Amylases enzymolysis of tuber starch granules for surface and internal structure observation under scanning electron microscopy

Research on starch structure can provide a theoretical basis for the modification reaction and application of starch. The surface blocklets and shell structure of starch granules determine their enzymolysis and swelling characteristics. At room temperature, three kinds of amylase (α-amylase, β-amylase and pullulanase) were used alone or in compound for three tuber starch (potato, sweet potato and cassava starch) hydrolysis respectively. The enzymolysis rate of starch granules was calculated, gelatinization enthalpy and relative crystallinity of three tuber starch granules before and after limited enzymolysis were calculated by differential scanning calorimetry and X-rays diffraction, respectively. The surface blocklets and shell structure of the enzymolyzed starch granules was observed by scanning electron microscopy. The results showed that the enzymolysis rate of α-amylase was higher than β-amylase and pullulanase. The non-reducing end and α-1,6 glucoside bond located mainly inside, while α-1,4 glucosidic bond located mainly in surface of starch granules. The gelatinization enthalpy and relative crystallinity of the enzymolyzed starch granules increased slightly, these physicochemical properties were close to those of the native starches. The results indicated that the structure of starch granules after enzymolysis was similar to that of native starch granules. Potato starch showed much lower enzymolysis rate (1.1%) than the other two starches (14.1%, 16.3%), and it had the strongest resistance to enzymolysis.In the reaction of single amylase, α-amylase could make the surface of three tuber starch granules appear scratches and cracks, and expose surface blocklets (diameter 29-73 nm) and inner shell structure (thickness 150-400 nm). The surface blocklets of potato starch granules were tightly packed; while those of sweet potato starch granules were loosely arranged with holes, and those of cassava starch granules showed collapses and cracks. The enzymolysis rate of potato starch was much lower than that of the other two starches. Potato starch had strong resistance to enzymes. The results indicated that structure of surface blocklets determined the enzymolysis characteristics of starch granules. Compared with sweet potato and cassava starches, the shell structure of potato starch was dense and orderly. This was consistent with the results of peak viscosity measurement. The pasting properties of three tuber starches (5%, w/w) were measured by using rapid viscosity analyzer (RVA), and the peak viscosity of potato starch (2216 mPa·s) was significantly higher than those of sweet potato and cassava starches (593 and 740 mPa·s). Potato starch granules swelled to a larger degree, indicating that the shell structure determines the expansion characteristics of starch.After β-amylase hydrolysis, only surface bolcklets of granules was observed. After pullulanase treatment, there was no obvious change for granules and no surface blocklets or shell were observed. When hydrolyzed with mixed amylase, the composite reaction of α-amylase was similar to that of single reaction. For example, after hydrolysis with combination of α-amylase and β-amylase, surface blocklets with sizes of 36-65, 32-61 and 32-52 nm appeared on the surface of potato, sweet potato and cassava starches, respectively. Potato and sweet potato starch showed shell structure (thickness 300-400 nm and 100-150 nm, respectively). Sweet potato and cassava starch appeared hollow shell structure (thickness 100-150 nm and 100-200 nm, respectively). Treatment with β-amylase or pullulanase, alone or mixed, could not show shell or outer shell structure for three tuber starch granules. Therefore, the single amylase hydrolysis of α-amylase (80 U/mL, room temperature, 12 h) could be used as a method to study surface blocklets and shell structure of starch granules simultaneously.