• Journal of fish pathology
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• v.20 no.3
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• pp.249-255
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• 2007

We investigated the phenotypic characteristics by using API20E, APIZYM and determined minimum inhibitory concentrations (MICs) of 7 antibiotics in motile aeromonads isolated from freshwater fishes in Korea and Japan, and 4 American Type Culture Collection (ATCC) strains. All isolates (n=7) were identified as motile Aeromonas species according to API20E test. Lysine decarboxylase activity and acid production from 4 different carbohydrates including mannitol, rhamnose, amygdalin and arabinose were observed in various strains. In enzymatic activities by APIZYM, all isolates showed negative reactions in valine and cystine arylamidases, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase and α-fucosidase. Although the intensities of each enzymatic activity were diverse in alkaline phosphatase, esterase-lipase, leucine arylamidase, β-galactosidase and N-acetyl-β-glucosaminidase, all isolates showed positive reactions. All isolates were resistant to ampicillin sodium (MIC>100㎍/ml), but sensitive to chloramphenicol (MIC≤1.6㎍/ml). However, recently isolated strains (AC9804, AC0202 and GMA0361) were commonly resistant to tetracycline (MIC=50㎍/ml). Furthermore, AC9804 was resistant to oxolinic acid (MIC=12.5㎍/ml). GMA0361 was resistant to kanamycin sulfate (MIC>100㎍/ml) and streptomycin sulfate (MIC>100 ㎍/ml).

• Korean Journal of Food Science and Technology
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• v.26 no.1
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• pp.81-87
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• 1994

Two different trypsin inhibitors, TRTI-1 and TRTI-2, were purified to near homogenity from Trichosanthes kirilowii root, by $0{\sim}90%$ saturated ammonium sulfate salting out, DEAE-Sephacel ion exchange chromatography, Sephadex G-50 gel filtration chromatography and trypsin-affinity chromatography. The molecular weight of TRTI-1 and TRTI-2 were estimated to be about 5,000 Da and 24,000 Da, respectively, by gel filtration and must be monomer and homodimer since they contain 4,000 Da and 10,000 Da each on SDS-polyacrylamide gel electrophoresis. TRTI-1 was stable after heating for at least 2 hr at $100^{\circ}C$ but TRTI-2 was completely inactivated after heating for 10 min at $90^{\circ}C$. When Bz-dl-Arg-pNA was used as a substrate of TPCK-treated trypsin, half-maximal inhibitions of TRTI-1 and TRTI-2 were observed at $0.8\;{\mu}M$ and 6\;${\mu}M$, repectively. Both TRTI-1 and TRTI-2 inhibited the hydrolysis of trypsin competitively and Km values were $0.97\;{\mu}M$ and $0.63\;{\mu}M$, respectively. Both TRTI-1 and TRTI-2 specifically inhibited trypsin but they did not inhibit other proteases tested, chymotrypsin, papain, elastase, collagenase, thermolysin, Nagarase, pepsin, and thrombin.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.38-45
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• 2013

In this study, euryhaline marine microorganism, Bacillus sp. strain EBW4 isolated from polychaete (Perinereis aibuhitensis) of Suncheon Bay was physiologically, biochemically and genetically characterized. Based on 16S rRNA sequence, EBW14 was found to share 98.25% similarity with Bacillus hemicentroti $JSM076093^T$, 97.96% similarity with Bacillus hwajinponensis SW-$72^T$ and 96.28% similarity with B. algicoa $KMM3737^T$, respectively. The temperature range for the growth of strain EBW4 was $4-40^{\circ}C$, NaCl concentration range 0-17% and pH range pH 5-9, revealing that EBW4 was euryhaline bacterium. Major fatty acids in strain EBW4 were composed of anteiso $C_{15:0}$ (48.2%), iso $C_{16:0}$ (12.1%), anteiso $C_{17:0}$ (11.6%) and iso $C_{14:0}$ (9.4%). EBW4 was found to have DNase, amylase, protease and lipase for the degradation of macromolecules such as DNA, carbohydrates, proteins, lipids, etc. The enzyme activities of alkaline phosphatase, esterase (C4), leucine arylamidase and ${\alpha}$-chymotrypsin were also found in strain EBW4. Analysis of the biodegradation ability of EBW4 for organic hydrocarbons under different salinity conditions using synthetic water waste revealed that EBW4 exhibited the ability to degrade organic hydrocarbons very quickly, suggesting strain EBW4 may be a good candidate for the application to various industries.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.5
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• pp.417-425
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• 2009

The potential utility of fish scales to the functional food industry has been investigated due to its antioxidant and antihypertensive characteristics. In this study, we report on the reactive oxygen species (ROS) scavenging and angiotensin I converting enzyme (ACE) inhibitory activities of gelatin hydrolysates processed from Branchiostegus japonicus scales, which are also high in protein content (about 46.1%). We prepared the enzymatic gelatin hydrolysates with four proteases (${\alpha}$-chymotrypsin, Alcalase, Neutrase and trypsin) from B. japonicus scale gelatin, which was prepared according to different reaction times, substrate/enzyme ratios and substrate concentrations. The enzymatic hydrolytic degrees of the gelatin increased time-dependently up to 6 hrs, while the Alcalase gelatin hydrolysates showed the highest hydrolysis degrees compared to the others. Furthermore, gelatin hydrolysates of Neutrase and ${\alpha}$-chymotrypsin showed the highest DPPH radical and $H_2O_2$ scavenging activities ($IC_{50}$ value; 9.18 mg/mL and 9.74 mg/mL), respectively. However, the activities were not significant (P<0.05). We also observed that the four gelatin hydrolysates significantly increased ACE inhibitory activities from approximately 20% to 60% (P<0.05), Among them, the Alcalase gelatin hydrolysates showed the higher ACE inhibitory activity ($IC_{50}$ value; 0.73 mg/mL) compared to the others. These results suggest that the enzymatic gelatin hydrolysates prepared from B. japonicus scales may possess a potentially useful function as an ACE inhibitory agent. As such, the utility of B. japonicus scales should be given due consideration for application in the functional food industry.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.5
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• pp.721-726
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• 2012

Lactic acid bacteria (LAB) are able to secrete antimicrobial peptides called bacteriocins, which inhibit other bacteria such as pathogenic microorganisms. Therefore, bacteriocin-producing starters can be used as natural biopreservatives for various foods. The objective of this study was to screen and characterize bacteriocin-producing LAB from Kimchi and to investigate their applicability as a starter in Kimchi fermentation. To screen bacteriocin-producing LAB, gram-positive and gram-negative bacteria were used as indicators. To measure the antimicrobial activities of isolates, agar well diffusion assay method was used. According to the results, bacteriocin produced by $Lb.$ $sakei$ B16 showed antimicrobial activity against $Listeria$ $monocytogenes$ ATCC 19115, $Escherichia$ $coli$ KCTC 1467, and$Lactobacillus$ $plantarum$ KTCT 3104. Furthermore, bacteriocin was very stable after treatment with high temperature and high and low pH, but its effects were inhibited by treatment with proteolytic enzymes such as trypsin, proteinase K, and ${\alpha}$-chymotrypsin, revealing their bacteriocin-like protein- based structure. These results suggest that $Lb.$ $sakei$ B16 and its bacteriocin are good candidates as a functional probiotic and natural biopreservative, respectively, in fermented foods.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Development and Reproduction
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• v.2 no.1
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• pp.29-37
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• 1998

To investigate the role of oviductal environment in early mammalian development, we examined the effects of bovine oviductal fluid (bOF) on the development of mouse 2-cell embryos in vitro. All of the embryos cultured in medium containing 5% or more of bOF underwent degeneration after 48 hr, whereas only 5% of embryos cultured in the absence of bOF degenerated. When bOF was heated at 65 \circ C for 30 min and then added to the culture medium, the embryotoxic effect of bOF was not removed at all such that none of the embryos remained alive after 48 hr. However, when bOF heated at 90 \circ C for 30 min was added to the culture, nearly most (95%) of embryos was alive. Similarly, pretreatment of bOF with 0.1% chymotrypsin for 1 hr or overnight following heating at 65 \circ C resulted in the development of 95.5% of mouse 2-cell embryos to early blastula after 48 hr culture in the presence of treated bOF. Interestingly addition of an anti-oxidant removed the evbryotoxic effect of bOF so that 91.0% of 2-cell embryos developed to morulae or blastulae in the presence of both 5% bOF and 10 mM of glutathione (GSH) after 48 hr culture. Neither oxidized form of GSH (GSSG) nor other antioxidants, however, could support the embryonic development in the presence of bOF. From these results, it is suggested that bOF contains a protein-like factor(s) which becomes embryotoxic by exposing in vitro, probably via oxidation reaction.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.6
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• pp.875-881
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• 1998

To develop functional food material with angiotensin converting enzyme (ACE) inhibitory peptides, muscle protein of anchovy, Engraulis japonica was hydrolyzed during 48 hrs by digestive pretenses such as pepsin, trypsin, $\alpha$-chymotrypsin, and commercial proteases such as papain, bromelain, complex enzyme, Elavourzyme, Novozym, Neutrase, Protamex and Alcalase. The only $50\%$ ethanol soluble hydrolysates were tested for inhibitory activity against ACE and yield of $50\%$ ethanol soluble peptide-nitrogen ($ESPN_{50}$). ACE inhibition effects and yield of $ESPN_{50}$ occurred as hydrolysis time increased to 8 hrs, Among those pretenses tested, hydrolysates by Alcalase and $\alpha$-chymohypsin had greater ACE inhibitory activity (80 and $74\%$, reipectively) with eletated levels of $ESPN_{50}$ (48 and 58 mg/ml, respectively), while Protamex hydrolysates had greater ACE inhibitory activities ($73\%$) with reduced levels of $ESPN_{50}$ (7.2mg/ml) than others. Amino acid compositions of $50\%$ ethanol solubles obtained from those hydrolysates were rich in glutamic acid, aspartic acid, cysteine and leucine.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.5
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• pp.842-849
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• 1997

This study was designed to investigate the antioxidative activity of enzymatic hydrolysates prepared from defatted anchor muscle by various pretenses. In these hydrolysates, the hydrolysates derived from pepsin and Protamex showed the strongest antioxidative activity. Enzymatic hydrolysates also showed the synergistic effects on antioxidative activity of $\alpha-tocopherol$, and almost no change in butylated hydroxytoluene (BHT). Peroxidation of metal ions $(Fe^{3+},\;Cu^{2+})$ was inhibited by enzymatic hydrolysates. Ten fractions (P-1 to P-10) were fractionated from the peptic hydrolysates by Amberlite IR-120 and Bio-gel P-2 column chromatography. The maximum antioxidative activity was observed in the traction P-2 (fraction No. $26\~31$). In amino acid composition of before and after hydrolysis of defatted anchovy muscle and the active fractions (P-2), contents of aspartic arid and glutamic acid were increased, but alanine, cysteine, tyrosine and phenylalanine were decreased.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.311-318
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• 2002

A new bacteriocin producing lactic acid bacteria having antagonistic activity against Lactobacillus plantarum, was isolated from Kimchi. It was identified as Leuconostoc mesenteroides, and designated as Leuconostoc mesenteroides B7. The bacteriocin from Leuconostoc mesenteroides B7 named as bacteriocin B7 was stable in the pH range $2.5{\sim}9.5$. Bacteriocin B7 was active over a wide temperature range from $4^{\circ}C$ to $120^{\circ}C$. It was inactivated by proteinase K, trypsin, ${\alpha}-chymotrypsin$, and protease treatments indicating its proteinous nature. Tricine-SDS-PAGE of the purified bacteriocin B7 showed the presence of a single band, having a molecular mass of about 3,500 dalton. Mixed culture of the producer and the indicator, Lb. plantarum KFRI 464 or Lb. delbruekii KFRI 347, increased production of bacteriocin B7. This result suggested the presence of bacteriocin inducing factor in the indicator strain. The inducing factor was localized in cell debris and intracellular faction of the indicator cell, Lb. plantarum KFRI 464. Treatment of the inducing factor with proteinase K destroyed inducing activity. This result strongly suggested that the inducing factor is a protein.