Periodic Report Summary 2 - WINESENSE (Research on extraction and formulation intensification processes for natural actives of wine) Europe is the world-leading producer of wine and grape must (60% of world production) Wine industry is an important sector for the EU economy and the development of new wine related products could lead a sustainable growth and development of the sector. Within the wine industry is essential to find new ways to expand the business. This project has improved the extraction process to catch most of the active parts of the polyphenols (presents in grapes), to achieve a suitable formulation for food, and cosmetics additives based on Solvent Free Microwave Extraction (SMFE), Microwave Assisted Extraction (MAE), and emulsion-template techniques combining high pressure and antisolvent effects, and to evaluate the possibilities for commercialization of products and processes developed. The extraction work package was aimed to develop an extraction technology by microwave processing, and to develop fundamental knowledge in the field of microwave extraction of compounds from natural products. Initial work was addressed to study conventional Solid-Liquid extraction (SLE), to be used as reference values in order to evaluate the efficiency of the developed microwave pretreatment. Next, two different microwave processing techniques were evaluated. The first one, SFME, proved inefficient, as it provided similar results to conventional extraction. However, the second technique, Microwave Assisted Extraction (MAE), led to positive results. The extraction procedure was optimized for the most influencing operation variables (solvent composition, solid/liquid ratio and absorbed energy). Optimal conditions led to 48% improvement in TPC extract yield, and a final dry product richness a 35% higher than with conventional extraction. In the case of anthocyanins, extract yield was improved by 85%. Bioactivity test performed with these improved extracts showed a 100 percent more pronounced cellular antioxidant activity against peroxyl radical damage. Final results were used to develop the design of a continuous microwave oven, in collaboration with researchers at Universidad Politécnica de Cartagena (Spain). This pilot plant scale oven has an installed power of 3 kW, and a capacity of 20 kg/h. The oven was finally assembled, and absorption efficiency was proved to be above 98%. That is a relevant result to minimize the energy consume and approach this technology to the market.The formulation (WP 2) aimed at providing a stable and highly concentrated formulation either in aqueous or solid form of model compounds with interesting properties and application in nutraceutical and cosmetic industry and the extract as a whole. Regarding formulation of quercetin, as a model water-insoluble compound, a stable quercetin suspension of 630ppm was produced by High Pressure Emulsion Template technique using lecithin (25g/L) as a carrier. In order to intensify the process of solvent removal, the use of SFEE (supercritical fluid extraction of emulsions) was studied as an alternative to solvent evaporation. The modeling of the SFEE process showed that density of supercritical carbon dioxide was the main parameter. Further, best quercetin formulations were processed to obtain a quercetin-lecithin powder by PGSS-drying with improved dermal permeability. About formulation of epigallocatechin gallate, as a model water-soluble compound, double emulsions experiments yielded low encapsulation. Therefore, solid formulations were produced by PGSS-drying with good encapsulation(>75%) for all carriers tested (OSA-modified starch, lecithin and beta-glucane). The formulation of the multicomponent polyphenol extract was performed with commercial product from MATARROMERA (Eminol ®). Double emulsions experiments produced low stability emulsions. Therefore, the formulation was focused on dry powder formulations. PGSS-drying formulations yielded high encapsulation (90%) but low recovery although allowed the processing of high sugar/ carbohydrate content extracts with respect to spray drying for low solid: carrier ratio (7000ppm vs 4000ppm). Further formulations were produced with conventional spray (related with WP5 ToK activities) using maltodextrine, whey protein isolate and pea protein as carries. Good encapsulation was also achieved (>80%). Programmed tasks have been totally fulfilled and results are reported in detail in deliverables 2.1 (updated) and 2.3 and disseminated in well-known meetings and journals. Further a complete study on the influence of carrier material (maltodextrine, whey protein isolate and pea protein) on the accelerated and long term stability of the product for 1:1 ratio (solid content of extract: carrier ratio) has been carried out. Also, gastrointestinal release was determined to show immediate but partial release. Extracts and formulations from WP1, WP2 and further extracts from wine lees that were received over the project timeframe, were analyzed throughout the project in terms of phytochemicals by colorimetric assays, HPLC-DAD-MS and LC-MS, in order to identify the main compounds. Additionally, wine lees are being pointed as a rich source of specific polyphenols compounds, which are not founded on grape marc residues. Wine lees from different fermentation steps were characterised regarding anthocyanins and anthocyanins-derivatives and antioxidant activities. MW-pretreatments were applied to the 1st fermentation wine lees and the extracts obtained characterised and compared to the extracts obtained for grape marc in WP1 in terms of total polyphenols content, antioxidant activities and anthocyanins and anthocyanins-derivatives. The results pointed out to antioxidant ingredients being important to validate the antioxidant activity for distinct applications: Nutraceutical application: Extracts and formulations from WP1, WP2 (before and after in vitro digestion) were analysed in terms of antioxidant activity by chemical assays (ORAC, HORAC) and further their cytotoxicity and the intracellular antioxidant activity were assayed on human intestinal epithelial cells (Caco-2 cells). The extracts evaluated for this application were only from grape marc obtained using MW pre—treatments. Formulations of epigallocatechin gallate (EGCG) and grape marc extract (obtained using MW) were also tested. Results have been published in well-known journals. Phytopharmaceutical application: Targeting the development of novel phytopharmaceutical product, solid lipid formulations using pressurized-based techniques and inverse microemulsion precipitation methodologies were prepared in collaboration with WP2. The main goal was to design a natural anti-colon cancer agent, in the form of a new nutraceutical formulation, containing the multicomponent grape marc extract obtained in WP1 or purified fractions from WP2, for application in chemotherapy. Unfortunately, the particulate systems prepared showed to be very cytotoxic not only for the colon cancer cells as well as for the cells that mimics the intestinal epithelia. Cosmetic applications. Extracts obtained from different winemaking waste streams (grape marc and wine lees), by different extraction procedures, were screened for bioactivity in several biological endpoints related to skin aging. For this purpose, all samples were characterized in terms of antioxidant activity in chemical assays (ORAC/HOSC/HORAC) and cell-based assays in keratinocytes (HaCaT) and fibroblasts (HFF). Inhibitory capacity towards specific enzymes, namely tyrosinase, elastase, and MMP-1, was evaluated. The project has allowed the mobility of 11 researchers, all of them improve his background significantly, and this project allows them to reach a better position. 3 of them in private companies as senior researchers, 4 postdoctoral positions in public research centers, and 3 of the reach a position to complete his Ph.D. and one in a University office of transfer of knowledge.About Impact drivers and detailed information about dissemination activities can be summarized i) Dissemination of WineSense at 72 Conferences ii) Presence at 12 Exhibition with WineSense poster and presentation iii) 66 publications through WineSense webpage iv) 3 publication through other webpages vi) Outreach activities: 3 training schools.For more information about WineSense project, visit our web site at http://winesense.eu/ or here are ways to contac us:María José Cocero Alonso Head of the group,mjcocero@iq.uva.esPhone: +34 983423174Departamento de Ingeniería Química y Tecnología del Medio Ambiente Escuela de Ingenierías Escuela de Ingenierías Industriales – Sede MergelinaUniversidad de ValladolidValladolid – 47011Spain Other report summaries Periodic Report Summary 1 - WINESENSE (Research on extraction and formulation intensification processes for natural actives of wine)
