Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae.

Abstract

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.