An enzymatic approach to quantify branching on the surface of starch granules by interfacial catalysis

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Enzymatically modified starch granules are useful in the food industry by endowing improved thermal properties, resistance to digestion and complexation capacity. However, it is of interest to correlate structural features on the granular surface with functional characteristics relevant to given applications. To meet this requirement, a method was developed to quantify the density of α-1,6 branch points on differently structured starch granules as based on interfacial enzyme catalysis. The branch points are attacked by pullulanase, a debranching enzyme, and the branch point density, as calculated from the kinetic attack site density (kinΓmax), was linked to the chain length distribution (CLD) of the released segments. The procedure involved a combination of conventional and inverse Michaelis–Menten (MM) kinetics for pullulanase degradation of native, branching enzyme- or 4-α-glucanotransferase-modified granular waxy and normal maize starch (WMS and NMS). The treatment by branching enzyme increased the branch point density for WMS from 1.7 to 3.3 nmol/g starch granules. CLD analysis indicated that 4-α-glucanotransferase catalyzed hydrolysis and/or cyclization on the surface of the granules, rather than disproportionation. The CLD data reflected the different spatial organization of amylopectin chains within WMS and NMS granules related to their different amylose contents of 0.7 and 20.7%, respectively. Scanning electron microscopy confirmed that the starch granules retained the morphology without prominent cracks or pores after pullulanase hydrolysis for the analysis of interfacial kinetics. Comparison with the corresponding gelatinized starches gave new insights into the connection between substrate structure and specificity of the two glucotransferases acting on the different starches.

OriginalsprogEngelsk
Artikelnummer109162
TidsskriftFood Hydrocolloids
Vol/bind146
Antal sider7
ISSN0268-005X
DOI
StatusUdgivet - 2024

Bibliografisk note

Funding Information:
The TuαGT modification of granular WMS (Fig. 1A) resulted in a 1.2-fold increase in A-chains and decreased B1, B2- and B3-chains by 1.1-, 1.1- and 1.8-fold, respectively, compared to native WMS. This resulted in a slight overall decrease in DPAve (Table 1). A similar trend was seen by TuαGT modification of NMS granules, where 1.3-fold higher and 1.2-fold lower proportions, respectively of A- and B1-chains were obtained relative to native NMS granules (Fig. 1B; Table 1). The CLD patterns for the corresponding native and modified gelatinized starches differed, as in case of WMS, both A- and B1-chains slightly decreased by 1.1-fold, while B2- and B3-chains increased by 1.2- and 1.1-fold, respectively. NMS exhibited a similar pattern, although its B1-chain content remained unchanged (Table 1). The CLD data supported the role of TuαGT catalyzing disproportionation between glucan chains, resulting in less short and more long chains. Notably, among the four different reactions catalyzed by TuαGT, only hydrolysis and cyclization will lead to an overall decrease in average chain length as found for the surface of modified granular starches (Table 1). Therefore, TuαGT apparently mainly catalyzed hydrolysis and/or cyclization of branch chains on the starch granules, but catalyzed disproportionation of the branch chains in gelatinized starches (Table 1).Karina Jansen (Department of Biotechnology and Biomedicine, Technical University of Denmark) is gratefully thanked for technical assistance. This work was supported by a China Scholarship Council, China (CSC) grant #202006790033 and the Technical University of Denmark, Denmark (YW), and by a China Scholarship Council, China (CSC) grant #202003250068 (YT). We are thankful to Cargill for providing waxy maize starch and to Archer Daniels Midland for providing normal maize starch.

Funding Information:
Karina Jansen (Department of Biotechnology and Biomedicine, Technical University of Denmark) is gratefully thanked for technical assistance. This work was supported by a China Scholarship Council, China ( CSC ) grant #202006790033 and the Technical University of Denmark, Denmark (YW), and by a China Scholarship Council, China ( CSC ) grant #202003250068 (YT). We are thankful to Cargill for providing waxy maize starch and to Archer Daniels Midland for providing normal maize starch.

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