2.7. Cryo-Scanning Electron microscopic observation of emulsion
gels and puree samples
Emulsion gels and puree samples were observed under cryo-SEM
(cryo-scanning electron microscope, Nano Imaging Facility Laboratory of
Carleton University, Ottawa, ON). The method of Liu & Lanier (2015) was
employed with slight modification. The internal portion of the fresh
emulsion gels and puree samples were cut into blocks of 0.5 cm, the
blocks were then frozen on a metal plate surrounded by liquid nitrogen
for 20 s. The block was placed on a copper specimen holding and observed
under a Cryo-SEM. The microscope was operated at 20 kV in low vacuum
mode (40 Pa), with the temperature at <−50 ℃.
2.8. Alpha-amylase
inhibitory activity
The α-amylase inhibitory activity of emulsion gels and puree samples was
carried out according to the standard method with minor modification
(Telagari & Hullatti, 2015; Bhutkar & Bhise, 2012). In a 96-well
plate, reaction mixture containing 20 μl α–amylase (1 U/ml) and 60 μl
of varying diluted sample solution was preincubated at room temperature
for 30 min. Then, 80 μl of 1% soluble starch in buffer (20 mM phosphate
buffer pH 6.9) was added as a substrate and incubated further at 37 °C
for 10 min; DNS color reagent (80 μl) was then added and boiled for 10
min. The absorbance of the resulting mixture was measured at 540 nm
using a Multiplate Reader (BioTek Cytation 5, Ottawa, Canada).
Psyllium husk (3%) and inulin emulsion gels (0, 5, 10, 15, and 20%)
were diluted in buffer to give a final concentration of 5 mg/ml. Diluted
emulsion samples were centrifuged (10,000 rpm, 2 min, room temperature),
and the clear supernatant was applied to the test. For meat puree
sample, a blank was prepared without sample and another without the
amylase enzyme, replaced by equal quantities of buffer (20 mM sodium
phosphate buffer, pH 6.9). Meat puree samples were diluted in buffer to
give a final concentration of 50 mg/ml and centrifuged (10,000 rpm, 2
min, room temperature). The clear supernatant was used for the test.
Puree sample (salmon, chicken, chicken stew or beef) without emulsion
incorporation was representative of the 100% enzyme activity.
Each experiment was performed in triplicates. The results were expressed
as percentage inhibition, calculated using the formula:
Inhibitory activity (%) = [(Absbank -
Abssample)/ Absbank]×100 (2)
Where Absbank is the absorbance of the bank and
Abssample is the absorbance of the sample.
2.9. Alpha -glucosidase
inhibitory activity
The α-glucosidase inhibitory activity of emulsion gels and puree samples
was carried out according to the standard method with minor modification
(Telagari & Hullatti, 2015; Picot et al., 2014). In a 96-well plate,
reaction mixture containing 50 μl phosphate buffer (0.1 mM, pH 6. 9), 10
μl alpha-glucosidase (1 U/ml), and 20 μl of varying diluted emulsion
gels was preincubated at 37 °C for 15 min. Then, 20 μl P-NPG (1 mM) was
added as a substrate and incubated further at 37 °C for 30 min. The
reaction was stopped by adding 50 μl
Na2CO3 (0.1 M). The absorbance of the
released p-nitrophenol was measured at 405 nm using Multiplate Reader
(BioTek Cytation 5, Ottawa, Canada). A blank was prepared without
diluted emulsion gels and another without the glucosidase enzyme,
replaced by equal quantities of buffer (0.1 mM phosphate buffer, pH
6.9). Psyllium husk (3%) and inulin emulsion gels (0, 5, 10, 15, and
20%) were diluted in buffer to give a final concentration of 5 mg/ml.
For meat sample, a blank was prepared without emulsion sample and
another without the glucosidase enzyme, replaced by equal quantities of
buffer (0.1 mM phosphate buffer, pH 6.9). Meat samples were diluted in
buffer to give a final concentration of 50 mg/ml and then were
centrifuged at 10,000 rpm for 2 mins at room temperature. After
centrifugation, the clear supernatant was applied to the test. Puree
sample (salmon, chicken, chicken stew or beef) without emulsion
incorporation was representative of the 100% enzyme activity. A blank
was prepared without sample and another without the glucosidase enzyme,
replaced by equal quantities of buffer (0.1 mM phosphate buffer, pH
6.9).
Each experiment was performed in triplicates. The results were expressed
as percentage inhibition, calculated using the formula:
Inhibitory activity (%) = [(Absbank -
Abssample)/ Absbank]×100 (3)
Where Absbank is the absorbance of the bank and
Abssample is the absorbance of the sample.
2.10. Statistical
analysis
Statistical analyses were performed with SAS Software (SAS Institute
Inc, Cary, NC). One-way analysis of variance (ANOVA) by Duncan’s
Multiple Range test was used to compare the mean values (α=0.05).
Differences were significant at p < 0.05.