CN108641988A - Lactobacillus plantarum NA136 and its application in alleviating non-alcoholic fatty liver disease - Google Patents

Abstract

The invention discloses plant lactobacillus NA136 and its application in alleviating non-alcoholic fatty liver disease. The present invention firstly provides Lactobacillus plantarum (Lactobacillus plantarum) NA136, the preservation number of which is CCTCC NO: M 2018112. The invention also protects the application of the plantarum lactobacillus NA136 in the preparation of products for treating and/or preventing non-alcoholic fatty liver. The inventors of the present invention found that Lactobacillus plantarum NA136 helps to reduce the occurrence of inflammation in the liver, and has outstanding effects on alleviating non-alcoholic fatty liver disease. Daily intake of Lactobacillus plantarum NA136 or its preparations can replace drugs to protect the integrity of liver function. The invention provides a new idea for clinical intervention and control of non-alcoholic fatty liver disease and other chronic liver diseases, and has broad application prospects and promotion value.

Description

Lactobacillus plantarum NA136 and its application in alleviating non-alcoholic fatty liver disease

technical field

The invention belongs to the field of biotechnology, in particular to plant lactobacillus NA136 and its application in alleviating non-alcoholic fatty liver disease.

Background technique

Non-alcoholic fatty liver disease (NAFLD) refers to liver pathology caused by alcohol and other clear liver damage factors, and is an acquired metabolic stress liver disease closely related to insulin resistance and genetic susceptibility. damage.

The main feature of non-alcoholic fatty liver disease is the accumulation of triglycerides in the liver. Excessive accumulation of triglycerides contributes to obesity and some metabolic syndromes.

From the initial non-alcoholic simple fatty liver, NAFLD can develop into non-alcoholic steatohepatitis (NASH), and even progress to later stages of liver fibrosis, liver cirrhosis and liver cancer.

NAFLD is associated with small intestinal bacterial overgrowth (SIBO), which produces large amounts of lipopolysaccharide (LPS) that damage the liver. These altered cytokines ultimately lead to a vicious cycle of inflammation in NAFLD.

Contents of the invention

The object of the present invention is to provide Lactobacillus plantarum NA136 and its application in alleviating non-alcoholic fatty liver disease.

The present invention firstly provides Lactobacillus plantarum NA136.

Lactobacillus plantarum NA136, the full name of Lactobacillus plantarum (Lactobacillus plantarum) NA136, has been preserved in the China Center for Type Culture Collection (CCTCC for short, address: China, Wuhan, Wuhan University) on March 11, 2018, and the preservation number is CCTCC NO: M 2018112.

The present invention also protects the application of plantarum lactobacillus NA136 in the preparation of products for treating and/or preventing fatty liver.

The invention also protects the application of the plantarum lactobacillus NA136 in the preparation of products for treating and/or preventing non-alcoholic fatty liver.

The present invention also protects the application of Lactobacillus plantarum NA136 in the preparation of products;

The function of the product is at least one of the following (a1) to (a39):

(a1) reducing liver fat content;

(a2) inhibit fat deposition in the liver;

(a3) reducing the level of free fatty acids in serum;

(a4) reducing the level of alanine aminotransferase in serum;

(a5) reducing the level of insulin in serum;

(a6) reduce the level of inflammatory factors in serum;

(a7) reducing the level of tumor necrosis factor-alpha in serum;

(a8) reducing the level of interleukin-6 in serum;

(a9) reducing the level of lipopolysaccharide in serum;

(a10) reducing the level of total cholesterol in serum;

(a11) lowering the level of triglycerides in serum;

(a12) reducing the level of low-density lipoprotein cholesterol in serum;

(a13) reducing body weight;

(a14) reducing liver fat content in patients with fatty liver;

(a15) Inhibiting intrahepatic fat deposition in patients with fatty liver;

(a16) reducing the level of free fatty acids in serum of fatty liver patients;

(a17) reducing the level of alanine aminotransferase in serum of fatty liver patients;

(a18) reducing the level of insulin in the serum of patients with fatty liver;

(a19) reducing the level of inflammatory factors in the serum of fatty liver patients;

(a20) reducing the level of tumor necrosis factor-alpha in serum of fatty liver patients;

(a21) reducing the level of interleukin-6 in serum of fatty liver patients;

(a22) reducing the level of lipopolysaccharide in the serum of patients with fatty liver;

(a23) reducing the level of total cholesterol in the serum of fatty liver patients;

(a24) reducing the level of triglycerides in serum of patients with fatty liver;

(a25) reducing the level of low-density lipoprotein cholesterol in the serum of fatty liver patients;

(a26) reducing the body weight of patients with fatty liver;

(a27) reducing liver fat content in patients with non-alcoholic fatty liver disease;

(a28) Inhibits intrahepatic fat deposition in patients with nonalcoholic fatty liver disease;

(a29) reducing the level of free fatty acids in serum of patients with non-alcoholic fatty liver disease;

(a30) reducing the level of alanine aminotransferase in serum of patients with nonalcoholic fatty liver disease;

(a31) reducing the level of insulin in the serum of patients with non-alcoholic fatty liver disease;

(a32) reducing the level of inflammatory factors in the serum of patients with non-alcoholic fatty liver;

(a33) reducing the level of tumor necrosis factor-alpha in serum of patients with non-alcoholic fatty liver disease;

(a34) reducing the level of interleukin-6 in the serum of patients with nonalcoholic fatty liver disease;

(a35) reducing the level of lipopolysaccharide in serum of patients with non-alcoholic fatty liver disease;

(a36) reducing the level of total cholesterol in the serum of patients with non-alcoholic fatty liver disease;

(a37) reducing the level of triglycerides in serum of patients with non-alcoholic fatty liver disease;

(a38) reducing the level of low-density lipoprotein cholesterol in the serum of patients with non-alcoholic fatty liver disease;

(a39) Reducing body weight in patients with nonalcoholic fatty liver disease.

The product is food, health product or medicine.

The invention also protects a product for treating and/or preventing fatty liver, the active ingredient of which is Lactobacillus plantarum NA136.

The invention also protects a product for treating and/or preventing non-alcoholic fatty liver, the active ingredient of which is Lactobacillus plantarum NA136.

The invention also protects a product whose active ingredient is Lactobacillus plantarum NA136;

The function of the product is at least one of the following (a1) to (a39):

(a1) reducing liver fat content;

(a2) inhibit fat deposition in the liver;

(a3) reducing the level of free fatty acids in serum;

(a4) reducing the level of alanine aminotransferase in serum;

(a5) reducing the level of insulin in serum;

(a6) reduce the level of inflammatory factors in serum;

(a7) reducing the level of tumor necrosis factor-alpha in serum;

(a8) reducing the level of interleukin-6 in serum;

(a9) reducing the level of lipopolysaccharide in serum;

(a10) reducing the level of total cholesterol in serum;

(a11) lowering the level of triglycerides in serum;

(a12) reducing the level of low-density lipoprotein cholesterol in serum;

(a13) reducing body weight;

(a14) reducing liver fat content in patients with fatty liver;

(a15) Inhibiting intrahepatic fat deposition in patients with fatty liver;

(a16) reducing the level of free fatty acids in serum of fatty liver patients;

(a17) reducing the level of alanine aminotransferase in serum of fatty liver patients;

(a18) reducing the level of insulin in the serum of patients with fatty liver;

(a19) reducing the level of inflammatory factors in the serum of fatty liver patients;

(a20) reducing the level of tumor necrosis factor-alpha in serum of fatty liver patients;

(a21) reducing the level of interleukin-6 in serum of fatty liver patients;

(a22) reducing the level of lipopolysaccharide in the serum of patients with fatty liver;

(a23) reducing the level of total cholesterol in the serum of fatty liver patients;

(a24) reducing the level of triglycerides in serum of patients with fatty liver;

(a25) reducing the level of low-density lipoprotein cholesterol in the serum of fatty liver patients;

(a26) reducing the body weight of patients with fatty liver;

(a27) reducing liver fat content in patients with non-alcoholic fatty liver disease;

(a28) Inhibits intrahepatic fat deposition in patients with nonalcoholic fatty liver disease;

(a29) reducing the level of free fatty acids in serum of patients with non-alcoholic fatty liver disease;

(a30) reducing the level of alanine aminotransferase in serum of patients with nonalcoholic fatty liver disease;

(a31) reducing the level of insulin in the serum of patients with non-alcoholic fatty liver disease;

(a32) reducing the level of inflammatory factors in the serum of patients with non-alcoholic fatty liver;

(a33) reducing the level of tumor necrosis factor-alpha in serum of patients with non-alcoholic fatty liver disease;

(a34) reducing the level of interleukin-6 in the serum of patients with nonalcoholic fatty liver disease;

(a35) reducing the level of lipopolysaccharide in serum of patients with non-alcoholic fatty liver disease;

(a36) reducing the level of total cholesterol in the serum of patients with non-alcoholic fatty liver disease;

(a37) reducing the level of triglycerides in serum of patients with non-alcoholic fatty liver disease;

(a38) reducing the level of low-density lipoprotein cholesterol in the serum of patients with non-alcoholic fatty liver disease;

(a39) Reducing body weight in patients with nonalcoholic fatty liver disease.

The product is food, health product or medicine.

Any of the above foods include but are not limited to: fermented fruits and vegetables containing the plantarum lactobacillus, fermented milk containing the plantarum lactobacillus, cheese containing the plantarum lactobacillus, milk-containing beverages containing the plantarum lactobacillus , milk powder containing the plantarum lactobacillus or other foods containing the plantarum lactobacillus. The food can specifically be a functional food, such as functional yogurt, functional bacterial agent and the like.

Any of the above-mentioned medicines includes but not limited to the following dosage forms: capsules, powders or tablets. The medicine may also include other pharmaceutically acceptable excipients or diluents. The method of preparing the powder can be freeze-drying or other techniques.

The recommended dosage of the Lactobacillus plantarum is as follows: once a day, the single dosage is 12×10 ⁹ CFU per kilogram of body weight.

The inventors of the present invention found that Lactobacillus plantarum NA136 helps to reduce the occurrence of inflammation in the liver, and has outstanding effects on alleviating non-alcoholic fatty liver disease. Daily intake of Lactobacillus plantarum NA136 or its preparations can replace drugs to protect the integrity of liver function. The invention provides a new idea for clinical intervention and control of non-alcoholic fatty liver disease and other chronic liver diseases, and has broad application prospects and promotion value.

Description of drawings

Figure 1 shows the detection results of insulin in serum.

Figure 2 is a photograph of HE slices of liver tissue.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Unless otherwise specified, the PBS buffers in the examples are all pH 7.0, 0.1M PBS buffers.

Solid BCP medium: containing yeast extract 2.5g/L, peptone 5g/L, glucose 5g/L, bromocresol purple 0.04g/L, agar 15g/L, and the balance is water; unless otherwise specified, pH=7.0 . Liquid BCP medium differs from solid BCP medium only in that agar is not added.

Solid MRS medium: containing peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, KH ₂ PO ₄ 2g/L, sodium acetate 5g/L, sodium citrate 5g/L, MgSO ₄ 7H ₂ O 0.2g/L, MnSO ₄ ·4H ₂ O 0.05g/L, Tween-80 1mL/L, agar 15g/L, glucose 20g/L, the balance is water; unless otherwise specified, pH=6.6. Liquid MRS medium differs from solid MRS medium only in that agar is not added.

Basic feed: Changchun Yisi Experimental Animal Technology Co., Ltd. High-fat feed (% represents g/100g): 75% of basic feed, 10% of lard, 10% of egg yolk powder, 5% of cholesterol (food grade, Zhengzhou Cangyu Chemical Products Co., Ltd.).

C57BL/6J mice: Changchun Yisi Experimental Animal Technology Co., Ltd., animal license number: SCXK (Ji)-2011-0004.

Total Cholesterol (TC) content in serum was detected by Shanghai Langton Mouse Total Cholesterol ELISA Kit (Product No. BPE20095). Triglyceride (TG) content in serum was detected by Shanghai Langton Mouse Triglyceride ELISA Kit (Product No. BPE20754). The high-density lipoprotein cholesterol (High Density Lipoprotein, HDL-C) content in the serum was detected by the Shanghai Longton mouse high-density lipoprotein cholesterol enzyme-linked immunosorbent assay kit (product number BPE20192). The level of low-density lipoprotein cholesterol (Low Density Lipoprotein, LDL-C) in serum was detected by Shanghai Langton mouse low-density lipoprotein cholesterol enzyme-linked immunosorbent assay kit (product number BPE20100). The level of insulin (Insulin, INS) in serum was detected by Shanghai Langton Mouse Insulin ELISA Kit (Product No. BPE20353). The level of tumor necrosis factor-α (tumor necrosis factor-α, TNF-α) in serum was detected by Shanghai Langdon Mouse Tumor Necrosis Factor-α ELISA Kit (Product No. BPE20220). The interleukin-6 (interleukin-6, IL-6) content in the serum was detected by the Shanghai Longton Mouse Interleukin-6 ELISA Kit (Product No. BPE20012). The level of lipopolysaccharide (LPS) in serum was detected by Jianglai biological mouse lipopolysaccharide enzyme-linked immunosorbent assay kit (product number JL20691). The level of alanine aminotransferase (ALT) in serum was detected by Jianglai Bio-Mouse Alanine Aminotransferase ELISA Kit (Product No. JL12668). Free Fat Acid (FFA) content in serum was detected by Jianglai Biological Mouse Free Fatty Acid ELISA Kit (Product No. JL11286).

The preparation method of fructose aqueous solution: dissolve 30g fructose in three-stage distilled water and use three-stage distilled water to make up to 100mL.

Lactobacillus plantarum K25 is independently isolated by the inventor of the present invention from Linggu mushroom. Lactobacillus plantarum K25 has good acid resistance, bile salt resistance, cell adhesion, and good blood lipid lowering and antioxidant effects. Documents that record Lactobacillus plantarum K25: Study on the effect of Lactobacillus plantarum K25 fermented milk on reducing serum cholesterol in mice, "Food Science", No. 07, 2012.

Embodiment 1, isolation, identification and preservation of bacterial strains

1. Isolation of strains

Sample material: traditional fermented sauerkraut.

In July 2016, traditional fermented sauerkraut was collected from Yanji City, Jilin Province. After homogenization, the BCP medium plate was repeatedly streaked and cultured. A single colony that produced a yellow circle was picked and inoculated on an MRS medium plate. Repeated streak culture and purification obtained multiple purely cultured strains.

The purely cultured strains were inoculated into liquid MRS medium for culture, then added 20% glycerol, and stored at -80°C.

Selection criteria: positive Gram stain, negative catalase test, negative indole reaction, negative hydrogen sulfide production test, negative gelatin liquefaction test, negative hydrolyzed starch test, and negative nitrate reduction test. Multiple strains of Lactobacillus were screened out from the purely cultured strains. One of the Lactobacillus plantarum strains was named NA136.

2. Identification of strains

Physiological and biochemical identification results of NA136: Gram positive, catalase negative; non-motile bacilli; able to grow at 15°C and 45°C; tolerant to 6.5% NaCl; not hydrolyzing starch, not liquefying gelatin, not producing hydrogen sulfide ; Fermentation of glucose produces acid without producing gas; the benzidine test is negative, the indole test is negative, and the acetylmethylmethanol test is positive.

NA136 grew uniformly and turbidly in MRS liquid medium, and the bacterial cells after long-term storage showed white precipitate.

The optimum growth temperature of NA136 is 37-42℃, and the optimum pH is 5.0-7.0.

Molecular identification results of NA136: the 16S rDNA sequence is shown in sequence 1 of the sequence listing.

The identification results showed that NA136 belonged to Lactobacillus plantarum.

3. Preservation of strains

Lactobacillus plantarum NA136 was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on March 11, 2018, and the deposit number is CCTCC NO: M2018112. Lactobacillus plantarum (Lactobacillus plantarum) NA136CCTCC NO: M2018112, referred to as Lactobacillus plantarum NA136.

Example 2, the alleviating effect of Lactobacillus plantarum NA136 on non-alcoholic fatty liver

1. Model success rate detection

The first group: 10 male C57BL/6J mice aged 8 weeks (23g-24g) were fed with high-fat diet and fructose aqueous solution for 56 consecutive days.

The second group: 10 8-week-old (23g-24g) male C57BL/6J mice were fed with basal feed and normal drinking water for 56 consecutive days.

After completing the above steps, the mice were sacrificed, the liver was separated, and HE stained sections were made (the method is the same as step five).

In the first group of 10 mice, the grade of hepatic steatosis was S1 (fat content: 5%-33% of liver weight).

In the second group of 10 mice, the grade of hepatic steatosis was S0 grade (fat content as liver weight: less than 5%).

The results showed that the success rate of non-alcoholic fatty liver animal model was 100% by adopting the method of "feeding with high-fat feed and 30% fructose aqueous solution for 56 consecutive days".

Two, group processing

Sixty 8-week-old (23g-24g) male C57BL/6J mice were subjected to adaptive feeding for 3 consecutive days (ie 3 days before the first day of the test), and then randomly divided into 4 groups, 15 mice in each group , and the grouping process is as follows (the whole test period is from the first day to the 112th day of the test):

Blank group (Control): From the 1st day to the 112th day of the test, they were fed with basic feed and ordinary drinking water; from the 57th day to the 112th day of the test, they were fed with normal saline once a day (per kilogram of body weight each time 12mL sterile saline);

Model group (Model): From the 1st day to the 56th day of the test, high-fat feed and fructose aqueous solution were used for feeding; For 112 days, intragastric administration of normal saline once a day (12 mL of sterile normal saline per kilogram of body weight each time);

Lactobacillus plantarum NA136 group (NA136 group): From the 1st day to the 56th day of the test, high-fat feed and fructose aqueous solution were used for feeding; From the 57th day to the 112th day, intragastric administration of bacterial solution once a day (every kilogram of body weight gives 12mL bacterial solution each time; the preparation method of the bacterial solution: suspend Lactobacillus plantarum NA136 with PBS buffer solution to obtain a concentration of Lactobacillus plantarum NA136 of 1.0×10 ⁹ CFU/mL bacterial solution).

Lactobacillus plantarum K25 group (K25 group): From the 1st day to the 56th day of the test, high-fat feed and fructose aqueous solution were used for feeding; From the 57th day to the 112th day, the bacterial solution was administered once a day (12 mL of the bacterial solution per kilogram of body weight each time; the preparation method of the bacterial solution: suspend Lactobacillus plantarum K25 with PBS buffer solution to obtain a K25 concentration of 1.0×10 ⁹ CFU/ mL of bacteria liquid).

3. Body weight measurement

During the test period, body weight was weighed every 14 days, and the results are shown in Table 1. Since the high-fat diet and fructose aqueous solution were fed for 8 consecutive weeks, the body weight of the mice in the model group, the body weight of the mice in the NA136 group, and the body weight of the mice in the K25 group were significantly higher than those in the blank group. After intragastric administration of the bacterial solution, the body weight of the mice in the NA136 group and the K25 group began to decrease continuously, and at the end of the 16th week, the body weight of the mice in the NA136 group was basically the same as that of the blank group. At the end of the 16th week, compared with the model group mice, the body weight of the mice in the K25 group decreased by 11.96%, and the body weight of the mice in the NA136 group decreased by 17.22%. The results showed that Lactobacillus plantarum NA136 could significantly reduce the body weight of mice with non-alcoholic fatty liver disease, and the effect was better than that of Lactobacillus plantarum K25.

Table 1 Effect of Lactobacillus plantarum NA136 on body weight of mice with nonalcoholic fatty liver disease

Body weight of blank group (g) Model group weight (g) K25 group weight (g) NA136 group body weight (g) initial weight 23.72±1.45 24.02±1.35 24.02±1.35 24.02±1.35 day 14 24.12±1.72 25.24±1.29 25.24±1.29 25.24±1.29 day 28 25.07±2.26 28.11±2.18 28.11±2.18 28.11±2.18 Day 42 26.45±1.81 29.89±1.25 29.89±1.25 29.89±1.25 Day 56 27.29±2.37 31.45±1.45 31.45±1.45 31.45±1.45 Day 70 27.68±1.75 33±1.41 31.21±0.98 30.54±1.65 Day 84 27.79±2.33 33.37±1.51 30.92±1.33 29.39±1.84 Day 98 27.89±1.68 33.99±2.37 30.45±1.54 28.91±2.15 Day 112 27.81±2.54 34.2±1.77 30.11±1.32 28.31±1.49

4. Detection of relevant indicators in serum

At the end of the test (the 113th day of the test), the mice in each group were taken, and the eyeballs were removed to get blood, centrifuged at 3000 rpm for 15 minutes after coagulation at room temperature for 60 minutes, and the supernatant (serum) was collected. Detection of total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, free fatty acids, alanine aminotransferase, insulin, tumor necrosis factor-α, interleukin-6, and lipopolysaccharide in serum content.

The detection results of blood lipid-related factors in serum are shown in Table 2. The content of TC, TG and LDL-C in the serum of mice in the model group were significantly higher than those in the blank group, and the content of HDL-C was significantly lower than that in the control group, which was due to the metabolic disorder in the mice caused by non-alcoholic fatty liver disease. Compared with the model group, the contents of TC, TG, and LDL-C in the serum of the mice in the NA136 group and the K25 group decreased, and the NA136 group improved the blood lipid level more significantly than the K25 group. The results showed that Lactobacillus plantarum NA136 could significantly improve the dyslipidemia in non-alcoholic fatty liver disease.

Table 2 Effect of Lactobacillus plantarum NA136 on blood lipids in mice with nonalcoholic fatty liver disease

Note: Compared with the model group, * has a significant difference (P<0.05), ** has a very significant difference (P<0.01).

The detection results of free fatty acids and alanine aminotransferase in serum are shown in Table 3. The FFA and ALT contents of the mice in the model group were significantly higher than those in the blank group. The increase of FFA content is a significant sign of obesity induced by high-fat food, and it is also an important inducement of non-alcoholic fatty liver disease. Elevated levels of ALT indicate that high-fat foods have caused liver damage. Compared with the model group, the FFA of mice in K25 group and NA136 group decreased by 46.41% and 58.77%, respectively. After comparison, it was found that Lactobacillus plantarum NA136 can better reduce FFA in serum. Compared with the model group, the ALT of K25 group and NA136 group decreased by 21.52% and 30.09%, respectively. It shows that Lactobacillus plantarum NA136 can effectively reduce the levels of FFA and ALT in serum. Based on the above results, Lactobacillus plantarum NA136 has a more obvious effect in alleviating non-alcoholic fatty liver disease than Lactobacillus plantarum K25.

Table 3 Concentrations of FFA and ALT in serum

blank group model group K25 group NA136 group FFA (μmol/L) 5.11±0.15 15.21±0.51 8.15±0.44 ^** 6.27±0.42 ^** ALT (ng/L) 14.52±2.62 24.63±5.71 19.33±2.56 ^** 17.22±4.73 ^*

Note: Compared with the model group, * has a significant difference (P<0.05), ** has a very significant difference (P<0.01).

The detection results of insulin in serum are shown in Figure 1. One of the pathogenic causes of nonalcoholic fatty liver disease is hyperinsulinemia. Oral administration of Lactobacillus plantarum NA136 can effectively alleviate the metabolic disorder in the body and reduce the serum insulin level (P<0.05).

The results of the concentration of each inflammatory factor in the serum are shown in Table 4. Compared with the blank group, the levels of TNF-α, IL-6 and LPS in the serum of the mice in the model group were significantly increased, indicating that lipopolysaccharides enter the blood circulation to stimulate the body's inflammatory response and increase the body's inflammation level. Compared with the model group, the levels of TNF-α, IL-6 and LPS of the mice in the NA136 group were decreased in different degrees. The results showed that Lactobacillus plantarum NA136 could significantly alleviate the inflammatory response induced by lipopolysaccharide and reduce the level of inflammatory factors in serum, thereby alleviating non-alcoholic fatty liver disease.

The concentration of each inflammatory factor in table 4 serum

group TNF-α(ng/L) IL-6(pg/mL) LPS (ng/L) blank group 202.82±27.73 33.48±6.44 175.21±22.89 model group 222.86±44.91 51.53±3.51 243.69±25.51 NA136 group 208.22±10.18 36.36±8.87 ^* 182.37±37.87 ^**

Note: Compared with the model group, * has a significant difference (P<0.05), ** has a very significant difference (P<0.01).

5. HE Slices of Liver Tissue

At the end of the experiment (the 113th day of the experiment), the mice in each group were taken, sacrificed, and the livers were separated. Take a piece of liver tissue with a thickness not exceeding 0.5 cm, and perform the following steps in sequence: put it in fixative solution for fixation, then soak it in water for 12 hours, then soak it in 75% alcohol solution for one time (lh), and then soak it in 85% alcohol solution for one time (1h), then soaked with 95% alcohol solution, 3 times (each time the solution is changed, each time 1h), then soaked with 100% alcohol 3 times (each time the solution is changed, each time 1h), and then soaked with xylene for 2 time (every change of solution, each 1h), then soaked with paraffin 3 times (each time to change the solution, each 2h); then soaked 2 times with xylene (each time to change the solution, each 5-10min), and then Soak with 100% alcohol for 1 time (1-2min), then soak with 95% alcohol solution for 1 time (1-2min), then soak with 85% alcohol for 1 time (1-2min each time), then soak with 75% alcohol solution Soak for 1 time (1-2min), then wash with distilled water; then stain with Mayer's hematoxylin for 1min, then rinse with warm water for 5-10min, then soak with 1% hydrochloric acid ethanol (take 2.75ml 36% hydrochloric acid and add ethanol to 100ml) for 2s, Then rinse with tap water for 30s; counterstain with eosin for 1-2min, then rinse with tap water for 30s, then soak once with 85% alcohol solution (1-2min), then soak twice with 95% alcohol solution (1-2min each time) ), then soaked 2 times with 100% alcohol (1-2min each time), then soaked 2 times with xylene (5-10min each time); then sealed with neutral gum.

The results are shown in Figure 2. A is the blank group, showing normal liver tissue, and intracellular vacuoles indicate the deposition of lipid droplets in the liver. B is the model group, a large number of lipid droplets are deposited in the liver, and the degree of fatty degeneration is severe. C is the NA136 group, compared with the model group, the deposition degree of lipid droplets in the liver was significantly reduced. The liver fat content (average value) of mice in the blank group was less than 5%. The liver fat content (average value) of the mice in the model group was about 45%. The liver fat content (mean value) of mice in NA136 group was less than 5%. The results showed that Lactobacillus plantarum NA136 improved the degree of hepatic steatosis and had an inhibitory effect on the occurrence of non-alcoholic fatty liver disease.

SEQUENCE LISTING

<110> Jilin Academy of Agricultural Sciences

<120> Lactobacillus plantarum NA136 and its application in alleviating non-alcoholic fatty liver disease

<130> GNCYX181112

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1050

<212>DNA

<213> Lactobacillus plantarum

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Claims (9)

1. Lactobacillus plantarum (Lactobacillus plantarum) NA136, its deposit number is CCTCC NO: M2018112. 2. The application of Lactobacillus plantarum NA136 according to claim 1 in the preparation of products for the treatment and/or prevention of fatty liver. 3. The application of the Lactobacillus plantarum NA136 according to claim 1 in the preparation of products for the treatment and/or prevention of non-alcoholic fatty liver. 4. the application of lactobacillus plantarum NA136 described in claim 1 in the preparation product; The function of the product is at least one of the following (a1) to (a39): (a1) reducing liver fat content; (a2) inhibit fat deposition in the liver; (a3) reducing the level of free fatty acids in serum; (a4) reducing the level of alanine aminotransferase in serum; (a5) reducing the level of insulin in serum; (a6) reduce the level of inflammatory factors in serum; (a7) reducing the level of tumor necrosis factor-alpha in serum; (a8) reducing the level of interleukin-6 in serum; (a9) reducing the level of lipopolysaccharide in serum; (a10) reducing the level of total cholesterol in serum; (a11) lowering the level of triglycerides in serum; (a12) reducing the level of low-density lipoprotein cholesterol in serum; (a13) reducing body weight; (a14) reducing liver fat content in patients with fatty liver; (a15) Inhibiting intrahepatic fat deposition in patients with fatty liver; (a16) reducing the level of free fatty acids in serum of fatty liver patients; (a17) reducing the level of alanine aminotransferase in serum of fatty liver patients; (a18) reducing the level of insulin in the serum of patients with fatty liver; (a19) reducing the level of inflammatory factors in the serum of fatty liver patients; (a20) reducing the level of tumor necrosis factor-alpha in serum of fatty liver patients; (a21) reducing the level of interleukin-6 in serum of fatty liver patients; (a22) reducing the level of lipopolysaccharide in the serum of patients with fatty liver; (a23) reducing the level of total cholesterol in the serum of fatty liver patients; (a24) reducing the level of triglycerides in serum of patients with fatty liver; (a25) reducing the level of low-density lipoprotein cholesterol in the serum of fatty liver patients; (a26) reducing the body weight of patients with fatty liver; (a27) reducing liver fat content in patients with non-alcoholic fatty liver disease; (a28) Inhibits intrahepatic fat deposition in patients with nonalcoholic fatty liver disease; (a29) reducing the level of free fatty acids in serum of patients with non-alcoholic fatty liver disease; (a30) reducing the level of alanine aminotransferase in serum of patients with nonalcoholic fatty liver disease; (a31) reducing the level of insulin in the serum of patients with non-alcoholic fatty liver disease; (a32) reducing the level of inflammatory factors in the serum of patients with non-alcoholic fatty liver; (a33) reducing the level of tumor necrosis factor-alpha in serum of patients with non-alcoholic fatty liver disease; (a34) reducing the level of interleukin-6 in the serum of patients with nonalcoholic fatty liver disease; (a35) reducing the level of lipopolysaccharide in serum of patients with non-alcoholic fatty liver disease; (a36) reducing the level of total cholesterol in the serum of patients with non-alcoholic fatty liver disease; (a37) reducing the level of triglycerides in serum of patients with non-alcoholic fatty liver disease; (a38) reducing the level of low-density lipoprotein cholesterol in the serum of patients with non-alcoholic fatty liver disease; (a39) Reducing body weight in patients with nonalcoholic fatty liver disease. 5. The application according to any one of claims 2 to 4, characterized in that: the product is food, health product or medicine. 6. A product for treating and/or preventing fatty liver, the active ingredient of which is Lactobacillus plantarum NA136 according to claim 1. 7. A product for treating and/or preventing non-alcoholic fatty liver, whose active ingredient is Lactobacillus plantarum NA136 according to claim 1. 8. A product whose active ingredient is Lactobacillus plantarum NA136 according to claim 1; The function of the product is at least one of the following (a1) to (a39): (a1) reducing liver fat content; (a2) inhibit fat deposition in the liver; (a3) reducing the level of free fatty acids in serum; (a4) reducing the level of alanine aminotransferase in serum; (a5) reducing the level of insulin in serum; (a6) reduce the level of inflammatory factors in serum; (a7) reducing the level of tumor necrosis factor-alpha in serum; (a8) reducing the level of interleukin-6 in serum; (a9) reducing the level of lipopolysaccharide in serum; (a10) reducing the level of total cholesterol in serum; (a11) lowering the level of triglycerides in serum; (a12) reducing the level of low-density lipoprotein cholesterol in serum; (a13) reducing body weight; (a14) reducing liver fat content in patients with fatty liver; (a15) Inhibiting intrahepatic fat deposition in patients with fatty liver; (a16) reducing the level of free fatty acids in serum of fatty liver patients; (a17) reducing the level of alanine aminotransferase in serum of fatty liver patients; (a18) reducing the level of insulin in the serum of patients with fatty liver; (a19) reducing the level of inflammatory factors in the serum of fatty liver patients; (a20) reducing the level of tumor necrosis factor-alpha in serum of fatty liver patients; (a21) reducing the level of interleukin-6 in serum of fatty liver patients; (a22) reducing the level of lipopolysaccharide in the serum of patients with fatty liver; (a23) reducing the level of total cholesterol in the serum of fatty liver patients; (a24) reducing the level of triglycerides in serum of patients with fatty liver; (a25) reducing the level of low-density lipoprotein cholesterol in the serum of fatty liver patients; (a26) reducing the body weight of patients with fatty liver; (a27) reducing liver fat content in patients with non-alcoholic fatty liver disease; (a28) Inhibits intrahepatic fat deposition in patients with nonalcoholic fatty liver disease; (a29) reducing the level of free fatty acids in serum of patients with non-alcoholic fatty liver disease; (a30) reducing the level of alanine aminotransferase in serum of patients with nonalcoholic fatty liver disease; (a31) reducing the level of insulin in the serum of patients with non-alcoholic fatty liver disease; (a32) reducing the level of inflammatory factors in the serum of patients with non-alcoholic fatty liver; (a33) reducing the level of tumor necrosis factor-alpha in serum of patients with non-alcoholic fatty liver disease; (a34) reducing the level of interleukin-6 in the serum of patients with nonalcoholic fatty liver disease; (a35) reducing the level of lipopolysaccharide in serum of patients with non-alcoholic fatty liver disease; (a36) reducing the level of total cholesterol in the serum of patients with non-alcoholic fatty liver disease; (a37) reducing the level of triglycerides in serum of patients with non-alcoholic fatty liver disease; (a38) reducing the level of low-density lipoprotein cholesterol in the serum of patients with non-alcoholic fatty liver disease; (a39) Reducing body weight in patients with nonalcoholic fatty liver disease. 9. The product according to any one of claims 5 to 7, wherein the product is food, health product or medicine.