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258 IV.Resultados y discusión. Capítulo III
solutions was measured spectrophotometrically using a UV-visible
spectrophotometer UVmini-1240 (SHIMADZU Europe Corporation,
Germany) at 340 nm after 2 min incubation at room temperature. The
proteolytic activity was expressed as the difference in absorbance of free
amino groups measured at 340 nm between fermented and non-fermented
almond samples.
2.7 Size-exclusion high-performance liquid chromatography analysis
(SEC-HPLC)
Fermented almond milk samples were analysed by SEC-HPLC in order
to obtain their unique peptide profiles. Digest inoculated samples were also
characterised on the SEC-HPLC, these samples were first subjected to a
heat treatment (80 ºC/20 min) in order to suppress any possible residual
enzymatic activities.
All HPLC analyses were performed in duplicate on Agilent 1200 HPLC
system (Agilent Technologies, Palo Alto, CA, U.S.A.). SEC screening of
samples was performed on a BioSep-SEC-S2000 (300 mm x 7.8 mm i.d.)
column with a Gel Filtration Chromatography guard column 4 x 3 mm
(Phenomenex, Cheshire, UK). Before injecting samples to the equipment,
fermented and non-fermented almond milk samples were filtered through
nylon membranes (0.45 m).
Two methods of analysis by SEC-HPLC were examined. The first is
specific for larger Mw peptides (roughly 17,000 - 700,000 Da). The
standards thyroglobulin, -globulin, myoglobin, BSA, ovalbumin, aprotinin,
uridine and sodium azide were used to prepare a calibration curve for this