The effect of frozen storage on the loss of extractability of natural actomyosin (NAM) with 0.6 M
NaCl (P1) was studied in hake (Merluccius merluccius), sardine (Sardina
pilchardus), and mixed
minces in varying proportions stored for up to 1 year at −20 °C. The objective was to ascertain the
types of bonds established among the proteins. The unextracted NAM was treated with different
extracting agents to cleave secondary bonds (2% SDS, 2% SDS + 8 M urea) and secondary and
disulfide bonds [2% SDS + 5% β-mercaptoethanol (ME)]. There was more NAM extracted from the
mixes with higher percentages of sardine, although this effect was weaker than might have been
expected from the percentages of sardine in the mixes. In hake, for up to 8 months of storage, the
bulk of the proteins not extracted in 0.6 M NaCl was extracted with SDS + urea or SDS + ME,
with extraction declining as storage progressed. This suggests that the amount of protein linked
by covalent bonds increased with time. In sardine and mixed lots, a high percentage of proteins
linked by covalent bonds was detected earlier, although no clear pattern emerged in terms of hake/sardine ratios. SDS−PAGE showed that treatment with SDS + urea and SDS + ME extracted
protein in the form of small aggregates that were retained in the stacking gel. These aggregates
were also found in the SDS extracts from the sardine and mixed minces.
Keywords: Hake; sardine; frozen storage; aggregates; solubilization