Total starch kit

The in vitro digestibility of nutrients was evaluated according to the procedure described by Świeca and Baraniak [8 (link)]. In vitro digestion was performed according to the methodology described by Minekus et al. [33 (link)]. For digestion, 2 mL of beverage was subjected. After digestion, the samples were mixed with an equal volume of methanol to stop enzyme action. The samples were centrifuged at 12,000× g at 4 °C for 20 min. The pellets were washed again with methanol. Undigested starch was determined using the Total Starch Kit. (Megazyme). Undigested proteins were determined in both the pellets after solubilization with 0.1 mol L⁻¹ NaOH and the supernatants using the Bradford method [30 (link)]. Nutrient digestibility was calculated as the ratio of undigestible starch/proteins to total starch/proteins using the equation:

Total starch (TS) was measured according to AACC method 76.13 (12 ) using a total starch kit from Megazyme (Wicklow, Ireland). Resistant starch (RS) was determined following the AACC method 32–40 (12 ) with the RS kit (Megazyme, Wicklow, Ireland).

Total carbon and nitrogen in freeze-dried powderized potato (potato flour) was determined using a Europa 20–20 isotope ratio mass spectrometer with an ANCA preparation system, comprising a combustion and reduction tube operating at 1,000°C and 600°C, respectively. Potato TAG was quantified by chloroform/methanol/water extraction, thin layer chromatography separation, fatty acid methylation and gas chromatography as described in Liu et al. (2017 (link)). Measurements of the total starch and sugar content of potato flour were as described in Liu et al. (2017 (link)). Cellulose was measured using the method of Updegraff (1969 (link)) with an additional digestion of the starch using α-amylase and amyloglucosidase (Total Starch Kit, Megazyme, Wicklow, Ireland). Soluble protein was extracted in buffer (Tris pH 7.5, 10 mM MgCl₂) and quantified by Bradford assay (Bradford, 1976 (link)).

Materials harvested at 12:00 pm from WT and transgenic plants were ground and baked at 80 °C for two days until a constant dry weight (DW). The dried sample 30 mg was dissolved in 0.7 mL of 80% ethanol, thoroughly vortexed and incubated at 70 °C for 2 h. Aliquots of 0.7 mL HPLC‐grade water and 0.7 mL chloroform were added to the sample. After shaking several times, the mixtures were centrifuged at 12 000 g for 10 min. The pellet was analysed for starch content while the supernatant was composed of soluble sugar. The starch pellet was washed three times with 80% ethanol, and the total starch content was analysed using total starch kit (Megazyme International Ireland Limited, Wicklow, Ireland). The aqueous supernatant of 0.7 mL was transferred into 1.5‐mL tube and mixed with 0.7 mL chloroform. After centrifugation at 12 000 g for 10 min, 0.5 mL of the supernatant was transferred to a glass tube for HPLC analysis of sugars. The sugar separation was carried out using the Agilent HPLC column (ZORBAX Carbohydrate column; 4.6 × 150 mm, 5 μm) with a differential refraction detector. The mobile phase consisted of 75% acetonitrile with a flow rate of 0.8 mL/min, and the column was kept at 35 °C. The sugar types were characterized according to the retention time of the standard references. The concentration in samples was calculated from the external standard curve.