Method for increasing probiotic genus in fish body by adding rare earth nano material

Abstract

The invention belongs to the technical field of healthy culture of fishes, and particularly relates to a method for increasing the content of probiotics in fish bodies by adding trace rare earth nano-materials, the fish is exposed in a nano cerium dioxide solution, and the abundance of the probiotics in the intestinal tracts of the fish can be remarkably improved; the nano cerium dioxide solution is obtained by adding nano cerium dioxide particles into a water body in which the fish is located; the exposure time is at least 3 days; replacing 1 / 2 of the nano cerium dioxide solution every two days; the trace rare earth nano material is added into a water environment, so that the abundance of probiotics in intestinal tracts of zebra fish can be improved, and the abundance of pathogenic bacteria can be reduced; therefore, the microflora structure composition in the intestinal tract of the zebra fish is improved. The method is simple, only nano cerium dioxide particles need to be added into a water body environment, mixing with feed is not needed, and the method is simple in component, low in cost and convenient to use; the method is suitable for large-scale popularization and application.

Description

Technical field:

[0001] The present invention belongs to the field of fish health breeding technology and biomedicine technology, and specifically relates to a method for increasing the abundance of probiotics in fish by adding rare earth nanomaterials. The abundance of probiotics in fish can be increased by adding nano cerium dioxide particles in water. 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. When they enter the water environment, they affect the physiological functions and growth and development of aquatic organisms.

[0003] In recent years, nanomaterials have been used more and more widely and have developed rapidly in various industrial and medical fields. Because the size of nanomaterials is much smaller than that of general macroscopic particles, certain properties of nanomaterials will change significantly compared with ordinary materials, so they have broad prospects in the field of new materials. Nano cerium dioxide particles (CeO2NPs) have attracted widespread attention due to their strong antioxidant properties. Studies have shown that CeO2NPs can reduce oxidative stress in mice by oxidative stress and improving mitochondrial function.

[0004] Probiotics refer to a class of active microorganisms that are beneficial to the host and colonize in the body and change the composition of the flora in a certain part of the host. Most of them colonize in the host's intestines, regulate the balance of intestinal flora, promote nutrient absorption and maintain intestinal health. The probiotics in the intestinal flora are of great significance to the normal physiological functions of the host, such as nutrition, immunity, and digestion. Studies have shown that probiotics in the intestinal flora can reduce the toxic effects of bisphenol A on zebrafish. Therefore, the intestinal microbiome is often considered to be one of the key factors in regulating host health. Summary of the invention:

[0005] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide a method for increasing probiotics in fish by adding trace amounts of rare earth nanomaterials to the water environment, thereby providing direction for the healthy breeding of aquatic organisms.

[0006] To achieve the above-mentioned purpose, the present invention provides a method for increasing probiotics in fish by adding trace rare earth nanomaterials. The fish is exposed to a nano-cerium dioxide solution and treated in a semi-static exposure manner for at least 3 days. The nano-cerium dioxide solution is obtained by adding nano-cerium dioxide particles to the water body where the fish is located, and the concentration of cerium in the solution is 1-1000 μg / L; 1 / 2 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 (in terms of cerium element). The fish is a wild-type zebrafish;

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

[0008] The present invention adds nano-cerium dioxide particles to the zebrafish growth environment and cultivates them for 26 days, and observes the changes in the abundance of probiotics in the zebrafish intestine under different concentrations of nano-cerium dioxide exposure conditions. Specifically, the nano-cerium dioxide particles are configured with a certain concentration of mother liquor, and the mother liquor is diluted by a certain multiple to obtain an exposure solution. A semi-static exposure method is adopted, and 1 / 2 volume of the treatment solution is replaced every two days. By setting a control group, the treatment groups are nano-cerium dioxide exposure solutions with cerium element concentrations of 1μg / L, 10μg / L, 100μg / L, and 1000μg / L, respectively, to observe the degree of change in the zebrafish intestinal flora. Treatment with different concentrations of nano-cerium dioxide exposure solutions can increase the abundance of probiotics in the zebrafish intestine and reduce the abundance of pathogenic bacteria; it is found that when the cerium element concentration in the zebrafish growth environment is 10-100μg / L, the abundance of probiotics in the zebrafish intestine can be significantly increased.

[0009] Compared with the prior art, the present invention increases the abundance of Cetobacterium, Reyranella and Rhodococcus in the zebrafish intestine by adding trace rare earth nanomaterials in the water environment. These bacteria play an important role in the production of amino acids, participation in various metabolic activities and enhancing the ability of organisms to adapt to environmental stress; the abundance of probiotics in the zebrafish intestine can be increased by treating the nano-cerium dioxide exposure liquid, and the abundance of pathogenic bacteria can be reduced; thereby improving the structure and composition of the microbial community in the zebrafish intestine. These results show that nano-cerium dioxide has a significant effect on the improvement of probiotics in the zebrafish intestine, and provides an important reference for fish health breeding. The method described in the present invention is simple, only nano-cerium dioxide particles need to be added to the water environment, no need to mix with feed, simple ingredients, low cost, and easy to use; the cerium element has a high reserve, the price is lower than other rare earth elements, and the nano-cerium dioxide preparation method is simple; it is suitable for large-scale promotion and use, and the market prospect is broad. Description of the drawings:

[0010] Figure 1The effects of different addition amounts of nano-cerium dioxide materials on the species composition of the zebrafish intestinal microbial flora at the phylum level and genus level, where A is the microbial flora phylum level and B is the microbial flora genus level.

[0011] Figure 2 This is a heat map of the effects of different amounts of nano-cerium dioxide materials on the genus level composition of zebrafish intestinal microorganisms.

[0012] Figure 3 The figure shows the effect of different addition amounts of nano-cerium dioxide materials on the diversity of zebrafish intestinal flora, where A is the Venn diagram of the intestinal flora of each treatment, and B is the chao1 diversity index bar chart of the intestinal flora of each treatment.

[0013] Figure 4 This is a heat map of the effects of different added amounts of nano-cerium dioxide materials on the function of zebrafish intestinal flora.

[0014] Figure 5 This is a graph showing the results of screening different species in the zebrafish intestinal flora composition with different addition amounts of nano-cerium dioxide materials, where A is a bar graph showing the LDA value distribution of the intestinal flora in each treatment, and B is a graph showing the evolutionary branches of the intestinal flora in each treatment. Specific implementation method:

[0015] The present invention will be described clearly and completely below in conjunction with the drawings in 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 creative work are within the scope of protection of the present invention.

[0016] Embodiment 1:

[0017] This embodiment relates to a method for increasing probiotics in fish by adding trace amounts of rare earth nanomaterials, wherein nano-cerium dioxide suspensions of different concentrations are added to a zebrafish production environment to expose the zebrafish to the nano-cerium dioxide environment, and specifically comprises the following steps:

[0018] (I) Preparation of Nano-Cerium Oxide Particles:

[0019] The synthesis steps are as follows: 1. Weigh 0.815g of hexahydrate cerium nitrate Ce(NO3)3·6H2O and add it to 10ml deionized water, and stir it magnetically for 10min to dissolve it; 2. Add the above solution dropwise into 40mL of a mixed deionized water solution containing 2.94g of trisodium citrate dihydrate and 0.96g of sodium hydroxide, and stir it magnetically for 30min; 3. Transfer the obtained solution into a 50mL polytetrafluoroethylene-lined stainless steel high-pressure hydrothermal autoclave, and perform hydrothermal reaction at 120°C for 24h; 4. After cooling to room temperature, wash it alternately with anhydrous ethanol and distilled water for 3 times, dry it, and calcine it 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 into 1 L of deionized water to obtain a nano-cerium dioxide suspension with a concentration of 61.4 mg / L as a mother solution, wherein the cerium element concentration was 50 mg / L, and the mother solution was sonicated for 30 minutes before use; when used, the mother solution was diluted to the required concentration according to a certain multiple to obtain an exposure solution;

[0021] (II) Zebrafish domestication and grouping:

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

[0023] The grouping scheme is as follows: after the acclimation is completed, the 60L glass cylinder is randomly divided into 5 groups, 40 in each group, and placed in 5 4L glass cylinders respectively; one group is the blank group (Control) without any intervention, using pure water as the growth environment; one group of growth environment is 1μg / L cerium exposure solution, that is, 80μL of mother solution is added to 4L pure water to obtain an exposure solution with a cerium concentration of 1μg / L (denoted as Ce_1); one group of growth environment is 10μg / L cerium exposure solution, that is, 800μL of mother solution is added to 4L pure water to obtain an exposure solution with a cerium concentration of 10μg / L (denoted as Ce_10); one group of growth environment is 100μg / L cerium exposure solution, that is, 8mL of mother solution was added to 4L of pure water to obtain an exposure solution with a cerium concentration of 100μg / L (referred to as Ce_100); a group of growth environments were 1000μg / L cerium exposure solution, that is, 80mL of mother solution was added to 4L of pure water to obtain an exposure solution with a cerium concentration of 1000μg / L (referred to as Ce_1000); half of the exposure solution was replaced every 48h in the above control group and the four treatment groups, food residues were cleaned regularly, and dead zebrafish were cleaned up in time; the water temperature was maintained at 26±1℃; shelled brine shrimp eggs were fed at 9:00 and 16:00 every day. After 26 days of exposure, the zebrafish were anesthetized with MS-222 and the intestinal tissue was obtained by dissection. After being quickly frozen in liquid nitrogen for 30min, it was stored in a -80℃ refrigerator.

[0024] (III) Sample collection:

[0025] The collection steps are as follows: 1. Sampling was performed after the formal exposure period of 26 days, and feeding was stopped one day before sampling. All zebrafish in each fish tank were taken out and placed under a dissecting microscope for dissection; before sampling, the zebrafish were anesthetized with MS-222 to minimize the pain of the zebrafish; after the complete intestine of the zebrafish was separated, it was placed in a sterile enzyme-free 2.0mL cryopreservation tube, quickly frozen with liquid nitrogen, and stored at -80℃ with numbering and recording for intestinal flora determination.

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

[0027] The abundance in the present invention is relative abundance, and the relative determination method of microorganisms or microbial flora is as follows: extract DNA from the collected intestinal sample and detect it; perform PCR amplification on the intestinal DNA, purify the product by magnetic beads, mix them in equal amounts according to the concentration of the PCR products, detect the PCR products after sufficient mixing and recover the target bands; construct a library, quantify the constructed library by Qubit and qPCR, and sequence the library after it is qualified; perform feature sequence denoising after shearing and filtering the data obtained by sequencing, perform ASVs (Amplicon Sequence Variants) cluster analysis, and perform species annotation and relative abundance analysis based on the ASV clustering results.

[0028] use High-Fidelity PCR Master Mix (New England Biolabs) was used to obtain the PCR products of intestinal tissues, and the first denaturation was performed at 98°C for 1 minute, followed by 30 cycles at 98°C (10s), 50°C (30s), and 72°C (30s), and finally kept at 72°C for 5 minutes. The V3-V4 variable region was PCR amplified using the F end (5'-CCTAYGGGRBGCASCAG-3') and the R end (5'-GGACTACNNGGGTATCTAAT-3').

[0029] 16S rRNA amplicon gene sequencing (16S rDNA Amplicon Sequencing) was performed at Novogene Company based on the Illumina NovaSeq sequencing platform to perform double-end sequencing on the library. First, the raw data obtained by sequencing was spliced ​​and filtered to obtain valid data; then, based on the valid data, noise reduction was performed using DADA2 to obtain the final ASVs. The classify-sklearn algorithm of QIIME2 was used to annotate each ASV using a pre-trained Naive Bayes classifier; based on the ASVs annotation results and the characteristic marks of each sample, the species abundance table at the phylum, genus, and other levels was obtained.

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

[0031] Effects of different amounts of nano-cerium dioxide materials on the species composition of zebrafish intestinal microbial flora at the phylum and genus levels Figure 1 As shown in the figure, the dominant species at the phylum level in the control group (Control) were Proteobacteria, Firmicutes, Actinobacteriota, and Cyanobacteria. The species composition of samples in each treatment group was similar, and the dominant species at the phylum level were Fusobacteriota, Proteobacteria, and Actinobacteriota. Figure 1 At the same time, the top ten bacterial genera in terms of relative abundance of intestinal microorganisms at the genus level were displayed, among which Cetobacterium and Reyranella in the treatment group were more abundant than those in the control group.

[0032] Heat map of the composition of zebrafish intestinal microorganisms at the genus level with different addition amounts of nano-cerium dioxide materials, such as Figure 2As shown. The horizontal axis in the figure is the sample grouping information, and the vertical axis is the species annotation information. The cluster tree on the left side of the figure is the species clustering tree; the value corresponding to the heat map is the Z value obtained after the relative abundance of each row of species is standardized, that is, the Z value of a sample in a certain category is the difference between the relative abundance of the sample in the category and the average relative abundance of all samples in the category divided by the standard deviation of all samples in the category. Figure 2 The results showed that the abundance of Cetobacterium, Reyranella and Rhodococcus in the treatment group was significantly higher than that in the control group. The study showed that Cetobacterium, as a high-abundance bacterium in the intestinal flora of fish, can activate the parasympathetic nervous system through its metabolite acetic acid, thereby promoting the sugar utilization ability of fish and playing an important regulatory role in fish health; Reyranella is believed to have a positive effect on the survival of adult fish in adverse environments; Rhodococcus has a variety of hydrolases and synthases, and can degrade a variety of exogenous substances to produce active compounds, which is of great significance in the adaptation of organisms to environmental stress.

[0033] According to the obtained characteristic sequence results, the common and unique sequences between the control group and each treatment group were generated in R by using the VebbDiagram() function, and the Venn diagram was drawn. The Alpha diversity index such as Chao1 was calculated using the QIIME2 software, and the Tukey and Kruskal-Wallis rank sum tests were used to analyze whether the species diversity differences between the groups were significant.

[0034] Effects of different amounts of nano-cerium dioxide materials on the diversity of zebrafish intestinal flora Figure 3 As shown. The results show that the total number of ASVs in the control group and the four treatment groups (Ce_1, Ce_10, Ce_100 and Ce_1000) is 398, 150, 236, 240 and 218 respectively. The total number of ASVs in the treatment groups is less than that in the control group, and the total number of ASVs in the five groups is 43. Nano-cerium dioxide solutions of different concentrations have a great influence on the chao1 index of zebrafish intestinal flora. When the cerium concentration is 1μg / L, the species richness of zebrafish intestine is significantly reduced compared with the control. This shows that the addition of nano rare earth materials has changed the composition structure of zebrafish intestinal flora.

[0035] Tax4Fun (V1.1.4) is an R software widely used for intestinal samples. Compared with PICRUSt, Tax4Fun can provide more accurate results. According to the functional annotations and abundance information of the samples in the database, the present invention selects the top 35 functions in abundance and their abundance information in each sample to draw a heat map, and clusters them from different functional levels.

[0036] The heat map of the effects of different concentrations of nano-cerium dioxide solutions on the functional annotation of zebrafish intestinal flora is shown in the figure. Figure 4 As shown. Figure 4 It can be seen that the results show that most of the ABC transporter family genes ABC-2.A (K01990), ABC-2.P (K01992), ABC.PE.S (K02035), ABC.FEV.P (K02015), ABC.SN.P (K02050), ABC.SN.A (K02049) were found in both the treatment group and the control group. These genes are mainly involved in the ABC transport system, which is related to the input and export of exogenous substances, drugs and internal and external toxins. In addition, it is worth noting that the oxidative stress-related entry K00799 was found, which is defined as glutathione-S-transferase (GST), which is mainly involved in glutathione metabolism. It was found that the Z value of the above genes was positively correlated with the concentration of CeO2NPs, indicating that after the exposure concentration exceeded 1μg / L, the expression of ABC transporters was abnormally upregulated with the increase of concentration, the synthesis and transportation of glycolipids was more active, and the oxidative stress-related pathways involved in GST were more active. It shows that the addition of nano-cerium dioxide material helps to improve the synthesis and transportation of glycolipids and the antioxidant system in the intestine of zebrafish. Provide direction for aquatic life and healthy breeding.

[0037] LEfSe (LDA Effect Size) is an analytical tool for discovering and interpreting high-dimensional biomarkers. It can be used for comparison of multiple groups. It emphasizes statistical significance and biological relevance, and can find biomarkers with statistical differences between groups. The statistical results of LEfSe include two parts, namely, the LDA value distribution bar graph and the evolutionary branch diagram (phylogenetic distribution). The present invention uses LEfSe software, and the LDA score is set to 4 to determine the bacterial groups with significant differences in abundance between groups.

[0038] Different concentrations of nano-cerium dioxide solution on the composition of zebrafish intestinal flora LDA and evolutionary branch diagram Figure 5 As shown. The biomarkers of each group were screened by LESFE linear discriminant analysis. In the control group, the main enriched ones were g_Stenoteophomonas, o_Xanthomonadales and f_Xanthomonadaceae; in the 1μg / L group, p_Fusobacteriota was significantly higher than that in the other groups; in the 10μg / L group, the abundance of c_Gammaproteobacteria was higher; in the 100μg / L group, p_Actinobacteriota and g_Rhodococcus were significantly different; in the 1000μg / L group, the abundance of p_Proteobacteria, g_Reyranella and o_Rhizobiales was higher.

[0039] In summary, as an important organ of zebrafish, intestinal flora plays an important role in regulating metabolism, host health and many other aspects. The present invention proves that different concentrations of nano-cerium dioxide solution can increase the beneficial bacteria Cetobacterium, Reyranella and Rhodococcus. These bacteria play an important role in promoting the utilization of fish intestinal glycolipids, degrading exogenous substances and improving the ability of organisms to resist stress.

Claims

1. A method for increasing probiotics in fish by adding rare earth nanomaterials, characterized in that: Exposing fish to a nano-cerium dioxide solution can significantly improve the abundance of probiotics in the fish's intestines; the nano-cerium dioxide solution is obtained by adding nano-cerium dioxide particles to the water in which the fish are located.

2. The method for increasing probiotics in fish by adding rare earth nanomaterials according to claim 1, characterized in that: The concentration of the nano-cerium dioxide solution is 1-1000 μg / L in terms of cerium element.

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

4. The method for increasing probiotics in fish by adding rare earth nanomaterials according to claim 1, characterized in that: The fish is zebrafish.

5. The method for increasing probiotics in fish by adding rare earth nanomaterials according to claim 1, characterized in that: The optimal concentration range of the nano-cerium dioxide solution is 10-100 μg / L in terms of cerium element.

6. The method for increasing probiotics 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, the light-dark ratio was 14h:10h, and the animals were fed molted brine shrimp eggs at 9:00 and 16:00 daily.

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