A highly effective cholesterol-lowering Lactobacillus paracasei IMAUJBC1 and its application

Abstract

The invention discloses a highly efficient cholesterol-lowering Lactobacillus paracasei I MAUJBC1 and its application, and relates to the technical field of functional microorganisms. The strain disclosed in the invention can alleviate weight gain caused by a high-fat diet, improve elevated blood lipid levels; reduce the expression level of cholesterol synthesis genes in the liver; significantly increase the expression level of cholesterol metabolism genes in the liver, and play a cholesterol-lowering role; and increase the abundance of beneficial bacteria in the intestinal flora, effectively improve the intestinal flora disorder caused by a high-fat diet, and play a role in alleviating hyperlipidemia.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganisms, and in particular to a strain of Lactobacillus paracasei IMAUJBC1 capable of effectively reducing cholesterol and application thereof. Background Art

[0002] High cholesterol levels in the human body are closely related to the development of cardiovascular diseases such as atherosclerosis. Studies have shown that blood cholesterol levels can be effectively reduced by changing dietary structure and drug treatment, but with people's increasing attention to diet therapy, it has become particularly important to find natural, effective and safe methods to regulate human cholesterol content. In recent years, probiotics have attracted much attention in the field of alleviating metabolic diseases, and the treatment of hypercholesterolemia is also a hot topic. Studies have shown that intestinal flora not only regulates human digestion, absorption and immune system, but also plays a key regulatory role in cholesterol metabolism. Probiotics have the potential to lower cholesterol. Some specific Lactobacillus paracasei or other intestinal bacteria can decompose bile acid, interfere with the absorption and excretion of the human intestine, and thus regulate the host's cholesterol level. Microbial metabolites also have a certain effect on cholesterol metabolism. Some intestinal flora regulate the host's metabolic activity, including the synthesis and degradation of cholesterol, by producing metabolites such as short-chain fatty acids. Supplementing specific Lactobacillus paracasei can regulate the structure of intestinal flora as a means of improving intestinal flora imbalance and degrading cholesterol. Research in this field is expected to provide new targets and strategies for the prevention and treatment of cholesterol-related diseases and provide a scientific basis for promoting human health. Summary of the invention

[0003] The invention aims to provide a highly efficient cholesterol-lowering Lactobacillus paracasei IMAUJBC1 and application thereof. The Lactobacillus paracasei IMAUJBC1 can improve intestinal flora imbalance and degrade cholesterol, thereby reducing human cholesterol level and reducing the risk of cardiovascular disease.

[0004] The purpose of the present invention can be achieved by the following technical solutions:

[0005] A highly effective cholesterol-lowering strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1, Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on December 21, 2021, with the deposit number CGMCC No.24176, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Application of Lactobacillus paracasei IMAUJBC1 with high cholesterol-lowering efficiency in the preparation of medicines for preventing and treating high cholesterol.

[0007] As a further embodiment of the present invention: the drug contains ≥1×10 7 CFU / g of Lactobacillus paracasei IMAUJBC1, or ≥1×10 9 CFU / mL of Lactobacillus paracasei IMAUJBC1.

[0008] As a further embodiment of the present invention, mammals can ingest the above-mentioned medicine through the gastrointestinal tract.

[0009] As a further embodiment of the present invention: preventing and treating high cholesterol includes at least one of the following functions:

[0010] (1) Improve lipid metabolism disorders caused by obesity;

[0011] (2) Alleviate hypercholesterolemia caused by obesity;

[0012] (3) Alleviate the increase in blood lipid levels caused by obesity;

[0013] (4) Improve intestinal flora disorders caused by obesity and increase the proportion of beneficial bacteria;

[0014] (5) Slow down weight gain.

[0015] Beneficial effects of the present invention:

[0016] The Lactobacillus paracasei IMAUJBC1 provided by the present invention alleviates the problems of weight gain and elevated blood lipid levels caused by a high-fat diet; reduces the expression level of cholesterol synthesis genes in the liver; significantly increases the expression level of genes related to cholesterol metabolism in the liver, thereby playing a cholesterol-lowering role; and increases the number of intestinal flora Lactobacillus ( Lactobacillus ), Akkermansia ( Akkermansia ), Bacteroides ( Bacteroides ) and other beneficial bacteria abundance, effectively improving the intestinal flora disorder caused by a high-fat diet and playing a role in alleviating hyperlipidemia. Therefore, the strain is used in a pharmaceutical composition for preventing and alleviating metabolic diseases such as obesity and high cholesterol, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the accompanying drawings.

[0018] Figure 1 This is the Gram staining image of Lactobacillus paracasei IMAUJBC1;

[0019] Figure 2 This is a graph showing the weight changes of mice over 10 weeks;

[0020] Figure 3 is the effect of Lactobacillus paracasei IMAUJBC1 on mouse serum TC;

[0021] Figure 4 is the effect of Lactobacillus paracasei IMAUJBC1 on serum LDL in mice;

[0022] Figure 5 The effect of Lactobacillus paracasei IMAUJBC1 on serum TG in mice;

[0023] Figure 6 is the effect of Lactobacillus paracasei IMAUJBC1 on mouse serum HDL;

[0024] Figure 7 The effect of Lactobacillus paracasei IMAUJBC1 on the expression of HMGCR gene in mouse serum and liver;

[0025] Figure 8 The effect of Lactobacillus paracasei IMAUJBC1 on the expression of AMPK gene in mouse serum and liver;

[0026] Fig. 9 The effect of Lactobacillus paracasei IMAUJBC1 on the expression of CYP7A1 gene in mouse serum and liver;

[0027] Fig.10 The effect of Lactobacillus paracasei IMAUJBC1 on the expression of FXR gene in mouse serum and liver;

[0028] Fig.11 The effect of Lactobacillus paracasei IMAUJBC1 on the intestinal flora of mice;

[0029] Fig.12 is the effect of Lactobacillus paracasei IMAUJBC1 on Akkermansia intestinal flora of mice;

[0030] Fig.13 The effect of Lactobacillus paracasei IMAUJBC1 on the intestinal flora Bacteroides of mice;

[0031] Note: * indicates significant differences between the groups represented (*: p<0.05; **: p<0.01; ***: p<0.001). DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1: Screening and preparation of Lactobacillus paracasei IMAUJBC1

[0034] It was derived from traditional fermented meat products in Inner Mongolia Autonomous Region, and was spread on MRS medium plates after gradient dilution and cultured at 37℃ for 24h. Single colonies were selected for purification and culture, and catalase test was performed on them. Gram-positive (G+) strains were identified by molecular biology. 2.5μL of PCR products of strain DNA were extracted and detected by 1% agarose gel electrophoresis. Samples with clear bands at 1500bp were sequenced with BLAST and NCBI data, and the results showed that they were Lactobacillus paracasei.

[0035] After DNA sequencing, the partial sequence of the 16S rRNA gene of Lactobacillus paracasei strain is:

[0036]

[0037] Characteristics of Lactobacillus paracasei IMAUJBC1:

[0038] (1) Bacterial characteristics: Gram-positive, short-rod bacteria, see Figure 1 .

[0039] (2) Colony characteristics: After culturing at 37°C for 24 h, the colonies appeared as white round protrusions.

[0040] (3) Physiological and biochemical characteristics: no production of bioamines, gas, viscosity or hydrogen sulfide.

[0041] The third generation bacterial suspension was cultured at 37°C for 24 h. After centrifugation and washing, it was mixed with freeze-drying protective agents such as trehalose, skim milk powder, sodium glutamate and maltodextrin, and then freeze-dried to prepare bacterial powder.

[0042] Example 2: Lactobacillus paracasei IMAUJBC1 has no toxic side effects on ICR mice

[0043] Lactobacillus paracasei IMAUJBC1 was prepared at a concentration of 1.0×10 9 Nine healthy male ICR mice weighing about 40-46 g were given a bacterial suspension of 0.5 mL / day by gavage after acclimating to the environment for one week. The mice were observed for one week and the mortality and body weight were recorded, as shown in Table 1.

[0044] Table 1: Body weight changes of mice in one week

[0045]

[0046] Example 3: Lactobacillus paracasei IMAUJBC1 slows down weight gain in mice fed a high-fat diet

[0047] Twenty-seven healthy male ICR mice weighing 40-46 g and aged 8 weeks were used. The mice were acclimatized for 1 week, modeled for 4 weeks, and intervened for 6 weeks. The experimental design is shown in Table 2.

[0048] Table 2: Animal experiment design

[0049]

[0050] The fat content of the high-fat diet was 60%, and body weight was recorded every three days.

[0051] At the end of the experiment, mice were fasted but not watered for 12 h, anesthetized with ether, and then killed by cervical dislocation. Blood was collected from the orbits, centrifuged at 2500 rpm and 4°C for 10 min, and the supernatant was collected and stored at -80°C. Organs and feces were removed with sterile forceps, weighed, and stored at -80°C.

[0052] The results of the weight test are as follows Figure 2 As shown, compared with the model group, Lactobacillus paracasei IMAUJBC1 reduced the weight of mice by 10.4%, which was close to the weight of the blank control group. This indicates that the Lactobacillus paracasei IMAUJBC1 of the present invention has the function of slowing down the weight gain of high-fat diet.

[0053] Example 4: Lactobacillus paracasei IMAUJBC1 reduces blood lipid levels in mice fed a high-fat diet

[0054] The ICR mice grouping, modeling and treatment methods were the same as in Example 3.

[0055] The experimental results are as follows Figure 3-6 The serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) of the model group mice increased significantly, while the TC, TG and LDL-C levels of the mice gavaged with Lactobacillus paracasei IMAUJBC1 decreased by 41%, 55.9% and 57.6%, respectively, indicating that IMAUJBC1 can improve the lipid levels of mice fed a high-fat diet. In addition, gavage with Lactobacillus paracasei IMAUJBC1 increased the content of serum high-density lipoprotein (HDL).

[0056] Example 5: Lactobacillus paracasei IMAUJBC1 reduces the expression level of cholesterol synthesis-related genes in the liver

[0057] The ICR mouse grouping, modeling and treatment methods are the same as in Example 3. The liver was removed and about 0.2 g of liver was weighed to extract total RNA. The concentration of RNA was determined using an ultraviolet spectrophotometer, and cDNA was synthesized using a reverse transcription kit. Real-time fluorescence quantitative PCR was used to determine the expression levels of mouse liver cholesterol synthesis genes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and adenylate-activated protein kinase (AMPK), and the internal reference gene was GAPDH. The primer sequences are shown in Table 3.

[0058] The PCR amplification procedure is as follows

[0059] (1) Pre-denaturation: 95°C, 30 s;

[0060] (2) PCR reaction: 95°C, 5 s; 57°C, 30 s; 72°C, 30 s; cycle 45 times.

[0061] (3) Melting curve analysis: 72°C, 600 s; 65°C, 60 s; 97°C, 1 s.

[0062] (4) Cooling: 37°C, 30 seconds.

[0063] The test results are as follows Figure 7 and Figure 8As shown in the figure, compared with the high-fat model group, oral administration of Lactobacillus paracasei IMAUJBC1 downregulated the expression of HMGCR gene by 45.84%, and significantly upregulated the expression of AMPK gene. HMGCR is the rate-limiting enzyme for cholesterol synthesis in the liver. AMPK inhibits the expression of HMGCR, thereby reducing the total cholesterol in the liver ( Figure 3 This indicates that Lactobacillus paracasei IMAUJBC1 can reduce the synthesis of liver cholesterol and reduce the accumulation of liver cholesterol.

[0064] Table 3: Gene primer sequence list

[0065]

[0066] Example 6: Lactobacillus paracasei IMAUJBC1 reduces the expression level of cholesterol catabolism-related genes in the liver

[0067] The grouping, modeling and treatment methods of ICR mice were the same as those in Example 3. The gene expression measurement methods of cholesterol 7α-hydroxylase (CYP7A1) and farnesoid X receptor (FXR) were the same as those in Case 5, and the gene primer sequences are shown in Table 3.

[0068] The test results are as follows Fig. 9 , 10 As shown. Compared with the high-fat model, oral administration of Lactobacillus paracasei IMAUJBC1 significantly upregulated the CYP7A1 gene by 2.14 times and the FXR gene by 1.49 times. FXR activates the expression of CYP7A1, and CYP7A1 promotes the decomposition of cholesterol to produce bile acid. This indicates that Lactobacillus paracasei IMAUJBC1 can promote the catabolism of cholesterol and reduce the accumulation of cholesterol in the liver.

[0069] Example 7: Regulatory effect of Lactobacillus paracasei IMAUJBC1 on intestinal flora of mice fed a high-fat diet

[0070] The grouping, modeling and treatment methods of ICR mice were the same as those in Example 3. After the experiment, the mouse feces were taken with sterile forceps and stored at -80°C. About 0.1 g of feces were weighed to extract total fecal bacterial DNA, and the intestinal flora was analyzed using Illumina sequencing technology.

[0071] The results of the mouse intestinal flora analysis experiment are as follows Figure 11-13 As shown. Oral administration of Lactobacillus paracasei IMAUJBC1 significantly increased the number of Lactobacillus genus in the intestinal flora of mice ( Lactobacillus ), Akkermansia ( Akkermansia ), Bacteroides ( Bacteroides) and the relative abundance of the MG group increased by 1.57, 3.72 and 1.49 times respectively compared with the MG group. A large number of studies have found that the above-mentioned intestinal bacterial genera are negatively correlated with liver cholesterol metabolism diseases. This shows that Lactobacillus paracasei IMAUJBC1 increases the abundance of beneficial bacteria in the intestine, can improve the intestinal flora disorder caused by a high-fat diet, and play a role in alleviating hypercholesterolemia.

[0072] The above is a detailed description of an embodiment of the present invention, but the content is only a preferred embodiment of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A cholesterol-lowering strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1, characterized in that The Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 21, 2021, with the deposit number CGMCC No.24176.

2. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei )Application of IMAUJBC1 in the preparation of medicines for preventing and treating hypercholesterolemia.

3. The use according to claim 2, characterized in that: The drug contains ≥1×10 7 CFU / g of Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1.

4. The use according to claim 2, characterized in that: The drug contains ≥1×10 9 CFU / mL of Lactobacillus paracasei ( Lacticaseibacillus paracasei )IMAUJBC1.

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