TECNOLOGÍA E INDUSTRIA DE LA PANIFICACIÓN – 2020 A

PRACTICA DE LABORATORIO N°4

EXTENSIÓN DE VIDA ÚTIL EN PRODUCTOS DE PANIFICACIÓN
I. OBJETIVOS.

• Revisar los principales mecanismos para extender la vida útil de productos panificados.
• Conocer el proceso industrial de fabricación de pan de molde.
• Relacionar los diferentes mecanismos para la extensión de la vida útil en términos de
conservación de las características texturales aplicados al proceso de fabricación de pan
de molde.

II. MATERIALES Y MÉTODOS

1. Material, Instrumental y Equipos

• Videos y papers selecionados y proporcionados por el docente

• Textos varios
• Computador con conexión a internet

2. Procedimiento

• Formar grupos de 4 personas

• Realizar la visualización del siguiente video seleccionado por el docente.
Fabricando Pan de molde: https://www.youtube.com/watch?v=0fIB0DX45JQ
• Realizar la lectura de los siguientes papers:
Gray, J. & Bemiller, J. (2003). Bread staling: Molecular basis and control. Comprehensive
Reviews in Food Science and Food Safety. 02, 473- 492. doi: 10.1111/1541-
4337.12064.
Fadda, C., Sanguinetti, A., Del Caro, A., Collar, C. & Piga, A. (2014). Bread staling:
Updating the view. Comprehensive Reviews in Food Science and Food Safety. 13,
1- 21. doi: 10.1111/1541-4337.12064.
• Preparar una lista de preguntas y comentarios para discusión post-visualización de los
videos.
• Responder el cuestionario planteado en base a la visualización realizada.
• Complementar y validar sus respuestas con fuentes bibliográficas confiables.
• Redactar su informe en forma grupal haciendo uso de estilo técnico APA para citas y
referencias bibliográficas.
• Remitir su informe grupal mediante plataforma DUTIC (7 días calendario hasta las
23.59hs)

III. CUESTIONARIO
1. Esquematice mediante un diagrama de flujo el proceso de producción industrial de
fabricación de pan de molde.
2. Describa los equipos y maquinarias que intervienen en cada etapa del proceso,
identificados en el diagrama de flujo.

MS. ENG. JULIO C. RODRÍGUEZ DÍAZ 1

 TECNOLOGÍA E INDUSTRIA DE LA PANIFICACIÓN – 2020 A

3. Según los papers, como influye el proceso de retrogradación del almidón y sus
reacciones conexas en el endurecimiento del pan y en la velocidad con el que éste se
da.
4. Cuáles son los principales ingredientes que pueden ser utilizados para retardar el
proceso de endurecimiento del pan.
5. El pan de molde referido en el video tiene un tiempo de vida útil entre 30 – 35 días.
a. ¿Cuál es la razón para prolongar la vida útil de este pan por todo este tiempo?
b. Analizar la declaración de ingredientes en el empaque de cualquier pan de molde
de larga duración (Ejm: Bimbo, Pyc u otro) y con ayuda del Codex Alimentarius
identificar las sustancias y aditivos que fueron usados y clasificarlos por
funcionalidad.
c. Una vez identificados los ingredientes y aditivos responsables por la prolongación
de la vida útil haga una breve revisión bibliográfica de las funciones de cada uno de
ellos y en base a información de papers, especificaciones técnicas, hojas de
aplicación y/o recomendaciones de fabricante, trate de identificar las posibles dosis
para cada uno de ellos.

MS. ENG. JULIO C. RODRÍGUEZ DÍAZ 2

 Bread Staling:
Molecular Basis
and Control
J.A. Gray and J.N. Bemiller

ABSTRACT: The molecular basis of staling is examined by reviewing what is known about the components of wheat flour,
factors that affect staling rate, and the various mechanisms that have been proposed. The conclusion reached is that bread
staling is a complex phenomenon in which multiple mechanisms operate. Polymer crystallizations with the formation of
supermolecular structures are certainly involved. The most plausible hypothesis is that retrogradation of amylopectin
occurs, and because water molecules are incorporated into the crystallites, the distribution of water is shifted from gluten
to starch/amylopectin, thereby changing the nature of the gluten network. The role of additives may be to change the
nature of starch protein molecules, to function as plasticizers, and/or to retard the redistribution of water between
components. Nothing more definite can be concluded at this time.

Introduction view of bread staling confined to a limited space cannot discuss

Although it has been studied for more than a century and a
half, bread staling has not been eliminated and remains responsi-
ble for huge economic losses to both the baking industry and the
consumer. Bechtel and others (1953) defined staling as “a term
which indicates decreasing consumer acceptance of bakery prod-
ucts caused by changes in crumb other than those resulting from
the action of spoilage organisms”. While an American Association
of Cereal Chemists Approved Method (AACC Method 74-30;
AACC 2000) quantifies staling organoleptically, many researchers
use the 1953 definition as a general definition and describe spe-
cific components of the complex staling process with specific
terms such as crumb firming, crust staling, and organoleptic stal-
ing (Kulp and Ponte 1981). In fact, the most widely used indicator
of staling is measurement of the increase in crumb firmness (see
“Rheological methods: Uniaxial compression” section), which is
the attribute most commonly recognized by the consumer. In this
review, the term “bread staling” is used to refer to the phenome-
non of “crumb firming” in white pan bread.
Bread is an unstable, elastic, solid foam, the solid part of which
contains a continuous phase composed in part of an elastic net-
work of cross-linked gluten molecules and in part of leached
starch polymer molecules, primarily amylose, both uncomplexed
and complexed with polar lipid molecules, and a discontinuous
phase of entrapped, gelatinized, swollen, deformed (wheat) starch
granules. Neither the bread system nor the staling process is un-
derstood well at the molecular level. Even simple bread dough
formulations contain several ingredients, which themselves may
contain several components, each of which may undergo chang-
es during the breadmaking process and during aging of the final
product. And just as bread is a complex, heterogeneous system,
the staling phenomenon seems to be complex, because investiga-
tion of hypotheses involving changes in 1 or 2 components have
failed to fully explained the process.
Because the literature on bread staling is so extensive, any re-
all available information, hypotheses, or conclusions; nor can it
give in-depth treatment to the aspects covered. It is believed, how-
ever, that most important pieces of known information, concepts,
principles, hypotheses, and conclusions are presented here.
Several previous reviews on staling (the process, the mecha-
nism, its measurement, and factors that affect it) have appeared
(referred to elsewhere and in the references), and 2 books (Hebe-
da and Zobel 1996; Chinachoti and Vodovotz 2000) are available
for a more thorough treatment. Discussions in the literature refer-
enced in this review will lead readers to additional information. A
brief review not elsewhere referenced in this review is that by
Guilbot and Godon (1984).
Physical and mechanical mixing, chemical reactions (including
enzyme-catalyzed reactions), and thermal effects (baking time and
temperature) are factors that influence the nature and properties of
the final product. This review focuses on antistaling agents, using
what is known about the mechanism of staling and factors that af-
fect staling rate as a basis for the discussion. It also focuses on
crumb staling, because crumb staling is of much greater concern
to the consumer than is crust staling and has been studied more.
Four things are called to the attention of the reader before be-
ginning: (1) Experimental work done on staling to date has in-
volved looking for correlations between staling (by whatever defi-
nition and measurement employed) and a change in the formula-
tion or process, but correlations do not necessarily prove a direct
cause-and-effect relationship; for example, addition of a surfactant
known to form a complex with amylose may increase shelf life,
but that does not necessarily mean that amylose complexation is
responsible for the increase in shelf life. The critical effect could
be on the structure of water, for example. (2) There is much infor-
mation on the effects of various additives and conditions on
starch gelation and retrogradation and complex formation in di-
lute and concentrated starch pastes. For the most part, that litera-
ture is neither presented nor discussed, even though the mecha-

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 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
nisms of retrogradation in concentrated amylopectin gels and
bread crumb are believed to be very similar, if not identical (Slade
and Levine 1987.) For a review of this literature as it pertains to
staling and that of the crystal structures in bread, the reader is re-
ferred to the review of Zobel and Kulp (1996). (3) When an
amount of an additive is stated, it is a percentage of the weight of
flour. (4) Abbreviations used include CP MAS (cross-polarization
magic-angle spinning), DSC (differential scanning calorimetry),
DTA (differential thermal analysis), MRI (magnetic resonance im-
aging), NMR (nuclear magnetic resonance), and Tg (glass transi-
tion temperature).
Molecular Basis of Staling
Components of wheat flour
To understand the mechanism of staling in breads, it is impor-
tant to understand the natures of the major components that make
up the system. Relationships of these components to staling are
described in Section 2.2. The role of water and additives in staling
are discussed in Sections 3 and 4.
A typical bread formula consists of the following ingredients:
flour (wheat), water, sugar, shortening, nonfat dried milk (or a sub-
stitute), salt, yeast, malt, a dough strengthener, a crumb softener, a
mold inhibitor (sodium propionate), and an oxidant (Hoseney
and Seib 1978). Wheat flour consists primarily of gluten, starch,
and “pentosans” (primarily arabinoxylans), all of which are impor-
tant contributors to the characteristics of the process and the final
product. Native flour lipids play an important role in breadmaking
(Morrison 1976), especially in their interaction with added short-
ening (Rogers and others 1988). Wheat flour has considerable ␣-
amylase activity and a minor amount of ␣-amylase activity.
States of the starch, gluten, and polar lipids in the 3 main stages
in the life of aged bread are outlined in Table 1.
Protein. Hydrated gluten is the continuous phase of wheat flour
doughs (Ponte and Faubion 1985; Davies 1986). During baking,
gluten is denatured, and protein-protein crosslinking occurs via
formation of disulfide bonds (Schofield 1986). The resulting net-
work, combined with partially gelatinized starch granules, is most
certainly responsible for the semirigid structure of baked products
(Blanshard 1988; Hoseney 1989).
Starch. Wheat flour contains 84 to 88% (db) starch. During
baking of bread dough, the starch granules are generally gelati-
nized (Table 1, footnote c), but little else other than restricted
swelling followed by collapse happens to them because of the
limited amount of water present in the dough system (Schoch
1965), so deformed wheat starch granules can be isolated from
the crumb (Hoseney and others 1978). [Note: When starch gran-
ules are heated in excess water, granules swell and some portion
of the amylose diffuses from the granules, concentrates in the in-
terstitial water between granules, and undergoes retrogradation.
The small amount of amylose that leaches from granules during
baking in the limited moisture system of bread dough retrogrades
upon cooling and rapidly becomes unextractable (Kim and
D’Appolonia 1977b,c); so even if amylose does leach from gran-
ules, by the time bread has completely cooled, any interstitial
amylose will have retrograded (that is, become insoluble) and is
unlikely, therefore, to play a major role in subsequent staling
events.] Even in the presence of excess water, monoglycerides
block the leaching of amylose molecules (Schoch 1965; see “Sur-
face-active lipids: Surfactants” section), so it can be assumed that
other surfactants would act in the same way, especially in the lim-
ited moisture system of bread. Therefore, freshly baked and
cooled bread is an elastic system containing swollen wheat starch
granules that are still largely intact, but may be deformed.
On the other hand, observations made with transmission elec-
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tron microscopy, led Bechtel and others (1978) to conclude that,
after baking, most starch granules were destroyed and most starch
molecules were part of the continuous phase, but separate from
protein strands.
Nonstarch polysaccharides. Arabinoxylans and arabinogalac-
tans (arabinogalactan-proteins) are the “pentosans” (more proper-
ly pentoglycans) of wheat flour. Arabinoxylans are divided into 2
classes (“water-soluble” and “water-insoluble”) and have been
much more extensively studied than have the arabinogalactans
(Loosveld and others 1997), because they are present in greater
concentrations and are believed to play a more important role in
both the preparation and the shelf-life of bakery products. Both
classes of arabinoxylans of hard wheat flours have been investi-
gated with regards to structure (Izydorczyk and others 1991; Izy-
dorczyk and Biliaderis 1995, 2000) and to differences in structure
as a function of cultivars (Izydorczyk and others 1991; Cleemput
and others 1993; Izydorczyk and Biliaderis 1993; Rattan and oth-
ers 1994). Their influence on breadmaking and bread quality is
still being debated (see “Mechanisms of staling: Role of pen-
tosans” section).
Mechanism of staling
Attention is called to another review on the mechanism of stal-
ing (Schiraldi and Fessas 2001). Bread staling falls into 2 catego-
ries: crust staling and crumb staling. Crust staling is generally
caused by moisture transfer from the crumb to the crust (Lin and
Lineback 1990), resulting in a soft, leathery texture and is general-
ly less objectionable than is crumb staling (Newbold 1976).
Crumb staling is more complex, more important, and less under-
stood. The firmness of bread varies with position within a loaf,
with maximum firmness occurring in the central portion of the
crumb (Short and Roberts 1971).
The key hindrance to development of a preventive strategy for
bread staling is the failure to understand the mechanism of the
process. Many investigations have examined the phenomenon of
crumb-firming, and many theories have been proposed and dis-
cussed in previous reviews (Herz 1965; Willhoft 1973; Zobel
1973; Maga 1975; Knightly 1977; Kulp and Ponte 1981; Zobel
and Kulp 1996). A cursory overview of the major theories on the
subject is presented here.
Amylopectin retrogradation. Katz (1928) proposed that starch
polymers retrogradation was responsible for staling of bread be-
cause his x-ray diffraction patterns of fresh bread were similar to
those of freshly gelatinized wheat starch, while the patterns of
stale bread were similar to those of retrograded starch. This find-
ing led to the hypothesis that a gradual change in the starch com-
ponents from amorphous to crystalline forms is important to the
staling process. Hellman and others (1954) provided evidence
that the rate of development of crystallinity in starch gels was simi-
lar to the rate of bread firming; but Dragsdorf and Varriano-Mar-
ston (1980) obtained evidence that the degree of crystallinity of
bread crumb was inversely related to its firmness and, therefore,
concluded that starch crystallization and bread firming were sepa-
rate processes.
Vodovotz and others (2002) detected no increase in molecular
rigidity, that is, decrease in molecular mobility, in an aged bread
sample (proton cross-relaxation NMR spectroscopy) that was con-
current with an increase in the amylopectin retrogradation endot-
herm (DSC). They concluded that “differences in molecular mobil-
ity could not be, therefore, due to recrystallized amylopectin and
may be attributed to the role of gluten [see “Mechanisms of stal-
ing: Role of flour protein” section] and/or redistribution of water
[see “Moisture migration: Moisture redistribution among compo-
nents” section] in the amorphous regions of the samples”.
Whether the fraction of starch that contributes to bread firming
is amylose or amylopectin also has been debated. The linear,
 Bread staling. . .
Table 1—States of critical components in various stages in the life of breada
Stage Starch
Dough Hydrated, intact granules.
Apb partially crystalline.
Amb amorphous
Fresh-baked, Granules in a spectrum of states.
but cooled, Some rather intact. Most gelatinizedc
bread and deformed/collapsed.
Starch-starch interactions both
within and between granules
Double-helical structure of Ap at least
partially lost. Perhaps some Ap molecules
partially or completely outside of granules
(Section 2.2.1).
Some Am partially or completely leached
from granules, putting some of it in the
continuous phase, where it is largely
insoluble. Some complexed with polar
lipid molecules. (Sections 2.2.2, 4.2).
Aged bread Retrograded Ap inside gelatinized
granules. Perhaps some outside of
granules (Sections 2.2.1, 4.1.1).
Am retrograded. Some complexed with
lipid. Probably little changed from fresh-
baked bread (Sections 2.2.2, 4.1.1).
a Based on best evidence available. Other views have been stated; see discussion.
bAp = amylopectin, Am = amylose
cGelatinization is the disruption of molecular order within starch granules as they are heated in the presence of water (Atwell and others 1988).
d Both macro-and microscopic redistribution of water occurs during aging.
more readily retrograded fraction, amylose, was suspected first
(see “Mechanisms of staling: Role of amylose” section). Evidence
from Katz (1928) suggested formation of side-by-side associations
of linear starch molecules in the B-type x-ray patterns of staled
bread and retrograded starch. Hixon (1943) speculated that, if a
waxy wheat variety were available, then bread made from that
flour might not stale since it would be essentially void of amylose.
Alsberg (1927, 1928) pointed out the well-known fact that heat-
ing stale bread above 50 °C can restore the loaf to its original
freshness. Since retrograded amylose will not melt at this tempera-
ture (Knightly 1977), amylopectin was suggested to be the fraction
of starch responsible for staling. Supporting evidence was pre-
sented when bread prepared from a synthetic flour composed of
waxy maize starch and nondevitalized gluten exhibited a normal
tendency to stale (Noznick and others 1946). Further, Schoch and
French (1947) found that the water-soluble material that could be
leached from bread crumb at 30 °C was predominantly amy-
lopectin. They hypothesized that progressive spontaneous aggre-
gations of amylopectin molecules was responsible for bread firm-
ing. Furthermore, they suspected that the contribution of the amy-
lose fraction to staling was negligible, since they believed it to be
retrograded/insolubilized during cooling. The important role of
amylopectin in starch retrogradation was confirmed by calorime-
try (Russell 1983a, b).
However, Hoseney and Miller (1998) have pointed out that
stale bread must be heated to about 100 ºC before its compress-
ibility approaches that of fresh bread (Ghiasi and others 1984)
and that, since retrograded amylopectin should have melted by
the time the temperature reached 60 ºC, retrogradation of amy-
lopectin cannot be the only factor affecting firming. Retrograded
waxy corn starch (5%) was added to a bread formula and found
to decrease gelatinization and to reduce the firming rate (Hibi
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Gluten Polar lipid
Hydrated. In the form of Free. Perhaps some
fibrils with adhering starch protein-lipid interactions.
granules in a continuous network.
Denatured. Crosslinked. Possible Some complexed with Am
formation of starch-gluten associations (inside and outside of granules).
(starch-gluten fibrils) during baking. Some free. Possible protein-lipid
interactions (Sections 2.2.6, 4.2).
Loss of water of hydration from gluten Unchanged from fresh-baked bread
network via transfer to starchd, which (?)
enables crystallization of Ap (Section 3.2).
2001). [Note: As pointed out above, the retrograded material
should have melted during baking so the effect would be one of
adding corn amylopectin.]
Toufeili and others (1999) found that an all-amylopectin Arabic
bread (made with waxy barley starch and cross-linked waxy bar-
ley starch in place of wheat starch) staled at a significantly faster
rate than did Arabic bread made with normal wheat starch, that a
low degree of starch crosslinking promotes recrystallization of
amylopectin [possibly by keeping polymer chains in close prox-
imity to one another], and that a higher degree of crosslinking de-
creased the staling rate [possibly by restricting granule swelling
and separation of polymer chains].
Most agree that there is at least a correlation between amy-
lopectin retrogradation/crystallization and staling, even though
the 2 events may not be part of the same process. Our conclusion
is that amylopectin retrogradation is part of the staling process,
but is not solely responsible for the observed changes in texture.
For information on associations of starch polymer molecules in
concentrated wheat starch (and other starch gels), see Keetels and
others (1993, 1995, 1996 a,b,c,d; Vodovotz and others 2002).
Role of amylose. While Schoch and French (1947) believed
that the linear fraction of starch had a negligible influence on
bread staling, there is evidence that amylose is involved in some
way. Due to its rapid rate of retrogradation, Hoseney and others
(1978) proposed that amylose was responsible for setting the ini-
tial crumb structure, but not involved in the staling process. Er-
lander and Erlander (1969) theorized that amylose-amylopectin
aggregation was responsible for the changes that occur during ag-
ing of bread crumb. Kim and D’Appolonia (1977c) found that the
solubility of amylose decreased markedly during the 1st d of
bread storage, while the solubility of amylopectin decreased
steadily over 5 d of storage. They also found that the amount of
3
 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
soluble amylopectin in fresh bread was 5 to 24 times the amount
of soluble amylose, indicating that little amylose was leached from
granules or that, by the time the bread had cooled to room tem-
perature, much of the amylose had become insoluble by retrogra-
dation, probably the latter. Ghiasi and others (1984) changed the
ratio of amylose to amylopectin in flour by using waxy barley
starch and also found that the amylose fraction was involved in
staling of bread through 1 d only. It has been suggested that the
role of amylose in bread staling may be merely one of diluting
amylopectin (Inagaki and Seib 1992), a conclusion reached from
a study of breads made with cross-linked waxy barley starch,
which staled at a faster rate than did control breads, even though
the experimental bread had less firmness after 6 h. Evidence
against a role for amylose in staling is that, while stale bread can
be refreshed by heating, amylose crystals (either of the V-type or
B-type) do not melt at the temperatures employed (Knightly 1977).
From an interesting microscopic examination, Hug-Iten and
others (1999) reported that, during baking, there was a separation
of amylose and amylopectin where amylose accumulated at gran-
ule centers, and that, upon aging, gelatinized granules regained
birefringence, with the most intense birefringence being observed
in the amylose-rich granule centers. [Note: It may have only ap-
peared that amylose accumulated at granule centers. Another
possible explanation is that amylose was lost by leaching from the
outer area.] They hypothesized that reorganization of intragranu-
lar amylose enhances the rigidity of starch granules during staling.
See also sections “Mechanisms of staling: Role of native lipids”
and “Surface-active lipids” for more on the role of amylose in
bread staling.
Relationship between crumb firming and starch retrograda-
tion. Alsberg (1927, 1928) proposed that bread staling could not
be completely attributed to starch retrogradation, since retrogra-
dation in pastes is a slower process than is staling. [Note: There is
reason to believe that retrogradation might occur more easily in
bread than in pastes, which are more often studied, because in
our opinion, since granules in baked bread are still largely intact,
although deformed because their swelling is limited by a deficien-
cy of water, the molecular chains in them are not completely dis-
engaged. Therefore, although there is some degree of crystalline
packing order disruption, it is much easier for chains, which are
still close to one another and still aligned similarly to what they
were in the native granule, to reassociate than it is for amylopectin
molecules in a cooked paste to realign and form an ordered struc-
ture. However, it is not known whether intragranular recrystalliza-
tion is related to staling. In this regard, surfactants that inhibit
granule swelling/gelatinization from occurring in the first place are
effective as antistaling agents.] Others have also questioned the
concept that amylopectin crystallization and bread firming are
one and the same, even though both may occur simultaneously
(Dragsdorf and Varriano-Marston 1980: Baik and Chinachoti
2000).
Dragsdorf and Varriano-Marston (1980) concluded that there is
not a cause-and-effect relationship between starch crystallization
and bread firming. Their results agreed with those from earlier
work by Zobel and Senti (1959), who observed an increase in
crystallinity from bacterial a-amylase addition along with the typi-
cal reduction in bread firming (see “Enzymes” section), and postu-
lated that the observed antistaling/antifirming effects of bacterial a-
amylases were the result of cleavage of interconnecting (amor-
phous) chains in the crystalline starch network.
Ghiasi and others (1984) also stated that the degree of retrogra-
dation/crystallization of starch molecules was not closely related
to the staling rate of bread. Neither did changes in starch crystal-
linity upon reheating bread (DSC monitoring) correlate well with
changes in staleness. Furthermore, it was suggested that the de-
gree of softening of stale bread was temperature-dependent, and
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because the relationship was biphasic, that at least 2 mechanisms
were responsible for the staling and refreshening of bread (Ghiasi
and others 1984).
However, using DTA to examine retrogradation in starch
(source unstated) pastes, McIver and others (1968) determined
that the calculated Avrami exponent and time constant were in
general agreement with values found for bread (Axford and Col-
well 1967) and, therefore, concluded that starch retrogradation is
the major factor in bread staling. Colwell and others (1969) found
that the role of starch crystallization in the firming of bread be-
comes progressively less important at storage temperatures above
21 °C (70 °F).
Others have concluded that starch plays a role in strengthening
the structure of bakery products that is at least equivalent to that
of gluten (Gambus 2000) and that starch retrogradation alone is
sufficient to cause bread firming (Morgan and others 1997).
Although considerable evidence has been presented that there
is not a direct cause-and-effect relationship between starch poly-
mer molecule retrogradation and crumb firming, most researchers
believe that starch retrogradation is part of the staling process. As
stated earlier, our conclusion is that amylopectin retrogradation
plays a significant, but not the only, role in the stalling process.
See also the “Surface-active lipids” section for evidence on the
relationship between starch polymer retrogradation and bread
staling.
Role of flour protein. Protein is another component that has
been studied for its role in bread staling. Kim and D’Appolonia
(1977b) and others have reported that flour protein content is an
important factor in the rate of bread staling. It has been suggested
by different investigators that protein (gluten) reduces the firming
rate of bread during staling, has no effect on the firming rate, and
is required for firming; that is, that staling is dependent on starch-
gluten interactions. It is now generally believed (Martin and others
1991) that starch-gluten interactions are somehow involved in the
firming process.
Steller and Bailey (1938) reported an inverse relationship be-
tween protein content and bread staleness upon storage, al-
though the 2 were not linearly correlated. Others have also found
that increasing the protein level resulted in decreased crumb firm-
ness and crumb firming rate (Bechtel and Meisner 1954a; Pren-
tice and others 1954; Callejo and others 1999). Bechtel and Meis-
ner (1954a) concluded that staling is a result of 2 separate pro-
cesses: staling during the 1st 2 to 3 d of storage is a result of
changes in the organization of starch polymer molecules; thereaf-
ter, staling is caused by loss of moisture from gluten. Prentice and
others (1954) explained that increasing the protein content would
tend to decrease any association between starch granules (swol-
len and embedded in the gluten network), thereby retarding
crumb firmness development. They also suggested that gluten
may serve as a moisture reservoir to buffer any changes in the hy-
dration capacity of starch. However, since high-loaf-volume
breads are generally softer than those of low volume, their results
are difficult to explain (Kulp and Ponte 1981).
Willhoft (1973) suggested that the antifirming activity of gluten
was due to either a dilution of starch or the effect of gluten enrich-
ment on loaf volume. Erlander and Erlander (1969) suggested that
starch-gluten interactions could prevent staling of bread, possibly
via hydrogen bonding between the amide groups of wheat glia-
din, glutenin, and possibly albumin and hydroxyl groups of
starch. They concluded that the ratio of starch to protein in the
dough is important in determining the rate of staling and suggest-
ed that some staling will occur no matter how much protein is
added.
Kim and D’Appolonia (1977b) also reported that the rate of
bread staling is inversely related to the protein content of the flour.
However, Avrami exponent values suggested that the basic staling
 Bread staling. . .
mechanism was not affected by protein content, suggesting that
the rate of staling is independent of protein quality. So they con-
cluded that the primary effect of protein in reducing staling is dilu-
tion of starch.
Several investigators have concluded that crumb firmness is not
significantly correlated to flour protein type or concentration (Pon-
te and others 1962; Leon and others 1997; Gerrard and others
2001). By examining a novel starch bread that contained no glu-
ten, Morgan and others (1997) suggested that starch retrograda-
tion alone is sufficient to effect bread firming.
Every and others (1998) suggested that, qualitatively, starch-
starch and starch-protein interactions are of equal importance to
the staling mechanism, but that quantitatively, starch-starch inter-
actions are more important since conventional wheat flour con-
tains about 85% starch (db). They hypothesized that gluten is not
essential to the firming process and that increasing bread firmness
results from chains of partially leached amylose and amylopectin
attached to swollen, partially gelatinized starch granules interact-
ing via hydrogen bonds with other starch granule remnants and,
to a lesser degree, with gluten fibrils. Reconstitution experiments
revealed that breads of equivalent specific loaf volume staled at
the same rate irrespective of protein type or concentration, but
other bread properties were altered by changes in the type or
concentration of protein (Gerrard and others 2001), lending sup-
port to the above hypothesis.
Maleki and others (1980) postulated that the flour component
primarily responsible for differences in staling rate is gluten and
that its role in staling is something other than dilution of starch.
Furthermore, they proposed that starch and water solubles were
not involved significantly in determining the rate of staling.
Martin and others (1991) proposed that bread firming is a result
of hydrogen bonding between gelatinized (partially pasted) starch
granules and the gluten network in bread tying together the con-
tinuous protein network and discontinuous granule remnants.
They theorized that the crosslinking interactions originate during
baking; then during aging, the crumb loses kinetic energy, and
both the number of interactions and their strength increases.
When reheated, bread freshness is restored because the
crosslinks (hydrogen bonds) and entanglements between gluten
and starch polymer molecules are easily broken. This theory is
congruent with results of Dreese and others (1988), who reported
that starch and gluten molecules interact during baking.
Gerrard and others (1997) suggested a modification to the hy-
pothesis of Martin and others (1991). They agreed with the hy-
pothesis that staling is a result of increasing interactions between
swollen starch granules and the gluten network. However, they
put forth the opinion that the decrease in firming rate in breads
made with a-amylase (see “Enzymes: ␣-amylases and debranch-
ing enzymes” section) as a dough additive is not the direct result
of starch hydrolysis products (dextrins and maltooligosaccha-
rides), some of which, they suggest, are nonspecifically associated
with the protein matrix, but a result of modification of swollen
starch granules in such a way that their interaction with the pro-
tein network is reduced (presumably either qualitatively or quanti-
tatively).
Rogers and others (1988) reported that, even though shortening
and native lipids have significant effects on bread staling, neither
have major effects on starch retrogradation. They suggested for-
mation of protein-lipid interactions.
Role of pentosans. As mentioned in the “Nonstarch polyosac-
charides” section, the influence of the so-called pentosans on
breadmaking and bread properties is not clear, although the sub-
ject has been examined extensively (Kulp 1968; D’Appolonia
1971, 1980; Hoseney 1984; Meuser and Sukow 1986; Jank-
iewicz and Michniewicz 1987; Roels and others 1993: Rattan
and others 1994; Krishnarau and Hoseney 1994; Izydorczyk and
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Biliaderis 1995; Biliaderis and Izydorczyk 1995; Cleemput and
others 1997). Water-soluble and -insoluble pentosans have been
reported both to retard staling and to have no effect on the staling
rate.
“Water-insoluble” pentosans. No differences in staling rate of
breads made with or without tailings (starch fraction containing
9% water-insoluble pentosans) was observed by a sensory panel
(Bechtel and Meisner 1954b). Neither did Prentice and others
(1954) observe any effect on crumb firming rate due to tailings, al-
though initial crumb firmness was decreased, probably due to the
high hydration capacity of pentosans. However, others found that
addition of water-insoluble pentosans resulted in a considerable
increase in loaf volume (Kulp 1968) and retardation of bread stal-
ing (Casier and others 1972, 1973; Denli and Ercan 2001). To
add more confusion, addition of insoluble pentosans was report-
ed to reduce bread quality, which could be overcome with addi-
tion of an optimum amount of pentosanase (Krishnarau and
Hoseney 1994). Such variable results may result from differences
in type, molecular weight, and/or concentration of the pentosans
present in the formulation.
“Water-soluble” pentosans. Contrary to the reports of less ben-
eficial effects of water-soluble pentosans (as compared to water-in-
soluble pentosans), reports which were not confirmed by Kulp
and Bechtel (1963) or Hoseney and others (1971), Michniewicz
and others (1992), like Jelaca and Hlynka (1972), found that wa-
ter-soluble pentosans had a significant positive effect on loaf vol-
ume and that water-insoluble ones did not, that water-soluble
pentosans retarded amylose aggregation, and that addition of wa-
ter-insoluble pentosans decreased susceptibility of bread crumb
to a-amylase. They suggested that the contradictory results ob-
tained when studying the effects of pentosans on loaf volume may
have originated in differences in baking characteristics of the
flours of various wheat cultivars, differences in chemical composi-
tion of pentosans, and/or the way pentosans were incorporated
into the dough. They further suggested that the reported reduction
in bread firmness upon storage when the dough was supplement-
ed with pentosans, as observed by Kim and D’Appolonia (1977d)
and others, may have been a direct consequence of a higher
moisture content of the system.
Interaction of pentosans with protein. It is possible that pen-
tosans can interact with wheat-flour components other than
starch. Jelaca and Hlynka (1972) proposed that pentosan-gluten
interactions were responsible for baking improvement effected by
pentosans. Based on the effects of actions of arabinoxylanases,
Cleemput and others (1997) suggested that there are associations
of arabinoxylans with proteins and/or other wheat components in
doughs.
Pentosans and starch retrogradation. The effect of pentosans
on starch retrogradation has been investigated using both starch
gels and bread itself. Gilles and others (1961) reported that water-
soluble pentosans found in the “soluble starch” extract of bread
crumb inhibited retrogradation of amylose and that, although the
pentosans affected some characteristics of the bread, staling rate
was not one of those characteristics. Kim and D’Appolonia
(1977a) found that pentosans had a definite effect on retarding
starch retrogradation in wheat starch gels, with the effect of water-
insoluble pentosans being more pronounced. They reported that
water-soluble pentosans reduced retrogradation by acting on
amylopectin, while water-insoluble pentosans reduced the degree
of retrogradation of both amylose and amylopectin. Similar results
were found when the effect of pentosans on staling was studied in
a bread system (Kim and D’Appolonia 1977d). Results indicated
that the basic mechanism of bread staling was unchanged; thus, it
was suggested that pentosans decreased the staling rate by reduc-
ing the amounts of starch components available for retrogradation
(Kim and D’Appolonia 1977d). However, others have concluded
5
 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
(based on calorimetry) that arabinoxylan-fortified breads exhibited
a greater rate of starch retrogradation (Biliaderis and Izydorczyk
1995), because of their higher moisture content (Rogers and oth-
ers 1988), while having softer crumbs than did controls.
Role of native lipids. Most reports on effects of lipids in prepar-
ing baked products discuss their effects on baking characteristics
(MacRitchie and Gras 1973; MacRitchie 1981), rather than on
crumb firming. The effects of native lipids are discussed briefly
here; added surfactants used as antistaling agents are discussed in
the “Surface-active lipids” section.
Flour lipid content has been shown to be inversely related to
loaf volume (Rogers and others 1988). Protein-lipid interaction
has been suggested as the mechanism. Shortening is known to
lower the firming rate of bread, but does not react with starch
(Rogers and others 1988). Therefore, the results suggest that native
flour lipids have an effect on the antifirming action of shortening.
While both native lipids and shortening affect firming rates signifi-
cantly, neither have significant effects on starch retrogradation.
Davidou and others (1996) reported that complexes between na-
tive lipids and amylose were formed within the 1st 2 d of storage
and that such complex formations appeared to reduce the maxi-
mum amount of starch retrogradation. However, thermodynamic
considerations indicate that amylose-lipid and amylose-surfactant
(see “Surface-active lipids” section) complexes are formed during
baking, since they form at temperatures higher than 60 °C and
melt at temperatures higher than 100 °C (Zobel and others 1988).
Summary. Bread staling is unquestionably a complex process.
While the mechanism of staling is still not understood, certain
ideas have been accepted, such as the important role of starch
retrogradation, specifically amylopectin retrogradation. Even so, it
is becoming increasingly evident that amylopectin retrogradation
alone is not responsible for bread staling, but it is unclear what
other bread components and processes contribute to the overall
staling process. Evidence has accumulated that gluten proteins
are important and that gluten-starch interactions play a role. Mois-
ture transfer (discussed in the “Moisture migration” section) seems
also to be involved in staling. In conclusion, it is probable that
several factors play a role in the bread firming process, but the
large volume of data that implicates amylopectin retrogradation as
a key factor, and the information that gluten is also involved can-
not be ignored.
Other Factors Affecting Staling Rate
Storage temperature
An interesting feature of bread is that the rate of staling has a
negative temperature coefficient (Colwell and others 1969). Thus,
the rate of bread staling is accelerated at lower storage tempera-
tures. Bread staling was correlated with starch recrystallization at
storage temperatures of –1, 10, and 21 °C, while the role of starch
crystallization in staling was diminished at higher temperatures
(32 and 43 °C).
Processes have been developed to quick-chill bakery products,
then allow them to stabilize to ambient conditions in order to re-
duce staling when the product is held at room temperature (Will-
iams and others 1995).
Freezing retarded firming, the effect being greater the longer the
frozen storage time. The effect of freezing was additive with the ef-
fect of monoglyceride addition (Malkki and others 1978).
Polymer crystal growth theory states that there are 3 phases to
polymer crystallization: nucleation, propagation, and maturation.
Slade and Levine (1987) and Marsh and Blanshard (1988) have
determined that amylopectin recrystallization, at least in concen-
trated pastes, is a nucleation-limiting process occurring at a tem-
perature above the glass transition temperature (Tg), or the glass
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transition temperature of the maximally freeze-concentrated
starch (Tg’) (about –5 °C) when the starch concentration is < 70%,
and below the melting temperature (Tm) of crystalline amylopectin
(about 60 °C). The maximum rate of nucleation occurs at tempera-
tures slightly greater than Tg (or Tg’ depending on concentration),
while the maximum rate of propagation occurs at a temperature
slightly less than the Tm of crystallized amylopectin. The retrogra-
dation rate of starch pastes held under isothermal conditions is
greatest at a temperature between the optimal temperatures for
nucleation and propagation (about 5 °C for a 50% paste) (Slade
and Levine 1987; Marsh and Blanshard 1988). The situation may
be somewhat different in bread, but temperature cycling is used to
accelerate the staling of bread in the production of croutons
(Slade and others 1987). The very fact that proper temperature cy-
cling is so effective in accelerating bread firming is strong support
for the involvement of starch polymer crystallization. The fact that
staled bread can be resoftened by reheating is additional support.
Slade and Levine (1987) also come to the conclusion that 4 °C
(refrigerator temperature) is the single optimum temperature be-
tween Tg and Tm that balances nucleation and crystallization and
that the melting temperature involved implicates amylopectin as
the polymer crystallizing.
Moisture migration
Water is involved in the following changes in the bread system:
drying out, moisture equilibration between crumb and crust, and
moisture redistribution between and among bread components
(Kulp and Ponte 1981). Drying out of the bread, as demonstrated
by Boussingault (1852), does not explain staling, but may acceler-
ate reactions leading to staling (MacMasters 1961). Thus, moisture
relationships within the crumb are important considerations when
studying bread staling.
Breadmakers in the U.S.A. are limited to 38% water for white
pan bread even though breads containing higher levels of mois-
ture generally stale more slowly (Kulp and Ponte 1981). This in-
verse relationship between moisture content and staling rate was
confirmed (Rogers and others 1988; He and Hoseney 1990),
even though the rate of starch retrogradation in bread was found
to be directly proportional to the moisture content (Rogers and
others 1988). Zeleznak and Hoseney (1986) confirmed that retro-
gradation in wheat starch gels was a function of the amount of
water present. They also reported that the moisture content of
bread is about optimal for amylopectin retrogradation and that
addition of either monoglycerides or shortening did not alter the
available moisture content.
Schiraldi and Fessas (2001) focus their review on water content
(on which the mobility of polymer chains is dependent), water ac-
tivity, water migration between phases, and the alveolar crumb
structure of bread. Their conclusion is that “The overall picture of
the crumb could be described as interpenetrated gels separated
by aqueous interphases which contain most of the low molecular
weight solutes. This water is rather mobile and can facilitate mutu-
al displacement of the incompatible gel phases, thus behaving as
a plasticizer, and can enhance the crumb-to-crust migration of
moisture. This local drying makes the walls of the crumb alveoli
more rigid, while the concurrent moisture increase within the
crust region is accompanied by a reduction of crispness even
when overall moisture loss is prevented by packing bread in
sealed bags (Piazza and Masi 1995). Along its way toward the
crust, water can contribute to a closer packing of the structure
through which it is moving, either within a given phase or at the
interphases, by tightening the sites able to form H bonds. This
would explain why refreshed bread softens when its temperature
has been raised above Tg, but then becomes harder than the start-
ing staled product, and why microwave-cooked or refreshed
bread shows a fast firming without significant enhancement of
 Bread staling. . .
amylopectin crystallization”.
Crumb-crust redistribution of moisture. As baked bread be-
gins to cool, a moisture gradient forms in the loaf (Piazza and
Masi 1995). Differences in vapor pressures between the crust and
the internal region of the loaf result in moisture migration from the
crumb to the crust (Stear 1990). Over time, the moisture content
in the center of the loaf decreases, while that in the external region
increases (Bechtel and others 1953).
Baik and Chinachoti (2000) found that bread stored with its
crust became significantly firmer than bread stored without its
crust and contained more recrystallized amylopectin, indicating
that moisture redistribution from crumb to crust plays a significant
role in firming, a conclusion confirmed by a loss in freezable wa-
ter in the crumb of bread stored with crust, which correlated with
changes in its thermomechanical profile.
Several NMR parameters correlate with crumb firming and are
believed to be related to both microscopic and macroscopic re-
distribution of water (Chen and others 1997b). Using NMR tech-
niques, it has been found that, as staling proceeds, the water in
bread becomes less and less mobile (Leung and others 1983;
Wynne-Jones and Blanshard 1986; Kim-Shin and others 1991;
Chen and others 1997a,b; Engelsen and others 2001). However,
Ruan and others (1996), using MRI, found that, as storage time of
sweet rolls increased, mobility of the less-mobile water fraction
decreased, while mobility of the more-mobile fraction increased.
Moisture redistribution among components. Transfer of mois-
ture from one constituent of the bread crumb to another is general-
ly accepted as a contributing factor in staling, possibly being re-
sponsible for the perceived dryness of stale bread (Senti and Dimler
1960). Water is a plasticizer, making the bread components more
flexible. Thus, as water is removed (from either gluten or starch or
both), increasing crumb firmness should occur. Whether staling in-
volves dehydration of gluten or starch has been studied extensively,
but is still unclear. However, the majority of evidence suggests a
gluten to starch transfer of water as the starch crystallizes.
Katz (1928) first suggested that, during staling, moisture was re-
leased from starch and taken up by gluten. Senti and Dimler (1960),
by studying equilibrium relative humidities, also suggested that
moisture transfer would likely occur from starch to gluten. Cluskey
and others (1959) reported a progressive drop in moisture-sorption
capacity for starch and lack of a change for gluten, indicating a
transfer of moisture from starch to gluten during aging.
In contrast, Alsberg and Griffing (1927) and Alsberg (1936) pos-
tulated that it was the gluten that hardened as result of moisture
loss to starch. This concept is supported by data of Bachrach and
Briggs (1947), who observed an increase in moisture-sorption ca-
pacity of gelatinized starch upon aging [contrary to the results of
Katz (1928) and Cluskey and others (1959)]. Further evidence
came from investigations by Willhoft and coworkers (Breaden and
Willhoft 1971; Willhoft 1971; Kay and Willhoft 1972), who re-
ported that gluten undergoes a 1st-order transformation resulting
in the release of water from gluten and absorption of this water by
retrograding starch.
The notions of “free” and “bound” water have been reported to
be of importance in altering the rate or extent of staling in bread
(Knjaginciev 1970). More recently, the use of NMR and a greater
understanding of the role and mechanism of starch polymer crys-
tallization have led to the conclusion that starch takes up water
from gluten upon aging of bread. Leung (1981) and Leung and
others (1983) proposed that, as starch changes to a more crystal-
line state, more water molecules become immobilized due to their
incorporation into crystal structures. Chen and others (1997a,b)
reported a decrease in water mobility in bread upon staling, in
agreement with results of others (Wynne-Jones and Blanshard
1986; Slade and Levine 1991), and concluded that the decrease
in water mobility was due to incorporation of water molecules re-
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leased from gluten into crystalline structure of starch that devel-
oped upon staling. [Note: The B structure has 36 water molecules
in the unit cell, whereas the A structure has only 8 (Sarko and Wu
1978).] Conversely, Kim-Shin and others (1991) proposed that the
redistribution of water occurs in the amorphous phase. The ratio
of starch to gluten (6:1) in bread crumb ensures that moisture
transfer to the starch would result in firming of the continuous glu-
ten phase (Willhoft 1971). It is important to keep in mind at all
times, however, that the change in the state of water cannot be
correlated directly to the retrogradation process (Wynne-Jones
and Blanshard 1986).
Levine and Slade (1990) and Slade and Levine (1991) present
thorough and well-documented evidence for the role of water in
the staling process. Their arguments are based upon the mecha-
nism of polymer crystallization, polymer crystallization kinetics as
a function of glass transition and melting temperatures, water as a
plasticizer, and sugars as antiplasticizers in the system. In their re-
view, Slade and Levine (1991) state essentially that “if adequate
packaging prevents simple moisture loss, the predominate mecha-
nism of staling in bread crumb is the time-dependent recrystalliza-
tion of amylopectin from the completely amorphous state of a
freshly heated product to the partially crystalline state of a stale
product, with concomitant formation of network junction zones,
redistribution of moisture via both microscopic and macroscopic
migration (Czuchajowska and Pomeranz 1989), and increased
textural firmness (Kulp and Ponte 1981; Russell 1983b; Russell
1987).” They further point out that there is evidence from studies
of starch gels/pastes that the rate and extent of amylopectin crys-
tallization depends on the mobility of its outer branches (Ring and
others 1987; Russell 1987; Marsh and Blanshard 1988; Slade
and Levine 1989, 1991) and on sample history, since the pro-
cesses that occur both during heating/baking and during aging/
storage are nonequilibrium processes (Ring and others 1987;
Slade and Levine 1989, 1991). [Note: Slade and Levine refer to
recrystallization of amylopectin, and indeed it is a recrystalliza-
tion. We have not used the term elsewhere in this review so as to
make it clear that amylopectin molecules do not recrystallize to
the same crystalline state that they were originally in nongelati-
nized granules.]
Amylopectin crystallization results in a partially crystalline, su-
permolecular structure containing disperse B-type crystalline re-
gions (Slade and Levine 1987). Incorporation of water molecules
into the crystal lattice occurs during formation of the B-type poly-
morph (Imberty and Perez 1988) and, thus, a redistribution of
moisture is effected. This process was demonstrated by a progres-
sive decrease in the percentage of “freezable” water as bread was
stored over 11 d (Slade and Levine 1991). The water molecules
that are part of the crystal lattice are not available for plasticiza-
tion, so the result is the perceived drier, firmer texture characteris-
tic of stale bread. So, all in all, amylopectin crystallization in bread
requires both microscopic and macroscopic redistribution of wa-
ter so that there is sufficient moisture present at the locus where
crystallization takes place to plasticize polymer chains so that
they are mobile enough for crystallization to occur and for incor-
poration into B-type crystal latices (Levine and Slade 1990; Slade
and Levine 1989, 1991).
It seems clear that moisture transfer between bread compo-
nents, specifically between gluten and starch, occurs as bread
ages. However, like other measurable changes in the nature of
bread components, the role, if any, of moisture and moisture re-
distribution in the staling process remains undetermined. (See
also “Mechanisms of staling: Role of pentosans” and “Carbohy-
drate ingredients” sections).
Processing factors
Effects of technological factors, which include manufacturing
7
 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
methods, formulas, and operational steps, on both loaf character-
istics and bread staling have been compiled by Kulp and Ponte
(1981) with information from the American Institute of Baking.
Swortfiguer (1971) and Maga (1975) also discuss these variables
in reviews.
Giovanelli and others (1997) showed that baking temperature
significantly affects bread staling. Bread baked at lower tempera-
tures stales at a slower rate in terms of both crumb hardening and
starch retrogradation. Higher baking temperatures led to in-
creased protein denaturation and starch granule disruption.
[Note: This is a little puzzling, since as long as there is water
present in the bread, the temperature inside the loaf cannot go
above the boiling temperature of that water, no matter what the
oven temperature. The oven temperature can, however, affect the
rate of temperature rise and, thus, the time at the maximum tem-
perature.] The authors suggested baking under slight vacuum to
achieve crumb cooking at temperatures < 100 °C, which may en-
hance the shelf life of bread.
In a study of the effects of processes, Axford and others (1968)
found that the rate and extent of staling decreased as the loaf vol-
ume increased in bread stored at the same temperature and that
breads made with the same dough ingredients, but by different
processes (and stored at the same temperature), underwent staling
at different rates because of differences in loaf volume.
Antistaling Additives
Enzymes
One strategy to reduce the rate of bread staling employs en-
zymes. The enzyme supplements labeled as amylases and pro-
teases are most commonly used in commercial baking (Miller and
others 1953; Waldt 1968, 1969; Martinez-Anaya 1998; Bowles
1996). The most useful enzymic approach to staling rate reduc-
tion has been the use of ␣-amylases, which catalyze a small
amount of hydrolysis of the starch. Proteases depolymerize gluten
proteins and modify baking characteristics. Nonamylolytic en-
zymes may also be active in the enzyme supplements (van Eijk
and Hille 1996). [Note: While many enzymes are useful in as-
pects of breadmaking other than in reducing crumb firmness,
only enzymes useful as antistaling agents are discussed below.]
␣ -Amylases and debranching enzymes. Numerous studies
have reported that the rates and degrees of firming in baked
goods can be reduced; and the texture, flavor, aroma, and general
qualities improved; by use of a-amylases. Fungal, cereal, and bac-
terial ␣-amylases all appeared to improve softness retention of
bread to an extent related to their heat stability (Conn and others
1950; Miller and others 1953). Fungal ␣-amylase was inactivated
by heat before acting on the starch. Although cereal (wheat or bar-
ley) a-amylases did not survive the baking process, they had time
to act on the swollen starch. A bacterial a-amylase was able to
partly survive the heat treatment (Amos 1955). [Note: after this
work was reported, intermediate thermostable bacterial ␣-amylas-
es became available. See below.] In any case, major ␣-amylase
activity takes place during baking after the starch is gelatinized
and becomes more susceptible to the enzyme (Ghiasi and others
1979); there is a specific temperature range and time in the bread-
making process when the enzyme is most active in degrading
starch (Martin 1989).
Waldt and Mahoney (1967) reported that, when bacterial a-
amylase was used, the freshness of 4-d-old bread was equivalent
to that of 2.0 to 2.5-d-old untreated bread, but it has been report-
ed that, when bacterial ␣-amylase derived from Bacillus subtilis is
used in a bread formulation, a gummy texture results (because it
can survive baking) (Hebeda and others 1991). Fungal a-amylases
(such as that from Aspergillus oryzae) are less thermostable than
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are bacterial ␣-amylases (Miller and others 1953).
Commercial ␣-amylases with intermediate thermostability char-
acteristics, known as intermediate-temperature-stable (ITS) en-
zymes, are now available. Though obtained from different micro-
bial sources, the various ITS enzymes exhibit similar thermostabil-
ity profiles (Hebeda and others 1991). ITS enzymes have thermo-
stabilities and temperature optima between those of fungal ␣-amy-
lases and conventional bacterial ␣-amylases. Fungal ␣-amylases
have temperature optima of 50 to 55 °C; bacterial ␣-amylases
have optimum activity near 75 °C. The maximum activity of ITS
enzymes occurs at about 65 to 70 °C. Thus, ITS enzymes have
optimal activity at or slightly above the gelatinization temperature
of wheat starch, but are inactivated by the 100 °C baking temper-
ature (Hebeda and others 1991).
Addition of Aspergillus ITS ␣-amylase increased the shelf life of
bread 38 to 75%. When the point in the process where enzyme is
added was optimized, the Aspergillus ITS enzyme increased shelf
life by as much as 200%. B. megaterium ITS ␣-amylase increased
shelf life by 15 to 33% (Hebeda and others 1991).
Rosell and others (2001) determined that commercial ␣-amylas-
es from different sources (wheat flour, malted barley, fungi, bacte-
ria) were strongly affected to different degrees by process condi-
tions and the presence of other ingredients in the dough.
Lent and Grant (2001), in a comparison of bagel ingredients (␣-
amylase, a modified food starch, xanthan, and a hydrated
monoglyceride), found that the a-amylase was the most effective
in retarding staling as determined by DSC analysis.
The mechanism of the antistaling effect of ␣-amylases has been
debated. At first, ␣-amylases were thought to affect the staling rate
of baked products via modifications of the structure of starch
(Maga 1975). Various techniques have shown that use of com-
mercial “antistaling” ␣-amylase preparations reduces both the rate
of starch retrogradation and the rate of crumb firming (Morgan
and others 1997; Champenois and others 1999). However, re-
sults from several studies indicate that the degree of starch crystal-
linity and the degree of firmness are not correlated (Champenois
and others 1999). Results from use of an “antistaling” a-amylase
and characterization of the properties of the resulting crumb by a
variety of techniques led Hug-Iten and others (2001) to conclude
that the antistaling effect of the enzyme preparation was due to its
ability to produce a partially degraded amylopectin that is less
prone to crystallize, and that its ability to produce partially de-
graded amylose is responsible for rapid formation of a partially
crystalline polymer network (in fresh bread) that resists later rear-
rangements.
Schultz and others (1952) suggested that the beneficial effect of
␣-amylase in reducing staling was due to production of low-mo-
lecular-weight dextrins that interfered with the retrogradation of
starch. Zobel and Senti (1959) also proposed that dextrins dis-
rupted the continuity of the starch network and reduced its rigidi-
ty. Akers and Hoseney (1994) agreed that dextrins produced from
␣-amylases are important in controlling the rate of bread firming.
They reported that ␣-amylases from different sources reduced the
rate of crumb firming to different degrees. They also extracted the
water-soluble hydrolysis products from aged crumb of breads
made with the different enzyme preparations, examined them by
HPLC, and found that certain peak areas were highly correlated
with a reduced rate of crumb firming and that other peaks were
highly correlated with an increased rate of crumb firming.
Leon and others (1997) also attributed the antifirming effect of
␣-amylases to hydrolysis products. Finding that incorporation of a
mixture of ␣-amylase and pullulanase caused bread to firm at a
faster rate, while use of the ␣-amylase alone retarded firming, Mar-
tin and Hoseney (1991) concluded that hydrolysis products of a
particular size were responsible for the reduced rate of firming.
Lin and Lineback (1990) found that a bacterial a-amylase pro-
 Bread staling. . .
duced mainly low-molecular-weight, branched dextrins of DP 19-
24 that either had less ability to retrograde, interfered with amy-
lopectin retrogradation, or interfered with whatever other interac-
tions are responsible for crumb firming.
Duran and others (2001) attributed the antistaling effect of ␣-
amylases to the production of maltooligosaccharides. Biliaderis
and Prokopowich (1994) found that maltose and maltotriose had
antiretrogradation effects on starch gels and proposed that the
chain ordering of amylopectin in sugar-containing starch gels is a
function of the compatibility of the sugar with the structure of wa-
ter. Solutes that fit well in the water structure retarded chain reor-
dering. On the other hand, solutes that disturb water structure
promoted ordering and aggregation of starch molecules. Maltotri-
ose, which was reported to be the most effective maltooligosac-
charide in impeding retrogradation, disturbs the structure of water
only slightly (Biliaderis and Prokopowich 1994).
Defloor and Delcour (1999) reported that starch hydrolysis
product preparations with average DPs of from 4 to 66 reduced
DSC staling endotherms in baked and stored bread doughs. They
attributed their antistaling effect to a reduction in starch recrystalli-
zation but did not speculate about a mechanism.
Martin and Hoseney (1991) proposed that low-molecular-
weight dextrins (maltooligosaccharides) produced by a-amylases
were directly responsible for the antistaling phenomenon ob-
served by enzyme addition. Their explanation was that the low-
molecular-weight products inhibited cross-link formation between
starch and gluten.
Min and others (1998) studied the effect of 2 novel antistaling
amylases. When added to bread, they produced selectively either
maltose and maltotriose or maltotetraose and maltotriose. Based
on the results, they postulated that maltotriose and maltotetraose
were directly responsible for retarding retrogradation in bread,
suggesting that these oligomers were of the right size to interfere
with starch-gluten interactions [theory of Martin and others (1991)
and Martin and Hoseney (1991) on the mechanism of staling].
Maltose was found to be less effective in bread staling prevention,
and it was suggested that its relatively small size and its ability to
diffuse easily might be the reason why it was less effective than
maltotriose or maltotetraose (Min and others 1998). Donnelly and
others (1973) reported that there is a slight decrease in moisture
adsorptive capacity as the molecular size of maltooligosaccha-
rides increases from DP 3 to DP 11, and that maltose was the ex-
ception, being less hygroscopic than was the DP 11 maltooli-
gosaccharide. This led Min and others (1998) to conjecture that
maltotriose and maltotetraose might hold water around starch
molecules and inhibit starch-starch interactions more than mal-
tose does.
Despite conclusions that dextrins directly affect staling in bread,
considerable evidence has been published to the contrary. Salem
and Johnson (1965) found, from experiments in which starch hy-
drolysis products were added to a bread dough formula, that cer-
tain maltooligosaccharides (such as maltohexaose and -heptaose,
as compared to glucose, maltose, and maltotriose, -tetraose, and -
pentaose) and dextrins increased the rate of crumb firming, in
contrast to results obtained when ␣-amylase was incorporated as
an additive. However, in contrast, Every and others (1992) found
that maltooligosaccharides of DP 3-10 correlated with a reduction
in firming rate, and Akers and Hoseney (1994) implied that starch
hydrolysis products of a size greater than maltoheptaose might
have antifirming properties.
There is a 3rd conclusion. Because added maltooligosaccha-
rides did not survive fermentation and because the presence of
maltooligosaccharides of a specific size class could not be corre-
lated with the firming rate of bread, Gerrard and others (1997)
concluded that maltooligosaccharides (DP 3-8) produced by ␣-
amylases are not themselves responsible for antistaling, but that
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their presence is simply correlated with a key modification of
starch granules that is related to reduced staling, possibly by re-
ducing gluten-starch interactions. Duedahl-Olesen and others
(1999) also reported that maltooligosaccharides of average DP up
to 20 had no effect on formation of the staling endotherm. Inter-
estingly, they did find that amylopectin recrystallization was re-
duced significantly when ␥-cyclodextrin (3%) was incorporated.
Slade and Levine (1987) and Levine and Slade (1990) studied
maltooligosaccharides of DP 3 to 8 at relatively high concentra-
tions (1:1, oligosaccharide:water). Their conclusion was that the
reported antistaling effect could be explained by an impact on the
Tg, resulting in a smaller ⌬T above Tg, which retarded the starch
crystallization process. Further, they reported a relationship be-
tween an increase in Tg and the degree of staling (Slade and Le-
vine 1991). The correlation of an increase in Tg during staling with
the firming of bread was confirmed by Jagannath and others
(1999a).
Dragsdorf and Varriano-Marston (1980) studied the effects of
barley malt, fungal ␣-amylase, and bacterial a-amylase on starch
crystallization and organization in staling breads using x-ray dif-
fraction (see also Akers and Hoseney 1994). Comparing stored
and fresh breads, they found that the degree of crystallinity of
breads baked with different enzyme sources was in the order bac-
terial ␣-amylase > cereal ␣-amylase > fungal ␣-amylase > control.
These results were in opposition to bread firming data, suggesting
to them that starch crystallization and bread firming are different
and separate processes. Their results agreed with those obtained
by Zobel and Senti (1959), who suggested that bacterial ␣-amylas-
es inhibit staling by breaking interconnecting chain associations
in the network of starch crystallites.
Retardation of bread staling, while avoiding a gummy mouth-
feel, was achieved by incorporating pullulanase with a cereal or
bacterial ␣-amylase in the dough (Carroll and others 1987). A
product produced by action of pullulanase or isoamylase on
starch, which the inventors refer to as low-molecular-weight amy-
lose, but which is in reality a mixture of released branch chains,
when added to a dough formulation, was reported to have an an-
tistaling action (Yoshida and others 1972).
All in all, it appears that starch hydrolysis products are involved
in inhibition of staling, but that the products must be of a unique
type, perhaps either maltotriose and maltotetraose or products
larger than those present in traditional maltodextrin preparations.
However, that the presence of such products is only correlated to
some other modification in starch (or another component) that is
the real determinant cannot be ruled out.
Lipases. Although Johnson and Welch (1968) patented lipase
formulations that retard staling in bread, the use of lipases for
breadmaking was virtually unknown until recently (Qi Si 1997).
Depending on the type of flour and the formula, addition of some
1,3-specific lipases resulted in more uniform crumb structure and
thus an improvement in crumb softness during storage (among
other dough conditioning improvements). Furthermore, these li-
pases were shown to be a replacement for shortening, although
no improvement in crumb elasticity was found.
Siswoyo and others (1999) found that, while use of a purified li-
pase alone retarded retrogradation in bread crumb, use of a com-
bination of a purified lipase and a purified a-amylase reduced ret-
rogradation to a much greater extent. [Note: Since commercial en-
zyme preparations are rather crude and probably contain both
activities, this combination could unknowingly be involved in the
antistaling activity.]
Qi Si (1997) suggested that the mechanism of retrogradation re-
tardation does not involve the most obvious explanation: hydrol-
ysis of lipids to monoglycerides, which are reported to have anti-
staling characteristics. [Note: Tri- and diacylglycerols do not de-
crease crumb firmness, but monoacylglycerols/monoglycerides
9
 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
do (see “Surface-active lipids” section). However, monoglycerides
lack the positive dough-conditioning effects produced by lipases,
and insufficient lipids are present in wheat flour to result in
enough monoglycerides to achieve an antistaling effect. Finally,
only 1st and 3rd position monoglycerides can complex with
starch, thus retarding staling; and the 1,3-specific lipase would
produce 2nd position monoglycerides (Qi Si 1997). There is no
evidence that an effect produced by the released free fatty acids
has been considered.]
Lipoxygenases. Lipoxygenase is reported to have a crumb soft-
ening effect when active in bread (van Eijk and Hille 1996). A ma-
jor source of lipoxygenase is from enzyme-active soy flour, a
common ingredient in breads. While action of the enzyme on the
structure of lipids could explain the crumb-softening effect, it is
also likely that changes in protein conformation via oxidation of
gluten are partially responsible (Daniels and others 1970; Frazier
and others 1973). Lipid peroxide intermediates produced by the
action of lipoxygenase on polyunsaturated lipids may react with
protein sulfhydryl groups to produce protein-bound lipids, which
may subsequently be released by oxidation of the protein.
Nonstarch polysaccharide-modifying enzymes. As discussed
in the “Mechanisms of staling: Role of pentosans” section, the in-
fluence of pentosans on bread properties, including the rate of
staling, is unclear. It is also unclear whether enzymes that degrade
nonstarch polysaccharides in bread have any effect on bread stal-
ing (van Eijk and Hille 1996). “Pentosanses” (hemicellulases) are
well-known dough conditioners in Europe and have reportedly
been used to increase loaf volume through improved dough ma-
chinability and overspring (Qi Si 1997). Fungal enzyme prepara-
tions with high endoxylanase, ␤-xylosidase, and ␣-L-arabinosi-
dase activities delayed bread staling considerably without affect-
ing porosity or loaf volume (Rodionova and others 1995).
Proteases. The role, if any, of proteases in the mechanism of
bread staling has not been investigated thoroughly. The purpose
of adding them to breads is to improve flavor profiles, flow char-
acteristics, machining properties, gas retention, and mixing time
(Barrett 1975; Mathewson 2000). However, given evidence that
protein has a significant role in the bread staling mechanism (Mar-
tin 1989; Martin and others 1991; Martin and Hoseney 1991), it
is likely that modification of the gluten network structure via en-
zyme-catalyzed proteolysis would have an effect on bread staling.
It is also possible that liberation of water molecules concurrent
with protein hydrolysis could enhance amylase activity (Schwim-
mer 1981). Alternatively, proteases could inhibit amylolysis if they
catalyzed the degradation of a-amylase molecules.
Sahlström and Bråthen (1996) reported that addition of a com-
mercial a-amylase product with protease activity resulted in a soft-
er crumb over a shorter time period compared with breads made
with ␣-amylase addition alone. [Note: Both ingredients most likely
contained proteases, though proteolytic activity was not tested.
The former product was marketed as a dual-function enzyme, and
it probably contained a significantly greater level of proteolytic ac-
tivity. Whether or not the reduction in crumb firmness was due to
its proteolytic activity is unknown].
Techniques for differential inactivation of ␣-amylase and pro-
tease from malted wheat and fungal sources were developed by
Miller and Johnson (1949). Results from their use led them to con-
clude that ␣-amylase alone might be less effective in creating im-
provements in texture as compared to addition of both protease (in
small amounts) and ␣-amylase activities (Johnson and Miller 1949).
They also concluded that, while ␣-amylase was the component of
malt mainly responsible for increasing crumb compressibility after
66 h of storage, protease alone (at low concentrations) increased
the compressibility of crumb over that of the controls.
Van Eijk and Hille (1996) concluded that, while the addition of
excess concentrations of proteolytic enzymes would certainly be
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crfsfsv2n1ms20010513-AF-af.p65 10
detrimental to the bread loaf, adding optimal levels of proteases to
breads might increase their shelf life. If so, the presence of con-
taminant levels of proteases in commercial ␣-amylase prepara-
tions might partially explain the currently unresolved mechanism
of antistaling (Gray and BeMiller 2001). In fact, since commercial
enzymes, including ␣-amylases, and enzyme blends have activi-
ties in addition to the stated one(s) (Silberstein 1961; Hebeda and
others 1991), the possibility that the presence of other activities
(that is, lipases, xylanases, and so on) in commercial enzyme
preparations might have an effect on bread staling cannot be
ruled out. Identification and characterization of such contaminant
activities would be useful.
Surface-active lipids
Most studies with lipids have been concerned with improving
functional properties of bread (D’Appolonia and Morad 1981).
Emulsifiers of various types are widely employed in the baking in-
dustry as dough strengtheners and/or crumb softeners (Kulp and
Ponte 1981), but their role in staling has not been established. Ex-
amples of surfactants used in breads as antistaling agents are pre-
sented briefly, followed by a review of research on the mechanism
of surfactants in reducing the rate of staling. Several more detailed
reviews of the use of emulsifiers in breadmaking, including a dis-
cussion on the role of emulsifiers as antistaling agents, have been
published (Knightly 1968, 1973, 1996; Morrison 1976; Krog
1981; Stampfli and Nersten 1995).
Most studies of amylose-lipid complexes involve complexes
formed in dilute solutions of amylose (see, for example, Biliaderis
and others 1985, 1986; Biliaderis and Galloway 1989; Biliaderis
and Seneviratne 1990; Seneviratne and Biliaderis 1991) and oc-
casionally in concentrated starch gels (see, for example, Biliaderis
and Tonogai 1991). Details of the structures of amylose-fatty acid
complexes, based on date from solid-state 13C CP/MAS and deu-
terium NMR, x-ray powder diffraction, and DSC analysis, have
been proposed (Lebail and others 2000).
Surfactants. Diacetyl tartaric acid esters of monoglycerides
(DATEM). DATEM surfactants (0.05%) were reported to be as ef-
fective as antistaling agents as SSL (see “Sodium stearoyl lactylate
(SSL)” section, below) or ethoxylated monoacylglycerols over 5 d
of storage (Rogers and Hoseney 1983), but to be less effective
[compared to monoglycerides (see “Polyoxyethylene monostear-
ate (POEMS)” section below)] in reducing retrogradation of amy-
lopectin and in forming complexes with amylose, while at the
same time reducing crumb firming (Krog and others 1989). It was
suggested that the antifirming properties of DATEM may be due to
changes in cell wall thickness and elasticity effected by it. It was
further reported that optimal reduction in firmness increase over
extended periods of storage can be achieved when DATEM is
used in combination with monoglycerides.
Lecithins. Lecithins have been reported to reduce staling and to
have the advantage of being amenable to modification for specific
applications (Forssell and others 1998). Soy lecithin hydrolyzate
effectively retarded crystallization in starch gels and bread staling.
Oat lecithin retarded staling significantly more than did soy leci-
thin, but did not affect crystallization in starch gels (Forssell and
others 1998).
Monoglycerides (MG). Most bakeries use mono- or diacylglyc-
erols to delay staling in bakery products (Huang and White 1993).
While Schoch and French (1947) first proposed the use of
monoglycerides (properly termed monoacylglycerols) in the form
of “superglycerinated shortening” to inhibit staling of bread, Hop-
per (1949) first reported their efficacy. Ofelt and others (1958)
confirmed the action of monoacylglycerols in decreasing crumb
firmness. Diacylglycerols (commonly called diglycerides) had no
effect on crumb firmness when added alone to replace lard and
showed no synergistic effects with monoacylglycerols. While ad-
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 Bread staling. . .
dition of monoglycerides may counteract staling of breads during
storage, an increased tendency to crumble may result (Malkki and
others 1978).
The mechanism of the antistaling effect of monoglycerides is
still unknown, but it is thought to be different from that of shorten-
ing (Rogers and others 1988), for monoacylglycerols can replace
shortening, but shortening cannot replace monoacylglycerols.
Krog and others (1989) concluded that reductions in crumb
firmness brought about by addition of monoglycerides were
probably the result of interactions with amylose rather than with
amylopectin. When relatively large amounts of monoglycerides
are used, essentially all released amylose can be complexed (as
measured by DSC); interactions with amylopectin are also in-
creased. At lower concentrations, monoglycerides interact prima-
rily with amylose because of competition between the 2 poly-
mers.
Polyoxyethylene monostearate (POEMS). POEMS, a reaction
product of ethylene oxide and stearic acid, was one of the first ad-
ditives reported to retard staling (Maga 1975). Favor and Johnson
(1947) demonstrated that POEMS (0.5 to 1.0%) dramatically re-
duced the firming rate of bread between the 1st and 3rd d. Other
results (Freilich 1948; Edelmann and Cathcart 1949; Edelmann
and others 1950; Skovholt and Dowdle 1950) confirmed that PO-
EMS was effective in reducing the rate of firming. Carson and oth-
ers (1950) theorized that POEMS retarded staling by 2 mecha-
nisms: (1) by insolubilizing amylose and (2) by interacting with
starch granules via hydrogen bonding.
Sodium stearoyl lactylate (SSL). Pisesookbunterng and
D’Appolonia (1983) found that, among various surfactants stud-
ied, SSL had the greatest binding affinity to starch. The anionic sur-
factant might also prevent protein denaturation. Calcium stearoyl
lactylate is less effective as a crumb softener, but is active.
Glycerol monostearate (GMS). GMS is used in many starch-
based food products to improve physical characteristics, includ-
ing the degree of softness after storage (Krog 1971).
Other surfactants. Other surfactants that are effective as anti-
staling agents include polyoxyethylene sorbitan monostearate
(Polysorbate 60), succinylated monoglycerides, and glycerol.
Novel surfactants or surfactant blends have been formulated for
use as antistaling agents in bakery products. A blend developed
by Knightly (1987) consisted of a hydrophilic lecithin and at least
one of the following: monoglyceride, lactic acid esterified
monoglyceride, succinic acid esterified monoglyceride, maleic
acid esterified monoglyceride, or edible salts of stearoyl lactylic
acid. This blend was claimed to both inhibit staling and to act as a
dough conditioner. Other antistaling surfactant blends were de-
veloped by Vidal and Gerrity (1979).
Mechanism of antistaling effect of surfactants. The mechanism
by which surfactants influence crumb firmness has been debated
and is discussed briefly in the following sections. Amylose-surfac-
tant, amylopectin-surfactant, and protein-surfactant interactions
have all been investigated, as has starch swelling in the presence
of added surfactants. Whether surfactants actually decrease the
rate of firming or produce softer breads that then stale at the same
rate as the control has been debated. Surfactants have multiple
properties, resulting in multiple functionalities, so definitive exper-
iments examining a cause-and-effect relationship with regards to
staling are difficult, if not impossible, to design.
In excess water, surfactants do not change the gelatinization
temperature, but they do delay pasting (Miller and others 1953).
Whether this is related to their functionality in breadmaking is un-
known. Knightly (1977) reported that surfactants had little to no
effect on initial crumb firmness, but did affect the firming rate dur-
ing storage, a finding in agreement with earlier reports (Favor and
Johnson 1947; Skovholt and Dowdle 1950; Hopper 1949; Edel-
mann and others 1950). Based on unpublished results from in-
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vestigations by Ponte and Titcomb (1971), Kulp and Ponte (1981)
concluded that a surfactant’s ability to retard firming is more im-
portant than an initial softening of crumb in freshly baked bread.
Amylose complexes. Details of fatty acid-amylose complexes
have been examined using x-ray diffraction, DSC, and electron
microscopy (Godet and others 1993b, 1995a, 1996). Using mo-
lecular modeling techniques, Godet and others (1993a,b, 1995b)
concluded that the hydrocarbon tails of complexed fatty acid
molecules are indeed inside the hydrated V helix (see below) with
the polar head group outside the lumen. Interactions of amylose
with over 20 surfactants were studied, and a “complexing index”
was calculated and assigned to each one (Krog 1971). Morad and
D’Appolonia (1980) demonstrated that incorporation (0.5%) of 5
commercial surfactants resulted in amylose-surfactant complexes.
It was found by Eliasson (1985) that the amount of amylose
leached from starch granules decreased in the presence of emul-
sifiers.
Numerous studies have dealt with the ability of polar lipids to
inhibit bread staling. Mikus and others (1946) suggested that a he-
lical complex formed between amylose and MG, thus effecting a
softer crumb, but without affecting the firming rate. Schoch (1965)
reached a similar conclusion.
Data from Lagendijk and Pennings (1970) provided evidence of
the relationship between amylose-lipid complexation and the in-
hibition of staling. They reported maximum complexation with
monopalmitin, which corresponded with the softest crumb after
48 and 72 h of storage and concluded that complexation reduces
the flexibility of amylose molecules and thereby reduces their ret-
rogradation.
Pisesookbunterng and D’Appolonia (1983) reported that sur-
factants (SSL, MDG, and 40% Poly-60 / 60% MDG blend) ad-
sorbed to the starch granule surface, preventing moisture uptake
by the starch from gluten during aging of bread. However, water
was able to migrate from crumb to crust. Firmness of fresh bread
was not affected by the surfactant, although firming rate during
storage was slowed. Xu and others (1992) confirmed these results.
On the other hand, no apparent relationship between amylose-
surfactant complex formation and reduction of crumb firming was
found by Osman and others (1961).
X-ray diffraction can be used to detect complex formation, as
well as crystallinity and crystal types in general (Zobel 1973). The
amylose V complex helix hydrate can be detected by measuring
the intensity of the characteristic 4.4Å diffraction line. Formation
of the B-type crystal structure is followed by measuring the inten-
sity of the 5.25Å spacing. The B structure, typical of retrograded
starch, is extended, unlike the tight V-form helix. Molecules in
these 2 forms do not cocrystallize (Zobel and Senti 1959). With
no surfactants present, bread that is freshly baked shows only V-
crystallinity due to amylose-lipid complexes formed with the na-
tive fatty acids in the starch granules (Zobel 1973), during dough
heating (Zobel and others 1988). Thus, a portion of the amylose is
insolubilized during the overall baking process. The remaining
pattern is an amorphous halo produced by gelatinized starch.
During bread aging, the amorphous starch crystallizes into (B-
type) crystals, while the V-type intensity (amylose-lipid complexes)
remains unchanged (Zobel and Senti 1959). With surfactant add-
ed, breads were softer after 3 d as measured by a compressimeter
(Zobel and Senti 1959). V-lines increased with surfactant addition,
indicating complexation between amylose and the adjuncts. But,
once again, complexation does not necessarily correlate with sur-
factant effectiveness in retarding staling (Osman and others 1961;
Zobel 1973).
Dragsdorf and Varriano-Marston (1980) suggested that surfac-
tants that produce a V complex hydrate structure (for example,
SSL) may either prevent migration of starch polymer molecules
from granules during baking, thus retarding crumb firming, or re-
11
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 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
duce the redistribution of water from gluten to starch and thus
prevent contraction and firming of the gluten phase.
Amylopectin complexes. If one accepts the considerable
amount of data pointing to amylopectin crystallization as a key
factor in bread staling, it would make sense that, to be effective
antistaling agents, surfactants must also interact with the amy-
lopectin fraction; but until recently, there was no direct evidence
for amylopectin-surfactant complexes. Zobel (1973) and Eliasson
and Ljunger (1988) alluded to possible amylopectin interaction
with surfactants, since breads with adjuncts showed diminished
B-type crystallinity. Similarly, Knightly (1973) explained that sur-
factants retarded staling by forming complexes with amylopectin,
but later (Knightly 1996) stated that complex formation is unrelat-
ed to bread firming.
While Krog (1971) reported limited interactions of monostearin
with amylopectin, Lagendijk and Pennings (1970) detected forma-
tion of amylopectin-monoglyceride complexes, the degree of
which increased in a linear fashion with fatty acyl chain length,
while still being much less than the amount of amylose complex
formation. Kulp and Ponte (1981) mentioned that the effects of
complexation could be intramolecular (outer branch associations)
or intermolecular (aggregation of polymers).
DeStefanis and others (1977) reported that SSL, succinylated
monoglyceride, and glyceryl monostearate complexed equally
with amylose and amylopectin. Interestingly, no binding of the 3
adjuncts was found during the sponge stage. As the dough devel-
oped, increasingly strong binding of adjuncts with protein was
discovered. After baking, however, the 3 surfactants were found
strongly associated with starch polymers, the temperature at
which most translocation took place being above 50 °C.
Finally, Biliaderis and Vaughan (1987) obtained direct evidence
of complexes of amylopectin (and amylose) with labeled fatty acid
molecules using electron spin resonance. Then, Gudmundsson
and Eliasson (1990) obtained additional evidence for amylopec-
tin-surfactant complexes using DSC and x-ray diffraction tech-
niques. They also found that the amount of complexed amylopec-
tin was a function of the amylopectin:amylose ratio, since amy-
lose molecules were more effective in forming complexes in com-
petition with amylopectin molecules for the surfactant molecules.
Finally, they determined that surfactant-starch polymer complex-
ation prevented amylose-amylopectin cocrystallization. Using
mutant corn starches, Villwock and others (1999) also provided
DSC evidence for the existence of amylopectin-surfactant interac-
tions in pastes and additional evidence that both hydrocarbon
chain length and the nature of the polar group affect complex for-
mation.
Effect on starch swelling. According to Ponte and others
(1973), the softening effect of surfactants is related to a reduction
in starch granule swelling, and the degree of granule swelling is
inversely related to crumb firmness. They concluded that surfac-
tants restrict granule swelling during baking by complexing with
amylose at the periphery of starch granules. Polar surfactants that
form strong complexes with amylose (for example, long-chain fat-
ty acids, MG, POEMS) restricted granule swelling and solubiliza-
tion of various starches over the pasting range 60 to 95 °C (Gray
and Schoch 1962). Sodium lauryl sulfate repressed hydration of
starches below 85 °C, but the complex dissociated at higher tem-
peratures.
Infrared spectroscopy was used to investigate whether surfac-
tants adhered to granule surfaces or entered granules (Finn and
Varriano-Marston 1981). SSL did not appear to interact with the
granule surface, while PGMS did.
Lord (1950) concluded that POEMS retarded staling by 2 mech-
anisms: (1) by a “shortening” action that softened the crumb and
(2) by reducing granule swelling, which resulted in an initial in-
crease in firmness, which changed little during storage. [Note: As
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mentioned earlier, less swelling means less disruption of crystal-
linity and other order within the granule, so there is less “gelati-
nized” starch to recrystallize, but that which is disordered can re-
crystallize more easily. Another possible effect is less leaching/mi-
gration of amylose so that there is less in the intergranular space
to retrograde. Even in excess water, monoglycerides prevent the
leaching of amylose molecules (Schoch 1965). Surfactants pre-
vent dissolution and leaching of amylose molecules, which may
be the factor responsible for the reduction in granule swelling,
and also complex with amylopectin molecules; and their antistal-
ing effect, which is correlated with a reduction in granule swell-
ing, is likely due to a reduction in starch polymer mobility after
complexation so that less crystallization can occur.
Interaction with protein. Willhoft (1973) hypothesized that the
antistaling effect of monoglycerides might be due to interaction
with gluten, and there is experimental evidence that supports this
hypothesis (Hoseney and others 1969; De Stefanis and others
1977; Quail and others 1991). It has been suggested that surfac-
tant molecules associated with gluten are released during baking
(DeStefanis and others 1977) and complex with leached starch
polymers in intergranular spaces (Conde-Petit and Escher 1994.)
Physical properties of surfactants. According to Kulp and Pon-
te (1981), the physical state of surfactants is an important factor in
their performance. Krog (1973) reported that amylose-monoglyc-
eride complexation ability decreased in the descending order of
monoglyceride physical states: ␣-type crystalline gel > ␤-type
crystalline hydrate > nonhydrated powder. ␣-Type monoglyceride
crystals pack so that polar groups are exposed to the water phase,
and thus have a greater tendency to form effective aqueous ad-
juncts (Wren 1968; Larsson 1968). ␤-Type crystals show no
marked antifirming effect unless first hydrated before use. Hydrat-
ed ␤-crystals are commonly known as a “coagel-foam” (Krog
1968).
Shortening
Shortening is quite effective in retarding bread crumb staling
and has, for many years, been used as an antistaling ingredient in
breads. Since shortening was shown to have no effect in defatted
bread, it was speculated that its effect is related to the native flour
lipids. Since shortening does not complex with starch, its mecha-
nism of antistaling action differs from that of monoglycerides (Rog-
ers and others 1988).
Carbohydrate ingredients
Roles of dextrins and maltooligosaccharides in staling were dis-
cussed in the section “Enzymes: ␣-amylases and debranching en-
zymes”. Roles of native water-soluble and water-insoluble pen-
tosans were discussed in the section “Mechanisms of staling: Role
of pentosans”. Use of hydrocolloids and modified starches and
effects of damaged starch are covered in this section. If it is ac-
cepted that moisture redistribution is a requirement for staling to
occur (see “Moisture migration” section), then it follows that any
ingredient that inhibits movement of moisture is a candidate for
reducing staling (Swortfiguer 1971).
Hydrocolloids/gums. Davidou and others (1996) found that,
among locust bean gum, alginate [presumably sodium alginate],
and xanthan, only locust bean gum reduced the rate of dehydra-
tion. However, any increased moisture content of breads, if the
moisture is available to the starch molecules, increases the rate of
retrogradation (Rogers and others 1988) (see sections “Mecha-
nisms of staling: Role of pentosans” and “Moisture migration:
Moisture redistribution among components”).
Schiraldi and others (1996a) studied the effects of added hydro-
colloids (pentosans, modified pentosans, galactomannans, whey
protein) and reported that guar and locust bean gums retarded
starch retrogradation, but did not have any clear antistaling activi-
1/27/2003, 3:40 PM
 Bread staling. . .
ty. They also found that all the hydrocolloids they used generally
improved quality and that those with higher water-holding capaci-
ty increased crumb firmness. In contrast, Davidou and others
(1996) reported that both degrees of crumb firmness and the rate
of staling during storage were reduced by addition of locust bean
gum, alginate [presumably sodium alginate], and xanthan. They
proposed that the gums modified the organization of the amor-
phous part of the crumb, perhaps by inhibiting gluten-starch inter-
actions, perhaps in the same manner as proposed for dextrins
(Martin and others 1991). They also reported that only locust
bean gum (of the 3 gums) effected water retention. Carboxymeth-
ylcellulose (CMC) and hydroxpropylmethylcellulose (HPMC)
(0.3%) also decreased initial firmness (Armero and Collar 1998).
No increases in the Avrami value were found. Hydrocolloid-glu-
ten entanglements or linkages were suggested.
Addition of psyllium husk powder/gum (2, 4, or 8%) decreased
the staling rate of bread as measured by compressibility and DSC
(Czuchajowska and others 1992). Moisture content remained
constant. Bread softness improved without increasing the possi-
bility of microbial deterioration. Lent and Grant (2001) found that
bagels containing added xanthan had slightly higher crumb mois-
ture contents and staled at a somewhat reduced rate (DSC). Addi-
tion of pectin increased the specific volume of bread and reduced
the rate of firming during storage (Kegoya-Yoshino 1997).
Replacement of 10% of the wheat flour with steamed oat flours
retarded bread staling without adversely affecting the loaf volume
(Zhang and others 1998). The reduction in the rate of staling was
attributed to the high water absorption capacity of the ␤-glucan in
oat flour, but oat starch has been found to retrograde at a slower
rate compared to other starches (White and others 1989).
Patents have been issued for the use of karaya gum (Andt 1966)
and what is called low-molecular-weight amyloses, but which in
reality are the branch chains of amylopectin released by the ac-
tion of an a-1,6-glucan hydrolase as antistaling agents (Yoshida
and others 1972).
It has also been reported (although not supported in the article
with published experimental data) that methylcellulose and hy-
droxpropylmethylcellulose extend the shelf life of baked products
via prevention of water loss during baking (Dziezak 1991). The
same report states that guar gum and xanthan gum function as an-
tistaling agents.
Damaged and modified starch. Modified starches have been
investigated as antistaling agents (Maga 1975). Tipples (1969) re-
ported that the use of 25 to 35% damaged wheat starch de-
creased the rate of staling, especially when malt was added and
the sponge-dough method was used.
On the 4th d of storage, breads containing 5% of a phosphory-
lated waxy maize starch were as fresh as a 1-d-old control bread
(Bergthaller and Stephan 1970). The water-holding capacity of the
bread was not affected by the starch phosphates.
Miscellaneous
Results of use of dairy ingredients in breads for antistaling pur-
poses have been inconsistent (Mannie and Asp 1999).
D’Appolonia (1984) reported that milk solids have little to no ef-
fect on bread staling, but do soften the crumb initially. Conversely,
others have suggested that nonfat dry milk solids retard staling
(Dubois and Dresse 1984). Acidic whey (concentrated or uncon-
centrated) retarded staling in Hamam (French-type) bread at 1%
whey solids (Yousif and others 1998). Neither acid casein or sweet
whey powder were found to reduce staling in bread significantly
(Erdogdu-Arnoczky and others 1996), while acid whey powder
did. Despite its high water-holding capacity, succinylated whey
protein concentrate did not prevent bread staling (Thompson and
Baker 1983).
L-Leucine n-alkyl esters slowed the staling rate of bread more
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effectively than did monostearin (Watanabe and others 1981).
Propylene glycol was found to be superior to glycerol, malto-
dextrins, gelatin, commercial ␣-amylase, and poly(propylene gly-
col) in antistaling activity (Jagannath and others 1998). 1,3-Bu-
tanediol and 1,3-heptanediol (0.5%) reduced the staling rate of
bread significantly, with 1,3-butanediol having the greatest effect
(Frankenfeld and others 1977).
Incorporation of durum wheat flour (25%) into a bread wheat
flour did not improve initial firmness, but did retard staling
through 4 d of storage (Boyacioglu and D’Appolonia 1994).
Breads made with triticale flour staled twice as fast as did bread
made with wheat flour (Tsen and others 1973).
Sato and others (1989) reported staling retardation during long-
term storage when methyl 3,6-anhydro-␣-D-glucopyranoside was
added to the formula.
Flavor Changes
Staled bread is considered unacceptable due to changed flavor.
A review on bread flavor is available (Lorenz and Maga 1972).
Processes for Acceleration of Staling
A process by which the staling rates of bread products can be
accelerated using a time-temperature-moisture protocol to pro-
duce croutons and dry crumbs at a faster rate has been patented
(Slade and others 1987). The staling process is accelerated via
temperature cycling. Bread is exposed alternately to a temperature
just above the glass transition temperature (maximum rate of nu-
cleation) and to a temperature just below the melting temperature
(maximum rate of crystal growth). Other patents for preparing
bread crumbs and/or croutons have been developed (Tu and oth-
ers 1986; Dyson and others 1980).
Summary of the Basis of Staling and Factors Affecting the
Rate of Staling
Bread staling is a complex phenomenon, certainly involving
multiple factors. Much has been learned about bread staling, and
application of this knowledge has led to considerable improve-
ments in shelf life. However, without knowledge of the precise
mechanism, addressing the problem of bread staling remains a
process of formulating and testing more and more hypotheses. It
is difficult to determine cause-and-effect relationships because in-
volvement of a constituent may be indirect and additives, other
changes in formulation, and process changes may alter more than
one property and the effects may cancel each other.
Retrogradation of starch molecules remains the most widely ac-
cepted factor contributing to bread staling, but it must be remem-
bered that there is also good evidence that there is no cause-and-
effect relationship between retrogradation and staling. While amy-
lopectin retrogradation is believed to play the major role, amylose
is now also thought to be involved. And while amylose-surfactant
complex formation has been a widely used strategy for reducing
bread staling, amylopectin complexes may also be important, not
necessarily related to an inhibition of retrogradation because ad-
ditives that retard starch retrogradation may not retard staling.
Moisture content and moisture transfer among bread compo-
nents is believed by many to be a significant factor contributing to
bread staling. Most evidence supports the concept that gluten
serves as a moisture reservoir from which water is transferred to
retrograding starch molecules. But the relative effects of dehydra-
tion of gluten and hydration of starch can only be surmised, as
can the degree of benefit from prevention of this type of moisture
transfer.
Evidence has accumulated for a major role of gluten in bread
13
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 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
staling. As mentioned above, moisture transfer from gluten to
starch might be involved in the staling process. Beyond this, it has
been proposed that gluten-starch crosslinks are responsible for
staling, but it has become increasingly clear that multiple mecha-
nisms operate during staling. Supermolecular structures of some
sort, perhaps both starch-starch and starch-protein interactions,
are certainly involved, with one or more of the starch or protein
components, including gelatinized granules, being incorporated
in the structures, and with either or both components of the struc-
ture possibly modified by interaction with polar lipid molecules.
As has been suggested, it is likely that both starch and gluten con-
tribute to staling with the process weighted towards starch retro-
gradation, since there is much more starch than protein in bread.
One theory states that bread firming is a result of hydrogen bond-
ing between gelatinized starch granules and the gluten network. It
could also involve hydrogen bonding between retrograded starch
molecules and the gluten network with retrogradation occurring
either before or after association of amylopectin and/or amylose
molecules with the protein network.
Additives that seem to have the greatest effect in reducing stal-
ing in bread are (in no special order) surfactants (complexing
agents), ␣-amylase, and hydrocolloids/gums, including modified
starch. The effect of adding ␣-amylase is most certainly indirect;
that is, the antistaling effect is due to in situ formation of starch
dextrins and/or maltodextrins. Processing protocols are also im-
portant.
There is the unmistakable conclusion that polymer crystalliza-
tion is involved in the staling process and that some, perhaps the
majority, of the crystallization involves amylopectin. Gluten, may
also be involved. The most plausible hypothesis is that amylopec-
tin retrogradation involves incorporation of water molecules into
the crystallites and that this requirement shifts the distribution of
water molecules between components, reducing the water associ-
ated with gluten and thereby changing the nature of the gluten
network. The role of surfactants may be to change the chemical
(for example, by ionic bonding to protein molecules) or physical
(for example, by complexing with starch polymer molecules) na-
ture of components involved in forming supermolecular struc-
tures so that associations are prevented or so that only less perfect
associations are formed. They might also function primarily as
plasticizers, lowering the glass-transition temperature (Tg) so that
the structure is not in a glassy state at room temperature. The fact
that propylene glycol is quite effective supports this latter idea.
Water is an effective plasticizer; and the fact that low-molecular-
weight carbohydrates, which hold water, are effective plasticizers
and antistaling agents, as are ␣-amylases which produce them, is
further support for the important role of Tg lowering. A water-hold-
ing effect of carbohydrates (such as maltodextrins, dextrins, pen-
tosans, and other gums/hydrocolloids that do not themselves be-
come involved in retrogradation or other polymer-polymer inter-
actions) may be involved; retardation of the movement/redistribu-
tion of water may be their mode of action. Although considerable
progress in dissecting the staling process has occurred, bread stal-
ing remains an intensively studied, yet not well understood, phe-
nomenon.
Methods for Measuring Degrees of Staleness
Probably because of the mystery that still surrounds the staling
process and because there appear to be so many facets to a pre-
sumably complex process, a variety of techniques have been em-
ployed to measure staling and/or to investigate the changes that
accompany it. Characteristics of bread crumb that have been
used as bases to determine the degree of staling are changes in
taste and aroma, increased hardness, increased opacity, increased
crumbliness, increased starch crystallinity, decreased absorptive
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capacity, decreased susceptibility to ␣-amylase, and decreased
soluble starch content (Geddes and Bice 1946). It is obvious that
no one method will completely measure or describe the degree of
staling as noticed by the consumer (Sidhu and others 1996). All
investigations of the mechanism and control of staling reported in
this review have employed one or more of the methods covered
briefly in this section to measure the rate and/or degree of staling.
Other reviews of bread staling measurement methods can be
found in Maga (1975), Kulp and Ponte (1981), and Ponte and
Ovadia (1996). Many of the methods used to measure bread stal-
ing are based on principles used to determine the extent of starch
retrogradation. Methods for measuring starch retrogradation have
been reviewed by Karim and others (2000).
Rheological methods
Uniaxial compression. As bread stales, the texture of the crumb
changes from a relatively soft, spongy texture to one that is firm
and crumbly. Hence, numerous compressibility methods have
been developed to quantify the firming of bread, which has been
shown to correlate with bread staling as measured by consumer
acceptability. Hence, compressibility measurements are most
commonly used to determine the degree of bread staleness. Com-
pressibility methods were used in most of the investigations men-
tioned throughout this review and include 2 of the 3 AACC-ap-
proved procedures to measure staleness (Maga 1975). Most mea-
sure the force applied to compress a sample a specific distance.
AACC Method 74-10A (AACC 2000) measures crumb firming
changes with a Baker’s Compressimeter, determining the force ap-
plied by use of a plunger to ensure uniform compression (Baker
and others 1987; Baker and Ponte 1987). AACC Method 74-09
(AACC 2000) uses the Instron Universal Testing Machine to deter-
mine the degree of firmness in white pan bread crumb. Baker and
others (1988) confirmed that a 25% compression depth (as speci-
fied in AACC Method 74-09) was the most effective method for
detecting significant differences in bread firmness due to staling.
Instron-type systems have advantages over the Baker’s Com-
pressimeter because the compression rate is linear, and thus,
force-time relationships can be directly converted to force-com-
pression curves (Kamel and others 1984). Most important in this
regard is that a correlation coefficient of 0.98 was found between
firmness measured as compressibility and sensory assessments of
the degree of staleness (Axford and others 1968).
Other instruments that measure compressibility, such the Preci-
sion Penetrometer (Kamel and others 1984), Texture Analyzer, Q-
Test, Wheat Research Institute Chomper (Baruch and Atkins
1989), Bloom Gelometer (Baker and others 1987), and the Gener-
al Foods Texturometer (Szczesniak and Hall 1975), can also be
used to quantify the extent of bread staling. The squeeze test,
which gives the perception of freshness of bread and is a reflec-
tion of textural properties of the crumb, is popular with consum-
ers (Kamel 1987).
Dynamometric methods have been developed to characterize
the rheological properties of bread slice surfaces (Kulp and Ponte
1981). Young’s modulus can be determined from results of studies
of compressive stress-strain relationships determined with instru-
ments such as the Instron Universal Testing Machine. Baruch and
Atkins (1989) found that, in a dynamic stress-strain curve, the ini-
tial slope, which is a measure of crumb flexibility, increased and
the peak height, which is an indicator of the strength of the gluten
network, decreased as staling progressed. Stress-strain results
were correlated to results of thermal analyses by Schiraldi and
others (1996b). The relationship between mechanical properties
of bread and crumb staling has been reviewed in detail (Vodovotz
and others 2001).
Pasting properties. Under the hypothesis that starch retrograda-
tion plays a significant role in bread staling, the Brabender Visco-
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 Bread staling. . .
Amylo-Graph, Rapid Visco Analyzer, and related instruments
have been used to measure the extent of starch gelatinization in
bread crumb slurries (Yasunaga and others 1968). Peak viscosity
changes were suggested as an index of staling because it was
thought that peak viscosity would decrease with age due to a
toughening effect on partially gelatinized starch granules during
staling. Based on results in which the outer 1-cm and 2-cm por-
tions of the crumb produced a lower peak viscosity than did the
center portion, it was concluded that starch granules in the crumb
center were less gelatinized than those in the crumb exterior. De-
spite reporting amylograph data that agreed with those of Yasuna-
ga and others (1968), Varriano-Marston and others (1980) con-
cluded that the amylograph does not indicate the degree of starch
swelling accurately in bakery products, but rather shows the sum
of the contributions of all macromolecules to the viscosity of the
bread slurry. Toufeili and others (1994) found that, as staling in-
creased, pastes made from Arabic bread changed from being vis-
coelastic solids (G” < G’) to elastoviscous liquids (G” > G’).
Thermal analysis
Thermal analysis has been used extensively to study starch ret-
rogradation as well as bread staling (Russell 1983a, b;
Czuchajowska and Pomeranz 1989; Le Meste and others 1992;
Schiraldi and others 1966a,b; Champenois and others 1995;
Vodovotz and others 1996; Baik and Chinachoti 2000), and its
use has been mentioned throughout this review. Of the thermoan-
alytical methods, differential scanning calorimetry (DSC) and dif-
ferential thermal analysis (DTA) have proven to be the most useful
in providing basic information on starch retrogradation (Karim
and others 2000). Because both measure the differential tempera-
ture or heat flow to or from a sample versus a reference material
as a function of time, both can be used to monitor such changes
as phase transitions, molecular conformational changes, interac-
tions with other components, and pyrolytic degradation of the
sample. Specialized DSC instruments, including modulated DSC
and polarization DSC, are also available (Schenz and Davis
1998).
When aged bread samples are heated in a DSC pan, an endot-
herm is observed as reordered amylopectin reaches its glass tran-
sition and/or melting temperature, and the enthalpy change asso-
ciated with this transition can be measured. Because the time
scales for endotherm development and for the increase in crumb
firmness are broadly similar in magnitude, DSC can be used to
measure the rate of bread staling quantitatively (Jagannath and
others 1999a). However, there are overlapping transitions over a
wide temperature range because of the variety of components
and range of structures present, which cause difficulty in analysis
(Vodovotz and others 2001).
DTA was used to investigate bread staling by Axford and Col-
well (1967). An endotherm peak, which was absent in fresh bread
samples, developed during storage, and the increases in peak
area were proportional to increases in bread firmness (Cornford
and others 1964). Because an increase in glass transition temper-
ature (Tg) of bread crumb stored for different times was correlated
(96.53%) with an increase in the degree of bread staling as mea-
sured by compression analysis, it was concluded that the mea-
surement of Tg during storage could be used to quantitatively pre-
dict the rate of staling (Jagannath and others 1999a). DSC studies
of starch can approximate gelatinization during baking, since in
both cases the gelatinized starch granules are swollen, but non-
disrupted (Jacobson and BeMiller 1998). Thus, the conditions of
gelatinization in the calorimeter more closely approximate those
encountered during baking than those encountered during starch
pasting. Unlike compressibility measurements, endotherm peak
development does not appear to be dependent on specific loaf
volume (Fearn and Russell 1982).
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Isothermal microcalorimety, a technique which is much more
sensitive and requires larger samples sizes than does convention-
al DSC (Karim and others 2000), has been used to study the early
stages of starch retrogradation and has been demonstrated to be
effective for examination of the antistaling effects of lipids and sur-
factants (Silverio and others 1996).
Other thermoanalytical instruments include thermogravimetric
analysis (TGA), thermomechanical analysis (TMA), and dynamic
mechanical analysis (DMA). TGA measures changes in the weight
of a sample as a function of temperature (Schenz and Davis
1998). While events such as volatization, dehydration, and chem-
ical reactions can be observed using TGA, other simple transitions
can be missed if no weight changes occur (Sperling 1992).
Schiraldi and others (1996b), using TGA, found that the release of
water upon heating bread corresponded to 2 main binding states,
and that the 2 fractions were dependent on the age of the bread.
TMA measures changes in penetration, extension, expansion,
or contraction as a function of temperature (Schenz and Davis
1998) and can be used to determine the Tg of a substance by de-
tecting a change in the thermal expansion coefficient (LeMeste
and others 1992). The deformation of a substance is measured
under nonoscillatory (static) load as the substance is subjected to
a controlled temperature program (Flynn 1990). LeMeste and oth-
ers (1992) developed a TMA method to measure the glass transi-
tion of white pan bread.
DMA measures the dynamic moduli and damping of a sub-
stance under oscillatory load as a function of temperature and fre-
quency as it is subjected to a controlled temperature program
(Flynn 1990). DMA has also been referred to as forced oscillatory
measurements, dynamic mechanical thermal analysis (DMTA),
dynamic thermomechanical analysis, and dynamic rheology (Me-
nard 1999). In DMA, as oscillatory stress is applied to the sample
in the bending or tensile mode of deformation, the lag of the re-
sulting oscillatory strain is measured. DMA is 1000 times more
sensitive in observing thermal transitions than is DSC (Vodovotz
and others 1996). DMA has been used to study staling profiles of
Indian unleavened breads by Jagannath and others (1999b), to in-
vestigate the effects of added hydrocolloids, pentosans, and solu-
ble proteins on bread staling (Schiraldi and others 1996a), and to
examine the effect of aging and drying on thermal transitions of
bread (Vodovotz and others 1996).
Infrared spectroscopy
Fourier transform infrared (FTIR) spectroscopy and near-infrared
(NIR) spectroscopy, which have the advantage of being noninva-
sive methods, have been used to monitor staling in bread (Wilson
and others 1991).
Fourier transform infrared (FTIR) spectroscopy. Because FTIR
spectroscopy measures the degree of short-range ordering in a
system, conformational changes brought about by starch retrogra-
dation can be monitored by analyzing the band-narrowing,
which is caused by a reduction in the range of conformations and
a smaller distribution of bond energies due to the system becom-
ing more ordered upon staling (Wilson and others 1991; Karim
and others 2000). Changes in band intensities in the 1300 to 800
cm-1 region correlate to conformational changes during starch ret-
rogradation. Peaks at 1047 cm–1, which relate to crystalline re-
gions of starch, and at 1022 cm–1, which are characteristic of
amorphous regions of starch, are of particular interest (Karim and
others 2000). Thus, starch retrogradation can be defined (in terms
of FTIR data) as an increase in the ratio of peak intensities at 1047
and 1022 cm–1 (Smits and others 1998).
Near infrared (NIR) reflectance spectroscopy. Radiation scat-
tering, which in the case of bread relates to the degree of crystal-
linity of amylopectin, can be measured by NIR absorbance, so
NIR can be used to follow the progress of bread staling (Wilson
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 CRFSFS: Comprehensive Reviews in Food Science and Food Safety
and others 1991). Starch molecules in bread are extensively hy-
drogen bonded (both intramolecularly and to water). Because the
absorption bands in reflected NIR give information about hydro-
gen bonding, NIR reflectance data can be used to detect changes
in the hydrogen bond network of a bread system during staling
(Iwamoto and others 1987; Wilson and others 1991).
Nuclear magnetic resonance (NMR) spectroscopy
NMR techniques that have been used to study bread staling
and NMR techniques that have been used to examine changes in
molecular mobilities in breads, include solid-state proton NMR,
deuterium NMR, 13C NMR with cross polarization and magic-an-
gle spinning (CP MAS), and pulsed NMR (Ruan and Chen 2000).
Many have used NMR methods to determine the states of water
in bread and to relate them to bread firming (Leung and others
1983; Wynne-Jones and Blanshard 1986; Kim-Shin and others
1991; Chen and others 1997a,b; Engelsen and others 2001).
Low-field proton NMR has been used preferentially to examine
bread staling since it can provide rapid determination of proton
mobility associated with different molecules (Ruan and Chen
2000). Theoretically, there is an equilibrium state in bread where
mobile (liquid phase) and immobile (solid phase) protons coexist.
Since physiochemical changes can effect a new equilibrium state,
NMR can be used to determine mobility changes during bread
staling. However, bread is always in a nonequilibrium state, and
therefore, its nature changes continuously.
A pulsed-NMR method was used to monitor molecular changes
that resulted in increases in firmness during aging of starch gels
and starch-based products (Seow and Teo 1996). Morgan and
others (1992) used 13C CP MAS NMR to determine crystalline sol-
id, amorphous solid, and liquid-like phases of fresh and stored
wheat starch gels. Other NMR techniques have also been used,
including the 17O NMR method developed by Kim-Shin and oth-
ers (1991), to monitor water mobility in bread.
Magnetic resonance imaging (MRI) maybe useful in investiga-
tions of the mobility of protons in breads during staling. Ruan and
others (1996) monitored moisture migration from crumb to crust
in sweet rolls by MRI during 5 d of storage and found that, with an
increase in storage time/staling, the mobility of the less-mobile
fraction of water decreased and the mobility of the more-mobile
fraction of water increased.
Using proton cross-relaxation NMR spectroscopy, Wu and
Eads (1993) determined that the starch polymer molecules in con-
centrated waxy maize starch gels could be divided into 3 classes,
characterized by their degree of molecular mobility, and that the
percentage of immobile molecules increased with time, while the
percentage of mobile molecules decreased. Using the same tech-
nique, Vodovotz and others (2002) found no change, that is, no
increase in rigidity, of an aged bread sample, even though there
was an increase in amylopectin retrogradation enthalpy (DSC).
X-ray crystallography
X-ray crystallography has been used to examine bread staling
(Zobel 1973), specifically the crystalline nature of the starch in the
system, which can be related to the firmness of the product
(Champenois and others 1995). Starch in freshly baked bread is
mostly amorphous, but slowly reorders during storage. The re-
crystallization is reflected in x-ray diffraction patterns (Karim and
others 2000). Therefore, x-ray crystallography can be used to de-
termine the molecular organization of starch in bread (Varriano-
Marston and others 1980). However, powder x-ray diffraction is
not particularly sensitive as compared with other techniques,
such as NMR and FTIR, which are able to detect even minor ex-
tents of recrystallization (Smits and others 1998).
X-ray crystallography has been compared with DSC for deter-
mining the increase in crystallinity during storage of Arabic bread
16 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 2, 2003
crfsfsv2n1ms20010513-AF-af.p65 16
(Sidhu and others 1997) and used in conjunction with DSC in the
analysis of the effect of various antistaling additives on wheat
bread (Jagannath and others 1998).
It has been concluded that there is not necessarily a cause-and-
effect relationship between starch crystallization and bread firm-
ing (Dragsdorf and Varriano-Marston 1980; Zobel and Senti
1959), emphasizing the need, when investigating bread staling,
for methods that are not limited to measuring changes in only 1
component.
Jagannath and others (1998) used wide-angle x-ray scattering
(WAXS) to measure the degree of staling.
Conductance and capacitance
It has been established that changes in bread resulting in staling
of the crumb are at least correlated with starch retrogradation and
moisture redistribution between gluten and starch, whether or not
there is any cause-and-effect relationship. Since free and bound
water differ in their dielectric constants, changes during staling
could cause a change in the electrical properties of bread crumb.
Kay and Willhoft (1972) found that retrogradation was accompa-
nied by changes in conductance and capacitance, indicating that
changes accompanying bread staling could be detected electri-
cally and, furthermore, could be described by an empirical equa-
tion identical in form with the Avrami equation. Zaussinger and
others (1975) obtained bread staling data in a similar fashion.
Microscopy
Transmitted and polarized light microscopies. Transmitted-
light and polarized-light microscopy have been utilized to moni-
tor changes in starch granules from bread before and after staling
(Hug-Iten and others 1999, 2001). Native starch granules are bire-
fringent and possess ‘Maltese crosses’ when viewed under polar-
ized light. Upon gelatinization, starch crystallites melt and order,
and birefringence is lost. During bread baking, starch granules
lose their Maltese crosses, but retain slight birefringence (Varri-
ano-Marston and others 1980) and granular identity. Upon aging,
the bread crumb regains some birefringence (which is not the
usual native starch granule birefringence, but does indicate
biopolymer ordering in the long, thin birefringent structures) due
to molecular reordering, except in ␣-amylase-containing bread
crumb, which contained more of the birefringent structures (as
compared to a control crumb made without a-amylase) initially,
which changed little with aging (Hug-Iten and others 2001).
Confocal laser scanning microscopy (CLSM). The advantage of
confocal laser scanning microscopy over other microscopies is its
ability to produce an image of the focal plane of interest (optical
section), which can be digitally reconstructed into a 3-dimension-
al image. CLSM has provided qualitative information about the
crumb structure of bread (Bugusu and others 2002). CLSM has
also been used to investigate changes in starch granules in bread
during staling (Vodovotz and Chinachoti 1998). However, it has
been reported that there were no differences in confocal images
of fresh and 10-d old bread, suggesting that the changes that oc-
cur during staling are submicroscopic, that is, molecular only.
[Note: Since, as bread stales, starch molecules become more crys-
talline and more opaque, reflectance confocal laser scanning mi-
croscopy (R-CLSM) might provide more precise 3-D information
on the changes in the starch fraction during staling. R-CLSM offers
the highest resolution of CLSM modes (Hibbs 2000), but to our
knowledge has not been applied to investigations of bread stal-
ing.]
Electron microscopy. Electron microscopy has not been used
to study bread staling, but certainly has promise. Both transmis-
sion and scanning electron microscopy have been used to inves-
tigate doughs (Aranyi and Hawrylewicz 1968; Khoo and others
1975; Bechtel and others 1978; Evans and others 1981), bread
1/27/2003, 3:40 PM
 Bread staling. . .
(Khoo and others 1975; Bechtel and others 1978), and pastes
(Fannon and BeMiller 1992 and references therein).
Sensory/organoleptic tests
Loss of flavor and aroma are among the most noticeable detri-
mental changes of bread upon staling. Reportedly, the decrease in
the acceptability of bread over 5 d of storage is correlated with a
reduction in aldehydes and an increase in ketones (Lorenz and
Maga 1972). The resulting flavor is one that has been described as
“bland” (Setser 1996). Changes in texture, of course, also accom-
pany the bread staling phenomenon and can be measured by
both uniaxial compression methods (Section 8.1.1) and sensory
evaluations.
AACC Method 74-30 (AACC 2000) involves panel ratings of a
sum of factors affecting overall staling (“appearance” or “feel” of
the crumb/crust, “taste” and “mouthfeel”, “firmness”, “flavor”, and
“texture” change, or any other important factor noted by a panel-
ist). A high correlation between measured crumb firmness and
staling as rated by panelists has been found (Cornford and others
1964; Axford and others 1968). Other examples of organoleptic
evaluations are discussed by Pomeranz and Shellenberger (1971)
and Setser (1996).
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crfsfsv2n1ms20010513-AF-af.p65 21
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MS 20010513 Submitted 9/18/01, Revised 8/12/02, Accepted 10/3/02, Received
10/5/02
Authors Gray and BeMiller are Ph.D. student and Professor, respectively,
with the Whistler Center for Carbohydrate Research, Dept. of Food Sci-
ence, Purdue Univ., West Lafayette, Ind., U.S.A. Direct inquiries to author
BeMiller (E-mail: bemiller@purdue.edu).
21
1/27/2003, 3:40 PM
Bread Staling: Updating the View
C. Fadda, A. M. Sanguinetti, A. Del Caro, C. Collar, and A. Piga

Abstract: Staling of bread is cause of significant product waste in the world. We reviewed the literature of the last
10 y with the aim to give an up-to-date overview on processing/storage parameters, antistaling ingredients, sourdough
technology, and measurement methods of the staling phenomenon. Many researchers have been focusing their interest
on the selection of ingredients able to retard staling, mainly hydrocolloids, waxy wheat flours (WWF), and enzymes, but
different efforts have been made to understand the molecular basis of bread staling with the help of various measurement
methods. Results obtained confirm the central role of amylopectin retrogradation and water redistribution within the
different polymers in determining bread staling, but highlighted also the importance of other flour constituents, such as
proteins and nonstarch polysaccharides. Data obtained with thermal, spectroscopy, nuclear magnetic resonance, X-ray
crystallography, and colorimetry analysis have pointed out the need to encourage the use of one or more of these
techniques in order to better understand the mechanisms of staling. Results so far obtained have provided new insight on
bread staling, but the phenomenon has not been fully elucidated so far.
Keywords: bread, shelf life, staling

Introduction
Bread stales and unfortunately it is a certainty and causes signif-
icant product waste all over the world (Collar and Rosell 2013).
Staling results in loss of important sensory parameters of bread, like
flavor and texture, and it is a consequence of a group of several
physical–chemical changes occurring during bread storage that
lead mainly to an increase of crumb firmness and loss of freshness
(Kulp and Ponte 1981; Gray and Bemiller 2003). Although the
staling mechanism has not been well established, the most impor-
tant causes responsible for this alteration are starch transformation,
starch–gluten interactions, and moisture redistribution (Schiraldi
and Fessas 2001).
Bread staling is being continuously studied and researchers have
been focusing their interest on mechanisms, factors, and mea-
surement, thus a huge body of literature is available, including a
number of reviews and book chapters dealing with the different
causes of bread staling and/or specific topics (Table 1). Most of the
reviews and book chapters do not cover all aspects dealing with
bread staling. A rather complete state of the art of molecular basis
and most of the factors influencing the quality of bread, as well
as of the main antistaling agents, have been, however, covered by
Gray and Bemiller (2003), while reviews published later focused
again only on specific aspects of bread staling, such as the influ-
ence of water, enzymes, frozen dough (FD), and partially baked
bread, waxy and high-amylose wheat (WT) starches and flours,
sourdough, and analytical methodology (Table 1). More than 300
papers have been published in international peer-reviewed jour-
nals since 2003 on this topic, thus we attempted to collect the most
important literature to give a new and up-to-date picture on bread
staling. In particular, this review will focus on new information
regarding the following aspects of bread staling: processing/storage
parameters, surface-active lipids, enzymes, carbohydrate ingredi-
ents, flours and other major ingredients, as well as new measure-
ment methods and sourdough technology. The review will take
into consideration only papers dealing with WT bread and not
with models such as diluted and concentrated starch pastes as well
as gluten-free bread. In the case of papers dealing with the effect
of different factors (such as storage temperature, ingredients, or
ingredients of different origin), we use hierarchic considerations
to select the proper section of discussion. Moreover, the reader has
to refer to the literature previously reported and in particular to
the paper of Gray and Bemiller (2003) and others that will be cited
for more general information, molecular basis, and mechanisms of
bread staling. As a general rule, only papers not cited by specific
or general reviews, which will be reported in the proper sections,
are discussed in this review. However, papers already cited in re-
views, but not properly discussed with regard to bread staling will
be reviewed again.

Main Ingredients Affecting Bread Staling

MS 20131894 Submitted 19/12/2013, Accepted 15/2/2014. Authors Fadda,
Sanguinetti, Del Caro, and Piga are with Dipto. di Agraria, Univ. degli Studi di
Flours
Sassari, Viale Italia 39/A, 07100, Sassari, Italy. Author Collar is with Cereal Flours other than WT or deriving from amylose-free WT flours
Group, Food Science Dept., Inst. de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), (waxy) have been extensively studied during this last decade. The
Avenida Catedrático Agustı́n Escardino 7, Paterna 46980, Valencia, Spain. Direct particular composition of some flours or the absence of amylose
inquiries to author Piga (E-mail: pigaa@uniss.it). (with its role on staling) have been proposed in the production of
The authors contributed equally to this work. All the authors do not have mixed flour breads in order both to improve nutritional aspects
conflict of interests. and bread aging.


C 2014 Institute of Food Technologists®

doi: 10.1111/1541-4337.12064 Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 473
Bread staling review . . .

Table 1– Topics regarding bread staling covered by reviews or book WT flour by HBGB flours are more nutritious than those replaced
chapters. by CB flours and much more than regular WT flour breads pre-
Topic Review Book or book chapter serving the sensory acceptance and improving bread keepability
Enzymes Amos 1955; Haros Bowles 1996.
during storage. The high β-glucan content of barley flour (BM)
and others 2002; has been shown to help in reducing the starch crystallization, thus
van der Mareel and delaying significantly the staling rate of bread when used at the
others 2002; Butt
and others 2008; 20% level, even if it increased the firmness of fresh product (Gujral
Goesaert and and others 2003). Moreover, when BM was used together with
others 2009. wet gluten and ascorbic acid they reduced both initial firmness
Fibers Sivam and others
2010. and staling rate, especially when the higher level of the 3 addi-
Freezing and partial Rosell and Gomez tives was used. Purhagen and others (2008) proposed that water
baking 2007; Selomulyo had a greater effect on bread staling as assessed by texture anal-
and Zhou 2007; Yi
2008. ysis (TA), with respect to amylopectin retrogradation measured
Fundamental causes Kulp and Ponte 1981; with differential scanning calorimetry (DSC), when normal or
Le Meste and others heat-treated BMs were supplemented at 2% or 4% levels. In fact,
1992.
Hydrocolloids Izydorczyk and Dexter Milani and Maleki although the retrogradation enthalpy of supplemented breads was
2008; Kohajdová 2012. higher than control breads, the firmness values of barley loaves
and Karovičová
2009; Kohajdová were significantly lower during 7 d of storage at room tempera-
and others 2009. ture. However, the authors suggested that this effect could not be
Methodologies Karim and others Ponte and Faubion simply explained by the higher amounts of water in barley for-
2000; Chung and 1985; Ponte and
others 2003; Liu Ovadia 1996; mulations, but by differences in the water-binding ability of flour
and Scanlon 2004; Vodovotz and formulations with BM or soluble fibers. Staling rate was retarded
Choi and others others 2001. in laboratory-produced breads by using pressure-treated BM, as
2010.
Pentosans Hoseney 1984. well as waxy and pregelatinized waxy barley starch at the 3% level
Polyols Bhise and Kaur 2013. (Purhagen and others 2011b). The best results in retarding crumb
Proteins Davies 1986. firmness were found for pretreated and pregelatinized additives,
Sodium chloride Beck and others
2012a. with respect to the other formulations, including control bread,
Sourdough Arendt and others regardless of the storage time, even if a higher amylopectin ret-
2007; Chavan and
Chavan 2011. rogradation was revealed. The authors explained this result with
Starch Miyazaki and others Alsberg 1928; Slade the increased water retention during storage of substituted formu-
2006. and Levine 1987; lations. Unfortunately, they did not manage to retard staling when
Slade and Levine
1989; Hung and the pregelatinized additives were used in an industrial baking trial.
others 2006. Vittadini and Vodovotz (2003) used thermal analysis to assess
Staling Herz 1965; Zobel Alsberg 1936; that soy flour may have a role in modulating bread staling. Results
1973; Maga 1975; Chinachoti and
Knightly 1977; Vodovotz 2000; indicated that replacing up to 40% of soy flour in the bread formu-
D’Appolonia and Pateras 2007; lation caused a significant decrease in amylopectin recrystallization
Morad 1981; Cauvain and Young as well as promoted moisture retention during storage, with re-
Hoseney and Miller 2008.
1998; Schiraldi and spect to control bread, thus leading to decreased staling. Lodi and
Fessas 2001; Gray Vodovotz (2008) studied the effect of the partial substitution of
and Bemiller 2003.
Surface-active lipids and Knightly 1973; WT flour with soy flour and the addition of raw ground almonds
shortenings Stampfli and (5%). The incorporation of almond increased the loaf-specific vol-
Nersten 1995; ume of bread and reduced the crumb firmness changes over a 10-d
Kohajdová and
others (2009). storage period, if compared to bread obtained with only soy, even
Water Choi and others 2008. Cauvain and Young if no differences in amylopectin recrystallization rate or formation
2008. of amylose–lipid complexes were detected between the 2 formu-
lations. The authors postulated that the addition of almond to soy
flour probably resulted in a stronger interaction between proteins
Non-WT flours. It raises a great deal of recent interest that minor of WT and soy, favored by the high lipid content of almonds.
cereals, ancient crops, and pseudocereals, besides WT, constitute On the other hand, the bread produced with only soy staled at
highly nutrient-dense grains with feasible breadmaking applica- a lower rate than control bread, due to a better homogeneous
tions despite the poor viscoelasticity they exhibit when mixed water distribution, as revealed by different thermal determina-
with water. tions and by magnetic resonance imaging (MRI) (Lodi and others
Salehifar and Shahedi (2007) have confirmed earlier the ben- 2007a, 2007b).
eficial effects found by Zhang and others (1998) using oat flour Watanabe and others (2004) reported that substitution of WT
in reducing firmness of breads stored at room and chill temper- flour with powdered pre-germinated brown rice (PBGR) was
ature for up to 3 d, provided a maximum 20% oat flour sub- able to reduce the staling rate of bread stored for 3 d at room
stitution is accomplished, in order not to impart a strong bitter temperature, with respect to both control formula and bread sup-
taste. plemented with ungerminated brown rice (BR). The replacement
The ability of high β-glucan barley (HBGB) flour compared of 10% to 20% PBGR resulted in delayed staling with respect to
with regular commercial barley (CB) to make highly nutri- BR sample, while 10% PGBR slowed starch retrogradation, com-
tious WT-blended breads has been recently discussed (Collar and pared to control loaves, but supplementation of 30% PGBR ac-
Angioloni 2014a). Mixed breads obtained by 40% replacement of celerated bread hardening. According to the authors, 10% PGBR

474 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Bread staling review . . .
addition enhanced softness of bread due to a certain amount of
starch granules being gelatinized during PGBR production, while
30% supplementation led to accelerated staling owing to the high
water content needed to obtain dough.
Mentes and others (2008) reported that substitution of WT flour
with ground flaxseed flour resulted in delayed staling of bread after
24 h in storage, with respect to control all–WT bread, as assessed
by a mechanical penetration test, but the authors did not give any
explanation of the probable causes. The best result was obtained
by using 15% flaxseed flour.
Wu and others (2009) studied the effect of potato paste sub-
stitution at 5% to 30% on hardness evolution of bread during a
3-d storage period and found that staling decreased in 1-d stored
samples obtained with 5% to 20% potato paste, with respect to
control breads, and they associated this with the differences in
water-binding capacities of potato paste and with interaction with
starch, thus affecting starch retrogradation.
Begum and others (2010) evidenced that bread obtained with
the use of 10% fermented cassava flour or 10% soy-fortified cassava
flour was softer after 3 d at room temperature, with respect to WT
bread (Note: the authors did not make an explanation for this
result and did not report the amount of soy used to fortify cassava
flour).
In a recent paper (Angioloni and Collar 2011), the suitability
of associated mixtures of minor/ancient cereals (rye, oat, Kamut R
WT, spelt WT) and pseudocereals (buckwheat) to obtain baked
goods with high characteristics was assessed in multigrain WT
flour highly replaced matrices. A quaternary blend of oat, rye,
buckwheat, and common WT flours (20:20:20:40 w/w/w/w)
without any additive and/or technological aid in the formulation
was proposed to make highly nutritious baked goods meeting
sensory standards and exhibiting a low staling rate during ageing.
The quality profile of binary mixtures of oat–WT (60:40 w/w),
millet–WT (40:60 w/w), and sorghum–WT (40:60 w/w) was sig-
nificantly improved in presence of some additives in terms of keep-
ability during storage, mainly for oat–WT blends, which stale at a
similar rate or even at lower rate than 100% WT breads (Angioloni
and Collar 2013). Dilution up to 20% of the basic rye/WT flour
blend by accumulative addition of amaranth, buckwheat, quinoa,
and teff flours (5% single flour) did positively impact both bread
keeping behavior during aging, and nutritional characteristics of
mixed bread matrices (Collar and Angioloni 2014b).
Waxy WT flours (WWF). Most of the research work on flour has
been focused, however, on the use of WWF, because, due to its lack
of amylose, WWF can reduce the initial phase of retrogradation
(Graybosch 1998). A comprehensive review on the production
and characteristics of WWF and waxy wheat starch (WWS) and
their application for food processing is that of Hung and others
(2006).
Baik and others (2003) suggested that the increased starch ret-
rogradation of bread crumb, as assessed by DSC, may not be the
cause of retarded staling during a period of 7 d in storage at 4 °C
in bread obtained with double-null partial WWF, with respect to
bread produced with hard red spring WT flours. They proposed
that the low amylose and high-protein contents of the waxy lines
were beneficial in retarding the increase in hardness. Peng and
others (2009) reported that the use of 15% WWF combined with
2 other WT flours was the optimal solution for retarding staling
up to 6 d without impairing bread quality, as revealed by sensory
analysis, if compared with the control. Data from Hung and others
(2007a) gave evidence of the relationship between the use of whole
WWF and delayed staling. Breads made with 30% and 50% whole

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 475
WWF substitution were softer up to 1 d in storage due to the
higher amount of water absorbed by the dough as well as the high
moisture content in breadcrumbs. In a further paper, the same
authors (Hung and others 2007b) by using 100% whole WWF
managed to delay staling of whole waxy bread up to 3 d by adding
40000 U/g of cellulase, due to the particular pentosans present in
the enzyme hydrolysate. Moreover, they obtained white WWF by
removing the bran and germ, and the resulting breadcrumbs kept
softer for 5 d, with respect to breadcrumbs from both the whole
regular and whole waxy WT, probably as a result of the enrich-
ment of the amylopectin fraction of the white WWF. Park and
Baik (2007) made a comparative test with WT genotypes of wild
type, partial waxy, and waxy starch, in order to study the influence
of starch amylose content on French bread performance of WT
flour. Their study evidenced that WT flours with reduced starch
amylose content allowed the production of breads with better re-
tained crumb moisture and delayed staling up to 48 h of storage,
probably because the greater crumb moisture resulted in a delay in
amylopectin retrogradation, even if DSC analysis did not evidence
significant differences in enthalpy values of the various WT geno-
types with different amylose content. Slowing the migration of
water from the gluten phase to the starch phase by WWF (5% to
30%) has been hypothesized as the cause of diminution of firmness
evolution, as determined with compression analysis (Mouliney and
others 2011).
The low amylose content of flours obtained from 2 new Japanese
WT varieties was related to reduced staling of bread, especially in
the first 48 h of storage at 20 °C, with respect to samples obtained
with 2 representative bread WT classes that are N. 1 Canada west-
ern red spring and hard red winter (Ito and others 2007). DSC
data of enthalpy and X-ray patterns evidenced a slow retrogra-
dation of starch gel in the bread obtained with the new varieties,
thus accounting for their softer texture that resulted in softness and
high cohesiveness of the loaves. Apparently different results were
found when replacing hard WT flours with 15% to 45% with two
hard WWF (Garimella Purna and others 2011). In fact, substi-
tution led to softer bread, but only at day 1 after baking, while
staling was not retarded during storage. The combination of less
amylose and more soluble starch from amylopectin characterizing
WWF could have resulted in a soft crumb structure on day 1 after
baking, while after 7 d the bread was as firm as the control, due
to a similar content of soluble starch, thus confirming a previous
study (Ghiasi and others 1984).
Yi and others (2009) studied the effect of partial WWF substi-
tution on staling of bread made from FD. They found that when
modulating WWF and water amounts it was possible to reduce the
staling rate, with respect to control formulations. The best com-
bination was 45% WWF replacement and 65% water. By using
pulsed hydrogen-1 nuclear magnetic resonance (1 H NMR) they
concluded that bread with higher WWF content held more water
and limited the movement of water from one domain to another.
Very recently, Lafaye and others (2013) obtained bread using
waxy durum flour and concluded that this flour acted as a unique
bread softener. The authors did not make any additional analysis
in order to suggest a satisfactory explanation of the antistaling
effect of this flour, however provided a well-described picture of
the possible causes leading to the beneficial effect of waxy flour
supplementation by summarizing literature results.
Carbohydrates
A consistent research activity has been carried out during the
last decade on the role of carbohydrate ingredients in reducing
Bread staling review . . .

Table 2– Main hydrocolloids proposed during the last decade for staling reduction.

Hydrocolloid class Hydrocolloid name Effect Suggested references

Cellulose HPMC Interaction with other bread Bell 1990; Collar and others 1999;
constituents and in particular with Barcenas and Rosell 2005;
water (retention capacity and Tavakolipour and Kalbasi-Ashtari
starch–gluten interactions) 2007.
Hemicellulose GG Inhibition of amylopectin Ribotta and others 2004b; Shalini
retrogradation and Laxi 2007.
LBG Increased loaf volume and improved Sharadanant and Khan 2003;
texture Selomulyo and Zhou 2007;
Angioloni and Collar 2009a.
KGM Hindering effect on macromolecular Sim and others 2011.
entanglements
Arabinoxylans and β-glucan Competition for water, limitation of Izydorczyk and Dexter 2008; Jacobs
starch swelling, and gelatinization and others 2008; Hager and others
2011.
Microbial XG Increased water absorption, Collar and others 1999; Mandala and
retardation of amylose Sotirakoglou 2005; Mandala and
retrogradation, gluten–starch others 2007; Shittu and others
interactions 2009.
Pectins Pectin, HMP Competition for water, reduction of Rosell and Santos 2010; Correa and
amylopectin recrystallization others 2012
Animal Chitosan Inhibition of crosslink formation Kerch and others 2010, 2012a,
between starch granules and 2012b.
protein fibrils
HPMC, hydroxypropyl methylcellulose; GG, guar gum; LAB, lactic acid bacteria; KGM, konjac glucomannan; XG, xanthan gum.

bread staling. Hydrocolloids, modified starches, dextrins, and mal-
tooligosaccharides and other fibers will be covered in this section.
Hydrocolloids. The antistaling effect of hydrocolloids (Table 2)
has been extensively studied and attributed to controlling and
maintaining the moisture content, stabilizing the dough, and in-
fluencing the crust structure (Davidou and others 1996; Collar and
others 1999; Rojas and others 1999; Mandala and Sotirakoglou
2005; Mandala and others 2007; Rosell and Gomez 2007). Some
interesting reviews focused on molecular structure, physicochem-
ical properties, and uses in food products of the whole class
of hydrocolloids as bread improvers (Kohajdová and Karovičová
2009) and more specifically of barley β-glucans and arabinoxylans
(Izydorczyk and Dexter 2008). A book chapter by Milani and
Maleki (2012) gives a classification of hydrocolloids and of their
functions, according to Hollingworth (2010).
The use of DSC allowed to establish that hydroxypropyl methyl-
cellulose (HPMC) and k-carragenan (K) decreased the retrograda-
tion enthalpy of amylopectin, thus retarding staling of part-baked
breads produced with an interrupted baking process and frozen
storage (Barcenas and others 2003). The latter results were, in
part, in contrast to what was reported previously by Sharadanant
and Khan (2003) who found a detrimental effect on bread firm-
ness evolution during storage of K-supplemented breads. In a later
paper, Barcenas and Rosell (2005) gave a more detailed explana-
tion of the possible cause of the antistaling effect of HPMC. The
authors, in fact, determined the microstructure of bread crumb by
cryo-SEM and found that HPMC use resulted in gas cells with
a more continuous surface and a thicker appearance, with respect
to the control. Thus, the presence of HPMC enfolded the other
bread constituents, with a consequent hindering of their interac-
tions and avoided some of the processes involved in bread staling.
The HPMC was suggested as the best antistaling ingredient also
for Lavash flat bread made with 2 different WT flours and stored
for 48 h (Tavakolipour and Kalbasi-Ashtari 2007). Similar results
on another flat bread, the Barbari, have recently been reported
by Maleki and others (2012) who found that hydrocolloids other
than HPMC, namely guar gum (GG), xanthan gum (XG), and
carboxymethylcellulose (CMC), reduced staling of bread up to 5
d, due to the limitation of water mobility that influenced the gela-
tinization process by decreasing the H, that was also reported by
Ghanbari and Farmani (2013) who revealed a significant antistaling
effect of K, especially when supplemented at 0.5%. Mandala and
Sotirakoglou (2005) suggested that the use of XG and GG in fresh
or microwave-heated bread after frozen storage was able to retain
water in the crumb and, consequently, moisture migration to the
crust, thus resulting in the crust to fail at greater deformation,
that is, the samples were less stiff. XG used at low concentrations,
on the other hand, improved the crumb viscoelastic properties on
defrosted and microwave-heated samples, probably by hindering
the deteriorating effects and avoiding the development of a spongy
structure during frozen storage, as suggested by Ferrero and oth-
ers (1993). Moreover, XG has been addressed to retard amylose
retrogradation, due to reduced amylose–amylose interactions. In
2 separate papers the effect of 4 different hydrocolloids was stud-
ied, namely XG, GG, locust bean gum (LBG), and HPMC on
staling retardation of dough bread (DB), par-baked (PB) bread,
and full-baked (FB) breads stored at chilling (Mandala and oth-
ers 2007) or frozen temperature (Mandala and others 2008) and
finally re-baked (DB and PB). The crust puncture test and re-
laxation test of the crumb revealed that XG addition resulted in
a significantly less firm crust on PB and FB breads after chilling
storage, with respect to the other samples. X had also the more
evident effects on crumb viscoelastic properties, as revealed by re-
laxation tests, as it gave PB breads with an elastic crumb, DB with
a more viscous crumb, and FB breads with an even more viscous
crumb (Mandala and others 2007). In the case of frozen sam-
ples (Mandala and others 2008), XG supplementation was able to
give a softer plastic crust, but only in PB breads, with respect
to control and other supplemented samples, probably due to the
thickening effect on the crumb walls associated with the air spaces
that resulted in a less rigid structure. Finally, the addition of XG to
formulations allowed PB and FB breads to have a more elastic
crumb when compared to the other samples, thus revealing that
this hydrocolloid is more efficient against crumb deterioration in
an FB product than in the DB, and highlighting a very different
behavior from that found during chilling storage (Mandala and
others 2007), in which FB breads presented a complete viscous
and deteriorated crumb when hydrocolloids were used. Shittu and
others (2009) reported that increasing the dosage of XG up to 2%
resulted in a major hindrance of gluten–starch interaction in the

476 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Bread staling review . . .
presence of hydrocolloid molecules, thus conferring a significantly
higher softness to fresh composite cassava–WT bread. They also
reported that crumb hardening and moisture loss followed a linear
sequence up to the 1% XG level, which, thus, was proposed as the
optimum concentration to reduce both phenomena, even if the
2% XG level best estimated the crumb firming rate. Shalini and
Laxmi (2007) investigated the effect of 4 different hydrocolloids,
HPMC, GG, K, and CMC on textural characteristics of Indian
chapatti bread stored at ambient or chilling temperature and evi-
denced that 0.75% w/w supplementation of GG gave the softest
bread and decreased the loss of extensibility up to 2 d in storage at
both temperatures, with respect to the control. The authors sug-
gested that GG has a softening effect, probably by an inhibition of
the amylopectin retrogradation, prevention of water release, and
polymer aggregation during refrigeration, as well as interference
during interchain–amylose association. In a further paper, Shalini
(2009) gave more explanations on the effects of GG on staling
parameters and found that moisture, water-soluble starch, and in
vitro digestibility enzyme contents in GG-incorporated chapatti
were higher than in the control chapatti at both storage temper-
atures. Smitha and others (2008), on the other hand, working
with another flat bread, the unleavened Indian parotta, found that
supplementation of hydrocolloids (gum arabic, GG, XG, CMC,
and HPMC) resulted in delayed staling 8 h after baking, with
respect to nonsupplemented breads. HPMC gave the best results
in terms of reduction of hardness, while XG was judged by pan-
elists as the best for preserving sensory attributes like softness and
chewiness. Angioloni and Collar (2009a) proposed the viability of
LBG and CMC blended with oligosaccharides, at a medium-high
substitution level, as very valuable sources of dietary fiber (DF)
for the baked goods with both “healthy” characteristics and ex-
tended shelf-life, due to reduced staling. These conclusions were
drawn after modeling the crumb firming kinetics parameters ob-
tained during storage with the Avrami equation. Moreover, good
relationships between the main parameters obtained with the dif-
ferent physical analyses (small dynamic and large static deformation
methods, viscometric pattern, and image analyses) performed on
raw materials and intermediate and final products were found.
The effect of sodium alginate (ALG) and konjac glucomannan
(KGM) supplementation at 0.2% and 0.8% w/w flour basis was
studied in terms of staling behavior of Chinese steamed bread by
Sim and others (2011) who reported that the higher supplemen-
tation dose of both hydrocolloids resulted in a significantly lower
staling rate up to 4 d, with respect to the control bread, probably
because of the hindering effect of gums on macromolecular en-
tanglements thus causing starch recrystallization delay. Wang and
others (2006) studied the effect of gluten hydrolysate (GHP)/λ-
carrageenan (λC) ratio on the increase in the bread crumb firm-
ness during storage and proposed that the changes occurring in
the amorphous part of the starch, when a concentration of 0.5%
GHP/γ C was used in the product formulation, thus significantly
delaying bread staling.
The use of hemicelluloses has been the topic of different studies
during the last 10 y. A penetrometric test revealed that supple-
mentation of 0.3%, 0.5%, or 0.7% hemicelluloses (extracted from
buckwheat) increased the penetration depth of the crumb after 72
h of storage at 30 °C, thus delaying crumb hardening, and resulted
in bread with a higher specific volume than the control during a
3-d storage period. The best results were in the order 0.5% > 0.3%
> 0.7% both for hardness and volume (P = 0.01) (Hromádková
and others 2007). Symons and Brennan (2004) reported that a β-
glucan-rich fraction (BGF) extracted from barley and incorporated

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 477
at 2.5% into a bread formulation reduced crumb staling after 1 d in
storage, as detected by TA, but they did not formulate any expla-
nation of the causes (Note: the discussion of data on firming is not
exhaustive, as the authors neither explained the rate of staling, nor
highlighted that there were no significant differences in firmness
between control and BGF-supplemented bread). The BGF gave,
moreover, breads with lower volume, confirming previous results
(Gill and others 2002). Jacobs and others (2008) gave interesting
new knowledge about the influence of bread production on bread
quality when fiber-rich fractions (FRF), enriched β-glucans, and
arabinoxylans from hull-less barley were used. They, while con-
firming the results of Symons and Brenan (2004), found that sup-
plementation of FRF (12% on flour basis, corresponding to 2.5 g
of arabinoxylans and β-glucans per 100 g of flour) and Xyn within
the sponge-and-dough (SAD) baking method, improved the loaf
volume, appearance, and crumb structure and resulted in crumb
hardness and staling rate similar to that of the control bread, while
other baking methods (Canadian short process, remix-to-peak)
gave negative results (Note: the main part of the paper deals with
a comparison of the 3 baking methods by using a 20% on flour
basis supplementation and the authors concluded that the qual-
ity of the 20% FRF-enriched SAD bread was equal to or better
than the remix-to-peak bread, but they neither presented a statis-
tical comparison between data of the 2 baking methods, nor did
they explain why they evaluated the impact of lower FRF addition
only with the SAD method). Skendi and others (2010) studied the
supplementation of 2 WT flours differing in bread making qual-
ity (poor and good) with two different-molecular-weight barley
β-glucan isolates (at 0%, 0.2%, 0.6%, 1.0%, and 1.4% w/w on
a flour dry weight basis) and found that the crumb hardness of
β-glucan–supplemented breads, measured after 24 h of storage,
decreased with its increasing level up to reaching a minimum, and
then with a reverse trend, however the values were always lower
than the control bread, if we ignore one sample. Moreover, the
antistaling effect was more pronounced up to 8 d in storage when
the higher-molecular-weight β-glucan isolate was used in both
flour types. The authors proposed that the beneficial effects found
could be ascribed either to the higher water retention capacity
and a possible inhibition of the amylopectin retrogradation of β-
glucan, as already suggested (Biliaderis and others 1995), or to the
increase of the total area of gas cells.
An increase in bread firmness with respect to control WT for-
mulation was, on the other hand, found by Hager and others
(2011) after addition of oat β-glucan, suggesting that this increase
in hardness might be ascribed to the increased water-binding ca-
pacity of the polysaccharide, thus hindering the development of
the gluten network. They also evidenced a consistent increase in
staling after the addition of the fat replacer inulin, thus confirm-
ing previous results (Wang and others 2002; O’Brien and others
2003; Poinot and others 2010) and in part in agreement with
the study of Peressini and Sensidoni (2009) who used 2 com-
mercial inulin products, with lower (ST) and higher (HP) degrees
of polymerization, to supplement 3 different WT flours, moder-
ately strong (MS) and weak (W), and found that the ST inulin
addition to MS flour significantly increased the volume and low-
ered bread firmness, with respect to the control. The authors
hypothesized that a delayed starch gelatinization during baking,
due to the presence of 12% solutes, and the significant reduc-
tion of dough water absorption of ST inulin, may explain this
result. The beneficial effect of inulin gel at 2.5% flour basis on
increasing the loaf volume and maintaining the hardness value,
with respect to a control bread, was also reported by O’Brien
Bread staling review . . .
and others (2003). In a very recent paper the antistaling effect
of substitution of WT flour with BM (28%, 56%, and 84%) or
β-glucan (1.5%, 3.0%, and 4.5%) on chapatti bread was assessed by
DSC (Sharma and Gujral 2014). Storage at 4 °C for 24 h induced
retrogradation in baked chapatties, as revealed by the increase in
enthalpy of melting (H), but it was concomitantly reduced up
to 44% or 64% when β-glucan or BM was used, respectively.
The authors proposed that BM supplementation increased the
levels of soluble as well as insoluble DF, with an increased water
absorption and change of the nature of the starch and protein,
thus preventing better the staling of chapatties, with respect to
loaves obtained with β-glucan alone, as suggested by Purhagen and
others (2012).
The use of pectin slowed crumb hardening in bread that was
part-baked and stored at chilling (PB) or subzero (PBF) temper-
atures for variable times (Rosell and Santos 2010). The authors
also revealed that PBF pectin-supplemented breads showed a sim-
ilar hardening trend, with respect to their conventionally baked
counterparts, as also demonstrated by using the Avrami equation.
Correa and others (2012) reported that the incorporation of high-
methoxyl pectin at 1% or 2% resulted in protection with respect
to staling, especially when salt was used in the formulation, as
it reduced the hardness values with respect to the control sam-
ple, as well as maintaining the chewiness. They proposed that the
improved specific volume of high-methoxyl pectin-supplemented
bread, which gave both a more cohesive and more resilient crumb
with a different alveolus structure, was the main reason for retarded
staling.
A certain interest during the last years has been focused also on
an animal hydrocolloid, chitosan, a nonbranched linear homopoly-
mer obtained from shrimp and other crustacean shells. Chitosan is
a water-soluble cationic polyelectrolyte, while most of the other
polysaccharides are neutral or negatively charged at acid pH. In
a first paper of Kerch and others (2008), addition of 2% chitosan
resulted in increased staling rate of bread, and the author, through
DSC analysis and SEM, suggested an increase in water migration
rate from crumb to crust and in dehydration rate both for starch
and gluten and a prevention of amylose–lipid complexation in
breads supplemented with chitosan.
In a following paper, Kerch and others (2010) proposed and
analyzed, with the aid of mechanical and DSC measurements,
the main possible mechanisms leading to staling in breads ob-
tained with supplementation of different chitosan and chitosan
oligosaccharides. They confirmed that staling was the result of
2 independent processes, the first during the first 2 d of stor-
age depended on changes in the organization of starch polymer
chains, and later on it was caused by loss of water by gluten.
They suggested also that chitosan increased the firming rate dur-
ing the 1st stage due to its ability to bind lipids and prevent
amylase–lipid complexation, while in the 2nd stage it was en-
hanced dehydration of gluten due to its water-binding ability. In
their work, however, they found that both chitosan oligosaccha-
rides and low-molecular-weight chitosan decreased significantly
the staling rate, if compared to middle-molecular-weight chitosan,
and they hypothesized that low-molecular-weight substances in-
hibited crosslink formations between starch granules and protein
fibrils which, in turn, are responsible for staling. Later on, Kerch
and others (2012a) demonstrated with DSC that when chitosan
was used in bread production by the straight-dough or the SAD
method it accelerated or slowed down the decrease of bound water
content during the 1st stage of staling, respectively, thus delaying
or accelerating staling during the first 2 to 3 d of storage (1st stage
478 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
of staling). In a further paper, they showed that supplementation
of ascorbic acid to chitosan-enriched bread resulted in a more pro-
nounced decrease of water content during baking in fresh bread
compared to the control bread (Kerch and others 2012b).
Modified and damaged starches. The use of modified starches
for retarding staling has been suggested since the 1990s, for their
ability to influence amylopectin crystallization (Inagaki and Seib
1992; Yook and others 1993; Toufeili and others 1999). Due to
the fact, however, that other linear fractions of starch may affect
retrogradation, an increased interest has been registered on cross-
linked starches, due to their ability to increase the gelatinization
temperature, setback viscosity, and decrease the transition enthalpy
of gelatinization (Zheng and others 1999; Woo and Seib 2002). A
well-focused review on this topic has been published by Miyazaki
and others (2006).
According to Leon and others (2006), the content of damaged
starch directly influences bread staling through the increase of
amylopectin recrystallization, as detected by DSC analysis. The
authors concluded that the limited use of damaged starch is a key
factor to control the quality of fresh bread and of its shelf-life,
in contrast to what was reported earlier by Tipples (1969). In a
paper of Miyazaki and others (2008), chemically modified tapioca
starches (MTS), but with different degrees of modification, have
been used to retard staling in breads obtained from FD, which
was subjected to one freeze–thaw cycle and 1-wk frozen storage.
Highly MTS retarded significantly the increase in firmness during
3 d of storage, thus confirming the results of previous papers, due
to the slow retrograding rate of amylopectin.
Dextrins and maltooligosaccharides. Dextrins are the product
of starch hydrolysis and, since bread staling has been attributed
partly to its retrogradation, shortening the starch chain length, as
obtained with particular α-amylases, results in reducing the rate of
staling.
Miyazaki and others (2004), using DSC, found that among
6 different dextrins (dextrose equivalent 3 to 40) used at 20%,
those with low molecular weight (DE 19, 25, and 40) at 2.5%
of substitution retarded retrogradation, as revealed by the H of
retrograded amylopectin, but did not delay staling during 3 d
of storage. They postulated that the antistaling mechanism fol-
lowing addition of dextrin differed from the retarding effect of
dextrin produced by α-amylase, as already reported (Akers and
Hoseney 1994; Morgan and others 1997). They also highlighted
that retrogradation is not related to water mobility in crumbs, as as-
sessed by the determination of water activity. An interesting study
involving the use of texture profile analysis (TPA), X-ray diffrac-
tion (XRD), and DSC reported that the use of β-cyclodextrin
(β-CD) resulted in retardation of bread staling during 35 d of
storage at 4 °C, as changes of some TPA indexes (hardness, co-
hesiveness, and springiness) were reduced (Tian and others 2009).
Data on hardness were fitted with the Avrami equation that evi-
denced a significant reduction of the rate constant (k), while in-
creasing the Avrami exponent, thus suggesting a retarded crumb-
firming kinetic for β-CD-supplemented bread. Moreover, data
of XRD showed a delay in changes of crystalline patterns occur-
ring in crust and crumb and this retardation was attributed to a
complex amylose–lipid β-CD, as observed by DSC, that resulted
in transformation of nucleation type and lowered rate of bread
staling.
Jakob and others (2012), studied for the first time, the beneficial
effects of different fructans produced by acetic acid bacteria on the
texture of bread. Out of 21 strains tested, 4 of them were able to
produce high amounts of exopolysaccharides (EPS), as detected

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Bread staling review . . .
by HPLC analysis, which elicited, when supplemented at 1% to
2% of flour basis, significantly the increase in bread volume and
retarded the hardness increase of crumb up to 1 wk of storage,
the highest differences being observed after addition of 2% sugar
polymer from Neoasaia chiangmaiensis. The authors proposed that
the functional properties of the tested EPS were due to their hy-
drocolloid character, allowing a high water retention, and due to
interactions between polysaccharides and other dough compo-
nents like gluten and starch, thus influencing the final structure of
the baked product. They, moreover, compared effects of EPS to
HPMC.
Other fibers. In this section the effect of DF other than pre-
viously defined hydrocolloids and dextrins on bread staling will
be reviewed. According to the Codex Alimentarius Commis-
sion (2009), DF are “Carbohydrate polymers with more than 10
monomeric units, which are not hydrolyzed by the endogenous
enzymes in the small intestine of humans” (ALINORM 09/32/26
2009). Recently dough properties of bread enriched with DF have
been reviewed and the reader is redirected to this paper for the
aspects dealing with the interaction of this component in dough
development and bread baking (Sivam and others 2010). Fibers
investigated during this last decade are of cereal or noncereal ori-
gin.
Maeda and Morita (2003) proposed the polishing of soft WT
grain from the outer layer in increments of 10% of total weight to
obtain flours with a high content of pentosans and damaged starch.
In particular, both water-soluble (WSP) and water-insoluble pen-
tosans (WISP) from the inner part of the WT grain were added to
the conventional flour and their effects on loaf volume and bread
staling were assessed. The results indicate that both pentosans gave
an increase in loaf volume and a significant decrease in staling up
to 3 d in storage, with respect to the control bread. The authors
presumed that the high viscous and gelling properties of WSP may
improve the strength of gluten and the retention of gas generated
in the dough.
Mandala and others (2009) studied the effect of different ingre-
dients (hydrocolloids, polydextrose, oat flour, inulin, and commer-
cial shortening) on crust firmness and crumb elasticity of breads
obtained after thawing and baking of FD (at subzero temperatures
for 1 wk) and PB breads, and found that inulin was the best of
them in reducing bread crust firmness, probably due to a better
moisture redistribution, even if fresh sample had the firmer crust.
Gomez and others (2003) found that the use of fibers of differ-
ent origin (cellulose, cocoa, coffee, pea, orange, and WT), while
increasing the crumb firmness of fresh bread with respect to the
control, reduced its evolution during 3 d of storage, and they
postulated that this effect may be attributed to the already demon-
strated water-binding capacity of fiber, which in turn reduced wa-
ter loss during storage, as well as the probable interaction between
fiber and starch, resulting in the delay of starch retrogradation. The
best effect in delaying the bread staling was noticed after 2 d by
using a short-length WT fiber.
Collar and others (2009) found a positive effect on reducing
staling rate during 16 d of storage of breads enriched with 2 kinds
of cocoa fiber, as assessed by hardness and chewiness fitted with
the Avrami equation, when increasing the dose of addition up
to 6%, especially when the formulation was supplemented with
alkalinized cocoa-soluble fiber, while over-dosage resulted in a
staling rate similar to that of the control breads.
Zhou and others (2009) correlated the reduction of starch
retrogradation after X-ray measurements and application of the
Avrami model with increasing levels, from 1% to 5%, of tea

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 479
polysaccharide, which was able to reduce the slope of the stal-
ing rate up to 9 times, with respect to the control. The authors
also found that the magnitude of bread staling retardation strongly
depended on the type of WT flour used.
Addition of butternut fiber at 10 g/kg of flour decreased the
staling rate of bread after 7 d of storage, as measured by compress-
ibility, DSC, and digital image analysis (Pla and others 2013). The
authors clearly showed that fiber extracted from the peel resulted
in a drastic reduction of the firmness value, suggesting a retarda-
tion of amylopectin retrogradation, more air occluded (cell area
100/total area), same number of particles (alveolus or gas cells) per
square centimeter, and higher mean size of particles, with respect
to the control bread. The authors concluded that the particular
composition of fiber extracted from peel, that is presence of lignin,
less-branched pectin chains, and significant higher protein content
than the other butternut fiber used, may have accounted for the
results obtained.
Lipids and shortenings
The role of surface-active lipids and shortenings has been well
described by Gray and Bemiller (2003), and later on by Kohajdová
and others (2009), thus we will report briefly the new knowledge
not or only partially covered by these 2 reviews focused mainly on
the use of mono or diacylglycerols alone or esterified (DATEM).
Collar (2003) suggested that individual and/or binary sup-
plementation of fat-monoglycerides (MGL) and sodium stearoyl
lactylate (SSL) to bread dough positively influenced the level of
the pasting parameters assessed by rapid visco-analysis (RVA) (peak
viscosity, pasting temperature, and setback during cooling) that are
associated with a significant delay in bread firming. Moreover, she
does not recommend binary use of MGL/CMC and SSL/CMC,
as the antagonistic effects of the pair gum/surfactant resulted in a
nullification of the benefits exerted by the individual emulsifiers.
Ribotta and others (2004a) evidenced the beneficial effect of
DATEM on retarding crumb firming at 4 and 20 °C aging tem-
perature of bread from both nonfrozen and FD, and they supposed
that the formation of complexes with amylose and amylopectin
inhibited the staling phenomenon. Sawa and others (2009) studied
the effects of a wide range of purified saturated and unsaturated
MGL at different concentrations on the crumb firmness evolu-
tion during bread storage and reported that the use of C16:0 and
C18:0 and cis- and trans- C18:1 resulted both in a lower crumb
firmness, even if depending on the baking process used, and in
delayed bread staling, when compared to control bread. They sug-
gested the interaction of MGL with amylose and amylopectin
as the main cause of the obtained results. Manzocco and others
(2012) proposed that a particular system morphology, as assessed
by proton density/mobility using MRI, was generated in bread in
which palm oil was replaced with a MGL–sunflower oil–water gel.
The morphology change resulted in an 81% reduction in bread
fat content as well as in a delay in bread staling during storage.
The incorporation of the gel resulted, in fact, in a reduced proton
density/mobility in comparison with standard formulation, thus it
was concluded that the physical architecture of the lipids used in
the formulation could contribute to modulate the retrogradation
rate. Smith and Johansson (2004) reported that the increase of solid
fat of a shortening containing fully hydrogenated soybean oil was
able to delay bread staling and they suggested that saturated tria-
cylglycerols acted in a similar way as saturated monoacylglycerols,
that was an interaction with amylopectin. Mnif and others (2012)
proposed a new biosurfactant obtained by Bacillus subtilis as anti-
staling agent and compared it to soy lecithin. The bioemulsifier
Bread staling review . . .
supplementation significantly reduced the bread staling over an
8-d period, depending on its amount, and the maximum decrease
of staling rate was obtained with a 0.05% biosurfactant addition,
which was also the dose that resulted in the highest bread-specific
volume. The authors suggested that the slower firming may be
ascribed to the capacity of the emulsifiers to form a complex
starch-emulsifier, which in turn delayed WT starch crystalliza-
tion. Additionally, the biosurfactant reduced the susceptibility to
microbial growth during bread storage.
Minor Ingredients Affecting Bread Staling
The functional effects on fermentation and bread baking of
whey protein and casein have been reported by Erdoghu-Arnozcki
and others (1996). Casein and whey, together with ALG and K
were used in an attempt to improve the quality of FD, specifically
to retard its quality loss during freezing time and after 3 freeze–
thaw cycles (Yun and Eun 2006). Bread made with milk proteins
and hydrocolloids were softer after 4 d in storage, with respect to
control bread, probably because of better moisture retention and
improved emulsification of these ingredients. Similar results were
obtained in a later paper of Shon and others (2009).
Addition of juices to WT bread formulations have been pro-
posed to ameliorate its nutritional profile (Batu 2005), as sweet-
eners and color enhancers, and to increase volume and extend
shelf-life (Matz 1989). Lasekan and others (2011) postulated that
the high concentration of monosaccharide of the pineapple juice
concentrate used at a 1.5% level in the formulation of white bread
interfered with protein–starch interaction and delayed staling, but
only after 1 d of storage. Sabanis and others (2009) studied the
effect of supplementation (at 50% level sucrose substitution) of
2 types of raisin juices, concentrated and dried on evolution of
crumb firmness of bread obtained with both bread WT and du-
rum WT. The dried juice decreased the WT starch gel rigid-
ity and retrogradation for the presence of glucose and fructose,
thus resulting in reduced staling after 2 d of storage, with re-
spect to control loaves, especially when durum WT flour was
used.
Tomato pomace has been suggested as a good source of hydro-
colloids and was thus proposed (0%, 1%, 3%, 5%, and 7%) for flat
bread production by Majzoobi and others (2011b), who detected
delayed bread staling up to 4 d of storage at 25 °C in tomato
pomace–supplemented bread, with respect to control sample, due
to the concomitant increase in volume and moisture content and
decreased starch retrogradation.
Surface coating treatments have been patented for improving
the quality of bakery products (Lang and others 1987; Lonergan
1999; Hahn and others 2001; Jacobson 2003; Casper and oth-
ers 2006, 2007). The main advantages proposed for glazing were
the improvement of flavor and appearance. The moisture barrier
exerted at the surface of baked products allows retaining and aid-
ing dough expansion during baking, thus resulting in a reduction
of surface defects, improvement of color, and higher baked vol-
ume. Recently, however, other beneficial effects of glazing have
been described. Jahromi and others (2012) studied the effect of
different glazing treatments, including natural substances, polyol,
sugar, and hydrocolloids on the staling rate of breads stored up to
12 d. Increased moisture and reduction of water movement have
been addressed as the main causes of delayed staling by differ-
ent glazing ingredients, mainly, water, egg yolk, propylene glycol,
and starch, while at intermediate storage periods (2, 5, and 8 d)
also other glazing substances significantly retarded the increase in
crumb firmness, with respect to control bread.
480 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
Chin and others (2012) focused their work also on crust behav-
ior following different glazing applications. Glazing with corn-
starch, skim milk, and egg white were able to reduce the rate of
moisture loss in bread crumb during 6 d of storage, thus reducing
the staling rate of glazed bread, with respect to the control. More-
over, glazing resulted in an increase in crust firmness, although the
moisture content of the crust increased, probably because the rate
of moisture migration from crust to the surrounding atmosphere
could be lower with respect to that from the crumb to the crust
region.
Sodium chloride impact on bread staling has recently been well
reviewed and ascribed mainly to the increased gas retention effect
of dough with NaCl that allows an increase in crumb porosity
and a consequent decrease in crumb firmness (Beck and others
2012a). The retrogradation effect ascribed to Na+ inclusion in
starch molecules during storage of bread has been suggested as
delaying staling (Beck and others 2012b). In particular, a decrease
in bread staling following the decrease in NaCl levels was shown.
Furthermore, a linear relationship between rheofermentometer
data, bread volume, and crumb firmness was demonstrated, thus
suggesting that the quality of bread could be predicted by gas
release measurement.
Enzymes
The role of enzymes on bread staling has been one of the
preferred topics during this last decade and along with quite recent
reviews (Haros and others 2002; Butt and others 2008; Goesaert
and others 2009), an important number of papers have appeared,
which will be discussed. Apart from the effects of amylases, an
increasing interest in transglutaminase, a protein modifier enzyme
and other nonstarch polysaccharide-modifying enzymes has been
recorded.
α-Amylases and transferases
The action of α-amylases in reducing bread staling has been the
topic of numerous studies (Gray and Bemiller 2003), and different
ways of action have been proposed. The paper of Goesaert and
others (2009) provided new knowledge on the α-amylase mode
of action and its antistaling activity. In particular, they found that
the maltogenic α-amylase from Bacillus stearothermophilus degraded
significantly the outer amylopectin branches, thus producing amy-
lopectin chains that are too short to crystallize. The result was the
prevention of a “permanent” (based on amylopectin crystallites
junction zones) amylopectin network, thus staling was delayed.
Maeda and others (2003) proposed that a particular thermostable
mutant, α-amylase (M77), purified from Bacillus amyloliquefaciens
F increased the specific volume of the bread and improved the
softness of bread crumb, when compared to the commercial exo-
type α-amylase Novamyl (NM). They also showed that softness
evolution of breadcrumb during storage was not correlated with
thermostability. Rao and Satyanarayana (2007) found that the ad-
dition of α-amylases produced by Geobacillus thermoleovorans to
WT flour improved the fermentation rate and decreased the vis-
cosity of dough, while increasing the volume and texture of bread,
moreover, it also increased its shelf-life by retarding staling, with
respect to control sample, but they did not give any explanation of
this beneficial effect. Jones and others (2008) managed to develop
a new maltogenic α-amylase from Bacillus sp. TS-25, formerly B.
stearothermophilus, which increased thermal stability and the possi-
bility to work at acidic pH values that are typical of sourdough and
rye breads. Kim and others (2006) reported that the addition of
a fungal α-amylase to polished flour resulted in an improvement

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Bread staling review . . .
of gas cell distribution and softness of breadcrumbs and delayed
staling, without lowering the loaf volume with regard to control
bread made with hard WT flour.
Blaszczack and others (2004) studied the effect of 2 α-amylases,
one of fungal and the other of bacterial origin, on the texture and
microstructure of bread. The two α-amylases resulted in different
microstructure of bread, with respect to control bread, as revealed
by light SEM, thus staling was delayed. The authors proposed
distinct antistaling mechanisms for the two α-amylases.
Xylanases (Xyns)
Xyns are enzymes able to retard bread staling, as reviewed by
Butt and others (2008), to which the reader is redirected.
A recombinantly produced Xyn B (XynB) from Thermotoga
maritima MSB8 retarded the staling of frozen PB bread (Jiang and
others 2008). When added to the formulation, the resulting bread
had a 40% reduction in crumb firmness and retarded staling, as
bread supplemented with XynB after 4 d of storage at 4 °C had the
same firmness as control bread after 1 d of storage. Data obtained
with DSC analysis showed that XynB was able to retard amy-
lopectin crystallization. Recently, Zheng and others (2011) found
the right dosage to be used for 2 GH 10 Xyns, a psychrophilic
(XynA from Glaciecola mesophila) and mesophilic one (EX1 from
Trichoderma pseudokoningii), with the aim to retard bread staling.
Both Xyns exhibited similar antistaling effects on the bread, but
while XynA proved to be more effective in reducing the firming
rate, the EX1 performed better in reduction of the initial bread
firmness. The optimal dosage of the psychrophilic Xyn was much
lower than that of the mesophilic counterpart, probably because
the temperatures used for dough preparation and proofing were in
the range of optimum activity of psychrophilic XynA, as otherwise
reported (Collins and others 2006).
Recent results of the application of a thermostable enzyme
cocktail from Thermoascus aurantiacus showed an antistaling effect
(Oliveira and others 2014). The main enzyme found on the cock-
tail was Xyn, xylose being the main product released through
enzyme activity after prolonged incubation, and its application at
35 units of Xyn/100 g significantly delayed staling of bread up to
10 d at 4 °C if compared to control loaves. On the basis of DSC
results (lower enthalpy) it was suggested that products deriving
from Xyn activity interfered with the reorganization of the amy-
lopectin and/or with the redistribution of water in the system,
with a consequent retrogradation reduction. Recently Ghoshal
and others (2013) suggested that the reduction of crystallization
and reduction of crystal growth in bread, as assessed by using n and
k parameters of the Avrami equation, was caused by Xyn addition
in whole-WT bread stored at 4 and 25 °C for 10 d, thus resulting
in delayed staling. Measurement of thermal properties confirmed
the beneficial effects of Xyn, as it lowered the endothermic peak
for staling and the change of enthalpy during storage, with respect
to control bread.
Enzyme mix
The difference in mode of action of the various enzymes has
been used recently by several authors, which depended on additive
or synergistic effects in order to retard staling.
Leon and others (2002) studied the effects of 2 commercial en-
zyme mixtures containing α-amylase and lipase activity on staling
rate. Both mixtures helped in slowing down the staling rate, espe-
cially the blend with the higher α-amylase activity. The beneficial
effect was attributed to a delay in amylopectin retrogradation and

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 481
to the formation of amylase–lipid complexes, both revealed by
DSC analysis.
The use of a microbial transglutaminase (protein-glutamine
γ -glutamyl transferase, Tgm), which catalyzes the formation of
ε-(γ glutamyl-)-lysine crosslinks in proteins via an acyl transfer
reaction (Motoki and Seguro 1998; Larre and others 2000), has
received a great deal of interest. Tgm with or without added
amylolytic (maltogenic bacterial α-amylase in granulate form
[NMYL]) or non-amylolytic (PTP) had beneficial effects on hard-
ness evolution of bread obtained with white (Collar and Bollain
2005) and whole-meal flour (Collar and others 2005). Bread soft-
ness was reduced up to 16%, with respect to control bread, when
interactive effects were tried, and the best combination was the
addition of NMYL to Tgm breads, ascribing this effect to the
relevant softening effect of NYML.
Gambaro and others (2006) proposed that the addition of a
mixture of α-amylase and Xyn was able to extend the shelf-life of
brown pan bread by retarding staling, as assessed by sensory and
instrumental analyses. They suggested that the mixture produces
low-molecular-weight dextrins with high water retention capacity,
and that could be partly responsible for the lower staling rate.
Moreover, they found a high correlation between both sensory
and instrumental parameters and staling rate.
Caballero and others (2007) studied the single and synergistic
effects of some gluten-crosslinking enzymes (Tgm, glucose oxidase
(GO), and laccase), and gluten-degrading enzymes (α-amylase,
Xyn, and protease) on bread staling. They found that α-amylase,
Xyn, and protease were able to lower significantly the staling effect
promoted by Tgm and proposed different mechanisms of action
for each enzyme. In particular, they suggested that α-amylase and
Xyn could have an effect on the dough polysaccharide fraction,
while the protease may counteract Tgm-action, by a simultaneous
action on the dough protein fraction.
Waters and others (2010) proposed that the highest Xyn and
α-amylase activities of 5 thermozyme cocktails with different hy-
drolytic enzyme profiles produced by Talaromyces emersonii resulted
in delayed staling. The enzyme cocktail B was the best in reduc-
ing crumb hardness evolution after 5 d of storage, with respect to
control bread.
Others
The oxidizing effect of GO was exploited for retarding staling
of bread (Bonet and others 2006). When used at a concentration
of 0.001%, GO delayed significantly the bread staling up to 12
d at 25 °C. The antistaling effect suggested was due to the large
amount of total pentosans produced by GO that can associate
with the glutenin macropolymer, thus leading to retention of high
amounts of water.
Associated Mixtures of Ingredients and/or Enzymes
In this section, we will summarize the results of the main studies
dealing with ingredients and/or technological aids not included in
the previous classes or combinations of different ingredients. An
interesting review on shelf-life improvement of polyols, to which
the reader is redirected, has recently been published (Bhise and
Kaur 2013).
Wang and others (2007) reported that, when 1% of WT GHP
was used, the hardness value of 3-d-old bread was equivalent to
that of 1-d-old control bread, probably for the higher, even if
not significantly, specific volume and moisture content of WT
GHP-supplemented sample.
Bread staling review . . .
Abu-Goush and others (2008) found a beneficial effect of
sodium-propionate in delaying staling of Arabic flat bread and cor-
related this result to moisture loss, starch retrogradation, and pro-
tein interaction effects, as revealed by near-infrared spectroscopy
data.
Shaikh and others (2008) tested 8 different antistaling agents on
unleavened chapatti bread and measured various staling parame-
ters such as moisture content, texture, water-soluble starch, in vitro
enzyme digestibility, enthalpy change, and sensory quality during
10 d of storage, at 4 and 29 °C. When comparing the effect of
the added ingredients the authors found that maltodextrin had
the highest rank at both temperatures, while the worst result was
exerted by glycerol monostearate, following the order: maltodex-
trin > GG > α-amylase > sorbitol > XG > SSL > propylene
glycol > glycerol monostearate. Moreover, when trying 6 combi-
nations, SSL + α-amylase gave the best texture values, suggesting
that α-amylase first breaks starch molecules, and then SSL forms
the complex with fragments derived from starch rupture.
The lowest amylopectin retrogradation of soy milk powder was
addressed as the cause of delayed staling rate in WT–soy bread
(Nilufer-Erdil and others 2012). This result was attributed to the
synergistic effect of soluble fiber and partly denatured soy proteins
and higher lipid content of the soy milk powder. The delay of
staling was confirmed by Instron firmness measurements, although
loss moduli revealed by dynamic mechanical analysis (DMA) did
not give significant differences of stiffness among formulations,
contrary to what had been reported previously by Vittadini and
Volovodtz (2003).
Jekle and Becker (2012) studied the effects of pH adjustment,
water, and sodium chloride addition in order to model bread tex-
ture and staling kinetics of bread crumb. By using the Avrami
equation and the firming rate, which gave a better square corre-
lation coefficient, the authors managed to predict the staling rate
as a function of pH, NaCl, and water addition. In particular, they
found an increase in the firming rate with increased NaCl concen-
tration and pH reduction and a decrease when water was added
to the dough, probably as the change in the volume of bread had
a better influence on the staling rate, with respect to the effect
of the chemicals, since the literature well correlated the specific
volume of breads with the firming rate (Axford and others 1968;
Russel 1983).
The addition of γ -polyglutamic acid (PGA) at 3 concentrations
(0.5, 1.0, and 5.0 g/kg, w/w) was suggested by Shyu and others
(2008) to evaluate its effect on staling of WT bread. The hardness
value of the 6-d 1.0 kg−1 PGA stored bread was less than the value
of control bread after 1 d, thus PGA significantly reduced staling
rate, as also demonstrated by the decrease in cohesiveness, which
was significantly delayed by the PGA addition.
Response surfaces and mathematical models were used by
Gomes-Ruffi and others (2012) to show the beneficial effect of
the contemporary addition of SSL and of the enzyme maltogenic
α-amylase (MALTO) on both the increase of bread volume and
the reduction of firmness, especially after 10 d of storage, when
the combination of 0.50 g SSL/100 g flour and 0.02 g MALTO/
100 g flour resulted in the same firmness value as the control at
day 1 of aging. The authors suggested that SSL formed complexes
with starch molecules, while MALTO reduced the molecular
weight of the starch molecules, thus reducing retrogradation.
Pourfarzad and Habibi-Naiaf (2012) used the positive results
in changing the hardening rate of Barbari bread obtained with an
antistaling liquid improver, made up of glycerol, SSL, and enzyme-
active soy flour, at different amounts, to test the consistency of
482 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
11 new mathematical staling models. They found that all models
presented high values, the best being the rational and the quadratic,
thus concluding that these models are suitable to simulate staling
kinetics. The best improver formulation contained 1.27% glycerol,
0.41% SSL, and 1.59% enzyme-active soy flour.
The plasticizing effect of the sorbitol on starch/gluten biopoly-
mers has been described by Pourfarzad and others (2011) as the
main reason of antistaling effect of soy-fortified bread for storage
times longer than 2 d and up to 5 d. The same effect was found
also for propylene glycol when used at 5 g/100 g flour.
Processing Factors Affecting Staling Rate
Researchers have focused their attention during the last 10 y
mainly on baking technology, process parameters, and storage tem-
perature, but other factors will also be reported.
Storage temperature. The effect of storage temperature on stal-
ing has been reported by different authors, and the main character-
istic is a negative dependence between staling rate and temperature
(Colwell and others 1969).
The consumer request to have “fresh” bread available at any
time of the day (Matuda and others 2005) has stimulated the
bakery industry to exploit freezing technology and this has driven
researchers to focus their attention mainly on effects of freezing
and frozen storage on bread staling, especially on dough and PB
samples.
A comprehensive picture up to 2008 on the effect of raw mate-
rial requirements, processing conditions, and baked bread quality
from FD and PB bread are reviewed by Rosell and Gomez (2007),
Selomulyo and Zhou (2007), and Yi (2008), to which the reader
is redirected.
Carr and others (2006) carried out a sensory comparison be-
tween frozen part-baked French bread (FPBFB) and fresh bread
during a week of frozen storage with daily inspections. The FPBFB
had a lower weight and specific volume, with respect to fresh
bread, but was rated better after 4 d of frozen storage by a con-
sumer acceptance test (difference from control test) with respect
to commercial brand bread. Moreover, data on texture and sen-
sory analysis of FPBFB stored for a week were similar to that of
fresh bread. Frozen storage of PB chappati, an Indian unleavened
flat bread, was beneficial for maintaining its quality (Gujral and
others 2008). In particular, the extensibility of PB chapatti after
rebaking was very similar to that of the fresh conventionally baked
sample. The main feature was that sample of PB bread stored
at ambient temperature or frozen (after thawing and rebaking),
showed a significant higher extensibility when compared to the
same sample of conventionally baked chapatti breads, thus giv-
ing loaves with better sensory quality than frozen conventionally
baked chapatties. Yi and Kerr (2009) highlighted the influence of
freezing rate (rate 1:15 °C/h, rate 2:33 °C/h, rate 3:44 °C/h, and
rate 4:59 °C/h), dough storage temperature (−10, −20, −30, and
−35 °C) and storage duration on bread quality. They found that
sample frozen at the lower freezing rates and stored at the higher
temperatures had higher specific volume, were softer, and were
lighter in color, but staled more easily, due probably to the higher
damage to the starch–gluten network at slower freezing rates (Yi
2008). They noted that response of gluten structure and yeast ac-
tivity to freezing rate and temperature should be balanced in order
to find the optimal freezing conditions. Aguirre and others (2011)
confirmed the existence of moisture equilibration between crumb
and crust during bread storage, and demonstrated that storage at
−18 °C resulted in very limited water movement when compared
to bread stored at 4 and 25 °C. As a consequence, water activity

C 2014 Institute of Food Technologists®
Bread staling review . . .
values were almost constant in bread stored for 23 d at −18 °C.
They showed that the starch molecules re-associate during storage
to give a new crystalline structure with a typical XRD B-type
structure and that storage at −18 °C, that is a temperature below
the glass-transition temperature (Tg ), slowed down but did not
stop the recrystallization speed, and only crystal growth occurred.
The effect of vacuum-cooling on the staling rate of sourdough
whole meal flour bread was assessed by Le-Bail and others (2011).
Vacuum-chilled bread showed higher moisture loss, crumb hard-
ness, and H of amylopectin crystals than conventionally cooled
bread. The authors concluded that the negative effects of the quick
vacuum-cooling is the result of the increased number formation
of amylopectin crystallites and, thus, of recrystallized amylopectin.
Ronda and others (2011) studied the effect of prolonged storage
time on staling of PB and fully baked (FB) breads. Three parame-
ters, namely moisture content, firmness, and starch retrogradation
as well as the Tg of the maximally freeze-concentrated state (Tg  ),
were considered to evaluate bread aging. The thawed and rebaked
PB bread showed significantly lower amylopectin H values than
that of FB bread, and this may partially explain the similarity of
PB bread with fresh bread. The authors evidenced the need to
select a proper frozen storage temperature, sufficiently lower than
Tg  . Frozen storage time, moreover, resulted in a significant de-
crease in firmness of PB bread crumb. Based on the obtained
results, the authors proposed that hardening of bread during stor-
age may not be related only to starch crystallization or water loss
and developed a regression study describing how the combined
effect of both variables could better explain the firming evolution.
Majzoobi and others (2011a) hypothesized that the higher mois-
ture content of Barbari PB flat breads after full baking was the cause
of delayed staling up to 72 h, with respect to control sample, and
proposed that bread crumb structure is formed completely during
the part-baking stage, while staling occurs in PB bread during
storage at ambient temperature, even if full-baking leads to the
disappearance of many signs of staling, thus the resulting bread has
softer texture. Finally, they suggest storing the part-baked bread at
frozen temperature for no more than 2 mo to reduce deterioration
of bread caused by the growth of ice crystals. In a subsequent pa-
per Majzoobi and others (2012) recommend the addition of 15%
WT germ for the general sensory improvement of Barbari bread,
although that did not manage to retard staling.
In 2 separate papers Karaoglu (2006) and Karaoglu and
Kotancilar (2006) evidenced the influence of par-baking on
quality of WT bran and white breads, respectively, supplemented
or not with calcium propionate, during chilling storage (4 °C)
up to 21 d. Both papers gave similar results, which were a softer
bread crumb, with respect to a control group, in breads PB for 10
min, rebaked, and stored for 7 and 14 d.
Sourdough fermentation
Sourdough fermentation has been known since ancient times
and, among the beneficial effects, reduction in staling has been
reported and recently discussed in 2 reviews (Arendt and others
2007; Chavan and Chavan 2011), to which the reader is redirected.
The different metabolites produced by lactic acid bacteria (LAB)
have proved to have a beneficial effect on texture and staling. EPS,
for example, are a valid and economic alternative to hydrocolloids,
while organic acids affect the protein and starch fractions and
reduce the pH that results in an increase in protease and amylase
activities of the flour, thus reducing staling.
Katina and others (2006a) managed to delay bread staling at 3
and 6 d of storage, with respect to white WT bread, by combin-

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 483
ing WT bran sourdough and an enzyme mix (α-amylase, Xyn,
and lipase). The crumb hardness of the supplemented bread after
6 d of storage was the same as that of white bread at day 1. The
authors used NMR, DSC, and microscopy to explain this result
and found fewer changes in amylopectin crystallinity and rigid-
ity of polymers in bran sourdough bread with enzymes, which
also showed starch granules much more swollen, with respect to
white bread, as a result of the higher water content and degrada-
tion of cell wall components. In another paper, Katina and others
(2006b) proposed the use of surface-response methodology to op-
timize sourdough process conditions aimed at improving flavor
and texture of WT bread. They found that combining flour with
low ash content, and optimizing sourdough fermentation time,
staling was reduced up to 4 d. The best result was, in particular,
obtained using Saccharomyces cerevisiae sourdough fermented bread
for 12 h at 32 °C and with flour ash content of 0.6 g/100 g. It
was also found that the fermentation time had an important linear
effect on softness of bread crumb. Finally, it was confirmed that
higher ash content of flour increased firmness in sourdough breads
fermented with Lactobacillus brevis, S. cerevisiae, or a combination
starter (Collar and others 1994). Plessas and others (2007) pro-
posed the use of sourdough with immobilized cells, as it resulted
in a 3-fold delay in staling, compared to the traditional compressed
baker’s yeast bread. The authors hypothesized that the retention of
higher moisture levels after baking and reduced moisture loss rates
are due to the more compact texture in breads obtained with the
suggested technique. In particular, they showed that sourdough
breads presented lower loaf volumes for the same loaf weights, and
fewer holes of higher size, with respect to conventional baker’s
yeast bread. Dal Bello and others (2007) confirmed that the higher
volume of bread produced by the sourdough fermentation activ-
ity of the antifungal strain Lactobacillus plantarum FST 1.7 and of
Lactobacillus sanfranciscensis LTH 2581, with respect to chemically
or nonchemically acidified bread, delayed crumb staling up to 3 d.
Additionally, the L. plantarum FST 1.7 revealed inhibitory activity
against Fusaria.
Fadda and others (2010) found that durum WT bread produced
with sourdough at a dose higher than 10% significantly lowered
and slowed crumb-firming kinetics, as assessed by TA and DSC
results, the latter used with the Avrami equation, provided gluten
and yeast were added.
Recently, Tamani and others (2013) associated the increased
EPS production during dough formation following the inocu-
lation of ropy LAB starter cultures (Lactobacillus delbrueckii subsp.
bulgaricus LB18; L. delbrueckii subsp. bulgaricus CNRZ 737, and L.
delbrueckii subsp. bulgaricus 2483) with increased bread volume and
reduced staling over 5 d of storage, with respect to the control
bread, while 1 nonropy LAB (Lactobacillus helveticus LH30) did not
result in beneficial effects. The authors suggested that the higher
levels of EPS obtained with LAB may have resulted in greater
water retention, leading to the softer crumb structure of these
breads, even if they evidenced that the EPS production did not
correlate with the extension of shelf-life, thus their effect was more
qualitative than quantitative.
Baking and fermentation
It has been reported that both baking time and temperature
affect the quality and staling rate of bread (Seetharaman and oth-
ers 2002). Patel and others (2005) studied the effects of the use
of different ovens and dough size, when baking at constant tem-
perature for varying times, on texture, thermal properties, and
pasting characteristics of products. Breads baked at the lower
Bread staling review . . .
heating rates had lower amylopectin recrystallization, rate of bread
firmness, and amount of soluble amylose. Similar results were ob-
tained by Mouneim and others (2012). Baking temperature and
time affected some physical properties of bread from composite
flour made by mixing cassava and WT flour at a ratio of 10:90
(w/w) as revealed by central composite rotatable experimental de-
sign (Shittu and others 2007). Both the baking temperature and
time, among others, influenced the dried crumb hardness, due to
the complex effect of temperature and time combination, but the
developed 2nd-order response surface regression equations could
not predict satisfactorily most of the measured properties, thus the
authors proposed further studies to optimize the cassava and WT
flour bread baking process. Three different heating temperatures
corresponding to 3 heating rates were also tested by Le-Bail and
others (2009) with an innovative protocol in which a degassed
piece of dough was baked in a miniaturized oven, in order to
compare it with traditional dough. Hardening of the crumb oc-
curred after retrogradation of amylopectin, as revealed by calori-
metric tests, and higher baking kinetics resulted in faster staling
rates. Additionally, the relative Young modulus, expressed as the
ratio of the modulus of the cellular crumb compared with the
modulus of the degassed crumb, was proportional to the square of
the relative density of the crumb. In a further paper Le-Bail and
others (2012), working with a degassed sourdough, confirmed the
previously obtained results and gave more explanation on the ef-
fect of prolonged baking on staling rate, that was an increase of
the amount of amylose leaching from the starch granule, lead-
ing to a higher Young’s modulus of the crumb at the end of
staling.
Different heating rates were recently associated with water va-
por permeability (WVP), effective moisture diffusivity (Deff ), and
sorption of bread crust and crumb (Besbes and others 2013). The
authors showed that baking at 240 °C gave both crust and crumb
with higher moisture diffusivity coefficient and that the crust had
a higher WVP than that of sample baked at 220 °C. They pro-
posed a more pronounced porosity of crumb and crusts of breads
baked at the higher temperature, as revealed by porosity values and
scanning electron microscopy (SEM) determinations, as the cause
of the obtained result. Purhagen and others (2012) concluded that
breads obtained with different fibers (fine durum, oat bran, rye
bran, and WT bran) baked in pan remained softer after 7 d of
storage, with respect to free-standing baked sample, and attributed
this to the lower specific volume of pan-baked breads due to their
high water content. Moreover, pan-baked loaves lost less water
during storage, with respect to free-standing sample, probably be-
cause of the smaller crust area of these loaves. The difference in
staling behavior between the 2 baking methods was not attributed,
however, to starch retrogradation, while the influence of fibers was
small, if compared to the baking method, thus confirming data ob-
tained in another paper in which other antistaling agents, namely
α-amilase, distilled monoglyceride, and lipase, were compared to
the baking method (Purhagen and others 2011a).
The effect of fermentation on the firming kinetics could not be
explained only by its effect on volume, but also with the presence
of different enzymes, such as amylases, proteases, or lipases that,
alone or in combination with other enzymes, may help in reducing
the firming rate in white or wholemeal bread, thus longer fermen-
tation times enhanced the action of the enzymes, with a resulting
reduction of the staling rate (Gomez and others 2008). The higher
the yeast dose, the higher the quantity of dough enzymes previ-
ously cited. Temperature of fermentation, on the other hand, had a
minor impact on bread staling. Moreover, the authors managed to
484 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
adjust the firmness parameters to simple curvilinear equations and
obtained high correlation coefficients (>90%). Ozkoc and others
(2009) compared different baking methods, namely conventional,
microwave, and infrared-microwave combination, in order to as-
sess staling kinetics of hydrocolloid-supplemented breads during
120 h of storage, by using several methods, namely TA, DSC, RVA,
X-ray, and Fourier transform infrared spectroscopy (FTIR). The
starch retrogradation of breads obtained with a combination oven
was similar to that of conventionally baked ones, as revealed by
H values and FTIR outputs, thus leading the authors to postu-
late that it was possible to produce breads by combination heating
with a staling rate similar to that of conventionally baked ones.
Moreover, data from RVA and X-ray showed that the rapid staling
rate typical of microwave baking can be mitigated by infrared-
microwave combination heating. As expected, the addition of a
XG–GG blend to the formulation retarded staling.
High-hydrostatic-pressure processing (HPP)
This unit operation may change structural and functional prop-
erties of proteins and cereal starches and is being investigated to
improve quality of breads made with flours alternative to WT.
In a fundamental study on the use of HPP to improve the
bread making performance of oat flour, Huttner and others (2010)
subjected oat batters to 3 levels of HPP (220, 350, and 500 MPa)
and the treated samples replaced untreated oat flour in an oat bread
recipe, by 10%, 20%, or 40%. Staling rate, as assessed by a TPA
crumb hardness test, was reduced when 10% to 40% oat batter
treated at 200 MPa was used, if compared to the control. The HPP
treatment at 200 MPa weakened the proteins, affected the moisture
distribution, and also influenced the interactions between proteins
and starch, which caused a decrease in the staling rate of the oat
bread. Opposite results were presented in another paper published
some months later (Vallons and others 2010). The authors replaced
2% or 10% of a sorghum bread recipe with sorghum batters HPP-
treated at 200 and 600 MP and found that breads containing 2%
sorghum treated at 600 MPa had slower staling rates than control.
More recently, Angioloni and Collar (2012) worked with fixed
amounts of oat, millet, and sorghum HPP-treated flours (350
MPa), which replaced (60% for oat, 40% for the other 2) WT
flour. Half of the control bread was prepared by applying HPP to
50% of WT flour. Results indicated that HPP-treated WT and
oat breads lowered final values of crumb hardness and Avrami
exponent, thus giving softer breads with slower staling kinetics,
with respect to control bread.
Measurement Methods
The results reviewed above refer to one or more measurement
methods to assess bread staling, but there has not been up to now
a methodology that allows a complete measurement of the staling
phenomenon to the same extent as that described by a consumer
(Sidhu and others 1996). Different specific reviews before that of
Gray and Bemiller (2003) have dealt with the methods used to
assess the rate and/or degree of staling such as those mentioned
by Maga (1975), Kulp and Ponte (1981), and Ponte and Ovadia
(1996). In most cases bread staling, apart from the more simple
and direct TA, is indirectly measured as the extent of starch ret-
rogradation, as also reviewed by Karim and others (2000). An
interesting review, moreover, revisited crumb texture evaluation
methods (Liu and Scanlon 2004), while another one summarized
the more frequently used analytical methodologies for assessing
bread staling (Choi and others 2010). In the following sections
the major reports dealing with new methodologies and/or new

C 2014 Institute of Food Technologists®
Bread staling review . . .

applications used to measure bread staling during the last 10 y will it was able to study accurately amylopectin retrogradation and to
be reviewed. obtain a very good correlation with DSC data when looking for
protein and temperature effects on amylopectin retrogradation de-
Thermal analysis velopment, even if it showed difficulty in measuring the changes of
Bollain and others (2005) proposed small dynamic defor- the amylase–lipid complex during storage. The authors proposed
mation and large static deformation methods to evaluate the 550, 970, 1155, 1395, and 1465 nm as important wavelengths of
thermodynamic and physical–mechanical changes of enzyme- NIRS and concluded that amylopectin retrogradation was prob-
supplemented white or whole bread during staling. They suc- ably the main factor in bread staling and that the amylase–lipid
cessfully detected rheological changes of bread, as influenced by complex contributed little to bread staling after 1 d of storage.
recipe and storage time, with dynamic thermomechanical analysis Cocchi and others (2005) coupled middle-infrared spectroscopy
(DTMA) in the compression mode. They detected that the onset (MIR) with principal component analysis (PCA) to follow bread
frequency (f0 ) and the rubbery or plateau moduli (E ) rose as the shelf-life in a rapid and affordable way. Spectra of breads stored up
bread aged in a similar way to the hardening and firming curves. to 7 d at ambient temperature were acquired in attenuated total
Moreover, relationships between the dynamic (DTMA) and static reflection mode with an FTIR spectrometer, normalized and then
(TA) methods were found. subjected to PCA. The authors revealed that the 1st PC increased
Ribotta and Le Bail (2007) used DSC and DMA to study bread with aging of samples and that the more influential variables on
staling. DSC evidenced water migration from the crumb to the PC1 corresponded to spectral regions attributed to typical starch
crust and changes of water properties as initial and onset temper- bond vibrations.
ature of ice melting decreased significantly after 1 d and freezable Pikus and others (2006) proposed for the first time the small-
water (FW) and unfreezable water (UFW) decreased and increased, angle X-ray scattering (SAXS) method to study bread staling. The
respectively, as a consequence of aging. DSC results suggested the authors, by using fresh dry and fresh water suspension samples,
existence of a possible 2nd transition, due to ice-melting transition found that bread staling is accompanied by significant electron
being diverted to lower temperatures. The authors proposed that a density changes, indicating that there were significant changes at
concomitant water migration from the crumb to the crust and an the nanoscale level during the staling process. They suggested,
incorporation of water molecules into the starch crystalline struc- by analyzing results obtained with the dynamics of the scattering
ture, developing after bread staling, may account for the decrease intensity changes in the bread samples, along with those of SAXS
in FW after 4 d of storage at 4 °C. Moreover, they suggested investigations on native starch, that SAXS scattering changes for
that some water molecules were incorporated in the crystalline the dry samples originated mainly from the gluten phase, while
lattice when starch crystallized. DMA analysis showed significant for water suspension samples they were mainly from the starch
changes in the thermo–mechanical profile of the crumb during matrix. The authors concluded that a comparison of results of
staling, as aged breads contracted at a lower rate during cool- SAXS with data obtained with other methods, on the same bread
ing, but they evidenced a greater deformation during freezing and sample, would be interesting.
higher retraction within the complete cooling–freezing cycle, thus Piccinini and others (2012) proposed, for the first time the
suggesting that the higher matrix rigidity, a consequence of the use of NIR Fourier-transform-Raman spectroscopy to monitor
higher amount of retrogradated starch, affected contraction capac- starch retrogradation in stored hard-WT bread and, with the help
ity. The authors postulated that interactions during the hydration of TA data, to follow bread staling for 20 d. The authors found,
of the gluten network might explain the latter phenomenon. by applying the 2D correlation analysis applied to the Raman
spectra of bread crumb during storage, that both the peak shift
Infrared spectroscopy and narrowing of the band at 480 cm−1 during retrogradation
Near-infrared reflectance spectroscopy (NIRS) was used to ob- correlated well with the crumb-firming data obtained using the
tain spectra during staling of bread and the results were compared stress relaxation tests and that during starch retrogradation a new
with those obtained by TA (Xie and others 2003). Results showed band peaking at 765 cm−1 appeared.
that NIRS spectra were highly correlated with firmness values
assessed with the more common TA. Moreover, the authors ev- NMR spectroscopy
idenced that NIRS measurements had a better correlation with Curti and others (2011) used 1 H NMR relaxometry and, for
storage time and also lower batch variability, with respect to TA- the first time in bread, the 1 H NMR fast field cycling (FFC) tech-
derived data, thus NIRS was suggested as a better tool than TA to nique to follow the changes in 1 H T1 relaxation in the 0.01 to 20
study bread aging, probably because NIRS may follow both phys- MHz frequency range, in order to check for the interactions of
ical and chemical changes occurring during the staling process, water molecules with paramagnetic and large-sized macromolec-
while TA was limited to the only aspect of firmness evolution. In ular system during bread staling. 1 H T1 relaxation data at 20 MHz
a further paper, Xie and others (2004) proposed the use of NIRS confirmed previous results, while studies conducted at a lower fre-
as a fundamental tool to study bread staling with the help of DSC, quency (0.52 MHz) evidenced, for the first time, the presence of
as well as the effects of starch, protein, and temperature (stor- two T1 proton populations, which were tentatively attributed to
age at 12.5 or 31.5 °C) on bread staling. DSC data showed that protons of the gluten domain at early storage times. The authors
temperature strongly affected the staling rate, while the protein suggested that the use of the 1 H NMR FFC technique at different
contribution was limited, if compared to temperature during 4 d frequencies may be an additional way for monitoring molecular
of storage. Using the enthalpy ratio between bread supplemented dynamics in bread and therefore a new valuable instrument to help
with starch and sample produced with starch–protein, it was pos- understand the bread staling phenomenon.
sible to conclude that protein might retard bread staling not only Bosmans and others (2013) used H NMR relaxometry, along
by diluting starch (Kim and D’Appolonia 1977; Every and others with DSC and wide-angle XRD, to better elucidate the relation-
1998), but also by interfering with amylopectin retrogradation. ship between biopolymer interactions, water dynamics, and crumb
NIRS was found to be very useful in studying bread staling, as texture evolution during 168 h of storage of bread. The NMR


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 485
Bread staling review . . .

analysis allowed finding 6 proton populations in bread crumb and ships between mechanical behavior of pan bread, supplemented
from the NMR profiles of bread crumb they were able to de- or not with an amylolytic enzyme, a nonamylolytic enzyme, and
duce the extent of formation of both amylopectin crystals and a combination of the 2, and loss of sensory quality during 20 d in
of crumb firmness. On the basis of data obtained, they con- storage. TPA at 40% and 80% was proposed for the first time as
cluded that the increase in crumb firmness of stored bread was well as a new penetration test. The authors positively correlated
caused by a combination of different events that were amylopectin hardness with sensory “difficulty in swallowing,” “crumbliness,”
retrogradation and the formation of a continuous, rigid, crystalline “hardness,” and “oral dryness,” and negatively correlated it with
starch network that included water in its structure. They also no- sensory “cohesiveness,” “softness,” and “size of soft zone,” while
ticed moisture migration from gluten to starch and from crumb to these parameters correlated well also for springiness and cohe-
crust, resulting in additional reduction of moisture in the gluten siveness detected at 80% TPA, thus evidencing that TPA values
network, with the consequence that the subsequent increase in obtained at the compressions resulted in greater sample distortion
stiffness contributed to the increase in crumb firmness. and gave information that was better correlated to sensory per-
ception. Finally, the analysis of the penetration profiles gave data
X-ray crystallography that were very useful to complement the TPA results, in order to
Del Nobile and others (2003) developed a mathematical model assess bread staling.
able to predict the starch retrogradation kinetics of durum WT Angioloni and Collar (2009b) suggested the complementar-
bread in order to link it to the crumb staling. Two equations were ities of instrumental static (TPA, firmness, and relaxation test)
proposed dealing with data obtained with wide-angle XRD (starch and dynamic (innovative oscillatory test) analyses with empirical
retrogradation) and compression tests (crumb firming process), and sensory characteristics in assessing commercial whole and white
related to samples held at 5 °C and 2 water activity values, in order bread quality during a 10-d storage period, although the 2 differ-
to accelerate the test. The proposed model fitted well with the ent approaches investigated the bread characteristics at molecular
obtained results; moreover, the authors evidenced that lowering or macroscopic level. In particular, the authors found that static re-
the water activity value resulted in a higher overall starch crystal laxation parameters initial force (F0 ), momentary force at time (t)
growth rate, due to the increase of the starch nucleation rate. F(t), constants related to stress decay k1(s) rate and residual stress at
X-ray patterns were studied with different methods, namely rel- the end of the experiment (k2t), and dynamic (stress) bread crumb
ative crystallinity, total mass crystallinity grade (TC), B-type mass rheological attributes were correlated well, thus both techniques
crystallinity grade, and V-type mass crystallinity grade, in order to were useful in evaluating crumb textural characteristics of fresh
increase knowledge of the relationship between starch crystallinity and staled breads. Moreover, the sensory attributes (softness) and
and bread staling during 7 d of storage at 4 °C (Ribotta and oth- the overall acceptability were negatively correlated with either dy-
ers 2004b). The authors pointed out that: (a) fresh baked bread namic stress or static F0 . The authors concluded that the obtained
contained only a V-type structure, while the B-type structure ap- results were quite promising for a proper bread crumb quality as-
peared after 24 h and increased during bread staling; (b) TC and sessment, as the novel proposed approaches gave data with better
relative crystallinity significantly increased during the first 24 h, accordance with consumer awareness.
then slightly decreased, thus indicating the appearance of the B-
type structure; (c) TC and relative crystallinity decreased at the end Electrical impedance
of aging, which is associated with an increased degree of ordering Bhatt and Nagaraju (2009) developed an instrument working
of the amorphous phase caused by staling. They suggested that with electrical impedance to assess the electrical properties of
staled bread showed reformation of the double helical structures WT bread crumb and crust, and they investigated changes in
of amylopectin and a reorganization, during aging, into crystalline electrical impedance behavior during 120 h of storage with the
regions that imparted rigidity. With this in mind, they concluded use of multichannel ring electrodes. Variations in crust capacitance
that amylopectin retrogradation is an essential step to consider to showed that there was a sharp increase in value after 96 h of storage
better understand bread staling. at 17.6% moisture content, so after that period a glass transition
Colorimetry occurred with a content of more than 17.6% of moisture at room
Popov-Raljić and others (2009) used, for the first time, an temperature. On the other hand, the resistance measurements of
MOM-color 100 tristimulus photo colorimeter, in CIE, CIELab, crumb showed a decrease during staling, thus revealing that the
ANLAB, and Hunter systems to correlate crust color changes and starch crumb recovered its crystallinity during the storage time
staling of bread of different compositions packed in polyethylene of 120 h. Data on crust capacitance and crumb resistance were
film during 3 d at 20 °C. The color of 3-d-stored bread sam- validated by results obtained with DSC analysis (variation in glass
ples was always lighter, as the stored breads showed higher average transition temperature and enthalpy). The authors concluded that
reflectance, with respect to just baked loaves. The authors hy- the proposed instrument was suitable for rapid and nondestructive
pothesized the moisture loss as the cause of this color change and, measurement of electrical properties of bread at different zones
by fitting the values of average reflectance with a curve describ- with minimum error, thus enabling to study staling at crust and
ing the dependence of average reflectance with storage time, they crumb simultaneously.
found a correlation coefficient of 0.99, thus they concluded that
the change in color is the direct consequence of staling (Note: it Mixed instrumentation
would be more useful to correlate crust color changes with objec- Primo-Martı́n and others (2007) gave new insight on staling of
tive bread staling measurements, such as hardness, more than with bread crust by using a wide range of measurement techniques,
time). namely, confocal scanning laser microscopy, wide-angle X-ray
powder diffraction, polarized light microscopy, solid-state 13C
Rheological methods cross-polarization-magic-angle spinning NMR, and DSC. The
Textural assessment of staling has been reviewed by Chung and authors found that baking resulted in gelatinization of only 60%
others (2003). Fiszman and others (2005) investigated the relation- of the crust starch, and this fraction retook its crystallinity after a

486 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Bread staling review . . .
long time, compared to crumb. The authors, thus, concluded that
staling of the crust cannot be ascribed to amylopectin retrograda-
tion that was measurable only after 2 d of storage, while loss of
bread crust freshness happened before 1 d of storage, as already
reported by Primo-Martı́n and others (2006).
A very interesting application was that proposed by Botre and
Garphure (2006) who used a tin oxide sensor array and self-
organized map (SOM)-based E-nose for analysis of volatile bread
aroma, in order to correlate the obtained data with bread freshness
and, thus, predict staling. Data obtained on bread stored for 5 d
at 25 °C over 3 wk and purchased by 3 producers showed that
the E-nose was able to predict freshness or staleness of bread with
an accuracy of up to 97%, when using data sets and the SOM
network of the same week, while this value dropped from 75% to
85% when considering the 3 wk. Moreover, when different bread
producers were considered, the accuracy value was again high and
ranged from 76% to 83%. The authors, thus, suggested that the
SnO2 gas sensor and SOM neural network-based electronic nose
was an attractive, low-price alternative for assessing bread freshness.
Lagrain and others (2012) considered bread crumb as a linear-
elastic, cellular solid with open cells in order to better understand
its mechanical properties at the fresh state and during storage,
when applying low stresses in the evaluation. They used static
compression of bread crumb and developed a new instrument
probe to determine the shear storage modulus by applying a si-
nusoidal shear force to the sample. Cellular structure evolution
during storage was assessed by digital image analysis, while a non-
contact ultrasound technique was used to measure crumb open
porosity and mean size of the intersections in the crumb cell walls.
Results of image and acoustic analyses showed that the original
crumb structure was not affected by staling and crumb physical
measurement confirmed this behavior, as the Poisson coefficient
ν obtained from texture data yielded a time-independent value.
Moreover, by changing gluten functionality with redox agents
(potassium bromate and glutathione) the authors found that the
increase in evolution of the normalized modulus, which was the
ratio between the Young’s modulus E and the crumb density ρ
(E/ρ), was independent from ρ, thus molecular changes in the
gluten protein network induced by the redox agents had effect on
crumb cell wall stiffening. Finally, changing starch properties with
a maltogenic exo-α-amylase, while reducing crumb stiffening dur-
ing 168 h of storage, as expected and as revealed by amylopectin
recrystallization (DSC), did not result in changes in the cellular
structure.
Conclusion
Bread staling continues to be responsible for huge food wastes
all over the world. The phenomenon is still far from being fully
elucidated, but this literature review of the last 10 y confirmed
existing theories and gave new insights. The text points out the
central role of starch and starch–gluten interactions at the ba-
sis of the staling mechanism and highlights the effect of dif-
ferent ingredients (hydrocolloids, enzymes, or WWS), as well
as the increased interest in dough or frozen PB bread for ex-
tending bread shelf-life. Despite new measurement techniques,
such as NIRS, NMR, and X-ray, which give novel and in-
teresting details on bread firming and also evidence of their
importance as complementary tools to traditional measurement
techniques, the real challenge still remains the knowledge of
the precise mechanism(s) of staling. Further efforts must be ex-
erted to explore and exploit the power of novel technologies in
bread processing, particularly the nonthermal technologies (high

C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 487
hydrostatic pressure, ultrasound processing, pulse-light technol-
ogy, and others), and their effects on the retardation of bread
staling.
Acknowledgments
The authors acknowledge the financial support of follow-
ing European Institutions: Regione Autonoma della Sardegna,
Legge 7, project title “Ottimizzazione della formulazione e
della tecnologia di processo per la produzione di prodotti da
forno gluten-free fermentati e non fermentati,” and Consejo
Superior de Investigaciones Cientı́-ficas (CSIC), and Ministerio
de Economı́-a y Competitividad (Project AGL 2011-22669) of
Spain.
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