CN116064286B - Lactobacillus helveticus ZJUIDS11 for improving nonalcoholic liver disease and application thereof - Google Patents
Lactobacillus helveticus ZJUIDS11 for improving nonalcoholic liver disease and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of food microorganisms, in particular to lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 for improving nonalcoholic liver diseases and application thereof. The invention discloses lactobacillus helveticus ZJUIDS11 with a preservation number of CGMCC No.24761. The invention also discloses application of the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 in preparing a product for improving the nonalcoholic liver disease.
Description
Technical Field
The invention relates to the technical field of food microorganisms, in particular to lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 for improving nonalcoholic liver diseases and application thereof.
Background
The incidence of nonalcoholic fatty liver disease (Non-alcoholic Fatty Liver Disease, NAFLD) increases year by year and tends to decrease in age, becoming one of the most common liver diseases worldwide. Today, as the incidence of obesity and diabetes increases, the prevalence of NAFLD is higher in most countries. NAFLD is a global important public health problem that can further develop into steatohepatitis (NASH), liver fibrosis and cirrhosis, and even liver cancer, severely compromising the health of people. However, no clinical medicine for safely and effectively treating NAFLD exists so far. How to effectively prevent NAFLD is an urgent problem to be solved in clinical medicine.
Alcoholic liver disease (alcoholic liver disease, ALD) differs from Non-alcoholic fatty liver disease (Non-alcoholic Fatty Liver Disease, NAFLD) in the factors that induce the disease; alcoholic fatty liver is a liver disease caused by long-term excessive drinking, and the initial stage of the alcoholic liver disease is shown as hepatic cell steatosis, so that the alcoholic liver disease is developed, and finally alcoholic liver cirrhosis and even liver cancer are caused, and the alcoholic liver disease can induce hepatic cell injury and even liver failure in a short time. Nonalcoholic fatty liver is a clinical case syndrome characterized by steatosis of the parenchymal cells of the liver without excessive alcohol consumption and other unequivocal factors of liver injury. Chronic metabolic stress liver injury, closely related to genetic and insulin resistance, has very similar changes in liver pathology to alcoholic liver disease, but the patient has no history of drinking or is drinking a small amount, insufficient to cause liver injury. Therefore, the current targeted drugs are different, for example, the drugs aiming at ALD are mainly metadoxine accelerating alcohol metabolism drugs and liver protecting drugs, while the drugs aiming at NAFLD are mainly used for improving insulin resistance and reducing blood lipid, and statin drugs are mainly used for reducing blood lipid and protecting liver.
Lactobacillus helveticus has a number of effects, among which studies have shown that: lactobacillus helveticus inhibits pathogens according to market competition with pathogens for nutrient elements, adjusts the diversity of gastrointestinal microorganisms, and generates an ecological natural barrier. 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 helveticus 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.
The currently known uses of lactobacillus helveticus are for the amelioration of alcoholic liver diseases, for example:
the invention of CN114381398A, "lactobacillus helveticus ZJUIDS12 with improved alcoholic liver disease and its application" informs: lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS12 is used to ameliorate alcoholic liver injury;
the invention of CN110241046A, a Lactobacillus helveticus capable of relieving alcoholic liver injury and application, informs: lactobacillus helveticus (Lactobacillus helveticus) L551 can effectively relieve liver injury caused by alcohol.
Disclosure of Invention
The invention aims to provide lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 with the function of improving nonalcoholic liver diseases and application thereof.
In order to solve the technical problems, the invention provides lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 with the preservation number of CGMCC NO.24761.
16S rDNA full sequence of ZJUIDS11 is SEQ ID No: 1.
The invention also provides application of the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 in preparing products for improving non-alcoholic liver disease; the product comprises bacterial powder.
The lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has stronger capacity of protecting liver injury; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capacity of improving in-vivo antioxidation; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capacity of reducing liver triglyceride; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capacity of reducing the synthesis of liver fatty acid; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capability of inhibiting inflammation; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capability of recovering the intestinal barrier function; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capability of promoting the synthesis of intestinal short-chain fatty acid; the lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention has the capability of improving intestinal flora.
The lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 provided by the invention can regulate and control the abundance of various intestinal probiotics in intestinal tracts, promote the synthesis of short-chain fatty acids in the intestinal tracts so as to recover the intestinal barrier function of high-fat diet injury, and in addition, the ZJUIDS11 can inhibit the synthesis pathway of liver fatty acids to reduce the deposition of liver triglyceride and improve the condition of liver injury through an antioxidant pathway.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a colony morphology of ZJUIDS 11;
FIG. 2 is a chart showing the morphology of ZJUIDS11 gram stained cells;
FIG. 3 is an electrophoretically identified map of 16S rDNA of ZJUIDS 11;
FIG. 4 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on mouse body weight, liver weight and liver mass ratio;
FIG. 5 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on H & E staining pattern of mouse liver slices and NAFLD Activity Score (NAS);
FIG. 6 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on Triglyceride (TG) in the liver of mice;
FIG. 7 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the invention on glutamic pyruvic transaminase and glutamic oxaloacetic transaminase (ALT and AST) in mouse plasma;
FIG. 8 is a graph showing the effect of Lactobacillus helveticus ZJUIDS11 on the glucose tolerance level of mice according to the present invention;
FIG. 9 is a graph showing the effect of Lactobacillus helveticus ZJUIDS11 on the insulin resistance level of mice according to the present invention;
FIG. 10 shows the effect of Lactobacillus helveticus ZJUIDS11 on mouse High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) according to the present invention;
FIG. 11 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on Malondialdehyde (MDA) and total superoxide dismutase (T-SOD) in the liver of mice;
FIG. 12 shows the effect of Lactobacillus helveticus ZJUIDS11 on inflammatory factor expression (IL-1. Beta. And TNF. Alpha.) in mouse plasma according to the present invention;
FIG. 13 shows the effect of Lactobacillus helveticus ZJUIDS11 on inflammatory gene expression (IL-1. Beta. And TNF. Alpha.) in the intestinal tract of mice according to the present invention;
FIG. 14 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on the zona tight junction protein genes (ZO-1 and Claudin-1) in the mouse intestinal tract;
FIG. 15 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on short chain fatty acids (propionic acid and butyric acid) in mouse faeces;
FIG. 16 shows the effect of Lactobacillus helveticus ZJUIDS11 on the mice intestinal flora PCOA according to the invention;
FIG. 17 shows the effect of Lactobacillus helveticus ZJUIDS11 according to the present invention on the abundance of mouse intestinal flora levels (Firmics and bacterioides).
FIG. 18 shows the effect of Lactobacillus helveticus ZJUIDS11 on the intestinal flora level in mice according to the present invention.
Note that: in fig. 4-18, control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet + lactobacillus helveticus ZJUIDS11 group (HFD + lactobacillus helveticus ZJUIDS11 group).
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1, screening and identification of lactobacillus helveticus ZJUIDS 11:
1. screening of Lactobacillus helveticus ZJUIDS11
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 up in an ultra-clean bench, ten-fold gradient dilution is carried out by using sterile physiological saline, and 10 is selected -6 、10 -7 、10 -8 Three dilution gradients, 100. Mu.L of each of which was plated on MRS agar medium and incubated at 37℃for 48h. 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 obtained strains were cultured in MRS broth containing 40% glycerolFreezing and preserving at-80deg.C.
2. Identification of Lactobacillus helveticus ZJUIDS11
2.1 colony characterization
After the isolated and purified lactobacillus helveticus ZJUIDS11 is cultured in MRS agar culture medium for 48h, the diameter is between 0.3 and 1.5mm, and the colony is round, the edge is neat, white and the surface is moist and smooth, as shown in figure 1.
2.2 morphology under microscope:
lactobacillus helveticus ZJUIDS11 colony smear: gram staining was positive, sporulation was absent, straight-ended, single, paired or short-chain, see figure 2.
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, taking PCR products after the PCR amplification, carrying out agarose gel detection and photographing, and the length of an amplified fragment is about 1.4 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 had over 99% homology with the identified 16S rDNA sequence of Lactobacillus helveticus. The sequence alignment result and physiological and biochemical result of the strain Lactobacillus helveticus ZJUIDS11 are combined, and the screened lactobacillus Lactobacillus helveticus ZJUIDS11 is determined to be Lactobacillus helveticus, and the result is shown in figure 3.
The strain ZJUIDS11 of the invention has the preservation name of Lactobacillus helveticus Lactobacillus helveticus and the preservation unit: china general microbiological culture Collection center, preservation address: beijing city, chaoyang district, north Chenxi lu 1, 3, accession number: CGMCC No.24761, and the preservation time is 2022, 04 and 24.
In the invention, the following components are added:
the preparation method of the ZJUIDS11 bacterial liquid comprises the following steps: the strain ZJUIDS11 stored in the glycerol tube is firstly streaked and activated on an MRS agar plate for 2 to 3 times, then single bacterial colony is selected and cultured in an MRS liquid culture medium for 18 to 24 hours at 37 ℃ in an expanding way, and the concentration of bacterial liquid reaches 10 9 ～10 10 About CFU/mL, as a bacterial suspension. In practice, the concentration can be adjusted in a conventional manner.
Example 2 lactobacillus helveticus ZJUIDS11 improves non-alcoholic liver disease:
1. experimental materials
Experimental animals: 24C 57BL/6 male mice were purchased from Shanghai Laike laboratory animal center and bred in Zhejiang university laboratory animal center and SPF environment.
Reagent: ALT kit (cat# C009-2 Nanjing institute of bioengineering), AST kit (cat# C010-2 Nanjing institute of bioengineering), free fatty acid kit (cat# A042-2-1 Nanjing institute of bioengineering), tissue triglyceride kit (cat# E1013 Beijing Priley Gene technologies Co., ltd.), malondialdehyde assay kit (cat# A003-1 Nanjing institute of bioengineering), total superoxide dismutase assay kit (cat# A001-1 Nanjing institute of bioengineering), catalase assay kit (cat# A001-1 Nanjing institute of bioengineering), IL-1. Beta. Elisa kit (cat# PI301, shanghai Biyun Co., ltd.), TNF-. Alpha.Elisa kit (cat# PI301, shanghai Bitian Co.).
2. The method comprises the following steps:
2.1 laboratory animal feeding
After one week of acclimation in SPF-graded animal laboratories, C57BL/6 mice were randomly divided into 3 groups of 8 weeks old, 8 each, 10% control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet+Lactobacillus helveticus ZJUIDS11 group (HFD+Lactobacillus helveticus ZJUIDS11 group).
10% control feed was produced by research diet company (code D12450J); 45% high fat diet was produced by research diet company (code D12451).
NFD group was fed with control feed for 12 weeks, HFD group and hfd+lactobacillus helveticus ZJUIDS11 group were fed with 45% high fat feed for 12 weeks, during which time hfd+lactobacillus helveticus ZJUIDS11 group was fed with 0.2mL of lactobacillus helveticus ZJUIDS11 (10 in 0.2mL of bacterial liquid) 1 time per stomach every day 9 ZJUIDS11 of CFU).
The feed eaten by the mice is changed into new feed every two days, 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, biochemical indexes such as ALT, AST and the like 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. Index measurement (conventional detection mode)
3.1 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.
3.2 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.
3.3 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.
3.4 plasma AST 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 AST/GPT activity unit.
3.5 sugar tolerance detection
Mice were fasted for 12h at week 10 of the feeding period, and 2.5g/kg glucose solution was intraperitoneally injected at body weight after the fasted period, blood samples were collected at 0, 30, 60, 120min after the intraperitoneal injection, respectively, and glucose content in blood was measured using a blood glucose meter.
3.6 insulin resistance detection
Mice were fasted for 4h at week 11 of the feeding period, and after the fasted period, 0.75U/kg of insulin solution was intraperitoneally injected per body weight, blood samples were collected at 0, 30, 60, 120min after the intraperitoneal injection, respectively, and glucose content in the blood was measured using a blood glucose meter.
3.7 plasma HDL-C detection
The plasma samples of the mice are directly sampled and measured according to the instruction of Nanjing to build HDL-C kit. 2.5 mu L of the sample to be measured is added to the measuring well, distilled water is added to the control well, and a calibration solution is added to the calibration well. 180 mu L of R1 is respectively added into a measuring hole, a control hole and a calibration hole, the mixture is uniformly mixed, incubation is carried out at 37 ℃ for 5min, an enzyme-labeled instrument is used for measuring an A1 value at 546nm wavelength, 180 mu L of R1 is added into the measuring hole, the control hole and the calibration hole, the mixture is uniformly mixed, incubation is carried out at 37 ℃ for 5min, the enzyme-labeled instrument is used for measuring an A2 value at 546nm wavelength, and the HDL-C concentration of a sample to be measured is calculated according to a calibrator.
3.8 plasma LDL-C assay
The plasma samples of mice were taken and assayed by direct sampling according to the instructions of Nanjing's as-built LDL-C kit. 2.5 mu L of the sample to be measured is added to the measuring well, distilled water is added to the control well, and a calibration solution is added to the calibration well. 180 mu L of R1 is respectively added into a measuring hole, a control hole and a calibration hole, the mixture is uniformly mixed, incubation is carried out at 37 ℃ for 5min, an enzyme-labeled instrument is used for measuring an A1 value at 546nm wavelength, 180 mu L of R1 is added into the measuring hole, the control hole and the calibration hole, the mixture is uniformly mixed, incubation is carried out at 37 ℃ for 5min, the enzyme-labeled instrument is used for measuring an A2 value at 546nm wavelength, and the concentration of LDL-C of a sample to be measured is calculated according to a calibrator.
3.9 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.
3.10 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.
3.11 plasma IL-1. Beta. TNF-a Elisa assay
Samples or standards of different concentrations were added to the corresponding wells at 100 μl/well, and the reaction wells were sealed with a sealing plate membrane (transparent) and incubated at room temperature for 120 minutes. For serum or plasma samples, 50. Mu.l of sample analysis buffer can be added followed by 50. Mu.l of sample; if the dilution ratio is large, the sample and the sample analysis buffer are added in equal amounts, and the insufficient amount is supplemented to 100. Mu.l with the standard dilution. Note the dilution of the recorded samples. Plates were washed 5 times and 100 μl/well of biotinylated antibody was added. The reaction wells were sealed with a sealing plate membrane (transparent) and incubated at room temperature for 60 minutes. Plates were washed 5 times and horseradish peroxidase-labeled strepitavidin 100 μl/well was added. The reaction wells were sealed with a sealing plate membrane (white), and incubated at room temperature for 20 minutes in the dark. The plate was washed 5 times, 100. Mu.l/well of the color developing agent TMB solution was added, the reaction well was sealed with a sealing plate film (white), and incubated at room temperature for 20 minutes in a dark place. The stop solution was added at 50. Mu.l/well, and the A450 value was measured immediately after mixing.
3.12 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 5-10 min;
(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) Adding corresponding reaction solution in the reverse transcription kit for premixing, adding a sample for reverse transcription, oscillating and centrifuging, and then placing into a PCR instrument, and setting corresponding PCR reaction conditions according to 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;
(12) 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;
(13) 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;
(14) The expression change of the target gene was calculated by the 2- ΔΔct method.
3.13 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.
3.14 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.
4. Experimental results:
the results in fig. 4 show that lactobacillus helveticus ZJUIDS11 can significantly reduce weight gain caused by high fat diets. The 12-week high-fat diet induced significantly higher body weight in mice than the normal group, while ZJUIDS11 intervention significantly reduced the body weight of mice that had been increased by the high-fat diet.
The results of the above-mentioned-3.1 "are shown in FIG. 5, and Lactobacillus helveticus ZJUIDS11 can significantly improve the liver pathological damage progress caused by high-fat diet. Model group H & E staining showed significant aggregation of lipid droplets in hepatocytes, and ZJUIDS11 staining showed fewer lipid droplets in hepatocytes, improving liver pathology.
As shown in fig. 6, the above-mentioned result of-3.2 "shows that lactobacillus helveticus ZJUIDS11 can significantly reduce the increase of liver TG caused by high fat diet, the content of triglyceride in the liver of 45% high fat feed group (HFD) mice is significantly increased relative to the control group (NFD), and the content of triglyceride in the liver of hfd+lactobacillus helveticus ZJUIDS11 group is significantly reduced relative to the HFD group, so that lactobacillus helveticus ZJUIDS11 is judged to be effective in reducing the content of triglyceride in the liver of mice.
The results of the above-mentioned-3.3 and 3.4 "are shown in FIG. 7, and Lactobacillus helveticus ZJUIDS11 can significantly reduce ALT and AST elevation caused by high fat diet. 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 of the above-mentioned-3.5 "are shown in FIG. 8, and Lactobacillus helveticus ZJUIDS11 can significantly improve glucose tolerance caused by high-fat diet.
The results of the above-mentioned-3.6 "are shown in FIG. 9, and Lactobacillus helveticus ZJUIDS11 can significantly improve insulin resistance caused by high-fat diet.
The results of the above-mentioned-3.7 and 3.8' are shown in FIG. 10, and Lactobacillus helveticus ZJUIDS11 can significantly reduce the increase of Low Density Lipoprotein (LDL) caused by high fat diet and increase the content of High Density Lipoprotein (HDL) in blood plasma.
The results of the above-mentioned-3.9 and 3.10 "are shown in FIG. 11, in which Lactobacillus helveticus ZJUIDS11 has a strong antioxidant ability, and the antioxidant enzymes in the natural antioxidant defense system of the organism can be cooperated with the antioxidants in the diet or medicine to remove the peroxides under normal conditions. Among the most important antioxidant enzymes include superoxide dismutase (SOD). SOD is responsible for the disambiguation of superoxide anions into hydrogen peroxide, thus 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 the above-mentioned-3.11 and 3.12' are shown in FIGS. 12, 13 and 14, and the results of FIGS. 12-13 show that Lactobacillus helveticus ZJUIDS11 has anti-inflammatory effect, and that by detecting the content of IL-1β and TNF-a in blood and measuring the gene expression of IL-1β and TNF-a in intestinal tract, the high fat diet can increase the expression of inflammatory factors in the intestinal tract environment, while Lactobacillus helveticus ZJUIDS11 can significantly inhibit inflammation, indicating that Lactobacillus helveticus ZJUIDS11 has anti-inflammatory ability. Furthermore, the results in fig. 14 show that lactobacillus helveticus ZJUIDS11 has the ability to improve intestinal mucosal barrier, tight junctions being the primary means of connection between intestinal epithelial cells, and plays an important role in maintaining 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. Among them, ZO-1 and Claudin 1 are important factors constituting the tight cell-cell connection, and the intervention of Lactobacillus helveticus ZJUIDS11 significantly restores the conditions of reduced gene expression of ZO-1 and Claudin 1 induced by high-fat diet.
The results of the above-mentioned-3.13 "are shown in FIG. 15, in which Lactobacillus helveticus ZJUIDS11 has the ability to promote the synthesis of short chain fatty acids in the intestinal tract, SCFA being the main metabolite produced by the fermentation of intestinal microorganisms. SCFA act as their metabolic end products, maintaining redox equivalents in the aerobically environment of the gut. SCFA are saturated fatty acids having 1-6 carbon atoms, wherein an increased content of propionic acid (C3) and butyric acid (C4) is beneficial for the growth of intestinal microorganisms. The results show that the intervention of lactobacillus helveticus ZJUIDS11 significantly increases the content of propionic acid and butyric acid in the intestinal tract.
The results of the above-mentioned 3.14 "are shown in FIGS. 16, 17 and 18, and the Lactobacillus helveticus ZJUIDS11 has the function of regulating intestinal flora, and at the gate level, the Lactobacillus helveticus ZJUIDS11 can significantly reduce the abundance of the fungus colony of the genus Firmides (Firmides) and increase the abundance of the fungus colony of the genus Bacteroides (Bacteroides), and furthermore, at the genus level, the Lactobacillus helveticus ZJUIDS11 can significantly reduce the abundance of Coriobacteriaceae UCG-002 and Rikenella eae RC9 gun group induced by high fat diet and significantly increase the abundance of the fungus colony of the genera Ruminococcaceae UCG-014, allobaculm and Candidatus Saccharimonas.
Description: in the invention, the (Lactobacillus helveticus) ZJUIDS12 and (Lactobacillus helveticus) L551 are used for replacing the Lactobacillus helveticus ZJUIDS11, and the rest are equivalent; the detection of liver TG was performed according to the above method, and found:
the content of triglyceride in liver of HFD+ZJUIDS12 group was not significantly reduced relative to HFD group, and similarly, the content of triglyceride in liver of HFD+L551 group was not significantly reduced relative to HFD group.
Whereas the content of triglycerides in the liver of hfd+zjuids11 group was significantly reduced relative to the HFD group (fig. 6).
Example 3 confirmation of acid resistance and bile salt resistance of lactobacillus helveticus ZJUIDS11 (see CN 114381398A): 1. acid resistance test
The Lactobacillus helveticus ZJUIDS11 single colony is picked up and cultured for 18 hours at 37 ℃ in an MRS liquid culture medium, and the amplified bacterial suspension is inoculated into the MRS liquid culture medium in an amount of 1 percent and cultured for 18 hours at 37 ℃. The culture broth was centrifuged at 8000r/min for 5min at 4℃to collect the cells, which were washed 2 times with PBS buffer (pH 6.8,0.1 mol/L). The cells were suspended in MRS liquid medium with pH adjusted to 3.0 in advance, and the initial viable count was adjusted to about 10 8 CFU/mL, cultured at 37℃for 3h. Counting living bacteria in 0h and 3h samples by adopting a pouring plate method, culturing the poured plates for 48h at 37 ℃, and measuring the survival rate, wherein the calculation formula of the survival rate is as follows:
in the above, N 0 Viable count (CFU/mL) for 0h of test strain; n (N) t The strain was tested for viable count (CFU/mL) for 3h.
2. Experiment of bile salt resistance
Inoculating activated lactobacillus helveticus ZJUIDS11 bacterial suspension into MRS liquid culture medium at 1%, culturing at 37deg.C for 18 hr, mixing with vortex, and correcting initial viable count to about 10 9 CFU/mL. The cells were inoculated in an amount of 10% into MRS liquid medium containing 0.3% (m/v) of bovine bile salt (control MRS liquid medium containing no bovine bile salt) and cultured at 37℃for 3 hours. The number of viable bacteria in the sample was then counted using the pour plate method. The poured plate was incubated at 37℃for 48h. The bile salt tolerance of the strain is expressed as the logarithm of the difference between the number of viable bacteria per milliliter of bile salt-containing medium at 3 hours and the number of viable bacteria in the non-bile salt-containing medium (log CFU/mL).
The acid and bile salt resistance was measured using lactobacillus rhamnosus (Lactobacillus acidophilus) ATCC53103 as a control.
As shown in Table 1, the acid and bile salt resistance of Lactobacillus helveticus ZJUIDS11 was significantly better than that of the control strain ATCC53103. Its survival rate in MRS medium at pH 3.0 is as high as 99.24%. The viable count still reaches 10 in the environment containing 0.3% of ox gall salt 8 CFU/mL above, it shows that it has better bile salt tolerance. Experiments prove that the lactobacillus helveticus ZJUIDS11 has higher gastrointestinal tract viability.
TABLE 1 results of acid and bile salt tolerance by strains
| Strain | Acid resistance (%) | Bile salt tolerance (DeltaLog CFU/mL) |
| Lactobacillus helveticus ZJUIDS11 | 99.24±0.61 | 1.25±0.24 |
| Lactobacillus rhamnosus ATCC53103 | 62.12±0.18 | 1.03±0.23 |
Probiotics must be able to withstand a range of adverse conditions in the gastrointestinal tract such as gastric acid and bile to survive their probiotic action. The lactobacillus helveticus ZJUIDS11 provided by the invention can grow and proliferate under the condition of pH 3.0, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. Meanwhile, lactobacillus helveticus ZJUIDS11 can tolerate bile salts, can survive in intestinal tracts, and further can effectively improve intestinal flora and further play a role in reducing blood sugar.
Example 4 confirmation of the hydrophobic ability of lactobacillus helveticus ZJUIDS11 (see CN 114381398A):
1. measurement of hydrophobicity
Preparation of cells referring to the above example, lactic acid bacteria pellet was washed twice with clean PBS buffer (0.1 mol/L, pH 6.8) and resuspended to OD 610 The absorbance of the obtained solution is about 0.5, and the lactobacillus suspension is obtained.
Thoroughly mixing 2ml lactobacillus suspension and 2ml xylene, shaking in 37 deg.C water bath for 5min, and measuring OD of water phase after 0 hr and 2 hr respectively 610 Absorbance values.
A 0 Absorbance value=0h, a t Absorbance of =th.
The results obtained are shown in Table 2 below.
TABLE 2 surface hydrophobicity of different strains (%)
2. Analysis of results
The hydrophobicity of lactobacillus helveticus ZJUIDS11 was measured to be 36.44% higher than that of the control standard strain. The strain has strong adhesion capability, can adhere to human intestinal tracts, and improves the health of intestinal flora.
Example 5 confirmation of antibiotic susceptibility of lactobacillus helveticus ZJUIDS11 (see CN 114381398A):
culturing for 18h at a concentration of about 10 7 CFU/mL Lactobacillus helveticus ZJUIDS11 bacterial suspension is added into sterile MRS agar culture medium cooled to about 45 ℃ according to the amount of 1%, fully mixed and quantitatively added into 15 mL/dish. After solidification, the drug sensitive paper is taken by forceps and placed on the culture medium. The culture dish is placed on the upper sideCulturing in an incubator at 37 ℃ for 24 hours. Paper sheets without antibiotics were used as blank. And measuring the diameter of the inhibition zone. Each was repeated three times.
The diameter of the antibiotic susceptibility zone of lactobacillus helveticus ZJUIDS11 is shown in table 3. Referring to the CLSI (2017) drug sensitivity test standard, it is available that lactobacillus helveticus ZJUIDS11 exhibits sensitivity to penicillin G, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, compound neonomine, chloramphenicol, and the like. Experimental results indicate that Lactobacillus helveticus ZJUIDS11 is sensitive to common antibiotics.
TABLE 3 results of sensitivity of Lactobacillus helveticus ZJUIDS11 to antibiotics
Note that: s, sensitivity; i, intermediation; r, drug resistance
With the wide application of antibiotics in clinical treatment, the drug resistance of lactobacillus is also more and more serious, and long-term intake of the drug-resistant lactobacillus can bring great difficulty to clinical treatment. The lactobacillus helveticus ZJUIDS11 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 6 confirmation of pathogenic bacteria inhibitory ability of lactobacillus helveticus ZJUIDS11 (see CN 114381398A):
the antibacterial activity of the lactic acid bacteria is measured by an international agar diffusion method. 10mL of LB agar medium was poured into a sterile dish and cooled to obtain a lower medium. Culturing for 18h to a concentration of about 10 7 CFU/mL indicator fungus suspension is added into the sterilized LB agar medium cooled to about 45 ℃ according to the amount of 1%, and the mixture is fully mixed and quantitatively added into 10 mL/dish. Placing the sterilized oxford cup on the cup. After the upper culture medium is condensed, the oxford cup is gently pulled out. Samples of lactobacillus helveticus ZJUIDS11 fermentation supernatant were dosed at 100 μl/well and PBS buffer (0.1 mol/L, pH 6.8) was used as a control. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times.
As shown in Table 4, the metabolites of Lactobacillus helveticus ZJUIDS11 have a certain inhibition effect on pathogenic bacteria such as staphylococcus aureus, escherichia coli, salmonella enteritidis, listeria monocytogenes and the like, and are superior to the antibacterial effect of ATCC53103. The metabolite of the strain has antibacterial property.
TABLE 4 results of the inhibition ability of strains against pathogenic bacteria
Staphylococcus aureus is the most common pathogen in suppurative infection of humans, some escherichia coli can cause severe diarrhea and septicemia, and some salmonella species can also cause food poisoning in humans. Bacteriocin, organic acid, hydrogen peroxide and other antibacterial substances generated by lactic acid bacteria metabolism can inhibit the growth of pathogenic bacteria singly or jointly. The metabolite of the Lactobacillus helveticus ZJUIDS11 provided by the invention has a certain antagonism to the three pathogenic bacteria, plays an important role in maintaining intestinal microecological balance, and plays a health promoting effect.
Example 7 confirmation of antioxidant capacity of lactobacillus helveticus ZJUIDS11 (see CN 114381398A):
1. total antioxidant capacity (FRAP method)
The method for measuring the total antioxidant capacity was slightly modified according to the method of Giuberti et al. To the ELISA plate, 150. Mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, mixed at V: V=10:1:1, as-prepared) and 20. Mu.L of sample were added, mixed with shaking, reacted at 37℃for 10min, and the absorbance of the solution at 593nm was measured. Substituting the absorbance measured by the sample into a ferrous sulfate standard curve, wherein the antioxidant capacity of the sample is expressed as ferrous sulfate equivalent (mu mol FeSO) 4 /mL sample). Each sample was repeated 3 times and averaged.
Ferrous sulfate standard curve: ferrous sulfate solutions with different mass concentrations (0 mu M, 50 mu M, 100 mu M, 200 mu M, 400 mu M, 600 mu M and 800 mu M) were prepared, the ferrous sulfate solutions with different molar concentrations, 10mM TPTZ buffer and 0.3M acetate buffer were mixed according to V: V=1:1:10, 170 mu L of the mixed solution was added to an ELISA plate, the reaction was carried out at 37℃for 10min, and the absorbance of the solution at 593nm was measured. And drawing a standard curve by taking absorbance as an ordinate and ferrous sulfate mass concentration as an abscissa, and measuring.
2. Reducing ability
Determination of reducing ability reference is made to the method of Lin et al with some modifications. 1mL of the sample was placed in a centrifuge tube, and 1mL of each of a PBS buffer solution and 1% (w/v) potassium ferricyanide solution, 0.2M, pH6.6, and the mixture was mixed. Water bath at 50 ℃ for 20min, and ice bath cooling. Then, 1mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 600 r/min for 5min, 1mL of the supernatant was collected, 1mL of 0.1% (w/v) ferric trichloride and 1mL of distilled water were added, and the mixture was mixed uniformly, allowed to stand for 10min, and absorbance was measured at 700 nm. The samples were replaced with PBS buffer or MRS broth as blank. Each sample was repeated 3 times and averaged.
Reducing power (%) = [ (As-Ab)/Ab ]. 100
Wherein: as—absorbance of the sample set;
ab—blank absorbance;
3. DPPH free radical scavenging ability
The method for measuring the DPPH radical scavenging ability was described with reference to Shimada et al and with some modifications. Preparing 1000mg/ml VC standard solution, and diluting to different concentration gradients (0-30 mu g/ml). Adding 100 mu L of a sample to be detected (or VC standard solution) and 100 mu L of 0.2mM DPPH ethanol solution (prepared by absolute ethanol, stored at 4 ℃ in a dark place and used at present) into an ELISA plate, shaking uniformly, keeping away from light at room temperature for 30min, and measuring the absorbance of the solution at 517 nm; 100 mu L of absolute ethyl alcohol is used to replace 100 mu L of DPPH ethanol solution to form a blank group; 100. Mu.L of PBS buffer (0.2M, pH6.6 PBS or MRS broth) was used as a control instead of 100. Mu.L of the sample to be tested, and the control was zeroed with 100. Mu.L of a mixture of PBS buffer (or MRS broth) and absolute ethanol. Each sample was repeated 3 times and averaged.
DPPH radical scavenging ability (%) = [1- (As-Ab)/Ac ] ×100
Wherein: as—absorbance of the sample set; ab—blank absorbance; ac-absorbance of control group.
The results obtained are set forth in Table 5 below:
table 5, antioxidant Activity of Lactobacillus helveticus ZJUIDS11
* Representing significant differences, P <0.05; * Representing significant differences, P <0.01.
As shown in Table 5, the total antioxidant capacity and DPPH free radical of the lactobacillus helveticus ZJUIDS11 fermentation supernatant obtained by the sieve of the invention are obviously higher than those of the standard strain ATCC53103, and the reducing capacity of the bacterial suspension is also higher than that of the control strain. Thus, the lactobacillus helveticus ZJUIDS11 fermentation supernatant and bacterial suspension have high-efficiency antioxidation capability.
Example 8 preparation of fatty liver-soothing powder Using Lactobacillus helveticus ZJUIDS11
1. Preparation of Lactobacillus helveticus ZJUIDS11 bacterial sludge
A single colony of Lactobacillus helveticus ZJUIDS11 is selected and inoculated into 50mL of MRS liquid culture medium, and placed in a 37 ℃ incubator for culture for 18 hours. Activated again in 250mL MRS liquid culture medium according to the inoculum size of 5%, and placed in a 37 ℃ incubator for 24 hours. Finally, the activated lactobacillus helveticus ZJUIDS11 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 helveticus ZJUIDS11 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 helveticus ZJUIDS11 bacterial powder
The lactobacillus helveticus ZJUIDS11 bacterial precipitate prepared above is fully and evenly mixed with the protective agent solution according to the proportion of 1:5. Pre-freezing at-40deg.C for 5 hr to uniformly freeze the powderAnd (3) vacuum freeze-drying the inner wall of the reactor for 18-20 hours to obtain lactobacillus helveticus ZJUIDS11 bacterial powder. After rehydration with physiological saline, the lactobacillus helveticus ZJUIDS11 bacterial powder is washed twice, and the viable count is 1.0x10 11 ～1×10 12 CFU/g。
Example 9 preparation of anti-hangover and liver-protecting oral liquid Using Lactobacillus helveticus ZJUIDS11
1. Preparation of Lactobacillus helveticus ZJUIDS11 bacterial powder
Referring to the above examples, the Lactobacillus helveticus ZJUIDS11 powder lyophilized powder was prepared, wherein the viable count of the powder was 1.0X10% 11 ～1×10 12 CFU/g。
2. 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.
3. Weighing according to the formula, and then crushing into particles.
4. Decocting the crushed product for 2h for the first time and 1.5h for the second time, and mixing decoctions.
5. Filtering, concentrating the filtrate under reduced pressure to obtain paste, and diluting.
6. Further filtering, adding 1g of fungus powder and 30ml of syrup to 50ml of sample, and adding water to 100ml
7. Stirring, and packaging.
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.
Claims (3)
1. Lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 is characterized in that the preservation number is cgmccno.24761.
2. Use of lactobacillus helveticus (Lactobacillus helveticus) ZJUIDS11 as claimed in claim 1 for the manufacture of a product for improving non-alcoholic liver disease, characterised in that: the product is fungus powder.
3. Use according to claim 2, characterized in that the bacterial powder has at least any one of the following functions:
protecting liver injury, reducing liver triglyceride, improving in vivo antioxidant capacity, reducing liver fatty acid synthesis, inhibiting inflammation, recovering intestinal barrier function, and improving intestinal flora.
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| JP2011201801A (en) * | 2010-03-25 | 2011-10-13 | Snow Brand Milk Products Co Ltd | Fatty liver preventive and/or inhibitor |
| CN109456909A (en) * | 2018-08-17 | 2019-03-12 | 甘肃普诺贝康生物科技有限责任公司 | One plant has the Lactobacillus helveticus for reducing cholesterol ability |
| WO2021075663A1 (en) * | 2019-10-14 | 2021-04-22 | 주식회사 종근당바이오 | Composition for preventing, ameliorating or treating non-alcoholic fatty liver disease comprising lactobacillus helvetica and bifidobacterium strains |
| CN114145461A (en) * | 2021-11-26 | 2022-03-08 | 广州能靓生物技术有限公司 | Probiotics composition for relieving non-alcoholic fatty liver |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011201801A (en) * | 2010-03-25 | 2011-10-13 | Snow Brand Milk Products Co Ltd | Fatty liver preventive and/or inhibitor |
| CN109456909A (en) * | 2018-08-17 | 2019-03-12 | 甘肃普诺贝康生物科技有限责任公司 | One plant has the Lactobacillus helveticus for reducing cholesterol ability |
| WO2021075663A1 (en) * | 2019-10-14 | 2021-04-22 | 주식회사 종근당바이오 | Composition for preventing, ameliorating or treating non-alcoholic fatty liver disease comprising lactobacillus helvetica and bifidobacterium strains |
| CN114145461A (en) * | 2021-11-26 | 2022-03-08 | 广州能靓生物技术有限公司 | Probiotics composition for relieving non-alcoholic fatty liver |
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