Synthesis of 1H-benzoxazine-2,4-diones from heterocyclic anhydrides: evaluation of antioxidant and antimicrobial activities

A facile one-step synthesis of 1H-benzoxazine-2,4-diones from heterocyclic anhydrides and TMSA was described. This paper determines their antimicrobial activity against nine human bacterial pathogens by the broth microdilution method; antioxidant activity by DPPH• inactivation and a ferric-reducing power assay; and toxicity by a brine shrimp, Artemia salina, assay. The 1H-benzoxazine-2,4-dione yields were in the range of 57 to 98%. The novel compound 1H-pyrazino[2,3-][1,3]oxazine-2,4-dione 4c showed the highest antioxidant capacity (DPPH 35.4% and FRAP 0.063 µmol TEs/µmol).

Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD50 141.25 µg protein/ml) and to mice injected intraperitoneally (LD50 201.8 ± 8.6 mg protein/kg), in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s) of a protein nature which may be involved in the chemical defense mechanism of this sea hare.

Infrequent p53 gene alterations in ulcerative colitis

The purpose of this study was to determine whether point mutations and loss of the p53 gene take place in ulcerative colitis which is histologically negative for dysplasia. DNA was extracted from 13 frozen rectal or colon biopsies and blood samples. Ulcerative colitis was classified histologically as active (10 cases) and inactive (3 cases). Exons 5-8 were amplified by PCR, treated with exonuclease and shrimp alkaline phosphatase and sequenced by the dideoxy chain termination method with the Sequenase Version 2.0 DNA sequencing kit. PCR products of intron 6 and exon 4 were digested with MspI and AccII, respectively, for RFLP analysis. No p53 gene mutation was detected in these cases. The number of informative patients for loss of heterozygosity (LOH) at the p53 intron 6 was high, 11 out of 12 (92%), whereas no LOH was observed. LOH affecting p53 exon 4 was not detected in lesions from 5 of 12 patients (42%). In ulcerative colitis, tumor progression is similar to that in sporadic colon cancer, and other oncogenes and tumor suppressor genes are likely to be mutated before the p53 gene.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Characterization of a hydrolyzed oil obtained from fish waste for nutraceutical application

The fish industry generates high volume of waste from fish oil that can have the extraction of its lipids used as nutraceuticals and foods. The objective of this study was to produce unsaturated fatty acids from industrialized fish oil by means of a differentiated hydrolysis process. The samples used were crude fish oil obtained from Campestre industry and characterized through physical-chemical parameters, according to AOCS: acidity, peroxide, saponification, iodine and percentage of free fatty acids and also obtained the fatty acid profile through derivatization method for gas chromatography. The results obtained for the oleochemical indices for refined oil were similar to the data found on the literature. The content of polyunsaturated fatty acids (PUFA) was found of 32,78%, with 9,12% of docosahexaenoic (DHA) and 10,36% of eicosapentaenoic (EPA), regarding monounsaturated fatty acids (MUFA) content was of 30,59% in the hydrolyzed fish oil in relation to refined (20,06%). Thus, it can be concluded that the hydrolysis process used for oils from fish-waste was satisfactory on the production of absolute yield of lipids in the process and significant preservation on the percentages of EPA and DHA, interesting on the production of nutraceuticals and nutrition of aquatic animals, including shrimp in captivity.

Characterization of antimicrobial resistance in Aeromonas and Vibrio isolated in Canada from fish and seafood

Plusieurs études ont examiné la sensibilité aux antimicrobiens chez les bactéries d’organismes provenant de produits issus de l’aquaculture ou de leur environnement. Aucune information n’est cependant disponible concernant la résistance aux antimicrobiens dans les bactéries de la flore de poissons ou de fruits de mer vendus au détail au Canada. C’est particulièrement vrai en ce qui a trait aux bactéries des genres Aeromonas et Vibrio, dont certaines espèces sont des agents pathogènes zoonotiques connus. Au cours de cette étude, la sensibilité aux antimicrobiens d’isolats d’Aeromonas spp. et de Vibrio spp. provenant de poissons et de crevettes domestiques et importés a été mesurée à l’aide de techniques de micro dilution en bouillon et/ou de diffusion sur disque. Les classes d’antimicrobiens examinés comprenaient les tétracyclines (TET), les inhibiteurs de la voie des folates (sulfadiméthoxine-triméthoprime, SXT), le florfenicol (FLO), et les quinolones (acide nalidixique / enrofloxacine, NA/ENO). Des valeurs seuils épidémiologiques pour Aeromonas et Vibrio ont été établies en utilisant la méthode d’interprétation normalisée des données de résistance provenant de diffusion sur disque. La recherche de gènes de résistance associés au profil de résistance des isolats a été effectuée en utilisant des PCRs et des puces ADN. Le nombre d’isolats résistants aux divers antimicrobiens parmi les 201 isolats d’Aeromonas et les 185 isolats de Vibrio étaient respectivement les suivants: TET (n=24 et 10), FLO (n=1 et 0), SXT (n=2 et 8), NA (n=7 et 5) et ENO (n= 5 et 0). Diverses associations de gènes tet(A), tet(B), tet(E), floR, sul1, sul2, et intI1 ont été détectées, les gènes tet(E), intI1, sul2 et tet(B) étant les plus communs. Les espèces d’Aeromonas et de Vibrio isolées de poissons au détail et de fruits de mer peuvent héberger une variété de gènes de résistance, bien que peu fréquemment. Le risque que représente ces gènes de résistance reste à évaluer en considérant le potentiel infectieux des bactéries, l’utilisation des ces agents antimicrobiens pour le traitement des maladies en aquaculture et en médecine humaine et leur rôle en tant que réservoir de la résistance antimicrobienne.

Standardization of Optimum Conditions for the Production of Glucosamine Hydrochiloride from Chitin

The study entitled standardization of optimum conditions for the production of glucosamine hydrochloride from chitin. Shellfish processing industries around the world turn out a significant quantity of head and shell as industrial waste. The waste must be removed immediately to prevent the contamination to the processing environment. The technique that are available for their disposal include ocean dumping, incineration or disposal of landfill sites. In this thesis the techniques and methods are used to process glucosamine hydrochloride from crustacean processing waste. Chitin is a nitrogenous polysaccharide, which is white, hard, inelastic, found in outer skeleton of insects, crabs, shrimp and lobsters and in the internal structures of other invertebrates. Glucosamine can be considered as a nutraceutical product by virtue of its properties. It is important for healthy skin, and plays a major role in the healing of surgical incisions and skin wounds. Deproteinisation of shrimp shell had significant effect on quality of chitin. Demineralization is also influences chitin quality. Solvents used for glucosamine hydrochloride affects the final yield and purity.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vihrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vihrio isolates, 16. 8'7(, were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

STANDARDIZATION OF PROCESS PARAMETERS FOR READY TO EAT FISH PRODUCTS IN INDIGENOUS POLYMER COATED TIN FREE STEEL CANS

In the present work, Indigenous polymer coated Tin Free Steel cans were analyzed fortheir suitability for thermal processing and storage of fish and fish products following standard methods. The raw materials used for the development of ready to eat thermally processed fish products were found to be of fresh condition. The values for various biochemical and microbiological parameters of the raw materials were well within the limits. Based on the analysis of commercial sterility, instrumental colour, texture, WB-shear force and sensory parameters, squid masala processed to F0 value of 8 min with a total process time of 38.5 min and cook value of 92 min was chosen as the optimum for squid masala in tin free steel cans while shrimp curry processed to F0 7 min with total process time of 44.0 min and cook value of 91.1 min was found to be ideal and was selected for storage study. Squid masala and shrimp curry thermally processed in indigenous polymer coated TFS cans were found to be acceptable even after one year of storage at room temperaturebased on the analysis of various sensory and biochemical parameters. Analysis of the Commission Internationale d’ Eclirage L*, a* and b* color values showed that the duration of exposure to heat treatment influenced the color parameters: the lightness (L*) and yellowness (b*)decreased, and the redness (a*) significantly increased with the increase in processing time or reduction in processing temperature.Instrumental analysis of texture showed that hardness-1 & 2 decreased with reduction in retort temperature while cohesiveness value did not show any appreciable change with decrease in temperature of processing. Other texture profile parameters like gumminess, springiness and chewiness decreased significantly with increase of processing time. W-B shear force values of mackerel meat processed at 130 °C were significantly higher than those processed at 121.1 and 115 °C. HTST processing of mackerel in brine helped in reducing the process time and improving the quality.The study also indicated that indigenous polymer coated TFS cans with easy openends can be a viable alternative to the conventional tin and aluminium cans. The industry can utilize these cans for processing ready to eat fish and shell fish products for both domestic and export markets. This will help in reviving the canning industry in India.

Antiviral property of marine actinomycetes against White Spot Syndrome Virus in penaeid shrimps

Aquaculture farms, particularly in Southeast Asia are facing severe crisis due to increasing incidences of White Spot Syndrome Virus (WSSV). Actinomycetes have provided many important bioactive compounds of high prophylactic and therapeutic value and are continually being screened for new compounds. In this communication, the results of a study made to determine the effectiveness of marine actinomycetes against the white spot disease in penaeid shrimps are presented. Twenty-five isolates of actinomycetes were tested for their ability to reduce infection due to WSSV among cultured shrimps. When these actinomycetes were made available as feed additives to the post-larvae of the black tiger shrimp Penaeus monodon for two weeks and challenged with WSSV, the post challenge survival showed variations from 11 to 83%. However, six isolates have shown to be the most potential candidates for further study.

INVESTIGATIONS ON THE STRESS EFFECTS OF PETROLEUM HYDROCARBONS ON METAPENAEUS DOBSON (MIERS)

The present investigation has addressed the effects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps. The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its effect on species, populations and communities,a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to dynamic steady-state systems in nature.

Metabolic Efficiency of Penaeus Indicus H.Milne Edwards Under Different Salinity Stress Conditions

The investigation was aimed at establishing the effect of salinity on the culture performance of Peneus Indicus in pokkali fields and also to find out the growth performance of the shrimp at varying salinities. The experiments were laid out at Rice Research Station, Vyttila of Kerala Agriculture University in three fields of area 1000 m2 each. The results of the experiment clearly establish that shrimps when stocked at higher salinity (20-25 ppt) for 45 days has given higher growth, survival and production than those stocked at lower salinity (10-15 ppt) in all the above parameters even when the culture experiment was maintained for longer periods in lower salinity. In the prolonged culture experiments conducted for 120 days in 10-25 ppt salinity, the results were poorer than the short period culture in higher salinity and the production values similar to lower saline culture. This clearly establishes the importance of salinity as an ecological factor which will have profound influence in shrimp farming operations.

Cell Culture Systems from Penaeus monodon: Development and Application

National Centre for Aquatic Animal Health, Cochin University of Science and Technology

Application of Fermentation Techniques in the Utilization of Prawn Shell Waste

The present study aimed at the utlisation of microbial organisms for the production of good quality chitin and chitosan. The three strains used for the study were Lactobacillus plantarum, Lactobacililus brevis and Bacillus subtilis. These strains were selected on the basis of their acid producing ability to reduce the pH of the fermenting substrates to prevent spoilage and thus caused demineralisation of the shell. Besides, the proteolytic enzymes in these strains acted on proteinaceous covering of shrimp and thus caused deprotenisation of shrimp shell waste. Thus the two processes involved in chitin production can be affected to certain extent using bacterial fermentation of shrimp shell.Optimization parameters like fermentation period, quantity of inoculum, type of sugar, concentration of sugar etc. for fermentation with three different strains were studied. For these, parameters like pH, Total titrable acidity (TTA), changes in sugar concentration, changes in microbial count, sensory changes etc. were studied.Fermentation study with Lactobacillus plantarum was continued with 20% w/v jaggery broth for 15 days. The inoculum prepared yislded a cell concentration of approximately 108 CFU/ml. In the present study, lactic acid and dilute hydrochloric acid were used for initial pH adjustment because; without adjusting the initial pH, it took more than 5 hours for the lactic acid bacteria to convert glucose to lactic acid and during this delay spoilage occurred due to putrefying enzymes active at neutral or higher pH. During the fermentation study, pH first decreased in correspondence with increase in TTA values. This showed a clear indication of acid production by the strain. This trend continued till their proteolytic activity showed an increasing trend. When the available sugar source started depleting, proteolytic activity also decreased and pH increased. This was clearly reflected in the sensory evaluation results. Lactic acid treated samples showed greater extent of demineralization and deprotenisation at the end of fermentation study than hydrochloric acid treated samples. It can be due to the effect of strong hydrochloric acid on the initial microbial count, which directly affects the fermentation process. At the end of fermentation, about 76.5% of ash was removed in lactic acid treated samples and 71.8% in hydrochloric acid treated samples; 72.8% of proteins in lactic acid treated samples and 70.6% in hydrochloric acid treated samples.The residual protein and ash in the fermented residue were reduced to permissible limit by treatment with 0.8N HCI and 1M NaOH. Characteristics of chitin like chitin content, ash content, protein content, % of N- acetylation etc. were studied. Quality characteristics like viscosity, degree of deacetylation and molecular weight of chitosan prepared were also compared. The chitosan samples prepared from lactic acid treated showed high viscosity than HCI treated samples. But degree of deacetylation is more in HCI treated samples than lactic acid treated ones. Characteristics of protein liquor obtained like its biogenic composition, amino acid composition, total volatile base nitrogen, alpha amino nitrogen etc. also were studied to find out its suitability as animal feed supplement.Optimization of fermentation parameters for Lactobacillus brevis fermentation study was also conducted and parameters were standardized. Then detailed fermentation study was done in 20%wlv jaggery broth for 17 days. Also the effect of two different acid treatments (mild HCI and lactic acid) used for initial pH adjustment on chitin production were also studied. In this study also trend of changes in pH. changes in sugar concentration ,microbial count changes were similar to Lactobacillus plantarum studies. At the end of fermentation, residual protein in the samples were only 32.48% in HCI treated samples and 31.85% in lactic acid treated samples. The residual ash content was about 33.68% in HCI treated ones and 32.52% in lactic acid treated ones. The fermented residue was converted to chitin with good characteristics by treatment with 1.2MNaOH and 1NHCI.Characteristics of chitin samples prepared were studied and extent of Nacetylation was about 84% in HCI treated chitin and 85%in lactic acid treated ones assessed from FTIR spectrum. Chitosan was prepared from these samples by usual chemical method and its extent of solubility, degree of deacetylation, viscosity and molecular weight etc were studied. The values of viscosity and molecular weight of the samples prepared were comparatively less than the chitosan prepared by Lactobacillus plantarum fermentation. Characteristics of protein liquor obtained were analyzed to determine its quality and is suitability as animal feed supplement.Another strain used for the study was Bacillus subtilis and fermentation was carried out in 20%w/v jaggery broth for 15 days. It was found that Bacillus subtilis was more efficient than other Lactobacillus species for deprotenisation and demineralization. This was mainly due to the difference in the proteolytic nature of the strains. About 84% of protein and 72% of ash were removed at the end of fermentation. Considering the statistical significance (P