US20200315211A1 - A feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or white spot syndrome (WSS), comprising a Bacillus subtilis strain as an active ingredient - Google Patents

A feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or white spot syndrome (WSS), comprising a Bacillus subtilis strain as an active ingredient Download PDF

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US20200315211A1
US20200315211A1 US16/461,348 US201816461348A US2020315211A1 US 20200315211 A1 US20200315211 A1 US 20200315211A1 US 201816461348 A US201816461348 A US 201816461348A US 2020315211 A1 US2020315211 A1 US 2020315211A1
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feed
bacillus subtilis
feed composition
shrimp
ahpnd
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Ji Eun Kim
Jee Eun HAN
Sung Hun Kim
Seo Hyung WOO
Jongsu EUN
Hayun JO
Jae Won Kim
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CJ CheilJedang Corp
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CJ CheilJedang Corp
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Priority claimed from PCT/KR2018/016893 external-priority patent/WO2019132605A1/ko
Assigned to CJ CHEILJEDANG CORPORATION reassignment CJ CHEILJEDANG CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HAN, Jee Eun, EUN, Jongsu, JO, Hayun, KIM, JAE WON, KIM, JI EUN, KIM, SUNG HUN, WOO, Seo Hyung
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes

Definitions

  • 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.
  • AHPND acute hepatopancreatic necrosis disease
  • WSS white spot syndrome
  • EMS Early mortality syndrome
  • AHPND acute hepatopancreatic necrosis disease
  • AHPNS acute hepatopancreatic necrosis syndrome
  • EMS Early mortality syndrome
  • AHPND acute hepatopancreatic necrosis disease
  • AHPNS acute hepatopancreatic necrosis syndrome
  • Insect toxins are produced by the expression of specific genes present in a specific plasmid of the bacteria (i.e., photorhabdus insect-related toxins; Pir toxin), and are easily moved around, that is, they have motility.
  • WSSV white spot syndrome virus
  • TSV Taura syndrome virus
  • IMNV infectious myonecrosis virus
  • AHPND began in China in 2009, and spread rapidly in Asian counties (e.g., Thailand, Malaysia, and Vietnam) within one year, and further occurred in Mexico to spread to other Central American countries, resulting in damage to most of the shrimp markets. South Korea also suffered great damage due to AHPND from 2015 to 2016, and studies are currently underway to prevent or manage the disease.
  • probiotics are defined as microbial preparations or components that assist the balance of microorganisms in the intestine, and have an etymological meaning that is opposite to antibiotics, which refers to an antibiotic material.
  • examples of the probiotics include lactic acid bacteria such as Lactobacillus and Bifidobacterium .
  • probiotics do not possess toxic genes against humans and animals, nor do they produce pathogenic substances, and thus are classified as GRAS (generally recognized as safe). Therefore, development of a feed additive using probiotics, the safety of which is demonstrated, has been actively accomplished.
  • Korean Patent Publication No. 10-2011-035554 discloses a mixed strain of novel CMB L1 of the genus Bacillus and CMB201 of the genus Lactobacillus , a food composition for anticancer and immunity enhancement using the same, and a microbial preparation having antibacterial activity.
  • Korean Patent No. 10-0977407 discloses an immune booster and feed additive for animals, containing lysates of Zygosaccharomyces bailii , which increase the various activities of neutrophils, the major phagocytic cells of animals, and enhances non-specific defense against attack inoculation by pathogenic bacteria.
  • the actual immunoactivity of the feed additive using probiotics is inadequate, and thus research on a feed additive using probiotics still having excellent immunoactivity is needed.
  • the present inventors have made intensive efforts to develop a shrimp feed supplemented with probiotics ( Bacillus sp.) for preventing shrimp AHPND. As a result, they have confirmed that when the feed composition supplemented with Bacillus subtilis is fed to a shrimp, the survival rate of a shrimp against AHPND infection (caused by a strain isolated from the affected area in Vietnam in 2013; Tran et. al. 2013.) or WSSV infection is improved, and that the growth rate and non-specific immunity of shrimp are not only increased but also the water quality is improved and the production of high-protein shrimp can be produced, thereby completing the present disclosure.
  • probiotics Bacillus sp.
  • 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.
  • AHPND acute hepatopancreatic necrosis disease
  • Another object of the present disclosure is to provide a method for preventing or treating acute hepatopancreatic necrosis disease, comprising administering the feed composition to a subject.
  • Still another object of the present disclosure is 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.
  • WSS white spot syndrome
  • Still another object of the present disclosure is to provide a method for preventing or treating white spot syndrome, comprising administering the feed composition to a subject.
  • composition of the present disclosure which comprises a Bacillus subtilis (KCCM11143P) strain as active ingredient, has antibacterial activity against Vibrio parahaemolyticus , which causes AHPND which is problematic in shrimp farming and antiviral activity against white spot syndrome virus, which causes WSS, and exhibits an effect of improving immunity of the shrimp hepatopancreas, and thus the composition of the present disclosure can be used as a shrimp feed composition or a feed additive.
  • KCCM11143P Bacillus subtilis
  • FIG. 1 is a graph showing the survival rate of shrimp infected with Vibrio parahaemolyticus.
  • FIG. 2 is a graph showing the analysis of the AHPND content in the shrimp hepatopancreas.
  • FIG. 3 is images showing the pathological features of the shrimp hepatopancreas.
  • FIG. 4 is graphs showing the growth rates of shrimps.
  • FIGS. 4( a ) to 4( d ) show the final body weight, the weight gain rate, the daily growth rate, and the feed conversion rate, respectively.
  • FIG. 5 is graphs analyzing the non-specific immunities of shrimps.
  • FIGS. 5( a ) to 5( e ) show the activities against macrophages, phenoloxidase, antiproteinase, lysozyme, and superoxide dismutase, respectively.
  • FIG. 6 is a graph showing the results of analysis of water quality in the breeding water with zero water exchange.
  • an aspect of the present disclosure provides 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.
  • AHPND acute hepatopancreatic necrosis disease
  • Bacillus subtilis is an aerobic bacterium that is not toxic and produces spores. Bacillus subtilis is widely distributed in nature such as in dry grass, soil, sewage, air, etc. This bacterium is widely used in industry because it produces enzymes to coagulate milk, saccharifies starch, and decomposes fat and oil. The optimal conditions for growth of the bacterium are pH of 7 to 8.5 and a temperature of 37° C. to 40° C. Due to the characteristics of a strain of the genus Bacillus , the strain does not possess toxic genes for humans and animals, is non-pathogenic, produces no pathogenic substances, and exhibits a rapid growth rate in vivo. Bacillus subtilis has an anaerobic habitat in the presence of glucose, etc., and endospores allow Bacillus to survive in extremely harsh environments such as high or low temperatures.
  • Bacillus subtilis may be a strain deposited with Accession No. KCCM11143P.
  • a feed composition comprising the Bacillus subtilis (KCCM11143P) was prepared.
  • the composition of the present disclosure may comprise the Bacillus subtilis (KCCM11143P), which has a bacterial count of 1 ⁇ 10 4 CFU to 1 ⁇ 10 11 CFU per gram of the total active ingredients.
  • the bacterial count may be 1 ⁇ 10 4 CFU/g to 1 ⁇ 10 10 CFU/g, and more specifically, the composition of the present disclosure comprises the Bacillus subtilis (KCCM11143P), which has the bacterial count of 1 ⁇ 10 8 to 1 ⁇ 10 10 CFU/g.
  • Bacillus subtilis contained in the feed composition as an active ingredient may comprise only Bacillus subtilis (KCCM11143P).
  • AHPND acute hepatopancreatic necrosis disease
  • EMS early mortality syndrome
  • AHPNS acute hepatopancreatic necrosis syndrome
  • AHPND is caused by Vibrio parahaemolyticus , a pathogenic bacterium present in seawater, which has a large number of infections in whiteleg shrimp accounting for about 96% of Korean farmed shrimps. Because of a high mortality rate in the early stages of life, it causes harm to shrimps, but it is harmless to humans.
  • Vibrio parahaemolyticus is a gram-negative bacillus 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 mass mortality in the shrimp farming industry.
  • AHPND acute hepatopancreatic necrosis disease
  • composition of the present disclosure may further comprise Bacillus pumilus, Bacillus licheniformis , or a combination thereof as an active ingredient.
  • Bacillus pumilus may be a strain deposited with Accession No. KCCM11144P
  • Bacillus licheniformis may be a strain deposited with Accession No. KCCM11270P.
  • prevention refers to any behavior resulting in suppression or delay of symptoms of shrimp AHPND by administering the composition according to the present disclosure, which comprises Bacillus subtilis.
  • treatment refers to any action resulting in alleviation of or full recovery from symptoms of shrimp AHPND by administering the composition according to the present disclosure, which comprises Bacillus subtilis (KCCM11143P).
  • the composition may include a known carrier or an additive which is acceptable for pharmaceutical, food, or feed use.
  • a probiotic preparation having antibacterial activity against Vibrio parahaemolyticus which comprises the Bacillus subtilis (KCCM11143P)
  • a feed mixture, etc. may be further comprised, but the present disclosure is not limited thereto.
  • the groups (BS Groups 1 and 2) in which the feed compositions (Examples 1 and 2) including the Bacillus subtilis (KCCM11143P) strain of the present disclosure is administered, could increase the disease resistance of shrimp against infection of Vibrio parahaemolyticus and significantly lower the toxin amount of AHPND in the shrimp hepatopancreas, compared to the groups (Control Groups 1 and 2), in which Comparative Example 1 (not including probiotics), and Comparative Example 2 (including commercially available probiotics (a mixed preparation of three Bacilli ( B. subtilis, B. pumilus, B. licheniformis ))) are administered.
  • the composition may be useful for increasing the non-specific immune response of shrimp or improving the immunity thereof.
  • the present disclosure provides a feed additive for shrimp farming, comprising the above-mentioned feed composition.
  • a known carrier or a stabilizer which is acceptable for pharmaceutical, food, or feed use may be added in addition to the above active ingredients.
  • all sorts of nutrients such as vitamin, amino acids, and minerals, antioxidants, and other additives may be added, whose shape may be convenient therefor, such as powder, granules, pellets, and suspensions.
  • the feed additive may be supplied alone or mixed with feed to non-ruminant animals.
  • the present disclosure provides a feed for shrimp farming, comprising the above-mentioned feed additive.
  • the Bacillus subtilis (KCCM11143P) strain of the present disclosure which is a gram-positive bacterium having a sporulation capacity, is preferably formulated in a spore form, but it is not limited thereto.
  • the feed of the present disclosure is not particularly limited, but any feed such as powder feed, solid feed, moist pellet feed, dry pellet feed, extruder pellet (EP) feed, and raw feed is available.
  • the Bacillus sp. forms endogenous spores, and thus is very stable against heat. Therefore, the Bacillus subtilis (KCCM11143P) of the present disclosure can be prepared separately as a feed additive form and then mixed with a feed, or can be prepared by directly adding to a feed when preparing the feed.
  • the Bacillus subtilis included in the feed of the present disclosure may be in a liquid or dry state, and preferably in the form of a dried 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 as 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.
  • the feed is used for aquaculture, and may further include, in addition to the Bacillus subtilis (KCCM11143P) of the present disclosure, conventional additives capable of increasing the preservability of the feed.
  • another aspect of the present disclosure provides a method for preventing or treating acute hepatopancreatic necrosis disease, comprising administering the feed composition of the present disclosure to a subject.
  • the term “subject” may refer to fish or crustaceans, the farming of which is possible, and which have or are at risk of developing acute hepatopancreatic necrosis disease, but the subject may refer to shrimp according to the objects of the present disclosure.
  • the feed is preferably supplied with the same amount and at the same feeding interval as conventional feeds.
  • the pathogenic bacterium refers to a bacterium that causes, in shrimp farming, mass mortality of shrimps by inducing AHPND, and specifically may refer to Vibrio parahaemolyticus.
  • causes of mass mortality in the shrimp farming include not only the Vibrio parahaemolyticus but also the infection by various viruses and the concentration of ammonia in breeding water.
  • ammonia in breeding water occurs as a metabolite of proteins such as shrimp feces, feed waste, etc., and it varies greatly with the increase of pH and water temperature.
  • Ammonia at a high concentration is a direct cause of acute death of shrimp, leading to mass mortality; and ammonia at a low concentration may lead to a reduction in the growth as well as feeding capacity and immunity of shrimp in the long term, which in turn can lead to the development of various diseases.
  • the breeding water of shrimp in which the feed composition of the present disclosure had been fed was collected and analyzed, and as a result, it was found that the total ammonia concentration in the breeding water was significantly lower than that of the control, and thereby the Bacillus subtilis (KCCM11143P) strain of the present disclosure could improve water quality of the shrimp-breeding water.
  • KCCM11143P Bacillus subtilis
  • the Bacillus subtilis (KCCM11143P) of the present disclosure can not only enhance the disease resistance of shrimp but can also inhibit the toxin of AHPND in the shrimp hepatopancreas. Therefore, by using the strain, the effect of preventing Vibrio parahaemolyticus that causes the disease can be obtained, and thereby shrimp can be farmed more safely.
  • 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.
  • WSS white spot syndrome
  • Bacillus subtilis Bacillus subtilis , prevention, and treatment are as described above.
  • the composition of the present disclosure may comprise the Bacillus subtilis (KCCM11143P), which has a bacterial count of 1 ⁇ 10 4 CFU to 1 ⁇ 10 11 CFU per gram of the total active ingredients.
  • the bacterial count may be 1 ⁇ 10 4 CFU/g to 1 ⁇ 10 10 CFU/g
  • the composition of the present disclosure comprises the Bacillus subtilis (KCCM11143P), which has the bacterial count of 1 ⁇ 10 8 CFU/g to 1 ⁇ 10 10 CFU/g.
  • WSSV white spot syndrome virus
  • the composition may include, in addition to the above strains contained as active ingredients, a known carrier or an additive which is acceptable for pharmaceutical, food, or feed use.
  • a probiotic preparation having antiviral activity against white spot syndrome virus which comprises the Bacillus subtilis (KCCM11143P)
  • a feed mixture, etc. may be further comprised, but the present disclosure is not limited thereto.
  • BS Group 1 in which the feed composition (Example 1) including the Bacillus subtilis (KCCM11143P) strain of the present disclosure is administered, could increase the disease resistance of shrimp against infection of white spot syndrome virus (WSSV), compared to the group (Control Group 1), in which Comparative Example 1 (not including probiotics) is administered.
  • BS Group 1 in which the feed composition (Example 1) including the Bacillus subtilis (KCCM11143P) strain of the present disclosure is administered, could enhance the disease resistance of shrimp against the complex infection of the WSSV and AHPND, compared to the group (Control Group 1), in which Comparative Example 1 (not including probiotics) is administered.
  • the present disclosure provides a feed additive for shrimp farming, comprising the above-mentioned feed composition.
  • a known carrier or a stabilizer which is acceptable for pharmaceutical, food, or feed use may be added in addition to the above active ingredients.
  • all sorts of nutrients such as vitamins, amino acids, and minerals, antioxidants, and other additives may be added, whose shape may be convenient therefor, such as powder, granules, pellets, and suspension.
  • the feed additive may be supplied alone or mixed with feed to non-ruminant animals.
  • the present disclosure provides a feed for shrimp farming, comprising the above-mentioned feed additive.
  • the Bacillus subtilis (KCCM11143P) strain of the present disclosure which is a gram-positive bacterium having a sporulation capacity, are preferably formulated in a spore form, but it is not limited thereto.
  • the feed of the present disclosure is not particularly limited, but any feed such as powder feed, solid feed, moist pellet feed, dry pellet feed, extruder pellet (EP) feed, and raw feed is available.
  • the Bacillus sp. forms endogenous spores, and thus is very stable against heat. Therefore, the Bacillus subtilis (KCCM11143P) of the present disclosure can be prepared separately as a feed additive form and then mixed with a feed, or can be prepared by directly adding to a feed when preparing the feed.
  • the Bacillus subtilis included in the feed of the present disclosure may be in a liquid or dry state, and preferably in the form of a dried 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 as 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.
  • the feed is used for aquaculture, and may further include, in addition to the Bacillus subtilis (KCCM11143P) of the present disclosure, conventional additives capable of increasing the preservability of the feed.
  • 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.
  • the terms of the present disclosure the white spot syndrome, prevention, treatment, and subject are as described above.
  • WSSV white spot syndrome virus
  • the Bacillus subtilis (KCCM11143P) of the present disclosure can obtain an effect of enhancing the resistance to white spot syndrome virus (WSSV), and thus shrimp can be farmed more safely.
  • WSSV white spot syndrome virus
  • a clear zone assay was performed. 0.5% agar (3 mL) and 100 ⁇ L of a shaking culture (2.0 ⁇ 10 9 CFU/mL) of pathogenic bacteria were mixed and seeded on a TSA+ medium to prepare top agar. Cultures of 12 kinds of Bacillus subtilis strains (those possessed by CJ CheilJedang and commercial strains), each in an amount of 10 ⁇ L, were dropped on top of the prepared top agar, cultured at 30° C. for 18 hours, and the presence/absence of clear zones was observed. The antibacterial activity of commercially available Bacillus subtilis and complex phage was evaluated together.
  • 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 (commercially + purchased, Company A, Korea) Complex phage ⁇ ++++: strong activity, +: presence of activity, ⁇ : no activity
  • the Bacillus subtilis 1 microorganism (CJBS-01) showed the most excellent antibacterial effect in vitro against Vibrio parahaemolyticus , which causes AHPND.
  • the microorganism showed antibacterial activity in vitro against a particular pathogen, the antibacterial activity observed is simply an in vitro effect and it does not necessarily mean that the ingestion of Vibrio parahaemolyticus by an animal will be able to provide the animal with immunity or a preventive effect against the particular pathogen.
  • the Bacillus subtilis 1 (CJBS-01) is a strain deposited to the Korean Culture Center of Microorganisms (KCCM) on Dec. 14, 2010, and was assigned Accession No. KCCM11143P.
  • a feed composition containing the Bacillus subtilis 1 (Accession No. KCCM11143P, hereinafter “BS”) selected in Preparation Example 1 was prepared.
  • compositions of Comparative Example 1 not containing Bacillus subtilis , Comparative Example 2 containing commercially-available Bacillus species i.e., a mixed preparation of three Bacillus species ( B. subtilis, B. pumilus , and B. licheniformis )
  • Example 2 containing the BS in an amount of 10 9 ⁇ 0.2 CFU/g were each mixed with fish oil and water, and prepared in the form of a pellet.
  • the feed compositions of Comparative Examples 1 and 2 and Examples 1 and 2 were dried at 25° C. for about 24 hours using a dryer and stored at ⁇ 20° C. until subsequent experiments.
  • the weight of the shrimp was measured every 2 weeks.
  • the evaluation items and the equations for calculation related to growth rate and feed efficiency are as follows:
  • Weight Gain (%) 100 ⁇ (final average body weight ⁇ initial average body weight)/average body weight
  • Feed Conversion Rate (%) amount of weight gain/amount of feed intake
  • the test of Vibrio parahaemolyticus attack on shrimp was performed over a total of two divided tests.
  • AHPND (EMS)-causing strains isolated in Vietnam in 2013 were used for the tests.
  • the attack test was carried out as follows: the feed compositions of Examples were fed to shrimp for two weeks, and then shrimp with the same weight (average weight: 2.32 g) were distributed into 4 replicates with 96 shrimps per group.
  • the bacteria were cultured at 30° C. with 150 rpm for 24 hours using the TSB + medium, and a suspension of Vibrio parahaemolyticus was immersed at a concentration 3.1 ⁇ 10 5 CFU/mL per tank.
  • the test feed was given three times a day (at 8:30, 13:30 and 18:30) in a divided dose in a restricted manner (10 to 12% of fish body weight), and the degree of mortality was observed for 70 hours.
  • the results are shown in Table 4 below.
  • the BS Group 1 which was provided with the feed composition containing the Bacillus subtilis 1 (BS) selected in Preparation Example 1, exhibited a higher survival rate in the attack test of Vibrio parahaemolyticus against shrimp, compared to Control Group 1 and Control Group 2.
  • the test of Vibrio parahaemolyticus attack on shrimps was carried out once.
  • AHPND (EMS)-induced strains isolated from Vietnam in 2013 were used for the test.
  • the attack test was carried out as follows: The feed compositions of the Examples were fed to shrimps for 4 weeks and then the shrimps with the same weight (average weight: 2.30 g) were distributed into 4 replicates with 96 shrimps per group.
  • the bacteria were cultured at 30° C. with 150 rpm for 24 hours using a TSB + medium, and a suspension of Vibrio parahaemolyticus was immersed at a concentration of 6.3 ⁇ 10 5 CFU/mL per tank.
  • the test feed was given three times a day (at 8:30, 13:30 and 18:30) in a divided dose in a restricted manner (10 to 12% of fish body weight), and the degree of mortality was observed for 70 hours.
  • the results are shown in Table 5 below.
  • the BS Groups 1 and 2 provided with the feed compositions of Examples 1 and 2 including Bacillus subtilis selected from Preparation Example 1 showed the survival rate higher than those of the Control Group 1 to which the feed composition of Comparative Example 1 is provided.
  • histopathological analysis was performed by the following method.
  • BS Group 1 which was provided with the feed composition containing the Bacillus subtilis according to the present disclosure, showed a significantly higher Ct value compared to Control Group 1 and Control Group 2, and in the hepatopancreas sampled at the 24 hour time-point of the attack test, the lowest level of AHPND toxin was detected in the BS Group 1. Additionally, at the termination time-point of the attack test (i.e., 193 h), the AHPND toxin was not detected in BS Group 2 and Control Group 2.
  • the feed composition containing the Bacillus subtilis according to the present disclosure can not only improve the disease resistance of shrimp to Vibrio parahaemolyticus infection, but can also significantly reduce the amount of AHPND toxin in the hepatopancreas of shrimp.
  • composition of each group in Table 6 was prepared by adding fish oil and water and mixing thereof, and prepared in the form of pellet.
  • the composition of each group in Table 6 was dried at 25° C. for about 24 hours using a dryer and stored at ⁇ 20° C. until subsequent experiments.
  • the weight of the shrimp was measured every 2 weeks.
  • the evaluation items and the equations for calculation related to growth rate and feed efficiency are as follows:
  • Weight Gain (%) 100 ⁇ (final average body weight ⁇ initial average body weight)/average body weight
  • Feed Conversion Rate (%) amount of weight gain/amount of feed intake
  • Test shrimp were weighed every 2 weeks and all of the test shrimp were fasted to reduce the stress of the shrimp 18 hours before the measurement.
  • the feed and impurities remaining in the water tanks were cleaned by siphoning and water exchange after 30 minutes of feeding, and 3 hours thereafter, the feces being excreted were collected using a siphon.
  • the collected samples were washed with distilled water, filtered through a filter paper, and stored in a ⁇ 40° C. low-temperature freezer until use as a sample for analysis.
  • test feed and the common ingredients of the feces were analyzed according to the AOAC (2005) method; the moisture contents by an atmospheric-pressure heating-drying method (125° C., 3 h) (Kejltec system 2300, Sweden); crude ash by direct incineration method (550° C., 4 h); crude proteins by an automated crude protein analyzer (Kejltec system 2300, Sweden); and crude lipids by the method of Folch et al. (1957).
  • Table 8 The results are shown in Table 8 below.
  • the amount of oxidative radical production by neutrophils during respiratory explosion was determined using the analysis method of Zhang et al. (2013).
  • hemolymph 50 ⁇ L was mixed with 200 ⁇ L of the Hank's balanced salt solution (HBSS) and allowed to react at 25° C. After 30 minutes, 100 ⁇ L of zymosan (0.1% Hank's solution) was added thereto and reacted at 37° C. for 2 hours. NBT solution (0.3%) was added thereto in an amount of 100 ⁇ L each time and reacted at 37° C. for 2 hours. 100% methanol (600 ⁇ L) was added thereto and the mixture was centrifuged at a rate of 6,500 rpm for 10 minutes. The supernatant was discarded and the pellet was washed 3 times with 70% methanol (100 ⁇ L) and dried for 5 minutes. Then, 2 M KOH (700 ⁇ L) and DMSO (800 ⁇ L) were added thereto, and the absorbance of the resultant was measured at 620 nm.
  • HBSS Hank's balanced salt solution
  • the GPx activity in serum was analyzed using the GPx kit (Biovision, Inc. California).
  • the cumene hydroperoxide a reaction mixture in which peroxide substrate (ROOH), glutathione reductase (GSSG-R), and reduced b-nicotinamide adenine denucleotide phosphate (NADPH) were mixed was used.
  • ROOH peroxide substrate
  • GSSG-R glutathione reductase
  • NADPH reduced b-nicotinamide adenine denucleotide phosphate
  • lysozyme which is an antibacterial enzymes involved in nonspecific (innate) immune responses, is an enzyme that exhibits antibacterial activity against various kinds of bacteria in a non-specific manner, rather than in a specific manner for a specific bacterium.
  • the antibacterial mechanism against pathogenic bacteria is the antibacterial action that hydrolyzes ⁇ -1,4-glucosidic bonds of peptidoglycan, which is a constituent of bacterial cell walls, thereby destroying bacterial cell walls.
  • Lysozyme is especially effective against gram-positive bacteria. Based on such a mechanism, lysozyme activity is widely used for analysis to measure non-specific immune responses in shrimp including fish.
  • immunostimulators e.g., ascorbic acid, ⁇ -glucan, probiotics, etc.
  • ascorbic acid e.g., ascorbic acid, ⁇ -glucan, probiotics, etc.
  • probiotics e.g., ascorbic acid, ⁇ -glucan, probiotics, etc.
  • the phenoloxidase (PO) activity was analyzed based on the method of Hernandez-Lopez et al. (1996).
  • phenoloxidase which is an enzyme that has an important role in the defense mechanism of the Crustacea, is present in the form of prophenoloxidase in blood cells and activated by the prophenoloxidase activating system.
  • the activated phenoloxidase produces opsonin, which promotes the phagocytosis of the blood cells and the coating action on the foreign antigen and participates in the blood clotting reaction. Accordingly, the phenoloxidase activity within the hemolymph is used as an important index of the innate immunity of shrimp.
  • the SOD activity was analyzed using the SOD assay kit (Sigma-Aldrich, 19160, St. Louis, USA).
  • a radical detector (20 ⁇ L) was added into a 96-well plate, and each blood sample (20 ⁇ L) was added to each well. Then, xanthine oxidase (20 ⁇ L) was added thereto and reacted for 20 minutes. The absorbance of the resultant was measured at 450 nm using the Microplate Reader (Thermo).
  • the antiprotease activity within the hemolymph was analyzed using the analysis method of Ellis (1990).
  • hemolymph (20 ⁇ L) and standard trypsin solution (20 ⁇ L; Type II-S, from porcine pancreas, Sigma-Aldrich, A2765, St. Louis, USA) were mixed and cultured at 22° C. for 10 minutes.
  • Phosphate buffer (200 ⁇ L; 0.1 M, pH 7.0) and azocasein (2%) (250 ⁇ L; Sigma-Aldrich) were added thereto, cultured at 22° C. for lhour, and trichloro acetic acid (500 ⁇ L; 10%) (TCA) was again added thereto, and cultured at 22° C. for 30 minutes.
  • the cultured solution was centrifuged (6000 g, 5 min), and the resultant (100 ⁇ L) was seeded into a 96-well plate, and 1 N NaOH (100 ⁇ L) was added thereto, and the absorbance of the resultant was measured at 430 nm using the Microplate Reader.
  • BS Group 1 and BS Group 3 showed significantly higher values, compared to Control Group 1 and Control Group 2; and in particular, BS Group 1 and BS Group 3 showed higher values compared to Control Group 1 by 27.7% and 28.8%, respectively.
  • the samples of culture water were collected from each water tank once every 5 days.
  • the samples were collected from the same location in each tank, and the level of dissolved oxygen (DO), salinity, pH, and the concentration of ammonia (NH4 + ) were measured.
  • DO was measured by a Thermo Scientific Orion Star A216 Benchtop Meter (Thermo Scientific)
  • the salinity was measured by a Master Refractometer (ATAGO).
  • ATAGO Master Refractometer
  • the pH was measured by a Seven Compact (METTLER TOLEDO), and the concentration of NH4 + was analyzed by the method according to Verdouw et al. (1978).
  • the test feed and the powder samples were subjected to ashing for 4 hours in an ashing furnace (550° C.), and the obtained samples were used for the analysis.
  • ashing furnace 550° C.
  • 5 mg to 10 mg of the powder samples 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.
  • the perchloric reagent (70%) was prepared by mixing 200 mL of nitric acid in 100 mL of distilled water, cooling the mixture and then mixing 200 mL of 70% perchloric acid thereto.
  • the glass test tube containing the sample and the perchloric reagent was placed in a heating plate, heated at 300° C. for 15 minutes, and then cooled to room temperature.
  • the pretreated sample was transferred to a 50 mL glass flask and quantified to 25 mL with triple-distilled water. Thereafter, the absorbance was measured at 350 nm 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 the sample analysis.
  • the dry matter and protein digestibility of the test feed were calculated by the following method:
  • ADC of dry matter (%) 100 ⁇ 100 ⁇ (% Cr 2 O 3 in diet/% Cr 2 O 3 in feces);
  • ADC of protein (%) 100 ⁇ 100 ⁇ (% Cr 2 O 3 in diet/% Cr 2 O 3 in feces) ⁇ (% protein in feces/% protein in diet)
  • the attack test was carried out such that the shrimps were attacked by white spot syndrome virus.
  • viruses isolated from whiteleg shrimps (Litopenaues vannamei) infected with WSSV were obtained from domestic farms in 2017 and used for the test.
  • the attack test was carried out as follows: the feed compositions of the Examples were given to shrimp for 6 weeks and then the shrimps having the same weight (average weight: 6.25 g) were placed into 4 replicates with 96 shrimps per group.
  • the inoculation concentration of the virus was 4.1 ⁇ 10 5 copies/ ⁇ L, and each shrimp was inoculated intramuscularly with 100 ⁇ L of virus using a syringe.
  • the final inoculation concentration per shrimp was 4.1 ⁇ 10 7 copies/ ⁇ L.
  • the test feed was given three times a day (at 8:30, 13:30 and 18:30) in a divided dose in a restricted manner (10% to 12% of fish body weight), and the degree of mortality was observed for 125 hours.
  • the results are shown in Table 12 below.
  • BS Group 1 provided with the feed composition of Examples 2 including Bacillus subtilis showed the survival rate higher than those of the control group 1 to which the feed composition of Comparative Example 1 was administered.
  • the attack test was carried out such that the shrimp were attacked by Vibrio parahaemolyticus and white spot syndrome virus.
  • Vibrio strain AHPND (EMS)-induced strains isolated from Vietnam in 2013 were used for the test.
  • white spot syndrome virus viruses isolated from whiteleg shrimps (Litopenaues vannamei) infected with WSSV were obtained from domestic farms in 2017 and used for the test.
  • the attack test was carried out as follows: the feed compositions of the Examples were given to shrimps for 6 weeks and then the shrimps having the same weight (average weight: 4.52 g) were placed into 4 replicates with 96 shrimps per group.
  • the inoculation concentration of the virus was 8.3 ⁇ 10 3 copies/ ⁇ L, and each shrimp was inoculated intramuscularly with 50 ⁇ L of virus using a syringe.
  • the final inoculation concentration per shrimp was 4.1 ⁇ 10 4 copies/ ⁇ L. Two days after the virus inoculation, the shrimp were infected with the Vibrio strain.
  • the bacteria were cultured at 30° C. with 150 rpm for 24 hours using a TSB + medium, and a suspension of Vibrio parahaemolyticus was immersed at a concentration of 1.3 ⁇ 10 5 CFU/mL per tank. After the immersion, the mortality of shrimps and their swimming states were confirmed every hour.
  • the test feed was given three times a day (at 8:30, 13:30 and 18:30) in a divided dose in a restricted manner (10% to 12% of fish body weight), and the degree of mortality was observed for 7 days. The results are shown in Table 13 below.
  • BS Group 1 provided with the feed composition of Example 1 including Bacillus subtilis showed the survival rate higher than those of Control Group 1 to which the feed composition of Comparative Example 1 was administered.
  • the feed compositions including Bacillus subtilis according to the present disclosure can increase the growth of whiteleg shrimp, feed efficiency, digestibility, quality of culture water and nonspecific immunity.
  • the present disclosure enables the production of high-protein whiteleg shrimp and thus can increase the marketability of the shrimp.

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