Application of Weissella mesenteroides in adsorption of metallic lead

Abstract

The present invention belongs to the field of microbial technology and discloses the application of Weissella paramesenteroides in the adsorption of metallic lead. The Weissella paramesenteroides is Weissella paramesenteroides SL7, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, with a deposit number of GDMCC No.62429. The Weissella paramesenteroides SL7 of the present invention has a strong tolerance to lead ions, is acid-resistant, can grow well in the pH range of 2.5 to 8.0, and can absorb lead ions in the presence of lead. 2+ The concentration ranges from 0 to 1000 mg / L and has a strong adsorption effect. Experiments show that it can reduce the lead content in the blood, liver, kidney and stomach of mice and relieve the pathological symptoms of lead poisoning mice. The Weissella mesenteroides SL7 is used to prepare food that reduces lead accumulation in the body or relieves lead poisoning, and has a very broad application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Weissella mesenteroides in adsorbing metallic lead. Background Art

[0002] Lead is a heavy metal that is extremely harmful to human health. In daily life, popcorn, canned food, preserved eggs, etc., or fruit and vegetable products produced from land contaminated with lead are prone to high lead content. Long-term lead exposure causes people to gradually increase their contact and absorption of lead, leading to lead accumulation in the body. Lead that enters the human body is easy to accumulate and difficult to be discharged, causing long-term damage to the human reproductive system, nervous system, etc. At the same time, due to the large-scale and intensive development of marine aquaculture, the aquaculture density has continued to increase. Due to factors such as urban development and industrial wastewater discharge, heavy metals have also become a common type of pollutant in offshore aquaculture waters and their organisms. Shellfish with high lead pollution levels are mainly Omi oysters, elegant scallops, and Chlamys farreri. The seasonal distribution generally shows a step-by-step increasing trend in spring, summer, autumn, and winter.

[0003] At present, for various physiological diseases caused by lead accumulation and lead poisoning, traditional treatment methods mainly use physical and chemical methods to remove lead, that is, calcium disodium edetate, sodium disulfide succinate and other integrators are injected for treatment to achieve the purpose of removing lead. However, these drugs are inefficient and have certain toxic side effects. Calcium disodium edetate is a broad-spectrum chelating agent with strong nephrotoxicity, and a large amount of trace elements that are indispensable to the human body, such as zinc, copper, manganese, iron, etc., will be excreted through urine when the drug is used. Sodium disulfide succinate may cause digestive system dysfunction and cause symptoms such as headache, nausea, and sore limbs. Because microorganisms are easy to cultivate, reproduce quickly, and have low costs, they have become the focus of heavy metal pollution control research, especially in soil, fresh water and food. Therefore, screening out microorganisms that can strongly adsorb heavy metal lead has great market prospects and significance.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the problems in the background technology, the purpose of the present invention is to provide the use of Weissella mesenteroides in the adsorption of metallic lead.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The application of Weissella paramesenteroides in the adsorption of metallic lead, wherein the Weissella paramesenteroides is Weissella paramesenteroides SL7, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, with the deposit number GDMCCNo:62429.

[0008] Preferably, metallic lead in the body or the environment is adsorbed.

[0009] Preferably, when adsorbing metallic lead in the body, the inoculation amount of Weissella mesenteroides is 1.5% to 3.5%.

[0010] Preferably, adsorbing metallic lead in the environment includes adsorbing metallic lead in water and adsorbing metallic lead in soil.

[0011] Preferably, when adsorbing metallic lead in water, the dosage of Weissella mesenteroides is 10-20 g / L; when adsorbing metallic lead in soil, the dosage of Weissella mesenteroides is 3%-6% of the soil mass.

[0012] Preferably, when adsorbing metallic lead in the body, Weissella mesenteroides is used as a fermentation agent to prepare food for direct consumption.

[0013] Preferably, the preparation of the leavening agent comprises the following steps:

[0014] The Weissella mesenteroides was inoculated into MRS broth medium and cultured at 35-37°C for 18-24 hours to obtain a fermentation liquid;

[0015] Concentrating the fermentation liquid, and adding skimmed milk powder and sucrose to the concentrated liquid to obtain a concentrated liquid; and

[0016] The concentrated liquid is subjected to spray drying under vacuum and low-temperature drying conditions.

[0017] Preferably, calculated by weight percentage of the concentrated liquid, the skimmed milk powder is 2-4% and the sucrose is 1-3%.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The Weissella mesenteroides SL7 of the present invention has a strong tolerance to lead ions. 2+ It has a strong adsorption effect in the concentration range of 0~1000mg / L. Experiments show that it can reduce the lead content in the blood, liver, kidney and stomach of mice, and can alleviate the pathological symptoms of lead poisoning mice. The Weissella mesenteroides SL7 is used to prepare foods that reduce lead accumulation in the body or alleviate lead poisoning, and has a very broad application prospect. At the same time, the application of the Weissella mesenteroides SL7 of the present invention in the environment reduces the harm to human health through the aquatic or soil ecosystem and the food chain food web from the source. The Weissella mesenteroides SL7 has a strong acid and alkali tolerance ability, is easy to cultivate, has a fast reproduction speed, has a strong lead adsorption ability, and has a low cost. It can make outstanding contributions to the adsorption of lead metal in the environment and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1ab are the culture characteristics and microscopic morphology of Weissella mesenteroides, respectively;

[0021] Figure 2 This is the phylogenetic tree of Weissella mesenteroides;

[0022] Figure 3 The growth of Weissella mesenteroides under different pH conditions;

[0023] Figure 4 The lead tolerance of Weissella mesenteroides was measured;

[0024] Figure 5 The lead adsorption of Weissella mesenteroides and the comparison strains;

[0025] Figure 6 The adsorption of Pb by Weissella mesenteroides at different temperatures 2+ The impact of

[0026] Figure 7 The figure shows the effect of Weissella mesenteroides on lead levels in various parts of the body of lead-exposed mice. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.

[0028] An embodiment of the present invention provides the use of Weissella paramesenteroides in the adsorption of metallic lead. The Weissella paramesenteroides is Weissella paramesenteroides SL7, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, with a deposit number of GDMCCNo:62429.

[0029] The Weissella mesenteroides-like bacteria can absorb metal lead in the body or in the environment. Specifically, the Weissella mesenteroides-like bacteria has the following properties:

[0030] (1) It has strong acid resistance and grows well in an environment with a pH of 3.0 to 8.0 and survives well in an environment with a pH of 2.5;

[0031] (2) When cultured in a lead-containing medium in vitro, it has a strong tolerance to lead ions;

[0032] (3) When cultured in a lead-containing aqueous solution in vitro, it has a strong adsorption capacity for lead ions;

[0033] (4) It has the effect of reducing the lead content in lead-exposed mice.

[0034] As some preferred implementation modes, when absorbing metallic lead in the body, the inoculation amount of Weissella mesenteroides is 1.5%~3.5%; when absorbing metallic lead in water, the dosage of Weissella mesenteroides is 10~20g / L; when absorbing metallic lead in soil, the dosage of Weissella mesenteroides is 3%~6%.

[0035] When adsorbing metallic lead in the body, Weissella mesenteroides is used as a fermentation agent to prepare food for direct consumption.

[0036] At this time, the preparation of the leavening agent comprises the following steps:

[0037] The Weissella mesenteroides was inoculated into MRS broth medium and cultured at 35-37°C for 18-24 hours to obtain a fermentation liquid;

[0038] Concentrating the fermentation liquid, and adding skimmed milk powder and sucrose to the concentrated liquid to obtain a concentrated liquid; and

[0039] The concentrated liquid is subjected to spray drying under vacuum and low-temperature drying conditions.

[0040] As some preferred embodiments, calculated by weight percentage of the concentrated liquid, the skimmed milk powder is 2-4% and the sucrose is 1-3%.

[0041] The application and performance of Weissella mesenteroides SL7 are described in detail with multiple specific examples below.

[0042] Example 1 Isolation, purification and identification of strains

[0043] 1.1 Isolation and screening of strains

[0044] The strain required by the present invention is isolated from Sichuan bacon. Under sterile conditions, 25g of the internal components of the bacon are taken, cut into pieces, put into a sterile homogenizing bag, added with 225ml of sterile physiological saline, and beaten in a beating homogenizer for 3min; a 10-fold gradient dilution is made, and a total of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 There are seven concentrations. Take 200 μl of sample and spread it on MRS medium containing 1.5% calcium carbonate. Do 3 replicates for each concentration. After standing for 30 min, invert and culture at 37°C for 48 h.

[0045] The colonies with obvious calcium dissolution circles were inoculated into MRS medium and purified by streaking until a single colony appeared and the colony morphology was consistent across the entire plate and under the microscope, and then the physiological and biochemical indices of the strains were determined; Gram-positive, non-H2S-producing, non-gas-producing, non-mucus-producing and myoglobin-positive strains were selected, and the screened strains were stored on slants at -4°C for later use.

[0046] 2) Screening of strains with nitrite degradation effects

[0047] The isolated strains were inoculated at 1% into MRS broth medium containing 150 μg / mL NaNO2, pH 6.0. After culturing at 35 °C for 24 h, the NaNO2 content in the medium was detected, and the nitrite degradation rate was used to indicate the ability of the strain to degrade nitrite.

[0048] The morphological characteristics of lactic acid bacteria on the MRS medium containing calcium carbonate: milky white colonies of various sizes and shapes, warm, with calcium-soluble circles and transparent circles. 12 strains of typical colonies with different shapes and larger transparent circles were selected from the strains on the MRS calcium carbonate medium, and the strains were purified until single colonies appeared and the morphology of the colonies on the entire plate and under the microscope was consistent. All strains were then transferred to slant surfaces and stored at -4°C, and sealed with glycerol for later use. The determination was carried out in accordance with GB5009.33-2016 "National Food Safety Standard Determination of Nitrite and Nitrate in Food". The results showed that the strain SL7 had the best effect in degrading nitrite, with a degradation rate of 98%, which was significantly higher than other strains. Therefore, strain SL7 was selected as an excellent strain. The culture characteristics and microscopic morphology of the SL7 strain are shown below. Figure 1 As shown in a and b.

[0049] 1.2 Molecular identification of strains

[0050] Bacterial genomic DNA extraction: Use TSINGKE plant DNA extraction kit (universal type) to extract bacterial DNA;

[0051] The extracted products were amplified by PCR, and the amplification system was: 1×TSE101 gold mix 45μL, 27 F 2μL, 1492R 2μL, Template 1μL;

[0052] PCR amplification reaction conditions: pre-denaturation at 98°C for 4 min; 38 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 15 s; repair and extension at 72°C for 10 min, and termination at 4°C.

[0053] The PCR product was sequenced by the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. The sequencing results were compared with the 16Sr DNA sequence in the GenBank gene database for homology analysis to identify the microbial species. Weissella paramesenteroides The nucleotide sequence of the strain is shown in SEQ ID NO: 1. The phylogenetic tree of the strain is shown in Figure 2 shown.

[0054] The strain Weissella paramesenteroides SL7 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, with the deposit number GDMCC No: 62429.

[0055] Example 2

[0056] (1) Acid resistance of strains

[0057] The strain SL7 was inoculated into MRS liquid culture medium and activated at 35°C and 120r / min for 18h. After the activation, the strain SL7 was inoculated into MRS liquid culture medium with different pH values ​​(3.0-9.0) at 1% and cultured at 35°C for 24h. The OD 600 At the same time, the same as the above culture method, the strain SL7 was activated and inoculated into pH 2.5 artificial gastric fluid at 1%, cultured at 35℃ for 24h, and the OD 600 Value, the result is Figure 3 .

[0058] The experimental results showed that strain SL7 had strong acid tolerance and good growth in the pH range of 3.0-8.0. 600 The value reached the highest value of 1.14 at pH 6.0, and the OD 600 The value is 0.52, which indicates good survival.

[0059] (2) Determination of lead tolerance of strains

[0060] The strain SL7 was inoculated into MRS liquid medium and activated at 35°C and 120r / min for 18h. After activation, it was inoculated into Pb 2+ The cells were cultured in MRS liquid medium with concentrations of 0 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, and 1400 mg / L at 35°C for 24 h, and the OD 600 Value, the result is Figure 4 .

[0061] like Figure 4It can be seen that the strain OD 600 The highest value was 1.13, indicating that the strain grew well, indicating that the strain Weissella mesenteroides SL7 had a strong lead tolerance. 2+ The increase in Pb concentration 2+ The toxicity and inhibitory effect on the strain gradually increased, and OD 600 The value is also gradually decreasing. 2+ When the concentration increased from 800mg / L to 1000mg / L, OD 600 The value dropped to 0.61. 2+ When the concentration increased to 1400 mg / L, OD 600 The value is 0.14, and the strain hardly grows, indicating that with the increase of Pb 2+ When the concentration exceeds 1400 mg / L, more and more bacteria are poisoned by lead and cannot grow.

[0062] (3) Experiment on the adsorption of heavy metal lead by bacterial strains

[0063] SL7 and the other 11 strains isolated in Example 1 were activated and cultured at 35°C for 18h, and centrifuged at 5000r / min for 10min to obtain bacterial cells, which were washed with sterile ultrapure water and repeated 3 times to prepare a 50g / L wet bacterial cell suspension for use. 100g / L lead nitrate mother solution was diluted to a 100ml volumetric flask to make the solution Pb 2+ The final concentration reached 50 mg / L and the pH value was adjusted to 6.0 for later use. In a 48-well deep-well plate, add the same volume of Pb 2+ Solution (50 mg / L, pH 6.0) was added to make the wet bacterial concentration reach 5 g / L. The culture was shaken in a deep-well shaker at 35°C, 200 r / min, for 4 h. The adsorption solution was centrifuged at 10,000 r / min for 10 min, and the supernatant was retained. Pb was determined using a flame atomic absorption spectrophotometer (AAS990, Beijing Puxi General Instrument Co., Ltd.) 2+ The remaining 11 strains were used as control strains. The results are as follows Figure 5 .

[0064] The calculation methods of lead adsorption rate and lead adsorption amount per unit mass of bacteria are as follows:

[0065] Adsorption rate (%) = [(M0-M1) / M0] × 100%

[0066] Unit bacterial adsorption capacity (mg / g) = (M0-M1) / m

[0067] Where: M0 is the Pb content in the initial solution 2+ The content of Pb in the solution after adsorption (mg); M1 is the content of Pb in the solution after adsorption 2+m is the amount of bacteria added (g).

[0068] Depend on Figure 5 It can be seen that compared with other test strains, the adsorption rate of lead ions by the Weissella mesenteroides SL7 of the present invention is the highest, which is 89.32%, and the effect is significantly better than other strains, and it has good adsorption capacity for lead ions.

[0069] (4) Effect of different temperatures on the adsorption of Pb by strains 2+ Impact

[0070] Take the same volume of Pb 2+ solution (50 mg / L, pH 6.0) into a 48-well deep-well plate, and 50 g / L of SL7 bacterial suspension was added to make Pb 2+ The final concentration of the bacteria in the solution reached 5 g / L. The samples were placed at 15°C, 25°C, 30°C, 35°C, and 45°C, and adsorbed in a deep-well shaker at 200 r / min for 4 h. The supernatant was then centrifuged at 10,000 r / min for 10 min and the Pb content was determined by flame atomic absorption spectrometry. 2+ Concentration, the results are as follows Figure 6 .

[0071] Depend on Figure 6 It can be seen that with the increase of temperature, the amount of lead adsorbed by strain SL7 shows an increasing trend. When the temperature is 15℃, the amount of lead adsorbed is relatively low, at 4.52mg / g; in the range of 15℃-35℃, the amount of lead adsorbed by strain SL7 has an obvious upward trend, with a large increase, and the amount of lead adsorbed by strain SL7 at 35℃ is 18.86mg / g. Between 35℃-50℃, the upward trend of lead adsorption by strain SL7 tends to be gentle, with a small change. When the temperature reaches 50℃, the adsorption amount basically reaches the maximum of 22.51mg / g, but high temperature is more likely to damage the surface structure of the bacteria, affecting the subsequent adsorption effect, so the temperature of 35℃ can be selected as the optimal adsorption temperature.

[0072] Example 3 Tolerance dose test of Weissella mesenteroides SL7 fed to mice

[0073] The freeze-dried powder of Weissella mesenteroides SL7 was resuspended in skim milk powder to a concentration of 1.0×10 9 cfu / mL bacterial suspension. Ten healthy male mice weighing about 20-30 g (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were gavaged with the bacterial suspension once a day and observed for one week. The mortality and body weight were recorded and the results were averaged. The results are shown in Table 1.

[0074] Table 1 Changes in body weight of mice fed with Weissella mesenteroides SL7

[0075] .

[0076] Among them: “-” means no mice died.

[0077] From the results in Table 1, we can see that the concentration of gavage is 1.0×10 9 cfu / mL of Weissella mesenteroides SL7 had no obvious effect on mice. Their body weight showed an upward trend, no mice died, and there were no pathological symptoms on the outside.

[0078] Example 4 Ability to alleviate lead toxicity in lead-exposed mice

[0079] Forty healthy male mice weighing 20-30 g (purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were randomly divided into four groups: a negative control group, a lead acetate model control group, a Weissella mesenteroides SL7 treatment group, and an edetate calcium disodium positive treatment control group.

[0080] Experimental setting: The negative control group was fed with ordinary drinking water; the other three groups were fed with 1 g / L lead acetate solution. After the lead exposure infection model was established, the Weissella mesenteroides SL7 treatment group was fed with 1.0×10 9 cfu / mL of Weissella mesenteroides SL7 skim milk suspension, and the positive control treatment group was intraperitoneally injected with 5g / L calcium disodium edetate every day.

[0081] After the experiment, blood was collected and the mice were killed. The lead content in the stomach, liver, kidney and blood was measured using a flame atomic absorption spectrophotometer. The results are as follows: Figure 7 .

[0082] Depend on Figure 7 It can be seen that by comparing the lead content in the blood, liver, kidney and stomach of each group of mice, it was found that the Weissella mesenteroides SL7 of the present invention can significantly reduce the lead content in the blood, liver, kidney and stomach of mice. Figure 7 The data show that the lead content in the blood, liver, kidney and stomach of mice in the Weissella mesenteroides SL7 treatment group is 0.36μg / g, 0.66μg / g, 1.95μg / g and 0.38μg / g respectively. Lead in the environment enters the human body through food and respiratory pathways, causing acute or chronic toxic effects on the digestive, nervous, respiratory and immune systems. Long-term accumulation in the body leads to anemia, myopathy and other diseases, and encephalopathy or death may occur in severe cases. It can be seen from the test results that the use of the Weissella mesenteroides SL7 bacterial agent of the present invention can reduce the lead content in the blood, liver and kidney tissues of lead-poisoned mice, relieve the body's oxidative stress and reduce the pathological symptoms of lead-poisoned mice, and has a health care function that promotes lead excretion. Therefore, we can prepare food by Weissella mesenteroides SL7 to achieve the process of reducing or eliminating the lead poisoning phenomenon in the body.

[0083] Example 5 Preparation of a dairy product containing Weissella mesenteroides SL7

[0084] The raw skim milk was sterilized at 95°C for 20 min, then cooled to 4°C, and then the Weissella mesenteroides SL7 starter prepared according to the embodiment was added to make the viable bacteria count reach 1.0×10 8 CFU / g or above, based on the mass of raw milk, the inoculation amount is 1.5%, and it is refrigerated and stored at 4°C, thus obtaining a dairy product containing live bacteria of Weissella mesenteroides SL7.

[0085] Example 6 Preparation of fruit and vegetable beverage containing Weissella mesenteroides SL7

[0086] Fresh vegetables were selected, washed and juiced, and then sterilized at high temperature for 2 seconds at 140°C, and then immediately cooled to about 37°C, and then inoculated with the Weissella mesenteroides SL7 bacterial agent prepared by the present invention to make the number of viable bacteria reach 1.0×10 8 CFU / g or above, based on the mass of the raw vegetable juice, the inoculation amount is 1.5%, and it is refrigerated and stored at a temperature of 4°C, thus obtaining a fruit and vegetable beverage containing live bacteria of Weissella mesenteroides SL7.

[0087] Example 7 Preparation of starter culture for producing dairy products, soy products and fruit and vegetable products using Weissella mesenteroides SL7

[0088] Weissella mesenteroides SL7 was inoculated in MRS medium at 2% inoculum weight of the medium, cultured at pH 6.8, 35°C for 24 h, centrifuged at 5000 r / min for 10 min, washed twice with pH 7.2 phosphate buffer, and resuspended with a protective agent to make the number of viable bacteria reach 1.0×10 8 CFU / g. The skimmed milk powder is 2-4% and the sucrose is 1-3%. Freeze drying is then performed to obtain the freeze-dried fermentation agent containing viable Weissella mesenteroides SL7 of the dairy products, bean products and fruit and vegetable products.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of Weissella mesenteroides in adsorbing metallic lead in water and soil, characterized in that: The Weissella paramesenteroides is Weissella paramesenteroides SL7, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 23, 2022, with the deposit number GDMCC No: 62429.

2. The use according to claim 1, characterized in that When adsorbing metallic lead in water, the dosage of Weissella mesenteroides used is 10~20g / L; when adsorbing metallic lead in soil, the dosage of Weissella mesenteroides used is 3%~6% of the soil mass.

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