M.R. Marshall

HPLC Method for Analysis of Free Amino Acids in Fish Using o-Phthaldialdehyde Precolumn Derivatization†

Precolumn derivatization applying o-phthaldialdehyde (OPA) was used to analyze free lysine, histidine, and ornithine, precursors of the respective biogenic amines cadaverine, histamine, and putrescine, which are considered indicators of fish quality and safety. This method uses 75% methanol to eliminate the use of strong acids as the extraction solution. Each analysis took 35 min, was reproducible, and allowed separation of primary amino acids in fish samples. A binary solvent delivery system coupled with a fluorescence detector and an Ultrasphere ODS column were utilized for HPLC separation. Linearity of the calibration curves was very good (r(2) = 0.99) for the amino acids of interest. Minimum concentrations of detection were 40 pmol/mL for histidine and lysine and 70 pmol/mL for ornithine. Average recoveries were 72% for lysine, 93% for histidine, and 98% for ornithine. This method used solvent gradient elution to study the levels of these analytes in mahi-mahi, bigeye tuna, and flounder.

Application of immunoassay in the food industry

Immunoassay techniques using the highly specific and sensitive nature of immunological reactions have been developed and applied in the food industry for detecting the naturally occurring constituents, antibiotics, pesticide residues, microorganisms, and fragments of microbial constituents related to food analysis, food production, food processing, and food safety. Both polyclonal and monoclonal antibodies are employed for the development of the various immunoassay systems, including enzyme-linked immunoassay (ELISA) and radioimmunoassay (RIA). Immunoassay techniques provide complementary and/or alternate approaches in reducing the use of costly, sophisticated equipment and analysis time, but still maintaining reliability and improved sensitivity. Immunoassay techniques in their most simple forms provide excellent screening tools to detect adulteration and contaminations qualitatively. The application of immunoassay techniques contributes tremendously to the quality control and safety of our food supply.

Isolation, purification and characterization of a trypsin from the pyloric ceca of mullet (Mugil cephalus)

1. n1. A protease classified as trypsin on the basis of its mol. wt of 24,000, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and the inhibitory effects of methyl sulfonyl fluoride (PMSF), SBTI, aprotinin and benzamidine on its activity was isolated from the pyloric ceca of mullet. n n2. n2. The enzyme exhibited optimal activity at pH 8 and a temperature of 55°C and was stable in the pH range of 7.5–9.0. The Arrhenius energy of activation (Ea) for BAPA hydrolysis by this enzyme was 7.4 kcal/mol. n n3. n3. The enzyme showed greater affinity for TAME (K′m = 0.19 mM) as a substrate than for BAPA (K′m = 0.49 mM), and preferential hydrolysis of ester bonds (Vmax = 2640 TAME units/μmmol enzyme) than amide bonds (Vmax = 400 BAPA units/μmol enzyme).

Astaxanthin Extraction from Crawfish Shells by Supercritical CO2 with Ethanol as Cosolvent

Abstract Astaxanthin is an important pigment in fish and shellfish aquaculture and increases the market value of fish such as salmon and trout. Commercially synthesized astaxanthin costs about

Comparison of two treatment methods on the purification of shrimp polyphenol oxidase

1,000/kg. Carotenoids have been extracted from crustacean wastes with organic solvents, but in many of the methods the pigments are degraded. Astaxanthin extraction yield from crawfish tail shells by supercritical CO2 was optimized for temperature, pressure, and moisture. The cosolvent ethanol (10% w) increased yield significantly. Temperature, pressure, and moisture content were other important independent variables. These were studied using an incomplete 33factorial (Box-Behnken) design. The equation of the model fitted to the data was extracted astaxanthin (mg/kg dry shell) = -169.3 + 5.36 T + 0.061 P + 7.32 M -0.0003 T P -0.10 T M + 0.0003 M P -0.02 T2 -0.000006 P2 -0.033M2, where T = temperature; P = pressure (psi); M = moisture. The maxima for moisture, pressure, and temperature were 13, 24 MPa, and 75, respectively.

Purification and characterization of a trypsin-like enzyme from the hepatopancreas of crayfish (Procambarus clarkii)

The effects of different stirring time and two treatment methods (salt and organic solvent) on the recovery of shrimp polyphenol oxidase (PPO) were investigated. Stirring for 30 min yielded maximal PPO recovery. With respect to PPO specific activity, yield and purification fold enhancement, the use of butanol treatment followed by Phenyl Sepharose CL-4B chromatography was shown to be better than ammonium sulphate fractionation and then Phenyl Sepharose chromatography. White shrimp PPO was more susceptible than pink shrimp PPO to inactivation during purification.

Phosphate Pretreatment on Smoke Adsorption of Cold Smoked Mullet (Mugil cephalus)

Abstract 1. 1. A trypsin-like enzyme was purified from the hepatopancreas of Louisiana swamp crayfish, Procambarus clarkii , by a combination of hydrophobic interaction chromatography, molecular sieving and ion-exchange chromatography. 2. 2. The enzyme had a molecular weight of 33.6 kDa on SDS-PAGE and an isoelectric point of 3.0. 3. 3. The enzyme exhibited optimal activity at pH 8.0 and an optimal temperature of 55°C. It was stable in the pH range of 7.5–9.0, but unstable above 55°C. 4. 4. The tryptic activity was inhibited by phenylmethyl sulfonyl fluoride and other well-established trypsin inhibitors. 5. 5. Immunological study showed that crayfish trypsin-like enzyme shared structural components with bovine trypsin.

Phenoloxidase forms of the Florida spiny lobster : immunological and spectropolarimetric characterization

Abstract Fresh mullet (Mugil cephalus) fillets dipped in brine for 30 min were cold smoked at 30°C, relative humidity 60-80%. Fillets treated with 5 and 10% sodium tripolyphosphate (STPP) or 5% NaCl had 1.20, 1.45, and 2.45% more moisture, respectively, than control. Treatment with 5 or 10% STPP both with 5% NaCl absorbed 1.25 and 0.95% more water, respectively; water loss occurred with fillets treated with 15% NaCl, 5 and 10% STPP plus 15% NaCl. Color (Hunter L value), reflecting smoke adsorption, was lightest for the control fillets, followed by the 5% salt treatment, and darkest (not significant at p > 0.05) for 5 and 10% STPP both with 5% salt. NaCl treated fillets had similar sensory saltiness as those treated with salt plus phosphate.

Enzyme Inactivation by Pressurized Carbon Dioxide

Abstract 1. 1. In vivo activation of cuticular phenoloxidase (PO) of Florida spiny lobster is apparently mediated by some mechanism other than tryptic activity. 2. 2. Endogenously activated (EAPO) and trypsin activated (TAPO) forms of Florida spiny lobster PO show differences in their secondary structure as evidenced by circular dichroism spectropolarimetry. 3. 3. EAPO and TAPO forms of Florida spiny lobster possess identical antigenic determinants.

Inhibition mechanism of kojic acid on polyphenol oxidase

Pectinesterase in orange juice, polyphenoloxidase in apple juice, and various polyphenoloxidases in model systems were used in the determination of the kinetics of inactivation of the enzymes by carbon dioxide treatment at different pressures, temperatures, pH values, and times. Controls were used for pressure, temperature and pH. Kinetics parameters are presented, effects of the treatment on the enzyme molecules examined, and implications to juice processing are discussed.