KR20090066410A - Feed additive composition containing Bacillus subtilis BCC1212 strain - Google Patents
Feed additive composition containing Bacillus subtilis BCC1212 strain Download PDFInfo
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- KR20090066410A KR20090066410A KR1020070133915A KR20070133915A KR20090066410A KR 20090066410 A KR20090066410 A KR 20090066410A KR 1020070133915 A KR1020070133915 A KR 1020070133915A KR 20070133915 A KR20070133915 A KR 20070133915A KR 20090066410 A KR20090066410 A KR 20090066410A
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- South Korea
- Prior art keywords
- bacillus subtilis
- strain
- feed
- composition
- addition
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Abstract
본 발명은 사료 첨가용 조성물에 관한 것으로서, 보다 상세하게는 바실러스 서브틸리스 BC1212 균주를 포함하는 사료 첨가용 조성물에 관한 것이다. 본 발명의 사료 첨가용 조성물은 종래 사료 첨가용으로 사용되는 미생물과는 달리 항균 활성을 갖는 항생물질을 생산하며, 효소 생산 능력에서도 종래 균주와는 다른 패턴을 갖고 있어 축산동물의 사료 효율 및 체중 증대에 유용하게 사용될 수 있다.The present invention relates to a feed additive composition, and more particularly to a feed additive composition comprising a Bacillus subtilis BC1212 strain. The feed composition of the present invention produces antibiotics having antimicrobial activity unlike microorganisms used for conventional feed addition, and has a different pattern from conventional strains in the ability to produce enzymes, thereby increasing feed efficiency and weight gain of livestock animals. It can be usefully used.
Description
본 발명은 사료 첨가용 조성물에 관한 것으로서, 보다 상세하게는 항생물질을 생산하는 바실러스 서브틸리스 종의 미생물을 포함하는 사료 첨가용 조성물에 관한 것이다.The present invention relates to a feed additive composition, and more particularly to a feed additive composition comprising a microorganism of Bacillus subtilis species that produce antibiotics.
가축 및 이들이 생산하는 생산품은 인류가 생존하기 위해 필요한 단백질의 중요한 공급원으로서 가축의 사육 및 이용의 발전은 인류의 건강과 생활의 질을 향상시키는데 있어 밀접한 관련을 갖는다. 가축을 사육하기 위한 사료는 현재 많은 부분에서 곡물이 사용되고 있는데, 최근 인구가 급격하게 증가함으로써 식량으로의 곡물의 수요가 증대되어 곡물을 사료의 원료로서 이용하기는 점점 어렵게 되었고 이러한 곡물 사료의 부족이 가축 사육의 발전에 제약이 되고 있다. 이에 사료 산업계에서는 곡물을 대체할 수 있는 사료의 개발 및 이용되는 사료의 효율을 극대화시키기 위한 많은 노력을 기울였으며, 동물의 소화기능 및 흡수율을 증진시킬 수 있는 사료 첨가용 조성물의 개발이 사료산업에서 중요한 위치를 차지하게 되었다. 사료 첨가용 조성물의 개발은 가축의 비육과 소화를 촉진시킴으로써 사료의 효율을 극대화시켜 사료 사용량을 줄일 수 있을 뿐만 아니라 빠른 증식과 비육으로 인해 고품질의 가축 생산을 가능하게 하여 농가의 소득 수준 향상에 기여할 수 있다는 점에서 그 중요성이 매우 크다고 할 수 있다.Livestock and the products they produce are an important source of protein for humankind's survival, and the development of livestock raising and utilization is closely linked to improving human health and quality of life. Grain is currently used in many parts of the feed for raising livestock, and the recent rapid increase in the population has increased the demand for grain for food, making it difficult to use grain as a feed source. It is limiting the development of livestock raising. Therefore, the feed industry has made a lot of efforts to maximize the efficiency of the feed and the feed used to replace the grain, the development of feed additive composition to improve the digestive function and absorption rate of animals in the feed industry It has taken an important position. The development of feed additives will not only reduce feed consumption by maximizing feed efficiency by promoting livestock fattening and digestion, but also contribute to improving farm household income levels by enabling high quality livestock production due to rapid growth and fattening. It can be said that the importance is very large in that it can be.
전통적으로 돼지 및 가금을 위한 동물 사료로는 주로 대두박, 참깨, 맥류, 볏짚 및 옥수수 등이 사용되어 왔으며, 최근에는 땅콩, 완두콩, 사탕무, 펄프, 곡물 부산물, 동물 내장 가루 및 어분 가루 등과 같은 대체 제품의 사용이 크게 증가되고 있다. 현재 축산농가에서 사용되는 동물 사료에는 아미노산, 무기염료, 비타민, 항생물질, 효소 등과 함께 생균제(살아있는 미생물제제)를 첨가하는데, 이는 영양보충, 소화 및 흡수 향상, 성장촉진, 질병예방 등과 같은 효과를 나타내기 위함이다. 그러나, 소화기계 질병이나 장기간에 걸친 항생제 사용 등으로 사육동물들의 건강을 유지하기가 쉽지 않은 실정이다. 또한, 가축에 있어서 장기간의 동물 약물 및 항생물질의 사용은 잔류 약물을 인간이 섭취함으로써 알레르기나 장내 정상 세균총의 변화를 일으키기 때문에 그의 용도가 제한되어 있으며, 그의 사용범위도 제한 및 감소되고 있다.Traditionally, animal feed for pigs and poultry has been used mainly for soybean meal, sesame seeds, wheat flour, rice straw and corn, and recently, alternative products such as peanuts, peas, sugar beets, pulp, grain by-products, animal intestine flour and fishmeal flour. The use of is greatly increased. Animal feed currently used in livestock farms is added with probiotics (living microbial agents) along with amino acids, inorganic dyes, vitamins, antibiotics, enzymes, etc., which help to improve nutrition, improve digestion and absorption, promote growth, and prevent disease. To indicate. However, it is not easy to maintain the health of breeding animals due to diseases of the digestive system or the use of antibiotics for a long time. In addition, long-term use of animal drugs and antibiotics in livestock is limited in their use because of the ingestion of residual drugs by humans causing changes in allergic or normal intestinal flora, and their range of use is also limited and reduced.
이와 같은 문제점들을 개선하기 위하여, 최근 사육 동물에서 발생하는 질병의 예방 및 치료를 위해 생균제로서 바실러스 나토, 유산 박테리아, 부틸산 박테리아, 락토바실러스 비피더스 등의 미생물이 연구되어 사용되고 있으나, 완전한 동물 질병 치료제로서 부족한 면이 많이 있다. 따라서, 동물에게 보다 안전할 뿐만 아 니라 동물의 장내에서 뛰어난 생존력을 나타낼 수 있는 새로운 생균제의 개발이 요구되고 있는 실정이다.In order to improve these problems, microorganisms such as Bacillus natto, lactic acid bacteria, butyric acid bacteria, Lactobacillus bifidus, etc. have been studied and used as probiotics for the prevention and treatment of diseases occurring in farm animals. There are many things that are lacking. Therefore, the development of new probiotics that are not only safer for animals but also show excellent viability in the intestines of animals are required.
이에, 본 발명자들은 동물사료 첨가제로 사용될 수 있는 미생물에 대한 광범위한 연구를 계속한 결과, 바실러스 서브틸리스 종에 속하는 미생물 균주로서 본 발명자들에 의해 분리된 바 있는 바실러스 서브틸리스 BC1212 균주가 종래 사육 동물에 미생물 첨가제로 사용되는 미생물과는 달리 항진균 및 항생활성을 갖는 물질을 생산할 뿐만 아니라, 다양한 효소생성 능력을 가져 사육 동물의 정장 및 소화 촉진 작용을 증가시킴으로써 사료 첨가용 생균제로 유용하게 사용될 수 있음을 밝힘으로써 본 발명을 완성하였다.Accordingly, the present inventors have continued extensive research on microorganisms that can be used as an animal feed additive, and as a result, the Bacillus subtilis BC1212 strain, which has been isolated by the present inventors as a microbial strain belonging to the Bacillus subtilis species, has been conventionally bred. Unlike microorganisms that are used as microbial additives in animals, it not only produces antifungal and anti-living substances, but also has various enzyme-generating ability, which can be useful as a probiotic for feed addition by increasing the suitability and digestion promoting action of a breeding animal. The present invention was completed by revealing.
본 발명의 목적은 바실러스 서브틸리스 BC1212 균주를 포함하는 사료 첨가용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for feed addition comprising Bacillus subtilis BC1212 strain.
상기 목적을 달성하기 위하여, 본 발명은 바실러스 서브틸리스 BC1212 균주를 포함하는 사료 첨가용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for feed addition comprising Bacillus subtilis BC1212 strain.
상기 바실러스 서브틸리스 BC1212 균주는 바실러스 속의 통성혐기성 박테이라군에 속하며, 항생물질인 화학식 1로 표시되는 설팩틴(surfactin) C를 생산한다.The Bacillus subtilis BC1212 strain belongs to the facultative anaerobic Bacteria family of the genus Bacillus, and produces sulfactin C represented by the antibiotic formula (1).
상기 바실러스 서브틸리스 BC1212 균주는 2004년 11월 8일자로 농업생명공학연구원에 기탁되어 있다(기탁번호: KACC 91141). The Bacillus subtilis BC1212 strain has been deposited with the Institute of Agricultural Biotechnology on November 8, 2004 (Accession No .: KACC 91141).
본 발명의 사료 첨가용 조성물의 제조에 사용되는 바실러스 서브틸리스 BC1212 균주는 공지의 발효기술에 의해 액체 배지 또는 고체 배지에서 배양될 수 있다. 배지로는 천연배지, 반합성배지 및 합성배지와 같은 각종 배지가 제한없이 사용될 수 있다. (ⅰ) 탄소원으로는 글루코스, 수크로스, 덱스트린, 글리세롤, 녹말, 몰라세 등이 사용될 수 있다; (ⅱ) 질소원으로는 펩톤, 육류 추출물, 효모 추출물, 건조된 효모, 대두 케이크, 우레아, 티오우레아, 암모늄염, 나이트레이트 및 기타 유기 또는 무기 질소-함유 화합물이 사용될 수 있다; (ⅲ) 무기염으로는 마그네슘, 망간, 포타슘, 칼슘, 철 등의 포스페이트, 나이트레이트, 카보네이트, 클로라이드 등이 사용될 수 있다. 그러나, 탄소원, 질소원 및 무기염은 반드시 상기 성분들에만 한정되는 것은 아니며, 그 외에도 아미노산, 비타민, 핵산 및 그와 관 련된 화합물들이 배지에 첨가될 수 있다.Bacillus subtilis BC1212 strain used in the preparation of the feed additive composition of the present invention can be cultured in a liquid medium or a solid medium by a known fermentation technique. As the medium, various mediums such as natural medium, semi-synthetic medium and synthetic medium can be used without limitation. (Iii) glucose, sucrose, dextrin, glycerol, starch, molasses and the like may be used as the carbon source; (Ii) nitrogen sources may include peptone, meat extract, yeast extract, dried yeast, soy cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds; (Iii) As inorganic salts, phosphates such as magnesium, manganese, potassium, calcium and iron, nitrates, carbonates, chlorides and the like can be used. However, the carbon source, nitrogen source and inorganic salts are not necessarily limited to the above components, in addition, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.
미생물은 공지의 발효 기술에 따라 적당한 온도에서 적당한 기간동안 배양되며, 바람직하게는 예를 들면 20℃∼50℃에서 12시간∼5일동안 배양된다. 본 발명에 따라 수득된 배양액은 반복 세척함으로써 분리된 세포와 함께 또는 배양액 단독으로 사용될 수 있다. 그 외에도 세포를 함유한 배양액 또는 분리된 세포는 첨가제를 가하거나 가하지 않은 채로 동결건조 또는 분무건조되어 제제화될 수 있다. 이렇게 수득된 제제는 본 발명의 사료 첨가제 또는 사료로 사용될 수 있다.The microorganism is incubated for a suitable period of time at a suitable temperature in accordance with known fermentation techniques, preferably for 12 hours to 5 days at 20 ℃ to 50 ℃, for example. The culture obtained according to the present invention can be used together with the cells isolated or by the culture alone by repeated washing. In addition, the culture medium or separated cells containing the cells can be formulated by lyophilization or spray drying with or without the addition of additives. The formulation thus obtained can be used as a feed additive or feed of the present invention.
본 발명에 따른 사료 첨가용 조성물을 제조하기 위하여, 배양액을 직접 제제화하거나 밀분, 녹말, 텍스트린 등의 희석제 및 곡류, 왕겨 및 탈지 쌀겨와 같은 겨, 오일 및 지방분이 풍부한 종자 케이크와 같은 사료용 원료와 함께 제제화될 수 있다. 또한, 배양액을 세척하여 세포를 분리한 후, 분리된 세포를 전술한 방법으로 제제화할 수 있다. 수득된 바실러스 서브틸리스 BC1212 균주의 살아있는 미생물 제제는 그를 함유한 사료형태로 또는 사료 첨가제 형태로 적당량 동물에 투여되어 체중 증가 및 촉진 성장을 시킨다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 사료 첨가용 조성물은 밀기울 1 내지 20 중량%, 쌀겨 1 내지 20 중량%, 대두박 1 내지 20 중량%, FeSO4 0.001 내지 0.1 중량%, MnSO4 0.001 내지 0.1 중량% 및 잔량의 물을 포함하며, 바람직하게는 밀기울 2.8 중량%, 쌀겨 4.9 중량%, 대두박 5.1 중량%, FeSO4 0.02 중량%, MnSO4 0.05 중량% 및 잔량(87.13 중량%)의 물을 포함한다.In order to prepare a composition for feed addition according to the present invention, the culture medium is formulated directly, or diluents such as wheat flour, starch, textrin, and the like, such as cereals, rice bran and skim rice bran, feed material such as bran, oil and fat-rich seed cake and Can be formulated together. In addition, after washing the culture solution to separate the cells, the isolated cells can be formulated by the method described above. The viable microbial preparation of the obtained Bacillus subtilis BC1212 strain is administered to an appropriate amount of animal in the form of a feed containing it or in the form of a feed additive to gain weight and promote growth. According to a preferred embodiment of the present invention, the composition for feed addition of the present invention is 1 to 20% by weight of bran, 1 to 20% by weight of rice bran, 1 to 20% by weight of soybean meal, FeSO 4 0.001 to 0.1% by weight, MnSO 4 0.001 to 0.1 wt% and balance water, preferably 2.8 wt% bran, 4.9 wt% rice bran, 5.1 wt% soybean meal, 0.02 wt% FeSO 4 , 0.05 wt% MnSO 4 , and balance (87.13 wt%) water. Include.
또한, 본 발명의 사료 첨가용 조성물은 건조 또는 액체 상태의 제제 형태일 수 있으며, 하나 이상의 효소 제제를 추가로 포함할 수 있다. 첨가되는 효소 제제는 건조 또는 액체 상태가 모두 가능하며, 리파제(lipase)와 같은 지방 분해효소, 피틴산(phytic acid)을 분해하여 인산염과 이노시톨인산염을 만드는 파이타제(phytase), 녹말과 글리코겐(glycogen) 등에 포함되어 있는 알파-1,4-글리코시드 결합(α-1,4-glycoside bond)을 가수분해하는 효소인 아밀라제(amylase), 유기인산에스테르를 가수분해하는 효소인 포스파타제(phosphatase), 셀룰로스(cellulose)를 분해하는 카르복시메틸셀룰라제(carboxymethylcellulase), 자일로스(xylose)를 분해하는 자일라나제(xylanase), 말토오스(maltose)를 두 분자의 글루코스(glucose)로 가수분해하는 말타제(maltase) 및 사카로스(saccharose)를 가수분해하여 글루코스-프룩토스(glucose-fructose) 혼합물을 만드는 전환효소(invertase) 등과 같은 당 생성 효소로 구성된 군으로부터 선택되어 사용될 수 있다.In addition, the feed composition of the present invention may be in the form of a dry or liquid formulation, it may further comprise one or more enzyme preparation. Enzyme preparations can be added in either dry or liquid form, lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and glycogen Amylase, an enzyme that hydrolyzes an α-1,4-glycoside bond, and phosphatase, cellulose, an enzyme that hydrolyzes an organophosphate ester carboxymethylcellulase to break down cellulose, xylanase to break down xylose, maltase to hydrolyze maltose into two molecules of glucose, and It may be selected and used from the group consisting of sugar producing enzymes such as invertase which hydrolyzes saccharose to form a glucose-fructose mixture.
또한, 본 발명의 조성물은 바실러스 서브틸리스 BC1212 균주 이외에 비병원성의 다른 미생물을 추가로 포함할 수 있다. 첨가할 수 있는 미생물로는 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능이 있는 락토바실러스 속(Lactobacillus sp.)의 미생물, 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 아스퍼질러스 오리자에(Aspergillus oryzae)와 같은 사상균(Slyter, L.L. J. Animal Sci. 1976, 43. 910-926) 및 사카로미세스 세레비지에(Saccharomyces cerevisiae)와 같은 효모(Jhonson, D.E et al. J. Anim. Sci., 1983, 56, 735-739; Williams, P.E.V. et al, 1990, 211) 등이 있으나, 이에 한정되는 것은 아니다.In addition, the compositions of the present invention may further comprise other non-pathogenic microorganisms in addition to the Bacillus subtilis BC1212 strain. The microorganisms that can be added include the microorganisms of Lactobacillus sp., Which have physiological activity and organic degradability under anaerobic conditions such as cow's stomach, increase the body weight of livestock, increase milk yield, and increase digestive absorption rate of feed. Filaments such as Aspergillus oryzae (Slyter, LLJ Animal Sci. 1976, 43. 910-926) and yeasts such as Saccharomyces cerevisiae (Jhonson, DE et al) J. Anim. Sci., 1983, 56, 735-739; Williams, PEV et al, 1990, 211), and the like.
각종 곡물 및 대두 단백을 비롯한 땅콩, 완두콩, 사탕무우, 펄프, 곡물 부산물, 동물 내장 가루 및 어분 가루 등과 같은 사료원료는 가공되지 않거나 또는 가공된 것을 적절히 사용할 수 있다. 가공 과정은 사료원료가 충진된 상태에서 가압 하에 일정한 배출구로 압축되는 공정으로 단백질의 경우에는 변성이 되어 이용성이 증가되는 압출 성형(extrusion)을 사용하는 것이 바람직하다. 압출 성형은 열처리 과정을 통해 단백질을 변성시키고, 항효소 인자를 파괴시키며, 단백질 분해효소의 활성을 분자적 구조변화에 의한 새로운 부위로의 효소적 노출에 의해 증가시키는 등의 장점을 갖는다. 또한, 대두 단백질과 같은 경우에는 압출 성형을 통해서 단백질의 소화율을 향상시키고 대두에 존재하는 단백질 분해효소의 저해제 중 하나인 트립신 저해제(trypsin inhibitor)와 같은 항 영양인자들을 불활성화시키며, 단백질 분해효소에 의한 소화율 향상을 증가시켜 대두 단백의 영양적 가치를 증가시킬 수 있다.Raw materials such as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera flour and fishmeal flour, including various grains and soy protein, may be appropriately used. The processing process is a process in which the feed material is compressed to a constant outlet under pressure in a state in which the feed material is filled, and in the case of protein, it is preferable to use an extrusion process in which the availability is increased. Extrusion has the advantages of denaturing proteins through heat treatment, destroying antienzyme factors, and increasing the activity of protease by enzymatic exposure to new sites by molecular structural changes. In addition, in the case of soy protein, the extrusion process improves the digestibility of the protein and inactivates antinutrients such as trypsin inhibitor, one of the inhibitors of protease present in soybean. Increasing digestion can increase the nutritional value of soy protein.
본 발명의 사료 첨가용 조성물이 사용될 수 있는 동물의 대표적인 예는 다음과 같다: 식용우, 젖소, 송아지, 돼지, 돼지새끼, 양, 염소, 말, 토끼, 개, 고양이 등과 같은 가축; 병아리, 알닭, 가정용 닭, 수탉, 오리, 거위, 칠면조, 메추라기, 작은새 등과 같은 가금류. 또한, 투여량은 투여될 동물의 종류, 나이 및 기타 사료 성분의 종류에 따라 변화되므로 일정하게 정의하기는 어렵지만, 일반적으로 본 발명의 사료 첨가용 조성물은 사료 1 ㎏당 살아있는 바실러스 서브틸리스 BC1212 102∼1015 세포, 바람직하게는 104∼1012 세포, 보다 바람직하게는 107∼1011 세포를 첨가한다.Representative examples of animals in which the composition for feed addition of the present invention can be used are as follows: cattle such as edible cattle, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats and the like; Poultry such as chicks, chickens, domestic chickens, roosters, ducks, geese, turkeys, quails, birds, etc. In addition, since the dosage varies depending on the type of animal to be administered, the age and the type of other feed components, it is difficult to define a constant, but in general, the feed composition of the present invention is a live Bacillus
본 발명의 바람직한 구현예에 따르면, 본 발명의 사료 첨가용 조성물에 사용되는 바실러스 서브틸리스 BC1212 균주는 산성 pH 조건, 특히 위산과 유사한 pH 1의 조건하에서도 뛰어난 생존력을 가지며(표 2 참조), 또한 담즙에서도 우수한 생존 및 성장 특성을 나타낸다. 이로부터, 상기 균주는 가축에 생균제로 사용할 경우 위 및 소장내에서도 생존하여 대장까지 안전하게 전달될 수 있음을 알 수 있다.According to a preferred embodiment of the present invention, the Bacillus subtilis BC1212 strain used in the feed composition of the present invention has excellent viability even under acidic pH conditions, especially
또한, 본 발명의 다른 바람직한 구현예에 따르면, 바실러스 서브틸리스 BC1212 균주는 종래에 생균제로 사용되고 있는 바실러스 서브틸리스 종의 다른 균주와는 전혀 다른 효소 활성을 나타낸다(도 5 참조). 바실러스 서브틸리스 BC1212는 알칼린 포스파타제(alkaline phosphatase) 및 산 포스파타제(acid phosphatase)에 대해 높은 활성을 나타내는데, 알칼린 포스파타제는 세균벽에서 분비되는 효소로서 세균이 외부환경에 대한 저항성을 가지는데 중요한 역할을 한다. 또한, 산 포스파타제는 인산분해효소로서 사료내의 인산 이용률을 증가시킬 수 있다. 산 및 염기 포스파타제는 인산화된 유기물로부터 인을 유리하는 역할을 하는 효소로서, 인은 생명체에서 필요로 하는 주요 무기물중의 하나로 에너지 대사(ATP) 및 생체 내 신호전달(kinase cascade)에 필수적인 인자 중에 하나이다. 바실러스 서브틸리스 BC1212 균주의 강한 포스파타제 활성은 장내에서 인산화된 단백질 및 당 등의 영양분으로부터 인을 유리하여 부족한 가축 체내에 인을 공급하며, 또한 포스파타 제는 BC1212 균주의 세포벽과 세포막 사이에 존재하여 인산화된 단백질 및 당 등의 영양분으로부터 생존에 필요한 유리된 인을 흡수하여 생존률을 증가시키게 된다(PNAS, 2004, 101, 7919-7924; Biochem, J., 1971, 125, 635-641). 이러한 결과는, 바실러스 서브틸리스 BC1212 균주의 강한 포스파타제 활성이 가축에게 부족하기 쉬운 영양성분인 인을 공급하고 BC1212 균주 자신은 생존에 필요한 인을 다량으로 흡수하여 생존률을 증가시킴으로써 항균 및 항바이러스 작용을 갖는 설펙틴의 생산 증가 효과와 더불어 가축의 장내 환경을 개선시키게 되고, 따라서 가축의 체중 증가를 보다 효과적으로 유도하게 된다는 것을 나타낸다.In addition, according to another preferred embodiment of the present invention, the Bacillus subtilis BC1212 strain exhibits enzymatic activity quite different from other strains of Bacillus subtilis species that are conventionally used as probiotics (see FIG. 5). Bacillus subtilis BC1212 has high activity against alkaline phosphatase and acid phosphatase.Alkaline phosphatase is an enzyme secreted from the bacterial wall and plays an important role in bacterial resistance to the external environment. Do it. In addition, acid phosphatase may increase phosphate utilization in feeds as phosphatase. Acid and base phosphatase are enzymes that release phosphorus from phosphorylated organics. Phosphorus is one of the major minerals required by life and is one of the essential factors for energy metabolism (ATP) and kinase cascade in vivo. . The strong phosphatase activity of the Bacillus subtilis BC1212 strain liberates phosphorus from phosphorylated proteins and sugars in the intestine to supply phosphorus in insufficient livestock bodies, and phosphatase is also present between the cell wall and the cell membrane of the BC1212 strain. Absorption of free phosphorus necessary for survival from phosphated proteins and sugars increases the survival rate (PNAS, 2004, 101, 7919-7924; Biochem, J., 1971, 125, 635-641). These results indicate that the strong phosphatase activity of the Bacillus subtilis BC1212 strain provides nutrients that are easily deficient in livestock, and the BC1212 strain itself absorbs large amounts of phosphorus necessary for survival to increase the survival rate. Together with the effect of increasing the production of sulfectin, it has been shown to improve the intestinal environment of the livestock, thus inducing more effective weight gain of the livestock.
또한, 본 발명의 다른 바람직한 구현예에 따르면, 상기 균주를 포함하는 사료를 급여할 경우에는 무첨가 대조군에 비해 사육 동물의 체중이 증가하며, 사료 효율 역시 무첨가 대조군에 비해 다소 높게 나타난다(표 3 참조). 아울러, 분변내 미생물 중 총혐기성균의 수는 차이를 나타내지 않으나, 바실러스 서브틸리스 BC1212 균주의 급여에 의해 분변내 바실러스 서브틸리스의 수는 증가하고, 반면 대장균의 수는 감소한다(표 4 참조).In addition, according to another preferred embodiment of the present invention, when feeding the feed containing the strain increases the body weight of the animal compared to the non-added control, the feed efficiency is also slightly higher than the non-added control (see Table 3) . In addition, the total number of anaerobic bacteria in fecal microorganisms does not show a difference, but the number of Bacillus subtilis in feces increases by the feeding of the Bacillus subtilis BC1212 strain, while the number of Escherichia coli decreases (see Table 4). ).
바실러스 서브틸리스 BC1212 균주를 포함하는 본 발명의 사료 첨가용 조성물은 종래 사료 첨가용으로 사용되는 미생물과는 달리 항균 활성을 갖는 설펙틴 C를 생산하며, 효소 생산 능력에서도 종래 기존 균주와는 다른 패턴을 갖고 있어 축산동물의 사료 효율 및 체중 증대 효과가 뛰어나다.The feed composition of the present invention comprising the Bacillus subtilis BC1212 strain produces sulfectin C having antimicrobial activity unlike microorganisms used for conventional feed addition, and has a different pattern from the conventional strain in the enzyme production capacity. It has excellent feed efficiency and weight gain effect of livestock animals.
이하, 본 발명을 실시예에 의해 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
단, 하기 실시예는 본 발명을 예시하기 위한 것이 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.
실시예Example 1: One: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주의 배양 및 Culture of the strain and 설펙틴의Sulfectin 분리 detach
설펙틴 생산 균주의 분리를 위해 된장 10 g을 멸균 증류수 100 ㎖에 녹인 후 그 된장 용액을 원액으로 하여 10분의 1씩 4번 희석하여 얻은 희석액 10 ㎖을 영양 아가 배지(nutrient agar, Difco Co. USA)에 1 ㎖씩 도말하여 28℃에서 48시간 배양하였다. 배양 후 형성된 균 집락 중 설펙틴 생산 균주를 확인하기 위하여 혈액이 함유된 영양 아가 배지에 이쑤시개를 사용하여 균체를 접종한 후 28℃에서 48시간 배양하였으며, 혈액을 분해하여 환이 생기는 균체를 선발하였다. 특히 그 환의 크기가 가장 큰 균주를 분리하여 설펙틴 생산을 위한 균주로 사용하였다. 균주 보관과 전배양을 위한 고체배지로는 영양 아가 배지를 사용하였으며, 액체 배지로 GYM 배지(glucose 4 g, Yeast extract 4 g, Malt extract 10 g, CaCO3 2g, pH 7.2)를 사용하여 설펙틴을 생산하였다. 배양 시간과 설펙틴의 생산관계를 확인하기 위해 동일한 배지 10 ㎖을 사용하여 전배양을 2-3일 한 후 본 배양을 위해 200 ㎖ 배지(500 ㎖ 프라스크)에 2.5%의 비율로 전배양한 배양액을 접종하여 28℃에서 200 rpm으로 7-8일간 배양하였다. 이때 배양시 균주의 성장과 설펙틴의 생산을 알아보기 위해 12시간 마다 배양액을 샘플링하였다. 성장 정도는 600 ㎚에서 흡광도(OD600)를 측정하여 비교하였고, 설펙틴 생산량은 표준물질(sigma S3523)을 사용하여 비교 측정하였다. 그 결과, 설펙틴의 생산과 균주의 성장은 모두 배양 80시간부터 급격히 증가하였으며 140시간 후에 최대값을 보이다가 180시간 이후에는 상당히 감소하여 유지되는 것으로 나타났다(도 1).10 g of doenjang was dissolved in 100 ml of sterile distilled water for the isolation of the sulfectin-producing strain, and 10 ml of the dilution obtained by diluting four times by tenths of the doenjang solution as a stock solution was added to the nutrient agar, Difco Co. USA) 1 ml each and incubated for 48 hours at 28 ℃. In order to identify the sulfectin-producing strain in the colonies formed after the culture, the cells were inoculated with a toothpick using a toothpick in a nutritive agar medium containing blood, and then cultured for 48 hours at 28 ° C. In particular, the strain with the largest size of the ring was isolated and used as a strain for sulfectin production. Nutrient agar medium was used as a solid medium for strain storage and preculture, and sulfectin using GYM medium (glucose 4 g, Yeast extract 4 g, Malt extract 10 g, CaCO 3 2 g, pH 7.2) as a liquid medium. Produced. In order to confirm the incubation time and the production of sulfectin, preculture was performed in 10 ml of the same medium for 2-3 days, and then pre-cultured at 2.5% in 200 ml medium (500 ml flask) for the main culture. The culture was inoculated and incubated at 28 rpm at 28 rpm for 7-8 days. At this time, the culture was sampled every 12 hours to determine the growth of the strain and the production of sulfectin. The degree of growth was compared by measuring the absorbance (OD 600 ) at 600 nm, the sulfectin production was measured using a standard (sigma S3523). As a result, the production of sulfectin and the growth of the strains both increased rapidly from 80 hours of culture and showed a maximum value after 140 hours, but significantly decreased after 180 hours (FIG. 1).
상기 균주의 정확한 동정을 위해 16S rDNA 유전자 분석법을 이용한 균주 동정(마이크로아이디, 서울대학교 유전공학특화창업보육센터 411호)을 실시하였으며, 이 때 리보좀(ribosomal) RNA의 2차 구조를 참고하여 서열을 배열한 후 % Similarity 값을 구함으로서 동정을 완료하였다. 결정된 염기서열의 수는 804 염기쌍으로서, 서열번호 1로 기재되는 염기서열을 갖는다. 결정된 염기서열을 바탕으로 리보좀 RNA의 2차 구조를 참고하여 % Similarity 값을 구하여 이를 분석한 결과, 16s RNA 염기서열의 상동성이 바실러스 서브틸리스 NRRL-B-23049T/AF074975와 99.75% 일치하였으며, 이로부터 본 발명자들은 상기 균주를 바실러스 서브틸리스에 속하는 "바실러스 서브틸리스 BC1212" 균주라 명명하고, 2004년 11월 8일자로 농업생명공학연구원에 기탁하였다(기탁번호: KACC 91141).16S rDNA for accurate identification of the strain Identification of strains using genetic analysis method (Micro ID, 411, Genetic Engineering Specialized Entrepreneurship Center, Seoul National University) was performed by arranging the sequences by referring to the secondary structure of the ribosomal RNA, and then calculating the% Similarity value. I completed the identification. The determined number of base sequences is 804 base pairs, having the base sequence set forth in SEQ ID NO: 1. Based on the determined nucleotide sequence, the% Similarity value was obtained by referring to the secondary structure of ribosomal RNA. As a result, the homology of the 16s RNA sequence was 99.75% identical to that of Bacillus subtilis NRRL-B-23049T / AF074975. From this, the inventors named the strain "Bacillus subtilis BC1212" strain belonging to Bacillus subtilis, and deposited on November 8, 2004 with the Institute of Agricultural Biotechnology (Accession Number: KACC 91141).
실시예Example 2: 2: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주의 항균 활성 측정 Determination of Antimicrobial Activity of Strains
실시예 1에서 분리된 바실러스 서브틸리스 BC1212 균주의 항균 활성을 확인 하기 위하여, 상기 균주를 영양 배지 (BD사, 미국)에서 24시간동안 배양한 후 원심분리하여 얻어진 배양 상등액을 HCl을 이용하여 pH 2 정도에서 침전시키고, 상기 침전물을 다시 원심분리하여 상등액은 버리고 침전물만 회수하였다. 얻어진 침전물을 에탄올로 2회 추출하여 수용성 불순물을 제거한 후, 에탄올에 녹는 분획만을 회수하였다.In order to confirm the antimicrobial activity of the Bacillus subtilis BC1212 strain isolated in Example 1, the culture supernatant obtained by incubating the strain for 24 hours in a nutrient medium (BD Co., USA) and centrifuging the pH using HCl The precipitate was precipitated at about 2 degrees and the precipitate was centrifuged again to discard the supernatant and recover only the precipitate. The obtained precipitate was extracted twice with ethanol to remove water soluble impurities, and only the fractions dissolved in ethanol were recovered.
다음으로, 활성 물질을 확인하기 위해 칼럼 크로마토그래피를 수행하였다. 먼저, 얻어진 에탄올 분획을 진공 증발기로 농축한 후, 5% 메탄올로 채워진 역상 C18 칼럼에 로딩(loading)하였고, 동일한 용매인 5% 메탄올로 용출하여 항균 물질이 포함된 분획만을 분리하였다. 다시 순도를 높이기 위해 세파덱스 LH-20 칼럼 크로마토그래피를 수행한 후 에탄올로 용출하였고, 각각의 분획을 LC/MS 및 NMR을 이용하여 화합물의 구조를 결정하였다.Next, column chromatography was performed to identify the active substance. First, the obtained ethanol fraction was concentrated on a vacuum evaporator, and then loaded into a reversed-phase C 18 column filled with 5% methanol, eluted with 5% methanol, the same solvent, to separate only the fraction containing the antimicrobial substance. In order to increase the purity again, Sephadex LH-20 column chromatography was performed and eluted with ethanol, and each fraction was determined by LC / MS and NMR to determine the structure of the compound.
그 결과, 바실러스 서브틸리스 BC1212 균주의 배양 상등액 중의 항균 활성을 나타내는 물질은 도 2와 같은 분리양상을 나타내었으며, 항균 활성 분획 중 제일 많은 양을 차지하고 있는 11분대의 물질을 순수분리하여 NMR 및 LC/MS를 이용하여 그 구조를 분석한 결과, 화학식 1로 표시되는 설팩틴 C (surfactin C)인 것을 확인하였다. 그 결과, 바실러스 서브틸리스 BC3125 균주의 배양 상등액 중의 항균 활성을 나타내는 물질은 도 2와 같은 분리양상을 나타내었으며, 항균 활성 분획 중 제일 많은 양을 차지하고 있는 11분대의 물질을 순수분리하여 NMR 및 LC/MS를 이용하여 그 구조를 분석한 결과, 화학식 1로 표시되는 설팩틴 C (surfactin C)인 것을 확인하였다.As a result, the material exhibiting the antimicrobial activity in the culture supernatant of the Bacillus subtilis BC1212 strain exhibited the separation phase as shown in FIG. 2, and the NMR and LC were purified by purely separating 11 components of the antimicrobial active fraction. As a result of analyzing the structure using / MS, it was confirmed that the sulfactin C (surfactin C) represented by the formula (1). As a result, the material showing the antimicrobial activity in the culture supernatant of the Bacillus subtilis BC3125 strain showed the separation pattern as shown in Figure 2, NMR and LC by pure separation of the 11 components of the antimicrobial activity fraction occupying the highest amount As a result of analyzing the structure using / MS, it was confirmed that the sulfactin C (surfactin C) represented by the formula (1).
또한, 분리된 설팩틴 C를 이용하여 질병 원인체로 작용하는 다양한 병원성 세균에 대한 항균 효과를 액체배지 희석법에 의해 측정하였다. 최소억제농도(MIC) 측정은 영양배지(BD사, 미국)에 설팩틴을 넣고 2배로 단계 희석한 후, 균의 최종 희석농도를 2.5×105 CFU/㎖로 하여 24시간 배양 후 세균의 발육유무를 육안으로 확인하였으며, 세균의 발육이 인정되지 않는 최소농도를 MIC로 결정하였다. 그 결과, 설팩틴 C는 주로 그람양성 세균에 대해 우수한 항균 활성을 나타내었으며, MRSA (methicillin resistant Staphylococcus aureus)로 알려져 있는 항생제 내성 세균에서 특히 기존 항생물질에 비해 높은 항균 활성을 나타내었다(표 1).In addition, the antiseptic effect of various pathogenic bacteria acting as a disease agent using the separated sulfactin C was measured by the liquid medium dilution method. In order to measure the minimum inhibitory concentration (MIC), add sulfatin to the nutrient medium (BD, USA), dilute it twice, and then develop the bacteria after 24 hours incubation with the final dilution concentration of 2.5 × 10 5 CFU / ml. The presence or absence was visually confirmed, and the minimum concentration at which no bacterial growth was recognized was determined by MIC. As a result, sulfactin C showed excellent antimicrobial activity mainly against Gram-positive bacteria, and showed higher antimicrobial activity than antibiotics, especially antibiotic resistant bacteria known as MRSA (methicillin resistant Staphylococcus aureus) (Table 1). .
상기에서, 실험에 사용된 미생물(a)들의 항생제 내성 활성은 다음과 같다:In the above, the antibiotic resistance activity of the microorganisms (a) used in the experiment is as follows:
- 스타필로코쿠스 아우레우스 KCCM 40511; 페니실린 및 젠타마이신 저항성Staphylococcus aureus KCCM 40511; Penicillin and gentamicin resistance
- 스타필로코쿠스 아우레우스 KCCM 41294; 테트라사이클린, 스트렙토마이신 및 매크롤라이드(macrolide) 저항성Staphylococcus aureus KCCM 41294; Tetracycline, streptomycin, and macrolide resistance
- 스타필로코쿠스 아우레우스 KCCM 40881; 페니실린 저항성Staphylococcus aureus KCCM 40881; Penicillin resistance
- 스타필로코쿠스 아우레우스 KCCM 11593; 페니실린 저항성Staphylococcus aureus KCCM 11593; Penicillin resistance
- 스타필로코쿠스 아우레우스 KCCM 40510; 메티실린(methicillin) 저항성Staphylococcus aureus KCCM 40510; Methicillin resistance
- 스타필로코쿠스 아우레우스 KCCM 11812; 페니실린 저항성.Staphylococcus aureus KCCM 11812; Penicillin resistance.
(b)는 감염동물에서 분리된 균주를 나타내며, SF는 설팩틴 C; AMOX는 아목사실린; COL은 콜리스틴; NFX는 노르플록사신; 및 STM은 스트렙토마이신을 나타낸다.(b) represents a strain isolated from an infected animal, SF is sulfactin C; AMOX is Amoxacillin; COL is colistin; NFX is norfloxacin; And STM represents streptomycin.
실시예Example 3: 3: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주의 배양 Cultivation of Strains
균주 보관과 전배양(seed) 접종을 위한 고체 배지로는 영양 아가 배지(BD사, 미국)를 사용하였으며, 액체 배지로는 분리 정제의 용이성을 위해 최소 배지를 사용하였다. 최소 배지의 조성은 4% 포도당, 30 mM KH2PO4, 30 mM Na2HPO4, 50 mM NH4NO3, 0.8 mM MgSO4, 2 mM FeSO4, 0.004 mM EDTA 및 0.007 mM CaCl2로 하였으며, 무기염류와 금속성분들은 각각 별도로 멸균하여 접종시 첨가하였다. 동일한 액체 배지에서 2-3일간 전배양한 다음, 균주의 농도를 2.5%로 맞추어 본 배양에 접종하였다. 본 배양에 사용한 배양액으로는 조성 A (1% 포도당, 3% 옥수수 전분, 1% 대두 밀(soybean meal), 0.5% 펩톤, 0.5% 효모 추출물, 0.2% CaCO3, 0.01% 소포제, 잔량의 물, 단위; wt/wt%)를 pH 6.75로 맞추어 사용하였으며, 140 rpm의 배양 회전 속도로 37℃에서 5일간 배양하여 바실러스 서브틸리스 BC1212 균주 및 그 배양액을 얻었다. 각각 24, 48, 72시간마다 배양액을 취한 후, HPLC를 이용하여 배지내 설팩틴 C의 양을 측정하였다.Nutrient agar medium (BD, USA) was used as a solid medium for strain storage and seed inoculation, and a minimal medium was used as a liquid medium for ease of separation and purification. The minimum media consisted of 4% glucose, 30 mM KH 2 PO 4 , 30 mM Na 2 HPO 4 , 50 mM NH 4 NO 3 , 0.8 mM MgSO 4 , 2 mM FeSO 4 , 0.004 mM EDTA and 0.007 mM CaCl 2 . , Inorganic salts and metal components were added separately at the time of inoculation. After 2-3 days preculture in the same liquid medium, the strain was inoculated into the culture at a concentration of 2.5%. The culture medium used for the culture included Composition A (1% glucose, 3% corn starch, 1% soybean meal, 0.5% peptone, 0.5% yeast extract, 0.2% CaCO 3 , 0.01% antifoam, residual water, Unit; wt / wt%) was used at pH 6.75 and cultured for 5 days at 37 ° C. at a culture rotation speed of 140 rpm to obtain Bacillus subtilis BC1212 strain and its culture. Cultures were taken every 24, 48 and 72 hours, respectively, and then the amount of sulfactin C in the medium was measured using HPLC.
또한, 현재 동물사료의 원료로 사용되고 있는 미강, 소맥피, 대두박, 폐당밀 등을 탄소원 및 질소원으로 사용할 수 있는지 여부를 확인하기 위하여, 각 원료의 C, H, O, N, S의 함량을 자동원소분석기(EA1110, CE Instrument)를 이용하여 분석하였으며, 탄소원 및 질소원을 각각 2% 및 5%로 고정한 후 조성 B (2.8% 밀기울, 4.9% 쌀겨, 5.1% 대두박, 0.02% FeSO4, 0.05% MnSO4, 잔량의 물, 단위; wt/wt%)를 이용하여 바실러스 서브틸리스 BC1212 균주를 배양하였다. 상기 조성 B는 pH를 6.75로 맞추어 사용하였으며, 140 rpm의 배양 회전 속도로 37℃에서 5일간 배양하여 바실러스 서브틸리스 BC1212 및 배양액을 얻었다. 각각 24, 48, 72시간마다 배양액을 취한 후, HPLC를 이용하여 배지내 설팩틴 C의 양을 측정하였다.In addition, in order to check whether rice bran, wheat buckwheat, soybean meal, waste molasses, etc., which are currently used as raw materials for animal feed, can be used as a carbon source and a nitrogen source, the contents of C, H, O, N, and S of each raw material are automatically adjusted. Analysis was performed using an elemental analyzer (EA1110, CE Instrument), and composition B (2.8% bran, 4.9% rice bran, 5.1% soybean meal, 0.02% FeSO 4 , 0.05% MnSO after fixing the carbon and nitrogen sources at 2% and 5%, respectively Bacillus subtilis BC1212 strain was cultured using 4 , remaining water, unit; wt / wt%). The composition B was used to adjust the pH to 6.75, and incubated at 37 ° C. for 5 days at a culture rotation speed of 140 rpm to obtain Bacillus subtilis BC1212 and the culture solution. Cultures were taken every 24, 48 and 72 hours, respectively, and then the amount of sulfactin C in the medium was measured using HPLC.
그 결과, 조성 A 및 조성 B 배양액을 이용하여 바실러스 서브틸리스 BC1212를 배양한 결과, 조성 B에서 높은 항균 활성을 나타내는 설팩틴 C를 많이 생산한다는 것을 확인하였다[도 3].As a result, the culture of Bacillus subtilis BC1212 using the composition A and composition B culture medium, it was confirmed that the production of a lot of sulfactin C showing a high antibacterial activity in composition B [Fig. 3].
실시예Example 4: 4: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주의 Strain pHpH 및 담즙 내성 And bile resistance
바실러스 서브틸리스 BC1212 균주의 소화 과정에서의 생존 능력을 평가하기 위하여, 산성 조건 및 담즙에 대한 내성을 조사하였다. 산성 조건하에서의 생존능력을 평가하기 위하여, 상기 균주를 액체 배지에 접종한 후 37℃에서 12시간 동안 정치 배양하여 균의 농도가 109 내지 1010 콜로니 형성 단위(colony forming unit, CFU)/㎖이 되도록 하였다. 배양된 균은 6,000 rpm으로 10분간 원심 분리하여 균을 수집하였고, 이를 멸균수로 2회 세척한 후 1/100로 농축하여 세포수가 약 1010 CFU/㎖이 되도록 하여 사용하였다. pH 1, 2, 3, 4 및 5로 조정된 완충액에 희석한 균을 접종하여 1, 2 및 4시간동안 37℃에서 배양한 후, 멸균수로 희석하여 트립티카제 소이 아가(trypticase soy agar, BD사, 미국)에 접종하여 콜로니를 계수하였다. 생존률(%)은 반응시간 후의 생균수를 초기접종시 세균수로 나누어 산출하였다.To assess the viability of the Bacillus subtilis BC1212 strain during digestion, the acidic conditions and resistance to bile were examined. In order to evaluate the viability under acidic conditions, the strain was inoculated in a liquid medium and then incubated at 37 ° C. for 12 hours to obtain a concentration of 10 9 to 10 10 colony forming units (CFU) / ml. It was made. The cultured bacteria were collected by centrifugation at 6,000 rpm for 10 minutes, and the cells were washed twice with sterile water and concentrated to 1/100 to give a cell number of about 10 10 CFU / ml. Inoculated with diluted bacteria in buffer adjusted to
그 결과, 바실러스 서브틸리스 BC1212는 산성 pH 조건하에서도 비교적 안정하게 생존하는 것을 알 수 있었다(표 2). 위산의 pH와 유사한 pH 1에서도 4시간동안 약 64%의 생존률을 나타내었으며, pH 2 이상에서는 4시간 처리시 85.7% 이상의 우수한 생존률을 나타내었다.As a result, it was found that Bacillus subtilis BC1212 survived relatively stable even under acidic pH conditions (Table 2).
다음으로, 담즙 내성을 평가하기 위하여 트립티카제 소이 배지에 0.3% 황소 담즙을 첨가한 배지에 바실러스 서브틸리스 BC1212를 접종하여 37℃에서 배양하면서, 12시간동안 분광광도기를 이용하여 550 ㎚ 파장에서의 흡광도에 의한 균의 생장 정도를 측정하였다.Next, in order to evaluate bile resistance, inoculated with Bacillus subtilis BC1212 in a medium to which 0.3% ox bile was added to trypticase soy medium and incubated at 37 ° C., using a spectrophotometer at 550 nm wavelength for 12 hours. The degree of growth of the bacteria by the absorbance of was measured.
그 결과, 0.3% 황소 담즙의 처리에 의해 초기 성장은 억제되었으나 6시간 이후에는 정상적으로 성장하였으며, 이로부터 바실러스 서브틸리스 BC3125 균주는 담즙에서도 우수한 생존 및 성장을 나타낸다는 것을 확인하였다(도 4).As a result, the initial growth was inhibited by the treatment of 0.3% bull bile, but after 6 hours, the growth was normal, and it was confirmed that the Bacillus subtilis BC3125 strain showed excellent survival and growth even in bile (FIG. 4).
상기 결과로부터, 바실러스 서브틸리스 BC1212 균주는 가축에 생균제로 사용할 경우 위 및 소장내에서도 생존하여 대장까지 전달될 수 있음을 확인하였다.From the above results, it was confirmed that Bacillus subtilis BC1212 strain can survive in the stomach and small intestine and be delivered to the large intestine when used as a probiotic in livestock.
실시예Example 5: 5: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주가 생산하는 효소 활성 Enzyme Activity Produced by Strains
바실러스 서브틸리스 BC1212 균주가 분비하는 각종 효소의 활성을 알아보기 위해 API ZYM 키트(bioMerieux, Montalieu-Vercieu, France)를 이용하였다. 바실러스 서브틸리스 BC1212 균주를 트립티카제 소이 배지에 접종하고 호기 조건으로 37℃에서 24시간 배양한 후 효소 활성을 측정하였다. 또한, 종래에 생균제로 사용되고 있는 바실러스 서브틸리스 나토와의 효소 활성을 비교하기 위해 바실러스 서브틸리스 나토(KCCM12027, 한국미생물균주은행, 한국)도 상기와 동일한 조건에서 배양한 후 효소 활성을 측정하였다. 효소 활성은 대조군과 비교하여 0점에서 5점까지 발색정도를 확인하여 상대적으로 평가하였다.API ZYM kit (bioMerieux, Montalieu-Vercieu, France) was used to determine the activity of various enzymes secreted by the Bacillus subtilis BC1212 strain. Bacillus subtilis BC1212 strain was inoculated in trypticase soy medium and incubated at 37 ° C. for 24 hours under aerobic conditions, and enzyme activity was measured. In addition, Bacillus subtilis NATO (KCCM12027, Korea microbial strain bank, Korea) was also cultured under the same conditions as above to compare the enzyme activity with Bacillus subtilis NATO, which is conventionally used as a probiotic. . Enzyme activity was relatively evaluated by confirming the color development from 0 to 5 points compared to the control.
그 결과, 바실러스 서브틸리스 BC1212는 종래에 생균제로 사용되고 있는 바실러스 서브틸리스 나토와는 전혀 다른 효소 활성을 나타내었다(도 5). 바실러스 서브틸리스 BC1212는 알칼린 포스파타제 및 산 포스파타제에 대해 높은 활성을 나타내었는데, 알칼린 포스파타제는 세균벽에서 분비되는 효소로서 세균이 외부환경에 대한 저항성을 가지는데 중요한 역할을 한다. 또한, 산 포스파타제는 인산분해효소로서 사료내의 인산 이용률을 증가시킬 수 있다.As a result, Bacillus subtilis BC1212 exhibited completely different enzymatic activity from Bacillus subtilis NATO conventionally used as a probiotic (FIG. 5). Bacillus subtilis BC1212 showed high activity against alkaline phosphatase and acid phosphatase. Alkaline phosphatase is an enzyme secreted from the bacterial wall and plays an important role in bacterial resistance to the external environment. In addition, acid phosphatase may increase phosphate utilization in feeds as phosphatase.
실시예Example 6: 6: 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주의 Strain 이유자돈에서의At weaners 증체Gain 효과 effect
이유자돈(Landrace×Yorkshire×Durok)은 충남대학교 부설 목장에서 4주령(평균 28일) 돼지를 총 20마리 구입하여 실험에 사용하였으며, 환경적인 스트레스를 제거하기 위해 한배 새끼인 돼지를 10마리씩 두 그룹으로 나누어 실시하였다. 대조군은 바실러스 서브틸리스 BC1212가 포함되지 않은 사료를 급여하였으며, 실험군은 실시예 1에서 분리된 바실러스 서브틸리스 BC1212 균주를 사료 ㎏ 당 0.1%로 첨가하여 4주간 급여하였다. 생산성 및 증체율을 측정하기 위해 입수시와 매주별 이유자돈의 체중을 측정하여 증체율을 계산하였으며, 사료 섭취율은 주간별로 누적 사료 섭취량으로 계산하였다. 사료 효율(%)은 체중 증가량을 사료 섭취율로 나누어 나타내었다.Weaning pigs (Landrace × Yorkshire × Durok) purchased a total of 20 four-week-old (average 28-day) pigs from a ranch attached to Chungnam National University and used them in the experiments. It was divided. The control group was fed a feed not containing Bacillus subtilis BC1212, the experimental group was fed for 4 weeks by adding the Bacillus subtilis BC1212 strain isolated in Example 1 at 0.1% per kg feed. In order to measure the productivity and the rate of increase, the weight gain was calculated by measuring the weight of weaning pigs at the time of acquisition and every week, and the feed intake rate was calculated as the cumulative feed intake weekly. Feed efficiency (%) was expressed by weight gain divided by feed intake rate.
다음으로, 바실러스 서브틸리스 BC1212 생균제의 급여에 의한 분변내 미생물의 변화를 알아보기 위해 실험 시작 후 마지막 28일에 각 시험군의 분변을 채취하였다. 채취한 분변을 혈액 배지에 접종하여 채취한 분변 중 총 혐기성 미생물의 집락수를 확인하였다. 또한, 분변내 바실러스 서브틸리스의 존재 유무를 확인하기 위해 각 시험군의 분변 시료를 바실러스 서브틸리스 감별 배지인 폴리믹신 피루베이트 달걀 난황 만니톨 브로모티몰 블루 아가(polymixin pyruvate egg yolk mannitol bromothymol blue agar, 옥소이드, 영국)에 접종하여 37℃의 혐기 조건에서 48시간동안 배양한 후 바실러스 서브틸리스의 집락수를 측정하였다. 또한, 이유자돈에서 설사를 유발하는 주요 세균인 대장균을 검출하기 위해 대장균 감별 배지인 맥콘키(MacConkey) 배지(BD사, 미국)에 접종하여 37℃의 호기 조건에서 24시간동안 배양한 후 유당을 분해하는 집락을 대장균군으로 추정하였다.Next, fecals of each test group were collected on the last 28 days after the start of the experiment in order to determine the change in the fecal microorganism due to the feeding of Bacillus subtilis BC1212 probiotics. The collected feces were inoculated into the blood medium to check the colony count of the total anaerobic microorganisms in the collected feces. Also, in order to confirm the presence of Bacillus subtilis in feces, the fecal samples of each test group were subjected to polymixin pyruvate egg yolk mannitol bromothymol blue agar. , Oxoid, UK) and incubated for 48 hours at 37 ℃ anaerobic conditions and the number of colonies of Bacillus subtilis was measured. In addition, in order to detect E. coli, a major bacterium that causes diarrhea in weaning pigs, it was inoculated in E. coli differentiating medium, MacConkey medium (BD company, USA), incubated for 24 hours at 37 ° C aerobic condition, and then lactose was decomposed. Colonies were estimated to be E. coli.
그 결과, 바실러스 서브틸리스 BC1212의 급여에 따른 체중변화는 무첨가 대조군에 비해 증가한 것으로 나타났으며, 사료 효율 역시 무첨가 대조군에 비해 바실러스 서브틸리스 BC1212 투여군에서 다소 높게 나타났다(표 3). 또한, 분변내 미생물 중 총혐기성균 수는 차이를 나타내지 않았으나, 바실러스 서브틸리스 BC1212의 급여에 의해 분변내 바실러스 서브틸리스의 수는 증가하였으며, 대장균의 수는 감소하였다(표 4).As a result, the weight change according to the feeding of Bacillus subtilis BC1212 was increased compared to the control group, and the feed efficiency was also slightly higher in the Bacillus subtilis BC1212 administration group than the control group (Table 3). In addition, the total number of anaerobic bacteria in fecal microorganisms did not show a difference, but the number of Bacillus subtilis in feces increased and the number of Escherichia coli decreased by feeding Bacillus subtilis BC1212 (Table 4).
제조예Production Example . . 바실러스Bacillus 서브틸리스Subtilis BC1212BC1212 균주를 포함하는 사료 첨가용 조성물의 제조 Preparation of a composition for feed addition containing a strain
본 발명자들은 바실러스 서브틸리스 BC1212 균주를 포함하는 본 발명의 사료 첨가용 조성물을 하기 표 5의 조성으로 제조하였다.We prepared a composition for feed addition of the present invention comprising Bacillus subtilis BC1212 strain with the composition of Table 5 below.
상기 표 5에서, 효소 제제는 파이타제, 셀룰라제, 자일라나제, 말타제 및 전환효소의 혼합제제를 사용하였고, 비병원성의 미생물로는 아스퍼질러스 오리자에를 사용하였다.In Table 5, the enzyme preparation used a combination of phytase, cellulase, xylanase, maltase, and converting enzyme, and Aspergillus Orissae was used as a non-pathogenic microorganism.
도 1은 균주의 성장과 설펙틴의 생산과의 상관관계를 보여주는 그래프로서,□는 균체의 성장, ●는 설펙틴 생산을 나타낸다.1 is a graph showing the correlation between the growth of the strain and the production of sulfectin, □ represents the growth of the cells, ● represents the sulfectin production.
도 2는 바실러스 서브틸리스 BC1212 균주의 배양 상등액 중에서 항균 활성을 나타내는 물질에 대한 액상크로마토그래피 자료이다.Figure 2 is a liquid chromatography data on the material showing the antimicrobial activity in the culture supernatant of Bacillus subtilis BC1212 strain.
도 3은 배지 조성물에 따른 바실러스 서브틸리스 BC1212 균주의 설팩틴 C 생산량을 비교한 그래프이다.Figure 3 is a graph comparing the sulfactin C production of Bacillus subtilis BC1212 strain according to the medium composition.
도 4는 0.3% 황소 담즙의 처리가 바실러스 서브틸리스 BC1212 균주의 성장에 미치는 영향을 평가한 그래프로서, "대조군"은 황소 담즙을 처리하지 않은 바실러스 서브틸리스 BC1212의 생장곡선, "황소 담즙 0.3%"는 0.3% 황소 담즙을 처리한 바실러스 서브틸리스 BC1212의 생장곡선을 나타낸다.Figure 4 is a graph evaluating the effect of 0.3% bull bile treatment on the growth of Bacillus subtilis BC1212 strain, "control" is the growth curve of Bacillus subtilis BC1212 untreated bull bile, "bull bile 0.3 % "Represents the growth curve of Bacillus subtilis BC1212 treated with 0.3% bull bile.
도 5는 바실러스 서브틸리스 BC1212 균주와 바실러스 서브틸리스 나토(KCCM12027) 균주의 효소 활성을 비교한 그래프이다.5 is a graph comparing the enzyme activity of Bacillus subtilis BC1212 strain and Bacillus subtilis NATO (KCCM12027) strain.
<110> B.N.C BIO PARM CO., LTD. <120> Composition for Prevention or Treatment of Osteoporosis Comprising Rubus Coreanus Miquel Extract <130> 2007-dpa-0151 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 804 <212> DNA <213> Bacillus subtilis BC1212 <220> <221> rRNA <222> (1)..(804) <223> 16S rDNA sequence of Bacills subtilis BC1212 <400> 1 gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag cttgctccct 60 gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120 tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca aacataaaag 180 gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240 ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360 tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420 gggaagaaca agtaccgttc gaatagggcg gtaccttgac ggtacctaac cagaaagcca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540 ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600 cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720 ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgatgag tgct 804 <110> B.N.C BIO PARM CO., LTD. <120> Composition for Prevention or Treatment of Osteoporosis Comprising Rubus Coreanus Miquel Extract <130> 2007-dpa-0151 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 804 <212> DNA <213> Bacillus subtilis BC1212 <220> <221> rRNA (222) (1) .. (804) <223> 16S rDNA sequence of Bacills subtilis BC1212 <400> 1 gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag cttgctccct 60 gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120 tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca aacataaaag 180 gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240 ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360 tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420 gggaagaaca agtaccgttc gaatagggcg gtaccttgac ggtacctaac cagaaagcca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540 ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600 cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720 ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgatgag tgct 804
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102771627A (en) * | 2011-05-09 | 2012-11-14 | 北京奕农顺丰生物技术有限公司 | Feed additive containing compound enzyme |
| KR101370942B1 (en) * | 2012-04-05 | 2014-03-12 | 씨제이제일제당 (주) | Novel Bacillus subtilis |
| KR101480028B1 (en) * | 2013-06-17 | 2015-01-07 | (주)창조바이오텍 | Novel microorganism bacillus subtilis sj-30 and additives for fish feeds containing it |
| CN104717968A (en) * | 2012-09-11 | 2015-06-17 | 达科他星都有限公司 | Nutritional supplement containing iron |
| CN106721009A (en) * | 2016-12-23 | 2017-05-31 | 山西大禹生物工程股份有限公司 | Growth promotion puies forward quality feed addictive and preparation method |
| KR20190079444A (en) * | 2017-12-27 | 2019-07-05 | 우림바이오 주식회사 | A novel Bacillus subtilis UB-156 for multi function |
| KR102176078B1 (en) * | 2020-06-09 | 2020-11-09 | 이정복 | Solid culture method to culture with new microbial bacillus subtilis BS300 strains with resolution to carbohydrates and proteins and fibres among organic matter |
| KR102411380B1 (en) * | 2021-06-28 | 2022-06-22 | 주식회사 프록스엔렘 | Novel bacillus subtilis strain with high productivity of surfactin and enzyme and use of the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102771627A (en) * | 2011-05-09 | 2012-11-14 | 北京奕农顺丰生物技术有限公司 | Feed additive containing compound enzyme |
| KR101370942B1 (en) * | 2012-04-05 | 2014-03-12 | 씨제이제일제당 (주) | Novel Bacillus subtilis |
| CN104717968A (en) * | 2012-09-11 | 2015-06-17 | 达科他星都有限公司 | Nutritional supplement containing iron |
| EP2895179A4 (en) * | 2012-09-11 | 2016-04-06 | Dakota Star Capital Llc | Nutritional supplement containing iron |
| US11478519B2 (en) | 2012-09-11 | 2022-10-25 | Cura Global Health (Bvi) Limited | Nutritional supplement containing iron |
| KR101480028B1 (en) * | 2013-06-17 | 2015-01-07 | (주)창조바이오텍 | Novel microorganism bacillus subtilis sj-30 and additives for fish feeds containing it |
| CN106721009A (en) * | 2016-12-23 | 2017-05-31 | 山西大禹生物工程股份有限公司 | Growth promotion puies forward quality feed addictive and preparation method |
| KR20190079444A (en) * | 2017-12-27 | 2019-07-05 | 우림바이오 주식회사 | A novel Bacillus subtilis UB-156 for multi function |
| KR102176078B1 (en) * | 2020-06-09 | 2020-11-09 | 이정복 | Solid culture method to culture with new microbial bacillus subtilis BS300 strains with resolution to carbohydrates and proteins and fibres among organic matter |
| KR102411380B1 (en) * | 2021-06-28 | 2022-06-22 | 주식회사 프록스엔렘 | Novel bacillus subtilis strain with high productivity of surfactin and enzyme and use of the same |
| WO2023277505A1 (en) * | 2021-06-28 | 2023-01-05 | 주식회사 프록스엔렘 | Bacillus subtilis strain having excellent production capacity of surfactin and enzyme, and use thereof |
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