Ice Recrystallization Inhibition Activity of Soy Protein Hydrolysates.

Identifying and developing ice recrystallization inhibitors from sustainable food proteins such as soy protein isolate (SPI) can lead to practical applications in both pharmaceutical and food industries. The objective of this study was to investigate the ice recrystallization inhibition (IRI) activity of SPI hydrolysates, and this was achieved by using an IRI activity-guided fractionation approach and relating IRI activity to interfacial molecular activity measured by vibrational sum frequency generation (VSFG). In addition, the impact of molecular weight (MW) and enzyme specificity was analyzed using three different proteases (Alcalase, trypsin, and pancreatin) and varying hydrolysis times. Using preparative chromatography, hydrolysates from each enzyme treatment were fractionated into five different MW fractions (F1-F5), which were then characterized by high-performance liquid chromatography (HPLC). All SPI hydrolysates had IRI activity, resulting in a 57-29% ice crystal diameter reduction when compared to native SPI. The F1 fraction (of 4-14 kDa) was most effective among all tested hydrolysates, while the lower MW peptide fractions lacked activity. One sample (SPI-ALC 20-F1) had a 52% reduction of ice crystal size at a lower concentration of 2% compared to the typical 4% used. SFG showed a difference in H-bonding and hydrophobic interactions of the molecules on the water/air interface, which may be linked to IRI activity. This study demonstrates for the first time the ability of SPI hydrolysates to inhibit ice crystal growth and the potential application of SFG to study molecular interaction at the interface that may help illustrate the mechanism of action.

Succinylation of zein and gelatin hydrolysates improved their ice recrystallization inhibition activity.

The goal of this research was to enhance the ice recrystallization inhibition (IRI) activity of zein and gelatin hydrolysates (ZH and GH, respectively) by succinylation modification. ZH was prepared by Alcalase treatment for 3 h and then modified by succinic anhydride (SA); whereas GH was made by Alcalase hydrolysis for 0.25 h and succinylated by n-octylsuccinic anhydride (OSA). After 0.5 h of annealing at -8 °C at 40 mg/mL, modified hydrolysates decreased the average Feret's diameter of ice crystal from 50.2 µm (polyethylene glycol, negative control) to 28.8 µm (SA modified ZH) and 29.5 µm (OSA modified GH) in comparison to the unmodified hydrolysates, which had the crystal size of 47.2 µm (ZH) and 45.4 µm (GH). Also, the two succinylated samples had altered surface hydrophobicity, which potentially contributed to their enhanced IRI activity. Our results indicate that succinylation of food-derived protein hydrolysates can improve their IRI activity.

A novel automated protocol for ice crystal segmentation analysis using Cellpose and Fiji.

Accurate measurement of ice crystal size is an essential step in quantitative ice recrystallization inhibition (IRI) analysis using the sucrose sandwiching assay (SSA) and splat assay (SA). Here, we introduce a novel method of measuring ice crystal size and shape using Fiji and Cellpose, an anatomical segmentation algorithm, to address the time-consuming and limited number of ice particle determination associated with the mean largest grain size measurement. This new automated approach, displaying rapid segmentation of ∼70 s per image, measures every ice crystal in an image field of view, consequently reducing bias introduced by subjectively selecting the largest crystals in an image. Consistent in determining a diverse set of crystal sizes and shapes, this method allows for the evaluation of ice crystals using Feret's diameter, a parameter that better accounts for irregular particle shape. This method provides new outputs such as standard deviation, particle size distributions of a population of ice crystals, and circularity to characterize and further provide insight into an analyte's IRI ability. Applicable to the SSA, the "shape descriptor" measurement can be used to quantify ice binding. This work presents a novel and accurate approach for ice crystal quantitative analysis.