Lactiplantibacillus plantarum PD01 for alleviating obesity and inflammatory response caused by microplastics and use thereof

Abstract

The present invention relates to the technical field of microorganisms. Specifically disclosed are Lactiplantibacillus plantarum PD01 and the use thereof. Lactiplantibacillus plantarum PD01 was deposited in the China General Microbiological Culture Collection Center on 22 April 2024, with the deposit number CGMCC No. 30386. The present invention demonstrates, by means of Caenorhabditis elegans and a mouse model, that Lactiplantibacillus plantarum PD01 reduces obesity and inflammatory responses caused by the microplastic intake by means of preventing microplastics from crossing the intestinal barrier.

Description

A strain of *Lactobacillus plantarum* PD01 that alleviates microplastic-induced obesity and inflammatory response and its application Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Lactobacillus plantarum PD01 and its applications. Background Technology

[0002] Although studies have shown that most microplastics are excreted through feces after ingestion, due to their extremely small size, some microplastic particles can still pass through the intestinal barrier, enter the bloodstream, and accumulate in tissues, organs, and body fluids, affecting fat absorption and metabolism and indirectly increasing the risk of obesity.

[0003] Some probiotic strains have been found to alleviate microplastic toxicity, mainly through their regulatory effects on the intestinal immune system. However, there are few reports of strains that can directly prevent microplastics from crossing the intestinal barrier and reduce the accumulation of microplastics in tissues. Technical solutions

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing a *Lactobacillus plantarum* PDO1 and its application. *Lactobacillus plantarum* PDO1 can prevent microplastics from crossing the intestinal barrier and entering tissues, reduce the accumulation of microplastics in the body, and alleviate obesity and inflammatory responses caused by microplastics.

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

[0006] The first objective of this invention is to provide a Lactiplantibacillus plantarum PD01, which was deposited on April 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30386.

[0007] A second objective of this invention is to provide the use of the above-mentioned *Lactobacillus plantarum* PD01 in the preparation of formulations for the prevention or treatment of obesity caused by microplastic ingestion.

[0008] The experimental results of this invention show that *Lactobacillus plantarum* PDO1 can reduce obesity and inflammatory responses caused by microplastic intake.

[0009] A third objective of this invention is to provide the use of the above-mentioned *Lactobacillus plantarum* PD01 in the preparation of drugs or foods for the prevention or treatment of obesity caused by microplastic ingestion.

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

[0011] Furthermore, the plant lactobacillus 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 and butyric acids.

[0014] The fourth objective of this invention is to provide a preparation for preventing or treating obesity caused by microplastic ingestion, comprising the above-mentioned *Lactobacillus plantarum* PDO1 cells and / or fermentation products, wherein the fermentation products are fermentation supernatant, which is obtained by filtration and sterilization after fermentation of *Lactobacillus plantarum* PDO1.

[0015] The fifth objective of this invention is to provide a drug containing the above-mentioned *Lactobacillus plantarum* PDO1 cells and / or fermentation products, wherein the fermentation products are fermentation supernatants, which are obtained by filtration and sterilization after fermentation of *Lactobacillus plantarum* PDO1.

[0016] The sixth objective of this invention is to provide a food product containing the above-mentioned *Lactobacillus plantarum* PDO1 cells and / or fermentation products, wherein the fermentation products are fermentation supernatant, which is obtained by filtering and sterilizing *Lactobacillus plantarum* PDO1 after fermentation.

[0017] As a specific example of fermentation supernatant, 20 mL of *Lactobacillus plantarum* PD01 bacterial culture was added to a blue-capped bottle containing 1 L of fresh MRS liquid culture medium. The cap was tightened, and the bottle was placed in a 37°C incubator for 24 hours. After incubation, the bacterial culture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected. It was then filtered through a 0.22 μm membrane for sterilization, or sterilized at 121°C for 20 minutes to obtain the fermentation supernatant. Beneficial effects

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

[0019] (1) The present invention isolates and screens a strain PD01 from adult fecal microbial samples, classifies it as Lactiplantibacillus plantarum, and deposits it at the China General Microbiological Culture Collection Center on April 22, 2024, with accession number CGMCC No. 30386. This strain PD01 reduces obesity and inflammatory response caused by microplastic intake by preventing microplastics from crossing the intestinal barrier.

[0020] (2) The present invention provides plant lactobacillus PD01, which can increase the intestinal barrier function and reduce microplastics passing through the intestine.

[0021] (3) The present invention provides plant lactobacillus PD01, which can reduce the number of microplastics entering the tissue and reduce the accumulation of microplastics in the body.

[0022] (4) The present invention provides that Lactobacillus plantarum PDO1 can produce short-chain fatty acids in vivo, thereby improving intestinal health. Attached Figure Description

[0023] Figure 1 shows the colony morphology of Bacillus plantarum PD01.

[0024] Figure 2 shows a Gram staining image of Lactobacillus plantarum PD01;

[0025] Figure 3 shows a comparison of the adsorption rates of microplastics by Bacillus plantarum PD01.

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

[0027] Figure 5 shows the results of acs-22 gene expression levels in Caenorhabditis elegans on different plates.

[0028] Figure 6 shows the expression levels of genes related to the intestinal barrier in mice.

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

[0030] Figure 8 shows the changes in short-chain fatty acid content in mouse feces;

[0031] Figure 9 shows the changes in mouse body weight after intervention with *Lactobacillus plantarum* PD01.

[0032] Figure 10 shows the expression levels of pro-inflammatory genes in the mouse colon. Embodiments of the present invention

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0034] This invention isolates a strain of *Lactiplantibacillus plantarum* from fecal samples of healthy adults, which can reduce obesity and inflammatory responses caused by microplastic intake by preventing microplastics from crossing the intestinal barrier. This strain is named PD01 and was deposited on April 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30386.

[0035] In this invention, the strain "PD01" is equivalent to "Lactobacillus plantarum PD01".

[0036] Example 1

[0037] Source, isolation and identification of Lactobacillus plantarum PD01.

[0038] Fecal samples from healthy adults were added to MRS broth medium for enrichment culture at 37°C under anaerobic conditions for 24 hours. The enriched culture was then diluted and spread onto MRS agar plates. Single colonies were picked and inoculated into liquid MRS broth medium for further culture at 37°C under anaerobic conditions for 24 hours. MRS medium is a well-known medium in this field, with a formulation of 10.0 g peptone, 8.0 g beef meal, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, and 1000 g water per liter, with a pH of 5.5-6.0.

[0039] The isolated *Lactobacillus plantarum* PD01 was cultured under anaerobic conditions at 37°C on MRS medium. Its colony morphology consisted of milky-white, round, opaque colonies with a smooth, moist surface, a raised center, and neat edges, as shown in Figure 1.

[0040] Gram staining was performed on the culture medium of *Lactobacillus plantarum* PD01. Microscopic examination showed that the strain was a Gram-positive bacillus, appearing as straight rods, singly, in pairs, or in chains, without flagella or spores. (See Figure 2).

[0041] Culture medium of Lactiplantibacillus plantarum PD01 was used to amplify the bacterial 16S rRNA gene fragment using PCR technology. After sequencing the amplified product, it was compared with the NCBI database. The comparison results showed that the strain was Lactiplantibacillus plantarum, and the sequencing sequence is shown in SEQ ID NO.1.

[0042] Example 2

[0043] Lactobacillus plantarum PD01 acid resistance and bile salt tolerance.

[0044] Take 1 mL of overnight culture of *Lactobacillus plantarum* PD01 and the commercial strain *Lactobacillus rhamnosus* GG (LGG), respectively, centrifuge at 4000 rpm for 5 min, remove the supernatant, resuspend in 1 mL of MRS liquid medium (pH 2.5), and incubate at 37℃ with shaking at 100 rpm for 3 h. Take the bacterial culture before and after incubation, dilute and plate, and calculate the viability count.

[0045] Take 1 mL of overnight cultured *Lactobacillus plantarum* PD01 and *Lactobacillus rhamnosus* LGG, centrifuge at 4000 rpm for 5 min, remove the supernatant, resuspend in 1 mL of MRS liquid medium containing 0.3% bile salts, and incubate at 37℃ with shaking at 100 rpm for 4 h. Take the bacterial culture before and after incubation, dilute and plate, and calculate the viability count.

[0046] Survival rate was calculated based on the number of viable cells: Survival rate (%) = N1 / N0 × 100%, where N0 is the number of viable cells before incubation and N1 is the number of viable cells after incubation. The survival rate results are shown in Table 1 below. After incubation for 3 hours in MRS liquid medium with a pH of 2.5, the survival rate of strain PD01 was 86.15%, comparable to the 90.43% survival rate 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%, comparable to the 78.17% survival rate of the control strain LGG, indicating that *Lactobacillus plantarum* PD01 has strong bile salt tolerance.

[0047] Table 1. Results of strain tolerance test

[0048]

[0049] Example 3

[0050] Adsorption capacity of Lactobacillus plantarum PD01 for microplastics.

[0051] Overnight cultured *Lactobacillus plantarum* PD01 and *Lactobacillus rhamnosus* LGG cultures were centrifuged at 6000 rpm for 5 minutes, the supernatant was discarded, and the cultures were washed twice with sterile PBS solution, then resuspended in sterile PBS solution and diluted to OD. 600=1.0. Take 100 μL of the diluted bacterial solution, centrifuge to remove the supernatant, add 1 mL of 30 μg / mL fluorescent PS microplastic solution with a particle size of 0.1 μm and resuspend. Incubate at 37℃ with shaking for 4 h. After incubation, centrifuge and collect the supernatant solution to detect the fluorescence value. The blank group only added 30 μg / mL fluorescent PS microplastic solution, without adding bacteria. After shaking incubation and centrifugation, collect the supernatant and detect the fluorescence value. Calculate the adsorption rate of microplastics in the solution according to the formula: Adsorption rate (%) = (fluorescence value of supernatant in blank group - fluorescence value of supernatant in experimental group) / fluorescence value of supernatant in blank group × 100%. *Lactobacillus plantarum* PD01 adsorbed 78.90% of the microplastics, while the control strain LGG only adsorbed 4.33% of the microplastics, indicating that *Lactobacillus plantarum* PD01 has a strong ability to adsorb and remove microplastics, as shown in Figure 3.

[0052] Example 4

[0053] Lactobacillus plantarum PD01 inhibits microplastics from crossing the intestinal barrier.

[0054] Wild-type *C. elegans* strain N2 was cultured on NGM agar plates containing *Escherichia coli* strain OP50. The culture temperature was 18°C. The NGM plate formulation contained 2.5 g peptone, 3 g sodium chloride, 5 mg cholesterol, 1 mmol calcium chloride, 1 mmol magnesium sulfate, 25 mL 1 mol / L PBS solution, 17 g 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 formulation contained 10 g trypone, 5 g yeast extract, 10 g sodium chloride per liter, and a pH of 7.4. The M9 solution used to wash *C. elegans* contained 3 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, 5 g sodium chloride, and 1 mmol magnesium sulfate per liter.

[0055] Using a cultured *E. coli* OP50 suspension, fluorescent PS microplastics with a particle size of 1 μm were diluted to 0.1 mg / mL. 100 μL of the diluted solution was dropped into the center of a freshly prepared NGM agar plate, gently shaken until evenly dispersed, and then incubated at 37°C for 12 h. Overnight cultures of *Lactobacillus plantarum* PD01 and *Lactobacillus rhamnosus* LGG were centrifuged, the supernatant was discarded, and the plates were resuspended in M9 solution to OD00. 600 =30. Take 50 μL of the resuspended bacterial solution and add it dropwise to the center of the NGM plate inoculated with E. coli and fluorescent PS microplastics. Gently shake to disperse and incubate at 37°C for 12 h. The blank control group is inoculated with only E. coli OP50 and fluorescent PS microplastics.

[0056] L1-stage Caenorhabditis elegans, after synchronization, were picked and transferred to the blank control group plates, plates inoculated with PD01 strain, and plates inoculated with LGG strain, and incubated at 18°C ​​for 72 h. The cultured nematodes from each group were collected, anesthetized with 1% tetraimidazole hydrochloride solution, transferred to a glass slide with an agar pad, and observed using a fluorescence microscope.

[0057] Figure 4 shows the distribution of fluorescent PS microplastics in *C. elegans* on different plates. The dashed line represents the intestinal wall, and the arrows indicate fluorescent PS microplastics that have penetrated the intestinal wall and entered the tissue. The figure shows that fluorescent PS microplastics were present in both the intestinal tract and tissue in the blank control group and the LGG strain group. In nematodes cultured on plates containing the PD01 strain, fluorescent PS microplastics were present only in the intestinal tract, and no fluorescent microplastics penetrated the intestinal tract to enter the tissue.

[0058] Example 5

[0059] Lactobacillus plantarum PD01 promotes the expression of intestinal barrier-related genes in Caenorhabditis elegans.

[0060] Studies have shown that deletion of the acs-22 gene in Caenorhabditis elegans makes it easier for PS microplastics to cross the intestinal barrier and enter tissues and organs, resulting in higher microplastic toxicity.

[0061] RNA was extracted from Caenorhabditis elegans on different plates in Example 4. The RNA was reverse transcribed into cDNA, and quantitative PCR analysis was performed using specific primers to detect the expression level of the intestinal barrier-related gene acs-22.

[0062] As shown in Figure 5, compared with the blank control group, feeding with strain PD01 increased the expression of the acs-22 gene, indicating that PD01 colonization enhanced the intestinal barrier function of Caenorhabditis elegans and prevented damage caused by microplastics entering tissues.

[0063] Example 6

[0064] Lactobacillus plantarum PD01 promotes the expression of intestinal barrier-related genes in mice.

[0065] To further verify the intestinal barrier-enhancing effect of *Lactobacillus plantarum* PD01, 6-week-old C57 mice were randomly divided into four groups of 10 mice each after 7 days of acclimatization. The model group was administered 1 mg of fluorescent PS microplastic solution by gavage daily, while the experimental strain group was administered 1 mg of fluorescent PS microplastic solution and 1×10⁻⁶ mg of PD01 by gavage daily. 9 CFU (Citrate Lactobacillus plantarum) PD01, mice in the control strain group were administered 1 mg of fluorescent PS microplastic solution and 1 × 10⁻⁶ mg of fluorescent PS microplastic solution by gavage daily. 9CFU-containing *Rhamnosus rhamnosus* LGG was administered to mice via gavage daily in the control group, along with an equal volume of physiological saline. The gavage intervention lasted for four weeks. After the intervention, mice were deprived of food and water for 16 hours, sacrificed, and their small intestine tissue was dissected. RNA was extracted, reverse transcribed into cDNA, and then 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).

[0066] As shown in Figure 6, compared with the control group, the expression levels of Claudin-1 and ZO-1 genes in the model group mice were significantly decreased, indicating that microplastic exposure caused damage to the intestinal barrier in mice. The intervention with the LGG strain in the control group partially upregulated the expression of Claudin-1 and ZO-1 genes. The experimental group, colonized with *Lactobacillus plantarum* PD01, significantly upregulated the expression of Claudin-1 and ZO-1 genes, enhancing the intestinal barrier function in mice.

[0067] Example 7

[0068] Lactobacillus plantarum PD01 reduces microplastics in mice.

[0069] Intervention with *Lactobacillus plantarum* PD01 enhanced the intestinal barrier function in mice, which may reduce the amount of microplastics entering mouse tissues. Since microplastics that cross the intestine can be widely distributed in various mouse body fluids and tissues, and their concentrations are extremely low, the amount of microplastics crossing the intestine cannot be directly assessed. Therefore, this embodiment uses quantitative analysis of the amount of microplastics excreted in mouse feces to confirm whether a reduced number of microplastics cross the intestine and enter tissues after strain intervention.

[0070] Feces from mice were collected during the 16-hour fasting and dehydration period following the intervention in Example 6. The feces were weighed and then mixed with PBS solution at a mass ratio of 1:20 (feces:PBS) to form a homogenate. Impurities were removed by filtering the homogenate through a 100 μm cell filter. The fluorescence signal intensity of the PS microspheres in the filtrate was detected using flow cytometry. The PS microsphere content in the filtrate was calculated by substituting the fluorescence intensity into a standard curve.

[0071] As shown in Figure 7, compared with the model group, *Lactobacillus plantarum* PD01 intervention increased the content of PS fluorescent microspheres in mouse feces, indicating that less fluorescent microplastics remained in the mice. This demonstrates that *Lactobacillus plantarum* PD01 alleviates microplastic uptake by preventing microplastics from crossing the intestinal barrier.

[0072] Example 8

[0073] Lactobacillus plantarum PD01 promotes the production of short-chain fatty acids.

[0074] Mice from the model group and experimental group of the PD01 strain in Example 6 were used for intervention. After the intervention, mouse feces were collected, weighed, and extracted with methanol. The content of short-chain fatty acids in the extract was determined by gas chromatography-mass spectrometry.

[0075] As shown in Figure 8, colonization of strain PD01 significantly increased the content of propionic acid and butyric acid.

[0076] Example 9

[0077] Lactobacillus plantarum PD01 alleviates obesity caused by microplastic intake.

[0078] The body weight of mice at the end of the intervention in Example 6 was recorded and compared.

[0079] As shown in Figure 9, compared with the control group (no microplastic intake), the model group mice showed a significant increase in body weight due to microplastic intake. However, compared with the model group, the PD01 group mice showed a significant decrease in body weight. This indicates that colonization of the PD01 strain can alleviate obesity induced by microplastic intake.

[0080] Example 10

[0081] Lactobacillus plantarum PD01 alleviates the inflammatory response caused by microplastics.

[0082] RNA was extracted from the mouse colon of Example 6, reverse transcribed into cDNA, and then quantitative PCR analysis was performed using specific primers to detect the expression levels of intestinal pro-inflammatory genes IL-1β, TNF-α, IFN-γ, and IL-6.

[0083] As shown in Figure 10, compared with the control group, microplastic intake induced colonic inflammation, and the expression levels of pro-inflammatory factors increased significantly. After colonization with strain PD01, the expression levels of pro-inflammatory factors decreased significantly and returned to normal levels, indicating that strain PD01 can alleviate the inflammatory response caused by microplastic intake.

[0084] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0085] 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 principles of the present invention should be included within the protection scope of the present invention. Industrial applicability

[0086] This disclosure provides *Lactobacillus plantarum* PDO1, which adsorbs microplastics and prevents them from crossing the intestinal barrier, thereby reducing obesity and inflammatory responses caused by microplastic intake. Therefore, the *Lactobacillus plantarum* PDO1 provided by this invention can be used to prepare pharmaceuticals or health foods that alleviate microplastic-induced obesity and inflammatory responses, and has industrial applicability.

Claims

1. The application of *Lactobacillus plantarum* PDO1 in reducing obesity and inflammatory responses induced by microplastic intake by inhibiting microplastics from crossing the intestinal barrier, characterized in that... The classification name of the plant lactobacillus PD01 is *Lactiplantibacillus plantarum*. It was deposited on April 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30386. The microplastics have a particle size of 100 nm; The plant lactobacillus PD01 has the ability to both enhance the intestinal barrier and adsorb microplastics in the small intestine exposed to microplastics.

2. The application as described in claim 1, characterized in that, The plant lactobacillus PD01 has the ability to promote the production of propionic and butyric acids in the small intestine exposed to microplastics.

3. A composition, characterized in that, The composition includes *Lactobacillus plantarum* with accession number CGMCC No. 30386.

4. The use of *Lactobacillus plantarum* PDO1 as described in any one of claims 1-2 or the composition as described in claim 3 in the preparation of a medicament for the prevention or treatment of obesity caused by microplastic ingestion.

5. A health food product, characterized in that, The health food includes the bacterial cells of *Lactobacillus plantarum* PD01 as described in any one of claims 1-2 or the composition as described in claim 3, and food-acceptable excipients.

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

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