Microstructures of Bread Dough and the Effects of Shortening on Frozen Dough

Biosci. Biotechnol. Biochem., 69 (2), 397 402, 2005 Microstructures of Bread Dough and the Effects of Shortening on Frozen Dough Shigeo AIBARA, 1;y Noriko OGAWA, 2 and Masaaki HIROSE 1 1 Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan 2 Faculty of Home Economics, Gifu Women s University, 80 Taromaru, Gifu 501-2529, Japan Received October 7, 2004; Accepted November 26, 2004 Three types of straight doughs different in combination of yeast and shortenings (RLS20, FTS20, and FTS80) were prepared, and the structure of the frozen doughs was examined under a microscope after staining protein or lipid droplets. Even after 2 months of frozen storage, distinct changes were not found in the gluten network of FTS80, although significant damages in the dough structures of FTS20 and RLS20 appeared after only one month of frozen storage. These results suggest that the gluten networks loosen and decrease in the water retention ability, and it may be concluded that the lipid is removed from the gluten protein due to the decrease in water in the continuous protein phase. The resulting product from the damage to the gluten matrix gave rise to fusion of lipid droplets and an increase in their size. Because of the difference in fatty acid composition, the lipids of shortening S80 are presumed to interact more strongly with gluten proteins and to keep the gluten matrix from damage in comparison with the lipids of shortening S20. Key words: frozen dough; shortening; lipid droplet; loaf volume; microstructures of bread dough Lowering of yeast fermentation and gas retention of bread dough have been pointed out as the two factors affecting loaf volume of frozen dough bread. 1) Even if one of these two abilities decreases, a sufficient loaf volume of the bread is fails to be secured. Particularly the ability of gas retention is associated with bread dough structures (gluten network); in other words, the expansibility of bread dough is depend on the relevant balance of the network structure of gluten proteins and lipids as a plasticizer. Proofing time of the regular yeast dough prolonged gradually depending on the period of frozen storage. However, using freeze-tolerant yeast improved retardation of proofing time and maintained almost the same level of proofing time as one week frozen storage-dough even after three or four months of frozen storage. 2) On the other hand, the frozen dough bread is not able to recover the loaf volume completely just by using frozen-tolerant yeast. These facts clearly indicated that the dough structures rather than yeast fermentability closely participated in the loaf volume of the frozen dough bread. Gluten protein and starch granules are badly damaged by long term frozen storage. Varriano-Marston et al. 3) have reported that water plays a role in maintaining bread dough structures, and Tipples 4) has reported that damaged starch granules which have separated act like a sponge in the presence of water, and possibly draw water away from the gluten network with more damage. The loss of ability of gluten proteins to retain water results in the separation of starch granules and brings about the deterioration of dough structures. From the viewpoint of the microstructure of bread dough, starch granules are embedded firmly in the intact gluten network, but once the gluten network has been damaged, starch granules exhibit a reticular pattern 5,6) and separate from the gluten network. Lipids derived from added shortening are classified into two groups from an aspect of the microstructure of bread dough: one is the lipids taken into the disperse phase as lipid droplets during mixing, and the other is the lipids interacting with gluten proteins, 7) as indicated in Fig. 1. The added shortening has a role as a plasticizer and closely relates to the expansion of the bread dough during the second fermentation and baking processes. 8) Many investigators have reported abundant data on the effects of shortenings on the bread-making of frozen and non-frozen doughs. 9 17) We noted in the previous paper that the fatty acid composition of the shortening is important for the expansibility of the frozen dough bread, and the size of lipid droplets in the disperse phase of the bread dough grew large when thawing frozen dough. 2) However, the details of deterioration of dough structures are not yet clear, and it is ambiguous how the shortening relates closely to the frozen dough expansion, or if some other factors are involved. In this paper, we observed microscopically the gluten matrix and lipid droplets of bread dough after long-term frozen storage for elucidation of the relationship y To whom correspondence should be addressed. Fax: +81-774-33-3004; E-mail: aibara@kais.kyoto-u.ac.jp Abbreviations: RLS, regular yeast and shortening; FTS, freeze-tolerant yeast and shortening

398 S. AIBARA et al. Fig. 1. Dough Composition. Classification of dough components according to Bloksma (1990). between the microstructures of the bread dough and a decrease in loaf volume of frozen dough bread. Materials and Methods Flour. The wheat flour used in his study was a commercial hard wheat flour ( Superking from Nisshin Flour Milling, Tokyo, Japan). Yeast. Two kinds of compressed, raw yeast, regular (RL) and freeze-tolerant (FT) yeast (Oriental Yeast, Tokyo, Japan) were bought and consumed in 2 weeks, and they were stored in a refrigerator at 4 C before use. Dough formulation. On a flour weight basis, water addition was 61%, compressed yeast was at 5%, sugar was added at 5%, salt at 2%, and plastic shortening at 5%. Water addition was a little less than the optimum amount due to preparation of the frozen straight dough. Two types of plastic shortening different in fatty acid composition were used, and they were named S20 and S80 according to degree of saturation of total fatty acids. 2) S20, containing about 75% of unsaturated fatty acid, oleic acid, was a soft type of shortening, but S80 was a hard type and contained considerable amounts (over 60%) of lauric and myristic acids. S80 was extremely hard and was pre-heated to 25 C for 10 min and smashed to a paste before use. Straight dough method. Straight dough was prepared using a mixing machine (Kanto mixer, HMS-30, Kanto Mixer Industry) according to the procedures of the previous study. 2) Three types of straight doughs different in the combination of yeast and shortenings (RLS20: regular yeast and shortening S20; FTS20: freeze-tolerant yeast and shortening S20; FTS80: freeze-tolerant yeast and shortening S80) were prepared. Freeze-and-thaw treatment. After divided the straight dough into 320 g portions, the pieces were sheet-molded (to a diameter of approximately 18 cm and 1.5 cm thick), and subjected to freeze treatment. The doughs, which had been formed into a disk slab shape, were frozen to 10 C on aluminum vats in an upright superfreezer (Super Freezer MDF-U281: Sanyo Electric, Japan) at 85 C for 40 min. Then the frozen dough was put into a sealed polyethylene bag and stored in a freezer (Medical Freezer MDF-U332, Sanyo) at 20 C. After a given period of frozen storage, each four pieces of the frozen dough was subjected to a two-step thawing in the sealed polyethylene bags. After the dough was thawed and re-rounded, the average temperature of the thawed dough before proofing was lower than that of the non-frozen dough before proofing. The total period of the freeze and thaw treatment was represented by 1M for 1 month of storage, 2M for 2 months of storage and so on, and the number of times of freeze-and-thaw treatment during storage was added to the period of the storage: 1M1, 2M1, 1M4, 2M4, and so on. 1M1 or 2M1 means that the dough was first thawed at the end of the frozen storage. In the case of 1M4, the freeze and thaw treatment was carried out every week, in 2M4 the treatment was applied every 2 weeks, and every 2 weeks of the last two months for 3 month frozen storage. Measurement of dough temperature. Measurement of dough temperature was carried out with a chromelalumel thermocouple equipped with a digital multimeter (Model 3457A, Hewlett-Packard). A sensor was inserted through the side to the center of the dough. Temperatures of the doughs in each step are indicated in Table 1. Measurement of loaf volume. Measurement of loaf volume was done after cooling the bread to room

Table 1. Changes in Temperature during the Process of Freezing Dough Flour- and water-temperatures should be kept at 20 and 8.5 C respectively, in order to adjust the dough temperature after full mixing to 23 C. Process Temperature ( C) After preliminary mixing for 5 min 12:0 0:6 After full mixing for 10 min 22:8 0:8 Frozen 20:0 Thawed in refrigerator 4:1 1:1 Thawed in electric fermentation cabinet 13:3 0:8 After bench resting 16:5 0:5 After second fermentation 27:8 0:4 temperature for 120 min. The rapeseed method was used. Microstructures of Frozen Bread Dough 399 Table 2. Statistics on the Size and Number of Lipid Droplets in Frozen Dough Name Mark Number Area Average Max. St. dev. in the of ratio of diameter diameter (mm) figure particles (%) (mm) (mm) FTS80 A 1992 3.7 1.457 1.061 7.693 FTS201M1 E 2788 4.8 1.368 1.080 7.701 FTS201M4 I 3948 7.3 1.400 1.123 14.171 FTS801M1 B 4225 5.1 1.223 0.930 7.981 FTS801M4 C 2953 6.2 1.245 0.964 7.912 RLS201M1 F 3434 5.7 1.296 1.103 21.012 RLS201M4 G 3815 8.0 1.438 1.262 13.221 FTS202M1 J 5241 5.7 0.998 0.958 11.443 FTS202M4 M 9340 8.3 0.953 0.799 9.370 FTS802M1 D 5683 5.7 1.145 0.676 7.374 FTS802M4 H 8267 6.7 0.899 0.777 9.979 RLS202M1 K 8570 8.7 0.961 0.920 10.041 RLS202M4 L 2996 8.5 1.545 1.601 14.267 FTS803M1 N 5869 6.3 1.093 0.827 10.183 Average 4937 6.5 1.215 1.006 10.881 Light microscopy. Small blocks were cut from the bread dough after bench resting and frozen in liquid nitrogen, and sectioned to 6 8 mm thick with a cryostat and then fixed on a slide glasses at room temperature with a gentle ventilation. Lipid in the bread dough was stained with Sudan IV and protein by the PAS method. Analysis of size distribution of lipid droplets. The size distribution of lipid droplets was analyzed using the program WinROOF. Lipid droplets were distinguished by color, and the minimum size was set to 0.370 mm. Results Distribution and size of lipid droplets in bread dough Lipid droplets, which look like small orange-colored particles under the optical microscope, as shown in Fig. 2, were distributed across almost the whole area in the gluten matrix. The area ratio of the lipid droplets occupied 3.7 8.7% (Table 2). Lipid particles grew large, to 10 to 20 mm, and the number of them increased in the regular yeast dough (RLS20) after 1 month of frozen storage (Fig. 2F), and the loaf volume of the bread decreased a little (Table 3). This suggests that the structure of the gluten network was altered by the freeze-and-thaw treatment and that the fusion of lipid particles in the frozen dough propagated by coming into frequent contact with them. Using freeze-tolerant yeast, the lipid droplets remained relatively small (Figs. 2B and 2E) and the loaf volume maintained the level of the fresh, non-frozen dough, but there was a tendency to increase in the frozen dough FTS20 (Fig. 2I) after 4 cycles of freeze-and-thaw treatment. The frozen dough of RLS20 treated with 4 cycles of freeze-and-thaw process did not have enough volume to bake the bread during the second fermentation (Fig. 2G and 2L, Table 3). It is suggested that the yeast could no longer show the fermentation ability after 4 cycles of the freeze-and-thaw treatment since the dough structure did Table 3. Specific Volume of Frozen Dough Bread with Various Shortenings Dough weight of one loaf was 320 g. The specific volume is expressed as the loaf volume divided by the dough weight. Times is the number of freeze-and-thaw processes. The dough was treated at the end of the frozen storage, but in the case of plural times, the dough was treated every week for 1 month, every two weeks for 2 months, and every two weeks of the last 2 months for 3 months frozen storage. means no experiment, and nd not possible to bake due to too long a second fermentation. Frozen Period RLS20 FTS20 FTS80 Week Times ml/g ml/g ml/g 0 0 5:273 0:023 5:317 0:051 5:267 0:019 1 1 5:331 0:031 2 1 5:286 0:060 5:297 0:092 3 1 5:328 0:078 4 1 5:193 0:036 5:297 0:045 5:254 0:096 4 4 nd 4:581 0:061 5:163 0:022 8 1 5:052 0:077 5:164 0:096 5:250 0:100 8 4 nd 4:501 0:053 5:032 0:035 12 1 4:819 0:095 5:047 0:076 5:123 0:057 12 4 nd 4:478 0:126 4:931 0:043 16 1 4:758 0:061 4:969 0:047 5:081 0:079 not suffer from such profound damages. Nevertheless, the ratio of lipid droplets area clearly increased (Table 2). In the case of using shortening S80 with freeze-tolerant yeast (FTS80), the size of the lipid droplets was not influenced even if repeating the freezeand-thaw treatment 4 times (Fig. 2C), and almost no changes in their distribution were found as compared to the fresh, non-frozen dough. In contrast, after 2 months of frozen storage, the area ratio of lipid droplets in the frozen doughs (RLS20 and FTS20) significantly increased, to 8% or more (Fig. 2K and 2M, Table 2), and the loaf volume decreased, even though the lipid droplets of the frozen dough (FTS80) remained small and the loaf volume had the normal value without repeating the freeze-and-thaw treatment (Fig. 2D). This indicated the protecting effects of

400 S. AIBARA et al. Fig. 2. Changes in Lipid Droplets in Frozen Dough Structures. A, FTS80-Non; B, FTS80-1M1; C, FTS80-1M4; D, FTS80-2M1; E, FTS20-1M1; F, RLS20-1M1; G, RLS20-1M4; H, FTS80-2M4; I, FTS20-1M4; J, FTS20-2M1; K, RLS20-2M1; L, RLS20-2M4; M, FTS20-2M4; N, FTS80-3M1. Fig. 3. Changes in Gluten Matrix in Frozen Dough Structures. A, FTS80-Non; B, FTS80-1M1; C, FTS80-1M4; D, FTS80-2M1; E, FTS20-1M1; F, RLS20-1M1; G, RLS20-1M4; H, FTS80-2M4; I, FTS20-1M4; J, FTS20-2M1; K, RLS20-2M1; L, RLS20-2M4; M, FTS20-2M4; N, FTS80-3M1. shortening S80 in comparison with shortening S20. After 2 months of frozen storage with 4 cycles of freezeand-thaw treatment, the lipid droplets of frozen dough FTS80 became large (Fig. 2H). After 3 months of frozen storage, the size of the lipid droplets resulted in almost the same level as the other frozen doughs (Fig. 2G and 2J), as shown in Fig. 2N.

Gluten matrix in the bread dough Gluten proteins in the dough were stained to a purple red color by the PAS method, as shown in Fig. 3. In the fresh, non-frozen dough, in which the gluten network extended well and stained to a deep purple red, starch granules lay embedded in the gluten matrix of the continuous protein phase (Table 1 and Fig. 3A). The structures of the gluten matrix did not change significantly after one month of frozen storage (Fig. 3B, 3E, and 3F). Therefore, the decrease in the loaf volume of frozen dough RLS20 is considered to result from a decrease in the fermentability of yeast. An indication of slight damage to the dough structures might have occurred in the size of the lipid droplets (Fig. 2F). Repeating the freeze-and-thaw treatment, the damage to the dough structures was slight in the frozen dough of FTS20, as shown in Fig. 3I. However, the damage clearly appeared in the regular yeast dough (Fig. 3G), and the color of the dough became thin and open seams were noticeable. After two months of frozen storage, starch granules of the frozen doughs (RLS20 and FTS20) became separate from the gluten matrix (Fig. 3J and 3K, respectively). Further starch granules were dissembled and the close connection of the dough structures disappeared by repeating the freeze-and-thaw treatment (Fig. 3L). In contrast, frozen dough FTS80 maintained a thick gluten matrix and kept a deep color (Fig. 3B, 3C, and 3D) and no indication of separation was recognized. Repeating the freeze-and-thaw treatment, however, starch granules of the frozen dough of FTS80 became separate after two months of frozen storage (Fig. 3H), and those of frozen dough FTS20 were almost completely destroyed (Fig. 3M). It is clear that the difference in the degree of damage to the dough structures was caused by the difference in the type of shortening. Thus, we considered that the effect of the shortening was attributable to the fatty acid composition of shortenings S20 and S80. 2) The reason baking was possible irrespective of the damage to the doughs after more than two months of frozen storage (Fig. 3J, 3K, 3M, and 3N) is the freeze-tolerant yeast. In the case of regular yeast, repeating the freeze-and-thaw treatment did take very long to obtain enough volume of the second fermentation for baking, and dough at one month (Fig. 3G) as well as two months of frozen storage (Fig. 3L) was not capable being baked. Discussion It was after 2 months of frozen storage of doughs RLS20 (Fig. 3K) and FTS20 (Fig. 3J) that significant damage to the gluten network appeared and the loaf volume of the frozen dough bread decreased. On the other hand, the size of the lipid droplets of RLS20 (Fig. 2F) was found to grow large after 1 month of frozen storage, and in the case of repeating the freezeand-thaw treatment, enlargement of lipid droplets occurred also in FTS20 (Fig. 2I) even after 1 month of Microstructures of Frozen Bread Dough 401 frozen storage, and RLS20 (Fig. 2G and 3G) could not be baked due to too long a second fermentation. However, no difference in the length of the second fermentation was found between one month- and two month-frozen storage doughs of FTS20 and FTS80. These facts suggest that the condition of the gluten network was closely related to the oven spring of the frozen dough as well as the freeze-tolerant yeast. Furthermore, the chemical property of the lipids interacting with gluten proteins as a plasticizer played a key role in protecting the gluten network from disintegration, taking into account that the effects of changes in the particle size of lipid droplets varied with the type of shortening. As mentioned with reference to the gluten network, that is, the interaction of gluten proteins with the lipids derived from the shortening on mixing, gluten protein is of high hydrophobicity and poor in solubility because of the high content of proline and glutamine in the amino acid composition. Thus, the interaction with the lipids is presumed to occur partly by sticking the fatty acid moiety of triacylglycerol to gluten proteins and partly by adhering to the surface of gluten proteins. If so, the middle chain saturated fatty acids are capable of forming a more stable interaction with gluten proteins than the long chain unsaturated fatty acid does. Therefore, the chemical structure of the fatty acid moiety of the lipids as the plasticizer is important to avoid their separation from the gluten proteins before disintegration of the dough structures. Further, we emphasize that the shortening possessing middle chain saturated fatty acids had good effects on expansibility of the frozen dough bread. Furthermore, the lack of water in the gluten matrix may be a trigger, taking into consideration that both the repetition of freeze-and-thaw treatment and the increase in the size of the lipid droplets were controlling factors involved in disruption of the gluten network. Water in the continuous protein phase of the frozen dough repeats changes in the states, ice and water, through the freezeand-thaw treatment, and then water is removed from gluten matrix every thawing time, and migrates to the damaged starch granules in the disperse phase. The lack of water in the gluten matrix makes it easy to remove the lipids interacting with the gluten proteins due to leading the hydrophobic environment in the gluten matrix, and lipid droplets re-arrange and fuse since the gluten matrix gives rise to a condition of less water content. Then the starch granules embedded in the gluten network are released from the gluten matrix, and the dough structures results in the fatal damage. Thus the lipid droplets grow large before the damage to the gluten network extends significantly. This is a possible reason why the size of the lipid droplets increases. Hydrophobicity in the gluten matrix increases due to a decrease in water with prolonging of the duration of frozen storage or repeating the freeze-and-thaw cycle, and it causes a fusion of the lipid droplets in the dough. Further, depletion of triacylglycerol from the gluten

402 S. AIBARA et al. protein by itself results in destruction of the gluten matrix. In conclusion, shortening S80 was able to lead the dough structures to a stable gluten network under the conditions mentioned above by making firmer interaction of the lipids with gluten protein than shortening S20 did, and by retaining water in the gluten matrix, since the former frozen dough FTS80 was rich in the middle chain saturated fatty acids. Acknowledgment We would like to thank Nisshin Flour Milling Co., Ltd., for the donation of bread flour Superking, Oriental Yeast Co., Ltd., for the donation of yeast, and Fuji Oil Co., Ltd., for shortening S80. We would also like to thank Mr. Seiji Fukusawa of the Mitani Corporation for the analysis of the size distribution of lipid droplets. References 1) Räsänen, J., Härkönen, H., and Autio, K., Freeze-thaw stability of prefermented frozen lean wheat doughs: effect of flour quality and fermentation time. Cereal Chem., 72, 637 642 (1995). 2) Aibara, S., Nishimura, K., and Esaki, K., Effects of shortening on the loaf volume of frozen dough bread. Food Sci. Biotechnol., 10, 521 528 (2001). 3) Varriano-Marston, E., Hsu, K. H., and Mahdi, J., Rheological and structural changes in frozen dough. Bakers Dig., 54, 32 (1980). 4) Tipples, K. H., The relation of starch damage to the baking performance of flour. Bakers Dig., 43, 28 (1969). 5) Berglund, P. T., Shelton, D. R., and Freeman, T. P., Comparison of two sample preparaion procedures for low-temperature scanning electron microscopy for frozen bread dough. Cereal Chem., 67, 139 140 (1990). 6) Berglund, P. T., Shelton, D. R., and Freeman, T. P., Frozen bread dough ultrastructure as affected by duration of frozen storage and freeze-thaw cycles. Cereal Chem., 68, 105 107 (1991). 7) Bloksma, A. H., Dough structure, dough rheology, and baking quality. Cereal Foods World, 35, 237 244 (1990). 8) Fu, J., Mulvaney, S. J., and Cohen, C., Effect of added fat on the rheological properties of wheat flour doughs. Cereal Chem., 74, 304 311 (1997). 9) Kline, L., and Sugihara, T. F., Factors affecting the stability of frozen bread doughs. I. Prepared by the straight dough method. Bakers Dig., 42, 44 (1968). 10) Bell, B. M., and Fisher, N., The binding of model shortenings during mixing of mechanically developed bread doughs from fresh and stored flours. J. Am. Oil Chem. Soc., 54, 479 483 (1977). 11) Bell, B. M., Daniels, D. G. H., and Fisher, N., The effects of pure saturated and unsaturated fatty acids on bread making and on lipid binding, using Chorleywood bread process doughs containing a Model Fat. J. Sci. Food Agric., 30, 1123 1130 (1979). 12) Bell, B. M., Daniels, D. G. H., and Fisher, N., Vacuum expansion of mechanically developed doughs at proof temperature. Effect of shortening. Cereal Chem., 58, 182 186 (1981). 13) Krog, N., Theoretical aspects of surfactants in relation to their use in breadmaking. Cereal Chem., 58, 158 164 (1981). 14) Addo, A., and Pomeranz, Y., Lipid binding and fatty acid distribution in flour, dough, and baked and steamed bread. Cereal Chem., 68, 570 572 (1991). 15) Hosomi, K., Nishio, K., and Matsumoto, H., Studies on frozen dough baking. I. Effects of egg yolk and sugar ester. Cereal Chem., 69, 89 92 (1992). 16) Inoue, Y., Sapirstein, H. D., and Bushuk, W., Studies on frozen doughs. IV. Effect of shortening systems on baking and rheological properties. Cereal Chem., 72, 221 226 (1995). 17) Silva, R. F., Uses of alpha-crystalline emulsifiers in the sweet goods industry. Cereal Foods World, 45, 405 411 (2000).