The frozen samples were removed from the freezer and rapidly
transferred to a refrigerated centrifuge (centrifuge Hettich model D-
7853, Tuttlingen, Germany) operated at 20 °C for 10 min at 4600 rpm
(2400 relative centrifuge force) to force the separation of solutes from
the frozen samples. After centrifugation, the concentrated solution
was collected, and the remaining frozen core was thawed so that the
soluble solids concentration was determined in both fractions. The concentration
of fractions Cf and Cs (solids in the molten frozen phase and
solution, respectively) obtained after assays was analyzed at ambient
temperature (approx. 22 °C) with an ATAGO refractometer (model
PAL-1, Tokyo, Japan) with a precision of ±0.1 °Brix. All measurements
were made in triplicate and each assay was performed six times with
two replicates.