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.