PhD in Food Science Cycle XXXII INSECT BIOREFINERY FOR GENERATING HIGH-ADDED-VALUE PRODUCTS SUCH AS PROTEIN AND CHITIN Coordinator: Chiara Dall’asta Promotors: Prof. dr. Stefano Sforza, University of Parma, Italy Dr. ir. Leen Bastiaens, Flemish Institute for Technological Research (VITO), Belgium PhD-student: Lise Soetemans Acknowledgments Waauw, four years have past, … already?! Time has really flown, but this is what happens when you are having a great time. Looking back, I am feeling very proud on what we were able to achieve, on the finished work and the (sometimes unexpected) results that I can present in this thesis. It was an unforgettable experience in many ways and I enjoyed every minute of it. I could not have presented this work without the support of my VITO-promotor Leen Bastiaens. She has taught me so much, for example on seeing the bigger picture, improving my scientific approach, writing and her suggestion always increased the quality of my work. But what I enjoyed the most was our collaboration that was always friendly with a bit of humor on the side, the open door and the joint, open- minded discussions to the next steps. I am very grateful for her advice, the encouragement and support I received during the PhD and her ideas that made this PhD successful. The quality of this thesis was also considerably improved thanks to the comments and suggestion of my University promotor Stefano Sforza. I want to thank him for his critical view on my work and his advice during my research. In addition, I am also very grateful for the opportunity to divide my time between VITO and the University of Parma. I got a taste of food science and was able to enrich my knowledge for which I am very thankful. In general, I would like the thank both my promotors for giving me the opportunity for personal and scientific growth and for the time and patience in correcting and improving the thesis. This PhD would be more limited without the useful and interesting input of the InDirect partners. They made the challenges in the PhD more valuable and it was nice to see the whole value-chain, starting from the selection of feed, to rearing, to fractionation and eventually applications tests. For this, I would like to thank all partners. In addition, the thesis was also incorporated and funded by BBI-JU under EU- Horizon2020 research and innovation program under GA-No720715, for which I am thankful. Time also flies when you are having fun, and fun we had. The REC team consists of eight fantastic people who all encouraged and helped me to reach my goal. It is easy to stay motivated and enthusiastic when your co-workers are always their to lend a hand, make a joke and once in a while listen to your complains. In my opinion, a good team is the foundations for making something difficult work and I would like to thank all members of the REC team for being my foundation throughout the PhD. Next to this amazing team, I was lucky to also be part of a sub-team of other PhD’s. Within this small group, I could find a great deal of encouragement, support, valuable advice, lots of laughter and friendship. Lastly, I would like to thank my family, friends and especially Dries for their patience and encouragement. Thank you for all the late night dinners, the time spent to listen to some of my complaints but also me being really enthusiastic about something and not able to stop speaking about it, the support and understanding. Especially to my parents and sister, who have no scientific background whatsoever but still kept trying to understand what I was doing and were interesting in learning every detail of my work. To Dries, who gave me the freedom to throw myself into this challenge and always cheered me up with some crazy goofiness. Table list Table 1-1 Applications of protein and lipid fractions ............................................... 36 Table 1-2 Chitin extraction from insect sources [119] ............................................. 43 Table 1-3 Examples of different deacetylation procedures [119]. .......................... 46 Table 2-1 Overview of all tested diets with the inclusion percentage of side-streams and a summary of the rearing evaluation. ............................................................... 60 Table 3-1 Extraction media and characteristics used in this study. ......................... 81 Table 3-2 Proximate composition of black soldier fly larvae juice .......................... 85 Table 3-3 Extraction NDY, yield and crude protein and lipid content on dry basis of the four extracted fractions (mean ± S.D., n =3). .................................................... 90 Table 4-1 Lipid and protein content (on dry matter basis) of all samples and subfractions. .......................................................................................................... 104 Table 5-1 Proximate composition of the samples, n=3. ........................................ 129 Table 5-2 Amino acid (AA) profile of the samples, n=2. ........................................ 131 Table 5-3 Characteristics of the samples, n=3. ...................................................... 132 Table 5-4 Minimal gelling concentration of the reference and LM samples, n=3, nd= not determined. ..................................................................................................... 138 Table 5-5 Summary of the TFP of the LM samples ................................................ 139 Table 6-1 Dry matter of BSF and control samples and chitin content in non-purified & purified samples, n=3. ........................................................................................ 151 Table 7-1 Overview of demineralization (DM) and deproteinization (DP) approaches applied. .................................................................................................................. 164 Table 7-2 Composition of the solid and liquid fractions after pre-treatment on dry matter basis (n =3). ................................................................................................ 167 Table 7-3 Results and composition of the residue obtained after demineralization on dry matter basis (n=2). ........................................................................................... 168 Table 7-4 Results related to traditional deproteinization approaches that were performed on residues obtained from DM-approach 2. ....................................... 170 Table 7-5 Chitin content and degree of deacetylation of raw chitin obtained by the alternative deproteinization/ deacetylation (n=3). ............................................... 173 Table 0-1 Composition of side-stream ingredients (on dry matter basis). ............ 218 Table 0-2 Protein content, amino acid profile and sum of essential amino acids of the diets on dry matter basis. E essential amino acids for insects. ....................... 220 Table 0-3 Lipid content, fatty acid profile and sum of essential fatty acids of the diets on dry matter basis ................................................................................................ 221 Table 0-4 Dry matter, protein content, amino acid profile, sum of the essential amino acids and chitin content of larvae on dry matter basis.......................................... 222 Table 0-5 Lipid content, fatty acid composition and sum of essential fattya acids of the larvae on dry matter basis. .............................................................................. 224 Figure List Figure 1-1 Sustainable insect rearing: resources needed to produce 1 kg of each protein source [9] .................................................................................................... 25 Figure 1-2 Life cycle of (a) the black soldier fly [31] and (b) the lesser mealworm [32] ................................................................................................................................. 28 Figure 1-3 Schematic presentation of the general procedure for extracting proteins from insects.............................................................................................................. 33 Figure 1-4 Schematic representation of (a) α-form and (b) ß-form of chitin [119] . 39 Figure 1-5 Schematic presentation of the isolation and conversion of chitin to chitosan .................................................................................................................... 42 Figure 1-6 Reaction mechanism of the deacetylation step [119]. ........................... 45 Figure 1-7 Insect value chain with an indication of the different research questions. ................................................................................................................................. 50 Figure 1-8 General overview of the thesis ............................................................... 54 Figure 2-1 Protein content (% on dry matter basis) of the larvae and their diet. Diet 2 (reference diet) is framed. .................................................................................... 63 Figure 2-2 Lipid content (% on dry matter basis) of the larvae and their diet. Diet 2 (reference diet) is framed. ....................................................................................... 64 Figure 2-3 (a) Average amino acid profile of LM larvae reared on the diets in the current study compared to data found in literature of the LM (Source 2: [169]). (b) Comparison of the essential amino acids in LM (measured in the current study) with YM and beef [6].......................................................................................................