Detailed description of the invention
Technical challenge
[9]
As a result of the present inventors making diligent efforts to develop shrimp feed with shrimp AHPND or probiotics (Bacillus sp.) for the prevention of white spot syndrome, the feed composition containing Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis was fed to shrimp In this case, AHPND infection (a strain isolated from Vietnam, isolated in 2013. Tran et. al. 2013.) or WSSV infection improves the survival rate of shrimp, increases the growth rate and non-specific immunity of shrimp, By confirming that the water quality is improved and the production of high protein shrimp is possible, the present invention was completed.
[10]
Means of solving the task
[11]
One object of the present invention is Bacillus subtilis strain, Bacillus pumilus strain and Bacillus licheniformis strain; Cultures thereof; Its concentrate; Or to provide a feed composition for the prevention or treatment of acute hepatic pancreatic necrosis (AHPND) comprising a dried product thereof as an active ingredient.
[12]
Another object of the present invention is to provide a method for preventing or treating acute hepatic pancreatic necrosis comprising administering the feed composition to an individual.
[13]
One object of the present invention is Bacillus subtilis strain, Bacillus pumilus strain and Bacillus licheniformis strain; Cultures thereof; Its concentrate; Or it is to provide a feed composition for the prevention or treatment of white spot syndrome (WSS) comprising the dried product as an active ingredient.
[14]
Another object of the present invention is to provide a method for preventing or treating white spot syndrome comprising administering the feed composition to an individual.
[15]
Effects of the Invention
[16]
In the present invention, Bacillus subtilis KCCM11143P strain, Bacillus pumilus KCCM11144P strain, and Bacillus licheniformis KCCM11270P strain have antibacterial activity against enteritis Vibrio bacteria that cause AHPND, which is a problem in shrimp farming. It has an antiviral activity against the white spot syndrome virus that causes, and an immunity enhancing effect of the liver and pancreas of shrimp, so it can be used as a shrimp feed composition or feed additive.
[17]
Brief description of the drawing
[18]
1 is a graph showing the survival rate of shrimp infected with enteritis Vibrio bacteria.
[19]
2 is a graph showing the analysis of AHPND content in shrimp liver pancreas.
[20]
Figure 3 is a diagram showing the pathological characteristics of the shrimp liver pancreas.
[21]
Figure 4 is a graph showing the growth rate of the shrimp, (a) to (d) each shows the final weight, weight gain, daily growth rate and feed conversion rate.
[22]
Figure 5 is a graph of the analysis of the non-specific immunity of shrimp, (a) to (e) each shows the activity against macrophages, phenol oxidase, anti-proteinase, lysozyme, and superoxide dismutase.
[23]
6 is a graph showing the results of water quality analysis in non-exchange breeding water.
[24]
Best mode for carrying out the invention
[25]
This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the specific description described below.
[26]
[27]
As an embodiment for solving the above problems, the present invention Bacillus subtilis strain, Bacillus pumilus strain and Bacillus licheniformis strain; Cultures thereof; Its concentrate; Or it provides a feed composition for the prevention or treatment of acute hepatic pancreatic necrosis (AHPND) comprising a dried product thereof as an active ingredient.
[28]
The term "Bacillus subtilus (Bacillus subtilus, Bacillus subtilis)" of the present invention is a type of aerobic bacteria that is not toxic and is a bacterium that produces spores. It is widely distributed in dry grass, soil, sewage, and air. It is widely used in industrial fields because it produces enzymes to coagulate milk, saccharify starch and decompose fats and oils. The optimum state of growth is pH 7~8.5, temperature 37~40℃. Due to the nature of the strain of the genus Bacillus, it does not possess toxic genes for humans and animals, and is not only non-pathogenic by itself, but also does not produce pathogenic substances, and exhibits a rapid growth rate in vivo. In an environment in which glucose is present, it lives anaerobic, and endospores allow Bacillus to survive in extremely bad environments such as high or low temperatures.
[29]
The term "Bacillus pumilus" of the present invention is a kind of gram-positive and aerobic bacteria, and produces spores. It is distributed in the soil or exists by forming colonies on the roots of some plants. Bacillus fumilus spores generally exhibit high resistance to environmental stresses, including exposure to UV light, desiccation and the presence of oxidizing agents such as hydrogen peroxide.
[30]
The term "Bacillus licheniformis" of the present invention is a bacterium commonly found in soil, which is Gram-positive and mesophilic. The optimum growth temperature is about 50°C, but it can survive even higher temperatures. Bacillus licheniformis can exist in the form of dormant spores to resist harsh environments.
[31]
In the present invention, the Bacillus subtilis may be a strain deposited with accession number KCCM11143P.
[32]
In the present invention, the Bacillus pumilus may be a strain deposited with accession number KCCM11144P.
[33]
In the present invention, the Bacillus licheniformis may be a strain deposited with accession number KCCM11270P.
[34]
In the present invention, a feed composition comprising the Bacillus subtilis KCCM11143P, the Bacillus pumilus KCCM11144P, and the Bacillus licheniformis KCCM11270P was prepared.
[35]
The composition of the present invention may include Bacillus subtilis KCCM11143P, the Bacillus pumilus KCCM11144P, and the Bacillus licheniformis KCCM11270P having a number of bacteria of 1 x 10 4 to 1 x 10 11 CFU per g of the total active ingredient , Specifically, it may be 1 x 10 4 to 1 x 10 10 CFU / g, more specifically 1 x 10 8 to 1 x 10 10 CFU / g of Bacillus subtilis KCCM11143P, the Bacillus pumilus KCCM11144P, and It includes the Bacillus licheniformis KCCM11270P.
[36]
For the purposes of the present invention, Bacillus subtilis included as an active ingredient in the feed composition may include only Bacillus subtilis KCCM11143P.
[37]
For the purposes of the present invention, Bacillus pumilus included as an active ingredient in the feed composition may include only Bacillus pumilus KCCM11144P.
[38]
For the purposes of the present invention, Bacillus licheniformis included as an active ingredient in the feed composition may include only Bacillus licheniformis KCCM11270P.
[39]
The term "acute hepatopancreatic necrosis (AHPND)" of the present invention refers to a disease named as early mortality syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS), when stocked in a farm. It collectively refers to mass mortality caused by pathogens within one day. There are many cases of infection in white-legged shrimp, which account for about 96% of farmed shrimp in Korea, and it is a disease caused by Vibrio parahaemolyticus , a pathogen that exists in seawater . Since the mortality rate is high in childhood, it causes fatal damage to shrimp, but is harmless to the human body. The " Vibrio parahaemolyticus " refers to a gram-negative bacillus belonging to the genus Vibrio, which causes acute food poisoning and enteritis in humans, and Vibriosis in fish. Recently, it was found to be the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND) that causes mass mortality in the shrimp farming industry.
[40]
The term "prevention" of the present invention refers to any action of suppressing or delaying symptoms caused by acute hepatic pancreatic necrosis (AHPND) of shrimp by administration of a composition comprising Bacillus subtilis according to the present invention.
[41]
The term "treatment" of the present invention refers to any action in which symptoms caused by acute hepatic pancreatic necrosis (AHPND) of shrimp are improved or cured by administration of a composition comprising Bacillus subtilis KCCM11143P according to the present invention.
[42]
In addition to the strain included as an active ingredient, the composition may contain known carriers or additives acceptable for pharmaceutical, food or feed. In the present invention, as a probiotic preparation having antibacterial activity against enteritis Vibrio bacteria, including Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM11144P, and Bacillus licheniformis KCCM11270P, a binder, emulsifier, added to prevent quality degradation, There are preservatives, and there are amino acids, vitamins, enzymes, flavors, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. added to feed to increase utility. In addition, it may further include a feed mixture, but is not limited thereto.
[43]
In one embodiment of the present invention, when the feed composition is fed to shrimp, as a result of confirming whether it can increase immunity against AHPND disease and exhibit a disease prevention effect, Bacillus subtilis KCCM11143P strain of the present invention, Bacillus pumilus Groups administered with feed compositions (Examples 5 and 6) containing KCCM11144P strain and Bacillus licheniformis KCCM11270P strain (BS+BP+BL groups 1 and 2) were Comparative Example 1 (not including probiotics) and Comparative Examples Compared to the group administered with 3 and 4 (including only Bacillus subtilis) (BS groups 3 and 4), it was possible to increase the disease resistance of shrimp against Vibrio parahaemolyticus infection, as well as in the liver pancreas of shrimp. It can significantly lower the amount of AHPND toxins.
[44]
In another embodiment of the present invention, as a result of performing non-specific immunity analysis of the feed composition containing the strain, macrophage (NBT) activity, glutathione peroxidase (GPx) activity, lysozyme activity, phenolic oxidase (PO) activity , It was confirmed that the superoxide dismutase (SOD) activity and the antiproteolytic enzyme activity were significantly higher than that of the comparative example, through which the composition may be useful for increasing the non-specific immune response or improving the immunity of shrimp.
[45]
The present invention provides a feed additive for shrimp farming comprising the aforementioned feed composition.
[46]
In addition to the above active ingredients, the feed additive of the present invention may add a known carrier or stabilizer that is acceptable for pharmaceutical, food, or feed. If necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants and other Additives and the like may be added, and the shape may be in a suitable state such as powder, granule, pellet, or suspension. In the case of supplying the feed additive of the present invention, it may be supplied alone or mixed with feed for a unit animal.
[47]
In addition, the present invention provides a feed for shrimp farming comprising the feed additives mentioned above.
[48]
Bacillus subtilis KCCM11143P strain, Bacillus pumilus KCCM1114P strain, and Bacillus licheniformis KCCM11270P strain of the present invention are Gram-positive bacteria capable of spore formation, so it is preferable to formulate in a spore form, but is not limited thereto. The feed of the present invention is not particularly limited, and any feed such as powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, and raw feed may be used.
[49]
As described above, the genus Bacillus has a characteristic that is very stable against heat by forming endospores. Therefore, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention can be prepared separately in the form of feed additives and mixed with feed or directly added during feed production. Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis in the feed of the present invention may be in a liquid or dry state, and preferably in a dry powder form. The drying method may be air drying, natural drying, spray drying, and freeze drying, but is not limited thereto. Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention may be mixed in a powder form in a component ratio of 0.05 to 10% by weight, preferably 0.1% to 1% by weight of the feed weight. Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis of the present invention may be mixed in a concentration ratio of 1 to 5: 1 to 5: 1 to 5. In addition, the feed is for aquaculture, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P and Bacillus licheniformis KCCM11270P of the present invention, in addition to conventional additives that can increase the preservability of the feed may be additionally included.
[50]
[51]
As another embodiment for solving the above problem, it is to provide a method for preventing or treating acute hepatic pancreatic necrosis comprising administering the feed composition of the present invention to an individual.
[52]
At this time, acute hepatic pancreatic necrosis, prevention and treatment of the present invention are as described above.
[53]
The term "subject" of the present invention may mean aquaculture fish or crustaceans that have or may develop acute hepatic pancreatic necrosis, but may mean shrimp for the purposes of the present invention.
[54]
The feed is preferably the same amount and grade supplied intervals with conventional feed and the pathogens by causing AHPND in the shrimp; refers to bacteria that cause the group mortality in shrimps, in particular, Vibrio parahaemolyticus ( vibrio parahaemolyticus ).
[55]
The causes of collective death in the shrimp farming include not only the enteritis Vibrio bacteria, but also infection by various viruses and ammonia concentration in the breeding water. Ammonia in the breeding water during shrimp farming is mainly produced as a protein metabolite such as shrimp excrement and feed waste, and varies greatly with the increase of pH and water temperature. High concentration of ammonia directly causes acute death of shrimp, leading to mass mortality. Even at low concentrations, in the long term, it may degrade shrimp's growth and feeding ability and lower immunity, resulting in various diseases. .
[56]
In one embodiment of the present invention, as a result of collecting the breeding water of the shrimp fed with the feed composition of the present invention and analyzing its water quality, it was confirmed that the total ammonia concentration was significantly lower than that of the control, so that the Bacillus of the present invention Subtilis KCCM11143P strain, Bacillus pumilus KCCM11144P strain, and Bacillus licheniformis KCCM11270P strain can improve the water quality of shrimp breeding water.
[57]
As described above, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention can not only enhance disease resistance of shrimp, but also suppress the amount of AHPND toxin in the liver pancreas of shrimp, When the strain is used, it is possible to obtain an effect of preventing enteritis Vibrio bacteria causing the disease, and to obtain an effect of enhancing resistance to the white spot syndrome virus, so that shrimp can be cultured more safely.
[58]
[59]
As another embodiment for solving the above problems, the present invention is a white spot comprising a Bacillus subtilis strain, a Bacillus pumilus strain, and a Bacillus licheniformis strain, a culture solution thereof, a concentrate thereof, or a dried product thereof as an active ingredient It provides a feed composition for the prevention or treatment of syndrome (WSS).
[60]
The terms of the present invention Bacillus subtilus (Bacillus subtilus, Bacillus bacillus), Bacillus pumilus, Bacillus licheniformis, prevention and treatment are as described above.
[61]
The composition of the present invention may include Bacillus subtilis KCCM11143P, the Bacillus pumilus KCCM11144P, and the Bacillus licheniformis KCCM11270P having a number of bacteria of 1 x 10 4 to 1 x 10 11 CFU per g of the total active ingredient , Specifically, it may be 1 x 10 4 to 1 x 10 10 CFU / g, more specifically 1 x 10 8 to 1 x 10 10 CFU / g of Bacillus subtilis KCCM11143P, the Bacillus pumilus KCCM11144P, and It includes the Bacillus licheniformis KCCM11270P.
[62]
The “white spot syndrome virus (WSSV)” is a virus that is widely distributed around the world, and has a tail-like attachment at the end of the viron, and has a rod-shaped capsid and envelope. It has a similar morphology to ovate bacillus, so it was called baculovirus or bacillus-like formed virus, but in recent years it has been named as whispovirus, a genetically new group of viruses. The virus is about 275 nm in length, about 120 nm in diameter, and consists of double-stranded DNA with a size of about 290 kb.
[63]
In addition to the strain included as an active ingredient, the composition may contain known carriers or additives acceptable for pharmaceutical, food or feed. As a probiotic preparation having antiviral activity against the white spot syndrome virus, including Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM11144P, and Bacillus licheniformis KCCM11270P in the present invention, a binder added to prevent quality degradation, There are emulsifiers and preservatives, and there are amino acids, vitamins, enzymes, flavoring agents, non-proteinaceous nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. added to feed for increased utility. In addition, it may further include a feed mixture, but is not limited thereto.
[64]
In one embodiment of the present invention, when the feed composition is fed to shrimp, as a result of confirming whether it can increase immunity against white spot syndrome and exhibit a disease prevention effect, Bacillus subtilis KCCM11143P strain of the present invention, Bacillus pu The group to which the feed composition (Examples 5 and 6) containing the Millers KCCM11144P strain and the Bacillus licheniformis strain KCCM11270P strain was administered (BS + BP + BL groups 1 and 2) was Comparative Example 1 (not including probiotics) and Compared to the group administered with the feed composition (Comparative Example 4) containing only Bacillus subtilis KCCM11143P strain (Comparative Example 4), the disease resistance of shrimp to white spot syndrome virus (WSSV) infection can be improved.
[65]
In another embodiment of the present invention, when the feed composition is fed to shrimp, it increases resistance to complex infections of white spot syndrome virus (WSSV) and acute hepatic pancreatic necrosis (AHPND) and improves survival rate. As a result of checking whether there is a result, the group administered with the feed composition (Examples 5 and 6) containing the Bacillus subtilis KCCM11143P strain, the Bacillus pumilus KCCM11144P strain, and the Bacillus licheniformis strain KCCM11270P strain of the present invention (BS+BP+BL group) 1 and 2) compared to the group administered with Comparative Example 1 (not including probiotics) and a feed composition containing only Bacillus subtilis KCCM11143P strain (Comparative Example 4) (BS group 4), for the combined infection of WSSV and AHPND. It can improve the disease resistance of shrimp.
[66]
The present invention provides a feed additive for shrimp farming comprising the aforementioned feed composition.
[67]
In addition to the above active ingredients, the feed additive of the present invention may add a known carrier or stabilizer that is acceptable for pharmaceutical, food, or feed. If necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants and other Additives and the like may be added, and the shape may be in a suitable state such as powder, granule, pellet, or suspension. In the case of supplying the feed additive of the present invention, it may be supplied alone or mixed with feed for a unit animal.
[68]
In addition, the present invention provides a feed for shrimp farming comprising the feed additives mentioned above.
[69]
Bacillus subtilis KCCM11143P strain, Bacillus pumilus KCCM1114P strain, and Bacillus licheniformis KCCM11270P strain of the present invention are Gram-positive bacteria capable of spore formation, so it is preferable to formulate in a spore form, but is not limited thereto. The feed of the present invention is not particularly limited, and any feed such as powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, and raw feed may be used.
[70]
As described above, the genus Bacillus has a characteristic that is very stable against heat by forming endospores. Therefore, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention can be prepared separately in the form of feed additives and mixed with feed or directly added during feed production. Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis in the feed of the present invention may be in a liquid or dry state, and preferably in a dry powder form. The drying method may be air drying, natural drying, spray drying, and freeze drying, but is not limited thereto. Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention may be mixed in a powder form in a component ratio of 0.05 to 10% by weight, preferably 0.1% to 1% by weight of the feed weight. In addition, the feed is for aquaculture, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P and Bacillus licheniformis KCCM11270P of the present invention, in addition to conventional additives that can increase the preservability of the feed may be additionally included.
[71]
[72]
As another embodiment for solving the above problems, it is to provide a method for preventing or treating white spot syndrome comprising administering the feed composition of the present invention to an individual.
[73]
At this time, the terms white spot syndrome, prevention, treatment and individual of the present invention are as described above.
[74]
In shrimp farming, the cause of mass death includes not only the above-described enteritis Vibrio bacteria, but also infection by various viruses. Specifically, the virus may refer to a white spot syndrome virus (WSSV).
[75]
As described above, Bacillus subtilis KCCM11143P, Bacillus pumilus KCCM1114P, and Bacillus licheniformis KCCM11270P of the present invention can obtain an effect of enhancing resistance to the white spot syndrome virus, so that shrimp can be cultured more safely.
[76]
Mode for carrying out the invention
[77]
Hereinafter, the present application will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited to these examples.
[78]
[79]
Preparation Example 1. Selection of probiotics having antibacterial activity
[80]
In order to select a strain having antibacterial activity against Vibrio parahaemolyticus , which causes shrimp AHPND , a growth inhibition assay was performed. Top-agar was prepared by mixing 100 μl of 0.5% agar 3 ml and shaking culture solution of pathogenic bacteria (2.0 × 10 9 CFU/ml) on TSA+ medium. 12 kinds of Bacillus subtilis (CJ self-owned strain and commercial strain) microbial culture solution were dropped onto the prepared top-agar, respectively, 10 μl, and the presence or absence of growth inhibition formed after culturing at 30° C. for 18 hours was observed. The antimicrobial activity of commercially available Bacillus subtilis and composite phage were evaluated together.
[81]
[Table 1]
Antibacterial evaluation target Vibrio parahaemolyticus (Vibrio parahaemolyticus)
Bacillus subtilis 1 (CJBS-01) ++++
Bacillus subtilis 2 (CJBS-02) +
Bacillus subtilis 3 (CJBS-03) -
Bacillus subtilis 4 (CJBS-04) -
Bacillus subtilis 5 (CJBS-05) +
Bacillus subtilis 6 (CJBS-06) -
Bacillus subtilis 7 (CJBS-07) -
Bacillus subtilis 8 (CJBS-08) -
Bacillus subtilis 9 (CJBS-09) +
Bacillus subtilis 10 (CJBS-10) -
Bacillus subtilis 11 (CJBS-11) -
Bacillus subtilis 12 (CJBS-12) -
Bacillus subtilis (purchased commercially, company A, Korea) +
Complex phage -
[82]
++++: active strong, +: active, -: active
[83]
As shown in Table 1, Bacillus subtilis 1 microorganism (CJBS-01) was the most excellent in antimicrobial effect against enteritis Vibrio bacteria, which is an AHPND-induced strain in vitro. However, even if it is a microorganism that shows antibacterial activity against a specific pathogen in vitro, it is only an in vitro effect, and when an animal ingests it, it cannot be considered to have an immunity or preventive effect against the pathogen.
[84]
Therefore, in the following experiment, when feeding the Bacillus subtilis 1 (CJBS-01) microorganism to shrimp, it was confirmed whether the effect of improving immunity against AHPND disease and preventing disease can be exhibited. In addition, it was also confirmed that the Bacillus subtilis 1 (CJBS-01) has an effect on the gain and digestibility of shrimp.
[85]
The Bacillus subtilis 1 (CJBS-01) is a strain deposited with the Korea Microbial Conservation Center on December 14, 2010 with the accession number KCCM11143P.
[86]
[87]
Preparation Example 2. Preparation of a shrimp feed composition containing Bacillus subtilis
[88]
A feed composition comprising Bacillus Subtilis 1 ( Bacillus Subtilis : accession number KCCM11143P, hereinafter referred to as'BS') selected in Preparation Example 1 was prepared.
[89]
Specifically, Comparative Example 1 not containing Bacillus subtilis, Comparative Example 2 containing commercially sold Bacillus genus ( B. subtilis, B. pumilus, B. licheniformis ), and the selection above. The composition of Example 1 containing 10 10 x 0.2 CFU/g of Bacillus subtilis 1 (BS) was mixed by adding fish oil and water, and then prepared in a pellet form. The feed compositions of Comparative Examples 1 and 2 and Example 1 were dried at 25°C for about 24 hours using a dryer and then stored at -20°C until the experiment.
[90]
[Table 2]
Ingredients (%) Comparative Example 1 Comparative Example 2 Example 1
Fish meal 40 40 40
SBM 44% South America 12.81 12.81 12.81
Squid liver powder 10 10 10
Wheat flour 25.61 25.61 25.61
Amygluten 110 3 3 3
Fish oil A/C 2 2 2
Amino acid 0.42 0.42 0.42
Vitamin/Mineral premix 5.96 5.96 5.96
Rice bran 0.2 0.18 0.00
Bacillus 3 species (10 10 x CFU/g) 0.00 0.2 0.00
BS (10 10 x CFU/g) 0.00 0.00 0.2
BS + BP (10 10 x CFU/g) 0.00 0.00 0.00
BS + BP +BL (10 9 x CFU/g) 0.00 0.00 0.00
Chemical composition (% dry matter)
Moisture 5.68 5.57 5.60
Crude protein 47.3 47.4 47.4
Crude lipid 7.31 7.47 7.49
Crude ash 6.01 6.18 6.12
[91]
[92]
Experimental Example 1. Evaluation of the preventive effect of feed composition on AHPND
[93]
1-1. Shrimp preparation and growth rate assessment
[94]
A total of 40 white-leg shrimp were prepared in a total of 30 fish per tank. Airstones were installed in all test tanks to maintain dissolved oxygen, and the breeding water temperature was maintained in the range of 28 to 32°C for the entire test period. Feed was limitedly supplied 4 times a day (4 to 12% of fish weight).
[95]
The weight of the shrimp was measured every two weeks, and the evaluation items and calculation formula related to growth rate and feed efficiency are as follows:
[96]

[97]

[98]

[99]
The batch of the experimental feed was carried out by a complete randomized design, and the growth and analysis results (SPSS Version 18.0) were statistically analyzed by one-way ANOVA using the program. The significance of the data values was compared with the mean ( P <0.05) by Duncan's multiple range test . Data were expressed as mean ± standard deviation (mean ± SD), and percentage data were calculated as arcsine deformation values ​​and statistically analyzed.
[100]
[Table 3]
Control 1 Control 2 BS Group 1
IBW 1 (g) 0.51±0.01 0.51±0.01 0.51±0.01
FBW 2 (g) 3.49±0.12 bc 3.80±0.22 ab 3.86±0.18 a
WG 3 (%) 592±18.9 c 655±39.3 ab 667±31.9 a
SGR 4 (%) 6.04±0.09 b 6.31±0.16 a 6.37±0.13 a
FCR 5 1.30±0.26 1.28±0.09 1.19±0.12
Survival (%) 80.0±3.33 75.6±6.94 71.1±6.94
[101]
1 IBW: Initial body weight
[102]
2 FBW: Final body weight
[103]
3 WG: Weight gain (gain rate) = [(final body weight-initial body weight)/ initial body weight] x 100
[104]
4 SGR: Specific growth ratio (% day -1 ) = [(log e final body weight-log e initial body weight)/days] x 100
[105]
5 FCR: Feed conversion ratio = dry feed fed/wet weight gain
[106]
[107]
As shown in Table 3, the feed composition of Example 1 containing Bacillus subtilis selected in Preparation Example 1 compared to the control groups 1 and 2 to which the feed composition of Comparative Examples 1 and 2 was administered was provided as a result of the feeding test. In group 1, it was confirmed that a significantly high growth rate was observed. In addition, it was confirmed that the group administered with Example 1 had a significantly higher daily growth rate compared to the group administered with Comparative Examples 1 and 2.
[108]
[109]
1-2. Vibrio enteritis attack test
[110]
The attack test of enterophilus Vibrio bacteria against shrimp was divided into two total. In the case of the Vibrio strain, the AHPND (EMS) induced strain isolated in Vietnam in 2013 was used for the test. In the case of the attack test, the feed composition of the example was supplied to the shrimp for 2 weeks, and then the same weight (average weight: 2.32 g) of shrimp was placed in 4 repetitions at 96 per group. The bacteria were cultured for 24 hours at 30 o C and 150 rpm using TSB + medium, and in 20 110 L acrylic water tanks (working volume 72.5 L) 2 x 10 9 CFU concentration (OD 1.7) of Vibrio bacteria suspension 30 ml was immersed. The mortality and swimming status of the shrimp were checked every 1 hour after immersion, and 95% water exchange was performed after 8 hours. The test feed was divided into three times a day (8:30, 13:30, and 18:30 hours) and limited supply (10 to 12% of the fish's weight), and mortality was observed for 70 hours. The results are shown in Table 4 below.
[111]
[Table 4]
Treatment Survival (%)
Trial
Control 1 50.0±16.0
Control 2 49.0±23.2
BS Group 1 67.7±28.9
[112]
As shown in Table 4, BS group 1 provided with a feed composition comprising Bacillus subtilis selected in Preparation Example 1 showed a higher survival rate compared to controls 1 and 2 in an attack test for shrimp of Vibrio Enteritis.
[113]
In addition, as shown in Fig. 1, in both tests, the movement of the shrimp became dull immediately after immersion of the Vibrio bacteria in both tests, and it was observed that the shrimp sinked in the bottom of the tank without swimming, and feed feeding was also active. Did not do it. At 8 hours after immersion, sudden death began to occur, and it was confirmed that the survival rate of BS group 1 was higher than that of the control group 1 or 2 by 17% or more.
[114]
[115]
1-3. Sample collection method and histopathological analysis method
[116]
Hepatic pancreas was isolated by randomly selecting 2 shrimps per group at the start (before infection), middle (infection), and end of the attack test for Shrimp of Vibrio Enteritis. Some of the isolated hepatic pancreas were stored directly in ethyl alcohol (100%) for quantitative real-time PCR (qPCR) analysis, and some were fixed in Davidson's fixative for 24 hours for histopathological examination and then ethyl alcohol (70%). Stored in.
[117]
More specifically, histopathological analysis was performed in the following manner. In order to minimize the destruction of the isolated liver pancreatic tissue, Davidson's fixative was injected into the liver pancreas of shrimp using a 1 ml syringe immediately after sampling in a water bath, and then separated. Then, it was fixed in a 1.5 ml Eppendorf tube containing Davison's fixative for 24 hours, stored in ethyl alcohol (70%), and used for analysis. After the fixation was completed organs were cut to a thickness of a shape (about 2 to 3 mm) suitable for tissue sample preparation, they were placed in a cassette and subjected to tissue treatment for 13 hours. Sections generated by cutting to a thickness of about 4 μm were collected using a brush, attached to the slides without wrinkles, and left in the air for about 5 minutes, followed by H&E staining. The stained slides were photographed 200 times with a microscope-specific program (TCapture, Tucen Photonics) using a phase contrast microscope (BX50, Olympus). Thereafter, qPCR analysis was performed on the amount of AHPND toxin in the sampled liver pancreas.
[118]
The results are shown in FIG. 2. Here, the lower the Ct value, the higher the amount of toxins.
[119]
AHPND toxin was not detected in all groups in the hepatic pancreas sampled before challenge test (0h), and AHPND toxin was detected in all test groups in the hepatic pancreas sampled at 10 hours when the number of dead individuals was the most. However, the BS group 1 provided with the feed composition containing Bacillus subtilis according to the present application showed significantly higher Ct values ​​compared to the controls 1 and 2, and in the liver pancreas sampled at 24 hours, the AHPND toxin in the BS group It was confirmed to detect the least. In addition, at the end of the challenge (193h), no AHPND toxin was detected in BS group 1 and control group 2.
[120]
In addition, the results of histopathological analysis of the hepatopancreas are shown in FIG. 3.
[121]
Normal tissue morphology was observed in all groups of hepatic pancreas sampled before challenge test (0h). The hepatic pancreas sampled after 10 hours was most severely damaged in the control group 1, and tissue necrosis was observed in the control group 2 and the BS group 1 as well. Hepatic pancreas sampled after 24 hours was more inflammatory than tissue necrosis caused by AHPND toxin. At the end of the challenge (193h), since 100% mortality had already occurred (37h) in control 1, sampling was not possible, and some inflammatory cells were observed in control 2 and BS groups.
[122]
From the above results, the feed composition containing Bacillus subtilis according to the present application can not only improve the disease resistance of shrimp against Vibrio parahaemolyticus infection, but also significantly lower the amount of AHPND toxin in the liver pancreas of shrimp. It was confirmed that it can be.
[123]
[124]
Experimental Example 2. Evaluation of growth rate and immunity according to the concentration of Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis
[125]
With reference to Preparation Example 2, Bacillus subtilis (KCCM11143P, hereinafter referred to as'BS'), Bacillus pumilus (KCCM11144P, hereinafter referred to as'BP') and Bacillus licheniformis (KCCM11270P, hereinafter referred to as'BP') selected in Preparation Example 1 Hereinafter, referred to as'BL'), feed compositions (Examples 1 to 5) having different concentrations were prepared. This is shown in Table 5 below.
[126]
[Table 5]
Ingredients (%) Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Example 4 Example 5
Fish meal 40 40 40 40 40 40 40
SBM 44% South America 12.81 12.81 12.81 12.81 12.81 12.81 12.81
Squid liver powder 10 10 10 10 10 10 10
Wheat flour 25.61 25.61 25.61 25.61 25.61 25.61 25.61
Amygluten 110 3 3 3 3 3 3 3
Fish oil A/C 2 2 2 2 2 2 2
Amino acid 0.42 0.42 0.42 0.42 0.42 0.42 0.42
Vitamin/Mineral premix 5.96 5.96 5.96 5.96 5.96 5.96 5.96
Rice bran 0.2 0.18 0.00 0.00 0.00 0.00 0.00
BS + BP + BL (10 10 x CFU/g) 0.00 0.2 0.00 0.00 0.00 0.00 0.00
BS (10 10 x CFU/g) 0.00 0.00 0.2 0.1 0.00 0.00 0.00
BS + BP (10 10 x CFU/g) 0.00 0.00 0.00 0.00 0.1 0.2 0.00
BS + BP +BL (10 9 x CFU/g) 0.00 0.00 0.00 0.00 0.00 0.00 0.2
Chemical composition (%, dry matter)
Moisture 5.59 5.80 5.78 5.71 5.71 5.71 5.71
Crude protein 47.3 48.6 49.2 48.9 48.1 48.5 48.5
Crude lipid 7.74 7.71 7.73 7.70 7.70 7.72 7.70
Crude ash 6.12 6.12 6.20 6.10 6.10 6.12 6.11
[127]
The composition of each group of Table 5 was mixed by adding fish oil and water, and then prepared in a pellet form, and the feed composition of each group was dried at 25° C. for about 24 hours using a dryer. , It was stored at -20 ℃ until the experiment.
[128]
[129]
2-1. Shrimp preparation and growth rate assessment
[130]
A total of 28 white shrimp were prepared in a total of 28 fish per tank. Airstone was installed in all the test tanks to maintain dissolved oxygen, and the breeding water temperature was maintained in the range of 28 to 32℃ throughout the test period. Feed was limitedly supplied 4 times a day for 8 weeks (6 to 12% of fish weight, initial average weight: 0.14 g).
[131]
The weight of shrimp was measured every two weeks, and the evaluation items and calculation formula related to growth rate and feed efficiency are as follows;
[132]

[133]

[134]

[135]
The results are shown in Table 6.
[136]
[Table 6]
Control 1 Control 2 BS Group 1 BS Group 2 BS+BP Group 1 BS+BP Group 2 BS+BP+BL Group 1
IBW 1 (g) 0.14±0.00 0.14±0.00 0.14±0.00 0.14±0.00 0.14±0.00 0.14±0.00 0.14±0.00
FBW 2 (g) 10.2±0.69 b 11.3±0.45 a 11.9±0.43 a 11.5±0.60 a 11.0±0.91 11.8±0.47 11.1±0.36
WG 3 (%) 7029±506 b 7969±413 a 8381±356 a 8085±464 a 7818±772 8151±202 7859±303
SGR 4 (%) 7.62±0.13 b 7.84±0.09 a 7.93±0.07 a 7.86±0.10 a 7.80±0.17 7.88±0.04 7.81±0.07
FCR 5 1.53±0.04 c 1.21±0.09 ab 1.11±0.22 a 1.13±0.12 a 1.36±0.06 1.21±0.05 1.33±0.08
Survival (%) 90.7±6.11 92.0±4.00 84.0±17.4 88.0±8.00 88.0±4.00 85.3±4.62 86.7±6.11
[137]
* (P <0.05)
[138]
1 IBW: Initial body weight
[139]
2 FBW: Final body weight
[140]
3 WG: Weight gain = [(final body weight-initial body weight)/ initial body weight] x 100 (gain rate)
[141]
4 SGR: Specific growth ratio (% day -1 ) = [(log e final body weight-log e initial body weight)/days] x 100
[142]
5 FCR: Feed conversion ratio = dry feed fed/wet weight gain
[143]
As shown in Table 6 and Figure 4, shrimp of all groups fed for 8 weeks showed a growth rate of about 7000% or more compared to before feeding. In particular, the final average weight of the BS group 1 provided with the feed composition of Example 1 and the BS group 2 provided with the feed composition of Example 2 was 15.7% and 16.7% higher than the final average weight of the control group 1. The final average weight of the BS+BP group 1 provided with the feed composition of Example 3, the BS+BP group 2 provided with the feed composition of Example 4 and the BS+BP+BL group 1 provided with the feed composition of Example 5 was the control group. Compared to the final average weight of 1, it was 7.8%, 15.6% and 8.8% higher, respectively. In addition, in the daily growth rate, BS group 1 and BS group 2 were 3.4% and 4.1% higher than that of control group 1, respectively. The daily growth rates of the BS+BP group 1, the BS+BP group 2, and the BS+BP+BL group 1 were 2.4%, 3.4%, and 2.5% higher than that of the control group 1, respectively. The feed efficiency of BS group 1 and BS group 2 was improved by 27.5% and 26.1%, respectively, compared to the feed efficiency of control group 1. The feed efficiency of BS+BP group 1, BS+BP group 2, and BS+BP+BL group 1 was 11.1%, 20.9%, and 13.1% higher than that of control group 1, respectively. However, there was no significant difference in the survival rate of shrimp in all groups.
[144]
[145]
2-2. Sample collection
[146]
The test shrimp were weighed every two weeks, and all test shrimp were fasted 18 hours before the measurement to reduce the stress of the test shrimp. After the final weight measurement, 7 shrimps per tank were randomly selected and anesthetized in ice water, and then hemolymp was collected using a syringe treated with ALSEVER'S solution. The collected hemolymp was used for the analysis of macrophage activity (NBT; nitroblue-tetrazolium activity), and the sample from which plasma was separated by a centrifuge (800 g, 10 minutes, 4°C) was used for non-specific immunity analysis. For the collection of feces of shrimp for digestibility analysis, the feed and foreign matter remaining in the tank were cleaned through siphoning and water exchange 30 minutes after feeding, and the waste excreted from 3 hours was collected with a siphon. The collected fraction was washed with distilled water, filtered using filter paper, and stored in a -40°C low temperature freezer until used as a sample for analysis.
[147]
[148]
2-3. Statistical analysis
[149]
The batch of the experimental feed was carried out by a complete randomized design, and the growth and analysis results (SPSS Version 18.0) were statistically analyzed by one-way ANOVA using the program. For the significant difference between the data values, the significance ( P <0.05) between the means was compared using Duncan's multiple range test . Data were expressed as mean ± standard deviation (mean ± SD), and percentage data were calculated as arcsine deformation values ​​and statistically analyzed.
[150]
[151]
2-4. General component analysis
[152]
Analysis of the test feed and the general components of the powder is carried out according to the AOAC (2005) method. Moisture is atmospheric pressure heating and drying method (125℃, 3h), the raw material is the direct conversation furnace method (550℃, 4h), and the protein is an automatic crude protein analyzer (Kejltec system 2300, Sweden), and fat was analyzed by Folch et al. It was analyzed according to the method of (1957), and is shown in Table 7.
[153]
[Table 7]
Control 1 Control 2 BS Group 1 BS Group 2 BS+BP Group 1 BS+BP Group 2 BS+BP+BL Group 1
Dry matter 24.9±0.35 24.5±0.10 23.7±0.39 23.7±0.35 25.1±0.36 24.3±0.23 24.3±0.16
Crude ash 12.9±2.34 12.8±0.12 12.3±0.33 14.5±0.22 14.8±3.47 13.9±1.46 14.6±3.60
Crude protein 76.5±3.46 b 82.0±1.67 a 84.3±2.85 a 83.9±2.31 a 79.4±2.23 ab 82.0±3.58 a 81.8±1.52 a
Crude lipid 5.37±0.96 5.45±1.15 5.09±0.28 5.26±1.05 5.29±0.12 5.40±0.53 5.59±0.73
[154]
(* P <0.05)
[155]
As shown in Table 7, the crude content and crude lipid content did not show a significant difference between the groups, but the crude protein content of the BS group 2 was significantly higher than that of the control group 1 and the control group 2. That is, when the feed composition according to the present application is provided to shrimp, it was confirmed that the protein content of the shrimp can be increased.
[156]
[157]
2-5. Non-specific immunity related analysis
[158]
2-5-1. NBT (Nitroblue tetrazolium) activity assay
[159]
To measure the amount of oxidative radicals produced by neutrophils during a respiratory explosion, Zhang et al. (2013) was applied.
[160]
Specifically, 50 μl of hemolymph was first mixed with a 200 μl HBSS (Hank's balanced salt solution) solution, and then reacted at 25°C. After 30 minutes, 100 μl of zymosan (0.1% Hank's solution) was added and reacted at 37° C. for 2 hours. After 100 μl of NBT solution (0.3%) was added, it was reacted at 37°C for 2 hours. 600 μl 100% methanol was added and centrifuged for 10 minutes (6500 rpm), the supernatant was discarded, washed 3 times with 100 μl 70% methanol, and dried for 5 minutes. Then, 700 μl of 2M KOH and 800 μl of DMSO were added, and the absorbance was measured at 620 nm.
[161]
[162]
2-5-2. Glutathione peroxidase (GPx) activity assay
[163]
To analyze the GPx activity in serum, a GPx kit (Biovision, Inc. California) was used.
[164]
Specifically, for Cumenehydroperoxide, a reaction mixture in which peroxide substrate (ROOH), glutathione reductase (GSSG-R) and NADPH (b-Nicotinamide Adenine Denucleotide Phosphate, Reduced) were mixed was used. First, 50 μl of a hemolymph sample was put into a microplate, 40 μl of the reaction mixture was added, and reacted for 15 minutes. Then, 10 μl of cumenehydroperoxide was added, and absorbance was measured at 340 nm. After 5 minutes, the absorbance was measured again at 340 nm, and the GPx activity was calculated as nmol/min/ml.
[165]
[166]
2-5-3. Lysozyme activity assay
[167]
Lysozyme activity assay was performed by Swain et al. (2007) was analyzed based on the method.
[168]
Specifically, lysozyme is one of the antimicrobial enzymes belonging to a non-specific (innate) immune response, and is an enzyme that exhibits non-specific antimicrobial activity against various bacteria rather than a specific antibacterial activity against specific bacteria. The antibacterial mechanism against pathogens exhibits antimicrobial action by destroying the bacterial cell wall by hydrolyzing the β-1,4-glucoside bond of peptidoglycan, a component of the bacterial cell wall. Lysozyme is particularly excellent in antibacterial effect against Gram-positive bacteria. Due to this mechanism, lysozyme activity is widely used as an analysis item to measure the non-specific immune response in shrimp including fish. Increased lysozyme activity in the hemolymph and tissue of the can be confirmed, and this result is interpreted as a result indicating an increase in non-specific immune response or an increase in immunity of fish (shrimp).
[169]
[170]
2-5-4. Phenoloxidase (PO) activity assay
[171]
PO activity assay was performed by Hernandez-Lopez et al. (1996) was analyzed based on the method.
[172]
Specifically, Phenoloxidase is an enzyme that plays a very important role in the defense mechanism of crustaceans. It exists in the form of prophenoloxidase in blood cells and is activated by the Prophenoloxidase activating system. Activated phenoloxidase produces opsonin, promotes phagocytic activity of blood cells and coating action against foreign antigens, and participates in the blood coagulation reaction. Therefore, phenoloxidase activity in hemolymph is used as an important indicator of innate immunity in shrimp.
[173]
[174]
2-5-5. Superoxide dismutases (SOD) activity assay
[175]
SOD activity was analyzed using the SOD assay kit (Sigma-aldrich, 19160, St. Louis, USA).
[176]
Specifically, after adding 20 μl of a radical detector to a 96-well plate, 10 μl of blood samples were added each. Thereafter, 20 μl xanthine oxidase was added and reacted for 20 minutes, and absorbance was measured at 450 nm using a microplate reader (Themo).
[177]
[178]
2-5-6. Antiproteases activity assay
[179]
Antiprotease activity in hemolymph was analyzed based on the analysis method of Ellis (1990).
[180]
Specifically, 20 μl of Hemolymph and 20 μl of a standard trypsin solution (Type II-S, from porcine pancreas, Sigma-aldrich, A2765, St. Louis, USA) were mixed, and then incubated at 22° C. for 10 minutes. Add 200 μl of phosphate buffer (0.1 M, pH 7.0) and 250 μl of azocasein (2%) (Sigma-Aldrich), incubate at 22° C. for 1 hour, and then add 500 μl of trichloro acetic acid (10%) (TCA) again. And incubated at 22° C. for 30 minutes. The cultured solution was centrifuged (6000 g , 5 minutes) and 100 μl was dispensed into a 96-well plate, and then 100 μl of NaOH (1 N) was added to measure the absorbance at 430 nm using a microplate reader. .
[181]
The results of 2-5-1 to 2-5-6, which were subjected to the non-specific immunity assay, are shown in Table 8 below.
[182]
[Table 8]
Control 1 Control 2 BS Group 1 BS Group 2 BS+BP Group 1 BS+BP Group 2 BS+BP+BL Group 1
NBT 1 1.65±0.15 c 1.72±0.08 bc 1.85±0.03 ab 1.79±0.13 ab 1.89±0.03 a 1.86±0.03 ab 1.83±0.06 ab
PO 2 0.213±0.008 b 0.249±0.031 a 0.254±0.015 a 0.249±0.009 a 0.270±0.023 a 0.250±0.011 a 0.252±0.006 a
Antiprotease 3 31.6±2.4 b 35.8±0.3 a 36.6±1.8 a 36.1±3.4 a 34.8±1.2 a 36.1±2.1 a 36.5±2.0 a
Lysozyme 4 6.40±1.04 b 8.57±0.50 a 8.43±0.98 a 8.08±1.26 ab 8.02±1.21 ab 7.89±1.02 ab 7.58±1.70 ab
SOD 5 67.3±4.6 b 73.1±3.3 ab 74.8±4.6 ab 72.8±7.7 ab 77.2±6.0 a 70.7±4.1 ab 74.1±8.3 ab
GPx 6 28.5±1.9 d 32.7±2.4 c 36.4±1.7 abc 36.7±2.3 a 34.3±2.6 abc 32.8±3.3 bc 36.6±2.3 ab
[183]
(* P <0.05)
[184]
1 Nitro blue tetrazolium; phagocytic activity (absorbance) macrophage activity
[185]
2 Phenoloxidase activity (absorbance) phenolic oxidase activity
[186]
3 Antiprotease (% inhibition) antiproteolytic enzyme
[187]
4 Lysozyme activity (μg ml -1 ) Lysozyme activity
[188]
5 Superoxide dismutase (% inhibition) Superoxide dismutase
[189]
6 Glutathione peroxidase (mU ml -1 ) Glutathione peroxidase
[190]
As shown in Table 8 and FIG. 5, the NBT activity and PO activity were in the control group 1 and the control group 2 in both the BS group 1, the BS group 2, the BS+BP group 1, the BS+BP group 2 and the BS+BP+BL group 1 Compared to the significantly higher value was shown, in particular, in the case of PO activity, BS group 1 showed a value 26.8% higher than that of the control group 1. The anti-proteinase activity was significantly higher in both the BS group 1, BS group 2, BS+BP group 2, and BS+BP+BL group 1 compared to the control group 1 and the control group 2, and in particular, the BS group 1 It was 15.8% higher than that of the control group 1. Lysozyme activity and SOD activity were significantly higher in BS group 1, BS group 2, BS+BP group 1, BS+BP group 2, and BS+BP+BL group 1 compared to control 1. The GPx activity was significantly higher than that of the control group 1 and the control group 2 in all of the BS group 1, BS group 2, BS+BP group 1, BS+BP group 2, and BS+BP+BL group 1, and in particular the BS group. It was confirmed that 1 and BS group 2 showed 27.7% and 28.8% higher values, respectively, compared to the control group 1.
[191]
[192]
2-6. Water quality analysis and no exchange experiment
[193]
During the 8-week feeding test period, samples of breeding water were collected from each tank once every 5 days and analyzed for water quality. Samples were collected at the same location in each tank, and dissolved oxygen (DO), salinity, pH, and ammonia (Ammonium; NH 4 + ) concentration were measured. DO was measured using a Thermo Scientific Orion Star A216 Benchtop Meter (Thermoscientific), and salinity was measured using a Master Refractometer (ATAGO). The pH was measured using Seven Compact (METTLER TOLEDO). NH 4 + is described in Verdouw et al. (1978) was used for analysis.
[194]
Vannamei ( L. vannamei ) with an average weight of 2.87 (±0.08) g were randomly placed in 14 96 L tanks in a total of 14 tanks in a zero water change method . There were 2 repetitions per test section, divided into 4 times a day (08:30, 12:00, 15:30 and 19:00 hours), and 12% of the body weight was fed. Verdouw et al. According to the method of (1978), it was collected once daily and the total ammonia concentration of the breeding water was measured for 10 days. The results are shown in Table 9.
[195]
[Table 9]
DO mgL -1 pH NH 4 + mgL -1
Mean 6.85 7.10 0.102
Max 7.21 6.78 0.154
Min 6.39 6.48 0.035
[196]
As shown in Table 9 and FIG. 6, interestingly, from the 7th day, compared to the control, all the test groups began to show lower results in the total ammonia concentration, and at the 10th day, all the test groups had significantly lower ammonia concentrations than the control. Showed.
[197]
[198]
2-7. Digestibility analysis
[199]
2-7-1. Indicator (Cr2O3) analysis
[200]
[201]
To analyze the chromium oxide content in test feed and manure, Divakaran et al. (2002) was used.
[202]
Specifically, the test feed and the minute sample were incinerated for 4 hours in an incinerator (550°C), and the obtained sample was used for analysis. First, in order to oxidize chromium oxide in mono-chromate form, 5 to 10 mg of a minute sample was weighed and transferred to a glass test tube. 4 ml of perchloric reagent (HClO 4 ) was added to the glass test tube containing the sample . Perchloric reagent (70%) was prepared by mixing 200 ml of nitric acid in 100 ml of distilled water, cooling, and then mixing 200 ml of 70% perchloric acid. The sample and the glass test tube to which the perchloric reagent was added were placed on a heating plate, heated at 300° C. for 15 minutes, and then allowed to cool at room temperature. The sample after the pretreatment was transferred to a 50 ml glass flask and quantified to 25 ml with 3rd distilled water. Then, the absorbance was measured at 350 nm using a spectrophotometer (Beckman DU-730). As for the measured absorbance, the chromium oxide content of the sample was calculated using a standard equation made of a standard solution pretreated as in sample analysis.
[203]
[204]
2-7-2. Dry and protein digestibility analysis
[205]
The dry matter and protein digestibility of the test feed was calculated by the following method;
[206]
ADC of dry matter (%) = 100-100 x (%Cr 2 O 3 in diet / %Cr 2 O 3 in feces);
[207]
ADC of protein (%) = 100-100 x (%Cr 2 O 3 in diet / %Cr 2 O 3 in feces) x (%protein in feces / %protein in diet)
[208]
Table 10 shows the results of digestibility analysis conducted after the feeding experiment.
[209]
[Table 10]
Control 1 Control 2 BS Group 1 BS Group 2 BS+BP Group 1 BS+BP Group 2 BS+BP+BL Group 1
ADCd (%) 1 85.6±0.7 c 87.4±0.6 ab 87.9±0.4 a 86.9±0.5 b 87.7±0.3 ab 87.9±0.3 a 88.1±0.4 a
ADCp (%) 2 93.5±0.3 b 95.0±0.2 a 95.2±0.2 a 94.8±0.2 a 95.0±0.1 a 95.1±0.1 a 95.1±0.2 a
[210]
Values ​​are mean of triplicate groups and presented as mean ± SD Values ​​with different superscripts in the same column are significantly different ( P <0.05).
[211]
1 Apparent digestibility coefficient of dry matter
[212]
2 Apparent digestibility coefficient of protein.
[213]
As shown in Table 10, in the dry matter digestibility and protein digestion rate, all test groups showed significantly higher results than the control group.
[214]
[215]
Preparation Example 3. Preparation of a shrimp feed composition containing Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis
[216]
Bacillus subtilis 1 selected in Preparation Example 1 ( Bacillus subtilis : accession number KCCM11143P, hereinafter referred to as'BS'), Bacillus pumilus (accession number KCCM11144P, hereinafter referred to as'BP') and Bacillus lichenipo A feed composition containing Miss (Bacillus licheniformis, hereinafter referred to as'BL') was prepared.
[217]
Specifically, Comparative Example 1 not including the Bacillus strains, Comparative Example 3 containing the BS 10 9 x 0.2 CFU / g, Comparative Example 4 containing the BS 10 10 x 0.2 CFU / g, the BS, The composition of Example 5 containing 10 9 x 0.2 CFU/g each of BP and BL and the composition of Example 6 containing 10 10 x 0.2 CFU/g each of BS, BP and BL was used in fish oil and water. After adding and mixing, it was prepared in a pellet form. The feed compositions of Comparative Examples 1, 3 and 4, and Examples 5 and 6 were dried at 25°C for about 24 hours using a dryer and then stored at -20°C until the experiment.
[218]
[Table 11]
Ingredients (%) Comparative Example 1 Comparative Example 3 Comparative Example 4 Example 5 Example 6
Fish meal 40 40 40 40 40
SBM 44% South America 12.81 12.81 12.81 12.81 12.81
Squid liver powder 10 10 10 10 10
Wheat flour 25.61 25.61 25.61 25.61 25.61
Amygluten 110 3 3 3 3 3
Fish oil A/C 2 2 2 2 2
Amino acid 0.42 0.42 0.42 0.42 0.42
Vitamin/Mineral premix 5.96 5.96 5.96 5.96 5.96
Rice bran 0.2 0.2 0.2 0.18 0.00
BS (10 9 x CFU/g) 0.00 0.2 0.00 0.00 0.00
BS (10 10 x CFU/g) 0.00 0.00 0.2 0.00 0.00
BS+BP+BL (10 9 x CFU/g) 0.00 0.00 0.00 0.2 0.00
BS+BP+BL (10 10 x CFU/g) 0.00 0.00 0.00 0.00 0.2
Chemical composition (% dry matter)
Moisture 5.68 5.61 5.60 5.71 5.71
Crude protein 47.3 47.4 47.4 48.5 48.5
Crude lipid 7.31 7.48 7.49 7.70 7.70
Crude ash 6.01 6.10 6.12 6.11 6.11
[219]
Experimental Example 3. Evaluation of the preventive effect of feed composition on AHPND
[220]
3-1. Shrimp preparation
[221]
A total of 40 white-leg shrimp were prepared in a total of 30 fish per tank. Airstones were installed in all test tanks to maintain dissolved oxygen, and the breeding water temperature was maintained in the range of 28 to 32°C for the entire test period. Feed was limitedly supplied 4 times a day (4 to 12% of fish weight).
[222]
[223]
3-2. Vibrio enteritis attack test
[224]
In order to confirm the antibacterial effect of the feed composition containing Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis, an attack test of enteritis Vibrio against shrimp was conducted once. In the case of the Vibrio strain, the AHPND (EMS) induced strain isolated in Vietnam in 2013 was used for the test. In the case of the attack test, the feed composition of the example was supplied to the shrimp for 4 weeks, and then the same weight (average weight: 2.30 g) of shrimp was placed in 4 repetitions at 96 per group. The bacteria were cultured for 24 hours at 30 o C and 150 rpm using TSB + medium, and the enteritis Vibrio suspension was immersed in a concentration of 6.3 x 10 5 CFU/ml per tank . The mortality and swimming status of the shrimp were checked every 1 hour after immersion, and 95% water exchange was performed after 8 hours. The test feed was divided into three times a day (8:30, 13:30, and 18:30 hours) and limited supply (10 to 12% of the fish's weight), and mortality was observed for 70 hours. The results for this are shown in Table 12 below.
[225]
[Table 12]
Treatment Survival (%)
Trial
Control 1 0.00±0.00
BS Group 3 8.93±10.7
BS Group 4 6.90±5.45
BS+BP+BL Group 1 15.6±11.6
BS+BP+BL Group 2 12.5±20.5
[226]
As shown in Table 12, BS + BP + BL groups 1 and 2 provided the feed compositions of Examples 5 and 6 containing Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis are In the attack test, compared to the control group 1 to which the feed composition of Comparative Example 1 was administered and the feed compositions of Comparative Examples 3 and 4 were provided, the survival rate was higher than that of BS groups 3 and 4.
[227]
[228]
Experimental Example 4. Evaluation of the preventive effect of feed composition against WSSV
[229]
In order to confirm the antiviral effect of the feed composition containing Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis, an attack test of white spot syndrome virus (WSSV) against shrimp was conducted. In the case of the white spot syndrome virus, a virus isolated from WSSV-infected white-legged shrimp ( Litopenaues vannamei ) in a domestic farm in 2017 was used. In the case of the attack test, the feed composition of the example was supplied to the shrimp for 6 weeks, and then the same weight (average weight: 6.25 g) of shrimp was placed in 4 repetitions at 96 per group. Virus inoculation concentration was 4.1 x 10 5 copies/ul, and 100ul per shrimp was inoculated into the shrimp muscle using a syringe. The final inoculation concentration per head is 4.1 x 10 7 copies/ul.
[230]
After inoculation, the mortality and swimming status of the shrimp were checked. The test feed was divided into three times a day (8:30, 13:30 and 18:30 hours) and limited supply (10 to 12% of the fish's weight), and the degree of mortality was observed for 125 hours. The results are shown in Table 13 below.
[231]
[Table 13]
Treatment Survival (%)
Trial
Control 1 23.21±3.57
BS Group 4 25.00±18.0
BS+BP+BL Group 1 41.1±17.9
BS+BP+BL Group 2 28.57±15.4
[232]
As shown in Table 13, BS + BP + BL groups 1 and 2 provided with the feed compositions of Examples 5 and 6 containing Bacillus subtilis, Bacillus pumils, and Bacillus licheniformis are white spot syndrome virus shrimp. In the attack test for, compared to the control group 1 to which the feed composition of Comparative Example 1 was administered and the BS group 4 to which the feed composition of Comparative Example 4 was provided, the survival rate was higher.
[233]
[234]
Experimental Example 5. Evaluation of the preventive effect of the feed composition against the complex infection of WSSV and AHPND
[235]
In order to confirm the antibacterial and antiviral effects of the feed composition containing Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis, an attack test was conducted against shrimps of Vibrio Enteritis and White Spot Syndrome Virus. In the case of the Vibrio strain, the AHPND (EMS) induced strain isolated in Vietnam in 2013 was used for the test. In the case of the white spot syndrome virus, a virus isolated from WSSV-infected white-legged shrimp ( Litopenaues vannamei ) in a domestic farm in 2017 was used. In the case of the attack test, the feed composition of the example was supplied to the shrimp for 6 weeks, and then the same weight (average weight: 4.52g) of shrimp was placed in 4 repetitions at 96 per group. The inoculation concentration of virus was 8.3 x 10 3 copies/ul, and 50ul per shrimp was inoculated into the shrimp muscle using a syringe. The final inoculation concentration per head is 4.1 x 10 4 copies/ul. After virus inoculation, 2 days later, Vibrio bacterial infection was performed.
[236]
The bacteria were cultured for 24 hours at 30 o C and 150 rpm using TSB + medium, and the enteritis Vibrio suspension was immersed in a concentration of 1.3 x 10 5 CFU/ml per tank . The mortality and swimming status of the shrimp were checked every 1 hour after immersion. The test feed was divided into three times a day (8:30, 13:30, and 18:30 hours) and limited supply (10 to 12% of the fish's weight), and the degree of mortality was observed for 7 days. The results are shown in Table 14 below.
[237]
[Table 14]
Treatment Survival (%)
Trial
Control 1 51.8±13.5
BS Group 4 62.5±14.7
BS+BP+BL Group 1 62.5±6.8
BS+BP+BL Group 2 66.1±28.8
[238]
As shown in Table 14, BS + BP + BL groups 1 and 2 provided with the feed compositions of Examples 5 and 6 containing Bacillus subtilis, Bacillus pumils, and Bacillus licheniformis are enteritis Vibrio and white spot syndrome In the virus attack test against shrimp, the survival rate was higher than that of the control group 1 to which the feed composition of Comparative Example 1 was administered and the BS group 4 to which the feed composition of Comparative Example 4 was administered.
[239]
[240]
Through the above examples, the feed composition comprising Bacillus subtilis, Bacillus pumilus, and Bacillus licheniformis according to the present application can increase the growth, feed efficiency, digestibility, breeding water quality and non-specific immunity of white shrimp. , In addition, it can be expected that the marketability of shrimp can be enhanced by enabling the production of high protein white leg shrimp.
[241]
[242]
Name of deposit institution: Korea Microorganism Conservation Center (overseas)
[243]
Accession number: KCCM11143P
[244]
Consignment date: 20101214
[245]
[246]
Name of deposit institution: Korea Microorganism Conservation Center (overseas)
[247]
Accession number: KCCM11144P
[248]
Consignment date: 20101214
[249]
[250]
Name of deposit institution: Korea Microorganism Conservation Center (overseas)
[251]
Accession number: KCCM11270P
[252]
Consignment Date: 20120322
[253]
[Amendment 14.08.2019 pursuant to Rule 91]　

[254]
[Amendment 14.08.2019 pursuant to Rule 91]　

[255]
[Amendment 14.08.2019 pursuant to Rule 91]　

[256]
[257]
[258]
Claims
[Claim 1]
Bacillus subtilis strain, Bavilus pumilus strain, and Bacillus licheniformis strain; Cultures thereof; Its concentrate; Or a feed composition for preventing or treating acute hepatic pancreatic necrosis (AHPND) comprising a dried product thereof as an active ingredient.
[Claim 2]
The feed composition of claim 1, wherein the Bacillus subtilis is KCCM11143P, the Bacillus pumilus is KCCM11144P, and the Bacillus licheniformis KCCM11270P.
[Claim 3]
The feed composition according to claim 1 or 2, wherein the Bacillus subtilis, the Bacillus pumilus, and the Bacillus licheniformis have a number of 1 x 10 4 to 1 x 10 11 CFU bacteria per g of the active ingredient .
[Claim 4]
The feed composition according to any one of claims 1 to 3, wherein the acute hepatic pancreatic necrosis (AHPND) is caused by Vibrio parahaemolyticus .
[Claim 5]
A method for preventing or treating acute hepatic pancreatic necrosis comprising administering the feed composition of any one of claims 1 to 4 to an individual.
[Claim 6]
The method of claim 5, wherein the subject is a shrimp.
[Claim 7]
Bacillus subtilis strain, Bacillus pumilus strain, and Bacillus licheniformis strain; Cultures thereof; Its concentrate; Or a feed composition for the prevention or treatment of white spot syndrome (WSS) comprising the dried product as an active ingredient.
[Claim 8]
The feed composition of claim 7, wherein the white spot syndrome (WSS) is caused by white spot virus (WSSV).
[Claim 9]
The feed composition according to claim 7 or 8, wherein the Bacillus subtilis is KCCM11143P, the Bacillus pumilus is KCCM11144P, and the Bacillus licheniformis KCCM11270P.
[Claim 10]
The method according to any one of claims 7 to 9, wherein the Bacillus subtilis, the Bacillus pumilus, and the Bacillus licheniformis have a number of 1 x 10 4 to 1 x 10 11 CFU per gram of active ingredient . , Feed composition.
[Claim 11]
A method of preventing or treating white spot syndrome, comprising administering the feed composition of any one of claims 7 to 10 to an individual.
[Claim 12]
The method of claim 11, wherein the subject is a shrimp.