Isolation and characterization of ice recrystallization inhibitory molecules from black soldier fly larvae

Authors: FOMICH, MADISON - University Of Tennessee; YUAN, YUAN - University Of Tennessee; Krishnan, Hari; DIA, VERMONT - University Of Tennessee; WANG, TONG - University Of Tennessee

Submitted to: Journal of Insects as Food and Feed
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/26/2025
Publication Date: 2/19/2025
Citation: Fomich, M., Yuan, Y., Krishnan, H.B., Dia, V., Wang, T. 2025. Isolation and characterization of ice recrystallization inhibitory molecules from black soldier fly larvae. Journal of Insects as Food and Feed. https://doi.org/10.1163/23524588-00001343.
DOI: https://doi.org/10.1163/23524588-00001343

Interpretive Summary: Insects have evolved and adapted in many ways to ensure survivability in different climates. One of these adaptations is their ability to survive in freezing conditions. Insects contain highly potent antifreeze proteins and ice-binding proteins. Understanding the important characteristics of ice recrystallization-active agents could lead to the development of new agents, which could then be used to prevent damage to frozen foods. In our study, we hypothesized that ice recrystallization inhibition active molecules present in Black Soldier Fly Larvae could have great commercial significance in the cryopreservation industry. In this study, we identified active molecules from the Black Soldier Fly Larvae albumin fraction and demonstrated very high ice recrystallization inhibition and ice-binding activity under various pH and NaCl concentrations. Interestingly, the major proteins in the Black Soldier Fly Larvae albumin were identified as cuticle proteins. This is the first time a cuticle protein has been found to possess ice recrystallization inhibition activity. Our study highlights the immense potential of Black Soldier Fly Larvae albumin as a cryoprotective additive. The results of our study could pave the way for the development of new ice recrystallization inhibition agents, which would be valuable for preserving foods.

Technical Abstract: Black solider fly larvae (BSFL) have demonstrated cold tolerance that suggests the presence of cryoprotective molecules. The objective of this research was to investigate if the proteins present in the BSFL have ice recrystallization inhibition (IRI) activity and how different environmental factors affect the activity. Osborne fractionation of the defatted BSFL was performed to separate the proteins based on solubility, then preparative size exclusion chromatography was used to fractionate the albumin fraction by molecular size to isolate IRI or ice binding proteins. The major proteins in the active fractions were identified by mass spectrometry, and molecular dynamic simulations were performed with two proteins identified to investigate their behaviors in an ice-water system. The main finding is the strong IRI activity of the water-soluble BSFL albumin fraction and the column fractionated fraction 1. This fraction had a 40.4-79.9% reduction in ice crystal size at 1% concentration and under a wide pH (3-9) and salt (10-200 mM NaCl) concentration. Pure proteins recovered were sequenced and dentified as cuticle proteins by mass spectrometry. One cuticle protein demonstrated strong H-bonding and structural flexibility by molecular dynamic simulations, explaining the IRI and ice binding activity. This is the first time BSFL protein is reported to possess IRI activity, and such protein extract can be feasibly obtained compared to other naturally occurring antifreezing proteins.