Application of lactobacillus helveticus ZJUIDS12 in preventing and treating pet injury caused by microplastics

Abstract

This invention belongs to the field of microbial technology, specifically relating to the application of *Lactobacillus helveticus* ZJUIDS12 in preventing and treating microplastic-induced injuries in pets. This invention discloses the application of *Lactobacillus helveticus* ZJUIDS12 in the preparation of drugs for preventing and treating microplastic-induced injuries in pets. The accession number for *Lactobacillus helveticus* ZJUIDS12 is CGMCC NO.23997. The prevention and treatment of microplastic-induced injuries in pets includes: preventing intestinal damage caused by microplastics and preventing fetal growth restriction caused by microplastics.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Lactobacillus helveticus ZJUIDS12 in the prevention and treatment of microplastic-induced injuries to pets. Background Technology

[0002] Microplastics (plastic particles with a diameter of less than 5 mm, MPs) have emerged as a new type of pollutant and have become an unavoidable part of the living environment and the pet industry. Commercial pet food is a significant source of exposure. Furthermore, companion animals, due to their chewing and licking habits, are at high risk of microplastic exposure. Studies have shown that in addition to causing direct physical abrasion, microplastics can also serve as a substrate for microbial colonization and biofilm formation, exhibiting unique biological characteristics. Diverse microbial communities, including potential pathogens, can form on microplastic surfaces, creating a pet-microplastic-microbe interaction pattern. Particularly in the gut, microplastics can significantly alter the structure and function of the microbial community, thereby affecting host health. Notably, the maternal gut microbiota during pregnancy is crucial for shaping offspring health. Disruptions in the maternal microbiome can severely interfere with offspring development, profoundly impacting long-term health. Fetal growth restriction not only manifests as low birth weight, poor vitality, and decreased survival rates in pups, but also foreshadows potential long-term health problems such as immune deficiencies, organ malformation, and an increased risk of metabolic diseases.

[0003] Pets are not only important companions to humans, but their health also affects the energy and financial investment of their owners. This issue concerns not only animal welfare and emotional value but also the economic benefits of the breeding industry. However, the current field of pet nutrition and health lacks targeted solutions to this problem.

[0004] Invention No. 202511040173.0, entitled "A Lactobacillus plantarum with the ability to alleviate enteritis and reduce polystyrene nanoplastic toxicity and its application," discloses a Lactobacillus plantarum (accession number CGMCCNO.31420). This Lactobacillus plantarum can promote the excretion of polystyrene nanoplastics (PS-NPs) in feces, reduce the accumulation of PS-NPs in organs, alleviate the toxicity of PS-NPs to various organs in mice, and significantly reduce the inflammatory response in mice. Specifically, Lactobacillus plantarum can reduce the accumulation of PS-NPs in organs, alleviate histopathological damage, inhibit inflammatory responses, alleviate enteritis, repair the intestinal barrier, and regulate liver metabolism. However, this Lactobacillus plantarum cannot alleviate intestinal flora imbalance caused by microplastics and cannot play a beneficial role in the adverse effects of microplastics on the placenta and fetus.

[0005] The inventor's earlier patent application CN114381398A, "Lactobacillus helveticus ZJUIDS12 with the ability to improve alcoholic liver disease and its application", only disclosed the application of Lactobacillus helveticus ZJUIDS12 (accession number CGMCCNO.23997) in the preparation of products that protect against liver damage. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a new use for Lactobacillus helveticus ZJUIDS12 for the prevention and treatment of microplastic-induced injuries to pets.

[0007] To address the aforementioned technical problems, this invention provides the application of Lactobacillus helveticus ZJUIDS12 in the preparation of a drug for preventing and treating microplastic-induced pet injuries. The accession number of Lactobacillus helveticus ZJUIDS12 is CGMCC NO.23997.

[0008] As an improvement to the application of the present invention, the prevention of microplastic-induced pet injury includes at least one of the following: prevention of microplastic-induced intestinal damage in pets, and prevention of microplastic-induced fetal growth restriction in pets.

[0009] In other words, Lactobacillus helveticus ZJUIDS12 can prevent and treat problems such as maternal intestinal damage, pregnancy health issues, and fetal growth restriction caused by pets ingesting microplastics.

[0010] As a further improvement to the application of the present invention:

[0011] Prevention of microplastic-induced intestinal damage in pets includes at least one of the following: reducing maternal intestinal damage during pregnancy, restoring intestinal barrier function, increasing the abundance and diversity of intestinal flora, and maintaining intestinal microecological health.

[0012] Prevention of microplastic-induced fetal growth restriction in pets includes at least one of the following: alleviating maternal placental oxidative stress damage and development during pregnancy, improving fetal survival rate, and reducing fetal growth restriction.

[0013] As a further improvement to the application of the present invention:

[0014] Lactobacillus helveticus ZJUIDS12 is used to prepare live bacterial preparations, bacterial powders, and other products. Specifically, Lactobacillus helveticus ZJUIDS12 is used to prepare live bacterial preparations and inactivated metabiotics. The live bacterial preparation has a viable count of 1.0 × 10⁻⁶. 9 ~1.0×10 11 CFU / g or 1.0×10 9 ~1.0×10 11CFU / ml.

[0015] This invention provides *Lactobacillus helveticus* ZJUIDS12, a strain that can prevent intestinal damage and fetal growth restriction caused by microplastics in pets. This strain also possesses the following capabilities: tolerance to the gastrointestinal environment, no antibiotic resistance in the culture medium, inhibition of harmful intestinal pathogens, and strong antioxidant properties. Therefore, *Lactobacillus helveticus* ZJUIDS12 of this invention can be widely used in the development of pet probiotics and the preparation of related products (including veterinary drugs).

[0016] In view of the current lack of research on how to prevent and treat the health of pet mothers and offspring from various plastics (especially microplastics), this invention aims to provide the application of Lactobacillus helveticus ZJUIDS12 in the preparation of drugs that prevent and treat microplastic-induced pet injuries, and to verify the application through animal models. This invention provides excellent bacterial strain resources for the development of probiotics in my country, and provides new theoretical basis for the development of effective drugs and therapeutic adjuvants to prevent microplastic toxicity.

[0017] This invention aims to alleviate intestinal, maternal, and offspring health problems caused by microplastics ingested by pets through probiotic intervention. It also provides new insights into the application of probiotics in relevant pet products. Attached Figure Description

[0018] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0019] Figure 1 The effect of Lactobacillus helveticus ZJUIDS12 et al. on the liver weight / body weight ratio (liver-to-body ratio) in pregnant mice;

[0020] Figure 2 The effects of Lactobacillus helveticus ZJUIDS12 et al. on the genes of inflammatory factors in the colon of pregnant mice: interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α);

[0021] Figure 3 The effects of Lactobacillus helveticus ZJUIDS12 and other bacteria on Claudin-1, Occludin, and ZO-1 in the intestines of pregnant mice were investigated.

[0022] Figure 4 Image of AB-PAS staining of colon sections from pregnant mice by Lactobacillus helveticus ZJUIDS12 et al.

[0023] Figure 5 The effects of Lactobacillus helveticus ZJUIDS12 and other bacteria on the α-diversity (Shannon index, Simpson index, Chao 1 index) and β-diversity (principal coordinate analysis) of gut microbiota in pregnant mice;

[0024] Figure 6 The effect of Lactobacillus helveticus ZJUIDS12 et al. on the phylum-level abundance (relative abundance) of gut microbiota in pregnant mice;

[0025] Figure 7 The effect of Lactobacillus helveticus ZJUIDS12 and other strains on the relative abundance of differentially abundant strains at the genus level in the gut microbiota of pregnant mice;

[0026] Figure 8 The effects of Lactobacillus helveticus ZJUIDS12 and other bacteria on fetal survival rate, fetal weight, and crown-rump diameter;

[0027] Figure 9 The effects of Lactobacillus helveticus ZJUIDS12 and other bacteria on fetal size;

[0028] Figure 10 The effects of Lactobacillus helveticus ZJUIDS12 et al. on H&E staining, placental weight in pregnant mice and labyrinth area;

[0029] Figure 11 The effects of Lactobacillus helveticus ZJUIDS12 and other bacteria on key developmental genes of the placenta in pregnant mice, namely Eomes, Ascl2, Fra1, and Hand1.

[0030] Figure 12 The effects of Lactobacillus helveticus ZJUIDS12 et al. on malondialdehyde (MDA) and total antioxidant capacity (T-AOC) levels in the placenta of pregnant mice under oxidative stress;

[0031] Figure 13 The effects of Lactobacillus helveticus ZJUIDS12 on placental metabolites in pregnant mice. Figure 13 middle: (A) is the principal component analysis plot, and (B) is the heatmap of total differential metabolite clustering.

[0032] Figure 14 The effect of *Lactobacillus plantarum* ZJUIDS04 in Comparative Example 1 on fetal survival rate, fetal weight, and crown-rump diameter;

[0033] Figure 15 To investigate the effect of *Lactobacillus plantarum* ZJUIDS04 in Comparative Example 1 on the levels of MDA and T-AOC in the placenta of pregnant mice under oxidative stress. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: The strain of this invention, ZJUIDS12, has the preservation name: Lactobacillus helveticus. Lactobacillus helveticusDepository institution: China General Microbiological Culture Collection Center, depository address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC NO.23997, deposit date: November 29, 2021.

[0035] Example 1: Determination of the effect of Lactobacillus helveticus ZJUIDS12 in preventing microplastic-induced intestinal damage and fetal growth restriction in animals.

[0036] 1.1 Laboratory Animals

[0037] Thirty-six female C57BL / 6J mice and 18 male mice were purchased from Zhejiang Vital River Laboratory Animal Co., Ltd. and housed in an SPF environment at the Laboratory Animal Center of Zhejiang University.

[0038] 1.2 Preparation of Lactobacillus helveticus ZJUIDS12 suspension

[0039] Lactobacillus helveticus ZJUIDS12, with accession number CGMCCNO.23997.

[0040] The ZJUIDS12 strain, preserved in glycerol tubes, was streaked onto MRS solid medium and incubated upside down at 37°C for 48 h. After three generations of activation, single colonies were inoculated into MRS liquid medium and incubated at 37°C for 18–24 h. The colonies were then centrifuged at 8000 rpm for 10 min, and the supernatant was discarded, retaining the bacterial cells. PBS buffer (0.01 M, pH 7.2–7.4) was added, vortexed, and centrifuged at 8000 rpm for 10 min. This process was repeated twice to wash the bacterial cells. The washed bacterial cells were resuspended in PBS buffer, and the cell concentration was adjusted to 10-1. 9 CFU / mL.

[0041] 1.3 Preparation of Microplastic Suspension

[0042] Hot oil at 110-120℃ was added to a polypropylene plastic box and kept at 60℃ for 20 min. Then, it was poured into a glass bottle, and hexane was immediately added to the bottle at a volume ratio of hot oil to hexane of 1:3. After mixing, the mixture was filtered through a 0.02 μm inorganic alumina membrane, and the residual oil on the alumina membrane was washed off with anhydrous ethanol. The polypropylene microplastics (MPs) on the inorganic alumina membrane were scraped off with a metal spoon and dried at 50℃ to constant weight to obtain polypropylene microplastics (the particle size was determined to be 44 nm-154 μm by a nanoparticle tracking analyzer and scanning electron microscopy). The polypropylene microplastics were added to a 0.1% (mass%) Tween 80 aqueous solution and sonicated to disperse the polypropylene microplastics evenly, resulting in a microplastic suspension with a concentration of 5 mg / mL.

[0043] 1.4 Animal Experiment Design

[0044] One week after acclimatization, female mice were paired with male mice at a 2:1 ratio, and pregnancy was confirmed via vaginal plugs, marking the start of pregnancy (GD0.5). After mating, the male mice were removed, and each pregnant mouse was placed individually in a standard cage. Mice with vaginal plugs were randomly divided into three groups (n=12): a blank control group (CTRL), a model group (MPs), and a Lactobacillus helveticus ZJUIDS12 intervention group (ZJUIDS12). It was confirmed that each group ultimately produced 6–9 pregnant mice.

[0045] From the onset of pregnancy (GD0.5), the model group and the ZJUIDS12 intervention group (GD0.5–16.5, gestational days 0.5–16.5) were administered 200 μL of microplastic suspension by gavage every other day, while the blank control group received an equal volume of 0.1% Tween 80 solution. Two hours later on the same day, the ZJUIDS12 intervention group was administered 10 μL of microplastic suspension by gavage. 9 200 μL of ZJUIDS12 bacterial culture (CFU / mL) was administered to the remaining groups, along with an equal volume of PBS buffer. All other housing conditions, such as temperature, humidity, light, and feed, were consistent (according to standard conditions). Animal welfare and experimental procedures were conducted in accordance with the guidelines of the Zhejiang University Laboratory Animal Management Regulations. Mice were fasted for 12 hours starting at GD 17.5 (water allowed), then euthanized. Samples were collected from the liver, placenta, colon, and cecum contents of pregnant mice.

[0046] 1.5 Efficacy evaluation indicators of Lactobacillus helveticus ZJUIDS12 intervention in preventing microplastic-induced intestinal damage in animals

[0047] 1.5.1 Mouse liver-to-body ratio index

[0048] Weigh the liver and body weight of the pregnant rats. The liver weight ratio is calculated as liver weight / body weight × 100%. The results are as follows: Figure 1 As shown, Lactobacillus helveticus ZJUIDS12 can significantly reduce the increase in maternal liver weight ratio caused by MPs.

[0049] 1.5.2 Indicators of Inflammatory Factors in Mouse Colon

[0050] The expression changes of inflammatory cytokines IL-10 and TNF-α were determined using real-time quantitative PCR (qPCR). Total RNA was extracted from mouse colon tissue, and its concentration was measured and adjusted to the same level using a micro-ultraviolet spectrophotometer. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. Subsequently, the relative expression levels of target gene mRNA were detected using 2×SYBR Green qPCR premix as the detection reagent and β-actin as the internal control gene.

[0051] The results are as follows Figure 2 Lactobacillus helveticus ZJUIDS12 significantly improved intestinal inflammation levels in pregnant mice. Explanation: The inflammatory cytokine TNF-α reflects the body's inflammation level, while IL-10 is an anti-inflammatory cytokine that exerts its anti-inflammatory effect by inhibiting the production of various pro-inflammatory factors, promoting the expression of anti-inflammatory factors, and regulating immune cell activity.

[0052] 1.5.3 Measurement of colonic barrier function in mice

[0053] The expression changes of tightly linked genes such as Claudin-1, Occludin, and ZO-1 were determined using qPCR. The specific steps are described in step 1.5.2.

[0054] The results are as follows Figure 3 Lactobacillus helveticus ZJUIDS12 has the ability to improve the intestinal mucosal barrier in pregnant mice. ZO-1, Claudin-1, and Occludin are important factors in the formation of tight junctions between cells. Intervention with Lactobacillus helveticus ZJUIDS12 significantly restored the MPs-induced decrease in the gene expression of ZO-1, Claudin-1, and Occludin.

[0055] The distal colon was preserved in 4% paraformaldehyde fixative, embedded in paraffin, fixed on a glass slide, and stained with Alcian blue-periodic acid-Schiff (AB-PAS) to assess the number of goblet cells in the colon.

[0056] The results are as follows Figure 4 AB-PAS staining in colon sections is mainly used to reflect the number of goblet cells and the thickness of the mucus layer. Goblet cells secrete mucin to form a mucus barrier. After treatment with *Lactobacillus helveticus* ZJUIDS12, compared to the model group, the number of goblet cells and mucus in the mouse colon tissue were significantly increased, indicating that the barrier function of mice treated with probiotics was significantly enhanced. Tight junctions are the main connection mode between intestinal epithelial cells and play an important role in maintaining the mechanical barrier and permeability of the intestinal mucosal epithelium. Tight junction proteins are important protein molecules that constitute the intestinal mucosal barrier and determine intestinal wall permeability, and have a significant impact on the composition and function of tight junctions.

[0057] 1.5.4 Sequencing of 16S rRNA genes of mouse gut microbiota

[0058] DNA was extracted from cecal contents samples. The V3-V4 region of 16S rRNA was amplified by PCR. The PCR products were pooled and purified, sequenced using an Illumina Novaseq 6000 platform, and the raw sequences were quality controlled using FASTP. Operational taxonomic units (OTUs) were constructed by binding sequences to clusters with greater than 97% sequence similarity using QIIME 2.

[0059] The results are as follows Figures 5-7 As shown.

[0060] Figure 5 The results showed that *Lactobacillus helveticus* ZJUIDS12 could significantly restore the decrease in gut microbiota abundance and diversity induced by phytotoxicants (MPs). Regarding α-diversity, the Chao1, Shannon, and Simpson indices in the model group were lower than those in the blank control group, indicating that MP intervention reduced the richness and diversity of the gut microbiota. Supplementation with *Lactobacillus helveticus* ZJUIDS12 significantly reversed this change. Furthermore, the PCoA plot showed clear separation between groups, indicating altered β-diversity in the gut microbiome.

[0061] Figure 6 The results showed that *Lactobacillus helveticus* ZJUIDS12 could restore the phylum-level intestinal flora dysbiosis induced by MPs in pregnant mice. *Lactobacillus helveticus* ZJUIDS12 could reduce the elevated ratio of Firmicutes (Bacillota) to Bacteroidota phylum induced in the model group.

[0062] Figure 7 The results showed that *Lactobacillus helveticus* ZJUIDS12 could restore the genus-level gut microbiota dysbiosis induced by MPs in pregnant mice. Compared with the control group, the abundance of *Eubacterium siraeum*, *Oscillibacter*, and *Alistipes* was significantly reduced in the model group, while the abundance of *Jeotgalicoccus* was significantly increased. Supplementation with *Lactobacillus helveticus* ZJUIDS12 significantly reversed these changes.

[0063] 1.6 Evaluation Indicators of the Efficacy of Lactobacillus helveticus ZJUIDS12 in Preventing Microplastic-Induced Fetal Growth Restriction

[0064] 1.6.1 Basic Fetal Information Statistics

[0065] The total number of surviving fetuses was counted, the fetal weights were measured, and the crown-rump diameter of the fetuses was measured. The results are as follows: Figure 8 As shown, polycystic ovaries (MPs) can lead to an increased number of resorbed fetuses, i.e., an increased fetal mortality rate, accompanied by fetal growth restriction. Lactobacillus helveticus ZJUIDS12 can reduce these adverse effects. Figure 9 This is a representative schematic diagram of each group of fetuses.

[0066] 1.6.2 Basic Indicators of Mouse Placenta

[0067] The placenta was weighed, and the leftmost placenta was preserved in 4% paraformaldehyde fixative, embedded in paraffin, fixed on a glass slide, stained with hematoxylin and eosin (H&E), and the area of ​​the placental labyrinth was calculated using ImageJ software.

[0068] The results are as follows Figure 10 Lactobacillus helveticus ZJUIDS12 can significantly improve placental weight loss and labyrinth shrinkage caused by MPs. The placental labyrinth is the core structure in the placenta for gas and substance exchange between the mother and fetus; a reduction in its area will decrease the exchange efficiency.

[0069] 1.6.3 Key Developmental Gene Indicators of Mouse Placenta

[0070] The expression changes of key placental development genes Eomes, Ascl2, Fra1, and Hand1 were determined using real-time quantitative PCR (qPCR). The specific steps are described in step 1.5.2.

[0071] The results are as follows Figure 11 Lactobacillus helveticus ZJUIDS12 can significantly improve placental developmental abnormalities caused by multiple myxobolus (MPs). Eomes, Ascl2, Fra1, and Hand1 genes jointly regulate early placental development, cell differentiation, and functional establishment. MP intervention leads to a significant decrease in the transcriptional levels of Eomes, Ascl2, Fra1, and Hand1 genes. Lactobacillus helveticus ZJUIDS12 can effectively mitigate this adverse effect.

[0072] 1.6.4 Mouse placental oxidative stress markers

[0073] Accurately weigh the placenta and add PBS buffer at a weight (g) : volume (mL) ratio of 1:9. Disrupt the tissue using an automated rapid sample homogenizer, then centrifuge and collect the supernatant (10% homogenate supernatant) for testing. Following the kit instructions from Nanjing Jiancheng Biotechnology Institute, detect malondialdehyde (MDA) and total antioxidant capacity (T-AOC) related to oxidative stress levels in mice.

[0074] The results are as follows Figure 12 The results show that *Lactobacillus helveticus* ZJUIDS12 can significantly reduce the elevated MDA level in the placenta induced by MPs, while simultaneously increasing the T-AOC level. This indicates that *Lactobacillus helveticus* ZJUIDS12 can significantly reduce placental oxidative stress damage induced by MPs.

[0075] 1.6.5 Non-targeted metabolomics assay of mouse placenta

[0076] This study utilizes liquid chromatography-mass spectrometry (LC-MS) for non-targeted metabolomics research. The experimental procedure mainly includes metabolite extraction from samples, LC-MS / MS detection, and data analysis. The raw mass spectrometry data is converted to mzXML format using ProteoWizard, followed by peak extraction, alignment, and retention time correction using XCMS software. Finally, the total peak area extracted from the samples is corrected, and peaks with a missing rate greater than 50% in each group are filtered. After correction and filtering, metabolite identification information is obtained by searching the Novogene local database. The identification results are standardized, and metabolites with a CV greater than 30% are filtered out. Multivariate statistical analysis is then performed on the metabolites, including principal component analysis (PCA) and hierarchical clustering (HCA).

[0077] The results are as follows Figure 13 It can be seen that the MPs group and the CTRL group are clearly separated, and the changes in placental metabolites can be partially reversed by the intervention of Lactobacillus helveticus ZJUIDS12. Furthermore, the clustering heatmap of total differential metabolites further confirms the beneficial effects of Lactobacillus helveticus ZJUIDS12.

[0078] Comparative Example 1: Lactobacillus plantarum ZJUIDS04 (CGMCC NO. 22609) was tested according to Example 1 above, and the results are as follows: Figure 14 and Figure 15 As shown, intervention with *Lactobacillus plantarum* ZJUIDS04 can improve fetal growth restriction and placental oxidative stress damage caused by polyps (MPs), but the effect is significantly less than that of *Lactobacillus helveticus* ZJUIDS12, and *Lactobacillus plantarum* ZJUIDS04 does not have the ability to improve fetal survival rate.

[0079] Example 2: Preparation of Lactobacillus helveticus ZJUIDS12 bacterial powder with the ability to prevent and treat plastic-induced intestinal damage and fetal growth restriction in pets.

[0080] A single colony of *Lactobacillus helveticus* ZJUIDS12 was inoculated into MRS liquid medium and incubated at 37°C for 24 h. Then, a 1% inoculum was transferred to fresh MRS liquid medium and incubated at 37°C for another 24 h. The resulting fermentation broth was centrifuged at 8000 r / min and 4°C for 15 min, the supernatant was discarded, and the bacterial precipitate was collected. The precipitate was washed twice with PBS buffer to obtain *Lactobacillus helveticus* ZJUIDS12 bacterial sludge.

[0081] Weigh 4.1 g of trehalose, 2.8 g of monosodium glutamate, and 8 g of sucrose, dissolve them in 15 mL of distilled water, filter the solution through a 0.22 μm microporous membrane for sterilization, and then add the solution to 85 mL of sterile water for later use. Dissolve 15 g of skim milk powder in distilled water to obtain a 15% (w / w) skim milk powder solution, sterilize it at 110℃, and set aside for later use. Mix the two solutions in a 1:1 volume ratio to obtain the lyophilization protection solution.

[0082] The prepared *Lactobacillus helveticus* ZJUIDS12 bacterial sludge was thoroughly mixed with the freeze-drying protectant at a ratio of 1:5 (g / ml). Pre-frozen at -70℃ for 2 hours, then removed and freeze-dried in a vacuum freeze dryer. The freeze dryer conditions were set to a vacuum of 5 Pa, a partition heating temperature of 20℃, and a cold trap temperature of -55℃. After freeze-drying for 48 hours, the mixture was crushed to obtain *Lactobacillus helveticus* ZJUIDS12 bacterial powder.

[0083] Example 3: Preparation of Lactobacillus helveticus ZJUIDS12 pet starter with anti-plastic intestinal damage to pets and prevention of fetal growth.

[0084] Lactobacillus helveticus ZJUIDS12 was inoculated into 10% sterilized skim milk and cultured at 37°C for 24 h until curdling. This process was repeated for two generations to obtain the mother starter culture. The mother starter culture was then inoculated into 10% skim milk at a 5% inoculation rate and cultured at 37°C for 24 h until curdling. At this point, the viable bacterial count reached 1×10⁻⁶. 9 CFU / mL yields the Lactobacillus helveticus ZJUIDS12 starter culture.

[0085] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The application of Lactobacillus helveticus ZJUIDS12 in the preparation of drugs for preventing and treating microplastic-induced injuries to pets, characterized by: The accession number for Lactobacillus helveticus ZJUIDS12 is CGMCC NO.23997.

2. The application according to claim 1, characterized in that: The prevention of microplastic-induced pet injury includes at least one of the following: prevention of microplastic-induced intestinal injury in pets, and prevention of microplastic-induced fetal growth restriction in pets.

3. The application according to claim 2, characterized in that: Prevention of microplastic-induced intestinal damage in pets includes at least one of the following: reducing maternal intestinal damage during pregnancy, restoring intestinal barrier function, increasing the abundance and diversity of intestinal flora, and maintaining intestinal microecological health. Prevention of microplastic-induced fetal growth restriction in pets includes at least one of the following: alleviating maternal placental oxidative stress damage and development during pregnancy, improving fetal survival rate, and reducing fetal growth restriction.

4. The application according to any one of claims 1 to 3, characterized in that: Lactobacillus helveticus ZJUIDS12 is used to prepare live bacterial agents and bacterial powders.

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