CN114317621B

CN114317621B - Lactobacillus metabolite and preparation method and application thereof

Info

Publication number: CN114317621B
Application number: CN202111203049.3A
Authority: CN
Inventors: 许商成; 李媛媛; 彭小红; 刘永生; 陈承志; 段郁; 王维; 付冠艳; 余沁; 刘聪; 段维霞; 孙亚培
Original assignee: First Affiliated Hospital Of Chongqing Medical College Chongqing Occupational Disease Control Center Chongqing Sixth People's Hospital
Current assignee: First Affiliated Hospital Of Chongqing Medical College Chongqing Occupational Disease Control Center Chongqing Sixth People's Hospital
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2024-03-12
Anticipated expiration: 2041-10-15
Also published as: CN114317621A

Abstract

The invention relates to the technical field of microbial preparations, in particular to a lactobacillus metabolite and a preparation method and application thereof. The lactobacillus metabolites were secreted by lactobacillus plantarum GDM 1.140. Inoculating lactobacillus plantarum GDM1.140 into a broth culture medium, and standing and culturing for 16 hours at 36 ℃ to obtain a metabolite with the effects of chelating heavy metals and resisting heavy metal poisoning. The technical problems that microbial agents are easy to inactivate and the time required for exerting the efficacy is long when the microbial agents are used for relieving heavy metal harm can be solved. The lactobacillus metabolite contains a large amount of effective small molecular components, can be used as a heavy metal chelating agent in environmental treatment and can be used as an antidote in the practice of treating biological heavy metal poisoning.

Description

Lactobacillus metabolite and preparation method and application thereof

Technical Field

The invention relates to the technical field of microbial preparations, in particular to a lactobacillus metabolite and a preparation method and application thereof.

Background

Heavy metal pollution refers to environmental pollution caused by heavy metals or compounds thereof, and is mainly caused by mining, waste gas emission, sewage irrigation, use of heavy metal exceeding products and other artificial factors, and the heavy metal pollution can cause heavy metal poisoning of people. Heavy metal poisoning refers to poisoning caused by heavy metal elements or compounds thereof having a relative atomic mass of more than 65, such as mercury poisoning, lead poisoning, etc. The heavy metal can irreversibly change the structure of the protein, thereby affecting the functions of tissue cells and further affecting the health of human bodies. Frequent exposure to heavy metal contaminated working environments (occupational heavy metal exposure) can lead to acute and chronic poisoning of multiple organs such as skin, liver, kidney and lung, cancers and the like. The occupational heavy metal exposure involves a plurality of people, the low-concentration composite exposure is widely existed, and the harm of the heavy metal is not ignored.

Chinese patent CN110628677a discloses a lactobacillus casei SYF-08 for adsorbing heavy metal lead and application thereof, wherein the lactobacillus casei SYF 08 is separated from healthy neonatal feces, has the capability of resisting lead ions and strongly adsorbing lead ions, and can stably fix and adsorb lead ions in the intestinal tract. The strain SYF 08 bacterial liquid can be applied to the preparation of food additives, foods or medicines with the efficacy of preventing and/or assisting in treating chronic lead poisoning. However, microbial formulations suffer from a number of disadvantages: microorganisms generally need a certain time to adapt to the environment (such as the environment in a human body) so as to play a role in removing heavy metals, and have slow effectiveness and are required to be used for a long time; the preservation conditions are severe, and the microorganisms are not properly preserved by a microorganism preparation producer or seller, so that the microorganisms die, and the quality of the microorganism preparation is difficult to ensure. There is a need to develop a small molecule antidote that can rapidly alleviate the harm of heavy metal ions and has better stability to cope with heavy metal pollution and related occupational exposure problems.

Disclosure of Invention

The invention aims to provide a lactobacillus metabolite to solve the technical problems that microbial agents are easy to inactivate and the time required for efficacy to be exerted is long when the microbial agents are used for relieving heavy metal harm.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a lactobacillus metabolite produced by the secretion of lactobacillus plantarum GDM 1.140.

The scheme also provides a preparation method of the lactobacillus metabolite, which is characterized in that: inoculating lactobacillus plantarum GDM1.140 into a broth culture medium, and standing and culturing at the temperature of 32-36 ℃ for 16-24 hours to obtain a culture system containing metabolites.

The scheme also provides application of the lactobacillus metabolite in chelating heavy metals.

The scheme also provides application of the lactobacillus metabolite in preparing the antidote for heavy metal poisoning.

The principle and the advantages of the scheme are as follows: the metabolite of lactobacillus plantarum GDM1.140 (ATCC 8014) has a large number of chemical chelating groups, and can chelate and precipitate heavy metal ions, thereby reducing heavy metal ion pollution and relieving the harm of the heavy metal ions to organisms. Experiments prove that the supernatant liquid of the scheme can not only directly chelate heavy metals in a liquid environment, but also act on cells and organisms. In vitro experiments show that the supernatant can relieve intestinal epithelial cell injury, restore the mitochondrial membrane potential of epithelial cells, reduce active oxygen generated by lead exposure and reduce the intracellular lead level. In vivo experiments show that after the mice are orally administrated with supernatant, the lead content in the blood of the mice and the damage of the ileum epithelial cells of the mice are obviously reduced. Therefore, the supernatant of the present protocol (i.e., the metabolite of lactobacillus plantarum GDM 1.140) can be used as a heavy metal chelator in environmental management, as well as an antidote in the practice of treatment of biological heavy metal poisoning.

In the technical scheme, the lactobacillus plantarum GDM1.140 can generate the metabolite with the effects of chelating heavy metals and detoxification only by being cultured in a common MRS broth culture medium, is very simple to operate and is suitable for industrial production. The inventor performs metabonomic analysis on the components of the supernatant, and finds that one part of the components contains a relatively large amount of compounds with the effect of chelating heavy metals; the other part contains substances including indole-3-lactic acid with effect of reducing inflammation of intestinal epithelial cells. Differential metabolites are enriched in organic acids and their derivatives (39%) and organic heterocyclic compounds (18%) etc. Compared with the common culture medium, the supernatant obtained by the scheme has good effect of chelating heavy metals.

The prior art reports on microorganisms with heavy metal pollution control and resistance to heavy metal toxicity have focused mainly on the microorganisms themselves and on the extracellular polysaccharides of the microorganisms. The microbial agent is easy to inactivate and preserve, and the microbial agent has slower action. While extracellular polysaccharides are part of the bacterial cell wall, or they surround the cell wall (like capsular polysaccharides), mainly composed of macromolecular substances. The process of extracting bacterial extracellular polysaccharide is complicated, and the application range is limited due to the large molecular weight. The supernatant of the scheme can be obtained by only culturing bacteria, and a large number of small molecule functional components contained in the supernatant can rapidly play a role.

Further, the lactobacillus plantarum GDM1.140 was cultured in MRS broth. Lactobacillus plantarum GDM1.140 is metabolized in MRS broth to yield a substance with chelated heavy metal components.

Further, one of the metabolites of lactobacillus includes penicillamine, 2-hydroxy-4-methylthiobutanoic acid and citric acid. The supernatant obtained by the scheme contains the three metabolites with the effect of chelating heavy metals.

Further, the culture system containing the metabolite is centrifuged, and then the supernatant is obtained by filtration. The supernatant fraction free of cells was obtained by centrifugation and filtration.

Further, the rotational speed of the centrifugal treatment is 5000g-10000g. The above centrifugation speed can be used to sufficiently separate the functional components from the bacterial cells in the supernatant.

Further, the filter membrane pore size of the filtration treatment was 0.22. Mu.m. The adoption of the filtering pore diameter can fully separate the functional components from the thalli in the supernatant

Further, the heavy metal is lead. Lead is an important component in heavy metal pollution and occupational exposure, and experiments prove that the supernatant of the scheme has good chelating and sedimentation effects on lead ions.

Drawings

FIG. 1 is a gram-stain microscopic image of Lactobacillus of example 1 after 16h of incubation.

FIG. 2 is a gram-stain microscopic image of Lactobacillus cultured for 24 hours in example 1.

FIG. 3 is a LC-MS quality control chart (A) of experimental example 1.

Fig. 4 is an LC-MS quality control chart (B) of experimental example 1.

Fig. 5 is an LC-MS quality control chart (C) of experimental example 1.

FIG. 6 is a LC-MS quality control chart (D) of experimental example 1.

FIG. 7 is a LC-MS quality control chart (F) of experimental example 1.

FIG. 8 is a LC-MS quality control chart (G) of experimental example 1.

FIG. 9 is a volcanic chart of Experimental example 1.

Fig. 10 is a cluster map (a) of experimental example 1.

Fig. 11 is a cluster map (B) of experimental example 1.

Fig. 12 is a cluster map (C) of experimental example 1.

Fig. 13 is a cluster map (D) of experimental example 1.

Fig. 14 is a cluster map (E) of experimental example 1.

FIG. 15 is a pie chart of up-regulated differential metabolite classification of Experimental example 1.

FIG. 16 is a graph of experimental results of supernatant fluid in experimental example 2 for relieving intestinal epithelial cell damage (representative of significant differences in lead damage compared to Control; representative of significant protectiveness in S1:20+Pb group compared to Pb group; p <0.05, statistical significance).

Fig. 17 is a graph of experimental results of the supernatant recovery of the mitochondrial membrane potential of epithelial cells of experimental example 2 (representative of significant differences in lead damage compared to Control; representative of significant protectiveness of s1:20+pb group compared to Pb group; p <0.05, statistical significance).

FIG. 18 is a graph of the results of experiments in which the supernatant of Experimental example 2 reduced active oxygen due to lead exposure (representative of significant differences in lead damage compared to Control; representative of significant protectiveness in S1:20+Pb group compared to Pb group; p <0.05, statistical significance; rosup is a positive Control in the kit).

FIG. 19 is a fluorescent chromogenic image of the supernatant of Experimental example 2 for reducing active oxygen generated by lead exposure.

FIG. 20 is a graph showing the results of examining the effect of lead acetate on intracellular lead levels in Experimental example 2.

FIG. 21 is a graph showing the results of examining the effect of lead acetate on intracellular lead levels in Experimental example 2.

FIG. 22 shows the results of the lead content test in the blood of the mice in Experimental example 3.

FIG. 23 shows the results of the distal ileum HE staining experiments in the mice of Experimental example 3.

FIG. 24 is an image before chelate sedimentation in Experimental example 4.

FIG. 25 is an image after chelate sedimentation in Experimental example 4.

FIG. 26 shows the results of atomic absorption spectrophotometry in a graphite furnace of Experimental example 4.

FIG. 27 shows the results of IR spectrum scan identification of the liquid phase after centrifugation in Experimental example 4.

FIG. 28 shows the results of IR spectrum scan identification of the solid phase after centrifugation in Experimental example 4.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated; the experimental methods used are all conventional methods; the materials used, etc., are all commercially available.

Example 1: acquisition of metabolites

Lactobacillus plantarum GDM1.140 (ATCC 8014) was purchased from the Guangdong province microorganism strain collection. Lactobacillus plantarum is preserved in glycerol and in a refrigerator at-80 ℃. Conventional activation and purification of glycerol bacteria is required before culturing lactobacillus plantarum and obtaining metabolites. The glycerol bacteria were removed and inoculated to the MRS medium at an inoculum size of 2% to activate the strain. After 16h of activation, the Lactobacillus plantarum was subjected to gram-color detection, and positive strains were picked up and streaked onto plates (MRS solid medium) for strain purification. After 24h streaking the plate, a single colony was grown on the plate and picked up into fresh broth (typically 1 single colony was inoculated in 50ml fresh MRS broth) at a temperature of 32-36℃C (optimal temperature 36 ℃)Static culture is carried out for 16h (optional range is 16-24 h) under the condition. Generally, bacterial count is performed after a period of time has elapsed since the cultivation, and it is generally necessary to ensure the number of cells in the bacterial liquid after the cultivation>10 ⁹ And/ml, so that the supernatant can be ensured to be fermented by enough thalli, and the function of better chelating heavy metals can be exerted. And centrifuging the whole culture system for 5min with 5000g (optionally ranging from 5000-10000 g), filtering with a 0.22 μm pore-size filter membrane to obtain supernatant containing a large amount of components with heavy metal chelating effect. In this embodiment, the broth medium (MRS medium) is a conventional medium of the prior art, which is commercially available directly, and the components thereof will not be described in detail.

In the process of obtaining the supernatant, the growth time of lactobacillus plantarum is controlled, so that most of lactobacillus plantarum is gram-positive bacillus, the most of lactobacillus plantarum under the lens (gram-stained) represents the best bacterial growth state, the metabolic products are stable (shown in figure 1), the bacterial liquid at the moment is centrifugally filtered for subsequent experiments, otherwise, the phenomenon of autolysis is easy to occur when the growth time of lactobacillus plantarum is too long (after 24 hours) (shown in figure 2), and most of lactobacillus plantarum is observed under the lens. And too short a growth time does not easily produce non-rich metabolites.

Experimental example 1: metabonomics identification of Lactobacillus plantarum metabolites

The supernatants obtained in example 1 were subjected to LC-MS (liquid chromatography-mass spectrometry) based metabonomics studies (n=6), which were carried out by qualified biochemical companies, using fresh broth medium (MRS medium) without lactobacillus culture as blank (n=6), with the following experimental results:

the LC-MS quality control chart is shown in figures 3-8, and the result of the separation detection process is credible. There were 225 up-regulated and 84 down-regulated differential metabolites compared to the supernatant and the blank (broth). Volcanic diagrams showing differential metabolites are seen in fig. 9, clustered heat maps are seen in fig. 10-14 (clustered heat maps are split into fig. 10-14 in sequence due to their oversized image).

The up-regulated differential metabolites were classified according to the human metabolic database, and as a result, referring to fig. 15, it was seen that the up-regulated differential metabolites were enriched in organic acids and their derivatives (39%), organic heterocyclic compounds (18%), and the like. More specifically, in fig. 15: organoheterocyclic compounds (organic heterocyclic compound) accounts for 18%; homogeneous non-metallic compounds 1%; organic acids and derivatives (organic acid and derivative thereof) is 39%; organic oxygen compounds (organic oxygen compound) accounts for 6%; benzenoides (Benzenoids) account for 14%; nucleic acids, nucleotides, and analogs account for 6%; lipids and lipid-like molecules account for 9%; alkaloids and derivatives (alkaloid and its derivatives) account for 2%; phenylpropanoids and polyketides (phenylpropane and polyketone) accounts for 2%; organic nitrogen compounds (organic nitrogen compound) accounts for 4%.

Through metabonomics analysis, up-regulated differential metabolic species have a relatively large amount of penicillamine, 2-hydroxy-4-methylthiobutanoic acid, and citric acid. The chemical formulas of the three substances are formula (1), formula (2) and formula (3) in sequence, and all the three substances have the effect of chelating heavy metals. See the following literature reports: penicillamine revisited: historic overview and review of the clinical uses and cutaneous adverse effects, R Ishak, O Abbas, american Journal of Clinical Dermatology, 2013 (DOI: 10.1007/s 40257-013-0022-z); poly (methacrylate citric acid) with good biosafety asnanoadsorbents of heavy metal ions, X Zhang, X Wang, H Qia, D Kong, Y Guo, colloids and surfaces B: biointerfaces,2019 (DOI: 10.1016/j. Colsurfb.2019.110656); metal chelates of2-hydroxy-4-methylthiobutanoic acid in animal feeding: preliminary investigations on stability and bioavailabilities, G predicteri, M Tegoni, E Cinti, G Leonardi, S Ferruzza, journal of Inorganic Biochemistry,2005 (DOI: 10.1016/S0162-0134 (03) 00067-9)

In addition, in the metabonomics analysis, it has been found that the supernatant contains a large amount of indole-3-lactic acid and the like, which have the function of reducing inflammation of intestinal epithelial cells.

The materials with the effects of chelating heavy metal ions, relieving verification and the like, which are found by metabonomics research, are listed in the table 1. The metabonomics study adopts the LC-MS means, and the retention time and the characteristic peak area of characteristic peaks of the functional substances in LC-MS detection are counted in table 1, so that the change condition of the substance content of supernatant fluid in a fresh culture medium after lactobacillus culture is judged. In Table 1, the peak area ratio is the average value of the characteristic peak areas of the specific substances of the 6 groups of supernatants divided by the average value of the characteristic peak areas of the specific substances of the 6 groups of controls, and reflects the change in the content of the substances before and after the culture of Lactobacillus. The p values in table 1 are t-tests performed on the characteristic peak areas of the specific substances of the 6 groups of supernatants and the characteristic peak areas of the specific substances of the 6 groups of blank controls, p < 0.005 indicating a significant difference; VIP > 1, the differential metabolite is considered significant and the numerical value is trusted. As can be seen from the data in Table 1, after the culture of Lactobacillus in broth, the contents of penicillamine, citric acid, 2-hydroxy-4-methylthiobutanoic acid and indole-3-lactic acid in the supernatant obtained by separation were significantly increased, and these substances were all substances having heavy metal chelating or anti-inflammatory effects.

Table 1: results of non-targeted metabonomics study of part of the efficacy substances

Experimental example 2: in vitro experiments

(1) Experiment for relieving intestinal epithelial cell injury by supernatant

The experiment uses HT-29 (a human colon cancer cell line, commonly used in vitro experiments in the intestinal tract) for the CCK-8 test. The supernatant was added to the cells 1h before the addition of lead ions to replace the cell culture medium,after incubation of the supernatant for 1h, lead ion treatment was performed. The concentration of lead ions was 2mM (lead concentration based on half lethal dose IC ₅₀ Determination) for 24 hours, CCK-8 was detected, and CCK-8 was detected according to the reagent instructions.

The results of the experiment are shown in FIG. 16, in order from left to right, a blank control (cells were not treated with pb), a positive control (cells were treated with pb but no supernatant was used), an experimental group 1 (S1:100+Pb, cells were treated with pb and 100-fold diluted supernatant was used), an experimental group 2 (S1:50+Pb, cells were treated with pb and 50-fold diluted supernatant was used), an experimental group 3 (S1:20+Pb, cells were treated with pb and 20-fold diluted supernatant was used). From the experimental results, the supernatant of the scheme is used for treating the intestinal epithelial cells under the lead stress, so that the damage of the lead stress can be relieved, and the cell survival rate can be increased.

(2) Experiment for recovering mitochondrial membrane potential of epithelial cells from supernatant

The supernatant and lead ions were treated in the same manner as in (1), and the mitochondrial membrane potential was measured by TMRM (tetramethyl rhodamine methyl ester, MCE HY-D0984) method, and the fluorescence intensity was decreased as the mitochondrial membrane potential was decreased.

The experimental results are shown in FIG. 17, in order from left to right, a blank control (cells were not treated with pb), a positive control (cells were treated with pb but no supernatant was used), experimental group 1 (S1:100+Pb, cells were treated with pb and 100-fold diluted supernatant was used), experimental group 2 (S1:50+Pb, cells were treated with pb and 50-fold diluted supernatant was used), experimental group 3 (S1:20+Pb, cells were treated with pb and 20-fold diluted supernatant was used). From the experimental results, the supernatant of the scheme is used for treating the intestinal epithelial cells under lead stress, so that the mitochondrial membrane potential of the epithelial cells can be recovered, and the mitochondrial damage can be relieved.

(3) Experiments of supernatant reduction of active oxygen due to lead exposure

The supernatant and lead ions were treated in the same manner as in (1), and the active oxygen was measured using a kit (cat No. S0033S) from Biyun Tian Biotechnology company as indicated in the specification.

The experimental results are shown in fig. 18 and 19. FIG. 18 shows, in order from left to right, a blank control (cells were not treated with pb), a positive control (cells were treated with pb but supernatant was not used), an experimental group 1 (S1:100+Pb, cells were treated with pb and supernatant diluted 100-fold), an experimental group 2 (S1:50+Pb, cells were treated with pb and supernatant diluted 50-fold), an experimental group 3 (S1:20+Pb, cells were treated with pb and supernatant diluted 20-fold). FIG. 19 is a fluorescent color image. From the experimental results, it is known that the supernatant of the present protocol can be used to treat intestinal epithelial cells under lead stress, so that the production of active oxygen in the cells can be significantly reduced.

(4) Determination of intracellular lead levels

The supernatant and lead ions were treated in the same manner as (1), and intracellular lead ions were measured using a Leadmium green probe (accession number Thermo Fisher Scientific A10024) and using an enzyme-labeled instrument and laser confocal detection.

As shown in fig. 20 and 21, it was found that the content of lead ions in the cells increased with increasing concentration of lead acetate, and that the content of lead ions in the cells could be reduced to a level lower than that of the blank control by treating the cells with the supernatant diluted 20 times.

Experimental example 3: animal experiment

A model of lead poisoning (subacute lead poisoning model, feeding time was two weeks) was constructed by adding 200mg/L of lead acetate to daily drinking water of mice using C57BL/6 mice as experimental animals. During two weeks of feeding mice with drinking water, 0.3ml of lactobacillus supernatant (required to ensure the number of cells in the cultured bacterial solution > 10) was infused regularly daily ⁹ Per ml, supernatant obtained as in example 1 for this bacterial solution), or equivalent lavage using control (MRS broth, water). The indices (behaviour of mice, blood lead content and ileal tissue damage) were then monitored. The detection result of the lead content in the blood of the mice is shown in figure 22, and the results are as follows from left to right: positive control (cells treated with Pb but without supernatant), experimental group 1 (s+pb, cells treated with Pb and with supernatant), experimental group 2 (mrs+pb, cells treated with Pb and with broth), blank control (without broth)Cells were treated with pb). From the experimental results, the supernatant treatment can significantly reduce the lead content in the blood of the mice.

The results of the experiments on the far-end ileum HE staining of mice are shown in FIG. 23, and it can be seen that the supernatant treatment can significantly reduce the damage of the ileum epithelial cells of the mice, but the pure use of broth culture medium has no effect of alleviating the damage.

Experimental example 4: chelation effect test

Lead acetate was added to the supernatant (using broth medium and water as controls) to give a lead ion concentration of 2mM, allowing the mixed solution to settle naturally, and starting at about 30min from the prepared solution, the supernatant group developed a white precipitate. The occurrence of precipitation was observed, and the experimental results are shown in fig. 24 and 25. Fig. 24 is an image before natural sedimentation, fig. 25 is an image after sedimentation, and fig. 24 and 25 are supernatant+lead, broth+lead, water+lead in this order from left to right. The experimental result shows that the supernatant and the lead group generate obvious precipitation, which indicates that the supernatant has chelation effect on lead ions, thereby further having the effect of removing the lead ions in the liquid environment.

The change in the lead ion concentration in fig. 24 and 25 was detected using a graphite oven atomic absorption spectrophotometer, and the supernatant adsorption rate (%) = [ (C) ₀ -C ₁ )/C ₀ ]×100％，C ₀ And C ₁ Representing the lead ion concentration in the initial solution and the adsorbed solution respectively), the experimental results are shown in fig. 26, which shows that the supernatant of the scheme has remarkable chelation effect on lead ions, thereby reducing the lead ion content in the liquid environment.

Centrifuging the supernatant and the lead group to obtain a centrifuged liquid phase and a centrifuged solid phase (precipitate), and respectively identifying by infrared spectrum scanning to confirm whether the supernatant and the lead group contain chelating groups. The experimental results are shown in FIGS. 27 and 28 (transmittance on the ordinate and wave number cm on the abscissa) ^-1 ) The supernatant+lead group contains a chelating group such as a carboxyl group, and FIG. 27 shows the results of infrared spectroscopic scanning identification of the liquid phase after centrifugation, and FIG. 28 shows the solid phase after centrifugation. Since the infrared spectrum scan sample is mostly solid, the sampleIn this experiment, the supernatant was liquid, and the natural precipitation of all the chelated lead ions could not be ensured, so the experimental tests of fig. 27 and 28 were performed.

The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. Use of a lactobacillus metabolite in the preparation of a heavy metal chelator or a heavy metal poisoning antidote, characterized in that the lactobacillus metabolite is prepared by the following method: inoculating lactobacillus plantarum GDMCC1.140 into a broth culture medium, and standing and culturing for 16 hours at the temperature of 36 ℃ to obtain a culture system containing metabolites; centrifuging 5000g of a culture system containing metabolites for 5min, and then filtering by a 0.22 mu m-pore filter membrane to obtain supernatant;

the metabolites of the lactobacillus include penicillamine, 2-hydroxy-4-methylthiobutanoic acid, citric acid and indole-3-lactic acid;

the heavy metal is lead.