Lactobacillus plantarum JXZ01 for reducing blood fat and application of lactobacillus plantarum JXZ01

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum JXZ01 for reducing blood fat and application of the lactobacillus plantarum JXZ01, the lactobacillus plantarum JXZ01 is preserved in China Center for Type Culture Collection on September 29th, 2024, the preservation address is No. 299 in eight road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC NO: M 20242117, the lactobacillus plantarum JXZ01 is named as the lactobacillus plantarum JXZ01, the survival state is survival, and the lactobacillus plantarum JXZ01 is a lactobacillus plantarum JXZ01. The sequence of 16S rDNA (ribosomal deoxyribonucleic acid) of the lactobacillus plantarum JXZ01 is as shown in SEQ ID NO. 1. The plant lactobacillus JXZ01 disclosed by the invention has a remarkable blood fat reducing effect, and has a good application prospect in preparation of medicines or health foods for reducing blood fat.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a lipid-lowering Lactobacillus plantarum JXZ01 and an application thereof. Background Art

[0002] Hyperlipidemia refers to a disease characterized by abnormal metabolism or function of fat in the body, high cholesterol (TC) and triglycerides (TG), or low high-density lipoprotein cholesterol (HDL-C). Studies have shown that a high-sugar and high-fat diet can increase blood lipids. In recent years, hyperlipidemia is an important factor causing cardiovascular and cerebrovascular diseases, and high cholesterol is the main cause of hyperlipidemia. In addition to genetic factors, unhealthy lifestyles such as lack of exercise, overeating, smoking and drinking are the main causes of hyperlipidemia. At present, the main methods for controlling and lowering blood lipid levels include dietary intervention and drug therapy. Commonly used clinical therapeutic drugs such as statins and fibrates may cause myopathy, liver damage and other problems if taken for a long time. Therefore, there is an urgent need to develop a new, safe and effective drug that can effectively lower blood lipid levels.

[0003] Zebrafish is a commonly used model organism, with a genetic similarity of more than 85% with humans. On the fifth day after fertilization, all organs in the zebrafish are basically fully developed and function similarly to those of humans. And the metabolism of fat in zebrafish is similar to that of humans. Therefore, zebrafish are fed with a high-sugar and high-fat diet, which rapidly increases the fat content in the zebrafish blood, thereby establishing a zebrafish hyperlipidemia model. Oil Red O is a fat-soluble azo dye that can specifically stain neutral triglycerides, lipids, and lipoproteins in tissues and cells; when the tissue is immersed in the dye solution, the dye leaves the dye solution and transfers to the lipids in the tissue, making the lipids in the tissue appear red.

[0004] Lactobacillus plantarum currently has a certain research basis in lowering blood lipids, but its survival rate in the human digestive tract is low, especially for traditional fermented lactic acid bacteria such as Lactobacillus bulgaricus and Streptococcus thermophilus. These strains often cannot tolerate the high acid and high osmotic pressure gastrointestinal fluid environment, resulting in most strains being killed when passing through the digestive tract, and only a small amount can enter the intestine, thus limiting the performance of its probiotic function. In contrast, Lactobacillus plantarum exhibits superior digestive fluid tolerance. Studies have shown that Lactobacillus plantarum can survive efficiently in a variety of digestive enzymes, low pH gastric juice and high osmotic pressure bile. For example, Lactobacillus plantarum can tolerate bile and exhibit good cholesterol degradation ability under conditions simulating the human gastrointestinal tract. Lactobacillus plantarum not only has strong tolerance in the digestive tract, but its metabolites such as short-chain fatty acids (SCFAs) can also lower blood pressure through a variety of mechanisms, including inhibition of angiotensin-converting enzyme activity, regulation of oxidized low-density lipoprotein signaling pathways, and association with intestinal barrier function.

[0005] Therefore, Lactobacillus plantarum is considered to be one of the important directions for future probiotic research and application due to its superior digestive juice tolerance. Summary of the invention

[0006] The invention aims to provide a lipid-lowering Lactobacillus plantarum JXZ01 and application thereof, which has good lipid-lowering performance and can be effectively applied to the preparation of lipid-lowering medicines or health foods.

[0007] To achieve the above-mentioned purpose, the first aspect of the present invention provides a lipid-lowering Lactobacillus plantarum JXZ01, which has been preserved by the China Center for Type Culture Collection on September 29, 2024, with a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a preservation number of CCTCC NO: M 20242117, named: Lactiplantibacillus plantarum JXZ01, and the survival status is alive.

[0008] Furthermore, the sequence of 16S rDNA of the Lactobacillus plantarum JXZ01 is shown in SEQ ID NO.1.

[0009] The second aspect of the present invention provides a use of the lipid-lowering Lactobacillus plantarum JXZ01 in the preparation of a lipid-lowering medicine or health food.

[0010] Furthermore, the medicine or health food is a capsule, tablet, pill, granule, powder or liquid preparation. Compared with the prior art, the beneficial effects of the present invention are:

[0011] 1. The present invention successfully constructed a hyperlipidemia zebrafish model. Compared with the model group, the plantarum Lactobacillus JXZ01 of the present invention can significantly reduce the fat content level in the hyperlipidemia zebrafish.

[0012] 2. Compared with the model group, the plant lactobacillus JXZ01 of the present invention can significantly increase the HDL content level in the lipid-positive group of zebrafish with hyperlipidemia, and significantly reduce the MDA, LDL, TC, and TG content levels.

[0013] 3. The plant lactobacillus JXZ01 of the present invention is isolated from the feces of healthy infants for the first time. It is the same species as the known plant lactobacillus but a different strain. It has a significant lipid-lowering effect and has a good application prospect in the preparation of lipid-lowering drugs or health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The result of BLAST comparison of 16S rDNA sequences of Lactobacillus plantarum JXZ01 and known Lactobacillus plantarum in Example 1 of the present invention is shown in FIG.

[0015] Figure 2 This is a diagram showing the observation results of the effect of Lactobacillus plantarum JXZ01 on the fat content in zebrafish in Example 3 of the present invention;

[0016] Figure 3 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on the fat content in zebrafish in Example 3 of the present invention;

[0017] Figure 4 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on the MDA content in zebrafish in Example 4 of the present invention;

[0018] Figure 5 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on the HDL content in zebrafish in Example 4 of the present invention;

[0019] Figure 6 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on the LDL content in zebrafish in Example 4 of the present invention;

[0020] Figure 7 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on TC content in zebrafish in Example 4 of the present invention;

[0021] Figure 8 This is a statistical result diagram of the effect of Lactobacillus plantarum JXZ01 on TG content in zebrafish in Example 4 of the present invention. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention. The following is a description of the principles and features of the present invention, and the examples cited are only used to explain the present invention and are not used to limit the scope of the present invention. All other embodiments obtained by those skilled in the art without making creative work belong to the scope protected by the present invention. In the description of the present invention, it should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer for reagents or instruments used are conventional products that can be purchased commercially.

[0023] Example 1. Acquisition and preservation of Lactobacillus plantarum JXZ01

[0024] The plant lactobacillus JXZ01 of the present invention is a lactic acid bacterium that can effectively reduce blood lipids and can be used as a component of medicine to improve the symptoms of hyperlipidemia in humans. The 16S rDNA sequence of the plant lactobacillus JXZ01 is shown in SEQ ID NO.1.

[0025] Lactobacillus plantarum JXZ0116S rDNA sequence (SEQ ID NO.1, 1446bp):

[0026] TAATACATGCAGTCGTACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGAGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGT ACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTAGGAACCAGCCGCCTAAGG.

[0027] The new strain of Lactobacillus plantarum JXZ01 of the present invention was isolated from the feces of a healthy infant in Guangzhou City, Guangdong Province, and the specific method is as follows:

[0028] Use a 50mL sterile centrifuge tube to collect fresh feces, transport it to the laboratory through cold chain, and transfer the sample to the biosafety cabinet after surface disinfection. According to the mass of the feces, add an appropriate amount of sterile water to fully dissolve it, take an appropriate amount of sample and spread it on the MRS culture plate, and culture it in the anaerobic workstation. After 48 hours, pick a single colony and inoculate it into a new MRS culture plate. As for the culture in the anaerobic workstation, refer to the "Bergey Bacterial Identification Manual" (8th Edition) and the "Fungal Classification and Identification Manual" to observe the growth status of the colony. The strain isolated after purification was named and numbered JXZ01. The growth status of the strain is that the colony is round, the surface is smooth, the edges are neat, and the milky white is opaque.

[0029] The plant lactobacillus JXZ01 was expanded and cultured. The isolated JXZ01 strain was molecularly identified using 16S rDNA universal primers (27F: AGAGTTTGATCCTGGCTCAG [SEQ ID NO.2]; 1492R: TACGGCTACCTTGTTACGACTT [SEQ ID NO.3]). The 16S rDNA sequence was sequenced by GeneWeizhi Biotechnology Co., Ltd. and the obtained 16S rDNA sequence was BLAST-aligned in the Genome database of NCBI. The alignment results are shown in Figure 1 The results showed that the homology between the 16S rDNA sequence of Lactobacillus plantarum JXZ01 and the known Lactobacillus plantarum was >99%, and analysis with homologous strains confirmed that JXZ01 was a different strain of the same species of Lactobacillus plantarum.

[0030] The plant lactobacillus JXZ01 of the present invention was preserved by the China Center for Type Culture Collection on September 29, 2024, with a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, a preservation number of CCTCC NO: M 20242117, and was named: Lactiplantibacillus plantarum JXZ01 (Lactiplantibacillus plantarum JXZ01), and the survival state was survival.

[0031] Example 2: Construction and testing methods of zebrafish hyperlipidemia model

[0032] This embodiment is used to provide a method for constructing a zebrafish hyperlipidemia model. The zebrafish used in the zebrafish hyperlipidemia model of this embodiment is an AB zebrafish, which is bred by Guangdong Xinxuan Testing Co., Ltd. The specific construction method is as follows.

[0033] 1. Experimental reagents, materials and equipment

[0034] 1) Reagents and Materials

[0035] The reagents and materials used in this example are shown in Table 1.

[0036] Table 1 Reagents and materials

[0037]

[0038] 2) Instruments and Equipment

[0039] The instruments and equipment used in this example are shown in Table 2.

[0040] Table 2 Instruments and equipment

[0041]

[0042]

[0043] 3) Test product information

[0044] The sample information used in this example is shown in Table 3.

[0045] Table 3 Test product information

[0046]

[0047] 2. Construction of zebrafish hyperlipidemia model

[0048] 1) Model construction: 5dpf wild-type AB zebrafish were randomly selected and placed in a 6-well cell culture plate; the experiment set up a normal group, a model group, a positive group (atorvastatin calcium), 10 4 CFU / mL Lactobacillus plantarum JXZ01 group, 10 5 CFU / mL Lactobacillus plantarum JXZ01 group and 10 6 CFU / mL plant lactobacillus JXZ01 group. The normal group was fed with E3 culture water and ordinary feed, and the other groups were fed with glucose solution and high-fat feed, and incubated in a 28.5℃ incubator for several days. The new solution was replaced every 24 hours, and the corresponding solution was replaced at a fixed time, and the feed and solution were replaced. After the solution was replaced, it was placed in the incubator for further incubation.

[0049] 2) Intervention: After the high-fat model was established, the normal group was fed with E3 aquaculture water and ordinary feed, the model group was fed with E3 aquaculture water and high-fat feed, the positive group was fed with atorvastatin calcium solution and high-fat feed, and the Lactobacillus plantarum JXZ01 group was fed with the corresponding solution (50 mL 10 4 CFU / mL bacterial solution, 10 5 CFU / mL bacterial solution, 10 6 CFU / mL bacterial solution) and high-fat feed, fed twice a day, replaced with new solution every day, and the corresponding solution change and feed and solution change were performed at fixed times. After the solution change was completed, the cells were placed in a 28.5℃ incubator for further incubation for 14 days.

[0050] 3. Test methods

[0051] 1) Detection of relative fat content (%): Using the Oil Red O staining method, after the intervention, the zebrafish were collected into a centrifuge tube, fixed with 4% paraformaldehyde, and stained with Oil Red O. The Image J software was used to analyze the zebrafish Oil Red O staining. The formula for calculating the relative fat content (%) in zebrafish is as follows:

[0052]

[0053] 2) Detection of MDA, HDL, LDL, TC, and TG content: After the intervention, the zebrafish in each group were washed with pure water, 70 zebrafish in each group were taken and divided into 2 tubes, respectively, and transferred to centrifuge tubes, so that the total volume of the liquid and fry in the centrifuge tubes was no more than 100 μL (operated on ice), and 300 μL of pre-cooled PBS was added to each tube, and then mechanically homogenized, centrifuged at 5000 rpm for 10 min at 4°C, and the supernatant was mixed. The absorbance value of each well was measured at the corresponding wavelength using a detection kit and an enzyme-labeled instrument to measure the content of malondialdehyde (MDA), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC), and triglyceride (TG).

[0054] 3) Data statistics: The experimental data were expressed as mean ± SEM and analyzed by t-test. Compared with the normal group: # P<0.05, ## P<0.01, ### P<0.001; compared with the model group: & P<0.05, && P<0.01 &&& P<0.001. Using one-way ANOVA, compared with the model group: * P<0.05, ** P<0.01, *** P<0.001.

[0055] Example 3: Effect of Lactobacillus plantarum JXZ01 on fat content in zebrafish

[0056] Based on the zebrafish test system and the method for detecting the relative fat content by Oil Red O staining described in Example 2, the effect of the plant lactobacillus JXZ01 of the present invention on the fat content in zebrafish was explored. The results are shown in Tables 4 and Figure 2-3 As shown. Among them, Figure 2 and Figure 3 They are the observation graph and statistical graph of the effect of Lactobacillus plantarum JXZ01 on the fat content in zebrafish (n=6).

[0057] Table 4 Statistical table of the effect of Lactobacillus plantarum JXZ01 on fat content in zebrafish (n=6)

[0058]

[0059] The depth of zebrafish oil red O staining reflects the fat content in zebrafish. Figure 2-3It can be seen that compared with the normal group, the zebrafish oil red O staining in the model group was darker and the relative fat content in the body was significantly increased (P<0.001), indicating that the hyperlipidemia model was successfully constructed in this test. Compared with the model group, the zebrafish oil red O staining in the positive group was lighter and the relative fat content in the body was significantly reduced (P<0.001), which is consistent with the clinical results, indicating that this experiment is effective. Compared with the model group, 10 4 The zebrafish in the Lactobacillus plantarum JXZ01 group had lighter oil red O staining and a significantly lower relative fat content (P<0.01). 5 CFU / mL and 10 6 The relative fat content in zebrafish of the CFU / mL Lactobacillus plantarum JXZ01 group was significantly reduced (P<0.001), indicating that the Lactobacillus plantarum JXZ01 of the present invention can reduce the fat content level in zebrafish with hyperlipidemia.

[0060] Example 4: Effects of Lactobacillus plantarum JXZ01 on the levels of MDA, HDL, LDL, TC and TG in zebrafish

[0061] Based on the zebrafish test system and the method for detecting the contents of MDA, HDL, LDL, TC and TG described in Example 2, the effects of the plantarum Lactobacillus JXZ01 of the present invention on the contents of malondialdehyde (MDA), high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (TC) and triglycerides (TG) in zebrafish were investigated. The results are shown in Tables 5 and 6. Figures 4 to 8 As shown. Among them, Figures 4 to 8 Statistical graphs showing the effects of Lactobacillus plantarum JXZ01 on the levels of MDA, HDL, LDL, TC and TG in zebrafish (n=3).

[0062] Table 5 Statistical table of the effects of Lactobacillus plantarum JXZ01 on the content of MDA, HDL, LDL, TC and TG in zebrafish (n=3)

[0063]

[0064] Table 5 and Figures 4 to 8 The results showed that compared with the normal group, the levels of MDA, LDL, TC, and TG in the zebrafish in the model group were increased and statistically significant (P<0.05), and the HDL content was significantly reduced (P<0.01), indicating that the hyperlipidemia model was successfully established. Compared with the model group, the levels of MDA, LDL, TC, and TG in the zebrafish in the positive group were decreased and statistically significant (P<0.05), and the HDL content was significantly increased (P<0.05), which was consistent with the clinical results, indicating that this experiment was effective. Compared with the model group, the highest concentration group of Lactobacillus plantarum JXZ01 (10 6CFU / mL) zebrafish MDA, LDL, TC, and TG levels were significantly reduced (P<0.001), and HDL content was significantly increased (P<0.01), indicating that Lactobacillus plantarum JXZ01 has potential lipid-lowering effect.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A lipid-lowering Lactobacillus plantarum JXZ01, characterized in that: The plant lactobacillus JXZ01 was preserved by the China Center for Type Culture Collection on September 29, 2024, with the preservation address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the preservation number being CCTCC NO:M 20242117, named: plant lactobacillus JXZ01, and the survival status being alive.

2. The lipid-lowering Lactobacillus plantarum JXZ01 according to claim 1, characterized in that The sequence of 16S rDNA of the Lactobacillus plantarum JXZ01 is shown in SEQ ID NO.

1.

3. Use of the lipid-lowering Lactobacillus plantarum JXZ01 as claimed in claim 1 or 2 in the preparation of lipid-lowering medicines or health foods.

4. The use of a lipid-lowering Lactobacillus plantarum JXZ01 according to claim 3, characterized in that: The medicine or health food is in the form of capsules, tablets, pills, granules, powders or liquid preparations.

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