Lactobacillus paracasei DT66 and application of lactobacillus paracasei DT66 in preparation of product with microplastic adsorption and removal functions

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus paracasei DT66 and application thereof in preparation of products with microplastic adsorption and removal functions. The invention provides a strain of Lactobacillus paracasei DT66, and the preservation number of the strain of Lactobacillus paracasei DT66 is GDMCC No: 63624. The strain of Lactobacillus paracasei DT66 has the preservation number of CGMCC No: 63624. The strain has good acid resistance and cholate resistance, and can be developed into edible probiotics; the microplastics can be adsorbed, discharge of plastics in the body is promoted and accelerated, microplastics residues in the intestinal tract are reduced, accumulated oxidative damage of the microplastics is reduced, and human intestinal tract health is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Lactobacillus paracasei DT66 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). 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. It is estimated that the weekly intake of microplastics by the human body can reach 5 g. Research shows that microplastics exist in human feces, blood, lung tissue and placenta.

[0003] 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. It can be seen that microplastic pollution has not only become a serious environmental problem, but also poses a potential threat to human health. Therefore, removing microplastics from the human body and reducing the content of microplastics are of great significance to the long-term health of the human body.

[0004] At present, there are only a few reports on using biological methods to reduce plastic pollution in the environment and water bodies. For example, some bacteria and fungi are 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 that can adsorb plastic particles. They attach to the surface of microplastics and can generate 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 the human gastrointestinal tract is not a suitable environment for the survival of such bacteria and fungi, making it difficult to apply their degradation and adsorption capabilities to the removal of human microplastics. Therefore, in order to reduce the accumulation of microplastics in the human body and reduce the health damage caused by microplastics to the human body, it is necessary to discover probiotics that can tolerate the gastrointestinal environment and adsorb and remove microplastics in this field. Summary of the Invention

[0006] The present invention aims to provide a strain of Lactobacillus paracasei DT66 with strong microplastic adsorption ability and potential for development into edible probiotics. The strain has good acid and bile salt resistance, can effectively adsorb microplastics and has an antioxidant effect. Therefore, the strain is developed into an edible probiotic, which is expected to achieve the adsorption of microplastics in the human body, accelerate the discharge and removal of microplastics, and reduce the damage of microplastics to the gastrointestinal tract.

[0007] The present invention's Lactobacillus paracasei DT66 is isolated from fish tea, and the strain is identified by bacterial morphology, physiology and 16S rRNA sequencing, and the result is Lactobacillus paracasei (Lacticaseibacillus paracasei), named Lactobacillus paracasei DT66. The strain has been preserved in Guangdong Microbial Culture Collection Center (Guangdong Microbial Culture Collection Center, GDMCC) on July 5, 2023, and the address of the preservation unit is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Postal Code 510070, Classification Name Lacticaseibacillus paracasei, Preservation Number: GDMCC No: 63624.

[0008] Lactobacillus paracasei DT66 has the following microbiological characteristics:

[0009] (1) Morphological characteristics

[0010] Gram staining is positive, and under light microscopy it is rod-shaped with round ends, and can exist in pairs or chains.

[0011] After culturing in MRS solid medium for 24 hours, round, convex-middle, smooth-edged, and bright-surfaced milky white colonies were formed.

[0012] (2)Physiological characteristics

[0013] Lactobacillus paracasei DT66 can grow in acidic or bile salt-containing culture media. The strain can effectively adsorb microplastics with an adsorption rate of over 70%. Lactobacillus paracasei DT66 has an antioxidant effect and can reduce the oxidative damage caused by microplastics. Lactobacillus paracasei DT66 can accelerate the excretion of microplastics in the intestines of mice and reduce the residual microplastics in the intestines.

[0014] Lacticaseibacillus paracasei widely exists in the human intestine and has a long and extensive history of use in food fermentation, especially in dairy product fermentation. Lacticaseibacillus paracasei has been included in the list of strains that can be used in food and 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 Lacticaseibacillus paracasei.

[0015] The present invention also provides a bacterial agent containing the above-mentioned Lacticaseibacillus paracasei DT66.

[0016] The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0017] Specifically, the liquid bacterial agent is a bacterial suspension containing the Lacticaseibacillus paracasei DT66 or a processed bacterial suspension; the solid bacterial agent is a product obtained by spray drying or freeze drying the bacterial suspension of the Lacticaseibacillus paracasei DT66 or the processed bacterial suspension.

[0018] The present invention also provides a preparation method of the above-mentioned bacterial agent, which includes the following steps: culturing the Lacticaseibacillus paracasei DT66 to prepare a bacterial suspension, or further preparing the bacterial suspension into a solid bacterial agent.

[0019] In the preparation method of the present invention, the medium for culturing the Lacticaseibacillus paracasei DT66 is MRS medium, and the culture conditions are anaerobic culture at 36 - 37 °C.

[0020] As a specific embodiment, the culture method of the Lacticaseibacillus paracasei DT66 is: inoculating the Lacticaseibacillus paracasei DT66 into the MRS medium. The composition of the MRS broth medium is: 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. The culture temperature is preferably 37 °C, and the culture time is preferably 24 h.

[0021] Based on the functions of the Lacticaseibacillus paracasei DT66 discovered in the present invention, the present invention further provides an application of the above-mentioned Lacticaseibacillus paracasei DT66 or bacterial agent in the preparation of products that can adsorb microplastics, promote the excretion of microplastics from the body, or reduce oxidative damage.

[0022] Preferably, reduce the oxidative damage caused by the accumulation of microplastics.

[0023] The present invention further provides an application of the above-mentioned Lacticaseibacillus paracasei DT66 or the bacterial agent in the preparation of food or medicine.

[0024] The product of the present invention can achieve the adsorption and removal of microplastics and reduce the oxidative damage caused by microplastics.

[0025] Furthermore, the present invention further provides a food or medicine, which comprises the above-mentioned Lacticaseibacillus paracasei DT66.

[0026] In addition to containing the Lacticaseibacillus paracasei DT66 of the present invention, the food may also contain a variety of other probiotics. The cooperation of various probiotics can further enhance the efficacy of the food.

[0027] The medicine also includes pharmaceutically acceptable excipients.

[0028] The medicine of the present invention can be prepared by existing conventional methods, such as by conventional mixing, granulation, sugar coating, dissolution or freeze-drying methods. It may contain a therapeutically effective amount of a pharmacologically active ingredient or may also contain one or more pharmaceutically acceptable carriers at the same time. The preferred administration route of the medicine of the present invention is oral administration. The dosage form can be sugar-coated tablets, tablets or capsules. The unit content of the active ingredient contained in a single dose in each dosage form does not necessarily need to constitute an effective amount by itself, and the required effective amount can be achieved by administering multiple dosage units.

[0029] When preparing a liquid medicine for an oral dosage form, any conventional medicinal medium can be used; in the case of preparing oral solid preparations, such as powders, capsules and tablets, carriers such as flavoring agents, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. can be used. The present invention preferably uses tablets and capsules that are easy to administer.

[0030] The beneficial effects of the present invention are at least as follows:

[0031] Compared with the prior art, the Lacticaseibacillus paracasei DT66 provided by the present invention has good acid and bile salt tolerance and can be developed into an edible probiotic; this strain can adsorb microplastics, increase the rate of microplastic excretion from the intestine in vivo, reduce the residue of microplastics in the intestine, and reduce the oxidative damage caused by the accumulation of microplastics, providing great benefits for human intestinal health. Description of the Drawings

[0032] Figure 1Colony morphology of Lactobacillus paracasei DT66 on MRS medium in Example 1.

[0033] Figure 2 Microscopic examination image of Lactobacillus paracasei DT66 in Example 1.

[0034] Figure 3 Survival rate of Lactobacillus paracasei DT66 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.

[0035] Figure 4 Survival rate of Lactobacillus paracasei DT66 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.

[0036] Figure 5 Photo of the adsorption and agglutination of Lactobacillus paracasei DT66 and PS fluorescent microspheres in solution in Example 4.

[0037] Figure 6 Adsorption rate of Lactobacillus paracasei DT66 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.

[0038] Figure 7 Electron microscopy image of the adsorption of Lactobacillus paracasei DT66 and PS fluorescent microspheres in Example 4. The magnification is 50000 times.

[0039] Figure 8 Scavenging rate of Lactobacillus paracasei DT66 on DPPH in Example 5. Error bars represent standard deviation, and data are from three repeated experiments.

[0040] Figure 9 Electron microscopy image of Lactobacillus paracasei DT66 after incubation with mixed microplastic particles in Example 6. The magnification is 2000 times.

[0041] Figure 10 Fluorescence imaging of Lactobacillus paracasei DT66 after incubation with mixed microplastic particles in Example 6. The green signal represents Lactobacillus paracasei DT66, and the red signal represents microplastic particles. The arrow indicates that Lactobacillus paracasei DT66 is adsorbed on the surface of microplastic particles. The magnification is 1000 times.

[0042] Figure 11Detection results of the accelerated transport of microplastics by the colonization of Lactobacillus paracasei DT66 in Example 7. In the figure, A is the in vivo imaging of the mouse intestine, and B is the excretion rate diagram. Error bars represent the standard deviation. The statistical analysis method is T-test, and * indicates that the p-value is less than 0.05.

[0043] Figure 12 Detection results of the residual amount of PS fluorescent microspheres in the ileum (A) and cecum (B) of mice in Example 8. Error bars represent the standard deviation. The statistical analysis method is ANOVA, ** indicates that the p-value is less than 0.01. *** indicates that the p-value is less than 0.001. **** indicates that the p-value is less than 0.0001. Detailed implementation manners

[0044] The preferred implementation manners of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments 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.

[0045] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources or prepared by conventional methods in this field unless otherwise specified.

[0046] Example 1 Isolation and identification of Lactobacillus paracasei DT66

[0047] 1. Isolation and identification of Lactobacillus paracasei DT66

[0048] 1.1 Sample source

[0049] The strain Lactobacillus paracasei DT66 used in the present invention was isolated from fish tea.

[0050] 1.2 Preparation of culture medium

[0051] The culture medium used for sample separation and strain screening is MRS medium, and the culture medium used for culturing Lactobacillus paracasei DT66 is MRS medium (pH 5.7 ± 0.2). The composition of MRS medium is shown in Table 1, and 1.5% agar is added to it to obtain MRS solid medium.

[0052] Table 1 MRS medium formula

[0053]

[0054]

[0055] 1.3 Isolation of strain

[0056] Put 1 g of the fish tea 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 and perform ten-fold serial dilution. Select 10 -4 、10 -5 、10 -6 、10 -7 For four dilution gradients, apply 100 μL of the bacterial suspension to a petri dish 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 dish into MRS liquid medium for culture. The species information of the isolated strain is determined by 16S rRNA sequencing.

[0057] 2. Identification of Lactobacillus paracasei DT66

[0058] 2.1 Colony characteristics

[0059] After culturing Lactobacillus paracasei DT66 in MRS solid medium for 24 h, round, convex in the middle, smooth at the edges, and bright milky white colonies are formed, as shown in Figure 1 .

[0060] 2.2 Morphology under the microscope

[0061] Colony smear of Lactobacillus paracasei DT66: Gram staining is positive, rod-shaped under a light microscope, round at both ends, and can exist in pairs or chains. See Figure 2 .

[0062] 2.3 16S rRNA identification

[0063] Identification unit: Tsingke Biotechnology Co., Ltd.

[0064] Identification sequence: See SEQ ID No.1.

[0065] Identification result: Compare the sequencing result with the NCBI database, and combine the comparison result with the physiological and biochemical results to determine that the strain is Lactobacillus paracasei.

[0066] Example 2 Acid tolerance detection of Lactobacillus paracasei DT66

[0067] The overall pH condition in the human stomach environment is strongly acidic. Therefore, the acid tolerance of the strain is an important indicator to evaluate whether it can survive and colonize in the gastric acid environment. The commercial strain Lactobacillus rhamnosus GG is a widely used probiotic with strong acid tolerance.

[0068] In the present invention, MRS medium with pH = 2.5 was used to verify the acid tolerance ability of Lactobacillus paracasei DT66. 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. Spread the diluted samples on MRS agar plates and perform colony counting after anaerobic culture at 37 °C for 16 h. 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 (control) is Lactobacillus rhamnosus GG.

[0069] After 3 hours of culture in the acidic medium, the survival rate of the control strain Lactobacillus rhamnosus GG was 84.56%, and the survival rate of Lactobacillus paracasei DT66 was 85.62% ( Figure 3 ), which is equivalent to the acid tolerance ability of the control strain, indicating that the strain of the present invention has strong acid tolerance ability and can survive in the gastric environment.

[0070] Example 3 Detection of bile salt tolerance of Lactobacillus paracasei DT66

[0071] After bacteria enter the intestine through 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 performance of Lactobacillus paracasei DT66 strain.

[0072] Inoculate the Lactobacillus paracasei DT66 bacterial solution into a 96-well deep well plate containing MRS medium and culture anaerobically 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 uL 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 uL of MTT solution. After adding the MTT solution, react in the dark at 37 °C 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-labeled instrument and calculate the survival rate. The survival rate = A1 / A0 × 100% (A1: absorbance value of the treatment group solution at 570 nm, A0: absorbance value of the control group solution at 570 nm). Measure the survival rate of the control strain Lactobacillus rhamnosus GG by the same method.

[0073] After culturing in 0.1% bile salt-MRS medium for 4 hours, the survival rate of the control strain Lactobacillus rhamnosus GG was 101.52%, and the survival rate of Lactobacillus paracasei DT66 was 101.50%( Figure 4 ). This indicates that Lactobacillus paracasei DT66 has the same bile salt tolerance as the control strain Lactobacillus rhamnosus GG and can survive in the small intestine.

[0074] Example 4 Determination of the effect of Lactobacillus paracasei DT66 on adsorbing microplastics

[0075] Inoculate Lactobacillus paracasei DT66 into MRS medium and culture anaerobically at 37°C for 24 h. The cultured bacterial solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 450 uL of sterile PBS buffer was added for washing twice. Then resuspend with PBS and adjust the bacterial solution concentration to 1×10 9 CFU / mL.

[0076] In the experimental group, take 100 uL of the Lactobacillus paracasei DT66 bacterial suspension into a 1.5 mL EP tube, add 900 uL of the PS fluorescent microsphere working solution (0.16 mg / mL, particle size 0.1 μm, Bestbio Company), mix well, and place it on a shaker for light-shielded oscillatory incubation 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 solution control group, take 100 μL of the Lactobacillus paracasei DT66 bacterial suspension and 900 μL of PBS into a 1.5 mL EP tube, mix well and incubate. After the incubation is over, take the incubation solution to observe and take pictures. The results are shown in Figure 5 .

[0077] From Figure 5 it can be seen that in the blank control group, the PS fluorescent microspheres do not self-aggregate; in the bacterial solution control group, Lactobacillus paracasei DT66 does not self-aggregate; in the experimental group, specific adsorption and aggregation flocs appear between Lactobacillus paracasei DT66 and the PS fluorescent microspheres.

[0078] Take the incubation solutions of the experimental group and the blank control group, centrifuge at 2000 rpm for 10 min, take 100 uL 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 paracasei 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 paracasei 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 inFigure 7 。

[0079] From Figure 6 It can be seen that the adsorption rate of the control strain is 15.33%, and its microplastic adsorption ability is poor. While the adsorption rate of Lactobacillus paracasei DT66 reaches 71.38%, showing a strong ability to adsorb microplastics. This indicates that the adsorption effect of Lactobacillus paracasei DT66 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 Lactobacillus paracasei DT66.

[0081] Example 5 Determination of the antioxidant capacity of Lactobacillus paracasei DT66

[0082] Lactobacillus paracasei DT66 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. The concentration of the bacterial solution 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 C solution was 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 reacted in the dark with shaking 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. Calculate the DPPH free radical scavenging rate. 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 on DPPH is 28.91%. Lactobacillus paracasei DT66 has certain antioxidant capacity, and its scavenging rate on DPPH is 22.91%, similar to that of vitamin C. Therefore, colonizing Lactobacillus paracasei DT66 can reduce the oxidative damage caused by microplastics to the host.

[0084] Example 6 Determination of the adsorption effect of Lactobacillus paracasei DT66 on mixed microplastics

[0085] To simulate and verify the adsorption effect of Lactobacillus paracasei DT66 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 1 mg / mL mixed microplastic suspension for adsorption effect detection.

[0086] Lactobacillus paracasei DT66 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 it was resuspended with PBS, and the bacterial solution concentration was adjusted to 1×10 9 CFU / mL. 100 μL of the Lactobacillus paracasei DT66 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 paracasei DT66 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 paracasei DT66 was taken, washed with PBS (for specific operations, see above), 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 bacteria labeled with FITC emitted green fluorescence. The red fluorescence in the figure was surrounded by green signals, indicating that Lactobacillus paracasei DT66 was adsorbed on the surface of the mixed microplastics.

[0089] Example 7: Accelerating the excretion of microplastics in mice by intragastric administration of Lactobacillus paracasei DT66

[0090] Six-week-old C57 mice were purchased. After one week of adaptive feeding, the experimental group was intragastrically administered 1×10 9 CFU of Lactobacillus paracasei DT66 daily, and the control group (Control) was intragastrically administered an equal volume of normal saline. After 7 consecutive days of intragastric administration, the mice were deprived of water and food for 16 h, then intragastrically administered 1 mg of PS fluorescent microspheres (10 mg / mL, particle size 5 μm, Besler Co., Ltd.). Twenty minutes later, the mouse intestine was 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 intestine, as shown in Figure 11 A. And the microplastic excretion rate was calculated. The calculation formula was: microplastic excretion rate = displacement length of PS fluorescent microspheres / intestinal length × 100%, as shown in Figure 11 B. It can be seen from the figure that after intragastric administration of Lactobacillus paracasei DT66, the PS fluorescent microspheres were transferred from the gastric end to the cecal end faster, and the microplastic excretion rate increased significantly by 14.86%.

[0091] Example 8: Reducing the residual microplastics in the body by intragastric administration of Lactobacillus paracasei DT66

[0092] 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 Co., Ltd.) every day, and at the same time, 1×10 9 CFU of Lactobacillus paracasei DT66 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 carried out continuously 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: weigh it and 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 oscillation, 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 fluorescence 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.

[0093] The results are shown inFigure 12 。In the control group of mice, a large amount of PS fluorescent microspheres remained in the ileum and cecum. Oral administration of Lactobacillus paracasei DT66 significantly reduced the residual amount of PS fluorescent microspheres in the ileum and cecum of mice. This indicates that oral administration of Lactobacillus paracasei DT66 reduces the residual amount of microplastics in mice.

[0094] In summary, a strain of Lactobacillus paracasei DT66 was isolated and screened in the present invention. This strain is acid-tolerant and bile-salt-tolerant, has the ability to colonize the stomach and small intestine, and can be applied to the development of edible probiotics. Lactobacillus paracasei DT66 has a strong ability to adsorb microplastics. Experimental data also prove the antioxidant ability of this strain, as well as the ability to accelerate the excretion of plastics in the body and reduce the residual amount of microplastics in the intestine. Thus, Lactobacillus paracasei DT66 is a strain suitable for the digestive tract environment and has broad application prospects in adsorbing microplastics, accelerating the excretion of microplastics from the body, and reducing the oxidative damage 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 strain of Lacticaseibacillus paracasei, characterized in that, It is Lactobacillus paracasei DT66 with the preservation number of GDMCC No: 63624.

2. A bacterial agent containing the Lactobacillus paracasei described in claim 1.

3. The preparation method of the microbial agent according to claim 2, characterized in that, It includes the following steps: preparing the Lactobacillus paracasei DT66 into a bacterial suspension through cultivation, or further preparing the bacterial suspension into a solid bacterial agent.

4. The preparation method according to claim 3, characterized in that, The culture medium for culturing the Lactobacillus paracasei DT66 is MRS medium, and the culture conditions are anaerobic culture at 36 - 37 °C.

5. The application of the Lactobacillus paracasei described in claim 1 or the bacterial agent described in claim 2 in the preparation of a product that can adsorb microplastics and / or promote the excretion of microplastics from the body.

6. The application of the Lactobacillus paracasei described in claim 1 or the bacterial agent described in claim 2 in the preparation of a product that can reduce oxidative damage.

7. The application of the Lactobacillus paracasei described in claim 1 or the bacterial agent described in claim 2 in the preparation of food or medicine.

8. A food, characterized in that, It contains the Lactobacillus paracasei described in claim 1.

9. A drug, characterized in that, It contains the Lactobacillus paracasei described in claim 1.

10. The drug according to claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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