 percent of stabilizer CMC, uniformly mixing, and adopting a two-stage homogenization method, wherein the diameter and the grain diameter of the melon pulp are 2-3 mu m by adopting a low pressure (15 MPa) and a high pressure (25 MPa) firstly;
(6) Sterilizing and cooling: preserving the heat of the blended composite fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃;
(7) Inoculating and fermenting: under aseptic condition, inoculating activated lactobacillus plantarum ZY08,the initial bacterial count is controlled at 10 7
CFU/mL. Fermenting at 37 deg.C for 24 hr;
(8) Post-ripening: after fermentation, placing the mixture in a refrigerator at 4 ℃ for 3 hours;
(9) Filling and refrigerating: after finishing the after-ripening, filling the mixture into a 250mL sterilized glass bottle, and sending the sterilized glass bottle to a refrigeration house for refrigeration.
Example 5 preparation of fatty liver-improving powder Using Lactobacillus plantarum ZY08
1. Preparation of lactobacillus plantarum ZY08 bacterial mud
The Lactobacillus plantarum ZY08 single colony is selected and inoculated into 50mL MRS liquid culture medium, and placed in a 37 ℃ incubator for culture for 18 hours. Activated again in 250mL of MRS broth at an inoculum size of 5%, and incubated in an incubator at 37 ℃ for 24 h. Finally, the activated lactobacillus plantarum ZY08 is subjected to high-density anaerobic culture in a 10L fermentation tank at an inoculum size of 5 percent, and is cultured for 18 hours at 37 ℃ and pH of 6.8. Then centrifuging at 8000r/min and 4deg.C for 15min, discarding supernatant, collecting bacterial precipitate, and rinsing bacterial cells with sterile phosphate buffer solution (pH 7.0) for 2 times. Obtaining lactobacillus plantarum ZY08 bacterial mud.
2. Preparation of protective agent
The lyoprotectant comprises 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride. Water was used as solvent. Sterilizing at 110deg.C.
3. Preparation of lactobacillus plantarum ZY08 bacterial powder
And fully and uniformly mixing the prepared lactobacillus plantarum ZY08 bacterial precipitate with a protective agent solution according to the proportion of 1:5. Pre-freezing for 5 hours at the temperature of minus 40 ℃ to uniformly freeze the lactobacillus plantarum ZY08 bacterial powder on the inner wall of the container, and then performing vacuum freeze drying for 18-20 hours to obtain the lactobacillus plantarum ZY08 bacterial powder. After rehydration with physiological saline, the lactobacillus plantarum ZY08 powder is washed twice, and the viable count is 1.0X10 11 ～1×10 12 CFU/g。
Example 6 preparation of probiotic milk powder for pets Using Lactobacillus plantarum ZY08
1. Preparation of lactobacillus plantarum ZY08 bacterial powder
Preparation of Lactobacillus plantarum ZY08 powder lyophilized powder according to reference example 5, wherein the viable count of the powder is 1.0X10 11 ～1×10 12 CFU/g。
2. Preparation of pet formula powder
Primary selection of raw materials: milk powder, fish meal, bone meal, cereal and vegetable oil, additives: vitamins, trace elements, functional factors and others;
(1) Automatic batching: throwing the obtained material raw materials into a material bin according to a formula;
(2) Crushing: crushing the weighed materials by a crusher;
(3) Mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniform mixing;
(4) Puffing: making the mixed materials into granular materials through a bulking machine;
(5) And (3) drying: drying the mixed materials by a dryer, wherein the temperature is controlled to be 65-70 ℃;
(6) And (3) classifying and screening: the stream was passed through a classifying screen with the particles controlled at 2.5-5 mm.
3. Preparation of probiotic formula powder for pets
Uniformly mixing the bacterial powder prepared in step 1 and the pet feed prepared in step 2 according to the proportion of 1:100, wherein the living bacteria leaving the factory in the final product are 10 8 CFU/g or more. After the product is filled, the product is put in storage for sale.
Example 7 preparation of an anti-hangover and liver-protecting oral liquid Using Lactobacillus plantarum ZY08
1. Preparation of lactobacillus plantarum ZY08 bacterial powder
Referring to example 5, a lactobacillus plantarum ZY08 fungus powder freeze-dried fungus powder is prepared, wherein the number of viable bacteria in the fungus powder is as follows:
1.0×10 11 ～1×10 12 CFU/g。
1. the formula comprises the following components: 10 parts of kudzuvine root, 10 parts of hovenia dulcis thunb, 3 parts of ginseng, 4 parts of medlar, 4 parts of hawthorn, 8 parts of glucose, 4 parts of xylitol, 2 parts of resistant starch and 3 parts of dried orange peel.
2. Weighing according to a formula, and then crushing into particles;
3. adding the crushed product into the decoction, decocting for 2 hours for the first time and 1.5 hours for the second time, and mixing the decoctions;
4. filtering, concentrating the filtrate under reduced pressure to obtain paste, and adding dilution;
5. further filtering, adding 1g of fungus powder and 30ml of syrup into each 50ml of sample, and adding water to 100ml;
stirring, and packaging.
Example 8 preparation of lactic acid bacteria solid beverage Using Lactobacillus plantarum ZY08
1. Preparation of lactobacillus plantarum ZY08 bacterial powder
Referring to example 5, a lactobacillus plantarum ZY08 fungus powder freeze-dried fungus powder is prepared, wherein the number of viable bacteria in the fungus powder is as follows:
1.0×10 11 ～1×10 12 CFU/g。
2. the formula comprises the following components: 50 parts of milk, 5 parts of sucrose, 1.5 parts of thickener (sodium carboxymethylcellulose 0.5 parts, oxidized hydroxypropyl starch 0.5 parts, carrageenan 0.2 parts, agar 0.2 parts, konjac gum 0.1 parts), 3 parts of prebiotics, 0.5 parts of essence-level pigment and 40 parts of water.
3. Milk and lactobacillus plantarum ZY08 bacterial powder are mixed and fermented (100 kg milk per gram bacterial powder);
4. mixing the fermented product with other ingredients, and homogenizing at 60deg.C/20 Mpa;
5. sterilizing the homogenized product, and putting the homogenized product into a cold storage for 12 hours to obtain solid beverage; or homogenizing, and spray drying or freeze drying to obtain solid powder;
6. the product is packaged and then used.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Sequence listing
<110> university of Zhejiang
<120> Lactobacillus plantarum ZY08 with effect of improving alcoholic liver injury and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Lactobacillus plantarum ZY08 (Lactobacillus plantarum ZY 08)
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ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgaacg aactctggta 60
ttgattggtg cttgcatcat gatttacatt tgagtgagtg gcgaactggt gagtaacacg 120
tgggaaacct gcccagaagc gggggataac acctggaaac agatgctaat accgcataac 180
aacttggacc gcatggtccg agtttgaaag atggcttcgg ctatcacttt tggatggtcc 240
cgcggcgtat tagctagatg gtggggtaac ggctcaccat ggcaatgata cgtagccgac 300
ctgagagggt aatcggccac attgggactg agacacggcc caaactccta cgggaggcag 360
cagtagggaa tcttccacaa tggacgaaag tctgatggag caacgccgcg tgagtgaaga 420
agggtttcgg ctcgtaaaac tctgttgtta aagaagaaca tatctgagag taactgttca 480
ggtattgacg gtatttaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 540
acgtaggtgg caagcgttgt ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt 600
aagtctgatg tgaaagcctt cggctcaacc gaagaagtgc atcggaaact gggaaacttg 660
agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag 720
aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg 780
gtagcaaaca ggattagata ccctggtagt ccataccgta aacgatgaat gctaagtgtt 840
ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 900
ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 960
gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 1020
agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 1080
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 1140
ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1200
caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 1260
gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 1320
tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1380
atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1440
gtcggtgggg taacctttta ggaaccagcc gcctaaggtg ggacagatga ttagggtgaa 1500
gtcgtacag 1509
Claims (5)
1. Lactobacillus plantarum ZY08 for improving alcoholic liver injury is characterized in that the Lactobacillus plantarum is @ or @ isLactobacillus plantarum) ZY08 was deposited in the China general microbiological culture Collection center, with accession number: CGMCC No.14847.
2. Use of lactobacillus plantarum ZY08 according to claim 1 for the preparation of functional food, characterized in that the functions are improving liver injury, improving antioxidant capacity in vivo, lowering triglycerides, improving intestinal mucosal barrier, promoting the synthesis of intestinal short chain fatty acids and improving intestinal flora.
3. The use according to claim 2, wherein the functional food comprises a fungus powder, a yoghurt, a milk powder and a fermented beverage.
4. Use of lactobacillus plantarum ZY08 according to claim 1 for the preparation of a health care product, characterized in that the health care function is to improve liver injury, improve antioxidant capacity in vivo, lower triglycerides, improve intestinal mucosal barrier, promote the synthesis of intestinal short chain fatty acids and improve intestinal flora.
5. The use according to claim 4, wherein the health product comprises a bacterial agent, an oral liquid or a solid beverage.
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| CN114196593B (en) * | 2021-12-30 | 2023-07-28 | 南昌大学 | Lactobacillus plantarum P101 with high antioxidant activity and application thereof |
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