Abstract: In this paper, ice-structuring proteins (AISPs) from alfalfa were extracted using ice spheres combined with phosphate buffer, and their amino acid composition and the properties and structures of the purified AISP fractions were identified. In order to study the effect of AISPs on the quality and structure of frozen wet gluten, AISPs was mixed with dry gluten powder in water for freezing treatment. The results showed that AISPs contained 17 amino acids with a ratio of hydrophilic amino acids to hydrophobic amino acids of 7:4. Two protein components with molecular masses of 34 and 52 kDa were obtained from the fractionation of AISPs, and their thermal hysteresis values were both 0.41 ℃. Schiff staining showed that the former was glycoprotein and the latter was non-glycoprotein, and the proportions of secondary structure in them were α-helix:β-sheet:β-turn:random coil = 13.19:60.21:12.21:19.16, and 16.87:39.41:26.69:17.03, respectively. The protective effect of AISPs on frozen wet gluten samples at ?40 ℃ was more significant than at ?18 ℃. With the increase in freezing time and the number of freezing-thawing cycles, the water-holding capacity of wet gluten protein decreased while the content of freezable water increased, and ω-gliadin was partially degraded. High-molecular-weight glutenin subunit (HMW-GS) was completely degraded. The B and C subunits of low-molecular-weight glutenin subunit (LMW-GS) in frozen samples were stable, while the D subunit increased. The frozen-thawed samples showed an increase in subunits B, C and D, especially for subunits B and C. After adding AISPs, the degradation of gluten protein components was alleviated to some extent. Scanning electronic microscopy (SEM) results showed that freezing treatment resulted in rough texture and enlarged pores in the wet gluten network, while the addition of AISPs made the gluten network clearer and more continuous.

Key words: alfalfa ice structuring protein; frozen wet gluten; freezable water content; molecular mass of protein; microstructure