Insects are considered a nutritionally valuable source of alternative proteins, and their efficient protein extraction is a prerequisite for large-scale use. The protein content is usually calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25. This factor overestimates the protein content, due to the presence of nonprotein nitrogen in insects. In this paper, a specific Kp of 4.76 ± 0.09 was calculated for larvae from Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens, using amino acid analysis. After protein extraction and purification, a Kp factor of 5.60 ± 0.39 was found for the larvae of three insect species studied. We propose to adopt these Kp values for determining protein content of insects to avoid overestimation of the protein content.

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins’ properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus.

Insects are a promising alternative protein source. One of the bottlenecks in applying insects in food is the fast darkening initiated during grinding. Besides enzymatic browning, non-enzymatic factors can cause off-colour formation, which differs between species. This study investigates the impact of iron, phenoloxidase, and polyphenols on off-colour formation in insect larvae. Hermetia illucens showed a blackish colour, whereas Tenebrio molitor turned brown and Alphitobius diaperinus remained the lightest. This off-colour formation appeared correlated with the iron content in the larvae, which was 61 ± 9.71, 54 ± 1.72 and 221 ± 6.07 mg/kg dw for T . molitor , A . diaperinus and H . illucens , respectively. In model systems, the formation of iron-L-3,4-dihydroxyphenylalanine (L-DOPA) bis- and tris-complexes were evidenced by direct injection into ESI-TOF-MS, based on their charges combined with iron isotope patterns. The reversibility of the binding of iron to phenolics, and thereby loss of blackening, was confirmed by EDTA addition. Besides complex formation, oxidation of L-DOPA by redox reactions with iron occurred mainly at low pH, whereas auto-oxidation of L-DOPA mainly occurred at pH 10. Tyrosinase (i.e. phenoloxidase) activity did not change complex formation. The similarity in off-colour formation between the model system and insects indicated an important role for iron-phenolic complexation in blackening.

Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom ( Agaricus bisporus , AbPPO) and PPO from potato ( Solanum tuberosum , StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate ( Ilex paraguariensis ) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones.

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Insects have been identified as excellent alternative source of proteins due to their high protein content. The acceptance of insects increases when used as ingredient in an invisible manner and hence grinding is necessary. Off-colour formation occurs upon grinding larvae, which can hamper their potential use as ingredient for food and feed. The aim of this thesis was to investigate potential of insect larvae as protein source, the mechanisms responsible for the browning or blackening of larvae during grinding, and its impact on protein functionality. This was investigated for the larvae of three species: Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. The specific Kp factor of 4.76±0.09 was determined for the three species to determine protein content based on nitrogen, and 5.60±0.39 after protein extraction and purification. Thus, the general Kp factor of 6.25, used until now, overestimated the protein content in insects. Off-colour formation upon grinding was caused by both enzymatic and non-enzymatic browning. Phenoloxidase was found to be mainly responsible for browning in T. molitor and likely in A. diaperinus, whereas iron-phenolic complexation likely contributed to the black colour in H. illucens. A model system of L-DOPA and iron was used to elucidate the structures of the iron-L-DOPA complexes by mass spectrometry. Enzymatic browning did not influence the solubility of the proteins of all three species. Upon in-vitro hydrolysis by pepsin and trypsin, soluble proteins from H. illucens were more digestible compared to those of T. molitor and A. diaperinus. Phenoloxidase activity during processing negatively affected in-vitro pepsin hydrolysis. Besides phenoloxidase activity, also endogenous proteases remained active at pH 8 in extracts of insect larvae. Summarizing, endogenous enzyme activities and iron complexation should be taken into account for future application, as well as the specific Kp factor to prevent overestimation of the protein content of insects. v 7 CONTENTS Chapter 1 General introduction Chapter 2 Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens Chapter 3 Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae Chapter 4 Iron-polyphenol complexes cause blackening upon grinding Hermetia illucens (black soldier fly) larvae Chapter 5 Effect of endogenous phenoloxidase on protein solubility and digestibility after processing of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens

Supporting information S1 Fig. Native PAGE stained with 3 mM L-DOPA (left) showed no active bands for extracts treated with sodium bisulfite from Tenebrio molitor (T s), Alphitobius diaperinus (A s) and Hermetia illucens (H s). A similar gel was stained with Coomassie (right). (TIF)