Journal of Cereal Science 52 (2010) 457e465

Contents lists available at ScienceDirect

Journal of Cereal Science

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

Shelf life extension of sliced wheat bread using either an ethanol emitter or an
ethanol emitter combined with an oxygen absorber as alternatives to chemical
preservatives
E. Latou, S.F. Mexis, A.V. Badeka, M.G. Kontominas*
Laboratory of Food Chemistry and Technology, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, the effect of active packaging [ethanol emitter (EE) or ethanol emitter combined
Received 26 May 2010 with an oxygen absorber (EE þ OA)] on shelf life extension of sliced wheat bread stored at 20  C was
Received in revised form investigated. Bread containing commercial preservatives (WP) and no preservatives (WOP) were taken as
12 July 2010
controls. Microbiological, physicochemical and sensory changes occurring in the product as a function of
Accepted 21 July 2010
treatment and storage time were monitored for 30 days. Counts for yeasts and molds were 5.1, 3.8, 2.0
and 2.0 log cfu/g and for Bacillus cereus 4.7, 2.5, 2.3 and 2.0 log cfu/g for WOP, WP, EE and EE þ OA
Keywords:
treatments respectively after 30 days of storage. Initial pH 6.3 and 6.4, for WP and WOP samples
Wheat bread
Shelf life extension
remained fairly constant irrespective of specific treatment. Aroma quality deterioration during storage
Ethanol emitter was due to the loss of volatile compounds and the formation of “off-flavors” through lipid oxidation.
Oxygen absorber Neither the EE nor the EE þ OA had an adverse effect on initial odor, taste and texture of bread. Based on
sensory (texture) and microbiological data, shelf life was ca. 4 days for samples WOP; 6 days for samples
WP; 24 days for samples containing the EE and at least 30 days for samples containing the EE þ OA.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Bread has always been one of the most popular food products
due to its superior nutritional and sensory characteristics being
consumed daily (Galic et al., 2009). Generally the shelf life of bread
is limited by several deterioration processes including fungal
growth (Nielsen and Rios, 2000), the loss of moisture and staling
(Del Nobile et al., 2003). According to Legan and Voysey (1991) in
a study performed on bakery products and their ingredients, 60% of
spoilage was attributed to molds (Penicillium spp and Aspergillus
niger) whereas yeasts accounted for only 15%. Besides the repelling
sight of visible growth, fungi are responsible for off-flavor devel-
opment, the production of mycotoxins as well as allergenic
compounds. These compounds may be formed even before mold
growth is visible (Nielsen and Rios, 2000). However even though
molds are destroyed during baking, recontamination may occur
during cooling and subsequent packaging causing the above
problems (Gali c et al., 2009).
In general, modified atmosphere packaging (MAP) has been
used for shelf life extension of a large variety of foodstuffs including
* Corresponding author. Tel.: þ30 2651008342; fax: þ30 2651008795.
E-mail address: mkontomi@cc.uoi.gr (M.G. Kontominas).
bakery products such as wheat bread (Rodríguez et al., 2000), rye
bread, hot-dog bread (Nielsen and Rios, 2000) and soy bread
(Fernandez et al., 2006). A problem associated with the MAP of
bakery products is that it is very difficult to reduce the oxygen
content within the package to a very low level due to a large
number of pores in the bread matrix which tend to trap oxygen
(Galic et al., 2009). One approach to overcome this problem is to use
oxygen absorbers inside the package. Oxygen absorbers have been
used to prevent discoloration of meats, inhibit rancidity in high fat
foods and mold growth in high water activity products (Berenzon
and Sam Saguy, 1998).
Besides oxygen absorbers, various companies manufacture and
sell products that release ethanol vapor into the packaged head-
space inhibiting the growth of molds, yeasts and bacteria. Ethanol
vapor generators may be combined with oxygen absorbers. Ethanol
emitters and/or oxygen absorbers have also been used for the shelf
life extension of bakery products such as sliced rye bread (Salminen
et al., 1996) and durum wheat bread (Del Nobile et al., 2003).
To the best of our knowledge there is no data available in the
literature regarding the combined use of an ethanol emitter and an
oxygen absorber for the preservation of sliced wheat bread. Based on
the above, the objective of the present work was to study the effect of
either an ethanol emitter (EE) or an ethanol emitter combined with

0733-5210/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2010.07.011
458 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465
an oxygen absorber (EE þ OA) for extending the shelf life of
sliced wheat bread. A second objective was to test the experimental
PET-SiOx//LDPE laminate, a high barrier material, for the preserva-
tion of sliced wheat bread.
2. Experimental
2.1. Packaging and storage
Wheat bread was produced according to standard commercial
practices by a local bread manufacturer using wheat flour, extrac-
tion rate 70%, bakers’ yeast and water. In case of commercial
samples, the following additives were used at a 1% total concen-
tration in wheat flour as dough improving agents: ascorbic acid
(E300), calcium carbonate (E170) and diacetyl-tartaric esters of
fatty acids (E472e). Baked loaves were cooled down to room
temperature for 60 min, transferred to the laboratory within 30 min
and sliced 2 cm thick. Bread composition was determined using
official AOAC methods (protein: 8.0%, fat: 1.7%, starch: 47.9%,
sugars: 1.8%, fiber: 2.7%, moisture: 39.0%).
Four lots of samples were prepared: Lot 1 comprised the control
samples (samples without chemical preservatives, WOP); Lot 2
consisted of commercial control samples with chemical preserva-
tives (WP). In lot 3 an ethanol emitter sachet (EE) was added to the
inner surface of the package of samples WOP while, in lot 4,
a sachet combining an ethanol emitter and an oxygen absorber
(EE þ OA) were used (Freund Ind. Co, Japan). All samples (150 g)
were packaged in high barrier PET-SiOx//LDPE pouches, 62 mm in
thickness and 4.0 mL/(m2 day atm) in oxygen permeability at 75%
RH, 25  C. Pouches were heat-sealed using a BOSS model N48
thermal sealer (BOSS, Bad Homburg, Germany). Size of OA/EE was
based on product weight and water activity (aw ¼ 0.95, measured
using a Novasina aw meter, Lachen, Switzerland). Samples were
then stored at ambient temperature (20  1  C intermittently
exposed to daylight/dark) for a period up to 30 days.
Three separate pouches from each treatment were removed for
analysis on day: 0, 3, 6 and 9 of storage for WOP samples; sampling
was continued up to day 15 for WP samples and up to day 30 for the
samples containing the EE and EE þ OA.
2.2. Microbiological analysis
The following groups of microflora were determined according
to official protocols (APHA, 2001): TVC, Yeasts/Molds, Enter-
obacteriaceae, Bacillus cereus and Clostridium spp. Anaerobic
conditions were achieved by the use of AnaeropackÒ GENbox Jars
combined with Pack-Anaero oxygen absorbers. All plates were
examined visually for typical colony types and morphological
characteristics associated with each growth medium. In addition,
the selectivity of each medium was checked routinely by Gram
staining and microscopic examination of smears prepared from
randomly selected colonies from all of the media.
2.3. Physicochemical analysis
2.3.1. Determination of the headspace gas composition
The headspace O2 composition was measured using a Dansensor
CheckMate 9900 gas space analyser (PBI Dansensor, Ringsted,
Denmark) after 4, 8, 12, 16 and 24 h of pouch sealing to determine
the time required for the oxygen absorber to reduce the oxygen
concentration inside the pouch to below 0.01%. Headspace ethanol
was determined by gas chromatography using an HP5890 GC
equipped with a FID. A SupelcowaxTM-10 column (30 m  0.32 mm
i.d.  0.5 mm film thickness) (Supelco, Bellefonte, USA) was used,
operated isothermally at 70  C with a helium flow rate of 1 ml/min.
The injector was operated in split mode (split ratio 10:1) at 260  C.
The detector temperature was 280  C. Ethanol concentrations were
reported as volume %.
2.3.2. Semi quantitative determination of volatile compounds
Volatiles were determined according to the method of Mexis
et al. (2009) using 1 g of sliced bread (crust and crumb) and 20 ml
of 4-ethyl-2-pentanone as an internal standard. A Divinylbenzene/
Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) fiber 50/30 mm
was used for volatiles’ separation.
2.3.3. pH determination
pH was determined using the method of AOAC (1995) after
appropriate modification (Goulas and Kontominas, 2005).
2.3.4. Texture
Objective texture analysis was performed using an Instron
Universal Testing Machine, model 4411 (Instron Corp., Bucks, UK).
Disks of bread crumbs (25 mm diameter  15 mm in thickness)
were cut from the central part of each bread slice.
The bread samples were analyzed in a double compression cycle
under the following conditions: crosshead speed 100 mm/min,
specimen compression depth 60% using a 5 cm diameter stainless
steel compression plunger.
The texture parameters considered were hardness (peak force of
the first compression cycle in N) and springiness (ratio of the time
duration of force input during the second compression to that
during the first compression, dimensionless) (Bianchi et al., 2008).
Data were recorded using the Bluehill software.
2.4. Sensory evaluation
Sensory evaluation (acceptability test) was carried out by a 51
member untrained panel (31 females and 20 males) consisting of
faculty and graduate students of the Department of Food Chem-
istry, University of Ioannina. Panelists were chosen using the
following criteria: ages between 22 and 60, non-smokers, without
reported cases of food allergies who consume bread daily. Sensory
data were collected between day 0 and day 30 of storage. After each
sampling day, samples were held at 0  C until sensory evaluation at
the end of storage. Approximately 10 g of bread samples were
placed in small plastic cups, coded with 3-digit random numbers
and tightly capped. Along with each set of four test samples,
a reference sample was presented to panelists consisting of sliced
bread taken from the same loaf that had been packed under
nitrogen and stored in the dark at 0  C up to 30 days. The samples
were allowed to stand for half an hour prior to evaluation to allow
equilibration of volatiles in the cup headspace. Sensory attributes
evaluated included odor, texture and taste. Scoring was carried out
on paper ballots using a 9 point hedonic scale where: 9 ¼ extremely
like and 1 ¼ extremely dislike for the evaluation of odor and taste
and 9 ¼ very soft and 1 ¼ very hard for evaluation of texture. A
score of 5 was taken as the lower limit of acceptability for odor,
taste and texture. Triplicate samples were evaluated for each
treatment on each sampling day. Bread slices were considered
unacceptable if one of triplicate samples showed visible signs of
mold growth or odor, taste or texture received a score less than 5.
2.5. Statistical analysis
The experiment was replicated twice with different bread samples
on different occasions (n ¼ 2  3 ¼ 6). Microbiological data were
transformed into logarithms of the number of colony forming units
(cfu/g) and were subjected to analysis of variance using the software
SPSS 16 for windows. Results are reported as mean values  standard
 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465
error (S.E.) (figures) or standard deviation (S.D) (table). Significance
was defined at p < 0.05.
3. Results and discussion
3.1. Microbiological changes
Yeasts and molds counts, TVC as well as B. cereus, for the four
different treatments as a function of storage time are shown
Fig. 1a, b and c.
A first observation to be made is that visible yeasts and mold growth
on the bread surface occurred when the yeasts and mold counts were
ca. 4 log cfu/g (Fig. 1a). Fernandez et al. (2006) and Rodríguez et al.
(2000) reported counts of 3.0 and 2.7 log cfu/g, respectively, for
yeasts and molds’ visible growth. A 4 log cfu/g yeasts and molds count
was reached after ca. 5 days for the samples WOP and 7e8 days for the
samples WP. Samples containing the EE and EE þ OA never reached
this population 4.0 log cfu/g even after 30 days of storage.
A second observation is that for the first 15 days of storage the
inhibition effect of the EE and EE þ OA was probably due to both the
antimicrobial activity of ethanol and the anoxic environment
created by the OA. After day 15 of storage, as the concentration of
ethanol dropped, possibly due to permeation through the pack-
aging material, a low growth of yeasts and molds was recorded in
samples containing the EE while in samples containing both the
EE þ OA, no yeasts and molds’ growth was recorded until day 30 of
storage probably due to the latter of the above two phenomena.
In contrast to our results, Fernandez et al. (2006) reported that
there was no significant difference in yeasts and molds’ counts of
air packaged samples of soy bread with and without chemical
preservatives (calcium propionate) during 12 days of storage at
21  C. Salminen et al. (1996) studied the effect of an ethanol emitter,
oxygen absorber and the combination of both on shelf life exten-
sion of rye bread slices and reported that air packaged bread
showed visible microbial growth on day 8; samples containing the
ethanol emitter on day 26e27, while in samples containing both an
oxygen absorber plus an ethanol emitter, no visible microbial
growth was observed during 42 days storage at 20  C. Finally, Del
Nobile et al. (2003) studied shelf life extension of durum wheat
bread using active packaging and reported that samples packaged
with an oxygen absorber did not show any visible sign of mold
growth during 38 days of storage while in air packaged samples
visible mold was observed after only 3 days of storage.
An initial TVC value of 3.0 log cfu/g (Fig. 1b) is indicative of an
acceptable quality product. Similar, aerobic plate counts (APC) have
also been reported by Franke et al. (2002) for pre-baked buns and
by Fernandez et al. (2006) for soy bread.
TVC exceeded the value of 7 log cfu/g, considered as the upper
microbiological limit for foods, as defined by the ICMSF (1986) on
day 7e8 for samples WOP; day 14e15 for samples WP; while for
both EE and EE þ OA samples, this limit was never reached during 30
days of storage. Similar to our results, Fernandez et al. (2006)
reported significant differences in TVC (ranging between 0.5 and
2 log cfu/g) for soy bread with and without calcium propionate,
during 12 days of storage. Likewise, Franke et al. (2002) reported that
pre-baked buns combined with an ethanol emitter recorded
a substantial increase in total mesophilic count after the first week of
storage and stabilized to 5e6 log cfu/g after 28 days of storage. In
contrast, control samples reached such values after 7 days of storage.
B. cereus is a gram-positive, spore-forming, motile, facultative
anaerobic bacterium causing both food spoilage in bread (ropy
texture) and possibly food poisoning (Thompson et al., 1993). On
day 0, B. cereus counts were under the method detection limit (2 log
cfu/g) and at the time of visible signs of mold growth, reached
4.0 log cfu/g for samples WOP and 2.7 log cfu/g for samples WP
459
(Fig. 1c). B. cereus counts reached 4.8 and 4.2 log cfu/g for samples
with EE and EE þ OA, respectively on day 30 of storage. According
to EFSA (2005), food borne diseases caused by B. cereus were
associated with concentrations of at least 5 log cfu/g, a value never
reached for any of the samples tested. In contrast to our results,
Franke et al. (2002) reported absence of B. cereus but high values
(5e6 log cfu/g) of Bacillus spp., probably Bacillus subtilis in pre-
baked buns combined with ethanol emitters after 32 days of
storage. Bailey and von Holy (1993) studied the contamination of
whole wheat bread by Bacillus spores during manufacture. On day 3
of storage a steep increase in Bacillus spore counts (6.38 log cfu/g)
was recorded. A similar increasing trend was also reported in the
present study for WOP and WP samples after days 3 and 6 of
storage respectively.
Enterobacteriaceae, a large group of facultative anaerobic
bacteria, used as hygiene indicators, remained below 1 log cfu/g for
all treatments during the entire storage period (data not shown).
Lastly, Clostridium spp. was absent in 25 g of sample (data not
shown).
3.2. Sensory analysis
The results for odor, taste and texture evaluation of bread slices
are presented in Fig. 2 a, b and c, respectively. These sensory
properties comprise highly significant criteria for the acceptability
of bread by consumers (Poinot et al., 2007). First of all, it is note-
worthy to mention that neither EE nor the combination of EE þ OA
affected (p > 0.05) the initial odor, taste and texture of sliced bread
while the addition of chemical preservatives (WP) resulted in
a product with higher hardness as compared to WOP samples.
According to Seiler and Russel (1991), at ethanol concentrations
2%, the alcoholic flavor is likely to be sensorily noted. In the
present study the highest ethanol concentration recorded in the
package headspace was 0.2%.
As a result of microbial growth, a slight sour off-odor was
detected by the sensory panel after day 4 and 5e6 for WOP and WP
samples respectively and day 26e27 of storage for EE samples,
respectively. In contrast, in case of EE þ OA samples (Fig. 2a), no off-
odor was recorded throughout storage, probably due to the absence
of oxygen inhibiting aerobic bacterial growth. Similar off-flavors
have been reported by Avital and Mannheim (1988) for air pack-
aged pita bread after 14 days of storage. Taste scores were similar to
those of odor.
Considering the value of 4-log cfu/g above,which there is visible
mold growth and an acceptability score of 5 for texture, it can be
stated that WOP and WP samples were rejected before visible mold
growth was recorded (Fig. 2c).
It is noteworthy to mention that even though chemical preser-
vatives increased sensorial life, they resulted in a harder product.
Salminen et al. (1996) reported that after 2 weeks of storage, rye
bread was described as dry and hard. This is in contrast with the
results of the present study according to which bread became hard
after ca. 5 days of storage.
Between, odor, taste and texture, the last proved to be the most
sensitive attribute for the evaluation of wheat bread. Based
primarily on texture scores but also on visible signs of molds and
a lower acceptability score of 5, shelf life was ca. 4 days for WOP
samples; 6 days for WP samples; 24 days for samples containing
the EE and at least 30 days for the samples containing the EE þ OA.
Based on sensory scores it can be stated that the ethanol emit-
ters performed better than chemical preservatives with regard to
shelf life extension of sliced wheat bread. However, the combina-
tion of ethanol emitter and oxygen absorber was the most effective
in extending product shelf life by at least 26 days as compared to
control samples.
460 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465

Fig. 1. Effect of active packaging and storage time on (a) yeasts and molds (b) TVC and (c) Bacillus cereus of sliced wheat bread stored at ambient temperature.
 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465 461

Fig. 2. Effect of active packaging and storage time on (a) odor (b) taste and (c) texture of sliced wheat bread stored at ambient temperature.
462 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465

Table 1
Effect of packaging and storage time on volatile compounds (mg/kg) of sliced wheat bread stored at ambient temperature at the time of sensory rejection.

Volatile compounds KILi KIEx Day 0 without Day 0 with Day 4 without Day 6 with Day 24 ethanol Day 30 sachet
chemical chemical chemical chemical emitter sachet combining an oxygen
preservatives preservatives preservatives preservatives absorber and an ethanol emitter
Alcohols
Ethanol 463 >500 0.40b  0.04 0.30a  0.02 0.70c  0.05 0.44b  0.03 4.80e  0.16 4.30d  0.22
3-Methyl-1-Butanol 743 739 0.20b  0.03 0.24b  0.07 0.13b  0.04 U.D.L U.D.L 0.06a  0.02
2-Methyl-1-Butanol 697 703 0.07a  0.01 0.05b  0.00 U.D.L U.D.L U.D.L U.D.L
2-Methyl-1-Propanol 609 610 0.02ab  0.00 0.02a  0.01 0.01a  0.00 0.01a  0.00 U.D.L U.D.L
2-Furanmethanol 808 811 0.02a  0.01 0.01a  0.00 0.04ab  0.01 0.09c  0.02 U.D.L 0.05b  0.01
1-Hexanol 862 859 0.03a  0.01 0.03a  0.01 0.04a  0.01 0.09b  0.01 0.05a  0.00 0.03a  0.01
1-Octen-3-ol 978 979 0.01a  0.00 0.01a  0.00 U.D.L 0.03b  0.01 U.D.L 0.02b  0.00
Benzeneethanol 1114 1115 0.05c  0.01 0.02b  0.00 0.06c  0.02 0.02b  0.00 0.01a  0.00 0.05c  0.01

Aldehydes
Hexanal 798 803 0.16a  0.02 0.17a  0.01 0.43c  0.05 0.39c  0.03 0.32b  0.02 0.38c  0.04
3-Methyl-Butanal 650 653 U.D.L 0.02a  0.01 U.D.L U.D.L 0.02a  0.01 U.D.L
2-Methyl-Butanal 660 660 U.D.L 0.03  0.00 U.D.L U.D.L U.D.L U.D.L
2-Furancarboxaldehyde 831 835 0.05a  0.02 0.06ab  0.01 U.D.L 0.03a  0.01 U.D.L 0.03a  0.01
Heptanal 901 903 0.07a  0.01 0.08a  0.02 0.28b  0.02 0.33c  0.02 0.29b  0.01 0.31b  0.03
2-Heptenal 990 990 U.D.L U.D.L 0.18b  0.04 0.22b  0.05 0.11a  0.00 U.D.L
Benzaldehyde 982 979 0.05  0.02 0.05ab  0.01 0.03a  0.01 0.03a  0.01 0.02a  0.00 0.03a  0.01
Octanal 1005 1005 U.D.L U.D.L U.D.L 0.01  0.00 U.D.L U.D.L
Benzeneacetaldehyde 1055 1057 U.D.L U.D.L U.D.L 0.02  0.00 U.D.L U.D.L
2-Octenal 1388 1382 U.D.L 0.01a  0.00 U.D.L 0.02a  0.01 U.D.L U.D.L
Nonanal 1104 1102 0.09a  0.02 0.12a  0.01 0.23b  0.06 0.25b  0.03 0.22b  0.04 0.26b  0.01
Nonenal 906 905 0.01a  0.00 0.02b  0.00 U.D.L U.D.L U.D.L U.D.L

Ketones
3-Hydroxy-2-Butanone 707 708 U.D.L 0.03  0.01 U.D.L U.D.L U.D.L U.D.L
1-(2-furanyl)-Ethanone 878 873 0.01a  0.00 0.01a  0.00 0.01a  0.00 0.02a  0.01 0.03ab  0.01 0.01a  0.00
5-methyl-3-Heptanone 888 882 U.D.L U.D.L U.D.L 0.01  0.00 U.D.L U.D.L

Esters
Acetic acid, ethyl ester 614 619 U.D.L U.D.L 0.06  0.01 0.05  0.01 0.07  0.00 0.05  0.00
Hexanoic acid, ethyl ester 996 995 U.D.L U.D.L U.D.L U.D.L 0.02  0.01 U.D.L
Octanoic acid, ethyl ester 1193 1195 0.02b  0.00 U.D.L 0.02b  0.00 U.D.L 0.01a  0.00 U.D.L

Organic acids
Acetic acid 606 605 0.05b  0.01 0.03a  0.00 0.07b  0.02 0.06b  0.01 0.08b  0.02 0.05b  0.01
Pentanoic acid 875 869 0.04ab  0.00 0.06ab  0.02 0.03a  0.02 0.01a  0.00 U.D.L 0.02a  0.01

Alkanes
Hexane 618 615 0.02a  0.00 0.03a  0.01 U.D.L U.D.L U.D.L U.D.L
2.2-Dimethyl-hexane 732 731 0.01a  0.00 U.D.L 0.05b  0.01 U.D.L U.D.L U.D.L
Cyclododecane 1439 1433 U.D.L U.D.L 0.02  0.00 U.D.L U.D.L U.D.L
Cyclopantane 1964 1962 U.D.L U.D.L 0.04a  0.01 0.08b  0.02 0.09b  0.03 0.07b  0.01

Aromatic hydrocarbons
Toluene 771 772 U.D.L U.D.L U.D.L U.D.L U.D.L 0.02  0.00

Pyrazines
2-methylpyrazine 838 838 U.D.L U.D.L 0.17b  0.02 0.14b  0.03 0.08a  0.02 0.06a  0.02

Furans
2-Pentyl-Furan 989 993 U.D.L 0.07b  0.02 U.D.L U.D.L 0.01a  0.00 0.04b  0.01

Pyrones
Maltol 1112 1110 U.D.L U.D.L U.D.L U.D.L U.D.L 0.33  0.14

U.D.L. ¼ under detection limit. Values are the mean of six determinations (n ¼ 6)  S.D. i a.b.c... Means with different superscripts in the same row are statistically different
(p < 0.05), KIEx ¼ Kovac Index experimentally determined data, KILi ¼ Kovac Index literature data Nist 05, Qf ¼ Quality factor, Quality factor ¼ % matching of the experimental
mass spectra against those found in the Wiley database Tentatively identified on the basis of the Wiley7, Nist 05 (J.Wiley & Sons Ltd., West Sussex, England).

3.3. Physicochemical changes
3.3.1. Head space gas composition
After 8 h of storage the O2 absorber reduced the O2 concentra-
tion inside the package to less than 0.01% and maintained this O2
value throughout the 30-day storage period (data not shown).
Initial ethanol concentration in package headspace (EE and EE þ OA
samples) was ca. 0.06%, reaching 0.2% after ca. 7 days. Between days
7 and 15, ethanol concentration remained fairly constant while
between days 15 and 30 it progressively decreased to less than
0.15% probably due to permeation through the package walls (data
not shown). Present results are in good agreement with those of
Salminen et al. (1996) regarding preservation of sliced rye bread
using an oxygen absorber and an ethanol emitter.
3.3.2. pH
The initial (day 0) pH of bread slices was 6.4 and 6.3 for samples
WOP and WP, respectively (data not shown).
Statistically insignificant (p > 0.05) changes were observed in
pH values of all sample WP and WOP treatments. Quílez et al.
(2006) reported initial pH values ranging from 5.6 to 6.3 for
baguette type samples of wheat bread bought from the Spanish
market. Rosenkvist and Hansen (1995) reported pH values of 5.7,
5.6 and 5.8 for white, wholemeal and laboratory baked white bread,
 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465
Fig. 3. Effect of active packaging and storage time on (a) hardness and (b) springiness of sliced wheat bread stored at ambient temperature.
respectively. Differences in pH recorded in various studies may be
related to different bread ingredients and baking processes.
3.3.3. Volatile compounds
A total number of 20 and 21 volatile compounds belonging to the
chemical classes of alcohols, aldehydes, ketones, esters, organic
acids, alkanes, aromatic hydrocarbons, pyrazines, furans and
pyrones were identified in sliced bread WOP and WP, respectively,
on day 0 (Table 1). (Table 1 presents selected data on bread volatiles,
corresponding to different treatments at time zero and at the point
of sensory rejection). Present results for the flavor profile of sliced
bread, are in good agreement with those of Bianchi et al. (2008),
Pozo-Bayón et al. (2006), Quílez et al. (2006). Aroma is the result
of enzymic activity during kneading, yeast fermentation as well as
lipid oxidation reactions and reactions taking place during baking,
mainly through Maillard and caramelization reactions (Pozo-Bayón
et al., 2006). For example, initially (day 0) identified compounds
such as 1-hexanol, hexanal, 1-octen-3-ol, heptanal, hexane,
nonanal and nonenal are products of lipid oxidation (Frankel, 1982);
3-methyl-butanal is a product of the Maillard reaction (Pozo-Bayón
et al., 2006) while ethanol, 3-methyl-1-butanol, 2-methyl-1-
butanol, 2-methyl-1-propanol, benzaldehyde and acetic acid are
products of yeast fermentation (Hansen and Schieberle, 2005).
As bread making involves baking, one would expect the presence
of compounds such as pyrazines (Maga, 1992) which were not
463
identified in the present study with the exception of 2-methylpyr-
azine. A possible explanation is that, after baking, pyrazines disappear
very rapidly via evaporation from the sample. According to Grosch
and Schieberle (1997) after 3 h at room temperature, there is a loss
of 47% of acetyl-pyrazine from bread. To the best of our knowledge this
is the first time that changes in volatile compounds of sliced bread
during medium term storage under active packaging are reported.
At the end of sensorial life, an increase in concentration of alde-
hydes such as hexanal, heptanal (p < 0.05) was recorded with
a parallel formation of esters such as acetic acid ethyl ester and
hydrocarbons such as cyclopentane. Esters formed are considered to
have a positive impact on bread aroma. Results of the present study
confirm the results of Adams and De Kimpe (2007) regarding
formation of 2-methylpyrazine by B. cereus. Samples in which
high counts of B. cereus were recorded (Fig. 1c), a higher content of
2-methylpyrazine was also recorded (Table 1). Compounds deter-
mined at higher concentrations included ethanol, hexanal and
nonanal. An increase in ethanol concentration was recorded in
control samples during storage while, as expected, a high ethanol
content was recorded in samples containing the ethanol emitter and
the combination of ethanol emitter plus the oxygen absorber. Plessas
et al. (2008) reported a decrease in ethanol concentration from
4.14 mg/g to traces during 5 days of storage of sourdough bread in air.
It must be noted, that deterioration of the aroma of sliced bread
is probably due to the loss of numerous volatile compounds during
464 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465
bread ageing as well as the formation of “off-flavors” occurring
from lipid oxidation during storage.
Compounds identified included ethanol (alcoholic odor),
3-methyl-1-butanol (malty odor), 2-methyl-1-butanol (fragrant
odor), 2-methyl-1-propanol (wine like odor), 1-hexanol (grass/
sweet/woody odor), benzeneethanol (rose/honey like odor), hexa-
nal (green/grass/fat odor), 3-methyl-butanal and 2-methyl-butanal
(malty odor), 2-heptanal (green/fatty odor), benzaldehyde (bitter
almond odor), octanal (fruity/soapy odor), 2-octenal (fatty/nutty/
waxy odor), nonanal (sweet/mellon odor), acetic acid (acid/
pungent/sour odor), pentanoic acid (sweaty/pungent odor), hexane
(alkane like odor), 2-methyl-pyrazine (burn/sweet odor), 2-pentyl-
furan (fruity odor), maltol (caramel odor) (Poinot et al., 2007; Pozo-
Bayón et al., 2006; Rychlik and Grosch, 1996; Schieberle and
Grosch, 1985). To the best to our knowledge, compounds such
as 2-furanmethanol, benzeneethanol, 2-furancarboxaldehyde,
1-(2-furanyl)-ethanone and 2.2-dimethyl-hexane are reported for
first time in the flavor profile of sliced wheat bread.
Mention should also be made to hexanal, the accumulation of
which is directly related to the development of oxidative off-flavors
having a very low odor threshold of 5 ng/g (Buttery et al., 1988). The
initial hexanal value of sliced bread was 0.17 mg/kg. At the end of
sensorial shelf life, hexanal content rose to ca. 0.43, 0.39, 0.32 and
0.38 mg/kg bread for samples WOP (day 4), samples WP (day 6),
samples containing the EE (day 24) and samples containing EE þ OA
(day 30), respectively. Based on the above data it is clear that the use
of the O2 absorber substantially (p < 0.05) inhibited lipid oxidation.
3.3.4. Mechanical properties
Results for hardness and springiness (four bread treatments) are
shown in Fig. 3a and b, respectively. Initial values of hardness and
springiness were 4.8 N and 7.4 mm for samples WOP and 12.5 N and
7.2 mm for samples WP, respectively. These findings are in good
agreement with those of sensory evaluation for texture which
showed an increase in hardness for the product with chemical
preservatives (WP).
At the time of sensory rejection, hardness rose to ca. 6.0, 17.1,
10.1 and 15.7 N (Fig. 3a), for samples WOP (day 4), samples WP (day
6), samples containing the EE (day 24) and samples containing
EE þ OA (day 30), respectively. Respective values for springiness
were 7.2, 6.7, 7.1 and 6.7 mm (Fig. 3b). While springiness values
were in close proximity for all treatments at the time of sensory
rejection, this was not the case for hardness values which varied
respectively between 6.0 and 17.1 N.
Similar results had been reported by Salminen et al. (1996) for
the effect of ethanol emitters and oxygen absorbers on hardness
and springiness of rye bread samples during storage for 6 weeks.
Likewise, Chiavaro et al. (2008) reported an increase in hardness
with a parallel decrease in cohesiveness and springiness for the
crumb of durum wheat sourdough bread during 8 days of storage at
25  C. Finally, He and Hoseney (1990) reported that regardless of
packaging treatment, the firmness of all bread samples increased
rapidly during the first 15 days of storage with the rate of increase
decreasing with time.
4. Conclusion
Data recorded in the present study clearly show that active
packaging in combination with a high barrier packaging material
(PET-SiOx//LDPE) can substantially extend the shelf life of sliced
bread. Based on sensory (texture) and microbiological data, shelf
life was ca. 4 days for WOP samples, 6 days for WP samples, 24 days
for samples containing the EE and at least 30 days for samples
containing the EE þ OA.
Acknowledgements
The authors would like to thank Mr. Motoki Osaki for providing
the ethanol emitter and ethanol emitter/oxygen absorbing sachets
(Freund Corporation, Tokyo, Japan) and Mr. C. Soukeras and
K. Rinopoulos for providing the bread samples.
References
Adams, A., De Kimpe, N., 2007. Formation of pyrazines and 2-acetyl-1-pyrroline by
Bacillus cereus. Food Chemistry 101, 1230e1238.
APHA, 2001. In: Downes, F.P., Ito, K. (Eds.), Compendium of Methods for the
Microbiological Examination of Foods, fourth ed. APHA, Washington DC, USA.
AOAC, 1995. Official Methods of Analysis. Association of Official Analytical Chemists,
Gaithersburg.
Avital, Y., Mannheim, C.H., 1988. Modified atmosphere packaging of pita (pocket)
bread. Packaging Technology and Science 1, 17e23.
Bailey, C.P., von Holy, A., 1993. Bacillus spore contamination associated with
commercial bread manufacture. Food Microbiology 10, 287e294.
Berenzon, S., Sam Saguy, I., 1998. Oxygen absorbers for extension of crackers shelf-
life. Lebensmittel Wissenschaft und Technologie 31, 1e5.
Bianchi, F., Careri, M., Chiavaro, E., Musci, M., Vittadini, E., 2008. Gas chromato-
graphic-mass spectrometric characterization of the Italian protected designa-
tion of origin “"Altamura” bread volatile profile. Food Chemistry 110, 787e793.
Buttery, R.G., Turnbaugh, J.G., Ling, L.C., 1988. Contribution of volatiles to rice aroma.
Journal of Agricultural and Food Chemistry 36, 1006e1009.
Chiavaro, E., Vittadini, E., Musci, M., Bianchi, F., Curti, E., 2008. Shelf life stability of
artisanally and industrially produced durum wheat sourdough bread (“Alta-
mura bread”). Lebensmittel Wissenschaft und Technologie 41, 58e70.
Del Nobile, M.A., Martoriello, T., Cavella, S., Giudici, P., Masi, P., 2003. Shelf life
extension of durum wheat bread. Italian Journal of Food Science 15, 383e393.
EFSA, 2005. Bacillus cereus and other Bacillus spp. in foodstuffs. The EFSA Journal
175, 1e45.
Fernandez, U., Vodovotz, Y., Courtney, P., Pascall, M., 2006. Extended shelf life of soy
bread using modified atmosphere packaging. Journal of Food Protection 69,
693e698.
Franke, I., Wijma, E., Bouma, K., 2002. Shelf life of pre-packed buns by an active
packaging ethanol emitter. Food Additives and Contaminants 19, 314e322.
Frankel, E., 1982. Volatile lipid oxidation products. Progress in Lipid Research 22,
1e33.
Gali  
c, K., Curic, D., Gabric, D., 2009. Shelf life of packaged bakery goods: a review.
Critical Reviews in Food Science and Nutrition 49, 405e426.
Goulas, A.E., Kontominas, M.G., 2005. Effect of salting and smoking-method on the
keeping quality of chub mackerel (Scomber japonicus): biochemical and sensory
attributes. Food Chemistry 93, 511e520.
Grosch, W., Schieberle, P., 1997. Flavor of cereal products e a review. Cereal
Chemistry 74, 91e97.
Hansen, A., Schieberle, P., 2005. Generation of aroma compounds during sourdough
fermentation: applied and fundamental aspects. Trends in Food Science and
Technology 16, 85e94.
He, H., Hoseney, R.C., 1990. Changes in bread firmness and moisture during long-
term storage. Cereal Chemistry 67, 603e605.
Legan, J.D., Voysey, P.A., 1991. Yeast spoilage of bakery products and ingredients.
Journal of Applied Bacteriology 70, 361e371.
Maga, J.A., 1992. Pyrazine update. Food Reviews International 8, 479e558.
Mexis, S.F., Badeka, A.V., Chouliara, E., Riganakos, K.A., Kontominas, M.G., 2009.
Effect of g-irradiation on the physicochemical and sensory properties of raw
unpeeled almond kernels (Prunus dulcis). Innovative Food Science and
Emerging Technologies 10, 87e92.
Nielsen, P.V., Rios, R., 2000. Inhibition of fungal growth on bread by volatile
components from spices and herbs, and the possible application in active
packaging, with special emphasis on mustard essential oil. International Journal
of Food Microbiology 60, 219e229.
Plessas, S., Bekatorou, A., Gallanagh, J., Nigam, P., Koutinas, A.A., Psarianos, C., 2008.
Evolution of aroma volatiles during storage of sourdough breads made by
mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii spp.
bulgaricus or Lactobacillus helveticus. Food Chemistry 107, 883e889.
Poinot, P., Grua-Priol, J., Arvisenet, G., Rannou, C., Semenou, M., Le Bail, A., Prost, C.,
2007. Optimisation of HS-SPME to study representativeness of partially baked
bread odorant extracts. Food Research International 40, 1170e1184.
Pozo-Bayón, M.A., Guichard, E., Cayot, N., 2006. Flavor control in baked cereal
products. Food Reviews International 22, 335e379.
Quílez, J., Ruiz, J.A., Romero, M.P., 2006. Relation between sensory flavor evaluation
and volatile and nonvolatile compounds in commercial wheat bread type
baguette. Journal of Food Science 71, S423eS427.
Rodríguez, M.V., Medina, L.M., Jordano, R., 2000. Effect of modified atmosphere
packaging on the shelf life of sliced wheat flour bread. Nahrung 44, 247e252.
Rosenkvist, H., Hansen, A., 1995. Contamination profiles and characterization of
Bacillus species in wheat bread and raw materials for bread production. Inter-
national Journal of Food Microbiology 26, 353e363.
 E. Latou et al. / Journal of Cereal Science 52 (2010) 457e465 465

Rychlik, M., Grosch, W., 1996. Identification and quantification of potent odorants
formed by toasting of wheat bread. Lebensmittel Wissenschaft und Technologie
29, 515e525.
Salminen, A., Latva-Kala, K., Randell, K., Hurme, E., Linko, P., Ahvenainen, R.,
1996. The effect of ethanol and oxygen absorption on the shelf life
of packed sliced rye bread. Packaging Technology and Science 9,
29e42.
Schieberle, P., Grosch, W., 1985. Identification of the volatile flavour compounds of
wheat bread crust-comparison with rye bread crust. Zeitschrift für Leb-
ensmittel-Untersuchung und Forschung 180, 474e478.
Seiler, D.A.L., Russel, N.J., 1991. Ethanol as a food preservative. In: Russel, N.J.,
Gould, G.W. (Eds.), Food Preservatives. AVI, New York, pp. 153e171.
Thompson, J.M., Dodd, C.E.R., Waites, W.M., 1993. Spoilage of bread by Bacillus.
International Biodeterioration and Biodegradation 32, 55e66.