A granule for inhibiting the growth of vibrio in an aquaculture water body and a method of making the same

Abstract

The application discloses a kind of granules for inhibiting the growth of vibrio in aquaculture water, comprising chitosan, poly-β-hydroxybutyric acid and sodium alginate.The chitosan inhibits and kills vibrio, and the growth of bacillus provides carbon source and energy for poly-β-hydroxybutyric acid.The growth of bacillus further inhibits vibrio, thereby obtaining granules containing chitosan, poly-β-hydroxybutyric acid and sodium alginate.Chitosan is a cationic polysaccharide, and sodium alginate is an anionic polysaccharide.The chitosan and sodium alginate attract each other to form a stable network structure, and the poly-β-hydroxybutyric acid is the core material, thereby obtaining the granules.All components of the present application are biosynthetic substances, which can specifically act on vibrio without negative effects on other bacterial flora, are non-toxic and harmless, safe for aquaculture animals and have immune-enhancing effects, can be used in all types of aquaculture water, are easy to use, low in cost, biodegradable and have great market potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquaculture, in particular to a granule for inhibiting the growth of Vibrio in aquaculture water and a preparation method thereof. BACKGROUND

[0002] Bacterial and viral diseases can cause serious economic losses in the process of aquaculture production. Among the pathogenic bacteria, some species in the genus Vibrio are among the most common and widespread pathogens. Most pathogenic Vibrio has strong invasiveness, adapts to the environment in the host body and grows and reproduces in the host body. Vibrio infection can occur suddenly in the process of breeding most cultured animals, especially when the water quality deteriorates, it rains, the temperature changes, and the like, the cultured animals are stressed, and the Vibrio in the water can multiply and replicate in large numbers, causing the spread and prevalence of Vibrio, posing a serious threat to the breeding activities. Moreover, humans can also be infected with serious Vibrio disease if they drink water contaminated with Vibrio or eat uncooked aquatic products with pathogenic Vibrio. Therefore, effectively preventing the growth of Vibrio in the breeding process is one of the keys to the success of breeding and the safety of aquatic products.

[0003] At present, there is no effective vaccine against Vibrio disease in cultured animals, and the overuse of antibiotics can lead to drug resistance and may also leave residues in aquatic products. Compounds containing available chlorine are often used to prevent and control Vibrio in the process of aquaculture production, but these chemical agents usually cannot selectively kill pathogenic bacteria, and while killing pathogenic bacteria such as Vibrio, they will also kill other bacteria in the water, including beneficial bacteria such as Bacillus, causing the destruction of the ecological balance of aquaculture and being not conducive to the growth of cultured animals.

[0004] Researchers and producers have tried environmentally friendly and safe methods, including:

[0005] Method 1: Improve the ability of farmed animals to resist vibrio by changing the feed components. For example, Chinese invention patent (CN110833128 A Artificial compound feed for improving the ability of penaeus vannamei to resist vibrio, preparation method and application) discloses an artificial compound feed for improving the ability of penaeus vannamei to resist vibrio, a preparation method and application. Chinese invention patent (CN108497158 A Biocontrol fermented feed for preventing and treating vibrio disease and its production method and production equipment) discloses a biocontrol fermented feed for preventing and treating vibrio disease. Chinese invention patent (CN1062074 Method for making ecological regulation bait additive for penaeus vannamei vibrio disease) discloses an ecological regulation bait additive for penaeus vannamei, which is prepared from bacteriostatic agent, growth promoter, biological active agent and carrier clinoptilolite. Chinese invention patent (CN112516269 A Plant extract composition for resisting vibrio disease and its preparation method and application) provides a plant extract composition for resisting vibrio disease and its preparation method and application, which is used by mixing the extracted components into the feed of penaeus vannamei. The infection of farmed animals with vibrio indicates that the types and concentrations of vibrio in the water body have caused diseases in farmed animals. Oral or mixed feed methods can relieve or treat disease reactions caused by infection with vibrio in a short time, but cannot handle vibrio in the farming water body, and cannot fundamentally solve the problem, so there is still a great risk in the farming process.

[0006] Method 2: Add probiotics or protozoa to the water body. Chinese invention patent (CN109329649A A compound microecological preparation for antagonizing penaeus vibrio and its preparation method) discloses a compound microecological preparation for antagonizing penaeus vibrio and its preparation method. Chinese invention patent (CN109880767B A strain for preventing or treating penaeus vibrio disease) discloses a strain for preventing or treating penaeus vibrio disease. The microbial community structure in the water body is determined by the environmental conditions of the water body itself. For a stable farming environment, the addition of external bacteria may have some effect in a short time, but the added probiotics have their own specific environmental requirements and ultimately cannot survive and reproduce due to the inadaptation to the farming water body environment. Therefore, the method of adding bacteria may be more effective for open farming mode, but the effect is limited for farming mode with less water exchange and stable water environment. And different farming objects and farming modes have different water environments, whether the added probiotics can adapt and play a role needs to be further confirmed. Chinese invention patent CN114042089A, a method for preventing and treating penaeus vibrio disease by using marine protozoan stenoprummum. This method is more environmentally friendly, but the survival of the target protozoan needs to be considered, and the protozoan comes from natural seawater, so the effect of preventing vibrio in freshwater and brackish water needs to be further verified.

[0007] Method 3: Add water quality regulator. Chinese invention patent (CN1062261 A Vibrio disease ecological balance water quality regulator) discloses a water quality regulator for preventing and treating shrimp bacterial disease in the water of shrimp pond by ecological regulation method, a composition of three components, A component includes inorganic effective chlorine compounds and organic effective chlorine compounds, B component includes alkyl sodium sulfate and alkyl sodium sulfonate; C component includes white carbon black silver complex. However, chlorine compounds cannot selectively kill bacteria. By regulating the method of water quality, it lacks pertinence. And the additives are mostly chemical synthetic substances, which have certain risks.

[0008] Chitosan is a natural alkaline cationic polysaccharide obtained by deacetylation of chitin, which can increase the permeability of the outer membrane of gram-negative bacteria. The pathogenic bacteria such as Vibrio in the aquaculture water are mostly gram-negative bacteria, which means that their cell walls are relatively thin. Therefore, chitosan can inhibit and kill gram-negative bacteria such as Vibrio. In addition, chitosan also has the functions of improving immune regulation ability and antioxidant function, and is listed in the national food additive use standard GB-2760, and is widely used in aquaculture and many industries. Sodium alginate is a natural polyanionic compound polysaccharide extracted from kelp or sargassum of brown algae, which is non-toxic, biocompatible and has good adhesion. It has been widely used in cell and drug carriers, pharmaceutical excipients. Chitosan can form a complex with alginate through electrostatic interaction. Since both of them are natural polymer materials, they are non-toxic, biocompatible, and the complexing process is mild. In the past decade, they have been mainly used as carriers for wrapping cells, macromolecular proteins, genes and other substances with poor stability and short biological half-life (Yu Yichen et al., 2005. Preparation of chitosan-alginate nanoparticles and their drug loading and release properties for BSA. Journal of Functional Polymers, 4:598-601; Chinese invention patent CN 108541866 A Cinnamyl-alginate-chitosan nanoparticles and preparation method thereof). Poly-β-hydroxybutyrate (PHB) can release β-hydroxybutyric acid in water, which is used as carbon source and energy source by probiotics such as Bacillus subtilis, which helps to maintain the balance of microbial community in water. SUMMARY

[0009] In view of the deficiencies in the existing technology for preventing and treating Vibrio in aquaculture, the present application provides a granule for inhibiting the growth of Vibrio in aquaculture water and a preparation method thereof. The granule is made of dissolved chitosan, sodium alginate and crushed poly-β-hydroxybutyrate as main components. The granule can specifically act on Vibrio without negative effects on other bacteria, is safe for cultured animals and has immune enhancing effect, can be used in all types of aquaculture water, is easy to use, has low cost and is biodegradable.

[0010] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0011] A granule for inhibiting the growth of Vibrio in aquaculture water, comprising chitosan, poly-β-hydroxybutyric acid and sodium alginate, wherein the chitosan inhibits and kills Vibrio, the poly-β-hydroxybutyric acid provides carbon source and energy for the growth of Bacillus, and the growth of Bacillus further inhibits Vibrio, so as to obtain the granule containing chitosan, poly-β-hydroxybutyric acid and sodium alginate.

[0012] As a preferred embodiment, the chitosan is a cationic polysaccharide, the sodium alginate is an anionic polysaccharide, the chitosan and the sodium alginate attract each other to form a stable network structure, and the poly-β-hydroxybutyric acid is a core material, so as to obtain the granule.

[0013] A preparation method of a granule for inhibiting the growth of Vibrio in aquaculture water, comprising the following steps:

[0014] Step (1) 1 mg of chitosan is completely dissolved in 5 ml of acetic acid buffer under magnetic stirring at room temperature to obtain a chitosan solution;

[0015] Step (2) 1 ml of sodium tripolyphosphate solution with a concentration of 1 mg / ml is mixed with 1 ml of sodium alginate solution with a concentration of 0.45 mg / ml to obtain a sodium tripolyphosphate-sodium alginate solution;

[0016] Both the two solutions in step (2) are prepared in the same 0.1 mol / L acetic acid buffer to ensure that the pH value remains unchanged in the subsequent preparation steps;

[0017] Step (4) poly-β-hydroxybutyric acid powder is added to the chitosan solution in step 1, and ultrasonic treatment is performed at 100 kHz for 5 minutes, and the mixture is fully mixed to obtain a chitosan suspension of poly-β-hydroxybutyric acid with a concentration of 1.0 mg / ml,

[0018] The sodium tripolyphosphate-sodium alginate solution is added to the chitosan-poly-β-hydroxybutyric acid solution, the concentration of poly-β-hydroxybutyric acid in the final suspension is 100 mg / L, and the suspension is magnetically stirred for 30 minutes to obtain chitosan, poly-β-hydroxybutyric acid and sodium alginate granules with a particle size of 450-1000 nm;

[0019] Step (5) the obtained granules are washed with distilled water and vacuum dried at room temperature for 2 days to completely remove the solvent molecules;

[0020] Step (6) the dried chitosan, poly-β-hydroxybutyric acid and sodium alginate granules are vacuum packaged and stored at room temperature for use.

[0021] As a preferred embodiment, the magnetic stirring in step (1) is 1000 rpm; the concentration of the acetic acid solution in step (1) is 0.1 mol / L, and pH = 5.2.

[0022] As a preferred embodiment, the chitosan in step (1) must be completely dissolved, otherwise it will cause uneven particle formation in the subsequent steps, affecting the quality of the final product, and in actual operation, it is necessary to ensure that the chitosan is completely dissolved, which can appropriately increase the stirring time or adjust the pH value of the buffer.

[0023] As a preferred embodiment, the concentration of the sodium tripolyphosphate solution in step (2) is 1 mg / ml; the concentration of the sodium alginate solution is 0.45 mg / ml.

[0024] The ratio of sodium tripolyphosphate to sodium alginate in step (2) is equal to 5:1; the ratio of chitosan to sodium alginate is equal to 11:1.

[0025] As a preferred embodiment, the stirring speed in step (4) is 750 rpm.

[0026] As a preferred embodiment, in step (4), the mixing speed of the sodium tripolyphosphate-sodium alginate solution needs to be controlled well to ensure uniform particle formation, and at the same time, the stirring time also needs to be long enough to ensure uniform particle composition.

[0027] As a preferred embodiment, in step (5), the use of ultrasonic treatment can make the β-hydroxybutyric acid ester uniformly dispersed in the chitosan solution, but attention should be paid to the time and intensity of the ultrasonic treatment to avoid excessive damage to the molecular structure of poly-β-hydroxybutyric acid ester.

[0028] As a preferred embodiment, in step (6), the washing of the particles needs to use distilled water that is pure enough and ensure complete washing, otherwise the impurities remaining on the surface of the particles will affect the quality of the final product.

[0029] Beneficial effects: The granules for inhibiting the growth of Vibrio in aquaculture water and the preparation method thereof, all components are biosynthetic substances, chitosan, poly-β-hydroxybutyric acid ester, and sodium alginate, which have good granulation performance and the best performance of killing Vibrio at the concentration of the present application. It can specifically act on Vibrio without negative effects on other bacterial flora, is non-toxic and harmless, is safe for farmed animals and has immune enhancing effect, can be used in all types of aquaculture water, is easy to use, has low cost, is biodegradable, and has great market potential. DETAILED DESCRIPTION

[0030] The following detailed description of the embodiments of the present application is based on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0031] A method for preparing a particle for inhibiting the growth of Vibrio in a cultured water body, comprising the following steps:

[0032] Step (1) 1000 mg of chitosan is weighed and completely dissolved in 5000 ml of acetic acid buffer (acetic acid solution concentration is 0.1 mol / L, pH = 5.2) under magnetic stirring (1000 rpm) at room temperature.

[0033] Step (2) 1000 ml of sodium tripolyphosphate solution (concentration is 1 mg / ml) and 1000 ml of sodium alginate solution (concentration is 0.45 mg / ml) are mixed to obtain the ratios of chitosan: sodium tripolyphosphate and chitosan: sodium alginate being 5:1 and 11:1 respectively.

[0034] Step (3) Both solutions are prepared in the same 0.1 M acetic acid buffer to ensure that the pH value remains unchanged in the subsequent preparation steps.

[0035] Step (4) At room temperature, the sodium tripolyphosphate-sodium alginate solution is mixed into the chitosan solution kept under magnetic stirring (750 rpm) at a rate of 1 ml per minute to form particles. The stirring is kept for 30 minutes to ensure that the composition of the obtained particles is uniform.

[0036] Step (5) Poly-β-hydroxybutyric acid powder is added to the chitosan solution, and ultrasonic treatment at 100 kHz is performed for 5 minutes to fully mix, obtaining a chitosan suspension with a concentration of 1.0 mg / ml of poly-β-hydroxybutyric acid. The sodium tripolyphosphate-chitosan solution is added to the chitosan-poly-β-hydroxybutyric acid solution, and the concentration of poly-β-hydroxybutyric acid in the final suspension is 100 mg / L. After the suspension is magnetically stirred for 30 minutes, chitosan, poly-β-hydroxybutyric acid and sodium alginate particles with a particle size of about 450-1000 nm are obtained.

[0037] Step (6) The obtained particles are washed with distilled water and vacuum dried at room temperature for 2 days to completely remove the solvent molecules.

[0038] Step (7) The dried chitosan, poly-β-hydroxybutyric acid and sodium alginate particles are vacuum packaged and stored at room temperature for use.

[0039] Application effect of the prepared chitosan-poly-β-hydroxybutyric acid-sodium alginate particles:

[0040] In 6 small sheds, earth pools, HDPE film, 18 m x 2.5 m x 1.5 m, water depth 1 m. Each breeding pool into the sea about 15 m 3 , supplemented with tap water to dilute salinity to 10. The first three days, 200 g of powder / m 3 , 100 g of glucose / m 3 , sufficient aeration, dissolved oxygen maintained at more than 6 mg / L, when the ammonia nitrogen and nitrite nitrogen is stable at 0.5 mg / L or less, put in SPF shrimp (0.75 ± 0.5) cm, stocking density of 700 PL / m 3 . The second day after the release of the feed white shrimp feed. Each day 8 times feeding, respectively, at 5:00, 8:00, 11:00, 13:00, 16:00, 19:00, 21:00, 24:00, according to the size of the shrimp to adjust the amount of feed. Water temperature during the breeding process 24 - 29℃, dissolved oxygen 6 mg / L or more, carbonate alkalinity 80 mg / L CaCO3 or so, pH above 8.2. The breeding process without water change. Daily determination of ammonia nitrogen and nitrite concentration, ammonia nitrogen to 2 mg / L, nitrite nitrogen to 0.5 mg / L, according to the 1 mg N to supplement 6 mg carbon to supplement glucose. Of which 3 breeding ponds according to 10 g chitosan-poly-β-hydroxybutyrate-sodium alginate particles per cubic breeding water, every 3 days whole pool splash once; another 3 breeding ponds without adding. Independent operation between the breeding ponds during the breeding process, all the instruments are fixed and regularly disinfected. During the breeding process, the water in the breeding pond is taken out after about 20 days and detected by TCBS agar medium to determine the number of vibrio.

[0041] During the breeding process, the shrimp with chitosan-poly-β-hydroxybutyrate-sodium alginate particles grew well and no obvious death was observed; the group without adding showed obvious death in the later period. After 98 days, the shrimp was collected and weighed, and the growth of the shrimp in the two test groups was shown in Table 1:

[0042]

[0043] The vibrio count: during the whole test process, the number of vibrio in the group with chitosan-poly-β-hydroxybutyrate-sodium alginate particles was significantly lower than that in the control group (Table 2).

[0044] Table 2 Effect of chitosan-poly-β-hydroxybutyrate-sodium alginate particles on the average number of vibrio (cfu / ml) in the breeding water:

[0045]

[0046] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method of preparing a granule for inhibiting the growth of Vibrio spp. in an aquaculture water body, characterized by, The method comprises the following steps: Step (1) 1 mg of chitosan is completely dissolved in 5 ml of acetic acid buffer solution under magnetic stirring at room temperature to obtain a chitosan solution; Step (2) 1 ml of a sodium tripolyphosphate solution with a concentration of 1 mg / ml is mixed with 1 ml of a sodium alginate solution with a concentration of 0.45 mg / ml to obtain a sodium tripolyphosphate-sodium alginate solution; Step (3) both the solutions in step (2) are prepared in the same 0.1 mol / L acetic acid buffer solution to ensure that the pH value remains unchanged in the subsequent preparation steps; Step (4) poly-β-hydroxybutyrate powder is added to the chitosan solution in step 1, and ultrasonic treatment is performed at 100 kHz for 5 minutes, and the mixture is fully mixed to obtain a chitosan suspension of poly-β-hydroxybutyrate with a concentration of 1.0 mg / ml, The sodium tripolyphosphate-sodium alginate solution is added to the chitosan-poly-β-hydroxybutyrate solution, and the concentration of poly-β-hydroxybutyrate in the final suspension is 100 mg / L, and the suspension is magnetically stirred for 30 minutes to obtain chitosan, poly-β-hydroxybutyrate and sodium alginate particles with a particle size of 450-1000 nm; Step (5) the obtained particles are washed with distilled water and vacuum dried at room temperature for 2 days to completely remove the solvent molecules; Step (6) the dried chitosan, poly-β-hydroxybutyrate and sodium alginate particles are vacuum packaged and stored at room temperature for use.

2. A method of preparing a granule for inhibiting the growth of Vibrio spp. in an aquaculture water body according to claim 1, characterised in that, The magnetic stirring in step (1) is 1000 rpm; the concentration of the acetic acid solution in step (1) is 0.1 mol / L, and the pH value is 5.

2.

3. The method for preparing particles for inhibiting the growth of Vibrio in aquaculture water according to claim 1, characterized in that, The chitosan in step (1) must be completely dissolved, otherwise it will cause uneven particle formation in the subsequent steps, affecting the quality of the final product. In actual operation, in order to ensure that the chitosan is completely dissolved, the stirring time is increased or the pH value of the buffer solution is adjusted.

4. The method of claim 1, wherein the particles are prepared by the steps of: a) providing a mixture of a biocide and a carrier; b) forming the mixture into particles; and c) drying the particles. The concentration of the sodium tripolyphosphate solution in step (2) is 1 mg / ml; the concentration of the sodium alginate solution is 0.45 mg / ml; The ratio of sodium tripolyphosphate to sodium alginate in step (2) is equal to 5:1; the ratio of chitosan to sodium alginate is equal to 11:

1.

5. The method of claim 1, wherein the particles are prepared by the steps of: a) providing a mixture of a biocide and a carrier; b) mixing the mixture to form a slurry; c) drying the slurry to form a powder; and d) milling the powder to form particles. The stirring speed in step (4) is 750 rpm.

6. The method of claim 1, wherein the particles are prepared by the steps of: a) providing a mixture of a biocide and a carrier; b) forming the mixture into a plurality of particles; and c) drying the particles. In step (4), the use of ultrasonic treatment can make β-hydroxybutyrate uniformly dispersed in the chitosan solution, but attention should be paid to the time and intensity of ultrasonic treatment to avoid damaging the molecular structure of poly-β-hydroxybutyrate.

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