In this study, we investigated antimicrobial peptide from the acidified muscle extract of Mytilus coruscus, which mostly inhabits China, Japan, and Korea, to develop a natural product-derived antibiotics substitution in terms of its abuse and restriction. Antimicrobial peptide was purified by $C_{18}$ reversed-phase high-performance liquid chromatography and was detected as having a molecular mass of 6,701 Da by MALDI-TOF/MS. The N-terminal amino acid sequence of the purified peak was obtained from edman degradation, and 20 identified residues shown 100% identity with the N-terminus region of sperm-specific protein and protamine-like PL-II/PL-IV precursor of Mytilus californianus. We also identified 60 open-reading frame (ORF) encoding amino acids with 183 bp of purified peptide based on the obtained amino acid residues. The amino acid sequence of ORF showed 100% and the nucleotide sequence revealed 97.2% identity with the protamine-like PL-II/PL-IV precursor of Mytilus californianus. Synthesized antimicrobial peptide showed antimicrobial activity against gram-positive bacteria, including Bacillus cereus (minimal effective concentration [MEC], $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), gram-negative bacteria including Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) and fungi, Candida albicans (MEC, $56.3{\mu}g/ml$). In addition, synthesized peptide showed stable activities under heat and salt conditions against gram-positive and gram-negative bacteria, but was inhibited by salt against only C. albicans. With these results, isolated peptide from M. coruscus could be an alternative agent to antibiotics for defending against pathogenic microorganisms, and helpful information to understand the innate immune system of marine invertebrates.
Song, Ki Cheol;Lee, Ka-Jeong;Yu, Hong-Sik;Mok, Jong-Soo;Kim, Ji Hoe;Lim, Keun-Sik;Lee, Mi-Ae;Kim, Mee-Hye
Korean Journal of Food Science and Technology
/
v.45
no.2
/
pp.241-247
/
2013
AOAC Mouse Bioassay Analysis (MBA) has been the gold standard for the analysis of paralytic shellfish poisoning toxin (PSP toxin) for more than 50 years. However, this method has inaccurate limit of quantification and cannot be used to determine toxic profiles. An HPLC method (PCOX) was optimized for Korean shellfish to establish an alternative or supplementary method for PSP analysis and was intended to be used for the official monitoring and regulation of food. The recovery rate of the PCOX method was 83.5-112.1% and the limit of quantification for total toxin was about $8.6{\mu}g$/100 g. A long-term comparison study showed a good correlation of the PCOX results with the AOAC MBA results: the correlation factors were 0.9534 and 0.9109 for oyster and mussel matrices, respectively. The PCOX method may be used as an alternative or supplementary method for AOAC MBA to monitor the occurrence of PSP and to analyze PSP toxin profile in oysters and mussels.
This study was aimed to classify the intertidal macrozoobenthic community status after 2 years of Sea Prince oil spill, and oil spill effects along oil spreading track from heavily impacted to unaffected reference site. Field sampling was initiated in late February and continued through November 1998 seasonally, after 2.5 years of oil spill. 7 rocky sampling sites were selected among coastal regions coated and/or affected by the Sea Prince spilled oil. Identified species was 158 species, 65 family, 24 order, 9 class, 5 phylum. Mollusca was the dominant faunal group comprising 100 species (63.3%), and followed by 38 species of Crustacea (24.1%), 12 species of Echinodermata (7.6%), 5 species of Porifera (3.2%), and 3 species of Cnidaria (1.9%). On Dugpo of Sori Island, the fewest species was collected from 28 species to 35 species seasonally among sampling stations. But far away Dugpo toward Gamak Bay, the number of species increased, collecting the maximum on Sohwoenggan Island. At the wreck site of Sori Island, especially the species number of attached animals such as poriferans and anthozoans was very low compared to another site. The density and biomass on the higher tidal zone increased toward the low affected sites, but biomass on middle tidal zone decreased. The invertebrate biomass of study area was dependent on the sessile animals. The major dominant species were small-sized barnacles, Chthmallus challengeri, periwinkles, Littorina brevicula, mussels, Septifer virgatus, and so on. The biomass of C. challengeri and L. brevicula on the higher tidal zone was highest in the wreck site of Sori Island and decreased further and further. However, mussels on the middle tidal zone showed the inverse trends because of the larger individual size of mussel inhabited in Sori Island than those of another sites. As a result of community analysis, the effect of oil spill was not found distinctly. Several ecological indices and cluster analysis did not show the meaningful variation with oil track despite of the conspicuous differences among tidal heights. These indicate that the macrozoobenthic community level of oil spreaded zone recovered in some degree after the Sea Prince oil spill accident, but population or individual levels of dominant sessile animals took more recovery times.
In 1987, the level of heavy metals were determined ina total of 200 samples of 9 species of shellfish of Korea. The samples were collected at the fish. markets by 10 Public Institute of Health. The samples were whelk (Buccinum striatiBBimum), oyster (Crassostrea gigas), ark shell(Tegillarca granesa), shartnecked clam (Venerupis semidecussta), hard clam (Meretrix lusoria), top shell (Turbo cornutus), abalone (Haliotis gigantea), ark shell (Scapharea broughtonii), sea-mussel (Mytilus conuscus gould), respectively. The levels of total mercury, lead, cadmium, arsenic, copper, zinc and manganese were determined. The total mercury levels were determined by mercury analyzer using the combustion gold amalgamation method. The arsenic level were determined by spectrophotometry using colorimetric sil ver diethyldithiocarbamate method after dry ash dige8tion of the samples with magnesium oxide and magnesium nitrate. The levels of other metals were determined by inductively coupled pluma spectrophotometry after wet digestion of the samples with nitric acid and su1furic acid. The results were summerized as follows; 1. The overallranges and mean(ppm) were; Hg, ND-O.221 (0.036); Pb, 0.05-1.51 (0.37); Cd, 0.02-1.86 (0.61); As, 0.5-3.97 (1.22); Cu, 0.14-54.16 (4.93); Zn, 7.40-207.17 (30.09); Mn, 0.13-s.72 (3.40). 2. The levels of all 6 metals were found to be below the maximum permissible Iimits set by the Japan lor mercury, the Netherland for lead the Hong Kong for cadmium. The Finland for Arsenic no statutory Iimits for Zn and Mn in shellfish in any countries. 3. The results show that all the 9 species of shellfish studied, none have accumulated levels dangerous enough to pose a health problem.roblem.
Kim, Yong-Uk;Myoung, Jung-Goo;Han, Kyeong-Ho;Kang, Chung-Bae
Korean Journal of Ichthyology
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v.4
no.2
/
pp.44-54
/
1992
The blenniid fish, Pictiblennius yatabei(Jordan et Snyder) is ditributed in the central and southern Japan and southern Korea. The fish grows to a maximum size of 9cm in total length, and it seems to be matured in two years. Sex dimorphisms of the fish appear in the crest and the anal fin. Five egg masses of the fish were collected in Tongyoung Bay near Kyeong Nam on June 16 and 20, 1991. The eggs were laid on inner surface of empty shells of the oyster, Crassotrea gigas and blue mussel, Mytilus edulis. Each egg mass was guarded by the male parent. Numbers of eggs in each of masses were as about 450~1000.The eggs were spherical but somewhat oval in shape and ranged from 0.72 to 0.80mm in longer axis and from 0.55 to 0.65mm in shorter axis. Each egg was provided with an adhesive pedestal. Hatching took place in 105 hours after formation of embyo at the water temperature varying from 20.0 to $21.4^{\circ}C$. Melanophores appearing on the yolk remarkably changed in form during the embryonic development.The newly hatched larvae, having 32~34 somites, were from 2.71 to 3.35mm in total length. The larvae absorbed the yolk material and oil globule completely in 7 days after hatching and became postlarvae. Nine days after hatching, the larvae averaged 5.35mm in TL and the caudal notochord flex at $45^{\circ}$ Total lengths of the larvae reached 6.00 and 6.58mm in 11 and 13 days after the hatching, respectively.
Inter- and intra-specific differences in removal activities, filtering rates (FR) and production of feces-and pseudo-feces (PF) between a native freshwater bivalve in Korea, Anodonta woodiana Lea and Unio douglasiae Griffith et Pidgeon, were compared using a continuous removal of organic matters (CROM) system. The CROM system comprised five steps; input of polluted water, control of water flow, mussel treatment, analysis of water quality and discharge of clean water. The study was designed to compare the removal activity of organic matters between A. woodiana and U. douglasiae, and the intra-specific differences between density and length in A. woordiana. Results clearly indicate that two kinds of mussels had obvious removal activities of seston in the eutrophic reservoir. First, if both are similar in shell length, there were no significant inter-specific differences in removal activity between A. woordiana and U. douglasiae (P>0.5), but FRs of U. douglasiae was relatively high due to low ash-fee dry weight. Second, if both are same in animal density, the smaller mussels (1$\sim$2 years old) showed a higher filtering rate and production of feces- and pseudo-feces and less release of ammonium than the larger mussels. Third, if both are same in biomass, FRs and PF of mussels were higher in the low-density tank than the high-density tank, While the Concentration of $NH_4$-N and $PO_4$-P released WRS similar to each other (P>0.5). Therefore, these results suggest that CROM system using a young bivalve A. woordiana can be applied to control the nuisance seston in eutrophic lake system, if a relevant species and density were selected. Additional pilot tests to optimize the age and density of domestic bivalves were needed for the generalization of CROM operation.
A study on field bioassay test using four species of commercially important shellfish was carried out to assess the effect of pollutants and determine the extent of marine environmental pollution of the Masan Bay from 9 to 15 August 1978. Water quality analysis and planktological examination of sea water were made during the experiment and the examination gave the following results. The water temperature was so high at 31.7$^{\circ}C$ in maximum and rather subject to change on weather condition of the land than on the effect of the water mass from outer bay. The range of DO,COD and SS at the stations were 0.3-7.08cc/l, 0.07-3.31ppm and 5.5-117ppm, respectively with the high values of COD and SS at the stations 7 and 1. The concentrations of the dissolved inorganic nitrogen in sea water, NH$\sub$4/-N, NO$\sub$2/-N, NO$\sub$3/-N, and PO$\sub$4/-P were 18.90-99.80, 2.48- 19.60, 13.00-39.00 and 1.04-14.0$\mu\textrm{g}$ at/l, respectively with decrease of their values in the outer part of the Bay. The high values mentioned above were caused by the sewage and industrial activities. The effects of organic waste are increased oxygen demand, nutrient concentration, turbidity and a higher input of pathogens, leading to structural changes in the marine ecosystems and to a considerable hazard to public health. The percentage composition of phytoplankton standing crop between diatom and dinoflagellate was characterized by making a difference between the two groups in respect of location: a decrease of diatom and a increase of dinoflagellate in numerical abundance toward inner part from outer part of the bay. Namely phytoplankton organisms were composed of 80% of diatom and 20% of dinoflagellate in outer bay, on the contrary, only 4% of diatom and 96% of dinoflagellate occupied by 94% of prorocentrum micans known as tolerant species to polluted reaas in the inner bay. On the occurrence and composition of zooplankton, there are two significant communities in the bay:one is characterized by the predominance of Oithona nana and the other by Favella sp They were composed of a range from 84% to 90% of the total organisms and monotonously constituted of themselves only at most inner station 3 even small numbers. From the results mentioned above, Oithona nana, Favella and prorocentrum micans recommed themselves as valuable indicators for judging the extent of the marine pollution.During the period of the biossays Mytilus edulis showed the highest mortality and Tapes japonica the lowest one between the four test species. The highest death rate by stations was found at most inner stations 3'and 4near Masan Free Export Zone with the most sensitive response and the lowest one occured at outer station 13 where no death specimen of oyster and arkshell was found during the whole test period.As for mussel,85 percent death rate appeared after 72 hours and 100percent rate after 120hours at station 4. It was found that the significant high mortality of the test shellfish mentioned above was caused by severe pollution with mainly organic pollutants from domestic sewage and industrial wastes from the results of too much higher concentrations of dissolved inorganic nitrogen especially ammonia-N,COD,SS and lack of dissolved oxygen,and furthermore occurrence and abundance composition of Prorcentrum,Favella and Oithona nana by stations, valuable indicator species of coastal pollution by orgnic and boilogical pollutants.
The purpose of this study was to determine the kinetics of paralytic shellfish poison (PSP) destruction at various temperature. The toxic digestive gland homogenate of blue mussel (Mytilus edulis), PSP crude toxin, gonyautoxin group and saxitoxin group were heated at temperature ranging from 90 to $120^{\circ}C$, and then the toxicities were measured in samples heated for various time intervals. The rate constant (k) of the toxic digestive gland homogenate, PSP crude toxin, gonyautoxin group and saxitoxin group were $3.28{\times}10^{-2},\;1.20{\times}10^{-2},\;5.88{\times}10^{-2}\;and\;2.58{\times}10^{-2}\;at\;120^{\circ}C$, respectively. The decimal reduction time (D-value) of the toxic digestive gland homogenate, PSP crude toxin, gonyautoxin group and saxitoxin group were 70, 192, 39 and 89 at $120^{\circ}C$, respectively. These results indicate that PSP crude toxin is most heat-stable of 4 types of PSP toxins and PSP toxin are more heat-stable than food poisoning bacteria and spores. The retorting condition to reduce PSP toxicity below quarantine limit ($80\;\mu\textrm{g}/100\;g$ in Korea and America, 4 MU/g in Japan) could be calculated by rate constant. For example, the digestive gland homogenate having a initial toxicity of $200\;\mu\textrm{g}/100\;g$ could have toxicity below quarantine limit when heated at $90^{\circ}C$ for 129 min., $100^{\circ}C$ for 82 min., $110^{\circ}C$ for 48 min. and $120^{\circ}C$ for 28 min. These results suggest that commercial retorting condition ($115^{\circ}C$ for 70 min) in Korea is enough to reduce toxicity below quarantine limit from initial toxicity of $200\;\mu\textrm{g}/100\;g$. From these results, the quarantine limit of PSP-infested shellfish for canning can be level up to raw score of $200\;\mu\textrm{g}/100\;g$.
This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.
Accuracy and precision of ID methods for different spike isotopes of 76Se, 77Se, and 78Se were compared for the analysis of Selenium using quadrupole ICP/MS equipped with Octopole reaction cell. From the analysis of Se inorganic standard solution, all of three spikes showed less than 1 % error and 1 % RSD for both short-term (a day) and long-term (several months) periods. They showed similar results with each other and 78Se showed was a bit better than 76Se and 77Se. However, different spikes showed different results when NIST SRM 1568a and SRM 2967 were analyzed because of the several interferences on the m/z measured and calculated. Interferences due to the generation of SeH from ORC was considered as well as As and Br in matrix. The results showed similar accuracy and precisions against SRM 1568a, which has a simple background matrix, for all three spikes and the recovery rate was about 80% with steadiness. The %RSD was a bit higher than inorganic standard (1.8 %, 8.6 %, and 6.3 % for 78Se, 76Se and 77Se, respectively) but low enough to conclude that this experiment is reliable. However, mussel tissue has a complex matrix showed inaccurate results in case of 78Se isotope spike (over 100 % RSD). 76Se and 77Se showd relatively good results of around 98.6 % and 104.2 % recovery rate. The errors were less than 5 % but the precision was a bit higher value of 15 % RSD. This clearly shows that Br interferences are so large that a simple mathematical calibration is not enough for a complex-matrixed sample. In conclusion, all three spikes show similar results when matrix is simple. However, 78Se should be avoided when large amount of Br exists in matrix. Either 76Se or 77Se would provide accurate results.
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