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IMMUNOLOGIC APPROACHES TO THE IDENTIFICATION OF FISH PROTEINS: TOWARD SPECIES-SPECIFIC “FIELD KITS” FOR STUDIES IN ECOLOGY AND SEAFOOD TECHNOLOGY D-R. Schult.z?, P.I. Arnold’, T.R. Capo2, C.B. Paris-Limouzy2, J-E. Serafy2, and W. J. Richards 3 1Department of Medicine University of Miami School of Medicine Miami, FL 33 10 1 2University of Miami Rosenstiel School of Marine & Atmospheric Science Miami, FL 33 149 and 3Southeast Fisheries Science Center National Oceanic & Atmospheric Administration Miami, FL 33 149, U.S.A. Introduction The use of immunological methods to investigate basic problems in oceanographic research and marine or freshwater ecology provides sensitive and specific tools with wide versatility and applicability (I, 11,12). Polyclonal and monoclonal antibody probes can be labeled with radioisotopes, fhrorochromes, enzymes, metals such as colloidal gold, and by biotinavidin technology for immtmoassays; the probes are detected microscopically, with automatic flow ~fl~metry devices, by electrophoretic methods, scintillation counters, and automated microplate readers (6). Although formerly used almost exclusively in clinical and basic medical research, increasing numbers of investigators are adapting these methods for studies in both applied and theoretical marine biology, and also seafood tech010gy. In this report we have used immunological techniques to investigate predator-prey relationships with recently released red drum fingerlings (Sciaenops ocellatus, Linnaeus), and to distinguish early life history specimens of specific species of the family Lutjanidae. The ~unological detection methods described here are also applicable for problems associated with identification of commercial seafood products and safety, and are valuable for preventing deliberate or unintentional substitution of inferior products for valuable fish species (4). ~mrnunolo~i methods to assess predation of hatchery-reared. juvenile red drum (S. ocellatus). During the last seven years, the University of Miami has been part of a cooperative stock enhancement program with Florida International University (Miami, FL), and the Florida Departmem of Environmental Protection. The purpose of the program has been to test the feasibility
of reestablishing and enhancing red drum populations in Biscayne Bay, Florida, because of the virtual disappearance of the species from coastal waters in this area. Since 1988, hundreds of thousands of hatchery-reared red drum fingerlings have been released at mainly two sites, Richenbacker Causeway (Hobie Beach) in north-central Biscayne Bay, and Matheson Hammock, located on the mangrove-lined western shore of central Biscayne Bay4. Our role in the red drum stock enhancement program has been to investigate predation of newly released fingerlings. The program appeared to be ideal for predation studies because after each release, the red drum fingerlings remained inshore in shallow aquatic vegetation close to the release site for at least 10 hours, facilitating the capture with a center-bag seine of a number of potential predators attracted to the site. Visual examination limited the identification of many of the specimens found in gut contents of the two major predators seined at both of the release sites, Sphyraena barracuda (Walbaum) (great barracuda) and StrongyZura notata (Poey) (redfin needlefish). Many of the ingested fish were partially proteolysed and could not be identified as to the species. Therefore, we have used immunological methods to develop reagents for assays to detect red drum in the gut contents of predators. Initially, a goat polyvalent antiserum was produced to soluble red drurn whole fish extract (anti-RDE serum) for use as the primary antiserum in Western blots. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with soluble extracts of 12 different species of fish found in Biscayne Bay (Table l), followed by Western blots with the anti- RDE serum. A red drum 80 kDa protein was selected that was not found in soluble extracts of the other fish, The protein was purified to homogeneity by sequential steps of solid ammonium sulfate fractionation and anionic and cationic exchange resins using the Fast Protein Liquid Chromatography system (FPLC) (11). The highly purified 80 kDa protein was a single chain polypeptide that reacted positively with periodic acid-Schiff reagent, indicating a glycoprotein. It was found in soluble extracts as two isoforms in small fish (ca. 1.5 cm.), but as a single protein from fish greater than 17 cm in length. The amino terminus of the 80 kDa protein was not blocked, and structural information is shown in Table 2. Further structural work for the purpose of developing assays of the protein (e.g., ELISA) was not carried out at this time. A goat antiserum was produced (anti-80 kDa gp) which was used as the primary antiserum in Western blots to test red drum fingerling extracts. Although both preimmune and immune goat sera reacted nonspecifically with extracts of many of the fish (e.g., silver jenny), adsorption of the antiserum with glutardehydeinsolubilized silver jenny extract resulted in an antiserum reactive only with the 80 kDa red drum protein and not with any proteins from the 12 species of fish and three invertebrate species selected for these studies (11). 4Rickenbacker Causeway (Hobie Beach): [25”44’50” (Lat.); 80°13’20” (Long.)]; Matheson Hammock: [25”‘44’15”(Lat.)J; 80°15’20” (Long.)].
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TABLE OF CONTENTS PAPERS & ABSTRACT
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Evaluation of On-board Handling Tec
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oduction of a Sardine Substitute Jo
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Effects of Temperature and Humidity
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Force. and the U.S. Coast Guard. Th
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for the program, showing that state
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12/18/92 such as diversion. The lat
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. Establish a formal database on th
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8. Standard reporting for seafood-b
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38. Education effort/materials on b
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egional business and industry may n
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PROJECT CODE: 95/PH/A10,13/P2 PROJE
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PROJECT CODE: 95/PH/A28/P1 PROJECT
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PROJECT CODE: 95/PH/A29/P3 PROJECT
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t PROJECT CODE: 95/PH/A37/P2 PROJEC
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investigate the problem. The ISSC w
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strategic plan for conference actio
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(4) advisory committee, and all con
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IRRADIATION TO ENSURE HYGIENIC QUAL
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food. Often, food manufacturers hav
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BACTERIA D-VALUE (kGy) E. coli 0.15
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ECONOMICS OF IRRADIATION OF FISH AN
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B. Market Testing of Irradiated Foo
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The Agreement on the Application of
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Table 1. Optimum radiation dose lev
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Loaharanu, P. 1973. Gamma irradiati
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EFFECTS OF LOW-DOSE GAMMA IRRADIATI
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diving bell and lowered into the wa
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12 1 Survival Curve for Standard Pl
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6 D value = 0.05 kGy ‘A. 0 / I I
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Dvalue=O.S9kGy 3.8 a’, 3.6 . 3.4
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8 0 . 0.1 0.2 0.3 0.4 0.S 0.6 rlrah
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Conclusion In this study, low dose
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processed and packaged product. A m
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Table l.Procedures schematic for ef
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(hydrogen peroxide) produced from t
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Johns, H.E. and Cunningham, J.R. 19
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k TBF#hoursCtime between split dose
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AUTOMATED OHMIC THAWING OF SHRIMP B
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A computer program was developed in
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RESULTS AND DISCUSSION The computer
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78 Quadrant 1 0 20 40 60 80 100 Tim
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FUTURE WORK The ohmic system perfor
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Using Edible Film to Improve Smoked
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LOG CFU / Sq cm 0 3 6 9 12, 15 18 2
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FIG-l.5 AE SMOKE LOG CFU / Sq cm FI
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FIG-2.1 PSYCHRQT SMOKE LOG CFU / Sq
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For samples stored at l0°C, no sig
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(3) 10 oz copolymer polyethylene ca
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Total Volatile Base Components of P
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Log Anaerobic Plate Counts of Packa
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0, 600 500 - :: 7 400 - 2 z 300 - k
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Log Aerobic Plate Counts of Package
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The variables that correlated well
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Woyewoda, A.D., S.J. Shaw, P.J. Ke,
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used to evaluate both heating and c
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minimize the chances for product re
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Part II Evaluation of the cooling p
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Table 1. Laboratory analyses on con
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REFERENCES APHA, 1984. Compendium o
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market and economic studies focusin
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The foreign demand for U.S. exports
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The survey of California-based seaf
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The Gulf butterfish (and harvestfis
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during the same period. The butterf
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48,693 mt/yr in 198690. The primary
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U.S. Landings consistently increase
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Christmas, J., D. Etzold, T. McIlwa
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Perkins, G. 1977. Potential for exp
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EFFECT OF WASHING WATER pH ON COLOR
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Figure 1, Whole Fish I Dehead (M-07
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138 Hematin concentration, expresse
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Table 1. Mean carotenoids, hematin,
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Table 2. Treatments Mean moisture v
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side). These adjustments are essent
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Siggia S. 1963. Methods for trace q
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work on combining acetates and phos
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untreated fresh fillets and treated
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Fig. 3. 10 0 m control tJ 0.1% MKP-
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negative bacteria such as Pseudomon
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concentrations up to 1% could be us
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Fey, M.S., and Regenstein, J.M. 198
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Schaack, M.M., and Marth E.H. 1988.
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MATERIALS AND METHODS Materials Liv
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. , FIGURE 1. ANAEROBIC PLATE COUNT
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il . --.-_._ 1+_- /: _- : _ _ _ _ _
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, I . ‘ r , r , c I, , -1 ’ I .
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I I ’ , ’ . I : , I ’ I 1 * :
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Reed, R. J., G. R. Ammerman, and T.
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Antioxidants can be added to increa
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Table 1. Effect of wash treatment o
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Table 2. Effect of antioxidant trea
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(1993) for whole dressed catfish. C
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Anonymous. 1992. Processing up 11%
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Sin&_&x, R. O. and Yu, T. C. 1958.
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1. Materials MATERIALS AND METHODS
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RESULTS AND DISCUSSION Aroma profil
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.z%M 5 =w B 14 29 39 4142 53 64 25
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.s CM 8 C s-t B W S' a .g CM $ C .-
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Marshall, G.A. Moody, M.W., Hackney
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AN OVERVIEW OF THE NMFS PRODUCT QUA
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STORAGE CHARACTERISTICS OF FROZEN B
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OPTIMUM CONDITIONS FOR THE PREPARAT
Page 212 and 213: 3 BUILDING STRATEGIC PARTNERSHIPS F
Page 214 and 215: for aquaculture supporting developm
Page 216 and 217: problem solving of complex regional
Page 218 and 219: DESIGN OF AN AUTOMAT EVALUATION DEV
Page 220 and 221: sides of the shrimp were averaged.
Page 222 and 223: Ammonia Measurements Figure 2 shows
Page 224 and 225: REFERENCES Balaban, M. O., Yeralan,
Page 226 and 227: QUALITIES OF FRESH AND PREVIOUSLY F
Page 228 and 229: For the samples stored for six mont
Page 230 and 231: stored for six month, had lower bac
Page 232 and 233: Table 1. Psychrotrophic bacterial p
Page 234 and 235: Table 7. Juiciness of refrigerated
Page 236 and 237: Table 12. Cohesiveness of baked ref
Page 238 and 239: Table 16. Shear force and centrifti
Page 240 and 241: SAS Institute Inc. 1987. “SAS/STA
Page 242 and 243: with a color chart. In this paper,
Page 244 and 245: TABLE 1 Comparison of Histamine Tes
Page 246 and 247: 11, Chassande, O., Renard, S., Barb
Page 248 and 249: FIGURE 1 Time Course for Histamine
Page 250 and 251: BACKGROUND A Rapid, Easily Used Tes
Page 252 and 253: We acknowledge with thanks the inte
Page 254 and 255: AOAC Add 1 ml Extract to 8 cm Prepa
Page 256 and 257: 248 . Fig. 4 pnitroaniline in HCl N
Page 258 and 259: 1 O .’ REFLECTANCE (arbitrary uni
Page 260 and 261: 552 650 600 Salt Effects in the Fig
Page 264 and 265: Table 1. Fish collected in Biscayne
Page 266 and 267: Based on unpublished data comparing
Page 268 and 269: 2. 3. 4. Hartmann, J.X., Poyer, J.C
Page 270 and 271: irradiated foods look like. After t
Page 272 and 273: silver carp Wpophthalmichthys molit
Page 274 and 275: ,$mory Analysis. Sensory data were
Page 276 and 277: Table 3. Percentage of panelists wh
Page 278 and 279: Table 5. Percent response of paneli
Page 280 and 281: McNulty, T, 1996. Personal communic
Page 282 and 283: in a stainless steel tank (SOL) wit
Page 284 and 285: Mineral Content Five macro-elements
Page 286 and 287: TBble 1 _ Yield of surimi made from
Page 288 and 289: Table 4 - Proximate composition of
Page 290 and 291: goatfish lizardfish, ponyfish, ther
Page 292 and 293: Table 7 - Amino acid composition of
Page 294 and 295: Gel Strength For surimi gels, stres
Page 296 and 297: 288 &f&odan, Y., Toyohara, H., and
Page 298 and 299: CONSUMER SURVEY OF POND RAISED CATF
Page 300 and 301: Accept&i&y scores of male and femal
Page 303 and 304: -I- -T-
Page 305 and 306: 100 90 80 70 60 50 40 30 20 10 0 Fi
Page 307 and 308: + 5:
Page 309 and 310: This study evaluated the feasibilit
Page 311 and 312: 1000000 100000 10000 9 1000 > 1 . T
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100000 PIs 10000 E 1000 z 8 100 UJ
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100000 10000 P 12 > 1000 10 1 � T
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THE EFFECT OF IONIZING Kvulnificus
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io Vulnificus: Past, Present, and F
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conce op on Vibrio vulnificus in 19
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LEVEL 4 L WA TEMPERATURE** TIME TO
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isregard include the education of o
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USE OF “COOL PASTEURIZATION” TO
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Survival rate of V: vuZnificus in o
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REFERENCES BAM. 1992. Bacteriologic
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0 To promote the cooperation and a
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SAFETY CONCERN IN THE USE OF MODIFI
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CONCLUSION According to some resear
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32 MATERIAL AND METHODS Product Pre
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FLOWCHART DIAGRAM FOR THE CANNING P
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Americans consume almost four pound
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pursuing such agreements and expect
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ABSTRACTS PROTECTION ACTIVPTIESOFTF
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Mildred Chaparro Food Science and T
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GENICALLY AND BLAST FROZEN PACKAGIN
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S TO ENSURE SEAFOOD SAFETY AND SEAF
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XYGEN-DE FRJZE ICALS AND LIPID PERO
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IIOTOXINS: PREVENTION, CONTROL SEAF
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ASPECTS OF CIGUATERA FIS POISONING
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BRETTANBMYCES CL-4 USSENII AS A CON
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USE OF ANTI ANTS IN FRESH AND FROZE
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INTERNATIONAL ASPECTS FOR HACCP E.
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