Lactobacillus plantarum DT55 and application thereof in preparation of product with microplastic adsorption and removal functions

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus plantarum DT55 and application thereof in preparation of products with microplastic adsorption and removal functions. The invention develops a new strain of Lactobacillus plantarum DT55, and the preservation number of the new strain of Lactobacillus plantarum DT55 is GDMCC No: 63623. The invention also discloses a preparation method of the new strain of Lactobacillus plantarum DT55, and the preservation number of the new strain of Lactobacillus plantarum DT55 is GDMCC No: 63623. The strain can adsorb micro-plastics, accelerate discharge of plastics in bodies, reduce micro-plastic residues in intestinal tracts of mice, reduce accumulated oxidative damage of the micro-plastics and reduce inflammatory response caused by the micro-plastics, and is expected to be developed into probiotic products to protect human health.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to a Lactobacillus plantarum DT55 and its application in the preparation of products with the function of adsorbing and removing microplastics. Background Art

[0002] With the wide 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 actions. Plastic particles with a particle size less than 5 mm are usually defined as microplastics (MPs), and plastic particles with a particle size 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. The materials of microplastics include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET), etc. They are widely present in air, water bodies and soil, and can be ingested by plankton, fish, birds, etc., and finally enter the human body through the food chain. Research shows that the weekly microplastic intake of the human body can reach 5 g, and microplastics exist in human feces, blood, lung tissue and placenta. Although there is currently no direct evidence to confirm the damage of microplastics to human health, a large number of studies have confirmed that microplastics can cause damage to the digestive system, respiratory system, immune system, nervous system and reproductive system of rodents and aquatic organisms. The microplastics accumulated in tissues cannot be removed, which can cause a large increase in reactive oxygen species, cause oxidative stress and produce toxic effects. Therefore, removing microplastics from the human body and reducing the content of microplastics are of great significance for the long-term health of the human body.

[0004] At present, there are only a few reports on methods for reducing plastic pollution in the environment and water bodies by biological methods. For example, some bacteria and fungi have been reported to have the ability to degrade plastics. They secrete cutinase, protease, esterase, lipase, etc. to decompose polymers into monomers or oligomers; another way to remove plastic particles is to use bacteria adsorption. Some bacteria can attach to the surface of microplastics and form a viscous biofilm. This viscous matrix can capture free microplastics, resulting in the bioaggregation of microplastics, thereby realizing the separation and removal of microplastics.

[0005] However, the existing bacteria and fungi that can degrade or adsorb microplastics are not edible strains, and these strains cannot tolerate the gastrointestinal environment, so their adsorption ability is difficult to be applied to the removal of human microplastics. In order to reduce the accumulation of microplastics in the human body and reduce the health damage caused by microplastics to humans, it is necessary to explore edible probiotics that can tolerate the gastrointestinal environment and can adsorb and remove microplastics in this field. Summary of the Invention

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

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

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

[0009] The Lactiplantibacillus plantarum DT55 strain of the present invention was isolated from the feces of healthy adults and identified by bacterial morphology, physiology, and 16S rRNA sequencing. The result was Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum DT55. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 5, 2023. The abbreviation of the depositary institution is GDMCC, the address of the depositary institution is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, postal code 510070. The taxonomic name is Lactiplantibacillus plantarum, and the deposit number is GDMCC No: 63623.

[0010] Lactiplantibacillus 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 round ends, and are arranged singly, in pairs, or in chains.

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

[0014] (2) Physiological characteristics

[0015] Lactiplantibacillus plantarum DT55 can grow in acidic or bile salt-containing media. This strain can effectively adsorb microplastics, with an adsorption rate exceeding 83%. Lactiplantibacillus plantarum DT55 has an antioxidant effect and can reduce the oxidative damage caused by microplastics. Moreover, Lactiplantibacillus plantarum DT55 can reduce the microplastic residue in the intestines of mice and reduce the inflammatory response caused by microplastics.

[0016] Lactiplantibacillus plantarum is widely present in fermented foods and is used as a starter culture and preservative in the food industry. Lactiplantibacillus plantarum has been included in the list of strains that can be used in foods. It has also obtained the GRAS (generally recognised as safe) certification from the US Food and Drug Administration (FDA) and is included in the Qualified Presumption of Safety (QPS) list promulgated by the European Food Safety Authority (EFSA). It is widely used in various probiotic foods and dietary supplements globally. Genomic research data, mouse experiments, and human clinical trials have all proven the safety of Lactiplantibacillus plantarum.

[0017] In a second aspect, the present invention provides a bacterial agent comprising the above-mentioned Lactiplantibacillus plantarum DT55.

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

[0019] In a third aspect, the present invention provides a food comprising the above-mentioned Lactiplantibacillus plantarum DT55 or the bacterial agent.

[0020] In a fourth aspect, the present invention provides a medicine comprising the above-mentioned Lactiplantibacillus plantarum DT55 or the bacterial agent.

[0021] Lactiplantibacillus plantarum DT55 of the present invention has good acid and bile salt tolerance. At the same time, the adsorption experiment of Lactiplantibacillus plantarum DT55 with microplastics proves that this strain can effectively adsorb microplastics and promote the excretion of microplastics from the body. In addition, Lactiplantibacillus plantarum DT55 also has antioxidant effects and the effect of reducing inflammatory responses. This strain can be developed into an edible probiotic, and it is expected to achieve the goal of adsorbing microplastics by intestinal bacteria in the human body, accelerating the excretion of microplastics, reducing microplastic damage, and protecting the gastrointestinal tract.

[0022] The medicine of the present invention also includes pharmaceutically acceptable excipients and is prepared by conventional methods in the art. It can be prepared by conventional mixing, granulation, sugar coating, dissolution, or freeze-drying methods and can also contain other active ingredients simultaneously.

[0023] In a fifth aspect, the present invention provides a preparation method of the above-mentioned bacterial agent. When the bacterial agent is a liquid bacterial agent, it includes the step of culturing Lactiplantibacillus 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.

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

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

[0026] Specifically, the above-mentioned method for culturing Lactiplantibacillus plantarum DT55 is to inoculate Lactiplantibacillus plantarum DT55 into MRS medium and culture it anaerobically at 37 °C for 24 h. The components of the MRS broth medium are: casein peptone 10.0 g / L, beef powder 10.0 g / L, yeast powder 4.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, Tween 80 1.0 g / L, pH = 5.7 ± 0.2, and 1.5% agar is added to the solid medium.

[0027] Based on the efficacy characteristics of this strain, in the sixth aspect, the present invention provides the application of the above-mentioned Lactiplantibacillus plantarum DT55 or bacterial agent in the preparation of foods or drugs.

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

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

[0030] In the eighth aspect, the present invention provides the application 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 the present invention are at least as follows:

[0032] Compared with the prior art, the new Lactiplantibacillus plantarum DT55 strain provided by the present invention has good acid and bile salt tolerance and is expected to be developed into an edible probiotic; and this strain can adsorb microplastics, accelerate the excretion of microplastics, reduce the residue of microplastics in the intestinal tract of the body, and reduce the oxidative damage and inflammatory response caused by microplastic accumulation, providing great benefits for human intestinal health. Description of the Drawings

[0033] Figure 1 Colony morphology of Lactobacillus plantarum DT55 on MRS medium in Example 1.

[0034] Figure 2 Microscopic examination image of Lactobacillus plantarum DT55 in Example 1.

[0035] Figure 3 Survival rate of Lactobacillus plantarum DT55 in acidic medium in Example 2. The statistical analysis method is T-test, and ns indicates that the p-value is greater than 0.05. Data are from three repeated experiments, and error bars represent standard deviation.

[0036] Figure 4 Survival rate of Lactobacillus plantarum DT55 in medium containing bile salts in Example 3. The statistical analysis method is T-test, and ns indicates that the p-value is greater than 0.05. Data are from three repeated experiments, and error bars represent standard deviation.

[0037] Figure 5 Photo of adsorption and agglutination of Lactobacillus plantarum DT55 and PS fluorescent microspheres in solution in Example 4.

[0038] Figure 6 Adsorption rate of Lactobacillus plantarum DT55 to PS fluorescent microspheres in solution in Example 4. Error bars represent standard deviation, and data are from three repeated experiments. The statistical analysis method is T-test, and **** indicates that the p-value is less than 0.0001.

[0039] Figure 7 Electron microscopy image of the adsorption of Lactobacillus plantarum DT55 and PS fluorescent microspheres in Example 4, with a magnification of 50,000 times.

[0040] Figure 8 Scavenging rate of DPPH by Lactobacillus plantarum DT55 in Example 5. Error bars represent standard deviation, and data are from three repeated experiments.

[0041] Figure 9 Electron microscopy image after incubation of Lactobacillus plantarum DT55 and mixed microplastic particles in Example 6, with a magnification of 2,000 times.

[0042] Figure 10 Fluorescence imaging after incubation of Lactobacillus plantarum DT55 and mixed microplastic particles in Example 6. The green signal represents Lactobacillus plantarum DT55, and the red signal represents microplastic particles. Arrows indicate that Lactobacillus plantarum DT55 is adsorbed on the surface of microplastic particles. The magnification is 1,000 times.

[0043] Figure 11Detection results of residual PS fluorescent microspheres in the ileum (A) and cecum (B) of mice in Example 7. Error bars represent standard deviation. 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 Detection results of immune factors in mouse serum (A) and ileum tissues (B-E) in Example 8. Error bars represent standard deviation. The statistical analysis method was ANOVA. ** indicates a p-value less than 0.01. *** indicates a p-value less than 0.001. **** indicates a p-value less than 0.0001. Detailed implementation manners

[0045] The preferred implementation manners of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources or prepared by conventional methods in the art.

[0047] Example 1 Isolation and identification of Lactiplantibacillus plantarum DT55

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

[0049] 1.1 Sample source

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

[0051] 1.2 Preparation of culture medium

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

[0053] Table 1 Formulation of MRS medium

[0054]

[0055]

[0056] 1.3 Isolation of strain

[0057] Put 1 g of healthy adult fecal sample into 10 mL of the MRS liquid medium prepared in Step 1.2, mix well, and culture at 36 °C for 24 h. Then, in a laminar flow hood, aspirate 1 mL of the enrichment solution, perform ten-fold serial dilution, and select 10 -4 、10 -5 、10 -6 、10 -7 Apply 100 μL of the bacterial suspensions of four dilution gradients onto Petri dishes containing sterile MRS solid medium, and culture statically at 36 °C under anaerobic conditions for 48 h - 72 h. After obvious single colonies are formed, use a high-throughput automated platform to automatically pick typical colonies from the Petri dishes into MRS liquid medium for culture. The species information of the isolated strains is determined by 16S rRNA sequencing.

[0058] 2. Identification of Lactiplantibacillus plantarum DT55

[0059] 2.1 Colony characteristics

[0060] After Lactiplantibacillus plantarum DT55 is cultured in MRS solid medium for 24 h, it forms round, convex colonies with smooth and regular edges and a moist, milky white surface, as shown in Figure 1 .

[0061] 2.2 Morphology under microscope

[0062] Colony smear of Lactiplantibacillus plantarum DT55: Gram staining is positive. Under a light microscope, the cells are rod-shaped with rounded ends, and are arranged singly, in pairs, or in chains. See Figure 2 .

[0063] 2.3 16S rRNA identification

[0064] Identification unit: Tsingke Biotechnology Co., Ltd.

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

[0066] Identification result: By comparing the sequencing results with the NCBI database and combining the comparison results with the physiological and biochemical results, it is determined that the strain is Lactiplantibacillus plantarum.

[0067] Example 2 Acid tolerance detection of Lactiplantibacillus plantarum DT55

[0068] The overall pH condition in the human gastric environment is strongly acidic. Therefore, the acid tolerance of strains is an important indicator for evaluating whether they can survive and colonize in the gastric acid environment. The commercial strain Lactobacillus rhamnosus GG is a probiotic that is widely used at present and has strong acid tolerance.

[0069] In the present invention, MRS medium with pH = 2.5 was used to verify the acid tolerance of Lactobacillus plantarum DT55. Take 1 mL of the bacterial solution, centrifuge at 4000 rpm for 10 min, discard the supernatant, then add 1 mL of PBS to wash once, and after centrifuging at 4000 rpm for 10 min, resuspend the precipitate with MRS medium with pH = 2.5. Incubate at 37 °C for 3 h, and sample at 0 h and 3 h respectively. After centrifuging the samples, resuspend them with PBS and dilute them serially. The diluted samples are spread on MRS agar plates and anaerobically cultured at 37 °C for 16 h, and then colony counting is performed. The survival rate calculation formula is: acid tolerance survival rate (%) = C1 / C0 × 100% (C0: the counting result at 0 h; C1: the counting result at 3 h). The control strain is Lactobacillus rhamnosus GG.

[0070] After culturing in the acidic medium for 3 hours, the survival rate of the control strain Lactobacillus rhamnosus GG was 84.56%. The survival rate of Lactobacillus plantarum DT55 was 83.90% ( Figure 3 ), which is equivalent to the acid tolerance of the control strain, indicating that this strain has strong acid tolerance and can survive in the gastric environment.

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

[0072] After bacteria enter the intestine from the stomach, the high concentration of bile salts in the small intestine will kill the bacteria. The residence time of food in the small intestine is generally 1 - 4 h. Therefore, in the present invention, 0.1% bile salt-MRS medium was used to verify the bile salt tolerance of Lactobacillus plantarum DT55 strain.

[0073] Inoculate the Lactobacillus plantarum DT55 bacterial solution into a 96-well deep well plate containing MRS medium and anaerobically culture at 37 °C for 24 h. Take 300 μL of the cultured bacterial solution, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 600 μL of MRS medium containing 0.1% bile salt, and resuspend and mix well. For the control group, take 100 μL of the resuspended solution and add 20 μL of MTT (thiazolyl blue) solution; for the treatment group, take 100 μL of the resuspended solution, incubate at 37 °C for 4 h, and then add 20 μL of MTT solution. After adding the MTT solution, react at 37 °C in the dark for 4 h. After the reaction is completed, centrifuge at 4000 rpm for 10 min and discard the supernatant. Add 100 μL of DMSO solution to each well, incubate at 37 °C with shaking for 10 min to completely dissolve and mix well the purple formazan produced by the reaction. After mixing, measure the absorbance of the solution at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the survival rate. The survival rate = A1 / A0 × 100% (A1: the absorbance value of the solution in the treatment group at 570 nm, A0: the absorbance value of the solution in the control group at 570 nm). Measure the survival rate of the control strain Lactobacillus rhamnosus GG in the same way.

[0074] After culturing in 0.1% bile salt-MRS medium for 4 hours, 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 the same bile salt tolerance as the control strain Lactobacillus rhamnosus GG and can survive in the small intestine.

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

[0076] Inoculate Lactobacillus plantarum DT55 into MRS medium and culture anaerobically at 37°C for 24 h. After culturing, centrifuge the bacterial liquid at 4000 rpm for 10 min, discard the supernatant, add 450 μL of sterile PBS buffer and wash twice. Then resuspend with PBS and adjust the bacterial liquid concentration to 1×10 9 CFU / mL. Take 100 μL of the Lactobacillus plantarum DT55 bacterial suspension into a 1.5 mL EP tube in the experimental group, add 900 μL of the PS fluorescent microsphere working solution (0.16 mg / mL, particle size 0.1 μm, Besler company), mix well, and place it on a shaker and incubate in the dark for 4 h. The incubation conditions are 37°C and 800 rpm. In the blank control group, take 100 μL of PBS and 900 μL of the PS fluorescent microsphere working solution into a 1.5 mL EP tube, mix well and incubate. In the bacterial liquid control group, take 100 μL of the Lactobacillus plantarum DT55 bacterial suspension and 900 μL of PBS into a 1.5 mL EP tube, mix well and incubate. After incubation, take the incubated solution to observe and take pictures. The results are shown in Figure 5 .

[0077] Take the incubated solutions of the experimental group and the blank control group, centrifuge at 2000 rpm for 10 min, take 100 μL of the supernatant, and measure the fluorescence intensity using an enzyme-linked immunosorbent assay (ELISA) reader. The parameters of the ELISA reader are: excitation wavelength, 494 nm; detection wavelength, 518 nm. Calculate the adsorption rate according to the fluorescence intensity value. The control strain is another strain of Lactobacillus plantarum obtained from the same batch of screening experiments, and the adsorption rate of the control strain is measured in the same way. The adsorption rate calculation formula is: adsorption rate (%) = (A1 - A2) / A1 × 100% (A1: fluorescence value of the blank control group, A2: fluorescence value of the Lactobacillus plantarum group), and the results are shown in Figure 6 . Take the precipitate after centrifugation of the experimental group, fix it with glutaraldehyde at 4°C overnight, then dehydrate it with a gradient of ethanol, dry it, and observe it with an electron microscope. The results are shown in Figure 7 .

[0078] From Figure 5 it can be seen that in the blank control group, the PS fluorescent microspheres do not self-aggregate; in the bacterial liquid control group, Lactobacillus plantarum DT55 does not self-aggregate; in the experimental group, specific adsorption and aggregation flocs appear between Lactobacillus plantarum DT55 and the PS fluorescent microspheres.

[0079] FromFigure 6 It can be seen that the adsorption rate of the control strain is 8.03%, with poor microplastic adsorption ability, while the adsorption rate of Lactobacillus plantarum DT55 reaches 83.90%, showing strong microplastic adsorption ability. This indicates that the adsorption effect of Lactobacillus plantarum DT55 on microplastics is strain-specific.

[0080] From Figure 7 It can be seen that under electron microscope observation, spherical PS fluorescent microspheres are adsorbed on the surface of rod-shaped Lactobacillus plantarum DT55.

[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 solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with 450 μL of sterile PBS buffer, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. In the experimental group, 500 μL of the bacterial suspension was added to 500 μL of 0.2 mmol / L 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution. The antioxidant vitamin C (Vc) was used as a positive control, 500 μL of 3 μg / mL vitamin solution was taken and added to 500 μL of 0.2 mmol / L DPPH ethanol solution. In the control group, 500 μL of PBS was added to 500 μL of 0.2 mmol / L DPPH ethanol solution. In the blank group, 500 μL of the bacterial suspension was added to 500 μL of absolute ethanol solution. After mixing, the reaction mixture was placed on a shaker at 30 °C and shaken in the dark for 30 min. After the shaking was completed, the reaction solution was centrifuged at 4000 rpm for 10 min, and 100 μL of the supernatant was taken to measure the absorbance at 517 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and the DPPH radical scavenging rate was calculated. The calculation formula is: DPPH scavenging rate (%) = [1 - (As - A0) / Ai] × 100% (As: fluorescence value of the experimental group; A0: fluorescence value of the blank group; Ai: fluorescence value of the control group). The results are shown in Figure 8 .

[0083] From Figure 8 It can be seen that the scavenging rate of vitamin C for DPPH is 47.90%, and Lactobacillus plantarum DT55 has certain antioxidant ability, with a scavenging rate of 34.12% for DPPH. Therefore, colonization of Lactobacillus plantarum DT55 can reduce the oxidative damage caused by microplastics to the host.

[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, in this example, microplastic powders of five common materials were mixed in equal proportions (polypropylene PP, polyethylene PE, polystyrene PS, polyethylene terephthalate PET, polycarbonate PC), resuspended in PBS solution containing 0.1% Tween-80, and formulated into 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 solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 450 μL of sterile PBS buffer was added for washing twice. Then PBS was added for resuspension, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. 100 μL of the Lactobacillus plantarum DT55 bacterial suspension was taken into a 1.5 mL EP tube, 900 μL of the above 1 mg / mL mixed microplastic suspension was added, and it was placed on a shaker and incubated in the dark with shaking for 4 h. The incubation conditions were 37 °C and 800 rpm. After incubation, the precipitate was taken for electron microscopy observation. The results are shown in Figure 9 . From Figure 9 it can be seen that under electron microscopy, short rod-shaped Lactobacillus plantarum DT55 was adsorbed on the surface of microplastic particles.

[0087] The above mixed microplastic powder was taken, 10 μg / mL nile red solution was added, and after incubation at 50 °C and 100 rpm for 1 hour, it was washed three times with PBS and resuspended in PBS to prepare a 1 mg / mL nile red-labeled mixed microplastic suspension.

[0088] Overnight-cultured Lactobacillus plantarum DT55 was taken, washed with PBS (for specific operations, see above), resuspended with 100 μM FITC solution, and incubated in the dark with shaking at 37 °C and 100 rpm for 0.5 h. After incubation, it was centrifuged, the bacterial precipitate was washed and resuspended with PBS, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. 100 μL of the bacterial suspension was taken into a 1.5 mL EP tube, 900 μL of the above 1 mg / mL nile red-labeled mixed microplastic suspension was added, and it was placed on a shaker and incubated in the dark with shaking for 4 h. The incubation conditions were 37 °C and 800 rpm. After incubation, the incubation solution was taken on a glass slide, dried, sealed with a mounting medium and a coverslip, and observed and photographed with a fluorescence microscope after fixation. The results are shown in Figure 10 . The mixed microplastic particles labeled with nile red emitted red fluorescence, and the bacteria labeled with FITC emitted green fluorescence. The red fluorescence in the figure was surrounded by green signals, indicating that Lactobacillus plantarum DT55 was adsorbed on the surface of the mixed microplastics.

[0089] Example 7 Reduction of in vivo microplastic residues by intragastric administration of Lactobacillus plantarum DT55

[0090] Six-week-old C57 mice were purchased. After one week of adaptive feeding, the mice in the experimental group were intragastrically administered 1 mg of PS fluorescent microspheres (10 mg / mL, particle size 5 μm, Besler Company) every day, and at the same time, 1×10 9 CFU of Lactobacillus plantarum DT55 were intragastrically administered every day. The mice in the control group were intragastrically administered 1 mg of PS fluorescent microspheres every day, and at the same time, an equal volume of normal saline was intragastrically administered every day. The mice in the blank control group (NC) were not intragastrically administered PS fluorescent microspheres and were only intragastrically administered an equal volume of normal saline. Intragastric administration was continued for 7 days. After the last intragastric administration of PS fluorescent microspheres, the mice were deprived of water and food for 16 h, then sacrificed, and the intestinal tissues of the mice were dissected to detect the residual amount of PS fluorescent microspheres. The detection method was as follows: Take the ileum or cecum tissue of the mice: After weighing, add 400 μL of lysis buffer (23 g / L Na2HPO4, 4.6 g / L NaH2PO4), grind it with a tissue grinder (60 Hz, 45 s, 4 2-mm steel beads), after grinding, add 40 μL of 50 g / L SDS and mix well by shaking, and then add 40 μL of Protein K (20 mg / mL). Incubate overnight at 37 °C, dilute with 400 μL of lysis buffer, aspirate the homogenate with a 1 mL syringe, and filter it through a 100 μm cell strainer into a 1.5 mL EP tube. Take 200 μL of the filtrate into a 96-well plate and detect the fluorescent microsphere signal with a flow cytometer. The detection parameters of the flow cytometer were that FSC was greater than 60000, the sample loading volume was 20 μL, and the detection channels were: B530, FITC-H.

[0091] The results are shown in Figure 11 . A large amount of PS fluorescent microspheres remained in the ileum and cecum of the control group mice. Intragastric administration of Lactobacillus plantarum DT55 significantly reduced the residual amount of PS fluorescent microspheres in the ileum and cecum of the mice. This indicates that intragastric administration of Lactobacillus plantarum DT55 reduced the residual amount of microplastics in the mice.

[0092] Example 8 Intragastric administration of Lactobacillus plantarum DT55 reduces the inflammatory response

[0093] Take the serum collected before sacrificing the mice and the dissected ileum tissues in Example 7, and detect the levels of immune factors by ELISA. The results are shown in Figure 12 . It can be seen from the figure that compared with the blank control group, PS fluorescent microspheres significantly reduced the content of the anti-inflammatory cytokine IL-10 in the serum and intestine, and significantly increased the content of the inflammatory cytokines TNF-α, IL-6 and IL-1β in the ileum. This indicates that PS fluorescent microspheres induced an inflammatory response in the mice. After intragastric administration of Lactobacillus plantarum DT55, the levels of inflammatory factors all significantly returned to normal levels. The content of IL-10 in the serum and intestine significantly increased, and the content of TNF-α, IL-6 and IL-1β in the ileum significantly decreased, indicating that intragastric administration of Lactobacillus plantarum DT55 can reduce the inflammatory response caused by microplastics.

[0094] In summary, a strain of Lactobacillus plantarum DT55 was isolated and screened in the present invention. This strain can tolerate acid and bile salts, has the ability to colonize the stomach and small intestine, and can be applied to the development of edible probiotics. Lactobacillus plantarum DT55 has a strong ability to adsorb microplastics. Experimental data also demonstrate the antioxidant ability of this strain, as well as the ability to reduce the residual microplastics in the intestine and reduce the inflammatory response. Thus, Lactobacillus plantarum DT55 is a strain suitable for the digestive tract environment and has broad application prospects in adsorbing microplastics, accelerating the excretion of microplastics, and reducing the oxidative damage and inflammatory response caused by microplastics.

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

Claims

1. A Lactiplantibacillus plantarum DT55, characterized in that, The preservation number is GDMCC No: 63623.

2. A bacterial agent, characterized in that, It contains Lactiplantibacillus plantarum DT55 described in claim 1.

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

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

5. A pharmaceutical product, characterized in that, It contains Lactiplantibacillus plantarum DT55 described in claim 1 or the bacterial agent described in claim 2 or 3.

6. The preparation method of 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 Lactiplantibacillus 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 Lactiplantibacillus plantarum DT55 is cultured, the medium used is MRS medium, and the culture conditions are anaerobic at 36 - 37 °C.

8. Use of Lactiplantibacillus plantarum DT55 described in claim 1 or the bacterial agent described in claim 2 or 3 in the preparation of food or medicine.

9. Use of Lactiplantibacillus plantarum DT55 described in claim 1 or the bacterial agent described in claim 2 or 3 in the preparation of a product that can reduce oxidative damage and / or inflammatory response.

10. Use of Lactiplantibacillus plantarum DT55 described in claim 1 or the bacterial agent described in claim 2 or 3 in the preparation of a product that can adsorb microplastics and / or promote the excretion of microplastics from the body.

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