A method for improving probiotic bacteria in fish by adding rare earth nanomaterials

Abstract

The present application belongs to the technical field of fish health breeding, and particularly relates to a method for increasing fish body probiotic genus by adding trace rare earth nanomaterials, which can significantly improve fish intestinal probiotic abundance by exposing fish to a nano cerium dioxide solution; the nano cerium dioxide solution is obtained by adding nano cerium dioxide particles to the water environment where the fish is located; the exposure time is at least 3 days; the nano cerium dioxide solution is replaced by 1 / 2 every two days; the present application can increase the abundance of probiotic genus in zebrafish intestinal tract and reduce the abundance of pathogenic bacteria genus by adding trace rare earth nanomaterials in the water environment, so as to improve the microbial community structure composition in the zebrafish intestinal tract. The method is simple, only needs to add nano cerium dioxide particles to the water environment, does not need to be mixed with feed, has simple components, low cost and convenient use, is suitable for large-scale popularization and use, and has wide market prospect.

Description

Technical fields:

[0001] This invention belongs to the fields of fish health aquaculture technology and biomedicine technology, specifically involving a method for increasing the abundance of beneficial bacteria in fish by adding rare earth nanomaterials. The method involves adding nano-cerium dioxide particles to water to increase the abundance of beneficial bacteria in fish. Background technology:

[0002] Rare Earth Elements (REEs) include 17 elements such as lanthanum, cerium, and praseodymium. Due to their unique physical and chemical properties, rare earth elements play an important role in the fields of electronics, energy, and materials. Furthermore, when they enter the aquatic environment, they affect the physiological functions and growth and development of aquatic organisms.

[0003] In recent years, nanomaterials have seen increasingly widespread applications and rapid development in various industrial and medical fields. Because nanomaterials are much smaller than ordinary macroscopic particles, they exhibit significant changes in certain properties compared to conventional materials, thus holding great promise in the field of new materials. Cerium dioxide nanoparticles (CeO2NPs) have attracted widespread attention due to their powerful antioxidant properties. Studies have demonstrated that CeO2NPs can alleviate oxidative stress in mice by reducing oxidative stress and improving mitochondrial function.

[0004] Probiotics are live microorganisms that colonize an organism and alter the composition of the gut microbiota in a specific part of the host, thus benefiting the host. Most of them colonize the host's gut, promoting nutrient absorption and maintaining gut health by regulating the balance of gut microbiota. Probiotics in the gut microbiota play an important role in the host's normal physiological functions, such as nutrition, immunity, and digestion. Studies have shown that probiotics in the gut microbiota can reduce the toxic effects of bisphenol A (BPA) on zebrafish. Therefore, the gut microbiota is often considered one of the key factors in regulating host health. Summary of the Invention:

[0005] To address the problems mentioned in the background section, the present invention aims to provide a method for enhancing the levels of beneficial bacteria in fish by adding trace amounts of rare earth nanomaterials to the aquatic environment, thus providing a direction for healthy aquaculture.

[0006] To achieve the above objectives, this invention provides a method for enhancing the levels of beneficial bacteria in fish by adding trace amounts of rare earth nanomaterials. The fish are exposed to a nano-cerium dioxide solution for at least 3 days using a semi-static exposure method. The nano-cerium dioxide solution is obtained by adding nano-cerium dioxide particles to the water in which the fish are located, and the concentration of cerium in the solution is 1-1000 μg / L. Half of the nano-cerium dioxide solution is replaced every two days. The optimal concentration range of the nano-cerium dioxide solution is 10-100 μg / L (based on cerium element). The fish used are wild-type zebrafish.

[0007] The nano-cerium dioxide particles are spherical nanoparticles prepared by a hydrothermal method using cerium nitrate hexahydrate as the cerium source.

[0008] This invention investigates the effects of adding cerium dioxide nanoparticles to the zebrafish growth environment and culturing them for 26 days on the abundance of beneficial bacteria in the zebrafish gut under different concentrations of cerium dioxide nanoparticle exposure. Specifically, a stock solution of cerium dioxide nanoparticles was prepared at a certain concentration, and the stock solution was diluted several times to obtain exposure solutions. A semi-static exposure method was used, with half the volume of the treatment solution being replaced every two days. A control group was set up, and the treatment groups were exposed to cerium dioxide nanoparticles at concentrations of 1 μg / L, 10 μg / L, 100 μg / L, and 1000 μg / L, respectively. The changes in the zebrafish gut microbiota were observed. Treatment with different concentrations of cerium dioxide nanoparticle exposure solutions all increased the abundance of beneficial bacteria and decreased the abundance of pathogenic bacteria in the zebrafish gut. It was found that a cerium concentration of 10-100 μg / L in the zebrafish growth environment significantly increased the abundance of beneficial bacteria in the zebrafish gut.

[0009] Compared with existing technologies, this invention increases the abundance of *Cetobacterium*, *Reyranella*, and *Rhodococcus* genera in the zebrafish gut by adding trace amounts of rare earth nanomaterials to the aquatic environment. These bacteria play important roles in amino acid production, participation in various metabolic activities, and enhancing the organism's ability to adapt to environmental stress. Treatment with nano-cerium dioxide exposure solution can increase the abundance of beneficial bacteria and decrease the abundance of pathogenic bacteria in the zebrafish gut, thereby improving the composition of the gut microbial community. These results indicate that nano-cerium dioxide has a significant effect on enhancing beneficial bacteria in the zebrafish gut, providing important reference for healthy fish farming. The method described in this invention is simple, requiring only the addition of nano-cerium dioxide particles to the aquatic environment without mixing with feed. It has a simple composition, low cost, and is easy to use. Cerium is abundant and cheaper than other rare earth elements, and the preparation method of nano-cerium dioxide is simple; it is suitable for large-scale promotion and has broad market prospects. Attached image description:

[0010] Figure 1The effects of different amounts of nano-cerium dioxide material on the species composition of zebrafish gut microbiota at the phylum and genus levels were investigated, where A represents the phylum level and B represents the genus level.

[0011] Figure 2 Heatmap of the genus-level composition of zebrafish gut microbiota for different amounts of nano-cerium dioxide material.

[0012] Figure 3 The effects of different amounts of nano-cerium dioxide material on the diversity of gut microbiota in zebrafish are shown in Figure A, where A is a Venn diagram of gut microbiota for each treatment and B is a bar chart of the chao1 diversity index of gut microbiota for each treatment.

[0013] Figure 4 Heatmap showing the effects of different amounts of nano-cerium dioxide material on the gut microbiota function of zebrafish.

[0014] Figure 5 The figure shows the results of differential species screening of zebrafish gut microbiota composition based on different amounts of nano-cerium dioxide materials. In the figure, A is a bar chart of LDA value distribution of gut microbiota in each treatment, and B is an evolutionary branching chart of gut microbiota in each treatment. Detailed implementation method:

[0015] The present invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1:

[0017] This embodiment relates to a method for enhancing the beneficial bacteria in fish by adding trace amounts of rare earth nanomaterials. Different concentrations of nano-cerium dioxide suspensions are added to the zebrafish's growth environment, exposing the zebrafish to the nano-cerium dioxide environment. The specific steps include:

[0018] (I) Preparation of nano-cerium dioxide particles:

[0019] The synthesis steps are as follows: 1. Weigh 0.815g of cerium nitrate hexahydrate Ce(NO3)3·6H2O and add it to 10ml of deionized water. Stir magnetically for 10min to dissolve it. 2. Add the above solution dropwise to 40mL of a mixed deionized water solution containing 2.94g of trisodium citrate dihydrate and 0.96g of sodium hydroxide. Stir magnetically for 30min. 3. Transfer the resulting solution to a 50mL stainless steel high-pressure hydrothermal reactor lined with polytetrafluoroethylene. React hydrothermally at 120℃ for 24h. 4. After cooling to room temperature, wash the solution three times alternately with anhydrous ethanol and distilled water, dry, and calcine to obtain nano-cerium dioxide particles.

[0020] The nano-cerium dioxide suspension was prepared as follows: 0.0614 g of the nano-cerium dioxide particles prepared above was weighed and added to 1 L of deionized water to obtain a nano-cerium dioxide suspension with a concentration of 61.4 mg / L, which was used as the mother liquor. The concentration of cerium element was 50 mg / L. The mother liquor was sonicated for 30 min before use. When using, the mother liquor was diluted to the required concentration by a certain multiple to obtain the exposure solution.

[0021] (II) Domestication and Grouping of Zebrafish:

[0022] The domestication steps are as follows: 1. Select wild-type zebrafish aged 3-4 months and purchase them from the Nanshan Flower and Bird Market in Qingdao; raise them in a 60L glass tank with circulating water, water temperature 26±1℃, pH=7.2±1, light-dark ratio 14h:10h, and feed them with molted brine shrimp eggs at 9:00 and 16:00 every day during the domestication period; raise them under the above conditions for one week, and after the zebrafish have no deaths and have adapted to the laboratory conditions, randomly select zebrafish for exposure experiments;

[0023] The grouping scheme is as follows: After acclimatization, the fry were randomly divided into 5 groups of 40 each from the 60L glass tanks, and placed in 5 4L glass tanks respectively. One group was the control group, which received no intervention and used pure water as the growth environment. Another group was grown in a 1μg / L cerium exposure solution, i.e., 80μL of stock solution was added to 4L pure water to obtain an exposure solution with a cerium concentration of 1μg / L (denoted as Ce_1). Another group was grown in a 10μg / L cerium exposure solution, i.e., 800μL of stock solution was added to 4L pure water to obtain an exposure solution with a cerium concentration of 10μg / L (denoted as Ce_10). A 100 μg / L cerium exposure solution was prepared by adding 8 mL of stock solution to 4 L of pure water, resulting in a cerium concentration of 100 μg / L (denoted as Ce_100). Another group received a 1000 μg / L cerium exposure solution, prepared by adding 80 mL of stock solution to 4 L of pure water, resulting in a cerium concentration of 1000 μg / L (denoted as Ce_1000). In both the Control group and the four treatment groups, half of the exposure solution was replaced every 48 hours. Food residue was regularly removed, and dead zebrafish were promptly removed. The water temperature was maintained at 26 ± 1℃. The zebrafish were fed molted brine shrimp eggs at 9:00 AM and 4:00 PM daily. After 26 days of exposure, the zebrafish were anesthetized with MS-222, and intestinal tissue was obtained through dissection. The tissue was flash-frozen in liquid nitrogen for 30 minutes and then stored at -80℃.

[0024] (III) Sample Collection:

[0025] The collection steps are as follows: 1. Sampling was conducted after the 26-day formal exposure period ended, with feeding stopped the day before sampling. All zebrafish in each tank were removed and dissected under a dissecting microscope; before sampling, the zebrafish were anesthetized with MS-222 to minimize their suffering; after isolating the complete zebrafish intestines, they were placed in sterile, enzyme-free 2.0mL cryovials, flash-frozen in liquid nitrogen, numbered, recorded, and stored at -80℃ for intestinal flora analysis.

[0026] (iv) Intestinal 16S amplicon analysis:

[0027] The abundance in this invention refers to relative abundance. The relative abundance determination method for microorganisms or microbial communities is as follows: DNA is extracted from collected intestinal samples and detected; the intestinal DNA is amplified by PCR, the product is purified by magnetic beads, and equal amounts of the PCR product are co-cultured according to the concentration of the PCR product. After thorough mixing, the PCR product is detected and the target band is recovered; a library is constructed, and the constructed library is quantified by Qubit and qPCR. After the library is qualified, it is sequenced; the sequencing data is cut, filtered, and subjected to noise reduction processing of characteristic sequences. ASVs (Amplicon Sequence Variants) cluster analysis is performed, and species annotation and relative abundance analysis are performed based on the ASV clustering results.

[0028] use Intestinal tissue PCR products were obtained using High-Fidelity PCR Master Mix (New England Biolabs). The tissue was first denatured at 98°C for 1 minute, followed by 30 cycles at 98°C (10 s), 50°C (30 s), and 72°C (30 s), and finally held at 72°C for 5 minutes. The V3-V4 variable region was amplified by PCR using the F-terminus (5'-CCTAYGGGRBGCASCAG-3') and the R-terminus (5'-GGACTACNNGGGTATCTAAT-3').

[0029] 16S rRNA amplicon sequencing (16S rDNA amplicon sequencing) was performed at Novogene using the Illumina NovaSeq sequencing platform to sequence the paired ends of the library. First, the raw sequencing data was assembled and filtered to obtain valid data. Then, based on the valid data, noise reduction was performed using DADA2 to obtain the final ASVs. The QIIME2 classify-sklearn algorithm was used to annotate each ASV using a pre-trained Naive Bayes classifier. Based on the ASV annotation results and the feature labels of each sample, a species abundance table at the phylum and genus levels was obtained.

[0030] The relative abundance of the above species is shown in the table below. Figure 1 As shown.

[0031] The effects of different amounts of nano-cerium dioxide materials on the phylum and genus-level species composition of zebrafish gut microbiota, such as Figure 1 As shown, the results indicated that the dominant species at the phylum level in the control group were Proteobacteria, Firmicutes, Actinobacteriota, and Cyanobacteria. The species composition of the samples in each treatment group was similar, with the dominant species at the phylum level being Fusobacteriota, Proteobacteria, and Actinobacteriota. Figure 1 The study also showcased the top ten genera in terms of relative abundance of gut microbiota at the genus level, with the abundance of Cetobacterium and Reyranella in the treatment group increasing compared to the control group.

[0032] Heatmaps of the genus-level composition of zebrafish gut microbiota with different amounts of nano-cerium dioxide materials, such as Figure 2As shown in the figure. The horizontal axis represents sample grouping information, and the vertical axis represents species annotation information. The clustering tree on the left side of the figure is a species clustering tree; the values ​​corresponding to the heatmap are the Z-values ​​obtained after standardizing the relative abundance of species in each row. That is, the Z-value of a sample in a certain category is the difference between the relative abundance of the sample in that category and the average relative abundance of all samples in that category, divided by the standard deviation of all samples in that category. Figure 2 The results showed that the abundance of *Cetobacterium*, *Reyranella*, and *Rhodococcus* bacteria in the treatment group was significantly upregulated compared to the control group. The study indicates that *Cetobacterium*, a highly abundant bacterium in the fish gut microbiota, can activate the parasympathetic nervous system through its metabolite acetic acid, thereby promoting sugar utilization in fish and playing an important regulatory role in fish health. *Reyranella* is believed to have a positive impact on the survival of adult fish in adverse environments. *Rhodococcus* possesses various hydrolases and synthases and can degrade various exogenous substances to produce bioactive compounds, which is significant in biological adaptation to environmental stress.

[0033] Based on the obtained characteristic sequence results, the VebbDiagram() function was used to generate common and unique sequences between the control group and each treatment group in R, and Venn diagrams were plotted. The QIIME2 software was used to calculate the chao1 et al. Alpha diversity index, and the Tukey and Kruskal-Wallis rank-sum tests were used to analyze whether the differences in species diversity between groups were significant.

[0034] The effects of different amounts of nano-cerium dioxide materials on the diversity of gut microbiota in zebrafish, such as Figure 3 As shown in the Veen plot, the total number of ASVs in the control group and the four treatment groups (Ce_1, Ce_10, Ce_100, and Ce_1000) were 398, 150, 236, 240, and 218, respectively. The total number of ASVs in the treatment groups was lower than that in the control group, and the total number of ASVs shared by all five groups was 43. Different concentrations of nano-cerium dioxide solution had a significant impact on the chao1 index of zebrafish gut microbiota. At a cerium concentration of 1 μg / L, the species richness of the zebrafish gut was significantly lower than that of the control. This indicates that the addition of nano-rare earth materials altered the composition and structure of the zebrafish gut microbiota.

[0035] Tax4Fun (V1.1.4) is an R software widely used for intestinal samples. Compared to PICRUSt, Tax4Fun can provide more accurate results. This invention selects the top 35 most abundant functions and their abundance information in each sample based on the functional annotations and abundance information of the samples in the database, plots a heatmap, and performs clustering at different functional levels.

[0036] The heatmap shows the effects of different concentrations of nano-cerium dioxide solutions on the functional annotation of zebrafish gut microbiota. Figure 4 As shown. From the appendix Figure 4 The results showed that most ABC transporter family genes, including ABC-2.A (K01990), ABC-2.P (K01992), ABC.PE.S (K02035), ABC.FEV.P (K02015), ABC.SN.P (K02050), and ABC.SN.A (K02049), were found in both the treatment and control groups. These genes are mainly involved in the ABC transport system and are related to the input and output of exogenous substances, drugs, and endotoxins. Furthermore, it is noteworthy that oxidative stress-related entry K00799 was found, defined as glutathione S-transferase (GST), which is mainly involved in glutathione metabolism. The Z-values ​​of these genes were found to be positively correlated with CeO2NPs concentration, indicating that after exposure concentrations exceeded 1 μg / L, ABC transporter expression was abnormally upregulated with increasing concentration, glycolipid synthesis and transport became more active, and the GST-involved oxidative stress-related pathways became more active. This suggests that the addition of nano-cerium dioxide materials helps improve the intestinal glycolipid synthesis and transport and the antioxidant system in zebrafish. To provide direction for aquatic organisms and healthy aquaculture.

[0037] LEfSe (LDA Effect Size) is an analytical tool for discovering and interpreting high-dimensional biomarkers. It can be used to compare multiple groups, emphasizing statistical significance and biological relevance, and is able to identify biomarkers with statistically significant differences between groups. LEfSe's statistical results include two parts: a bar chart of LDA value distribution and an evolutionary clade plot (phylogenetic distribution). This invention uses LEfSe software with an LDA score set to 4 to identify bacterial taxa with significant differences in abundance between groups.

[0038] Differential species screening LDA and evolutionary clade diagram of zebrafish gut microbiota composition by different concentrations of nano-cerium dioxide solutions are shown below. Figure 5 As shown in the figure. LESFE linear discriminant analysis was used to screen biomarkers in each group. In the control group, g_Stenoteophomonas, o_Xanthomonadales, and f_Xanthomonadaceae were mainly enriched. In the 1 μg / L group, p_Fusobacteriota was significantly higher than in the other groups. In the 10 μg / L group, c_Gammaproteobacteria had a high abundance. In the 100 μg / L group, p_Actinobacteriota and g_Rhodococcus showed significant differences. In the 1000 μg / L group, p_Proteobacteria, g_Reyranella, and o_Rhizobiales had high abundance.

[0039] In summary, the gut microbiota, as an important organ in zebrafish, plays a vital role in regulating metabolism and host health. This invention demonstrates that different concentrations of nano-cerium dioxide solutions can increase the number of beneficial bacteria such as *Cetobacterium*, *Reyranella*, and *Rhodococcus*. These bacteria play an important role in promoting the utilization of glycolipids in the fish gut, degrading exogenous substances, and enhancing the organism's resistance to stress.

Claims

1. A method for increasing the number of beneficial bacteria in fish by adding rare earth nanomaterials, characterized in that, Exposing fish to a nano-cerium dioxide solution can significantly improve the abundance of beneficial bacteria in the fish gut; the nano-cerium dioxide solution is obtained by adding nano-cerium dioxide particles to the water in which the fish are located; the concentration of the nano-cerium dioxide solution, calculated as cerium, is 1-1000 μg / L; the fish is zebrafish.

2. The method for improving the beneficial bacteria in fish by adding rare earth nanomaterials according to claim 1, characterized in that, The exposure time is at least 3 days; half of the nano-cerium dioxide solution is replaced every two days.

3. The method for improving the beneficial bacteria in fish by adding rare earth nanomaterials according to claim 1, characterized in that, The concentration range of nano-cerium dioxide solution, calculated as cerium element, is 10-100 μg / L.

4. The method for improving the beneficial bacteria in fish by adding rare earth nanomaterials according to claim 1, characterized in that, During the exposure period, the water temperature was 26±1℃, pH=7.2±1, and the light-dark ratio was 14h:10h. The shrimp were fed with molted brine shrimp eggs at 9:00 and 16:00 daily.

Images