Breadmaking Properties of Frozen-and-Thawed
Bread Dough

2015
Naomi Morimoto

Contents
Page

Chapter 1

Introduction

1

Chapter 2

Breadmaking Properties of Frozen-and-Thawed BreadDough

Object8

Abstract

8

Materials and Methods

8

Results and Discussion

10

Conclusion

15

Chapter 3

Restoration of Breadmaking Properties to Frozen Dough by
Addition of Sugar and Yeast and Subsequent Processing

Object17

Abstract

17

Materials and Methods

18

Results and Discussion

23

Conclusion

29

iChapter 4

Effect of Xanthan Gum on Improvement of Bread Heightand Specific Volume upon Baking with Frozen and Thawed
Dough

Object31

Abstract

31

Materials and Methods

32

Results and Discussion

35

Conclusion

44

Chapter 5

Centrifuged Liquid of Frozen-and-Thawed Bread Dough

Object46

Abstract

46

Materials and Methods

46

Results and Discussion

48

Conclusion

55

Chapter 6

Conclusions

57

List of Publications

59

Acknowledgments

60

ii

List of Abbreviation
SV

Specific volume

HM pectin

High Methoxyl pectin

LM pectin

Low Methoxyl pectin

CMC

Carboxymethyl celluloseiii

Chapter 1
Introduction
Frozen dough is widely used in the breadmaking industry, and it is generally
believed that the use of frozen dough will increase in the future. The use of frozen
dough has the advantage of allowing breadmaking to be discontinuous, obviating theneed for the process to continue uninterrupted from start to finish. However, there are
two problems with the use of frozen dough that hinder its use in the breadmaking
industry. One is related to the effects of freezing on baker’s yeast and has been studied
by many researchers,1) resulting in the development of freeze-resistant yeast strains.
Kline and Sugihara proposed that release of disulfide-reducing substances into thedough from dead yeast cells caused a decrease in gassing power and gradual loss ofdough strength. 2) Progress being made in yeast biochemistry and the underlying genetic
elements responsible for freeze-tolerance traits may soon offer opportunities to exploit
the tools of biotechnology to create improved strains of yeast for frozen dough.3)
Another problem is related to deterioration in the bread dough itself caused by thefreezing, and this also has been studied by many researchers. According to
Variano-Marston et al.,4) the structure of gluten in frozen dough was damaged byformation of ice crystals. And Wolt and D’Appolonia suggested that protein quality(strength) was important for bread production from frozen dough. 5) Inoue and Bushuk
6,7)

and Inoue et al.8) reexamined the extensigraphic patterns of frozen doughs andconfirmed the weakening of doughs containing yeast observed by Variano-Marston etal.4) during frozen storage and successive freeze-thaw cycles. However, the mechanismof this deterioration has not been determined. Therefore, we investigated to clarify thecause of the deterioration of breadmaking properties. First, we observed the surface ofthe frozen-and-thawed dough and we noticed that the dough was wet.
Thus in chapter 2, we tried to collect free water oozed from frozen-and-thawed

1dough and to show the relationship between free water oozed from frozen-and-thawed
dough and the dough’s breadmaking properties.
The reason is unclear why depression of breadmaking properties such as breadheight (mm) and specific volume (cm 3/g) caused by freezing and thawing of breaddough occurs.9) Berglund et al. observed that changes in water distribution occurred
during extended frozen storage and freeze-thaw cycles, and the observed changes in theultrastructure of the starch granules and gluten may contribute to the extended proof
times and reduced loaf volumes of frozen bread doughs. 10) Lu and Grant showed thatwater separates from the protein and starch components in frozen dough andaccumulates into a pool that subsequently crystallizes and that during prolonged frozen
storage the amount of freezable water in the frozen doughs increases significantly. 11)
Zounis et al. observed that the cause of disruption apparently was a change in the ice
crystal structure as indicated by the appearance and increase in the size of angular voids
in doughs during frozen storage. 12) Bot reported that in gluten stored at -15℃, the watercontent in the gluten phase decreased by around 1% over the first three weeks, and thesame changes occurred in dough stored at either -15 or -25℃ as in gluten at -15℃. 13)
Naito et al. reported that scanning electron microscopy images of grain pore walls thatwere washed with distilled water (20℃) clearly showed that gluten fibrils forming theskeletal framework of pore walls were cut and became coarse and nonuniform strings
and that many knots were generated on gluten fibrils from freeze damage. 14) Esselink et
al. reported that, at the macroscopic level, ice crystals are not evenly distributed overthe molded dough, nor are the gas cells homogenously distributed throughout the dough.
15)

Nicolas et al. indicated that after thawing, gluten microstructure exhibited a structure

similar to that of fresh gluten, with the structure looking like a sponge (a fine gluten
structure with tiny water pockets). 16) Seguchi et al. examined the relationship between
depression of breadmaking properties and increase in the amount of the centrifugedliquid from frozen and thawed dough and reported that the amount of centrifuged liquidfrom bread dough increased by freezing and thawing (in Chapter 2). 17)2

In chapter 3, we studied the cause of these phenomena that were reported in
Chapter 2. To better understand which breadmaking processes are affected by freezing
and thawing and why freezing and thawing cause depression of breadmaking properties,
we inserted a freezing step into the bread dough process (mix, proof, mold, proof, andbake). We inserted the freezing step after mixing of wheat flour, sugar, salt, and water(bread dough A); after mixing of dough A plus yeast (bread dough B); and after first
proof, mold, and second proof of dough B (bread dough C). We further investigatedwhether acceptable breadmaking properties could be obtained when sugar and yeast
were added to the thawed bread dough C. To research the effect of freezing on breaddough rheology, we further performed extension tests of dough by pulling the surface ofthe bread dough under reduced pressure.
It is generally known that breadmaking properties are markedly deteriorated by thefreezing/thawing of bread dough; that is, bread height (mm) and specific volume(cm3/g) decrease to far lower levels.7,9,10,18-20) The amount of extruded liquid fromthawed bread dough upon centrifugation was increased by freezing/thawing, and thedeterioration of breadmaking properties by freezing is closely related to this increase in
the volume of exuded liquid.17) The equally distributed water in the dough matrix
migrated in order to form larger water crystals upon freezing; therefore, the amount ofwater necessary to make a bread cell wall was decreased by freezing, resulting in thedeterioration of breadmaking properties.21) Ice formation and growth occur
preferentially at the gas pore interface under dough freezing conditions.22) It wasobserved that the changes in the microstructure of the gluten network that are induced
by cryodehydration were reversible upon thawing. However, since the thawed doughwas remixed with sugar and yeast, and re-fermented as it was, normal breadmaking
properties could be obtained. Thus, it was suggested that the damage caused byfreezing/thawing to wheat flour components, such as water-soluble, gluten, prime starch,and tailing fractions, was small and it was shown that the deterioration due tofreezing/thawing of yeast was also small.21) Therefore, the cause of deterioration of3

bread baked with freezing/thawing was due to the loss of water from the frozen dough.
Simmons et al. reported that soy ingredients stabilized bread dough during frozen
storage. 23) Sharadanant and Khan reported that the addition of gums (gum arabic,
carboxymethyl cellulose (CMC), kappa (κ) carrageenan, and locust bean gum) to afrozen dough formulation helps to immobilize water and reduce the formation of large
ice crystals that damage the gluten network. 24) The way of these improvements seemed
to represent our conclusion in Chapter 3 which the cause of deterioration of bread bakedwith freezing/thawing was not dough’s components but the loss of water from frozen
dough.
Thus, in chapter 4, we tried to use polysaccharides because they have a goodabsorption. The aim of this Chapter was to prevent bread deterioration fromfreezing/thawing by adding polysaccharides to the dough. To stop the loss of water fromfrozen dough, 13 kinds of polysaccharide (locust bean gum, guar gum, xanthan gum,tamarind seed gum, native gellan gum, dextrin, LM pectin, fermented cellulose CMC,
κ-carrageenan, konjac glucomannan, HM pectin, ι-carrageenan, and λ-carrageenan)
were examined. Breadmaking tests using these polysaccharides and determinations of
extruded liquid from the dough upon centrifugation were performed. When xanthan
gum was mixed in frozen bread dough, remarkable breadmaking properties wereobtained. The mechanism of the improvement in breadmaking properties due to xanthan
gum is also discussed in chapter 4.
We studied the relationship between free water oozed from frozen-and-thawed
dough and the dough’s breadmaking properties (in chapter 2). Furthermore, in chapter 5,
we examined which components of bread dough (sugar, salt, and yeast) affect theamount of centrifuged liquid, and we also studied the possibility that fermented
products affect that. From these findings, we continued to investigate the relationshipbetween amount of liquid centrifuged from dough and the presence of salt andfermented products.

4

References
1) Kulp K: 1995. Biochemical and biophysical principles of freezing. Pages 63-89 in:Frozen and Refrigerated Doughs and Batters. K. Kulp, ed. Am. Assoc. Cereal
Chem.: St. Paul, MN.
2) Kline L, Sugihara TF: Factors affecting the stability of frozen bread doughs. I.
Prepared by straight dough method. Baker’s Digest 46:44-50, 1968.
3) Casey GP, Foy JJ: 1995. Yeast performance in frozen doughs and strategies for
improvement. Pages 19-51 in: Frozen and Refrigerated Doughs and Batters. K. Kulp,
ed. Am. Assoc. Cereal Chem.: St. Paul, MN.
4) Variano-Marston E, Hsu KH, Mhadi J: Rheological and structural changes in frozen
dough. Baker’s Digest 54:32-34, 41, 1980.
5) Wolt MJ, D’Appolonia BL: Factors involved in the stability of frozen dough. I. The
influence of yeast reducing compounds on frozen-dough stability. Cereal Chem
61:209-212, 1984.
6) Inoue Y, Bushuk W: Studies on frozen doughs. I. Effects of frozen storage andfreeze-thaw cycles on baking rheological properties. Cereal Chem 68:627-631,
1991.
7) Inoue Y, Bushuk W: Studies on frozen doughs. II. Flour quality requirements forbread production from frozen dough. Cereal Chem 69:423-428, 1992.
8) Inoue Y, Sapirstein HD, Takayanagi S, et al: Studies on frozen doughs. III. Somefactors involved in dough weakening during frozen storage and thaw-freeze cycles.
Cereal Chem 71:118-121, 1994.
9) Selomulyo VO, Zhou W: Frozen bread dough: Effects of freezing storage and doughimprovers. J Cereal Sci 45:1-17, 2007.
10) Berglund PT, Shelton DR, Freeman TP: Frozen bread dough ultrastructure asaffected by duration of frozen storage and freeze-thaw cycles. Cereal Chem
68:105-107, 1991.
11) Lu W, and Grant LA: Effects of prolonged storage at freezing temperatures on starch

5and baking quality of frozen doughs. Cereal Chem 76:656-662, 1999.
12) Zounis S, Quail KJ, Wootton M, et al: Studying frozen dough structure using
low-temperature scanning electron microscopy. J Cereal Sci 35:135-147, 2002.
13) Bot A: Differential scanning calorimetric study on the effects of frozen storage on
gluten and dough. Cereal Chem 80:366-370, 2003.
14) Naito S, Fukami S, Mizokami Y, et al: Effect of freeze-thaw cycles on the gluten
fibrils and crumb grain structures of breads made from frozen doughs. Cereal Chem
81:80-86, 2004.
15) Esseliznk EFJ, van Aalst H, Maliepaard M, et al: Long-term storage effect in frozen
dough by spectroscopy and microscopy. Cereal Chem 80:396-403, 2003.
16) Nicolas Y, Smit RJM, van Aalst H, et al: Effect of storage time and temperature on
rheological and microstructural properties of gluten. Cereal Chem 80:371-377,
2003.
17) Seguchi M, Nikaidoo S, Morimoto, N: Centrifuged liquid and breadmaking
properties of frozen-and thawed bread dough. Cereal Chem. 80:264-268, 2003.
18) Kenny S, Wehrle K, Dennehy T: Correlations between empirical and fundamental
rheology measurements and baking performance of frozen bread dough. Cereal
Chem 76: 421-425, 1999.
19) Le Bail A, Grinand C, Le Cleach S, et al: Influence of storage conditions on frozen
French bread dough. J Food Eng 39: 289-291, 1999.
20) Nilufer D, Boyacioglu D, Vodovocz Y: Functionality of soymilk powder and its
components in fresh soy bread. J Food Sci 73: C275-C281, 2008.
21) Morimoto N, Seguchi M: Restoration of breadmaking properties to frozen dough byaddition of sugar and yeast and subsequent processing. Cereal Chem 88: 409-413,
2011.
22) Baier-Schenk A, Handschin S, von Schonau M, et al: In situ observation of thefreezing process in wheat dough by confocal laser scanning microscopy (CLSM):
Formation of ice and changes in the gluten network. J Cereal Sci 42: 255-260, 2005.

623) Simmons AL, Smith KB, Vodovotz Y: Soy ingredients stabilize bread dough duringfrozen storage. J Cereal Sci 56: 232-238, 2012.
24) Sharadanant R, and Khan K: Effect of hydrophilic gums on the quality of frozen
dough: electron microscopy, protein solubility, and electrophoresis studies. Cereal
Chem 83: 411-417, 2006.

7

Chapter 2
Breadmaking Properties of Frozen-and-Thawed Bread Dough
ObjectWe studied the relationship between free water oozed from frozen-and-thawed
dough and the dough’s breadmaking properties.

Abstract
Breadmaking properties (bread height (mm) and specific volume (cm3/g)) showedmarked deterioration when bread dough was frozen and stored at -20℃ for one day.
However, these properties of bread dough baked after storage for three to six days were
not further deteriorated as compared with that baked after one day of storage. A large
amount of liquid was oozed from the frozen-and-thawed bread dough. The liquid wasseparated from the bread dough by centrifugation (38,900 g for 120 min at 4℃), andcollected by tilting the centrifuge tube at an angle of 45°for 60 min. There was a strong
correlation between the amount of centrifuged liquid and breadmaking properties (breadheight and specific volume). The mechanism responsible for the oozing of liquid infrozen-and-thawed bread dough was studied.

Materials and Methods
Wheat flour
Seven different wheat flours were used in this experiment: Camellia (Nisshin Flour
Milling Co. Ltd., Japan), Red Knight and Alps (Nittofuji Flour Milling Co. Ltd., Japan),Midoriuzumaki (Masuda Flour Milling Co. Ltd., Japan), Horoshiri (Ebetsu Flour
Milling Co. Ltd., Japan), and Kitakami and Nanbujigona (Fukin Flour Milling Co. Ltd.,
Japan). Protein conversion was N×5.7. Ash was determined by Approved Method 08-01
at 14.0% mb.1)

8

Preparation of bread dough and freezing-and-thawing, and baking were as reported
in Seguchi et al.2) Baking absorption of each flour was determined in a farinograph with
300 g of flour (Approved Method 54-21).1) Freeze-resistant yeast (Kaneka Co. Ltd.,
Japan) was used in this experiment. Wheat flour (290 g), compressed yeast (8.7 g),sugar (14.5 g), salt (2.9 g), and water (estimated from farinograph at 500 BU) weremixed in a computer-controlled National Automatic Bread Maker (SD-BT6, Matsushita
Electric Ind. Co. Ltd., Japan) with the first proof for 2 hr 20 min at 30℃. The time
consisted of 15 min for the first mixing, 50 min of rest, 5 min for the second mixing,
and 70 min of fermentation. The bread dough was divided into 120 g pieces, rounded,molded, and placed in baking pans (Approved Method 10-10A).1) The bread dough was
further proofed for 22 min 15 sec at 38℃. This was the complete bread dough. The
baking pan, in which the bread dough (120 g) was placed, was put into a plastic filmbag, frozen, and stored at -20℃ for one, three, and six days. For the thawing, thebaking pan was placed in a refrigerator at 4℃ for 16 hr, which was adequate for breaddough to thaw completely. After thawing, one part of the bread dough was used forbread-making and the other was subjected to determination of free water oozed frombread dough. The thawed dough was baked at 210℃ for 30 min in a model DN-63
oven (Yamato Scientific Co. Ltd., Japan). After baking, the bread was removed from thepan and cooled for 1 hr at a room temperature of 26℃ and relative humidity of 43%.
Bread height (mm), weight (g), and volume (cm 3) were measured, and the crumb grains
were evaluated visually. As a control, nonfrozen and unstored bread dough of each flour
was subjected to same breadmaking.
The amount of liquid oozed from bread dough was determined. The thawed breaddough was removed from its pan. No water drops were observed in the pan or plastic
film bag. The dough was centrifuged at 38,900 g for 120 min at 4℃ (Hitachi Himac
CR 21G, HITACHI KOKI Co. Ltd., Japan), after which the centrifuge tube was titled at
an angle of 45° for 60 min in a cold room (4℃), and the supernatant (centrifuged liquid)
was collected in a dropwise manner and recorded as mL/100 g of dough. The

9centrifuged liquid after dialysis against water was subjected to protein and carbohydrate
determinations by the method of Lowry et al.3) and the phenol sulfuric acid method,respectively.

Statistical analysis
A statistical software package (SPSS Inc., Chicago, IL.) was used for statisticalanalyses. Experiments were repeated four times and the results were averaged. Analysis
produced significant F values by analysis of variance, followed by Duncan’s multiple
range test for comparison of means.

Results and Discussion
Breadmaking properties from frozen-and-thawed bread doughCamellia, Red Knight, Alps, Midoriuzumaki, Horoshiri, Kitakami, andNanbujigona wheat flours were used in this experiment. The protein and ash contents ofthese wheat flours are listed in Table 1. Table 2 lists the changes in breadmaking
properties (bread height (mm) and specific volume (cm3/g)) with increasing storage
time of the bread dough at -20℃. These breadmaking properties showed marked
deterioration after one day frozen storage of the bread dough compared with nonfrozen
and unstored bread dough. However, no further deterioration was observed after three tosix days of frozen storage. Figure 1 shows the appearance of sectioned breads bakedfrom bread doughs stored at 0 (nonfrozen), one, three, and six days made from Camellia
and Red Knight wheat flours.

10

11

< Camellia >Frozen storage (days)< Red Knight >Frozen storage (days)

Fig. 1. Appearance of sectioned bread baked from bread doughs of Camellia and Red
Knight wheat flours after 0 (nonfrozen), 1, 3 or 6 days in frozen storage.

12

13

Centrifuged liquid in frozen-and-thawed bread doughIt was qualitatively observed that liquid oozed from dough was increased byfreezing-and-thawing, and the increased amount of liquid may be related to thedeterioration of breadmaking properties baked with frozen-and-thawed bread dough.
Then, we thought that it was necessary to measure the amount of the oozed liquid frombread dough by centrifugation. Relevant studies are presented in the literature.4-8)
However, to measure a similar amount of the liquid that naturally oozed from dough, we
excluded the high gravity of ultracentrifugation conditions. We designed acentrifugation condition of 38,900 g for 120 min at 4℃ to obtain the amount of thecentrifuged liquid. Resulting centrifugation was performed at steady state. The
centrifuged liquid from nonfrozen and unstored bread dough was rather viscous andcontained high protein (12.2-14.3 mg/mL) and carbohydrate (48.6-55.3 mg/mL)
concentrations (Table 3). The protein and carbohydrate composition of the centrifugedliquid was not so affected by the centrifugation parameters. After one day of frozen
storage, the amount of free water obtained from thawed dough samples was markedly
increased, and the amounts from doughs stored for three to six days were slightly
increased or decreased (Table 2). All wheat flours showed the same trends. There was amarked correlation between the increase in the amount of liquid centrifuged fromfrozen-and-thawed doughs and the deterioration in their breadmaking properties (Table4). The deterioration of the breadmaking properties of frozen-and-thawed bread doughs
compared with nonfrozen and unstored doughs may be due to decreases in the waterbinding ability of a bread dough caused by freezing-and-thawing.

14

Conclusion
The breadmaking properties (bread height (mm) and specific volume (cm3/g)) of
breads baked from frozen-and-thawed doughs were markedly deteriorated. At the sametime, the exudation of liquid from thawed bread dough upon centrifugation increases.
And we showed that there was a strong correlation between the amount of centrifugedliquid and breadmaking properties.

15

References
1) American Association of Cereal Chemists. 2000. Approved Methods of the AACC,
10th ed. Methods 08-01, 10-10A, and 54-21. The Association: St. Paul, MN.
2) Seguchi M, Hayashi M, Matsumoto H: Effect of gaseous acetic acid on doughrheological and breadmaking properties. Cereal Chem 74:129-134, 1997.
3) Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin
phenol reagent. J. Biol. Chem. 193: 265-275, 1951
4) Mauritzen CM, Stewart PR: The ultracentrifugation of doughs made from wheat
flour dough. Aust J Biol Sci 18:173-189, 1965.
5) Mauritzen CM, Stewart PR: The disulphide and sulphydryl content of fractions
prepared by the ultracentrifugation of doughs made from wheat flour. Aust J BiolSci 19:1111-1123, 1966.
6) MacRitchie F: The liquid phase of dough and its role in baking. Cereal Chem
53:318-326, 1976.
7) Larsson H, Eliasson AC: Phase separation of wheat flour dough studied byultracentrifugation and stress-relaxation I. Influence of water content. Cereal Chem
73:18-24, 1996.
8) Rasanen J, Blanshard JMV, Siitari-Kauppi M, et al: Water distribution in frozen lean
wheat flour doughs. Cereal Chem 74:806-813, 1997.

16

Chapter 3
Restoration of Breadmaking Properties to Frozen Dough by Additionof Sugar and Yeast and Subsequent Processing
ObjectWe tried to clarify the cause of the deterioration of breadmaking properties that
correlate with the increase of centrifuged liquid from frozen-and-thawed dough.

Abstract
We prepared bread dough A (a mixture of wheat flour, sugar, salt, and water), breaddough B (a mixture of bread dough A and yeast), and bread dough C (first proofed,
molded, and second proofed bread dough B) and froze them at -20℃ for six days.
They were thawed at 4℃ for 16 hr and subjected to their breadmaking processes. The
results indicated that breadmaking properties (bread height (mm) and specific volume(cm3/g)) after bread dough A and B processes were the same as those of control breaddough (unfrozen dough). However, in the case of bread dough C, the resulting breadshowed depression of the properties. The amount of centrifuged liquid from thawed
bread dough C increased. Sugar was added to thawed bread dough C (bread dough C-1),
and then yeast was further added to bread dough C-1 (bread dough C-2), and they were
subjected to the breadmaking process. The results showed that the breadmaking
properties of bread dough C-2 were the same as those of the control. It was further
found that when the first proof step in the bread dough C-2 process was omitted, thebreadmaking properties were depressed. Frozen and thawed bread dough C was packed
into a plastic tube, and extension of the dough was compared with that of control doughunder reduced pressure. Bread dough C extended to 50 mm, compared with 70 mm forcontrol dough. First proof, mold, and second proof steps of dough C-2 caused it toextend to the same height as control dough. It was concluded that the increased amount17

of the separated liquid in thawed dough C caused depression of breadmaking properties
resulting from lack of water in the appropriate places to provide the expected properties,
but these properties could be restored to the levels of control bread dough by theaddition of sugar and yeast following the first proof, mold, and second proof steps.

Materials and Methods
Wheat flour
Four wheat flours were used in this experiment: Camellia (Nisshin Flour Milling
Co. Ltd., Japan), Alps (Nittofuji Flour Milling Co. Ltd., Japan), and Haruyokoi and
Kitanokaori (Maeda Grain Farm Products, Tokachi, Japan). General analysis of theseflours is given in Table 1. Protein conversion was N × 5.7 (Approved Method46-10.01).1) Ash was determined by AACC International Approved Method 08-01.01 at
14.0% mb.1)

Preparation of control (unfrozen) and frozen-and-thawed bread doughs andbreadmaking
Breadmaking was performed according to Seguchi et al.2) Baking absorption offlour was determined in a farinograph with 300 g of flour (Approved Method54-21.01).1) Freeze-resistant yeast (Kaneka Co. Ltd., Japan) was used in this experiment.

18

Wheat flour (290 g), compressed yeast (8.7 g), sugar (14.5 g), salt (2.9 g), and water(estimated from farinograph at 500 BU) were mixed in a computer-controlled National
Automatic Bread Maker (SD-BT6, Matsushita Electric Ind. Co. Ltd., Japan), with thefirst proof for 2 hr 20 min at 30℃. The time consisted of 15 min for the first mixing, 50
min of rest, 5 min for the second mixing, and 70 min of fermentation. The bread doughwas divided into 120 g pieces, rounded, molded, and placed in baking pans (Approved
Method 10-10.03).1) The bread dough was further proofed for 22 min 15 sec at 38℃.
The baking pan in which the bread dough (120 g) was placed was put into a plastic filmbag, frozen, and stored in a freezer (Rotary Chest MF-C29B-H, Mitsubishi Electric Ind.
Co. Ltd., Japan) at -20℃. For the thawing, the baking pan was placed in a refrigerator(SF-B100LS-AC, Toshiba Home Technology Corporation Co. Ltd., Japan) at 4℃ for
16 hr, which was adequate for the bread dough to thaw completely. After thawing, onepart of the bread dough was used for breadmaking, and the other was subjected todetermination of free water exuding from the dough. The thawed dough was baked at
210℃ for 30 min in a model DN-63 oven (Yamato Scientific Co. Ltd., Japan). Afterbaking, the bread was removed from the pan and cooled for 1 hr at a room temperatureof

26℃ and relative humidity of 43%. Bread height (mm), weight (g), and volume(cm3) were measured, and the crumb grains were evaluated visually.

Determination of liquid exudate from bread doughThe thawed bread dough was removed from its pan. No water drops were observedin the pan or plastic film bag. The dough was centrifuged at 38,900 g for 120 min at 4℃(Hitachi Himac CR 21G, HITACHI KOKI Co. Ltd., Japan), after which the centrifuge
tube was titrated at an angle of 45°for 60 min in a cold room (4℃), and thesupernatant (centrifuged liquid) was collected in a dropwise manner and recorded as mL
per 100 g of dough.3)

19

Effect of freezing of yeast
Compressed yeast (8.7 g) was suspended in 20 mL of water and frozen at -20℃
for six days. After thawing at 4℃ for 16 hr, it was used in breadmaking with Camellia
flour.

Preparation of various bread doughs (A, B, C, C-1, and C-2) and subsequent
processes
Various bread doughs (A, B, C, C-1, and C-2) were prepared, and breadmaking
was performed (Figs. 1 and 2). In the case of bread dough A, wheat flour (290 g), sugar(14.5 g), salt (2.9 g), and water were mixed for 2 min at room temperature and frozen at-20℃ for six days. After thawing, the dough was mixed with yeast (8.7 g) for 1 min,first proofed for 2 hr 20 min, molded, second proofed for 22 min 15 sec, and baked at
210℃ for 30 min. In the case of bread dough B, dough A was mixed with yeast (8.7 g)
for 1 min and then frozen. After the dough thawed, first proof for 2 hr 20 min, mold,and second proof were performed, and the bread was baked. In the case of dough C,
dough B was first proofed, molded, second proofed for 22 min 15 sec, and then frozen.
After thawing, it was baked. In the cases of doughs C-1 and C-2, frozen dough C was
thawed and mixed with sugar (14.5 g) for 1 min (dough C-1) or with sugar (14.5g) andyeast (8.7 g) (dough C-2) and then first proofed, molded, second proofed, and baked(Fig. 2). Dough C-2 was further tested with the first proof omitted. Measurements ofbread height (mm), weight (g), and volume (cm 3) were as described in the second
section under Materials and Methods. Freezing and frozen storage equipment were
described in the same section.

Bread dough extension in a vacuum
First proofed bread dough was divided into 60 g pieces, rounded, and squeezed
into a plastic tube (5.0 cm diameter, 16 cm height, and 3 mm thickness) and sealed witha rubber stopper (no. 19, diameter 4.8 cm) from the bottom.2) The bread dough in the20plastic tube was frozen at -20℃ for six days and thawed. The plastic tube containing
dough was set up in a glass case (Bell Jar VKU-1000, Kiriyama Co.,Tokyo) (21.5 cm
diameter, 30.0 cm height). The pressure in the glass case was reduced by an aspirator(Handy Aspirator WP-15, Yamato Scientific Co. Ltd., Tokyo) at room temperature for
420 sec. until reaching -76 cm Hg. The height (mm) of bread dough in the plastic tube
was equivalent to the volume of the dough and was measured with a cathetometer(PCTM-300, Pika Co. Ltd., Tokyo) from outside the glass case.

Statistical analysis
A statistical software package (SPSS Inc., Chicago, IL.) was used for statisticalanalyses. Experiments were repeated two times and the results were averaged.
Significant F values by analysis of variance were followed by Duncan's multiple range
test for comparison of means.

21

22

Results and Discussion
Effect of freezing of yeast on breadmaking properties
The effects of frozen and thawed yeast with or without water on breadmaking areindicated in Table 2, in which the breadmaking properties such as bread height (mm)and specific volume (cm3/g) were almost the same as the control (unfrozen breaddough). As indicated in the Materials and Methods section, not dry yeast but
compressed yeast (moisture content 64.9 %) was used throughout the experiments.

23

Effects of freezing step in the breadmaking process
Dough A (a mixture of wheat flour, sugar, salt, and water) was frozen at -20℃ forsix days. After thawing at 4℃ for 16 hr, it was mixed with yeast and processed (Fig. 1).
The results are shown in Table 3. Breadmaking properties (% vs. control) for Camellia
wheat flour (bread height 100% and SV 99%), Alps (102 and 100), Haruyokoi (107 and115), and Kitanokaori (108 and 109) were each almost the same as the control.
Centrifuged liquid in Camellia (116), Alps (105), Haruyokoi (111), and Kitanokaori (64),were not much larger than each control. It was found that the wheat flour fraction suchas water solubles, gluten, prime starch, and tailings4) in bread dough was not affected byfreezing and thawing, and the amount of centrifuged liquid did not increase by much.
Next, dough B (a mixture of dough A plus yeast) was frozen. After thawing, it wasprocessed (Fig. 1). The results（Table 3）showed the same breadmaking performance forCamellia (bread height 101 and SV 104), Alps (106 and 102), Haruyokoi (104 and 113),
and Kitanokaori (109 and 106) as for the control, and the amount of centrifuged liquidincreased or decreased in Camellia (146), Alps (106), Haruyokoi (119), and Kitanokaori(71) compared with each control. It was found that yeast in bread dough was notaffected by freezing and thawing, and the amount of centrifuged liquid did not increaseby much.
After thawing of frozen dough C, breadmaking was performed (Fig. 1). The results(Table 3) revealed large decreases in breadmaking performance with Camellia (bread24

height 75 and SV 76), Alps (78 and 71), Haruyokoi (81 and 85), and Kitanokaori (88and 85) compared with each control. The amount of centrifuged liquid significantly
increased in Camellia (167), Alps (128), Haruyokoi (122), and Kitanokaori (372)compared with each control. From these results it was concluded that the effect offreezing and thawing of bread dough on breadmaking properties (doughs A and B) was
small, but when processing was performed before freezing (dough C), the depression
was large. That is, freezing of yeast and wheat flour fractions in bread dough was not
related to breadmaking properties, but freezing after processing (proof and mold) was
highly related to breadmaking properties. Bread dough C was damaged by freezing andthawing; that is, water separated from dough C, crystallized on freezing, and afterthawing was not absorbed back into the dough. The dehydrated dough C lost its baking
extensibility because moisture moved from the gluten matrix by freezing and thawing ofbread dough, and acceptable breadmaking properties were not obtained.

Addition of sugar and yeast to thawed bread dough following first proof, mold, andsecond proof
We prepared doughs C-1 and C-2 with Camellia wheat flour and added sugar or
sugar and yeast to the thawed dough C and processed them (Fig. 2). The results areshown in Table 4. Dough C-1 showed depressed breadmaking properties (bread height62.30 mm (80％) and SV 2.24 cm3/g (68%)), which indicated that yeast in the thawed
dough C did not use added sugar and generate CO2. So we added yeast to the thawed
dough C and processed; however, the depression of the baking properties was not
improved (data not shown here). Dough C-2 gave acceptable breadmaking properties(83.09 mm (107%) and 3.53 cm3/g (107%)), similar to control (77.80 mm (100%) and3.31 cm3/g (100%)). It was assumed that the yeast in dough C lost its ability of CO 2
generation even if sugar was added (dough C-1); however, the addition of yeast andsugar in dough C-2 could generate CO2, resulting in the same breadmaking properties as
those of the control.

25

26

The reason dough C-2 showed acceptable breadmaking properties may be the CO2
generation by the addition of yeast and the repetition of the first proof, mold, andsecond proof steps. It is thus possible that the depression caused by freezing andthawing in bread dough C may have resulted from loss of yeast activity and a change ofcharacteristics such as extensibility of the gluten matrix in bread dough.

Necessity of first proof step in dough C-2 processing
Next we examined whether the first proof step was necessary in bread dough C-2
processing (Fig. 2). Dough C-2 was treated with a second proof only. The results (Table5) show that, in this case, acceptable breadmaking properties could not be obtained(bread height 58.35 mm (75%) and SV 2.24 cm3/g (68%)) compared with the control(77.80 mm (100%) and 3.31 cm3/g (100%)). It was assumed that because of theseparation of water from thawed dough C, the gluten matrix in the thawed dough C did
not recover to the unfrozen dough level even if yeast and sugar were added. However,the first proof, mold, and second proof steps of dough C-2 could give acceptablebreadmaking properties. If yeast and sugar were added to thawed bread dough C after
the initial first proof, mold, and second proof steps, the bread dough showedbreadmaking properties similar to those of the control.

27

Low extension of bread dough related to freezing and thawing
To examine the effect of freezing and thawing on bread dough rheology, thecontrol bread dough, frozen and thawed dough C, and first proofed, molded, and second
proofed dough C-2 were examined under reduced pressure (0 to -76 cm Hg) (Fig. 3).
With reduction of the pressure in the glass case, the volume of the dough increased, andthe height (mm) of the dough in the plastic tube increased. The heights of the control
and of the first proofed, molded, and second proofed dough C-2 increased from 30.0
mm to 72.8 mm after 120 sec. However, frozen and thawed dough C did not increase to
more than 50.0 mm. Since water was separated from the thawed dough, the growth ofthe dough was lower than that of the control. However, the growth of first proofed,
molded, and second proofed dough C-2 was restored to the control level. The
rheological properties of a wheat flour dough are extremely sensitive to water content.5)
This was reflected in the recovery of the breadmaking properties of dough C-2.
Czuchajowska et al. reported that loaf volume is more affected by a water level that is
too low than by one that is too high. 6)28

Conclusion
Freezing of yeast and wheat flour fractions in bread dough was not related tobreadmaking properties, but freezing after processing (proof and mold) was highly
related to the breadmaking properties. This indicated that acceptable breadmaking
properties were not obtained because moisture moved from the gluten matrix byfreezing and thawing of bread dough. Thus, we could improve the properties to
acceptable levels by the addition of sugar and yeast to the thawed dough and thenrepeating first proof, mold, and second proof steps.

29References
1) AACC International. 2010. Approved Methods of Analysis, 11th Ed. Methods
08-01.01, 46-10.01, 10-10.03, and 54-21.01. Available online only. AACC
International: St. Paul, MN.
2) Seguchi M, Hayashi M, Matsumoto H: Effect of gaseous acetic acid on doughrheological and breadmaking properties. Cereal Chem 74:129-134, 1997.
3) Seguchi M, Nikaidoo S, Morimoto N: Centrifuged liquid and breadmaking
properties of frozen-and thawed bread dough. Cereal Chem 80:264-268, 2003.
4) Ozawa M, Seguchi M: Effect of dry-heated prime starch and tailings fractions inwheat flour on pancake springiness. Cereal Chem 85:626-628, 2008.
5) Eliasson AC, and Larsson K: Cereals in breadmaking, first edition, Eliasson AC and
Larsson K (eds.), Marcel Dekker, New York, 1993, 265.
6) Czuchajowska Z, Pomeranz Y, Jeffers HC: Water activity and moisture content ofdough and bread. Cereal Chem 66:128-132, 1989.

30

Chapter 4
Effect of Xanthan Gum on Improvement of Bread Height and SpecificVolume upon Baking with Frozen and Thawed Dough
ObjectFrom findings of chapter 3, we supposed that acceptable level of the breadmaking
propterties may be obtained by preventing the movement of water. Thus, we tried to use
polysaccharides. The aim of this Chapter was to prevent bread deterioration fromfreezing/thawing by adding polysaccharides to the dough.

Abstract
Wheat flour was preliminarily mixed with 5% polysaccharides (locust bean gum,guar gum, xanthan gum, tamarind seed gum, native gellan gum, dextrin, LM pectin,
fermented cellulose CMC, konjac glucomannan, HM pectin, κ-carrageenan,
ι-carrageenan, and λ-carrageenan), and then mixed with water, salt, sugar, and yeast,
followed by proofing. Next, the proofed bread dough was divided into two, frozen at-20°C for 6 days, and then thawed at 4°C for 16 hr. One part of the dough was baked at
210°C for 30 min, and the other was subjected to determination of the extruded liquidfrom the thawed dough upon centrifugation. As the results, frozen/thawed control(wheat flour) dough presented a lower bread height (mm), lower specific volume(cm3/g), and a higher volume of exuded liquid than unfrozen dough. When
polysaccharides such as guar gum, xanthan gum, and tamarind seed gum were mixed
with the flour prior to making the bread dough, similar or greater bread height andspecific volume than those of bread baked with unfrozen control dough were obtained.
The greatest increases in bread height and specific volume on supplementation ofpolysaccharides were observed when xanthan gum was used. In order to clarify why
xanthan gum markedly improved the breadmaking properties, oven-spring tests wereconducted for 10, 15, 20, and 30 min in a 210°C oven. The breadmaking properties31

(bread height) of baking with frozen/thawed control dough increased until 10 min in the
210°C oven and then stopped. However, for the bread with xanthan gum, these variables
did not stop increasing.

Materials and Methods
Wheat flour and polysaccharides
Wheat flour (Camellia) was donated by Nisshin Flour Milling Co. Ltd., Japan.
Protein conversion was carried out using the formula N x 5.7 (Approved Method 46-10,
AACC International).1) Ash was determined by the AACC International method (08-01)
on a 14.0% moisture basis.1) Locust bean gum, guar gum, xanthan gum, tamarind seedgum, native gellan gum, dextrin, LM pectin, fermented cellulose CMC, κ-carrageenan,
konjac glucomannan, HM pectin, ι-carrageenan, and λ-carrageenan were donated by
San-Ei Gen F.F.I. Co. (Tokyo, Japan). Reagent-grade xanthan gum from Xanthomonas
campestris was purchased from Tokyo Chemical Industry Co. Ltd., Japan.

Breadmaking methods: Preparation of frozen/thawed bread dough andbreadmaking
Breadmaking was performed according to Seguchi et al.2) Baking absorption offlour was determined in a Farinograph with 300 g of flour (AACC International method54-21).1) In this experiment, wheat flour was mixed with 5% polysaccharides and water
absorption (%) was determined. The dosage level of polysaccharides (5%) exceededcurrent industry practice (0.5%) by tenfold. Specifically to clarify the effect of dosed
polysaccharides in this experiment, the dosage level of polysaccharides was increased to
5% instead of 0.5%. The breadmaking properties (bread height (mm) and specificvolume (cm3/g)) increased linearly from 0 to 5% dosage, and clear dosage effects could
be observed at 5% dosage. Freezing-resistant yeast (Kaneka Co. Ltd., Japan) was usedin this experiment. Wheat flour (290 g), compressed yeast (8.7 g), sugar (14.5 g), salt
(2.9 g), and water (estimated from the Farinograph at 500 BU) were mixed in a32

computer-controlled National Automatic Bread Maker (SD-BT6, Matsushita ElectricInd. Co. Ltd., Japan) with the first proof for 2 hr 20 min at 30°C. The time consisted of
15 min for the first mixing, 50 min of rest, 5 min for the second mixing, and 70 min of
fermentation. The bread dough was divided into 120 g pieces, rounded, molded, andplaced on baking pans (AACC International method 10-10A).1) The bread dough was
further proofed for 22 min 15 sec at 38°C. The baking pan on which the bread dough(120 g) was placed was put into a plastic film bag, frozen, and stored in a freezer(Rotary Chest MF-C29B-H, Mitsubishi Electric Ind. Co. Ltd., Japan) at -20°C. For thethawing, the baking pan was placed in a refrigerator (SF-B100LS-AC, Toshiba Home
Technology Corporation Co. Ltd., Japan) at 4°C for 16 hr, which was adequate for thebread dough to thaw completely. After thawing, one part of the bread dough was used
for breadmaking and the other was subjected to determination of the amount of liquidthat exuded from the dough upon centrifugation. The thawed dough was baked at 210°C
for 30 min in a model DN-63 oven (Yamato Scientific Co. Ltd., Japan). After baking,the bread was removed from the pan and cooled for 1 hr at room temperature (26°C)
and 43% relative humidity. Bread height (mm), weight (g), and volume (cm 3) were
measured, and the crumb grain was evaluated visually. Numerous other factors were
influenced by the use of polysaccharides. Since bread height and specific volume arehighly related to other factors and thus provide equivalent results, we determined breadheight and specific volume in this experiment.

Measurement of bread temperature using an infrared thermometer
Bread temperature was measured using an infrared thermometer (ThermalView
XHR 32-RA0350; pixels: 320 x 256, ViewOhre Imaging Co. Ltd., Tokyo, Japan). Breaddough was baked at 210°C for 0, 5, 10, and 30 min, removed from the oven, cut in halfwith a knife, and photographed using a thermal camera within 30 sec. The thermal
image was saved as a bitmap image, and the temperature of the surface of sectioned
bread was measured from a color bar.

33

Effects of frozen storage time of bread dough supplemented with 5% xanthan gum
on breadmaking properties
Bread dough supplemented with 5% xanthan gum was frozen and stored at -20°C
for 1, 3, and 6 days, thawed at 4°C for 16 hr, and baked at 210°C for 30 min.

Effects of baking time on the oven-spring and shrinkage (%) of bread
supplemented with 5% xanthan gum, guar gum, and tamarind seed gum
After thawing at 4°C for 16 hr, frozen/thawed bread dough supplemented with 5%
xanthan gum, guar gum, and tamarind seed gum was baked in an oven (210°C) for 10,
15, 20, and 30 min, and oven-spring (bread height) was measured after being left tostand for 60 min at room temperature.
To measure shrinkage (%) of bread, bread dough (30 g) was put into a glass bottlewith a size scale on it and subjected to baking at 210°C for 5, 10, 15, 20, 25, and 30 min.
Bread height (mm) was read from the bottle. Shrinkage (%) of bread at each time point
was calculated as follows: (bread height (mm) just after removal from the oven – breadheight (mm) at 60 min after removal from the oven) ÷ bread height (mm) just after
removal from the oven × 100 = shrinkage (%) of bread.

Determination of exuded liquid from bread dough upon centrifugation
The thawed bread dough (120 g) was removed from its pan and divided into quarters,
each of which was placed in a separate 38-mL plastic centrifuge tube (2.9 cm diameter x
10.3 cm length) and centrifuged at 38,900 g for 120 min at 4°C (Hitachi Himac CR
21G; HITACHI KOKI Co. Ltd., Japan), after which the centrifuge tubes were tilted at
an angle of 45° for 60 min in a cold room (4°C), and total supernatant (centrifugedliquid) was collected and recorded as mL/100 g dough.3)

34

Statistical analysis
A statistical software package (SPSS Inc., Chicago, IL) was used for statisticalanalyses. Experiments were repeated twice and the results were averaged. Whensignificant F values were obtained by analysis of variance, Duncan's multiple range test
was carried out for comparison of means.

Results and Discussion
The effect of polysaccharides in frozen/thawed dough on the breadmaking
properties
Wheat flour was mixed with various polysaccharides at 5%, and Brabender
Farinograph tests of the flour were performed. The optimum level of water addition isrelated to the composition of the flour, and therefore it is necessary to determine thisoptimum level for each type of flour. The Brabender Farinograph is commonly used todetermine water absorption. The water absorption of flour is described as the amountnecessary to bring the dough to a specified consistency (normally 500 Brabender units,
BU) at the point of optimum development. The content of polysaccharides has beenshown to affect water absorption.4) Water absorption of the mixed flour is shown in
Table 1. From these results, the amount of additional water (mL) needed forbreadmaking was calculated (Table 1). Water absorption was optimized using a
Farinograph and ranged from 61.4 to 106.5%. Types of dough with a wide range ofwater absorption were indicated; however, the specified consistency (500 BU) with theaddition of various levels of water was obtained in dough mixing in breadmaking, andcomparable types of bread could be obtained with a fixed mixing time for all of theflour with different polysaccharides. The mixtures were then used to make bread doughand frozen at -20°C for 6 days. After thawing at 4°C for 16 hr, they were used forbreadmaking and determination of the amount of exuded liquid from the bread doughupon centrifugation (Table 2).

35

The breadmaking properties of control (wheat flour) bread dough worsened byfreezing/thawing: bread height, from 81.50 mm to 62.05 mm; and specific volume, from
3.63 to 2.59 cm3/g. When the 13 kinds of polysaccharide were mixed in the dough, thesame tendency was observed in every breadmaking process (Table 2). It is generally
known that breadmaking properties such as bread height (mm) and specific volume(cm3/g) deteriorate upon freezing/thawing. To stop this deterioration in bread bakedwith frozen/thawed control (wheat flour) bread dough, we added various
polysaccharides to the flour prior to freezing, as described below, and aimed to obtainthe same level of breadmaking properties as in unfrozen control breadmaking. The
results indicated that bread baked with frozen/thawed bread dough supplemented withguar gum (bread height, 77.22 mm; and specific volume, 3.19 cm 3/g), xanthan gum(105.20 mm and 4.67 cm3/g), and tamarind seed gum (78.21 mm and 3.53 cm 3/g)

36

exhibited similar or greater bread height and specific volume than bread (81.50 mm and3.63 cm3/g) baked with unfrozen control dough. Bread baked with dextrin (65.17 mm
and 2.89 cm3/g) or konjac glucomannan (65.97 mm and 2.89 cm 3/g)-supplemented
frozen/thawed dough was smaller than that baked with unfrozen control bread dough.
Other polysaccharide-supplemented frozen/thawed dough also produced smaller breadthan unfrozen control dough. The results of breadmaking upon supplementation with
polysaccharides varied (Table 2). We looked for types of bread with similar or superiorbreadmaking properties compared with that baked with unfrozen control dough, andidentified those supplemented with three polysaccharides: guar gum, xanthan gum, or
tamarind seed gum.
The amount of extruded liquid (mL/100 g dough) from unfrozen or frozen/thawed
dough upon centrifugation is presented in Table 2. For unfrozen and frozen/thawed
control dough, the levels were 3.27 and 10.08 mL/100 g dough, respectively, which aresimilar to values reported previously.3) Similarly, when the 13 kinds of polysaccharide
were mixed in the bread dough that was then centrifuged, exuded liquid was alsomeasured. When guar gum, xanthan gum, tamarind seed gum, native gellan gum,fermented cellulose CMC, HM pectin, -carrageenan, and -carrageenan were added,
the amounts of exuded liquid were small or zero in both unfrozen and frozen/thawed
bread dough, owing to their high water absorption. Remarkably improved breadmaking
properties were observed when guar gum, xanthan gum, and tamarind seed gum wereadded, and almost no centrifuged liquid was exuded from such thawed dough uponcentrifugation. When native gellan gum, fermented cellulose CMC, HM pectin, ι-, and- carrageenan were added to dough, high water absorption was found in both unfrozen
and frozen/thawed dough; however, deteriorated breadmaking properties were exhibitedby frozen/thawed dough compared to unfrozen control dough (Table 2). When locustbean gum, dextrin, and -carrageenan were added, the amounts of exuded liquid were
the same between unfrozen and frozen/thawed control bread dough.

37

38

On the other hand, when LM pectin or konjac glucomannan was added, higher levels of
exuded liquid in both unfrozen and frozen/thawed bread dough were observed uponcentrifugation: LM pectin: unfrozen dough, 17.50 mL/100 g dough, and frozen/thawed
dough, 20.67 mL/100 g dough; and konjac glucomannan: unfrozen dough, 5.38 mL/100
g dough, and frozen/thawed dough, 12.58 mL/100 g dough.
Seguchi et al. indicated a strong relationship between the increase of exuded liquidfrom bread dough (wheat flour) by freezing/thawing and the deterioration ofbreadmaking properties. 3) However, in these experiments, when guar gum, xanthangum, tamarind seed gum, ι-carrageenan, and λ-carrageenan were added, exuded liquid
upon centrifugation (38,900 g) from unfrozen and frozen/thawed bread dough could not
be observed due to higher water absorption. However, more intense centrifugation could
produce exuded liquid from these types of dough and differences in the volumes of
exuded liquid were observed between unfrozen and frozen/thawed dough. Xanthan gum
showed the largest contribution to improving the breadmaking properties, likely due tothe chemical structures of the polysaccharide. When xanthan gum-supplemented doughwas frozen/thawed, breadmaking properties deteriorated, although they were superior tothose of unfrozen control dough.

Change of freezing/thawing effects on xanthan gum-supplemented bread doughThe breadmaking properties (bread height (Fig. 1a) and specific volume (Fig. 1b))
upon baking with xanthan gum-supplemented bread dough deteriorated from 0 to 1, 3,
and 6 days (frozen storage temperature, -20°C) of freezing (open circle); however, theseproperties were consistently superior to the breadmaking properties upon baking with
control (wheat flour) dough throughout the 6 days of freezing (closed circle). It was thus
shown that the addition of xanthan gum to bread dough could stop the deterioration ofbreadmaking properties caused by freezing/thawing. Figure 2 shows the change in theamount of exuded liquid from control and xanthan gum-supplemented bread doughs
upon centrifugation throughout the 6 days of freezing. For the control bread dough, the39amount of exuded liquid upon centrifugation was increased by freezing/thawing;
however, that of xanthan gum-supplemented frozen/thawed bread dough did notincrease due to the strong water absorption. Although there is a close relationshipbetween the deterioration of breadmaking properties (Fig. 1a and b) and the increase of
exuded liquid (Fig. 2) in control dough, as reported previously,3) in the case of 5%
xanthan gum-supplemented dough, the precise amount of exuded liquid uponcentrifugation was not measured, so the relationship between the deterioration ofbreadmaking properties and the increase of exuded liquid was not determined.

40

Measurement of bread temperature using infrared thermometry
We measured the temperature of bread just after its removal from the oven using an
infrared thermometer, which indicated the surface temperature of bread crumbs from acolor image. The color image of the bottom and top of bread dough showed light bluish(30~35°C) and bluish (below 25°C) features at room temperature (Fig. 3A). At 5 min of
baking (Fig. 3B), the center (deep blue) of the image of bread crumbs indicated atemperature of below 40°C, at which yeast could survive and produce CO2 gas; however,
the temperature of the upper surface (yellowish~reddish) was around 75-80°C, at whichyeast could not survive. At 10 min of baking (Fig. 3C), the temperature of the center(greenish~reddish) of the image of bread crumbs was 65-80°C, at which yeast would bekilled, and the swelling of bread dough was terminated. At 30 min of baking (Fig. 3D),

41

the bread crumb temperature (reddish) was more than 80°C, at which the gelatinizationof starch and denaturation of gluten protein ceased, followed by formation of the final
bread structures.

Oven-spring by guar gum-, xanthan gum-, or tamarind seed gum-supplemented
bread dough and shrinkage upon removal from the oven
Frozen/thawed guar gum-, xanthan gum-, or tamarind seed gum-supplemented
dough was baked for 30 min at 210°C. The bread was removed from the oven at 10, 15,
20, and 30 min of baking, and bread height was measured after 60 min at room
temperature. In frozen/thawed guar gum-, xanthan gum-, or tamarind seed42

gum-supplemented dough the yeast would be killed by the heat, and all doughs arelikely to stop swelling at 10 min of baking (Fig. 3C); however, in the case of xanthan
gum-supplemented dough, it continues to increase in height (Fig. 4). Xanthan
gum-supplemented bread showed shrinkage of 34.2% (at 10 min), 13.4% (at 15 min),
4.1% (at 20 min), and 0% (at 30 min). On the other hand, control (wheat flour) or theother doughs showed almost no shrinkage at the same time point; that is, the breadheights at 10 min of baking were maintained at 30 min of baking. Xanthan
gum-supplemented bread at 65-80°C appeared to continue to increase in terms of its
height until 30 min of baking; however, guar gum- or tamarind seed gum-supplemented
bread did not increase in this regard with more than 10 min of baking, owing todifferences in the chemical structures. Among the 13 kinds of polysaccharide, xanthan
gum was the most effective to prevent the deterioration of breadmaking properties
exhibited by frozen/thawed control dough. Xanthan gum is a polysaccharide derived
from the bacterial coat of Xanthomonas campestris. It is produced by the fermentationof glucose, sucrose, or lactose by this bacterium. It functions to protect the bacterial cellfrom drying, heating, cooling, and other adverse conditions. Xanthan gum can also help
bread dough to endure adverse conditions associated with freezing. To ascertain theeffect of xanthan gum on the improvement of bread baked with frozen/thawed dough,reagent-grade xanthan gum was used in the same manner. Table 2 shows that the same
results could be obtained in bread by using reagent-grade xanthan gum.

43

Conclusion
13 kinds of polysaccharide were added to dough, and breadmaking tests were
performed after freezing/thawing. As a result, xanthan gum, guar gum, and tamarindseed gum were shown to prevent the deterioration of breadmaking properties by
freezing/thawing; in particular, xanthan gum could ensure the remarkable recovery ofbread from freezing/thawing effects.

44

References
1) American Association of Cereal Chemists. 2000. Approved Methods of the AACC,
10th Ed. Methods 08-01, 10-10A, 46-10, and 54-21. The Association: St. Paul, MN.
2) Seguchi M, Hayashi M, Matsumoto H: Effect of gaseous acetic acid on doughrheological and breadmaking properties. Cereal Chem 74: 129-134, 1997.
3) Seguchi M, Nikaidoo S, Morimoto N: Centrifuged liquid and breadmaking
properties of frozen-and-thawed bread dough. Cereal Chem 80: 264-268, 2003.
4) Eliasson AC, and Larsson K: Cereals in breadmaking, first edition, Eliasson AC and
Larsson K (eds.), Marcel Dekker, New York, 1993, 261-263, 325-326.

45

Chapter 5
Centrifuged Liquid of Frozen-and-Thawed Bread Dough
ObjectWe studied the relationship between free water oozed from frozen-and-thawed
dough and the dough’s breadmaking properties (in chapter 2). Thus, we studied which
components of bread dough (sugar, salt, and yeast) affect the amount of centrifugedliquid, and we also studied the possibility that fermented products affect that.

Abstract
The presence of yeast and salt in bread dough was suggested to be closely related
to the amount of centrifuged liquid, and fermented products particularly had a large
effect on the amount of centrifuged liquid.

Materials and Methods
Wheat flour
Seven different wheat flours were used in this experiment: Camellia (Nisshin Flour
Milling Co. Ltd., Japan), Red Knight and Alps (Nittofuji Flour Milling Co. Ltd., Japan),Midoriuzumaki (Masuda Flour Milling Co. Ltd., Japan), Horoshiri (Ebetsu Flour
Milling Co. Ltd., Japan), and Kitakami and Nanbujigona (Fukin Flour Milling Co. Ltd.,
Japan). Protein conversion was N×5.7. Ash was determined by Approved Method 08-01
at 14.0% mb.1)
The amount of liquid oozed from bread dough was determined. The thawed breaddough was removed from its pan. No water drops were observed in the pan or plastic
film bag. The dough was centrifuged at 38,900 g for 120 min at 4℃ (Hitachi Himac
CR 21G, HITACHI KOKI Co. Ltd., Japan), after which the centrifuge tube was titled at
an angle of 45° for 60 min in a cold room (4℃), and the supernatant (centrifuged liquid)46

was collected in a dropwise manner and recorded as mL/100 g of dough. The
centrifuged liquid after dialysis against water was subjected to protein and carbohydrate
determinations by the method of Lowry et al.2) and the phenol sulfuric acid method,respectively. Various combined bread doughs were prepared. Salt (2.9 g) and Camellia
wheat flour (290 g), sugar (14.5 g) and flour, yeast (8.7 g) and flour, yeast, sugar, andflour, yeast, salt, and flour, sugar, salt, and flour, and yeast, sugar, salt, and flour(complete) were blended, respectively, and were mixed with 210 mL of water in a breadmaker. They were then treated in the same manner as above. All dough samples werefrozen-and-thawed after one day of storage, and the amount of liquid centrifuged fromeach was determined. Protein and carbohydrate in the liquid were also determined asabove.
Cultured fluid/flour dough were prepared, and centrifuged liquid was determined.
Compressed yeast (4.35 g), sugar (7.25 g), and salt (1.45 g) were mixed with 105 mL ofwater in 300 mL conical flasks and incubated at 35℃ for 140 min with slight shaking.
This was the complete cultured fluid. Then, mixtures were boiled for 5 min to kill all theyeast and cooled with tap water. Camellia wheat flour (145 g) was mixed with above
whole cultured fluid in a farinograph bowl at 30℃ for 10 min and referred to as
cultured fluid/flour dough. The cultured fluid/flour dough represented dough withpresence of yeast and fermentation products. Cultured fluid/flour dough (120 g) was
centrifuged at 38,900 g at 4℃ for 120 min. The centrifuge tubes were titled at an angleof 45° for 60 min in a cold room (4℃), and centrifuged liquid was collected. Protein
and carbohydrate in the liquid were also determined as above. Various combined
cultured fluid/flour doughs were prepared with salt (1.45 g) and Camellia wheat flour(145 g), sugar (7.25 g) and flour, yeast (4.35 g) and flour, yeast, sugar, and flour, yeast,
salt, and flour, sugar, salt, and flour, and yeast, sugar, salt, and flour (complete) weremixed with 105 mL of water, respectively, and treated in the same manner as above. All
cultured fluid/flour doughs were frozen-and-thawed after one day of storage, and theliquid centrifuged from them was determined. Protein and carbohydrate in the liquid47

were also determined as above.
Time course of liquid centrifuged from cultured fluid/flour dough was determinedby incubating cultured fluids at 35℃ for 0, 70, 140, 210, and 280 min, and were treatedas above. The relationship between incubation time of cultured fluid and the amount of
the centrifuged liquid was investigated.

Statistical analysis
A statistical software package (SPSS Inc., Chicago, IL.) was used for statisticalanalyses. Experiments were repeated four times and the results were averaged. Analysis
produced significant F values by analysis of variance, followed by Duncan’s multiple
range test for comparison of means.

Results and Discussion
Effects of bread dough components on the amount of centrifuged liquid
It is necessary to determine which components of bread dough affect the amount ofcentrifuged liquid. Figure 1 lists the bread dough components (except for wheat flour)and their combinations, and shows the effects of the presence of each component andtheir combinations on the amount of centrifuged liquid. When the amount of centrifugedliquid from the nonfrozen and unstored complete bread dough was set as 100%, thenearest value 85%, was obtained in the nonfrozen and unstored doughs with the
combination of yeast and salt, followed by 42% with the combination of salt and sugar,
and 27% with salt alone. Others were all < 5%. From these results, we concluded thatsalt was an important component in bread dough for the centrifuged liquid. Yeast also
contributed to the centrifuged liquid when it was mixed with salt and, in this case, yeast
could use wheat carbohydrates as a carbon source even if sugar is absent, and thefermented products could contribute to the amount of centrifuged liquid. These changes
in the amount of the centrifuged liquid with bread dough components were also
observed in frozen-and-thawed dough samples. When samples were frozen-and-thawed48

after one day of storage, each bread dough showed almost the same trends. However, incase of yeast and yeast + sugar bread doughs, the amount of liquid centrifuged from thefrozen-and-thawed dough was greater than those from nonfrozen and unstored doughs.
To make clear the mechanism of centrifuged liquid from bread dough in the presence ofsalt and yeast, a new experiment with cultured fluid/flour dough, in which wheat flour
was excluded in the incubation period of 2 hr 20 min and added afterward, wasdesigned. The amounts of protein and carbohydrate in a centrifuged liquid from eachcombined bread dough with or without freezing-and-thawing were determined andcompared with the amount of centrifuged liquid from each (Table1). Results of these
correlation coefficients (r) indicates that the amount of the protein and carbohydrate ineach bread dough is highly related to the amount of the centrifuged liquid, and suggeststhat there are no higher or lower extraction of protein and carbohydrate in eachcentrifuged liquid. In bread dough, wheat flour was mixed in the bread dough andincubated for 2 hr 20 min at 38℃ and wheat flour enzymes such as amylases, proteases
could be activated in dough. So, it was possible that those enzymes in wheat flour could
affect the amount of centrifuged liquid. However, the amount of the resulting
centrifuged liquid from the nonfrozen complete cultured fluid (yeast + sugar +salt)/flour dough was almost the same as that from nonfrozen complete bread dough(yeast + sugar + salt + flour), which indicated that the enzymes in wheat flour were notrelated to the amount of liquid released. Then, it was ascertained that the presence of
wheat flour in incubation time did not relate to the amount of centrifuged liquid. So,
cultured fluid/flour dough was used in the experiments described below.

Centrifuged liquid from cultured fluid/flour doughAs various bread dough samples were listed in Fig.1, the various cultured
fluid/flour dough made with various components (yeast, salt, and sugar) and their
combinations are listed, and the amounts of the liquid centrifuged from each respective
nonfrozen and unstored cultured fluid/flour dough sample are indicated in Fig. 2. The

49

greatest difference in the amount of centrifuged liquid between nonfrozen and unstoredcultured fluid/flour dough and bread dough with the same combinations of components
was observed when yeast and salt were combined (yeast + salt) (Figs.1. and 2). In
cultured fluid/flour dough, the amount of centrifuged liquid from nonfrozen andunstored cultured fluid/flour dough was ~36% compared with nonfrozen and unstoredcomplete cultured fluid/flour dough (100%) (Fig.2), while in nonfrozen and unstored
bread dough made with yeast and salt, it was ~85% compared with nonfrozen andunstored complete bread dough (100%) (Fig.1). Sugar was omitted in both cases, so it
was suggested that yeast was able to use wheat carbohydrates in bread dough where theyeast was visible, but could not use yeast in the cultured fluid/flour dough because
wheat flour was omitted in incubation time. These observations suggested that the
products of fermentation may be key materials in increasing the amount of centrifugedliquid. When they were frozen-and-thawed after one day of storage, each dough sample
showed almost the same trends. Yeast + sugar cultured fluid/flour dough increased
remarkably by freezing and thawing. Salt alone or in combination with sugar in thecultured fluid/flour doughs showed higher levels of centrifuged liquid than the othercombinations examined for both nonfrozen and frozen-and-thawed doughs. The
amounts of protein and carbohydrate in the centrifuged liquid from each combined
cultured fluid/flour dough, with or without freezing-and-thawing were determined andcompared with the amount of centrifuged liquid (Table 1). Results of these correlation
coefficients (r) indicates that the amount of protein and carbohydrate in each cultured
fluid/flour dough is highly related to the amount of the centrifuged liquid, and suggeststhat there are also no higher or lower extraction results of protein and carbohydrate ineach centrifuged liquid.

50

51

52

Effects of fermented products and salt in cultured fluid/flour dough on centrifugedliquidIt has not yet been determined whether the liquid centrifuged from cultured
fluid/flour dough was increased by the presence of increased yeast metabolic products.
So, the relationship between incubation time (0, 70, 140, 210, and 280 min) of cultured
fluid and the amount of the centrifuged liquid was investigated. In Fig. 3B, the amountof centrifuged liquid increased linearly with incubation time of cultured fluid, whichsuggested that the increase in free water was due to increases in the amounts offermented products made by the yeast. Furthermore, the effect of level of salt additionon the centrifuged liquid was also observed (Fig. 3A, C, and D). When salt level wasincreased twofold in cultured fluid (Fig. 3A), the amount of centrifuged liquid wasincreased to a greater extent than that from normal level salt cultured fluid/flour dough(Fig. 3B). However, the centrifuged liquid increased even when incubation time waszero, which demonstrated an increasing effect of salt alone. When salt was omitted fromthe cultured fluid (Fig. 3D), no increase in amount of centrifuged liquid was observed
for any level of cultured fluid incubation time. However, when salt was added to thecultured fluid at the same time as mixing with wheat flour (Fig. 3C), the amount ofcentrifuged liquid from the dough was very similar to that from doughs made withnormal cultured fluid as indicated in Fig. 3B. These observations indicated thatfermented products are present after each cultured fluid incubation time without salt andhave the ability to increase the amount of liquid centrifuged from doughs. However, nocentrifuged liquid was observed without the presence of salt.

53Effect of Salt in Cultured Fluid/Flore Dough on Centrifuged Liquid
The relationship between salt and centrifuged liquid was examined in more detail.
As shown in Fig. 4, the addition of salt to cultured fluid at the same time as mixing withwheat flour increased the amount of centrifuged liquid to 3.63 g/145 g of wheat flour,
while further addition was associated with a gradual decrease. These observations
suggested that when salt was added to the doughs, loosely bound water was moved fromflour to salt, while further addition of salt may change the nature of the flour andsalt-bound water was reabsorbed into the wheat flour. Addition of 29 g of salt resulted
in complete loss of liquid release. The amount of protein and carbohydrate incentrifuged liquid from each salt adding cultured fluid/flour dough were determined,
and compared with the amount of centrifuged liquid. The correlation coefficients (r)54(Table 2) indicates high value between them, which indicates that the concentrations of
the extracted protein and carbohydrate did not change by salt addition.

Conclusion
Salt was an important component in bread dough for the centrifuged liquid. And
under the presence of salt, the amount of centrifuged liquid increased linearly withincubation time of cultured fluid, which suggested that the increase in free water was
due to increases in the amounts of fermented products made by the yeast.

55

References
1) American Association of Cereal Chemists. 2000. Approved Methods of the AACC,
10th Ed. Methods 08-01, 10-10A, and 54-21. The Association: St. Paul, MN.
2) Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the folin
phenol reagent. J. Biol. Chem. 193: 265-275, 1951.

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Chapter 6
Conclusions
The breadmaking properties (bread height (mm), and specific volume (cm 3/g)) of
breads baked from frozen-and-thawed doughs were markedly deteriorated. Centrifugedliquid which was observed on the frozen-and-thawed dough samples could bequantitatively analyzed, and high inverse correlations between the breadmaking
properties and centrifuged liquid were present (Chapter 2).

Water in frozen bread dough separated by thawing, and the amount of centrifugedliquid was higher than in controls. The thawed dough showed depressed breadmaking
properties. However, we could improve the properties to acceptable levels by theaddition of sugar and yeast to the thawed dough and then repeating first proof, mold,and second proof steps. The frozen and thawed dough showed smaller extension thanthe control under reduced pressure; however, the dough showed the same extension as
the control when the dough was reformed by the addition of sugar and yeast following
processing (Chapter 3).

It is generally known that breadmaking properties such as bread height (mm) andspecific volume (cm3/g) deteriorate upon freezing/thawing. At the same time, theexudation of liquid from thawed bread dough upon centrifugation increases. To preventthe deterioration of breadmaking properties by freezing/thawing, 13 kinds ofpolysaccharide were added to dough, and breadmaking tests were performed after
freezing/thawing. As a result, xanthan gum, guar gum, and tamarind seed gum wereshown to prevent the deterioration of breadmaking properties by freezing/thawing; inparticular, xanthan gum could ensure the remarkable recovery of bread fromfreezing/thawing effects (Chapter 4).

57

There is evidence that the amount of centrifuged liquid is highly related to both salt
and fermented products in the bread dough (Chapter 5).

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List of Publications
1) M. Seguchi, S. Nikaidoo, and N. Morimoto: Centrifuged liquid and breadmaking
properties of frozen-and-thawed bread dough. Cereal Chemistry 80: 264-268, 2003.
2) N. Morimoto and M. Seguchi: Restoration of breadmaking properties to frozen
dough by addition of sugar and yeast and subsequent processing. Cereal Chemistry
88: 409-413, 2011.
3) N. Morimoto, A. Tabara, and M. Seguchi: Effect of xanthan gum on improvementof bread height and specific volume upon baking with frozen and thawed dough.
Food Science and Technology Research 21: 309-316, 2015.
4) 森元直美, 田原彩, 瀬口正晴：冷凍ドウの製パン性劣化の原因と再ミックスによる製パン性回復について. 神戸女子大学家政学部紀要 48: 1-16, 2015.

59

Acknowledgments
I wish to express my deepest gratitude to my chief instructor, Prof. Dr. Masaharu
Seguchi, Graduate School of Home Economics, Kobe Women’s University, for his
valuable teaching and endless encouragement which have been indispensable to thecompletion of the present thesis.
I would like to offer my heartfelt thanks to the members of the supervisorycommittee, Prof. Dr. Masahiro Goto, Prof. Dr. Chihiro Yamane, and Assoc. Prof. Dr.
Mariko Kimura, Graduate School of Home Economics, Kobe Women’s University, andProf. Dr. Yasuki Matsumura, Graduate School of Agriculture, Kyoto University; without
their assistance, passionate guidance, and professional advice, this study would not havebeen successful.
I gratefully acknowledge to Aya Tabala and Seiko Nikkaidoo, for their help on
several subjects in this paper.
Special thanks to all members of Faculty of Home Economics, Kobe Women’s
University for their support.
Finally, I wish to express my gratitude to my family and friends for theirunderstanding and support.

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