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ThesisItem Open Access STUDIES ON NUTRITIONAL, STRUCTURAL, FUNCTIONAL AND STABILITY CHARACTERISTICS OF BAKERY PRODUCTS FORMULATED WITH SURIMI POWDER(Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, 2019) A. BRITA NICY, M.F.Sc.; Dr. P. VELAYUTHAM; Dr. R. JEYA SHAKILA; Dr. N. NEETHISELVAN; Dr. S. BALASUNDARIThis research was aimed to evaluate the physico-chemical and nutritional qualities of bakery products such as biscuits, cake and bread produced from itoyori surimi in the form of freeze dried and oven dried powder, based on proximate composition, effects of surimi powder incorporation and storage stability. Freeze dried and oven dried surimi powder were used in production of bakery products by replacing 10%, 20% and 30% of maida in chocolate biscuit and chocolate cake and 5%, 10% and 15% of maida in bread to ensure the quality and acceptability of the bakery products. Based on the results obtained from sensory and nutritional evaluation of bakery products fortified with surimi powder, 10% surimi powder incorporated bakery products were organoleptically accepted and the nutritional quality of products significantly (p<0.05) improved. The proximate composition of biscuits with freeze dried surimi (FDS) and oven dried surimi (ODS) powder were estimated and the results showed that moisture, ash, fat, protein and carbohydrates contents in control biscuits and biscuit incorporated with 10% FDS powder and 10% ODS powder were 2.19 ± 0.09%, 1.26 ± 0.05%, 25.26 ± 0.23%, 6.22 ± 0.09% and 65.07 ± 0.27%, respectively, 2.38 ± 0.06%, 1.73 ± 0.06%, 24.83 ± 0.04%, 9.21 ± 0.11% and 61.85 ± 0.15%, respectively and 2.01 ± 0.08%, 1.52 ± 0.06%, 24.90 ± 0.03%, 9.00 ± 0.09% and 62.56 ± 0.08%, respectively. Mineral content and amino acid composition were also analysed. The scores for overall acceptability of control biscuit, biscuit incorporated with 10% FDS and biscuit incorporated with 10% ODS powder were 8.90 ± 0.31, 8.83 ± 0.38 and 8.83 ± 0.38, respectively. The proximate composition of bread incorporated with FDS powder and that incorporated with ODS powder were estimated and the results showed that the moisture, ash, fat, protein and carbohydrates content in control bread were 20.49 ± 0.07%, 0.30 ± 0.00%, 3.46 ± 0.02%, 8.06 ± 0.08% and 67.69 ± 0.07%, respectively, while in bread incorporated with 10% FDS powder were 20.45 ± 0.19%, 0.73 ± 0.06%, 3.62 ± 0.03%, 13.36 ± 0.04% and 61.84 ± 0.23%, respectively, and in bread incorporated with 10% ODS powder were 20.73 ± 0.16%, 0.63 ± 0.06%, 3.54 ± 0.02%, 13.21 ± 0.11% and 61.89 ± 0.28%, respectively. The scores for overall acceptability of bread incorporated with FDS powder on an average for control bread, bread incorporated with 10% FDS powder and 10% ODS powder were 8.77 ± 0.43, 8.47 ± 0.51 and 8.03 ± 0.61, respectively. The proximate composition of cake with FDS and ODS powder were estimated and the moisture, ash, fat, protein and carbohydrates contents in control cake and cake incorporated with 10% FDS powder and 10% ODS powder were 22.46 ± 0.56%, 0.37 ± 0.06%, 28.49 ± 0.10%, 6.11 ± 0.05% and 42.58 ± 0.64, respectively, 22.13 ± 0.15%, 0.80 ± 0.10%, 28.82 ± 0.16%, 8.13 ± 0.06% and 40.12 ± 0.32%, respectively and 22.53 ± 0.11%, 0.80 ± 0.10%, 28.21 ± 0.06%, 8.03 ± 0.03% and 40.44 ± 0.19%, respectively. The scores for overall acceptability of cakes incorporated with 10% FDS and ODS powder on an average for control cake, cake incorporated with 10%, FDS powder and 10% ODS powder were 8.87 ± 0.35, 8.60 ± 0.50, and 8.63 ± 0.49, respectively. Scanning electron microscopic images and FTIR spectra of control bakery products and that incorporated with surimi powder were used to analyze the structural and functional properties, respectively of bakery products. Storage stability of control bakery product and that incorporated with surimi powder were analyzed by estimating the peroxide value, thiobarbituric acid value, free fatty acid value and microbial quality during storage and all parameters were within the acceptable limits during the entire storage period. The sensory evaluation of control bakery products, bakery products incorporated with FDS powder and that incorporated with ODS powder were estimated during storage. The scores for appearance, colour, odour, taste, mouth feel, flavour, texture and overall acceptability of all the products decreased during their storage period. The obtained results also showed the supplementation of surimi powder into flour used for making bakery products at level up to 10% improved the nutritional parameters of the final bakery products. Moreover, inclusion of oven dried surimi powder in bakery products have almost similar acceptability as compared to bakery products incorporated with freeze dried surimi powder. Therefore, from the present study, it is evident that the incorporation of dried surimi in food products for fortification, especially of the bakery products up to the concentration of 10% can be recommended for commercial production.ThesisItem Open Access HOST IMMUNE RESPONSES TO RANAVIRUS INFECTION IN MARINE AND FRESHWATER FISHES(Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, 2018) B. SIVARAMAN, M.F.Sc.; Dr. K. RIJI JOHN; Dr. M. ROSALIND GEORGE; Dr. A. SRINIVASAN; Dr. K. VEERABHADRANInfectious diseases have led to the most devastating problem in aquaculture sector. Viral diseases are one of the major challenges to aquaculturists because it is very difficult to control once they occur in the system. Outbreaks of the viral diseases due to various improper farm managements lead to a greater economic loss in aquaculture production. Prevention is the only way to control disease incidence caused by viral pathogens. Incorrect use of antibiotics and chemical drugs in aquaculture is associated with several deleterious side effects. Instead of using antibiotics and chemical drugs, vaccination is an effective tool in controlling, preventing, protecting and recovery of fish from virus diseases in cultured fish contributing to sustainability in the aquaculture sector. Therefore, the development of viral vaccines against emergent viral diseases is crucial for promoting successful aquaculture production. In this study, experimental inactivated similar damselfish virus (SRDV) vaccine and recombinant major capsid protein vaccine were prepared and immunogenicity and protection against ranavirus infection of the vaccine were investigated in koi carp (Cyprinus carpio), seabass (Lates calcarifer) and similar damselfish (Pomacentrus similis). Firstly, experimental inactivated SRDV vaccine was prepared and expression profiles of immune related genes against virus challenge of the vaccine were investigated in koi carp. Formalin-inactivated virus at 4°C for 2 days exhibited no typical clinical symptoms of SRDV infection and no mortality following challenge at 28-day post-vaccination. The immune response was induced by intraperitoneal injection of koi carp with formalin-inactivated vaccine with added Quil-A® adjuvant. The expression levels of genes including IRF-7 in spleen of koi immunized with vaccine added Quil-A® adjuvant was highly up-regulated (52.2 fold) at 24 h post infection of the virus but in kidney it showed down-regulation (0.4 fold). Interestingly, the highest level of up-regulation (16 fold) was recorded at 96 h post infection of SRDV. Like IRF-7, highest up-regulation of IL-10 gene was observed in spleen of koi immunized with vaccine added Quil-A® adjuvant at 24 post infection of the virus while kidney was showed down-regulation. This experiment provides strong evidence to show the expression of immune related genes (IRF-7 and IL-10) which is up-regulated during the ranavirus infection. It also suggests that Quil-A® adjuvant enhances the immune response of the vaccine candidates. In the next experiment, expression profiles of immune related genes against virus challenge against formalin-inactivated SRDV vaccine were investigated in seabass. The immune response was induced by intra-peritoneal injection with inactivated viral vaccine added Quil-A® adjuvant. The expression levels of IRF-7 in the kidney and spleen of seabass immunized with vaccine added Quil-A® adjuvant was highly up-regulated (3 and 4.8 fold) at 24 h post challenge of the virus and it continued to be up-regulated (7.23 and 7.49 fold) at 48 hpi. IL-10 was slightly up- regulated (1.09 and 1.87 fold) in kidney and spleen at 24 h post infection. Like IRF-7, the expression of IL-10 showed continued up-regulation (2.23 and 4.81 fold) in kidney and spleen at 48 h post virus challenge. Expression profiles of immune related genes (IRF-7 and IL-10) in the kidney and spleen of seabass immunized with vaccine added adjuvant were up-regulated at 48 hpi of the virus. In comparison, spleen of seabass immunized with vaccine added adjuvant showed highest expression profiles than kidney. This study also provided an evidence for the presence of expression profiles of immune-related genes during the SRDV infection. The study also strongly suggests that Quil-A® adjuvant enhances the immune response of the vaccine candidates. Development of recombinant MCP vaccine was carried out to assess its efficacy in fish. Recombinant MCP gene inserted into cloning vector pTZ57R/T was confirmed by colony PCR. Subsequently, the plasmid was extracted from MCP gene inserted vector pTZ57R/T and confirmed by agarose gel electrophoresis. The MCP gene of SRDV containing BamHI and XhoI as restriction sites was amplified from viral DNA with specific primer set and confirmed by agarose gel electrophoresis with the target size of 1416bp. The gel purified PCR product was double digested with the said enzymes and ligated into pTriEx1.1 vector followed by transformation in E.coli DH5α competent cell. Plasmids from two clones namely pTriEx-MCP-1416-1 and pTriEx-MCP-1416-3 were transformed into E. coli BL21 (DE-3) pLacI. Two separate colonies obtained after transformation of pTriEx-MCP-1416-1 and pTriEx- MCP-1416-3 E. coli BL21 (DE-3) pLacI were tested for the expression of MCP gene. The polyclonal antibody produced from rabbit against the purified protein of SRDV has neutralised the homologous virus infectivity in vitro. The crude protein mixture obtained from the colony of E. coli BL21 (DE-3) strain transformed with MCP inserts was used to assess the efficacy in similar damselfish and koi carp by intra-peritoneal injection. After challenge with SRDV, damselfish immunized with recombinant protein showed the low level of protection with of relative percentage survival (RPS) value of 18.8% while vaccine added Quil-A® adjuvant showed RPS of 26%. No specific mortality was found in all treated groups of koi including control group-B which had received virus alone. Therefore, further investigations are required for the development of effective recombinant protein vaccine and assess its efficacy against SRDV challenge in marine and freshwater fishes.ThesisItem Open Access AUTHENTICATION OF PRODUCTS FROM SNAPPER USING DNA BASED TECHNIQUES(Fisheries College and Research Institute, Thoothukudi, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, 2018) B. SIVARAMAN, M.F.Sc.; Dr. G. JEYASEKARAN; Dr. R. JEYA SHAKILA; Dr. D. SUKUMAR; Dr. S. AANANDSeafood adulteration is an important issue for food authorities, because species substitution and mislabeling present the major commercial frauds in seafood market sector around the world. In this study, different PCR based methods such as PCR-RFLP, PCR-SSCP, PCR-FINS and PCR-AFLP protocols were developed to authenticate nine different snapper species viz., Lutjanus fulvus, L. gibbus, L. lemniscatus, L. argentimaculatus, L. rivulatus, L. quinquelineatus, L. fulviflamma, L. madras and L. decussatus of commercial importance in the region of Thoothukudi, South India. For authentication analysis, fresh snappers and three processed snapper products such as frozen, cooked and fried snappers were used. Four different mitochondrial genome regions such as cytochrome b, 12S rRNA, 16S rRNA and hyper variable D-loop region were chosen as the molecular targets for authentication. Four sets of primers viz., Fish-CytB-F/Fish-CytB-R, 16S-SGS-F/16S-SGS-R, 12SU-F/12SU-R and Fish-DL-F/Fish-DL-R to amplify cytb, 16S rRNA, 12S rRNA and D-loop regions,respectively were designed using bioinformatics tools such as NCBI GenBank database, CLUSTAL OMEGA and BioEdit. The PCR amplified the respective targets successfully and the amplicons were sequenced, analysed using BLAST tool in NCBI and 24 sequences of different species of South Indian coast were deposited in GenBank database. All the PCR amplicons were used for developing a suitable PCR-RFLP protocol to authenticate nine snapper species. Webcutter software was used for in silico analysis to choose the suitable restriction enzymes for the respective mitochondrial region. Suitable restriction enzymes selected were Cfr13I, HaeIII and Tsp509I for cyt b region, Mn1I, MspAI and Sau3AI for 16S rRNA, BseDI, Mn1I and MspAI for 12S rRNA and Tsp509I for D-loop region. PCR-RFLP performed with these restriction enzymes on the four regions showed that cytb could differentiate only two snappers viz., L. rivulatus and L. decussatus with the enzyme, Cfr13I; and L. madras and L. fulvus from others with the enzyme, HaeIII. The 16S rRNA region differentiated only L. quinquelineatus from other five species with the enzyme, Mn1I while Sau3AI differentiated L. rivulatus from other species. The 12S rRNA region differentiated L. madras, L. quinquelineatus and L. decussatus from the other snapper species with the enzyme, BseDI. But, PCR-RFLP was very successful with D-loop region that differentiated all the nine species with a single enzyme, Tsp509I. The PCR-RFLP pattern obtained for the frozen, cooked and fried snappers were also similar to that of fresh snappers proving that the protocol was suitable for processed products. PCR-SSCP performed on the target regions viz., cytb, 12S rRNA and 16S rRNA could not differentiate all the snapper species; but few species were differentiated randomly. On the other hand, SSCP of D-loop region differentiated all the nine snapper species with the newly designed reverse primer, DLFR for the forward primer, Fish-DL-F that produced a 360 bp D-loop fragment. PCR-FINS was used to construct the phylogenetic trees separately for 12S rRNA and D-loop sequences of snapper species using the Neighbor-Joining method. All the snapper species got placed in separate clusters with bootstraps values from 51 to 100, enabling differentiation by the 12S rRNA and D-loop region sequences without any ambiguity. PCR-AFLP developed using single pre-selective primer, EcoRI, and three selective primers viz., ACC, ACG and ACT also differentiated all the nine snapper species yielding specific AFLP markers that can further be used to develop or study unique regions of individual snapper species of importance. In this study, among the four DNA based methods viz., PCR-RFLP, PCR-SSCP, PCR-FINS and PCR-AFLP tried for the authentication of nine processed snapper products, PCR-SSCP, PCR-FINS and PCR-AFLP methods could differentiate all the snapper species. However, these methods are not widely employed due to various difficulties in field application. For instance, FINS needs an expensive DNA sequencer for routine sequencing, while AFLP involves complicated procedures besides giving high inter and intra-species variation posing difficulty in the interpretation of the results. Likewise, PCR-SSCP also requires skilled persons due to stringent procedure in running electrophoresis. Hence, PCR-RFLP is a better choice for adaptation by the regulatory agencies. In this study, PCR-RFLP protocol targeting D-loop region of mitochondria was found more reliable by means of repeatability and low cost in authentication of fresh as well as the processed snapper products within 8 h, thereby add on to its suitability for regulatory purposes.