Lactobacillus plantarum DT88 and application thereof in preparation of micro-plastic adsorption and removal products

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum DT88 and application of the lactobacillus plantarum DT88 in preparation of micro-plastic adsorption and removal products. The lactobacillus plantarum DT88 provided by the invention is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the lactobacillus plantarum DT88 is GDMCC No: 63626. The lactobacillus plantarum DT88 provided by the invention has the advantages that the lactobacillus plantarum DT88 is preserved in the Guangdong Microbial Culture Collection Center; the bacterial strain has good acid resistance and cholate resistance, can tolerate gastrointestinal tract environment, has efficient micro-plastic adsorption capacity, has oxidation resistance, can reduce oxidative damage caused by micro-plastic accumulation, can promote in-vivo micro-plastic discharge, reduces micro-plastic residues in intestinal tracts, reduces inflammatory response caused by micro-plastics, and has the advantages of being safe and reliable. The probiotic strain has the potential of being developed into an edible probiotic product, and has the effects of adsorbing micro-plastics, accelerating discharge of the micro-plastics and reducing damage of the micro-plastics as intestinal bacteria in a human body.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Lactobacillus plantarum DT88 and its application in the preparation of products for microplastic adsorption and removal. Background Art

[0002] Plastic waste generated from plastic products is decomposed into tiny particles in the environment. Generally, plastic particles with a particle size less than 5 mm are microplastics (MPs), and among them, plastic particles with a particle size less than 0.1 μm are nanoplastics (NPs). Microplastics are widely present in air, water bodies, and soil, and can be ingested by plankton, fish, birds, etc., and ultimately enter the human body through the food chain. This not only causes environmental pollution but also poses a potential threat to human health. A large number of studies have confirmed that microplastics can damage 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, leading to oxidative stress and toxic effects. Therefore, removing microplastics from the body and reducing the microplastic content are of great significance to human health.

[0003] At present, there is no report on any method capable of removing microplastics from the human body. There are a few reports on using biological methods to reduce plastic pollution in the environment and water bodies. 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. In addition, there are also some bacteria with the ability to capture and adsorb microplastics. These 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 achieving the separation and removal of microplastics. However, the bacteria and fungi that have been reported to be able to degrade or adsorb microplastics are all inedible microorganisms, and the bacteria that adsorb microplastics in environments such as water usually require a long time to form a biofilm for adsorbing microplastics, and at the same time, such bacteria are difficult to tolerate the gastrointestinal environment. Therefore, the above methods are difficult to be used for removing microplastics from the human body. Summary of the Invention

[0004] The present invention provides a Lactobacillus plantarum DT88 and its application in the preparation of products for microplastic adsorption and removal.

[0005] The present invention provides Lactobacillus plantarum DT88, which was deposited in Guangdong Microbial Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070) on July 5, 2023, and was classified and named Lactiplantibacillus plantarum, and the preservation number was GDMCC No: 63626.

[0006] In the present invention, Lactobacillus plantarum DT88 strain is isolated from fish tea, and the strain is identified by bacterial morphology, physiology and 16SrRNA sequencing, and the result is Lactobacillus plantarum (Lactiplantibacillus plantarum), which is named Lactobacillus plantarum DT88.

[0007] Lactobacillus plantarum DT88 has the following microbiological characteristics:

[0008] (1) Morphological characteristics

[0009] Gram staining is positive, and under light microscopy the cells are rod-shaped with round ends, and they are arranged individually or in pairs or chains.

[0010] After culturing in MRS solid medium for 24 h, round, convex, smooth-edged, and moist milky white colonies were formed.

[0011] (2)Physiological characteristics

[0012] Lactobacillus plantarum DT88 can grow in acidic or bile salt-containing culture media. The strain can effectively adsorb microplastics with an adsorption rate of 79%. Lactobacillus plantarum DT88 has an antioxidant effect and can reduce the oxidative damage caused by microplastics. Lactobacillus plantarum DT88 can accelerate the excretion of microplastics from the intestines of mice, reduce the residual microplastics in the intestines, and reduce the inflammatory response caused by microplastics.

[0013] Lactobacillus plantarum DT88 can be cultured by the following culture method: inoculating Lactobacillus plantarum DT88 into MRS broth medium and anaerobic culture at 37°C for 24 hours. The components of the MRS broth medium are: 10.0 g / L casein digest, 10.0 g / L beef powder, 4.0 g / L yeast powder, 2.0 g / L ammonium 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 potassium dihydrogen phosphate, 1.0 g / L Tween 80, pH 5.7±0.2, and 1.5% agar is added to the solid culture medium.

[0014] Lactobacillus plantarum is widely present in fermented foods and is often used as a starter or preservative in the food industry. Lactobacillus 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 Lactobacillus plantarum.

[0015] The present invention provides a microbial preparation comprising the above-mentioned Lactobacillus plantarum DT88.

[0016] Preferably, in the above-mentioned microbial preparation, Lactobacillus plantarum DT88 exists in the form of viable bacteria.

[0017] The above-mentioned microbial preparation can be a solid preparation (such as bacterial powder) or a liquid preparation.

[0018] The present invention provides a method for preparing the above-mentioned microbial preparation, the method comprising the step of culturing Lactobacillus plantarum DT88.

[0019] Preferably, the culturing is anaerobic culturing at 35 - 37 °C.

[0020] Preferably, the culturing is carried out using MRS broth medium. After the culturing is completed, the bacterial liquid is collected and further prepared into a microbial preparation.

[0021] Based on the functions of Lactobacillus plantarum DT88, the present invention provides the following applications of this strain.

[0022] The present invention provides the application of the above-mentioned Lactobacillus plantarum DT88 or the microbial preparation in the preparation of a product for adsorbing microplastics and / or promoting the excretion of microplastics.

[0023] Lactobacillus plantarum DT88 has good acid and bile salt tolerance characteristics, can well tolerate the in vivo gastrointestinal environment, and thus exerts the effects of adsorbing microplastics, promoting the excretion of microplastics, and antioxidation in vivo.

[0024] In the above application, the product is preferably a food, a drug, or a feed. The food is preferably a health food.

[0025] The present invention provides the application of the above-mentioned Lactobacillus plantarum DT88 or the microbial preparation in adsorbing and / or removing microplastics in the environment.

[0026] Lactobacillus plantarum DT88 can also play the functions of adsorbing microplastics and promoting the removal of microplastics in the in vitro environment, and can be used for the adsorption and / or removal of microplastics in the environment, such as water, soil, etc.

[0027] The present invention provides the use of the Lactobacillus plantarum DT88 or the microbial preparation in the preparation of antioxidant products.

[0028] Lactobacillus plantarum DT88 has the ability to scavenge free radicals, can play an antioxidant function, and reduce oxidative damage caused by microplastics. Lactobacillus plantarum DT88 has both the functions of adsorbing microplastics and antioxidation. On the one hand, it can promote the excretion of microplastics in the body, and on the other hand, it can reduce the oxidative damage caused by residual microplastics in the body, and can effectively reduce the adverse effects caused by the accumulation of microplastics on the body from the above two aspects.

[0029] In the above application, the product is preferably a food, a drug or a feed. The food is preferably a dietary supplement or a health food.

[0030] The present invention provides the use of the Lactobacillus plantarum DT88 or the microbial preparation in the preparation of foods, drugs or feeds.

[0031] The present invention provides a food, which contains the Lactobacillus plantarum DT88 or the microbial preparation described above.

[0032] The present invention provides a drug, which contains the Lactobacillus plantarum DT88 or the microbial preparation described above.

[0033] The present invention provides a feed, which contains the Lactobacillus plantarum DT88 or the microbial preparation described above.

[0034] In addition to containing the Lactobacillus plantarum DT88 or the microbial preparation, the above drugs, foods, and feeds may also contain raw materials or excipients permitted in the fields of drugs, foods, and feeds. Among them, the excipients permitted in the pharmaceutical field include fillers, excipients, lubricants, wetting agents, diluents, etc. The preparation types of drugs can be solid preparations (such as powders, granules, capsules, tablets, etc.) or liquid preparations (such as oral liquids, etc.).

[0035] The present invention provides a microplastic adsorbent, which contains the Lactobacillus plantarum DT88 or the microbial preparation described above.

[0036] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention at least include: Lactobacillus plantarum DT88 has good acid and bile salt tolerance, can tolerate the gastrointestinal environment, and has the potential to be an edible probiotic; this strain can efficiently adsorb microplastics, accelerate the excretion and clearance of microplastics, and has antioxidant capacity, which can reduce the oxidative damage caused by microplastic accumulation. This strain can increase the rate of microplastic excretion from the intestine, reduce the residual microplastics in the intestine, and reduce the inflammatory response caused by microplastics in the body. It is expected to be developed into an edible probiotic product to play the role of adsorbing microplastics, accelerating the excretion of microplastics, reducing microplastic damage, and protecting gastrointestinal health as an intestinal bacterium in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is the colony morphology diagram of Lactobacillus plantarum DT88 on MRS medium in Example 1.

[0039] Figure 2 It is the microscopic examination diagram of Lactobacillus plantarum DT88 in Example 1.

[0040] Figure 3 It is the survival rate result of Lactobacillus plantarum DT88 in acidic medium in Example 2. Among them, ns indicates that the p value is greater than 0.05 (the data comes from three repeated experiments, and the error bars represent the standard deviation). The statistical analysis method is t test.

[0041] Figure 4 It is the survival rate result of Lactobacillus plantarum DT88 in bile salt-containing medium in Example 3. Among them, ns indicates that the p value is greater than 0.05 (the data comes from three repeated experiments, and the error bars represent the standard deviation). The statistical analysis method is t test.

[0042] Figure 5 It is the adsorption and agglutination result of Lactobacillus plantarum DT88 and PS fluorescent microspheres in the solution in Example 4.

[0043] Figure 6 It is the adsorption rate result of Lactobacillus plantarum DT88 to PS fluorescent microspheres in the solution in Example 4. Among them, **** indicates that the p value is less than 0.0001 (the data comes from three repeated experiments, and the error bars represent the standard deviation). The statistical analysis method is t test.

[0044] Figure 7Electron micrograph of the adsorption of Lactobacillus plantarum DT88 and PS fluorescent microspheres in Example 4, magnification 50,000 times.

[0045] Figure 8 Results of the scavenging rate of DPPH by Lactobacillus plantarum DT88 in Example 5. Among them, the data are from three repeated experiments, and the error bars represent the standard deviation.

[0046] Figure 9 Electron micrograph of the adsorption of Lactobacillus plantarum DT88 and mixed microplastic particles in Example 6, magnification 2,000 times.

[0047] Figure 10 Fluorescence imaging of the adsorption of Lactobacillus plantarum DT88 and mixed microplastic particles in Example 6. Among them, the green signal represents Lactobacillus plantarum DT88, and the red signal represents microplastic particles; the arrow indicates that Lactobacillus plantarum DT88 is adsorbed on the surface of the microplastic particles; magnification 1,000 times.

[0048] Figure 11 Lactobacillus plantarum DT88 colonization accelerates microplastic transport in Example 7. Among them, A is the in vivo imaging map of the mouse intestine, and B is the excretion rate map; the error bars represent the standard deviation, the statistical analysis method is T-test, and * represents a p-value less than 0.05.

[0049] Figure 12 Detection of the residual amount of PS fluorescent microspheres in the ileum (A) and cecum (B) of mice in Example 8. Among them, the error bars represent the standard deviation, the statistical analysis method is ANOVA, ** represents a p-value less than 0.01, *** represents a p-value less than 0.001, and **** represents a p-value less than 0.0001.

[0050] Figure 13 Detection of immune factors in mouse serum (A) and ileum tissue (B-E) in Example 9. Among them, the error bars represent the standard deviation, the statistical analysis method is ANOVA, ** represents a p-value less than 0.01, *** represents a p-value less than 0.001, and **** represents a p-value less than 0.0001.

[0051] Figure 3 、 Figure 4 、 Figure 6 、 Figure 11 、 Figure 12 、 Figure 13 In it, Control represents the control, Figure 12 、 Figure 13 In it, NC represents the blank control. Detailed implementation method

[0052] To make the objectives, technical solutions and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts belong to the scope of protection of the present invention.

[0053] Example 1 Isolation and Identification of Lactobacillus plantarum DT88

[0054] 1. Isolation and Identification of Lactobacillus plantarum DT88

[0055] 1.1 Sample Source

[0056] The Lactobacillus plantarum DT88 of the present invention is isolated from fish tea.

[0057] 1.2 Preparation of Culture Medium

[0058] The culture medium used for sample isolation is BBL medium, and MRS medium is used for culturing Lactobacillus plantarum DT88.

[0059] The components of BBL medium are shown in Table 1, and the components of MRS medium are shown in Table 2. Adding 1.5% agar makes it a solid medium.

[0060] Table 1 Formulation of BBL Medium

[0061]

[0062] Table 2 Formulation of MRS Medium

[0063]

[0064]

[0065] 1.3 Isolation of Strains

[0066] Put 1 g of fish tea sample into 10 mL of the BBL liquid medium prepared in step 1.2, mix well and culture at 36°C for 24 h. Then, in the ultra-clean workbench, pipette 1 mL of the enrichment solution and perform ten-fold serial dilution. Select 10 -4 、10 -5 、10 -6 、10 -7 For the four dilution gradients of bacterial solutions, apply 100 μL to the culture dishes containing sterile BBL solid medium, and culture statically at 36°C under anaerobic conditions for 48 h - 72 h. After obvious single colonies are formed, use the high-throughput automation platform to automatically pick typical colonies from the culture dishes into the BBL liquid medium for culture. The isolated strains are identified for their genus and species information through 16S rRNA sequencing.

[0067] 2. Identification of Lactobacillus plantarum DT88

[0068] 2.1 Colony characteristics

[0069] After culturing Lactobacillus plantarum DT88 in MRS solid medium for 24 hours, round, convex, smooth-edged, and moist milky white colonies were formed. Figure 1 .

[0070] 2.2 Morphology under microscope

[0071] Colony smear of Lactobacillus plantarum DT88: Gram staining is positive, and the cells are rod-shaped under light microscopy, with round ends, and single cells or arranged in pairs or chains. Figure 2 .

[0072] 2.3 16S rRNA identification

[0073] Identification unit: Qingke Biotechnology Co., Ltd.

[0074] Identification sequence: as shown in SEQ ID NO.1.

[0075] Identification results: The sequencing results were compared with the NCBI database, and the comparison results were combined with the physiological and biochemical results to determine that the strain was Lactobacillus plantarum.

[0076] Lactobacillus plantarum DT88 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070) on July 5, 2023, and was classified and named Lactiplantibacillus plantarum, with the preservation number GDMCC No: 63626.

[0077] Example 2 Acid resistance test of Lactobacillus plantarum DT88

[0078] The overall pH condition in the human stomach environment is highly acidic, so the acid resistance of the strain is an important indicator for evaluating whether it can survive and colonize in the stomach acid environment. The commercial strain Lactobacillus rhamnosus GG is currently a widely used probiotic with strong acid resistance.

[0079] In this example, an MRS medium with a pH of 2.5 was used to verify the acid tolerance of Lactobacillus plantarum DT88. Take 1 mL of the bacterial solution, centrifuge it at 4000 rpm for 10 min, discard the supernatant, then add 1 mL of PBS for washing once. After centrifuging at 4000 rpm for 10 min, the precipitate was resuspended with the MRS medium at pH = 2.5. Incubate at 37 °C for 3 h, and samples were taken at 0 h and 3 h respectively. After centrifuging the samples, they were resuspended with PBS and serially diluted. The diluted samples were spread on MRS agar plates and anaerobically cultured at 37 °C for 16 h, followed by colony counting. The survival rate calculation formula is: Acid tolerance survival rate (%) = C1 / C0 × 100% (C0: counting result at 0 h; C1: counting result at 3 h). The control strain was Lactobacillus rhamnosus GG.

[0080] After culturing in the acidic medium for 3 hours, the survival rate of the control strain Lactobacillus rhamnosus GG was 84.56%, and the survival rate of Lactobacillus plantarum DT88 was 62.53% ( Figure 3 ), showing no significant difference compared with the control strain and being able to survive in the gastric environment.

[0081] Example 3 Detection of the bile salt tolerance of Lactobacillus plantarum DT88

[0082] 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.

[0083] Therefore, in this example, a 0.1% bile salt - MRS medium was used to verify the bile salt tolerance of Lactobacillus plantarum DT88 strain. The Lactobacillus plantarum DT88 bacterial solution was inoculated into a 96 - deep well plate containing MRS medium and anaerobically cultured at 37 °C for 24 h. Take 300 μL of the cultured bacterial solution, centrifuge it at 4000 rpm for 10 min, discard the supernatant, add 600 μL of the 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 the blue - 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. Survival rate = A1 / A0 × 100% (A1: absorbance value of the solution in the treatment group at 570 nm, A0: absorbance value of the solution in the control group at 570 nm). The survival rate of the control strain Lactobacillus rhamnosus GG was measured using the same method.

[0084] 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 DT88 was 100.9%( Figure 4 ). This indicates that Lactobacillus plantarum DT88 has the same bile salt tolerance as the control strain Lactobacillus rhamnosus GG and can survive in the small intestine.

[0085] Example 4 Determination of the effect of Lactobacillus plantarum DT88 on adsorbing microplastics

[0086] Inoculate Lactobacillus plantarum DT88 into MRS medium and anaerobically culture 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. For the experimental group, take 100 μL of the Lactobacillus plantarum DT88 bacterial suspension into a 1.5 mL EP tube, add 900 μL of the PS fluorescent microsphere working solution (0.16 mg / mL, particle size 0.1 μm, Besler Co., Ltd.), mix well, place it on a shaker and incubate in the dark for 4 h. The incubation conditions are 37 °C and 800 rpm. For 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. For the bacterial liquid control group, take 100 μL of the Lactobacillus plantarum DT88 bacterial suspension and 900 μL of PBS into a 1.5 mL EP tube, mix well and incubate. After incubation, take the incubation solution, observe and take pictures. The results are shown in Figure 5 .

[0087] Take the incubation solutions of the experimental group and the blank control group, centrifuge at 2000 rpm for 10 min, take 100 μL of the supernatant, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the fluorescence intensity. 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 results are shown in Figure 6 . 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). Take the precipitate after centrifugation, fix it overnight at 4 °C with glutaraldehyde, then dehydrate it stepwise with ethanol, dry it and observe it with an electron microscope. The results are shown in Figure 7 .

[0088] It can be seen from Figure 5 that in the blank control group, the PS fluorescent microspheres do not self-aggregate; in the bacterial liquid control group, Lactobacillus plantarum DT88 does not self-aggregate; in the experimental group, specific adsorption and aggregation flocs appear between Lactobacillus plantarum DT88 and the PS fluorescent microspheres.

[0089] It can be seen fromFigure 6 It can be seen that the adsorption rate of the control strain was 8.03%, with poor microplastic adsorption ability, while the adsorption rate of Lactobacillus plantarum DT88 reached 79.78%, showing strong microplastic adsorption ability. This indicates that the adsorption effect of Lactobacillus plantarum DT88 on microplastics is strain-specific.

[0090] From Figure 7 It can be seen that under electron microscope observation, spherical PS fluorescent microspheres were adsorbed on the surface of Lactobacillus plantarum DT88.

[0091] Example 5 Determination of the antioxidant capacity of Lactobacillus plantarum DT88

[0092] Lactobacillus plantarum DT88 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. 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, it was placed on a shaker at 30 °C and reacted with light-shielding oscillation for 30 min. After the oscillation ended, 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-labeling instrument. The DPPH radical scavenging rate was calculated. The calculation formula was: 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).

[0093] From Figure 8 It can be seen that the scavenging rate of the antioxidant vitamin C for DPPH was 47.90%. Similar to vitamin C, Lactobacillus plantarum DT88 had certain antioxidant ability, and the scavenging rate for DPPH was 38.79%. Therefore, colonizing Lactobacillus plantarum DT88 can reduce the oxidative damage caused by microplastics to the host.

[0094] Example 6 Determination of the adsorption effect of Lactobacillus plantarum DT88 on mixed microplastics

[0095] To simulate and verify the adsorption effect of Lactobacillus plantarum DT88 on microplastics in the natural environment, 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 1 mg / mL mixed microplastic suspension for adsorption effect detection.

[0096] Lactobacillus plantarum DT88 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 DT88 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 with light avoidance and oscillation for 4 h. The incubation conditions were 37 °C and 800 rpm. After incubation, the precipitate was taken for electron microscope observation. The results are shown in Figure 9 . From Figure 9 it can be seen that under electron microscope observation, short rod-shaped Lactobacillus plantarum DT88 was adsorbed on the surface of microplastic particles.

[0097] The above-mentioned mixed microplastic powder was taken, 10 μg / mL nile red solution was added, incubated at 50 °C and 100 rpm for 1 hour, washed three times with PBS, and resuspended in PBS to prepare a 1 mg / mL nile red-labeled mixed microplastic suspension.

[0098] The overnight-cultured Lactobacillus plantarum DT88 was taken, washed with PBS, resuspended with 100 μM FITC solution, and incubated with light avoidance and oscillation 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 with light avoidance and oscillation 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 nile red-labeled mixed microplastic particles emitted red fluorescence, and the FITC-labeled bacteria emitted green fluorescence. The red fluorescence in the figure was surrounded by green signals, indicating that Lactobacillus plantarum DT88 was adsorbed on the surface of the mixed microplastics.

[0099] Example 7 Lactobacillus plantarum DT88 gavage accelerates the excretion of microplastics in mice

[0100] Six-week-old C57 mice were purchased. After one week of adaptive feeding, the experimental group was intragastrically administered 1×10 9 CFU of Lactiplantibacillus plantarum DT88 every day, and the control group was intragastrically administered an equal volume of normal saline. After 7 consecutive days of intragastric administration, the mice were fasted and watered for 16 h, then intragastrically administered 1 mg of PS fluorescent microspheres (10 mg / mL, particle size 5 μm, BestBio Co., Ltd.). After 20 min, the mouse intestines were dissected, and a live imaging instrument (excitation filter wavelength: 520 nm, emission filter wavelength: 480 nm) was used to take pictures to observe the position of PS fluorescent microspheres in the intestines. The results are shown in Figure 11 A. The microplastic excretion rate was calculated. The calculation formula was microplastic excretion rate = displacement length of PS fluorescent microspheres / intestinal length × 100%. The results are shown in Figure 11 B. It can be seen from the figure that after intragastric administration of Lactiplantibacillus plantarum DT88, the PS fluorescent microspheres transferred from the gastric end to the cecal end faster, and the microplastic excretion rate increased significantly by 14.17%.

[0101] Example 8: Intragastric administration of Lactiplantibacillus plantarum DT88 reduces microplastic residues in vivo

[0102] 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, BestBio Co., Ltd.) every day, and at the same time, 1×10 9 CFU of Lactiplantibacillus plantarum DT88 every day. The mice in the control group (Control) 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 fasted and watered 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 with a tissue grinder (60 Hz, 45 s, 4 2-mm steel balls), add 40 μL of 50 g / L SDS after grinding and mix well by shaking, and 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 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 FSC greater than 60000, the sample loading volume was 20 μL, and the detection channels were: B530, FITC-H.

[0103] The results are shown in Figure 12。In the control group, a large amount of PS fluorescent microspheres remained in the ileum and cecum of the mice. Oral administration of Lactobacillus plantarum DT88 significantly reduced the residual amount of PS fluorescent microspheres in the ileum and cecum of the mice. This indicates that oral administration of Lactobacillus plantarum DT88 reduced the residual amount of microplastics in the mice.

[0104] Example 9 Oral administration of Lactobacillus plantarum DT88 reduces inflammatory response

[0105] Take the serum collected before sacrificing the mice in Example 8 and the dissected ileum tissues, and detect the levels of immune factors by ELISA. The results are shown in Figure 13 。As can be seen from the figure, 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 oral administration of Lactobacillus plantarum DT88, the levels of inflammatory factors returned to normal levels. The content of IL-10 in the serum and intestine increased significantly, and the content of TNF-α, IL-6 and IL-1β in the ileum decreased significantly, indicating that oral administration of Lactobacillus plantarum DT88 can reduce the inflammatory response caused by microplastics.

[0106] In summary, a strain of Lactobacillus plantarum DT88 was isolated and screened in the present invention. This strain is acid-resistant and bile salt-resistant, and has the ability to colonize the stomach and small intestine, and can be applied to the development of edible probiotics. Lactobacillus plantarum DT88 has a strong ability to adsorb microplastics. Experimental data also prove that this strain has antioxidant ability, can increase the rate of microplastic excretion from the intestine in vivo, reduce the residual amount of microplastics in the intestine, and reduce the inflammatory response caused by microplastics. Thus, Lactobacillus plantarum DT88 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 caused by microplastics.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactiplantibacillus plantarum DT88, characterized in that, The Lactiplantibacillus plantarum DT88 is deposited in the Guangdong Provincial Microbial Culture Collection Center under the deposit number GDMCC No: 63626.

2. A microbial preparation comprising the Lactiplantibacillus plantarum DT88 as claimed in claim 1.

3. The preparation method of the microbial agent according to claim 2, characterized in that, The method comprises the step of culturing the Lactiplantibacillus plantarum DT88.

4. Use of the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2 in the preparation of a product for adsorbing microplastics and / or promoting the excretion of microplastics.

5. Use of the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2 in the adsorption and / or removal of microplastics in the environment.

6. Use of the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2 in the preparation of an antioxidant product.

7. Use of the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2 in the preparation of food, medicine or feed.

8. A food product, characterized in that, The food comprises the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2.

9. A drug, characterized in that, The medicine comprises the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2.

10. A microplastic adsorbent, characterized in that, The microplastic adsorbent comprises the Lactiplantibacillus plantarum DT88 as claimed in claim 1 or the microbial preparation as claimed in claim 2.

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