Fernando García-Carreño

Nutritive value of squid and hydrolyzed protein supplement in shrimp feed

Though some protein sources like squid and protein hydrolysates are assumed as growth enhancers for shrimp, little is known about the biochemical basis of this phenomenon. Low, heat-dried squid (Dosidicus gigas) (SQ) and two commercial protein hydrolysates from fish (FH) and krill (Euphasia sp.) (KH) were assayed in feeding trials with Penaeus vannamei. Feeds were prepared with the tested proteins at 3%, 9%, and 15% of the total crude protein. A total of nine experimental feeds plus a commercial one as control (C32) were tried. Additionally, digestibility in vivo and in vitro was evaluated. Survival was not different among groups. Weight gain of shrimp and total and specific proteolytic activity for trypsin and chymotrypsin were affected by type and quantity of supplemented protein. In vivo and in vitro digestibilities were also influenced by the source and quantity of the protein supplement. Shrimp fed feed with FH at 3% protein supplementation grew more than those fed with higher supplementations. Groups fed SQ had similar results as those fed FH, and gained more weight when fed the lowest SQ quantity. SDS-PAGE showed a large concentration of small peptides in SQ, which may explain results similar to FH. KH enhanced shrimp growth at all supplementations and had a lower degree of hydrolysis (DH) than FH. SQ also demonstrated good growth performance, but better at the lower supplementation, probably because of the presence of small peptides and possibly free amino acids from protein hydrolyzed by endogenous enzymes in the squid mantle. We conclude that hydrolyzed protein is a good supplement for shrimp feeds, but it must meet specific requirements for adequate assimilation.

Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei

We investigated the effect of starvation as a stimulant of the digestive system on digestive proteinase activities in the white shrimp Penaeus vannamei. The starved organisms were sampled periodically according to the molting stage and compared with a continuously fed group. Molting stage was included as an independent variable. Most analyzed variables, except for trypsin, were more affected by starvation than by molting, indicating that starvation is a stimulant that masks the effect of molting and showing that food or alimentary stress is more conspicuous than physiological ones. We found that starvation is a stimulant that surpasses the effect of molting, and because it affects the activity of digestive proteinases, studies of starving organisms in combination with tools of molecular biology, can be a helpful working model in the understanding of mechanisms of regulation of digestive enzyme activity. In the starved organisms, trypsin and chymotrypsin activities were similar, suggesting dependence of one to the other. Changes in proteolytic activities and the number of protein bands in electrophoresis showed evidence of synthesis regulation in the midgut gland of white shrimp.

Invertebrate trypsins: a review

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.

The digestive proteases of langostilla (pleuroncodes planipes, decapoda): their partial characterization, and the effect of feed on their composition

Abstract 1. 1. Three methods of recuperating and preserving enzyme activity from freshly-caught langostilla were assessed. In the pressing and acetone extract methods, the recovered specific activity was similar. 2. 2. Protease activity was higher between 6.5 and 8 pH, and was sensitive to high temperatures. 3. 3. In PAGE and serine inhibition assays, one fraction resembled bovine trypsin. 4. 4. The composition of proteins and molecules bearing protease activity from the hepatopancreas and stomach of both fed and starved animals was similar, indicating proteases are not induced but constitutive.

Protein digestion in penaeid shrimp: digestive proteinases, proteinase inhibitors and feed digestibility

Protein is the most abundant ingredient in both natural and prepared diets of penaeid shrimp. The assessment of protein digestion through the developmental stages of penaeids may contribute to the development of more suitable feeding schedules for their cultivation. Among the techniques to study protein digestion, detection and characterization of digestive proteinase inhibitors in w proteinaceous feed ingredients can be achieved by substrate- sodium dodecyl sulphate polyacryl- x . amide gel electrophoresis SDS-PAGE . In vitro assays of protein digestibility are also useful tools when testing alternative protein sources in the formulation of shrimp feeds. The present . article reviews three methods that have been used to assess protein digestion: 1 detection and characterization of proteinase activity and proteinaceous proteinase inhibitors by substrate-SDS- . . PAGE, 2 quantification of proteinase activity, and 3 in vitro evaluation of digestibility of dietary protein sources by shrimp proteinases. A compilation of previously reported and unpub- lished data on some aspects of penaeid protein digestion is presented. Trypsin activity of Litopenaeus schmitti varied considerably during larval and postlarval development, showing the highest value at protozoea III. The molecular weight of digestive proteinases from early stages of Farfantepenaeus paulensisalso differed from the adult pattern, and some activity bands could be characterized as trypsin in adult F. paulensis. The digestive proteinase pattern of adult Farfante- penaeus californiensis, F. paulensis, L. schmitti and Litopenaeus ˝annamei in SDS-PAGE showed clear differences among these species, which may be evidence of a species-specific pattern of protein digestion. In vitro evaluation of digestibility of aquafeeds can be achieved by the pH-stat method, which can help in the choice for alternative protein sources. Moreover, the quality

pH-stat method to predict protein digestibility in white shrimp (Penaeus vannamei)

Abstract In aquaculture, feeding trials are the most dependable method of measuring nutritive value of feeds. However, these methods are time consuming, expensive, and the results may be affected by environmental factors. In vitro methods to evaluate protein digestibility are rapid and allow close observation of how far bonds are cleaved using only small amounts of raw materials. We developed a pH-stat assay using an enzyme fraction from shrimp hepatopancreas. We used the assay to evaluate the degree of hydrolysis (DH) and estimate protein quality in menhaden, anchovy, white fish, tuna waste, soybean protein, and langostilla meals. Data obtained by the pH-stat method were compared with those obtained by chemical analysis and an in vivo apparent protein digestibility (APD) assay. The pH-stat method predicted the protein quality better than chemical analysis. The APD of the experimental diets ranged from 64–91%. A significant correlation (P

Characterization of fish acid proteases by substrate-gel electrophoresis

Several analytical techniques based upon the use of substrate-polyacrylamide gel electrophoresis were evaluated to achieve characterization of aspartate proteases in fish stomach. Since aspartate proteases of fish are more stable at high pH than mammalian pepsins, the most accurate technique for activity assessment is electrophoresis at neutral pH and revealing of such activity at low pH with hemoglobin as substrate. The technique is suitable for characterization of proteases and in comparative assessment of acid protease activity in different sparids.

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting.

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.

Digestive proteinases of Brycon orbignyanus (Characidae, Teleostei): characteristics and effects of protein quality

Juvenile piracanjuba, Brycon orbignyanus, in the wild consume protein from both plant and animal sources. Digestion of protein in piracanjuba begins in the stomach with pepsin, at low pH, and is followed by hydrolysis at alkaline pH in the lumen of the intestine. The digestive system in piracanjuba was evaluated to characterize the enzymes responsible for the digestion of feed protein and their composition. The gastric tissue synthesizes pepsin and the intestine tissues trypsin and chymotrypsin. Operational variables were evaluated and defined for future studies of the digestive system physiology. The enzymatic activity in the intestine and the relative concentration of enzymes were heavily influenced by the composition of the feed and the feeding regime, as detected by substrate-SDS-PAGE. Piracanjuba possess a mechanism of enzyme adaptation responding to food quality and regime, by varying the amount and composition of digestive proteases. This is a requisite study to determine the enzymes digesting protein in food and their characteristics and to gain some clues about the possible regulation mechanisms of enzyme synthesis in piracanjuba.

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.