• 한국축산식품학회지
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• 제39권5호
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• pp.804-819
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• 2019

Ezine cheese is a non-starter and long-ripened cheese produced in the Mount of Ida region of Canakkale, Turkey, with a protected designation of origin status. Non-starter lactic acid bacteria (NSLAB) have a substantial effect on the quality and final sensorial characteristics of long-ripened cheeses. The dominance of NSLAB can be attributed to their high tolerance to the hostile environment in cheese during ripening relative to many other microbial groups and to its ability to inhibit undesired microorganisms. These qualities promote the microbiological stability of long-ripened cheeses. In this study, 144 samples were collected from three dairies during the ripening period of Ezine cheese. Physicochemical composition and NSLAB identification analyses were performed using both conventional and molecular methods. According to the results of a 16S rRNA gene sequence analysis, 13 different species belonging to seven genera were identified. Enterococcus faecium (38.42%) and E. faecalis (18.94%) were dominant species during the cheese manufacturing process, surviving 12 months of ripening together with Lactobacillus paracasei (13.68%) and Lb. plantarum (11.05%). The results indicate that NSLAB contributes to the microbiological stability of Ezine cheese over 12 months of ripening. The isolation of NSLAB with antimicrobial activity, potential bacteriocin producers, yielded defined collections of natural NSLAB isolates from Ezine cheese that can be used to generate specific starter cultures for the production of Ezine cheese (PDO).

• 한국응용과학기술학회지
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• 제37권6호
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• pp.1535-1544
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• 2020

본 연구의 목적은 3년 이상 숙성된 재래식 된장으로부터 아민 산화 효소를 생산하는 프로바이오틱 바실러스균을 분리 동정하는 것이다. 시료로부터 분리된 바이오제닉 아민(biogenic amines, BA) 생성균은 Bacillus sp. TS09, Bacillus licheniformis TS17, Bacillus subtilis TS19, Bacillus cereus TS23, Bacillus sp. TS30, Bacillus megaterium TS31, B. subtilis TS44, Bacillus coagulans TS46 및 Bacillus amyloliquefaciens TS59 등으로 동정되었다. 한편 동일한 시료로부터 분리된 B. subtilis TS04와 TS50은 인공 위액 및 담즙액에 대한 저항성, 장내 상피세포에 대한 부착능 및 BA 생성균(Bacillus sp. TS30 및 B. subtilis TS44)에 대한 박테리오신 생산 등의 프로바이오틱 활성을 나타내었다. 게다가 B. subtilis TS04와 TS50 균주가 생산한 아민 산화 효소에 의하여 카다베린, 푸트레신 및 티라민의 생성량을 감소시킬 수 있었으므로 이들은 BA 중독 위험을 낮출 수 있는 프로바이오틱스 소재로서의 활용 가치가 높을 것으로 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제18권8호
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• pp.1199-1208
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• 2005

Abatement of greenhouse gas emitted from ruminants and promotion of biogas energy from animal effluent were comprehensively examined in each anaerobic fermentation reactor and animal experiments. Moreover, the energy conversion efficiency of biomass energy to power generation were evaluated with a gas engine generator or proton exchange membrane fuel cell (PEMFC). To mitigate safely rumen methanogenesis with nutritional manipulation the suppressing effects of some strains of lactic acid bacteria and yeast, bacteriocin, $\beta$1-4 galactooligosaccharide, plant extracts (Yucca schidigera and Quillaja saponarea), L-cysteine and/or nitrate on rumen methane emission were compared with antibiotics. For in vitro trials, cumulative methane production was evaluated using the continuous fermented gas qualification system inoculated with the strained rumen fluid from rumen fistulated Holstein cows. For in vivo, four sequential ventilated head cages equipped with a fully automated gas analyzing system were used to examine the manipulating effects of $\beta$1-4 galactooligosaccharide, lactic acid bacteria (Leuconostoc mesenteroides subsp. mesenteroides), yeast (Trichosporon serticeum), nisin and Yucca schidigera and/or nitrate on rumen methanogenesis. Furthermore, biogas energy recycled from animal effluent was evaluated with anaerobic bioreactors. Utilization of recycled energy as fuel for a co-generator and fuel cell was tested in the thermophilic biogas plant system. From the results of in vitro and in vivo trials, nitrate was shown to be a strong methane suppressor, although nitrate per se is hazardous. L-cysteine could remove this risk. $\beta$1-4 galactooligosaccharide, Candida kefyr, nisin, Yucca schidigera and Quillaja saponarea are thought to possibly control methanogenesis in the rumen. It is possible to simulate the available energy recycled through animal effluent from feed energy resources by making total energy balance sheets of the process from feed energy to recycled energy.

• 한국식품위생안전성학회지
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• 제37권2호
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• pp.97-106
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• 2022

L. monocytogenes는 그람양성의 대표적인 식중독균 중 하나로 치명률이 대단히 높으며 대부분의 식중독균과 달리 저온에서도 생육 가능하여 냉장 보관된 식품에서도 식중독을 발생시킬 수 있다. 이에 따라 식품의 제조, 가공 및 유통과정에서 다양한 물리, 화학적 방법이 사용되고 있지만, 이러한 방법은 식품의 품질 변화를 초래하고 합성보존제에 대한 소비자의 인식 등으로 적용에 한계가 있을 수 있다. 따라서 본 연구에서는 식품의 미생물적 안전성 향상을 위해 김치에서 분리된 LAB의 항리스테리아 활성을 분석하여 천연항균제로서 활용 가능성을 평가하였다. Agar overlay 방법으로 김치에서 분리된 총 36종(Lactobacillus속, Weissella속, Lactobacillus속, Lactococcus속)의 유산균에 대한 항리스테리아 활성을 분석한 결과 L. lactis NJ 1-10과 NJ 1-16이 가장 항리스테리아 활성이 높은 것으로 나타났다. 항리스테리아 활성을 정량적으로 분석하기 위해 NJ 1-10과 NJ 1-16을 각각 L. monocytogenes와 BHI broth에서 공동 배양한 결과, 20시간 만에 L. monocytogenes를 3.0 log CFU/mL 감소시켜 검출한계 이하까지 균수가 감소하였다. 두 LAB균주 모두 24개의 L. monocytogenes 혈청형에 대해 저해환의 크기는 조금씩 다르지만 모두 항리스테리아 활성을 보였다. NJ 1-10과 NJ 1-16의 부분 정제된 박테리오신 모두 proteinase-K 처리에서 항리스테리아 활성이 소실되어 항균물질이 단백질의 박테리오신임을 확인하였다. 부분 정제된 박테리오신의 열에 대한 안정성은 NJ 1-10과 NJ 1-16 모두 60℃ 및 80℃에서 비교적 안정했지만, 100℃에서 60분과 121℃에서 15분 처리로 활성이 완전히 소실되었다. pH의 안정성의 경우, pH 4.01에서 활성이 가장 안정하였고 pH가 높아질수록 그 활성이 감소하는 경향을 나타내었으나, 활성이 완전하게 소실되지는 않았고, 유기용매 안정성은 acetone, ethanol, methanol에 비교적 안정한 활성을 보였으나 chloroform 처리 시 활성의 정도가 감소하였지만 완전히 소실되지는 않았다. 따라서 본 연구의 결과, NJ 1-10과 NJ 1-16이 생산하는 박테리오신은 L. monocytogenes를 효과적으로 저감시켰으며, 열, pH, 유기용매에 대해 비교적 안정하여 식품에 존재하는 리스테리아균 제어를 위한 천연항균제로서의 잠재적인 가능성이 있음을 확인하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.285-294
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• 2011

The abatement of methane emission from ruminants is an important global issue due to its contribution to greenhouse gas with carbon dioxide. Methane is generated in the rumen by methanogens (archaea) that utilize metabolic hydrogen ($H_2$) to reduce carbon dioxide, and is a significant electron sink in the rumen ecosystem. Therefore, the competition for hydrogen used for methanogenesis with alternative reductions of rumen microbes should be an effective option to reduce rumen methanogenesis. Some methanogens parasitically survive on the surface of ciliate protozoa, so that defaunation or decrease in protozoa number might contribute to abate methanogenesis. The most important issue for mitigation of rumen methanogenesis with manipulators is to secure safety for animals and their products and the environment. In this respect, prophylactic effects of probiotics, prebiotics and miscellaneous compounds to mitigate rumen methanogenesis have been developed instead of antibiotics, ionophores such as monensin, and lasalocid in Japan. Nitrate suppresses rumen methanogenesis by its reducing reaction in the rumen. However, excess intake of nitrate causes intoxication due to nitrite accumulation, which induces methemoglobinemia. The nitrite accumulation is attributed to a relatively higher rate of nitrate reduction to nitrite than nitrite to ammonia via nitroxyl and hydroxylamine. The in vitro and in vivo trials have been conducted to clarify the prophylactic effects of L-cysteine, some strains of lactic acid bacteria and yeast and/or ${\beta}$1-4 galactooligosaccharide on nitrate-nitrite intoxication and methanogenesis. The administration of nitrate with ${\beta}$1-4 galacto-oligosaccharide, Candida kefyr, and Lactococcus lactis subsp. lactis were suggested to possibly control rumen methanogenesis and prevent nitrite formation in the rumen. For prebiotics, nisin which is a bacteriocin produced by Lactococcus lactis subsp. lactis has been demonstrated to abate rumen methanogenesis in the same manner as monensin. A protein resistant anti-microbe (PRA) has been isolated from Lactobacillus plantarum as a manipulator to mitigate rumen methanogenesis. Recently, hydrogen peroxide was identified as a part of the manipulating effect of PRA on rumen methanogenesis. The suppressing effects of secondary metabolites from plants such as saponin and tannin on rumen methanogenesis have been examined. Especially, yucca schidigera extract, sarsaponin (steroidal glycosides), can suppress rumen methanogenesis thereby improving protein utilization efficiency. The cashew nutshell liquid (CNSL), or cashew shell oil, which is a natural resin found in the honeycomb structure of the cashew nutshell has been found to mitigate rumen methanogenesis. In an attempt to seek manipulators in the section on methane belching from ruminants, the arrangement of an inventory of mitigation technologies available for the Clean Development Mechanism (CDM) and Joint Implementation (JI) in the Kyoto mechanism has been advancing to target ruminant livestock in Asian and Pacific regions.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.149-153
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• 2001

To investigate the mode of bactericidal action for antimicrobial peptide, pediocin, synthetic and mutant pediocins were prepared by direct chemical synthesis. Native pediocin was purified from Pedio-coccus acidilactici M and its conformational structure and bactericidal functions were analyzed and compared to synthetic pediocin. Schematic mode of pediocin actions, how pediocin binds on the target cell membrane, penetrates and makes tunnel are proposed. For these purposes, primary and secondary structures of pediocin was analyzed and disulfide bond assignment was also done. The pediocin purified from P. acidilactici M had high effective bactericidal ability against gram positive bacteria, especially Listeria monocytogenes and was very stable at extreme pHs and even at high temperatures such as autoclaving temperature (121$^{\circ}C$). Pediocin was consisted of 44 amino acids with four cysteines. Novel synthetic peptides were achieved by solid phase peptide synthesis(SPPS) method. To explain the function of cysteine in C-terminal region, mutant pediocin, Ped[C24A＋C44A], was synthesized and their structural and biological functions were analyzed. Second mutant pediocin, Ped[KllE], was prepared to explain the function of lysine at 11 of N-terminal part of pediocin, especially loop of $\beta$-sheet, and to predict the initial binding site of pediocin. The native and synthetic pediocins was showed random coil conformation by spectropolarimetry in moderate conditions. This conformation was observed in extreme conditions such as high temperature and low and high pHs, also. Circular dichroism(CD) data also showed the existence of $\beta$-turn structure in N-terminal part both native and synthetic pediocins. A structural model for pediocin predicts that 18 amino acids in the N-terminal part of the peptide assume a three-strand $\beta$-sheet conformation. This random coil in C-terminal part of pediocin was converted to folding structure, helix structure, in nonpolar solvents such as alcohol and TFE. The disulfide bond between $^{9}$ Cys and $^{14}$ Cys was concrete and inevitable, however, evidences of disulfide bond between $^{24}$ Cys and $^{44}$ Cys was not. Data of Ped[C24A＋C44A], pediocin mutant showed that $^{44}$ Cys was required during killing the target cells but not inevitable, since Ped[C24A＋C44A] still have bactericidal activity but much less than native pediocin. Another pediocin mutant, Ped[KllE], had still bactericidal activity, was controversial to propose that positive charge like as $^{11}$ Lys in loop or hinge in bacteriocin bound or helped to binding to microorganism with electrostatic interaction between cell membrane especially teichoic acid and positive amino acid nonspecifically. The conformation of pediocin among native, synthetic and mutant pediocins did not show big difference. The conformations between oxidized and reduced pediocin were almost similar regardless of native or synthetic.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.225-236
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• 2013

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at $30^{\circ}C$. Interestingly, antibacterial activity remained unchanged after heating at $121^{\circ}C$ for 45 min, 24 h storage in temperature range of $70^{\circ}C$ to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

• 한국식품저장유통학회지
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• 제6권4호
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• pp.514-520
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• 1999

김치의 저장성과 기증성 향상을 위한 기초 연구로 서, 유기게르마늄(Ge-132)과 올리고당(프럭토)을 함유하는 배추김치의 생즙액에 3가지 젖산균으로 조합한 스타트를 첨가하여 3$0^{\circ}C$에서 발효시키면서, pH, 산도, $CO_2$생성량, 환원당 및 젖산균수의 변화를 조사하였다. 게르마늄 첨가구는 박테리오신을 생산하는 SNF-13 분리 젖산균주와 E. faecium의 혼합배양(I)에서 유기산과 $CO_2$생성량이 가장 적었으며, 스타트유래의 짖산균수 증식을 촉진시켰고, 환원당 소비량은 가장 많았다. 올리고당 첨가구는 4가지의 혼합배양에서 모두 각각의 대조구 및 게르마늄 첨가구보다 훨씬 유기산 및 $CO_2$생성량이 많았으며, 김치유래의 젖산균수 증식을 촉진시켰다. 유기게르마늄은 박테리오신을 생산하거나 게르마늄 저농도에서 생육이 촉진되는 내산성의 유산균과 혼합병용하면, 김치의 기능성 증진(저장성, 생리활성)의 미네랄로서 활용가능성이 높을 것으로 판단된다.