Pharmaceutical composition and its application in preparing medicine for preventing and treating red bottom in breeding of baby snails

Abstract

The application discloses a kind of pharmaceutical compositions. The pharmaceutical composition includes A agent and B agent;The A agent includes Vibrio parahaemolyticus bacteriophage, Vibrio harveyi bacteriophage, Vibrio alginolyticus bacteriophage and Bacteriophage of Achromobacter nematophilus;The B agent includes bacitracin, short bacitracin, polymyxin and nisin. The present application can effectively inhibit and solve the "red bottom" phenomenon that appears in the bottom of pool in the process of breeding of snail, improve the success rate of breeding of snail.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of snail breeding, and more particularly relates to a pharmaceutical composition and application thereof in preparing a medicine for preventing and treating red bottom in the process of breeding abalone. BACKGROUND

[0002] Abalone is one of the important marine biological resources. Due to its high economic value, the artificial breeding of abalone has developed rapidly. At present, the main artificially bred abalone species in China are Rapana venosa (Gmelin) (also known as Rapana venosa), Rapana venosa (Gmelin) (also known as Rapana venosa), and Rapana venosa (Gmelin) (also known as Rapana venosa). Babylonia areolata Babylonia areolata Due to market demand, the scale and area of abalone breeding have been expanding year by year, and abalone has become one of the main varieties of seawater breeding in the south.

[0003] However, in recent years, bacterial and fungal diseases have occurred frequently in the process of large-scale and high-density intensive breeding, which has seriously restricted the development of the industrialization of Rapana venosa. In particular, during the middle and late stages of abalone breeding, multiple harmful fungal and bacterial colonies often appear on the bottom of the breeding pond, forming a red circle, which is referred to as the "red bottom" phenomenon. The "red bottom" phenomenon greatly affects the survival rate of abalone breeding and the subsequent metamorphosis process of abalone seedlings. The planktonic larvae of abalone metamorphose and fall to the bottom, resulting in a mortality rate of over 95%.

[0004] For the "red bottom" phenomenon in the process of abalone breeding, "Research Progress of Breeding, Nutrition and Disease of Rapana venosa", Chen Meng, Animal Science College of Zhongkai Agricultural Engineering College, discloses that the main disease pathogens of abalone identified so far can be divided into two categories: one is bacterial disease, including swollen proboscis disease caused by Vibrio anguillarum var., proboscis edema disease caused by Vibrio harveyi, acute death syndrome (also known as back-flip syndrome) caused by Vibrio harveyi or Vibrio tapetis, Vibrio outbreak, foot swelling, pustule disease, and respiratory tube inflammation; the other is parasitic disease, including ciliates disease caused by Conchophthas sp. during the planktonic period, shell meat separation disease (also known as shedding disease) caused by copepods during the juvenile / spine period, and monosporidium disease caused by monosporidium var. during the growing period.

[0005] However, there is currently no suitable treatment for the "red bottom" phenomenon. It is an urgent technical problem to find a safer and more environmentally friendly disease control method, provide a product and method that can effectively prevent the occurrence of red bottom in the process of abalone breeding and improve the success rate of abalone breeding. SUMMARY

[0006] In view of the above technical problems, the primary purpose of the present application is to provide a pharmaceutical composition, which uses A agent and B agent in combination, can effectively inhibit and solve the "red bottom" phenomenon on the bottom of the breeding pond in the process of abalone breeding, and improve the success rate of abalone breeding. ​

[0007] The second object of the present application is to provide a preparation method of the pharmaceutical composition.

[0008] The third object of the present application is to provide an application of the pharmaceutical composition in preparing a medicine for increasing the amount of breeding snails or the success rate of breeding.

[0009] The fourth object of the present application is to provide an application of the pharmaceutical composition in preparing a medicine for preventing and / or treating red bottom in the breeding process of the snail.

[0010] The fifth object of the present application is to provide a breeding method of the snail.

[0011] In order to achieve the above objects, the present application is implemented by the following technical solutions.

[0012] The pharmaceutical composition comprises an A agent and a B agent; the A agent comprises Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage and Bdellovibrio bacteriovorus; and the B agent comprises bacitracin, gramicidin, polymyxin and nisin.

[0013] Further, the pharmaceutical composition further comprises a C agent; the C agent comprises Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans.

[0014] In the present application, the A agent grows and reproduces in the pool, can cause lysis of pathogenic bacteria, reduce the density of pathogenic bacteria, thereby reducing or avoiding the opportunity of infection or disease of pathogenic bacteria, and achieves the purpose of treatment and prevention of diseases, and the prevention and treatment effect is better under the combined action of multiple phages.

[0015] Further, the B agent is all a polypeptide substance, has broad-spectrum antibacterial and bactericidal effects. Firstly, the B agent affects the outer membrane of sensitive bacteria, the amino group of the annular polypeptide part produces electrostatic interaction with the 2-valent cation binding point of the bacterial outer membrane lipopolysaccharide, the integrity of the outer membrane is destroyed, the fatty acid part of the drug can penetrate the outer membrane, and then the permeability of the cytoplasmic membrane is increased, leading to the outflow of small molecules such as phosphoric acid and nucleosides in the cytoplasm, causing cell dysfunction and even death. The multiple peptides in the present application can enhance the drug effect.

[0016] Further, the C agent is all spore bacteria, and its effects include: ① competition, which is more advantageous in space sites, and can quickly and massively multiply and colonize in the water body of the seedling pond, effectively prevent the propagation of pathogenic microorganisms, interfere with the invasion of planktonic larvae by pathogenic microorganisms, and destroy the colonization of pathogenic microorganisms on larvae, thereby achieving the effect of inhibiting bacteria and controlling diseases. ② Fusion bacteria, which can be adsorbed on the hyphae of pathogenic fungi and grow together with the hyphae. During the growth process, lytic substances are produced to digest the hyphae. Generally, it will cause the hyphae to break, disintegrate or cytoplasm to digest; or the secondary metabolites will dissolve the cell wall of pathogenic spores, resulting in cell wall perforation, deformation, etc. ③ Production of antibacterial substances, which can produce antibiotic substances that can inhibit the growth of bacteria, viruses, fungi and pathogens.

[0017] The present application can more effectively inhibit and solve the "red bottom" phenomenon of the octopus seedling process pond bottom and the problem of metamorphosis and death of the planktonic larvae of the octopus by using the specific combination of the A agent, the B agent and the C agent, and can significantly improve the success rate of the octopus seedling and the amount of octopus seedlings. The pharmaceutical composition provided by the present application has the advantages of low safety and can be widely promoted on a large scale.

[0018] Preferably, the mass ratio of the Vibrio parahaemolyticus phage, the Vibrio harveyi phage, the Vibrio alginolyticus phage and the Bdellovibrio bacteriovorus is 1-2:1-2:1-2:3-5.

[0019] Preferably, the mass ratio of the bacitracin, gramicidin, polymyxin and nisin is 1:2-4:1-3:1.

[0020] Preferably, the mass ratio of the Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans is 3-4:0.8-1.5:3-4:2-3:1.5-2:1-2:1.

[0021] Preferably, the mass ratio of the A agent, the B agent and the C agent is 1-3:2-4:5-8.

[0022] Preferably, the pharmaceutical composition further comprises a carbon source and / or a nitrogen source.

[0023] Preferably, the mass ratio of the carbon source and the nitrogen source is 1-2:1-1.5.

[0024] Preferably, the mass ratio of the total amount of the A agent, the B agent and the C agent to the synergist is 4-14:1. More preferably, the mass ratio is 8-12:1; further preferably, the mass ratio is 10:1.

[0025] Preferably, the carbon source is selected from one or more of bicarbonate, glucose or acetate. More specifically, the bicarbonate includes but is not limited to sodium bicarbonate and the like; the acetate includes but is not limited to sodium acetate and the like.

[0026] Preferably, the nitrogen source is selected from one or more of ammonia, ammonium salt, nitrate or urea. More specifically, the ammonium salt includes but is not limited to ammonium sulfate, ammonium chloride and the like.

[0027] Further, the present application claims to protect the preparation method of the pharmaceutical composition, comprising the following steps:

[0028] (1) mixing Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage and Bdellovibrio phage to obtain A agent;

[0029] (2) mixing bacitracin, gramicidin, polymyxin and nisin to obtain B agent;

[0030] (3) mixing Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans to obtain C agent; more specifically, the temperature of the sterile environment is 25-30℃.

[0031] (4) mixing the carbon source and the nitrogen source in proportion to obtain a synergist;

[0032] (5) mixing A agent, B agent, C agent and synergist to obtain the pharmaceutical composition.

[0033] Preferably, in the step (1), mixing is carried out under normal temperature and sterile conditions.

[0034] Preferably, in the step (2), mixing is carried out under normal temperature and sterile conditions.

[0035] Preferably, in the step (3), mixing is carried out under sterile environment conditions.

[0036] Preferably, in the step (4), mixing is carried out under normal temperature and pressure conditions.

[0037] Further, the present application claims to protect the application of the pharmaceutical composition in the preparation of the drug for improving the amount of abalone larvae or the success rate of abalone breeding.

[0038] Further, the present application claims to protect the application of the pharmaceutical composition in the preparation of the drug for preventing and / or treating red bottom during abalone breeding.

[0039] Specifically, in the present application, the success rate of abalone breeding refers to the ratio of the amount of abalone larvae to the total number of abalone planktonic larvae.

[0040] Specifically, in the present application, the red bottom refers to a red circle composed of multiple harmful fungal and / or bacterial colonies appearing on the bottom of the breeding tank of the bubble snail.

[0041] Further, the present application claims a breeding method of the bubble snail, which adopts the above-mentioned pharmaceutical composition for breeding the bubble snail.

[0042] Preferably, during the breeding process, the water temperature is maintained at 20-30℃, the dissolved oxygen is 5-7mg / L, and the pH is 8.2-8.5.

[0043] In some specific embodiments, the above-mentioned pharmaceutical composition can be used for preventive breeding of the bubble snail immediately after the egg capsule hatches, or the above-mentioned pharmaceutical composition can be used for therapeutic breeding of the bubble snail planktonic larvae after the red bottom phenomenon appears.

[0044] In some embodiments, when prevention is performed, the dosage of the pharmaceutical composition is 0.5-1.5ppm with 1-5 days as a cycle. Further preferably, the dosage of the pharmaceutical composition is 0.6-1.2ppm.

[0045] In some embodiments, when treatment is performed, the pharmaceutical composition is used once a day for 2-4 days, and the dosage is 3-15g / m 3 . Further preferably, the dosage is 5-10g / m 3 .

[0046] Specifically, the bubble snail is of the genus Bolinus (Bolinus), including but not limited to Bolinus cornutus (Bolinus cornutus), Bolinus coruscus (Bolinus coruscus), and the like. Babylonia areolata Babylonia areolata Babylonia areolata

[0047] Compared with the prior art, the present application has the following beneficial effects:

[0048] The present application provides a pharmaceutical composition, which can more effectively inhibit and solve the red bottom phenomenon appearing on the bottom of the breeding tank during the breeding process of the bubble snail, and the problem of metamorphosis and bottom death of the bubble snail larvae; can significantly reduce the mortality of the bubble snail larvae after infection, improve the body immunity of the snail larvae, and increase the amount of snail larvae and the success rate of breeding. The pharmaceutical composition provided by the present application has the advantages of low safety, and can be widely promoted on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Figure 4 is a schematic diagram of the breeding tank with reduced water level in Example 4, in which the red bottom phenomenon appears and some bubble snail larvae die on the bottom.

[0050] Figure 2 Figure 5 is a schematic diagram of the breeding tank after the red bottom phenomenon is treated and the breeding is successful in Example 4. DETAILED DESCRIPTION​​​

[0051] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0052] The phrase "early facial disc larva stage" refers to the development stage of the snail larva in which two residual yolk blocks are clearly visible, two oval facial discs are symmetrical, and the edges are densely covered with cilia.

[0053] The phrase "mid facial disc larva stage" refers to the development stage of the snail larva in which the yolk blocks disappear, and obvious features such as eyes, forelegs, primary shell, and a pair of antennae are present.

[0054] The phrase "late facial disc larva stage" refers to the development stage of the snail larva in which the head has a rostrum, the water tube, and the gill filament are clearly differentiated, and 1-2 rows of red-brown pigment spots appear on the spiral layer.

[0055] Example 1 A pharmaceutical composition

[0056] (1) Under sterile conditions at room temperature, Vibrio parahaemolyticus phage A3S, Vibrio harveyi phage Viha10, Vibrio alginolyticus phage HH109, and Bdellovibrio NA7 (all of the above strains are from Shanghai Xinyilai Biological Technology Co., Ltd.) are mixed according to a mass ratio of 1:1:1:4 to obtain A agent;

[0057] (2) Under sterile conditions at room temperature, bacitracin, gramicidin, polymyxin, and nisin are mixed according to a mass ratio of 1:4:3:1 to obtain B agent;

[0058] (3) Under sterile conditions at 25-30°C, Bacillus subtilis BS-01, Bacillus velezensis YFI-4, Bacillus licheniformis JHD-Z2, Bacillus pumilus DS 3, Bacillus coagulans NR372, Bacillus megaterium NCT-2, and Bacillus circulans L38 (all of the above strains are from the China General Microbiological Culture Collection Center) are mixed according to a mass ratio of 4:1:4:3:2:1.5:1 to obtain C agent;

[0059] (4) Under normal temperature and pressure, sodium bicarbonate and ammonium chloride are mixed according to a mass ratio of 2:1 to obtain an auxiliary agent;

[0060] (5) Under normal temperature and pressure, A agent, B agent, and C agent are mixed under sterile conditions at room temperature according to a mass ratio of 2:2:7, and then the mixture and the auxiliary agent are mixed under sterile conditions at room temperature according to a mass ratio of 10:1 to obtain a pharmaceutical composition.

[0061] Example 2 A pharmaceutical composition

[0062] The difference between this embodiment and embodiment 1 is that:

[0063] The A agent is mixed by Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage, Bdellovibrio bacteriovorus according to the mass ratio of 2:2:1:3;

[0064] The B agent is mixed by bacitracin, gramicidin, polymyxin and nisin according to the ratio of 1:3:2:1.

[0065] The C agent is mixed by Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium, Bacillus circulans according to the mass ratio of 3:1:3:2:2:2:1.

[0066] Example 3 A pharmaceutical composition

[0067] The difference between this embodiment and embodiment 1 is that:

[0068] The A agent is mixed by Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage, Bdellovibrio bacteriovorus according to the mass ratio of 1.5:2:1.5:4;

[0069] The B agent is mixed by bacitracin, gramicidin, polymyxin and nisin according to the mass ratio of 1:4:2.5:1.

[0070] The C agent is mixed by Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium, Bacillus circulans according to the mass ratio of 4:1:3.5:2.5:2:1.5:1.

[0071] Example 4 Treatment test of red bottom in conch larviculture process

[0072] The egg sacs of abalone sp. were purchased from Wenchang, Hainan, and the attached materials and sludge were washed with seawater. The infected egg sacs with white and red color were removed, and the egg sacs were placed in the incubation basket. The egg sacs were placed in the larviculture pond at 4 kg per pond. The specifications of the larviculture pond were 3 m x 4 m x 1 m, and the water depth was 0.8 m. The water temperature was 28℃ when entering the pond. The pond was emptied once a day before hatching, and the hatching density was maintained at 0.1 per milliliter.

[0073] During the cultivation, the water was continuously aerated, and 1 / 3 of the water was replaced every day in the early disc larva stage, 1 / 2 of the water was replaced every day in the early disc larva stage, and 3 / 4 of the water was replaced every day in the early disc larva stage. In the early stage, only golden algae were fed, in the middle stage, golden algae were mainly fed, and auxiliary chlorella was fed, and in the later stage, golden algae, chlorella and flat algae were mixed according to the bait situation. The feeding of golden algae was started at 3000 pieces per milliliter per day, and gradually increased to 1.7 million pieces per milliliter per day in the later stage; the feeding of chlorella and flat algae was adjusted according to the amount of stomach contents of the planktonic larvae. The feeding was increased from 2 times per day at the beginning to 4 times per day in the later stage. At the same time, spirulina powder was fed as an auxiliary, and the feeding amount was determined according to the actual situation, 2-3 grams / m 3 , 2-3 times a day.

[0074] On the 10th day of cultivation, the rearing pond was full of red circles (i.e. the "red bottom" phenomenon appeared, as shown in Figure 1 ), and the pharmaceutical composition prepared in Example 1 was poured into the whole pond at 5 grams / m 3 (based on the volume of the water body) once a day, and the red circles on the bottom of the pond disappeared (as shown in Figure 2 ) after 3 days of continuous pouring.

[0075] Example 5 Treatment test of red bottom in the process of breeding Babylonia areolata

[0076] The difference between this example and Example 4 is that the treatment is started on the 11th day, and the pharmaceutical composition prepared in Example 2 is used. The amount, frequency and time of the pharmaceutical composition are the same as those in Example 4.

[0077] Example 6 Prevention test of red bottom in the process of breeding Babylonia areolata

[0078] The difference between this example and Example 4 is that the pharmaceutical composition prepared in Example 1 is added as soon as the Babylonia areolata planktonic larvae hatch from the egg sac, and 1 ppm (based on the volume of the water body) is added every 2 days until the last day of the rearing.

[0079] Example 7

[0080] The difference between this example and Example 4 is that the pharmaceutical composition used only contains A agent and B agent.

[0081] Comparative Example 1

[0082] The difference between this comparative example and Example 4 is that no treatment measures are taken, i.e. the pharmaceutical composition prepared in Example 1 is not used.

[0083] On the 11th day, the density of Babylonia areolata planktonic larvae in the water body of the rearing pond gradually decreased, and on the 13th day, there were almost no Babylonia areolata planktonic larvae in the rearing pond, and the Babylonia areolata planktonic larvae died and settled on the bottom of the pond, resulting in failure of the rearing cultivation.

[0084] Comparative Example 2

[0085] The difference between the present comparative example and Example 4 is that the pharmaceutical composition used only contains A agent and C agent.

[0086] Comparative Example 3

[0087] The difference between the present comparative example and Example 4 is that the pharmaceutical composition used only contains B agent and C agent.

[0088] Test Example

[0089] After the end of the egg hatching, five-point sampling method is used in each of the rearing ponds, and water samples are taken at the four corners and the middle of the rearing pond to fill 250 ml measuring cylinders, the number of the Babylonia areolate planktonic larvae in each 250 ml measuring cylinder is counted, the average value is converted to obtain the average number of Babylonia areolate planktonic larvae per 250 ml, multiplied by 4 and 1000 to convert to obtain the number of Babylonia areolate planktonic larvae per cubic water body, and then multiplied by the volume of the rearing water body 9.6 cubic meters to obtain the total number of Babylonia areolate planktonic larvae in the rearing pond.

[0090] The number of snail seedlings: 50 grams of Babylonia areolate seedlings are randomly taken for three times, and the average value is counted, then divided by 50 to obtain the snail seedling specification (ind / gram), and multiplied by the weight (gram) to obtain the number of snail seedlings (ind).

[0091] The calculation formula is as follows:

[0092] The total number of Babylonia areolate planktonic larvae = the sum of the number of larvae in 5 times of 250 ml measuring cylinders / 5*4*1000*9.6;

[0093] The number of snail seedlings = the average number of 50 grams of Babylonia areolate seedlings for three times / 50*gram number;

[0094] The rearing success rate = the number of snail seedlings / the total number of Babylonia areolate planktonic larvae*100%;

[0095] The test data of the above examples and comparative examples are shown in Table 1.

[0096] Table 1

[0097]

[0098] From the above Examples 4-7, it can be seen that the pharmaceutical composition provided by the present application can more effectively inhibit and solve the red bottom phenomenon appearing in the rearing process of Babylonia areolate, and improve the rearing success rate of Babylonia areolate.

[0099] And from Example 4, Example 7, Comparative Examples 1-3, it can be seen that when A agent, B agent and C agent are combined to form a pharmaceutical composition for use, excellent synergistic effect is obtained, which can significantly improve the number of snail seedlings, the rearing success rate and reduce the number of days for the disappearance of red bottom. However, the use of any two components in combination is difficult to achieve the technical effect of the present application.

[0100] The foregoing examples are illustrative only and are not intended to limit the scope of the methods described herein. The appended claims are intended to claim as broad a range as possible as the inventors can conceive of at the time of filing. The examples presented herein are intended to demonstrate the inventors' knowledge of the present application and are not intended to limit the scope of the claims. Some of the numerical ranges recited in the claims are inclusive of the integers within the defined range. Unless otherwise specified, all ranges include endpoints.

Claims

1. The use of a pharmaceutical composition in the preparation of a medicament for increasing the seedling yield or seedling success rate of *Bambusa multiplex*, or in the preparation of a medicament for preventing and / or treating red discoloration during the seedling stage of *Bambusa multiplex*, characterized in that, The pharmaceutical composition is composed of A agent, B agent, C agent and synergist; The A agent is composed of Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage and Bdellovibrio bacteriovorus; The B agent is composed of bacitracin, gramicidin, polymyxin and nisin; The C agent is composed of Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans; The mass ratio of Vibrio parahaemolyticus phage, Vibrio harveyi phage, Vibrio alginolyticus phage and Bdellovibrio bacteriovorus is 1-2:1-2:1-2:3-5; The mass ratio of bacitracin, gramicidin, polymyxin and nisin is 1:2-4:1-3:1; The mass ratio of Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans is 3-4:0.8-1.5:3-4:2-3:1.5-2:1-2:1; The mass ratio of A agent, B agent and C agent is 1-3:2-4:5-8; The synergist is carbon source and nitrogen source; the mass ratio of the total amount of A agent, B agent and C agent to synergist is 4-14:

1.

2. Use according to claim 1, characterized in that, The mass ratio of carbon source and nitrogen source is 1-2:1-1.

5.

3. Use according to claim 1, characterized in that, The carbon source is selected from one or more of bicarbonate, glucose or acetate; the nitrogen source is selected from one or more of ammonia water, ammonium salt, nitrate or urea.

4. The use according to claim 1, characterized in that, During the process of breeding, the water temperature is maintained at 20-30℃, the dissolved oxygen is 5-7mg / L, and the pH is 8.2-8.5.

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