• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1004-1008
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• 2004

An extracellular acid phytase from Pseudomonas fragi Y9451 was purified to homogeneity from the culture supernatant by salting-out, DEAE-Sepharose column chromatography, CM-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 74 kDa on gel filtration and 54 kDa and 25 kDa on SDS-PAGE, suggesting that the native enzyme was a heterodimeric protein. The purified enzyme was most active at pH 4.5 and $70^{\circ}C$ and fairly stable from pH 4.0- 6.0. It was specific for phytate and exhibited a $K_{m}$ value of 27 mM (sodium phytate, pH 4.5, $50^{\circ}C$). The phytase activity was strongly inhibited (at maximum by 87%) by $Fe^{3+},\;Cu^{2+},\;Fe^{2+}$, and $Zn^{2+}$ at 5 mM concentration, and greatly inhibited by $Ca^{2+}$ at 10 mM concentration. However, EDTA notably stimulated the phytase activity at 10 mM concentration. With optimum pH and stability, Pseudomonas fragi phytase could be a potential candidate for animal feed applications.

• Applied Biological Chemistry
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• 제46권4호
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• pp.291-298
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• 2003

축산폐수가 방류되는 단위동물 밀집사육장 인근의 하천수로부터 phytase를 생산하는 세균들 중 활성이 가장 우수한 균주를 분리하여, Pseudomonas fragi Y9451로 동정, 명명하였다. 탄소원으로 fructose를 첨가할 경우에 phytase의 생산능이 가장 우수하였고, 배지에 7% 첨가할 경우에 72시간 배양 후 효소활성이 1,078 mU/ml로 최대의 phytase 생산능을 보였다. 또한, 질소원으로는 nutrient broth(NB)가 적합하였으며, NB를 3%의 농도로 첨가할 경우에 96시간 배양 후 효소활성이 1,425 mU/ml까지 향상되었다. 인산원인 $KH_2PO_4$, phytate는 첨가 농도가 높을수록 phytase 생산은 억제되었으며, 무기염으로 $MgSO_4$와 $CaCl_2$를 첨가하는 경우는 첨가농도와 상관없이 phytase의 생산을 현저히 억제하였다. 산업생산을 목적으로 하는 scale-up에 있어서의 통기교반 효과는 phytase의 생산량에 큰 영향을 미치는 것으로 나타났다.

• Bulletin of the Korean Chemical Society
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• 제16권5호
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• pp.401-406
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• 1995

The P. fragi lipase overexpressed in E. coli as a fusion protein of 57 kilodalton (kDa) has been purified through glutathione-agarose affinity chromatography by elution with free glutathione. The general properties of the purified GST-fusion protein were characterized by observing absorbance of released p-nitrophenoxide at 400 nm which was hydrolyzed from the substrate p-nitrophenyl palmitate. The optimum condition was observed at 25 $^{\circ}C$, pH 7.8 with 0.4 ${\mu}g$ of protein and 1.0 mM substrate in 0.6% (v/v) TritonX-100 solution. Also the lipase was activated by Ca+2, Mg+2, Ba+2 and Na+ but it was inhibited by Co+2 and Ni+2. pGEX-2T containing P. fragi lipase gene as expression vector was named pGL191 and used as a template for the site-directed mutagenesis by sequential PCR steps. A Ser-His-Asp catalytic triad similar to that present in serine proteases may be present in Pseudomonas lipase. Therefore, the PCR fragments replacing Asp217 to Arg and His260 to Arg were synthesized, and substituted for original fragment in pGL19. The ligated products were transformed into E. coli NM522, and pGEX-2T harboring mutant lipase genes were screened through digestion with XbaI and StuI sites created by mutagenic primers, respectively. No activity of mutant lipases was observed on the plate containing tributyrin. The purified mutant lipases were not activated on the substrate and affected at pH variation. These results demonstrate that Asp217 and His260 are involved in the catalytic site of Pseudomonas lipase.

• 한국식품위생안전성학회지
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• 제14권4호
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• pp.346-351
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• 1999

본 연구에서는 발효 식품의 제조에 유용한 항균성 물질을 생산하는 유산균 starter의 개발의 일환으로 장내 상존균으로 알려진 Lactobacillus acidophilus group유산균인 Lactobacillus amylovorus IMC-1 균주가 생산한 항균성 물질의 식품오염세균에 대한 항균 활성을 검토하였다. 내몽골 원산 치즈에서 분리된 L. amylovorus IMC-1 균주는 탈지유배지에서 37$^{\circ}C$에서 72시간 배양하였을 때 최대로 항균성 물질을 생산하였으며, 더 이상의 배양은 항균성 물질의 생산에 영향을 미치지 않았다. 겔 여과후의 항균성 물질은 식품오염세균인 Bacillus subtilis IFO 3025, staphylococcus aureus IAM 1011, Listeria monocytogenes VTU 206, Escherichia coli RB. 및 Pseudomonas fragi IFO 3458등과 같은 모든 피검균에 대하여 20units/ml 첨가로 항균활성을 나타내었다. 그리고 이 물질은 B. subtilis, E. coli및 Ps. fragi에 대해서는 살균 작용을 나타내었으며, Staphy. aureus와 L. monocytogenes에 대해서는 정균 작용을 보였다. 그 살균 작용은 용균 작용에 기인한 것임이 밝혀졌다. 또한 이 물질은 유기산, 과산화수소 및 단백질성 물질이 아님이 밝혀졌다.

• 한국동물위생학회지
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• 제44권3호
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• pp.133-140
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• 2021

This study was aimed to investigate the prevalence and antimicrobial resistance of Pseudomonas spp. isolated from bovine mastitis milk samples. A total of 50 (4.9%) Pseudomonas spp. was isolated from 1,023 samples, those collected between 2018 and 2021, derived from 110 dairy farms. The prevalence of the identified species of Pseudomonas isolates was as follows; P. aeruginosa (70.0%), P. fluorescens (14.0%), P. putida (10.0%), P. fragi (4.0%), and P. chlororaphis (2.0%). Most of somatic cell counts in the quarter milk carrying Pseudomonas spp. were less than 3,000,000 cell/ml (90.0%). The isolates of Pseudomonas spp. showed high susceptibility to cefepime (98.0%), ciprofloxacin (98.0%), ceftazidime (96.0%), and colistin (96.0%). The rate of antibiotic resistance in the isolates was highest to ceftiofur (92.0%), followed by the resistance rate to chloramphenicol (86.0%) and trimethoprim/sulphamethoxazole (80.0%). In addition, there is a remarkable difference in antimicrobial resistance pattern among Pseudomonas species. P. aeruginosa and P. putida showed a similar resistance pattern, whereas P. fluorescens showed exceptionally lower resistance to trimethoprim/sulphamethoxazole and chloramphenicol than that of the other species. This study showed that prevalence of Pseudomonas spp. other than P. aeruginosa were 30.0% in bovine mastitis milk, and the occurrence rate of antibiotic resistance were similar or higher level, compared with the previous reports on the mastitisderived Pseudomonas spp. isolated in Korea.

• 한국식품과학회지
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• 제31권6호
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• pp.1613-1618
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• 1999

영귤(Citrus sudachi) 중의 항균성 물질을 검색하기 위하여 영글 과즙 및 과피를 hexane, acetone, ethyl acetate과 methanol로 추출하여 그램 양성 세균 14종과 그램 음성 세균 4종을 사용하여 항균 효과를 측정한 결과 영귤 과즙 추출물은 과피 추출물보다 항균 효과가 우수하였으며, 그 중 영귤 J-A추출물이 가장 강한 항균 활성을 나타내었다. 특히 B. subtilis, E. coli O157 : H7에 대하여 강한 항균력을 나타내었다. 영귤J-A추출물의 첨가 농도(0, 0.5, 1, 1.5, 2. 2.5%)에 따른 항균 효과를 조사한 결과, 모든 실험균이 1%(v/v) 첨가 농도 미만에서 생육이 지연되었으며, B. subtilis는 0.5%, P. fragi는 1%, L. Monocytogenes와 E. coli는 0.5%, P. fragi는 1%, L. monocytogenes와 E. coli O157 : H7은 1.5%, P. acnes와 S. typhimurium은 2%, S. aureus는 2.5% 첨가 농도에서 생육이 정지되었다. 영귤 J-A 추출물의 항균력은 동정되지 않은 천연항균 물질과 citric acid의 상승효과에 기인하는 것으로 판단되었다.

• 한국환경보건학회지
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• 제22권4호
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• pp.109-121
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• 1996

It was intended to investigate the effect of the microbiological kitchen hygiene such as dishclothes and scrubbers. The 8indicator organisms (standard plate counts, coliform, heterotroph, enterococcus, staphylococcus, heat-stable bacteria, psychrotroph, Pseudomonas aeruginosa) were detected highly in dishwaters, dishcloth and scrubber. Coliform and Staphylococcus aureus were appeared on dishcloth dominantly than the scrubber, and the scrubbers were intruded by hetrotrophs and psychrotrophs numerously than dishclothes. The germ-resistant sponge inhibited the growth of the most of test strain, and appeared the about 100% reduction rate after 24 hr, but did not affect Pseudomonas aeruginosa and P. fragi so typically after 24 hr. The anti-microorganism durability of germ-resistant sponge, treated with food soil, was maintained by 10 days, the early stage strain density was founded in 20 days, and the strains grew after 30 days.

• 한국식품위생안전성학회지
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• 제35권2호
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• pp.170-176
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• 2020

생육, 습식 숙성육, 건식 숙성육을 종류별 3종을 구입하여 냉장 상태로 보관하면서 pH, 휘발성 염기질소(VBN), 미생물 정량, 우점균을 분석하였다. 초기 생육, 건식 숙성육의 중온균은 3.3-3.9 Log CFU/g, 습식 숙성육은 경우 5.4 Log CFU/g 이였으나, 냉장으로 18일 보관 후 생육과 건식 숙성육에 존재하는 중온균은 6.1-6.4 Log CFU/g로 증가하였다. 습식 숙성육의 경우 Lactic acid bacteria (LAB)는 냉장 보관시 4.5-6.0 Log CFU/g으로 나타났으나, 건식숙성육에서는 검출되지 않았다. 저장 기간이 길어짐에 따라 중온균, 저온균, LAB, 효모 및 곰팡이 수가 증가하였으나, 식품매개 병원성 미생물은 검출되지 않았다. 식육의 오염 및 부패 판단은 7 Log CFU/g 이상으로 규정하고 있어 본 연구 결과에 의하면 12일 이상 냉장 보관하였을 경우 6-7 Log CFU/g으로 기준을 초과하였다. 이 때 VBN이 평균 15 mg%으로 부패의 초기 단계로 판단 할 수 있었다. 냉장 보관에 따른 우점 미생물은 다양한 양상으로 나타냈다. 생육에서는 초기 우점균으로 S. saprophyticus가 분석되었으나, VBN이 증가함에 따라 Carnobacterium divergens가 우점하는 양상을 나타냈다. 습식 숙성육에서 Carnobacterium divergens가 냉장보관 초기에 우점 미생물로 분석되었으나, 이후 Lactobacillus sakei가 우점균으로 분석되었다. 건식 숙성육의 경우 Dermacoccus nishinomiyaensis가 냉장보관 초기에 우점하였으나 이후 Pseudomonas fragi가 우점균으로 변화하였다. 부패의 초기 단계에서 특정 박테리아의 역할 외에도 부패, 향기 분석 및 숙성 육류의 성분 변화에 대한 연구를 수행하여 숙성 육류의 안전한 유통 및 보관에 활용할 수 있다고 판단된다.

• 한국축산식품학회지
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• 제39권2호
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• pp.209-221
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• 2019

In this study, the changes in microbial communities of lamb meat packaged in the air (plastic tray, PT) and in a vacuum pouch (VAC) were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) during the storage at $4^{\circ}C$. For the PT lamb, the total viable count (TVC) was $10^7CFU/g$ on Day 5, and the dominated bacteria were Pseudomonas fragi, P. fluorescens, and Acinetobacter spp. For the VAC lamb, the TVC was $10^7CFU/g$ on Day 9, and the dominated bacteria were lactic acid bacteria, including Carnobacterium divergens, C. maltaromaticum, and Lactococcus piscium. One strain of Pseudomonas spp. also appeared in VAC lamb. The relative abundance of Enterobacteriaceae in VAC lamb was higher than that PT lamb, indicating a more important role of Enterobacteriaceae in spoilage for VAC lamb than that of PT lamb. The microbial compositions changed faster in the lamb stored in a PT than that stored in a VAC, and microbial community compositions of the late storage period were largely different from those of the early storage period for both the conditions. The findings of this study may guide improve the lamb hygiene and prolong the shelf life of the lamb.

• Asian-Australasian Journal of Animal Sciences
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• 제31권4호
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• pp.585-594
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• 2018

Objective: This study was to determine the bacterial diversity and monitor the community dynamic changes during storage of vacuum-packaged sliced raw beef as affected by Lactobacillus sakei and Lactobacillus curvatus. Methods: L. sakei and L. curvatus were separately incubated in vacuumed-packaged raw beef as bio-protective cultures to inhibit the naturally contaminating microbial load. Dynamic changes of the microbial diversity of inoculated or non-inoculated (control) samples were monitored at $4^{\circ}C$ for 0 to 38 days, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Results: The DGGE profiles of DNA directly extracted from non-inoculated control samples highlighted the order of appearance of spoilage bacteria during storage, showing that Enterbacteriaceae and Pseudomonas fragi emerged early, then Brochothrix thermosphacta shared the dominant position, and finally, Pseudomonas putida showed up became predominant. Compared with control, the inoculation of either L. sakei or L. curvatus significantly lowered the complexity of microbial diversity and inhibited the growth of spoilage bacteria (p<0.05). Interestingly, we also found that the dominant position of L. curvatus was replaced by indigenous L. sakei after 13 d for L. curvatus-inoculated samples. Plate counts on selective agars further showed that inoculation with L. sakei or L. curvatus obviously reduced the viable counts of Enterbacteraceae, Pseudomonas spp. and B. thermosphacta during later storage (p<0.05), with L. sakei exerting greater inhibitory effect. Inoculation with both bio-protective cultures also significantly decreased the total volatile basic nitrogen values of stored samples (p<0.05). Conclusion: Taken together, the results proved the benefits of inoculation with lactic acid bacteria especially L. sakei as a potential way to inhibit growth of spoilage-related bacteria and improve the shelf life of vacuum-packaged raw beef.