A microbial premix product and its application in aquaculture

Abstract

This invention discloses a microbial premix product and its application in aquaculture, belonging to the field of microbial technology. The microbial premix product includes *Bacillus lysinibacillus* and *Lactobacillus rhamnosus*. *Bacillus lysinibacillus* macroides is deposited at the China Center for Type Microbiology & Culture Collection (CCTCC), Wuhan University, Hubei Province, with accession number CCTCC No. M20241816; *Lactobacillus rhamnosus* is also deposited at the same location, Wuhan University, Hubei Province, with accession number CCTCC No. M20241815. The microbial premix product of this invention helps promote the growth of sea cucumbers and can significantly reduce their cadmium content.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial premixed product and its application in aquaculture. Background Technology

[0002] In the applicant's earlier application CN2025106181719 (invention title: A Lysinibacillus macroides that degrades Alternaria toxins and its application), a Lysinibacillus macroides is described, which can effectively reduce and degrade Alternaria toxins. The Lysinibacillus macroides is deposited at the China Center for Type Culture Collection, located at Wuhan University, Hubei Province, with accession number CCTCC No. M20241816.

[0003] In the applicant's earlier application CN2025106170771 (invention title: A strain of Lactobacillus rhamnosus that alleviates the toxicity of heavy metal cadmium and its application), a strain of Lactobacillus rhamnosus with good tolerance and removal ability to heavy metal cadmium is described. The Lactobacillus rhamnosus is deposited at the China Center for Type Microbiology, located at Wuhan University, Hubei Province, with accession number CCTCCNo.M20241815.

[0004] Further research by the applicant revealed that combining the lysine-containing Bacillus and Lactobacillus rhamnosus not only further improves the cadmium tolerance of sea cucumbers but also enhances their growth efficiency, thus completing this invention.

[0005] It should be noted that the full text of prior applications CN2025106181719 and CN2025106170771 is incorporated into this invention and is part of the content of this invention. The applicant reserves the right to include the entire contents of prior applications CN2025106181719 and CN2025106170771 in the specification of this invention and use them as the basis for modifications during subsequent examination. Summary of the Invention

[0006] Based on the above reasons, this invention proposes a microbial premixed product and its application in aquaculture, which helps promote the growth of sea cucumbers and can significantly reduce the cadmium content of sea cucumbers. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0007] This invention relates to a microbial premixed product comprising *Lysinibacillus macroides* and *Lactobacillus rhamnosus*, wherein the *Lysinibacillus macroides* is deposited at the China Center for Type Microbiology & Culture Collection (CCTCC), Wuhan University, Hubei Province, with accession number CCTCC No. M20241816; and the *Lactobacillus rhamnosus* is deposited at the same location, Wuhan University, Hubei Province, with accession number CCTCC No. M20241815.

[0008] In a preferred embodiment of the present invention, the weight ratio of the lysine-containing Bacillus to the rhamnosine-containing Lactobacillus is 1:2-4; preferably 1:3.

[0009] In a preferred embodiment of the present invention, the microbial premixed product is a lyophilized powder.

[0010] Another aspect of the present invention relates to an aquaculture feed containing the aforementioned microbial premix product.

[0011] In a preferred embodiment of the present invention, the content of the microbial premixed product in the aquaculture feed is 0.05-0.15% by weight.

[0012] Another aspect of the present invention relates to the application of the above-mentioned microbial premixed products in promoting the growth of sea cucumbers and / or reducing the cadmium content of sea cucumbers.

[0013] Beneficial effects

[0014] The beneficial effects of this invention are: the microbial premix product of this invention helps promote the growth of sea cucumbers and can significantly reduce the cadmium content of sea cucumbers. Detailed Implementation

[0015] 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.

[0016] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0017] Example 1:

[0018] 1. Isolation and preservation of Lysinibacillus macroides:

[0019] The inventors of this application discovered that Alternaria toxin was never detected in the intestines of turbot from some of the fish raised at the Yantai Laizhou Aquaculture Experimental Farm. Through further research, a strain of Lysinibacillus macroides was isolated from the intestines of the aforementioned fish and named LY015. This microbial strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Hubei Province, with accession number CCTCCNo.M20241816 and deposit date of August 19, 2024.

[0020] 2. Strain identification

[0021] 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 products were 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 strain LY015 is *Lysinibacillus macroides* using MK757660.1 (NCBI) as the standard. The 16S rDNA gene sequence is shown in SEQ ID NO. 1.

[0022]

[0023] Example 2

[0024] 1. Isolation and preservation of Lactobacillus rhamnosus:

[0025] The inventors of this application isolated and extracted a strain of Lacticaseibacillus rhamnosus from the intestines of healthy sea cucumbers, named LRM2-4. This microbial strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Hubei Province, with accession number CCTCCNo.M20241815 and deposit date of August 19, 2024.

[0026] 2. Strain identification

[0027] 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 products were 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*, and its 16S rDNA gene sequence is shown in SEQ ID NO. 1.

[0028] ACTTTGTCCCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGT

[0029] GTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC

[0030] ACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTT

[0031] GCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGT

[0032] CTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGG

[0033] GCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTAC

[0034] TAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGG

[0035] ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAT

[0036] TTTGCCCCCGAAGGGGAAACTGCATCTCTCAGGTGATCAAAAGATGTCAAGACCT

[0037] GGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGG

[0038] GCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAAT

[0039] GCTTAATGCGTTAGCTGCGGCACGCTGAAGGGGAAACCCTCCAACACCTAGCATT

[0040] CATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTC

[0041] GAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCC

[0042] ATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCA

[0043] AGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGA

[0044] CTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGGC

[0045] ACCCACCAATTACCGCGGGTGGTTGGGGCCCAAATTAACCCGGGGTTTCTTGGGG

[0046] GGAAACCCCCCACCCACAAAAAGAAACTCG

[0047] 3. Experiment on the strain's tolerance to cadmium

[0048] 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.

[0049] Example 3: Preparation of microbial premix products

[0050] 100g of Bacillus lysine LY015 fermentation broth (containing 10g of Bacillus lysine LY015) 8 CFU / g (dry matter content of 10%) and 300g of Lactobacillus rhamnosus LRM2-4 fermentation broth (Lactobacillus rhamnosus dry matter content of LRM2-4 is 10%). 8 A mixture of CFU / g (with a dry matter content of 8.3%) was freeze-dried to obtain a microecological premixed product.

[0051] As a control, lyophilized powders of Bacillus lysine LY015 fermentation broth and Lactobacillus rhamnosus LRM2-4 fermentation broth were used directly.

[0052] Example 4: Sea Cucumber Feeding Experiment

[0053] (1) 0.1% by weight of microecological premix product, Bacillus lysine LY015 fermentation broth and Lactobacillus rhamnosus LRM2-4 fermentation broth were added to the sea cucumber diet.

[0054] (2) Select sea cucumbers with an initial weight of about 8g. After 60 days of feeding, record the weight of the sea cucumbers and test their cadmium content. Calculate the weight gain rate of the sea cucumbers using the following formula:

[0055] Weight gain rate = (W-W0) / W0×100.

[0056] Where: W: final body weight (g), W0: initial body weight (g)

[0057] Table 1: Weight gain and cadmium content of sea cucumbers after feeding with different diets

[0058] Control group (without added diet) 15.5 93.8 0.163 Microbial premix product 21.3 166.3 0.072 Bacillus lysinus LY015 16.1 101.3 0.158 Lactobacillus rhamnosus LRM2-4 18.2 127.5 0.121

[0059] The experimental results show that Bacillus lysine-containing bacteria (LY015) and Lactobacillus rhamnosus (LRM2-4) alone did not significantly increase the weight of sea cucumbers, while the microbial premix significantly promoted sea cucumber growth. Furthermore, although Lactobacillus rhamnosus (LRM2-4) alone could reduce the cadmium content of sea cucumbers, the microbial premix significantly reduced the cadmium content.

[0060] 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. The application of a microbial premix product in promoting the growth of sea cucumbers and reducing the cadmium content of sea cucumbers, characterized in that: The microbial premixed product includes Bacillus lysine and Lactobacillus rhamnosus, wherein Bacillus lysine (… Lysinibacillus macroides The *Lactobacillus rhamnosus* described is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Hubei Province, with accession number CCTCC No. M20241816. Lactobacillus rhamnosus The sample is deposited at the China Center for Type Microbiology, located at Wuhan University, Hubei Province, with accession number CCTCCNo.M20241815; the weight ratio of the lysine-containing Bacillus and the rhamnosine-containing Lactobacillus is 1:2-4.

2. The application according to claim 1, characterized in that: The weight ratio of the lysine-containing Bacillus to the rhamnosine-containing Lactobacillus is 1:

3.

3. The application according to claim 1 or 2, characterized in that: The microbial premixed product is a freeze-dried powder.