Gel physical propertiesAgar (1.5% w

Gel physical properties
Agar (1.5% w/w in water) was hydrated during 4 h
at room temperature in closed flasks with stirring.
Afterwards, heating proceeded at 90°C for 30 min until
homogeneous viscous solutions were obtained. The hot
agar solutions were placed in glass test tubes, cooled
and allowed to gel overnight at room temperature to
give cylindrical gel specimens (1.6 cm diameter¥
10 mm height).
The mechanical studies were carried out in an
Instron Universal Testing Machine (Model 3345,
Instron, Norwood, MA, USA). Compression tests were
performed until fracture under two different rates of
compression: 1 mm min
-1
and 1 mm s
-1
. The texture
profile analysis (TPA) was performed at a rate of compression of 1 mm min
-1
and a deformation of 20%, in
order to allow reproducibility in the results. A cylindrical probe (3.0 cm diameter¥1.0 cm height) was
used in all assays. The following parameters were
obtained for all the samples according to Rosenthal
(1999): (i) gel strength (ratio between the fracture
force at compression and the area of the sample,
expressed in g cm
-2
); (ii) the elasticity index (quotient
between the time needed to attain the higher force in
the second compression and the time necessary for
attaining the maximum in the first compression-TPA
assay); (iii) resilience (the ratio of the work between
the withdrawal and the work during the first
compression-TPA assay); and (iv) adhesiveness (negative area calculated from the curve during the first
retraction of probe-TPA assay, expressed in g min
-1
).
Elasticity refers to the ability of a gel to regain its
original shape once the probe is removed, while resilience is the amount of energy it can store while being
compressed without creating a permanent distortion.
The adhesiveness is the capacity of the gel to overcome the attractive forces with the substrate it
contacts.
Data for analyzed W70.1 samples were expressed as
means of duplicates.
Rheological characterization
The W70.1 product (0.0050 g) of autumn, winter,
spring and summer harvests was suspended in deionized water and vortexed until complete hydration in
order to get a 0.5% w/w final concentration. Then,
systems were stored at 25°C for 18 h to attain swelling
equilibrium before measurement.
Rheological characterization was accomplished
using a RheoStress RS600, controlled stress rheometer
(Haake, Karlsruhe, Germany) equipped with a PP35
serrated parallel plate (Haake, Karlsruhe) geometry
(35 mm-diameter). A gap size of 500mm was set, and
data points were recorded at steady-state.
Amplitude sweeps were first performed in order to
determine the linear viscoelastic range (LVR). Storage
(G′) and loss (G′′) shear moduli as well as strain were
recorded as a function of stress, at a constant frequency
of 0.1 Hz and at 25°C in the LVR without affecting gel
structure (evaluationsat rest). The constant stress value
to use in the subsequent frequency sweeps was chosen
according to the linear viscoelastic range previously
determined for each agarose sample. Each mechanical
spectrum was then obtained at the selected constant
stress value, recording G′,G′′, and Tand(loss tangent) as
a function of increasing angular frequency (w), after
reaching steady state condition for each point.
Determinations were repeated at least twice for each
sample.
Transmission electron microscopy
Four seasonG. gracilissamples were fixed at 5°C for
12 h in 0.1 M Na-cacodylate buffer (pH 7.4) containing 0.25 M sucrose, 3% glutaraldehyde and 1.5%
paraformaldehyde. Fixation was followed by a series of
rinses in the buffer with gradually decreasing concentrations of sucrose. Then, the samples were post-fixed
in 2% OsO4 in the buffer, dehydrated in acetone and
infiltrated in Spurr’s resin. Thin sections were stained
with aqueous uranyl acetate followed by lead citrate
and observed in a JEOL 100CX-II transmission electron
microscope operated at 80 kV.
RESULTS
The composition of the aqueous extracts RTW, W70.1
and W90.1 obtained from the four season algal material
is depicted in Table 1. Due to their low yield, only
composition and monosaccharide analyses were performed for products W70.2–3 and W90.2–3 (Table 2).
Regardless of the harvest season, higher agar yield
was obtained after the first extraction at 70°C. Four
season W70.1 fractions did not differ in their composition or in their sulfation degree (between 4% and
6%). High glucose molar percentages would suggest
higher floridean starch content in summer plants.
Accordingly, transmission electron microscopy (TEM)
observations of summer specimens showed abundant
floridean starch granules when compared to autumn
ones (Fig. 1). This increment in starch content was
concomitant with lower nitrogen content in water
(Table 3). On the other hand, higher nitrogen and phosphate content in water coincided with important thylakoidal development in algal cortical cells (Fig. 1a) and
higher protein content in the products extracted from
autumn-winter plants (Table 1).
The highest gel strength for spring or summer agar
was in agreement with high elasticity and resilience
indexes for these samples (Table 4). On the contrary,