Lactobacillus plantarum strain dt77 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 particularly relates to a lactobacillus plantarum DT77 and application of the lactobacillus plantarum DT77 in preparation of products with micro-plastic adsorption and removal functions. The lactobacillus plantarum DT77 provided by the present application is preserved in the Guangdong Microbial Digital Culture Collection Center, and the preservation number is GDMCC No: 63625. The lactobacillus plantarum DT77 has good acid and bile salt resistance, and can resist the gastrointestinal environment. The strain can efficiently adsorb micro-plastics, accelerate the discharge and removal of micro-plastics, has antioxidant capacity, can reduce the oxidative damage caused by micro-plastic accumulation, can reduce the micro-plastic residues in the intestinal tract, and reduce the inflammatory response caused by micro-plastics, and is expected to be developed into edible probiotic products to play the functions of adsorbing micro-plastics, accelerating the discharge of micro-plastics, reducing the damage of micro-plastics, and protecting the gastrointestinal tract.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus plantarum DT77 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 decomposes into tiny particles through physical, chemical, and biological processes. Plastic particles with a diameter of less than 5 mm are generally defined as microplastics (MPs), and those with a diameter of less than 0.1 μm are generally defined as nanoplastics (NPs). Microplastics are made from materials such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), and polyethylene terephthalate (PET).

[0003] Currently, microplastic pollution has not only become a serious environmental problem but also poses a potential threat to human health. Microplastics are widely present in the 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 found in human feces, blood, lung tissue, and placenta. Although there is currently no direct evidence to prove 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 producing toxic effects. Therefore, the harm of microplastics to human health is receiving increasing attention, and promoting the excretion of microplastics from the body and reducing microplastic levels is of great significance for long-term human health.

[0004] Currently, no method has been reported to remove microplastics from the human body; only some biological methods have been reported to reduce plastic pollution in water and other environments. For example, some bacteria and fungi have been reported to have the ability to degrade plastics by secreting keratinase, protease, esterase, and lipase to break down polymers into monomers or oligomers. Another method for removing plastic particles is to utilize bacteria to adsorb them. Some bacteria have the ability to capture microplastics; they attach to the surface of microplastics and can form a sticky biofilm. This sticky matrix can capture free microplastics, leading to microplastic bioaggregation, thereby achieving the separation and removal of microplastics. However, the existing bacteria and fungi capable of degrading or adsorbing microplastics are all inedible strains, and these bacteria are difficult to tolerate the gastrointestinal environment. Therefore, the above-mentioned bacteria or fungi have no practical application value in removing microplastics from the human body.

[0005] To reduce the accumulation of microplastics in the human body and mitigate the health damage caused by microplastics, it is necessary to discover bacteria that can tolerate the gastrointestinal environment and efficiently adsorb and remove microplastics. Summary of the Invention

[0006] This invention provides a strain of Lactobacillus plantarum DT77 and its application in the preparation of products with microplastic adsorption and removal functions.

[0007] This invention provides Lactiplantibacillus plantarum DT77, which was deposited on July 5, 2023, at the Guangdong Microbial Culture Collection Center (GDMCC, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China), classified as Lactiplantibacillus plantarum, with accession number GDMCC No: 63625.

[0008] In this invention, the Lactobacillus plantarum DT77 strain was isolated from sauerkraut. The strain was identified by bacterial morphology, physiology and 16S rRNA sequencing, and the result was Lactobacillus plantarum, named Lactobacillus plantarum DT77.

[0009] Lactobacillus plantarum DT77 has the following microbiological characteristics:

[0010] (1) Morphological characteristics

[0011] 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.

[0012] After culturing in MRS solid medium for 24 hours, round, convex, smooth-edged, moist, milky-white colonies are formed.

[0013] (2)Physiological characteristics

[0014] Lactobacillus plantarum DT77 can grow in acidic or bile-containing media. This strain can effectively adsorb microplastics, with an adsorption rate exceeding 80%. Lactobacillus plantarum DT77 has antioxidant effects, reducing oxidative damage caused by microplastics. Lactobacillus plantarum DT77 can reduce microplastic residues in the mouse intestine and reduce the inflammatory response caused by microplastics.

[0015] Lactobacillus plantarum DT77 can be cultured using the following method: Inoculate Lactobacillus plantarum DT77 into MRS broth medium and culture anaerobically at 37℃ for 24 hours. The MRS broth medium consists of: casein digest 10.0 g / L, beef meal 10.0 g / L, yeast extract 4.0 g / L, triammonium 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 added to the solid medium.

[0016] Lactobacillus plantarum is widely found in fermented foods and is commonly used in the food industry as a starter culture or 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 (QPS) list 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] The present invention provides a microbial preparation comprising the above-described Lactobacillus plantarum DT77.

[0018] Preferably, in the microbial preparations described above, Lactobacillus plantarum DT77 exists in the form of live bacteria.

[0019] The microbial preparations described above may be solid preparations (e.g., bacterial powder) or liquid preparations.

[0020] The present invention provides a method for preparing the above-described microbial preparation, the method comprising the step of culturing the *Lactobacillus plantarum* DT77.

[0021] Preferably, the culture is an anaerobic culture at 35-37°C.

[0022] Preferably, the culture is carried out using MRS broth medium. After the culture is completed, the bacterial solution is collected and further prepared into a microbial preparation.

[0023] Based on the functions of Lactobacillus plantarum DT77, the present invention provides any of the following applications of this strain.

[0024] The present invention provides the use of the Lactobacillus plantarum DT77 or the microbial preparation in the preparation of products for adsorbing microplastics and / or promoting the expulsion of microplastics.

[0025] Lactobacillus plantarum DT77 has good acid and bile salt resistance, and can well tolerate the gastrointestinal environment in the body, thus exerting the effects of adsorbing microplastics, promoting the excretion of microplastics, and anti-oxidation in the body.

[0026] In the above applications, the product is preferably food, medicine, or feed. The food is preferably health food.

[0027] This invention provides the application of the Lactobacillus plantarum DT77 or the microbial preparation in adsorbing and / or removing microplastics in the environment.

[0028] Lactobacillus plantarum DT77 can also adsorb and promote 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 and soil.

[0029] This invention provides the application of the Lactobacillus plantarum DT77 or the microbial preparation in the preparation of antioxidant products.

[0030] Lactobacillus plantarum DT77 possesses free radical scavenging capabilities, enabling it to exert antioxidant functions and reduce oxidative damage caused by microplastics. Lactobacillus plantarum DT77 simultaneously adsorbs microplastics and has antioxidant properties. On the one hand, it promotes the excretion of microplastics from the body; on the other hand, it reduces oxidative damage caused by residual microplastics, effectively mitigating the adverse effects of microplastic accumulation on the body from both perspectives.

[0031] In the above applications, the product is preferably food, medicine, or feed. The food is preferably a dietary supplement or health food.

[0032] This invention provides the use of the Lactobacillus plantarum DT77 or the microbial preparation in the preparation of food, pharmaceuticals or feed.

[0033] The present invention provides a food product containing the above-mentioned Lactobacillus plantarum DT77 or the microbial preparation.

[0034] The present invention provides a medicine comprising the above-described Lactobacillus plantarum DT77 or the microbial preparation described above.

[0035] The present invention provides a feed comprising the above-described Lactobacillus plantarum DT77 or the microbial preparation.

[0036] In addition to containing *Lactobacillus plantarum* DT77 or the aforementioned microbial preparations, the aforementioned pharmaceuticals, foods, and feeds may also contain raw materials or excipients permitted in the pharmaceutical, food, and feed industries. Among these, excipients permitted in the pharmaceutical field include fillers, excipients, lubricants, wetting agents, diluents, etc. The formulation type of the pharmaceutical product may be a solid dosage form (e.g., powder, granules, capsules, tablets, etc.) or a liquid dosage form (e.g., oral liquid, etc.).

[0037] The present invention provides a microplastic adsorbent comprising the above-described Lactobacillus plantarum DT77 or the microbial preparation.

[0038] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention include at least the following: Lactobacillus plantarum DT77 has good acid and bile salt resistance, can tolerate the gastrointestinal environment, and has the potential to be used as an edible probiotic; this strain can efficiently adsorb microplastics, accelerate the excretion and removal of microplastics, and has antioxidant capacity, which can reduce oxidative damage caused by microplastic accumulation. In vivo, this strain can reduce microplastic residues in the intestine and reduce the inflammatory response caused by microplastics, and is expected to be developed into an edible probiotic product to exert the effects of adsorbing microplastics, accelerating the excretion of microplastics, reducing microplastic damage, and protecting the gastrointestinal tract. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum DT77 on MRS medium in Example 1.

[0041] Figure 2 This is a microscopic image of Lactobacillus plantarum DT77 from Example 1.

[0042] Figure 3 The results show the survival rate of Lactobacillus plantarum DT77 in acidic culture medium in Example 2. ns indicates a p-value greater than 0.05 (data from three replicate experiments, error bars represent standard deviations). The statistical analysis method was t-test.

[0043] Figure 4 The results show the survival rate of Lactobacillus plantarum DT77 in bile salt-containing medium in Example 3. ns indicates a p-value greater than 0.05 (data from three replicate experiments, error bars represent standard deviations). The statistical analysis method was t-test.

[0044] Figure 5 This is a photograph of Lactobacillus plantarum DT77 adsorbing and agglomerating with PS fluorescent microspheres in solution in Example 4.

[0045] Figure 6The adsorption rate of PS fluorescent microspheres in solution by Lactobacillus plantarum DT77 in Example 4 is shown. In this figure, **** indicates a p-value less than 0.0001 (data from three repeated experiments, error bars represent standard deviations). The statistical analysis method is ttest.

[0046] Figure 7 The image shows an adsorption electron microscope image of Lactobacillus plantarum DT77 and PS fluorescent microspheres in Example 4, magnified 50,000 times.

[0047] Figure 8 The results of DPPH scavenging rate by Lactobacillus plantarum DT77 in Example 5 are shown. The data are from three replicate experiments, and the error bars represent the standard deviation.

[0048] Figure 9 The image shows an adsorption electron microscope image of Lactobacillus plantarum DT77 and mixed microplastic particles in Example 6, magnified 2000 times.

[0049] Figure 10 The image shows the adsorption fluorescence imaging of Lactobacillus plantarum DT77 and mixed microplastic particles in Example 6. In the image, the green signal represents Lactobacillus plantarum DT77, and the red signal represents microplastic particles. The arrows indicate that Lactobacillus plantarum DT77 is adsorbed on the surface of the microplastic particles. The magnification is 1000x.

[0050] Figure 11 The residual amount of PS fluorescent microspheres in the ileum (A) and cecum (B) of mice in Example 7 was detected. The error bar represents the standard deviation. The statistical analysis method was ANOVA. ns indicates a p-value greater than 0.01, and **** indicates a p-value less than 0.0001.

[0051] Figure 12 The results for the detection of immune factors in mouse serum (A) and ileum tissue (BE) in Example 8 are shown below. Error bars represent standard deviations. The statistical analysis method is ANOVA. * indicates a p-value less than 0.05, ** 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.

[0052] Figure 3 , Figure 4 , Figure 6 , Figure 11 , Figure 12 In the middle, "Control" represents the control. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0054] Example 1: Isolation and Identification of Lactobacillus plantarum DT77

[0055] 1. Isolation and identification of Lactobacillus plantarum DT77

[0056] 1.1 Sample Source

[0057] The *Lactobacillus plantarum* DT77 of this invention was isolated from sauerkraut.

[0058] 1.2 Preparation of culture medium

[0059] The culture medium used for sample separation was BBL medium, and MRS medium was used for Lactobacillus plantarum DT77 culture.

[0060] 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 will produce a solid medium.

[0061] Table 1 BBL culture medium formulation

[0062]

[0063] Table 2 MRS culture medium formulation

[0064]

[0065] 1.3 Isolation of strains

[0066] Add 1g of sauerkraut sample to 10mL of BBL 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 petri dishes containing sterile BBL 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 petri dishes and culture them in BBL liquid medium. The species information of the isolated strains was determined by 16S rRNA sequencing.

[0067] 2. Identification of Lactobacillus plantarum DT77

[0068] 2.1 Colony characteristics

[0069] After culturing *Lactobacillus plantarum* DT77 on MRS solid medium for 24 hours, it formed round, convex, smooth-edged, moist, milky-white colonies. Figure 1 .

[0070] 2.2 Microscopic morphology

[0071] Lactobacillus plantarum DT77 colony smear: Gram-positive; under light microscopy, cells are rod-shaped with rounded ends, arranged singly or in pairs or chains. See Figure 2 .

[0072] 2.3 Identification of 16S rRNA

[0073] Testing organization: Qingke Biotechnology Co., Ltd.

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

[0075] 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.

[0076] Lactiplantibacillus plantarum DT77 was deposited on July 5, 2023, at the Guangdong Microbial Culture Collection Center (GDMCC, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China), classified as Lactiplantibacillus plantarum, with accession number GDMCC No: 63625.

[0077] Example 2: Acid resistance test of Lactobacillus plantarum DT77

[0078] 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.

[0079] This example uses MRS medium at pH 2.5 to verify the acid tolerance of *Lactobacillus plantarum* DT77. 1 mL of bacterial culture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the sample 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.

[0080] After 3 hours of incubation in acidic medium, the survival rate of the control strain *Lactobacillus rhamnosus* GG was 84.56%, and the survival rate of *Lactobacillus plantarum* DT77 was 95.38%. 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.

[0081] Example 3: Detection of bile salt tolerance in Lactobacillus plantarum DT77

[0082] After bacteria enter the intestines from the stomach, the high concentration of bile salts in the small intestine kills them. Food typically stays in the small intestine for 1–4 hours.

[0083] Therefore, this embodiment uses 0.1% bile salt-MRS medium to verify the bile salt tolerance of *Lactobacillus plantarum* DT77 strain. *Lactobacillus plantarum* DT77 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 mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. 100 μL of DMSO solution was added to each well, and the mixture was incubated at 37°C with shaking for 10 min to completely dissolve and mix the generated blue-purple formazan. After mixing, the absorbance of the solution at 570 nm was measured using an ELISA reader, and the survival rate was calculated. Survival rate = A1 / A0*100% (A1: absorbance of the experimental 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.

[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* DT77 was 114.4%. Figure 4 This indicates that *Lactobacillus plantarum* DT77 has comparable bile salt tolerance to the control strain *Lactobacillus rhamnosus* GG and can survive in the small intestine.

[0085] Example 4: Detection of the adsorption effect of Lactobacillus plantarum DT77 on microplastics

[0086] Lactobacillus plantarum DT77 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* DT77 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* DT77 bacterial suspension and 900 μL of PBS were added to a 1.5 mL EP tube and incubated. After incubation, the incubation solutions were observed and photographed. The results are shown below. Figure 5 .

[0087] 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. The adsorption rate of the control strain was measured using the same method, and the results are shown below. Figure 6 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 precipitate after centrifugation was fixed overnight at 4°C with glutaraldehyde, then subjected to gradient dehydration with ethanol. After drying, the precipitate was observed under an electron microscope. The results are shown in the figure. Figure 7 .

[0088] 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 DT77 did not self-aggregate; in the experimental group, Lactobacillus plantarum DT77 and PS fluorescent microspheres showed specific adsorption and agglomeration of flocculent material.

[0089] 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* DT77 reached 80.47%, demonstrating a strong ability to adsorb microplastics. This indicates that the adsorption effect of *Lactobacillus plantarum* DT77 on microplastics is strain-specific.

[0090] from Figure 7 As can be seen from electron microscopy, spherical PS fluorescent microspheres were adsorbed on the surface of Lactobacillus plantarum DT77.

[0091] Example 5: Determination of the antioxidant capacity of Lactobacillus plantarum DT77

[0092] Lactobacillus plantarum DT77 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 culture 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 incubated at 30℃ in the dark for 30 min. After incubation, 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. The DPPH free radical scavenging rate was calculated. 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).

[0093] The results are as follows Figure 8 As shown, the antioxidant vitamin C scavenged 47.90% of DPPH. Similar to vitamin C, *Lactobacillus plantarum* DT77 also possesses certain antioxidant capabilities, scavenging 35.21% of DPPH. Therefore, colonizing *Lactobacillus plantarum* DT77 can reduce the oxidative damage to the host caused by microplastics.

[0094] Example 6: Determination of the adsorption effect of Lactobacillus plantarum DT77 on mixed microplastics

[0095] To simulate and verify the adsorption effect of Lactobacillus plantarum DT77 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, and polycarbonate PC), resuspended in PBS solution containing 0.1% Tween-80, and prepared into a mixed microplastic suspension of 1 mg / mL for adsorption effect detection.

[0096] Lactobacillus plantarum DT77 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* DT77 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 h 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 DT77 is adsorbed on the surface of microplastic particles.

[0097] 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.

[0098] Overnight cultured *Lactobacillus plantarum* DT77 was washed with PBS, resuspended in 100 μM FITC solution, and incubated at 37°C with shaking at 100 rpm in the dark for 0.5 h. After incubation, the bacterial pellet was centrifuged, washed with PBS, resuspended, and the bacterial concentration was adjusted 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 DT77 is adsorbed on the surface of the mixed microplastics.

[0099] Example 7: Gavage administration of Lactobacillus plantarum DT77 reduces microplastic residues in the body.

[0100] 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 (Chemical Oxygenae Plantarum) DT77 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 vortexed to mix. 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.

[0101] See results Figure 11 In the control group, a large amount of PS fluorescent microspheres remained in the ileum and cecum of mice. After gavage with *Lactobacillus plantarum* DT77, the amount of PS fluorescent microspheres remaining in the ileum decreased, but not significantly; while the amount of PS fluorescent microspheres remaining in the cecum decreased significantly. This indicates that gavage with *Lactobacillus plantarum* DT77 reduced the residue of microplastics in mice.

[0102] Example 8: Gavage administration of Lactobacillus plantarum DT77 reduces inflammatory response

[0103] 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* DT77, all inflammatory factors returned to normal levels, and the levels of IL-10 in serum and intestine significantly increased, while the levels of TNF-α, IL-6, and IL-1β in the ileum significantly decreased, indicating that gavage administration of *Lactobacillus plantarum* DT77 can reduce the inflammatory response induced by microplastics.

[0104] In summary, this invention has isolated and screened a strain of *Lactobacillus plantarum* DT77. This strain is acid- and bile-tolerant, and has the ability to colonize the stomach and small intestine, making it suitable for the development of edible probiotics. *Lactobacillus plantarum* DT77 exhibits a strong ability to adsorb microplastics, and experimental data also demonstrates its antioxidant capacity. Furthermore, this strain can reduce microplastic residues in the intestine and alleviate inflammatory responses caused by microplastics in vivo. Therefore, *Lactobacillus plantarum* DT77 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 caused by microplastics.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum DT77, characterized in that, It is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 63625.

2. A microbial preparation, characterized in that, The microbial preparation comprises the Lactiplantibacillus plantarum DT77 of claim 1.

3. The method for preparing the microbial preparation according to claim 2, characterized in that, The method comprises the step of culturing the Lactiplantibacillus plantarum DT77.

4. Use of the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2 in the preparation of a product for adsorbing microplastics and / or promoting the excretion of microplastics.

5. Use of the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2 in adsorbing and / or removing microplastics in the environment.

6. Use of the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2 in the preparation of an antioxidant product.

7. Use of the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2 in the preparation of food or feed.

8. A food product, characterized by, The food comprises the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2.

9. A microplastic adsorbent, characterized by, The microplastic adsorbent comprises the Lactiplantibacillus plantarum DT77 of claim 1 or the microbial preparation of claim 2.

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