• Journal of Microbiology and Biotechnology
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• v.14 no.5
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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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• v.46 no.4
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• pp.291-298
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• 2003

A bacterial strain producing a high level of an extracellular phytase was isolated from livestock waste water, identified as a strain of Pseudomonas fragi and designated as Pseudomonas fragi Y9451. Under the phytase production medium, the activity of phytase reached the highest level after 120 hours of incubation. On the effect of carbon sources on the phytase production, the most favorable carbon source for phytase production was fructose. As for the effect of nitrogen sources, high levels of phytase activity were detected in the medium containing nutrient broth as the nitrogen source. Free $PO_4^{3-}$ inhibited phytase production with increasing concentration of $KE_2PO_4$ and phytate in the media. The addition of $CaCl_2$ and $MgSO_4$ also resulted in the inhibition of phytase production. To investigate the effect of aeration on the phytase production, different volumes of culture broth in Erlenmeyer flasks were incubated in rotary shaker at the speed of 200 rpm. As a result, a high level of phytase activity was detected at small volume of culture broth as compared to larger volume because of its more aerobic condition.

• Bulletin of the Korean Chemical Society
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• v.16 no.5
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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.

• Journal of Food Hygiene and Safety
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• v.14 no.4
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• pp.346-351
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• 1999

To develop a lactic starter to produce antimicrobial substance for inhibiting the growth of a variety of foodborne spoilage bacteria in fermented foods, we investigated the anti-bacterial effect of the antibacterial substance, produced by Lactobacillus amylovorus IMC-1, against foodborne spoilage strains, and its sensitivity on the treatment of proteolytic enzymes. L. amylovorus IMC-1, which was isolated from a traditional cheese in Inner Mongolia, produced a maximum amount of antibacterial substance in the skim milk medium after 72 h incubation at 37$^{\circ}C$, and further incubation resulted in the same activity. The substance obtained from gel filtration inhibited all strains used such as Bacillus subtilis IFO 3025, Staphylococcus aureus IAM 1011, Listeria monocytogenes VTU 206, Escherichia coli RB, and Pseudomonas fragi IFO 3458 at the concentration of 20 units/ml. This substance was found to show bactericidal action against B. subtilis, E. coli, and Ps. fragi, and bacteriostatic activity against both Staph. aureus and L. monocytogenes. The bactericidal action was due to cellular Iysis. The substance is not organic acid, hydrogen peroxide and proteinaceous compound.

• Korean Journal of Veterinary Service
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• v.44 no.3
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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.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1613-1618
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• 1999

Extracts of Citrus sudachi juice and peel were obtained by several organic solvents such as hexane, acetone, ethyl acetate and methanol. Their antimicrobial spectrum were determined against 14 strains of gram positive and 4 strains of gram negative bacteria by paper disk method and minimal inhibitory concentration (MIC) was also obtained. Antimicrobial activities of solvent extracts from Citrus sudachi juice showed stronger than those of solvent extracts from peel. Acetone extract from juice showed the strongest antimicrobial activity among extracts, but the hexane extract did not show antimicrobial activities on tested target strains. The MIC was differant among tested strains; i.e. 0.5% (v/v) to Bacillus subtilis, 1% to Pseudomonas fragi, 1.5% to Listeria monocytogenes and Escherichia coli O157:H7, 2% to Propionibacterium acnes and Salmonella typhimurium, and 2.5% to Staphylococcus aureus.

• Journal of Environmental Health Sciences
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• v.22 no.4
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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.

• Journal of Food Hygiene and Safety
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• v.35 no.2
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• pp.170-176
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• 2020

We investigated the pH, volatile basic nitrogen (VBN), microbial changes and dominant microbes in raw beef, wet-aging beef, and dry-aging beef after the meat had been stored in a refrigerator. The count of mesophilic bacteria was 3.3-3.9 log CFU/g in raw beef and dry-aging beef, and 5.4 log CFU/g in wet-aging meat. After 18 days of refrigeration, the mesophilic bacterial count in raw and aging beef increased to 6.1-6.4 log CFU/g. In wet-aging beef, the number of lactic acid bacteria increased from 4.5 log CFU/g to 6.0 log CFU/g at refrigeration temperature. However, lactic acid bacteria were not detected in dry aging beef. Major foodborne pathogens such as Salmonella spp, Listeria monocytogenes, and Escherichia coli (EHEC) were not detected. Based on the legal standard for mesophilic bacteria count, the estimated shelf-life of aged beef was less than 12 days and the average VBN was 15 mg%. The dominant microorganisms varied between the different types of meat. In raw meat, Staphylococcus saprophyticus was the dominant microorganism, and as the VBN increased, Carnobacterium divergens dominated. In wet-aging beef, Carnobacterium divergens dominated during the initial days of refrigeration after which the number of Lactobacillus sakei increased. Dermacoccus nishinomiyaensis was initially the dominant microbe in dry-aging beef, after which Pseudomonas fragi dominated. In addition to the role of specific bacteria in the early stage of decay, it is thought that microorganisms can be utilized for safe distribution and storage of matured meats by conducting research on changes in rot, fragrance analysis, and changes of ingredients in matured meats.

• Food Science of Animal Resources
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• v.39 no.2
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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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• v.31 no.4
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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.