LWT - Food Science and Technology 42 (2009) 14741483

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Combined effects of freezing rate, storage temperature and time on bread dough
and baking properties
Jinhee Yi a, William L. Kerr b, *
a
b

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA

Department Food Science and Technology, University of Georgia, Athens, GA 30602, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 October 2008
Received in revised form
13 May 2009
Accepted 15 May 2009

This study compares the effects of freezing temperature and rate as well as storage temperature and time
on the quality of frozen dough. Yeasted bread dough was frozen using four freezing rates (1969  C/h),
then stored at 10, 20, 30, or 35  C for up to 180 days. Dough strength diminished with longer
storage time and higher storage temperatures. Cryo-SEM showed that dough stored at 30 and 35  C
had the least damaged gluten network. NMR studies showed that more rapidly frozen dough, and that
stored at lower temperatures had lower transverse relaxation (T2) times (910 ms). However, dough
stored at 20  C displayed the highest yeast activity among samples. Bread loaf volume decreased with
storage time, and bread made from dough stored at 20  C showed the highest loaf volume. Breads
produced from 30 and 35  C stored dough displayed less change in the texture prole during storage
as well as less change in T2 values. Response surface analysis showed that optimal properties occurred at
freezing rates of around 1941  C/h and storage temperatures of 15 to 20  C.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Frozen dough
Stickiness
Extensibility
Bread volume

1. Introduction
Bread made from frozen dough has become an increasingly
popular alternative to that made directly from unfrozen dough.
Frozen dough can be manufactured in large quantities off-site, and
then shipped to local restaurants or retail operations, saving on
both equipment and labor costs. In recent years, the quality of these
products has improved owing to advances in technology and
formulation, but there is room for additional improvement. Problems associated with frozen dough include long proof time, low
volume, poor texture, and variable performance (Kenny, Wehrle,
Dennehy, & Arendt, 1999). Some of the poorer quality can be
attributed to diminished yeast activity, the characteristics of the
yeast and their survival after freezing (Baguena, Soriano, Martinezanaya, & Debarber, 1991; El-Hady, ElSamahy, Seibel, & Brummer,
1996; Hino, Takano, & Tanaka, 1987; Hsu, Hoseney, & Seib, 1979;
Ribotta, Leon, & Anon, 2003; Wolt & DAppolonia, 1984). In order to
improve performance, frozen dough processors may add extra
yeast, use short or no-time dough processing procedures, mix
ingredients at relatively low temperatures, or incorporate new
strains of freeze-tolerant yeasts.

* Corresponding author. Tel.: 1 706 542 1085; fax: 1 706 542 1050.
E-mail address: wlkerr@uga.edu (W.L. Kerr).
0023-6438/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2009.05.017

During freezing, there are also deleterious effects on dough

structure. Electron microscopy studies (Esselink, Aalst, Maliepaard,
Henderson, et al., 2003) showed a breakdown of the reticular
gluten network when subjected to freezing and thawing, and
related these to changes in the rheological properties of the dough.
Ice recrystallization and water migration in the dough also affects
the dough structure (Bache & Donald, 1998; Rojas, Rosell, Benedito
de Barber, Perez-Munuera, & Lluch, 2000). Extended frozen storage
alters the gluten protein matrix (Berglund, Shelton, & Freeman,
1991; Varrianomarston, Hsu, & Mahdi, 1980), results in dough
weakening, and causes less gas retention (Autio & Sinda, 1992; Hsu
et al., 1979; Inoue & Bushuk, 1991). In addition, injury to yeast
membranes caused by freezing and thawing release cellular
chemicals that have deleterious effects on the dough structure.
However, researchers have different opinions on the effects of
specic leachates, mainly reducing compounds such as glutathione, on the gluten network (Casey & Foy, 1995; Inoue & Bushuk,
1991; Wolt & DAppolonia, 1984).
While several researchers have studied the effects of storage
time or temperature on yeast viability and dough structure
(El-Hady et al., 1996; Havet, Mankai, & Le Bail, 2000; Mazur, 1961),
less has been done on the inuence of freezing rate. Especially
in frozen dough preparations, where freezing and sometimes
prolonged frozen storage intervene between dough formation and
bread baking, several factors still have not been fully investigated
(Giannou & Tzia, 2007). In the present study, we examined the

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

combined effects of freezing rate, storage temperature and storage

time on dough and bread quality. The dough properties were
evaluated based on dough extensibility and adhesiveness, and the
viability of yeast assayed through measurements of gas production.
Both volume and rmness were measured in the nished breads.
Direct examination of the dough in the frozen state was accomplished using cryogenic scanning electron microscopy (cryo-SEM),
allowing visualization of the ultra-structure of gluten-starch
association and the state of gluten strands in the network. We also
used time-domain NMR to investigate changes in water dynamics
(Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Roman-Gutierrez,
Guilbert, & Cuq, 2002; Ruan et al., 1999).
2. Materials and methods
2.1. Dough preparation and freezing
Bread dough was prepared from enriched, bleached hard winter
wheat our (Organic Select Artisan Flour, King Arthur Company,
Norwich, Vermont) with 11.5 g/100 g protein content; Bakers dry
yeast (Fleischmanns Yeast, Quebec, Canada); Dominion pure cane
sugar (Dixie Crystal, Savannah, GA); non-iodized salt (Morton
International, Inc., Chicago, IL); and soybean oil (Wesson Vegetable
Oil, ConAgra Foods, Omaha, Nebraska). The basic formulation was
(per 100 g our weight): water (60 g/100 g), sugar (5 g/100 g), oil
(5 g/100 g), yeast (3 g/100 g), and salt (2 g/100 g). The yeast was
prehydrated with the water, and then all of the dough ingredients
were placed in a 6-quart, 575 W mixer (Kitchen Aid Professional
600 Series, St. Joseph, MI) and mixed for 2 min at 120 rpm with
a paddle, and for 8 min at 178 rmp with a dough hook. Once the
dough was formed, it was separated into samples of 50 g, and
shaped by hand into approximately 50 mm diameter circular slabs.
Dough pieces were frozen in a built-in forced-air blast freezer
located at the UGA Department of Food Science and Technology in
Athens, GA. As part of the experimental design, the freezer was
adjusted to give four different freezing rate schemes, as shown by
the temperature-time plots in Fig. 1. Specic freezing times and
rates depend on the conditions and nal temperature. The average
rate (and time) to reach 30  C were: Rate 1: 19  C/h (2.53 h); Rate
2: 41  C/h (1.18 h); Rate 3: 55  C/h (0.88 h); and Rate 4: 72  C/h
(0.67 h). In terms of the time to go from the initial freezing point to
30  C, these times were: Rate 1: 2.27 h; Rate 2: 0.95 h; Rate 3:
0.62 h; and Rate 4: 0.45 h.
After freezing to the desired temperature, dough samples were
vacuum-packaged in plastic bags and placed at four different frozen
storage temperatures (10, 20, 30 or 35  C), and stored for 30,

1475

60, 90, and 180 days. At each sampling period, 3 replications of the
16 treatments were withdrawn from the freezers and placed in an
environmental chamber (Model HEC10R, HotPack, Warminster, PA)
at 8  C and 35% RH in order to thaw. Thawed dough pieces were
removed from the package, then placed in a chamber at 36  C and
85% RH for proong. Dough samples were baked in a 5-rack gas
convection oven (Blodgett BLD-DFG100, Burlington, Vermont) at
180  C for 15 min. After baking, bread samples were allowed to cool
for approximately 1 h prior to subsequent measurements.
2.2. Dough extensibility and adhesiveness
Extensibility of the dough was measured using a texture
analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) with
a modied Kieffer extensibility rig and 5 kg load cell. Approximately 50 g dough samples were molded into strips approximately
7 mm in diameter and 60 mm in length. All samples were left to
rest on a grooved plate at 8  C for 20 min and 90% RH prior to
testing (Anderssen, Bekes, Gras, Nikolov, & Wood, 2004). The dough
was pulled at a crosshead speed of 3.3 mm/s. The resistance to
extension (maximum force) and extensibility (distance to break)
were calculated from the forcedeformation curves. One advantage
of the Kieffer dough extensibility rig is that it uses a micro-extension method involving a very small sample size. It correlates highly
with methods such as the extensigraph as indicated by baking
performance (Kieffer, Wieser, Henderson, & Graveland, 1998;
Sharadanant & Khan, 2003; Suchy, Lukow, & Ingelin, 2000).
Adhesiveness was measured using the texture analyzer with
a modied ChenHoseney stickiness rig, with conditions as
described by Chen and Hoseney (1995). The sample was placed in
a cylindrical cell on the base of the texture analyzer, which was
then enclosed by a lid with a perforated hole. A small amount of
dough was extruded through the hole. The upper cylindrical probe
was brought in contact with the exposed dough to adhere to it, and
the probe was pulled away from the base at a speed of 1.7 mm/s.
Both the maximum force and the area under the forcedeformation
curve required to separate the probe from the test sample were
used as measures of adhesiveness.
2.3. Cryo scanning electron microscopy
A Jeol JSM-5410 scanning electron microscope with a CT-500C
cryo-unit (Oxford Instruments, Oxfordshire, UK) was used to
investigate the microstructure of the frozen bread dough. Each
frozen dough sample was placed on the cryo-specimen holder,
placed in liquid nitrogen, and then transferred to the cryo-unit in
the frozen state. The dough specimens were fractured, sublimated
(10 min at 70  C) and sputter coated with gold (4 min at 0.2 kPa).
The prepared dough specimen was transferred to the microscope
where it was observed at 15 kV and 120  C. Micrographs were
taken at 500, 1000 and 2000 magnication.
2.4. NMR measurements of dough

Fig. 1. Freezing protocols for frozen bread dough. Average rate (and time) to reach
30  C. Rate 1: 19  C/h (2.53 h); Rate 2: 41  C/h (1.18 h); Rate 3: 55  C/h (0.88 h); and
Rate 4: 72  C/h (0.67 h).

Proton relaxation measurements were made using a 20 MHz

Proton (1H) NMR spectrometer (Resonance Instruments, Whitney,
UK). Approximately 5 g dough samples were taken from the center
of the thawed dough. Three replicates were taken from each piece
of dough. Each sample was placed in a 10 mm diameter glass tube
then covered with paralm. The glass tube was placed in a 10  C
bath for 20 min. After 20 min, the glass tubes with dough specimens were placed in 18 mm diameter NMR tubes. Transverse (T2)
relaxation curves were developed using the CPMG pulse sequence:
90x(s-180y  s-echo)n. Acquisition parameters were set to a 90
pulse of 4.2 ms and a recycle delay of 2 s. A pulse spacing (s) of

1476

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

100 ms was chosen to exclude the fast decaying solid-like signal. All
measurements were made at 25  C  1  C. Relaxation curves were
analyzed using the WinFIT multi-exponential routine (Resonance
Instruments, Whitney, UK).
2.5. Yeast activity
Yeast activity was measured using AACC method 89-01 (2000)
with slight modication. Dough pieces (10 g) and 100 g water were
placed into a 250 ml glass reaction vessel, capped, and placed in
a water bath at 36  1  C. A hose was connected to the vessel,
leading to a volumetric manometer. Gassing power was determined from the volume of carbon dioxide gas trapped after
180 min. Each 10 g sample contained 0.163 g dry yeast.

Table 1
Maximum resistance to extension of frozen bread dough (force, g) after thawing.
Values for unfrozen dough: 69.98 g.
Storage day

Freezing rate

10  C

20  C

30  C

35  C

30

Rate
Rate
Rate
Rate

1
2
3
4

49.68op
51.19pq
52.19r
52.75s

53.28st
54.01t
55.60vw
56.05wx

54.33tu
54.19t
56.99y
56.41xy

54.73uv
55.25v
55.74w
56.02wx

60

Rate
Rate
Rate
Rate

1
2
3
4

47.01kl
47.62m
48.89no
48.70n

49.62o
50.38p
52.62rs
53.38st

51.77q
51.86qr
52.65rs
52.74s

51.11pq
52.25rs
51.45q
52.22rs

90

Rate
Rate
Rate
Rate

1
2
3
4

43.78h
44.84hi
45.11i
45.71j

45.45ij
46.34jk
47.31lm
47.30lm

45.33i
47.50m
47.73
46.64k

45.38
47.73m
47.02kl
47.32lm

180

Rate
Rate
Rate
Rate

1
2
3
4

29.17a
29.54ab
29.49a
29.71ab

30.15bc
33.68fg
32.64def
32.39de

31.99cd
33.19fg
32.82ef
32.67ef

32.01d
33.06f
33.12fg
32.13de

2.6. Bread volume

Loaf volume and weight were determined after baking and
cooling for 1 h at ambient temperature (AACC 10-05, 2000). A
container was lled with a known volume of rapeseed. The amount
of seed displaced when the loaf was introduced was measured in
a graduated cylinder, and measured the loaf volume.
2.7. Bread crumb rmness
The rmness of the bread samples was determined using
a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale,
NY) with a 36 mm radius cylinder probe according to AACC Method
74-10A (2000). Three center slices of each loaf were measured at
25  C. The compression rate was set at 1.7 mm/s. Firmness was the
force required to compress the slices (20 mm thick) by 40% strain.
2.8. Statistical analysis
ANOVA was used to analyze all data, based on a three-way
mixed design. Fishers LSD was used to determine signicant
differences between samples. A p-value of less than 0.05 was
considered signicant. A response surface methodology was used
to examine regions of minimal or maximal response, particularly to
see if there were similar regions of optimal response for the various
response variables. Models surfaces were t using linear, squared,
and crossed terms using the JMP 7 software (SAS, Cary, NC).
Contour plots were developed to where the response reached 90%
of its minimum or maximum value.
3. Results and discussion
3.1. Dough extensibility and adhesiveness

Storage temperature

Values followed by different superscript letters are signicantly different at p < 0.05.

of frozen dough. Freezing rate 2 produced dough with the highest

extensibility, while those from freezing rate 1 had the lowest
extensibility. Dough from freezing rate 1 also had lower MRE than
those from other freezing rates.
Frozen storage temperature also had some effect on dough
extensibility. With lower frozen storage temperature (closer to
35  C), the extensibility increased but there was no signicant
difference between rates 3 and 4 (the fastest rates). The interaction
between the freezing rate and frozen storage temperature determined extensibility and MRE. For the rst 30 days, dough prepared
at freezing rate 4 (the fastest freezing) and stored at 35  C had the
highest extensibility (81.0 mm); dough prepared at freezing rate 1
and stored at 10  C had the lowest extensibility (74.45 mm). After
180 days, dough prepared at freezing rate 2 and stored at 35  C had
the highest extensibility (71.39 mm); those produced at freezing
rate 1 and stored at 10  C had the lowest extensibility (49.94 mm).
Decreases in maximum resistance, and in extensibility may
indicate deterioration in the gluten network. Extensibility has been
attributed to the gluten structure, and particularly to high molecular weight glutenins (Belton, 1999; Bushuk & Macritchie, 1989;
Table 2
Extensibility of frozen bread dough (distance to peak, mm) after thawing. Values for
unfrozen dough: 84.99 mm.
Storage day

Freezing rate

Storage temperature
10  C

Tables 1 and 2 show the maximum resistance to extension

(MRE, in g force) and extensibility (in mm) of dough subject to
different freezing and storage conditions. For the control (a freshly
made, unfrozen dough), MRE was 69.98 g with an extensibility of
84.99 mm. All dough samples subject to freezing had lower resistance and extensibility than the control. Both the MRE and the
extensibility decreased with time from 0 to 180 days of frozen
storage. For example, maximum resistance ranged from 49.68 to
56.99 g at 30 days, and from 29.17 to 33.68 g at 180 days. Similarly,
extensibility ranged from 74.45 to 81.00 mm at 30 days, and from
49.94 to 71.39 mm at 180 days. Storage time had a greater inuence
on extensibility than temperature. For samples prepared at freezing
rate 1 (the slowest rate), and held for 30 days, extensibility varied
from 74.45 mm (at 10  C) to 76.84 mm (at 35  C). After 180 days
of storage, extensibility ranged from 49.94 mm (at 10  C) to
70.04 mm (at 35  C). Freezing rate also affected the extensibility

qr

20  C
vw

30  C
wx

35  C

30

Rate
Rate
Rate
Rate

1
2
3
4

74.45
76.12uv
75.86tuv
75.22rs

76.23
78.10AB
77.42xy
77.69yzA

77.19
79.58BC
80.18CD
79.95CD

76.84vw
80.92D
80.99D
81.00D

60

Rate
Rate
Rate
Rate

1
2
3
4

69.81hi
72.90no
71.64lmn
70.96kl

73.37nop
76.09uv
75.23rs
75.30rs

75.28rs
77.45xy
76.93w
77.30wx

73.46op
78.81AB
77.49xy
77.93zA

90

Rate
Rate
Rate
Rate

1
2
3
4

63.94c
67.10e
66.66de
65.75d

72.59mn
73.58op
73.74pq
74.63qr

73.94pq
75.76tu
74.93rs
75.63st

74.71r
75.81tuv
75.15rs
75.42st

180

Rate
Rate
Rate
Rate

1
2
3
4

49.94a
52.43b
52.00b
52.42b

67.59ef
69.24g
68.38f
69.66gh

69.19g
71.07kl
70.02hij
70.78jkl

70.04ij
71.39lmn
70.30ij
70.48jk

Values followed by different superscript letters are signicantly different at p < 0.05.

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

Lindsay & Skerritt, 1999). While disulde bonds help to maintain

the gluten network and provide resistance to stretching, several
theories have been developed to explain dough elasticity (Belton,
1999; MacRitchie & Laandra, 1997). Our results indicate that
relatively rapid freezing and low storage temperatures result in the
least changes to the gluten structure. Most deleterious were slow
freezing rates, high storage temperature, and prolonged storage
times. Rapid freezing and low storage temperatures promote the
creation of a greater number of small ice crystals, due to enhanced
nucleation and crystal growth rates. Low freezing rate promotes the
growth of relatively large crystals. It has been suggested that ice
crystals physically disrupt and damage the gluten network, and
dehydrate the gluten so as to cause irreversible structural changes.
Extensibility determines the ability of the dough to extend
during gas production by yeast during proong. Excessively high
extensibility results in weak and slack dough, which collapses
during the proong stage or while baking in the oven (Sharadanant
& Khan, 2003). MRE measures the ability of the dough to retain gas
and subsequently to form springy bread. A very low resistance to
extension results in poor gas retention and lower loaf volume. A
very high resistance to extension also results in a lower loaf volume
because the tough dough is not capable of proong to an optimum
height with the gas produced by the yeast.
Contrary to our ndings, Inoue and Bushuk (1991) reported that
extensibility increased with storage time for one week but no clear
trend was observed. However, Sharadanant and Khan (2003)
reported that extensibility increased with extended storage time
and maximum resistance to extension decreased with storage time.
Table 3 shows the adhesiveness of dough samples prepared at
different freezing rates, storage temperatures and storage times.
Adhesiveness of unfrozen dough was 69.08 g. Adhesiveness of all
samples from frozen dough were higher than that of control,
indicating more sticky dough. In general, dough adhesiveness
increased with storage time. For example, at 30 days adhesiveness
ranged from 74.77 to 80.77 g, while at 180 days values ranged from
93.25 to 115.63 g. Storage temperature was not a signicant factor
for samples stored at 30 and 60 days, but was for samples stored at
90 and 180 days. At 90 and 180 days, samples stored at lower
temperatures were less adhesive.
Freezing rate was also a signicant factor for adhesiveness,
except for samples stored at 30 days. Samples prepared at freezing
rate 2 were less adhesive; those at freezing rate 1 had the highest
adhesiveness. In general, adhesiveness values followed a similar
Table 3
Adhesiveness of frozen bread dough (tension force, g) after thawing. Values for
unfrozen dough: 69.08 g.
Storage day

Freezing rate

Storage temperature
10  C
fg

20  C
cd

30  C

35  C

ef

30

Rate
Rate
Rate
Rate

1
2
3
4

79.47
75.04b
77.49c
78.73def

78.03
77.20c
81.00hi
80.77ghi

79.30
78.22def
80.10gh
79.28ef

78.07cd
75.12b
74.77a
77.82cd

60

Rate
Rate
Rate
Rate

1
2
3
4

82.78klm
77.88cd
82.21jk
82.01jk

88.39mn
78.81ef
83.37lm
82.37jkl

87.70mn
78.16de
80.30gh
82.00jk

83.15lm
81.63ij
82.41kl
80.93hi

90

Rate
Rate
Rate
Rate

1
2
3
4

108.45BC
105.86zA
107.84B
106.24A

97.48x
91.99p
92.36qr
92.37qr

94.37tu
90.83o
91.97p
92.00pq

93.15rs
91.63p
92.41qr
93.43rs

180

Rate
Rate
Rate
Rate

1
2
3
4

115.63E
114.47DE
115.52E
113.74D

109.60C
98.83y
108.69BC
105.32z

97.23x
93.25rs
94.44uv
94.25tu

95.19vw
94.34tu
94.02st
93.46rst

Values followed by different superscript letters are signicantly different at p < 0.05.

1477

dependence as extensibility. Samples stored at 35  C for short

times (30 days) were more extensible (with higher force) and less
adhesive. Samples stored for long time at low temperature (10  C)
were less extensible (with lower maximum force) and were more
adhesive.
Adhesiveness is related to the force required to separate
a material from a dissimilar surface. As discussed previously,
freezing of dough and frozen storage may cause separation of water
from the gluten network, particularly as water is redistributed due
to ice recrystallization. This water may be more feely assessable at
surfaces, leading to increased adhesiveness.
Samples prepared at freezing rate 2 were less adhesive than
those at other freezing rates. Several researchers have reported that
dead yeast cells produce reducing substances that cause disruption
of molecular bonds in the gluten network. It will be shown in
a subsequent section that highest yeast activity incurred for
samples prepared at freezing rate 2. One possibility is that fewer
reducing substances are produced at freezing rate 2, as there are
more viable yeast cells, and consequently less deterioration of the
gluten network. This in turn results in less migration of water after
freezing and thawing, and thus less adhesiveness.
3.2. Microstructure of frozen bread dough
Cryo-SEM was used to visualize the microstructure of frozen
dough (in the frozen state). The frozen dough structure at 90 days
showed the typical structure of starch granules embedded in
a gluten network (Figs. 25). All frozen dough samples had voids
among the gluten network and starch granules. The gluten strands
composing the network appeared to be quite damaged after 90 days.
Dough produced at freezing rate 1 and 2 (the slowest rates) were
less uniform and had thinner strands. Dough produced at freezing
rates 3 and 4 (more rapid freezing) showed less disrupted structure.
Storage temperature had a more notable effect on the gluten
network in the dough. With lower storage temperature (closer to
35  C), the dough was more intact and had a more uniform gluten
network. Regardless of freezing rate, dough stored at 35  C had
less disrupted gluten strands than that stored at 10  C.
Microstructural observations help explain the observed rheological properties of dough. As noted, dough stored at higher
temperatures and for longer times was less extensible, and more
adhesive, than dough stored at low temperatures for shorter times.
Lower extensibility can be attributed to the thinner, more disrupted
bonding in the gluten. As previously discussed, this disrupted
structure is also less able to hold water, and thus more adhesive.
Dough microstructure has been investigated by several
researchers. Ribotta, Perez, Leon, and Anon (2004) studied the
effect of emulsiers on frozen bread dough and described the
effects of extended frozen storage on the protein matrices. After
long storage time, the gluten matrix was quite damaged. Rojas et al.
(2000) described dough as a continuous gluten matrix, with starch
granules scattered among the protein network. Discontinuities
were observed in the lax matrix surrounding the starch granules
under greater magnication. Berglund et al. (1991) found that after
24 weeks frozen storage, the gluten matrix had thinner strands,
seemed more disrupted, and was separated from the starch granules. Nicolas et al. (2003) observed that during freezing, ice crystals
appear to compress the gluten, leading to a signicant phase
separation between the gluten and ice.
3.3. NMR relaxometry
Pulsed 1H NMR was used to investigate the relaxation characteristics of dough systems subject to different freezing rate and
storage conditions. Table 4 shows the transverse relaxation times

1478

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

Fig. 2. Cryo scanning electron microscopy dough pieces stored for 90 days at 10  C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.

for the thawed dough samples. The T2 relaxation time increased

somewhat with increased frozen storage time at all storage
temperatures. At 30 days, T2 values ranged from 15.39 to 16.45 ms,
while at 180 days T2 values ranged from 17.56 to 18.71 ms. In most
cases, the T2 times were smallest for samples at freezing rate 2.
Frozen storage temperature also had a signicant, but small, effect
on transverse relaxation times. Samples stored at lower

temperature had somewhat shorter T2 values. For example, at 30

days samples held at 10  C had values ranging from 16.28 to 16.42,
while at 35  C values ranged from 15.39 to 15.93 ms.
The amount and state of water play an important role in the
preparation and properties of wheat our dough and their products. Ruan et al. (1999) used several methods for presentation and
analysis of relaxation time measurement of protons in dough, and

Fig. 3. Cryo scanning electron microscopy dough pieces stored for 90 days at 20  C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

1479

Fig. 4. Cryo scanning electron microscopy dough pieces stored for 90 days at 30  C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.

suggested there is a continuous distribution of protons having

different relaxation times in heterogeneous systems such as dough
(Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Esselink, Aalst,
Maliepaard, Henderson, et al., 2003; Ruan et al., 1999). Timedomain NMR techniques have demonstrated that at high water
contents, water is no longer bound in the physical sense. However,

the T2 relaxation times of this water population are still small due
to hydrogen exchange with surface hydroxyl groups. In gluten,
generally much longer T2 relaxation times are observed, and this
has been attributed to a higher degree of water mobility (Cherian &
Chinachoti, 1996; Esselink, Aalst, Maliepaard, & Duynhoven, 2003;
Esselink, Aalst, Maliepaard, Henderson, et al., 2003).

Fig. 5. Cryo scanning electron microscopy dough pieces stored for 90 days at 35  C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.

1480

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

Table 4
Transverse relaxation time (T2, ms) of frozen bread dough after thawing.
Storage day

Freezing rate

Storage temperature
10  C

20  C

30  C

35  C

30

Rate
Rate
Rate
Rate

1
2
3
4

16.42
16.35ef
16.28d
16.35ef

16.32
16.01bc
16.25cd
16.45f

16.01
16.35ef
16.37ef
16.21cd

15.93
15.52a
15.66ab
15.39a

60

Rate
Rate
Rate
Rate

1
2
3
4

16.91j
16.76h
16.82hi
16.89ij

16.98jk
16.59g
16.87ij
16.93j

16.84i
16.43f
16.79h
17.06kl

16.73h
16.42f
16.80h
16.62g

90

Rate
Rate
Rate
Rate

1
2
3
4

17.84o
18.01pq
18.20rs
17.61m

17.96pq
17.65m
17.82o
17.67mn

17.50m
17.85o
17.15l
17.51m

17.80o
17.73n
17.60m
17.84o

180

Rate
Rate
Rate
Rate

1
2
3
4

18.59tu
18.46t
18.71w
18.54t

17.56m
18.15r
18.30s
18.03q

18.65uv
18.31st
18.00pq
17.89op

18.00pq
17.78no
17.62m
17.91pq

Values followed by different superscript letters are signicantly different at p < 0.05.

In general, longer relaxation times indicate the presence of more

mobile water, or at least water that has less restricted rotational
freedom. In our studies, T2 values were greatest at 180 days storage
at 10  C, and least at 30 days storage at 35  C. At 180 days at
10  C, we also found the least dough extensibility and maximum
dough stickiness, while at 30 days storage at 35  C we found
maximum extensibility and the least stickiness. This suggests that
changes in T2 values are most related to changes in the gluten and
starch network. As discussed previously, greater changes in the
gluten structure and separation of water occur at high storage
temperatures and longer storage times. This greater mobility of
water once removed from the vicinity of gluten could well explain
the increased T2 values.
3.4. Yeast activity and gas production
Table 5 presents the total gas production for thawed dough
slurries after 180 min at 36  C. Yeast activity in frozen dough
produced under all freezing and storage conditions was lower than
that for an unfrozen control. For example, total gas was 112 ml/g

Table 5
Yeast activity (ml CO2 gas/g dry yeast) in frozen dough after thawing. Values for
unfrozen dough: 147.0 ml/g dry yeast.
Storage day

Freezing rate

Storage temperature
10  C
qr

20  C
s

30  C
q

35  C

30

Rate
Rate
Rate
Rate

1
2
3
4

75.6
84.0s
61.2mn
58.8lm

86.4
95.4t
64.8op
60.0no

74.4
74.4q
57.0lm
55.2k

73.2q
73.2q
55.2k
56.4klm

60

Rate
Rate
Rate
Rate

1
2
3
4

64.8op
72.0q
54.0k
48.6gh

75.6qr
84.6s
56.4klm
51.6ij

63.0no
64.2nop
49.2hi
49.2hi

61.2mn
62.4n
46.8fg
48.0gh

90

Rate
Rate
Rate
Rate

1
2
3
4

51.0ij
56.4klm
43.2ef
40.8d

55.8kl
63.0no
50.4hij
50.4hij

48.6gh
55.8kl
42.6ef
41.4de

47.4fgh
54.6k
41.4de
41.4de

180

Rate
Rate
Rate
Rate

1
2
3
4

43.2ef
46.8fg
36.6bc
34.2a

47.4fgh
51.6ij
41.4de
39.6bcd

41.4de
47.4fgh
36.0ab
35.4a

40.8d
46.2f
34.8a
36.0ab

Values followed by different superscript letters are signicantly different at p < 0.05.

dry yeast for unfrozen dough as compared to 95.4 ml/g for frozen
dough produced under freezing rate 2 and stored at 20  C for 30
days. In general, the total gas production decreased with longer
storage periods. The highest gas production at 30 days was
obtained from frozen dough produced at freezing rate 2 and stored
at 20  C storage temperature (95.4 ml/g), and the lowest from
dough produced at freezing rates 3 and 4 (most rapid freezing) and
stored at 30 or 35  C (55.257.0 ml/g). Dough frozen at rates 1
and 2 (relatively slow freezing) and stored at 10 and 20  C had
higher gas production for all frozen dough samples. This same
relative gas production was maintained for all storage periods.
However, the total gas volume obtained was lower at longer storage
periods at all freezing rates and storage temperatures. The highest
gas volume for frozen dough was 84.6 ml/g at 60 days storage,
63.0 ml/g at 90 days, and 51.6 ml/g ml at 180 days; the lowest
volume was 46.8 ml/g at 60 days, 40.8 ml/g at 90 days, and 34.2 ml/
g at 180 days. Greatest volumes were obtained from dough
produced at freezing rate 2.
In almost all cases, maximum gassing power was observed for
dough samples held at 20  C, particularly at freezing rates 2 or 1
(41  C or 19  C/h). This temperature is often recommended for
storage in industrial settings, although the reasons for this are
somewhat empirical (Trevedi, Hauser, Nagodawithana, & Reed,
1989). It has been observed by several researchers that yeast
activity is diminished by freezing and storage (Autio & Sinda, 1992;
El-Hady et al., 1996; Neyreneuf & Delpuech, 1993; Wolt & DAppolonia, 1984). Fluctuations in temperature have been found to be
especially damaging (Berglund et al., 1991; El-Hady et al., 1996;
Inoue & Bushuk, 1991). In addition, yeast may be less viable after
freezing (Baguena et al., 1991).
Several factors inuence yeast activity in frozen dough,
including the inuence of temperature on metabolism, osmotic
stress incurred as ice forms, the ability of yeast to form osmoregulating or other protective compounds, and the effects that
freeze-damage of the gluten network may play in limiting diffusion
of nutrients and byproducts to and from metabolizing yeast. Lower
temperature per se should improve the survival of yeast. However,
in the frozen state, temperature determines the amount of ice
present in the system, and the rate of freezing determines how the
freezing process ensues. Early work (Mazur, 1970; Mazur &
Schmidt, 1968) showed that optimal survival of Saccharomyces
cerevisiae occurs at cooling rates of 7  C/min. It was suggested that
water in yeast cells remains supercooled down to 10  C, as the cell
membrane prevents extracellular ice from propagating into the cell.
At relatively slow freezing rates this allows time for water to diffuse
from the cytoplasm to external spaces, due to the osmotic gradient.
Concentration of intercellular solutes and electrolytes leads to
dehydration and changes in ionic strength and pH that affect the
metabolic functions of the cell and integrity of the cell membrane.
At rates faster than 10  C/min, less time is allowed for cell dehydration, and intercellular freezing is reached more quickly. This also
contributes to freeze concentration of solutes in the cell. Interestingly, much of the cell water is frozen at 20  C.
One might expect that faster freezing rates and lower storage
temperatures would be more benecial to yeast survival.
However, Mazur (1970) postulated that in these conditions,
intercellular ice is subject to recrystallization during warming, and
that this leads to direct damage of cell membranes. Alternately,
Muldrew and McGann (1990) speculated that rapid cooling leads
to greater osmotic pressure gradients across the membrane,
rupturing the membrane and allowing ice to propagate into the
cell. In any event, thawing is also a critical determinant of yeast
survival. For example, survival in slowly cooled-slowly warmed
systems is greater than in slowly cooled-rapidly warmed systems
(Mazur & Schmidt, 1968).

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

The direct effect of temperature on yeast cell membranes

also cannot be discounted. Lower temperature can lead to phase
transitions and loss of uidity of the lipid bilayer, and alter proteinbilayer interactions (Morris & Clark, 1987). Thermotropic transitions have been measured in a variety of unicellular organisms and
various lesions observed during freezing. The membrane can
become leaky at low temperature. For example, liposomal models
progressively released more glucose at temperatures between
0 and 25  C (Morris & McGrath, 1981). The amount released and
response to temperature depended on the lipid composition of the
membrane.
A few other factors should also be considered in understanding
survival and activity of yeast in frozen dough. First, most of the
above studies are done in isolated systems. Yeast in dough will be
subject to a different physical and osmotic environment than in
compressed yeast (Ribotta et al., 2003). Optimal freezing rates
in 2dough are typically below 2  C/min as compared to 7  C/min in
isolated yeast cultures (Gelinas, Deaudelin, & Grenier, 1995). In our
work, rates around 0.861.2  C/min provided optimal yeast activity.
In addition, S. cerevisiae have developed mechanisms to combat
stress conditions. The accumulation of trehalose in the cell seems to
be particularly important in combating the osmotic stresses of
freezing and drying, as well as stabilizing the cell membrane (Hino,
Mihara, Nakashima, & Takano, 1990; Majara, Oconnor-Cox, & Axcell,
1996; Van Dijck, Colavizza, Smet, & Thevelein, 1995). Indeed, much
work is ongoing in producing S. cerevisiae with elevated trehalose,
as by disrupting the ATH1 gene that produces trehalose-hydrolyzing enzyme (Kim, Alizadeh, Harding, Hafner-Gravink, & Klionsky, 1996). In addition, membrane proteins such as aquaporin also
help transport water across the yeast cell membrane, and it is
thought that this controlled efux of water during the freezing
process reduces intracellular ice crystal formation and resulting cell
damage (Tanghe et al., 2002). Rapid freezing may limit the ability of
yeast to adapt to freezing stress. On the whole, relatively slow
freezing rates are optimal for maintaining yeast activity after
thawing (El-Hady et al., 1996; Havet et al., 2000; Lorenz, 1974).
3.5. Bread volume
Table 6 shows the volume of bread made from the various frozen
dough treatments. The volume of bread made from unfrozen
control dough was 137.8 ml. All breads made from frozen dough

1481

had lower volumes. Typically, greatest loaf volumes were attained

from dough frozen at rate 2. However, freezing rate was not a strong
determinant for loaf volume. For example, after 10  C and 30 days,
loaf volume varied only from 111.7 to 113.3 ml, while at 35  C it
varied from 91.7 to 94.7 ml. Similarly, for dough stored for 180 days
at 10  C loaf volume varied from 86.3 to 90.0 ml. Greatest overall
loaf volume (119.0 ml) was seen with bread made from dough
frozen at rate 2, and stored at 20  C for 30 days, the same
conditions found for greatest yeast activity. Lowest volumes were
found with bread made from dough frozen at rates 3 or 4 and stored
at 30 or 35  C (83.784.0 ml at 180 days). There was no significant difference between freezing rate 3 and 4 for all storage times.
Loaf volumes for all breads decreased with increased dough
storage time. For example, at 10  C volumes decreased from 111.7
to 113.3 ml at 10  C and 30 days to 86.390.0 ml after 180 days. At
storage temperatures of 30 and 35  C, the effect of storage time
was less pronounced. After 180 days, bread volumes from samples
frozen at rate 2, and held at 10 or 20  C, were slightly higher
than the other treatments, but there were no signicant differences
amongst volumes for all other samples held for 180 days.
Storage temperature also had a major inuence on subsequent
loaf volume. In general, samples held at 20  C produced bread
with greatest loaf volume, although storage temperature effects
interacted with storage time as described above. Again, this
seems to reect, at least in part, the effects of treatment on yeast
activity.

3.6. Bread crumb rmness

Table 7 shows crumb rmness for bread made from frozen
dough. Measured rmness was 4.03 N for bread made from
unfrozen control dough. For that made from frozen/thawed dough,
crumb rmness increased signicantly with frozen storage time of
the dough. For example, at 20  C rmness values ranged from
5.99 to 6.88 N at 30 days, and from 7.76 to 8.04 N at 180 days. In
general, bread made from dough frozen at rates 1 and 2 was less
rm than bread made from dough frozen at rates 3 and 4 for all
storage times. The storage temperature also had a signicant effect
on bread rmness. All dough samples stored at 10 and 20  C
produced bread that was less rm than other samples for all storage
times. At 30 and 60 days, bread made from dough stored at 10  C
was less rm than that from dough stored at 20  C.

Table 6
Volume of bread (ml) made from frozen dough. Values of bread from unfrozen
dough: 137.8 ml.

Table 7
Firmness (force, N) of bread made from frozen bread dough. Values of bread from
unfrozen dough: 4.03 N.

Storage day

storage day

Freezing rate

Storage temperature
10  C
w

20  C

Freezing rate

35  C
no

Storage temperature
10  C

tu

35  C

1
2
3
4

111.7
113.3xy
113.3xy
112.7wx

115.3
119.0A
113.3xy
113.0wxy

94.3
95.3pq
93.7mno
94.7op

94.0
94.7op
91.7jk
92.0jkl

30

Rate
Rate
Rate
Rate

1
2
3
4

5.00
4.56a
5.78c
5.91c

6.12
5.99cd
6.47e
6.88fg

10.60
10.76tu
10.46stu
10.38rs

9.05no
8.92no
9.33p
9.52pqr

60

Rate
Rate
Rate
Rate

1
2
3
4

108.7tu
109.3uv
109.0u
108.0t

113.3x
116.3z
111.7w
112.7wx

92.3kl
93.7mno
91.3ij
91.7jk

92.3kl
93.3mn
91.3ij
91.3ij

60

Rate
Rate
Rate
Rate

1
2
3
4

5.93c
5.87c
6.47e
6.16de

6.80f
6.64f
6.90g
6.95gh

11.81wx
12.18yz
12.29yzA
11.74w

11.40v
11.68vw
11.42v
11.75w

90

Rate
Rate
Rate
Rate

1
2
3
4

104.7s
107.3t
101.0r
102.0r

109.0u
112.0w
107.7t
107.7t

92.0jkl
92.7lm
88.7e
88.7e

90.3ghi
90.7hi
88.3de
88.7e

90

Rate
Rate
Rate
Rate

1
2
3
4

8.00kl
7.71k
9.07op
8.86mno

7.28hij
7.12hi
6.96gh
7.35ij

12.26yz
12.36zA
12.04xy
12.40zAB

12.39zA
11.91wxy
11.92wxy
12.38zA

180

Rate
Rate
Rate
Rate

1
2
3
4

88.7e
91.3ij
87.0c
87.7cd

88.0cde
89.3fg
85.3ab
84.0a

86.0b
89.0ef
84.0a
83.7a

180

Rate
Rate
Rate
Rate

1
2
3
4

8.03kl
8.04kl
7.76k
8.04kl

12.48AB
12.73CD
12.34zA
12.94CD

12.91CD
12.77BCD
12.60BC
12.82CD

8.78mn
8.46lm
10.18rs
9.73qr

cd

30  C

Rate
Rate
Rate
Rate

Values followed by different superscript letters are signicantly different at p < 0.05.

ab

20  C

30

88.3de
90.0gh
86.3bc
87.7cd

30  C

Values followed by different superscript letters are signicantly different at p < 0.05.

1482

J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 14741483

optimize bread quality. Prolonged storage times lead to detrimental

changes in both gluten structure and yeast viability.

References

Fig. 6. Contour plots for elasticity (E), yeast activity (Y), and bread volume (V) at 90% of
their maximum value; and adhesiveness (A) and rmness (F) at 90% of their minimum
value. Arrows show direction toward more optimal values.

One usually assumes that a less rm, tenderer bread is most

desirable for consumers. As noted, rmness of bread from the control
was lower than that from frozen dough. Crumb rmness arises from
the integrity of the gluten network, the degree of crosslinking, the
amount of gas incorporated, and from other structural components
of the bread. Ribbotta, Perez, Leon & Anon (2004) reported similar
results to the ones reported in this work with frozen storage
temperature. With longer frozen storage time, the rmness
increased, which may come from depolymerization of glutenin and
higher retrogradation of amylopectin in bread made from frozen
dough.

3.7. Response surface

Fig. 6 shows a response surface for the various quality
measurements as a function of freezing rate and frozen storage
temperature. The overlapping contour lines show each variable at
90% of their optimal levels, with arrows pointing to more optimal
regions. Both volume and rmness depended primarily on storage
temperature, and storage temperatures below 20  C were most
benecial. Elasticity and adhesiveness depended on both storage
temperature and freezing rate, and generally lower temperatures
and higher freezing rates were benecial. Yeast activity also
depended on temperature and freezing rate, but in this case lower
temperatures and slower freezing rates were more benecial. An
optimal region for all attributes (shaded in grey) occurs at temperatures approximately between 15 and 20  C and centered
around freezing rate 2.

4. Conclusions
The quality of bread made from frozen dough depends on the
rate of freezing, storage temperature, and the length of time stored.
Faster freezing and lower storage temperatures promote less
damage to the gluten network, thus help retain elastic properties of
the dough. However, relatively lower freezing rates and storage
temperatures promote yeast viability and gassing power. As such,
a compromise in freezing rate and storage temperature is needed to

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