Application of picria fel-terrae in relieving bean pulp induced enteritis, regulating intestinal flora and inhibiting bacteria

Abstract

The invention discloses application of picria fel-terrae in relieving fish food-borne intestinal inflammation, regulating intestinal flora and inhibiting bacteria, based on a zebra fish enteritis model induced by soybean meal for 9 days, 0.5-0.2% o of dry whole herb of picria fel-terrae is added into feed, so that aggregation of neutrophils and macrophages in middle and rear intestines can be relieved, and the food-borne intestinal inflammation of zebra fish can be relieved; meanwhile, a 16S rRNA (ribosomal Ribonucleic Acid) gene sequencing result shows that the intestinal flora homeostasis of the fish with the food-borne intestinal inflammation can be regulated by adding 1% o of the dried picria fel-terrae herb into the feed; an aeromonas hydrophila in-vitro bacteriostasis experiment shows that the picria fel-terrae aqueous extract has a remarkable bacteriostasis effect on the aeromonas hydrophila, and an aeromonas hydrophila in-vivo bacteriostasis experiment shows that the bean pulp feed added with 1% o of dry whole picria fel-terrae can remarkably improve the survival rate of zebra fish after toxin attacking by the aeromonas hydrophila.

Description

Technical Field

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

[0002] With the increase in the annual output of aquaculture, the breeding cost has also increased accordingly. Cheaper plant-derived protein feeds have begun to be added. Soybean meal is an important choice of plant protein source, and the anti-nutritional factors therein can induce intestinal inflammation [1] , which limits the addition amount of plant-derived protein feeds in the feed.

[0003] With the increase in the annual output of aquaculture, the harm of aquaculture pathogens to aquaculture is also increasing. Aeromonas hydrophila is a Gram-negative bacterium, a zoonotic bacterium widely distributed in nature, which can cause bacterial septicemia in various fish, resulting in extremely high mortality and huge economic losses [2-4] .

[0004] Antibiotics are the main means of resistance to aquaculture pathogens. Due to the abuse of antibiotics, drug-resistant bacteria have been selected. In the context of the blue transformation and the international ban on antibiotics, the development of new antibiotic alternatives and new aquaculture technologies has been greatly promoted. Natural medicinal plant resources have been widely used in preventing intestinal inflammation caused by the addition of plant-derived protein feeds and controlling aquaculture pathogens. Traditional Mongolian and Tibetan medicines such as Hippohgae rhamnoides, natural substances such as sinomenine and gallic acid can alleviate soybean meal-induced intestinal and liver inflammation [5-7] ; more antibacterial active substances have been found in Origanum vulgare [8] , and the ethanol and methanol extracts of Astragalus membranaceus and Dendrobium officinale have significant inhibitory effects on the growth of Salmonella typhimurium, Shigella flexneri and Bacillus succinogenes [9] , and feeding Glycyrrhiza uralensis at a concentration higher than 0.05 g / kg reduced the mortality rate under the infection of Yersinia ruckeri in rainbow trout

[10] .

[0005] Picria felterrae Lour. is a plant of the Scrophulariaceae family, which can treat wind-heat cold, sore throat, mumps, furuncle, diarrhea, dysentery, hemorrhoids, eczema, snake bites, traumatic injuries, etc.

[11] . Currently, existing studies have shown that Picria felterrae has anti-inflammatory effects such as inhibiting ear swelling in mice induced by xylene and increasing the permeability of peritoneal capillaries in mice induced by acetic acid

[12] , and has antibacterial effects against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus hemolyticus type B [13,14] . Picria felterrae can significantly inhibit the permeability of peritoneal capillaries in mice

[15] such inflammatory effects, but there is a lack of research on fish foodborne intestinal inflammation, and its resistance to Aeromonas hydrophila has not been reported. Summary of the Invention

[0006] The object of the present invention is to provide a new use of Picria felterrae, including its application in the preparation of drugs for inhibiting Aeromonas hydrophila infection in fish, its application in the preparation of drugs for alleviating fish foodborne intestinal inflammation, and its application in the preparation of products for improving the intestinal flora of fish with foodborne intestinal inflammation.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] Application of Picria felterrae in the preparation of drugs for alleviating 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. Picria felterrae was added at 0.5‰, 1‰, and 2‰ respectively to the feed in which 50% of the protein source was replaced by soybean meal. Juvenile innate immune cell imaging analysis was performed on 9 dpf zebrafish to examine its anti-inflammatory effect. 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 Picria felterrae to the soybean meal feed could alleviate the aggregation of neutrophils and macrophages in the mid and posterior intestines to a certain extent. And 2‰ Picria felterrae had the best effect on alleviating neutrophil aggregation, and 0.5‰ Picria felterrae had the best effect on alleviating macrophage aggregation. Therefore, Picria felterrae has a relieving effect on fish foodborne intestinal inflammation.

[0009] Application of Picria felterrae in the preparation of products 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 1‰ Picria felterrae 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 Picria felterrae on the intestinal flora. The results showed that adding Picria felterrae could regulate the intestinal flora of fish with foodborne intestinal inflammation. At the genus level, the abundances of Bacteroides, Akkermansia, and Parabacteroides increased, improving the intestinal homeostasis of fish with foodborne intestinal inflammation.

[0010] Application of Picria felterrae in the preparation of drugs for inhibiting Aeromonas hydrophila infection in fish: In a specific embodiment of the present invention, the antibacterial effect of the water extract of Picria felterrae on Aeromonas hydrophila was preliminarily verified by measuring the inhibition zone. The minimum inhibitory concentration of Picria felterrae against Aeromonas hydrophila was 12.5 mg / ml, and the minimum bactericidal concentration was 25 mg / ml. Further, after feeding 3-month-old zebrafish with 1‰ Picria felterrae 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 adult zebrafish. The results showed that adding 1‰ Picria felterrae to the soybean meal feed could significantly improve the survival rate of zebrafish under Aeromonas hydrophila infection.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] The present invention firstly proposes the application of Picria fel-terrae Lour. in preventing and treating diet-induced enteritis in zebrafish caused by soybean meal. Adding 0.5‰ - 2‰ of Picria fel-terrae Lour. to the soybean meal feed can significantly relieve the aggregation of inflammatory cells in the mid and hind guts of juvenile zebrafish, improve the immunity of fish while saving feed costs, and provide the possibility for the application of anti-inflammatory feed additives in more economic fish species.

[0013] The present invention firstly proposes the application of Picria fel-terrae Lour. in regulating the intestinal flora of zebrafish. After feeding zebrafish with soybean meal feed supplemented with 1‰ of Picria fel-terrae Lour. for 2 weeks, compared with the soybean meal feed, the abundances of Bdellovibrionales, Desulfobacterota, and Myxococcota are significantly increased, providing more application options for feed additives for regulating the intestinal flora of fish.

[0014] The present invention firstly proposes the application of Picria fel-terrae Lour. in inhibiting Aeromonas hydrophila, providing a new alternative antibiotic feed additive for further preventing the infection of fish-related pathogenic bacteria. In addition, the Picria fel-terrae Lour. used has the advantages of being a natural medicinal plant, being environmentally friendly, non-toxic, cost-effective, and easy to handle as a herbal feed.

[0015] The present invention provides a coping strategy for enteritis induced during the process of replacing fish meal with soybean meal in fish feed, while regulating the intestinal flora of fish and inhibiting bacteria, realizing multiple functions of fish feed additives at low cost, and having feasible economic benefits. Description of the Drawings

[0016] Figure 1 It is a fluorescence imaging result diagram of intestinal neutrophils and macrophages of juvenile zebrafish (9 dpf) after adding Picria fel-terrae Lour. to the soybean meal feed.

[0017] Figure 2 It is a heat map of relative abundances at the phylum level for intestinal 16S rRNA gene sequencing of adult zebrafish fed with soybean meal feed supplemented with 1‰ of Picria fel-terrae Lour. for two weeks. The abscissa FM represents the fish meal group, SBM represents 50% soybean meal replacing fish meal, and PFL represents the 50% soybean meal replacing fish meal group supplemented with 1‰ of Picria fel-terrae Lour.

[0018] Figure 3 It is a heat map of relative abundances obtained by intestinal 16S rRNA gene sequencing at the genus level after feeding adult zebrafish with soybean meal feed supplemented with 1‰ of Picria fel-terrae Lour. for two weeks. The abscissa FM represents the fish meal group, SBM represents 50% soybean meal replacing fish meal, and PFL represents the 50% soybean meal replacing fish meal group supplemented with 1‰ of Picria fel-terrae Lour.

[0019] Figure 4 It is an inhibition zone diagram of Picria fel-terrae Lour. against Aeromonas hydrophila.

[0020] Figure 5 Growth curve of Picria felterrae and Aeromonas hydrophila co - culture.

[0021] Figure 6 Survival rate graph of adult zebrafish after feeding with a diet containing 1‰ Picria felterrae in soybean meal for six weeks and then challenged with Aeromonas hydrophila for 10 days. Specific implementation manners

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation to the present invention. All molecular biology operation methods involved in the embodiments, if not specifically stated, are conventional methods well known to those skilled in the art.

[0023] The following are the experimental materials, reagents and equipment involved in the embodiments:

[0024] 1. Biological materials: AB wild - type zebrafish (Danio rerio) used in this experiment, transgenic zebrafish lines, 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 - fluorescence line was obtained by crossing two single - color fluorescence lines. The strains used in this experiment were provided by the National Aquatic Germplasm Resource Bank - Sub - bank of Special Aquatic Animal Germplasm Resources. Among them, the Aeromonas hydrophila is coded as Aer - 028 and was isolated from the liver of grass carp in 2008.

[0025] 2. Reagents and consumables: Low - melting - point agarose (UltraPureTM LMP Agarose, invitrogen) for imaging and fixing fry, MS - 222 (Sigma Aldrich), dry whole herb of Picria felterrae (purchased from Yudu), tryptone soy broth (TSB, Solarbio), confocal bottom culture dish (20 mm in diameter, biosharp), 12 cm sterile glass culture dish (120 mm in diameter). Other reagents such as absolute ethanol and other conventional reagents are all from Sinopharm reagents.

[0026] 3. Instruments and equipment: Zebrafish independent single - rack circulating breeding system (Tecnplast); Laser confocal microscopy imaging system (SP8, Leica) for fluorescence imaging analysis; Heating rod (purchased from Zhongshan Xinshili Electric Appliance Co., Ltd.).

[0027] Example 1 Application of Picria felterrae as a feed additive in alleviating soybean - meal - induced enteritis in zebrafish 1. Experimental feed formula and preparation method of adding Picria felterrae in the zebrafish model

[0028] Table 1 Modeling of Zebrafish with Soybean Meal and Feed Formulation with Picria felterrae Added

[0029]

[0030]

[0031] Note: 1. FM: fish meal group; 50SBM: soybean meal group; 0.5‰PFL: group with 0.5‰ Picria felterrae added; 1‰PFL: group with 1‰ Picria felterrae added; 2‰PFL: group with 2‰ Picria felterrae added; For various vitamins in the vitamin additive in the formula, refer to NRC, 1993.

[0032] (1) Pass fish meal, soybean meal, corn starch, wheat flour, cellulose, mineral premix, vitamin premix, etc. through a 60-mesh sieve. For the larger particles in fish meal and soybean meal, crush them with a pulverizer and then pass through a 60-mesh sieve;

[0033] (2) Weigh fish meal, soybean meal, corn starch, wheat flour, cellulose, mineral premix, vitamin premix, and Picria felterrae drug respectively according to the requirements of the weight percentages of each component in the feed formula;

[0034] (3) Stir and mix the weighed fish meal, soybean meal, and wheat flour evenly;

[0035] (4) After fully stirring and mixing cellulose, mineral premix, vitamin premix, and Picria felterrae evenly, add corn starch and mix evenly. Then add the evenly mixed mixture to the mixture in step (3) and continue to stir until evenly mixed;

[0036] (5) Weigh fish oil according to the formula table, add it to the mixture in step (4), rub open the large oil drops and stir well until the fish oil is evenly distributed in the mixture;

[0037] (6) Pass the above mixture through a 60-mesh sieve. Crush the larger particles with a pulverizer and then pass through a 60-mesh sieve until no particles remain;

[0038] (7) Add a certain proportion of water to the mixture in step (6) and stir to make the mixture in a state where it can be formed into a ball by hand and scattered when put down;

[0039] (8) Use a double-screw granulator with a 2-mm diameter, 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%;

[0040] (9) The dried feed is pulverized and passed through a 60-mesh sample sieve to obtain the corresponding powdered feed, which is stored in a sub-tube of a -20°C refrigerator for future use.

[0041] 2. Preparation of Tg(lyz:DsRED2); Tg(mpeg1:EGFP) fry

[0042] (1) One week in advance, the transgenic zebrafish Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) that are separately cultured by gender are fed to satiety. The breeding water temperature is controlled 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, and other conditions refer to the requirements provided by the National Zebrafish Breeding Center (http: / / www.zfish.cn);

[0043] (2) Adult fish of the Tg(lyz:DsRED2) and Tg(mpeg1:EGFP) fluorescent strains, one female and one male, are separated by a mating fish tank overnight. The partition is removed the next morning, and they are allowed to naturally chase and lay eggs for 1 hour. The fish eggs are collected and dispensed into glass culture dishes 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 culturing zebrafish fry is as follows:

[0044] Table 2 Stock solution of 30×Danieau’s buffer

[0045]

[0046] (3) Incubate in an illumination incubator (28 ± 1°C, light 14 h, darkness 10 h), change the water every day and pick out the dead white fish eggs. On the 5th day after fertilization (5 dpf), the fry are dispensed and the subsequent modeling experiment is started.

[0047] 3. Feeding plan for zebrafish fry SBMIE modeling

[0048] (1) After the double transgenic zebrafish Tg(lyz:DsRED2); Tg(mpeg1:EGFP) are hatched for 5 days (5 pdf), they are dispensed into round culture dishes with a diameter of 150 mm, 30 tails per dish. The experiment is divided into five groups, namely: Group 1: FM, Group 2: 50SBM, Group 3: 0.5‰ PFL, Group 4: 1‰ PFL, Group 5: 2‰ PFL, and each group has three biological replicates;

[0049] (2) Feed each group with the corresponding feed. When feeding, first dissolve the feed in 0.3× Danieau’s buffer, and feed twice a day. The feeding time periods are: 9:00 - 9:30 and 16:00 - 16:30. After feeding for half an hour, change to 0.3× Danieau’s buffer culture solution;

[0050] (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 subsequent in vivo imaging analysis of the fry.

[0051] 4. In vivo imaging method for enteritis and remission degree of zebrafish larvae

[0052] (1) Prepare 25×100 mL of MS-222 stock solution (400 mg of Tricaine powder, 97.9 mL of sterilized water, 2.1 mL of 1M Tris-HCl with pH = 9.0) with sterile water and store it at 4°C. Dilute its working solution to 1× with dd H2O; Prepare 0.5% (w / v) low melting point agarose with dd H2O and let it stand at room temperature for fixing the fry for imaging;

[0053] (2) Aspirate the corresponding group of larvae into 1× MS-222 to anesthetize a large number of fry for the next experiment;

[0054] (3) Heat the prepared 0.5% low melting point agarose in a microwave oven until it dissolves and keep its temperature at 29°C; Just cover the bottom of the confocal culture dish (20 mm) with 0.5% low melting point agarose, put the anesthetized fry into the 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;

[0055] (4) Take pictures of the fixed fry 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;

[0056] (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 is about 10 microns) in the mid- and hindgut, and perform statistical analysis with GraphPad Prism 7.0.

[0057] In this example, first, by crossing two homozygous transgenic marker lines, Tg(lyz:DsRED2) and Tg(mpeg1:EGFP), Tg(lyz:DsRED2; Tg(mpeg1:EGFP)) double-fluorescent fish fry containing neutrophils with red fluorescent markers and macrophages with green fluorescent markers can be obtained. Under the feeding of zebrafish larvae with soybean meal and Picria felterrae feeds, an imaging model of the impact of a foodborne enteritis model on acute inflammatory responses can be rapidly established, and Picria felterrae, a component that can alleviate the corresponding foodborne enteritis, can be evaluated based on the soybean meal feed.

[0058] On the 4th day of feeding the feed during the modeling of foodborne enteritis (SBMIE), that is, at 9 days post-fertilization (9 dpf), as Figure 1 shown, in the FM group, SBM group, and different concentrations of Picria felterrae groups, neutrophils and macrophages with corresponding fluorescent markers appeared in the mid and hindgut regions. The numbers of fluorescently labeled cells in the mid and hindgut of the FM group, SBM group, and different concentrations of Picria felterrae groups were counted. Compared with the FM group, more fluorescently labeled neutrophils and macrophages were found in the SBM group, indicating that feeding soybean meal feed can cause acute inflammatory responses. Compared with the SBM group, after adding 0.5‰ PFL, 1‰ PFL, and 2‰ PFL to the soybean meal feed, the aggregation of neutrophils and macrophages in the mid and hindgut can be alleviated to a certain extent. Among them, adding 2‰ PFL to the soybean meal feed has the best effect on alleviating neutrophil aggregation, and adding 0.5‰ PFL to the soybean meal feed has the best effect on alleviating macrophage aggregation.

[0059] In summary, in this example, the foodborne enteritis model established by feeding different feeds at the innate immune stage and using imaging methods can more clearly reflect the acute inflammatory responses of juvenile fish. After feeding the feed supplemented with Picria felterrae, the aggregation of neutrophils and macrophages in the mid and hindgut can be alleviated, and the acute inflammatory response in the mid and hindgut can be reduced, indicating that Picria felterrae has a mitigating effect on fish foodborne intestinal inflammation at the innate immune stage.

[0060] Example 2 Application of Picria felterrae as a feed additive in regulating the intestinal flora of zebrafish

[0061] After 3-month-old zebrafish were trained to eat with fish meal for one week, they were fed with the fish meal (FM), 50% soybean meal replacing fish meal (SBM), and 50% soybean meal replacing fish meal feed containing 1‰ Picria felterrae described in Example 1 at 2% of their body weight for 2 weeks. Then, the whole intestines of each group were randomly sampled, with 5 replicates in each group. After sampling, they were quickly frozen in liquid nitrogen and stored at -80 °C for 16S rRNA gene sequencing of the intestinal flora.

[0062] As Figure 2As shown, at the phylum level, compared with the soybean meal group, the Bdellovibrionota (P<0.001), Desulfobacterota (P<0.01), and Myxomycota (P<0.05) increased in the Picria felterrae group. Some studies have shown that Bdellovibrio can be used as an antibiotic alternative and has a positive impact on the microbial community. Desulfobacterota is positively correlated with weight gain in mice. Myxomycota is related to genes involved in glucose and lipid metabolism. In addition, the abundance of Myxomycota is lower in patients with human ankylosing spondylitis than in healthy people.

[0063] As Figure 3 shown, at the genus level, compared with the soybean meal group, ZOR006 decreased, Bacteroides increased, Akkermansia increased, and Parabacteroides increased in the Picria felterrae group. ZOR006 belongs to Firmicutes and is related to the host's metabolic disorders and inflammatory diseases. Bacteroides and Parabacteroides belong to Bacteroidetes. Bacteroides makes important contributions to the synthesis of γ-aminobutyric acid and taurine in the intestine; Parabacteroides can promote host health by regulating the immune system, alleviating inflammation, regulating host metabolism, and secreting metabolites. Akkermansia belongs to the genera intensively studied in Verrucomicrobia, and Akkermansia is related to lipid metabolism and maintaining intestinal homeostasis. In this experiment, Picria felterrae improved the intestinal homeostasis of zebrafish at 3 months old by regulating the intestinal flora.

[0064] Example 3 In vitro antibacterial effect of Picria felterrae on Aeromonas hydrophila

[0065] The disc diffusion method was used to determine the size of the inhibition zone of Picria felterrae against Aeromonas hydrophila. The steps are as follows:

[0066] 1. Weigh 5 g of ground dry whole herb of Picria felterrae, add 100 ml of dd H2O and soak for 24 h. Boil the soaked Picria felterrae and keep it boiling for 30 min. Filter it through four layers of gauze, add 100 ml of dd H2O to the filter residue, boil it again and keep it boiling for 30 min, filter it through four layers of gauze, collect the two filtrates, centrifuge at 2000 rpm for 10 min to remove the remaining residue, heat-concentrate the supernatant to 25 ml, sterilize it by high pressure, and store it in a refrigerator at 4℃ for later use.

[0067] 2. Sub-pack the above-mentioned medicinal liquid to immerse the filter paper discs for 24 h, transfer the filter paper discs to a petri dish, and take them out after drying in an oven at 65℃ for 12 h.

[0068] 3. Place the filter paper discs on the TSB solid medium coated with Aeromonas hydrophila, and after culturing at 29℃ for 12 h, measure the size of the inhibition zone with a ruler.

[0069] AsFigure 4 As shown, the antibacterial circle of the aqueous extract of Picria felterrae Lour. at 200 mg / ml against Aeromonas hydrophila was 6.1 mm (the diameter of the filter paper was 6 mm), that is, at this concentration, Picria felterrae Lour. had a certain antibacterial effect on Aeromonas hydrophila.

[0070] Example 4 Determination of the minimum inhibitory concentration and minimum bactericidal concentration of Picria felterrae Lour. against Aeromonas hydrophila

[0071] The gradient dilution method was used to determine the minimum inhibitory concentration and minimum bactericidal concentration of Picria felterrae Lour. against Aeromonas hydrophila:

[0072] 1. Aeromonas hydrophila was aseptically inoculated into TSB medium and cultured at 29 °C for 12 h. The bacterial concentration was measured by spread plate using the 10-fold dilution method and diluted to 10 7 CFU / ml with the corresponding medium.

[0073] 2. 10 μl of the bacterial solution was added to 1 ml of the medium containing the aqueous extract of Picria felterrae Lour. at different concentrations. The concentrations of the aqueous extract of Picria felterrae Lour. were 100 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, 6.25 mg / ml, and 3.125 mg / ml respectively. After culturing at 29 °C and 180 rpm for 12 h, the results were observed.

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

[0075] The minimum inhibitory concentration of Picria felterrae Lour. against Aeromonas hydrophila was 12.5 mg / ml, and the minimum bactericidal concentration was 25 mg / ml.

[0076] Example 5 Effect of Picria felterrae Lour. on the growth of Aeromonas hydrophila

[0077] 10 7 CFU / ml of Aeromonas hydrophila was aseptically inoculated into TSB medium supplemented with the aqueous extract of Picria felterrae Lour. (final concentration 6.25 mg / ml) at a volume ratio of 1 / 100 and cultured at 29 °C and 180 rpm. Bacterial solutions were taken every 1 h after 4 h of culturing, and the absorbance at a wavelength of 600 nm was measured.

[0078] As Figure 5 shown, the aqueous extract of Picria felterrae Lour. at 6.25 mg / ml had varying degrees of inhibitory effects on Aeromonas hydrophila.

[0079] Example 6 In vivo antibacterial effect of Picria felterrae Lour. against Aeromonas hydrophila

[0080] After one week of training the 3-month-old zebrafish with fish meal, the zebrafish were continuously fed for 6 weeks at 2% of the average body weight with the fish meal (FM) described in Example 1, 50% soybean meal replacing fish meal (SBM), and 50% soybean meal replacing fish meal feed containing 1‰ Picria felterrae (PFL). After the feeding ended, 30 zebrafish were randomly selected from each group and placed into 3 water tanks (27 cm × 16 cm × 13 cm) containing 3 L of aerated water with Aeromonas hydrophila (1.0×10 5 CFU / ml), 10 fish in each tank, and the water temperature was maintained at 28°C. The challenge experiment lasted for 10 days. During the experiment, one-third of the water was changed every day and the corresponding bacterial solution was added, and the number of dead zebrafish was recorded every 24 hours. The results are as Figure 6 shown. The PFL group could increase the survival rate of zebrafish under Aeromonas hydrophila infection, and the survival rate (73.3%) after 10 days of challenge was slightly higher than that of the FM group (70.0%) and significantly higher than that of the SBM group (43.3%). It indicates that Picria felterrae can significantly inhibit the infection of Aeromonas hydrophila to zebrafish.

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Claims

1. Use of Picria felterrae in the preparation of a drug for inhibiting Aeromonas hydrophila infection in fish.

2. Use of Picria felterrae in the preparation of a drug for alleviating foodborne intestinal inflammation in fish.

3. Use of Picria felterrae in the preparation of a product for improving the intestinal flora of fish with foodborne intestinal inflammation.

4. The application according to claim 3, wherein Enhance the abundances of Bacteroides, Parabacteroides, and Akkermansia.

Images