Lactobacillus plantarum strain dt55 and application thereof in preparation of product with microplastic adsorption and removal function

Abstract

The present application relates to the field of microbial technology, and specifically discloses a lactobacillus plantarum DT55 and its application in preparing products with micro-plastic adsorption and removal functions.A new lactobacillus plantarum (Lactiplantibacillus plantarum) DT55 is developed, and the preservation number is GDMCC No:63623.The strain can adsorb micro-plastics, accelerate the discharge of plastics in the body, reduce the micro-plastic residues in the intestinal tract of mice, reduce the oxidative damage caused by micro-plastic accumulation, and reduce the inflammatory response caused by micro-plastics, and is expected to be developed into a probiotic product to protect human health.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus plantarum DT55 and its application in the preparation of products with microplastic adsorption and removal functions. Background Technology

[0002] With the widespread use of plastic products, a large amount of plastic waste is discarded into the environment and decomposed into tiny particles under physical, chemical and biological processes. Plastic particles with a diameter of less than 5 mm are usually defined as microplastics (MPs), and plastic particles with a diameter of less than 0.1 μm are usually defined as nanoplastics (NPs).

[0003] Microplastic pollution has become a serious environmental problem and also poses a potential threat to human health. Microplastics are made of materials such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), and polyethylene terephthalate (PET). They are widely present in air, water, and soil, and can be ingested by plankton, fish, birds, and other organisms, ultimately entering the human body through the food chain. Studies have shown that the human body can ingest up to 5g of microplastics per week, and microplastics are present in human feces, blood, lung tissue, and placenta. Although there is currently no direct evidence confirming the harmful effects of microplastics on human health, numerous studies have confirmed that microplastics can damage the digestive, respiratory, immune, nervous, and reproductive systems of rodents and aquatic organisms. Microplastics accumulated in tissues cannot be eliminated, leading to a significant increase in reactive oxygen species, causing oxidative stress and toxic effects. Therefore, eliminating microplastics from the human body and reducing their levels is of great importance for long-term human health.

[0004] Currently, there are only a few reports on biological methods for reducing plastic pollution in the environment and water bodies. For example, some bacteria and fungi have been reported to have the ability to degrade plastics by secreting keratinase, protease, esterase, and lipase, breaking down polymers into monomers or oligomers. Another way to remove plastic particles is through bacterial adsorption. Some bacteria can attach to the surface of microplastics and form sticky biofilms. This sticky matrix can capture free microplastics, leading to the bioaggregation of microplastics, thereby achieving the separation and removal of microplastics.

[0005] However, existing bacteria and fungi capable of degrading or adsorbing microplastics are not edible strains, and these strains cannot tolerate the gastrointestinal environment. Therefore, their adsorption capacity is difficult to apply to the removal of microplastics from the human body. In order to reduce the accumulation of microplastics in the human body and reduce the health damage caused by microplastics, there is a need in this field to discover edible probiotics that can tolerate the gastrointestinal environment and adsorb and remove microplastics. Summary of the Invention

[0006] One of the objectives of this invention is to develop a new type of edible probiotic that is expected to remove microplastics from the human body and reduce oxidative damage.

[0007] To achieve this objective, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a strain of Lactiplantibacillus plantarum DT55, which has the accession number GDMCC No:63623.

[0009] The *Lactobacillus plantarum* strain DT55 of this invention was isolated from the feces of healthy adults. The strain was identified by bacterial morphology, physiology, and 16S rRNA sequencing, and was named *Lactobacillus plantarum* DT55. This strain was deposited on July 5, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, 510070, China. Its classification name is *Lactobacillus plantarum*, and its accession number is GDMCC No: 63623.

[0010] Lactobacillus plantarum DT55 has the following microbiological characteristics:

[0011] (1) Morphological characteristics

[0012] Gram staining is positive. Under a light microscope, the cells are rod-shaped with rounded ends, and they are arranged as single cells or in pairs or chains.

[0013] After culturing in MRS solid medium for 24 hours, round, convex, smooth-edged, moist, milky-white colonies are formed.

[0014] (2)Physiological characteristics

[0015] Lactobacillus plantarum DT55 can grow in acidic or bile-containing media. This strain can effectively adsorb microplastics, with an adsorption rate exceeding 83%. Lactobacillus plantarum DT55 has antioxidant effects, reducing oxidative damage caused by microplastics. Furthermore, Lactobacillus plantarum DT55 can reduce microplastic residues in the mouse intestine and decrease the inflammatory response caused by microplastics.

[0016] Lactobacillus plantarum is widely found in fermented foods and is used in the food industry as a starter culture and preservative. It is listed in the list of microbial strains that can be used in food and is included in the European Union's Quality Professionals List (QPS) issued by the European Food Safety Authority (EFSA). It has also received GRAS (generally recognized as safe) certification from the US Food and Drug Administration (FDA) and is widely used globally in various probiotic foods and dietary supplements. Genomic research data, mouse experiments, and human clinical trials have all demonstrated the safety of Lactobacillus plantarum.

[0017] Secondly, the present invention provides a microbial agent comprising the aforementioned Lactobacillus plantarum DT55.

[0018] The bacterial agent of the present invention is a solid bacterial agent or a liquid bacterial agent.

[0019] Thirdly, the present invention provides a food product containing the aforementioned Lactobacillus plantarum DT55 or a bacterial agent.

[0020] Fourthly, the present invention provides a medicine comprising the above-mentioned Lactobacillus plantarum DT55 or a bacterial agent.

[0021] The *Lactobacillus plantarum* DT55 strain of this invention exhibits good acid and bile salt resistance. Furthermore, adsorption experiments on microplastics demonstrate that this strain can effectively adsorb microplastics and promote their excretion from the body. In addition, *Lactobacillus plantarum* DT55 also possesses antioxidant effects and reduces inflammatory responses. This strain can be developed into an edible probiotic, potentially achieving the goal of adsorbing microplastics by intestinal bacteria, accelerating microplastic excretion, reducing microplastic damage, and protecting the gastrointestinal tract.

[0022] The pharmaceutical products of this invention also include pharmaceutically acceptable excipients and are prepared using methods conventional in the art. They can be prepared by conventional mixing, granulation, sugar coating, dissolving, or freeze-drying methods, and may simultaneously contain other active ingredients.

[0023] Fifthly, the present invention provides a method for preparing the above-mentioned bacterial agent, wherein when the bacterial agent is a liquid bacterial agent, the method includes the step of culturing Lactobacillus plantarum DT55 to obtain a bacterial suspension; and when the bacterial agent is a solid bacterial agent, the method further includes the step of drying the bacterial suspension.

[0024] The solid bacterial agent is a bacterial suspension of Lactobacillus plantarum DT55 or a product obtained by spray drying or freeze drying of a treated bacterial suspension using conventional methods in the art.

[0025] In the preparation method of this invention, when Lactiplantibacillus plantarum DT55 is cultured, the culture medium used is MRS medium, and the culture conditions are anaerobic at 36-37°C.

[0026] Specifically, the above-mentioned method for culturing *Lactobacillus plantarum* DT55 involves inoculating *Lactobacillus plantarum* DT55 into MRS medium and anaerobically culturing it at 37°C for 24 hours. The MRS broth medium comprises: 10.0 g / L casein digest, 10.0 g / L beef meal, 4.0 g / L yeast extract, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 1.0 g / L Tween 80, pH 5.7 ± 0.2, and 1.5% agar added to the solid medium.

[0027] Based on the efficacy characteristics of this strain, in a sixth aspect, the present invention provides the application of the above-mentioned Lactiplantibacillus plantarum DT55 or its inoculum in the preparation of food or pharmaceuticals.

[0028] In a seventh aspect, the present invention provides the use of the above-mentioned Lactiplantibacillus plantarum DT55 or bacterial agent in the preparation of products that can reduce oxidative damage and / or inflammatory responses.

[0029] Preferably, it reduces oxidative damage and / or inflammatory response caused by microplastic accumulation.

[0030] Eighthly, the present invention provides the use of the above-mentioned Lactiplantibacillus plantarum DT55 or bacterial agent in the preparation of products that can adsorb microplastics and / or promote the excretion of microplastics from the body.

[0031] The beneficial effects of this invention are at least as follows:

[0032] Compared with existing technologies, the novel *Lactobacillus plantarum* DT55 strain provided by this invention has better acid and bile salt resistance and is expected to be developed into an edible probiotic. Moreover, this strain can adsorb microplastics, accelerate the excretion of microplastics, reduce the residue of microplastics in the intestine and reduce oxidative damage and inflammatory response caused by microplastic accumulation, thus providing great benefits to human intestinal health. Attached Figure Description

[0033] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum DT55 on MRS medium in Example 1.

[0034] Figure 2 This is a microscopic image of Lactobacillus plantarum DT55 from Example 1.

[0035] Figure 3 The survival rate of *Lactobacillus plantarum* DT55 in acidic medium in Example 2 is shown. The statistical analysis method was the T-test, where ns represents a p-value greater than 0.05. Data are from three replicate experiments, and the error bar represents the standard deviation.

[0036] Figure 4 The survival rate of *Lactobacillus plantarum* DT55 in bile salt-containing medium in Example 3 is shown. The statistical analysis method was the T-test, where ns represents a p-value greater than 0.05. Data were obtained from three replicate experiments, and the error bar represents the standard deviation.

[0037] Figure 5 This is a photograph of Lactobacillus plantarum DT55 adsorbing and agglomerating with PS fluorescent microspheres in solution in Example 4.

[0038] Figure 6 Example 4 shows the adsorption rate of PS fluorescent microspheres in solution by *Lactobacillus plantarum* DT55. Error bars represent standard deviations, and data were obtained from three replicate experiments. Statistical analysis was performed using the T-test, with **** indicating a p-value less than 0.0001.

[0039] Figure 7 This is an adsorption electron microscope image of Lactobacillus plantarum DT55 and PS fluorescent microspheres in Example 4, magnified 50,000 times.

[0040] Figure 8 Example 5 shows the DPPH scavenging rate of *Lactobacillus plantarum* DT55. Error bars represent standard deviations, and data are from three replicate experiments.

[0041] Figure 9 The image shown is an electron microscope image of Lactobacillus plantarum DT55 and mixed microplastic particles after incubation in Example 6, at a magnification of 2000x.

[0042] Figure 10 This is a fluorescence image of *Lactobacillus plantarum* DT55 and mixed microplastic particles after incubation in Example 6. Green signals represent *Lactobacillus plantarum* DT55, and red signals represent microplastic particles. Arrows indicate that *Lactobacillus plantarum* DT55 is adsorbed on the surface of the microplastic particles. Magnification is 1000x.

[0043] Figure 11The results show the residual PS fluorescent microspheres in the mouse ileum (A) and cecum (B) in Example 7. Error bars represent standard deviations. The statistical analysis method was ANOVA. * indicates a p-value less than 0.05. ** indicates a p-value less than 0.01. **** indicates a p-value less than 0.0001.

[0044] Figure 12 The results of immune factor detection in mouse serum (A) and ileum tissue (BE) in Example 8 are shown. Error bars represent standard deviations. The statistical analysis method is ANOVA, where ** indicates a p-value less than 0.01, *** indicates a p-value less than 0.001, and **** indicates a p-value less than 0.0001. Detailed Implementation

[0045] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0047] Example 1: Isolation and Identification of Lactobacillus plantarum DT55

[0048] 1. Isolation and identification of Lactobacillus plantarum DT55

[0049] 1.1 Sample Source

[0050] The strain Lactobacillus plantarum DT55 used in this invention was isolated from the feces of healthy adults.

[0051] 1.2 Preparation of culture medium

[0052] The medium used for sample isolation and strain screening was MRS medium, and the medium used for culturing Lactobacillus plantarum DT55 was MRS medium (pH 5.7 ± 0.2). The composition of MRS medium is shown in Table 1. Adding 1.5% agar makes it MRS solid medium.

[0053] Table 1 MRS culture medium formulation

[0054]

[0055]

[0056] 1.3 Isolation of strains

[0057] Add 1g of healthy adult fecal sample to 10mL of the MRS liquid culture medium prepared in step 1.2, mix well, and incubate at 36℃ for 24h. Then, in a clean bench, take 1mL of the enrichment solution and perform a tenfold serial dilution. Select 10... -4 10 -5 10 -6 10 -7 Four dilution gradients of bacterial suspension, 100 μL each, were spread onto culture dishes containing sterile MRS solid medium and incubated under anaerobic conditions at 36°C for 48-72 h until obvious single colonies were formed. Then, a high-throughput automated platform was used to automatically pick typical colonies from the culture dishes and culture them in MRS liquid medium. The species information of the isolated strains was determined by 16S rRNA sequencing.

[0058] 2. Identification of Lactobacillus plantarum DT55

[0059] 2.1 Colony characteristics

[0060] After culturing *Lactobacillus plantarum* DT55 in MRS solid medium for 24 hours, it formed round, convex, smooth-edged, moist, milky-white colonies. Figure 1 .

[0061] 2.2 Microscopic morphology

[0062] Lactobacillus plantarum DT55 colony smear: Gram-positive; under light microscopy, cells appear rod-shaped with rounded ends, arranged singly or in pairs or chains. See Figure 2 .

[0063] 2.3 Identification of 16S rRNA

[0064] Testing organization: Qingke Biotechnology Co., Ltd.

[0065] Identification sequence: see SEQ ID No. 1.

[0066] Identification results: By comparing the sequencing results with the NCBI database and combining the comparison results with physiological and biochemical results, the strain was identified as Lactiplantibacillus plantarum.

[0067] Example 2: Acid resistance test of Lactobacillus plantarum DT55

[0068] The overall pH environment of the human stomach is highly acidic; therefore, the acid resistance of a bacterial strain is an important indicator for assessing its ability to survive and colonize in the acidic environment of the stomach. The commercially available strain *Lactobacillus rhamnosus* GG is a widely used probiotic with strong acid resistance.

[0069] In this invention, the acid tolerance of *Lactobacillus plantarum* DT55 was verified using MRS medium at pH 2.5. 1 mL of bacterial culture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the culture was washed once with 1 mL of PBS. After centrifugation at 4000 rpm for 10 min, the precipitate was resuspended in MRS medium at pH 2.5. The culture was incubated at 37°C for 3 h, with samples taken at 0 h and 3 h. After centrifugation, the samples were resuspended in PBS and serially diluted. The diluted samples were then plated on MRS agar plates and anaerobically cultured at 37°C for 16 h before colony counting. The survival rate was calculated using the formula: Acid tolerance survival rate (%) = C1 / C0 × 100% (C0: 0 h count result; C1: 3 h count result). The control strain was *Lactobacillus rhamnosus* GG.

[0070] After 3 hours of incubation in acidic medium, the survival rate of the control strain *Lactobacillus rhamnosus* GG was 84.56%, while the survival rate of *Lactobacillus plantarum* DT55 was 83.90%. Figure 3 The acid resistance of this strain was comparable to that of the control strain, indicating that this strain has strong acid resistance and can survive in the stomach environment.

[0071] Example 3: Detection of bile salt tolerance in Lactobacillus plantarum DT55

[0072] After bacteria enter the intestines from the stomach, the high concentration of bile salts in the small intestine kills them. Food typically remains in the small intestine for 1–4 hours. Therefore, this invention uses 0.1% bile salt-MRS medium to verify the bile salt tolerance of the *Lactobacillus plantarum* DT55 strain.

[0073] Lactobacillus plantarum DT55 bacterial suspension was inoculated into 96-well plates containing MRS medium and anaerobically cultured at 37°C for 24 h. 300 μL of the cultured suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 600 μL of MRS medium containing 0.1% bile salts was added, followed by resuspending and mixing. For the control group, 100 μL of the resuspended suspension was added to 20 μL of MTT (thiazolyl blue) solution; for the treatment group, 100 μL of the resuspended suspension was incubated at 37°C for 4 h, followed by the addition of 20 μL of MTT solution. After adding MTT solution, the reaction was carried out at 37°C in the dark for 4 h. After the reaction, the plates were centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. 100 μL of DMSO solution was added to each well, and the plates were incubated at 37°C with shaking for 10 min to completely dissolve and mix the generated blue-purple formazan. The absorbance at 570 nm was measured using a microplate reader to calculate the survival rate. Survival rate = A1 / A0 × 100% (A1: absorbance of the treatment group solution at 570 nm, A0: absorbance of the control group solution at 570 nm). The survival rate of the control strain Lactobacillus rhamnosus GG was determined using the same method.

[0074] After 4 hours of culture in 0.1% bile salt-MRS medium, the survival rate of the control strain *Lactobacillus rhamnosus* GG was 101.5%, and the survival rate of *Lactobacillus plantarum* DT55 was 113.4%. Figure 4 This indicates that *Lactobacillus plantarum* DT55 has comparable bile salt tolerance to the control strain *Lactobacillus rhamnosus* GG and can survive in the small intestine.

[0075] Example 4: Determination of the adsorption effect of Lactobacillus plantarum DT55 on microplastics

[0076] Lactobacillus plantarum DT55 was inoculated into MRS medium and anaerobically cultured at 37°C for 24 h. The cultured bacterial suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the suspension was washed twice with 450 μL of sterile PBS buffer. The suspension was then resuspended in PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL. In the experimental group, 100 μL of *Lactobacillus plantarum* DT55 bacterial suspension was added to a 1.5 mL EP tube, along with 900 μL of PS fluorescent microsphere working solution (0.16 mg / mL, 0.1 μm particle size, Basell). The mixture was then incubated on a shaker in the dark for 4 hours at 37°C and 800 rpm. In the blank control group, 100 μL of PBS and 900 μL of PS fluorescent microsphere working solution were added to a 1.5 mL EP tube and incubated. In the bacterial control group, 100 μL of *Lactobacillus plantarum* DT55 bacterial suspension and 900 μL of PBS were added to a 1.5 mL EP tube and incubated. After incubation, the incubation solution was observed and photographed. Results are shown below. Figure 5 .

[0077] Incubation solutions from the experimental group and blank control group were centrifuged at 2000 rpm for 10 min, and 100 μL of the supernatant was collected for fluorescence intensity measurement using a microplate reader. The microplate reader parameters were: excitation wavelength, 494 nm; detection wavelength, 518 nm. The adsorption rate was calculated based on the fluorescence intensity values. The control strain was another *Lactobacillus plantarum* strain screened in the same batch of screening experiments, and its adsorption rate was measured using the same method. The adsorption rate was calculated using the formula: Adsorption rate (%) = (A1 - A2) / A1 × 100% (A1: fluorescence value of the blank control group, A2: fluorescence value of the *Lactobacillus plantarum* group). The results are shown in the table below. Figure 6 The precipitate from the experimental group was centrifuged, fixed overnight with glutaraldehyde at 4°C, then dehydrated using a gradient of ethanol, dried, and observed under an electron microscope. The results are shown in [Figure number missing]. Figure 7 .

[0078] from Figure 5 It can be seen that in the blank control group, PS fluorescent microspheres did not self-aggregate; in the bacterial culture control group, Lactobacillus plantarum DT55 did not self-aggregate; in the experimental group, Lactobacillus plantarum DT55 and PS fluorescent microspheres showed specific adsorption and agglomeration of flocculent matter.

[0079] from Figure 6 As can be seen, the adsorption rate of the control strain was 8.03%, indicating poor microplastic adsorption capacity, while the adsorption rate of *Lactobacillus plantarum* DT55 reached 83.90%, demonstrating a strong ability to adsorb microplastics. This indicates that the adsorption effect of *Lactobacillus plantarum* DT55 on microplastics is strain-specific.

[0080] from Figure 7 As can be seen from electron microscopy, the surface of the rod-shaped Lactobacillus plantarum DT55 is adsorbed with spherical PS fluorescent microspheres.

[0081] Example 5: Determination of the antioxidant capacity of Lactobacillus plantarum DT55

[0082] Lactobacillus plantarum DT55 was inoculated into MRS medium and anaerobically cultured at 37°C for 24 h. The cultured bacterial suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the suspension was washed twice with 450 μL of sterile PBS buffer to adjust the bacterial concentration to 1 × 10⁻⁶. 9 CFU / mL. For the experimental group, 500 μL of bacterial suspension was added to 500 μL of 0.2 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) ethanol solution. Antioxidant vitamin C (Vc) was used as a positive control; 500 μL of 3 μg / mL vitamin C solution was added to 500 μL of 0.2 mmol / L DPPH ethanol solution. For the control group, 500 μL of PBS was added to 500 μL of 0.2 mmol / L DPPH ethanol solution. For the blank group, 500 μL of bacterial suspension was added to 500 μL of anhydrous ethanol solution. After mixing, the mixture was placed in a shaker at 30℃ in the dark for 30 min. After shaking, the reaction solution was centrifuged at 4000 rpm for 10 min, and 100 μL of the supernatant was measured at 517 nm using a microplate reader to calculate the DPPH free radical scavenging rate. The calculation formula is: DPPH scavenging rate (%) = [1 - (As - A0) / Ai] × 100% (As: fluorescence value of experimental group; A0: fluorescence value of blank group; Ai: fluorescence value of control group). The results are shown in […]. Figure 8 .

[0083] from Figure 8 It can be seen that the antioxidant vitamin C has a DPPH scavenging rate of 47.90%, while Lactobacillus plantarum DT55 has a certain antioxidant capacity, with a DPPH scavenging rate of 34.12%. Therefore, colonizing Lactobacillus plantarum DT55 can reduce the oxidative damage to the host caused by microplastics.

[0084] Example 6: Determination of the adsorption effect of Lactobacillus plantarum DT55 on mixed microplastics

[0085] To simulate and verify the adsorption effect of Lactobacillus plantarum DT55 on microplastics in the natural environment, this embodiment mixed microplastic powders of five common materials in equal proportions (polypropylene PP, polyethylene PE, polystyrene PS, polyethylene terephthalate PET, and polycarbonate PC), resuspended them in PBS solution containing 0.1% Tween-80, and prepared a mixed microplastic suspension of 1 mg / mL for adsorption effect detection.

[0086] Lactobacillus plantarum DT55 was inoculated into MRS medium and anaerobically cultured at 37°C for 24 h. The cultured bacterial suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the suspension was washed twice with 450 μL of sterile PBS buffer. The suspension was then resuspended in PBS, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL. 100 μL of *Lactobacillus plantarum* DT55 suspension was transferred to a 1.5 mL EP tube, and 900 μL of the above 1 mg / mL mixed microplastic suspension was added. The tube was then incubated on a shaker in the dark for 4 hours at 37°C and 800 rpm. After incubation, the precipitate was collected for electron microscopy observation. Results are shown below. Figure 9 .from Figure 9 As can be seen from electron microscopy, short rod-shaped Lactobacillus plantarum DT55 is adsorbed on the surface of microplastic particles.

[0087] Take the above mixed microplastic powder, add 10 μg / mL Nile Red solution, incubate at 50℃ and 100 rpm for 1 hour, wash three times with PBS, resuspend in PBS, and prepare a 1 mg / mL Nile Red labeled mixed microplastic suspension.

[0088] Take the overnight cultured *Lactobacillus plantarum* DT55, wash with PBS (see above for details), resuspend in 100 μM FITC solution, and incubate at 37°C with shaking at 100 rpm in the dark for 0.5 h. After incubation, centrifuge, wash and resuspend the bacterial pellet with PBS, and adjust the bacterial concentration to 1 × 10⁻⁶. 9 CFU / mL. Take 100 μL of bacterial suspension into a 1.5 mL EP tube, add 900 μL of the above-mentioned 1 mg / mL Nile Red-labeled mixed microplastic suspension, and incubate on a shaker in the dark for 4 h at 37 °C and 800 rpm. After incubation, transfer the incubation solution to a glass slide, dry it, mount it with mounting medium and coverslip, fix it, and observe and photograph it using a fluorescence microscope. Results are shown below. Figure 10 The mixed microplastic particles labeled with Nile Red emitted red fluorescence, while the bacteria labeled with FITC emitted green fluorescence. The red fluorescence in the image is surrounded by green signals, indicating that Lactobacillus plantarum DT55 is adsorbed on the surface of the mixed microplastics.

[0089] Example 7: Gavage administration of *Lactobacillus plantarum* DT55 reduces microplastic residues in the body.

[0090] Six-week-old C57 mice were purchased and, after a week of acclimatization, the experimental group mice were administered 1 mg of PS fluorescent microspheres (10 mg / mL, 5 μm particle size, Basell) by gavage daily, along with 1 × 10⁻⁶ microspheres by gavage daily. 9 CFU (Cytobacterium plantarum) DT55 was administered to control mice via gavage at a dose of 1 mg of PS fluorescent microspheres daily, along with an equal volume of physiological saline daily. The blank control (NC) mice received only an equal volume of physiological saline, without PS fluorescent microspheres. This gavage regimen was continued for 7 days. After the last administration of PS fluorescent microspheres, mice were deprived of food and water for 16 hours, then sacrificed. Intestinal tissue was dissected to detect residual PS fluorescent microspheres. The detection method was as follows: Ileum or cecal tissue was collected from mice, weighed, and 400 μL of lysis buffer (23 g / L Na2HPO4, 4.6 g / L NaH2PO4) was added. The tissue was then ground using a tissue homogenizer (60 Hz, 45 s, 4 2 mm steel balls). After grinding, 40 μL of 50 g / L SDS was added and mixed thoroughly. Finally, 40 μL of Protein K (20 mg / mL) was added. Incubate overnight at 37°C, dilute with 400 μL of lysis buffer, and use a 1 mL syringe to draw up the homogenate. Filter the homogenate through a 100 μm cell filter into a 1.5 mL EP tube. Transfer 200 μL of the filtrate to a 96-well plate and detect the fluorescent microsphere signal using flow cytometry. Flow cytometry parameters were: FSC > 60000, loading volume 20 μL, and detection channels: B530, FITC-H.

[0091] See results Figure 11 In the control group, a large amount of PS fluorescent microspheres remained in the ileum and cecum of mice. Gavage administration of *Lactobacillus plantarum* DT55 significantly reduced the amount of PS fluorescent microspheres remaining in the ileum and cecum of mice. This indicates that gavage administration of *Lactobacillus plantarum* DT55 reduced the residue of microplastics in mice.

[0092] Example 8: Oral administration of *Lactobacillus plantarum* DT55 to reduce inflammatory response

[0093] Serum and ileum tissue collected from the mice in Example 7 before sacrifice were used to detect immune factor levels by ELISA. Results are shown below. Figure 12 As shown in the figure, compared with the blank control group, PS fluorescent microspheres significantly reduced the levels of the anti-inflammatory cytokine IL-10 in serum and intestine, and significantly increased the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β in the ileum, indicating that PS fluorescent microspheres induced an inflammatory response in mice. After gavage administration of *Lactobacillus plantarum* DT55, the levels of inflammatory factors significantly returned to normal, the levels of IL-10 in serum and intestine significantly increased, and the levels of TNF-α, IL-6, and IL-1β in the ileum significantly decreased, indicating that gavage administration of *Lactobacillus plantarum* DT55 can reduce the inflammatory response induced by microplastics.

[0094] In summary, this invention has isolated and screened a strain of *Lactobacillus plantarum* DT55. This strain is acid- and bile-tolerant, and possesses the ability to colonize the stomach and small intestine, making it suitable for the development of edible probiotics. *Lactobacillus plantarum* DT55 exhibits a strong ability to adsorb microplastics, and experimental data have also demonstrated its antioxidant capacity, as well as its ability to reduce microplastic residues in the intestine and decrease inflammatory responses. Therefore, *Lactobacillus plantarum* DT55 is a strain well-suited to the digestive tract environment and has broad application prospects in adsorbing microplastics, accelerating microplastic excretion, and reducing oxidative damage and inflammatory responses caused by microplastics.

[0095] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum DT55, characterized in that, The accession number is GDMCC No:63623.

2. A microbial agent, characterized in that, It includes Lactobacillus plantarum DT55 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent can be a solid bacterial agent or a liquid bacterial agent.

4. A food product, characterized in that, It contains Lactobacillus plantarum DT55 as described in claim 1 or the bacterial agent as described in claim 2 or 3.

5. A medicine, characterized in that, It contains Lactobacillus plantarum DT55 as described in claim 1 or the bacterial agent as described in claim 2 or 3.

6. The method for preparing the microbial agent according to claim 2 or 3, characterized in that, When the bacterial agent is a liquid bacterial agent, it includes the step of culturing Lactobacillus plantarum DT55 to obtain a bacterial suspension; when the bacterial agent is a solid bacterial agent, it further includes the step of drying the bacterial suspension.

7. The preparation method according to claim 6, characterized in that, When culturing Lactobacillus plantarum DT55, the culture medium used is MRS medium, and the culture conditions are anaerobic at 36~37℃.

8. The use of Lactobacillus plantarum DT55 as described in claim 1 or the microbial agent as described in claim 2 or 3 in the preparation of food.

9. The use of Lactobacillus plantarum DT55 as described in claim 1 or the bacterial agent as described in claim 2 or 3 in the preparation of products that can reduce oxidative damage and / or inflammatory responses.

10. The use of Lactobacillus plantarum DT55 as described in claim 1 or the bacterial agent as described in claim 2 or 3 in the preparation of products that can adsorb microplastics and / or promote the excretion of microplastics from the body.

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