Application of Oocystis borgei in repairing the imbalance of fish intestinal microecology

Abstract

The present invention discloses the application of Oocystis borgei in repairing the microecological imbalance of fish intestines, especially in the preparation of products for repairing the microecological imbalance of fish intestines caused by antibiotics. In the present invention, Oocystis borgei is used as a rapid repair product for the microecological imbalance of fish intestines caused by antibiotics, and its functions include: rapidly restoring the structure and function of the fish intestinal microbial community, promoting the restoration of the composition of resistance genes in the intestinal microbial community, restoring the length of fish intestinal villi, and regulating the expression of genes related to apoptosis and proliferation in fish intestines to achieve rapid repair of the microecological imbalance of fish intestines caused by antibiotics. When Oocystis borgei is prepared into a product with a rapid repair effect on the microecological imbalance of fish intestines caused by antibiotics, it has good application prospects. As a rapid repair product for the microecological imbalance of fish intestines caused by antibiotics, Oocystis borgei functions include: rapidly restoring the structure and function of the fish intestinal microbial community, promoting the restoration of the composition of resistance genes in the intestinal microbial community, restoring the length of fish intestinal villi, and regulating the expression of genes related to apoptosis of fish intestinal villi, the expression of genes related to proliferation of fish intestinal villi, and the expression of genes related to the inhibition of fish intestinal inflammatory response to achieve rapid repair of the microecological imbalance of fish intestines caused by antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of Oocystis borgei, and specifically relates to the application of Oocystis borgei in repairing the microecological imbalance of fish intestines. Background Art

[0002] The antibiotic florfenicol is a veterinary chloramphenicol-specific broad-spectrum antibiotic developed and widely used in the 1980s, and is also a commonly used antibiotic in farmed fish. The extensive use of florfenicol will lead to problems such as bacterial drug resistance and adverse animal reactions, and will also seriously affect public safety. How to reduce the harm caused by florfenicol exposure is a new topic for improving the health of farmed fish and other organisms and even humans. As a model organism, the research and results carried out on medaka can be widely promoted.

[0003] Oocystis borgei is a green alga isolated from a shrimp high-level pond, which has characteristics such as a suitable population growth rate, strong adaptability and stress resistance. Directing it into the shrimp farming environment forms a stable ecological niche, is not easy to cause algal collapse, can optimize the microalgae community structure in the aquaculture water body, reduce the content of nitrogen pollutants and heavy metals in the water body, inhibit the outbreak of pond blue algae, and effectively maintain the ecological balance of the farming system for a long time, accelerating the material cycle in the water body and purifying the farming environment. However, there is currently no research on adding algae to repair the microecological imbalance of fish intestines, especially the microecological imbalance of fish intestines caused by antibiotics. At the same time, the fish intestinal flora is closely related to the distribution, enrichment and transmission of antibiotic resistance genes (ARGs), and there is no report on the impact of adding algae on resistance genes. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of Oocystis borgei in repairing the microecological imbalance of fish intestines.

[0005] The above object of the present invention can be achieved by the following technical solutions: the application of Oocystis borgei in repairing the microecological imbalance of fish intestines.

[0006] Furthermore, the present invention relates to the application of the Oocystis borgei in preparing a product for repairing the microecological imbalance of fish intestines caused by antibiotics.

[0007] Optionally, the antibiotic is florfenicol.

[0008] Optionally, the fish is medaka.

[0009] Optionally, the product is a concentrated solution of Oocystis borgei.

[0010] As a preferred embodiment of the present invention, the Oocystis borgei concentrated liquid is prepared by the following method: Culturing with a culture medium indoors under the culture conditions of a temperature of 25 ± 3°C, natural light supplemented with 50 - 100 W fluorescent lamps for 12 - 24 h of illumination, continuously aerating and culturing for 5 - 7 days, and then obtaining concentrated algal cells through the natural sedimentation method, namely the Oocystis borgei concentrated liquid.

[0011] As a better embodiment of the present invention, the Oocystis borgei concentrated liquid is prepared by the following method: Culturing with a culture medium indoors under the culture conditions of a temperature of 25 ± 3°C, natural light supplemented with 70 W fluorescent lamps for 24 h of illumination, continuously aerating and culturing for 5 - 7 days, and then obtaining concentrated algal cells through the natural sedimentation method, namely the Oocystis borgei concentrated liquid.

[0012] Optionally, the culture medium is Zhanshui 107 - 3 culture solution or f / 2 culture solution prepared with natural seawater.

[0013] Optionally, during the breeding process, the water body is stirred 3 - 6 times a day at fixed points, and after culturing for 5 - 8 days, the Oocystis borgei concentrated liquid is supplemented to the initial inoculation concentration.

[0014] More preferably, during the breeding process, the water body is stirred 3 times a day at fixed points, and after culturing for 7 days, the Oocystis borgei concentrated liquid is supplemented to the initial inoculation concentration.

[0015] Optionally, in the fish breeding water body, Oocystis borgei is added to quickly repair the intestinal microecological imbalance of fish caused by antibiotics.

[0016] Optionally, the inoculation density of the Oocystis borgei is 3.6×10 4 ~1.2×10 5 cells / mL.

[0017] Furthermore, the inoculation density of the Oocystis borgei is 3.6×10 4 ~1.2×10 5 cells / mL, and 50 - 60 mg / L NaNO3 and 5 - 7 mg / L KH2PO4 are added as nutrient salts.

[0018] Optionally, adding Oocystis borgei to the fish breeding water body of the present invention can quickly restore the structure and function of the fish intestinal microbial community.

[0019] The test results in Example 1 show that for the intestinal microecological damage of medaka caused by adding the antibiotic florfenicol, after inoculating the Oocystis borgei concentrated liquid, the recovery time can reach the effect of natural recovery for 28 days when it is 14 days, and adding Oocystis borgei can assist the rapid recovery of the microbial community structure and function.

[0020] Optionally, adding Oocystis borgei to the fish culture water body of the present invention can promote the recovery of the composition of resistance genes in the intestinal microbial community.

[0021] The test results in Example 2 show that: by analyzing the intestinal microbial resistance genes (ARGs) of six groups of medaka, it can be known that the treatment with florfenicol significantly changes the composition of resistance genes in the intestinal microbial community of medaka, while adding Oocystis borgei can promote the recovery of the abundance of some resistance genes to the NC level.

[0022] Optionally, adding Oocystis borgei to the fish culture water body of the present invention can restore the intestinal villus length of fish.

[0023] The test results in Example 3 show that: compared with the FF group treated with the antibiotic florfenicol, the intestinal villus length of the FFR1 group only recovered by 2.36% through self-regulation, while the FFROB1 group recovered by 42.37% compared with the FF group after adding the Oocystis borgei concentrate, with the best recovery effect.

[0024] Optionally, adding Oocystis borgei to the fish culture water body of the present invention can quickly repair the imbalance of the fish intestinal microecology caused by antibiotics by regulating the expression of apoptosis-related genes and intestinal villus proliferation-related genes in the fish intestine.

[0025] The results in Example 4 of the present invention show that adding algae can affect the expression of apoptosis-related genes and promote the expression of genes related to intestinal cell proliferation.

[0026] Generally speaking, as a product for quickly repairing the imbalance of the fish intestinal microecology caused by antibiotics, Oocystis borgei in the present invention has the following functions: quickly restoring the structure and function of the fish intestinal microbial community, promoting the recovery of the composition of resistance genes in the intestinal microbial community, restoring the intestinal villus length of fish, and regulating the expression of apoptosis- and proliferation-related genes in the fish intestine to quickly repair the imbalance of the fish intestinal microecology caused by antibiotics.

[0027] Therefore, the present invention can prepare Oocystis borgei into a product with a rapid repair effect on the imbalance of the fish intestinal microecology caused by antibiotics.

[0028] The present invention has the following advantages:

[0029] (1) By continuously exposing medaka to antibiotics in the present invention, after adding the Oocystis borgei concentrate, especially 14 days after adding the Oocystis borgei concentrate, it can help medaka accelerate the recovery of the structure and function of the intestinal microbial community;

[0030] (2) Adding the Oocystis borgei concentrate in the present invention, especially 14 days after adding the Oocystis borgei concentrate, can help improve the composition of resistance genes in the intestinal microbe of medaka, reduce the horizontal transmission of resistance genes, and help medaka restore the intestinal microecology.

[0031] (3) The addition of Oscillatoria bogoriensis concentrated solution, especially 14 days after the addition of Oscillatoria bogoriensis concentrated solution, can help restore the intestinal microecology of medaka by promoting the recovery of intestinal villus length.

[0032] (4) The addition of Oscillatoria bogoriensis concentrated solution, especially 14 days after the addition of Oscillatoria bogoriensis concentrated solution, can restore the intestinal microecology of medaka by regulating the expression of genes related to intestinal tissue apoptosis and proliferation. Description of the Drawings

[0033] The present invention will be further described below with reference to the drawings in conjunction with the embodiments.

[0034] Figure 1 It is a result graph for analyzing the diversity of each group (NC group, FF group, FFR1 group, FFR2 group, FFROB1 group and FFROB2 group) in Example 1 to compare the microbial community structure and function of each group. Among them, (A) graph is the box plot of Chao1 index - measuring species abundance; (B) graph is the box plot of Shannon index - measuring species diversity; (C) graph is the Venn diagram at the genus level; (D) graph is the PCA; (E) graph is the bar chart of species composition at the genus level; (F) graph is the box plot of Firmicutes / Bacteroidetes ratio (F / B); (G) graph is the bar chart of the distribution of Carbohydrate-active enzymes (CAZy) (top 20); (H) graph is the bar chart of the distribution of virulence factors (top 20); (I) graph is the bar chart of the distribution of pathogen-host interaction targets (top 20);

[0035] Figure 2 It is the analysis of the composition of antibiotic resistance genes for each group (NC group, FF group, FFR1 group, FFR2 group, FFROB1 group and FFROB2 group) in Example 2. Among them, (A) graph is the bar chart of the distribution of antibiotic resistance genes; (B) graph is the bar chart of the distribution of MGEs;

[0036] Figure 3 It is the biological phenotype experimental analysis of the intestinal tissues of each group (NC group, FF group, FFR1 group, FFR2 group, FFROB1 group and FFROB2 group) in Examples 3 - 4. Among them, (A) graph is the HE staining section of the intestinal tissue; (B) graph is the statistical chart of the significant differences in intestinal villus length among groups; (C) graph is the relative expression level analysis of intestinal-related genes by qRT-PCR. Detailed Embodiments

[0037] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0039] The experimental methods used in the following implementation methods are all conventional experimental methods without special instructions.

[0040] The terms used in the following implementation methods and examples generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0041] Example 1

[0042] 1. Oocystis culture

[0043] Oocystis borgei is sourced from the Algae Resources Development and Aquaculture Environment Ecological Restoration Laboratory of Guangdong Ocean University. It is cultured indoors using the Zhanshui 107-3 medium (Table 1). The culture conditions are a temperature of 25 ± 3 °C, natural light supplemented by fluorescent lamps (70W) for 24-hour illumination, and continuous aeration culture. After 5-7 days, the algal cells are concentrated by the natural sedimentation method to obtain the concentrated Oocystis borgei solution.

[0044] Table 1 Composition of Zhanshui 107-3 medium

[0045] Component Mother liquor concentration Dosage <![CDATA[NaNO3]]> 80 g / L 1 mL / L <![CDATA[KH2PO4]]> 8 g / L 1 mL / L <![CDATA[C6H5FeO7]]> 10 g / L 0.2 mL / L Natural seawater 1L

[0046] Oocystis borgei can also be cultured by other conventional methods in the art, such as the concentrated Oocystis borgei solution obtained by culturing with f / 2 culture solution.

[0047] The f / 2 culture solution includes: 1 mL of f / 2 basic component solution and 1000 mL of artificial seawater.

[0048] f / 2 basic component solution: 74.8 mg of sodium nitrate, 4.4 mg of sodium dihydrogen phosphate, made up to 1000 mL with ultrapure water. Autoclaved at 121 °C for 30 min and reserved for use.

[0049] 2. Adding the concentrated Oocystis solution to the fish culture water body

[0050] Add the concentrated Oocystis borgei solution to the fish tank, and the inoculation density is about 3.6×10 4 cells / mL (OD 680=(0.03 ± 0.002), 50 mg / L of NaNO3 and 5 mg / L of KH2PO4 were added as nutrients. The experiment lasted for 14 days, and the water body was stirred 3 times a day at fixed points. After 7 days of cultivation, the bottom of the fish tank was siphoned and a concentrated solution of Oocystis borgei was supplemented once.

[0051] 3. Antibiotic exposure and recovery test:

[0052] After the medaka was immersed in florfenicol for 2 hours, the water containing the antibiotic was removed, and the treatment was repeated for 14 days to obtain the FF group. Subsequently, half of the fish in the FF group were allowed to recover naturally, which was the FFR group. Among them, the group that recovered naturally for 14 days was the FFR1 group, and the group that recovered naturally for 28 days was the FFR2 group; the other half of the FF group was added with the microalgae Oocystis borgei and lived together according to the method in 2 to form the FFROB group. Among them, the experimental groups that lived with Oocystis borgei for 14 days and 28 days were named the FFROB1 group and the FFROB2 group respectively. The fish without any treatment were defined as the NC group. There were a total of six groups, namely the FF group, the FFR1 group, the FFR2 group, the FFROB1 group, the FFROB2 group, and the NC group.

[0053] Results: After treatment with florfenicol, significant changes occurred in the Alpha diversity, species composition, and community function of the intestinal microbiota of medaka, while the addition of Oocystis borgei had a beneficial effect on this imbalance. The Chao1 and Shannon indices of the FFROB1 group were significantly lower than those of the FFR1 group ( Figure 1 Figures A and B in it); there were 388 common genera among the six groups. The FFR1 group and the FFROB2 group had the most unique species genera, with 287 and 160 genera respectively, while the FFROB1 group, the FFR2 group, the FF group, and the NC group had fewer unique species genera and the species composition was relatively close ( Figure 1 Figure C in it); according to the PCA clustering (genus level) analysis of the intestinal microbiota composition of the six groups (PC1 = 64.28%, PC2 = 23.97%), the FFR1, FFROB2, and FF groups were separated from the NC, FFR2, and FFROB1 groups. It was known from the ANOISM test that there were significant differences among the six groups of samples ( Figure 1 Figure D in it); further research found that after 14 days of recovery by adding Oocystis borgei, the microbiota of the medaka would recover by itself, mainly manifested as: (1) the species composition at the genus level of the FFR2 group and the FFROB1 group was the most similar; (2) Vibrio, whose abundance increased after treatment with florfenicol, was significantly downregulated in the FFR2 group and the FFROB1 group ( Figure 1 Figure E in it); (3) in addition, among the groups, the F / B value of the FFR1 group was the lowest (0.36 ± 0.14), while the FFROB1, FFROB2, and FFR2 groups recovered to the level of the NC group ( Figure 1 Figure F in it).

[0054] Furthermore, for the intestinal microecological imbalance of medaka caused by the addition of florfenicol, the FFROB1 group treated with the concentrated solution of Oocystis borgei at an inoculation density of about 3.6×10 4 cells / mL (OD 680 = 0.03±0.002) for 14 days can assist the rapid recovery of the microbial community function. By comparing the Carbohydrate-Active enZYmes (CAZy) database, it was found that CE3 in carbohydrate esterases (CEs) accounted for the largest proportion in the FFROB1 group, and the composition of carbohydrate-active enzymes in the FFROB1 group was most similar to that in the FFR2 group ( Figure 1 Figure G therein); on the one hand, by comparing the Virulence Factor Database (VFDB), the prediction of virulence factors in the Oocystis borgei addition group recovered to the NC level ( Figure 1 Figure H therein); on the other hand, by comparing the PHI (Pathogen-Host Interactions) database, the proportion of the I1S8E5 target in the Oocystis borgei addition group was higher than that in other groups and was the highest 28 days after the addition of oocysts ( Figure 1 Figure I therein).

[0055] Therefore, for the intestinal microecological imbalance of medaka caused by the antibiotic florfenicol, the recovery effect of the FFROB1 group treated with the concentrated solution of Oocystis borgei at an inoculation density of about 3.6×10 4 cells / mL (OD 680 = 0.03±0.002) for 14 days was equivalent to that of the FFR2 group that naturally recovered without algae addition for 28 days, that is, the addition of Oocystis borgei could assist the rapid recovery of the microbial community structure and function.

[0056] Example 2

[0057] As in Example 1, the algae were added in the ways of 1, 2, and 3, and the composition of the antibiotic resistance genes (ARGs) in the intestinal microbiota of six groups of medaka was analyzed as shown in Figure 2 shown, as shown in Figure 2 Figure A therein, the resistance gene AAC(6')-Is was downregulated after antibiotic treatment, and the FFR2 and FFROB2 groups recovered to the NC level; abcA was downregulated in the FF, FFR1, and FFR2 groups, the highest in the FFROB1 group and higher than the NC level, while the FFROB2 group was equivalent to the FFR2 group; PER-5 was absent in the FF and FFR1 groups, that is, it was significantly reduced after florfenicol treatment, while the proportion of PER-5 recovered in the FFR2 and FFROB2 groups and was significantly upregulated in the FFROB2 group (p<0.05); adeF, which was upregulated in the FF and FFR1 groups, recovered to the NC level in the FFR2 and FFROB1 groups; in the FFEOB1 group with the addition of Oocystis borgei, the proportions of abcA, tet(Y), etc. increased significantly and were higher than those in other groups. In addition, as shown in Figure 2As shown in Figure B, the compositions of the mobile genetic elements (MGEs) in the FFR2 and FFROB1 groups were the most similar and had the lowest abundances, indicating that the incidence of ARG horizontal transfer events mediated by MGEs in these two groups was the lowest.

[0058] Therefore, florfenicol treatment significantly altered the composition of resistance genes in the intestinal microbiota of medaka, and the addition of *Oocystis borgei* could promote the recovery of the resistance gene composition to the NC level.

[0059] Example 3

[0060] As in the implementation method of Example 1, the effects of treatment with the antibiotic florfenicol on the intestinal villus length of medaka and the recovery after adding algae were investigated, and the results are as Figure 3 shown.

[0061] Figure 3 The results in Figures A and B show that:

[0062] (1) Compared with the NC group, the intestinal villus length of the FF group after florfenicol treatment was significantly reduced;

[0063] (2) The intestinal villus length of the FFROB1 group was the longest, followed by the FFROB2 group, indicating that adding algae could promote the growth of intestinal villi. Among them, the intestinal villus length of the FFROB1 group was significantly higher than that of the FF, FFR1, and NC groups (p < 0.05) and higher than that of the FFR2 group;

[0064] (3) Compared with the FFROB1 group, the intestinal villus length of the FFROB2 group showed a decreasing trend, indicating that 14 days of algae addition treatment was an optimal time period;

[0065] (4) Compared with the FF group, the intestinal villus length of medaka that naturally recovered for 14 days (FFR1) only recovered by 2.36%, while the group with 14 days of *Oocystis borgei* addition (FFROB1) could recover by 42.37%, showing the best recovery effect.

[0066] Therefore, the rapid repair of fish intestinal microecological imbalance also includes helping to restore the intestinal villus length of fish.

[0067] Example 4

[0068] Treat as in the method of Example 1, and take intestinal tissues of each group for qRT-PCR analysis.

[0069] The results are as Figure 3As shown in Figure C, the relative expression levels of apoptosis-related genes Bax and bcl2 / 1 in the FFROB1 group were the highest; the bcl2 / 1 / Bax ratios in the NC, FF, FFROB1, FFROB2, FFR1, and FFR2 groups were 1.29±0.1287, 2.84±0.5978, 1.59±0.1648, 2.28±0.3325, 1.87±0.1923, and 2.18±0.4222, respectively. Among them, the ratio in the NC group was the lowest, and the FFROB1 group was closest to the NC level; in addition, the expression level of the apoptosis effector subclass Caspase protease Caspase-3 encoding gene Casp3a was the highest in the FFOBR1 group and was significantly upregulated compared with the FFR2 group (p<0.05). The above indicates that adding algae can affect the expression of apoptosis-related genes.

[0070] Regarding the genes related to cell growth and proliferation, Axin2 affects the contact inhibition signal by downregulating the stability of β-catenin, (1) causing epithelial cells that should have stopped dividing to continue dividing; (2) affecting the adherens junctions of cells and increasing the cell migration ability. This study found that the relative expression level of Axin2 in the FF group treated with florfenicol for 14 days was the lowest; compared with the NC group, the FFROB1 group was significantly upregulated. The above indicates that adding algae can promote the expression of genes related to cell proliferation, thereby promoting the growth and proliferation of intestinal villus cells.

[0071] Therefore, adding algae in the present invention can rapidly repair the imbalance of the fish intestinal microecology caused by antibiotics by regulating the expression of genes related to apoptosis and proliferation of fish intestinal villi, etc.

[0072] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. Application of Oocystis borgei in the preparation of a product for repairing the reduction of intestinal villus length in fish caused by antibiotics, wherein the antibiotic is florfenicol, the fish is Oryzias latipes, and the product is an Oocystis borgei concentrated solution.

2. The application according to claim 1, wherein: The Oocystis borgei concentrated solution is prepared by the following method: Culturing is carried out indoors using a culture medium, and the culture conditions are a temperature of 25 ± 3 °C, natural light supplemented with 50 - 100 W fluorescent lamps for 12 - 24 h of illumination, and continuous aeration culture for 5 - 7 days. Then, concentrated algal cells, namely the Oocystis borgei concentrated solution, are obtained by the natural sedimentation method.

3. The application according to claim 2, characterized in that: The culture medium is Zhanshui 107 - 3 culture solution or f / 2 culture solution prepared with natural seawater. The Zhanshui 107 - 3 culture solution includes the following components: 80 g / L NaNO3 1 mL, 8 g / L KH2PO4 1 mL, 10 g / L C6H5FeO7 0.2 mL, and 1 L of natural seawater. The f / 2 culture solution includes: 1 mL of f / 2 basic component solution and 1000 mL of artificial seawater. f / 2 basic component solution: Sodium nitrate 74.8 mg, sodium dihydrogen phosphate 4.4 mg, made up to 1000 mL with ultrapure water, and sterilized at 121 °C under high - pressure steam for 30 min for standby.

4. The application according to claim 1, characterized in that: Add the Oocystis borgei concentrated solution to the fish culture water body.

5. The application according to claim 4, wherein: The inoculation density of the Oocystis borgei is 3.6×10 4 ~1.2×10 5 cells / mL.

6. The application according to claim 5, wherein: The inoculation density of the Oocystis borgei is 3.6×10 4 ~1.2×10 5 cells / mL, and 50-60 mg / L NaNO3 and 5-7 mg / L KH2PO4 are added as nutrient salts.

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