Journal of Cereal Science 53 (2011) 291e297

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Journal of Cereal Science

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Antioxidants and shelf life of whole wheat bread

Sidsel Jensen a, c, Henrik Ostdal b, Leif H. Skibsted c, Anette K. Thybo a, *
a
Department of Food Science, Faculty of Agricultural Sciences, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark
b
Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark
c
Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

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

Article history: Industrially produced bread normally operates with a shelf life of several weeks at room temperature and
Received 25 May 2010 indications of storage-related off-flavour development as a consequence of lipid oxidation have been
Received in revised form suggested. The present study has tested enrichment of whole wheat bread with a-tocopherol or
21 January 2011
commercially used rosemary extracts for production of bread loaves with higher oxidative stability and
Accepted 24 January 2011
hence better overall sensory quality. Bread quality was evaluated by sensory profiling, determination of
antioxidative capacity, determination of lipid hydroperoxides as primary oxidation products, and analysis
Keywords:
of volatile compounds including secondary lipid oxidation products. Enrichment of bread with a-
Whole wheat bread
Storage
tocopherol resulted in higher degrees of rancid aroma and flavour in fresh samples, which was explained
Antioxidants by higher levels of secondary oxidation products, whereas enrichment of bread with rosemary extracts
did not have any effect. Accordingly, application of antioxidants cannot, based on the current data, be
recommended for achievement of bread with improved sensory properties during storage.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction of volatiles have been performed (Chiavaro et al., 2008; Schieberle

Modern food production necessitates a certain shelf life and
longer shelf life is often considered as a competitive advantage due
to more flexible and centralised production, longer display time
during retail, and increased convenience for the consumer. Extended
shelf life is achieved by control of key reactions decisive for shelf life
and therefore other reactions normally considered to be of less
importance may become important in determining shelf life.
Shelf life of bread products are normally limited by bread
firming and microbial growth (Fernandez et al., 2006). Currently,
bread producers can, by controlling these parameters, achieve
products with a shelf life of up to 4 weeks (Sargent, 2008).
Management of firming rate and microbial activity means that
other parameters like flavour and aroma may become the limiting
factor for shelf life of baked products (Holtekjoelen et al., 2008;
Jensen et al., 2010, in press).
The aroma and flavour of bread has long been acknowledged to
influence the desirability of baked products and a vast number of
studies employing sensory analysis and chemical characterisation
Abbreviations: ANOVA, Analysis of variance; DM, Dry matter; GCeMS, Gas
chromatography mass spectroscopy; ORAC, Oxygen radical absorbance capacity; PC,
Principal component; PLS, Partial least squares; POV, Peroxide value; TEAC, Trolox
equivalent antioxidant capacity.
* Corresponding author. Tel.: þ45 8999 3405; fax: þ45 8999 3495.
E-mail address: Anette.Thybo@agrsci.dk (A.K. Thybo).
and Grosch, 1992; Seitz et al., 1998). Most of these studies concern
flavour and aroma of fresh bread, and only very few studies touch
upon changes of these parameters in bread products as a conse-
quence of storage. Chiavaro et al. (2008) found a decrease in the
total content of volatiles after 4 days of storage and Schieberle and
Grosch (1992) detected a decrease in the concentration of selected
volatiles after 8 days of storage, and concluded this decrease to be
of utmost importance for the acceptability of the bread product in
question. Significant changes in aroma, flavour, and taste of wheat
bread and whole wheat bread during 3 weeks of storage were
previously reported together with a build up of hexanal and hep-
tanal (Jensen et al., accepted for publication). Formation of primary
lipid oxidation products as precursors for these volatiles indicates
that oxidative reactions probably have a high impact on the flavour
and aroma profile of bread with extended shelf life (Jensen et al.,
2011b).
Effective control of lipid oxidation in food includes reduction in
loss of antioxidants naturally present in the raw materials by
modification in processing steps, elimination of metal contamina-
tion, addition of antioxidants, and implementation of appropriate
packaging technologies (Fernandez et al., 2006; Frankel, 1996;
Paradiso et al., 2008). Use of synthetic antioxidants in food prod-
ucts has for many years been an effective and relatively cheap way
to achieve more oxidative stable products (Pokorny, 1991).
However, synthetic antioxidants such as butylated hydroxyanisole
and butylated hydroxytoluene may present a risk of carcinogenesis

0733-5210/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2011.01.010
292 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297
which is why the interest in natural antioxidants has been inten-
sified (Yanishlieva and Marinova, 1992; IARC, 1986a, 1986b).
Apart from ascorbic acid normally considered being an antiox-
idant, but which in bread is used to achieve increased dough
stability and bread volume (Grosch and Wieser, 1999), addition of
antioxidants is not common for bread products. Enrichment of
bread dough with antioxidants represents a strategy for production
of bread loaves with a higher degree of oxidative stability and
hence better overall sensory quality during storage, but only few
studies concerning addition of antioxidants to bread products are
available. Peng et al. (2010) studied the effect of grape seed extracts
on the antioxidative capacity in bread made from refined wheat
flour and found the oxidative status of the enriched bread to be
greatly enhanced. These bread loaves were further evaluated to
have a more desirable crust colour when compared with control
bread prepared without the addition of grape seed extract. Other
than the composition of the bread matrix, the effect of an anti-
oxidative extract in food products depends on the type of antioxi-
dants present in the extract and their solubility in the specific food
matrix. Furthermore, the concentration of the individual antioxi-
dants and their combined effect are important as well as the
compatibility of the antioxidants with the processing and pack-
aging methods employed. Benefits in bread production with one
type of antioxidative extract is, due to the above mentioned factors,
not necessarily valid for other antioxidative extracts or for other
types of bread.
The present study investigates the effect of adding naturally
occurring antioxidants, a-tocopherol and two different rosemary
extracts, to bread and monitoring the effect of these on bread
quality during extended storage. a-Tocopherol is a lipophilic anti-
oxidant naturally present in wheat, particularly in wheat bran
(Zhou et al., 2004), while the antioxidative effect of rosemary
extracts have been assigned to the high content of carnosol and
carnosic acid (Aruoma et al., 1992). Bread quality was evaluated by
sensory profiling, changes in antioxidative capacity, formation of
primary oxidation products, and analysis of volatiles.
2. Materials and methods
2.1. Types of bread and preparation
Whole wheat bread was made from stone milled fine whole
wheat flour (min. 14% protein) and the ingredients listed in Table 1.
All ingredients were obtained from commercial suppliers in EU
except from stone milled fine whole wheat flour and azodicarbo-
namide, which was obtained from commercial suppliers in the US.
Table 1
Ingredients per kilo whole wheat flour [g].
Ingredients [g/kg whole wheat flour]
Water 629.5
Salt 20.0
Glucose syrup 80.0
Cane syrup 40.0
Soy oil 20.0
Yeast 50.0
sodium stearoyl-2-lactylate 5.0
Ascorbic acid 0.06
Calcium propionate 2.5
Mono and di-glycerides 10.0
Azodicarbonamide 0.04
Novamyl 10.000 BG 0.25
Fungamyl Super MA 0.04
a-Tocopherol 1.0
Rosemary extract (fat soluble) 1.0
Rosemary extract (water dispersible) 1.0
The bread was prepared according to a sponge and dough proce-
dure and the four different bread varieties were prepared differing
with respect to antioxidative additives: ‘a-tocopherol’, ‘fat soluble
rosemary extract’, ‘water dispersible rosemary extracts’ and ‘no
additive’. The fourth bread prepared without any antioxidative
additive was included as a control sample. a-Tocopherol (>97) was
purchased from SigmaeAldrich (SigmaeAldrich Inc., Steinheim,
Germany) and fat soluble and water dispersible rosemary extracts
(GUARDIANTM Rosemary Extract 201 and GUARDIANTM Rosemary
Extract 202) were kindly donated by Danisco (Danisco A/S, Brab-
rand, Denmark). The rosemary extracts were practically flavourless
and contained 96% natural rosemary extract, 4% phenolic di-
terpenes (compounds present in rosemary extract), together with
propylene glycol, E1520. In addition, GUARDIANTM Rosemary
Extract 202 also contained polyoxyethylene (20) sorbitan mono-
oleate, E433. The list of ingredients for each of the bread varieties
are given in Table 1.
Novamyl 10.000 BG and Fungamyl Super MA were both from
Novozymes (Novozymes A/S, Bagsvaerd, Denmark). Novamyl is
a maltogenic amylase and Fungamyl Super is a blend of fungal
a-amylase and xylanase. Microbial growth was prevented by addi-
tion of propanoic acid and by surface treatment with a 6% calcium
sorbate solution of the baked bread. After surface treatment, the
bread was further baked for 1 min to evaporate excess water from
the sorbate treatment. Moisture loss during baking was not deter-
mined in the present baking experiment, but for this type of bread
a moisture loss of 11e13% is normal. After cooling, the bread was
packed in commercially used plastic bags (polyethylene, 25 mm,
Multiline A/S, Soroe, Denmark) with high oxygen-permeability.
Bread was prepared over a five week period providing bread stored
for 0, 1, 2, 3, 4, and 5 weeks. All samples were analysed by sensory
analysis and GC measurements at the same dates. Bread loaves were
stored at room temperature (approximately 22  C) and samples
stored for 4 and 5 weeks were only subjected to chemical analysis
due to safety reasons (microbial status was not evaluated, but no
visible mould growth was observed). For analysis of the overall
antioxidative capacity and peroxide value, approximately 40 g of
bread crumb was frozen rapidly by immersing samples directly into
liquid nitrogen. The frozen bread pieces were ground and vacuum
packed in non-transparent oxygen-impermeable aluminium foil
bags (PETP12/ALU9/LLDPE75, Dansico Flexible, Horsens, Denmark)
in sample portions of approximately 3 g. The foil bags were stored
at 80  C until the dates of analyses. Samples were frozen on the
same dates as the sensory analyses and the GCeMS measurements
were carried out. All bread loaves were produced by the same baker
to ensure uniformity in the baking procedure.
Control a-Tocopherol Rosemary (lipophilic) Rosemary (hydrophilic)
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x x x x
x
x
x
 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297 293

2.2. Sensory analysis 2.4. Evaluation of the oxidative status

The samples of bread crumb used for sensory analysis consisted The overall antioxidative capacity analysed by the TEAC and
of bread sliced to a thickness of approximately 1.2 cm using a slicing ORAC assay together with analysis of lipid hydroperoxides (POV)
machine (Gourmet Food Slicer, MAS 6108, Bosch, Stuttgart, were performed as described by Jensen et al. (2011b).
Germany) and divided into quarters. The end pieces of each bread
loaf, approximately 5 cm, were discarded. One serving consisted of 2.5. Statistics
two quarters of a bread slice, free of crust, put in a transparent
plastic container and labelled with a three-digit number. The Data were analysed for significant effects of type of antioxidant
sensory evaluation was conducted by a sensory panel consisting of added and storage time using univariate analysis of variance
8 experienced assessors (7 females/1 male, aged from 42 to 55). The (ANOVA) (SAS Institute, version 9.1, Cary, NC). For the sensory data
sensory profiling was performed in a sensory evaluation laboratory, analysis, the interaction between sample and assessor was used as
which fulfil requirements according to international standards random effect, and thereby, level and range differences between
(ASTM, 1986; ISO, 1988). individual assessors were modelled (Brockhoff and Skovgaard,
The sensory evaluation started with a panel discussion, where the 1994). Statistical significance was defined at P < 0.05. Principal
assessors agreed on a consensus list of attributes and on a definition component analysis (PCA) was used to interpret the structure in the
of each attribute. Fifteen attributes were included in the list: 6 aroma large amount of volatile compounds while partial least squares
related attributes (nasal perception), 6 flavour related attributes (oral (PLS) regression was used to study the prediction of sensory data
and retro nasal perception), and 3 taste and mouth-feel related (Y-data) from chemical measurements (X-data). Uncertainty testing
attributes (oral perception and physical sensation in the oral cavity). by a jack-knife procedure was applied to identify variables of
The attributes and matching descriptions are listed in Table 2. All highest importance in the predictive model (Martens and Martens,
assessors were experienced in evaluating plant based food products 2000). Multivariate data analyses were performed using
on a weekly basis, and most of the assessors had experience in Unscrambler (version 9.6, CAMO Software A/S, Oslo, Norway) and
evaluating whole wheat bread and therefore the training sessions LatentiX (version 2.00, Latent5 Aps, Copenhagen, Denmark). Full
were limited to four times 2 h sessions. After each training session, cross validation was used as validation criterion and all data were
assessors received feedback on their performance with the aim of auto-scaled.
improving and standardising the panel’s discriminative power.
For each of the four bread varieties (control, addition of a- 3. Results and discussion
tocopherol, addition of fat soluble rosemary extract or addition of
water dispersible rosemary extract) the assessors evaluated bread 3.1. Sensory analysis
stored for: ‘0 weeks’, ‘1 week’, ‘2 weeks’, and ‘3 weeks’ in triplicates
over a period of three days giving a total of 16 samples per test day. Results from the univariate ANOVA showed a significant effect of
The assessors evaluated the samples at individual speed on an assessors for all tested attributes, which is a common observation in
unstructured 15.0 cm line scale anchored on the left by ‘low’ and on sensory evaluation and can be ascribed to level differences in the
the right with the term ‘high’. Ratings were registered directly on scaling (Brockhoff and Skovgaard, 1994; Naes and Langsrud, 1998)
a computerised registration system (Fizz software, 2.30C, Bio- (data not shown). The panel was able to distinguish between
systemes, Couternon, France). samples according to storage time and less pronounced between
types of antioxidant added. Main effect of storage time was obtained
2.3. Analysis of volatile compounds for 80% of the attributes, emphasising that the sensory quality of
bread is highly dependent on storage time and that aroma, flavour,
Head space collection of volatile compounds from whole wheat and taste are in fact important characteristics when shelf life of
bread crumb was conducted and analysed by GCeMS as described bread is to be determined. Fig. 1 illustrates sensory mean data across
by Jensen et al. in press. Verification of compounds suggested by the replicates and assessors for each bread variety. It is obvious that
MS database (NIST, 1998) was made by comparison of the relative storage affects the sensory profiles and that especially the sensory
retention indices and MS of authentic reference compounds. profile of non-stored (0 weeks) bread displays a huge difference
Authentic compounds were supplied by SigmaeAldrich (Sigmae compared to bread stored for 1, 2, and 3 weeks.
Aldrich Inc., Steinheim, Germany). A significant effect of antioxidant addition was displayed for the
aroma attributes: ‘dough’, ‘bran’, and ‘rancid’, and for rancid flavour.
Table 2 Especially bread enriched with a-tocopherol differed from the
Sensory attributes and descriptions. remaining samples (Fig. 1) as these samples were generally evalu-
Category Attribute Description ated to have a lower intensity of dough, bran, and dust aroma and
Aroma Dough Freshly made wheat dough a higher intensity of rancid aroma and flavour. a-Tocopherol has
Bran Bran, cereal-like been found to enhance the sensory and chemical stability in various
Rancid Rancid linseed food products (Paradiso et al., 2008; Warner and Moser, 2009). In
Acid Propanoic acid a study concerning the stability of vitamin E in wheat flour, wheat
Spice Mild spicy, roasted
Dust Dry
flour with high vitamin E content was found to delay the formation
Flavour Dough Freshly made wheat dough of hexanal compared to flour with a lower content of vitamin E
Sweet aromatic Toffee, malt-like (Nielsen and Hansen, 2008). Hexanal is commonly used as an
Rancid Rancid linseed indicator of lipid oxidation as it contributes to the rancid flavour.
Dust Dry
The antioxidant/pro-oxidant nature of a-tocopherol depends,
Bran Bran, cereal-like
Spice Mild spicy, roasted, cocoa however, on the concentration, and a pro-oxidative effect has been
Taste and Bitter Bitter observed especially when a-tocopherol has been added in rela-
Mouth-Feel Prickly Prickly sensation on the tip of the tongue tively high concentrations (Huang et al., 1994). The rancid flavour of
Astringency Pungent feeling on the side of the tongue the present bread enriched with a-tocopherol suggests a pro-
and pain-like feeling in the jaw
oxidative effect of a-tocopherol and might be a result of the
294 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297

Fig. 1. Spider plot of sensory scores (0 (low intensity)-15 (high intensity)) of control bread and bread enriched with a-tocopherol, and with a fat soluble (L) and water dispersible (H)
rosemary extract, respectively. Means are calculated across replicates and assessors. A_: Aroma attributes; F_: Flavour attributes.

relatively high concentration of a-tocopherol (1 g kg1 flour). This
conclusion is supported by analyses performed by Park et al. (2002)
and Ochi et al. (1993), who observed a pro-oxidative effect of
tocopherols when added in concentrations of 100e200 mg kg1 to
heat treated cereal products such as cookies and rice snacks.
3.2. Analysis of volatiles
Thirty five different volatiles were identified and verified in the
present study. The concentrations of all verified volatile
compounds for the four bread varieties are given in Table 3. The
volatile compounds can be divided into seven classes: ‘acids’,
‘alkanes’, alcohols’, ‘aldehydes’, ‘esters’, ‘furans’, ‘ketones’, and
‘terpenes’. Statistical analysis showed significant effect of the
interaction between storage and bread variety for the total content
of acids (P ¼ 0.048). Furthermore, a significant main effect of
storage was found for the decreasing content of alcohols, alde-
hydes, alkanes, furans, and terpenes (P < 0.0001). In addition,
aldehydes were found to be significantly affected by bread variety
(P ¼ 0.041). Higher concentrations of aldehydes were found in fresh
samples of bread enriched with a-tocopherol compared to the
other bread varieties. Especially, the secondary oxidation products
hexanal (V12) and heptanal (V15) were higher in fresh samples of
these bread loaves indicating a higher degree of lipid oxidation
already during baking of bread enriched with a-tocopherol.
A multivariate interpretation of the volatile components (Fig. 2)
confirmed the results from the ANOVA as it is evident that bread
samples could be separated according to effect of storage time
along PC1 (PC1 explains 51% of variance) and less pronounced
according to effect of antioxidative additives along PC2 (PC2
explains 10% of variance). This differentiation of samples in accor-
dance with storage was in good agreement with results from the
sensory analysis. Fresh samples of bread are positioned in the
opposite direction of PC1 compared to the more stored samples,
with a few exceptions (a-tocopherol stored for 1 and 2 weeks). A
differentiation of bread enriched with a-tocopherol and bread
enriched with rosemary extracts are seen along PC2. No obvious
differences in volatile profiles between fat soluble and water
dispersible rosemary extracts were found. The efficiency of fat
soluble and water dispersible antioxidants is known to differ
significantly when tested in similar food systems (Frankel, 1996)
and a differentiation of samples according to type of rosemary
extract could therefore be expected. However, the present data
showed no difference between the two tested rosemary extracts
which could be explained by the active ingredients in the rosemary
extracts being the same, and that these ingredients generally did
not have an effect on the chemical stability of bread. The difference
in solubility of the two extracts is exclusively obtained by addition
of the emulsifier polyoxyethylene (20) sorbitan monooleate, E433,
to the water dispersible rosemary extract.
 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297 295

Table 3
Maximum and minimum values for the concentration [ng g1 of bread] of verified volatile compounds in control bread and bread enriched with a-tocopherol and a fat soluble
(L) and water dispersible (H) rosemary extracts.

No Volatiles Control a-Tocopherol Rosemary (L) Rosemary (H) Sign. variety

Min-Max
Acid 28 Acetic acid 0.129e0.336 0.150e0.263 0.118e0.225 0.130e0.234
31 Propanoic acid 25.618e43.362 25.366e39.995 28.737e37.492 27.112e40.623
32 2-Methylpropanoic acid 2.039e3.087 2.127e2.997 2.134e2.762 1.995e2.851
33 Butanoic acid 0.145e0.211 0.118e0.196 0.176e0.292 0.164e0.216
Alkane and 1 Octane 0.059e0.571 0.078e0.691 0.060e0.541 0.044e0.426
alcohol 8 Ethanol 731.713e1352.534 618.571e1406.702 654.456e1508.114 592.502e1491.608
10 1-Propanol 4.011e6.748 3.995e6.777 4.055e6.930 3.726e7.331
13 2-Methyl-1-propanol 93.071e180.345 95.596e180.886 93.648e179.107 70.941e204.239 **
17 3-Methyl-1-butanol 28.235e77.777 27.892e77.834 28.529e73.020 22.172e84.544
24 Hexanol 1.006e2.114 0.856e2.141 0.917e1.974 0.669e2.163
25 3-Ethoxy-1-propanol 0.212e0.349 0.218e0.296 0.213e0.284 0.206e0.283
35 Phenylethanol 1.904e2.192 1.627e2.089 1.704e2.260 1.654e2.152
Aldehyde 2 2-Methylpropanal 0.196e2.100 0.237e2.582 0.184e1.657 0.188e1.478
6 2-Methylbutanal 0.156e3.617 0.187e5.954 0.155e2.920 0.164e3.071
7 3-Methylbutanal 0.698e11.597 1.055e14.375 0.781e8.213 0.900e8.729
12 Hexanal 1.845e3.867 2.690e4.758 1.616e3.747 1.555e4.006 ***
15 Heptanal 0.271e1.050 0.318e1.157 0.229e0.499 0.192e1.062 *
26 Nonanal 0.337e0.518 0.138e0.505 0.277e0.680 0.310e0.523 *
30 Benzaldehyde 0.746e1.013 0.601e1.099 0.678e0.956 0.558e0.963
Ester 4 Ethylacetate 1.274e3.809 1.126e2.141 1.250e2.512 1.228e2.863
19 Ethylhexanoate 0.210e0.502 0.375e0.656 0.253e0.468 0.248e0.548 *
23 Ethyllactate 0.166e0.608 0.312e0.662 0.151e0.667 0.296e0.610
27 Ethyloctanoate 0.418e0.896 0.376e0.708 0.440e0.777 0.340e0.722
Furan 3 2-Methylfuran 0.107e0.204 0.105e0.281 0.073e0.261 0.050e0.129
18 2-Pentylfuran 0.158e0.853 0.175e0.955 0.146e0.726 0.141e0.757
20 2-Methyltetrahydro3furanone 0.090e0.188 0.099e0.247 0.082e0.156 0.074e0.144 *
29 Furfural 0.330e1.255 0.267e1.593 0.287e0.925 0.210e0.857
34 Furfuryl alcohol 0.187e0.613 0.240e0.471 0.217e0.470 0.182e0.431
Ketone 5 2-Butanone 0.414e0.778 0.343e0.760 0.366e0.727 0.282e0.646
9 2,3-Butandione 4.95e6.291 3.637e7.481 4.470e5.706 4.357e7.244
11 2,3-Pentandione 0.137e0.698 0.067e0.898 0.123e0.659 0.108e0.706
14 2-Heptanone 0.177e0.235 0.242e0.390 0.096e0.559 0.136e0.264
21 3-Hydroxy-2-butanone 16.596e27.173 23.925e28.379 21.732e25.685 21.569e25.945
22 1-Hydroxy-2-propanone 0.763e1.823 0.577e1.494 2.395e3.576 1.155e1.947 ***
Terpene 16 Limonene 0.048e0.145 0.050e0.173 0.044e0.174 0.070e0.191

Fig. 2. Principal component analysis bi-plot on volatile compounds in whole wheat bread (control) and whole wheat bread enriched with a-tocopherol and fat soluble (L) and water
dispersible (H) rosemary extracts. Volatiles (V) are designated by the number of the individual compound given in Table 3. The digits at the end of the sample name states the time
of storage [0e5 weeks].
296 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297
A substantial part of the volatiles were associated with the fresh
samples of bread, independent of antioxidant added. Furfuryl
alcohol (V34) and butanoic acid (V33) increased in concentration
during storage while all other volatiles decreased as a consequence
of storage. Furfuryl alcohol was found to increase in whole wheat
bread crumb and decrease in whole wheat bread crust during a 3
week period of storage in a previous study conducted by the
present authors (Jensen et al., accepted for publication), suggesting
that furfuryl alcohol migrates from crust to crumb during storage.
3.3. Oxidative status
The measurement of lipid hydroperoxides (POV) was performed
to follow the progress in lipid oxidation. No changes in POV was
observed during storage, but a significant difference (P < 0.0001)
was observed for the type of antioxidant added. A substantially
higher level of hydroperoxides was present in bread enriched with
a-tocopherol compared to the three other bread varieties
(0.7e0.9 mE H2O2 g1 of bread enriched with a-tocopherol, based
on dry matter content (DM) compared to 0.0e0.2 mE H2O2 g1 of
bread for control bread and bread enriched with rosemary extracts,
DM). Similar results were obtained in a study performed by Terao
and Matsushita (1986), who found an increase in the concentra-
tion of hydroperoxides for edible oil enriched with a-tocopherol.
The elevated level of hydroperoxides can be explained by break-
down of lipid hydroperoxides induced by reactive species of
a-tocopherol (a-tocopherol radical). The accumulation of hydro-
peroxides observed in the present study right after baking can thus
be explained by an increase in the concentration of a-tocopheroxyl
as a result of the elevated temperatures during the baking process.
Comparison of Fig. 1 and Table 3 shows that the increased level
of lipid hydroperoxides in bread with added a-tocopherol was also
accompanied by increased rancidity score in the sensory evalua-
tion. A study by Haakansson and Jägerstad (1990) showed that
vitamin E loss in whole wheat flours started immediately after flour
and water were mixed due to lipoxygenase activity and the loss
increased as the temperature of the flour-water slurry increased.
Another study determined the content of tocopherols to decrease
by one third during baking (Ranhotra et al., 2000). Vitamin E loss is
equal to formation of a-tocopheroxyl radicals and a-tocopheroxyl
Fig. 3. Partial least squares (PLS) regression with only significant important variables of 4 different bread varieties (Control bread, bread enriched with a-tocopherol, fat soluble (L)
and water dispersible (H) rosemary extracts) and with 4 different storage times. Score plot (a) and loading plot (b) with chemical data as X-variables and sensory data as Y-variables.
Sensory aroma attributes (normal), flavour attributes (italic), and taste and mouth-feel (underlined). The digit in sample name indicates storage time [0e3 weeks].
radicals have been shown to act as pro-oxidants as mentioned
above. The high concentration of lipid hydroperoxides in bread
enriched with a-tocopherol is accordingly suggested to be a result
of a pro-oxidative activity of oxidized tocopherols during bread
making.
The overall antioxidative capacity was evaluated by the TEAC
assay and the ORAC assay. The antioxidative capacity in bread
samples when measured by ORAC assay was between 17.4 and
19.8 mg trolox equivalents g1 crumb, DM. This was significantly
higher than the level measured by TEAC, which showed the anti-
oxidative capacity to be between 4.8 and 6.8 mg trolox equivalents
g1 crumb, DM. Both assays exhibited significance for the interac-
tion between storage time and bread variety, emphasising that the
development in overall antioxidative capacity during storage
depended on bread variety.
3.4. Correlation of sensory and chemical results
To study the correlation between sensory and chemical
(GCeMS, POV, ABTS, and ORAC) data a multivariate PLS regression,
based on prediction of the sensory data (Y-variables) from the
chemical data (X-variables) was performed (Fig. 3).
The regression plot shows that 1) most of the aroma attributes
are spanned by PC1 together with a large amount of volatile
compounds, 2) a few flavour attributes are spanned by PC2 together
with fewer volatile compounds, and 3) another large group of
attributes related to flavour, taste, and mouth-feel are located near
origio and are thus not explained by any of the volatile compounds.
For PC1, the dough aroma, mainly presented in the fresh bread
samples, was positively correlated with a large number of volatiles
like ethanol (V8), 1-propanol (V10), 2,3-pentadione (V11),
2-methyl-1-propanol (V13), limonene (V16), 3-methyl-1-butanol
(V17), hexanol (V24), and ethyloctanoate (V27) and inversely
correlated to furfuryl alcohol (V34) which, on the contrary, corre-
lated with the aroma attributes: ‘rancid’, ‘spicy’, and ‘aromatic
sweet’. This means that most of the sensory aroma attributes are
correlated to volatile compounds, and that volatility has a great
impact on the perceived changes of bread aroma. In PC2, a positive
correlation was found between two flavour attributes: ‘bran’ and
‘dough’, mainly found in samples added rosemary extract, and
 S. Jensen et al. / Journal of Cereal Science 53 (2011) 291e297
ethyloctanoate (V27), which was inversely correlated to ‘rancid’
flavour. The remaining flavour, taste, and mouth-feel attributes
being: ‘prickly’, ‘bitter’, ‘spicy’, ‘astringent’, ‘acid’, and ‘dust’ are less
correlated to any of the aroma compounds and the TEAC. This
indicates that the volatile compounds are not the main contributors
to the perceived flavour, taste, and mouth-feel of bread and prob-
ably non-volatile compounds are responsible for changes of these
attributes.
4. Conclusion
The present study showed that the tested antioxidative addi-
tives did not improve the sensory quality and stability of bread.
Changes in the sensory profile and in the content of volatile
compounds in bread enriched with a-tocopherol or rosemary
extracts were, like for the control bread, mainly ascribed to the
effect of storage time whereas the effect of bread enrichment was
less pronounced. Most volatile compounds were present in higher
concentrations in the fresh samples of bread, emphasising that
volatility is an important contributor to changes, especially changes
related to aroma, during storage. Fresh samples of bread enriched
with a-tocopherol had higher concentrations of hydroperoxides
and secondary lipid oxidation products. These samples shared
some of the same sensory characteristics as stored control samples
indicting that lipid oxidation is responsible for these less favourable
sensory notes like rancid aroma and flavour, bitter taste, and
astringency. The analysis of the antioxidative capacity showed that
the relatively small changes in antioxidative capacity during
storage depended on the type of additive included in the bread
recipe.
Acknowledgements
The authors gratefully acknowledge the excellent technical
assistance from Nina Eggers and Birgitte Foged, the great help from
Lone R. Borum in the sensory lab as well as the work performed by
Bo B. Madsen, who baked the large number of breads forming the
basis of the present work. The investigation is a part of a Ph.D. study
supported financially by Novozymes, University of Copenhagen,
and the Research School FOOD Denmark.
References
Aruoma, O.I., Halliwell, B., Aeschbach, R., Löligers, J., 1992. Antioxidant and pro-
oxidant properties of active rosemary constituents: carnosol and carnosic acid.
Xenobiotica 22, 257e268.
ASTM, 1986. Physical Requirements, Guidelines for Sensory Evaluation Laboratories,
STP 913. American Society for Testing and Materials, PA.
Brockhoff, P.M., Skovgaard, I.M., 1994. Modelling individual differences between
assessors in sensory evaluations. Food Quality and Preferences 5, 215e224.
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’’). LWT 41, 58e70.
Fernandez, U., Vodovotz, Y., Courtney, P., Pascall, M.A., 2006. Extended shelf life of
soy bread using modified atmosphere packaging. Journal of Food Protection 69,
693e698.
Frankel, E.N., 1996. Antioxidants in lipid foods and their impact on food quality.
Food Chemistry 57, 51e55.
297
Grosch, W., Wieser, H., 1999. Redox reactions in wheat dough as affected by ascorbic
acid. Journal of Cereal Science 29, 1e16.
Haakansson, B., Jägerstad, M., 1990. The effect of thermal inactivation of lip-
oxygenase on the stability of vitamin E in wheat. Journal of Cereal Science 12,
177e185.
Holtekjoelen, A.K., Baevre, A.B., Roedbotten, M., Berg, H., Knutsen, S.H., 2008.
Antioxidant properties and sensory profiles of breads containing barley flour.
Food Chemistry 110, 414e421.
Huang, S.-W., Frankel, E.N., German, J.B., 1994. Antioxidant activity of a- and
g-tocopherols in bulk oils and in oil-in-water emulsions. Journal of Agricultural
and Food Chemistry 42, 2108e2114.
IARC, 1986a. Butylated hydroxyanisole (BHA). IARC Monographs on the evaluation of
the Carcinogenic risks of chemicals to Humans. Some naturally occurring and
synthetic food components. Furocoumarins and Ultraviolet Radiation 40, 123e159.
IARC, 1986b. Butylated hydroxytoluene (BHT). IARC Monographs on the evaluation
of the Carcinogenic risk of chemicals to Humans., some naturally occurring and
synthetic food components. Furocoumarins and Ultraviolet Radiation 40,
161e206.
ISO, 1988. International Standard 8589, Sensory Analysis - General Guidance for the
Design of Test Rooms. Ref. no. ISO 8589:1988 (E). International Organization for
Standardization, Genéve, Switzerland.
Jensen, S., Oestdal, H., Thybo, A.K., 2010. Sensory profiling of changes in wheat and
whole wheat bread during a prolonged period of storage. Journal of Sensory
Studies 25, 231e245.
Jensen, S., Oestdal, H., Skibsted, L. H., Larsen, E., Thybo, A. K., Chemical changes in
wheat pan bread during storage and how it affects the sensory perception of
aroma, flavor, and taste. Journal of Cereal Science, in press.
Jensen, S., Oestdal, H., Clausen, M.R., Andersen, M., Skibsted, L.H., 2011b. Oxidative
stability in whole wheat bread during storage. LTW e Food Science and Tech-
nology 44, 637e642.
Martens, H., Martens, M., 2000. Modified Jack-knife estimation of parameter
uncertainty in bilinear modelling by partial least squares regression (PLSR).
Food Quality and Preference 11, 5e16.
Naes, T., Langsrud, O., 1998. Fixed or random assessors in sensory profiling? Food
Quality and Preferences 9, 145e152.
Nielsen, M.M., Hansen, Aa, 2008. Stability of vitamin e in wheat flour and whole
wheat flour during storage. Cereal Chemistry 85, 716e720.
NIST, 1998. Mass Spectral Database, Standard Reference Database 1. National
Institute of Standards and Technology, Gaithersburg, Maryland, US.
Ochi, T., Tsuchiya, K., Ohtsuka, Y., Aoyama, M., Maruyama, T., Niiya, I., 1993.
Synergistic antioxidant effects of organic acids and their derivatives with
tocopherols on cookies. Nippon Shokuhin Kogyo Gakkaishi 40, 393e399.
Paradiso, V.M., Summo, C., Trani, A., Caponio, F., 2008. An effort to improve the shelf
life of breakfast cereals using natural mixed tocopherols. Journal of Cereal
Science 47, 322e330.
Park, Y.S., Kim, Y.S., Shin, D.H., 2002. Antioxidative effects of ethanol extracts from
Rhus verniciflua Stoke on Yukwa (Oil popped rice snack) base during storage.
Journal of Food Science 67, 2474e2479.
Peng, X., Ma, J., Cheng, K.-W., Jiang, Y., Chen, F., Wang, M., 2010. The effects of grape
seed extract fortification on the antioxidant activity and quality attributes of
bread. Food Chemistry 119, 49e53.
Pokorny, J., 1991. Natural antioxidants for food use. Trends in Food Science &
Technology 2, 223e227.
Ranhotra, G.S., Gelroth, J.A., Okot-Kotber, B.M., 2000. Stability and dietary contri-
bution of vitamin E added to bread. Cereal Chemistry 77, 159e162.
Sargent, K., 2008. A softer“ approach to improving the quality of refrigerated bakery
products. Cereal Foods World 53, 301e305.
Schieberle, P., Grosch, W., 1992. Changes in the concentration of potent odorants
during storage of white bread. Flavour and Fragrance Journal 7, 213e218.
Seitz, L.M., Chung, O.K., Rengarajan, R., 1998. Volatiles in selected commercial
breads. Cereal Chemistry 75, 847e853.
Terao, J., Matsushita, S., 1986. The peroxidizing effect of a-tocopherol on autoxi-
dation of methyl linoleate in bulk phase. Lipids 21, 255e260.
Warner, K., Moser, J., 2009. Frying stability of purified mid-oleic sunflower oil tri-
acylglycerols with added pure tocopherols and tocopherol mixtures. Journal of
the American Oil Chemists’ Society 86, 1199e1207.
Yanishlieva, N.V., Marinova, E.M., 1992. Inhibited oxidation of lipids. 1. Complex
estimation and comparison of the antioxidative properties of some natural and
synthetic antioxidants. Fett Wissenschaft Technologie 94, 374e379.
Zhou, K., Su, L., Yu, L.L., 2004. Phytochemicals and antioxidant properties in wheat
bran. Journal of Agri-cultural and Food Chemistry 52, 6108e6114.