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Unicellular Protein: Isolation, Techno-Functionality and Digestibility’ 2018 Unicellular protein: isolation, techno-functionality and digestibility Emma Teuling 2018 Unicellular protein: isolation,techno-functionality and digestibility Emma Hier komt de tekstvoor de rug; hoe dikker rug, groter tekst Unicellular protein Isolation, techno-functionality and digestibility++ Emma Teuling Propositions 1. For microalgae, protein accessibility dominates protein digestibility. (this thesis) 2. The emulsifying ability of proteins as determined by the critical protein concentration is affected by the volume fraction of oil. (this thesis) 3. Substrate specificity of lytic polysaccharide monooxygenase subclasses cannot be predicted by their amino acid sequence similarities. 4. Promoting the home use of antiseptic hand soaps puts public health at risk. 5. The new norms (2014) of the Dutch mental healthcare association stimulate abuse of the DSM IV classification to benefit patients in their insurance coverage. 6. The difficulties in forming majority cabinets in The Netherlands are not related to the number of active political parties. Propositions belonging to the doctoral thesis: ‘Unicellular protein: isolation, techno-functionality and digestibility’ Emma Teuling Wageningen, 18th May 2018. Unicellular protein: isolation, techno- functionality and digestibility Emma Teuling Thesis committee Promotor Prof. Dr H. Gruppen Professor of Food Chemistry Wageningen University & Research Co-promotors Dr P.A. Wierenga Assistant professor, Laboratory of Food Chemistry Wageningen University & Research Dr J.W. Schrama Associate professor, Aquaculture and Fisheries Group Wageningen University & Research Other members Prof. Dr M.H.M. Eppink, Wageningen University & Research Prof. Dr P. Bossier, Ghent University, Ghent, Belgium Dr A.F.B. van der Poel, Wageningen University & Research Dr F. van de Velde, NIZO Food Research, Ede, The Netherlands This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Unicellular protein: isolation, techno- functionality and digestibility Emma Teuling Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 18 May 2018 at 4 p.m. in the Aula. Emma Teuling Unicellular protein: isolation, techno-functionality and digestibility 170 pages. PhD thesis, Wageningen University, Wageningen, The Netherlands (2018) With references, with summary in English. ISBN: 978-94-6343-853-7 DOI: 10.18174/444729 ABSTRACT Unicellular, photosynthetic microalgae and cyanobacteria are potential new protein sources for both food and feed. Variations between these sources are reflected in chemical variations (e.g. in the chemical composition) and in structural variation (e.g. in cell wall robustness). The aim of this thesis was to understand how chemical and structural variations between unicellular photosynthetic sources affect the application of unicellular protein as techno-functional ingredients and as a fish feed ingredient. To study the applicability of unicellular protein as techno-functional ingredient in food, proteins were extracted and isolated from the cyanobacterium Arthrospira (spirulina) maxima and the microalgae Nannochloropsis gaditana, Tetraselmis impellucida and Scenedesmus dimorphus. Chemical variations observed between photosynthetic unicellular sources were reflected in variations in protein extractability. The isolates (62–77% w/w protein) varied in protein solubility as a function of pH and ionic strength, especially at pH < 4.0. Isolates from N. gaditana, T. impellucida and A. maxima were able to form emulsions (d3,2 0.2–0.3 µm) at pH 8.0. The amount of each isolate needed to form emulsions varied between the isolates (9–74 mg protein / mL oil), but was within the range of proteins from both similar (photosynthetic) sources (algae and sugar beet leaves) and other protein sources (dairy, legume and egg). Minor differences were observed in the pH dependence of flocculation amongst the isolate stabilized emulsions. To study the applicability of microalgae and cyanobacteria as dietary protein sources for fish, Chlorella vulgaris, S. dimorphus, N gaditana, and A. maxima biomass was incorporated in fish feed (30% inclusion) and fed to Nile tilapia and African catfish. The cell walls of these unicellular sources used were quantified to vary in their robustness to mechanical degradation (structural variations). Although protein digestibility varied between the unicellular sources (ranging from 67–83%), the protein digestibility did not relate to the variations in unicellular cell wall robustness. There was no difference between both fish species regarding the nutrient digestibilities of the unicellular sources. Subjecting N. gaditana biomass to treatments that decrease its cell wall integrity increased in vitro accessibility of microalgae nutrients up to 4 times. The increased in vitro accessibility correlated with an increased in vivo digestibility of protein in Nile tilapia, confirming that nutrient accessibility plays an important role in the nutrient digestibility of microalgae in fish. In conclusion, chemical variations observed between photosynthetic unicellular sources were reflected in variations in protein extractability. Structural variations between the sources were reflected in variations in in vivo protein accessibility and subsequent protein digestibility. TABLE OF CONTENTS Abstract Chapter 1 General Introduction 1 Chapter 2 Comparison of protein extracts from various unicellular 27 green sources Chapter 3 Characterizing emulsion properties of microalgal and 55 cyanobacterial protein isolates Chapter 4 Effect of cell wall characteristics on algae nutrient 77 digestibility in Nile tilapia and African catfish Chapter 5 Increasing algae nutrient accessibility increases algae 103 nutrient digestibility in Nile tilapia Chapter 6 General Discussion 133 Summary 157 Acknowledgements 161 About the author 163 General introduction General Introduction. 1 1 By 2050 the world population is expected to increase by 30% compared to 2017, reaching approximately 9.8 billion 1. To feed this number of people, food and feed production need to be increased by 50 percent in this time period 2. A significant issue in both the food and feed production is the availability of protein(rich) sources. Consequently, there is an interest in exploring alternative protein sources. These alternative protein sources should be produced in a sustainable way, not competing with current food and feed production and in addition have a reasonable protein digestibility relative to its price in comparison to current sources used in feed formulation. One class of alternative protein sources that has the potential to meet these requirements are the unicellular, photosynthetic microalgae and cyanobacteria. For application in food products, the microalgal and cyanobacterial proteins need to be extracted and isolated to be free of colour and highly soluble. Such levels of purification are not necessarily required for the application in feed. The isolation procedure required for food yields a considerable side stream. This side stream contains many valuable nutrients, of which the focus will be on proteins in this thesis. When using these insoluble (or membrane-bound) protein fractions in feed, and the soluble isolates in food, both the feed and food markets can be supplied with algal or cyanobacterial protein. In this thesis, both fields of application of proteins from unicellular sources (various microalgae and one cyanobacterium) are explored. The first aim of this thesis is to provide a better understanding of the extent to which photosynthetic unicellular sources differ in their chemical and protein composition, and how these differences affect protein digestibility in fish, protein extractability and techno-functionality of the protein isolates obtained. For unicellular protein digestibility in fish, the effect of cell wall hardness, protein accessibility and fish species on unicellular protein digestibility in fish was elucidated. MICROALGAE AND CYANOBACTERIA AS PROTEIN SOURCES Protein contents Microalgae and cyanobacteria encompass a wide variety of photosynthetic unicellular organisms, ranging from marine prokaryotes to freshwater eukaryotes and belonging to over eleven phyla 3. The only microalgae and cyanobacteria currently holding a GRAS status as food ingredients are Dunaliella bardawil, Chlorella vulgaris and Arthrospira (spirulina) platensis 4. These sources are also already applied industrially in feed: in aquaculture hatcheries (for molluscs, crustaceans and zooplankton) 5. They are, however, not yet commercially applied as a protein source in formulated fish feeds. Microalgae that do not hold the GRAS status but are applied commercially in aquaculture hatcheries are from the genera Nannochloris, Haematococcus, Tetraselmis, Pyramimonas, Nannochloropsis, 1 Chapter 1 Isochrysis, Pavlova (Monochrysis) and Schizochytrium 6. In addition, microalgae from the genus Scenedesmus have been studied extensively in food and feed related research. The protein contents of these microalgae and cyanobacteria vary greatly, between 12 and 72% (w/w on DM) (Table 1). This variation is partially due to growing conditions, seasonal conditions and due to differences between species within each genus 7, 8 (Table 1). Although variation
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