Medicinal preparation and application thereof in improving seedling raising success rate of babylonia lutosa

Abstract

The invention discloses a medicinal preparation and application thereof in improving the success rate of babylonia breeding. The pharmaceutical preparation comprises a spore microbial agent and composite polypeptide, the spore microbial agent comprises bacillus subtilis, bacillus velezensis, bacillus licheniformis, bacillus pumilus, bacillus coagulans, bacillus megaterium and bacillus circulans; the composite polypeptide comprises bacitracin, brevibacterium peptide, polymyxin and nisin. The medicinal preparation can remarkably improve the seedling culture success rate of babylonia lutosa.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compositions, and more specifically, relates to a pharmaceutical preparation and an application thereof in improving the success rate of babylonica seedling cultivation. Background Art

[0002] Dongfeng conch is commonly known as flower conch, prosperous conch, and yellow conch. It is also known as phoenix conch in Taiwan, China. It is distributed in tropical and subtropical regions. It is a carnivorous shallow-sea benthic shellfish. It tastes delicious and is a kind of high-protein, low-fat precious seafood that meets modern nutritional needs.

[0003] There are more than ten species of babylonia in the world, all of which are edible. Currently, only three species, namely, Babylonia square, Babylonia muda and Babylonia formosanus, are put into aquaculture. my country is the world's leading country for aquaculture of babylonia. At present, aquaculture of babylonia in China is mainly distributed in the coastal areas south of Fujian. Among them, Hainan has become the most suitable region for the cultivation and breeding of babylonia seedlings in my country due to its unique geographical environment and climatic conditions.

[0004] However, in recent years, bacterial and fungal diseases have frequently occurred during large-scale and high-density intensive breeding, which has seriously restricted the development of the factory-based breeding industry of Babylonia areolata. In the early, middle and late stages of Babylonia snail breeding, multiple red circles composed of harmful fungi and bacterial colonies often appear at the bottom of the breeding pond, which is called the "red bottom" phenomenon. The "red bottom" phenomenon greatly affects the success rate of Babylonia snail planktonic larvae breeding.

[0005] Patent Publication No. CN115141264A discloses antimicrobial peptides and their preparation and application. The antimicrobial peptides are prepared by fermenting the crayfish shell with tropical Candida and Bacillus licheniformis, which can effectively inhibit Vibrio. Adding it as a feed additive to the basic feed for aquaculture can effectively reduce the harm caused by Vibrio to farmed animals. Patent Publication No. CN106922997A discloses a pig feed additive with high-efficiency gastrointestinal mucosal repair function, which includes active probiotics, including Enterococcus faecium, Bacillus subtilis, Lactobacillus, Bifidobacterium longum, and Streptococcus thermophilus, which significantly improves the specific and nonspecific immune functions of the pig herd and reduces the mortality rate.

[0006] However, the above-mentioned additives are only used in the breeding process of farmed animals or pigs, and are not completely practical for the breeding of Babylonia snails. Moreover, they can only eliminate diseases caused by Vibrio to a certain extent, but have limited effect on improving the "red bottom phenomenon" that occurs during the breeding process of Babylonia snails. Summary of the invention

[0007] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a pharmaceutical preparation, which includes a spore-forming agent and a composite polypeptide, and the pharmaceutical preparation can effectively improve the success rate of Babylonia snail seedling cultivation.

[0008] The second object of the present invention is to provide a method for preparing a pharmaceutical preparation.

[0009] The third object of the present invention is to provide a pharmaceutical preparation for use in the preparation of a drug for improving the success rate of Babylonia snail seedling cultivation.

[0010] The fourth object of the present invention is to provide a pharmaceutical preparation for use in preparing a drug for preventing and / or treating red bottom in the process of raising seedlings of Babylonia snails.

[0011] The fifth object of the present invention is to provide a method for raising and cultivating baby cones.

[0012] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0013] A pharmaceutical preparation comprises a spore-forming agent and a composite polypeptide; the spore-forming agent comprises Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans; the composite polypeptide comprises bacitracin, gramicidin, polymyxin and nisin.

[0014] The inventors found through research that spore-forming agents have more advantages in spatial site competition, and can achieve antibacterial and disease control effects through rapid and large-scale reproduction and colonization, as well as destroying the colonization of pathogenic microorganisms on the larvae of the snails; in addition, spore-forming agents can also produce bacteriolytic substances and antibacterial substances, further inhibiting the reproduction of harmful bacteria. The inventors found through research that under the combined action of a specific combination of spore-forming agents, the red bottom phenomenon that appears at the bottom of the pond during the breeding process of the snails can be significantly solved, and the success rate of breeding of the snails can be significantly improved.

[0015] Furthermore, the composite polypeptide can affect the outer membrane of sensitive bacteria, destroy the integrity of the outer membrane, and cause bacterial cell dysfunction and even death. The inventors have found through research that the specific combination of composite polypeptides of the present invention can enhance the effect of medication and significantly solve the red bottom phenomenon that occurs at the bottom of the pond during the breeding process of Babylonia snails.

[0016] The present invention can synergistically play an antibacterial and bactericidal role through the above-mentioned specific combination of spore-forming agents and specific combination of composite polypeptides, improve the body immunity of snail seedlings, improve the infection and disease problems in the breeding process of Dongfeng snails, and greatly improve the success rate of Dongfeng snail seedlings. The pharmaceutical preparation provided by the present invention has the advantages of safety and convenience in use, and can be promoted on a large scale.

[0017] Preferably, the mass ratio of the Bacillus subtilis, Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium or Bacillus circulans is 3-4:0.8-1.5:3-4:2-3:1.5-2:1-2:1; more preferably, the mass ratio is 4:1:4:3:2:1.5:1.

[0018] Preferably, the mass ratio of bacitracin, gramicidin, polymyxin and nisin is 1:2 to 4:1 to 3:1. Further preferably, the mass ratio of bacitracin, gramicidin, polymyxin and nisin is 1:4:3:1.

[0019] Preferably, the mass ratio of the spore-forming agent to the composite polypeptide is 5 to 8: 2 to 4. Further preferably, the mass ratio of the spore-forming agent to the composite polypeptide is 6 to 7: 2 to 3.

[0020] Preferably, the pharmaceutical preparation further comprises a synergist; the synergist is a carbon source and / or a nitrogen source.

[0021] Preferably, the synergist comprises a carbon source and a nitrogen source.

[0022] Preferably, 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, ammonium salt, nitrate or urea. More specifically, the bicarbonate includes but is not limited to sodium bicarbonate, etc.; the acetate includes but is not limited to sodium acetate, etc. More specifically, the ammonium salt includes but is not limited to ammonium sulfate, ammonium chloride, etc.

[0023] Furthermore, the present invention claims a method for preparing a pharmaceutical preparation, comprising the following steps:

[0024] (1) mixing Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans to prepare a spore-forming agent;

[0025] (2) mixing bacitracin, gramicidin, polymyxin and nisin to prepare a composite polypeptide;

[0026] (3) Mixing the spore-forming agent and the composite polypeptide to prepare a pharmaceutical preparation.

[0027] Preferably, in step (1), mixing is carried out under a sterile environment.

[0028] Preferably, in step (2), mixing is carried out under a sterile environment at room temperature.

[0029] Preferably, in step (3), mixing is carried out under a sterile environment at room temperature.

[0030] In some specific embodiments, the present invention provides another method for preparing a pharmaceutical preparation, further comprising the following steps: mixing a carbon source and a nitrogen source at normal temperature and pressure to prepare a synergist; and mixing the synergist, a spore-forming agent and a composite polypeptide to prepare a pharmaceutical preparation.

[0031] Furthermore, the present invention seeks to protect the use of the pharmaceutical preparation in the preparation of a drug for improving the success rate of raising babylonica seedlings.

[0032] Specifically, in the present invention, the success rate of raising baby snails refers to the ratio of the number of juvenile snails divided by the number of initial face disc larvae.

[0033] Furthermore, the present invention seeks to protect the use of the pharmaceutical preparation in the preparation of a drug for preventing and / or treating red bottom in the process of raising seedlings of Babylonia snails.

[0034] Specifically, the red bottom refers to: a red circle composed of multiple harmful fungi and / or bacterial colonies appearing at the bottom of the snail nursery pond.

[0035] Furthermore, the present invention seeks to protect a method for cultivating seedlings of Babylonia snails, which uses the above-mentioned pharmaceutical preparation to cultivate seedlings of Babylonia snails.

[0036] Preferably, during the seedling cultivation process, the water temperature is maintained at 20-30°C, the dissolved oxygen is 5-7 mg / L, and the pH is 8.2-8.5.

[0037] Preferably, the above-mentioned drug preparation can be used for preventive seedling cultivation immediately after the hatching of the egg capsules of the Dongfeng snail, and can also be used for therapeutic seedling cultivation of the Dongfeng snail planktonic larvae that have already shown the red bottom phenomenon.

[0038] Preferably, in prevention, the dosage of the drug preparation is 0.5 to 1.5 ppm. More preferably, the dosage of the drug preparation is 0.8 to 1.2 ppm. The drug preparation is used once every 1 to 3 days.

[0039] Preferably, during treatment, the dosage of the drug preparation is 3 to 15 g / m 3 More preferably, the dosage of the drug preparation is 5 to 10 g / m 3 The pharmaceutical preparation is used at least once a day for 2 to 4 consecutive days.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention can synergistically play an antibacterial and bactericidal role through the above-mentioned specific combination of spore-forming agents and specific combination of composite polypeptides, improve the body immunity of snail seedlings, improve the infection and disease problems in the breeding process of Dongfeng snails, and greatly improve the success rate of Dongfeng snail seedlings. The pharmaceutical preparation provided by the present invention has the advantages of safety and convenience in use, and can be promoted on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the dead Babylonia snail larvae that sank to the bottom of the seedling barrel after the seedling cultivation failed in Comparative Example 1.

[0043] Figure 2 This is a schematic diagram of the emptying of the seedling barrel with the bottom turning red after the seedling cultivation failed in comparative example 1.

[0044] Figure 3 This is a schematic diagram of the Dongfeng snail larvae that died without treatment after the seedling cultivation failed in comparative example 1 and a red bottom appeared. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Example 1 Preparation of pharmaceutical preparations

[0047] A pharmaceutical preparation is prepared according to the following method:

[0048] (1) Under sterile environmental conditions at 25 to 30° C., Bacillus subtilis BS-01, Bacillus Velezii YFI-4, Bacillus licheniformis JHD-Z2, Bacillus pumilus DS3, Bacillus coagulans NR372, Bacillus megaterium NCT-2, and Bacillus circulans L38 (all of the above strains are from the China Center for the Collection of Microbiological Cultures) are mixed in a mass ratio of 4:1:4:3:2:1.5:1 to prepare a spore-forming agent;

[0049] (2) under normal temperature and sterile conditions, bacitracin, gramicidin, polymyxin and nisin were mixed in a mass ratio of 1:4:3:1 to prepare a composite polypeptide;

[0050] (3) At room temperature and pressure, sodium bicarbonate and ammonium chloride are mixed in a mass ratio of 2:1 to prepare a synergist;

[0051] (4) At room temperature and pressure, the spore-forming agent and the composite polypeptide are mixed at a mass ratio of 7:2 under aseptic conditions at room temperature to obtain a mixture; then the mixture and the synergist are mixed at a mass ratio of 10:1 under aseptic conditions at room temperature to prepare a pharmaceutical preparation.

[0052] Example 2 Preparation of pharmaceutical preparations

[0053] The difference between Example 2 and Example 1 is:

[0054] The mass ratio of Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus brevis, Bacillus coagulans, Bacillus megaterium, and Bacillus circulans is 3:1:3:2:2:2:1;

[0055] The mass ratio of bacitracin, gramicidin, polymyxin, and nisin was 1:3:2:1;

[0056] The spore agent and the composite polypeptide are mixed at a mass ratio of 2:1 under sterile conditions at room temperature.

[0057] Example 3 Preparation of pharmaceutical preparations

[0058] The difference between Example 3 and Example 1 is:

[0059] The mass ratio of Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus brevis, Bacillus coagulans, Bacillus megaterium, and Bacillus circulans is 4:1:3.5:2.5:2:1.5:1;

[0060] The mass ratio of bacitracin, gramicidin, polymyxin, and nisin was 1:4:2.5:1;

[0061] The spore agent and the composite polypeptide are mixed at a mass ratio of 2:1 under sterile conditions at room temperature.

[0062] Example 4 Treatment test of red bottom in the process of raising baby snails

[0063] Purchase the egg capsules of Dongfeng Snail from Wenchang, Hainan. Rinse the attachments and sludge on the surface of Dongfeng Snail egg capsules with seawater, remove the whitish and reddish infected egg capsules, and put them in the hatching basket. Put 4 jin of egg capsules in each seedling barrel. The specifications of the seedling barrel are 1m in diameter, 1.5m in height, 1.3m in water depth, and the water temperature is 28℃ when entering the barrel. Invert the barrel once a day before hatching. When it is close to hatching, it will not be moved anymore, and the hatching density is maintained at 0.1 / ml. Continuously aerate during the cultivation period, change 1 / 3 of the water every day during the early face disc larvae stage, 1 / 2 of the water every day during the middle face disc larvae stage, and 3 / 4 of the water every day during the late face disc larvae stage.

[0064] In the early stage, all golden algae are fed, in the middle stage, golden algae are mainly fed, supplemented by horn algae, and in the later stage, golden algae, horn algae, and flat algae are mixed according to the bait situation. The feeding of golden algae is 3,000 / ml per day at the beginning, and gradually increases to 17,000 / ml per day in the later stage; horn algae and flat algae are fed as appropriate according to the amount of stomach contents of the planktonic larvae of the baby snails. Feeding is increased from 2 times a day at the beginning to 4 times a day in the later stage. At the same time, spirulina powder is fed as an auxiliary, 2-3 grams / m 3, feed 2 to 3 times a day.

[0065] On the 10th day of cultivation, it was found that the seedling barrel was covered with red circles (i.e., a "red bottom" phenomenon appeared). The drug preparation prepared in Example 1 was used at a rate of 5 g / m 3 (The dosage of the drug preparation is based on the volume of the water body) Spray once a day for 3 consecutive days, and the red circle at the bottom of the seedling barrel will disappear.

[0066] Example 5 Treatment test of red bottom in the process of raising baby snails

[0067] The difference between Example 5 and Example 4 is that on the 9th day of cultivation, the seedling barrel is full of red circles, and the drug preparation prepared in Example 2 is used. The dosage, frequency and time of the drug preparation are the same as those in Example 4.

[0068] Example 6 Prevention test of red bottom in the process of raising baby snails

[0069] The difference between Example 6 and Example 4 is that:

[0070] When the planktonic larvae of Babylonia snails hatched from the egg capsules, the drug preparation prepared in Example 1 was added once every 2 days, with 1 ppm added each time (based on the volume of the aquaculture water body) until the last day of seedling cultivation.

[0071] Example 7

[0072] The difference between this embodiment and embodiment 4 is that:

[0073] The drug preparation only contains spore-forming agents and complex polypeptides, but no synergist.

[0074] Comparative Example 1

[0075] The difference between this comparative example and Example 4 is that no treatment measures are taken and the pharmaceutical preparation prepared in Example 1 is not used.

[0076] Starting from the 11th day, the density of the floating larvae of the baby snails in the water of the seedling barrel gradually decreased. By the 14th day, almost no floating larvae of the baby snails could be seen. The baby snails died and sank to the bottom of the barrel, indicating that the seedling cultivation failed (e.g. Figures 1 to 3 shown).

[0077] Comparative Example 2

[0078] The difference between this comparative example and Example 4 is that:

[0079] The drug preparation contains only spore-forming agents.

[0080] Comparative Example 3

[0081] The difference between this comparative example and Example 4 is that:

[0082] The drug preparation contains only the complex polypeptide.

[0083] Comparative Example 4

[0084] The difference between this comparative example and Example 4 is that:

[0085] The spore-forming agent in the drug preparation contains only Bacillus licheniformis.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 4 is that:

[0088] The complex polypeptide in the pharmaceutical preparation contains only bacitracin.

[0089] Test Case

[0090] Number of initial veliger larvae: After the egg sacs are hatched, use a 200 ml beaker to randomly sample three times from each nursery barrel, count the total number of planktonic larvae of Babylonia snails in the three sampling beakers, convert the average value, and then divide it by 200 to get the average number of planktonic larvae of Babylonia snails per ml of nursery water, and then multiply it by the volume of nursery water to get the number of initial veliger larvae in the nursery barrel.

[0091] Number of juvenile snails: Randomly take 20 grams of juvenile snails of Dongfeng Snail for three times and count them. Divide the total number of juvenile snails for three times by 3 and then by 20 to get the average number of juvenile snails per gram. Multiply it by the weight (g) to get the number of juvenile snails.

[0092] Seedling raising success rate: The seedling raising success rate is obtained by dividing the number of juvenile snails by the number of initial disc larvae.

[0093] The test data of the above embodiments and comparative examples are shown in Table 1 below.

[0094] Table 1

[0095] Group Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Seedling success rate% 72.9 73.5 71.4 47.1 / 23.3 25.1 26.6 25.7 Days for red background to disappear / d 3 3 / 4 / 7 7 6 6

[0096] As can be seen from Examples 4 to 7 above, the use of the pharmaceutical preparation provided by the present invention can synergistically play an antibacterial and bactericidal role, improve the body immunity of snail seedlings, improve the infection and disease problems in the breeding process of Dongfeng snails, greatly improve the success rate of Dongfeng snail seedlings, and shorten the number of days for the red bottom phenomenon to disappear. The seedling success rate of the pharmaceutical preparation used in the present invention is ≥47.1%, and the number of days for the red bottom to disappear is ≤4 days. Furthermore, the seedling success rate is ≥71.4%, and the number of days for the red bottom to disappear is ≤3 days.

[0097] It can be seen from Example 4, Example 7, Comparative Example 1, Comparative Example 2 and Comparative Example 3 that when the spore-forming agent and the composite polypeptide are combined to form a pharmaceutical preparation for use, there is an excellent synergistic effect, which greatly improves the success rate of raising baby snails and accelerates the number of days for the red background to disappear. It is difficult to achieve the technical effect of the present invention by using only one of the components.

[0098] It can be seen from Example 4, Comparative Example 4 and Comparative Example 5 that when one bacterium or peptide in a spore-forming agent or a composite polypeptide is used in combination with another component, it is difficult to achieve the technical effect of the present invention.

[0099] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A pharmaceutical preparation, It is characterized in that Including spore-forming agents and complex peptides; The spore-forming agents include Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans; The composite polypeptide comprises bacitracin, gramicidin, polymyxin and nisin.

2. The pharmaceutical preparation according to claim 1, It is characterized in that The mass ratio of the Bacillus subtilis, Bacillus Velez, 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.

3. The pharmaceutical preparation according to claim 1, It is characterized in that The mass ratio of bacitracin, gramicidin, polymyxin and nisin is 1:2-4:1-3:

1.

4. The pharmaceutical preparation according to claim 1, It is characterized in that The mass ratio of the spore agent to the composite polypeptide is 5-8:2-4.

5. The pharmaceutical preparation according to claim 1, It is characterized in that The pharmaceutical preparation further comprises a synergist; the synergist is a carbon source and / or a nitrogen source.

6. The pharmaceutical preparation according to claim 5, It is 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.

7. A method for preparing the pharmaceutical preparation according to any one of claims 1 to 6, It is characterized in that The following steps are involved: (1) mixing Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus megaterium and Bacillus circulans to prepare a spore-forming agent; (2) mixing bacitracin, gramicidin, polymyxin and nisin to prepare a composite polypeptide; (3) Mixing the spore-forming agent and the composite polypeptide to prepare a pharmaceutical preparation.

8. Use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a drug for improving the success rate of Babylonia snail seedling cultivation.

9. Use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a drug for preventing and / or treating red bottom in the process of raising baby snails.

10. A method for raising seedlings of Babylonia spp. It is characterized in that The drug preparation according to any one of claims 1 to 6 is used to cultivate baby snails.

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