Lactobacillus rhamnosus for alleviating cadmium toxicity and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus rhamnosus that alleviates the toxicity of heavy metal cadmium and its application. Background Technology

[0002] Chinese authorized invention patent CN116987638B discloses an efficient method for treating heavy metal cadmium based on heavy metal passivation strains. This method uses Bacillus belye YB03 as a cadmium-resistant microorganism. During the strain cultivation process, a specific inducer is added to enhance the adsorption capacity of the bacteria for 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 produce beneficial effects on animals after ingestion. Some are common in the animal gut microbiota, such as Lactobacillus, Bifidobacterium, and Bacillus, while others, like yeast, are less common. Probiotics must be able to colonize the animal's gastrointestinal tract and exert their beneficial effects within the gut environment. With increasing understanding of the link between animal gut microbiota and various diseases, probiotics have gained increasing attention as an effective method for regulating gut microbiota. The beneficial functions of probiotics in animals include: mediating the immune system's defense against pathogens; directly inhibiting or killing pathogens; protecting intestinal fluid, protecting and improving the intestinal barrier, and alleviating inflammatory effects; and maintaining the balance of the animal's gut microbiota. Lactobacillus rhamnosus and its compound preparations, by affecting the TLR-2 / MyD88 signaling pathway, enable mesenchymal stem cells expressing cyclooxygenase-2 to reposition in intestinal crypts, thereby reducing radiation damage and improving intestinal 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 as one of the most easily accumulated toxins in the body. However, current treatments for diseases caused by cadmium poisoning all have certain toxic side effects. Therefore, the use of probiotics with cadmium removal capabilities is a safer and more effective option.

[0005] Chinese authorized invention patent CN113046276B discloses a breast milk-derived *Lactobacillus rhamnosus* and its applications, with the preservation number CGMCC NO.21815. *Lactobacillus rhamnosus* AUH2101, derived from healthy Chinese breast milk, not only produces high concentrations of lactic acid and hydrogen peroxide—two antibacterial substances—and is sensitive to most antibiotics, but also exhibits gastrointestinal tolerance and good tolerance and removal ability for the heavy metal cadmium. This gives it excellent antibacterial potential, low potential risk, and good cadmium removal function, making it a promising candidate for development and application in the food industry.

[0006] However, the removal capacity of the aforementioned lactobacillus rhamnosus from breast milk for the heavy metal cadmium still has room for improvement. In order to provide a lactobacillus rhamnosus that can more effectively alleviate the toxicity of cadmium, the inventors of this application conducted research and completed this invention accordingly. Summary of the Invention

[0007] Based on the above reasons, this invention proposes a strain of *Lactobacillus rhamnosus* that alleviates the toxicity of the heavy metal cadmium and its application. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0008] One aspect of this invention relates to a strain of Lactobacillus rhamnosus, specifically Lactobacillus rhamnosus LRM2-4, which is deposited at the China Center for Type Microbiology, Wuhan University, Hubei Province, with accession number CCTCC No. M20241815.

[0009] Another aspect of the present invention relates to the application of the aforementioned Lactobacillus rhamnosus in alleviating the toxicity of the heavy metal cadmium.

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

[0011] Beneficial effects

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

[0013] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] Unless otherwise specified, all reagents involved in the embodiments of this 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 strain of Lacticaseibacillus rhamnosus LRM2-4 from the intestines of healthy sea cucumbers, named it LRM2-4, and deposited it at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Hubei Province, with accession number CCTCC No. M20241815 and deposit date of August 19, 2024.

[0018] 2. Strain identification

[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 detected by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Bomeide Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis, after sequence comparison and identification, confirmed that *Lactobacillus rhamnosus* LRM2-4 is indeed *Lactobacillus rhamnosus* LRM2-4, and its 16S rDNA gene sequence is shown in SEQ ID NO. 1.

[0020] ACTTTGTCCCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGT

[0021] GTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC

[0022] ACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTT

[0023] GCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGT

[0024] CTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGG

[0025] GCATGATGATTTGACGTCATCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCTTAC

[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 the strain's tolerance to cadmium

[0040] Weigh 27.47g of Cd(NO3)2·4H2O and dissolve it in 100mL of sterile distilled water to prepare a cadmium aqueous solution stock solution with a cadmium concentration of 100g / L. After filtration through a 0.45μm filter membrane, store it in a refrigerator at 4℃ for later use. Prepare MRS solid culture media with different cadmium concentrations, making the cadmium ion concentrations 5, 10, 50, 100, 200, 500, 1000 and 2000 mg / L respectively. Divide each culture medium into two equal areas. Inoculate one area with Lactobacillus rhamnosus LRM2-4, leaving one area empty as a control. Incubate at 37℃ aerobically for 48-72h and observe and record the growth of each bacterial species. Meanwhile, MRS broth media containing cadmium concentrations of 5, 10, 50, 100, 200, 500, 1000, and 2000 mg / L were prepared. Overnight bacterial cultures were inoculated at a 1% inoculum rate and cultured at 37°C for 20 hours. The absorbance (OD) of each culture at 600 nm was then measured. 600nm The minimum inhibitory concentration (MIC) of Lactobacillus rhamnosus LRM2-4 in cadmium-containing MRS solid medium and cadmium-containing MRS broth was obtained to evaluate its cadmium tolerance. The results showed that Lactobacillus rhamnosus LRM2-4 could tolerate a cadmium concentration of 2000 mg / L on solid medium and a cadmium concentration of 100 mg / L in MRS broth, demonstrating good cadmium tolerance.

[0041] 4. Cadmium adsorption capacity of the strain

[0042] Lactobacillus rhamnosus LRM2-4 was inoculated onto MRS medium and cultured at 37℃ for 48 h. Single colonies were picked and inoculated into MRS broth, cultured at 37℃ for 20 h, and then 10 mL of bacterial culture was centrifuged at 12000g for 20 min to collect the bacterial cells. The cells were washed twice with sterile ultrapure water and weighed. A 100 g / L cadmium solution stock solution was diluted with sterile water to prepare a 50 mg / L Cd solution. 2+ An aqueous solution was added to the centrifuged bacterial cells to achieve a final concentration of 1 g / L, and the mixture was incubated at 37°C in a shaker for 60 min. The adsorbed bacterial cells were then centrifuged at 8000 g for 20 min to precipitate the solution. The supernatant was collected, and residual Cd in the solution after adsorption was detected using inductively coupled plasma atomic emission spectrometry (ICP-AES). 2+ The concentration was calculated, and the cadmium removal rate (repeated 3 times and averaged) was used to characterize the cadmium adsorption capacity of the strain. The results showed that Lactobacillus rhamnosus LRM2-4 could remove cadmium at a rate of 12.35%, indicating that the strain has a good cadmium adsorption capacity.

[0043] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A strain of Lactobacillus rhamnosus, characterized in that: The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus LRM2-4 is deposited at the China Center for Type Microorganisms, located at Wuhan University, Hubei Province, with accession number CCTCC No. M20241815.

2. The application of Lactobacillus rhamnosus as described in claim 1 in alleviating the toxicity of heavy metal cadmium.

3. A probiotic product, characterized in that: The probiotic product contains Lactobacillus rhamnosus as described in claim 1.

4. The probiotic product according to claim 3, characterized in that: The probiotic products are dairy products, soy products, fruit and vegetable products, or live bacteria preparations.