CN111096459B - Application of lactobacillus plantarum LP33 in preparation of product for preventing lead poisoning - Google Patents
Application of lactobacillus plantarum LP33 in preparation of product for preventing lead poisoning Download PDFInfo
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- CN111096459B CN111096459B CN201911131328.6A CN201911131328A CN111096459B CN 111096459 B CN111096459 B CN 111096459B CN 201911131328 A CN201911131328 A CN 201911131328A CN 111096459 B CN111096459 B CN 111096459B
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Abstract
The invention discloses a preservation number of CCTCC NO: the Lactobacillus plantarum LP33(Lactobacillus plantarum LP33) of M2019594 and the application of the Lactobacillus plantarum in the preparation of health-care food and medicines for promoting lead excretion have important practical significance for developing functional health-care products, enriching the types of fermentation products and establishing a Lactobacillus strain resource library, and simultaneously bring new hopes for preventing lead poisoning.
Description
Technical Field
The invention relates to an application of lactobacillus plantarum in preparation of a product for preventing lead poisoning.
Background
Lead (Pb) is a toxic heavy metal element that is not essential to the human body, which is widely present in nature, and can cause many acute and chronic diseases. Lead is widely used in the manufacture of lead storage batteries, wire sheaths, lead pipes, chemical reaction vessels, pigments, pesticides and the like. Millions of tons of lead are consumed every year all over the world, only less than 25 percent of lead can be recycled, and the remaining 75 percent of lead can enter the environment through waste water, waste gas, waste residues and the like, so that pollution is generated, and animal and plant as well as human health are harmed. Most of the lead and its compounds infect through the respiratory and digestive tracts, a small part through the skin, and about 40% of the lead is absorbed through the respiratory tract. Lead enters a human body, is absorbed, transported and redistributed, and then is accumulated in different organs, so that the lead load is increased, finally 90-95% of lead is mainly deposited in bones, the half-life period in the bones can reach 25 years, the toxicity of the lead is continuously caused, a plurality of systems such as nerves, hematopoiesis, cardiovascular, immunity, liver, kidney, bones and the like are involved, children in the growth and development period are particularly sensitive to lead exposure, and the damage caused by the lead exposure cannot be completely repaired. Lead exposure is a serious public health problem in many countries and, despite efforts to control and reduce the environmental pollution caused by lead, contact lead pollution remains a serious global problem, especially in public health, where lead exposure in the occupational environment is always of the greatest concern. Clinically, the typical acute lead poisoning cases are rare, and chronic injuries caused by long-term low-level lead contact are common, so that the acute lead poisoning cases are widely concerned.
At present, chelating agents are mainly used for promoting lead discharge in vivo, and the chelating agents commonly used clinically mainly comprise dimercaptosuccinic acid (DMSA), dimercaptopropanol, edetic acid and penicillamine, wherein the DMSA has good water solubility and fat solubility, high bioavailability, can reduce the absorption of the gastrointestinal tract to lead, increase the lead discharge, effectively reduce the level of blood lead and reduce the damage of the lead to the organism, and is generally used clinically. However, DMSA has various toxic and side effects in application, and oral administration can cause digestive tract symptoms and rash; in addition, the chelating agent is mainly used for acute lead poisoning, and cannot completely repair tissue damage caused by lead.
Most lactic acid bacteria are considered to be nonpathogenic, safe microorganisms, are involved in numerous metabolic activities in the body, and have important probiotic properties. Such as relieving lactose intolerance, lowering cholesterol, improving immune function, resisting oxidation, and inhibiting adhesion of certain pathogens. Heavy metal exposure can cause oxidative stress injury to organisms, damage intestinal barriers and change intestinal permeability, so that heavy metals can be absorbed by the organisms through the intestinal barriers. Researches find that the lactic acid bacteria have good antioxidant activity, can repair oxidative stress damage caused by various factors to the intestinal tract, and improve the barrier function of the intestinal tract of a host. Thereby protecting intestinal barrier of host and inhibiting absorption of heavy metal in intestinal tract, reducing accumulation of heavy metal in organism, and eliminating heavy metal harm. As people expect to reduce heavy metal pollution from the perspective of higher safety and higher efficiency, lactic acid bacteria are increasingly concerned as probiotics of food safety level, and the property of adsorbing heavy metals is concerned. If lactobacillus is added into food containing heavy metal to remove the heavy metal as heavy metal adsorbent or to supplement and ingest the heavy metal in vivo as diet, the heavy metal is preemptively combined with the heavy metal before being absorbed by intestinal tract, and the heavy metal is discharged out of the body through feces, so that the toxic damage of the heavy metal to the body can be inhibited. Therefore, lactic acid bacteria have great potential as heavy metal adsorbents.
Disclosure of Invention
The invention aims to provide the application of a lactobacillus strain which has strong resistance of digestive tract and good antioxidation effect and can prevent or treat lead poisoning in developing functional health products, foods or medicines and the like;
the invention also aims to provide a pharmaceutical composition, food, health-care product and food additive containing the lactobacillus plantarum.
Through research, the invention provides the following technical scheme:
the invention discloses an application of Lactobacillus plantarum LP33, and an application of Lactobacillus plantarum LP33 in preparation of food for preventing lead poisoning, wherein the preservation number of Lactobacillus plantarum LP33(Lactobacillus plantarum LP33) is CCTCC NO: m2019594.
The invention also discloses an application of the Lactobacillus plantarum LP33, and an application of the Lactobacillus plantarum LP33 in preparing a health-care product for preventing lead poisoning, wherein the preservation number of the Lactobacillus plantarum LP33(Lactobacillus plantarum LP33) is CCTCC NO: m2019594.
The invention also discloses an application of lactobacillus plantarum LP33, and an application of lactobacillus plantarum LP33 in preparation of a medicine for preventing or treating lead poisoning, wherein the preservation number of lactobacillus plantarum LP33(Lactobacillus plantarum LP33) is CCTCC NO: m2019594.
The invention further discloses a pharmaceutical composition for treating or preventing lead poisoning, which contains a compound with a preservation number of CCTCC NO: a pharmaceutically effective dose of M2019594 of lactobacillus plantarum LP 33.
The invention further discloses a food for preventing lead poisoning, which contains the following components in the preservation number of CCTCC NO: m2019594 Lactobacillus plantarum LP 33.
The invention further discloses a health product for preventing lead poisoning, which contains the following components in the preservation number of CCTCC NO: m2019594 Lactobacillus plantarum LP 33.
The invention further discloses a food additive for preventing lead poisoning, which contains a food additive with a preservation number of CCTCC NO: m2019594 Lactobacillus plantarum LP 33.
According to a strain list available for food, which is published by the Ministry of health in 2010, 37 strains are selected from a laboratory strain bank for in vitro screening. Through lead adsorption experiments, model artificial gastric juice and bile salt tolerance experiments and strain antioxidant experiments, comprehensive comparison shows that the lactobacillus plantarum LP33 is the most potential target strain for relieving lead toxicity injury, the lead ion clearance rate of the lactobacillus plantarum LP33 in a 50mg/L lead ion solution reaches 55.63%, the survival rate of the lactobacillus plantarum LP33 in artificial gastric juice with pH of 3.0 is 104.08%, the growth rate of the lactobacillus plantarum in 0.30% bile salt is 20.86%, and the lactobacillus plantarum has a good antioxidant effect. The surface of the fungus body can adsorb lead ions through a scanning electron microscope; the observation of a transmission electron microscope shows that not only the surface of the thalli can adsorb lead ions, but also part of the lead ions enter the thalli.
The invention examines the prevention effect of lactobacillus plantarum LP33 on chronic lead poisoning. The results show that the treatment of lactobacillus plantarum LP33 can obviously reduce the blood lead of chronic lead-exposed rats, obviously improve the inflammatory cell infiltration phenomenon of liver tissues, obviously reduce delta-ALAD, AChE, ALT, AST, BUN and CRE in serum, reduce the MDA level of each tissue, recover the GSH level, CAT and SOD activity of each tissue, obviously reduce the levels of proinflammatory factors TNF-alpha, IL-1 beta and IL-6, obviously increase the level of immunosuppressive cytokine IL-10, and up-regulate the mRNA expression of Nrf2, NOQ1, HO-1, SOD and CAT. These results indicate that lactobacillus plantarum LP33 can ameliorate chronic lead exposure-induced oxidative damage by activating the Nrf2-ARE pathway. Therefore, the lactobacillus plantarum LP33 can be used for preparing health-care food and medicines for preventing lead poisoning.
The invention has the beneficial effects that: the invention utilizes the Chinese characteristic lactobacillus resource strain library to screen out a lactobacillus strain, namely lactobacillus plantarum LP33, which has strong resistance of digestive tract and good antioxidation effect and can prevent lead poisoning, has important practical significance for developing functional health care products, enriching the types of fermentation products and establishing the lactobacillus strain resource library, and simultaneously brings new hope for preventing lead poisoning.
Preservation information
The preservation unit: china center for type culture Collection; address: wuhan university in Wuhan, China; the preservation date is as follows: 8 month 1 in 2019; the preservation number is: CCTCC NO: m2019594; and (3) classification and naming: lactobacillus plantarum LP33(Lactobacillus plantarum LP 33).
Drawings
FIG. 1 shows the colony morphology and gram-stain results of the isolated strains.
FIG. 2 shows that the adsorption capacity of the strain for lead ions was tested under the conditions of lead ion concentrations of 50mg/L (A) and 500mg/L (B).
FIG. 3 API50CH reaction results for Lactobacillus plantarum LP 33.
FIG. 4 shows scanning electron microscope and energy dispersive X-ray spectroscopy scanning results of lead adsorption of Lactobacillus plantarum LP 33.
FIG. 5 transmission electron microscope scanning results of lead adsorption by Lactobacillus plantarum 33.
FIG. 6 weekly body weight of rats in each group
FIG. 7 Effect of Lactobacillus plantarum LP33 on blood lead content in chronic lead-exposed rats.
FIG. 8 effect of Lactobacillus plantarum LP33 on delta-ALAD in serum and AchE in brain tissue of chronic lead-exposed rats.
FIG. 9 Effect of Lactobacillus plantarum LP33 on ALT, AST, CRE and BUN in serum of chronic lead-exposed rats.
FIG. 10 pathological section observation of liver tissue.
FIG. 11 Effect of Lactobacillus plantarum LP33 on cytokines in serum of chronic lead-exposed rats.
FIG. 12 Effect of Lactobacillus plantarum LP33 on the expression of relevant mRNA in liver tissue of chronic lead-exposed rats.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Screening of lactobacillus capable of adsorbing lead ions and research of adsorption mechanism of lactobacillus
1 materials of the experiment
According to the List of strains available for food issued by the Ministry of health, 37 strains were selected from the bacterial bank of the Soxhlet laboratory of the university of southwest food science institute for subsequent experiments. The strain of the strain stock is obtained by separating traditional fermented milk products in high-altitude areas such as Hongyuan, Qinghai, Xinjiang and the like and traditional fermented vegetables in Chongqing areas.
2 method of experiment
2.1 activation and culture of lactic acid bacteria
The strain preserved in the ampoule tube of the laboratory is inoculated in MRS liquid culture medium, incubated for 18h in a constant temperature incubator at 37 ℃, inoculated in the MRS liquid culture medium at a ratio of 2% (v/v) for activation, and morphologically observed by gram staining. The strain was activated for two generations and then used in subsequent experiments.
2.2PCR amplification of 16S rDNA sequences
And extracting the DNA of the strain by using a bacterial genome DNA extraction kit. PCR amplification is carried out by adopting a 25 mu L reaction system, and agarose gel electrophoresis is used for detection after the reaction is finished. The qualified samples were sent to Huada Gene science and technology Co., Ltd for sequencing, and the sequencing results were subjected to homology comparison analysis by BLAST program in NCBI.
2.3 determination of lead adsorption Capacity of Strain
After all activated strains are subjected to amplification culture at 37 ℃ for 18h, thalli are obtained by centrifuging at 8000 Xg for 10min, the thalli obtained by centrifuging are cleaned twice by using sterilized ultrapure water, and the centrifuging process is repeated to obtain the lactobacillus thalli. A certain amount of lead acetate was weighed, dissolved in ultrapure water to give a final concentration of lead ions of 50mg/L, and sterilized by filtration through a 0.22um filter. The cells obtained by centrifugation were suspended in the above lead solution so that the concentration of lactic acid bacteria therein became 1g/L of the wet cell weight. The p H value of the bacterial suspension obtained above was adjusted to 6.0, and the suspension was incubated at 37 ℃ for 1 hour with shaking, and then centrifuged at 8000 Xg for 10min to obtain a supernatant, and the lead concentration of the supernatant was measured using an atomic absorption spectrophotometer. The adsorption capacity of the lactic acid bacteria to lead ions can be calculated by the following formula:
in the formula:
C O concentration of lead ions in the initial solution
C 1 Concentration of lead ions in the solution after adsorption of the Strain
According to the results of the adsorption capacity of the strains under the lead ion condition of 50mg/L, lactic acid bacteria with the lead ion adsorption capacity of more than 50% are selected from 37 lactic acid bacteria strains, 8 primary-screened strains are re-suspended in the lead ion concentration condition of 500mg/L for re-screening, and the adsorption capacity is further evaluated.
2.4 determination of the ability of the strains to withstand simulated gastrointestinal fluids
2.4.1 determination of the survival Rate of the Strain in Artificial gastric juice at pH3.0
The separated strain is cultured for 18h at 37 ℃, the thalli are collected by centrifugation under the conditions of 3000r/min and 15min, and the thalli are washed by sterile normal saline and then are resuspended into bacterial suspension. Mixing the obtained bacterial suspension with artificial gastric juice (0.2% NaCl, 0.35% pepsin 1: 10000, pH is adjusted to 3.00 with 1mol/L HCl) at a volume ratio of 1: 9, culturing at 37 deg.C for 3h, measuring viable count of 0h and 3h respectively by plate coating method, and calculating the survival rate of the strain in artificial gastric juice with pH of 3.00 according to formula (1).
In the formula:
c-survival,%;
m 1 -viable count of 3h, CFU/mL;
m 2 -0 h viable count, CFU/mL.
2.4.2 determination of the growth efficiency of the strains in 0.3% bile salts
The separated strain is cultured for 18h at 37 ℃, inoculated in MRS-THIO culture medium containing 0.00 percent and 0.30 percent of bovine bile salt by the inoculum size of 2 percent respectively, the growth rate of the separated strain is measured after the separated strain is cultured for 24h at 37 ℃, and the growth rate of the separated strain in the bile salt is calculated according to the formula (2) by taking the liquid culture medium without inoculated bacterial liquid as a blank control.
In the formula:
c-growth rate,%;
A 0 blank control OD 600nm A value;
A 1 culture Medium OD containing 0.00% bile salts 600nm A value;
A 2 culture Medium OD containing 0.30% bile salts 600nm The value is obtained.
2.5 determination of the antioxidant Activity of lactic acid bacteria
Selecting the bacterial strain with good performance and lead ion rate more than or equal to 45 percent and growth rate more than 10 percent in 0.30 percent of bile salt to carry out the next step of antioxidant capacity determination.
2.5.1 preparation of lactic acid bacteria samples
The cultured lactobacillus bacterial solution was centrifuged at 8000 Xg for 20min to obtain bacterial cells, washed twice with PBS (p H ═ 7.2), resuspended in PBS, and the bacterial solution concentration was adjusted to OD 600 The value is 1.0, and two 5mL bacterial suspensions are respectively taken for standby. One group of the cells is taken as intact cells, the other group of the cells is placed in an ice bath for 10min of ultrasonic disruption (amplitude transformer phi 6; ultrasonic on for 4 s; ultrasonic off for 4 s; power for 46%), then the cells are centrifuged for 10min at 8000r/min and 4 ℃, and supernatant is taken and put in a sterile centrifuge tube, thus obtaining the cell-free extract.
2.5.2 determination of the ability of lactic acid bacteria to scavenge the free radical of diphenyltrinitrohydrazine
According to the method of the reference document, 1mL of a bacterial liquid supernatant and 1mL of a cell-free extract are respectively taken, 1mL of a 0.2mmol/L DPPH-absolute ethanol solution is added, the mixture is fully mixed and reacted in a dark place for 30min, the mixture is centrifuged at 6000r/min at 4 ℃ for 10min, and the supernatant is absorbed and placed at a wavelength of 517nm to measure the light absorption value. The PBS solution is used as a control group to replace the sample solution to be detected, and the calculation formula is as follows:
in the formula:
d, the absorbance value of a sample to be detected;
D 0 -blank absorbance value.
2.5.3 determination of the ability of lactic acid bacteria to scavenge hydroxyl radicals
According to the literature, the supernatant of the bacterial liquid and the cell-free culture are respectively and uniformly mixed with 1mL of 2.5 mmol/L1, 10-phenanthroline solution and sterile PBS solution (p H ═ 7.4), and then 2.5mmol/L Fe is added 2 SO 4 Solution 1m L, mix well and add 1mL 20mmol/L H 2 O 2 The solution is put into a water bath at 37 ℃ for acting for 1 h. After the water bath is finished, centrifuging at 8000r/min for 10min, sucking the supernatant, and measuring the light absorption value at 536nm wavelength. Replacing the sample solution to be detected with PBS solution as blank control, and replacing H with PBS solution 2 O 2 The solution was used as a negative control and the calculation formula was as follows:
in the formula:
h, the absorbance value of a sample to be detected;
H 0 represents the blank control absorbance value;
H 1 representing the absorbance value of the negative control.
2.5.4 measurement of the reducing ability of lactic acid bacteria
0.5m L of PBS buffer solution (0.2mol/L, p H ═ 6.6) and 0.5m L potassium ferricyanide (mass fraction is 1%) are added into a sample to be tested of 0.5m L, mixed uniformly, placed in a thermostatic waterbath kettle at 50 ℃ for 20min, and placed in ice water for cooling. Adding 0.5m L trichloroacetic acid (mass fraction of 10%), centrifuging at 4000rpm for 10min, collecting 1m L supernatant, adding 1m L distilled water and 1m L ferric trichloride (mass fraction of 0.1%), mixing, reacting for 10min, and measuring light absorption value at 700 nm; PBS was used as a blank instead of the test sample.
In the formula:
A S -absorbance value of the sample to be tested;
A 0 -blank absorbance value.
According to the results, the lactobacillus with excellent lead adsorption capacity, gastrointestinal fluid tolerance simulation capacity and in-vitro oxidation resistance is screened out for subsequent tests.
2.6API kit identification
The separated strain is cultured for 18h at 37 ℃, the thalli are collected by centrifugation under the conditions of 3000r/min and 15min, and the thalli are washed by sterile normal saline and then are resuspended into bacterial suspension. The procedure was performed with reference to the API kit instructions.
2.7 scanning Electron microscope Observation and energy Spectrum scanning before and after lead ion adsorption by Lactobacillus plantarum LP33
Performing an experiment of adsorbing lead ions by LP33 according to the method in 2.3, fixing the centrifuged thallus in 2.5% glutaraldehyde solution at 4 deg.C overnight, and rinsing the sample with 0.1M phosphate buffer solution with pH of 7.0 for three times, each time for 15 min; fixing the sample with 1% osmate solution for 1-2 h; osmate waste was carefully removed and the samples were rinsed three times for 15min each with 0.1M, pH7.0 phosphate buffer; the samples were dehydrated with graded concentrations of ethanol (including five concentrations, 30%, 50%, 70%, 80%, 90% and 95%) for 15min each and then treated twice with 100% ethanol for 20min each. The sample was treated with a mixture of ethanol and isoamyl acetate (V/V. 1/1) for 30min and then with pure isoamyl acetate for 1h or left overnight. Drying at critical point. And (6) coating and observing. The treated sample was observed in a scanning electron microscope.
In addition to not requiring the lead ion adsorption experiment, the blank sample preparation was also performed as described above. Changes in the cell morphology of the cells in the sample were observed using a Scanning Electron Microscope (SEM) and the elemental composition was analyzed using an energy dispersive spectrometer (EDX) connected to it.
2.8 Transmission Electron microscope sample Observation before and after lead ion adsorption by Lactobacillus plantarum LP33
Carrying out an experiment of adsorbing lead ions by LP33 according to the method in 2.3, fixing the thalli obtained by centrifuging in 2.5% glutaraldehyde solution at 4 ℃ overnight, pouring out the fixing solution after the fixation is finished, and rinsing the sample with 0.1M phosphate buffer solution with pH of 7.0 for three times, 15min each time; fixing the sample with 1% osmate solution for 1-2 h; osmate waste was carefully removed and the samples were rinsed three times for 15min each with 0.1M, pH7.0 phosphate buffer; dehydrating the sample with ethanol solution with gradient concentration (including five concentrations of 30%, 50%, 70%, 80%, 90% and 95%), treating for 15min each concentration, and treating with 100% ethanol for 20 min; finally, the treatment is carried out for 20min by using pure acetone. The sample was treated with a mixture of embedding medium and acetone (V/V-1/1) for 1 h; the sample was treated with a mixture of embedding medium and acetone (V/V-3/1) for 3 h; treating the sample with pure embedding agent overnight; embedding the sample after the permeation treatment, and heating the sample at 70 ℃ overnight to obtain the embedded sample. Slicing the sample in an ultrathin slicer to obtain 70-90nm slices, staining the slices with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10min respectively, and observing in a transmission electron microscope
In addition to not requiring the lead ion adsorption experiment, the blank sample preparation was also performed as described above. Observation of changes in cell morphology of bacterial cells in samples Using Transmission Electron microscope (SEM)
2.9 statistical analysis
Each test was performed in 3 replicates and the data were expressed as "mean. + -. standard deviation", and the analysis of variance was performed with SPSS20, with P <0.05 being statistically significant.
3 results and analysis
3.1 colony morphology and cell morphology of the Strain
After being activated, 37 strains form a single colony in an MRS culture medium, the colony morphology is almost consistent, most of the colony is round and white, and the surface is smooth and moist. The purple cell morphology was observed under a microscope after gram staining, and the cells were judged as gram-positive bacteria (G) + ). Among them, the colony morphology and gram staining of the 33-numbered strain are shown in FIG. 1.
3.2 PCR amplification results of 16S rDNA sequence of Strain
The PCR amplified sequence of the strain numbered 33 was identified as Lactobacillus plantarum by NCBI alignment.
3.3 adsorption Capacity of lactic acid bacteria on lead ions
When the lactic acid bacteria capable of relieving lead toxicity is screened, the lactic acid bacteria should have strong lead ion adsorption capacity, so that the lactic acid bacteria can adsorb lead ions in advance before the lead ions enter the intestinal tract of a host to be absorbed and then discharge the lead ions out of the body along with excrement, the absorption of the intestinal tract on the lead ions is reduced, and the accumulation of the lead ions in various organs of the host is reduced. The adsorption capacity of 37 experimental lactic acid bacteria for lead ions was expressed as the adsorption rate (%) for lead ions. As shown in FIG. 2A, when the lead ion concentration was 50mg/L, there was a large difference in the lead ion adsorption capacity of the different strains (from 16.93% to 55.63%). Under the condition, strains with the adsorption rate of more than 50 percent are selected for a re-screening experiment, and the strains are respectively as follows: lactobacillus fermentum 1, 5, 6, 12 and lactobacillus plantarum 7, 26, 30, 33. The results showed that the aluminum ion adsorption capacity of these 8 strains of lactic acid bacteria generally decreased when the initial concentration of lead ions was increased to 500mg/L (FIG. 2B), but Lactobacillus plantarum LP33 still showed the strongest lead ion adsorption capacity (26.83%).
3.4 tolerance of lactic acid bacteria to simulated gastrointestinal fluids
One of the main functions of gastrointestinal fluids is to absorb digestion of food and nutrients, and secondly, it contains a large amount of enzymes and bile salts which can destroy the structure of cell membranes and is the first barrier to prevent microorganisms from entering the gastrointestinal tract. Therefore, the ideal functional strain should have good acid and bile salt resistance, and can ensure that the strain can successfully pass through the gastric acid environment to reach the intestinal tract and can continuously maintain the activity in the intestinal tract. According to the research, the tolerance of lactic acid bacteria is tested by selecting artificial gastric juice with pH value of 3.0 and bile salt concentration of 0.3% according to the pH value and bile salt concentration of gastric juice and intestinal juice in normal human physiological environment and the retention time of food in the stomach and intestinal tract, so that the bacterial strain with better tolerance is screened out. The research result shows that the bacterium 33 shows good bile salt and gastric acid resistance, the survival rate of the bacterium 33 in artificial gastric juice reaches 104.08%, the growth rate of bile salt reaches 20.86% in 0.30%, the survival rate of the bacterium 2 in artificial gastric juice is 78.69%, and the growth rate of bile salt is only 5.56% in 0.30%.
3.5 antioxidant capacity of lactic acid bacteria
3.5.1 ability of lactic acid bacteria to scavenge DPPH free radicals
DPPH is a stable artificially synthesized free radical taking nitrogen as a center, when a free radical quencher or an antioxidant reacts with the free radical quencher or the antioxidant, a solution is converted from dark purple to light yellow or colorless, so that the free radical scavenging condition in a sample can be quantitatively detected by measuring the change of the absorbance value at 517nm, and the free radical scavenging capability of the sample to be detected is further evaluated. The clearance rate of DPPH of the bacteria 33 whole cell suspension is higher than that of a cell-free extracting solution by 23.20 percent. For cell-free extracts, bacteria 33 showed the highest DPPH clearance (13.63%).
3.5.2 lactic acid bacteria ability to scavenge hydroxyl radicals
Hydroxyl radical (. OH) is a strongly oxidizing ROS that can disrupt the permeability of biological cell membranes and cause oxidative damage to DNA, disrupting normal cell function. Therefore, the hydroxyl radical scavenging ability is an important index for evaluating the antioxidant activity of the lactic acid bacteria. The complete cell suspension of the bacterium 33 shows the strongest hydroxyl free radical clearance rate (30.81%), and the clearance rate of the bacterium 33 is higher than 25% and obviously higher than other strains (P <0.05) in the case of a cell-free extracting solution.
3.5.3 reducing power of lactic acid bacteria
The reducing ability mainly means that some enzyme substances of redox reaction and some non-enzyme compounds with antioxidant ability not only inhibit the generation of ROS, but also can control Fe 2+ And the transition metal ions, thereby effectively preventing the generation of oxidation reaction, which is called reducing power. Therefore, the reducing ability is often selected as an index for evaluating the antioxidant ability of the strain. The bacterium 33 has the strongest reduction capability, and the reduction capability of the whole cell suspension or the cell-free extracting solution is more than 90 percent and is obviously superior to that of other strains (P < 0.05).
3.6 Biochemical characterization of the best resistant Strain
And comprehensively comparing the lead adsorption capacity, the tolerance of artificial gastric juice and bile salt and the antioxidant evaluation result of the strain, wherein the strain with the number of 33 is the best resistant strain. Phenotypic identification at the lactobacillus species level is based primarily on carbohydrate fermentation assays. The API50CH kit was identified by the utilization of 49 different carbohydrates by the strain.
Figure 3 shows the API50CH reaction results for strain No. 33. Table 1 shows the results of fermentation tests of strain number 33 on 49 carbohydrates. As can be seen from fig. 3 and table 1, among the 49 carbon sources tested, the 33 numbered strain can utilize 25 of the carbohydrates. Finally identified by API lab plus system, the strain with number 33 is Lactobacillus plantarum (Lactobacillus plantarum), with an ID value of 95.00% and a T value of 0.36. The ID value of the strain does not reach more than 99.0 percent, which indicates that the strain is a new variety of the lactobacillus plantarum.
TABLE 1 fermentation test results of bacterium 33 on 49 carbohydrates
3.7 Electron microscopy and energy Spectroscopy scanning of LP33 lead adsorption
As shown in FIG. 2, compared with the normal blank control bacteria (FIG. 4A), the results of scanning electron microscopy show that some new irregular particles appear on the surface of the bacteria after the lead ion adsorption test, and some particles are aggregated. Meanwhile, we also found that the rupture of the thallus occurs. The energy dispersive X-ray spectroscopy (EDX) was used to perform spectral scanning on the partial regions before and after the adsorption of lead ions, and the results showed that the cell scanning spectrum of the lead ion exposed group was significantly higher than the lead element peaks (fig. 4C and D) of the blank control group. As shown in table 2, the lead element proportion of the lead ion exposed group reached 0.18% in terms of element atomic percentage, whereas the lead element proportion of the blank group was only 0.03%. This result further confirmed the adsorption of lead ions by Lactobacillus plantarum LP33 cells.
TABLE 2 atomic percentages of elements of the thalli before and after EDX scanning lead adsorption
The result of the transmission electron microscope is shown in fig. 3, after the adsorption is completed, obvious sediments appear at the periphery of the thallus, and partial sediments are found in the protoplast (fig. 5B); similar deposits did not appear around the bacteria in the blank control group (FIG. 5A). This result is consistent with the scanning electron microscope results.
Second, the relieving effect of lactobacillus plantarum on lead poisoning rats
1 materials of the experiment
The strain is as follows: lactobacillus plantarum LP33(LP33) is deposited at the China center for type culture Collection (deposit number: M2018592) and the college of food science, university in southwest (deposit number: LP 33).
Experimental animals: 30 male SD rats of 4-6 weeks old are purchased from the Experimental animals center of Chongqing medical university, and the license numbers are as follows: SCXK (Yu) 2018-.
2 method of experiment
2.1 Experimental animal grouping and handling
TABLE 3 animal experimental design protocol
Note: ordinary water: the lead-free common drinking water is freely drunk by rats; lead water: dissolving lead acetate trihydrate into drinking water to enable the concentration of the lead acetate trihydrate to reach 500mg/l, and enabling the lead acetate trihydrate to be freely drunk by mice; LP 33: the concentration of the bacterial suspension is 1X 10 9 CFU/ml, the gavage dosage is determined according to gavage 0.1ml per 100g of body weight; physiological saline: the gavage dose is determined by gavage at 1ml per 100g of body weight. The experiment lasted 8 weeks.
As shown in table 3, 30 SD rats were randomly divided into 3 groups of normal group (control), model group (Pb only), and bacteria 33 group (Pb + LP33), 10 of each group, and the experiment was started one week after acclimation, with an experimental period of 8 weeks. The mental state, hair color, diet, activity and the like of the mice were observed. Rat body weights were measured periodically weekly.
2.2 sample Collection and preservation
After 8 weeks of experiment, the rats were fasted and deprived of water for 18h, then weighed, and blood was taken from the eyeballs after ether stunning. Taking 2ml of whole blood for detecting blood lead, putting the whole blood into a heparin sodium tube, and turning the whole blood upside down and mixing the whole blood with the heparin sodium tube uniformly to be detected. Centrifuging the rest blood at 4 deg.C and 3000r/min for 10min to collect serum, and storing at-80 deg.C. Taking blood, removing cervical vertebra, killing rat, quickly dissecting and taking out liver, kidney, brain and heart of rat, removing fat and connective tissue around viscera, washing surface blood stain of viscera with physiological saline, drying with filter paper, weighing, recording data, and calculating viscera coefficient. Cutting soybean liver tissue, fixing in 10% formalin solution for HE staining, and labeling the rest part at-80 deg.C.
2.3 determination of the lead content in blood
Transferring the blood sample to an erlenmeyer flask soaked with 20% nitric acid overnight, adding 10ml of concentrated nitric acid, standing overnight, and then placing on an adjustable electric hot plate for digestion until the digestive juice is colorless and transparent or slightly yellowish. And (4) after cooling, fixing the volume, and measuring the content of the lead in the sample by using a flame atomic absorption spectrophotometer.
2.4 liver tissue section (H & E staining)
Liver tissues were fixed in 10% formalin for 24H, stained with H & E (Hematoxylin and eosin staining, Hematoxylin-eosin staining), and then the pathological morphology of rat liver tissues was observed by means of an upright microscope, comprising the following steps:
(1) fixing: cutting liver tissue of soybean size, and fixing in 10% formalin solution for 24 hr;
(2) embedding: after fixation and dehydration, embedding by adopting paraffin;
(3) slicing: cutting the slices into 0.4um slices by a paraffin slicer;
(4) tabletting: pasting the slices with the thickness of 0.4um on a glass slide, and drying by adopting a baking sheet machine;
(5) dewaxing: dewaxing in xylene (I) for 5min, dewaxing in fresh xylene (II) for 5min again, then using absolute ethyl alcohol for 5min, 95% ethyl alcohol for 2min, 80% ethyl alcohol for 2min, 70% ethyl alcohol for 2min, and finally washing with distilled water for 2 min;
(6) placing the slices in hematoxylin staining solution for staining for 2-8min, washing off excessive staining solution with tap water, and then differentiating in differentiation solution (1% hydrochloric acid alcohol) for 30s, and soaking in tap water for 15 min;
(7) placing the slices in eosin staining solution for 1-2min, washing off excessive staining solution with tap water, and soaking in tap water for 5 min;
(8) placing the slices in 95% ethanol solution (I), 95% ethanol solution (II), anhydrous ethanol (I), anhydrous ethanol (II), xylene carbolic acid (3: 1), xylene (I), and xylene (II) in sequence, reacting for 1min, sealing with neutral gum, and observing under microscope.
2.5 measurement of Delta-ALAD in serum and AchE levels in brain tissue
The content of delta-ALAD in the large serum and the AchE level in the brain tissue homogenate were measured according to the ELISA kit instructions.
2.6 determination of antioxidant index of serum and tissue
And measuring the T-SOD enzyme activity, CAT enzyme activity, GSH enzyme activity and MDA content in the rat serum and tissues according to the conventional biochemical kit specification.
2.7 measurement of inflammatory factors, lipopolysaccharide and liver and kidney injury indexes in serum
The contents of LPS, TNF-alpha, IL-1 beta, IL-6, IL-10, ALT, AST, BUN and CRE in rat serum were determined according to the ELISA kit instructions.
2.8qRT-PCR determination of mRNA expression of related genes in tissues
Ileal and liver total RNA was extracted according to Trizol (Invitrogen, carlsbad, ca, usa) instructions; then 1 mu L of RNA sample of 1 mu L is taken, 1 mu L of (oligo) primer dT and 10 mu L of sterile ultrapure water are added, the mixture reacts for 5min at 65 ℃, after the Reaction is finished, 1 mu L of RiblockRNase Inhibitor, 2 mu L of 100mM dNTP mix, 4 mu L of 5 × Reaction buffer and 1 mu L of reverse AidM-mu/v RT are added into the Reaction system, and after the mixture is mixed uniformly, cDNA is synthesized under the conditions of 42 ℃, 60min, 70 ℃ and 5 min; the purity and concentration of the total cDNA were measured by a ultramicro-spectrophotometer, and the cDNA concentration of each sample was adjusted to the same level (1. mu.g/. mu.L). Then, the target gene was reverse transcribed and amplified with the primer sequences shown in Table 4, and the reaction was carried outThe conditions are as follows: pre-denaturation at 95 ℃ for 10 min; 40 cycles of 95 deg.C, 15s, 60 deg.C, 1min, 72 deg.C, 30 s; finally, GAPDH was used as an internal reference gene, through 2 -ΔΔCT Calculating the relative expression amount of the target gene.
TABLE 4 primer sequences
2.9 data analysis
The data were analyzed for significance using one-way anova in sps 17.0 (p <0.05) and the final results are expressed as mean ± standard deviation (x ± s). The figures used herein were made by Graphpad software.
3 results and analysis of the experiments
3.1 general Condition Observation of rats during the course of the experiment
During the whole experiment, the rats in each group eat normally, the shape, appearance and color of the excrement of the rats in each group are not obviously abnormal, and the rats do not die. From week 3, rats with plumbum group showed clinical symptoms of anorexia, listlessness, dull, somnolence, etc., and the fur gradually lost luster; the rats in the fungus 33 group also have the symptoms of anorexia, hypoactivity, listlessness and the like, but the fur changes little; the normal group of rats had no abnormal condition. As shown in fig. 6, the body weights of the mice in the model group and the normal group were significantly different from each other from week 4 (P <0.05), and the mice in the bacteria 33 group and the rats in the model group were also significantly different from each other at week 8 (P < 0.05).
3.2 Effect of Lactobacillus plantarum LP33 on blood lead content
The lead content in rat blood is shown in FIG. 7. The lead content was much higher in the lead-exposed group than in the normal group, the blood lead content was significantly lower in the Pb + LP33 and Pb + LF2 groups than in the model group and the lead content was significantly lower in the Pb + LP33 group than in the Pb + LF2 group (P < 0.05). The research result shows that the lactobacillus plantarum LP33 can remarkably reduce the lead content in the blood of the chronic lead-exposed mice.
3.3 Effect of Lactobacillus plantarum LP33 on the organ coefficient of chronic lead-exposed rats
The organ coefficient is the ratio of the weight of a certain organ to the body weight of an experimental animal, and is a commonly used index in toxicological experiments. As shown in table 5, the organ coefficients of the heart were decreased in both the model group and the bacteria 33 group compared to the normal group, and there was a significant difference between the model group and the normal group, and there was no significant difference between the bacteria 33 group and the other two groups (P < 0.05). Similarly, the brain organ coefficients of the model group and the bacterium 33 group are reduced compared with those of the control group, the lead staining group and the normal group have significant difference, and the bacterium 33 group and the other two groups have no significant difference (P < 0.05). In contrast, the organ coefficients of the kidney were elevated in both the model group and the bacteria 33 group compared to the normal group, and significant differences were observed between the model group and the bacteria 33 group and the normal group (P < 0.05). There was no significant change in liver coefficients between groups (P < 0.05).
TABLE 5 organ coefficients
3.4 Effect of Lactobacillus plantarum LP33 on delta-ALAD in serum and AchE in brain tissue of chronic lead-exposed rats.
Lead is absorbed and rapidly enters blood, and the synthesis of heme is inhibited by inhibiting the activity of delta-aminolevulinic acid dehydratase (delta-ALAD). As shown in FIG. 8A, the levels of delta-ALAD in the serum of the lead-exposed rats were significantly decreased compared to the normal group and the bacteria 33 group, and were significantly different from the normal group (P < 0.05). The Lactobacillus plantarum LP33 group significantly elevated the level of delta-ALAD in the serum of lead-exposed rats (P < 0.05).
Acetylcholinesterase (ACh E) can catalyze the hydrolysis of acetylcholine into active metabolites, choline and acetic acid, is a key enzyme involved in cholinergic neurotransmission, and the activity of the enzyme is an intuitive reflection of the metabolic condition and level change condition of acetylcholine in brain tissues, so the determination of ACh E activity is one of indexes for elucidating whether the nervous system functions normally or not. Lead exposure obviously promotes the activity enhancement of ACh E, thereby lifting the accelerated decomposition of acetylcholine, influencing the transmission of nervous system transmitters, breaking the internal dynamic balance of brain tissues, and finally destroying the behavior of normal nervous system to induce learning and memory ability and the like to generate abnormity. However, the gavage lactobacillus plantarum 33 significantly reduced ACh E activity (fig. 8B) (P < 0.05).
3.5 Effect of Lactobacillus plantarum LP33 on the Oxidation index of serum and various tissues in chronic lead-exposed rats
One of the major mechanisms of lead poisoning is the induction of oxidative stress states. As shown by the results in tables 5 and 6, chronic lead ion exposure significantly increased MDA levels in serum, liver, kidney, heart and brain tissues, and decreased the corresponding GSH, SOD, CAT levels (P < 0.05). Lead exposure induces the production of active oxygen, and the reduction and excessive consumption of enzyme activities important in body oxidative defense systems such as GSH, SOD and CAT are major causes of lead-induced body oxidative stress states. The MDA level of the Lactobacillus plantarum LP33 serum, liver, kidney, heart and brain tissues is high, and the GSH level, CAT and SOD activity of each tissue are recovered, so that the strain can effectively relieve the oxidative damage caused by lead exposure.
TABLE 5 Effect of Lactobacillus plantarum LP33 on the oxidative index in the serum of lead-exposed mice
TABLE 6 Effect of Lactobacillus plantarum LP33 on the oxidative index in lead-exposed mouse tissues
3.6 Effect of Lactobacillus plantarum LP33 on ALT, AST, CRE and BUN in serum of chronic lead-exposed rats
As shown in the results of fig. 9, chronic lead exposure increased ALT, AST, BUN and CRE levels in rat serum. ALT and AST are important indicators of liver injury, while BUN and CRE are considered markers of kidney injury. The lactobacillus plantarum 33 treatment can significantly improve the above-mentioned indexes, and these results indicate that the lactobacillus has a protective effect on liver and kidney damage caused by chronic lead ion exposure.
3.7 Effect of Lactobacillus plantarum LP33 on histopathological morphology of liver in chronic lead-exposed rats
Morphological changes of colon tissues were observed by HE staining, and the results are shown in fig. 10. The normal group of rats has complete liver tissue structure, clear shape, orderly radial arrangement of liver cells, normal structure and clear level; chronic lead exposure resulted in pathological damage to the rat liver, inflammatory cell infiltration of the liver, loss of hepatic lobule structural integrity, and steatosis compared to the normal group. Treatment with Lactobacillus plantarum LP33 significantly ameliorated the pathological lesions described above. The histopathological results further prove that the lactobacillus plantarum LP33 has a certain protective effect on liver injury caused by lead ion exposure.
3.8 Effect of Lactobacillus plantarum LP33 on cytokines in serum of chronic lead-exposed rats
Cytokines are small molecular proteins or polypeptides which are stimulated by immune cells (and partial non-immune cells) to synthesize the substances with immunoregulation and physiological effect functions, the cytokine level change of intestinal epithelial cells is closely related to the immune state of the intestinal epithelial cells, and the abnormal cell level often represents the inflammatory reaction or other physiological disorders of the intestinal tract. TNF-alpha, IL-1 beta, IL-6 are widely recognized as pro-inflammatory cytokines secreted by body tissues, including the intestinal tract. IL-10 is widely recognized as an immunosuppressive cytokine that plays a major role in the intestinal tract to suppress inflammatory responses and maintain immune homeostasis. In this experiment, lead exposure resulted in significantly increased levels of cytokines such as TNF-alpha, IL-1 beta, IL-6, etc. (FIG. 11), suggesting that lead toxicity may cause inflammatory responses in intestinal cells. And the lactobacillus plantarum LP33 with gastric perfusion remarkably reduces the level of the proinflammatory cytokine and also remarkably increases the level of immunosuppressive cytokine IL-10, which proves that the lactobacillus plantarum LP33 can really have the function of relieving the inflammatory response induced by lead exposure. Numerous studies have shown that probiotics have the effect of modulating the intestinal immune system and suppressing inflammatory responses, which may be one of the mechanisms by which lactobacillus plantarum LP33 affects cytokine levels; on the other hand, the lead adsorption capacity and the antioxidation effect of the strain can weaken the toxic effect of lead exposure on intestinal cells, thereby inhibiting inflammatory reaction.
3.9 Effect of Lactobacillus plantarum LP33 on the expression of related mRNA in liver tissue of chronic lead-exposed rats
The Keap1-Nrf2-ARE signal path is the most important antioxidant signal path in the organism. When oxidative stress occurs or is stimulated by other chemical substances, the Nrf2 is dissociated from the Keap1 through phosphorylation, the Nrf2 enters the cell nucleus and is combined with a promoter ARE sequence of a related gene to further induce the expression of downstream target genes regulated and controlled by the gene, such as heme oxygenase 1(HO-1), reduced coenzyme I/II quinone oxidoreductase 1(NQO1), superoxide dismutase (SOD), Catalase (CAT) and the like, and the like ARE activated by antioxidant and detoxifying enzymes, so that various cytoprotective effects of antioxidant stress, anti-inflammation, anti-apoptosis and the like ARE achieved.
FIG. 12 shows the effect of Lactobacillus plantarum LP33 on Nrf2, NQO1, HO-1, SOD1, SOD2, and CAT mRNA expression in liver tissue. As can be seen from the figure, except that the liver tissues of the model mice are slightly higher than those of the normal group in Nrf2, NQO1 and HO-1, the expression of SOD1, SOD2 and CAT mRNA is obviously lower than that of the normal group. After being treated by lactobacillus plantarum LP33, the expression of Nrf2, NQO1, HO-1, SOD1, SOD2 and CAT mRNA is obviously improved compared with that of a model group. Therefore, the lactobacillus plantarum LP33 can improve the oxidative damage caused by chronic lead poisoning by activating the Keap1-Nrf2-ARE signal pathway.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (2)
1. The application of lactobacillus plantarum LP33 is characterized in that: application of lactobacillus plantarum LP33 in preparation of medicine for preventing or treating lead poisoning, wherein lactobacillus plantarum LP33 (A) (B)Lactobacillus plantarum LP33) The preservation number of (A) is CCTCC NO: m2019594.
2. A pharmaceutical composition for preventing or treating lead poisoning, characterized in that: the medicine composition contains a medicine with a preservation number of CCTCC NO: a pharmaceutically effective dose of M2019594 of lactobacillus plantarum LP 33.
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