Method to improve hatching rate of Artemia using Exiguabacterium genus strain

Abstract

The present invention relates to a method for improving the hatching rate of Artemia using Exiguobaterium spp., wherein a strain of Exiguobaterium spp. is inoculated into an Artemia cyst culture tank and cultured together with Artemia cysts, thereby improving the hatching rate of Artemia, which can be utilized for the mass production of Artemia, a live food for fry.

Description

Technology Field

[0001] The present invention relates to the genus Exiguabacterium ( Small battery This relates to a method for improving the hatching rate of Artemia using strains of spp.) and, more specifically, to a method for improving the hatching rate of Artemia using strains of the genus Exiguabacterium ( Small battery This invention relates to a method for improving the hatching rate of Artemia by inoculating a strain of Artemia spp. and culturing it together with Artemia cysts. Background Technology

[0003] Since the early survival rate of fry is crucial for mass-producing high-quality individuals in aquaculture, various live feed formulations are being used instead of artificial feed to improve the health of the fry. Live feed consists of whiteleg shrimp ( Penaeus vannamei It increased the survival rate of fry by 12.5%, and also showed higher growth rates and energy efficiency compared to artificial feed. In addition, catfish ( Clarias batrachus It induced high growth rates and body weight in the fry of ) and also improved the survival rate of rainbow trout fry.

[0004] Generally, phytoplankton and small zooplankton, particularly rotifers, are suitable for Stage 1 fry feed (4–7 dph after hatching), while small aquatic crustaceans (copepods, Artemia) are suitable for Stage 2 fry feed (7–14 dph) and have been widely used in fry aquaculture. Providing live food plays an important role in supplying the necessary nutrients and bacterial load to larvae, but carelessness can introduce pathogenic bacteria into the fry culture medium. Lactococcus garvieae, Tenacibaculum discolor and Vibrio Some fish pathogens, such as spp., have been found in rotifers before being fed to fish, and some antibiotic-resistant bacteria that can cause disease in hosts have been isolated from Artemia.

[0005] Since such pathogen invasions deplete the host's nutritional resources, destroy beneficial bacterial populations, and disrupt the gut balance, it is essential to regulate the microbiome of live prey to alleviate bacterial infections in the host organism.

[0006] Furthermore, the microbial communities inhabiting the gastrointestinal tracts of fish fry are influenced by the bacterial composition of live food and the quality of the water used during feeding; these communities play a crucial role in the health of the host fish by aiding food digestion, regulating the immune system, and producing beneficial metabolites. To ensure the consistent production of healthy fish fry, a comprehensive understanding of the microbial communities in live food and the rearing water is essential.

[0007] Brine shrimp belonging to the genus Artemia are widely distributed from tropical to temperate regions, and in extreme environments, they protect their young by producing dormant cysts encased in a protective membrane. There are different species of Artemia capable of producing cysts, for example, Artemia franciscana ( Artemia franciscana ), Artemia persimilis ( Artemia very similar ), Artemia urmiana ( Artemia urmiana ), Artemia Tunisian ( Artemia tunisian ), Artemia tibetiana ( Artemia tibetana ), Artemia sinica ( Chinese brine shrimp ) and Artemia partogenetica ( Artemia parthogenetica These cysts can hatch in a more appropriately controlled environment, so they have been used as live aquaculture feed worldwide.

[0008] As mentioned above, the inventors [use] Exiguabacterium genus among the microbial groups of Artemia ( Small batteryThe present invention has been completed regarding a method to improve the hatching rate of Artemia cysts using strains spp.). Prior art literature

[0010] Korean Patent Publication No. 10-1333901 (2013.11.21.)

[0011] Wilkes Walburn J, Wemheuer B, Thomas T, Copeland 535 E, O'Connor W, Booth M, Fielder S, 536 Egan S, Microb Biotechnol. 2019 Mar, 12(2): 275-288. [DOI: 10.1111 / 1751-7915.13323]Rani M, Kumar S, Gupta RK, Yadava NK, Int j fish aquat sci. 2020, 8:360-36. The problem to be solved

[0012] The object of the present invention is the genus Exiguabacterium ( Small battery The purpose is to provide a method to improve the hatching rate of Artemia using strains (spp.).

[0013] The object of the present invention is an Exiguabacterium genus characterized by improving the hatching rate of Artemia ( Small battery It is to provide spp.) strains.

[0015] The technical problems that the invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the invention belongs from the description below. means of solving the problem

[0017] To solve the above problem, the present invention relates to the genus Exiguabacterium ( Small battery A step of inoculating and culturing a strain of spp.) into a culture medium; and the cultured Exiguabacterium genus ( Small batteryA method for improving the hatching rate of Artemia is provided, characterized by including the step of inoculating an Artemia cyst culture tank with a strain of spp.) to hatch Artemia.

[0018] The above genus Exiguabacterium ( Small battery The step of inoculating and culturing the spp.) strain into a medium involves the above Exiguabacterium genus ( Small battery It is characterized by inoculating a strain of spp.) and culturing it at 29 to 31°C for 2 to 4 days.

[0019] The hatching rate of the above Artemia is the above cultured Exiguabacterium ( Small battery It is characterized by the fact that the strain (spp.) improves for 17 to 25 hours after inoculating it into an Artemia cyst culture tank.

[0020] The hatching rate of the above Artemia is the above inoculated Exiguabacterium genus ( Small battery It is characterized by an improved hatching rate due to the spp.) strains forming colonies on the surface of Artemia cysts.

[0022] In addition, the present invention is characterized by improving the hatching rate of Artemia, and the genus Exiguabacterium ( Small battery Provides spp.) strains.

[0023] The above Artemia is Artemia franciscana , Artemia very similar , Artemia urmiana , Artemia tunisian , Artemia tibetana , Chinese brine shrimp and Artemia parthogenetica It is characterized by being one type selected from a group consisting of

[0024] The above genus Exiguabacterium ( Small battery spp.) strains Exiguobacterium oxidotalerans, Exiguobacterium acetylicum, Exiguobacterium indicum, Exiguobacterium enclense, Exiguobacterium antercticum, Exiguobacterium soli, Exiguobacterium undae, Exiguobacterium sibiricum, Exiguobacterium artemiae and Exiguobacterium flavidum It is characterized by being one type selected from a group composed of Effects of the invention

[0026] The present invention relates to the genus Exiguabacterium ( Small battery This relates to a method for improving the hatching rate of Artemia using strains of the spp.) strain, wherein Exiguabacterium genus ( Small battery By inoculating the spp.) strain and culturing it together with Artemia cysts, the hatching rate of Artemia is improved, which can be utilized for the mass production of Artemia, a live food for fry.

[0028] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description in the claims. Brief explanation of the drawing

[0030] Fig. 1 is Artemia franciscana The genus Exiguabacterium, the dominant species for ( Small battery spp.) strains and the genus Cyclobacter ( Psychrobacter This is a graph analyzing the hatching rates of Artemia by spp.) strains. Figure 2 shows the genus Exiguabacterium ( Small battery This is a phylogenetic tree classifying strains (spp.). Fig. 3 is Artemia tibetana In the cyst culture tank Exiguabacterium genus ( Small battery Inoculate with the spp.) strain for 18 hours Artemia tibetana This is a graph analyzing the hatching rate of. Fig. 4 is Artemia tibetana In the cyst culture tank Exiguabacterium genus ( Small battery Inoculate with the spp.) strain for 24 hours Artemia tibetana This is a graph analyzing the hatching rate of. Fig. 5 is Artemia franciscana In the cyst culture tank Exiguabacterium genus ( Small battery Inoculate with the spp.) strain for 18 hours Artemia franciscana This is a graph analyzing the hatching rate of. Fig. 6 is Artemia franciscana In the cyst culture tank Exiguabacterium genus ( Small batteryInoculate with the spp.) strain for 24 hours Artemia franciscana This is a graph analyzing the hatching rate of. Fig. 7 is Exiguobacterium oxidotalerans After inoculating the strain into an Artemia cyst culture tank, the formed on the surface of the Artemia cyst Exiguobacterium oxidotalerans This is a diagram showing a strain colony as an SEM image. Fig. 8 shows the genus Exiguabacterium ( Small battery This is a schematic diagram of a method to improve the hatching rate of Artemia using spp.). Specific details for implementing the invention

[0031] The terms used in this specification will be briefly explained, and the invention will be described in detail.

[0032] The terms used in this invention have been selected based on currently widely used general terms while considering their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall context of the invention.

[0033] When a part of a specification is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0034] Embodiments of the present invention are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0035] Specific details regarding the problem to be solved by the present invention, the means for solving the problem, and the effects of the invention are included in the embodiments and drawings described below. The advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described below in detail together with the accompanying drawings.

[0036] Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

[0038] The present invention relates to the genus Exiguabacterium ( Small battery A step of inoculating and culturing a strain of spp.) into a culture medium; and the cultured Exiguabacterium genus ( Small battery A method for improving the hatching rate of Artemia is provided, characterized by including the step of inoculating an Artemia cyst culture tank with a strain of spp.) to hatch Artemia.

[0039] The above genus Exiguabacterium ( Small battery The step of inoculating and culturing the spp.) strain into a medium involves the above Exiguabacterium genus (in marine broth 2216 (MB;DB)) in a marine liquid medium. Small battery The strain of spp.) can be inoculated and cultured at 29 to 31°C for 2 to 4 days, preferably in a marine broth (marine broth 2216 (MB;DB)) medium containing the genus Exiguabacterium ( Small battery spp.) strains can be inoculated and cultured at 30°C for 3 days.

[0040] The step of hatching the above Artemia is the cultured Exiguabacterium genus ( Small battery Hatching of Artemia can be induced by inoculating the spp.) strain into an Artemia cyst culture tank and culturing it together with Artemia cysts.

[0041] The hatching rate of the above Artemia is the above cultured Exiguabacterium ( Small batteryThe spp.) strain can be enhanced for 17 to 25 hours after inoculation into an Artemia cyst culture tank, preferably the cultured Exiguabacterium genus ( Small battery spp.) strains can be enhanced for 18 to 24 hours after inoculation into an Artemia cyst culture tank.

[0042] The hatching rate of the above Artemia is the above inoculated Exiguabacterium genus ( Small battery The hatching rate may be improved because the spp.) strains form colonies on the surface of the Artemia cyst.

[0043] The hatching rate of the above Artemia can be improved to 10 to 50%.

[0045] In addition, the present invention is characterized by improving the hatching rate of Artemia, and the genus Exiguabacterium ( Small battery Provides spp.) strains.

[0046] The above Artemia is Artemia franciscana , Artemia very similar , Artemia urmiana , Artemia tunisian , Artemia tibetana , Chinese brine shrimp and Artemia parthogenetica It is preferable that it be one type selected from the group consisting of, Artemia tibetana or Artemia franciscana It is more desirable to select one type from.

[0047] The above genus Exiguabacterium ( Small battery spp.) strains Exiguobacterium oxidotalerans, Exiguobacterium acetylicum, Exiguobacterium indicum, Exiguobacterium enclense, Exiguobacterium antercticum, Exiguobacterium soli, Exiguobacterium undae, Exiguobacterium sibiricum, Exiguobacterium artemiae and Exiguobacterium flavidum It is preferable that it be one type selected from the group composed of, Exiguobacterium acetylicum and Exiguobacterium oxidotalerans It is more desirable to select one of the types.

[0049] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0051] <실시예 1> 아타미아 이스트 부화

[0052] Commercially available Artemia cysts were used, and the hatching of the cysts was carried out according to the manufacturer's experimental method. 3.75 g of Artemia cysts were inoculated into 1.5 L of water containing 34 psu (practical salinity units) and hatched by incubating at 28°C for 24 hours under artificial natural light of 2000 lux.

[0054] <실시예 2> 엑시구아박현리움 웈 ( Small battery spp.)

[0055] Exiguabacterium genus ( Small battery The strains of the genus Exiguabacterium were inoculated into marine broth 2216 (MB;DB) medium and cultured at 30°C for 3 days. The cultured strains of Exiguabacterium were then inoculated into an Artemia cyst culture tank and cultured with Artemia cysts for 18 to 24 hours under the same culture conditions as in Example 1 to induce hatching, after which the hatching rate was measured. All experiments were repeated three times. For the analysis of the hatching rate, hatched individuals were identified using a microscope, and statistical analysis was performed using Graphpad software (Inc. La Jolla, CA).

[0057] <비교예>

[0058] Artemia cysteine, genus Exiguabacterium ( Small battery Without inoculating the spp.) strain, incubation was carried out for 18 to 24 hours under the same incubation conditions as in Example 1 above.

[0060] <실호예 1> Artemia franciscana 에 대한 우점종의 comparison

[0061] Microbiome analysis confirmed microbial differences among Artemia species, and among them, those with a high hatching rate Artemia franciscana The dominant species is the genus Exiguabacterium ( Small battery spp.) strains and the genus Cyclobacter ( Psychrobacter It was found to be a strain of spp.) Artemia franciscana The dominant species of the genus Exiguabacterium ( Small battery spp.) strains and the genus Cyclobacter ( Psychrobacter Artemia by spp.) strains The hatching rate was checked as shown in Fig. 1. Cyclobacter genus ( Psychrobacter Exiguabacterium genus (than spp.) strains ( Exiguobaterium It was confirmed that the hatching rate of Artemia by the spp.) strain was higher.

[0062] Exiguabacterium genus with a high hatching rate of the above Artemia ( Exiguobaterium The strains (spp.) were classified as shown in Figure 2, and subsequently classified into Group I and Group II to evaluate their efficacy in improving the hatching rate of Artemia.

[0064] <Experimental Example 2> Exiguabacterium genus ( Exiguobaterium using spp.) strains Artemia tibetiana Improvement of hatching rate

[0065] Exiguabacterium genus ( Exiguobaterium spp.) strains were tested as in Examples 1 and 2 above. Artemia tibetiana The hatching rate was checked. When Artemia were hatched for 18 hours, as shown in Fig. 3, Exiguobacterium mexicanum The remaining strains excluding (strain MEBiC 9034) Exiguobacterium acetylicum (strain MMD254), Exiguobacterium oxidotalerans (strain MEBiC 10663), Exiguobacterium aurantiacum(strain MEBiC 8563) and Exiguobacterium profundum by strains (strain MEBiC 12417) Artemia tibetiana It was confirmed that the hatching rate of improved. In particular, Exiguobacterium oxidotalerans (strain MEBiC 10663) cultured together with strain Artemia tibetiana It was confirmed that the hatching rate was improved by more than 30% compared to the control.

[0066] In addition, when Artemia was hatched for 24 hours, as shown in Fig. 4, Exiguobacterium acetylicum (strain MMD254), Exiguobacterium oxidotalerans (strain MEBiC 10663), Exiguobacterium profundum By strains (strain MEBiC 12417) Artemia tibetiana The hatching rate increased, showing a higher rate than when Artemia was hatched for 18 hours. In particular Exiguobacterium acetylicum (strain MMD254) cultured together Artemia tibetiana It was confirmed that the hatching rate of the control increased the highest, by more than 40%.

[0068] <Experimental Example 3> Exiguabacterium genus ( Exiguobaterium Artemia Franciscana using spp.) strains ( Artemia franciscana Improvement of ) hatching rate

[0069] Exiguabacterium genus ( Exiguobaterium spp.) strains were tested as in Examples 1 and 2 above. Artemia franciscana The hatching rate was checked. When Artemia were hatched for 18 hours, as shown in Fig. 5, Exiguobacterium oxidotalerans cultured with strain MEBiC 10663 Artemia franciscana It was confirmed that the hatching rate was improved only in [location], and even when hatching Artemia was performed for 24 hours Exiguobacterium oxidotalerans cultured with strain MEBiC 10663 Artemia franciscana It was confirmed that the hatching rate was maintained only in [the area].

[0070] Experimental Example 2 Artemia tibetiana Rather than the improved hatching rate efficacy of Artemia franciscana The low efficacy of the improved hatching rate is Artemia franciscana Exiguabacterium, the dominant species of microorganisms in the body ( Exiguobaterium This is likely because spp.) strains already exist.

[0071] The results of the above Experiment Examples 2 and 3 are of the genus Exiguabacterium of Group II (Group II) shown in Fig. 2 ( Exiguobaterium It can be seen that spp.) strains mainly improve the hatching rate of Artemia.

[0073] <Experimental Example 4> Exiguabacterium genus improving the hatching rate of Artemia ( Exiguobaterium Colonization of Artemia cyst surfaces of spp.) strains

[0074] Exiguobacterium oxidotalerans The strain (strain MEBiC 10663) as in Examples 1 and 2 above Artemia franciscana When inducing hatching by culturing with cysts, the surface of the Artemia cysts was examined using a scanning electron micrograph (SEM) as shown in Fig. 7. The Artemia cysts that improved the hatching rate Exiguobacterium oxidotalerans It was confirmed that the strain MEBiC 10663 formed colonies on the surface of the cyst. This is the above Exiguobacterium oxidotalerans It is believed that the improvement in hatching rate was induced through interaction with the surface of Artemia cysts as microorganisms such as strain MEBiC 10663 formed a colony.

[0076] As specific parts of the present invention have been described in detail above, it is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention shall be defined by the appended claims and their equivalents. The scope of the invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and their equivalents shall be interpreted as being included within the scope of the invention.

Claims

Claim 1 Exiguabacterium genus ( Exiguobaterium A step of inoculating a strain of spp.) into a marine broth medium and culturing it at 29 to 31°C for 2 to 4 days; and the cultured Exiguabacterium genus ( Exiguobaterium A method for improving the hatching rate of Artemia, characterized by comprising the step of inoculating an Artemia cyst culture tank with an Artemia strain (spp.) and hatching Artemia for 17 to 25 hours. Claim 2 Deleted Claim 3 Deleted Claim 4 In paragraph 1, the hatching rate of the Artemia is the inoculated Exiguabacterium genus ( Exiguobaterium A method for improving the hatching rate of Artemia, characterized by the hatching rate being improved due to the formation of colonies of spp.) strains on the surface of Artemia cysts. Claim 5 Artemia franciscana , Artemia persimilis , Artemia urmiana , Artemia tunisian , Artemia tibetiana , Artemia sinica and Artemia parthogenetica Improving the hatching rate of Artemia, one species selected from a group consisting of, and Exiguobacterium oxidotalerans, Exiguobacterium acetylicum, Exiguobacterium indicum, Exiguobacterium enclense, Exiguobacterium antercticum, Exiguobacterium soli, Exiguobacterium undae, Exiguobacterium sibiricum, Exiguobacterium artemiae and Exiguobacterium flavidum Exiguabacterium genus characterized as being one species selected from the group composed of ( Exiguobaterium spp.) strains. Claim 6 Deleted Claim 7 Deleted

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