Journal of the Science of Food and Agriculture

J Sci Food Agric 81:513518 (online: 2001)

Effect of toasting time on the browning of sliced

bread
Antonio Ramrez-Jimenez, Belen Garca-Villanova and Eduardo Guerra-Hernandez*
Departamento de Nutricion y Bromatologa, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja, 18012
Granada, Spain

Abstract: Slices of wheat bread were toasted for different times until a distinct intensity of brown
colour was reached. Two assays were carried out: prolonged toasting times (560 min) and reduced
toasting times (0.55 min). The browning indicators (furosine, available lysine, hydroxymethylfurfural
(HMF), colour and absorbance at 284 and 420 nm) were determined. The precision of all indicators
used was high (CV < 4%). No furosine or HMF was detected in the dough before baking. The furosine
content increased until 7 min (299 mg per 100 g protein) and then decreased to 2.9 mg per 100 g protein at
60 minutes. For the rst toasting times (0.5, 1 and 2 min) the furosine content decreased slightly.
Available lysine reached losses of 50% after 25 min of heating. The toasting of bread increased HMF
values from 12 to 2025 mg kg 1 for the assay at prolonged times of heating and from 1.3 to 4.2 mg kg 1 at
reduced times (0.55 min). The HMF content decreased (1000 mg kg 1) when the sliced bread was
toasted until it burnt. Colour (DE, 100 L*) and absorbance at 284 and 420 nm always increased. High
linear correlations (r2 > 0.860) were obtained between browning indicators and time (A284/time,
A420/time, 100 L*/time and HMF/time).
# 2001 Society of Chemical Industry

Keywords: browning indicators; toasting; sliced bread

INTRODUCTION

A pleasant avour and colour are produced when

slices of wheat bread are toasted, corresponding to the
organoleptic characteristics of bread crust.1 The
chemical reactions involved in this process are
essentially the Maillard reaction and caramelisation.
Both depend on the type of reagent, temperature,
water activity and pH.
Maillard reactions are favoured in systems with an
intermediate moisture content, temperatures above
50 C and pH 47 (ie in the pH range of food),
producing changes in colour (melanoidins), avour
(aldehydes and ketones), functional properties and
nutritional value (blocking or destruction of lysine).2
Caramelisation depends on direct degradation of
sugars and requires more drastic conditions (temperatures >120 C, pH < 3 or pH > 9 and low Aw).3
The manufacture of bread involves baking a loaf at
200 C for 50 minutes, then cutting the loaf into slices.
The sliced bread can be consumed fresh or toasted. In
this process, therefore, the Maillard reaction and
caramelisation may occur simultaneously.4
Evaluation of the early stages of the Maillard
reaction can be achieved by the determination of
furosine (e-N-(furoylmethyl)-L-lysine), an amino acid
formed during acid hydrolysis of the Amadori compounds fructosyl-lysine, lactulosyl-lysine and maltulo-

syl-lysine produced by reaction of e-amino groups of

lysine with glucose, lactose and maltose.5 Furosine
determination has been applied to cereal products to
monitor the extent of non-enzymatic browning in
pasta and bakery products6 and commercial baby
cereals7 and to control baby cereal processing.8
Lysine is limiting in cereal proteins. This deciency
can be further aggravated by losses from browning
reactions during processing. Available lysine can be
utilised for metabolism and is distinguished from total
lysine, which includes damaged or bound lysine. It is
suggested that lysine is not used biologically unless the
e-amino group is free.9 The method using 1-uoro2,4-dinitrobenzene (FDNB) is the most widely used10
and there is reason to believe that lysine that does not
react with FDNB is not nutritionally available.11
Available lysine has been measured to evaluate the
effect of heating on the protein quality of the following
cereal products: pasta,12 noodles,13 breads,14 cereal
akes15 and infant cereals.16
Hydroxymethylfurfural (HMF) is formed from the
degradation of hexoses heated in acid solution, even in
mild acid solution,3 and is also an intermediate
product in the Maillard reaction.17 HMF has been
used to control the drying of pasta18 and baking of
cookies19 and in baby20 and breakfast21 cereals.
Brown pigments with low and high molecular

* Correspondence to: Eduardo Guerra-Hernandez, Departamento de Nutricion y Bromatologa, Facultad de Farmacia, Universidad de
Granada, Campus Universitario de Cartuja, 18012 Granada, Spain
E-mail: ejguerra@ugr.es
(Received 28 July 2000; accepted 7 December 2000)

# 2001 Society of Chemical Industry. J Sci Food Agric 00225142/2001/$30.00

513

A Ramrez-Jimenez et al

weights are formed in the advanced stages of the

browning reactions and are assumed to be watersoluble up to a molecular size of 100 kDa.22 The
absorbance at 420 nm is a classic browning index that
is very useful in sugar and sugaramino acid model
systems. Moreover, A420 has been applied to the
kinetics of bread baking.23 The brown colour index in
solid products has been applied to follow the modelling of bread crust browning kinetics during baking,4
the toasting of wheat bread1 and the twin-screw
extrusion of corn and soy products.24
Little information has been found about these
indicators in breads. Rychlik and Grosch1 studied
the avour components of toasted wheat bread by a
complex methodology. However, studies on toasted
bread with simpler indicators to evaluate the process of
toasting have not been reported.
This paper describes the behaviour of browning
indicators during the process of toasting bread for
different times in order to give industry some useful
indicators for evaluating and controlling this process.
In addition, the study shows the inuence of toasting
time on the loss of lysine.
MATERIALS AND METHODS
Apparatus

. Elrepho 2000 reectance spectrophotometer (Datacolor SA, Spain).

. Perkin Elmer model 551S UV/VIS spectrophotometer (Norwalk, CT, USA).
. Konik model 500A liquid chromatograph, Konik
model 200 variable-wavelength UV detector
(Barcelona, Spain) and Hewlett Packard model
3394A integrator (Avondale, PA, USA).
. Perkin Elmer model 250 liquid chromatograph
(Norwalk, CT, USA) with Waters plus 717 autosampler (Milford, MA, USA) and Perkin Elmer
model 235 diode array detector (Norwalk, CT,
USA). Data were collected by a 1020 software data
system (Perkin Elmer, Norwalk, CT, USA).
Reagents

Analytical reagent grade chemicals were used.

. A standard stock solution containing 200 mg l 1
5-(hydroxymethyl) furfural (Merck, Darmstadt,
Germany) was used to prepare the working standard
solutions (0.020.5 mg l 1).
. A standard stock solution containing 1.2 mg ml 1
furosine (Neosystem Laboratoire, Strasbourg,
France) was used to prepare the working standard
solution.
. A standard stock solution containing 1.256 mg l 1
HCl 8.1 M DNP-lysine (Sigma Chemical Co) was
used to prepare the working standard solution (1 ml
with water to 10 ml).
. Lysine derivative reagent: 1-uoro-2-4-dinitrobenzene (FDNB) solution (Sigma Chemical Co)
in 3% ethanol.
514

Sample preparation

Commercial samples of sliced bread were made by a

bread-making company. The bread formula and
conditions used by the company were: wheat our
(50 kg), water (27 kg), baker's yeast (2 kg), NaCl
(1 kg), previously fermented dough (5 kg) and doughconditioning agents; fermentation at 30 C for 60 min;
and baking at 200 C for 50 min. The 700 g loaves were
cut into 10 mm thick slices weighing 25 g each. The
slices were toasted in the laboratory in a Moulinex
model T-90 toaster (Spain). Two assays were carried
out: prolonged toasting timeswhole (crust and crumb)
bread slices were toasted for between 5 and 60 min;
reduced toasting timesbread slices without crust were
toasted for between 0.5 and 5 min. The toasted bread
slices of both assays were ground in a Wiley mill to pass
through a 40-mesh screen and stored in screw-capped
bottles at 40 C. Each assay (prolonged and reduced
toasting times) was performed on bread slices from
different loaves in duplicate samples. All determinations were carried out in duplicate.
Colour

The colour of the toasted and untoasted bread samples

was measured in a reectance spectrophotometer
using the CIE L*, a*, b* colour system, where L* is
lightness, a* is redness and b* is yellowness.20 The
results are expressed as 100 L* and the colour
difference (DE) between the untoasted bread and the
toasted samples was determined according to the
following
equation:25
DE = (DL2 Da2 Db2)1/2,
where DL is the brightness difference, Da is the redness
difference and Db is the yellowness difference. The
samples were lyophilised prior to the analysis.
Hydroxymethylfurfural (HMF)

HMF determination was performed by HPLC following the Garca-Villanova et al method.21 A Konik
liquid chromatograph was used and the HMF was
separated in a reverse phase C18 column (Spherisorb
S5 ODS2, 250 mm  4 mm id; Sugelabor, Spain).
HMF was extracted by shaking and centrifugation
with deionised water, and the supernatants were
claried with Carrez solutions.
A284 and A420

The aqueous extracts obtained from HMF extraction21 were measured at 284 and 420 nm in a UV/VIS
spectrophotometer using as a reference material the
solution obtained from untoasted bread. The solutions
measured at A284 had to be diluted with water before
determination.
Furosine

Furosine determination was performed by the

method described by Guerra-Hernandez and Corzo.7
A Perkin Elmer HPLC was used and the furosine was
obtained by hydrolysis with HCl, puried with a
Sep-pak C18 cartridge (Millipore) and separated by
ion pair reverse phase chromatography using a
J Sci Food Agric 81:513518 (online: 2001)

Browning of sliced bread

Table 1. Colour parameters at prolonged toasting times

Time (min)
0
5
7
10
14
18
21
25
30
40
60

Table 2. Browning parameter correlations

a*

b*

L*

100 L*

DE

2.2
2.6
4.1
3.6
7.0
6.3
10.3
9.8
11.5
13.6
14.1

15.1
16.7
20.1
18.5
23.8
22.8
29.4
29.0
30.4
31.3
30.0

84.1
81.6
80.6
78.8
74.6
72.2
67.7
67.1
60.9
54.4
48.9

15.9  0.17
18.4  0.19
19.4  0.17
21.2  0.18
25.4  0.19
27.8  0.17
32.3  0.15
32.9  0.15
39.1  0.13
45.6  0.12
51.1  0.11

3.0  0.03
6.4  0.06
6.4  0.06
13.8  0.13
14.7  0.14
22.0  0.35
23.3  0.13
29.3  0.32
35.7  0.35
40.0  0.40

n = 4.

Spherisorb ODS2 5 mm column (250 mm  4.6 mm

id; Phenomenex, Torrance, USA).
Available lysine with FDNB (DNP-L)

Available lysine was determined by Carpenter's

method10 as modied by Booth.11 e-DNP lysine was
determined by spectrophotometric measurement at
435 nm after hydrolysis of the FNDB derivate.
Moisture, protein and reducing sugar
determinations

Moisture was determined by a gravimetric method

(AOAC method 925.10).26 Protein was determined by
the Kjeldahl method (AOAC method 920.87).26
Reducing sugars were determined by a titrimetric
method (AOAC method 939.03).26
The results for the different parameters are expressed on a dry matter basis.
Statistical analysis

The Sigma package (Horus Hardware SA, Madrid,

Spain) was applied to study parameter correlations.

RESULTS AND DISCUSSION

Colour

The reproducibility of the refractometer colour

method was studied on bread (n = 7). The coefcients
of variation (CV) were 2.92%, 2.28% and 0.30% for
a*, b* and L* respectively. The colour parameters
considered by other authors were DE for bread4 and
corn and soy extruded products24 and 100 L* for
pasta.18 Fernandez-Artigas et al 20 applied both parameters to processed baby cereals.
Assay at prolonged toasting times
Table 1 illustrates the effects of toasting on the colour
values. The browning index (DE) for toasted bread
increased with heat treatment time in comparison with
untoasted bread. The linear correlation between DE
and time was r2 = 0.913 (Table 2). When 100 L*
values were considered, the linear correlation was
r2 = 0.962 (Table 2).
The absorbance at 420 nm measures the colour of
J Sci Food Agric 81:513518 (online: 2001)

Correlations a
100 L*/time
DE/time
HMF/time
Furosine/time
DNP-L/time
A284 /time
A420 /time
HMF/100 L*
Furosine/100 L*
DNP-L/100 L*
HMF/furosine
DNP-L/furosine
DNP-L/HMF
A284 /A420
A284 /HMF
A420 /100 L*
A420 /DE
a

Assay at prolonged
toasting times

Assay at reduced
toasting times

(r2)

(r2)

0.962
0.913
0.866
0.712
0.694
0.927
0.939
0.871
0.834
0.788
0.612
0.748
0.515b
0.967
0.964
0.935
0.901

0.919
0.835
0.952
0.610b
0.903

0.954
0.530c
0.755b
0.639b
0.491c
0.855

All correlations have a value P < 0.01; b P < 0.05; c P < 0.1.

water-soluble compounds and has also been applied in

kinetic studies of baked dough.23 The reproducibility
of A420 was studied on sliced bread at two times of
toasting (5 and 40 min). The coefcients of variation
(CV) were 3.56% (n = 7) and 4.04% (n = 7) respectively. These values increased during heat treatment
(Table 3). The linear correlation between A420 and
time was r2 = 0.939 (Table 2). The relation between
100 L* (ground bread sample) and A420 (watersoluble compounds) was r2 = 0.935 (Table 2).
Assay at reduced toasting times
Table 4 shows the effects of toasting on the colour
values. The colour increased most at 4 and 5 min. The
100 L* value (13.9) at t = 0 was lower than for the
assay at prolonged toasting times, since this sample did
not contain the crust. The linear correlation between
DE and time was r2 = 0.835, while r2 was 0.919 for the
correlation 100 L*/time (Table 2).

Table 3. Absorbance at prolonged toasting timesa

Time (min)
5
7
10
14
18
21
25
30
40
60

A420

A284

0.010  0.0004
0.025  0.001
0.026  0.001
0.029  0.001
0.032  0.001
0.057  0.002
0.069  0.003
0.132  0.005
0.140  0.006
0.198  0.008

0.034  0.0002
0.153  0.001
0.209  0.002
0.520  0.005
0.682  0.007
0.741  0.008
1.323  0.02
2.260  0.04
2.910  0.06
3.328  0.07

n = 4.
a
The toasted samples were measured against an
untoasted sample.

515

A Ramrez-Jimenez et al

times and 1.3 mg kg 1 HMF for the bread used in the

assay at reduced toasting times. The different HMF
values are due to the presence of crust in the sample
with higher content.

Table 4. Colour parameters at reduced toasting times

Time (min)
0
0.5
1
2
3
4
5

a*

b*

L*

100 L*

DE

0.65
0.65
0.70
0.70
0.75
1.15
1.40

13.40
13.50
13.50
13.55
13.55
14.65
15.65

86.15
86.07
85.75
85.73
85.56
85.22
84.57

13.85  0.07
13.93  0.04
14.25  0.04
14.27  0.08
14.44  0.08
14.78  0.13
15.43  0.17

0.14  0.02
0.42  0.03
0.45  0.10
0.62  0.09
1.64  0.15
2.88  0.04

Assay at prolonged toasting times

The toasting of sliced bread (25 g) for 10 min more
than doubled the HMF content (26 mg kg 1) (Table
5). The HMF content increased by approximately
2000 mg kg 1 after toasting for 40 min (Table 5). The
linear correlation between HMF and time was
r2 = 0.866 (Table 2). After 14 min, when the loss of
moisture is very slight, the values ranged from 78 to
2024 mg kg 1. Using an HMF value determined by
HPLC and by applying the coefcient of molar
absorptivity ( = 16 830), it is possible to calculate a
theoretical absorbance value at 284 nm for the HMF
determined by HPLC. After 30 min the contribution
of the HMF to the A284 value was 74%, at 40 min it
was 97% and at 60 min it was 94%. After 30 min of
toasting, HMF is probably produced by caramelisation and is presumably not a result of the Maillard
reaction.

n = 4.

The 100 L* index showed a better correlation with

time than did DE. Therefore 100 L* may be a more
suitable parameter in such studies. The absorbance at
420 nm is a simple measure for the browning index of
toast and could be useful for rapid control of the
toasting process.
HMF

The identity and purity of the chromatographic peak

were conrmed by diode array detection. The precision (n = 7) for bread with 3.41 mg kg 1 HMF was
1.57% (CV), increasing to 2.60% for high concentrations (176.1 mg kg 1). No HMF was detected in the
dough. This suggests that the increase in this parameter during the heat-processing step may be a useful
indicator. The baking of the bread loaf was performed
at 200 C for 50 min and produced 11.8 mg kg 1 HMF
for the bread used in the assay at prolonged toasting

Time
(min)

Table 5. HMF, furosine and available lysine

contents at prolonged toasting times
(expressed on a dry matter basis)

0
5
7
10
14
18
21
25
30
40
60
a

516

0
0.5
1
2
3
4
5
a

Furosine
HMF (mg kg 1)
11.8  0.23
14.8  0.28
20.7  0.31
26.0  0.30
78.1  0.78
168.4  1.59
195.3  1.75
408.7  3.74
1096.4  7.60
1853.7  10.1
2024.8  15.3

mg kg

242.7
290.1
326.4
289.6
201.6
144.0
86.4
36.0
18.0
7.2
3.2

DNP-L

mg per 100 g protein a

222.7  4.0
266.1  4.7
299.4  4.9
265.7  4.4
185.0  3.1
132.1  2.2
79.3  1.0
33.0  0.7
16.5  0.3
6.6  0.1
2.9  0.1

g kg

protein a

17.5  0.3
13.7  0.2
13.6  0.5
13.5  0.3
11.4  0.5
11.5  0.4
10.4  0.3
8.9  0.2
8.2  0.4
8.3  0.5
8.5  0.4

Loss (%)

21.7
22.3
22.9
34.9
34.3
40.6
49.1
53.1
52.6
51.4

N  5.70; n = 4.

Time
(min)

Table 6. HMF, furosine and available lysine

contents at reduced toasting times
(expressed on a dry matter basis)

Assay at reduced toasting times

The HMF values increased with the heating from
1.3 mg kg 1 (t = 0) to 4.2 mg kg 1 (t = 5 min) (Table
6). Thus an increase in HMF content can still be
observed when the toasting times are reduced. The

Furosine
1

HMF (mg kg )
1.3  0.02
1.6  0.02
1.6  0.02
1.9  0.03
2.6  0.04
3.2  0.05
4.2  0.06

mg kg
106.5
72.3
62.3
100.6
110.5
122.2
138.9

mg per 100 g protein

97.8  1.7
66.3  1.3
57.2  1.5
92.4  1.1
101.4  0.8
112.2  1.0
127.4  1.5

DNP-L
a

g kg

protein a

19.6  0.01
19.0  0.21
19.0  0.01
18.9  0.13
17.4  0.21
17.4  0.22
17.1  0.17

Loss (%)

3.1
3.1
3.6
11.2
11.2
12.8

N  5.70; n = 4.

J Sci Food Agric 81:513518 (online: 2001)

Browning of sliced bread

linear correlation between HMF and time was

r2 = 0.952 (Table 2).
A study of the HMF content in commercial toasted
sliced breads27 determined values between 13 and
90 mg kg 1, with a mean value of 30 mg kg 1. Our
corresponding values were obtained at 1014 min of
toasting. Toasting times of 520 min (frequently used
for this type of bread) show a linear increase with a
shallow slope. A steeper slope can be observed
between 20 and 40 min. The HMF value at 60 min
was slightly higher than at 40 min. Heat treatment
until the product was nearly burnt reduced the HMF
value to 1337 mg kg 1.
A high correlation between HMF and 100 L*
(colour index) was found in the study at reduced
toasting times (r2 = 0.954). A good correlation
(r2 = 0.871) was also found during the assay at
prolonged toasting times. Colour index and HMF
are adequate indicators to control the toasting time.
Studies carried out on the toasting of single and
mixed wheat-based and rice-based ours gave values
of 15 mg kg 1 HMF.20 In commercial breakfast
cereals21 the HMF values ranged from 4 to 193 mg
kg 1. Acquistucci and Bassotti28 and Resmini et al 18
found 7.0 and 0.45 mg kg 1 HMF respectively during
pasta drying.
The reproducibility of the A284 value was studied
with seven samples of sliced bread at two times of
toasting (5 and 40 min). The coefcients of variation
(CV) were 0.68% and 2.10% respectively. The
absorbance of water-soluble compounds at 284 nm
(Table 3) showed a linear increase with toasting time
(r2 = 0.927). The correlation between A284 and HMF
was 0.964 (Table 2). A284 is a rapid and sensitive
parameter and could be an adequate browning index
for such samples.
Furosine

Assay at prolonged toasting times

The furosine content in baked bread (t = 0) was
223 mg per 100 g protein (Table 5). Toasting the
sliced bread produced an increased furosine value after
5 and 7 min of treatment. Furosine levels fell after
10 min. The reducing sugars were determined in the
samples, and the content was between 4.72% for
baked bread (t = 0) and 1.68% for toasted sliced bread
(t = 60 min). The reducing sugar content when the
furosine value began to decrease was 3.5%. The
highest loss of moisture was reached at 1014 min of
heat treatment. Furosine levels began to decrease
when the moisture content fell below 10%. The linear
correlation between furosine and time was r2 = 0.712
(Table 2).
Assay at reduced toasting times
The furosine value at t = 0 was 97.8 mg per 100 g
protein (Table 6). Heating for 0.5 and 1 min produced
a decrease in furosine values, but from 2 min the
furosine content increased. The correlation between
furosine and time showed an r2 value of 0.610.
J Sci Food Agric 81:513518 (online: 2001)

Studies carried out by the authors8 on wheat our

toasted at 140 C showed an increase in furosine
content from 10.6 to 14.3 mg per 100 g protein.
During this heat treatment the moisture content fell
from 13.2% to 1.7% after toasting. A study with two
toasting temperatures showed furosine values of 15.4
and 11.1 mg per 100 g protein at 140 and 150 C
respectively. The results obtained from the two studies
(our and toasted bread) are basically similar. Early
stages of the Maillard reaction are not favoured when
the water content is very low, even if there are
precursors. The furosine degradation was linear until
25 min of heating (r2 = 0.994).
Available lysine with FDNB

Available lysine was determined in untoasted bread to

assess the heat damage produced in the toasting of
sliced bread. The losses of lysine during heating were
determined with respect to sliced bread before toasting
(Tables 5 and 6).
Assay at prolonged toasting times
The contents of available lysine decreased from
17.5 g kg 1 protein (t = 0) to 8.5 g kg 1 protein
(t = 60 min) (Table 5). From 25 min the losses of
lysine were 50%, and subsequently no increase was
observed. The correlation between available lysine and
time was r2 = 0.694 (Table 2).
Assay at reduced toasting times
Available lysine contents decreased at reduced times of
heating from 19.6 to 17.1 g kg 1 protein. The losses of
lysine ranged from 3.1% to 12.8% at 5 min of heating
(Table 6).
Losses of lysine of around 40% have been found in
bread toasted for 20 min which exhibited good
organoleptic quality.
Considerable heat damage has been reported in
heated cereal products such as pasta, breads, breakfast
cereals and biscuits.5 Lightly toasted extruded wheat
breakfast cereals showed no decrease in lysine, while
darkly toasted ones suffered a 50% loss of available
lysine.29 During the industrial production of wheatbased and rice-based baby cereals, available lysine was
affected by the toasting and drying processes. Considerable reduction was observed as a result of the
roller-drying process.16

CONCLUSIONS

Studies of the relationship between browning indicators and times of prolonged toasting (Table 2) showed
high correlations of A420, A284, 100 L* and HMF
with time. In studies at reduced toasting times,
available lysine also showed a high correlation with
time. HMF is the best indicator of browning at usual
toasting times. Absorbance measurement is a simple
and rapid method and therefore could be a useful
technique for rapid control of the industrial process.
When moisture loss during heating is greatest, the
517

A Ramrez-Jimenez et al

HMF content increases considerably, while that of

furosine falls sharply. During this heating stage,
caramelisation could be the predominant reaction.
The loss of lysine reached about 40% when the bread
exhibited good organoleptic quality.

15
16

17

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