Original papers
Increase in the Viscosity of Soft-Flour Batter by Weak Direct-Current Processing
2014 Volume 20 Issue 4 Pages 815-819
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2014 Volume 20 Issue 4 Pages 815-819
Effect of direct current on the functional properties of batter made from soft-wheat flour was studied to establish a new processing method for food. Soft-wheat batter was processed with direct current (30 mA) and was separated into anode side and cathode side from the middle. The anode and cathode side samples were each kneaded six times for thirty seconds per. Batter viscosity was measured with a rotation viscometer after each kneading. After kneading, the viscosity of the anode side sample greatly increased with increasing electrical processing period. To elucidate the cause of this change, the processed batter was lyophilized and then extracted with water. The amount of extracted protein detected by the Bradford method increased with increasing period of electro-processing, especially in the anode side sample. Sodium dodecyl sulfate gel electrophoresis showed the increase of protein bands was attributable to glutenin macropolymer (GMP) in the extracted substance. Microscopic observation showed that the solubilized protein re-combined to make a protein matrix that captured starch granules.
Wheat flour is one of the most important food materials, and is used as a main material for breads, cakes, noodles etc. This is based on the ability of wheat protein to form gluten during dough formation. Dough properties have been studied widely, especially in bread making (Bloksma and Bushuk, 1988). The major proteins of wheat form a gluten network during kneading in various wheat foods. In dough systems, gluten proteins provide the main structural matrix (Bloksma and Bushuk, 1988).
Electrochemical methods, as opposed to electric thermal processing, have seldom been applied to food production systems. The electrolytic reduction of soybean protein was used to improve its functional properties (Komeyasu and Miura, 1981; Miura and Komeyasu, 1982). Another usage was the acidification of soybean-water-extract to obtain acid precipitated protein (Bazinet et al., 1998). Recently, we reported that tofu (soybean curd) could be produced by electrochemical processing without coagulation salt (Kamata et al., 2004). The electrochemical method could be a new and effective technique for the food industry because it is simple, easy to control and does not require chemical agents.
The viscosity of batter or dough has a large effect on the quality of various products such as bread and noodles. Thus, we examined ways to change the viscosity without using direct chemical treatment. To date, in an effort to improve the physical properties of flour using a method other than direct chemical reaction, treatment by acetic acid vapor is one of few examples (Seguchi et al., 1997; Hayashi and Seguchi, 2001). This paper describes a new electrochemical method to improve the wheat flour properties without the use of chemicals.
Materials Commercial soft wheat flour obtained from Nippon Flour Mills Co., Ltd. (Tokyo, Japan) was used throughout this work. Commercial semi-hard flour was obtained from Shiroishi Kosan Inc. (Miyagi, Japan). All reagents were of the highest grade.
Preparation of wheat batter We used ice made from tap water as the water for batter making because of its relatively low electrical resistance compared to distilled water. The ice was crushed with a home crusher. The crushed ice was mixed with wheat flour in equal amounts. In this method, mixing was performed without kneading. As a result, we could prevent the formation of gluten by mixing at this stage; therefore, interpretation of the experimental results was simplified.
Electrical processing of wheat batter The batter was placed in a polypropylene container (135 × 102 × 68 mm) and titanium rod (8ϕ × 100 mm) electrodes were inserted. Eight titanium rods were used in each electrode (Fig. 1). The sample was processed at a direct current of 30 mA (from one to three hours) with a power supply (NC-1010, Nihon Eido Co., Ltd., Tokyo, Japan), and then the sample was divided into half at the center. The temperature of the batter at the start of treatment was about 0°C because of the contained ice. Low power (0.3 – 3.7 W) was used in electrical treatment, and the temperature-rise of the sample reached a maximum of 31°C. The sample was lyophilized for use in various chemical analyses after rheological measurement.
The electrical processing device. A: side view of the apparatus. B: titanium rod electrodes. Distance of the electrodes from center to center of rods: a, 24 mm; b, 30 mm; c, 32 mm.
Rheological properties of the electro-processed batter The slurry from each electrode side (100 g) was placed in a plastic container. The viscosity of the batter was measured by a rotation viscometer (PASTE METER MODEL PM-1; Malcom, Tokyo, Japan). The batter was kneaded with a kitchen kneader (Smart Blender SKH-A with kneader unit SKH-N100X, Tiger Corporation, Osaka, Japan) six times for 30 seconds per. Viscosity was measured three times after each kneading. Three independent measurements were carried out for each condition. Means and standard deviations are provided in the figures.
Electrophoresis Sodium dodecyl sulfate (SDS) gel electrophoresis (Laemmeli, 1970) was performed using a commercial pre-made gradient gel (5 – 20%, ET-520L; ATTO, Tokyo, Japan) for 1.5 hour at 100 V. The samples were reduced using 2-mercaptoethanol.
Protein extraction Electro-processed samples were lyophilized and ground without kneading. A portion (200 mg) of the sample powder was dispersed in 5 mL of water. Extraction was performed under shaking for 30 minutes. The Bradford method (Bradford, 1976) was used to detect protein amounts from three samples of two independent experiments. Mean values and standard deviations were calculated. Bovine serum albumin was used as a standard protein for the method.
Microscopy The electro-processed and kneaded batter was lyophilized. A portion (10 mg) of wheat flour or the treated sample was dispersed in 1 mL of water. One drop of the dispersion was dried onto a glass slide, allowing the proteins in the solution to adhere. The proteins on the slide were stained with 0.1% Coomassie Brilliant Blue G-250 (Nakarai Tesque, Kyoto, Japan) solution. The slide was gently washed with water and allowed to dry. A microscope (CS-T15; Carton Optics, Tokyo, Japan) with a CCD camera was used for observation.
Viscosity changes during electro-processing The slurry from each electrode side (100 g) was placed in a plastic container. The sample was kneaded six times at 30 seconds per. Viscosity was measured between each kneading. Figure 2 shows a comparison of viscosity between the electro-processed batter at the anode side and the non-treated batter made from semi-hard flour. Figure 3 shows a comparison of viscosity between the electro-processed batters at the cathode side. The viscosity of the batter treated for the shortest duration (1 hour) did not change with kneading. As the electro-processing duration increased, the viscosity of the sample at the anode side increased (Fig. 2); however, no change in the cathode side sample was observed (Fig. 3). The final viscosity of the batter treated for 3 hours at the anode side approximated that of the semi-hard wheat batter.
Viscosity of the electro-processed batter at the anode side.
Viscosity of the electro-processed batter at the cathode side. Standard deviations were omitted for visual simplification.
Extraction of gluten protein from electro-processed batter The lyophilized batter was extracted with water. Figure 4 shows the extracted protein detected by the Bradford method. Electro-processing increased the quantity of extracted protein. Almost 22% of the total protein was extracted at the anode side with 1 hour treatment.
Amount of extracted protein after electro-processing.
The batter temperature ranged from 12 to 17°C during the 1-hour electrical treatment, and 3 hours thereafter was 31°C. On the other hand, the temperature of the batter made with ice was about 0°C, even after 3-hour standing without electrical treatment. When a sample was treated with alternating current (20 V AC) using the same apparatus with a slidac for reference, the temperature reached about 30°C after 2 hours. However, there was no change in the amount of extracted protein.
Electrophoresis The electro-processed wheat flour sample was lyophilized and extracted with water. The extract was relyophilized and then analyzed using SDS electrophoresis. Figure 5 shows the extracted protein analysis at the anode side in relation to electro-processing time. When samples are not subjected to electro-processing, only a low molecular weight protein subunit is extracted. There is an increase in high molecular weight subunits (80,000 – 90,000) with increasing electro-processing time. These bands appear to be high molecular weight subunits of glutenin (HMW-GS; Dupont et al., 2007). On the other hand, at the cathode side, the HMW-GS bands did not appear and no alterations in electrophoretic patterns with processing time were observed (data not shown).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted protein from the lyophilized batter treated at the anode side. C: Sample treated under identical conditions without electro-processing. N: Soft flour without any treatment. HMW-GS: High molecular weight glutenin subunit. LMW-GS: Low molecular weight glutenin subunit.
Microscopy The kneaded and lyophilized batter was dispersed with water. The dispersion was placed on a glass slide and fixed by air-drying. The fixed protein was stained with Coomassie Brilliant Blue. Figure 6 shows images of the treated wheat flour. Without electro-processing, most dyed protein appears to attach to the surface of starch granules like a membrane. The existence of a similar membrane has been reported by Yoshino et al. (2005). Part of this protein membrane appears to detach from the granule. On the other hand, after electro-processing, solubilized protein was found on the glass slide, especially on the anode side. The protein may capture many starch granules in its matrix. Therefore, it was suggested that a certain wheat protein was extracted from the surface of the starch granules into the water as a result of electro-processing. The solubilized protein might re-combine to form a protein matrix under mixing.
Microscopic images of starch granules of the lyophilized batter treated with direct current. Scale bar: 100 µm. Arrows indicate: A, protein membrane and detached membrane; B, large and small starch granules; C, re-combined matrix of the solubilized protein.
Various discussions have been conducted regarding the formation of wheat flour dough. The most important constituents are the gluten-forming proteins. The gluten proteins have a strong aggregation tendency, resulting from the hydrogen-bonding potential of the unusually large numbers of glutamine side chains. The aggregation results in formation of the network structure as well as protein insolubility. The hydrophobic bonding of many nonpolar side chains may also contribute to the insolubility. However, the possibility of incomplete interaction of the gluten protein exists, thereby resulting in a certain degree of solubility when the batter or dough is kneaded. Proteins on starch granules may cross-link more efficiently with gluten proteins if a proportion of the proteins are in the water phase. Solubilization of wheat protein by electro-processing has been proved by a number of methods. This was an unexpected effect of the processing.
From the electrophoresis result, a high molecular weight glutenin subunit (HMW-GS) was shown to dissolve in water by the reaction. HMW-GS typically connect to each other through disulfide bonding. In addition, this complex is combined with low molecular weight glutenin subunits (LMW-GS) through disulfide bonding, forming the glutenin macropolymer (GMP; Wieser et al., 1988). When the disulfide bond was maintained, the GMP band settled on the top of the electrophoresis gel. Thus, it was postulated that the disulfide bonds were not broken by electro-processing. Therefore, it was thought that intact GMP was solubilized. Although the protein solubilized, the viscosity of the batter subjected to one-hour treatment did not increase. On the other hand, the main extraction of GMP began at the second hour, with a concomitant increase in viscosity. Therefore, the existence of solubilized GMP was considered to have influenced the viscosity increase greatly.
It is known that wheat with identical total protein content can produce flours that behave quite differently in baking operations, and in many instances these differences are attributable to qualitative differences in the gluten proteins (Halverson and Zeleny, 1988). In this experiment, however, total protein content and gluten protein quality did not appear to change during electro-processing, except with respect to solubility. Only the changes in solubility of the protein may be a possible cause of viscosity differences. Part of the reason of the change is the pH change during electro-processing. Seguchi et al. (1997) showed that dough viscosity greatly increased by lowering the dough pH. The treated batter at the anode side showed lower pH (5.6) than that of non-treated batter (pH 5.9). However, this may be an insufficient explanation for the phenomena. The isoelectric points of the gluten proteins generally fall in the pH range 6–9 (Wrigley, 1968a, 1968b), where these proteins are not soluble. At pH values lower than 4 or 5, they are moderately soluble (Wrigley and Biets, 1988). Therefore, the pH change to this level may be too small to solubilize GMP.
Figure 7 is a hypothetical representation that explains this phenomenon. Typical batter dough development is understood as follows (Auger et al., 2007). The viscosity curve of batter is characterized by a lag phase where the baseline apparent viscosity is minimal and stable as a function of mixing time. The lag phase is followed by a sudden and progressive increase in apparent dough viscosity until a maximum value corresponding to the optimal dough development time is reached.
Hypothesis of GMP dissolution effect on increase of dough viscosity. A: Electrical treatment. B: Kneading.
During the lag phase, proteins mainly exist as lumps in a typical batter. It is possible that these lumps and the protein membrane that detaches from the starch granules are related. The dough status during the lag phase may be viewed as a suspension of gluten lumps dispersed in a water/starch medium. In the middle of the viscosity increase the gluten lumps are larger and linked together with gluten strands. Full gluten strand development is achieved when all gluten lumps have disappeared and gluten proteins have been transformed into thin strands.
The mixing time represent the status of the lag phase, and it is shown that viscosity is increased during this phase by electro-processing. In this case, part of the gluten lump solubilizes and is thought to be distributed in the water/starch medium. Protein interaction is thought to occur during mixing of the medium, and the solubilized protein may form a protein matrix that includes starch granules. The formation of such a matrix may be the cause of the increased viscosity. The extracted GMP molecules may have a greater chance to interact with each other in water and this may cause a denser gluten network.
Thus, it is thought that the solubilization of GMP plays a large role in the increased batter viscosity at the anode side. On the other hand, the soluble protein level did not increase at the cathode side. Notably, starch granules without a protein membrane are also often microscopically observed at the cathode side. Thus, the cathode side sample also has the possibility of protein solubilization. The migration of dissolved proteins to the anode side is one possibility. Further studies are required to elucidate this subject.
Food processing by electrical treatment has excellent advantages in the operation of food production lines. The technique is simple, easy to control and does not require chemical agents. Additionally, this method may enable the use of inexpensive soft flour for the manufacture of Japanese noodles instead of semi-hard flour. Therefore, upon confirmation of safety, it is expected to become an important food-processing method in the future.
Acknowledgment We wish to thank Rumi Sawada for technical assistance in these experiments.
