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 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.

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.