Use of bifidobacterium longum for the preparation of a microplastic toxicity mitigant and compositions

Abstract

The present application belongs to the field of microbial technology, and particularly relates to the use of Bifidobacterium longum for preparing a microplastic toxicity alleviating agent and a composition. The Bifidobacterium longum is Bifidobacterium longum subsp. infantis with a preservation number of CCTCC NO: M 20221253. Research shows that both live bacteria and inactivated bacteria of the strain can reduce the in vivo accumulation of microplastics, thereby inhibiting the toxic effects such as decreased movement ability, weakened reproductive ability, and shortened lifespan caused by the accumulation of microplastics. Moreover, oral administration of the inactivated bacteria does not need to consider the killing effect of stomach acid on live bacteria. Therefore, the Bifidobacterium longum can be used for oral administration to alleviate the related toxicity caused by the intake and accumulation of microplastics.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the use and composition of Bifidobacterium longum in the preparation of microplastic toxicity mitigators. Background Technology

[0002] With the widespread use of plastic products globally, plastic fragments further degrade into nanoplastics (≤100 nm) and microplastics (≤5 μm) due to ultraviolet radiation, biodegradation, and physical wear. These degradation products have become an increasingly serious environmental and health challenge. Nanoplastics (typically 0.001–0.1 μm in size) are particularly vulnerable due to their smaller size, making them more likely to penetrate biological barriers and accumulate in tissues, inducing a range of toxicities caused by microplastic accumulation, including oxidative stress and impaired reproductive capacity. Studies have shown that nanoplastics can accumulate in the food chain and be introduced into food through the air, further entering the human body through ingestion, respiration, and other routes, posing potential systemic health risks. Currently, biological interventions for nanoplastic exposure remain relatively limited, making the development of safe and effective active formulations crucial.

[0003] In recent years, some live probiotics, such as *Bifidobacterium animalis* subsp. *lactobacter* B01 (see CN118240690A), *Lactobacillus plantarum* CCFM1372 (see CN118460399A), and *Bifidobacterium breve* M16V (see CN114767720A), have been found to reduce microplastic accumulation in the gut by regulating gut microbiota, thereby reducing the toxicity caused by deaccumulation. However, probiotics need to be orally ingested into the gastrointestinal tract to exert their clearance effect on microplastic accumulation and subsequent activity. The survival rate of live bacteria is severely weakened in the acidic environment of the stomach, making their application extremely difficult and requiring special formulation methods to ensure improved tolerance of live bacteria to gastric acid. Research on the mitigating effect of non-live bacteria on microplastic accumulation is relatively limited. Summary of the Invention

[0004] To address the aforementioned problems, this invention has discovered that a strain of *Bifidobacterium longum* possesses a good effect in mitigating the cumulative toxicity of microplastics. Therefore, this invention provides the use of a strain of *Bifidobacterium longum* in the preparation of microplastic toxicity mitigating agents and related compositions. The technical solution of this invention is as follows: The use of Bifidobacterium longum in the preparation of microplastic toxicity mitigators, wherein the Bifidobacterium longum is the infant subsp. Bifidobacterium longum. Bifidobacterium longumsubsp. infantis Y46 The accession number is CCTCC NO: M20221253, the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is August 9, 2022, and the taxonomic name is... Bifidobacterium longum subsp.infantisPreferably, the microplastic is a nanoscale polystyrene microplastic. Preferably, the microplastic toxicity includes the toxicity caused by the microplastic to marine invertebrates, fish, and terrestrial mammals (including humans).

[0005] Preferably, the *Bifidobacterium longum* is inactivated *Bifidobacterium longum* and / or live *Bifidobacterium longum*. Inactivated *Bifidobacterium longum* has the advantage of not requiring special formulation protection against the killing effect of gastric acid, meaning that the mitigation effect on microplastic toxicity can be achieved solely through inactivated bacteria.

[0006] Preferably, the microplastic toxicity mitigator is an oral formulation.

[0007] Preferably, the microplastic toxicity mitigator is a microplastic toxicity mitigator that improves the decline in motor ability caused by microplastic exposure.

[0008] Preferably, the microplastic toxicity mitigator is a microplastic toxicity mitigator that improves the increase in reactive oxygen species levels and / or the decrease in resistance to oxidative stress caused by microplastic exposure.

[0009] Preferably, the microplastic toxicity mitigator is a microplastic toxicity mitigator that improves reproductive capacity reduced by microplastic exposure.

[0010] Preferably, the microplastic toxicity mitigator is a microplastic toxicity mitigator that improves the shortened lifespan caused by microplastic exposure.

[0011] Preferably, the microplastic toxicity mitigating agent is a microplastic toxicity mitigating agent that reduces the accumulation of microplastics in the body.

[0012] An oral composition for mitigating microplastic toxicity, the oral composition containing *Bifidobacterium longum* and pharmaceutically acceptable excipients, wherein the *Bifidobacterium longum* is *Bifidobacterium longum* subsp. infantis. Bifidobacterium longumsubsp. infantis Y46 The accession number is CCTCC NO: M 20221253, the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is August 9, 2022, and the taxonomic name is... Bifidobacterium longumsubsp. infantis The Bifidobacterium longum is an inactivated Bifidobacterium longum and / or a live Bifidobacterium longum.

[0013] Beneficial effects:

[0014] This invention uses nematodes as an animal model and found that both live and inactivated *Bifidobacterium longum* subsp. infantis BI_Y46 can reduce the accumulation of microplastics in vivo, thereby inhibiting the toxic effects of microplastic accumulation, such as decreased motility, reduced reproductive capacity, and shortened lifespan. Furthermore, oral administration of inactivated bacteria eliminates the need to consider the bactericidal effect of gastric acid on live bacteria, thus reducing the difficulty of formulation development and eliminating the need for special inclusion complexes to protect the bacteria from gastric acid. The mitigating effect of *Bifidobacterium longum* subsp. infantis BI_Y46 on microplastic accumulation toxicity was also unexpectedly significantly better than that of the control strain. Attached Figure Description

[0015] Figure 1 The results show the effects of different live bacteria on the head oscillation frequency and body bending frequency of nematodes. Figure 2 The results show the effects of different live bacteria on the oviposition rate of nematodes induced by nano-polystyrene; Figure 3 The results show the effects of different live bacteria on the shortened lifespan of nematodes induced by nano-polystyrene; Figure 4 The results show the effects of different live bacteria on the accumulation of nano-polystyrene in nematodes. Figure 5 The results show the effects of different heat-inactivated bacteria on the head-shaking frequency of nematodes. Figure 6 The effects of different heat-inactivated bacteria on ROS levels in nematodes and the results of DCFH-DA probe detection; Figure 7 The results show the effects of different heat-inactivated bacteria on the oxidative stress resistance of nematodes induced by nano-polystyrene; Figure 8 The results show the effects of different heat-inactivated bacteria on the oviposition rate of nematodes induced by nano-polystyrene; Figure 9 The results show the effects of different heat-inactivated bacteria on the shortened lifespan of nematodes induced by nano-polystyrene; Figure 10 The results show the effects of different heat-inactivated bacteria on the accumulation of nano-polystyrene in nematodes. Detailed Implementation

[0016] The technical solution and effects of the present invention will be shown and described below with reference to specific embodiments and accompanying drawings. The embodiments are only for illustrating the technical solution of the present invention and are not considered as limiting the scope of protection. The letters a, b, and c used to indicate significant differences between groups in the accompanying drawings have the following meanings: when these letters are different between groups, it indicates that there is a significant difference between the groups (…). P <0.05). In the examples, h and d, which are related to time, are time units representing hours and days, respectively.

[0017] In the following examples, the wild-type N2 nematode of *Caenorhabditis elegans* was derived from *Caenorhabditis Genetics Centre* (CGC); *Escherichia coli* (… E. coli OP50 (hereinafter referred to as OP50) originated from Fujian Shangyuan Biotechnology Co., Ltd.; *Bifidobacterium longum* was identified as *Bifidobacterium longum* subsp. *infantii* (published in CN115354009A; accession number CCTCC NO: M20221253, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 9, 2022, and taxonomically named...). Bifidobacterium longumsubsp. Infantis Named Bifidobacterium longumsubsp. infantis Y46 (BI_Y46) is from our laboratory; Bifidobacterium longum infantis subspecies M63 ( Bifidobacterium longum subsp. infantisM63 Both *Bifidobacterium longum* subsp. infantis ATCC15697 and *Bifidobacterium longum* subsp. infantis M63 were obtained from our laboratory, while *Bifidobacterium longum* subsp. infantis ATCC15697 were purchased from the China Microbial Culture Collection Center.

[0018] The culture media and main solutions used in the following examples were prepared as follows: (1) NGM solid culture medium: Stock solutions were prepared as follows: 1M magnesium sulfate solution (pH 6.0, 246.47 g magnesium sulfate diluted to 1 L); 1M calcium chloride solution (pH 6, 110.99 g anhydrous calcium chloride diluted to 1 L); 5 mg / mL cholesterol solution (5 g cholesterol diluted to 1 L with anhydrous ethanol); and potassium dihydrogen sulfate solution (pH 6, 136.00 g potassium dihydrogen sulfate diluted to 1 L). All solutions were filtered through a 0.22 μm sterile membrane and stored at 4°C for later use.

[0019] NGM solid medium (1L): 3g sodium chloride, 2.5g peptone, 17g agar, and sterile water were added to a final volume of 972mL. The mixture was then autoclaved at 121℃ for 15min. After cooling to 55℃, 25mL potassium dihydrogen phosphate solution, 1mL 1M calcium chloride solution, 1mL 1M magnesium sulfate solution, and 1mL 5mg / mL cholesterol solution were added. The mixture was thoroughly mixed and then poured into sterile petri dishes.

[0020] (2) LB liquid culture medium (1L): Dissolve 10g sodium chloride, 10g peptone, and 5g yeast extract in deionized water and bring the volume to 1L. Sterilize at 121℃ for 15min before use.

[0021] (3) M9 buffer (1L): Dissolve 7g disodium hydrogen phosphate, 3g potassium dihydrogen phosphate, 5g sodium chloride, and 0.25g magnesium sulfate in deionized water and bring the volume to 1L. Sterilize at 121℃ for 15min before use.

[0022] (4) Nematode lysis buffer: Mix 4M sodium hydroxide solution (1.6g sodium hydroxide dissolved in sterile water and then brought to a final volume of 10mL) and sodium hypochlorite solution at a volume ratio of 8:7 and shake well. Prepare and use immediately.

[0023] (5) MRSc liquid culture medium (1L): 10g peptone, 5g beef extract, 20g glucose, 4g yeast extract, 2g dipotassium hydrogen phosphate, 5g anhydrous sodium acetate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 2g triammonium citrate, 1mL Tween 80, 0.5g L-cysteine ​​hydrochloride, dissolved in deionized water, and brought to a final volume of 1L. Sterilize at 121℃ for 15 minutes before use.

[0024] (6) Microplastic solution: Disperse microplastics, i.e., nano-polystyrene (average particle size 0.1 μm), in M9 buffer to prepare a stock solution of 1000 mg / L. Sterilize with UV for 1 h before use, mix with Escherichia coli OP50, and dilute to the required concentration.

[0025] Example 1: Test strains, culture conditions, and grouping This study used *Bifidobacterium longum* subsp. *infantica*. Bifidobacterium longumsubsp. infantis Y46 Bifidobacterium longum infantis subsp. M63 (hereinafter referred to as BI_M63) and Bifidobacterium longum infantis subsp. ATCC15697 (hereinafter referred to as BI_15697) were used as test strains.

[0026] BI_Y46, BI_M63, and BI_15697 were activated in MRSc liquid medium and cultured anaerobically at 37°C for 18 hours. After three generations of continuous culture, they were used as test strains. E. coli OP50 (hereinafter referred to as OP50), used as the standard food for nematodes, was activated in LB liquid medium and cultured at 37°C and 225 rpm for 12 hours. *Bifidobacterium longum* subsp. *infantitidis* was stored at -80°C using 25% glycerol as a cryoprotectant for later use. E. coli OP50 is stored at -80°C with 30% glycerol as a cryoprotectant.

[0027] L4 stage nematodes were treated with different treatments (the treatments were spread on NGM medium): First, the cultured Bifidobacterium longum subsp. infantis bacterial suspension was centrifuged (8000×g, 4℃, 5min), the supernatant was removed, and then the suspension was washed 2-3 times with sterile M9 buffer solution. The bacterial concentration was then adjusted to 0.2mg / μL (wet weight) as a bacterial concentrate for later use. Finally, the L4 stage nematodes were divided into groups for treatment: the control group (con group) received 10 mg OP50; the microplastic group received 1000 mg / L microplastic (PS) + 10 mg OP50; the heat-inactivated BI_Y46 group (HK_BI_46) received 5 mg OP50 + 5 mg HK_BI_46 + 1000 mg / L PS; the non-heat-inactivated BI_Y46 group (BI_Y46 group) received 5 mg OP50 + 5 mg BI_46 + 1000 mg / L PS; the heat-inactivated BI_M63 group (HK_BI_M63) received 5 mg OP50 + 5 mg HK_BI_M63 + 1000 mg / L PS; and the non-heat-inactivated BI_M63 group (BI_M63 group) received 5 mg OP50 + 5 mg BI_M63 + 1000 mg / L PS. PS treatment; the heat-inactivated BI_15697 group (hereinafter referred to as HK_BI_15697) was treated with 5mg OP50 + 5mg HK_BI_15697 + 1000mg / L PS; the non-heat-inactivated BI_15697 group, i.e., the BI_15697 group, was treated with 5mg OP50 + 5mg BI_15697 + 1000mg / L PS. The nematodes were spread on NGM plates and allowed to dry before use. All nematodes in each group were cultured for 5 days to adulthood after the addition of the test substance before subsequent testing. In each group, the OP50 dosage was based on the dry weight of the bacterial cells (lyophilization protectants, etc., were not included in the dosage). All heat-inactivated *Bifidobacterium longum* subsp. *infantitidis* were inactivated using conventional heat inactivation processes (95°C, 90 min) and no viable bacteria were detected.

[0028] In each group of PS-containing treatments, the microplastic content was 100 mg / g NGM medium.

[0029] Example 2: Cultivation, treatment, and effect determination of nematodes This embodiment is a specific experiment based on the strains and groups used in Example 1. The experimental procedures and tests are all standard procedures and tests in the art, and can be found in the literature: [1]BRENNER S. The genetics of Caenorhabditis elegans[J]. Genetics, 1974, 77(1):71-94. [2]Zhao, Dan, et al. Antioxidant and antiaging activity of fermentedcoix seed polysaccharides on Caenorhabditis elegans. Nutrients 15.11 (2023):2474. [3]Guo, Xiuhuan, et al . "Antioxidant and anti-aging activities ofLongan crude and purified polysaccharide (LP-A) in nematode Caenorhabditiselegans." International journal of biological macromolecules 267 (2024):131634. [4]Chen, Wen, et al . "Cyanidin-3-O-glucoside promotes stress tolerance and lifespan extension of Caenorhabditis elegans exposed to polystyrene viaDAF-16 pathway." Mechanisms of Aging and Development 207 (2022): 111723. 1.1 Preparation of bacteria for feeding nematodes Live bacteria: Three subspecies of Bifidobacterium longum were cultured and passaged separately. After centrifugation (8000×g, 5min, 4℃), the supernatant was removed, the precipitate was collected, washed twice with M9 buffer, and the bacterial concentration was adjusted to 0.2mg / μL (wet weight) to prepare live bacteria for feeding nematodes.

[0030] Heat-inactivated bacteria: Three subspecies of Bifidobacterium longum were cultured and passaged separately. After centrifugation (8000×g, 5min, 4℃), the supernatant was removed, the precipitate was collected, washed twice with M9 buffer, and the bacterial concentration was adjusted to 0.2 mg / μL (wet weight). The bacteria were then heat-treated at 95℃ for 30min to prepare heat-inactivated bacteria for feeding nematodes.

[0031] The above-treated live Bifidobacterium longum subsp. infantis and heat-inactivated bacteria were mixed in Escherichia coli OP50 culture medium at a volume ratio of 1:1 to prepare nematode feeding plates exposed with nano-PS.

[0032] 1.2 Nematode Synchronization Nematodes in their oviposition stage were flushed from NGM medium with M9 solution into 15 mL centrifuge tubes and centrifuged (8000×g, 1 min, 4℃) to collect the nematodes. The supernatant was discarded, and 350 μL of nematode lysis buffer (prepared using 4M NaOH aqueous solution and 10% NaClO aqueous solution at a volume ratio of 8:7) was added. The mixture was vortexed for 3 min as the lysis condition, followed by the addition of 12 mL of sterile M9 buffer, and then centrifuged (8000×g, 3 min, 4℃) for washing. After centrifugation, the supernatant was discarded, leaving approximately 1 mL of nematodes. This was washed three times with sterile M9 buffer. The supernatant was discarded, and 10 mL of M9 solution was added and the mixture was incubated overnight at 20℃. After overnight incubation, the mixture was centrifuged (8000×g, 3 min, 4℃), and the supernatant was discarded. 10 μL of the nematodes was then added to NGM medium containing OP50 and incubated upside down at 20℃ for 48 h. The nematodes obtained at this stage were all L4 nematodes and could be used for subsequent experiments.

[0033] Each group of L4 stage nematodes was cultured using the grouping treatment method corresponding to Example 1 for subsequent testing.

[0034] 1.3 Cryopreservation of Nematodes The nematodes were cultured to a starvation state, washed with M9 buffer into 800 μL of cryovials, and 800 μL of 30% glycerol was added. The nematodes and glycerol were mixed thoroughly, frozen at -20°C overnight, and then stored at -80°C.

[0035] 1.4 Nematode resuscitation Take out the frozen nematodes, thaw them at room temperature, centrifuge (3000×g, 1min, 4℃), discard the supernatant, take 20 μL and add it to the center of an NGM plate containing OP50, and observe whether there are nematode crawling tracks. If there are crawling tracks, the revival is successful.

[0036] 1.5 Measurement of head swaying and body bending in nematodes Nematodes were exposed from the L4 stage to day 5 of adulthood. On day 5 of adulthood, the head wagging frequency was measured. The nematodes were placed on an NGM plate without bacterial solution, and M9 buffer was added. The nematodes were allowed to move freely in the M9 for 1 min, and the head wagging frequency was recorded over 30 s. The experiment was repeated 3 times.

[0037] After rinsing the nematodes three times with M9 solution, they were dropped onto NGM plates without bacterial solution. After the liquid was absorbed, the movement behavior of the nematodes was measured, and the number of times the body bent within 20 seconds was counted.

[0038] 1.6 Determination of Nematode Body Length and Size Nematodes were exposed from the L4 stage until day 5 of adulthood, at which time body length was measured. Nematodes were anesthetized with 5 mM levamisole solution and placed on 2% agarose-coated slides. Images of the nematodes were acquired using a fluorescence microscope, and quantitative analysis was performed using ImageJ software.

[0039] 1.7 Measurement of ROS levels in nematodes On day 5 of adult nematodes, nematodes from each group were washed three times with M9 buffer into 2 mL centrifuge tubes. The nematodes were then transferred to new 2 mL centrifuge tubes, and 250 μM of the DCFH-DA probe (Reactive Oxygen Spectroscopy (ROS) assay kit, Nanjing Jiancheng Biotechnology Co., Ltd., catalog number E004-1-1) was added. The tubes were incubated at 37°C in the dark for 2 h, washed three times with M9 buffer, and fixed onto a 2% agarose pad on a glass slide using 5 mM levamisole hydrochloride solution. Fluorescence images of the nematodes were obtained using a Leica fluorescence microscope under unsaturated exposure conditions, and the green fluorescence was quantified using ImageJ software.

[0040] 1.8 Determination of lipofuscin accumulation in nematodes On day 5 of adult nematodes, the nematodes were washed three times with M9 buffer into 2 mL centrifuge tubes and fixed onto 2% agarose pads on glass slides with 5 mM levamisole hydrochloride solution. Fluorescent images of the nematodes were obtained using a Leica fluorescence microscope. The blue fluorescence was quantified using ImageJ software.

[0041] 1.9 Nematode lifespan experiment Bifidobacterium longum subsp. infantis culture medium was mixed with Escherichia coli OP50 culture medium at a 1:1 volume ratio to prepare nematode feeding plates (see section 1.1 of this example). Nematodes were exposed to this growth environment from stage L4 until death, with stage L4 recorded as day 0. The nematodes were transferred to fresh NGM medium daily, and the number of surviving and dead nematodes was recorded. Nematodes that escaped from the NGM medium or were damaged by external forces were recorded as lost nematodes. Each group had three replicates.

[0042] 1.10 Nematode Heat Stress Resistance Experiment Twenty nematodes that were 5 days old and were in adulthood were randomly selected and exposed to a constant temperature environment of 35°C. The number of surviving nematodes was counted every hour.

[0043] 1.11 Assay for Hydrogen Peroxide Stress Resistance in Nematodes Twenty nematodes were randomly selected on day 5 after reaching adulthood and transferred to sterile NGM solid plates containing 10 mM hydrogen peroxide. The number of nematodes surviving was counted every hour.

[0044] 1.12 Determination of nematode oviposition Nine nematodes in the L4 stage after synchronization were transferred to three fresh NGM solid culture media every 24 hours until reproduction was roughly completed. The transferred plates were then cultured for another 48 hours and the number of larvae was counted.

[0045] 1.13 Determination of Nanoparticle Polystyrene Accumulation in Nematodes The polystyrene nanospheres were fluorescent microspheres with red fluorescence. On day 5 of adult nematodes, the nematodes were washed three times with M9 buffer into 2 mL centrifuge tubes and fixed onto 2% agarose pads on glass slides with 5 mM levamisole hydrochloride solution. Fluorescent images of the nematodes were obtained using a Leica fluorescence microscope. The red fluorescence was quantified using ImageJ software.

[0046] 2. Statistical Analysis All experiments were performed in triplicate. The mean and standard deviation were calculated using SPSS software, and significance analysis and analysis of variance were performed. The statistical significance level was set to [value missing]. P <0.05, plotted using GraphPad Prism 10 software.

[0047] Example 3: Results of the determination of microplastic toxicity by different strains or heat-inactivated strains This embodiment presents the specific experimental results of Embodiment 2.

[0048] 1. Results of the determination of the toxicity of different live bacteria to microplastics 1.1 The effect of different live bacteria on the reduction of nematode motility induced by nano-polystyrene To evaluate the mitigating effects of different live bacteria on the damage to nematode locomotion caused by nanoplastics, this study analyzed the head swaying frequency and body bending frequency as indicators. It was found that all three live bacteria could significantly improve the locomotion of nematodes and alleviate the reduction in head swaying and body bending frequency caused by nanoplastic treatment from L4 stage to day 5 of adulthood.

[0049] The results are as follows Figure 1As shown, the head twitching frequencies within 30 seconds for the Con group, PS group, BI_Y46 group, BI_M63 group, and BI_15697 group were 45.67 ± 4.81 times / 30s, 30.58 ± 2.64 times / 30s, 39.33 ± 3.35 times / 30s, 34.96 ± 3.57 times / 30s, and 36.42 ± 3.32 times / 30s, respectively; and the body flexion frequencies within 20 seconds were 11.63 ± 1.81 times / 20s, 7.63 ± 1.13 times / 20s, 10.33 ± 1.32 times / 20s, 8.63 ± 1.13 times / 20s, and 9.03 ± 1.03 times / 20s, respectively. Statistical analysis showed significant differences between each live bacteria intervention group and the single PS treatment group (…). P <0.05 indicates that all three live bacteria can effectively alleviate the decline in nematode motility induced by nano-polystyrene and increase the frequency of head swaying and body bending. Among the intervention groups, the BI_Y46 group showed the greatest recovery in both motility frequencies, with head swaying and body bending frequencies recovering by 28.6% and 35.4% respectively (compared to the PS group). This indicates that BI_Y46 has the most prominent alleviating effect on the motility toxicity of nano-PS, and its effect on head swaying and body bending frequencies is significantly better than the other two Bifidobacterium infantis subspecies (Bifidobacterium longum). P <0.05).

[0050] 1.2 The effect of different live bacteria on reducing the oviposition rate of nematodes induced by nano-polystyrene Egg production directly reflects the reproductive capacity of nematodes, and the reproductive system is highly sensitive to toxic damage. Microplastics may reduce egg production by interfering with the endocrine function, germ cell development, or energy metabolism of nematodes. To investigate the mitigating effect of live bacteria on reproductive damage induced by polystyrene nanoparticles (PS), this study statistically analyzed the changes in egg production of nematodes over 5 consecutive days.

[0051] like Figure 2 As shown, exposure to nano-PS significantly inhibited the reproductive capacity of nematodes. In the early oviposition stage (day 1), the oviposition count on day 1 was 53.00 ± 2.31 in the BI_Y46 group, 56.67 ± 2.73 in BI_M63, and 54.00 ± 3.22 in BI_15697. Compared with the PS group (48.33 ± 2.73), all three live bacteria interventions significantly increased oviposition. P <0.05. There was no significant difference between day 2 and day 3. By day 4 of spawning, the number of eggs laid in the PS group further decreased to 27.33 ± 2.96, while the BI_Y46 group (41.33 ± 2.91) and the BI_M63 group (45.67 ± 3.67) were still able to significantly recover their egg-laying levels. P<0.05The BI_15697 group (26.00 ± 3.51 eggs) showed no significant difference from the PS group. By day 5, only the BI_Y46 group (3.33 ± 0.33 eggs) still had a significantly higher egg production than the PS group (1.67 ± 0.33 eggs). P <0.05, and there was no significant difference compared with the Con group ( P >0.05); there was no statistically significant difference between the BI_M63 and BI_15697 groups and the PS group.

[0052] The above results indicate that the three live bacteria can alleviate nano-PS-induced reproductive damage in nematodes to varying degrees. Among them, BI_Y46 and BI_M63 showed more sustained protective effects in the mid-to-late stages of reproduction, while the alleviating effect of BI_15697 was relatively limited.

[0053] 1.3 The effect of different live bacteria on improving the lifespan reduction of nematodes induced by nano-polystyrene To investigate the mitigating effects of different live bacteria on the toxicity of polystyrene nanoparticles (PS), this study assessed the survival status of nematodes through a lifespan experiment. For example... Figure 3 As shown, compared with the Con group, PS exposure significantly shortened the mean lifespan, median lifespan, maximum lifespan, and survival rate of nematodes. The survival curves of all live bacteria intervention groups shifted significantly to the right compared with the PS group, indicating that BI_Y46, BI_M63, and BI_15697 can alleviate the toxicity of PS to nematodes to varying degrees.

[0054] Specific data analysis shows that: In terms of mean lifespan, the PS group had a mean lifespan of (12.80 ± 0.43) days, while the BI_Y46, BI_M63, and BI_15697 groups had mean lifespans of (16.25 ± 0.17) days, (14.61 ± 0.49) days, and (13.54 ± 0.40) days, respectively, representing increases of 26.95%, 14.14%, and 5.78% compared to the PS group. Statistical analysis showed that the mean lifespans of the BI_Y46 and BI_M63 groups were significantly higher than those of the PS group. P <0.05, and the average lifespan of the BI_Y46 group was significantly higher than that of the other groups ( P <0.05); there was no significant difference between the BI_15697 group and the PS group.

[0055] In terms of median lifetime, the PS group had (13.67 ± 0.33) days, while the BI_Y46, BI_M63, and BI_15697 groups had (17.67 ± 0.33) days, (16.33 ± 0.33) days, and (15.00 ± 0.58) days, respectively, representing increases of 29.26%, 19.46%, and 9.73%. The BI_Y46 and BI_M63 groups had significantly longer lifetimes than the PS group.P <0.05), BI_Y46 was also significantly higher than that of the Con group; there was no significant difference between the BI_15697 group and the PS group.

[0056] Regarding maximum lifespan, the PS group had a lifespan of (19.67 ± 0.33) days, while the intervention groups had lifespans of (23.00 ± 0.58) days, (24.33 ± 0.33) days, and (22.33 ± 0.88) days, respectively, representing increases of 16.93%, 23.69%, and 13.52%. All live bacteria groups showed significantly higher lifespans than the PS group. P <0.05), and there was no statistically significant difference from Con.

[0057] In summary, all three live bacteria could improve the shortened lifespan of nematodes induced by PS. BI_Y46 and BI_M63 significantly prolonged the mean, median, and maximum lifespan simultaneously, demonstrating remarkable effects. While BI_15697 prolonged the maximum lifespan, its effect on improving the mean and median lifespan was limited. BI_Y46 showed a significant and outstanding advantage in improving the mean lifespan, significantly outperforming BI_M63 and BI_15697.

[0058] 1.4 Effects of different live bacteria on the accumulation of nano-polystyrene in nematodes Nanoparticles of polystyrene (PS) are fluorescent microspheres exhibiting red fluorescence. To investigate the effect of live bacteria on the accumulation of PS in nematodes, this study utilized their autofluorescence properties and employed a Leica inverted fluorescence microscope to observe and quantitatively analyze the accumulation of PS within nematodes. The results are as follows: Figure 4 As shown, compared with the PS group (100.0%), all three live bacteria interventions reduced the fluorescence intensity in nematodes. Among them, the relative fluorescence intensity of the BI_Y46 group decreased to 69.38 ± 4.66%, and the difference was significant compared with the PS group. P <0.05; while the BI_M63 group (93.88 ± 8.71%) and BI_15697 group (91.75 ± 6.16%) showed a decreasing trend, there was no statistically significant difference compared with the PS group. P >0.05).

[0059] The results indicate that strain BI_Y46 can significantly reduce the accumulation of nano-PS in nematodes, while strains BI_M63 and BI_15697 did not show a significant reduction in accumulation.

[0060] 2. Investigation on the protective effects of different heat-inactivated bacteria against nematodes exposed to nano-polystyrene 2.1 The effect of different heat-inactivated bacteria on the reduction of nematode motility induced by nano-polystyrene To investigate the effects of heat-inactivated bacteria on the movement behavior of polystyrene nanoparticles (PS) induced by nematodes, the head-shaking frequency (30 s) of adult nematodes was measured on day 5. The results are as follows: Figure 5 As shown, the PS group had 30.29 ± 2.90 cycles, and all heat-sterilization treatment groups were significantly higher than the PS group. P The effective values ​​were <0.05, specifically: HK_BI_Y46 group 39.21 ± 3.53 times, HK_BI_M63 group 35.46 ± 2.70 times, and HK_BI_15697 group 36.25 ± 3.21 times. Among them, the improvement effect of HK_BI_Y46 group was the most significant, significantly higher than that of HK_BI_15697 group and HK_BI_M63 group.

[0061] 2.2 Effects of different heat-inactivated bacteria on improving ROS levels in nematodes induced by nano-polystyrene To further investigate the potential mechanism by which heat-inactivated bacteria alleviate the toxicity of polystyrene nanoparticles (PS), the levels of reactive oxygen species (ROS) in nematodes were measured. The results are as follows: Figure 6 As shown, compared with the Con group, the relative fluorescence intensity of ROS in the PS group was significantly increased to 144.5 ± 8.31%, indicating that it successfully induced oxidative stress in nematodes. All heat-inactivated bacterial treatments significantly reversed this trend. P <0.05). Among them, the HK_BI_Y46 group had the lowest ROS level, at 63.07 ± 3.98%, which was significantly better than the HK_BI_M63 group, HK_BI_15697 group and Con group.

[0062] 2.3 The effect of different heat-inactivated bacteria on improving the reduced resistance to oxidative stress induced by nano-polystyrene in nematodes To investigate whether heat-inactivated bacteria can enhance the survival ability of nematodes under direct chemical oxidation stress, the survival of nematodes in 10 mM hydrogen peroxide solution was further examined.

[0063] The results are as follows Figure 7 As shown, survival curve analysis revealed that exposure to nano-polystyrene (PS) significantly weakened the oxidative stress resistance of nematodes. Mortality occurred earliest in this group, starting on day 3, and the curve showed the fastest rate of decline. In contrast, the survival curves of the Con group and the three heat-inactivated bacteria intervention groups all exhibited a significant rightward shift, meaning the onset of mortality was delayed until day 4, and the overall survival rate decline was significantly slower than in the PS group. Statistical analysis confirmed that the survival curves of these four groups were significantly better than those of the PS group. P<0.05 indicates that heat-sterilized bacteria treatment has the potential to improve PS-induced oxidative stress vulnerability. Almost all nematodes in the PS group died by day 10. Among the intervention groups, the HK_BI_Y46 group showed the best performance, with a survival rate of 23.33 ± 3.33%, significantly higher than the PS group, HK_BI_M63 group, and HK_BI_15697 group. P <0.05. The survival rates of the HK_BI_M63 group and the HK_BI_15697 group were 8.33 ± 4.41% and 5.00 ± 2.89%, respectively. Although these rates were numerically higher than those of the PS group, statistical analysis showed that the difference was not statistically significant. P >0.05).

[0064] 2.4 The effect of different heat-inactivated bacteria on reducing oviposition rate in nematodes induced by nano-polystyrene To assess the long-term effects of heat-inactivated bacteria on reproductive function impairment in nematodes induced by polystyrene nanoparticles (PS), the daily oviposition rate was systematically monitored throughout the reproductive period.

[0065] The results are as follows Figure 8 As shown, during the first two days of the reproductive period, the daily oviposition rate of nematodes in each group remained at a similar level, and statistical analysis showed no significant difference. P >0.05). This indicates that neither PS exposure nor heat-inactivated bacteria intervention affected the basal oviposition capacity of nematodes in the initial reproductive stage. However, from day 3 onwards, significant differences between treatments began to appear. The reproductive capacity of nematodes in the PS group showed a significant decline, with the daily oviposition rate decreasing to 43.00 ± 0.58. On days 3, 4, and 5, the daily oviposition rate of the HK_BI_Y46 group was significantly higher than that of the PS group, HK_BI_M63 group, and HK_BI_15697 group ( P <0.05), with no statistically significant difference from the Con group; while the HK_BI_M63 group had a significantly higher oviposition rate on day 5 than the PS group, but it was still significantly lower than the Con group. The HK_BI_15697 group had no significant difference in oviposition rate on days 3, 4, and 5 compared to the PS group.

[0066] 2.5 The effect of different heat-inactivated bacteria on improving the lifespan reduction of nematodes induced by nano-polystyrene To systematically evaluate the long-term effects of heat-inactivated Bifidobacterium on the overall health and survival of nematodes induced by polystyrene nanoparticles (PS), survival curves and key lifespan parameters of nematodes were determined in the Con group, PS exposure group, and each heat-inactivated bacteria intervention group. Lifespan, as the ultimate indicator for assessing the cumulative effects of environmental toxicity and the protective efficacy of intervention measures, can comprehensively reflect whether the improvements in various physiological functions observed in the early stages ultimately translate into a survival advantage.

[0067] The results are as follows Figure 9 As shown, survival curve analysis revealed that, compared to the PS group, the survival curves of all three heat-inactivated bacteria intervention groups exhibited a significant rightward shift. This means that throughout the entire observation period, the survival rate of nematodes in the intervention groups was consistently higher than that in the PS group, and the time of death was significantly delayed. P <0.05).

[0068] Statistical analysis results showed that, in terms of mean lifespan and median lifespan, the mean and median lifespan of nematodes in all heat-inactivated bacteria groups were significantly longer than those in the PS group. P <0.05), among which the HK_BI_Y46 group showed the best results, with its average lifetime significantly higher than that of the HK_BI_M63 and HK_BI_15697 groups, and also significantly higher than that of the Con group. The median lifetime of the HK_BI_Y46 group was significantly higher than that of the HK_BI_15697 group, and also significantly higher than that of the Con group. The maximum lifetime of the HK_BI_Y46 group was significantly higher than that of the HK_BI_M63 and HK_BI_15697 groups, and also reached a level that was not significantly different from that of the Con group.

[0069] 2.6 Effects of different heat-inactivated Bifidobacteria on the accumulation of nano-polystyrene in nematodes To investigate the potential mechanism by which heat-inactivated bacteria alleviate the toxicity of polystyrene nanoparticles (PS), quantitative analysis of PS fluorescence in nematodes was performed using a Leica inverted fluorescence microscope. By comparing the overall relative fluorescence intensity of nematodes in different treatment groups, the accumulation level of polystyrene nanoparticles in their bodies could be assessed.

[0070] The results are as follows Figure 10 As shown. Quantitative analysis showed that among the three heat-inactivated bacteria intervention groups, only the HK_BI_Y46 group showed a significant effect in reducing PS accumulation, with its relative fluorescence intensity decreasing to 59.45 ± 2.51%, which was statistically significant compared with the PS group. P <0.05. The relative fluorescence intensities of the HK_BI_M63 group and the HK_BI_15697 group were 94.81 ± 9.76% and 98.56 ± 9.77%, respectively. Statistical analysis confirmed that there was no significant difference between these two groups and the PS group. P >0.05).

[0071] This indicates that HK_BI_Y46 can effectively reduce the accumulation of nano-polystyrene by nematodes or promote its clearance, while the HK_BI_M63 and HK_BI_15697 strains have very limited clearance effects on nano-polystyrene.

Claims

1. Use of Bifidobacterium longum for the preparation of a microplastic toxicity mitigant, characterized in that, The Bifidobacterium longum is the Bifidobacterium longum infantis subspecies (Bifidobacterium longum infantis). Bifidobacterium longum subsp. infantis ) Y46 The accession number is CCTCCNO: M 20221253, the depositary institution is the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is August 9, 2022, and the taxonomic name is... Bifidobacterium longum subsp. infantis The *Bifidobacterium longum* is an inactivated *Bifidobacterium longum* subsp. *infantii*; the microplastic toxicity mitigator is a microplastic toxicity mitigator that improves reproductive capacity reduced by microplastic exposure, and the microplastic is nanoscale polystyrene microplastic.

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