CN113509514A

CN113509514A - Composition for inhibiting vibrio and preparation method and application thereof

Info

Publication number: CN113509514A
Application number: CN202110481766.6A
Authority: CN
Inventors: 朱宇嘉; 辛年香; 朱盛山
Original assignee: Guangzhou Pujing Biological Technology Co ltd
Current assignee: Guangzhou Pujing Biological Technology Co ltd
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-10-19

Abstract

The invention relates to a composition for inhibiting vibrios and a preparation method and application thereof, belonging to the technical field of natural active products. The active ingredients of the composition are prepared from the following raw materials in parts by weight: 30-40 parts of rosemary, 30-40 parts of centella, 25-35 parts of tea, 15-25 parts of loropetalum chinense leaves and 15-25 parts of Chinese violet. The composition has good activity to various vibrios. Can effectively inhibit and kill in-vivo and in-vitro vibrios, has low minimum inhibitory concentration (<0.5mg/mL) to common vibrios in aquaculture, and does not find insensitive strains. After 1 hour of the prawn drug administration, intestinal tract and hepatopancreatic vibrio are basically inhibited and killed. Compared with antibiotics such as amoxicillin and the like, the continuous medication of the composition for over 14 days can inhibit and kill vibrio, and the quick-acting advantage of the composition is obvious.

Description

Composition for inhibiting vibrio and preparation method and application thereof

Technical Field

The invention relates to the technical field of natural active products, in particular to a composition for inhibiting vibrios and a preparation method and application thereof.

Background

Vibriosis is one of the most common problems in the process of prawn cultivation, and is considered to be one of the important reasons for hindering the development of prawn cultivation industry in China or abroad no matter in the past or at present, and no ideal medicine is available at present.

The Ministry of agriculture advocates the research on the action of Chinese herbal medicines in the aquatic industry and the development of Chinese herbal medicines capable of resisting aquatic diseases. The antibacterial effect of the aquatic antibacterial Chinese patent medicine in the market is not ideal, and the main reasons are that the formula is random, the processing is coarse, the administration mode is unreasonable, and the product efficacy is exaggerated. Therefore, in order to make the aquaculture industry develop healthily and sustainably, it is necessary to develop a Chinese herbal medicine with low toxic and side effects, no residue and less drug resistance to pathogens.

The invention provides a Chinese medicinal composition for preventing and treating vibriosis of prawns, which is prepared from Chinese lobelia, loropetalum chinensis leaf and at least one of astragalus root, vitex flower, elephantopus scaber flower, coptis root leaf and scutellaria baicalensis. The Chinese medicinal composition can reduce death of prawn and improve survival rate in prawn animal experiment. However, in the process of medication, the effect is found to be poor for some types of vibrios, and the problem that the composition is difficult to feed due to large dosage is existed.

Therefore, a new traditional Chinese medicine formula is urgently needed to be found, and the traditional Chinese medicine formula can play an effective prevention and treatment role for most types of vibrio prawns.

Disclosure of Invention

In view of this, there is a need to provide a composition for inhibiting Vibrio, which has a better activity against various types of Vibrio.

The composition for inhibiting vibrios comprises the following active ingredients in parts by weight: 30-40 parts of rosemary, 30-40 parts of centella, 25-35 parts of tea, 15-25 parts of loropetalum chinense leaves and 15-25 parts of Chinese violet.

The composition for inhibiting vibrio has good effect, small dosage and good food calling property.

In one embodiment, the active ingredients of the composition are prepared from the following raw materials in parts by weight: 35 parts of rosemary, 35 parts of asiatic pennywort herb, 30 parts of tea, 20 parts of loropetalum chinense leaves and 20 parts of Chinese violet.

The invention also discloses a preparation method of the composition for inhibiting vibrios, which comprises the following steps:

extraction: weighing the raw materials according to the formula ratio, crushing, adding 70-80% methanol aqueous solution by volume percentage as an extraction solvent according to the amount of 4-14ml solvent/1 g raw materials, performing reflux extraction for 15-60min, filtering, taking filter residues, repeatedly extracting for 1-3 times, combining filtrates, and concentrating to obtain an extract;

and (3) purification: collecting the above extract, monitoring with antibacterial activity experiment, and separating and purifying active components.

In one embodiment, the specific separation and purification method of the purification step is as follows: and (3) adding acid liquor into the filtrate obtained by extraction to adjust the pH value to 5.0-6.0, adding ethyl acetate for extraction, and recovering the solvent to obtain the compound.

Experiments prove that the obtained product has the best vibrio inhibition effect by purifying with the method, and has the advantages of simple extraction method and low cost.

In one embodiment, the acid solution is acetic acid.

In one embodiment, the purification step employs a solvent extraction method, which specifically comprises the following steps: and (4) concentrating the filtrate obtained in the extraction step to obtain an extract, and adding ethyl acetate for extraction to obtain the extract.

In one embodiment, the purification step is a membrane separation method, and specifically comprises the following steps: collecting the filtrate, concentrating, adding acid solution to adjust pH to 5.0-6.0, adding ethanol to make ethanol content of 65-75%, centrifuging, collecting supernatant, passing through 0.01-0.02 μm ultrafiltration membrane, collecting filtrate, recovering solvent, and concentrating.

The invention also discloses application of the composition for inhibiting vibrios in preventing and treating vibriosis of prawns.

In one embodiment, the vibrio comprises: vibrio parahaemolyticus, Vibrio harveyi, Aeromonas hydrophila, Vibrio vulnificus, Vibrio anguillarum, Vibrio fluvialis and Vibrio cholerae.

The invention also discloses a feed for preventing and treating the vibriosis of the prawns, which comprises the composition for inhibiting the vibriosis, wherein the content of the composition is 4-10 wt%.

Compared with the prior art, the invention has the following beneficial effects:

the composition for inhibiting vibrios can effectively inhibit and kill in-vivo and in-vitro vibrios, has low minimum inhibitory concentration (<0.5mg/mL) to common vibrios in aquaculture, and does not find insensitive strains. After 1 hour of the prawn drug administration, intestinal tract and hepatopancreatic vibrio are basically inhibited and killed. Compared with antibiotics such as amoxicillin and the like, the continuous medication of the composition for over 14 days can inhibit and kill vibrio, and the quick-acting advantage of the composition is obvious.

After the composition is used, the activity of the hepatopancrease of the prawns can be obviously improved, compared with the situation that the antibiotic has toxicity to the hepatopancrease of the prawns, the prawns can be poisoned and the hepatopancrease is necrotized after the antibiotic is continuously used, so that the cotton prawns and the iron prawns commonly known in the industry are formed, and the safety advantage of the HCV compound is obvious.

The antibiotic causes the bacterial drug resistance which is a focus of international attention, the composition of the invention is a pure Chinese herbal medicine preparation, and the drug resistance of vibrio to the composition is not found so far.

After the composition is used in the south three island prawn culture bases of the Union of China and the industrialized prawn culture bases of the Zhongshan Haiteng, no medicine residue is found after the detection of the prawn.

Drawings

FIG. 1 is a linear fitting equation of absorbance to MIC in example 1;

FIG. 2 shows the results of the experiment for inhibiting the growth of hepatopancreas in example 2;

FIG. 3 shows the results of the intestinal bacteriostasis test in example 2;

FIG. 4 shows the diagnosis results of a shrimp in example 3;

FIG. 5 shows the diagnosis results of example 3 cases of shrimp;

FIG. 6 shows the results of example 3, case one day;

FIG. 7 shows the results of example 3, case two, one day;

FIG. 8 shows the results of example 3, two days after the administration of one drug;

FIG. 9 shows the results of example 3, two days after two administrations;

FIG. 10 shows the results of all the experimental ponds of example 3.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The starting materials used in the following examples are all commercially available unless otherwise specified, and the procedures used in the following examples are all conventional procedures unless otherwise specified.

The process conditions in the following examples were optimized and all followed by pharmacodynamic evaluation, the pharmacodynamic evaluation method used was as follows:

establishing a pharmacodynamic evaluation method.

1. And (5) testing strains.

Vibrio alginolyticus (Vibrio alginolyticus, V.a); vibrio Parahaemolyticus (VibBrio Parahaemolyticus, V.p); vibrio harveyi (VibBrio harveyi, V.h); aeromonas hydrophila (Aeromonas hydrophila, A.h); vibrio vulnificus (Vibrio vulgaris, V.v); vibrio anguillarum (Vibrio anguillarum, V.g); vibrio fluvialis (Vibrio fluvialis, V.f); vibrio cholerae (Vibrio cholerae, V.c);

2. experimental methods.

2.1 in vitro antibacterial experiments.

2.1.1 bacteriostatic ring test (K-B paper method).

2.1.1.1 preparation of culture medium.

2216E nutrient broth culture medium and 2216E nutrient agar culture medium are weighed according to prescription, poured into a triangular flask, added with a certain amount of distilled water, stirred uniformly and sealed, sterilized under high pressure (121 deg.C, 30min), sterilized and refrigerated for use.

2.1.1.2 recovering and culturing the strain.

And (3) performing streak culture on the strains subjected to refrigeration preservation on an agar plate for 24h, selecting well-grown single colonies, inoculating the single colonies into a bacteria shaking tube filled with a 2216E nutrient broth culture medium, and culturing in a constant-temperature incubator at 37 ℃ until the logarithmic phase.

2.1.1.3 inoculation.

And measuring the OD value of the bacterial suspension at the wavelength of 600nm by using 2216E nutrient broth as a blank control liquid, and calculating the bacterial content. Sucking bacteria liquid, putting it in plate, adding 2216E nutritive agar, and mixing to make bacteria concentration 5X 10⁵CFU/ml。

2.1.1.4 preparing the drug sensitive tablet.

Selecting Xinhua No. 1 qualitative small disc filter paper with the diameter of 5mm, autoclaving (121 ℃, 30min), and drying in a warm box. Dripping a certain amount of liquid medicine into each filter paper sheet, and drying in an incubator at 37 ℃.

2.1.1.5 the medicine-sensitive tablet is applied topically.

The drug sensitive tablets are stuck on the surface of an agar plate containing bacteria, the distance between the centers of the paper tablets is about 24mm, the distance between the paper tablets and the edge of the plate is about 15mm, and the distance between the paper tablets is equal, and the drug sensitive tablets are cultured for 240 hours in a constant temperature incubator at 37 ℃.

2.1.1.6 interpretation of results.

The diameter of the zone was measured with a vernier caliper, and the average was calculated by repeating the measurement 3 times.

And (3) judging standard: the diameter of the inhibition zone is less than 7mm, the sensitivity is low at 7-12 mm, the sensitivity is moderate at 12-17 mm, and the sensitivity is high at >17 mm.

2.1.2 Minimal Inhibitory Concentration (MIC) assay (microdilution)

2.1.2.1 and (3) preparing the medicine.

The tested medicine is compounded with proper solvent into 0.8g/ml liquid medicine, which is autoclaved and diluted in two times lower concentration to prepare 11 kinds of medicine diluent liquid with gradient concentration.

2.1.2.2 and preparing a culture medium.

2.1.2.3 recovering and culturing the strain, the strain preserved by cold storage is first streaked on agar plate for recovery, and then the single colony of well-grown bacterium plate is picked up for inoculation.

The strain is inoculated into a shake culture tube filled with 2216E nutrient broth culture medium, and is placed in a constant temperature incubator to be cultured to the logarithmic phase at 37 ℃.

2.1.2.4.

Zeroing 2216E nutrient broth as blank control liquid, measuring OD value of bacterial suspension at 600nm wavelength, calculating bacteria content, and diluting to 1 × 10⁶CFU/ml bacterial suspension.

2.1.2.5 incubation with added sample.

Sucking 100 μ l of each of the liquid medicine and the bacterial suspension into the 1 st to 11 th wells of a 96-well plate according to aseptic technique, adding 100 μ l of each of the prepared solvent and the bacterial suspension into the 12 th well as taking negative control, and culturing at 37 ℃ in a constant temperature incubator for 20 h.

2.1.2.6 interpretation of results

And (4) observing by naked eyes, wherein the hole with the lowest concentration of the medicine has no bacteria growth, and the MIC value of the test bacterium is obtained. For those with difficulty in judgment, the culture medium in the well is streaked on a sterile agar plate, and the result is read after culturing at 37 ℃ for 20 hours.

2.1.3 Minimum Bactericidal Concentration (MBC) experiments.

2.1.3.1-1.2.1.3.6 the operation steps are the same as those of MIC experiment.

2.1.3.7 incubation of sterile well cultures.

Sucking 0.1ml of culture of each test well with the minimum inhibitory concentration of the drug and without bacterial growth, transplanting the culture onto a sterile agar plate, uniformly coating, and culturing in a constant-temperature incubator at 37 ℃ for 24 h.

2.1.3.8 interpretation of results

The MBC value of the drug is determined when the bacterial count in the plate medium is less than 5 colonies.

Example 1

A method for preparing composition for inhibiting Vibrio is optimized.

1. And (4) prescription.

35 parts of rosemary, 35 parts of asiatic pennywort herb, 30 parts of tea, 20 parts of loropetalum chinense leaves and 20 parts of Chinese violet.

2. And (4) extracting.

Weighing the raw materials according to the formula amount, crushing the raw materials into coarse powder, adding 6 times of 75% methanol aqueous solution in volume percent according to the amount of 1g of the raw materials added with 6ml of solvent, performing reflux extraction for 45min, filtering, taking filter residue, adding 6 times of 75% methanol aqueous solution in volume percent, extracting for 45min, filtering, taking filter residue again, adding 4 times of 75% methanol aqueous solution in volume percent, extracting for 15min, filtering, combining filtrate, recovering the solvent, and concentrating to obtain extract, namely the extract.

3. And (5) purifying.

3.1 solvent separation method

Extracting the extract with acetone, ethyl acetate and butanol, respectively, recovering solvent from each organic layer under reduced pressure to obtain corresponding components, which are respectively defined as part 1, part 2 and part 3.

Taking the different parts, carrying out in-vitro bacteriostasis experiments according to the method, measuring the diameter of a bacteriostasis ring, an MIC value and an MBC value, and comparing and extracting to obtain the bacteriostasis effect of the product.

The above-mentioned materials of each part were prepared into 0.8g/ml (crude drug) solution. The results of the in vitro bacteriostasis experiment on the diameter, MIC value and MBC value of the bacteriostasis ring are shown in the following table.

TABLE 1 bacteriostatic ring diameter (mm) for different solvent extractions

TABLE 2 MIC values (mg/ml) for different solvent extractions

TABLE 3 MBC values (mg/ml) for different solvent extractions

From the above results, the antibacterial activity of the part 2 (ethyl acetate extract) is the strongest, and the diameter of the inhibition ring is 12.35-20.40 mm, the MIC value is 1.56-12.50 mg/ml, and the MBC value is 1.56-12.50 mg/ml. The extracts of the part 1 and the part 3 show weaker antibacterial activity, and have obvious difference compared with the part 2.

3.2 Membrane separation

Taking the extract obtained in the previous step, adding a proper amount of acid, respectively adjusting the pH value to 5.0, 5.5 and 6.0, then adding ethanol to enable the alcohol content of the solution to be 70%, centrifuging at a high speed, taking supernatant to respectively flow through ultrafiltration membranes of 0.01 mu m and 0.02 mu m, recovering ethanol from filtrate, concentrating, quantifying the extract, carrying out in-vitro bacteriostasis experiments on the concentrated solution of each filter membrane according to the method, measuring the diameter, MIC value and MBC value of a bacteriostasis ring, comparing the bacteriostasis effects of different membrane separation methods, and obtaining the results of the diameter, MIC value and MBC value of the bacteriostasis ring through the in-vitro antibiosis experiments, wherein the results are shown in the following table.

TABLE 4 bacteriostatic ring diameter (mm) for different membrane separation methods

TABLE 5 MIC values (mg/ml) for different membrane separation methods

TABLE 6 MBC values (mg/ml) for different membrane separation methods

From the above results, it can be seen that in 6 groups of drugs with different treatment methods, the pH value is 5.5, the antibacterial activity of the drug group separated by a 0.02 μm membrane is strongest, the diameter of the bacteriostatic ring is 15.10-22.34 mm, the MIC value is 0.78-12.50 mg/mL, and the MBC value is 0.78-12.50 mg/mL; the pH value is 6.0, the diameter of the bacteriostatic ring is 14.84-21.92 mm, the MIC value is 0.78-25.0 mg/mL, and the MBC value is 0.78-25.0 mg/mL after the drug separation by a 0.02 mu m membrane.

3.3 acid extraction-organic solvent extraction combination

Through investigation and comparison of the various separation methods, the activity of the part 2 extracted by ethyl acetate in the solvent separation method is strongest, indicating that the effective active ingredient is a weak substance; the membrane separation method uses pH5.5 pretreatment, and the antibacterial activity of 0.02 μm membrane separation drug group is strongest, indicating that the effective active component is a certain weak acidic small molecular substance.

Finally, the pretreatment is carried out by using nitrous acid (H acid), carbonic acid (J acid) and acetic acid (D acid), the pH is adjusted to 5-6, and then ethyl acetate is used for extraction and purification.

Finally, the purification mode is determined as follows:

taking the combined filtrate obtained in the extraction step, adding a proper amount of different acid solutions, adjusting the pH value to 5-6, respectively extracting, respectively recovering solvents of each organic layer under reduced pressure to obtain corresponding parts 2, performing an in vitro bacteriostasis experiment according to the method, measuring the diameter of a bacteriostasis ring, the MIC value and the MBC value, and comparing the bacteriostasis effects before and after purification, wherein the results are shown in the following table.

TABLE 10 bacteriostatic ring diameter (mm) for different acid treatment methods

TABLE 11 MIC values (mg/ml) for different acid treatment methods

TABLE 12 MBC values (mg/ml) for different acid treatment methods

The results show that the bacteriostatic action of the acid-treated and ethyl acetate-purified product is obviously better than that before the treatment, wherein the bacteriostatic action of the acid-purified product is strongest, and finally, a method combining acidification with acetic acid (acetic acid) and extraction with ethyl acetate is proposed.

Example 2

In this example, a pharmacodynamic experiment was conducted using the composition prepared under the optimum extraction and purification conditions in example 1 (ethyl acetate extraction after acidification) as a sample.

1. Minimum Inhibitory Concentration (MIC) test method and results.

1.1 Experimental methods.

The experimental steps of the minimum inhibitory concentration are as follows:

(1) diluting the liquid medicine (liquid medicine bacteriostasis zone experimental preparation): taking 12 EP tubes, adding 1mL of water into each tube, adding 1mL of liquid medicine into the first tube, uniformly mixing, sucking 1mL of liquid medicine into the second tube, uniformly mixing, sequentially diluting to 11 EP tubes, and taking the 12 th tube without adding medicine as a blank control. (all reagents were processed in the same operation)

(2) Adding a bacterial liquid: and sucking 100 mu L of diluted vibrio parahaemolyticus liquid to 96 plates, and marking 12 holes.

(3) Adding liquid medicine: sucking 100 mu L of liquid medicine with 12 concentration gradient, sequentially adding the liquid medicine into a 96-plate, and uniformly mixing the liquid medicine with the liquid medicine.

(4) And (3) incubation: and placing the mixture in an incubator at 37 ℃ for incubation for 16-24 hours.

(5) Scribing: the incubation was removed, 5. mu.L of the solution was aspirated, and streaked.

(6) Culturing: culturing in an incubator at 37 ℃ for 16-24 hours, taking out and observing the result.

The other Vibrio species were all the same as above.

1.2 results of the experiment

The results of the experiments with the lowest inhibitory concentration are shown in the following table, wherein No. 1 (amount of drug substance) and No. 2 (amount of extract) are the results of the composition obtained in example 1, No. 3 is the experimental results of sanhuangsan, No. 4 and No. 5 are the previous experiments of the present inventors, and the composition disclosed in chinese patent document CN106924449A is subjected to the control experiment, wherein No. 4 is the amount of drug substance and No. 5 is the amount of extract.

TABLE 13 Minimum Inhibitory Concentration (MIC) test results table

2. In vivo antibacterial effect test

2.1 Experimental methods

And throwing a net at the center of the shrimp pond to obtain the shrimps. Selecting 9 shrimps with plump intestinal tracts in the net, peeling off the head and the chest beetles, picking off the hepatopancreas and the intestinal tracts respectively, placing the hepatopancreas and the intestinal tracts respectively in clean 10mL beakers, mashing the prawns, pouring 2mL of clear water respectively, stirring the mixture evenly, taking 0.1mL of tissue fluid respectively, dripping the tissue fluid into a TCBS culture dish respectively, coating the solution evenly by using an L-shaped rod, covering and marking the solution.

The plate was placed in a constant temperature incubator and incubated at 37 ℃. After 24h, they were removed and the amount of vibrio on the medium was observed and recorded.

500g of the vibrio-inhibiting composition prepared in example 1 was mixed with 10000g of feed 5000-.

Feeding the uniformly mixed pesticide materials in the whole pond, throwing the net after 60min, fishing out the shrimps, and selecting 9 shrimps with plump intestinal tracts in the net. The liver pancreas and the intestinal tract were dissected and the amount of vibrio therein was measured in the same manner as the control group.

2.2 results of the experiment

The results of the inhibition of the liver, pancreas and intestine are shown in fig. 2-3, respectively, wherein fig. 2 is the result of the inhibition of the liver, pancreas and intestine, and fig. 3 is the result of the inhibition of the intestine. The data of the results of the experiments are shown in the following table.

TABLE 14 in vivo bacteriostatic results

From the above results, it can be seen that the composition of the present invention can better inhibit vibrio prawn.

3. Challenge test

The test was carried out on a Han-dynasty shrimp breeder farm in Zhanjiang. 1000 tails of shrimp larvae used in the test have the body length of about 6-8 centimeters, the source of the shrimp larvae is a high-quality breeding field of the provincial prawn, and the results are negative when WSSV, TSV, IHHNV, EMS and EHP are detected during feeding of the shrimp larvae. One week of temporary culture before the experiment begins, the survival rate of the shrimps is not less than 98% for formal experiments.

The implementation place is in the Han-dynasty prawn fine breeding field in Zhanjiang city. The experimental shrimps used in each sample are cultured in an independent water bucket, the specification is 40 liters per shrimp, 25 liters of water is added, and 20 tails of the shrimps are fed in each bucket. Each barrel is provided with an aeration device, a sewage suction pipe and a thermometer, aeration is carried out for 24 hours during the experiment, the water temperature is controlled to be 27-32 ℃, and shrimp feed is regularly and quantitatively fed every day.

The bacterial strain used for infection is vibrio parahaemolyticus, which is a pathogenic bacterial strain which is purified and identified from a fish disease research laboratory of the south China sea aquatic research institute and is subjected to virulence rejuvenation. After the bacterial liquids with various concentrations used for infection are completed in the aquatic animal quarantine laboratory in Zhanjiang city, the bacterial liquids are transported to an infection experiment site within 1 hour for infection experiments.

Before the formal experiment, a preparation experiment needs to be carried out, and the optimal experimental conditions, such as feeding conditions (including feeding amount and frequency, water quality and temperature monitoring and the like), infection dosage, feeding time and the like, are searched for so as to ensure the successful completion of the formal experiment. 1.2 x 10 design of equal logarithmic spacing⁵、1.2×10⁶、1.2×10⁷、1.2×10⁸、1.2×10⁹CFU/ml total 5 infection dose, according to the selected infection route infection pre-test, observed record in 10 days of the test subject mortality. Each set of 3 buckets was operated in parallel.

TABLE 15 toxicity test result table

Under the same test environment, the infection dose is 1.4 multiplied by 10⁷CFU/ml for infection. Group a is a high dose group (10 wt% of the composition), group B is a medium dose group (5 wt% of the composition), and group C is a low dose group (2.5 wt% of the composition). Group D was a control drug group, and a known drug having a definite drug effect similar to the test drug, florfenicol bait, was administered orally at a dose of 30 mg/kg body weight. Group E is a positive control group and was not administered after infection. Group F is a blank control group: no infection and no administration. Each set of 3 buckets was operated in parallel.

TABLE 16 infection test results Table

Note: control group, no drug was given after infection. Group F is a blank control group: no infection and no administration.

The experiments show that the composition prepared by the invention has better vibrio inhibiting effect.

Example 3

In this example, a clinical suitability study was conducted using the composition prepared under the optimum extraction and purification conditions in example 1 as a sample.

1. Time and place of experiment

From 5 months in 2020 to 12 months in 2020, 2 naturally occurring shrimps were selected for comparison of pre-and post-drug effects from the 185-mouth freshwater pond treated as shown in the following table.

TABLE 17 basic data of test sample pond

One pond in Bai Jiu village in Daao town of New river city and one pond in Xin Sha village in Muzhou town are selected, and the two tests are respectively recorded as case one and case two. The test sample pool basis data is shown in the table below.

TABLE 18 Water quality basic data for the test (Water quality kit determination)

TABLE 19 basic data of test sample pond

2. Diagnosis of shrimp

Case one: the feed intake of the prawns in the shrimp pond is reduced within 2 days 6 months in 2020; the material platform has long feces, a large amount of white (floating) feces exist at the air port under the water surface, and the length is 3-8 cm; the liver of the sick shrimp becomes red and the white membrane disappears. In 10 days after 6 months, the food consumption is reduced from 22kg after one meal and 15kg after 2 hours, and the food is not consumed after 4 hours; the dead shrimps are fished from the bottom of the aerator for 2 times every day, and the total weight is 4 kg. A TCBS culture dish is used for detecting that the hepatopancreatic tissue fluid contains a large amount of Vibrio flavus, as shown in figure 4, wherein A is schematic of necrotizing symptom, B is schematic of leucorrhea symptom, C is vibrio detection result, D is schematic of body surface symptom of sick shrimp, and E is schematic of hepatopancreatic symptom of sick shrimp.

Case two: the feed intake of the prawns in the shrimp pond is reduced within 10 months and 2 days in 2020; the activity of the sick shrimps is weakened, and the body color is dark; part of the body is red, and a small amount of excrement is grown on the material table; the coverage rate of the hepatopancreatic white membrane of the sick shrimps is about 60 percent; the dead shrimps are fished from the bottom of the aerator for 2 times every day, and the total amount is 1 kg. And when the meal is eaten for 5 days in 10 months, the food consumption is reduced from 28kg of meal and 24kg of meal after 2 hours, and the meal is not eaten after 2 hours and is eaten for three meals every day. The TCBS culture dish is used for detecting that the hepatopancreatic tissue fluid has a large amount of vibrio flavus. Specifically, as shown in fig. 5, a is indicative of symptoms of death, B is indicative of symptoms of white feces, C is the result of vibrio detection, D is indicative of body surface symptoms of diseased shrimps, and E is indicative of hepatopancreas symptoms of diseased shrimps.

3. And (6) obtaining the result.

3.1 one-time administration results.

In case one, the activity of the prawns is enhanced, no feces are found on the material table, the white membrane of the liver is gradually recovered, and the jejunum, the stomach and the fluorescent prawns are reduced. The amount of dead shrimps discharged is reduced from 10kg to 4kg, and 20kg of feed is eaten after one meal, and the dead shrimps are eaten after 2 hours. FIG. 6 shows the case after drug administration, where A is the body surface condition and B is the anatomy condition.

In case two, the activity of the prawns is enhanced, the net throwing observation shows that the content of the prawns in jejunum, jejunum and intestine is reduced from 30% to 20%, the total amount of the discharged penaeus vannamei died is reduced from 7kg to 2kg, the white membranes of the liver become gradually clear, the body color becomes transparent, the prawns eat 23kg of feed after one meal, and the prawns eat the feed after 2 hours. FIG. 7 shows the case of case two after administration, where A is the body surface condition and B is the anatomy condition.

3.2 number of Vibrio administered once.

By adopting the method, the quantity of the vibrio in the hepatopancreas and the intestinal tract tissue fluid of the prawns is detected, and the results are shown in the table.

TABLE 20 case-hepatopancreas, intestinal vibrio counts

TABLE 21 example amounts of arcus matter in the pancreas and intestine of the second liver

3.3 two days to cure vibriosis

In case one, the activity of the prawns is enhanced, no excrement grows on a material table, the number of jejunum stomachs and the number of the litopenaeus vannamei observed by throwing a net is less than 10%, white membranes of livers of the prawns are clear, livers of the prawns have blood color, body color changes to be transparent, about 1kg of the penaeus vannamei killed by stealing is discharged from a sewage discharge port, and the penaeus vannamei eaten after eating a meal is 27kg and less than 2 h. FIG. 8 shows the case after drug administration, where A is the body surface condition and B is the anatomy condition.

In case two, the activity of the prawns is enhanced, and the body color becomes transparent. No jejunum stomach prawn is observed by throwing the net, and about 10 prawn with intestinal obstruction (about 100 prawn salvaged by throwing the net) are observed. The coverage rate of the white membranes of the livers of the prawns is more than 70 percent, 25kg of feed is eaten in one meal, and the prawns are eaten after 2 hours. FIG. 9 shows the case of case two after administration, where A is the body surface condition and B is the anatomy condition.

The evaluation is carried out according to 2 grades of cure and non-cure. After the medicine is taken for 48 hours, the clinical symptoms disappear, the normal state of the food intake is recovered, and the patients with less or no increase of the number of the thieves are judged to be cured; after the medicine is taken for 48 hours, the vibrio disease is not obviously improved or the vibrio with aggravation is judged to be not cured. The day of diagnosis was counted as D0, the next day as D1, and so on. The results of observing D0, D1, D2 and D3 and counting the treatment effect in the experimental pond of Table 17 are shown in FIG. 10.

The results showed that most (113) of all the test ponds were dosed on the day at diagnosis, and the remaining (72) were dosed at D1 because the day feeding time was missed due to late diagnosis. As can be seen from the experimental results, most of the shrimp ponds (85%) were cured (D2) after two days of drug administration, and 14 of the uncured 28 shrimp ponds continued until D3 disappeared or declined, and another 14 were uncured at D3. By D3, the total cure rate was 92.4%.

The experimental results show that the composition prepared by the invention can be suitable for different shrimp ponds in different regions, and has the advantages of wide applicability, good effect and high cure rate.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The composition for inhibiting vibrios is characterized in that the effective components of the composition are prepared from the following raw materials in parts by weight: 30-40 parts of rosemary, 30-40 parts of centella, 25-35 parts of tea, 15-25 parts of loropetalum chinense leaves and 15-25 parts of Chinese violet.

2. The composition for inhibiting vibrio according to claim 1, wherein the effective components of the composition are prepared from the following raw materials in parts by weight: 35 parts of rosemary, 35 parts of asiatic pennywort herb, 30 parts of tea, 20 parts of loropetalum chinense leaves and 20 parts of Chinese violet.

3. The method for preparing the composition for inhibiting vibrio according to claim 1 or 2, which comprises the steps of:

4. The method for preparing a composition for inhibiting Vibrio according to claim 3, wherein the purification step comprises the following steps: adding acid solution into the filtrate to adjust pH to 5.0-6.0, adding ethyl acetate for extraction, and recovering solvent.

5. The method for preparing a composition for inhibiting Vibrio according to claim 4, wherein the acid solution is acetic acid.

6. The method for preparing the composition for inhibiting vibrio according to claim 1, wherein the purification step adopts a solvent extraction method, and specifically comprises the following steps: and (4) concentrating the filtrate obtained in the extraction step to obtain an extract, and adding ethyl acetate for extraction to obtain the extract.

7. The method for preparing a composition for inhibiting Vibrio according to claim 1, wherein the purifying step is a membrane separation method, and comprises the following steps: collecting the filtrate, adding acid solution to adjust pH to 5.0-6.0, adding ethanol to make ethanol content of the solution 65-75%, centrifuging, collecting supernatant, passing through 0.01-0.02 μm ultrafiltration membrane, collecting filtrate, and recovering solvent.

8. The use of the composition for inhibiting vibrio according to claim 1 or 2 for the control of vibriosis in prawns.

9. The use of claim 8, wherein the vibrio comprises: vibrio parahaemolyticus, Vibrio harveyi, Aeromonas hydrophila, Vibrio vulnificus, Vibrio anguillarum, Vibrio fluvialis and Vibrio cholerae.

10. A feed for preventing and treating vibriosis of prawns, comprising the composition for inhibiting vibrio according to claim 1 or 2, wherein the content of the composition is 4-10 wt%.