阅读说明：本技术 包括作为活性成分的枯草芽孢杆菌菌株的用于预防或治疗急性肝胰腺坏死病（ahpnd）或白斑综合征（wss）的饲料组合物 (Feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or White Spot Syndrome (WSS) comprising Bacillus subtilis strain as active ingredient ) 是由 金智恩 韩知垠 金星勋 禹谞衡 殷钟秀 曹厦昀 金载沅 于 2018-12-28 设计创作，主要内容包括：本公开涉及用于预防或治疗急性肝胰腺坏死病(AHPND)或白斑综合征(WSS)的饲料组合物,包括作为活性成分的枯草芽孢杆菌(KCCM11143P)菌株、其培养基、其浓缩物、或其干物质。饲料组合物展现出抵抗引起虾AHPND的副溶血弧菌的抗菌活性和抵抗引起WSS的白斑综合征病毒(WSSV)的抗病毒活性。(The present disclosure relates to a feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or White Spot Syndrome (WSS), comprising a bacillus subtilis (KCCM11143P) strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof, as an active ingredient. The feed composition exhibits antibacterial activity against Vibrio parahaemolyticus that causes AHPND in shrimp and antiviral activity against WSS-causing White Spot Syndrome Virus (WSSV).)

1. A feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND), comprising as an active ingredient a Bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof.

2. The feed composition of claim 1, wherein the bacillus subtilis is KCCM 11143P.

3. The feed composition of claim 1 or 2, wherein the bacillus subtilis has a bacteria count of 1x 10⁴To 1x 10¹¹CFU per g of said active ingredient.

4. The feed composition according to any one of claims 1 to 3, wherein the feed composition further comprises as an active ingredient Bacillus pumilus, Bacillus licheniformis or a combination thereof.

5. The feed composition according to any one of claims 1 to 4, wherein the acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus.

6. A method for preventing or treating acute hepatopancreatic necrosis disease, comprising administering to a subject the feed composition of any one of claims 1 to 5.

7. The method of claim 6, wherein the subject is a shrimp.

8. A feed composition for preventing or treating White Spot Syndrome (WSS), comprising a Bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof as an active ingredient.

9. The feed composition according to claim 8, wherein the White Spot Syndrome (WSS) is caused by White Spot Syndrome Virus (WSSV).

10. The feed composition according to claim 8 or 9, wherein the bacillus subtilis is KCCM 11143P.

11. The feed composition of any one of claims 8-10, wherein the bacillus subtilis has a bacteria count of 1x 10⁴To 1x 10¹¹CFU per g of said active ingredient.

12. The feed composition according to any one of claims 8 to 11, wherein the feed composition further comprises as an active ingredient bacillus pumilus, bacillus licheniformis or a combination thereof.

13. A method for preventing or treating white spot syndrome, comprising administering to a subject the feed composition of any one of claims 8 to 12.

14. The method of claim 13, wherein the subject is a shrimp.

Technical Field

The present disclosure relates to a feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or White Spot Syndrome (WSS), comprising a bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof, as an active ingredient.

Background

Early Mortality Syndrome (EMS), acute hepatopancreas necrosis disease (AHPND), and acute hepatopancreas necrosis syndrome (AHPNS) are rapidly developing diseases in shrimp farming, and vibrio parahaemolyticus is the causative agent of these diseases; that is, the insect toxin produced by Vibrio parahaemolyticus results in 100% lethality within 6 hours or within a week (Tran et al 2013 Dis aqua Org105: 45-55). Insect toxins are produced by the expression of specific genes present in specific plasmids of bacteria (i.e., photorhabdus insect-related toxins; Pir toxins) and are susceptible to movement around, i.e., they have motility. In this regard, these diseases spread faster than viral shrimp diseases such as White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), infectious myonecrosis virus (IMNV), etc., which have recently received attention. AHPND started in china in 2009 and spread rapidly in asian countries (e.g. thailand, malaysia and vietnam) within a year and further occurred in mexico to spread to other central america countries, causing losses to most of the shrimp market. Korea also suffers from a tremendous loss of AHPND from 2015 to 2016, and research is currently being conducted to prevent or control diseases.

Recently, therapeutic agents for the treatment of existing pathogenic bacteria have been limited by breeding-related regulations due to consumer demand for safe products and production strategies for continuous breeding. Therefore, in shrimp farming, not only the quality of seed commodities (seeds) and the farming method but also important factors for controlling diseases are considered.

Meanwhile, probiotics are defined as microbial preparations or components contributing to microbial balance in the intestinal tract, and have a word-derived meaning opposite to antibiotics, which means antibacterial substances. Representatively, examples of the probiotic include lactic acid bacteria such as lactobacillus and bifidobacterium. Furthermore, probiotics do not have toxic genes against humans and animals, nor do they produce pathogenic substances, and are therefore classified as GRAS (generally regarded as safe). Therefore, the development of a feed additive using probiotics, the safety of which is proven, has been actively completed.

As an example, korean patent laid-open No. 10-2011-035554 discloses a novel mixed strain of CMB L1 of bacillus and CMB201 of lactobacillus, an anticancer and immunity-enhancing food composition using the same, and a microbial preparation having antibacterial activity. Further, korean patent No. 10-0977407 discloses an immunopotentiator and a feed additive for animals, which contains lysate of Zygosaccharomyces bailii (Zygosaccharomyces bailii), which increases various activities of neutrophils, major phagocytes of animals, and enhances non-specific defense against invasive inoculation by pathogenic bacteria. However, the actual immune activity of the feed additive using probiotics is insufficient, and thus research on the feed additive using probiotics still having excellent immune activity is required.

Disclosure of Invention

Technical problem

The present inventors have made a great effort to develop shrimp feed supplemented with probiotics (bacillus) to prevent shrimp AHPND. As a result, they have confirmed that when a shrimp is fed with a feed composition supplemented with Bacillus subtilis, the survival rate of shrimp against AHPND infection (caused by a strain isolated from the affected area of Vietnam in 2013; Tran et al 2013) or WSSV infection is improved, and not only the growth rate and nonspecific immunity of shrimp are improved, but also the water quality is improved, and the production of high-protein shrimp can be obtained, thereby completing the present disclosure.

Technical scheme

An object of the present disclosure is to provide a feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND), comprising a bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof as an active ingredient.

It is another object of the present disclosure to provide a method for preventing or treating acute hepatopancreatic necrosis disease, comprising administering to a subject a feed composition.

It is still another object of the present disclosure to provide a feed composition for preventing or treating White Spot Syndrome (WSS), comprising a bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof as an active ingredient.

It is still another object of the present disclosure to provide a method for preventing or treating white spot syndrome, comprising administering to a subject a feed composition.

Advantageous effects

The composition of the present disclosure, which has antibacterial activity against vibrio parahaemolyticus, which causes AHPND causing problems in shrimp farming, and antiviral activity against white spot syndrome virus, which causes WSS, and exhibits the effect of improving the immunity of the hepatopancreas of shrimps, comprises bacillus subtilis (KCCM11143P) strain as an active ingredient, and thus can be used as a shrimp feed composition or a feed additive.

Drawings

FIG. 1 is a graph showing the survival rate of shrimps infected with Vibrio parahaemolyticus.

FIG. 2 is a graph of an analysis of AHPND content in shrimp hepatopancreas.

Fig. 3 is an image showing pathological features of a shrimp hepatopancreas.

FIG. 4 is a graph showing the growth rate of shrimp. Fig. 4(a) to 4(d) show final body weight, weight gain rate, daily growth rate and feed conversion rate, respectively.

FIG. 5 is a graph analyzing nonspecific immunity of shrimp. FIGS. 5(a) to 5(e) show the activity against macrophage, phenol oxidase, antiprotease, lysozyme and superoxide dismutase, respectively.

FIG. 6 is a graph showing the results of analyzing the quality of culture water with zero water exchange.

Best mode for carrying out the invention

Hereinafter, the present disclosure will be described in detail. Meanwhile, each of the explanations and exemplary embodiments disclosed herein may be applied to other explanations and exemplary embodiments. That is, all combinations of the various factors disclosed herein are within the scope of the present disclosure. Furthermore, the scope of the present disclosure should not be limited by the specific disclosure provided below.

In order to achieve the above objects, aspects of the present disclosure provide a feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND), comprising a bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof, as an active ingredient.

As used herein, the term "Bacillus subtilis" is a non-toxic and spore-forming aerobic bacterium. Bacillus subtilis is widely distributed in nature, such as in hay, soil, sewage, air, and the like. This bacterium is widely used in industry because it produces enzymes that coagulate milk, saccharified starch, and decomposed fats and oils. The optimal conditions for bacterial growth are a pH of 7 to 8.5 and a temperature of 37 ℃ to 40 ℃. Due to the characteristics of bacillus strains, the strains do not have toxic genes to humans and animals, are nonpathogenic, do not produce pathogenic substances, and exhibit rapid growth rates in vivo. Bacillus subtilis has an anaerobic habitat in the presence of glucose or the like, and endospores allow bacillus to survive in extremely severe environments such as high or low temperatures.

In the present disclosure, the bacillus subtilis may be the strain deposited under accession number KCCM 11143P.

In the present disclosure, a feed composition comprising bacillus subtilis (KCCM11143P) was prepared.

The compositions of the present disclosure may include bacillus subtilis (KCCM11143P) with a bacterial count of 1x 10⁴CFU to 1x 10¹¹CFU per gram of total active ingredient. In particular, the bacterial count may be 1x 10⁴CFU/g to 1x 10¹⁰CFU/g, and more specifically, the compositions of the present disclosure comprise Bacillus subtilis (KCCM11143P) with a bacterial count of 1x 10⁸CFU/g to 1x 10¹⁰CFU/g。

For the purpose of the present disclosure, the bacillus subtilis contained as an active ingredient in the feed composition may include only bacillus subtilis (KCCM 11143P).

As used herein, the term "acute hepatopancreatic necrosis disease (AHPND)" refers to a disease named Early Mortality Syndrome (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS), collectively referred to as mass mortality (mass death) caused by pathogens within 30 days of placement in aquafarms. AHPND is caused by vibrio parahaemolyticus, a pathogenic bacterium present in seawater, causing a large number of infections in white leg shrimps, accounting for about 96% of korean cultured shrimps. It is harmful to prawn due to high mortality in the early life, but it is not harmful to humans.

Vibrio parahaemolyticus is a gram-negative bacterium belonging to the genus Vibrio, which causes acute food poisoning and enteritis in humans and causes vibriosis in fish. Recently, Vibrio parahaemolyticus has been identified as a causative bacterium of acute hepatopancreatic necrosis disease (AHPND), which causes population death in shrimp farming.

The compositions of the present disclosure may further comprise bacillus pumilus, bacillus licheniformis, or a combination thereof as an active ingredient. The Bacillus pumilus may be the strain deposited under accession number KCCM11144P, and the Bacillus licheniformis may be the strain deposited under accession number KCCM 11270P.

As used herein, the term "prevention" refers to any action that results in the suppression or delay of symptoms of shrimp AHPND by administering a composition comprising bacillus subtilis according to the present disclosure.

As used herein, the term "treatment" refers to any action that results in the symptomatic relief or complete recovery of the AHPND of prawns by administering a composition comprising bacillus subtilis (KCCM11143P) according to the present disclosure.

The composition may comprise, in addition to the above-mentioned strains contained as active ingredients, known carriers or additives acceptable for pharmaceutical, food or feed use. In the present disclosure, as a probiotic preparation having antibacterial activity against vibrio parahaemolyticus, it includes bacillus subtilis (KCCM11143P) to which a binder, an emulsifier, a preservative, and the like are added to prevent deterioration (deterioration) in the quality of the probiotic preparation; and the feed may be added with amino acids, vitamins, enzymes, flavorings, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. to increase the effectiveness of the probiotic preparation. In addition thereto, a feed mixture, etc. may be further included, but the present disclosure is not limited thereto.

In the embodiments of the present disclosure, as a result of confirming whether feeding of a feed composition to shrimp would improve immunity against AHPND and exhibit a disease prevention effect, it was confirmed that the groups (BS groups 1 and 2) to which the feed composition comprising a bacillus subtilis (KCCM11143P) strain of the present disclosure (examples 1 and 2) was administered can improve disease resistance of shrimp against vibrio parahaemolyticus infection and significantly reduce the amount of toxin of AHPND in the hepatopancreas of shrimp, compared to the groups (control groups 1 and 2) to which comparative example 1 (excluding probiotics) and comparative example 2 (including commercially available probiotics (mixed preparation of three bacillus (bacillus subtilis, bacillus pumilus, bacillus licheniformis))) were administered.

In other embodiments of the present disclosure, as a result of analyzing non-specific immunity of a feed composition including the above-mentioned strain, it was found that macrophage (NBT) activity, glutathione peroxidase (GPx) activity, lysozyme activity, Phenol Oxidase (PO) activity, superoxide dismutase (SOD) activity and anti-protease activity were significantly higher than those of the comparative examples. Based on this result, it can be seen that the composition can be used to increase the nonspecific immune response of shrimp or to increase its immunity.

The present disclosure provides a feed additive for shrimp farming comprising the above feed composition.

In the feed additive of the present disclosure, in addition to the above-mentioned active ingredients, known carriers or stabilizers acceptable for pharmaceutical, food, or feed use may be added. If necessary, all kinds of nutrients such as vitamins, amino acids and minerals, antioxidants, and other additives may be added, and the shape thereof may be convenient such as powder, granule, pellet and suspension. When a feed additive according to the present disclosure is supplied, the feed additive may be supplied to a non-ruminant animal alone or mixed with feed.

In addition, the present disclosure provides a feed for shrimp farming, which includes the above-described feed additive.

The bacillus subtilis (KCCM11143P) strain of the present disclosure, which is a gram-positive bacterium having sporulation ability, is preferably formulated in a spore form, but is not limited thereto. The feed of the present disclosure is not particularly limited, and any feeds such as powder feed, solid feed, wet pellet feed, dry pellet feed, Extruder Pellet (EP) feed, and raw feed (raw feed) may be used.

As mentioned above, bacillus forms endospores and is therefore very stable to heat. Accordingly, bacillus subtilis (KCCM11143P) of the present disclosure may be prepared in the form of a feed additive and then mixed with a feed, respectively, or may be prepared by directly adding to a feed at the time of preparing the feed. The bacillus subtilis included in the feed of the present disclosure may be in a liquid or dry state, and is preferably in the form of a dry powder. The drying process may be performed by air drying, natural drying, spray drying, and freeze drying, but is not limited thereto. The bacillus subtilis (KCCM11143P) of the present disclosure may be mixed into a powder form in an amount of 0.05 to 10% by weight, preferably 0.1 to 1% by weight, based on the weight of the feed. In addition, the feed is used for aquaculture, and in addition to the bacillus subtilis (KCCM11143P) of the present disclosure, the feed may further include conventional additives capable of improving the preservability of the feed.

In order to achieve the above objects, another aspect of the present disclosure provides a method for preventing or treating acute hepatopancreatic necrosis disease, comprising administering to a subject a feed composition of the present disclosure.

Herein, acute hepatopancreatic necrosis disease, prevention and treatment of the present disclosure are as described above.

As used herein, the term "subject" may refer to a fish or crustacean (the farming of which is possible) and which has or is at risk of developing acute hepatopancreatic necrosis, but which may refer to a shrimp according to the objects of the present disclosure.

The feed is preferably supplied in the same amount and at the same feeding intervals as conventional feed. In addition, pathogenic bacteria refer to bacteria causing population death of shrimp by inducing AHPND in shrimp farming, and specifically may refer to vibrio parahaemolyticus.

In shrimp farming, the causes of population death include not only Vibrio parahaemolyticus but also infections caused by various viruses and the ammonia concentration in the farming water. In shrimp farming, ammonia in the farming water appears as a metabolite of proteins such as shrimp excrements, feed wastes, and the like, and it greatly changes with the increase in pH and water temperature. High concentrations of ammonia are a direct cause of acute death in shrimp, leading to population death; and low concentrations of ammonia can lead to shrimp growth and decreased feeding ability and immunity in the long term, which in turn can lead to the development of various diseases.

In an embodiment of the present disclosure, culture water of shrimp, in which the feed composition of the present disclosure has been fed, is collected and analyzed, and as a result, it is found that the total ammonia concentration in the culture water is significantly lower than that of a control, so that the bacillus subtilis (KCCM11143P) strain of the present disclosure can improve the water quality of the water in which the shrimp are cultured.

As described above, bacillus subtilis (KCCM11143P) of the present disclosure can not only enhance disease resistance of shrimp but also inhibit the toxin of AHPND in shrimp hepatopancreas. Therefore, by using the strain, an effect of preventing vibrio parahaemolyticus causing diseases can be obtained, thereby enabling shrimp to be cultured more safely.

In order to achieve the above objects, still another aspect of the present disclosure provides a feed composition for preventing or treating White Spot Syndrome (WSS), comprising a bacillus subtilis strain, a culture medium thereof, a concentrate thereof, or a dry matter thereof as an active ingredient.

The terms bacillus subtilis, prophylaxis and treatment of the present disclosure are as described above.

The compositions of the present disclosure may include bacillus subtilis (KCCM11143P) with a bacterial count of 1x 10⁴CFU to 1x 10¹¹CFU per gram of total active ingredient. In particular, the bacterial count may be 1x 10⁴CFU/g to 1x 10¹⁰CFU/g, and more specifically, the compositions of the present disclosure comprise Bacillus subtilis (KCCM11143P) with a bacterial count of 1x 10⁸CFU/g to 1x 10¹⁰CFU/g。

As used herein, the term "White Spot Syndrome Virus (WSSV)" refers to a virus that is widely distributed around the world. Since WSSV has a tail attachment at the end of virions and has a form similar to bacillus ovorans in which rod-shaped capsids and envelopes are present, it is considered to be a baculovirus or bacillus-like formed virus; however, recently, it has been renamed as whispo virus (whispovir), which is a genetically new group of viruses. The virus was about 275nm in length, about 120nm in diameter, and consisted of double-stranded DNA of about 290kb in size.

The composition may comprise, in addition to the above-mentioned strains contained as active ingredients, known carriers or additives acceptable for pharmaceutical, food or feed use. In the present disclosure, as a probiotic preparation having an antiviral activity against white spot syndrome virus, it includes bacillus subtilis (KCCM11143P) added with a binder, an emulsifier, a preservative, etc. to prevent deterioration of the quality of the probiotic preparation; and the feed is supplemented with amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. to improve the efficacy of the probiotic preparation. In addition thereto, a feed mixture, etc. may be further included, but the present disclosure is not limited thereto.

In the embodiments of the present disclosure, as a result of confirming whether feeding of a feed composition to shrimps would increase resistance to White Spot Syndrome Virus (WSSV) and exhibit a disease prevention effect, it was confirmed that the disease resistance of shrimps against White Spot Syndrome Virus (WSSV) infection could be increased compared to the group (control group 1) to which comparative example 1 (not including probiotics) was administered (BS group 1) to which the feed composition including bacillus subtilis (KCCM11143P) strain (example 1) of the present disclosure was administered.

In other embodiments of the present disclosure, as a result of confirming whether resistance to complex (complex) infection of White Spot Syndrome Virus (WSSV) and acute hepatopancreatic necrosis disease (AHPND) is improved and a survival rate-enhancing effect is exhibited when the feed composition is fed to shrimps, it was confirmed that disease resistance of shrimps against complex infection of WSSV and AHPND can be enhanced compared to a group (control group 1) to which comparative example 1 (not including probiotics) is administered (BS group 1) to which the feed composition including bacillus subtilis (KCCM11143P) strain (example 1) of the present disclosure is administered.

The present disclosure provides a feed additive for shrimp farming comprising the feed composition described above.

In the feed additive of the present disclosure, known carriers or stabilizers acceptable for pharmaceutical, food or feed use may be added in addition to the above-mentioned active ingredients. If necessary, all kinds of nutrients such as vitamins, amino acids and minerals, antioxidants, and other additives may be added, and the shape thereof may be convenient such as powder, granule, pellet and suspension. When a feed additive according to the present disclosure is supplied, the feed additive may be supplied to a non-ruminant animal alone or mixed with feed.

In addition, the present disclosure provides a feed for shrimp farming, comprising the above feed additive.

The bacillus subtilis (KCCM11143P) strain of the present disclosure, which is a gram-positive bacterium having sporulation ability, is preferably formulated in a spore form, but is not limited thereto. The feed of the present disclosure is not particularly limited, and any feeds such as powder feed, solid feed, wet pellet feed, dry pellet feed, Extruder Pellet (EP) feed, and raw feed are usable.

As mentioned above, bacillus forms endospores and is therefore very stable to heat. Accordingly, bacillus subtilis (KCCM11143P) of the present disclosure may be prepared in the form of a feed additive and then mixed with a feed, respectively, or may be prepared by directly adding to a feed at the time of preparing the feed. The bacillus subtilis included in the feed of the present disclosure may be in a liquid or dry state, and is preferably in the form of a dry powder. The drying process may be performed by air drying, natural drying, spray drying, and freeze drying, but is not limited thereto. The bacillus subtilis (KCCM11143P) of the present disclosure may be mixed into a powder form in an amount of 0.05 to 10% by weight, preferably 0.1 to 1% by weight, based on the weight of the feed. In addition, the feed is used for aquaculture, and in addition to the bacillus subtilis (KCCM11143P) of the present disclosure, the feed may further include conventional additives capable of improving the preservability of the feed.

In order to achieve the above objects, still another aspect of the present disclosure provides a method for preventing or treating white spot syndrome, comprising administering the feed composition of the present disclosure to a subject.

Herein, the terms white spot syndrome, prevention, treatment and subject of the present disclosure are as described above.

In shrimp farming, the causes of population death include not only Vibrio parahaemolyticus but also infections caused by various viruses. In particular, the virus may be referred to as White Spot Syndrome Virus (WSSV).

As described above, bacillus subtilis (KCCM11143P) of the present disclosure may obtain the effect of enhancing resistance to White Spot Syndrome Virus (WSSV), thereby enabling shrimp to be cultured more safely.

Detailed Description

Hereinafter, the present disclosure will be described in detail by way of exemplary embodiments. However, these exemplary embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Preparation example 1 selection of probiotic bacteria with antibacterial Activity

In order to select a strain having antibacterial activity against vibrio parahaemolyticus which induces shrimp AHPND, a clear zone (clear zone) test was performed. 0.5% agar (3mL) and 100. mu.L of a shaking culture (2.0X 10) of pathogenic bacteria were added⁹CFU/mL) were mixed and inoculated on TSA + medium to prepare upper (top layer, top) agar. Cultures of 12 Bacillus subtilis strains (held by CJCheilJedang and commercial strains), each in an amount of 10. mu.L, were dropped on the upper part (top ) of the prepared upper agar, cultured at 30 ℃ for 18 hours, and the presence/absence of the transparent region was observed. Commercially available bacillus subtilis and composite phage were evaluated together for antibacterial activity.

[ Table 1]

++++: strong activity, +: presence of activity, -: is inactive

As shown in Table 1 above, Bacillus subtilis 1 microorganism (CJBS-01) showed the most excellent in vitro antibacterial effect against Vibrio parahaemolyticus causing AHPND. However, although this microorganism exhibits in vitro antibacterial activity against a particular pathogen, the antibacterial activity observed is only an in vitro effect, and it does not necessarily mean that ingestion of Vibrio parahaemolyticus by an animal will provide immunity to the animal or a prophylactic effect against a particular pathogen.

Therefore, in the following experiments, it was examined whether the Bacillus subtilis 1(CJBS-01) microorganism resulted in exhibiting an improvement in immunity and a disease prevention effect against AHPND disease in shrimps when fed to the shrimps. In addition, it was also examined whether Bacillus subtilis 1(CJBS-01) had an effect on weight gain and digestibility.

Bacillus subtilis 1(CJBS-01) is a strain deposited at 14.12.2010 in Korea Culture Collection (KCCM) and designated as KCCM 11143P.

Preparation example 2 preparation of Bacillus subtilis-containing feed composition for shrimp

A feed composition containing Bacillus subtilis 1 (accession No. KCCM11143P, hereinafter referred to as "BS") selected in preparation example 1 was prepared.

Specifically, comparative example 1 containing no Bacillus subtilis, comparative example 2 containing commercially available Bacillus species (i.e., a mixed preparation of three Bacillus species (Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis)), contained the selected amount of 10¹⁰EXAMPLE 1 Bacillus subtilis 1(BS) at 0.2CFU/g and a content of 10⁹X 0.2CFU/g BS the composition of example 2 was each mixed with fish oil and water and prepared in pellet form. The feed compositions of comparative examples 1 and 2 and examples 1 and 2 were dried at 25 ℃ for about 24 hours using a dryer and stored at-20 ℃ until the subsequent experiments.

[ Table 2]

Experimental example 1 evaluation of the preventive Effect of the feed composition against AHPND

1-1 evaluation of shrimp preparation and growth Rate

A total of 40 tanks were prepared, each of which was thirty white leg shrimps. All experimental water tanks were equipped with air stones (air stores) to maintain dissolved oxygen and the water tank temperature was maintained in the range of 28 ℃ to 32 ℃ throughout the experiment. The feed (4% to 12% of the fish weight) is fed 4 times a day with limited feeding.

Shrimp weight was measured every 2 weeks. The evaluation items and the calculation equation related to growth rate and feed efficacy are as follows:

weight gain (%)

100 × (final average body weight-initial average body weight)/average body weight

Feed conversion (%). amount of weight gain/amount of feed intake

Specific daily growth rate (% day)^-1)＝

100×(log_eFinal body weight log_eInitial body weight)/days

To distribute the experimental feed, a fully randomized design was performed and the results of growth and analysis were statistically analyzed by one-way ANOVA using the SPSS version 18.0 program. Significant differences in data values were compared between Duncan's multiple range tests (P < 0.05). Data are expressed as mean ± SD, while percentage data are calculated as arcsine transformed values and statistically analyzed.

[ Table 3]

	Control group 1	Control group 2	BS group 1
				IBW1(g)	0.51±0.01	0.51±0.01	0.51±0.01
FBW2(g)	3.49±0.12bc	3.80±0.22ab	3.86±0.18a
				WG3(％)	592±18.9c	655±39.3ab	667±31.9a
SGR4(％)	6.04±0.09b	6.31±0.16a	6.37±0.13a
				FCR5	1.30±0.26	1.28±0.09	1.19±0.12
Survival rate (%)	80.0±3.33	75.6±6.94	71.1±6.94

¹IBW: initial body weight

²FBW: final body weight

³WG: weight gain (%) - (final weight-initial weight)/initial weight]×100

⁴SGR: specific growth rate per day (% day)^-1)＝[(log_eFinal body weight log_eInitial body weight)/days]× 100

⁵FCR: feeding foodFeed conversion ratio dry feed/wet weight gain

As shown in table 3, as a result of the feeding test, it was confirmed that BS group 1 provided with the feed composition of example 1 containing bacillus subtilis 1(BS) selected in preparation example 1 exhibited a significantly higher growth rate than control group 1 and control group 2 provided with the feed compositions of comparative example 1 and comparative example 2, respectively. In addition, it was confirmed that the daily specific growth rate of the group provided with the feed composition of example 1 was significantly higher than that of the group provided with the feed compositions of comparative example 1 and comparative example 2, respectively.

1-2. test of infestation of Vibrio parahaemolyticus prawn 1

The challenge of vibrio parahaemolyticus prawns was tested in a total of two separate tests. In the case of the vibrio strain, ahpnd (ems) -causing strain isolated in vietnam in 2013 was used for the test. The invasion test was performed as follows: the shrimp were fed the feed composition of the example for two weeks, and then the same weight (average weight: 2.32g) of shrimp was distributed into 4 replicates, with 96 shrimp per group. By TSB⁺Culture medium, at 30 ℃, 150rpm bacteria 24 hours, and will be concentration of 3.1x 10⁵Each tank was impregnated with a suspension of CFU/mL of Vibrio parahaemolyticus. After immersion, the survival rate and swimming state of the shrimp were confirmed every hour, and after 8 hours, 95% of water was replaced. Three times a day (at 8:30, 13:30 and 18: 30), test feed (10 to 12% of fish body weight) was given in divided doses in a limited manner, and the degree of death was observed for 70 hours. The results are shown in table 4 below.

[ Table 4]

As shown in table 4 above, BS group 1 provided with the feed composition containing bacillus subtilis 1(BS) selected in preparation example 1 exhibited higher survival rates in the challenge test of vibrio parahaemolyticus prawns, compared to control group 1 and control group 2.

In addition, as shown in fig. 1, it was generally observed in the above two tests that after the immersion of vibrio parahaemolyticus, the shrimp moved slowly, and they sat on the bottom of the water tank without swimming movements, and their feed intake was also not active. After 8 hours of immersion, rapid death of the shrimp began to occur and the survival rate of BS group 1 was at least 17% higher compared to control group 1 or control group 2.

1-3. test of infestation of Vibrio parahaemolyticus prawn 2

The invasion test of the vibrio parahaemolyticus prawn is carried out once. In the case of the vibrio strain, the test was performed using an ahpnd (ems) -induced strain isolated from vietnam in 2013. The invasion test was performed as follows: the shrimp was fed with the feed composition of the example for 4 weeks, and then the same weight (average weight: 2.30g) of shrimp was distributed into 4 replicates, with 96 shrimp per group. Using TSB⁺The bacteria were cultured in medium at 30 ℃ for 24 hours at 150rpm and 6.3X 10⁵Each tank was impregnated with a suspension of Vibrio parahaemolyticus at a concentration of CFU/mL. After immersion, the survival rate of the shrimp and its swimming state were confirmed every hour, and after 8 hours, 95% of water was replaced. Three times a day (at 8:30, 13:30 and 18: 30), test feed (10% to 12% of fish body weight) was given in divided doses in a limited manner, and the degree of death was observed for 70 hours. The results are shown in Table 5 below.

[ Table 5]

As shown in table 5, in the challenge test of vibrio parahaemolyticus prawn, the BS groups 1 and 2 provided with the feed compositions of examples 1 and 2 including the bacillus subtilis selected in preparation example 1 showed higher survival rates than the control group 1 provided with the feed composition of comparative example 1.

1-4. methods of collecting samples and methods of histopathological analysis

At various time points of the beginning (before infection), the middle time (infection) and completion of the challenge test for vibrio parahaemolyticus prawns, two prawns were randomly selected per group and their hepatopancreas were extracted. One portion of the extracted hepatopancreas was stored in ethanol (100%) for quantitative real-time pcr (qpcr) analysis, while the other portion was fixed in Davidson fixative (Davidson's) for 24 hours and then stored in ethanol (70%) for histopathological analysis.

More specifically, the histopathological analysis was performed by the following method.

To minimize damage to the extracted hepatopancreas tissue, Davidson's fixative was injected into the shrimp hepatopancreas using a 1mL syringe immediately after sampling each water tank and the hepatopancreas were extracted. Each extracted hepatopancreas was then fixed in 1.5mL Eppendorf tubes containing Davidson's fixative for 24 hours, stored in ethanol (70%), and used for analysis. After fixation was completed, organs were trimmed to a thickness of about 2mm to about 3mm in size to be suitable for preparing tissue samples and inserting them into cassettes (cases), and their tissues were processed for 13 hours. These tissues were thinly cut to a thickness of about 4 μm, and the resulting specimens were collected with a brush, attached to each slide glass without any wrinkles, left in the air for about 5 minutes, and then subjected to H & E staining. After staining was completed, the slides were photographed at 200 magnification using a phase contrast microscope (BX50, Olympus) using a specialized program for microscopy (TCapture, Tucen Photonics). qPCR analysis was then performed on the amount of AHPND toxin present in the sampled hepatopancreas.

The results are shown in FIG. 2, where lower Ct values indicate higher amounts of toxin.

In the hepatopancreas sampled at the time point before the invasion test (i.e., 0 hour), no AHPND toxin was detected in all groups, whereas in the hepatopancreas sampled at the time point of 10 hours of the invasion test (i.e., 10 hours; when the number of dead subjects was the highest), AHPND toxin was detected in all samples. However, BS group 1 provided with the feed composition comprising bacillus subtilis according to the present disclosure showed significantly higher Ct values compared to control group 1 and control group 2, and the lowest level of AHPND toxin was detected in BS group 1 in the hepatopancreas sampled at the 24 hour time point of the invasion test. In addition, no AHPND toxin was detected in BS group 2 and control group 2 at the termination time point of the challenge test (i.e., 193 hours).

In addition, the results of histopathological analysis of the hepatopancreas are shown in fig. 3.

In the hepatopancreas sampled at the time point before the invasion test (i.e., 0 hour), abnormal tissues were observed in all groups. In the hepatopancreas sampled at the 10-hour time point, the damage level in the control group 1 was shown to be the most severe, and the progress of tissue necrosis was observed in the BS group 1 and the control group 2. In the hepatopancreas sampled at the 24 hour time point, inflammation was observed, rather than tissue necrosis caused by AHPND toxin. In the hepatopancreas sampled at the termination time point of the invasion test (i.e., 193 hours), sampling was impossible due to 100% death occurring at the time point of 37 hours in the control group 1, while some inflammatory cells were observed in the BS group and the control group 2.

From the above results, it was confirmed that the feed composition containing bacillus subtilis according to the present disclosure can not only improve the disease resistance of shrimp against vibrio parahaemolyticus infection, but also significantly reduce the amount of AHPND toxin in the hepatopancreas of shrimp.

Experimental example 2 evaluation of growth rate and Immunity according to the concentration of Bacillus subtilis

Considering the results in preparation example 2, feed compositions (examples 1 to 3) containing bacillus subtilis (KCCM11143P, hereinafter, "BS") selected in preparation example 1 at various concentrations were prepared. The results are shown in table 6 below.

[ Table 6]

The compositions of the groups in table 6 were prepared by adding fish oil and water and mixing them, and were prepared in pellet form. The compositions of each set in table 6 were dried at 25 ℃ for about 24 hours using a desiccator and stored at-20 ℃ until subsequent experiments.

2-1 evaluation of shrimp preparation and growth Rate

30 white leg shrimps were prepared in each of the total 28 water tanks. All experimental water tanks were equipped with air stones to maintain dissolved oxygen and the temperature of the water tanks was maintained in the range of 28 ℃ to 32 ℃ throughout the experiment. The feed was fed 4 times a day for 8 weeks with limited supply (6% to 12% of the weight of the fish, initial body weight: 0.14 g).

Shrimp weight was measured every 2 weeks. The evaluation items and calculation equations relating to growth rate and feed efficiency were as follows:

weight gain (%)

100 × (final average body weight-initial average body weight)/average body weight

Feed conversion (%). amount of weight gain/amount of feed intake

Specific daily growth rate (% day)^-1)＝

100×(log_eFinal body weight log_eInitial body weight)/days

The results are shown in table 7 below.

[ Table 7]

	Control group 1	Control group 2	BS group 1	BS group 3
					IBW1(g)	0.14±0.00	0.14±0.00	0.14±0.00	0.14±0.00
FBW2(g)	10.2±0.69b	11.3±0.45a	11.9±0.43a	11.5±0.60a
					WG3(％)	7029±506b	7969±413a	8381±356a	8085±464a
SGR4(％)	7.62±0.13b	7.84±0.09a	7.93±0.07a	7.86±0.10a
					FCR5	1.53±0.04c	1.21±0.09ab	1.11±0.22a	1.13±0.12a
Survival rate (%)	90.7±6.11	92.0±4.00	84.0±17.4	88.0±8.00

*(P<0.05)

¹IBW: initial body weight

²FBW: final body weight

³WG: weight gain (%) - (final weight-initial weight)/initial weight]×100

⁴SGR: specific growth rate per day (% day)^-1)＝[(log_eFinal body weight log_eInitial body weight)/days]×100

⁵FCR: dry feed/wet weight gain on feed conversion

As shown in table 7 and fig. 4, all groups of shrimps fed for 8 weeks showed growth rates of more than 7,000% compared to those before feeding. Specifically, the final average body weight of BS group 1 and the final average body weight of BS group 3 were 15.7% and 16.7% greater than those of control group 1, respectively. In addition, regarding the specific growth rate per day, the values of BS group 1 and BS group 3 showed increases of 3.4% and 4.1%, respectively, compared to the value of control group 1. The feed efficiency of BS group 1 and BS group 3 was improved by 27.5% and 26.1%, respectively, compared to the feed efficiency of control group 1. However, there was no significant difference in the survival rate of shrimp in all groups.

2-2. sample Collection

Test shrimps were weighed every 2 weeks and all test shrimps were fasted 18 hours before the measurement to reduce stress (stress) of the shrimps.

After measuring the final weight, 7 shrimps were randomly selected from each tank and anesthetized in ice water, and hemolymph was collected using a syringe treated with Alsever's solution. Collected hemolymph was analyzed for Nitro Blue Tetrazolium (NBT) activity, where a sample of serum was separated using a centrifuge (800g, 10 min, 4 ℃) for non-specific immunity.

Regarding the collection of shrimp feces for digestibility analysis, the remaining feed and impurities in the water tank were cleaned by siphoning and water exchange after feeding for 30 minutes, and the excreted feces were collected using a siphon tube 3 hours thereafter. The collected samples were washed with distilled water, filtered through filter paper, and stored in a low temperature freezer at-40 ℃ until used as analytical samples.

2-3 statistical analysis

For the distribution of experimental feeds, a fully randomized design was performed and growth and results analyzed by one-way ANOVA statistics using the SPSS version 18.0 program. Significant differences in data values were compared between duncan multiple range assays (P < 0.05). Data are expressed as mean ± SD, while percentage data are calculated as arcsine transformed values and statistically analyzed.

2-4 analysis of conventional Components

The conventional ingredients of the test feed and feces were analyzed according to the AOAC (2005) method; the moisture content was analyzed by means of an atmospheric pressure heat drying method (125 ℃, 3h) (Kejtec System 2300, Sweden); the coarse ash was analyzed by direct incineration (550 ℃,4 h); crude protein was analyzed by an automated crude protein analyzer (kejtec system 2300, sweden); and crude lipids were analyzed by the method of Folch et al (1957). The results are shown in table 8 below.

[ Table 8]

	Control group 1	Control group 2	BS group 1	BS group 3
					Dried substance	24.9±0.35	24.5±0.10	23.7±0.39	23.7±0.35
Coarse ash content	12.9±2.34	12.8±0.12	12.3±0.33	14.5±0.22
					Crude protein	76.5±3.46b	82.0±1.67a	84.3±2.85a	83.9±2.31a
Crude lipid	5.37±0.96	5.45±1.15	5.09±0.28	5.26±1.05

(*P<0.05)

As shown in table 8, there was no significant difference between groups with respect to the content of crude ash and crude lipid. However, the content of crude protein in BS group 2 was significantly higher compared to the content of crude protein in control group 1 and control group 3. That is, it was confirmed that when the feed composition according to the present disclosure was provided to shrimp, the protein content of shrimp could be increased.

2-5 analysis relating to non-specific Immunity

2-5-1 analysis of Azobenzothiazole (NBT) Activity

The amount of oxidative radicals produced by neutrophils during respiratory explosion (respiratory explosion) was determined using the analytical method of Zhang et al (2013).

Specifically, hemolymph (50. mu.L) was first mixed with 200. mu.L of Hank's Balanced Salt Solution (HBSS) and allowed to react at 25 ℃. After 30 minutes, 100. mu.L of zymosan (0.1% Hank's solution) was added thereto and reacted at 37 ℃ for 2 hours. NBT solution (0.3%) was added thereto in an amount of 100. mu.L each time and reacted at 37 ℃ for 2 hours. 100% methanol (600. mu.L) was added thereto and the mixture was centrifuged at 6,500rpm for 10 minutes. The supernatant was discarded, and the pellet (pellet) was washed 3 times with 70% methanol (100 μ L) and dried for 5 minutes. Then, 2M KOH (700. mu.L) and DMSO (800. mu.L) were added thereto, and the absorbance of the resultant was measured at 620 nm.

2-5-2 assay of glutathione peroxidase (GPx) Activity

GPx activity in serum was analyzed using GPx kit (Biovision, inc. california).

Specifically, as cumene hydroperoxide, a reaction mixture in which a peroxide substrate (ROOH), glutathione reductase (GSSG-R) and reduced b-Nicotinamide Adenine Dinucleotide Phosphate (NADPH) are mixed is used. First, a hemolymph sample (50. mu.L) was added to a small plate (microplate), and a reaction mixture (40. mu.L) was added thereto and reacted. Then, cumene hydroperoxide (10. mu.L) was added thereto, and after 5 minutes, the absorbance of the resultant was measured at 340 nm. GPx activity was calculated as nmol/min/mL.

2-5-3 analysis of lysozyme Activity

Lysozyme activity assays were analyzed based on the method of Swain et al (2007).

Specifically, lysozyme is an antibacterial enzyme involved in nonspecific (innate) immune responses, and is an enzyme that exhibits antibacterial activity against various bacteria in a nonspecific manner rather than in a specific manner for specific bacteria.

The antibacterial mechanism against pathogenic bacteria is an antibacterial action that hydrolyzes the beta-1, 4-glycosidic bond of peptidoglycan, which is an integral part of the bacterial cell wall, thus destroying the bacterial cell wall. Lysozyme is particularly effective against gram-positive bacteria. Based on this mechanism, lysozyme activity is widely used in assays to measure non-specific immune responses in shrimp (including fish). When immunostimulants (e.g., ascorbic acid, β -glucan, probiotics, etc.) are added to shrimp feed, it can be confirmed that lysozyme activity is increased in hemolymph and tissues of shrimp, and these results are interpreted as increasing nonspecific immune response or enhancing immunity of fish (shrimp).

2-5-4 analysis of Phenol Oxidase (PO) Activity

Phenol Oxidase (PO) activity was analyzed based on the method of Hernandez-Lopez et al (1996).

Specifically, phenol oxidizing enzymes are enzymes having an important role in the defense mechanism of crustaceans, which exist in the form of phenol oxidizing enzymes in blood cells and are activated by a prophenol oxidizing enzyme activation system. Activated phenol oxidizing enzymes produce opsonins, which promote phagocytosis and coating of blood cells with foreign antigens and participate in the blood coagulation reaction. Therefore, the phenoloxidase activity in hemolymph is used as an important index of the innate immunity of shrimp.

2-5-5 analysis of superoxide dismutase (SOD) Activity

SOD activity was analyzed using SOD assay kit (Sigma-Aldrich, 19160, St. Louis, USA).

Specifically, a free radical detector (20 μ L) was added to the 96-well plate, and each blood sample (20 μ L) was added to each well. Then, xanthine oxidase (20. mu.L) was added thereto and reacted for 20 minutes. The absorbance of the resultant was measured at 450nm using a Microplate Reader (Thermo).

2-5-6 analysis of the Activity of the antiprotease

The antiprotease activity in hemolymph was analyzed using the assay of Ellis (1990).

Specifically, hemolymph (20. mu.L) and a standard trypsin solution (20. mu.L; type II-S from porcine pancreas, Sigma-Aldrich, A2765, St. Louis, USA) were mixed and incubated at 22 ℃ for 10 minutes. Phosphate buffer (200. mu.L; 0.1M, pH7.0) and azocasein (2%) (250. mu.L; Sigma-Aldrich) were added thereto, incubated at 22 ℃ for 1 hour, and trichloroacetic acid (500. mu.L; 10%) (TCA) was again added thereto and incubated at 22 ℃ for 30 minutes. The culture solution was centrifuged (6000g, 5 minutes), and the resultant (100. mu.L) was inoculated into a 96-well plate, and 1N NaOH (100. mu.L) was added thereto, and the absorbance of the resultant was measured at 430nm using a Microplate Reader.

The results of Experimental examples 2-5-1 to 2-5-6 in which non-specific immunological analysis was performed are shown in Table 9 below.

[ Table 9]

	Control group 1	Control group 2	BS group 1	BS group 3
					NBT1	1.65±0.15c	1.72±0.08bc	1.85±0.03ab	1.79±0.13ab
PO2	0.213±0.008b	0.249±0.031a	0.254±0.015a	0.249±0.009a
					Anti-protease3	31.6±2.4b	35.8±0.3a	36.6±1.8a	36.1±3.4a
Lysozyme4	6.40±1.04b	8.57±0.50a	8.43±0.98a	8.08±1.26ab
					SOD5	67.3±4.6b	73.1±3.3ab	74.8±4.6ab	72.8±7.7ab
GPx6	28.5±1.9d	32.7±2.4c	36.4±1.7abc	36.7±2.3a

(*P<0.05)

¹Nitro blue tetrazolium; phagocytic Activity (Absorbance)

²Phenol oxidase activity (absorbance)

³Anti-protease (% inhibition)

⁴Lysozyme activity (μ g mL)^-1)

⁵Superoxide dismutase (% inhibition)

⁶Glutathione peroxidase (mU mL)^-1)

As shown in table 9 and fig. 5, both BS group 1 and BS group 3 showed significantly higher values compared to control group 1 and control group 2 with respect to NBT activity and PO activity; and specifically, in the case of PO activity, the value of BS group 1 showed 26.8% higher than that of control group 1. With respect to the anti-protease activity, both BS group 1 and BS group 3 showed significantly higher values compared to control group 1 and control group 2; and specifically, the value of BS group 1 showed 15.8% higher than that of control group 1. Both BS group 1 and BS group 3 showed significantly higher activity with respect to lysozyme activity and SOD activity compared to control group 1. With respect to GPx activity, both BS group 1 and BS group 3 values showed significantly higher compared to control group 1 and control group 2; and specifically, the values of BS group 1 and BS group 3 showed 27.7% and 28.8% higher, respectively, compared to control group 1.

2-6. analysis of Water quality and zero Water exchange experiment

Culture water samples were collected from each water tank every 5 days during 8 weeks of the feeding experiment. Samples were collected from the same location in each tank and Dissolved Oxygen (DO), salinity, pH and ammonia (NH) were measured₄ ⁺) The level of concentration. DO was measured by Thermo Scientific origin Star A216 Benchtop Meter (Thermoscientific) and salinity was measured by MasterRefractometer (ATAGO). The pH is measured by Seven Compact (METTLER TOLEDO) and the NH is analyzed by the method according to Verdouw et al (1978)₄ ⁺The concentration of (c).

Twelve white leg shrimps (penaeus vannamei) with an average weight of 2.87 (+ -0.08) g were randomly placed into each 96L tank of a total of 14 tanks using a zero-change water method. Each test group had two replicates and four times a day, the shrimps were given test feed in divided doses (at 08:30, 12:00, 15:30 and 19: 00) of 10% of their body weight. According to Verdouw et al (1978), water samples were collected once a day and the total ammonia concentration in the water samples was measured for 10 days. The results are shown in table 10.

[ Table 10]

	DO mgL-1	pH	NH4 +mgL-1
				Mean value of	6.85	7.10	0.102
Maximum value	7.21	6.78	0.154
				Minimum value	6.39	6.48	0.035

As shown in table 10 and fig. 6, interestingly, it was found that all the test groups started to show a lower total ammonia concentration than the control group from day 7, and all the test groups showed a significantly lower ammonia concentration than the control group at day 10.

2-7 analysis of digestibility

2-7-1. indicator (Cr)₂O₃) Analysis of (2)

For the analytical testing of the chromium oxide content in the feed and the powder, the method according to Divakaran et al (2002) was used.

Specifically, the test feed and the powder sample were subjected to ashing in an ashing furnace (550 ℃) for 4 hours, and the obtained sample was used for analysis. First, to oxidize chromium oxide to mono-chromic acidSalt form, 5mg to 10mg of powder sample are weighed and transferred into a glass test tube. 4mL perchloric acid reagent (HClO)₄) Add to a glass tube containing the sample. A perchloric acid reagent (70%) is prepared by mixing 200mL of nitric acid in 100mL of distilled water, cooling the mixture, and then mixing 200mL of 70% perchloric acid thereto. The glass tube containing the sample and perchloric acid reagent was placed in a hot plate, heated at 300 ℃ for 15 minutes, and then cooled to room temperature. The pretreated specimens were transferred to a 50mL glass flask and quantified to 25mL with triple distilled water. Thereafter, the absorbance was measured at 350nm using a spectrophotometer (Beckman DU-730). The measured absorbance was used to calculate the chromium oxide content of the sample using a standard equation prepared from a pretreated standard solution as in sample analysis.

2-7-2 analysis of Dry matter and protein digestibility

Protein digestibility of dry matter and test feed was calculated by the following method:

ADC (%) of dry matter 100-₂O₃Cr% in feces₂O₃)；

ADC (%) for protein 100-₂O₃Cr% in feces₂O₃) x (% protein in feces/% protein in diet)

The results of the digestibility analysis performed at the end of the feeding experiment are shown in table 11.

[ Table 11]

Values are the mean of triplicate and expressed as mean ± s.d. The values in the same column with different superscripts are significantly different (P < 0.05).

¹Apparent digestibility coefficient of dry matter

²Apparent digestibility coefficient of protein

As shown in table 11, all test groups showed significantly higher dry matter digestibility and protein digestibility than the control group.

Experimental example 3 evaluation of the preventive Effect of the feed composition on WSSV

In order to confirm the antiviral activity of the feed composition comprising bacillus subtilis, an invasion test was performed such that shrimps were invaded by white spot syndrome virus. In the case of white spot syndrome virus, virus isolated from white leg shrimp (penaeus vannamei) infected with WSSV was obtained from domestic farms (domestic farms) in 2017 and used for testing. The invasion test was performed as follows: the feed compositions of the examples were administered to the shrimps for 6 weeks, and then the shrimps having the same weight (average weight: 6.25g) were placed in 4 replicates of 96 shrimps per group. Inoculation concentration of virus was 4.1X 10⁵One copy/. mu.L, and 100. mu.L of virus was used to inoculate each shrimp intramuscularly using a syringe. Final inoculum concentration per shrimp was 4.1x 10⁷Copies/. mu.L.

After inoculation, mortality and motility of the shrimp was confirmed. Three times a day (at 8:30, 13:30 and 18: 30), test feed (10% to 12% of fish body weight) was given in divided doses in a limited manner, and the degree of mortality was observed for 125 hours. The results are shown in table 12 below.

[ Table 12]

As shown in table 12, in the invasion test for white spot syndrome virus of shrimp, BS group 1 provided with the feed composition including bacillus subtilis of example 2 showed higher survival rate than control group 1 (the feed composition of comparative example 1 was administered to control group 1).

Experimental example 4 evaluation of the preventive Effect of the feed composition on Simultaneous infection with WSSV and AHPND

In order to confirm the antiviral activity of the feed composition comprising bacillus subtilis, an invasion test was performed such that shrimp were invaded by vibrio parahaemolyticus and white spot syndrome virus. In the case of the vibrio strain, the test was performed using an ahpnd (ems) -induced strain isolated from vietnam in 2013. In the case of white spot syndrome virus, from infection with WSSIsolated virus from V white leg shrimp (penaeus vannamei) was obtained from domestic farms in 2017 and used for testing. The invasion test was performed as follows: the feed compositions of the examples were administered to the shrimps for 6 weeks, and then the shrimps having the same weight (average weight: 4.52g) were placed in 4 replicates of 96 shrimps per group. Inoculation concentration of virus 8.3X 10³One copy/. mu.L and intramuscularly inoculated with 50. mu.L of virus using a syringe. Final inoculum concentration per shrimp was 4.1x 10⁴Copies/. mu.L. Two days after virus inoculation, the shrimp were infected with a vibrio strain.

Using TSB⁺The bacteria were cultured in medium at 30 ℃ for 24 hours at 150rpm and 1.3X 10⁵Each tank was impregnated with a suspension of Vibrio parahaemolyticus at a concentration of CFU/mL. After the immersion, the mortality and swimming state of the shrimp were confirmed every hour. Three times a day (at 8:30, 13:30 and 18: 30), test feed (10% to 12% of fish body weight) was given in divided doses in a limited manner, and the degree of mortality was observed for 7 days. The results are shown in table 13 below.

[ Table 13]

As shown in table 13, in the invasion test of vibrio parahaemolyticus and white spot syndrome virus for shrimp, BS group 1 provided with the feed composition including bacillus subtilis of example 1 showed a higher survival rate than control group 1 (the feed composition of comparative example 1 was administered to control group 1).

Based on the above examples, the feed composition including bacillus subtilis according to the present disclosure may improve the growth, feed efficiency, digestibility, quality of culture water, and nonspecific immunity of white leg shrimps. Further, it is expected that the present disclosure can produce high-protein white leg shrimps, so that marketability of the shrimps can be improved.

Accession number

The preservation organization: korean Collection of microorganisms (International Depositary Authority)

The preservation number is as follows: KCCM11143P

The preservation date is as follows: 20101214

The preservation number is as follows: KCCM11144P

The preservation date is as follows: 20101214

The preservation number is as follows: KCCM11270P

The preservation date is as follows: 20120322