Application of rhizoma osmundae in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria

Abstract

The invention discloses an application of Osmunda japonica in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria, based on a 9-day soybean meal induced zebra fish enteritis model, 2% o of Osmunda japonica is added, so that aggregation of neutrophils and macrophages in middle and posterior intestines can be relieved, and zebra fish food-borne intestinal inflammation can be relieved; meanwhile, by means of intestinal flora sequencing, it is found that by adding 2% o of rhizoma osmundae, the intestinal flora of the fish with the food-borne intestinal inflammation can be improved; the result of an aeromonas hydrophila in-vitro bacteriostasis experiment shows that the rhizoma osmundae aqueous extract has a remarkable effect of inhibiting aeromonas hydrophila, has an inhibition zone and has a certain bacteriostasis effect, and a zebra fish in-vitro challenge experiment shows that the survival rate of zebra fish can be increased by adding 2 per thousand of rhizoma osmundae.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture drugs, and particularly relates to the application of Osmunda japonica rhizome in the preparation of drugs for relieving fish foodborne intestinal inflammation, regulating intestinal flora and inhibiting bacteria. Background Art

[0002] Aquatic products are one of the important sources of protein for humans. China ranks among the top in the world in terms of aquaculture production and economic benefits. With the continuous development and growth of the aquaculture industry in China, some economic resistances affecting the aquaculture industry have emerged. One of the most influential types is that when dealing with aquaculture diseases such as bacterial infections and viral infections, a large amount of antibiotics were initially used in the industry. Although certain results were achieved in the initial disease control, due to the abuse of antibiotics, residues of antibiotics were detected in many aquatic products, which is not conducive to people's health. Therefore, people actively research products to replace antibiotics in the field of aquaculture disease prevention and control. It has been found that many Chinese herbal medicine extracts have played a very good role in the prevention and control of aquaculture diseases, and the treatment process is green and pollution-free. It is a very good product to replace antibiotics for disease prevention and control. Since then, the application of Chinese herbal medicine preparations in the aquaculture industry has developed by leaps and bounds.

[0003] Among aquaculture animal bacterial diseases, the common bacterial pathogen Aeromonas hydrophila is a Gram-negative bacterium that is widely distributed in various water bodies. It is a human-animal-fish zoonotic pathogen that can cause aquatic organisms to suffer from septicemia, diarrhea, enteritis and other diseases, bringing great losses to the aquaculture industry and seriously affecting the development of the aquaculture industry.

[0004] Osmunda japonica rhizome is the rhizome and the base of the petiole of Osmunda japonica Thunb, a plant of the genus Osmunda in the family Osmundaceae. It is produced in Gansu, Shandong, Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Henan, Hubei, Hunan, Guangdong, Guangxi, Sichuan, Guizhou, Yunnan and other places. Several commonly used Dryopteris rhizomes are included in the Chinese Pharmacopoeia (2015 Edition), namely Dryopteris crassirhizoma, Osmunda japonica, Woodwardia japonica, Lunathyrium acrostichoides, etc. Modern pharmacological research shows that the n-butanol and ethyl acetate extraction parts of Osmunda japonica can inhibit the swelling of the auricles of mice caused by xylene, suggesting that Osmunda japonica has an inhibitory effect on acute inflammatory swelling and is an effective anti-inflammatory part, and there is no report on its effect on enteritis. Some studies have shown that Dryopteris has a strong inhibitory effect on Shigella dysenteriae, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, etc., and also has an inhibitory effect on Staphylococcus aureus. Summary of the Invention

[0005] The purpose of the present invention is to provide a new use of Osmunda japonica rhizome, including its application in the preparation of drugs for inhibiting the infection of Aeromonas hydrophila in fish, its application in the preparation of drugs for relieving fish foodborne intestinal inflammation, and its application in the preparation of products for improving the intestinal flora of fish with foodborne intestinal inflammation.

[0006] To achieve the above object, the present invention adopts the following technical measures:

[0007] Application of Osmunda japonica Thunb. in preparing a drug for relieving fish foodborne intestinal inflammation: In a specific embodiment of the present invention, a foodborne enteritis model was established by feeding juvenile zebrafish with a feed in which 50% of the protein source was replaced by soybean meal. 0.5‰, 1‰, and 2‰ of Osmunda japonica Thunb. were respectively added to the feed in which 50% of the protein source was replaced by soybean meal. Imaging analysis of innate immune cells of juvenile zebrafish at 9 dpf was performed to examine the effect of improving inflammation. The results showed that, compared with the group fed with the feed in which 50% of the protein source was replaced by soybean meal, adding Osmunda japonica Thunb. to the soybean meal feed could alleviate the aggregation of neutrophils and macrophages in the mid and hindgut to a certain extent. The effect of 1‰ of Osmunda japonica Thunb. in alleviating neutrophil aggregation was the best, and the effect of 2‰ of Osmunda japonica Thunb. in alleviating macrophage aggregation was the best. Therefore, Osmunda japonica Thunb. has a relieving effect on fish foodborne intestinal inflammation.

[0008] Application of Osmunda japonica Thunb. in preparing a product for improving the intestinal flora of fish with foodborne intestinal inflammation: In a specific embodiment of the present invention, after feeding 3-month-old zebrafish with a protein source feed, a feed in which 50% of the protein source was replaced by soybean meal, and a 50% soybean meal replacement protein source feed containing 2‰ of Osmunda japonica Thunb. for 2 weeks, random samples of the whole intestine of each group were taken for 16S rRNA gene sequencing to determine the regulatory effect of Osmunda japonica Thunb. on the intestinal flora. The results showed that adding Osmunda japonica Thunb. could regulate the intestinal flora of fish with foodborne intestinal inflammation. At the genus level, the abundances of Faecalibacterium, Brevibacillus, and Bacteroides were increased, improving the intestinal homeostasis of fish with foodborne intestinal inflammation.

[0009] Application of Osmunda japonica Thunb. in preparing a drug for inhibiting Aeromonas hydrophila infection in fish: In a specific embodiment of the present invention, the antibacterial effect of the water extract of Osmunda japonica Thunb. on Aeromonas hydrophila was preliminarily verified by measuring the antibacterial zone of Osmunda japonica Thunb. against Aeromonas hydrophila. The minimum inhibitory concentration of Osmunda japonica Thunb. against Aeromonas hydrophila was 50 mg / ml, and the minimum bactericidal concentration was 100 mg / ml. Further, after feeding 3-month-old zebrafish with a 50% soybean meal replacement protein source feed containing 1‰ of Osmunda japonica Thunb. for 6 weeks, a 10-day Aeromonas hydrophila challenge experiment was performed on the zebrafish to determine the in vivo anti-Aeromonas hydrophila ability of Osmunda japonica Thunb. in adult zebrafish. The results showed that 2‰ of Osmunda japonica Thunb. could significantly improve the survival rate of zebrafish under Aeromonas hydrophila infection.

[0010] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0011] By establishing a foodborne enteritis model induced by soybean meal, it was found that compared with the soybean meal group, the aggregation of neutrophils and macrophages in the mid and hind guts of the Osmunda japonica group was reduced, reflecting a decrease in its inflammation level, which confirmed the role of Osmunda japonica in alleviating foodborne enteritis. At the same time, the results of 16S rRNA gene sequencing showed that Osmunda japonica increased the proportion of flora related to the homeostasis of the intestinal flora, resulting in a certain degree of improvement in the homeostasis level of the intestinal flora.

[0012] The present invention first proposes the application of Osmunda japonica in inhibiting Aeromonas hydrophila, providing a new alternative antibiotic feed additive for further preventing the infection of fish-related pathogenic bacteria. The Osmunda japonica used is a natural medicinal plant, and as a feed additive ingredient, it has the advantages of being green, environmentally friendly, safe, pollution-free, having good effects, and low cost. Brief Description of the Drawings

[0013] Figure 1 It is a fluorescence imaging result diagram of neutrophils and macrophages in the intestine of zebrafish larvae at 9 dpf after adding Osmunda japonica to the soybean meal feed.

[0014] Figure 2 It is the relative abundance at the phylum level of 16S rRNA gene sequencing in the intestines of different feed groups.

[0015] Figure 3 It is the relative abundance at the genus level of 16S rRNA gene sequencing in the intestines of different feed groups.

[0016] Figure 4 It is an inhibition zone diagram of Osmunda japonica against Aeromonas hydrophila.

[0017] Figure 5 It is the growth curve of the co-culture of Osmunda japonica and Aeromonas hydrophila.

[0018] Figure 6 The survival rates of each group after challenge with Aeromonas hydrophila. Detailed Embodiments

[0019] The present invention will be further described below in conjunction with the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. All molecular biology operation methods involved in the embodiments are conventional methods well known to those skilled in the art unless otherwise specified.

[0020] The following are the experimental materials, reagents and instrument equipment involved in the embodiments:

[0021] 1. Biomaterials: The wild-type AB zebrafish (Danio rerio) and transgenic zebrafish lines used in this experiment, including Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), were all purchased from the China Zebrafish Resource Center (http: / / zfish.cn / ). The Tg(lyz:DsRED2);Tg(mpeg1:EGFP) double-fluorescent line was obtained by crossing two single-color fluorescent lines.

[0022] 2. Reagents and consumables: Low melting point agarose (UltraPureTM LMP Agarose, invitrogen), 4% paraformaldehyde (barsharp), MS-222 (Sigma Aldrich), Osmunda japonica (purchased from Shandu), confocal bottom culture dishes (20 mm in diameter, biosharp), 12 cm sterile glass culture dishes (120 mm in diameter). Other reagents such as analytical pure alcohol and xylene, etc., are all national pharmaceutical reagents.

[0023] 3. Instruments and equipment: Zebrafish independent single-frame circulating breeding system (Tecnplast); The fluorescence imaging analysis equipment is a laser confocal microscopy imaging system (SP8, Leica); Heating rod (purchased from Zhongshan Xinshili Electric Appliance Co., Ltd.).

[0024] Example 1 Application of Osmunda japonica as a feed additive in alleviating soybean meal-induced foodborne enteritis

[0025] 1. Experimental feed formula and preparation method of adding Osmunda japonica in the zebrafish model

[0026] Table 1 Zebrafish soybean meal modeling and Osmunda japonica-added feed formula

[0027]

[0028] Note: 1. FM: fish meal group; 50SBM: soybean meal group; 0.5‰OJT: group added with 0.5‰ Osmunda japonica; 1‰OJT: group added with 1‰ Osmunda japonica; 2‰OJT: group added with 2‰ Osmunda japonica; For various vitamins in the vitamin additive in the formula, refer to NRC, 1993.

[0029] (1) Fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, etc. are passed through a 60-mesh sieve. The fish meal and soybean meal contain larger particles such as fish bones and bean skins, which are crushed with a grinder and then passed through a 60-mesh sieve. For vitamins and minerals with smaller content: VD3, VK3, VB12, thiamine, VB6, folic acid, copper sulfate and sodium selenite, they need to be diluted 20 times to prepare the premix; each kilogram of the mineral mixture contains: magnesium sulfate (MgSO4·2H2O) 60.530g, ferrous sulfate (FeSO4·H2O) 23.110g, copper sulfate (CuSO4·5H2O) 0. 0.010g, zinc sulfate (ZnSO4·H2O) 0.620g, manganese sulfate (MnSO4·H2O) 1.640g, potassium iodide (KI) 0.070g, sodium selenite (NaSeO3) 0.005g, adjusted to 1kg with microcrystalline cellulose; each kilogram of the above vitamin premix contains: vitamin B1 (Thiamin) 0.05g, vitamin B2 (Riboflavin) 0.55g, vitamin B6 (pyridoxine) 0.59g, vitamin B12 (cyanocobalamine) 0.83g, pantothenic acid (pantothenic acid) 2.89 g of dapoxetine, 0.40 g of folic acid, 19.39 g of inositol, 2.24 g of niacin, 4.91 g of biotin, 7.16 g of ascorbic acid, 2.40 g of vitamin A, 0.40 g of vitamin D, 12.55 g of vitamin E, and 0.80 g of vitamin K, adjusted to 1 kg with microcrystalline cellulose;

[0030] (2) Weighing fish meal, soybean meal, corn starch, wheat flour, microcrystalline cellulose, mineral premix, vitamin premix, and Rhizoma Cyperi in proportion to the weight percentage of each component in the feed formula;

[0031] (3) Stir and mix the weighed fish meal, soybean meal, and wheat flour thoroughly;

[0032] (4) After the microcrystalline cellulose, mineral premix, vitamin premix, and Osmanthus fragrans are fully stirred and mixed, corn starch is added and mixed evenly, and the mixed mixture is added to the mixture in step (3) and stirred continuously until the mixture is evenly mixed;

[0033] (5) Weigh fish oil according to the recipe, add it to the mixture in step (4), rub the large oil droplets apart, and stir thoroughly until the fish oil is evenly distributed in the mixture;

[0034] (6) Pass the above mixture through a 60-mesh sieve. Crush the larger particles with a pulverizer and then pass them through a 60-mesh sieve until no particles remain.

[0035] (7) Add a certain proportion of water to the mixture in step (6) and stir it to make the mixture in a state where it can be formed into a ball by hand and scattered when put down.

[0036] (8) Use a twin-screw granulator with a diameter of 2 mm. Control the extrusion pressure, corresponding rotation speed and temperature. After preheating, slowly add the mixture in step (7) into the granulator. Place the granulated feed in a pre-prepared dryer for drying. Control the temperature of the dryer at 55 - 60 °C and dry the moisture to about 10%.

[0037] (9) Crush the dried feed, pass it through a 60-mesh sub-sample sieve to obtain the corresponding powdered feed, and store it in a refrigerator at -20 °C in separate tubes for later use.

[0038] 2. Preparation of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) fry

[0039] (1) One week in advance, feed the transgenic zebrafish Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) that are separately cultured by male and female to satiety. Control the breeding water temperature at 28 ± 0.5 °C, the light time is 14 h, and the darkness is 10 h. The environment of the zebrafish breeding system is pH 7.0 - 8.0, salinity 0.25 - 0.50 ‰, dissolved oxygen 5 - 8 mg / L, total ammonia nitrogen less than 0.02 mg / L. Other conditions refer to the requirements provided by the National Zebrafish Breeding Center (http: / / www.zfish.cn);

[0040] (2) Separate one female and one male adult fish of the Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) fluorescent strains with a mating fish tank overnight. Remove the partition the next morning and let them naturally chase and spawn for one hour. Collect the fish eggs and divide them into 12 cm × 5 cm glass culture dishes, about 100 eggs per dish, to obtain embryos with double fluorescence labeling of Tg(lyz:DsRED2); Tg(mpeg1:EGFP). The stock solution formula of 30× Danieau’s buffer used for cultivating zebrafish larvae is as follows:

[0041] Table 2 Stock solution of 30× Danieau’s buffer

[0042]

[0043] (3) Incubate in a light incubator (28 ± 1°C, 14 h of light, 10 h of darkness), change water daily and pick out dead white fish eggs. On the 5th day of cultivation (5 dpf), prepare for the sub-packaging of fry and start the subsequent modeling experiments.

[0044] 3. Feeding protocol for the SBMIE modeling of zebrafish larvae

[0045] (1) After the double transgenic zebrafish Tg(lyz:DsRED2); Tg(mpeg1:EGFP) hatches to 5 days (5 pdf), sub-pack it into a 150 mm diameter round sterile culture dish, with 30 tails per dish. The experiment is divided into five groups, namely: Group 1: FM, Group 2: 50SBM, Group 3: 0.5‰ OJT, Group 4: 1‰ OJT, Group 5: 2‰ OJT. Each group has three biological replicates.

[0046] (2) Feed each group with the corresponding feed. First, dissolve the feed in 0.3× Danieau’s buffer. Feed twice a day, and the feeding time period is: 9:00 - 9:30, 16:00 - 16:30. After feeding for half an hour, change to 0.3× Danieau’s buffer culture solution.

[0047] (3) After the feeding is completed on the 9th day (9 dpf), replace all of the 0.3× Danieau’s buffer to keep the culture dish clean and tidy for the subsequent in vivo imaging analysis of fry.

[0048] 4. In vivo imaging method for enteritis and remission degree of zebrafish larvae

[0049] (1) Prepare 25×100 mL of MS-222 stock solution (400 mg of Tricaine powder, 97.9 ml of deionized water, 2.1 ml of 1M Tris-HCL pH = 9.0) with sterile water, store it at 4°C, and dilute its working solution to 1× MS-222 working solution with deionized water; prepare 1% (w / v) low melting point agarose with deionized water and let it stand at room temperature for fixing fry imaging.

[0050] (2) Anesthesia: Inhale the corresponding group of juvenile fish into 1× MS-222 working solution and anesthetize for about 30 s until a large number of fry are in an anesthetized state.

[0051] (3) Fixation: Heat the 1% low melting point agarose prepared in (1) with a microwave oven until it dissolves. When its temperature is maintained at 29°C, add the anesthetized fry in (2) to a 20 mm diameter confocal culture dish, add the corresponding low melting point agarose solution, quickly adjust the posture of the fry so that the enlarged part of the foregut and the mid- and hindgut are not covered by the yolk, and keep the fry lying on its side at the bottom of the confocal culture dish.

[0052] (4) Imaging: As soon as possible within 2 hours, take frontal and lateral photos of all the fixed fry in (3) under a laser confocal microscope (SP8, Leica). Set the software and simultaneously complete imaging in three channels: red fluorescence (excitation light 638 nm), green fluorescence (excitation light 488 nm), and white light under a 10× objective lens.

[0053] (5) Statistical analysis: After data collection, use Leica Application Suite X and ImageJ software to process the image data and count the number of fluorescent cells (cell size approximately 10 microns) in the mid- and hindgut. Use GraphPad Prism 7.0 for statistical analysis.

[0054] In this example, first, by crossing two homozygous transgenic marker strains, Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), fry of Tg(lyz:DsRED2);Tg(mpeg1:EGFP) containing both red and green fluorescent markers can be obtained, enabling the evaluation of two very important immune cells involved in innate immunity, neutrophils (red) and macrophages (green), in a single strain. By formulating and feeding powdered feed, a foodborne enteritis model can be rapidly established at the juvenile fish level to study the impact on acute inflammatory responses, and the effect of Osmunda japonica Thunb., a component that can alleviate the corresponding foodborne enteritis, can be evaluated based on soybean meal feed.

[0055] According to the modeling strategy for the innate immune stage, on the 9th day (9 dpf), as Figure 1 shown, fluorescently labeled neutrophils and macrophages appeared in the mid- and hindgut regions of the FM group, SBM group, and different concentrations of Osmunda japonica Thunb. groups. The number of fluorescently labeled cells in the mid- and hindgut regions of the FM group, SBM group, and different concentrations of Osmunda japonica Thunb. groups was counted. Compared with the FM group, it was found that more fluorescently labeled neutrophils and macrophages aggregated in the SBM group, indicating that feeding soybean meal feed can cause acute inflammatory responses. Compared with the SBM group, after adding 0.5‰ OJT, 1‰ OJT, and 2‰ OJT to the soybean meal feed, the aggregation of neutrophils and macrophages in the mid- and hindgut can be alleviated to a certain extent. The effect of adding 1‰ OJT soybean meal feed in alleviating neutrophil aggregation is the best, and the effect of adding 2‰ OJT soybean meal feed in alleviating macrophage aggregation is the best.

[0056] The results of this experiment showed that, based on the 9dpf zebrafish enteritis model induced by soybean meal, in the group fed with 2‰ OJT soybean meal feed, the reduction effect on the aggregation of macrophages in the mid and hindgut was the best, and the reduction effect on the aggregation of neutrophils in the mid and hindgut was also extremely significant, indicating that the addition of Osmunda japonica Thunb. in the feed could alleviate the aggregation of neutrophils and macrophages in the mid and hindgut and relieve zebrafish intestinal inflammation.

[0057] Example 2 Effect of Osmunda japonica Thunb. on the composition of intestinal flora in zebrafish SBMIE modeling

[0058] Three cylinders of wild-type zebrafish with the same number (N = 60) and the same body size were separately fed twice a day with 2‰ OJT, FM, and SBM (the same as in Example 1). In the circulating water system, after stopping the water supply and feeding for half an hour at 8:00 am and 5:00 pm every day, the circulating water system was then turned on. After two weeks of feeding, their intestines were taken for 16S rRNA gene sequencing.

[0059] The results of intestinal flora sequencing analysis are as Figure 2 and Figure 3 shown:

[0060] At the phylum level, compared with SBM, in OJT: the proportion of Proteobacteria decreased, and the proportion of Fusobacteria increased. Some studies have shown that there are many pathogenic bacteria in Proteobacteria, such as Escherichia coli, Escherichia, Salmonella, Vibrio, etc. Fusobacteria is the dominant phylum in the intestine and can improve host metabolism. At the phylum level, compared with FM, in OJT: the proportions of Firmicutes, Proteobacteria, and Bacteroidetes all showed an upward trend. Firmicutes, Proteobacteria, and Bacteroidetes play important roles in the nutrient absorption and immune response of the host intestine. At the genus level, compared with SBM, in OJT: the proportion of ZOR0006 decreased significantly. Some studies have shown that ZOR0006 belongs to the family Erysipelotrichaceae, and this family is related to the metabolic disorders and inflammatory diseases of the host. The proportion of Faecalibacterium increased, and it is an important producer of butyric acid and has an anti-inflammatory effect. At the genus level, compared with FM, in OJT: Brevibacillus and Bacteroides returned to normal levels. Brevibacillus has long been used as a probiotic, and Bacteroides can promote the degradation of polysaccharides and help release energy from dietary fiber and starch.

[0061] Based on the above analysis, Osmunda japonica Thunb. increased the proportion of flora related to the intestinal flora homeostasis, resulting in a certain degree of improvement in the intestinal flora homeostasis level.

[0062] Example 3: Effect of Osmunda japonica Thunb. in vitro on inhibiting Aeromonas hydrophila

[0063] The strains used in the present invention were provided by the National Aquatic Biological Germplasm Resource Bank - the Sub - bank of Special Aquatic Animal Germplasm Resources. Among them, the coding of Aeromonas hydrophila is Aer - 028, which was isolated from the liver of grass carp in 2008.

[0064] 1. Activation of the strain: Take out the frozen bacterial liquid from the - 80°C refrigerator. After it melts and is mixed evenly, add 10 ml of LB liquid medium into a test tube, inoculate 100 μL of the bacterial liquid into it with a pipette gun, and culture it overnight at 800 rpm in a constant temperature shaker at 37°C until the medium in the test tube becomes turbid.

[0065] 2. Cultivation of single colonies: Prepare LB solid medium and sterilize it in a high - temperature and high - pressure sterilizer. After it cools to an appropriate temperature, pour the plate. Dip the bacterial liquid with an inoculation needle and use the method of three - zone streaking to culture single colonies. Culture them overnight in a constant temperature incubator at 37°C until single colonies grow.

[0066] 3. Inoculation of single colonies: Prepare a test tube containing 10 mL of LB liquid medium. Use sterilized forceps to pick up a small pipette tip, pick up a single colony with the front end, and vertically put it into the test tube. Culture it overnight at 800 rpm in a constant temperature shaker at 37°C until the medium in the test tube becomes turbid.

[0067] 4. Preparation of the water extract of Osmunda japonica Thunb.: Take 5 g of Osmunda japonica Thunb., soak it in 100 mL of distilled water for 24 h, then boil it for 30 min. Filter it with four - layer gauze, collect the drug residue and repeat the above operation twice. Centrifuge the collected liquid at 2000 rpm for 10 min, take the supernatant, boil and concentrate all the supernatant to 25 ml, sterilize it in a high - pressure steam sterilizer, and place it in a 4°C refrigerator for standby.

[0068] 5. Preparation of the drug - sensitive test paper: Take the drug - sensitive test paper in a sterile operating table and soak it in the original solution of Osmunda japonica Thunb. (concentration: 200 mg / ml) for 24 h. After taking it out, place it in a petri dish and dry it in an oven.

[0069] 6. Take 100 μL of Aeromonas hydrophila bacterial liquid (10 7 CFU / ml) and spread it on a TSB plate. Place the drug - sensitive test paper on the TSB plate. After culturing at 29°C for 12 h, use a scale to measure the size of the inhibition zone.

[0070] The results are as Figure 4 shown. The water extract of Osmunda japonica Thunb. at a concentration of 200 mg / ml has a certain antibacterial effect on Aeromonas hydrophila.

[0071] Example 4: Determination of the minimum inhibitory concentration and minimum bactericidal concentration of Osmunda japonica Thunb. against Aeromonas hydrophila

[0072] 1. In a sterile operating table, inoculate according to the volume ratio of Aeromonas hydrophila: TSB liquid medium = 1:1000, and culture at 29 °C for 12 h in a constant temperature incubator. The bacterial concentration is determined by the ten-fold serial dilution and spread plate method, and diluted to 10 7 CFU / ml with the corresponding medium.

[0073] 2. Add 10 μl of Aeromonas hydrophila bacterial solution to 1 ml of medium containing different concentrations of aqueous extract of Osmunda japonica Thunb. The concentrations of the aqueous extract of Osmunda japonica Thunb. are 100 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, and 3.125 mg / ml respectively. Observe the results after culturing at 29 °C and 180 rpm for 12 h.

[0074] 3. The minimum inhibitory concentration is the minimum concentration at which the culture medium is not visibly turbid to the naked eye. The samples that are visibly non-turbid to the naked eye are further streaked on a plate and cultured at the corresponding temperature for 12 h. The minimum concentration without bacterial growth is the minimum bactericidal concentration.

[0075] The minimum inhibitory concentration of Osmunda japonica Thunb. against Aeromonas hydrophila is 50 mg / ml, and the minimum bactericidal concentration is 100 mg / ml.

[0076] Example 5 Effect of Osmunda japonica Thunb. on the growth of Aeromonas hydrophila

[0077] Inoculate 10 7 CFU / ml of Aeromonas hydrophila aseptically into TSB medium supplemented with aqueous extract of Osmunda japonica Thunb. (final concentration 12.5 mg / ml) at a volume ratio of 1 / 100, and culture at 29 °C and 180 rpm. Take the bacterial solution every 1 h after 4 h of culture, and measure the absorbance at a wavelength of 600 nm.

[0078] As Figure 5 shown, the aqueous extract of Osmunda japonica Thunb. at 12.5 mg / ml has a certain inhibitory effect on Aeromonas hydrophila. Example 6 Effect of Aeromonas hydrophila challenge on the survival rate of each group

[0079] First, to determine the LD50 (median lethal concentration), after feeding adult zebrafish at three months of age with fish meal at 2% of the average body weight for one week of training, set 1×10 5 CFU / ml, 1.5×10 5 CFU / ml, 2×10 5 CFU / ml, 2.5×10 5 CFU / ml, 3×10 5The challenge experiment was carried out on Aeromonas hydrophila with a concentration gradient of CFU / ml to determine the median lethal concentration. Three parallel fish tanks (length × width × height: 27 cm × 16 cm × 13 cm) were set up for each concentration. A heating rod was placed inside to keep the water temperature at 28 °C. 10 adult zebrafish were placed in each parallel. The death situation of zebrafish was recorded after 10 days of challenge. The final test results showed that the median lethal concentration of Aeromonas hydrophila for zebrafish was 1×10 5 CFU / ml.

[0080] Then, the challenge experiment of Aeromonas hydrophila on zebrafish was carried out. The FM group, SBM group, and OJT group (50% soybean meal replacing fish meal feed supplemented with 2‰ Osmunda japonica) were set up according to the method described in Example 1. After three-month-old adult zebrafish were trained to eat with fish meal at 2% of the average body weight for one week, they were continuously fed with the feeds of each group for six weeks. Three parallel fish tanks (length × width × height: 27 cm × 16 cm × 13 cm) were set up for each group. A heating rod was placed inside to keep the water temperature at 28 °C. 10 zebrafish were placed in each parallel. The Aeromonas hydrophila bacterial solution was added to each group to make the final concentration reach 1×10 5 CFU / ml. One-third of the water was changed every morning and the bacterial solution concentration was replenished. The death situation of each group was observed and recorded. As Figure 6 shown: The 10-day survival rates of the FM group, SBM group, and OJT group were 70%, 43%, and 53.33% respectively. The survival rate of the OJT group was significantly increased compared with that of the SBM group. The results showed that Osmunda japonica could effectively inhibit the infection of Aeromonas hydrophila and had a certain improvement on the survival rate level of zebrafish.

Claims

1. Application of Osmunda japonica Thunb. in preparing a drug for relieving fish foodborne intestinal inflammation.

2. Application of Osmunda japonica Thunb. in preparing a product for improving the intestinal flora of fish with foodborne intestinal inflammation.

3. The application according to claim 3, wherein Enhancing the abundances of Faecalibacterium, Brevibacillus, and Bacteroides.

4. Application of Osmunda japonica Thunb. in preparing a drug for inhibiting Aeromonas hydrophila infection in fish.

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