Isolation and application of a lead adsorption strain of lactobacillus mucosus

Abstract

The application discloses a lead adsorption fermentation lactobacillus muciaginous separation and screening and application, and belongs to the field of lead adsorption based on microbial technology. The lactobacillus plantarum RS7 of the application has good acid production and acid resistance, and the highest lead adsorption rate in the lead-containing MRS liquid culture medium reaches 95%. Meanwhile, the lactobacillus plantarum RS7 has good tolerance and adsorption capacity to lead ions in vitro, can reduce the lead content in a preserved egg, and the lead removal rate reaches about 60% after 48 hours. The fermentation lactobacillus muciaginous RS7 is used in fermentation food and other some lead-polluted sewage environments with lead removal function, and has very wide application prospect.

Description

Technical Field

[0001] This invention relates to the isolation, screening, and application of a lead-adsorbing fermenting Lactobacillus mucinus, belonging to the field of lead adsorption based on microbial technology. Background Technology

[0002] Lead contamination in food primarily originates from human pollution. Lead, as a heavy metal, is highly toxic and permeable, with a very low natural degradation rate, making its harmless disposal difficult. During food processing, some traditional methods or equipment may introduce lead contamination. For example, in the traditional production of preserved eggs, lead oxide is often used as a food additive to improve its shape. Some traditional popcorn machines have lids and chambers made of lead-containing cast iron, which can easily volatilize and enter the popcorn during closed heating. Additionally, some food containers and packaging materials, such as food cans made with lead-soldered materials, lead-containing plastic packaging, and lead-containing food additives, can all lead to food contamination and harm human health.

[0003] Currently, lead removal methods include physical, chemical, and biological methods. In recent years, microbial adsorption has been widely used in the treatment of heavy metal wastewater and soil due to its advantages such as ease of use, high removal efficiency, abundant resources, low cost, and few byproducts. Bacteria are the most abundant microorganisms on Earth. Studies have shown that many types of bacteria, such as Lactobacillus, Pseudomonas, Bacillus subtilis, and Enterobacter leucovorin, and their products have a strong binding force with metal ions. The phosphoproteopolysaccharides in the cell walls of Gram-positive bacteria and the lipopolysaccharides on the outer layer of peptidoglycan in the cell walls of Gram-negative bacteria both carry a strong negative charge. This charge characteristic enables them to adsorb heavy metal ions. Moreover, due to the high affinity between the structural components of the bacterial outer membrane and metal ions, metal ions are easily adsorbed by bacteria. This adsorption not only reflects the chemical characteristics of bacterial cell wall components but also reveals the potential treatment capacity of bacteria for heavy metal pollutants in the environment.

[0004] Lactic acid bacteria (LAB) are non-pathogenic, safe Gram-positive bacteria capable of fermenting carbohydrates and producing large amounts of lactic acid. LAB participates in numerous metabolic activities in the human body, improving intestinal microbiota and intestinal function, lowering serum cholesterol levels, enhancing immune function, preventing and inhibiting tumor metabolism, improving food metabolism and digestion, improving vitamin metabolism, and delaying aging. They also enhance food flavor. In recent years, heavy metal contamination in food has attracted increasing attention. Besides their unique health benefits, lactic acid bacteria also possess the ability to adsorb heavy metals. Compared to traditional heavy metal removal methods, the removal effect of lactic acid bacteria has garnered more attention, leading to a growing number of researchers focusing on the adsorption of heavy metals by lactic acid bacteria. Man Zhaohong et al. screened lead-resistant bacteria from the intestinal contents and excrement of broiler chickens for the removal of heavy metal lead. The JT1 strain showed the highest lead removal capacity at 66.95%. Halttumen et al. isolated *Bifidobacterium longum*, *Lactobacillus fermentum*, and *Bifidobacterium lactis*, all of which effectively removed lead, with *Bifidobacterium longum* achieving a lead removal of 175.7 mg / g. Ma Hongmei et al. analyzed the effects of three different lactic acid bacteria—*Lactobacillus deuterans*, *Streptococcus thermophilus*, and *Enterococcus faecium*—on the adsorption of lead ions in seawater. These studies indicate that lactic acid bacteria capable of adsorbing heavy metals have the potential to remove heavy metals from the human body, thereby reducing the harm caused by heavy metals. However, the currently reported lactic acid bacteria exhibit poor lead adsorption capacity and low survival rate in high-concentration lead environments, and the types of lead salts they adsorb are limited. More superior lactic acid bacteria are needed to improve this situation. Therefore, it is essential to screen out a type of lactic acid bacteria that has excellent tolerance and adsorption capacity to high concentrations of lead, demonstrate its good lead removal effect in different forms of lead-containing fermented foods, and develop practical applications for these lactic acid bacteria. Summary of the Invention

[0005] The purpose of this invention is to provide strains that can survive in high-concentration lead environments and have a strong adsorption effect on various forms of lead.

[0006] [Technical Solution]

[0007] The present invention is achieved through the following technical solution.

[0008] This invention relates to a lactic acid bacterium, identified as *Limosilactobacillus fermentum* RS7 based on its morphological characteristics, culture properties, and physiological and biochemical features. This strain was deposited on August 15, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 64983). The address of the depository is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The *Limosilactobacillus fermentum* RS7 can be used to remove lead from substrates such as feed, soil, and culture media. It can tolerate lead concentrations up to 250 mg / L. Under pH 6 conditions, it exhibits a lead adsorption rate of over 50% for a 100 mg / L lead acetate solution, and a lead acetate removal rate of 74.17% in a 100 mg / L solution.

[0009] In one embodiment, the fermenting Lactobacillus mucinus RS7 of the present invention has the following properties:

[0010] (1) It is acid-resistant and grows well in environments with pH 3.0-8.0, and survives well in environments with pH 2.5;

[0011] (2) When cultured in lead-containing culture medium in vitro, it has good tolerance to lead ions;

[0012] (3) When incubated in an aqueous solution containing lead in vitro, it has a good adsorption capacity for lead ions;

[0013] (4) It has the potential to reduce the lead content in lead-containing fermented foods and promotes lead removal from lead-containing fermented foods mainly through lead adsorption.

[0014] In one embodiment, and in some embodiments of the present invention, the base sequence of the 16S rRNA gene of the fermenting Lactobacillus mucinus RS7 is shown in SEQ ID NO.1.

[0015] In one embodiment, the fermenting Lactobacillus mucinus RS7 tolerates a bile salt concentration of 0.3%.

[0016] In one embodiment, the fermenting Lactobacillus RS7 has good acid resistance and can grow normally between pH 3 and 7.

[0017] In one embodiment, the fermented Lactobacillus mucinus RS7 has a good acid-producing capacity, and fermentation for 12 hours can reduce the acid content from the initial 6.31 to 4.1.

[0018] In one embodiment, Lactobacillus fermentum RS7 exhibits excellent lead tolerance, with a survival rate of over 50% in a solution containing 100 mg / L of lead ions.

[0019] In one embodiment, Lactobacillus fermentans RS7 has a good lead adsorption capacity and can be used to remove lead metal elements from a substrate, which can be lead-containing fermented food, soil, feed, culture medium and other substances that need to remove lead.

[0020] The present invention also provides a microbial preparation containing the aforementioned fermenting Lactobacillus mucinus RS7.

[0021] In one embodiment, the viable count of *Lactobacillus mucinus* RS7 in the microbial preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0022] The present invention also provides a product containing the aforementioned fermented Lactobacillus mucinus RS7, or the aforementioned microbial preparation.

[0023] In one embodiment, the product includes food, medicine, and health products.

[0024] In one embodiment, the product comprises a food additive; preferably, the product comprises a heavy metal adsorbent.

[0025] In one embodiment, the product includes a lead adsorbent.

[0026] The present invention also provides a method for adsorbing lead, wherein the fermented Lactobacillus mucinus RS7 or the microbial preparation is added to a lead-containing matrix for cultivation.

[0027] In one embodiment, the lead-containing matrix includes a solid matrix, a liquid matrix, or a semi-solid matrix.

[0028] In one embodiment, the type of lead includes lead acetate, lead oxide, or lead nitrate.

[0029] In one embodiment, the adsorption conditions are: pH 3-7 and adsorption time of at least 24 hours.

[0030] The present invention also provides the application of the aforementioned fermented Lactobacillus mucinus RS7, or the aforementioned microbial preparation, in the removal of lead from a matrix, the matrix including food, soil, feed, and culture medium.

[0031] Furthermore, in some embodiments of the present invention, the pH of the matrix is ​​controlled to be 3-7.

[0032] Furthermore, in some embodiments of the present invention, the pH of the matrix is ​​controlled to be 5.5-6.3.

[0033] The study of this invention found that the fermenting Lactobacillus RS7 has a good lead adsorption rate under pH conditions of 3-7. For example, under pH conditions of 6, its lead adsorption rate exceeds 70%, indicating that fermenting Lactobacillus RS7 has great application potential.

[0034] The present invention also provides the application of the aforementioned fermented Lactobacillus mucinus RS7, or the aforementioned microbial preparation, in the preparation of lead adsorbents.

[0035] Beneficial effects:

[0036] This invention provides a strain of *Lactobacillus fermentans* RS7, which exhibits good acid resistance, bile salt resistance, and lead resistance. It can adsorb lead ions from solutions containing lead acetate, lead nitrate, and lead oxide. The lead removal rate reaches 74.17% in a solution with a lead ion concentration of 100 mg / L and 95% in a solution with a lead ion concentration of 50 mg / L.

[0037] This strain demonstrates excellent lead adsorption effects on lead-containing fermented foods and lead-contaminated water bodies. Finally, the fermented *Lactobacillus myxobin* RS7 can be prepared into a highly active microbial preparation, conveniently applied to lead-contaminated substrates, and can also be used as a lead adsorbent, alone or in combination with similar adsorbents, to reduce lead content in food and the environment.

[0038] Preservation of biological materials

[0039] A strain of *Limosilactobacillus fermentum* RS7, taxonomically named *Limosilactobacillus fermentum*, was deposited on August 15, 2024, at the Guangdong Provincial Center for Microbiological Culture Collection (GDMCC) with accession number GDMCC No: 64983. The address of the depository is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0040] Figure 1 This is a plate isolation diagram of lactic acid bacteria.

[0041] Figure 2 The results are Gram staining results for lactic acid bacteria.

[0042] Figure 3 Degradation rate of lead by different strains.

[0043] Figure 4Acid resistance of the selected strains.

[0044] Figure 5 Phylogenetic tree of lactic acid bacteria RS7 based on 16S rDNA genes.

[0045] Figure 6 Scanning electron microscope images of the strain before (left) and after (right) lead adsorption.

[0046] Figure 7 The lead removal rate of strain RS7 in preserved eggs. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] The adsorption rate of lead ions by the strains involved in the following examples was calculated according to the following formula:

[0050] Adsorption rate (%) = [(A0-A1) / A0] × 100%;

[0051] In the formula, A0 represents the initial lead ion concentration of the solution, and A1 represents the lead ion concentration of the solution after adsorption. The lead ion concentration is detected by a flame atomic absorption spectrophotometer.

[0052] Example 1: Screening and identification of fermented Lactobacillus mucinus RS7

[0053] 1. Isolation and screening of lead-adsorbed lactic acid bacteria

[0054] 1 mL of fermented food slurry such as kimchi, yogurt, and Yakult was added to 30 mL of MRS liquid medium and cultured at 37°C and 120 rpm for 48 h to enrich lactic acid bacteria. The enriched liquid medium was then streaked onto MRS solid medium and incubated upside down at 37°C for 48 h. Single colonies were then isolated and purified three times. Figure 1 The obtained strains were subjected to catalase test and Gram staining microscopy to preliminarily determine whether they were lactic acid bacteria.

[0055] Single colonies of the target strains obtained from the initial plate screening were picked and transferred to 30 mL of MRS liquid medium. The culture was then placed in a shaking incubator at 37°C and 120 rpm for 48 h. Finally, a 1% inoculum was created (initial inoculum size was 10...). 6Lead (CFU / ml) was inoculated into 30 mL of MRS liquid medium containing 100 mg / L lead acetate and cultured in a shaking incubator for 48 h. An uninoculated MRS liquid medium containing an equal amount of lead was used as a control. The culture was then centrifuged three times at 8000 r / min for 20 min using a high-speed refrigerated centrifuge. The lead content in the supernatant was determined by flame atomic absorption spectrometry. The lead degradation rate of the test strains was calculated based on the lead content in the initial MRS liquid medium. The results showed that different strains had different lead adsorption rates, with strain RS7 exhibiting the highest adsorption rate of 74.17% (CFU / ml). Figure 3 Therefore, we identified this strain of bacteria.

[0056] 2. Identification of lactic acid bacteria

[0057] (1) Gram staining

[0058] Select a single colony of lactic acid bacteria, mix it in 10 μL of sterile aqueous solution, spread it on a sterile glass slide, fix it quickly in a flame, and after cooling, perform Gram staining according to the procedure. Observe under an inverted microscope with an oil immersion microscope to confirm that it is a Gram-positive bacterium. Figure 2 ).

[0059] (2) Biological identification of 16S rRNA

[0060] After overnight culture of the selected lead-tolerant strains, genomic DNA of the lactic acid bacteria was extracted from the bacterial culture according to the instructions of the DNA extraction kit. The extracted DNA was used as a template for polymerase chain reaction (PCR) amplification using universal primers. The forward primer was 27F (5'-TGAGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (5'-TACGGCTACCTTGTTACGAC-3'), both synthesized by Shanghai Sangon Biotech Co., Ltd. The PCR amplification system consisted of 25 μL of 2X SanTaq PCR Mix (containing blue dye), 2 μL of DNA template, 2 μL of forward primer 27F, 2 μL of reverse primer 1492R, and sterilized ddH2O to a final volume of 50 μL. After thorough mixing and brief centrifugation, the reaction tubes were placed in a PCR instrument for the reaction. The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by PCR cycling: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min. After amplification, the PCR tubes were stored at 4℃. A small amount of the PCR amplification product was then subjected to electrophoresis on a 1% agarose gel to observe the amplification results. The amplification product was then sent for sequencing to Shanghai Sangon Biotech Co., Ltd. After sequencing, the sequencing results were aligned to the BLAST sequence on NCBI, and a gene phylogenetic tree was constructed using MEGA11.0 with the nearest neighbor-joining method.

[0061] The 16S rRNA gene sequencing results are as follows (SEQ ID NO.1):

[0062]

[0063] See the phylogenetic tree constructed based on the 16S rRNA gene sequence. Figure 5 ,according to Figure 5 The results show that RS7 is a strain of Lactobacillus fermentans, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 15, 2024, with accession number GDMCC NO: 64983.

[0064] Example 2 Characterization of the properties of fermented Lactobacillus mucinus RS7

[0065] (1) Acid resistance of fermenting Lactobacillus mucinus RS7

[0066] The acid resistance of RS7, which exhibits high lead adsorption capacity after secondary screening, was determined, and the results are as follows: Figure 4 As shown in the figure, RS7 can grow normally between pH 4 and 7. At pH less than 3, both strains can hardly grow normally; therefore, lactic acid bacteria cannot grow normally in a low-acid environment (pH < 3).

[0067] The enhanced tolerance of lactic acid bacteria may be achieved through the production of ammonia and arginine metabolism to maintain a suitable intracellular pH. Arginine decarboxylase and urease pathways are two relatively common pathways for NH3 production in Gram-positive bacteria. + In a low-acid environment, lactic acid bacteria mainly produce ammonia through the arginine decarboxylase pathway (A-DI), thereby increasing the acidity inside the cell and maintaining the pH balance inside the cell.

[0068] (2) Bile salt tolerance of fermenting Lactobacillus mucinus RS7

[0069] Table 1. Bile salt tolerance of the screened strains

[0070]

[0071] The bile salt tolerance of RS7, which showed high lead adsorption capacity after secondary screening, was determined, as shown in Table 1. The growth time of strain RS7 in MRS was 1.13 h, while the growth time in MRS with 0.3% bile salts was 1.67 h, which was 0.54 h slower than in MRS, indicating that RS7 has a certain tolerance to bile salts.

[0072] (3) Maximum lead tolerance of Lactobacillus fermentum RS7

[0073] The *Lactobacillus fermentans* RS7 strain screened in Example 1 was inoculated into 20 mL of MRS liquid medium at an inoculum size of 1% and cultured for 18 h. The absorbance was adjusted to 1 (OD). 600Then, at an inoculation rate of 1% (v / v), the bacteria were inoculated into MRS liquid medium containing lead acetate at final concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L, respectively. After incubation at 37°C for 72 h, the results were observed to determine the maximum lead concentration that *Lactobacillus fermentum* RS7 could tolerate. When the lead concentration exceeded 250 mg / L (Table 2), the survival rate of *Lactobacillus fermentum* RS7 was 0; within the lead concentration range of 10–100 mg / mL, the survival rate of *Lactobacillus fermentum* RS7 was above 46%.

[0074] Table 2 Survival rate of Lactobacillus fermentans RS7 under different lead concentrations

[0075]

[0076] (4) Adsorption of lead ions by fermented Lactobacillus mucinus RS7

[0077] The lactic acid bacteria screened in Example 1 were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. Then, 1% of the culture was inoculated into MRS liquid medium containing 100 mg / ml lead acetate (initial inoculation concentration was 10). 6 The lactic acid bacteria fermentation broth (CFU / mL) was incubated in a shaker at 37℃ for 48 h. The broth was then centrifuged at 8000 rpm for 20 min, and the centrifugation was repeated three times. The supernatant was collected and the lead ion concentration was determined using a flame atomic absorption spectrophotometer. The precipitate was collected for subsequent electron microscopy.

[0078] The results showed that the fermentation rate of Lactobacillus mucinus RS7 for lead adsorption from 100 mg / L lead acetate solution was 74.17%. Figure 3 This adsorption effect is far superior to that of Lactobacillus rhamnosus IBRC-M 10782, which has been reported in the literature. Furthermore, we increased the lead acetate solution concentration to 150 mg / L and measured the adsorption rate of Lactobacillus fermentum RS7. The results showed that the adsorption rate could still reach 42% at this concentration, which is also higher than the adsorption rate of Lactobacillus fermentum HNU312 for lead at 150 mg / L (27.8%), demonstrating the superiority of Lactobacillus fermentum RS7 in lead adsorption.

[0079] Furthermore, the adsorption effect of *Lactobacillus fermentum* RS7 under different conditions was investigated. Subsequently, the lead acetate concentration was reduced to 50 mg / L, and the lead adsorption rate was examined after 24 h and 48 h. The results showed that the lead adsorption rate of *Lactobacillus fermentum* RS7 reached 89% after 24 h and 95% after 48 h, indicating that the adsorption effect of *Lactobacillus fermentum* RS7 on lead was better when the lead concentration decreased.

[0080] (5) Mechanism of lead degradation by fermenting Lactobacillus mucinus RS7

[0081] Heavy metal Pb 2+ Both treated (bacterial cells collected after lead adsorption in step 4) and untreated bacterial cells were collected after centrifugation at 5000×g for 10 min. The cells were then fixed in 2.5% glutaraldehyde overnight at 4°C. After fixation, the cells were rinsed three times with 0.1 mol / L PBS solution (pH 7.2) for 10 min each time. The cells were then dehydrated with a gradient of ethanol (50%, 70%, 80%, 90%, 95%, 100%) for 15 min each time. Excess ethanol was replaced with isoamyl acetate, and the cells were then frozen at -80°C for 24 h. After drying using a freeze dryer, the cells were observed under a scanning electron microscope.

[0082] Figure 6 The images show scanning electron micrographs of the bacterial strain before and after lead adsorption. (a) shows the strain before lead adsorption, where the bacteria are rod-shaped, exhibiting good cell growth, a smooth and intact cell surface, and clear outlines. (b) shows the bacterial cells after lead adsorption, where the cells are wrinkled and deformed, some becoming elongated, and particulate matter adsorbed onto the cell surface. This may be related to lead toxicity; heavy metals alter the hydrophobicity and charge of the cell surface, thereby changing the cell structure. Simultaneously, when subjected to heavy metal stress, the strain detoxifies through ion exchange and surface deposition of particulate matter, resulting in surface adsorption. Therefore, extracellular precipitation may be one of the important pathways for tolerant strains to adsorb heavy metals.

[0083] Example 3: Application of lead-adsorbing lactic acid bacteria on lead-containing preserved eggs

[0084] Lead-containing preserved eggs (containing lead oxide, 20 mg / kg) were deshelled. The preserved eggs and MRS broth were added to a juicer at a ratio of 1:2 (m / v, g / mL). The juice was extracted and centrifuged at 10000×g for 20 min. The supernatant was transferred to a 50 mL Erlenmeyer flask and sterilized at 121℃ for 15 min to obtain preserved egg MRS mixed culture medium. Lactobacillus mucilaginosus RS7 was fermented in a clean bench at a concentration of 10... 6The lead removal rate was compared between four groups at 0h, 24h, 48h, and 72h. The inoculum was inoculated into the MRS mixed medium of preserved eggs at a concentration of CFU / mL and cultured in a shaker at 37℃ and 120r / min.

[0085] The results are as follows Figure 7 As shown, strain RS7 achieved a lead removal rate of 28.68% from preserved eggs at 24 hours, and a removal rate of 58.88% at 48 hours, more than double the removal rate at 24 hours, indicating that RS7 has a good lead removal ability from preserved eggs. At 72 hours, the lead removal rate reached 63.51%, almost the same as the removal rate at 48 hours, indicating that the adsorption capacity of strain RS7 for lead in preserved eggs had approached its limit at this time, and also indicating that the growth of the strain had reached the stationary phase.

[0086] Example 4: Application of lead-adsorbing lactic acid bacteria in lead-containing rice

[0087] Rice (lead acetate, lead nitrate, 10 mg / kg) was ground into rice flour using a universal grinder. The rice flour and MRS broth were mixed in a 1:2 ratio and sterilized at 121°C for 15 minutes to obtain rice-MRS medium. Lactobacillus mucilaginosus RS7 was then fermented at 10... 6 Inoculation with CFU / mL was performed on sterilized rice-MRS medium, with rice-MRS medium without the target strain serving as a blank control. The culture was carried out at 37℃ for 72 h, with three replicates. Under aseptic conditions, 1 mL of fermentation broth was collected at 0 h, 24 h, 48 h, and 72 h, and the lead concentration was measured using a flame atomic absorption spectrophotometer to calculate the lead removal rate. The results showed that strain RS7 achieved a lead removal rate of 31% from rice at 24 h and reached 45% at 48 h, indicating that fermented *Lactobacillus mucilaginosus* RS7 also exhibits good lead adsorption capacity for rice.

[0088] Example 5: Application of lead-adsorbing lactic acid bacteria in lead-containing wastewater

[0089] Fermented Lactobacillus mucinus RS7 at 10 6 An inoculum of CFU / mL was inoculated into wastewater containing lead (lead nitrate, 10-30 mg / L). Under aseptic conditions, 1 mL of liquid was collected at 0h, 24h, 48h, and 72h, and the lead concentration was measured using a flame atomic absorption spectrophotometer to calculate the lead removal rate. The results showed that strain RS7 achieved a lead removal rate of 40% from the wastewater at 24h and reached 65% at 48h, indicating that *Lactobacillus fermentans* RS7 also has a good adsorption capacity for lead in wastewater.

[0090] Example 6: Application of lead-adsorbing lactic acid bacteria in lead-containing fish

[0091] The fish was minced into fish paste using a crusher (lead acetate, 2-4 mg / kg). The fish paste and sterile water were mixed in a 1:2 ratio and sterilized at 121°C for 15 minutes. Lactobacillus mucinus RS7 was then fermented at 10... 6 An inoculum of CFU / mL was inoculated into sterilized fish surimi liquid, with fish surimi liquid of the same treatment but without the target strain serving as a blank control. The mixture was incubated at 37℃ for 72 h, with three replicates. Under aseptic conditions, 1 mL of fermentation broth was collected at 0 h, 24 h, 48 h, and 72 h, and the lead concentration was measured using a flame atomic absorption spectrophotometer to calculate the lead removal rate. The results showed that the fermentation of *Lactobacillus mucinus* RS7 removed 40% of the lead from the fish surimi at 24 h and reached 65% at 48 h, indicating that *Lactobacillus mucinus* RS7 also has good lead adsorption capacity for lead-contaminated fish.

[0092] Example 7: Application of lead-adsorbing lactic acid bacteria in lead-contaminated soil

[0093] Soil contaminated with lead (containing lead acetate and lead nitrate, with a total content of 10-30 mg / kg) was dissolved in sterile water at a certain ratio (1:10, m / v). Then, certain nutrients such as carbon source (10% w / w glucose) and nitrogen source (5% w / w peptone) were added. Finally, *Lactobacillus mucilaginosus* RS7 was fermented at 10... 6 An inoculum of CFU / mL was inoculated into lead-containing soil solution. Lead-containing soil without the target strain was used as a blank control. Under aseptic conditions, 1 mL of the liquid was collected at 0h, 24h, 48h, and 72h, and the lead concentration was measured using a flame atomic absorption spectrophotometer to calculate the lead removal rate. The results showed that *Lactobacillus fermentum* RS7 achieved a lead removal rate of 35% at 24h and 55% at 48h, indicating that *Lactobacillus fermentum* RS7 also has a good adsorption capacity for lead in soil.

[0094] Example 8: Preparation of fermented Lactobacillus mucinus RS7 microbial preparation

[0095] The preparation method of fermented Lactobacillus mucinus RS7 microbial preparation (liquid) involves activating and culturing the preserved RS7 strain on MRS solid medium at 37°C for 24-48 hours, then inoculating it into an Erlenmeyer flask containing MRS culture medium and culturing it at 37°C for another 24-48 hours. This flask is then used for inoculation of a seed tank, which is also inoculated with sterilized MRS culture medium and cultured at 37°C for 72 hours. The final viable count reaches 102. 9 CFU / mL is sufficient.

[0096] A method for preparing a solid microbial preparation of *Lactobacillus mucinus* RS7 is as follows: The preserved RS7 strain is activated and cultured on MRS solid medium at 37°C for 24-48 hours. Then, it is inoculated into an Erlenmeyer flask containing MRS culture medium and cultured at 37°C for 24-48 hours. This inoculation is then used for seed tank inoculation. The seed tank is also inoculated with sterilized MRS culture medium and cultured at 37°C for 72 hours. After centrifugation, the supernatant is discarded to obtain bacterial cells. Skim milk (10%, m / v) is used as a protectant and mixed with the bacterial cells at a 1:1 (v / m) ratio. The mixture is then freeze-dried for 48-72 hours. The final solid microbial preparation has a viable count of not less than 10⁻⁶. 9 CFU / g.

[0097] Example 9: Preparation of lead adsorbent

[0098] The microbial preparation containing a certain amount of microorganisms from Example 8 was mixed with a powder prepared from a fibrous matrix such as pineapple peel and sugarcane bagasse at a certain ratio (1:1). Then, prebiotics (1% inulin, 1% sodium alginate, and 1% chitosan) were added. After mixing, the mixture was stored at 4°C. The prepared microbial agent contained no less than 10 viable bacteria. 7 CFU / g or CFU / mL.

[0099] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum RS7, the fermenting Lactobacillus mucinus RS7, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 15, 2024, with accession number GDMCC NO: 64983.

2. A microbial preparation containing the fermenting Lactobacillus RS7 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The number of Lactobacillus fumarate RS7 in the microbial preparation is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. A method for adsorbing lead, characterized in that, The fermentation of Lactobacillus mucinus RS7 as described in claim 1, or the microbial preparation as described in claim 2 or 3, is added to a lead-containing matrix for cultivation, characterized in that the culture is carried out at pH 3-7 for at least 24 hours.

5. The method as described in claim 4, characterized in that, The lead-containing matrix includes a solid matrix, a liquid matrix, or a semi-solid matrix; the type of lead includes lead acetate, lead nitrate, or lead oxide.

6. The use of the fermented *Lactobacillus mucinus* RS7 according to claim 1, or the microbial preparation according to claim 2 or 3, in the removal of lead from a matrix, characterized in that... The substrate may include food, soil, feed, or culture medium.

7. The use of the fermented Lactobacillus mucinus RS7 according to claim 1, or the microbial preparation according to claim 2 or 3, in the preparation of lead adsorbents.

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