Resistant starch assay kit

The in vitro digestibility of starch was evaluated on the basis of total starch content (TS) and potentially resistant starch (RS) determined after digestion in vitro [40 (link)]. Starch contained in the residues remaining after the in vitro digestion was hydrolyzed with thermostable α-amylase and amyloglucosidase according to AOAC Official Method 996.11 (Resistant Starch Assay Kit, Megazyme) [41 (link)]. Starch digestibility in vitro was calculated as follows:
where: SD = in vitro digestibility of starch, TS = total starch content, and RS = content of starch after in vitro digestion

The content of resistant starch (RS) of native and hydrothermally modified starch samples were determined according to the analysis procedure provided by the Resistant Starch Assay Kit (Megazyme International Ireland Ltd. Co., Wicklow, Ireland).

The resistant starch content was determined with Resistant Starch Assay kit (Megazyme, Bray, Ireland) with the description of Association of Official Analytical Chemists (AOAC) 2002.02.

The dog food samples were analyzed without pre-drying. To determine dry matter (DM) content, the samples were dried at 103 °C for 16 h, and then placed in a desiccator to cool before weighing³⁶ . For ash determination, samples were incinerated at 550 °C for three hours and then cooled in a desiccator before weighing. The Kjeldahl method³⁷ was applied to determine nitrogen content, using a 2020 digester and a 2400 Kjeltec analyzer (FOSS Analytical A/S, Hilleröd, Denmark). Crude protein was then calculated as N × 6.25. Crude fat was analyzed in accordance with Commission Directive EC/152/2009³⁸ , on a Soxtec extraction unit (FOSS Analytical A/S, Hilleröd, Denmark). Crude fiber was analyzed as previously described³⁹ . Gross energy (GE) was measured on a Parr isoperobol Bomb Calorimeter 6300 (Parr Instrument Company, Moline, Illinois, USA). Total dietary fiber (TDF) as well as soluble and insoluble dietary fiber were analyzed in accordance with AOAC 991.43 using a Total Dietary Fiber Assay Kit (Megazyme, Bray, Ireland), while resistant and non-resistant starch were analyzed according to AOAC 2002.02 using a Resistant Starch Assay Kit (Megazyme, Bray, Ireland).

Granule-bound starch synthase I enzymatic activity was determined using the GBSSI enzymatic activity assay kit (Solarbio Science & Technology, Beijing, China) according to the manufacturer′s instructions. Total starch, amylose, and resistant starch contents in GP and M2-1105 were determined using the total starch (AA/AMG) Assay Kit, Amylose/Amylopectin Assay Kit, and Resistant Starch Assay Kit (Megazyme, Wicklow, Ireland), respectively. Protein content was determined using the automatic Kjeldahl apparatus equipment (Foss Analytical Kjeltec™ 8400 series), as previously described (Yang et al., 2019 (link)). Each sample was analyzed in triplicate.

The resistant starch (RS) content of the samples was measured according to the resistant starch assay kit from Megazyme International (Wicklow, Ireland) (AACC method 32-40). Total starch (TS) was measured using the Total starch assay kit from Megazyme (AACC method 76-13).