Final Report Summary - LEANGREENFOOD (Enzyme technology for Lean and Green food processing) The LEANGREENFOOD network has educated young scientists to rethink current, established food processes and utilise new technology for socially and environmentally responsible management of natural resources in a world of tightening food supplies and climate change. The network focus is on improved yields of biomasses, decreased water and energy consumption, and lower use of chemicals. This includes development of new food processes, in which enzymatic conversions are performed in highly concentrated systems, chemical extraction is replaced by enzymatic extraction, and where novel analytical tools are developed for characterization of raw materials, intermediates, and the final products from these new processes. Consumer perceptions of the foods produced by applying the novel technology are also studied.Use of enzymes to extract and modify food ingredients based on starch, pectin and proteins, reduces the emission of chemicals such as nitric acid in the pectin industry, and limits the use of water and energy in starch and protein refinery processes. By catalysing chemical processes, enzymes are the basis for green production in which the use of unwanted chemicals can be limited or even excluded. LEANGREENFOOD research has proved there is a large potential to increase efficiency and reduce energy and water consumption by applying enzyme technology and by optimising the enzymatic properties and reaction conditions making enzymes the basis for lean food production systems. Thus, enzyme technology is one of the keys towards a more sustainable food production. LEANGREENFOOD has produced a platform of trained scientists and required technology, from which new food production systems meeting global challenges related to sustainability and consumer expectations can be developed.LEANGREENFOOD is subdivided into 6 work packages with separate, yet interconnected objectives including scientific and complementary training (WP1-2), reduction of chemicals (WP3), development of novel analytical technologies (WP4), reduction of energy and water (WP5), and consumer perception of new technology (WP6).The scientific training activities provided the LEANGREENFOOD fellows with knowledge of methods, theories and models at advanced levels and emphasised a multidisciplinary approach. Highly qualified international faculty was engaged from both academia and industry, and where possible and relevant also experts outside the network were involved. Training activities were interactive and ‘hands-on’ allowing adequate interaction between the participants and faculty promoting independent development of solutions to research questions. Parts of the training programme were specifically developed for the network combining existing approaches to training in food science with a new paradigm of environmental awareness to face the actual challenges of the food production. The activities had the format of intensive courses, and were open to researchers - mainly PhD students - from outside the network. The training also included complementary skills like project management, presentation techniques towards the scientific community and the general public, collaborative research and transfer of knowledge between different fields of research. Also, ethics in food science and global food production were focus areas in order to secure a mind-set keeping sustainability and responsibility at the centre of scientific thinking among the researchers. Excursions to industrial partners in Europe and China including lectures from the industry were completed in order for the fellows to be able to see their own research in an industrial context and to be able to relate to the real problems faced by industry in the production of foods. Intercultural communication and collaboration skills were promoted through secondments and training periods in another country than place-of-work, and this also stimulated exchange of knowledge between different scientific communities.The reduction in use of chemicals in the production of hydrocolloids and oligosaccharides by use of specific enzymes and pressure tuning for extraction and modification of food ingredients has shown to be an environmentally friendly process. Mono-component enzymes with only one enzymatic activity towards pectin, proteins, or xyloglucan have been expressed and characterized, demonstrating that it was possible to substitute classical acid-based extraction of waste stream from sunflower peels, citrus peels and sugar beet pulp with enzymatic catalysis and obtain pectin products and oligosaccharides with specific functional properties. The raw materials, the enzymatic process and the final products, pectin and oligosaccarides have been analysed by a range of new state-of-the-art techniques developed in WP4. Chemical synthesis of the rhamnogalacturonan I oligosaccharides have been accomplished to test towards enzymes and antibodies acting on plant polysaccharides. Pressure tuning of enzymes acting on lime peel extracted pectin polymers showing that the combined use of high pressure and enzymes adds a novel dimension to biocatalysis reactions as being environmentally friendly and sustainable.The novel analytical methods in LEANGREENFOOD comprised developments of microbial biosensors based on bioluminescence and ice nucleation showing the novelty of using biosensors for analysis of carbohydrates in foods. Further, a novel, green method for methyl esters determination in foods based on automated microfluidic analytical methods has been accomplished. A genetically modified glucose oxidase with improved thermal stability has been obtained using the approaches of site-directed mutagenesis and directed evolution in order to construct biosensors with increased lifetime for medical and food applications. Immobilisation of beta-galactosidase using cellulose or chitin as a carbohydrate-binding module has been performed to improve the stability and catalytic properties of the enzyme. The introduction of the concept Temporal Evolution Profiling for assessment of enzyme activity has proven to be successful as a fast and reliable way for measuring enzymatic carbohydrate modification either in biomass pre-treatment or during pectin extraction. A novel immuno-glycan microarray technology for the high-throughput screening of glucosyl hydrolsases (GHs) was developed, and the method was optimized and validated resulting in a filed patent entitled: ‘High throughput screening of glucosyl hydrolase enzymes’. The significant advantage of this method is that GH activities can be determined even when many substrates are mixed together. Moreover, this technology can be used to screen already identified enzymes (purified or non-purified) and previously uncharacterised enzymes.Methods based on enzymatic fingerprinting were assessed for their ability to reveal some structural characteristics of lemon pectins. The use of a combination of endopolygalacturonase II and pectin lyase degraded pectin into oligomers that were analysed by different chromatography techniques showing a discrimination of pectins with different patterns of methyl-esterification. The pattern of cleavage of a xyloglucanase analysed by LC-MS showed that the enzyme specifically cleaves xyloglucan polymer between two consecutive X units.The reduction of water and energy consumption in WP5 focused on enzymatic hydrolysis processes of food biopolymers in extremely highly concentrated systems by the use of extreme process conditions. The effect of high solid loadings on various physical and chemical properties of the solutions resulted in a concentration effect, with a slower hydrolysis rate at higher solid concentrations. The solubilization of high molecular weight arabinoxylans and degradation of these arabinoxylans has been modelled using the specificity of the endo-xylanases and combinatorial application of industrially used enzyme preparations revealed synergistic potential towards arabinoxylan degradation. Purification and identification of individual enzymes present in synergistically active preparations demonstrated high activity of an acetyl xylan esterase together with crude, fungal preparations. The development of synergistically active enzyme mixtures that show high rates of arabinoxylan conversion is a useful strategy to improve the hydrolysis at high solids loadings using less water and energy, which makes the hydrolysis more sustainable. Further, models have been established for starch hydrolysis that can predict the outcomes of the investigated reactions at various conditions of substrate concentrations and temperatures. A model for maltodextrin hydrolysis is under construction.Mapping of consumer attitudes in Europe and China to new lean and green techniques potentially implemented by the food and ingredient industries as result of the research and development activities in LEANGREENFOOD were investigated in WP6. The underlying mechanism of how consumers in China and Denmark create liking or disliking for new food technologies was explained by the moderating effect of social trust in the two countries. The impact of situational factors on process-related product attributes like process benefit, environmental benefit, and food technology concluded that in particular the interaction of time pressure and priming of environmental values appear to determine people’s emphasis on process related product attributes.To sum up, LEANGREENFOOD has trained highly skilled scientists who can rethink the food production systems to meet global challenges related to sustainability and consumer expectations. Access to further information about LEANGREENFOOD can be found at http://www.leangreenfood.eu/ or can be obtained from the coordinator Karsten Olsen, Department of Food Science, University of Copenhagen (firstname.lastname@example.org).