Lactobacillus rhamnosus for relieving toxicity of heavy metal cadmium and application of lactobacillus rhamnosus

Abstract

The invention belongs to the technical field of microorganisms, and discloses lactobacillus rhamnosus capable of relieving toxicity of heavy metal cadmium and application of the lactobacillus rhamnosus. The lactobacillus rhamnosus is preserved in the China Center for Type Microbiological Culture Collection, the preservation address is Wuhan University, Hubei province, and the preservation number is CCTCC No.M20241815. The lactobacillus rhamnosus is named lactobacillus rhamnosus. The lactobacillus rhamnosus provided by the invention is derived from intestinal tracts of healthy stichopus japonicus, has good heavy metal cadmium tolerance and removal capacity, and is expected to be developed and applied to the healthy food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus rhamnosus strain capable of alleviating the toxicity of heavy metal cadmium and an application thereof. Background Art

[0002] China's authorized invention patent CN116987638B discloses an efficient treatment method for heavy metal cadmium based on heavy metal passivation strains. This method uses Bacillus Velezii YB03 as a cadmium-resistant microorganism. Specific inducers are added during the strain cultivation process to enhance the bacteria's ability to adsorb heavy metals. The heavy metal adsorption carrier has excellent adsorption performance for heavy metal cadmium, with high adsorption efficiency and short time.

[0003] Probiotics are a class of microorganisms that have beneficial effects on animals when ingested. These include species commonly found in the animal gastrointestinal flora, such as Lactobacillus, Bifidobacterium, and Bacillus, as well as less common species, such as yeast. Probiotics must be able to colonize the animal gastrointestinal tract and exert their beneficial effects within the GI tract. With increasing awareness of the link between the animal gastrointestinal flora and various diseases, probiotics are gaining increasing attention as an effective method for modulating the gastrointestinal flora. Probiotics have been shown to mediate immune defense against pathogens; directly inhibit or kill pathogens; protect intestinal fluid, improve the intestinal barrier, alleviate inflammation, and maintain a balanced gastrointestinal microbiome. Lactobacillus rhamnosus and its combination formulations, by affecting the TLR-2 / MyD88 signaling pathway, relocalize mesenchymal stem cells expressing cyclooxygenase-2 to intestinal crypts, thereby reducing radiation damage and improving crypt survival.

[0004] Cadmium is a heavy metal that is extremely harmful to human health. It is widely and stably present in the environment and is known to be the most easily accumulated toxin in the body. However, current medications for diseases caused by cadmium poisoning have certain toxic side effects. Therefore, the use of probiotics with cadmium-removing properties is a safer and more effective option.

[0005] Authorized Chinese invention patent CN113046276B discloses a breast milk-derived Lactobacillus rhamnosus strain and its applications. The strain is deposited under the CGMCC No. 21815. Lactobacillus rhamnosus AUH2101, derived from healthy Chinese breast milk, produces high concentrations of lactic acid and hydrogen peroxide, two antibacterial substances, and is sensitive to most antibiotics. It also exhibits excellent gastrointestinal tolerance and strong cadmium removal capacity, resulting in excellent antibacterial potential, low potential risk, and excellent cadmium removal, making it promising for development and application in the food industry.

[0006] However, the ability of the above-mentioned breast milk-derived Lactobacillus rhamnosus to remove heavy metal cadmium still needs to be further improved. In order to provide a Lactobacillus rhamnosus that can more effectively alleviate the toxicity of heavy metal cadmium, the inventors of this application conducted research and completed the present invention accordingly. Summary of the Invention

[0007] Based on the above reasons, the present invention proposes a strain of Lactobacillus rhamnosus for alleviating heavy metal cadmium toxicity and its application. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0008] One aspect of the present invention relates to a Lactobacillus rhamnosus strain. The Lactobacillus rhamnosus strain (Lacticaseibacillus rhamnosus LRM2-4) is a strain of the China Collection of Typical Microorganisms, with a preservation address of Wuhan University in Hubei Province and a preservation number of CCTCC No. M20241815.

[0009] Another aspect of the present invention relates to the use of the above-mentioned Lactobacillus rhamnosus in alleviating the toxicity of heavy metal cadmium.

[0010] Another aspect of the present invention relates to a probiotic product, which contains the above-mentioned Lactobacillus rhamnosus; preferably, the probiotic product is a dairy product, a soy product, a fruit and vegetable product, or a live bacteria preparation.

[0011] Beneficial effects

[0012] The beneficial effects of the present invention are as follows: the Lactobacillus rhamnosus provided by the present invention is derived from the intestines of healthy sea cucumbers, has good heavy metal cadmium tolerance and removal ability, and is expected to be developed and applied in the health food industry. DETAILED DESCRIPTION

[0013] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0014] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0015] Example 1:

[0016] 1. Isolation and preservation of Lactobacillus rhamnosus LRM2-4:

[0017] The inventors of this application isolated and extracted a Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus LRM2-4) strain from the intestine of healthy sea cucumbers, named LRM2-4. The microbial strain was deposited in the China Center for Type Microorganism Collection, with the deposit address being Wuhan University, Hubei Province. The deposit number is: CCTCC No. M20241815, and the deposit date is August 19, 2024.

[0018] 2. Identification of bacterial species

[0019] Genomic DNA was extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit. Using the genomic DNA of the test strain as a template, the target fragment was amplified using the universal primers 27F / 1492R for the bacterial 16S rRNA gene. The PCR product was analyzed by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Biomed Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis confirmed that Lactobacillus rhamnosus LRM2-4 was Lactobacillus rhamnosus LRM2-4. The 16S rDNA gene sequence is shown in SEQ ID NO. 1.

[0020] ACTTTGTCCCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGT

[0021] GTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC

[0022] ACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTT

[0023] GCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGT

[0024] CTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGG

[0025] GCATGATGATTTGACGTCATCCCCACCTTTCCTCCGGTTTGTCACCGGCAGTCTTAC

[0026] TAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGG

[0027] ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAT

[0028] TTTGCCCCCGAAGGGGAAACTGCATCTCTCAGGTGATCAAAAGATGTCAAGACCT

[0029] GGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGG

[0030] GCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAAT

[0031] GCTTAATGCGTTAGCTGCGGCACGCTGAAGGGGAAACCCTCCAACACCTAGCATT

[0032] CATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTC

[0033] GAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCC

[0034] ATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCA

[0035] AGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGA

[0036] CTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGGC

[0037] ACCCACCAATTACCGCGGGTGGTGGGGCCCAAATTAACCCGGGGTTTCTTGGGG

[0038] GGAAACCCCCCACCCACAAAAAGAAACTCG

[0039] 3. Experiment on strain tolerance to cadmium

[0040] Weigh 27.47 g of Cd(NO₃)₂·4H₂O and dissolve it in 100 mL of sterile distilled water to prepare a cadmium aqueous solution with a cadmium concentration of 100 g / L. The solution was filtered through a 0.45 μm filter and stored in a refrigerator at 4°C until use. MRS solid culture media with varying cadmium concentrations were prepared, with the cadmium ion concentrations ranging from 5, 10, 50, 100, 200, 500, 1000, and 2000 mg / L. Each culture medium was divided into two equal sections, one of which was inoculated with Lactobacillus rhamnosus LRM2-4, while the other section was left empty as a control. The culture media were aerobically incubated at 37°C for 48–72 h, and the growth of each bacterial species was observed and recorded. At the same time, MRS broth containing cadmium concentrations of 5, 10, 50, 100, 200, 500, 1000 and 2000 mg / L was prepared, and the overnight culture solution was inoculated at a 1% inoculum volume. After inoculation, the culture solution was cultured at 37°C for 20 h, and the absorbance value (OD) of each culture solution at 600 nm was measured. 600nm The minimum inhibitory concentration (MIC) of Lactobacillus rhamnosus LRM2-4 in cadmium-containing MRS solid medium and cadmium-containing MRS broth medium was obtained to evaluate cadmium tolerance. The results showed that Lactobacillus rhamnosus LRM2-4 could tolerate 2000 mg / L cadmium concentration on solid medium and 100 mg / L cadmium concentration in MRS broth medium, indicating good cadmium tolerance.

[0041] 4. Cadmium adsorption capacity of strains

[0042] Lactobacillus rhamnosus LRM2-4 was inoculated on MRS medium and cultured at 37℃ for 48h. A single colony was picked and inoculated into MRS broth. After culture at 37℃ for 20h, 10mL of bacterial solution was centrifuged at 12000g for 20min to collect the bacteria. The bacteria were washed twice with sterile ultrapure water and weighed. 100g / L cadmium solution was prepared with sterile water to a concentration of 50mg / L Cd. 2+ The aqueous solution was added to the bacterial cells obtained by centrifugation to a final concentration of 1 g / L, and the cells were cultured in a shaking incubator at 37°C for 60 min. The adsorbed bacterial cells were precipitated by centrifugation at 8000 g for 20 min, and the supernatant was collected. The residual Cd in the solution after lactic acid bacteria adsorption was detected using an inductively coupled plasma spectrometer. 2+ The cadmium removal rate was calculated (the average value was taken after 3 repetitions) to characterize the cadmium adsorption capacity of the strain. The results showed that the cadmium removal rate of Lactobacillus rhamnosus LRM2-4 could reach 12.35%, indicating that the strain had good cadmium adsorption capacity.

[0043] The above describes the preferred embodiments of the present invention, which is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A Lactobacillus rhamnosus strain, characterized by: The Lactobacillus rhamnosus LRM2-4 is deposited in the China Center for Type Microorganism Collection, Wuhan University, Hubei Province, with a deposit number of CCTCC No. M20241815.

2. Use of the Lactobacillus rhamnosus according to claim 1 in alleviating heavy metal cadmium toxicity.

3. A probiotic product, characterized in that: The probiotic product contains the Lactobacillus rhamnosus according to claim 1.

4. The probiotic product according to claim 3, characterized in that: The probiotic product is a dairy product, a soy product, a fruit and vegetable product or a live bacteria preparation.