CN117431188A - Lactobacillus plantarum ZJUIDS14 with function of improving nonalcoholic liver diseases and application thereof - Google Patents
Lactobacillus plantarum ZJUIDS14 with function of improving nonalcoholic liver diseases and application thereof Download PDFInfo
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Classifications
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- 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
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- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
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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
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Abstract
The invention provides lactobacillus plantarum ZJUIDS14 for improving non-alcoholic liver disease and application thereof, and the classification of the lactobacillus plantarum ZJUIDS14 is named as follows: lactiplantibacillus plantarum, accession number: CGMCC No.28091. The strain of the invention can promote the synthesis of short-chain fatty acid in intestinal tracts by regulating and controlling the abundance of various intestinal probiotics in the intestinal tracts, thereby recovering the intestinal barrier function of high-fat diet injury. In addition, ZJUIDS14 and extracellular polysaccharide produced by the same can inhibit the synthesis of liver fatty acid, reduce liver triglyceride deposition, and improve liver injury through antioxidant. The strain has the functions of protecting liver injury, improving in vivo antioxidation, reducing liver triglyceride, reducing liver fatty acid synthesis, recovering intestinal barrier function, promoting intestinal short chain fatty acid synthesis, improving intestinal flora and the like, and can be applied to preparing functional foods such as bacterial powder, yoghourt or milk powder and the like.
Description
Technical Field
The invention belongs to the technical field of food microorganisms, and particularly relates to lactobacillus plantarum ZJUIDS14 for improving non-alcoholic liver disease 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 in most asian countries, including china, has exceeded 25%. NAFLD is not only a global important public health problem in the 21 st century, but also a common chronic liver disease in China, which can further develop into steatohepatitis (NASH), liver fibrosis and cirrhosis or even liver cancer, and seriously endanger 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.
The lactobacillus plantarum belongs to one of lactobacillus and has multiple effects, wherein research shows that the lactobacillus plantarum inhibits germs and adjusts the diversity of gastrointestinal microorganisms according to market competition with germs on nutrient elements, so that 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 a lactobacillus plantarum ZJUIDS14 for improving non-alcoholic fatty liver disease, wherein the classification name of the lactobacillus plantarum ZJUIDS14 is as follows: lactiplantibacillus plantarum, which was deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.28091. The full sequence of 16s rDNA of the lactobacillus plantarum ZJUIDS14 (Lactiplantibacillus plantarum) 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 ZJUIDS14 for the preparation of functional food products.
It is a further object of the present invention to provide the use of the extracellular polysaccharide ZJUIDS14-EPS produced by the Lactobacillus plantarum ZJUIDS14 for the preparation of functional foods, said functions being protection of liver injury, improvement of in vivo antioxidant, reduction of liver triglycerides.
The lactobacillus plantarum ZJUIDS14 provided by the invention has strong liver injury protection capability.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capacity of improving in-vivo antioxidation.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capacity of reducing liver triglyceride.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capacity of reducing the synthesis of liver fatty acid.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capability of recovering the intestinal barrier function.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capability of promoting the synthesis of intestinal short-chain fatty acid.
The lactobacillus plantarum ZJUIDS14 provided by the invention has the capability of improving intestinal flora.
The food is fungus powder, yogurt, and milk powder.
The food specifically comprises functional fermented yoghourt, functional fermented fruit and vegetable juice, non-alcoholic fatty liver fungus powder relieving and probiotic milk powder for pets.
The lactobacillus plantarum ZJUIDS14 provided by the invention regulates and controls the abundance of various intestinal probiotics (blautia, lachnoclothridium and the like) in intestinal tracts, and promotes the synthesis of intestinal short-chain fatty acids so as to recover the intestinal barrier function of high-fat diet injury. In addition, ZJUIDS14 and extracellular polysaccharide produced by the same can inhibit the synthesis of liver fatty acid, reduce liver triglyceride deposition, and improve liver injury through antioxidant.
Drawings
FIG. 1 shows the effect of Lactobacillus plantarum ZJUIDS14 of the present invention on mouse body weight, liver weight and liver mass ratio
FIG. 2 is a graph showing H & E staining of mouse liver sections by Lactobacillus plantarum ZJUIDS14 according to the present invention.
FIG. 3 shows NAFLD activity score of Lactobacillus plantarum ZJUIDS14 of the present invention in the liver of mice
(NAS) and Triglycerides (TG).
FIG. 4 shows the effect of Lactobacillus plantarum ZJUIDS14 of the present invention on glutamic-oxaloacetic and glutamic-pyruvic transaminases (AST and ALT) in mouse plasma.
FIG. 5 shows the effect of Lactobacillus plantarum ZJUIDS14 of the present invention on Triglyceride (TG), free Fatty Acids (FFA), total Cholesterol (TC), high density lipoprotein (HDL-c), low density lipoprotein (LDL-c) in mouse plasma.
FIG. 6 shows the effect of Lactobacillus plantarum ZJUIDS14 on hepatic glucose tolerance levels in mice according to the invention.
FIG. 7 shows the effect of Lactobacillus plantarum ZJUIDS14 on the insulin resistance level of mice according to the present invention.
FIG. 8 shows the effect of Lactobacillus plantarum ZJUIDS14 of the present invention on Malondialdehyde (MDA) and total superoxide dismutase (T-SOD) in the liver of mice.
FIG. 9 shows the effect of Lactobacillus plantarum ZJUIDS14 of the present invention on lipid synthesis genes (FATP 2, FABP2 and CD 36), inflammation-related genes (IL-1. Beta. And TNF. Alpha.) and antibacterial peptide Genes (GRAMP) in the intestinal tract of mice.
FIG. 10 shows the effect of Lactobacillus plantarum ZJUIDS14 according to the invention on the zona tight junctions (ZO-1 and Claudin-1) in the intestinal tract of mice.
FIG. 11 shows the effect of Lactobacillus plantarum ZJUIDS14 according to the invention on short chain fatty acids (acetic acid, propionic acid and butyric acid) in mouse faeces.
FIG. 12 shows the effect of Lactobacillus plantarum ZJUIDS14 according to the invention on the diversity of intestinal flora a and beta diversity of mice.
FIG. 13 shows the effect of Lactobacillus plantarum ZJUIDS14 on the abundance of the intestinal flora gate in mice according to the invention.
FIG. 14 shows the effect of Lactobacillus plantarum ZJUIDS14 on the abundance of different strains of the intestinal flora level of mice according to the invention.
FIG. 15 shows the effect of Lactobacillus plantarum ZJUIDS14 exopolysaccharide according to the invention on mouse body weight, liver weight and liver mass ratio.
FIG. 16 shows the effect of Lactobacillus plantarum ZJUIDS14 exopolysaccharide on Triglyceride (TG) in the liver of mice according to the invention.
FIG. 17 shows the effect of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on glutamic-oxaloacetic and glutamic-pyruvic transaminases (AST and ALT) in mouse plasma.
FIG. 18 shows the effect of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on Triglyceride (TG), free Fatty Acids (FFA), total Cholesterol (TC), high density lipoprotein (HDL-c), low density lipoprotein (LDL-c) in mouse plasma.
FIG. 19 shows the effect of Lactobacillus plantarum ZJUIDS14 exopolysaccharide on hepatic glucose tolerance levels in mice according to the invention.
FIG. 20 shows the effect of Lactobacillus plantarum ZJUIDS14 exopolysaccharide on insulin resistance levels in mice according to the invention.
Note that: in fig. 1-14, the control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet + lactobacillus plantarum ZJUIDS14 group (HFD + ZJUIDS14 group).
Note that: in FIGS. 15-20, control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet+Lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group (HFD+ZJUIDS 14-EPS group).
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 ZJUIDS 14:
1. screening of Lactobacillus plantarum ZJUIDS14
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 ℃.
2. Identification of Lactobacillus plantarum ZJUIDS14
2.1 colony characterization
After the lactobacillus plantarum ZJUIDS14 is cultured in an MRS agar medium for 48 hours, the diameter is between 0.3 and 1.5mm, and the colony is round, neat in edge, white and moist and smooth in surface.
2.2 morphology under microscope:
lactobacillus plantarum ZJUIDS14 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. The sequence comparison result and the physiological and biochemical result of the strain lactobacillus plantarum ZJUIDS14 are combined, and the screened lactobacillus plantarum ZJUIDS14 is determined to be lactobacillus plantarum.
Example 2 lactobacillus plantarum ZJUIDS14 improves non-alcoholic liver disease:
1. experimental animals: c57BL/6 Male mice, 32, purchased from Shanghai Laek laboratory animal center, company license number: SCXK 2013-0016, and is fed into the laboratory animal center of Zhejiang university, SPF environment.
2. 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).
3. Experimental animal feeding
After C57BL/6 mice were acclimatized to the SPF-grade animal laboratory for one week, C57BL/6 mice at 8 weeks of age were randomly divided into 3 groups of 8 animals each, each of which was a control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet+lactobacillus plantarum ZJUIDS14 group (hfd+zjuids 14 group).
NFD group was fed with control diet for 12 weeks, HFD group and hfd+lactobacillus plantarum ZJUIDS14 group were fed with 45% high fat diet for 12 weeks, during which time hfd+lactobacillus plantarum ZJUIDS14 group was fed with 0.2mL of lactobacillus plantarum ZJUIDS14 (10) 1 time per stomach every day 9 CFU/solvent for physiological saline).
The feed consumed 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, 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.
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 sugar tolerance detection
Mice were intraperitoneally injected with 2.5g/kg glucose solution at body weight after 12h fasting, blood samples were collected at 0, 30, 60, 120min after intraperitoneally injection, respectively, and glucose content in the blood was measured using a blood glucose meter.
4.6 insulin resistance detection
Mice were intraperitoneally injected with 0.75U/kg glucose solution at body weight after 4h fasting, blood samples were collected at 0, 30, 60, 120min after intraperitoneal injection, respectively, and glucose content in the blood was measured using a blood glucose meter.
4.7 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.
4.8 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.
4.9 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.
4.10 Western blotting (Western blotting)
And adding a proper amount of RIPA buffer solution supplemented with protease and phosphatase inhibitor into liver tissue or cells, crushing and cracking, centrifuging at 12000rpm at 4 ℃, centrifuging for 15min, taking the supernatant to measure the protein concentration, and preparing a protein sample so that the protein content in each milliliter of sample is equal. Protein samples were loaded into SDS-PAGE gels and transferred onto PVDF membranes. The membrane was blocked with 5% skim milk in TBST and then allowed to react with the antibody at 4 ℃ for 12 hours. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Finally, the immunoreactivity of protein expression was observed with a chemiluminescent kit. Immunoblots were quantified by measuring the density of each band with Image-J.
4.11 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) 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;
(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.
4.12 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.
4.13 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. 16srRNA 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 ZJUIDS14 can significantly reduce weight, liver weight and liver weight ratio increase caused by high fat diets.
The results in fig. 2 show that lactobacillus plantarum ZJUIDS14 can significantly improve the progress of liver pathological damage caused by high-fat diets. Model group H & E staining showed significant aggregation of lipid droplets in hepatocytes, and ZJUIDS14 staining showed fewer lipid droplets in hepatocytes, improving liver pathology.
The results in fig. 3 show that lactobacillus plantarum ZJUIDS14 can significantly reduce the increase of liver NAS and 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), NAS is significantly increased, and the content of triglyceride in the liver and NAS in the hfd+lactobacillus plantarum ZJUIDS14 group are significantly reduced relative to the HFD group, so that lactobacillus plantarum ZJUIDS14 is judged to be effective in reducing the content of triglyceride and NAS in the liver of mice.
The results of fig. 4 show that lactobacillus plantarum ZJUIDS14 significantly reduced ALT and AST elevation caused by high fat diets. 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. 5 demonstrate that the triglyceride, free fatty acid and total cholesterol levels were significantly higher in the model group than in the control group, indicating that the model group rats had developed non-alcoholic fatty liver. Compared with the model group, the liver tissue triglyceride, free fatty acid and total cholesterol levels of the ZJUIDS14 experimental group are significantly lower than those of the model group, which indicates that the ZJUIDS14 can relieve nonalcoholic fatty liver. 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. 6 show that lactobacillus plantarum ZJUIDS14 can significantly improve glucose tolerance caused by high-fat diets.
The results in fig. 7 show that lactobacillus plantarum ZJUIDS14 can significantly improve insulin resistance caused by high-fat diets.
The results in fig. 8 demonstrate that lactobacillus plantarum ZJUIDS14 is able to significantly reduce elevated MDA levels in the liver caused by high fat diets, which can result in elevated T-SOD levels. Normally, antioxidant enzymes in the natural antioxidant defense system of organisms act synergistically with antioxidants in diets or drugs to scavenge peroxides. SOD is one of the most important antioxidant enzymes responsible for the differentiation of superoxide anions into hydrogen peroxide. 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 in fig. 9 demonstrate that lactobacillus plantarum is able to significantly improve intestinal lipid metabolism and inflammation levels. Lipid synthesis genes (FATP 2, FABP2 and CD 36) play an important role in lipid synthesis, and when these genes are elevated, the body's lipid synthesis increases. The inflammatory factors IL-1. Beta. And TNF. Alpha. Can reflect the inflammatory level of the body. Compared with the common feed group, the high-fat feed has the advantages that the levels of lipid synthesis genes (FATP 2, FABP2 and CD 36), inflammation-related genes (IL-1 beta and TNF alpha) and antibacterial peptide Genes (GRAMP) are improved, and the lactobacillus plantarum ZJUIDS14 has the advantages that the levels of the genes are obviously reduced, so that the lactobacillus plantarum ZJUIDS14 can improve the lipid synthesis and the increase of the inflammation level caused by the high-fat feed.
The results of fig. 10 demonstrate that lactobacillus plantarum ZJUIDS14 has the ability to improve intestinal mucosal barrier, tight junctions being the primary means of attachment between intestinal epithelial cells, and play 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. Wherein ZO-1 and Claudin-1 are important factors for forming cell-cell tight connection, and the interference of the lactobacillus plantarum ZJUIDS14 significantly restores the condition of reduced gene expression of ZO-1 and Claudin-1 induced by high-fat diet.
The results in FIG. 11 show that Lactobacillus plantarum ZJUIDS14 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 the lactobacillus plantarum ZJUIDS14 obviously increases the contents of acetic acid, propionic acid and butyric acid in intestinal tracts.
The results in fig. 12 show that lactobacillus plantarum ZJUIDS14 has the function of restoring the abundance of intestinal flora. The chao1 index reflects the abundance of the intestinal flora, the higher the index, the higher the abundance of the intestinal flora. Compared with the common feed group, the high-fat feed reduces the abundance of intestinal flora of mice, and the lactobacillus plantarum ZJUIDS14 obviously improves the Chao1 index, which indicates that the lactobacillus plantarum ZJUIDS14 can restore the abundance of intestinal flora after the period.
The results in FIG. 13 show that Lactobacillus plantarum ZJUIDS14 has the function of regulating intestinal flora, and at the portal level, lactobacillus plantarum ZJUIDS14 can significantly increase Proteus (Proteus) and cyanobacterium (Cyanobacteria) and reduce actinomycete (Actinobacterium) flora abundance.
The results in FIG. 14 show that Lactobacillus plantarum ZJUIDS14 has the function of regulating intestinal flora, and at the genus level, lactobacillus plantarum ZJUIDS14 can significantly increase the flora abundance of the genus Coprostanoligenes group, ruminococcaceae UCG-014,Allobaculum,and Ruminiclostridium 1 and decrease the flora abundance of the genus Roseburia.
The results show that the lactobacillus plantarum ZJUIDS14 can effectively reduce the weight, liver fat accumulation and liver injury of a high-fat diet mouse, and improve the glucose metabolism and fat metabolism of the mouse. In addition, the lactobacillus plantarum ZJUIDS14 can improve intestinal mucosa barrier, promote intestinal short-chain fatty acid synthesis and regulate intestinal flora.
Example 3 lactobacillus plantarum ZJUIDS14 extracellular polysaccharide improves non-alcoholic liver disease:
1. separation and purification of lactobacillus plantarum ZJUIDS14 extracellular polysaccharide
Lactobacillus plantarum ZJUIDS14 was cultured in MRS medium at 37℃for 24 hours. Cells were removed by centrifugation (8,000Xg, 4 ℃,15 min) and the supernatant was collected. Trichloroacetic acid was then added to the supernatant to a final concentration of 4% (w/v), stirred overnight at 4℃and the precipitated protein was isolated by centrifugation (12,000Xg, 4 ℃, 15). After separation of the proteins 3 volumes of ethanol were added to the supernatant to precipitate EPS. After 12 hours of incubation, EPS was collected by centrifugation (12,000Xg, 15 min). The precipitate was dissolved in deionized water, then dialyzed (MW cut-off 3,500 Da) and lyophilized. Using a Toxin Sensor TM The purified exopolysaccharide was assayed for endotoxin concentration by a chromogenic limulus kit (Genscript) and the next experiment was performed using exopolysaccharide without endotoxin.
2. Experimental animals: c57BL/6 Male mice 24, purchased from Shanghai Laek laboratory animal center, company license number: SCXK 2013-0016, and is fed into the laboratory animal center of Zhejiang university, SPF environment.
3. Experimental animal feeding
After C57BL/6 mice were acclimatized to the SPF-grade animal laboratory for one week, C57BL/6 mice at 8 weeks of age were randomly divided into 3 groups of 8 animals each, each of which was a control diet group (NFD group), 45% high fat diet group (HFD group), 45% high fat diet+lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group (hfd+zjuids 14-EPS group).
NFD group was fed with control diet for 12 weeks, HFD group and hfd+lactobacillus plantarum ZJUIDS14-EPS group were fed with 45% high fat diet for 12 weeks, during which time hfd+lactobacillus plantarum ZJUIDS14 group was gavaged 1 time per day with 0.2ml of ZJUIDS14 extracellular polysaccharide (80 mg/kg, solvent is physiological saline).
The feed consumed 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, 1% pentobarbital is used for intraperitoneal injection for anesthesia, the blood of the inferior vena cava is taken for measuring ALT, AST and FFA, and the liver and intestinal tissues of the mice are taken for relevant index measurement.
4. The experimental method comprises the following steps: same as in example 3.
Experimental results
The results of fig. 15 show that lactobacillus plantarum ZJUIDS14 exopolysaccharide can significantly reduce weight, liver weight and liver weight ratio increase caused by high fat diet.
The results in fig. 16 show that the lactobacillus plantarum ZJUIDS14 extracellular polysaccharide can significantly reduce the liver TG elevation caused by the high fat diet, the triglyceride content in the liver of the high fat mice is significantly increased relative to the control group, and the triglyceride content in the liver of the hfd+ lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group is significantly reduced relative to the HFD group, so that the lactobacillus plantarum ZJUIDS14 extracellular polysaccharide can effectively reduce the triglyceride content in the liver of the mice.
The results of fig. 17 show that lactobacillus plantarum ZJUIDS14 exopolysaccharide significantly reduced plasma ALT and AST elevation caused by high fat diets.
The results in fig. 18 demonstrate that the triglyceride, free fatty acid and total cholesterol levels were significantly higher in the model group than in the control group. Compared with the model group, the liver tissue triglyceride, free fatty acid and total cholesterol levels of the ZJUIDS14 extracellular polysaccharide group are significantly lower than those of the model group, which indicates that the ZJUIDS14 extracellular polysaccharide of the lactobacillus plantarum can improve the non-alcoholic fatty liver induced by high-fat diet.
The results in fig. 19 show that lactobacillus plantarum ZJUIDS14 extracellular polysaccharide can significantly improve glucose tolerance caused by high-fat diet.
The results in fig. 20 show that lactobacillus plantarum ZJUIDS14 extracellular polysaccharide can significantly improve insulin resistance caused by high-fat diet.
The results show that the lactobacillus plantarum ZJUIDS14 extracellular polysaccharide can effectively reduce the weight, liver fat accumulation and liver injury of a high-fat diet mouse, and improve the glucose tolerance and insulin resistance of the mouse.
Example 4 preparation of functional fermented yoghurt Using Lactobacillus plantarum ZJUIDS14
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 ZJUIDS14 strain is inoculated in a test tube containing sterilized skim milk (12% w/v) under aseptic condition, and cultured at 37deg.C for 20 hr. 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 ZJUIDS14 is inoculated with the inoculation amount of 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 5 preparation of functional fermented fruit and vegetable juice Using Lactobacillus plantarum ZJUIDS14
1. Process flow of fermented fruit and vegetable juice
Raw material cleaning, flash evaporation, pulping, blending, homogenizing, sterilizing, cooling, inoculating, sealing fermentation, after-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 pumpkin: water (weight ratio) =1: 1, gradually grinding pumpkin and water into colloid mill, and performing coarse grinding and fine grinding 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, the activated lactobacillus plantarum ZJUIDS14 is inoculated, and 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 6 preparation of non-alcoholic fatty liver disease alleviating powder Using Lactobacillus plantarum ZJUIDS14
1. Preparation of lactobacillus plantarum ZJUIDS14 bacterial mud
The single colony of the lactobacillus plantarum ZJUIDS14 is selected and inoculated into 50mL of MRS liquid culture medium, and the culture is carried out in a 37 ℃ incubator 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 plantarum ZJUIDS14 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 ZJUIDS14 bacterial sludge.
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 ZJUIDS14 bacterial powder
And fully and uniformly mixing the prepared lactobacillus plantarum ZJUIDS14 bacterial precipitate with a protective agent solution according to the ratio of 1:5. Pre-freezing for 5 hours at the temperature of minus 40 ℃ to uniformly freeze the lactobacillus plantarum ZJUIDS14 bacterial powder on the inner wall of the container, and then performing vacuum freeze drying for 18-20 hours to obtain the lactobacillus plantarum ZJUIDS14 bacterial powder. After rehydration with physiological saline, the lactobacillus plantarum ZJUIDS14 strain powder is washed twice, and the viable count is 1.0x10 11 ～1×10 12 CFU/g。
Example 7 preparation of probiotic milk powder for pets Using Lactobacillus plantarum ZJUIDS14
1. Preparation of lactobacillus plantarum ZJUIDS14 bacterial powder
Preparation of Lactobacillus plantarum ZJUIDS14 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;
automatic batching: throwing the obtained material raw materials into a material bin according to a formula;
crushing: crushing the weighed materials by a crusher;
mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniform mixing;
puffing: making the mixed materials into granular materials through a bulking machine;
and (3) drying: drying the mixed materials by a dryer, wherein the temperature is controlled to be 65-70 ℃;
and (3) classifying and screening: passing the stream through a classifying screen with the particles controlled at 2.5-5 mm;
3. preparation of probiotic formula powder for pets
The bacterial powder prepared in the step 1 and the pet feed prepared in the step 2 are mixed according to the following steps: 100, and the viable bacteria leaving the factory in the final product is 10 8 CFU/g or more. After the product is filled, the product is put in storage for sale.
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 (5)
1. A lactobacillus plantarum ZJUIDS14 with improved non-alcoholic liver disease, characterized in that the classification of lactobacillus plantarum ZJUIDS14 is named: lactiplantibacillus plantarum, accession number: CGMCC No.28091.
2. The use of lactobacillus plantarum ZJUIDS14 according to claim 1 for the preparation of functional foods, characterized in that the functions are protecting liver injury, improving in vivo antioxidant, reducing liver triglycerides, reducing liver fatty acid synthesis, restoring intestinal barrier function, promoting intestinal short chain fatty acid synthesis, improving intestinal flora, and the strain has good probiotic properties and safety.
3. Use of the extracellular polysaccharide ZJUIDS14-EPS produced by lactobacillus plantarum ZJUIDS14 according to claim 1 for the preparation of functional foods, characterized in that the function is protection of liver injury, improvement of in vivo antioxidant and reduction of liver triglycerides.
4. Use according to claim 2 or 3, characterized in that the food product is a bacterial powder, yoghurt or milk powder.
5. Use according to claim 2 or 3, characterized in that the food product comprises a functional fermented yoghurt, a functional fermented fruit and vegetable juice, a non-alcoholic fatty liver bacteria powder relief, a probiotic milk powder for pets.
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