Lactobacillus plantarum ZJUIDS14 with the ability to improve non-alcoholic liver disease and its application

Abstract

The present invention provides Lactobacillus plantarum ZJUIDS14 and its application for improving non-alcoholic liver disease, which is classified and named as: Lactiplantibacillus plantarum, and the preservation number is: CGMCC NO.28091. The strain of the present invention regulates the abundance of various intestinal probiotics in the intestine and promotes the synthesis of intestinal short-chain fatty acids to restore the intestinal barrier function damaged by a high-fat diet. In addition, ZJUIDS14 and the extracellular polysaccharides produced by it can inhibit the liver fatty acid synthesis pathway to reduce liver triglyceride deposition, and improve liver damage through antioxidant pathways. The strain has the functions of protecting liver damage, improving antioxidant effects in the body, reducing liver triglycerides, reducing liver fatty acid synthesis, restoring intestinal barrier function, promoting intestinal short-chain fatty acid synthesis, and improving intestinal flora, and can be used in the preparation of functional foods such as bacterial powder, yogurt or milk powder.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms, and particularly relates to Lactobacillus plantarum ZJUIDS14 capable of improving non-alcoholic liver disease and applications thereof. Background Art

[0002] The incidence of non-alcoholic fatty liver disease (NAFLD) has increased year by year, and is showing a trend of younger age, becoming one of the most common liver diseases in the world. Today, with the increasing incidence of obesity and diabetes, the prevalence of NAFLD in most Asian countries, including China, has exceeded 25%. NAFLD is not only an important global public health issue in the 21st century, but has also become a common chronic liver disease in my country. The disease can further develop into fatty liver hepatitis (NASH), liver fibrosis and cirrhosis, and even liver cancer, seriously endangering people's health. However, there is no safe and effective clinical drug for the treatment of NAFLD so far. How to effectively prevent and treat NAFLD is an urgent problem that clinical medicine needs to solve.

[0003] Lactobacillus plantarum belongs to a type of lactic acid bacteria and has multiple functions. Studies have shown that Lactobacillus plantarum inhibits bacteria and adjusts the diversity of gastrointestinal microorganisms through competition with bacteria for nutrients, forming an ecological barrier. Its metabolites lactic acid and virucoid have the function of inhibiting the reproduction of other harmful bacteria in the gastrointestinal tract, maintaining and ensuring the optimal composition of beneficial bacteria and the stability of this composition, blocking the colonization and invasion of pathogens, inhibiting the growth of pathogens and harmful microbial species and the adhesion of toxins. Lactobacillus plantarum has the excellent effect of reducing blood cholesterol levels and reducing the incidence of cardiovascular diseases. Experiments have confirmed that Lactobacillus plantarum has the ability to reduce substances and blood cholesterol. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus plantarum ZJUIDS14 that improves non-alcoholic fatty liver disease. The classification name of the Lactobacillus plantarum ZJUIDS14 is: Lactiplantibacillus plantarum, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on August 3, 2023, with a deposit number of: CGMCCNO.28091. The 16s rDNA full sequence of Lactobacillus plantarum ZJUIDS14 (Lactiplantibacillus plantarum) provided by the present invention is shown in SEQ ID No.1.

[0005] Another object of the present invention is to provide the use of the Lactobacillus plantarum ZJUIDS14 in the preparation of functional foods.

[0006] Another object of the present invention is to provide the use of the extracellular polysaccharide ZJUIDS14-EPS produced by the plant lactobacillus ZJUIDS14 in the preparation of functional foods, wherein the functions are to improve antioxidant capacity in the body and reduce liver triglycerides.

[0007] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to improve the anti-oxidation in vivo.

[0008] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to reduce liver triglyceride.

[0009] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to reduce the synthesis of fatty acids in the liver.

[0010] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to restore the intestinal barrier function.

[0011] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to promote the synthesis of intestinal short-chain fatty acids.

[0012] The Lactobacillus plantarum ZJUIDS14 provided by the present invention has the ability to improve intestinal flora.

[0013] The food is bacterial powder, yogurt and milk powder.

[0014] The food specifically includes functional fermented yogurt, functional fermented fruit and vegetable juice, and pet probiotic milk powder.

[0015] The plant lactobacillus ZJUIDS14 provided by the present invention regulates the abundance of various intestinal probiotics (blautia, lachnoclostridium, etc.) in the intestine and promotes the synthesis of intestinal short-chain fatty acids to restore the intestinal barrier function damaged by a high-fat diet. In addition, ZJUIDS14 and the exopolysaccharides it produces can inhibit the liver fatty acid synthesis pathway, reduce liver triglyceride deposition, and improve liver damage through an antioxidant pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of Lactobacillus plantarum ZJUIDS14 on body weight, liver weight and liver-to-body ratio of mice

[0017] Figure 2 This is an H&E staining image of mouse liver sections by Lactobacillus plantarum ZJUIDS14 of the present invention.

[0018] Figure 3 The present invention shows the effect of Lactobacillus plantarum ZJUIDS14 on NAFLD activity score (NAS) and triglyceride (TG) in mouse liver.

[0019] Figure 4 The present invention shows the influence of Lactobacillus plantarum ZJUIDS14 on aspartate aminotransferase and alanine aminotransferase (AST and ALT) in mouse plasma.

[0020] Figure 5 The invention discloses the effect of Lactobacillus plantarum ZJUIDS14 on triglyceride (TG), free fatty acid (FFA), total cholesterol (TC), high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in mouse plasma.

[0021] Figure 6 The present invention shows the influence of Lactobacillus plantarum ZJUIDS14 on the glucose tolerance level of mice.

[0022] Figure 7 The invention discloses the effect of the Lactobacillus plantarum ZJUIDS14 on the insulin resistance level of mice.

[0023] Figure 8 The present invention shows the effect of Lactobacillus plantarum ZJUIDS14 on malondialdehyde (MDA) and total superoxide dismutase (T-SOD) in mouse liver.

[0024] Fig. 9 The invention discloses the effects of Lactobacillus plantarum ZJUIDS14 on lipid synthesis genes (FATP2, FABP2 and CD36), inflammation-related genes (IL-1β and TNFα) and antimicrobial peptide genes (GRAMP) in the intestine of mice.

[0025] Fig.10 The present invention shows the influence of Lactobacillus plantarum ZJUIDS14 on tight junction proteins (ZO-1 and Claudin-1) in the intestine of mice.

[0026] Fig.11 This is the effect of the plant lactobacillus ZJUIDS14 of the present invention on short-chain fatty acids (acetic acid, propionic acid and butyric acid) in mouse feces.

[0027] Fig.12 This is the effect of the plant lactobacillus ZJUIDS14 of the present invention on the α diversity and β diversity of the intestinal flora of mice.

[0028] Fig.13 This is the effect of the Lactobacillus plantarum ZJUIDS14 of the present invention on the abundance of the phylum level of the intestinal flora of mice.

[0029] Fig.14 This is the effect of the Lactobacillus plantarum ZJUIDS14 of the present invention on the abundance of differential strains at the genus level in the intestinal flora of mice.

[0030] Fig.15The invention discloses the effects of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on the body weight, liver weight and liver-to-body ratio of mice.

[0031] Fig.16 The present invention shows the effect of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on triglyceride (TG) in mouse liver.

[0032] Fig.17 The invention discloses the influence of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on aspartate aminotransferase and alanine aminotransferase (AST and ALT) in mouse plasma.

[0033] Fig.18 The invention discloses the influence of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on triglyceride (TG), free fatty acid (FFA), total cholesterol (TC), high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) in mouse plasma.

[0034] Fig.19 The invention discloses the influence of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on the glucose tolerance level of mice.

[0035] Fig. 20 The invention discloses the influence of the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 on the insulin resistance level of mice.

[0036] Note: Figure 1-14 There were three groups: control diet group (NFD group), 45% high-fat diet group (HFD group), and 45% high-fat diet + Lactobacillus plantarum ZJUIDS14 group (HFD+ZJUIDS14 group).

[0037] Note: Figure 15-20 There were three groups: control feed group (NFD group), 45% high-fat feed group (HFD group), and 45% high-fat feed + Lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group (HFD+ZJUIDS14-EPS group). DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0039] Example 1 Screening and identification of Lactobacillus plantarum ZJUIDS14:

[0040] 1. Screening of Lactobacillus plantarum ZJUIDS14

[0041] 1.1 Sample source

[0042] The strain used in the present invention is isolated from feces of healthy breast-fed infants in Hangzhou.

[0043] 1.2 Isolation and purification of strains

[0044] Take about 5g of fresh fecal sample and collect it with a sterile tube, and immediately send it to the laboratory for strain isolation. Take 1g of sample and put it into 9mL of MRS broth medium, vortex and mix it, and then enrich and culture it at 37℃ for 48h; then take 1mL of enrichment solution in the clean bench, dilute it tenfold with sterile saline, select three dilution gradients of 10-6, 10-7, and 10-8, and take 100μL of bacterial solution from each gradient and apply it on MRS agar medium, and culture it at 37℃ for 48h. After the culture is completed, select a plate with 50-150 single colonies from the agar medium, pick a typical colony, and purify it by streaking it on the MRS agar plate several times until the colony morphology on the entire plate is consistent, and pick a single colony to MRS broth medium for bacterial enrichment culture. The obtained strains are all frozen at -80℃ in MRS broth medium containing 40% glycerol.

[0045] 2. Identification of Lactobacillus plantarum ZJUIDS14

[0046] 2.1 Colony characteristics

[0047] After culturing Lactobacillus plantarum ZJUIDS14 on MRS agar medium for 48 hours, the colonies were round in diameter with neat white edges and a moist and smooth surface, ranging from 0.3 to 1.5 mm.

[0048] 2.2 Morphology under microscope:

[0049] Colony smear of Lactobacillus plantarum ZJUIDS14: Gram stain positive, non-spore-forming, round-ended straight rods, single, paired or in short chains.

[0050] 2.3 16S rDNA identification

[0051] The target strain genomic DNA was extracted with an Ezup column bacterial genomic DNA extraction kit, and the extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. The PCR experiment of 16S rDNA was performed using bacterial universal primers 27F and 1492R. After the PCR reaction amplification was completed, the PCR product was taken for agarose gel detection and photography, and the amplified fragment length was about 1.2kbp. The PCR product was sent to Huada Gene Co., Ltd. for sequencing, and the result was shown in SEQ ID NO.1. The BLAST sequence comparison was performed on the NCBI website, and the result showed that the sequence had a homology of more than 99% with the 16S rDNA sequence identified by plant lactobacillus. The sequence comparison results of the strain plant lactobacillus ZJUIDS14 were combined with the physiological and biochemical results to determine that the screened lactic acid bacteria plant lactobacillus ZJUIDS14 was plant lactobacillus.

[0052] Example 2 Lactobacillus plantarum ZJUIDS14 improves non-alcoholic liver disease:

[0053] 1. Experimental animals: 32 C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center, company license number: SCXK (Shanghai) 2013-0016, and were kept in the Experimental Animal Center of Zhejiang Chinese Medical University in an SPF environment.

[0054] 2. Reagents: ALT kit (Cat. No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Cat. No.: C010-2 Nanjing Jiancheng Bioengineering Institute), free fatty acid kit (Cat. No.: A042-2-1 Nanjing Jiancheng Bioengineering Institute), tissue triglyceride kit (Cat. No.: E1013 Beijing Prilai Gene Technology Co., Ltd.), malondialdehyde assay kit (Cat. No.: A003-1 Nanjing Jiancheng Bioengineering Institute), total superoxide dismutase assay kit (Cat. No.: A001-1 Nanjing Jiancheng Bioengineering Institute).

[0055] 3. Animal husbandry

[0056] After one week of adaptation feeding in an SPF-level animal laboratory, C57BL / 6 mice at age of 8 weeks were randomly divided into 3 groups, with 8 mice in each group, namely, control feed group (NFD group), 45% high-fat feed group (HFD group), and 45% high-fat feed + Lactobacillus plantarum ZJUIDS14 group (HFD+ZJUIDS14 group).

[0057] The NFD group was fed with a control diet for 12 weeks, and the HFD group and HFD+Lactobacillus plantarum ZJUIDS14 group were fed with a 45% high-fat diet for 12 weeks. During this period, the HFD+Lactobacillus plantarum ZJUIDS14 group was gavaged once every other day with 0.2 mL of Lactobacillus plantarum ZJUIDS14 (10 9 CFU / unit, the solvent was normal saline).

[0058] The feed consumed by the mice was replaced with new feed every two days, and the weight of the mice was recorded weekly to detect changes. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneally, blood was drawn from the inferior vena cava to measure ALT, AST and FFA, and liver and intestinal tissues of the mice were taken for measurement of relevant indicators.

[0059] 4. Index determination

[0060] 4.1 Plasma ALT detection

[0061] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the determination wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0062] 4.2 Plasma AST detection

[0063] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the determination wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0064] 4.3 Plasma FFA detection

[0065] Add 4 μL of double distilled water to the blank well, 4 μL of standard to the calibration well, and 4 μL of sample to the sample well. Add 200 μL of reagent 1 to the three wells. Mix well, incubate at 37°C for 5 min, read the absorbance value A1, add 50 μL of reagent 2 to the three wells, mix well, incubate at 37°C for 5 min, read the absorbance value A2, calculate the value of A2-A1, and use two-point calibration calculation.

[0066] 4.4 H&E staining of liver

[0067] Fresh liver tissue of animals was fixed with 4% paraformaldehyde, dehydrated with graded alcohol after fixation, and paraffin embedded after permeabilization with xylene. The embedded paraffin was sliced, and 4 μm slices were taken for H&E staining, dewaxed with xylene, and gradually dehydrated with ethanol: xylene (I) 5min; xylene (II) 5min; 100% ethanol 2min; 95% ethanol 1min; 80% ethanol 1min; 75% ethanol 1min; washed with distilled water for 2min. Hematoxylin staining for 5min, rinsed with water, differentiated with hydrochloric acid ethanol for 30s, soaked in water for 15min, and placed in eosin solution for 2-3min. Conventional dehydration, transparent, and sealing: 95% ethanol (I) 30s; 95% ethanol (II) 30s; 100% ethanol (I) 30s; 100% ethanol (II) 1min; xylene 15min; neutral resin sealing.

[0068] 4.5 Glucose tolerance test

[0069] After fasting for 12 h, mice were intraperitoneally injected with 2.5 g / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 min after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0070] 4.6 Insulin resistance testing

[0071] After 4 hours of fasting, the mice were intraperitoneally injected with 0.75 U / kg of glucose solution according to their body weight. Blood samples were collected at 0, 30, 60, and 120 minutes after the intraperitoneal injection, and the glucose content in the blood was measured using a blood glucose meter.

[0072] 4.7 Detection of liver TG

[0073] Weigh 50 mg of liver accurately and add lysate at a ratio of 1 mg liver to 20 μL lysate. Use an automatic sample rapid grinder to break up the tissue, then let it stand for 10 minutes, take an appropriate amount of supernatant and transfer it to a 1.5 mL centrifuge tube, and perform the following steps. The remaining lysate can be used for protein quantification by the BCA method. Heat the supernatant in a 70°C metal bath for 10 minutes. Centrifuge at room temperature for 5 minutes at 2000 rpm, and take the supernatant for TG determination. Take 10 μL of supernatant in a 96-well plate, and dilute the 4 mM glycerol standard to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μmol / L, and take 10 μL of each dilution in a 96-well plate, and prepare the working solution according to the instructions of the Beijing Prilai Liquid TG Kit. Add 190 μL of working solution to the sample, incubate at 37°C for 15 minutes, and measure the OD value at a working wavelength of 550 nm.

[0074] 4.8 Malondialdehyde (MDA) detection

[0075] Accurately weigh the liver tissue, add 9 times the volume of physiological saline according to the ratio of weight (g): volume (mL) = 1:9, cut the tissue into pieces, prepare homogenate in an ice water bath, centrifuge at 3000 rpm for 10 min, and take the supernatant, i.e. 10% homogenate supernatant for testing. Add 0.1 mL of anhydrous ethanol to the blank tube, add 0.1 mL of 10 nmol / mL standard to the standard tube, add 0.1 mL of test sample to the assay tube and control tube, add 0.1 mL of reagent 1 to each of the four tubes, mix well, add 3 mL of reagent 2 application solution to the four tubes, add 1 mL of reagent 3 application solution to the blank tube, standard tube, and assay tube, and add 1 mL of 50% glacial acetic acid to the control tube. Mix well, incubate at 95℃ for 40 min, take out and cool, then centrifuge at 3500-4000 r / min for 10 min to take the supernatant, and measure the OD value at 532 nm. Calculate the MDA content between each group according to the calculation formula.

[0076] 4.9 Detection of superoxide dismutase (T-SOD)

[0077] Take 10% liver homogenate supernatant for testing. Add 1mL of reagent 1 application solution to two tubes, add 0.05mL of sample to the test tube, add 0.05mL of distilled water to the control tube, add 0.1mL of reagent 2, reagent 3, and reagent 4 application solution to the two tubes respectively, mix thoroughly with a vortex mixer, incubate at 37℃ for 40min, and add 2mL of color developer to the two tubes respectively. Mix well, leave at room temperature for 10min, and measure the OD value at a wavelength of 550nm.

[0078] 4.10 Western blotting

[0079] Add an appropriate amount of RIPA buffer supplemented with protease and phosphatase inhibitors to the liver tissue or cells for disruption and lysis, centrifuge at 12000rpm at 4℃ for 15min, take the supernatant to determine the protein concentration and prepare the protein sample so that the protein content contained in each milliliter of sample is equal. The protein sample was loaded into the SDS-PAGE gel and transferred to the PVDF membrane. 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 at room temperature for 1 hour. Finally, the immunoreactivity of protein expression was observed with a chemiluminescence kit. Immunoblotting was quantified by measuring the density of each band with Image-J.

[0080] 4.11 Real-time PCR

[0081] (1) Take out the tissue from the -80°C freezer and place it in an ice box. Use an electronic balance to weigh about 0.02 g of tissue into an EP tube and precool the centrifuge at 4°C.

[0082] (2) Add 1 mL of Trizol and 3 steel beads to the EP tube containing the tissue block, grind with a grinder, remove the liquid, pour out the steel beads, and react at room temperature for 10 min;

[0083] (3) Add 200 μL of chloroform to the EP tube, shake vigorously for 30 seconds, and place on ice for 5-10 minutes;

[0084] (4) After standing, place the centrifuge tube in a 4°C centrifuge at 12,000 rpm for 15 min;

[0085] (5) Pipette the aqueous phase (supernatant) of the centrifuged sample into a new 1.5 mL centrifuge tube;

[0086] (6) Add an equal volume of isopropanol solution to the centrifuged solution, gently invert to mix, and let stand at -20°C for 20 min;

[0087] (7) Take out the sample from -20°C and centrifuge it at 12000 rpm and 4°C for 15 min;

[0088] (8) After centrifugation, discard the supernatant to obtain a white (or colorless and transparent) precipitate. Add 100-300 μL of pre-cooled 75% ethanol prepared with DEPC water along the inner wall of the centrifuge tube and wash 2-3 times.

[0089] (9) After discarding the liquid, air-dry at room temperature for about 15 min, add 20-50 μL of pre-cooled DEPC water to the centrifuge tube to dissolve the air-dried precipitate (RNA) at the bottom, and store in a -20°C refrigerator for later use;

[0090] (10) Use an ultra-micro UV spectrophotometer to measure the RNA concentration, record the results and calculate the sample loading amount for each group;

[0091] (11) As shown in Table 1, add the corresponding reaction solution in the reverse transcription kit to premix, add the sample for reverse transcription, shake and centrifuge, place in the PCR instrument, and set the corresponding PCR reaction conditions according to the reverse transcription kit. Store the cDNA sample reverse transcribed by the reverse transcription instrument in a -20°C refrigerator for later use;

[0092] (12) Use a 0.2 mL fluorescent quantitative PCR eight-tube concatenation, add the reactants, use cDNA as a template, and use a fluorescent quantitative PCR amplification instrument to amplify the experimental target gene and perform fluorescent quantitative PCR determination;

[0093] (13) The mixed reactants were shaken and mixed, and after centrifugation, the eight-tube strip was placed in a qRT-PCR reactor. The PCR reaction program was set as follows: pre-denaturation: 94°C, 5 min; denaturation: 94°C, 30 s, 60°C, 30 s, 72°C, 30 s, 40 cycles; extension: 72°C, 5 min, stored at 4°C;

[0094] (14) The expression changes of the target genes were calculated using the 2–ΔΔCT method.

[0095] 4.12 Determination of short-chain fatty acids

[0096] The segmented colon slices were squeezed with sterile forceps, and the contents were removed and stored in -80°C cryopreservation tubes. The colon contents were diluted fivefold with ultrapure water and vortexed for 3 minutes. Next, the suspension was allowed to stand for 5 minutes and then centrifuged at 4°C, 5000×g for 20 minutes. One milliliter of the supernatant was mixed with 20 μL of chromatography-grade phosphoric acid, and the mixture was injected into a chromatographic bottle through a 0.45 μm membrane filter for gas chromatography analysis. The gas chromatograph consisted of an AOC-20S automatic sampler and a GC-2010 equipped with a flame ionization detector. Nitrogen was used as the carrier gas with a flow rate of 3 ml / min. An SH-stabilized wax high-polarity column was installed on the gas chromatograph, the sample injection volume was 0.2 μL, the split injection ratio was 50, and the injection temperature was 200°C. Ethyl acetate was injected between each sample as a blank solvent to eliminate any memory effects. The initial column temperature was set at 80 °C and maintained for 1 min, then increased to 170 °C at a rate of 8 °C / min, and then immediately increased to 220 °C at a rate of 20 °C / min and maintained for 4 min. The total time was 18.75 min. Finally, the SCFAs content was calculated according to the SCFA standard curve using the external standard method.

[0097] 4.13 16S rRNA sequencing analysis of intestinal flora

[0098] The collected colon content samples were subjected to total DNA isolation and 16s rRNA high-throughput sequencing technology by Hangzhou Mingke Biotechnology. 16s rRNA was amplified in the V3-V4 region, the amplicon was purified with the QIA fast PCR purification kit, and the sequencing was performed by the Illumina Novaseq platform (PE300), and the original sequence was quality controlled by UPARSE. Operational taxonomic units (OTUs) were constructed by binding sequences into clusters with sequence similarity greater than 97% using QIIME.

[0099] 5. Experimental results:

[0100] Figure 1 The results show that Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase in body weight, liver weight and liver weight ratio caused by a high-fat diet.

[0101] Figure 2 The results showed that Lactobacillus plantarum ZJUIDS14 can significantly improve the pathological damage process of the liver caused by a high-fat diet. H&E staining of the model group showed that lipid droplets were significantly aggregated in hepatocytes, while ZJUIDS14 staining showed fewer lipid droplets in hepatocytes, improving the pathological state of the liver.

[0102] Figure 3 The results showed that Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase of liver NAS and TG caused by a high-fat diet. The triglyceride content in the liver of mice in the 45% high-fat diet group (HFD) was significantly increased compared with the control group (NFD), and NAS was significantly increased, while the triglyceride content and NAS in the liver of the HFD+Lactobacillus plantarum ZJUIDS14 group were significantly reduced compared with the HFD group. Therefore, it is judged that Lactobacillus plantarum ZJUIDS14 can effectively reduce the triglyceride content and NAS in the liver of mice.

[0103] Figure 4 The results show that Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase of ALT and AST caused by a high-fat diet. ALT, or alanine aminotransferase, is mainly present in the cytoplasm of liver cells. The intracellular concentration is 1000-3000 times higher than that in serum. As long as 1% of liver cells are destroyed, the serum enzyme can be doubled. Therefore, alanine aminotransferase is recommended by the World Health Organization as the most sensitive detection indicator of liver function damage. A decrease in ALT means a decrease in liver damage. AST, or aspartate aminotransferase, is also called aspartate aminotransferase. AST is mainly distributed in the mitochondria of liver cells and is also one of the sensitive indicators of liver cell damage.

[0104] Figure 5 The results showed that the levels of triglycerides, free fatty acids and total cholesterol in the model group were significantly higher than those in the control group, indicating that the rats in the model group had developed non-alcoholic fatty liver. Compared with the model group, the levels of triglycerides, free fatty acids and total cholesterol in the liver tissue of the ZJUIDS14 experimental group were significantly lower than those in the model group, indicating that Lactobacillus plantarum ZJUIDS14 can alleviate non-alcoholic fatty liver. Free fatty acids (FFA) are substances decomposed from triglycerides. Under normal circumstances, the content in plasma is very small. The increase in free fatty acids will change the permeability of the mucosa and cause damage to the mucosa. In addition, the excessive intake of free fatty acids by the liver exceeds the oxidation of fatty acids by the liver mitochondria, which will promote the increase of triglycerides and aggravate the formation of fatty liver.

[0105] Figure 6 The results showed that Lactobacillus plantarum ZJUIDS14 could significantly improve glucose tolerance caused by a high-fat diet.

[0106] Figure 7The results showed that Lactobacillus plantarum ZJUIDS14 could significantly improve the insulin resistance caused by a high-fat diet.

[0107] Figure 8 The results showed that Lactobacillus plantarum ZJUIDS14 could significantly reduce the elevated MDA levels in the liver caused by a high-fat diet and increase the T-SOD levels. Under normal circumstances, the antioxidant enzymes in the natural antioxidant defense system of the organism can work synergistically with antioxidants in the diet or drugs to remove peroxides. SOD is one of the most important antioxidant enzymes, responsible for the dismutation of superoxide anions into hydrogen peroxide. On the other hand, the level of lipid peroxidation product MDA is detected to indirectly determine the severity of free radical attack on cells.

[0108] Fig. 9 The results showed that Lactobacillus plantarum could significantly improve intestinal lipid metabolism and inflammation levels. Lipid synthesis genes (FATP2, FABP2 and CD36) play an important role in lipid synthesis. When these genes are increased, the body's lipid synthesis increases. Inflammatory factors IL-1β and TNFα can reflect the body's inflammation level. Compared with the common feed omics, high-fat diet increased the levels of lipid synthesis genes (FATP2, FABP2 and CD36), inflammation-related genes (IL-1β and TNFα) and antimicrobial peptide genes (GRAMP), while Lactobacillus plantarum ZJUIDS14 significantly reduced the levels of these genes, indicating that Lactobacillus plantarum ZJUIDS14 can improve the increase in lipid synthesis and inflammation caused by high-fat diet.

[0109] Fig.10 The results showed that Lactobacillus plantarum ZJUIDS14 has the ability to improve the intestinal mucosal barrier. Tight junctions are the main connection between intestinal epithelial cells and play an important role in maintaining the mechanical barrier and permeability of the intestinal mucosal epithelium. Tight junction proteins are important protein molecules that constitute the intestinal mucosal barrier and determine the permeability of the intestinal wall. They have a great influence on the composition and function of tight junctions. Among them, ZO-1 and Claudin-1 are important factors that constitute tight junctions between cells. Lactobacillus plantarum ZJUIDS14 intervention significantly restored the decrease in gene expression of ZO-1 and Claudin-1 induced by a high-fat diet.

[0110] Fig.11The results showed that Lactobacillus plantarum ZJUIDS14 has the ability to promote the synthesis of short-chain fatty acids in the intestine. SCFA is the main metabolite produced by intestinal microbial fermentation. SCFA, as the final product of its metabolism, maintains the redox equivalents in the aerobic environment of the intestine. SCFA is a saturated fatty acid with 1-6 carbon atoms. The most abundant SCFA (~95%) are acetic acid (C2), propionic acid (C3) and butyric acid (C4). Intervention with Lactobacillus plantarum ZJUIDS14 significantly increased the content of acetic acid, propionic acid and butyric acid in the intestine.

[0111] Fig.12 The results showed that Lactobacillus plantarum ZJUIDS14 has the function of restoring the abundance of intestinal flora. The chao1 index reflects the abundance of intestinal flora. The higher the index, the higher the abundance of intestinal flora. Compared with the ordinary feed group, the high-fat diet reduced the abundance of intestinal flora in mice, while Lactobacillus plantarum ZJUIDS14 significantly increased the chao1 index, indicating that Lactobacillus plantarum ZJUIDS14 can restore the abundance of intestinal flora.

[0112] Fig.13 The results showed that Lactobacillus plantarum ZJUIDS14 has the function of regulating intestinal flora. At the phylum level, Lactobacillus plantarum ZJUIDS14 can significantly increase the abundance of Proteobacteria and Cyanobacteria, and reduce the abundance of Actinobacteria.

[0113] Fig.14 The results showed that Lactobacillus plantarum ZJUIDS14 has the function of regulating intestinal flora. At the genus level, Lactobacillus plantarum ZJUIDS14 can significantly increase the abundance of Coprostanoligenes group, Ruminococcaceae UCG-014, Allobaculum, and Ruminiclostridium 1, and reduce the abundance of Roseburia.

[0114] The above results show that Lactobacillus plantarum ZJUIDS14 can effectively reduce the body weight, liver weight, liver fat accumulation and liver damage of mice fed a high-fat diet, and improve the glucose metabolism and lipid metabolism of mice. In addition, Lactobacillus plantarum ZJUIDS14 can also improve the intestinal mucosal barrier, promote the synthesis of intestinal short-chain fatty acids, and regulate intestinal flora.

[0115] Example 3: Extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 improves non-alcoholic liver disease:

[0116] 1. Isolation and purification of extracellular polysaccharides from Lactobacillus plantarum ZJUIDS14

[0117] Lactobacillus plantarum ZJUIDS14 was cultured in MRS medium at 37°C for 24 hours. The cells were removed by centrifugation (8,000×g, 4°C, 15min) 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°C, and the precipitated protein was separated by centrifugation (12,000×g, 4°C, 15). After separating the protein, 3 volumes of ethanol were added to the supernatant to precipitate EPS. After incubation for 12 hours, EPS was collected by centrifugation (12,000×g, 15min). The precipitate was dissolved in deionized water, then dialyzed (MW cut-off 3,500Da) and lyophilized. Using ToxinSensor TM The endotoxin concentration of the purified exopolysaccharide was detected by a chromogenic horseshoe crab kit (Genscript), and endotoxin-free exopolysaccharide was used for the following experiments.

[0118] 2. Experimental animals: 24 C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center, company license number: SCXK(Shanghai)2013-0016, and were kept in the Experimental Animal Center of Zhejiang Chinese Medical University in an SPF environment.

[0119] 3. Animal husbandry

[0120] After one week of adaptation feeding in an SPF-level animal laboratory, C57BL / 6 mice at age of 8 weeks were randomly divided into three groups, with 8 mice in each group, namely, control feed group (NFD group), 45% high-fat feed group (HFD group), and 45% high-fat feed + Lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group (HFD+ZJUIDS14-EPS group).

[0121] The NFD group was fed with a control diet for 12 weeks, and the HFD group and the HFD+Lactobacillus plantarum ZJUIDS14-EPS group were fed with a 45% high-fat diet for 12 weeks. During this period, the HFD+Lactobacillus plantarum ZJUIDS14 group was gavaged with 0.2 mL of ZJUIDS14 exopolysaccharide (80 mg / kg, the solvent was normal saline) every other day.

[0122] The feed consumed by mice was replaced with new feed every two days, and the weight of mice was recorded every week to detect changes. After feeding, mice were anesthetized with 1% pentobarbital intraperitoneal injection, blood was drawn from the inferior vena cava to measure ALT, AST and FFA, and liver and intestinal tissues of mice were taken for determination of relevant indicators.

[0123] 4. Experimental method: Same as Example 3.

[0124] Experimental Results

[0125] Fig.15The results show that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase in body weight, liver weight and liver weight ratio caused by a high-fat diet.

[0126] Fig.16 The results showed that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase of liver TG caused by a high-fat diet. The triglyceride content in the liver of mice in the high-fat group was significantly increased compared with the control group, while the triglyceride content in the liver of the HFD+Lactobacillus plantarum ZJUIDS14 extracellular polysaccharide group was significantly decreased compared with the HFD group. Therefore, it is judged that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 can effectively reduce the triglyceride content in the liver of mice.

[0127] Fig.17 The results show that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 can significantly reduce the increase of plasma ALT and AST caused by a high-fat diet.

[0128] Fig.18 The results showed that the levels of triglycerides, free fatty acids and total cholesterol in the model group were significantly higher than those in the control group. Compared with the model group, the levels of triglycerides, free fatty acids and total cholesterol in the liver tissue of the ZJUIDS14 exopolysaccharide group were significantly lower than those in the model group, indicating that the exopolysaccharide of Lactobacillus plantarum ZJUIDS14 can improve non-alcoholic fatty liver induced by a high-fat diet.

[0129] Fig.19 The results showed that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 could significantly improve the glucose tolerance caused by high-fat diet.

[0130] Fig. 20 The results showed that the extracellular polysaccharide of Lactobacillus plantarum ZJUIDS14 can significantly improve the insulin resistance caused by a high-fat diet.

[0131] The above results indicate that the extracellular polysaccharides of Lactobacillus plantarum ZJUIDS14 can effectively reduce the body weight, liver weight, liver fat accumulation and liver damage of mice fed a high-fat diet, and improve the glucose tolerance and insulin resistance of mice.

[0132] Example 4 Preparation of functional fermented yogurt using Lactobacillus plantarum ZJUIDS14

[0133] 1. Yogurt processing technology:

[0134] Raw materials → preheating → homogenization → mixing → sterilization → cooling → preparation and inoculation → fermentation → post-ripening → refrigeration

[0135] 2. Operation points

[0136] (1) Raw materials: 2L full fat UHT sterilized milk or fresh milk;

[0137] (2) Preheating: Place in a container and heat to 63°C;

[0138] (3) Homogenization: Pour the mixture into a homogenizer and homogenize at a pressure of 15-25 MPa), pour the mixture into an iron can, add 100 g of sugar, and sterilize in a 90°C water bath for 10 min;

[0139] (4) Preparation: Add ingredients to milk and dissolve;

[0140] (5) Sterilization: Sterilize the sweetened milk in a water bath at 90°C for 10 min;

[0141] (6) Cooling: After sterilization, the milk is cooled to 40-50°C for later use;

[0142] (7) Preparation of starter culture: Lactobacillus plantarum ZJUIDS14 was inoculated into a test tube containing sterilized skim milk (12%, w / v) under a sterile environment and cultured at 37°C for 20 hours. The inoculation volume for each subculture was 2-4% (v / v), and the culture was subcultured 2-3 times to restore vitality and stored in a refrigerator at 4°C;

[0143] (8) Inoculation and fermentation: Under sterile conditions, inoculate the activated Lactobacillus plantarum ZJUIDS14 at an inoculation rate of 2-4% (v / v). Ferment at a constant temperature of 42°C for 6-10 hours;

[0144] (9) Post-ripening: After fermentation, place in a refrigerator at 4°C for 12-24 hours;

[0145] (10) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

[0146] Example 5 Preparation of functional fermented fruit and vegetable juice using Lactobacillus plantarum ZJUIDS14

[0147] 1. Processing process of fermented fruit and vegetable juice

[0148] Raw materials → cleaning → flash steaming → beating → mixing → homogenization → sterilization →

[0149] Cooling → inoculation → closed fermentation → post-ripening → filling → refrigeration

[0150] 2. Operation points

[0151] (1) Raw materials: fresh pumpkin and dragon fruit;

[0152] (2) Cleaning and cutting: Clean, peel (remove the pumpkin pulp), and cut into small pieces;

[0153] (3) Flash evaporation: The enzyme is inactivated by flash evaporation for 0.5 to 1 min at 121°C and the exhaust is rapid;

[0154] (4) Pulping: According to the ratio of pumpkin to water (weight ratio) = 1:1, gradually put the pumpkin and water into a colloid mill and grind them, performing coarse grinding and fine grinding once each. Pulp the dragon fruit with a pulper until the pulp is uniform and free of lumps;

[0155] (5) Mixing and homogenizing: 15% pumpkin juice and 30% pitaya juice are added, and the soluble solid content is adjusted to 10° Brix with sucrose. 0.2% stabilizer CMC is added and mixed evenly. A two-stage homogenization method is used, first low pressure (15 MPa) and then high pressure (25 MPa), so that the diameter of the melon pulp particles is 2 to 3 μm;

[0156] (6) Sterilization and cooling: The prepared composite fruit and vegetable juice is kept at 100°C for 10 min and then cooled to about 40°C;

[0157] (7) Inoculation and fermentation: Under sterile conditions, inoculate the activated Lactobacillus plantarum ZJUIDS14, and control the initial bacterial count to 10 7 CFU / mL. Fermented at 37℃ for 24h;

[0158] (8) Post-ripening: After fermentation, place in a 4°C refrigerator for 3 h;

[0159] (9) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

[0160] Example 6: Preparation of bacterial powder for alleviating non-alcoholic fatty liver disease using Lactobacillus plantarum ZJUIDS14

[0161] 1. Preparation of Lactobacillus plantarum ZJUIDS14 bacterial slurry

[0162] Pick a single colony of plant lactobacillus ZJUIDS14 and inoculate it in 50mL MRS liquid culture medium, and place it in a 37°C incubator to cultivate for 18h. Activate it again in 250mL MRS liquid culture medium at 5% inoculum, and place it in a 37°C incubator to cultivate for 24h. Finally, the activated plant lactobacillus ZJUIDS14 is cultured in a 10L fermenter at a high density anaerobic culture with a 5% inoculum, and cultivated for 18h at 37°C and pH 6.8. Afterwards, centrifuge for 15min at 8000r / min and 4°C, discard the supernatant, collect the bacterial precipitation, and rinse the bacterial body twice with sterile phosphate buffer (pH 7.0). You can get plant lactobacillus ZJUIDS14 bacterial mud.

[0163] 2. Preparation of Protective Agent

[0164] The freeze-dried protective agent contains 15% skim milk powder, 5% trehalose, 3% sodium glutamate, 1% glycerol, and 0.5% cysteine ​​hydrochloride. Water is used as the solvent. Sterilize at 110°C for use.

[0165] 3. Preparation of Lactobacillus plantarum ZJUIDS14 powder

[0166] The prepared Lactobacillus plantarum ZJUIDS14 bacterial precipitate was thoroughly mixed with the protective agent solution at a ratio of 1:5. It was pre-frozen at -40°C for 5 hours to freeze evenly on the inner wall of the container, and then vacuum-freeze-dried for 18 to 20 hours to obtain Lactobacillus plantarum ZJUIDS14 bacterial powder. After rehydration with physiological saline, it was washed twice, and the number of viable bacteria in the Lactobacillus plantarum ZJUIDS14 bacterial powder was measured to be 1.0×10 11 ~1×10 12 CFU / g.

[0167] Example 7 Preparation of pet probiotic milk powder using Lactobacillus plantarum ZJUIDS14

[0168] 1. Preparation of Lactobacillus plantarum ZJUIDS14 powder

[0169] Referring to Example 5, freeze-dried powder of Lactobacillus plantarum ZJUIDS14 was prepared, and the number of viable bacteria in the powder was 1.0×10 11 ~1×10 12 CFU / g.

[0170] 2. Preparation of pet formula powder

[0171] Primary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil, additives: vitamins, trace elements, functional factors, others;

[0172] Automatic batching: put the obtained raw materials into the material bin according to the formula;

[0173] Crushing: crush the weighed materials through a crusher;

[0174] Mixing: Add vegetable oil and trace elements to the crushed materials and put them into the mixer to mix evenly;

[0175] Puffing: The mixed materials are made into granular materials through the puffing machine;

[0176] Drying: The mixed materials are dried in a dryer with the temperature controlled at 65-70 degrees;

[0177] Grading screening: The logistics is passed through the grading screen, and the particles are controlled at 2.5-5 mm;

[0178] 3. Preparation of pet probiotic formula powder

[0179] The bacterial powder prepared in 1 and the pet feed prepared in 2 are mixed evenly in a ratio of 1:100. The live bacteria in the final product are 8 CFU / g or above. After filling, the product is stored in the warehouse for sale.

[0180] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ZJUIDS14 that improves non-alcoholic liver disease, characterized in that: The classification name of the plant lactobacillus ZJUIDS14 is: Lactiplantibacillus plantarum , deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC NO. 28091.

2. The use of Lactobacillus plantarum ZJUIDS14 according to claim 1 in preparing functional foods, characterized in that: The functions are to improve antioxidant capacity in the body, reduce liver triglycerides, reduce liver fatty acid synthesis, restore intestinal barrier function, promote intestinal short-chain fatty acid synthesis, improve intestinal flora, and the strain has good probiotic properties and safety.

3. The use of the exopolysaccharide ZJUIDS14-EPS produced by Lactobacillus plantarum ZJUIDS14 according to claim 1 in the preparation of functional foods, characterized in that: The functions are improving antioxidant activity in the body and lowering liver triglycerides.

4. The use according to claim 2 or 3, characterized in that: The food is bacterial powder, yogurt or milk powder.

5. The use according to claim 2 or 3, characterized in that: The food includes functional fermented yogurt, functional fermented fruit and vegetable juice, and pet probiotic milk powder.

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