KR101480028B1 - Novel microorganism bacillus subtilis sj-30 and additives for fish feeds containing it - Google Patents

Novel microorganism bacillus subtilis sj-30 and additives for fish feeds containing it Download PDF

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KR101480028B1
KR101480028B1 KR20130073873A KR20130073873A KR101480028B1 KR 101480028 B1 KR101480028 B1 KR 101480028B1 KR 20130073873 A KR20130073873 A KR 20130073873A KR 20130073873 A KR20130073873 A KR 20130073873A KR 101480028 B1 KR101480028 B1 KR 101480028B1
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정철연
오남순
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(주)창조바이오텍
공주대학교 산학협력단
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Abstract

본 발명은 신규미생물 바실러스 서브틸러스 SJ-30를 포함하고 있는 어류용 사료첨가제에 관한 것이다.
본 발명의 어류용 사료첨가제는 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내며, 서열번호 1로 표현되는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P를 유효성분으로 포함하여 구성된다.
본 발명에 의해, 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 및 이를 포함하여 열안정성 및 내산성을 갖으며 어병세균(Streptococcus parauberis)에 대해 방제효과를 갖는 어류용 사료첨가제를 제공하게 된다.The present invention relates to a feed additive for fish comprising a novel microorganism Bacillus subtilis SJ-30.
The feed additive for fish according to the present invention comprises an effective ingredient of a novel microorganism Bacillus subtilis SJ-30 strain KCTC18249P which exhibits an antibacterial activity against Streptococcus parauberis (SEQ ID NO: 1) .
According to the present invention, there is provided a novel microorganism Bacillus subtilis SJ-30 strain showing antimicrobial activity against Streptococcus parauberis , and having a thermostability and acid resistance including Streptococcus parauberis , Thereby providing a feed additive for fish having a controlling effect.

Description

신규미생물 바실러스 서브틸러스 ＳＪ-30 및 이를 포함하고 있는 어류용 사료첨가제 {NOVEL MICROORGANISM BACILLUS SUBTILIS SJ-30 AND ADDITIVES FOR FISH FEEDS CONTAINING IT}TECHNICAL FIELD [0001] The present invention relates to a novel microbial Bacillus subtilis SJ-30 and a fish feed additive comprising the microbial Bacillus subtilis SJ-30 and a fish feed additive comprising the microbial Bacillus subtilis SJ-

본 발명은 신규미생물 바실러스 서브틸러스 SJ-30 및 이를 포함하고 있는 어류용 사료첨가제에 관한 것이다.The present invention relates to a novel microorganism Bacillus subtilis SJ-30 and a feed additive for fish comprising the same.

현재 어류 양식장에서는 밀집양식으로 인한 질병 증가, 영양상태 불균형 등과 같은 요인들로 인하여 어류의 성장저하 및 폐사가 발생되는 문제가 심각하게 대두되고 있다.Currently, in fish farms, problems such as increased disease caused by dense farming, nutritional imbalance and other factors are causing serious problems such as fish growth and mortality.

이에 사료에 첨가제를 첨가하여 상기 문제를 해결토록 하는데, 통상적으로 사용되는 사료첨가제로는 항생제, 비타민제, 아미노산 공급제, 광물질 공급제, 효소제, 호르몬제 등이 있다. Additives are added to the feed to solve the above problems. Commonly used feed additives include antibiotics, vitamins, amino acid feeders, mineral feeders, enzymes, and hormones.

그러나, 상기의 사료첨가제들은 과다 급이시 양식어류의 약품에 대한 내성이 증가하여 약제 남용을 야기시킬 수 있으며, 이로 인하여 양식된 어류에는 상기 유해물질들이 축적되어 어류 섭취시 인체에 유해할 수 있는 유해물질을 간접적으로 섭취할 수 있다는 점이 수산물의 안전성 면에서 문제점을 갖게 된다. However, the above-mentioned feed additives may cause drug abuse due to an increase in tolerance to chemicals of cultured fish in the case of excessive feeding, so that the harmful substances accumulate in the cultured fish, The fact that indirect consumption of harmful substances has a problem in the safety of marine products.

이에, 이를 해소하기위해 하기와 같은 여러 연구들이 시행된바 있다. In order to solve this problem, several studies have been carried out as follows.

관련 선행기술로는 한국등록특허 제10-057771호(바실러스 폴리퍼멘티루스, 바실러스 리케니포르미스 및 사카로마이세스 세로비지에를 포함하는 어류사료첨가용 조성물) 및 한국등록특허 제10-860111(신규 미생물 트라우스토카이트리움 에스피.케이제이에스-1, 바실러스 폴리퍼멘티쿠스 케이제이에스-2 및 이들과 바실러스 리케니포르미스를 포함하고 있는 사료첨가제 및 사료)이 공개되어 있다. Related prior arts include Korean Patent No. 10-057771 (a composition for adding a fish feed containing Bacillus polyfermentus, Bacillus licheniformis and Saccharomyces cerevisiae) and Korean Patent No. 10-860111 (Feed additives and feedstuffs comprising the new microbial strains Truace, Tokyintium, Escherichia, Kayeses-1, Bacillus polyfermenticus kayseres-2 and their and Bacillus licheniformis).

그러나, 현재까지 어병을 일으키는 세균에 대한 방제효과를 나타내는 천연항생제에 대하여 보고된 바가 없다. However, there has been no report on natural antibiotics which shows the control effect against bacteria causing fish diseases to date.

본 발명에서는 상기의 문제점을 해결하기 위해, 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물을 제공하려는 목적이 있다.In order to solve the above problems, it is an object of the present invention to provide a novel microorganism exhibiting antibacterial activity against a fish microbial strain ( Streptococcus parauberis ).

또한, 상기 신규미생물을 포함하고 있는 어류용 사료첨가제를 제공하려는 목적도 있다.It is also an object of the present invention to provide a feed additive for fish comprising the new microorganism.

본 발명의 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P는 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내며 서열번호 1로 표현되는 신규미생물인 것이 특징이다.The novel microorganism Bacillus subtilis SJ-30 strain KCTC18249P of the present invention is a novel microorganism exhibiting an antimicrobial activity against a fish bacterium ( Streptococcus parauberis ) and represented by SEQ ID NO: 1.

본 발명의 어류용 사료첨가제는 상기 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P를 유효성분으로 포함한 것이 특징이다.The fish feed additive of the present invention is characterized by containing the new microorganism Bacillus subtilis SJ-30 strain KCTC18249P as an active ingredient.

상기 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P는 미생물의 균체, 농축물, 배양액 또는 그 건조물로 이루어진 것이 특징이다.The new microorganism Bacillus subtilis SJ-30 strain KCTC18249P is characterized by comprising microbial cells, a concentrate, a culture solution or a dried product thereof.

상기 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P는 글루코오스 1~3 중량% 첨가한 배지에서 12~24시간동안 배양된 것이 특징이다.The new microorganism Bacillus subtilis SJ-30 strain KCTC18249P is characterized in that it is cultured for 12 to 24 hours in a medium supplemented with 1 to 3% by weight of glucose.

상기 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P는 열안정성 및 내산성을 갖는 것이 특징이다.The novel microorganism Bacillus subtilis SJ-30 strain KCTC18249P is characterized by having heat stability and acid resistance.

본 발명에 의해, 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 및 이를 포함하여 열안정성 및 내산성을 갖으며 어병세균(Streptococcus parauberis)에 대해 방제효과를 갖는 어류용 사료첨가제를 제공하게 된다.According to the present invention, there is provided a novel microorganism Bacillus subtilis SJ-30 strain showing antimicrobial activity against Streptococcus parauberis , and having a thermostability and acid resistance including Streptococcus parauberis , Thereby providing a feed additive for fish having a controlling effect.

도 1은 선발균주인 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주의 생육도를 나타낸 도면이다.
도 2는 선발균주인 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 배양액의 배양시간 및 당농도에 따른 pH변화를 나타낸 도면이다.
도 3은 선발균주인 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주의 내산성을 나타낸 도면이다. Fig. 1 is a diagram showing the growth of Bacillus subtilis strain SJ-30, which is a starting strain.
FIG. 2 is a graph showing changes in pH of culture medium of Bacillus subtilis SJ-30 strain, which is a starting strain, according to culture time and sugar concentration.
FIG. 3 is a graph showing the acid resistance of a selected strain, Bacillus subtilis SJ-30.

본 발명에 대하여 보다 상세히 설명하면 다음과 같다.The present invention will be described in more detail as follows.

본 발명에서는 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물을 도출해내기 위해 보유 중인 Bacillus 균종과 변이주를 대상으로 항균활성도를 측정하여 항균활성도가 비교적 높다고 판단되는 균주를 선발하였으며, 그 결과 6개의 변이주를 포함하여 총 42개의 균주를 선발 하였다. In the present invention, a strain having a relatively high antibacterial activity was selected from Bacillus species and mutant strains, which were found to be novel microorganisms showing antimicrobial activity against a fish bacterial strain ( Streptococcus parauberis ) A total of 42 strains including mutant strains were selected.

이 중 항균활성이 높다고 판단되는 균주를 최종적으로 선택하여 배양조건에 따른 항균활성, 균주의 열안정성, 내산성 및 내담즙산성을 분석하여, 어류용 사료첨가제의 유효성분으로 적합한 SJ-30균주를 확인하였다. Among them, strains determined to have high antimicrobial activity were finally selected to analyze the antimicrobial activity, the thermal stability, the acid resistance and the bile acid resistance according to the culture conditions, and confirmed the SJ-30 strain suitable as an effective ingredient of the feed additive for fish Respectively.

상기 선발된 SJ-30 균주는 16S rRNA 유전자 염기서열로 계통 유전학적 분석을 실시한 바, SJ-30 균주는 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주임을 확인하였다. The selected SJ-30 strain was subjected to a genetic analysis based on a 16S rRNA gene sequence, and the SJ-30 strain was found to be a Bacillus subtilis SJ-30 strain.

이에 본 발명자는 상기 균주를 "바실러스 서브틸러스(Bacillus subtilis) SJ-30균주"로 명명하고, 이 균주를 한국생명공학연구원 미생물자원센터에 기탁하여 2013년 6월 17일 수탁번호 KCTC18249P를 부여받았다.Therefore, the present inventor named the strain as " Bacillus subtilis SJ-30 strain" and deposited this strain on the microorganism resource center of the Korea Research Institute of Bioscience and Biotechnology, and received the deposit number KCTC18249P on June 17, 2013 .

상기 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P는 서열번호 1로 기재되는 염기서열 전체 또는 그것의 일부로 이루어지며, 1430bp의 오픈 리딩 프레임(ORF)으로 구성된다.The Bacillus subtilis SJ-30 strain KCTC18249P consists of the entire nucleotide sequence shown in SEQ ID NO: 1 or a part thereof and is composed of an open reading frame (ORF) of 1430 bp.

이러한 상기 균주는 미생물 제제의 형태로 이루어지기도 하며, 이때 상기 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주는 배양액의 형태로 함유된다.Such a strain may be in the form of a microorganism preparation, wherein the Bacillus subtilis strain SJ-30 is contained in the form of a culture solution.

설명하면, 본 발명에 있어서의 용어 '배양액'은 미생물 생장에 적절한 배지에 일정 농도의 포자를 접종하여 배양기에서 일정 기간 동안 미생물이 생장하도록 유도한 후 수득된, 균체, 균체가 생장하면서 분비한 물질, 및 생장에 필요한 영양성분이 혼합된 액으로서, 불순물을 걸러내는 과정을 거쳐 제조할 수 있다.The term " culture medium " in the present invention means a microorganism which is inoculated with a certain concentration of spores in a medium suitable for microorganism growth, inducing the microorganisms to grow for a certain period of time in an incubator, , And a nutrient component necessary for growth, and can be produced by a process of filtering off impurities.

본 발명의 용어 '방제'는 일반적으로 식물 병원체 감염을 예방, 감소 또는 근절시키는 것을 포함하며, 감염에 있어서의 예방 또는 감소는 비처리된 기주식물에 대하여 통계적으로 현저한 차이가 있음을 의미한다.The term " control " of the present invention generally includes preventing, reducing or eradicating plant pathogenic infections, wherein the prevention or reduction in infection means that there is a statistically significant difference for untreated host plants.

이에, 본 발명의 어병세균(Streptococcus parauberis) 방제효과를 갖는 어류용 사료 첨가제에 상기 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주를 배양액의 형태로 함유될 경우 상기 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주가 생장할 수 있도록 글루코오스 1~3 중량% 첨가한 배지에서 12~24시간동안 배양하여 수득된 배양액을 사용하는 것이 좋다. Therefore, when the Bacillus subtilis SJ-30 strain is contained in the form of a culture solution in the feed additives for fish having the effect of controlling the Streptococcus parauberis of the present invention, the Bacillus subtilis , It is preferable to use the culture obtained by culturing in a medium containing 1 to 3% by weight of glucose for 12 to 24 hours so that the SJ-30 strain can grow.

상기 미생물 제제는 미생물의 안정적인 제제화를 목적으로 수화제, 입제 또는 캡슐화의 형태로 제조될 수 있다.The microorganism preparation may be prepared in the form of a wettable powder, granules or encapsulation for the purpose of stable formulation of microorganisms.

이와 같이 본 발명은 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P 및 이를 포함하여 어병세균(Streptococcus parauberis)에 대해 방제효과를 갖는 어류용 사료첨가제를 제공하게 된다.
Thus, the present invention is for fish having a controlling effect for Fish Pathology bacteria novel microorganism Bacillus sub-blocks bus (Bacillus subtilis) SJ-30 strain KCTC18249P and Fish Pathology, bacteria (Streptococcus parauberis) including this represents the anti-bacterial activity against (Streptococcus parauberis) Feed additives.

이하, 본 발명에 대하여 실시예를 통하여 상세히 설명하나, 이들이 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto.

<실시예 1> 어병세균(Streptococcus parauberis)에 대한 항균활성 측정 및 바실러스(Bacillus) 균주 선발Example 1 Measurement of Antibacterial Activity against Streptococcus parauberis and Selection of Bacillus Strain

1) 실험방법1) Experimental method

ⓛ 항균활성방법Ⓛ Antimicrobial activity method

항균성 측정을 위해 TSB 사면배지에 접종하여 30℃에서 24시간 활성화 한 후 TSB 액체배지에 접종하여 12시간 종균배양 하였다(37℃, 180 rpm). For antimicrobial activity, TSB slant medium was inoculated and incubated at 30 ° C for 24 hours, then inoculated on TSB liquid medium and cultured for 12 hours (37 ° C, 180 rpm).

종균배양한 균주는 다시 TSB에 글루코오스(glucose)가 1% 첨가된 배지에 접종하여 24시간동안 같은 조건으로 본배양 하였으며, 그 후 배양액을 원심분리(12,000 rpm, 25℃, 15분)하여 상징액을 취해 제균(0.45㎛ filter)한 것을 조항균물질로하여 어병세균에 대해 디스크확산방법(disc diffusion method)으로 항균성을 측정하였다. The strain was cultivated in the same condition for 24 hours after inoculation on the culture medium containing 1% of glucose added to TSB. After that, the culture was centrifuged (12,000 rpm, 25 ° C, 15 minutes) (0.45 占 퐉 filter) was used as a antibacterial substance, and the antibacterial activity of the fish microbes was measured by a disc diffusion method.

어병세균 현탁액 2 ml와 BHI soft agar(0.6% agar) 8 ml를 혼합한 후 미리 준비한 BHI agar 배지에 중층하였으며, 실온에 정치시켜 배지를 굳힌 후 항균활성 측정용 배지로 사용하였다. 2 ml of fish microbial suspension and 8 ml of BHI soft agar (0.6% agar) were mixed and layered on a BHI agar medium prepared beforehand. After incubation at room temperature, the medium was used as a medium for measuring the antimicrobial activity.

핀셋을 이용하여 페이퍼 디스크(paper disc, diameter 8 mm)를 항균활성 측정용 배지에 얹은 후, 조항균물질을 50 ㎕를 떨어뜨리고 30℃, 24시간 동안 배양하면서 paper disc 주위에 어병균에 대한 생육저해환(clear zone)의 생성유무를 관찰하여 생육저해환의 크기(mm)를 측정하였다.
Using a tweezers, a paper disc (diameter 8 mm) was placed on a culture medium for antimicrobial activity, and then 50 μl of the antibacterial substance was dropped. After incubation at 30 ° C. for 24 hours, growth inhibition The size (mm) of the growth inhibition ring was measured by observing whether a clear zone was formed or not.

② 프로테아제(Protease) 활성도 확인방법② How to check protease activity

아조카제인(Azocasein)을 이용한 protease 분석은 다음 방법에 따라 측정하였다. 배양액(180 rpm, 37℃, 24 hr) 2 mL를 원심분리(8,000 rpm, 25℃, 10 min)한 후 상징액을 50배 희석하였다. Protease analysis using azocasein was performed according to the following method. 2 mL of the culture (180 rpm, 37 ° C, 24 hr) was centrifuged (8,000 rpm, 25 ° C, 10 min) and the supernatant was diluted 50-fold.

희석액 0.15 mL에 버퍼(1 M MES-NaOH, pH 6.5) 0.05mL, H2O 0.05 mL 및 0.25 mL의 2% azocasein 용액을 첨가한 후 항온수조에서 반응시켰다(37℃, 30 min). 0.05 mL of buffer (1 M MES-NaOH, pH 6.5), 0.05 mL of H 2 O and 0.25 mL of 2% azocasein solution were added to 0.15 mL of the diluted solution, and the mixture was reacted in a constant temperature bath (37 ° C., 30 min).

그 후 과염소산(perchloric acid, HClO4, 7%) 1 mL를 첨가하여 반응을 중지시켰으며, 원심분리(12,000 rpm, 4℃, 10 min) 하여 취한 상징액 1 mL에 10 N NaOH를 0.15 mL 첨가한 후 430nm에 흡광도를 측정하였다.Then, 1 mL of perchloric acid (HClO 4, 7%) was added to stop the reaction. 0.1 mL of 10 N NaOH was added to 1 mL of the supernatant obtained by centrifugation (12,000 rpm, 4 ° C, 10 min) The absorbance was measured at 430 nm.

Figure 112013057387165-pat00001

Figure 112013057387165-pat00001

③ γ-GTP 활성도 확인③ Confirmation of γ-GTP activity

γ-GTP의 활성도 측정은 γ-GTP 측정용 키트(AM-158, 아산제약(주))를 이용하여 측정하였다(5-아미노 살리실산법 protocol sheet 05-03-25 참조, 아산제약(주)). The activity of γ-GTP was measured using a kit for measuring γ-GTP (AM-158, ASAN PHARMACEUTICAL CO., LTD.) (see protocol sheet 05-03-25, ASAN PHARMACEUTICAL CO. .

배양액(180 rpm, 37℃, 24 hr) 2 mL를 원심분리(8000 rpm, 25℃, 10 min)하여 취한 상징액을 50배 희석하여 시료로 사용하였다.The supernatant was diluted 50 times by centrifugation (8000 rpm, 25 ° C, 10 min) and 2 mL of the culture solution (180 rpm, 37 ° C, 24 hr)

항온수조(37℃)에서 5분간 예열한 기질액 1 mL에 시료액 0.02 mL를 넣고 37℃에서 20분간 반응시킨 후 정색시약을 3 mL를 넣고 잘 혼합하여 실온에서 10분간 반응시켰다. Add 0.02 mL of the sample solution to 1 mL of the substrate solution preheated for 5 minutes in a constant-temperature water bath (37 ° C), react at 37 ° C for 20 minutes, add 3 mL of the color reagent, mix well and react at room temperature for 10 minutes.

대조구는 시료액 대신 증류수 0.02 mL를 넣고 상기와 같은 방법으로 반응시켰다. As a control, 0.02 mL of distilled water was added instead of the sample solution, and the reaction was carried out in the same manner as described above.

반응 후 시료를 잘 혼합하여 60분 이내에 635nm에서 흡광도를 측정하고, 주어진 계산식(γ-GTP 분석 protocol, (주)아산제약)과 비교하여 γ-GTP활성도(mU/mL)를 나타내었다. The absorbance at 635 nm was measured within 60 minutes after the reaction and the γ-GTP activity (mU / mL) was compared with the given equation (γ-GTP assay protocol, Asan Pharmaceutical Co., Ltd.).

Figure 112013057387165-pat00002

Figure 112013057387165-pat00002

2. 실험결과 2. Experimental results

보유균주에 대한 항균성실험은 Bacillus subtilis 42개 균주에 대하여 실시하였으며, 그 결과 상기 하기 표 1에 나타나 있듯이, Bacillus subtilis 8개 균주가 어병세균에 대하여 항균성을 나타내었다.The antimicrobial activity against the strains was carried out on 42 strains of Bacillus subtilis. As shown in Table 1, 8 strains of Bacillus subtilis showed antibacterial activity against the fish microbes.

변이균주Mutant strain 종Bell 클리어 존
(mm)Clear zone
(mm) 투명도
(Transparey)transparency
(Transparey) 프로테아제 활성(U/mL)Protease activity (U / mL) γ-GTP활성
(mU/mL)
γ-GTP activity
(mU / mL)
SJ-4SJ-4 B.subtilisB. subtilis 12.61±0.3712.61 + - 0.37 좋음good 263.4263.4 2482.52482.5 SJ-25SJ-25 B.subtilisB. subtilis 12.37±0.1512.37 + - 0.15 좋음good 264.1264.1 2542.12542.1 SJ-26SJ-26 B.subtilisB. subtilis 11.51±0.3011.51 + - 0.30 좋음good 215.4215.4 2098.92098.9 SJ-30SJ-30 B.subtilisB. subtilis 12.09±0.7612.09 + - 0.76 좋음good 278.1278.1 2698.12698.1 SJ-31SJ-31 B.subtilisB. subtilis 12.57±0.2612.57 ± 0.26 좋음good 247.2247.2 23852385 SJ-32SJ-32 B.subtilisB. subtilis 12.34±0.3512.34 + - 0.35 좋음good 204204 2145.22145.2 SJ-33SJ-33 B.subtilisB. subtilis 12.93±0.5412.93 + - 0.54 좋음good 263.4263.4 2869.42869.4 SJ-34SJ-34 B.subtilisB. subtilis 11.36±0.1411.36 + - 0.14 좋음good 213.2213.2 2213.52213.5

상기 표 1에 나타나 있는 균주들 중 항균성이 양호하다고 판단되는 Bacillus subtilis SJ-30 균주를 선발하여 균주의 배양특성 및 조항균물질에 대한 이화학적인 특성을 조사하였다
 Bacillus subtilis SJ-30 strain, which is considered to have good antimicrobial activity among the strains shown in Table 1, was selected to investigate the culture characteristics and physicochemical properties of the bacteria

<실시예 2> 배양조건에 따른 선발균주의 생육과 항균활성확인 Example 2: Growth and antimicrobial activity of selected strain according to culture conditions

1. 검토선발균주의 생육도 및 pH 측정방법1. Evaluation of the growth and pH of the selected strain

균주의 생육은 배양액 1 mL을 취해 분광광도기로 600 nm에서 측정하였으며, pH는 pH 메타(meter)를 이용하여 배양액의 pH를 측정하였다
The growth of the strain was measured at 600 nm using a spectrophotometer in 1 mL of the culture, and the pH of the culture was measured using a pH meter

2. 실험결과 2. Experimental results

도 1에 나타나 있듯이, 선발한 균주의 생육은 배양 후 12시간째 2.5~2.6 (흡광도, 600nm)의 생육도를 나타냈다. 어병세균에 대한 항균성은 균종간 다소 차이가 있으나 24시간 배양 후 획득한 조항균물질에 의한 투명환이 약 10~12 mm로, 그 후 배양시간이 길어짐에 따른 투명환의 크기차이는 미미하였다. As shown in Fig. 1, the growth of the selected strains showed a growth rate of 2.5 to 2.6 (absorbance, 600 nm) at 12 hours after the cultivation. The antimicrobial activity against fish microbes was somewhat different, but the difference in the size of the transparent rings was small as the incubation time was increased to about 10 ~ 12 mm after 24 h incubation.

또한, 도 2와 하기 표 2에 나타나 있듯이 당농도에 따른 투명환의 크기도 1%첨가군과 3%첨가군이 비슷하게 나타났다. In addition, as shown in Fig. 2 and Table 2, the sizes of the transparent rings according to sugar concentration were similar to those of 1% addition group and 3% addition group.

글루코오스농도(%)Glucose concentration (%) 배양시간(h)Culture time (h) 1212 2424 3636 4848 6060 7272 00 9.219.21 9.099.09 10.4810.48 10.0610.06 9.689.68 9.689.68 1One 11.4211.42 12.2812.28 12.1612.16 11.4111.41 12.0812.08 12.7112.71 22 12.2512.25 12.3712.37 12.5412.54 12.3112.31 12.5512.55 12.5512.55 33 10.9310.93 11.2211.22 11.5911.59 12.2512.25 12.2112.21 12.2212.22

이에, 상기 실시예 1에서 선발한 Bacillus subtilis SJ-30 균주는 글루코오스 1~3 중량% 첨가한 배지에서 12~24시간동안 배양하는 것이 가장 적합한 생육조건임을 알 수 있었다.
Thus, the Bacillus The subtilis strain SJ-30 was found to be the most suitable growth condition for 12 to 24 hours in the medium supplemented with 1 to 3% by weight of glucose.

<실시예 5> 조항균물질의 열안정성, 내산성 및 내담즙산성<Example 5> The thermal stability, acid resistance and bile acid resistance

1. 실험방법1. Experimental Method

조항균물질의 열안정성은 상기 조항균물질을 60℃, 90℃에서 30분, 그리고 121℃에서 15분 동안 각각 처리한 후, 조항균물질의 항균활성을 측정하였다.The antimicrobial activity of the antibacterial substance was measured after treating the antibacterial substance at 60 ° C, 90 ° C for 30 minutes and 121 ° C for 15 minutes, respectively.

균주의 내산성을 측정하기 위해 0.05 M 인산 나트륨(sodium phosphate) 완충용액(pH 7.0)을 HCl을 사용하여 pH 7, 4, 3, 2로 조정하였으며, 조정된 0.05 M sodium phosphate에 균주를 107 cfu/ml 정도의 농도로 각각 접종한 후 37℃에서 2시간 동안 방치한 다음 PCA agar배지에 도말하여 배양한 후 생균수를 측정하여 생존율(%)을 다음 식에 의하여 계산하였다.0.05 M sodium phosphate buffer solution (pH 7.0) was adjusted to pH 7, 4, 3 and 2 with HCl to adjust the acid resistance of the strain. To the adjusted 0.05 M sodium phosphate, 10 7 cfu / ml, followed by incubation at 37 ° C for 2 hours. The cells were plated on PCA agar medium, and viable cells were counted and the survival rate (%) was calculated by the following formula.

Figure 112013057387165-pat00003
Figure 112013057387165-pat00003

조항균물질의 내산성은 조항균물질을 NaOH와 HCl로 pH 2.0에서 10.0까지 조정한 후 상온에서 1시간 방치하고, 항균성을 측정하였다.The antimicrobial activity of the antibacterial substance was measured by adjusting the pH of the antibacterial substance with NaOH and HCl to 10.0 and then left at room temperature for 1 hour.

내담즙산성은 담즙산(bile acid)이 각각 0, 0.05, 0.1, 0.3, 0.5%씩 함유된 PCA배지에 동일한 농도(106)로 희석한 균주를 도말하여 30℃에서 24시간동안 배양하였다. 그리고 배양 후 나타난 집락을 계수하였다.
Myobiliary acidity was determined by culturing the strains diluted at the same concentration (10 6 ) in PCA medium containing bile acid (0, 0.05, 0.1, 0.3, 0.5%) at 30 ° C for 24 hours. The colonies were counted after incubation.

2. 실험결과2. Experimental results

하기 표 3에 나타나 있듯이, TSB배지에 glucose를 1% 첨가한 배지 각각의 균주를 접종하여 24시간동안 배양한 후 획득한 조항균물질에 대한 열안정성을 검토하였다. As shown in Table 3, the thermostability of the obtained bacteria was examined after culturing for 24 hours inoculated with each strain of the culture medium containing 1% glucose in the TSB medium.

종Bell 온도(℃)Temperature (℃) 대조군Control group 6060 9090 121121 Bacillus subtilis SJ-30 Bacillus subtilis SJ-30 클리어존(mm)Clear zone (mm) 15.5315.53 13.5313.53 12.0312.03 11.7511.75 투명도(Transparency)Transparency 가장좋음Best 나쁨Poor 나쁨Poor 나쁨Poor

상기 표 3과 같이, 온도가 증가함에 따라 대조군에 비해 투명환의 크기와 선명도가 다소 감소하는 경향을 보였으나, 90℃ 이상의 열처리 후에도 항균성을 나타내었다. As shown in Table 3, as the temperature was increased, the size and clarity of the transparent ring tended to decrease somewhat as compared with the control group, but the antibacterial activity was also exhibited after the heat treatment at 90 ° C or higher.

또한, 도 3에 나타나 있듯이, 선발균주의 내산성을 나타내는 생존율 pH 4일 때 18~28%로 감소하였으며, pH 2에서 균종에 따라 4~12%의 생존율을 나타냈다. 조항균물질의 내산성은 하기 표 4에서와 같이 pH 6~10까지는 투명환이 생성되어 항균력을 나타냈으나, pH 5~2구간에서는 투명환이 생성되지 않았다. In addition, as shown in FIG. 3, the survival rate showing the acid resistance of the selected strain was decreased to 18-28% at pH 4, and 4-12% at pH 2 depending on the species. As shown in the following Table 4, the acid resistance of the antibacterial substance was shown to be the antimicrobial activity due to the formation of a transparent ring at pH 6 to 10, but no transparent ring was formed at pH 5 to 2.

pH
pH
종Bell Bacillus subtilis SJ-30 Bacillus subtilis SJ-30 22 발견되지 않음Not found 33 발견되지 않음Not found 44 발견되지 않음Not found 55 발견되지 않음Not found 66 11.01±0.5511.01 + - 0.55 77 11.69±0.4611.69 + - 0.46 88 11.72±0.5111.72 + - 0.51 99 10.97±0.2210.97 ± 0.22 1010 11.40±0.2611.40 ± 0.26

또한, 하기 표 5에 나타나 있듯이, 대조군과 동일한 희석배수(106)로 희석한 균주를 bile acid 농도를 달리한 배지에 도말하여 배양한 결과 bile acid를 첨가한 배지에서는 균체집락을 관찰할 수 없었다. In addition, as shown in Table 5 below, when the strain diluted with the same dilution ratio (10 6 ) as that of the control group was cultured on a culture medium having different bile acid concentrations, no culture of bacteria was observed in the medium supplemented with bile acid .

Bile acid(%)
Bile acid (%)
종Bell Bacillus subtilis SJ-30 Bacillus subtilis SJ-30 00 성장함Grown 0.050.05 성장안함Do not grow 0.10.1 성장안함Do not grow 0.30.3 성장안함Do not grow 0.50.5 성장안함Do not grow

이와같이, 본 발명은 어병세균(Streptococcus parauberis)에 대한 항균활성을 나타내는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P 및 이를 포함하여 열안정성 및 내산성을 갖으며 어병세균(Streptococcus parauberis)에 대해 방제효과를 갖는 어류용 사료첨가제를 제공하게 된다.Thus, the invention Fish Pathology, bacteria (Streptococcus parasitic &lt; / RTI &gt; microorganism Bacillus subtilis subtilis ) SJ-30 strain KCTC18249P and its thermostability and acid resistance, and the strains of Streptococcus Parauberis ) as a feed additive for fish.

상기의 본 발명은 바람직한 실시예를 중심으로 살펴보았으며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명의 본질적 기술 범위 내에서 상기 본 발명의 상세한 설명과 다른 형태의 실시예들을 구현할 수 있을 것이다. 여기서 본 발명의 본질적 기술범위는 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be possible. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

한국생명공학연구원Korea Biotechnology Research Institute KCTC18249PKCTC18249P 2013061720130617

<110> changjobiotech KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> NOVEL MICROORGANISM BACILLUS SUBTILIS SJ-30 AND ADDITIVES FOR FISH FEEDS CONTAINING IT <130> p-201306 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1430 <212> DNA <213> Bacillus subtilis SJ-30 <400> 1 cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60 gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120 attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180 gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240 cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300 tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360 tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420 cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480 gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540 gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600 atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660 ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720 agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780 ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840 cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900 acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960 gttagccgtg gctttctggt taggtaccgt caaggtaccg ccctattcga acggtacttg 1020 ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080 ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140 ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgttgcctt 1200 ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260 gaagccacct tttatgtttg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320 ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380 ctaacatcag ggagcaagct cccatctgtc cgctcgactg catgtatagc 1430 <110> changjobiotech          KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> NOVEL MICROORGANISM BACILLUS SUBTILIS SJ-30 AND ADDITIVES FOR          FISH FEEDS CONTAINING IT <130> p-201306 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1430 <212> DNA <213> Bacillus subtilis SJ-30 <400> 1 cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca aactctcgtg 60 gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120 attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga actgagaaca 180 gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc cattgtagca 240 cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300 tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa gatcaagggt 360 gt; cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag aggatgtcaa 480 gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc gcttgtgcgg 540 gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc ggagtgctta 600 atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact catcgtttac 660 ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc 720 agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct acgcatttca 780 ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt ttccaatgac 840 cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg cgagcccttt 900 acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960 gttagccgtg gctttctggt taggtaccgt caaggtaccg ccctattcga acggtacttg 1020 ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg cggcgttgct 1080 ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta ggagtctggg 1140 ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca tcgttgcctt 1200 ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta agtggtagcc 1260 gaagccacct tttatgtttg aaccatgcgg ttcaaacaac catccggtat tagccccggt 1320 ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac ccgtccgccg 1380 ctaacatcag ggagcaagct cccatctgtc cgctcgactg catgtatagc 1430

Claims (5)

삭제delete 스트렙토코커스 파라우베리스(Streptococcus parauberis)에 대한 항균활성을 나타내며, 서열번호 1로 표현되는 신규미생물 바실러스 서브틸러스(Bacillus subtilis) SJ-30균주 KCTC18249P를 배양한 후 획득한 배양액을 유효성분으로 포함하되,
상기 배양액은 열안정성 및 내담즙산성을 갖는 것이 특징인, 어류용 사료첨가제.A culture solution obtained by culturing a new microorganism Bacillus subtilis SJ-30 strain KCTC18249P, which exhibits an antibacterial activity against Streptococcus parauberis and is represented by SEQ ID NO: 1, as an active ingredient, ,
Wherein the culture liquid has heat stability and bile acid resistance. 삭제delete 제2항에 있어서,
상기 배양은 글루코오스 1~3 중량% 첨가한 배지에서 12~24시간동안 이루어지는 것임을 특징으로 하는,
어류용 사료첨가제.
3. The method of claim 2,
Wherein the culture is performed in a medium containing 1 to 3% by weight of glucose for 12 to 24 hours.
Feed additives for fish.
삭제delete
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