Postbiotic for preventing or treating lead poisoning as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a metagen for preventing or treating lead poisoning and a preparation method and application thereof.The metagen for preventing or treating lead poisoning is obtained by conducting fermentation culture on paenibacillus bovis BD3526 in a bran culture medium and then inactivating the paenibacillus bovis BD3526, the bacillus subtilis BD3526 comprises inactivated thalli of the bacillus subtilis BD3526 and a metabolite of the bacillus subtilis BD3526. The prepared metagen has the effects of remarkably promoting lead excretion, reducing lead accumulation in the liver or kidney and reducing the content of blood lead.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a postbiotic for preventing or treating lead poisoning, and a preparation method and application thereof. Background Art

[0002] Lead (Pb) is a typical heavy metal pollutant. Unlike organic pollutants, it is difficult to degrade, volatilize, and decompose. This property makes lead easily enriched in the food chain, posing a serious threat to human health through exposure routes such as the food chain and drinking water. Lead is particularly sensitive to children's growth and development, causing adverse effects such as hyperactivity, attention deficit, and developmental delay. According to the American Academy of Pediatrics (AAP), blood lead concentrations greater than 50 μg / L in preschool children (i.e., children aged 1 to 5 years) can cause hyperactivity, attention deficit, and low birth weight. Currently, traditional lead-removing chelating drugs (such as dithiothreitol and sodium edetate) are mostly used to treat patients with high blood lead levels. However, significant side effects and safety issues limit their use. Therefore, there is an urgent need to find safe and effective alternative interventions to alleviate high blood lead levels.

[0003] Currently, the use of probiotics as a substitute for traditional chelation therapy has become a research hotspot due to their high safety, lack of side effects, and strong heavy metal binding ability. However, existing research on the role of postbiotics in reducing heavy metal emissions in the body is relatively limited. Most studies have used live bacteria, and the chemical culture media required to prepare these postbiotics are expensive and complex. Research on the role of postbiotics prepared using low-cost agricultural byproducts as culture media in reducing heavy metal emissions in the body is still lacking, and there are relatively few available strains and their metabolites. This is a problem that technicians in this field urgently need to solve. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a postbiotic for preventing or treating lead poisoning, and a preparation method and use thereof, so as to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides a postbiotic for preventing or treating lead poisoning, wherein the postbiotic is obtained by inactivating and fermenting Paenibacillus bovis BD3526.

[0006] The present invention also provides use of the above-mentioned postbiotics in preparing products for preventing and / or treating lead poisoning.

[0007] The present invention also provides a product for preventing and / or treating lead poisoning, wherein the product contains the above-mentioned postbiotics and additives.

[0008] The present invention also provides a method for preparing postbiotics, which comprises the following steps:

[0009] a) activating Paenibacillus BD3526 and transferring the activated strain to a seed culture medium to obtain a seed solution;

[0010] b) inoculating the seed solution obtained in step a) into a fermentation medium for fermentation to obtain a fermentation liquid;

[0011] c) performing an inactivation treatment on the fermentation liquid obtained in step b) to obtain the postbiotics.

[0012] As described above, the postbiotic for preventing or treating lead poisoning of the present invention, and its preparation method and use, have the following beneficial effects:

[0013] The postbiotics prepared by Paenibacillus BD3526 in the bran culture medium of the present invention have the effect of reducing the lead content in the blood, liver and kidneys of lead model mice, especially the protective effect on the kidneys is more obvious, and at the same time, the lead content in the feces in the colon of mice can be significantly increased; in addition, the postbiotics prepared by Paenibacillus BD3526 in the bran culture medium have the effect of reducing the lead content in the whole blood and kidneys of the lead model mouse treatment group, and the effect of reducing blood lead is more obvious, and at the same time, the lead content in the feces in the colon of mice can be significantly increased, indicating that the Paenibacillus BD3526 postbiotics can reduce the level of lead accumulation in the tissues of lead-exposed mice and suggest that the intestine has the ability to excrete lead.

[0014] Under the same conditions, the postbiotics of Bacillus BD3526 were more effective in excreting lead in mice than those prepared from the control strain in bran culture medium. This indicates that the postbiotic products of this strain have good market application prospects. Biomaterial deposit information The Paenibacillus sp. BD3526 strain of the present invention was deposited with the General Microbiology Center of the China Culture Collection Administration (CGMCC) on October 14, 2013, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit number of this strain is CGMCC No. 8333. This strain was taxonomically designated Paenibacillus sp., later defined as Paenibacillus bovis, and named BD3526. DETAILED DESCRIPTION

[0015] The present invention first provides a postbiotic for preventing or treating lead poisoning. The postbiotic is obtained by inactivating Paenibacillus bovis BD3526 after fermentation and culture.

[0016] The deposit number of the Paenibacillus sp. BD3526 is CGMCC No. 8333, and the deposit information of the strain has been recorded in the prior art document CN 103740618 A.

[0017] In certain embodiments of the present invention, before inactivation, the number of viable bacteria of Paenibacillus BD3526 in the culture system after fermentation culture is ≥2.32×10 8 CFU / mL.

[0018] In a preferred embodiment of the present invention, before inactivation, the number of viable bacteria of Paenibacillus BD3526 in the culture system after fermentation culture is 2.32×10 8 CFU / mL~1.76×10 9 CFU / mL.

[0019] In certain embodiments of the present invention, the postbiotics include inactivated Paenibacillus BD3526 bacteria and Paenibacillus BD3526 metabolites.

[0020] In certain embodiments of the present invention, the postbiotic further comprises one or more of a cell wall component of Paenibacillus BD3526, a cell membrane component of Paenibacillus BD3526, and / or a cell content component of Paenibacillus BD3526.

[0021] In certain embodiments of the present invention, the cell wall components of the Paenibacillus BD3526 include peptidoglycan, teichoic acid or surface protein; the cell membrane components include phospholipids, lipopolysaccharide or membrane protein; and the cell content components include enzymes, antimicrobial peptides or coenzymes and antioxidants.

[0022] In certain embodiments of the present invention, the fermentation culture medium is a bran culture medium.

[0023] The present invention also provides use of the above-mentioned postbiotics in preparing products for preventing and / or treating lead poisoning.

[0024] In certain embodiments of the present invention, the product has any one or more of the following effects:

[0025] 1) Promote lead excretion;

[0026] 2) Reduce lead accumulation in the liver or kidneys;

[0027] 3) Reduce blood lead levels.

[0028] The present invention also provides a product for preventing and / or treating lead poisoning, wherein the product contains the above-mentioned postbiotics and additives.

[0029] In certain embodiments of the present invention, the product for preventing and / or treating lead poisoning contains the above-mentioned postbiotic as the sole active ingredient or one of the active ingredients.

[0030] In certain embodiments of the present invention, the additive is selected from excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

[0031] In certain embodiments of the present invention, products for preventing and / or treating lead poisoning include, but are not limited to, drugs, pharmaceutical compositions, drug combinations, reagents, kits, foods, and health products.

[0032] The dosage form or state of the product in the present invention is not limited. For example, when the product is a medicine, a pharmaceutical composition, or a combination of medicines, the dosage form can be one or more of a solution, an injection, a spray, a nasal drop, an aerosol, a powder spray, a tablet, a capsule, and a granule. The above-mentioned various dosage forms of the medicine can be prepared according to conventional methods in the pharmaceutical field.

[0033] The present invention also provides a method for preparing postbiotics, which comprises the following steps:

[0034] a) activating Paenibacillus BD3526 and transferring the activated strain to a seed culture medium to obtain a seed solution;

[0035] b) inoculating the seed solution obtained in step a) into a fermentation medium for fermentation to obtain a fermentation liquid;

[0036] c) performing an inactivation treatment on the fermentation liquid obtained in step b) to obtain the postbiotics.

[0037] In certain embodiments of the present invention, step a) further comprises expanding the culture of the activated bacteria.

[0038] In certain embodiments of the present invention, in step a), the seed culture medium is a skim milk medium.

[0039] Furthermore, based on the mass of the skim milk culture medium, the content of skim milk powder in the skim milk culture medium is 3-6 wt.%. The content of skim milk in the skim milk culture medium is selected from any of the following ranges: 3-4 wt.%, 4-5 wt.%, and 5-6 wt.%.

[0040] In the present invention, the skim milk culture medium is prepared by mixing skim milk powder with water.

[0041] In certain embodiments of the present invention, in step b), the fermentation medium is bran medium.

[0042] Furthermore, based on the mass of the bran culture medium, the bran content in the bran culture medium is 2-6 wt. %, and the bran content in the bran culture medium is selected from any of the following ranges: 2-3 wt. %, 3-4 wt. %, 4-5 wt. %, or 5-6 wt. %.

[0043] Furthermore, the method further comprises gelatinizing the bran culture medium.

[0044] Furthermore, the gelatinization treatment temperature is 85-100°C.

[0045] Furthermore, the gelatinization treatment time is 3 to 15 minutes.

[0046] In certain embodiments of the present invention, in step b), the inoculum size is 1-5% based on the volume of the fermentation medium. The inoculum size is selected from any of the following ranges: 1-2%, 2-3%, 3-4%, 4-5%. Preferably, the inoculum size is 3-4%.

[0047] In certain embodiments of the present invention, in step b), the culture is aerobic culture.

[0048] In certain embodiments of the present invention, in step b), the culture temperature is 28-33°C.

[0049] In certain embodiments of the present invention, in step b), the culture is shaker culture.

[0050] Furthermore, the shaking table has a speed of 150 to 300 rpm.

[0051] In certain embodiments of the present invention, in step b), the fermentation is carried out to a pH of 5.4 to 5.7.

[0052] In certain embodiments of the present invention, in step c), the inactivation method is selected from any one of thermal inactivation, chemical inactivation or biological inactivation.

[0053] Furthermore, the inactivation method is heat inactivation.

[0054] In certain embodiments of the present invention, step c) further comprises centrifugation after the inactivation treatment.

[0055] Furthermore, the centrifugal speed is 500-1000 rpm. The centrifugal speed is selected from any of the following ranges: 500-600 rpm, 600-700 rpm, 700-800 rpm, 800-900 rpm, 900-1000 rpm.

[0056] Furthermore, the centrifugation time is 3 to 10 minutes. The centrifugation time is selected from any of the following ranges: 3 to 5 minutes, 5 to 7 minutes, 7 to 9 minutes, and 9 to 10 minutes.

[0057] In certain embodiments of the present invention, step c) further comprises drying the fermentation broth after the inactivation treatment.

[0058] Furthermore, the drying temperature is 85-100° C. The drying temperature is selected from any one of the following ranges: 85-90° C., 90-95° C., and 95-100° C.

[0059] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific 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.

[0061] When the embodiments provide 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 may 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 understanding of the prior art by those skilled in the art and the description 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 may also be used to implement the present invention.

[0062] The "room temperature" mentioned in the present invention refers to the temperature of the operating room where the experiment is carried out, which is generally 15-25° C. Unless otherwise specified, the reagents used in the examples were of analytical grade and purchased from Sinopharm Group.

[0063] Example 1 Preparation of postbiotic A from Paenibacillus BD3526

[0064] 1. Materials and Methods

[0065] a) Preparation of fermented seeds:

[0066] The lyophilized powder of Paenibacillus BD3526 was dissolved in sterile distilled water, and a loop of the bacteria was streaked onto TPY solid medium with an inoculating loop. The bacteria were cultured aerobically at 30°C for 48 h and then taken out. A single colony was picked out with an inoculating loop and placed in 20 mL of 4 wt.% skim milk medium (skim milk powder was purchased from Fonterra Trading (Shanghai) Co., Ltd.). The bacteria were cultured in a shaker at 30°C and 180 rpm for 18 h and then taken out to obtain the seeds for fermentation. The bacterial concentration of the seed liquid was 4.8×10 8 cfu / mL.

[0067] b) Preparation of bran culture medium:

[0068] The bran was evenly dispersed in distilled water to obtain a 3% bran solution by mass, which was heated to boiling on a hot plate and gelatinized for 5 minutes with frequent stirring, followed by sterilization at 121°C for 15 minutes and cooling to room temperature to obtain a bran culture medium of the desired concentration.

[0069] 2. Preparation of Postbiotic A Samples:

[0070] The skim milk seed liquid of Paenibacillus BD3526 obtained in step a) was aseptically inoculated into the bran medium prepared in step b) at an inoculum amount of 2% (v / v), and fermented at 29°C for 55 h. The pH was 5.6, and the number of viable bacteria of Paenibacillus BD3526 in the culture liquid was 7.40×10 8 CFU / mL, the fermentation broth was inactivated in boiling water for 30 minutes, cooled, and centrifuged at 1000 rpm for 5 minutes to remove the bran sediment. The resulting fermentation culture was dried at 95°C to obtain postbiotic sample A.

[0071] Example 2 Preparation of postbiotic B from Paenibacillus BD3526

[0072] 1. Materials and Methods

[0073] a) Preparation of fermented seeds: same as in Example 1.

[0074] b) Preparation of bran culture medium:

[0075] The bran was evenly dispersed in distilled water to obtain a 3% bran solution by mass, which was heated to boiling on a hot plate and gelatinized for 10 minutes with frequent stirring, followed by sterilization at 121°C for 20 minutes and cooling to room temperature to obtain a bran culture medium of the desired concentration.

[0076] 2. Preparation of postbiotic sample B:

[0077] The skim milk seed liquid of Paenibacillus BD3526 obtained in step a) was aseptically inoculated into the bran medium prepared in step b) at an inoculum amount of 3% (v / v), and fermented at 30°C for 72 h. The pH was 5.7, and the number of viable bacteria of Paenibacillus BD3526 in the culture liquid was 1.36×10 9 CFU / mL, the fermentation broth was inactivated in boiling water for 30 minutes, cooled, and centrifuged at 1000 rpm for 5 minutes to remove the bran sediment. The resulting fermentation culture was dried at 85°C to obtain postbiotic sample B.

[0078] Example 3: Effect of Paenibacillus BD3526 Postbiotics on Alleviating Blood Lead Levels in Lead-Poisoned Mice

[0079] 3.1 Preparation of experimental reagents:

[0080] 1) Paenibacillus BD3526 postbiotic A sample solution: Obtain a Paenibacillus postbiotic A sample as in Example 1, and prepare a 15 mg / ml postbiotic A sample solution using sterile water;

[0081] 2) Standard strain Paenibacillus hunanensis 10718 (deposit number: CGMCC No. 1.8907) postbiotic sample solution: Paenibacillus hunanensis 10718 was fermented and cultured as in Example 1 to prepare a Paenibacillus hunanensis 10718 postbiotic sample, which was then prepared into a 15 mg / ml postbiotic sample solution using sterile water;

[0082] 3) Non-inactivated Paenibacillus BD3526 live bacterial inoculum: Paenibacillus BD3526 obtained by fermentation in bran culture medium was resuspended in 10 wt.% skim milk at a volume of 1 / 10 of the culture medium. The resuspended bacterial solution was freeze-dried to obtain a live bacterial sample with a bacterial activity of 5.1×10 9 CFU / g;

[0083] 4) Prepare a lead ion solution: Weigh approximately 1473 mg of lead acetate trihydrate and dissolve it in deionized water to a volume of 1 L.

[0084] 3.2 ICR mouse grouping and experimental design:

[0085] Thirty healthy male ICR mice weighing 20-25 g were randomly divided into five groups, each containing six mice: a blank group, a lead exposure model group, a BD3526 live bacteria group, a BD3526 postbiotic A group, and a 10718 postbiotic group. The following treatments were performed according to the groupings:

[0086] Blank group: mice drank 5.5 mL of blank drinking water once a day for 42 consecutive days;

[0087] Lead exposure model group: Mice drank 5.3 mL of drinking water containing 0.8 g / L of lead ions and were gavaged with 0.2 mL of sterile water once a day for 42 consecutive days.

[0088] BD3526 live bacteria group: mice drank 5.3 mL of drinking water containing 0.8 g / L of lead ions every day and were gavaged with drugs at a dose of 1 × 10 9 CFU / kg (bw) of Paenibacillus sp. BD3526 live bacteria inoculum, once daily for 42 consecutive days;

[0089] BD3526 postbiotic A group: Mice drank 5.3 mL of drinking water containing 0.8 g / L of lead ions daily and were gavaged with 150 mg / kg (bw) of Paenibacillus BD3526 postbiotic A solution once daily for 42 consecutive days.

[0090] 10718 postbiotic group: Mice drank 5.3 mL of drinking water containing a lead ion concentration of 0.8 g / L every day and were gavaged with 150 mg / kg (bw) of Hunan Paenibacillus 10718 postbiotic solution once a day for 42 consecutive days.

[0091] The above-mentioned microbial agents and solvents were all prepared with sterile water.

[0092] 3.3 Results Analysis

[0093] 24 hours after the last administration of the drug in the experiment, blood was collected from the mice's orbits (the mice were allowed to eat normally but not water for 12 hours before blood collection). The collected blood was placed in an anticoagulant tube and stored at -20°C for subsequent detection of blood lead concentration.

[0094] 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, 10 ml of a 1% volume fraction Triton X-100 solution was placed in 1 L of a 0.5% concentration of HNO3 solution to prepare a diluent. Subsequently, the refrigerated blood sample was taken out and returned to room temperature. After thorough shaking and mixing, 100 μl of the blood sample was transferred to a 15 mL polyethylene centrifuge tube, 10 mL of the prepared diluent was added, and after shaking and mixing, the sample was measured using an ICP-MS instrument (PerkinElmer NexION 2000). The specific results are shown in Table 1:

[0095] Table 1 Lead concentration in whole blood of ICR mice in each group ( n=6)

[0096] Group Pb concentration (μg / L) blank 2.91±3.03 Lead exposure model 493.8±70.3 BD3526 live bacteria 415.2±28.2 BD3526 Postbiotic A 428.3±35.9 10718 Postnatal 472.8±69.1

[0097] As shown in Table 1, after the six-week experimental period, the whole blood lead concentrations of mice in all model groups were higher than those in the blank group. Both the live Paenibacillus BD3526 bacteria group and the Paenibacillus BD3526 postbiotic A group reduced the whole blood lead concentration in lead-poisoned mice, with the live Paenibacillus BD3526 bacteria and Paenibacillus BD3526 postbiotic A groups showing the greatest efficacy. Following intervention with the Paenibacillus BD3526 postbiotic A solution, the whole blood lead concentration of mice was lower than that in the lead-exposed model group, with a decrease rate of 13.3%, demonstrating excellent lead-lowering capacity. Compared with the postbiotic sample prepared from the control strain Hunan Paenibacillus 10718, Paenibacillus BD3526 postbiotic A demonstrated superior blood lead-lowering capacity after six weeks of experimental intervention, indicating that BD3526 postbiotic A has significant potential for reducing whole blood lead concentrations in lead-poisoned mice.

[0098] Example 4: The preventive and alleviating effects of Paenibacillus BD3526 postbiotics on lead accumulation in the liver and kidneys of lead-poisoned mice

[0099] 4.1 Preparation of experimental reagents: same as 3.1.

[0100] 4.2 ICR mouse grouping and experimental design: same as 3.2.

[0101] 4.3 Result Analysis:

[0102] 24 h after the last administration of the drug, all mice were killed and liver and kidney tissue samples were taken.

[0103] Following the animal tissue digestion method described in Liang Yanqiu et al.'s "ICP-MS Determination of Lead, Cadmium, and Arsenic in Animal Liver," the tissues were rinsed with deionized water, dried in a 60°C oven, and then ground into a powder. After nitric acid digestion, the lead content in the tissues was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The results are shown in Table 2:

[0104] Table 2 Lead content in liver and kidney tissues of ICR mice in each group ( n=6)

[0105] Group Liver lead content (μg / g dry weight) Kidney lead content (μg / g dry weight) blank 0.0278±0.0112 0.0654±0.0243 Lead exposure model 3.34±1.47 10.6±4.45 BD3526 live bacteria 2.30±0.60 7.56±1.13 BD3526 Postbiotic A 2.50±0.35 6.38±1.48* 10718 Postnatal 2.94±0.78 8.26±1.61

[0106] Note: * indicates significant difference compared with the lead exposure model group, p<0.05

[0107] The results showed that after a 6-week experimental period, the lead content in the liver and kidney tissues of mice in the lead exposure model group was higher than that in the blank group. The lead content in the liver and kidney tissues of mice in the live bacteria group of Paenibacillus BD3526 and the postbiotic A group of Paenibacillus BD3526 were significantly lower than those in the lead exposure model group. The liver lead content in the postbiotic A group decreased by 25.2%, and the kidney lead content decreased by 58.0% (p < 0.05), showing a significant lead-lowering effect. In the liver group, the live bacteria group had the best effect, and the postbiotic A group had a better effect. In particular, the protective effect of Paenibacillus BD3526 postbiotic A on the kidneys was more obvious. In addition, compared with the postbiotic samples prepared from the control strain Hunan Paenibacillus 10718, after 6 weeks of experimental intervention, the postbiotic A of Paenibacillus BD3526 had a better ability to reduce lead accumulation in liver and kidney tissues, which further confirmed the superiority of Paenibacillus BD3526 postbiotic A in reducing lead accumulation in the liver and kidneys of lead-poisoned mice.

[0108] Example 5: The promoting effect of Paenibacillus BD3526 postbiotics in preventing fecal excretion in lead-poisoned mice

[0109] 5.1 Preparation of experimental reagents:

[0110] The preparation methods of Paenibacillus BD3526 postbiotic A sample solution and Hunan Paenibacillus 10718 postbiotic sample solution are the same as 3.1.

[0111] 5.2 ICR mouse grouping and experimental design:

[0112] Twenty-four healthy male ICR mice weighing 20-25 g were randomly divided into four groups, each containing six mice, including a blank group, a lead exposure model group, a BD3526 postbiotic A group, and a 10718 postbiotic group.

[0113] The treatment method for mice in each group was the same as 3.2.

[0114] 5.3 Results Analysis

[0115] 24 hours after the last administration of the drug, all mice were killed and pelleted feces samples were collected from the colon of the mice.

[0116] Following the animal tissue digestion method described in Liang Yanqiu et al.'s "ICP-MS Determination of Lead, Cadmium, and Arsenic in Animal Liver," the tissues were dried in a 60°C oven and then ground into powder. After nitric acid digestion, the lead content in feces was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The results are shown in Table 3:

[0117] Table 3 Lead content in feces of ICR mice in each group ( n=6)

[0118] Group Pb concentration (μg / g dry weight) blank 162.6±243.9 Lead exposure model 1220.1±399.7 BD3526 Postbiotic A 1612.1±675.3 10718 Postnatal 1226.6±493.4

[0119] As shown in Table 3, after 6 weeks of the experiment, the lead content in the feces of mice in the lead exposure model group was significantly higher than that in the blank group. The lead content in the feces of mice in the Paenibacillus BD3526 postbiotic A group was higher than that in the lead exposure model group. In addition, compared with the postbiotic sample prepared from the control strain Hunan Paenibacillus 10718, Paenibacillus BD3526 postbiotic A had a better ability to promote fecal lead excretion after 6 weeks of experimental intervention.

[0120] Example 6 Effect of Paenibacillus BD3526 Postbiotics on Alleviating Blood Lead Levels in Lead-Poisoned Mice

[0121] 6.1 Preparation of experimental reagents:

[0122] Paenibacillus BD3526 postbiotic B sample solution: Obtain a Paenibacillus postbiotic B sample as in Example 1, and prepare a 15 mg / ml postbiotic B sample solution using sterile water;

[0123] The preparation method of Hunan Paenibacillus 10718 solution is the same as 3.3.

[0124] 6.2 C57BL / 6 Mouse Grouping and Experimental Design

[0125] Twenty-four healthy male C57BL / 6 mice weighing 20-25g were randomly divided into two groups after one week of adaptive feeding: a blank group (6 mice) and an experimental group (18 mice). The blank group drank 5.1ml of blank drinking water daily, while the experimental group was given 5.1ml of drinking water containing 0.8g / L lead ions daily for 6 weeks to establish the model. After 6 weeks of modeling, the experimental group was divided into 3 groups, as follows:

[0126] Pb model group: mice were gavaged with 0.2 mL of sterile water once a day for 28 consecutive days;

[0127] BD3526 postbiotic group B: Mice were orally administered with 200 mg / kg (bw) BD3526 postbiotic solution once a day for 28 consecutive days;

[0128] 10718 postbiotic group: Mice were orally administered with 200 mg / kg (bw) 10718 postbiotic solution once daily for 28 consecutive days;

[0129] During the 4-week experimental period, the mice in the blank group were gavaged with 0.2 mL of sterile water once a day for 28 consecutive days.

[0130] 6.3 Results Analysis

[0131] 24 hours after the last administration of the drug in the experiment, blood was collected from the mice's orbits (the mice were allowed to eat normally but not water for 12 hours before blood collection). The collected blood was placed in an anticoagulant tube and stored at -20°C for subsequent detection of blood lead concentration.

[0132] 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, 10 ml of a 1% volume fraction Triton X-100 solution was placed in 1 L of a 0.5% concentration HNO3 solution to prepare a diluent. Subsequently, the refrigerated blood sample was taken out and returned to room temperature. After thorough shaking and mixing, 100 μl of the blood sample was transferred to a 15 mL polyethylene centrifuge tube, 10 mL of the prepared diluent was added, and after shaking and mixing, the sample was measured using an ICP-MS instrument (PerkinElmer NexION 2000). The specific results are shown in Table 4:

[0133] Table 4 Lead concentration in whole blood of C57BL / 6 mice in each group ( n=6)

[0134] Group Pb concentration (μg / L) blank 2.62±0.46 Pb exposure model 87.1±11.7 BD3526 Postbiotic B 70.1±7.0* 10718 Postnatal 85.3±10.3

[0135] *Indicates significant difference compared with the model group, p < 0.05.

[0136] As shown in Table 4, after six weeks of lead acetate modeling and four weeks of experimentation, the whole blood lead concentrations of the experimental group mice were higher than those of the control group. The whole blood lead concentrations of mice treated with postbiotic B were significantly lower than those in the Pb model group (p < 0.05), with a decrease rate of 18.0%. Furthermore, compared with the postbiotic sample prepared from the control strain Hunan Paenibacillus 10718, postbiotic B from Paenibacillus BD3526 demonstrated a superior ability to lower blood lead levels.

[0137] Example 7: Effect of Paenibacillus BD3526 postbiotics on alleviating renal lead poisoning in lead-poisoned mice

[0138] 7.1 Preparation of experimental reagents is the same as 6.1.

[0139] 7.2 C57BL / 6 mouse grouping and experimental design are the same as 6.2.

[0140] 7.3 Results Analysis

[0141] 24 h after the last administration of the drug, all mice were killed and kidney tissue samples were taken.

[0142] Following the animal tissue digestion method described in Liang Yanqiu et al.'s "ICP-MS Determination of Lead, Cadmium, and Arsenic in Animal Liver," the tissue was first rinsed with deionized water, dried in a 60°C oven, and then ground into a powder. After nitric acid digestion, the lead content in kidney tissue was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The specific results are shown in Table 5:

[0143] Table 5 Lead content in kidney tissue of C57BL / 6 mice in each group ( n=6)

[0144] Group Kidney lead content (μg / g) blank 0.0625±0.091 Pb exposure model 4.16±0.660 BD3526 Postbiotic B 3.61±0.599 10718 Postnatal 3.98±0.74

[0145] As shown in Table 5, after six weeks of lead acetate exposure and four weeks of treatment, the lead levels in the liver and kidney tissues of the experimental group mice were higher than those in the control group. Following intervention with the postbiotic B solution, the lead level in the kidney tissue of the mice was significantly lower than that in the lead-exposed group, with a decrease rate of 11.3%. Furthermore, compared with the postbiotic sample prepared from the control strain Hunan Paenibacillus 10718, the postbiotic B from Paenibacillus BD3526 exhibited a significantly greater ability to reduce lead accumulation in kidney tissue.

[0146] Example 8: Effect of Paenibacillus BD3526 postbiotics on fecal excretion in the treatment of lead poisoning in mice

[0147] 8.1 Preparation of experimental reagents is the same as 6.1.

[0148] 8.2 C57BL / 6 mouse grouping and experimental design are the same as 6.2.

[0149] 8.3 Results Analysis

[0150] 24 hours after the last administration of the drug, all mice were killed and pelleted feces samples were collected from the colon of the mice.

[0151] Following the animal tissue digestion method described in Liang Yanqiu et al.'s "ICP-MS Determination of Lead, Cadmium, and Arsenic in Animal Liver," the tissues were dried in a 60°C oven and then ground into powder. After nitric acid digestion, the lead content in feces was determined using an ICP-MS instrument (PerkinElmer NexION 2000). The results are shown in Table 6:

[0152] Table 6 Lead content in feces of C57BL / 6 mice in each group ( n=6)

[0153] Group Lead content in feces (μg / g) blank 0.609±0.442 Pb exposure model 2.96±2.56 BD3526 Postbiotic B 8.81±4.05* 10718 Postnatal 4.24±1.39

[0154] Note: * indicates significant difference compared with the Pb exposure model group, p<0.05.

[0155] The results showed that after 6 weeks of lead acetate modeling and 4 weeks of experimentation, the lead content in the granular feces in the colon of mice in the Pb model group was higher than that in the blank group. After intervention with the postbiotic B solution, the lead content in the mouse feces was significantly higher than that in the model group (p<0.05), an increase of 189%, suggesting that the Paenibacillus BD3526 postbiotic has the ability to promote intestinal excretion of lead. In addition, compared with the postbiotic sample prepared from the control strain Hunan Paenibacillus 10718, the Paenibacillus BD3526 postbiotic B has a better ability to improve fecal lead excretion in mice.

[0156] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent 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 apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A postbiotic for preventing or treating lead poisoning, characterized in that: The postbiotics are obtained by inactivating Paenibacillus bovis BD3526 after fermentation and culture; the preservation number of Paenibacillus bovis BD3526 is CGMCC No.8333.

2. The postbiotic according to claim 1, wherein Before inactivation, the number of viable bacteria of Paenibacillus BD3526 in the culture system after fermentation culture is ≥2.32×10 8 CFU / mL.

3. The postbiotic according to claim 1, wherein The postbiotics include inactivated bacteria of Paenibacillus BD3526 and metabolites of Paenibacillus BD3526.

4. The postbiotic according to any one of claims 1 to 3, characterized in that The postbiotics further include one or more of the cell wall components of Paenibacillus BD3526, the cell membrane components of Paenibacillus BD3526, and / or the cell content components of Paenibacillus BD3526; preferably, the cell wall components include peptidoglycan, teichoic acid, or surface protein; preferably, the cell membrane components include phospholipids, lipopolysaccharides, or membrane proteins; preferably, the cell content components include enzymes, antimicrobial peptides, coenzymes, and antioxidants; And / or, the culture medium for the fermentation culture is bran culture medium.

5. Use of the postbiotic according to any one of claims 1 to 4 in the preparation of a product for preventing and / or treating lead poisoning.

6. The use according to claim 5, characterized in that The product has any one or more of the following effects: 1) Promote lead excretion; 2) Reduce lead accumulation in the liver or kidneys; 3) Reduce blood lead levels.

7. A product for preventing and / or treating lead poisoning, characterized in that: The product contains the postbiotics and additives described in any one of claims 1 to 4.

8. The method for preparing postbiotics according to any one of claims 1 to 4, wherein: The preparation method comprises the following steps: a) activating Paenibacillus BD3526 and transferring the activated strain to a seed culture medium to obtain a seed solution; b) inoculating the seed solution obtained in step a) into a fermentation medium for fermentation to obtain a fermentation liquid; c) performing an inactivation treatment on the fermentation liquid obtained in step b) to obtain the postbiotics.

9. The preparation method according to claim 8, wherein In step a), any one or more of the following features are included: a1) Expanding and culturing the activated bacteria; a2) the seed culture medium is a skim milk culture medium; preferably, the content of skim milk powder in the skim milk culture medium is 3 to 6 wt.% based on the mass of the skim milk culture medium; And / or, in step b), including any one or more of the following features: b1) the fermentation medium is a bran medium; preferably, the bran content in the bran medium is 2 to 6 wt.% based on the mass of the bran medium; preferably, the bran medium is gelatinized; more preferably, the gelatinization temperature is 85 to 100° C.; more preferably, the gelatinization time is 3 to 15 minutes; b2) fermenting and culturing until the pH reaches 5.4 to 5.7; b3) the inoculum volume is 1 to 5% based on the volume of the fermentation medium; b4) the culture is aerobic; b5) the culture temperature is 28-33° C.; b6) The culture is carried out on a shaking platform; preferably, the shaking speed is 150 to 300 rpm.

10. The preparation method according to claim 8, characterized in that In step c), any one or more of the following features are included: c1) the inactivation method is selected from any one of thermal inactivation, chemical inactivation or biological inactivation; preferably, the inactivation method is thermal inactivation; c2) performing centrifugation after the inactivation treatment; preferably, the centrifugation speed is 500-1000 rpm; preferably, the centrifugation time is 3-10 minutes; c3) drying the inactivated fermentation liquid; preferably, the drying temperature is 85-100°C.