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Plant leaves are recognised as a potential source for food applications based on their nutritional profile and interesting technological properties of leaf components, and based on the large availability of plant leaves in agricultural waste streams. Besides proteins, leaves have a rich nutritional profile (e.g. dietary fibres, minerals and secondary metabolites) and consist of complex biological structures (e.g. chloroplastic membranes) that can be explored as novel fractions that ultimately broaden the use of leaves. The overall aim of this thesis is to explore green leaves as a food source, with emphasis on neglected leaf fractions. This thesis describes a processing approach that aims at separating/generating enriched- functional fractions rather than pure components, and highlights the implications for value creation out of green leaves. The extraction of leaf membrane proteins is investigated using a proteomics extraction method, while the properties of other valuable leaf components (complexes and fibres) are analysed for techno-functional applications. Furthermore, the feasibility of leaves as a food source is studied at an industrial scale, considering large scale processing and options for leaf stabilisation. The extraction of proteins from sugar beet leaves is evaluated in Chapter 2 by using a traditional heat coagulation method. The heat treatment is thought to precipitate the insoluble proteins together with fibres, chlorophyll and other components, resulting in a green curd. Therefore, the distribution of soluble and insoluble proteins was followed along the extraction process to discern the effect of the heating step on protein fractionation. This study showed that both soluble and insoluble protein distribute almost evenly over the leaf fractions juice, pulp, supernatant and final pellet. The even distribution of the proteins was attributed to the anatomy of leaves and their biological function, which is predominantly the enzymatic activity related to photosynthesis instead of protein storage, which occurs in other plant tissues. This chapter further concludes that striving for high purity severely compromises the yield, and consequently results in inefficient use of the leave proteins. Chapter 3 describes the application of proteomic analytical extraction protocols to analyse the fractionation behaviour of leaf proteins. This analysis lead to the translation into food- grade processes based on four fundamental extraction steps: (1) tissue disruption, (2) enzymatic inhibition, (3) removal of interfering compounds, and (4) protein fractionation and purification. Part of these extraction steps can be translated into food-grade alternatives, while the processing conditions determine the potential properties for food of the final products. Nevertheless, it was concluded that harsh and/or non-food grade conditions were required to isolate the leaf membrane proteins with high purity. Those results were explained by the fact that membrane proteins are heterogeneous w.r.t. charge, hydrophobicity, post- translational modification and complexation, leading to non-selective behaviour when compared with a single pool of proteins. Given the large challenges in isolating membrane proteins from leaves, we studied another approach in which green leaves are considered as a source of naturally structured elements that have relevant techno-functional properties for food products, like the chloroplastic membranes (i.e. thylakoid membranes) and cellulose-rich fibres. Chapter 4 describes the properties of thylakoid membranes and their emulsifying mechanism. These membranes showed surface active properties and their adsorption kinetics were typical for large molecules or soft particles. The thylakoid fragments can effectively stabilise emulsion droplets, even though aggregation was observed already during emulsion preparation and increased with increased thylakoid concentration. Both composition and structure make thylakoid membranes suitable as a biobased material for food and pharma applications. To continue exploring valuable fractions from leaves, Chapter 5 reports on the interfacial behaviour of cellulose-rich particles obtained from leaf pulp. Cellulosic particles were produced from the pulp obtained after leaf pressing. The particles spontaneous adsorption onto the oil-water interface and interfacial behaviour similar to that of solid particles. Addition of cellulosic particles to oil-in-water emulsions resulted in stable emulsions above a particle concentration of 0.1 w/v%, although phase separation was observed. The particle fines (0.04 – 1.0 µm) stabilised the droplet interface, while large particles formed a network in the continuous phase and rendered a top (green) phase in the emulsions. Finding applications for leaf side streams, like leaf pulp, broadens the options for total leaf processing and contributes to resource use optimisation. A sustainability assessment of leaf processing is discussed in Chapter 6, considering the challenges that may appear at industrial scale. The seasonal availability of sugar beet plants implies the need of processing large amounts of biomass within a short time due to their high moisture content (85 - 90%) and their sensitivity to spoilage. Processing options were evaluated on their resource use efficiency in terms of energy requirement and exergy indicators. A decentralised process constitutes a good option compared to freezing, since solid side streams can be directly returned the land, leaving nutrients to the soil, and reducing transportation loads. With a decentralised process, freezing of the leaves becomes unnecessary; the leaf juice is transported while chilled, resembling the transportation of fresh milk that is also chill-transported from the farm to a central factory. Chapter 7 concludes this thesis with a general discussion of the main findings. An integrated process for leaf valorisation is described, which combines the production of functional fractions with the production of bulk products such as protein-rich and fibre-rich fractions. A compilation of data on protein yield and protein purity of fractions obtained from protein crops (e.g. soy, lupine beans, pulses) and from photosynthetic active tissues (e.g., leaves, algae, duckweed) is included. Protein crops reach 50 - 60% protein yield with a protein purity of ~ 90%, whereas leaves and other photosynthetic active tissues reach similar protein purity (60 – 80 w/w% protein) but at much lower yields (10%). We hypothesize that the low yields are due to the small length scale in which protein is structured inside the leaves and the lack of protein storage anatomy in these tissues. Therefore, we conclude that leaf valorisation requires non-conventional approaches that go beyond higher extraction yields but that consider a complete use of the biomass.

Sugar beet leaves for functional ingredients