Application of a cyclic (L-leucine-D-proline) dipeptide and its aquatic antibacterial agent

Abstract

The present invention relates to an application of a cyclic (L-leucine-D-proline) dipeptide and an aquatic antibacterial agent thereof, wherein the application is as an aquatic antibacterial agent. The application of a cyclic (L-leucine-D-proline) dipeptide of the present invention is specifically to uniformly mix it with aquaculture inputs in a certain ratio, and prepare it as an aquatic antibacterial agent; then put it into use at a certain frequency, that is, it can be achieved in the aquaculture process. Effectively inhibit the growth of Vibrio anguillarum and Staphylococcus aureus in the water body, effectively prevent and control aquatic organisms from being infected with Vibrio anguillarum and Staphylococcus aureus, and then effectively avoid the impact of Vibrio anguillarum on aquatic product output, and effectively avoid the increase in subsequent processing costs caused by the infection of aquatic raw materials with Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, in particular to the application of a cyclic (L-leucine-D-proline) dipeptide and an aquatic antibacterial agent thereof. Background Art

[0002] Vibrio anguillarum is a Gram-negative bacterium with flagella, no capsule, no spore formation, motile, oxidase-positive, and sensitive to O / 129. It has multiple routes of infection, mainly including the skin, gills, lateral line, and intestines. Specifically, Vibrio anguillarum is a conditionally pathogenic bacterium. When aquaculture animals are in adverse environmental conditions, encounter adverse stimulation or are injured, it will induce the occurrence of diseases, affecting aquaculture production.

[0003] Staphylococcus aureus, also known as "golden Staphylococcus", belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is a common foodborne pathogenic microorganism that is almost ubiquitous and can exist in the entire aquatic product cycle, including aquaculture, processing, transportation, and sales. Under appropriate conditions, Staphylococcus aureus can produce enterotoxins and cause food poisoning. Therefore, it is very important to inhibit Staphylococcus aureus for aquatic products.

[0004] Currently, the main method for preventing and controlling bacterial diseases in aquatic animals is the use of antibiotics. However, long-term use of antibiotics leads to the emergence of drug-resistant strains and destroys the microecological balance of the water body. Therefore, it is necessary to find a more effective, green and safe compound to prevent and control Vibrio anguillarum.

[0005] According to existing research, cyclic (L-leucine-D-proline) dipeptide is 1×10 －5 mol·L, it can effectively inhibit MPP+-induced PC12-syn cell damage. There is no report on the antibacterial effect of cyclic (L-leucine-D-proline) dipeptide in aquatic products. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide an application of a cyclic (L-leucine-D-proline) dipeptide, specifically, mixing it evenly with aquaculture inputs in a certain proportion to prepare it as an aquatic antibacterial agent; then putting it into use at a certain frequency, that is, it can effectively inhibit the growth of Vibrio anguillarum and Staphylococcus aureus in the water body during the aquaculture process, effectively prevent and control aquatic organisms from being infected with Vibrio anguillarum and Staphylococcus aureus, and thus effectively avoid the impact of Vibrio anguillarum on aquatic production, and effectively avoid the increase in subsequent processing costs caused by the infection of aquatic raw materials with Staphylococcus aureus.

[0007] The invention discloses an application of a cyclic (L-leucine-D-proline) dipeptide, wherein the application is for preparing an aquatic antibacterial agent.

[0008] The application of the cyclic (L-leucine-D-proline) dipeptide of the present invention can prevent aquatic products from being infected by bacteria through the antibacterial effect of the cyclic (L-leucine-D-proline) dipeptide, thereby ensuring the quality of aquatic products.

[0009] Furthermore, the application method comprises the following steps: directly adding the cyclic (L-leucine-D-proline) dipeptide to aquaculture inputs, and mixing them evenly to prepare an aquaculture antibacterial agent.

[0010] Furthermore, the content of the cyclic (L-leucine-D-proline) dipeptide is 50-100 mg / kg.

[0011] Furthermore, the aquatic product is one or more of salmon, rainbow trout, eel, sweetfish, sea bass, cod, turbot, flounder, yellow croaker and shrimp.

[0012] Furthermore, the aquaculture inputs include one of aquatic feed, additives, water conditioners and water.

[0013] Furthermore, the application method includes using the aquatic antibacterial agent three times a day.

[0014] Furthermore, the bacteria are Vibrio anguillarum and Staphylococcus aureus.

[0015] The present invention also provides an aquatic antibacterial agent, comprising cyclic (L-leucine-D-proline) dipeptide.

[0016] Furthermore, the content of cyclic (L-leucine-D-proline) dipeptide in the aquatic antibacterial agent is 50-100 mg / kg.

[0017] For better understanding and implementation, the present invention is further described in detail below in conjunction with specific examples and accompanying drawings. The following examples are intended only to illustrate the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the structural formula of cyclo(L-leucine-D-proline) dipeptide;

[0019] Figure 2 Schematic diagram of the chromatography results of cyclo(L-leucine-D-proline) dipeptide;

[0020] Figure 3 Schematic diagram of ESI-MS results of cyclo(L-leucine-D-proline) dipeptide;

[0021] Figure 4 This is a schematic diagram of the antibacterial activity experimental results of Vibrio anguillarum;

[0022] Figure 5 Schematic diagram of the antibacterial activity experimental results of Staphylococcus aureus;

[0023] Among them, CK: methanol; P: Amp; DL: cyclo(D-leucine-L-proline) dipeptide; DD: cyclo(D-leucine-D-proline) dipeptide; cyclo(L-leucine-L-proline) dipeptide; cyclo(L-leucine-D-proline) dipeptide. DETAILED DESCRIPTION

[0024] The cyclic (L-leucine-D-proline) dipeptide of the present invention is obtained by chemical synthesis, and its structural formula is as follows Figure 1 As shown, the crude cyclic (L-leucine-D-proline) dipeptide synthesized was purified and identified by HPLC, and the purified cyclic (L-leucine-D-proline) dipeptide was analyzed by ESI-MS.

[0025] The process and results of HPLC purification and identification are as follows: The crude product of chemically synthesized cyclo(L-Leucine-D-Proline) dipeptide was subjected to HPLC chromatography, wherein the solvent phase A was 0.1% (v / v) trifluoroacetic acid and the solvent was chromatographically pure water; the solvent phase B was trifluoroacetic acid and the solvent was chromatographically pure acetonitrile; the elution procedure was 15% B phase elution for 0.01 min, 40% B phase elution for 25 min, then 100% B phase elution for 40 min, and finally 15% B phase elution for 50 min, all at 1 mL / min. The chromatographic results of the cyclo(L-Leucine-D-Proline) dipeptide are as follows: Figure 2 As shown, there is a single peak at 12 to 14 minutes, and the purity of cyclic (L-leucine-D-proline) dipeptide reaches 98.451%.

[0026] The process and results of ESI-MS analysis are as follows: the purified cyclic (L-leucine-D-proline) dipeptide was dissolved in a mixed solution of 5% acetic acid + 8% acetonitrile + 87% pure water and filtered, and then subjected to ESI-MS analysis. The analysis results are as follows Figure 3 As shown, the relative molecular mass of cyclo(L-leucine-D-proline) dipeptide is 210.28, which is consistent with the theory.

[0027] Example 1

[0028] This example provides an application example of cyclic (L-leucine-D-proline) dipeptide as an aquatic antibacterial agent in the culture process of whiteleg shrimp.

[0029] The implementation location of this embodiment is a breeding base in Sanjiao Town, Zhongshan City. After repeated breeding of whiteleg shrimp, the breeding base developed serious bacterial diseases in the whiteleg shrimp. After microbial technology, the Vibrio anguillarum in the water reached 3×10 3 cfu / ml, the current survival rate of shrimp is about 50%. This embodiment selected one acre of shrimp pond for application.

[0030] The breeding method is as follows: the water body is disinfected before stocking, and then 30,000 shrimp fry are stocked per mu, for a total of 120,000 shrimp fry, for conventional breeding. During the breeding process, a mixture of aquatic feed and cyclic (L-leucine-D-proline) dipeptide is fed 3 times per day, wherein the content of cyclic (L-leucine-D-proline) dipeptide is 50 mg / kg, and the breeding time is 3 months.

[0031] After 3 months, the survival rate of the whiteleg shrimp was calculated by observing their appearance and weighing their mass. The survival rate was calculated as follows: survival rate = number of surviving shrimps / total number of shrimps × 100%.

[0032] The whiteleg shrimp of this example had a survival rate of 65.3%. They had a transparent body, a slender line, clean legs, and thick antennae. They also had strong jumping ability and a relatively high individual weight, reaching 24 per jin (100 grams). Ten shrimp were randomly sampled and tested for PCR, and no Staphylococcus aureus was detected in any of them.

[0033] Example 2

[0034] This example provides an application example of cyclic (L-leucine-D-proline) dipeptide as an aquatic antibacterial agent in the culture process of whiteleg shrimp.

[0035] The implementation location of this embodiment is a breeding base in Sanjiao Town, Zhongshan City. After repeated breeding of whiteleg shrimp, the breeding base developed serious bacterial diseases in the whiteleg shrimp. After microbial technology, the Vibrio anguillarum in the water reached 3×10 3 cfu / ml, the current survival rate of shrimp is about 50%. This embodiment selected one acre of shrimp pond for application.

[0036] The breeding method is as follows: the water body is disinfected before stocking, and then 30,000 shrimp fry are stocked per mu, for a total of 120,000 shrimp fry, for conventional breeding. During the breeding process, a mixture of aquatic feed and cyclic (L-leucine-D-proline) dipeptide is fed 3 times per day, wherein the content of cyclic (L-leucine-D-proline) dipeptide is 100 mg / kg, and the breeding time is 3 months.

[0037] After 3 months, the survival rate of the whiteleg shrimp was calculated by observing their appearance and weighing their mass. The survival rate was calculated as follows: survival rate = number of surviving shrimps / total number of shrimps × 100%.

[0038] The survival rate of the whiteleg shrimp in this example was 85.2%. The shrimp had a transparent body, a slender line, clean legs, and thick antennae. They also had strong jumping ability and a relatively high individual shrimp weight, reaching 24 per jin. Ten shrimp were randomly sampled and tested for PCR, and no Staphylococcus aureus was detected in any of them.

[0039] Comparative Example 1

[0040] This example provides an example of aquaculture without using antibacterial agents.

[0041] The implementation location of this embodiment is a breeding base in Sanjiao Town, Zhongshan City. After repeated breeding of whiteleg shrimp, the breeding base developed serious bacterial diseases in the whiteleg shrimp. After microbial technology, the Vibrio anguillarum in the water reached 3×10 3 cfu / ml, the current survival rate of shrimp is about 50%. This example selected a shrimp pond of 1 mu with similar environmental conditions to the shrimp pond in Example 1 for application.

[0042] The breeding method of the control group was as follows: the water was disinfected before stocking, and then 30,000 shrimp fry were stocked per mu, for a total of 120,000 shrimp, for conventional breeding. During the breeding process, aquatic feed was fed 3 times a day, and the breeding time was 3 months.

[0043] After three months, the survival rate of the whiteleg shrimp was calculated, their appearance was observed, and their weight was weighed. The survival rate was calculated using the following formula: survival rate = number of surviving shrimp / total number of shrimp × 100%. The statistical results of survival rate, appearance, and weight are shown in Table 2 below.

[0044] The survival rate of the whiteleg shrimp in this example was 55%. They had a darker body with thick, black lines, weak jumping ability, and a light weight of 35 per jin (100 grams). Ten shrimp were randomly sampled and tested by PCR, and two of them tested positive for Staphylococcus aureus.

[0045] Comparative Example 2

[0046] In this example, sterile methanol, Amp (ampicillin) solution, cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide, and cyclo(L-leucine-D-proline) dipeptide were tested for their antibacterial activity against Vibrio anguillarum.

[0047] The experimental method is:

[0048] Dissolve cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide, and cyclo(L-leucine-D-proline) dipeptide in methanol to prepare a 100 mg / L cyclodipeptide methanol solution, which is sterilized by filtration through a 0.22 μm filter membrane.

[0049] Vibrio anguillarum was cultured to an OD600 of 1.5. The solution was then spread onto LB medium, and six filter paper discs (6 mm diameter) were placed equidistantly in the center of the medium to create six dotted areas. The LB liquid medium consisted of 10 g of tryptone, 5 g of yeast extract, and 7 g of sodium chloride, diluted to 1 L with distilled water. Solid LB medium was supplemented with 1.5% agar.

[0050] Add 10uL of sterile methanol, 10mg / mL Ampicillin solution, 100mg / L cyclic (D-leucine-L-proline) dipeptide, 100mg / L cyclic (D-leucine-D-proline) dipeptide, 100mg / L cyclic (L-leucine-L-proline) dipeptide, and 100mg / L cyclic (L-leucine-D-proline) dipeptide to the six sample spots respectively; place the samples in a 30°C incubator for 1 day, observe, and measure the diameter of the inhibition zone.

[0051] The results of the antibacterial activity test of Vibrio anguillarum are as follows Figure 4 As shown, the substance corresponding to CK is methanol; the substance corresponding to P is 10 mg / mL Ampicillin, and the diameter of the inhibition zone is 8 mm; the substance corresponding to DL is 100 mg / L cyclic (D-leucine-L-proline) dipeptide, and the diameter of the inhibition zone is 5.5 mm; the substance corresponding to DD is 100 mg / L cyclic (D-leucine-D-proline) dipeptide, and the diameter of the inhibition zone is 5 mm; the substance corresponding to LL is 100 mg / L cyclic (L-leucine-L-proline) dipeptide, and there is no inhibition zone; the substance corresponding to LD is 100 mg / L cyclic (L-leucine-D-proline) dipeptide, and the diameter of the inhibition zone is 8 mm. It is not difficult to see that cyclic (L-leucine-D-proline) dipeptide has a significant antibacterial effect on Vibrio anguillarum, and its inhibition zone diameter is 8 mm, which is comparable to the antibacterial effect of the traditional antibiotic ampicillin (Amp). Therefore, cyclic (L-leucine-D-proline) dipeptide can be used to prevent and treat fish and shrimp aquatic diseases caused by Vibrio anguillarum.

[0052] Comparative Example 3

[0053] In this example, antibacterial activity experiments against Staphylococcus aureus were conducted on sterile methanol, Amp (ampicillin) solution, cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide, and cyclo(L-leucine-D-proline) dipeptide.

[0054] The experimental method is:

[0055] Dissolve cyclo(D-leucine-L-proline) dipeptide, cyclo(D-leucine-D-proline) dipeptide, cyclo(L-leucine-L-proline) dipeptide, and cyclo(L-leucine-D-proline) dipeptide in methanol to prepare a 100 mg / L cyclodipeptide methanol solution, which is sterilized by filtration through a 0.22 μm filter membrane.

[0056] Staphylococcus aureus was cultured to an OD600 of 1.5. The solution was then spread onto LB medium. Six filter paper discs (6 mm diameter) were placed equidistantly in the center of the medium, forming six dotted areas. The LB liquid medium consisted of 10 g of tryptone, 5 g of yeast extract, and 7 g of sodium chloride, diluted to 1 L with distilled water. For solid LB medium, 1.5% agar was added.

[0057] Add 10 μL of sterile methanol, 10 mg / mL Ampicillin solution, 100 mg / L cyclic (D-leucine-L-proline) dipeptide, 100 mg / L cyclic (D-leucine-D-proline) dipeptide, 100 mg / L cyclic (L-leucine-L-proline) dipeptide, and 100 mg / L cyclic (L-leucine-D-proline) dipeptide to the six sample areas respectively; place the samples in a 30°C incubator for 1 day, then observe and measure the diameter of the inhibition zone.

[0058] The results of the antibacterial activity test on Staphylococcus aureus are as follows Figure 5As shown, the substance corresponding to CK is methanol; the substance corresponding to P is 10 mg / mL Ampicillin, and the diameter of the inhibition zone is 12 mm; the substance corresponding to DL is 100 mg / L cyclic (D-leucine-L-proline) dipeptide, and the diameter of the inhibition zone is 5 mm; the substance corresponding to DD is 100 mg / L cyclic (D-leucine-D-proline) dipeptide, and the diameter of the inhibition zone is 2.5 mm; the substance corresponding to LL is 100 mg / L cyclic (L-leucine-L-proline) dipeptide, and the diameter of the inhibition zone is 2 mm; the substance corresponding to LD is 100 mg / L cyclic (L-leucine-D-proline) dipeptide, and the diameter of the inhibition zone is 8 mm. It is not difficult to see that cyclic (L-leucine-D-proline) dipeptide has a significant antibacterial effect on Staphylococcus aureus. The diameter of its inhibition zone is 8 mm, which is two-thirds of the traditional antibiotic ampicillin (Amp). Therefore, cyclic (L-leucine-D-proline) dipeptide can be used to prevent the growth of Staphylococcus aureus in aquaculture, affect the quality of aquatic products, and increase the subsequent aquatic product processing costs.

[0059] By comparing the results of Example 1, Example 2 and Comparative Example 1, it can be seen that the use of cyclic (L-leucine-D-proline) dipeptide improves the survival rate of shrimp, reduces the mortality rate of shrimp, and improves its vitality and quality; in particular, when the content of cyclic (L-leucine-D-proline) dipeptide is 100 mg / kg, the survival rate of shrimp is as high as 85.2%, which greatly improves the survival rate of shrimp.

[0060] The present invention first discovered that cyclic (L-leucine-D-proline) dipeptide has a significant inhibitory effect on Vibrio anguillarum and Staphylococcus aureus and applied it to aquatic antibacterial agents. Specifically, it has a significant inhibitory effect on shrimp stealth disease, yellow gill disease, etc., providing a new prevention and treatment method for aquatic diseases caused by Vibrio anguillarum, reducing the content of Staphylococcus aureus in aquatic products, and helping to ensure the food safety of aquatic products. In addition, the cyclic (L-leucine-D-proline) dipeptide of the present invention can be isolated from Bacillus B1032 derived from mangroves, which allows the aquatic antibacterial agent of the present invention to be extracted from natural plants, without pollution to the environment and harmless to humans and animals. Among them, Bacillus B1032 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 2020498.

[0061] The above-described embodiments merely represent several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, and the present invention is intended to encompass such modifications and variations.

Claims

1. Application of a cyclic (L-leucine-D-proline) dipeptide in the preparation of an aquatic antibacterial agent, characterized in that: The bacteria are Vibrio anguillarum and Staphylococcus aureus. The content of the cyclic (L-leucine-D-proline) dipeptide in the aquatic antibacterial agent is 50-100 mg / kg. The structural formula of the cyclic (L-leucine-D-proline) dipeptide is as follows: 。 2. The use of a cyclic (L-leucine-D-proline) dipeptide in the preparation of an aquatic antibacterial agent according to claim 1, characterized in that: The application comprises the following steps: directly adding the cyclic (L-leucine-D-proline) dipeptide to aquaculture inputs, and mixing them evenly to prepare an aquaculture antibacterial agent.

3. The use of a cyclic (L-leucine-D-proline) dipeptide in the preparation of an aquatic antibacterial agent according to claim 1, characterized in that: The aquatic product is one or more of salmon, rainbow trout, eel, sweetfish, sea bass, cod, turbot, flounder, yellow croaker and shrimp.

4. The use of a cyclic (L-leucine-D-proline) dipeptide in the preparation of an aquatic antibacterial agent according to claim 2, characterized in that: The aquaculture input is one of aquatic feed, water conditioner and water.

5. The use of a cyclic (L-leucine-D-proline) dipeptide in the preparation of an aquatic antibacterial agent according to claim 2, characterized in that: The aquaculture input is an additive.

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