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Abstract Black soldier 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 identified 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. 

Abstract Soy protein hydrolysates have demonstrated moderate ice recrystallization inhibition (IRI) activity, but the properties of the unhydrolyzed protein contributing to this activity are not well known. The objective of this research was to identify the main protein processing factors important to the varying IRI activities observed. Three possible modification reactions were applied to soy protein isolate (SPI): the Maillard reaction that can occur in soy flour before protein isolation, heat denaturation of the fully defatted protein, and heat denaturation of the protein with the presence of residual lipid, mainly polar phospholipids. These modified proteins were hydrolyzed by Alcalase protease (for 2 min) to produce hydrolysates, which were characterized by HPLC and FTIR to investigate how molecular weight (MW) and changes in secondary structure relate to IRI activity. A multilinear regression with the parameters of modification type, surface hydrophobicity, secondary structure profile, and average MW of the hydrolysates was used to investigate their roles in reducing ice crystal size. It was discovered that polar lipids present in the soy hydrolysate samples and MW were the only significant factors (p < 0.05) for IRI activity of SPI hydrolysates which had an ice crystal size reduction from 22.0% to 36.7%. This study demonstrates for the first time a protein hydrolysate‐phospholipid interaction can produce IRI active molecules.