WO2015053419A1 - 누에 조성물 및 애벌레에 주사액을 주입하는 장치 - Google Patents

누에 조성물 및 애벌레에 주사액을 주입하는 장치 Download PDF

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Publication number
WO2015053419A1
WO2015053419A1 PCT/KR2013/008995 KR2013008995W WO2015053419A1 WO 2015053419 A1 WO2015053419 A1 WO 2015053419A1 KR 2013008995 W KR2013008995 W KR 2013008995W WO 2015053419 A1 WO2015053419 A1 WO 2015053419A1
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Prior art keywords
silkworm
injection
composition
larvae
larva
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PCT/KR2013/008995
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English (en)
French (fr)
Inventor
이상훈
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그린테코 주식회사
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Priority to KR1020167011930A priority Critical patent/KR102222051B1/ko
Priority to PCT/KR2013/008995 priority patent/WO2015053419A1/ko
Publication of WO2015053419A1 publication Critical patent/WO2015053419A1/ko

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    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
    • 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
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • 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
    • A61K39/0003—Invertebrate antigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/31—Details
    • A61M5/315—Pistons; Piston-rods; Guiding, blocking or restricting the movement of the rod or piston; Appliances on the rod for facilitating dosing ; Dosing mechanisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the present invention is a composition for silkworm immunity induction and thereby induced immunity.
  • silkworms with added antimicrobial activity more specifically to silkworm immune response-inducing compositions that induce antimicrobial protein expression produced by humoral immune responses to silkworms, and to silkworms with added antibacterial activity by inducing immunity.
  • the present invention relates to a composition containing a silkworm in which an antibacterial peptide is fortified and a method for producing the same.
  • the present invention relates to a device for injecting larvae.
  • [4] silkworm larvae, larvae of the silkworm moth (Bombyx mori): a silkworm that hatches from eggs 10 to 14 days after the onset of awakening. Thus, it is black and has a lot of hair and is about 5 mm in the shape similar to ants.
  • Silkworms are sewn from the earth and sand from the 5th to 6th day of age, and after 12 to 13 days, the moths become silkworms.
  • the silk industry produces silk using cocoons.
  • He is called a sleeper ( ⁇ ⁇ ⁇ ). He is said to have a good taste, taste, and no poison.
  • the silkworm powder manufactured by freeze drying is a silkworm powder manufactured by freeze drying
  • Health functional food (Korean Patent Publication No. 2010-0020463) is developed as a representative, and it is a health functional food containing silkworm powder as a main ingredient such as glycemic control, immune enhancement, and anti-fatigue.
  • silkworms themselves can be used as a powder or silkworms can be used to extract food or
  • Growth-promoting antibiotics are antibiotics that are supplemented with low levels of feed for the purpose of promoting livestock growth, inhibiting intestinal harmful bacteria, preventing diseases, and improving the breeding environment.Another treatment antibiotic is also used to treat diseases in case of abnormal symptoms and diseases of livestock. It is an antibiotic that is administered at a high level under the prescription of a veterinarian.
  • a method of producing recombinant antibodies from silk glands is disclosed.
  • the purpose of the present invention is to provide a silkworm immunosuppressive composition that induces antimicrobial protein expression produced by humoral immune reaction of silkworm and silkworm with antimicrobial activity induced by immunity.
  • the present invention provides a composition containing silkworms enriched with antibacterial peptides having various effects such as improved productivity, maintenance of health, inhibition of enteric harmful bacteria, and immunomodulation, and a method of manufacturing the same.
  • the present invention also provides a device for injecting an immune-inducing substance into a larva that can be used as an antibiotic to produce large quantities of natural antibiotics.
  • composition for inducing silkworm immune reaction of the present invention to achieve the above object is
  • Lactobacillus lysate extracts from which proteins are removed are characterized by inducing antimicrobial protein expression, which is produced by humoral immune response of silkworms, including active ingredients.
  • the protein-lactic acid bacteria lysate extract is free of 0.5 ⁇ 5 mg per 1ml of the total composition.
  • the lactic acid bacteria may be Lactobacillus plantarum.
  • the lactic acid bacteria lysate extract from which the protein is removed is a cell membrane component.
  • the lactic acid bacteria lysate extract from which the protein is removed is obtained by lysing the cells of the lactic acid bacteria by sonication and then removing the protein with butane.
  • the manufacturing method of the silkworm immune response inducing composition of the present invention comprises the steps of culturing the lactic acid bacteria; After obtaining, the second step of preparing a lactobacillus suspension by putting a buffer; the third step of pulverizing the cells of the lactic acid bacteria by the ultrasonic decomposition of the lactic acid bacteria suspension to prepare a lactic acid bacteria cell lysate; extracted by adding butanol to the lactic acid cell lysate After the centrifugation, the fourth step of removing the supernatant to obtain a protein-removed inferior solution; The fifth supernatant is first freeze-dried to prepare a freeze-dried product; the freeze-dried product is dissolved in water, and then dialyzed, and then the second freeze-dried to remove the protein-derived lactic acid bacterium extract step 6 And, a seventh step of producing a silkworm immune reaction composition comprising the protein removed lactic acid bacteria lysate extract.
  • the method of preparing silkworms having immunity-induced immunity and preparing antimicrobial activity according to the present invention comprises the steps of preparing a silkworm immune response-inducing composition; It is a feature that includes;
  • the silkworm oil layer is characterized in that it is 5 days and 5 days.
  • the prepared silkworm immune response composition is characterized by injecting 50 to 60 per layer of silkworm into the body cavity through the back.
  • the antimicrobial activity is characterized by antibacterial activity against Salmonella Enteritidis and Escherichia coli, which are the main pathogens in poultry.
  • Silkworms are used as natural antibiotics for preventing bacterial diseases and promoting animal growth or as feed additives for preventing bacterial diseases and promoting animal growth.
  • a method for preparing a silkworm infused with an immune-inducing substance comprising the step of producing a composition by adding the silkworm.
  • composition further comprises an excipient, and in 100 parts by weight of the total composition
  • the silkworm may be 1 to 50 parts by weight, and 50 to 99 parts by weight of the excipient.
  • the excipient may include any one or more of powder, limestone, ground corn cob, corn gluten feed and soybean.
  • the silkworm may be added to the composition in powder or liquid form, and the composition may be in powder or pellet form.
  • the preparing of the silkworm may be performed by using a microwave.
  • Drying silkworms may be further included.
  • the composition may be a feed of one or more animals of chicken, duck, goose, pheasant, pig, cow, goat, dog and cat.
  • the silkworm may be added to the composition in a ratio of 0.001 to 0.1% by weight of the total composition.
  • a caterpillar support on which a caterpillar to be injected is placed;
  • a caterpillar moving part which moves the caterpillar support part in a predetermined direction;
  • an injection liquid injection part for injecting a injection liquid into a caterpillar placed on the caterpillar support part;
  • a caterpillar separation part for separating the caterpillar injected with the injection liquid from the caterpillar support part
  • a device for injecting is provided.
  • the caterpillar separation unit the caterpillar adsorption unit for coupling with the caterpillar;
  • the larvae may include an adsorption pumping unit for pumping air of the larvae adsorption unit to be adsorbed to the larvae adsorption unit.
  • the adsorption pumping unit may stop the pumping of the air when the larvae adsorption unit is combined with the larvae to separate the larvae from the larvae support unit.
  • the injection liquid injection unit the cylinder portion for reciprocating the inner piston; and coupled to the cylinder portion, the injection needle is coupled to one end and the injection liquid is received in the body to inject the injection liquid to the larvae It may contain a syringe.
  • the present embodiment may further include an injection pump for pumping the injection liquid into the syringe when the syringe inserts the needle into the larvae.
  • the direction in which the larva moving part moves and the direction in which the injection liquid injection unit injects the injection liquid into the larva may be different from each other.
  • the injection may include an immune-inducing substance.
  • the larvae can be silkworm larvae.
  • the injection liquid injection unit may inject the injection liquid into the rear body cavity of the larvae.
  • the caterpillar is placed before the caterpillar support part is placed.
  • the caterpillar deactivator may further include a caterpillar deactivator.
  • the number of the injection amount injection unit of the present embodiment may be a plurality of
  • Injection solution injection unit can be injected into the larvae by adjusting the amount of the injection.
  • Animals are provided with a silkworm immune-induced composition that induces the production of antimicrobial proteins and thereby induces immunity to provide silkworms with antimicrobial activity.
  • the composition chicken As a result of addition to the composition chicken, it has various effects such as improvement of productivity, maintenance of health, inhibition of intestinal harmful bacteria, and regulation of immunity, and it can be used as a new antibiotic replacement substance as it is comparable or excellent in comparison with antibiotics for promoting growth.
  • the device for injecting the larvae according to the present invention has the effect of injecting an immune-inducing substance into the larva that can be used as an antibiotic to produce a large amount of natural antibiotics.
  • FIG. 1 is a diagram showing a method for producing lactic acid bacteria lysate extract as an active ingredient of the silkworm immune reaction composition for silkworm immunity of the present invention.
  • FIG. 2 is a diagram showing a mass production process of immune-induced silkworm silkworms by immunization with inoculum injection prepared in Example 1.
  • FIG. 2 is a diagram showing a mass production process of immune-induced silkworm silkworms by immunization with inoculum injection prepared in Example 1.
  • FIG. 3 is a diagram showing the antimicrobial activity results of E. coli of randomly selected immune-induced silkworm body fluids.
  • FIG. 4 is a diagram showing the results of antimicrobial activity against silkworm humoral proteins in Escherichia coli in order to analyze the silkworm immune induction efficiency according to the inoculation of the candidate immune inducer (control, Example 1, Comparative Example).
  • FIG. 5 shows the results of antimicrobial activity against silkworm body fluid proteins in Escherichia coli to analyze the silkworm immune induction efficiency according to the injection concentration and immune induction time of the immuno-induced injection (lactic acid bacteria lysate extract) of Example 1.
  • FIG. This is a drawing showing.
  • Example 6 shows Salmonella of the immune-induced silkworm powder extract of Example 2 (Salmonella).
  • FIG. 7 is a diagram showing the results of antimicrobial activity against Escherichia coli of the immuno-induced silkworm powder extract of Example 2.
  • FIG. 7 is a diagram showing the results of antimicrobial activity against Escherichia coli of the immuno-induced silkworm powder extract of Example 2.
  • FIG. 8 is a diagram showing an antimicrobial peptide having a silkworm fortified according to an embodiment of the present invention.
  • FIG. 10 shows a silkworm powder fortified with an antibacterial peptide according to an embodiment of the present invention.
  • FIG. 11 is a dried silkworm powder dried powder according to an embodiment of the present invention.
  • FIG. 14 is a block diagram of an apparatus for injecting injection liquid into a larva according to an embodiment of the present invention.
  • 15 is a side view of an apparatus for injecting injection liquid into a larva according to an embodiment of the present invention.
  • 16 is a plan view of a device for injecting injection liquid into a larva according to an embodiment of the present invention.
  • FIG. 17 is a side view of the injection liquid injector injecting the injection liquid into the caterpillar according to the embodiment of the present invention
  • FIG. 18 is a photograph of the internal organs and body cavities of silkworm larvae.
  • the present invention is intended to prevent bacterial diseases occurring in humans and animals as natural antibiotics, or to develop a natural antibiotic for promoting animal growth.
  • lactobacillus, lactobacillus cell wall and lactobacillus lysate extract as an immune-induced candidate for the production of anti-bacterial silkworms was injected into the silkworm body cavity at various concentrations. It was confirmed that the lactic acid bacteria lysate extract showed the best antibacterial activity.
  • the inventors of the present invention have developed a silkworm immune response composition containing silkworm protein lysate extract from which the protein has been removed. By inducing protein expression, silkworms with added antibacterial activity are produced.
  • the lactic acid bacteria which are called 3 ⁇ 4 acid bacteria, can be used for all lactic acid bacteria that ferment saccharides to obtain energy and produce a large amount of lactic acid.
  • Lactobacillus plantamm is preferably used. .
  • the cultured lactic acid bacteria are centrifuged and the supernatant is removed to obtain the cells. Then, a buffer is added to prepare a lactic acid bacteria suspension.
  • the buffer having a pH of 4.7 to increase the glycosylation rate of the lactic acid bacteria suspension.
  • Lactic Acid Bacteria by Crushing Lactic Acid Bacteria Cells Prepare cell lysate.
  • the sonication is preferably carried out for 0.5 to 1 hour so that sonication is performed.
  • Butanol is added to the lactic acid bacteria cell lysate, followed by centrifugation. The supernatant is removed to obtain a protein-free supernatant.
  • the butanol is preferably extracted by adding the same amount as the lactic acid bacteria lysate, and the centrifugation is also centrifuged at 8,000 rpm for 30 minutes to minimize the cell membrane components of the cultured lactic acid bacteria.
  • the same supernatant and the same amount of butanol are added to the prepared supernatant, and the above extraction process is repeated one or three times to remove the protein.
  • Steps 5 and 6 Preparation of Lactobacillus lysate extract from which protein is removed after freeze drying
  • the bottom liquid is subjected to the first step of freezing drying to prepare a freeze dried product, and then the freeze dried product. After dissolving with water, dialysis and then freeze-dried to prepare a protein-lactic acid bacteria lysate extract.
  • the lactic acid bacteria lysate extract from which the protein is removed is a cell membrane component.
  • the protein-lactic acid bacteria lysate extract prepared as described above is to be included as an active ingredient of silkworm immune reaction induction composition, and is used for preventing bacterial diseases and promoting animal growth or preventing natural diseases or bacterial diseases and promoting animal growth.
  • the protein-derived lactic acid bacteria lysate extract preferably contains 0.5 to 5 mg per 1 ml of the total composition.
  • the present invention confirmed that silkworms with antimicrobial activity can be produced in large quantities using the composition for immunoreaction of the present invention as described above.
  • freeze-dried lactic acid bacteria lysate extract was sterilized at lmg / ml concentration.
  • Immunization-induced immune-induced silkworms were immunized by injecting immunized reaction composition of the present invention prepared in the form of injection solution in saline into the body cavity through the back of silkworm larva at the age of 5 to 5 at 50-60 per layer. Know that production is possible have.
  • the silkworms of this invention-free silkworm were found to exhibit excellent antimicrobial activity against Salmonella Enteritidis and Escherichia coli, the major poultry pathogens that cause poultry diseases and gastrointestinal damage.
  • the composition is useful as a pharmaceutical composition or feed composition that is harmless to humans and is effective for preventing and treating bacterial diseases occurring in humans or plants.
  • a pharmaceutically acceptable carrier when used as the pharmaceutical composition, a pharmaceutically acceptable carrier may be additionally contained, and the pharmaceutically acceptable carrier may be
  • lactose textulose, sucrose, sorbitol, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose , Water , Syrup , Methyl Cellulose,
  • Methyl hydroxybenzoate, propyl hydroxybenzoate, talc, stearic acid magnesium and mineral oil but not limited to these.
  • the above components may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.
  • the pharmaceutical composition may be administered parenterally, and subcutaneous injection or topical administration through the skin (dermal dermal administration) is preferred, and the suitable dosage is formulation method, administration method, age, congestion, sex, and morbidity of the patient. It can be prescribed in various ways by factors such as food, time of administration, route of administration, rate of excretion and reaction sensation.
  • compositions may be prepared in unit dosage form by preparation using pharmaceutically acceptable carriers and / or excipients, in accordance with methods which may be readily available by those skilled in the art to which the invention pertains. Or it can be manufactured by embedding it in a multi-capacity container.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets, injections, or capsules, and may further include a dispersant or stabilizer.
  • the silkworm with antimicrobial activity of the present invention is used as a feed composition
  • it is used as a feed composition for animals such as mammals, algae, crustaceans, amphibians, fish, and preferably mammals.
  • feed compositions are properly blended with feedstocks and provided as feeds, which include grains, crudes, vegetable oils, animal feeds, other feedstocks, and tablets.
  • daily feeding of animal feed containing silkworms with antimicrobial activity of the present invention can prevent infections of pathogenic microorganisms.
  • Example 1 Selection and Preparation of Injection Preparation for Production of Immune-Induced Silkworms with Antibacterial Activity
  • Lactobacillus plantarum with a cell membrane structure similar to Gram-positive bacteria was cultured and used.
  • Extracts containing Peptidoglycan and Lipoteichoic Acid (LTA) were prepared.
  • the cells were harvested by centrifugation for 10 minutes, suspended in 50 ml of 0.1 M sodium citrate buffer (pH 4.7), and then disrupted by sonication for 1 hour.
  • the chamber was extracted by stirring for 30 minutes at (24), and then centrifuged at 8,000 rpm for 30 minutes.
  • Protein-free cell lysates were freeze-dried at -40 to 24 hours.
  • Dialysis was performed in tertiary distilled water for 60 hours and tertiary distilled water was exchanged every 12 hours.
  • the freeze-dried product was measured and used in silkworm induction test in 15, 10, 5, 1 and 0.5 mg / ml in various concentrations.
  • Lactobacillus MRS liquid medium The lactic acid bacteria cultured in Lactobacillus MRS liquid medium were obtained by centrifugation, and the cells were freeze-dried.
  • Example 2 Mass Production and Dry Powder Production of Immune-Induced Silkworms with Antibacterial Activity
  • the extract (Example 1) was dissolved in saline sterilized to lmg / ml concentration to prepare an injection solution.
  • the silkworm oil layer of 5-year-old silkworms grown on mulberry leaves was inactivated by immersion for 10 minutes and 15 minutes, and then immunized with a body cavity injection through the back of 50ul of injection solution using an autoinjector as shown in FIG.
  • Immunization-induced injections were administered to approximately 60,000 silkworms for the purpose of mass production of immune-induced silkworms with increased antimicrobial activity.
  • the body fluid was heat-treated at 90 to 10 minutes, and then separated from the supernatant after centrifugation at 13,000 rpm and 4 conditions for 10 minutes.
  • the antimicrobial activity assay was compared to the size of the inhibitory zone that indicates the growth inhibition of pathogens by visualizing the antibacterial activity against pathogenic Escherichia coli (E. coli KACC 1039) through a radiation diffusion assay.
  • the viable bacterial inoculation showed a very low antimicrobial activity, and in the case of the lactobacillus cell wall inoculation, the high mortality was shown when the injection concentration was 0.5 mg / ml or more and 0.5 mg / ml or less. At concentration, mortality decreased but low antimicrobial activity.
  • Lactobacillus lysate extract was found to be the best inducer for silkworm immunity.
  • Lactobacillus plantarum lysate extract was found to be suitable.
  • Silkworm (Gumokjam) was inoculated into the body cavity through the oil layer at day 5 of 5 years old, and fluids were collected from 10 larvae at 6, 10, 18, and 22 hours after induction of immunity and used for the antibacterial activity test against Escherichia coli. .
  • the collected body fluid was heat-treated at 90 to 10 minutes and centrifuged at 13,000 rpm and 4 conditions for 10 minutes to extract the humoral protein.
  • the antibacterial activity assay was compared to the size of the inhibitory zone that represents the pathogen growth inhibition by visualizing the antibacterial activity against Escherichia coli (E. coli KACC 1039) on a medium by using a radiation diffusion assay. The experiment was repeated three times.
  • the antibacterial activity test on the dried immune-induced silkworm powder prepared in Example 2 was carried out on Salmonella Enteritidis and Escherichia coli, which are pathogens related to poultry diseases and gastrointestinal damage.
  • an extract containing antimicrobial protein was prepared from silkworm powder.
  • the separated supernatant was heat-treated at 90 to 10 minutes and centrifuged at 13,000 rpm and 4 conditions for 10 minutes to obtain an extract containing antimicrobial protein.
  • the separated extract was freeze-dried at 90 degrees and weighed.
  • the extract was dissolved in a constant concentration of 0.01% acetic acid and used for assay of antibacterial activity.
  • FIG. 8 shows an antimicrobial peptide having a silkworm fortified according to an embodiment of the present invention.
  • the composition of the present embodiment may be a feed composition, a food additive, a cosmetic additive, or a cultured feed composition such as fish.
  • the following description will focus on the case where the composition of the convenience of description is a feed composition.
  • the feed composition of the present embodiment may be a feed of animals such as chickens, ducks, geese, pheasants, pigs, cattle, goats, dogs, and cats.
  • Silkworms contain a variety of physiologically active substances such as cecropin, GABA, and rutin. Antibacterial, antioxidant, hepatic and renal function improvement, toxicity prevention, fatigue It has various physiological activity effects such as recovery and nerve stabilization. In particular, antibacterial, antioxidant, antiviral, insecticidal, cell regeneration, and immunomodulatory effects such as cecropin and enbocin, which are antibacterial peptide components of silkworm, are applied to the cursor industry. Very large value.
  • the feed composition according to the present embodiment may be implemented in the form of a powder or a pellet having a predetermined volume, and silkworms may be added to the feed composition in powder or liquid form.
  • the explanation focuses on the case where it is added.
  • Each step to be described below may be performed by a feed composition manufacturing apparatus.
  • the processes described for each step need to be performed in a time-series sequence. It is, of course, possible that this process would fall within the scope of the present invention, provided that the subject matter of the present invention is satisfied even if the order of execution of each stage is changed.
  • step S110 the silkworm injected with the immune-inducing substance is powdered.
  • Lactobacillus plantarum which has a cell wall structure similar to a pathogenic Gram-positive bacterium, was used for the development of an injection agent for silkworm induction.
  • L. plantarum n-butanol extract (pLTA) containing L. plantarum probiotics, L. plantarum cell wall extract (pCW), and cell membrane component lipoteichoic acid (LTA) as a candidate source of immune induction were prepared for silkworm immune-inducing substance selection. Used.
  • the L. plantarum cell wall was prepared according to the following method. First, the frozen and dried strains were dissolved in sterile distilled water, heated at 100 to 20 minutes, and then centrifuged at l and 500 g for 10 minutes to destroy them. The bacteria were removed, and the supernatant was centrifuged again at 6,500 g for 30 minutes. The precipitated cell walls were collected, washed three times with distilled water, and lyophilized, and then used for LTA extraction and silkworm induction test.
  • L. plantarum LTA extract (pLTA) was also prepared according to the following method.
  • L. plantarum was prepared in the LTA-containing extract using n-butanol extraction from a cell well. Freeze dried cell well ⁇ sodium citrate buffer (pH 4.7) was ground to a throat Enoch supersonic crusher, the same Hanyang n -b u t an0 I have added the following fact
  • aqueous solution containing various concentrations of L. plantarum bacteria, cell wall extract (pCW) and LTA extract (pLTA) was inoculated into the body cavity at 5 days old silkworms.
  • the silkworm body fluids were collected by the time of induction of immunity (6 hours, 10 hours, 22 hours) and the antibacterial activity against Escherichia coli was evaluated.
  • L. plantarum LTA extract (pLTA) was found to be the best.
  • the antimicrobial activity of the L. plantarum LTA extract was determined according to the inoculation concentration and induction time to establish the optimal silkworm immune induction conditions.
  • Silkworms were inoculated into the silkworm body at 10, 5, 1, and 0.5 mg / ml concentrations, and silkworm blood lymphocytes were harvested at 6, 10, 18 and 22 hours after induction of immunity and used for the evaluation of antimicrobial activity against Escherichia coli.
  • the analysis showed the best antibacterial activity at concentrations of 5 mg / ml and lmg / ml and the highest antibacterial activity in the samples induced with immunization for 18 hours after inoculation.
  • the injection concentration can be determined to be lmg / ml and the induction time to immunity is less than 10 hours and less than 22 hours.
  • the immunoinducing substance may be injected into a growing silkworm larvae.
  • the present embodiment may be equipped with a device for injecting an immune-inducing substance into the silkworm larvae.
  • Injection part which injects the injection containing immunity-inducing substance into the caterpillar support part which is placed in the caterpillar support part, injection part which injects the injection liquid into the caterpillar, injection part injecting the injection liquid into the injection part injection part which inactivates the injection part
  • a larval reservoir which is a means for storing the larvae that have been injected.
  • the larval deactivator can be a tank of cold water, and the caterpillar is deactivated, such as fainting momentarily when immersed in cold water, so that the caterpillar support can easily support a slow moving larvae.
  • the silkworm in which the immune-inducing substance is injected can be dried by a hot air drying method.
  • the hot air drying method is a method of drying silkworms by using hot air having a predetermined temperature.
  • the silkworm in which the immune-inducing substance is injected in step SU4 may be dried by the microwave drying method.
  • the equilibrium point and the time of emergence when using the microwave are about 40 minutes. Also, after analyzing the general components of hot silk drying and drying silk using a microwave oven, the difference between them is not shown. Therefore, the most efficient drying method in the short term is analyzed as using microwave.
  • the antimicrobial activity test was performed by E. coli via radiodiffusion analysis (RDA).
  • ML35 ML35 was analyzed for antimicrobial activity, and for the comparison of antimicrobial activity, the size of inhibition zone indicating pathogen growth inhibition was compared.
  • the antimicrobial activity was highest in the sample dried with microwave (MW) and the sample according to hot air drying showed different results depending on the temperature.
  • step S120 a powdered silkworm is added to the feed to prepare a feed composition.
  • the silkworm powder is preliminarily added to the feed to add a trace amount to the feed.
  • excipient which is a substance used in the formulation, in which excipients may have a fixed dose and weight in order to provide a firmness or shape suitable for the drug, or when the quantity of the subject is small. It is easy to handle and can be added as a purpose. It can be powder, limestone, ground corn cob, corn gluten feed, soybean mill run.
  • 1 to 50 parts by weight of silkworms and 50 to 99 parts by weight of excipients may be included with respect to 100 parts by weight of the total feed composition.
  • the silkworm-derived antimicrobial peptide-containing feed composition was tested for chickens. Investigation of the additive benefits of the silkworm powder-containing feed composition developed for chickens For 1 week old chicken broiler chickens were subjected to a five-week specification test. Example 1, 2, 3, 4 added to the 0.05, 0.10 or 50% level and silkworm powder, which induced the production of the antibacterial peptide by injecting immuno-inducing substance, was added to the level of .01, 0.05, 0.10 or 0.50%. Examples 5, 6, 7, 8 were placed. All the examples did not include feed antibiotics and anticoccidium.
  • Table 4 shows a comparison of broiler productivity.
  • the weight gain and weight gain at the end of 5 weeks were increased by 0.95 to 3.54% compared to Comparative Example 1.
  • Example 5 wherein the antimicrobial peptide was overexpressed by immunoinduction
  • the weight gain effect was higher than that of the general silk powders of Examples 1, 2, 3, and 4.
  • the feed requirements were also reduced in all the examples in which the silk powder was fed compared to Comparative Example 1.
  • the feed composition was able to improve productivity by improving the weight gain and feed rate, and in terms of productivity, the appropriate level of addition was 0.01 ⁇ 0.50 for general silk powder and 0.01 ⁇ 0.05% for immune-induced silkworm powder.
  • Table 5 compares the relative weights of the main organs of broilers.
  • Table 6 compares the biochemical composition of blood.
  • Table 7 shows the comparison of total microbiota changes in the intestinal tract. Reduction compared to comparative example 1 in all examples in which enteric harmful bacteria E. coli and Salmonella spp. In addition, it did not affect the intestinal beneficial bacteria, lactic acid-producing bacteria, and had a positive effect on the stabilization of intestinal microbiota.
  • Table 8 shows the results of comparing the antioxidant and stress related factors in blood. Total antioxidant activity was higher in all of the Examples than in the Comparative Examples, and as the level of silkworm powder was increased, the total antioxidant activity was increased.
  • cortisol content which is known as a typical stress hormone
  • ⁇ Cortisol which is released in large amounts during stress, disease infection, and inflammatory reaction, has a basic metabolic rate, protein synthesis and degradation, It can affect immunity reactions, body continuity, etc., and negatively affect long-term productivity as well as long-term health.
  • Silkworm powder lowers the amount of cortisol in the blood, similar to antibiotics. The stress control effect was confirmed.
  • Table 9 shows the effects of silkworm powder supplementation on broiler white blood cell structure.
  • the multinucleated / lymphocyte ratio which is used as a stress indicator, of course, increases in acute infection or inflammatory reaction, and the nucleus / lymphocyte ratio is significant compared to Comparative Example 1, which is an antibiotic-free supplement in the embodiment in which the general silk and immune-induced silkworm powders are added.
  • the feed composition containing silkworm-derived antibacterial peptides according to the present invention was found to inhibit the growth of harmful bacteria and intestinal secretion of Escherichia coli, Salmonella, etc. and to reduce their biological immune response to them. .
  • the injection liquid injection device 100 includes a caterpillar support 101, a caterpillar mover 102, Injection solution injection part (103), caterpillar separation part (104), caterpillar
  • It may include a deactivator 105, a caterpillar storage 106, and a controller 107.
  • the present invention has the feature of injecting an immune-inducing substance into a larva that can be used as an antibiotic for producing large quantities of natural antibiotics.
  • the present embodiment is a functional larvae including an antimicrobial substance, for example, silkworm.
  • a device for injecting an immune-inducing substance into silkworm larvae is provided.
  • This embodiment is applicable to general caterpillars (mild, dongae, silkworms, slugs, etc.), and for the sake of convenience, the following description will focus on a device for producing large quantities of silkworm caterpillars. Process procedures such as powder processing to produce are obvious to those with ordinary knowledge in the technical field to which the present invention belongs, and explanations thereof will be omitted.
  • the caterpillar support portion 101 is a portion where the caterpillar to be injected is placed.
  • the caterpillar support portion 101 fixes the caterpillar to a specific position in order to inject the injection liquid into the caterpillar, which will be described later.
  • the caterpillar support 101 is a structure that can support a caterpillar, in a specific form.
  • the caterpillar support 101 places the caterpillar on a planar support, and the caterpillar by the push of the injection liquid injection portion 102 in the opposite direction where the injection liquid injection portion KB is located.
  • Blocking means to block movement of the larvae can be fixed.
  • a predetermined groove into which the larvae may be inserted may be formed in the larval support 101.
  • the number of the larvae support 101 may be plural, and a single larvae may be inserted into each of the larval support 101. have.
  • the larvae moving part 102 moves the larvae support part 101 in a predetermined direction.
  • the caterpillar support unit 101 may have a groove formed in each unit as described above, and the entire shape formed by each unit may be a chain or a caterpillar shape, and the caterpillar moving unit 102 may be a caterpillar support unit 101.
  • the caterpillar mover 102 can be a motor.
  • This injection liquid injection section 103 injects the injection liquid into the larvae placed on the larval support section 101.
  • the injection liquid injection unit 103 may include a syringe for receiving the injection liquid therein.
  • the injection liquid injection unit 103 is provided in a specific portion of the larvae placed on the larval support unit 10 1. Injection solution can be injected ⁇
  • the injection solution injection section 103 can inject the injection solution into the body cavity of the caterpillar's back side.
  • FIG. 18 a silkworm larvae peeled off is shown pinned to the pin.
  • FIG. 18 (a) shows a state in which the intestinal organ 410, which is a digestive organ, is located at the back of the larval cavity 420.
  • 18 (b) shows a state in which the intestine 410 is removed from the dorsal part of the larval body cavity 420. In a normal state, the intestine 410 is in the back part of the caterpillar.
  • the needle is inserted into the lower part of the intestinal 410 position at the back of the caterpillar, i.e., the back of the caterpillar and the body cavity 420 which is 80% of the total body thickness from the caterpillar.
  • Immuno-inducing substances may be nontoxic to the body and have the effect of regulating immune capacity.
  • immuno-inducing substances may be bioreactive substances (BRM).
  • BRM bioreactive substances
  • AHCC Active Hexose Correlated Compound
  • Polysaccarides, Polysaccaride peptides, Nucleosides, Triterpenoids, and the like, and various other substances may be applied to the present invention as a reverse inducer.
  • the caterpillar separator 104 separates the larvae injected with the injection solution from the caterpillar support 101.
  • the caterpillar separator 104 separates the caterpillar from the caterpillar support 101 by pushing the caterpillar in the extension direction of the groove.
  • the caterpillar by changing the upper surface of the groove in the direction of gravity to drop the caterpillar in the direction of gravity
  • the larvae separation unit 104 includes a larvae adsorption unit (not shown) that combines with the larvae and an adsorption pumping unit (not shown) that pumps the air of the larvae adsorption unit so that the larvae are adsorbed to the larvae adsorption unit.
  • the larvae can be larvaized in various positions and directions by utilizing the attraction force by air pumping.
  • the pumping of the air may stop the larvae from the larvae adsorption unit. Can be dropped to 106 and stored.
  • the caterpillar deactivator 105 may be a tank containing cold water, and the caterpillar is deactivated by being instantaneously stunned when immersed in the cold water, so that the caterpillar support 101 is easily supported by the moving caterpillar. can do.
  • the larval deactivator 105 may be an ice, a refrigerator, an anesthetic, or the like.
  • the caterpillar storage unit 106 is a means for storing a caterpillar in which the injection liquid is injected by the injection liquid injection unit 103 and the injection processing is completed.
  • the caterpillar storage unit 106 is a container for holding the caterpillar. The larvae that have escaped from the larval support 101 can be moved to and stored in the larval storage 106.
  • the method of moving the larvae to the larvae storage unit 106 may be implemented in various ways, for example, when the larvae support unit 101 is formed in a structure that puts the larvae on the support of the planar shape as described above
  • the larvae can be moved to the larval reservoir 106 by raising or lowering the plane to the side so that the larvae fall downward.
  • the larvae support 101 can absorb the larva with a vacuum pump. When supported, the operation of the vacuum pump may be stopped and the caterpillar may be moved to the caterpillar storage unit 106 by dropping the caterpillar in a straight direction.
  • the caterpillar storage unit 106 may be located below the caterpillar separation unit 104.
  • the larvae may be stored as larvae after the treatment by dropping to the larva storage unit 130 after the injection solution is completed.
  • the larvae placed on the larval deactivation unit 103 is driven by a specific push means.
  • the push means may move the larva by pushing the larva one by one toward the larval support 101 while the larva is accommodated in a tubular shape.
  • control unit 107 The control unit 107, the above-described caterpillar support 101, the larva moving unit 102, injection
  • the injection section 103, the larval separator 104, the larval deactivator 105, and the larva storage 106 interlock with each other to control each functional section so as to perform its own unique functions.
  • the specific function of the control unit 107 may vary depending on the specific embodiment.
  • Blocks generally showing a device for injecting injection liquid into the larva above
  • 15 is a view of an apparatus for injecting injection liquid into a larva according to an embodiment of the present invention.
  • 16 is a plan view of a device for injecting injection liquid into a caterpillar according to an embodiment of the present invention.
  • the caterpillar support part 111, the caterpillar moving part 112, and the injection liquid injection part 113 are shown.
  • the larvae separating part 114, the device support part 121, and the larvae 220 are shown.
  • the difference with the above-mentioned is mainly focused.
  • the injection liquid injection unit 113 is one, but this embodiment may include a plurality of injection liquid injection units 113.
  • each injection liquid injection unit 113 The injection can be done independently, as described below, or in conjunction with the same timing and action.
  • the plurality of larvae supporting parts 1 1 are combined in a caterpillar shape and rotated by the larval moving parts 112 to move the larvae 220 which are coupled to the larval supporting parts 111 in a predetermined direction.
  • the device support 121 can support the entire device, including the caterpillar support 111 and the caterpillar mover 112.
  • the injection liquid injection portion 113 is located on the upper side of the larval support portion 111, and the injection liquid
  • the injection liquid is injected into the larvae located in front of the injection unit 113.
  • the injection unit 113 may adjust the amount of injection liquid injected, that is, the injection liquid.
  • the injection unit 113 may inject the same amount of injection solution for each larvae, or inject a predetermined amount of injection solution corresponding to the size of the larvae.
  • the injection direction of the injection into the larvae may be different from each other.
  • the larvae are supported in parallel with the larva support 111 to be moved laterally, and the injection solution 113 is directed to the larvae toward the extension direction. Injection solution can be injected.
  • the caterpillar separating unit 114 separates the larvae injected with the injection solution from the larval support unit i n and stores the larvae in the larval storage unit 106 as described above.
  • FIG. 17 is a side view of the injection portion injection portion of the apparatus for injecting the injection liquid into the larva according to the embodiment of the present invention. Referring to FIG. 17, the upper frame 110, the cylinder portion 116, the syringe portion 118, Injection fluid inlet tube 119 and lower frame 120 are shown.
  • the injection liquid injecting unit 113 is formed of a body with a frame, specifically, with the ground
  • Parallel upper frame 110, lower frame 120 supporting the upper frame 110 can form a body.
  • the larvae can be firmly coupled to the above-described rear part of the larval support 111.
  • the needle can inject the injection liquid into the rear body cavity as described above by puncturing the cavities of the larvae.
  • the injection needle according to the present embodiment may be a needle generally having a through-hole formed therein, or a needle having an external defect formed therein to accommodate the injection solution.
  • a spiral needle is formed on the outer surface of the needle.
  • a plurality of grooves are formed or extended in a direction different from the direction in which the needle is extended, and a plurality of grooves are formed to have a predetermined distance from each other, or a plurality of grooves are extended in the same direction as the extended direction of the needle.
  • a groove may be formed or a groove extending to have a predetermined pattern, for example, a 'W' shape, or the like may be formed.
  • a concave ball groove is formed on the outer surface of the injection needle.
  • Various types of grooves can be formed, in which case the injections can be accommodated in the above-mentioned grooves so that the injections can be injected into the larvae or buried in the larvae even in the case of larvae that can not be inserted normally.
  • It can consist of a needle.
  • the injection solution injection section 113 applies the injection solution to the larvae. It may further comprise a caterpillar fixation (not shown) that secures the caterpillar to inject correctly.
  • the caterpillar fixation may also include caterpillar contacts (not shown) that are elongated in correspondence with the size and / or shape of the caterpillar.
  • the caterpillar contact is a means of making actual contact with the caterpillar, thereby securing the caterpillar to the caterpillar support 111. Inject the larvae accurately
  • the cylinder portion 116 has an internal piston reciprocatingly coupled to the syringe portion 118 at one end.
  • the length of the cylinder portion 116 and the push length of the syringe portion 118 can be specified to such an extent that injection liquid can be injected into the larvae.
  • the operation of the cylinder portion 116 can be determined manually or automatically. For example, if the user presses the corresponding push button with a push button that pushes the syringe portion 118 in the larval direction, the cylinder portion (116) is activated, the syringe section 118 can move to the caterpillar a predetermined length, inject the injection, and then return back to stop the initial state.
  • the cylinder section 116 can be operated to inject the injection liquid into the larvae.
  • the above-described control unit 107 is formed by the caterpillar support section 111 where the larva to be injected is combined with
  • the syringe section 118 can be controlled to move to the caterpillar immediately or after a specific time, for example, one to two seconds.
  • the syringe portion 118 is coupled to one side of the cylinder portion 116, and at one end a needle
  • the embodiment is connected to the syringe portion (U 8) and the injection liquid inlet pipe (119),
  • the syringe portion 118 may further include an injection pump (not shown) that pumps the injection liquid into the syringe portion (8) when the syringe needle is inserted into the larvae.
  • the operation method of the device for injecting the injection liquid into the larva according to the present invention is various It can be embodied in the form of program instructions that can be executed by computer means and recorded on a computer readable medium. That is, the recording medium can be a computer readable recording medium having recorded thereon a program for executing each step described above on the computer.
  • the instructions may be specially designed and configured for the present invention or may be known and available to those skilled in the computer software art.
  • Examples of computer readable media include magnetic media, such as hard disks, floppy disks, and magnetic tape, Optical Media such as CD-ROMs, DVDs, Magnetic-Optical Media such as Floppy Optical Disks, and ROMs, RAM, Flash Memory, etc.
  • Hardware devices specifically configured to store and execute program instructions are included.
  • each of the above-described components may be implemented as one physically adjacent component or may be implemented as different components. In the latter case, each component may be controlled by being adjacent or located in different zones.
  • the invention may be equipped with a separate control means or control room for controlling each component to control each component by wire or wireless.

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Abstract

본 발명은 누에 면역반응 유도용 조성물 및 이에 의해 면역이 유도되어 항균활성이 부가된 누에에 관한 것으로서, 보다 상세하게는 누에의 체액성 면역반응으로 생성되는 항균성 단백질 발현을 유도하는 누에 면역반응 유도용 조성물 및 이에 의해 면역이 유도되어 항균활성이 부가된 누에를 제공한다.

Description

명세서
발명의명칭:누에조성물및애벌레에주사액을주입하는장치 기술분야
[1] 본발명은누에면역반웅유도용조성물및이에의해면역이유도되어
항균활성이부가된누에에관한것으로서，보다상세하게는누에의체액성 면역반응으로생성되는항균성단백질발현을유도하는누에면역반응유도용 조성물및이에의해면역이유도되어항균활성이부가된누에에관한것이다.
[2] 또한，본발명은항균펩타이드가강화된누에를함유하는조성물및그제조 방법에관한것이다.
[3] 본발명은애벌레에주사액을주입하는장치에관한것이다.
배경기술
[4] 누에는한자로잠 (霞)이라고하며 ,누에나방 (Bombyx mori,문화어：누에나비 )의 애벌레로서 ,알깨기시작후 10 ~ 14일만알에서부화한누에는 1령누에또는 개미누에라하여,까맣고털이많고약 5 mm정도로개미와비슷한형태가 특징이며， 3령이지나야지만누에특유의형태가나타난다.
[5] 누에가 5령이된지 5~6일째부터토사구에서실을뽑아고치를짓게되는데 고치상태에서 12~13일이지나면나방이되며，양잠산업에서는고치를이용하여 실크를생산하고있다.
[6] 동의보감에나와있는천연자양강정식품인누에번데기는한방에서는
잠용자 (震踊子)라고부르며,성질이고르고맛이달며독이없어,풍과여원것을 다스린다고하였다.
[7] 최근국내의누에와관련된산업시장은전통적인양잠산업인누에고치
생산에서벗어나，누에를소재로하는기능성식품과천연물의약품과관련된 기술개발이활발하게이루어지고있는데,동결건조로제조된누에분말은
1995년부터상품화되기시작하였으며， 5령 3일누에의알콜추출물을생강,계피, 타우린과함께흔합한기능성항당뇨음료 (한국등록특허제 10-0354151호)，누에 번데기유래의지방산을유효성분으로하는고지혈증예방및치료
조성물 (한국둥록특허제 10-0732727호)，누에체액추출물을이용한
건강기능식품 (한국공개특허제 2010-0020463호)등이대표적으로개발되어 있으며 ,혈당조절제，면역증강,항피로등효능으로누에분말을주성분으로한 건강기능식품이다수출시되었다.
[8] 그러나，누에자체를분말화하여사용하거나누에를추출하여식품또는
약품의소재로사용되어져왔을뿐，누에를동물에발생하는세균성질환예방및 동물성장촉진용소재로활용하기위해누에의면역올유도하는방법에대한 연구는이루어진바가없다. [10] 또한，최근소비자의경제수준향상으로인해건강에대한관심이고조되면서 축산물의양적측면보다질적측면이강조되고있으며，축산물의품질향상및 안전성확보는국내축산업의경쟁력확보는물론국민건강을위해더욱 중요시되고있다.또한지속가능한친환경순환식축산의중요성이더욱 커지면서유휴농산물및국내부존자원이용을통해축산업의긍정적모습을 부각시킬필요가있다.이로인해최근국내부존자원을활용한고품질안전 축산물을생산하기위한연구가활발히진행되고있다.
[11] 국내잠업은 1976년을정점으로급격히감소하였으며 1995년을기점으로 누에고치생산을거의중단되고,현재는건조누에나동충하초등의기능성 양잠산물생산으로전환하여잠업을유지하고있다.그러나국내누에사육 /생산 기술및잠재력은타곤층에비해축적되어있어안정적인생산시장확보및 고부가가치상품개발시안정적인생산및공급이가능하다.대한민국공개특허 제 10-2000-0025517호를참조하면,누에의배설물을이용하여사료조성물을 제조하는기술이개시된다ᅳ
[12] 하지만，축산분야에서는누에나누에유래항균펩타이드를가축에적용한 사례및연구가많지않아실제로적용하기가어려워그이용가치및활용범위가 낮은실정이다.
[13]
[14] 또한,동물용항생제는성장촉진용과치료용으로사용되어왔으며,오늘날 집약적및기업적축산으로대규모화되는데주도적인역할을하였다.
성장촉진용항생제는가축의성장촉진，장내유해균억제，질병예방，사육환경 개선등의목적으로저수준으로사료내첨가급여되는항생제이다.또한， 치료용항생제는가축의이상증후및질병발생시질병치료를목적으로수의사 처방하에고수준으로투여되는항생제이다.
[15] 하지만,최근에가축사료내항생제오남용으로인한내성균출현，축산물내 항생제잔류，가축의질병저항성약화，분뇨로유출된항생제잔류물로인한 생태계오염등의문제가대두되고있다.
[16] 우리나라는축산물 1톤생산시항생제 0.91kg사용으로뉴질랜드 0.04kg,미국 0.15kg,일본 ().36kg에비해매우높은편이다.또한，식품의약품안전청의 2009년 조사에따르면,축산물내항생제내성균조사결과,대장균
53.6 69.3% (테트라사이클린，암피실린)，장구균 62.4~68.9% (테트라사이클린， 엔트로마이신),포도상구균 67% (페니실린)， 3가지계열이상의항생제내성을 보인다제내성균비율이 39%차지하였다.
[17] 따라서항생제오남용방지,소비자의축산물위생렙흔轨요구,국내축산 경쟁력제고를위한항생제사용규제강화및무항생제축산물인증정책 추진에따른대책마련이시급한실정이다.
[18] 최근항생제오남용에따른내성균주를퇴치할수있는새로운작용기작을 가지는천연항생제개발이모색되고있다.누에를포함한곤충은불량환경에서 오랜진화의역사를통해자기방어를위한강력한항균물질을보유하고있어， 화학항생제를대체할수있는천연항생제개발소재로써매우우수한것으로 파악된다.
[19] 선행기술인대한민국공개특허제 2008-0083111호 (항체를생산하는
트랜스제닉누에와그의제조방법)에따르면，트랜스제닉누에의
견사선으로부터재조합항체를생산하는방법이개시된다.
[20] 하지만,종래기술에따르면,천연항생펩타이드를가진곤층을대량으로
유도하기위한장치개발은미흡한실정이다.
발명의상세한설명
기술적과제
[21] 본발명의목적은누에의체액성면역반웅으로생성되는항균성단백질발현을 유도하는누에면역반웅유도용조성물및이에의해면역이유도되어 항균활성이부가된누에를제공하기위한것이다.
[22] 또한,본발명은생산성향상,건강유지，장내유해균억제,면역조절등다양한 효과를가지는항균펩타이드가강화된누에를함유하는조성물및그제조 방법을제공한다.
[23] 또한，본발명은천연항생제를대량으로생산하기위하여항생제로사용될수 있는애벌레에면역유도물질을주입하는위한장치를제공한다.
[24] 본발명이이루고자하는기술적과제들은이상에서언급한기술적과제들로 제한되지않으며,언급되지않은다른기술적과제들은아래의기재로부터본 발명이속하는기술분야에서통상의지식을가진자에게명확하게이해될수 있을것이다.
과제해결수단
[25] 상기목적을달성하기위한본발명의누에면역반응유도용조성물은
단백질이제거된유산균파쇄액추출물을유효성분을포함하여누에의체액성 면역반웅으로생성되는항균성단백질발현을유도하는것이특징이다.
[26] 상기단백질이제거된유산균파쇄액추출물은상기조성물전체 1ml당 0.5~5 mg을포함하는것이특징이다.
[27] 상기유산균은락토바실러스플란타륨 (Lactobacillus plantarum)인것이
특징이다.
[28] 상기단백질이제거된유산균파쇄액추출물은세포막성분인
펩티도글리칸 (Peptidoglycan)및리포테이코익산 (lipoteichoic acid, LTA)이포함된 것이특징이다.
[29] 상기단백질이제거된유산균파쇄액추출물은초음파분해로상기유산균의 세포를파쇄한후,부탄을로단백질을제거하여얻은것이특징이다.
[30] 본발명의누에면역반응유도용조성물의제조방법은유산균을배양하는 제 1단계;상기배양된유산균을원심분리한후상등액을제거하여균체를 획득한다음,버퍼를넣어유산균현탁액을제조하는제 2단계;상기유산균 현탁액을초음파분해로인해유산균의세포를파쇄하여유산균세포파쇄물을 제조하는계 3단계;상기유산균세포파쇄물에부탄올을첨가하여추출한다음 원심분리한후，상등액을제거하여단백질이제거된하등액을얻는제 4단계; 상기하등액은 1차동결건조하여동결건조물을제조하는제 5단계;상기 동결건조물을물로녹인후,이를투석한다음 2차동결건조하여단백질이 제거된유산균파쇄액추출물을제조하는제 6단계및,상기단백질이제거된 유산균파쇄액추출물올포함하는누에면역반웅유도용조성물을제조하는 제 7단계；를포함하는것이특징이다.
[31] 본발명의면역이유도되어항균활성이부가된누에의제조방법은상기의누에 면역반응유도용조성물을준비하는단계;및，상기준비한누에면역반웅 유도용조성물을누에유층의체강에주입하는단계;를포함하는것이특징이다.
[32] 상기누에유층은 5령 5일인것이특징이다.
[33] 상기준비한누에면역반응유도용조성물은상기누에 1층당 50~60를배면을 통한체강에주입하는것이특징이다.
[34] 본'발명의항균활성이부가된누에는상기의항균활성이부가된누에의
제조방법에의해면역이유도되어제조되는것이특징이다.
[35] 상기항균활성은가금장내주요병원균인살모넬라균 (Salmonella Enteritidis) 및대장균 (Escherichia coli)에대한항균활성인것이특징이다.
[36] 본발명의항균활성이부가된누에의사용방법은상기항균활성이부가된
누에를세균성질환예방및동물성장촉진용천연항생제또는세균성질환예방 및동물성장촉진용사료첨가제로사용되는것이특징이다.
[37]
[38] 본발명의일측면에따르면,면역유도물질을주입한누에를준비하는단계; 및상기누에를첨간하여조성물을제조하는단계를포함하는항균펩타이드가 강화된누에를함유하는조성물제조방법이제공된다.
[39] 여기서，상기조성물은부형제를더포함하며 ,전체조성물 100중량부에
대하여，상기누에 1내지 50중량부,상기부형제 50내지 99중량부가될수있다.
[40] 또한,상기부형제는말분，석회석,분쇄옥수수속대，옥수수글루텐피드및대두 중어느하나이상을포함할수있다.
[41] 여기서,상기누에는분말또는액상으로상기조성물에첨가될수있으며，상기 조성물은분말또는팰렛형상이될수있다.
[42] 여기서,상기누에를준비하는단계는,마이크로웨이브를이용하여상기
누에를건조하는단계를더포함할수있다.
[43] 여기서，상기조성물은닭，오리,거위,꿩,돼지，소，염소,개및고양이중어느 하나이상의동물의사료가될수있다.
[44] 또한，상기누에는전체조성물의질량대비 0.001내지 0.1%의비율로상기 조성물에첨가될수있다. [46] 본발명의일측면에따르면，주사액이주입될애벌레가놓이는애벌레지지부; 상기애벌레지지부를소정의방향으로이동시키는애벌레이동부;상기애벌레 지지부에놓인애벌레에주사액을주입하는주사액주입부;및상기주사액이 주입된애벌레를상기애벌레지지부로부터분리하는애벌레분리부를 포함하는애벌레에주사액을주입하는장치가제공된다.
[47] 여기서,상기애벌레분리부는，상기애벌레와결합하는애벌레흡착부;및
상기애벌레가상기애벌레흡착부에흡착되도록상기애벌레흡착부의공기를 펌핑하는흡착펌핑부를포함할수있다.
[48] 또한，상기흡착펌핑부는상기애벌레흡착부가상기애벌레와결합되어상기 애벌레를상기애벌레지지부로부터분리시키는경우상기공기의펌핑을 중단할수있다.
[49] 여기서，상기주사액주입부는，내부피스톤이왕복운동을하는실린더부;및 상기실린더부에결합하며,일단에주사바늘이결합하고몸체에상기주사액이 수용되어상기애벌레에상기주사액을주입하는시린지부를포함할수있다.
[50] 또한,본실시예는상기시린지부가상기애벌레에주사바늘을꽂을때상기 주사액을상기시린지부에펌핑하는주사액펌핑부를더포함할수있다.
[51] 여기서，상기애벌레이동부가이동하는방향과상기주사액주입부가상기 애벌레에주사액을주입하는방향은서로다를수있다.
[52] 또한，상기주사액은면역유도물질을포함할수있다.
[53] 여기서，상기애벌레는누에애벌레가될수있다.
[54] 여기서，상기주사액주입부는상기애벌레의배면부체강에상기주사액을 주입할수있다.
[55] 또한,본실시예는상기애벌레를상기애벌레지지부에놓기전에상기
애벌레를비활성화시키는애벌레비활성화부를더포함할수있다.
[56] 또한,본실시예의상기주사액주입부의개수는복수가될수있으며,상기
주사액주입부는상기주사액의양을조절하여상기애벌레에주입할수있다. 발명의효과
[57] 이상에서설명한바와같이본발명에의해，누에의체액성면역반웅으로
생성되는항균성단백질발현을유도하는누에면역반응유도용조성물및이에 의해면역이유도되어항균활성이부가된누에가제공됨에따라동물
성장촉진용천연항생제및세균성질환예방사료첨가제의개발이가능하게 된다.
[58] 또한，본발명에서개발한항균펩타이드가강화된누에를함유하는사료
조성물올닭에첨가급여한결과생산성향상,건강유지,장내유해균억제，면역 조절등다양한효과가있었으며，성장촉진용항생제와비교할때대등하거나 우수한결과를보임으로추후신규항생제대체물질로서의사용이가능하다. [59] 또한,본발명에따른애벌레에주사액을주입하는장치는항생제로사용될수 있는애벌레에면역유도물질을주입하여천연항생제를대량으로생산할수 있는효과가있다.
도면의간단한설명
[60] 도 1은본발명의누에면역반웅유도용조성물의유효성분인유산균파쇄액 추출물제조방법도를나타낸도면이다.
[61] 도 2는실시예 1에서제조된면역유도용주사제접종을통한면역유도누에 대량생산과정을나타낸도면이다.
[62] 도 3은임의로선별된면역유도누에체액의대장균에대한항균활성결과를 나타낸도면이다.
[63] 도 4는후보면역유도제의주사접종 (대조구,실시예 1，비교예)에따른누에 면역유도효율을분석하기위해대장균을대상으로누에체액성단백질에대한 항균활성결과를나타낸도면이다.
[64] 도 5는실시예 1의면역유도용주사제 (유산균파쇄액추출물)의주사농도및 면역유도시간에따른누에면역유도효율을분석하기위해대장균을대상으로 누에체액성단백질에대한항균활성결과를나타낸도면이다.
[65] 도 6은실시예 2의면역유도누에분말추출물의살모넬라균 (Salmonella
Enteritidis)에대한항균활성결과를나타낸도면이다.
[66] 도 7은실시예 2의면역유도누에분말추출물의병원성대장균 (Escherichia coli)에대한항균활성결과를나타낸도면이다.
[67]
[68] 도 8은본발명의실시예에따른항균펩타이드가강화된누에를함유하는
조성물의제조방법을나타낸흐름도.
[69] 도 9는본발명의실시예에따른항균펩타이드가강화된누에분말을열풍
건조법으로제조시온도및시간별중량변화를나타낸그래프.
[70] 도 10은본발명의실시예에따른항균펩타이드가강화된누에분말을
마이크로웨이브건조법으로제조시시간별중량변화를나타낸그래프.
[71] 도 11은본발명의실시예에따른항균펩타이드가강화된누에분말건조
방법에따른누에분말항균활성비교도면.
[72] 도 12와도 13은본발명의실험결과를나타낸도면.
[73]
[74] 도 14는본발명의실시예에따른애벌레에주사액을주입하는장치의블록 구성도.
[75] 도 15는본발명의실시예에따른애벌레에주사액을주입하는장치의측면도.
[76] 도 16은본발명의실시예에따른애벌레에주사액올주입하는장치의평면도.
[77] 도 17은본발명의실시예에따른애벌레에주사액을주입하는장치중주사액 주입부의측면도. [78] 도 18은누에애벌레의내장및체강위치를촬영한사진.
발명의실시를위한최선의형태
[79] 이하,본발명의바람직한실시예를상세하게설명하며，본발명의요지를
불필요하게흐릴수있는공지기능및구성에대한상세한설명은생략한다.
[80] 이하에서설명될실시예는크게세가지로구분되며，각실시예를설명하는 도면은순서대로제시되고,각도면에도시된구성요소의참조번호는각 실시예를설명하는도면에상웅하여이해해야한다.
발명의실시를위한형태
[81] . 본발명은천연항생제로써인체및동물에발생하는세균성질환을예방하거나 '동물성장촉진용천연항생제개발을위한목적으로，단백질이제거된유산균 파쇄액추출물을유효성분을포함하여누에의체액성면역반웅으로생성되는 항균성단백질발현을유도하는누에면역반웅유도용조성물및이에의해 면역이유도되어항균활성이부가된누에를제공한다.
[82] 설명하면,항균성이부가된누에생산을위해서면역유도후보물질로유산균， 유산균세포벽및유산균파쇄액추출물을제조하여다양한농도로누에체강에 주사하여면역유도시간에따른항균활성을검정한결과유산균파쇄액추출물 주사접종에서가장우수한항균활성을나타냄을확인하였다.
[83] 이에본발명의발명자들은상기단백질이제거된유산균파쇄액추출물을 포함하는누에면역반웅유도용조성물을개발하게된것이며,이들조성물을 누에에주입하면누에의체액성면역반웅으로생성되는항균성단백질발현을 유도함으로써항균활성이부가된누에가생산된다.
[84] 이하,상기의누에면역반웅유도용조성물의제조방법에대하여구체적으로 설명하면다음과같다.
[85] 1.제 1단계 ;유산균배양
[86] 먼저유산균을배양한다.
[87] 상기유산균으로는일명 ¾산균이라고불리우는것으로서，당류를발효하여 에너지를획득하고다량의락트산을생성하는모든유산균은사용이가능하나, 바람직하게는락토바실러스플란타륨 (Lactobacillus plantamm)를사용한다.
[88] 2.제 2단계;유산균현탁액제조
[89] 상기배양된유산균을원심분리한후상등액올제거하여균체를획득한다음, 버퍼를넣어유산균현탁액을제조한다.
[90] 이때,상기배양된유산균의세포막성분을최소화하기위해 8,000 rpm에서
30분동안원심분리하는것이좋다,
[91] 또한,상기버퍼로는유산균현탁액의당화율을높이기위해 pH가 4.7인버퍼
0.1M sodium citrate buffer를사용하는것이좋다.
[92] 3.제 3단계;유산균세포파쇄물제조
[93] 상기유산균현탁액을초음파분해로인해유산균의세포를파쇄하여유산균 세포파쇄물을제조한다.
[94] 이는，하기단백질제거를위해유산균의세포가파쇄된상태여야하는
것으로서상기배양된유산균의세포막성분을최소화하면서,상기
초음파분해 (sonication)가이루어지도록상기초음파분해는 0.5~1시간동안 이루어지는것이좋다.
[95] 4.제 4단계；단백질이제거된하둥액제조
[96] 상기유산균세포파쇄물에부탄올을첨가하여추출한다음원심분리한후, 상등액을제거하여단백질이제거된하등액을얻는다.
[97] 이때,상기부탄올은상기유산균세포파쇄물과동일한양을넣어추출하는 것이좋으며,상기원심분리또한상기배양된유산균의세포막성분을최소화 하기위해 8,000 rpm에서 30분동안원심분리하는것이좋다.
[98] 또한，단백질제거율을높이기위해상기제조된하등액에하등액과동일양의 부탄올을첨가하여상기와같은추출과정을 1-3회이상반복하여단백질을 제거하기도한다.
[99] 5.제 5및 6단계;동결건조후단백질이제거된유산균파쇄액추출물제조 [100] 상기하둥액은 1차동결건조하여동결건조물을제조하는제 5단계를거친후， 상기동결건조물을물로녹인후,이를투석한다음 2차동결건조하여단백질이 제거된유산균파쇄액추출물을제조한다.
[101] 이때,상기단백질이제거된유산균파쇄액추출물에는세포막성분인
펩티도글리칸 (Peptidoglycan)및리포테이코익산 (lipoteichoic acid, LTA)이 포함되어있다.
[102] 6.누에면역반응유도용조성물제조
[103] 이렇게제조된상기단백질이제거된유산균파쇄액추출물은누에면역반웅 유도용조성물의유효성분으로포함하도록하는것으로서,세균성질환예방및 동물성장촉진용천연항생제또는세균성질환예방및동물성장촉진용 사료첨가제로사용되는하기항균활성을나타내는누에를제조하기위해상기 단백질이제거된유산균파쇄액추출물은상기조성물전체 1ml당 0.5~5 mg을 포함하는것이적합하다.
[104] 이는 0.5mg미만으로함유될경우본발명이목적하는누에면역유도능을 나타내기어려우며, 5mg을초과하여함유될경우에는누에에주입시상기 단백질이제거된유산균파쇄액추출물의과도한주입량으로인해누에성장에 오히려악영향을미칠우려가있게된다.
[105] 또한,본발명은상기와같은본발명의면역반웅유도용조성물을이용하여 항균활성이부가된누에를대량생산할수있음을확인하였다.
[106] 설명하면,동결건조된유산균파쇄액추출물을 lmg/ml농도로멸균된
식염수에녹여주사액의형태로제조된본발명의면역반응유도용조성물을 누에 1층당 50~60씩 5령 5일째누에유충의배면을통한체강에주사하여 면역화함으로서항균성이부가된면역유도누에를대량생산이가능함을알수 있다.
[107] 이렇게생성된본발명의항균성이무가된누에는가금질병및위장관손상을 유발하는가금장내주요병원균인살모넬라균 (Salmonella Enteritidis)및 대장균 (Escherichia coli)에탁월한항균활성을나타냄을확인한바，비록 구체적인실시예로제시되지는않았지만，인체에무해하면서인체또는 식물체에발생하는세균성질환예방및치료에효과적인약학적조성물또는 사료용조성물로유용하게이용가능함이당업자에게당연할것이다.
[108] 이때，상기약학적조성물로사용할경우에는약학적으로허용되는담체가 추가로함유될수도있는데,약제학적으로허용되는담체는제제시에
통상적으로이용되는것으로서，락토스,텍스트로스，수크로스，솔비를,만니를， 전분,아카시아고무,인산칼슴,알기네이트,젤라틴，규산칼슘,미세결정성 셀를로스,폴리비닐피롤리돈,셀를로스，물，시럽，메틸셀를로스,
메틸히드록시벤조에이트,프로필히드록시벤조에이트,활석,스테아르산 마그네슴및미네랄오일등을포함하나,이에한정되는것은아니다.
[109] 또한,상기성분들이외에윤활제，습윤제，감미제 ,향미제 ,유화제 ,현탁제， 보존제등을추가로포함할수있다.
[110] 상기약학적조성물은비경구로투여할수있고，피하주입또는피부를통한 국소투여 (경피투여)가바람직하며,적합한투여량은제제화방법,투여방식， 환자의연령，체증,성,병적상태,음식,투여시간，투여경로，배설속도및반웅 감웅성과같은요인들에의해다양하게처방될수있다.
[111] 이러한약학적조성물은당해발명이속하는기술분야에서통상의지식을가진 자가용이하게실시할수있는방법에따라，약제학적으로허용되는담체 및 /또는부형제를이용하여제제화함으로써단위용량형태로제조되거나또는 다용량용기내에내입시켜제조될수있다.
[112] 이때제형은오일또는수성매질중의용액，현탁액또는유화액형태이거나 엑스제,분말제,과립제,정제,주사제또는캅샐제형태일수도있으며，분산제 또는안정화제를추가적으로포함할수있다.
[113] 또한，본발명의항균활성이부가된누에를사료용조성물로사용할경우， 포유류，조류,파층류,양서류,어류등의동물,바람직하게는포유류에대한 사료용조성물로사용된다.
[114] 이러한사료용조성물은사료원료에적절하게배합하여사료로제공되는데， 이때，사료원료로서는곡물류，조강류，식물성유박류,동물성사료원료,기타 사료원료，정제품등을들수있다.
[115] 특히,본발명의항균활성이부가된누에가함유된동물용사료를동물에게 일상적으로섭취시킬경우병원성미생물에대한감염등올예방할수도있게 된다.
[116] 이하,본발명에대하여실시예및실험예를통하여상세히설명하나，이들이본 발명의범위를제한하는것은아니다. [117] <실시예 1및비교예 >항균활성이부가된면역유도누에생산을위한주사제 선발및제조
[118] 누에체액성면역반응을유도하기위한주사제로인체및동물에안전하고
그람양성균과유사한세포막구조를지닌유산균 (Lactobacillus plantarum)을 배양하여사용하였다.
[119]——즉 항균성—이-강화된ᅳ누에ᅳ생—산을ᅳ위해서—면—역유도一후 —물¾^쨍ᅳ균 7¾1쥰 세포벽및유산균파쇄액추출물을제조하여다양한농도로누에체강에주사한 다음면역유도시간에따른항균활성검정을통하여면역유도효율분석을 실시하였다.
[120] 1)실시예 1;유산균파쇄액추출물제조 (도 1에제시됨)
[121] 초음파분해및부탄올추출을통하여세포막성분인
펩티도글리칸 (Peptidoglycan)및리포테이코익산 (lipoteichoic acid, LTA)이포함된 추출물을제조하였다. ,
[122] 즉，락토바실러스 MRS액체배지에서배양된유산균을 4에서 8,000rpm으로
10분간원심분리를통하여균체를획득한다음, 0.1M sodium citrate buffer(pH 4.7) 50 ml를넣어현탁한뒤,초음파분해 (sonication)를 1시간동안수행하여세포를 파쇄하였다.
[123] 상기파쇄액에서단백질을제거하기위해서동일한부피의부탄을을첨가한 후,실은 (24)에서 30분간교반을통하여추출한다음， 8,000rpm에서 30분간 원심분리하여분획하였다.
[124] 상등액을버리고하등액만획득한다음동일한부피의부탄을을넣고위와
같은추출을반복하였다.
[125] 단백질이제거된세포파쇄물은 -40에서 24시간동안동결건조하였다.
[126] 상기동결건조물을멸균된증류수 10 ml를넣고녹인다음，이를투석막에넣고
3차증류수에서 60시간동안투석하였고， 3차증류수는 12시간마다교환하였다.
[127] 투석을마친샘플은 -40에서 24시간동안동결건조하여무게를측정하였다.
[128] 측정된상기동결건조물은 15， 10, 5, 1및 0.5mg/ml등다양한농도로멸균된 식염수에녹여누에면역유도시험에사용하였다.
[129] 2)비교예:유산균세포벽분리
[130] 락토바실러스 MRS액체배지에서배양된유산균을원심분리를통하여균체를 획득한다음,동결건조하였다.
[131] 동결건조된균체를 10배량의살균된증류수에현탁한후 100에서 20분간
가열한다음 l,500g에서 10분간원심분리하여파괴되지않은균을제거하였고， 상등액을다시 6,500g에서 30분간원심분리하였다.
[132] 침전된세포벽을수집하여증류수로 3회세척한다음동결건조하여무게를 측정한후멸균된식염수에일정한농도로녹여누에면역유도시험에
사용하였다.
[133] [134] <실시예 2>항균활성이부가된면역유도누에대량생산및건조분말제조 [135] 면역유도누에대량생산을위하여동결건조된유산균파쇄액
추출물 (실시예 1)을 lmg/ml농도로멸균된식염수에녹여주사액을제조하였다.
[136] 뽕잎으로사육된 5령 5일째누에유층은 10ᅳ 15분간침수시켜비활성화시킨 다음,도 2에서보는바와같이자동주사기를이용하여주사액 50ul를배면을 통한체강주사로면역화하였다.
[137] 항균활성이증가된면역유도누에를대량생산올위하여약 6만마리누에를 대상으로면역유도용주사액을접종하였다.
[138] 주사접종한누에는뽕잎을급이하여 18-24시간동안면역유도한다음
-20에서냉동보관하였다.
[139] 임의로선별한면역유도된누에로부터체액을채취하여상기의방법으로
체액성단백질을분리한후,대장균에대한항균활성검정을통하여면역유도 유무를확인하였다 (도 3).냉동보관된면역유도누에는 70에서 24시간동안열풍 건조한다음분말화하였다.이후항균활성검정에사용하였다.
[140] <실험예 1>누에면역유도용주사제선발올위한항균활성검정
[141] 1)실험방법
[142] 누에면역유도주사제선발을위하여다양한농도의유산균 (대조구),유산균 세포벽 (비교예)및유산균파쇄액추출물 (실시예 1)을누에유층체강에주사하여 면역유도한후유도시간별로누에체액을채취하여항균활성을검정하였다.
[143] 즉,후보유도물질이함유된 50수용액을누에 (금옥잠) 5령 5일째유층배면을 통하여체강에주사접종한후유도시간별 (6시간, 10시간, 22시간)로각각유층 10마리로부터체액을채취하였다.
[144] 체액은 90에서 10분간열처리한다음 13,000rpm, 4조건에서 10분간원심분리 후상등액을분리하여항균활성검정에사용하였다.
[145] 항균활성검정은방사선확산분석법 (radial diffusion assay)을통하여병원성 대장균 (E. coli KACC 1039)에대한항균활성을배지상에서가시화하여병원균 생육억제를나타내는 Inhibition zone의크기로비교분석하였다.
[146] 2)실험결과
[147] 상기실험결과도 4에나타나있듯이,생균주사접종의경우대체로매우낮은 항균활성을나타냈었으며,유산균세포벽주사접종에서는주사농도 0.5mg/ml 이상인경우높은치사율을보였으며 0.5mg/ml이하농도에서는치사율은 감소하였지만낮은항균활성을나타냈었다.
[148] 또한，유산균파쇄액추출물주사접종시 5~10mg/ml주사농도에서 10시간 경과후가장높은항균활성을보였다.
[149] 따라서누에면역유도를위한유도물질로유산균파쇄액추출물이가장우수한 것으로확인되었다.
[150] 즉,항균성이증가된누에를생산하기위한누에면역유도용주사제로
유산균 (Lactobacillus plantarum)파쇄액추출물이적합함을알수있었다. [151] <실험예 2>면역유도누에대량생산을위한유도조건구명
[152] 1)실험방법
[153] 최적의누에면역유도조건을수립하기위해서누에면역유도용주사제로
선정된유산균파쇄액추출물 (실시예 1)의주사농도및유도시간에따른 면역유도효율을대장균에대한항균활성분석을통하여검정하였다.
[154] 즉^ Γ면—역—유도ᅭ주자 Γ"^산—균¾—액—추출불 ^T0^7r ^mg/mT¥로^ 멸균된식염수에녹여주사액을제조한다음,각농도별주사액 50을
누에 (금옥잠) 5령 5일째유층배면을통하여체강에주사접종하였으며면역유도 후 6， 10, 18및 22시간경과에따라각각유충 10마리로부터체액을채취하여 대장균에대한항균활성검정에사용하였다.
[155] 채취된체액은 90에서 10분간열처리한다음 13,000rpm, 4조건에서 10분간 원심을통하여체액성단백질을추출하였으며이후항균활성검정에
사용하였다.항균활성검정은방사선확산분석법 (radial diffusion assay)을통하여 병원성대장균 (E. coli KACC 1039)에대한항균활성을배지상에서가시화하여 병원균생육억제를나타내는 Inhibition zone의크기로비교분석하였다.이상의 실험은 3회반복하여수행하였다.
[156] 2)실험결과
[157] 도 5에나타나있듯이 , 5mg/ml와 lmg/ml주사농도에서가장우수한
항균활성을보였으면주사접종후 18시간면역유도된시료에서가장높은 항균활성을나타냄을확인하였다.
[158] 따라서항균성이증가된면역유도누에생산올위해서면역유도주사제의적정 농도는 lmg/ml로면역유도시간은 18시간으로결정하였다.
[159] <실험예 3>면역유도누에분말의항균활성검정
[160] 1)실험방법
[161] 실시예 2에서제조된건조된면역유도누에분말에대한항균활성검정은가금 질병및위장관손상관련병원균인살모넬라균 (Salmonella Enteritidis)및 대장균 (Escherichia coli)을대상으로실시하였다.
[162] 먼저누에분말로부터항균성단백질이포함된추출물을제조하였다.
[163] 즉,누에분말에 10배부피의 0.3%아세트산용액을넣어현탁한뒤,실온에서
1시간동안교반을통하여추출한다음 4,000rpm에서 30분간원심분리하여 상등액을획득하였다
[164] 상기분리된상등액은 90에서 10분간열처리한다음 13,000rpm, 4조건에서 10분간원심분리하여항균성단백질이포함된추출물을획득하였다.
[165] 분리된추출물은 90동결건조하여무게를측정한다음멸균된 0.01%아세트산 용액에일정한농도로녹여항균활성검정에사용하였다.
[166] 살모넬라균 (S. Enteritidis)및대장균 (E. coli)을 LB배지에서배양하여얻은 1 105 세포와다양한농도의누에분말추출물을섞은후 37에서 2시간동안배양한 다음，배양액을 LB아가풀레이트에도말하여균주를가시화시켰다. [167] 2)실험결과
[168] 도 6에나타나있듯이 ,살모넬라균 (S. Enteritidis)에 0.01%아세트산을처리한 대조구에서는많은수의콜로니를발견할수있었으나 2000ppm농도의 면역유도누에분말추출물을첨가한경우에균이조금자랐으며 4000ppm 농도의경우에는균의성장이완전히억제되어콜로니가보이지않음을 확인하였다.
[169] 또한，도 7에나타나있듯이,대장균 (E. coli)에 0.01%아세트산을처리한
대조구에서는많은수의콜로니를발견할수있었고 2000ppm농도의면역유도 누에분말추출물을첨가한경우에는균의성장이완전히억제되어콜로니가 보이지않음을확인하였다.
[170] 상기의결과로부터，본연구에서생산된면역유도누에분말은가금질병및 위장관손상을유발하는살모넬라균 (S. Enteritidis)및대장균 (E. coli)에탁월한 항균활성을나타냄을확인하였다.
[171] 이상에서설명한바와같이본발명에의해，누에의체액성면역반웅으로
생성되는항균성단백질발현을유도하는누에면역반응유도용조성물및이에 의해제조되어항균활성이부가된누에가제공됨에따라동물성장촉진용 천연항생제및세균성질환예방사료첨가제의개발이가능하게됨을알수 있었다.
[172]
[173] 또한,본발명의다른실시예를설명하면다음과같다.
[174] 도 8은본발명의실시예에따른항균펩타이드가강화된누에를함유하는
조성물의제조방법을나타낸흐름도이다.본실시예의조성물은사료조성물， 식품첨가물,화장품첨가물，어류등의양식장사료조성물등이될수있다. 이하에서는설명의편의상조성물이사료조성물인경우를중심으로설명한다.
[175] 본실시예의사료조성물은닭,오리，거위,꿩 ,돼지，소,염소,개및고양이등 동물의사료가될수있다.
[176] 누에에는세크로핀 (cecropin),가바 (-aminobutyric acid, GABA),투틴 (rutin)등 다양한성분의생리활성물질이존재하며，항균，항산화，간및신장기능개선， 독성방지，피로회복,신경안정등다양한생리활성효과를발휘한다.특히 누에의항균펩타이드성분인세크로핀,엔보신 (enbocin)등의항균,항산화， 항바이러스,살충,세포재생，면역조절효과는커서그산업적이용가치가매우 크다.
[177] 또한，본실시예에따른사료조성물은분말또는소정의부피를가진팰렛 형상으로구현될수있으며,누에는분말또는액상으로사료조성물에첨가될 수있다.이하에서는누에가분말화되어사료조성물에첨가되는경우를 중심으로설명한다.
[178] 이하에서서술할각단계는사료조성물제조장치가주체가되어수행할수 있다.각단계별로기술되는과정은반드시시계열적인순서대로수행될필요는 없으며,각단계의수행순서가바뀌어도본발명의요지를층족한다면이러한 과정은본발명의권리범위에속할수있음은물론이다.
[179] 단계 S110에서,면역유도물질을주입한누에를분말화한다.여기서，누에 면역유도를위한주사제개발을위해서병원성그람양성균과유사한세포벽 구조를지닌유산균 Lactobacillus plantarum을배양하여사용하였다.즉,면역 유도원후보물질로 L. plantarum생균， L. plantarum cell wall추출물 (pCW)및 세포막성분인 lipoteichoic acid(LTA)가포함된 L. plantarum n-butanol 추출물 (pLTA)을제조하여누에면역유도물질선발에사용하였다.
[180] L. plantarum cell wall(pCW)은다음방식에따라제조하였다.먼저，동결건조된 균주를살균된증류수에녹인후 100에서 20분간가열한다음 l,500g에서 10분간 원심분리하여파괴되지않은균을제거하였고,상등액을다시 6,500g에서 30분간원심분리하였다.침전된 cell wall을수집하여증류수로 3회세척한다음 동결건조하였고,이후 LTA추출및누에면역유도시험에사용하였다.
[181] 또한， L. plantarum LTA추출물 (pLTA)은다음방식에따라제조하였다. L. plantarum cell well로부터 n-butanol추출법을사용하여 LTA가함유된추출물을 제조하였다.동결건조된 cell well을 αΐΜ sodium citrate buffer(pH 4.7)에녹인후 초음속파쇄기로분쇄하였고，동일한양의 n-butan0I을첨가한다음실은에서
30분간교반을통하여추출하였다.추출후 10,000 rpm에서 30분간원심분리한 다음상등액을회수하였고, evaporate및살균된증류수로투석한다음동결 건조하였다.동결건조된 LTA가함유된추출물은무게를측정한다음,살균된 식염수에녹여누에면역유도시험에사용하였다.
[182] 도 9를참조하면，다양한농도의 L. plantarum생균， cell wall추출물 (pCW)및 LTA추출물 (pLTA)이함유된 50ul수용액을 5령 5일누에 (금옥잠)체강에주사 접종한다음,면역유도시간 (6시간, 10시간, 22시간)별누에체액을채취하여 대장균에대한항균활성을검정하였다.
[183] 항균활성분석결과，생균주사접종의경우대체로매우낮은항균활성을 나타냈었으며, cell well주사면역유도에서는주사농도가 0.5mg/ml이상인경우 높은치사율을보였으며 0.5mg/ml이하농도에서는치사율은감소하였지만낮은 항균활성을나타냈었다. pLTA주사접종시 5~10mg/ml주사농도에서 10시간 경과후가장높은항균활성을보였다ᅳ따라서누에면역유도를위한유도물질로
L. plantarum LTA추출물 (pLTA)이가장우수한것으로확인되었다.
[184] 도 10을참조하면，최적의누에면역유도조건을수립하기위해서선정된 L. plantarum LTA추출물 (pLTA)의접종농도및유도시간에따른항균활성을 검정하였다. 10, 5, 1, 0.5mg/ml농도로누에체강에주사접종하였으며면역유도 후 6， 10, 18， 22시간경과에따라누에혈림프를채취하여대장균에대한 항균활성검정에사용하였다.항균활성분석에서 5mg/ml와 lmg/ml농도에서 가장우수한항균활성을보였으며주사접종후 18시간면역유도된시료에서 가장높은항균활성을나타냈었다.따라서누에면역유도를위한주사조건으로 주사농도는 lmg/ml,면역유도시간은 10시간초과 22시간미만으로결정할수 있다.
[185] 면역유도물질은성장하고있는누에애벌레에주입될수있다.본실시예는 누에애벌레에면역유도물질을주입하기위한장치를구비할수있다.예를 들면，주입장치는주사액이주입될애벌레가놓이는부분인애벌레지지부, 애벌레지지부에놓인애벌레에면역유도물질을포함하는주사액을주입하는 주사액주입부，애벌레에게주사액을주입시키기편리하도록애벌레를 비활성화시키는애벌레비활성화부，주사액주입부에의해주사액이주입되어, 주입처리가완료된애벌레를보관하는수단인애벌레저장부를포함할수있다.
[186] 여기서，애벌레비활성화부는차가운물을담은수조가될수있으며 ,애벌레는 차가운물에담기는경우순간적으로기절하는등비활성화되므로，애벌레 지지부는움직임이둔한애벌레는수월하게지지할수있다.
[187] 단계 S112에서,면역유도물질을주입한누에는열풍건조법으로건조시킬수 있다.열풍건조법은소정의온도를가진열풍을이용하여누에를건조시키는 방법이다.
[188] 또한，다른실시예에따르면，단계 SU4에서，면역유도물질을주입한누에는 마이크로웨이브건조법으로건조시킬수있다.
[189] 열풍기의온도별 (60, 70, 80, 90)과마이크로웨이브오본을이용하여
누에 (1.8kg)를투입하여매시간질량을측정하여더이상중량의변화가없는 상태 (평형점)의최종시간과중량을측정한후，온도별일반성분의차이 분석 (분석항목：수분，회분,조지방,조단백，조섬유)과항균력시험을
시행하였다.
[190] 도 11을참조하면，열풍건조법에의한온도별 (60, 70, 80， 90)누에상태를
비교하였다ᅳ초기중량 1800g의누에를열풍건조하였을때,평형점이나타난 시점은 60에서대략 34시간의시간이소요되었으며 , 70에서 26시간， 80 25시간, 90에서는 24시간이소요된것으로나타났다.또한,평형점에도달하였을시，그 중량은 60에서 319.9g, 70에서 329g, 80에서 323g, 90에서 315g인것으로 나타났다.이는총중량 1800g에서평형점에도달하였을시，평균중량이
17%임을나타낸다.
[191] 도 12를참조하면，마이크로웨이브를이용할때평형점이나타난시점은대략 40분정도이다.또한，은도별열풍건조와마이크로웨이브오븐을이용한 건조누에의일반성분을분석해본결과그유의차가나타나지않았다.따라서 단기간에가장효율적인건조방법은마이크로웨이브를활용한방법인것으로 분석된다.
[192] 표 1과표 2를참조하면,일정시간이지난후,건조누에의일반성분을분석한 결과 (유의차검정 )，조성비율간유의차가없는것으로나타났다.이는누에의 열풍건조시시간과건조비용을고려할시 60의낮은온도보다 90에시행되는 누에건조가경제성이높은것으로나타난것임을알수있고,종합적으로는열풍 보다마이크로웨이브를활용한건조가더효율적이라는것을알수있었다. 수분보정후의성분분석에서도비슷한결과를나타낸다.
[193] 표 1
[Table 1]
Figure imgf000017_0001
[194]
[195] 표 2
[Table 2]
Figure imgf000017_0002
[196] 또한,각분말시료 0.5^1으3%아세트산 5ml을첨가하고,실온에서 1시간동안 교반한후 10,000rmp, 4조건에서 20분간원심분리후상등액분리하고，상등액을 lml로농축한다음항균활성검정을수행하였다.
[197] 항균활성검정은방사선확산분석법 (RDA)을통하여병원성대장균 (E. coli
ML35)에대한항균활성분석하였으며,항균활성비교분석을위해서，병원균 생육억제를나타내는 Inhibition zone의크기로비교분석하였다.
[198] 도 13을참조하면, Zone크기를비교분석한결과마이크로웨이브 (MW)로 건조한시료에서항균활성이가장높고열풍건조에따른시료는온도에따라 다른결과를나타내었다.
[199] 상술한바에따르면，누에의건조에열풍을활용할경우，누에분말의
항균활성을높이려면낮은온도로적용을해야하지만,건조시간이길어져 건조비용의중가를초래할수있다.하지만,마이크로웨이브를활용할경우， 누에분말의항균활성이높게나타나고,건조시간도열풍과비교하여크게 감소하는바，열풍건조보다마이크로웨이브를이용한누에건조가더
효율적이라고분석된다.
[200] 단계 S120에서，분말화된누에를사료에첨가하여사료조성물을제조한다.이 경우누에분말은미량원료를사료에군일하게첨가하기위하여예비
배합하는데쓰이는물질인부형제 (carrier,浮衡劑)와섞일수있다.여기서, 부형제는약제에적당한굳기나형상올주기위해서，또는주제 (主齊 1ᅵ)의양이 적은경우에일정용량,중량을주어취급하기쉬운크기로할목적으로 첨가되는성분으로서，말분,석회석,분쇄옥수수속대,옥수수글루텐피드，대두 mill run등이될수있다.
[201] 여기서,전체사료조성물 100중량부에대하여，누에 1내지 50중량부,부형제 50내지 99중량부가포함될수있다.
[202]
[203] 이상에서항균펩타이드가강화된누에를함유하는조성물의제조방법을
설명하였으며，이하에서는첨부도면을참조하여，본발명에따른
항균펩타이드가강화된누에를함유하는조성물의효과를실험한결과를 설명한다.본발명이이러한실시예에한정되지않음은물론이다.
[204]
[205] 본실시예에따른사료조성물의성능을시험하기위하여 ,닭에대한누에유래 항균펩타이드함유사료조성물첨가급여를시험하였다.닭에대해개발된누에 분말함유사료조성물의첨가급여효과를조사하기위하여 1일령육계 병아리를대상으로 5주간사양시험을실시하였다.비교예 1은항생제무첨가구, 비교예 2는항생제 (아빌라마이신 10ppm+살리노마이신 60ppm)첨가구， 실시예는일반누에분말을 0.01， 0.05, 0.10또는으50%수준으로첨가한실시예 1, 2， 3, 4와면역유도물질을주입하여항균펩타이드를생산을유도한누에 분말을 .01， 0.05， 0.10또는 0.50%수준으로첨가한실시예 5, 6, 7， 8을두었다. 모든실시예에는사료용항생제및항콕시듐제를첨가하지않았다.
[206] 표 3
[Table 3]
Figure imgf000019_0001
[207] 표 4는육계생산성을비교한것이다.모든실시예에서비교예 1에비해 5주 종료체중및증체량이 0.95~3.54 %증가하였다.톡히면역유도에의해 항균펩타이드가과발현된실시예 5, 6， 7， 8의경우일반누에분말의급여한 실시예 1， 2， 3, 4에비해높은체중증가효과가나타났다.사료요구율역시 비교예 1에비해누에분말을급여한모든실시예에서 2ᅳ82~5.08 «¾가량 개선되었다.이를통해본발명에따른누에유래항균펩타이드함유
사료조성물은증체량및사료요구율개선을통해생산성향상효과가우수하며, 생산성측면에서적정첨가수준은일반누에분말은 0.01~0.50 ,면역유도누에 분말은 0.01 ~0.05 %내외인것을확인할수있었다.
[208] 표 4
[Table 4]
Figure imgf000020_0001
[209] 육계에대한누에및면역유도누에분말의생체안전성을확인하기위해
장기의상대적증량과혈액생화학조성을조사한결과，육계에 5주간
0.01-0.50%수준으로사료내첨가급여하여도간，신장등주요장기발달과 혈액내간및손상지표에영향을미치지않는다는것을확인할수있었다.
[210] 표 5는육계주요장기의상대적중량을비교한것이다.일반누에분말및
면역유도누에분말의사료내첨가급여가육계의주요장기발달 (간,비장,췌장, 신장， F낭)에미치는영향을조사한결과,비교예 1, 2를비롯한전실시예에서 차이가관찰되지않았으며，육계에일반누에분말및면역유도누에분말을 5주간사료내 0.01~0.50%수준으로첨가급여하여도주요장기발달에는영향을 미치지않는다는것을확인할수있었다.
표 5
[Table 5]
Figure imgf000021_0001
[212] 표 6은혈액내생화학조성을비교한것이다.간및신장의손상여부를
나타내고신규 (사료첨가제의독성여부를판단할수있는지표로이용되는 요소질소 (BUN, blood urea nitrogen),크레아티닌 (creatinine),총단백질 (total protein),알부민 (albumin),글로불린 (globulin), AST(aspartate aminotransferase)를 조사한결과，실시예에있어서비교예 1에비해간및신장손상지표인 AST및 ALT수치가감소하였다.그외요소질소，크레아티닌，총단백질,알부민， 글로불린에서는큰차이가관찰되지않았다.이를통해누에유래항균펩타이드 함유사료조성물이항산화,항염증작용을통해세포및조직의손상을 감소시키는한편생체면역반웅을조절하고가축에독성이나부작용역시 나타내지않아사료첨가제로서사용할수있음을확인할수있었다.
[213] 표 6
[Table 6]
Figure imgf000022_0001
[214] 표 7은맹장내미생물총변화를비교한결과이다.장내유해균인대장균및 살모넬라ᅳ수가일반누에분말및면역유도누에분말함유사료조성물을첨가 급여한모든실시예에서비교예 1에비해감소하는것을확인하였다.또한장내 유익균인젖산생성균에는영향을미치지않아장내미생물총안정화에 긍정적인영향을미치는것을확인할수있었다.
[215] 표 7
[Table 7]
Figure imgf000022_0002
[216] 표 8은혈액내항산화및스트레스관련인자를비교한결과이다.혈액 총항산화활성은모든실시예가비교예에비해높은활성을보였으며누에 분말의첨가수준이증가할수록총항산화활성증가하는경향을나타냈다. 대표적인스트레스호르몬으로알려진코티졸함량을분석한결과，비교예 1에 비해모든실시예에서감소하는결과를확인할수있었다ᅳ스트레스，질병감염， 염증반응시다량분비되는코티졸은기초대사율,단백질합성및분해, 면역반웅，체내항상성등에영향을미쳐가축건강은물론장기적으로 생산성에도부정적인영향을미칠수있다.누에분말은항생제와유사하게혈액 내코티졸함량을낮추어가축건강은물론생산성에긍정적인영향을미친다는 것과스트레스제어효과가있음을확인할수있었다.
[217] 표 8
[Table 8]
Figure imgf000023_0001
[218] 누에분말첨가급여가육계백혈구조성에미치는영향을표 9에제시하였다. 급성감염이나염증반웅시증가하는다핵구 (heterophil)는물론스트레스지표로 사용되는다핵구 /림프구비율은일반누에분말및면역유도누에분말을첨가 급여한실시예에서항생제무첨가구인비교예 1에비해유의하게감소하거나 감소하는경향을보였다.본발명에따른누에유래항균펩타이드함유 사료조성물은장관내대장균,살모넬라등의유해균증식및이들의독소 분비를억제하여이들에대한생체면역반웅을감소시킨것으로판단된다.
[219] 표 9
[Table 9]
비교 비교 실시 실시 실시 실시 실시 실시 실시 실시 예 1 예 2 예 1 예 2 예 3 예 4 예 5 예 6 예 7 예 8 백혈구 (K/ L) 23.04 20.58 23.12 23.72 22.46 24.36 24.36 26.64 24.34 29.42 다핵구 (K/ L) 8.16 7.05 7.69 7.93 7.09 7.63 7.63 7.82 7.10 9.10 림프구 (K/ L) 11.94 11.45 11.88 12.16 11.13 12.74 12.74 13.48 12.83 15.22 다핵구 /림프구 0.68 0.62 0.65 0.65 0.64 0.60 0.60 0.58 0.55 0.60 [220] 그외본발명의실시예에따른항균펩타이드가강화된누에를함유하는 조성물및그제조방법에대한구체적인구성물，제조장치등에대한구체적인 설명은본발명이속하는기술분야의통상의지식을가진자에게자명한 사항이므로생략하기로한다.
[221]
[222] 또한,본발명의다른실시예를설명하면다음과같다.
[223] 도 14는본발명의실시예에따른애벌레에주사액을주입하는장치의블록 구성도이다.도 14를참조하면,주사액주입장치 (100)는애벌레지지부 (101), 애벌레이동부 (102),주사액주입부 (103),애벌레분리부 (104),애벌레
비활성화부 (105)，애벌레저장부 (106),제어부 (107)를포함할수있다.
[224] 본발명은천연항생제를대량으로생산하기위하여항생제로사용될수있는 애벌레에면역유도물질을주입하는특징이있다.구체적으로본실시예는 항균물질을포함하는기능성애벌레,예를들면,누에를사료첨가제로활용하기 위하여，누에애벌레에면역유도물질을주입하는장치를제공한다.
[225] 본실시예는일반적인애벌레 (밀엄,동애등에,누에,굼뱅이등)에적용가능하며, 이하에서는설명의편의상누에애벌레를대량으로생산하기위한장치를 중심으로설명한다.애벌레로부터천연항생제를생성하기위한분말처리등의 공정절차는본발명이속하는기술분야의통상의지식을가진자에게자명한 사항이므로，이에대한설명은생략한다.
[226] 애벌레지지부 (101)는주사액이주입될애벌레가놓이는부분이다.애벌레 지지부 (101)는후술할주사액주입부 (103)가애벌레에주사액을주입하기 위해서애벌레를특정위치에고정시킨다.
[227] 애벌레지지부 (101)는애벌레를지지할수있는구조라면，특정한형태에
한정되지않고본발명에적용가능하다.예를들면,애벌레지지부 (101)는 애벌레를평면형상의지지대위에놓고,주사액주입부 (KB)가위치한반대 방향에주사액주입부 (102)의푸쉬에의한애벌레의이동을차단하는차단 수단이마련되어애벌레를고정시킬수있다.
[228] 애벌레지지부 (101)에는애벌레가삽입되는소정의홈이형성될수있다.이 경우애벌레지지부 (101)의개수는복수가될수있으며，각각의애벌레 지지부 (101)에애벌레가한마리씩삽입될수있다.
[229] 애벌레이동부 (102)는애벌레지지부 (101)를소정의방향으로이동시킨다. 예를들면，애벌레지지부 (101)는상술한바와같이각각의유닛에홈이 형성되고,각유닛이형성하는전체형상은체인또는캐터필러형상이될수 있으며，애벌레이동부 (102)는애벌레지지부 (101)를회전시킬수있다.예를 들면,애벌레이동부 (102)는모터가될수있다.
[23이 주사액주입부 (103)는애벌레지지부 (101)에놓인애벌레에주사액을
주입한다.주사액주입부 (103)는주사액을내부에수용하는주사기를포함할수 있다주사액주입부 (103)는애벌레지지부 (101)에놓인애벌레의특정부분에 주사액을주입할수있다ᅳ예를들면，주사액주입부 (103)는애벌레의배면부 체강에주사액을주입할수있다.주사액이주입되는부분은애벌레의
배면부에서,전체높이의 80%부위까지의부분이될수있다.
[231] 도 18을참조하면，껍질을벗긴누에애벌레가핀에꽂힌상태가도시된다.도 18의 (a)는소화기관인내장 (410)이애벌레체강 (420)의등부위에위치한상태를 도시하며,도 18의 (b)는내장 (410)을애벌레체강 (420)의등부위에서벗겨낸 상태를도시한다.정상적인상태에서내장 (410)은애벌레의등부위에
위치하므로，주사바늘은애벌레의배면부에서내장 (410)위치의아래부분,즉, 애벌레의밑면인배면부와그로부터전체몸통두께의 80%가되는체강 (420)에 삽입되어주사액을주입할수있다.
[232] 주사액은면역유도물질을포함할수있다.면역유도물질은생체에독성이 없으면서면역능력을조절하는효과를가지는물질이될수있다.예를들면， 면역유도물질은생체반응조절물질 (BRM： Biological Response Modifier)이될 수있으며，구체적으로， AHCC(Active Hexose Correlated Compound),
Polysaccarides, Polysaccaride peptides, Nucleosides, Triterpenoids등이될수있고, 이외에도다양한물질이면역유도물질로서본발명에적용될수있음은 물론이다.
[233] 애벌레분리부 (104)는주사액이주입된애벌레를애벌레지지부 (101)로부터 분리시킨다.애벌레분리부 (104)는애벌레를상기홈의연장방향으로밀어서 애벌레지지부 (101)로부터분리시키거나,상기홈의상면을중력이향하는 방향으로변경하여애벌레를중력방향으로떨어뜨림으로써애벌레
지지부 (101)로부터분리시키는등다양한방식을이용할수있다.
[234] 예를들면,애벌레분리부 (104)는애벌레와결합하는애벌레흡착부 (미도시)및 애벌레가애벌레흡착부에흡착되도록애벌레흡착부의공기를펌핑하는흡착 펌핑부 (미도시)를포함할수있다.이러한실시예에따르면，공기펌핑에의한 흡착력을이용함으로써 ,다양한위치및방향에서애벌레를애벌레
지지부 (101)로부터분리시킬수있다.
[235] 여기서，흡착펌핑부는애벌레흡착부가애벌레와결합되어애벌레를애벌레 지지부 (101)로부터분리시키는경우공기의펌핑을중단하여애벌레를애벌레 흡착부로부터이탈시킬수있다.이후애벌레는후술할애벌레저장부 (106)에 낙하하여저장될수있다.
[236] 애벌레비활성화부 (105)는애벌레를애벌레지지부 (101)에놓기전에
애벌레에게주사액을주입시키기편리하도록애벌레를비활성화하는수단이다. 예를들면，애벌레비활성화부 (105)는차가운물을담은수조가될수있으며, 애벌레는차가운물에담기는경우순간적으로기절하는등비활성화되므로, 애벌레지지부 (101)는움직임이둔한애벌레는수월하게지지할수있다.
[237] 또한,다른실시예에따르면,애벌레비활성화부 (105)는얼음,냉장고，마취제 등이될수도있다. [238] 애벌레저장부 (106)는주사액주입부 (103)에의해주사액이주입되어，주입 처리가완료된애벌레를보관하는수단이다.예를들면,애벌레저장부 (106)는 애벌레를담는용기가될수있으며,애벌레지지부 (101)로부터이탈한애벌레가 애벌레저장부 (106)로이동하여보관될수있다.
[239] 여기서,애벌레가애벌레저장부 (106)로이동하는방법은다양하게구현될수 있으며,예를들면，애벌레지지부 (101)가상술한바와같이애벌레를평면 형상의지지대위에놓는구조로형성된경우해당평면이기울어지거나 측면으로이동하여애벌레가아래방향으로떨어지게함으로써애벌레를 애벌레저장부 (106)로이동시킬수있다.또한，다른실시예에따르면，애벌레 지지부 (101)가진공펌프로애벌레를흡착하여지지하는경우진공펌프의 작동이정지하여애벌레를직하방향으로낙하시킴으로써애벌레저장부 (106)로 이동시킬수도있다.
[240] 애벌레저장부 (106)는애벌레분리부 (104)의하방에위치할수있으며 ,
애벌레는주사액주입처리가완료된후애벌레저장부 (130)로낙하하여처리후 애벌레로서보관될수밌다.
[241] 여기서,애벌레비활성화부 (103)에놓인애벌레는특정푸쉬수단에의해
하나씩상술한애벌레지지부 (101)에놓일수있다.예를들면,푸쉬수단은관 형상에애벌레가일렬로수용된상태에서애벌레지지부 (101)방향으로 애벌레를한마리씩밀어서이동시킬수있다.
[242] 제어부 (107)는상술한애벌레지지부 (101)，애벌레이동부 (102)，주사액
주입부 (103)，애벌레분리부 (104),애벌레비활성화부 (105)，애벌레저장부 (106)가 서로연동하면서자신의고유한기능을수행하도록각기능부를제어한다. 제어부 (107)의구체적인기능은특정실시예에따라달라질수있다.
[243]
[244] 이상에서애벌레에주사액을주입하는장치를일반적으로도시한블록
구성도를설명하였으며，이하에서는첨부도면을참조하여 ,본발명에따른 애벌레에주사액을주입하는장치를구체적인실시예를기준으로설명하기로 한다.본발명이이러한실시예에한정되지않음은물론이다.
[245]
[246] 도 15는본발명의실시예에따른애벌레에주사액을주입하는장치의
측면도이고，도 16은본발명의실시예에따른애벌레에주사액을주입하는 장치의평면도이다.도 15와도 16을참조하면，애벌레지지부 (111)，애벌레 이동부 (112)，주사액주입부 (113),애벌레분리부 (114),장치지지부 (121), 애벌레 (220)가도시된다.이하에서는,상술한바와의차이점을위주로설명한다.
[247] 도 15및도 16을참조하면,주사액주입부 (113)가하나인경우가도시되지만, 본실시예는복수의주사액주입부 (113)를포함할수있다.이경우각주사액 주입부 (113)는후술할바와같이주사액을주입하는기능을독립적으로 수행하거나또는동일한시점및동작으로서로연동하여수행할수있다. [248] 본실시예는복수의애벌레지지부 (11 )가캐터필러형상으로결합하며 , 애벌레이동부 (112)에의해회전함으로써，애벌레지지부 (111)에결합한 애벌레 (220)를소정의방향으로이동시킨다.장치지지부 (121)는애벌레 지지부 (111)및애벌레이동부 (112)를포함한전체장치를지지할수있다.
[249] 주사액주입부 (113)는애벌레지지부 (111)의상측면에위치하며,주사액
주입부 (113)의전방에위치한애벌레에주사액을주입한다.주사액
주입부 (113)는주입되는주사액의양을조절할수있다.즉,주사액
주입부 (113)는애벌레마다동일한양의주사액을주입하거나또는애벌레의 크기등에상응하여미리설정되는양의주사액을주입할수있다.
[250] 여기서,애벌레이동부 (112)가이동하는방향과주사액주입부 (113)가
애벌레에주사액을주입하는방향은서로다른방향이될수있다.예를들면， 애벌레는애벌레지지부 (111)에병렬로지지되어측방향으로이동되며,주사액 주입부 (113)는애벌레가연장방향을향하여주사액을주입할수있다.
[251] 애벌레분리부 (114)는주사액이주입된애벌레를애벌레지지부 (i n)로부터 이탈시켜서상술한바와같이애벌레저장부 (106)에저장시킨다.
[252] 도 17은본발명의실시예에따른애벌레에주사액을주입하는장치중주사액 주입부의측면도이다.도 17을참조하면，상부프레임 (110)，실린더부 (116)， 시린지부 (118)，주사액유입관 (119)，하부프레임 (120)이도시된다.
[253] 주사액주입부 (113)는프레임으로몸체가형성되며，구체적으로，지면과
평행한상부프레임 ( 110),상부프레임 ( 110)을지지하는하부프레임 ( 120)이 몸체를형성할수있다.
[254] 애벌레는상술한배면부가애벌레지지부 (111)에견고하게결합할수있다.이 경우주사바늘은애벌레의체강부분을찔러서상술한바와같이배면부체강에 주사액이주입되도록할수있다.
[255] 여기서，본실시예에다른주사바늘은통상적으로내부에관통홀이형성된 주사바늘이거나또는외부에흠이형성되어주사액이수용되는바늘이될수 있다.후자의경우,주사바늘의외면에나선형의홈이형성되거나，주사바늘의 연장된방향과다른방향으로연장되며서로소정의간격을가지는복수의홈이 형성되거나,주사바늘의연장된방향과같은방향으로연장되며서로소정의 간격을가지는복수의훔이형성되거나,소정의패턴，예를들면, ' W'형상등의 패턴을가지도록연장되는홈이형성될수있다.이외에도본실시예는주사 바늘의외면에오목한볼형상의홈이형성되는등다양한형태의홈이형성될 수있다.이경우상술한홈에주사액이수용되어통상의주사바늘이삽입될수 없는애벌레에도홈에수용된주사액을애벌레의내부에주입하거나또는 외부에묻힐수있다.
[256] 그리고짧은시간에많은애벌레에주액액을주입하기위해복수이상의
주사바늘로구성될수있다.
[257] 또한，다른실시예에따르면,주사액주입부 (113)가애벌레에주사액을 정확하게주입하도록애벌레를고정시키는애벌레고정부 (미도시)를더포함할 수있다.예를들면,애벌레고정부는애벌레지지부 (111)가주사액
주입부 (113)의전방등에위치하는경우애벌레가애벌레지지부 (111)에의해 지지된상태에서애벌레에결합하여애벌레가움직이지못하도록함으로써， 주사바늘이애벌레에정확하게삽입되도록한다.애벌레고정부는주사액 주입부 (113)와결합할수있으며,상하운동이가능한구조로형성될수있다. 또한，애벌레고정부는애벌레의크기및 /또는형상에상응하여연장된애벌레 접촉부 (미도시)를포함할수있다.애벌레접촉부는애벌레와실제접촉하는 수단으로서，애벌레를애벌레지지부 (111)에밀착고정시킴으로써,애벌레에 주사액이정확하게주입되도록할수있다ᅳ
[258] 실린더부 (116)는일단에시린지부 (118)와결합하는내부피스톤이왕복
운동하여애벌레흡착부 (114)에결합된애벌레에주사액이주입되도록한다. 실린더부 (116)의길이및시린지부 (118)의푸쉬길이등은애벌레에주사액이 주입될수있는정도로특정될수있다.
[259] 실린더부 (116)의작동방식은수동또는자동으로정해질수있다.예를들면， 시린지부 (118)를애벌레방향으로밀어내는푸쉬버튼을구비하여사용자가 해당푸쉬버튼을누르는경우실린더부 (116)가작동하여시린지부 (118)가미리 정해진길이만큼애벌레에게이동하고주사액을주입한후다시뒤로돌아와서 초기상태로정지할수있다.
[260] 또한,다른실시예에따르면，애벌레지지부 (111)의움직임에연동하여
실린더부 (116)가작동하여애벌레에게주사액올주입할수있다.이경우 애벌레에안정적으로주사액을주입하기위해서，상술한제어부 (107)는 주사액이주입될애벌레가결합한애벌레지지부 (111)가시린지부 (118)의 전방에위치하는경우,바로또는특정시간,예를들면, 1내지 2초후에 시린지부 (118)가애벌레에게이동하도록제어할수있다.
[261] 시린지부 (118)는실린더부 (116)의일측에결합하며，일단에주사바늘이
결합하고몸체에주사액이수용되어애벌레에주사액을주입한다.
[262] 또한,본실시예는시린지부 ( U 8)와주사액유입관 (119)으로연결되며，
시린지부 (118)가애벌레에주사바늘을꽂을때주사액을시린지부 (Π8)에 펌핑하는주사액펌핑부 (미도시 )를더포함할수있다.
[263]
[264] 그외본발명의실시예에따른애벌레에주사액을주입하는장치에대한 구체적인시스템구성，임베디드시스템, O/S등의공통플랫폼기술과통신 프로토콜， I/O인터페이스등인터페이스표준화기술및엑추에이터,배터리, 카메라,센서등부품표준화기술등에대한구체적인설명은본발명이속하는 기술분야의통상의지식을가진자에게자명한사항이므로생략하기로한다.
[265]
[266] 본발명에따른애벌레에주사액을주입하는장치의동작방법은다양한 컴퓨터수단을통하여수행될수있는프로그램명령형태로구현되어컴퓨터 판독가능매체에기록될수있다.즉，기록매체는컴퓨터에상술한각단계를 실행시키기위한프로그램을기록한컴퓨터로읽을수있는기록매체가될수 있다.
Figure imgf000029_0001
명령은본발명을위하여특별히설계되고구성된것들이거나컴퓨터 소프트웨어당업자에게공지되어사용가능한것일수도있다.컴퓨터판독가능 기록매체의예에는하드디스크，플로피디스크및자기테이프와같은자기 매체 (Magnetic Media), CD-ROM, DVD와같은광기록매체 (Optical Media), 풀롭티컬디스크 (Floptical Disk)와같은자기-광매체 (Magneto-Optical Media),및 름 (ROM),램 (RAM),플래시메모리등과같은프로그램명령을저장하고 수행하도록특별히구성된하드웨어장치가포함된다.
[268] 또한，상술한각구성요소는물리적으로인접한하나의부품으로구현되거나 서로다른부품으로구현될수도있다.후자의경우각구성요소는인접하거나 또는서로다른구역에위치하여제어될수있으며，이경우본발명은각구성 요소를제어하는별도의제어수단또는제어실을구비하여유선또는무선으로 각구성요소를제어할수도있다.
산업상이용가능성
[269] 상기의본발명은바람직한실시예를중심으로살펴보았으며 ,본발명이
속하는기술분야에서통상의지식을가진자는본발명의본질적기술범위 내에서상기본발명의상세한설명과다른형태의실시예들을구현할수있을 것이다.여기서본발명의본질적기술범위는특허청구범위에나타나있으며， 그와동등한범위내에있는모든차이점은본발명에포함된것으로해석되어야 할것이다.

Claims

청구범위
[청구항 1] 단백질이제거된유산균파쇄액추출물을유효성분을포함하여 누에의체액성면역반옹으로생성되는항균성단백질발현을 유도하는，누에면역반웅유도용조성물.
[청구항 2] 제 1항에있어서,
상기단백질이제거된유산균파쇄액추출물은상기조성물전체 lml당 0.5-5 mg을포함하는，누에면역반웅유도용조성물.
[청구항 3] 제 1항에있어서,
상기유산균은락토바실러스플란타륨 (Lactobacillus plantarum)인 것이특징인,누에면역반응유도용조성물.
[청구항 4] 제 1항에있어서，
상기단백질이제거된유산균파쇄액추출물은세포막성분인 펩티도글리칸 (Peptidoglycan)및리포테이코익산 (lipoteichoic acid, LTA)이포함된것이특징인，누에면역반응유도용조성물.
[청구항 5] 제 1항에있어서,
상기단백질이제거된유산균파쇄액추출물은초음파분해로 상기유산균의세포를파쇄한후，부탄올로단백질을제거하여 얻은것이특징인,누에면역반웅유도용조성물.
[청구항 6] 면역유도물질을주입"한누에를준비하는단계;및
상기누에를첨가하여조성물을제조하는단계를포함하는 항균 ¾타이드가강화된누에를함유하는조성물제조방법 .
[청구항 7] 청구항 6에있어서，
상기조성물은부형제를더포함하며，전체조성물 100중량부에 대하여,상기누에 1내지 50중량부，상기부형제 50내지
99중량부인것을특징으로하는항균펩타이드가강화된누에를 함유하는조성물제조방법 .
[청구항 8] 청구항 7에있어서,
상기부형제는말분，석회석，분쇄옥수수속대，옥수수글루텐피드 및대두중어느하나이상을포함하는항균펩타이드가강화된 누에를함유하는조성물제조방법.
[청구항 9] 청구항 6에있어서,
상기누에는분말또는액상으로상기조성물에첨가되는것을 특징으로하는항균펩타이드가강화된누에를함유하는조성물 제조방법 .
[청구항 10] 청구항 6에있어서,
상기조성물은분말또는팰렛형상인것을특징으로하는 항균펩타이드가강화된누에를함유하는조성물제조방법 . 주사액이주입될애벌레가놓이는애벌레지지부;
상기애벌레지지부를소정의방향으로이동시키는애벌레 이동부;
'상기애벌레지지부에놓인애벌레에주사액을주입하는주사액 주입부；및
상기주사액이주입된애벌레를상기애벌레지지부로부터 분리하는애벌레분리부를포함하는애벌레에주사액을주입하는 장치.
청구항 n에있어서，
상기애벌레분리부는，
상기애벌레와결합하는애벌레흡착부;및
상기애벌레가상기애벌레흡착부에흡착되도록상기애벌레 흡착부의공기를펌핑하는흡착펌핑부를포함하는애벌레에 주사액을주입하는장치.
청구항 12에있어서,
상기흡착펌핑부는상기애벌레흡착부가상기애벌레와 결합되어상기애벌레를상기애벌레지지부로부터분리시키는 경우상기공기의펌핑을중단하는것을특징으로하는애벌레에 주사액을주입하는장치.
청구항 11에있어서，
상기주사액주입부는,
내부피스톤이왕복운동을하는실린더부；및
상기실린더부에결합하며，일단에주사바늘이결합하고몸체에 상기주사액이수용되어상기애벌레에상기주사액을주입하는 시린지부를포함하는애벌레에주사액을주입하는장치 .
청구항 14에있어서，
상기시린지부가상기애벌레에주사바늘을꽂올때상기 주사액을상기시린지부에펌핑하는주사액펌핑부를더포함하는 애벌레에주사액을주입하는장치.
PCT/KR2013/008995 2013-10-08 2013-10-08 누에 조성물 및 애벌레에 주사액을 주입하는 장치 WO2015053419A1 (ko)

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Families Citing this family (4)

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Publication number Priority date Publication date Assignee Title
KR102121810B1 (ko) * 2018-05-16 2020-06-12 대한민국 누에의 면역반응 유도방법
KR102036457B1 (ko) * 2018-08-09 2019-10-24 그린테코 주식회사 대량의 동애등에 면역유도 및 천연 항생제의 제형화 방법
KR102374291B1 (ko) * 2019-09-10 2022-03-14 강릉원주대학교산학협력단 천연 포졸란을 포함하는 사료 첨가제 조성물
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05227967A (ja) * 1992-02-17 1993-09-07 Katakura Kogyo Kk カイコからの有用タンパク質の製造方法
JPH0951742A (ja) * 1995-08-11 1997-02-25 Toray Ind Inc 蚕への接種方法
KR20080042386A (ko) * 2006-11-09 2008-05-15 동아대학교 산학협력단 누에유충 생체에서 직접 외래단백질을 발현하는 재조합베큘로바이러스를 제작하는 기술
JP2010133975A (ja) * 2000-05-11 2010-06-17 Genome Soyaku Kenkyusho:Kk 獲得免疫機構を有する生物に感染する病原微生物に対し抗菌活性を有する化合物を自然免疫機構のみを有する昆虫類の幼虫を利用してスクリーニングする方法、および該抗菌活性を自然免疫機構のみを有する昆虫類の幼虫を利用して評価する方法
KR20130096367A (ko) * 2012-02-22 2013-08-30 강원도 양잠 산물을 포함하는 발효성 곤충 천연사료 및 이의 제조방법

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101471558B1 (ko) * 2012-11-02 2014-12-15 그린테코 주식회사 애벌레에 주사액을 주입하는 장치
KR101533602B1 (ko) * 2013-06-24 2015-07-06 대한민국 누에 면역반응 유도용 조성물 및 이에 의해 면역이 유도되어 항균활성이 부가된 누에

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05227967A (ja) * 1992-02-17 1993-09-07 Katakura Kogyo Kk カイコからの有用タンパク質の製造方法
JPH0951742A (ja) * 1995-08-11 1997-02-25 Toray Ind Inc 蚕への接種方法
JP2010133975A (ja) * 2000-05-11 2010-06-17 Genome Soyaku Kenkyusho:Kk 獲得免疫機構を有する生物に感染する病原微生物に対し抗菌活性を有する化合物を自然免疫機構のみを有する昆虫類の幼虫を利用してスクリーニングする方法、および該抗菌活性を自然免疫機構のみを有する昆虫類の幼虫を利用して評価する方法
KR20080042386A (ko) * 2006-11-09 2008-05-15 동아대학교 산학협력단 누에유충 생체에서 직접 외래단백질을 발현하는 재조합베큘로바이러스를 제작하는 기술
KR20130096367A (ko) * 2012-02-22 2013-08-30 강원도 양잠 산물을 포함하는 발효성 곤충 천연사료 및 이의 제조방법

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