KR20210052342A - Multi-functional probiotics for marine fish and uses thereof - Google Patents
Multi-functional probiotics for marine fish and uses thereof Download PDFInfo
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- KR20210052342A KR20210052342A KR1020200142570A KR20200142570A KR20210052342A KR 20210052342 A KR20210052342 A KR 20210052342A KR 1020200142570 A KR1020200142570 A KR 1020200142570A KR 20200142570 A KR20200142570 A KR 20200142570A KR 20210052342 A KR20210052342 A KR 20210052342A
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Abstract
Description
본 발명은 다기능 어류용 생균제 및 이의 용도에 관한 것으로, 더욱 상세하게는 내염성을 가지는 내열성 젖산 생성균과 저온활성 셀룰라제 분비균의 복합균주를 이용하여 한약재와 크릴 새우의 혼합물을 발효시키는 단계를 포함하여 제조한 어류 질병에 대한 항균력을 가지는 다기능 어류용 생균제에 관한 것이다.The present invention relates to a multifunctional probiotic for fish and its use, and more particularly, comprising fermenting a mixture of herbal medicinal herbs and krill shrimp using a complex strain of heat-resistant lactic acid-producing bacteria and low-temperature active cellulase-secreting bacteria having salt resistance. It relates to a multifunctional probiotic for fish having antibacterial activity against the prepared fish diseases.
수산양식산업이 증가하고, 수중 생태계 및 기후 변화 등의 환경이 변화함에 따라 예상하지 못했던 어류질병이 발생함은 물론, 기존의 어류질병의 발생 및 확산 속도가 증가하고 있다. 국립수산과학원 및 대한수산질병관리사회에서 2012년 우리나라 대표적인 해산, 담수산 양식어종을 대상으로 양식장 피해발생 현황을 조사한 바에 의하면, 넙치 27.9%, 조피볼락 13.6%, 무지개송어 17.1%, 흰다리새우 71.2% 및 전복 10.7% 등 대부분의 양식어종별에서 10% 이상 피해가 발생하고 있음을 보고하였다. 제주 양식넙치의 경우 폐사로 인한 피해액이 2007년 320억원에서 2008년 294억원, 2009년 376억원, 2010년 513억원으로 생산량 대비 피해량 및 피해금액이 증가하고 있는 것으로 보고되어, 효과적인 어류질병의 예방 및 치료 방법이 요구되고 있는 실정이다.As the aquaculture industry increases and environmental changes such as aquatic ecosystems and climate change, unexpected fish diseases occur, as well as the rate of occurrence and spread of existing fish diseases. According to a survey by the National Institute of Fisheries Science and the Korean Fisheries Disease Management Association in 2012 on the damage to farms for representative marine and freshwater aquaculture fish species in Korea, flounder 27.9%, rockfish 13.6%, rainbow trout 17.1%, and white-legged shrimp 71.2%. It was reported that more than 10% of damage occurred in most farmed fish species such as abalone and 10.7%. In the case of Jeju farmed flounder, the damage caused by mortality was reported to increase from 32 billion won in 2007 to 29.4 billion won in 2008, 37.6 billion won in 2009, and 51.3 billion won in 2010. This is an effective prevention of fish diseases and There is a demand for treatment methods.
양식어류의 질병예방 및 치료를 위한 가장 일반적인 방법으로는 항생제 투여 등의 화학적인 방법과 백신요법이 있다. 그러나, 항생제 요법은 오남용에 따른 내성균의 출현 및 체내 잔류 문제로 인한 사회적, 생태적인 심각한 문제를 유발하여 사용이 제한되고 있는 추세이며, 백신요법의 경우 양식어류는 군집을 대상으로 하기 때문에 백신투여가 매우 번거롭고 많은 인력이 필요한 단점이 있다. 또한, 세균성 질병의 복합적 감염에 따른 효과적인 백신의 개발이 어려워 어류질병 예방과 치료를 위한 대책으로서는 한계가 있다.The most common methods for disease prevention and treatment of farmed fish are chemical methods such as antibiotic administration and vaccine therapy. However, the use of antibiotic therapy is limited due to the emergence of resistant bacteria due to abuse and serious social and ecological problems due to residual problems in the body. It is very cumbersome and requires a lot of manpower. In addition, it is difficult to develop effective vaccines for complex infections of bacterial diseases, so there is a limit as a countermeasure for the prevention and treatment of fish diseases.
이에 기존의 문제점에 대한 대책으로 천연 자원인 미생물 자원을 이용한 프로바이오틱스(probiotics, 생균제)를 양어사료에 첨가한 친환경적인 요법에 관심과 연구가 많아지고 있다. 또한, 정부가 신규 항생물질의 허가를 규제하는 현실을 감안할 때 양식어류에 대해 항생물질 남용 억제 및 대체할 수 있는 생균제 개발로 어류의 장내 유익세균총 형성, 성장촉진 및 질병예방, 수질오염 억제 등을 목적으로 하는 어류용 생균제는 필요성이 높아지고 있는 상황이다.Accordingly, as a countermeasure against the existing problems, interest and research are increasing in eco-friendly therapy in which probiotics (probiotics) using microbial resources, which are natural resources, are added to fish feed. In addition, given the reality that the government regulates the approval of new antibiotics, the development of probiotics that can suppress and replace the abuse of antibiotics for aquaculture fishes will help to promote the formation of beneficial bacteria in the intestine of fish, promote growth and prevent diseases, and control water pollution. The need for probiotics for fish for the purpose is increasing.
프로바이오틱스는 숙주건강에 유익한 효과를 나타내는 살아있는 미생물로서 다양한 종류의 정상 장내 세균총에서 병원성균에 대해 증식을 억제하는 항균력이 있으며, 정상 장내 세균총의 균형을 유지하고 병원성 세균증식을 억제하는데 중요한 역할을 한다. 프로바이오틱스의 유용한 효과는 주로 사람과 가축에 대한 실험보고 내용이 대부분이나, 어류와 다른 수생생물에서도 프로바이오틱스에 의한 선천적 면역반응 향상과 병원성 미생물의 억제 효과가 일부 보고되었다. 수산양식에서 사용되는 프로바이오틱스는 바실러스 종(Bacillus sp.), 락토바실러스 종(Lactobacillus sp.), 사카로마이세스 종(Saccharomyces sp.) 및 엔테로코커스 종(Enterococcus sp.) 등이 있다. 어류양식에 사용되는 유산균 생균제는 일반적으로 락토바실러스 또는 스트렙토코커스(Streptococcus) 균주가 주로 사용되고 있는데, 이는 어류 장내 병원균 증식억제, 면역력 증대에 영향을 주는 것으로 알려져 있기 때문이다. 몇 가지 어류를 대상으로 한 연구에서 바실러스 균주를 급여하였을 때 성장과 사료효율 증가가 보고되었으며, 특히 바실러스 균주는 셀룰라제(cellulase), 프로테아제(protease) 및 리파제(lipase) 등과 같은 효소 분비능에 초점을 맞추어 많이 연구되고 있다. 다만, 사람 및 가축에 사용되는 생균제는 최적발육온도가 37℃로서, 15-25℃에서 생육하는 양식어류에는 부적절하며, 해수 양식어의 경우 높은 염(salt) 농도에서도 견딜 수 있는 특성의 생균제가 필수적이다.Probiotics are living microorganisms that exhibit beneficial effects on host health, and have antibacterial activity that inhibits the proliferation of pathogenic bacteria in various types of normal intestinal flora, and plays an important role in maintaining the balance of the normal intestinal flora and inhibiting the growth of pathogenic bacteria. The useful effects of probiotics are mostly reported in experiments on humans and livestock, but some have been reported to improve innate immune responses and suppress pathogenic microorganisms by probiotics in fish and other aquatic organisms. Probiotics used in aquaculture include Bacillus sp., Lactobacillus sp., Saccharomyces sp., and Enterococcus sp. Lactobacillus or Streptococcus strains are generally used as probiotics for lactic acid bacteria used in fish farming, because they are known to affect the growth of pathogens in the fish intestine and increase immunity. Studies on several fish have reported increased growth and feed efficiency when feeding Bacillus strains. In particular, Bacillus strains focus on the ability to secrete enzymes such as cellulase, protease, and lipase. It has been studied a lot. However, probiotics used in humans and livestock have an optimal growth temperature of 37℃, which is inappropriate for farmed fish growing at 15-25℃. In the case of seawater farmed fish, probiotics with characteristics that can withstand high salt concentrations It is essential.
남극 크릴(krill)은 난바다곤쟁이목(Euphausiacea)에 속하는 갑각류로 새우와 비슷하게 생긴 동물플랑크톤으로, 새우와는 관련이 없다. 최대 5cm까지 자라며 이들은 고래, 펭귄, 일부 상어, 물범, 심지어 인간에게 중요한 식량원으로, 흰수염고래는 하루에 약 400만 마리나 되는 크릴을 먹으며, 거대한 떼를 지어 나타나기도 해 가끔 바다를 온통 붉게 물들인다. 크릴은 세계 곳곳에 존재하지만, 특히 남극해에서 중요한 역할을 한다. 미래 식량자원으로서 유럽과 일본에서 고급요리에 사용되는 자연 청정식품이고, 크릴밀(krill meal)의 사료적 가치로서 기호성이 좋으며, 미네랄, 인지질, 불포화 지방산, 키틴, 필수 아미노산이 풍부하다.Antarctic krill is a crustacean belonging to the order Euphausiacea . It is a shrimp-like zooplankton, not related to shrimp. They grow up to 5 cm tall and are important food sources for whales, penguins, some sharks, seals, and even humans.Bluhead whales eat about 4 million krill a day, sometimes appearing in large swarms, sometimes coloring the seas red. . Krill exist all over the world, but play an important role, especially in the Antarctic Ocean. It is a natural clean food used in high-end cooking in Europe and Japan as a future food resource, has good palatability as a feed value of krill meal, and is rich in minerals, phospholipids, unsaturated fatty acids, chitin, and essential amino acids.
한편, 한국공개특허 제2010-0045758호에는 '장내효소활성, 내산성, 내담즙성이 우수한 신규 락토바실러스 펜토서스 PL-11 균주와 이를 이용한 어류용 프로바이오틱스'가 개시되어 있고, 한국공개특허 제2015-0024115호에는 '바실러스 서브틸러스 및 파지 혼합물을 유효성분으로 함유하는 어류용 프로바이오틱스 조성물'이 개시되어 있으나, 본 발명의 다기능 어류용 생균제 및 이의 용도에 대해서는 기재된 바가 없다.On the other hand, Korean Patent Publication No. 2010-0045758 discloses'new Lactobacillus pentosus PL-11 strain excellent in intestinal enzyme activity, acid resistance, and bile resistance and probiotics for fish using the same.' 0024115 discloses a'probiotic composition for fish containing a mixture of Bacillus subtilis and phage as an active ingredient', but there is no description of the multifunctional probiotic for fish of the present invention and its use.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 생산성 증대 및 어류질병에 대한 면역성 증진 효과가 우수한 기능성 양어용 생균제를 개발하기 위해, 50℃에서도 생장 가능한 내열성 젖산 생성 바실러스와 저온(10℃)에서 활성을 나타내는 셀룰라제를 분비하는 바실러스를 이용하여 대두박, 크릴밀(krill meal) 및 복합 한약재(어성초, 지황, 당귀, 약콩 및 감초)를 포함하는 조성물의 최적 발효 조건을 확립하였고, 확립된 최적 발효 조건으로 제조된 생균제를 이용하여 넙치에 사료 첨가제로 급이시킨 결과, 무처리구 대비 상기 생균제를 급이한 실험군에서 스트레스 저감 및 면역 증대 효과가 있음을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above requirements, and the present inventors developed a functional probiotic for fish farming with excellent productivity enhancement and immunity to fish diseases, and heat-resistant lactic acid-producing bacillus and low temperature (10 ℃), using Bacillus secreting cellulase that exhibits activity at, establish and establish optimal fermentation conditions for a composition containing soybean meal, krill meal, and complex herbal medicinal materials (Eoseongcho, Rhizome, Angelica, Yakbean and Licorice). As a result of feeding the flounder with a feed additive using the probiotic prepared under the optimal fermentation conditions, the present invention was completed by confirming that there is an effect of reducing stress and increasing immunity in the experimental group fed the probiotic compared to the untreated group.
상기 과제를 해결하기 위해, 본 발명은 대두박, 크릴밀(krill meal) 및 복합 한약재를 포함하는 혼합물을 내열성 젖산 생성균과 저온활성 셀룰라제 분비균의 복합 균주를 이용하여 고상발효시키는 단계를 포함하는, 다기능 어류용 생균제(probiotics)의 제조방법을 제공한다.In order to solve the above problems, the present invention comprises the step of solid-phase fermentation of a mixture containing soybean meal, krill meal and a complex herbal medicine using a complex strain of heat-resistant lactic acid-producing bacteria and low-temperature active cellulase-secreting bacteria, Provides a method of manufacturing multifunctional fish probiotics.
또한, 본 발명은 상기 방법으로 제조된 다기능 어류용 생균제를 제공한다.In addition, the present invention provides a probiotic for multifunctional fish prepared by the above method.
또한, 본 발명은 상기 다기능 어류용 생균제를 첨가한 사료를 양식 어류에 급이시키는 단계를 포함하는, 양식 어류의 생산성 및 면역력 증대 방법을 제공한다.In addition, the present invention provides a method for increasing productivity and immunity of farmed fish, comprising the step of feeding the feed to which the multifunctional fish probiotic is added to the farmed fish.
종래 어류용 생균제의 셀룰라제는 넙치 양어사육 수온인 21~24℃에서 효소활성 여부가 불분명한데 반해, 본 발명에서 사용된 바실러스 리케니포르미스(Bacillus licheniformis) DK42 균주의 셀룰라제는 20℃에서도 활성이 유지되어 양어사육 수온에서 조섬유 분해가 용이한 장점이 있다. 또한, 본 발명에서는 액상 어류용 생균제 발효조건의 확립을 통해 농도 1.0x108 cfu/㎖ 이상의 생균수 확보로 산업적 효율성을 확립하였으며, 안정성에서 중요한 포자형성율이 거의 100%에 가까워 1년 이상 장기보관이 가능하게 되었다. 또한, 본 발명에 따른 생균제는 양식 어류의 생산성 증대뿐만 아니라 개체의 면역력 또한 증진시킬 수 있으므로, 무항생제, 유기농, 친환경 브랜드의 양어 어류 생산이 가능할 수 있다.The cellulase of conventional probiotics for fish is unclear whether or not the enzyme is active at 21~24℃, which is the water temperature for halibut fish farming, whereas the cellulase of the Bacillus licheniformis DK42 strain used in the present invention is active even at 20℃. This maintains the advantage of easy decomposition of crude fiber at aquaculture breeding water temperature. In addition, in the present invention, industrial efficiency was established by securing the number of viable cells at a concentration of 1.0x10 8 cfu/ml or higher through establishment of fermentation conditions for liquid fish probiotics. This became possible. In addition, since the probiotic according to the present invention can increase the productivity of farmed fish as well as enhance the immunity of the individual, it is possible to produce aquaculture fish of an antibiotic-free, organic, eco-friendly brand.
도 1은 DNS 환원당 정량법을 통한 바실러스 리케니포르미스(Bacillus licheniformis) DK42 균주가 분비하는 셀룰라제에 대한 온도별 활성 측정 결과이다.
도 2는 1 %(w/v) Skim milk 평판배지를 이용한 프로테아제 활성 측정 결과로, (a)는 바실러스 리케니포르미스 DK42 균주, (b)는 바실러스 코아귤런스(B. coagulans) TM3 균주의 결과이다.
도 3은 본 발명의 어류용 생균제 제조 순서를 보여준다.
도 4는 생균제 함량에 따른 넙치의 온도자극 후 코르티졸 농도 분석 결과(a)와 생균제 첨가 함량별 각 실험군의 혈 중 리소자임 활성 분석 결과(b)이다.1 is a result of temperature-specific activity measurement for cellulase secreted by Bacillus licheniformis DK42 strain through DNS reducing sugar quantification method.
Figure 2 is a 1% (w / v) Skim milk plate medium using the protease activity measurement results, (a) is Bacillus licheniformis DK42 strain, (b) is Bacillus coagulans (B. coagulans ) TM3 strain of the strain of TM3. It is the result.
Figure 3 shows the production sequence of the probiotic for fish of the present invention.
4 is a result of analysis of cortisol concentration after temperature stimulation of flounder according to the content of probiotics (a) and results of analysis of lysozyme activity in blood of each experimental group according to the amount of probiotic added (b).
본 발명의 목적을 달성하기 위하여, 본 발명은 대두박, 크릴밀(krill meal) 및 복합 한약재를 포함하는 혼합물을 내열성 젖산 생성균과 저온활성 셀룰라제 분비균의 복합 균주를 이용하여 고상발효시키는 단계를 포함하는, 다기능 어류용 생균제(probiotics)의 제조방법을 제공한다.In order to achieve the object of the present invention, the present invention comprises the step of solid-phase fermentation of a mixture comprising soybean meal, krill meal and a complex herbal medicine using a complex strain of heat-resistant lactic acid-producing bacteria and low-temperature active cellulase-secreting bacteria. It provides a method of manufacturing multifunctional fish probiotics.
본 발명에 따른 다기능 어류용 생균제의 제조방법에 있어서, 상기 내열성 젖산 생성균은 젖산을 생성하며 45~55℃에서 생장이 가능하며 프로테아제 활성을 갖는 바실러스 코아귤런스(Bacillus coagulans) 균주이고, 저온활성 셀룰라제 분비균은 10~20℃의 저온에서 활성을 갖는 셀룰라제를 분비하며 프로테아제 활성을 갖는 바실러스 리케니포르미스(Bacillus licheniformis) 균주일 수 있으며, 보다 바람직하게는 각각 바실러스 코아귤런스 TM3 균주 및 바실러스 리케니포르미스(Bacillus licheniformis) DK42 균주(기탁번호: KACC 91410P)일 수 있다.In the method for producing a probiotic for multifunctional fish according to the present invention, the heat-resistant lactic acid-producing bacteria produce lactic acid, can grow at 45 to 55°C, and are Bacillus coagulans strains having protease activity, and are low-temperature active cellulosic The secretory bacterium may be a Bacillus licheniformis strain that secretes cellulase having activity at a low temperature of 10 to 20°C and has protease activity, more preferably Bacillus coagulation TM3 strain and Bacillus, respectively. It may be a licheniformis (Bacillus licheniformis ) DK42 strain (accession number: KACC 91410P).
상기 바실러스 코아귤런스(Bacillus coagulans) TM3 균주는 단국대학교 생명공학대학 생명자원학부 강대경 교수로부터 제공받은 것으로, 유산균으로 분류되었을 정도로 다량의 젖산을 생산하면서도 고온(50℃)에서 생장이 가능하며, 포자를 형성하는 특징이 있다. 항생제 대체제로 효과적인 소재로 알려진 유산균은 내열성이 약하기 때문에, 유통 중 생존율이 떨어지는 단점이 있으나, 본 발명의 생균제는 내열성의 젖산 생성균인 바실러스 코아귤런스 TM3 균주를 포함하고 있어, 이러한 문제점을 보완할 수 있는 장점이 있다.The Bacillus coagulans TM3 strain was provided by Professor Dae-Kyung Kang of the Department of Biological Resources, College of Biotechnology, Dankook University, and produces a large amount of lactic acid as classified as a lactic acid bacterium, and can be grown at high temperatures (50°C), and spores There are features that form. Lactobacillus known as an effective material as an antibiotic substitute has a disadvantage of low survival rate during distribution because of poor heat resistance, but the probiotic of the present invention contains Bacillus coagulation TM3 strain, which is a heat-resistant lactic acid-producing bacterium, and thus can compensate for these problems. There is an advantage.
또한, 상기 바실러스 리케니포르미스(Bacillus licheniformis) DK42 균주(기탁번호: KACC 91410P)는 10℃의 저온에서 온도의 변화에 따른 최대 활성의 50%의 활성을 나타내는 셀룰라제를 분비하는 것이 특징으로, 한국등록특허 제1062309호에 특성이 상세히 기술되어 있다. 어류용 바실러스 프로바이오틱스는 셀룰라제(cellulase), 프로테아제(protease) 또는 아밀라제(amylase)와 같은 소화효소 능력이 우수한 균주가 사용되고 있으나, 어류 체온에서의 효소 활성 여부는 불확실하다. 그러나, 본 발명의 생균제는 10℃의 저온에서도 활성을 가지는 셀룰라제를 분비하는 바실러스 리케니포르미스 DK42 균주를 포함하고 있어, 어류용 사료 성분에서 조섬유 분해가 용이한 장점이 있다.In addition, the Bacillus licheniformis (Bacillus licheniformis) DK42 strain (accession number: KACC 91410P) is characterized by secreting cellulase, which exhibits 50% of the maximum activity according to temperature change at a low temperature of 10°C, The characteristics are described in detail in Korean Patent No. 1062309. Bacillus probiotics for fish are strains with excellent digestive enzyme ability such as cellulase, protease, or amylase, but the enzyme activity at fish body temperature is uncertain. However, the probiotic of the present invention contains Bacillus licheniformis DK42 strain that secretes cellulase having activity even at a low temperature of 10°C, and thus has the advantage of easy decomposition of crude fiber in feed ingredients for fish.
본 발명의 제조방법에 있어서, 상기 크릴밀(krill mill)은 식품 크릴새우를 가공하고 남은 부산물 즉, 크릴박을 의미한다.In the manufacturing method of the present invention, the krill mill means a by-product that is left after processing the food krill, that is, krill meal.
또한, 본 발명에 따른 다기능 어류용 생균제의 제조방법에 있어서, 상기 복합 한약재는 어성초, 지황, 당귀, 약콩 및 감초의 혼합물일 수 있고, 바람직하게는 어성초 분말, 지황 분말, 당귀 분말, 약콩 분말 및 감초 분말이 동량으로 혼합된 혼합물일 수 있으나, 이에 제한되지 않는다.In addition, in the method for producing a probiotic for multifunctional fish according to the present invention, the complex herbal medicine may be a mixture of Eoseongcho, Rehmannia, Angelica, Yakbean and Licorice, and preferably Eoseongcho powder, Rehmannia powder, Angelica powder, Yakbean powder and It may be a mixture in which the licorice powder is mixed in the same amount, but is not limited thereto.
본 발명의 일 구현 예에 따른 다기능 어류용 생균제의 제조방법은 구체적으로,The method for producing a probiotic for multifunctional fish according to an embodiment of the present invention is specifically,
(a) 효모 추출물, 포도당 및 미네랄로 이루어진 액상 배지에서 젖산을 생성하는 내열성 바실러스 코아귤런스(Bacillus coagulans) 균주와 저온활성 셀룰라제를 분비하는 바실러스 리케니포르미스(B. licheniformis) 균주의 복합 균주를 30~35℃에서 45~50시간 동안 배양하는 단계;(a) A complex strain of a heat-resistant Bacillus coagulans strain that produces lactic acid in a liquid medium consisting of yeast extract, glucose and minerals, and a Bacillus licheniformis strain that secretes low-temperature active cellulase. Incubating at 30 to 35° C. for 45 to 50 hours;
(b) 대두박, 크릴밀(krill meal) 및 복합 한약재를 포함하는 혼합물을 살균한 후 상기 (a) 단계의 복합 균주 배양액을 3~7 %(w/v) 접종하여 30~35℃에서 20~28시간 동안 고상발효시키는 단계;(b) After sterilizing the mixture containing soybean meal, krill meal, and complex herbal medicinal materials, 3-7% (w/v) of the complex strain culture solution of step (a) was inoculated at 30-35℃ for 20~ Solid-phase fermentation for 28 hours;
(c) 상기 (b) 단계의 고상발효물을 30~35℃에서 70~75시간 동안 숙성시키는 단계; 및(c) aging the solid fermented product of step (b) at 30 to 35° C. for 70 to 75 hours; And
(d) 상기 (c) 단계의 고상발효물의 숙성물을 48~52℃에서 열풍 건조한 후 분쇄하는 단계;를 포함할 수 있고, 더욱 구체적으로는,(d) pulverizing the aged product of the solid fermented product of step (c) after hot air drying at 48 to 52° C., and more specifically,
(a) 효모 추출물 2.4 g/L , 포도당 0.4 g/L, 황산마그네슘 0.096 g/L, 염화칼륨 0.5 g/L, Ca(NO3)2 0.032 g/L, MnCl2 0.002 g/L 및 FeSO4·7H2O 0.055 g/L로 이루어진 액상 배지에서 젖산을 생성하는 내열성 바실러스 코아귤런스(B. coagulans) TM3 균주와 저온활성 셀룰라제를 분비하는 바실러스 리케니포르미스(B. licheniformis) DK42 균주의 복합 균주를 32℃에서 48시간 동안 배양하는 단계;(a) Yeast extract 2.4 g/L, glucose 0.4 g/L, magnesium sulfate 0.096 g/L, potassium chloride 0.5 g/L, Ca(NO 3 ) 2 0.032 g/L, MnCl 2 0.002 g/L and FeSO 4 7H 2 O In a liquid medium consisting of 0.055 g/L, a complex of a heat-resistant Bacillus coagulans TM3 strain that produces lactic acid and a Bacillus licheniformis DK42 strain that secretes low-temperature active cellulase Culturing the strain at 32° C. for 48 hours;
(b) 혼합물 총 중량 기준, 94~96 중량%의 대두박, 1~5 중량%의 크릴밀 및 1~5 중량%의 복합 한약재를 포함하는 혼합물을 살균한 후 상기 (a) 단계의 복합 균주 배양액을 4~12 %(w/v) 접종하여 32℃에서 24시간 동안 고상발효시키는 단계;(b) Based on the total weight of the mixture, after sterilizing a mixture containing 94 to 96% by weight of soybean meal, 1 to 5% by weight of krill meal, and 1 to 5% by weight of a complex herbal medicine, the complex strain culture solution of step (a) 4-12% (w/v) inoculation and solid-state fermentation at 32° C. for 24 hours;
(c) 상기 (b) 단계의 고상발효물을 32℃에서 72시간 동안 숙성시키는 단계; 및(c) aging the solid fermented product of step (b) at 32° C. for 72 hours; And
(d) 상기 (c) 단계의 고상발효물의 숙성물을 50℃에서 열풍 건조한 후 분쇄하는 단계;를 포함할 수 있으나, 이에 제한되지 않는다.(d) pulverizing the aged product of the solid fermented product in step (c) after hot air drying at 50° C., but is not limited thereto.
본 발명은 또한, 상기 방법으로 제조된 다기능 어류용 생균제를 제공한다. 본 발명에 따른 다기능 어류용 생균제는 내열성 젖산 생성균인 바실러스 코아귤런스(Bacillus coagulans) TM3 균주와 저온활성 셀룰라제 분비균인 바실러스 리케니포르미스(B. licheniformis) DK42 균주를 유효성분으로 포함하고 있어, 유통 중 생존율이 떨어지는 단점이 보완되었으며, 사료 성분에서 조섬유 분해가 용이한 장점이 있다.The present invention also provides a probiotic for multifunctional fish prepared by the above method. The multifunctional probiotic for fish according to the present invention contains Bacillus coagulans TM3 strain, which is a heat-resistant lactic acid-producing bacteria, and Bacillus licheniformis DK42 strain, which is a low-temperature active cellulase secreting bacteria, as active ingredients. , The disadvantage of poor survival rate during distribution has been compensated, and there is an advantage of easy decomposition of crude fiber in feed ingredients.
본 발명의 "생균제" 즉. 프로바이오틱스는 살아 있는 균 즉, 사람이나 동물이 섭취했을 때 위장관에 머물러 생존할 수 있는 미생물로서 특정 병리 상태를 예방하거나 치료할 수 있는 효과가 있는 미생물 제제를 말한다. 일반적으로 프로바이오틱스는 장내 세균 총의 이상 발효에 의하여 야기되는 제반 증상을 치료하고 개선하는 효과가 있으며 사람 및 동물에 투여되면 장내의 소화관 벽에 밀집, 정착하여 유해 미생물이 정착하지 못하게 하는 작용을 하며 유산을 생성하여 장내 pH를 낮추어서 유해 미생물의 증식을 억제한다.The "probiotic" of the present invention, ie. Probiotics are living bacteria, that is, microorganisms that can survive by staying in the gastrointestinal tract when ingested by humans or animals, and refer to microbial preparations that can prevent or treat specific pathological conditions. In general, probiotics have the effect of treating and ameliorating all symptoms caused by abnormal fermentation of the intestinal flora. When administered to humans and animals, probiotics are concentrated and settled in the walls of the digestive tract in the intestine, preventing harmful microorganisms from settling. It lowers the pH of the intestine by producing it and suppresses the proliferation of harmful microorganisms.
본 발명에 따른 상기 생균제는 당업계에 공지된 방법에 따라 다양한 제형과 방법으로 제조 및 투여될 수 있다. 예를 들어, 대두박, 크릴밀 및 복합 한약재를 포함하는 혼합물을 바실러스 코아귤런스(B. coagulans) TM3 균주와 바실러스 리케니포르미스(B. licheniformis) DK42 균주의 복합 균주를 이용하여 고상발효시킨 발효물은 약제학적 분야에서 통상적으로 사용되는 담체와 혼합하여 산제(powder), 액제(liquids and solutions), 정제(tablet), 캡슐(capsule), 현탁제(suspension) 또는 과립제(granule) 등의 형태로 제조되어 투여될 수 있다. 상기 담체로는 예를 들어, 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료 등일 수 있으나, 이에 제한되지 않는다. 또한, 투여 용량은 투여 대상의 체내에서의 활성성분의 흡수도, 불활성률, 배설속도, 투여 대상의 연령, 성별, 축종, 상태 및 질병의 중증 정도 등에 따라 적절히 선택할 수 있다.The probiotic according to the present invention can be prepared and administered in various formulations and methods according to methods known in the art. For example, a mixture containing soybean meal, krill wheat, and complex herbal medicinal herbs was fermented in solid phase using a complex strain of Bacillus coagulans TM3 strain and B. licheniformis DK42 strain. Water is mixed with carriers commonly used in the pharmaceutical field to form powders, liquids and solutions, tablets, capsules, suspensions, or granules. It can be prepared and administered. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent, but is not limited thereto. In addition, the dose to be administered can be appropriately selected according to the degree of absorption, inactivation rate, excretion rate of the active ingredient in the body of the administration subject, the age, sex, species, condition, and severity of disease of the administration subject.
본 발명의 일 구현 예에서, 어류 사양실험(대상: 넙치)을 통한 장내균총 분석 결과, 생균제를 급여하지 않고 일반 사료만 급여한 대조구에 비해 사료와 생균제를 함께 급여한 처리구에서 해양생물에서 가장 대표적인 병원균인 비브리오 속이 속하는 비브리오과(Vibrionaceae)가 7배 이상 낮게 나타났으며, 유산균에 속하는 락토바실러스 속이 2배 가량 높게 나타나, 장내 병원성 세균의 억제 효과가 우수함을 확인할 수 있었다.In one embodiment of the present invention, as a result of analysis of intestinal flora through fish feeding experiment (object: flounder), the most representative in marine organisms in the treatment group fed with feed and probiotics compared to the control group fed only general feed without feeding probiotics. Vibrionaceae (Vibrionaceae) belonging to the genus Vibrio, which is a pathogen, appeared to be 7 times lower, and the genus Lactobacillus, which belongs to lactic acid bacteria, appeared twice as high, and it was confirmed that the inhibitory effect of pathogenic bacteria in the intestine was excellent.
본 발명은 또한, 상기 다기능 어류용 생균제를 첨가한 사료를 양식 어류에 급이시키는 단계를 포함하는, 양식 어류의 생산성 및 면역력 증대 방법을 제공한다.The present invention also provides a method for increasing productivity and immunity of farmed fish, comprising the step of feeding the feed to which the multifunctional fish probiotic is added to the farmed fish.
본 발명의 양식 어류의 생산성 및 면역력 증대 방법에 있어서, 상기 다기능 어류용 생균제는 바람직하게는 사료 중량 대비 0.3~0.7 중량%로 첨가되는 것일 수 있고, 더욱 바람직하게는 사료 중량 대비 0.5 중량%로 첨가되는 것일 수 있으나, 이에 제한되지 않는다. 상기 다기능 어류용 생균제의 사료 내 첨가 함량이 높아지면 오히려 성장율과 사료효율이 감소하는 경향을 보였다.In the method of increasing the productivity and immunity of cultured fish of the present invention, the probiotic for multifunctional fish may be preferably added in an amount of 0.3 to 0.7% by weight based on the weight of feed, and more preferably in an amount of 0.5% by weight based on the weight of the feed. It may be, but is not limited thereto. When the added content of the multifunctional fish probiotics in the feed increased, the growth rate and feed efficiency tended to decrease.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
실시예 1. 내열성 젖산 생성균 및 저온활성 셀룰라제 분비균의 복합 균주의 특성Example 1. Characteristics of complex strains of heat-resistant lactic acid-producing bacteria and low-temperature-active cellulase-secreting bacteria
본 발명에서 사용된 내열성 젖산 생성균인 바실러스 코아귤런스(Bacillus coagulans) TM3 균주는 곡물, 식품, 분뇨 등의 다양한 자원으로부터 균주를 스크리닝하여 내열성 젖산 생성균을 예비선발하였으며, 16S rRNA 유전자 분석을 통하여 선발된 균주가 Bacillus coagulans로 동정되었다. Bacillus coagulans TM3 strain, a heat-resistant lactic acid-producing bacteria used in the present invention, was preselected for heat-resistant lactic acid-producing bacteria by screening strains from various resources such as grain, food, and manure, and selected through 16S rRNA gene analysis. The strain was identified as Bacillus coagulans.
Nutrient Broth를 이용하여 32℃ 또는 50℃ 조건에서 상기 바실러스 코아귤런스 TM3 균주의 생장 특성을 분석하였다. 그 결과, 배양 48시간 후 32℃ 조건에서는 6.0x107 cfu/㎖의 균수를 보였으며, 50℃ 조건에서는 1.8x107 cfu/㎖의 균수를 보여, 온도에 따른 차이를 확인할 수 없었다. 상기 결과를 통해, 바실러스 코아귤런스 TM3 균주가 내열성이 있음을 알 수 있었다.Nutrient Broth was used to analyze the growth characteristics of the Bacillus coagulation TM3 strain at 32°C or 50°C. As a result, in the culture after 48 hours 32 ℃ conditions showed a bacterial count of 6.0x10 7 cfu / ㎖, the 50 ℃ condition showing the number of bacteria 1.8x10 7 cfu / ㎖, could determine the differences in temperature. Through the above results, it was found that the Bacillus coagulation TM3 strain has heat resistance.
또한, 본 발명의 균주는 어류용 생균제로 이용될 것이므로, 바닷물 농도에서의 적응력을 알아보기 위해, 바실러스 코아귤런스 TM3 균주와 바실러스 리케니포르미스(Bacillus licheniformis) DK42 균주의 내염성을 조사하였다. Nutrient broth에 각 0, 2, 3, 4 또는 5%의 염화나트륨을 첨가한 다음 32℃에서 24시간 배양한 결과, 접종 직후에 두 균주 각각 7.6x105 cfu/㎖, 3.0x106 cfu/㎖에서 시작하여 24시간 후까지 0% 내지 5% 염화나트륨 농도 모두에서 108 cfu/㎖ 수준을 유지하여 염 농도에 따른 처리구간 차이는 볼 수 없었으며, 내열성 젖산 생성균인 바실러스 코아귤런스 TM3 균주가 저온활성 셀룰라제 분비균인 바실러스 리케니포르미스 DK42 균주보다 약간 높은 생장수준을 보이는 것으로 확인되었다.In addition, since the strain of the present invention will be used as a probiotic for fish, in order to examine the adaptability in seawater concentration, the salt resistance of Bacillus coagulation TM3 strain and Bacillus licheniformis DK42 strain was investigated. Nutrient broth was added 0, 2, 3, 4 or 5% sodium chloride and then incubated for 24 hours at 32°C. Immediately after inoculation, the two strains started at 7.6x10 5 cfu/ml and 3.0x10 6 cfu/ml respectively. Therefore, until 24 hours later, the level of 10 8 cfu/ml was maintained at both 0% to 5% sodium chloride concentration, so no difference in treatment intervals according to the salt concentration was observed, and Bacillus coagulence TM3 strain, a heat-resistant lactic acid-producing bacteria, was low-temperature active cellulosic. It was confirmed that the growth level was slightly higher than that of the secretory bacteria Bacillus licheniformis DK42 strain.
(cfu/㎖) B. coagulans TM3
(cfu/ml)
일반적으로 어류용 사료에서는 단백질 함량이 높으며, 동물성/식물성 단백질이 모두 사용되고 있다. 특히 식물성 사료인 대두박은 저렴한 가격으로 인해 전 세계적으로 양어사료에 이용되고 있다. 따라서 어류용 사료 내 대부분을 차지하는 단백질과 식물성 단백질에 함유된 조섬유의 분해는 양식 어류의 소화률에 영향을 미치게 된다. 이에 본 발명의 균주에 대하여 셀룰라제(cellulase), 프로테아제(protease)의 분비 유무를 기질(Carboxymethyl cellulose, Skim milk)을 사용하여 확인하였다.In general, fish feed has a high protein content, and both animal and vegetable proteins are used. In particular, soybean meal, which is a vegetable feed, is being used in aquaculture feed all over the world due to its low price. Therefore, the decomposition of crude fiber contained in protein and vegetable protein, which accounts for most of fish feed, affects the digestibility of farmed fish. Accordingly, the presence or absence of secretion of cellulase and protease in the strain of the present invention was confirmed using a substrate (Carboxymethyl cellulose, Skim milk).
셀룰라제 활성은 조효소액을 만들어 DNS 환원당 정량법을 통하여 측정하였으며, 그 결과 바실러스 리케니포르미스 DK42 균주가 셀룰라제를 분비하며, 20℃의 저온에서도 활성을 띄는 저온활성 셀룰라제를 분비하는 것이 확인되었다(도 1). 프로테아제 활성은 1 %(w/v) Skim milk 배지에 균주의 배양상등액을 이용한 agar well diffusion 방법으로 측정하였으며, 그 결과 바실러스 리케니포르미스 DK42 균주와 바실러스 코아귤런스 TM3 균주 모두 투명환을 형성하며 프로테아제 활성이 확인되었다(도 2).Cellulase activity was measured through the determination of DNS reducing sugar by making a crude enzyme solution. As a result, it was confirmed that Bacillus licheniformis DK42 strain secreted cellulase and secreted low-temperature active cellulase, which is active even at a low temperature of 20°C. (Fig. 1). Protease activity was measured by the agar well diffusion method using the culture supernatant of the strain in 1% (w/v) Skim milk medium. As a result, both the Bacillus licheniformis DK42 strain and the Bacillus coagulation TM3 strain form a transparent ring. Protease activity was confirmed (Fig. 2).
실시예 2. 바실러스 코아귤런스(Example 2. Bacillus coagulation ( B. coagulansB. coagulans ) TM3과 바실러스 리케니포르미스() TM3 and Bacillus licheniformis ( B. licheniformisB. licheniformis ) DK42의 복합 균주 최적 발효 조건) Optimal fermentation conditions for complex strains of DK42
2-1. 액상 배지별 복합균주의 생육 특성 분석2-1. Analysis of growth characteristics of complex strains by liquid medium
바실러스 코아귤런스(B. coagulans) TM3 균주(이하, TM3)와 바실러스 리케니포르미스(B. licheniformis) DK42 균주(이하, DK42)의 복합 균주를 DK 또는 DK2 배지를 이용하여 배양시키며 생장 특성을 확인하였다. A complex strain of Bacillus coagulans TM3 strain (hereinafter, TM3) and Bacillus licheniformis (B. licheniformis ) DK42 strain (hereinafter, referred to as DK42) was cultured using DK or DK2 medium to obtain growth characteristics. Confirmed.
그 결과, 세포의 생장은 각각 1.8x109 과 2.1x109 cfu/㎖로 유사한 것으로 나타났으나, 포자형성의 경우 각각 1.3x108과 2.1x109 cfu/㎖로 DK 배지가 포자생성에 적합함을 알 수 있었다(표 3).As a result, cell growth is 1.8x10 9 and It was found to be similar at 2.1x10 9 cfu/ml, but in the case of sporulation, 1.3x10 8 and It was found that the DK medium was suitable for sporulation at 2.1×10 9 cfu/ml (Table 3).
배지DK2
badge
cellTM3+DK42
cell
sporeTM3+DK42
spore
배지DK
badge
cellTM3+DK42
cell
sporeTM3+DK42
spore
2-2. 고상배지에 염 첨가에 따른 복합균주의 배양 분석2-2. Analysis of culture of complex strains according to salt addition to solid medium
TM3과 DK42 혼합균주로 고상배양 시 포자형성에 도움을 주는 염 첨가에 따른 배양의 차이를 비교하였다. 고상배양시 TM3과 DK42 혼합균주 배양액을 스타터로 사용하였으며, 상기 TM3과 DK42 혼합균주 배양액은 DK 배지를 이용하여 32℃에서 2일간 배양한 것을 사용하였다.The differences in culture according to the addition of salts that help sporulation during solid phase culture with TM3 and DK42 mixed strains were compared. In solid phase culture, the TM3 and DK42 mixed strain culture solution was used as a starter, and the TM3 and DK42 mixed strain culture solution was cultured at 32° C. for 2 days using DK medium.
그 결과, 고상배양은 대부분 1일 배양 후 109 cfu/g 이상 수준을 유지하였으나, 5일 건조 후 대부분 108 cfu/g 수준으로 감소하였으나, 소맥피 단독처리구와 소맥피+대두박+염 처리구에서만 109 cfu/g 수준을 유지하였다.As a result, most of the solid-phase culture maintained a level of 10 9 cfu/g or higher after 1 day culture, but after drying for 5 days, most decreased to the level of 10 8 cfu/g, but only in wheat skin alone treatment and wheat skin + soybean meal + salt treatment. The 10 9 cfu/g level was maintained.
또한, 유산을 생성하는 Bacillus coagulans의 선택배지로서 BCP(Bromo Cresol Puple) Agar를 사용하여 TM3를 검출한 결과 유의성은 인정할 수 없었으며, 전반적으로 DK42 균주와 어울려 잘 배양되는 것으로 판단되었다. 포자수의 경우 배양이 끝나는 시점에서 계수를 실시하였으며, 전반적으로 건조 과정 중 포자수가 증가하는 것으로 나타났다. 이는 건조온도가 50℃ 이하에서 이루어진 관계로 해당 온도에서 잘 자랄수 있는 TM3 균주의 포자가 형성된 것에 기인한 것으로 판단되었다. In addition, as a result of detecting TM3 using BCP (Bromo Cresol Puple) Agar as a selective medium for Bacillus coagulans producing lactic acid, the significance was not recognized, and it was determined that the overall culture was well suited with the DK42 strain. In the case of the number of spores, counting was performed at the end of the culture, and it was found that the number of spores increased during the drying process. This was determined to be due to the formation of spores of the TM3 strain that can grow well at the temperature, as the drying temperature was made at 50°C or less.
소맥피50%4. Soybean meal 50%+
50% wheat blood
소맥피50%+염6. Soybean meal 50%+
50% wheat blood + salt
2-3. 고상배지에 크릴 첨가에 따른 복합균주의 배양 분석2-3. Culture analysis of complex strains according to krill addition to solid medium
DK42 및 TM3 혼합균주로 고상배양 시 소맥피 또는 대두박에 크릴밀을 10%, 30%, 50% 첨가하고, 이에 따른 배양의 차이를 비교하였다. 그 결과, 고상배양 1일 배양 후 DK42 및 TM3 혼합균주의 세포수는 대부분 109 cfu/g 수준을 유지하였으나, 크릴 함량이 높아질수록, 건조시간이 경과할수록 DK42 및 TM3 혼합균주의 세포수는 감소하는 경향을 보여 높은 함량의 크릴이 균주의 성장을 억제하는 경향을 보였다. 특히 크릴 50% 첨가의 경우 108 cfu/g 수준으로 감소하는 경향을 보여 현장의 대량배양에 참고하기로 하였다.In solid culture with DK42 and TM3 mixed strains, 10%, 30%, and 50% of krill meal were added to wheat bark or soybean meal, and the differences in culture were compared. As a result, the number of cells of the mixed strains DK42 and TM3 remained mostly 10 9 cfu/g level after 1 day of solid culture culture, but the number of cells of the mixed strains DK42 and TM3 decreased as the krill content increased and the drying time elapsed. The high content of krill showed a tendency to inhibit the growth of the strain. In particular, the addition of 50% krill showed a tendency to decrease to the level of 10 8 cfu/g, so it was decided to refer to mass cultivation in the field.
또한, 포자수는 크릴 함량이 높아질수록, 건조시간이 경과할수록 107 cfu/g 수준까지 매우 크게 감소하는 경향을 확인할 수 있었다.In addition, as the number of spores increased, as the krill content increased and the drying time elapsed, it was confirmed that the number of spores decreased very significantly to the level of 10 7 cfu/g.
2-4. 고상배지에 한약재 첨가에 따른 복합균주의 배양 분석2-4. Analysis of the culture of complex strains according to the addition of herbal medicinal herbs to solid medium
TM3 및 DK42 혼합균주로 한약재(어성초, 지황, 당귀, 약콩 및 감초를 동량으로 혼합한 혼합물)가 5% 첨가된 고상배지의 발효패턴을 조사한 결과, 초기 고상배양은 DK42 균주가 이끌어 가고 있음을 확인하였으며, 건조과정을 거치면서 살아있는 세포는 많이 사멸하였으나 포자수는 107 cfu/g 수준을 유지하고 있음을 보였다. 배지 곡물은 소맥피보다 대두박에서 가장 효과가 좋은 것으로 나타났다.As a result of investigating the fermentation pattern of the solid medium containing 5% herbal medicines (a mixture of Eoseongcho, Reichstag, Angelica, Yakbean and Licorice) as a mixed strain of TM3 and DK42, it was confirmed that the initial solid culture was led by the DK42 strain During the drying process, a lot of living cells died, but the number of spores was maintained at a level of 10 7 cfu/g. Medium grains were found to be the most effective in soybean meal than wheat skin.
상기 결과를 통해, 본 발명자는 크릴(2.5%), 복합 한약재(2.5%) 및 대두박(95%)을 주성분으로 한 고상배지 조성물에 바실러스 코아귤런스(Bacillus coagulans) TM3 균주와 바실러스 리케니포르미스(B. licheniformis) DK42 균주의 복합균주를 접종하여 어류용 생균제를 제조하였다. Through the above results, the present inventors found that Bacillus coagulans TM3 strain and Bacillus licheniformis in a solid medium composition containing krill (2.5%), complex herbal medicine (2.5%) and soybean meal (95%) as main components. ( B. licheniformis ) A probiotic for fish was prepared by inoculating a complex strain of DK42 strain.
실시예 3. 다기능 어류용 생균제를 이용한 사양 실험Example 3. Specification experiment using probiotics for multifunctional fish
상기 실시예 2를 통해 최적화시킨 발효조건으로 제조한 어류용 생균제(도 3)를 이용하여 넙치 사양 실험을 진행하였다.A flounder specification experiment was carried out using a probiotic for fish (FIG. 3) prepared under the fermentation conditions optimized through Example 2 above.
넙치는 평균 전장 22±1.0cm, 중량 105±1.1g을 대상으로 8주간 실험에 사용하였으며, 실험에 사용한 사료는 동물성 단백질 함량이 73% 이상인 조단백 54% 및 조지방 6% 이상의 상품용 넙치사료(표 7)를 생균제 첨가실험에 사용하였다. 어류용 생균제는 0.5%, 1.0% 또는 2.0%의 비율로 사료에 첨가하여 넙치에 공급하였다. 대조군은 생균제를 첨가하지 않고 사료만 제공하였다.Flounder was used for 8 weeks with an average length of 22±1.0cm and a weight of 105±1.1g, and the feed used in the experiment was commercial flounder feed with an animal protein content of more than 73%, crude protein of 54% and crude fat of 6% or more (Table 7) was used in the experiment of adding probiotics. Probiotics for fish were added to the feed in a ratio of 0.5%, 1.0% or 2.0% and supplied to the flounder. The control group provided only feed without adding probiotics.
사료는 매일 2회 반복 공급하였고 잔여 사료는 건져내 계수한 후 무게를 환산하여 사료공급량을 계산하였으며, 실험 종료 후 어류용 생균제 농도에 따른 실험구간 별로 전장 및 체중을 측정한 후, 사료공급량과 성장을 통해 성장률과 사료효율 관련 지표수치 도출하였다. 5일 간격으로 수질측정 위해 수온, pH, 용존산소, 암모니아, 질산염, 아질산염을 측정하였다. 실험 결과는 SPSS 20 (Chicago, IL, USA)을 이용하여 one-way ANOVA test를 수행하여 분산 가정이 되는 실험구는 Duncan's test, 등 분산 가정이 안되는 실험구는 Games-Howell로 평균 간의 유의성을 95% 수준에서 검정하였다.Feed was repeatedly supplied twice a day, and the remaining feed was counted and weight was converted to calculate the feed supply amount.After the experiment was completed, the total length and weight were measured for each experimental section according to the concentration of probiotics for fish, and the feed supply amount and growth were measured. Through this, index values related to growth rate and feed efficiency were derived. Water temperature, pH, dissolved oxygen, ammonia, nitrate, and nitrite were measured at intervals of 5 days. The experimental result is a one-way ANOVA test using SPSS 20 (Chicago, IL, USA), and Duncan's test for the experimental group that has a variance assumption, and Games-Howell for the experimental group that does not have a variance assumption, with 95% significance between the means. It was tested at.
실험종료 후 모든 실험구간에서 폐사는 확인되지 않아 전 구간 생존율은 100%로 나타났다. 최종 중량은 생균제를 0.5% 첨가한 실험군이 평균 7137.7g, 생균제를 1.0% 첨가한 실험군이 7099.3g 순으로 높았으며, 생균제 2.0% 첨가와 무첨가 대조군은 각각 6936.8, 6984.5g로 2% 첨가 실험군에서 오히려 다소 낮은 최종중량이 확인되었다. 성장률(WG)과 일간성장율(SGR) 또한, 생균제 0.5% 첨가 실험군이 126.8%와 1.46%로 다른 실험군보다 유의하게 높았으며, 생균제 1.0% 실험군이 126.4%와 1.44%로 0.5% 첨가 실험군 다음으로 높았으나 다른 실험구간들과 유의한 차이를 보이진 않는 것으로 나타났다(표 8).After the end of the experiment, death was not confirmed in all experimental sections, so the survival rate for all sections was 100%. The final weight was higher in the experimental group with 0.5% probiotics, on average, 7137.7g, and the experimental group with 1.0% probiotics in the order of 7099.3g, respectively.The 2.0% and no probiotics were 6936.8 and 6984.5g, respectively. A somewhat lower final weight was identified. The growth rate (WG) and daily growth rate (SGR) were also significantly higher in the experimental group with 0.5% probiotics, 126.8% and 1.46%, compared to the other experimental groups, and the 1.0% probiotic experiment group, 126.4% and 1.44%, the second highest after the experimental group with 0.5% However, it was found that there was no significant difference from other experimental sections (Table 8).
groupExperimental
group
2 Specific growth rate = [ln(final body weight) - ln(initial body weight)] × 100 / days reared.
3 Survival rate = (the number of initial fish - the number of dead fish) / the number of initial fish × 100. 1 Weight gain = (final body weight-initial body weight) / initial body weight × 100.
2 Specific growth rate = [ln(final body weight)-ln(initial body weight)] × 100 / day reared.
3 Survival rate = (the number of initial fish-the number of dead fish) / the number of initial fish × 100.
사료계수(FCR)는 생균제 2.0% 첨가 실험군에서 1.55로 가장 유의하게 높은 값을 보였으며, 일일사료급이량(DFI) 또한 생균제 2.0% 첨가 실험군에서 2.07%로 다른 실험군보다 유의하게 높게 나타났다. 사료효율(FE)은 생균제 2% 첨가 실험군에서 64.7%로 가장 낮게 나타났으며, 대조군, 생균제 0.5% 첨가군, 생균제 1% 첨가군에서는 유의한 차이가 없었다. 단백질효율비(PER)는 사료효율과 동일한 경향으로 확인되었고, 실험결과 생균제 0.5% 첨가 실험군에서 성장률 126.8%로 유의수준 95%에서 가장 좋은 결과를 보였으며, 대조군 대비 약 5.2% 증가하여 본 발명에 따른 다기능 어류용 생균제(probiotics)의 첨가가 성장에 영향을 미치는 것을 확인할 수 있었다. 다만, 함량이 높아지면 오히려 성장률과 사료효율이 감소하는 경향을 보여 적절한 농도조절이 필요할 것으로 판단되었다(표 9).The feed coefficient (FCR) was significantly higher at 1.55 in the experimental group with 2.0% probiotic added, and the daily feed amount (DFI) was also significantly higher than the other experimental groups at 2.07% in the experimental group with 2.0% probiotic added. Feed efficiency (FE) was the lowest at 64.7% in the experimental group with 2% probiotic, and there was no significant difference in the control group, the group with 0.5% probiotic, and the group with 1% probiotic. Protein efficiency ratio (PER) was confirmed to be the same trend as feed efficiency, and as a result of the experiment, the growth rate was 126.8% in the experimental group with 0.5% probiotic, showing the best result at the significance level of 95%, and increased by about 5.2% compared to the control group. It was confirmed that the addition of multifunctional fish probiotics had an effect on growth. However, as the content increased, the growth rate and feed efficiency tended to decrease, so it was determined that proper concentration control was necessary (Table 9).
groupExperimental
group
2 Daily feed intake = feed intake × 100 / [(initial body weight + final body weight + dead fish weight) × days reared /2)].
3 Feed efficiency = (wet weight gain / feed intake) × 100.
4 Protein efficiency ratio = (final body weight - initial body weight) / (protein contents percentages (54%) × feed intake / 100). 1 Feed conversion ratio = feed intake / (final body weight-initial body weight).
2 Daily feed intake = feed intake × 100 / [(initial body weight + final body weight + dead fish weight) × days reared /2)].
3 Feed efficiency = (wet weight gain / feed intake) × 100.
4 Protein efficiency ratio = (final body weight-initial body weight) / (protein contents percentages (54%) × feed intake / 100).
또한, 사육 실험이 종료된 실험어는 혈액을 채취해 리소자임(lysozyme) 활성 분석 실험에 사용하였으며, 생균제 첨가에 따른 스트레스 저감 효과를 확인하기 위해 스트레스의 척도인 코르티졸(cortisol)의 수준을 분석하였다. 이를 위해, 실험종료 3일 후 각 실험군 별로 10 마리를 무작위 선별하여 자극온도 25, 28, 31℃의 2 ton 수조로 옮기고 24 및 48시간 동안 노출시킨 뒤 실험군 당 3 마리씩, 한 마리당 2 ㎖씩 채혈하였다. 혈액은 4℃에서 30분 보관 후 8,000 rpm에서 10분간 원심분리하여 혈장을 분리하였으며, 혈장 코르티졸 농도는 Fish ELISA Cortisol kit (Cusabio Biotech, China)를 사용하여 분석하였고, 모든 실험은 3반복 수행하였다.In addition, blood was collected from the experimental fish after the breeding experiment was completed and used in the lysozyme activity assay experiment, and the level of cortisol, a measure of stress, was analyzed to confirm the effect of reducing stress according to the addition of probiotics. To this end, 3 days after the end of the experiment, 10 animals were randomly selected for each experimental group, transferred to a 2 ton tank with stimulation temperatures of 25, 28, and 31℃, exposed for 24 and 48 hours, and then collected 3 animals per experimental group and 2 ml per animal. I did. Blood was stored at 4° C. for 30 minutes and then centrifuged at 8,000 rpm for 10 minutes to separate plasma. Plasma cortisol concentration was analyzed using Fish ELISA Cortisol kit (Cusabio Biotech, China), and all experiments were performed in triplicate.
혈액 내 코르티졸 농도 분석 결과, 24시간 노출되었을 때 25℃, 28℃, 31℃ 모두 생균제 첨가 농도별 실험군 간 유의한 차이는 보이지 않았으나 0.5% 첨가 실험군에서 다른 농도보다 코르티졸 농도의 완만한 상승 경향이 나타났으며, 48시간 이후에는 유의한 차이가 발생하는 것을 알 수 있었다. 또한, 생균제 0.5% 첨가 실험군은 31℃ 48시간에서 다른 실험군과 비교하여 유의하게 낮은 코르티졸 농도를 보여 스트레스 저감효과가 우수함을 알 수 있었다(표 10 및 도 4a). 특히, 수온 28℃에서는 스트레스가 적어 실험군간 혈중 코르티졸 농도의 유의차가 없으나, 수온 31℃에서는 실험군간 뚜렷한 차이를 보였다.As a result of analysis of blood cortisol concentration, when exposed for 24 hours, there was no significant difference between the experimental groups at each concentration of probiotics added at 25°C, 28°C, and 31°C. However, in the 0.5% added experimental group, the cortisol concentration tended to increase more slowly than other concentrations. It was found that a significant difference occurred after 48 hours. In addition, the experimental group added with 0.5% probiotic showed significantly lower cortisol concentration compared to the other experimental groups at 31° C. 48 hours, indicating that the stress reducing effect was excellent (Table 10 and FIG. 4A). In particular, there was no significant difference in blood cortisol concentration between the experimental groups due to less stress at a water temperature of 28°C, but there was a clear difference between the experimental groups at a water temperature of 31°C.
또한, 리소자임 활성 분석결과 대조군이 6.8±2.8로 가장 낮았으며, 생균제 1% 또는 2% 첨가 실험군은 각각 8.9±2.5, 11.1±3.7로 대조군보다 높았으나 유의한 차이를 보이지 않았고, 생균제 0.5% 첨가 실험군에서 19.7±4.7로 다른 실험군보다 유의하게 높아 생균제를 0.5% 첨가한 사료급이 실험군의 면역력 증대효과가 확인되었다(표 11 및 도 4b).In addition, as a result of analysis of lysozyme activity, the control group was the lowest at 6.8±2.8, and the experimental group with 1% or 2% probiotics was higher than the control at 8.9±2.5 and 11.1±3.7, respectively, but there was no significant difference, and the experimental group with 0.5% probiotic was added. At 19.7±4.7, it was significantly higher than that of the other experimental groups, and the effect of increasing the immunity of the experimental group was confirmed in the feed supplemented with 0.5% probiotics (Table 11 and FIG. 4b).
Claims (8)
(a) 효모 추출물, 포도당 및 미네랄로 이루어진 액상 배지에서 젖산을 생성하며 프로테아제 활성을 갖는 내열성 바실러스 코아귤런스(Bacillus coagulans) 균주와 저온활성 셀룰라제를 분비하며 프로테아제 활성을 갖는 바실러스 리케니포르미스(Bacillus licheniformis) 균주의 복합 균주를 30~35℃에서 45~50시간 동안 배양하는 단계;
(b) 대두박; 크릴밀(krill meal);과 어성초, 지황, 당귀, 약콩 및 감초로 이루어진 복합 한약재;를 포함하는 혼합물을 살균한 후 상기 (a) 단계의 복합 균주 배양액을 접종하여 30~35℃에서 20~28시간 동안 고상발효시키는 단계;
(c) 상기 (b) 단계의 고상발효물을 30~35℃에서 70~75시간 동안 숙성시키는 단계; 및
(d) 상기 (c) 단계의 고상발효물의 숙성물을 48~52℃에서 열풍 건조한 후 분쇄하는 단계;를 포함하는 것을 특징으로 하는 다기능 어류용 생균제의 제조방법.The method of claim 1,
(a) Bacillus licheniformis , which produces lactic acid in a liquid medium consisting of yeast extract, glucose and minerals, secretes a heat-resistant Bacillus coagulans strain having protease activity and low-temperature active cellulase, and has protease activity ( Bacillus licheniformis ) culturing the complex strain of the strain for 45 to 50 hours at 30 to 35 ℃;
(b) soybean meal; After sterilizing the mixture containing krill meal; and a complex herbal medicine consisting of Eoseongcho, Reichstag, Angelica, Yakbean and Licorice; and inoculating the complex strain culture solution of step (a), 20-28 at 30-35℃ Solid-phase fermentation for a period of time;
(c) aging the solid fermented product of step (b) at 30 to 35° C. for 70 to 75 hours; And
(d) pulverizing the aged product of the solid fermented product of step (c) after hot air drying at 48 to 52°C.
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| KR20170027650A (en) * | 2016-03-02 | 2017-03-10 | 대한민국(관리부서:국립수산과학원) | Winter feed formula for olive flounder |
| KR101971984B1 (en) * | 2018-10-05 | 2019-04-24 | 한석주 | Mixed probiotics using heat-treated whole fat soybean, method for preparing the same, and method for producing high quality Korean native steers meat by using the mixed probiotics |
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| KR20170027650A (en) * | 2016-03-02 | 2017-03-10 | 대한민국(관리부서:국립수산과학원) | Winter feed formula for olive flounder |
| KR101971984B1 (en) * | 2018-10-05 | 2019-04-24 | 한석주 | Mixed probiotics using heat-treated whole fat soybean, method for preparing the same, and method for producing high quality Korean native steers meat by using the mixed probiotics |
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