In this study, a safety evaluation was conducted to confirm if the Enterococcus faecium CKDB003 strain obtained by selection from a mixed fermentation of fruit and milk is suitable for use as a probiotic. The MIC value for the 10 antibiotics specified in the EFSA guidance was below the acceptable cut-off value. The antibiotic resistance genes aac(6')-li, eatAv, and msr(C) exist by whole genome sequencing, but are in the chromosome and not in the plasmid, thus confirming that there is no possibility of transmission to other microorganisms. It was confirmed that cytolysin (cylA, cylB, cylI, cylL-l, cylL-s, cylM, cylR1, cylR2), aggregation substance (asa1, asp1), collagen adhesion (ace), enterococcal surface protein (esp), endocarditis antigen (efaA), hyaluronidase (hyl) and gelatinase (gelE) were not present in the genome by examining the genes of factors related to virulence. Also, the biochemical analysis showed no toxic enzyme activities, and no virulence genes were detected by the PCR method. Thus, the E. faecium CKDB003 strain can be safely used as a health functional food probiotic, based on the results of the safety assessment.

박테리오신은 미생물이 생산하는 단백질성의 항균물질이다. 본 연구에서는 Enterococcus faecium CJNU 2008 균주로부터 생산되는 박테리오신에 대한 일부 특성을 규명 하였다. 부분 정제 박테리오신은 열처리(100℃ 30분, 121℃ 15분) 및 유기용매(메탄올, 에탄올, 아세톤, 아세토니트릴, 클로로포름)에 대한 안정성이 우수하였으며 효소처리의 경우 Lipase와 α-Amylase에 대해서는 안정하였으나 Protease 처리에서 활성이 소실되었다. 이는 E. faecium CJNU 2008 균주가 생산하는 항균물질이 단백질성의 박테리오신임을 추가적으로 증명하는 것이다. 병원성 세균인 Listeria monocytogenes 균주를 지시균으로 사용했을 때 박테리오신은 살균(bactericidal)의 작용양상을 보였다. Tricine-SDS-PAGE를 이용한 박테리오신의 분자량은 6.5 kDa 이하로 확인되었다. 부분 정제된 박테리오신을 이용하여 HPLC법을 활용한 정제를 수행하였으며 크로마토그램 상에서 단일 피크를 얻었을 수 있었다. 앞으로 정제된 박테리오신은 생화학적 분석 등에 활용할 계획이다.

Probiotics are defined as advantageous microorganisms to human when they are ingested. However, without any protection, the viability of microbes and their adhesive ability to surface of colon decreases through acidic condition such as stomach and intestines. Therefore, many studies have been conducted to figure out to enhance not only the viability of probiotics, but also its adhesion for increasing effect of probiotics. In this study, extrusion method was conducted to encapsulate Enterococcus faecium. E. faecium-alginate solution was injected to CaCl2 solution with regular side air injection. To prevent coagulation of beads, stirring was conducted in CaCl2 solution and encapsulated alginate-Ca2+ microspheres were produced. For optimal encapsulation condition, air pressure was 100 mbar, flow rate of E. faecium solution was 0.02 ml/h and stirring rate was 200 rpm. For mucoadhesive ability, Monolayer of HT-29 cells used as a colon cell and encapsulated cells were inoculated and incubated in 37℃, 5% CO2/95% air atmosphere for 1 h. Encapsulation efficiency of the encapsulation method used in this study was 98.2%. For mucoadhesive test, the concentration of inoculated E. faecium was 9.9×108 CFU/ml and the concentration of adhered E. faecium was 1.6×106 CFU/ml. In conclusion, encapsulation efficiency of extrusion method was high enough to be accepted for this study, however, alginate-Ca2+ microspheres revealed lower adhesive ability compared to expectation. Therefore, it needs further studies to increase adhesive ability with other polymers.

기능성 발효유로 알려진 케피어로부터 박테리오신 생산 유산균주(CJNU 2008)를 선발하였다. CJNU 2008 균주는 16S rRNA 유전자 염기서열 분석을 통해 Enterococcus faecium으로 동정되어 E. faecium CJNU 2008로 명명하였다. E. faecium CJNU 2008균주의 배양액 및 부분정제 농축액은 Listeria monocytogenes와 Staphylococcus aureus와 같은 병원성 세균을 포함하여 몇몇 균주들에 대해서 항균활성을 나타내었다. E. faecium CJNU 2008 균주의 박테리오신 생산을 위한 배양 최적조건은 MRS broth, 30℃ 및 pH 7.0으로 확인되었다.

We selected lactic acid bacteria to reduce cholesterol levels in vitro and in vivo. Among the 140 strains tested, a strain ID9201 was shown to reduce cholesterol in the spent medium over 30% and to increase cholesterol in cell pellet. A strain ID9201 isolated from breast milk-fed Korean infant feces was identified as Enterococcus faecium on the basis of morphological analysis, biochemical analysis (API kit), and 16S rDNA sequence analysis. A freeze-dried powder of the strain was fed to Sprague-Dawley(SD) rats with high cholesterol diet for 6 wk. In the control group fed cholesterol enriched diet without probiotics, the serum cholesterol increased by 1.9 times in comparison with the initial value. Among the test group, ID9201 group showed the lowest cholesterol increase, 1.5 times of initial serum cholesterol, as in the blank group fed normal diet without probiotics. Based on these data, we assumed that ID9201 inhibited intestinal absorption of dietary cholesterol.

Characteristics and immuno-modulatory effects of Enterococcus (E.) faecium JS1-8 isolated from Kimchi were investigated for potential probiotic use. We measured their acid, bile, and heat tolerances, adhesion properties in Caco-2 cells, antimicrobial activity against pathogenic bacteria, and bacteriocin-like substance-producing activity. Immuno-modulatory effects of E. faecium JS1-8 were measured by determination of nitric oxide (NO), nuclear factor-κB (NF-κB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) production in RAW 264.7 cells or RAW BLUE cells. JS1-8 survived at pH 2.0 for 2 hr and showed tolerance to 0.3% oxgall bile salt, and it survived after exposure for 5 min at 80°C. JS1-8 showed high antimicrobial inhibition zones to Staphylococcus aureus (460 mm), Listeria monocytogenes (310 mm), Salmonella enteritidis (280 mm), and E. coli (150 mm). Bacteriocin-like substance produced by JS1-8 showed a broad spectrum of activity against Listeria monocytogenes KCCM 40307 and Lactobacillus sake KCCM 40264. Low concentration (1 × 107 CFU/mL) of heat-killed E. faecium JS1-8 induced statistically higher production of NO than Lactobacillus rhamnosus GG (LGG), which is a well-known immuno-modulatory lactic acid bacteria. Low and high (5 × 107 CFU/mL) concentrations of JS1-8 induced statistically higher production of NF-κB than that produced by LGG. We also found that JS1-8 increased production of pro-inflammatory cytokines such as IL-1β and TNF-α in a concentration-dependent manner. As a result, E. faecium JSI-8 could be used as a useful probiotic for controlling pathogens and enhancing host immune responses.

이 연구는 자돈의 분변에서 분리된 Enterococcus faecim 193이 생산한 박테리오신을 부분정제하고 그 특성을 파악하여 천연 식품보존제 또는 첨가제로서의 이용가능성을 알아보고자 실시되었다. Enterococcus faecium 193의 배양시간에 따른 항균활성을 측정한 결과 항균활성은 3시간째부터 나타나기 시작하여 12시간째에 1280 AU/ml로 최대 활성을 나타내었다. Enterocin 193은 단백질 분해효소(trypsin, pepsin, 및 proteinase K)의 처리에 의해 항균활성이 소실되었으나 넓은 범위의 pH (1-11)와 열처리(121℃, 15분)에 비교적 안정하였다. 또한 50% 농도의 acetone, acetonitrile, chloroform 및 ethanol 등 유기용매에 안정하였으나, 50% methanol을 처리할 경우 항균활성이 50% 감소하였다. 이 박테리오신은 황산암모늄 침전, 소수성 크로마토그래피와 젤 여과 크로마토그래피에 의해 정제되었고 specific activity는 14.8배 증가하였다. SDS-PAGE에 의해 측정한 이 단백질의 분자량은 약 5 kDa이었다.

To produce rapidly the traditional Kanjang soy sauce-like product with rich flavors, lactic acid bacteria of Enterococcus spp. isolated from Chungkukjang was used as one of starter cultures. Among 119 Enterococcus spp., eight strains were selected by protease-secreting activities and identified as four E. faecium, three E. faecalis, and one E. gallinarium. The strains showed low resistances toward eight antibiotics and had no resistant genes to the vancomycin. Especially, E. faecium O24 was cultivated well on 5% NaCl medium that was selected for further study as the starter. E. faecium O24 grew well on the steamed soybean and the counts increased by ten times overnight, which produced mostly 80 mg% glutamic acid and aspartic acid as the seasoning amino acids on the product. Various organic acids including principal lactic acid were also produced. Flavors of maltol and guaiacol, typical soy-sauce flavor, were produced in the mixed cultures of Zygosaccharomyces rouxii and Candida versatilis. Therefore, E. faecium O24 could be a starter of soybean fermentation for soy sauce-like product with rich flavors rapidly.

There are many researches about the contribution of virginiamycin use in animals to quinupristin/dalfopristin (Q/D) resistance in humans. In this study, the prevalence and mechanisms of streptogramin resistance in Enterococcus faecium from chickens were investigated. A total of 170 E. faecium isolates from 38 chicken farms was tested for antimicrobial susceptibility. Eleven (6.5%) E. faecium isolates were found to be resistant to Q/D. The vatE and ermB genes were detected in 2 (18%) and 6 (55%) of Q/D-resistant E. faecium respectively. By using pulsed-field gel electrophoresis (PFGE), 6 distinct PFGE patterns were identified. These data indicate that Q/D resistance among E. faecium from chickens remains low despite the long history of virginiamycin use. However, we have to concern over antimicrobial resistance in bacteria originated from animals, including the possibility of transfer of resistance genes from animal to human.

우리나라 고유의 전통발효 식품인 메주로부터 항리스테리아 활성을 나타내는 유산균을 분리한 후 동정한 결과 E. faecium MJ5-14로 명명되었으며, 이들의 박테리오신은 MRS broth중에 37??에서 12-18시간 배양하는 동안 최대의 활성을 나타내었다. L. monorcytogenes KCTC 3569와 KCTC 3710 및 L. ivanovii subsp. ivanovii에 대한 박테리오신의 활성은 640 BU/mL이었고, L. innocua ATCC 33090, L. ivanovii ATCC 19119, L. grayi KCTC 3581 및 L. seeliger KCTC 3591에 대한 활성은 1280 BU/mL으로 나타났다. L. monocytogenes KCTC 3569와 E. faecium MJ5-14을 혼합 배양한 경우 L. monocytogenes의 감소 효과는 25?? 이하 보다는 E. faecium MJ5-14 박테리오신 생산 최적 온도인 37℃에서 더 높게 나타났으며, f. Jhrrf'um MJ5-1꾸T 박테리오신 항균 효과는 1. mouoryrogenrrKCTC 3569의 초기 균수와 첨가한 박테리오신의 양에 따라 좌우되어 교. mouocyrogrurr의 초기 균수가 적고, 박테리오신의 첨가량이 많을수록 항균효과가 높아짐을 될인하였다. 또한 박테리오신 70 BU/mL첨가에 의한 항균 효과는 균 증식이 활발한 대수증식기 중반 이후 보다는 유도기 내지는 대수증식기 초반부에 더 크게 나타났다. 한편,박테리오신 25 BU/CL을 첨가에 의해선 고. HororyrDfrurr KCfC 3569세포벽의 일부가 얇아rl고, 50 BU/DL 이상의 농도에 의해선 세포막이 파괴되면서 세포 내용물의 유출 현상이 관찰되었다.

상업적 복합균주와 효모를 sourdough starter로 사용하여V5가지의 시료를 다음과 같이 준비하였다. 제빵용 효모만을V사용한 control군, 상업용 복합균주를 사용한 LAB 1군, 상업용 복합균주와 효모로 각각 발효한 후 합친 LAB 2군, 상업용 복합균주와 효모를 함께 사용하여 발효한 LAB 3군, 상업용 복합균주와 효모를 사용하여 각각 발효하여 합친 후 다시 10시간 발효한 LAB 4군을 사용하여 제조한 발효식빵의 제빵특성은 다음과