Neutral calcium-activated proteases from European sea bass (Dicentrarchus labrax L.) muscle: polymorphism and biochemical studies

Calcium-dependent proteinases or calpains were studied in fish muscle. Hydrophobic chromatography, followed by anion-exchange chromatography of the soluble fraction of sea bass white muscle proteins, resulted in three peaks of calcium-dependent protease activity at neutral pH (A, B and C). They are all neutral cysteine calcium-activated proteinases and can, therefore, be classified as calpain-like enzymes. From the Ca2+ concentration required for activity, A is a mu-calpain, and B and C are m-calpains. They share many properties with calpains from other vertebrate cells but differ in native mass, subunit composition, and the unusual numbers in which they are present. Their specific pattern of expression throughout the year could be of great importance to the resulting rate and extent of degradation of fish flesh after death.     

Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

The diatom Haslea ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine”, a blue-green pigment of which the chemical nature still remains unknown. This pigment is responsible for the greening of oyster gills. Here, we report a new method for extraction and purification of intracellular (accumulated in the apex of the cell) and extracellular (released into the external medium) forms of the pigment. Intracellular marennine is obtained by extraction from blue algal pellets with a carbonate buffer. The extract is then centrifuged and filtered. Extracellular marennine is obtained by clarification of blue-coloured culture medium. Both extracts are then purified by a semi-preparative process, using ultrafiltration through membranes and anion-exchange chromatography. This procedure allows us to produce native pigment displaying the degree of purity required to enter upon the molecular characterisation of marennine. By this process, about 35% of the initial amount of pigment can be recovered. If necessary, this method could be easily scaled up to a larger production system to accommodate potential industrial applications.     

Determination of the nutritional value of proteins obtained from Ulva armoricana

The in vitro digestibility of Ulva armoricana proteins by trypsin, chymotrypsin and human intestinal juice was determined to evaluate their nutritional value. The amino acid composition of the protein fraction and its changes during a sampling period from October to February were also studied. Some differences in the protein pattern shown by SDS PAGE were found in different months, such as the presence of a 54 kDa protein in February. The protein fraction is composed mainly of aspartic and glutamic acids (24–35% of protein fraction, according to season) and the essential amino acids constitute 27–36% of the total fraction. The efficiencies of trypsin and chymotrypsin in Ulva protein digestion are comparable. Only four proteins with apparent molecular weights of 86, 68, 40, and 29 KDa are digested by these proteolytic systems. The proteins from the October sample were more sensitive to chymotrypsin than those from the February sample. For instance, two proteins with apparent molecular weights of 100 and 67 kDa were weakly digested by chymotrypsin in the February extract, were fully digested in the October sample. The February sample differed from two others in the presence of glycosylated proteins, most of which have apparent molecular weights higher than 43 KDa. With the October sample, the activity of human intestinal juice was more effective than two other proteolytic systems. This is especially evident with a 27 kDa protein, which was only partially digested by the intestinal liquid and not digested by chymotrypsin or trypsin. However, human intestinal juice in the February apparently did not attack the 27 kDa protein. These data suggest a change in protein structure making it less sensitive to human intestinal juice. The glycosylation of protein extract, which was especially marked in February, could explain the differences in behaviour of U. armoricana proteins in response to the digestive action of human enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutritional value of proteins from edible seaweed Palmaria palmata (dulse)

Palmaria palmata (Dulse) is a red seaweed that may be a potential protein source in the human diet. Its protein content, amino acid composition, and protein digestibility were studied with algae collected every month over a 1-year period. Significant variations in protein content were observed according to the season: The highest protein content (21.9 +/- 3.5%) was found in the winter-spring period and the lowest (11.9 +/- 2.0%) in the summer-early autumn period. Most of the essential amino acids were present throughout the year. After 6-hour in vitro digestion in a cell dialysis using porcine pepsin and porcine pancreatin, the digestibility of proteins from Palmaria palmata crude powder, represented by dialyzed nitrogen, was estimated at 29.52 +/- 1.47%. Relative digestibility was 56%, using casein hydrolysis as 100% reference digestibility. In vitro digestibility of proteins extracted in water was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis using either bovine trypsin, bovine chymotrypsin, pronase from Streptomyces griseus, or human intestinal juice. Dulse proteins were hydrolyzed to a limited extent, which confirmed a rather low digestibility. Hydrolysis rate was higher with trypsin and lower with chymotrypsin compared with the two other enzymatic systems, pronase and intestinal juice, respectively. The association of algal powder and protein extract to casein and bovine serum albumin, respectively, produced a significant decrease in the hydrolysis rate of the standard proteins. In conclusion, the digestibility of Palmaria palmata proteins seems to be limited by the algae non-proteic fraction.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Identification by SDS PAGE of green seaweeds ( Ulva and Enteromorpha) used in the food industry

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.     

Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata

Proteins have been extracted from the edible seaweeds Ulva rigida Agardh and Ulva rotundata Bliding using classical or enzymatic procedures. The protocols using NaOH under reductive conditions or a two-phase system (PEG/K₂CO₃) produced the best protein yields. The cleavage or the limitation of the linkages between proteins and polysaccharides caused by these experimental conditions probably explains the efficiency of these protocols. In SDS PAGE, the protein fraction obtained after NaOH extraction from U. rotundata is characterised by the presence of three major bands with apparent molecular weights of 45 600, 31 800 and 18 600. The protein fraction from U. rigida presents two specific bands with apparent molecular weights of about 27 000 and 12 000. These fractions are mainly rich in aspartic and glutamic acids, alanine, glycine and contain few hydroxyproline residues (0.91–2.44% total amino acid content). The use of cellulase does not significantly improve the extraction of algal proteins in comparison with the blank procedure (without enzymes). The weak accessibility of the substrates in the intact cell wall could explain these experimental data. The improvement of protein yield after the use of the polysaccharidase mixture (-glucanase, hemicellulase, cellulase) partially confirms this hypothesis.     

Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.     

Seasonal composition of lipids, fatty acids, and sterols in the edible red alga Grateloupia turuturu

Composition of lipids, sterols, fatty acids (FA), and phospholipids in the edible Rhodophyta Grateloupia turuturu from Britanny, France, was investigated over four seasons in order to identify compounds with potential benefits in health and nutrition. The lipid content was found to vary from 3.3 to 4.1 % dry weight. No marked variations were observed for glycolipids accounting for 42.3–46.8 %, whereas neutral lipids and phospholipids fluctuated from 20.1 % (summer) to 41.8 % (winter), and 11.2 % (winter) to 33.4 % (summer), respectively. Polyunsaturated FA of the total lipids were found from 20.4 % (winter) to 31.1 % (summer), including 20:5 ω3 acid as the major one (up to 16.3 % in summer). Phosphatidylcholine (20.0–43.7 %) and phosphatidylserine (24.6–37.5 %) were the dominant phospholipids in all seasons. Compounds of interest were identified in minor amounts such as squalene, α-tocopherol, phytonadione (vitamin K₁), cholesteryl formate, cholest-4-en-3-one, and cholesta-4,6-dien-3-one. Cholesterol was the major sterol with a lower content in spring and summer.     

An evaluation of methods for quantifying the enzymatic degradation of red seaweed Grateloupia turuturu

For better exploitation of the red seaweed Grateloupia, enzymatic digestion of the thallus may be a way to increase access to metabolites of industrial interest. With this aim, we have tried to find a method to quantify the efficiency of enzymatic digestion. Vegetative algal material was treated with polysaccharidases (Onozuka R-10 cellulase, agarase, and Ultraflo L mixture). The proportion of degraded surface area was determined by microscopic measurement of the residue surface using imaging software and compared with the analysis of carbohydrates and R-phycoerythrin released in the incubation solution. Both the reducing carbohydrate concentration and percentage of degraded surface area appeared the most reliable methods to study enzymatic efficiency. The amount of solubilized total carbohydrates, and particularly that of R-phycoerythrin, showed non-specific variations, so no conclusions could be drawn. The application of this procedure to the screening of the efficient digestion of Grateloupia material demonstrated that cell walls were only partially digested by polysaccharidase enzymes alone. The Ultraflo L mixture and Onozuka R-10 cellulase produced a greater degradation of Grateloupia tissues and a higher release of reducing carbohydrates, whereas agarase did not display any specific action. Thus, the proposed procedure based on the quantification of residue surface area seems to be an accurate method to analyze enzymatic digestion. Other tests using different concentrations and combinations of enzymes are now required.