Application of lactobacillus paracasei composition in relieving liver metabolic disorder

Abstract

The invention relates to application of a lactobacillus paracasei composition in relieving liver metabolic disorder, and belongs to the technical field of microorganisms. The invention discloses application of a lactobacillus paracasei composition containing lactobacillus paracasei JN-8 and E-10 in preparation of a product for relieving liver metabolic disorder caused by high methionine diet, the ratio of viable bacteria of two strains in the product is 1: 1, the range of the viable count of single bacteria is 1 * 10 < 8 >-1 * 10 < 10 > CFU / mL, and the two strains have a synergistic effect in a viable bacteria suspension form. According to the composition, the expression of lipid metabolism abnormality related genes such as Dgat1, Htgl, Srep1, Acc1, Aoah and the like is inhibited, and the levels of liver inflammatory factors such as Il-6, Il-10, Tlr-4 and the like are reduced, so that the liver metabolic imbalance and inflammatory response are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to the application of Lactobacillus paracasei compositions in alleviating liver metabolic disorders. Background Art

[0002] A high-sulfur diet (high-methionine diet, HMD) is a dietary pattern dominated by animal-derived foods. Its typical feature is that by consuming a large amount of foods rich in sulfur-containing amino acids such as meat, eggs, and dairy products, the daily methionine intake significantly exceeds the standard value of 10-13 mg / kg body weight recommended by the WHO. This dietary structure shows a rapid popularization trend in contemporary society. According to epidemiological surveys, the average daily methionine intake of adults in developed countries has generally reached 2-3 times the recommended value. Its prevalence stems from both the promotion of high-protein diets by the fast-food culture and the excessive pursuit of muscle-building diets by fitness enthusiasts. Although methionine, as an essential amino acid, plays an important role in protein synthesis and methyl donor metabolism, excessive intake will disrupt the sulfur metabolism homeostasis in the body, leading to the accumulation of homocysteine and abnormal generation of hydrogen sulfide (H2S), and further causing oxidative stress and mitochondrial dysfunction.

[0003] It is worth noting that long-term maintenance of HMD will trigger multi-system metabolic disorders. At the liver level, excessive methionine inhibits the expression of the key enzyme CPT1A for lipid β-oxidation by activating the S-adenosylmethionine (SAM) synthesis pathway, resulting in abnormal deposition of triglycerides and fatty degeneration. In the intestine, the toxic effect of sulfur metabolism by-products on goblet cells will reduce the thickness of the mucus layer. At the same time, H2S overload can directly damage the tight junction structure between intestinal epithelial cells, promoting the translocation of lipopolysaccharide (LPS) and activating the Toll-like receptor 4 (TLR4) signaling pathway, forming systemic low-grade inflammation. Preclinical studies have confirmed that 8 weeks of HMD intervention can increase the levels of serum Il-6 and Tnf-α in experimental animals by 2-3 times, and is accompanied by a significant decrease in the α-diversity index of the gut microbiota.

[0004] In this context, lactic acid bacteria (LAB) have become the focus of intervention studies due to their multi-target regulatory properties. Specific strains such as Lactobacillus rhamnosus GG (LGG) can inhibit the overgrowth of hydrogen sulfide-producing bacteria (such as Desulfovibrio) by secreting antimicrobial peptides, while promoting the colonization of beneficial bacteria such as Bifidobacterium. Its metabolites (such as short-chain fatty acids) can improve liver lipid metabolism by activating the AMPK pathway and repair the intestinal mechanical barrier by upregulating the expression of tight junction proteins such as Occludin and Zo-1. More studies have pointed out that Lactobacillus plantarum CCFM8610 can regulate bile acid metabolism through the FXR-SHP pathway and reduce liver fat deposition. However, the current research on probiotic intervention in HMD-induced liver metabolic disorders is still unclear and urgently needs further exploration and experiments. The synergistic effects between different strains have also lacked attempts. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of a Lactobacillus paracasei composition in alleviating liver metabolic disorders. The present invention discloses the application of a Lactobacillus paracasei composition comprising Lactobacillus paracasei JN-8 and E-10 in the preparation of a product for alleviating liver metabolic disorders caused by a high methionine diet. The viable bacteria ratio of the two strains in the product is 1:1, and the viable bacteria count range of a single strain is 1×10 8 -1×10 10 CFU / mL, and they act synergistically in the form of a viable bacteria suspension.

[0006] The first object of the present invention is to provide an application of a Lactobacillus paracasei composition in the preparation of a product for alleviating liver metabolic disorders caused by a high methionine diet. The Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10. The preservation number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the preservation number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

[0007] Furthermore, the product contains a viable bacteria suspension of Lactobacillus paracasei JN-8 and a viable bacteria suspension of Lactobacillus paracasei E-10.

[0008] Furthermore, the viable bacteria ratio of Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10 in the product is 1:1.

[0009] Furthermore, the viable bacteria count of Lactobacillus paracasei JN-8 in the product is 1×10 8 -1×10 10 CFU / mL, and the viable bacteria count of Lactobacillus paracasei E-10 is 1×10 8 -1×10 10 CFU / mL.

[0010] Preferably, the viable count of Lactobacillus paracasei JN-8 in the product is 1×10 8 CFU / mL.

[0011] Preferably, the viable count of Lactobacillus paracasei E-10 in the product is 1×10 8 CFU / mL.

[0012] The second object of the present invention is to provide a product for alleviating liver metabolic disorders caused by a high-methionine diet, the product contains a Lactobacillus paracasei composition, the Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10, the preservation number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the preservation number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

[0013] Furthermore, the viable ratio of Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10 in the product is 1:1.

[0014] The third object of the present invention is to provide the application of a Lactobacillus paracasei composition in the preparation of a product for reducing the expression level of genes related to abnormal lipid metabolism, the Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10, the preservation number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the preservation number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

[0015] Furthermore, the genes related to abnormal lipid metabolism include one or several of the fatty acid storage enzyme-encoding gene Dgat1, the hepatic lipase-encoding gene Htgl, the sterol regulatory element-binding protein-1-encoding gene Srbep1, the acetyl-CoA carboxylase-1-encoding gene Acc1, and the acylcarboxylic acid hydrolase-encoding gene Aoah.

[0016] The fourth object of the present invention is to provide the application of a Lactobacillus paracasei composition in the preparation of a product for reducing the expression level of liver inflammation-related factors, the Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10, the preservation number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the preservation number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

[0017] Furthermore, the liver inflammation-related factors include one or several of Il-10, Il-6, and Tlr-4.

[0018] The beneficial effects of the present invention:

[0019] The Lactobacillus paracasei composition of the present invention has the following advantages: (1) It synergistically regulates the lipid metabolism pathway, significantly inhibits the expression of key genes such as Dgat1 and Htgl, and reduces abnormal lipid accumulation; (2) It reduces the expression of pro-inflammatory factors Il-6, Il-10 and the inflammatory signal molecule Tlr-4, and alleviates liver inflammatory damage; (3) In response to the metabolic disorders induced by a high-methionine diet, it restores liver metabolic homeostasis through multi-target intervention. This composition has significant potential in the preparation of functional preparations or drugs, providing an efficient and safe microbial solution for the prevention and treatment of diet-related liver diseases.

[0020] Biomaterial preservation

[0021] Lactobacillus paracasei E-10 was deposited at the General Microbiological Center of the China National Committee for Culture Collection of Microorganisms on June 21, 2021. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 22744.

[0022] Lactobacillus paracasei JN-8 was deposited at the General Microbiological Center of the China National Committee for Culture Collection of Microorganisms on June 21, 2021. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 22746. Description of the drawings

[0023] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein:

[0024] Figure 1 It shows the changes in serum biochemical indexes of mice in the embodiments of the present invention. Among them, A shows the change in the content of TG in the serum of mice, B shows the change in the content of LDL in the serum of mice, C shows the change in the content of HDL in the serum of mice, D shows the change in the content of ALT in the serum of mice, and E shows the change in the content of AST in the serum of mice;

[0025] Figure 2 It shows the results of changes in the expression levels of genes related to liver lipid metabolism in mice in the embodiments of the present invention. Among them, A shows the change in the relative expression level of the Srbep-1 gene, B shows the change in the relative expression level of the Acc-1 gene, C shows the change in the relative expression level of the Dgat-1 gene, D shows the change in the relative expression level of the Htgl gene, and E shows the change in the relative expression level of the Aoah gene;

[0026] Figure 3Results of the changes in the expression levels of inflammation-related factors in the mouse liver in the embodiments of the present invention. Among them, A is the change in the expression level of Il-10, B is the change in the expression level of Il-6, and C is the change in the expression level of Tlr-4;

[0027] Figure 4 Results of the changes in the expression levels of hydrogen sulfide and its related genes in the mouse liver tissue in the embodiments of the present invention. Among them, A is the change in the hydrogen sulfide level in the mouse liver, and B is the change in the relative expression level of the Cse gene;

[0028] Figure 5 Results of H&E staining and F4 / 80 labeling in the histopathological analysis of the mouse liver tissue in the embodiments of the present invention. Detailed implementation manners

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0030] The culture media and culture methods involved in the following embodiments are as follows:

[0031] Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10 are respectively cultured in the following culture media: 8-12 g / L peptone, 8-12 g / L beef extract, 3.5-5.5 g / L yeast powder, 18-22 g / L glucose, 5 g / L anhydrous sodium acetate, 2.6 g / L dipotassium hydrogen phosphate trihydrate, 0.2 g / L magnesium sulfate heptahydrate, 2 g / L ammonium citrate dibasic, 0.05 g / L manganese sulfate, 1.0 mL / L Tween 80, and 10-20 g / L calcium carbonate. After culturing until the OD 600 is in the range of 0.6-0.9, they are mixed and used.

[0032] Example: Comparison of the effects of various lactic acid bacteria on liver metabolic disorders induced by a high-sulfur diet

[0033] BALB / c male mice weighing 18-22 g are selected and randomly divided into 4 groups (8 mice in each group):

[0034] (1) Control group (PURE): Free access to a normal purified diet (NRC-95, methionine 0.82%, cystine 0.35%, the feed is from Nantong Trofi Feed Technology Co., Ltd.);

[0035] (2) High-sulfur diet group (SAA): Free access to a high-sulfur diet (methionine content 2.46%, cystine 0.35%, the feed is from Nantong Trofi Feed Technology Co., Ltd.);

[0036] (3) High-sulfur diet + Lactobacillus paracasei JN-8 (JN-8): Freely fed a high-sulfur diet, and intragastrically administered with a bacterial agent prepared from 1×10 8 CFU of Lactobacillus paracasei JN-1 every day;

[0037] (4) High-sulfur diet + Lactobacillus paracasei E-10 (E-10): Freely fed a high-sulfur diet, and intragastrically administered with a bacterial agent prepared from 1×10 8 CFU of Lactobacillus paracasei E-10 every day;

[0038] (5) Purified diet + Lactobacillus fermentum mixture (JE): Freely fed a purified diet, and intragastrically administered with a bacterial agent prepared from 1×10 8 CFU of a mixture of Lactobacillus paracasei (JN-8:E-10 = 1:1) every day.

[0039] During the experiment, all mice were freely fed and given water, continuously fed for 28 days. The control group and the high-sulfur diet group were intragastrically administered with an equal amount of normal saline every day. The health status and food intake were recorded every day, and the body weight of the mice was recorded every 7 days. After 28 days of feeding, the mice were fasted for 12 h, and blood and liver were collected for subsequent analysis.

[0040] The changes in serum biochemical indexes of mice were as Figure 1 shown. The high-sulfur diet could cause significant increases in high-density lipoprotein HDL ( Figure 1 C), low-density lipoprotein LDL ( Figure 1 B), alanine aminotransferase ALT ( Figure 1 D) and aspartate aminotransferase AST ( Figure 1 E) in the serum of mice. However, after the intervention of lactic acid bacteria, there were obvious decreases, and the effect of the Lactobacillus paracasei mixture (JE) was the most significant. The high-sulfur diet led to a significant decrease in triglyceride TG ( Figure 1 A). However, after the intervention of the Lactobacillus paracasei mixture (JE), it was significantly decreased, indicating that the Lactobacillus paracasei mixture (JE) has a certain protective effect on the liver.

[0041] The results of the relative expression levels of lipid metabolism-related genes in the liver of mice were as Figure 2 shown. Compared with the purified diet group, in the high-sulfur diet group, the gene encoding fatty acid storage enzyme Dgat1 ( Figure 2 C), the gene encoding hepatic lipase Htgl ( Figure 2 D), the gene encoding sterol regulatory element binding protein-1 Srbep1 ( Figure 2 A), the gene encoding acetyl-CoA carboxylase-1 Acc1 ( Figure 2 B) and the gene encoding acyl-carboxylic acid hydrolase Aoah ( Figure 2The relative expression levels of (E) all increased significantly and decreased significantly after the intervention of lactic acid bacteria. This indicates that a high-methionine diet may trigger changes in metabolic processes such as lipid synthesis, storage, and hydrolysis, leading to upregulation of the expression of these genes, thereby promoting fat accumulation and metabolic disorders. The intervention of lactic acid bacteria plays a certain role in metabolic regulation by inhibiting fatty acid synthesis, promoting fat metabolism, and reducing fat storage, which helps to alleviate the lipid metabolism disorders caused by a high-methionine diet. The effect of the Lactobacillus paracasei mixture (JE) is more significant.

[0042] The changes in the relative expression levels of cancer-related factors in the livers of mice are as Figure 3 shown. A high-sulfur diet led to a significant increase in the anti-inflammatory factor Il-10 ( Figure 3 A) in the liver, an increase in the expression of the inflammatory factor Tlr-4 ( Figure 3 C) and the pro-inflammatory factor Il-6 ( Figure 3 B). However, the intervention of the Lactobacillus paracasei mixture (JE) could significantly restore the expression of these genes to normal levels and reduce the liver inflammatory response.

[0043] The detection results of hydrogen sulfide-related indicators in the livers of mice are as Figure 4 shown. A high-sulfur diet led to an increase in the expression of the gene Cse encoding caffeoyl shikimate esterase related to hydrogen sulfide production in the livers of mice ( Figure 4 B), and at the same time, the content of hydrogen sulfide in the liver tissue increased ( Figure 4 A). This may exacerbate oxidative stress and liver damage. The intervention of the Lactobacillus paracasei mixture (JE) could most significantly reduce the expression of these genes and reduce the hydrogen sulfide level, thereby alleviating the liver damage caused by a high-sulfur diet.

[0044] The results of pathological analysis of the livers of mice are as Figure 5 shown. The high-sulfur diet group caused infiltration of inflammatory cells and lipid vacuoles in the livers of mice, while the intervention of the Lactobacillus paracasei mixture (JE) could significantly restore liver inflammation and lipid vacuoles. At the same time, observing the results of immunohistochemical F4 / 80 staining, it was found that the same high-sulfur diet caused an increase in brown-like in the liver, but the intervention of the Lactobacillus paracasei mixture (JE) could also be significantly alleviated, indicating that the Lactobacillus paracasei mixture (JE) has the function of alleviating liver inflammation caused by a high-sulfur diet.

[0045] In summary, Lactobacillus paracasei E-10 can downregulate AST, ALT, and LDL indicators to protect the liver; regulate the genes related to hydrogen sulfide production in the liver to control the concentration of hydrogen sulfide and regulate the detoxification ability of the liver; downregulate lipid metabolism-related genes such as Dgat1, Htgl, Srbep1, Acc1, and Aoah to alleviate liver lipid metabolism disorders; inhibit the level of pro-inflammatory factors and alleviate liver inflammation.

[0046] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. Use of Lactobacillus paracasei composition in the preparation of a product for relieving liver metabolic disorders caused by a high-methionine diet, characterized in that: The Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10. The deposit number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the deposit number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

2. The application according to claim 1, characterized in that: The product contains a live bacteria suspension of Lactobacillus paracasei JN-8 and a live bacteria suspension of Lactobacillus paracasei E-10.

3. The application according to claim 1, wherein: The ratio of the live bacteria of Lactobacillus paracasei JN-8 to Lactobacillus paracasei E-10 in the product is 1:

1.

4. The application according to claim 1, wherein: The viable count of Lactobacillus paracasei JN-8 in the product is 1×10 8 -1×10 10 CFU / mL, and the viable count of Lactobacillus paracasei E-10 is 1×10 8 -1×10 10 CFU / mL.

5. A product for alleviating liver metabolic disorders caused by a high-methionine diet, characterized in that: The product contains a Lactobacillus paracasei composition, which includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10. The deposit number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the deposit number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

6. The product according to claim 5, wherein: The ratio of the live bacteria of Lactobacillus paracasei JN-8 to Lactobacillus paracasei E-10 in the product is 1:

1.

7. Use of the Lactobacillus paracasei composition in the preparation of a product for reducing the expression level of genes related to abnormal lipid metabolism, characterized in that: The Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10. The deposit number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the deposit number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

8. The application according to claim 7, wherein: The lipid metabolism disorder-related genes include one or more of the fatty acid storage enzyme-encoding gene Dgat1, the hepatic lipase-encoding gene Htgl, the sterol regulatory element-binding protein-1-encoding gene Srbep1, the acetyl-CoA carboxylase-1-encoding gene Acc1, and the acylcarboxylic acid hydrolase-encoding gene Aoah.

9. Use of the Lactobacillus paracasei composition in the preparation of a product for reducing the expression level of liver inflammation-related factors, characterized in that: The Lactobacillus paracasei composition includes Lactobacillus paracasei JN-8 and Lactobacillus paracasei E-10. The deposit number of Lactobacillus paracasei E-10 is CGMCC NO.22744, and the deposit number of Lactobacillus paracasei JN-8 is CGMCC NO.22746.

10. The application according to claim 9, wherein: The liver inflammation-related factors include one or more of Il-10, Il-6, and Tlr-4.

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