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Vol. 2, 2003—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 1© 2003 Institute of Food Technologists

Bread Staling:Molecular Basis

and ControlJ.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 breadstaling is a complex phenomenon in which multiple mechanisms operate. Polymer crystallizations with the formation ofsupermolecular structures are certainly involved. The most plausible hypothesis is that retrogradation of amylopectinoccurs, and because water molecules are incorporated into the crystallites, the distribution of water is shifted from glutento starch/amylopectin, thereby changing the nature of the gluten network. The role of additives may be to change thenature of starch protein molecules, to function as plasticizers, and/or to retard the redistribution of water betweencomponents. Nothing more definite can be concluded at this time.

IntroductionAlthough 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 theconsumer. Bechtel and others (1953) defined staling as “a termwhich indicates decreasing consumer acceptance of bakery prod-ucts caused by changes in crumb other than those resulting fromthe action of spoilage organisms”. While an American Associationof Cereal Chemists Approved Method (AACC Method 74-30;AACC 2000) quantifies staling organoleptically, many researchersuse the 1953 definition as a general definition and describe spe-cific components of the complex staling process with specificterms such as crumb firming, crust staling, and organoleptic stal-ing (Kulp and Ponte 1981). In fact, the most widely used indicatorof staling is measurement of the increase in crumb firmness (see“Rheological methods: Uniaxial compression” section), which isthe attribute most commonly recognized by the consumer. In thisreview, 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 whichcontains a continuous phase composed in part of an elastic net-work of cross-linked gluten molecules and in part of leachedstarch polymer molecules, primarily amylose, both uncomplexedand complexed with polar lipid molecules, and a discontinuousphase of entrapped, gelatinized, swollen, deformed (wheat) starchgranules. Neither the bread system nor the staling process is un-derstood well at the molecular level. Even simple bread doughformulations contain several ingredients, which themselves maycontain several components, each of which may undergo chang-es during the breadmaking process and during aging of the finalproduct. 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 havefailed to fully explained the process.

Because the literature on bread staling is so extensive, any re-

view of bread staling confined to a limited space cannot discussall available information, hypotheses, or conclusions; nor can itgive 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 availablefor a more thorough treatment. Discussions in the literature refer-enced in this review will lead readers to additional information. Abrief review not elsewhere referenced in this review is that byGuilbot and Godon (1984).

Physical and mechanical mixing, chemical reactions (includingenzyme-catalyzed reactions), and thermal effects (baking time andtemperature) are factors that influence the nature and properties ofthe final product. This review focuses on antistaling agents, usingwhat is known about the mechanism of staling and factors that af-fect staling rate as a basis for the discussion. It also focuses oncrumb staling, because crumb staling is of much greater concernto 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 directcause-and-effect relationship; for example, addition of a surfactantknown to form a complex with amylose may increase shelf life,but that does not necessarily mean that amylose complexation isresponsible for the increase in shelf life. The critical effect couldbe on the structure of water, for example. (2) There is much infor-mation on the effects of various additives and conditions onstarch 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 andbread crumb are believed to be very similar, if not identical (Sladeand Levine 1987.) For a review of this literature as it pertains tostaling and that of the crystal structures in bread, the reader is re-ferred to the review of Zobel and Kulp (1996). (3) When anamount of an additive is stated, it is a percentage of the weight offlour. (4) Abbreviations used include CP MAS (cross-polarizationmagic-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 flourTo understand the mechanism of staling in breads, it is impor-

tant to understand the natures of the major components that makeup the system. Relationships of these components to staling aredescribed in Section 2.2. The role of water and additives in stalingare 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, amold inhibitor (sodium propionate), and an oxidant (Hoseneyand 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 finalproduct. 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 stagesin the life of aged bread are outlined in Table 1.

Protein. Hydrated gluten is the continuous phase of wheat flourdoughs (Ponte and Faubion 1985; Davies 1986). During baking,gluten is denatured, and protein-protein crosslinking occurs viaformation of disulfide bonds (Schofield 1986). The resulting net-work, combined with partially gelatinized starch granules, is mostcertainly responsible for the semirigid structure of baked products(Blanshard 1988; Hoseney 1989).

Starch. Wheat flour contains 84 to 88% (db) starch. Duringbaking of bread dough, the starch granules are generally gelati-nized (Table 1, footnote c), but little else other than restrictedswelling followed by collapse happens to them because of thelimited amount of water present in the dough system (Schoch1965), so deformed wheat starch granules can be isolated fromthe crumb (Hoseney and others 1978). [Note: When starch gran-ules are heated in excess water, granules swell and some portionof 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 duringbaking in the limited moisture system of bread dough retrogradesupon cooling and rapidly becomes unextractable (Kim andD’Appolonia 1977b,c); so even if amylose does leach from gran-ules, by the time bread has completely cooled, any interstitialamylose will have retrograded (that is, become insoluble) and isunlikely, therefore, to play a major role in subsequent stalingevents.] Even in the presence of excess water, monoglyceridesblock the leaching of amylose molecules (Schoch 1965; see “Sur-face-active lipids: Surfactants” section), so it can be assumed thatother surfactants would act in the same way, especially in the lim-ited moisture system of bread. Therefore, freshly baked andcooled bread is an elastic system containing swollen wheat starchgranules that are still largely intact, but may be deformed.

On the other hand, observations made with transmission elec-

tron microscopy, led Bechtel and others (1978) to conclude that,after baking, most starch granules were destroyed and most starchmolecules were part of the continuous phase, but separate fromprotein strands.

Nonstarch polysaccharides. Arabinoxylans and arabinogalac-tans (arabinogalactan-proteins) are the “pentosans” (more proper-ly pentoglycans) of wheat flour. Arabinoxylans are divided into 2classes (“water-soluble” and “water-insoluble”) and have beenmuch more extensively studied than have the arabinogalactans(Loosveld and others 1997), because they are present in greaterconcentrations and are believed to play a more important role inboth the preparation and the shelf-life of bakery products. Bothclasses 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 structureas a function of cultivars (Izydorczyk and others 1991; Cleemputand others 1993; Izydorczyk and Biliaderis 1993; Rattan and oth-ers 1994). Their influence on breadmaking and bread quality isstill being debated (see “Mechanisms of staling: Role of pen-tosans” section).

Mechanism of stalingAttention 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 generallycaused by moisture transfer from the crumb to the crust (Lin andLineback 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 thecrumb (Short and Roberts 1971).

The key hindrance to development of a preventive strategy forbread staling is the failure to understand the mechanism of theprocess. Many investigations have examined the phenomenon ofcrumb-firming, and many theories have been proposed and dis-cussed in previous reviews (Herz 1965; Willhoft 1973; Zobel1973; Maga 1975; Knightly 1977; Kulp and Ponte 1981; Zobeland Kulp 1996). A cursory overview of the major theories on thesubject is presented here.

Amylopectin retrogradation. Katz (1928) proposed that starchpolymers retrogradation was responsible for staling of bread be-cause his x-ray diffraction patterns of fresh bread were similar tothose of freshly gelatinized wheat starch, while the patterns ofstale 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 thestaling process. Hellman and others (1954) provided evidencethat 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 ofbread 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 molecularrigidity, that is, decrease in molecular mobility, in an aged breadsample (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 andmay 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 firmingis amylose or amylopectin also has been debated. The linear,


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Bread staling. . .

more readily retrograded fraction, amylose, was suspected first(see “Mechanisms of staling: Role of amylose” section). Evidencefrom Katz (1928) suggested formation of side-by-side associationsof linear starch molecules in the B-type x-ray patterns of staledbread and retrograded starch. Hixon (1943) speculated that, if awaxy wheat variety were available, then bread made from thatflour 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 originalfreshness. Since retrograded amylose will not melt at this tempera-ture (Knightly 1977), amylopectin was suggested to be the fractionof starch responsible for staling. Supporting evidence was pre-sented when bread prepared from a synthetic flour composed ofwaxy maize starch and nondevitalized gluten exhibited a normaltendency to stale (Noznick and others 1946). Further, Schoch andFrench (1947) found that the water-soluble material that could beleached 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 beretrograded/insolubilized during cooling. The important role ofamylopectin in starch retrogradation was confirmed by calorime-try (Russell 1983a, b).

However, Hoseney and Miller (1998) have pointed out thatstale 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 bythe time the temperature reached 60 ºC, retrogradation of amy-lopectin cannot be the only factor affecting firming. Retrogradedwaxy corn starch (5%) was added to a bread formula and foundto decrease gelatinization and to reduce the firming rate (Hibi

2001). [Note: As pointed out above, the retrograded materialshould have melted during baking so the effect would be one ofadding corn amylopectin.]

Toufeili and others (1999) found that an all-amylopectin Arabicbread (made with waxy barley starch and cross-linked waxy bar-ley starch in place of wheat starch) staled at a significantly fasterrate than did Arabic bread made with normal wheat starch, that alow degree of starch crosslinking promotes recrystallization ofamylopectin [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 swellingand separation of polymer chains].

Most agree that there is at least a correlation between amy-lopectin retrogradation/crystallization and staling, even thoughthe 2 events may not be part of the same process. Our conclusionis 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 inconcentrated wheat starch (and other starch gels), see Keetels andothers (1993, 1995, 1996 a,b,c,d; Vodovotz and others 2002).

Role of amylose. While Schoch and French (1947) believedthat the linear fraction of starch had a negligible influence onbread staling, there is evidence that amylose is involved in someway. 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-amylopectinaggregation was responsible for the changes that occur during ag-ing of bread crumb. Kim and D’Appolonia (1977c) found that thesolubility of amylose decreased markedly during the 1st d ofbread storage, while the solubility of amylopectin decreasedsteadily over 5 d of storage. They also found that the amount of

Table 1—States of critical components in various stages in the life of breada

Stage Starch Gluten Polar lipid

Dough Hydrated, intact granules. Hydrated. In the form of Free. Perhaps someApb partially crystalline. fibrils with adhering starch protein-lipid interactions.Amb amorphous granules in a continuous network.

Fresh-baked, Granules in a spectrum of states. Denatured. Crosslinked. Possible Some complexed with Ambut cooled, Some rather intact. Most gelatinizedc formation of starch-gluten associations (inside and outside of granules).bread and deformed/collapsed. (starch-gluten fibrils) during baking. Some free. Possible protein-lipid

interactions (Sections 2.2.6, 4.2).

Starch-starch interactions bothwithin and between granules

Double-helical structure of Ap at leastpartially lost. Perhaps some Ap moleculespartially or completely outside of granules(Section 2.2.1).

Some Am partially or completely leachedfrom granules, putting some of it in thecontinuous phase, where it is largelyinsoluble. Some complexed with polarlipid molecules. (Sections 2.2.2, 4.2).

Aged bread Retrograded Ap inside gelatinized Loss of water of hydration from gluten Unchanged from fresh-baked breadgranules. Perhaps some outside of network via transfer to starchd, which (?)granules (Sections 2.2.1, 4.1.1). enables crystallization of Ap (Section 3.2).

Am retrograded. Some complexed withlipid. Probably little changed from fresh-baked bread (Sections 2.2.2, 4.1.1).

aBased on best evidence available. Other views have been stated; see discussion.bAp = amylopectin, Am = amylosecGelatinization is the disruption of molecular order within starch granules as they are heated in the presence of water (Atwell and others 1988).dBoth macro-and microscopic redistribution of water occurs during aging.




soluble amylopectin in fresh bread was 5 to 24 times the amountof soluble amylose, indicating that little amylose was leached fromgranules 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 theratio of amylose to amylopectin in flour by using waxy barleystarch and also found that the amylose fraction was involved instaling of bread through 1 d only. It has been suggested that therole of amylose in bread staling may be merely one of dilutingamylopectin (Inagaki and Seib 1992), a conclusion reached froma study of breads made with cross-linked waxy barley starch,which staled at a faster rate than did control breads, even thoughthe experimental bread had less firmness after 6 h. Evidenceagainst a role for amylose in staling is that, while stale bread canbe refreshed by heating, amylose crystals (either of the V-type orB-type) do not melt at the temperatures employed (Knightly 1977).

From an interesting microscopic examination, Hug-Iten andothers (1999) reported that, during baking, there was a separationof amylose and amylopectin where amylose accumulated at gran-ule centers, and that, upon aging, gelatinized granules regainedbirefringence, with the most intense birefringence being observedin the amylose-rich granule centers. [Note: It may have only ap-peared that amylose accumulated at granule centers. Anotherpossible explanation is that amylose was lost by leaching from theouter 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 inbread staling.

Relationship between crumb firming and starch retrograda-tion. Alsberg (1927, 1928) proposed that bread staling could notbe completely attributed to starch retrogradation, since retrogra-dation in pastes is a slower process than is staling. [Note: There isreason to believe that retrogradation might occur more easily inbread than in pastes, which are more often studied, because inour 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 crystallinepacking order disruption, it is much easier for chains, which arestill close to one another and still aligned similarly to what theywere in the native granule, to reassociate than it is for amylopectinmolecules 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 inhibitgranule swelling/gelatinization from occurring in the first place areeffective as antistaling agents.] Others have also questioned theconcept that amylopectin crystallization and bread firming areone and the same, even though both may occur simultaneously(Dragsdorf and Varriano-Marston 1980: Baik and Chinachoti2000).

Dragsdorf and Varriano-Marston (1980) concluded that there isnot a cause-and-effect relationship between starch crystallizationand bread firming. Their results agreed with those from earlierwork by Zobel and Senti (1959), who observed an increase incrystallinity 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 relatedto the staling rate of bread. Neither did changes in starch crystal-linity upon reheating bread (DSC monitoring) correlate well withchanges in staleness. Furthermore, it was suggested that the de-gree of softening of stale bread was temperature-dependent, and

because the relationship was biphasic, that at least 2 mechanismswere responsible for the staling and refreshening of bread (Ghiasiand others 1984).

However, using DTA to examine retrogradation in starch(source unstated) pastes, McIver and others (1968) determinedthat the calculated Avrami exponent and time constant were ingeneral agreement with values found for bread (Axford and Col-well 1967) and, therefore, concluded that starch retrogradation isthe major factor in bread staling. Colwell and others (1969) foundthat the role of starch crystallization in the firming of bread be-comes progressively less important at storage temperatures above21 °C (70 °F).

Others have concluded that starch plays a role in strengtheningthe structure of bakery products that is at least equivalent to thatof gluten (Gambus 2000) and that starch retrogradation alone issufficient to cause bread firming (Morgan and others 1997).

Although considerable evidence has been presented that thereis not a direct cause-and-effect relationship between starch poly-mer molecule retrogradation and crumb firming, most researchersbelieve that starch retrogradation is part of the staling process. Asstated earlier, our conclusion is that amylopectin retrogradationplays a significant, but not the only, role in the stalling process.

See also the “Surface-active lipids” section for evidence on therelationship between starch polymer retrogradation and breadstaling.

Role of flour protein. Protein is another component that hasbeen studied for its role in bread staling. Kim and D’Appolonia(1977b) and others have reported that flour protein content is animportant factor in the rate of bread staling. It has been suggestedby different investigators that protein (gluten) reduces the firmingrate of bread during staling, has no effect on the firming rate, andis required for firming; that is, that staling is dependent on starch-gluten interactions. It is now generally believed (Martin and others1991) that starch-gluten interactions are somehow involved in thefirming 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 foundthat 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 ofchanges in the organization of starch polymer molecules; thereaf-ter, staling is caused by loss of moisture from gluten. Prentice andothers (1954) explained that increasing the protein content wouldtend to decrease any association between starch granules (swol-len and embedded in the gluten network), thereby retardingcrumb firmness development. They also suggested that glutenmay serve as a moisture reservoir to buffer any changes in the hy-dration capacity of starch. However, since high-loaf-volumebreads are generally softer than those of low volume, their resultsare difficult to explain (Kulp and Ponte 1981).

Willhoft (1973) suggested that the antifirming activity of glutenwas due to either a dilution of starch or the effect of gluten enrich-ment on loaf volume. Erlander and Erlander (1969) suggested thatstarch-gluten interactions could prevent staling of bread, possiblyvia hydrogen bonding between the amide groups of wheat glia-din, glutenin, and possibly albumin and hydroxyl groups ofstarch. They concluded that the ratio of starch to protein in thedough is important in determining the rate of staling and suggest-ed that some staling will occur no matter how much protein isadded.

Kim and D’Appolonia (1977b) also reported that the rate ofbread 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 thatthe 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 notsignificantly correlated to flour protein type or concentration (Pon-te and others 1962; Leon and others 1997; Gerrard and others2001). 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 tothe 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 notessential to the firming process and that increasing bread firmnessresults from chains of partially leached amylose and amylopectinattached 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 experimentsrevealed that breads of equivalent specific loaf volume staled atthe same rate irrespective of protein type or concentration, butother bread properties were altered by changes in the type orconcentration of protein (Gerrard and others 2001), lending sup-port to the above hypothesis.

Maleki and others (1980) postulated that the flour componentprimarily responsible for differences in staling rate is gluten andthat its role in staling is something other than dilution of starch.Furthermore, they proposed that starch and water solubles werenot involved significantly in determining the rate of staling.

Martin and others (1991) proposed that bread firming is a resultof hydrogen bonding between gelatinized (partially pasted) starchgranules and the gluten network in bread tying together the con-tinuous protein network and discontinuous granule remnants.They theorized that the crosslinking interactions originate duringbaking; then during aging, the crumb loses kinetic energy, andboth the number of interactions and their strength increases.When reheated, bread freshness is restored because thecrosslinks (hydrogen bonds) and entanglements between glutenand starch polymer molecules are easily broken. This theory iscongruent with results of Dreese and others (1988), who reportedthat 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 betweenswollen starch granules and the gluten network. However, theyput forth the opinion that the decrease in firming rate in breadsmade with a-amylase (see “Enzymes: �-amylases and debranch-ing enzymes” section) as a dough additive is not the direct resultof starch hydrolysis products (dextrins and maltooligosaccha-rides), some of which, they suggest, are nonspecifically associatedwith the protein matrix, but a result of modification of swollenstarch 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 shorteningand native lipids have significant effects on bread staling, neitherhave 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 onbreadmaking and bread properties is not clear, although the sub-ject has been examined extensively (Kulp 1968; D’Appolonia1971, 1980; Hoseney 1984; Meuser and Sukow 1986; Jank-iewicz and Michniewicz 1987; Roels and others 1993: Rattanand others 1994; Krishnarau and Hoseney 1994; Izydorczyk and

Biliaderis 1995; Biliaderis and Izydorczyk 1995; Cleemput andothers 1997). Water-soluble and -insoluble pentosans have beenreported both to retard staling and to have no effect on the stalingrate.

“Water-insoluble” pentosans. No differences in staling rate ofbreads made with or without tailings (starch fraction containing9% 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 thehigh hydration capacity of pentosans. However, others found thataddition of water-insoluble pentosans resulted in a considerableincrease in loaf volume (Kulp 1968) and retardation of bread stal-ing (Casier and others 1972, 1973; Denli and Ercan 2001). Toadd 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 andHoseney 1994). Such variable results may result from differencesin type, molecular weight, and/or concentration of the pentosanspresent 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 Kulpand Bechtel (1963) or Hoseney and others (1971), Michniewiczand 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-solublepentosans retarded amylose aggregation, and that addition of wa-ter-insoluble pentosans decreased susceptibility of bread crumbto a-amylase. They suggested that the contradictory results ob-tained when studying the effects of pentosans on loaf volume mayhave originated in differences in baking characteristics of theflours of various wheat cultivars, differences in chemical composi-tion of pentosans, and/or the way pentosans were incorporatedinto the dough. They further suggested that the reported reductionin 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 highermoisture content of the system.

Interaction of pentosans with protein. It is possible that pen-tosans can interact with wheat-flour components other thanstarch. Jelaca and Hlynka (1972) proposed that pentosan-gluteninteractions were responsible for baking improvement effected bypentosans. Based on the effects of actions of arabinoxylanases,Cleemput and others (1997) suggested that there are associationsof arabinoxylans with proteins and/or other wheat components indoughs.

Pentosans and starch retrogradation. The effect of pentosanson starch retrogradation has been investigated using both starchgels and bread itself. Gilles and others (1961) reported that water-soluble pentosans found in the “soluble starch” extract of breadcrumb inhibited retrogradation of amylose and that, although thepentosans affected some characteristics of the bread, staling ratewas not one of those characteristics. Kim and D’Appolonia(1977a) found that pentosans had a definite effect on retardingstarch retrogradation in wheat starch gels, with the effect of water-insoluble pentosans being more pronounced. They reported thatwater-soluble pentosans reduced retrogradation by acting onamylopectin, while water-insoluble pentosans reduced the degreeof retrogradation of both amylose and amylopectin. Similar resultswere found when the effect of pentosans on staling was studied ina bread system (Kim and D’Appolonia 1977d). Results indicatedthat the basic mechanism of bread staling was unchanged; thus, itwas 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




(based on calorimetry) that arabinoxylan-fortified breads exhibiteda greater rate of starch retrogradation (Biliaderis and Izydorczyk1995), 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 oncrumb firming. The effects of native lipids are discussed brieflyhere; added surfactants used as antistaling agents are discussed inthe “Surface-active lipids” section.

Flour lipid content has been shown to be inversely related toloaf volume (Rogers and others 1988). Protein-lipid interactionhas been suggested as the mechanism. Shortening is known tolower the firming rate of bread, but does not react with starch(Rogers and others 1988). Therefore, the results suggest that nativeflour 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 storageand that such complex formations appeared to reduce the maxi-mum amount of starch retrogradation. However, thermodynamicconsiderations indicate that amylose-lipid and amylose-surfactant(see “Surface-active lipids” section) complexes are formed duringbaking, since they form at temperatures higher than 60 °C andmelt 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, certainideas have been accepted, such as the important role of starchretrogradation, specifically amylopectin retrogradation. Even so, itis becoming increasingly evident that amylopectin retrogradationalone is not responsible for bread staling, but it is unclear whatother bread components and processes contribute to the overallstaling process. Evidence has accumulated that gluten proteinsare important and that gluten-starch interactions play a role. Mois-ture transfer (discussed in the “Moisture migration” section) seemsalso to be involved in staling. In conclusion, it is probable thatseveral factors play a role in the bread firming process, but thelarge volume of data that implicates amylopectin retrogradation asa key factor, and the information that gluten is also involved can-not be ignored.

Other Factors Affecting Staling Rate

Storage temperatureAn 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 atstorage temperatures of –1, 10, and 21 °C, while the role of starchcrystallization 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 thefrozen 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 topolymer crystallization: nucleation, propagation, and maturation.Slade and Levine (1987) and Marsh and Blanshard (1988) havedetermined 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

transition temperature of the maximally freeze-concentratedstarch (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 temperatureslightly less than the Tm of crystallized amylopectin. The retrogra-dation rate of starch pastes held under isothermal conditions isgreatest at a temperature between the optimal temperatures fornucleation and propagation (about 5 °C for a 50% paste) (Sladeand Levine 1987; Marsh and Blanshard 1988). The situation maybe somewhat different in bread, but temperature cycling is used toaccelerate 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 supportfor the involvement of starch polymer crystallization. The fact thatstaled 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 andthat the melting temperature involved implicates amylopectin asthe polymer crystallizing.

Moisture migrationWater is involved in the following changes in the bread system:

drying out, moisture equilibration between crumb and crust, andmoisture redistribution between and among bread components(Kulp and Ponte 1981). Drying out of the bread, as demonstratedby Boussingault (1852), does not explain staling, but may acceler-ate reactions leading to staling (MacMasters 1961). Thus, moisturerelationships within the crumb are important considerations whenstudying bread staling.

Breadmakers in the U.S.A. are limited to 38% water for whitepan 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 wasconfirmed (Rogers and others 1988; He and Hoseney 1990),even though the rate of starch retrogradation in bread was foundto be directly proportional to the moisture content (Rogers andothers 1988). Zeleznak and Hoseney (1986) confirmed that retro-gradation in wheat starch gels was a function of the amount ofwater present. They also reported that the moisture content ofbread is about optimal for amylopectin retrogradation and thataddition of either monoglycerides or shortening did not alter theavailable 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 crumbstructure of bread. Their conclusion is that “The overall picture ofthe crumb could be described as interpenetrated gels separatedby aqueous interphases which contain most of the low molecularweight solutes. This water is rather mobile and can facilitate mutu-al displacement of the incompatible gel phases, thus behaving asa plasticizer, and can enhance the crumb-to-crust migration ofmoisture. This local drying makes the walls of the crumb alveolimore rigid, while the concurrent moisture increase within thecrust region is accompanied by a reduction of crispness evenwhen overall moisture loss is prevented by packing bread insealed bags (Piazza and Masi 1995). Along its way toward thecrust, water can contribute to a closer packing of the structurethrough which it is moving, either within a given phase or at theinterphases, by tightening the sites able to form H bonds. Thiswould explain why refreshed bread softens when its temperaturehas been raised above Tg, but then becomes harder than the start-ing staled product, and why microwave-cooked or refreshedbread 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 andMasi 1995). Differences in vapor pressures between the crust andthe internal region of the loaf result in moisture migration from thecrumb to the crust (Stear 1990). Over time, the moisture contentin the center of the loaf decreases, while that in the external regionincreases (Bechtel and others 1953).

Baik and Chinachoti (2000) found that bread stored with itscrust became significantly firmer than bread stored without itscrust and contained more recrystallized amylopectin, indicatingthat moisture redistribution from crumb to crust plays a significantrole in firming, a conclusion confirmed by a loss in freezable wa-ter in the crumb of bread stored with crust, which correlated withchanges in its thermomechanical profile.

Several NMR parameters correlate with crumb firming and arebelieved 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 inbread 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 ofsweet rolls increased, mobility of the less-mobile water fractiondecreased, 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 Dimler1960). Water is a plasticizer, making the bread components moreflexible. Thus, as water is removed (from either gluten or starch orboth), 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 agluten 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 thatmoisture transfer would likely occur from starch to gluten. Cluskeyand others (1959) reported a progressive drop in moisture-sorptioncapacity for starch and lack of a change for gluten, indicating atransfer 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 moistureloss to starch. This concept is supported by data of Bachrach andBriggs (1947), who observed an increase in moisture-sorption ca-pacity of gelatinized starch upon aging [contrary to the results ofKatz (1928) and Cluskey and others (1959)]. Further evidencecame from investigations by Willhoft and coworkers (Breaden andWillhoft 1971; Willhoft 1971; Kay and Willhoft 1972), who re-ported that gluten undergoes a 1st-order transformation resultingin the release of water from gluten and absorption of this water byretrograding starch.

The notions of “free” and “bound” water have been reported tobe of importance in altering the rate or extent of staling in bread(Knjaginciev 1970). More recently, the use of NMR and a greaterunderstanding of the role and mechanism of starch polymer crys-tallization have led to the conclusion that starch takes up waterfrom gluten upon aging of bread. Leung (1981) and Leung andothers (1983) proposed that, as starch changes to a more crystal-line state, more water molecules become immobilized due to theirincorporation into crystal structures. Chen and others (1997a,b)reported a decrease in water mobility in bread upon staling, inagreement with results of others (Wynne-Jones and Blanshard1986; Slade and Levine 1991), and concluded that the decreasein water mobility was due to incorporation of water molecules re-

leased from gluten into crystalline structure of starch that devel-oped upon staling. [Note: The B structure has 36 water moleculesin the unit cell, whereas the A structure has only 8 (Sarko and Wu1978).] Conversely, Kim-Shin and others (1991) proposed that theredistribution of water occurs in the amorphous phase. The ratioof starch to gluten (6:1) in bread crumb ensures that moisturetransfer to the starch would result in firming of the continuous glu-ten phase (Willhoft 1971). It is important to keep in mind at alltimes, however, that the change in the state of water cannot becorrelated directly to the retrogradation process (Wynne-Jonesand Blanshard 1986).

Levine and Slade (1990) and Slade and Levine (1991) presentthorough and well-documented evidence for the role of water inthe staling process. Their arguments are based upon the mecha-nism of polymer crystallization, polymer crystallization kinetics asa function of glass transition and melting temperatures, water as aplasticizer, and sugars as antiplasticizers in the system. In their re-view, Slade and Levine (1991) state essentially that “if adequatepackaging 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 afreshly heated product to the partially crystalline state of a staleproduct, with concomitant formation of network junction zones,redistribution of moisture via both microscopic and macroscopicmigration (Czuchajowska and Pomeranz 1989), and increasedtextural firmness (Kulp and Ponte 1981; Russell 1983b; Russell1987).” They further point out that there is evidence from studiesof starch gels/pastes that the rate and extent of amylopectin crys-tallization depends on the mobility of its outer branches (Ring andothers 1987; Russell 1987; Marsh and Blanshard 1988; Sladeand 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 torecrystallization of amylopectin, and indeed it is a recrystalliza-tion. We have not used the term elsewhere in this review so as tomake it clear that amylopectin molecules do not recrystallize tothe 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 moleculesinto the crystal lattice occurs during formation of the B-type poly-morph (Imberty and Perez 1988) and, thus, a redistribution ofmoisture is effected. This process was demonstrated by a progres-sive decrease in the percentage of “freezable” water as bread wasstored over 11 d (Slade and Levine 1991). The water moleculesthat 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 breadrequires both microscopic and macroscopic redistribution of wa-ter so that there is sufficient moisture present at the locus wherecrystallization takes place to plasticize polymer chains so thatthey are mobile enough for crystallization to occur and for incor-poration into B-type crystal latices (Levine and Slade 1990; Sladeand Levine 1989, 1991).

It seems clear that moisture transfer between bread compo-nents, specifically between gluten and starch, occurs as breadages. However, like other measurable changes in the nature ofbread components, the role, if any, of moisture and moisture re-distribution in the staling process remains undetermined. (Seealso “Mechanisms of staling: Role of pentosans” and “Carbohy-drate ingredients” sections).

Processing factorsEffects of technological factors, which include manufacturing




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 variablesin reviews.

Giovanelli and others (1997) showed that baking temperaturesignificantly affects bread staling. Bread baked at lower tempera-tures stales at a slower rate in terms of both crumb hardening andstarch 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 waterpresent in the bread, the temperature inside the loaf cannot goabove the boiling temperature of that water, no matter what theoven temperature. The oven temperature can, however, affect therate of temperature rise and, thus, the time at the maximum tem-perature.] The authors suggested baking under slight vacuum toachieve 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 thatbreads made with the same dough ingredients, but by differentprocesses (and stored at the same temperature), underwent stalingat different rates because of differences in loaf volume.

Antistaling Additives

EnzymesOne 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 andothers 1953; Waldt 1968, 1969; Martinez-Anaya 1998; Bowles1996). The most useful enzymic approach to staling rate reduc-tion has been the use of �-amylases, which catalyze a smallamount of hydrolysis of the starch. Proteases depolymerize glutenproteins and modify baking characteristics. Nonamylolytic en-zymes may also be active in the enzyme supplements (van Eijkand 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 studieshave reported that the rates and degrees of firming in bakedgoods can be reduced; and the texture, flavor, aroma, and generalqualities improved; by use of a-amylases. Fungal, cereal, and bac-terial �-amylases all appeared to improve softness retention ofbread to an extent related to their heat stability (Conn and others1950; Miller and others 1953). Fungal �-amylase was inactivatedby heat before acting on the starch. Although cereal (wheat or bar-ley) a-amylases did not survive the baking process, they had timeto act on the swollen starch. A bacterial a-amylase was able topartly survive the heat treatment (Amos 1955). [Note: after thiswork was reported, intermediate thermostable bacterial �-amylas-es became available. See below.] In any case, major �-amylaseactivity takes place during baking after the starch is gelatinizedand becomes more susceptible to the enzyme (Ghiasi and others1979); there is a specific temperature range and time in the bread-making process when the enzyme is most active in degradingstarch (Martin 1989).

Waldt and Mahoney (1967) reported that, when bacterial a-amylase was used, the freshness of 4-d-old bread was equivalentto 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 isused in a bread formulation, a gummy texture results (because itcan survive baking) (Hebeda and others 1991). Fungal a-amylases(such as that from Aspergillus oryzae) are less thermostable than

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 �-amylaseshave temperature optima of 50 to 55 °C; bacterial �-amylaseshave optimum activity near 75 °C. The maximum activity of ITSenzymes occurs at about 65 to 70 °C. Thus, ITS enzymes haveoptimal activity at or slightly above the gelatinization temperatureof 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 ofbread 38 to 75%. When the point in the process where enzyme isadded was optimized, the Aspergillus ITS enzyme increased shelflife by as much as 200%. B. megaterium ITS �-amylase increasedshelf 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 hydratedmonoglyceride), found that the a-amylase was the most effectivein retarding staling as determined by DSC analysis.

The mechanism of the antistaling effect of �-amylases has beendebated. At first, �-amylases were thought to affect the staling rateof 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 rateof starch retrogradation and the rate of crumb firming (Morganand 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 (Champenoisand others 1999). Results from use of an “antistaling” a-amylaseand characterization of the properties of the resulting crumb by avariety of techniques led Hug-Iten and others (2001) to concludethat the antistaling effect of the enzyme preparation was due to itsability to produce a partially degraded amylopectin that is lessprone to crystallize, and that its ability to produce partially de-graded amylose is responsible for rapid formation of a partiallycrystalline 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 ofstarch. 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 therate of crumb firming to different degrees. They also extracted thewater-soluble hydrolysis products from aged crumb of breadsmade with the different enzyme preparations, examined them byHPLC, and found that certain peak areas were highly correlatedwith a reduced rate of crumb firming and that other peaks werehighly 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 amixture of �-amylase and pullulanase caused bread to firm at afaster rate, while use of the �-amylase alone retarded firming, Mar-tin and Hoseney (1991) concluded that hydrolysis products of aparticular 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. Biliaderisand Prokopowich (1994) found that maltose and maltotriose hadantiretrogradation effects on starch gels and proposed that thechain ordering of amylopectin in sugar-containing starch gels is afunction 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 structurepromoted 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 wateronly slightly (Biliaderis and Prokopowich 1994).

Defloor and Delcour (1999) reported that starch hydrolysisproduct preparations with average DPs of from 4 to 66 reducedDSC staling endotherms in baked and stored bread doughs. Theyattributed 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-amylaseswere directly responsible for the antistaling phenomenon ob-served by enzyme addition. Their explanation was that the low-molecular-weight products inhibited cross-link formation betweenstarch and gluten.

Min and others (1998) studied the effect of 2 novel antistalingamylases. When added to bread, they produced selectively eithermaltose and maltotriose or maltotetraose and maltotriose. Basedon the results, they postulated that maltotriose and maltotetraosewere directly responsible for retarding retrogradation in bread,suggesting that these oligomers were of the right size to interferewith 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 todiffuse easily might be the reason why it was less effective thanmaltotriose or maltotetraose (Min and others 1998). Donnelly andothers (1973) reported that there is a slight decrease in moistureadsorptive 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 thatmaltotriose and maltotetraose might hold water around starchmolecules 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. Salemand 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, incontrast to results obtained when �-amylase was incorporated asan additive. However, in contrast, Every and others (1992) foundthat maltooligosaccharides of DP 3-10 correlated with a reductionin firming rate, and Akers and Hoseney (1994) implied that starchhydrolysis products of a size greater than maltoheptaose mighthave antifirming properties.

There is a 3rd conclusion. Because added maltooligosaccha-rides did not survive fermentation and because the presence ofmaltooligosaccharides 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

their presence is simply correlated with a key modification ofstarch 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 upto 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) studiedmaltooligosaccharides of DP 3 to 8 at relatively high concentra-tions (1:1, oligosaccharide:water). Their conclusion was that thereported antistaling effect could be explained by an impact on theTg, resulting in a smaller �T above Tg, which retarded the starchcrystallization 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 withthe firming of bread was confirmed by Jagannath and others(1999a).

Dragsdorf and Varriano-Marston (1980) studied the effects ofbarley malt, fungal �-amylase, and bacterial a-amylase on starchcrystallization and organization in staling breads using x-ray dif-fraction (see also Akers and Hoseney 1994). Comparing storedand fresh breads, they found that the degree of crystallinity ofbreads 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, suggestingto them that starch crystallization and bread firming are differentand separate processes. Their results agreed with those obtainedby Zobel and Senti (1959), who suggested that bacterial �-amylas-es inhibit staling by breaking interconnecting chain associationsin the network of starch crystallites.

Retardation of bread staling, while avoiding a gummy mouth-feel, was achieved by incorporating pullulanase with a cereal orbacterial �-amylase in the dough (Carroll and others 1987). Aproduct produced by action of pullulanase or isoamylase onstarch, 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 involvedin inhibition of staling, but that the products must be of a uniquetype, perhaps either maltotriose and maltotetraose or productslarger than those present in traditional maltodextrin preparations.However, that the presence of such products is only correlated tosome other modification in starch (or another component) that isthe real determinant cannot be ruled out.

Lipases. Although Johnson and Welch (1968) patented lipaseformulations that retard staling in bread, the use of lipases forbreadmaking was virtually unknown until recently (Qi Si 1997).Depending on the type of flour and the formula, addition of some1,3-specific lipases resulted in more uniform crumb structure andthus an improvement in crumb softness during storage (amongother dough conditioning improvements). Furthermore, these li-pases were shown to be a replacement for shortening, althoughno 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 bothactivities, this combination could unknowingly be involved in theantistaling 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




do (see “Surface-active lipids” section). However, monoglycerideslack the positive dough-conditioning effects produced by lipases,and insufficient lipids are present in wheat flour to result inenough monoglycerides to achieve an antistaling effect. Finally,only 1st and 3rd position monoglycerides can complex withstarch, thus retarding staling; and the 1,3-specific lipase wouldproduce 2nd position monoglycerides (Qi Si 1997). There is noevidence that an effect produced by the released free fatty acidshas 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, acommon ingredient in breads. While action of the enzyme on thestructure of lipids could explain the crumb-softening effect, it isalso likely that changes in protein conformation via oxidation ofgluten are partially responsible (Daniels and others 1970; Frazierand others 1973). Lipid peroxide intermediates produced by theaction of lipoxygenase on polyunsaturated lipids may react withprotein sulfhydryl groups to produce protein-bound lipids, whichmay subsequently be released by oxidation of the protein.

Nonstarch polysaccharide-modifying enzymes. As discussedin the “Mechanisms of staling: Role of pentosans” section, the in-fluence of pentosans on bread properties, including the rate ofstaling, is unclear. It is also unclear whether enzymes that degradenonstarch polysaccharides in bread have any effect on bread stal-ing (van Eijk and Hille 1996). “Pentosanses” (hemicellulases) arewell-known dough conditioners in Europe and have reportedlybeen 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 ofbread staling has not been investigated thoroughly. The purposeof 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 thatprotein has a significant role in the bread staling mechanism (Mar-tin 1989; Martin and others 1991; Martin and Hoseney 1991), itis 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 concurrentwith protein hydrolysis could enhance amylase activity (Schwim-mer 1981). Alternatively, proteases could inhibit amylolysis if theycatalyzed 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 madewith �-amylase addition alone. [Note: Both ingredients most likelycontained proteases, though proteolytic activity was not tested.The former product was marketed as a dual-function enzyme, andit probably contained a significantly greater level of proteolytic ac-tivity. Whether or not the reduction in crumb firmness was due toits proteolytic activity is unknown].

Techniques for differential inactivation of �-amylase and pro-tease from malted wheat and fungal sources were developed byMiller 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 (insmall amounts) and �-amylase activities (Johnson and Miller 1949).They also concluded that, while �-amylase was the component ofmalt mainly responsible for increasing crumb compressibility after66 h of storage, protease alone (at low concentrations) increasedthe compressibility of crumb over that of the controls.

Van Eijk and Hille (1996) concluded that, while the addition ofexcess concentrations of proteolytic enzymes would certainly be

detrimental to the bread loaf, adding optimal levels of proteases tobreads 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 mechanismof antistaling (Gray and BeMiller 2001). In fact, since commercialenzymes, including �-amylases, and enzyme blends have activi-ties in addition to the stated one(s) (Silberstein 1961; Hebeda andothers 1991), the possibility that the presence of other activities(that is, lipases, xylanases, and so on) in commercial enzymepreparations might have an effect on bread staling cannot beruled out. Identification and characterization of such contaminantactivities would be useful.

Surface-active lipidsMost 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 andPonte 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 mechanismof surfactants in reducing the rate of staling. Several more detailedreviews of the use of emulsifiers in breadmaking, including a dis-cussion on the role of emulsifiers as antistaling agents, have beenpublished (Knightly 1968, 1973, 1996; Morrison 1976; Krog1981; Stampfli and Nersten 1995).

Most studies of amylose-lipid complexes involve complexesformed in dilute solutions of amylose (see, for example, Biliaderisand others 1985, 1986; Biliaderis and Galloway 1989; Biliaderisand Seneviratne 1990; Seneviratne and Biliaderis 1991) and oc-casionally in concentrated starch gels (see, for example, Biliaderisand Tonogai 1991). Details of the structures of amylose-fatty acidcomplexes, based on date from solid-state 13C CP/MAS and deu-terium NMR, x-ray powder diffraction, and DSC analysis, havebeen 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 dof 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 thesame time reducing crumb firming (Krog and others 1989). It wassuggested that the antifirming properties of DATEM may be due tochanges in cell wall thickness and elasticity effected by it. It wasfurther reported that optimal reduction in firmness increase overextended periods of storage can be achieved when DATEM isused in combination with monoglycerides.

Lecithins. Lecithins have been reported to reduce staling and tohave the advantage of being amenable to modification for specificapplications (Forssell and others 1998). Soy lecithin hydrolyzateeffectively 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 andothers 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 ofmonoglycerides (properly termed monoacylglycerols) in the formof “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 crumbfirmness. Diacylglycerols (commonly called diglycerides) had noeffect on crumb firmness when added alone to replace lard andshowed no synergistic effects with monoacylglycerols. While ad-



dition of monoglycerides may counteract staling of breads duringstorage, an increased tendency to crumble may result (Malkki andothers 1978).

The mechanism of the antistaling effect of monoglycerides isstill unknown, but it is thought to be different from that of shorten-ing (Rogers and others 1988), for monoacylglycerols can replaceshortening, but shortening cannot replace monoacylglycerols.

Krog and others (1989) concluded that reductions in crumbfirmness brought about by addition of monoglycerides wereprobably the result of interactions with amylose rather than withamylopectin. When relatively large amounts of monoglyceridesare used, essentially all released amylose can be complexed (asmeasured 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 reactionproduct 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. Otherresults (Freilich 1948; Edelmann and Cathcart 1949; Edelmannand 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 withstarch granules via hydrogen bonding.

Sodium stearoyl lactylate (SSL). Pisesookbunterng andD’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 stearoyllactylate 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 foruse as antistaling agents in bakery products. A blend developedby Knightly (1987) consisted of a hydrophilic lecithin and at leastone of the following: monoglyceride, lactic acid esterifiedmonoglyceride, succinic acid esterified monoglyceride, maleicacid esterified monoglyceride, or edible salts of stearoyl lactylicacid. This blend was claimed to both inhibit staling and to act as adough conditioner. Other antistaling surfactant blends were de-veloped by Vidal and Gerrity (1979).

Mechanism of antistaling effect of surfactants. The mechanismby which surfactants influence crumb firmness has been debatedand is discussed briefly in the following sections. Amylose-surfac-tant, amylopectin-surfactant, and protein-surfactant interactionshave all been investigated, as has starch swelling in the presenceof added surfactants. Whether surfactants actually decrease therate of firming or produce softer breads that then stale at the samerate as the control has been debated. Surfactants have multipleproperties, resulting in multiple functionalities, so definitive exper-iments examining a cause-and-effect relationship with regards tostaling are difficult, if not impossible, to design.

In excess water, surfactants do not change the gelatinizationtemperature, 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 noeffect on initial crumb firmness, but did affect the firming rate dur-ing storage, a finding in agreement with earlier reports (Favor andJohnson 1947; Skovholt and Dowdle 1950; Hopper 1949; Edel-mann and others 1950). Based on unpublished results from in-

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 complexeshave been examined using x-ray diffraction, DSC, and electronmicroscopy (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 acidmolecules are indeed inside the hydrated V helix (see below) withthe polar head group outside the lumen. Interactions of amylosewith over 20 surfactants were studied, and a “complexing index”was calculated and assigned to each one (Krog 1971). Morad andD’Appolonia (1980) demonstrated that incorporation (0.5%) of 5commercial surfactants resulted in amylose-surfactant complexes.It was found by Eliasson (1985) that the amount of amyloseleached from starch granules decreased in the presence of emul-sifiers.

Numerous studies have dealt with the ability of polar lipids toinhibit bread staling. Mikus and others (1946) suggested that a he-lical complex formed between amylose and MG, thus effecting asofter crumb, but without affecting the firming rate. Schoch (1965)reached a similar conclusion.

Data from Lagendijk and Pennings (1970) provided evidence ofthe relationship between amylose-lipid complexation and the in-hibition of staling. They reported maximum complexation withmonopalmitin, which corresponded with the softest crumb after48 and 72 h of storage and concluded that complexation reducesthe 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 uptakeby the starch from gluten during aging of bread. However, waterwas able to migrate from crumb to crust. Firmness of fresh breadwas not affected by the surfactant, although firming rate duringstorage 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 wasfound by Osman and others (1961).

X-ray diffraction can be used to detect complex formation, aswell as crystallinity and crystal types in general (Zobel 1973). Theamylose V complex helix hydrate can be detected by measuringthe intensity of the characteristic 4.4Å diffraction line. Formationof the B-type crystal structure is followed by measuring the inten-sity of the 5.25Å spacing. The B structure, typical of retrogradedstarch, is extended, unlike the tight V-form helix. Molecules inthese 2 forms do not cocrystallize (Zobel and Senti 1959). Withno 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 doughheating (Zobel and others 1988). Thus, a portion of the amylose isinsolubilized during the overall baking process. The remainingpattern 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 moleculesfrom granules during baking, thus retarding crumb firming, or re-

Bread staling. . .




duce the redistribution of water from gluten to starch and thusprevent contraction and firming of the gluten phase.

Amylopectin complexes. If one accepts the considerableamount of data pointing to amylopectin crystallization as a keyfactor in bread staling, it would make sense that, to be effectiveantistaling agents, surfactants must also interact with the amy-lopectin fraction; but until recently, there was no direct evidencefor amylopectin-surfactant complexes. Zobel (1973) and Eliassonand Ljunger (1988) alluded to possible amylopectin interactionwith surfactants, since breads with adjuncts showed diminishedB-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 monostearinwith amylopectin, Lagendijk and Pennings (1970) detected forma-tion of amylopectin-monoglyceride complexes, the degree ofwhich increased in a linear fashion with fatty acyl chain length,while still being much less than the amount of amylose complexformation. Kulp and Ponte (1981) mentioned that the effects ofcomplexation could be intramolecular (outer branch associations)or intermolecular (aggregation of polymers).

DeStefanis and others (1977) reported that SSL, succinylatedmonoglyceride, and glyceryl monostearate complexed equallywith amylose and amylopectin. Interestingly, no binding of the 3adjuncts was found during the sponge stage. As the dough devel-oped, increasingly strong binding of adjuncts with protein wasdiscovered. After baking, however, the 3 surfactants were foundstrongly associated with starch polymers, the temperature atwhich most translocation took place being above 50 °C.

Finally, Biliaderis and Vaughan (1987) obtained direct evidenceof complexes of amylopectin (and amylose) with labeled fatty acidmolecules using electron spin resonance. Then, Gudmundssonand 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. Usingmutant corn starches, Villwock and others (1999) also providedDSC evidence for the existence of amylopectin-surfactant interac-tions in pastes and additional evidence that both hydrocarbonchain 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 reductionin starch granule swelling, and the degree of granule swelling isinversely related to crumb firmness. They concluded that surfac-tants restrict granule swelling during baking by complexing withamylose at the periphery of starch granules. Polar surfactants thatform 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 (Grayand Schoch 1962). Sodium lauryl sulfate repressed hydration ofstarches 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 andVarriano-Marston 1981). SSL did not appear to interact with thegranule 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

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 spaceto retrograde. Even in excess water, monoglycerides prevent theleaching of amylose molecules (Schoch 1965). Surfactants pre-vent dissolution and leaching of amylose molecules, which maybe 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 aftercomplexation so that less crystallization can occur.

Interaction with protein. Willhoft (1973) hypothesized that theantistaling effect of monoglycerides might be due to interactionwith gluten, and there is experimental evidence that supports thishypothesis (Hoseney and others 1969; De Stefanis and others1977; 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 starchpolymers 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 intheir performance. Krog (1973) reported that amylose-monoglyc-eride complexation ability decreased in the descending order ofmonoglyceride physical states: �-type crystalline gel > �-typecrystalline hydrate > nonhydrated powder. �-Type monoglyceridecrystals 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 nomarked antifirming effect unless first hydrated before use. Hydrat-ed �-crystals are commonly known as a “coagel-foam” (Krog1968).

ShorteningShortening is quite effective in retarding bread crumb staling

and has, for many years, been used as an antistaling ingredient inbreads. Since shortening was shown to have no effect in defattedbread, it was speculated that its effect is related to the native flourlipids. Since shortening does not complex with starch, its mecha-nism of antistaling action differs from that of monoglycerides (Rog-ers and others 1988).

Carbohydrate ingredientsRoles 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: Roleof pentosans”. Use of hydrocolloids and modified starches andeffects of damaged starch are covered in this section. If it is ac-cepted that moisture redistribution is a requirement for staling tooccur (see “Moisture migration” section), then it follows that anyingredient that inhibits movement of moisture is a candidate forreducing 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 themoisture is available to the starch molecules, increases the rate ofretrogradation (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, wheyprotein) and reported that guar and locust bean gums retardedstarch retrogradation, but did not have any clear antistaling activi-



ty. They also found that all the hydrocolloids they used generallyimproved 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 rateof staling during storage were reduced by addition of locust beangum, alginate [presumably sodium alginate], and xanthan. Theyproposed 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 locustbean 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%) decreasedthe staling rate of bread as measured by compressibility and DSC(Czuchajowska and others 1992). Moisture content remainedconstant. Bread softness improved without increasing the possi-bility of microbial deterioration. Lent and Grant (2001) found thatbagels 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 reducedthe rate of firming during storage (Kegoya-Yoshino 1997).

Replacement of 10% of the wheat flour with steamed oat floursretarded bread staling without adversely affecting the loaf volume(Zhang and others 1998). The reduction in the rate of staling wasattributed to the high water absorption capacity of the �-glucan inoat flour, but oat starch has been found to retrograde at a slowerrate 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 inreality are the branch chains of amylopectin released by the ac-tion of an a-1,6-glucan hydrolase as antistaling agents (Yoshidaand others 1972).

It has also been reported (although not supported in the articlewith published experimental data) that methylcellulose and hy-droxpropylmethylcellulose extend the shelf life of baked productsvia prevention of water loss during baking (Dziezak 1991). Thesame report states that guar gum and xanthan gum function as an-tistaling agents.

Damaged and modified starch. Modified starches have beeninvestigated 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 andthe 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 thebread was not affected by the starch phosphates.

MiscellaneousResults 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 sweetwhey powder were found to reduce staling in bread significantly(Erdogdu-Arnoczky and others 1996), while acid whey powderdid. Despite its high water-holding capacity, succinylated wheyprotein concentrate did not prevent bread staling (Thompson andBaker 1983).

L-Leucine n-alkyl esters slowed the staling rate of bread more

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 ofbread significantly, with 1,3-butanediol having the greatest effect(Frankenfeld and others 1977).

Incorporation of durum wheat flour (25%) into a bread wheatflour did not improve initial firmness, but did retard stalingthrough 4 d of storage (Boyacioglu and D’Appolonia 1994).Breads made with triticale flour staled twice as fast as did breadmade 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 wasadded to the formula.

Flavor ChangesStaled bread is considered unacceptable due to changed flavor.

A review on bread flavor is available (Lorenz and Maga 1972).

Processes for Acceleration of StalingA 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 viatemperature cycling. Bread is exposed alternately to a temperaturejust 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 preparingbread 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 theRate of Staling

Bread staling is a complex phenomenon, certainly involvingmultiple factors. Much has been learned about bread staling, andapplication of this knowledge has led to considerable improve-ments in shelf life. However, without knowledge of the precisemechanism, addressing the problem of bread staling remains aprocess of formulating and testing more and more hypotheses. Itis difficult to determine cause-and-effect relationships because in-volvement of a constituent may be indirect and additives, otherchanges in formulation, and process changes may alter more thanone 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, amyloseis now also thought to be involved. And while amylose-surfactantcomplex formation has been a widely used strategy for reducingbread staling, amylopectin complexes may also be important, notnecessarily 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 tobread staling. Most evidence supports the concept that glutenserves as a moisture reservoir from which water is transferred toretrograding starch molecules. But the relative effects of dehydra-tion of gluten and hydration of starch can only be surmised, ascan the degree of benefit from prevention of this type of moisturetransfer.

Evidence has accumulated for a major role of gluten in bread

Bread staling. . .




staling. As mentioned above, moisture transfer from gluten tostarch might be involved in the staling process. Beyond this, it hasbeen proposed that gluten-starch crosslinks are responsible forstaling, but it has become increasingly clear that multiple mecha-nisms operate during staling. Supermolecular structures of somesort, perhaps both starch-starch and starch-protein interactions,are certainly involved, with one or more of the starch or proteincomponents, including gelatinized granules, being incorporatedin 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. Itcould also involve hydrogen bonding between retrograded starchmolecules and the gluten network with retrogradation occurringeither before or after association of amylopectin and/or amylosemolecules with the protein network.

Additives that seem to have the greatest effect in reducing stal-ing in bread are (in no special order) surfactants (complexingagents), �-amylase, and hydrocolloids/gums, including modifiedstarch. The effect of adding �-amylase is most certainly indirect;that is, the antistaling effect is due to in situ formation of starchdextrins 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 themajority, of the crystallization involves amylopectin. Gluten, mayalso be involved. The most plausible hypothesis is that amylopec-tin retrogradation involves incorporation of water molecules intothe crystallites and that this requirement shifts the distribution ofwater molecules between components, reducing the water associ-ated with gluten and thereby changing the nature of the glutennetwork. 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 perfectassociations are formed. They might also function primarily asplasticizers, lowering the glass-transition temperature (Tg) so thatthe structure is not in a glassy state at room temperature. The factthat 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 plasticizersand antistaling agents, as are �-amylases which produce them, isfurther 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 considerableprogress 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 StalenessProbably 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 thataccompany it. Characteristics of bread crumb that have beenused as bases to determine the degree of staling are changes intaste and aroma, increased hardness, increased opacity, increasedcrumbliness, increased starch crystallinity, decreased absorptive

capacity, decreased susceptibility to �-amylase, and decreasedsoluble starch content (Geddes and Bice 1946). It is obvious thatno one method will completely measure or describe the degree ofstaling as noticed by the consumer (Sidhu and others 1996). Allinvestigations of the mechanism and control of staling reported inthis review have employed one or more of the methods coveredbriefly in this section to measure the rate and/or degree of staling.Other reviews of bread staling measurement methods can befound in Maga (1975), Kulp and Ponte (1981), and Ponte andOvadia (1996). Many of the methods used to measure bread stal-ing are based on principles used to determine the extent of starchretrogradation. Methods for measuring starch retrogradation havebeen reviewed by Karim and others (2000).

Rheological methodsUniaxial compression. As bread stales, the texture of the crumb

changes from a relatively soft, spongy texture to one that is firmand crumbly. Hence, numerous compressibility methods havebeen developed to quantify the firming of bread, which has beenshown to correlate with bread staling as measured by consumeracceptability. Hence, compressibility measurements are mostcommonly 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 firmingchanges with a Baker’s Compressimeter, determining the force ap-plied by use of a plunger to ensure uniform compression (Bakerand 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 andothers (1988) confirmed that a 25% compression depth (as speci-fied in AACC Method 74-09) was the most effective method fordetecting 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 thisregard is that a correlation coefficient of 0.98 was found betweenfirmness measured as compressibility and sensory assessments ofthe 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 Atkins1989), Bloom Gelometer (Baker and others 1987), and the Gener-al Foods Texturometer (Szczesniak and Hall 1975), can also beused 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 characterizethe rheological properties of bread slice surfaces (Kulp and Ponte1981). Young’s modulus can be determined from results of studiesof compressive stress-strain relationships determined with instru-ments such as the Instron Universal Testing Machine. Baruch andAtkins (1989) found that, in a dynamic stress-strain curve, the ini-tial slope, which is a measure of crumb flexibility, increased andthe peak height, which is an indicator of the strength of the glutennetwork, decreased as staling progressed. Stress-strain resultswere correlated to results of thermal analyses by Schiraldi andothers (1996b). The relationship between mechanical propertiesof bread and crumb staling has been reviewed in detail (Vodovotzand others 2001).

Pasting properties. Under the hypothesis that starch retrograda-tion plays a significant role in bread staling, the Brabender Visco-



Amylo-Graph, Rapid Visco Analyzer, and related instrumentshave been used to measure the extent of starch gelatinization inbread crumb slurries (Yasunaga and others 1968). Peak viscositychanges were suggested as an index of staling because it wasthought that peak viscosity would decrease with age due to atoughening effect on partially gelatinized starch granules duringstaling. Based on results in which the outer 1-cm and 2-cm por-tions of the crumb produced a lower peak viscosity than did thecenter portion, it was concluded that starch granules in the crumbcenter 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 starchswelling accurately in bakery products, but rather shows the sumof the contributions of all macromolecules to the viscosity of thebread 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 analysisThermal 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 itsuse 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 usefulin providing basic information on starch retrogradation (Karimand others 2000). Because both measure the differential tempera-ture or heat flow to or from a sample versus a reference materialas a function of time, both can be used to monitor such changesas phase transitions, molecular conformational changes, interac-tions with other components, and pyrolytic degradation of thesample. Specialized DSC instruments, including modulated DSCand polarization DSC, are also available (Schenz and Davis1998).

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 timescales for endotherm development and for the increase in crumbfirmness are broadly similar in magnitude, DSC can be used tomeasure the rate of bread staling quantitatively (Jagannath andothers 1999a). However, there are overlapping transitions over awide temperature range because of the variety of componentsand 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 breadsamples, developed during storage, and the increases in peakarea were proportional to increases in bread firmness (Cornfordand 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 studiesof starch can approximate gelatinization during baking, since inboth cases the gelatinized starch granules are swollen, but non-disrupted (Jacobson and BeMiller 1998). Thus, the conditions ofgelatinization in the calorimeter more closely approximate thoseencountered during baking than those encountered during starchpasting. Unlike compressibility measurements, endotherm peakdevelopment does not appear to be dependent on specific loafvolume (Fearn and Russell 1982).

Isothermal microcalorimety, a technique which is much moresensitive and requires larger samples sizes than does convention-al DSC (Karim and others 2000), has been used to study the earlystages of starch retrogradation and has been demonstrated to beeffective for examination of the antistaling effects of lipids and sur-factants (Silverio and others 1996).

Other thermoanalytical instruments include thermogravimetricanalysis (TGA), thermomechanical analysis (TMA), and dynamicmechanical analysis (DMA). TGA measures changes in the weightof a sample as a function of temperature (Schenz and Davis1998). While events such as volatization, dehydration, and chem-ical reactions can be observed using TGA, other simple transitionscan be missed if no weight changes occur (Sperling 1992).Schiraldi and others (1996b), using TGA, found that the release ofwater 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 Davis1998) and can be used to determine the Tg of a substance by de-tecting a change in the thermal expansion coefficient (LeMesteand others 1992). The deformation of a substance is measuredunder nonoscillatory (static) load as the substance is subjected toa 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 oscillatorymeasurements, dynamic mechanical thermal analysis (DMTA),dynamic thermomechanical analysis, and dynamic rheology (Me-nard 1999). In DMA, as oscillatory stress is applied to the samplein the bending or tensile mode of deformation, the lag of the re-sulting oscillatory strain is measured. DMA is 1000 times moresensitive in observing thermal transitions than is DSC (Vodovotzand others 1996). DMA has been used to study staling profiles ofIndian 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 toexamine the effect of aging and drying on thermal transitions ofbread (Vodovotz and others 1996).

Infrared spectroscopyFourier 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 (Wilsonand others 1991).

Fourier transform infrared (FTIR) spectroscopy. Because FTIRspectroscopy measures the degree of short-range ordering in asystem, 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 anda smaller distribution of bond energies due to the system becom-ing more ordered upon staling (Wilson and others 1991; Karimand others 2000). Changes in band intensities in the 1300 to 800cm-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 ofamorphous regions of starch, are of particular interest (Karim andothers 2000). Thus, starch retrogradation can be defined (in termsof FTIR data) as an increase in the ratio of peak intensities at 1047and 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, soNIR can be used to follow the progress of bread staling (Wilson

Bread staling. . .




and others 1991). Starch molecules in bread are extensively hy-drogen bonded (both intramolecularly and to water). Because theabsorption bands in reflected NIR give information about hydro-gen bonding, NIR reflectance data can be used to detect changesin the hydrogen bond network of a bread system during staling(Iwamoto and others 1987; Wilson and others 1991).

Nuclear magnetic resonance (NMR) spectroscopyNMR techniques that have been used to study bread staling

and NMR techniques that have been used to examine changes inmolecular 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 waterin bread and to relate them to bread firming (Leung and others1983; Wynne-Jones and Blanshard 1986; Kim-Shin and others1991; Chen and others 1997a,b; Engelsen and others 2001).Low-field proton NMR has been used preferentially to examinebread staling since it can provide rapid determination of protonmobility associated with different molecules (Ruan and Chen2000). Theoretically, there is an equilibrium state in bread wheremobile (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 breadstaling. However, bread is always in a nonequilibrium state, andtherefore, its nature changes continuously.

A pulsed-NMR method was used to monitor molecular changesthat resulted in increases in firmness during aging of starch gelsand starch-based products (Seow and Teo 1996). Morgan andothers (1992) used 13C CP MAS NMR to determine crystalline sol-id, amorphous solid, and liquid-like phases of fresh and storedwheat 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 andothers (1996) monitored moisture migration from crumb to crustin sweet rolls by MRI during 5 d of storage and found that, with anincrease in storage time/staling, the mobility of the less-mobilefraction of water decreased and the mobility of the more-mobilefraction of water increased.

Using proton cross-relaxation NMR spectroscopy, Wu andEads (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 thepercentage of immobile molecules increased with time, while thepercentage of mobile molecules decreased. Using the same tech-nique, Vodovotz and others (2002) found no change, that is, noincrease in rigidity, of an aged bread sample, even though therewas an increase in amylopectin retrogradation enthalpy (DSC).

X-ray crystallographyX-ray crystallography has been used to examine bread staling

(Zobel 1973), specifically the crystalline nature of the starch in thesystem, which can be related to the firmness of the product(Champenois and others 1995). Starch in freshly baked bread ismostly amorphous, but slowly reorders during storage. The re-crystallization is reflected in x-ray diffraction patterns (Karim andothers 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 isnot 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

(Sidhu and others 1997) and used in conjunction with DSC in theanalysis of the effect of various antistaling additives on wheatbread (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 Senti1959), emphasizing the need, when investigating bread staling,for methods that are not limited to measuring changes in only 1component.

Jagannath and others (1998) used wide-angle x-ray scattering(WAXS) to measure the degree of staling.

Conductance and capacitanceIt has been established that changes in bread resulting in staling

of the crumb are at least correlated with starch retrogradation andmoisture redistribution between gluten and starch, whether or notthere is any cause-and-effect relationship. Since free and boundwater differ in their dielectric constants, changes during stalingcould 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 thatchanges 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 andothers (1975) obtained bread staling data in a similar fashion.

MicroscopyTransmitted 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 granuleslose 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 theusual native starch granule birefringence, but does indicatebiopolymer ordering in the long, thin birefringent structures) dueto molecular reordering, except in �-amylase-containing breadcrumb, which contained more of the birefringent structures (ascompared 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 ofconfocal laser scanning microscopy over other microscopies is itsability to produce an image of the focal plane of interest (opticalsection), which can be digitally reconstructed into a 3-dimension-al image. CLSM has provided qualitative information about thecrumb structure of bread (Bugusu and others 2002). CLSM hasalso been used to investigate changes in starch granules in breadduring staling (Vodovotz and Chinachoti 1998). However, it hasbeen reported that there were no differences in confocal imagesof 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 informationon the changes in the starch fraction during staling. R-CLSM offersthe highest resolution of CLSM modes (Hibbs 2000), but to ourknowledge has not been applied to investigations of bread stal-ing.]

Electron microscopy. Electron microscopy has not been usedto 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 others1975; Bechtel and others 1978; Evans and others 1981), bread



(Khoo and others 1975; Bechtel and others 1978), and pastes(Fannon and BeMiller 1992 and references therein).

Sensory/organoleptic testsLoss of flavor and aroma are among the most noticeable detri-

mental changes of bread upon staling. Reportedly, the decrease inthe acceptability of bread over 5 d of storage is correlated with areduction in aldehydes and an increase in ketones (Lorenz andMaga 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 byboth uniaxial compression methods (Section 8.1.1) and sensoryevaluations.

AACC Method 74-30 (AACC 2000) involves panel ratings of asum of factors affecting overall staling (“appearance” or “feel” ofthe 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 andstaling as rated by panelists has been found (Cornford and others1964; Axford and others 1968). Other examples of organolepticevaluations are discussed by Pomeranz and Shellenberger (1971)and Setser (1996).

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MS 20010513 Submitted 9/18/01, Revised 8/12/02, Accepted 10/3/02, Received10/5/02

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 authorBeMiller (E-mail: [email protected]).


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