KR101434220B1 - Novel lactic acid bacteria having inhibitory activity against multidrug resistant bacteria - Google Patents

Novel lactic acid bacteria having inhibitory activity against multidrug resistant bacteria Download PDF

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KR101434220B1
KR101434220B1 KR1020130031835A KR20130031835A KR101434220B1 KR 101434220 B1 KR101434220 B1 KR 101434220B1 KR 1020130031835 A KR1020130031835 A KR 1020130031835A KR 20130031835 A KR20130031835 A KR 20130031835A KR 101434220 B1 KR101434220 B1 KR 101434220B1
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하남주
이강오
김경제
이도경
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삼육대학교산학협력단
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Abstract

The present invention relates to a novel lactic acid bacteria having an inhibitory activity against a multidrug resistant bacteria and, more particularly, to a novel lactic acid bacteria of the Bifidobacterium pseudocatenulatum SPM 1309 having an inhibitory activity against the multidrug resistant bacteria of the Acinetobacter baumannii and the Pseudomonas aeruginosa.

Description

다제내성균에 대한 저해 활성을 갖는 신규 유산균 {Novel lactic acid bacteria having inhibitory activity against multidrug resistant bacteria} [0001] The present invention relates to novel lactic acid bacteria having inhibitory activity against multidrug resistant bacteria,

본 발명은 다제내성균에 대한 저해 활성을 갖는 신규 유산균에 대한 것으로 보다 상세하게는 다제내성균인 아시네토박터 바우마니(Acinetobacter baumannii)와 슈도모나스 에루지노사(Pseudomonas aeruginosa)에 대해 저해 활성을 갖는 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum ) SPM 1309에 관한 것이다.
The present invention relates to a novel lactic acid bacterium having an inhibitory activity against multidrug-resistant bacteria. More particularly, the present invention relates to a novel lactic acid bacterium having an inhibitory activity against multidrug-resistant bacteria such as Acinetobacter baumannii ) and Pseudomonas aeruginosa Bifidobacterium < / RTI > ( Bifidobacterium < RTI ID = 0.0 > Pseudocatenulatum ) SPM 1309.

아시네토박터 바우마니(Acinetobacter baumannii)와 슈도모나스 에루지노사(Pseudomonas aeruginosa)는 주요한 병원감염 병원균(nosocomial pathogen)으로 광범위한 항생제 내성(resistance)을 나타낼 뿐이 아니라, 치료기간 중에도 항생제에 대해 새로운 내성을 발달시킨다. Acinetobacter baumannii and Pseudomonas spp. aeruginosa is a major nosocomial pathogen that not only has broad antibiotic resistance but also develops new resistance to antibiotics during treatment.

이런 내성을 갖는 박테리아에 의한 감염은 치료에 있어 선택할 수 있는 항생제의 종류가 매우 적거나 없기 때문에 더 높은 치사율을 갖는다. 미국의 병원감염 감시 시스템(National Nosocomial Infections Surveillance system: NNIS)에 따르면, 이런 항생제 내성 병원균들의 저항성은 증가 추세에 있으며 이런 병원균들을 조절할 수 있는 새로운 전략이 절실히 필요하다.Infection by bacteria with this resistance has a higher mortality rate because there are very few or no classes of antibiotics to choose from for treatment. According to the National Nosocomial Infections Surveillance System (NNIS), the resistance of these antibiotic resistant pathogens is on the rise and a new strategy to control these pathogens is urgently needed.

과거 수십년간 인간의 몸에 이득이 되는 미생물 그룹인 프로바이오틱(probiotic)에 대한 연구는 다방면에서 이루어졌다. 프로바이오틱은 영양 대사에 필수적인 역할을 수행하고, 항생 물질을 생산하며 이에 내성 박테리아가 생기지 않는 병원균 성장 억제 효과를 갖는다. 유산균(lactic acid bacteria = lactic acid bacteria + bifidobacterium + bacillus)은 그 대표적인 균으로 인간의 건강에 이득이 되고, 영양학적으로도 약리학적으로도 도움이 되는 균으로 전통 식품에서의 이용을 비롯해 산업화되어서도 이용된다. 이런 프로바이오틱 제품들의 수요는 전세계적으로 지속적으로 증가하고 있으며 이에 프로바이오틱에 대한 연구 역시 활발한 모습을 보여주고 있다.Research on probiotic, a group of microorganisms that have benefited the human body for decades, has been done in many directions. Probiotics play an essential role in nutritional metabolism, produce antibiotics, and have pathogenic growth inhibition that does not result in resistant bacteria. Lactic acid bacteria (lactic acid bacteria + bifidobacterium + bacillus) is a typical bacterium that is beneficial to human health and is helpful in nutrition and pharmacology. It is used in traditional foods and industrialized do. Demand for these probiotic products is continuously increasing worldwide, and research on probiotics is also active.

비피도박테리아(bifidobacteria)는 이런 유산균류의 하나로 유아의 장내 미생물중에서는 큰 비중을 차지하고 있으나 성인이 되면 그 수가 감소하는 것으로 알려져 있다. 인체에서 비피도박테리아는 (i) 위장관(gastrointestinal tract; GI tract) 건강의 개선; (ii) 영양분의 합성과 생체내 이용성 강화; (iii) 면역체계 강화; (iv) 혈중 콜레스테롤 강하; (v) 병원균 억제 및 파괴; (vi) 특정 암의 위험성 감소와 같은 다양한 유용성으로 인간에게 유익한 미생물로 알려져 있다(한국 공개 특허 제2004-0022386호). Bifidobacteria are one of these lactic acid bacteria, and they account for a large portion of infant intestinal microorganisms, but they are known to decrease in adults. Bifidobacteria in the human body are (i) improving the health of the gastrointestinal tract (GI tract); (ii) synthesis of nutrients and enhancement of bioavailability; (iii) strengthening the immune system; (iv) blood cholesterol lowering; (v) pathogen inhibition and destruction; (vi) a variety of usability such as a reduction in the risk of a specific cancer (Korean Patent Publication No. 2004-0022386).

많은 연구들에서 비피도박테리아는 병원성 미생물에 대한 항생 작용을 갖는 것으로 알려져 있지만, 구체적인 종류 및 작용에 대해서는 그다지 알려진 바가 없다.
In many studies, bifidobacteria are known to have antibiotic action against pathogenic microorganisms, but the specific types and actions of them are not well known.

본 발명은 다제내성균에 대한 저해 활성을 갖는 신규한 유산균을 제공하는 것을 목적으로 한다.
It is an object of the present invention to provide a novel lactic acid bacterium having inhibitory activity against multidrug-resistant bacteria.

본 발명은 상기와 같은 과제를 해결하기 위하여 다제내성균에 대한 저해 활성을 갖는 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum ) SPM 1309(KCTC 12385 BP) 을 제공한다. DISCLOSURE OF THE INVENTION In order to solve the above-mentioned problems, the present invention provides a Bifidobacterium ( Bifidobacterium) Pseudocatenulatum ) SPM 1309 (KCTC 12385 BP).

본 발명에 있어서, 상기 다제내성균은 아시네토박터 바우마니(Acinetobacter baumannii)인 것을 특징으로 한다. In the present invention, the multi-drug resistance microorganism is characterized by being Acinetobacter baumannii .

본 발명에 있어서, 상기 다제내성균은 슈도모나스 에루지노사(Pseudomonas aeruginosa) 인 것을 특징으로 한다.In the present invention, the multi-drug resistance microbe is Pseudomonas aeruginosa .

본 발명에 있어서, 상기 다제내성균은 시프로플록사신(ciprofloxacin), 토브라마이신(tobramycin), 티게사이클린(tigecycline), 겐타마이신(gentamicin), 메로페넴(meropenem) 및 세프타지딤(ceftazidime)에 대해서 내성을 가지는 것을 특징으로 한다. In the present invention, the multi-drug resistant microorganism is resistant to ciprofloxacin, tobramycin, tigecycline, gentamicin, meropenem and ceftazidime. .

본 발명은 또한, 본 발명에 의한 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum ) SPM 1309 (KCTC 12385 BP) 또는 이의 배양물을 유효 성분으로 하는 다제내성균에 의해 유발되는 감염증의 치료 또는 예방용 조성물을 제공한다. The invention also Bifidobacterium pseudo category nyul ratum (Bifidobacterium according to the invention Pseudocatenulatum) provides SPM 1309 (KCTC 12385 BP), or treating or preventing composition of the infection is caused by multi-drug resistant bacteria to the culture thereof as an active ingredient.

본 발명은 또한, 본 발명에 의한 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum ) SPM 1309 또는 이의 배양물을 유효 성분으로 하는 항균 조성물을 제공한다.
The invention also Bifidobacterium pseudo category nyul ratum (Bifidobacterium according to the invention Pseudocatenulatum ) SPM 1309 or a culture thereof as an active ingredient.

본 발명에 의한 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum) SPM 1309는 다제내성균인 아시네토박터 바우마니(multidrug resistant Acinetobacter baumanii), 슈도모나스 에루지노사(multidrug resistant Pseudomanos aeruginosa.)에 대해 우수한 항균 활성을 나타내고, 이에 따라 항균 조성물, 감영증 치료 또는 예방용 조성물에 응용할 수 있다.
The Bifidobacterium pseudocatenulatum SPM 1309 according to the present invention is a multidrug resistant Acinetobacter multidrug resistant bacterium, baumanii), exhibit excellent antibacterial activity against Rouge labor (multidrug resistant Pseudomanos aeruginosa.) Pseudomonas, and thus can be applied to the antimicrobial composition, Gamyeong increase the treatment or prevention composition.

도 1은 본 발명의 일 실시예에 의하여 다제내성 슈도모나스 에루지노사(multidrug resistant Pseudomanos aeruginosa.; MDRPA)에 대해 항균성을 갖는 비피도박테리아를 선별한 결과를 나타낸다.
도 2는 본 발명의 일 실시예에 의하여 다제내성 아시네토박터 바우마니(multidrug resistant Acinetobacter baumanii; MDRAB)에 대해 항균성을 갖는 비피도박테리아를 선별한 결과를 나타낸다.
도 3은 본 발명의 일 실시예에 의하여 선별된 비피도박테리움 슈도카테뉼라툼(Bifidobacterium pseudocatenulatum) SPM1309의 저해 활성 물질을 비교한 결과를 나타낸다.
도 4는 본 발명의 일 실시예에 의하여 선별된 비피도박테리움 슈도카테뉼라툼(Bifidobacterium pseudocatenulatum) SPM1309의 성장 억제(growth inhibition) 효과를 비교한 결과를 나타낸다. 1 is labor Rouge (multidrug resistant Pseudomanos in multidrug-resistant Pseudomonas by one embodiment of the present invention aeruginosa .; MDRPA). ≪ / RTI >
Figure 2 is a schematic diagram of a multidrug-resistant Acinetobacter sp. ≪ RTI ID = 0.0 > baumanii ; MDRAB). ≪ / RTI >< RTI ID = 0.0 > Bifidobacterium < / RTI >
Figure 3 is a graphical representation of Bifidobacterium < RTI ID = 0.0 > ( Bifidobacterium < / RTI > pseudocatenulatum ) SPM1309.
Figure 4 is a graphical representation of Bifidobacterium < RTI ID = 0.0 > ( Bifidobacterium < / RTI > pseudocatenulatum ) SPM1309. The results are shown in FIG.

이하에서는 본 발명을 실시예에 의하여 더욱 상세히 설명한다. 그러나, 본 발명이 이하의 실시예에 의하여 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited by the following examples.

<< 실시예Example 1>  1> 다제내성균의Multidrug-resistant 분리 detach

삼육 의료원의 다양한 임상 현장에서 수집된 검체-가래, 소변, 상처, 혈액, 대변, 가관 세척액, intubation tube-에서 슈도모나스 에루지노사(Pseudomonas aeruginosa)와 아시네토박터 바우마니(Acinetobacter baumannii)를 분리해 냈다. 분리된 것들은 테스트 하기 전까지 20% 글리세롤이 첨가된 Nutrient broth(Difco, USA)에서 -70℃로 보관되었다.Collected from a variety of clinical site of the medical center Sahmyook sample - sputum, urine, wounds, blood, feces, glass cleaning solution, in the intubation tube- Rouge labor Pseudomonas (Pseudomonas aeruginosa) and Acinetobacter baumannii (Acinetobacter baumannii . Separations were stored at -70 ° C in Nutrient broth (Difco, USA) supplemented with 20% glycerol until tested.

임상에서 분리된 검체에 대해서 Clinical Laboratory and Standards Institute의 agar dilution method에 따라 6가지 항생제(시프로플록사신(ciprofloxacin), 토브라마이신(tobramycin), 티게싸이클린(tigecycline), 겐타마이신(gentamicin), 메로페넴(meropenem), 세프타지딤(ceftazidime))에 대해서 내성을 가진 슈도모나스 에루지노사(multidrug resistant Pseudomanos aeruginosa.; MDRPA)로서 CPA-1 내지 CPA-4 및 다제내성을 가진 아시네토박터 바우마니(multidrug resistant Acinetobacter baumanii; MDRAB)로서 CAB-1 내지 CPA-4를 분리하고, 각각의 병원균의 항생제에 대한 최소억제 농도(minimum inhibitory concentration; MIC)를 측정한 결과를 아래 표 1에 나타내었다.For clinical isolates, six antibiotics (ciprofloxacin, tobramycin, tigecycline, gentamicin, meropenem) were administered according to the agar dilution method of the Clinical Laboratory and Standards Institute ), Joseph ride dim (ceftazidime)) Rouge labor (multidrug resistant Pseudomonas Pseudomanos with resistance to aeruginosa .; MDRPA) as CPA-1 to CPA-4 and multidrug-resistant Acinetobacter baumanii ; MDRAB), and the minimum inhibitory concentration (MIC) of antibiotics of each pathogen was measured. The results are shown in Table 1 below.

strainsstrains MIC(㎍/ml)MIC ([mu] g / ml) CIPCIP TOBTOB TIGTIG GENGEN MEMMEM CAZCAZ PseudomonasPseudomonas aeruginosaaeruginosa CPA-1CPA-1 >64> 64 >128> 128 128128 >128> 128 >128> 128 128128 CPA-2CPA-2 3232 >128> 128 128128 >128> 128 >128> 128 128128 CPA-3CPA-3 6464 >128> 128 128128 >128> 128 128128 3232 CPA-4CPA-4 6464 >128> 128 6464 6464 >128> 128 128128 AcinetobacterAcinetobacter baumanniibaumannii CAB-1CAB-1 >128> 128 >128> 128 88 >128> 128 1616 6464 CAB-2CAB-2 6464 >128> 128 44 >128> 128 1616 >128> 128 CAB-3CAB-3 >128> 128 3232 44 3232 1616 128128 CAB-4CAB-4 >128> 128 >128> 128 44 >128> 128 1616 >128> 128

<< 실시예Example 2> 유산균의 분리 2> Isolation of lactic acid bacteria

비피도박테리아(bifidobacteria)의 분리는 건강한 20~30대 한국인 분변 채취물로부터 이루어졌다. 분변 샘플을 희석한 뒤, 5% 양 혈액이 함유된 선택적.blood liver agar(Nissui Pharm, Japan)에 파종(seeding)되었다. 이를 Bactron Anaerobic Chamber (Sheldon Manufacturing Inc., USA)에서 37℃ 혐기성 상태(90% N2, 5% H2, 5% CO2)로 48시간 동안 배양하고, 형성된 갈색 혹은 reddish-brown의 직경 2-3 mm 의 집락(colony)을 동정(identification)하였다. Bifidobacteria were isolated from healthy 20 ~ 30 Korean fecal samples. Fecal samples were diluted and seeded in a selective .blood liver agar (Nissui Pharm, Japan) containing 5% sheep blood. The cells were cultured in a Bactron Anaerobic Chamber (Sheldon Manufacturing Inc., USA) for 48 hours in an anaerobic state (90% N 2 , 5% H 2 , 5% CO 2 ) at 37 ° C. and the diameter of the formed brown or reddish- A colony of 3 mm was identified.

선별된 집락에 대해서 fructose-6-phosphate phosphoketolase(F6PPK) 테스트 결과 비피도박테리아로 확인되었다(결과 미도시). Fructose-6-phosphate phosphoketolase (F6PPK) test for selected colonies confirmed Bifidobacterium as the result (not shown).

분리해낸 Bifidobacterium spp .의 level을 실험하기 위해 Bio leaders (Daejeon, Korea)로 업계에서 공지된 통상적인 방법인 16S rRNA sequencing(Steven D. Mahlen and Jill E. Clarridge III, 2009, Site and Clinical Significance of Alloscardovia omnicolens and Bifidobacterium Species Isolated in the Clinical Laboratory, Journal of Clinical Microbiology: 47(10): 3289-3293)을 수행하고 아래 표2와 같이 B. adolescentis 9개 균주, B. longum 3개 균주, B. pseudocatenulatum 2개 균주를 동정하였다. Isolated Bifidobacterium spp . 16S rRNA sequencing (Steven D. Mahlen and Jill E. Clarridge III, 2009, Site and Clinical Significance of Alloscardovia omnicolens and Bifidobacterium Species Isolated), which is a conventional method known in the art as a Bio leaders (Daejeon, Korea) in the Clinical Laboratory, Journal of Clinical Microbiology: 47 (10): 3289-3293), and as shown in Table 2 below, B. adolescentis 9 strains, 3 B. longum strains, and 2 B. pseudocatenulatum strains were identified.

speciesspecies OriginOrigin sexsex AgeAge BifidobacteriumBifidobacterium adolescentisadolescentis SPM0212SPM0212 FemaleFemale 2121 SPM0214SPM0214 FemaleFemale 2121 SPM0308SPM0308 FemaleFemale 2222 SPM1005SPM1005 MaleMale 2525 SPM1601SPM1601 MaleMale 2020 SPM1604SPM1604 MaleMale 2020 SPM1605SPM1605 MaleMale 2020 SPM1606SPM1606 MaleMale 2020 SPM1608SPM1608 MaleMale 2020 BifidobacteriumBifidobacterium . . LongumLongum SPM1205SPM1205 FemaleFemale 2222 SPM1206SPM1206 FemaleFemale 2222 SPM1207SPM1207 FemaleFemale 2222 BifidobacteriumBifidobacterium . . PseudocatenulatumPseudocatenulatum SPM1204SPM1204 FemaleFemale 2222 SPM1309SPM1309 MaleMale 2424

<< 실시예Example 3>  3> 다제내성균에Multidrug resistant bacteria 대한 항균 활성을 가진 유산균의 선발 Selection of lactic acid bacteria with antimicrobial activity against

상기 실시예 2에서 분리된 Bifidobacterium spp들은 혐기성 조건하에 일반 혐기성배지(general anaerobic medium; GAM) broth (Nissui Pharm. Co. Ltd., Japan)에서 37℃로 48시간 동안 배양되었다. The Bifidobacterium spp isolated in Example 2 was cultivated in a general anaerobic medium (GAM) broth (Nissui Pharm. Co. Ltd., Japan) at 37 ° C for 48 hours under anaerobic conditions.

상기 박테리아 현탁액(suspension)들은 상기 세포 배양액을 PBS로 세척(washing)하고 4,000rpm에서 10분간 원심분리해 수집하고, 같은 완충용액(buffer)으로 재현탁(resuspend)했다. 비피도박테리아 추출물(bifidobacteria cell extract)은 최종 농도(final concentration)가 1.0×109 colony forming unit (CFU)/ml이 되도록 맞춰진 후, 6분간 초음파 분쇄(sonication)-amplitude 100%-되었다. 세포 추출물과 배양 상층액(supernatant)들은 0.2㎛ 필터로 여과처리했다.
The bacterial suspensions were washed with PBS, collected by centrifugation at 4,000 rpm for 10 minutes, and resuspended in the same buffer. Bifidobacteria extract Cell extracts were adjusted to a final concentration of 1.0 × 10 9 colony forming units (CFU) / ml and sonicated for 10 min. Cell extracts and culture supernatants were filtered with a 0.2 μm filter.

<< 실시예Example 4>  4> 다제내성균에Multidrug resistant bacteria 대해 항균 활성을 가진 유산균의 선발 Selection of lactic acid bacteria with antimicrobial activity against

상기 실시예 3에서 분리된 비피도박테리아들의 상기 실시예 1에서 분리해 낸 다제내성균에 대한 항균 활성(antimicrobial activity)을 측정하였다.The antimicrobial activity of the bifidobacteria isolated in Example 3 against the multidrug-resistant bacteria isolated in Example 1 was measured.

상기 실시예 3에서 분리된 항균 활성을 가진 유산균의 25% 세포 추출물(cell extract) 혹은 배양 상층액(supernatant)이 함유된 Mueller-Hinton broth에서 상기 실시예 1에서 분리한 다제내성균을 37℃로 24시간 동안 배양하면서, 성장 억제 효과를 측정하였다. 성장 억제 효과는 변형 액체배지희석법(modified broth microdilution method)으로 흡광도를 측정하였다. 대조군으로는 동량의 PBS나 GAM broth가 함유된 Mueller-Hinton broth에서 배양한 동일한 다제내성균의 배양체를 배양하면서 흡광도를 측정하였다. 구체적으로는 ELISA reader(Molecular Devices, USA)로 625 nm 영역에서 흡수율을 측정하고 그 결과를 도 1, 도 2에 나타내었다. In the Mueller-Hinton broth containing the 25% cell extract or the supernatant of the lactic acid bacteria having the antibacterial activity isolated in Example 3, the multi-drug resistant bacteria isolated in Example 1 were cultured at 37 ° C The growth inhibitory effect was measured while culturing for a period of time. The growth inhibition effect was measured by the modified broth microdilution method. As a control, the absorbance was measured while culturing the same multi-drug resistance cultured in Mueller-Hinton broth containing the same amount of PBS or GAM broth. Specifically, an absorption rate was measured in an area of 625 nm with an ELISA reader (Molecular Devices, USA), and the results are shown in FIGS. 1 and 2. FIG.

도 1 및 도 2에서 본 발명에 의한 SPM 1309가 다제내성을 가진 슈도모나스 에루지노사 (Multidrug Resistant Pseudomonas aeruginosa; MDRPA) CPA-1내지 CPA-4와 아시네토박터 바우마니(Multidrug Resistant acinetobacter baumannii; MDRAB) CAB-1내지 CAB-4에 대해 가장 높은 성장 저해 효과를 나타내는 균주인 것을 확인할 수 있다. 1 and 2, the SPM 1309 according to the present invention is a multidrug-resistant Pseudomonas aeruginosa (Multidrug Resistant) Pseudomonas aeruginosa ; MDRPA) CPA-1 to CPA-4 and Multidrug Resistant acinetobacter baumanni i; MDRAB) CAB-1 to CAB-4.

본 발명자는 상기 유산균을 '비피도박테리움 슈도카테뉼라툼 SPM1309'라 명명하여 한국생명공학연구원 생물자원센터(주소:305-333 대전광역시 유성구 어은동 52번지)에 2013년 3월 25일자로 기탁하였다(기탁번호: KCTC 12385BP).
The present inventor deposited the lactic acid bacterium as 'Bifidobacterium pseudocatenulatumum SPM1309' on Mar. 25, 2013 at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology (Address: 52, Eudun-dong, Yuseong-gu, Daejeon) (Accession No .: KCTC 12385BP).

<< 실시예Example 5> 선발된 유산균의 항균 활성 물질 조사 5> Investigation of antibacterial activity of selected lactic acid bacteria

<< 실시예Example 5-1> 항균 활성 물질의 분자량 조사 5-1> Investigation of molecular weight of antimicrobial active substance

본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309에서 항균성을 나타내는 물질(antimicrobial compound)을 분리하기 위해 배양 상층액(supernatant)을 Amicon® Ultra-0.5 Centrifugal Filter Device(Millipore, USA)로 분자량이 10kDa보다 작은 것을 F1, 10kDa보다 큰 것을 F2로 분리하였다. In order to separate antimicrobial compounds from Bifidobacterium pseudomonas sp. SPM1309 according to the present invention, the culture supernatant was diluted with an Amicon Ultra-0.5 Centrifugal Filter Device (Millipore, USA) at a molecular weight of 10 kDa F1 was separated by F2, and those larger than 10 kDa were separated by F2.

상기 F1, F2 에 대해서 상기 실시예 4에서 설명한 것과 동일한 방법으로 성장 억제 효과를 측정하고 그 결과를 도 3에 나타내었다. The growth inhibition effect was measured for F1 and F2 in the same manner as described in Example 4, and the results are shown in Fig.

도 3에서 분자량이 10kDa보다 작은 분획물에서는 항균 활성이 나타났으며 분자량이 10kDa보다 큰 화합물에서는 항균 활성이 관찰되지 않았다.
In FIG. 3, fractions having a molecular weight of less than 10 kDa exhibited antimicrobial activity, whereas those having a molecular weight of more than 10 kDa did not exhibit antimicrobial activity.

<< 실시예Example 5-2> 항균 활성 물질의 열에 대한 안정성 조사 5-2> Investigation of the stability of antimicrobial active substance against heat

상기 실시예 5-1에서 분리된 비피도박테리움 슈도카테뉼라툼 SPM1309의 항균 물질(antimicrobial compound)의 열안정성(heat stability)을 시험하기 위해 배양 상층액(supernatant)을 70℃에서 10분간 가열하고, 상기 실시예 4에서 설명한 것과 동일한 방법으로 성장 억제 효과를 측정하였으며, 그 결과를 도 3에 heated로 나타내었다.To test the heat stability of the antimicrobial compound of Bifidobacterium pseudomonas brotum SPM1309 isolated in Example 5-1, the culture supernatant was heated at 70 ° C for 10 minutes , The growth inhibition effect was measured in the same manner as described in Example 4, and the result was shown as heated in FIG.

도 3에서 heated는 70℃에서 10분간 가열하였어도 항균 활성에 영향이 없어 본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309의 활성 물질이 열안정성을 나타냄을 알 수 있다.
In FIG. 3, heated does not affect the antimicrobial activity even when heated at 70 ° C for 10 minutes, indicating that the active ingredient of Bifidobacterium pseudomonas sp. SPM1309 according to the present invention exhibits thermal stability.

<< 실시예Example 6>  6> 다제내성균에Multidrug resistant bacteria 대한 항균 특성 시험 Antibacterial properties test

본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309의 항생작용(antibiotic action) 특성을 확인하기 위해 비피도박테리움 슈도카테뉼라툼 SPM1309의 배양 상층액(supernatant)이 함유된 Mueller-Hinton broth에서 다제내성균을 48시간 동안 배양하면서, 혼탁도의 변화를 통해 다제내성균의 증식 여부를 관찰했다. In order to confirm the antibiotic action characteristics of Bifidobacterium pseudomonas putanum lapumum SPM1309 according to the present invention, in a Mueller-Hinton broth containing culture supernatants of Bifidobacterium pseudomonas latifum SPM1309, The resistant bacteria were cultured for 48 hours, and the proliferation of the multi-drug resistant bacteria was observed through the change of turbidity.

이후 본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309의 배양상층액이 함유된 배지에서 배양된 다제내성균을 다시 SPM1309 배양상층액이 함유되지 않은 동일한 배지에 접종해 증식 여부를 관찰했다. 대조군으로는 SPM1309가 포함되지 않고 동량의 GAM broth가 함유된 Mueller-Hinton broth에서 배양한 동일한 다제내성균의 배양체를 이용했다. Then, the multi-drug resistant bacteria cultured in the medium containing the culture supernatant of Bifidobacterium pseudomonas fallumum SPM1309 according to the present invention was again inoculated into the same medium not containing the SPM1309 culture supernatant to observe whether it proliferated. As control, the same multi-drug resistant cultures cultured in Mueller-Hinton broth containing SPM1309 but containing the same amount of GAM broth were used.

도 4에서 보는 바와 같이 본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309의 배양상층액이 포함되지 않고 동량의 PBS나 GAM broth가 함유된 Mueller-Hinton broth에서 배양한 대조군의 경우 24시간 동안 다제내성균이 증식하여 혼탁도가 크게 증가하는데 비해, 본 발명에 의한 본 발명에 의한 비피도박테리움 슈도카테뉼라툼 SPM1309의 배양상층액이 함유된 실험군은 다제내성을 가진 슈도모나스 에루지노사 (Multidrug Resistant Pseudomonas aeruginosa; MDRPA)와 아시네토박터 바우마니(Multidrug Resistant acinetobacter baumannii; MDRAB)에 강한 성장 억제 효과를 나타내어 24시간동안 혼탁도의 변화가 일어나지 않았다.As shown in FIG. 4, in the case of the control group cultured in Mueller-Hinton broth containing no culture supernatant of Bifidobacterium pseudomonas brotium SPM1309 according to the present invention and containing the same amount of PBS or GAM broth, In contrast, the experimental group containing the culture supernatant of Bifidobacterium pseudoacetate catenulatum SPM1309 according to the present invention according to the present invention increased the susceptibility to multidrug resistance (Multidrug Resistant Pseudomonas aeruginosa ; MDRPA) and Multidrug Resistant acinetobacter baumanni i; MDRAB) showed no growth inhibition for 24 hours.

그러나, 24시간 동안 증식이 되지 않았던 실험군의 배양액을 SPM1309의 배양상층액이 함유되지 않은 평판 배지에 다시 접종했을 때에는 군락이 형성되었으며, 도 4에서 보는 바와 같이 실험군의 배양액도 48시간 이후에는 혼탁도가 증가되었다.However, when the culture medium of the test group which had not been proliferated for 24 hours was inoculated again on a plate medium containing no culture supernatant of SPM1309, colonies were formed, and as shown in Fig. 4, .

이는 본 발명에 의한 비피도박테리움 슈도카테라툼 B. pseudocatenulatum SPM1309의 항생작용이 살균작용(bactericidal action)이 아니라 정균작용(bacteriostatic action)을 하는 것을 알 수 있다.This Bifidobacterium pseudo car TB tomb according to the present invention, B. pseudocatenulatum It can be seen that the antibiotic action of SPM1309 is not a bactericidal action but a bacteriostatic action.

한국생명공학연구원Korea Biotechnology Research Institute KCTC12385BPKCTC12385BP 2013032520130325

Claims (6)

다제내성균에 대한 저해 활성을 갖는 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum) SPM 1309 (KCTC 12385 BP)으로,
상기 다제내성균은 아시네토박터 바우마니(Acinetobacter baumannii) 또는 슈도모나스 에루지노사(Pseudomonas aeruginosa)인 것을 특징으로 하는 것인 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum) SPM 1309.
Bifidobacterium pseudocatenulatum SPM 1309 (KCTC 12385 BP), which has inhibitory activity against multidrug-resistant bacteria,
Wherein the multidrug resistant microorganism is Acinetobacter baumannii or Pseudomonas aeruginosa. Bifidobacterium pseudocatenulatum SPM 1309 is characterized in that the multidrug resistant microorganism is Acinetobacter baumannii or Pseudomonas aeruginosa.
삭제delete 삭제delete 제 1 항에 있어서,
상기 다제내성균은 시프로플록사신(ciprofloxacin) 및 토브라마이신(tobramycin), 티게싸이클린(tigecycline), 겐타마이신(gentamicin), 메로페넴(meropenem), 세프타지딤(ceftazidime) 항생제에 대해서 내성을 가지는 것인 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum ) SPM 1309.
The method according to claim 1,
Wherein said multidrug resistant microbes are resistant to ciprofloxacin and tobramycin, tigecycline, gentamicin, meropenem, ceftazidime antibiotics, Bifidobacterium &lt; RTI ID = 0.0 &gt; Pseudocatenulatum ) SPM 1309.
제 1 항에 의한 비피도박테리움 슈도카테뉼라툼(Bifidobacterium Pseudocatenulatum) SPM 1309 또는 이의 배양물을 유효 성분으로 하는 아시네토박터 바우마니(Acinetobacter baumannii) 또는 슈도모나스 에루지노사(Pseudomonas aeruginosa)에 의한 감염의 치료 또는 예방용 조성물.
The use of Bifidobacterium pseudocatenulatum SPM 1309 or a culture thereof as an active ingredient for the treatment of infection with Acinetobacter baumannii or Pseudomonas aeruginosa according to claim 1 &Lt; / RTI &gt;
삭제delete
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20030215467A1 (en) * 1999-01-15 2003-11-20 Enterprise Ireland (Trading As Bioresearch Ireland And National University Of Ireland, Cork. Bifidobacteriumin the treatment of inflammatory disease
KR100610379B1 (en) * 2004-08-26 2006-08-10 하남주 Novel lactic acid bacteria having immune enhancement activity
KR101164512B1 (en) * 2009-12-09 2012-07-11 삼육대학교산학협력단 Probiotic composition for animal comprising bifidobacterium pseudocatenulatum spm1204 or its culture

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030215467A1 (en) * 1999-01-15 2003-11-20 Enterprise Ireland (Trading As Bioresearch Ireland And National University Of Ireland, Cork. Bifidobacteriumin the treatment of inflammatory disease
KR100610379B1 (en) * 2004-08-26 2006-08-10 하남주 Novel lactic acid bacteria having immune enhancement activity
KR101164512B1 (en) * 2009-12-09 2012-07-11 삼육대학교산학협력단 Probiotic composition for animal comprising bifidobacterium pseudocatenulatum spm1204 or its culture

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