Application of Lactobacillus paracasei FMBL L23249 FJX in removal of heavy metals and prevention and treatment of heavy metal pollution

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of lactobacillus paracasei FMBL L23249 FJX in removal of heavy metals and prevention and treatment of heavy metal pollution, the lactobacillus paracasei FMBL L23249 FJX is preserved in China Center for Type Culture Collection on June 26, 2023, and the preservation number is CCTCC NO: M 20231097; the lead adsorption rate can reach 99.87%, and the cadmium adsorption rate can reach 82.74%; the strain has good tolerance when the lead ion concentration is 0-300mg / L, and the strain still can survive when the cadmium ion concentration is 300mg / L; an inhibition effect is achieved on enteropathogenic bacteria; acid-resistant and cholate-resistant capabilities, oxidation resistance and antibiotic resistance are also realized; the method can be used for preparing adsorbents for highly adsorbing heavy metal lead and cadmium, medicines for relieving toxicity of lead and / or cadmium and inhibiting pathogenic bacteria, fermented foods, health-care products, food additives and feeds, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of Lactobacillus paracasei FMBL L23249 FJX in removing heavy metals and preventing and controlling heavy metal pollution. Background Art

[0002] Heavy metals are elements with high density and high atomic mass. They are non-biodegradable. If absorbed by the human body or other animals and plants, they will seriously endanger the healthy growth of the human body and other animals and plants. Lead is the second most excessive heavy metal found in the world. It can enter the human body through food (65%), water (20%) and air (15%), causing neurological dysfunction and kidney disorders. Cadmium is mainly caused by human activities, metal smelting, chemical fuel combustion and sewage sludge; cadmium is highly toxic. Cadmium poisoning mainly produces various harmful effects on cell molecules through oxidation-antioxidation imbalance, thus leading to hypertension, renal tubular dysfunction, fractures and cancer. Excessive cadmium content can also cause serious damage to the nervous system, hematopoietic system, liver and reproductive function of poultry and livestock.

[0003] At present, methods for removing heavy metals from contaminated systems include physical remediation, chemical precipitation, dialysis, ion exchange, reverse osmosis, solvent extraction, etc. However, most of these methods are costly and harmful to the environment. Biological methods have become a hot topic in the research of removing heavy metal pollution due to their advantages such as high efficiency, non-toxicity, environmental protection and economy. Biological methods mainly use bacteria, fungi, yeast, etc. as adsorbents to adsorb heavy metals. For example, invention patent CN110628678B discloses a heavy metal-resistant copper-greedy bacteria that can tolerate heavy metal copper and improve the absorption of Cu. 2+ The germination rate of contaminated seeds and the promotion of Cu 2+ The growth of polluted seedlings can effectively remove copper ions in the environment and can be used as Cu 2+ It is used as a contaminated soil remediation agent, but it cannot be used for heavy metal poisoning in humans or other animals.

[0004] In view of the above technical problems, those skilled in the art screened strains from probiotics that can absorb / tolerate heavy metals, have multiple probiotic properties in the human body, and promote and maintain the health of intestinal flora, thereby playing a beneficial role in the health of the host. For example, invention patent CN102296046B discloses recombinant food-grade lactic acid bacteria that can be used to prevent chronic heavy metal poisoning. A recombinant engineered bacterium was obtained by fusion expression of glutathione S-transferase, small molecule ubiquitin-related modifier mature peptide and human type IA metallothionein. The recombinant lactic acid engineered bacteria can continuously express a fusion protein containing human type IA metallothionein during the fermentation process, and the protein has the ability to bind heavy metals Pb, Cd and Hg in the living environment.

[0005] The inventor isolated a strain of Lactobacillus paracasei FMBL L23249 FJX from the human intestinal tract during the research process, and unexpectedly found that the Lactobacillus paracasei FMBL L23249 FJX had high adsorption to heavy metals lead and cadmium, had good tolerance at a lead ion concentration of 0-300mg / L, and could still survive at a cadmium ion concentration of 300mg / L. In addition, the prepared Lactobacillus paracasei FMBL L23249 FJX had acid resistance, bile salt resistance, antioxidant capacity, and antibacterial effects. The invention can be used to prepare an adsorbent for high adsorption of heavy metals lead and cadmium, a medicine, fermented food, health products, food additives, feed or microbial preparations, etc., for alleviating the toxicity of lead and / or cadmium and inhibiting pathogenic bacteria, and has a wide range of application prospects. Summary of the invention

[0006] The primary purpose of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same or a fermentation liquid thereof in removing heavy metals. The Lactobacillus paracasei FMBLL23249FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231097.

[0007] The second object of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of a medicament for preventing and treating heavy metal poisoning. The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

[0008] Preferably, the heavy metal poisoning is chronic heavy metal poisoning.

[0009] The third object of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of cosmetics for preventing and treating heavy metal poisoning. The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

[0010] Preferably, the heavy metal is cadmium and / or lead.

[0011] The fourth object of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of a starter, a fermented food, a health product, a food additive or a dietary supplement. The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

[0012] The fifth object of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of feed or feed additives. The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231097.

[0013] The sixth object of the present invention is to provide an application of Lactobacillus paracasei FMBLL23249FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of an antibacterial drug. The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231097.

[0014] The invention has the following beneficial effects: the invention provides application of lactobacillus paracasei FMBL L23249 FJX in adsorbing heavy metals or in preparing medicines, foods, health products or cosmetics for preventing and treating heavy metal poisoning; the lactobacillus plantarum has strong lead and cadmium adsorption capacity, the lead adsorption rate can reach 99.87%, and the cadmium adsorption rate is 82.74%; the lactobacillus paracasei FMBL L23249 FJX has good tolerance at a lead ion concentration of 0-300 mg / L, and can still survive at a cadmium ion concentration of 300 mg / L; has an inhibitory effect on diarrheagenic Escherichia coli, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli and Listeria monocytogenes; has acid and bile salt resistance; has the ability to utilize carbohydrates and good antioxidant capacity; can be used for preparing an adsorbent for high adsorption of heavy metals lead and cadmium, medicines for alleviating lead and / or cadmium toxicity and inhibiting pathogenic bacteria, fermented foods, health products, food additives, feed or microbial preparations, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Colony image of strain FMBL L23249 FJX

[0016] Note: a. Cell morphology; b. Cell morphology after Gram staining

[0017] Figure 2 Phylogenetic tree of Lactobacillus paracasei FMBL L23249 FJX

[0018] Figure 3 Adsorption rate of heavy metals lead and cadmium by different strains

[0019] Figure 4 FJX infrared spectrum scanning results of Lactobacillus paracasei FMBL L23249 before and after lead and cadmium adsorption DETAILED DESCRIPTION

[0020] The following embodiments are for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples, unless otherwise specified, are all routine laboratory methods, and the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents. The quantitative tests in the following examples are all repeated three times, and the results are averaged.

[0021] The medium formula used is as follows:

[0022] MRS liquid medium: peptone 10g; beef extract 10g; yeast extract 5g; glucose 20g; Tween 80 1mL; K2HPO42g; sodium acetate 5g; diammonium hydrogen citrate 2g; MgSO4·7H2O 0.58g; MnSO4·4H2O 0.25g, deionized water 1000mL;

[0023] LAMVAB solid culture medium (1L): peptone 10g; beef extract 8g; yeast extract 4g; glucose 20g, Tween 801mL; K2HPO4 2g; sodium acetate 5g, diammonium hydrogen citrate 2g; MgSO4·7H2O 0.2g; MnSO4·4H2O 0.05g; agar 20g; L-cysteine ​​hydrochloride 0.5g; vancomycin hydrochloride 20mg, sterilized at 115℃ for 20min.

[0024] M17 broth medium; M17 solid medium (M17 broth medium plus 2% agar);

[0025] PYG medium: peptone 10.0 g, yeast extract 5.0 g, glucose 1.0 g, agar 20.0 g, deionized water 1000 mL;

[0026] TSA medium: 15.0 g tryptone, 5.0 g soy peptone, 5.0 g sodium chloride, 15 g agar, 1000 mL deionized water;

[0027] Nutrient broth agar medium: peptone 5.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar 20.0 g, deionized water 1000 mL.

[0028] Example 1: Isolation and identification of strains

[0029] 1. Strain Isolation

[0030] The strains were isolated from fresh feces of Uygur people in Xinjiang according to the conventional method, and a strain of Lactobacillus paracasei was isolated and named FMBL L23249 FJX.

[0031] 2. Strain Identification

[0032] Place the collected fresh feces in a sampling tube. Take 1g of feces sample and inoculate it into 9mL of MRS liquid culture medium, mix thoroughly, and dilute the feces sample to 10 -3 , 10 -4 , 10 -5 , and spread on LAMVAB agar medium and cultured at 37°C for 24-48 hours. After picking the suspected lactic acid bacteria colonies and purifying them three times, the isolates were preserved in MRS liquid medium supplemented with 25% glycerol and stored at -20°C.

[0033] PCR amplification of the groEL gene was performed as shown in Table 1. After the PCR reaction, the product was subjected to 1.2% agarose gel electrophoresis to observe the molecular size, and the appropriate PCR product was selected and sent to the company for sequencing. The returned sequencing results were uploaded to the NCBI database for BLAST comparison. After the comparison, the sequence of the corresponding genus was obtained from the database, and the phylogenetic tree was established using MEGA11.0.

[0034] Table 1 PCR amplification of groEL gene

[0035]

[0036] The bacterial strain FMBL L23249 FJX has the following colony characteristics and microscopic characteristics: Figure 1 The phylogenetic tree is shown in Figure 2As shown. After identification, the strain FMBL L23249 FJX was obtained as Lactobacillus paracasei, with a Latin name of Lactobacillus paracasei, and was named Lactobacillus paracasei FMBL L23249 FJX. Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097, and the deposit address is: Wuhan University, Wuhan, China, Tel: 027-68754052.

[0037] In the following examples, Lacticaseibacillus paracasei FMBL L23249FJX is abbreviated as Lacticaseibacillus paracasei FMBL L23249 FJX.

[0038] Example 2: Adsorption capacity of Lactobacillus paracasei FMBLL23249 FJX on heavy metals lead and cadmium

[0039] 1. Test strains

[0040] Lactobacillus plantarum FMBL L23290 WP, Lactobacillus paracasei FMBL L23249 FJX, Lactobacillus paracasei FMBL L23284 WP, Lactobacillus rhamnosus FMBL L23136 YWT, and Lactobacillus mucilaginosus reuteri were all isolated from feces of healthy people. Among them, Lactobacillus plantarum FMBL L23290 WP, Lactobacillus paracasei FMBL L23284 WP, Lactobacillus rhamnosus FMBL L23136 YWT, and Lactobacillus mucilaginosus reuteri were all deposited in the Food Microbiology and Biotechnology Research Center, School of Food Science, Shihezi University.

[0041] 2. Adsorption experiment

[0042] (1) The strain to be tested was inoculated into liquid MRS medium and cultured at 37°C for 24 h for activation.

[0043] (2) The activated strain in step (1) was centrifuged at 8000 rpm and 4°C for 20 min to collect the bacterial precipitate, and the bacterial precipitate was washed three times with PBS buffer (pH = 7.4) to obtain the bacterial precipitate to be tested.

[0044] (3) Prepare the bacterial precipitate obtained in step (2) into a 50g / L bacterial suspension; prepare lead nitrate and cadmium nitrate into 50mg / L lead ion solution and cadmium ion solution; add the bacterial suspension to 50mg / L lead and cadmium ion solutions respectively, so that the final concentration of the bacteria in the solution is 5g / L; place it in a shaker at 150rpm and 37℃ for 1h, centrifuge and take the supernatant, and determine the content of lead and cadmium in the supernatant by atomic spectrophotometry. Calculate the adsorption rate by the following formula. C0 is the content of lead and cadmium ions in the adsorbed lead solution, C I is the content of lead and cadmium ions in the solution after adsorption.

[0045]

[0046] 3. Results

[0047] The results are as follows Figure 3 As shown, the adsorption rate of lead by each strain was between 55% and 100%, and the adsorption rate of cadmium was between 70% and 90%. Different strains had obvious differences in their adsorption capacity for heavy metal ions. Lactobacillus paracasei FMBLL23249 FJX had the best adsorption effect on heavy metals, with an adsorption rate of 99.87% for lead ions and 82.74% for cadmium ions, which were significantly higher than those of other strains.

[0048] Example 3 Effects of different concentrations of lead and cadmium ions on the growth of Lactobacillus paracasei FMBLL23249 FJX 1. Experimental process

[0049] (1) Filter the lead nitrate and cadmium nitrate solutions through a filter membrane and add them into MRS liquid culture medium to prepare liquid culture medium with lead ion concentrations of (50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) and cadmium ion concentrations of (50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L), respectively.

[0050] (2) Add the activated Lactobacillus paracasei FMBL L23249 FJX to the liquid culture medium prepared in step (1) at an inoculum volume of 2%. Cultivate at 37°C for 24 hours and measure the OD with a spectrophotometer. 600 The liquid culture medium without heavy metal ions was used as the control.

[0051] Table 2 Activity of Lactobacillus paracasei FMBL L23249 FJX in cadmium-containing medium

[0052]

[0053]

[0054] Table 3 Activity of Lactobacillus paracasei FMBL L23249 FJX in lead-containing medium

[0055]

[0056] 2. Results

[0057] The results of the effects of different lead and cadmium ion concentrations on the growth of Lactobacillus paracasei FMBL L23249 FJX are shown in Tables 2 and 3. The results show that the growth of Lactobacillus paracasei FMBL L23249 FJX is basically unaffected when the lead ion concentration is in the range of 0-300 mg / L, and it has good tolerance. The growth of Lactobacillus paracasei FMBL L23249 FJX is basically unaffected in the range of heavy metal cadmium ion concentration of 0-100 mg / L; when the cadmium ion concentration is ≥200 mg / L, the growth of Lactobacillus paracasei FMBL L23249 FJX gradually weakens with the increase of cadmium ion concentration, but it is significantly higher than other strains; when the cadmium ion concentration is 300 mg / L, Lactobacillus paracasei FMBL L23249 FJX can still survive and has a certain tolerance to cadmium ions.

[0058] Example 4: Infrared spectroscopy to detect the related groups of lead and cadmium ion adsorption by Lactobacillus paracasei FMBL L23249 FJX

[0059] 1. Experimental Procedure

[0060] (1) Prepare lead nitrate and cadmium nitrate into 50 mg / L lead ion solution and cadmium ion solution respectively, sterilize and set aside.

[0061] (2) Activate Lactobacillus paracasei FMBL L23249 FJX and collect the cells by centrifugation. Wash the cell precipitate three times with PBS buffer and add it to the lead ion solution and cadmium ion solution of step (1) to make the final cell concentration in the solution 5 g / L. Incubate at 37° C. for 1 h on a shaker at 150 rpm, and collect the cell precipitate by centrifugation.

[0062] (3) Freeze-dry the bacterial precipitate from step (2). Grind the dried bacterial powder and potassium bromide at a ratio of 1:50, mix and press into thin sheets. Measure using a Fourier transform infrared spectrometer. -1 The sample is scanned within the range and its spectrum is recorded.

[0063] 2. Results

[0064] The results are as follows Figure 4As shown, it can be seen that the spectral peak intensity increased before and after lead and cadmium stress, and the position of the characteristic peak changed, which indicates that functional groups such as carbonyl, phosphate, and hydroxyl groups on the bacteria are related to the ability to adsorb heavy metals.

[0065] Example 5: Determination of probiotic properties of Lactobacillus paracasei FMBLL23249 FJX

[0066] 1. Acid and bile salt resistance

[0067] The activated lactobacilli cultured for 24 hours were centrifuged (10000rpm, 5min, 4°C), the supernatant was discarded, and the bacterial precipitate was collected. Then, it was added into MRS liquid culture medium with different pH values ​​(2.5, 3, 3.5, 4) and different bile salt concentrations (0.05%, 0.1%, 0.5%), and placed at 37°C for 0h and 4h before sampling and coating. After 48h, the live bacteria were counted and the survival rate under different pH conditions was calculated.

[0068] Table 4 Acid resistance test of Lactobacillus paracasei FMBL L23249 FJX

[0069] strain pH=2.5 pH = 3 pH=3.5 pH = 4 Lactobacillus paracasei FMBLL23249FJX 23±1.47％ 29.04±1.22％ 36.45±2.43％ 40.55±1.47％

[0070] Table 5 Bile salt tolerance test of Lactobacillus paracasei FMBL L23249 FJX

[0071] strain 0.05% Bile Salt 0.1% Bile Salts 0.5% Bile Salts Lactobacillus paracasei FMBLL23249FJX 51.64±0.88％ 22.54±1.16％ 1.93±0.33％

[0072] As can be seen from Tables 4 and 5, Lactobacillus paracasei FMBL L23249 FJX can survive under the conditions of pH 4-pH 2.5. The survival rate was 40.55±1.47% under the condition of pH 4, and the survival rate was still more than 23±1.47% under the condition of pH 2.5; the survival rate reached 51.64±0.88% at a bile salt concentration of 0.05%. This shows that Lactobacillus paracasei FMBL L23249 FJX has good acid and bile salt resistance, and the good acid and bile salt resistance can enable the strain to reach the colonization site, thereby surviving, growing and playing a probiotic role in the gastrointestinal tract.

[0073] 2. Antibacterial experiment

[0074] The Oxford cup method was used to determine the antibacterial activity of the metabolites of Lactobacillus plantarum FMBL L23220 CLL, with Escherichia coli (10411), Escherichia coli (21530), Escherichia coli (10421), Salmonella typhimurium (10420), Listeria monocytogenes (LS1) and Salmonella enteritidis serotype (SM1) as indicator bacteria (see Table 6 for purchase sources). The strain was inoculated into MRS medium at an inoculum rate of 2%, cultured at 37°C for 24h, centrifuged at 8000rpm for 10min, and the supernatant was taken and filtered for sterilization to prepare a cell-free supernatant. The indicator bacteria were inoculated at 10 6 The CFU / mL concentration was coated on the corresponding solid culture medium, and a sterile Oxford cup was placed vertically on the culture dish coated with pathogenic bacteria, and 0.2 mL of cell-free supernatant was added to the Oxford cup. The culture dish was placed at 4°C for diffusion for 4 hours, and then placed in a 37°C incubator for 24 hours, and the diameter of the inhibition zone was measured.

[0075] Table 6 Source of indicator bacteria

[0076]

[0077]

[0078] Table 7 Determination of antibacterial ability of Lactobacillus paracasei FMBL L23249 FJX

[0079]

[0080] Note: ≤15+; 15<inhibition zone diameter≤20++; 20<inhibition zone diameter≤25+++; inhibition zone diameter>25+++; no inhibition zone -.

[0081] As shown in Table 7, Lactobacillus paracasei FMBL L23249 FJX has good antibacterial ability against enteropathogenic Escherichia coli (10411), enterotoxigenic Escherichia coli (10421), enterohemorrhagic Escherichia coli (21530), Salmonella enterica subspecies Typhimurium (10420), and Salmonella enterica subspecies (SM1).

[0082] 3. Drug sensitivity test

[0083] The disc diffusion method (KB method) was used. The information of the drug sensitivity sheet was as follows. The strain was inoculated into sterilized liquid MRS medium at a 2% inoculum and cultured at 37°C for 24 hours. The activated strain was diluted and spread (the bacterial concentration was 10 7 -10 8CFU / mL), place the drug-sensitive paper evenly on it with sterile tweezers, culture at 37℃ for 24h, record the size of the drug-sensitive sheet inhibition zone diameter, repeat the test three times, take the average value, and judge the sensitivity of the strain to the drug-sensitive sheet according to the diameter of the inhibition zone. The experimental results were determined according to the CISI data and drug sensitivity classification labeling formulated by the American Clinical Laboratory Standards Committee, and the results are shown in Table 8.

[0084] Table 8 Drug sensitive tablet information

[0085]

[0086]

[0087] Table 9 Drug sensitivity test of Lactobacillus paracasei FMBL L23249 FJX

[0088]

[0089] Note: R: resistant; I: moderately sensitive; S: sensitive

[0090] In order to ensure the safety of edible probiotics for human body, the drug resistance of strains has become an important indicator for the in vitro safety assessment of probiotic strains. As shown in Table 9, Lactobacillus paracasei FMBL L23249 FJX is sensitive to rifampicin, tetracycline, minocycline, penicillin, levofloxacin, ampicillin, cefotaxime, and chloramphenicol; and is resistant to gentamicin, streptomycin, oxacillin, and cefoxitin.

[0091] 4. Antioxidant capacity determination

[0092] The cultured bacterial solution was centrifuged (10000 rpm, 10 min, 4°C) to collect the bacterial cells, washed three times with PBS buffer, and then prepared into a bacterial suspension. The cell-free supernatant is the supernatant filtered through a 0.22 μm microporous filter membrane.

[0093] (1) Determination of the ability to scavenge diphenyltrinitrophenylhydrazine free radical (DPPH)

[0094] Prepare 0.2mM DPPH-methanol solution, mix the supernatant or bacterial suspension with 0.2mM DPPH-methanol solution in a ratio of 1:1, react in the dark for 60 minutes, and measure the OD517 nm value of the supernatant. Set up three sets of repeated experiments.

[0095]

[0096] Where: D0: absorbance of 1mL PBS buffer at OD517 nm; D1: absorbance of 1mL sample solution + 1mL DPPH-methanol solution at 517nm; D2: absorbance of 1mL sample solution + 1mL anhydrous methanol.

[0097] (2) Determination of ABTS removal ability

[0098] Prepare 7mmol / LABTS working solution: Accurately measure 15mL of 3.8g / L ABTS solution (prepared with deionized water) and add 0.264Ml of potassium persulfate aqueous solution (molar concentration is 140mmol / L), mix well and place in the dark at room temperature for 12-16h. Adjust the working solution OD734 nm to 0.7±0.02 with anhydrous ethanol, take the sample to be tested and mix it with the adjusted ABTS working solution in a ratio of 1:6, place it at room temperature for 30min, and measure the absorbance at 734nm. Set up three sets of repeated experiments.

[0099]

[0100] Where Ac is the absorbance of an equal volume of water; A is the absorbance of the sample solution.

[0101] Table 10 Antioxidant capacity of Lactobacillus paracasei FMBL L23249 FJX

[0102]

[0103] As shown in Table 10, the order of scavenging ability of Lactobacillus paracasei FMBL L23249 FJX for DPPH free radicals is bacterial suspension > supernatant, and the order of scavenging ability for ABTS is bacterial suspension > supernatant.

[0104] In the following examples, the inventors prepared the Lactobacillus paracasei FMBL L23249 FJX into different types of bacterial agents or foods.

[0105] Application Example 1: Preparation of Lactobacillus paracasei FMBLL23249 FJX bacterial agent

[0106] Preparation of culture medium: Use water and culture medium raw materials to prepare a medium containing 20 g / L of glucose, 8 g / L of beef extract, 10 g / L of yeast extract, 9 g / L of NaCl, 1 mL / L of Tween 80, 2 g / L of K2HPO4, 5 g / L of sodium acetate, 2 g of diammonium hydrogen citrate, 0.58 g of MgSO4·7H2O, and 0.25 g of MnSO4·4H2O, and adjust the pH to 6.8 to obtain the culture medium.

[0107] Preparation of protective agent: Use water and protective agent raw materials to prepare a protective agent containing 120 g / L skim milk powder, 20 mL / L glycerol, 22 g / L maltodextrin, 60 g / L trehalose, and 22 g / L galacto-oligosaccharide.

[0108] Lactobacillus paracasei FMBL L23249 FJX was inoculated at 2% in the above culture medium sterilized at 115°C for 15 min; the culture medium contained 20 g / L glucose, 8 g / L beef extract, 10 g / L yeast extract, 9 g / L NaCl, 1 mL / L Tween 80, 2 g / L K2HPO4, 5 g / L sodium acetate, 2 g diammonium hydrogen citrate, 0.58 g MgSO4·7H2O, 0.25 g MnSO4·4H2O, and the pH was adjusted to 6.8. The culture was cultured at 37°C for 24 h, washed twice with pH 7.2 PBS buffer, and then resuspended with a protective agent to a concentration of 10 11 CFU / mL. The protective agent contains 120g / L skim milk powder, 20mL / L glycerol, 22g / L maltodextrin, 60g / L trehalose, and 22g / L galacto-oligosaccharide. Then, the suspension is pre-cultured at a temperature of 37°C for 60min, and then freeze-dried to obtain the Lactobacillus paracasei FMBL L23249 FJX bacterial agent.

[0109] Application Example 2: Preparation of fermented milk using Lactobacillus paracasei FMBL L23249 FJX of the present invention

[0110] After sugar is dissolved in fresh milk, it is homogenized at a temperature of 60 DEG C and a pressure of 20MPa, and then sterilized at a temperature of 90-95 DEG C for 5-8 minutes. When the temperature drops to 35 DEG C, mixed bacteria consisting of Lactobacillus paracasei FMBL L23249 FJX, commercial dry powder starter Lactobacillus bulgaricus and thermophilic streptococcus are added, and the mass ratio of the mixed bacteria is 1:1:1. The inoculation amount of the mixed bacteria is 0.03-2.0% of the weight of the fresh milk. The mixture is mixed, and the mixture is fermented at a temperature of 37 DEG C. After the milk is coagulated, the mixture is refrigerated at a temperature of 4 DEG C for 16 hours to obtain the fermented milk.

[0111] Application Example 3: Preparation of fruit and vegetable beverages containing Lactobacillus paracasei FMBLL23249 FJX

[0112] Fresh fruits and vegetables were selected and washed and squeezed for juice, and then sterilized at high temperature, and then immediately cooled to about 37°C, and the Lactobacillus paracasei FMBL L23249 FJX bacterial agent prepared by the present invention was added to make its concentration reach 10 6 CFU / mL or more, and refrigerate at 4°C to obtain a fruit and vegetable beverage containing live bacteria of Lactobacillus paracasei FMBL L23249 FJX of the present invention. Application Example 4: Preparation of microcapsules and capsule products containing Lactobacillus paracasei FMBL L23249 FJX

[0113] The Lactobacillus paracasei FMBL L23249 FJX of the present invention was cultured in MRS medium for 24 hours, centrifuged at 4°C and 8000 r / min for 10 minutes, the cells were collected and washed twice with sterile saline, and the cells were resuspended with sterile saline to a final cell concentration of 2×10 10 The bacterial suspension is added to a solution containing 2.5% sodium alginate and 1% pectin, and the mixture is fully stirred to uniformly disperse the cells in the sodium alginate solution, and then the mixed solution is squeezed into a solidifying solution containing 2% calcium chloride and chitosan to form micelles, and the micelles are statically solidified for 30 minutes, and the micelles are collected by filtration, and the collected micelles are freeze-dried to obtain microcapsules containing Lactobacillus paracasei FMBL L23249 FJX of the present invention, and the microcapsules are loaded into medicinal capsules currently sold on the market to obtain the capsule product.

[0114] Application Example 5: Preparation of plant milk containing Lactobacillus paracasei FMBL L23249 FJX by using the present invention

[0115] Soybeans, almonds, and other raw materials are soaked at 80°C for 2 hours, peeled, and the soaking water is drained, and the pulp is ground with boiling water and kept at 80-85°C for 12-15 minutes. The obtained slurry is filtered with a 200-mesh screen to remove the residue and then boiled. The supernatant obtained after the solids are removed by centrifugation is cooled to about 37°C, and the Lactobacillus paracasei FMBLL23249FJX of the present invention is inoculated to make its concentration reach 10 6 CFU / mL or above, and refrigerated storage at a temperature of 4°C to obtain a plant milk containing the FMBLL23249FJX live bacteria of the present invention.

[0116] Application Example 6: Preparation of probiotic milk tablets containing Lactobacillus paracasei FMBL L23249 FJX

[0117] 5.96 parts by weight of the Lactobacillus paracasei FMBL L23249 FJX bacterial agent prepared by a freeze-drying method, 65.0 parts by weight of whole milk powder, 7.92 parts by weight of white granulated sugar, 2.5 parts by weight of magnesium stearate and 18.0 parts by weight of skim milk powder are respectively weighed, mixed with 1.0 part by weight of water, prepared into wet granules by a conventional method, and then tabletted by a tabletting machine, dried in a dryer, and packaged to obtain the probiotic milk tablets of the present invention.

[0118] In summary, the present invention provides an application of Lactobacillus paracasei FMBL L23249 FJX in adsorbing heavy metals or in preparing medicines, foods, health products or cosmetics for preventing and treating heavy metal poisoning. The Lactobacillus paracasei FMBL L23249 FJX has high adsorption to heavy metals lead and cadmium, with an adsorption rate of lead reaching 99.87% and an adsorption rate of cadmium reaching 82.74%. The strain has good acid and bile resistance, drug resistance, self-aggregation and hydrophobicity. The strain has good intestinal pathogenic Escherichia coli (10411) and enterotoxigenic Escherichia coli (104 The invention has an inhibitory effect on enterohemorrhagic Escherichia coli (21530), Salmonella enterica subspecies Typhimurium serotype (10420), and Salmonella enterica subspecies (SM1); and is resistant to gentamicin, streptomycin, oxacillin, and cefoxitin; and has antioxidant capacity; and can be used to prepare adsorbents with high adsorption of heavy metals lead and cadmium, drugs for alleviating the toxicity of lead and / or cadmium and inhibiting pathogenic bacteria, fermented foods, health products, food additives, feed or microbial preparations, etc., and has broad application prospects.

Claims

1. Application of Lactobacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same or a fermentation liquid thereof in removing heavy metals, characterized in that: The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

2. Use of Lactobacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same or a fermentation liquid thereof in the preparation of a medicament for preventing and treating heavy metal poisoning, characterized in that: The Lactobacillus paracasei FMBL L23249FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231097.

3. The use according to claim 2, characterized in that The heavy metal poisoning is chronic heavy metal poisoning.

4. Use of Lactobacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same or a fermentation liquid thereof in the preparation of cosmetics for preventing and treating heavy metal poisoning, characterized in that: The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231097.

5. The use according to any one of claims 1 to 4, characterized in that: The heavy metals are cadmium and / or lead.

6. Use of Lactobacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same, or a fermentation liquid thereof in the preparation of a starter, a fermented food, a health product, a food additive or a dietary supplement, characterized in that: The Lactobacillus paracasei FMBL L23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

7. Use of Lacticaseibacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same or a fermentation liquid thereof in the preparation of feed or feed additives, characterized in that: The Lactobacillus paracasei FMBLL23249 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231097.

8. Use of Lacticaseibacillus paracasei FMBL L23249 FJX, a bacterial agent containing the same or a fermentation liquid thereof in the preparation of an antibacterial drug, characterized in that: The Lactobacillus paracasei FMBL L23249FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231097.

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