CN112616862A

CN112616862A - Compound plant extract for inhibiting vibrio parahaemolyticus in water environment and preparation method and application thereof

Info

Publication number: CN112616862A
Application number: CN202110019291.9A
Authority: CN
Inventors: 黄锦炉
Original assignee: Qingyuan Haibei Biological Technology Co ltd
Current assignee: Qingyuan Haibei Biological Technology Co ltd
Priority date: 2021-01-07
Filing date: 2021-01-07
Publication date: 2021-04-09
Anticipated expiration: 2041-01-07
Also published as: CN112616862B

Abstract

The invention belongs to the technical field of water environment bacteriostat, and discloses a composite plant extract for vibrio parahaemolyticus in water environment, which is mainly prepared from the following components in percentage by mass: 35-55% of longzhu grass extract, 30-42% of roughhaired holly root extract, 5-12% of gynura bicolor flower extract and 5-12% of dandelion extract. 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 on shrimp bodies, has an obvious inhibition effect on vibrio parahaemolyticus when being splashed with water for use, is not interfered by water organic matters, can effectively inhibit the abnormal reproduction of vibrio parahaemolyticus in the culture of 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, and has good safety on pelteobagrus fulvidraco, channel catfish and catfish.

Description

Compound plant extract for inhibiting vibrio parahaemolyticus in water environment and preparation method and application thereof

Technical Field

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

Background

Vibrio parahaemolyticus is one of the members of pathogenic vibrio, gram-negative bacteria, Vibrio of Vibrionaceae, is mostly arc-shaped, rod-shaped or oval-shaped under an electron microscope, has flagella and capsules, has no spores, is widely distributed in salt-containing water bodies such as seawater and saline lakes, and the like, well grows on a culture medium with the salt content of 3 percent, does not grow when no salt exists, and is also called halophilic bacteria. The optimal growth temperature of the bacterium is 37 ℃, so summer is a season of diseases caused by the bacterium. Practice shows that the infection of vibrio parahaemolyticus in high-temperature seasons causes the jejunum and the jejunum of the prawns to cause forced harvest, and the method is one of the main reasons of the low-salinity mode prawn culture mode of the Kyobo soil pond and the high-salinity prawn culture mode of the coastal high-level pond. Therefore, the occurrence risk of the vibrio parahaemolyticus disease is reduced, and the success rate of healthy prawn culture is improved.

In production, regular use of a compliance disinfectant and use of a beneficial microbial ecological agent are common external means for reducing abnormal reproduction of vibrio parahaemolyticus in a water body, and oral antibiotics are common internal means for preventing and treating vibrio parahaemolyticus. However, for prawn culture modes with obvious salinity difference such as an earth pond and an elevated pond, the means of regularly using a compliant disinfectant and using a beneficial microbial ecological agent to prevent and control vibrio parahaemolyticus in a water body also has some disadvantages, such as the rise of drug resistance of vibrio parahaemolyticus, the problem of cost increase caused by the use of a large amount of a guaranteed effect, the problem of water body algal phase damage caused by the use of a large amount of a guaranteed effect, and the like, so that a component for inhibiting the vibrio parahaemolyticus in the water body with higher safety, effectiveness and lower cost is sought, and the prawn culture mode has higher health promotion economic value and social value.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks of the prior art, the present invention firstly provides a composite plant extract for inhibiting vibrio parahaemolyticus in an aqueous environment.

The second object of the present invention is to provide a method for preparing the above composite plant extract for inhibiting vibrio parahaemolyticus in an aqueous environment.

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

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

a composite plant extract for inhibiting Vibrio parahaemolyticus in water environment comprises, by weight, 35-55% Callicarpa Formosanae extract, 30-42% Prunus mume extract, 5-12% Aster Lasiocladi flower extract, and 5-12% herba Taraxaci extract.

Preferably, the composite plant extract for inhibiting the vibrio parahaemolyticus in the aqueous environment comprises, by weight, 45% of tequila extract, 38% of roughhaired holly root extract, 9% of bidens pilosa flower extract and 8% of dandelion extract.

Preferably, the calliopsis longituba extract is a fermentation concentrate after enzymolysis, the enzyme holly extract is a fermentation concentrate after pretreatment, the gynura bicolor flower extract is a fermentation concentrate after pretreatment, and the dandelion extract is a fermentation concentrate after enzymolysis.

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

s1, cutting the callicarpa bodinieri into short pieces, baking for 6-10h at the temperature of 45-50 ℃, sieving to obtain crushed materials, taking 100 parts of callicarpa bodinieri crushed materials according to the mass percentage, fully mixing with 1200 parts of 600-1200 parts of water, adding 5000-15000IU of cellulase and papain compound, stirring in a sealed and interval mode at the temperature of 35-40 ℃, and storing for 48-72 h;

s2, unsealing, adding 400 portions of water and 600 portions of spore bacteria liquid culture medium, adding 10-18 portions of spore bacteria (2-6) gamma 10⁵cfu, stirring in a closed and spaced mode, obtaining a culture when the OD value of the culture solution is 6.5-7.5, centrifuging the culture to remove residues, collecting supernate, and concentrating the collected supernate to 10% -30% of the original volume to obtain concentrated solution, namely the tequila extract.

Wherein the Callicarpa gigantea is root-containing whole plant of Gynura procumbens of Compositae, preferably root-containing whole plant of annual Callicarpa procumbens.

Wherein, the water in the large-dragon pearl grass extract is preferably sterile distilled water.

Wherein, the concentration adopts stirring-free ultrafiltration concentration, and 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 bacillus is preferably bacillus polymyxa, and other bacillus such as bacillus subtilis, bacillus licheniformis and the like cannot replace the functions and effects of the bacillus polymyxa.

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

As a preferred embodiment, the tequila extract is prepared by a method comprising the following steps: cutting the macrorrhiza japonica into short pieces, baking for 6-9h in an environment at 45-48 ℃, sieving by a 20-mesh sieve to obtain crushed materials, taking 100 parts of the macrorrhiza japonica crushed materials by mass percent, fully mixing with 1200 parts of water, then adding 7000-15000IU of cellulase and papain complex, stirring in a closed and interval manner in an environment at 36-40 ℃, storing for 52-72h, unsealing, adding 600 parts of 450-1200 parts of water, adding 10-16 parts of spore bacteria liquid culture medium, adding 10-16 parts of spore bacteria (3-6) 10 x⁵cfu, stirring under sealed and spaced conditions, collecting culture with OD of 6.8-7.5, centrifuging, removing residue, collecting supernatant, and placing the collected supernatant into a stirring-free ultrafiltration device under operating pressure of 0.45-0.5Mpa and membrane flux of 50-60L/m²H is a technical parameter, and when the supernatant is ultrafiltered and concentrated to 20-30% of the original volume, the obtained concentrated solution is the tequila extract.

As a most preferred embodiment, the tequila extract is prepared by a method comprising the following steps: cutting the macrorrhiza japonica into short pieces, baking for 8 hours in an environment of 47 ℃, sieving by a 20-mesh sieve to obtain crushed materials, taking 100 parts of the macrorrhiza japonica crushed materials by mass percent, fully mixing the crushed materials with 1000 parts of water, then adding 12000IU of cellulase and papain compound, stirring in a closed and interval mode in an environment of 38 ℃, storing for 68-72 hours, unsealing, adding 500 parts of water, 15 parts of spore-adding liquid culture medium, 5 gamma 10 of spore-adding bacteria⁵cfu, stirring under sealed and spaced conditions, collecting culture with OD of 7.2, centrifuging, removing residue, collecting supernatant, and loading into stirring-free ultrafiltration device with operating pressure of 0.45Mpa and membrane flux of ultrafiltration membraneAt 60L/m²H is a technical parameter, and when the supernatant is ultrafiltered and concentrated to 28% of the original volume, the obtained concentrated solution is the tequila extract.

The spore liquid culture medium can be a commercially available spore liquid culture medium, such as imported bacillus media base.

Wherein, the compound of the cellulase and the papain is characterized by (50-60 percent) by the active component percentage: (40% -50%)

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

s1, carrying out first-pass soaking on the roughhaired holly in 3.0-4.5mmol/L sodium hydroxide solution for 5-10 seconds, taking out, and drying at the ambient temperature of 30-35 ℃ to obtain a pretreated roughhaired holly slice;

s2, sieving the pretreated roughhaired holly slices to obtain roughhaired holly primary micro powder, mixing the roughhaired holly primary micro powder, water, a spore liquid culture medium and spore bacteria, carrying out closed culture at 35-40 ℃ until the OD value is 5.5-6.5 to obtain a culture, carrying out centrifugal deslagging, collecting supernatant, sterilizing, cooling, taking out, concentrating the supernatant subjected to high-temperature sterilization to 10% -25% of the original volume, and obtaining a concentrated solution, namely the roughhaired holly extract, wherein the roughhaired holly primary micro powder: water: the weight volume ratio of the spore bacteria liquid culture medium is 1: (6-12): (2-4), the addition amount of the spore bacteria is (3-8) x 10 added into each mass part of the rough powder of the holly⁶cfu。

Wherein, the dried stem of the roughhaired holly is preferably 2-2.5 m high, while the dried stem of the roughhaired holly can not be replaced by the root of the roughhaired holly.

The mixing of the roughhaired holly, the water, the spore bacteria liquid culture medium and the spore bacteria is not limited by the adding sequence, for example, the roughhaired holly and the spore liquid culture medium are mixed firstly, then the spore bacteria are added and mixed, and finally the water is added and mixed uniformly.

Further, the water in the above roughhaired holly extract is preferably sterile distilled water.

Further, the above-mentioned bacillus is preferably bacillus polymyxa, bacillus subtilis, bacillus licheniformis or the like which cannot functionally replace the bacillus polymyxa isolated in the present invention.

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

As a preferred embodiment, the roughhaired holly extract is prepared by a method comprising the following steps: cutting flos Ilicis Asprellae into slices with thickness of 2-3mm, placing in 3.5-4.5Mmol/L sodium hydroxide solution, performing first-pass soaking for 5-8 seconds, taking out the sliced flos Ilicis Asprellae, oven drying at 30-35 deg.C to obtain pretreated sliced flos Ilicis Asprellae, sieving the pretreated sliced flos Ilicis Asprellae with 20 mesh sieve to obtain primary micropowder, mixing the primary micropowder with spore liquid culture medium and spore bacteria, hermetically culturing at 35-40 deg.C until OD value is 5.8-6.5 to obtain culture, centrifuging at 25-28 deg.C to remove residue, collecting supernatant, packaging, sterilizing at 121 deg.C under high pressure for 15min, cooling, taking out, centrifuging to remove residue, then loading the collected supernatant into a stirring-free ultrafiltration device at the ambient temperature of 25-30 ℃, the operation pressure is 0.4-0.5 Mpa, and the membrane flux of the ultrafiltration membrane is 60L/m.²H is a technical parameter, and the obtained concentrated solution is concentrated to 18-20% of the original volume to obtain the concentrated solution, namely the roughhaired holly extract.

As a further preferred embodiment, the holly extract is prepared by a method comprising the steps of: slicing flos Ilicis Asprellae into slices with thickness of 2-3mm, subjecting to one-pass soaking in 3.8Mmol/L sodium hydroxide solution environment for 5-8 s, taking out, oven drying at 35 deg.C to obtain pretreated flos Ilicis Asprellae slices, sieving with 20 mesh sieve to obtain flos Ilicis Asprellae primary micropowder, mixing the flos Ilicis Asprellae primary micropowder with spore liquid culture medium and spore bacteria, culturing at 38 deg.C under sealed condition to OD value of 6.5 to obtain culture, centrifuging at 26 deg.C to remove residue, collecting supernatant, subpackaging, sterilizing at 121 deg.C under high pressure for 15min, cooling, taking out, centrifuging to remove residue, collecting supernatant at 28 deg.C under sealed conditionLoading the solution into a stirring-free ultrafiltration device with an operating pressure of 0.45Mpa and a membrane flux of 60L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 20% of the original volume to obtain the concentrated solution, namely the roughhaired holly extract.

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

s1, removing impurities and dust from the preprocessed gynura bicolor flowers, then carrying out quick-freezing, pre-puffing and vacuum deoiling treatment to obtain gynura bicolor flower dehydrated and dried substances, sieving and crushing the dried substances to obtain gynura bicolor flower crushed substances;

s2, crushing the gynura bicolor into powder: water: mixing a spore liquid culture medium and spore bacteria, carrying out closed culture at 35-40 ℃ until the OD value is 4.0-6.5 to obtain a culture, filtering and collecting the culture, centrifuging at 3000-plus 5000rpm for 20-30min, discarding the centrifuged supernatant, collecting the centrifuged sediment, adding water in an amount which is 6-10 times the mass of the centrifuged sediment to fully dissolve the sediment, subpackaging and sealing, carrying out autoclaving at 121 ℃ for 15min, cooling and taking out the sediment, centrifuging at 800-plus 1200rpm for 5-10min, removing residues to leave the centrifuged supernatant, concentrating the collected supernatant to 10-25% of the original volume, and obtaining a concentrated solution, namely the gynura bicolor extract. Wherein, the crushed material of the gynura bicolor: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: (6-12): (2-4) mixing, wherein the addition amount of the spore bacteria is (3-8) gamma 10 added into each part by mass of the gynura bicolor pollen crushed material⁶cfu。

Wherein, the gynura bicolor flower is preferably fresh gynura bicolor petals, which means that the fresh gynura bicolor flower is removed, and the flower receptacle, the flower crown, the calyx and the remaining petal part after the flower receptacle are removed.

The operation steps of the pretreated gynura bicolor flower are as follows, preferably, fresh gynura bicolor petals are picked, soaked in 1.2-1.5Mmol/L ammonia water solution with the mass volume ratio of 2-3 times, taken out at the ambient temperature of 25-30 ℃ for 24-36h, and drained, and the water content of the pretreated gynura bicolor flower is 8% -10%.

The mixing of the pretreated gynura bicolor flower, the water, the spore liquid culture medium and the spore bacteria is not limited by the adding sequence, for example, the pretreated gynura bicolor flower and the spore liquid culture medium are mixed firstly, then the spore bacteria are added and mixed, and finally the water is added and mixed uniformly.

Further, the quick-freezing is preferably carried out by quickly freezing the pretreated gynura bicolor flower at-20 to-40 ℃ for 10 to 12 hours.

Further, the pre-puffing is to fry and puff the quick-frozen pre-treated gynura bicolor flowers for 3-6 min under the conditions of vacuum degree of 90-95 KPa and temperature of 70-80 ℃.

Further, the vacuum deoiling is to place the pre-treated gynura bicolor flowers after being fried at the rotating speed of 150-250 r/min for deoiling for 6-15 min.

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

Further, the above-mentioned Bacillus is preferably Bacillus curvatus, Bacillus subtilis, Bacillus licheniformis or the like which cannot functionally replace the Bacillus curvatus isolated in the present invention.

As a preferred embodiment, the gynura bicolor flower extract is prepared by a method comprising the following steps: removing impurities and dust from the pretreated gynura bicolor flower, quickly freezing, pre-puffing and performing vacuum deoiling treatment to obtain a gynura bicolor flower dehydrated dry substance, sieving the dry substance with a 50-60-mesh sieve for crushing to obtain gynura bicolor pollen crushed substance, and crushing the gynura bicolor pollen crushed 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 22-28min, discarding supernatant, collecting centrifugal sediment, dissolving in 7-10 times of the centrifugal sediment in water, packaging, sealing, autoclaving at 121 deg.C for 15min, cooling, taking out, centrifuging at 800-1100rpm for 7-10min, removing residue to obtain supernatant, adding the collected supernatant into non-stirring type ultra-high pressure mediumConcentrating the extract to 15-25% of the original volume by using a filter device to obtain a concentrated solution, namely the gynura bicolor flower extract. Wherein, the crushed material of the gynura bicolor: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: (8-12): (3-4) mixing, wherein the addition amount of the spore bacteria is (5-8) gamma 10 added into each mass part of the gynura bicolor pollen crushed material⁶cfu。

As a more preferred embodiment, the gynura bicolor flower extract is prepared by a method comprising the following steps: removing impurities and dust from the pretreated gynura bicolor flower, quickly freezing, pre-puffing and performing vacuum deoiling treatment to obtain a gynura bicolor flower dehydrated dry substance, sieving the dry substance with a 50-mesh sieve for crushing to obtain gynura bicolor pollen crushed substance, and crushing the gynura bicolor pollen crushed substance: water: mixing a spore liquid culture medium and spore bacteria, carrying out closed culture at 38 ℃ until the OD value is 6.3 to obtain a culture, filtering and collecting the culture at 26 ℃, centrifuging at 5000rpm for 25min, discarding the centrifuged supernatant, collecting the centrifuged sediment, adding 9 times of the mass of the centrifuged sediment into water to fully dissolve the sediment, subpackaging and sealing, carrying out autoclaving at 121 ℃ for 15min, cooling, taking out the cooled sediment, centrifuging at 1000rpm for 8min, removing residues to obtain the centrifuged supernatant, adding the collected supernatant into a stirring-free ultrafiltration device, and concentrating the supernatant to 20% of the original volume to obtain a concentrated solution, namely the gynura bicolor extract. Wherein, the crushed material of the gynura bicolor: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 10: 3.5, the adding amount of the spore bacteria is 6.5 gamma 10 added into each mass part of the mallotus philippinensis crushed powder⁶cfu。

Wherein, the quick-freezing is preferably carried out by quickly freezing the gynura bicolor flowers at-30 ℃ for 12 h.

Wherein the pre-puffing is to fry and puff the quick-frozen gynura bicolor flowers for 5min under the conditions of vacuum degree of 90KPa and temperature of 80 ℃.

Wherein the vacuum deoiling is to deoil the fried gynura bicolor flower for 6min under the condition of the rotating speed of 200 r/min.

Wherein the water in the above-mentioned Echinacea purpurea extract is preferably sterile distilled water.

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

s1, taking 100 parts of dandelion, drying until the water content is less than or equal to 4%, frying with slow fire for 30-60min, sieving with a 20-mesh sieve to obtain dandelion primary micro powder, fully mixing the dandelion primary micro powder with 800 parts of 600 plus materials of water, then adding 9000IU cellulose of 5000 plus materials, stirring at intervals in an enclosed manner at the temperature of 35-40 ℃, and storing for 48-72 h;

s2, unsealing, adding 400 portions of water and 500 portions of spore bacteria liquid culture medium 10-18 portions of spore bacteria (2-6) gamma 10⁵cfu, stirring in a closed and interval manner, obtaining a culture when the OD value of the culture solution is 6.2-7.2, centrifuging the culture to remove residues, collecting supernate, and concentrating the collected supernate to 10% -30% of the original volume to obtain concentrated solution, namely the dandelion extract.

Among them, the dandelion is preferably a non-root-containing dandelion plant in a non-flowering period of one year.

Among them, the cellulase is preferably β -1, 4-glucan-4-glucan hydrolase.

Wherein the water in the herba Taraxaci extract is preferably sterile distilled water.

Furthermore, the above-mentioned bacillus is preferably bacillus polymyxa, and other bacillus such as bacillus subtilis, bacillus licheniformis and the like cannot replace the function and function of the bacillus polymyxa isolate of the invention.

As a preferred embodiment, the dandelion extract is prepared by a method comprising the following steps: taking 100 parts by mass of dandelion, placing the dandelion in an environment with the temperature of 35-45 ℃ for drying until the water content is less than or equal to 4%, frying the dandelion for 30-60min by slow fire, passing the dandelion through a 20-mesh automatic grinding machine to obtain the dandelion primary micro powder, fully mixing the dandelion primary micro powder with 800 parts of 600-plus-one water, and then adding 5000-plus-one waterUnder 35-40 deg.C, stirring in sealed and spaced manner, storing for 48-72h, unsealing, adding 400-500 parts of water, 15-18 parts of spore-adding liquid culture medium, and spore-adding bacteria (4-6) gamma 10⁵cfu, stirring in a closed and spaced mode, obtaining a culture when the OD value of a culture solution is 6.8-7.0, centrifuging the culture to remove residues, then loading collected supernate into a stirring-free ultrafiltration device at the ambient temperature of 26-30 ℃, wherein the operation pressure is 0.4-0.5 Mpa, and the membrane flux of the ultrafiltration membrane is 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 dandelion extract.

As a more preferred embodiment, the dandelion extract is prepared by a method comprising the following steps: 100 parts of dandelion are taken according to the mass part, the dandelion is placed in an environment with the temperature of 35-45 ℃ to be dried until the water content is less than or equal to 4 percent, the dandelion is fried for 40min by slow fire, the dandelion primary micro powder is obtained by a 20-mesh automatic grinding machine, the dandelion primary micro powder is fully mixed with 800 parts of water, 7000IU cellulase is added, the dandelion primary micro powder is stirred in a closed and interval mode in an environment with the temperature of 36 ℃, the dandelion primary micro powder is stored for 52h, the dandelion is unsealed, 450 parts of water is added, 16 parts of spore added bacteria liquid culture medium is added⁵cfu, stirring under sealed and spaced conditions, collecting culture solution OD 7.0 to obtain culture, centrifuging to remove residue, and loading collected supernatant into stirring-free ultrafiltration device at ambient temperature of 28 deg.C under operating pressure of 0.45Mpa and membrane flux of 60L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 20% of the original volume, and the obtained concentrated solution is the dandelion extract.

The invention also provides a preparation method of the composite plant extract, which is characterized in that the components are mixed according to the proportion at the temperature of 25-30 ℃, the ultrasonic emulsification is uniform, the obtained mixed solution is the composite plant extract, and preferably, the composite plant extract is mixed at the temperature of 28 ℃.

The invention also provides the use of the composite plant extract for inhibiting nutritionAbnormal reproduction of Vibrio parahaemolyticus in the upper, middle and bottom layers of the aquatic water body. When the content of the vibrio parahaemolyticus in the culture water body is less than or equal to 10³cfu/mL, the invention is used for splashing water for 50mL per mu per meter, and has good inhibition effect on vibrio parahaemolyticus in the culture water body within 4-8 h; when the content of the vibrio parahaemolyticus in the culture water body is 10⁵-10⁶cfu/mL, the invention is used for splashing 100mL of chemical water according to the volume of each mu per meter, and has good inhibition effect on vibrio parahaemolyticus in the culture water body within 4-8 h.

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

the invention provides a composite plant extract for vibrio parahaemolyticus in water environment, which has the advantages that firstly, the composite plant extract has a special killing effect on the vibrio parahaemolyticus, and meanwhile, the safety concentration range of shrimp bodies is larger, and algae in water bodies are not damaged.

Secondly, under the same dosage, the composite plant extract has no obvious inhibition effect on Vibrio harveyi, Vibrio alginolyticus and the like which are the same genus of Vibrio, and has no effect on a plurality of members of the aeromonas of the enterobacteriaceae family of shrimp source.

Finally, the composite plant extract of the invention is composed of a callicarpa bodinieri extract, a holly extract, a gynura bicolor flower extract and a dandelion extract, and the four components have obvious compatibility and synergism and have good inhibition effect on vibrio parahaemolyticus in the culture water body.

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 vibrio parahaemolyticus in an aqueous environment is prepared by weighing 45% of longzhu grass extract, 38% of roughhaired holly extract, 9% of gynura bicolor flower extract and 8% of dandelion extract in percentage by weight. Mixing the above components at 28 deg.C, and performing conventional ultrasonic emulsification to obtain mixed solution as the composite plant extract.

Wherein, the tequila extract is obtained by the following method: cutting the macrorrhiza japonica into short pieces, baking for 8 hours in an environment of 47 ℃, sieving by a 20-mesh sieve to obtain crushed materials, taking 100 parts of the macrorrhiza japonica crushed materials by mass percent, fully mixing the crushed materials with 1000 parts of water, then adding 12000IU of cellulase and papain compound, stirring in a closed and interval mode in an environment of 38 ℃, storing for 68-72 hours, unsealing, adding 500 parts of water, 15 parts of spore-adding liquid culture medium, 5 gamma 10 of spore-adding bacteria⁵cfu, stirring under sealed and spaced conditions, collecting culture with OD value of 7.2, centrifuging, removing residue, collecting supernatant, and placing the collected supernatant into a stirring-free ultrafiltration device with operating pressure of 0.45Mpa and membrane flux of 60L/m²H is a technical parameter, and when the supernatant is ultrafiltered and concentrated to 28% of the original volume, the obtained concentrated solution is the tequila extract.

Wherein the holly extract is prepared by the method comprising the following steps: slicing flos Ilicis Asprellae into slices with thickness of 2-3mm, subjecting to one-time filtration soaking in 3.8mmol/L sodium hydroxide solution environment for 5-8 s, taking out the flos Ilicis Asprellae slices, oven drying at 35 deg.C to obtain pretreated flos Ilicis Asprellae slices, sieving with 20 mesh sieve to obtain flos Ilicis Asprellae primary micropowder, mixing the flos Ilicis Asprellae primary micropowder with spore liquid culture medium and Bacillus polymyxa, culturing at 38 deg.C under sealed condition to OD value of 6.5 to obtain culture, centrifuging at 26 deg.C to remove residue, collecting supernatant, sub-packaging under sealed condition at 121 deg.C, autoclaving for 15min, cooling, taking out, centrifuging to remove residue, and loading the collected supernatant into stirring-free ultrafiltration device at 28 deg.C to operate pressure of 0.M 45, and membrane flux of ultrafiltration membrane of 60L/m²H is a technical parameter, obtainedConcentrating to obtain a concentrated solution with the original volume of 20%, and obtaining the concentrated solution which is the holly root extract. Wherein, the roughhaired holly primary micro powder: water: the weight volume ratio of the spore bacteria liquid culture medium is 1: 8: 3, the addition amount of the bacillus polymyxa is that 5 x 10 is added into each mass part of the rough powder of the holly⁶cfu。

The gynura bicolor flower extract is prepared by a method comprising the following steps: removing impurities and dust from the pretreated gynura bicolor flower, quickly freezing, pre-puffing and performing vacuum deoiling treatment to obtain a gynura bicolor flower dehydrated dry substance, sieving the dry substance with a 50-mesh sieve for crushing to obtain gynura bicolor pollen crushed substance, and crushing the gynura bicolor pollen crushed substance: water: mixing a spore liquid culture medium and spore bacteria, carrying out closed culture at 38 ℃ until the OD value is 6.3 to obtain a culture, filtering and collecting the culture at 26 ℃, centrifuging at 5000rpm for 25min, discarding the centrifuged supernatant, collecting the centrifuged sediment, adding 9 times of the mass of the centrifuged sediment into water to fully dissolve the sediment, subpackaging and sealing, carrying out autoclaving at 121 ℃ for 15min, cooling, taking out the cooled sediment, centrifuging at 1000rpm for 8min, removing residues to obtain the centrifuged supernatant, adding the collected supernatant into a stirring-free ultrafiltration device, and concentrating the supernatant to 20% of the original volume to obtain a concentrated solution, namely the gynura bicolor extract. Wherein, the crushed material of the gynura bicolor: water: spore liquid culture medium is prepared by mixing the following components in a weight-volume ratio of 1: 10: 3.5, the adding amount of the spore bacteria is 6.5 gamma 10 added into each mass part of the mallotus philippinensis crushed powder⁶cfu。

Wherein the dandelion extract is prepared by a method comprising the following steps: 100 parts of dandelion are taken according to the mass part, the dandelion is placed in an environment with the temperature of 35-45 ℃ to be dried until the water content is less than or equal to 4 percent, the dandelion is fried for 40min by slow fire, the dandelion primary micro powder is obtained by a 20-mesh automatic grinding machine, the dandelion primary micro powder is fully mixed with 800 parts of water, 7000IU cellulase is added, the dandelion primary micro powder is stirred in a closed and interval mode in an environment with the temperature of 36 ℃, the dandelion primary micro powder is stored for 52h, the dandelion is unsealed, 450 parts of water is added, 16 parts of spore added bacteria liquid culture medium is added⁵cfu, stirring under sealed and intermittent conditions, collecting culture solution OD 7.0 to obtain culture, centrifuging to remove residue, collecting supernatant, placing into stirring-free ultrafiltration device at ambient temperature of 28 deg.C under operating pressure of 0.45Mpa,the membrane flux of the ultrafiltration membrane is 60L/m²H is a technical parameter, and the obtained concentrated solution is concentrated to 20% of the original volume, and the obtained concentrated solution is the dandelion extract.

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

Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Aeromonas hydrophila: all provided by the subject group.

Potassium hydrogen persulfate: the content of potassium hydrogen persulfate is 50 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

Inoculating Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio harveyi into TSB nutrient broth containing 0.5% sodium chloride, culturing at 28 deg.C for 16-20h, centrifuging to collect thallus, turbidifying the thallus with turbidimetric tube to estimate the concentration of the thallus, diluting the thallus with PBS to 1 × 10⁴～5×10⁴cfu/mL of bacterial suspension.

Inoculating Aeromonas hydrophila into TSB nutrient broth, culturing at 28 deg.C for 16-20h, centrifuging to collect thallus, turbidifying the thallus with McLeod turbidimetric tube to estimate the concentration of thallus, and diluting the thallus 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 was placed in a 20 + -2 deg.C water bath for 10min, 0.5mL of each reaction solution was transferred to 4.5mL of fresh broth, and cultured at 28 deg.C for 24h, and the results were 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 the following table show that the MIC of the composite plant extract to Vibrio parahaemolyticus is 0.125mg/L, the MIC is far lower than the MIC of oxone to Vibrio parahaemolyticus by 2mg/L, and the MIC to Vibrio alginolyticus, Vibrio harveyi and Aeromonas hydrophila is 2mg/L, 1mg/L and 1mg/L respectively. Therefore, the composite plant extract has good inhibition effect on vibrio parahaemolyticus, and the inhibition effect on vibrio alginolyticus, vibrio harveyi and aeromonas hydrophila is weaker than that of potassium hydrogen persulfate.

TABLE 1

The experimental data in Table 2 show that the sterilization rate of the composite plant extract to Vibrio parahaemolyticus is over 100% when the concentration of the composite plant extract is 0.25mg/L, the sterilization rate to Vibrio parahaemolyticus is still 100% when the concentration of the composite plant extract is 0.125mg/L, and the sterilization rate to Vibrio parahaemolyticus is over 70% when the concentration of the composite plant extract is 0.0625 mg/L; on the other hand, when the concentration of the composite plant extract is 1ppm, the sterilization rate of the composite plant extract on Vibrio alginolyticus, Vibrio harveyi and Aeromonas hydrophila is 0.

TABLE 2

Experimental example 2 inhibition of Vibrio parahaemolyticus by composite plant extract formulation compatibility of the present invention

1. Test materials

1.1 preparation of Complex plant extracts

The compound plant extract of the present invention used in this experiment was named Y and its preparation method was the same 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, the technical idea of single-component deletion or simultaneous deletion of two components or simultaneous deletion of three components and equivalent substitution of the deleted components by PBS is adopted to prepare the composite plant extract with single deletion of the callicarpa bodinieri extract, the composite plant extract with single deletion of the holly extract, the composite plant extract with single deletion of the gynura bicolor extract and the holy flower extract, the composite plant extract with simultaneous deletion of the callicarpa bodinieri extract and the dandelion extract, the composite plant extract with simultaneous deletion of the gynura bicolor extract and the gynura bicolor flower extract, the composite plant extract with simultaneous deletion of the gynura bicolor extract and the, The composite plant extract containing the gynura bicolor flower extract and the dandelion extract which are simultaneously deleted is respectively prepared into a composite plant extract only containing a callicarpa bodinieri extract, a composite plant extract only containing a holly flower extract, a composite plant extract only containing the gynura bicolor flower extract and a composite plant extract only containing the dandelion extract, and the prepared composite plant extracts are sequentially named as A, B, C, D, E, F, G, H, K, L, M, N, P, Q.

2. Test method

2.1 preparation of bacterial suspension

Inoculating Vibrio parahaemolyticus in TSB nutrient broth containing 0.5% sodium chloride, culturing at 28 deg.C for 16-20h, centrifuging to collect thallus, turbidifying the thallus with turbidimetric tube to estimate the thallus concentration, and diluting the thallus with PBS to 1 × 10⁴～5×10⁴cfu/mL of bacterial suspension.

2.2 preparation of Compound plant extract

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

2.3MIC determination and result determination

3. Test results

The test data in Table 3 show that the MIC of the composite plant extract to Vibrio parahaemolyticus is 0.125mg/L, the MIC of oxone to Vibrio parahaemolyticus is 2mg/L, and the MICs of the individual components of the callicarpa macrogola extract, the Ilicis Asprella extract, the Aster Lasiocladi flower extract and the dandelion extract to Vibrio parahaemolyticus are much higher than the MIC of the composite plant extract of the invention, which indicates that when the callicarpa macrogola extract, the Ilicis Asprella extract, the Aster Lasiocladi flower extract and the dandelion extract exist as single components, the inhibition effect to Vibrio parahaemolyticus is seriously weakened compared with. When the dandelion extract in the composite plant extract is singly absent, the minimum MIC of the dandelion extract to the vibrio parahaemolyticus is 0.5mg/L, and the dandelion extract is obviously superior to the MIC of the dandelion extract to the vibrio parahaemolyticus when the longzhu extract, the holly extract and the gynura bicolor flower extract are singly absent respectively. When the two components of the composite plant extract are simultaneously deleted, the MIC of the vibrio parahaemolyticus in the test group containing the dandelion extract which is simultaneously deleted is higher than that in the test group containing the large dragon grass extract, the holly root extract and the gynura bicolor flower extract which is simultaneously deleted. Therefore, as the composite plant extract with good inhibition effect on vibrio parahaemolyticus, the callicarpa bodinieri extract, the holly extract and the gynura bicolor flower extract are common main components of the composite plant extract with the effect of inhibiting vibrio parahaemolyticus, and the dandelion extract is an auxiliary synergistic component of the composite plant extract for inhibiting vibrio parahaemolyticus of the other three extract components.

TABLE 3

Experimental example 3 inhibitory effect of the composite plant extract of the present invention on Vibrio parahaemolyticus in water bodies of different depths

1. Test water source

Chlorine was exposed for 24h of tap water.

2. Test method

2.1 expanding culture of strains

Selecting Vibrio parahaemolyticus colony from inclined surface of seed batch, inoculating in TSB nutrient broth containing 0.5% sodium chloride, shake-culturing at 28 deg.C for 20-24h, centrifuging to collect thallus, turbidifying the thallus with turbidimetric tube to estimate the thallus concentration, diluting the thallus with PBS to 10¹¹cfu/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 vibrio parahaemolyticus suspension into each test parallel group at the environmental temperature of 25-28 ℃ until the vibrio parahaemolyticus content of each water body of A, B, C, D test parallel groups 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 tank 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 potassium persulfate into the B series test parallel group until the final concentration is 2ppm, and setting the C series test parallel group as a blank control group without adding any substance.

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 and is dripped 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 28 ℃ for culturing for 20-24h, and the colony number of vibrio parahaemolyticus 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.

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 model formation in the water environment with bacteria was successful, the content of Vibrio parahaemolyticus in the water environment at the upper layer, the middle layer and the lower layer at different time points in the water environment was kept increased, and the content of Vibrio parahaemolyticus in the water environment at the upper layer, the middle layer and the lower layer at different time points was relatively close. Compared with a blank control group, the B-series test group added with potassium persulfate has the advantages that the content of vibrio parahaemolyticus in the water bodies of the upper layer, the middle layer and the lower layer of the water body at different time points is obviously reduced, and the content of the vibrio parahaemolyticus in the water bodies of different water layers is relatively close, so that the potassium persulfate solution with the concentration of 2ppm has a good inhibition effect on the vibrio parahaemolyticus in the water body, but the effect of completely inhibiting the vibrio parahaemolyticus cannot be achieved, and the content of the vibrio parahaemolyticus in the water bodies of the upper layer, the middle layer and the lower layer of the water body is increased to a certain extent compared with the content of the vibrio parahaemo. The content of vibrio parahaemolyticus in the water body of the B-series test group added with the composite plant extract is extremely obviously reduced in the water body 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, the composite plant extract has complete inhibition effect on vibrio parahaemolyticus in upper and middle water bodies and certain inhibition effect on vibrio parahaemolyticus in lower water bodies, and the effect is obviously superior to that of a B-series test group added with potassium persulfate; in the stage of 32-64h, the content of vibrio parahaemolyticus in the upper, middle and lower water bodies in the water environment is only slightly increased compared with that in the first 16 h.

In conclusion, the composite plant extract has obvious inhibition effect on vibrio parahaemolyticus in water bodies of different depths in water environment, has better inhibition effect on vibrio parahaemolyticus in upper and middle water bodies, is far better than a potassium persulfate test group with concentration 15 times higher than that of the composite plant extract, and has an inhibition effect on vibrio parahaemolyticus in upper and middle water bodies within 1-64h after use, which is still obviously estimated in a blank control group and a potassium hydrogen persulfate treatment group.

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 model formation in the water environment with bacteria is successful, the content of vibrio parahaemolyticus in the water environment at the upper layer, the middle layer and the lower layer of the water body is kept to be increased at different time points in the C-series test group without any disinfectant, and the content of the vibrio parahaemolyticus in the water environment at the upper layer, the middle layer and the lower layer of the water body at different time points is relatively close to that of the water body at. 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 vibrio parahaemolyticus in the water body at the upper layer and the middle layer of the water body at different time points is reduced remarkably; wherein, in the stage of 1-16h, after the composite plant extract is used, the composite plant extract has 100% inhibition effect on vibrio parahaemolyticus in water bodies of an upper layer, a middle layer and a lower layer (except for a sampling point of 1 h), and is worthy of pointing out that the composite plant extract is combined with polymers, the initial first 2h can reduce the content level of 0.2% and below of the vibrio parahaemolyticus when the vibrio parahaemolyticus is molded, and when the polymerization is completely settled to the bottom, in the stage of 4-16h, the composite plant extract is combined with polymers, the composite plant extract is expressed as having 100% inhibition effect on the vibrio parahaemolyticus in the lower layer water; in the stage of 32-64h, the content of vibrio parahaemolyticus in the upper, middle and lower water bodies in the water environment is only 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 on the vibrio parahaemolyticus in the upper, middle and lower water bodies.

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

TABLE 5

Experimental example 4 repair of Vibrio parahaemolyticus-derived Red feet by the Compound plant extract of the present invention

1. Test materials

1.1 test strains

Vibrio parahaemolyticus: all provided by the subject group.

1.2 test consumables

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

Potassium hydrogen persulfate: the content of the potassium hydrogen persulfate is 50 percent.

Test south american white control: the standard is 500 plus or minus 5 counts/jin, the appearance has no any visible abnormal pathological symptoms, and the random spot examination and dissection of the white shrimps with 3 percent of the same batch of shrimp groups can not find the phenomenon of the infection of the hepatopancreatic vibrio parahaemolyticus.

1.3 preparation of Complex plant extracts

2. Test method

2.1 expanding culture of strains

Selecting a vibrio parahaemolyticus colony from a seed batch inclined plane, inoculating the vibrio parahaemolyticus colony in TSB nutrient broth containing 0.5% of sodium chloride, carrying out shake cultivation for 20-24h at 28 ℃, centrifugally collecting thalli, carrying out turbidimetry on the bacteria liquid through a turbidimetric tube, and estimating the bacteria concentration, diluting the thalli to 10 by PBS¹¹cfu/mL of bacterial suspension as mother liquor.

2.2 construction of model for testing fish in vitro immersion infection

On the 1 st day of the test, 600 shrimps are randomly fished from the white shrimps to be used for the test, and no macroscopic abnormal pathological symptoms exist through detecting carapace, feet, tentacle and the like, so that the use requirements of the test white shrimps constructed by the infection model are met. Evenly distributing 600 test white shrimps into 12 aquariums pre-filled with 300L of tap water with 24h of chlorine exposure, putting 50 white shrimps into each aquarium, respectively naming the 12 aquariums as A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2 and D3, and statically culturing for 12-18h, and sequentially adding Vibrio parahaemolyticus mother liquor into 9 aquariums until the final content of Vibrio parahaemolyticus in each aquarium is 10⁴And (3) observing the number of red feet of the white shrimps in each aquarium in a cfu/L period of 36-48h, and if the proportion of the shrimp body number of the red feet of the white shrimps in each aquarium is more than 10 percent and the number of the red feet of the white shrimps 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 Compound plant extract on Red feet caused by infection of raw Fish in vitro

From 48h of the test, the composite plant extract of the invention was added to the A series (A1, A2, A3), the B series (B1, B2, B3) and the C series (C1, C2, C3) in the order of final concentration of 0.125ppm and 0.25ppm, and oxone was added to the D series (D1, D2, D3) in the order of final concentration of 2ppm, and the corresponding aquaria were 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 with 24 hours of chlorine exposure, whether the red feet of the white shrimps disappear or are subjected to infectious diffusion is observed for 48 hours, so that the repairing effect of the composite plant extract on the red feet caused by the external infection of the white shrimps is judged, and the effect judgment standard is as follows:

repairing red feet: counting the single white shrimps with red feet, when the focus symptoms of the red feet symptoms of the single white shrimps are completely disappeared, if the focus symptoms appear in the disinfectant treatment group, the added substances are considered to have the repairing effect of repairing the side hemolytic vibrio red feet of the white shrimps; if the white shrimp appears in the blank group, the white shrimp is considered to have self-healing effect.

Note 1: in the above formula, x represents A, B, C; the number of repairs represents the total number of red leg repairs for the group of white shrimps; the initial number of red feet represents the total number of red feet present in the group counted when the control group was successfully molded.

Note 2: the number of red legs counted in each test white shrimp is 6.

3. Test results

3.1 test results of the model for infection by external soaking of white shrimps

As shown in table 6 below, the red-toe occurrence ratios of the test white shrimps in each group were 21.3%, 20.7% and 20.7%, respectively, and the red-toe number of the test white shrimps in each group was more than 2. Therefore, the test proves that the in-vitro white shrimp soaking infection model is successfully constructed and can be used as a pathological model for evaluating the red foot repairing effect of different substances in the next step.

TABLE 6

3.2 repair of Red feet caused by in vitro infection of various groups of white shrimps

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 disease symptoms of the red feet of the test white shrimps with red feet do not disappear and tend to spread infection; when the test white shrimps soaked in 2ppm potassium hydrogen persulfate are soaked, 16.6 percent of the red-foot disease focus symptoms of the test white shrimps with red feet disappear and are repaired; when the test white shrimps soaked in the composite plant extract of 0.125ppm are soaked, 94.4 percent of the test white shrimps with red feet have 94.4 percent of symptoms of the red feet focus disappearance and are repaired; the test white shrimps soaked in 0.25ppm of the composite plant extract showed 100% of the symptoms of red-foot lesions of the test white shrimps which showed red feet disappeared and were repaired.

In conclusion, compared with the blank group and the 2ppm potassium hydrogen persulfate soaking group, the compound plant extract has the advantage of extremely obvious repair effect on the red leg of the prawn caused by the in vitro infection of the vibrio parahaemolyticus. The red spot repair rate of the compound plant extract can reach 94.4 percent by using 83 milliliters of the compound plant extract according to the water depth of 1 mu per meter and by using the dosage of 0.125ppm to 94.4 percent of the red spot repair rate; and the use dosage of 0.25ppm for the red foot repair rate of 100 percent, according to the water depth of 1 mu per meter, 167 milliliters of the composite plant extract can achieve the effect of the red foot repair rate of 100 percent.

TABLE 7

Note: represents the self-healing rate of shrimp bodies.

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

1.2 temporary rearing and grouping of test fishes

Selecting 600 healthy white shrimps, dividing the white shrimps into 6 groups, dividing each group into 100 groups, arranging two parallel groups under each group, and randomly naming 50 groups of the two parallel groups as A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1 and F2.

Selecting 600 healthy penaeus diamondra, dividing the penaeus diamondra into 6 groups, 100 groups, arranging two parallel groups under each group, and 50 groups under each parallel group, wherein the groups are randomly named as H1, H2, J1, J2, K1, K2, L1, L2, M1, M2, N1 and N2.

Selecting 600 healthy prawns and dividing the prawns into 6 groups, 100 groups, two parallel groups under each group, and 50 groups under each parallel group, wherein the groups are randomly named 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 hour, 12 hours and 24 hours, the survival conditions of the test prawns in the test water bodies with the final concentrations of 0ppm, 0.625ppm, 1.25ppm, 2.5ppm, 5ppm and 10ppm are respectively evaluated, and the safety of the compound plant extract on different test prawns 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 Table 9 below, the safety evaluation results at various time points showed that the composite plant extracts had a safe concentration of 5mg/L or less for Penaeus vannamei, Pandalus amansi and Penaeus sodalifolius. When the final concentration is 10mg/L, the safety of the compound plant extract to white shrimp is similar to that of the amantadine and the grass shrimp.

TABLE 9

The results of the comprehensive test examples 1-5 show that the composite plant extract has good application prospects of repair and treatment on vibrio parahaemolyticus in the aquaculture water body of the white shrimps and red feet of the white shrimps infected by the vibrio parahaemolyticus under a proper dosage, and can enhance the inhibition effect of the composite plant extract on the vibrio parahaemolyticus in the middle-layer water body and the lower-layer water body by compounding the polymer with a sedimentation effect.

Claims

1. A composite plant extract for inhibiting vibrio parahaemolyticus in water environment is characterized by comprising, by weight, 35-55% of tequila extract, 30-42% of roughhaired holly extract, 5-12% of bidens pilosa flower extract and 5-12% of dandelion extract.

2. The composite plant extract for inhibiting vibrio parahaemolyticus in water environment as claimed in claim 1, comprising 45% of tequila extract, 38% of roughhaired holly extract, 9% of bidens pilosa extract and 8% of dandelion extract in percentage by weight.

3. The composite plant extract for inhibiting vibrio parahaemolyticus in aqueous environment according to claim 2, wherein the tequila extract is a fermentation concentrate after enzymolysis, the enzyme hillock extract is a fermentation concentrate after pretreatment, the gynura bicolor extract is a fermentation concentrate after pretreatment, and the dandelion extract is a fermentation concentrate after enzymolysis.

4. The composite plant extract for inhibiting vibrio parahaemolyticus in aqueous environment according to claim 3, wherein the preparation method of the tequila grandiflora extract comprises:

5. The composite plant extract for inhibiting vibrio parahaemolyticus in aqueous environment according to claim 3, wherein the holly extract is prepared by the method comprising:

6. The composite plant extract for inhibiting vibrio parahaemolyticus in aqueous environment according to claim 3, wherein the preparation method of the extract of the flower of gynura bicolor comprises:

7. The composite plant extract for inhibiting vibrio parahaemolyticus in aqueous environment according to claim 3, wherein the dandelion extract is prepared by the method comprising:

8. The complex plant extract for inhibiting Vibrio parahaemolyticus in an aqueous environment according to any one of claims 4 to 7, wherein the concentration conditions are: the temperature is 25-30 ℃,the operation pressure is 0.3-0.6Mpa, and the membrane flux of the 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 25-30 ℃, and the mixture is ultrasonically emulsified to be uniform, and the obtained mixture 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 reproduction of Vibrio parahaemolyticus in the upper, middle and bottom layers of aquatic water.