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Investigation of antifreeze proteins as cryoprotectants for ling cod (Ophiodon elongatus) mince and natural actomyosin Liceaga, Andrea

Abstract

Cryoprotectants used commercially to minimize changes in texture and protein properties during freezing and frozen storage of fish typically impart an undesirable sweet taste. Since fish antifreeze proteins (AFP) are known to modify and suppress ice crystal growth, the objective of this study was to evaluate AFP as alternative cryoprotectants for frozen ling cod mince and natural actomyosin (NAM). Mince from ling cod was subjected to freeze-thaw abuse in the absence (control) or presence of AFP (5, 10, 50 or 500 ppm), AFP (50 ppm) with 0.3% phosphates, polyols (4% sucrose + 4% sorbitol or 8% trehalose), or AFP (50 ppm) with polyols (2% sucrose + 2% sorbitol). Freeze-thawed mince with AFP showed higher textural hardness, less salt extractable protein, and higher expressible moisture. These samples also formed a layer of ice crystals, which was not observed in polyol blends or control. Differential scanning calorimetry showed more unbound water in mince with AFP, while Raman spectroscopy indicated increased prevalence of β-sheet and random coil structures at the expense of α-helix. AFP failed to prevent loss of Ca-ATPase activity in NAM following freeze-thaw abuse. AFP solutions were evaluated at ambient and subzero (-0.5, -1.8, -4.0°C) temperatures using Raman spectroscopy. AFGP had small peaks near 1620 and 1674cm⁻¹ attributed to polyproline type-II helix and extended/unordered β-structures, respectively, and a strong band at 1070cm⁻¹ assigned to backbone C-C, C-N stretching and carbohydrate vibrations. Sharpening of the amide I band near 1645cm⁻¹ for AFPI at subzero temperatures showed strengthening of α-helix upon cooling. Strong hydrophobic interactions from aliphatic amino acids were seen at -0.5°C, and hydrogen-bonding and involvement of methyl groups were implicated at subzero temperatures. Frequency shifts in the O-H stretching band of water were observed in the presence of AFP at subzero temperatures. AFP did not prevent ice recrystallization or protein denaturation in fish mince during freeze-thawing. Conformational changes of AFP were observed at subzero temperatures, especially at -0.5°C. This information could be useful to study future applications of AFP in situations where intense ice crystallization formation will be desired or applications such as chemical adjuvants to cryosurgery.

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