KR102393124B1 - Method for preparing fermented insect feed additive comprising Ptecticus tenebrifer and insect chitin, and fermented insect feed additive prepared by the same - Google Patents
Method for preparing fermented insect feed additive comprising Ptecticus tenebrifer and insect chitin, and fermented insect feed additive prepared by the same Download PDFInfo
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- KR102393124B1 KR102393124B1 KR1020210004354A KR20210004354A KR102393124B1 KR 102393124 B1 KR102393124 B1 KR 102393124B1 KR 1020210004354 A KR1020210004354 A KR 1020210004354A KR 20210004354 A KR20210004354 A KR 20210004354A KR 102393124 B1 KR102393124 B1 KR 102393124B1
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- insect
- chitin
- feed additive
- fermented
- streptomyces
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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
- A23K10/20—Animal feeding-stuffs from material of animal origin
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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
-
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/07—Bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12R2001/225—Lactobacillus
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- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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Abstract
Description
본 발명은 사료첨가제의 제조방법 및 그에 따라 제조된 사료첨가제에 관한 것으로, 더욱 상세하게는 곤충 재료를 이용하여 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법 및 그에 따라 제조된 사료첨가제에 관한 것이다.The present invention relates to a method for manufacturing a feed additive and to a feed additive prepared thereby, and more particularly, to a method for manufacturing a feed additive comprising insect chitin fermented using an insect material, and to a feed additive manufactured thereby. .
과거 관상용이나 학습용으로 주로 키우던 곤충이 최근 들어 가축 사료 원료와 사람이 섭취하는 단백질원 등으로 주목 받으면서 국내 곤충 시장이 빠르게 성장하고 있다.Insects, which were mainly raised for ornamental purposes or learning in the past, have recently been attracting attention as raw materials for livestock feed and protein sources consumed by humans, and the domestic insect market is growing rapidly.
한 연구보고서에 따르면 굼벵이로 불리는 ‘흰점박이꽃무지’ 애벌레를 비롯해 ‘갈색거저리’, ‘귀뚜라미’ 등 사람이 먹을 수 있는 식용곤충의 경우 2011년까지만 해도 시장조차 형성되지 않았지만 2018년 시장 규모가 430억원으로 성장했다. 2020년에는 508억원에 달하였다. 또한 갈색거저리, 귀뚜라미, 동애등에 등 사료용 곤충 시장은 2011년 25억원에서 2018년 170억원으로 성장했고, 2020년에는 220억원 이상으로 확대되었다. According to a research report, edible insects such as 'brown mealworm' and 'cricket', as well as 'white-spotted caterpillar' called slugs, as well as edible insects that can be eaten by humans, did not even form a market until 2011, but the market size was 430 in 2018. grew into billions of dollars. In 2020, it reached 508 billion won. In addition, the market for feed insects, such as brown mealworm, cricket, and Dongae, grew from KRW 2.5 billion in 2011 to KRW 17 billion in 2018, and expanded to more than KRW 22 billion in 2020.
최근 식용 곤충 리스트에 새로 포함된 아메리카왕거저리를 비롯해 메뚜기, 누에번데기, 백강잠, 갈색거저리유충(고소애), 쌍별귀뚜라미(쌍별이), 흰점박이꽃무지 유충(꽃벵이), 장수풍뎅이 유충(장수애) 등은 40 내지 70%가 양질의 단백질로 구성될 정도로 우수한 식품원료다. 혐오스러운 생김새와 달리 맛도 우수하다. 대표적인 곤충이 ‘고소애’로 알려진 갈색거저리다. 갈색거저리는 새우와 같은 ‘고소한 맛’이 특징이다. 감칠맛을 내기 위해 사용하는 소고기, 다시마, 멸치, 새우 등과 비교해도 결코 뒤지지 않을 정도다. 일반적으로 곤충 단백질에는 20가지 아미노산이 골고루 들어 있는데 이들 아미노산 중 고소한 맛을 내는 글루탐산(Glutamic acid)이 가장 많기 때문이다.Mealworm caterpillars, grasshoppers, silkworm pupa, white worms, brown mealworm larvae (Ko So-ae), twin-spotted cricket larvae (flower larvae), and long-lived beetle larvae (long-lived beetle larvae) ), etc., are excellent food raw materials to the extent that 40 to 70% of them are composed of high-quality protein. Contrary to its disgusting appearance, the taste is also excellent. A representative insect is the brown mealworm, also known as 'Gosoae'. Brown mealworm is characterized by a 'savory taste' similar to that of shrimp. Compared to beef, kelp, anchovies, and shrimp, which are used for flavor, it is not inferior to anything else. In general, insect protein contains 20 amino acids, and glutamic acid, which gives a savory taste, is the most among these amino acids.
한편, 곤충은 가축이나 물고기 사료 원료로 경제성이 우수하고 친환경적이다. 유엔식량농업기구(FAO)가 2013년부터 10년 동안 진행한 세계 식용·사료곤충에 대한 연구·조사결과를 보면 1kg의 단백질을 얻기 위해 소는 10kg의 사료를 먹어야 하지만 곤충은 1.7kg의 사료만 먹어도 될 정도로 생산성이 우수하다. 또한 소와 곤충의 온실가스 배출량과 물 사용량 비율도 각각 2850:1과 1500:1일 정도로 친환경적이다. 세계 경작지의 33%가 가축 사료용 작물 생산에 이용되고, 사료용 작물 경작지 확대를 위해 매년 브라질 아마존과 같은 크기의 산림이 파괴된다는 점을 고려하면 곤충 단백질의 유용성을 판단할 수 있다. 이밖에 식용 및 사료로 사용되는 곤충의 경우 곡물의 껍질이나 찌꺼기를 주요 먹이로 하기 때문에 인간과 먹이 경쟁을 하지 않는다는 점도 매력적이다.On the other hand, insects are economical and eco-friendly as raw materials for livestock or fish feed. According to the results of research and survey on world edible and forage insects conducted by the Food and Agriculture Organization of the United Nations (FAO) for 10 years from 2013, cows need to eat 10 kg of feed to get 1 kg of protein, whereas insects only need 1.7 kg of feed. It is so productive that it can be eaten. In addition, the ratio of greenhouse gas emissions and water consumption of cattle and insects is 2850:1 and 1500:1, respectively, which is eco-friendly. Considering that 33% of the world's arable land is used for the production of crops for livestock feed, and that forests the size of the Brazilian Amazon are destroyed every year to expand the arable land for feed crops, the usefulness of insect proteins can be judged. In addition, it is attractive that insects used for food and feed do not compete for food with humans because their main food is grain husks or offal.
곤충사육전문기업인 CIEF는 최근 국립수산과학원과 동애등에를 원료로 일반 배합사료보다 영양가가 많고, 기능성이 우수한 양식 넙치(광어)용 친환경 곤충배합사료를 개발했다. CIEF는 하루 4톤(t)쯤의 동애등에를 생산할 수 있는데 가공 처리한 음식물 잔반을 이용해 생산비가 적게 들고, 면역물질인 라우릭산을 다량 함유한 것이 특징이다.CIEF, a specialized insect breeding company, recently developed an eco-friendly insect feed for farmed halibut (flounder) that has more nutritional value and superior functionality than general formula feed, using dongae, etc. as raw materials with the National Institute of Fisheries Science. CIEF can produce about 4 tons (t) of Dongae, etc. per day, and it is characterized by low production costs by using processed food leftovers and contains a large amount of lauric acid, an immune substance.
실제 국립수산과학원이 곤충배합사료와 일반 배합사료를 비교 분석한 결과, 곤충배합사료를 먹인 넙치는 배합사료를 먹인 넙치보다 중량은 17%, 생존율은 20% 높은 것으로 나타났다. 육질 분석 결과에서도 기억력 향상에 도움이 되는 DHA 등이 곤충 배합사료를 먹인 넙치에 더 많았다. 해외에서도 곤충 단백질을 물고기 사료용 원료로 활용하려는 움직임이 일고 있다. 프랑스 옌섹트와 네덜란드 '프로틱스(Protix)'가 대표적인 기업이다. 옌섹트는 곤충 단백질 옌밀과 곤충 오일 옌오일을 개발한 뒤 현재 다양한 양식장에서 테스트를 진행 중이다.In fact, as a result of comparative analysis by the National Institute of Fisheries Research on the compound feed and the general compound feed, it was found that the weight and survival rate of halibut fed the compound feed was 17% and the survival rate was 20% higher than that of halibut fed the compound feed. According to the meat quality analysis result, DHA, which is helpful in improving memory, was found to be higher in halibut fed an insect compound feed. Overseas, there is a movement to use insect protein as a raw material for fish feed. Jensect of France and Protix of the Netherlands are representative companies. After developing the insect protein yenmeal and the insect oil yen oil, Yansect is currently testing it in various farms.
한편, 키틴은 N-아세틸-D-글루코사민(GlcNAc)가 β-1,4 결합한 직쇄상 다당류로서 셀룰로오스와 아주 비슷한 화학구조와 결정구조를 가진 무코 다당류이다. 키틴은 생체 내 소화성, 생체적합성, 표피세포 성장촉진인자의 자극작용 등을 가진 저 독성 물질임에도 불구하고 거의 이용되지 못하고 오로지 키토산 중간물질로서 위치를 유지하고 있는 것은 일반 용매에는 녹지 않아 성형가공이 어렵기 때문이다. 따라서 키틴질을 용해시킬 수 있는 새로운 용매들이나 그 시스템을 개발하고 있는 실정이다.On the other hand, chitin is a linear polysaccharide in which N-acetyl-D-glucosamine (GlcNAc) is bonded to β-1,4, and is a mucopolysaccharide with a chemical structure and crystal structure very similar to that of cellulose. Although chitin is a low toxic substance with in vivo digestibility, biocompatibility, and stimulating action of epidermal cell growth promoting factors, it is rarely used. because it is Therefore, new solvents or systems capable of dissolving chitin are being developed.
본 발명의 목적은 곤충을 원료로 하면서 곤충 키틴질을 키틴 분해 미생물을 이용한 적절한 발효 공정을 통하여 면역 및 항병력 기능을 강화시킬 수 있는 사료첨가제의 제조방법과 그에 따라 제조된 사료첨가제를 제공하는 데 있다.It is an object of the present invention to provide a method for producing a feed additive capable of enhancing immunity and anti-military function through an appropriate fermentation process using insect chitin as a raw material while using a chitin-degrading microorganism, and a feed additive manufactured accordingly.
본 발명의 일 측면에 따르면,According to one aspect of the present invention,
(a) 건조된 곤충을 착유하여 지방을 제거한 후 분쇄하여 곤충탈지분말을 제조하는 곤충재료 전처리 단계;(a) milking dried insects to remove fat and pulverizing them to prepare insect degreasing powder;
(b) 곤충 번데기의 탈피각이 첨가된 배지에서 키틴 분해 미생물을 배양하여 키틴 분해 미생물 배양액을 제조하는 1차 발효단계; 및(b) a primary fermentation step of culturing chitin-degrading microorganisms in a medium to which the molting shells of insect pupae are added to prepare a chitin-degrading microorganism culture solution; and
(c) 상기 곤충탈지분말을 포함하는 배지에 상기 키틴 분해 미생물 배양액 및 발효균을 혼합하여 배양하는 2차 발효단계;를 포함하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법이 제공된다.(c) a secondary fermentation step of culturing by mixing the chitin-degrading microorganism culture solution and the fermented bacteria in a medium containing the insect defatted powder; a method for producing a feed additive containing fermented insect chitin is provided.
상기 곤충은 아메리카동애등에, 동애등에, 풍뎅이, 파리, 흰점박이 꽃무지, 갈색거저리, 누에, 메뚜기 및 귀뚜라미 중에서 선택된 1종 이상일 수 있다.The insect may be at least one selected from the group consisting of stinging insects, such as A. larvae, stinging insects, scarabs, flies, white-spotted beetles, brown mealworms, silkworms, grasshoppers, and crickets.
단계 (b)에서 상기 곤충 번데기는 파리, 아메리카동애등에, 동애등에, 풍뎅이, 흰점박이 꽃무지, 갈색거저리 및 누에 중에서 선택된 1종 이상일 수 있다.In step (b), the insect pupae may be at least one selected from among flies, American larvae, etc., caterpillars, white-spotted beetles, brown mealworms, and silkworms.
단계 (b)에서 상기 키틴 분해 미생물은 바실러스 서브틸리스(Bacillus subtilis), 바실러스 리체니포미스 (Bacillus licheniformis), 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquefaciens) 및 바실러스 투링기엔시스(Bacillus thuringiensis), 락토바실루스 사케이(Lactobacillus sakei), 류코노스톡 시트륨(Leuconostoc citreum), 바이셀라 코리엔시스(Weissella koreensis)중에서 선택된 어느 하나일 수 있다.In step (b), the chitin-degrading microorganism is Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Bacillus thuringiensis, Lactobacillus It may be any one selected from sake (Lactobacillus sakei), leuconostoc citreum, and Weissella koreensis.
단계 (b)의 1차 발효단계는 액상 배양에 의해 수행될 수 있다.The primary fermentation step of step (b) may be performed by liquid culture.
단계 (c)에서 상기 곤충탈지분말을 포함하는 배지는 밀기울, 올리고당, 키토산, 미강 중에서 선택된 1종 이상을 포함할 수 있다.In step (c), the medium containing the insect defatted powder may include at least one selected from bran, oligosaccharide, chitosan, and rice bran.
단계 (c)에서 상기 발효균은 광합성균, 방선균, 유산균 및 고초균이 혼합된 복합 발효균; 및 1종 이상의 유산균을 포함하는 추가 유산균;을 포함할 수 있다.In step (c), the fermenter is a complex fermenter in which photosynthetic bacteria, actinomycetes, lactic acid bacteria and Bacillus subtilis are mixed; and additional lactic acid bacteria including one or more lactic acid bacteria; may include.
또는, 단계 (c)에서 상기 발효균은 단백질 발효균일 수 있다.Alternatively, in step (c), the fermenter may be a protein fermenter.
상기 복합 발효균에 포함되는 상기 광합성균은 로도슈도모나스 팔루스트리스(Rhodopseudomonas palustris), 로도박터 스패로이데스(Rhodobacter sphaeroides) 및 로도박터 캡슐라투스(Rhodobacter capsulatus) 중에서 선택된 1종 이상일 수 있다.The photosynthetic bacteria included in the complex fermentation bacteria may be one or more selected from Rhodopseudomonas palustris, Rhodobacter sphaeroides and Rhodobacter capsulatus.
상기 복합 발효균에 포함되는 상기 방선균은 스트렙토마이세스 그리세우스(Streptomyces griseus), 스트렙토마이세스 코엘리콜라(Streptomyces coelicolor), 스트렙토마이세스 멜라노스포로파시엔스(Streptomyces melanosporofaciens), 스트렙토마이세스 아베르미틸리스(Streptomyces avermitilis), 사카로폴리스포라 에리트레아(Saccharopolyspora erythraea), 스트렙토마이세스 퓨세티우스(Streptomyces peucetius) 및 스트렙토마이세스 아쿨레오라투스(Streptomyces aculeolatus) 중에서 선택된 1종 이상일 수 있다.The actinomycetes included in the complex fermentation bacteria are Streptomyces griseus, Streptomyces coelicolor, Streptomyces melanosporofaciens, Streptomyces avermitili It may be at least one selected from Streptomyces avermitilis, Saccharopolyspora erythraea, Streptomyces peucetius, and Streptomyces aculeolatus.
상기 복합 발효균에 포함되는 상기 고초균은 바실러스 서브틸리스(Bacillus subtilis) 및 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquefaciens) 중에서 선택된 1종 이상일 수 있다.The Bacillus subtilis contained in the complex fermentation bacteria may be at least one selected from Bacillus subtilis and Bacillus amyloliquefaciens.
상기 복합 발효균에 포함되는 상기 유산균은 락토바실러스 플란타럼(Lactobacillus plantarum) 일 수 있다.The lactic acid bacteria included in the complex fermentation bacteria may be Lactobacillus plantarum.
상기 추가 유산균은 엔테로코커스 패칼리스(Enterococcus faecalis), 락토바실러스 애시도필러스(Lactobacillus acidophilus) 및 비피도박테리움 비피덤 (Bfidobacterium bifidum) 중에서 선택된 1종 이상일 수 있다.The additional lactic acid bacteria may be at least one selected from Enterococcus faecalis, Lactobacillus acidophilus, and Bfidobacterium bifidum.
단계 (c)에서 상기 단백질 발효균은 바실러스 리체니포미스(Bacillus licheniformis) 일 수 있다.In step (c), the protein fermenting bacteria may be Bacillus licheniformis.
본 발명의 다른 하나의 측면에 따르면,According to another aspect of the present invention,
상기 제조방법에 따라 제조된 발효된 곤충키틴을 포함하는 사료첨가제가 제공된다.There is provided a feed additive comprising the fermented insect chitin prepared according to the manufacturing method.
본 발명의 다른 또 하나의 측면에 따르면,According to another aspect of the present invention,
상기 발효된 곤충키틴을 포함하는 사료첨가제를 포함하는 사료조성물이 제공된다.There is provided a feed composition comprising a feed additive comprising the fermented insect chitin.
상기 사료조성물은 가축용, 가금류용, 양어용 및 반려동물용 중에서 선택된 어느 하나의 용도로 사용될 수 있다.The feed composition may be used for any one purpose selected from among livestock, poultry, aquaculture and companion animals.
본 발명의 발효된 곤충키틴을 포함하는 사료첨가제는 곤충을 원료로 하면서 곤충 키틴질을 키틴 분해 미생물을 이용한 적절한 발효 공정을 통하여 종래 곤충분말을 포함하는 사료첨가제에 비하여 면역 및 항병력 기능이 향상된 사료첨가제의 제조방법과 그에 따라 제조된 사료첨가제를 제공하는 데 있다.The feed additive containing fermented insect chitin of the present invention is a feed additive with improved immunity and anti-military function compared to feed additives containing conventional insect powder through an appropriate fermentation process using insect chitin as a raw material while using insect chitin as a chitin-degrading microorganism. An object of the present invention is to provide a manufacturing method and a feed additive manufactured accordingly.
도 1은 실험예에 사용된 사육장 및 각 실험군의 사진이다.
도 2는 실험예 1의 평균 증체량 측정 결과이다.
도 3은 실험예 1의 사료 요구율 측정 결과이다.
도 4는 실험예 1의 생산지수 측정 결과이다.
도 5는 실험예 2의 단백질 함량 측정 결과이다.
도 6은 실험예 2의 수분 함량 측정 결과이다.
도 7은 실험예 2의 지방 함량 측정 결과이다.
도 8은 실험예 2의 회분 함량 측정 결과이다.
도 9는 실험예 2의 pH 측정 결과이다.
도 10은 실험예 3의 라이소자임 활성 정도를 분석 결과이다.
도 11은 실험예 4의 MTT 분석 결과이다.
도 12는 실험예 5의 IL-1β 측정 결과이다.
도 13은 실험예 5의 IL-2 측정 결과이다.
도 14는 실험예 5의 IL-12 측정 결과이다.
도 15는 실험예 5의 TNF-a 측정 결과이다.
도 16은 실험예 5의 IFN-gamma 측정 결과이다.
도 17 내지 도 19는 실험예 6의 부검한 닭의 내장 사진이다.
도 20 내지 도 24는 실험예 6에서 각각 분변(feces), 간(liver), 비장(spleen), 윤활낭(bursa), 맹장(cecum)에서 S. gallinarum의 수를 측정한 결과이다.
도 25 내지 도 27은 실험예 6에서 림프구아집단(lymphocyte subpopulation) 분석 결과를 나타낸 것이다.1 is a photograph of the kennel and each experimental group used in the experimental example.
2 is an average weight gain measurement result of Experimental Example 1. FIG.
3 is a measurement result of the feed demand rate of Experimental Example 1.
4 is a measurement result of the production index of Experimental Example 1.
5 is a result of measuring the protein content of Experimental Example 2.
6 is a measurement result of the moisture content of Experimental Example 2.
7 is a result of measuring the fat content of Experimental Example 2.
8 is an ash content measurement result of Experimental Example 2.
9 is a pH measurement result of Experimental Example 2.
10 is an analysis result of the degree of lysozyme activity of Experimental Example 3.
11 is an MTT analysis result of Experimental Example 4.
12 is an IL-1β measurement result of Experimental Example 5;
13 is an IL-2 measurement result of Experimental Example 5;
14 is an IL-12 measurement result of Experimental Example 5.
15 is a TNF-a measurement result of Experimental Example 5.
16 is an IFN-gamma measurement result of Experimental Example 5.
17 to 19 are photographs of the intestines of the autopsied chicken of Experimental Example 6.
20 to 24 are results of measuring the number of S. gallinarum in feces, liver, spleen, bursa, and cecum in Experimental Example 6, respectively.
25 to 27 show the results of analysis of lymphocyte subpopulation in Experimental Example 6.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.
이하, 본 발명의 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법에 대해 설명하도록 한다.Hereinafter, a method for manufacturing a feed additive containing fermented insect chitin of the present invention will be described.
먼저, 건조된 곤충을 착유하여 지방을 제거한 후 분쇄하여 곤충탈지분말을 제조한다(단계 a; 곤충재료 전처리). First, the dried insects are milked to remove fat and then pulverized to prepare an insect degreasing powder (step a; insect material pretreatment).
상기 곤충은 아메리카동애등에, 동애등에, 풍뎅이, 파리, 흰점박이 꽃무지, 갈색거저리, 누에, 메뚜기 및 귀뚜라미 중에서 선택된 1종 이상인 것이 바람직하고, 더욱 바람직하게는 아메리카동애등에 또는 동애등에를 사용할 수 있다.The insect is preferably at least one selected from the group consisting of stinging insects, stinging insects, scarabs, flies, white-spotted caterpillars, brown mealworms, silkworms, grasshoppers, and crickets, and more preferably, it is possible to use locusts or locusts, etc. .
상기 곤충탈지분말의 지방 함량은 20wt% 이하가 되도록 착유하는 것이 바람직하고, 더욱 바람직하게는 10wt% 이하가 되도록 착유할 수 있다. 지방 함량이 20wt% 보다 높은 경우 발효 후 면역 기능이나 항병력 기능이 저하될 수 있다.The fat content of the insect defatted powder is preferably milked so that it is 20 wt% or less, and more preferably, it can be milked so that it is 10 wt% or less. If the fat content is higher than 20wt%, the immune function or anti-military function may decrease after fermentation.
상기 곤충탈지분말의 입도는 50 내지 200㎛가 되도록 분쇄하는 것이 바람직하고, 더욱 바람직하게는 바람직하게는 100 내지 150㎛ 되도록 분쇄할 수 있다. 곤충탈지분말의 입도를 50㎛ 미만으로 분쇄하기에는 공정 설비에 추가 비용이 발생할 수 있고, 200㎛를 초과하는 경우에는 이후 미생물 처리시 발효 효율이 저하될 수 있다.The particle size of the insect degreasing powder is preferably pulverized to 50 to 200 μm, more preferably 100 to 150 μm. When the particle size of the insect degreasing powder is pulverized to less than 50 μm, additional costs may be incurred in the process equipment, and if it exceeds 200 μm, fermentation efficiency may be lowered during subsequent microbial treatment.
한편, 곤충 번데기의 탈피각이 첨가된 배지에서 키틴 분해 미생물을 배양하여 키틴 분해 미생물 배양액을 제조한다(단계 b: 1차 발효).Meanwhile, a chitin-degrading microorganism culture solution is prepared by culturing a chitin-degrading microorganism in a medium to which the molting shell of an insect pupae is added (step b: primary fermentation).
파리, 아메리카동애등에, 동애등에, 풍뎅이, 흰점박이 꽃무지, 갈색거저리 및 누에 중에서 선택된 1종 이상일 수 있으나, 본 발명의 범위가 여기에 한정되지 않으며 주변에서 용이하게 구할 수 있고 키틴 분해 미생물의 영양분이 될 수 있는 곤충 번데기의 탈피각은 모두 적용할 수 있다.It may be one or more species selected from flies, American ferrets, etc., stinging insects, scarabs, white-spotted beetles, brown mealworms, and silkworms, but the scope of the present invention is not limited thereto, and nutrients of chitin-degrading microorganisms The moult angle of the insect pupae that can be this can be applied to all.
1차 발효는 액상의 혐기발효로 수행되는 것이 바람직하다.The primary fermentation is preferably carried out as anaerobic fermentation in liquid phase.
단계 (b)에서 상기 키틴 분해 미생물은 바실러스 서브틸리스(Bacillus subtilis), 바실러스 리체니포미스 (Bacillus licheniformis), 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquefaciens) 및 바실러스 투링기엔시스(Bacillus thuringiensis), 락토바실루스 사케이(Lactobacillus sakei), 류코노스톡 시트륨(Leuconostoc citreum), 바이셀라 코리엔시스(Weissella koreensis)중에서 선택된 어느 하나인 것이 바람직하고, 더욱 바람직하게는 바실러스 서브틸리스(Bacillus subtilis) 또는 바실러스 리체니포미스 (Bacillus licheniformis) 일 수 있다.In step (b), the chitin-degrading microorganism is Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Bacillus thuringiensis, Lactobacillus Sake (Lactobacillus sakei), leuconostoc citreum, preferably any one selected from Weissella koreensis, more preferably Bacillus subtilis (Bacillus subtilis) or Bacillus lichenipo Miss (Bacillus licheniformis).
상기 곤충 번데기의 탈피각은 상기 배지에 0.5 내지 2wt%로 첨가될 수 있고, 바람직하게는 1 내지 1.5wt% 첨가될 수 있다.The molting shell of the insect pupae may be added to the medium in an amount of 0.5 to 2 wt%, preferably 1 to 1.5 wt%.
1차 발효단계는 액상 배양에 의해 수행되는 것이 바람직하다.The primary fermentation step is preferably performed by liquid culture.
1차 발효단계는 25 내지 35℃에서 수행될 수 있고, 바람직하게는 27 내지 33℃, 더욱 바람직하게는 29 내지 32℃, 더욱 더 바람직하게는 29 내지 31℃에서 수행될 수 있다. 이와 같은 온도 범위에서 발효가 가장 효율적으로 이루어질 수 있다.The primary fermentation step may be carried out at 25 to 35 °C, preferably at 27 to 33 °C, more preferably at 29 to 32 °C, even more preferably at 29 to 31 °C. Fermentation can be performed most efficiently in this temperature range.
이후, 상기 곤충탈지분말을 포함하는 배지에 상기 키틴 분해 미생물 배양액 및 발효균을 혼합하여 배양한다(단계 c: 2차 발효).Thereafter, the chitin-degrading microorganism culture solution and the fermenter are mixed and cultured in a medium containing the insect defatted powder (step c: secondary fermentation).
상기 곤충탈지분말을 포함하는 배지는 밀기울, 올리고당, 키토산, 미강 중에서 선택된 1종 이상을 포함할 수 있다.The medium containing the insect defatted powder may include at least one selected from bran, oligosaccharide, chitosan, and rice bran.
선택적으로, 상기 발효균은 광합성균, 방선균, 유산균 및 고초균이 혼합된 '복합 발효균', 및 1종 이상의 유산균을 포함하는 '추가 유산균'을 포함하거나, 또는 상기 발효균은 단백질 발효균일 수 있다.Optionally, the fermenter may include a 'complex fermenter' in which photosynthetic bacteria, actinomycetes, lactic acid bacteria and Bacillus subtilis are mixed, and 'additional lactic acid bacteria' including one or more lactic acid bacteria, or the fermenter may be a protein fermenter.
상기 복합 발효균에 포함되는 상기 광합성균은 로도슈도모나스 팔루스트리스(Rhodopseudomonas palustris), 로도박터 스패로이데스(Rhodobacter sphaeroides) 및 로도박터 캡슐라투스(Rhodobacter capsulatus) 중에서 선택된 어느 하나인 것이 바람직하고, 더욱 바람직하게는 로도슈도모나스 팔루스트리스(Rhodopseudomonas palustris) 일 수 있다.The photosynthetic bacteria included in the complex fermentation bacteria are preferably any one selected from Rhodopseudomonas palustris, Rhodobacter sphaeroides and Rhodobacter capsulatus, more preferably It may be Rhodopseudomonas palustris.
상기 복합 발효균에 포함되는 상기 방선균은 스트렙토마이세스 그리세우스(Streptomyces griseus), 스트렙토마이세스 코엘리콜라(Streptomyces coelicolor), 스트렙토마이세스 멜라노스포로파시엔스(Streptomyces melanosporofaciens), 스트렙토마이세스 아베르미틸리스(Streptomyces avermitilis), 사카로폴리스포라 에리트레아(Saccharopolyspora erythraea), 스트렙토마이세스 퓨세티우스(Streptomyces peucetius) 및 스트렙토마이세스 아쿨레오라투스(Streptomyces aculeolatus) 중에서 선택된 어느 하나일 수 있다.The actinomycetes included in the complex fermentation bacteria are Streptomyces griseus, Streptomyces coelicolor, Streptomyces melanosporofaciens, Streptomyces avermitili It may be any one selected from Streptomyces avermitilis, Saccharopolyspora erythraea, Streptomyces peucetius, and Streptomyces aculeolatus.
상기 복합 발효균에 포함되는 상기 고초균은 바실러스 서브틸리스(Bacillus subtilis) 및 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquefaciens) 중에서 선택된 1종 이상일 수 있다.The Bacillus subtilis contained in the complex fermentation bacteria may be at least one selected from Bacillus subtilis and Bacillus amyloliquefaciens.
상기 복합 발효균에 포함되는 상기 유산균은 락토바실러스 플란타럼(Lactobacillus plantarum)일 수 있다.The lactic acid bacteria included in the complex fermentation bacteria may be Lactobacillus plantarum.
상기 추가 유산균은 엔테로코커스 패칼리스(Enterococcus faecalis), 락토바실러스 애시도필러스(Lactobacillus acidophilus) 및 비피도박테리움 비피덤 (Bfidobacterium bifidum) 중에서 선택된 1종 이상인 것이 바람직하고, 더욱 바람직하게는 상기 3종의 유산균 조합일 수 있다.The additional lactic acid bacteria is preferably at least one selected from Enterococcus faecalis, Lactobacillus acidophilus and Bfidobacterium bifidum, more preferably the three It may be a combination of lactic acid bacteria.
단계 (c)에서 상기 단백질 발효균은 바실러스 리체니포미스(Bacillus licheniformis)인 것이 바람직하다.In step (c), the protein fermenting bacteria is preferably Bacillus licheniformis.
2차 발효단계는 40 내지 50℃에서 수행되는 것이 바람직하고, 더욱 바람직하게는 42 내지 47℃, 더욱 더 바람직하게는 44 내지 46℃에서 수행될 수 있다. 이와 같은 온도 범위에서 발효가 가장 효율적으로 이루어질 수 있다.The secondary fermentation step is preferably carried out at 40 to 50 ℃, more preferably 42 to 47 ℃, even more preferably it may be carried out at 44 to 46 ℃. Fermentation can be performed most efficiently in this temperature range.
2차 발효단계는 함수율 45 내지 55wt%의 조건에서 수행되는 것이 바람직하다.The secondary fermentation step is preferably carried out under conditions of 45 to 55 wt% of moisture content.
또한, 본 발명은 상술한 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법에 따라 제조된 발효된 곤충키틴을 포함하는 사료첨가제를 제공한다.In addition, the present invention provides a feed additive comprising fermented insect chitin prepared according to the method for producing a feed additive comprising fermented insect chitin described above.
또한, 본 발명은 상기 발효된 곤충키틴을 포함하는 사료첨가제를 포함하는 사료조성물을 제공한다.In addition, the present invention provides a feed composition comprising a feed additive comprising the fermented insect chitin.
상기 사료조성물은 가축용, 가금류용, 양어용 및 반려동물용 중에서 선택된 어느 하나의 용도로 사용될 수 있다.The feed composition may be used for any one purpose selected from among livestock, poultry, aquaculture and companion animals.
이하, 본 발명의 실시예를 들어 설명하나 본 발명의 범위가 실시예에 한정되는 것은 아니다.Hereinafter, an embodiment of the present invention will be described, but the scope of the present invention is not limited to the embodiment.
[실시예][Example]
실시예 1: 곤충키틴 발효사료첨가제 제조 Example 1: Preparation of insect chitin fermented feed additive
(1) 곤충재료 전처리(곤충탈지분말 제조)(1) Insect material pretreatment (manufacture of insect defatting powder)
본 출원인의 등록특허 제10-1838201호에 개시된 대량생산 시스템과 그에 따른 생산방법을 이용하여 아메리카동애등에(Hermetia illucens)를 생산하였다. 생산된 아메리카동애등에를 멸균기능을 포함한 건조시스템에서 마이크로웨이브로 처리하여 사멸시킨 후 60℃로 열풍건조하여 수분함량이 10wt% 이하가 되도록 건조시켜 건조된 동애등에를 얻었다. 건조된 동애등에에서 지방 성분을 분리하기 위하여 착유기(SCHNECKENPRESSE AP 08, GERMANY)를 이용하여 착유함으로써 지방 성분이 제거된 동애등에를 얻었으며, 이를 사이클론밀 400SS형 분쇄장치를 이용하여 분쇄입도 150㎛ 미만의 동애등에 탈지분말을 제조하였다.Hermetia illucens was produced using the mass production system and production method according to the mass production system disclosed in Patent Registration No. 10-1838201 of the present applicant. The produced American Dongae was treated with microwave in a drying system including a sterilization function to be killed, and then dried with hot air at 60° C. so that the moisture content was 10wt% or less, and dried Dongae was obtained. In order to separate the fat component from the dried dongae, milked using a milking machine (SCHNECKENPRESSE AP 08, GERMANY) to obtain dongae from which the fat has been removed. Skim powder was prepared in Dongae et al.
(2) 1차 발효(키틴 분해 미생물 배양액 제조)(2) Primary fermentation (production of chitin-degrading microorganism culture solution)
파리번데기의 탈피각을 1wt% 첨가된 제한배지(defined medium)에 키틴 분해 미생물인 바실러스 서브틸리스(Bacillus subtilis)를 1wt%로 접종하여 30℃, 150rpm 조건으로 5일 동안 액상 배양하여 키틴 분해 미생물 배양액을 제조하였다.Bacillus subtilis, a chitin-degrading microorganism, was inoculated at 1wt% in a defined medium containing 1wt% of the molting shells of the fly pupa, and the chitin-degrading microorganisms were cultured in a liquid phase at 30℃, 150rpm for 5 days. A culture solution was prepared.
(3) 2차 발효(곤충 키틴 발효)(3) Secondary fermentation (insect chitin fermentation)
(1)에서 제조된 동애등에 곤충 분말 9kg, (2)에서 제조된 키틴 분해 미생물 배양액 6.5ℓ를 혼합하였다.9 kg of insect powder and 6.5 liters of chitin-degrading microorganism culture solution prepared in (2) were mixed with Dongae etc. prepared in (1).
여기에, 로도슈도모나스 팔루스트리스(Rhodopseudomonas palustris)(광합성균) + 스트렙토마이세스 sp.(Streptomyces sp.)(방선균) + 락토바실러스 플란타넘(Lactobacillus plantarum)(유산균) + 바실러스 아밀로리큐파시엔시스(Bacillus amyloliquefaciens)(고초균)가 동일 중량비율로 혼합된 균주가 혼합된 '복합 발효균' 1kg을 첨가하였다.Here, Rhodopseudomonas palustris (photosynthetic bacteria) + Streptomyces sp. (actinomycetes) + Lactobacillus plantarum (Lactobacillus) + Bacillus amyloliquefaciensis ( Bacillus amyloliquefaciens) (Bacillus subtilis) 1kg of 'composite fermenter' mixed in the same weight ratio was added.
또한, 스트렙토코커스 패칼리스(Streptococcus faecalis) + 락토바실러스 애시도펠리스(Lactobacillus acidophilus) + 비피도박테리움 비피덤(Bifidobacterium bifidum)가 동일 중량비율로 혼합된 '추가 유산균' 11g을 첨가한 후 함수율 50%가 되도록 수분을 조절하여 45℃에서 7일 동안 교반하면서 발효시켰다.In addition, after adding 11 g of 'additional lactic acid bacteria' in which Streptococcus faecalis + Lactobacillus acidophilus + Bifidobacterium bifidum are mixed in the same weight ratio, the moisture content is 50% It was fermented while stirring at 45 °C for 7 days by controlling the moisture so that the
이후 발효물은 함수율 10wt% 이하가 되도록 건조시켜 곤충키틴 발효사료첨가제를 제조하였다.Thereafter, the fermented product was dried to have a moisture content of 10 wt% or less to prepare an insect chitin fermented feed additive.
실시예 2: 곤충키틴 발효사료첨가제 제조Example 2: Preparation of insect chitin fermented feed additive
'복합 발효균'및 '추가 유산균'을 첨가하는 것 대신에 단백질 분해 미생물로 바실러스 리체니포르미스(Bacillus licheniformis)를 첨가하여 2차 발효시킨 것을 제외하고는 실시예 1과 동일한 조건으로 곤충키틴 발효사료첨가제를 제조하였다.Insect chitin fermented feed under the same conditions as in Example 1, except for secondary fermentation by adding Bacillus licheniformis as a proteolytic microorganism instead of adding 'complex fermented bacteria' and 'additional lactic acid bacteria' Additives were prepared.
비교예 1: 곤충키틴 사료첨가제 제조Comparative Example 1: Preparation of insect chitin feed additive
1차 발효 및 2차 발효를 수행하지 않고, 실시예 1의 (1)에서 제조된 동애등에 분말을 그대로 사료 첨가제로 사용하였다.Without performing the primary fermentation and secondary fermentation, the powder of Dongae etc. prepared in (1) of Example 1 was used as a feed additive.
[실험예][Experimental example]
실험동물은 입사 일주일 전에 사육사를 포르말린으로 완전 훈증 소독 후, 후드를 이용하여 완전 환기를 실시하였고, 소독 후 1일령의 육계(Ross broiler) 병아리 70수를 전남대학교 수의과대학 실험동물 사육사로 입식하였다. 사육장 내 온도는 28 ~ 30℃, 상대습도는 45 ~ 55%의 조건으로 유지시켰으며, 외부공기의 유입을 차단하기 위해 후드를 통해서만 환기를 실시하였다. 병아리 입사 후에는 사료와 물에 자유롭게 접근할 수 있도록 해주었다.Experimental animals were completely fumigated with formalin one week before entering the company, and then completely ventilated using a hood. After disinfection, 70 1-day-old Ross broiler chicks were stocked with the laboratory animal breeder of Chonnam National University College of Veterinary Medicine. The temperature inside the kennel was maintained at 28 ~ 30℃ and relative humidity of 45 ~ 55%, and ventilation was performed only through the hood to block the inflow of outside air. The chicks were given free access to feed and water after entry.
1일령 Ross 육계 70마리를 7개의 군으로 나누어 농도별로 비교예 1의 곤충키틴 사료첨가제(C군), 실시예 1의 곤충키틴 발효사료첨가제(W군), 실시예 2의 곤충키틴 발효사료첨가제(B군)를 급여하여 그에 대한 효과를 평가하였다. 급여 후 매일 사료 섭취량을 측정하였으며, 7일에 한 번씩, 급여 21일까지 체중 측정을 실시하였다. 실험에 사용된 사육장 및 각 실험군의 사진을 도 1에 나타내었다.70 1-day-old Ross broilers were divided into 7 groups and the insect chitin feed additive of Comparative Example 1 (group C), the insect chitin fermented feed additive of Example 1 (group W), and the insect chitin fermented feed additive of Example 2 by concentration (Group B) was fed and its effect was evaluated. Feed intake was measured every day after feeding, and body weight was measured once every 7 days until the 21st day of feeding. The kennel used in the experiment and photos of each experimental group are shown in FIG. 1 .
구체적인 실험군은 아래와 같다.The specific experimental group is as follows.
- ND군: 일반사료 급여군- ND group: general feed group
- C2군: 비교예 1의 곤충키틴 사료첨가제 2wt% 급여군- C2 group: 2wt% feeding group of insect chitin feed additive of Comparative Example 1
- C4군: 비교예 1의 곤충키틴 사료첨가제 4wt% 급여군-C4 group: 4wt% feeding group of insect chitin feed additive of Comparative Example 1
- W2군: 실시예 1의 곤충키틴 발효사료첨가제 W 2wt% 급여군- W2 group: Example 1 insect chitin fermented feed additive W 2wt% feeding group
- W4군: 실시예 1의 곤충키틴 발효사료첨가제 W 4wt% 급여군- W4 group: Example 1 insect chitin fermented feed additive W 4wt% feeding group
- B2군: 실시예 2의 곤충키틴 발효사료첨가제 B 2wt% 급여군- B2 group: Example 2 insect chitin fermented feed additive B 2wt% feeding group
- B4군: 실시예 2의 곤충키틴 발효사료첨가제 B 4wt% 급여군- B4 group: Example 2 insect chitin fermented feed additive B 4wt% feeding group
실험예 1: 생산성 증가 효과 분석(평균 증체량, 사료 요구율, 생산지수 측정)Experimental Example 1: Analysis of productivity increase effect (average weight gain, feed demand rate, production index measurement)
육계의 체중 및 사료섭취량을 측정한 후, 평균 증체량과 생산지수를 확인하였으며, 이를 통해 사료 요구율을 계산하였다.After measuring the body weight and feed intake of the broiler, the average weight gain and production index were checked, and the feed requirement was calculated through this.
[식 1][Equation 1]
사료 요구율 = 평균사료섭취량(kg)/평균출하중량(kg)Feed demand rate = Average feed intake (kg) / Average shipment weight (kg)
[식 2][Equation 2]
생산지수 = 육성율(%) x 평균출하 체중(kg)/사료요구율Production index = growth rate (%) x average weight shipped (kg) / feed demand rate
[식 3][Equation 3]
육성율 = 출하수수/입추수수Growth rate = number of shipments / number of harvests
계산된 평균 증체량(g)은 도 2에 나타내었고, 사료 요구율은 도 3에 나타내었으며, 생산지수는 도 4에 나타내었다.The calculated average weight gain (g) is shown in FIG. 2 , the feed demand rate is shown in FIG. 3 , and the production index is shown in FIG. 4 .
도 2에 따르면, ND군에 비해 C군, W군 및 B군에서 평균 증체량이 더 높은 것으로 나타났으며, 실시예 1의 곤충키틴 발효사료첨가제를 급여한 W군이 B군 보다 평균 증체량이 조금 더 높은 것으로 나타났다.According to Figure 2, compared to the ND group, it was found that the average weight gain was higher in group C, group W and group B, and group W fed the insect chitin fermented feed additive of Example 1 had a slightly higher average weight gain than group B. appeared to be higher.
도 3에 따르면, ND군에 비해 C군, W군 및 B군에서 사료 요구율이 다소 감소한 것으로 나타났으며, B군에서 사료 요구율이 사료 요구율이 가장 낮은 것으로 나타났다.According to FIG. 3 , compared to the ND group, it was found that the feed demand rate was slightly decreased in group C, group W, and group B, and the feed demand rate was the lowest in group B.
도 4에 따르면, ND군에 비해 C군, W군 및 B군에서 생산지수가 더 높게 측정되었으며, 실시예 1의 곤충키틴 발효사료첨가제를 급여한 W군이 B군 보다 생산지수가 더 높게 측정되었고, 2wt% 급여군보다 4wt% 고농도 급여군에서 생산지수가 더 높은 것으로 나타났다.According to FIG. 4, the production index was higher in group C, group W and B than in the ND group, and the W group fed the insect chitin fermented feed additive of Example 1 had a higher production index than group B. It was found that the production index was higher in the 4wt% high concentration group than in the 2wt% pay group.
실험예 2: 생산성 증가 효과 분석(급여 후 도체 특성 및 육질에 미치는 영향 분석)Experimental Example 2: Analysis of the effect of increasing productivity (analysis of effects on carcass characteristics and meat quality after feeding)
비교예 1의 곤충키틴 사료첨가제 및 실시예 1 또는 실시예 2의 곤충키틴 발효사료첨가제 급여 후 도체특성 및 육질에 미치는 영향을 규명하기 위하여 실험군인 닭고기의 일반성분을 분석하였다.After feeding the insect chitin feed additive of Comparative Example 1 and the insect chitin fermented feed additive of Example 1 or Example 2, the general components of the experimental group chicken were analyzed to determine the effect on carcase characteristics and meat quality.
구체적으로, 사양시험 종료 후 닭의 가슴살 부위를 적출하여 단백질, 수분, 지방, 회분 및 pH를 분석하여 그 결과를 도 5 내지 도 9에 각각 나타내었다. 닭고기의 일반성분 분석은 농업기술실용화재단 종합분석 서비스센터에서 실시되었다. Specifically, after the completion of the feeding test, the chicken breast was extracted and analyzed for protein, moisture, fat, ash and pH, and the results are shown in FIGS. 5 to 9, respectively. The analysis of general components of chicken was conducted at the Agricultural Technology Commercialization Foundation's Comprehensive Analysis Service Center.
이에 따르면, 단백질, 수분 함량 및 pH에서는 실험군간 유의적인 차이는 나타나지 않았으며, 지방 함량의 경우에는 ND 군에 비해 C4, W4, B2, B4군에서 감소하는 경향을 보였으며, 특히, 실시예 2의 곤충키틴 발효사료첨가제를 고농도로 급여한 B4군에서는 가장 낮은 지방 함량을 나타내었다.According to this, there was no significant difference between the experimental groups in protein, water content and pH, and in the case of fat content, there was a tendency to decrease in the C4, W4, B2, and B4 groups compared to the ND group, in particular, Example 2 B4 group fed with insect chitin fermented feed additive at high concentration showed the lowest fat content.
또한, 회분의 경우 ND 군에 비해 W2, W4, B4 군에서 감소하는 경향을 보였고 특히, 실시예 2의 곤충키틴 발효사료첨가제를 고농도로 급여한 B4군에서는 가장 낮은 회분 함량을 나타내었다.In addition, in the case of ash, the W2, W4, and B4 groups showed a tendency to decrease compared to the ND group, and in particular, the B4 group fed the insect chitin fermented feed additive of Example 2 at a high concentration showed the lowest ash content.
실험예 3: 면역 증강 효과 분석(라이소자임 활성 측정)Experimental Example 3: Immune enhancing effect analysis (lysozyme activity measurement)
곤충키틴 사료첨가제 및 곤충키틴 발효사료첨가제의 급여에 의한 대식세포 활성화를 측정하기 위하여 라이소자임 활성을 측정하였다.Lysozyme activity was measured to measure macrophage activation by feeding insect chitin feed additive and insect chitin fermented feed additive.
라이소자임(Lysozyme)은 항균성 효소로 그람양성균의 세포벽을 파괴해 세균을 죽임으로써 선천 면역을 구성하는 요소이다. 비교예 1의 곤충키틴 사료첨가제, 실시예 1 또는 실시예 2의 곤충키틴 발효사료첨가제의 급여에 의한 대식세포 활성화 정도의 확인은 혈청을 이용한 라이소자임 활성 분석(lysozyme activity assay)을 통해 실시하였다. 이를 위해 급여 21일 후 실험군의 육계로부터 분리한 혈청을 Micrococcus lysodeikticus와 반응시킨 후 540nm에서 흡광도를 측정하였으며, 최종적으로 측정한 흡광도를 이용해 라이소자임 활성 정도를 분석하여 그 결과를 도 10에 나타내었다.Lysozyme is an antibacterial enzyme that destroys the cell wall of Gram-positive bacteria and kills bacteria, and is a component of innate immunity. The confirmation of the degree of macrophage activation by feeding the insect chitin feed additive of Comparative Example 1, the insect chitin fermented feed additive of Example 1 or Example 2 was carried out through a lysozyme activity assay using serum. To this end, after 21 days of feeding, the serum separated from the broiler in the experimental group was reacted with Micrococcus lysodeikticus, and the absorbance was measured at 540 nm, and the degree of lysozyme activity was analyzed using the finally measured absorbance, and the results are shown in FIG. 10 .
이에 따르면, 그 결과 ND 군과 비교해 C4, W2, W4, B2, B4 군에서는 유의적인 라이소자임 활성의 증가효과를 나타냈으며(*p<0.05, ***p<0.001), 특히 실시예 1과 2의 곤충키틴 발효사료첨가제를 급여한 W2, W4, B2, B4 군에서는 현저히 높은 라이소자임 활성 증가가 나타났고, 2wt%의 저농도 급여군에서도 매우 높은 라이소자임 활성 증가가 나타났다. 즉, 본 발명의 곤충키틴 발효사료첨가제의 급여는 대식세포를 활성화하여 육계의 선천면역성을 증가시키는데 현저한 효과가 있음을 확인할 수 있다.According to this, as a result, compared to the ND group, the C4, W2, W4, B2, B4 group showed a significant increase in lysozyme activity (*p<0.05, ***p<0.001), especially Examples 1 and 2 In the W2, W4, B2, and B4 groups fed with insect chitin fermented feed additive, a significantly high increase in lysozyme activity was observed, and a very high increase in lysozyme activity was also observed in the low concentration feeding group of 2 wt%. That is, it can be confirmed that the feeding of the insect chitin fermented feed additive of the present invention has a remarkable effect in increasing the innate immunity of broilers by activating macrophages.
실험예 4: 면역 증강 효과 분석(림프구 증식능 측정)Experimental Example 4: Immune enhancing effect analysis (lymphocyte proliferative capacity measurement)
곤충키틴사료첨가제 및 곤충키틴발효사료첨가제의 급여에 의한 림프구 증식능 효과 확인하였다.Lymphocyte proliferation effect was confirmed by feeding of insect chitin feed additive and insect chitin fermented feed additive.
사료첨가제 급여 21일 후, 닭의 비장림프구를 1 X 106 cells 용량으로 96 well plate에 시딩(seeding)한 후, T cell mitogen인 concanavalin a (ConA)를 처리하여 36시간 배양을 실시하였다. 배양 후에는 Thiazolyl Blue Tetrazolium Bromide(MTT)를 넣고 다시 4시간 배양을 실시하였으며, 최종적으로 540nm에서 흡광도 측정을 하여 그 결과를 도 11에 나타내었다.After 21 days of feeding the feed additive, chicken splenic lymphocytes were seeded in a 96-well plate at a dose of 1
이에 따르면, 림프구 증식능은 비교예 1의 곤충키틴 사료첨가제 급여군, 실시예 1 또는 실시예 2의 곤충키틴 발효사료첨가제 급여군에서 증가하는 경향을 나타내었으며, 특히 실시예 1의 곤충키틴 발효사료첨가제 급여군인 W4군(*p<0.05)과 실시예 2의 곤충키틴 발효사료첨가제 급여군인 B4군(**p<0.01)은 ND 그룹에 비교하여 유의적인 증가를 나타내었다. 즉, 본 발명의 곤충키틴 발효사료첨가제의 급여는 림프구의 증식을 강화하는데 매우 효과적이며 발효되지 않은 곤충키틴 사료첨가제 보다 우수한 효과를 나타냄을 알 수 있다.According to this, the lymphocyte proliferation ability showed a tendency to increase in the insect chitin feed additive feeding group of Comparative Example 1, the insect chitin fermented feed additive feeding group of Example 1 or Example 2, in particular, the insect chitin fermenting feed additive of Example 1 The W4 group (*p<0.05) and the B4 group (**p<0.01) fed the insect chitin fermented feed additive of Example 2 showed a significant increase compared to the ND group. That is, it can be seen that the feeding of the insect chitin fermented feed additive of the present invention is very effective in enhancing the proliferation of lymphocytes and exhibits a superior effect than the non-fermented insect chitin feed additive.
실험예 5: 면역 증강 효과 분석(면역 사이토카인 발현 평가)Experimental Example 5: Immune enhancing effect analysis (immune cytokine expression evaluation)
곤충키틴사료첨가제 및 곤충키틴발효사료첨가제의 급여에 의한 면역 사이토카인 발현 변화를 관찰하였다.Changes in immune cytokine expression by feeding of insect chitin feed additive and insect chitin fermented feed additive were observed.
구체적으로, 비장림프구에서 mRNA 사이토카인 발현 수준(cytokine expression level) 확인을 위해 분리한 비장림프구를 1 × 106 cells 용량으로 12 well plate에 seeding 한 후, T cell mitogen인 concanavalin a (ConA)를 처리하여 36시간동안 배양하였다. 배양 후에는 다시 cell을 수거하여 RNA extraction kit를 이용한 RNA 분리를 실시하였고, 최종적으로 분리한 RNA는 cDNA 합성 후 Real-time PCR에 사용하였다. Specifically, spleen lymphocytes isolated to check mRNA cytokine expression level in splenic lymphocytes were seeded in a 12 well plate at a capacity of 1 × 10 6 cells, and then treated with concanavalin a (ConA), a T cell mitogen. and incubated for 36 hours. After incubation, the cells were collected again and RNA was isolated using an RNA extraction kit, and the finally isolated RNA was used for real-time PCR after cDNA synthesis.
구체적으로, cDNA를 template로 하여 MyiQTM real-time PCR detection system (Bio-165 Rad, Hercules, CA, USA)을 이용하여 Interleukin (IL)-1β, IL-2, IL-12, TNF-a, IFN-gamma 수준을 측정하였고, 그 결과를 각각 도 12 내지 도 16에 나타내었다.Specifically, Interleukin (IL)-1β, IL-2, IL-12, TNF-a, IFN using MyiQTM real-time PCR detection system (Bio-165 Rad, Hercules, CA, USA) using cDNA as a template -gamma levels were measured, and the results are shown in FIGS. 12 to 16, respectively.
도 13에 따르면, IL-2의 경우 ND 군에 비해 C4, W2, W4, B2, B4 군에서 고도의 유의적인 증가(***p<0.001)를 나타내었다.According to FIG. 13 , in the case of IL-2, the C4, W2, W4, B2, and B4 groups showed a highly significant increase (***p<0.001) compared to the ND group.
도 14에 따르면, IL-12의 경우 ND 군에 비해 C4와 W2 군에서 유의적인 증가(*p<0.05), B2, B4군에서 유의적인 증가(**p<0.01), 특히 W4군에서 고도의 유의적인 증가(***p<0.001)를 나타내었다. 즉, 본 발명의 실시예 1의 곤충키틴 발효사료첨가제가 가장 우수한 효과를 나타내었다.According to FIG. 14 , in the case of IL-12, a significant increase (*p<0.05) in the C4 and W2 groups compared to the ND group, a significant increase in the B2 and B4 groups (**p<0.01), especially in the W4 group, high showed a significant increase (***p<0.001). That is, the insect chitin fermented feed additive of Example 1 of the present invention showed the most excellent effect.
도 15에 따르면, TNF-a의 경우 ND 군에 비해 C4와 W2 군에서 유의적인 증가(*p<0.05,**p<0.01), W4, B2, B4 군에서는 고도의 유의적인 증가(***p<0.001)를 나타내었다. 즉, 본 발명의 곤충키틴 발효사료첨가제가 발효하지 않은 곤충키틴 사료첨가제에 비하여 효과가 더 우수하였다.According to FIG. 15 , in the case of TNF-a, a significant increase (*p<0.05, **p<0.01) in the C4 and W2 groups compared to the ND group, a highly significant increase in the W4, B2, and B4 groups (** *p<0.001). That is, the insect chitin fermented feed additive of the present invention was more effective than the non-fermented insect chitin feed additive.
도 16에 따르면, IFN-gamma에서 ND 군에 비해 C4와 B4 군은 유의적인 증가(**p<0.01)를, C2, W2, W4, B2 군에서 고도의 유의적인 증가(***p<0.001)를 나타내었다.According to FIG. 16, compared to the ND group in IFN-gamma, the C4 and B4 groups showed a significant increase (**p<0.01), and the C2, W2, W4, and B2 groups showed a highly significant increase (***p< 0.001) was shown.
이와 같은 결과에 따르면, 곤충키틴 사료첨가제 및 곤충키틴 발효사료첨가제의 급여는 육계의 면역반응 사이토카인의 분비량을 증가시켜 면역성을 향상시킬 수 있으며, 특히 본 발명의 실시예 1 또는 실시예 2의 곤충키틴 발효사료첨가제 급여군에서 면역 사이토카인 발현량이 대체로 높게 측정되었다.According to these results, the feeding of insect chitin feed additive and insect chitin fermented feed additive can improve immunity by increasing the secretion of immune response cytokines of broilers, especially the insects of Example 1 or Example 2 of the present invention In the group fed with chitin fermented feed additive, the expression level of immune cytokines was generally high.
실험예 6: 항병력 효과 분석Experimental Example 6: Analysis of the anti-history effect
급여 후 21일 차에 각 실험군에 살모넬라 갈리나룸 (Salmonella Gallinarum)의 인공 감염을 실시하였다. 인공 감염에 사용한 S. gallinarum의 균주는 SG3001이며, 영양배지(nutrient broth)에 37℃에서 오버나이트로 배양하여 1x109 cfu/㎖의 농도로 마리당 5㎖씩 경구 접종하였다. S. gallinarum 공격 접종 이후에는 매일 임상증상 평가 및 생존율 확인을 실시하였으며, 공격 접종 14일 이후에 안락사하여 부검을 실시하여 병리학적 병변확인을 실시하였다.On the 21st day after feeding, artificial infection of Salmonella Gallinarum was performed in each experimental group. The strain of S. gallinarum used for artificial infection is SG3001, and it was cultured overnight at 37° C. in a nutrient broth and orally inoculated at a concentration of 1x10 9 cfu/ml by 5 ml per animal. After S. gallinarum challenge inoculation, clinical symptoms were evaluated and survival rate was checked every day. After 14 days of attack inoculation, euthanasia was performed and an autopsy was performed to confirm pathological lesions.
부검한 닭의 내장 사진을 도 17 내지 도 19에 나타내었으며, 도 17은 ND, C2, C4군의 사진이고, 도 18은 W2, W4군의 사진이며, 도 19는 B2, B4군의 사진이다. The autopsied chicken intestine photos are shown in Figures 17 to 19, Figure 17 is a photograph of the ND, C2, and C4 groups, Figure 18 is a photograph of the W2, W4 groups, and Figure 19 is a photograph of the B2, B4 groups. .
이에 따르면, 대부분의 감염된 닭의 간에서 살모넬라 감염증에 의한 전형적인 증상 중에 하나인 회백색 반점(white spot)을 관찰할 수 있었고, 비장의 비대와 맹장 부위에서 충혈 증상을 관찰할 수 있었다. 실험군별로 소견을 살펴보면, ND 군에서 살모넬라 감염시에 특이적으로 관찰되는 회백색 반점이 간 전체에서 관찰되었지만, 사료첨가제를 투여한 C, W, B군의 간에서는 회백색 반점이 ND군에 비해 적적은 수로 관찰되었으며, 살모넬라 감염에 대한 병변의 정도가 현저히 감소하는 경향을 나타내었다. 즉, 부검 후 육안소견으로 곤충키틴 발효사료첨가제의 급여는 육계에서 살모넬라 감염에 대한 저항성을 증가시키는 효과를 보였다.According to this, white spots, one of the typical symptoms of Salmonella infection, could be observed in the livers of most infected chickens, and hypertrophy of the spleen and hyperemia were observed in the cecum. Looking at the findings by experimental group, gray-white spots, which were specifically observed during Salmonella infection, were observed throughout the liver in the ND group, but fewer gray-white spots were observed in the livers of groups C, W, and B treated with feed additives compared to the ND group. number was observed, and the degree of lesion for Salmonella infection showed a tendency to significantly decrease. That is, after the autopsy, the feeding of insect chitin fermented feed additive showed the effect of increasing resistance to Salmonella infection in broilers.
한편, 공격 접종후 재분리되는 살모넬라 세균수 조사를 위하여 S. gallinarum 공격접종 이후 14일째에 모든 육계를 안락사한 후 부검을 실시하여 분변(feces)과 살모넬라의 대표적인 표적 장기인 간(liver), 비장(spleen), 윤활낭(bursa), 맹장(cecum)에서 S. gallinarum의 수를 측정하였다. 구체적으로, 샘플 1g을 취하여 멸균된 PBS 9㎖ 에 넣고 vortexing한 다음 적정 농도까지 계단 희석 후 Macconkey plate에 희석액 0.1㎖를 접종하여 37℃에서 1-2일간 배양 후 집락을 계수하였다. 집락 수는 확산 집락이 없고 30-300개의 집락이 있는 평판을 선택하여 계수하였다. 이에 따른 살모넬라 균수 측정 결과를 도 20 내지 도 24에 나타내었다. 도 20은 분변(feces), 도 21은 간(liver), 도 22는 비장(spleen), 도 23은 윤활낭(bursa), 도 24는 맹장(cecum)에서 측정된 살모넬라 균수 측정결과이다. On the other hand, in order to investigate the number of Salmonella bacteria re-isolated after attack inoculation, all broilers were euthanized on the 14th day after S. gallinarum challenge inoculation, and then an autopsy was performed. The number of S. gallinarum was measured in the spleen, bursa, and cecum. Specifically, 1 g of a sample was placed in 9 ml of sterilized PBS, vortexed, and serially diluted to an appropriate concentration. Then, 0.1 ml of the diluted solution was inoculated on a Macconkey plate, and incubated at 37° C. for 1-2 days, colonies were counted. The number of colonies was counted by selecting plates with no diffuse colonies and 30-300 colonies. Accordingly, the results of the measurement of the number of Salmonella bacteria are shown in FIGS. 20 to 24 . 20 is a feces (feces), FIG. 21 is a liver (liver), FIG. 22 is a spleen (spleen), FIG. 23 is a bursa, and FIG.
도 20에 따르면, 닭의 분변 속 살모넬라의 그룹 간의 차이는 ND 군과 비교하여 C4, W2, W4, B2, B4 군에서 유의적으로 감소했다(*p<0.05, **p<0.01).According to FIG. 20, the difference between the groups of Salmonella in chicken feces was significantly reduced in the C4, W2, W4, B2, B4 groups compared to the ND group (*p<0.05, **p<0.01).
도 21에 따르면, 닭의 간 조직에서 살모넬라 검출량은 ND 군에 비해 C2, C4, W2, W4, B2, B4 군에서 유의적으로 감소하였다(*p<0.05, **p<0.01, ***p<0.001).According to FIG. 21, the detection amount of Salmonella in the liver tissue of chickens was significantly decreased in the C2, C4, W2, W4, B2, and B4 groups compared to the ND group (*p<0.05, **p<0.01, *** p<0.001).
도 22에 따르면, 닭의 비장 조직에서 살모넬라 검출량은 그룹 간 유의적인 차이가 보이지 않았다.According to FIG. 22 , there was no significant difference between the groups in the amount of Salmonella detected in the spleen tissue of chickens.
도 23에 따르면, 닭의 윤활낭 조직에서 살모넬라 검출량은 ND 군에 비해 C4, B4 군에서 유의적으로 감소하였다(*p<0.05).According to FIG. 23 , the detection amount of Salmonella in the bursa tissue of chickens was significantly decreased in the C4 and B4 groups compared to the ND group (*p<0.05).
도 24에 따르면, 닭의 맹장 조직에서 살모넬라 검출량은 ND 군에 비해 C2, C4, W2, W4, B2, B4 군에서 유의적으로 감소하였다(**p<0.01, ***p<0.001).According to FIG. 24, the amount of Salmonella detected in the cecal tissue of chickens was significantly decreased in the C2, C4, W2, W4, B2, and B4 groups compared to the ND group (**p<0.01, ***p<0.001).
따라서, 곤충키틴 사료첨가제 및 곤충키틴 발효사료첨가제의 급여는 육계의 분변, 간, 비장, 맹장, 윤활낭 조직에서 살모넬라의 재분리율을 감소시킴을 확인하였다. 즉, 이는 곤충키틴 유래의 사료첨가제의 급여는 살모넬라 감염에 대한 항병력을 증가시키는 효능을 나타내고, 고농도의 사료첨가제 급여군에서 고도의 유의성을 나타내었다.Therefore, it was confirmed that the feeding of insect chitin feed additive and insect chitin fermented feed additive reduced the re-isolation rate of Salmonella from broiler feces, liver, spleen, cecum, and bursa tissue. That is, the feeding of insect chitin-derived feed additives showed the efficacy of increasing the anti-military force against Salmonella infection, and showed a high level of significance in the high concentration feed additive feeding group.
한편, S. gallinarum 공격 접종 이후 육계에서 림프구아집단(lymphocyte subpopulation) 분석을 실시하였다. S. gallinarum 공격 접종 이후 14일째에 부검을 실시하여 비장을 채취한 후, 비장림프구 분리 및 이를 이용한 림프구아집단을 확인하였다.Meanwhile, analysis of lymphocyte subpopulation was performed in broilers after S. gallinarum challenge inoculation. On the 14th day after S. gallinarum challenge inoculation, an autopsy was performed and the spleen was collected, and the splenic lymphocytes were isolated and the lymphocyte subpopulation using the spleen was confirmed.
구체적으로, 비장림프구에서 CD3+CD4+CD8-, CD3+CD4-CD8+ 림프구(lymphocyte)의 비율 측정을 위해 1 X 106 용량으로 희석된 비장림프구를 PE/Cy7 anti-chicken CD3 (T cell marker), FITC anti-chicken CD4 (help T cell marker)와 PE anti-chicken CD8 (cytotoxic T cell marker)의 항체로 암실에서 30분간 반응시켜 염색하였다. 반응 후에 2회 원심 세척하고 PBS 1㎖를 분주하여 유세포분석기 (BD Accuri Flow Cytometry, BD Biosciences, USA)를 이용하여 CD3+CD4+CD8-, CD3+CD4-CD8+ 림프구(lymphocyte)의 비율을 측정하여 그 결과를 도 25 내지 도 27에 나타내었다.Specifically, to measure the ratio of CD3+CD4+CD8-, CD3+CD4-CD8+ lymphocytes in splenic lymphocytes, splenic lymphocytes diluted in 1 X 10 6 doses were used as PE/Cy7 anti-chicken CD3 (T cell marker). , FITC anti-chicken CD4 (help T cell marker) and PE anti-chicken CD8 (cytotoxic T cell marker) antibodies were reacted for 30 minutes in the dark for staining. After the reaction, centrifugal washing was performed twice, 1 ml of PBS was dispensed, and the ratio of CD3+CD4+CD8-, CD3+CD4-CD8+ lymphocytes was measured using a flow cytometer (BD Accuri Flow Cytometry, BD Biosciences, USA). The results are shown in FIGS. 25 to 27 .
이에 따르면, helper T cell(CD4)의 경우 ND군은 14.25%, C2군은 18.72%, C4군은 21.59%, W2군은 21.34%, W4군은 23.01%, B2군은 21.49%, B4군은 22.35%를 나타냈으며, cytotoxic T cell(CD8)의 경우 ND군은 8.51%, C2군은 7.28%, C4군은 7.42%, W2군은 6.14%, W4군은 6.83%, B2군은 6.64%, B4군은 4.87%를 나타내었다. 따라서 ND 군에 비해 비장세포의 CD4와 CD8의 비율(ratio)은 C4, W2, W4, B2, B4 군에서 통계적으로 유의적으로 그 수치가 증가함을 확인할 수 있었으며(*p<0.05, **p<0.01, ***p<0.001), 특히 실시예 1 또는 실시예 2의 곤충키틴 발효사료첨가제 급여군인 W2, W4, B2, B4군에서 높은 수치를 나타내었다.According to this, in the case of helper T cells (CD4), 14.25% in ND group, 18.72% in C2 group, 21.59% in C4 group, 21.34% in W2 group, 23.01% in W4 group, 21.49% in B2 group, 21.49% in B4 group. 22.35%, and for cytotoxic T cells (CD8), ND group was 8.51%, C2 group was 7.28%, C4 group was 7.42%, W2 group was 6.14%, W4 group was 6.83%, B2 group was 6.64%, B4 group showed 4.87%. Therefore, compared to the ND group, it was confirmed that the ratio of CD4 and CD8 of splenocytes increased statistically and significantly in the C4, W2, W4, B2, and B4 groups (*p<0.05, ** p<0.01, ***p<0.001), especially in the W2, W4, B2, B4 groups, which are the insect chitin fermented feed additive feeding groups of Example 1 or Example 2, showed high values.
이상의 결과를 종합해보면, 살모넬라에 공격 접종 후 C4, W2, W4, B2, B4 군에서 T cell의 CD4/CD8의 비율이 증가함을 보였음. 일반적으로 T cell의 CD4/CD8의 비율이 증가한 경우는 면역력이 증강되었다는 것을 의미한다. 특히 발효되지 않은 비교예 1의 곤충키틴 사료첨가제 급여군 보다 실시예 1 또는 2의 곤충키틴 발효사료첨가제 급여군의 면역 활성이 더욱 증가하는 것을 나타났다.Combining the above results, it was shown that the CD4/CD8 ratio of T cells increased in C4, W2, W4, B2, and B4 groups after challenge inoculation with Salmonella. In general, when the ratio of CD4/CD8 of T cells is increased, it means that immunity is enhanced. In particular, it was shown that the immune activity of the insect chitin fermented feed additive-fed group of Example 1 or 2 was further increased than the non-fermented insect chitin feed additive-fed group of Comparative Example 1.
이상, 본 발명의 실시예들에 대하여 설명하였으나, 해당 기술 분야에서 통상의 지식을 가진 자라면 특허청구범위에 기재된 본 발명의 사상으로부터 벗어나지 않는 범위 내에서, 구성 요소의 부가, 변경, 삭제 또는 추가 등에 의해 본 발명을 다양하게 수정 및 변경시킬 수 있을 것이며, 이 또한 본 발명의 권리범위 내에 포함된다고 할 것이다.In the above, although embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or add components within the scope that does not depart from the spirit of the present invention described in the claims. The present invention may be variously modified and changed by, etc., and this will also be included within the scope of the present invention.
Claims (17)
(b) 곤충 번데기의 탈피각이 첨가된 배지에서 키틴 분해 미생물을 배양하여 키틴 분해 미생물 배양액을 제조하는 1차 발효단계; 및
(c) 상기 곤충탈지분말을 포함하는 배지에 상기 키틴 분해 미생물 배양액 및 발효균을 혼합하여 배양하는 2차 발효단계;를 포함하고,
단계 (c)에서 상기 발효균은 광합성균, 방선균, 유산균 및 고초균이 혼합된 복합 발효균; 및 3종의 추가 유산균;을 포함하거나, 또는
단계 (c)에서 상기 발효균은 단백질 분해 미생물이고,
상기 복합 발효균은 상기 발효균은 로도슈도모나스 팔루스트리스(Rhodopseudomonas palustris), 스트렙토마이세스 sp.(Streptomyces sp.), 락토바실러스 플란타넘(Lactobacillus plantarum) 및 바실러스 아밀로리큐파시엔시스(Bacillus amyloliquefaciens)를 포함하고,
상기 추가 유산균은 스트렙토코커스 패칼리스(Streptococcus faecalis), 락토바실러스 애시도펠리스(Lactobacillus acidophilus) 및 비피도박테리움 비피덤(Bifidobacterium bifidum)를 포함하고,
상기 단백질 분해 미생물은 바실러스 리체니포르미스(Bacillus licheniformis)인 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.(a) milking dried insects to remove fat and pulverizing them to prepare insect degreasing powder;
(b) a primary fermentation step of culturing chitin-degrading microorganisms in a medium to which the molting shells of insect pupae are added to prepare a chitin-degrading microorganism culture solution; and
(c) a secondary fermentation step of culturing by mixing the chitin-degrading microorganism culture solution and fermenting bacteria in a medium containing the insect defatted powder;
In step (c), the fermenter is a complex fermenter in which photosynthetic bacteria, actinomycetes, lactic acid bacteria and Bacillus subtilis are mixed; and three additional lactic acid bacteria; or
In step (c), the fermenter is a proteolytic microorganism,
The complex fermentation bacteria include Rhodopseudomonas palustris, Streptomyces sp., Lactobacillus plantarum and Bacillus amyloliquefaciens. ,
The additional lactic acid bacteria include Streptococcus faecalis, Lactobacillus acidophilus and Bifidobacterium bifidum,
The proteolytic microorganism is a method for producing a feed additive comprising a fermented insect chitin, characterized in that the Bacillus licheniformis (Bacillus licheniformis).
상기 곤충은 아메리카동애등에, 동애등에, 풍뎅이, 파리, 흰점박이 꽃무지, 갈색거저리, 누에, 메뚜기 및 귀뚜라미 중에서 선택된 1종 이상인 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
The method for producing a feed additive containing fermented insect chitin, characterized in that the insect is at least one selected from the group consisting of stinging insects, etc., stinging insects, scarabs, flies, white-spotted beetles, brown mealworms, silkworms, grasshoppers and crickets.
단계 (b)에서 상기 곤충 번데기는 파리, 아메리카동애등에, 동애등에, 풍뎅이, 흰점박이 꽃무지, 갈색거저리 및 누에 중에서 선택된 1종 이상인 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
In step (b), the insect pupae is a fermented insect chitin, characterized in that at least one selected from among flies, American larvae, egrets, scarabs, white spotted flower beetles, brown mealworms and silkworms. Method for producing a feed additive containing chitin .
단계 (b)에서 상기 키틴 분해 미생물은 바실러스 서브틸리스(Bacillus subtilis), 바실러스 리체니포미스 (Bacillus licheniformis), 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquefaciens) 및 바실러스 투링기엔시스(Bacillus thuringiensis), 락토바실루스 사케이(Lactobacillus sakei), 류코노스톡 시트륨(Leuconostoc citreum), 바이셀라 코리엔시스(Weissella koreensis)중에서 선택된 어느 하나인 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
In step (b), the chitin-decomposing microorganism is Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus thuringiensis, Lactobacillus Sake (Lactobacillus sakei), leuconostoc citreum (Leuconostoc citreum), Weissella koreensis (Weissella koreensis), characterized in that any one selected from the method of manufacturing a feed additive containing fermented insect chitin.
단계 (b)의 1차 발효단계는 액상 배양에 의해 수행되는 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
The primary fermentation step of step (b) is a method for producing a feed additive containing fermented insect chitin, characterized in that performed by liquid culture.
단계 (c)에서 상기 곤충탈지분말을 포함하는 배지는 밀기울, 올리고당, 키토산, 미강 중에서 선택된 1종 이상을 포함하는 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
In step (c), the medium containing the insect defatted powder is a method of manufacturing a feed additive containing fermented insect chitin, characterized in that it contains at least one selected from bran, oligosaccharide, chitosan, and rice bran.
상기 복합 발효균에 포함되는 상기 방선균은 스트렙토마이세스 그리세우스(Streptomyces griseus), 스트렙토마이세스 코엘리콜라(Streptomyces coelicolor), 스트렙토마이세스 멜라노스포로파시엔스(Streptomyces melanosporofaciens), 스트렙토마이세스 아베르미틸리스(Streptomyces avermitilis), 사카로폴리스포라 에리트레아(Saccharopolyspora erythraea), 스트렙토마이세스 퓨세티우스(Streptomyces peucetius) 및 스트렙토마이세스 아쿨레오라투스(Streptomyces aculeolatus) 중에서 선택된 1종 이상인 것을 특징으로 하는 발효된 곤충키틴을 포함하는 사료첨가제의 제조방법.The method of claim 1,
The actinomycetes included in the complex fermentation bacteria are Streptomyces griseus, Streptomyces coelicolor, Streptomyces melanosporofaciens, Streptomyces avermitili S (Streptomyces avermitilis), Saccharopolyspora erythraea (Saccharopolyspora erythraea), Streptomyces peucetius (Streptomyces peucetius) and Streptomyces aculeolatus (Streptomyces aculeolatus) characterized in that at least one fermented insect selected from A method for manufacturing a feed additive containing chitin.
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