Lactobacillus delbrueckii subsp. Bulgaricus, microbial inoculum containing lactobacillus delbrueckii subsp. Bulgaricus, additive containing lactobacillus delbrueckii subsp. Bulgaricus, application of lactobacillus delbrueckii subsp. Bulgaricus and method for relieving pollution

Abstract

The invention relates to the field of microorganisms, and discloses lactobacillus delbrueckii subsp. Bulgaricus, a fungicide and an additive containing the lactobacillus delbrueckii subsp. Bulgaricus, application of the fungicide and the additive and a pollution relieving method. The preservation number of the lactobacillus delbrueckii subsp. Bulgaricus is CCTCC (China Center For Type Culture Collection) NO: M 20251218, and the microbial agent comprises the lactobacillus delbrueckii subsp. Bulgaricus, the lactobacillus delbrueckii subsp. Bulgaricus and the microbial inoculum provided by the invention can be used for preparing food additives for preventing and / or relieving microplastic-induced body injury, reducing cadmium pollution of food and inhibiting food-borne enterobacter sakazakii, can provide a new scheme for infant food safety and health protection, and have relatively high application value.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a *Lactobacillus delbrueckii* subsp. bulgaricus, bacterial agents and additives containing this *Lactobacillus delbrueckii* subsp. bulgaricus, their applications, and methods for mitigating contamination. Background Technology

[0002] With the development of the food industry and the spread of environmental pollutants, food safety issues have received increasing attention, especially the safety of infant and toddler food, which has become a focus of the industry.

[0003] Microplastics (MPs), a widespread environmental pollutant, can enter the food chain through various pathways, including water sources, soil, and packaging materials, and have been detected particularly in infant formula and complementary foods. Once ingested, microplastics can accumulate in the gut and cause a range of physiological damages, such as cytotoxicity and oxidative stress, posing a potential threat to the delicate digestive and immune systems of infants and young children. Currently, the removal of microplastics from food relies primarily on physical filtration or chemical adsorbents. However, physical methods can easily damage the nutritional components of food, while chemical adsorbents may leave harmful residues, failing to meet the safety requirements for infant formula and complementary foods.

[0004] Meanwhile, cadmium (Cd), a highly toxic heavy metal, can contaminate food through absorption by crops and contact with processing equipment. Its presence in infant formula can severely harm the nervous system, bone development, and kidney function. Existing cadmium reduction technologies mainly include chemical precipitation and ion exchange, but these methods often require complex process control and may introduce secondary pollution, making them unsuitable for direct addition to food.

[0005] Furthermore, Cronobacter sakazakii is a common pathogen in infant formula, and its contamination can lead to serious illnesses such as neonatal sepsis and meningitis, with a high mortality rate. Current control measures mainly rely on high-temperature sterilization or the addition of antibiotics. However, high temperatures can destroy the active nutrients in the formula, and the overuse of antibiotics may induce antibiotic resistance in bacteria, while also affecting the balance of the infant's gut microbiota, thus presenting significant limitations.

[0006] Probiotics, as a green and safe biological agent, have gained widespread recognition in the food industry for their functions such as regulating intestinal flora and enhancing immunity. However, current probiotic preparations mostly focus on a single function, such as only inhibiting certain types of pathogenic bacteria or having only a weak adsorption capacity for specific pollutants, and cannot simultaneously address the combined risks of microplastics, cadmium pollution, and Cronobacter sakazakii infection. Furthermore, most probiotics have low survival rates in the digestive environment of stomach acid and bile salts, making it difficult for them to function stably in the intestines, thus limiting their practical application in food contamination control.

[0007] Therefore, developing a probiotic preparation that combines the functions of adsorbing microplastics, degrading cadmium, inhibiting Cronobacter sakazakii, and is tolerant of the digestive environment and suitable as an additive for infant food has become an urgent technical problem to be solved in the current food industry and infant health field. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems of existing technologies, which cannot simultaneously address the combined risks of microplastics, cadmium pollution, and Cronobacter sakazakii infection, and the limited functionality and weak resistance to the digestive environment of existing probiotic preparations, making them unsuitable for the food safety needs of infants and young children. This invention provides *Lactobacillus delbrueckii* subsp. bulgaricus, a probiotic preparation containing *Lactobacillus delbrueckii* subsp. bulgaricus and its additives, as well as their applications and methods for mitigating pollution. This probiotic preparation can synergistically address microplastic-induced bodily damage, food cadmium pollution, and the threat of foodborne Cronobacter sakazakii, and is resistant to the digestive environment, making it suitable as a probiotic preparation for use as an additive in infant and young children's food, thus ensuring the safety of infant and young children's food consumption.

[0009] To achieve the above objectives, the present invention provides a strain of *Lactobacillus delbrueckii* subsp. bulgaricus (… Lactobacillus delbruekii subsp bulgaricus The Lactobacillus delbrueckii subsp. bulgaricus has the accession number CCTCC NO: M 20251218.

[0010] A second aspect of the present invention provides a microbial agent comprising Lactobacillus delbrueckii subsp. bulgaricus as described above.

[0011] A third aspect of the present invention provides an additive containing *Lactobacillus delbrueckii* subsp. *bulgaricus* as described above or an agent as described above.

[0012] The fourth aspect of the present invention provides the use of at least one of the previously described Lactobacillus delbrueckii subsp. bulgaricus, the previously described bacterial agent, and the previously described additive in mitigating microplastic pollution, mitigating cadmium pollution, and inhibiting at least one of Cronobacter sakazakii.

[0013] The fifth aspect of the present invention provides a method for mitigating pollution, the method comprising contacting at least one of the previously described Lactobacillus delbrueckii subsp. bulgaricus, the previously described bacterial agent, and the previously described additive with at least one of the following: a material contaminated with microplastics, a material contaminated with cadmium, and a material contaminated with Cronobacter sakazakii.

[0014] Through the above technical solution, the present invention provides a *Lactobacillus delbrueckii* subsp. *bulgaricus* (…). Lactobacillus delbruekii subsp bulgaricus This compound can be used to mitigate microplastic pollution, cadmium pollution, and inhibit Cronobacter sakazakii. It also possesses excellent acid and bile salt resistance, allowing it to survive stably in the digestive environment. When combined with Bifidobacterium breve... Bifidobacterium breve ) and Lactobacillus reuteri ( Limosilactobacillus reuteri When used in combination, it can work together to combat microplastics, cadmium and Cronobacter sakazakii contamination, alleviate the damage to the body caused by these contaminants, and is a probiotic preparation that can tolerate the digestive environment and is suitable as an additive for infant food, ensuring the safety of infant food consumption.

[0015] Biological Preservation The strain provided by this invention is classified and named Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve It was deposited on July 22, 2024 at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M20241663).

[0016] The strain provided by this invention is classified as *Lactobacillus delbrueckii* subsp. *bulgaricus* (…). Lactobacillus delbruekii subsp bulgaricus It was deposited on May 29, 2025 at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20251218).

[0017] The strain provided by this invention is classified and named *Lactobacillus reuteri* (…). Limosilactobacillus reuteri It was deposited on July 28, 2025 at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20251715). Attached Figure Description

[0018] Figure 1 This is a graph showing the acid resistance of the three probiotic strains (ZZ409, JP006 and YS401) in Example 2; Figure 2 This is a graph showing the bile salt tolerance of the three probiotic strains (ZZ409, JP006 and YS401) in Example 3. Figure 3 This is a graph showing the adsorption capacity of the bacterial agent on microplastics in Example 4; Figure 4 This is a graph showing the effect of the bacterial agent in Example 5 on the degradation of cadmium ions; Figure 5 This is a diagram showing the inhibitory effect of the bacterial agent in Example 6 on Cronobacter sakazakii; Figure 6 This is a graph showing the effect of the bacterial agent on cell survival rate induced by MPs in Example 7; Figure 7 This is a diagram showing the effect of the bacterial agent in Example 8 on the production of ROS in HT-29 cells induced by MPs; Figure 8 This is a graph showing the effect of MPs on the activity of antioxidant enzymes in HT-29 cells in Example 9. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] The first aspect of this invention provides a strain of Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbruekii subsp. bulgaricus The Lactobacillus delbrueckii subsp. bulgaricus has the accession number CCTCC NO: M 20251218.

[0021] The *Bifidobacterium breve*, *Lactobacillus delbrueckii* subsp. bulgaricus*, and *Lactobacillus reuteri* strains provided by this invention were isolated from the feces of healthy infants (collected in Nanjing). The isolation of these strains can be performed using conventional methods for isolating new strains in the art, such as using the streak plate method to pick single colonies, repeatedly purifying them, extracting the genome, and performing PCR identification.

[0022] The Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbruekii subsp bulgaricus The sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M20251218; deposit date: May 29, 2025).

[0023] The inventors have discovered that the Lactobacillus delbrueckii subsp. bulgaricus, with accession number CCTCCNO: M 20251218 provided by this invention, possesses excellent acid and bile salt resistance and can survive stably in the digestive environment; it also has the effect of mitigating microplastic and cadmium pollution and significantly inhibiting Cronobacter sakazakii.

[0024] A second aspect of the present invention provides a microbial agent comprising Lactobacillus delbrueckii subsp. bulgaricus as described above.

[0025] According to the present invention, preferably, the bacterial agent further contains *Bifidobacterium breve* and / or *Lactobacillus reuteri*; more preferably, the bacterial agent further contains *Bifidobacterium breve* and *Lactobacillus reuteri*; more preferably, the preservation number of the *Bifidobacterium breve* is CCTCC NO: M 20241663, and the preservation number of the *Lactobacillus reuteri* is CCTCC NO: M20251715.

[0026] The short bifidobacteria ( Bifidobacterium breve The sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20241663; deposit date: July 22, 2024).

[0027] The Lactobacillus reuteri ( Limosilactobacillus reuteri The sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20251715; deposit date: July 28, 2025).

[0028] The microbial agent described in this invention is not a simple superposition of the functions of the three, but rather strengthens the chain protection mechanism of "alleviating microplastic pollution - alleviating cadmium pollution - inhibiting Cronobacter sakazakii" by means of metabolic complementarity, effectively solving the problems of the limitation of single microbial agent function and poor synergy of non-specific strain combinations in the prior art.

[0029] According to the present invention, preferably, the content ratio of *Bifidobacterium breve*, *Lactobacillus delbrueckii* subsp. bulgaricus, and *Lactobacillus reuteri* in the microbial agent is 1:0.1-10:0.1-10, more preferably 1:0.2-5:0.2-5. The inventors have found that this embodiment ensures a balance and synergistic effect among the various microbial species, with their content within the specified range to meet the needs of different application scenarios. This not only enhances the functionality of the microbial agent provided by the present invention in addressing the potential risks of microplastic, cadmium contamination, and *Cronobacter sakazakii* infection in infant food, but also improves its applicability and stability, providing a more comprehensive and reliable guarantee for the safety of infant food.

[0030] According to the present invention, preferably, the concentrations of *Bifidobacterium breve*, *Lactobacillus delbrueckii* subsp. bulgaricus, and *Lactobacillus reuteri* in the bacterial agent are 1×10⁻⁶. 8CFU / mL or higher. The inventors have found that this preferred embodiment ensures the stability and effectiveness of the microbial agent provided by this invention in infant formula, and it is gut-friendly to infants, causing no adverse reactions and providing a safer intestinal microecological environment for infants.

[0031] A third aspect of the present invention provides an additive containing *Lactobacillus delbrueckii* subsp. *bulgaricus* as described above or an agent as described above.

[0032] The additive can be used as an additive in any product containing microplastics, cadmium, or Cronobacter sakazakii, and can be applied in the food, health product, cosmetic, aquaculture, environmental and other fields.

[0033] According to the present invention, preferably, the additive is a food additive. More preferably, the food is infant food. More preferably, the infant food is selected from infant formula milk powder and / or infant complementary food. Compared with conventional food additives, the additive provided by the present invention is not only adapted to the special environment of the infant digestive system (such as acid and bile salt resistance), but also specifically addresses the problem of combined contamination of microplastics, cadmium, and Cronobacter sakazakii in infant food, thereby protecting the liver and kidneys of infants from the toxicity of combined contamination of microplastics, cadmium, and Cronobacter sakazakii. This targeted and precise application design breaks through the limitation of the existing technology where the function of additives does not match the needs of infant food.

[0034] The fourth aspect of the present invention provides the use of at least one of the previously described Lactobacillus delbrueckii subsp. bulgaricus, the previously described bacterial agent, and the previously described additive in mitigating microplastic pollution, mitigating cadmium pollution, and inhibiting at least one of Cronobacter sakazakii.

[0035] The fifth aspect of the present invention provides a method for mitigating pollution, the method comprising contacting at least one of the previously described Lactobacillus delbrueckii subsp. bulgaricus, the previously described bacterial agent, and the previously described additive with at least one of the following: a material contaminated with microplastics, a material contaminated with cadmium, and a material contaminated with Cronobacter sakazakii.

[0036] This invention uses a live bacteria concentration of 1×10 7 -5×10 9 The dosage of CFU / g (based on the total mass of the material) is applied by direct mixing, suspension spraying, or addition after carrier encapsulation, upon contact with the material containing the contaminants; wherein the concentration of microplastic contamination in the material is 0.05-20 mg / kg, the concentration of cadmium contamination is 0.2-10 mg / kg, and the concentration of Cronobacter sakazakii contamination is 1×10⁻⁶ mg / kg. 3 -5×10 6CFU / g. During the contact process, depending on the characteristics of the material (such as solid food, liquid matrix, etc.), either static or stirring treatment can be selected to promote the full interaction between the strain and / or inoculant and / or additives and contaminants.

[0037] According to the present invention, preferably, the contact conditions include: a temperature of 2-37°C, a time of 4-6 h, and a pH of 3-7. Through extensive experimental verification, the inventors have shown that under the above-mentioned preferred contact conditions, *Lactobacillus delbrueckii* subsp. bulgaricus, the bacterial agent, or the additives described above can fully exert their effects on microplastics, cadmium, and *Cronobacter sakazakii*. When the temperature is between 2-37°C, the activity of the strain can be well maintained. A contact time of 4-6 h is sufficient for the strain to fully interact with the contaminated material, achieving the adsorption of microplastics, the degradation of cadmium, and the inhibition of *Cronobacter sakazakii*. The pH range of 3-7 simulates the acidic or alkaline environment that may exist in food, especially infant food, and the strain can still maintain good performance. This method of mitigating pollution through biological action is not only highly efficient and targeted but also environmentally friendly.

[0038] The present invention will be described in detail below through embodiments.

[0039] In the following examples, *Cronobacter sakazakii* was purchased from the American Type Culture Collection (ATCC); HT-29 cells were purchased from the American Type Culture Collection (ATCC); Caco-2 cells were purchased from the American Type Culture Collection (ATCC); superoxide dismutase (SOD) kits were purchased from Nanjing Jiancheng Biotechnology Institute; catalase (CAT) kits were purchased from Nanjing Jiancheng Biotechnology Institute; and RPMI-1640 / DMEM medium was purchased from Jiangsu Kaiji Biotechnology. All other materials and reagents were commercially available.

[0040] MRS liquid culture medium composition: protein 10 g / L, beef meal 5 g / L, yeast powder 4 g / L, glucose 2 g / L, Tween 80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L.

[0041] MRS solid culture medium composition: protein powder 10 g / L, beef meal 5 g / L, yeast powder 4 g / L, glucose 2 g / L, Tween 80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar powder 18 g / L.

[0042] RPMI-1640 / DMEM medium contains 100 IU / mL penicillin and 100 μg / mL streptomycin, but does not contain antibiotics.

[0043] Example 1: Materials and Methods Bifidobacterium breve ( Bifidobacterium breve Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbruekii subsp bulgaricus ) and Lactobacillus reuteri ( Limosilactobacillus reuteri All samples were isolated from stool samples of healthy, breastfed infants at Nanjing Gulou Hospital. The specific operational procedures are described in detail below: S1. Collect fresh fecal samples and quickly place them in a dedicated preservation solution to ensure sample viability. These samples are then sent to the laboratory for immediate filtration and serial dilution to ensure accurate subsequent cultures. Next, the treated samples are evenly spread onto MRS agar plates and incubated in a 37°C anaerobic workstation. The gas composition of this workstation is 85% nitrogen, 10% hydrogen, and 5% carbon dioxide; the specific model is ELECTROTEK AW 400TG TWO GAS VERSION. After 48 hours of anaerobic incubation, single colonies are picked and inoculated into freshly prepared MRS liquid medium for further culture. Subsequently, streak plating is performed again, and single colonies are picked from each streak. To ensure the acquisition of pure bacterial strains, the above steps are repeated three times. After the final single colony pick and inoculation into fresh MRS liquid medium, the strain is allowed to enter the logarithmic growth phase. At this point, the bacterial culture is collected and centrifuged to obtain bacterial cells. The obtained bacterial cells were resuspended in sterile PBS buffer and 25% glycerol was added for bacterial preservation to ensure the long-term activity of the strain.

[0044] S2. Collect the culture media of the three pure bacterial strains mentioned above and centrifuge them to collect bacterial cells. Add the collected bacterial cells to bacterial lysis buffer to allow for complete lysis and extract DNA. Use the extracted DNA as a template for PCR amplification, collect the PCR amplification products, and perform sequencing analysis. Assemble the sequencing data and then perform sequence alignment analysis using the BLAST tool in the NCBI database. Based on the sequence alignment results, one strain was identified as *Bifidobacterium breve*, with a similarity range of 99.9% to 100.0%, and was named ZZ409; another strain was *Lactobacillus delbrueckii* subsp. bulgaricus*, with a similarity also between 99.9% and 100.0%, and was named JP006; and the third strain was *Lactobacillus reuteri*, with a similarity also between 99.9% and 100.0%, and was named YS401. Through these steps, three target bacterial strains were successfully identified and named.

[0045] Bifidobacterium breve ( Bifidobacterium breveThe sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20241663; deposit date: July 22, 2024).

[0046] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbruekii subsp bulgaricus The sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20251218; deposit date: May 29, 2025).

[0047] Lactobacillus reuteri ( Limosilactobacillus reuteri The sample is deposited at the China Center for Type Culture Collection (Address: Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan, Hubei Province, 430072, China; accession number: CCTCC NO: M 20251715; deposit date: July 28, 2025).

[0048] Example 2: Acid Resistance Assessment S1. Sterilize and cool the MRS liquid culture medium. Inoculate the three probiotic strains (Bifidobacterium breve ZZ409, Lactobacillus delbrueckii subsp. bulgaricus JP006, and Lactobacillus reuteri YS401) prepared in Example 1 into the sterilized and cooled MRS liquid culture medium at an inoculation rate of 1% by volume. Incubate at a constant temperature of 37°C until the strains reach the late logarithmic growth phase to ensure that the strains are in an active state.

[0049] S2. The bacterial strains cultured in step S1 were inoculated into MRS liquid medium with a pH of 3, which was adjusted using 0.1 N HCl. After inoculation, these cultures were placed in an anaerobic workstation at 37°C for further cultivation. The gas composition of this anaerobic workstation was 85% nitrogen, 10% hydrogen, and 5% carbon dioxide to ensure an anaerobic environment; the equipment used was an ELECTROTEK AW 400TG TWO GAS VERSION. During cultivation, bacterial culture samples were taken at 0 hours and 3 hours for plating.

[0050] The plate count method was used to evaluate the survival of each strain at different time points. The specific calculation formula is as follows: Final survival rate (%) = [Number of viable bacteria in MRS liquid medium at 3 h (log CFU / mL) / Number of viable bacteria in MRS liquid medium at 0 h (log CFU / mL)] × 100. This formula allows for the accurate determination of the survival rate of each strain in an acidic environment, as shown in the results. Figure 1 As shown.

[0051] Depend on Figure 1 It was found that the three probiotic strains (Bifidobacterium breve ZZ409, Lactobacillus delbrueckii subsp. bulgaricus JP006, and Lactobacillus reuteri YS401) all exhibited good tolerance in an acidic environment with a pH of 3, indicating that they also possess the ability to survive stably in the digestive system of infants and young children. This acid-resistant characteristic ensures that the various strains in the probiotic agent can synergistically maintain a sufficient quantity and activity in the digestive system of infants and young children, thereby fully exerting their functions in combating microplastic-induced damage, reducing cadmium levels, and inhibiting Cronobacter sakazakii, providing comprehensive food safety assurance for infants and young children.

[0052] Example 3: Evaluation of bile salt tolerance The three probiotic strains prepared in Example 1 (Bifidobacterium breve ZZ409, Lactobacillus delbrueckii subsp. bulgaricus JP006, and Lactobacillus reuteri YS401) were inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C until the end of the logarithmic growth phase. Subsequently, each probiotic strain was transferred to MRS liquid medium containing 2.5% (w / w) porcine bile salts and cultured in a 37°C anaerobic workstation (gas composition: 85% nitrogen, 10% hydrogen, 5% carbon dioxide; model: ELECTROTEK AW 400TG TWO GAS VERSION). Bacterial culture was plated at 0 h and 3 h, and the survival rate of each strain was assessed using the plate count method. The calculation formula is as follows: Final survival rate (%) = [Number of viable bacteria in MRS liquid medium at 3 h (log CFU / mL) / Number of viable bacteria in MRS liquid medium at 0 h (log CFU / mL)] × 100.

[0053] The survival rate calculation results are as follows: Figure 2 As shown, by Figure 2It was found that the survival rates of *Lactobacillus delbrueckii* subsp. bulgaricus JP006, *Bifidobacterium breve* ZZ409, and *Lactobacillus reuteri* YS401 all showed a significant increasing trend, exceeding the initial inoculation levels, demonstrating their stability and adaptability in the simulated high bile salt environment of the human gut. This result not only strongly supports the survival and application of the microbial agents provided by this invention as food additives in the intestinal environment, but also lays a solid foundation for their potential application in preventing or mitigating bodily damage caused by microplastics, cadmium pollution, and *Cronobacter sakazakii*.

[0054] Example 4: Evaluation of the Adsorption Capacity of Microplastics (MPs) The three probiotic strains prepared in Example 1 (Bifidobacterium breve ZZ409, Lactobacillus delbrueckii subsp. bulgaricus JP006, and Lactobacillus reuteri YS401) were inoculated into MRS liquid medium at a 1% (v / v) inoculum and cultured at 37°C until the end of the logarithmic phase. The culture was then centrifuged at 6000 r / min for 15 min. The bacterial precipitate was washed twice with PBS and the bacterial suspension was resuspended. JP006, ZZ409, and YS401 were then mixed in a 1:1:1 ratio to prepare bacterial agent LBL, wherein the concentrations of JP006, ZZ409, and YS401 were all 2 × 10⁻⁶. 9 CFU / mL, stored at 4℃ for later use. Transfer 100 μL of LBL bacterial culture to a 1.5 mL EP tube, mix with 900 μL of sterile PBS or PS fluorescent microplastic working solution (0.1 mg / mL, particle size 5 μm), and co-incubate at 37℃ for 4 h for adsorption, then centrifuge at 6000 r / min for 10 min. Transfer the supernatant to a 96-well plate and measure the fluorescence intensity at an excitation wavelength of 488 nm. Microplastic (MPs) adsorption rate (%) = [(C0 - C1) / C0] × 100% (C0: initial MPs content in the sample; C1: MPs content measured in the supernatant of the experimental group). The control group consisted of a prepared solution with the same MPs concentration.

[0055] Adsorption results are as follows Figure 3 As shown, by Figure 3 It can be seen that the bacterial agent LBL provided in this embodiment has an extremely strong adsorption capacity for MPs, and the adsorption rate is much higher than that of the control group. This result not only verifies the feasibility of LBL as a biosorbent, but also provides strong support for its promotion in practical applications.

[0056] Example 5 Assessment of the mitigation capacity of cadmium (Cd) pollution Take 1 mL of the bacterial solution LBL prepared in Example 4 (JP006, ZZ409 and YS401 are 2×10⁻⁶ respectively). 9The bacterial culture (CFU / mL) was mixed with 5 mL of PBS solution containing Cd ions (100 μg / mL CdCl2), and allowed to stand at room temperature for 6 h. The culture was then centrifuged at 0 h and 6 h, and the supernatant was collected. The supernatant was analyzed by atomic absorption spectrometry to determine the residual Cd concentration. Cd degradation (%) = [(C0 - C1) / C0] × 100% (C0: initial C ions in the sample). d Content; C1: Cd content measured in the supernatant of the experimental group. The control group consisted of a prepared solution with the same Cd concentration.

[0057] Degradation effect such as Figure 4 As shown, by Figure 4 It is evident that the microbial agent LBL has a significant degradation effect on Cd, substantially reducing the Cd concentration in the solution. Compared with the control group, the Cd content in the experimental group was greatly reduced, demonstrating the great potential of the microbial agent LBL provided by this invention in treating heavy metal pollution.

[0058] Example 6 Evaluation of inhibitory effect on Cronobacter sakazakii The Oxford cup double-layer plate method was used to test the bacterial culture of LBL prepared in Example 4 (which contained JP006, ZZ409, and YS401, with concentrations of 2×10⁻⁶ for the three bacterial strains). 9 The antibacterial effect was evaluated using CFU / mL. Specifically, the compound bacterial solution sample was placed in an anaerobic workstation at 37°C for 24 hours. After incubation, the diameter of the formed inhibition zone was precisely measured and recorded (in millimeters). For comparative analysis, a control group consisting of normally growing *Cronobacter sakazakii* was included in the experiment.

[0059] Experimental results are as follows Figure 5 As shown, through Figure 5 Analysis clearly shows that the LBL bacterial solution exhibits a significant inhibitory effect on Cronobacter sakazakii, with the diameter of its inhibition zone being significantly larger than that of the control group. This result fully demonstrates that the LBL bacterial solution possesses strong antibacterial activity, effectively proving its inhibitory effect on Cronobacter sakazakii. This discovery provides solid data support and theoretical basis for the practical application of LBL in the food industry, indicating that LBL has the potential to be developed into a highly efficient and reliable Cronobacter sakazakii inhibitor, thus playing an important role in ensuring the safety of infant and young children's food, and possessing significant application prospects and social significance.

[0060] Example 7 Assessment of mitigation of MPs-induced cytotoxicity Cell viability and cell proliferation assays were performed to study the bacterial culture of LBL prepared in Example 4 (JP006, ZZ409 and YS401 were 2×10⁻⁶ each). 9The effect of CFU / mL on mitigating the toxicity of MPs on HT-29 and Caco-2 cells. The bacterial agent LBL was centrifuged and the supernatant was discarded. The cells were washed three times with PBS and then resuspended to the original concentration in RPMI-1640 / DMEM medium (without 100 IU / mL penicillin-100 μg / mL streptomycin).

[0061] HT-29 / Caco-2 cells were digested, centrifuged, and resuspended in 1 mL of culture medium. 20 μL of the cell suspension was used for cell counting. The cell suspension was diluted to 100,000 cells / well with RPMI-1640 / DMEM complete medium (containing 100 IU / mL penicillin and 100 μg / mL streptomycin). After thorough mixing, 1 mL of the cell suspension was added to each well of a 24-well plate and incubated. Once cells adhered, the culture medium was discarded, and the cells were washed three times with PBS buffer. Transwell chambers (Corning, USA) were placed in the wells. 1 mL of 100 μg / mL MPs solution was added to the lower chamber, and 200 μL of LBL suspension was added to the upper chamber. A control group was established, consisting only of RPMI-1640 / DMEM medium and no MPs or LBL suspension. Each group was tested in triplicate. Incubate in an incubator (37℃, 5% CO2) for 24 h, and then test using a cell proliferation and toxicity assay kit (Albatross, Guangzhou, China).

[0062] Test results as follows Figure 6 As shown, in HT-29 and Caco-2 cell models, compared to groups exposed only to microplastics (MPs), the addition of the bacterial agent LBL (MPs+LBL group) significantly improved cell survival. For Caco-2 cells, MP exposure decreased cell survival, while the MPs+LBL group showed significantly higher cell survival than the MPs group; HT-29 cells showed a similar trend, with the MPs+LBL group exhibiting better cell survival than the MPs-only treatment group. This indicates that LBL can effectively alleviate microplastic-induced cytotoxicity, has a protective effect on intestinal cells (using HT-29 and Caco-2 as models), and helps maintain cell viability and proliferation capacity, providing cellular-level experimental evidence for its application in addressing microplastic-induced damage to the body.

[0063] Example 8: Determination of Levels of Reactive Oxygen Species (ROS) To investigate the effect of MPs stimulation on ROS production in HT-29 cells and whether LBL could alleviate ROS production under MPs stimulation, 2 × 10⁻⁶ LBL bacterial suspensions (JP006, ZZ409, and YS401) prepared in Example 4 were used. 9Centrifuge (CFU / mL) and discard the supernatant. Wash the cells three times with PBS and then resuspend them to the original concentration in RPMI-1640 / DMEM medium (without 100 IU / mL penicillin-100 μg / mL streptomycin).

[0064] HT-29 / Caco-2 cells were digested, centrifuged, and resuspended in 1 mL of RPMI-1640 / DMEM medium. 20 μL of the cell suspension was aspirated for cell counting. The cell suspension was diluted to 100,000 cells / well with RPMI-1640 / DMEM complete medium (containing 100 IU / mL penicillin and 100 μg / mL streptomycin). After thorough mixing, 1 mL of the cell suspension was added to each well of a 24-well plate and incubated. Once cells adhered, the medium was discarded, and the cells were washed three times with PBS buffer. Transwell chambers (Corning, USA) were placed in the wells. 1 mL of 100 μg / mL MPs was added to the lower chamber, and 200 μL of the corresponding LBL suspension was added to the upper chamber. A control group was established without MPs and LBL suspension. Each group was tested in triplicate. Incubate in an incubator (37℃, 5% CO2) for 24 h, and determine reactive oxygen species using a reactive oxygen species detection kit (Beyotime, Shanghai, China).

[0065] Test results as follows Figure 7 As shown, by Figure 7 It was found that the intracellular ROS level of HT-29 cells significantly increased under MP stimulation, indicating that MPs can induce cellular oxidative stress. However, the addition of LBL suspension significantly reduced the intracellular ROS level of HT-29 cells, and the inhibitory effect on ROS became more pronounced with increasing LBL concentration. This result clearly demonstrates that LBL has excellent antioxidant capacity, effectively alleviating MP-induced cellular oxidative stress, reducing intracellular ROS levels, and thus protecting cells from oxidative damage. This finding further confirms the great potential of LBL in preventing or mitigating microplastic-induced damage to the body.

[0066] Example 9: Determination of Intracellular Antioxidant Enzymes The intracellular major antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), were measured to assess the cellular oxidative stress status and antioxidant defense capacity of HT-29 cells under MP stimulation. Two × 10⁻⁶ LBL bacterial cultures (JP006, ZZ409, and YS401) prepared in Example 4 were used. 9 Centrifuge (CFU / mL) and discard the supernatant. Wash the cells three times with PBS and then resuspend them to the original concentration in RPMI-1640 / DMEM medium (without 100 IU / mL penicillin-100 μg / mL streptomycin).

[0067] HT-29 cells were digested, centrifuged, and resuspended in 1 mL of culture medium. 20 μL of the cell suspension was used for cell counting. The cell suspension was diluted to 100,000 cells / well with RPMI-1640 / DMEM complete medium (containing 100 IU / mL penicillin and 100 μg / mL streptomycin). After thorough mixing, 1 mL of the cell suspension was added to each well of a 24-well plate and incubated. Once cells adhered, the culture medium was discarded, and the cells were washed three times with PBS buffer. Transwell chambers (Corning, USA) were placed in the wells. 1 mL of 100 μg / mL PS-MPs was added to the lower chamber, and 200 μL of the corresponding LBL suspension was added to the upper chamber. A control group (without MPs and LBL suspension) was included. Each group was tested in triplicate. Incubate in an incubator (37℃, 5% CO2) for 24 h, and detect the results using the superoxide dismutase assay kit and catalase assay kit from Nanjing Jiancheng Biotechnology Institute (Nanjing Jiancheng, Jiangsu, China).

[0068] Test results as follows Figure 8 As shown, by Figure 8 It was found that under MP stimulation, the activities of superoxide dismutase (SOD) and catalase (CAT) in HT-29 cells both showed a decreasing trend, reflecting that the cellular antioxidant defense system was challenged when responding to MP-induced oxidative stress. However, when LBL suspension was added, the activities of SOD and CAT in HT-29 cells were significantly increased, and this effect became more pronounced with increasing LBL concentration. This result strongly demonstrates that LBL can not only effectively alleviate MP-induced cellular oxidative stress but also enhance the cellular antioxidant defense capacity, thereby further protecting cells from oxidative damage. This discovery further emphasizes the enormous application potential of LBL in preventing or alleviating microplastic-induced damage to the body, and also provides strong scientific evidence for its use as a novel green and healthy food additive.

[0069] Example 10 The microbial agent I was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the methods provided in Examples 4, 5, and 6. The microbial agent I was prepared according to the method provided in Example 4, except that the ratio of the three bacteria JP006, ZZ409, and YS401 was adjusted to 1:0.1:10. The test results are shown in Table 1.

[0070] Comparative Example 1 The microbial agent II was tested according to the method provided in Example 10 for its adsorption capacity (adsorption rate %) of microplastics (MPs), degradation capacity (degradation rate %) of cadmium (Cd), and inhibitory effect on Cronobacter sakazakii (mm). The microbial agent II was prepared according to the method provided in Example 4, except that the ratio of the three bacteria JP006, ZZ409 and YS401 was adjusted to 1:0.001:20. The test results are shown in Table 1.

[0071] Example 11 The microbial agent III was tested according to the method provided in Example 10 for its adsorption capacity (adsorption rate %) of microplastics (MPs), degradation capacity (degradation rate %) of cadmium (Cd), and inhibitory effect (mm) on Cronobacter sakazakii. The microbial agent III was prepared according to the method provided in Example 4, except that Bifidobacterium breve was replaced with commercially available Bifidobacterium breve purchased from Minsheng Zhongke Jiayi (Shandong) Biotechnology Co., Ltd. The test results are shown in Table 1.

[0072] Example 12 The microbial agent V was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the method provided in Example 10. The microbial agent V was prepared according to the method provided in Example 4, except that Lactobacillus reuteri was replaced with commercially available Lactobacillus reuteri purchased from Shanghai Bohu Biotechnology Co., Ltd. The test results are shown in Table 1.

[0073] Comparative Example 2 The microbial agent IV was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the method provided in Example 10. The microbial agent IV was prepared according to the method provided in Example 4, except that Lactobacillus delbrueckii subsp. bulgaricus was replaced with commercially available Lactobacillus delbrueckii subsp. bulgaricus, purchased from Shanghai Fuxiang Biotechnology Co., Ltd. The test results are shown in Table 1.

[0074] Example 13 The microbial agent V was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the method provided in Example 10. The microbial agent V was prepared according to the method provided in Example 4, except that the application of Bifidobacterium breve was omitted. The test results are shown in Table 1.

[0075] Example 14 The microbial agent V was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the method provided in Example 10. The microbial agent V was prepared according to the method provided in Example 4, except that the application of Bifidobacterium breve and Lactobacillus reuteri was omitted. The test results are shown in Table 1.

[0076] Comparative Example 3 The microbial agent V was tested for its microplastic (MPs) adsorption capacity (adsorption rate %), cadmium (Cd) degradation capacity (degradation rate %), and inhibitory effect on Cronobacter sakazakii (mm) according to the method provided in Example 10. The microbial agent V was prepared according to the method provided in Example 4, except that the application of Lactobacillus delbrueckii subsp. bulgaricus was omitted. The test results are shown in Table 1.

[0077] Table 1

[0078] As can be seen from the detailed experimental results shown in Table 1, the bacterial agent prepared by the method provided in this invention exhibits significant advantages.

[0079] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbruekii subsp bulgaricus ), characterized in that, The preservation number of the Lactobacillus delbrueckii subsp. bulgaricus is CCTCC NO: M 20251218.

2. A microbial agent, characterized in that, The bacterial agent comprises *Lactobacillus delbrueckii* subsp. *bulgaricus* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent also contains Bifidobacterium breve and / or Lactobacillus reuteri; Preferably, the microbial agent further contains Bifidobacterium breve and Lactobacillus reuteri; Preferably, the preservation number of the Bifidobacterium breve is CCTCC NO: M 20241663; Preferably, the Lactobacillus reuteri has the preservation number CCTCC NO: M 20251715.

4. The microbial agent according to claim 3, characterized in that, The content ratio of Bifidobacterium breve, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus reuteri in the bacterial agent is 1:0.1-10:0.1-10, preferably 1:0.2-5:0.2-5.

5. The microbial agent according to claim 3 or 4, characterized in that, The concentrations of *Bifidobacterium breve*, *Lactobacillus delbrueckii* subsp. bulgaricus, and *Lactobacillus reuteri* in the bacterial agent were 1×10⁻⁶. 8 CFU / mL or higher.

6. An additive, characterized in that, The additive contains the Lactobacillus delbrueckii subsp. bulgaricus as described in claim 1 or the bacterial agent as described in any one of claims 2-5.

7. The additive according to claim 6, characterized in that, The additive is a food additive; Preferably, the food is infant food; Preferably, the infant food is selected from infant formula milk powder and / or infant complementary food.

8. The use of at least one of the following: Lactobacillus delbrueckii subsp. bulgaricus according to claim 1, the bacterial agent according to any one of claims 2-5, and the additive according to claim 6 or 7, in mitigating microplastic pollution, mitigating cadmium pollution, and inhibiting at least one of Cronobacter sakazakii.

9. A method for mitigating pollution, characterized in that, The method comprises contacting at least one of the following: Lactobacillus delbrueckii subsp. bulgaricus as described in claim 1, the microbial agent as described in any one of claims 2-5, and the additive as described in claim 6 or 7, with at least one of the following: microplastic contaminated material, cadmium contaminated material, and Cronobacter sakazakii contaminated material.

10. The method according to claim 9, characterized in that, The contact conditions include: a temperature of 25-37°C, a time of 4-6 hours, and a pH of 3-7.

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