Messenger RNA based assay for RT-PCR detection of viable Salmonella and Vibrio cholerae from fresh and processed finfish and shellfish

• A RT-PCR assay for the detection of Salmonella serovars using five specific mRNA genes hto, invA, hns, himA, and fimA was developed. Bioinoculation studies carried out proved the developed RT-PCR assay could detect the presence of enteric serovar Typhimurium in fresh and processed finfish and shellfish using a specific mRNA gene invA (275bp).
• A RT-PCR assay was developed for the detection of different strains of Vibrio cholerae using specific mRNA genes rpoA, ctxA, tsf, groEL and rtxA. Bioinoculation studies carried out proved the developed RT-PCR assay could detect cholerae O139 (SG24) in fresh and processed finfish and shellfish using the specific mRNA gene ctxA (308bp).
• RT-PCR assays developed could detect both the organism within 5 min of pre-enrichment in fresh and cooked finfish and shellfish. They were also detected by RT-PCR after 30 days in dried fish products. But, in the case of frozen products, only cholerae was detected after 30 days.

Development of database for Indian seafood safety and traceability based on AFLP markers

• AFLP-PCR based analysis was done for the authentication of five commercially important tuna species namely Euthynnus affinis, Auxis thazard, Katsuwonus pelamis, Thunnus albacares, and Thunnus obesus. Genomic DNA extracted from fresh tuna was subjected to restriction digestion using EcoR1 and Mse1 enzymes followed by ligation with adaptors by using T4 DNA Ligase. Pre-amplification was done by using primer having one selective nucleotide and selective amplification was done using primers having three selective nucleotides.
• Different species specific AFLP banding patterns were obtained for fresh tuna in the primer combination EcoR1-AGA/Mse1-CTG. Amplified products were then analyzed by 2% metaphor agarose gel electrophoresis and their molecular sizes were determined by Alpha Innotech software.
• Species specific AFLP marker for different species of tuna are 67 bp for Euthynnus affinis, 113 bp for Auxis thazard, 590 bp for Katsuwonus pelamis, 131 bp for Thunnus albacares and 450 bp  for Thunnus obesus. This AFLP marker can be used to authenticate commercially important tuna species available in Tamil Nadu.

Development of PCR-RFLP method for identification of fish products from different species of sardines

• Authentication of sardine fish products made from five species of sardines viz., Sardinella longiceps, S. gibbosa, S. albella, S. fimbriata and sirm by PCR-RFLP method. Mitochondrial cyt b gene was amplified from all the products using the primers C-CB28df and C-CB341R having a product size of 147 bp, which was found to be specific for the sardines belonging to the genus, Sardinella
• Amplified DNA fragment on subsequent digestion with Hinfi and MnII restriction enzymes yielded unique RFP patterns for the individual sardine species. longiceps was distinguished by the presence of no major bands. S. gibbosa had a single major band at 107 bp and no band >50bp. S. albella showed two clear bands at 107 bp and 80 bp. S. fimbriata had two major band with one major band at 107 bp. S. sirm had two bands with one major band at 75 bp.
• PCR-RFLP patterns of the cooked, chilled, frozen and salt-dried sardines also exhibited the same pattern as that noticed with raw sardines. The developed PCR-RFLP method can therefore be used for authentication of sardine species on commercial scale, even for processed products.

Antioxidative potential of the squid protein hydrolysate

• Antioxidative potential of squid protein hydrolysates (SPH) prepared using endogenous pepsin and trypsin extracted from seer fish was evaluated in comparison with commercial enzymes. Endogenous pepsin or trypsin were efficient than commercial enzymes in the hydrolysis. Pepsinogen SPH acts rapidly than pepsin SPH. Antioxidative properties viz. DPPH and ABTS activities were related to the presence of hydrophobic amino acids. Superoxide anion radical scavenging activity was based on the molecular conformation of peptides, while reducing power correlated well with the degree of hydrolysis (DH). Metal chelating abilities were influenced by the presence of suitable metal binding sites.
• Peptides of alcalase SPH fractionated based on different MWCO filters viz. 30KDa, 10KDa, 5KDa and 3KDa were examined for their amino acid composition. The proportion of aromatic and aliphatic amino acids increased with the decreasing molecular weights. The proportion of acidic and basic amino acids decreased with decreasing molecular weight of peptides. The proportions of neutral and cyclic amino acids were almost constant in all molecular weight peptides.
• SPH derived with commercial enzymes viz. pepsin, trypsin and alcalase showed DH of 8-15% within 150 min of reaction. Minimum inhibitory concentration (IC₅₀) values exhibited by alcalase SPH for DPPH was 0.87 mg/ml; ABTS was 1.40 mg/ml; hydroxyl radicals was 0.2 mg/ml; superoxide anion radicals was 6.12 mg/ml; metal chelating ability was 0.8 mg/ml; and reducing power was 0.5 mg/ml. Pepsin SPH do not chelate metals. Trypsin SPH did not scavenge superoxide anion radicals.
• Peptides with desirable antioxidative potential can thus be prepared by choosing appropriate enzymes as well as desirable concentration of hydrolysates.

Impact of heat processing methods on the health beneficial omega-3 fatty acids of sardines

• Sardine curry was examined for the changes in fatty acid composition particularly, omega 3 fatty acids when subjected to cooking for varying time duration. The dominant fatty acids in the fish curry were lauric acid, myristic acid, linoleic acid and linolenic acids that had been derived through the migration from the cooking oil and ingredients used for curry preparation. There was a reduction in EPA from 9.61% to 2.29% in 20 min boiled sardine curry and to 0.49% in 30 min boiled sardine curry. The DHA content reduced from 14.72% to 2.86% in 20 min. boiled sardine curry and to 0.42% in 30 min boiled sardine curry. However, sardine pieces in curry retained 56% of EPA and 51% of DHA even after boiling for 20 min.
• Effect of shallow and deep frying of sardine (Sardinella gibbosa) in different cooking oils viz sunflower, groundnut and gingelly oils was investigated. There was an increase in fat content upon frying. Fat content was low in deep fried sardines than shallow fried ones. Cholesterol decreased in sardines shallow and deep fried in sunflower and gingelly oil but increased in sardines shallow fried in groundnut oil. Oleic and linoleic acids were the predominant fatty acids. The destruction EPA and DHA was more in shallow fried sardines than deep fried ones.
• Effect of microwave cooking on the fatty acid composition of sardine was investigated. Fatty acid profiles of microwave cooked sardines at different time period of 20, 30 and 40 sec were compared with that of raw and sardines boiled to 10 min. The retention of EPA and DHA were higher in sardine microwave cooked for 20 sec.

Establishment of chemical residue monitoring laboratory for fish in Tamil Nadu

• LC MS/MS equipment for the detection and quantification of antibiotics and pesticide residue was purchased. Methods for analysis were being developed for several antibiotics viz. chloramphenicol, nitrofurans, oxytetracyclines and sulfonamides.
• Construction of the 1^st floor in the existing Fish Quality and Monitoring Certification Centre was completed. Required laboratory furnitures have been purchased to set up the laboratory for NABL accreditation.