阅读说明：本技术 一种用于抑制水环境迟缓爱德华氏菌的复合植物提取物及其制备方法和应用 (Composite plant extract for inhibiting Edwardsiella tarda in water environment and preparation method and application thereof ) 是由 黄锦炉 于 2021-01-07 设计创作，主要内容包括：本发明属于水体环境抑菌剂技术领域,公开了一种用于水环境迟缓爱德华氏菌的复合植物提取物,该复合植物提取物主要由以下质量百分比的组分制成：紫背草鲜叶提取物55％-65％、马齿苋提取物26％-35％、金不换茎提取物12％-18％。与传统GMP消毒剂安全性相比,本发明复合植物提取物属于环境友好型物质,对无鳞鱼的安全浓度范围大,化水泼洒使用时,其对迟缓爱德华氏菌有明显的抑制效果,且不会受水体有机物干扰,使用剂量≤5mg/L时,可有效抑制养殖水体的上层、中层和底层养殖中的迟缓爱德华氏菌的异常繁殖,且对黄颡鱼、斑点叉尾鮰和沟鲇的安全性良好。(The invention belongs to the technical field of water environment bacteriostat, and discloses a composite plant extract for Edwardsiella tarda in water environment, which is mainly prepared from the following components in percentage by mass: 55-65% of the extract of the fresh leaves of the gynura bicolor, 26-35% of the extract of the purslane and 12-18% of the extract of the stem of the Stephania delavayi Diels. Compared with the safety of the traditional GMP disinfectant, the composite plant extract disclosed by the invention belongs to an environment-friendly substance, has a large safe concentration range for non-scaly fishes, has an obvious inhibiting effect on Edwardsiella tarda when being splashed with water for use, is not interfered by water organism, can effectively inhibit the abnormal propagation of Edwardsiella tarda in the culture of the upper layer, the middle layer and the bottom layer of a culture water body when the using dose is less than or equal to 5mg/L, and has good safety for yellow catfishes, channel catfishes and catfishes.)

1. The composite plant extract for inhibiting Edwardsiella tarda in an aqueous environment is characterized by comprising, by weight, 55-65% of an extract of fresh leaves of gynura bicolor, 26-35% of an extract of purslane, and 12-18% of an extract of Stephania delavayi.

2. The composite plant extract for inhibiting aquatic environment edwardsiella tarda according to claim 1, which comprises 55-60% of the fresh leaf extract of gynura bicolor, 25-30% of the purslane extract and 15-18% of the Stephania delavayi stem extract in percentage by weight.

3. The composite plant extract for inhibiting aquatic environment edwardsiella tarda according to claim 2, wherein the fresh leaf extract of gynura bicolor is a fermentation concentrate after vinegar processing, the purslane extract is a concentrate after purslane is fermented and roasted with hot salt, and the Stephania delavayi extract is a concentrate after the Stephania delavayi is pretreated and fermented.

4. The composite plant extract for inhibiting the aquatic environment edwardsiella tarda according to claim 3, wherein the preparation method of the gynura bicolor fresh leaf extract comprises the following steps:

s1, preprocessing the gynura bicolor fresh leaves: mixing the fresh leaves of Gynura bicolor with salt, grinding, mashing, mixing with vinegar, sealing and mixing at 33-35 deg.C for 8-10 days while stirring for 2-5min every 3-4 hr to obtain fresh leaves of Gynura bicolor processed with vinegar;

s2, mixing the fresh leaves of the vinegar-processed gynura bicolor with water, a lactobacillus liquid culture medium and lactobacillus,hermetically culturing at 33-37 deg.C until OD value is 6.0-7.5 to obtain culture, centrifuging the culture to remove residue, collecting supernatant, ultrafiltering and concentrating to 10-30% of original volume to obtain concentrated solution, i.e. the fresh leaf extract of herba Aleuritis Fortunei; wherein, the vinegar processed gynura bicolor fresh leaves: water: the weight volume ratio of the lactobacillus liquid culture medium is 8-10: 16-30: 3-6, the addition amount of lactobacillus is (2-6) x 10 added to each mass part of the fresh leaves of the vinegar-processed gynura bicolor⁵cfu。

5. The composite plant extract for inhibiting the aquatic environment edwardsiella tarda according to claim 3, wherein the purslane extract is prepared by the following steps:

s1, fermentation: mixing purslane, water, a mycobacterium liquid culture medium and mycobacteria, and carrying out closed culture at 35-40 ℃ until the OD value is 3.0-4.5 to obtain a culture, wherein the purslane: water: the weight-volume ratio of the mycobacterium liquid medium is 1: 6-12: 2-4, the addition amount of the mycobacteria is (3-8) x 10 added to each part of purslane by mass⁶cfu；

S2, roasting with hot salt: filtering and collecting fermented purslane in the culture at 35-40 ℃, placing the purslane on a hot pot heated to 70-85 ℃, mixing with 1/4-1/3 parts by mass of compound salt, stir-frying for 30-50min, collecting the purslane containing salt when the compound salt is completely dissolved, placing the purslane containing salt at 40-45 ℃ and drying until the water content is less than or equal to 8%, mixing with 3-4 parts by mass of water, sterilizing at high pressure, cooling, taking out, and centrifuging to remove residues;

s3, concentration: and (3) performing ultrafiltration concentration on the supernatant collected after centrifugation to 10-25% of the original volume to obtain a concentrated solution, namely the purslane extract.

6. The composite plant extract for inhibiting the aquatic environment Edwardsiella tarda according to claim 5, wherein the composite salt S2 is a mixture of potassium chloride, magnesium sulfate and sodium sulfate, and the mixing mass ratio of the potassium chloride, the magnesium sulfate and the sodium sulfate is 20-40: 50-65: 20-30.

7. The composite plant extract for inhibiting the aquatic environment edwardsiella tarda according to claim 3, wherein the preparation method of the Stephania delavayi extract comprises the following steps:

s1, obtaining a supernatant A: squeezing the Stephania delavayi Diels to obtain Stephania delavayi Diels juice, reserving the Stephania delavayi Diels squeezed meal, centrifuging the Stephania delavayi Diels juice, collecting the precipitate, fermenting the precipitate with spore bacteria, water and spore liquid culture medium for 4-6 days, and centrifuging the fermentation liquor to obtain supernatant A;

s2, preprocessing the Stephania delavayi Diels pressed meal to obtain Stephania delavayi Diels crushed material;

s3, obtaining a supernatant B: crushing the Stephania delavayi Diels: water: mixing spore liquid culture medium and spore bacteria, hermetically culturing at 35-40 deg.C until OD value is 4.0-6.5 to obtain culture, filtering at 25-28 deg.C to collect culture, centrifuging to collect sediment, dissolving in 6-10 times of the centrifugal sediment by adding water, packaging, sealing, autoclaving, and removing residue to obtain centrifugal supernatant B; replacing stems of Stephania delavayi Diels and crushing: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 6-12: 2-4, the addition of spore bacteria is (3-8) gamma 10 added into each part of crushed Stephania delavayi Diels⁶cfu；

S4, mixing the supernatant A and the supernatant B, and performing ultrafiltration concentration to obtain a concentrated solution, namely the Stephania delavayi Diels extract.

8. The composite plant extract for inhibiting Edwardsiella tarda in an aqueous environment according to any one of claims 4 to 7, wherein the concentration conditions are as follows: the temperature is 25-30 deg.C, the operation pressure is 0.3-0.6Mpa, and the membrane flux of ultrafiltration membrane is 50-100L/m²·h。

9. The method for preparing the composite plant extract as claimed in any one of claims 1 to 3, wherein the components are mixed in proportion at 15-40 ℃, and the obtained mixed solution is the composite plant extract.

10. The use of the composite plant extract as claimed in any one of claims 1 to 3, wherein the composite plant extract is used for inhibiting abnormal propagation of Edwardsiella tarda in the upper, middle and bottom layers of an aquaculture water body.

Technical Field

The invention relates to the technical field of water environment bacteriostat, and particularly relates to a composite plant extract for inhibiting Edwardsiella tarda in a water environment, and a preparation method and application thereof.

Background

The Edwardsiella tarda is a gram-negative pathogenic bacterium with larger harm, the size of the bacterium is about 1-4 mu m, the bacterium is facultative anaerobic, no spore is formed, periphytic flagella is taken as a movement organ and commonly exists in the global fresh water and seawater environment, the Edwardsiella tarda can grow at 4-10 ℃, the optimal growth temperature is 25-32 ℃, the Edwardsiella tarda does not grow at 42 ℃ or above, and the pH value ranges from 5.5 to 9. Can grow in the salt content of 0-4%.

The Edwardsiella tarda has strong pathogenicity on scale-free breeding varieties such as eels, catfishes, flounders, frogs and the like, diseases caused by the Edwardsiella tarda enter fish bodies by adhering to baits or invade the fish bodies through digestive tracts, gills or injured epidermis of the fish are classified as three animal epidemic diseases, the disease mainly occurs at the water temperature of more than 15 ℃, and the disease occurs in a peak period at the water temperature of 25-30 ℃. In the actual culture, in areas without scales such as Guangdong, Hubei and Sichuan, the common phenomena are as follows: when conditions such as water body temperature, organic matters and the like are suitable for rapid propagation of the Edwardsiella tarda, the pathogenic risk of the Edwardsiella tarda to channel catfish, catfishes and pelteobagrus fulvidraco is greatly improved, once the Edwardsiella tarda enters a fish body to cause morbidity, effective feeding time can be prolonged, the medicine cost is increased, and the medicine residue risk is caused by the use of chemical medicines.

The propagation risk of the delayed Edwardsiella tarda is cut off by inhibiting the abnormal propagation of the delayed Edwardsiella tarda in a water body, the propagation risk is one of common prevention and control means, and chlorine dioxide, povidone iodine, potassium salt and other common disinfectant commodities have good safety and sterilizing capability, but are easily influenced by the content of organic matters in the water body, so that the sterilizing effect is generally difficult to achieve by using the disinfectant in a conventional dosage when the delayed Edwardsiella tarda occurs, and the total use cost of 2-3 disinfection treatment courses in a single disease period is very high when the dosage is increased to have the sterilizing effect, so that the use will of farmers is greatly reduced; in the research report of screening and inhibiting Edwardsiella tarda for different Chinese herbal medicine decoction, although part of the Chinese herbal medicine decoction can be definitely inhibited from bacteria, the outstanding problems are that the dosage is too high when the Chinese herbal medicine decoction is converted into production application, the corresponding use cost is too high, and the value converted into the production application is lower. Therefore, based on effectively reducing the infection risk of the Edwardsiella tarda, the green safe disinfectant which can effectively inhibit the abnormal propagation of the Edwardsiella tarda in the culture water environment more than the traditional disinfectant commodity and the related traditional Chinese medicine boiled water reported in research is developed for the two dimensions of the standard effective dosage and the effective speed, not only meets the requirement of effectively preventing and controlling the infection of the Edwardsiella tarda in the healthy culture of the scaleless fish, but also has very important technical innovation value.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention firstly provides a composite plant extract for inhibiting Edwardsiella tarda in a water environment.

The second purpose of the invention is to provide a preparation method of the composite plant extract for inhibiting the Edwardsiella tarda in the water environment.

The third purpose of the invention is to provide the application of the composite plant extract for inhibiting the Edwardsiella tarda in the water environment.

The purpose of the invention is realized by the following technical scheme:

a composite plant extract for inhibiting Edwardsiella tarda in water environment comprises, by weight, 55-65% of an extract of fresh leaves of Gynura bicolor, 26-35% of an extract of purslane, and 12-18% of an extract of Stephania delavayi.

Preferably, the composite plant extract for inhibiting the aquatic environment edwardsiella tarda comprises, by weight, 55-60% of a fresh leaf extract of gynura bicolor, 25-30% of a purslane extract and 15-18% of a Stephania delavayi stem extract.

As a most preferred embodiment, the composite plant extract for inhibiting the aquatic environment Edwardsiella tarda comprises 56 percent of the extract of the fresh leaves of the gynura bicolor, 26 percent of the extract of the purslane and 18 percent of the extract of the Stephania delavayi.

Preferably, the gynura bicolor fresh leaf extract is a fermentation concentrate after being processed by vinegar.

Preferably, the preparation method of the gynura bicolor fresh leaf extract comprises the following steps:

s1, preprocessing the gynura bicolor fresh leaves: mixing, grinding and mashing the gynura bicolor fresh leaves and salt (the mass ratio of the gynura bicolor fresh leaves to the salt is 1: 80-1: 100), mixing the mashed materials with vinegar according to the mass volume ratio of 1: (10-15), placing in an environment with room temperature of 33-35 ℃ for closed mixing for 8-10 days, and stirring for 2-5min every 3-4h to obtain vinegar-processed gynura bicolor fresh leaves;

s2, mixing vinegar-processed fresh leaves of Gynura bicolor with water, lactobacillus liquid culture medium and lactobacillus, hermetically culturing at 33-37 deg.C until OD value is 6.0-7.5 to obtain culture, centrifuging the culture to remove residue, collecting supernatant, ultrafiltering and concentrating to 10-30% of original volume to obtain concentrated solutionIs the extract of the fresh leaves of the gynura bicolor; wherein, the vinegar processed gynura bicolor fresh leaves: water: the weight volume ratio of the lactobacillus liquid culture medium is 8-10: 16-30: 3-6, the addition amount of lactobacillus is (2-6) x 10 added to each mass part of the fresh leaves of the vinegar-processed gynura bicolor⁵cfu。

Wherein, the mixing of the fresh leaves of the vinegar-processed gynura bicolor, the water, the lactic acid bacteria liquid culture medium and the lactic acid bacteria is not limited in the adding sequence, for example, the fresh leaves of the vinegar-processed gynura bicolor are mixed with the water, then the lactic acid liquid culture medium is added for mixing, and finally the lactic acid bacteria is added for mixing.

Wherein the concentration conditions are as follows: the temperature is 25-30 deg.C, the operation pressure is 0.3-0.6Mpa, and the membrane flux of ultrafiltration membrane is 50-100L/m²·h。

Preferably, the gynura bicolor fresh leaves in the gynura bicolor fresh leaf extract are freshly picked gynura bicolor leaves which are baked at 45 ℃ until the water content is 40% -50%, and rotten leaves, dead leaves, curly yellow leaves, insect leaves and the like are deleted during fresh picking.

Further, the water in the above-mentioned fresh gynura bicolor leaf extract is preferably sterile distilled water.

Further, the lactic acid bacteria are preferably leuconostoc dextranicum, lactococcus faecalis, lactococcus casei and the like which cannot replace the leuconostoc dextranicum of the present invention.

Further, the above culture is centrifuged preferably at 5000rpm, for example 5000rpm for 10 minutes.

As a more preferred embodiment, the preparation method of the gynura bicolor fresh leaf extract comprises the following steps:

mixing the gynura bicolor fresh leaves and (1/100-1/80) salt according to mass percent, fully grinding into a smashed material, mixing the smashed material with a mature vinegar solution according to a mass volume ratio of 1: (10-12), placing in a room temperature of 33-35 ℃, hermetically mixing for 8-10 days, keeping stirring for 3-5min every 3-4h, taking out the vinegar-processed gynura bicolor fresh leaves, mixing the vinegar-processed gynura bicolor fresh leaves with water, a lactobacillus liquid culture medium and lactobacillus, hermetically culturing at 33-37 ℃ until the OD value is 6.4-7.5 to obtain a culture, centrifuging the culture to remove residues, collecting the supernatant, and collecting the collected gynura bicolor fresh leaves at the ambient temperature of 25-30 DEG CPutting the supernatant into a stirring-free ultrafiltration device, and taking the operating pressure of 0.4-0.5 Mpa and the membrane flux of an ultrafiltration membrane of 60L/m2 & h as technical parameters to obtain a concentrated solution which is concentrated to 10-30% of the original volume, namely the gynura bicolor fresh leaf extract; wherein, the fresh leaves of the gynura bicolor processed by vinegar: water: the weight-volume ratio of the lactobacillus liquid culture medium is (8-10): (16-25): (4-6), wherein the addition amount of the lactic acid bacteria is (4-6) x 10 added to each mass part of the vinegar-processed gynura bicolor fresh leaves⁵cfu。

As a most preferred embodiment, the preparation method of the gynura bicolor fresh leaf extract comprises the following steps:

mixing the gynura bicolor fresh leaves and (1/100) salt according to mass percentage, fully grinding into a smashed material, mixing the smashed material with a mature vinegar solution according to the mass volume ratio of 1: 10, placing the mixture in an environment with the room temperature of 33-35 ℃ for closed mixing for 8-10 days, keeping stirring for 4-5min every 3-4h, taking out the fresh leaves of the vinegar-processed gynura bicolor, mixing the fresh leaves of the vinegar-processed gynura bicolor with water, a lactobacillus liquid culture medium and lactobacillus, performing closed culture at the temperature of 33-37 ℃ until the OD value is 7.0-7.5 to obtain a culture, centrifuging the culture to remove residues, collecting supernatant, then placing the collected supernatant into a stirring-free ultrafiltration device at the environment temperature of 28 ℃, and setting the operation pressure to be 0.5Mpa and the membrane flux of the ultrafiltration membrane to be 60L/m²H is a technical parameter, and the obtained concentrated solution concentrated to 20% of the original volume is the gynura bicolor fresh leaf extract. Wherein, the fresh leaves of the gynura bicolor processed by vinegar: water: the weight volume ratio of the lactobacillus liquid culture medium is 8: 22: 5, the adding amount of the lactobacillus is 5.5 x 10 added to each mass part of the fresh leaves of the vinegar-processed gynura divaricata⁵cfu。

Wherein the lactobacillus liquid culture medium can be a commercially available lactobacillus liquid culture medium, such as imported Lactic-Lactobacillus Midium.

Wherein the herba Portulacae extract is the concentrate of herba Portulacae (whole plant without root) after fermentation and hot salt roasting.

Preferably, the preparation method of the purslane extract comprises the following steps:

s1, fermentation: mixing herba Portulacae, water, mycobacterial liquid culture medium and mycobacteria, and sealing at 35-40 deg.CAnd closing the culture until the OD value is 3.0-4.5 to obtain a culture, wherein the weight ratio of the purslane: water: the weight-volume ratio of the mycobacterium liquid medium is 1: 6-12: 2-4, the addition amount of the mycobacteria is (3-8) x 10 added to each part of purslane by mass⁶cfu；

S2, roasting with hot salt: filtering and collecting fermented purslane in the culture at 35-40 ℃, placing the purslane on a hot pot heated to 70-85 ℃, mixing with 1/4-1/3 parts by mass of compound salt, stir-frying for 30-50min, collecting the purslane containing salt when the compound salt is completely dissolved, placing the purslane containing salt at 40-45 ℃ and drying until the water content is less than or equal to 8%, mixing with 3-4 parts by mass of water, sterilizing at high pressure, cooling, taking out, and centrifuging to remove residues;

s3, concentration: and (3) performing ultrafiltration concentration on the supernatant collected after centrifugation to 10-25% of the original volume to obtain a concentrated solution, namely the purslane extract.

Preferably, the compound salt of S2 is a mixture of potassium chloride, magnesium sulfate and sodium sulfate, and the mixing mass ratio of the potassium chloride, the magnesium sulfate and the sodium sulfate is 20-40: 50-65: 20-30.

The above-mentioned purslane, water, mycobacteria liquid culture medium and mycobacteria are mixed without any restriction on the order of addition, for example, the purslane and mycobacteria liquid culture medium are mixed first, then the mycobacteria are added and mixed, finally water is added and mixed uniformly.

Wherein the concentration conditions are as follows: the temperature is 25-30 deg.C, the operation pressure is 0.3-0.6Mpa, and the membrane flux of ultrafiltration membrane is 50-100L/m²·h。

Further, the water in the purslane extract is preferably sterile distilled water.

Further, the above culture is centrifuged preferably at 3000rpm, for example 3000rpm for 10 minutes.

As a more preferred embodiment, the preparation method of the purslane extract comprises the following steps: mixing herba Portulacae, water, mycobacteria liquid culture medium and mycobacteria, hermetically culturing at 35-40 deg.C until OD value is 3.5-4.5 to obtain culture, filtering at 33-35 deg.C to collect fermented herba Portulacae in the culture, placing on a hot pot heated to 75-85 deg.C, and mixing with 1/4-1/3 parts by weight of compoundMixing salt, frying for 30-50min, collecting salt containing herba Portulacae when the compound salt is completely dissolved, oven drying at 43-45 deg.C until the water content is less than or equal to 8%, mixing with 3-4 weight parts of water, subpackaging, sealing at 121 deg.C, autoclaving for 15min, cooling, taking out, centrifuging to remove residue, placing the collected supernatant into a stirring-free ultrafiltration device at 25-30 deg.C with an operating pressure of 0.4-0.5 Mpa and a membrane flux of 60L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 18-20% of the original volume, and the obtained concentrated solution is the purslane extract. Wherein, the purslane: water: the weight-volume ratio of the mycobacterium liquid medium is 1: (8-12): (3-4), the addition amount of the mycobacteria is (5-8) x 10 added to each part of purslane by mass⁶cfu。

As a most preferred embodiment, the preparation method of the purslane extract comprises the following steps: mixing herba Portulacae, water, mycobacteria liquid culture medium and mycobacteria, hermetically culturing at 35-40 deg.C until OD value is 4.0 to obtain culture, filtering and collecting herba Portulacae fermented in the culture at 35 deg.C, placing on a hot pot heated to 85 deg.C, mixing with 1/4-1/3 parts by weight of compound salt, parching for 30-50min, collecting herba Portulacae containing salt when the compound salt is completely dissolved, oven drying at 45 deg.C until water content is less than or equal to 8%, mixing with 4 parts by weight of water, subpackaging, sterilizing at 121 deg.C under high pressure for 15min, cooling, taking out, centrifuging to remove residue, placing the collected supernatant into a stirring-free ultrafiltration device at 30 deg.C under operating pressure of 0.5 and membrane flux of 60L/m Mpa of ultrafiltration membrane²H is a technical parameter, and the obtained concentrated solution is concentrated to 18% of the original volume, and the obtained concentrated solution is the purslane extract. Wherein, the purslane: water: the weight-volume ratio of the mycobacterium liquid medium is 1: 10: 3, the addition amount of the mycobacteria is 8 gamma 10 added into each part of purslane by mass⁶cfu。

The spore liquid culture medium can be a commercially available spore liquid culture medium, such as imported Bacillus Mediumbase.

In the composite plant extract, the Stephania delavayi Diels extract is a concentrate of the Stephania delavayi Diels after pretreatment and fermentation.

Preferably, the preparation method of the Stephania delavayi Diels extract comprises the following steps:

s1, obtaining a supernatant A: squeezing the Stephania delavayi Diels to obtain Stephania delavayi Diels juice, reserving the Stephania delavayi Diels squeezed meal, centrifuging the Stephania delavayi Diels juice, collecting the precipitate, fermenting the precipitate with spore bacteria, water and spore liquid culture medium for 4-6 days, and centrifuging the fermentation liquor to obtain supernatant A;

s2, preprocessing the Stephania delavayi Diels pressed meal to obtain Stephania delavayi Diels crushed material;

s3, obtaining a supernatant B: crushing the Stephania delavayi Diels: water: mixing spore liquid culture medium and spore bacteria, hermetically culturing at 35-40 deg.C until OD value is 4.0-6.5 to obtain culture, filtering at 25-28 deg.C to collect culture, centrifuging to collect sediment, dissolving in 6-10 times of the centrifugal sediment by adding water, packaging, sealing, autoclaving, and removing residue to obtain centrifugal supernatant B; replacing stems of Stephania delavayi Diels and crushing: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 6-12: 2-4, the addition of spore bacteria is (3-8) gamma 10 added into each part of crushed Stephania delavayi Diels⁶cfu；

S4, mixing the supernatant A and the supernatant B, and performing ultrafiltration concentration to obtain a concentrated solution, namely the Stephania delavayi Diels extract.

Wherein, the preprocessing of step S2 is: the squeezed Stephania delavayi Diels meal is wrapped by a film to be naked and sealed, and then is subjected to quick freezing, pre-puffing and vacuum deoiling treatment to obtain a Stephania delavayi Diels dehydrated dry substance, and the dry substance is sieved by a 40-60 mesh sieve to be crushed to obtain a Stephania delavayi Diels crushed substance.

The above-mentioned mixture of the crushed Stephania delavayi Diels, water, the spore bacteria liquid culture medium and the spore bacteria is not limited in order of addition, and for example, the crushed Stephania delavayi Diels and the spore liquid culture medium are mixed first, then the spore bacteria is added and mixed, finally the water is added and mixed uniformly.

Wherein, the preferred plant of the Stephania delavayi Diels is a complete plant with the full length (without root length) not less than 70cm, and the Stephania delavayi Diels are obtained by removing roots, fresh leaves and stamen receptacle.

WhereinThe concentration conditions are as follows: the temperature is 25-30 deg.C, the operation pressure is 0.3-0.6Mpa, and the membrane flux of ultrafiltration membrane is 50-100L/m²·h。

Further, the quick freezing is preferably carried out by quickly freezing the stem of the bare gold at-20 to-40 ℃ for 10 to 12 hours.

Further, the pre-puffing is to fry and puff the quick-frozen stem of the preserved stem of the naked gold for 3-6min under the conditions of vacuum degree of 90-95KPa and temperature of 70-80 ℃.

Further, the vacuum deoiling is to place the gold exchange stem after the frying under the condition of the rotation speed of 150-.

Further, the water in the above Stephania delavayi Diels extract is preferably sterile distilled water.

Further, the above-mentioned bacillus is preferably clostridium polyminerans, bacillus subtilis, bacillus licheniformis and the like which cannot functionally replace clostridium polyminerans of the present invention.

As a more preferred embodiment, the preparation method of the permuted stem extract comprises: the preparation method comprises the steps of squeezing 100 parts by weight of Stephania delavayi Diels to collect Stephania delavayi Diels juice, reserving the Stephania delavayi Diels squeezed meal, centrifuging the squeezed Stephania delavayi Diels juice to remove supernatant, collecting centrifugal sediment, fully fermenting the sediment with spore bacteria, water and a culture medium for 5-6 days, taking fermentation, centrifuging to remove the sediment, taking centrifugal supernatant, and marking the centrifugal supernatant as supernatant A. Wrapping the squeezed Stephania delavayi Diels with a film, sealing, quickly freezing, pre-puffing, and vacuum deoiling to obtain Stephania delavayi Diels dehydrated dry substance, sieving the dry substance with a 50-60 mesh sieve, pulverizing to obtain Stephania delavayi Diels pulverized substance, and pulverizing the Stephania delavayi Diels pulverized substance: water: mixing spore liquid culture medium and spore bacteria, culturing at 36-40 deg.C under sealed condition until OD value is 5.0-6.5 to obtain culture, filtering at 26-28 deg.C to collect culture, centrifuging at 4000-5000rpm for 25-30min, discarding supernatant, collecting centrifugal deposit, dissolving 8-10 times of the centrifugal deposit in water, packaging, sealing, autoclaving at 121 deg.C for 15min, cooling, taking out, centrifuging at 800-1100rpm for 6-10min, removing residue to obtain supernatant B, mixing the collected supernatant A and supernatant B, adding stirring-free type ultra-high pressure medium, and mixingConcentrating the filtrate to 10-20% of the original volume to obtain a concentrated solution, namely the Stephania delavayi Diels extract. Wherein, the crushed substances of the stem of Stephania delavayi Diels: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: (8-10): (2-3) mixing, wherein the addition amount of the spore bacteria is (5-7) gamma 10 added into each part of the crushed material of the stem of Stephania delavayi Diels by mass⁶cfu。

As a most preferred embodiment, the preparation method of the permuted stem extract comprises: the preparation method comprises the steps of squeezing 100 parts by weight of Stephania delavayi Diels to collect Stephania delavayi Diels juice, reserving the Stephania delavayi Diels squeezed meal, centrifuging the squeezed Stephania delavayi Diels juice to remove supernatant, collecting centrifugal sediment, fully fermenting the sediment with spore bacteria, water and a culture medium for 5-6 days, taking fermentation, centrifuging to remove the sediment, taking centrifugal supernatant, and marking the centrifugal supernatant as supernatant A. The squeezed Stephania delavayi Diels are wrapped by a film to be naked and sealed, and then are subjected to quick-freezing, pre-puffing and vacuum deoiling treatment to obtain a Stephania delavayi Diels dehydrated dry substance, the dry substance is sieved by a 60-mesh sieve to be crushed to obtain a Stephania delavayi Diels crushed substance, and the Stephania delavayi Diels crushed substance is crushed: water: mixing spore liquid culture medium and spore bacteria, culturing at 38 deg.C under sealed condition until OD value is 6.5 to obtain culture, filtering at 26 deg.C, collecting culture, centrifuging at 5000rpm for 25min, discarding centrifuged supernatant, collecting centrifuged sediment, dissolving in 9 times of the centrifuged sediment in water, packaging, sealing, autoclaving at 121 deg.C for 15min, cooling, taking out, centrifuging at 900rpm for 8min, removing residue to obtain centrifuged supernatant B, mixing collected supernatant A and supernatant B at 28 deg.C, and loading into stirring-free ultrafiltration device with operating pressure of 0.4Mpa and membrane flux of 50L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 20 percent of the original volume to obtain the concentrated solution, namely the Stephania delavayi Diels extract. Wherein, the crushed substances of the stem of Stephania delavayi Diels: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 9: 3, mixing the materials, wherein the adding amount of the spore bacteria is 6 x 10 added into each mass part of the crushed material of the stem of the Stephania delavayi Diels⁶cfu。

The spore liquid culture medium can be a commercially available spore liquid culture medium, such as imported Bacillus Mediumbase.

Wherein, the quick freezing is preferably carried out by quickly freezing the stem of the nude gold-wrapped bag at-30 ℃ for 12 h.

Wherein the pre-puffing is to fry and puff the quick-frozen stem of the dead gold under the condition of vacuum degree of 90KPa and temperature of 80 ℃ for 5 min.

Wherein, the vacuum deoiling is to deoil the fried golden stem for 6min under the condition of the rotating speed of 200 r/min.

Wherein the water in the Stephania delavayi Diels extract is preferably sterile distilled water.

The invention also provides a preparation method of the composite plant extract, which is to mix the components in proportion at 15-40 ℃, and the obtained mixed solution is the composite plant extract.

Preferably, the mixing temperature of the complex extract is 25-30 deg.C, and more preferably, the mixing temperature of the complex extract is 28 deg.C.

The invention also provides an application of the composite plant extract, and the composite plant extract is used for inhibiting the abnormal propagation of Edwardsiella tarda on the upper layer, the middle layer and the bottom layer of a culture water body; when the dosage is less than or equal to 5mg/L, the abnormal propagation of the Edwardsiella tarda in the upper layer, middle layer and bottom layer cultivation of the cultivation water body can be effectively inhibited, the safety on the yellow catfish, the channel catfish and the ditch catfish is good, and when the final dosage is 10mg/L, the safety on the yellow catfish, the channel catfish and the ditch catfish is obviously reduced.

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

compared with the safety of the traditional GMP disinfectant, the composite plant extract belongs to an environment-friendly substance, has a wide range of safe concentration for the alepidote fish, and has no interference on the inhibition effect of the edwardsiella tarda by water organisms when used for splashing water. Compared with the broad-spectrum effectiveness of the traditional GMP disinfectant, the action spectrum of the compound extract has special effect on Edwardsiella tarda, and the compound extract can be used for inhibiting the abnormal reproduction of the Edwardsiella tarda in the aquaculture water environment, so that the attack force of the Edwardsiella tarda in the water environment on scale-free fish bodies is reduced; under the same dosage, the composite plant extract of the invention has no obvious low-concentration inhibition effect on escherichia coli and the like which are enterobacteriaceae, and has no obvious inhibition effect on aeromonas hydrophila of aeromonas of vibriaceae, flavobacterium columniformis and the like of myxobacter. From the aspect of formula compatibility, the composite plant extract disclosed by the invention is composed of a gynura bicolor fresh leaf extract, a purslane extract and a Stephania delavayi stem extract, the three components have obvious compatibility and synergism, and have a good inhibition effect on Edwardsiella tarda in a culture water body, but have no repair effect on local color fading caused by the fact that Edwardsiella tarda is infected by non-scaly fish.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

A composite plant extract for inhibiting Edwardsiella tarda in an aqueous environment is prepared by weighing 56 parts by weight of a fresh leaf extract of gynura bicolor, 26 parts by weight of a purslane extract and 18 parts by weight of a Stephania delavayi stem extract, and mixing the components at 28 ℃ to obtain a mixed solution, namely the composite plant extract.

Wherein, the gynura bicolor fresh leaf extract is obtained by the following method:

(1) mixing the gynura bicolor fresh leaves and salt according to the mass percentage (1/100), fully grinding into a mash, mixing the mash with a mature vinegar solution according to the mass volume ratio of 1: 10 mixing, placing in an environment of room temperature of 33-35 ℃ for sealed mixing for 8-10 days, keeping stirring for 4-5min every 3-4h, taking out the vinegar-processed gynura bicolor fresh leaves;

(2) mixing the vinegar-processed folium Aleuritoidis with water, lactobacillus liquid culture medium (weight volume ratio is 8: 22: 5) and lactobacillus (the addition amount of lactobacillus is 5.5 gamma 10 per mass part of vinegar-processed folium Aleuritoidis⁵cfu), culturing at 33-37 deg.C under sealed condition until OD value is 7.0-7.5 to obtain culture, centrifuging the culture to remove residue, collecting supernatant, and loading the collected supernatant into stirring-free ultrafiltration device at 28 deg.C under operating pressure of 0.5Mpa and membrane flux of 60L/m²H is a technical parameter, and the obtained concentrated solution concentrated to 20% of the original volume is the gynura bicolor fresh leaf extract.

The purslane extract is prepared by a method comprising the following steps: mixing herba Portulacae, water, mycobacteria liquid culture medium and mycobacteria, hermetically culturing at 35-40 deg.C until OD value is 4.0 to obtain culture, filtering and collecting herba Portulacae fermented in the culture at 35 deg.C, placing on a hot pot heated to 85 deg.C, mixing with 1/4-1/3 parts by weight of compound salt, parching for 30-50min, collecting herba Portulacae containing salt when the compound salt is completely dissolved, oven drying at 45 deg.C until water content is less than or equal to 8%, mixing with 4 parts by weight of water, subpackaging, sterilizing at 121 deg.C under high pressure for 15min, cooling, taking out, centrifuging to remove residue, and loading the collected supernatant into a stirring-free ultrafiltration device at 30 deg.C under 0.5Mpa of operating pressure and 60L/m of membrane flux of ultrafiltration membrane²H is a technical parameter, and the obtained concentrated solution is concentrated to 18% of the original volume, and the obtained concentrated solution is the purslane extract. Wherein, the purslane: water: the weight-volume ratio of the mycobacterium liquid medium is 1: 10: 3, the addition amount of the mycobacteria is 8 gamma 10 added into each part of purslane by mass⁶cfu。

Wherein the Stephania delavayi Diels extract is prepared by the method comprising the following steps: the preparation method comprises the steps of squeezing 100 parts by weight of Stephania delavayi Diels to collect Stephania delavayi Diels juice, reserving the Stephania delavayi Diels squeezed meal, centrifuging the squeezed Stephania delavayi Diels juice to remove supernatant, collecting centrifugal sediment, fully fermenting the sediment with spore bacteria, water and a culture medium for 5-6 days, taking fermentation, centrifuging to remove the sediment, taking centrifugal supernatant, and marking the centrifugal supernatant as supernatant A. Will be provided withThe squeezed Stephania delavayi Diels are wrapped by a film to be naked and sealed, and then are subjected to quick-freezing, pre-puffing and vacuum deoiling treatment to obtain a Stephania delavayi Diels dehydrated dry substance, the dry substance is sieved by a 60-mesh sieve to be crushed to obtain a Stephania delavayi Diels crushed substance, and the Stephania delavayi Diels crushed substance is crushed: water: mixing spore liquid culture medium and spore bacteria, culturing at 38 deg.C under sealed condition until OD value is 6.5 to obtain culture, filtering at 26 deg.C, collecting culture, centrifuging at 5000rpm for 25min, discarding centrifugation to collect centrifugal sediment, dissolving in 9 times of water, packaging, sealing, sterilizing at 121 deg.C under high pressure for 15min, cooling, taking out, centrifuging at 900rpm for 8min, removing residue to obtain centrifugal supernatant (labeled as supernatant B), mixing collected supernatant A and supernatant B at 28 deg.C, and loading into stirring-free ultrafiltration device at operating pressure of 0.4Mpa and membrane flux of 50L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 20 percent of the original volume to obtain the concentrated solution, namely the Stephania delavayi Diels extract. Wherein, the crushed substances of the stem of Stephania delavayi Diels: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 9: 3, mixing the materials, wherein the adding amount of the spore bacteria is 6 x 10 added into each mass part of the crushed material of the stem of the Stephania delavayi Diels⁶cfu。

Experimental example 1 comparison of inhibitory action of the composite plant extract of the present invention on common aquatic pathogenic bacteria

1. Test materials

1.1 test strains

Edwardsiella tarda, aeromonas hydrophila, flavobacterium columnare: all provided by the subject group.

Povidone iodine: is povidone iodine solution with the content of 10 percent.

1.2 test consumables

TSB medium, petri dish, test tube, distilled water, etc.

1.3 composite plant extracts

The preparation method of the composite plant extract used in the experiment is the same as that of example 1.

2. Test method

2.1 preparation of bacterial suspension

Will delay EdwardInoculating bacteria, Aeromonas hydrophila and Flavobacterium columnare into TSB nutrient broth, culturing at 30 deg.C for 16-20h, centrifuging to collect bacteria, turbidifying the bacteria liquid with McLeod turbidimetric tube to estimate bacteria concentration, diluting the bacteria with PBS to 1 × 10⁴-5×10⁴cfu/mL of bacterial suspension.

2.2 preparation of Compound plant extract

The composite plant extract was diluted to the test concentration with sterile water.

2.3MIC determination and result determination

The test concentration of 4.5mL of the composite plant extract and 0.5mL of the bacterial suspension were mixed well, PBS was used as a control group instead of the disinfectant, and PBS and no bacterial suspension was added as a blank group. After the reaction solution is placed in a water bath kettle at 20 +/-2 ℃ for reaction for 10min, 0.5mL of each group of reaction solution is taken and transferred into 4.5mL of fresh broth, and the culture is carried out for 24h at 30-35 ℃, and the result is observed.

If the broth was turbid, indicating bacterial growth, positive (+) and clear, indicating sterile growth, negative (-); the control group should be cloudy and the blank group should be clear. The disinfectant concentration of the clear lowest disinfectant concentration group is the MIC value for that disinfectant.

2.4 measurement of Sterilization Rate and determination of results

The test concentration of 4.5mL of the composite plant extract and 0.5mL of the bacterial suspension were mixed uniformly, PBS was used as a control group instead of the disinfectant, and PBS was used as a blank group without adding the bacterial suspension. Placing the reaction solution in a water bath kettle at 20 + -2 deg.C for reaction for 10min, coating 100uL of each group of reaction solution, culturing 3 groups in parallel at 30-35 deg.C for 24 hr, and counting the sterilization rate.

The number of control group bacteria is more than 100, and the bacteria concentration of the bacteria suspension reaches 1 × 10⁴-3×10⁴cfu/mL; blank group should be grown aseptically; the killing effect of the disinfectant on bacteria was evaluated by calculating the bactericidal rate:

the sterilization rate (%) (number of surviving bacteria in control group-number of surviving bacteria in test group) × 100/number of surviving bacteria in control group).

3. Test results

The test data in Table 1 show that the MIC of the composite plant extract to Edwardsiella tarda is 0.063-0.125mg/L, the MIC is far lower than the MIC of povidone iodine to Edwardsiella tarda by 2mg/L, the MICs to Aeromonas hydrophila and Flavobacterium columnare respectively 8mg/L and 4mg/L, and the MICs are respectively lower than the MICs of povidone iodine to Aeromonas hydrophila by 4mg/L and the MICs of Flavobacterium columnare 2 mg/L. Therefore, the composite plant extract has good inhibition effect on Edwardsiella tarda, and the inhibition effect on Aeromonas hydrophila and Flavobacterium columnare weaker than that of povidone iodine.

TABLE 1

The test data in Table 2 show that when the concentration of the composite plant extract is 0.063mg/L, the sterilization rate of the composite plant extract to Edwardsiella tarda is unstable, the highest sterilization rate can reach 100 percent, and the lowest sterilization rate can reach 80 percent; when the concentration of the composite plant extract is more than or equal to 0.125mg/L, the sterilization rate of the composite plant extract on Edwardsiella tarda exceeds 100 percent; on the other hand, when the concentration of the composite plant extract is 2mg/L, the sterilization rate of the composite plant extract to aeromonas hydrophila and flavobacterium columnare is 0.

TABLE 2

Experimental example 2 inhibiting effect of compound plant extract formula compatibility on Edwardsiella tarda

1. Test materials

1.1 preparation of Complex plant extracts

The compound plant extract of the present invention used in this experiment, designated as A, was prepared in the same manner as in example 1.

1.2 preparation of Compound plant extract lacking in formulation

Based on the formula of the composite plant extract prepared in example 1, a composite plant extract with a single deletion of a fresh leaf extract of gynura bicolor, a composite plant extract with a single deletion of a purslane extract and a composite plant extract with a single deletion of a stem extract of Stellaria sinensis, a composite plant extract with a single deletion of a fresh leaf extract of purslane and a composite plant extract with a single deletion of a stem extract of Stellaria sinensis, a composite plant extract with a simultaneous deletion of a fresh leaf extract of Stellaria sinensis and a stem extract of Stellaria sinensis, a composite plant extract with a simultaneous deletion of a fresh leaf extract of purslane and a stem extract of Stellaria sinensis and a composite plant extract with a simultaneous deletion of a stem extract of purslane and a stem extract of Stephania sinensis are prepared by a technical idea of single-component.

Mixing the fresh leaves of the gynura bicolor, the purslane and the stephania sinica diels with distilled water with the mass volume ratio of 10 times, boiling the mixture in slow fire until 1ml of liquid medicine is equal to 1g of the weight of the original medicine, and naming the boiled liquid of the fresh leaves of the gynura bicolor, the boiled liquid of the purslane and the boiled liquid of the stephania sinica diels as H, K, L serving as a control group.

2. Test method

2.1 preparation of bacterial suspension

The same as in experimental example 1.

2.2 preparation of Compound plant extract

The test composite plant extracts A, B, C, D, E, F, G, H, K, L were each diluted to test concentrations using sterile water.

2.3MIC determination and result determination

The test concentration of 4.5mL of the composite plant extract and 0.5mL of the bacterial suspension were mixed well, PBS was used as a control group instead of the disinfectant, and PBS and no bacterial suspension was added as a blank group. After the reaction solution is placed in a water bath kettle at 20 +/-2 ℃ for reaction for 10min, 0.5mL of each group of reaction solution is taken and transferred into 4.5mL of fresh broth, and the culture is carried out for 24h at 30-35 ℃, and the result is observed.

If the broth was turbid, indicating bacterial growth, positive (+) and clear, indicating sterile growth, negative (-); the control group should be cloudy and the blank group should be clear. The disinfectant concentration of the clear lowest disinfectant concentration group is the MIC value for that disinfectant.

3. Test results

The test data in Table 3 show that the MIC of the composite plant extract to Edwardsiella tarda is 0.63-0.125mg/L, the MIC of povidone iodine to Edwardsiella tarda is 4mg/L, and the MIC of the single components of the fresh leaf extract of gynura bicolor, the purslane extract and the Stephania delavayi stem extract to Edwardsiella tarda is far higher than that of the composite plant extract disclosed by the invention, which indicates that when the fresh leaf extract of gynura bicolor, the purslane extract and the Stephania delavayi stem extract exist as a single component, the inhibitory effect on Edwardsiella tarda is seriously weakened compared with that of the composite plant extract. When the fresh gynura bicolor leaf extract in the composite plant extract is singly absent, the MIC of the fresh gynura bicolor leaf extract to Edwardsiella tarda is increased to 4 mg/L; when the purslane extract in the composite plant extract is absent and the Stephania delavayi extract in the composite plant extract is absent, the MICs of the purslane extract and the Stephania delavayi extract in the composite plant extract are both 1 mg/L. When the composite plant extract is simultaneously deleted in two components, the MIC of the test group containing the gynura bicolor fresh leaf extract and the MIC of the test group containing the purslane extract with the other two components simultaneously deleted are higher than that of the test group containing the stephania japonica extract with the other two components simultaneously deleted, so that the gynura bicolor fresh leaf extract is the main component of the composite plant extract with the function of inhibiting the stephania bicolor while the purslane extract is the secondary effective component, and the stephania japonica extract composite plant extract has the auxiliary synergistic component for inhibiting the stephania bicolor.

The MICs of the gynura bicolor fresh leaf decoction, the purslane decoction and the Stephania delavayi decoction for Edwardsiella tarda are 16mg/L, 16mg/L and 32mg/L when the gynura bicolor fresh leaf decoction, the purslane decoction and the Stephania delavayi decoction are used independently.

TABLE 3

Experimental example 3 inhibiting effect of the composite plant extract on Edwardsiella tarda in water bodies with different depths

1. Test water source

The yellow catfish pond mouth has Edwardsiella tarda diseases and the daily mu average death rate of more than 1 percent.

Chlorine was exposed for 24h of tap water.

2. Test method

2.1 expanding culture of strains

Selecting Edwardsiella tarda colonies from a seed batch inclined plane, inoculating the Edwardsiella tarda colonies in TSB nutrient broth, performing shake culture at 30 ℃ for 20-24h, centrifugally collecting thalli, performing turbidimetry on the thalli through a Mach turbidimeter tube to estimate the concentration of the thalli, and diluting the thalli to 10 by PBS¹¹cfu/mL of bacterial suspension as mother liquor.

2.2 construction of quantitative bacteria-carrying small water body environment model

12 aquariums with the volume of 1 cubic meter and the height of 2 meters are fixed on a flat ground and are respectively named as A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2 and D3, wherein the A1, the A2 and the A3 are arranged into an A series test parallel group, the B1, the B2 and the B3 are arranged into a B series test parallel group, the C1, the C2 and the C3 are arranged into a C series test parallel group, the D1, the D2 and the D3 are arranged into a D series test parallel group, all the test groups and consumables used in the test are thoroughly sterilized by chlorine dioxide spray, the aquariums are preloaded with isothermal sterilized tap water for 24 hours, the water depth is 1.5 meters, and the aquariums are placed in an indoor environment with ultraviolet ray sterilization.

Respectively adding the Edwardsiella tarda suspension into each group of parallel water bodies of the experiment at the ambient temperature of 25-28 ℃ until the content of the Edwardsiella tarda in each group of water bodies of A, B, C, D series of parallel water bodies is 10⁴cfu/L, continuously supplying oxygen, and maintaining the dissolved oxygen content of the test water body not less than 7mg/L in the whole process. And (4) finishing the test operation, namely, successfully constructing the quantitative bacteria-carrying small water body environment model.

The volume area 20 cm away from the upper water surface is defined as the upper water environment layer, the volume area which is expanded upwards and downwards by equal amount and is 20 cm away from the center line of the water level of the aquarium is defined as the middle water environment layer, and the volume area which is expanded upwards by 20 cm away from the water surface of the bottom layer is defined as the lower water environment layer.

2.3 test of bacteriostatic effect of the composite plant extract of the invention on different depths of the water environment with bacteria

Selecting A, B, C series test parallel groups at the environment temperature of 25-28 ℃, after the molding is successful, installing water pipes at the upper layer, the middle layer and the lower layer of the water environment of the aquarium in a mode of fixing the aquarium wall, standing for 2h, adding the composite plant extract of the invention into the A series test parallel group until the final concentration is 0.125ppm, adding povidone iodine into the B series test parallel group until the final concentration is 2ppm, and setting a blank control group without adding any substance into the C series test parallel group.

Respectively extracting 1ml of water samples of the upper layer, the middle layer and the lower layer of the water environment from each parallel group by water pipes within 1h, 2h, 4h, 8h, 16h, 32h and 64h, wherein 1ml of the water sample of the upper layer of the water environment is regarded as a water sample, 1 water sample is diluted by 2-4 times, 100 mu L of the water sample is taken from 2-time dilution of 1 water sample, dropwise added and coated on a plate, standing is carried out for 5min, each water sample is made into 3 parallel samples, all the plates coated with the water samples are placed in an incubator at 35 ℃ for culturing for 20-24h, and the colony number of the Edwardsiella tarda on the plate is observed and recorded.

The colony counting result judgment standard is as follows: counting, selecting a plate with the colony number between 30 and 300, if two dilutions are between 30 and 300, determining the plate according to the ratio of the two dilutions according to the requirements of the national standard method, taking the average number when the ratio is less than or equal to 2, and taking the smaller number when the ratio is greater than 2, wherein the formula is as follows:

the bacterial concentration of each original water sample is equal to the dilution multiple multiplied by the bacterial colony count of the corresponding plate under the dilution multiple;

if the colony number is less than 30, the data is recorded statistically.

2.4 test of bacteriostasis effect of the combined active substance of the composite plant extract of the invention on different depths of the environment with bacteria

Selecting A, C, D series of parallel test groups at the environment temperature of 25-28 ℃, wherein A and C groups share the test group with 2.3, after the molding is successful, arranging water guide pipes at the upper layer, the middle layer and the lower layer of the water environment of the aquarium in a mode of fixing the tank wall, standing for 2h, adding the composite plant extract of the invention into the A series of parallel test groups until the final concentration is 0.125ppm, adding the composite plant extract of the invention and the high molecular anionic polymer into the D series of parallel test groups, wherein the compounding concentrations are 0.125ppm and 0.2ppm, and adding no substance into the C series of parallel test groups to be set as a blank control group.

Respectively extracting 1ml of water samples of the upper layer, the middle layer and the lower layer of the water environment from each parallel group by water pipes within 1h, 2h, 4h, 8h, 16h, 32h and 64h, wherein 1ml of the water sample of the upper layer of the water environment is regarded as a water sample, 1 water sample is diluted by 2-4 times, 100 mu L of the water sample is taken from 2-time dilution of 1 water sample, dropwise added and coated on a plate, standing is carried out for 5min, each water sample is made into 3 parallel samples, all the plates coated with the water samples are placed in an incubator at 35 ℃ for culturing for 20-24h, and the colony number of the Edwardsiella tarda on the plate is observed and recorded.

The colony counting result judgment standard is as follows: as above.

3. Test results

3.1 the bacteriostatic effect of the composite plant extract of the invention on different depths of the water environment with bacteria

As shown in Table 4, after the water environment with bacteria was successfully molded, the content of Edwardsiella tarda in the water environment at the upper layer, the middle layer and the lower layer of the water was kept increased at different time points in the C-series test group without any disinfectant, and the content of Edwardsiella tarda in the water environment at the upper layer, the middle layer and the lower layer was relatively close to that at different time points. Compared with a blank control group, the content of the Edwardsiella tarda in the water environment of the B-system test group added with the povidone iodine is obviously reduced at different time points in the water body, and the content of the Edwardsiella tarda in the water body of the B-system test group is relatively close to that of the water body of the middle layer and the lower layer, so that the 2ppm povidone iodine solution has a good inhibition effect on the Edwardsiella tarda in the water body, but still can not achieve the effect of completely inhibiting the Edwardsiella tarda, and the content of the Edwardsiella tarda in the water environment of the upper layer, the middle layer and the lower layer is increased to a certain extent compared with that of the Edwardsiella tarda in the first 16 hours in a period. The content of Edwardsiella tarda in the water bodies of the B-series test group added with the composite plant extract is extremely obviously reduced in the water bodies of the upper layer, the middle layer and the lower layer of the water environment at different time points; after the composite plant extract is used in the stage of 1-16h, the composite plant extract has a complete inhibition effect on Edwardsiella tarda in the upper and middle water bodies, has a certain inhibition effect on Edwardsiella tarda in the lower water body, and has an effect obviously superior to that of a B-series test group added with povidone iodine; in the stage of 32-64h, the content of Edwardsiella tarda in the upper, middle and lower water bodies of the water environment is slightly increased compared with that in the first 16 h.

In conclusion, the composite plant extract has obvious inhibition effect on Edwardsiella tarda in water bodies of different depths in water environment, has better inhibition effect on Edwardsiella tarda in upper and middle layers of water bodies, and is far superior to a povidone iodine test group with the concentration 15 times higher than that of the composite plant extract.

TABLE 4

3.2 bacteriostatic effect of the combined active substance of the composite plant extract on different depths of the water environment with bacteria

As shown in the following table 5, after the water environment with bacteria is successfully molded, the content of Edwardsiella tarda in the water environment at the upper layer, the middle layer and the lower layer of the water body is kept increasing at different time points by the C-series test group without adding any disinfectant, and the content quantity in the water body at the upper layer, the middle layer and the lower layer is relatively close to that at different time points. Compared with a blank control group, the D-series test group added with the composite plant extract compound polymer has the advantages that the content of Edwardsiella tarda in the water environment at the upper layer and the middle layer is reduced remarkably at different time points; after the composite plant extract is used, the composite plant extract has a complete inhibition effect on Edwardsiella tarda of upper and middle water bodies, and the inhibition effect on Edwardsiella tarda of lower water bodies is related to the settling position of a polymer, and the composite plant extract is worthy of pointing out that the composite plant extract is combined with the polymer, the initial first 2 hours can be reduced to 5 percent of the bacteria content level during molding and below, and when the polymer is completely settled to the bottom, the composite plant extract is combined with the polymer for 4-16 hours, so that the composite plant extract is represented as complete inhibition on Edwardsiella tarda of lower water; in the stage of 32-64h, the content of Edwardsiella tarda in the upper, middle and lower water bodies of the water environment is slightly increased compared with that in the first 16 h. Compared with a B-series test group added with the composite plant extract, the compound of the invention can enhance the complete inhibition effect of the composite of the invention on Edwardsiella tarda in the middle and lower water bodies.

In conclusion, the composite plant extract has an obvious inhibiting effect on Edwardsiella tarda in water bodies of different depths in a water environment, and the complete inhibiting effect and the continuous inhibiting time of the composite plant extract on the Edwardsiella tarda in the middle-layer and lower-layer water bodies can be enhanced by compounding the composite plant extract with a polymer.

TABLE 5

Experimental example 4 repairing effect of the composite plant extract of the invention on Edwardsiella tarda-derived body surface fading spots

1. Test materials

1.1 test yellow catfish: the specification is 20 +/-3 g/tail, no visible abnormal pathological symptoms exist on the body surface, and no visible abnormal pathological symptoms of internal organs are found by randomly sampling and dissecting pelteobagrus fulvidraco with the same fish school of 3 percent.

2. Test method

2.1 expanding culture of the strain: the same as in Experimental example 3.

2.2 construction of model for testing fish in vitro immersion infection

On the 1 st day of the experiment, 600 fish tails are randomly fished from raw fish to be used for the experiment, and the fish is determined to meet the use requirements of the experiment fish constructed by the infection model through detecting that no macroscopic abnormal pathological symptoms exist on the body surface, the oral cavity and the fin rays. The 600 fish test raw fish were randomly distributed evenly over 12 aquaria filled with 300L of tap water exposed to chlorine for 24h, each aquarium having 50 fish, the 12 aquaria being designated A1, A2, A3, B1, B2, B3, C1, C2, C3. D1, D2 and D3, standing for 12-18h, adding sodium dodecyl sulfate as a detergent into each aquarium until the final mass concentration is 0.2%, when test fish in each aquarium is soaked for 5 minutes, the denatured mucus on the surface of the test fish begins to locally fall off, changing all water in each aquarium into isothermal tap water of 24h of oxygenated chlorine, standing for 2-3h, and sequentially adding Edwardsiella tarda mother liquor into 12 aquariums until the final content of Edwardsiella tarda in each aquarium is 10⁴And (3) observing the quantity of red spots appearing on the body surface, the fin lines and the oral cavity of the raw fish in each aquarium in a cfu/L period of 36-48h, and if the ratio of the fish tail number of the red spots appearing on the body surface, the fin lines and the oral cavity of the raw fish in each aquarium is more than 10 percent, and the quantity of red spot focuses of the raw fish with the red spots appearing on the body surface, the fin lines and the oral cavity in each aquarium is not less than 2, determining that the in-vitro soaking infection model is successfully constructed.

During the test period, oxygen is continuously added to keep the water temperature of each aquarium at 28-30 ℃, the dissolved oxygen content of the water is not lower than 7mg/L, and no feed is added. Wherein A1, A2 and A3 are set as a series A test parallel group, B1, B2 and B3 are set as a series B test parallel group, C1, C2 and C3 are set as a series C test parallel group, and D1, D2 and D3 are set as a series D test parallel group.

2.3 repair action of Complex plant extract on Red spots caused by infection of live Fish in vitro

From 48h of the test, the composite plant extract of the invention was added to the A series (A1, A2, A3) and the B series (B1, B2, B3) in the order of final concentrations of 0.195ppm and 0.39ppm, povidone-iodine was added to the C series (C1, C2, C3) in the order of final concentration of 2ppm, and the D series (D1, D2, D3) were set as a blank control group and the corresponding aquarium was not added with any disinfectant. The test groups and the blank control group are kept for 12 hours without changing water, then all the water in each aquarium is changed into isothermal tap water of 24 hours of oxygenation and chlorination, whether the fish body discoloration spot area stops diffusing or not is observed for 48 hours, so that the repair effect of the composite plant extract on the discoloration spots caused by the external infection of the pelteobagrus fulvidraco is judged, and the effect judgment standard is as follows:

repairing the fading spots: by taking the pelteobagrus fulvidraco with a single tail fading spot, when the single tail fading spot of the pelteobagrus fulvidraco is completely normal, if the single tail fading spot of the pelteobagrus fulvidraco appears in a disinfectant treatment group, the added substances are regarded as having the effect of inhibiting the delayed Edwardsiella tarda infection and diffusion; if the fish body is found in the blank group, the fish body is regarded as self-healing.

Note: in the above formula, x represents A, B, C; the total amount of color fading spots of the yellow catfish; the initial number of faded spots represents the total number of faded spots of the group counted when the molding of the control group is successful;

3. test results

3.1 test results of the model for in vitro immersion infection of Fish

As shown in table 6 below, the proportions of the spots on the body surface of each group of tested pelteobagrus fulvidraco are respectively 15.3%, 16.7%, 16.0% and 14.7%, and the number of the focus of the spots on the body surface of each group of tested pelteobagrus fulvidraco with the spots on the body surface is more than or equal to 2. Therefore, the construction of the yellow catfish in-vitro soaking infection model in the test is successful, and the yellow catfish in-vitro soaking infection model can be used as a pathological model for evaluating the repairing effect of different substances on the fading spots in the next step.

TABLE 6

3.2 repair results of red spots caused by in vitro infection of various groups of pelteobagrus fulvidraco

As shown in table 7 below, under the same test conditions, after the self-molding was successful, it was observed after 48 hours: the blank control group does not use any disinfectant, and the test pelteobagrus fulvidraco body surface fading spot stove with fading spots presents obvious outward diffusion pathological change trend; the test pelteobagrus fulvidraco soaked in 2ppm of povidone iodine has the defect that the repair rate of the focus of the discoloring spots on the body surface of the test pelteobagrus fulvidraco is only 3.5%; the test pelteobagrus fulvidraco soaked in the composite plant extracts of 0.125ppm and 0.25ppm has the defect that the repair rate of the focus of the faded spots on the body surface of the test pelteobagrus fulvidraco with the faded spots is only 52.4 percent and 52.3 percent respectively; therefore, the test yellow catfish soaked in the composite plant extracts of 0.125ppm and 0.25ppm is soaked in the test yellow catfish, the repair degree of the test yellow catfish body surface fading spot diseases with fading spots is superior to that of a control group and a polyketone iodine group, and the repair rate of the fading spots is still less than 60%.

TABLE 7

Note: represents the self-healing rate of the fish body.

Experimental example 5 evaluation of safety of the composite plant extract of the present invention to aquatic animals

1. Test materials and methods

1.1 preparation of Complex plant extracts

The preparation method of the composite plant extract used in the experiment is the same as that of example 1.

1.2 temporary rearing and grouping of test fishes

The healthy yellow catfish 600 tails are selected and divided into 6 groups, each group has 100 tails, two parallel groups are arranged under each group, and 50 tails of each parallel group are randomly named as A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 and F2.

The method comprises the steps of selecting 600 tails of healthy channel catfishes, dividing the channel catfishes into 6 groups, 100 tails of each group, arranging two parallel groups below each group, and randomly naming 50 tails of each parallel group as H1, H2, J1, J2, K1, K2, L1, L2, M1, M2, N1 and N2.

Selecting 600 tails of healthy silurus asotus, dividing the healthy silurus asotus into 6 groups, 100 tails of each group, arranging two parallel groups under each group, and randomly naming 50 tails of each parallel group as P1, P2, Q1, Q2, R1, R2, S1, S2, T1, T2, V1 and V2.

1.3 preparation of gradient concentration of composite plant extract and safety evaluation

The compound plant extract of the invention is taken to be dissolved, and the corresponding test components are distributed as the following table 8 according to the final concentration requirements of 0, 0.625ppm, 1.25ppm, 2.5ppm, 5ppm and 10 ppm.

TABLE 8

Under three time dimensions of 0, 12h and 24h, survival conditions of test fishes in test water bodies with final concentrations of 0, 0.625ppm, 1.25ppm, 2.5ppm, 5ppm and 10ppm are respectively evaluated, and safety of the compound plant extract on different test fishes is evaluated through the following formula.

1.4 other consumables

A certain amount of distilled water and a proper amount of measuring cup.

2. Test results

As shown in the following table 9, the safety evaluation results at different time points show that the safety concentration of the composite plant extract on the yellow catfish, the channel catfish and the catfish is less than or equal to 2.5 mg/L. When the final concentration of the compound plant extract is 5-10mg/L, the safety of the compound plant extract to the yellow catfish is better than that to channel catfish and catfish.

TABLE 9

The results of the comprehensive test examples 1-5 show that the composite plant extract can effectively inhibit the abnormal propagation of Edwardsiella tarda in the aquaculture water body and reduce the risk of fish body infection; if the polymer with sedimentation function is compounded for use, the inhibition effect of the composite plant extract on the Edwardsiella tarda in the middle and lower water bodies can be further enhanced; meanwhile, the composite plant has about 50 percent of repairing effect on body surface fading spots caused by the infection of the Edwardsiella tarda, and the disinfection and bacteriostasis operation of a water body on the Edwardsiella tarda can be performed in the early stage of epidemic by utilizing the characteristic that the composite plant has low dose and quick effect, so that the risk of loss caused by body surface lesion caused by overhigh water content of the bacterium is reduced, and the application value is very clear.