Staling of Bread: Role of Amylose and Amylopectin and Influence of Starch‐Degrading Enzymes

The present investigation aims at understanding the mechanism of bread firming during staling. Changes in the starch fraction due to the addition of amylases and their influence on the texture of bread crumb were studied during aging and after rebaking of stale bread. Pan bread was prepared by a conventional baking procedure. The influence of three different starch‐degrading enzymes, a conventional α‐amylase, a maltogenic α‐amylase, and a β‐amylase were investigated. The mechanical properties of bread were followed by uniaxial compression measurements. The microstructure was investigated by light microscopy, and starch transformations were assessed by differential scanning calorimetry (DSC) and wide‐angle X‐ray powder diffraction. Firming of bread crumb and crystallization of starch are not necessarily in agreement in systems with added amylases. Reorganization of both starch fractions, amylopectin and amylose, and the increase of starch network rigidity due to increase of polymer order are important during aging. Starch‐degrading enzymes act by decreasing the structural strength of the starch phase; for instance, by preventing the recrystallization of amylopectin or by reducing the connectivity between crystalline starch phases. On the other hand, starch‐degrading enzymes may also promote the formation of a partly crystalline amylose network and, by this, contribute to a kinetic stabilization of the starch network. Based on the results, a model for bread staling is proposed, taking into account the biphasic nature of starch and the changes in both the amylose and amylopectin fraction.     

Determining the Role of Starch in Flour Tortilla Staling Using α‐Amylase

Effects of α‐amylase modification on dough and tortilla properties were determined to establish the role of starch in tortilla staling and elucidate the antistaling mechanism of this enzyme. Control and amylase‐treated tortillas were prepared using a standard bake test procedure, stored at 22°C, and evaluated over four weeks. Amylase improved shelf‐stability of tortillas. The enzyme also produced a significant amount of dextrins and sugars, decreased loss of amylose solubility, and weakened starch granules. Amylopectin crystallinity increased with time, but was similar for the control and treated tortillas. Staling of tortillas appears to mainly involve the starch in the amorphous phase. As such, amylase activity does not significantly interfere with amylopectin crystallization. It is proposed that amylase partially hydrolyzed the dispersed starch (i.e., mostly amylose), starch bridging the crystalline region, and protruding amylopectin branches. Starch hydrolysis decreases the rigid structure and plasticized polymers during storage. The flexibility of tortillas results from the combined functionalities of the amylose gel and amylopectin solidifying the starch granules during storage. Protein functionality may also be involved in tortilla staling, but this needs further research.     

Using Visible and Near‐Infrared Reflectance Spectroscopy and Differential Scanning Calorimetry to Study Starch,Protein, and Temperature Effects on Bread Staling

Starch, protein, and temperature effects on bread staling were investigated using visible and near‐infrared spectroscopy (NIRS) and differential scanning calorimetry (DSC). Bread staling was mainly due to amylopectin retrogradation. NIRS measured amylopectin retrogradation accurately in different batches. Three important wavelengths, 970 nm, 1,155 nm, and 1,395 nm, were associated with amylopectin retrogradation. NIRS followed moisture and starch structure changes when amylopectin retrograded. The amylose‐lipid complex changed little from one day after baking. The capability of NIRS to measure changes in the retrograded amylose‐lipid complex was limited. Two important wavelengths, 550 nm and 1,465 nm, were key for NIRS to successfully classify the starch‐starch (SS) and starch‐protein (SP) bread based on different colors and protein contents in SS and SP. Low temperature dramatically accelerated the amylopectin retrogradation process. Protein retarded bread staling, but not as much as temperature. The starch and protein interaction was less important than the starch retrogradation. Protein hindered the bread staling process mainly by diluting starch and retarding starch retrogradation.     

Effect of Partial Gelatinization and Lipid Addition on α‐Amylolysis of Barley Starch Granules

The effect of partial gelatinization with and without lipid addition on the granular structure and on α‐amylolysis of large barley starch granules was studied. The extent of hydrolysis was monitored by measuring the amount of soluble carbohydrates and the amount of total and free amylose and lipids in the insoluble residue. Similarly to the α‐amylolysis of native large barley starch granules, lipid‐complexed amylose (LAM) appeared to be more resistant than free amylose and amylopectin. Partial gelatinization changed the hydrolysis pattern of large barley starch granules; the pinholes typical of α‐amylase‐treated large barley starch granules could not be seen. Lipid addition during partial gelatinization decreased the formation of soluble carbohydrates during α‐amylolysis. Also free amylose remained in the granule residues and mostly amylopectin hydrolyzed into soluble carbohydrates. These findings indicate that lysophospholipid (LPL) complexation with amylose occurred either during pretreatment or after hydrolysis, and free amylose was now part of otherwise complexed molecules instead of being separate molecules. Partial gelatinization caused the granules to swell somewhat less during heating 2% starch‐water suspensions up to 90°C, and lipid addition prevented the swelling completely. α‐Amylolysis changed the microstructure of heated suspensions. No typical twisting of the granules was seen, although the extent of swelling appeared to be similar to the reference starch. The granules with added LPL were partly fragmented after hydrolysis.     

Starch Hydrolyzing Enzymes for Retarding the Staling of Rice Bread

Previous attempts have been made to obtain gluten‐free bread of acceptable quality for bread specific volume and crumb texture. Rice bread is a good alternative to celiac patients, but it has a very rapid staling during storage. Rice starch is more prone to retrograde during storage than wheat starch, and the special hydrophobic nature of the rice proteins requires specific enzymes to be used in the rice bread process. To retard rice bread staling, two different starch hydrolyzing enzymes (α‐amylase of intermediate thermostability and cyclodextrin glycoxyl transferase [CGTase]) have been tested and their effect on fresh bread quality and staling during storage has been evaluated. The addition of α‐amylase improved bread specific volume and crumb firmness but very sticky textures were obtained. The addition of CGTase produced even higher specific volume and similar crumb firmness with better texture. Both enzymes decreased the ability of amylopectin to retrograde during storage. The firming kinetic was lowered by the α‐amylase but not the limiting firmness, while the rice crumb from CGTase firmed quickly with a very short range of firmness increase. Results revealed that the starch hydrolysis brought about by the α‐amylase was not sufficient to retard staling. CGTase was considered a better antistaling agent because of its starch hydrolyzing and cyclizing activity.     

Starch Retrogradation and Firming of Bread Containing Hydroxypropylated,Acetylated, and Phosphorylated Cross‐Linked Tapioca Starches for Wheat Flour

The present investigation aims at understanding the role of chemically modified starch on the firmness of fresh or stale bread. Bread was prepared from wheat flour or substituted wheat flour that contained 18% chemically modified tapioca starch and 2% vital gluten. Hydroxypropylated tapioca starch (HTS), acetylated tapioca starch (ATS), phosphorylated cross‐linked tapioca starch (PTS), and native tapioca starch (NTS) were tested. Bread prepared from the substituted flour with PTS showed a firmer texture on the day of baking compared with bread prepared from NTS, HTS, and ATS. PTS retained its granular structure in the gluten network after baking and seemed to play the role of filler particles in the gluten matrix, thereby increasing firmness of fresh bread crumb. Bread prepared from the substituted flour with HTS or ATS firmed at a lower rate and showed a lower endothermic melting enthalpy of amylopectin after three days of storage compared with NTS or PTS. These findings suggest that the staling of bread containing chemically modified tapioca starch involves recrystallization of amylopectin.     

Dough and Baking Properties of High‐Amylose and Waxy Wheat Flours

The dough properties and baking qualities of a novel high‐amylose wheat flour (HAWF) and a waxy wheat flour (WWF) (both Triticum aestivum L.) were investigated by comparing them with common wheat flours. HAWF and WWF had more dietary fiber than Chinese Spring flour (CSF), a nonwaxy wheat flour. Also, HAWF contained larger amounts of lipids and proteins than WWF and CSF. There were significant differences in the amylose and amylopectin contents among all samples tested. Farinograph data showed water absorptions of HAWF and WWF were significantly higher than that of CSF, and both flours showed poorer flour qualities than CSF. The dough of WWF was weaker and less stable than that of CSF, whereas HAWF produced a harder and more viscous dough than CSF. Differential scanning calorimetry data showed that starch in HAWF dough gelatinized at a lower temperature in the baking process than the starches in doughs of WWF and CSF. The starch in a WWF suspension had a larger enthalpy of gelatinization than those in HAWF and CSF suspensions. Amylograph data showed that the WWF starch gelatinized faster and had a higher viscosity than that in CSF. The loaves made from WWF and CSF were significantly larger than the loaves made from HAWF. However, the appearance of bread baked with WWF and HAWF was inferior to the appearance of bread baked with CSF. Bread made with WWF became softer than the bread made with CSF after storage, and reheating was more effective in refreshing WWF bread than CSF bread. Moreover, clear differences in dough and bread samples were revealed by scanning electron microscopy. These differences might have some effect on dough and baking qualities.     

Observations on the α‐Amylolysis Pattern of Some Waxy Maize Starches from Inbred Line Ia453

Maize starches of the endosperm mutants waxy (wx), dull:waxy (duwx), and amylose‐extender:dull:waxy (aeduwx) from inbred line Ia453 lack amylose. However, in addition to high molecular weight (HMW) amylopectin, the duwx and aeduwx starches contained 40 and 80%, respectively, intermediate branched material of low molecular weight (LMW). As gelatinized, the amylopectin of the wx starch was easily hydrolyzed into small dextrins by the α‐amylase of B. amyloliquefaciens, but components of duwx and aeduwx possessed partial resistance to amylolytic attack. Residual material of intermediate size obtained by a 4‐hr α‐amylolysis could not be separated from LMW dextrins by fractional precipitation in methanol. It is suggested that this material possessed a more regularly branched structure, in which the d ‐glucosyl chain segments were too short to allow α‐amylase action. The granular starches of duwx and aeduwx genotypes were initially considerably more resistant than the wx sample to α‐amylase attack. This was possibly due to an altered structure in the amylopectin component or the high content of intermediate material in the former granules.     

Bread Quality of Spelt Wheat and Its Starch

Flours from five spelt cultivars grown over three years were evaluated as to their breadbaking quality and isolated starch properties. The starch properties included amylose contents, gelatinization temperatures (differential scanning calorimetry), granule size distributions, and pasting properties. Milled flour showed highly variable protein content and was higher than hard winter wheat, with short dough‐mix times indicating weak gluten. High protein cultivars gave good crumb scores, some of which surpassed the HRW baking control. Loaf volume was correlated to protein and all spelt cultivars were at least 9–51% lower than the HRW control. Isolated starch properties revealed an increase in amylose in the spelt starches of 2–21% over the hard red winter wheat (HRW) control. Negative correlations were observed for the large A‐type granules to bread crumb score, amylose level, and final pasting viscosity for cultivars grown in year 1999 and to pasting temperature in 1998 samples. Positive correlations were found for the small B‐ and C‐type granules relative to crumb score, loaf volume, amylose, and RVA final pasting viscosity for cultivars grown in 1999, and to RVA pasting temperature for samples grown in 1998. The environmental impact on spelt properties seemed to have a greater effect than genetic control.     

Effects of Mutant Thermostable α‐Amylases on Rheological Properties of Wheat Dough and Bread

Thermostable mutant α‐amylases (21B, M111, and M77) with various degrees of thermostability were purified from Bacillus amyloliquefaciens F and used as improvers for breadmaking. Test baking with the mutant enzymes was conducted using the long fermentation sponge‐dough method. Addition of an appropriate amount of mutant α‐amylases to the ingredients distinctly increased the specific volume of the bread and improved the softness of breadcrumb as compared with the addition of Novamyl (NM), an exo‐type α‐amylase. M77 was the most effective in retarding the staleness of breadcrumb. The softness of breadcrumb during storage, however, was not correlated with the thermostability. All mutant α‐amylases weakened the mixing property of the dough, whereas they strengthened the property of fermented dough. Especially, M77 and NM had different effects on the dough properties, but their bread qualities were similar to each other. The strong tolerance of M77 dough to the long baking process might be due to the production of hydrolyzed starches, oligosaccharides in the range of maltopentaose to maltohexaose, as compared with NM. Therefore, in the light of present findings, these mutant α‐amylases are possible substitutes for NM as bread improvers.     

Effect of Different Enzymes on the Textural Stability of Shelf‐Stable Bread

Three enzyme systems (2 amylase‐based and 1 protease‐based) were tested in shelf‐stable bread to determine effectiveness in preserving texture during storage for eight weeks. Each enzyme was tested in formulations without glycerol or with 6% glycerol. Bread samples were analyzed to determine physical properties (crumb density, crust‐to‐crumb ratio, rate of moisture distribution from crumb to crust), mechanical properties (modulus, and a parameter [C1] describing resistance to high levels of deformation obtained by fitting stress‐strain data to a three‐parameter function), and thermal properties (thermal stability and enthalpy of transitions) as a function of storage time. Mechanical properties were further analyzed to predict asymptotic firmness. Bread firmness after storage as evaluated in terms of modulus and C1 were lower in all enzyme‐added systems, the effect of protease being the most significant. Enzymes had less effect on glycerol‐containing systems with no apparent trend. The breads had complex thermal behavior and exhibited multiple transitions. Both amylase preparations in the presence of glycerol reduced the amount of starch recrystallization.     

Activity‐Guided Fractionation to Identify Blue Wheat (UC66049 Triticum aestivum L.) Constituents Capable of Inhibiting In Vitro Starch Digestion

The inhibition or delay of starch digestion by dietary compounds could help manage postprandial blood glucose levels. The objective of this study was to identify constituents from whole grain blue wheat capable of decreasing α‐amylase‐catalyzed starch digestion. An activity‐guided fractionation approach based on liquid chromatography was used to identify solvent‐ and alkaline‐extractable blue wheat constituents reducing α‐amylase‐mediated starch digestion in vitro. Fatty acids, potentially released from cell wall polymers by alkaline hydrolysis, inhibited the digestion of amylose, probably through the formation of amylose‐lipid complexes. However, the degradation of amylopectin was not affected by fatty acids. In addition, 1‐(3,5‐dihydroxyphenyl)heneicosan‐2‐one, a 5‐(2′‐oxoalkyl)resorcinol, was found to reduce starch digestion. However, because the digestion of both amylopectin and amylose was reduced, the inhibition mechanism was different from that of fatty acids. Further research is needed to evaluate whether this component also reduces starch digestion in vivo. Other phenolic compounds of blue wheat such as anthocyanins or hydroxycinnamates were not identified as major starch digestion inhibitors by using the activity‐guided fractionation approach.     

Effects of Heat-Moisture Treatment and Lipids on Gelatinization and Retrogradation of Maize and Potato Starches

Effects of heat-moisture treatment (HMT) and lipids on the structure and gelatinization of maize and potato starches were studied, and the retrogradation process of 20% HMT starch gels was also investigated. Maize starch was physically modified by HMT or by defatting. Potato starch was physically modified by HMT or by adding monoglycerides. The X-ray pattern of the HMT maize starch was assigned to a combination of A and V patterns, which indicated that HMT formed crystallized amylose complexes and recrystallized amylose in maize starch granules. However, the X-ray pattern of defatted maize starch did not change for HMT, so the lipids originally existing in starch granules were important to the formation of new crystallites during this treatment. Differential scanning calorimetry (DSC) results suggested that weaker structures in amylopectin crystallites were more susceptible to degradation after HMT, while crystallized amylose complexes developed thermal stability after treatment. The amylose contents increased with increasing degree of HMT, which suggested that the newly created amylose arose from exterior linear chains of amylopectin degraded by the treatment. Investigation of retrogradation process showed that HMT significantly promoted retrogradation of starch gels, especially the initiation of recrystallization.     

Impact of maltodextrins and antistaling enzymes on the differential scanning calorimetry staling endotherm of baked bread doughs

Different concentrations (1.2-3.6%) of maltodextrin preparations with average degrees of polymerization (DP) varying between 4 and 66 reduced the differential scanning calorimetry (DSC) staling endotherm in baked and stored (7 days, 23 degrees C) bread doughs from 3.4 mJ/mg to values within a 3.0-1.9 mJ/mg range. Commercial enzymes used in industrial practice as antistaling agents for bread also reduced amylopectin retrogradation. This suggested that the maltodextrins used are promising antistaling components and that the staling of bread and amylopectin retrogradation are related phenomena. In addition, the results obtained suggest that starch hydrolysis products resulting from enzymic attack may well be responsible for the antistaling effect induced by antistaling enzymes.     

Resistance to α‐Amylase Digestion in Four Native High‐Amylose Maize Starches

Native and processed high‐amylose maize starch (HAMS) is an important source of resistant starch (RS). The objectives of this work were to use an in vitro procedure to estimate the RS content of native granules from a series of ae‐containing HAMS genotypes, and to examine the nature of the α‐amylase resistant starch (ARS). By the method of Englyst et al (1992), RS for ae V, ae VII, ae su2, and ae du were estimated to be 66.0, 69.5, 69.5, and 40.6%, respectively. By transmission electron microscopy, most of the residual granules from ae V, ae VII, and ae su2 showed little evidence of digestion. Partially digested granules had a radial digestion pattern in the interior and an enzyme‐resistant layer near the surface. Size and chain‐length profile of constituents of ARS were similar to those of the native HAMS (unlike type 3 RS), consistent with complete hydrolysis in susceptible granule regions. Between crossed polarizers, many iodine‐stained native and residual HAMS granules had blue centers and pink exteriors, which may be due to a difference in orientation of the amylose‐iodine complexes in the exterior. Four granule color types were observed for ae du, differing in enzyme resistance. The high‐enzyme resistance of native HAMS granules may result from altered granule organization, which appears to vary among and within granules from ae‐containing genotypes.     

Influence of Amylose Content on Properties of Wheat Starch and Breadmaking Quality of Starch and Gluten Blends

The effects of amylose content on thermal properties of starches, dough rheology, and bread staling were investigated using starch of waxy and regular wheat genotypes. As the amylose content of starch blends decreased from 24 to 0%, the gelatinization enthalpy increased from 10.5 to 15.3 J/g and retrogradation enthalpy after 96 hr of storage at 4°C decreased from 2.2 to 0 J/g. Mixograph water absorption of starch and gluten blends increased as the amylose content decreased. Generally, lower rheofermentometer dough height, higher gas production, and a lower gas retention coefficient were observed in starch and gluten blends with 12 or 18% amylose content compared with the regular starch and gluten blend. Bread baked from starch and gluten blends exhibited a more porous crumb structure with increased loaf volume as amylose content in the starch decreased. Bread from starch and gluten blends with amylose content of 19.2–21.6% exhibited similar crumb structure to that of bread with regular wheat starch which contained 24% amylose. Crumb moisture content was similar at 5 hr after baking but higher in bread with waxy starch than in bread without waxy starch after seven days of storage at 4°C. Bread with 10% waxy wheat starch exhibited lower crumb hardness values compared with bread without waxy wheat starch. Higher retrogradation enthalpy values were observed in breads containing waxy wheat starch (4.56 J/g at 18% amylose and 5.43 J/g at 12% amylose) compared with breads containing regular wheat starch (3.82 J/g at 24% amylose).     

Effects of Varying Weight Ratios of Large and Small Wheat Starch Granules on Experimental Straight‐Dough Bread

One commercial bread wheat flour with medium strength (11.3% protein content, 14% mb) was fractionated into starch, gluten, and water solubles by hand‐washing. The starch fraction was separated further into large and small granules by repeated sedimentation. Large (10–40 μm diameter) and small (1–15 μm diameter) starch fractions were examined. Flour fractions were reconstituted to original levels in the flour using composites of varying weight percentages of starch granules: 0% small granules (100% large granules), 30, 60, and 100% (0% large granules). A modified straight‐dough method was used in an experimental baking test. Crumb grain and texture were significantly affected. The bread made from the reconstituted flour with 30% small granules and 70% large granules starch had the highest crumb grain score (4.0, subjective method), the highest peak fineness value (1,029), and the second‐highest elongation ratio (1.55). Inferior crumb grain scores and low fineness and elongation ratios were observed in breads made from flours with starch fractions with 100% small granules or 100% large granules. As the proportion of small granules increased in the reconstituted flour, it yielded bread with softer texture that was better maintained than the bread made from the reconstituted reference flour during storage.     

α-Amylolysis of Large Barley Starch Granules

The α-amylolysis of large (volume average 16 μm) barley starch granules was studied by measuring the amount of carbohydrates solubilizing during hydrolysis, and the changes in morphology and molecular structure of the granule residues by scanning electron microscopy, particlesize analysis, size-exclusion chromatography, X-ray diffraction, and differential scanning calorimetry. X-ray diffraction showed that, in the earlier stages of α-amylolysis, both amorphous and crystalline parts of the granules were equally solubilized. More extensive hydrolysis caused a gradual decrease in A-type crystallinity and degradation of the granular structure. Scanning electron microscopy revealed that hydrolysis proceeded through pinholes, and pitted and partially hollow granule residues were formed. The lipid-complexed amylose was less susceptible to α-amylolysis than free amylose and amylopectin. Lipid-complexed amylose started leaching out of the granule residues only after half of the starch had solubilized due to the α-amylase treatment. Even though scanning electron microscopy indicated that there were intact granules left throughout the hydrolysis, the results obtained suggested that α-amylolysis of large barley starch granules proceeded rather evenly among the granules.     

Model approach to starch functionality in bread making

We used modified wheat starches in gluten-starch flour models to study the role of starch in bread making. Incorporation of hydroxypropylated starch in the recipe reduced loaf volume and initial crumb firmness and increased crumb gas cell size. Firming rate and firmness after storage increased for loaves containing the least hydroxypropylated starch. Inclusion of cross-linked starch had little effect on loaf volume or crumb structure but increased crumb firmness. The firming rate was mostly similar to that of control samples. Presumably, the moment and extent of starch gelatinization and the concomitant water migration influence the structure formation during baking. Initial bread firmness seems determined by the rigidity of the gelatinized granules and leached amylose. Amylopectin retrogradation and strengthening of a long-range network by intensifying the inter- and intramolecular starch-starch and possibly also starch-gluten interactions (presumably because of water incorporation in retrograded amylopectin crystallites) play an important role in firming.     

Changes in Distribution of Isoflavones and β‐Glucosidase Activity During Soy Bread Proofing and Baking

Bread made partially with soy may represent a viable alternative for increasing soy consumption in populations consuming Western diets. The potential health‐promoting activity of soy isoflavones may depend on their abundance and chemical form. The objective of this study was to characterize the changes in isoflavone distribution and β‐glucosidase activity during the soy breadmaking process. Soy bread ingredients were combined and mixed to form a dough that was subsequently proofed at 48°C for 1–4 hr and baked at 165°C for 50 min to produce breads. The isoflavone composition and β‐glucosidase activity in bread ingredients, doughs, and breads were monitored. Soy ingredients and wheat flour (not bread yeast) were the major contributors of the β‐glucosidase activity in bread. No degradation of isoflavones was observed during breadmaking but the isoflavone distribution was largely altered. Proofing and baking have important but different roles in changing the isoflavone distribution. Proofing converted isoflavone β‐glucosides to aglycones by highly specific β‐glucosidase activity. Thermal treatment during baking significantly decreased the isoflavone malonylglucosides and increased isoflavone β‐glucosides. Enzyme activity during proofing and the balance between formation and deconjugation of isoflavones during baking determine the isoflavone content and composition in the final product.