Antifirming properties of amylases in bread crumb were evaluated in straight dough breadmaking and related to the amylolytically modified starch structure. Amylase properties and action mechanisms determine starch structure in the breads and, hence, how amylopectin recrystallization, starch network formation, water redistribution, and water mobility occur during breadmaking and storage. A bacterial endo-alpha-amylase mainly hydrolyzed the longer starch polymer chains internally. It thus reduced the number of connections between the crystallites in the starch networks, resulting in a softer bread crumb. However, because the enzyme had only little impact on the outer amylopectin chains, amylopectin recrystallization and the concomitant water immobilization presumably were not hindered. The loss of plasticizing water as a result of recrystallization presumably reduces the flexibility of the gluten network and results in poor crumb resilience. In contrast, in breadmaking, the Bacillus stearothermophilus maltogenic alpha-amylase acted as an exoacting amylase with more pronounced endoaction at higher temperatures. This enzyme caused extensive degradation of the crystallizable amylopectin side chains and thus limited amylopectin recrystallization and network formation during storage. As a result, it prevented the incorporation of water in the amylopectin crystallites. In this way, the different starch and gluten networks kept their flexibility, resulting in a softer crumb with good resilience.

Several decades ago, the first reports on differences in action pattern between amylases from different sources indicated that the starch polymers are not degraded in a completely random manner. We here give an overview of different action patterns of amylases on amylose and amylopectin, focusing on the so-called multiple attack action of the enzymes. Nowadays, the multiple attack action is generally an accepted concept to explain the differences in amylase action pattern. However, the pancreatic α-amylase remains one of the few enzymes known with a considerable level of multiple attack action. Despite some recent studies, the molecular mechanism of the multiple attack action is still largely unclear. Probably, the degree to which the active site architecture and binding properties allow both the reorganization (sliding) of the substrate in the active site and the stabilisation of the productive enzyme/substrate complex mainly determine the multiple attack action of amylases.

The action pattern of several amylases was studied at 35, 50, and 70 °C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus α-amylase (BStA) and porcine pancreatic α-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme−substrate complex minus one), the fungal α-amylase from Aspergillus oryzae a low DMA, and the α-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of α-amylases is influenced by structural parameters of the starch substrate.

The effects of Bacillus subtilis, porcine pancreatic and Aspergillus oryzae α-amylases, sweet potato β-amylase and Bacillus stearothermophilus maltogenic amylase (BStA) on the rheological properties (measured with a Rapid Visco Analyser) of partially damaged wheat starch were studied and the accompanying changes in starch molecular properties were analysed by high-performance size exclusion chromatography. Pasting and gelation of starch slurries (with an increased level of damaged starch) were significantly affected by the supplemented amylases and greatly depended on the mode of action and properties of the enzymes added. In general, at low endo-amylase concentrations, peak, hot paste and cold paste viscosities were more reduced for enzyme-supplemented partially damaged starch than for enzyme-supplemented native wheat starch, demonstrating the significance of damaged starch levels in determining amylase functionality. Higher dosages of thermostable amylases ruled out most of the differences between amylase-supplemented native starch and partially damaged starches, except for BStA. Furthermore, the (limited) endo-action of BStA determines to a great extent the rheological properties of the starch paste. These results contribute to a better understanding of (maltogenic) amylase functionality in processing (damaged) starch-containing foods.

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