Bibliografisk note

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This work was supported by China Scholarship Council (CSC) grant # 202003250068 (YT) and # 202006790033 (YW) and HIAMBA - grain, flour, bread & bakery products preventing type 2 diabetes” Innovation Fund Denmark , Project 9067-00004A (YZ).

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The approximate values of the apparent saturation coverage (adsΓmax) of GA for the three maize starch types waxy maize starch (WMS, 0% AM), normal maize starch (NMS, 26.0% AM) and high amylose maize starch (HAMS, 72.2% AM) (Fig. 2) can be used to estimate the density of binding sites of GA on different granular surfaces. Higher density of adsorption binding sites was observed for WMS (20 nmol/g) when compared with NMS (13 nmol/g) and HAMS (11 nmol/g) (Table S1). This might suggest higher catalytic efficiency of GA acting on WMS when compared with NMS and HAMS, supporting the view that WMS typically has low amylolytic resistance (Hu, Zhao, Duan, Linlin, & Wu, 2004). However, the higher density of binding sites on HAMS than on NMS (Fig. 2A) was inconsistent with the higher amylolytic resistance of HAMS than NMS reported previously (H. Li, Gidley, & Dhital, 2019; Zhong, Tai, et al., 2022) as well as the high K½, kcat and low KM values found for HAMS under conventional (high substrate) conditions (Fig. 3 and Table S1). The attack site density (kinΓmax) of these starches calculated from the combined conventional and inverse M-M kinetics revealed interesting features related to HAMS, expected to confer high hydrolytic resistance. For WMS, the value of kinΓmax i.e. the maximal density of productive binding sites, was 20 nmol/g, accounting for 97% of the binding sites, indicating that almost all binding sites on WMS granules were productive. Correspondingly, the amounts of attack sites on NMS were 7 nmol/g, accounting for the 53% of binding sites and only 22% of binding sites on HAMS (2.6 nmol/g) were productive attack sites. It can therefore be argued that, while the enzyme binds readily to the surface of HAMS, it is only able to form productive complexes to a very limited extent. It should, however, be noted that the conventional M-M data (excess substrate) revealed that the HAMS granules (expected to possess high hydrolytic resistance), showed the highest K½, kcat and lowest KM values while the inverse approach revealed an exceptionally low amount of attack sites. In agreement with our previous study on cellulose degradation (Kari et al., 2017), the inverse maximum rate provides a better indicator for the overall efficacy of hydrolysis of the solid starch substrate than the conventional maximum rate. This is substantiated by the parameter invVmax (=kcat kinΓmaxmassS0), which provides combined information on the catalytic rate and the ability to recognize the attack sites (kinΓmax), while convVmax (kcat E0) solely reflected maximal turnover and hence did not identify the overall efficacy. This documents clearly that the differential hydrolytic resistance of the different starch types for GA is dependent on the relative amounts of enzyme and substrate. Only when GA was present in excess, HAMS showed high resistance relative to the other two starch granule types, an effect mainly attributed to the low density of attack sites on the HAMS granules. Hence, our data provide evidence for the degradative resistance of granular starch for enzyme excess situations which are likely reflecting in vivo conditions (Betts et al., 1991; Woolnough et al., 2008; Zotter et al., 2017) and typically used in vitro starch digestion analysis (C. Li et al., 2020).The surface topography and morphology of WMS, NMS and HAMS and their digestion residuals as monitored by FE-SEM (Fig. 5), revealed that the degree of disruption of the original granular integrity during digestion decreased with increased AC. A few porous starch granules with large pore size were found among the WMS granular population digested for 40 min, and these granules were completely reorganized, disrupted and transformed into an aggregated structure after 240 min of degradation. Many porous starch granules were also found for NMS digested for 40 min, however, the pore size was smaller than found for WMS. For NMS, the pore size increased and only some of the granules were disrupted after 240 min of digestion. For HAMS, the granular structure was slightly disrupted after 40 min, and only few hydrolytic pores were found, however, the pore number increased after 240 min degradation (Fig. 5). As monitored by CLSM imaging using FITC-labelled GA, GA remained at the granular surface only for the native starches. After 40 and 240 min of digestion, hydrolytic pores (100–500 nm) were generated, which exceed the size of GA protein (∼6–12 nm) (Kramer, Gunning, Morris, Belshaw, & Williamson, 1993). Consequently, GA could migrate/diffuse to internal surface regions of the starch granules. which helps explaining the increased density of binding sites (Fig. 4). For the HAMS, the high hydrolytic resistance at excess GA (Fig. 3D and F), can be explained by HAMS’ low disruption level. We also found that GA was still mainly located on the surface regions of the granules (Fig. 5), reflecting that the increase in binding sites during digestion was less when compared with WMS and NMS. Hence, the FE-SEM morphological data and the CLSM imaging of GA localization both support the notion of increased granular binding sites of GA during digestion. This is attributed to the generation of porous structures, increased surface area, and eventually, increased GA binding density to internal surfaces of the granules.This work was supported by China Scholarship Council (CSC) grant #202003250068 (YT) and #202006790033 (YW) and HIAMBA - grain, flour, bread & bakery products preventing type 2 diabetes” Innovation Fund Denmark, Project 9067-00004A (YZ).

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