Development of a sandwich vertical flow immunogold assay for rapid detection of oxytetracycline residue in fish tissues.

A rapid multiplex silver enhanced 'sandwich vertical flow immunogold assay (SVIA)' based on disposable porous filter-membrane was developed for on-site detection of oxytetracycline (OTC) residues in fish tissues. Artificial antigens were synthesized to raise selective anti-OTC rabbit antiserum and highly specific monoclonal antibody (mAb). The assay consists of three layers. The first layer was composed of immobilized anti-OTC rabbit antiserum (capture antibody) onto the membrane, the second layer was the molecule of interest (OTC) and the third layer was refined with detector immunogold labeled mAb. The sensitivity of the silver enhanced SVIA was as low as 2 ng mL-1 (within 4 min) representing a 125-fold increase in sensitivity over the HRP labeled sandwich vertical flow assay. The reliability of developed technique was co-evaluated with HPLC and validated using 115 field fish samples from different regions of southern India. The results demonstrated that SVIA has the high potentiality to be established as a semi-quantitative routine on-site screening tool.

Angiotensin I-converting enzyme (ACE) inhibitory activity and structural properties of oven- and freeze-dried protein hydrolysate from fresh water fish (Cirrhinus mrigala).

The angiotensin I-converting enzyme (ACE) inhibitory activity and structural properties of oven-dried (OD-FPH) and freeze-dried (FD-FPH) protein hydrolysates derived from fresh water fish (Cirrhinus mrigala) muscle, using papain, were investigated. Amino acid profiles indicated a higher proportion of hydrophobic residues in OD-FPH and hydrophilic residues in FD-FPH samples. Fourier transform infrared (FT-IR) spectra revealed random coil structure in OD-FPH and ß-sheet in FD-FPH samples. The approximate molecular weight of peptides in OD-FPH and FD-FPH was in the range of 7030-339Da. The IC50 values for ACE inhibition by OD-FPH and FD-FPH samples were found to be 1.15 and 1.53mg of proteinml(-1), respectively. The ACE-inhibitory activity of OD-FPH was more stable (during sequential digestion, using pepsin and pancreatin) than that of FD-FPH sample. The study suggested that the ACE inhibitory activity of protein hydrolysate was not affected by oven-drying.

Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes.

Effect of ice storage on the functional properties of proteins from a few species of fresh water fish (Indian major carps) with special emphasis on gel forming ability.

In the present study the effect of ice storage on physico-chemical and functional properties of proteins from Indian major carps with special emphasis on gel forming ability have been assessed for a period of 22 days. The solubility profile of proteins in high ionic strength buffer and calcium adenosine triphosphatase (ATPase) enzyme activity reduced significantly (p < 0.05), while that of total volatile base nitrogen (TVB-N) increased significantly (p < 0.05) at the end of 22 days of ice storage. The major protein fraction showed association-dissociation-denaturation phenomenon during ice storage as revealed by gel filtration profile and viscosity measurements. The gel forming ability of three fish species both in fresh and during different periods of ice storage was assessed by measuring the gel strength of heat induced gel. Among the three species the gel strength of the gel obtained from Catla catla and Cirrhinus mrigala was higher (586 and 561 g.cm) than the gel obtained from Labeo rohita (395 g.cm) in fresh condition. The gel forming ability of three species was significantly affected (p < 0.05) during ice storage. The TVB-N values of fish meat as a function of ice storage was within the prescribed limit up to 17 days of the ice storage.

Visco-elastic and flow properties of gelatin from the bone of freshwater fish (Cirrhinus mrigala).

The average yield of gelatin from the bone of freshwater fish (Cirrhinus mrigala) was 6.13%. The fluorescence spectra revealed maximum emission at 303 nm indicating the exposure of chromophores to bulk solvent. The amino acid profile of gelatin revealed a higher proportion of glycine and imino acids. The bloom strength of gelled gelatin was 159.8 g. The average molecular weight of fish bone gelatin was 281 kDa as determined by gel filtration technique. The dynamic oscillatory test of gelatin solution as a function of time and temperature revealed gelling and melting temperatures of 8.0 °C and 17.0 °C, respectively. The flow behavior of gelatin solution as a function of concentrations and temperatures revealed non-Newtonian behavior with pseudo-plastic phenomenon. The Herschel-Bulkley and Casson models were found suitable to study the flow behavior. The emulsion capacity (EC) of gelatin was inversely proportional to its concentration.

Effect of ice storage on the functional properties of proteins from shark (Scoliodon laticaudus) meat.

Ice storage characteristics of Scoliodon laticaudus with reference to physicochemical and functional properties have been assessed. Total nitrogen content reduced to a value of 3.90 g/100 g from an initial value of 4.27 g/100 g of meat. Moisture content did not show much variation. The initial nonprotein nitrogen content which was 1.09% w/w of meat reduced to 0.98% during ice storage. Urea content of shark meat reduced by 25% during 12 days of ice storage. The increase in total volatile base nitrogen of shark meat was more than twofold at the end of 12 days of ice storage. Solubility of proteins showed an initial increase reaching a value of 87.9%, further reduced with ice storage. Gel filtration profile and SDS-PAGE pattern indicated aggregation of protein fractions and also showed dissociation or cleavage to small molecular weight fractions. Slope of reduced viscosity curve as a function of protein concentration decreased with ice storage period. Emulsion capacity of proteins from shark meat reduced from a value of 0.18 mL oil/mg protein to 0.14 mL oil/mg protein after 12 days of ice storage. Gel forming ability of shark meat monitored by dynamic viscoelastic measurement, gel strength, expressible water content and folding test showed that shark meat has got excellent ability to form gel and this property was marginally reduced during ice storage.

Effect of water washing of shark (Scoliodon laticaudus) meat on the properties of proteins with special reference to gelation.

The effect of water washing of shark meat on the properties of proteins has been investigated. The contents of low-molecular-weight proteins and urea were reduced significantly with three washing cycles. The gel forming ability showed marked improvement with the number of washing cycles. The dynamic viscoelastic behavior of washed and unwashed meat revealed a structure build-up reaction that was more pronounced in the washed meat. The concentration of myosin heavy chain of washed meat increased as revealed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Addition of urea at biological concentration (approximately 250 mM) to the washed meat reduced the gel forming ability significantly as compared to unwashed meat. The emulsion capacity showed an increase with the number of washing cycles.