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AimsThis study aims to assess the use of marine lactic acid bacteria (LAB) to reduce Vibrio parahaemolyticus levels during oyster depuration process.Methods and resultsThe inhibitory effect of 30 marine LAB strains against V. parahaemolyticus strains was evaluated by in vitro assays. A total of three positive strains (Latilactobacillus sakei SF1583, Lactococcus lactis SF1945, and Vagococcus fluvialis CD264) were selected for V. parahaemolyticus levels reduction during oyster depuration. Pacific oysters Crassostrea gigas were artificially and independently contaminated by four GFP-labelled V. parahaemolyticus strains (IFVp201, IFVp69, IFVp195, and LMG2850T) at 105 CFU ml−1 and then exposed by balneation to 106 CFU ml−1 of each LAB strains during 24 h, at 19°C. Quantification of V. parahaemolyticus in haemolymph by flow cytometry revealed variations in natural depuration of the different V. parahaemolyticus strains alone. Furthermore, the addition of LABs improved up to 1-log bacteria ml−1 the reduction of IFVp201 concentration in comparison to the control condition.ConclusionsAlthough further optimizations of procedure are needed, addition of marine LABs during oyster depuration may be an interesting strategy to reduce V. parahaemolyticus levels in Crassostrea gigas.

Recent advances in glycobiotechnology show that bacterial exopolysaccharides (EPS) presenting glycosaminoglycan (GAG)-like properties can provide a valuable source of bio-active macromolecules for industrial applications. The HE800 EPS, named diabolican, is a marine-derived anionic high-molecular-weight polysaccharide produced by

Serratia sp. cause food losses and waste due to spoilage; it is noteworthy that they represent a dominant population in seafood. The main spoilage associated species comprise S. liquefaciens, S. grimesii, S. proteamaculans and S. quinivorans, also known as S. liquefaciens-like strains. These species are difficult to discriminate since classical 16S rRNA gene-based sequences do not possess sufficient resolution. In this study, a phylogeny based on the short-length luxS gene was able to speciate 47 Serratia isolates from seafood, with S. proteamaculans being the main species from fresh salmon and tuna, cold-smoked salmon, and cooked shrimp while S. liquefaciens was only found in cold-smoked salmon. The genome of the first S. proteamaculans strain isolated from the seafood matrix (CD3406 strain) was sequenced. Pangenome analyses of S. proteamaculans and S. liquefaciens indicated high adaptation potential. Biosynthetic pathways involved in antimicrobial compounds production and in the main seafood spoilage compounds were also identified. The genetic equipment highlighted in this study contributed to gain further insights into the predominance of Serratia in seafood products and their capacity to spoil.

Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain Alteromonas infernus have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that A. infernus produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.

Bacteria have developed a unique strategy to survive in extreme environmental conditions through the synthesis of an extracellular polymeric matrix conferring upon the cells a protective microenvironment. The main structural component of this complex network constitutes high-molecular weight hydrophilic macromolecules, namely exopolysaccharides (EPS). EPS composition with the presence of particular chemical features may closely be related to the specific conditions in which bacteria evolve. Deep-sea hydrothermal vent bacteria have already been shown to produce EPS rich in hexosamines and uronic acids, frequently bearing some sulfate groups. Such a particular composition ensures interesting functional properties, including biological activities mimicking those known for glycosaminoglycans (GAG). The aim of the present study was to go further into the exploration of the deep-sea hydrothermal vent IFREMER (French Research Institute for Exploitation of the Sea) collection of bacteria to discover new strains able to excrete EPS endowed with GAG-like structural features. After the screening of our whole collection containing 692 strains, 38 bacteria have been selected for EPS production at the laboratory scale. EPS-producing strains were identified according to 16S rDNA phylogeny. Chemical characterization of the obtained EPS highlighted their high chemical diversity with the presence of atypical compositional patterns. These EPS constitute potential bioactives for a number of biomedical applications, including regenerative medicines and cancer treatment.

International audience; Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.

Vibrio diabolicus, a marine bacterium originating from deep-sea hydrothermal vents, produces the HE800 exopolysaccharide with high value for biotechnological purposes, especially for human health. Its genome was sequenced and analyzed; phylogenetic analysis using the core genome revealed V. diabolicus is close to another deep-sea Vibrio sp. (Ex25) within the Harveyi clade and Alginolyticus group. A genetic locus homologous to the syp cluster from Vibrio fischeri was demonstrated to be involved in the HE800 production. However, few genetic particularities suggest that the regulation of syp expression may be different in V. diabolicus. The presence of several types of glycosyltransferases within the locus indicates a capacity to generate diversity in the glycosidic structure, which may confer an adaptability to environmental conditions. These results contribute to better understanding exopolysaccharide biosynthesis and for developing new efficient processes to produce this molecule for biotechnological applications.

International audience; Alteromonas infernus bacterium isolated from deep-sea hydrothermal vents can produce by fermentation a high molecular weight exopolysaccharide (EPS) called GY785. This EPS described as a new source of glycosaminoglycan-like molecule presents a great potential for pharmaceutical and biotechnological applications. However, this unusual EPS is secreted by a Gram-negative bacterium and can be therefore contaminated by endotoxins, in particular the lipopolysaccharides (LPS). Biochemical and chemical analyses of the LPS extracted from A. infernus membranes have shown the lack of the typical LPS architecture since 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), glucosamine (GlcN), and phosphorylated monosaccharides were not present. Unlike for other Gram-negative bacteria, the results revealed that the outer membrane of A. infernus bacterium is most likely composed of peculiar glycolipids. Furthermore, the presence of these glycolipids was also detected in the EPS batches produced by fermentation. Different purification and chemical detoxification methods were evaluated to efficiently purify the EPS. Only the method based on a differential solubility of EPS and glycolipids in deoxycholate detergent showed the highest decrease in the endotoxin content. In contrast to the other tested methods, this new protocol can provide an effective method for obtaining endotoxin-free EPS without any important modification of its molecular weight, monosaccharide composition, and sulfate content.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.

Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and this contrasts with muscle of mammalian meats. The tenderization can be partly attributed to the acid lysosomal cathepsins and cytosolic neutral calcium-activated calpains. In this study, these proteases from fish and bovine muscles were quantified and compared. The cathepsin B and L activities were in more important amounts in sea bass white muscle than in bovine muscle. On the other hand, cathepsin D activity was 1.4 times higher in meat that in fish muscle, while cathepsin H was negligible in both muscles. Calpain activities were similar in both types of muscle. Moreover, calpastatin (calpain endogenous inhibitor) level is 3.9 times higher in sea bass white muscle. These differential activities are considered in relation to their probable involvement in post mortem degradation of muscle. (c) 2006 Elsevier Ltd. All rights reserved.

High-pressure processing is a nonthermal technique ensuring food product safety and enabling a longer shelf life. The purpose of this experiment was to evaluate the effect of high pressure on the main proteolytic enzymes involved in fish muscle degradation during storage. Enzymes were extracted with sarcoplasmic proteins from Dicentrarchus labrax sea bass white muscle. Activity of cathepsins B, D, H, and L was quantified in protein extract, whereas calpain activity was evaluated after isolation from its endogenous inhibitor. High-pressure processing up to 500 MPa enhanced the activity of cathepsin B, H, and L, whereas the activity of cathepsin D increased up to 300 MPa and decreased above 300 MPa. With regard to calpain activity, high-pressure processing led to a decrease of activity, which was zero above 400 MPa. We suggest a leading explanation based on simultaneous deactivation of enzymes and an increase of liberation from lysosomes for cathepsins and on dissociation of subunits for calpains.

The variations in the amounts of milli-calpain and its specific inhibitor in the white muscle of sea bass (Dicentrarchus labrax) during storage at 4 degreesC for up to 7 days were determined after separation by hydrophobic chromatography on a Phenyl Sepharose vel. There was a significant decline in post-slaughter m-calpain activity with an important inter-individual variability in the rate of decrease of the total activity. In contrast with the calpastatin of mammalian post mortem muscles, calpastatin remained constant within fish muscles after death. The initial levels of protease and inhibitor activities, and their behaviour through post mortem storage. are discussed and implications for the mechanism of tenderisation of fish muscle are suggested. (C) 2003 Elsevier Ltd. All rights reserved.

Marine biotechnology still remains a new and emergent science, which is closely linked to marine biodiversity and to the technological capacities of investigating more atypical ecosystems. Marine microorganisms show a unique biodiversity since they have to adapt to various marine environmental conditions such as low or high temperatures, alkaline or acidic water, high pressure, and limited nutrients. Marine natural products, especially marine polysaccharides, are attracting more and more attention. Microbial polysaccharides are of growing interest for many sectors of industry, resulting in isolation of new exopolysaccharide (EPS )-producing bacteria. The diversity of these polysaccharides arises from the structural variations (glycosidic bonds, side branching chains, monosaccharidic content) controlled through a genetic basis. A lower molecular weight and functionalized derivatives together with the native form of the polysaccharide have been shown to possess a variety of biotechnological activities. Therefore, the biophysical and biological properties have made them useful in many pharmaceutical, food, and industrial applications. This chapter gives information on EPS -producing bacteria from the marine environment, as well as on the carbohydrate molecule they produce, including the chemical composition or structure when available, the putative pathways of biosynthesis, and the potential applications in industry with a focus on healthcare and glycosaminoglycan-like compounds.

Although polysaccharides are ubiquitous and the most abundant renewable bio-components, their studies, covered by the glycochemistry and glycobiology fields, remain a challenge due to their high molecular diversity and complexity. Polysaccharides are industrially used in food products; human therapeutics fall into a more recent research field and pharmaceutical industry is looking for more and more molecules with enhanced activities. Glycosaminoglycans (GAGs) found in animal tissues play a critical role in cellular physiological and pathological processes as they bind many cellular components. Therefore, they present a great potential for the design and preparation of therapeutic drugs. On the other hand, microorganisms producing exopolysaccharides (EPS) are renewable resources meeting well the actual industrial demand. In particular, the diversity of marine microorganisms is still largely unexplored offering great opportunities to discover high value products such as new molecules and biocatalysts. EPS-producing bacteria from the marine environment will be reviewed with a focus on marine-derived EPS from bacteria isolated from deep-sea hydrothermal vents. Information on chemical and structural features, putative pathways of biosynthesis, novel strategies for chemical and enzymatic modifications and potentialities in the biomedical field will be provided. An integrated approach should be used to increase the basic knowledge on these compounds and their applications; new clean environmentally friendly processes for the production of carbohydrate bioactive compounds should also be proposed for a sustainable industry.

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.

Polysaccharides are ubiquitous in animals and plant cells where they play a significant role in a number of physiological situations e.g. hydration, mechanical properties of cell walls and ionic regulation. This review concentrates on heparin-like entities from marine procaryotes and eukaryotes. Carbohydrates from marine prokaryotes offer a significant structural chemodiversity with novel material and biological properties. Cyanobacteria are Gram-negative photosynthetic prokaryotes considered as a rich source of novel molecules, and marine bacteria are a rich source of polysaccharides with novel structures, which may be a good starting point from which to synthesise heparinoid molecules. For example, some sulphated polysaccharides have been isolated from gamma-proteobacteria such as Alteromonas and Pseudoalteromonas sp. In contrast to marine bacteria, all marine algae contain sulphated wall polysaccharides, whereas such polymers are not found in terrestrial plants. In their native form, or after chemical modifications, a range of polysaccharides isolated from marine organisms have been described that have anticoagulant, anti-thrombotic, anti-tumour, anti-proliferative, anti-viral or anti-inflammatory activities. In spite of the enormous potential of sulphated oligosaccharides from marine sources, their technical and pharmaceutical usage is still limited because of the high complexity of these molecules. Thus, the production of tailor-made oligo- and polysaccharidic structures by biocatalysis is also a growing field of interest in biotechnology.

Calpains (calcium-activated neutral proteases) of sea bass (Dicentrarchus labrax L.) muscle may participate in the degradation of muscle tissue during postmortem storage. These enzymes are regulated by calpastatin, their endogenous specific inhibitor. The objective of this study was to evaluate the changes encountered by the calpain system during the postmortem storage of fish muscle after high-pressure treatment. From 100 MPa, high-pressure treatment of purified calpains results in a loss of their activity as well as in the dissociation of the heterodimeric form. In muscle, the high-pressure processing decreases the initial activity of calpain. This loss in activity may be due to an inactivation by a change of structure. Initial calpastatin activity is not modified by the high-pressure treatment, but it decreases during the storage from the beginning for a treatment at 300 MPa after which calpastatin is stable during 2 d. Therefore, this study also suggests that high-pressure treatment could be a useful way to improve fish flesh quality.

Aims: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid, quantitative microplate fluorescent test. Methods and Results: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96 wells polystyrene microplate. The 4'6-diamidino-2-phenylindole (DAPI) dye was used to quantify adhered bacteria in each well. Total measured fluorescence in wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 µm2 and quantifying 2 x 107 to 2 x 108 bacteria adhered per cm2. The antifouling properties of three commercial surface active agents and chlorine were tested by this method in prevention of adhesion and also in detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on agent concentration showed a sigmoid shape like dose-response curve. Conclusions: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance and impact of the study: In contrast with usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on antifouling properties of products, especially the first steps of marine biofilm formation.

Introduction : Quality attributes of fish flesh, including food safety, organoleptic features, nutritional quality and aptitude to industrial transformation, influence consumption and acceptability of fish as food. Fish sensorial changes and texture properties are closely linked to freshness. Along with ante mortem muscle biochemistry, postmortem biochemical processes are directly linked to final quality attributes. The understanding of postmortem mechanisms is a prerequisite for an accurate control of the quality of commercialized fish by the identification of objective markers or indicators. Postmortem tenderization is one of the most important quality attributes of fish muscle. Loss of freshness is due to a complex combination of biochemical, chemical and physical processes, and is followed by muscle spoilage due to microbiological contamination. Autolytic modifications include protease action on proteins and connective tissue, and also fat hydrolysis. Tenderization is enzymatic in nature; physio-chemical conditions (pH, osmotic pressure) may modulate the proteolytic action of endogenous enzymes. Our studies have given additional information regarding the proteolytic mechanisms involved in postmortem disorganization of fish muscle by characterizing the myofibrillar components undergoing proteolysis 1-3 as well as by studying the involved proteases especially calpains 4;5 and their effects in vitro on myofibrillar proteins 6. The knowledge of the relationship between this disorganization, loss of freshness, and textural degradation should be improved in order to identify potential freshness indicators, and develop methods to improve quality. The purpose of this paper is to give a broad overview of quality changes in fish and the possible mechanisms responsible.

The effects of a milli(m)-calpain isolated from the white muscle of sea bass (Dicentrarchus labrax L) and commercial cathepsins B, D and L, used in combination on the myofibrillar and sarcoplasmic proteins were examined. Protein digestion was first performed by the endogenous m-calpain, (luring 2 h before the addition of a mixture of cathepsins B, D and L and a further incubation up to 22 h. Calpain degraded a 27 kDa sarcoplasmic component as well as myosin heavy chain, a-actinin, desmin and a 32 kDa component from the myofibrillar fraction. A 97 kDa component and the assumed creatine kinase-aldolase doublet were degraded during the incubation of sarcoplasmic proteins with the cathepsin mixture while, among the myofibrillar proteins, myosin, actin, tropomyosin, and the 22 kDa component were digested by the same enzymatic preparation. Myofibril-bound a-actinin and tropomyosin were released into the soluble fraction by m-calpain and the cathepsins mixture. Some smaller fragments were also produced. This appears to be the result of the cumulative action of each protease, as already investigated in our previous studies, except for the degradation of the sarcoplasmic 97 kDa component. It was only degraded if the cathepsins were mixed; this indicates that the three cathepsins functioned synergistically on muscle proteins. No difference in the proteolytic effect of cathepsins was observed, whether calpain was acting first or not. This work showed that calpain did not modify any muscle protein prior to later hydrolysis by the catheptic proteinases. (C) 2004 Elsevier Ltd. All rights reserved.

High pressure is an innovative non-thermal food-preservation technology. We studied the effect of high-pressure treatment up to 500 MPa for 5 min on physical characteristics of sea bass fillets after 0, 7, and 14 d of refrigerated storage. Color results exhibited an increase of lightness and a slight change of hue, which might be imperceptible in cooked fish. High-pressure treatment induced a decrease of exudation and water-holding capacity. Pressure treatment above 300 MPa provoked higher fish hardness after storage than in untreated sample, proving the ability of high pressure to improve textural quality of chilled, stored fish fillet. These assessments were corroborated with microstructure observations. We showed that high-pressure treatment at 500 MPa allowed, after 7 d of storage, a total aerobic count equivalent to that of untreated fresh fish fillet to be obtained. Thus, high pressure might be considered to be a technology able to improve safety and textural quality of fresh fish fillets.

EPS biochemical characterization and observation by epifluorescencemicroscopy. Development of a marine biofilmformation model onto a polystyrene microtiterplate using the “O’Tooleand Koltermethod”[1] adapted with the well known fluorescent DNA staining DAPI