Application of lactobacillus mucilaginosus B44 in preparation of blood lead reducing product or lead toxicity relieving product

Abstract

The invention relates to the technical field of food biology, in particular to application of lactobacillus mucus B44 to preparation of products for reducing blood lead or reducing lead toxicity. The preservation number of the lactobacillus mucus B44 in the invention is CGMCC (China General Microbiological Culture Collection Center) No.17321. Compared with other existing strains, the lactobacillus mucilaginosus B44 provided by the invention has better adsorption capacity on lead ions in water. After the bacterial powder prepared by the invention is taken, the content of lead in blood, livers and kidneys in lead poisoning model mice and rats can be effectively reduced, particularly, the bacterial powder has an obvious protection effect on the kidneys, and has an obvious protection effect on the livers and kidneys of the rats; by adopting the microbial inoculum or fermented food for fermenting the heavy metal lead poisoning in the lactobacillus mucus B44, the source and the variety of microorganisms for treating or preventing the heavy metal lead poisoning by adopting a micro-ecological microbial inoculum are widened, and the lactobacillus mucus B44 has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of food biotechnology, in particular to application of fermented mucus lactobacillus B44 in preparing a product for reducing blood lead or alleviating lead toxicity. Background Art

[0002] Lead (Pb) pollution is a common heavy metal pollution. Heavy metal pollution is different from organic pollutants. It is difficult to degrade, volatilize and decompose. It is more likely to cause serious harm to human health through food chain enrichment. Especially for children, lead is more sensitive to children's growth and development, and can cause children's hyperactivity, attention deficit, and developmental delay. At present, the methods for reducing heavy metal lead in the body include traditional lead chelating agents (such as dithiothreitol, sodium calcium edetate, etc.) and nutritional intervention. However, due to the side effects and safety issues of chelation therapy, it is not recommended as a medication method for children, the elderly and other groups. Therefore, it is urgent to find a safe and effective alternative intervention therapy to alleviate the risk level of high blood lead in people with high exposure to heavy metal lead in my country.

[0003] Lead exposure is mainly through oral ingestion and nasal inhalation. Lead particles (diameter > 2.5 μm) deposited in the nasopharynx and bronchial ciliary areas will enter the gastrointestinal tract through mucociliary action and then be absorbed by the intestine. In addition, it has been reported that some heavy metals in the blood will return to the intestinal cavity through the bile through the enterohepatic recirculation process, and then be quickly absorbed by intestinal epithelial cells and return to body fluids and the liver. This shows that the intestine is the target of lead exposure, and blocking lead absorption in the intestine helps reduce the toxic effects of lead on human health.

[0004] Replacing traditional chelation therapy with safe probiotics has gradually become a research hotspot because of its safety, lack of side effects, and strong ability to bind heavy metals. At present, most researchers believe that lactic acid bacteria have the ability to adsorb and remove heavy metals because their bacterial surface is rich in active groups such as carboxyl and amino groups, and lactic acid bacteria can inhibit the toxic effects of heavy metals such as oxidative damage, dysbacteriosis, and intestinal barrier damage in the intestine. However, most studies use lactic acid bacteria that do not produce exopolysaccharides, such as Lactobacillus plantarum and Lactobacillus rhamnosus, which do not produce exopolysaccharides. For lactic acid bacteria that produce exopolysaccharides, such as Limosilactobacillus fermentum, there are few studies on biological emission reduction and in vivo toxicity reduction. The extracellular polysaccharides produced by the bacteria are rich in negatively charged groups such as hydroxyl and carboxyl groups, have good ability to adsorb or chelate multivalent metal ions, and have the ability to alleviate the toxicity of heavy metal lead in the body. However, the research on the effective use of lactic acid bacteria producing extracellular polysaccharides to reduce lead emissions in the body is still blank, and there are few available strains. This is a key problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of fermented Lactobacillus mucilaginosus B44 in the preparation of a product for reducing blood lead or reducing lead toxicity, so as to solve the problems in the prior art.

[0006] To achieve the above purpose and other related purposes, the present invention provides the use of fermented Lactobacillus mucilaginosus B44 in preparing a product for adsorbing heavy metal ions. The deposit number of the fermented Lactobacillus mucilaginosus B44 is CGMCC No.17321.

[0007] Preferably, the heavy metal ion adsorption product is a lead toxicity reduction product; the lead toxicity reduction product has any one or more of the following functions:

[0008] 1) Reduce blood lead concentration;

[0009] 2) Reduce lead concentration in the liver;

[0010] 3) Reduce lead concentration in the kidneys.

[0011] The present invention also provides a method for preparing a bacterial agent for adsorbing heavy metal lead, the preparation method comprising the following steps:

[0012] 1) inoculating the fermented mucus lactobacillus B44 in the aforementioned use into MRS medium for fermentation and cultivation;

[0013] 2) Separating and collecting the bacterial cells cultured in step 1), mixing them with a bacterial cell protective agent and then drying them to obtain a bacterial agent that adsorbs heavy metal lead.

[0014] The present invention also provides a bacterial agent for adsorbing heavy metal lead obtained by the above-mentioned preparation method.

[0015] As described above, the use of the fermented Lactobacillus mucilaginosus B44 of the present invention in the preparation of a product for reducing blood lead or a product for reducing lead toxicity has the following beneficial effects:

[0016] 1) After incubation in a lead-containing culture medium in vitro, it has good tolerance to lead ions, and the tolerance to lead ions is ≥300 mg / L;

[0017] 2) Compared with the control strain, after incubation in an aqueous solution containing lead in vitro, it has a better adsorption effect on lead ions, with an adsorption capacity of ≥1.58 mg Pb / g dry bacterial weight;

[0018] 3) Compared with the lead poisoning model mice, after taking the bacterial powder of the present invention, the blood lead concentration of the lead poisoning mice was reduced by at least 27.6%, the lead content in the liver was reduced by at least 26.5%, and the lead content in the kidney was reduced by at least 43.4%;

[0019] 4) Compared with the lead poisoning model rats, after taking the bacterial powder of the present invention, the blood lead concentration of the lead poisoning rats was reduced by at least 49.6%, and the lead content in the liver and kidney was reduced by 44.25% and 24.4% respectively compared with the model group. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It shows the growth curve of the lead tolerance of the fermented Lactobacillus mucilaginosus B44 of the present invention.

[0021] Figure 2 Shown are transmission electron microscope photos of the fermented mucus Lactobacillus B44 bacteria of the present invention that adsorb lead, wherein (a) is the B44 bacteria, and (b) is the B44 bacteria after adsorbing lead.

[0022] Figure 3 Shown are energy spectrum scanning photos of the Lactobacillus mucilaginosus B44 bacteria adsorbing lead of the present invention, wherein (a) is the mapping diagram of the bacteria element energy spectrum before B44 adsorption, (b) is the scanning energy spectrum diagram before B44 adsorption, (c) is the mapping diagram of the bacteria element energy spectrum after B44 adsorbs lead, and (d) is the scanning energy spectrum diagram after B44 adsorbs lead. DETAILED DESCRIPTION

[0023] The invention provides use of Limosilactobacillus fermentum B44 in preparing a product for adsorbing heavy metal ions.

[0024] The fermented mucus lactobacillus B44 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration CGMCC, with a deposit number of CGMCC No.17321, and a deposit address of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0025] In some specific embodiments, the adsorbed heavy metal ions are lead toxicity reduction products.

[0026] In some embodiments, the lead toxicity reduction product has any one or more of the following functions:

[0027] 1) Reduce blood lead concentration;

[0028] 2) Reduce lead concentration in the liver;

[0029] 3) Reduce lead concentration in the kidneys.

[0030] In some specific embodiments, in the heavy metal ion adsorption product, based on the total mass of the heavy metal ion adsorption product, the content of the fermented mucus lactobacillus (Limosilactobacillus fermentum) B44 is 10 6 -10 11 CFU / g. More specifically, the content of the fermented mucus lactobacillus (Limosilactobacillus fermentum) B44 is 10 6 -10 7 CFU / g, 10 7 -10 8 CFU / g, 10 8 -10 9 CFU / g, 10 9 -10 10 CFU / g or 10 10 -10 11 CFU / g.

[0031] In some specific embodiments, the heavy metal ion adsorption product further includes a pharmaceutically acceptable carrier or medium.

[0032] Furthermore, the acceptable carriers and media are, for example, sterile water or physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc. Moreover, other low molecular weight polypeptides, proteins such as serum albumin, gelatin or immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars or carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol or sorbitol, etc. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (such as ethanol), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50), etc. can be used in combination.

[0033] As used herein, the dosage form of the composition is selected from: injection, tablet, pill, capsule, lozenge, elixir, powder, granule, syrup, solution, tincture, aerosol, powder spray, or suppository. Those skilled in the art can select a suitable preparation form according to the mode of administration. For example, the preparation form suitable for oral administration can include but is not limited to pills, tablets, chewables, capsules, granules, solutions, drops, syrups, aerosols or powder sprays, etc. For another example, the preparation form suitable for parenteral administration can include but is not limited to solutions, suspensions, reconstituted dry preparations or sprays, etc. For another example, the preparation form suitable for rectal administration can usually be a suppository.

[0034] Among them, tablets, lozenges, pills, capsules, etc. may also contain the following components: binders, such as gum, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin, or flavorings, such as mint, wintergreen oil or cherry flavorings, may be added. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the above substances. Various other substances may be present in the form of coatings or used to improve the physical form of the unit dosage form. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Syrups or elixirs may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, pigments and flavorings, such as cherry or orange flavorings. Any substance used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the dosage. Additionally, the active compound may be incorporated into sustained-release preparations or formulations.

[0035] In some specific embodiments, the heavy metal ion adsorption product has the following functions: adsorbing heavy metal ions in water.

[0036] In some specific embodiments, the heavy metal ion is selected from one or more of copper ion, lead ion, zinc ion, tin ion, nickel ion, cobalt ion, antimony ion, mercury ion, cadmium ion or bismuth ion. Preferably, the heavy metal ion is lead ion.

[0037] The present invention also provides a method for preparing a bacterial agent for adsorbing heavy metal lead, the preparation method comprising the following steps:

[0038] 1) inoculating the fermented Lactobacillus fermentum B44 in the aforementioned application into MRS medium for fermentation and cultivation;

[0039] 2) Separating and collecting the bacterial cells cultured in step 1), mixing them with a bacterial cell protective agent and then drying them to obtain a bacterial agent that adsorbs heavy metal lead.

[0040] In some specific embodiments, the culturing method in step 1) is anaerobic culturing.

[0041] In some embodiments, the fermentation temperature in step 1) is 25-45° C. Specifically, the temperature is 25-27° C., 27-30° C., 30-33° C., 33-35° C., 35-37° C., 37-40° C., 40-42° C. or 42-45° C. Preferably, the temperature is 30-42° C.

[0042] In some specific embodiments, the fermentation time in step 1) is 6-36 hours. Specifically, the fermentation time is 6-8 hours, 8-12 hours, 12-16 hours, 16-20 hours, 20-24 hours, 24-30 hours or 30-36 hours. Preferably, the fermentation time is 8-24 hours.

[0043] In some specific embodiments, the separation and collection process in step 2) is centrifugation. Specifically, the centrifugal force of the centrifugation is 6000-15000g; or, the centrifugation time is 5-30min. More specifically, the centrifugal force is 6000-7000g, 7000-8000g, 8000-10000g, 10000-12000g, 12000-13000g or 13000-15000g; or, the centrifugation time is 5-8min, 8-10min, 10-15min, 15-20min, 20-25min, 25-27min or 27-30min. Preferably, the centrifugal force is 8000-12000g, and the centrifugation time is 10-25min.

[0044] In some specific embodiments, the cell protective agent in step 2) is skim milk. Specifically, based on the volume of skim milk, the mass volume ratio (w / v) of the mass g of milk powder in the skim milk to the volume ml of skim milk is 5-15%. More specifically, the mass volume ratio is 5-7%, 7-8%, 8-10%, 10-13%, 13-14% or 14-15%. Preferably, the mass volume ratio is 8-13%.

[0045] In some specific embodiments, in step 2), the mass of the bacterial agent is used as the basis, and the number of viable bacteria of fermented Lactobacillus mucilaginosus B44 in the bacterial agent is 1×10 6 CFU / g or more. Specifically, the number of viable bacteria is 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g or 1×10 9 CFU / g or more. Preferably, the number of viable bacteria is 1×10 9 CFU / g and above.

[0046] The present invention also provides a bacterial agent for adsorbing heavy metal lead obtained by the above-mentioned preparation method.

[0047] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0048] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0049] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0050] Example 1: Experiment on the tolerance of fermented Lactobacillus mucilaginosus B44 to lead ions

[0051] Add 0.480g of Pb(NO3)2 solid powder to 1L of water to obtain a 300mg / L lead ion aqueous solution. Use the above solution to dissolve the MRS liquid culture medium dry powder to obtain a 300mg / L lead ion MRS liquid culture medium. MRS culture medium is a culture medium well known to those skilled in the art, which contains tryptone, yeast extract, glucose, sodium acetate, diammonium citrate, Tween 80, magnesium sulfate, manganese sulfate, and a pH of 6.2 to 6.4.

[0052] Similarly, MRS liquid culture medium containing lead ion concentrations of 0 mg / L, 20 mg / L, 50 mg / L, 150 mg / L, and 300 mg / L was prepared according to the method described above. After high temperature sterilization at 118°C for 15 min, it was used for later use. The fermentation mucus lactobacillus B44 entering the stable period was inoculated into the above MRS liquid culture medium at 1% of the inoculum amount of the lead-containing MRS liquid culture medium, and the OD was measured every 1 h using a fully automatic microbial growth analyzer. 600 The value is obtained as shown in the following Figure 1 Growth curve of fermentative Lactobacillus mucilaginosus B44 is shown.

[0053] The results showed that fermented Lactobacillus mucilaginosus B44 had good tolerance to lead ions, and its tolerance to lead ions was ≥300 mg / L.

[0054] Example 2: In vitro adsorption experiment of lead ions in water by fermented Lactobacillus mucilaginosus B44, other lactic acid bacteria and Bifidobacterium

[0055] Through in vitro incubation experiments with lead-containing aqueous solutions, the adsorption capacity of 10 bacterial strains, including fermented Lactobacillus mucilaginosus B44 and the control strains Lactobacillus paracasei BD5115 (CGMCC No.20045), Lactobacillus rhamnosus KF7 (CGMCC No.6430), Lactobacillus rhamnosus B6 (CGMCC No.13310), Lactobacillus rhamnosus LGG, Lactobacillus plantarum ST-III (CGMCC No.0847), Lactobacillus plantarum RT-6 (CGMCC No.26778), Lactobacillus plantarum CCFM8610 (CGMCC No.6077), Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus casei ATCC334, on lead ions in water were analyzed at the same dry bacteria concentration. After purification and activation culture of these 10 strains of lactic acid bacteria and bifidobacterium, the above bacteria were scraped from MRS or TPY solid culture medium with a sterile cell scraper, the moisture content of each bacteria was first determined, and the bacteria were fully suspended with sterile water, prepared into a dry bacteria concentration of 1g / L, and resuspended in 20mg / L lead ion aqueous solution for adsorption experiment, oscillated at 37°C and 200rpm for 1h, centrifuged at 6000rpm for 15min, and the supernatant was collected. The supernatant solution was filtered through a 0.22μm filter membrane and measured using an inductively coupled plasma emission mass spectrometer.

[0056] Table 1 In vitro adsorption of lead ions compared with control strains

[0057]

[0058] The results showed that B44 bacteria had a higher adsorption efficiency for lead ions in water than common lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus paracasei, as well as Bifidobacterium animalis.

[0059] Example 3: In vitro adsorption experiment of lead ions in water by fermented Lactobacillus mucilaginosus B44

[0060] Through in vitro lead-containing aqueous solution incubation experiments, the adsorption capacity of lead ions in water by fermentation Lactobacillus mucilaginosus B44 and control strain Lactobacillus casei subspecies ATCC334 was analyzed at the same dry bacteria concentration. After purification and activation culture of the two lactic acid bacteria, the bacteria were scraped from the MRS solid culture medium with a sterile cell scraper, the water content of each bacteria was first determined, and after being fully suspended with sterile water, the dry bacteria concentration was prepared to 1g / L, and resuspended in 50mg / L lead ion aqueous solution for adsorption experiments, 37℃, 200rpm oscillation reaction for 1h, 6000rpm centrifugation for 15min, and the supernatant and bacterial mud were collected. After freeze-dried treatment, the bacterial mud was weighed, and 2.5mL concentrated nitric acid (superior grade) was added to digest it in a graphite digester at 130℃ for 30min, the tube was rinsed twice with deionized water, and the volume was fixed to 5mL. The supernatant and digestion solution were measured by inductively coupled plasma emission mass spectrometry.

[0061] Table 2 In vitro adsorption of lead ions compared with standard strains

[0062]

[0063] The results showed that compared with the control strain Lactobacillus casei subsp. casei ATCC334 and the fermented Lactobacillus mucilaginosus LF-SCHY34 in the literature, the fermented Lactobacillus mucilaginosus B44 had better adsorption ability for lead ions after incubation in a lead-containing aqueous solution in vitro, with the highest adsorption capacity reaching 28.5 mg / g dry bacterial weight and the adsorption rate reaching 81.25%, which was significantly higher than the control strain ATCC334 and the fermented Lactobacillus mucilaginosus LF-SCHY34.

[0064] Example 4: In vitro adsorption experiment of lead ions in water by fermented Lactobacillus mucilaginosus B44

[0065] Through in vitro lead-containing aqueous solution incubation experiments, the adsorption capacity of lead ions in water by fermentation of Lactobacillus mucilaginosus B44 and control strain Lactobacillus casei subspecies ATCC334 was analyzed at the same wet bacteria concentration. After purification and activation culture of the two lactic acid bacteria, the bacteria were scraped from the MRS solid culture medium with a sterile cell scraper, fully suspended with sterile water, prepared to a wet bacteria concentration of 7.5g / L, and resuspended in 10 and 20mg / L lead ion aqueous solution for adsorption experiments, 37℃, 200rpm oscillation reaction for 1h, 6000rpm centrifugation for 15min, and the bacterial mud was collected. After freeze-drying, the dry weight of the bacterial mud was weighed, and 2.5mL concentrated nitric acid (superior grade) was added to digest it in a graphite digester at 130℃ for 30min, the tube was rinsed with deionized water twice, the volume was fixed to 5mL, and the inductively coupled plasma emission mass spectrometer was used for determination.

[0066] Table 3 In vitro adsorption of lead ions compared with standard strains

[0067]

[0068] Compared with the control strain, the adsorption capacity for lead ions was greater after incubation in aqueous lead solution in vitro.

[0069] Example 4: Transmission electron microscopy spectroscopy analysis of lead ion adsorption in water by fermented Lactobacillus mucilaginosus B44

[0070] According to the method of Example 2, the bacterial cells were scraped off from the MRS solid culture medium with a sterile cell scraper, the water content of each bacterium was first determined, and after being fully suspended with sterile water, the dry bacterial concentration of 1g / L was prepared, and the adsorption experiment was performed by resuspending in a 150mg / L lead ion aqueous solution, 37°C, 200rpm oscillation reaction for 1h, 6000rpm centrifugation for 15min, and the bacterial mud was collected. After being rinsed 3 times with sterilized ultrapure water, 2.5% glutaraldehyde was immediately added under the same centrifugal conditions to fix for at least 6h, and placed at 4°C overnight. Subsequently, 0.1M PB was used for washing 4 times, and 30% / 50% / 70% / 90% ethanol was used for gradient dehydration, and after the same gradient replacement of acetone, 0.3mL pure resin was added for fixation, and it was placed in a 60°C oven for polymerization for 48h. Finally, the sample was sliced ​​by an ultrathin slicer, and observed and analyzed by a material-type transmission electron microscope combined with an energy spectrometer.

[0071] Figure 2 These are transmission electron micrographs of lead adsorption by fermented Lactobacillus mucilaginosus B44, where (a) is the B44 bacteria and (b) is the B44 bacteria after lead adsorption.

[0072] From the attached Figure 2 The results show that the outer circle of B44 bacterial cells shows a bright white mucus layer. The samples after B44 bacteria adsorbed lead can be seen compared with the normal group.

[0073] B44 bacteria can absorb a large amount of lead ions, which are mainly distributed on the periphery of bacterial cells, with a small amount enriched inside the bacteria.

[0074] Figure 3The energy spectrum scanning photos of the fermented mucus lactobacillus B44 bacteria adsorbing lead, where (a) is the mapping diagram of the bacteria element spectrum before B44 adsorption, (b) is the scanning spectrum diagram before B44 adsorption, (c) is the mapping diagram of the bacteria element spectrum after B44 adsorbs lead, and (d) is the scanning spectrum diagram after B44 adsorbs lead. Tables 3 and 4 are the scanning spectrum element change tables before and after B44 lactic acid bacteria adsorb lead ions. From the results of Table 3, it can be seen that compared with the normal group, the C and N element contents on the surface of the lactic acid bacteria after lead adsorption are reduced, and the Pb and O element contents are increased. From the results of Table 4, it can be seen that after unified data conversion, compared with the control strain fermented mucus lactobacillus LF-SCHY34 in the literature, the mass fraction and atomic percentage change values ​​of lead in the bacteria before and after lead adsorption of fermented mucus lactobacillus B44 are significantly higher than those of other lactic acid bacteria, indicating that fermented mucus lactobacillus B44 has a good ability to adsorb lead ions.

[0075] Table 4 Changes in scanning energy spectrum before and after fermentation of Lactobacillus mucilaginosus B44 adsorbs lead ions

[0076]

[0077]

[0078] Table 5 Scanning spectrum change values ​​before and after fermentation of mucus lactobacillus adsorbs lead ions

[0079]

[0080] Example 6: Effect of fermented Lactobacillus mucilaginosus B44 on the alleviation of lead acetate-induced blood lead in ICR mice

[0081] The experimental data are expressed as mean ± standard deviation. Indicates, where n represents the number of valid samples. SPSS 21.0 software was used to analyze the data. One-way ANOVA statistical method was used for significance test when the data were in accordance with normal distribution; KW statistical method was used for significance test when the data were not in accordance with normal distribution.

[0082] The freeze-dried powder of fermented Lactobacillus mucilaginosus B44 was resuspended in skim milk to a concentration of 1.0×10 9 CFU / mL bacterial suspension. Eighteen healthy male ICR mice weighing 20-25g were randomly divided into three groups, each containing 6 mice, including a negative control group, a lead exposure model group, and a fermented mucus lactobacillus B44 intervention group. According to the grouping, the blank group mice drank blank drinking water normally; the model group and B44 group mice drank drinking water containing lead ions at a concentration of 0.8g / L. The negative control group and model group mice were gavaged with 0.2ml of blank skim milk once a day for 42 consecutive days; the B44 group mice were gavaged with 0.2ml of a concentration of 1.0×10 9CFU / mL of B44 bacterial suspension was administered once a day for 42 consecutive days. 24 hours after the last administration of the experiment, blood was collected from the eye socket (water was not allowed 12 hours before eating) and stored in anticoagulant tubes at -20°C. The blood was used to detect blood lead concentration.

[0083] The blood samples were tested in accordance with the requirements of the national standard "Determination of Lead in Blood Part 2: Inductively Coupled Plasma Mass Spectrometry" (GBZ / T 316.2-2018). The specific steps are as follows: First, place 10 ml of 1% Triton X-100 solution in 1L nitric acid solution (0.5%) to prepare a diluent. Subsequently, take out the refrigerated blood sample and return it to room temperature. After shaking thoroughly, transfer 100 μl of blood sample into a 15 mL polyethylene centrifuge tube, add 10 mL of 0.01% TritonX-100 in 0.5% HNO3 diluent, shake and then use ICP-MS instrument (PerkinElmer NexION 2000) for determination. The specific results are as follows:

[0084] Table 6 Lead concentration in whole blood of ICR mice in each group. n=6)

[0085]

[0086]

[0087] ***, compared with the blank group, P < 0.001; ###, compared with the model group, P < 0.001.

[0088] The results showed that after 6 weeks of the experiment, the lead concentration in the whole blood of the mice in the modeling group was higher than that in the blank group (P < 0.001). The lead concentration in the whole blood of the mice after intervention with the fermented Lactobacillus mucosa B44 solution was lower than that in the model group (P < 0.001), with a decrease rate of 27.6%. Example 7: The alleviating effect of fermented Lactobacillus mucosa B44 on lead accumulation in the liver and kidney of ICR mice induced by lead acetate

[0089] The experimental data are expressed as mean ± standard deviation. Indicates, where n represents the number of valid samples. SPSS 21.0 software was used to analyze the data. One-way ANOVA statistical method was used for significance test when the data were in accordance with normal distribution; KW statistical method was used for significance test when the data were not in accordance with normal distribution.

[0090] The freeze-dried powder of fermented Lactobacillus mucilaginosus B44 was resuspended in skim milk to a concentration of 1.0×10 9CFU / mL bacterial suspension. Eighteen healthy male ICR mice weighing 20-25g were randomly divided into three groups, each containing 6 mice, including a negative control group, a lead exposure model group, and a fermented mucus lactobacillus B44 intervention group. According to the grouping, the blank group mice drank blank drinking water normally; the model group and B44 group mice drank drinking water containing lead ions at a concentration of 0.8g / L. The negative control group and model group mice were gavaged with 0.2ml of blank skim milk once a day for 42 consecutive days; the B44 group mice were gavaged with 0.2ml of a concentration of 1.0×10 9 CFU / mL of B44 bacterial suspension was administered once a day for 42 consecutive days. 24 hours after the last administration of the experiment, all mice were killed and liver and kidney tissue samples were taken.

[0091] According to the animal tissue digestion method described in the reference Liang Yanqiu et al. "ICP-MS Determination of Lead, Cadmium and Arsenic in Animal Liver", the tissue was first rinsed with deionized water, dried in an oven at 60°C, and then ground into powder. After nitric acid digestion, the lead content in the tissue was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The specific results are as follows:

[0092] Table 7 Lead content in liver and kidney tissues of ICR mice in each group ( n=6)

[0093]

[0094] ***, compared with the blank group, P < 0.001; #, compared with the model group, P < 0.05.

[0095] The results showed that after 6 weeks of the experiment, the lead concentrations in the liver and kidneys of the mice in the modeling group were higher than those in the blank group (P < 0.001). After intervention with fermented mucus lactobacillus B44 liquid, the lead content in the liver and kidney tissues of the mice was significantly reduced, especially the lead concentration in the kidney was significantly lower than that in the model group (P < 0.05), and the lead content in the liver and kidney decreased by 26.5% and 43.4% respectively compared with the model group. In addition, through data comparison, it was found that the lead content in the kidney was higher than that in the liver. This shows that the kidney protection of lead-poisoned mice is more significant after intervention with fermented mucus lactobacillus B44 liquid.

[0096] Example 7: Effect of fermented Lactobacillus mucilaginosus B44 on the alleviation of lead acetate-induced blood lead in SD rats

[0097] The experimental data are expressed as mean ± standard deviation Indicates, where n represents the number of valid samples. SPSS 21.0 software was used to analyze the data. One-way ANOVA statistical method was used for significance test when the data were in accordance with normal distribution; KW statistical method was used for significance test when the data were not in accordance with normal distribution.

[0098] The freeze-dried powder of fermented Lactobacillus mucilaginosus B44 was resuspended in skim milk to a concentration of 1.0×10 9 CFU / mL bacterial suspension. Thirty-six healthy male SD rats weighing 150-200g, SPF grade, were randomly divided into 3 groups, each containing 12 SD rats, including a negative control group, a lead exposure model group, and a fermented mucus lactobacillus B44 intervention group. According to the grouping, the blank group rats drank blank drinking water normally; the model group and B44 group rats drank an aqueous solution containing 400 mg / L lead ions (accurately weighed 736.5 mg of PbAc2·3H2O, added sterile water to dissolve, fixed to 1L, and mixed). The B44 group rats were fed with 5×10 9 CFU / kg (BW) was gavaged with B44 bacterial suspension once a day for 42 consecutive days; the negative control group and model group rats were gavaged with the same dose of blank skim milk once a day for 42 consecutive days. 24 hours after the last administration of the experiment, blood was collected from the orbit (water was not allowed for 12 hours before eating), stored in anticoagulant tubes at -20℃, and the blood was used to detect blood lead concentration.

[0099] The blood samples were tested in accordance with the requirements of the national standard "Determination of Lead in Blood Part 2: Inductively Coupled Plasma Mass Spectrometry" (GBZ / T 316.2-2018). The specific steps are as follows: First, place 10 ml of 1% Triton X-100 solution in 1L nitric acid solution (0.5%) to prepare a diluent. Subsequently, take out the refrigerated blood sample and return it to room temperature. After shaking thoroughly, transfer 100 μl of blood sample into a 15 mL polyethylene centrifuge tube, add 10 mL of 0.01% TritonX-100 in 0.5% HNO3 diluent, shake and then use ICP-MS instrument (PerkinElmer NexION 2000) for determination. The specific results are as follows:

[0100] Table 8 Lead concentration in whole blood of SD rats in each group. n=12)

[0101]

[0102] ***, compared with the blank group, P < 0.001; ###, compared with the model group, P < 0.001.

[0103] The results showed that after 6 weeks of the experiment, the lead concentration in the whole blood of SD rats in the modeling group was higher than that in the blank group (P < 0.001). After intervention with fermented Lactobacillus mucosa B44 liquid, the lead concentration in the whole blood of SD rats was lower than that in the model group (P < 0.001), and the decrease rate reached 49.6%.

[0104] Example 8: The alleviating effect of fermented Lactobacillus mucilaginosus B44 on lead acetate-induced lead accumulation in the liver and kidney of SD rats

[0105] The experimental data are expressed as mean ± standard deviation Indicates, where n represents the number of valid samples. SPSS 21.0 software was used to analyze the data. One-way ANOVA statistical method was used for significance test when the data were in accordance with normal distribution; KW statistical method was used for significance test when the data were not in accordance with normal distribution.

[0106] The freeze-dried powder of fermented Lactobacillus mucilaginosus B44 was resuspended in skim milk to a concentration of 1.0×10 9 CFU / mL bacterial suspension. Thirty-six healthy male SD rats weighing 150-200g, SPF grade, were randomly divided into 3 groups, each containing 12 SD rats, including a negative control group, a lead exposure model group, and a fermented mucus lactobacillus B44 intervention group. According to the grouping, the blank group rats drank blank drinking water normally; the model group and B44 group rats drank an aqueous solution containing 400 mg / L lead ions (accurately weighed 736.5 mg of PbAc2·3H2O, added sterile water to dissolve, fixed to 1L, and mixed). The B44 group rats were fed with 5×10 9 CFU / kg (BW) was gavaged with B44 bacterial suspension once a day for 42 consecutive days; the negative control group and model group rats were gavaged with the same dose of blank skim milk once a day for 42 consecutive days. 24 hours after the last administration of the experiment, all rats were killed and liver and kidney tissue samples were taken.

[0107] According to the animal tissue digestion method described in the reference Liang Yanqiu et al. "ICP-MS Determination of Lead, Cadmium and Arsenic in Animal Liver", the tissue was first rinsed with deionized water, dried in an oven at 60°C, and then ground into powder. After nitric acid digestion, the lead content in the tissue was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The specific results are as follows:

[0108] Table 9 Lead content in liver and kidney tissues of SD rats in each group ( n=12)

[0109]

[0110] ***, compared with the blank group, P < 0.001; **, compared with the blank group, P < 0.01; ##, compared with the model group, P < 0.01; #, compared with the model group, P < 0.05.

[0111] The results showed that after 6 weeks of the experiment, the lead concentrations in the liver and kidney of the modeling group rats were higher than those in the blank group (P < 0.001). After intervention with fermented mucus lactobacillus B44 liquid, the lead content in the liver tissue of SD rats was significantly lower than that in the model group (P < 0.01), and the lead concentration in the kidney tissue was significantly lower than that in the model group (P < 0.05). The lead content in the liver and kidney decreased by 44.25% and 24.4% respectively compared with the model group. In addition, data comparison showed that the lead content in the kidney of SD rats was higher than that in the liver, which was consistent with the results of Example 6. This shows that intervention with fermented mucus lactobacillus B44 liquid has a significant protective effect on the liver and kidney of lead-poisoned SD rats.

[0112] In summary, the fermented Lactobacillus mucilaginosus B44 in the present invention has a better adsorption effect on lead ions in water than other lactic acid bacteria under the same conditions; in addition, the fermented Lactobacillus mucilaginosus B44 has the effect of reducing the lead content in the blood, liver and kidney of lead-exposed mice. In addition, the fermented Lactobacillus mucilaginosus B44 also has the effect of reducing the lead content in the blood, liver and kidney of lead-exposed rats. Based on this, it is shown that the strain has a good market application prospect.

[0113] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of fermented mucus lactobacillus (Limosilactobacillus fermentum) B44 in preparing a product for adsorbing heavy metal ions, wherein the preservation number of the fermented mucus lactobacillus (Limosilactobacillus fermentum) B44 is CGMCC No.17321.

2. The use according to claim 1, characterized in that The heavy metal ion adsorption product is a product that reduces lead toxicity; Preferably, the lead toxicity reduction product has any one or more of the following functions: 1) Reduce blood lead concentration; 2) Reduce lead concentration in the liver; 3) Reduce lead concentration in the kidneys.

3. The use according to claim 1, characterized in that In the heavy metal ion adsorption product, based on the total mass of the heavy metal ion adsorption product, the content of the fermented mucus lactobacillus (Limosilactobacillus fermentum) B44 is 10 6 -10 11 CFU / g.

4. The use according to claim 1, characterized in that The heavy metal ion adsorption product also includes pharmaceutically acceptable carriers and media.

5. The use according to claim 1, characterized in that: The heavy metal ion adsorption product has the following functions: adsorbing heavy metal ions in water.

6. The use according to claim 5, characterized in that The heavy metal ion is selected from one or more of copper ion, lead ion, zinc ion, tin ion, nickel ion, cobalt ion, antimony ion, mercury ion, cadmium ion or bismuth ion; preferably, the heavy metal ion is lead ion.

7. A method for preparing a bacterial agent for adsorbing heavy metal lead, characterized in that: The preparation method comprises the following steps: 1) inoculating and culturing Limosilactobacillus fermentum B44 in MRS medium, wherein the fermentation Lactobacillus fermentum B44 has a deposit number of CGMCC No. 17321; 2) Separating and collecting the bacterial cells cultured in step 1), mixing them with a bacterial cell protective agent and then drying them to obtain a bacterial agent that adsorbs heavy metal lead.

8. The preparation method according to claim 7, characterized in that: The preparation method also includes one or more of the following features: 1) The culture method in step 1) is anaerobic culture; II) the fermentation temperature in step 1) is 25-45°C; III) The fermentation time in step 1) is 6-36 hours.

9. The preparation method according to claim 7, characterized in that: The preparation method also includes one or more of the following features: IV) The separation and collection process in step 2) is centrifugation; preferably, the centrifugal force of the centrifugation is 8000-12000g, and the centrifugation time is 10-25min; V) in step 2), the cell protective agent is skim milk; preferably, based on the volume of skim milk, the mass volume ratio (w / v) of the mass g of milk powder in the skim milk to the volume ml of skim milk is 5-15%; VI) In step 2), the mass of the bacterial agent is used as the basis, and the number of viable bacteria of fermented Lactobacillus mucilaginosus B44 in the bacterial agent is 1×10 6 CFU / g or more; preferably, the number of viable bacteria is 1×10 9 CFU / g and above.

10. A bacterial agent for adsorbing heavy metal lead obtained by any one of the preparation methods of claims 7 to 9.

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