Thermal degradation of red cabbage (Brassica oleracea L. var. Capitata f. rubra) anthocyanins in a water model extract under accelerated shelf-life testing.

Red cabbage (RC) represents a source of anthocyanins acylated with hydroxycinnamic acids (HCA) that are described to enhance their stability. Nevertheless, data about their thermal degradation are still controversial. Our aim was to comprehensively analyse the degradation kinetics of individual RC anthocyanins in a model aqueous extract treated at 40 °C × 30 days to simulate severe but realistic storage conditions. Free anthocyanins and radical-scavenging capacity showed different kinetics. The results confirm the high stability of RC anthocyanins (t1/2: 16.4-18.4 days), although HPLC analyses of each molecule displayed distinct kinetics with t1/2 from 12.6 to 35.1 days. In particular, the sinapoyl acylation negatively affected the stability of the anthocyanins, while the forms monoacylated with glycosyl p-coumaric and ferulic acids exhibited higher stability. In conclusion, our results indicate that acylation is not a prerogative of stability, as this is instead more dependent on specific acylation patterns and the glycosylation of HCA.

Nanoencapsulation of Anthocyanins from Red Cabbage (Brassica oleracea L. var. Capitata f. rubra) through Coacervation of Whey Protein Isolate and Apple High Methoxyl Pectin.

Encapsulation is a valuable strategy to protect and deliver anthocyanins (ACNs), phenolic compounds with outstanding antioxidant capacity but limited stability. In this study, coacervation was used to encapsulate an ACN-rich red cabbage extract (RCE). Two agri-food by-product polymers, whey protein isolate (WPI) and apple high-methoxyl pectin (HMP), were blended at pH 4.0 in a specific ratio to induce the formation of nanoparticles (NPs). The process optimisation yielded a monodispersed population (PDI < 0.200) of negatively charged (-17 mV) NPs with an average diameter of 380 nm. RCE concentration influenced size, charge, and antioxidant capacity in a dose-dependent manner. NPs were also sensitive to pH increases from 4 to 7, showing a progressive breakdown. The encapsulation efficiency was 30%, with the retention of ACNs within the polymeric matrix being influenced by their chemical structure: diacylated and/or C3-triglucoside forms were more efficiently encapsulated than monoacylated C3-diglucosides. In conclusion, we report a promising, simple, and sustainable method to produce monodispersed NPs for ACN encapsulation and delivery. Evidence of differential binding of ACNs to NPs, dependent on specific acylation/glycosylation patterns, indicates that care must be taken in the choice of the appropriate NP formulation for the encapsulation of phenolic compounds.

Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity.

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products.

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility.

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 µg/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 µg/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to Nω-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs.

Effects of Combination Treatments with Astaxanthin-Loaded Microparticles and Pentoxifylline on Intracellular ROS and Radiosensitivity of J774A.1 Macrophages.

Radiation-induced fibrosis (RIF) is a serious, yet incurable, complication of external beam radiation therapy for the treatment of cancer. Macrophages are key cellular actors in RIF because of their ability to produce reactive oxidants, such as reactive oxygen species (ROS) and inflammatory cytokines that, in turn, are the drivers of pro-fibrotic pathways. In a previous work, we showed that phagocytosis could be exploited to deliver the potent natural antioxidant astaxanthin specifically to macrophages. For this purpose, astaxanthin encapsulated into µm-sized protein particles could specifically target macrophages that can uptake the particles by phagocytosis. In these cells, astaxanthin microparticles significantly reduced intracellular ROS levels and the secretion of bioactive TGFß and increased cell survival after radiation treatments. Here we show that pentoxifylline, a drug currently used for the treatment of muscle pain resulting from peripheral artery disease, amplifies the effects of astaxanthin microparticles on J774A.1 macrophages. Combination treatments with pentoxifylline and encapsulated astaxanthin might reduce the risk of RIF in cancer patients.

Allergens from Edible Insects: Cross-reactivity and Effects of Processing.

PURPOSE OF REVIEW: The recent introduction of edible insects in Western countries has raised concerns about their safety in terms of allergenic reactions. The characterization of insect allergens, the sensitization and cross-reactivity mechanisms, and the effects of food processing represent crucial information for risk assessment. RECENT FINDINGS: Allergic reactions to different insects and cross-reactivity with crustacean and inhalant allergens have been described, with the identification of new IgE-binding proteins besides well-known pan-allergens. Depending on the route of sensitization, different potential allergens seem to be involved. Food processing may affect the solubility and the immunoreactivity of insect allergens, with results depending on species and type of proteins. Chemical/enzymatic hydrolysis, in some cases, abolishes immunoreactivity. More studies based on subjects with a confirmed insect allergy are necessary to identify major and minor allergens and the role of the route of sensitization. The effects of processing need to be further investigated to assess the risk associated with the ingestion of insect-containing food products.

Allergenicity assessment of the edible cricket Acheta domesticus in terms of thermal and gastrointestinal processing and IgE cross-reactivity with shrimp.

The allergenic potency of the cricket Acheta domesticus, a promising edible insect, has never been assessed. This work aims to study the immunoreactivity of Acheta domesticus, and its cross-reactivity with the shrimp Litopenaeus vannamei, assessing the effect of cooking and gastrointestinal digestion on their allergenic properties. Different cricket proteins were detected by immunoblotting with shrimp-allergic patients' sera. Tropomyosin was identified as the most relevant IgE-binding protein, and its cross-reactivity with shrimp tropomyosin was demonstrated by ELISA. While shrimp tropomyosin showed scarce stability to gastric digestion, cricket tropomyosin withstood the whole digestion process. The sarcoplasmic calcium-binding protein, specifically detected in shrimp, showed exceptional stability to gastrointestinal digestion. IgE-binding proteins in a model of enriched baked products were partially protected from proteolysis. In conclusion, the ingestion of A. domesticus proteins poses serious concerns to the Crustacean-allergic population. The high stability of tropomyosin may represent a risk of primary sensitization and clinical cross-reactivity.

Phagocytosis of Astaxanthin-Loaded Microparticles Modulates TGFß Production and Intracellular ROS Levels in J774A.1 Macrophages.

Radiation-induced fibrosis is a serious long-lasting side effect of radiation therapy. Central to this condition is the role of macrophages that, activated by radiation-induced reactive oxygen species and tissue cell damage, produce pro-inflammatory cytokines, such as transforming growth factor beta (TGFß). This, in turn, recruits fibroblasts at the site of the lesion that initiates fibrosis. We investigated whether astaxanthin, an antioxidant molecule extracted from marine and freshwater organisms, could help control macrophage activation. To this purpose, we encapsulated food-grade astaxanthin from Haematococcus pluvialis into micrometer-sized whey protein particles to specifically target macrophages that can uptake material within this size range by phagocytosis. The data show that astaxanthin-loaded microparticles are resistant to radiation, are well-tolerated by J774A.1 macrophages, induce in these cells a significant reduction of intracellular reactive oxygen species and inhibit the release of active TGFß as evaluated in a bioassay with transformed MFB-F11 fibroblasts. Micro-encapsulation of bioactive molecules is a promising strategy to specifically target phagocytic cells and modulate their own functions.

Identification and Characterization of IgE-Reactive Proteins and a New Allergen (Cic a 1.01) from Chickpea (Cicer arietinum).

SCOPE: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. METHODS AND RESULTS: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. CONCLUSION: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin; however, a ß-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments were much higher in cell wall-deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: Effects on stability and color properties.

The research of antioxidants and natural pigments to replace synthetic molecules is increasingly considering wastes from plant food supply chains. Red chicory (RCH) and red cabbage (RCA) are rich sources of polyphenols (PP), especially anthocyanins, well know natural pigments possessing strong antioxidant capacity and beneficial health effects. The aim of this work was to compare different solvents for PP extraction and to evaluate the effect of spray-drying encapsulation using modified starch on PP, antioxidant capacity (AOC) and color properties. Methanol:water (70:30) showed the best extraction capacity, while ethanol:water (70:30) extracts displayed the highest thermal stability. Ethanol:water extracts were spray-dried with a yield of 95-99% for both crops, while the efficiency of PP encapsulation was 79% (RCA) and 88% (RCH). Encapsulation improved retention of PP and AOC upon thermal treatment (RCH: 20-30%, RCA: 44-55%) without altering color properties. This process can be employed for the development of functional foods and supplements.

Development and Characterization of Astaxanthin-Containing Whey Protein-Based Nanoparticles.

Astaxanthin (ASX) is a carotenoid of great interest due to its potential health benefits. However, its use in the food, feed, and pharmaceutical fields is limited due to low bioavailability, poor stability during thermochemical treatments, susceptibility to oxidation, and poor organoleptic characteristics. The aim of this work was to develop a method to stabilize astaxanthin extracted from the microalgae Haematococcus pluvialis (H.p.) and to improve its nutritional and functional properties through nanoencapsulation. Nanoparticles (NPs) were produced by emulsification-solvent evaporation technique starting from H.p. oleoresin using whey proteins concentrate (WPC) as stabilizer. The efficiency of encapsulation was 96%. The particle size (Z-average) was in the range of 80-130 nm and the superficial charge (measured as zeta-potential) was negative (-20 to -30 mV). The stability of the NPs upon resuspension in water was assayed through a panel of stress tests, i.e., extreme pH, UV radiation, Fe3+ exposition, and heating at 65 °C, that always showed a superior performance of encapsulated ASX in comparison to oleoresin, even if NPs tended to precipitate at pH 3.5-5.5. Simulated gastroenteric digestion was conducted to study the release of ASX in physiological conditions, and showed a maximum bioaccessibility of 76%, with 75% ASX converted into the more bioavailable free form. The collected data suggest that NPs might have possible future applications as supplements for human and animal diets.

Effects of microencapsulation by ionic gelation on the oxidative stability of flaxseed oil.

Flaxseed oil is a major source of omega-3 polyunsaturated fatty acids (PUFAs), as it contains nearly 50% of alpha-linolenic acid. For this reason it is highly susceptible to auto-oxidation. The aim of the work was to increase the stability of flaxseed oil by a microencapsulation process based on ionic gelation through vibrating-nozzle extrusion technology, using pectin as shell material. Two different drying systems, passive air drying (AD) and fluid bed (FB), were compared. The results show that the encapsulation efficiency is very high (up to 98%). Besides being approximately 20-fold faster, FB gives beads showing on average higher payload (76% vs 68%) and lower peroxide value (9.64 vs 21.33) than the AD. An accelerated test carried out on FB-dried beads shows that the oxidative stability of encapsulated oil is 13-fold higher than bulk oil (PV FB: 20 vs PV oil: 260), demonstrating the protecting effect of microencapsulation.

Multi-approach metabolomics analysis and artificial simplified phytocomplexes reveal cultivar-dependent synergy between polyphenols and ascorbic acid in fruits of the sweet cherry (Prunus avium L.).

Fruits of the sweet cherry (Prunus avium L.) accumulate a range of antioxidants that can help to prevent cardiovascular disease, inflammation and cancer. We tested the in vitro antioxidant activity of 18 sweet cherry cultivars collected from 12 farms in the protected geographical indication region of Marostica (Vicenza, Italy) during two growing seasons. Multiple targeted and untargeted metabolomics approaches (NMR, LC-MS, HPLC-DAD, HPLC-UV) as well as artificial simplified phytocomplexes representing the cultivars Sandra Tardiva, Sandra and Grace Star were then used to determine whether the total antioxidant activity reflected the additive effects of each compound or resulted from synergistic interactions. We found that the composition of each cultivar depended more on genetic variability than environmental factors. Furthermore, phenolic compounds were the principal source of antioxidant activity and experiments with artificial simplified phytocomplexes indicated strong synergy between the anthocyanins and quercetins/ascorbic acid specifically in the cultivar Sandra Tardiva. Our data therefore indicate that the total antioxidant activity of sweet cherry fruits may originate from cultivar-dependent interactions among different classes of metabolite.

Tomato cystine-knot miniproteins possessing anti-angiogenic activity exhibit in vitro gastrointestinal stability, intestinal absorption and resistance to food industrial processing.

The cystine-knot miniproteins present in tomato fruit (TCMPs) have been shown to exert anti-angiogenic effects by inhibiting endothelial cell migration and to display resistance to gastrointestinal proteolytic attack. To better define the pharmacological potential of TCMPs, their oral bioavailability and their resistance to industrial processing must be assessed. To explore the intestinal transport of TCMPs we used the differentiated Caco-2 cells model. After 24h incubation, 37.73±9.34% of TCMPs crossed the epithelium, without altering the integrity of the cell layer. To assess the effects of the industrial processing on the biochemical features and the biological activity of TCMPs, we developed a method for purifying the proteins from tomato paste. The tomato-paste purified TCMPs retained the resistance to gastrointestinal digestion and the inhibitory activity towards endothelial cell migration. Our previous and present results collectively demonstrate that TCMPs possess interesting features for drug development.

Production of stable food-grade microencapsulated astaxanthin by vibrating nozzle technology.

Astaxanthin is a carotenoid known for its strong antioxidant and health-promoting characteristics, but it is also highly degradable and thus unsuited for several applications. We developed a sustainable method for the extraction and the production of stable astaxanthin microencapsulates. Nearly 2% astaxanthin was extracted by high-pressure homogenization of dried Haematococcus pluvialis cells in soybean oil. Astaxanthin-enriched oil was encapsulated in alginate and low-methoxyl pectin by Ca2+-mediated vibrating-nozzle extrusion technology. The 3% pectin microbeads resulted the best compromise between sphericity and oil retention upon drying. We monitored the stability of these astaxanthin beads under four different conditions of light, temperature and oxygen exposition. After 52weeks, the microbeads showed a total-astaxanthin retention of 94.1±4.1% (+4°C/-light/+O2), 83.1±3.2% (RT/-light/-O2), 38.3±2.2% (RT/-light/+O2), and 57.0±0.4% (RT/+light/+O2), with different degradation kinetics. Refrigeration, therefore, resulted the optimal storage condition to preserve astaxanthin stability.

Grapevine Downy Mildew Plasmopara viticola Infection Elicits the Expression of Allergenic Pathogenesis-Related Proteins.

BACKGROUND: Downy mildews are a group of microorganisms belonging to the Chromista kingdom that can infect specific plants. When growing on plant tissues these microbes can elicit the expression of pathogenesis-related proteins (PRs), a group of stress-induced proteins frequently described as allergens in many plant species. Our aim was to verify by a proteomic approach whether the allergic reactions experienced by a farmer working in a vineyard infected by Plasmopara viticola (Pv), the etiological agent of downy mildew, are elicited by PRs expressed by the grapevine upon infection or by allergens present in Pv. METHODS: A skin prick test and prick-to-prick test with infected field grapevine leaves and control leaves were carried out. Field leaves and ad hoc Pv-inoculated leaves were compared by SDS-PAGE and IgE-immunoblotting with extracts from control leaves and Pv sporangia. IgE-binding proteins were further separated by two-dimensional electrophoresis and the positive spots analyzed by nanoHPLC-Chip and tandem mass spectrometry (MS/MS) for identification. RESULTS: Only infected leaves showed IgE-binding protein bands at 42 and 36 kDa. This agreed with the positive skin prick test experienced by the patient only with the infected leaves extract. Two-dimensional electrophoresis followed by MS/MS analysis led to the identification of PR-2 (ß-1,3-glucanase) and harpin-binding protein 1 as putative allergens, the latter having never been reported before. CONCLUSION: The results indicate that Pv infection might represent a new source of plant allergens.

Study on the Immunoreactivity of Triticum monococcum (Einkorn) Wheat in Patients with Wheat-Dependent Exercise-Induced Anaphylaxis for the Production of Hypoallergenic Foods.

Wheat [Triticum aestivum (T.a.)] ingestion can cause a specific allergic reaction, which is called wheat-dependent exercise-induced anaphylaxis (WDEIA). The major allergen involved is ω-5 gliadin, a gluten protein coded by genes located on the B genome. Our aim was to study the immunoreactivity of proteins in Triticum monococcum (einkorn, T.m.), a diploid ancestral wheat lacking B chromosomes, for possible use in the production of hypoallergenic foods. A total of 14 patients with a clear history of WDEIA and specific immunoglobulin E (IgE) to ω-5 gliadin were enrolled. Skin prick test (SPT) with a commercial wheat extract and an in-house T.a. gluten diagnostic solution tested positive for 43 and 100% of the cases, respectively. No reactivity in patients tested with solutions prepared from four T.m. accessions was observed. The immunoblotting of T.m. gluten proteins performed with the sera of patients showed different IgE-binding profiles with respect to T.a., confirming the absence of ω-5 gliadin. A general lower immunoreactivity of T.m. gluten proteins with scarce cross-reactivity to ω-5 gliadin epitopes was assessed by an enzyme-linked immunosorbent assay (ELISA). Given the absence of reactivity by SPT and the limited cross-reactivity with ω-5 gliadin, T.m. might represent a potential candidate in the production of hypoallergenic bakery products for patients sensitized to ω-5 gliadin. Further analyses need to be carried out regarding its safety.

A cystine-knot miniprotein from tomato fruit inhibits endothelial cell migration and angiogenesis by affecting vascular endothelial growth factor receptor (VEGFR) activation and nitric oxide production.

SCOPE: Cystine-knot miniproteins are bioactive molecules with a broad range of potential therapeutic applications. Recently, it was demonstrated that two tomato cystine-knot miniproteins (TCMPs) exhibit in vitro antiangiogenic activity on human umbilical vein cells. The aim of the present study was to investigate the effects of a fruit-specific cystine-knot miniprotein of tomato on in vitro endothelial cell migration and in vivo angiogenesis using a zebrafish model. METHODS AND RESULTS: The cystine-knot protein purified from tomato fruits using gel filtration LC and RP-HPLC inhibited cell migration when tested at 200 nM using the wound healing assay, and reduced nitric oxide formation probed by 4-amino-5-methylamino-27-difluorofluoscescin diacetate. RT-PCR and Western blot analyses demonstrated that vascular endothelium growth factor A dependent signaling was the target of TCMP bioactivity. Angiogenesis was inhibited in vivo in zebrafish embryos treated with 500 nM TCMP. CONCLUSION: Our results demonstrate that cystine-knot miniproteins present in mature tomato fruits are endowed with antiangiogenic activity in vitro and in vivo. These molecules may confer beneficial effects to tomato dietary intake, along with lycopene and other antioxidants. Further investigation is warranted to explore the potential of these compounds as model scaffolds for the development of new drugs.

Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.

The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl ß-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry.