Food Research International 36 (2003) 357363

www.elsevier.com/locate/foodres

Eect of freezing and frozen storage on the gelatinization and

retrogradation of amylopectin in dough baked in a
dierential scanning calorimeter
Pablo D. Ribottaa,*, Alberto E. Leona, Mara Cristina Anonb
a

Qumica Biologica. Facultad de Ciencias Agropecuarias, Universidad Nacional de Cordoba,

Av. Valparaso s/n, C.C 509, Cordoba (5000), Argentina
b
Centro de Investigacion y Desarrollo en Criotecnologa de Alimentos (CONICET, UNLP),
calle 47 y 116, C.C. 553, La Plata (1900), Argentina
Received 1 August 2001; accepted 29 May 2002

Abstract
Frozen and non-frozen dough were baked in a dierential scanning calorimeter (DSC) pan (heated in the calorimeter at temperatures similar to those of the center of the crumb during baking) and were aged at dierent temperatures. Gelatinized dough
(DSC-baked dough) was heated again in the DSC. This methodology permitted us to study the eects of dough freezing and frozen
storage on gelatinization and retrogradation of starch. During storage of frozen doughs at 18  C an increase in the gelatinization
enthalpy after 150 day of storage was observed. At 230 days of frozen storage a decrease in the onset temperature and an increase in
the gelatinization temperature range was also detected. An increase of starch retrogradation with time of storage in frozen conditions was observed. During the aging of dough baked in DSC, a higher retrogradation temperature range was detected together
with a faster retrogradation of starch at low temperature of aging.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Frozen dough; Starch; Gelatinization; Retrogradation

1. Introduction
There has been an important development in the
manufacturing of frozen bakery products based on the
availability of fresh products in the market. Both the
ingredients and processing conditions have a stronger
inuence on this sort of production process than on the
traditional technique.
Several problems arising from the production of frozen dough have been described: (1) weakening of the
dough; (2) diculties derived from freezing yeast; (3)
decrease in the retention capacity of CO2; and (4) higher
fermentation times, lowering in loaf volume and deterioration in texture of end products (Casey & Foy, 1995;
Dubois & Blockolsky, 1986; Inoue & Bushuk, 1991,
1992; Inoue, Sapirstein, Takayanagi, & Bushuk, 1994;
Neyreneuf & Van Der Plaat, 1991; Wang & Ponte,
* Corresponding author. Fax: +54-351-4334118.
E-mail address: [email protected] (P.D. Ribotta).

1994; Wolt & Dappolonia, 1984). The development of

new technologies regarding the production and
formulation of bakery products led to an improvement
in the quality of the aforementioned products.
The aging of bakery products results in the loss of
consumer acceptability originating in alterations dierent from those caused by spoilage microorganisms
(Betchel, 1955). These involve increase in crumb rmness, changes (usually loss) in avor and aroma, and
loss of crispiness (Cauvain, 1998). Loss of moisture as
well as starch retrogradation are two of the basic
mechanisms operating in the rming of the crumb.
Bread rming is mainly caused by recrystalization of the
starch fraction, involving amylopectin chains (Schoch &
French, 1947; Zobel & Kulp, 1996). Others authors
(Martin, Zeleznak, & Hoseney, 1991) have suggested
that the main reason for the rming of bread is the formation of hydrogen bonds between gluten and starch
granules. Little information is available on the inuence
of dough freezing and frozen storage on the starch

0963-9969/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0963-9969(02)00227-2

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P.D. Ribotta et al. / Food Research International 36 (2003) 357363

retrogradation in bread. Kulp (1995) reported that the

involvement of starch in frozen dough quality seems to
be minor, but indicated that movement of moisture
among the our and ingredient components takes place.
Dierential scanning calorimetry (DSC) constitutes a
useful tool for the study of rst-order transitions, like
starch gelatinization and retrogradation, during baking
and storage of bakery products (Chinachoti, Kim-Shim,
Mari, & Lo, 1991; Donovan, 1977; Giashi, Hoseney, &
Varriano-Marston, 1983; Leon, Duran, & Barber, 1997;
Zanoni, Smaldone, & Schiraldi, 1991; Zeleznak & Hoseney, 1986).
Leon et al. (1997) have developed a new approach to
study changes during the dough-baking process and
bread storage. The approach was based on a direct
observation of starch changes during the baking process
and storage by DSC. Bread doughs were heated in a
DSC pan at similar temperatures to that of the center of
the crumb during baking. These authors reported that
the retrogradation rate of starch increased when the
storage dough was prolonged to 30 days.
In the present work, we extended this methodology to
analyze the eect of dough freezing and frozen storage
on the gelatinization and retrogradation of starch. The
inuence of frozen storage dough on properties of
starch in bread baked in DSC and aging at 4 and 20  C
was also analyzed.

2. Materials and methods

2.1. Dough samples
A stock dough was prepared with the following
recipe: our (protein 13.2%, water 11.8%, ash 0.7%)
100%, compressed yeast 3%, NaCl 1.8%, sodium propionate 0.2%, ascorbic acid 0.015%, and water (64%).
Yeast and salt were separately dissolved in water before
addition to the dough. The remaining ingredients were
added as solids. This recipe is currently employed in our
country for the preparation of bread and its simplicity
allows a clear observation of changes occurring during
the processing of frozen dough. Ingredients were mixed
in a Philips HR 1495 mixer (Philips, Argentina) for 2
min and rested for 15 min in a fermentation cabinet at
30  C and 70% rh. The resulting dough was degassed
and hand molded and treated as described below.
2.2. Dierential scanning calorimetry
Analyses were performed in a Polymer Laboratories
Calorimeter (Rheometric Scientic Ltd, England).
Transition temperatures and enthalpies were obtained
with the PL-V5.41 software. The equipment was calibrated with Indium and an empty double pan was used
as a reference. At least, two determinations per dough

lot were made

mean  S.D.

and

results

were

expressed

as

2.2.1. Starch gelatinization

Samples (1520 mg) were weighed in DSC pans and
hermetically closed. One set of pans was frozen to
18  C for 1, 60, 150 and 230 days. Another set of pans
was heated in the calorimeter at temperatures similar to
those of the center of the crumb during baking (control
sample) after the fermentation period (45 min at 30  C).
Frozen samples were defrosted and fermented at 30  C
for 2 h and then heated in the calorimeter (DSC-baked
dough). To simulate the temperature prole in the center of the crumb during baking, calorimeter scan conditions were set as follows: samples were kept at 30  C for
2 min, heated from 30 to 110  C with a heating rate of
11.7  C/min and nally kept at 110  C for 5 min (Leon
et al., 1997).
Onset temperature (To) and gelatinization temperature range (
Tg), as well as the enthalpy of starch
gelatinization (
Hg) (expressed as mJ/mg of sample) of
bread doughs were determined.
2.2.2. Retrogradation of amylopectin
In this case we studied the eect of storage time in
frozen conditions followed by an aging at a xed time
and temperature after baking (7 days at 20  C). The
eect of aging time at two temperatures (4 and 20  C)
after a single storage time (60 days) 18  C on the retrogradation of amylopectin was also analyzed. The
degree of amylopectin retrogradation was calculated as

Hretrogradation/
Hmax, where
Hmax is the value of the
enthalpy of starch gelatinization obtained for a fresh
sample (control sample) run in the DSC from 30 to
130  C at 10  C/min.
2.2.3. Eect of storage in frozen conditions
To analyze amylopectin retrogradation as a function
of storage time at 18  C, frozen dough samples in
DSC pans were maintained at 18  C for 1, 60, 150 and
230 days. After the corresponding storage time, pans
were baked in the DSC as described previously and kept
at 20  C for 7 days. Then, samples were analyzed again
in a DSC from 10 to 130  C at 10  C/min. Fresh nonfrozen doughs were used as controls. At least two replicates were analyzed. Results were expressed as mean
values  S.D.
2.2.4. Eect of aging
Dough samples were frozen in DSC pans, as described previously and kept 60 days at 18  C. Pans were
baked in the DSC as described and cooled to store for
dierent times (1, 2, 4, 6 and 7 days) at two temperatures (4  C  1 and 20  C  2). Finally, DSC-baked
dough samples were heated again in the calorimeter
from 10 to 130  C at 10  C/min. Fresh non-frozen

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P.D. Ribotta et al. / Food Research International 36 (2003) 357363

doughs were used as controls. At least, two replicates

were analyzed. Results were expressed as mean
values  S.D.
2.3. Statistical analysis
Data obtained were statistically evaluated by variance
analysis (ANOVA). Means comparison was done by the
LSD Fisher test at a level of 0.05. Both were carried out
using the statistical analysis package INFOSTAT
(Facultad de Ciencias Agropecuarias, UNC, Argentina).

3. Results and discussion

3.1. Inuence of dough freezing on starch gelatinization
Fig. 1 shows a thermogram obtained from a frozen
sample which was stored at 18  C and then defrosted,
fermented and baked in the DSC. A curve with a
shoulder corresponding to the gelatinization (G endotherm) and to the fusion of the most stable crystallites
(F endotherm) resulting from the low level of water in
the sample were observed. The endotherm corresponding to the dissociation of the amylose-lipid complex was
not detected due to the heating program we chose. As
stated previously, this program mimics the process of
baking. Similar thermograms were obtained in previous
studies employing the same experimental design for
unfrozen fermented and unfermented doughs (Leon et
al., 1997) and for a wheatourwater mixture with
similar water content to our formulation (Jovanovich,
Zamponi, Lupano, & Anon, 1992).
Table 1 shows the inuence of frozen storage of
dough on the gelatinization of starch during baking in
the DSC. The freezing process slightly decreased the To
but the storage in frozen conditions did not have a signicant eect (P > 0.05) on the onset temperature and

the gelatinization temperature range until to 150 days.

At 230 days of frozen storage To decreased and the
gelatinization temperature range of starch increased.
The results obtained were similar to those reported by
Autio and Sinda (1992) who found dierences in the
onset temperature, between frozen and non-frozen
doughs stored at a single temperature.
Regarding the enthalpy (
Hg) involved during gelatinization and melting of starch crystals, the results
obtained showed signicant dierences (P < 0.05)
between non-frozen dough and dough stored in frozen
conditions at 150 and 230 days. An increase of
Hg
with the storage time at 18  C was also observed
(P < 0.05).
3.2. Eect of dough storage at
retrogradation of amylopectin

18  C on the

Frozen samples, which were stored for dierent periods and then baked in DSC pans as discussed previously, were stored at 20  C for 7 days in order to
analyze starch retrogradation. DSC-baked doughs were
submitted to a second DSC scan after storage. The DSC
Table 1
Eects of dough freezing and stored at 18  C on onset temperatures
(To), gelatinization temperature ranges (
Tg) and gelatinization
enthalpies (
Hg)
Frozen storage (day) To ( C)
Control
(non-frozen dough)
1b
60
150
230

Tg ( C)

Hg (mJ/mg)

61.710.14 ca 41.360.08 pq

3.750.01 tv

60.640.14
60.420.53
60.390.09
57.820.06

3.600.07
3.720.09
3.900.01
4.110.10

bc
b
b
a

40.460.30
38.830.24
39.381.71
43.740.05

p
p
p
q

t
tv
v
x

a
Values followed by the same letter are not signicantly dierent
(P<0.05).
b
Time required for dough core reach 18  C.

Fig. 1. Dierential scanning calorimetry (DSC) thermogram of frozen, thawed, fermented and DSC-baked dough. Two overlapped endotherms
corresponding to gelatinization (G) and to the fusion of the most stable crystallites (F).

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P.D. Ribotta et al. / Food Research International 36 (2003) 357363

thermograms obtained (Fig. 2) showed two well-dened

endotherms corresponding to the retrogradation of
amylopectin (R) and melting of the amyloselipid complex (C). Table 2 depicts the values of To, retrogradation temperature range and
H of amylopectin
retrogradation in the dierent DSC-baked dough samples. Results obtained showed that the longer the time
the dough remained stored in frozen conditions, the
more pronounced was the starch retrogradation
(P < 0.05). The same tendency was observed previously
for dough frozen and stored at 18  C for 1 month and
aged at room temperature (Leon et al., 1997). On the
other hand, a decrease of To and an increase of the retrogradation temperature range were found only at 150
and 230 days of storage. It is generally known that
starch retrogradation, measured by DSC, is aected by
dierent factors, such as moisture, lipid content and
other ingredients (Biliaderis, 1990). Zeleznak and Hoseney (1986) showed that the retrogradation enthalpy in
starch gels (50% moisture) and breads (3040% moisture) depended on the water content of samples during
storage. The magnitude of the amylopectin melting
peak was controlled by the water availability during
retrogradation, while the water content during gelatinization had little eect. Lu and Grant (1999) showed that
the amount of freeezable water in frozen dough
increased with time of storage in frozen conditions.
These results, in coincidence with Berglund, Shelton, and
Freeman (1991) clearly indicate that there is a redistribution of total water present in the system during frozen
storage. Moreover, a recrystallization of ice-crystals probably occurs during frozen storage leading to the formation of a lower number of crystals of greater size. Both
processes (dislocation of water and ice-recrystallization)

could induce changes in the structure and arrangement

of amylose and amylopectin. According to the results
of the present work, such changes would be reected
during starch gelatinization and retrogradation.

H of melting of the amyloselipid complex
decreased as the time of storage increased, in dough
stored at 18  C, but it was dicult to quantify the
enthalpy changes due to the non-gaussian prole of
these endotherms.
3.3. Eect of storage at two temperatures on the
kinetics of amylopectin retrogradation in baked bread
dough
Thermograms obtained from frozen doughs baked in
the DSC and stored at 20  2  C and 4 1  C showed
two typical endotherms corresponding to amylopectin
retrogradation and melting of the amylopectin-lipid
complex. Samples aging for 1.5 h showed only one
Table 2
Eects of dough freezing and stored at 18  C on onset temperatures
(To), retrogradation temperature ranges (
Tr) and retrogradation
enthalpies (
Hr)
Frozen storage (day) To ( C)
Control
(non-frozen dough)
1b
60
150
230

Tr ( C)
a

Hr (mJ/mg)

47.981.21 a

30.160.65 pq

1.250.02 v

50.940.94
52.110.55
46.810.25
45.700.24

25.342.84
26.240.01
30.541.65
31.270.48

0.790.01
0.880.05
1.530.11
2.530.03

b
b
a
a

p
pq
pq
q

t
t
x
y

a
Values followed by the same letter are not signicantly dierent
(P<0.05).
b
Time required for dough core reach 18  C.

Fig. 2. DSC thermogram of a DSC-baked dough aged for 7 days at 20 3  C. The endotherm showed two well-dened peaks corresponding to
amylopectin retrogradation (R) and melting of the amyloselipid complex (C).

361

P.D. Ribotta et al. / Food Research International 36 (2003) 357363

endotherm that could be assigned to the fusion of the

amylopectin-lipid complex. Tables 3 and 4 present
values of onset temperatures, retrogradation temperature ranges and enthalpies calculated for all DSC-baked
dough samples. Onset temperature was lower for DSCbaked dough obtained from doughs stored at 18  C

for 2 months than for doughs that were not frozen,

aging at 4  C (P< 0.05). DSC-baked dough aging at 4  C
had lower onset temperature than DSC-baked dough
aging at 20  C either in frozen or fresh doughs
(P < 0.05). DSC-baked dough aging at 4  C had higher
retrogradation temperature range than DSC-baked

Table 3
Eects of dough frozen storage and DSC-baked dough aging temperature on onset temperatures (To), retrogradation temperature ranges (
Tr) and
retrogradation enthalpies (
Hr)
Aging time (days)

1
2
4
6
7
a

Non-frozen dough
DSC-baked dough aging at 4  C
To ( C)

Tr ( C)

40.730.42efa
39.891.99de
38.500.08cd
37.311.71bc
38.050.93cd

31.98 0.34klm
34.28 5.46lmn
36.52 1.48mnop
39.74 5.88pq
34.92 2.04lmno

Frozen dough (60 days)

DSC-baked dough aging at 4  C

Hr (mJ/mg)
1.13 0.06s
1.76 0.59tu
2.32 0.56vw
2.64 0.59wx
3.29 0.23yz

To ( C)

Tr ( C)

35.721.64ab
35.370.23a
35.530.00ab
34.930.42a
34.650.37a

41.660.83q
39.321.10opq
39.160.44opq
38.520.02nopq
38.800.81nopq

Hr (mJ/mg)
2.10 0.12uv
2.89 0.07xy
3.27 0.07yz
3.46 0.04z
3.68 0.03z

Values followed by the same letter are not signicantly dierent (P<0.05).

Table 4
Eects of dough frozen storage and DSC-baked dough aging temperature on onset temperatures (To), retrogradation temperature ranges (
Tr) and
retrogradation enthalpies (
Hr)
Ageing time (day)

1
2
4
6
7
a

Non-frozen dough
DSC-baked dough aging at 20  C
To ( C)

Tr ( C)

46.710.40ga
46.110.56g
46.150.89g
45.650.31g
45.830.57g

32.902.69klm
29.171.08k
32.192.91klm
32.180.93klm
31.812.18kl

Frozen dough (60 days)

DSC-baked dough aging at 20  C

Hr (mJ/mg)
0.94 0.09s
1.30 0.06st
1.82 0.15tuv
2.03 0.09uv
2.18 0.06uvw

To ( C)

Tr ( C)

42.201.36f
45.920.86g
46.490.48g
46.880.00g
45.830.71g

34.210.69lmn
29.251.52k
28.670.62k
29.370.00k
29.521.63k

Hr (mJ/mg)
1.110.19s
1.320.18st
1.920.07uv
2.190.10uvw
2.160.21uvw

Values followed by the same letter are not signicantly dierent (P<0.05).

Fig. 3. Eects of frozen storage and aging temperature on degree of retrogradation occurring in DSC-baked doughs. Fresh dough (), and dough
frozen and stored for 60 days at 18  C (). DSC-baked dough aged at 4 1  C () and at 202  C (- - -).

362

P.D. Ribotta et al. / Food Research International 36 (2003) 357363

dough aging at 20  C either in frozen or fresh doughs

(P < 0.05), except for the rst day of aging in non-frozen
doughs. On the other hand, in DSC-baked dough stored
more than two days at 20  C, retrogradation temperature range was lower for frozen doughs that for nonfrozen one (P < 0.05). DSC-baked dough stored at 4  C
did not show the same trend.
Changes in
H of amylopectin retrogradation in
DSC-baked dough were always higher (P < 0.05) in
samples stored more than 1 day at 4  C than at 20  C
either fresh or stored in frozen conditions. These results
agreed with those that for temperatures between 1 and
43  C the rate of starch retrogradation is higher at low
temperature (Biliaderis, 1990). Doughs that were stored
frozen for 60 days and aging after baking in the DSC at
4  C had a higher
H of retrogradation than doughs,
which were not stored frozen. In dough stored frozen
and non-frozen, the extent of retrogradation of amylopectin in DSC-baked dough increased with decreasing
temperature of storage (Fig. 3). Faster retrogradation of
amylopectin in DSC-baked dough obtained from dough
stored in frozen conditions for 60 days was observed in
comparison to DSC-baked dough obtained from nonfrozen doughs. This phenomenon was more evident at
4  C.

4. Conclusions
The methodological approach employed (DSC simulation of freezingstorage in frozen conditions and
bakingaging) allowed the analysis of changes occurring
in starch. This method may allow the simulation of the
changes in bread. Results indicated that freezing and
storage of doughs at 18  C modied starch properties.
These modications were slightly reected during the
gelatinization process of starch, but they had an
important inuence in both the rate of retrogradation
and on the amount of amylopectin that retrograded.
We also observed that the rate of retrogradation of
amylopectin in doughs, which were previously baked in
the DSC, was higher at lower aging temperatures. This
dierence was more evident in frozen doughs, which
were stored in frozen conditions. Our results were independent from the loss of water that may be occurring in
the sample on the basis that we worked with pans hermetically closed that prevented evaporation during the
assay.

Acknowledgements
The authors wish to acknowledge the nancial support of Agencia Nacional de Promocion Cientca y
Tecnologica, contrato de prestamo BID 1201/OC-AR
No. PICT 09-07321 and CONICET.

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