Lactobacillus plantarum PD01 and its application

Abstract

The present invention relates to the field of microbial technology, and specifically discloses Lactobacillus plantarum PD01 and its application. Lactiplantibacillus plantarum ) PD01, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 22, 2024, with the deposit number CGMCC No. 30386. The present invention uses Caenorhabditis elegans and mouse models to prove that Lactobacillus plantarum PD01 reduces obesity and inflammatory responses caused by microplastic intake by preventing microplastics from passing through the intestinal barrier.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Lactobacillus plantarum PD01 and applications thereof. Background Art

[0002] Although studies have shown that most microplastics will be excreted from the body through defecation after being ingested, due to the extremely small size of microplastic particles, some microplastics can still pass through the intestinal barrier, enter the blood, and accumulate in tissues, organs, and body fluids, affecting the absorption and metabolism of fat and indirectly increasing the risk of obesity.

[0003] It has been found that some probiotic strains can alleviate the toxicity of microplastics, mainly through their regulatory effects on the intestinal immune system. There are few reports of strains that can directly prevent microplastics from passing through the intestinal barrier and reduce the accumulation of microplastics in tissues. Summary of the invention

[0004] The purpose of the present invention is to provide a Lactobacillus plantarum PD01 and its application in view of the above-mentioned deficiencies in the prior art. Lactobacillus plantarum PD01 can prevent microplastics from passing through the intestinal barrier and entering tissues, reduce the accumulation of microplastics in the body, and alleviate obesity and inflammatory reactions caused by microplastics.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first object of the present invention is to provide a plant lactobacillus ( Lactiplantibacillus plantarum ) PD01, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 22, 2024. The address of the depository is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30386.

[0007] The second objective of the present invention is to provide the use of the above-mentioned Lactobacillus plantarum PD01 in the preparation of a preparation for preventing or treating obesity caused by ingestion of microplastics.

[0008] The experimental results of the present invention show that Lactobacillus plantarum PD01 can reduce obesity and inflammatory responses caused by microplastic intake.

[0009] The third objective of the present invention is to provide the use of the above-mentioned Lactobacillus plantarum PD01 in the preparation of medicines or foods for preventing or treating obesity caused by ingestion of microplastics.

[0010] Furthermore, the Lactobacillus plantarum PD01 alleviates obesity and inflammatory responses caused by microplastic intake by preventing microplastics from passing through the intestinal barrier.

[0011] Furthermore, the Lactobacillus plantarum PD01 has the ability to adsorb and remove microplastics.

[0012] Furthermore, the Lactobacillus plantarum PD01 has the ability to enhance the intestinal barrier.

[0013] Furthermore, the Lactobacillus plantarum PD01 promotes the production of propionic acid and butyric acid.

[0014] The fourth object of the present invention is to provide a preparation for preventing or treating obesity caused by ingestion of microplastics, containing the bacteria and / or fermentation products of the above-mentioned Lactobacillus plantarum PD01, wherein the fermentation product is a fermentation supernatant, and the fermentation supernatant is obtained after fermentation of Lactobacillus plantarum PD01 and filtration and sterilization.

[0015] The fifth object of the present invention is to provide a medicine containing the bacteria and / or fermentation products of the above-mentioned Lactobacillus plantarum PD01, wherein the fermentation product is a fermentation supernatant, and the fermentation supernatant is obtained after Lactobacillus plantarum PD01 is fermented and filtered and sterilized.

[0016] The sixth object of the present invention is to provide a food containing the bacteria and / or fermentation products of the above-mentioned Lactobacillus plantarum PD01, wherein the fermentation product is a fermentation supernatant, and the fermentation supernatant is obtained after Lactobacillus plantarum PD01 is fermented and filtered and sterilized.

[0017] As a specific example of the fermentation supernatant, 20 mL of Lactobacillus plantarum PD01 bacterial solution was added to a blue-capped bottle containing 1 L of fresh MRS liquid culture medium, the bottle cap was tightened, and the bottle was placed in a 37° C. incubator for 24 hours. After the cultured bacterial solution was centrifuged at 4000 rpm for 5 minutes, the supernatant was taken, and sterilized by 0.22 μm membrane filtration or sterilized at 121° C. for 20 minutes to obtain the fermentation supernatant.

[0018] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0019] (1) The present invention isolated and screened a strain PD01 from an adult fecal microbial sample and classified it as Lactobacillus plantarum Lactiplantibacillus plantarum , was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 22, 2024, with the deposit number CGMCC No. 30386. The strain PD01 reduces obesity and inflammatory responses caused by microplastic intake by preventing microplastics from passing through the intestinal barrier.

[0020] (2) The present invention provides Lactobacillus plantarum PD01, which can increase the intestinal barrier function and reduce the passage of microplastics through the intestine.

[0021] (3) The present invention provides Lactobacillus plantarum PD01, which can reduce the amount of microplastics entering tissues and reduce the accumulation of microplastics in the body.

[0022] (4) The present invention provides Lactobacillus plantarum PD01, which can produce short-chain fatty acids in vivo and improve intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of the colony morphology of Lactobacillus plantarum PD01;

[0024] Figure 2 This is a Gram staining picture of Lactobacillus plantarum PD01;

[0025] Figure 3 This is a comparison chart of the adsorption rate of microplastics by Lactobacillus plantarum PD01;

[0026] Figure 4 The distribution of fluorescent PS microplastics in Caenorhabditis elegans on different plates;

[0027] Figure 5 The results of the expression level of acs-22 gene in Caenorhabditis elegans on different plates are shown in the figure;

[0028] Figure 6 This is the result diagram of the expression level of genes related to the intestinal barrier in mice;

[0029] Figure 7 The results of PS fluorescent microsphere content in the feces of mice in each group;

[0030] Figure 8 This is a graph showing changes in the content of short-chain fatty acids in mouse feces;

[0031] Fig. 9 This is a graph showing the weight changes of mice after the intervention with Lactobacillus plantarum PD01;

[0032] Fig.10 This is a graph showing the expression levels of pro-inflammatory genes in mouse colon. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0034] The present invention isolates a strain of Lactobacillus plantarum from fecal samples of healthy adults, which can reduce obesity and inflammatory response caused by microplastic intake by preventing microplastics from passing through the intestinal barrier. Lactiplantibacillus plantarumThe strain, named PD01, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 22, 2024. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30386.

[0035] The strain "PD01" in the present invention is equivalent to "Lactobacillus plantarum PD01". Example 1

[0036] Origin, isolation and identification of Lactobacillus plantarum PD01.

[0037] Take a stool sample from a healthy adult and add it to MRS broth medium for enrichment culture. The enrichment culture conditions are 37°C, anaerobic, and static culture for 24 hours. Dilute the enriched culture solution and spread it on an MRS agar plate. After picking a single colony, inoculate it into liquid MRS broth medium for culture. The culture conditions are 37°C, anaerobic, and static culture for 24 hours. MRS medium is a medium well known to technicians in this technical field. Its formula is that each liter of medium contains 10.0 grams of peptone, 8.0 grams of beef powder, 4.0 grams of yeast powder, 20.0 grams of glucose, 2.0 grams of dipotassium hydrogen phosphate, 2.0 grams of diammonium hydrogen citrate, 5.0 grams of sodium acetate, 0.2 grams of magnesium sulfate, 0.04 grams of manganese sulfate, 1.0 grams of Tween 80, 1000 grams of water, and a pH value of 5.5-6.0.

[0038] The isolated plant lactobacillus PD01 has a suitable culture condition of 37°C, anaerobic, and MRS medium. Its colony morphology is milky white round colonies, opaque, smooth and moist surface, convex in the center, and neat edges. Figure 1 shown.

[0039] Gram staining of the culture fluid of Lactobacillus plantarum PD01 showed that the strain was a Gram-positive bacillus, in the form of a straight rod, in single, paired or chain form, without flagella and spores. Figure 2 shown.

[0040] The culture medium of Lactobacillus plantarum PD01 was taken and the 16S rRNA gene fragment of the bacteria was amplified by PCR technology. The amplified product was sequenced and compared with the NCBI database. The comparison results showed that the strain was Lactobacillus plantarum. Lactiplantibacillus plantarum , the sequencing sequence is shown in SEQ ID NO.1. Example 2

[0041] Acid resistance and bile salt tolerance of Lactobacillus plantarum PD01.

[0042] Take 1 mL of overnight cultured Lactobacillus plantarum PD01 and commercial strain Lactobacillus rhamnosus GG (LGG), centrifuge at 4000 rpm for 5 min, remove the supernatant, add 1 mL of MRS liquid medium with a pH value of 2.5 to resuspend, and place it in a shaking incubator at 37°C and 100 rpm for 3 h. Take the bacterial solution before and after incubation, dilute and plate to calculate the number of viable bacteria.

[0043] Take 1 mL of overnight cultured Lactobacillus plantarum PD01 and Lactobacillus rhamnosus LGG respectively, centrifuge at 4000 rpm for 5 min, remove the supernatant, add 1 mL of MRS liquid medium containing 0.3% bile salts to resuspend, and place in a shaking incubator at 37°C and 100 rpm for 4 h. Take the bacterial solution before and after incubation respectively, dilute and plate to calculate the number of viable bacteria.

[0044] The survival rate was calculated based on the number of viable bacteria, survival rate (%) = N1 / N0 × 100%, N0 is the number of viable bacteria before incubation, and N1 is the number of viable bacteria after incubation. The survival rate results are shown in Table 1 below. After incubation for 3 hours in MRS liquid medium with a pH value of 2.5, the survival rate of strain PD01 was 86.15%, which was equivalent to the survival rate of 90.43% of the control strain LGG, indicating that strain PD01 has strong acid resistance. After incubation for 4 hours in MRS liquid medium containing 0.3% bile salts, the survival rate of strain PD01 was 73.16%, which was equivalent to the survival rate of 78.17% of the control strain LGG, indicating that Lactobacillus plantarum PD01 has strong bile salt tolerance.

[0045] Table 1. Results of strain tolerance test

[0046] Example 3

[0047] Adsorption capacity of Lactobacillus plantarum PD01 on microplastics.

[0048] Take the overnight cultured Lactobacillus plantarum PD01 and Lactobacillus rhamnosus LGG bacterial suspensions, centrifuge at 6000rpm for 5 minutes, remove the supernatant, wash twice with sterile PBS solution, and resuspend and dilute with sterile PBS solution to OD 600Equal to 1.0. Take 100μL of the diluted bacterial solution, centrifuge to remove the supernatant, add 1mL of 30 μg / mL fluorescent PS microplastic solution with a particle size of 0.1 micron and resuspend it, and shake and culture at 37℃ for 4h. After the culture is completed, centrifuge and take the supernatant solution to detect the fluorescence value. The blank group only added 30 μg / mL fluorescent PS microplastic solution without bacteria. After shaking culture and centrifugation, the supernatant was collected to detect the fluorescence value. The adsorption rate of microplastics in the solution was calculated according to the formula: Adsorption rate (%) = (blank group supernatant fluorescence value-experimental group supernatant fluorescence value) / blank group supernatant fluorescence value × 100%. Lactobacillus plantarum PD01 adsorbed 78.90% of microplastics, and the control strain LGG only adsorbed 4.33% of microplastics, indicating that Lactobacillus plantarum PD01 has a strong ability to adsorb and remove microplastics. Figure 3 shown. Example 4

[0049] Lactobacillus plantarum PD01 blocks microplastics from crossing the intestinal barrier.

[0050] The wild-type C. elegans N2 strain was raised on NGM agar plates containing the E. coli OP50 strain. The culture temperature was 18°C. The NGM plate formula contained 2.5g of peptone, 3g of sodium chloride, 5mg of cholesterol, 1mmol of calcium chloride, 1mmol of magnesium sulfate, 25mL of 1mol / L PBS solution, 17g of agar, and a pH of 6.0 per liter. E. coli OP50 was cultured in LB liquid medium at 37°C for 24 hours. The LB medium formula was: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride per liter, and a pH of 7.4. The M9 solution formula used to wash C. elegans was: 3g of potassium dihydrogen phosphate, 6g of disodium hydrogen phosphate, 5g of sodium chloride, and 1mmol of magnesium sulfate per liter.

[0051] Use the cultured E. coli OP50 bacterial solution to dilute the fluorescent PS microplastics with a particle size of 1 micron to 0.1 mg / mL, draw 100 μL of the diluted solution and drop it in the center of the freshly prepared NGM agar plate, shake it gently until the liquid is evenly dispersed, and then place it in a 37°C incubator for 12 hours. Take the overnight cultured Lactobacillus plantarum PD01 and Lactobacillus rhamnosus LGG bacterial solutions, centrifuge and remove the supernatant, and resuspend them in M9 solution to OD 600 =30. Take 50 μL of the resuspended bacterial solution and drop it into the center of the NGM plate inoculated with E. coli and fluorescent PS microplastics. After gently shaking to disperse, place it at 37°C for 12 hours. The blank control group was inoculated with only E. coli OP50 and fluorescent PS microplastics.

[0052] The synchronized L1-stage Caenorhabditis elegans were picked and placed on the blank control plate, the plate inoculated with the PD01 strain, and the plate inoculated with the LGG strain, and cultured at 18°C ​​for 72 h. The cultured nematodes in each group were collected, anesthetized with 1% tetramisole hydrochloride solution, and transferred to a slide with an agar pad for observation under a fluorescence microscope.

[0053] like Figure 4 As shown, the distribution of fluorescent PS microplastics in Caenorhabditis elegans on different plates is shown. The dotted line represents the intestinal wall, and the fluorescence indicated by the arrow represents the fluorescent PS microplastics that pass through the intestinal wall and enter the tissue. As can be seen from the figure, in the blank control group and the LGG strain group, fluorescent PS microplastics are present in the intestine and tissues. For nematodes cultured on plates containing the PD01 strain, fluorescent PS microplastics are present in the intestine, and no fluorescent microplastics pass through the intestine into the tissue. Example 5

[0054] Lactobacillus plantarum PD01 promotes the expression of intestinal barrier-related genes in Caenorhabditis elegans.

[0055] Studies have shown that after the loss of the acs-22 gene in Caenorhabditis elegans, PS microplastics can more easily pass through the intestinal barrier and enter tissues and organs, causing higher microplastic toxicity.

[0056] RNA was extracted from Caenorhabditis elegans on different plates in Example 4, and after reverse transcription into cDNA, quantitative PCR analysis was performed using specific primers to detect the expression level of the intestinal barrier-related gene acs-22.

[0057] like Figure 5 As shown, it can be seen that compared with the blank control group, feeding the PD01 strain increased the expression of the acs-22 gene, which indicates that PD01 colonization enhances the intestinal barrier function of Caenorhabditis elegans and prevents the damage caused by microplastics entering the tissues. Example 6

[0058] Lactobacillus plantarum PD01 promotes the expression of intestinal barrier-related genes in mice.

[0059] To further verify the strengthening effect of Lactobacillus plantarum PD01 on the intestinal barrier, 6-week-old C57 mice were randomly divided into four groups, with 10 mice in each group, after 7 days of adaptation. The model group was gavaged with 1 mg of fluorescent PS microplastic solution every day, and the experimental strain group was gavaged with 1 mg of fluorescent PS microplastic solution and 1×10 9 CFU Lactobacillus plantarum PD01, mice in the control group were gavaged daily with 1 mg fluorescent PS microplastic solution and 1×10 9CFU rhamnosus LGG, and the blank group was gavaged with an equal amount of saline every day. The gavage intervention lasted for four weeks. After the intervention, the mice were deprived of water and food for 16 hours, and the small intestinal tissue was dissected after being killed to extract RNA. After reverse transcription of RNA into cDNA, quantitative PCR analysis was performed using specific primers to detect the expression levels of intestinal barrier-related genes Claudin-1 and ZO-1 (tight junction protein 1).

[0060] like Figure 6 As shown, it can be seen that compared with the blank group, the expression levels of Claudin-1 and ZO-1 genes in the model group mice decreased significantly, indicating that microplastic exposure caused intestinal barrier damage in mice. The LGG strain intervention in the control group partially upregulated the expression of Claudin-1 and ZO-1 genes. The experimental group was colonized with Lactobacillus plantarum PD01, which significantly upregulated the expression of Claudin-1 and ZO-1 genes and enhanced the intestinal barrier function of mice. Example 7

[0061] Lactobacillus plantarum PD01 reduces microplastics in mice.

[0062] Intervention with Lactobacillus plantarum PD01 enhanced the intestinal barrier function of mice, which may reduce the number of microplastics entering mouse tissues. Since microplastics that pass through the intestine may be widely distributed in a variety of mouse body fluids and tissues, and the content is extremely low, the number of microplastics that pass through the intestine cannot be directly assessed. Therefore, this example quantifies the number of microplastics excreted in mouse feces to confirm whether fewer microplastics pass through the intestine and enter tissues after strain intervention.

[0063] The feces of mice in Example 6 were collected during the 16-hour fasting period after the intervention was completed, and the PBS solution was added at a mass ratio of feces: PBS = 1:20 after weighing. After homogenization, impurities were filtered out with a 100 μm cell filter. The fluorescence signal intensity of the PS microspheres in the filtrate was detected by flow cytometry. The fluorescence intensity was substituted into the standard curve and converted to obtain the PS microsphere content in the filtrate.

[0064] like Figure 7 As shown, it can be seen that compared with the model group, Lactobacillus plantarum PD01 intervention increased the content of PS fluorescent microspheres in mouse feces, indicating that fewer fluorescent microplastics remained in the mice. This proves that Lactobacillus plantarum PD01 alleviates microplastic intake by preventing microplastics from passing through the intestinal barrier. Example 8

[0065] Lactobacillus plantarum PD01 promotes the production of short-chain fatty acids.

[0066] The model group mice and the experimental group PD01 strain intervention mice in Example 6 were taken, and the mouse feces were collected after the intervention, weighed and extracted with methanol, and the content of short-chain fatty acids in the extract was determined by gas chromatography-mass spectrometry.

[0067] The results are as follows Figure 8 As shown, colonization with PD01 strain significantly increased the contents of propionate and butyrate. Example 9

[0068] Lactobacillus plantarum PD01 alleviates obesity induced by microplastic ingestion.

[0069] The body weights of mice at the end of the intervention in Example 6 were recorded for comparative analysis.

[0070] The results are as follows Fig. 9 As shown in the figure, compared with the blank group that did not ingest microplastics, the mice in the model group gained weight significantly due to ingestion of microplastics. However, compared with the model group, the weight of mice in the PD01 group decreased significantly. This indicates that colonization with the PD01 strain can alleviate obesity caused by microplastic ingestion. Example 10

[0071] Lactobacillus plantarum PD01 alleviates the inflammatory response caused by microplastics.

[0072] The mouse colon in Example 6 was taken, RNA was extracted, and after reverse transcription into cDNA, quantitative PCR analysis was performed using specific primers to detect the expression levels of intestinal pro-inflammatory related genes IL-1β, TNF-α, IFN-γ, and IL-6.

[0073] The results are as follows Fig.10 As shown in the figure, compared with the blank group, microplastic ingestion induced colon inflammation and the expression level of pro-inflammatory factors increased significantly. After colonization with the PD01 strain, the expression level of pro-inflammatory factors decreased significantly and returned to normal levels, indicating that the PD01 strain can alleviate the inflammatory response caused by microplastic ingestion.

[0074] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0075] 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. The use of Lactobacillus plantarum PD01 in preventing microplastics from passing through the intestinal barrier to reduce obesity and inflammatory response caused by microplastic intake, characterized in that: The classification name of the Lactobacillus plantarum PD01 is Lactiplantibacillus plantarum, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on April 22, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30386; The particle size of the microplastic is 100 nm; The plant lactobacillus PD01 has the ability to enhance the intestinal barrier and adsorb microplastics in the small intestine exposed to microplastics.

2. The use according to claim 1, characterized in that The Lactobacillus plantarum PD01 has the ability to promote the production of propionate and butyrate in the small intestine exposed to microplastics.

3. A composition, characterized in that The composition comprises Lactobacillus plantarum with a deposit number of CGMCC No. 30386.

4. Use of the Lactobacillus plantarum PD01 according to any one of claims 1 to 2 or the composition according to claim 3 in the preparation of a medicine for preventing or treating obesity caused by ingestion of microplastics.

5. A health food, characterized in that: The health food comprises the bacteria of Lactobacillus plantarum PD01 according to any one of claims 1 to 2 or the composition according to claim 3, and a food-acceptable auxiliary material.

6. A drug, characterized in that The drug comprises the bacteria of Lactobacillus plantarum PD01 according to any one of claims 1-2 or the composition according to claim 3, and pharmaceutically acceptable excipients.

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