Maltose formation from wheat flour with different degrees of damaged starch

Abstract

Six types of wheat flour with different degrees of damaged starch were used to prepare dough by adding 3.0 times the amount of water to that of flour on a weight basis. The amount of maltose formed during rest at 60 °C increased sharply at the early stage of rest and then asymptotically approached a constant value, M_∞. The initial amount of maltose M₀ was proportional to the degree of damaged starch, but M_∞ was independent of the degree of damaged starch, 0.291 ± 0.015 g maltose/g flour (mean ± S.D.). The possible amount of maltose to be formed, M_∞−M, was normalized by the difference between M_∞ and M₀, (M_∞−M)/(M_∞−M₀), which could be expressed by the Weibull equation as a function of the resting time. Both the rate constant k and the shape coefficient n did not depend on the degree of damaged starch; k = 1.19 ± 0.16 h⁻¹ and n = 0.601 ± 0.091, respectively.

Introduction

Bread made from wheat flour has been widely consumed by humans since ancient times (Mondal and Datta, 2008). It is made from wheat flour, water, salt, and yeast (essential ingredients), and fats and oils, sugar, emulsifiers, and enzymes are added as additional ingredients (Goesaert et al., 2005). Bread manufacturing methods are classified on the basis of how the raw materials are mixed (Mondal and Datta, 2008). In the Yudane method, dough is produced by kneading a part of the wheat flour with boiling water and further kneading it with all the remaining ingredients (Yamauchi et al., 2014). Bread produced by this method is popular in Japan because of its mild sweetness and unique texture (Yamauchi et al., 2014; Yamada et al., 2015). These characteristics are derived from maltose formed through the hydrolysis of starch by endogenous β-amylase in wheat flour (Yamauchi et al., 2014; Yamada et al., 2015).

Maltose plays an important role in determining the bread quality. Maltose not only sweetens but also serves as a substrate for fermentation by yeast to produce carbon dioxide and other aromatic substances (Mondal and Datta, 2008; Makhoul et al., 2015). Maltose is also associated with the crust color of baked bread (Bowles, 1996; Heinz et al., 2005) and the staling process (Katsuta et al., 1992; Yamada et al., 2015). In this way, research on bread has mainly focused on the quality of baked bread. On the other hand, few studies have focused on maltose formation during bread making.

The activity of endogenous β-amylase and the state of starch, that is, the degree of starch gelatinization, affect maltose formation. Starch gelatinization is in turn affected by both the degree of damaged starch and the amount of water added (Eliasson and Larsson, 1993; Putri et al., 2020). In the bread making process, it is difficult to alter parameters such as the amount and activity of endogenous β-amylase and the amount of water added. The temperature at which the dough is rested cannot be altered drastically. In this context, we focused on the degree of damaged starch. Damaged starch reduces the handleability of bread dough and swelling of bread (Barrera et al., 2007; Liu et al., 2014; Putri et al., 2020). On the other hand, damaged starch increases water sorption and facilitates hydrolysis in the presence of the enzyme α-amylase (Barrera et al., 2007), which may increase the amount of maltose formed.

In this study, dough was prepared using wheat flour with different degrees of damaged starch, and the amount of maltose formed in the dough was measured over time during its rest at 60 °C. The data were kinetically analyzed to examine the effect of the degree of damaged starch on maltose formation.

Materials and Methods

Materials    Six wheat flour samples (designated as A to F) with different degrees of damaged starch were supplied by the Nisshin Seifun Group. The degree of damaged starch was measured by the supplier according to the AACC method 76-30.02 (1969) (Table 1). In this method, the degree of damaged starch is defined in units of mg maltose/10-g flour based on the amount of sugar produced during storage at 30 °C in the presence of fungal α-amylase. Glucose (reagent grade) and maltotriose (chemical grade) were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). Maltose monohydrate (reagent grade) was purchased from Nacalai Tesque (Kyoto, Japan).

Table 1. Degree of damaged starch of flours used

Sample	A	B	C	D	E	F
Degree of damaged starch [mg/10-g flour]	187	212	323	373	603	622

Differential scanning colorimetry measurement    Wheat flour (2.0–2.5 mg) and water, whose amount was 2.5 times the weight of flour, were accurately weighed into an aluminum cell treated with Alodine (SSC000C009, Hitachi High-Technologies, Tokyo). The cell was crimp-sealed and allowed to stand at 5 °C for 30 min or longer. Differential scanning colorimetry (DSC) measurements were then performed using a DSC7020 differential scanning calorimeter (Hitachi High-Technologies Corporation, Tokyo) in the temperature range of 5 °C–95 °C at a heating rate of 5 °C/min. The DSC curve was analyzed to estimate the onset, peak, and conclusion temperatures for gelatinization as well as the gelatinization enthalpy.

Maltose formation    Wheat flour (30 g) was placed in a beaker. To this was added 90 g of distilled water (3.0 times the weight of wheat flour). The mixture was mixed with a spatula at room temperature (ca. 25 °C). A lump of the sample was transferred to a test tube and rested at 60 °C in a thermostated water bath (Thermo Minder SD, Titech, Saitama, Japan). The test tube was removed from the bath over time, and distilled water (weight measuring 6.5 times the weight of the flour) was added to the tube. The contents in the tube were homogenized (T-25 digital homogenizer, Ika Japan, Osaka) at 1.6 × 10⁴ rpm for 2 min under cooling with ice. Then, the mixture was centrifuged at 5.4 × 10³ rpm for 10 min at 4 °C (LX-120, Tomy Seiko, Tokyo), and the supernatant was filtered through a 0.45-µm membrane filter (RephiQuik Sylinge Filter, Rephile Bioscience, MA, USA) to obtain the sample for analysis. The sample was stored in a freezer until analysis. Maltose in the sample was determined by high-performance liquid chromatography (HPLC). The HPLC system consisted of a pump (LC-20AD, Shimadzu), a size exclusion chromatographic column (YMC-Pack Diol-60; 8.0 × 500 mm, YMC, Kyoto), an IR detector (RID-10A, Shimadzu), and a data processor (Chromatopack C-R8D, Shimadzu). Distilled water was used as the mobile phase at a flow rate of 1.0 mL/min. The peak that appeared at an elution time of 15.5 min was identified as maltose.

The maltose eluted from the Diol-60 column was fractionated, and the fraction was further applied to a polyamine-based silica gel column, Cosmosil Sugar-D (3.0 × 150 mm, Nacalai Tesque) to confirm the absence of glucose and maltotriose. The eluent was a mixture of acetonitrile and water (80:20 v/v) at a flow rate of 0.5 mL/min.

Results and Discussion

DSC analysis    The DSC curves of the samples with different degrees of damaged starch were measured to estimate the onset, peak, and conclusion temperatures for starch gelatinization. The gelatinization enthalpies of the samples were also estimated by integrating the curves (Fig. 1). Although the degree of damaged starch did not significantly affect the onset and peak temperatures of gelatinization, the peak temperature of gelatinization shifted slightly to higher values as the degree of damaged starch increased. On the other hand, gelatinization enthalpy tended to be smaller as the degree of damaged starch increased. This tendency was consistent with those reported by Stevens and Elton (1971) and León et al. (2006). These facts indicate that damaged starch has a lower enthalpy than that of undamaged starch for water absorption. Stevens and Elton (1971) stated that the loss of crystallinity, which was inferred from the magnitude of gelatinization enthalpy by DSC analysis, became more apparent in the early stages of damage than did the susceptibility to enzyme action.

Fig. 1.

(□) Onset, (○) peak, and (△) conclusion temperatures and (●) gelatinization enthalpy, ΔH, of wheat flour with different degrees of damaged starch

Maltose formation    Figure 2 shows the change in the amount of maltose formed M over time when doughs made from wheat flour with different degrees of damaged starch were rested at 60 °C. Dough prepared from wheat flour with a high degree of damaged starch tended to have a large amount of maltose initially. Regardless of the degree of damaged starch, the amount of maltose formed rapidly increased in the early stage of storage at 60 °C and gradually approached a certain value, M_∞, with time. The applicability of the Weibull equation (Eq. 1) to the change in M over time was examined:   

where M is the amount of maltose formed at time t, k is the rate constant, and n is the shape constant. The parameters M_∞, M₀, k, and n were determined using the Solver function of Microsoft Excel^® so that the sum of the residual squares of the M value calculated by Eq. 1 and the experimentally observed M value were minimized. Although M₀ had been measured, it was regarded as an unknown parameter and was treated in the same way as were other data points. The solid curves in Fig. 3 are the calculated values of the change in M over time by substituting the estimated parameters into Eq. 1. Since the calculated curves represented the measured values well regardless of the degree of starch damage, the maltose formation process during the resting period of the dough at a constant temperature can be empirically expressed by Eq. 1.

Fig. 2.

Maltose formation in doughs prepared from wheat flour with different degrees of damaged starch during rest at 60 °C. Symbols: (△) sample A, (□) sample B, (▷) sample C, (○) sample D, (◇) sample E, (▽) sample F. The solid curves were calculated based on Eq. 1.

Fig. 3.

Effects of the degree of damaged starch on (△) the initial amount of maltose M₀, (▽) the maximum amount of maltose M_∞, (○) the rate constant k, and (□) the shape constant n for maltose formation during rest at 60 °C. The solid line for M₀ was drawn according to Eq. 2.

Figure 3 shows the effects of the degree of damaged starch on the parameters M_∞, M₀, k, and n. The initial amount of maltose M₀ was proportional to the degree of damaged starch d, and the two were related by the following equation:   

As mentioned above, the degree of damaged starch is defined based on the amount of sugar produced from flour dough held at 30 °C. Since the dough was prepared at room temperature (about 25 °C) in this study, M₀ would reflect the amount of maltose formed from the damaged starch during dough preparation.

M_∞ was almost constant regardless of the degree of damaged starch, 0.291 ± 0.015 g maltose/g-flour. Since M_∞ reflects the amount of starch that has been gelatinized at 60 °C and hydrolyzed to maltose, the dependence of M_∞ on the degree of damaged starch was inconsistent. Since the starch content of wheat flour is approximately 70% (based on weight) (Goesaert et al., 2005) and integrating the DSC curve in terms of temperature indicates that about half of the starch is gelatinized at 60 °C, the estimated M_∞ would be reasonable.

Both the rate constant k and the shape constant n were almost independent of the degree of damaged starch, 1.19 ± 0.16 h⁻¹ and 0.601 ± 0.091, respectively. The almost constant value of n suggested that the mechanism by which maltose was formed through hydrolysis of starch by β-amylase was not affected by the degree of damaged starch. Although M₀ depended on the degree of damaged starch, M_∞ was considerably larger than M₀. Therefore, the amount of starch substrate that is hydrolyzed to maltose during rest at 60 °C, M_∞−M₀, can be approximated to be almost constant. This is presumed to be the reason why the rate constant was almost constant.

Prediction of maltose formation    Because of the four parameters in Eq. 1, three other than M₀ did not depend on the degree of damaged starch; the change in maltose formed over time can be predicted based on Eq. 1. Although M_∞−M₀ could be approximated to be almost constant, M₀ would significantly affect the amount of maltose produced in the early stage of the rest. Therefore, considering the dependence of M₀ on the degree of damaged starch, the change in M was calculated according to Eqs. 1 and 2 and compared with that in the measured M. As shown in Fig. 4, the plots of the observed M values against the calculated values were located diagonally. This indicates a good agreement between the measured M values and those calculated using the averaged M_∞, k, and n values and based on the dependence of d on the degree of damaged starch.

Fig. 4.

Comparison of calculated values M_cal and observed ones M_obs for the amount of maltose formed during rest at 60 °C. The key is the same as that presented in Fig. 2 legend.

M₀ does not depend on the storage temperature of the dough and can be predicted from the degree of damaged starch. M_∞ can be estimated from the starch content of wheat flour and the ratio of the area from low temperature to the rest temperature of the DSC curve to the total area. Furthermore, since maltose is formed through hydrolysis of starch by β-amylase, it can be assumed that the shape constant n is temperature-independent. On the other hand, the rate constant k depends on the temperature, and in the temperature range where the thermal denaturation of β-amylase can be ignored, k becomes larger as the rest temperature increases. Therefore, although there is a restriction of a constant moisture content of dough, if the temperature dependence of k is clarified, the maltose formation behavior in dough prepared from wheat flour with different degrees of damaged starch can be predicted during its isothermal rest. Verification of the validity of this prediction is a future task.

Conclusions

Dough prepared from wheat flour with different degrees of damaged starch was rested at 60 °C, and the effect of the degree of damaged starch on the amount of maltose formed during the rest was investigated. The initial amount of maltose was proportional to the degree of damaged starch, but the maximum amount of maltose formed was independent. The maltose formation process could be expressed by the Weibull equation, and neither the rate constant nor the shape constant depended on the degree of damaged starch.

Acknowledgements    This study was carried out as part of the project of the joint research on cereal science by Kyoto University, Gifu University, and the Nisshin Seifun Group, Inc.

Conflict of interest    The authors have no conflicts of interest to declare.

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