Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus / Caracterização de enzimas digestivas do linguado brasileiro de cativeiro Paralichthys orbignyanus

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.

Caracterización bioquímica, fisicoquímica y funcional de turbot (Scophthalmus maximus) y estudio de sus modificaciones durante el almacenamiento a 4ºC / Biochemical, physicochemical and functional characterization of turbot (Scophthalmus maximus). Study of the changes occurring during the 4ºC storage

El turbot es un pez plano (Scophthalmus maximus), es uno de los peces que se adapta muy bien al cultivo intensivo en granja. Es necesario conocer sus propiedades fisicoquímicas-bioquímicas-tecnológicas y cómo éstas cambian durante su almacenamiento una vez que han sido cosechados. El objetivo de este estudio fue evaluar la composición centesimal, propiedades bioquímicas, fisicoquímicas y funcionales, y cómo se ven influidas por el almacenamiento a 4ºC durante 16 días. Se determinó su composición proximal, electroforesis en geles de poliacrilamida desnaturantes (PAGE-SDS), estabilidad térmica proteica a través de calorimetría diferencial de barrido (DSC). Se evaluó pH, nitrógeno básico volátil total (NBV-T), pérdida por goteo, textura, capacidad de retención de agua (CRA), emulsificación (CE) y gelificación (CG). El análisis proximal fue: humedad 76,3%; proteínas 19,6%; lípidos 2,71%; cenizas 1,3%. Se encontraron dos transiciones térmicas (DSC) a 47,5°C (miosina) y 76,9ºC (actina). PAGE-SDS mostraron perfiles correspondientes a proteínas miofibrilares y sarcoplasmáticas y durante el almacenamiento no se vieron alterados El pH cambió desde 5,9 a 6,6; las NBV-T variaron desde 20,0 a 39,5 mg NBVT/100 g músculo al día 16. CRA presentó valores de 10,5% y llegando al final a 58,1%, sin embargo no presentó CG. La CE y CRA aumentaron por el almacenamiento debido a cambios conformacionales de las proteínas. El esfuerzo de compresión fluctuó entre 111,2 a 106,0 N; el esfuerzo de cizalla fue disminuyendo en el tiempo de almacenamiento desde 148,6 a 95,2 N. La pérdida por goteo varió entre 1,7% a 2,5%. El estudio de almacenamiento refrigerado por 16 días, permitió concluir que el parámetro de frescura de NBV-T, sobrepasa el límite permitido de comercialización a los 14 días de almacenamiento, determinando de esa forma su vida útil.

The turbot (Scophthalmus maximus) is a kind of flatfish that is well adapted to intensive farm culture. After the harvest it is necessary to know the physicochemical, biochemical and technological properties and if during the refrigerated storage, changes of these properties are developed. The main objective of the study was the assessment of the proximal composition, the biochemical, physicochemical and functional properties, and how they are influenced during the 16 days storage at 4º C. Parameters such as centesimal composition, PAGE-SDS, and protein thermal stability through a DSC were carried out. pH, total volatile base-nitrogen (TVBN) dripping, texture, holding water capacity (WHC), emulsification (EC) and gelification (GC) were also determined. Results for the proximal composition were: humidity 76,3%; fat content 2,71%; proteins 19,6%; and ashes 1,3%. Two different thermal transitions at 47,5°C (myosin) and 76,9°C (actin) were observed. The PAGE-SDS showed profiles corresponding to myofibrilars and sarcoplasmatic proteins, which presented no changes during the storage. pH experimented an increase from 5,9 to 6,6; TVB-N showed a variation from 20,0 to 39,5 mg TVB-N/100 g of muscle at the day 16. The WHC started with a 10,5% and ended in 58,1%; no GC was observed. The increase of the EC and WHC during the storage was due to conformational changes of proteins. The compression force presented a fluctuation between 111,2 and 106,0 N and the shear strain decreased during the storage from 148,6 to 95,2 N. The dripping showed a variation between 1,7% and 2,5%. According to the results of the storage during 16 days at 4ºC, the NBV-T content determined a shelf life of 14 days.

Structure-antimicrobial activity relationship between pleurocidin and its enantiomer

To develop novel antibiotic peptides useful as therapeutic drugs, the enantiomeric analogue of pleurocidin (Ple), which is a well known 25-mer antimicrobial peptide, was designed for proteolytic resistance by D-amino acids substitution. The proteolytic resistance was confirmed by using HPLC after the digestion with various proteases. To investigate the antibiotic effect of L- and D-Ple, the antibacterial activity and hemolytic effect were tested against human erythrocytes. The D-Ple showed a decreased antibacterial activity and a dramatically decreased hemolytic activity compared with L-Ple. The hemolytic effect of analogue was further confirmed by using calcein leakage measurement with liposome. To elucidate these results, the secondary structure of the peptides was investigated by using circular dichroism spectroscopy. The results revealed that D-Ple, as well as L-Ple, had typical ?-helical structures which were mirror images, with a different helicity. These results suggested that the discrepancy of the structure between the two peptides made their antibacterial activity distinct.

Role of pepsin in modifying the allergenicity of bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) fish.

The effect of pepsin digestion on the allergenicity of raw and thermally processed (boiled and fried) fish muscle extracts of two widely consumed fishes bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) was studied. Sere were collected from 110 patients who were hypersensitive to fish, as evidenced by their clinical history, symptoms and positive skin-prick test results. The various extracts after digestion with pepsin at different times of incubation were tested for specific IgE-binding activity by ELISA and immunoblotting with patients' sera. All the extracts of both the fishes retained their allergenicity as evidenced by ELISA and immunoblotting. In bhetki, maximum allergenicity was found in the pepsin-digested fried extract, whereas similar treatment decreased the allergenicity in fried mackerel. Results showed that raw as well as thermally processed allergens of both the fishes maintained strong allergenicity, even after digestion with pepsin for different time periods. The study revealed that the fish proteins played an important role in manifestation of allergy, due to their stable structure, which was retained even after pepsin and heat treatment.

Isolation of a haemorrhagic protein toxin (SA-HT) from the Indian venomous butterfish (Scatophagus argus, Linn) sting extract.

A haemorrhagic protein toxin (SA-HT) was isolated and purified from the spine extract of the Indian venomous butterfish, S. argus Linn, by two step ion exchange chromatography. The toxin was homogeneous in native and SDS-PAGE gel. SDS-molecular weight of the toxin was found to be 18.1 +/- 0.09 kDa. SA-HT produced severe haemorrhage on stomach wall but devoid of cutaneous haemorrhage. UV, EDTA, trypsin, protease, cyproheptadine, indomethacin, acetylsalicylic acid and BW755C treatment significantly antagonized the haemorrhagic activity of SA-HT. The toxin produced dose and time dependent oedema on mice hind paw, which was significantly encountered by cyproheptadine, indomethacin and BW755C. SA-HT increased capillary permeability on guinea pig dorsal flank. On isolated guineapig ileum, rat fundus and uterus, SA-HT produced slow contraction which was completely antagonised by prostaglandin blocker SC19220. On isolated rat duodenum, SA-HT produced slow relaxation. SA-HT significantly increased plasma plasmin, serum MDA level and decreased serum SOD level indicating the possible involvement of cyclooxygenase and lipooxygenase pathway.